Accepted Manuscript Short Review Applications of Quantum Dots with Upconverting Luminescence in Bioimaging Yunyun Chen, Hong Liang PII: DOI: Reference:

S1011-1344(14)00103-1 http://dx.doi.org/10.1016/j.jphotobiol.2014.04.003 JPB 9712

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

27 December 2013 1 April 2014 7 April 2014

Please cite this article as: Y. Chen, H. Liang, Applications of Quantum Dots with Upconverting Luminescence in Bioimaging, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/ j.jphotobiol.2014.04.003

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Applications of Quantum Dots with Upconverting Luminescence in Bioimaging Yunyun Chen1 and Hong Liang1,2,* 1

Materials Science and Engineering, Texas A&M University, College Station, TX 77843-3123, USA

2

Department of Mechanical Engineering, Texas A&M University, College Station, TX 77843-3123,

USA *

Email: [email protected]

Abstract

Quantum dots (QDs) have attracted great attention in recent years due to their promising applications in bioiamging. Compared with traditional ultraviolet excitation of QDs, near-infrared laser (NIR) excitation has many advantages, such as being less harmful, little blinking effects, zero autofluorescence and deep penetration in tissue. Composing QDs with upconverting properties is promising to enable NIR excitation. This article provides a review of QDs with upconverting luminescence and their applications in bioimaging. Based on the mechanisms of luminescence, discussion will be divided into four groups: nanoheterostructures/mixtures of QDs and upconverting nanoparticles, graphene quantum dots, lanthanide-doped QDs, and double QDs. The content includes synthetic routes, upconverting luminescence properties, and their applications in bioimaging.

Keywords QDs, upconverting luminescence, bioimaging

1. Introduction 1

Quantum dots (QDs) are semiconductor nanocrystals that have narrow, symmetric and size-tunable emission spectra, broad absorption spectra, high level of brightness and photostability, large Stokes shift, long fluorescence lifetime, and the ability to be conjugated with proteins.[1-4] These unique electronic and optical properties of QDs have received great interests in bio-applications.[4-6] Since Chan et al. and Bruchez et al. reported the modification of QDs water-soluble and bio-conjugating, [7, 8] QDs have been widely used in cell labeling, vivo imaging, single particle tracking in living cells, drug delivery, and cancer detection and therapy.[9-12] Currently, QDs have been tested in living cells, mice, and monkeys, but there have been no clinical cases.[13, 14] The use of QDs in biological applications for clinical cases still has some limitations. The potentially hazardous and toxic nature of QDs, like cadmium-based QDs, will affect human health.[15, 16] The blinking of a single QD crystal will result in losing the signal of QDs when using the single particle tracking and labeling in living cells.[17-19] Furthermore, quantum dots are normally excited with a higher energy ultraviolet (UV) light and emit a lower energy visible light, which is a Stokes shift phenomenon. It is unsafe to the sample with a long time exposure under a UV light. It will result in autofluorescence because of biological structures. [20, 21]

To solve these pitfalls, making use of the upconverting luminescence (UCL), an anti-Stokes shift phenomenon, is a potential solution. It is a process in which two or more lower energy photons are absorbed, leading to emitting higher energy. In the last decade, upconverting nanoparticles (UCNPs), particularly lanthanide (Ln)-doped nanocrystals, have been widely reported.[22-24] UCNPs have narrow emission spectra, high chemical stability, as well as strong brightness.[18, 25] Upon anti-Stokes shift feature, the near-infrared (NIR)-to-visible UNCPs emit higher energy in visible light wavelength by absorbing the lower energy, the NIR laser energy.[26, 27] This results in the absence of blinking effects, and zero autofluorescence.[28, 29] In addition, in the biological applications, the optical window in 2

biological tissue, as known as the NIR window, is a significant factor needs to be considered. It determines the range of wavelength where the light can penetrate deeply and efficiently in tissue. The range of wavelength in the NIR window is approximately from 650nm to 1400nm wavelength. That contains both first NIR window (650-950nm) and second NIR window (1000-1350nm).[30-32] When excitation and emission lights are in the NIR window, it will be more effective for the nanoparticles to be excited by the excitation light. In addition, the signals will be much stronger for detection. Compared with UV excitation, the NIR diffuses and penetrates more rapidly and deeply into cells and tissues.[33, 34]

To date, the focus has been on finding and modifying QDs in order to engage them with UCL properties [35-37]. Compared with UV excitation QDs, the modified QDs can be excited by an NIR laser. According to the mechanism of UCL, these upconverting QDs can be classified into four categories: nanoheterostructures/mixtures of QDs and UCNPs, graphene quantum dots (GQDs), Lndoped QDs, and double QDs. This paper will review these four types of QDs in aspects of their synthesis, UCL properties and bioimaging.

2. Methods and Upoconversion Luminescence Properties

2.1. Nanoheterostructures/Mixtures of QDs and UCNPs

The nanoheterostructures/mixtures (NH/Ms) consist of both QDs and UCNPs. The heterostructure can be core-shell structured [38, 39] or the QDs in situ growth on the UCNPs [37], as Table 1 shows. The

mixtures

are

mixing

QDs

and

UCNPs

together.

[40]

3

Table 1. The summary of nanoparticles, synthesis and schematic of heterostructures1 Nanoparticles

Synthesis

CdSe/NaYF4:Yb,Er

NaYF4:Yb,Er : Thermal decomposition

Schematic of Heterostructure

Ref. [37]

Heterostucture: Grown on UCNPs QDs/SiO2/NaYF4:Yb,Er/Tm NaYF4:Yb,Er/Tm: Thermal decomposition

[38]

Heterostucture: Microemulsion

CdSe/SiO2/ β-NaYF4:Yb,Er

NaYF4:Yb,Er: Thermal decomposition

[39]

Heterostucture: Sensitization

2.1.1. Synthetic Method The synthesis of NH/Ms has two steps. The first step is to synthesize UCNPs. Several methods have been used to produce UCNPs. Wang et al. used the hydrothermal/solvothermal method. [41] Yi et al. UCNPs with the co-precipitation method [42]. Ehlert et al. and Boyer et al. synthesized UCNPs with the thermal decomposition method [43, 44]. The various approaches have been summarized elsewhere.[45]

1

Figures from listed references are used with permission.

4

The second step is to generate heterostructures. The UCNPs are used as seeds for QDs synthesis and growth.[37] Others used SiO2 or TiO2, which conjunct with QDs, to modify and coat on the surface of UCNPs.[38]

For the mixtures, the following step is to synthesize QDs and mix with UCNPs in a solution with or without sonication. Jin et al. reviewed the methods to synthesize QDs. [2, 46]

2.1.2. UCL Properties

The NH/Ms are the combination of QDs and UCNPs in a system, which use QDs as energy acceptors and UCNPs as energy donors. The fluorescence emission band of UCNPs overlaps the absorption band of QDs; resulting UCNPs transfer their emission energy to QDs via a fluorescence resonance energy transfer (FRET) or energy re-absorption. The way to transfer energy from UCNPs to QDs greatly depends on the distance between UCNPs and QDs. If the distance is less than 100 Å, the preferred procedure is FRET, which transfers energy in the form of phonons or lattice vibrations. Otherwise, the alternative is re-absorption, which transfers energy as photons.

Bednarkiewicz et al. reported on the study of mixing CdSe QDs with NaYF4:Yb,Er UCNPs. The UCNP absorbs two lower NIR photons energy, and emits higher energy which is absorbed by QD by FRET process (Fig 1.a). As the emission spectrum shows (Fig 1.b), UCNPs without QDs emission spectrum (green line) has a stronger intensity at 520-560nm wavelength. The stronger energy intensity was emitted when electrons transfer from 2H11/2 to 4I15/2, and 4S3/2 to 4I15/2. After QDs mixed with UCNPs, the emission spectrum (red line) has one more peak at 585nm, and the emission intensity at 520-560nm becomes weaker. This is the reason that absorption spectrum (light gray line) of QDs overlaps with the

5

green line. QDs absorb the energy emitted by UCNPs at 520-560nm, and emit at 585nm wavelength, which is around the emission spectrum of CdSe QDs (dark gray line).

Fig. 1. a. Schematic energy transfer process; b. emission spectrum of QDs (dark gray line), absorption coefficient of QDs (light gray line), UCNPs (green line, UCNP region on the inset picture) and UCNPs with QDs (red one, UCNP + QD region on the inset picture); insert is fluorescence image (with permission).[40] For other NH/M particles’ excitation and emission information are listed in Table 2. All of them are reported as FRED-based particles.

Table 2. Summary of nanoparticles, excitation wavelength, emission peaks without QDs and emission peaks with QDs

6

Particles

Excitation wavelength (nm) 980

Emission peaks without QDs (nm)

Emission Peaks with QDs (nm)

Ref.

524, 542, 660

524, 542, 660, 634

[37]

QDs/SiO2/NaYF4:Yb,Tm

980

450, 479, 510, 640

450, 479, 605

[38]

CdSe/SiO2/ β-NaYF4:Yb,Er

980

522, 540, 653

522, 540, 653

[39]

CdSe + NaYF4:Yb,Er

976

521, 539, 654

521, 539, 654, 585

[40]

CdSe/NaYF4:Yb,Er

2.2. Graphene QDs

Recently, carbon dots have gained much attention for bioimaging.[47] The subclass of carbon dots, carbon quantum dots (C-Dots) and graphene QDs (GQDs), are all reported with UCL performance.[48, 49] Compared with C-Dots, GQDs are easier synthesized. Because of the complex synthesis processes of C-Dots, they are difficult for mass process.[50] In this case, the GQDs will be discussed. GQDs have outstanding characteristics which QDs or UNCPs lack. They have low toxicity for bio-applications, and the raw materials are cheap, unlike rare earth elements.[51] Based on the 2D layered structure, GQDs display certain performance the same as graphene, including large surface area and surface π-π conjunction with different ligands or functional groups. In addition, GQDs have size and color tunable features, quantum confinement, and edge effects, which graphene particles do not have. Recently, the UCL of GQDs have been researched and investigated.

2.2.1. Synthetic Method

The common approaches of using GQDs can be classified into two types: the top-down method and the bottom-up method.[52] Shen et al. presented a schematic diagram of two types of synthesis processes (Fig 2).[51] Pan et al. cut graphene sheet into GQDs (top-down) with hydrothermal method.[53] Zhang et al. synthesized GQDs with electrochemical method by top-down approaches. [54] 7

Liu et al. used the self-assembly method to build up graphite, then used Hummer’s method [55], functionalized and reduced graphene oxide nanosheet to obtain GQDs with uniform morphology. Several methods have been reviewed by Li et al..[56] In addition, it is significant to synthesize stable colloidal GQDs with tunable and controllable size in order to obtain tunable fluorescence GQDs.[57] The distributions of GQDs diameters are mainly from 3nm to 20nm.[51]

Fig. 2 . Schematic diagram of two common methods for synthesis of GQDs (with permission).[51] 2.2.2. UCL Properties

Shen et al. reported the GQDs emission spectra, which were excited by the different excitation energies, from 600nm to 980nm. And the emission peaks are at the range from 450nm to 550nm (Fig 3). The mechanism of UCL is proposed by Shen et al. as shown in Fig 4. Because of quantum confinement, GQDs with large size (left) have smaller band gap than GQDs with a smaller size (right). The energy transfer is considered between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). In UCL process, when the π bond absorbs excitation energy, an electron is exited to the LUMO. When this electron goes back to the σ bond, it will generate higher energy. 8

Fig. 3. a. Emission spectrum excited at 980nm laser. Inset: photograph of the GQDs solution luminesced by 980nm laser; b. emission spectra excited by 600-800nm laser (with permission).[50]

Fig. 4. UCL energy transfer process of GQDs (with permission).[50] 2.3. Ln-doped QDs

Both Ln elements and QDs have excellent luminescence features. Many researchers investigate the photoluminescence of Ln-doped QDs.

2.3.1. Synthetic Method

Several methods can synthesize Ln-doped QDs. Planelles-Aragó et al. used the sol-gel method.[58] Luo et al. synthesized with the sol-gel solvothermal method. [59] Wang et al produced with the polysaccharide template.[60]

9

2.3.2. UCL Properties

Owing to the Ln elements, Ln-doped QDs absorb two low energy photons and emit the higher energy, such as TiO2: Er, TiO2:Er/Yb [59, 61], ZrO2:Er, ZrO2:Er/Yb[62-64], ZnO:Er[65], and CaS:Er[66]. For TiO2:Er, Er absorbs two lower NIR energy photons and is exited to 4I11/2 and 4F7/2 levels; while for TiO2: Er/Yb, the sensitizer, Yb3+, will absorb the photon and transfer the energy to the activator, Er3+, which is excited to 4I11/2, 4F9/2 and 4F7/2 levels shown in fig. 5. When the TiO2:Er and TiO2: Er/Yb are excited by the NIR laser (975nm), they will emit the energy in visible green (2H11/2 ( 4S3/2 ) → 4I15/2) and red wavelength (4F9/2→ 4I15/2). (Fig. 6)

Fig. 5. UCL energy transfer process of TiO2:Er and TiO2: Er/Yb (with permission).[59]

Fig. 6 The emission spectra of Er/TiO2 and Er/Yb/TiO2 (with permission).[59]

10

2.4. Double QDs

The double QDs are based on the band-gap engineering in order to modify the QDs with UCL properties. Deutsch et al. reported a letter investigating the UCL properties of double QDs. Double QDs is a nanoheterocrystal (NHC), which fabricates different types of QDs together to generate double potential wells for holes (Fig 7). CdSe(Te)/CdS forms a core-rod nanocrystal. CdSe grows at tip of CdS rod.

Fig. 7. Schematic depiction of dual emitting quantum dot and TEM image of double QDs nanocrystal (with permission).[35] In current research, many types of NHCs have been studied. Donega reviewed various colloidal NHCs, seen in Fig 8, which have numerous types. And based on the distinct structure, NHC QDs exhibit diverse affections. Although no more NHC QDs were reported with UCL properties at room temperature or with high quantum yield, they have great potential to obtain UCL performance by designing the hertostuctures and band band-gap engineering. The double QDs reported by Deutsch’s group is a good case to explain how the UCL happened.

11

Fig. 8. Schematic depiction of various NHCs structure (with permission).[67] 2.4.1. Synthetic Method

Various methods can be used to form the NHC[67, 68], such as the solvothermal method[69] and the sol-gel method[70].

2.4.2. UCL Properties.

From Deutsch’s group report, NHCs were luminesced with 690nm laser, and emitted with571nm wavelength and 692nm wavelength. The absorption of NHCs was 400nm wavelength. (Fig. 9)

Fig. 9. The absorption spectrum (blue) and emission spectrum (red) of double QDs (with permission).[35]

12

Two possible mechanisms could explain UCL. The core first absorbs one lower energy photon (690nm). To the ‘intraband’ mechanism (Fig 10 left), the intraband absorbs the second lower energy photon, and a hot hole is formed (1). The hot hole was cross the barrier to form a hole (2). Then the exiton is annihilated to ground state of the end CeSe. To the ‘Auger mediated’ mechanism (Fig 10 right), the second photon is absorbed by the core again (1), which leads to the formation of the hot hole by Auger recombination (3). The following steps are the same.

Fig. 10. Two possible UCL mechanism of double QDs (with permission).[35] 3. Bioimaging

During the last decade, owing to the fantastic optical and electric properties, QDs have sparked vast interest in the biomedical research field, which were modified by organic shell and functional surface ligands to make QDs water-soluble, stable, oriented and target-specific.[71-73] Vast work has been done to test fluorescent properties of QDs in living cells or in mice.[13, 74] The QDs achieved or behaved with UCL properties has a great potential for bioimaging. The advantages and disadvantages of four types will be reviewed and discussed.

13

3.1. Nanoheterostructures/Mixtures of QDs and UCNPs

Nanoheterostructures/Mixtures (NH/Ms) are excited with NIR wavelength energy, which is less harmful to the living cell, non-autofluorescence, and penetrates deeply in tissue to luminescence the samples. With Ln elements, whose 4f orbit can be with at most seven unpaired electrons, NH/Ms have a potential application to be used in magnetic resonance image (MRI). In addition, Bednarkiewicz’s group measured photoluminescence quantum yield of 23%.[40] However, Ln element is expensive. In addition, when doing the imaging, toxicity needs to be considered, for QDs with heavy metal would be harmful to human health.

The FRET process of NH/Ms can be demonstrated with the emission lifetime measurement. Bednarkiewicz et al. reported the lifetimes of UCNPs with QDs and without QDs are different, 130.4 µs and 153.1 µs. (Fig. 11) This is because some UCNPs excitons are annihilated with the energy transfer as phonon or lattice vibration, for the excitation of QDs. Then, the lifetimes of energy donors, UCNPs, get short once with energy accepters, QDs, in the solution. Based on the differences of the fluorescence time of the particles, FRET indicates the potential for using in bioimaging with fluorescence lifetime imaging technique. FRET based probes have been widely investigated in bioimaging application.[75-77]

14

Fig. 11. The lifetimes of UCNPs with QDs (black) or without QDs (red) (with permission). 3.2. GQDs

GQDs have three extraordinary features. They are non-/low-toxicity, made of cheap raw materials, and able to connect with more ligands or functional groups. Yang et al. reviewed nano graphene in biomedicine. A typical approach is to use nano-graphene as a platform and carrier.[78] Several papers reported using GQDs for bioimaging, but they mainly used down-conversion (UV excitation), which is harmful to the living cells, for the long time under a UV laser.[54, 79]

Zhu et al. used GQDs with NIR excitation to do the living cellular imaging in mouse osteoblastic cell line (Fig. 12). The excitation wavelength of GQDs was 808nm, and the detection wavelength was in the range of 490-550nm. The outstanding attribute was that GQDs could go into the membranes of cells without any more decorating. Even though much progress has been achieved, many issues need to be considered. One challenge is to get higher quantum yields.[80] Shen et al. reported UCL quantum yield of GQD is 7.4%.[51]

15

Fig. 12. GQDs upconverting cellular imaging. a. the washed cells under bright field. b. under 808nm laser excitation (with permission).[80] 3.3. Ln-doped QDs

Ln-doped QDs have potential for bioimaging and biolabel approaches.[81, 82] Ln elements are usually used for MRI.[45] However, some drawbacks need to be solved. The fluorescence of Ln exhibits on the surface or near the surface. The most challenge in bioimaging is that the intensity of fluorescence is not strong at room temperature. It is possible that the trivalent Ln ions are not easy to dope into QDs deeply.[58, 59, 83, 84]

3.4 Double QDs

Double QDs, NHCs, obtain UCL properties by bad-gap engineering. The double QDsare excited by the 690nm laser and emit both orange and red. Excitation and emission lights within the NIR window are excellent in assisting bioimaging. The quantum yield is 42%. Compared with Ln elements and GQDs, double QDs are size-tunable.

16

Fig. 13. The set of emission spectra of a single dot (with permission).[35] Despite the above mentioned advantages, the double QDs are reported blinking. The emission spectra were measured through a single nanocrystal. Each emission spectrum was integrated spectrum for 0.4 seconds. Both orange wavelength and red emission were changing all the time. (Fig. 13) In addition, CdSe QDs are high toxic.[35] For biological applications, they need to be modified to reduce the toxicity.[85]

3.5 Potential Bioimaging Applications

The advantages and disadvantages of four types of QDs are listed in Table 3. Applications of these QDs are discussed in the following. Except GQDs, no more studies of other three types QDs have been reported in bioimaging application with their UCL performance.

Table 3. Summary of advantages and disadvantages of four types of QDs Advantages

NH/Ms Excitation and emission in NIR window FRET application MRI possible Non-autofluorescence

GQDs Excitation in NIR window Non or low toxicity Non-autofluorescence Multi-conjunctions Cheap raw materials

Ln-doped QDs Excitation and emission in NIR window MRI possible Non-autofluorescence

Double QDs Excitation and emission in NIR window Non-autofluorescence High quantum yields

17

High quantum yields

Disadvantages

Toxicity Ln-element expensive

Low quantum yields

Toxicity Ln-element expensive

Toxicity

Blinking

Zrazhevskiy et al. reviewed the general steps to design traditional QDs probes in bio-applications (Fig. 14), which can be learned and imitated by UCL QDs.[86] The first step is to design the QDs core. The second step is to coat QDs with a shell, which can make QDs water soluble, stable [87] and less toxic for living cells[88]. The third step is to modify QDs bio-functional via decorating QDs with different ligands, which makes QDs targeted for specific applications, such as drug delivery, and cancer therapy [89, 90].

Fig. 14. The general three steps to design QDs for bio-applications (with permission).[86]

For NH/Ms and Ln-doped QDs, with the Ln elements, they can be used with MRI. Ln elements have drawn much attention with upconverting luminescence and magnetic resonance properties.[91-93] The most common used Ln element for MRI is gadolinium [94, 95], as trivalent gadolinium ion has extreme seven unpaired electrons at the 4f orbital. Thus, when coating the shell on QDs, the trivalent gadolinium ion can be doped in the shell, or at first step, it can be co-doped in the particles.

4. Summary and Future Perspectives 18

In this review, the mechanisms, performance, and applications of upconverting luminescence of QDs are discussed. With upconverting luminescence properties, QDs are excited with an NIR laser, which will reduce some pitfalls appeared in traditional QDs. With NIR excitation, QDs overcome the disadvantages of autofluorescence and longtime exposure under UV laser. Through those the NIR light can penetrate deeply in tissues. In addition, GQDs are non-/low- toxic and non-blinking. However, successful application in the human body is still a course needed to be studied and investigated. For future applications in bioimaging, many issues need to be considered. For instance, the hydrophobity of the double QDs requires functionalization with hydrophilic ligands as bioimaging in cells or tissues needs aqueous solution. The methods to make it hydrophilic need to be developed. Further understanding in four types of QDs with UCL properties for biological imaging needs to be obtained. These include: effective targeting to pathogens by functional ligands; working condition requirements for QDs, such as the reticuloendothelial system (RES); avoiding antibody attacking QDs; reducing toxicity of QDs; and stronger signal to obtain exactly information in vivo. Based on these requirements to decorate four types of QDs is still an issue need to be considered and improved.

Acknowledgement

Authors wish to acknowledge Drs. Jaime Grunlan, Igor Roshchin, and Elizabeth Cosgriff-Hernandez for suggestions.

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Highlights • QDs modified with upconverting luminescence properties could overcome the disadvantages of traditional QDs in bioimaging. • The upconverting QDs were reviewed and categorized into four groups. • QDs with upconverting luminescence are suitable for bioimaging.

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