Covalent Monofunctionalization ofPeptide-Coated Quantum Dots forSingle-Molecule AssaysSamuel Clarke,† Fabien Pinaud,† Oliver Beutel,‡ Changjiang You,‡ Jacob Piehler,‡ andMaxime Dahan*,†

†Laboratoire Kastler Brossel, CNRS UMR 8552, Departement de Physique et Biologie, Ecole Normale Superieure,Universite Pierre et Marie Curie (Paris6), 46 rue d’Ulm 75005 Paris, France, and ‡Universitat Osnabruck,Fachbereich Biologie, Barbarastrasse 11 49076 Osnabruck Germany

ABSTRACT Fluorescent probes for biological imaging of single molecules (SM) have many stringent design requirements. In thecase of quantum dot (QD) probes, it remains a challenge to control their functional properties with high precision. Here, we describethe simple preparation of QDs with reduced size and monovalency. Our approach combines a peptide surface coating, stable covalentconjugation of targeting units and purification by gel electrophoresis. We precisely characterize these probes by ensemble and SMtechniques and apply them to tracking individual proteins in living cells.

KEYWORDS Quantum dots, single molecules, particle tracking, cell imaging, fluorescent probes

Single-molecule (SM) fluorescent imaging techniquesand tools represent a powerful approach for investi-gating the behavior of individual biomolecules in vitro

and in live cells.1 Yet, the conditions imposed by biologicalmilieus necessitate the design of photostable and highbrightness SM probes. Fluorescent quantum dots (QDs) areinorganic semiconductor nanoparticles (∼2-10 nm) thatmeet these criteria and overcome numerous other opticallimitations associated with common organic probes.2,3 Assuch, they have found numerous applications for imagingSM events.4,5 In vitro, this includes assays for studying thedynamics of enzymes on DNA,6 the function of motorproteins7 or biosensing via Forster resonance energy trans-fer (FRET),8-10 among others. Long-term, single QD tracking(SQT) of biomolecules in living cells is providing fresh insightinto the organization of the plasma membrane,11 the traf-ficking of receptor ligands,12 the dynamics of neurotrans-mitter receptors13 and many other open questions in cellbiology.5

Thus far, commercial QD probes conjugated to multiplecopies (∼8-12) of proteins like streptavidin (SAV) or anti-bodies have enabled the majority of QD experiments at theSM level. Their wide availability and compatibility withvarious targeting schemes has favored their use in manyapplications. These probes are generally constructed fromQDs encapsulated in amphiphilic polymers or micelles,whichhasbeenapopularroutetoobtainbiocompatibility.14-18

While this design facilitates good conjugate stability and

enables specific targeting, it suffers from high multivalencyand a large hydrodynamic diameter (HD) in the range∼25-40 nm,19-22 depending on the particular surfacecoating and conjugated proteins. Already, several exampleshave shown how the large size of these QD probes can alterthe kinetics and equilibrium of molecular interactions,23

influence the diffusion of receptors in the cell membrane,24,25

prohibit access to size-restricted cellular regions22,24 andlimit FRET efficiency toward acceptors in close proximity.9,10

At the same time, the multivalency of QD probes has beenshown to trigger undesirable cell signaling events by cross-linking target receptors22 and to complicate the analysis ofdata from SM assays.8,10,26

In recent years, the challenge of designing improved QDprobes for SM imaging has resulted in new surface coatings,conjugation strategies and purification techniques. For in-stance, compact surface coatings have been developed toreduce the size, but also maintain effective shielding againstnonspecific interactions. Poly(ethylene glycol) (PEG) has beenshown as a key ingredient to reduce nonspecific interactions27,28

and has been incorporated into dihydrolipoic acid (DHLA)derivatives22,28-30 and compact polymers30-32 to produceQDs with a HD in the range of 8-18 nm. Similarly, surfacecoatings based on PEGylated peptides containing 6-20amino acid residues can also yield QDs with a HD of∼10-12 nm,11,33-35 having the added benefit that they areavailable from commercial vendors rather than requiringchemical synthesis. A purification technique to prepare QDswith reduced valency has also emerged,22,34,36,37 which wasinitially applied to prepare gold nanoparticles with definednumbers of DNA.38 It relies on stochastic conjugation of largetargeting units to the QD surface and then separation and

* To whom correspondence should be addressed. E-mail: maxime.dahan@lkb.ens.fr.Received for review: 03/8/2010Published on Web: 04/30/2010

pubs.acs.org/NanoLett

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purification of discrete stoichiometries of the complex viagel electrophoresis (GE). This technique has now beenapplied to prepare monovalent QD probes integrating DHLAor peptide surface coatings and a single copy of SAV,22 asingle chain antibody22 or a microtubule associated pro-tein,34 achieving a final probe HD of just 13-15 nm. Thesedesigns applied a noncovalent conjugation strategy relyingon the self-assembly of polyhistidine-tagged targeting pro-teins to divalent ions at the nanoparticle surface, which hasthe advantage that the proteins can be site-specificallyattached.39-41 However, a disadvantage is that the conjuga-tion is noncovalent, possibly leading to unstable com-plexes31 or resulting in the dissociation of the protein fromthe QD over time (off-rate, koff ∼ 10-4 s-1).40

Here, we report several advances in the design of reduced-size and valency QD probes for SM imaging. We show thatthe combination of a compact peptide surface coating, stablecovalent conjugation of biotin or SAV targeting units and GEpurification can yield monofunctional QD probes with highconjugate stability (>1 week), and a HD of ∼11-15 nm,which represents a reduction in diameter by a factor of 2compared to commercial QD probes prepared with anamphiphilic polymer coating. Importantly, our design re-duces the technical complexity associated with QD probepreparation, which should allow researchers from a widerange of backgrounds to apply them in different applications.

Using ensemble methods, we precisely characterize the size,activity, valency, and stability of the QD probes. We also usea SM assay to directly count the number of functionalbinding sites on individual QDs and we apply these probesfor tracking the diffusion of individual proteins in themembrane of living cells.

Design and Synthesis of pQDs. To prepare compact andbiocompatible QDs, we selected a surface coating based onengineered, two-domain peptides of ∼20 amino acid resi-dues in length.33,42 These peptides contain a cysteine-richadhesive domain that binds to the QD surface, cysteine, andlysine terminal amino acids that serve as reactive sites forconjugation and PEG to minimize nonspecific interactions(Supporting Information). Next, we exchanged the nativehydrophobic surfactants of green- (λem ) 520 nm) or red-emitting (λem ) 605 nm) CdSe/ZnS QDs with these peptides.The resulting peptide-coated QDs (pQDs) were highly solublein various aqueous buffers and they retained the character-istic optical properties of the original hydrophobic QDs(Supporting Information Figure S1), with a modest decreasein quantum yield from 34 to 25% (Supporting Information).

We determined the size of the pQDs by electron micros-copy (EM) after negative staining with uranyl acetate (Figure1a). The inorganic core/shell CdSe/ZnS nanoparticle wasrevealed by regions of high contrast, while the organicpeptide coating appeared as regions of lower contrast sur-

FIGURE 1. Physical characterization of peptide-coated QDs and a covalent conjugation scheme. (a) Histograms for the diameter of streptavidin(SAV) (N ) 301, 3.0 nm) obtained by cryo-EM (inset) and green-emitting pQD520 (N ) 141, 6.9 nm), red-emitting pQD605 (N ) 315, 9.4 nm),and commercial cQD-SAV probes (N ) 309, 23.1 nm) obtained by EM following negative staining (inset). In the QD samples, the organicsurface coating appears as a low contrast halo surrounding the core/shell nanoparticle. Scale bar ) 25 nm. (b) Conjugation scheme for pQDs.Scheme I: Bifunctional PEG linkers containing a thiol-reactive maleimide and a small targeting unit (e.g., biotin) are covalently conjugated tocysteine residues on the pQDs. Scheme II: Maleimide-activated antibodies or proteins (e.g., streptavidin) are formed with the bifunctionalcross-linker SMCC and covalently conjugated to cysteine residues on the pQDs.

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rounding the nanoparticle. The diameter of green-emittingpQDs was 6.9 ( 1.4 nm, while that of red-emitting pQDswas 9.4 ( 1.6 nm, owing to the larger nanoparticle size (∼5versus 2.5 nm). In both samples, the peptide coating was∼2 nm thick, contributing ∼4 nm to the overall diameter.For comparison, we also examined commercial, SAV-conjugated QD probes (cQD-SAV, λem ) 655 nm) containingan amphiphilic polymer-based surface coating. The cQD-SAVwere 23.1 ( 3.7 nm in diameter, more than twice the sizeof the pQDs, because of both a larger nanoparticle size andthicker surface coating (∼8 nm in diameter). Finally, we usedcyro-EM to determine the diameter of wild-type streptavidin(SAV), which was found to be 3.0 ( 0.6 nm (Figure 1a).

Covalent Conjugation of Discrete Numbers of TargetingUnits. Having established the compact size of the pQDs, wetested the ability to covalently conjugate discrete numbersof targeting units (Figure 1b). First, we selected biotin, toenable specific targeting toward SAV and avidin variants.Because of its small size (240 Da), direct conjugation of biotinto the pQDs did not enable us to separate different stoichi-ometries of the complex via GE (data not shown). Toincrease the effective size of biotin, we used a long, flexiblelinker between the biotin and the pQDs. A 10 kDa PEG linkercontaining a single biotin on one end and a single maleimideon the other end (biotin-PEG-mal) was selected to enableconjugation to the pQDs via thiol groups of cysteine residuesin the peptide coating. The pQDs were titrated with increas-ing amounts of the biotin-PEG-mal and analyzed by gelelectrophoresis (GE) after a short incubation period (Figure2a). In the absence of the biotin-PEG-mal, the pQDs migratedin a narrow band in the gel toward the positive electrode,consistent with their expected net negative charge.33 Theaddition of increasing amounts of biotin-PEG-mal led to theformation of new, discrete bands in the gel with a lowermobility than the unconjugated pQDs. These bands were

assigned to pQDs conjugated to different stoichiometries ofbiotin via the linker (N ) 0,1 and 2). The biotin on the endof the linker remained highly active after the conjugation,because the addition of a 50-fold molar excess of SAV to thebulk mixture led to a quantitative shift in migration of onlythe pQD bands containing biotin (N g 1) (Figure 2b). Wefurther explored the effect of linker length on the ability toresolve discrete bands by GE following conjugation to thepQDs. PEG-maleimide linkers of length 5, 10, and 20 kDawere reacted with the pQDs in a 3-fold molar excess. Anobvious trend emerged during GE of these samples; thedistance between the discrete bands in the gel increasedwith the linker length (Figure 2c). Clear separation betweenbands could not be seen when the linker was 5 kDa in length,imposing a practical size limit.

Given the positive results obtained with a small targetingunit, we extended the GE technique to prepare pQDs withdiscrete numbers of proteins. We selected the wild-type SAVprotein (∼56 kDa) to enable specific targeting of the probestoward biotinylated biomolecules. First, we prepared amaleimide-activated SAV by reacting free amines on theprotein with a 3-fold molar excess of the bifunctional cross-linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Figure 1b). As with the biotin-PEG-mal,titration of the SAV-maleimide with the pQDs led to theformation of discrete bands during GE of the samples,confirming their successful conjugation to the peptide coat-ing. We selected a ratio of SAV-maleimide to produce a bulkmixture of pQDs containing 0, 1, and 2 copies of SAV (Figure3a). To test the activity of the conjugated SAV, we reactedthis bulk mixture with a 50-fold molar excess of a largebiotinylated target, a 10 kDa biotin-PEG. Analysis by GErevealed a quantitative shift in migration of only the pQDbands containing SAV (N g 1), indicating a high level ofspecific activity retained by the SAV. We could purify thedifferent stoichiometries of the complex from the bulkmixture by cutting the desired bands out of the gel andeluting the pQDs from the agarose. Reanalysis of the samplescontaining 1 (pQD-SAV1) or 2 (pQD-SAV2) of the proteins perQD showed that they could be recovered with very highpurity (Figure 3b). Importantly, the purified pQD-SAV1 wasstable over storage periods exceeding one week at 4 °C, withno apparent dissociation of the SAV from the nanoparticlesurface, even in at nanomolar concentrations (Figure 3c).

Ensemble Characterization of pQD-SAV. We then de-termined the contribution of discrete numbers of SAV to theoverall hydrodynamic diameter (HD) of pQDs by both size-exclusion chromatography (SEC) and fluorescence correla-tion spectroscopy (FCS). In the case of SEC, we detected thefluorescent signal from the pQDs as they eluted from thechromatography column (Figure 3d) and determined theirsize with a set of protein standards of known HD (SupportingInformation Figure S2). For the unconjugated pQDs, the HDwas determined to be 14.0 nm. This measurement is ∼4 nmlarger than the diameter obtained by EM (9.6 nm) and

FIGURE 2. Formation of discrete stoichiometries of pQDs with biotinvia a linker. (a) Gel electrophoresis (GE) of pQDs covalently conju-gated to increasing amounts of a bifunctional 10 kDA biotin-PEG-maleimide. Discrete bands represent pQDs with N ) 0, 1, and 2copies of biotin. (b) GE of a bulk mixture of biotin-conjugated pQDsbefore (left) and after (right) the addition of excess SAV. There is alarge shift in migration for only bands corresponding to Ng 1 copiesof biotin, indicating the high level of specific activity retained bythe biotin at the end of the linker following conjugation to the pQDs(c) Effect of PEG linker length on the separation of differentconjugate stoichiometries during GE. Discrete bands are resolvedin the case of the 10 and 20 kDA PEG-maleimide, but not for the 5kDA PEG- maleimide linker. All samples were loaded at the startposition and the direction of applied voltage is indicated.

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accounts for hydrodynamic and hydration effects in solution.In the case of pQD-SAV1, conjugation of a single SAVincreased the HD by ∼1.6 to 15.6 nm. Additional HDmeasurements on these samples were also performed usingFCS, where the mean HD was calculated from autocorrela-tion functions (Figure 3e). These results were consistent withthe SEC measurements with a HD of 12.8 nm for theunconjugated pQDs and 16.0 nm for pQD-SAV1, represent-ing an increase of 3.2 nm due to the SAV. In addition, weused SEC and FCS to measure the HD of a broader sampleset, including pQDs with 2 copies of the SAV and cQD-SAV(Supporting Information Figure S2). These results indicatea progressive increase in HD of the pQDs following conjuga-tion of discrete copies of SAV, and also highlight the fact thatpQD-SAV1 is less than half the HD of the cQD-SAV probes(∼15 nm versus 35 nm).

Single-Molecule Characterization of pQD-SAV. For thepurpose of measuring the precise number of biotin-bindingsites on pQD-SAV1, we designed a SM counting assay basedon the stepwise photobleaching of a biotinylated organicfluorophore.43,44 We synthesized a biotin derivative contain-

ing a single Alexa647 dye (biotin-A647, λem ) 673 nm) andmixed it in 100-fold molar excess with pQD-SAV1 beforeanalyzing and purifying the complex by GE (Figure 4a). Ingels, binding of biotin-A647 to pQD-SAV1 was confirmed bycolocalization of their fluorescent emission, while the excessunbound biotin-A647 migrated at a faster rate than the QD-dye complex. We confirmed that the binding of the dye wasspecific to the SAV and not the QD surface or coating,because it was eliminated by coincubation of pQD-SAV1 andbiotin-A647 in the presence of a 1000-fold molar excess offree biotin (Figure 4a). We then purified the QD-dye complexby extracting the colocalized region from the gel and depos-ited it on glass coverslips at nanomolar concentrations forfurther characterization.

To analyze this hybrid QD-dye complex, we used SMimaging by dual color total internal reflection fluorescence(TIRF). Individual QD-dye complexes, attached to the cov-erslip, were excited at 488 or 633 nm and the respectivefluorescent emission of pQD-SAV1 and biotin-A647 wereseparated and detected on different halves of the sameEMCCD camera (Figure 4b, Supporting Information Figure

FIGURE 3. Synthesis and characterization of pQDs conjugated to discrete copies of SAV. (a) GE of pQDs after covalent conjugation to SAV-maleimide (left lane). In this bulk mixture, there is a distribution of pQDs containing N ) 0, 1, 2 copies of SAV. After reaction with a biotinylatedtarget (right lane) there is a large shift in migration for bands with Ng 1, indicating the high level of specific activity retained by the pQD-SAVconjugates. (b) GE of pQDs conjugated to 0 (pQD605), 1 (pQD-SAV1), and 2 (pQD-SAV2) copies of SAV, which were purified from the bulkmixture. The SAV-conjugated pQDs retain their original position in the gel, indicating the good conjugate stability of the isolated samples. (c)GE showing the stability of the pQD-SAV1 after ∼1 week of storage at a concentration of 100 nM. No dissociation of the SAV from the surfaceof the pQDs is apparent, which would otherwise appear as a second band at the N ) 0 position. All samples were loaded at the start positionand the direction of applied voltage is indicated. (d) Size-exclusion chromatography (SEC) of streptavidin (elution at 10.3 mL, HD ) 4.8 nm),pQD605 (8.1 mL, 14.0 nm), pQD-SAV1 (7.9 mL, 15.6 nm), and pQD-SAV2 (7.7 mL, 17.1 nm) showing the size-effect of SAV conjugation. V0 isthe exclusion volume and VB is the void volume. HDs of the pQDs were determined by comparing the sample elution volume to a standardcurve formed from a set of protein standards. (e) Normalized autocorrelation functions, obtained by fluorescence correlation spectroscopy(FCS) of a fluorescein standard (HD ) 1.3 nm), pQD605 (12.8 nm), pQD-SAV1 (16.0 nm), pQD-SAV2 (17.0 nm), and cQD-SAV (34.8 nm). Theblack lines are raw data and the colored lines are the corresponding fit. HDs were determined by the Stokes-Einstein relation using thediffusion time at the inflection point of the curve.

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S1). Excitation of the pQD-SAV1 at 488 nm produced dif-fraction-limited fluorescent spots in the QD channel with thecharacteristic intermittency (blinking) of individual QDs.Under these conditions, many correlated spots also ap-peared in the dye channel, resulting from QD FRET to biotin-A647 (Supporting Information Figure S3). However, toaccurately count the number of biotin-A647 per pQD-SAV1,we applied direct excitation at 633 nm and collected time-

lapse images in the dye channel until all dye molecules hadphotobleached (∼30 s, Figure 4c). Individual QD-SAV1 werehighly colocalized (∼90%) with the dye molecules as verifiedby excitation at 488 nm (Figure 4c). In the dye channel,intensity time traces from individual spots showed consecu-tive photobleaching steps as expected from single or fewmolecules of biotin-A647 bound to pQD-SAV1 (Figure 4d).Counting the photobleaching steps permitted an evaluationof the number of biotin-binding sites on the pQD-SAV1. Weclassified ∼250 of the QD-dye complexes based on thenumber of observed steps and determined the distributionof the biotin-binding sites on individual pQD-SAV1 to be 1(41%), 2 (40%), 3 (15%), and 4 (4%) (Figure 4e). This resultsupports the notion that each pQD is conjugated to a singlecopy of the SAV protein, and that some the 4 biotin-bindingsites of the tetrameric, wild-type SAV are not accessible. Asa control, we found the distribution of steps associated withthe free biotin-A647 dye to be 1 (94%) and 2 (6%), asexpected (Supporting Information Figure S4). Alternatively,we determined the distribution of biotin-binding sites usinga histogram analysis of the maximum intensity of spots inthe dye channel, which yielded results similar to the step-counting analysis (Supporting Information Figure S4).

We also tested the effect of substituting the wild-type SAVwith a monovalent variant containing a single biotin-bindingsite (mSAV)45 and prepared pQD-mSAV1, a probe containinga single mSAV. When reacted with biotin-A647, we foundthat in contrast to the wild-type pQD-SAV1, these pQD-mSAV1 were colocalized with the dye only ∼20% of thetime, suggesting that the binding site on mSAV was notaccessible in most cases. Nevertheless, for the pQD-mSAV1

that were colocalized with the dye, the distribution of biotin-binding sites was very similar to the free dye control, 1(89%) and 2 (11%), confirming our ability to achieve QDmonovalency (Figure 4e). Overall, data from pQD-SAV1 andpQD-mSAV1 demonstrate that we can finely tune the valencyof pQD probes.

SQT of Single Membrane Proteins in Living Cells.Finally, we evaluated the performance of pQD-SAV1 forspecific targeting and tracking of a membrane protein inliving HeLa cells using wide-field epifluorescent microscopy.As a model system we used HeLa cells expressing cyanfluorescent protein (CFP) anchored to the plasma membranethrough the transmembrane domain of platelet-derivedgrowth factor receptor (AP-CFP-TM) (Figure 5a).17,22,28,46

This CFP fusion has a N-terminal 15 amino acid acceptorpeptide (AP) tag that can be site-specifically biotinylated withbiotin ligase (BirA). The cells were cotransfected with plas-mids for AP-CFP-TM and BirA and CFP expression wasdetected in 50-60% of the cells 24-48 h post-transfection(Figure 5b). When these cells were further incubated for 5min with nanomolar concentrations of pQD-SAV1, specificQD labeling at the plasma membrane of only AP-CFP-TMexpressing cells was observed (Figure 5b). As a furthercontrol, no QD labeling was observed when BirA was omit-

FIGURE 4. Counting binding sites on SAV-conjugated pQDs via anorganic dye reporter. (a) GE of a mixture of pQD-SAV1 and biotin-A647 (left lane) and a control when a 1000-fold molar excess of freebiotin was added to the mixture (right lane). The yellow bandindicates colocalization of pQD-SAV1 (green) and biotin-A647 (red).The QD-dye complex was purified from unbound dye by extractingthe yellow region of the gel. Samples were loaded at the startposition and the direction of applied voltage is indicated. (b) Totalinternal reflection fluorescence (TIRF) excitation scheme for the QD-dye complex. The pQDs are directly excited at 488 nm and biotin-A647 is independently and directly excited at 633 nm. The emissionof the QDs and dye are separated and detected on two halves of thesame camera. (c) TIRF microscopy of single QD-dye complexesnonspecifically attached to a glass coverslip. Images are a maximumintensity projection of two image sequences acquired sequentiallyfor biotin-A647 with 633 nm excitation (right, dye channel) and thenfor pQD-SAV1 with 488 nm excitation (left, QD channel). A high levelof colocalization (∼90%) was observed between the fluorescentspots in the dye and QD channels (white circles). Scale bar ) 2 µm.(d) Typical intensity time traces of spots in the dye channel. Singleand multistep photobleaching is observed up to 4 unique steps(arrows). (e) Distribution of biotin-binding sites for pQD-SAV1 andpQD-mSAV1, prepared with wild-type (top, N ) 256) or monovalent(bottom, N ) 57) SAV protein, respectively. These distributions weredetermined from the number of photobleaching steps observed forspots in the dye channel.

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ted during the transfection (data not shown). For labeledcells, the density of QDs at the cell surface was low, andallowed for the identification of single QDs diffusing in themembrane (Figure 5c, Supporting Information Movie S1).From the motion of individual pQD-SAV1, we reconstructedthe trajectories of the diffusing proteins by Gaussian fitting(localization accuracy ∼50 nm) in each frame of the imagesequence (Figure 5c). Using mean square displacement(MSD) analysis of individual trajectories we determined themean diffusion coefficient (D) for AP-CFP-TM to be 0.037µm2/s (standard error: 0.032-0.042) at 25 °C (Figure 5d).As an alternative analysis, we pooled together the individualtrajectories and determined D using the probability distribu-tion of square displacements (PDSD).11,47 This analysisidentified fast, D ) 0.053 µm2/s (68%), and slow, D ) 0.007µm2/s (32%), populations of diffusing proteins (SupportingInformation Figure S5). As a comparison, the cQD-SAVprobe was targeted to AP-CFP-TM and tracked in the samemanner, producing nearly identical diffusion coefficients(Supporting Information Figure S5), an indication that bothQD designs perform equally well in this application.

Discussion. For the design of our QD probes, we selecteda peptide surface coating because it generates highly com-pact and biocompatible QDs, but also because it does notrequire the complicated chemical synthesis demanded by

other compact surface coatings.28,29,31 In fact, our probesare quickly and easily prepared from commercially availablecomponents, which is an important advantage to this design.

Using gel electrophoresis, we observed heterogeneity inthe number of attached targeting units following theirconjugation to the pQDs, which is due to the presence ofmultiple reactive cysteine residues on the QD surface. Atleast, for a low average ratio of targeting units per pQD, thedistribution in the stoichiometry following conjugation wasconsistent with Poisson statistics21,26 (Supporting Informa-tion Table S1). Deviation from Poisson statistics at higherratios is possibly a result of the limited number of reactivesites for conjugation and the fact that multiple bindingcannot be considered as independent events. Importantly,we could easily separate and purify the pQDs containingexactly one targeting unit by GE, which is the desiredstoichiometry for many applications. Our choice of biotinand SAV targeting units comes from their widespread usein biology, their compatibility with a variety of targetingschemes and the extremely high affinity and long half-lifeof the binding interaction. We have also applied the GEtechnique to prepare pQDs conjugated to a single copy ofIgG antibodies or Fab fragments (data not shown) and inprinciple, it should be applicable to a wide range of otherlarge biomolecules (DNA, enzymes, proteins, etc.), as long

FIGURE 5. Specific targeting and tracking of individual proteins in living cells. (a) Scheme for the plasma membrane-anchored cyan fluorescentprotein fusion (AP-CFP-TM). The acceptor peptide (AP) tag is site-specifically biotinylated by coexpression of biotin ligase (BirA). (b) Wide-field epifluorescence microscopy of living HeLa cells after incubation with nanomolar concentrations of pQD-SAV1. Specific QD labeling isobserved on the plasma membrane of cells expressing the biotinylated AP-CFP-TM, as seen from colocalizing CFP and QD signals. Brightspots are individual QDs. The QD channel was color-coded for intensity as indicated. Scale bar ) 20 µm. (c) Maximum intensity projectionimage of a image sequence showing the membrane diffusion of selected pQD-SAV1 targeted to AP-CFP-TM (top) and their correspondinghigh-resolution tracking trajectories obtained by frame-by-frame Gaussian fitting, localization and linking between frames (bottom). (d)Histogram of the diffusion coefficients (D) for AP-CFP-TM obtained from analysis of the mean-square displacement (MSD) curve for N ) 72trajectories. A threshold for immobile QDs was determined from measurements on pQDs attached directly to the coverslip.

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as their conjugation alters the electrophoretic mobility of thepQDs. On the basis of our experiments with PEG linkers, wedetermined a size threshold of ∼10 kDA, below which it isnot possible to separate discrete stoichiometries using GE.Nevertheless, smaller molecules (biotin, receptor ligands,trisNTA,17 etc.) can be attached to the end of a linker andtheir stoichiometry controlled in a similar fashion.37

The choice of a covalent conjugation strategy allowed usto prepare QD probes with very good conjugate stability,which is an improvement over other noncovalent conjuga-tion approaches such as those using polyhistidine-taggedproteins,39-41 which may dissociate from the QD surface.31

During characterization of pQD-SAV1, we measured the HDto be ∼15 nm, which is less than half that of the commercialcQD-SAV probe. This difference becomes significant interms of volume, because pQD-SAV1 occupies ∼8 times lessspace than cQD-SAV. A further decrease in pQD-SAV1 size(diameter reduced by ∼4 nm) could be achieved by usingthe smaller green-emitting pQDs. In addition to their re-duced size, we have shown that pQD-SAV1 has a lowvalency, as confirmed by the presence of a single SAV at theQD surface and limited access to its biotin-binding sites.Indeed, we found that near the surface of QDs, only 1 or 2of the 4 biotin-binding sites in the wild-type SAV are easilyaccessible after the conjugation. Because a large excess(∼100-fold) of biotinylated dye is used and given the strongaffinity of the biotin-streptavidin pair, the heterogeneity innumber of bound dyes most likely reflects the number ofaccessible binding sites on the surface of a single QD. Thissuggests that steric hindrance from the peptide coating orthe nanoparticle surface limits access to some of the SAVbinding sites. Substituting wild-type SAV with mSAV allowedus to further reduce the number of biotin-binding sites tojust one, effectively achieving a monovalent pQD probe.Since many of the pQD-mSAV1 (∼ 80%) did not bind to anybiotin molecules, a future improvement could be the site-specific conjugation of mSAV, to ensure that its single biotin-binding site is oriented away from the QD surface, withincreased accessibility.

We demonstrated the suitability of pQD-SAV1 for SQTstudies in living cells, an application that can benefit fromreduced size and monovalent QD probes, especially whenapplying them in crowded cellular environments, wherelarger QD probes have limited access.22,24 While diffusionof proteins on the 2D plasma membrane is largely indepen-dent of probe size, this is not the case in the cell cytosol, orthe nucleus where diffusion is presumably in 3D.48 Thus, oursmall probes could facilitate targeting and tracking of intra-cellular targets as techniques for delivering QDs inside cellsare further optimized.49 Surface or solution-based in vitroassays should also benefit from the reduced-size and va-lency. For example, SM FRET measurements employingcommercial QD probes as donors have previously sufferedfrom poor energy transfer efficiency, partially because thethick surface coating increases the distance of the acceptors

from the nanoparticle surface.9,10 In these applications,pQDs could potentially increase FRET efficiency while en-suring a 1:1 stoichiometry between donor QD and acceptordye. Aside from biological assays, monovalent peptide-coated nanoparticles could, in principle, enable the con-trolled bottom-up assembly and spatial organization of morecomplex nanostructures.50

Acknowledgment. We thank Thomas Le Saux (FCS), EricLarquet (EM), Geraldine Gouzer (transfection), and MariyaBarch (data analysis) for assistance during various experi-ments and Alice Ting of MIT for providing plasmids forexpression of AP-CFP-TM, BirA-ER, and the streptavidinvariants. S.C. acknowledges support from postdoctoral fel-lowships through Universite Pierre et Marie Curie and theFondation pour la Recherche Medicale. F.P. acknowledgesfinancial support from a Marie Curie-Intra-European Fellow-ship under contract number MEIF-CT-2006-040210, and anEMBO long-term fellowship. M.D. acknowledges supportfrom the Fondation pour la Recherche Medicale, AgenceNationale pour la Recherche (Grant ANR-05-PNANO-045),the Human Frontier Science Program (Grant RGP0005/2007)and the centre C’Nano Ile de France. We are grateful toRegion Ile-de-France for convention SESAME 2000 E 1435and to C’Nano Ile-de-France for supporting cryo-EM atIMPMC.

Supporting Information Available. Supporting Methods,Supporting Figures S1-S5, Supporting Movie S1, and Sup-porting Table S1. This material is available free of charge viathe Internet at http://pubs.acs.org.

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