Nanodiamonds and silicon quantum dots: ultrastable and biocompatible luminescent nanoprobes for long-term bioimaging.

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Nanodiamonds and silicon quantum dots: ultrastable and biocompatible luminescent nanoprobes for long-term bioimaging M. Montalti,* A. Cantelli and G. Battistelli Fluorescence bioimaging is a powerful, versatile, method for investigating, both in vivo and in vitro, the complex structures and functions of living organisms in real time and space, also using super-resolution techniques. Being poorly invasive, fluorescence bioimaging is suitable for long-term observation of biological processes. Long-term detection is partially prevented by photobleaching of organic fluorescent probes. Semiconductor quantum dots, in contrast, are ultrastable, fluorescent contrast agents detectable even at the single nanoparticle level. Emission color of quantum dots is size dependent and nanoprobes emitting in the near infrared (NIR) region are ideal for low back-ground in vivo imaging. Biocompatibility of nanoparticles, containing toxic elements, is debated. Recent safety concerns enforced the search for alternative ultrastable luminescent nanoprobes. Most recent results demonstrated that optimized silicon quantum dots (Si QDs) and fluorescent nanodiamonds (FNDs) show almost no photobleaching in a physio-

Received 15th December 2014

logical environment. Moreover in vitro and in vivo toxicity studies demonstrated their unique biocompatibility.

DOI: 10.1039/c4cs00486h

Si QDs and FNDs are hence ideal diagnostic tools and promising non-toxic vectors for the delivery of therapeutic cargos. Most relevant examples of applications of Si QDs and FNDs to long-term bioimaging

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are discussed in this review comparing the toxicity and the stability of different nanoprobes.

1. Introduction Versatility of design, as well as unique optical and electronic properties, make nanosized materials ideal platforms for the Department of Chemistry ‘‘G. Ciamician’’, University of Bologna, Via Selmi 2, 40126, Bologna, Italy. E-mail: [email protected]

M. Montalti

Marco Montalti received his PhD in Chemical Sciences in 2001 from the University of Bologna, after being a research assistant at Tulane University. In 2002 he started his independent research career in the field of luminescent silica and metal nanoparticles for sensing and bioimaging. The main research topic of his group is the design, production, and characterization of ultrabright, biocompatible and stimuli-responsive luminescent nanostructures for application to in vitro and in vivo bioimaging.

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development of specifically tailored, multifunctional, luminescent contrast agents for structural and functional imaging of biological systems, both in vitro and in vivo.1–17 Convergent contributions, from different research areas related to material and life sciences, made available a wide variety of individual nano-objects, generically designated as nanoparticles (NPs), dispersible in a physiological environment, and that, for some

Andrea Cantelli was born in Bologna in 1988. He received his Master’s degree in Chemistry in 2012 from the University of Bologna, working on gold nanoparticles for manipulation of autoreactive T-cells, in collaboration with the group of Prof. Molly Stevens of the Imperial College of London. Currently he is pursuing his PhD in the research group of Prof. Marco Montalti. His research is focused on the design, producA. Cantelli tion and characterization of bright, NIR emitting gold nanoparticles for in vivo bioimaging and theranostic applications.

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properties and applications, surpassed the performances of traditional molecular contrast agents.18,19 Although full potential of these nanoprobes, applied to biosciences, is still unexplored, concerns about their safety for the environment and the human health are increasing together with their diffusion.20–22 Intrinsic NP toxicity finds therapeutic applications,23,24 nevertheless, awareness of the possible risks related to uncontrolled effects of NPs led the scientific community to reconsider them under a new light: balancing performances and biocompatibility. The interactions of NPs with living cells and organisms25 involve parameters other than the simple composition, such as size,26 shape,27 structure and surface chemistry.15,28–31 It is hence problematic to identify a general model32 for predicting the toxicity of a given family of nanomaterials, to which typically belong a wide range of different systems.33 Even the definition of markers suitable to quantify the biological damage of NPs,34 as well as the optimization of strategies to manage the possible related risks, are demanding tasks.35 In this framework, biological application of some categories of semiconductor quantum dots (QDs) raised increasing safety concerns because of contents of hazardous elements such as cadmium and lead.36–44 The actual risks related to these NPs, as well as their ability to release their toxic components, are still debated,45 the search for structures with alternative composition and similar imaging performances has been enforced.46 Group II–VI (e.g. CdSe, CdS and CdTl) and II–VI QDs (e.g. PbSe and PbS) are commercially available materials that have been used for many years as imaging probes, both in vitro and in vivo,47 in virtue of their unique photophysical and photochemical features.48 Photoluminescence (PL) of QDs is generated by the quantum confinement effect and their emission maximum can be tuned by changing the size of the nanocrystals (NCs). Moreover their narrow emission bands are easily distinguishable in multiplexed detection. Because of the nature of the involved electronic transition, QDs absorb light in a broad spectral window and multicolor imaging can be achieved by simultaneous excitation of different probes at a single wavelength. These NPs also show high PL brightness49,50 which results from the combination of

G. Battistelli

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Giulia Battistelli was born in Terni in 1989. She received her Master’s degree in Chemistry in 2012 from the University of Bologna where she is currently pursuing her PhD in the group of Prof. Marco Montalti. Her research interests include the design of stimuli-responsive fluorescent nanoparticles, based on doped silica and organic materials, for bioimaging applications. More in the detail, her work is focused on the development of new methods for the production and characterization of ultrabright and biocompatible nanoprobes.

Fig. 1 The NIR window is ideally suited for in vivo imaging because of minimal light absorption by hemoglobin (o650 nm) and water (4900 nm). Reprinted with permission from ref. 51. Copyright 2001, Macmillan Publishers Ltd.

excellent PL quantum yields and high molar absorption coefficients, also in the near-infrared spectral window. Excitation and detection in this region are ideal for in vivo imaging and phototherapy since, as shown in Fig. 1,51 biological tissues and fluids are more transparent to NIR photons.52 The most unique feature of QDs is, anyway, their extraordinary photostability that allows long-term observation even at the single emitter level.42 Luminescent probes with prolonged chemical and photochemical stability promise to become extraordinary tools for investigating biological processes permitting several days up to months lasting detections. Although, systems sharing these features are quite rare. In the perspective of long-lasting observation, toxicity of the probes assumes a meaning other than pure safety, since it may interfere with the reliability of the observation itself. This makes the requirements for biocompatibility even more severe. Biocompatibility and biodegradability of nanostructured silicon have been investigated since the end of the last century.53,54 Silicon is a common trace element in humans, in the form of orthosilicic acid, which is a biodegradation product efficiently excreted from the body through the urine.55,56 From the environmental point of view silicon is the most abundant electropositive element in the Earth’s crust and unprotected silicon nanostructures are readily oxidized to inert species upon exposure to oxygen and humidity.57,58 Intrinsic benign biodegradability and biocompatibility of silicon are ideal features for the design of non-toxic nanoprobes. Nevertheless, the implicit low stability of silicon under physiological conditions delayed its application to bioimaging. Preparation of Si NCs that withstand the contact with a physiological environment, without degrading or losing their optical and electronic properties, was indeed the real challenge in the development of silicon based luminescent nanoprobes for bio-applications.59,60 Recently, several examples of water compatible, strongly luminescent, Si QDs have been reported. In contrast to bulk silicon, that is a weakly luminescent, indirect band gap semiconductor,


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these Si NCs showed features comparable to direct band gap semiconductor NCs. Moreover the use of Si QDs for long-term bioimaging has been demonstrated, both in vitro and in vivo, and it will be treated in this review article. Most recent results demonstrated that Si QDs, if properly designed and surface protected, exhibit chemical and photochemical stabilities superior to metal based NCs. Moreover the low toxicity of Si QDs on living organism has been demonstrated by very detailed in vivo and in vitro studies also considering long-term effects of these NPs in complex animal models such as monkeys. In contrast to nanocrystalline silicon, diamond is well known for its extreme chemical stability. Although silicon and diamond share the same crystalline structure, arising from tetrahedral sp3 hybridization, they have completely different optical and electronic properties, diamond being an insulator. The production of diamond in the form of nanocrystals was pursued both via top-down and bottom-up approaches and it was mainly aimed to the application of the nanopowders as abrasive. The potential application of nanodiamonds (NDs) as diagnostic and therapeutic agents was explored in view of their high chemical inertness and biocompatibility. In this framework NDs61–75 showed, as a major drawback, a marked tendency to form large aggregates in an aqueous environment and the lack of any bright intrinsic PL. Observation that exposition of NDs to ion beams, followed by thermal annealing and oxidative treatment, yielded to stable, water dispersible, strongly fluorescent NPs with unique photostability represented a real breakthrough. Moreover, after activation, these fluorescent NDs (FNDs) emit in the far red region and they are hence suitable for low-background in vivo detection. As expected based on the characteristics of the bulk material recent results also demonstrated an unprecedented high biocompatibility of FNDs both in vitro and in vivo. 1.1

Challenges in applying Si QDs and FNDs to bioimaging

Si QDs and FNDs share unique chemical, photophysical and photochemical features, such as: high brightness, emission color tunability extended to the NIR region, and an extreme photostability. For these properties both classes of NPs are, from the application point of view, interesting biocompatible alternatives to metal containing QDs in bioimaging. Integration of Si QDs and FNDs into nanoprobes for longterm bioimaging requires an accurate and sophisticated design that takes into consideration the mechanisms of interaction of NPs with living organisms. At the cellular level, as schematized in Fig. 2,76 surface functionalization, final probe size and shape play a fundamental role in determining the mechanism of internalization of NPs,77 as well as the degree and reversibility of their accumulation in the cellular compartments and their cytotoxicity.78,79 For in vivo application, the same morphological and physicochemical parameters affect the circulation time of the probes in the body, their interaction with the reticuloendothelial system, their biodistribution and activity, the kinetics of accumulation and elimination and their toxicity. As shown in Fig. 380 conjugation of the probes to biomolecules, able to target specific receptors, is an efficient strategy to drive the probes towards selected cells, tissues or organs. As an alternative, passive targeting has been


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Fig. 2 Cellular uptake pathways for nanoparticles. Several routes were discovered for cellular NP uptake, including phagocytosis (A), macropinocytosis (B), clathrin-mediated endocytosis (C), caveolin-mediated endocytosis (D), and non-clathrin- and non-caveolin-mediatedendocytosis (E). Parameters that regulate cellular uptake (F). Reproduced with permission from ref. 76. Copyright 2013, American Chemical Society.

proposed as a very general approach to deliver diagnostic or therapeutic cargos to cancer tissue exploiting the enhanced permeability and retention (EPR) effect.80 The ability to control the relevant physicochemical parameters that regulate the short and long-term interaction of nanomaterials with living organisms entail, as a simple but fundamental requirement, the stabilization of the NPs against aggregation. Colloidal stabilization of Si QDs and FNDs, in a physiological environment, represented a major challenge for their application as contrast agents to bioimaging. Successful strategies to achieve stable aqueous dispersions of these NPs, which are strongly prone to aggregate, will be discussed. Si NCs suffer, as a further complication, an intrinsic chemical instability in a biological environment. This together with the difficulties in producing a significant amount of material with controlled features delayed their use for bioimaging with respect to other semiconductor QDs. As will be discussed in next sections important breakthroughs yielded functionalized Si NCs with unprecedented stability whose use for long-term PL imaging was demonstrated both in vitro and in vivo. 1.2

Ultrastability versus biocompatibility

Long-term PL bioimaging requires the development of chemically and photochemically extremely stable contrast agents:

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Fig. 3 Passive uptake vs. active targeting. (A) Untargeted NPs with size up to a few hundred nanometers do not normally accumulate in healthy tissues, (B) the enhanced permeation and retention (EPR) effect. NPs accumulate in tissues with abnormally permeable vessels, (C) large particles, 10–50 mm are mechanically retained, (D) active targeting of nanocarriers coated by affinity. Reproduced with permission from ref. 80. Copyright 2014, American Chemical Society.

properly modified Si QDs and FNDs meet these requirements. Nevertheless, ultrastability of NPs is, at least in principle, in part incompatible with their low impact on the target organism and hence with a long-term lack of toxicity. The implicit risk of producing ultrastable NPs is to create objects that living organisms cannot degrade even after they have terminated their function as signaling moieties. Although the experimental results that we will discuss in next sections demonstrated almost univocally the safety of Si QDs and NDs, their actual, irreversible, partial accumulation in the living organisms was not excluded and in some cases it was confirmed. Probes with programmable, long-term stability may, in principle, be safer than ultra-stable ones and, in this perspective, the poor stability of silicon to oxidation has been exploited to achieve probes with a controlled degree of bio-degradability. 1.3

Towards background-free long-term bioimaging

FNDs show, as a very unique feature, an unusually strong response of their PL to magnetic and electromagnetic stimuli. Modulation of PL of FNDs with magnetic fields or microwaves promises to become a powerful tool for minimizing background, especially for in vivo detection. The fundamentals and the first demonstrations of FNDs based, background-free imaging systems will be discussed. 1.4

Overview

In this review article, we will analyse the recent experimental reports that demonstrated the applicability of Si QDs and FNDs as ultrastable, highly biocompatible, luminescent nanoprobes for long-term bioimaging, both in vitro and in vivo (Sections 7–10). Actual biocompatibility of the NPs will be assessed by combining in vitro and in vivo toxicity data. PL stability, in comparison

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with other probes, will be discussed based on the available results. In the case of FNDs, very promising and very recent methods for background-free bioimaging, based on non-optical signal modulation, will be highlighted. In Section 2 the origin of the PL of Si NCs and FNDs will be surveyed in order to identify the parameters that regulate the photophysical properties of the NPs in view of the design of ultrastable nanoprobes. Considering the fundamental importance of the synthetic design and surface chemistry in determining the features of nanomaterials, methods of synthesis and functionalization of Si QDs and FNDs will be examined in detail in Sections 3–6. In the final part (Section 11) the two classes of materials will be directly compared with the objective of finding out the issues of possible improvement and the challenges for the future research.

2 Photophysical properties of Si QDs and FNDs Diamond and crystalline silicon exhibit the same sp3 lattice structure but they have very different electronic properties. When reduced to nanometric size, these materials share important chemical, photophysical and photochemical features that make them, for some aspects, superior to metal containing semiconductor QDs for bioimaging. Nevertheless, because of the substantial differences in the origin of their PL they also show fundamental dissimilarities. In particular, fluorescence of FNDs is due to the presence of local defects and, at least in principle, it is not expected to be affected by the crystal size. Different emission colors of FNDs are related to the presence of diverse emitting defects but not to their size. In contrast, with some exceptions that will be discussed, PL of Si NCs arises from


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quantum confinement of charge carriers, and light emission properties are directly correlated with the NC diameters.


2.1

Photophysical properties of Si QDs

Bulk silicon is an indirect band gap semiconductor81 and it exhibits very weak PL as well as a long exciton-hole recombination time.57–60 This feature, together with the difficulties of preparing significant amounts of Si NCs, stable in an aerated, aqueous environment, delayed the development of Si NPs for bio-applications with respect to other QDs composed of semiconductors strongly emitting at the bulk level. Emission of QDs82 arises from quantum confinement83 of the excitons. This phenomenon originates discrete density of states with a sizedependent energy gap. The quantum confinement effect is relevant when the size of the NCs becomes smaller than the exciton Bohr radius (4.2 nm for silicon) and it causes the energy of the optical transitions to be dependent not only on the properties of the hosting material, but strongly on its size.83–87 Theoretical investigation of exciton quantum confinement in silicon, based on the effective-mass approximation, predicted an indirect-to-direct conversion of the character of the optical transition for Si crystals, when reduced to nanometric size.83 According to this model, the radiative lifetime of excitons is strongly size dependent, going from millisecond to nanosecond when the diameter is reduced from B3 nm to B1 nm.83 Although permitted transition with decay time of the order of nanoseconds has been reported for Si QDs, their actual origin is still debated.81 Freestanding Si NCs used for bio-applications are indeed far from being ideal QDs and the interpretation of their photophysical properties is complicated. In perfect QDs, the hosting material is confined into an ideal infinite, wider band-gap

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matrix, with an identical lattice structure. In the case of CdSe, for example, the structural continuity is achieved by incorporating the core material in a shell of ZnS.88 The surface of the freestanding Si NCs used as bioprobes is typically terminated by carbon connected organic residuals, oxygen or thin silica layers. Hence, these Si NCs are surrounded by a monolayer, or a thin shell, rather than by a long range, electron confining matrix. Further complexity arises from the fact that under a certain size limit, the concept of crystallinity becomes looser due to an insufficient number of atoms and the lack of long-range order.89 Experimental and theoretical results revealed a fundamental influence of the surface termination90,91 and of surface states14,92 on the photophysical properties of Si NCs.89 As shown in Fig. 4,81 the rate of the electron–hole recombination, for nano-sized silicon, is strongly dependent on the surface terminating atoms. The overlap of the hole and electron wave-functions is much larger in the case of carbon termination with respect to hydrogen capped NPs. In the case of oxygen terminated NCs, PL is dominated by surface states that show a characteristic long time, low energy emission in the microsecond decay region as shown in Fig. 5.81 The complexity of the photophysical behavior of Si NCs becomes evident when it is considered that NPs prepared with different methods, apparently structurally and dimensionally identical, were reported to show radically different PL responses.93 In general, NCs prepared using high-temperature methods show PL features that agree with the effective mass approximation, while those prepared via solution methods exhibit, almost size independent blue emission. Dasog et al. analyzed this apparent dichotomy and they identified the origin of the blue emission in the presence of trace nitrogen and oxygen

Fig. 4 Schematic illustration of the dominant radiative channels in H-, O- and C-terminated Si QDs. Hole and electron density in the lowest excited state are depicted in red and blue, respectively. Left: in H-terminated Si QDs, slow radiative rate PL originates from phonon-assisted recombination. Centre: in O-terminated Si QDs, slow PL is O-defect-related. Right: in C-terminated Si QDs, the radiative rate is dramatically enhanced as a result of direct phonon-less character of the recombination. Reprinted with permission from ref. 81. Copyright 2013, Macmillan Publishers Ltd.


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Fig. 5 PL from C- and O-terminated Si QDs. (a) PL spectra of C-terminated (green), partially oxidized (orange) and heavily oxidized (red) Si QDs. The oxidized sample shows two contributions around 1.85 and 2.4 eV, whereas the C-terminated shows only the latter band. (b) PL decays recorded at 1.85 eV (red) with decay tPL B 12 ms and at 2.4 eV with decay of tPL B 4 ns. (c) Schematic illustration of carrier recombination processes. Reprinted with permission from ref. 81. Copyright 2013, Macmillan Publishers Ltd.

even at the parts per million level.93 Defects and surface chemistry, hence, play a fundamental role together with size in controlling the photophysical properties of Si QDs.94,95 As will be shown, depending on the synthetic approach, Si NCs present a variable degree of porosity, different surface termination and a number of defects and impurities, as well different size and size distributions. Emission energy of polydispersed Si QDs is strongly dependent on the excitation wavelength as shown in Fig. 6. The observed spectral shift results from a combination of size dependencies of the bandgap energy, absorption cross-section and radiative rate.81 As a result of these factors, a time dependent shift of the spectrum during fluorescence decay is observed as shown in Fig. 6. A similar effect, but not due to NP polydispersity, was reported in the case of Si QDs with sizes of 2.5, 3, 3.5, 4, 4.5 and 5.5 nm, embedded in a SiO2 matrix. For these NCs, a short living emission due to the radiative recombination of non-equilibrium electron–hole pairs, in a process that does not involve phonons, was observed.96 As schematized in Fig. 7,96 this fast emission occurs immediately after excitation, from not thermally relaxed (hot) states and it is blue shifted with respect to the delayed thermalized PL. The short-lived hot PL band increases in intensity and shifts to longer wavelength for smaller nanocrystal sizes. 2.1.1 Emission color of Si QDs. Emission due to excitonhole recombination in quantum confined materials shows a narrow, symmetric spectral band centered at a wavelength which can be tuned by changing the crystal size in a wide range. Experimental evidence of optically allowed band gaps97,98 and emission wavelength tunability in low-dimensional silicons was reported for hydrogen terminated crystals99 with diameters ranging from

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1.0 to 3.7 nm. These NPs showed bright luminescence with color shifting from blue, to green, to red by increasing the size. Interestingly the smallest crystals were estimated to be clusters of just 29 (1.0 nm) and 123 (1.67 nm) atoms. Kang et al. prepared a set of highly monodisperse hydrogen terminated Si QDs (B1, B2, B3, and B4 nm) with a narrow size distribution using an electrochemical method.100,101 The NPs exhibited the size dependent PL spectra reported in Fig. 8. The red shift of the PL band, with increasing Si QD size, and the larger band energy than the Si indirect band gap of 1.1 eV demonstrate distinctly the quantum confinement effect on the band gap energy in Si quantum dots smaller than 4 nm. The same authors investigated the effect of controlled oxidation of their 3 nm H terminated Si QDs. This process was exploited to tune the Si core size, and it yielded Si QDs with fine wavelengthtunable PL as shown in Fig. 9.102,103 Size dependent color emission was reported by English et al.104 who investigated the photophysics of a set of octanol capped Si NCs (d = 1.0–10 nm), at the single particle level, measuring PL quantum yields up to 23% and nanosecond decay times consistent with a direct band gap transition even for NCs as large as 6.5 nm. Emission band tunability was also demonstrated for Si NCs prepared by laser pyrolysis and with plasma based methods upon etching with HF–HNO3 mixtures and surface stabilization. Recently, Dasog et al. demonstrated that the PL of Si QDs, prepared by high temperature decomposition of hydrogen silsesquioxane (HSQ), can be effectively tuned across the entire visible spectral region, without changing particle size, via rational variation of the surface moieties as shown in Fig. 10.105 The surface-state dependent emission showed short-lived excited-states and higher relative PL quantum yields compared to materials of equivalent size


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Fig. 6 Tunable PL of butyl-terminated Si QDs. (a) Si QDs upon different excitation wavelengths, (b) fast (4 ns) PL lifetime detected at 2.6 eV and spectral dependence of PL decays. (c) The corresponding PL spectra of the Si QDs plotted together with optical density. Reprinted with permission from ref. 81. Copyright 2013, Macmillan Publishers Ltd.

Fig. 7 Right: schematization of the transition involved in the fast emission arising from thermally hot and relaxed states. Left: hot and thermalized PL spectra are shown as continuous line and dots, respectively, as a function of the Si NCs size. Reprinted with permission from ref. 96. Copyright 2010, Macmillan Publishers Ltd.

exhibiting emission originating from the band gap transition. Strong excitation wavelength dependence of the emission spectrum of Si NCs been reported in some cases.106 A recent approach


proposed to control the PL properties of Si NCs by controlled doping of the NCs with other elements107 or by changing the shape to give nanorods.108

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Fig. 8 (a) Schematic for the POM-assisted electrochemical etching process. (b) TEM image of a set of SiQDs and their corresponding luminescence colors under UV irradiation (inset). (c) Typical PL spectra of SiQDs with sizes ranging from 1 to 4 nm. Reproduced with permission from ref. 100. Copyright 2007, American Chemical Society.

Luminescence quantum yield of Si NCs in biological probes typically ranges from 5% to 30%, nevertheless, water-dispersible silicon nanoparticles with quantum yields up to 75% were prepared by chemical surface modification.109 An original strategy to control Si QDs emission was proposed by Zhang et al. who exploited the short range exciton coupling of Si QDs at an inter-dot distance lower than 5 nm, after incorporation into nanospheres, in combination with Si NC surface modification.110,111 Functionalization of Si QDs with organic fluorophores has also been proposed to obtain hybrid materials with tailored photophysical properties exploiting excitation energy transfer112–116 processes.117,118 These materials maintain only in part the characteristic properties of Si QDs. 2.1.2 Absorption. The molar absorption coefficient (e) is also important in determining the actual brightness of luminescent contrast agents. In the low concentration probe regime, typical of luminescence based imaging techniques, PL signal intensity is proportional to the NP absorbance.49,50 Nevertheless, a precise determination of e for NPs is, in general, not trivial since it implies the ability of counting the number of particles per volume unit.119 Hessel et al. discussed the electronic absorption properties of Si NCs produced by high temperature decomposition of HSQ dispersed in toluene and having a diameter in the 3 to 12 nm range.120 The absorbance spectra of these NPs were essentially featureless compatibly with an indirect band-gap in this size range. The absorption cross-section was strongly size and wavelength dependent: for example at 400 nm the molar absorption coefficients were e = 3 104 M 1 cm 1 for the 3 nm NCs and 41 106 M 1 cm 1 for the 12 nm NCs. At 650 nm, these same NPs showed much weaker absorption e = 4 102 M 1 cm 1 and e = 2 105 M 1 cm 1 for the smaller and larger respectively. The last value is comparable to the molar absorption coefficients of direct band-gap NIR emitting QDs (2–5 105 M 1 cm 1 at

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the excitonic maxima). For smaller NCs of 1.5 nm Holmes et al.121 reported an e value of B1 104 M 1 cm 1 at 400 nm. 2.1.3 Photoblinking. Fluorescence blinking is a typical behavior of semiconductor QDs that limits to some extent their application for long time single particle tracking but which is also useful to distinguish single dots from aggregates in imaging experiments.42 Most studies about Si QDs report no photoblinking of these materials. Nevertheless these data are often relative to NPs containing several dots or aggregates. Photoblinking was indeed reported in the case of experiments carried out at the single dot level.104 2.2

Photophysical properties of FNDs

In this review article we discuss the application of FNDs to bioimaging. The photoactive parts of these NPs are structural defects in the sp3 carbon lattice. Although the nanometric emitting diamond cores can present different kinds of surface termination, including reconstruction to give sp2 graphitic like shells, in FNDs this shell is not directly involved in originating light emission. Recently, a large variety of very interesting carbon nanomaterials, composed of emitting graphene like structures, have been proposed for bioimaging. Properties of these carbon or graphene dots,122–125 as well as of other carbon based nanomaterials and their application as contrast agents for bioimaging have been recently reviewed and will not be discussed here.126–129 The most relevant effect of reducing size of diamonds to the nanometric scale is a huge increase of the fraction of surface atoms with respect to the bulk ones.61,66,74 The nature of the surface hence strongly affects the thermodynamical stability of the nanomaterials and parameters such as size and the surface termination, usually neglected on the macro scale, have to be take into consideration in the phase diagram at the nanometric level.61 Surface energies, indeed, are fundamental in the stabilization of nanocrystalline diamonds.


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Fig. 9 (a) TEM images of B3 nm H terminated Si QDs. (b–d) TEM images of Si QDs after oxidation of 0.5, 3.5, and 24 h, respectively. Insets are the corresponding fluorescent microscope and HRTEM images. (e) Photograph (under UV light) of H terminated Si QDs (left, red emission) and water-soluble Si QDs (yielding seven distinct emission colours). (f) PL spectra of H terminated Si QDs (curve 1) and oxidizes Si QDs (curves 2 to 8) after 0.5, 1.5, 3.5, 6, 9, 14, and 24 h oxidation, respectively (excitation wavelength: 360 nm). Reproduced with permission from ref. 102. Copyright 2009, John Wiley and Sons.

Fig. 10 Photograph of 3.4 nm Si NCs functionalized with various surface groups dispersed in toluene, under UV illumination. From left to right the Si NC functionalization: blue, dodecylamine; blue-green, acetal; green, diphenylamine; yellow, TOPO; orange, dodecyl (air); red, dodecyl (inert). Reproduced with permission from ref. 105. Copyright 2014, American Chemical Society.

In the case of hydrogen terminated structures it was calculated that diamonds smaller than 3 nm in diameter are energetically


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favored over graphitic structures.130 Good stability of small –H terminated diamonds, called diamandoids, was confirmed by the possibility of separating them from other components of oil, where they are present in traces, exploiting their extraordinary resistance to pyrolysis.131 For sizes of about 3 nm, particles with bare, reconstructed surfaces arising from transition from the sp3 to sp2 lattice132 become thermodynamically more stable than those with hydrogenated surfaces.133 This effect favors the formation of buckydiamonds (onion-like graphitic shells with diamond cores) for larger clusters.134 Transmission electron microscope (TEM) images of purified NDs confirm that they consist of polyhedra diamond cores of sp3 carbon atoms, partially coated by a graphitic shell or amorphous carbon. The surface presents dandling bonds, as well as functional groups, mostly deriving from oxidation by atmospheric oxygen as schematized in Fig. 11.61 From the optical point of view, NDs are strongly scattering objects because of their high refractive index but do not absorb light and do not show any luminescence unless they present structural crystal defects.135 Interestingly diamonds may host over 500 kinds of photon absorbing defects (color centers) some of which emit as bright single-photon sources at room temperature.136 2.2.1 Origin of the emission of FNDs. Whereas the emission of Si NCs results from quantum confinement effects, NDs show PL suitable for bio-imaging only when they contain defects in the structure that produce localized excited states upon light absorption. In almost all the cases of FNDs used for bio-imaging, emission of light arises from the presence of nitrogen atoms,137 as dopants, in the carbon sp3 structure; a significant exception is represented by silicon defect containing NDs.138–140 Nitrogen atoms originate a set of structurally and electronically different local defects62,141–144 that absorb incident light and emit at characteristic wavelengths.145,146 Nitrogen content is hence a fundamental criterion in the choice of the diamond precursors for FND preparation, since it limits the maximum density of color centers achievable in the materials, and consequently their potential brightness, as well as the probability of having defects containing two or more adjacent N atoms.147 Nitrogen content of NDs, on the other hand, depends on their production method. NDs are created either by top-down processes, as from the fragmentation of micrometric synthetic or natural diamonds, or less frequently, by bottom up approaches. In general, diamonds containing nitrogen impurities are classified as type I while those with no measurable trace of N as type II.148,149 Only type I diamonds hence are suitable for producing luminescent probes. Moreover type I diamonds are sub-classified into type Ia, containing N atoms up to 3000 ppm mainly in the form of aggregates (most natural diamonds belong to this category), and type Ib containing less nitrogen and in the form of isolated atoms (most artificial diamonds). As for analogous doped materials the brightness of FNDs is dependent on the average number of emitting centers per particle.49 This parameter is controlled by crating artificially the color sites in the synthetic or natural diamonds by radiation damaging with ion beams followed by thermal annealing. The former process produces stationary vacancies of carbon atoms

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Fig. 11 Structure of a single ND. (a) Schematic model of a single 5 nm ND after oxidative purification. The diamond core is covered by a layer of surface functional groups, which stabilize the particle by terminating the dangling bonds. (b, c) Close-up views of two regions of the NDs shown in (a). (b) The majority of surface atoms are terminated with oxygen-containing groups (c; oxygen atoms are shown in red, nitrogen in blue). Some hydrocarbon chains (green) and hydrogen (white) terminations are also seen. (d) Transmission electron micrograph, whereas most have a rounded shape, as shown in a. In the inset the fast Fourier transform of the micrograph. Reprinted with permission from ref. 61. Copyright 2012, Macmillan Publishers Ltd.

in the lattice that at 600 1C or above, become mobile and are trapped by nitrogen atoms to form color centers. The actual chemical structure of the formed defects depends on the richness of N atoms themselves in the starting material. Vacancies in diamonds migrate primarily in their neutral charge state, with an activation energy of 2.3 eV while the color centers can exist either in the negatively charged state or in the neutral state.142 More in general defect related transitions in diamonds show characteristic zero phonon lines (ZPL) both in the absorption and luminescence spectrum that give information about their energies. In the absence of nitrogen atoms indeed, simple vacancies of carbon atoms in the diamond lattice (center V0 or GR1) are non-paramagnetic color centers that adsorb light in the NIR region (ZPL = 741.2 nm). When excited these sites deactivate mainly via non-radiative pathways showing a PL quantum yield of B1% (t = 2.55 ns) which makes them suitable for photothermal and photoacoustic150–153 but not for fluorescence imaging. Starting from materials doped with a suitable level of nitrogen atoms, it becomes possible to prepare FNDs emitting in the specific spectral region spanning from the NIR to green or blue depending on the nature of the

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created color centers. Nevertheless, because of the origin of the emission, differently from Si QD,s the PL maximum of FNDs cannot be tuned continuously. 2.2.2 Red-NIR emitting FNDs. The most interesting color centers for fluorescence imaging are the negatively charged nitrogen-vacancies (NV centers) which are produced by ion beam damaging followed by thermal annealing, in type Ib diamonds together with their neutral counterpart NV0.154 As shown in Fig. 12154 these centers correspond to the presence of a substitutional nitrogen atom adjacent to a carbon atom vacancy and show distinct ZPL at 637.6 nm and 575.4 nm for the negative and neutral defects respectively. Because of their NIR component, NV centers PL can be detected in vivo at a useful tissue depth. Studies of charge state conversion of single nitrogen-vacancy defects hosted in NDs demonstrated that the amount of negatively charged defects, with respect to its neutral counterpart, increases with the size of the NDs and it is enhanced when graphitic defects on the surface, created during the thermal annealing, are removed by thermal oxidation in air154 or oxygen.155 Oxidation also causes a decrease of the NC size.156 Negative centers are dominating in oxidized NDs


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Fig. 12 Normalized PL spectra of single NV and NV0 curve color centers in NDs. The zero-phonon line (*) symbols of NV (637 nm), NV0 (575 nm). The inset shows the atomic structure of the NV defect, consisting of a substitutional nitrogen atom N associated with a vacancy V in an adjacent lattice site of the diamond crystalline matrix. Reproduced with permission from ref. 154. Copyright 2010, American Physical Society.

being more than 80% of the total ones in type Ib NDs with diameters larger than 20 nm. NV centers show a unitary quantum yield, an excited state lifetime of B12 ns and they can be excited in the visible to give red-NIR emission. The electronic transition involved in the emission is the spin-allowed 3E - 3A2 one. The associated absorption band shows a broad phonon sideband at B560 nm. As a very unique feature, the emission of NV centers is strongly sensitive to magnetic and electromagnetic fields as well as to temperature.157,158 In fact, as schematized in Fig. 13159 the three degenerated spin microstates of the triplet state are split into two energy levels with ms = 0 and ms = 1 even in the absence of an external magnetic field as an effect of the crystal field and of the onsite magnetic interaction.160 As a consequence, two different emission pathways are possible that have been demonstrated to differ by B20% in efficiency.161 Hence, a change in the population of the two sub-levels, due for example to temperature changes or microwave irradiation, causes fluorescence intensity variations. Transition between the two spin states occurs at 2.87 GHz and is shifted in the presence of an external magnetic field as shown in Fig. 13. Magnetic sensitivity of NV defect emission together with their extraordinary photostability make these centers unique single spin individually optically readable magnetometers.162 Modulation of the NV center emission, by microwaves and magnetic fields, has been proposed as a basis for new in vivo, high contrast imaging methods in which signal analysis is exploited to isolate the modulated emission of the NDs from the magnetic insensitive background.160 Thanks to their high photostability, NV defects could be investigated at the single emitter level. Gruber et al. observed individual nitrogen-vacancy defect centers in diamonds with roomtemperature scanning confocal optical microscopy proving that resulting fluorescence is uniquely photostable without any photoblinking.163 Excited color centers, as in the case of organic fluorophores, can be involved in bimolecular processes that cause their non-radiative deactivation via excitation energy transfer.


Fig. 13 (a) Splitting of the electronic transitions of NV color centers in the absence of external magnetic field. (b) Frequency of transition between the magnetic sublevels. (c) Effect of an external magnetic field on the transition frequency. (d) Modulation of the fluorescence by microwave. Reproduced with permission from ref. 159. Copyright 2013, American Chemical Society.

Such processes involving external moieties,117,164 other color centers, or surface defect146 are strongly dependent on the donor– acceptor distance and hence on the depth of the location of the defect in the crystalline structure and they may cause some degree of emission intermittency.147 Bradac et al. demonstrated this effect by progressive etching of the ND host. As summarized in Fig. 14 they observed that the NV centers are induced to switch from latent, through continuous, to intermittent or ‘‘blinking’’ emission states as shown.147 Photoblinking has also been reported in the case of direct observation of nitrogen-vacancy centers in discrete 5 nm detonation NDs.144 Also in this case blinking resulted to be dependent on the nature of the surface of the NDs62 revealing the importance of the positioning of the defects with respect to the surface in determining the fluorescence properties.18,19,165,166 Non-optical modulation of NV color center emission has been exploited for high-resolution optical techniques.167 Stimulated emission depletion (STED) microscopy,168 schematized in Fig. 15,169 has turned out to be highly suitable for imaging NV centers. Hell and coworkers achieved, in diamonds, resolutions down to 6 nm170 and in conjunction with solid immersion, a resolving power of 2.4 nm.171 As shown in Fig. 16169 even multiple adjacent defects located in single NDs were imaged individually.169 Emission sensitivity to magnetic fields162,172–174 was also exploited in DESM (deterministic emitter switch microscopy). By modulating the fluorescence brightness, via magnetic resonance techniques, super-resolution imaging of N-V centers with localization down to 12 nm across a 35 35 mm2 matrix was possible.175

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Fig. 14 Analysis of the NV centers fluorescence after progressive step-treatment of the hosting NDs. The sample composition and the fluorescence of the NV centers are different at each progressive phase of the treatment. The quantity Zem is the fraction of emitting NDs. The quantity Zblink is the fraction of emissive NDs that show blinking. Reproduced with permission from ref. 147. Copyright 2013, John Wiley and Sons.

Fig. 15 (a) Energy levels of the triplet (left) and singlet states (right) of the NV center and the applied excitation (EXC), de-excitation (STED) and microwave (mw) fields and the detected fluorescence (FLUO). (b) Sketch of the NV center in the diamond matrix. (c) STED microscope for investigations of the resolution limit of single NV centers in diamond nanocrystals of subwavelength size (APD: avalanche photodiode in single photon counting mode), including the microwave excitation antenna next to the sample, and a Hanbury Brown and Twiss time intensity correlation measurement setup on the detection path, after the pinhole, consisting of a 50/50 beamsplitter (BS), two APD, a delay box and a coincidence counting module. The top right inset shows the combined confocal and AFM colocalization setup used for precharacterization. Reproduced with permission from ref. 169. Copyright 2013, American Chemical Society.

Conversion of neutral NV centers in NV was proposed to involve a second N atom as an electron donor.176 Optically detected magnetic resonance (ODMR) of a NV center within an individual B45 nm NDs was proposed to investigate the composition and spin dynamics of the particle-hosted spin bath. These studies confirmed the presence of nitrogen donors and of a second, yet-unidentified, class of paramagnetic centers.176 Schell et al. analyzed the excited state decay of a single nitrogen vacancy center in NDs to acquire information on the local density of optical states at the nanoscale177 by fluorescence lifetime imaging microscopy (FLIM). The effect of the coupling of this atomic-size emitter to plasmonic nanostructures was also studied164 by quantitatively mapping the nearfield coupling between the NV center and a flake of graphene, in three dimensions, with nanoscale resolution. In a similar approach, the coupling of the emitting centers to nanofabricated gold antennas was exploited to optically control the NV excited lifetime.178

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As shown in Fig. 17 the emission spectrum of NV emission matches well the transparency windows of living organism and it is hence well suited for in vivo optical imaging.179 2.2.3 Green emitting FNDs. Other luminescent defects can be produced in nitrogen rich diamonds when, as in type Ia diamonds, the N atoms are clustered in the structure. As shown in Fig. 18180 the complex N–V–N (A aggregate or H3 center) shows a ZPL at about 504 nm and a maximum absorption in the blue at B470 nm.181,182 This center emits exceptionally stable green fluorescence at 531 nm with a PL quantum yield close to 1, an excited state lifetime t = 16 ns and no photoblinking.180 Wee et al. prepared green emitting NDs for biological applications that contained, according to their calculations, a H3 center density of 1.7 1018 centers per cm3 (10 ppm or 2.8 10 3 M)183 and a molar absorption coefficient of 1.4 104 M 1 cm 1 at 450 nm. Green luminescence has also been reported in the case of detonation NDs stabilized with hydrophilic sodium benzenesulfonate groups covalently grafted onto the surface.184


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Fig. 16 (a) Subdiffraction resolution STED image and (b) corresponding vertically binned STED image profile of a diamond particle with B100 nm diameter showing five isolated NV centers (red curve: Lorentzian fit). (c) SEM image of the same ND and (d) overlay of the STED image and the SEM image illustrating the relative dimensions. (e) Confocally recorded ESR spectrum of the same ND showing five distinct ESR line pairs corresponding to the five NV centers (each line pair fitted with a double Lorentzian in a separate color). Reproduced with permission from ref. 169. Copyright 2013, American Chemical Society.

Fig. 17 Comparison of the fluorescence spectrum (red curve) of NV centers in FNDs with the near-infrared (NIR) window of biological tissues. The black, dark gray, and light gray curves are the absorption spectra of H2O, oxygenbound hemoglobin (HbO2), and hemoglobin (Hb), respectively. Reproduced with permission from ref. 179. Copyright 2012, Elsevier.

2.2.4 Blue emitting FNDs. Weaker emission in the blue is typical of another kind of defect, namely the N3 center, which is composed of three nitrogen atoms (B aggregate) surrounding a vacancy.142 Formation of complex defects related to the N3 center in NDs can be enhanced by plasma immersion and focused ion beam implantation methods. In this approach,


Fig. 18 Representative PL spectra of NDs containing H3 and H4 centers. Sharp ZPLs at 503 nm and 496 nm indicate that the PL of ND1 black and ND2 red is derived from H3 and H4, respectively. Inset: PL decays of the corresponding NDs. Solid curves are best fits to a two-exponential mode. Reproduced with permission from ref. 180. Copyright 2011, AIP Publishing LLC.

first He+ ions are used to create carbon atom vacancies in the diamond structure. This is followed by the introduction of molecular N2+ ions and heat treatment in a vacuum at 750 1C

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to initiate vacancy diffusion. Final heat treatment at 450 1C in a flowing air atmosphere was used to purify the surface.185 2.2.5 Other color centers in FNDs. Embedded siliconvacancy (Si-V) color centers were created in NDs, grown by microwave plasma-enhanced chemical vapor deposition (CVD), using silane as a source of Si impurity atoms. Si-V centers have a strong narrow-band PL at room temperature. PL intensity of the 738 nm ZPL depends on the silane concentration in the feed-gas mixture.139,140 Si-V color centers in NDs have been recently proposed as markers in the near infrared for cathodoluminescence imaging.186 Ni-related optical centers in diamonds were created by implanting high purity CVD diamond samples with Ni and N and thermal annealing. Samples, implanted with Ni, showed a new Ni-related PL peak centered at 711 nm, a doublet at 883/885 nm and an excited state lifetime to be 11.6 ns.187 Both silicon and nickel related color centers show promising interesting features for potential application to bioimaging. 2.3 Optical and PL properties of Si QDs and FNDs: critical issues The investigation of the optical and PL properties of Si QDs and FNDs is a rich and hot research topic. As mentioned, optical transitions in ideal Si QDs arises from quantum confinement effects while in FNDs they are related to local excited defects. Although, an exhaustive and univocally accepted interpretation of the photophysical and photochemical behavior of these two families of nanomaterials has not been reached yet. Critical issues arise from the variability of the structures of the nanosystems that largely change depending on their synthetic design. For Si QDs, as shown in Fig. 5–7 dual emission, as well as excitation and time dependent PL have been reported. The origin of these phenomena, and more in general the role of the surface in the origin of the PL of Si QDs, is still debated. To better understand the nature of the dual emission, Hannah et al. investigated the effect of pressure on the PL of alkaneterminated, plasma synthesized Si NCs.188 The correlation of X-ray diffraction and PL measurements revealed multiple phase transitions, associated with PL red shifts, which matched the electronic transitions from the conduction to the valence band of bulk crystalline silicon. According to the authors, these results, supported by calculations, suggested that the emission low-energy (red) component, frequently attributed to defects, arises from core-states that remain highly indirect despite a quantum confinement. More recently, the same authors examined ultrafast PL, generated by plasma-grown, colloidal silicon NCs, as a function of both particle size and lattice crystallinity. They finally reported that the high-energy (400–600 nm, 2–3 eV) PL component consists of two decay processes with distinct time scales.189 The fastest PL exhibits an B30 ps decay largely independent of the particle size. Having recorded a nearly identical emission for amorphous particles, the authors associated this feature with the presence of an amorphous silicon fraction in all the measured samples, rather than to phononless electron–hole recombination. This conclusion was partially counterargumented by Kusova et al.190 who had previously investigated in detail the role of the

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core in the generation of the fast blue PL of oxidized Si NCs.191 These studies had showed that the low-energy component of the PL underwent a much lower red-shift with respect to the high-energy band upon rising the temperature from 4 K to 296 K. Moreover, also the fast decay time of the blue component of the emission had been reported to be rather temperature independent. According to the authors, these results demonstrated that the fast blue emission arises from corerelated quasi-direct radiative recombination governed by slowly thermalizing photoholes. The apparent incompatibility of the two interpretations was partially attenuated by Hannah et al. who underlined the difficulty of determining an ‘‘ultimate’’ interpretation of the photophysical behavior of Si QDs irrespective of the synthetic method, surface termination, strain, etc.192 A detailed study on the role of surface defects in Si NC emission was recently reported by Wang et al.193 Analogous difficulties and complexities, which arise from the difficulty of classifying in a systematic way the large variety of newly synthesized carbon based nanomaterials, recently complicated the interpretation of the optical behavior of NDs.194 Although this review article is focused on the application to bioimaging of FNDs prepared either from diamond powders or detonation techniques, other emitting carbon NPs having a diamond-like structure have been described. In this framework, Xiao et al. recently discussed the emission of ND colloids produced by microsecond laser irradiation of a suspension of carbon powders in organic solvents.195 The PL of these NPs strongly differs from the one reported for NV and related defects (discussed in previous sections) showing a peak wavelength in the 400–600 nm range which is largely excitation dependent. Moreover, these laser produced NDs adsorb mostly UV light and uncorrelated excitation and absorption spectra. Interestingly, analogous features have been reported for other carbon based NPs such as carbon dots.126–129 As an origin of the fluorescence of laser produced NDs, the authors proposed the presence of functional groups such as OH, ketone and ester carbonyl groups. Remarkably, a similar hypothesis had been suggested for the interpretation of the PL of graphene oxide (GO).196 Shang et al., in fact, suggested electron– hole recombination from the bottom of the conduction band and nearby localized states to a wide-range valance band as an origin of GO fluorescence. In terms of molecular orbitals, these authors concluded that dominant fluorescence was originated from the electronic transitions among/between the non-oxidized carbon regions and the boundary of oxidized carbon atom regions, containing C–O, CQO and OQC–OH groups. Recently, a very similar PL emission mechanism has also been suggested as a common origin of the typical green fluorescence of carbon nanodots (C-dots) synthesized by electrochemical ablation and small molecule carbonization, as well as graphene quantum dots (GQDs) fabricated by solvothermally cutting graphene oxide.197 Hence, quite different carbon based nanomaterials display surprisingly similar photophysical behaviors. Xiao et al. demonstrated that a reversible, ND to carbon onion, phase transformation occurs when colloidal suspensions of NDs are laser irradiated at ambient temperature and pressure.198


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In particular, NDs are first transformed into carbon onions driven by the laser-induced high temperature in which an intermediary bucky diamond phase is observed. This result confirmed that transition from sp3 to sp2 carbon atoms occurs quite easily at the nanoscale. Moreover, the presence of diamond-like sp3 carbon insertions in C-dots mostly characterized by extended sp2 regions was also reported.128 In conclusion, the borderline between graphitic and diamond nanostructures becomes partially vague in nanostructures: the co-presence of different carbon allotropic forms complicates the interpretation and the modelling of the photophysical behavior of NDs as well as of other carbon based nanomaterials. Nevertheless, this issue can be in large part addressed though a detailed classification of NPs which take into particular consideration their synthetic origin.

3 Synthesis of Si QDs Numerous synthetic strategies have been proposed for the preparation of Si QDs. Only those methods that have been actually demonstrated suitable to produce NPs stable in a physiological environment, and usable as luminescent contrast agents for bioimaging, will be discussed in detail in this section. These methods are listed in Table 1 where the main features of the resulting Si QDs, as well as some general aspects of the production processes themselves (e.g. process safety), are summarized. We would like to stress that surface passivation is essential to obtain Si QDs stable in a biological environment and that all the Si nanoprobes used in bioimaging are surface functionalized. A partial exception is represented by some porous silicon NPs produced by electrochemical etching. These NPs are simply covered by a spontaneously grown silica layer and they decompose relatively fast under physiological conditions. Other possible preparative approaches, still not demonstrated suitable for luminescent bioprobe design, include sonochemical synthesis,206 electrochemical reduction,207–209 laser ablation210–214 and mechanochemical methods.215,216 Photolithography has also been proposed to prepare porous Si nanovectors for biological application.217–220 Although these materials share very interesting features for application to drug delivery and controlled release, they show no intrinsic PL and they will not be discussed in this review article.

Table 1

3.1

Laser pyrolysis (LP)

Laser pyrolysis of SiH4 is an effective, continuous process, which yields gram-scale quantities of highly pure, loosely agglomerated Si NCs with controlled primary particle size and size distribution.221–223 First attempts produced particles showing little or no visible PL.224,225 Huisken et al.226 obtained luminescent particles, but with small PL efficiency, and studied the effect of the aging in air of the hydrogen terminated Si QDs, as well as the effect of surface etching with HF, on the PL features.226–228 As shown in Fig. 19229 the PL quantum efficiency of Si NCs was found to increase, with a time constant of 8 days, from zero to a terminal, saturation value of approximately 65%, as an effect of air oxidation.229 Luminescent NCs were prepared by doping the silicon matrix with germanium during the synthesis.230 Large scale production was achieved by Li et al. who designed and optimized a flow reactor, schematized in Fig. 19,199 suitable to produce, initially non luminescent, crystalline silicon NPs with a B5 nm diameter at a rate of up to 200 mg h 1.199 As prepared NPs become strongly fluorescent after etching with HF–HNO3 mixtures. Moreover, etching reduces the size of the NPs and, as shown in Fig. 20,231 it was used to tune efficiently the emission color of the Si NCs. The observed marked dependency of the PL energy on the NC size proved that the emission was originated by the quantum confinement effect. Dependent on the etching time, materials with PL maximum ranging from 850 nm to 550 nm were prepared. Nevertheless, when dispersed in methanol and water the Si NCs luminescence was reported to undergo to a considerable decrease in intensity, and to a blue shift of the emission band. Stability of the PL as well as NC dispersibility in aqueous solution were hence improved by passivation of the hydrogen terminated Si surface with organic ligands by hydrosilylation.200 Application of NCs synthesized by laser pyrolysis to bioimaging has been reported both in vitro232 and in vivo231 by Prasad and Swihart. These authors also investigated the long-term toxicity, and the fate in the body, of these materials in model animals up to the level of primates.233 Although their studies do not fully exclude possible negative effects of very heavy dose of Si NCs in a specific animal model, they clearly demonstrated the high biocompatibility and the applicability of these materials to long-term bioimaging.

Comparison of the different synthetic methods for Si NCs production

Method

PL QYa (%)

LP199,200 EE56 EE (POM)100 ME201,202 HT120 P203 MW (Si NW)204 MW (TEOS)205

2–25 10 15–25 10 5–25 20–30 15–18 20–25

t — B10 ms — 3–17 ns B100 ms — —

Emission colorb

Emission originc

Band widthd

Size (nm)/ dispersione

Crystallinity/ aggregation

Cost/ expertize

Yield

B-NIR R-NIR B-NIR B-G R-NIR B-NIR R B

QC QC, SS QC SS QC QC QC QC, SS

n b n b n n n b

5–10/m 120–160/p 1–4/m 1.4–10/m 3–12/m 2–8/m 3.1/m 2.2/m

+/ +/+ +/ +/ +/ +/ +/ +/

+/+ /+ /+ / +/+ +/+ +/ /

20–200 mg h Modest Modest High Modest 0.1–10 g h 1 100 mg 100 mg

Reproducibility f/ risksg 1

+/+(HF) +/+(HF) +/+(HF) +/+(SiH4) +/+(HF) +/+(HF) +/ +/

a

PL QY in aqueous environment. b B = blue, G = green, R = red. c QC = quantum confinement, SS = surface states. d n = narrow, b = broad. m = monodispersed, p = polydispersed. f As difficulty to reproduce the synthetic conditions and set-up. g Use of hazardous reactants (e.g. HF) or conditions. e


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Fig. 19 Left: integrated PL signal and PL efficiencies of Si NCs prepared by laser pyrolysis as a function of time during exposure to atmospheric oxygen. Right: apparatus for the production of Si QDs by laser pyrolysis. Reproduced with permission from ref. 229. Copyright 2011, AIP Publishing LLC. Reproduced with permission from ref. 199. Copyright 2003, American Chemical Society.

Fig. 20 Luminescent Si QDs prepared by laser pyrolysis followed by size reduction via chemical etching. Reproduced with permission from ref. 231. Copyright 2011, American Chemical Society.

3.2

Electrochemical and chemical etching (EE)

Electrochemical etching14,54,55 is another synthetic approach that has been largely exploited to prepare luminescent Si NPs for bioimaging. As schematized in Fig. 21,56 this process is based on the initial formation of a film of porous silicon234 by the electrochemical oxidation of a commercial silicon wafer under controlled current conditions (e.g. 200 mA cm 2 for 150 s) in an aqueous HF/ethanol electrolyte.56 Detachment of the porous silicon layer can be achieved by applying a second,

Fig. 21 Synthesis of Si NCs by electrochemical etching. Reprinted with permission from ref. 56. Copyright 2009, Macmillan Publishers Ltd.

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less intense, current pulse (e.g. 4 mA cm 2 for 250 s) that produces a freestanding hydrogen terminated film which is fragmentized in deionized water by sonication. Weak initial NIR luminescence of the produced porous Si NCs increases of almost an order of magnitude upon aging the silicon dispersion in water for 2 week. PL of the resulting NPs is in large part due to surface defects and it shows quite a long excited state lifetime (B10 ms) suitable for time gated imaging. Moreover, these NPs are easily biodegradable, and they dissolved, when inoculated into a mouse, into components, mostly silicic acid, which are renally cleared causing no evident toxicity.235 Biodegradation time could be controlled by changing the size and porosity of the NPs, features that mostly depend on the current density applied during the synthesis. Aging of the NPs, prepared by electrochemical etching, in air exposed water suspension, was reported to lead to the formation of an hydrated silica shell that could be functionalized, as in the case of silica NPs,10,50 with alkoxysilane functional reagents.235 As a drawback, electrochemical etching produces a very polydisperse suspension of aggregates of Si NCs mixed to silicon fragments that need separation. Hence this method is not suitable to synthesize individual, small sized, Si QDs. Kang et al. improved the control of the electrochemical synthetic process by using polyoxymetalate (POM) as a catalyzer.100,103 In this way, they produced H-terminated highly monodispersed Si QDs, with controlled average size in the 1–4 nm range, which required no further separation.236 Emission of these Si NCs arises from the quantum confinement effect and, as shown in Fig. 8, PL color is strongly dependent on the size of the NPs. The same authors investigated the effect of controlled oxidation of their 3 nm H-terminated Si QDs. They proved that this process permitted to decrease the Si core size, and, as a


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consequence, to tune finely the PL wavelength maximum of the produced core–shell NPs as shown in Fig. 9.102,103 Organic capping of the surface has also been proposed, as an alternative to oxidation, to improve the luminescence properties, as well as the solvent compatibility and stability, of NCs prepared by electrochemical etching.237,238 Kusumi et al. recently proposed chemical etching239 as an alternative to the electrochemical process. Etching of a piece of a single-crystal silicon wafer was performed into two steps first exposing only an edge of the crystal to an HF–HNO3 and then immersing the entire piece in a HF–HNO3 water solution. After rinsing with deionized water and vacuum drying, Si NCs, still bound to the Si wafer and bearing –OH surface groups, were functionalized with a mercaptotrimethoxysilane before being mechanically detached by scraping the surface with a razor blade and dispersed in water by sonication. Final NCs bear thiols as surface groups and they were used for bioconjugation to maleimide derivatives. This method allowed us to prepare small individual, stabilized Si NCs but with a quite low production yield. 3.3

Microemulsion (ME)

Solution chemistry has been proposed as a convenient approach to Si QD synthesis.106,240–242 Colloidal Si NCs can be prepared in microemulsion by chemical reduction of silicon

Fig. 22 Reaction scheme for synthesis of silicon quantum dots by microemulsion followed by hydrosilylation. Reproduced with permission from ref. 244. Copyright 2011, Royal Society of Chemistry.

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precursors in the presence of surfactants. These serve as templating agents and they are essential to improve the control of the particle size and the size dispersion.243,244 The microemulsion process, schematized in Fig. 22, directly yields liquid dispersions of Si NCs that can be easily processed to obtain water suspended bioprobes. Nevertheless Si NC microemulsion syntheses is less attractive with respect to liquid phase methods developed for the production of other NPs (e.g. silica NPs) when process safety is considered. Because of the high reactivity of the reagents and the formation of hazardous byproducts such as pyrophoric SiH4, the reaction has to be carried out in a glove box in the complete absence of water and oxygen.245 As a drawback the microemulsion method permitted us to prepare only blue to greenish emitting Si NCs mostly absorbing UV light and hence not suitable for in vivo imaging applications. Possible utilized silicon sources were silicon alogenides, alcoxide or Zinnt salts (e.g. Mg2Si) that were dispersed in the inverse micelles and reduced with metal hydrides in the complete absence of water. Shiohara et al. investigated the effect of the hydride reducing agent on the size and optical properties244 of the final Si NCs. They concluded that NCs synthesized using the strongest hydride reducing agent showed the smallest size and narrowest size distribution. PL spectra of these Si NPs, with 1.6 nm and 2.5 nm diameter, are shown in Fig. 23.244 Their emission spectra are strongly dependent on the excitation wavelength but not on the NC size suggesting that PL arises from surface defect states rather than from the quantum confinement effect. Wilcoxon et al. used either nonionic aliphatic polyethers or quaternary ammonium cationic micelles in octane and LiAlH4 as a reductant of SiCl4 to prepare highly crystalline Si NCs with visible light emission and sizes of 1.8–10 nm.201 Recently Linehan et al. used a similar method to synthesize highly monodisperse Si NCs with average diameters ranging from 2 to 6 nm, controlling the size simply by changing the cationic quaternary ammonium salts used as surfactants.246 PL of Si QDs prepared by microemulsion disappears on the minute time scale when they are exposed to water and to atmospheric oxygen. Surface functionalization with allylamine allowed Warner et al.202 to produce hydrophilic 1.4 0.3 nm size Si QDs with a stable PL quantum yield (B10%) in water, showing emission

Fig. 23 (a) UV-vis absorbance spectra of the silicon nanocrystals with the size range of 1.6 nm, red line; and 2.5 nm, black line. (b) Photoluminescence spectra of 1.6 nm silicon nanocrystals. (c) Photoluminescence spectra of 2.5 nm silicon nanocrystals. Reproduced with permission from ref. 244. Copyright 2011, Royal Society of Chemistry.


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maximum at 480 nm and a full-width at half maximum height of 70–80 nm. Being the average excited state lifetime as short as 4 ns the emission was attributed to a direct band gap transition. Direct stabilization with organic surface groups was achieved in microemulsion synthesis using allyl trichlorosilane both as the surfactant and the reactant.247 Si QDs prepared by this method were reasonably monodispersed and could be modified via thiolene click reactions. An analogous result was achieved using a mixture of silicon tetrachloride and hexyltrichlorosilane in apolar solvents.248 Resulting coated particles could be readily dispersed in water to form clear and stable aqueous solutions. Lin et al.249 readapted a previously reported synthetic approach,250 based on the reduction of Mg2Si with Br2, to obtain water compatible Si QDs simply by treating the final product with butanol. Fourier transform infrared (FTIR) spectra revealed that while 1-butanol was not capped on the surface of the silicon NCs, silicon dioxide characteristic peaks were clearly detected. These observations led the authors to conclude that oxidation of the surface was responsible for the good water dispersibility of the NPs. Oxide-passivated Si NCs exhibited, in water, blue PL with a quantum yield of about 12% that was quite stable over a six month period. The emission decay time was in the nanosecond range and hence compatible with a direct band gap transition. Recently bluegreen luminescent octoxy capped Si NCs with 3 to 7 nm diameters were synthesized via homogeneous reduction of SiCl4 with the

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crown ether alkalide K+(15-crown-5)2K in THF.251 The concentration of SiCl4 was found to be the most important parameter governing the particle size and size distribution. Octoxy capped particles were stable under an ambient atmosphere for at least one month. Reduction of SiCl4 with Na(naphthalide)252 in glyme was also proposed to prepare chloride terminated Si NCs that after surface modification show bright temperature dependent blue PL with a quantum yield as high as 75%.109 Also in this case emission was originated from defect states. 3.4

High temperature processes (HT)

High temperature decomposition of silicon precursors is effective for the synthesis of size-controlled, oxide-embedded and freestanding, Si NCs with PL spanning from the visible to the NIR range. Synthesis can start from controlled hydrolysis and polycondensation of trichlorosilane (HSiCl3) to yield (HSiO1.5)n sol–gel glasses. Their thermal processing in 5% H2/95% Ar causes decomposition and diffusion mediated formation, growth, and crystallization of SiO2-embedded Si NCs. Alternatively, thermal treatment can be applied directly to commercial solid hydrogen silsesquioxane.253 Hessel et al. reported the preparation of monodisperse Si NCs with diameters ranging from 3 nm to 12 nm (Fig. 24) based on this approach.120 Control of the size and of the emission wavelength (in the 700–1100 nm range, Fig. 25) was achieved by modifying the temperature process peak. Acidic

Fig. 24 (A–G) TEM images of alkene-passivated Si nanocrystals generated by HSQ decomposition at the indicated temperatures. (H) Synthetic pathway from HSQ to alkyl passivated Si nanocrystals. Reproduced with permission from ref. 120. Copyright 2012, American Chemical Society.

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Fig. 26 Setup for plasma based synthesis of Si NC. Right: plasma chamber and picture of the reacting plasma. Left: setup for on the flight functionalization of the Si NCs. Reproduced with permission from ref. 254. Copyright 2007, John Wiley and Sons.

Fig. 25 Room-temperature PL (lexc = 420 nm) and PLE (measured at emission maximum) spectroscopy of alkane-stabilized Si nanocrystals dispersed in toluene. Reproduced with permission from ref. 120. Copyright 2012, American Chemical Society.

etching with HF was necessary to liberate the dots from the silica matrix formed during the synthesis. These Si NCs showed a quite high molar absorption coefficient in the red region (650 nm), up to e = 2 105 M 1 cm 1, and a PL quantum yield of B8%. Holmes et al. produced highly crystalline, organic-monolayer passivated Si NCs, with size dependent emission peaks, by thermally degrading diphenylsilane in a supercritical fluid in the presence of octanol at 500 1C and 345 bar.121 The Si QDs had diameters from 1.0 to 10 nm and they were sterically stabilized by octanol bound through Si–O bonds. Depending on the size, these Si NCs emitted with peaks in the 520 nm to 680 nm range with a PL quantum yield of B6% and sub-nanosecond excited state lifetimes.104 When investigated at the single dot level these NPs presented a narrow emission line as well as stochastic single-step ‘‘blinking’’ behavior. Emission maxima of the Si QDs prepared by high temperature methods are strongly related to the NP diameter, as expected in the case of the quantum confinement effect originated PL. Nevertheless, recently105 Dasog et al. reported that emission of Si NCs, synthesized at high temperature, can be tuned across the visible spectrum via surface group modification. In this particular case, emission was independent of the NC size and hence originated by surface states. 3.5

Plasma-assisted synthesis (P)

Considerable advantages of the synthesis of Si NCs in nonthermal plasmas are: first the electrical charging of the NPs,


which helps avoiding particle agglomeration, and then, the selective heating.254–256 Using this method red to NIR emitting Si QDs with diameter between 2 and 8 nm were prepared from a controlled flux of a SiH4/N2 on time scales of a few milliseconds upon localized irradiation with radio waves using the setup schematized in Fig. 26.255 Due to the abundance of atomic hydrogen in the discharge, the process produced hydrogen terminated surfaces255,257 that were rapidly oxidized in the atmosphere. As prepared Si NCs were not luminescent, because of the presence of surface quenching states, but they acquired intense red-orange PL after a few minutes of exposure to air either as a powder or as an ethanol dispersion. This PL switching on was due to the passivation of interface states by the formation of a native silicon oxide. The effect of HF etching on the silicon oxide shell covering the surface, followed by roomtemperature atmospheric re-oxidation, has been investigated as a possible strategy to tune the Si NC color emission. Indeed the process caused a severe quenching of the fluorescence. Etching, in fact, was reported to cause a restructuration of the surface that led to a decrease in the incorporation of oxygen during subsequent re-oxidation. A smaller ratio of O to Si in the newly produced silicon oxide shell is known to produce a higher density of quenching defects.258,259 As schematized in Fig. 26, surface passivation of Si NCs produced in the plasma reactor was directly achieved by flowing the crystals in a secondary furnace saturated with 1-dodecene yielding Si QDs dispersible in toluene with almost no aggregation. The plasma set-up yielded 14–52 mg h 1 of luminescent Si NCs.254 Production yields were considerably increased by Gupta et al. who obtained Si QDs in the 5 to 8 nm range at a rate of 0.1–10 g h 1 in a specifically designed microwave reactor.203 As shown in Fig. 27 these authors succeeded in controlling the emission wavelength, across the entire visible spectrum, by combining controlled etching in a HF–HNO3 mixture to surface stabilization via hydrosilylation. These results confirmed that emission of these Si QDs prepared in on thermal-plasma is

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Fig. 27 (a) Representation of the sequential size reduction of plasma prepared Si–NPs during the etching procedure. (b) Normalized PL emission spectra and pictures of the corresponding red (etching time 2 min), orange (9 min), yellow (11 min), green (13 min), and blue samples. Reproduced with permission from ref. 203. Copyright 2009, John Wiley and Sons.

originated by quantum confinement effects. Shen et al. used plasma-assisted decomposition of SiBr4 to prepare NPs that depending on the pressure in the plasma chamber give emission ranging from blue to yellow and stable quantum yields of up to 24% in ethanol.260 3.6

Microwave-assisted processes in solution (MW)

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comparison to microemulsion, is the possibility of preparing red emitting materials. It is the case of Si NCs produced by microwave assisted decomposition of Si nanowires (NWs) in the presence of glutaric acid as a stabilizing agent.204 Reaction occurs in about 15 minutes and it yields Si NCs with a size of 3.1 0.7 nm that are shown in HRTEM images as well-resolved lattice planes of B0.16 nm spacing demonstrating the excellent crystalline structures (Fig. 29).204 These Si NCs show a broad unstructured absorption spectrum and an emission peak at B660 nm with a PL quantum yield of B15% largely independent of the pH in the 1–10 interval. Moreover these NPs are extremely photostable when excited in an aqueous environment either in comparison to organic molecules (fluorescein) or CdTe quantum dots. Finally Si QDs prepared by MW degradation of Si NWs are capped by a large number of surface COOH groups, detectable by FTIR spectroscopy, suitable for bioconjugation according to conventional protocols. As schematized in Fig. 28, blue fluorescent Si NCs (PL QY = 20–25%) can be prepared on a large-scale by microwave irradiation of aminopropyltriethoxysilane (APTES) in a facile one-pot process that yields B0.1 g Si QDs of small sizes (B2.2 nm) in 10 min.205 The as-prepared particles feature favourable biocompatibility, and robust photo- and pH-stability. The strong fluorescence of the Si NPs was attributable to both the quantumsize and ligand-related effect.205

4 Functionalization of Si QDs

Microwave heating has been proposed as an interesting tool to synthesize Si NCs in solution using much less reactive and potentially hazardous precursors than in the case of microemulsion. As a unique advantage, microwave-assisted synthesis can be carried out in an aqueous environment, under mild conditions, giving, as in the example of Fig. 28,205 directly water dispersed Si NCs in a one-pot method. Further advantage, in

Surface functionalization of Si QDs plays different fundamental roles in their application to bioimaging. Surface termination influences the chemical resistance of Si NCs, PL stability as well as NP dispersibility in aqueous medium. Surface chemistry is also critical for bioconjugation. Recently simultaneously doping with phosphorus and boron has been proposed as an interesting alternative to ligand stabilization for improving Si NC stability in water.107 Nevertheless surface modification is still the most effective and largely explored method to transfer the features of Si QDs to biological environments. A judicious choice of the passivating agents permits us also to modulate the photophysical properties of the Si NCs themselves. The effect of surface groups on the PL feature can arise from the creation of a surface localized excited state as well as from more subtle phenomena. For example, structural relaxation after excitation of the Si QDs regulates their Stoke’s shift, namely the energetic separation between the absorption and emission bands. Such an energy varies not only with the particle size, but also with the degree of surface passivation, and the nature of the passivating species. Large Stoke’s shift may be useful to reduce the overlap between the excitation and detection spectral windows, and hence to increase the signal to noise ratio. Nevertheless Stoke’s shift needs to be minimized for maximizing the PL efficiency of the QDs.90

Fig. 28 Schematic illustration of microwave assisted synthesis of Si NCs (A) reaction precursor; (B) 21 nuclei; and (C) 4 small-size and (D) 1 largesize NCs. Reproduced with permission from ref. 205. Copyright 2013, American Chemical Society.

4.1

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Surface oxidation and etching

Surface defect states have been reported to quench the luminescence of Si QDs synthesized by laser pyrolysis199 and plasma


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Fig. 29 TEM and HRTEM images of Si NCs prepared by microwave degradation of Si nanowires. Absorption and PL spectra of the as-prepared Si QDs. Photostability comparison of FITC, CdTe QDs, and as-prepared Si QDs. Reproduced with permission from ref. 204. Copyright 2011, American Chemical Society.

assisted methods255 and to reduce considerably the emission efficiency of NCs produced by electrochemical etching.235 Oxidation and chemical etching with hydrofluoric acid was proposed as a process for removing quenching defects from Si NC surfaces improving their PL properties, as well as to reduce the size of the NCs with the objective of controlling the emission maximum wavelength. Different strategies were optimized depending on the starting materials using mostly HNO3 or atmospheric oxygen as oxidizers. Experimental reports show that oxidation by environmental oxygen has a benign effect on the luminescence properties of NCs synthesized by non-thermal plasma procedures which exhibit intense photoluminescence only after exposure to air.255 In the case of NCs prepared by laser pyrolysis etching in the HF–HNO3 mixture was preferred to obtain luminescent NCs with controlled emission wavelength. Surface oxidation, anyhow, occurs spontaneously upon exposition of the silicon surface, typically hydrogen terminated after synthesis or HF treatment, to atmospheric oxygen. The complex nature of the Si/SiO2 interface has been deeply investigated both from the theoretical and the experimental point of view demonstrating that its structure is affected by the oxidation conditions and it is crucial in determining the chemical, optical and electronic properties of Si NCs.200,221,222,261 Formation of a silica layer increases the colloidal stability of Si NCs in water.262 Nevertheless simple stabilization by surface


oxidation does not provide long-term resistant luminescent materials as achievable with other capping agents. On the other hand, controlled biodegradation of Si QDs may become valuable in their application to in vivo imaging to avoid irreversible accumulation of the NPs in the organism. In this perspective Park et al. optimized the conditions of electrochemical etching of silicon wafers, to produce Si NPs with controlled porosity and, as shown in Fig. 30, controlled excited state lifetimes. Modulation of the porosity was exploited by these authors to regulate the rate of degradation of the particles in a physiological environment.56 NIR emission of the Si NPs was activated by aging them in an air equilibrated aqueous environment. Oxidation was reported to produce235 a mesoporous silica shell that improves the loading capability for drug delivery applications. Oxidation in an oxygen rich environment has also been proposed to produce blue emitting Si nanoemitters starting from red emitting NPs also by producing thickness controlled silica layers. The propensity of Si NCs towards oxidation has also been reported to depend on the NC size: first-principles computations carried out predicted that methyl- and siloxanecoated silicon quantum dots with diameters in the range of 1.2–2 nm offer a superb resistance to oxidation.263 Although controlled oxidation can be advantageously exploited for tuning the properties of Si NCs during their synthetic development; in a biological environment oxidation causes Si NC

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Fig. 30 Characterization of PEG-conjugated luminescent porous Si NPs. (a) Hydrodynamic diameter, surface chemistry and emission lifetime of Si NPs prepared at the indicated etch current densities. (b) Schematic diagram depicting the PEG surface chemistry on the Si NPs. Photograph of PEG–Si NPs in water, obtained under ultraviolet light illumination. (c) Absorbance and steady-state photoluminescence spectrum of PEG-Si NPs. (d) Normalized photoluminescence intensity-time trace for PEG-Si NPs after pulsed excitation. (e) Fluorescence lifetimes of commonly used fluorophores and tissue autofluorescence. Reprinted with permission from ref. 235. Copyright 2013, Macmillan Publishers Ltd.

degradation and luminescence fading. Hence robust surface passivation is essential for producing Si QDs suitable for longterm bioimaging. 4.2

Covalent functionalization

Oxidation of Si NCs in the bio-environment can be prevented by suitable surface functionalization. Hydrosilylation is surely the most widely explored reaction for silicon surface functionalization. Recently other Si NCs surface termination reactions for covalent passivation of Si QDs have been proposed and they will be discussed, together with hydrosilylation, in this section. 4.2.1 Hydrosilylation. Stabilization of the Si NCs surfaces against atmospheric oxidation is typically achieved via chemically catalyzed or photoactivated hydrosilylation. Depending on the chosen stabilizing alkene either the dispersibility of the QDs in organic solvent or in aqueous medium can be improved. Moreover the use of functional species bearing a second reactive group, such as a primary amine or a carboxylate, has been proposed to produce NPs directly suitable for bioconjugation. Chemical and photochemical hydrosilylation processes were compared by Shiohara et al. who investigated the capping of Si NCs, prepared in microemulsion, with allylamine by using either a platinum catalyst (hexachloroplatinic acid) or UV-irradiation.244 Photochemical processes presented relevant advantages with respect to the chemical one. Formation of platinum NPs, which occurred as a result of the catalyzer degradation in the thermal reaction, was avoided. Moreover UV treatment enabled a shorter capping time of 4 hours compared to the 12 hours needed for the platinum catalyst. Rosso-Vasic et al. demonstrated that the nature of the capping agent used in hydrosilylation had also an effect on the emission wavelength of the produced Si NCs. As a result, Si dot emission

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could be tuned from the UV to the blue spectral region, in a controllable fashion, by only changing the alkyl spacer length of the capping reagent.264 The effect on the optical properties of Si QDs modified by hydrosilylation was reported to depend on the s–p conjugation length by comparing octyl-, (trimethylsilyl)vinyl-, and 2-phenylvinyl-capped NPs.265 In view of the development of fully biocompatible probes Prasad suggested the use of ethyl undecylenate as a capping agent, this additive being permitted for direct addition to food for human consumption.233,266 Resulting NCs required encapsulation in micelles for dispersion in an aqueous environment. Recently, as shown in Fig. 31, microwave assisted hydrosilylation was also proposed as an alternative to the conventional processes offering, as advantages, simpler workup procedures, shorter reaction times, milder conditions, and environmental friendliness.236 Efficiency of the MW approach was demonstrated in the case of various surface functionalities such as alkyl, alcohol, and carboxylic acid. MW capped Si QDs showed good PL properties and very good stability in organic or aqueous media. PEGylation is a well-established approach to the design of water dispersible NPs with long circulation time in the vascular system. Sudeep et al. prepared PEG capped Si NCs by hydrosilylation. They functionalized PEG-1100 monomethyl ether with 1-undecenyl bromide to introduce a terminal alkene group suitable for hydrosilylation.267 Summarizing, hydrosilylation is a very versatile reaction it is suitable to graft several different groups on the Si NC surface via formation of Si–C bonds. Nevertheless, as will be shown, further non-covalent functionalization of the alkylated NPs has been in some cases preferred to improve the water stability and biocompatibility of Si NCs for bioimaging applications.


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Fig. 31 Microwave assisted hydrosilylation. (a) Absorption and PL spectra of decene-, undecylenyl alcohol-, and undecenoic acid-functionalized Si QDs. PL is measured using ultraviolet excitation (l = 365 nm). (b) Photographs of SiQDs functionalized with 1-decene (left) dispersed in hexane, and with undecylenyl alcohol (middle) and undecenoic acid (right) dispersed in water under 365 nm irradiation or ambient light. Reproduced with permission from ref. 236. Copyright 2012, John Wiley and Sons.

4.2.2 Other reactions. Using functional alkene as acrylic acid (AA), reactions different from hydrosilylation may occur, in the presence of Si QDs, upon light irradiation, which lead to the formation of Si–O rather than Si–C bonds. He et al. demonstrated by modelling that the grafting of AA to silicon via the carboxylic group is favored upon blue light irradiation. Calculation results are summarized in Fig. 32 and they also predicted that later UV irradiation of the previously passivated Si NCs led to acrylate polymerization rather than to hydrosilylation. The authors hence developed a two-step synthetic process, schematized in Fig. 32, which yielded a water dispersible polymeric nanosphere where Si NCs were incorporated into a poly-AA matrix.237 Glutaric acid has also been used as a stabilizer that binds covalently to Si NCs obtained by microwave induced degradation of preformed silicon nanowires.204 Covalent functionalization of Si NCs bearing surface termination different than hydrogen has been reported. Silica covered NPs can be modified, analogous to silica NPs,268–272 with alcoxysilane reagents. This approach was applied to terminate with primary amines the surface of Si NCs using APTES. Surface amine reactivity was hence exploited to introduce PEG chains via peptide type coupling and copper(I)-catalyzed azide– alkyne cycloaddition.273 Other reagents suitable for the modification of oxidized Si NCs are chlorosilanes. This reaction was used to graft alkyl chains to the surface of Si QDs prepared in non-thermal plasma after their wet oxidation to produce surface –OH groups.274 Halogenide Si NCs were functionalized by reaction with amine. In particular this process allowed the production of ultrabright, water-dispersible, Si NPs. After the reaction, as shown in Fig. 33, the PL quantum yield of the starting chloride covered NCs, prepared by microemulsion reduction of SiCl4 with sodium naphthalide, increased from 8% up to 75%.109


Functionalization caused also a shift of the emission maximum from about 400 nm to about 500 nm according to the mechanism schematized in Fig. 34. After functionalization, emission arises from the population of the newly formed emitting surface states resulting from grafting. In contrast in the pristine unmodified, NPs non-radiative trap states cause PL quenching. Interestingly, the surface modified Si NCs showed a strongly temperature dependent emission that allowed their use as nanosized luminescent thermometers. A surface modification based approach to photophysical properties control was extended to a large variety of Si NCs to tune their emission color (see Fig. 8) taking advantage of the reactions schematized in Fig. 35.105 4.3

Non-covalent functionalization

Dispersion of Si NCs in an aqueous environment may take advantage of incorporation into micellar systems. Simple surfactants were used to transfer hydrophobic Si NCs prepared in microemulsion to an aqueous environment. Pluronic F127 is a large (12 kDa) neutral surfactant that has been reported to stabilize in an aqueous environment Si NCs prepared in non-thermal plasma. Luminescent and stable micelles were produced without preventive covalent surface capping of the Si NCs.275,276 Pluronic F127 was also used as a dispersing agent for Si NCs produced by laser pyrolysis and capped with ethyl undecylenate. This polaxamer was chosen proposed in view of future clinical application of the luminescent NPs, since Pluronic F127 was approved by FDA as an injectable material for use in the human body.233 In a different approach hydroxy terminations of the polymer were oxidized to carboxylic groups for bioconjugation.266 Cross-linking of Pluronic F127 micelles, after loading with decyl-terminated Si NCs produced via chemical etching, was achieved by growing silica in the inner hydrophobic

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Fig. 32 (a) Schematic representation showing potential energy profiles for Si–C and Si–O bond formation along with B3LYP/6-31G relative energies of the reactants (R), transition states (T), and products (P). Red lines indicate the new Si–C, Si–O, Si–H, and C–H bonds in the addition reaction. (b) Optimized structures involved in the formation of the PAA chain on the surface of the Si QD. (c) Schematic representation showing the synthesis of silicon-based nanospheres. Reproduced with permission from ref. 237. Copyright 2008, John Wiley and Sons.

core of the micelles. This approach was proposed to improve the micelle stability preventing their disaggregation upon dilution.277 Phospholipid micelles were used by Swihart and Prasad to encapsulate Si NCs prepared by laser pyrolysis and capped by organic low polar residuals via hydrosilylation. As an advantage, phospholipid micelles are more stable than those formed by conventional detergents: they have a low critical micelle concentration and high kinetic stability. Incorporation of the Si QDs occurred simply by premixing the phospholipids and the stabilized NCs in chloroform, evaporating the solvents and redispersing the solid residual in water. Bioconjugation of the final micelles was achieved by pre-functionalizing a fraction of the terminal phosphate groups with folate or biotin through a suitable cross-linker as shown in Fig. 36.232

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4.4

Bioconjugation

Most of the Si NCs developed for bioimaging applications are prepared as amino, carboxylate or thiol terminated NPs. Protocols suitable to conjugate such functionalities to biomolecules are well established and they have been the central topic of comprehensive recent review articles.29,278 Most typical bioconjugation reactions are schematized in Fig. 37. Coupling of amine to carboxylic group is achieved in an aqueous solvent after activation of the poorly reactive carboxylate. Conjugation of biomolecules to Si NCs by pure electrostatic interaction has been reported for allylamine capped NPs.279,280 In the case of micelles stabilized Si NCs, functionalization of the surfactant constituting the micelles themselves was proposed as an indirect method for targeting the Si NPs. Depending on the system


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Fig. 33 Surface-modified water-dispersible Si NPs with ultrabright fluorescence. (a) Scheme of solution reduction synthesis. (b) PL spectra of Si NPs before and after surface modification (lex = 360 nm, 20 1C). (c) Normalized PL and UV-visible absorption spectra of the two surface-modified Si NPs. Insets: photographs of dilute sample solutions dispersed with the ca-Si NPs (left) and di-Si NPs (right). From left to right are photographs of sample solution under sunlight in black background, under normal conditions, and under 365 nm UV photoexcitation, respectively. Reproduced with permission from ref. 109. Copyright 2013, American Chemical Society.

Fig. 34 TCSPC and femtosecond time-resolved PL techniques to unravel the mechanism leading to ultrabright fluorescence. (a) TCSPC spectra of Cl–Si NPs, di-Si NPs, and ca-Si NPs (lex = 365 nm; lem = emission maximum wavelength, 20 1C). (b) PL decay of di-Si NPs measured at different emission wavelengths. (c) Femtosecond time resolved PL data of di-Si NPs and ca-Si NPs. (d) Scheme of the surface modification process leading to ultrabright PL. Reproduced with permission from ref. 109. Copyright 2013, American Chemical Society.

pre-functionalization of the surfactant with the target molecule or post-functionalization of the final Si NCs loaded micelle was preferred. A very peculiar approach to bio-conjugation was reported by Zhong et al. who grafted IgG to Si NCs, already prepared by microwave irradiation, by applying a further low power


Fig. 35 Scheme of surface medication reactions to tune the PL of Si NCs. Reproduced with permission from ref. 105. Copyright 2014, American Chemical Society.

microwave pulse after addition of the protein, which once bound to the NPs also acted as a stabilizing agent against aggregation.281

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Fig. 36 Schematic of surface functionalization of Si QDs followed by micellar encapsulation and a table indicating the compounds (R1) grafted onto the Si NPs and the functional groups (R2) commercially available on the phospholipids. Reproduced with permission from ref. 232. Copyright 2008, American Chemical Society.

5 Preparation of NDs and FNDs The real challenge in producing PL nanoprobes based on FNDs is to obtain stable aqueous colloidal suspension of these NPs that are known to show a very strong propensity towards aggregation. For this reason, as already discussed for Si QDs, surface chemistry control is a major issue in the application of FNDs to bioimaging. As will be shown, compatibility with an aqueous environment is in part favored by the surface modification to which the NDs are subjected during the process undertaken for making them fluorescent. In fact, at the end of this multi-step procedure aimed at the creation of emitting color centers in the NCs, oxidation of the ND surface yields carboxylate terminated NPs. Most NDs used for bioimaging are prepared from commercial diamond powders resulting from high-energy ball milling of highpressure high-temperature (HPHT) diamond microcrystals.282 These materials have a nitrogen content suitable for the creation of NV color centers emitting in the far red. Green emitting NDs, on the other hand, are prepared by pulverizing natural, nitrogen rich diamonds.180 Also NDs synthesized by detonation techniques61,283 have been proposed as PL contrast agents. More in general, a large variety of methods has been reported for ND production such as: laser ablation,198,284 plasma-assisted chemical vapor deposition (CVD),285 autoclave synthesis from

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supercritical fluids,286 ion irradiation of graphite,287 chlorination of carbides,288 electron irradiation of carbon onions289 and ultrasound cavitation.290 Nevertheless, considered that the properties of NDs are strongly dependent on the preparative methods, and, in particular, on the presence of dopant elements, the applicability of these alternative synthetic methods to the design of PL nanoprobes for bioimaging needs to be fully demonstrated. The general approach to the production of bright fluorescent NDs (FNDs) compatible with a physiological environment and suitable for bioconjugation is schematized in Fig. 38. Two alternative strategies have been proposed. In the former one (from left to right in Fig. 38), non-fluorescent NDs, obtained either by size reduction of microdiamonds or detonation processes, are irradiated with an ion beam and thermally annealed at high temperature in a vacuum to produce color centers. The graphitic shell formed after the thermal treatments is hence removed by a combination of dry and wet oxidation to enhance the ND brightness and water dispersibility thanks to the formation of surface carboxylate groups. In the second alternative approach, (from right to left in Fig. 38) fluorescence activation is carried out directly on the microsized diamonds which are then pulverized into NDs. Following this last strategy NDs are not directly exposed to the thermal treatment and, as an advantage, they present less graphitic surface residuals. Nevertheless purification by oxidation is still needed to remove residual impurities from the


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Fig. 37 Scheme of the chemical reactions typically used for bioconjugation. Readapted with permission from ref. 278. Copyright 2013, American Chemical Society.

Fig. 38 Schematic representation of the two main strategies for the preparation of aqueous dispersions of FNDs. Micro-sized diamonds (Ds) are reduced in size (from left to right) and ion beam damaged and then thermally treated to create colour centres in the activated NDs (ANDs). Thermal annealing produces a surface graphitic layer which is eliminated by oxidation producing surface carboxylic groups together with other functions (anhydride and hydroxyl groups, not shown). In the alternative approach (from right to left). Microdiamonds are first activated to produce fluorescent diamonds (FDs) which are then reduced in size and oxidated.

milling process and to create the surface carboxylic groups. A major drawback of post activation milling is that it requires facilities for the mechanical size reduction while the former method can be applied directly on commercial, industrially produced nanopowders.


5.1

Diamond nanopowders

NDs are commercially available in the form of nanopowders industrially produced from the crushing of synthetic micrometric diamonds, obtained by high-pressure high-temperature (HPHT) methods, or, less frequently, by fracturing larger

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natural diamond grains of poor quality.187 The most common size reduction technique is to mill diamonds with hard balls (e.g. made of steel) in a rotating cylinder. In the process, the reduction ratio of diamonds should be kept low to increase the efficiency of milling. As a consequence, several ball mills are used in stages to pulverize diamonds to very fine sizes Ball milling may be effective in reducing the size of diamonds, but it produces NDs which are irregular in shape. The presence of sharp edges has been demonstrated to be crucial in determining the interaction of the NDs with biological targets.291 The sharp corners may be chipped off by using a jet mill that forces two streams of micron diamonds to collide each other. After jet milling, the chipped diamond is mixed with much debris and impurities derived from the interior of the milling machine. A separation technique allow us to obtain monodispersed diamond nanopowders but the size of the commercial products at the current stage can be hardly reduced below 10 nm.61 5.2

Detonation NDs

Smaller NDs can be prepared by detonation processes that yield aggregates of NDs with sizes of 4–5 nm embedded in a detonation soot composed of other carbon allotropes and impurities.61,292 An explosive mixture (typically trinitrotoluene and hexogen in a mass ratio ranging from 40 : 60 to 70 : 30) having an overall negative oxygen balance provides both a source of carbon and energy for the conversion.293,294 The carbon yield of the process is 4–10% and the crude detonation solid product contains, under optimal conditions, about 75% in weight of aggregates of diamond NPs. The detonation chamber reaches temperature as high as 3000–4000 1C and pressure of 20–30 GPa. Since graphite is the most stable allotropic form of carbon at ambient temperature and pressure, the fast cooling of the detonation products is crucial to avoid transformation of the diamond into graphitic phase. Cooling may be achieved either under dry or wet conditions, filling the detonation chamber with an inert gas or water respectively. Formation of graphite, and other carbon allotropes, cannot be completely avoided, as well as the presence in the crude detonation ‘‘soot’’ of metals and oxide residuals deriving from the ignition charge or from the degradation of the detonation chamber. Detonation NDs for their 2–10 nm small size have also been referred to as ultradispersed,292,294 ultrananocrystalline diamonds.293 or single-digit NDs. Actually, detonation NDs form extremely tight core aggregates that stand a conventional disintegration technique (e.g. ultrasonication).295 Ozone-purified 3–5 nm detonation NDs296 form stable acidic hydrosol (pH = 1.6–2, z-potential = 50 to 100 mV) that can be disaggregated by milling with ceramic microbeads (ZrO2 or SiO2) or by microbead-assisted ultrasonic disintegration.297 Contamination with bead material and generation of graphitic layers on the NDs surface are major drawback of this method.298 As a consequence further purification299 of the primary 4–5 nm NPs is needed and it causes in some cases re-aggregation of NPs.298 Contamination can be avoid by dry milling, using low-cost, abundant and cheap milling media, such as water-soluble salts and sugars.300 A different method exploited the annealing of ND aggregates in hydrogen

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gas to separate their core particles. Dispersion of these NPs into water via high power ultrasound and high speed centrifugation yielded a monodisperse ND colloid, with exceptional long time stability in a wide range of pH, and with high positive zeta potential (460 mV).301 Although detonation NDs promise to become ideal substrates for the development of sub 10 nm size contrast agents, at the present stage major limitations to their application are the low efficiency of the color center formation144 and the lower photostability with respect to other NDs.62 5.3

Color center creation in NDs

Fluorescence of as prepared NDs is weak because of the presence of few color centers. Production of the color defects involves the use of ion beam sources in high vacuum and can be hence accomplished only on dry materials.302 Conventionally, the N-V defect centers in diamonds were produced by bombarding the material with a high-energy (typically 2 MeV) electron beam, from a van de Graaff accelerator,303,304 or a proton (B3 MeV).182,305 Irradiation was followed by annealing at elevated temperatures (typically 800 1C).306 As a drawback, this process required sophisticated, potentially hazardous, costly equipment not frequent in most research laboratories. A medium-energy (40 keV) He+ beam was hence proposed by Chang et al. as a convenient alternative to produce high concentration of color centers in a setup that, according to the authors, can be installed and operated safely in ordinary laboratories. As shown in Fig. 39306 the low energy irradiation treatment produced FNDs with an acceptable PL brightness when compared to the particles produced under high energy ion damaging. One further advantage of helium ions is their chemically inertness. As a consequence they do not alter the photophysical properties of the produced FNDs. Moreover a 40 keV He+ ion beam can be generated by a radiofrequency ion source and a single ion can create up to 40 vacancies as it penetrates diamonds,307 with respect to the 0.1 and 13 vacancies generated by 2 MeV electron and 3 MeV proton, respectively.308,309 Independently of the ion source utilized for diamond irradiation, the exposed NPs need to undergo thermal annealing to induce vacancy migration and actual color center formation. As a side effect, thermal annealing causes surface degradation, mostly in the form of graphitization. Surface graphite layers are detrimental to the photophysical properties of the FNDs and they have to be eliminated by oxidation in oxygen, air, ozone or nitric acid mixture in order to obtain bright materials. Reduction of the ND size during dry oxidation of the surface has been visualized by AFM as shown in Fig. 40.147 A critical point during the exposure of the diamond nanopowders to the ion beam is the actual depth of penetration of the ions into the diamond structure. This parameter depends on the energy of the ionized particles, on the size of the crystals, and on the thickness of the layer of NDs which is irradiated. Considering this last point, although pre-dispersion of the NDs in an aqueous environment is not required for activation, it can become advantageous to achieve a homogeneous distribution of the defects in the ND layer exposed to irradiation. In particular, deposition of the NDs as a dense monolayer on amino-silanized silicon substrates before He+ beam irradiation


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It is important to note that the overall PL activation process, namely irradiation followed by thermal annealing and oxidation, induces substantial surface chemical modification and should not be carried out on pre-functionalized NDs. In fact, any functional group or biomolecule bound to the NPs beforehand the PL activation steps would be greatly damaged. As a consequence, as a general strategy, surface functionalization and bioconjugation are performed in the very last stage of nanoprobe preparation. 5.4

Fig. 39 (a) Fluorescence spectra of 35 nm FNDs suspended in water prepared with either 40 keV He+ or 3 MeV H+ irradiation. Inset: fluorescence image of a 35 nm FND suspension excited by 532 nm laser light. (b) Fluorescence intensities of FNDs as a function of particle size at three different laser powers. Inset: fluorescence time trace (intensity normalized) of a 25 nm FND. Reprinted with permission from ref. 306. Copyright 2008, Macmillan Publishers Ltd.

was proposed to enhance the uniformity of the color center distribution.310

NDs brightness: number of color centers

Brightness of individual NDs depends on the fluorescence quantum yield of the created emitting defects, as well as on their molar absorption coefficient, and on number of color centers produces per particle. Although as for other doped emitting materials, quenching due to inter-defect interactions cannot be ruled out, this process has not been investigated in detail in NDs. The average molar absorption coefficient of the color centers, on the other hand, was not determined accurately also because of the high scattering efficiency of these materials but it has been estimated to be roughly around 104 M 1 cm 1 and hence about one order of magnitude less than best absorbing organic dyes. The number of color centers produced in each ND particle depends on the irradiation conditions during activation, in particular on ion energies and dosage, but also on the size and nature of the NDs themselves. The few estimation values reported for the quantification of the number of emitting defects in FNDs show indeed a large variability. For example in the case of NCs with 100 nm diameters a number of color centers that vary from one to a few tens311 up to the surprising value of 1 104 of defects per particle have been calculated.312 The last parameter that affects FND brightness is the fluorescence quantum efficiency of the color centers. This is in part dependent on the localization of the defects themselves with respect to the diamond surface which is often covered by

Fig. 40 AFM time-lapse images of the same substrate area containing several NDs at subsequent oxidation steps. Left to right: before treatment; 2.5 h after treatment at 600 1C in air; 5 h after treatment at 600 1C in air. The size reduction of individual NDs is readily observable at each treatment step. Reproduced with permission from ref. 147. Copyright 2013, John Wiley and Sons.


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residual graphite or defects. It has been reported that quenching effects, including graphite-shell quenching and impurity quenching, on negatively charged nitrogen-vacancy centers in fluorescent NDs can reduce the fluorescence quantum yield and bring about multi-exponential decay fluorescence to FNDs.311,313


6 Surface modification of NDs 6.1

Oxidation of NDs

Oxidative processes are used in the production of a FNDs based contrast agent into three essential stages. The first one is the purification of the industrial starting materials. The second step is the removal of the surface graphitic layer produced by ion beam damage and thermal annealing. Finally, oxidation is exploited to produce the surface carboxylate groups that allow the dispersion of FNDs in water as well as their functionalization. The opportunity of an initial purification of NDs depends on their origin since they may present very different purity. NDs are purified, at the industrial level, using strong liquid acidic or alkaline oxidants also in combination with high temperatures. Residuals of graphite and other non-diamond carbon allotropes, metals and other impurities are removed with HNO3, mixtures of H2SO4 and HNO3, K2Cr2O7 in H2SO4, KOH/KNO3, Na2O2, HNO3/ H2O2 under pressure, or HClO4.61,62 Treatment with HF is rarely used to eliminate traces of silica. As synthesized detonation NDs contain a large fraction of non-diamond carbon structures removable by milder purification processes. Surface reduction in a hydrogen atmosphere is revealed to be only partially efficient,301 in contrast exposition to air or ozone-enriched air at elevated temperatures has been demonstrated to increase the diamond content in detonation soot to 495 wt%.296,314,315 At the laboratory level high temperature etching in a HNO3–H2SO4 mixture is often used as a pre-treatment of commercial NDs. Oxidation leads to a modification of the ND surface producing mainly anhydrides and carboxylic acids yielding materials water dispersible and suitable for functionalization according to conventional bioconjugation protocols. 6.2

Covalent functionalization of FNDs

Carboxylic groups impart to FNDs a reasonable dispersibility in a physiological environment as well as an acceptable colloidal stability. Nevertheless covalent modification has been proposed to improve stability in biological environments as well as to increase the loading capability for the delivery of therapeutic cargos. Covalent functionalization with PEG was achieved by dispersing dry carboxylated NDs in DMF and refluxing them in the presence of thionyl chloride (SOCl2) to produce acyl-chloride surface groups that were then reacted with PEG-4000. The PEG shell was then loaded with doxorubicin for cellular delivery.316 In a different approach, amino terminated NDs were prepared by treating the NPs with APTES. The alkoxysilane groups reacted with the residual hydroxyls on the surface of the NDs, and partially polymerized to form an aminosilane film.317 Further growth of a silica shell, starting from this initial aminosilane layer, was proposed to improve the surface smoothness of initially prickly NDs in order to investigate, by comparison, the role of sharp

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NPs edges in endocytosis and exocytosis processes.291 In particular NDs were treated with APTES in water suspension and then centrifuged and redispersed in ethanol. Because of the presence of the thin layer of polymerized silane on the ND surface, successive condensation of the silica precursor (TEOS), under pH controlled hydrolysis conditions, started from the ND surface leading to the formation of a shell rather than to newly formed pure silica NPs. It is worth underling that although the amorphous shell has a covalent structure the nature of the binding at the silica carbon interface is not completely clarified and it expected to have, at least in part, an electrostatic character because of the presence of carboxylic and ammonium residuals. Covalent conjugation of bio-active molecules to NDs in an aqueous environment takes advantage of the rich chemistry of COOH and of well-defined coupling protocols. Poly-L-lysine coating of FNDs for example was conducted by dispersing the NPs in water in the presence of EDC and NHS to activate surface carboxyl groups. The activated FNDs were separated by centrifugation, re-suspended in phosphate buffer saline, and reacted with poly-L-lysine for surface grafting with amino groups.318 Resulting FNDs were also used as staring materials to produce dextran or BSA functionalized NPs by coupling the terminal amines to carboxylated dextran or BSA activated with EDC and NHS. Covalent functionalization of detonation NDs with lysine has also been proposed to reduce the propensity to aggregation in liquid formulation media.319 Protected lysine was first modified by attaching 1,3-diaminopropane as a spacer to the carboxylic group and conjugated to oxidized detonation NDs. The modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and a particle size of approximately 20 nm. Transferrin was conjugated to NDs directly by activating the COOH groups with EDC and NHS.317,320 The same strategy was proposed for functionalizing NDs with BmK CT, a key chlorotoxin-like peptide isolated from scorpion venom,321 and to conjugate mitochondrial antibodies to detonation NDs.322 Attachment of biomolecules to NDs previously included into a silica matrix was achieved by functionalizing the molecule bearing a carboxylate group with APTES in order to introduce a terminal triethoxysilane unit that was then grafted onto the silica surface as schematized in Fig. 41.323 6.3

Non-covalent functionalization of NDs

Functionalization and stabilization of NDs was also achieved by simple adsorption of large water compatible molecules or incorporation into micelles. Inclusion in SDS has been reported to improve colloidal stability of carboxylic terminated NDs.303 Encapsulation of NDs into silica has been proposed as a method to render the particle surface biocompatible, stable, and suitable for bioconjugation by introducing surface amino groups.323 Growth of a silica shell surrounding NDs can be achieved without pre-functionalization of the surface with APTES in the presence of surfactants. Controlled hydrolysis and condensation of TMOS in the presence of CTAB is known to produce mesoporous silica. As shown in Fig. 42, in the presence of NDs this process leads to the formation of a porous silica shell around the carbon NPs rather than to pure silica nanostructures.175 Further modification of the core–shell structure was achieved by adsorbing a


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Fig. 41 (a) NDs in a solution of TEOS are trapped in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) multilamellar vesicles (MLVs) that can range in size from 500 to 10 000 nm. Ultrasonication breaks the MLVs into small unilamellar vesicles (SUVs) with nominal diameters of B100 nm. TEOS is converted into silica, catalyzed by triethylamine (TEA). After dialysis, sodium dodecyl sulfate (SDS) wash breaks up the liposomes to free the coated NDs that are purified by further dialysis. (b) Conjugation of a ligand to the amine of APTES and use of the remaining silane moieties of APTES to form a linkage with the surface of the particle. Reproduced with permission from ref. 323. Copyright 2013, American Chemical Society.

of NDs for in vivo imaging applications. Biotin–streptavidin recognition is a strong non covalent interaction at the basis of many bio-conjugation. Biotinylated antibodies were bound to NDs by covering them with a lipidic bilayer containing a fraction of biotinylated lipidic units and using streptavidin as a cross-linker as shown in Fig. 43.325 In a different approach NDs were initially incorporated into large (0.5–100 mm) multilamellar vesicles that were broken by ultrasonication to give uni-lamellar 100 nm vesicles that are used as a template for the growth of a silica shell starting from TEOS and using triethylamine as a catalyzer After vesicle disruption and surface functionalization with APTES the NDs were conjugated to biotin as schematized in Fig. 41.323

7 Si QDs for in vitro imaging

Fig. 42 TEM images of ND and ND@MSN. (a) Pure ND cores as imaged 40 by TEM (b) individual ND@MSN imaged by high-resolution TEM (HRTEM). Corresponding (c) bright field and (d) dark field image revealing the presence of a crystalline ND core. Reproduced with permission from ref. 175. Copyright 2013, Royal Society of Chemistry.

poly(ethylene glycol)–poly(ethylene imine) copolymer. Use of NDs to vectorize si-RNA into the cells requires their modification with cationic polymers such as polyallylamine and polyethylenimine.324 These polymers adsorb efficiently on the negatively charged surface of oxidized NDs and bind the anionic RNA allowing cell transfection upon NP internalization. Other transfecting agents such as poly(propylene imine), dendrimers, liposomes and protamine sulfate that are absorbed onto carboxylated surfaces have been used to improve the cellular uptake of antibody covalently modified NDs.322 Surface adsorption of BSA179 has also been proposed to enhance the biocompatibility


Si QDs show unstable PL and poor colloidal stability when dispersed in a physiological environment without suitable protection. PL fading typically occurs within a few days after the exposure to aqueous medium. Chemical, photochemical and colloidal Si NC stabilization, essential for bioimaging application, requires a proper synthetic design and a specific surface modification. Considering that the properties of final Si NCs strongly depend on their preparation, in this section we will discuss the most relevant examples of application of Si QDs to in vitro bioimaging, by classifying the probes according to their preparative method. The features of all the systems have been summarized in Table 2. 7.1

Si QDs from laser pyrolysis

Stabilization in an aqueous environment of Si NCs prepared by laser pyrolysis199 was first achieved by Li and Ruckenstein via UV-induced graft polymerization of acrylic acid.326 These authors demonstrated, as a proof of principle, the use of the resulting red emitting NPs for in vitro imaging of Chinese hamster ovary cells (CHO). NPs showed, under UV irradiation, a PL quantum yield as high as 24% and a photostability higher than organic fluorophores. Chemical stability, in contrast, was only modest

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Fig. 43 NDLPs are synthesized by rehydration of lipid thin films containing cholesterol and biotinylated lipids with concentrated ND solutions. Hybrid particles are then targeted using biotinylated antibodies and streptavidin crossbridges. Reproduced with permission from ref. 325. Copyright 2013, John Wiley and Sons.

and a 20% decrease of the PL signal was observed after storage of the Si NP solution for 5 days in the dark. Actual toxicity of the nanoprobes on living cells was not investigated. Moreover labelling experiments were performed after fixation of the cells with paraformaldehyde and hence not on living cells. Biocompatibility of Si QDs prepared by laser pyrolysis, as well as their use for targeted bioimaging, were demonstrated by Erogbogbo et al. who incorporated these NCs into transferrin functionalized phospholipid micelles.232 The transferrin receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and it is efficiently internalized.336 The final probes, with diameters ranging from 50 to 120 nm, were prepared in several steps starting from 5–10 nm Si NCs produced by CO2 laser pyrolysis of silane. TEM images, shown in Fig. 44, demonstrate that tens of Si QDs, with a well-defined crystalline structure, are incorporated inside each individual micelle. Prior to the inclusion in the micelles, the Si NCs underwent acidic etching in a HNO3– HF mixture,221 a treatment used to de-agglomerate them and reduce their size in order to control their emission color, as well as to improve the PL QY. Increasing the etching time, PL evolved from red, to yellow, to green, as expected in the case of emission originated by the quantum confinement effect. Passivation of the Si surface via photo-activated hydrosilylation yielded organically capped hydrophobic NCs that were finally incorporated in phospholipid micelles as schematized in Fig. 33. As a relevant advantage of this synthetic approach, the final probes showed an almost pH and temperature independent luminescence and a shelf stability longer than one month. Nevertheless, the actual PL quantum yield of 2–4%, even if still suitable for fluorescence imaging, was quite modest and considerably lower than the one measured in organic solvent for the capped NCs before encapsulation in the micelles (17%). For bio-conjugation, transferrin was coupled to the nanoprobes prepared starting from phospholipids bearing a PEG-2000 chain terminated with a carboxylate. Confocal fluorescence images (Fig. 44) showed that the transferrin terminated probes were internalized by human pancreatic cancer cell (Panc-1) originating a characteristic localized red emission. In order to investigate

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the specificity of the binding, the authors performed a parallel labelling experiment using methoxy terminated nanoprobes as reference non bioconjugated systems. Although non-biofunctionalized NPs were not taken up by the cancer cells, the specific recognition of the transferrin receptors was only partially proved, since, as shown in Fig. 44, also amino terminated NPs associated with the cancer cells. Moreover no comparative experiments blocking the transferrin receptors themselves were performed in order to clarify the actual mechanism of internalization. Preliminary investigation of the cytotoxicity of these probes demonstrated their good biocompatibility: an average cell viability of about 95%, after 24 h incubation at the concentration used for bioimaging (8 mg ml 1), was measured testing the cellular metabolic activity by MTS assay. Further studies proved that the inhibitory particle concentration corresponding to 50% cell viability (IC50) was 4500 mg ml 1 for the micelle based nanoprobes, after 24 h incubation, and hence considerably higher than 20 mg ml 1 and 11 mg ml 1 measured for CdTe and CdHgTe QDs under the same exposition conditions.231,233 These results confirmed the expected higher biocompatibility of Si NCs with respect to metal based semiconductor QDs. Nanoprobe size is one of the parameters that strongly affect its interaction with living systems. Phospholipid micelles contained a large number of Si NPs and showed quite a large hydrodynamic radius. The same authors proposed a complementary synthetic approach to produce water dispersible individual small Si NCs. In this case, Si QDs made by laser pyrolysis were functionalized via hydrosilylation after acidic etching.327 Undecylenic acid was chosen as capping agent because of its low toxicity (5000 mg kg 1 in mice). Bioconjugation of the resulting carboxylate terminated small water-dispersible Si QDs to transferrin, antimesothelin and folic acid was explored for targeted fluorescence imaging of Panc-1 cells. These cells, in fact, have been reported to have receptors for all three biomolecules.337,338 Confocal images of Panc-1 cells incubated with the bioconjugated QDs proved the internalization of the NPs. Moreover, in the case of folic acid, specificity of the binding to the corresponding receptors was demonstrated by presaturating the binding sites with excess folic acid: no Si QD uptake by the cells was observed under these



EE EE

EE EE EE EE

EE CE ME ME ME HT HT HT P P P MW MW MW EE

EE

He et al.237 He et al.328

Wang et al.238 Ge et al.10329 Osminkina et al.330 Wang et al.236

Ahire et al.280 Nishimura et al.239 Rosso-Vasic et al.264 Shiohara et al.331 Shiohara et al.244 Henderson et al.332 Chiu et al.333 Xu et al.334 Shen et al.275 Ohta et al.276 Ohta et al.335 He et al.204 Zhong et al.281 Zhong et al.205 Park et al.56

Gu et al.235

LP LP

Styrene/octadecene/ethyl undecylenate (UV) Ethyl undecylenate (UV) Ethyl undecylenate (UV)

Oxidation

Acrylic acid (blue/UV) Acrylic acid (blue/UV) photo-oxidation AA (UV) Organic peroxide — Decene/undecylenyl alcohol/undecenoic acid Allylamine (T) 3-MPTS Allylamines Allylamine, 1,5-hexadiene Allylamine (UV/Pt) 1-Octadecene (T) 1-Decene (T) MAA/MPEG — Allylamines Allylamine (Pt) Glutaric acid — APTES citrate Oxidation

Acrylic acid (UV) Styrene/octadecene/ethyl undecylenate (UV) Undecylenic acid

Covalentb

Functionalization

F127 Modified F127 (COOH)

Phospholipid micelles

PEG-silane

Mannose ester — — — — Stearic acid NPs CTAB, PAA Doxorubicin Pluronic F127 Pluronic F127 — — — — Dextran/doxorubicin

— — — —

— —

— Phospholipid micelles PEG/COOH —

Non covalent

— Folic acid, anti-mesothelin, anti-claudin-4

RGD peptide

—

Concananvalin A Transferrin (C) — — — — — — — — — IgG IgG IgG —

— — — —

Lysine/folic acid/antimesothelin/apo-transferrin — IcG

— Transferrin/folate/biotin

Biomolecules

60 175

60–160

143–160

16 4.1 1.6 3–4 1.6/2.5 100 120 40–110 20–40 65/215/32/270 65 3–4 40 4 126

11 1.6–3.5 40–300 4

60/120/200 2–10

— 1–10

—

—

— — 1–13 — — — o7.0 — — — — 1–10 4–12 4–11 —

b

800 (36) 650

800 (10) 5–12 103 450–900 (5–10)

464 (11.5) 680 (8) 385–475 (11–13) B 3 450 325–400 955 (24) 133 103 580 (4–5) 550 450 (33) 450 (33) 450 660 (15) 660 (18) 460 (20–25) 800 (10)

600 450 (8) 755 650–680

600 (15–20) 510 (25)

650 (B5)

610 (25) 650 (2–4)

lmax/nm (QY %) t/ns

UV = ultraviolet irradiation, blue = blue

Mice/monkeys Balb C mouse

Panc-1 mice

MCF-7 NRK BV2 HepG2/HeLa MCF-7 MDA 435 N2a HeLa HUVEC HUVEC HUVEC HeLa HeLa HeLa MDA-MB-435 mouse Mice

HHL5/HepG2/3T3-L1 HeLa CF2Th HeLa

HEK2-273T HEK293

o7.0 2–13 — 2–10 — —

Panc-1

CHO Panc-1

Target

1–10

5.5 2–12

o10 50–120 26/47

pH

Size (nm)

LP = laser pyrolysis, EC = electrochemical etching, ME = microemulsion, HT = high temperature, P = plasma, MW = microwave, CE = chemical etching. irradiation, Pt = platinum catalyzed, T = high temperature.

a

Liu et al.233 May et al.266

LP

LP

Erogbogbo et al.327

Erogbogbo et al.

LP LP

Li and Ruckenstein326 Erogbogbo et al.232

231

Synthesis

a

Summary of the properties of the Si NCs based luminescent contrast agents discussed in this review article

Authors

Table 2


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Fig. 44 Top: TEM images of micelle-encapsulated Si QDs: all of the silicon QDs contained in a single micelle (left); and silicon QDs visible within the micelle (right). Bottom: confocal microscopic visualization of live pancreatic cancer cells treated with (left) amine-terminated micelle encapsulated Si QDs, (right) Tf-conjugated micelle-encapsulated Si QDs. Overlapped to the transmission images. Reproduced with permission from ref. 232. Copyright 2008, American Chemical Society.

conditions. On the other hand, aspecific binding was detected for lysine terminated Si NCs that associated with the cells without being internalized. In cell viability assays the nanoprobes showed an IC50 4 500 mg ml 1 for a 24 h incubation period confirming the high biocompatibility of this material. From the dimensional point of view, although these NPs exhibited at DLS hydrodynamic diameters of 20–50 nm, and then smaller than the previously discussed micellar systems, they still consisted of small aggregates of particles and cannot be considered individually dispersed Si NCs. 7.2

Si QDs from electrochemical etching

The use of Si NCs, prepared by electrochemical etching, for in vitro and in vivo bioimaging, was proposed by Park et al. after they observed a significant increase of the PL of these NPs upon aging in water for 2 weeks.56 During this activation step, silicon oxide growth on the initially H-terminated porous silicon surface produced an enhanced emission that the authors attributed both to the quantum confinement effect and to the presence of emitting defects localized at the Si–SiO2 interface.94,261,339 Interestingly these Si NC aggregates self-destructed in a mouse model into renally cleared components in a relatively short period of time with no evidence of toxicity. Thanks to their emission in the NIR region, they were also used for in vivo imaging, as it will be discussed in Section 8. Applicability to in vitro bioimaging was demonstrated in the case of HeLa cells that, after 2 h incubation with the NPs, showed significant luminescence due to non-specific cellular uptake. The NPs were prepared by electrochemical etching of single-crystal silicon

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wafers in ethanolic HF solution. The synthetic conditions were optimized to provide NP size and porosity suitable for loading of therapeutics and long in vivo circulation times, while maintaining an acceptable degradation rate. Optimal performances were reported for B126 nm mesoporous NPs (the diameter was measured both by SEM and DLS) showing pore sizes of 5–10 nm. The Si NPs showed a PL quantum yield of 10% in the 650–900 region nm upon UV excitation (Fig. 45) and a photostability superior to NIR emitting cyanine dyes (Cy5.5 and cy7). Moreover DLS and atomic emission spectroscopy measurements shown in Fig. 45 demonstrated that these NPs undergo complete degradation under physiological conditions (PBS, pH 7.4, 37 1C) after 8 h. During this time lapse their PL gradually disappeared while the PL maximum shifted toward higher energy. Because of their biodegradability and high porosity (demonstrated by TEM and BET analysis these) NPs revealed promising drug loading and releasing properties that were investigated in the case of the anti-cancer drug doxorubicin (DOX). Loading of DOX increased the zeta potential of the NPs from 52 to 39 mV and, as shown in Fig. 45, a relatively slow release of the drug was observed at physiological pH and temperature, reaching significant levels within 8 h. Cell viability of the unloaded NPs, determined by measuring the esterase activity with the calcein assay, demonstrated the low cytotoxicity of the NP formulation: after 48 h incubation at a concentration up to 200 mg ml 1 no significant mortality of HeLa cells was observed. In contrast, the cytotoxic effect of DOX on the MDAMB-435 human carcinoma cell was enhanced when loaded in the Si NPs. MTT assay, in fact, revealed a higher cellular mortality for the same DOX concentration (in the range 1–10 mg ml 1) in the presence of the Si NPs with respect to the drug alone. Biodegradability is an interesting feature for drug release application as well as for the safe elimination of Si NPs from living organisms. As a drawback it prevents long-term bioimaging and it reduces the storage life of the nanoprobes. A weakness of the synthetic method proposed by Sailor is the production of a very polydispersed population of silicon fragments whose size distribution can be narrowed only by combined filtration and centrifugation. Biodegradable Si QDs were also prepared by mechanical grinding of electrochemically produced porous silicon.330 Osminkina et al. demonstrated that these NPs penetrate the cytoplasm in the case of CF2Th cells showing an emission at about 750 nm showing no cytotoxic effect up to the concentration of 100 mg ml 1 in the dark. Surprisingly, according to these authors, these NPs behave as an efficient photo-generator of singlet oxygen and they show relevant photo-toxicity, which make them suitable for photodynamic therapy of cancer. Kang et al.100 developed an electrochemical method, based on the use of polyoxometalates (POMs), that, in contrast to the Sailor’s group one,56 produced highly monodisperse small Si QDs (1, 2, 3, and 4 nm diameter) with a narrow size distribution and that required no separation. In order to increase their resistance against degradation these Si QDs were incorporated into polyacrylic-acid nanospheres, yielding probes suitable for bioconjugation and immunofluorescent cell imaging.237


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Fig. 45 (a) Schematic diagram depicting the structure and in vivo degradation process for the (biopolymer-coated) NPs. (b) SEM image of Si NPs (the inset shows the porous nanostructure of one of the nanoparticles). The scale bar is 500 nm (50 nm for the inset). (c) PL emission and absorbance spectra (d) appearance of silicon in solution (by ICP-OES) and PL intensity from a sample of Si NPs incubated in PBS solution as a function of time. (e) Release profile depicting per cent of DOX from DOX-Si NPs released into a PBS solution as a function of time C. (f) Cytotoxicity towards MDA-MB-435 human carcinoma cells. Reprinted with permission from ref. 56. Copyright 2009, Macmillan Publishers Ltd.

The surface modification strategy was based on calculations performed by using a classic method based on hybrid density functional theory. Theoretical results predicted that the photoreaction of hydrogen terminated Si NCs with acrylic acid favored the formation of Si–O bonds under blue (l = 490 nm), and the creation of Si–C bonds under UV (l = 360 nm) irradiation respectively. Hence, Si dots were first irradiated with blue light, in the presence of acrylic acid, to passivate the Si surface, and then, they were exposed to UV radiation to polymerize acrylic acid. The overall process produced the nanospheres shown in the TEM images of Fig. 46. The polymeric NPs presented a controllable diameter in the range from 60 nm to about 200 nm and they contained hundreds of Si QDs with crystalline structure per particle. Nanospheres also showed a FL QY of about 15–20%, hence more than six times higher than the one measured for the starting Si NCs before incorporation (2–3%). Moreover the NP emission was stable after 6 month storage in air. The as-prepared nanospheres were used as cellular probes to label HEK293T human kidney cells.237 Images acquired with laser scanning confocal fluorescence microscopy, at different observation time during continuous scanning, are shown in Fig. 46. NPs showed a superior photoluminescence stability compared to molecular dyes and to II/VI QDs in aqueous solution. Moreover, almost no photobleaching of the labelled cells was observed after 20 minute irradiation. Favorable biocompatibility was also reported for these NPs. In fact, cell viability determined by MTT assay, after 24 h incubation of HEK2-273T cells at the same concentration used for cellular imaging experiments, remained roughly 100%. According to the authors, the high photostability and shelf stability of these materials are due to


Fig. 46 Top: TEM images of the as-prepared nanospheres with a size of approximately 120 nm. HRTEM image of single Si QDs inside the as-prepared nanospheres (right). Bottom left: temporal evolution of fluorescence of the HEK293T cells labeled with the as-prepared nanospheres (red images) and FITC (green images). Bottom right: photostability comparison of fluorescent II/VI QDs (CdTe QDs and CdTe–CdS–ZnS core–shell–shell QDs) and the as-prepared nanospheres (right). Reproduced with permission from ref. 237. Copyright 2008, John Wiley and Sons.

the polymeric protective shell that shields the Si dots from molecular quenching and oxidation. Although the possible bioconjugation of these NPs, after activation with EDC according to standard protocols, had been anticipated in the original work, the authors later reported that a major drawback of these nanoprobes is, indeed, the lack of stability at the pH required

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Fig. 47 Absorption and PL spectra of oxidized Si nanosphere O-SiNSs (a). PL spectra of samples with an identical optical density of 0.10 (b). The insets display the corresponding optical micrographs of the sample under UV irradiation. Photostability comparison (c and d). Reproduced with permission from ref. 328. Copyright 2009, American Chemical Society.

for the reaction and more in general at pH higher than 7.0. As shown in Fig. 47 this effect becomes extremely deleterious in the case of polymeric particles with sizes of a few tens of nanometer. In order to eliminate this drawback, the authors modified their probes in a further synthetic step. Nanospheres, with 35 nm of diameter, were thermally oxidized in an O2 saturated aqueous environment at 80 1C.328 As show in Fig. 47328 oxidation caused a change of the emission color, from orange to green, and a rise of the PL QY to about 25%. Most importantly, the pH stability of the nanospheres, which before oxidation degraded at pH 4 7 completely losing their luminescence, was strongly increased in the pH range 2–10. Thanks to this improved pH resistance, oxidized NPs, containing about 100 silicon dots per particles, could be readily conjugated with goat-antimouse IgG in PBS buffer (pH B 7.4), maintaining very bright and stable fluorescence for over 1 month. The bioconjugates were successfully applied in immunofluorescent imaging of HEK293T cells. Although oxidation of the nanospheres enlarged their pH stability window, producing materials that in MTT assay presented the same low cytotoxicity of the pristine probes, the oxidative approach has a major weakness. In fact, oxidation alters the emission color of the probe and, although in principle emission wavelength can be controlled by changing the size of the embedded Si QDs, other emission colors different from green have not been demonstrated. Moreover, after oxidation emission is expected to be originated by surface defect states rather than by the quantum confinement effect and hence to be size independent. Acrylic acid was also used by Wang et al. to stabilize the surface of Si QDs produced by electrochemical etching238,250,339 In a more traditional approach, with respect to the two-step, irradiation wavelength dependent one previously reported,237 grafting of the acrylic group to the silicon surface was achieved

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via simple photochemical hydrosilylation, hence forming C–Si bonds under UV irradiation. The authors investigated the biological effects of their Si NPs on cell growth and proliferation, both in human and mouse cell lines, after incubation up to 48 h and up to a probe concentration of 200 mg ml 1. There was no evidence of in vitro cytotoxicity on the cells exposed to polyacrylic acid terminated Si NPs when assessed by cell morphology, cell proliferation and viability, and DNA damage assays. The uptake of the Si NCs both by HepG2 and 3T3-L1 cells was investigated by confocal microscopy and flow cytometry. The process showed a clear time-dependence at high concentrations. Reconstructed 3-D confocal microscope images exhibited that Si NPs were evenly distributed throughout the cytosol rather than attached to outer membranes. Emission of the Si NPs was centered at 600 nm and reasonably stable within one month storage in the dark. PL QY and pH stability of the NPs were not discussed. As an advantage these Si NPs show a rather small hydrodynamic diameter (o10 nm) that, according to the authors, may allow them to diffuse more readily though the tumor tissue’s interstitial space in a potential application as drug carriers in cancer therapy. Si NCs based bioprobes with small hydrodynamic radius were also prepared by Ge et al. who proposed a different synthetic approach to water dispersible carboxylic acid terminated Si NCs. Functionalization of the surface of hydrogen terminated NCs, prepared by electrochemical etching, was achieved using an organic peroxide, via radical thermal formation.329 Surface grafting was confirmed by XPS and the produced Si NCs showed in TEM images a diameter ranging from 1.6 to 3.5 nm, in agreement with the average hydrodynamic diameter of about 2.6 nm measured by DLS. Si QDs displayed, when dispersed in an aqueous environment, quite strong and stable PL in the visible spectrum with an emission quantum yield of 8%. Moreover, after storage in air for 16 days, without any additional protection, 70% of the initial PL intensity was retained. No obvious decrease in cell vitality was observed for HeLa cells cultured in the presence of 20 mg mL 1 of the carboxylated NCs. Confocal microscope visualization of the treated cells showed bright blue emission from the Si QDs distributed uniformly into the cytoplasm. As an advantage of the radical surface termination, small Si NPs could be prepared. Nevertheless, their storage stability and alkaline pH resistance were moderate. Moreover, as reported by the authors, NP samples were quite polydisperse in size and only blue emission was achieved. A different strategy for stabilizing electrochemically produced Si QDs was proposed by Wang et al. who developed a microwaveassisted hydrosilylation method for the preparation of stable fluorescent probes for cellular imaging starting from Si NCs obtained by conventional electrochemical etching. A remarkable advantage of this process was the faster rate of hydrosilylation with respect to photochemical or thermal modification. Moreover a higher surface coverage is achieved guaranteeing a significantly increased chemical stability of the produced materials.236 The high versatility of the process was demonstrated by introducing various terminal functionalities, such as alcohol, alkyl groups, and carboxylic acid. The authors demonstrated the use of


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the as-prepared particles for long-term intracellular fluorescent imaging proving their excellent cell compatibility. Hydrosilylation was also proposed by Ahire et al. for functionalizing Si QDs, produced by electrochemical etching, with carbohydrates, molecules that play a critical role in cell recognition. Hydrosilylation was exploited to produce amine surface groups that were then coupled to D-mannopyranoside acid. The average diameter of the crystal core of the mannose functionalized particles was 5.5 nm while the hydrodynamic diameter was 16 nm.280 The NPs exhibited a PL quantum yield of 11.5% and a good stability over two weeks. Internalization of the functionalized NCs and their distribution throughout the cytoplasm were demonstrated in the case of MCF-7 human breast cancer cells. It is well known that mannose has a strong binding affinity towards lactin. In a non-cancerous cell line, galactoside binding galactines-4, -7, and -8 are absent. However, these galactines are present in the MCF-7 cancer cells. Hence, according to the authors, internalization proved the specific interaction of the mannose units on the surface of the Si NCs with the cell receptor demonstrating the applicability of their probe for selective drug delivery to cancer cells. Nevertheless, no blank experiments using non bioconjugated Si NPs or non-cancer target cells were performed in order to demonstrate the actual specificity of the recognition. Nishimura et al. proposed chemical etching, as an alternative to the electrochemical one, to develop biocompatible, red-emitting Si NCs. The NPs, with a 4.1 nm hydrodynamic diameter, were used to image single receptor molecules in the plasma membrane of living cells (using a transferrin receptor) for Z10 times longer than with other probes.239 The Si NCs were prepared by chemical etching of a silicon wafer and functionalized when still bound to the bulk silicon with mercaptosilane. Finally the dots were detached by mechanical scraping and conjugated to transferrin precisely at a 1 : 1 ratio. Thanks to their approach the authors were able to observe the internalization process of receptor molecules at the single-molecule level unrevealing spatial variations of molecular diffusivity on the scale of 1–2 mm. As an advantage chemical etching and NC functionalization with mercaptosilane, produced small size NCs stable as individual particles in water. Nevertheless, in the final preparative step, the Si NCs are liberated from the polished side of the wafer by mechanical scraping and the overall yield of the synthetic process, not discussed in detail, is expected to be rather low. 7.3

Si QDs from microemulsion

Microemulsion techniques yield easily processable liquid suspensions of Si QDs. Hence these methods are very convenient to prepare Si QDs for bioimaging. Rosso-Vasic et al. developed a set of, very stable and bright emitting, amine-terminated, Si NCs featuring tunable emission from the UV to the blue spectral region. Emission maximum wavelength was controlled simply by changing the length of the alkyl amine stabilizer.264 The emission quantum yields were about 12% for all synthesized particles and the excited state lifetimes were in the nanoseconds range, as expected in the case of direct band gap transitions. The Si NPs showed in TEM images, a spherical shape and a


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homogeneous size distribution (1.6 0.2 nm) compatible with the hydrodynamic diameter measured by fluorescence correlation spectroscopy (1.4 nm). Moreover the NCs exhibited an exceptional stability over a wide pH range (1–13) and at high temperatures. The amino capped Si NCs were synthesized from hydrogen terminated Si QDs prepared by202 reducing Si(OCH3)4 with LiAlH4 by hydrosilylation with allylamines of different lengths. The resulting NPs have been demonstrated to be highly suitable for bioimaging studies. They were readily taken up by BV2 nerve cells where they located in the cytosol and did not tend to relocate to the nuclei. Moreover, proliferation of stained BV2 cells was observed and revealed that newly formed cells were also labelled by the fluorescent NPs, suggesting that the toxicity of these materials is minimal. Thanks to this feature, according to the authors, it becomes possible to stain multiple cell generations by only staining the mother cells. On the other hand, labeling experimental conditions were quite unusual. A very high NP concentration (7 mg ml 1) and a quite short incubation time (5 minutes) were used. Moreover although different emission colors were achieved, the PL bands were, in all the cases, quite broad, strongly excitation wavelength dependent and located in a relatively small spectral windows. Because of all these features, the obtained set of probes is not ideal for multiplexed detection. As advantage the amino terminated Si NPs are suitable, at least in principle, for bioconjugation. A more detailed investigation of the cytotoxicity of Si NPs prepared by microemulsion was reported by Shiohara et al. in the case of Si QDs functionalized with reactive surface groups such as amines, diols and epoxides.331 The Si NCs showed, in the TEM images, an average diameter ranging from 3.4 nm to 3.7 nm. Cell viability assay (MTT assay) was applied for cytotoxicity evaluation in human skin fibroblasts (WS1) and lung epithelial cells (A549). Epoxide terminated Si QDs were reported to be cytotoxic at a concentration of 112 mg mL 1 (IC50) while diol terminated Si NCs toxicity manifested at 448 mg mL 1. The more pronounced toxicity of the epoxy terminated NPs was, according to the authors, in agreement with the higher reactivity of these groups with respect to diols. Cytotoxicity on liver cells, which has the function to reduce toxicity by albumin secretion or glucuronate conjugation, was also evaluated in the case of amineterminated Si QDs. The results showed that IC50 of amineterminated silicon quantum dots in the hepatoma cell line (HepG2) was 100 mg mL 1, which is similar to the one of the diol-terminated silicon quantum dots in WS1 cells. The authors hence concluded that the use of Si QDs in biological application is safe below the 100 mg mL 1 concentration threshold. The prepared Si QDs where then used for bioimaging. HeLa cells became bright blue fluorescent, after incubation with the nanoprobes, the emission being distributed uniformly into the cytoplasm. The same authors optimized the microemulsion based process comparing different reducing agents and hydrosilylation methods244 and they concluded that the strongest reducing agent yields the smallest size and narrowest size distribution. Moreover photochemical hydrosilylation is preferable to thermal one since it is more efficient and it does not produce undesired residual products. Cellular uptake of the

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strong source of noise under such operative conditions (UV excitation, blue detection).


7.4

Fig. 48 (a) Fluorescent image of the cells after incubating with silicon quantum dots for 6 hours monitoring at 420 nm to 475 nm (excited at 405 nm), (b) fluorescent image of the same sample after marking the lysosome with Lyso Tracker monitoring between 510 and 550 nm (excited at 488 nm). (c) Overlapped image of (a, b) and transmitted image, (d) fluorescent image of the cells incubated for 24 hours with silicon quantum dots. The monitoring wavelengths were 420 nm to 475 nm (excited at 405 nm), (e) fluorescent image of the same sample after marking the lysosome with Lyso Tracker monitoring between 510 and 550 nm (excited at 488 nm). (f) Overlapped image of (d), (e) and transmitted image. Reproduced with permission from ref. 244. Copyright 2011, Royal Society of Chemistry.

optimized 1.6 nm silicon quantum dots by living cells was studied with confocal fluorescence microscopy to identify the distribution of any silicon quantum dots within the cells. Breast cancer MCF-7 cells were used in this study showing that the amine terminated silicon NCs accumulated in lysosome but not in nuclei and could be used as bio-markers to monitor cancer cells over long timescales (Fig. 48). Allylamine modified Si QDs prepared in microemulsion were also used by Chinnathambi et al. to follow the delivery of cytosine–phosphate–guanine oligodeoxynucleotides, molecules that stimulate the immune system and that can be applied to infectious disease and allergy treatments and cancer therapy, into peripheral blood mononuclear cells.279 As a general consideration, although microemulsion methods are convenient for the simple preparation of water compatible Si NCs they yield only blue-greenish emitting NCs that show reasonably bright emission only upon UV irradiation. These photophysical features are not ideal for bioimaging applications since biological sample autofluorescence becomes a

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Si QDs from high temperature methods

Contrary to microemulsion, high temperature process yields red, NIR emitting Si NCs suitable for low background detection in bioimaging. Henderson et al. proposed the incorporation into stearic acid NPs as a method to disperse in a physiological environment Si NCs prepared via high temperature decomposition of the silicon precursor.332 Alkyl stabilized Si NCs, emitting in the NIR region (lmax B 750 nm), were synthesized by hydrolysis and poly-condensation of HSiCl3 that yields (HSiO1.5)n sol–gel glasses. Subsequent thermal processing led to their decomposition and to the diffusion-mediated formation, growth, and crystallization of Si NCs embedded in a SiO2 matrix. Acidic etching of the silica shell with HF yielded hydrogen terminated Si NCs that were passivated by hydrosilylation with 1-octadecene HRTEM images of the octadecyl-capped NCs showing spherical particles with an average diameter of 4.4 0.8 nm and a lattice spacing of 0.31 nm, consistent with the (111) crystal plane of diamond-structure silicon. The Si NCs were finally loaded within the matrix of PEG-stearate NPs to ensure aqueous colloidal stability and biocompatibility. The final probes were applied to in vitro labeling of human MDA435 breast cancer cells. After incubation with the NPs and extensive washing, cells were imaged under a confocal microscope. NIR emission was clearly observed within the cells demonstrating efficient cellular uptake. For the octadecyl-capped Si NCs a decay with a lifetime as long as t = 133 ms was measured that was attributed to an indirect bandgap transition involving the participation of lattice vibrations for radiative recombination. Thanks to the long excited state lifetime, these NPs are promising contrast agents for time gated bioimaging. In this perspective investigation of the cytotoxicity of the material, not considered by the authors, should be taken into account. Moreover, although before incorporation into the stearic acid NPs, the Si NCs presented an extraordinary high PL QY in the NIR (26%), to what extent such a feature was maintained in the final water dispersed probe, as well as the stability of the emission are not discussed by the authors. Good storage stability in water, in contrast, was demonstrated for similar NCs by Chiu et al. who developed a surface modification strategy that yielded Si NCs that preserved red PL in water for more than 6 months.333 The Si QDs, liberated from the oxide matrix, were functionalized via hydrosilylation and phase transferred to water by using the surfactant CTAB without altering the red PL. According to the authors CTAB played a double role in providing stable, aqueous, red-emitting Si NPs by forming a hydrophobic barrier between the Si NPs and water and providing aqueous colloidal stability via the polar head group. Preservation of the aqueous red emission of these Si NPs was observed in N2a cells when imaged by fluorescence microscopy. A drawback of these NPs was the, pH and wavelength dependent, quite modest reported PL QY (4%). Xu et al. proposed the incorporation into a water compatible shell as a method to stabilize in an aqueous environment


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Si QDs prepared via high temperature methods also increasing the drug loading ability of the NPs. The nanocrystalline silicon core was enveloped in a dual shell structure having an inner part based on a poly(methacrylic acid) and a polyethylene glycol outer layer. Produced nanocarriers had sizes ranging from 40 to 110 nm in a simulated physiological environment.334 The loading efficiency of model drug doxorubicin (DOX) was approximately 6.1–7.4 wt% and the drug release was pH controlled. Fluorescent imaging studies showed that the nanocarriers could be used as a tracker at the cellular level while cytotoxicity studies demonstrated that DOX-loaded core–shell particles showed high anticancer activity against HeLa cells. Indeed studies on the HeLa cells demonstrated also a moderate cytotoxicity of the unloaded probe that caused a decrease of cell viability of about 20% and 40% after 24 h incubation at concentration 8 and 128 mg mL 1 respectively. 7.5

Si QDs from the plasma-assisted process

Dispersion in water of hydrophobic Si NCs synthesized via plasma-assisted decomposition of SiBr4260 was achieved by Shen et al. using Pluronic F127 as a surfactant. This method produced Si NC aggregates able to selectively label the endoplasmic reticulum (ER) in live cells.275 Pluronics are neutral triblock surfactant polymers, made of a central PPG section and two terminal PEG chains. They form, in water, micelles where the PPG core constitutes a low polar nano-environment. TEM images of the Si QDs, dispersed in water after F127 treatment, show the Si QDs in the form of uniform and small aggregates of 20–40 nm diameter whose presence in solution was confirmed by DLS analysis. The PL of the Si QDs quantum yield increased to about 33% immediately after F127-treatment becoming about twice the one of the as synthesized NCs. Moreover, the NCs retained 80% of the initial PL intensity after UV irradiation for 3 hours. The nano-aggregates showed almost no cytotoxicity in cell viability assay on human umbilical vein endothelial cells (HUVECs) at a concentration up to 1000 mg mL 1 after

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48 h incubation. Si NCs treated cells were imaged by confocal fluorescence microscopy. High-magnification revealed that a network structure compatible with the ER was selectively labeled in the cell. The selectivity for the ER was proved by colocalization experiments. This result was the first example of specific intracellular localization of Si QDs. The same authors extended their approach, based on the use of Si QD aggregates, to label specific cellular compartments in human umbilical vein endothelial cells, producing a set of probes that targeted different cellular organelles such as lysosomes, endoplasmic reticulum, cytosol and nuclei. As shown in Fig. 49 selectivity was tuned by changing the size of the aggregates and their surface properties.276 In particular, size of the aggregates was controlled from ca. 30 to 270 nm diameter and either covalent passivation allylamine via hydrosilylation or non-covalent stabilization with Pluronic F-127 was used to modify the surface chemistry. In fixed cells allylamine-terminated Si QDs were reported to selectively label cell nuclei, while NPs treated by the amphiphilic block copolymer uniformly labeled the cytosol. A different response was observed in live HUVECs where allylamine-modified Si QDs selectively labeled lysosomes, whereas F127-treated Si QDs showed size-dependent intracellular localization: F127-treated NP aggregates with a small diameter of ca. 30 nm selectively targeted the endoplasmic reticulum and those with a large diameter of ca. 270 nm labeled lysosomes. Amino-terminated particles presented the highest cytotoxicity to HUVECs. In the case of the smallest aggregates a decrease of cell viability of about 50% was observed after 24 h incubation at 100 mg mL 1 concentration. Ohta et al. also investigated the kinetics of uptake and removal of their amino terminated probes by HUVECs via a confocal laser scanning microscope335 observing a gradual increase of the number of the internalized particles during the time till a plateau is reached. After washing and exposure to fresh, Si QDs free, culture medium, a fraction of the particles was gradually released via exocytosis but a large number of

Fig. 49 Confocal microscopic images of live HUVECs labeled with Si NCs. HUVECs were also labeled with Lyso-Tracker/ER Tracker. In each panel, the left and central rows represent the fluorescent image from Si-QDs and Lyso-Tracker/ER-Tracker, respectively, and the right row represents the merged image. The scale bar is 10 mm. Reproduced with permission from ref. 276. Copyright 2012, Royal Society of Chemistry.


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Fig. 50 Schematic of cellular uptake and removal processes of Si QDs in the kinetic model. ka, kd, ken, krec, Kd,in represent rate constants for each step. P0, P, Pin represent the number density of Si QDs in dispersion, the number density of Si QDs bound to surface receptors and the number density of internalized Si QDs in cell, respectively. Rs and Rin are the number density of receptors on the plasma membrane and cell interior, respectively. The inset shows the dissociation equilibrium between Si QD aggregates and receptors in the endosome. [P_R], [P], [R] represent the concentration of complexes of Si QD aggregates and receptors, the concentration of free Si-QDs and the ‘‘effective’’ concentration of free receptors in the endosome, respectively. Reproduced with permission from ref. 335. Copyright 2012, Elsevier.

them was accumulated into the cells. The authors used the kinetic model schematized in Fig. 50,335 based on the mass balance of Si QDs and receptors in a cell, to fit the experimental data and they demonstrated that the dissociation constant between receptors and the particles in the endosome was a determinant factor for accumulation in cells. It is quite surprising that, although color-tunable emission is one of the interesting feature of Si NCs prepared via non thermal plasma, only examples of water dispersed NPs with broad emission in the blue-green region have been reported. 7.6

Si QDs from microwave assisted methods

Direct production of Si NCs in an aqueous environment offers unique opportunities in the design of silicon based nanoprobes for bioimaging. He et al. developed a one-pot microwave assisted method to synthesize small Si NCs directly in water.204 In this approach silicon nanowires and glutaric acid are used as reactants to produce Si QDs that exhibit excellent aqueous dispersibility, robust photo- and pH-stability and strong luminescence centered at 660 nm (PL QY B 15%). In the TEM images the dots appeared as spherical particles with good monodispersity (d = 3.11–0.65 nm). Moreover, the well-resolved lattice planes of B0.16 nm spacing in the HRTEM images demonstrated the excellent crystalline structure. DLS analysis provided a hydrodynamic diameter (4.2 nm) for the particles in aqueous dispersion that was compatible with the TEM observations. Dots were conjugated to a goat anti-mouse antibody via EDC/NHS cross-linking reaction and used for targeted

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Fig. 51 Photos of immunofluorescent cell imaging captured by laser scanning confocal microscopy. (a) Left: microtubules of HeLa cells are distinctively labeled by the Si QDs–protein bioconjugates. Middle: brightfield image. Right: superposition of fluorescence and transillumination images. (b–d) Stability comparison of fluorescence signals of HeLa cells labeled by Si QDs (b), CdTe QDs (c), and FITC (d). Scale bar = 5 mm. Reproduced with permission from ref. 204. Copyright 2011, American Chemical Society.

immunological labeling of HeLa cells. Confocal fluorescence images of the labeled cells taken at different timed during continuous scanning are shown in Fig. 51. Microtubules of the HeLa cells are distinctively targeted by the silicon bioconjugates and show almost no photobleaching even after 2 hours observation. In contrast fluorescence of the cells treated with CdTe and FITC completely disappeared after 25 and 3 minutes respectively. Cytotoxicity studies demonstrated that the NPs do not decrease the viability of HeLa cells after 48 h incubation with the silicon based probes at the concentration used for the immunofluorescence experiments. The same authors modified their silicon nanowire based synthesis to achieve direct NC bioconjugation via a two steps microwave-assisted method that allowed them to prepare strongly fluorescent (QY = 18%), photo- and pH-stable, protein functionalized Si QDs. Silicon nanowires were first break up into Si NPs in a specialized microwave reactor at high reaction temperature (180–200 1C).281 Modification with the protein immunoglobulin G


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Fig. 52 Cell images obtained by using laser-scanning confocal microscopy. (a) Microtubules are specifically targeted by the prepared Si NPs, showing strong red fluorescence signals. (b) As a control, pure Si NPs, i.e. Si NPs without IgG proteins as ligands, are nonspecifically absorbed by HeLa cells. The red signals are distributed in the whole cellular region. Scale bar = 10 mm. Insets present one single cell imaged using the prepared Si NPs (a) and pure Si NPs (b). Left panel shows 488 nm excitation images, middle panel shows bright field images, right panel shows superposition of fluorescence and transillumination images. Reproduced with permission from ref. 281. Copyright 2012, John Wiley and Sons.

was then carried out under mild microwave employing shorttime (ca. 5–10 min) and at a low-temperature (30 1C) to avoid protein degradation. The as-prepared Si NCs showed excellent aqueous dispersibility and biospecific properties and they were directly employed for immunofluorescent cellular targeting of HeLa cells, without requiring additional bioconjugation. As shown in Fig. 52, confocal microscopy images demonstrated the specific targeting of the microtubules of the cells as well as the extreme photostability of the fluorescence that allowed continuous acquisition for 3 hours without any evidence of photobleaching. The microwave methods proposed by Lee and He are very convenient and they produce water dispersed ultraphotostable red emitting Si NPs with small size that as a further advantage can be stored for months without changing their properties. A weakness of this approach, on the other hand, is that it makes use of preformed nanowires as starting materials. Considering this limitation the same authors developed alternative synthetic methods, also based on microwaves irradiation, to obtain Si NCs from an easily accessible low-cost reagent. The new large-scale synthetic strategy for facile one-pot aqueous synthesis of silicon nanoparticles yielded B0.1 g Si NPs of small sizes (B2.2 nm) in 10 min using APTES as a silicon source and microwave irradiation.205 As-prepared particles feature strong blue fluorescence (PL QY of 20–25%), favorable biocompatibility, and robust photo- and pH-stability. According to the authors these features are due to the plentiful surface-covered amino groups, which act as a protective shell around the NPs. These same functional groups are suitable for conjugation with protein (e.g., goat-antimouse IgG) via traditional EDC/NHS cross-linking reaction, i.e., the amino groups of ligands on Si NPs readily react with the carboxylic acid groups of the antibody via NHS


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Fig. 53 Photos of immunofluorescent cell images captured by laser scanning confocal microscopy. (a) Left: nuclei are distinctively labeled by Si NPs (excitation: 405 nm, detection window: 450–510 nm); middle: microtubules are distinctively labeled by FITC (excitation: 488 nm, detection window: 515–550 nm); and right: superposition of the two fluorescence images. (b) Time-dependent stability comparison of fluorescence signals of HeLa cells labeled by SiNPs (blue) and FITC (green). Scale bar = 5 mm. Reproduced with permission from ref. 205. Copyright 2013, American Chemical Society.

and EDC as zero length cross-linkers. As shown in Fig. 53,205 the resulting probes are particularly suitable for long-term cellular imaging due to their high photostability allowing, in a typical imaging experiment where they are used to stain the nuclei of HeLa cells, continuous observation of stable fluorescent signals for 60 minutes. Also in this case MTT assay showed no relevant decrease of HeLa cell viability after 48 h incubation with the NPs at the concentration used for the immunofluorescence. Nevertheless, as in the case of the Si NCs prepared starting from the Si NWs the authors did not perform a detailed investigation of the effect of the NPs dosage on the toxicity.

8 Si QDs for in vivo imaging In vivo imaging was demonstrated, in the case of Si NPs prepared by electrochemical etching, by the group of Sailor while Swihart and Prasad proposed the use of nanoprobes obtained via laser pyrolysis for the same application. Both approaches will be discussed in this section by comparing the features of the two categories of nanoprobes, also summarized in Table 2. Si NCs prepared by electrochemical etching of porous silicon, undergo fast, partially controllable, biodegradation in living organism. Such a process favors organism washing out and it is

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expected to guarantee low toxicity. As a disadvantage, these porous materials cannot be used for long-term imaging. In contrast long-term in vivo detection of the more stable nanoprobes prepared via laser pyrolysis was demonstrated. The in vivo toxicity of both the sets of probes was investigated in very detail in mice and in the case the Si QDs are produced by laser pyrolysis it was investigated in the monkey model. 8.1

In vivo imaging with Si NCs from electrochemical etching

Park et al. demonstrated the application to in vivo bioimaging of the luminescent porous Si NPs schematized in Fig. 45 that we already discussed for in vitro detection. These Si NPs can carry a drug payload and they exhibit an intrinsic NIR photoluminescence that enables monitoring of both their accumulation and degradation in tissues.56 Moreover they self-destructed in a mouse model into renally cleared components in a relatively short period of time with no evidence of toxicity. The Si NPs were prepared by electrochemical etching of single-crystal silicon wafers in ethanolic HF solution and underwent activation of luminescence in aqueous dispersion. Thanks to the NIR emission the NPs enabled the whole-body fluorescence imaging of nude mice although a significant fraction of the bare NPs were removed by renal clearance immediately after the intravenous injection. The retention time was increased by coating the NPs with dextran by simple physisorption. Biodistribution and histological studies of the organs collected from the same mice 24 h after injection were consistent with the whole-body fluorescence imaging data. These results indicated that the use of intrinsic luminescent properties of Si NPs enables the non-invasive monitoring of their bi distribution and degradation in a live animal as well as the microscopic observation of their localization in the organs. Injection of the Si NP formulation (20 mg kg 1) into a nude mouse bearing an MDA-MB-435 tumor resulted in passive accumulation of the nanomaterial in the tumor, as revealed in the near-infrared fluorescence image as shown in Fig. 54.56 The ex vivo fluorescence images and histology confirm the presence of dextran coated Si NPs in the tumor. Similar to some of the other near-infrared-emitting

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semiconductor quantum dots the brightness of Si NP decreases with longer excitation wavelengths. Nevertheless, according to the authors, it was sufficient to enable their observation in internal organs using a conventional fluorescence imaging system. Recently the same research group reported a different application of photoluminescent porous silicon nanoparticles340 that exploits their unusually long emission lifetime (5–13 ms) for timegated imaging of tissues in vivo, completely eliminating shorterlived (10 ns) emission signals from organic chromophores or tissue autofluorescence demonstrating improvement of the signal to background contrast ratio by 450-fold in vitro and by 420-fold with a non-optimized setup. Results are summarized in Fig. 55.235 Time-gated imaging of porous silicon nanoparticles, covalently functionalized with PEG as schematized in Fig. 30, accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The authors also demonstrated that the decay time of the probe can be modulated by changing the synthetic conditions as reported in Fig. 30 and discussed the potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes. Fluorescence decays of Fig. 55 demonstrate that emission from organic molecules can be removed from images by time gated acquisition. In particular emission from a solution of the organic dye Cy 3.5 was observed to be comparable to the one of the PEG terminated Si NPs in the steady state mode but it disappeared in the time gated images. The same detection selectivity was demonstrated in the mouse where time gated acquisition also allowed to eliminate the background strongly increasing the signal to noise ratio. 8.2

In vivo imaging with Si NCs from laser pyrolysis

Erogbogbo et al. prepared targeted Si QD probes for multiple cancerrelated in vivo applications,231 including tumor vasculature targeting, sentinel lymph node (SLN) mapping, and multicolor NIR imaging in live mice. These probes undergo enzymatic degradation, evade

Fig. 54 Representative fluorescence images of a mouse bearing an MDA-MB-435 tumour. The mouse was imaged using a Cy5.5 excitation filter and an ICG emission filter at the indicated times after intravenous injection of Si NPs (20 mg kg 1). Note that a strong signal is observed in the tumour, indicating significant passive accumulation in the tumour by the enhanced permeability and retention (EPR) effect. Reprinted with permission from ref. 56. Copyright 2009, Macmillan Publishers Ltd.

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Fig. 55 Comparison of time-resolved fluorescence characteristics of Si NPs and the organic fluorophore Cy3.5. (a) Steady-state fluorescence images the two emitters in microtubes (b) Normalized intensity decay of the two samples the vertical orange bar depicts the time window used for time gated acquisition. (c) Time gated image of the microtubes: the image of the Cy3.5 sample almost completely disappears. (d) Bright field image of a nude mouse injected subcutaneously with Si NPs (arrow) and Cy3.5 (arrowhead). Fluorescence imaging of the mouse in steady state (e) and time resolved (f) mode. (g) PL decays of Si NPs and Cy3.5. Reprinted with permission from ref. 235. Copyright 2013, Macmillan Publishers Ltd.

uptake by the reticulo endothelial system (RES), maintain stability in the acidic tumor microenvironment, and produce bright and stable luminescence. These authors achieve dispersibility and functionalization required for in vivo imaging by implementing the synthetic strategy they previously proposed for in vitro imaging probe design.232 Exploiting PEGylated micelle encapsulation and bioconjugation the authors produced bright, targeted nanospheres with stable luminescence and long (440 h) tumor accumulation time in vivo. In particular the emission color of the probe could be tuned from the visible to the NIR by controlled etching of laser-synthesized Si NPs followed by covalent linking of organic ligands to their surfaces using photo-initiated hydrosilylation. TEM images of the final micelle-encapsulated Si QDs showed spherical aggregates of crystalline particles with 50 to 120 nm overall diameter. Conjugation to RGD peptides was exploited to achieve active targeting of avb3 integrins that are known to be overexpressed in the tumor vasculature. As shown in Fig. 56, Si QDs could be detected in vivo one month after injection. Nude mice bearing subcutaneously implanted Panc1tumors were intravenously injected with the RGD targeted probe following the NIR emission distribution as a function of time. The luminescence intensity at the tumor site increased with time up to 40 h namely for a time much longer (810 times) than observed in the case of single CdSe and CdTe QDs. In a control


analogous experiment with non-bioconjugated probes only a very weak luminescence was observed that was interpreted by the authors as resulting from the enhanced permeation and retention (EPR) effect schematized in Fig. 3. This observation demonstrated the essential role of the peptides in the uptake mechanism. Biodistribution studies also showed that the aspecific uptake in the liver and spleen was reduced for the targeted particles, compared to the untargeted ones. Micelles stabilized NPs were also used to successfully identify SLN as shown in Fig. 57.231 The probes, injected subcutaneously in the paw of a mouse in fact, traveled through the lymphatics and migrated to an axillary location indicating the position of the SLN. The authors also demonstrated the applicability of their probes for in vivo multiplex imaging in the red-NIR spectral region, and they studied in vivo distribution, clearance kinetics, and toxicity of the non-bioconjugated micelle-encapsulated Si QDs after their intravenous injection into healthy mice. As far as their effect on the mice health is concerned, no significant changes in body weight, in eating, drinking, exploratory behavior, activity, or physical features (e.g., hair, color) were observed during four weeks after subministration suggesting that the formulation was nontoxic in vivo at a dosage as high as 380 mg kg 1. Moreover the overall luminescence signal decreased as a function of time indicating, according to the authors, that the Si QDs

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Fig. 56 Luminescence imaging of BALB/c mice injected with MSiQD. All images were acquired at the same instrument settings, at different time points postinjection. Autofluorescence is shown in green (left panels) and the QD signal is shown in red (center panels). The right panels show overlaid autofluorescence and QDs luminescence images. Uptake in the liver and spleen was observed. Reproduced with permission from ref. 231. Copyright 2011, American Chemical Society.

Fig. 57 Sentinel lymph node imaging following localization of Si QDs in an axillary position. Autofluorescence is coded in green, and the unmixed Si QD signal is coded in red. Reproduced with permission from ref. 231. Copyright 2011, American Chemical Society.

were degraded in the liver and the spleen in two months. Histology results for tissues (lung, heart, spleen, kidney, and liver) also showed no signs of overt toxicity (tissue degeneration or necrosis) at 380 mg per kg. These results were confirmed by serum analysis considering the markers which are commonly used to evaluate kidney, renal and hepatic functions. A comparative study with Cd-based QDs on a human pancreatic cancer (Panc-1) cell line at 24 h post-treatment showed that the inhibitory particle concentrations corresponding to 50% cell viability (IC50) were 20 mg mL 1 and 11 mg mL 1 for CdTe and CdHgTe in Panc-1 cells, respectively, compared to 4500 mg mL 1 for Si. These results clearly demonstrate the low cytotoxicity of the silicon based probes when compared to other QDs.

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Recently the same authors developed a new set of silicon based luminescent probes suggesting a synthetic approach that is expected to produce even more biocompatible materials. In particular their strategy was based on the replacement of all the components of the probe, with the exception of the silicon emitters, with FDA approved materials.233 The FDA-approved components used in this formulation were ethyl undecylenate, which is a food additive permitted for direct addition to food for human consumption, and Pluronic F-127, which is approved as an injectable material for use in the human body. Response of two animal models, mice and monkeys (Rhesus macaques), to intravenous injection of an aggressive dose (200 mg kg 1) of Pluronic-encapsulated silicon quantum dots, was followed for three months. In particular Rhesus macaques were chosen because of their availability and genetic similarity to humans, for quantum dot translation to the clinic. In the case of mice the confocal imaging 3 days or 1 week post-injection clearly revealed particles localized in the liver, spleen, and kidneys. ICP-MS showed that the concentration of silicon in the lymph and kidneys declined over the 14 week time period, while the liver and spleen retained a significant fraction of the silicon injected, even after 14 weeks. There is no evidence of the biodegradability of silicon that was expected based upon previous studies of other forms of nanostructured silicon. Physiological and behavioral parameters of the treated population were normal when compared to a reference untreated one throughout the 3 month evaluation period. Moreover no clear signs of infection or allergic or toxic reactions that could be attributed to the nanoparticles were found by analyzing blood chemistry of the animals. In contrast the histology images of the livers of treated mice revealed significant, unexpected, alterations attributable to the effects of the treatment with the Si QD formulation. The observed effects included inflammation, proliferation of Kupffer cells, multifocal cholestasis, and spotty necrosis of hepatic cells. The pilot study of the silicon QD formulation was then extended to Rhesus macaques in order to verify that whether this liver pathology could be reproduced in the primate model. Also for the monkeys no significant differences in the body masses were observed between treated and untreated animals and the eating, drinking, grooming, exploratory behavior, physical features, neurological status, and urination were not distinguishable in the two populations during the 3 month evaluation period. As for mice, blood chemistry parameters of the monkeys, including indicators of kidney and liver function, showed no signs of infection or allergic or toxic reactions that could be attributed to the NPs. Surprisingly, differently to what reported for mice, the histological images of explanted tissues of monkeys showed no signs of NPs induced changes. In particular the kidneys, liver, and spleen that are the expected sites of NP accumulation reported no signs of disease or damage. May et al. demonstrated that carboxyl-terminated Pluronic can be used as an effective coating for Si QD stable aqueous dispersions The resulting carboxyl-terminated Pluronic-blockcopolymer-encapsulated Si QDs have been used as diagnostic agents for pancreatic cancer.266 When bound to the Si surface


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these amphiphiles did not quench or spectrally modify the luminescence of the QDs. Moreover Pluronic coating conferred to the Si QDs good stability at physiological pH and temperature, while thanks to the presence of the terminal carboxylate units that also play an important role in the electrostatic stabilization of the NPs, the NC surfaces could be readily modified to incorporate cancer targeting molecules. In vivo imaging was performed by injecting micelle-encapsulated Si QDs intravenously into a Balb/c mouse, which was then sacrificed after 24 h. The mouse was dissected, and the organs were imaged to investigate the distribution and accumulation of the Si NCs within the body. The results of the in vivo experiments showed that QDs were not present in the heart, lungs, or the brain. In addition, Si QDs encapsulating micelles were found only in the liver and the spleen, because of the clearing of the QDs by the reticuloendothelial system. This study further proved that Si QDs can be used to target tumors in cancer treatments.

9 FNDs for in vitro bioimaging A major difficulty in using FNDs for bioimaging is to avoid their aggregation in a physiological environment. Nevertheless, internalization of FNDs as small aggregates or as individual objects has been reported. Hence, as it will be shown in this section, the usability of these FNDs has been demonstrated both for untargeted and for targeted cellular imaging. Dosage dependent cytotoxicity data, mostly based on cell viability assays, will be compared in order to assess the safety of FNDs in vitro in relation to morphological and physicochemical parameters. Variability of the effects of exposition to FNDs on different families of cells, distinguishing in particular cancer and non cancer ones, will be discussed. Experiments aimed to clarify the mechanism of cellular uptakes as well as the kinetics of the process will be discussed also in consideration of the application of FNDs as theranostic vectors. Finally we will review some innovative optical imaging strategies that exploit the sensitivity of FND emission to non-optical stimuli for local temperature detection in cells as well as for background free imaging. Also the opportunity of functionalizing NDs with fluorescent molecules and detecting FND cathodoluminescene will be shortly analyzed. 9.1

FNDs for non-targeted in vitro bioimaging

Biocompatibility of FNDs, as well as their application as bright and photostable fluorescent biomarkers for in vitro imaging, was first demonstrated by Yu et al. in the case of 293T human kidney cells.312 These authors prepared the fluorescent nanoprobes starting from synthetic type Ib diamond powders with a nominal size of 100 nm. After liquid oxidative purification, the NDs were deposited on a silicon wafer and irradiated by a 3 MeV proton beam. Annealing of the ion-irradiated film at 800 1C in a vacuum for 2 hours yielded the FNDs. According to the authors, under their experimental conditions (irradiation dose of 5 1015 ions per cm2) a concentration of nitrogen-vacancy defect centers of 1 107 centers per mm3 was created in the


Fig. 58 (a) Bright field and (b) epifluorescence images of FND. Both images were obtained with a 40 objective. (c) Fluorescence spectra of annealed FNDs with (red) or without (blue) proton beam irradiation. The excitation was made at 510–560 nm, and the emission was collected at a wavelength of 4590 nm. (d) Photostability tests of FND (red) and fluorescent polystyrene nanospheres (blue) excited under the same conditions. The fluorescence intensity was obtained by integrating over the wavelength range of 590–900 nm for each sample. Reproduced with permission from ref. 312. Copyright 2015, American Chemical Society.

FNDs, corresponding to the production of 1 104 vacancies per 100 nm particle. Bright field and epifluorescence images of the FNDs are shown in Fig. 58 (a and b respectively) together with their fluorescence spectrum (c). It is interesting to note that the emission wavelength range of the produced FND in the 600– 900 nm window is ideal for low background in vitro imaging and, as it will be shown in the next section, for in vivo imaging since it matches well the optimal transparency window of biological tissues shown in Fig. 1. The extreme photostability of the FNDs was demonstrated by comparing their emission with the one of 100 nm red fluorescent polystyrene carboxylated nanospheres containing 104 dye equivalents per particle under continuous excitation with a Hg lamp as shown in Fig. 58(d). No sign of photobleaching was observed, for FNDs, even after 8 h excitation; by contrast, the polymeric beads photobleached within 0.5 h under the same excitation conditions. Interaction of FNDs with living cells was investigated by incubating the NPs with 293T human kidney cell. Uptake of the FNDs was confirmed by the vertical cross-sectional images of the cells acquired using a confocal fluorescence microscope shown in Fig. 59. According to the authors, observation of the bright red spots inside the reddish envelope was indicative of FND translocation through the cell’s membrane, possibly by endocytosis, but also of NP aggregation. Cytotoxicity of the FNDs was also investigated by MTT assay that, as shown in the inset of Fig. 59, demonstrated only a modest decrease of cell viability at a FND dose of 400 mg ml 1 corresponding to 1 1011 particles per ml. These results clearly demonstrated a low toxicity for FNDs and that because of their composition and chemical inertness they do not release any toxic chemicals even in harsh environments.

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Fig. 59 Confocal fluorescence images of a single 293T human kidney cell after FND uptake. The cross-sectional image in each three-dimensional scan (as indicated by the yellow dashed square) has a vertical thickness of 0.25 mm and an area of 42 42 mm2. The bright red spots correspond to FND. Inset: cytotoxicity tests of FND with the 293T cells and the MTT reduction assay. Reproduced with permission from ref. 312. Copyright 2015, American Chemical Society.

Nevertheless the viability test for incubation times longer than 3 hours was not performed and the long-term effect on cellular development of FNDs was not examined in that work. Considering the detrimental effect of aggregation on the performance of FNDs for bioimaging, especially in view of single FND observation,163 the same authors proposed the replacement of the 100 nm probes with smaller diamond particles that they demonstrated to be detectable, as bright and photostable individually fluorescent biomarkers, in cancer cells.341 For probe preparation, commercial type Ib NDs, with a nominal size of 35 nm, were purified by acidic oxidative treatment and exposed to proton-beam irradiation followed by thermal annealing to generate luminescent NV defect centers. Wet oxidation of the surface allowed to remove the graphitic structures formed during the thermal treatment and to create carboxylate surface groups without deteriorating the photophysical properties of the particles. The 35 nm diameter, COOH terminated, FNDs were incubated with HeLa cells and the uptake process was investigated by fluorescence microscopy. Translocation of the FNDs through the cellular membrane was observed. Most of the uptaken NDs were seen to distribute in the cytoplasm where they were individually tracked341 presenting a reasonable stability against aggregation. The authors demonstrated that fluorescence of a single 35 nm FND is significantly brighter and considerably more photostable than that of a single dye molecule such as Alexa Fluor 546 under the same excitation conditions. The latter dye photobleached in the range of 10 s at a laser power density of 104 W cm 2, whereas the FNDs showed no sign of photobleaching even after 5 min of continuous excitation. Furthermore, no fluorescence blinking was observed within a time resolution of 1 ms. Comparing these results with the ones relative to 100 nm NDs, the authors concluded that long-term photostability and absence fluorescence blinking

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are size independent properties of FNDs and estimated that their method would create about 30 emitting defects in a hypothetic 10 nm ND. Quite different results, about the interaction of red emitting FNDs with living HeLa cells, were reported by Neugart et al. who investigated the motional dynamics of FNDs in aqueous dispersion and in the cellular system.303 In this case a sample of Ib-type diamond powder (B50 nm) was irradiated with highenergy (1 MeV) electrons and annealed at 800 1C. Removal of surface contaminants deriving by annealing, was achieved by purifying the NCs in a boiling mixture of sulfuric and perchloric acid. The number of NV centers per crystal was measured via fluorescence intensity and fluorescence antibunching experiments. A statistical analysis revealed that every 50 nm crystal contains on average two NV centers. This result is in stark contrast with the high density of NV centers previously reported for ion beam damaged NDs. Fluorescence correlation spectroscopy, used to measure the diffusion constants of the emitting species, demonstrated a pH dependent aggregation tendency. According to the authors FNDs could be stabilized and observed as individual NPs in solution only in the presence of a surfactant (SDS). Confocal fluorescence images demonstrated that these FNDs strongly interact with HeLa cells but also that most of them are simply adsorbed on the cellular membrane. About 6% of the total particles (estimated from fluorescence imaging) were internalized and immobilized inside the cell in a short time suggesting an endosomal pathway for the up-taking. Only rare cases of freely diffusing single FNDs, showing a diffusion constant compatible with the 50 nm size of the primary particles could be observed. Analogous to red-NIR emitting FNDs, also green emitting ones were used for in vitro imaging.183 Green FNDs containing a high concentration of N–V–N (H3) centers were prepared by radiation damage of type Ia natural diamond nanocrystallites with a nitrogen concentration in the range of 900 ppm and size of 350, 140, 70, and 50 nm. Irradiation was performed either using a medium power (40 keV) He+ beam or a high power (3 MeV) H+ beam and it was followed by thermal annealing at 800 1C. Irradiation and annealing of the NDs led to the emergence of a broad absorption band at B470 nm, corresponding to the phonon sideband of the electronic transition of the H3 center. Measuring the integrated absorption coefficient of the zero-phonon line (at 503 nm) of this center at liquid nitrogen temperature (in the case of a millimetric sized crystal) the density of H3 centers was calculate to be 1.7 10 3 centers per nm3 (or 10 ppm). The H3 centers emitted green light upon illumination by a blue laser. Application of the nanomaterial as a fluorescent cellular marker was demonstrated by confocal fluorescence microscopy that revealed that the 70 nm sized particles were internalized by live HeLa cells as shown in Fig. 60.183 9.2

FNDs for targeted in vitro bioimaging

FNDs for targeted cellular imaging were synthesized by Weng320 et al. by conjugating transferrin to the carboxylate surface groups of 100 nm diameter red emitting diamond NPs. Diamonds were irradiated with a 2.5 MeV ion beam and thermally annealed to


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Fig. 60 Uptake of green FNDs by HeLa cells: (a) differential interference contrast image of a single cell and (b) epifluorescence image of the same cell with its nucleus stained in blue by Hoechst dye 33258. In this experiment, the cells were incubated with FNDs for 5 h at a particle concentration of 10 mg mL 1. Time traces of FNDs and 100 nm green fluorescent polystyrene beads excited under the same conditions. Reproduced with permission from ref. 183. Copyright 2009, Elsevier.

emit bright fluorescence in the 550–800 nm region upon excitation at 514.5 nm. According to the authors these spectral features allowed them to eliminate the interference of cellular auto-fluorescence at 520–650 nm by detecting fluorescence at 660 nm. Transferrin receptors are over-expressed on HeLa cells and the transferrin functionalized diamond nanoprobes were expected to be internalized via a receptor-mediated uptake mechanism. The process was investigated by confocal microscopy and, in order to demonstrate the nature and the specificity of the uptake, two populations of HeLa cells were investigated, one of which had been pre-saturated with simple transferrin. As shown in Fig. 61,320 internalization of the FNDs was observed only in the case of the unsaturated cells, indicating that the transferrin receptors play a fundamental role in the trans-membrane permeation of the probe. Based on the results of this experiment, the authors concluded that not only internalization of the FNDs was mediated by transferrin uptake, but also that transferrin activity was not altered after conjugation to the FNDs. Moreover the lack of quenching of the FNDs color centers by the iron ions complexed into the protein was interpreted as a proof of a large distance existing between the metal ions and the emissive centers compatible with a localization of the defect deeply inside the crystal lattice rather than on the surfaces. Also detonation NDs have been proposed for targeted fluorescent cellular bioimaging184 Mkandawire et al. prepared fluorescent cellular biomarkers by conjugating detonation NDs, to antibodies and using 4th generation dendrimers, cationic liposomes and protamine sulphate as transfecting agents. The size of the starting NDs was about 5 nm in diameter and the detonation product was purified and functionalized by strong acidic oxidation to achieve the formation of surface carboxylate groups suitable for bioconjugation using conventional protocols.322 9.3

Cytotoxicity of FNDs

After preliminary demonstration of the short-term low toxicity of FNDs produced from synthetic diamond powders312 by the


Fig. 61 Comparison of confocal fluorescence images of HeLa cells treated with carboxylated FND and FND–Tf bioconjugates; (a) the left two brightfield images are HeLa cells treated, respectively, with carboxylated FNDs and FND–Tf bioconjugates, and the right one image is a HeLa cell presaturated with free transferrin for 1 h before being treated with FND–Tf bioconjugates; (b) corresponding confocal fluorescence images obtained upon excitation at 514.5 nm; (c) overlays of bright and fluorescence images. Reproduced with permission from ref. 320. Copyright 2009, Elsevier.

Chang’s group, cytotoxicity of FNDs was investigated more in detail considering parameters such as the incubation time, nature of the cells, method of production and functionalization of the diamond particles, as well as their shape and size.349 Schrand et al. assessed the cytotoxicity of detonation NDs, ranging in size from 2 to 10 nm, as resulting from the synthesis or after termination with carboxylate or sulfonate groups. Assays of cell viability based on mitochondrial function (MTT) and luminescent ATP production350 demonstrated that, independently of the functionalization, NDs were not toxic to a variety of cell types (including neuroblastoma and macrophages) and that they did not produce significant reactive oxygen species. Moreover, the authors confirmed that cells can grow on ND-coated substrates without morphological changes compared to control populations. Viability assays were performed after 24 h incubation of cells with NDs. Yet, the effect of NDs dose higher than 100 mg ml 1 was not investigated. Faklaris et al.351 extended the cytotoxicity study to incubation time of 48 h and to a ND concentration of 480 mg ml 1 in the case of 25 nm size FNDs prepared from Ib-type diamond powders. Although the authors concluded that the diamond NPs were non-toxic to HeLa cells, MTT assays showed a decrease of cell viability of about 20% and 30%, at a probe concentration of 53 and 480 mg ml 1 respectively, after the 48 h incubation. Although such cytotoxicity levels are quite modest they were not completely in agreement with the previously reported extreme biocompatibility of FND. This partial contradiction

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led Vairakkannu et al. to re-examine the issue of FNDs cytotoxicity.352 These authors performed the MTS assay on HeLa cells at 48 h after incubation with different concentrations of FNDs and they reported the absence of cytotoxicity of FNDs on HeLa cells at a concentration up to 200 mg ml 1 in the culture. Moreover they investigated the possibility to extend the use of FNDs in research applications with tissue stem and progenitor cells. They choose two cell models, 3T3-L1 pre-adipocytes and 489.2 osteoprogenitors, which can undergo adipogenesis and osteogenesis, respectively, when they are switched to an appropriate differentiation inducing medium. FND-treated cells showed, at 14 days after differentiation induction, no significant difference with respect to untreated cultures. Weng et al. compared the toxicity on HeLa cells of Type Ib 140 nm FNDs (irradiated with 2.5 MeV proton) after functionalization either with transferrin or amino groups.317 Both transferrin and amine functionalization enforced the inhibition of the cellular growth (about 50% after 24 h), an effect that was not observed for carboxylated FNDs.317 Moreover transferrin functionalized NDs showed a moderate phototoxicity on HeLa cells when irradiated with a 532 nm continuous laser. Indeed the experiment was carried out under powerful light exposure.

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In fact, the green laser itself was reported to kill the untreated HeLa cells after 10 min exposure at 75 W cm 2 while the same effect on transferrin NDs treated cells was observed using less than half the energy required for untreated cells. The actual mechanism of the process was demonstrated by Raman spectroscopy that revealed an increase of the temperature of the FNDs cluster during irradiation as expected in the case of the photothermal effect. Nevertheless, as expected for highly luminescent materials such as FNDs, the heating effect was modest since absorbed energy is mostly released via radiative pathways. More recently the biocompatibility of FNDs with neurons has been investigated in consideration of the potential use of this material for drug delivery in the nervous system.71 Data shown in Fig. 62 indicate that FNDs show almost no neuronal toxicity up to a concentration of 250 mg ml 1. Nevertheless time-lapse live cell imaging showed a reduction of neurite length due to the spatial hindrance of FNDs on advancing axonal growth cone. The authors concluded that although FNDs exhibited indeed low neuronal toxicity, the interference with neuronal morphogenesis should be taken into consideration when applications involve actively growing neurites as in the case of nerve regeneration.

Fig. 62 Fluorescent nanodiamonds did not cause cytotoxicity in dissociated hippocampal or dorsal root ganglion neuron cultures. (A) Images of dissociated hippocampal neurons treated with various concentrations of FNDs 4 hours after seeding and incubated for 3 days in vitro. Images on the top row show DAPI-stained nuclei, merged images on the bottom row show FND (red), neuron-specific b-III-tubulin (green), and DAPI-stained nuclei (blue). Scale bars represent 50 mm. (B) Quantification of neuron number per image field of FND treated hippocampal neurons. (C) Images of dissociated DRG neurons treated with various concentrations of FNDs after seeding and incubated for 2 days in vitro. Arrows point to the nuclei of DRG neurons. Scale bars represent 20 mm. (D) Quantification of neuron number per image field of FND treated DRG neurons. All quantification data were obtained from 3 independent repeats and normalized to the group without FND. No statistically significant difference between control group and FND-treated groups can be detected. p 0.05, by one-way ANOVA. Both bar graphs are expressed as mean SEM. Reprinted with permission from ref. 71. Copyright 2014, Macmillan Publishers Ltd.

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9.4

Kinetics and mechanisms of internalization of FNDs

Weng et al. compared the kinetics of the uptake of FNDs by HeLa cells (irradiated with 2.5 MeV proton) after functionalization either with transferrin or amino groups.317 Both the probes were prepared starting from carboxylated NDs using, in the case of the amino derivative, APTES to produce a layer of about 10 nm of organo-silica on the particle surface. During the cellular uptake, transferrin bearing particles reached their saturation values with a half-time of about 0.8 hour, about twice faster than the amino terminated FNDs. The authors concluded that the receptormediated endocytosis was more effective than the endocytic process involving surface electrostatic interactions. The internalization pathways of 25 nm FNDs were investigated by Faklaris et al. in HeLa cancer cells with endosomal marking and colocalization analyses.351 A rather low degree of colocalization was observed between early endosomes and the FND emission. In particular, after 2 h of incubation colocalization was observed for about 21% of the particles with an error margin of 6% analyzing 20 cells and 256 internalized FNDs. The authors proposed two possible interpretations of this result: either the FNDs do not enter the cells by endocytosis, or they are liberated early from the endosomes in the cytosol or in the lysosomes. In a later study, the same authors analyzed in detail the internalization mechanism of FNDs with 46 nm hydrodynamic diameter in living HeLa cell.342 The FNDs were produced starting from large (150–200 mm) type Ib diamonds that were irradiated with an electron beam (8 MeV) and reduced in size by nitrogen jet milling and ball milling under argon after thermal annealing at 800 1C in a vacuum. Blocking selectively the different possible internalization pathways (see Fig. 2) by using specific drugs, the authors concluded that FNDs enter the cancer cells mainly by clathrin-mediated endocytosis. Intracellular localization of FNDs was analyzed by immunofluorescence and TEM. Results confirmed that smallest particles appeared to be free in the cytosol. This study revealed also an effect of the ND size on their internalization by cancer cells. A high degree of colocalization between vesicles and the biggest nanoparticles or aggregates was in fact detected. Internalization of FNDs is expected to takes place with different efficiency and mechanism in different target cells. Perevedentseva et al. investigated this issue by comparing the interaction of FNDs with cancer and non-cancer cells.343 In particular internalization of 100 nm FNDs by A549 lung man adenocarcinoma cell, Beas-2b non tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell was studied, comparing the dependence on dose and time of the uptake by confocal fluorescence imaging and Raman imaging methods. The main mechanism of internalization by cells was confirmed to be via clathrin-dependent endocytosis, both for cancer and non-cancer cells. Moreover the uptake was quantitatively larger in the case of the cancer cells. Shape is another factor that regulates the interaction of NPs with cellular systems. According to Chu et al., the effect of morphology of FNDs is so relevant to determine independently


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their cellular fate.291 This result was demonstrated using FNDs with a physical size distribution of tens to hundreds of nanometers and an aspect ratio close to 1 but that exhibited in HRTEM irregular shapes and in most cases at least 1–2 sharp corners as shown in Fig. 63.291 Combination of TEM and confocal microscopy revealed, using fluorescence based co-localization techniques, that FNDs having irregular shapes with sharp corners escaped from endosome to cytoplasm shortly (a couple of hours on average) after their cellular uptake, and could hardly exit the cells afterwards accordingly to the mechanism schematized in Fig. 64.291 In contrast round-shaped NDs, obtained by direct chemical etching of the prickly ones, demonstrated stable endosomal residence and effective cellular excretion. 9.5

FNDs for drug delivery

Combination of luminescent properties of FNDs with therapeutic agent delivery has been explored as a strategy for the production of biocompatible multifunctional materials. Alhaddad et al. investigated the internalization pathways of cationic red emitting FNDs in the Ewing Sarcoma Cell Model and exploited the NPs as vectors for the delivery of siRNA in view of theranostic applications.324 SiRNAs are powerful therapeutics commonly used for the specific inhibition of gene expression that require vectorization to facilitate cell penetration and to prevent siRNA degradation by nucleases. The nanovectors were prepared from commercial 50 nm size NDs purified by strong acidic oxidation. The NDs, irradiated with a 13.9 MeV electron beam and then oxidized in air at 550 1C to remove surface graphite, formed after thermal annealing in a vacuum.155 After a final wet oxidative step, which produced carboxylate surface groups, the NPs were conjugated to two different cationic polymers, polyallylamine and a branched low-molecular weight (800 Da) polyethylenimine, by simple electrostatic adsorption. The same kind of interaction was exploited to load the positively charged nanovectors with the anionic siRNA to give final theranostic probes of 120–130 nm size. Oncogene silencing was observed only in the case of polyethylenimine. The origin of such a specificity was investigated by the authors by following the cell penetration by a combination of fluorescence and electronic microscopy. Using drugs to selectively inhibit the different internalization pathways they demonstrate that siRNA gene silencing occurred only if the siRNA–NDs complex followed the macropinocytosis route. Receptor mediated uptake was also exploited to design a ND based probe for glioma cell specific labelling using BmK CT, a key chlorotoxin-like peptide isolated from the venom of a scorpion, to covalently functionalize the surface of –COOH terminated FNDs.321 Internalization of the bioconjugates into rat C6 glioma cells were confirmed by confocal fluorescence imaging. In order to establish a potential therapeutical action of their probes the authors investigated the inhibition of cell migration by in vitro wound healing assay demonstrating a modest increase of the effect for the probe with respect to the peptide or the bare NDs alone. Covalent functionalization with PEG has been proposed a strategy to increase the dispersibility and stability of 140 nm

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Fig. 63 Intracellular translocation of sharp-shaped NDs from membrane bounded vesicles and stable residence in cytoplasm. (a) High resolution TEM image showing the sharp corner of a typical ND. (c–g) Representative confocal microscopy images showing the fluorescent signals of endosome markers (green) and NDs (red) in HepG2 cells after cell incubation with NDs in serum-free medium for (c) 1 hour, (d) 2 hours, (e) 4 hours, (f) 8 hours and (g) 10 hours. The yellow spots show spatial overlap between NDs and endosomes (as marked by white arrows). The observed low overlap for longer incubation durations indicates that the NDs escaped the endosomes. (h–l) Representative confocal microscopy images showing the fluorescent signals of lysotracker (green) and NDs (red) in HepG2 cells after cell incubation with NDs in serum-free medium for (h) 1 hour, (i) 2 hours, (j) 4 hours, (k) 8 hours and (l) 10 hours. Very low spatial overlap between NDs and lysosomes was observed, indicating that most NDs escaped the endosomes before the lysosomes were formed. Reprinted with permission from ref. 291. Copyright 2014, Macmillan Publishers Ltd.

red emitting NDs in a physiological environment. The carboxylate surface group of the oxidized NDs was activated with SOCl2 and reacted with OH terminated PEG-4000. The PEG shell was also exploited for loading the probes with doxorubicin hydrochloride for its cellular delivery.316 Enhanced deliver of the drug into the human liver cancer cells (HepG2) via a clathrin-dependent endocytosis pathway was observed using the nanovector. The uptake half-life of the drug was approximately two times for the nanoparticles with respect to that of free molecules. Confocal fluorescence microscopy images showed that DOX detached from composites inside the cytoplasm could migrate and enter the nucleolus to inhibit the cellular growth. Moreover in vitro dialysis determination and imaging experiments indicated that the composites had a slow and sustained drug release capability. The survival rate of tumor-bearing mice treated with ND–DOX was four times greater than that of mice treated with free doxorubicin and similar to the one of mice treated with NDs alone. Histopathological analysis showed that neither the NDs nor ND–DOX were toxic to the kidneys, liver, or spleen in contrast with the wellknown toxic effects of free doxorubicin on the kidneys and liver. Further, both the bare NDs and ND–DOX could suppress tumor growth effectively. In a similar approach a bifunctional composite material was developed resulting from the inclusion of 50 nm NDs in a porous silica shell to increase the loading

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ability for drug delivery.175 Silica coating improved also the dispersibility of the NDs in an aqueous environment with subsequent enhanced uptake by HeLa cells. Further surface modification of the nanocomposite by absorption of a poly(ethylene glycol)–poly(ethylene imine) copolymer improved its ability in intracellular delivery of poorly soluble agents. 9.6

FNDs for stimuli responsive imaging

Sensitivity of FND emission to temperature was recently exploited to design a nano-thermometer capable of sub-degree temperature resolution over a large temperature range as well as suitable for integration within a living system.158 Kucsko et al. used this approach to detect temperature variations as small as 1.8 mK in an ultrapure bulk diamond sample and to investigate local thermal environment changes caused by the heating of a laser irradiated gold NP on length scales as short as 200 nm in a prototypical setup. The same heating process was hence investigated into a single human embryonic fibroblast by introducing both FNDs and gold NPs as shown in Fig. 65.158 The temperature effect in FNDs is to change the population of the two electronic sublevels with different magnetic components (ms = 0 and ms = 1) in which the triplet ground state of the nitrogen vacancy defect is split because of the magnetic interaction in the crystal even in the absence of an external magnetic field. Since the two states show a different luminescence efficiency a difference in


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Fig. 64 Schematics of intracellular trafficking of nanoparticles with different morphological features. Left: a nanoparticle with low sharpness enters the cell via endocytosis with the endosome as the vehicle, stably resides in the endosome, which evolves into the lysosome by endosomal maturation, and finally exits the cell via exocytosis with the lysosome as the vehicle. Right: a sharp-shaped nanoparticle enters the cell via endocytosis with the endosome as the vehicle and escapes the endosome by rupturing the endosomal membrane before the lysosome is formed. Without a lysosome as the vehicle, the nanoparticle has very low excretion rate and will stay in the cytoplasm for a long time. Reprinted with permission from ref. 291. Copyright 2014, Macmillan Publishers Ltd.

their population causes a change in the emission intensity. Nevertheless to achieve high temperature sensitivity possible effects due to local magnetic field on the probes had to be ruled out. This was achieved by using a sequence of four microwave irradiation and fluorescence measurements that were combined according to an algorithm that allowed magnetic field independent temperature determination. The cellular experiments demonstrated the submicrometer measurement of an intracellular heat gradient, without damaging the cell itself, if irradiation is controlled to avoid excessive heating. Extended illumination, in contrast, led to the death of the cell as confirmed by a standard live/dead fluorimetric assay. According to the authors this last result demonstrated that ND thermometry may enable the optimization of nanoparticle-based photothermal therapies. Used FNDs contained B260 NV centers (assuming a density of NVCs 0.3 ppm per carbon atom).353 Igarashi et al.160 exploited the sensitivity of FND emission to microwaves to design a method that successfully removes autofluorescence of biological tissues. This technique, defined as optically detected magnetic resonance (ODMR), was demonstrated, as a proof of principle, for a wide variety of living systems from cells to mice. The setup used for ODMR is schematized in Fig. 66160 and it is based on an inverted fluorescence microscope equipped with an electron-multiplying charge-coupled device (EMCCD) for photon detection. In this method, no external static magnetic field is applied, but a microwave irradiation system is used to manipulate occupation of the spin states. First tests of the technique carried out on individual FNDs on a glass slide as shown in Fig. 66. By scanning the wavelength of the MW irradiation, a decrease of the PL intensity of the NPs at a resonance frequency of 2.87 GHz, expected for the S = 0, S = 1 transition, was observed. Modulation of the PL was then achieved by alternately switching on and off the MW source at the resonance peak. During each cycle a single integrated PL image was acquired. Since only the signal of the NDs responds


to the modulation, PL arising from the background (or other fluorophores) was easily eliminated by image processing using a selective imaging protocol (SIP). In vitro ODMR was demonstrated for living HeLa cells containing both NDs and fluorescent beads. An acquisition time of B320 ms was sufficient to acquire high-quality selective images of FNDs with a diameter of B170 nm with a complete removal of the signal produced by the fluorescent beads. Although ODMR and other imaging methods based on modulation of FNDs PL via non optical stimuli need further technical development, they promise to become unique tools for real-rime background-free detection both in vitro and in vivo. 9.7

Fluorophore conjugated NDs

Peculiar photostability of NDs arises from color center emission. Nevertheless examples of imaging based on organic fluorophores bound to NDs as probes have been reported. Since in these systems fluorescence is not produced directly by the NDs they will not be discussed exhaustively in this review article and only a few recent representative examples will be considered. Triple negative breast cancers are among the most aggressive breast cancer subtypes because they do not express the estrogen receptor, progesterone receptor and Her2 that are commonly targeted with breast cancer therapies. Nevertheless since they do typically overexpress epidermal growth factor receptor Moore et al. developed novel ND–lipid hybrid particles targeted to EGFR325 that were used to specifically deliver imaging and therapeutic molecules to triple negative breast cancers cells MDA-MB-231 in vitro and in vivo. NPs were synthesized by rehydration of lipid thin films containing cholesterol and biotinylated lipids in the presence of amino-functionalized NDs obtained from a commercial powder after oxidation to carboxylate followed by reduction to –OH and coverage with APTES. A fraction of the NH2 groups was coupled to the succinimidyl esters of AlexaFluor 488, AlexaFluor 555 or Xenolights CF750.

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Fig. 65 Nanoscale thermometry in cells. (a) Confocal scan of a single cell under laser excitation (532 nm), with collection at wavelengths greater than 638 nm. The cross marks the position of the gold nanoparticle used for heating, and circles represent the location of the NDs (NV1 and NV2) used for thermometry. The dotted line outlines the cell membrane. (b) Measured change in temperature at the positions of NV1 and NV2 relative to the incident laser power applied to the gold nanoparticle. Dashed lines are linear fits to the data. (c) Fluorescence scan of stained cells (live/dead assay) with excitation at 494 or 528 nm and emission at 515 nm (green; live cells) and 617 nm (red; dead cells). The bar graph shows the temperature of a single ND (circle) with local heat applied at two different locations (crosses). (d) Confocal fluorescence scans of an individual cell under varying illumination power. Cell death is indicated by the penetration of ethidiumhomodimer-1 through the cell membrane, staining the nucleus. At low laser powers, the cell remains alive; cell death occurs as laser induced heating is increased. Reprinted with permission from ref. 158. Copyright 2013, Macmillan Publishers Ltd.

Hybrid particles were then targeted using biotinylated antibodies and streptavidin crossbridges. The authors used the resulting NDs as vectors for mediated epirubicin delivery observing a markedly increased efficacy with respect to direct treatment with epirubicin. Hybrid NDs appeared to be non-toxic. In a different approach, fluorescent proteins were conjugated to NDs in a study aimed to investigate the phototherapeutic action of the carbon NPs. According to Chu et al., the trace nitroso groups present in NDs can be photolyzed by laser irradiation to release nitrogen monoxide, creating high internal pressure in cells and inducing selective cell death with potential therapeutic application.354 This feature has been exploited by conjugating carboxylated NDs to growth hormone, a molecule that, together with its antagonists, have been proposed as cancer cell-targeting unit.354 Green fluorescence protein was also bound to the complex. The starting NDs had a diameter of about 5 nm and the carboxylate surface groups were activated by EDC/S-NHS for coupling to give final complexes showing at DLS and hydrodynamic diameter of approximately 9 nm. Co-localization of the scattering of the NDs with GFP fluorescence was reported as a demonstration of the complex formation. Three-dimensional reconstitution of the confocal microscopy images of A549 cells treated with the ND complex showed that the probe did not

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penetrate into the cells being localized on the cell membrane. The ND complex induced selective damage, after laser irradiation at 532 nm, decreased the viability of the A549 cells to 20% while only a minor effect was observed upon irradiation in the absence of the ND complex. 9.8

Cathodoluminescence of FNDs

A further possible application of FNDs to fluorescence bioimaging is based on optical excitation. Spatial resolution of fluorescence microscopy is limited to hundreds of nanometer when conventional optical excitation is performed. Excitation with a focused electron beam has been proposed as a higher resolution alternative for multi-color, imaging of NDs in living cells. The resulting emission has been referred to as cathodoluminescence (CL)355 and it is detectable with nanoscale resolution. Green- and red-light-emitting NDs were employed for two-color imaging and were observed simultaneously in HeLa cells with a spatial resolution of B100 nm.356 Silicon-vacancy (Si-V) color centers138,357 were also included into the set of known CL-emitting defects in NDs, increasing the potential for wavelengthselective multiplexing, and providing a convenient CL marker in the near-infrared window.186 Drawbacks of electron beam excitation to biological structure imaging are low photon count


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Fig. 66 Selective imaging of NDs in vitro and inside HeLa cells. (a) Time chart of fluorescence excitation at 532 nm, MW irradiation and image acquisition used in the selective imaging protocol (SIP), along with the expected profiles of non-NVC and NVC fluorescence. (b) Time courses of observed fluorescence intensity of the ND and the fluorescent bead indicated by the red and blue circles in c, respectively. (c, d) Fluorescence images of FNDs and fluorescent microspheres (0.11 mm in diameter) dispersed on a coverslip obtained without (c) and with SIP (d). (e–g) Selective imaging of an ND inside HeLa cells. Bright-field image (e), conventional fluorescence image (f), and SIP image (g) are shown. Reproduced with permission from ref. 160. Copyright 2012, American Chemical Society.

rates and rapid signal degradation due to the destruction of biomolecules. Hence CL is not compatible with long-term, non invasive, detection.

10 FNDs for long-term in vivo imaging Thanks to their photochemical and chemical inertness, and in virtue of their emission in the NIR region, FNDs could be detected for very long time lapses during in vivo imaging experiments. This permitted us to investigate their long term biodistribution and fate in the organs of living animals as well as to analyse their possible toxic effects. Recent studies univocally demonstrated the lack of toxicity of FNDs on mice and the usability of these NPs to track very sophisticated processes such as steam cell differentiation in living animals. Most relevant studies in this field will be reviewed in this section considering the different investigated animal models. Finally we will analyse the promising performance of recently proposed experimental set up that exploits non-optical modulation of FNDs by magnetic and electromagnetic stimuli to abate the background signal. 10.1

Long-term in vivo imaging in C. elegans

Applicability of FNDs to long-term in vivo optical imaging was demonstrated by Mohan et al.318 who explored the long-term interaction of red emitting FNDs with the translucent model hermaphrodite organism Caenorhabditis elegans (C. elegans). These authors demonstrated the lack of toxicity of these materials after incorporation, by analyzing parameters such as life span,


brood size and ROS levels. Alteration of feeding behavior and reproduction of the animals were also considered. C. elegans is a suitable model to study biological molecular processes since its genome has been completely sequenced. Moreover, this worm has a simple and well-defined anatomy which arises from the organization of 959 cells into complex structures, which include intestine, muscle, hypodermis, gonad, and nerve systems. Carboxylated FNDs were produced by oxidation of a commercial synthetic type Ib diamond powder that was irradiated with a 40 keV He+ beam and thermally annealed.306 After surface functionalization by electrostatic adsorption of poly-Llysin, the amine terminated NPs were conjugated either to carboxy dextran or BSA, according to conventional bioconjugation protocols. Resulting bio-functionalized NPs, as well as the bare carboxylated FNDs, were incorporated into C. elegans either by feeding or injection to the gonad. Effects of the FND surface functionalization, as well as of the method of subministration, on the physiological response of the animal model were investigated. In the case of the oral administration, epifluorescence in vivo images, shown in Fig. 67 and 68,318 proved that both carboxylated and functionalized NPs were incorporated by the worms. Hence, the FNDs were not regarded as repellent by the organisms during the feeding process. The nature of the NP surface, on the other hand, strongly affected the biodistribution and the fate of the probe in the model animal. As shown in Fig. 68, bare FNDs accumulated in the lumen of the digestive system in an amount of about 1 106 particles per worm. After incorporation, these NPs were excreted out within one hour, if the worm was fed with Escherichia coli, but they

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Fig. 67 Epifluorescence and epifluorescence/DIC-merged images of wild-type C. elegans. (A) An untreated young adult. (B, C) Worms fed with bare FNDs for 2 h (B) and 12 h (C). The FNDs stayed inside the gut and were not excreted out when the worms were deprived of food. Excretion of FNDs occurred upon feeding with E. coli. (D, E) Worms were provided with E. coli for 20 min (D) and 40 min (E), after being fed with bare FNDs for 2 h. Almost, if not all, FNDs were excreted out within 1 h. The upper panels in (B–E) show the epifluorescence images; (A) and the lower panels in (B–E) show the epifluorescence/DIC-merged images. Anterior is left and dorsal is up in all figures. Scale bar is 50 mm. Reproduced with permission from ref. 318. Copyright 2010, American Chemical Society.

Fig. 68 Epifluorescence/DIC-merged images of wild-type C. elegans fed with bioconjugated FNDs. (A, B) Worms fed with dextran-coated FNDs (A) and BSAcoated FNDs (B) for 3 h. FNDs can be seen to be localized within in the intestinal cells (blue solid arrows) and a few stay in the lumen (yellow dash arrow). (C, D) Worms fed with dextran-coated (C) and BSA-coated (D) FNDs for 3 h and recovered on to E. coli bacterial lawns for 1 h. In both cases, the FNDs staying in the lumen are excreted out, whereas the ones localized in the cells retain. Insets: 100 magnified images of the FNDs within the intestinal cells. Anterior is left and dorsal is up in all the figures. Scale bars are 50 mm. Reproduced with permission from ref. 318. Copyright 2010, American Chemical Society.

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persisted in the organism even after 12 hours if the animals were not fed. Thanks to the high resistance of the emitters to photobleaching and chemical degradation, even in the presence of robust digestive enzyme of the gut, the entire digestive tract of the worm was imaged for more than 48 hours showing no translocation of the FNDs to the intestinal cells. A completely different fate was observed for BSA and dextran coated FNDs. As shown in Fig. 68, these NPs, in fact, were readily internalized by the intestinal cells where they remained located even 24 h after recovery of the worms on nutrient bacteria. According to the authors, the different biodistribution and fate observed for the two classes of FNDs, resulting from the combination of two different effects. First, bioconjugation stabilizes the FNDs in the physiological medium reducing their tendency towards aggregation, a phenomenon that, in the case of bare FNDs, causes the formation of large particles which are not internalized by the intestine cells. Small functionalized FND aggregates, in contrast, are readily taken up via endocytosis by the same cells. The second effect is the activation of receptor mediated endocytosis pathways by the biomolecules. Interestingly, no toxicity upon internalization of the FNDs into the intestinal cells was noticed considering parameters such as the lifespan and number of progeny per warm. Moreover the level of ROS of the animal treated with the NDs perfectly matched those of a control, untreated, population. Worms exposed to FNDs showed also no gross behavior defect. Finally, the authors investigated the influence of the same NPs on the formation of new embryos, by microinjecting them into the distal gonads of gravid hermaphrodites. Fluorescence images revealed that FNDs were first incorporated into the oocytes and then, the NPs persisted both in the fertilized zygotes and in the final embryos, without inducing any apparent alteration in their growth and development.318 Kuo et al. extended the study on C. elegans to the intercellular transport of yolk lipoproteins in the animal, by using fluorescence lifetime imaging microscopy (FLIM).345 As shown in Fig. 69, FLIM allowed these authors to remove background fluorescence by time-gated acquisition, with an important decrease of the signal to noise ratio. The yolk lipoproteins in the nematode are similar to human serum low-density lipoproteins (LDLs), serving as an intercellular transporter of fat molecules and cholesterol. To study this fundamental transport process, FNDs were first coated with yolk lipoprotein complexes, and then microinjected into the intestinal cells of the living organism. Real-time imaging acquired over a time period of more than 50 min by FLIM revealed the secretion of the biofunctionalized FNDs from the intestine to the pseudocoelomic space, followed by transporting into oocytes and subsequent accumulation in the multicellular embryos derived from the oocytes. These data confirmed the generally accepted mechanism for the energy supply process that involves yolk lipoproteins. Moreover, the results proved the value of FNDs as biomolecular nanocarriers for intercellular transport, cell-specific targeting, and long-term imaging applications in vivo. 10.2

Long-term in vivo imaging in mice and rats

Vaijayanthimala et al. investigated the long-term stability and biocompatibility of red emitting 100 nm FNDs in rats and mice,


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Fig. 69 Observation of GFP::YLC-FNDs in C. elegans by FLIM. (a) A fluorescence decay time trace of 100 nm FNDs suspended in water. The area shaded in magenta represents the fluorescence signal collected at the gating time (s) longer than 10 ns. (b) Bright-field, (c) confocal fluorescence, (d) FLIM, (e) time-gated fluorescence at s 4 10 ns, and (f) merged bright-field and time-gated fluorescence images of a worm microinjected with GFP::YLC-FNDs at the distal gonad. A blue arrow indicates the site of injection. Anterior is left and dorsal is up in all figures. Scale bar is 50 mm. Reproduced with permission from ref. 345. Copyright 2013, Elsevier.

as well as the potential use of these materials for sentinel lymph node mapping.179 The carboxylate terminated surface of the FNDs was functionalized with BSA by simple electrostatic absorption. In a first experiment bare FNDs were subcutaneously injected into a rat to evaluate the long-term stability of the fluorescence signal. As shown in Fig. 70179 stable emission was detected over the surprisingly long period of 37 days post-dosing. This observation demonstrated that FNDs are robust in vivo and potentially useful as long-term imaging agents for living animals. In an additional study the same NPs were administrated via intraperitoneal injection. In stark contrast to what observed after the subcutaneous injection, the intensity of the far-red fluorescence, in this case, gradually decreased and disappeared in 6 min. According to the authors, fluorescence signal fading resulted indeed from the gradual dispersion of the FNDs throughout the peritoneal cavity rather

than from actual degradation or digestion. This conclusion was motivated by the detection of the FNDs in the organs and tissues explanted from the animals treated with the NPs. Lastly, in a very long-term experiment, mice were observed over a 5 months period after intradermal injection, into the right foot paw, of BSA conjugated FNDs. Combination of in vivo and ex vivo fluorescence imaging, as well as transmission electron microscopy, showed that FND particles were drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice as shown in Fig. 71.179 The physiological response of the animals to the FND administration was analyzed by measuring water and fodder consumption, body weight, and organ index. Observations revealed no significant alterations with respect to a control mice population. The lack of toxicity for dosages as high as 75 mg kg 1 body weight of FNDs was confirmed by histopathological analysis of various tissues and organs.

Fig. 70 Long-term stability test of FNDs in a rat after s.c. injection. Images of the same rat were acquired over a time period of more than 37 days. White arrows indicate the site of FND injection. Reproduced with permission from ref. 179. Copyright 2012, Elsevier.


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Fig. 71 In vivo and ex vivo lymph node imaging of a nude mouse after i.d. injection of BSA-coated FNDs. (a) In vivo image showing accumulation of the FND particles in the right axillary lymph node (indicated by the blue arrow) on day 8. Note that most of the injected FND particles remain trapped at the injection site. (b) Ex vivo fluorescence image of four extracted lymph nodes, where ALN1 and ALN2 are the lymph nodes located at the right and left axillary, respectively, and BLN1 and BLN2 are the lymph nodes located at the right and left brachial regions, respectively. Reproduced with permission from ref. 179. Copyright 2012, Elsevier.

Considering such an extraordinary biocompatibility Wu et al. investigated the application of FNDs to track how lung stem cells incorporate and regenerate themselves in vivo over time in a mouse model.344 These cells are potentially useful for regenerative therapy in repairing damaged or lost lung tissues. The authors used FNDs, in combination with fluorescenceactivated cell sorting, FLIM and immunostaining, to identify

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transplanted CD45 CD54+CD157+ lung stem/progenitor cells in vivo, and to track their engraftment and regenerative capabilities with single-cell resolution. FNDs with a diameter of B100 nm were produced by radiation damage of type Ib diamond powders using a 40 keV He+ ion beam, followed by thermal annealing at 800 C, air oxidation at 450 1C and purification in concentrated H2SO4/HNO3. Isolated pulmonary cells were labeled by incubation with the FNDs at an optimized dose of 100 mg ml 1, determined by flow cytometric analysis. Immediately after labelling, the mean fluorescence intensity of the cells was 45-fold greater than the one of un-labelled, control, cells and it decreased of about 50% every 48 h because of FND redistribution resulting from cell division. When injected into the tail veins of adult healthy normal mice, FND labeled cells localized in the lungs, and not in other organs, demonstrating their maintained activity and the actual possibility of tracking them in living animals. Nevertheless, weather this result was clear in flow cytometric analysis, direct imaging of the ex vivo tissues by confocal microscopy was complicated by the strong background signal arising from the fluorescence of haematoxylin and eosin dyes used to stain the samples for morphological analysis. The signal to noise ratio was strongly improved by FLIM exploiting time-gated acquisition. As shown in Fig. 72,344 time gated imaging allowed us to distinguish clearly the FND emission from the background. Distribution of the fluorescent cells in the pulmonary tissues was compared in the case of healthy and naphthalene-injured mice showing

Fig. 72 Representative FLIM, TGF and bright-field H&E staining images of the same lung tissue sections from mice. The merged H&E and TGF images show that the FND-labelled cells (denoted by black arrows) are primarily located in the subepithelium of bronchiolar airways. Scale bar, 50 mm. Co-localization examination of FND-labelled LSCs and macrophages in a typical lung tissue section immunostained with macrophage-specific antigen F4/80 and haematoxylin counterstain. Reprinted with permission from ref. 344. Copyright 2013, Macmillan Publishers Ltd.

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Fig. 73 Top and centre: representative FLIM, TGF and bright-field H&E staining images of the same lung tissue sections, showing the location of FND-labelled LSCs (denoted by white and black arrows) in terminal bronchioles of the lungs. Bottom: FLIM and H&E/TGF images of the lung tissue section of a naphthalene-injured mouse on day 7, showing engraftment of the transplanted FND-labelled LSCs (denoted by white and black arrows) to terminal bronchioles in cluster form. Scale bars, 50 mm. Reprinted with permission from ref. 344. Copyright 2013, Macmillan Publishers Ltd.

different localization. In the healthy animals labelled cells were primarily located in the subepithelium of bronchiolar airways. In contrast, in the case of injured mice, time-gated fluorescence images (shown in Fig. 73)344 displayed engraftment of the transplanted FND-labelled cells to terminal bronchioles also in cluster form. Moreover, lung epithelia of the injured mice were restored more rapidly in the population of transplanted animals with respect to a control one treated with a saline solution. These results allowed the author to reach the relevant conclusion that FND labelling permitted quantitative assessment of the distribution of transplanted cells in tissue. Moreover, thanks to the excellent chemical stability and photostability of the nanomaterial, labelling does not alter the cells’ properties of self renewal and differentiation into type I and type II pneumocytes. More recently Huang et al.71 studied the compatibility of FNDs on the nervous system in mice. These authors injected intracranially 10 mL of FNDs at a concentration of 100 mg mL 1 into the hippocampi of post-weaned juvenile rats. After the injection, the body weight, fodder and water consumption were assessed on a daily basis for one week. There were no significant differences in these parameters between FND- and salineinjected rats. The authors also performed a behavioral test to determine whether there were more subtle effects of FNDs on


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the hippocampus. The novel object recognition test (NORT)358 is based on the natural preference of rats to explore novel objects more than familiar ones. It has been shown that drugs which can damage the hippocampus lowered the discriminating index of NORT. Interestingly neither the procedure of intracranial injection nor the injection of FNDs to the hippocampus altered the discriminating index of NORT. According to the authors these results suggested that FNDs did not interfere with the general function of the hippocampus in live animals. All the in vivo experiments carried out on rats and mice, discussed in this section, demonstrated the extreme chemical and photochemical stability of FNDs in living organism, as well as a unique long-term biocompatibility of these materials. Surprisingly Lin et al. reported FND toxic effects in the case of incorporation into much simpler animal models.346 These authors compared the interaction of commercial carboxylated FNDs of different nominal sizes, 5 and 100 nm, with Protozoa organism Paramecium caudatum and Tetrahymena thermophila. Fluorescence imaging experiments shown that the FNDs could be excreted by the microorganisms, which partially preserved functioning such as division. Nevertheless, significant toxicity was observed following the growth rate of the microorganism as a function of probe concentration and size. In particular smaller FNDs resulted to be more toxic. These results, which are in contrast with the well documented high biocompatibility of NDs, may be in part influenced by the poor colloidal stability of the produced diamond particles that were reported to form aggregates with hydrodynamic diameter of several hundreds of nm.347 10.3 Background-free in vivo imaging by ND fluorescence modulation A very unique feature of NV defects in FNDs is that their fluorescence intensity depends on their spin states.160,161 Magnetic sensitivity of FND emission has been proposed as a powerful instrument to develop new in vivo imaging methods with a reduced background and, as a consequence, high contrast. Igarashi et al.160 used ODMR to remove autofluorescence in whole-body imaging of C. elegans. Conventional imaging methods are affected by autofluorescene which arises throughout the animal body and which is markedly intense in the intestine. As shown in Fig. 74,160 MW modulation allowed the authors to selectively detect the signals from NDs localized in the intestinal lumen strongly reducing the background emission. Most of the NDs localized in the gut lumen but a few NPs were observed absorbed into intestinal cells, which emitted weaker fluorescence than those remaining in the intestinal lumen. In a demonstrative application to mice NDs injected at a depth of 0.4–0.5 mm could be visualized completely erasing the background signals as shown in Fig. 75.160 Hegyi et al. demonstrated the use of NDs for molecular imaging by optical detected electron spin resonance as a proof of principle.159 This method is suitable for 3-D, background free, optical tomography. The triplet ground state of NV defects

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Fig. 74 Selective imaging of NDs inside C. elegans. (a–c) Bright-field/fluorescence merged images of a living wild-type C. elegans fed with NDs. A conventional fluorescence image is shown in red. An image obtained by PL modulation is shown in green (b). The merged image between (a) and (b) is shown in (c). (d–f) A magnified view of the intestine of C. elegans. Bright-field (d), conventional fluorescence (e), and PL modulated (f) images are displayed. Red and blue circles indicate the positions of a ND in the intestinal lumen and autofluorescence, respectively (g) ODMR spectrum of the ND indicated by the red circle in parts (e) and (f). (h) Time courses of fluorescence intensity for the ND and autofluorescence indicated by the red and blue circles in part (e), respectively. Reproduced with permission from ref. 160. Copyright 2012, American Chemical Society.

in NDs is weakly coupled to the diamond lattice, it has long spin relaxation and spin coherence lifetimes (or narrow electron spin resonance linewidths), even at room temperature.359 The authors developed a prototype imaging system using permanent magnets to create a magnetic field-free point. Only NDs localized near the field-free point are resonant with a microwave field at 2.869 GHz and respond to microwave modulation. Thus, when the microwave source is ON a decrease of the fluorescence, by an amount proportional to the ND concentration at the field-free point, is measured with respect to the intensity detected when the modulation signal is OFF. By sweeping the field-free point across an organism and tracking the changes in fluorescence, a quantitative map of the ND concentration as a function of position can be obtained. To image a 2D slice or a 3D volume of the ND concentration within an organism, the authors scanned the field-free point across it in two or three dimensions. Images were then combined using a standard reconstruction algorithm, as in computed

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tomography. The applicability of the imaging setup was demonstrated in the case of ND phantom inserted in chicken breast. Magnetic sensitivity of NDs emission allowed Sarkar et al. to develop a wide field in vivo background free imaging method based on magnetic modulation reducing the signal-tobackground ratio for in vivo imaging up to 100-fold.348 Fluorescence modulation was achieved mechanically by alternately positioning and removing a permanent magnet close to the sample, in a commercial imaging system, to induce a local field that oscillate as a square wave from 0 G to 100 G with a 0.1 Hz frequency. Processing of the acquired images by lock-in detection of the fluorescence led to a strong background decrease in imaging FNDs in the sentinel lymph nodes of a mouse after injection into the front pad of the animal. According to the reported examples, although technological improvements are needed, non-optical modulation of FND emission is a very promising approach to background free, in vivo, optical bioimaging.


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Fig. 75 Selective imaging of ND aggregates inside a nude mouse. (a) Photograph of the nude mouse lying on the sample plate. The MW coil was placed just underneath the mouse. (b, c) Enlarged fluorescence images of the mouse obtained without (b) and with (c) SIP (see Fig. 66). The SIP image clearly indicates that the signal from the area of the red circle is attributed to NDs, whereas that from the area in the blue circles is not. (d) Time courses of fluorescence intensity for the ND aggregate and autofluorescence marked in b and c. The repeated slow fluorescence variation is due to mouse respiration, whereas the rapid modulation laid on top of the slow variation seen in fluorescence from NDs aggregate arises from SIP with MW irradiation turned on and off every 10 ms. (e) ODMR spectrum of the ND aggregate indicated by a red circle in parts b and c. Reproduced with permission from ref. 160. Copyright 2012, American Chemical Society.

11 Comparing synthesis, properties and bioimaging applications of Si NCs and FNDs Recent results, relative to the application of both FNDs and Si QDs as luminescent contrast agents for bioimaging, demonstrated that these materials equal, and in some aspects surpass, metal based quantum dots in terms of photostability and usability for long-term detection. In this section we will summarize and compare the different features of the two families of nanomaterials also considering the synthetic accessibility as well as physical– chemical properties. The different issues analyzed in this section are summarized in Table 4.

11.1

Comparing synthesis of Si QDs and FNDs

Synthesis of both Si NCs and FNDs is, with a few exceptions, less direct and less practical than most methods developed for the preparation of other luminescent nanoprobes proposed for bioimaging such as, for example, silica NPs268–272 or carbon dots.126–129 The preparative effort, anyway, is often rewarded by the extraordinary photostability, both in vivo and in vitro, of the final materials. As discussed in Section 3, the preparation of Si QDs is quite a complex procedure which requires, in most cases, dedicated setup, the use of hazardous reactants, a controlled atmosphere, harsh conditions and several synthetic steps. An important


exception is represented by microwave based methods in solution, since they allow the direct production of water dispersed Si NCs. On the other hands, some of these processes start from preformed nanosilicon (e.g. nanowires) which requires a non-trivial preliminary synthetic step. Microwave assisted synthesis from APTES is a very convenient method, but it permits only the preparation of blue emitting NCs. Research for new preparative strategies is surely fundamental for future Si NC development. FND synthesis also requires dedicated production facilities, as discussed in detail in Section 5. Nevertheless ND synthesis is nowadays carried out, on a quite large scale, at the industrial level for applications different than bioimaging. A large variety of commercial ND powders and suspensions are available. The most critical point in the production of FND is the color center creation, since it requires the use of ion beam irradiation. Research in alternative methods for activation of NDs would surely strongly enlarge the accessibility of FNDs and their application in bioimaging. 11.2

Size of Si QDs and FNDs

For biological application size is very important since it is one of the parameters that control the interaction of the probes with the biological targets, as well as their fate in the living organisms. In particular sub 10 nm probes are expected to be renally cleared and to accumulate less in the body with respect to larger NPs.56 Although NDs with sizes of about 5 nm can be prepared by the detonation technique, these materials strongly

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Table 3

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Summary of the properties of the FNDs based luminescent contrast agents discussed in this review article

Authors

ND typea Size (nm)

Fluorescence activation Termination

Fu et al.341 Neugart et al.303 Faklaris et al.342 Wee et al.181 Perevedentseva et al.343 Weng et al.320 Weng et al.317 Chu et al.291 Mkandawire et al.322

H Ib H Ib H Ib N Ia H Ib H Ib H Ib H Ib D

35 50 46 50/70/140/350 100 100 144/166/222 35/150 30–50

3 MeV H+ 1 MeV e 8 MeV e 40 keV He+ n.r. 2.5 MeV H+ 2.5 MeV H+ n.r. n.r.

Alhaddad et al. Fu et al.321 Wang et al.316 Prabhakar et al.175 Wu et al.344 Kucsko et al.158 Moore et al.325 Mohan et al.318

H H H H H H H H

Ib Ib Ib Ib Ib Ib Ib Ib

110/120 140 140 50 100 100 — 120

Kuo et al.345 Lin et al.346 Simpson et al.347 Vaijayanthimala et al.179

H H H H

Ib Ib Ib Ib

100 100 130 100

Hegy et al.159 Sarkar et al.348 Igarashi et al.160

H Ib H Ib H Ib

100 100 170

324

a

–COOH –COOH, SDS –COOH –COOH –COOH –COOH –COOH/–NH2 APTES TEOS PPI, dendrimer, liposomes, protamine sulfate 13.9 MeV e PEI, PAI n.r. –COOH n.r. –COOH, PEG 4000 2 MeV e Porous silica/PEG_PEI + 40 keV He –COOH n.r. — Fluorophores Lipid film 40 keV He+ –COOH, polylis, CMDX dextran 3 MeV H+ –COOH n.r. –COOH 2 MeV e –COOH 3 MeV H+ –COOH 40 keV He+ 40 keV He+ –COOH n.r. TEOS 4.6 MeV e —

H Ib: high pressure high temperature, type Ib. N Ia natural, type Ia. D: detonation.

aggregate in aqueous environments. Hence, as summarized in Table 3, imaging contrast agents based on FNDs exhibit sizes ranging from a few tens to hundreds of nanometers. Utilization of single digit NDs as individual NPs, and not as aggregates, for bioimaging is one of the challenging issues in their future application. In the case of Si QDs, as shown in Table 2, examples of stabilization of individual small NPs in a biological environment have been reported. Nevertheless in most cases larger nanosystems containing tens to hundreds Si NCs have been proposed as probes for bioimaging. 11.3

Toxicity and safety of Si NCs and FNDs

As discussed in the introductive part, a definitive assessment of the safety of a full class of nanomaterials on the human health and the environment is a hard task.22 Toxicity of nanomaterials can be difficultly generalized without considering, as critical variables, their synthetic origin, size, shape and surface functionalization and charge.79,360,361 Long-term toxicity in mice and rats has been investigated for both kinds of nanomaterials. Only in the case of Si NCs the study has been extended to the level of primates. Several behavioral, functional and anatomical parameters have been considered as indicators of possible deleterious effects. No evidence of toxicity was reported for FNDs in mice at a dosage of up to 75 mg kg 1 body mass, neither in the living animals, nor in the histopathological examination. In contrast, alterations in the tissues of the liver were observed in mice, in the case of Si NCs, at heavy dosage as high as 380 mg kg 1. Surprisingly these effects were not detected in monkeys treated

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b

Biomolecules

Target

Colorb

— — — — — Transferrin Transferrin Antibody

HeLa HeLa HeLa HeLa A549/HFL-1/Beas-2b HeLa HeLa HepG2, HCT116, LCC6 HeLa

R R R G G R R R G

si-RNA BmK CT DOX — — — Antibody BSA

NIH/3T3 C6 Glioma HepG2 HeLa Lung stem WS1 MDA-MB-231 C. elegans

R R R R R R m R

Yolk lipoproteins C. elegans — Protozoa BSA Drosophila melanogaster BSA Rat, mouse — — —

R R R R

Chicken breast R Mouse R HeLa, C. elegans, mouse R

R: red. G: green. m: multi-color.

with the same nanomaterials although, also in this case, elemental analysis revealed accumulation of silicon in the tissues. At the cellular level, carboxylated FNDs do not affect cell viability up to concentration in the 100–400 mg ml 1 range and show in all cases an IC50 4 500 mg ml 1. Exceptions were reported in the case of surface modified FNDs. As far as Si QDs are concerned, variability of the synthetic methodologies produces a more complicated scenario. Nevertheless the data we discussed proved that Si QDs, if properly functionalized, are highly biocompatible with an IC50 4 500 mg ml 1 compared to 20 mg ml 1 and 11 mg ml 1 measured for CdTe and CdHgTe QDs under the same conditions.231,233 Summarizing, FNDs and Si QDs can be considered highly biocompatible materials both in vitro and in vivo although some toxic effects, at high dose subministration, were observed. Environmental safety of Si QDs and FNDs has been poorly investigated.362–365 Nevertheless actual environmental concern about these materials arises more from the procedures and the chemicals used for their synthesis than from their actual impact on the ecosystems. Examples of risks associated with the production of Si QDs and FNDs are the possible release of concentrated HF or of the strongly oxidizing mixture. 11.4 Photostability and long-term detectability of Si QDs and FNDs Long-term detection of the PL of FNDs and Si QDs is possible thanks to the chemical and photochemical stability in a physiological environment of the optimized materials. In vitro, FNDs show a practically unlimited photostability under continuous


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Chem Soc Rev Table 4

Direct comparison of the main features of Si QDs and FNDs


Features Brightness (absorption, e) Brightness (PL, f) PL decay times PL tunability Photostability Long term stability in vivo Toxicity Biodegradability Fate: body clearance Size control Synthetic methods a

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Si QDs

FND a

Good in the UV-vis, moderate in the NIR Moderate in the UV-vis, good in the NIRa In the ns or ms range Good in a wide range of wavelengths Extraordinary Extraordinary Very low Controllable Not completea Down to few nanometers Few example of facile processesa

Depends on the number of color centera Good In the ns range Limited to some wavelengthsa Extraordinary Extraordinary Very low Not reporteda Needs investigationa Down to tens of nanometers because of aggregationa PL activation requires radiation damagea

Issues of potential improvement.

irradiation. A signal decrease was reported, in the case of cell replication, as an effect of dilution of the NPs in the new cells. Si NCs exhibit, if properly designed, a photostability higher than cadmium based QDs. Nevertheless partial photobleaching is observed after several hours under continuous and strong-power excitation. Whether this long-term PL fading results from chemical or photochemical degradation has not been investigated in detail. Chemical stability of Si NCs, as aqueous dispersion, has been largely increased in the last few years and NPs with shelf stabilities of several months have been prepared. For in vivo bioimaging PL detectability time of FNDs is mostly related to the kinetics of accumulation, redistribution and elimination in the organs and tissues rather than to their actual chemical and photochemical stability. FNDs were detected in vivo in mice for time periods as long as 37 days but only when injected intradermally. In the case of Si QDs in vivo their PL could be clearly detected in liver and spleen of a mouse model still 1 month after NP injection. 11.5

Brightness of Si QDs and FNDs

Brightness is a very important feature for luminescent probes since it regulates the detection sensitivity.366 Color centers in FNDs show a PL quantum yield that, in the case of NV defects, are unitary. Nevertheless, the few data available about the molar extinction coefficient of these emitters indicate values which are one order of magnitude lower than organic dyes. The presence of surface graphitic carbon has also been reported to cause partial PL quenching, reducing the FND brightness. Super bright FNDs can be, in principle, prepared by creating a large number of emitting defects; nevertheless the effect of the inter-defect interactions on the PL has not been explored in detail yet. Moreover, the actual number of color centers produced in the NDs under the different activation conditions is still not well established. In the case of Si NCs, although PL quantum yield as high as 75% has been reported, the actual emission efficiencies in a biological environment are typically much lower (see Table 2). Improving this feature is surely one of the important challenges in future development of Si NCs for bioimaging. Molar absorption coefficients of Si QDs are strongly size dependent and they markedly decrease with increasing wavelength. As an advantage, this feature allows simultaneous excitation of different Si QDs based probes for multicolor detection.


Nevertheless, the absence of strong, narrow absorption peaks in the visible/NIR region becomes a drawback for imaging applications in which specific laser excitation in such a spectral region is preferable. This issue may be partially addressed in the future using hybrid materials in which excitation energy adsorbed by organic chromophores is funneled to Si NCs via excitation energy transfer processes. 11.6

PL tunability in Si QDs and FNDs

Wide tunability of the emission of Si NCs has been demonstrated exploiting both the quantum confinement effect and surface modification. Si QDs show strong NIR emission, suitable for low background bioimaging, nevertheless their light absorption efficiency dramatically decreases at high wavelengths. Excitation in the NIR for in vivo imaging is hence typically performed on the tail of the absorption band. The variety of the emission colors is less broad for FNDs their PL being related to localized states with peculiar, not tunable, features. NV centers show emission in the far red suitable for in vivo detection. Nevertheless these centers absorb light in the green region, which is acceptable but no ideal for in vivo applications. Considering the wide variety of defect centers in NDs, the creation of new color centers is a promising approach to the future production of materials with intense excitation bands in the NIR spectral window. 11.7

PL modulability for background-free imaging with FNDs

Response of FND luminescence to magnetic fields has been demonstrated to be a unique, powerful feature for background elimination in bioimaging and it promises to become more and more important in the development of new high sensitivity and high contrast imaging systems. Because of the different nature of optical transitions in Si QDs the future availability of silicon based NPs with similar sensitivity of the PL to magnetic field is unlikely, at least considering the present state of the art. 11.8 Si QDs and FNDs: critical issues for their bioapplications Summarizing the issues discussed in this section, we can outline some key problems still not completely solved that hamper to some degree the application of Si QDs and FNDs to bioimaging.

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Issues that need improvement are shortly listed in Table 4. Very shortly, preparation of the materials as stable, individual NPs in a physiological environment is still a delicate issue as well as a complete and fully predictable control of the optical and PL features. Chemical and photochemical stabilities are indeed extraordinary for best performing materials, but they cannot still be considered as general features. A last very important point that needs further investigation is the fate and the long term toxicity of both classes of nanomaterials in view of their possible future clinical application.

12 Conclusions and outlook Recent reports of the applications of both Si QDs and FNDs to in vitro and in vivo imaging demonstrated their very unique features, especially as far as biocompatibility and stability are concerned. Nevertheless, several characteristics of these NPs still need development, and progress in this research field is expected to have a strong impact on optical bioimaging techniques. Challenges for the future include a better and detailed understanding of the sub-structure of both Si QDs and FNDs. In fact, although a huge effort has been devoted to understand the effect of the morphological and chemical features on the optical properties of these NPs, the models used for their description are still incomplete. Local changes in the composition, degree of crystallinity and structure, as well as the presence of a large fraction of surface atoms are just the most critical variables of a wide set of parameters that have been demonstrated to play a dramatic role in controlling the properties of these materials. Considering such a complexity, understanding and taking into account the chemical and structural variability that arises from different preparative approaches and post-synthetic treatments are still challenging. As for other nanomaterials, a partial simplification of this scenario could result from a more systematic classification of both Si QDs and FNDs in more specific sub-categories, especially taking into account their origin. Presently a large variety of quite different materials are collected inside the same classes of NPs making tricky a generalization of their description. Development of new convenient and mild preparative approaches for both Si QDs and FNDs is also desirable. Dedicated setup and skills required for the synthesis of these materials partially hampered their diffusion with respect to other easily accessible nanomaterials. In conclusion, although examples of ultrastable NPs suitable for very long term bioimaging have been reported, both Si QDs and FNDs are far from being fully developed and optimized nanoprobes and we believe that their unique features will motivate future efforts for the achievement of new extraordinary materials.

Acknowledgements We gratefully acknowledge financial support from ERC (‘‘MOSAIC’’ Starting Grant 259014).

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