Occurrence of Förster Resonance Energy Transfer between Quantum Dots and Gold Nanoparticles in the...

Published: September 19, 2011

r 2011 American Chemical Society 20840 dx.doi.org/10.1021/jp204456k | J. Phys. Chem. C 2011, 115, 20840–20848

ARTICLE

pubs.acs.org/JPCC

Occurrence of F€orster Resonance Energy Transfer between QuantumDots and Gold Nanoparticles in the Presence of a BiomoleculeGopa Mandal, Munmun Bardhan, and Tapan Ganguly*

Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India

1. INTRODUCTION

Fluorescent semiconductor nanoparticles have drawn consid-erable interest due to their size-dependent optical properties1

and their vast applications in photonics. In nanoparticles a largepercentage of the atoms are in the surface, rather than in the bulkphase. Nanoparticles can be made from a wide variety ofmaterials including CdS, ZnS, Cd3P2, and PbS, to name a few.The nanoparticles frequently display photoluminescence andsometimes display electroluminescence.2�7 Additionally, somenanoparticles can form self-assembled arrays.8,9 Because ofthose favorable properties, nanoparticles are being extensivelystudied for use in optoelectronic displays. Recently, there hasbeen a growing interest in the study of nanoparticles in biomo-lecules such as proteins, DNA, and peptides for biophotonicapplications.10�13 The luminescent quantum dots promise to bean attractive alternative for biolabeling and biosensing applica-tions. They emit bright and steady fluorescence. Among a varietyof semiconductor nanoparticles, CdS has been intensivelystudied.14�17 Nanocrystalline semiconductor materials such asPbS and CdS have attracted considerable attention due to theirunique properties, which are not present in bulk materials.18�20

CdS, in particular, has been extensively studied due to itspotential applications such as field effect transistors, light-emit-ting diodes, photocatalysis, and biological sensors.21�23

On the other hand bovine serum albumin (BSA),most abundantprotein in plasma, is a commonly used reagent in biological study. Itplays an important role in many physiological functions as it is themajor soluble protein constituent of the circulatory system. It isresponsible for drug deposition and efficacy.24 Recently, thesebiomolecules are utilized in many applications apart from beingthe building block of “life”.25 Recent addition to their repertoire isthe conjugation with nanoparticles (NPs). BSA, a commonly usedreagent in biological studies, has long been used as a capping agentfor various nanoparticles.26�29 These bioconjugate nanoparticlesare of great importance because of their potential applications inluminescence tagging, imaging, medical diagnostics, and mostimportantly as biosensors as well as for assembling hybrid pro-tein�NP units for molecular electronics. Several studies on the

Received: May 12, 2011Revised: August 13, 2011

ABSTRACT: In this paper the interactions of gold nanoparticles (Au NPs) andbovin serum albumin�gold nanoconjugates (BSA�GNPs) with cadmiumsulfide quantum dots (CdS QDs) are investigated by using steady-state andtime-resolved spectroscopic techniques under physiological conditions (pH ∼ 7).From the analysis of the steady-state and time-resolved fluorescence quenchingof CdS QDs in aqueous solution in the presence of BSA�GNPs it has beeninferred that fluorescence resonance (F€orster type) energy transfer (FRET) isprimarily responsible for the quenching phenomenon. But in the presence of onlyAu NPs the fluorescence quenching of CdS NPs is primarily static in nature.Thus, it is apparent that, in the presence of BSA (in the case of the bionano-conjugate system), FRET becomes operative between CdSQDs and AuNPs present in the BSA�GNPs bionanoconjugate, whereasin the absence of this biomolecule direct contact between CdS and Au NPs facilitates the formation of ground-state complex. Asshown from the high-resolution transmission electron microscopy (HRTEM) images of the bionanoconjugate, formation of a thinBSA layer around the Au NPs, situated at the core, inhibits the CdS QDs to come in contact with the Au NPs. In theCdS�bionanoconjugate system, CdS and Au NPs become separated by a distance of ∼17 ( 2 Å, as observed from HRTEMmeasurement. It may be presumed that when Au NPs are present in the bionanoconjugate system, the system may suffer someconformational changes which facilitates the energy transfer process to occur within the CdS QDs and the Au NPs. Furtherinvestigations with similar systems would be necessary to make unequivocal assertion of this phenomenon. From the determinationof the thermodynamic parameters it is apparent that the effect of van der Waals interaction is responsible for the interaction of CdSQDs with AuNPs to form ground-state complex. The effect of CdSNPs on the conformation of BSA�GNPs has been examined byanalyzing CD spectra. Though the observed results demonstrate some conformational changes in the bionanoconjugate in thepresence of CdSNPs, the secondary structure of the conjugate, predominantly of theα-helix, is found to retain its identity. This typeof interaction between QDs and Au NPs in a protein-conjugated form provides a new insight for design and the development ofFRET-based bionanosensors.

20841 dx.doi.org/10.1021/jp204456k |J. Phys. Chem. C 2011, 115, 20840–20848

The Journal of Physical Chemistry C ARTICLE

bioconjugate nanoparticles have been reported. Protein conjugationhas also been used as a strategy for increasing colloidal stability,30�32

conferring biochemical activity,33,34 and enhancing biocom-patibility35�37 in various nanoparticles systems.

The present article reports the interactions of CdS quantumdots (QDs) with bovin serum albumin�gold nanoconjugates(BSA�GNPs) and Au NPs by absorption and time-resolvedfluorescence spectroscopy. Additionally, the conformationalchanges of BSA�GNPs conjugates by CdS NPs were analyzedby circular dichroism (CD) spectra. The primary aim of our workis to compare and to reveal the nature of interactions of CdS NPswith BSA�GNPs conjugates and Au NPs. To elucidate the truepicture, the detailed spectroscopic investigations on such inter-actions have been done.

2. EXPERIMENTAL METHODS

2.1. Materials. Bovine serum albumin (fatty acid free) waspurchased from Aldrich and directly used from the package.

The procedure of synthesis and characterization of CdS NPs isdescribed elsewhere.38 NPs are routinely coated with inorganicshells to impart solubility in biological media. The NP coatingcan isolate the reactive metal ions of the core from the cell andcan thus prevent oxidative stress. Encapsulation of CdS or CdSeQDs with a zinc sulfide shell helps them to be soluble in waterand decreases their toxicity dramatically.39�42 But here tosolubilize the CdS NP we use MPA (HSCH2CH2COOH) ascapping agent, not ZnS.We have used phosphate buffer for preparing solutions through-

out the course of investigations. The pH of phosphate buffer used inthe experiment is 7. The buffer solution is prepared in Milliporewater. The solvent is tested before use to check any unwantedimpurity emission in the concerned wavelength region.2.1.1. Preparation of BSA�GNPs. The procedure for BSA�

GNPs preparation was described previously.43a,b The isoelectricpoint of BSA is between 4.5 and 4.9.44�46 Therefore, in thephosphate buffer solution at pH value ∼7.0, the BSA moleculehas a significant number of negative ions attached on the surface,

Figure 1. (A) TEM picture of CdS NPs; (B) magnified view of a single CdS NP; (C) BSA�Au conjugate (inset, magnified view of BSA�Aunanoconjugate); (D) BSA�Au�CdS conjugate; white arrows indicate the thickness of BSA; red spheres show CdS QDs; (E) Au�CdS conjugate; thesky blue sphere shows the Au NP, and the red spheres show CdS QDs; (F) magnified view of BSA�Au�CdS nanoconjugate.



which forms the attractive Columbic interaction between theprotein and the Au NPs.2.2. Apparatus. At the ambient temperature (296 K) steady-

state UV�vis absorption and fluorescence emission spectra ofdilute solutions (10�5 to 10�7 M) of the samples were recordedusing 1 cm path length rectangular quartz cells by means of anabsorption spectrophotometer (Shimadzu UV�vis 2401PC)and F-4500 fluorescence spectrophotometer (Hitachi), respec-tively. Fluorescence lifetime measurements were carried out bythe time-correlated single-photon counting (TCSPC) methodusing a Horiba Jobin Yvon Fluorocube. For fluorescence lifetimemeasurements the samples were excited at 375 nm using apicosecond diode (IBH Nanoled-07). The emission was col-lected at a magic-angle polarization using a Hamamatsu micro-channel plate photomultiplier (2809U). The TCSPC setupconsists of an Ortec 9327 CFD and a Tennelec TC 863 TAC.The data is collected with a PCA3 card (Oxford) as a multi-channel analyzer. The typical full width at half-maximum (fwhm)of the system response is about 238 ps. The channel width is12 ps/channel. The fluorescence decays were deconvoluted usingIBH DAS6 software. The goodness of fit has been assessed overthe full decay including the rising edge with the help of statisticalparameters χ2 and Durbin�Watson (DW). CD has been re-corded by a JASCOCD spectrometer, model J-815-150S, using a0.1 cm path length quartz cell in a wavelength range between 200and 260 nm. The size and dispersivity of the nanoparticles weredetermined from a high-resolution transmission electron micro-graph (HRTEM) (JEOL, model JEM-2010) in which sampleswere dropped onto a copper grid covered with a thin film ofamorphous carbon.

3. RESULTS AND DISCUSSION

3.1. UV�Vis Absorption and Steady-State FluorescenceStudies. Figure 1 shows the HRTEM images of CdS, BSA�Auconjugate, BSA�Au�CdS nanoconjugates, and Au-conjugatedCdS QD nanoparticles. It is seen from the TEM images(Figure 1, parts D and F) that Au NPs being surrounded byBSA protein form a core (Au)�shell (BSA)-type structure andCdS QDs are attached to this core�shell structure type con-jugate. It is seen from the TEM images that the BSA moleculesprevent the Au NPs from interacting with CdS QDs directly. Butin the absence of protein, the HRTEM image (Figure 1E) clearly

demonstrates that CdS QDs are directly connected with thesurface of AuNPs. From the TEM image, it is seen that the size ofCdS QDs is 2�5 nm.From UV�vis absorption studies an absorption band peaking

at around 415 nm is observed (Figure 2) for CdS QDs, whichindicates the narrow distribution of the nanoparticles. It is well-known that the diameter of the particles is related with theabsorption edge,47�49 andwemeasured the size of the nanoparticlesusing the equation 2RCdS = 0.1/(0.1338� 0.0002345λe) nm,wherethe absorption edge is λe and it is calculated from the intersection ofthe sharply decreasing region of the spectrum with the baseline.47

The estimated particles size is 3.4 nm for the 444 nm absorptionedge. This calculated size is of slightly lower value than that of mostof the particles observed from the TEM picture.Figure 2 shows the regular decrement of UV�vis absorption

spectra of CdS QDs near the domain of 415 nm in the presenceof increasing concentration of Au NPs at pH ∼ 7. On the otherhand, the UV�vis absorption spectra of CdS QDs remain more-or-less unchanged in the presence of BSA�GNPs nanocompo-site (not shown). In the former case the decrement is presumablydue to the formation of ground-state complex that resulted fromthe intermolecular interactions.Fluorescence behavior of CdS QDs, measured by the excita-

tion at 375 nm, in the presence of varying concentrations ofBSA�GNPs and at pH∼ 7 is shown in Figure 3. After excitationat 375 nm, the two emission peaks (Figure 3) at 437 and 576 nmare observed for CdS QDs. The peak at∼437 nm corresponds tothe band�band and 576 nm is due to the lattice defect emissionfrom host CdS, respectively.50�54 At 415 nm excitation, only onepeak at 576 nm is observed, but the intensity of the peak isrelatively strong (inset of Figure 3) compared to that observedfor 375 nm excitation. Here, the observed photoluminescence(PL) broadening may be due to the ensemble of different sizes ofcolloidal nanoparticles. BSA�GNPs bioconjugate causes quenching

Figure 2. Steady-state UV�vis absorption spectra of CdS NPs in thepresence of increasing concentration of GNPs (concentration of CdSNPs∼2� 10�6 M); [GNPs] ranges from (1) 0 to (2) 6.64� 10�7, (3)1.3 � 10�6, and (4) 3.28 � 10�6 M.

Figure 3. Fluorescence emission spectra (λex = 375 nm) of CdS NPs inthe presence of increasing concentration of BSA�GNPs (concentrationof CdS NPs ∼2 � 10�6 M); [BSA�GNPs] ranges from (0) 0 to (1)6.64� 10�7, (2) 1.3� 10�6, (3) 1.98� 10�6, (4) 2.63� 10�6, and (5)3.28 � 10�6 M at 298 K. Inset: fluorescence emission spectra (λex =415 nm) of CdS NPs in the presence of increasing concentration ofBSA�GNPs (concentration of CdS NPs ∼2 � 10�6 M); [BSA�GNPs] ranges from (0) 0 to (1) 6.64� 10�7, (2) 1.3� 10�6, (3) 1.98�10�6, (4) 2.63 � 10�6, and (5) 3.28 � 10�6 M at 298 K.



of fluorescence emission of CdS NPs. To reveal the nature of thequenching mechanism of the fluorescence emission band of CdSQDs peaking at about 437 nm in the presence of BSA�GNPs, theStern�Volmer (SV) relation has been applied.The Stern�Volmer equation is given by55

f0=f ¼ 1 þ Ksv½Q �¼ 1 þ kqτ0½Q �

where f0 and f denote the steady-state fluorescence emissionintensities of the fluorescer in the absence and in the presence ofquencher, respectively. KSV is the Stern�Volmer quenchingconstant, and [Q] is the concentration of quencher (BSA�GNPs). kq is the bimolecular quenching rate constant. τ0 is thefluorescence lifetime of the free fluorescer, i.e., in the absence ofany quencher.The observed linearity in SV plots (Figure 4a) suggests that

the quenching may be of either pure dynamic or pure staticnature.55 Generally, deviations from linearity indicate the mixingof both dynamic and static quenching processes. However, to

locate the true nature of the quenching, time-resolved fluores-cence measurements are made (vide infra). It is observed fromthe measurements that the fluorescence lifetime of the CdS QDsis gradually shortened with increasing concentrations of BSA�GNPs. This observation clearly demonstrates the dynamicnature of quenching.3.2. Time-Resolved Fluorescence Studies.The fluorescence

lifetimes of CdS QDs are measured in the absence and presenceof BSA�GNPs. The decay of emission was monitored at 440 nm.The best-fitted curves for the decays are obtained by using athree-exponential fit. The lifetime data have been given inTable 1. CdS emission consists of a range of lifetimes whichare almost independent of the average cluster size.56 It is well-known that photoexcited electrons of CdS NPs can decay byradiative or nonradiative pathways, and the quantum efficiency isgiven by η = τR

�1/(τR�1 + τNR

�1), where τR�1 and τNR

�1 arethe radiative and nonradiative surface recombination, respec-tively. Matsuura et al.57 suggested that the fast component comesfrom the free-exciton emission, while the slow componentoriginates from the bound exciton emission at shallow localizedstates of CdS NPs. O’Neil et al.58 also reported that the decays ofCdS NPs are composed of two distinct time regimes. They havedescribed the slow and fast components by a distribution kineticmodel and thermal repopulation mechanisms.The average emission lifetime, Æτæ, was calculated by the

following expression59

τh i ¼ ∑ aiτi2=∑ aiτi

where ai and τi denote the preexponential factor and thecorresponding lifetime, respectively. The fluorescence decaysof CdS QDs in the presence of different concentrations ofBSA�GNPs are triple-exponential in nature. From the com-puted average decay time by using the above eq it reveals thatthere is a shortening of decay time of CdS NPs in the presence ofbionanoconjugate.But no change in lifetime of CdSQDswith the addition of only

Au NPs (or BSA) is observed. Ghali60 reported the static natureof the BSA�CdS ground-state complex. In the present case itmay be concluded from the unperturbed fluorescence lifetime ofCdS in the presence of Au NPs that emission quenching of CdSQDs is primarily static in nature and initiated mainly due toground-state complex formation between CdS and Au. Thepossibility of ground-state complex formation is also apparentfrom the UV�vis spectra, as discussed above. The static nature ofthe CdS�Au complex is most likely due to the specific interac-tion of gold with sulfur61�66 in the ground state.But in the presence of the bionanoconjugate of Au and protein

BSA, the quenching of CdS fluorescence emission is turned to bedynamic in nature. Time-resolved fluorescence spectroscopydemonstrates that the emission quenching of CdS QDs occurs

Figure 4. (a) Stern�Volmer plot of quenching of CdS NPs byBSA�GNPs at different temperatures. (b) Stern�Volmer (SV) plotfrom fluorescence lifetime measurements (time-resolved) of a mixtureof CdS NPs and BSA�GNPs.

Table 1. Time-Resolved Fluorescence Data (fi ∼FractionalContribution) of a Mixture of CdS QDs and BSA�GNPsConjugate at Ambient Temperature (λex ∼ 375 nm)

concn of

conjugate (M) τ1 (ns) f1 τ2 (ns) f2 τ3 (ps) f3 Æτæ (ns)

0 3.41 0.36 28.9 0.39 115.6 0.25 26.35

6.64 � 10�7 3.34 0.35 27.4 0.31 66.7 0.34 24.45

1.98 � 10�6 3.36 0.35 26.01 0.27 62.3 0.38 22.70

2.63 � 10�6 3.11 0.33 23.05 0.26 55.36 0.41 19.50



primarily due to the involvement of F€orster resonance energytransfer from CdS QDs to Au of BSA�GNPs (vide infra).3.3. Temperature Effect. The temperature-dependent stud-

ies on fluorescence quenching of CdS NPs in the presence ofBSA�GNPs have been carried out. The SV plots at differenttemperatures are shown in Figure 4a. The plots are linear innature. The kq values at different temperatures are estimated tobe 4.2� 1012 (288K), 6.4� 1012 (298K), and 8.7� 1012 (308 K)M�1 s�1, respectively. The observation of regular increase ofbimolecular quenching rate with temperature suggests in favor ofthe dynamic nature of quenching of CdSQDs fluorescence in thepresence of BSA�GNPs. As the bimolecular quenching rateconstants are much greater than the maximum collisionquenching constant of various kinds of quenchers to fluor-escers (∼2 � 1010 M�1 s�1, which is the diffusion-controlledlimit of the rate constant), it is apparent that the quenchingoccurs via Coulombic resonance interaction. Such types ofquenching constants exceeding the diffusion-controlled valuewere reported earlier.67�71

Therefore, the high magnitude of the observed kq in this casecould be due to the occurrences of primarily energy transferfrom CdS NPs (E = 2.99 eV) to Au (E = 2.34 eV) ofBSA�GNPs bionanoconjugate. The possibility of occurrenceof another nonradiative process, intermolecular photoinducedelectron transfer, appears to be slim within CdS and Au NPs asin the presence of the latter NPs, the quenching in thefluorescence emission of CdS QDs appears to be static innature (as discussed above). However, the possibility of theoccurrence of electron transfer from CdS to Au has beendiscussed by Kamat and Shanghavi,72 but in their compositenanocluster system.Moreover, as f0/f ≈ τ0/τ for a concentration of the quencher

as derived from both the SV plots constructed from steady-stateand time-resolved fluorescence (Figure 4b), the dynamic natureof the quenching is further substantiated.73,74 The lack offormation of ground-state complex is observed, as discussed insection 3.1, between CdS and Au NPs when the latter is presentwithin the bionanoconjugates.Similar emission quenching phenomena are observed in the

case of exciting CdS in the presence of only Au NPs and BSA.The observed linearity in both the cases indicates to only onetype of quenching, pure dynamic or static. From the SV relationof the linear plots of f0/f against [Q ] in case of CdS�AuNPs, theSV quenching constant (KSV) and bimolecular quenching rateconstant (kq) are computed at different temperatures. Thekq values at different temperatures are estimated to be 1.4 � 1013

(288 K), 9.5 � 1012 (298 K), and 7.2 � 1012 (308 K) M�1 s�1,respectively. This observation demonstrates the static nature offluorescence quenching, and the increased temperature appearsto decrease the stability of the ground-state complex. So it can beconcluded that the quenching mechanism of CdS NPs in thepresence of only Au NPs is initiated by ground-state complexformation55 rather than by dynamic collision. The same result isobtained in the case of fluorescence emission quenching of CdSby BSA as reported earlier.60

3.4. F€orster Resonance Energy Transfer. The QD-basedF€orster resonance energy transfer (FRET) is a significantphenomenon in fluorescence spectroscopy. This process pos-sesses the ability of tuning of emission properties with changingsize of QDs for efficient energy transfer within various organicdyes and proteins. FRET has various applications in biologicalsystems such as photosensitization.55,75 It involves the transfer

of excited-state energy from an initially excited donor to anacceptor and is used for measuring molecular distances ordonor�acceptor proximity. FRET is an important tool forinvestigating various biological phenomena involving energytransfer and is extensively used to investigate the structure,conformation, spatial distribution, and assembly of complexproteins.75 Thus, energy transfer could be used as a “spectro-scopic ruler” for the measurement of distances between thedonors and acceptors.Since the fluorescence spectra of CdS NPs overlap consider-

ably with the absorption spectra of BSA�GNPs, it furthersubstantiates our presumption that the fluorescence quenchingoccurs due to energy transfer from CdS NPs to Au ofBSA�GNPs. The energy transfer efficiency has been studiedaccording to the F€orster nonradiative energy transfer theory. Theenergy transfer efficiency (E) is not only related to the center-to-center distance (r) between the acceptor and donor but also tothe F€orster critical energy transfer distance (R0)

E ¼ R06

R06 þ r6

ð1Þ

where R0 is the F€orster’s critical transfer distance when thetransfer efficiency is 50%.

R06 ¼ 8:8� 10�25k2N�4ΦJ ð2Þ

where k2 is the spatial orientation factor of the dipole, N is therefractive index of the medium, Φ is the fluorescence quantumyield of the donor, J is the effect of the spectral overlap betweenthe fluorescence emission spectrum of the donor CdS QDs andthe absorption spectrum of the acceptor BSA�GNPs, whichcould be computed by the equation

J ¼

Z ∞

0FðλÞεðλÞλ4 dλ

Z ∞

0FðλÞ dλ

ð3Þ

where F(λ) is the fluorescence intensity of the donor at wave-length range λ and ε(λ) is the molar absorption coefficient of theacceptor at λ.In aqueous solution k2 = 2/3, N = 1.335, andΦ = 0.1134 for

CdS NPs. Using these we get the following values: J = 4.8 �10�14 M�1 cm3, R0 = 31.6 Å, r = 37.6 Å. In calculation of “r”, thecenter�center distance between QD and Au NPs of the bio-conjugate system (which was observed as 37.6 Å), we haveneglected the distance of the bond of MPA involved because it isconsiderably smaller (<3 Å) than the distance 37.6 Å whichincludes the shell thickness of BSA (17 Å) along with the radii ofthe reacting components, CdS QD and Au.Thus, the linker between CdS and BSA�Au NPs in the true

sense is MPA, whose length being considerably smaller com-pared to the sum of the radii of the reacting components CdS andAu along with the protein shell thickness has been neglected incomputing the center-to-center distance.Since the average distance 0.5R0 < r < 1.5R0

32,75,76 energytransfer takes place with some probability. The energy transferefficiency from CdS NPs to bionanoconjugate is estimatedaccordingly: ET = 1 � τDA/τD where τDA is the decay time ofCdSNPs in the presence of BSA�GNPs and τD is the decay timeof CdS NPs without BSA�GNPs. The calculated energy transferefficiency is 26%.



It is interesting to note that energy transfer is found to occurfrom CdS QDs to BSA�GNPs conjugate but not in the case ofthe CdS�AuNP system. It is to be mentioned in this connectionthat in the bionanoconjugate system, Au NPs being situated inthe core position are protected from CdS QDs by the thinenvelop (shell type) of BSA protein, as evidenced from theHRTEM image (Figure 1D). No direct contact is possiblebetween QDs and Au NPs. It is evident that QDs and Au NPsare within a short distance (edge-to-edge distance ∼17 ( 2 Å,from TEMmeasurements), and in such a situation occurrence ofenergy transfer from the donor CdS QD to acceptor Au, presentin the BSA�Au bionanoconjugate system (Scheme 1) is appar-ent. It may be presumed that when Au NPs are present in thebionanoconjugate system, the system may suffer some confor-mational changes through orientation which possibly facilitatesthe energy transfer process to occur. Further investigations withsimilar systems would be necessary to make unequivocal asser-tion of this phenomenon.On the other hand, in the absence of protein, due to direct

contact between CdS and Au NPs (Figure 1E) formation ofground-state complex is evident as observed from absorption,static nature fluorescence quenching, and time-resolved experi-ments (Scheme 2).3.5. Number of Binding Sites and Binding Constant. To

reveal the nature of interactions of CdS NPs with BSA�GNPs

and Au NPs the binding constant value and binding site arecalculated from the following equation:

logF0 � F

F

� �¼ log Kb þ n log½Q �

whereKb is the binding constant, which can be determined by theslope of the log[((F0 � F)/F)] versus log[Q] curve as shown inFigure 5. The values for binding constant Kb and binding sites nare obtained from the intercept and slope of Figure 5 (Kb = 4.3�104 M�1, n = 0.97) at 298 K, whereas for Au NPs the bindingconstant and binding sites areKb = 1.6� 105M�1, n = 1.05 at thesame temperature. Tables 2 and 3 show the results at differenttemperatures for BSA�GNPs and Au NPs, respectively. The R2

values are larger than 0.99, and the standard deviation is less than0.05. From Table 3 it can be shown that Kb and n decrease withincrease in temperature which indicates to the formation of anunstable compound. But in the case of BSA�GNPs, Kb and nincrease with increase in temperature (Table 2).3.6. Thermodynamic Parameters and Nature of the Bind-

ing Forces. In general, the interactions between small moleculesand biological macromolecules may result from four types offorces: hydrogen bonds, van der Waals force, electrostaticinteractions, and hydrophobic interaction, etc. According tothe data of enthalpy change and entropy change, the model ofinteraction can be concluded as77,78

(1) ΔH > 0 and ΔS > 0, hydrophobic forces;(2) ΔH < 0 and ΔS < 0, van der Waals interactions and

hydrogen bonds;(3) ΔH < 0 and ΔS > 0, electrostatic interaction.If the enthalpy change does not vary significantly over the

temperature change studied, then its value and that of entropycan be calculated from the van’t Hoff equation:

ln K ¼ ΔSR

�ΔHRT

where K is analogous to the binding constant at the correspond-ing temperature andR is the gas constant. The free energy changecan be obtained from the following relationship:

ΔG ¼ ΔH � TΔS ¼ � RT ln K

Scheme 1. Schematic Diagram of the FRET Binding Assay

Scheme 2. Schematic Diagram of Ground-State ComplexFormationa

a For clarity, only one semiconductor�metal complex is shown. The tworeaction pathways are denoted by I and II.

Figure 5. Plot to measure the binding constants of the CdS NPs toBSA�GNPs at 298 K.



According to the above two eqs the values of ΔG, ΔH, and ΔSwere obtained and shown in Tables 2 and 3. From the negativevalue of ΔG it appears that the process of interaction isspontaneous. In case of interaction of CdS QDs and Au NPs(Table 3) the negative enthalpy (ΔH) and negative entropy(ΔS) values indicate the van der Waals interaction may beresponsible for the formation of ground-state complex.3.7. Circular Dichroism Spectra. To explore the structural

change of BSA�GNPs, a CD experiment at room temperaturehas been carried out in the absence and in the presence of CdSQDs (Figure 6). The addition of nanoparticles in the bionano-conjugate causes very minor α helicity changes in the CDspectra.The CD results are expressed in terms of mean residue

ellipticity (MRE) in deg cm2 mol�1 according to the followingequation.79

½θλ� ¼ θ

10nlc

where c is the concentration of BSA�GNPs in g/mL, θ is theobserved rotation in deg, l is the path length in cm, and n is thenumber of amino acid residues of protein (583 for BSA).

The α-helix contents of BSA have been calculated from MREvalues at 208 nm using the following equation:80

α-helix ð%Þ ¼ �½θ�208 � 400033000� 4000

where [θ]208 is the observed MRE value at 208 nm, 4000 is theMRE of the β-form and random coil conformation cross at208 nm, and 33 000 is theMRE value of a pureα-helix at 208 nm.According to the above equations the percentage of α-helix of

BSA�GNPs is computed, and as can be seen the percentage ofhelicity of BSA�GNPs is 14.52% in the absence of CdSQDs, andin the presence of CdS QDs, it decreases to 14.27%. As thechange inα-helical content of BSA�GNPs is not significant evenat optimum concentration of CdS, i.e., when BSA�GNPs aredispersed at high concentration of CdS, denaturation does notoccur, and the bionanoconjugate could retain most of its helicalstructure.43b This observation strongly indicates that the bindingof CdS QDs to BSA�GNPs induces some conformationalchanges in the conjugate. But the secondary structure of proteinis predominantly of α-helix which is very essential for thepreparation of protein-based assemblies of nanoconjugates. Asno significant structural deformation occurred, the biologicalactivity, the activity of immune response of the protein, andthe biocompatibility of the protein�nanoconjugate remain assuch. So bioconjugate Au NPs could pave the way to designnew optical-based materials having significant biomedicalapplications.81

3.8. Comparison between CdS�BSA�Au and CdS�AuSystems. In the case of the CdS�BSA�Au conjugate systemthe TEM images show that the BSA molecules prevent the AuNPs from interacting with CdS QDs directly. But in the absenceof protein, the HRTEM image (Figure 1E) clearly demonstratesthat CdS QDs are directly connected with the surface of Au NPs.In both cases the fluorescence spectra of CdS QDs werequenched in addition of the quencher. From time-resolved fluo-rescence data it reveals that there is a shortening of decay time ofCdS NPs (showing the quenching process is dynamic in nature)in the presence of bionanoconjugate, but in the case of only AuNPs there is no change in lifetime data (static in nature) of thefluorescers CdS QDs. From Table 3 it can be shown that thebinding constant (Kb) decreases with increase in temperature,

Table 2. Binding Constant Kb, Binding Sites n, and Relative Thermodynamic Parameters for CdS NPs with BSA�GNPsConjugate at Different Temperatures

T Kb (M�1) n R2 a SDb ΔH (kJ mol�1) ΔG (kJ mol�1) ΔS (J mol�1 K�1)

288 1.7� 103 0.67 0.99606 0.02086 �23.37

298 4.3� 104 0.97 0.98594 0.05713 146 �29.25 588.1

308 5.8� 104 0.98 0.99888 0.01615 �35.13a R2 is the correlation coefficient. b SD is the standard deviation.

Table 3. Binding Constant Kb, Binding Sites n, and Relative Thermodynamic Parameters for CdS NPs with Au NPs Interaction atDifferent Temperatures

T Kb (M�1) n R2 a SDb ΔH (kJ mol�1) ΔG (kJ mol�1) ΔS (J mol�1 K�1)

288 7.2� 105 0.96 0.99212 0.02154 �32.02

298 1.6� 105 1.05 0.99866 0.04013 �84.15 �32.21 �181

308 7.4� 104 1.2 0.99152 0.05015 �28.40a R2 is the correlation coefficient. b SD is the standard deviation.

Figure 6. CD spectra of BSA�GNPs conjugate (concentration ofBSA�GNPs∼2� 10�5 M) in the presence of increasing concentrationof CdS NPs; concentration of CdS NPs ranges from (1) 0 to (2) 8 �10�5 and (3) 1.5 � 10�4 M.



which indicates the formation of an unstable compound(CdS�Au). But in the case of CdS�BSA�GNPs, Kb increaseswith increase in temperature (Table 2). These observationsfurther substantiate our inference of occurrence of dynamicquenching in the case of the latter and the presence of staticmode in the case of the former.Moreover, it is interesting to note that energy transfer is found

to occur from CdS QDs to BSA�GNPs conjugate (FRETefficiency∼26%) but not in the case of the CdS�Au NP system(no FRET). In the latter case the observed experimental findingsdemonstrate that the formation of ground-state complex is onlyresponsible for the observed fluorescence quenching of CdS QDs.

4. CONCLUSIONS

In the present investigation the interactions of CdS QDs withthe BSA�GNPs and Au NPs are studied by the differentspectroscopic methods including UV�vis, steady-state andtime-resolved fluorescence, and CD in phosphate buffer at pH∼ 7. The shortening of the decay time as observed from time-resolved fluorescence and steady-state fluorescence quenchingbehavior of CdS QDs in the presence of BSA�GNPs is observedto be due to primarily the efficient FRET between CdS QDs andAu NPs. In this bionanoconjugate system, Au NPs being situatedin the core position are protected from CdS QDs by the thinenvelop (shell type) of BSA protein, as evidenced from HRTEMimages. Thus, CdS and Au NPs being separated by a shortdistance (∼17 ( 2 Å, from HRTEM measurement) in theCdS�bionanoconjugate system facilitates the energy transferprocess rather than formation of ground-state complex for whichthe direct contact is necessary. It may be presumed that when AuNPs are present in the bionanoconjugate system, the systemmaysuffer some conformational changes which facilitates the energytransfer process to occur within CdS QDs and Au NPs present asa core within the shell of BSA. Further investigations with similarsystems would be necessary to prove conclusively this presump-tion. The results of CD spectra indicate that the secondarystructure of the bionanoconjugate undergoes marginal changesin the presence of CdS NPs. As no significant structuraldeformation occurred, the biological activity, the activity ofimmune response of the protein, and the biocompatibility ofthe protein�nanoconjugate remain as such. So bioconjugate AuNPs could pave the way to design new optical-based materials,and the observed properties of the nanoassemblies are capable ofdeveloping FRET-based bionanosensors for their potentialapplications.

’AUTHOR INFORMATION

Corresponding Author*Phone: +91 33 2473 4971/3073, ext 253. Fax: +91 33 24732805. E-mail: [email protected], [email protected].

’ACKNOWLEDGMENT

The financial assistance provided by the Council of Scientificand Industrial Research (CSIR), New Delhi, India to G.M. in theform of NET-CSIR fellowships is gratefully acknowledged. Theauthors also thank Mr. Subrata Das of the Department ofSpectroscopy for assisting in the time-resolved fluorescencemeasurements. Thanks to Mr. Santanu Jana of the Departmentof Materials Science for helping to synthesize QDs. Thanks are

also due to Professor P. K. Das of Biological Chemistry for help-ing us in the measurements of CD spectra. T.G. specially thanksthe Department of Science and Technology (DST), New Delhi,India, for the Nano-Mission project (no. SR/NM/NS-51/2010)for supporting the various research works conducted in hisphotophysics and photochemistry group.

’REFERENCES

(1) Henglein, A. Ber. Bunsen-Ges. Phys. Chem. 1995, 99, 903.(2) Dabbousi, B. O.; Bawendi, M. G.; Onitsuka, O.; Rubner, M. F.

Appl. Phys. Lett. 1995, 66, 1316.(3) Colvin, V. L.; Schlamp,M.C.; Alivisatos, A. P.Nature 1994, 370, 354.(4) Zhang, L.; Coffer, J. L. J. Phys. Chem. B 1997, 101, 6874.(5) Artemyev, M. V.; Sperling, V.; Woggon, U. J. Appl. Phys. 1997,

81, 6975.(6) Huang, J.; Yang, Y.; Xue, S.; Yang, B.; Liu, S.; Shen, J. Appl. Phys.

Lett. 1997, 70, 2335.(7) Artemyev, M. V.; Sperling, V.;Woggon, U. J. Cryst. Growth 1998,

184/185, 374.(8) Shen, M. Y.; Goto, T.; Kurtz, E.; Zhu, Z.; Yao, T. J. Phys.: Condens.

Matter 1998, 10, L171.(9) Zhang, B. P.; Yasuda, T.; Wang, W. X.; Segawa, Y.; Edamatsu, K.;

Itoh, T.; Yaguchi, H.; Onabe, K. Mater. Sci. Eng. 1998, B51, 127.(10) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos,

A. P. Science 1998, 281, 2013.(11) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016.(12) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.;

Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630.(13) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher,

A. M. Nature 2000, 405, 665.(14) Spanhel, L.; Haase, M.; Weller, H.; Henglein, A. J. Am. Chem.

Soc. 1987, 109, 5649.(15) Hirai, T.; Okubo, H.; Komasawa, I. J. Phys. Chem. B 1999,

103, 4228.(16) Chae,W. S.; Ko, J. H.; Hwang, I.W.; Kim, Y. R.Chem. Phys. Lett.

2002, 365, 49.(17) Orii, T.; Kaito, S.; Matsuishi, K.; Onari, S.; Arai, T. J. Phys.:

Condens. Matter 2002, 14, 9743.(18) Henglein, A. Chem. Rev. 1989, 89, 1861.(19) Fukuoka, A.; Sakamoto, Y.; Guan, S.; Inagaki, S.; Sugimoto, N.;

Fukushima, Y.; Hirahara, K.; Iijima, S.; Ichikawa, M. J. Am. Chem. Soc.2001, 123, 3373.

(20) Petit, C.; Pilleni, M. P. J. Phys. Chem. 1988, 92, 2282.(21) Alivisatos, A. P. Science 1996, 271, 933.(22) Kolvin, V. L.; Schlamp, M. C.; Alivisatos, A. P. Nature 1994,

370, 354.(23) Klein, D. L.; Roth, R.; Lim, A. K. L.; Alivisatos, A. P. Nature

1997, 389, 699.(24) Olson, R. E.; Christ, D. D.Annu. Rep. Med. Chem. 1996, 31, 327.(25) Murawala, P.; Phadnis, S. M.; Bhonde, R. R.; Prasad, B. L. V.

Colloids Surf., B 2009, 73, 224.(26) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.;

Franzen, S. Langmuir 2005, 21, 9303.(27) Yang, L.; Xing, R.; Shen, Q.; Jiang, K.; Ye, F.; Wang, J.; Ren, Q.

J. Phys. Chem. B 2006, 110, 10534.(28) Mamedova, N. N.; Kotov, N. A.; Rogach, A. L.; Studer, J. Nano

Lett. 2001, 1, 281.(29) Elkin, T.; Jiang, X.; Taylor, S.; Lin, Y.; Gu, L.; Yang, H.; Brown,

J.; Collins, S.; Sun, Y.-P. ChemBioChem 2005, 6, 640.(30) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B

1998, 102, 9404.(31) Feng, J.-J.; Zhoa, G.; Xu, J.-J.; Chen, H.-Y. Anal. Biochem. 2005,

342, 280.(32) Yang, T.; Li, Z.; Wang, L.; Guo, C.; Sun, Y. Langmuir 2007,

23, 10533.



(33) Srivastava, S.; Verma, A.; Frankamp, B. L.; Rotello, V. M. Adv.Mater. 2005, 17, 617.(34) Katz, E.; Willner, I.; Wang, J. Electroanalysis 2004, 16, 19.(35) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.;

Sundar, V. C.; Mikulec, F. B.; Bawendi, M. G. J. Am. Chem. Soc. 2000,122, 12142.(36) Goldman, E. R.; Anderson, G. P.; Tran, P. T.; Mattoussi, H.;

Charles, P. T.; Mauro, J. M. Anal. Chem. 2002, 74, 841.(37) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat.

Biotechnol. 2003, 21, 47.(38) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183.(39) Rzigalinski, B. A.; Strobl, J. S. Toxicol. Appl. Pharmacol. 2009,

238, 280.(40) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004,

4, 11.(41) Bakalova, R.; Zhelev, Z.; Jose, R.; Nagase, T.; Ohba, H.;

Ishikawa, M.; Baba, Y. J. Nanosci. Nanotechnol. 2005, 5, 887.(42) Chan, W.; Shiao, N.; Lu, P. Toxicol. Lett. 2006, 167, 191.(43) (a) Liu, L.; Zheng, H. Z.; Zhang, Z. J.; Huang, Y. M.; Chen,

S. M.; Hu, Y. F. Spectrochim. Acta, Part A 2008, 69, 701. (b) Mandal, G.;Bardhan, M.; Ganguly, T. Colloids Surf., B 2010, 81, 178.(44) Chaiyasut, C.; Tsuda, T. Chromatography 2001, 22, 91.(45) Song, J. H.; Kim, W.-S.; Park, Y. H.; Yu, E. K.; Lee, D. W. Bull.

Korean Chem. Soc. 1999, 20, 1159.(46) Combes, C.; Rey, C.; Freche, M. J. Mater. Sci.: Mater. Med.

1999, 10, 153.(47) Moffitt, M.; Eisenberg, A. Chem. Mater. 1995, 7, 1178.(48) Rong, H.; Xue-feng, Q.; Jie, Y.; Hong-an, X.; Li-juan, B.;

Zi-Kang, Z. Colloids Surf., A 2003, 220, 151.(49) Hasselbarth, A.; Eychmuller, A.; Weller, H. Chem. Phys. Lett.

1993, 203, 271.(50) Uchihara, T.; Kato, H.; Miyagi, E. J. Photochem. Photobiol., A

2006, 181, 86.(51) Savchuk, A. I.; Fediv, V. I.; Voloshchuk, A. G.; Savchuk, T. A.;

Bacherikov, Y. Y.; Perrone, A. Mater. Sci. Eng., C 2006, 26, 809.(52) Deng, D.-W.; Yu, J.-S.; Pan, Y. J. Colloid Interface Sci. 2006,

299, 225.(53) Chahbouna, A.; Roloa, A. G.; Filonovicha, S. A.; Gomes,

M. J. M. Sol. Energy Mater. Sol. Cells 2006, 90, 1413.(54) Cheng, C.; Xu, G.; Zhang, H.; Wang, H.; Cao, J.; Ji, H. Mater.

Chem. Phys. 2006, 97, 448.(55) Lakowich, J. R. Principles of Fluorescence Spectroscopy; Plenum

Press: New York, 1999; p 267.(56) Chestnoy, N.; Harris, T. D.; Hull, R.; Brus, L. E. J. Phys. Chem.

1986, 90, 3393.(57) Matsuura, D.; Kanemitsu, Y.; Kushida, T.; White, C. W.; Budai,

J. D.; Meldrum, A. Appl. Phys. Lett. 2000, 77, 2289.(58) O’Neil, M.; Marohn, J.; McLendon, G. J. Phys. Chem. 1990,

94, 4356.(59) James, D. R.; Liu, Y.-S.; De Mayo, P.; Ware, W. R. Chem. Phys.

Lett. 1985, 120, 460.(60) Ghali, M. J. Lumin. 2010, 130, 1254.(61) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir

1996, 12 (16), 3763.(62) Hostetler, M. J.; Green, S. J.; Stokes, J. J.; Murray, R. W. J. Am.

Chem. Soc. 1996, 118 (17), 4212.(63) Brust, M.; Flink, J.; Schiffrin, D. J.; Kiely, C. J. Chem. Soc., Chem.

Commun. 1995, 1655.(64) Johnson, S. R.; Evans, S. D.; Brydson, R. Langmuir 1998, 14

(23), 6639.(65) Chen, S.; Murray, R. W. Langmuir 1999, 15 (3), 682.(66) Leff, D. V.; Brandt, L.; Heath, J. R.Langmuir 1996, 12 (20), 4723.(67) Wei, Y.-L.; Li, J.-Q.; Dong, C.; Shuang, S.-M.; Liu, D.-S.; Huie,

C. W. Talanta 2006, 70, 377.(68) Cui, F.-L.; Wang, J.-L.; Cui, Y.-R.; Li, J.-P. Anal. Chim. Acta

2006, 571, 175.(69) Kandagal, P. B.; Seetharamappa, J.; Ashoka, S.; Shaikh, S. M. T.;

Manjunatha, D. H. Int. J. Biol. Macromol. 2006, 39, 234.

(70) Lu, J.-Q.; Jin, F.; Sun, T.-Q.; Zhou, X.-W. Int. J. Biol. Macromol.2007, 40, 299.

(71) Bian, H.; Li, M.; Yu, Q.; Chen, Z.; Tian, J.; Liang, H. Int. J. Biol.Macromol. 2006, 39, 291.

(72) Kamat, P. V.; Shanghavi, B. J. Phys. Chem. B 1997, 101, 7675.(73) De, A. K.; Sinha, S.; Nandy, S. K.; Ganguly, T. J. Chem.Soc.,

Faraday Trans. 1998, 94, 1695.(74) Murakami,M.;Ohkubo,K.;Mandal, P.;Ganguly,T.; Fukuzumi, S.

J. Phys. Chem. A 2008, 112, 635.(75) Sepulveda-Becirra, M. A.; Ferreira, S. T.; Strasser, R. J.; Gerzan-

Rodriguz, W.; Beltrain, C.; Gomez-Phyou, A.; Darszon, A. Biochemistry1996, 35, 15915.

(76) Valeur, B. Molecular Fluorescence: Principles and Application;Wiley Press: New York, 2001; p 250.

(77) Ross, P. D.; Subhramanian, S. Biochemistry 1981, 20, 3096.(78) Bhattacharya, R.; Patra, C. R.; Wang, S.; Lu, L.; Yaszemski,

M. J.; Mukhopadhyay, D.; Mukherjee, P. Adv. Funct. Mater. 2006,16, 395.

(79) Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, 2714.(80) Lu, Z. X.; Cui, T.; Shi, Q. L.Molecular Biology, 1st ed.; Science

Press: Beijing, China, 1987; pp 79�82.(81) Brandes, N.; Welzel, P. B.; Werner, C.; Kroh, L. W. J. Colloid

Interface Sci. 2006, 99, 56.

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