Semiconductor Quantum Dots in Bioanalysis - American Chemical

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Semiconductor Quantum Dots in Bioanalysis: Crossing the Valley of Death Colloidal semiconductor quantum dots (QDs) have evolved beyond scientific novelties and are transitioning into bona fide analytical tools. We describe the burgeoning role of QDs in many different fields of bioanalyses and highlight the advantages afforded by their unique physical and optical properties. W. Russ Algar,†,§ Kimihiro Susumu,‡ James B. Delehanty,† and Igor L. Medintz†,* †

Center for Bio/Molecular Science and Engineering, Code 6900 and ‡Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory 4555 Overlook Avenue, South West, Washington, DC 20375, United States § College of Science, George Mason University, 4400 University Drive, Fairfax, Virginia 22030, United States them hydrophilic and biofunctional, we also examine how this important aspect has developed. Cumulatively, this allows us to assess whether QDs remain a scientific curiosity or have arrived as an important bioanalytical research tool.

Robert Gates

he ominous phrase, “crossing the valley of death” often refers to the period during commercialization of a new scientific product that occurs between its initial launch and sustained adoption by targeted consumers, where unsuccessful technologies never emerge from this chasm. In another context, the “valley of death” can refer to the divide between basic and translational research. Both connotations aptly describe the current point in the development of luminescent semiconductor nanocrystals, or quantum dots (QDs), as bioanalytical tools. After describing their relevant photophysical attributes, we look at the progress QDs have made in the field of bioanalysis over the past decade. Representative examples of bioanalytical applications that highlight the unique capabilities of QDs are discussed, ranging from multiplexed assays and biological sensing to intracellular labeling and theranostics. Given that the use of QDs in biological applications is directly dependent on the chemistries that make

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r 2011 American Chemical Society

’ INTRODUCTION OF QUANTUM DOTS TO BIOLOGY Brus and colleagues at Bell Laboratories first reported colloidal QDs in 1983.1 While many researchers were interested in fabricating quantum nanostructures, Brus and Steigerwald developed a bottom-up chemical synthesis that was in contrast to more favored top-down epitaxial approaches.2 However, it was not until the mid-1990s that Bawendi, Guyot-Sionnest, and others pioneered refined synthetic methods that provided access to nearly monodisperse, nanocrystalline QDs.3
5 The unique photophysical properties of QDs rapidly catalyzed broad scientific interest that, for biological applications, was ignited by two seminal studies published in 1998: Chan and Nie6 along with Bruchez et al.7 prepared water-soluble, photoluminescent (PL) CdSe/ZnS core/shell QDs and utilized them for cellular imaging. The total citations of all papers noted above1
7 grew from 1 031 at the close of 1999 to >13 800 citations by May 2011.8 Almost half of these citations are credited to refs 6 and 7 reflecting widespread interest in using QDs for bioapplications. Alivisatos, another QD pioneer, recently noted how remarkable the transition has been from early skepticism about these materials to fanfare in the 21st century.9 ’ OPTICAL PROPERTIES It is the photophysical properties of QDs that have inspired more than a decade of research toward biological applications.10 QD PL is narrow and symmetric, with an approximately Gaussian profile presenting a full-width-at-half-maximum of 25
35 nm. Brightness arises from high quantum yields (QY ≈ 0.2
0.9) and a large physical cross-section that yields strong one-photon absorption (ε ≈ 104
106 M
1 cm
1) and remarkably high two-photon absorption cross sections (σTPA ≈ 103
104 GM). Published: September 19, 2011 8826

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Analytical Chemistry Absorption is also broad, extending from the emission band and increasing into the near UV. However, the most striking feature of QD emission is that, for a given material, the PL wavelength (i.e., color) can be tuned continuously across a broad spectral range through quantum confinement and control of nanocrystal size. Quantum confinement is the term used to describe changes in electronic properties as the number of atoms in a crystal becomes small. In a semiconductor characterized by a filled valence band (VB) and empty conduction band (CB) separated by an energy gap, electrons are treated as extended wave functions that fit within the crystal and the periodic constraints of its lattice. For large numbers of atoms, many wave functions are allowed and differences in their energies are very small, resulting in bulk band structure. As the crystal becomes small, many of these wave functions no longer satisfy the nanocrystal constraints, allowed states shift away from the band edges, and the density of states decreases to yield discrete quantum confined states with an energy gap that increases with decreasing crystal size. This is shown schematically in Figure 1 for a size series of CdSe QDs. The onset of quantum confinement coincides with crystal dimensionality below the exciton Bohr radius, which is the preferred electron
hole separation, for a semiconductor material (∼5 nm for CdSe). Confinement of an exciton to a nanocrystal with such dimensionality forces the electron and hole closer to one another, increasing the exciton binding energy. The latter is directly correlated with the PL emission wavelength of a QD. In the case of CdSe, the energy gap can be tuned from 1.9 to 2.8 eV and PL varied from 650 to 450 nm as the nanocrystal size decreases from ∼7 to 2 nm. To a first approximation, quantum confinement effects in QDs can be summarized as a physical manifestation of the particle-in-a-box model taught in quantum mechanics. Compared to molecular fluorophores (QY ≈ 0.05
0.9; ε ≈ 103
105 M
1 cm
1; σTPA ≈ 101
103 GM), QDs are brighter and more resistant to photobleaching. These properties afford longer observation times and more reliable measurements in complex samples, such as cells or tissues, where efficient twophoton excitation minimizes background autofluorescence and enhances spatial resolution. QDs also offer greater capacity for multiplexing than molecular fluorophores, which arises from: (1) broad flexibility in excitation wavelength, including the ability to simultaneously excite multiple colors of QD at a single wavelength; (2) greater spectral resolution and available bandwidth by virtue of narrow PL; (3) convenient synthesis of a series of discrete colors (Figure 1B) from a common QD core material using different growth times and temperatures (cf. different molecular structures). An initial concern was that the “blinking” or fluorescence intermittency of a single QD, wherein it fluctuates between bright and dark states over a broad range of time scales (10
6
101 s) with a power law-dependence, would be detrimental to tracking, colocalization, and other single-molecule spectroscopy experiments. However, since blinking is not observed in ensembles or aggregates of QDs, it now routinely serves as confirmation of single QD tracking and contributes toward super-resolution imaging. In addition to their optical properties, QDs also provide extra value due to their nontrivial surface area and ability to participate in charge-transfer (CT) or (dipolar) resonance energy transfer. These interactions can modulate QD PL and allow transduction of biomolecular binding events or enzymatic activity.11,12 Furthermore, the large QD surface area provides a nanoscaffold that can be used to carry biomolecular probes or molecular

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Figure 1. (A) Cartoon and TEM image of a CdSe/ZnS QD. (B) Cartoon, photograph, and PL spectra illustrating progressive color changes of CdSe/ZnS with increasing nanocrystal size. (C) Qualitative changes in QD energy levels with increasing nanocrystal size. Band gap energies, Eg, were estimated from PL spectra. Conduction (CB) and valence (VB) bands of bulk CdSe are shown for comparison. The energy scale is expanded as 10E for clarity.

cargos. The former provides opportunities for higher avidity targeting (e.g., antigens, receptors) and sensing; the latter allows for biomolecular delivery (e.g., drugs, genes).11,13 More interesting near-term applications are expected to combine superior QD optical properties with these extra values to create multifunctional active nanomaterials.11,13

’ QUANTUM DOT MATERIALS CdSe/ZnS nanocrystals have been the most utilized type of QD in biological applications. They consist of a nanocrystalline CdSe core overcoated with a few atomic layers of ZnS. Although faceted, the CdSe core is roughly spherical in shape. Growth of a ZnS shell enhances and protects the optical properties of the core in four critical ways:3
5 (1) passivating the core to isolate it from 8827

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Figure 2. Approximate PL emission ranges and core sizes for different QD materials. Estimated from refs 3
5, 10, 64 and therein.

the surrounding microenvironment, (2) truncating the core size without breaking crystal structure or introducing defects, (3) providing a larger potential energy barrier for exciton confinement within the core, and (4) protection against degradation. The prevalence of CdSe/ZnS QDs arises from the refined synthetic protocols available, PL across the visible region of the spectrum that overlaps with many common organic dyes/ fluorescent proteins, and access to established methods for chemical/biomolecular derivatization.10,14 Many other QD materials have also been developed, although none yet enjoy the same widespread biological use as CdSe/ZnS. In general, QDs are between 1 and 10 nm in size and composed of group IV (e.g., Si), III
V (e.g., InP), and II
VI (e.g., CdTe) semiconductors or alloys thereof. Figure 2 shows how selection of a core material determines the range of QD PL colors that can be tuned via nanocrystal size, with different materials emitting across portions of the near-ultraviolet, visible, and near-infrared spectrum. Alloyed materials (e.g., CdSexTe1
x) further provide the opportunity to tune PL emission as a function of both size and composition.15 Band offsets and lattice parameters of the shell material as well as structural variations such as core
shell
shell QDs16 and thick-shell “giant” QDs17 can further shift or enhance emission and suppress blinking, respectively. Although CdSe/ ZnS QDs are primarily used in the examples of bioanalyses we describe below, the fundamental concepts, and often chemistries, can be applied to a wide range of different QD materials. The use of alternative materials is already emerging in imaging applications and will gradually become more prevalent in bioanalyses.

’ SURFACE FUNCTIONALIZATION OF QUANTUM DOTS Most high-quality QDs are synthesized using hot-injection methods based on the pyrolysis of organometallic precursors in nonpolar coordinating solvents.3
5 Native QDs are insoluble in aqueous media, and it was the development of hydrophilic QD coatings that enabled utility in biology. In the first iteration, Chan et al. and Bruchez et al. replaced the native QD ligands with hydrophilic coatings of mercaptoacetic acid (MAA) ligands and silica, respectively.6,7 Dubertret et al. later used an alternative approach of encapsulating the native QDs within phospholipid

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micelles.18 However, since the efficacy of QDs in a biological application is critically dependent on coating properties, the liabilities of these initial methods required continued development of QD coatings. Important criteria for an ideal QD coating include (1) high-affinity for the QD surface, (2) long-term colloidal stability across a broad range of pH and ionic strengths, (3) capacity for bioconjugation, (4) minimization of hydrodynamic size, and (5) biocompatibility (nontoxic/nonimmunogenic) with low nonspecific binding. Thiol-ligands and amphiphilic polymers (amphipols) are the most prevalent types of QD coating available. As shown in Figure 3A,B, all coatings conserve two essential design elements: a moiety that anchors to the QD surface and a hydrophilic functionality for aqueous dispersion. The selection of these groups determines the degree to which a QD coating can approach the criteria listed above. Figure 3C
E shows a series of ligand/amphipol structures that summarize the progression of key design elements used in coatings. The simplest embodiment of a coating is MAA (Figure 3C,i), a small molecule ligand with a proximal thiol group to bind the QD surface and a distal carboxylate group to provide aqueous colloidal stability. However, electrostatically driven colloidal stability of MAA-coated QDs is limited to basic pH and low ionic strength, while long-term stability is compromised by gradual desorption of ligands from the surface. To improve long-term stability, Mattoussi et al. pioneered the use of dihydrolipoic acid (DHLA, Figure 3C,iv), a bidentate, small molecule, dithiol ligand that binds the QD more tightly.19 To provide aqueous dispersion approximately independent of pH and ionic strength, the DHLA was appended with a poly(ethylene glycol) (PEG) oligomer (Figure 3C,v). PEG also improves biocompatibility and minimizes nonspecific binding. Significant success has been achieved with CdSe/ZnS QDs coated with PEG-appended DHLA as well as derivatives that incorporate a variety of distal functional groups.20 In the latter, three distinct modules of the ligand structure separately address the QD anchoring, solubility, and bioconjugation requirements (Figure 3C,vii). Combining robust anchoring and strongly hydrophilic moieties yields QDs with nearly universal aqueous solubility; a PEGylated ligand with a tetradentate thiol moiety (Figure 3C,ix) was recently developed to provide long-term stability between pH 1
14 and at ionic strengths as high as 2 M.21 An important goal in developing coatings is minimizing the QDs hydrodynamic size while still retaining stability and biocompatibility. Thus, zwitterionic ligands (Figure 3C,iii,vi) are emerging as alternatives to larger PEG ligands as they can provide colloidal stability across biologically relevant ranges of pH/ionic strength, minimize nonspecific interactions and concurrently provide more compact hydrodynamic dimensions.22,23 In general, the disadvantage of a ligand exchange is a decrease in QY relative to the native QDs in organic solvent. The exchange of the hydrophobic synthetic coordinating ligands with a hydrophilic ligand through mass-action (i.e., ligand exchange) can potentially result in unpassivated sites that promote PL quenching. Although refinements of ligand-exchange methods can limit quenching,24,25 the best strategy for preparing aqueous QDs with a minimal decrease in QY is by encapsulation of the native QD inside an amphipol. Analogous to the use of phospholipid micelles, the association of an amphipol with the QD is driven by hydrophobic interactions between the native ligands and interdigitated aliphatic polymer side chains. For example, poly(acrylic acid) grafted with alkyl amines, or an alternating 8828

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Figure 3. (A) Ligand binding at the QD surface. (B) Association of an amphipol (blue) with the native QD ligands (red). (C) Ligand chemistries (i) thioalkyl acids, (ii) PEGylated ligands, (iii) zwitterionic ligands, (iv) dihydrolipoic acid ligands and (v) PEGylated, (vi) zwitterionic and (vii) modular derivatives thereof, (viii) multidentate charged and (ix) multidentate PEGylated ligands. (D) Amphipol coatings (i) phospholipid micelles, (ii) hydrophilic polymer backbones grafted with alkyl chains, (iii) triblock copolymers, and (iv) alternating copolymers that hydrolyze to acids or (v) are grafted with PEG chains. (E) Copolymers with pendant PEG oligomers and (i) dithiol or (ii) imidazole groups. Discrete moieties for (a) QD binding, (b) solubility, and (c) bioconjugation are identified where applicable (green). The arrows illustrate a conceptual progression and not synthetic pathways or chronological development. 8829

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copolymer of maleic anhydride and a long-chain terminal alkane, can provide charge stabilization in aqueous solution26,27 or be further grafted with aminated PEG oligomers (Figure 3D).28,29 The trade-off for an amphipol coating is a relatively large hydrodynamic diameter. However, coatings have recently been developed where the hydrophobic side chains are replaced with pendant QD-coordinating groups such as an imidazole or dithiol (Figure 3E).30,31 This represents a convergence of ligand coatings with the functional diversity of copolymer synthesis, exploiting direct coordination to the QD to decrease hydrodynamic dimensions while using discrete building blocks to modularly address the criteria for an ideal coating.

’ QUANTUM DOT BIOCONJUGATION Bioconjugate chemistry is essential to most biological applications of QDs, and the resulting structure
function relationship cannot be understated. The tools most widely used for preparing QD bioconjugates borrow from conventional protein labeling chemistry and generally target amine, carboxyl, and thiol groups via carbodiimide coupling, succinimidyl esters, and maleimides.14 However, researchers are recognizing that these chemistries are poorly controlled and prone to cross-linking/aggregation along with mixed conjugate valence, all of which result in reduced biomolecular activity and mixed avidity.14 Here again, there are several ideal design criteria for QD bioconjugates:14 (1) control over the number of biomolecules per QD (i.e., conjugate valence), (2) control over biomolecular orientation and position relative to the QD, (3) conjugate stability (or selective lability), (4) retention of biological activity, and (5) reproducibility and homogeneity. Conjugate valence can be important in identifying and correlating single binding events or, alternatively, improving binding avidity. Biomolecular orientation determines the accessibility of binding or catalytic sites, while the position relative to the QD can be important in distance-dependent processes such as F€orster resonance energy transfer (FRET) or charge transfer (CT). Stable conjugation is important to ensure longevity in nonequilibrium conditions, although controlled release may be desirable in theranostic applications. One of the most successful alternatives to labeling QDs with covalent or biotin
avidin chemistries is based on dative selfassembly.14 Biomolecules appended with polyhistidine tags spontaneously coordinate, via metal-affinity interactions driven by histidine’s imidazole side-chain, directly to ligand-coated CdSe/ZnS QDs. This strategy has been utilized with recombinant proteins, synthetic peptides, and modified oligonucleotides.14 Polyhistidine assembly is also compatible with ligand or amphipol coatings that can chelate nickel(II) mutually with the histidine residues in a manner akin to immobilized metal ion affinity chromatography.14,32 In addition to self-assembly, intein-mediated chemical ligation and several enzyme-catalyzed ligation chemistries have recently been demonstrated for QD bioconjugation; the latter include the use of biotin ligase, acyl carrier proteins, and the HaloTag system.14 However, the greatest utility arguably lies in developing highly chemoselective and bioorthogonal chemical reactions for use with aqueous dispersions of QDs, such as strainpromoted azide-alkyne cycloaddition, alkene-tetrazine ligation, hydrazone and oxime ligation, and the Staudinger ligation. Importantly, the combination of chemoselectivity and bioorthogonality may allow multiple reaction chemistries to be used (in series or in parallel) to prepare QDs that display several different biomolecular functions, where each has a controlled and well-defined linkage.

Figure 4. (A) Composite experimental PL spectra (green) with deconvolved contributions of eight different QD colors and the overall model fit (black). (B) QD-barcode immunoassay system: (i) measurement platform, (ii) PL spectra for three simple barcodes and inset showing an image of the corresponding QD-doped microspheres (scale bar = 15 μm), and (iii) two-color barcode (orange arrows) for probe identification and blue dye emission (blue arrow) to signal target (e.g., HIV antigen) binding. Figures adapted from refs 34 and 41. Copyright 2007 and 2009, respectively, American Chemical Society.

One of the biggest hindrances to advancing QDs in bioanalysis appears to be the degree to which developing coatings and bioconjugate reactions has lagged behind the interest in harnessing their optical properties. Fortunately, bioconjugate chemistry is beginning to catch up to the needs of specific QD applications (see ref 14 for a recent review).

’ MULTIPLEXED ASSAYS USING QUANTUM DOTS Despite the generally superior brightness of QDs, one-to-one substitution for a molecular fluorophore in an in vitro assay may not be particularly advantageous. The greater impact of QDs is in multiplexed assays, where their optical properties can simplify and expand diagnostic panels. The ability to excite multiple QDs with one excitation source and/or select an arbitrary QD color, greatly facilitates technical implementation of multiplexing. 8830

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Analytical Chemistry Similarly, narrow QD emission enables spectral encoding of different analyte signals rather than spatial encoding of different biomolecular probes. This is complemented by the availability of Gaussian peak-fitting algorithms that can resolve overlapping QD PL profiles, although often unnecessary with three or fewer spectrally resolved QD colors. For example, Goldman et al. developed a four-plex toxin immunoassay for simultaneously detecting ricin, staphylococcal enterotoxin B, shiga-like toxin I, and cholera toxin within the same assay sample using QDantibody conjugates as reporters; each toxin was associated with a different color of QD.33 Such a compact and parallel multiplexed measurement is not easily achieved with conventional, broadly emitting organic fluorophores. To further highlight the potential of spectrally multiplexed formats, it was recently demonstrated that eight-different QD PL profiles could be simultaneously resolved across the visible spectrum (Figure 4A),34 potentially providing access to multiplexing levels well beyond the capability of molecular fluorophores. The addition of more QD colors across a broader spectral range (i.e., 450
850 nm) could enable 12
15 detection channels. As reviewed by Pumera,35 there has been much interest in using QDs and other nanomaterials within miniaturized diagnostic platforms that have intrinsic analytical advantages of their own.36,37 In particular, QD optical properties are synergistic with developing nano/microfluidic assays that are based on spectral multiplexing rather than complex patterning of channels. For example, Jokerst et al. demonstrated proof-of-concept by replacing spatially resolved detection in chip-based immunoassays with spectrally resolved detection.38 This was accomplished using one microsphere coderivatized with different antibody probes and labeled with two colors of QD reporter. The convergence of microtechnology and QDs has also been applied to highly multiplexed bioanalyses using QD barcodes. At the cost of requiring single-particle analysis, barcodes offer the potential for “deep” multiplexing by combining PL color and intensity to spectrally encode biomolecular probes.39,40 Typically, between two and four different colors of QD are incorporated into a microparticle at controlled ratios to generate a library of barcodes. The overall multiplexing capacity is determined by the number of QD colors, m, and intensity levels, N, as Nm
1 and reach ∼102 for two- and three-color systems at ten- and fiveresolvable intensity levels, respectively.34,40 While barcodes with molecular fluorophores are typically limited to two color dimensions and require dual-wavelength excitation, QDs enable suspension arrays that require only single wavelength excitation and less intensity resolution by using more colors. The Chan Laboratory recently demonstrated QD-barcode assay panels for immunogenic41 (Figure 4B) and genetic42 biomarkers of globally prevalent blood-borne infections, including HIV, hepatitis B/C, syphilis, and malaria in up to a nine-plex format. These diagnostic panels used either a microfluidic chip or flow cytometer for analysis with optical detection, required sample volumes of only 100
200 μL and 10
60 min to complete the assay, and provided better sensitivity than methods currently approved by the U.S. Food and Drug Administration.

’ BIOSENSING WITH QUANTUM DOTS We make a distinction between QD-based assays and biosensors in that the latter exploit the QD as a scaffold for the assembly of biomolecular probes and provide a form of active signal-transduction based on modulation of QD PL intensity (rather than changing

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Figure 5. (A) QDs are good FRET donors for fluorescent protein (FP), dye, and gold nanoparticle (Au NP) acceptors. The dashed circle represents an arbitrary F€orster distance (R0) measured from the QD center. The scale at the right indicates how R0 proportionally increases as the number of proximal acceptors (a) increases. Conversely, QDs can function as acceptors for terbium complexes and bioluminescent luciferase donors. (B) Qualitative spectral overlap (shaded) for a 625 nm emitting CdSe/ZnS QD as (i) donor to fluorescent dye acceptor (Alexa Fluor 647, A647) and (ii) acceptor to a terbium chelate donor. (C) CT quenching is an alternative method of modulating QD PL: (i) an electron acceptor (e.g., quinone) has an unoccupied energy level intermediate in energy to the 1Sh and 1Se band-edge states to which the excited QD transfers an electron, (ii) an electron donor (e.g., ruthenium phenanthroline) has an occupied intermediate energy level and transfers an electron to the QD. Charge transfer inhibits radiative recombination of the exciton. Both redox active species are illustrated as peptides conjugates.

the number of QDs interrogated). Although CT-based sensing is growing in interest (vide infra), FRET is still the most common mechanism for modulating QD PL and perhaps the best characterized (Figure 5). QDs are ideal FRET donors and can be excellent acceptors when paired with appropriate donors.11,12 Although the F€orster model was originally derived for energy 8831

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Figure 6. (A) FRET detection of proteolysis: (i) 550 nm emitting QDs assembled with a fluorescent protein, mCherry, through a terminal peptidyl caspase-3 cleavage site and polyhistidine tag to yield FRET; caspase-3 activity disrupts FRET. (ii) PL changes vs mCherry acceptor valence per QD donor. (iii) Analysis of caspase-3 Michaelis
Menten kinetic parameters with the mCherry-peptidyl substrate. (B) Example of a FRET QD-acceptor fiveplex. Time-gated (50
450 μs) PL spectrum showing Tb-donor FRET-sensitized 529 (blue), 565 (green), 604 (orange), 653 (red), and 712 nm (brown) QD emission. Reprinted with permission from ref 43. Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA. (C) Immobilized QDs for DNA FRET sensing: (i) green, 530 nm emitting QDs (gQD) and red, 622 nm emitting QDs (rQD) immobilized on an optical fiber and coated with NeutrAvidin (NA)/bovine serum albumin (BSA) and biotinylated probe oligonucleotides. QDs transfer energy to corresponding acceptor dyes (Cy3 and Alexa Fluor 647, A647) when probe hybridizes with the target. (ii) PL spectra for two-plex detection. Parts A and C were adapted from refs 45 and 52. Copyright 2009 American Chemical Society.

transfer between two molecules approximated as point dipoles, it has been repeatedly verified as a good empirical description for FRET with QD donors and molecular chromophore acceptors.12 Dipole
dipole energy transfer between QDs and gold (or other metal) nanoparticles is conceptually similar but is not mechanistically FRET. Nevertheless, these interactions are also very useful since they appear to extend the range of energy transfer to significantly larger distances than classical FRET. An increasing number of researchers are interested in exploiting QDs as FRET donors since they offer several intrinsic advantages that are cumulatively unavailable to molecular fluorophores in the same role. These include (1) high QYs to maximize F€orster distances, (2) tuning of FRET efficiency by

controlling the number of acceptors assembled in a centrosymmetric array around each QD (Figure 5A). This is conceptually summarized by eq 1, where E is efficiency, r is the separation distance between the donor and a equivalent acceptors, and R0 is the characteristic F€orster distance (donor
acceptor separation corresponding to 50% efficiency). In contrast, molecular fluorophores do not provide an intrinsic interface for the assembly of multiple acceptors. E¼

aR0 6 r 6 þ aR0 6

ð1Þ

(3) Narrow and size-tunable QD PL that allows tuning and optimization of the spectral overlap integral (Figure 5B) while 8832

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Analytical Chemistry minimizing crosstalk between measurement channels for donor/ acceptor emission. This is not possible with the fixed and broader, red-tailed emission of molecular fluorophores. (4) Flexibility in wavelength selection for QD excitation, thereby allowing minimal direct excitation of acceptors. This can also be achieved through much more efficient two-photon excitation of QDs compared to dye acceptors. (5) Ready implementation of multiplexed FRET configurations since different QD colors can be simultaneously excited at a common wavelength far removed from their respective PL emissions. While the use of QDs as donors is quite general, QDs are only suitable acceptors for a small set of potential donor pairings. FRET requires an excited state donor in close proximity to a ground state acceptor. However, the PL decay time of QDs (>10 ns) is generally longer than that of most molecular fluorophores (microseconds), or molecular fluorophores that can be excited by nonoptical means, for example, chemically as in bioluminescence. As demonstrated by Hildebrandt43 and Rao,32,44 QD acceptors offer several advantages over their molecular counterparts including (1) large spectral overlap integrals due to the broad and strong QD absorption (Figure 5B), (2) FRETsensitized acceptor PL exhibiting a large bathochromic shift compared to the donor emission, potentially maximizing spectral resolution and sensitivity toward low FRET efficiencies, (3) access to multiplexed configurations with a common donor and multiple spectrally distinct QD acceptors (see also Figure 6B). Whether the QD is a donor or acceptor, transduction is generally driven by associative/dissociative biomolecular interactions that alter the proximity needed for efficient FRET. In practice, this is measured as changes in donor quenching, FRETsensitized acceptor PL, or the ratio thereof. For example, protease activity was recently monitored by the disruption of a QD-FRET complex. Boeneman et al. quantitatively measured the activity of caspase-3 (a cysteine protease important in apoptosis and down regulated in many cancers) by using QD donors self-assembled with mCherry fluorescent protein acceptors (Figure 6A).45 The mCherry displayed an N-terminal peptide linker incorporating a caspase-3 recognized cleavage sequence between the protein and a polyhistidine tag used for QD attachment. Further, changes in FRET measured for the QD-mCherry assembly following proteolysis could be converted into units of enzyme activity. Peptidyl substrate bridges between a QD and a fluorescent dye acceptor46 or a bioluminescent luciferase donor32 have been similarly used to detect the activity of other proteases. The QD-FRET sensing paradigm is rapidly growing, and a variety of assemblies have been utilized for detecting dozens of diverse bioanalytical targets, including toxins, explosives, cofactors, nutrients, metabolites, drugs, ions, nucleic acids, proteins, and enzymatic activity.11,12 Multiplexed detection is also possible with FRET sensing by combining different QD donors with a common broad-spectrum dark quencher or multiple fluorescent dye acceptors.12 Alternatively, luciferase44 or lanthanide43 donors can be paired with different colors of QD acceptor (Figure 6B). An important advantage of QD-FRET is that each QD can operate as a selfcontained analytical platform suitable for both in vitro and intracellular sensing at detection levels ranging from single assemblies to macroscopic ensembles. Moreover, the QD surface is amenable to the assembly of multiple functional components.

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For example, coderivatization of QD-fluorescent protein assemblies with cell-penetrating peptides enabled their intracellular delivery via endocytic uptake.47 Combining signal transduction with a nanoscaffold for carrying targeting moieties and other molecular cargos is the primary motivation behind developing theranostic and other “smart” bioanalytical probes, a challenge perhaps ideally suited to QDs.13 The development of reusable, in vitro biosensors offering high-throughput or continuous monitoring represents another evolution of QD technology. Research in this area has been limited by the challenge of immobilizing QDs in a functional manner. In a biosensor configuration where QDs are integrated and nondisposable, the QDs must be immobilized at an interface but remain accessible for further bioconjugation, biomolecular binding, and efficient PL modulation. Immobilization provides opportunities for near-field optical interrogation and other enhancement strategies, while potentially representing a “greener” approach to the use of QD materials. Biomolecular interfacial tethers, such as proteins, peptides, and oligonucleotides have been used to immobilize QDs and create FRET-based sensors targeting nutrients and nucleic acids.48
50 Oligonucleotide tethers further provide programmable and reversible immobilization of QDs through hybridization.50 As an alternative to biomolecular tethers, an interfacial ligand exchange method for QD immobilization was developed as the basis for assembling QD-FRET nucleic acid sensors.51 One such example is shown in Figure 6C, where two colors of QD donor were coimmobilized and derivatized with two different oligonucleotide probe sequences to enable two-plex detection upon hybridization with acceptor-dye labeled targets.52 Similar to assays, the impact of QDs on immobilized biosensors is expected to be in tandem with lab-on-a-chip devices that are ideally suited to exploit their advantages for spectral multiplexing in parallel with automated handling of small volumes, improved stringency, and/or other advantages inherent to microanalytical platforms.35

’ CELL STUDIES AND THERANOSTICS In the study of cellular processes, there is a continuous need for improved methods of molecular tracking and correlation of multiple intracellular events in real-time. Fluorescence microscopy is capable of sensitive detection at rapid frame-rates with excellent spatial resolution but is limited by the poor brightness (requiring long integration times) and photobleaching (resulting in short observation times) of most molecular fluorophores. In contrast, a bright and photostable QD can enable spatial and temporal tracking of a single emitter over longer time scales and enable novel experimental formats. In an elegant example of exploiting single-QD tracking analysis, Lowe et al. performed experiments to reveal the translocation of receptor-cargo complexes through the nuclear pore complex (NPC), which is a critical eukaryotic cellular process.53 QDs were modified with ∼40 copies of the importin-β binding (IBB) domain of snurportin-1 (the import receptor for small nuclear ribonucleoproteins), and the rejection or import of single QD cargos by the NPC in HeLa cells was observed with time-resolved imaging (Figure 7A). The authors were able to develop a model of a multistep translocation process comprising (i) cargo capture at the outer NPC, (ii) sizeselective filtering of cargos within the NPC channel, and (iii) translocation with cargo release into the nucleus. QD-bioconjugates have been similarly applied to revealing the dynamics of G-protein-coupled receptor endosomal trafficking (Figure 7B),54 8833

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Figure 7. (A) Bright-field image of a HeLa cell nucleus with PL images of single 605 nm emitting QD-snurportin IBB cargos (boxed) overlaid in red. Frames from single particle tracking (at right) reveal arrival from cytoplasm and departure of cargo into the nucleus. Figure adapted with permission from ref 53. Copyright 2010 Macmillan Publishers Ltd. (B) Kinetic monitoring of 655 nm emitting QD-tagged serotonin receptors. Plasma membrane labeled green and QD-receptor complexes within endosomes appear red (scale bar = 10 μm). (C) Image of a live neuronal cell axon with four endosomes labeled red using 605 nm emitting QD-NGF assemblies. Figures adapted with permission from refs 54 and 55. Copyright 2007 and 2010, respectively, National Academy of Sciences U.S.A. (D) Monitoring APP trajectory and dimerization in neuronal cell membranes by labeling with mixtures of 655 nm emitting (red) and 585 nm emitting (green) QDs: endocytosis (/), internalized monomers and dimer (ellipse), external dimer (boxed). Figure adapted with permission from ref 56. Copyright 2009 American Society for Biochemistry and Molecular Biology. (E) QD-aptamer conjugates for the targeted delivery of Dox to prostate cancer cells: (i) upon endocytosis, Dox is released intracellularly and both QD and Dox PL are turned “ON”; images showing QD (green) and Dox (red) PL (ii) immediately and (iii) 1.5 h after delivery (scale bar = 20 μm). Figure adapted from ref 57. Copyright 2007 American Chemical Society. (F) Two-photon images of QD-labeled dendritic cells (red) inside draining lymph nodes. T-cells are dye labeled (green). Figure adapted with permission under the Creative Commons Attribution License from ref 58. Copyright 2008 the authors.

resolving the stop-and-go retrograde trajectory of nerve-growth factor-containing endocytic vesicles along axons (Figure 7C),55 and following the trajectory/dimerization of amyloid precursor protein (APP) in the plasma membrane over several minutes (Figure 7D).56 Clearly, reliably tracking discrete molecular events over long time periods allows the observation of subtle yet important mechanistic details that are otherwise lost in the ensemble. In a theranostic context, QDs can be used as a potent targeting and visualization probe while concurrently carrying a therapeutic payload, thus highlighting the added value of the QD surface area. Bagalkot et al. developed a QD-aptamer-doxorubicin (Dox) conjugate capable of simultaneously targeting prostate cancer cells and signaling drug delivery in vitro (Figure 7E).57 The aptamer was selected to bind prostate specific membrane antigen, and this stimulated subsequent endocytosis and uptake of the QD conjugates by target cells. Intracellular release of the Dox, which had quenched the 490 nm emitting QDs within the assembly via FRET, generated a light up signal that could be

directly monitored. Importantly, this QD-based architecture minimally interfered with the cytotoxicity of Dox toward cancer cells but substantially decreased cytotoxicity toward healthy cells. In another embodiment that exploited QD-surface area with high-avidity display and strong PL, Sen et al. decorated QDs to both stimulate and track dendritic cells in lymph nodes during an immune response (Figure 7F). 58 Streptavidinconjugated 655 nm emitting QDs were decorated with an average of 10 biotinylated ovalbumin (ova) as an antigen. Following subcutaneous injection into BALB/c mice, T cells clustered around the dendritic cells that had bound the QD-ova conjugates and allowed tracking of these clusters over time. They also found a significant upregulation of immune markers in comparison to controls exposed to equivalent amounts of free antigen. In this case, the QD served a multitiered role as (i) an efficient-central NP scaffold, (ii) an antigen delivery system, (iii) a high-avidity antigen construct, (iv) a primer of an augmented in vivo immune response, and (v) a long-term PL tracer of dendritic/T-cell clusters.58 8834

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Analytical Chemistry The finite volume of the QD can also offer extra value and new opportunities. In a prominent example, Zhang et al. recognized that both the tracking capability and size offered by single QDs were ideal for elucidating the vesicular secretion of neurotransmitters, which is a process essential to efficient neuronal communication.59 “Kiss-and-run” (K&R) is a mode of membrane fusion and retrieval that occurs without collapse of the neurotransmitter containing vesicle into the plasma membrane. Although prior evidence suggested K&R occurs in mammalian nerve terminals, the use of fluorescent dyes for tracking yielded poor signal-tonoise ratios that did not unambiguously differentiate K&R from other mechanisms. To this end, individual synaptic vesicles were loaded with single 605 nm emitting CdSe/ZnS QDs that were simultaneously small enough to fit into the endocytic vesicle lumen (∼24 nm), yet large enough to be unable to traverse putative K&R fusion pores (∼1
5 nm). Tracking the QDs, in combination with a small but measurable change in PL (15%) between vesicular and extracellular pH (5.5 vs 7.3), allowed multiple rounds of K&R and vesicle reuse to be monitored, demonstrating not only that K&R occurred but that its prevalence relative to other mechanisms was condition dependent.

’ QUANTUM DOT TOXICITY Concerns about the potential toxicity of QDs are pervasive and generally rooted in the Cd constituent of the CdSe/ZnS QDs most prominently used in research. However, toxicity does not hedge only on the inclusion or elimination of Cd. Assessments of QD toxicity must consider all three material layers of the QD, the core, shell, and organic coating, and quality thereof, highlighting the challenge associated with the lack of a single, standard, and reproducible QD structure. Further, the context of each application is equally important since it is both dose and exposure that effectively determine toxicity. Botrill and Green60 have recently reviewed many aspects of this complex debate, which might be perceived as the largest hill in the “valley of death.” Pragmatically, potential QD toxicity does not impact the development of in vitro bioanalyses, where QDs are handled with the same precautions as other hazardous materials. In the context of ex vivo cellular studies, toxicity is only relevant insofar as it causes a perturbation over the time scale of experiments. The growth of high-quality ZnS shells around heavy metal chalcogenide cores and the use of biocompatible coatings generally overcome this challenge. In vivo applications are tremendously complicated by QD circulation, accumulation, degradation, and clearance within an organism and beyond our scope here. Nonetheless, to put the debate into a simplified context based purely on toxicity, chemotherapeutic agents and radioactive contrast agents are hardly benign but are nonetheless part of clinical practices by virtue of the greater benefits than liabilities afforded in specific contexts—should not QDs be given similar consideration if they can pass regulatory review for a particular application? ’ LOOKING AHEAD As an alternative label to molecular dyes, QDs are already relatively well established; the current state-of-the-art is primarily limited by the nuances of bioconjugation chemistry. Although still a challenge for bioanalytical development, available chemistries are rapidly improving, allowing the advantages of QD surface area to be maximized.14 QD-based assays and biosensors will undoubtedly progress to fill a niche

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within in vitro diagnostics; however, QDs have the opportunity to make a far-reaching impact in cellular bioanalysis. The necessary foundation has already been laid in developing QDbased transduction strategies for a variety of (bio)analytes. Although the design of “active” QD probes has been largely monopolized by FRET, sensing configurations based on CT quenching of QD PL by proximal redox-active complexes (e.g., ruthenium phenanthroline, quinones; Figure 5C) have been recently developed and include an intracellular pH sensor.11,61,62 CT quenching acts analogously to a dark quencher in FRET but does not require spectral overlap and has a stronger distance dependence. Further developments could ultimately lead to the use of QD-CT quenching as a powerful signal transduction modality for probing redox-active metabolic processes. The most pressing challenge for intracellular sensing is reliably delivering active QD constructs to the cellular cytosol. Facilitated delivery methods that decorate QDs with peptides, proteins, or small molecules to stimulate endocytosis hold the most longterm promise.63 However, in most cases the QDs are taken up into the endolysomal system and tend to remain sequestered there. Robust methods must be developed to mediate intracellular uptake and delivery of QDs (with associated cargos) to the cellular cytosol and other targeted subcellular sites. It will be equally critical to evaluate to what degree biomolecular probes and targeting moieties coassembled with the QD will remain active. In the short term, active delivery methods (e.g., microinjection) may be more suitable for evaluating the activity, specificity, longevity, and side effects of QD probes within the cytosol.13,63 In summary, a fervent pace of research into developing new QD materials, surface coatings, bioconjugate chemistries, and applications continues more than a decade after the introduction of QDs to bioanalysis. No longer nascent, the field is still in an important period of transition across “the valley of death” that will ultimately define the range of bioapplications for which QDs will be utilized. In the sense that QDs are still being newly introduced to many researchers, they do remain a scientific curiosity. It should be noted that there remains a learning curve for initial users that has likely tempered the growth of QDs in bioanalysis relative to its full potential. The cost of commercial QD materials is not prohibitive but can be sufficient to curtail projects that suffer early setbacks. Synthesis of QDs in-house provides much greater flexibility but is a substantial time and resource investment that often motivates collaborations between groups with separate expertise in materials and applications. Moreover, since QD compositions and synthesis are not strictly defined and properties are sensitive to defects, a common hurdle is rationalizing batch-to-batch irreproducibility with a common QD material. Quality control for QD size, shape, elemental composition, and PL properties are important for ensuring the continuity of materials between batches and studies. In a sense, each laboratory thus traverses its own “valley of death” as it works to develop a set of standard protocols and emerge with a desired QD application. More sophisticated applications require the navigation of more complex valleys; however, this is supported by an ever growing array of tools and knowledge that results in progressively greater impact. Clearly, QDs have developed into bona fide tools for multidisciplinary bioanalytical research and assay development, with unrivaled capabilities arising from a unique combination of their optical, electronic, and physical characteristics. 8835

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’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ BIOGRAPHY Russ Algar is a postdoctoral researcher with Igor Medintz at the Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory through the College of Science at George Mason University. Trained as an analytical chemist, his current research interests include developing multiplexed and multifunctional nanoparticle systems for optical diagnostics, quantitative intracellular measurements, and theranostics; he is also interested in biophysics of molecular recognition at nanoparticle bioconjugates. He can be contacted by e-mail at russ. [email protected]. Kimihiro Susumu is a research chemist in the Optical Sciences Division at the Naval Research Laboratory through Sotera Defense Solutions. His research has focused on the development of biocompatible quantum dots for biosensing and imaging technologies. Specifically, he has focused on the design and synthesis of a series of multifunctional surface coating ligands to enhance the biocompatibility of quantum dots and gold nanoparticles. Contact Kimihiro Susumu at the Naval Research Laboratory, Washington, DC 20375; e-mail [email protected]. James B. Delehanty is a Research Biologist at the Naval Research Laboratory. Trained as a molecular and cellular biologist, his research interests include the development of nanoparticle-based constructs for biological sensing and imaging. A primary area of current research is the development of facile, noninvasive strategies for the delivery of nanoparticles to targeted regions/structures within mammalian cells for the purpose of intracellular sensing, imaging, monitoring, and actuation. He can be contacted at the U.S. Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375; e-mail [email protected]. Igor L. Medintz is a Research Biologist at the Naval Research Laboratory. His formal training is in molecular and cellular biology. Current research interests include developing chemistries to bridge the nanoparticle-bioconjugate interface, elucidating how nanoparticle-bioconjugates can function in a cooperative manner and understanding how nanoparticles engage in energy transfer. Contact him at the Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375; e-mail [email protected]. ’ ACKNOWLEDGMENT The TEM image in Figure 1 was kindly provided by Eunkeu Oh, U.S. Naval Research Laboratory. W.R.A. is grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC) for support through a postdoctoral fellowship. The authors acknowledge the NRL-NSI, ONR, DTRA, and DARPA for financial support. ’ REFERENCES (1) Rossetti, R.; Nakahara, S.; Brus, L. E. J. Chem. Phys. 1983, 79, 1086–1088. (2) Steigerwald, M. L.; Alivisatos, A. P.; Gibson, J. M.; Harris, T. D.; Kortan, R.; Muller, A. J.; Thayer, A. M.; Duncan, T. M.; Douglass, D. C.; Brus, L. E. J. Am. Chem. Soc. 1988, 110, 3046–3050.

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