Available online at www.sciencedirect.com

ScienceDirect Mind your P’s and Q’s: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications Melissa Massey, Miao Wu, Erin M Conroy and W Russ Algar Semiconductor quantum dots (QDs) and semiconducting polymer nanoparticles (Pdots) are brightly emissive materials that offer many advantages for bioanalysis and bioimaging, and are complementary to revolutionary advances in fluorescence technology. Within the context of biological applications, this review compares the evolution and different stages of development of these two types of nanoparticle, and addresses current perceptions about QDs. Although neither material is a wholesale replacement for fluorescent dyes, recent trends have demonstrated that both types of nanoparticle can excel in applications that are often too demanding for fluorescent dyes alone. Examples discussed in this review include single particle tracking and imaging, multicolor imaging and multiplexed detection, biosensing, point-of-care diagnostics, in vivo imaging and drug delivery. Addresses Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada Corresponding author: Algar, W Russ ([email protected])

Current Opinion in Biotechnology 2015, 34:30–40 This review comes from a themed issue on Nanobiotechnology Edited by Igor L Medintz and Matthew Tirrell

http://dx.doi.org/10.1016/j.copbio.2014.11.006 0958-1669/# 2014 Elsevier Ltd. All rights reserved.

Introduction Fluorescence remains one of the most powerful and versatile tools for biological research. Over the past two decades, advances in fluorescence methods have been revolutionary, as evident from the awarding of the 2014 Nobel Prize in Chemistry for single molecule spectroscopy and super-resolution imaging [1]. The development of new fluorescent materials has been complementary to these methods, as well as a multitude of other fluorescence assay and imaging applications. Of these new materials, two of the most promising have been colloidal semiconductor nanocrystals, or ‘quantum dots’ (QDs), and semiconducting polymer nanoparticles, or ‘polymer dots’ (Pdots). These nanoparticles (NPs) have several capabilities in common but are at different stages Current Opinion in Biotechnology 2015, 34:30–40

of their development and proliferation. Here, we highlight trends in the utilization of these materials as biological probes, addressing recent perceptions, progress, current limitations, and applications.

Bright emitters A summary comparison of QDs, Pdots and fluorescent dyes is presented in Table 1, and several advantages of QDs and Pdots are illustrated in Figure 1. Interested readers can refer to comprehensive reviews written on these NPs for detailed discussions of their properties [2,3,4,5]. Both materials offer bright emission, which, by virtue of their larger molar absorption coefficients, can exceed that of fluorescent dyes by an order of magnitude or more, and endure longer because of their greater resistance to photobleaching. Compositionally, the two materials differ significantly: QDs are nanocrystallites of inorganic semiconductors, with core/shell structures of II–VI materials such as CdSe/ZnS, CdSeS/ZnS and CdTe/ZnS being the most common in biological applications [4,5]; Pdots are nanoparticulate aggregates of p-conjugated organic semiconducting polymers such as those based on fluorene, phenylene, thiophene, and benzothiadiazole monomers and derivatives, typically in a glassy phase, and as the primary constituent of the NP [2,3]. As multi-chromophore systems, Pdots are often brighter per NP than QDs and, indeed, most of their advantages over QDs and fluorescent dyes arise from the high number density of chromophore/fluorophore units per NP. QDs have an advantage in that they typically provide emission that is more spectrally narrow and more precisely tunable. Being hydrophobic, neither class of NP is intrinsically compatible with biologically relevant media, and hydrophilic surface functionalization is required for aqueous dispersion, colloidal stabilization, and bioconjugation [2,3,4,5]. At present, bioconjugate chemistry is better developed with QDs [6], and a broader range of applications in bioanalysis and imaging have been reported across a greater number of discipline boundaries.

QDs: from breakthrough to boom to skepticism Following breakthrough demonstrations of cellular labeling in 1998 [7,8], research toward biological applications of QDs exploded between 2000 and 2010. QDs were ported to nearly every application where fluorescent dyes were the norm, with mixed levels of success, while www.sciencedirect.com

Polymer dots and quantum dots in biotechnology Massey et al. 31

Table 1 Comparison of Pdots, QDs, and fluorescent dyes. Values represent typical ranges for common materials Pdots

QDs

Fluorescent dyes

Doping b Passivation Diameter c Size polydispersity Aqueous dispersion d

p-Conjugated polymer (MW 104–105 Da) Molecules, other NPs – 5–50 nm Moderate to high Direct functionalization, amphiphiles

Inorganic nanocrystal (102–105 atoms) Ions Inorganic shell(s) 4–10 nm Low Bifunctional ligands, amphiphiles

Organic molecule (MW 102–103 Da) – – 1 nm – –

Optical property Transition e Chromophore units Abs. coefficient f Abs. FWHM g Two-photon cross-section f Quantum yield Emission lifetime h Emission FWHM i Blinking j Number of photons k

p–p* 102–10 5 107–108 M 1 cm 50–200 nm 103–107 GM 0.1–0.6 100 ps to 1 ns 50–100 nm Size dependent 108–10 9

VB–CB 1 105–107 M 1 cm 150–300 nm 103–104 GM 0.1–0.9 10–50 ns 25–40 nm Typical 107–10 8

p–p*, nb–p* 1 104–105 M 1 cm 30–80 nm 101–102 GM 0.05–0.9 1–10 ns 30–80 nm Some 104–10 6

Physical property Composition a

1

1

1

a

MW, molecular weight. Discrete entities within the NP phase. c Geometric diameter of NP (core/shell for QDs); hydrodynamic diameter varies with functionalization. d Method(s) for colloidal stabilization in biologically relevant media. e Electronic transition associated with photon absorption and emission; VB, quantum confined valence band state; CB, quantum confined conduction band state; nb, non-bonding. f Values increase with increasing NP size. g FWHM, full-width-at-half maximum. h Pdots and QDs often have multiexponential lifetimes. i Pdots and fluorescent dyes have asymmetric, red-tailed emission underestimated by the FWHM. j Only Pdots < 10 nm in size exhibit significant blinking. k Number of photons emitted by a single NP/molecule before photobleaching. b

driving remarkable progress in the development of robust, biocompatible coatings for QDs and bioconjugate chemistries [9]. However, in recent years, there has been some skepticism about QDs, in large part from dogged concerns about toxicity, and pessimism that QDs are not superior to fluorescent dyes in practice. These notions, although not without some merit, are imprecise. The putative toxicity of QDs is too often oversimplified to the presence of cadmium in the most prolific QD materials. In reality, toxicity is a complex issue with a notable disconnect between studies with cell models and those with animal models: cytotoxicity associated with QDs does not directly translate into toxicity in animal models [10,11]. Although it is prudent to remain cautious about the prospect of in vivo administration of QDs to humans, the potential toxicity of QDs is not a compelling argument against their use as cellular or in vivo probes for fundamental studies on model systems, but rather a directive to use high-quality and well-characterized materials, which many studies have shown to be non-cytotoxic at doses and exposures relevant to experiments (for recent examples, see [12,13]). Moreover, it is impractical to argue that QDs should be avoided for in vitro assays because of their ‘toxic’ cadmium content — the amount of QDs in a www.sciencedirect.com

typical assay (10 11 mol QD per sample, or 100 mg Cd) is no more cadmium than the average North American or European adult consumes as part of their weekly diet (175 mg) [14]. These arguments also neglect vibrant research on alternative QD materials such as silicon [15– 17], InP/ZnS [18,19], Ag2S [20], and copper-based materials [21,22], among others. Research on these materials is motivated, in part, by the elimination of heavy metals, but also by shifting QD emission out of the visible spectrum and into the first (ca. 650–950 nm) or second (ca. 1000–1300 nm) near-infrared (NIR) window for biological imaging. The caveat to the foregoing is that there should still be bona fide reasons to utilize QDs instead of fluorescent dyes or other materials, and their advantages should be considered on a case-by-case basis. The field has long outgrown the notion that QDs are universally better than fluorescent dyes, and has rather matured to the point where there is, quite appropriately, increasing focus on targeting QDs to applications where their properties are uniquely suitable.

QDs shine on With highly sensitive fluorescence detection methods more accessible than ever, the potential edge in sensitivity Current Opinion in Biotechnology 2015, 34:30–40

32 Nanobiotechnology

Figure 1

(a) Inorganic/Organic Interface Self-assembled monolayers High-density of functional groups

Light Absorption Spectrally very broad Large cross-sections

Light Emission Continuously tunable Spectrally narrow Low photobleaching rates Good quantum yields Energy transfer

QDs

Nanocrystal Structure Small size Monodisperse

(b)

Light Absorption Spectrally broad Very large cross-sections Multiple chromophores

Pdots

Light Emission High emission rates Good quantum yields Very low photobleaching rates Energy transfer

Hydrophobic interior Molecular cargo Composite materials Permeable

Polymer Structure Pendant functionalization Functional monomer units Low intrinsic toxicity Current Opinion in Biotechnology

Advantageous properties and features of QDs and Pdots.

that a 1:1 substitution of a QD for a fluorescent dye will provide in an ensemble measurement is almost a moot point, particularly since dyes remain more widely available and easier to utilize. Rather, QDs have their greatest value in technically demanding and value-added applications such as single particle tracking (SPT) and long-term imaging, multiplexed assays/multicolor imaging, as multifunctional probes, and, most recently, in assays with consumer electronics rather than laboratory instrumentation. Recent examples of these applications are highlighted below. For imaging and SPT, the bright and robust emission from QDs permits the detection of a greater number of photons, which translates into more precise localization of single particles and tracking over longer periods of time. SPT with QDs has recently been used to visualize the infection of cells by avian influenza virus [23], and the dynamics of cell-surface molecules in cultured cells [24,25] and brain slices [26], among many other examples. The well-known emission intermittency (i.e. blinking) of Current Opinion in Biotechnology 2015, 34:30–40

QDs can be a challenge for tracking, but can be avoided by using giant-shell non-blinking QDs, which have enabled three-dimensional tracking of IgE receptor dynamics with live cells over several minutes [27]. On the other hand, the blinking of QDs has been recently exploited for three-dimensional super-resolution imaging of the distribution of epidermal growth factor receptors of breast cancer cells [28]. In vitro singleparticle assays have been reported on the basis of colocalizing two colors of QD [29] and charge-transfer quenching [30], complementing the long-standing development of single-particle Fo¨rster resonance energy transfer (FRET) assays [4]. QDs are outstanding for multiplexed detection and imaging because multiple colors can be excited simultaneously, with good spectral separation between the excitation and emission wavelengths, and their emission detected with minimal crosstalk because of the narrow width of their emission spectra. This multiplexing www.sciencedirect.com

Polymer dots and quantum dots in biotechnology Massey et al. 33

capability is further enhanced when combined with spectral imaging, as has been demonstrated for immunohistological staining of tissue sections with QD–antibody conjugates [31–34]. In these applications, the brightness of the QDs is also advantageous, not necessarily because the instrumentation would have difficulty detecting a fluorescent dye-labeled antibody, but because the QDs provide a higher signal-to-background ratio versus the autofluorescence of the tissue, leading to superior sensitivity versus fluorescent dyes [35]. In vivo staining of cancer markers is also possible, as recently demonstrated in the colon of a mouse model, albeit still with ex vivo imaging but ultimately amenable to endoscopy [36]. Spectral imaging has also been applied to a single cell 25-plex analysis that utilized five rounds of immunolabeling with five colors of QD and denaturable probes (Figure 2a) [37], and highspeed spectral imaging SPT of eight colors of QD bound to a cell membrane has been reported (Figure 2b) [38]. Even without spectral imaging, QDs remain powerful tools for multicolor SPT (up to 3 colors) of receptors on live cells [39]. In concert with their optical properties, the functionalizable surface area of QDs also provides new opportunities. Multivalent QD bioconjugates can yield enhanced binding avidity [40], enhanced rates of enzyme activity [41], and engage in a geometric progression of layer-by-layer biological assembly to yield dramatic levels of signal amplification [42]. Moreover, the combination of multiple biorecognition elements with multiple emissive materials allows the creation of multifunctional vectors. Recent examples include the use of QD/fluorescent dye-based concentric Fo¨rster resonance energy transfer (cFRET) configurations for spectrally multiplexed detection of protease activity and activation [43], or distinguishing between protease activity and concentration (Figure 3a) [44]. cFRET systems featuring energy transfer relays between QDs, fluorescent dyes, and luminescent lanthanide complexes have also been developed for spectrotemporally multiplexed assays for DNA hybridization [45] and proteolytic activity [46], as well as the construction of NP-based biophotonic logic gates [47,48]. Finally, the brightness of QDs is promising for point-ofcare diagnostic methods that utilize mass-produced consumer devices rather than sophisticated scientific instrumentation. A particularly illustrative example is the demonstration that quantitative and multiplexed FRET-based assays can be carried out with low-cost, low-power light sources and a smartphone camera (Figure 3b) [49,50]. Here, the broad and strong absorption of the QD permits use of a violet light-emitting diode for excitation without crosstalk in the detection channels, which were defined by the built-in red/green/blue color filters of the smartphone camera. The weaker absorption and broader emission from fluorescent dyes precludes their direct use in this sort of application. www.sciencedirect.com

Pdots: entering their boom years To date, the development of Pdots has somewhat mirrored the early development of QDs, but is not yet as extensive. First used for biological applications in 2008 [51], Pdots have had a growth spurt over the past few years, with increasing materials development and refinement with proof-of-concept applications such as SPT with fixed cells [52], immunofluorescent labeling of cultured cancer cells [53,54] or blot-style assays [55], along with cell viability assays that show no significant toxicity [2,3]. These efforts are similar to the early porting of QDs to standard applications of fluorescent dyes [56–58]. One indicator of the relative nascency of Pdots is the limited variety of bioconjugate chemistries that have been employed with these NPs. Most studies have utilized stalwart chemistries such as carbodiimide coupling and streptavidin–biotin binding to produce bioconjugates. In the coming years, there will likely be much greater use of chemoselective reactions; for example, disulfide exchange and hydrazone ligation, enzyme and intein-mediated ligation, and ‘click’ chemistries such as azide-alkyne cycloaddition [59], Staudinger–Bertozzi ligation and Diels–Alder reactions, to name only a few [6]. Interestingly, the physical nature of Pdots provides a solution to the challenge of generating monovalent NPs — a single semiconductor polymer chain with a terminal functional group can be collapsed into a small (5 nm) Pdot [60]. This method avoids isolation via gel electrophoresis, as is typically required for monovalent QD bioconjugates [61,62]. Whereas QDs are hard NPs, Pdots are soft NPs and their interior can be doped with both molecular and other NP materials to form composite materials with new optical or multimodal properties. For example, doping Pdots with NIR fluorophores [63–65,66], QDs [67], or Eu(III) complexes [68] yields more spectrally narrow, redshifted emission, and, in the case of Eu(III), long-lived excited states to permit time-gated measurements with improved signal-to-background ratios. Alternatively, boron-dipyrromethene (BODIPY) fluorophores [53,54] and Eu(III) complexes [69] can be incorporated directly into the constituent polymer chains (Figure 4a). In each case, Pdots act as antennae that absorb excitation light and efficiently transfer the excitonic energy to the dopant emitters, increasing their apparent brightness. Other dopants have included photochromic spiropyran dyes to generate photoswitchable Pdots that may be useful for super-resolution imaging [70], gold NPs to provide both dark-field and fluorescence contrast [71], and iron oxide NPs to enable magnetic manipulation [71]. The above developments are conceptually similar to composite materials of QDs that have been, and continue to be investigated, although the hard character of QDs Current Opinion in Biotechnology 2015, 34:30–40

34 Nanobiotechnology

Figure 2

(a)

(i) Step 1: Staining

Step 2: HSI

Step 3: De-staining

RGB Ch5

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150 100 50

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QD800 QD800 fit QD705 QD705 fit QD655 QD655 fit QD625 QD625 fit QD605 QD605 fit QD585 QD585 fit QD565 QD565 fit QD525 QD525 fit

700

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Current Opinion in Biotechnology

Recent examples of multiplexing with QDs. (a) Immunostaining of cell surface biomarkers with five different colors of QD and hyperspectral imaging (HSI), with subsequent de-staining and re-staining: (i) assay steps; (ii) representative 5-color image (scale bar = 50 mm); (iii) validation of 5color staining. Adapted with permission from Macmillan Publishers Ltd: Nature Communications, Ref. [37], copyright 2013. (b) Hyperspectral SPT with QDs: (i) single particle emission spectra for 8 colors of QD; (ii) measured trajectories for the different colors of individual QDs. Adapted from Ref. [38] under the Creative Commons Attribution License.

Current Opinion in Biotechnology 2015, 34:30–40

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Polymer dots and quantum dots in biotechnology Massey et al. 35

Figure 3

(ii)

A647 Sub(A647) peptide

FRET

Cy3

cleavage site

HD1p/cHD1(Cy3) aptamer hybrid His6 FRET

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0.8 0.6 0.4 0.2 0.0 0 4 8 12 16 [Argatroban] (µM)

[THR], [TRP], [LYZ] (µM)

DHLA-PEG

Inactive THR THR-like activity

Peptide Response (a.u.)

Active THR

0.8

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[TRP] (µm) 0.0

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[THR] (µM) 2.0,1.0, 0.50,0.25

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(b) Time Series

Protease 1 Protease 2

2

FRET (i)

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Smartphone Image Acquisition

Red QD

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Protease 3 FRET Quencher

Microtiter plate UV lamp

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Plate reader RGB imaging

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[Protease] Current Opinion in Biotechnology

Recent examples of novel assay formats with QDs. (a) cFRET probe for the parallel detection of thrombin activity and concentration: (i) probe design and sensing mechanism; (ii) data showing discrimination between thrombin (THR), trypsin (TRP; an enzyme with thrombin-like activity), and lysozyme (LYZ; a non-proteolytic enzyme); (iii) data showing that the aptamer response to thrombin concentration is unaffected by argatroban, a reversible inhibitor, whereas the peptide response to thrombin activity is affected. Adapted with permission from Ref. [44]. Copyright 2014 American Chemical Society. (b) Multiplexed, FRET-based homogeneous assays for proteolytic activity using the red-green-blue (RGB) color filters of a smartphone camera: (i) assay format; (ii) representative smartphone images for four samples at five time points; and (iii) comparison of replicate assays between a fluorescent plate reader and smartphone RGB imaging. Reprinted with permission from [50]. Copyright 2014 American Chemical Society.

typically requires synthesis of ‘dumbbell’ structures [72], ion doping [73–75], or codoping of a polymer or silica carrier matrix with QDs and another functional NP [76,77]. Polymer coatings with pendant functionality www.sciencedirect.com

can also be utilized, as in the case of Eu(III)–QD conjugates for time-gated biosensing [78] and photoswitchable QDs coupled to photochromic dyes [79]. From the perspective of composite materials, the permeable nature Current Opinion in Biotechnology 2015, 34:30–40

36 Nanobiotechnology

Figure 4

488 laser excitation

(a)

Intra-chain energy transfer

S

S N

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(i) C8H17

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C8H17 HOOC

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(p s–1 cm–2 sr–1 0.8 0.6 0.4 0.2 0 Current Opinion in Biotechnology

Recent advances with Pdots. (a) Semiconducting polymer with incorporated BODIPY units for more spectrally narrow emission: (i) polymer structure and energy transfer pathway; (ii) electron microscope image of Pdots; (iii) emission spectra for three different BODIPY-Pdots; (iv) flow cytometry data showing that Pdots (520P) are brighter than QDs (QP); and (v) immunolabeling of MCF-7 cells. Adapted with permission from Ref. [53]. Copyright 2013 American Chemical Society. (b) Combined CRET/FRET Pdot probe for oxidative and nitrosative stress: (i) design and sensing mechanism; (ii) chemiluminescence and fluorescence images of a mouse model with injected probe following administration of the indicated doses of isoniazid (INH). Adapted with permission from Macmillan Publishers Ltd: Nature Biotechnology, Ref. [66], copyright 2014.

and hydrophobic interior of Pdots is a versatile combination. Many drug delivery applications with Pdots should be expected in the near future because hydrophobic therapeutics can be carried within the interior of the Pdot [80], with delivery via passive release (e.g. as demonstrated with another new material, fluorescent liquid crystal NPs [81]) or through active release by Current Opinion in Biotechnology 2015, 34:30–40

mixing pH-responsive or enzyme-degradable polymers with semiconducting polymers [82]. Enzymatic degradation of Pdots has also been used as the basis for ‘light up’ sensing of proteolytic activity, where hydrolysis of crosslinks between polymer chains alleviates selfquenching of fluorescence [83]. Although leaching of dopant can be a concern for biological imaging www.sciencedirect.com

Polymer dots and quantum dots in biotechnology Massey et al. 37

applications, permeability can be a benefit for sensing applications as it allows small analytes (e.g. ions) to access indicator dyes within the polymer matrix [64]. Further considering Pdots for chemical and biological sensing, one recent strategy is ratiometric turn-on probes where the Pdot is functionalized with a dye that has emission sensitive to an analyte of interest, but the Pdot itself is insensitive to that analyte. Two such examples include Pdot probes with pendant terbium ions for detection of a bacterial spore biomarker [84] and Pdots with a coupled fluorescent dye for pH sensing [85], the latter of which has a QD analog [86]. Alternatively, analyteresponsive groups can be incorporated into the semiconducting polymer, a recent example of which is the addition of an iridium complex to a Pdot for oxygen sensing and as a potential photosensitizer for photodynamic therapy [87]. Finally, there is a growing and impressive array of in vivo applications of Pdots. For example, Chiu and colleagues used Pdots for imaging brain tumors in mice models [88], and Rao’s group developed NIR dye-doped Pdots that self-illuminate through bioluminescence resonance energy transfer (BRET), demonstrating their utility for sentinel lymph node mapping (SLNM) [89]. Interestingly, this group is also well-known for developing BRET-based self-illuminating QDs [90], and SLNM was one of the first proof-of-concept applications of NIR-emitting QDs [91]. Rao and coworkers have also developed CRET/FRET-based Pdot probes for oxidative and nitrosative stress [65,66], including measurement of peroxide and peroxynitrite in the liver of a mouse model with overdoses of acetaminophen and isoniazid (Figure 4b) [66].

Conclusions and outlook Pdots and QDs are brilliantly bright NP probes with established, emerging and prospective biological applications. Over the past 15 years, QDs have grown to excel in several roles that are often too demanding for fluorescent dyes, including multiplexing and SPT among other applications, and are now also establishing themselves in novel roles that are inaccessible to dyes, such as those where their scaffold capability is needed in combination with their optical properties. Dismissing QDs for reasons of ‘toxicity’ will result in lost opportunities to use these materials for advances in both fundamental and applied biological science. Certainly, new QD materials with no risk of toxicity and optical properties comparable to the best Cd-based QDs would be a tremendous benefit to the field; however, it is equally important to facilitate the proliferation of high-quality CdSe/ZnS QDs and similar materials in biological research by making them more accessible and as easy to use as fluorescent dyes. More recently, Pdots have come to the fore, and, although still exploring their prospective application space, are a www.sciencedirect.com

very promising addition to the fluorescence toolbox. Just as QDs did not replace fluorescent dyes, Pdots will replace neither QDs nor fluorescent dyes, but can be anticipated to grow into significant roles. With brightness greater than QDs, Pdots may lower the technical barrier to single particle fluorescence and may also have value for point-of-care diagnostics. Moreover, with no signs of significant toxicity (thus far) and a hydrophobic core, NIR-emitting Pdots are exciting candidates for drug delivery and theranostics. As with QDs, it will be important to make Pdots readily available and easy to use for a broad range of researchers. The next generation of fluorescence methods in biological research will greatly benefit from using QDs and Pdots to their full potential, which can be expected to catalyze new breakthroughs in biological research.

Acknowledgements The authors acknowledge support of their research program by the University of British Columbia, the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Canada Foundation for Innovation (CFI). W.R.A. is grateful for a Canada Research Chair (Tier 2) and a Michael Smith Foundation for Health Research Scholar Award.

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for interrogating the dynamics of protein complexes in living cells. ACS Chem Biol 2013, 8:320-326. 26. Biermann B, Sokoll S, Klueva J, Missler M, Wiegert JS, Sibarita JB, Heine M: Imaging of molecular surface dynamics in brain slices using single-particle tracking. Nat Commun 2014, 5:3024. 27. Keller AM, Ghosh Y, DeVore MS, Phipps ME, Stewart MH,  Wilson BS, Lidke DS, Hollingsworth JA, Werner JH: 3Dimensional tracking of non-blinking ‘giant’ quantum dots in live cells. Adv Funct Mater 2014, 24:4796-4803. A demonstration of how QDs specially synthesized with a very thick shell to eliminate blinking can be used for 3D tracking of IgE-FceRI receptors associated with live cells for more than 1 min. 28. Wang Y, Fruhwirth G, Cai E, Ng T, Selvin PR: 3D super-resolution  imaging with blinking quantum dots. Nano Lett 2013, 13:52335241. A contrast to the previous reference, where the blinking of QDs is not a problem to be avoided but enables three-dimensional super-resolution imaging of epidermal growth factor receptors on the membrane and inside of cancer cells. 29. Liu J, Yang X, Wang K, Qang Q, Liu W, Wang D: Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes. Nanoscale 2013, 5:11257. 30. Opperwall SR, Divakaran A, Porter EG, Christians JA, DenHartigh AJ, Benson DE: Wide dynamic range sensing with single quantum dot biosensors. ACS Nano 2012, 6:8078-8086. 31. Liu J, Lau SK, Varma VA, Moffitt RA, Caldwell M, Liu T, Young AN, Petros JA, Osunkoya AO, Krogstad T et al.: Molecular mapping of tumor heterogeneity on clinical tissue specimens with multiplexed quantum dots. ACS Nano 2010, 4:2755-2765. 32. Liu J, Lau SK, Varma VA, Kairdolf BA, Nie S: Multiplexed detection and characterization of rare tumor cells in Hodgkin’s lymphoma with multicolor quantum dots. Anal Chem 2010, 82:6237-6243. 33. Peng CW, Liu XL, Chen C, Liu X, Yang XQ, Pang DW, Zhu XB, Li Y: Patterns of cancer invasion revealed by QDs-based quantitative multiplexed imaging of tumor microenvironment. Biomaterials 2011, 32:2907-2917. 34. Xu H, Xu J, Wang X, Wu D, Chen ZG, Wang AY: Quantum dotbased, quantitative, and multiplexed assay for tissue staining. ACS Appl Mater Interfaces 2013, 5:2901-2907. 35. Rakovich TY, Mahfoud OK, Mohamed BM, Prina-Mello A, CrosbieStaunton K, VanDenBroeck T, DeKimpe L, Sukhanova A, Baty D, Rakovich A et al.: Highly sensitive single domain antibody quantum dot conjugates for detection of HER2 biomarker in lung and breast cancer cells. ACS Nano 2014, 6:5682-5695. 36. Park Y, Ryu YM, Jung Y, Wang T, Baek Y, Yoon Y, Bae SM, Park J,  Hwang S, Kim J et al.: Spraying quantum dot conjugates in the colon of live animals enabled rapid and multiplex cancer diagnosis using endoscopy. ACS Nano 2014, 8:8896-8910. A recent example of the use of QD-immunoconjugates and multispectral imaging for staining of cellular biomarkers for immunohistological analysis, with in vivo administration of the QDs. The format is amenable to endoscopic analysis and fluorescence measurements, and is a potential clinical application for QDs in the long-term. 37. Zrazhevskiy P, Gao X: Quantum dot imaging platform for single cell molecular profiling. Nat Commun 2013, 4:1619. A demonstration of 5-color imaging for single cell analysis, enhanced by bioconjugate chemistry that enables multiple rounds of immunostaining and de-staining for a 25-plex analysis or higher with additional cycles. 38. Cutler PJ, Malik MD, Liu S, Byars JM, Lidke DS, Lidke KA: Multicolor quantum dot tracking using a high-speed hyperspectral  line-scanning microscope. PLOS ONE 2013, 8:e64320. A proof-of-concept demonstration of how hyperspectral imaging can be combined with the optical properties of QDs to enable highly multiplexed single particle tracking. 39. Clausen MP, Arnspang EC, Ballou B, Bear JE, Lagerholm BC: Simultaneous multi-species tracking in live cells with quantum dot conjugates. PLOS ONE 2014, 9:e97671. www.sciencedirect.com

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Mind your P's and Q's: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications.

Semiconductor quantum dots (QDs) and semiconducting polymer nanoparticles (Pdots) are brightly emissive materials that offer many advantages for bioan...
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