ARTICLE pubs.acs.org/JPCC
€ rster Resonance Energy Transfer between Quantum Occurrence of Fo Dots and Gold Nanoparticles in the Presence of a Biomolecule Gopa Mandal, Munmun Bardhan, and Tapan Ganguly* Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India ABSTRACT: In this paper the interactions of gold nanoparticles (Au NPs) and bovin serum albumingold nanoconjugates (BSAGNPs) with cadmium sulfide quantum dots (CdS QDs) are investigated by using steady-state and time-resolved spectroscopic techniques under physiological conditions (pH ∼ 7). From the analysis of the steady-state and time-resolved fluorescence quenching of CdS QDs in aqueous solution in the presence of BSAGNPs it has been inferred that fluorescence resonance (F€orster type) energy transfer (FRET) is primarily responsible for the quenching phenomenon. But in the presence of only Au 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 bionanoconjugate system), FRET becomes operative between CdS QDs and Au NPs present in the BSAGNPs bionanoconjugate, whereas in the absence of this biomolecule direct contact between CdS and Au NPs facilitates the formation of ground-state complex. As shown from the high-resolution transmission electron microscopy (HRTEM) images of the bionanoconjugate, formation of a thin BSA layer around the Au NPs, situated at the core, inhibits the CdS QDs to come in contact with the Au NPs. In the CdSbionanoconjugate system, CdS and Au NPs become separated by a distance of ∼17 ( 2 Å, as observed from HRTEM measurement. It may be presumed that when Au NPs are present in the bionanoconjugate system, the system may suffer some conformational changes which facilitates the energy transfer process to occur within the CdS QDs and the Au NPs. Further investigations with similar systems would be necessary to make unequivocal assertion of this phenomenon. From the determination of the thermodynamic parameters it is apparent that the effect of van der Waals interaction is responsible for the interaction of CdS QDs with Au NPs to form ground-state complex. The effect of CdS NPs on the conformation of BSAGNPs has been examined by analyzing CD spectra. Though the observed results demonstrate some conformational changes in the bionanoconjugate in the presence of CdS NPs, the secondary structure of the conjugate, predominantly of the α-helix, is found to retain its identity. This type of interaction between QDs and Au NPs in a protein-conjugated form provides a new insight for design and the development of FRET-based bionanosensors.
1. INTRODUCTION Fluorescent semiconductor nanoparticles have drawn considerable interest due to their size-dependent optical properties1 and their vast applications in photonics. In nanoparticles a large percentage of the atoms are in the surface, rather than in the bulk phase. Nanoparticles can be made from a wide variety of materials including CdS, ZnS, Cd3P2, and PbS, to name a few. The nanoparticles frequently display photoluminescence and sometimes display electroluminescence.27 Additionally, some nanoparticles can form self-assembled arrays.8,9 Because of those favorable properties, nanoparticles are being extensively studied for use in optoelectronic displays. Recently, there has been a growing interest in the study of nanoparticles in biomolecules such as proteins, DNA, and peptides for biophotonic applications.1013 The luminescent quantum dots promise to be an attractive alternative for biolabeling and biosensing applications. They emit bright and steady fluorescence. Among a variety of semiconductor nanoparticles, CdS has been intensively studied.1417 Nanocrystalline semiconductor materials such as PbS and CdS have attracted considerable attention due to their unique properties, which are not present in bulk materials.1820 r 2011 American Chemical Society
CdS, in particular, has been extensively studied due to its potential applications such as field effect transistors, light-emitting diodes, photocatalysis, and biological sensors.2123 On the other hand bovine serum albumin (BSA), most abundant protein in plasma, is a commonly used reagent in biological study. It plays an important role in many physiological functions as it is the major soluble protein constituent of the circulatory system. It is responsible for drug deposition and efficacy.24 Recently, these biomolecules are utilized in many applications apart from being the building block of “life”.25 Recent addition to their repertoire is the conjugation with nanoparticles (NPs). BSA, a commonly used reagent in biological studies, has long been used as a capping agent for various nanoparticles.2629 These bioconjugate nanoparticles are of great importance because of their potential applications in luminescence tagging, imaging, medical diagnostics, and most importantly as biosensors as well as for assembling hybrid proteinNP units for molecular electronics. Several studies on the Received: May 12, 2011 Revised: August 13, 2011 Published: September 19, 2011 20840
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Figure 1. (A) TEM picture of CdS NPs; (B) magnified view of a single CdS NP; (C) BSAAu conjugate (inset, magnified view of BSAAu nanoconjugate); (D) BSAAuCdS conjugate; white arrows indicate the thickness of BSA; red spheres show CdS QDs; (E) AuCdS conjugate; the sky blue sphere shows the Au NP, and the red spheres show CdS QDs; (F) magnified view of BSAAuCdS nanoconjugate.
bioconjugate nanoparticles have been reported. Protein conjugation has also been used as a strategy for increasing colloidal stability,3032 conferring biochemical activity,33,34 and enhancing biocompatibility3537 in various nanoparticles systems. The present article reports the interactions of CdS quantum dots (QDs) with bovin serum albumingold nanoconjugates (BSAGNPs) and Au NPs by absorption and time-resolved fluorescence spectroscopy. Additionally, the conformational changes of BSAGNPs conjugates by CdS NPs were analyzed by circular dichroism (CD) spectra. The primary aim of our work is to compare and to reveal the nature of interactions of CdS NPs with BSAGNPs conjugates and Au NPs. To elucidate the true picture, the detailed spectroscopic investigations on such interactions have been done.
2. EXPERIMENTAL METHODS 2.1. Materials. Bovine serum albumin (fatty acid free) was purchased from Aldrich and directly used from the package.
The procedure of synthesis and characterization of CdS NPs is described elsewhere.38 NPs are routinely coated with inorganic shells to impart solubility in biological media. The NP coating can isolate the reactive metal ions of the core from the cell and can thus prevent oxidative stress. Encapsulation of CdS or CdSe QDs with a zinc sulfide shell helps them to be soluble in water and decreases their toxicity dramatically.3942 But here to solubilize the CdS NP we use MPA (HSCH2CH2COOH) as capping agent, not ZnS. We have used phosphate buffer for preparing solutions throughout the course of investigations. The pH of phosphate buffer used in the experiment is 7. The buffer solution is prepared in Millipore water. The solvent is tested before use to check any unwanted impurity emission in the concerned wavelength region. 2.1.1. Preparation of BSAGNPs. The procedure for BSA GNPs preparation was described previously.43a,b The isoelectric point of BSA is between 4.5 and 4.9.4446 Therefore, in the phosphate buffer solution at pH value ∼7.0, the BSA molecule has a significant number of negative ions attached on the surface, 20841
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Figure 2. Steady-state UVvis absorption spectra of CdS NPs in the presence of increasing concentration of GNPs (concentration of CdS NPs ∼2 106 M); [GNPs] ranges from (1) 0 to (2) 6.64 107, (3) 1.3 106, and (4) 3.28 106 M.
which forms the attractive Columbic interaction between the protein and the Au NPs. 2.2. Apparatus. At the ambient temperature (296 K) steadystate UVvis absorption and fluorescence emission spectra of dilute solutions (105 to 107 M) of the samples were recorded using 1 cm path length rectangular quartz cells by means of an absorption spectrophotometer (Shimadzu UVvis 2401PC) and F-4500 fluorescence spectrophotometer (Hitachi), respectively. Fluorescence lifetime measurements were carried out by the time-correlated single-photon counting (TCSPC) method using a Horiba Jobin Yvon Fluorocube. For fluorescence lifetime measurements the samples were excited at 375 nm using a picosecond diode (IBH Nanoled-07). The emission was collected at a magic-angle polarization using a Hamamatsu microchannel plate photomultiplier (2809U). The TCSPC setup consists of an Ortec 9327 CFD and a Tennelec TC 863 TAC. The data is collected with a PCA3 card (Oxford) as a multichannel analyzer. The typical full width at half-maximum (fwhm) of the system response is about 238 ps. The channel width is 12 ps/channel. The fluorescence decays were deconvoluted using IBH DAS6 software. The goodness of fit has been assessed over the full decay including the rising edge with the help of statistical parameters χ2 and DurbinWatson (DW). CD has been recorded by a JASCO CD spectrometer, model J-815-150S, using a 0.1 cm path length quartz cell in a wavelength range between 200 and 260 nm. The size and dispersivity of the nanoparticles were determined from a high-resolution transmission electron micrograph (HRTEM) (JEOL, model JEM-2010) in which samples were dropped onto a copper grid covered with a thin film of amorphous carbon.
3. RESULTS AND DISCUSSION 3.1. UVVis Absorption and Steady-State Fluorescence Studies. Figure 1 shows the HRTEM images of CdS, BSAAu
conjugate, BSAAuCdS nanoconjugates, and Au-conjugated CdS QD nanoparticles. It is seen from the TEM images (Figure 1, parts D and F) that Au NPs being surrounded by BSA protein form a core (Au)shell (BSA)-type structure and CdS QDs are attached to this coreshell structure type conjugate. It is seen from the TEM images that the BSA molecules prevent the Au NPs from interacting with CdS QDs directly. But in the absence of protein, the HRTEM image (Figure 1E) clearly
Figure 3. Fluorescence emission spectra (λex = 375 nm) of CdS NPs in the presence of increasing concentration of BSAGNPs (concentration of CdS NPs ∼2 106 M); [BSAGNPs] ranges from (0) 0 to (1) 6.64 107, (2) 1.3 106, (3) 1.98 106, (4) 2.63 106, and (5) 3.28 106 M at 298 K. Inset: fluorescence emission spectra (λex = 415 nm) of CdS NPs in the presence of increasing concentration of BSAGNPs (concentration of CdS NPs ∼2 106 M); [BSA GNPs] ranges from (0) 0 to (1) 6.64 107, (2) 1.3 106, (3) 1.98 106, (4) 2.63 106, and (5) 3.28 106 M at 298 K.
demonstrates that CdS QDs are directly connected with the surface of Au NPs. From the TEM image, it is seen that the size of CdS QDs is 25 nm. From UVvis absorption studies an absorption band peaking at around 415 nm is observed (Figure 2) for CdS QDs, which indicates the narrow distribution of the nanoparticles. It is wellknown that the diameter of the particles is related with the absorption edge,4749 and we measured the size of the nanoparticles using the equation 2RCdS = 0.1/(0.1338 0.0002345λe) nm, where the absorption edge is λe and it is calculated from the intersection of the sharply decreasing region of the spectrum with the baseline.47 The estimated particles size is 3.4 nm for the 444 nm absorption edge. This calculated size is of slightly lower value than that of most of the particles observed from the TEM picture. Figure 2 shows the regular decrement of UVvis absorption spectra of CdS QDs near the domain of 415 nm in the presence of increasing concentration of Au NPs at pH ∼ 7. On the other hand, the UVvis absorption spectra of CdS QDs remain moreor-less unchanged in the presence of BSAGNPs nanocomposite (not shown). In the former case the decrement is presumably due to the formation of ground-state complex that resulted from the intermolecular interactions. Fluorescence behavior of CdS QDs, measured by the excitation at 375 nm, in the presence of varying concentrations of BSAGNPs and at pH ∼ 7 is shown in Figure 3. After excitation at 375 nm, the two emission peaks (Figure 3) at 437 and 576 nm are observed for CdS QDs. The peak at ∼437 nm corresponds to the bandband and 576 nm is due to the lattice defect emission from host CdS, respectively.5054 At 415 nm excitation, only one peak at 576 nm is observed, but the intensity of the peak is relatively strong (inset of Figure 3) compared to that observed for 375 nm excitation. Here, the observed photoluminescence (PL) broadening may be due to the ensemble of different sizes of colloidal nanoparticles. BSAGNPs bioconjugate causes quenching 20842
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Table 1. Time-Resolved Fluorescence Data (fi ∼Fractional Contribution) of a Mixture of CdS QDs and BSAGNPs Conjugate at Ambient Temperature (λex ∼ 375 nm) concn of τ1 (ns)
f1
τ2 (ns)
f2
τ3 (ps)
0
3.41
0.36
28.9
0.39
115.6
0.25
26.35
6.64 107
3.34
0.35
27.4
0.31
66.7
0.34
24.45
1.98 106
3.36
0.35
26.01
0.27
62.3
0.38
22.70
2.63 106
3.11
0.33
23.05
0.26
55.36
0.41
19.50
conjugate (M)
Figure 4. (a) SternVolmer plot of quenching of CdS NPs by BSAGNPs at different temperatures. (b) SternVolmer (SV) plot from fluorescence lifetime measurements (time-resolved) of a mixture of CdS NPs and BSAGNPs.
of fluorescence emission of CdS NPs. To reveal the nature of the quenching mechanism of the fluorescence emission band of CdS QDs peaking at about 437 nm in the presence of BSAGNPs, the SternVolmer (SV) relation has been applied. The SternVolmer equation is given by55 f0 =f ¼ 1 þ Ksv ½Q ¼ 1 þ kq τ0 ½Q where f0 and f denote the steady-state fluorescence emission intensities of the fluorescer in the absence and in the presence of quencher, respectively. KSV is the SternVolmer quenching constant, and [Q] is the concentration of quencher (BSA GNPs). kq is the bimolecular quenching rate constant. τ0 is the fluorescence lifetime of the free fluorescer, i.e., in the absence of any quencher. The observed linearity in SV plots (Figure 4a) suggests that the quenching may be of either pure dynamic or pure static nature.55 Generally, deviations from linearity indicate the mixing of both dynamic and static quenching processes. However, to
f3
Æτæ (ns)
locate the true nature of the quenching, time-resolved fluorescence measurements are made (vide infra). It is observed from the measurements that the fluorescence lifetime of the CdS QDs is gradually shortened with increasing concentrations of BSA GNPs. This observation clearly demonstrates the dynamic nature of quenching. 3.2. Time-Resolved Fluorescence Studies. The fluorescence lifetimes of CdS QDs are measured in the absence and presence of BSAGNPs. The decay of emission was monitored at 440 nm. The best-fitted curves for the decays are obtained by using a three-exponential fit. The lifetime data have been given in Table 1. CdS emission consists of a range of lifetimes which are almost independent of the average cluster size.56 It is wellknown that photoexcited electrons of CdS NPs can decay by radiative or nonradiative pathways, and the quantum efficiency is given by η = τR1/(τR1 + τNR1), where τR1 and τNR1 are the radiative and nonradiative surface recombination, respectively. Matsuura et al.57 suggested that the fast component comes from the free-exciton emission, while the slow component originates from the bound exciton emission at shallow localized states of CdS NPs. O’Neil et al.58 also reported that the decays of CdS NPs are composed of two distinct time regimes. They have described the slow and fast components by a distribution kinetic model and thermal repopulation mechanisms. The average emission lifetime, Æτæ, was calculated by the following expression59 hτ i ¼
∑ ai τi 2 = ∑ ai τi
where ai and τi denote the preexponential factor and the corresponding lifetime, respectively. The fluorescence decays of CdS QDs in the presence of different concentrations of BSAGNPs are triple-exponential in nature. From the computed average decay time by using the above eq it reveals that there is a shortening of decay time of CdS NPs in the presence of bionanoconjugate. But no change in lifetime of CdS QDs with the addition of only Au NPs (or BSA) is observed. Ghali60 reported the static nature of the BSACdS ground-state complex. In the present case it may be concluded from the unperturbed fluorescence lifetime of CdS in the presence of Au NPs that emission quenching of CdS QDs is primarily static in nature and initiated mainly due to ground-state complex formation between CdS and Au. The possibility of ground-state complex formation is also apparent from the UVvis spectra, as discussed above. The static nature of the CdSAu complex is most likely due to the specific interaction of gold with sulfur6166 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 be dynamic in nature. Time-resolved fluorescence spectroscopy demonstrates that the emission quenching of CdS QDs occurs 20843
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The Journal of Physical Chemistry C primarily due to the involvement of F€orster resonance energy transfer from CdS QDs to Au of BSAGNPs (vide infra). 3.3. Temperature Effect. The temperature-dependent studies on fluorescence quenching of CdS NPs in the presence of BSAGNPs have been carried out. The SV plots at different temperatures are shown in Figure 4a. The plots are linear in nature. The kq values at different temperatures are estimated to be 4.2 1012 (288 K), 6.4 1012 (298 K), and 8.7 1012 (308 K) M1 s1, respectively. The observation of regular increase of bimolecular quenching rate with temperature suggests in favor of the dynamic nature of quenching of CdS QDs fluorescence in the presence of BSAGNPs. As the bimolecular quenching rate constants are much greater than the maximum collision quenching constant of various kinds of quenchers to fluorescers (∼2 1010 M1 s1, which is the diffusion-controlled limit of the rate constant), it is apparent that the quenching occurs via Coulombic resonance interaction. Such types of quenching constants exceeding the diffusion-controlled value were reported earlier.6771 Therefore, the high magnitude of the observed kq in this case could be due to the occurrences of primarily energy transfer from CdS NPs (E = 2.99 eV) to Au (E = 2.34 eV) of BSAGNPs bionanoconjugate. The possibility of occurrence of another nonradiative process, intermolecular photoinduced electron transfer, appears to be slim within CdS and Au NPs as in the presence of the latter NPs, the quenching in the fluorescence emission of CdS QDs appears to be static in nature (as discussed above). However, the possibility of the occurrence of electron transfer from CdS to Au has been discussed by Kamat and Shanghavi,72 but in their composite nanocluster system. Moreover, as f0/f ≈ τ0/τ for a concentration of the quencher as derived from both the SV plots constructed from steady-state and time-resolved fluorescence (Figure 4b), the dynamic nature of the quenching is further substantiated.73,74 The lack of formation of ground-state complex is observed, as discussed in section 3.1, between CdS and Au NPs when the latter is present within 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 one type of quenching, pure dynamic or static. From the SV relation of the linear plots of f0/f against [Q ] in case of CdSAu NPs, the SV quenching constant (KSV) and bimolecular quenching rate constant (kq) are computed at different temperatures. The kq values at different temperatures are estimated to be 1.4 1013 (288 K), 9.5 1012 (298 K), and 7.2 1012 (308 K) M1 s1, respectively. This observation demonstrates the static nature of fluorescence quenching, and the increased temperature appears to decrease the stability of the ground-state complex. So it can be concluded that the quenching mechanism of CdS NPs in the presence of only Au NPs is initiated by ground-state complex formation55 rather than by dynamic collision. The same result is obtained in the case of fluorescence emission quenching of CdS by BSA as reported earlier.60 € rster Resonance Energy Transfer. The QD-based 3.4. Fo F€orster resonance energy transfer (FRET) is a significant phenomenon in fluorescence spectroscopy. This process possesses the ability of tuning of emission properties with changing size of QDs for efficient energy transfer within various organic dyes and proteins. FRET has various applications in biological systems such as photosensitization.55,75 It involves the transfer
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of excited-state energy from an initially excited donor to an acceptor and is used for measuring molecular distances or donoracceptor proximity. FRET is an important tool for investigating various biological phenomena involving energy transfer and is extensively used to investigate the structure, conformation, spatial distribution, and assembly of complex proteins.75 Thus, energy transfer could be used as a “spectroscopic ruler” for the measurement of distances between the donors and acceptors. Since the fluorescence spectra of CdS NPs overlap considerably with the absorption spectra of BSAGNPs, it further substantiates our presumption that the fluorescence quenching occurs due to energy transfer from CdS NPs to Au of BSAGNPs. The energy transfer efficiency has been studied according to the F€orster nonradiative energy transfer theory. The energy transfer efficiency (E) is not only related to the center-tocenter distance (r) between the acceptor and donor but also to the F€orster critical energy transfer distance (R0) E¼
R0 6 R0 þ r 6 6
ð1Þ
where R0 is the F€orster’s critical transfer distance when the transfer efficiency is 50%. R 0 6 ¼ 8:8 1025 k2 N 4 ΦJ
ð2Þ
where k2 is the spatial orientation factor of the dipole, N is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor, J is the effect of the spectral overlap between the fluorescence emission spectrum of the donor CdS QDs and the absorption spectrum of the acceptor BSAGNPs, which could be computed by the equation Z ∞ FðλÞεðλÞλ4 dλ ð3Þ J ¼ 0 Z ∞ FðλÞ dλ 0
where F(λ) is the fluorescence intensity of the donor at wavelength range λ and ε(λ) is the molar absorption coefficient of the acceptor at λ. In aqueous solution k2 = 2/3, N = 1.335, and Φ = 0.1134 for CdS NPs. Using these we get the following values: J = 4.8 1014 M1 cm3, R0 = 31.6 Å, r = 37.6 Å. In calculation of “r”, the centercenter distance between QD and Au NPs of the bioconjugate system (which was observed as 37.6 Å), we have neglected the distance of the bond of MPA involved because it is considerably smaller ( 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 entropy can be calculated from the van’t Hoff equation: ΔS ΔH R RT where K is analogous to the binding constant at the corresponding temperature and R is the gas constant. The free energy change can be obtained from the following relationship: ln K ¼
ΔG ¼ ΔH TΔS ¼ RT ln K 20845
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Table 2. Binding Constant Kb, Binding Sites n, and Relative Thermodynamic Parameters for CdS NPs with BSAGNPs Conjugate at Different Temperatures
a
T
Kb (M1)
n
R2 a
SDb
288
1.7 103
0.67
0.99606
0.02086
298 308
4.3 104 5.8 104
0.97 0.98
0.98594 0.99888
0.05713 0.01615
ΔH (kJ mol1)
ΔG (kJ mol1)
ΔS (J mol1 K1)
23.37 29.25 35.13
146
588.1
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 at Different Temperatures
a
T
Kb (M1)
n
R2 a
SDb
288
7.2 105
0.96
0.99212
0.02154
298
1.6 105
1.05
0.99866
0.04013
308
7.4 104
1.2
0.99152
0.05015
ΔH (kJ mol1)
ΔG (kJ mol1)
84.15
32.21
ΔS (J mol1 K1)
32.02 181
28.40
R2 is the correlation coefficient. b SD is the standard deviation.
The α-helix contents of BSA have been calculated from MRE values at 208 nm using the following equation:80 α-helix ð%Þ ¼
Figure 6. CD spectra of BSAGNPs conjugate (concentration of BSAGNPs ∼2 105 M) in the presence of increasing concentration of CdS NPs; concentration of CdS NPs ranges from (1) 0 to (2) 8 105 and (3) 1.5 104 M.
According to the above two eqs the values of ΔG, ΔH, and ΔS were obtained and shown in Tables 2 and 3. From the negative value of ΔG it appears that the process of interaction is spontaneous. 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 be responsible for the formation of ground-state complex. 3.7. Circular Dichroism Spectra. To explore the structural change of BSAGNPs, a CD experiment at room temperature has been carried out in the absence and in the presence of CdS QDs (Figure 6). The addition of nanoparticles in the bionanoconjugate causes very minor α helicity changes in the CD spectra. The CD results are expressed in terms of mean residue ellipticity (MRE) in deg cm2 mol1 according to the following equation.79 ½θλ ¼
θ 10nlc
where c is the concentration of BSAGNPs in g/mL, θ is the observed rotation in deg, l is the path length in cm, and n is the number of amino acid residues of protein (583 for BSA).
½θ208 4000 33000 4000
where [θ]208 is the observed MRE value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33 000 is the MRE value of a pure α-helix at 208 nm. According to the above equations the percentage of α-helix of BSAGNPs is computed, and as can be seen the percentage of helicity of BSAGNPs is 14.52% in the absence of CdS QDs, and in the presence of CdS QDs, it decreases to 14.27%. As the change in α-helical content of BSAGNPs is not significant even at optimum concentration of CdS, i.e., when BSAGNPs are dispersed at high concentration of CdS, denaturation does not occur, and the bionanoconjugate could retain most of its helical structure.43b This observation strongly indicates that the binding of CdS QDs to BSAGNPs induces some conformational changes in the conjugate. But the secondary structure of protein is predominantly of α-helix which is very essential for the preparation of protein-based assemblies of nanoconjugates. As no significant structural deformation occurred, the biological activity, the activity of immune response of the protein, and the biocompatibility of the proteinnanoconjugate remain as such. So bioconjugate Au NPs could pave the way to design new optical-based materials having significant biomedical applications.81 3.8. Comparison between CdSBSAAu and CdSAu Systems. In the case of the CdSBSAAu conjugate system the TEM images show that the BSA molecules prevent the Au NPs from interacting with CdS QDs directly. But in the absence of protein, the HRTEM image (Figure 1E) clearly demonstrates that CdS QDs are directly connected with the surface of Au NPs. In both cases the fluorescence spectra of CdS QDs were quenched in addition of the quencher. From time-resolved fluorescence data it reveals that there is a shortening of decay time of CdS NPs (showing the quenching process is dynamic in nature) in the presence of bionanoconjugate, but in the case of only Au NPs there is no change in lifetime data (static in nature) of the fluorescers CdS QDs. From Table 3 it can be shown that the binding constant (Kb) decreases with increase in temperature, 20846
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The Journal of Physical Chemistry C which indicates the formation of an unstable compound (CdSAu). But in the case of CdSBSAGNPs, Kb increases with increase in temperature (Table 2). These observations further substantiate our inference of occurrence of dynamic quenching in the case of the latter and the presence of static mode in the case of the former. Moreover, it is interesting to note that energy transfer is found to occur from CdS QDs to BSAGNPs conjugate (FRET efficiency ∼26%) but not in the case of the CdSAu NP system (no FRET). In the latter case the observed experimental findings demonstrate that the formation of ground-state complex is only responsible for the observed fluorescence quenching of CdS QDs.
4. CONCLUSIONS In the present investigation the interactions of CdS QDs with the BSAGNPs and Au NPs are studied by the different spectroscopic methods including UVvis, steady-state and time-resolved fluorescence, and CD in phosphate buffer at pH ∼ 7. The shortening of the decay time as observed from timeresolved fluorescence and steady-state fluorescence quenching behavior of CdS QDs in the presence of BSAGNPs is observed to be due to primarily the efficient FRET between CdS QDs and Au NPs. In this bionanoconjugate system, Au NPs being situated in the core position are protected from CdS QDs by the thin envelop (shell type) of BSA protein, as evidenced from HRTEM images. Thus, CdS and Au NPs being separated by a short distance (∼17 ( 2 Å, from HRTEM measurement) in the CdSbionanoconjugate system facilitates the energy transfer process rather than formation of ground-state complex for which the direct contact is necessary. It may be presumed that when Au NPs are present in the bionanoconjugate system, the system may suffer some conformational changes which facilitates the energy transfer process to occur within CdS QDs and Au NPs present as a core within the shell of BSA. Further investigations with similar systems would be necessary to prove conclusively this presumption. The results of CD spectra indicate that the secondary structure of the bionanoconjugate undergoes marginal changes in the presence of CdS NPs. As no significant structural deformation occurred, the biological activity, the activity of immune response of the protein, and the biocompatibility of the proteinnanoconjugate remain as such. So bioconjugate Au NPs could pave the way to design new optical-based materials, and the observed properties of the nanoassemblies are capable of developing FRET-based bionanosensors for their potential applications. ’ AUTHOR INFORMATION Corresponding Author
*Phone: +91 33 2473 4971/3073, ext 253. Fax: +91 33 2473 2805. E-mail: tapcla@rediffmail.com,
[email protected].
’ ACKNOWLEDGMENT The financial assistance provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, India to G.M. in the form of NET-CSIR fellowships is gratefully acknowledged. The authors also thank Mr. Subrata Das of the Department of Spectroscopy for assisting in the time-resolved fluorescence measurements. Thanks to Mr. Santanu Jana of the Department of Materials Science for helping to synthesize QDs. Thanks are
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also due to Professor P. K. Das of Biological Chemistry for helping us in the measurements of CD spectra. T.G. specially thanks the 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 his photophysics and photochemistry group.
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