fçrster resonance energy transfer

DOI: 10.1002/cphc.200500217

Fçrster Resonance Energy TransferInvestigations Using Quantum-DotFluorophoresAaron R. Clapp,[a] Igor L. Medintz,[b] and Hedi Mattoussi*[a]

1. Introduction: Fçrster ResonanceEnergy Transfer

Fçrster (or fluorescence) resonance energy transfer (FRET) in-volves the nonradiative transfer of excitation energy from anexcited donor fluorophore, D, (after absorption of a higher-energy photon) to a ground-state acceptor fluorophore, A,brought in close proximity, which can radiatively emit a lower-energy photon.[1,2] FRET processes are driven by dipole–dipoleinteractions and depend on the degree of spectral overlap be-tween donor photoluminescence (PL) and acceptor absorption,and on the sixth power of the separation distance betweenthe donor and acceptor pair, r.[2] Within the Fçrster formalism,the rate of nonradiative energy transfer is given by Equa-tion (1),

kDA ¼ B� QDItDr6

¼ 1tD

� �� R0

r

� �6

ð1Þ

where tD is the excited-state radiative lifetime of the donorand R0 is the Fçrster separation distance corresponding to arate of FRET equaling the rate of radiative decay (kDA=1/tD). R0

is a function of the refractive index of the medium, nD; Avoga-dro number, NA; the donor PL quantum yield, QD; the overlapintegral, I ; and a parameter, kp, that depends on the relativeorientation of the donor and acceptor dipoles; it is expressedas in Equation (2):

R0 ¼9000 ln 10ð Þk2

pQD

NA128p5n4D

I

!1=6

ð2Þ

The spectral overlap integral, I, defined as in Equation (3),

I ¼Z

JðlÞdl ¼Z

PLD�corr lð Þ � l4 � eA lð Þdl ð3Þ

is a quantitative measure of the donor–acceptor spectral over-lap over all wavelengths l, where PLD�corr and eA represent thedonor emission (normalized dimensionless spectrum) and ac-ceptor absorption extinction coefficient spectrum, respectively.The above expressions imply that a quantum-yield measure-ment is necessary for deriving an accurate estimate of the Fçr-ster distance. The FRET efficiency, E, defined as in Equation (4):

E ¼ kDA

kDA þ t�1D

¼ R60

R60 þ r6

ð4Þ

accounts for the fraction of excitons transferred from donor toacceptor nonradiatively. This can be experimentally measuredby monitoring changes in the donor or/and acceptor fluores-cence intensities, or changes in the fluorescent lifetimes of the

Fçrster resonance energy transfer (FRET), which involves the non-radiative transfer of excitation energy from an excited donor fluo-rophore to a proximal ground-state acceptor fluorophore, is awell-characterized photophysical tool. It is very sensitive to nano-meter-scale changes in donor–acceptor separation distance andtheir relative dipole orientations. It has found a wide range of ap-plications in analytical chemistry, protein conformation studies,and biological assays. Luminescent semiconductor nanocrystals(quantum dots, QDs) are inorganic fluorophores with unique op-tical and spectroscopic properties that could enhance FRET as ananalytical tool, due to broad excitation spectra and tunablenarrow and symmetric photoemission. Recently, there have been

several FRET investigations using luminescent QDs that focusedon addressing basic fundamental questions, as well as develop-ing targeted applications with potential use in biology, includingsensor design and protein conformation studies. Herein, we pro-vide a critical review of those developments. We discuss some ofthe basic aspects of FRET applied to QDs as both donors and ac-ceptors, and highlight some of the advantages offered (and limi-tations encountered) by QDs as energy donors and acceptorscompared to conventional dyes. We also review the recent devel-opments made in using QD bioreceptor conjugates to designFRET-based assays.

[a] Dr. A. R. Clapp, Dr. H. MattoussiUS Naval Research LaboratoryOptical Sciences Division, Code 5611, Washington, DC 20375 (USA)Fax: (+1)202-404-8114E-mail : [email protected]

[b] Dr. I. L. MedintzUS Naval Research LaboratoryCenter for Bio/Molecular Science and EngineeringCode 6900, Washington, DC 20375 (USA)

ChemPhysChem 2006, 7, 47 – 57 < 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 47

fluorophores. During FRET, energy transfer manifests as a de-crease in donor fluorescence intensity and shortening of its ex-citon lifetime; this may in turn be coupled with enhancementof the acceptor fluorescence and increase of its exciton life-time. For additional details on the comprehensive derivation ofenergy transfer formalism, as described in the Fçrster theory,and a description of the methods for monitoring FRET, the in-terested reader is referred to a few specific references.[1, 2] In aconfiguration where a single donor interacts with multiple ac-ceptors brought in close proximity, we have extended/modi-fied the above analysis in the case where all acceptors areequally separated from a central donor, and derived an expres-sion of the FRET efficiency, Equation (5):

E n; rð Þ ¼ nR60

nR60 þ r6

ð5Þ

where n is the number of acceptors surrounding a singledonor.[3, 4]

FRET is a powerful photophysical reporting technique. It hasbeen extensively used in a variety of in vivo and in vitro bio-logical investigations, including the monitoring of DNA hybridi-zation and sequencing, protein conformation studies, diffusiondynamics, and the monitoring of intracellular receptor–ligandbinding and cellular membrane dynamics.[2, 5–8] The power ofthis technique derives from its high intrinsic sensitivity to smallchanges (0.5–10 nm) in the separation distance and orientationbetween the donor and acceptor dipoles, which has led to thecharacterization of FRET in several reports as a “spectroscopicruler”.[2]

2. Luminescent Quantum Dots versus OrganicFluorophores: Background

Most of the biological investigations using FRET for signaltransduction have used two types of fluorophores: molecular(or organic) dyes and genetically encoded fluorescent pro-teins.[6] Emissive fluorophores can act as donors and acceptors,while “dark” quenching dyes can function as acceptors only.However, the use of these types of fluorophores has severallimitations, including low resistance to chemical and photode-gradation and low photobleaching thresholds. Their broad ab-sorption/emission spectra limit the number of possible pairingchoices, and thus multiplexing capabilities. Furthermore, varia-tion of the absorption and/or emission spectra requires theuse of distinct molecular labels with the associated syntheticand conjugation constraints. Fluorescent proteins have the ad-vantage of being genetically encoded and allow, for example,easier intracellular loading, manipulation, and expression of flu-orescence-based sensing and imaging in live cells, but tend tohave low quantum yields and broad absorption/emission spec-tra.[9] Proteins containing fluorescent amino acids (tryptophan,tyrosine, and phenylalanine) have also been used as FRETdonors.[2]

Quantum-dot (QD) fluorophores have unique optical andspectroscopic properties that offer a compelling alternative totraditional fluorophores in several fluorescence-based biologi-

cal applications (e.g. , fluoroimmuno- and FRET-based assays).Unique advantages offered by QD fluorophores include broadtunable absorption spectra with high molar extinction coeffi-cients (�10–100Hthat of organic dyes), narrow symmetric PLspectra (full width at half maximum �25–40 nm) spanning theUV to near-IR with high quantum yields, exceptional resistanceto photo- and chemical degradation, high photobleachingthresholds and large achievable energy separation between ex-citation and emission lines (i.e. large effective “Stokesshifts”).[10–14] Two properties are particularly appealing for de-veloping FRET-based investigations: 1) the ability to tune thefluorescent emission (for example, as a function of core size forbinary CdSe nanocrystals),[10–12] which can allow better controlof the spectral overlap with a particular acceptor,[3, 4] and 2) theability to excite mixed QD populations at a single wavelengthfar removed (>100 nm) from their respective emissions whichcould allow extensive multiplexing.[10–12]

Herein, we review the latest developments geared towardsusing QDs to design and implement FRET-based investigations,with some emphasis on those having biological applications.In particular, we address the advantages offered and limita-tions encountered by QDs as energy donors and acceptors.

In applying the Fçrster formalism to analyze the nonradia-tive transfer of excitation energy in systems involving QD fluo-rophores (e.g. from an excited QD to a proximal ground-statedye acceptor), we approximate the QD excited state (or exci-ton) as an oscillating (point) dipole. This approximation is de-rived from the combination of two factors: 1) strong overlapof the electron and hole carrier wavefunctions in the nanocrys-tal (dot size is smaller than the bulk Bohr exciton size), and 2)the spatial extent of the carrier wavefunctions as well as theseparation between electron and holes carriers inside thenanocrystals are much smaller than the wavelength of light.Furthermore, the treatment of nonradiative energy transferwithin the Fçrster formalism (dipole–dipole interactions) hasprovided excellent description of most experimental data col-lected from systems using QD fluorophores alone or in con-junction with conventional dyes.[3,4, 15–17]

3. QD Fluorophores and FRET Investigations

3.1. QDs as FRET Donors

The first demonstration that CdSe QDs (core only), as other or-ganic fluorophores, can engage in efficient nonradiativeenergy transfer was reported by the Bawendi group.[15] The au-thors prepared thin films made of mixed close-packed QDs,grown by using high-temperature solution chemistry,[10] of twodifferent diameters: �38.5 L (PL maximum at �555 nm) func-tioning as exciton donors and larger QDs (�62 L diameter,with PL maximum at �620 nm) serving as energy acceptors.Solutions containing populations of these nanocrystals wereprepared and used to spin-cast films of close-packed inter-penetrating networks of donor–acceptor nanocrystals. Steady-state fluorescence data (collected at room temperature and at10 K) showed that there was a clear decrease in the PL fromthe 38.5-L QDs along with an increase in acceptor (62-L QDs)

48 www.chemphyschem.org < 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemPhysChem 2006, 7, 47 – 57

H. Mattoussi et al.

www.chemphyschem.org

contribution when exciting to the blue edge of the first ab-sorption maximum of the smaller-size nanocrystals (Figure 1).Time-resolved fluorescence experiments corroborated thesteady-state results where a decrease in the PL lifetime of the

donor and an increase in the acceptor PL lifetime were meas-ured; data were successfully analyzed within the Fçrster for-malism.[15] In subsequent studies, the authors showed that in apopulation (with a finite size distribution) of close-packedCdSe QDs, there is shift of the PL maximum wavelength com-pared to the wavelength of their dilute solution counterparts,which was attributed to FRET from small- to larger-size nano-crystals. They further confirmed that the shift is more pro-nounced when the size distribution becomes broader.[16]

In a more recent study, Crooker et al. investigated FRET dy-namics in close-packed films of mixed-size and gradient (lay-ered) assemblies of CdSe (core only) and CdSe–ZnS core–shellQDs.[17] In addition to measuring a red shift in the PL fromclose-packed films of core and core–shell QDs compared tothe PL of their solution counterparts (as reported in ref. [16]),the authors employed time-resolved and spectrally-resolvedfluorescence to probe the exciton decay acquired at specificenergies (narrow wavelength windows) and showed that thePL decay was not constant along the emission energy spec-trum. For a close-packed population of nanocrystals (averagecore radius=12 L and shell=9 L), a short (1.9 ns) initial PLdecay was measured at high energy (smaller-size nanocrystals),and it progressively increased with decreasing energy (or in-creasing QD size) to �22 ns at the lowest energy probed (seeFigure 2).[17] In contrast, a single average lifetime of �24 nswas measured for a dilute solution of the same QD population.

They then fabricated close-packed films made of a mixture ofthree disparate-size TOP/TOPO-capped CdSe populations(radius=17, 18, and 21 L) and compared the PL dynamics insuch samples to that of their single-population film counter-

Figure 1. Absorption and emission spectra of 38.5-L (�555-nm emissionpeak) and 62-L (�620-nm emission peak) CdSe QD close-packed solids at a)RT and at b) 10 K. PL spectra of the mixed system (solid lines) dispersed insolution at c) RT and d) 10 K and close-packed in the solid films at e) RT andf) 10 K. The system was made of 18% of 62-L dots mixed with 82% of 38.5-L nanocrystals ; an excitation of 2.762 eV (�448 nm) was used. Dotted linesare the relative quantum yields for 38.5-L dots in a pure film and for 62-Ldots in the mixed film when excited to the red (2.143 eV, �578 nm) of the38.5-L dot absorption edge at e) RT and f) 10 K. Reproduced from ref. [15]with permission from the American Physical Society.

Figure 2. a) PL decays from a close-packed film of a single population ofCdSe–ZnS nanocrystals with an average core CdSe radius of 12.4 L and aZnS shell of 9 L at the energies specified in the inset. The inset showssteady-state PL spectra from the close-packed film (solid) and original dilutesolution of the same QDs (dashed). b) Dynamic redshift of the peak emis-sion; the inset shows the PL spectra at the specified times. Reproducedfrom ref. [17] with permission from the American Physical Society.

Figure 3. a) Steady-state PL spectra from close-packed solid films of 17-L(~), 18-L (*) and 21-L (*) CdSe colloidal QDs, along with the spectrum froma film made of an equal mixture of the three populations of QDs (&). b) Thecorresponding PL lifetimes versus energy for each sample shown in (a). Re-produced from ref. [17] with permission from the American Physical Society.

ChemPhysChem 2006, 7, 47 – 57 < 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemphyschem.org 49

Quantum-Dot Fluorophores


parts (PL spectra are shown in Figure 3a). They found that theemission from the close-packed solid mixture collapses into asingle spectrum strongly weighted towards low energies orlarge sizes, while in a mixed dilute solution the PL spectrum isa composite sum of the three PL spectra. Furthermore, efficientFRET is seen in the three close-packed films of each popula-tion, through a pronounced decrease in PL lifetime from lowto high energies, but the blue decay (700 ps) was � threetimes faster than in the CdSe–ZnS dots discussed above, a fea-ture attributed to the smaller donor–acceptor separation dis-tance in core-only QDs. When the three-size QDs are combinedin a ‘‘mixed’’ film, they measured short PL lifetimes (700–800 ps) throughout the high-energy range (covering the twosmaller sizes), whereas at low energies longer lifetimes weremeasured, roughly mimicking the behavior of the film with thelargest size QDs.

These studies demonstrated that QDs could potentially func-tion both as FRET donors and acceptors. In addition, for QDpopulation of a particular size, the rate of FRET is due primarilyto the availability of nearby viable acceptor dots. Center-to-center separation distances for these QD films are only slightlylarger than R0 values, which confirms that significant FRETwould occur in these systems.

Following the early demonstrations of water-soluble QDs aspotentially useful fluorophores for developing biologicalassays,[18–20] interest in developing assays based on QDs andFRET naturally followed. Willard et al. reported the first biologi-cally relevant FRET investigation using QDs as energydonors.[21] Water-soluble CdSe–ZnS QDs were first conjugatedto biotinylated bovine serum albumin (bBSA, with �11 bBSAper QD) via a thiol linkage, although free bBSA was still presentin solution. In a separate reaction, StreptAvidin (SAv) was cova-lently labeled with tetramethylrhodamine (TMR) and used asacceptor. The two subunits were allowed to react in phosphatebuffered saline (PBS) to form QD–bBSA–SAv–TMR complexesvia avidin–biotin interactions. Emission spectra were measuredfor each subunit separately and for the complex as a functionof SAv–TMR concentration. They reported a decrease in theQD PL and an increase in the TMR PL as the molar ratio ofSAv–TMR to QD increased from 0 to 11 (presumably about thenumber of bound bBSA per QD), and attributed their results toFRET between QDs and TMR on the SAv. No time-resolved flu-orescence experiments were carried out and no estimates forthe separation distance or for R0 were provided for this QD–dye pair. Approximate estimates of these values using availableexperimental data indicate that R0 and r are �54 L and 100 L(bBSA minor axis �60 L, SAv minor axis �48 L), respectively,suggesting that low FRET efficiencies would be expected withthis QD–protein–dye configuration.

In a context similar to the one used by Kagan et al. butadapted to a biological setting, a recent study reported thatby using green-emitting CdTe QDs conjugated to anti-BSA anti-body and red-emitting QDs conjugated to BSA, assembled to-gether via receptor–ligand interactions, resulted in quenchingof the green QDs and an enhancement in the luminescence ofthe red QDs. Results were again attributed to FRET betweenQDs of different sizes brought together by specific interac-

tions.[22] This configuration produces a rather large center-to-center separation distance between donor and acceptor, andin the absence of time-resolved fluorescence data interpreta-tion based only on FRET may not be fully warranted.

Our group took a more systematic approach to understand-ing FRET between CdSe–ZnS QDs and proximal dyes. For this,QD donors capped with dihydrolipoic acid (DHLA) were conju-gated to engineered proteins containing site-specifically la-beled dye acceptors and appended with either a C-terminaloligohistidine (His), or a basic leucine zipper (zb) attach-ment;[3, 4,20,23,24] Figure 4A schematically depicts the bioconju-gate structure. In such configuration, each QD is surroundedby a fixed number of proteins (e.g. , �5, 10 or 15 maltose-binding proteins, MBPs); the proteins adhere onto the nano-crystal surface through either electrostatic self-assembly (forMBP–zb) or via metal-affinity coordination between the QDsurface and C-terminal histidine (for MBP-His).[3,4, 20,23,24] Thetotal number of MBP bound to a QD remained fixed while wevaried the number of dye-labeled proteins. More importantly,such configuration maintains the average distance betweenthe QD center and the proximal dyes in each conjugate fixed.The proteins have the same anchoring point for attachment tothe QD, which ensured consistent protein orientation anddonor–acceptor distances. These studies utilized different size/color QDs whose emission maxima were varied to tune/opti-mize the degree of spectral overlap with the acceptor absorp-tion (see Figure 4B). Steady-state fluorescence measurementsshowed that as the fraction of dye-labeled proteins per QD in-creased the QD PL systematically decreased.[3,4,24] In the casewhere Cy3 (an emitting dye) was the acceptor a concomitantenhancement of Cy3 PL was measured (Figure 4C–D).[3] Similar-ly, time-resolved fluorescence experiments on these self-assem-bled QD–protein–dye conjugates showed a progressive de-crease of the QD exciton lifetime with increasing dye-to-QDratio (Figure 4E).[3] These two sets of observations confirm thatefficient nonradiative exciton transfer between QD donors anddye acceptors is realized by using dye-labeled proteins self-as-sembled on QD surfaces, and that the overall behavior is con-sistent with the Fçrster formalism.

We further analyzed the FRET data collected from QD PLloss (or dye PL gain) with increasing Cy3-to-QD ratio in eachconjugate using Equation (5) to extract estimates of the sepa-ration distance, r, at each ratio n.[3] Moreover, compilation ofthe values at each ratio (derived for each QD–Cy3 pair) provid-ed an average distance with a statistical error. We found thatthe average values for r were generally consistent with thoseanticipated by using the radius of the QD inorganic core, thedimension of the proteins used, and assuming a close ap-proach between QD and protein–dye in a self-assembly proc-ess.

In a recent study, Grecco et al. used commercially availableCdSe–ZnS QDs emitting at 585 nm coated with a thick passi-vating and functionalized polymer layer and conjugated toSAv as FRET donors with proximal AlexaFluor 594-biocytin;specific interactions between biocytin and SAv brought the ac-ceptor in close proximity to the QD center.[25] In particular, theymeasured FRET efficiencies that increased (though linearly)


H. Mattoussi et al.


with Alexa 594-to-QD ratio, similar to what we reported inref. [3, 4] . However, reported efficiencies were much higherthan what was anticipated from estimates of R0 and the rela-tively large separation distances involved. Furthermore, giventhe multivalency of the receptors on the QDs, accurate controlover donor–acceptor separation distance in their system couldnot be achieved. The authors attributed these observations to

possible aggregate formation and overestimation of the initialQD concentration.[25]

3.2. QDs as FRET Acceptors

In principle, QDs should be excellent FRET acceptors due totheir large absorption cross-section, which can be an order of

Figure 4. A) Schematic representation of a QD–protein–dye assembly (not drawn to scale). The QD consists of a CdSe–ZnS core–shell capped with DHLA. Thecharged carboxyl on the DHLA keeps the colloid nanocrystals dispersed in solution at basic pH. Also depicted are representative proteins (MBP, in this case)that are immobilized on the QD via metal-affinity coordination. Site-specific dye labeling of the MBP allows a quasi-symmetrical arrangement of Cy3, at afixed distance, r, from the QD center. B) Normalized absorption spectra (eA) of Cy3 dye and photoemission spectra of the three CdSe–ZnS core–shell QD solu-tions. The inset shows plots of the resulting overlap functions J(l) [Eq. (3)] and highlights the effects of tuning the QD emission on the degree of spectraloverlap with Cy3 dye. C) Evolution of the photoluminescence spectra derived from titrating 510-nm emitting QDs with an increasing ratio, n, in QD–MBP–Cy3assemblies. Spectra have been deconvoluted and corrected for direct excitation contribution to acceptor emission; this allows discrimination in QD donorloss and Cy3 acceptor gain. D) Integrated intensities for both QD and Cy3 versus ratio n. E) Series of false-color time–wavelength intensity images showingthe intensity of: free MBP–Cy3, top series; 510-nm QDs with 10 unlabeled MBP per QD, middle series; and 510 nm QDs with 3 MBP/7 MBP–Cy3 on the QDsurface, bottom series; as recorded by a CCD camera at 2-ns intervals following an initial 90-ps laser pulse. Horizontal axis represents wavelength; dashedlines mark the maximal emission for the QD (at 510 nm) and the dye (at 570 nm). Adapted from ref. [3] with permission from the American Chemical Society.




magnitude larger than that of organic dyes.[3,13] Studies byKagan and Crooker demonstrated that QDs can transferenergy nonradiatively between populations of differentsizes,[15–17] but their ability to accept excited-state energy fromorganic dyes or other donor fluorophores was unclear. In a bio-logical context (using QD bioconjugates), there are few exam-ples of QDs functioning as energy acceptors in the literature.Mamedova et al. described the use of 1:1 conjugates of BSAand 580-nm emitting CdTe QDs and reported substantialenergy transfer from the native tryptophan (Trp) residues inBSA to attached QDs; the system was excited at 290 nm.[26]

However, it is widely known that addition of surface-boundprotein alone can significantly influence the PL of QDs in solu-tion irrespective of any potential FRET interaction.[4,20,26,27] Inthe absence of suitable control experiments, it is not clearwhether the above observations could unequivocally be attrib-uted to FRET or other phenomena such as protein-induced sur-face passivation of QD trapping states.

We explored the use of QDs as FRET acceptors in our QD–MBP system by labeling MBP with various organic dyes thatemit to the blue edge of the QDs.[28] Excitation wavelengthsthat maximize dye excitation and emission also resulted insubstantial direct excitation of the QD acceptors due to theirbroad absorption spectra. Using AlexaFluor 488 (AF488) andCy3 dyes as donor fluorophores and 555-, 570-, and 590-nm-emitting QDs as acceptors, we found no evidence of nonradia-tive energy transfer from dye to QDs. Steady-state and time-re-solved experiments showed no apparent change in fluores-cence behavior from either donor or acceptor, as comparedwith the fluorescence of the independent control samples.Similar to the above study,[26] we also attempted Trp-to-QDFRET by exciting QD-bound MBP at 280 nm (each MBP mole-cule contains �120 Trp residues). Our results showed thatthere was no evidence of Trp quenching or significant QD PLenhancement when MBP was placed near the QD surface. Asexpected, the addition of MBP alone was found to be solely re-sponsible for observed QD PL enhancement (due to surfacepassivation effects), which suggests that FRET was absent inthis system even though donor–acceptor separation distanceswere favorable (r�1.3HR0).

[28]

Our results were attributed to the slow decay rate of theQDs combined with a strong direct excitation.[28] Lifetimes ofsoluble CdSe–ZnS QDs were reported to vary between 3 and15 ns depending on the conditions used.[3,15, 17,28] To testwhether relative donor–acceptor lifetimes were important pa-rameters for efficient FRET, we replaced the above organic fluo-rophore with a long-lifetime Ru-bpy isothiocyanate (ITC) metalchelate dye covalently attached to the MBP. Steady-state datawere inconclusive, due to a pronounced overlap in donor andacceptor PL spectra and low Ru-bpy quantum yield. However,time-resolved fluorescence data showed a substantial decreasein Ru-bpy lifetime when bound to the QD surface, indicatingFRET from Ru-bpy to the central QD (see Figure 5).[28] Althoughnot fully conclusive, the results provide strong evidence thatthe lifetime of the acceptor relative to that of the donor playsa significant factor for generating efficient FRET for a givendonor–acceptor pair.

The above findings contrasted with results from two recentstudies suggesting that luminescent QDs can be effectiveenergy acceptors with polymer and quantum well (QW)donors. In the first study, Acherman et al. reported efficientnonradiative energy transfer to QDs in a three-layer hetero-structure made of an InGaN QW donor and an adjacent layermade of close-packed CdSe–ZnS QDs.[29] The QD layer is depos-ited on top of the QW by using the Langmuir–Blodgett techni-que, either directly or separated by a thin GaN cap layer. Thesystem was excited with a 200-fs laser pulse at 266 nm (thirdharmonic of a Ti-sapphire laser). The authors first demonstrat-ed that the QW photoemission has a quadratic dependence onthe excitation power, while its lifetime decay was independentof that power, which indicates that the InGaN QW is character-ized by free-carrier recombination not bound excitons, due toweak electron–hole interactions (Figure 6A). This implies thatthe energy-transfer rate of carriers from the QW to QD layer

Figure 5. Fluorescence intensity plotted as a function of delay time follow-ing a 90-ps excitation pulse: A) MBP–Ru-bpy–ITC alone and B) MBP–Ru-bpy–ITC-610–QD conjugate. The insets are time–wavelength intensity plots foreach sample with the same false-color intensity scale. A fit of the datashown in (A) provides a long exciton lifetime of 420 ns, whereas in (B) twodistinct contributions can be isolated: a relatively short lifetime (attributedto the QDs and a long lifetime attributed to the Ru-bpy–ITC. The shortdecay time (�7 ns) derived from the data by the fitting procedure is identi-cal to the one for the MBP–QD-only sample, whereas the longer decay time(�160 ns) is about half of the lifetime measured for MBP–Ru-bpy–ITC alone.Adapted from ref. [28] with permission from the American Chemical Society.


H. Mattoussi et al.


varies as 1/d4 (where d is the separation distance between theQW and QD layer centers). Time-resolved measurementsshowed a power-dependent shortening of the QW PL lifetimein the presence of the QD layer in comparison with the QWlifetime alone. Furthermore, changes in the PL decay rates forthe two-layer structure compared to the rates of the QW alone(DG=GQW with NC(QD) layer�GQW alone) varied linearly (in a semilogar-ithmic plot) with the electron–hole carrier density (neh), butwere consistently smaller in the presence of the cap GaN layer.Using a single layer of QDs deposited on a glass substrate as acontrol, they also showed that at high excitation power thethree-layer structure provided a larger PL intensity than thecontrol sample with saturation occurring for both sample andcontrol. The difference in PL signal also increased with laserpower before saturation (Figure 6C). The authors attributedthese observations to efficient nonradiative energy transfer be-tween the QW and the top QD layer. Energy transfer is moreefficient because of its inverse fourth-power dependence onthe separation distance (E�1/d4), as expected for a donorsystem with unbound (or very weakly bound) electron–holecarriers. This results in a fast energy-transfer rate compared

with the radiative and nonradiative decay rates of the QW exci-tation energy. The authors further suggest that the systemcould be modified to allow for electrical excitation of the QW,followed by energy transfer and emission from the QD layer.[29]

In the second report, the authors performed low-tempera-ture (at 18 K) steady-state and time-resolved fluorescence ex-periments using thin films made of a blue fluorescing polymermatrix doped with QDs at low loading concentrations; with aQD PL centered at 560 nm and a broad polymer fluorescencewith a composite peak between 420 and 500 nm, R0ffi56 L forthis pair. Samples were excited with a short (2 ps) laser pulseat 390 nm (the second harmonic of a mode-locked Ti-sapphirelaser).[30] Time-resolved measurements, limited to time scalesbelow 1 ns, indicated that as QD concentration increased ashortening of the polymer exciton lifetime along with an appa-rent lengthening of the QD excitation lifetime could be meas-ured, which was attributed to an efficient FRET from polymerto QDs. Analysis of the efficiencies extracted from the time-re-solved data, allowing for exciton diffusion contribution in therate equations (since the entire polymer matrix is the donor),provided an experimental value for R0 of 80 L, about twice thevalue estimated from the overlap integral (R0ffi56 L). This im-plies that higher than expected FRET efficiencies were meas-ured for this system. The authors attributed this difference toan error in estimating the polymer molecular weight and den-sity. Steady-state fluorescence showed a clear contributionfrom the QDs at the larger concentration, but the data couldnot be corrected for a direct excitation contribution, whichmakes an interpretation based only on FRET ambiguous.

It is difficult to provide an accurate and objective compari-son between the findings discussed in these studies withthose described above in ref. [28] . The QW donor used inref. [29] has different carrier characteristics than our organicdyes, and the energy-transfer rates predicted for that systemare higher than those predicted for single donor–acceptorpairs (Equation (3)). In ref. [30] , the time-resolved data werecollected at low temperature by using a shorter excitationpulse and analyzed within a short time window (t<1 ns),whereas in ref. [28] data were collected at room temperature,by using a longer excitation pulse (90 ps) and analyzed over alonger time window (0.5 ns< t<50 ns with 0.5 ns resolution).Additional studies are necessary to clarify the issues associatedwith QDs as energy acceptors with organic donors. Better con-trol samples of dots only and dye or polymer only should beused in both steady-state and time-resolved experiments. Ex-ploring the effects of donor lifetime on the occurrence of FRETand its efficiencies would also be extremely useful in providingbetter understanding of these systems.

4. Bio-Inspired FRET-Based Applications UsingQD Donors

4.1. Use of FRET to Derive QD–Protein-ConjugateConfiguration

A property of crucial importance when assembling QD–biore-ceptor conjugates is the final bioreceptor orientation within

Figure 6. Experimental observations of QW-to-nanocrystal(NC)-layer energytransfer. a) Normalized PL dynamics of the isolated QW (black solid line) atcarrier density of neh=3H012 cm�2 in comparison with QW PL dynamicsmeasured for the QW/QD structure at neh=3H012 cm�2 (grey dotted line)and at neh=10H012 cm�2 (grey dashed line). b) The difference between theinitial PL decay rates measured for the isolated QW and the QW/QD struc-ture (DG=GQW with NC�GQW without NC) versus QW carrier density for samplesbased on the capped and the uncapped QW. c) Time-integrated nanocrystalPL intensity versus pump fluence for the nanocrystal monolayer assembledon a glass substrate and on top of a capped QW. Reproduced from ref. [29]with permission from Nature Publishing Group.




the conjugate, since this has ramifications on the conjugate bi-ological functionality; proteins that are oriented on the QDheterogeneously will manifest a mixed functional avidity andmay not perform optimally in biological assays. With this inmind, we set out to derive the optimal configuration of ourself-assembled conjugates using FRET between a central QDdonor and a dye acceptor whose position in the protein wasselectively controlled.[31] His-terminated MBP mutants express-ing six individual and unique cysteine residues spread acrossthe two protein lobes were site-specifically labeled with rhoda-mine red (RR) and immobilized on the QDs (Figure 7A). QD–MBP–RR conjugates with different RR-to-QD ratios (as descri-bed above) were used and average distances (ri) from the QDcenter to each RR location on the protein were derived fromFRET data. Applying the concepts developed for a global posi-

tioning system with its spherical coordinate symmetry, butadapted to our nanoscacle QD–protein conjugate (nanoscaleGPS), a least squares minimization of the deviations betweenthe experimental distances (ri, derived from the FRET data) andthose derived from the protein PDB crystallographic structureswas carried out for all six dye locations. This model assumed aprotein that is free to translate on the QD surface and free torotate around the conjugate polar axis originating at the QDcenter. We were able to accurately determine the optimal con-figuration of the self-assembled QD–MBP conjugates (Fig-ure 7B). A critical result from this analysis was that MBP self-as-sembles onto QD surfaces with a preferred “homogeneous”conformation where the C-terminus His tail in each MBPcomes in close contact with (coordinates to) the QD, while thebinding pocket faces away from that surface, thus preservingprotein function in the conjugate.[31]

This approach of using self-assembly combined with FRET toderive protein conformation in a functional bioconjugate couldbe applied to a wide range of QD–protein conjugates. The de-rived information could potentially be used to infer proteinstructure as it self-assembles on the nanocrystals and its ulti-mate functionality.

4.2. Photochromic Switching Based on QD FRET

Using a QD donor and photochromic dye acceptor pair, wetested the ability of such system to realize modulation of FRETrates induced by dye photochromicity (“photochromic FRET”),and use that to control QD emission.[32] To do this, we labeledMBP with a sulfo-N-hydroxysuccinimide activated photochro-mic BIPS molecule (1’,3-dihydro-1’-(2-carboxyethyl)-3,3-dimeth-yl-6-nitrospiro[2H-1-benzopyran-2,2’-(2H)-indoline]) at a dye-to-MBP ratio of �5. BIPS-labeled MBPs were self-assembled onthe QDs and the ability of BIPS to modulate QD photolumines-cence was tested by switching the BIPS from the colorless spi-ropyran (SP nonabsorbing) to the colored merocyanine (MCenergy absorbing). FRET efficiency for this system was meas-ured following irradiation with white light (>500 nm) and UVlight (�365 nm), which respectively generate the SP and MCforms of the dye. A FRET efficiency of �60% was measured inthe presence of the MC form compared to �10 % for the SPform. This system has also shown the benefit of arraying multi-ple acceptors around a single donor, where starting with adonor–acceptor pair having a modest overlap (R0ffi38.5 L) andimmobilizing 20 MBPs (�100 dyes) on each QD producedlarge experimental FRET efficiencies.[32]

4.3. QD–Protein-Sensing Assemblies Based on FRET

Building on our understanding of optimal FRET conditions forQD–protein–dye conjugates, we constructed a prototypesensor for the specific detection of the nutrient sugar maltosein solution using a QD–MBP conjugate. This sensing assemblyused QDs as both energy donors and structural scaffolds toarray multiple protein receptors (see Figure 8).[4] The equilibri-um dissociation constant for maltose, KD, derived from satura-tion data agreed well with literature values for the solution-

Figure 7. A) 3-D representation of an MBP protein with the labeled residueshighlighted and numbered. The location of the His tail is also shown. B) Re-fined arrangement/orientation of MBP–His as it self-assembles on the sur-face of a QD. Side view presenting the structure of the final refined MBP ori-entation with all six dye locations highlighted in red. The refined distancesfrom each of these dyes to the center of the spheroid are shown in yellow.Adapted from ref. [31] with permission from the Proceedings of the NationalAcademy of Sciences.


H. Mattoussi et al.


phase-behavior of wild-type MBP.[4] Time-resolved fluorescencedata collected for this system showed a decrease in the QDlifetime when the dye-labeled analog was bound to MBP, andrecovery of the QD lifetime when maltose was added to thesystem. The nanosensor also shows high specificity by re-sponding only to sugars having the MBP-recognized a-1,4-glu-cosidic linkage, which proves that QD-bound proteins maintaintheir intrinsic binding properties.[4]

A second sensing assembly based on FRET and QD energydonors targeted the explosive TNT.[33] For this, an antibodyfragment that recognizes TNT (TNB2-45) expressing a C-termi-

nal 12-His sequence was allowed to prebind an analog of TNT(TNB which is conjugated to the dark quenching dye BHQ10)and then attached to the QD surface (similar to the schemeshown in Figure 8A). Again, due to excellent spectral overlapand close proximity of the QD donor to the BHQ10-quenchingacceptor dye, a progressive and BHQ10-dependent quenchingof QD fluorescence is measured. As TNT is added to the assaysolution, it competes for binding to the antibody fragmentand the QD fluorescence increases as TNB–BHQ10 is displacedin a concentration-dependent manner. Antibody-fragment spe-cificity for explosive analogs of TNT was retained in this QDsensing configuration.[33] Both of these studies demonstratethat QDs not only function as efficient FRET donors, but alsoas scaffolds for assembling the sensor elements.

4.4. QD FRET To Probe DNA Replication and Telomerization

Replication of DNA is an important natural process that occursin live cells before they divide. It was shown that replicationon solid surfaces could be employed to amplify DNA detec-tion.[34] QDs conjugated to oligonucleotides have been used asFRET donors to monitor DNA replication and telomerizationprocesses in solution.[35] CdSe–ZnS QDs were attached to athiol-terminated DNA primer (sequence: 5’-HS-(CH2)6-CCCCCACGTTGTAAAACGACGGCCAGT-3’), then incubated withthe complementary sequence M13fDNA to allow for hybridi-zation. Addition of polymerase (to initiate replication) mixedwith dNTPs and dUTP labeled with Texas Red (TR–dUTP) per-mitted to follow the dynamics of DNA replication with time, bymonitoring changes in the QD and dye emissions. Replicationof the DNA progressively brings TR–dUTP complexes specifical-ly in close proximity to the QD center, resulting in time-de-pendent FRET between the QD donor and proximal TR accept-ors. In particular, FRET data show that the replication processstarts fast (nearly linearly with time) and saturates after 1 hour(Figure 9). The authors further applied FRET-induced quench-ing of QD emission to detect the dynamics of DNA telomeriza-tion, by incubating the QD–DNA primer conjugates with dNTPmixtures that include TR-labeled dUTP and telomerase.[35] FRETefficiencies were relatively weak for this system due the ratherlarge separation distance between QD and acceptors involved;further, the nature of this system does not allow accurate con-trol over separation distance.

4.5. QDs in Photodynamic Medical Therapy

Photodynamic therapy is a process in which an excited photo-sensitizing agent transfers its excitation energy to a nearbyoxygen molecule to form a reactive singlet oxygen (1O2). Thishighly reactive species causes cytotoxic reactions in cells or tis-sues, which has made the process a useful therapeutic tool totreat cancerous tissue and cells. The technique is highly selec-tive because only tissues that are simultaneously exposed tothe photosensitizer and photoexcitation in the presence ofoxygen are affected. In a preliminary study, QDs were linked toa known silicon photosensitizer (Pc4) through alkyl aminegroups. Photosensitization of the Pc4 alone is limited to the

Figure 8. A) Schematic description of functions and properties of a solution-phase QD–MBP–His-sensing assembly targeting maltose. Each QD is sur-rounded by an average of �10 MBP moieties. b-CD (beta-cyclodextrin)analog attached to a QSY-9 quenching dye is prebound in the MBP bindingpocket. Formation of QD–MBP–b-CD–QSY-9 complex brings the dye in closeproximity to the QD center and results in substantial FRET quenching of QDemission due to nonradiative energy transfer from QD to QSY-9; the sensoris in the initial “off” state. B) Added maltose displaces b-CD–QSY-9 awayfrom the QD donor, which results in a concentration-dependent increase inQD emission. The saturation curve (derived from the titration of QD–MBPconjugate (preassembled with 1 mm b-CD–QSY-9) with increasing concentra-tions of maltose is shown. Adapted from ref. [4] with permission fromNature Publishing Group.




window between 550 and 630 nm (absorption peak of thePc4). Taking advantage of the broad excitation spectra of QDs,the authors showed that one can achieve photosensitization ofthe Pc4 via FRET between the QD center and proximal Pc4groups; the system was excited at 488 nm, far from the ab-sorption peak of Pc4.[36] Furthermore, due to the short donor–acceptor separation distance high FRET efficiencies and thushigh degrees of photosensitization were achieved. The samestudy has also shown that QDs alone can generate 1O2 in theabsence of Pc4, detected by measuring and analyzing emissionat 1270 nm; yields from this process were much lower thatthose generated with Pc4, however.

4.6. Additional Uses of QD-Based FRET

There have been other FRET-based investigations (bio-inspiredand otherwise) using QDs as energy donors where authorshave, for example, arrayed multiple acceptors around a singlenanocrystal or selected the excitation line over a broad rangeof wavelengths.[37–43] They include the use of QDs as excitondonors and scaffolds for DNA molecular beacons, wherechanges in FRET rates to dyes attached at the beacon endwere assessed, and use of FRET modulation to demonstrate aninhibition assay of binding between SAv–QDs and proximal bi-otinylated Au nanoparticles in the presence of molecular inhib-itors.[37,38] There is also growing interest in realizing FRET at thesingle-molecule level, where microcopy (optical and scanningprobe) techniques combined with spectroscopy are used toimage individual QDs interacting with proximal dyes or Aunanoparticle quenchers. In one study, AFM tips were function-alized with QDs, and their potential as highly localized FRETimaging systems was demonstrated by bringing a dye accept-or close to the tip and measuring distinctive FRET signals.[39] Inanother study, the authors used double-stranded DNA as a“rigid” spacer to tune the distance between a QD and proximalAu nanoparticles and reported distance-dependent quenchingof QD emission.[40] Energy transfer between lipid membranes,or between QDs and dye-labeled proteins assembled on a sur-face have also been investigated.[41,42] Demonstration of QD-

based FRET using lipid mem-branes and surface-immobilizedassemblies have potential usefor monitoring protein interac-tions and dynamics near cellularmembranes,[41] or constructingsurface-tethered sensing assem-blies using QDs as a scaffoldsand exciton donors.[42]

5. Summary andConclusions

Herein, we reviewed the devel-opments and progress made inthe past few years using lumi-nescent QDs as energy donorsand acceptors in FRET investiga-

tions, both in purely spectroscopic and biological contexts. Asenergy donors, QDs offer several unique advantages comparedto conventional dyes. These include the ability to tune thedegree of spectral overlap with a given dye acceptor by, for ex-ample, varying the nanoparticle size for binary materials. In ad-dition, due to their large size (comparable to that of a protein)QDs provide a unique configuration where multiple dye-la-beled bioreceptors can be arrayed around a single nanoparti-cle, which enhances the effective FRET cross-section, even for amodest overlap (i.e. , small Fçrster distance). Both of theseproperties increase the measured FRET rates and improve thesignal-to-noise ratio. Further, the possibility of arraying severalreceptors with different properties/functionalities on QDdonors provides the potential for multiplexing.

These results indicate that FRET applications using QDscould see significant use both in biological and nonbiologicalcontexts. Accurate derivation of protein conformation andsensor design represent only two of a long list of potential ap-plications. QD-based FRET nanosensors will be particularly ap-pealing for intracellular sensing, where their high photobleach-ing thresholds and substantial reduction in direct excitation ofdye and fluorescent protein acceptors could permit the moni-toring of intracellular processes over longer periods of time.FRET-based uses of QDs will potentially find applications in themonitoring of a wide array of intra- and extracellular indica-tors; success will still depend on the ability to design easy andreproducible means of delivering compact QD–bioreceptorconjugates specifically to subcellular regions and cell mem-branes. This could also provide a very useful tool to investigateprotein dynamics and interactions in vivo over extended peri-ods of time. Further development of FRET assays using QDsand QD conjugates tethered to surfaces could open up thepossibility of designing and implementing regenerable sensingassemblies and devices.

However, there remain limitations to using these inorganicfluorophores in FRET-based investigations, most notably in bio-logical applications. Due to their large and broad excitationspectra and long exciton lifetimes, they may not be useful asacceptor fluorophores. Their relatively large size and the need

Figure 9. A) changes in emission spectra upon the time-dependent DNA replication. Before addition of TR–dUTP,orange; after 1, 30, and 60 min of replication, black, purple, and blue, respectively. B) Time-dependent intensity ofthe QD and TR during replication: decrease of QD emission (&), increase of dye contribution (*). Adapted fromref. [35] with permission from the American Chemical Society.


H. Mattoussi et al.


for surface functionalization can increase the overall dimen-sions of the bioconjugates. Despite the substantial progressmade in the last several years to prepare water-compatibleand functional well-dispersed QDs, problems remain includingaggregation, limited pH stability, and large QD–bioreceptorconjugate sizes, that still need to be solved. Unfortunately, afew publications reporting FRET with luminescent QDs lackednecessary control experiments to prove unequivocally thatenergy transfer was the only means of interaction and notother uncontrolled processes. As progress is made in preparingadditional QD materials and designing additional QD bioconju-gates with good control over conjugate architecture and func-tionality, better understanding of energy transfer applied to lu-minescent QDs and further refinement of FRET-based QD sens-ing assemblies that are specific will be developed.

Acknowledgements

The authors acknowledge NRL and ONR for support. A.R.C. is sup-ported by a National Research Council Fellowship through theNaval Research Laboratory. H.M. acknowledges Drs. A. Ervin andL. Chrisey at the Office of Naval Research (ONR) and A. Krishnanat DARPA for research support.

Keywords: bioconjugates · fluorescence · FRET (fluorescenceresonance energy transfer) · nanocrystals · quantum dots

[1] T. Fçrster, Discuss. Faraday Soc. 1959, 27, 7–17.[2] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed. , Kluwer

Academic, New York, 1999.[3] A. R. Clapp, I. L. Medintz, J. M. Mauro, B. R. Fisher, M. G. Bawendi, H. Mat-

toussi, J. Am. Chem. Soc. 2004, 126, 301–310.[4] I. L. Medintz, A. R. Clapp, H. Mattoussi, E. R. Goldman, J. M. Mauro, Nat.

Mater. 2003, 2, 630–638.[5] E. A. Jares-Erijman, T. M. Jovin, Nat. Biotech. 2003, 11, 1387–1395.[6] A. Miyawaki, Developmental Cell 2003, 4, 295–305.[7] P. Wu, L. Brand, Anal. Biochem. 1994, 218, 1–13.[8] V. V. Didenko, Biotechniques 2001, 31, 1106–1121.[9] M. Fehr, W. B. Frommer, S. Lalonde, Proc. Natl. Acad. Sci. USA, 2002, 99,

9846–9851.[10] C. B. Murray, D. J. Norris, M. G. Bawendi, J. Am. Chem. Soc. 1993, 115,

8706–8715.[11] M. A. Hines, P. Guyot-Sionnest, J. Phys. Chem. B 1996, 100, 468–471.[12] B. O. Dabbousi, J. Rodriguez Viejo, F. V. Mikulec, J. R. Heine, H. Mattoussi,

R. Ober, K. F. Jensen, M. G. Bawendi, J. Phys. Chem. B 1997, 101, 9463–9475.

[13] C. A. Leatherdale, W. K. Woo, F. V. Mikulec, M. G. Bawendi, J. Phys. Chem.B 2002, 106, 7619–7622.

[14] J. K. Jaiswal, H. Mattoussi, J. M. Mauro, S. M. Simon, Nat. Biotechnol.2003, 21, 47–51.

[15] C. R. Kagan, C. B. Murray, M. Nirmal, M. G. Bawendi, Phys. Rev. Lett. 1996,76, 1517–1520.

[16] a) C. R. Kagan, C. B. Murray, M. G. Bawendi, Phys. Rev. B 1996, 54, 8633–8643; b) C. R. Kagan, Ph. D. Dissertation, Massachusetts Institute of Tech-nology, 1997.

[17] S. A. Crooker, J. A. Hollingsworth, S. Tretiak, V. I. Klimov, Phys Rev. Lett.2002, 89, 186802.

[18] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Science 1998,281, 2013–2015.

[19] W. C. W. Chan, S. Nie, Science 1998, 281, 2016–2018.[20] H. Mattoussi, J. M. Mauro, E. R. Goldman, G. P. Anderson, V. C. Sundar,

F. V. Mikulec, M. G. Bawendi, J. Am. Chem. Soc. 2000, 122, 12142–12150.[21] D. M. Willard, L. L. Carillo, J. Jung, A. Van Orden, Nano Lett. 2001, 1,

469–474.[22] S. Wang, N. Mamedova, N. A. Kotov, W. Chen, J. Studer, Nano Lett. 2002,

2, 817–822.[23] J. F. Hainfeld, W. Liu, C. M. R. Halsey, P. Freimuth, R. D. Powell, J. Struct.

Biol. 1999, 127, 185–198.[24] P. T. Tran, E. R. Goldman, G. P. Anderson, J. M. Mauro, H. Mattoussi, Phys.

Stat. Sol. B 2002, 229, 427–432.[25] H. E. Grecco, K. A. Lidke, R. Heintzmann, D. S. Lidke, C. Spagnuolo, O. E.

Martinez, E. A. Jares-Erijman, T. M. Jovin, Microscopy Res. Technique2004, 65, 169–179.

[26] N. M. Mamedova, N. A. Kotov, A. L. Rogach, J. Studer, Nano Lett. 2001, 1,281–286.

[27] K. Hanaki, A. Momo, T. Oku, A. Komoto, S. Maenosono, Y. Yamaguchi, K.Yamamoto, Biochem. Biophys. Res. Comm. 2003, 302, 496–501.

[28] A. R. Clapp, I. L. Medintz, B. R. Fisher, G. P. Anderson, H. Mattoussi, J. Am.Chem. Soc. 2005, 127, 1242–1250.

[29] M. Achermann, M. A. Petruska, S. Kos, D. L. Smith, D. D. Koleske, V. I.Klimov, Nature 2004, 429, 642–646.

[30] M. Anni, L. Manna, R. Cingolani, D. Valerini, A. Creti, M. Lomascolo, App.Phys. Lett. 2004, 85, 4169–4171.

[31] I. L. Medintz, J. H. Konnert, A. R. Clapp, I. Standish, M. E. Twigg, H. Mat-toussi, J. M. Mauro, J. R. Deschamps, Proc. Nat. Acad. Sci. USA, 2004,101, 9612–9617.

[32] I. L. Medintz, S. A. Trammell, H. Mattoussi, J. M. Mauro, J. Am. Chem. Soc.2004, 126, 30–31.

[33] E. R Goldman, I. L. Medintz, J. L. Whitley, A. Hayhurst, A. R. Clapp, H. T.Uyeda, J. R. Deschamps, M. E. Lassman, H. Mattoussi, J. Am. Chem. Soc.2005, 127, 6744–6751.

[34] F. Patolsky, Y. Weizmann, I. Willner, J. Am. Chem. Soc. 2002, 124, 770–771.

[35] F. Patolsky, R. Gill, Y. Weizmann, T. Mokari, U. Banin, I. Willner, J. Am.Chem. Soc. 2003, 125, 13918–13919.

[36] A. C. Samia, X. Chen, C. Burda, J. Am. Chem. Soc. 2003, 125, 15736–15737.

[37] J. H. Kim, D. Morikis, M. Ozkan, Sensors Actuators B 2004, 102, 315–319.[38] E. Oh, M. Y. Hong, D. Lee, S. H. Nam, H. C. Yoon, H. S. Kim, J. Am. Chem.

Soc. 2005, 127, 3270–3271.[39] Y. Ebenstein, T. Mokari, U. Banin, J. Phys. Chem. B 2004, 108, 93–99.[40] Z. Gueroui and A. Libchaber, Phys Rev. Lett. 2004, 93, 166108.[41] J. A. Kloepfer, N. Cohen, J. I. Nadeau, J. Phys. Chem. B 2004, 108, 17042–

17049.[42] K. E. Sapsford, I. L. Medintz, J. P. Golden, J.R Deschamps, H. T. Uyeda, H.

Mattoussi, Langmuir 2004, 20, 7720–7728.[43] F. MUller, S. Gçtzinger, N. Gaponik, H. Weller, J. Mlynek, O. Benson, J.

Phys. Chem. B 2004, 108, 14527–14534.

Received: April 18, 2005Published online on December 21, 2005




fçrster resonance energy transfer

Documents