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Evolution of biofunctional semiconductor nanocrystals: a calorimetric investigation† Debasmita Ghosh, Somrita Mondal, Chandra Nath Roy and Abhijit Saha* Semiconductor nanomaterials have found numerous applications in optoelectronic device fabrication and in platforms for drug delivery and hyperthermia cancer treatment, and in various other biomedical fields because of their high photochemical stability and size-tunable photoluminescence (PL). However, little attention has been paid to exploring the energetics of formation of these semiconductor nanoparticles. We demonstrate that formation of nanocrystals with biofunctionalization supported by widely used groups, BSA and cysteine, is an exothermic spontaneous process driven by enthalpy. The whole energetics of the reaction shows that formation of smaller particles is favored with lower synthesis temperature. Further, it is shown that the thermodynamics of nanoparticle formation is
Received 22nd May 2013, Accepted 14th October 2013
strongly influenced by the conformation of the protein matrix. We also demonstrate that protein
DOI: 10.1039/c3cp52158c
smaller organic thiol groups. The favorable enthalpy of formation compensates unfavorable entropy,
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resulting in favorable Gibbs free energy. Thus, this study can open up new avenues for establishing a thermodynamic basis for the design of nanosystems with new and tunable properties.
supported formation of nanocrystals is thermodynamically more favorable compared to that involving
Introduction A plethora of methodologies has emerged in recent years to fabricate semiconductor nanocrystalline particles or quantum dots due to their unique electronic and optical properties arising from quantum confinement effects.1–5 The ability to tune the optical absorption–emission properties of semiconductor nanoparticles (NPs) by simple variation in nanoparticle size is particularly attractive in the facile band-gap engineering of materials6 and the growth of quantum dots.7 Recently, these materials have made tremendous inroads into diverse biomedical applications, such as biosensing,8 cellular imaging,9 drug delivery,10 etc. Further, conjugation of biological molecules to semiconductors has added a new dimension to nanoparticle research.11 The surface of the nanoparticles is extremely sensitive to environmental changes12 and can be modified with suitable molecules to make them biocompatible and amenable to several biological manipulations.13 Thus, it is imperative to synthesize these materials through aqueous routes with suitable biofunctionalization. So methods that allow for tuning the shape of these nanoparticles in a controlled way are of significant interest. However, the design
UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8 Bidhannagar, Kolkata 700 098, India. E-mail: [email protected]; Fax: +91-33-2335 7008; Tel: +91-33-2335 1866 † Electronic supplementary information (ESI) available. Optical characterization of the nanoparticles are given in the ESI. See DOI: 10.1039/c3cp52158c
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of nanosystems with new and tunable properties requires understanding the phenomena that influence cluster size and composition. The formation of nanocrystals involves crystallization processes which consist of, typically, supersaturation, nucleation and crystal growth.14 The nature of the nucleation and growth of a crystal is governed by the thermodynamic equilibria via the reactant precursors.15 A close insight into the binding process is of significant and practical interest, since it provides the fundamental know-how for development of design strategies of nanocluster architecture. The isothermal calorimetry (ITC) is the only direct sensitive method to measure the heat change during molecular association at constant temperature and has thus emerged as the primary tool for characterizing interactions in quantitative terms of thermodynamic parameters. ITC allows the simultaneous determination of the equilibrium binding constant (Ks) and thus the standard Gibbs free energy change (DG), the enthalpy change (DH), the entropy change (DS), as well as the stoichiometry (n) of the association event. ITC has been successfully used to study the interactions of protein–protein,16 protein–DNA,17 and protein–lipid18 in biological systems. Recently, this technique has been applied to understand the complex interactions between nanoparticles and protein molecules.19,20 However, little attention has been paid to understand the energetics of the formation of nanoparticles. There have been a few calorimetric studies on interactions between two sets of metallic nanoparticles leading to the formation of bimetallic metal particles.21 The change in
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enthalpy in the formation of metal nanoparticles has been investigated by several groups.22 In the majority of these studies, the nature of heat of the reactions (exothermic or endothermic or mixed) during the formation of the nanoparticles has been monitored. However, the thermodynamics of formation of semiconductor CdS nanoparticles has been reported recently by Jiang et al.23 In that study, formation of CdS nanoparticles without having capping groups has been followed in two solvents. Therefore, there has been a need to look into a more comprehensive thermodynamic study on the evolution of nanoparticles in real synthesis terms. This has spurred our interest to investigate the thermodynamics of formation of biofunctionalized CdS nanocrystals as a model system by ITC. Among the semiconducting nanoparticles, CdS nanoparticles seem to be one of the well characterized systems and cysteine amino acid and bovine serum albumin (BSA) are the widely used groups for biofunctionalization or biomimetic processes.24–29 Hence, in the present investigation, energetics of the formation of CdS nanocrystals with biofunctionalization groups such as cysteine amino acid and bovine serum albumin (BSA) through the aqueous route has been emphasized. Our previous investigations have demonstrated that protein conformation controls the size and quality of the nanoparticles synthesized through the biomimetic route.30 Circular dichroism spectroscopy has revealed that BSA adopts different conformations at different pH which in turn controls the particle size. Hence, here attempt has been made to investigate how the thermodynamics influencing the evolution of the nanocrystals are affected by the protein conformation. Further, several thiols have been extensively employed for synthesizing nanoparticles as stabilizers and giving the opportunity to vary the functional groups at the surface and, thus, regulating the chemical behavior of the particles. This opens the field of performing chemistry with the preformed NPs. Cysteine, in particular, has been used as it moderates the growth process and imparts bio-functionality to the particle surface. Thus, it is interesting to see how cysteine and BSA can regulate the thermodynamics of formation of CdS NPs. The variation of thermodynamic functions under different parameters, such as temperature, pH of solution, concentration, etc., was monitored and the possible reasons for the change are also discussed.
ions (2 10 3 M) and BSA of appropriate amount was prepared and purged with nitrogen for few minutes prior to titration with sulfide. The pH of BSA solution was maintained with phosphate– citrate buffer prepared by dissolving requisite amounts of di-sodium hydrogen phosphate and tri-sodium citrate. Nitrogen gas was passed slowly over the surface of the protein solution to avoid any structural change. Isothermal calorimetry Isothermal titration calorimetry (ITC) was performed using an isothermal titration calorimeter (ITC200) from Microcal, Northampton (MA, USA) with the normal cell (200 mL). In a typical titration, with stirring at 500 rpm, 2 mL of 20 mmol aqueous solution of Na2S (2 10 3 M) taken in a syringe was injected at equal intervals of 1 min into the Cys-Cd(II) or BSA-Cd(II) aqueous solution (2 mmol) which was taken in the sample cell with 200 ml capacity. A total of 40 mL of Na2S solution was added to Cys-Cd(II) or BSA-Cd(II) solution. Titration consisted of an injection of 2 mL of the sodium sulfide solution into the Cys-Cd(II) or BSA-Cd(II) solution for a total number of 20 injections. Control experiments were carried out for sodium sulfide to determine the heats of dilution. The thermal responses of the dilution were then subtracted to obtain the heat of binding. From the known concentrations of sodium sulfide and Cys-Cd(II) or BSA-Cd(II) solution, the affinity and enthalpy changes upon binding were derived from a simple fit to the data model using the software ORIGIN (Microcal ITC200, Northampton, MA).
Characterization The formation of CdS nanocrystals was monitored by absorption (Perkin-Elmer UV-VIS-NIR) and luminescence spectrometry (Perkin Elmer LS 55). The experimental setup was similar to that of the calorimetric measurements (the volume ratio, concentrations of the components and temperature were identical and spectra were recorded at different time intervals after the addition of the individual portions of sulfide). The end product withdrawn from the ITC cell under different synthesis conditions was also characterized. Determination of average particle size and distribution
Experimental details Materials CdCl2 and Na2S were purchased from Seisco Research Laboratory. L-Cysteine hydrochloride was purchased from Merck, Germany. BSA was obtained from Sigma-Aldrich. All chemicals used were of analytical grade or of high purity available. Milli-Q water (Millipore) was used as solvent. The precursor solutions were prepared following the typical method of preparation as reported earlier.3,7 An aqueous solution of Cd(II) ions (2 10 3 M) and cysteine (5 10 3 M) was prepared. The pH of the solution was adjusted to 11.2–11.8 (using the Jenway 3345 ion meter) by adding NaOH (0.1 M) and nitrogen was bubbled through the solution for few minutes prior to titration. Another aqueous solution containing Cd(II)
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Semiconductor nanoparticles exhibit a blue shift in the absorption spectra as the size is reduced below the characteristic Bohr exciton diameter of the bulk material.31 The quantitative relationship between absorption spectra and particle size is now well understood.32 The band gap (Eg) was calculated from absorption onset (lonset) in the UV-Vis absorption spectra of each nanoparticle solution using the relation Eg = hc/lonset, where h is the Planck’s constant and c is the speed of light. The average size of nanoparticles (d) was obtained using the correlation of band gap shift (DEg = Eg(nanocrystal) Eg(bulk)) and particle size deduced by tight-binding approximation32 (eqn (1)). DEg = a1e
d/b1
+ a2e
d/b2
(1)
The values of the parameters for CdS nanoparticles are a1 = 2.83, b1 = 8.22, a2 = 1.96, b2 = 18.07. The relative percentage
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distribution of particle sizes within nanocrystal solution was determined from the absorption spectra by following the method reported earlier.33,34 It was demonstrated earlier that the particle size determined by this optical method was found to be in good agreement with the size obtained from TEM measurements.
Results and discussion In the studied model system producing the CdS nanocrystals, various synthetic parameters, the nature of the reaction medium, and the interplay between them affect the nanocrystal formation. In a synthetic batch, thermodynamic equilibriums can exist between the reactant monomers and the CdS nanocrystals produced. The concentration of the reactant monomers together with temperature may affect both the chemical reaction kinetics and the thermodynamic equilibriums. Also, the reaction medium affects the reactivity of the reactant precursors leading to the formation of the reactant monomers, as well as the thermodynamic properties, such as solubility and chemical stability, of nanocrystals produced. The affinity is indeed the change in free energy with its sign changed. Hence a high affinity means an important decrease in the free energy upon binding. Anyway, the condition for the spontaneous evolution of a system is that DG o 0. In order to understand the thermodynamics of the evolution of biofunctional semiconductor nanoparticles, we have measured the change in heat associated with the growth of CdS nanoparticles in different conditions by carrying out ITC measurements. The heat change associated with growth is exothermic in all the conditions studied. Similar to the evolution of Au nanoparticles,35 the observed heat change as a function of time indicating the growth process in semiconductor nanocrystals appears to be sigmoidal in nature. The sigmoidal growth model suggests that there are three stages of the total process, nucleation, growth and saturation. Interestingly, the heat change pattern with the growth of the semiconductor particles is reverse to what was observed in
metal nanoclusters. In the case of Au nanoparticles, heat change decreases with the growth of the particles,35 while we observe that heat change increases with growing particles and reaches almost zero in semiconductor nanocrystals. In the present study, the experimental data (corrected with blank titration of S2 ions) determined under all reaction conditions employed satisfactorily fit into the typical single site binding model of commercially available software (ORIGIN, Microcal, Northampton, MA) suggesting that one type of interaction is involved in the formation of semiconductor nanocrystals, presumably, electrostatic or non-covalent in nature.36,37 Thus, this fit can provide an overall estimate of the thermodynamic parameters. In order to determine the heat of dilution, sulfide at the same concentration as in the formation reaction was titrated into water. The interaction of Cys-Cd(II) or BSA-Cd(II) with S2 ions resulted in a relatively large exothermic enthalpy change. The obtained parameter values from the fitting process were only marginally changed with the subtraction of the blank titration. Since the heat of dilution of S2 was very small, the values of the overall heat change in the Cys-CdS or BSA-CdS formation reactions were reported without subtraction. It is evident from the calorimetric measurements that the reaction of S2 ions with Cys-Cd(II) or BSA-Cd(II) involves one site binding between the two entities. The binding constants (K), reaction stoichiometry (n), and enthalpy changes (DH) were determined from the curve fitting analyses. The Gibbs free energy changes (DG) and entropy changes (DS) were calculated using the standard thermodynamic equations DG = RT ln K and DG = DH TDS.38 Fig. 1 illustrates the calorimetric profile of the binding of Cys-Cd(II) with sulfide. In order to investigate the effect of temperature on nanoparticle formation, the ITC was done at different temperatures: 25, 35, 55 and 75 1C (Fig. 1). Fig. 1: A1, B1, C1 and D1 show raw ITC curves resulting from the injections of Na2S solution into Cys-Cd(II) solution. Each negative peak shown in the heat signal curves from Cys-Cd(II)–sulfide (Fig. 1: A1, B1, C1 and D1) represents an exothermic process, which
Fig. 1 ITC analyses for the complexation of Cys-Cd(II) with sulfide at different temperatures A1, A2 at 25 1C; B1, B2 at 35 1C; C1, C2 at 55 1C and D1, D2 at 75 1C. A1, B1, C1, D1 are ITC profiles for Cys-Cd(II) binding of sulfide and A2, B2, C2, D2 are plots of heat evolved (mcal) against the molar ratio of sulfide to Cd(II) corresponding to the ITC.
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denotes the heat released in one injection of sulfide into the Cd(II) solution as a function of time. During the initial injection cycles, the exothermic peak heights are roughly similar, indicating complete complexation between sulfide ensuing into the sample cell and cysteine-Cd(II) during these cycles. As the injections progress, the exothermicity of the peaks decreases because of depleting available binding sites. Toward the end of the injection cycles, it is observed that little binding takes place and that the cysteine-Cd(II)–sulfide complex has already formed. Fig. 1: A2, B2, C2 and D2 are plots of the integrated heat response obtained from the raw data plotted as a function of the ratio of [sulfide]/[Cd(II)]. Fig. 1: A2, B2, C2 and D2 would thus correspond to binding isotherms of CdS nanoparticle formation. The data points reflect the experimental injection heats, while the solid line reflects the calculated fit of the data with a model for a single set of identical sites.39 The absorption and luminescence spectra of the end product of the titration are shown in Fig. 2. It is observed that UV-Vis and PL spectra represent the characteristic spectral bands of CdS nanocrystals which are similar to those observed in earlier work.7 To illustrate, typical UV-Vis and PL spectra of cysteine capped CdS NPs synthesized following the usual procedure are given in the ESI† (Fig. S1). These results suggest that the observed thermogram actually represents heat changes involved in the evolution of CdS nanocrystals. The average particle size of CdS NPs synthesized at different temperatures was determined from absorption onset using the correlation with bandgap as described in the Characterization section. It is shown that as the temperature is increased, the absorption peak and the onset of the nanocrystals shift to a higher
Fig. 2
Table 1
wavelength. With the increase in synthesis temperature from 25 1C to 75 1C, the average size of CdS nanoparticles increased from 3 to 5 nm and size distribution increased from 8% to 20%. The thermodynamic parameters obtained from our ITC experiments at different temperatures are summarized in Table 1. Inspection of these thermodynamic data reveals that the value of K decreases with the increase in synthesis temperature which corresponds to the rule that the higher temperature goes against the exothermic reactions. The temperature dependence of thermodynamic parameters is similar to what was observed in the formation of bare CdS nanocrystals at two temperatures by Jiang et al.23 However, the difference between their reported values of thermodynamic parameters and our observed values signifies the role of surface capping groups in the nanocrystal formation process. The value of DS increases (less negative) as temperature increases. It was shown earlier that growth of cysteine capped CdS NPs is governed by the ‘Ostwald ripening’ mechanism.7,40 The smaller particles are thermodynamically less stable because of their higher surface free energy (Es). Thus, there exists a stability gradient and, as the solution is heated, there is a slow diffusion of materials from smaller particles to the surface of larger particles. As a result, the particle size increases continuously during growth. As particle size increases with increasing temperature, entropy becomes more positive indicating an increase in randomness. This suggests more randomness at higher temperature, which may be due to the de-focusing in the particle size distribution. It has been shown by D. Alloyeau et al.41 that for small NPs, the surface energy (Es) is not negligible and the overall energy should be treated as G = H TS + Es. Since the increase in
Absorbance (a) and luminescence (b) spectra showing the formation of cysteine capped CdS nanoparticles at different temperatures.
Thermodynamic parameters determined from calorimetric measurements of temperature dependent formation of cysteine-CdS nanoparticles
Temperature (1C)
n
K (M 1)
25 35 55 75
0.9 1.10 1.08 1.06
(8.09 (1.59 (799 (302
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DH (kJ mol 1) 0.05) 103 0.11) 103 72.6) 11.3)
50.04 44.00 41.81 34.65
1.04 1.18 1.04 1.83
DS (kJ mol 0.09 0.08 0.07 0.05
1
deg 1)
DG (kJ mol 1) 22.29 19.00 18.22 16.52
TDS (kJ mol 1) 27.75 25.00 23.60 18.13
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Fig. 3
Paper
(a) Absorption and (b) luminescence spectra of cysteine capped CdS NPs during the progress of calorimetric titration.
synthesis temperature led to an increase in particle size, it was expected that DG should be more negative for a given enthalpy and entropy. However, the observed less negative values of DG in the formation of nanocrystals in question with the rise in temperature suggest that the entropy and enthalpy contributions outweigh the decrease in the surface energy of the nanoparticles formed at higher temperature. The n value of 1 indicates that each sulfide ion is able to bind on a single site of the cadmium thiolate complex, presumably with the Cd(II) site, leading to the formation of CdS nanoparticles in water. In order to investigate how the properties of nanoparticles evolve at a given temperature (55 1C) during calorimetric titration, UV-Vis and PL spectra were recorded at different time intervals. From Fig. 3a, the shift of the absorption peak and onset of the nanocrystals to a higher wavelength is proof for particle growth. The average particle size, calculated by tight binding approximation as described earlier, increases with the time of progress of reactions (from 3.4 to 4.6 nm). The size distribution of the nanocrystals was also determined as described in the Characterization section. It was observed that size distribution increases from 10% to 15% with the progress of reaction. Fig. 3b shows the PL spectra at different time intervals. The determination of FWHM in PL spectra also shows that the distribution of particles increases with the progress of reaction. Studies on the thermodynamics of nanomaterial formation in the BSA matrix lead to significant advances in our understanding of its growth mechanism, as well as the correlation between thermodynamic data and structure. The characteristic absorption and luminescence spectra of the as-synthesized BSA encapsulated CdS nanocrystals are shown in ESI† (Fig. S2a). Fig. 4 illustrates the raw data and fitted curves for binding of sulfide to BSA-Cd(II) solution. The binding isotherm indicates that the binding process is also exothermic in nature. Excellent fit of the data is achieved for a model with single site binding. The thermodynamic parameters obtained from our ITC experiments are n = 1.26, K = 3.31 104 M 1, DH = 95.23 kJ mol 1, DS = 0.23 kJ mol 1 deg 1, DG = 25.78 kJ mol 1 and TDS = 69.43 kJ mol 1. The evolution of particle growth as a function of protein concentration was followed. The variation of thermodynamic
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Fig. 4
ITC analysis for the reaction of BSA-Cd(II) with sulfide at 25 1C.
parameters for formation of CdS nanoparticles synthesized at different concentrations of BSA (2, 3 and 4 mg ml 1) at pH 6 is given in Table 2. It is observed that the value of DS increases (becomes less negative) and the value of DG decreases (becomes more negative) with the increase of BSA concentration. In our earlier work,30 it was shown that the size of CdS nanoparticles decreased with increasing concentration of BSA from 2 mg ml 1 to 4 mg ml 1. As the size of the NPs decreases, randomness of particles, i.e. the DS value, increases with increasing BSA concentration. This was further supported by the fact that the size of gold particles decreased with increasing concentration of capping thiol groups.42 It can be argued that in order to stabilize nanocrystals in solution, this energy per capping group must be sufficiently negative. This signifies that since it is energetically unfavorable for metal or semiconductor particles to be brought to the interface, the energy of bringing the capping group to the interface must overcompensate. BSA has reversible conformational isomerization in different pH conditions. BSA has two conformers, such as N (normal) and F (fast) at pH 6 and 4 respectively (Fig. 5).43 When the pH
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Table 2
PCCP Thermodynamic parameters determined from calorimetric measurements of formation of BSA-CdS nanoparticles at different concentrations of BSA
BSA concentration (mg ml 1)
n
K (M 1)
4 3 2
1.34 1.40 1.18
(4.72 0.24) 104 (3.15 0.12) 104 (2.48 0.06) 104
DH (kJ mol 1) 92.46 1.56 96.23 1.15 100.83 2.30
DS (kJ mol 0.21 0.23 0.25
1
deg 1)
DG (kJ mol 1) 27.00 26.00 25.07
TDS (kJ mol 1) 65.46 70.23 75.76
Fig. 5 The two conformers of BSA, which are N (normal) and F (fast) at pH 6 and 4, respectively.
value is lower than 4.7, the normal conformer (N) of BSA (at pH 6) undergoes an expansion leading to the formation of relatively open structured F conformer. In our earlier work,30 it was demonstrated that these conformational variations result in the formation of different sizes of semiconductor nanoparticles with varying quality. To investigate the effect of protein conformation on the thermodynamics of CdS nanoparticle formation in the BSA matrix, the titration of sulfide in BSA-Cd(II) solution was done at two different pHs (pH 6 and 4) of BSA (Fig. 6). The characteristic absorption and luminescence spectra of the as-synthesized CdS nanocrystals are shown in Fig. 7. It is observed that the average particle size varies with the pH of BSA solution (3 nm at pH 6 and 4.5 nm at pH 4). Thermodynamic parameters obtained from the ITC study are given in Table 3. The binding constant K and DS values increase but DG and n decrease with the increasing pH of BSA. Since the observed data fit into the single independent site
Fig. 6
Fig. 7 Absorption and luminescence spectra (inset) of BSA encapsulated CdS NPs at two different pH of BSA solution.
model, it is apparent that the binding of sulfide ions on the Cd-BSA complex occurs at the cationic Cd(II) site. However, the observed n value of 4 at pH 4 suggests that there has been an increase in the available binding sites for sulfide ions due to the conformational change in BSA from N to F form. As CdS formation plays the dominant role, the DH value does not change significantly. Further, we had observed earlier that NPs bound with the N form of BSA (at pH 6) have a smaller size and particles bound with the F form (at pH 4) have a larger size due to the expanded conformation of BSA at the latter pH.
ITC analyses for the reaction of BSA-Cd(II) with sulfide at different pH of BSA, A = pH 6, B = pH 4.
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Table 3
Paper Thermodynamic parameters determined from calorimetric measurements of pH dependent formation of BSA-CdS nanoparticles
pH
n
K (M 1)
6 4
1.26 3.81
(3.31 0.09) 104 (1.13 0.22) 103
DH (kJ mol 1) 95.23 2.40 108.07 4.24
As the size of NPs at pH 6 is smaller than the particle size at pH 4, randomness is greater at pH 6 than that at pH 4. This results in a greater DS value at pH 6 than at pH 4. A closer examination of the thermodynamic quantities determined in the present investigation reveals that the formation of cysteine or BSA mediated CdS nanoparticles characterizes a favorable enthalpy change (DH o 0), which is offset partially by unfavorable entropy loss (DS o 0), contributing overall negative free energy changes (DG). The thermodynamics of complexation depend on two simultaneous processes featuring noncovalent interaction (including electrostatic, hydrophobic, hydrogen bonding, and Van der Waal’s interactions) and solvent reorganization. From an enthalpic consideration, the noncovalent interaction is exothermic (DHintrinsic o 0) while the solvent reorganization is endothermic (DHdesolv > 0).19 The observed negative enthalpy suggests that the intrinsic bond formation between Cd(II) associated with BSA or cysteine and inorganic sulfide plays a predominant role in the complex formation. Water molecules at interfaces can sometimes enhance the complementarity of the interacting surfaces; however, the negative entropy changes do not necessarily indicate that the hydration of the CysCdS or BSA-CdS interface remains unchanged or increases in comparison with that of the free Cys-Cd(II) or BSA-Cd(II) and sulfide. In addition, an unfavorable contribution to the entropy change to a greater extent in BSA-CdS formation in comparison with that in cysteine-CdS may arise from the conformational restriction of the surface groups in BSA. When the entropy increase due to desolvation is not large enough to compensate the entropy loss owing to solute freedom reduction, overall unfavorable entropy changes are observed for the binding of Cys-Cd(II) or BSA-Cd(II) with sulfide. However, in the present case, a dominating contribution to the unfavorable entropy is that the nanocluster formation follows the nucleation process induced by initial monomer formation. In all the cases of formation of Cys-CdS or BSA-CdS, it is observed that the contribution of DH to the free energy DG is greater than the one in TDS. Therefore, we may infer that the interaction between Cys-Cd(II) or BSA-Cd(II) and sulfide is an enthalpy driven process. However, if we compare the formation processes of CdS nanocrystals with cysteine and BSA functional groups under their optimum synthesis conditions, greater negative free energy and higher binding affinity suggest more efficient stabilization of nanoparticles by BSA. It is apparent from the tables that favorable enthalpy changes in Cys-Cd(II)–sulfide interactions are always balanced by entropic loss, i.e., enthalpy–entropy compensation.44–49 Although no explicit relationship between the enthalpic and the entropic terms can be deduced from fundamental thermodynamics, the compensation effect has been observed
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DS (kJ mol
1
deg 1)
DG (kJ mol 1)
0.23 0.30
25.78 17.42
TDS (kJ mol 1) 69.45 90.65
universally in host–guest complexation.50 Inoue et al. have carried out quantitative correlation analyses of compensatory enthalpy–entropy relationships using a wide variety of molecular recognition systems.49,51 To analyze the compensation, if any, the TDS value is linearly correlated with the DH value to give eqn (2). When eqn (3) is incorporated into the Gibbs–Helmholtz equation followed by the differential, eqn (3) is obtained TDS = aDH + TDS0
(2)
a) jDH
(3)
jDG = (1
According to eqn (3), the slope (a) of DH TDS plots reflects the contribution of enthalpic gains (jDH) induced by alterations in host, guest, and/or solvent to the free energy change (jDG), as some enthalpy has been cancelled by the accompanying entropic loss (jDS).43 The intercept (TDS0) represents the inherent complex stability (DG) obtained at DH = 0. By employing this correlation approach, the entropy changes (TDS) are plotted (Fig. 7) against corresponding enthalpy changes (DH) for the formation of BSA-CdS and cysteine-CdS nanomaterials studied. As shown in Fig. 8, an excellent linear relationship is obtained in the case of BSA-CdS or Cys-CdS for these thermodynamic quantities with a correlation coefficient of 0.99, slope a = 0.96, and intercept TDS0 = 17.69 kJ mol 1. Since the intercept value is not as high as in the case of protein–protein interaction, it may be assumed that the contribution of the desolvation effect is minimal. On the other hand, the low intercept value is consistent with earlier reported results on interactions of protein–nonpeptide ligand or macromolecule– small organic molecule.19
Fig. 8 Plot of entropy (TDS) versus enthalpy (DH) for the formation of biofunctionalized CdS nanoparticles.
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Conclusions We have determined the different thermodynamic parameters and showed how these can influence the evolution of semiconductor nanocrystals. It is also demonstrated that enthalpy predominantly contributes to the formation processes and protein conformation can play a significant role in nanostructure formation and stabilization. A whole protein molecule can be a more effective capping group compared to an amino acid, even if it contains metal affinic thiol groups. It is observed that the evolution process is not favored by entropy, but favorable enthalpy compensates entropy loss leading to favorable Gibbs free energy. It is established from thermodynamic consideration that lower synthesis temperature can help in the formation of smaller particles. This investigation is possibly the first endeavor to shed light on the basic energetics of semiconductor nanocrystal formation, which may facilitate, in future, the development of a strategy for designing biofunctional nanostructured materials with tunable and optimal properties.
Acknowledgements One of the authors (D. Ghosh) is grateful to CSIR, Govt of India, for the award of senior Research Fellowship. Both authors (S. Mondal and C. N. Roy) are thankful to the University Grants Commission for NET fellowship. We sincerely appreciate the valuable suggestions of one of the reviewers.
Notes and references 1 Q. J. Y. Sun, A. Wang, L. S. Li, D. Wang, T. Zhu, J. Xu, C. Yang and Y. Li, Nat. Photonics, 2007, 1, 717; L. E. Brus, J. Chem. Phys., 1984, 80, 4403. 2 P. E. Lippens and M. Lannoo, Phys. Rev. B: Condens. Matter Mater. Phys., 1989, 39, 10935. 3 M. V. Ramakrishna and R. A. Friesner, J. Chem. Phys., 1991, 95, 8309. 4 A. P. Alivisatos, Science, 1996, 271, 933. 5 H. Weller, Angew. Chem., Int. Ed. Engl., 1993, 32, 41. 6 J. H. Fendler and F. Meldrum, Adv. Mater., 1995, 7, 607. 7 A. Priyam, A. Chatterjee, S. C. Bhattacharya and A. Saha, J. Cryst. Growth, 2007, 304, 416. 8 R. Gill, L. Bahshi, R. Freeman and I. Willner, Angew. Chem., Int. Ed., 2008, 47, 1676. 9 Y. Zhu, Z. Li, M. Chen, H. M. Cooper, G. Q. Lu and Z. Ping Xu, Chem. Mater., 2012, 24, 421. 10 N. Gaponik, I. L. Radtchenko, G. B. Sukorukov and A. L. Rogach, Langmuir, 2004, 20, 1449. 11 C. T. Dameron, R. N. Reese, R. K. Mehra, A. R. Kortan and P. J. Carroll, Nature, 1989, 338, 596; E. Dujardin and S. Mann, Adv. Mater., 2002, 14, 775; A. Schroedter, H. Weller, R. Eritja, W. E. Ford and M. J. Wessels, Nano Lett., 2002, 2, 1363. 12 K. Boldt, O. T. Bruns, N. Gaponik and A. Eychmuller, J. Phys. Chem. B, 2006, 110, 1959.
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13 N. N. Mamedova, N. A. Kotov, A. L. Rogach and J. Studer, Nano Lett., 2001, 1, 281; P. Alivisatos, Nat. Biotechnol., 2004, 22, 47. 14 K. F. Kelton, Solid State Phys., 1991, 45, 75. 15 K. Yu, M. Z. Hu, R. Wang, M. L. Piolet, M. Frotey, Md. B. Zaman, X. Wu, D. M. Leek, Y. Tao, D. Wilkinson and C. Li, J. Phys. Chem. C, 2010, 114, 3329. 16 M. M. Pierce, C. S. Raman and B. T. Nall, Methods, 1999, 19, 213. 17 A. Kunne, M. Sieber, D. Meierhans and R. K. Allemann, Biochemistry, 1998, 37, 4217. 18 M. R. Wenk and J. Seelig, Biochemistry, 1998, 37, 3909. 19 M. De, C. C. You, S. Srivastava and V. M. Rotello, J. Am. Chem. Soc., 2007, 129, 10747. 20 S. Lindman, I. Lynch, E. Thulin, H. Nilsson, K. A. Dawson and S. Linse, Nano Lett., 2007, 7, 914. 21 N. Toshima, M. Kanemaru, Y. Shiraishi and Y. Koga, J. Phys. Chem. B, 2005, 109, 16326. 22 R. Patakfalvi and I. Dekany, J. Therm. Anal. Calorim., 2005, 79, 587; A. Meister, S. Drescher, I. Mey, M. Wahab, G. Gesche, V. M. Garamus, G. Hause, H. J. Mogel, A. Janshoff, S. Papp, L. Korosi, B. Gool, T. Dederichs, P. Mela, M. Moller and I. Dekany, J. Therm. Anal. Calorim., 2010, 101, 865; M. Kanemaru, Y. Shiraishi, Y. Koga and N. Toshima, J. Therm. Anal. Calorim., 2005, 81, 523. 23 J. Jiang, Z. Huang, Q. Xiao, Y. Liu, Y. Mi and Y. Li, Thermochim. Acta, 2010, 503, 136. 24 A. V. Singh, B. M. Bandgar, M. Kasture, B. L. V. Prasad and M. Sastry, J. Mater. Chem., 2005, 15, 5115. 25 A. V. Singh, R. Patil, M. B. Kasture, W. N. Gade and B. L. V. Prasad, Colloids Surf., B: Biointerfaces, 2009, 69, 239. 26 L. Yang, R. Xing, Q. Shen, K. Jiang, F. Ye, J. Wang and Q. Ren, J. Phys. Chem. B, 2006, 110, 10534. 27 L. Yang, H. Yang and Z. Yang, et al., J. Phys. Chem. B, 2008, 112, 9795. 28 D. Qin, X. Ma, L. Yang, L. Zhang, Z. Ma and J. Zhang, J. Nanopart. Res., 2008, 10, 559. 29 J. Zhang, X. Ma, Y. Guo, L. Yang, Q. Shen, H. Wang and Z. Ma, Mater. Chem. Phys., 2010, 119, 112. 30 D. Ghosh, S. Ghosh, S. Mondal and A. Saha, J. Mater. Chem., 2012, 22, 699. 31 W. C. W. Chan and S. Nie, Science, 1998, 281, 2016; M. Bruchez, M. Moronne, P. Gin, S. Weiss and A. P. Alivisators, Science, 1998, 281, 2013. 32 S. Sapra and D. D. Sarma, Phys. Rev. B: Condens. Matter Mater. Phys., 2004, 69, 125304. 33 R. Viswanatha and D. D. Sarma, Chem.–Eur. J., 2006, 12, 180. 34 A. Priyam, S. Ghosh, S. C. Bhattacharya and A. Saha, J. Colloid Interface Sci., 2009, 333, 195. 35 K. Biswas, N. Varghese and C. N. R. Rao, Small, 2008, 4, 649. 36 A. Gourishankar, S. Shukla, K. N. Ganesh and M. Sastry, J. Am. Chem. Soc., 2004, 126, 13186. 37 C.-C. You, M. De, G. Han and V. M. Rotello, J. Am. Chem. Soc., 2005, 127, 12873. 38 S. Mondal, S. Ghosh, D. Ghosh and A. Saha, J. Phys. Chem. C, 2012, 116, 9774.
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39 E. Merabet and G. Ackers, Biochemistry, 1995, 34, 8554. 40 A. L. Smith, Particle growth in suspensions, Academic Press, London, 1983, p. 3. ´vot, G. Le Bouar, T. Oikawa, C. Langlois, 41 D. Alloyeau, Y. Pre A. Loiseau and C. Ricolleau, Phys. Rev. Lett., 2010, 105, 255901. 42 D. V. Leff, P. C. Ohara, J. R. Heath and W. M. Gelbart, J. Phys. Chem., 1995, 99, 7036. 43 D. C. Carter and J. X. Ho, Adv. Protein Chem., 1994, 45, 153; S. Mandal, S. K. Arumugam, S. D. Adyanthaya and M. Sastry, J. Mater. Chem., 2004, 14, 43. 44 K. N. Houk, A. G. Leach, S. P. Kim and X. Zhang, Angew. Chem., Int. Ed., 2003, 42, 4872.
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45 W. Linert, L.-F. Han and I. Likovits, Chem. Phys., 1989, 139, 441. 46 K. Sharp, Protein Sci., 2001, 10, 661. 47 D. H. Williams, E. Stephens, D. P. O’Brien and M. Zhou, Angew. Chem., Int. Ed., 2004, 43, 6596. 48 J. D. Dunitz, Chem. Biol., 1995, 2, 709. 49 M. V. Rekharsky and Y. Inoue, Chem. Rev., 1998, 98, 1875. 50 L. Liu and Q.-X. Guo, Chem. Rev., 2001, 101, 673; D. J. Winzor and C. M. Jackson, J. Mol. Recognit., 2006, 19, 389. 51 Y. Inoue and T. Wada, in Advances in Supramolecular Chemistry, ed. G. W. Gokel, JAI Press, Greenwich, CT, 1997, vol. 4, pp. 55–96.
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Evolution of biofunctional semiconductor nanocrystals: a calorimetric investigation† Debasmita Ghosh, Somrita Mondal, Chandra Nath Roy and Abhijit Saha* Semiconductor nanomaterials have found numerous applications in optoelectronic device fabrication and in platforms for drug delivery and hyperthermia cancer treatment, and in various other biomedical fields because of their high photochemical stability and size-tunable photoluminescence (PL). However, little attention has been paid to exploring the energetics of formation of these semiconductor nanoparticles. We demonstrate that formation of nanocrystals with biofunctionalization supported by widely used groups, BSA and cysteine, is an exothermic spontaneous process driven by enthalpy. The whole energetics of the reaction shows that formation of smaller particles is favored with lower synthesis temperature. Further, it is shown that the thermodynamics of nanoparticle formation is
Received 22nd May 2013, Accepted 14th October 2013
strongly influenced by the conformation of the protein matrix. We also demonstrate that protein
DOI: 10.1039/c3cp52158c
smaller organic thiol groups. The favorable enthalpy of formation compensates unfavorable entropy,
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resulting in favorable Gibbs free energy. Thus, this study can open up new avenues for establishing a thermodynamic basis for the design of nanosystems with new and tunable properties.
supported formation of nanocrystals is thermodynamically more favorable compared to that involving
Introduction A plethora of methodologies has emerged in recent years to fabricate semiconductor nanocrystalline particles or quantum dots due to their unique electronic and optical properties arising from quantum confinement effects.1–5 The ability to tune the optical absorption–emission properties of semiconductor nanoparticles (NPs) by simple variation in nanoparticle size is particularly attractive in the facile band-gap engineering of materials6 and the growth of quantum dots.7 Recently, these materials have made tremendous inroads into diverse biomedical applications, such as biosensing,8 cellular imaging,9 drug delivery,10 etc. Further, conjugation of biological molecules to semiconductors has added a new dimension to nanoparticle research.11 The surface of the nanoparticles is extremely sensitive to environmental changes12 and can be modified with suitable molecules to make them biocompatible and amenable to several biological manipulations.13 Thus, it is imperative to synthesize these materials through aqueous routes with suitable biofunctionalization. So methods that allow for tuning the shape of these nanoparticles in a controlled way are of significant interest. However, the design
UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8 Bidhannagar, Kolkata 700 098, India. E-mail: [email protected]; Fax: +91-33-2335 7008; Tel: +91-33-2335 1866 † Electronic supplementary information (ESI) available. Optical characterization of the nanoparticles are given in the ESI. See DOI: 10.1039/c3cp52158c
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of nanosystems with new and tunable properties requires understanding the phenomena that influence cluster size and composition. The formation of nanocrystals involves crystallization processes which consist of, typically, supersaturation, nucleation and crystal growth.14 The nature of the nucleation and growth of a crystal is governed by the thermodynamic equilibria via the reactant precursors.15 A close insight into the binding process is of significant and practical interest, since it provides the fundamental know-how for development of design strategies of nanocluster architecture. The isothermal calorimetry (ITC) is the only direct sensitive method to measure the heat change during molecular association at constant temperature and has thus emerged as the primary tool for characterizing interactions in quantitative terms of thermodynamic parameters. ITC allows the simultaneous determination of the equilibrium binding constant (Ks) and thus the standard Gibbs free energy change (DG), the enthalpy change (DH), the entropy change (DS), as well as the stoichiometry (n) of the association event. ITC has been successfully used to study the interactions of protein–protein,16 protein–DNA,17 and protein–lipid18 in biological systems. Recently, this technique has been applied to understand the complex interactions between nanoparticles and protein molecules.19,20 However, little attention has been paid to understand the energetics of the formation of nanoparticles. There have been a few calorimetric studies on interactions between two sets of metallic nanoparticles leading to the formation of bimetallic metal particles.21 The change in
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enthalpy in the formation of metal nanoparticles has been investigated by several groups.22 In the majority of these studies, the nature of heat of the reactions (exothermic or endothermic or mixed) during the formation of the nanoparticles has been monitored. However, the thermodynamics of formation of semiconductor CdS nanoparticles has been reported recently by Jiang et al.23 In that study, formation of CdS nanoparticles without having capping groups has been followed in two solvents. Therefore, there has been a need to look into a more comprehensive thermodynamic study on the evolution of nanoparticles in real synthesis terms. This has spurred our interest to investigate the thermodynamics of formation of biofunctionalized CdS nanocrystals as a model system by ITC. Among the semiconducting nanoparticles, CdS nanoparticles seem to be one of the well characterized systems and cysteine amino acid and bovine serum albumin (BSA) are the widely used groups for biofunctionalization or biomimetic processes.24–29 Hence, in the present investigation, energetics of the formation of CdS nanocrystals with biofunctionalization groups such as cysteine amino acid and bovine serum albumin (BSA) through the aqueous route has been emphasized. Our previous investigations have demonstrated that protein conformation controls the size and quality of the nanoparticles synthesized through the biomimetic route.30 Circular dichroism spectroscopy has revealed that BSA adopts different conformations at different pH which in turn controls the particle size. Hence, here attempt has been made to investigate how the thermodynamics influencing the evolution of the nanocrystals are affected by the protein conformation. Further, several thiols have been extensively employed for synthesizing nanoparticles as stabilizers and giving the opportunity to vary the functional groups at the surface and, thus, regulating the chemical behavior of the particles. This opens the field of performing chemistry with the preformed NPs. Cysteine, in particular, has been used as it moderates the growth process and imparts bio-functionality to the particle surface. Thus, it is interesting to see how cysteine and BSA can regulate the thermodynamics of formation of CdS NPs. The variation of thermodynamic functions under different parameters, such as temperature, pH of solution, concentration, etc., was monitored and the possible reasons for the change are also discussed.
ions (2 10 3 M) and BSA of appropriate amount was prepared and purged with nitrogen for few minutes prior to titration with sulfide. The pH of BSA solution was maintained with phosphate– citrate buffer prepared by dissolving requisite amounts of di-sodium hydrogen phosphate and tri-sodium citrate. Nitrogen gas was passed slowly over the surface of the protein solution to avoid any structural change. Isothermal calorimetry Isothermal titration calorimetry (ITC) was performed using an isothermal titration calorimeter (ITC200) from Microcal, Northampton (MA, USA) with the normal cell (200 mL). In a typical titration, with stirring at 500 rpm, 2 mL of 20 mmol aqueous solution of Na2S (2 10 3 M) taken in a syringe was injected at equal intervals of 1 min into the Cys-Cd(II) or BSA-Cd(II) aqueous solution (2 mmol) which was taken in the sample cell with 200 ml capacity. A total of 40 mL of Na2S solution was added to Cys-Cd(II) or BSA-Cd(II) solution. Titration consisted of an injection of 2 mL of the sodium sulfide solution into the Cys-Cd(II) or BSA-Cd(II) solution for a total number of 20 injections. Control experiments were carried out for sodium sulfide to determine the heats of dilution. The thermal responses of the dilution were then subtracted to obtain the heat of binding. From the known concentrations of sodium sulfide and Cys-Cd(II) or BSA-Cd(II) solution, the affinity and enthalpy changes upon binding were derived from a simple fit to the data model using the software ORIGIN (Microcal ITC200, Northampton, MA).
Characterization The formation of CdS nanocrystals was monitored by absorption (Perkin-Elmer UV-VIS-NIR) and luminescence spectrometry (Perkin Elmer LS 55). The experimental setup was similar to that of the calorimetric measurements (the volume ratio, concentrations of the components and temperature were identical and spectra were recorded at different time intervals after the addition of the individual portions of sulfide). The end product withdrawn from the ITC cell under different synthesis conditions was also characterized. Determination of average particle size and distribution
Experimental details Materials CdCl2 and Na2S were purchased from Seisco Research Laboratory. L-Cysteine hydrochloride was purchased from Merck, Germany. BSA was obtained from Sigma-Aldrich. All chemicals used were of analytical grade or of high purity available. Milli-Q water (Millipore) was used as solvent. The precursor solutions were prepared following the typical method of preparation as reported earlier.3,7 An aqueous solution of Cd(II) ions (2 10 3 M) and cysteine (5 10 3 M) was prepared. The pH of the solution was adjusted to 11.2–11.8 (using the Jenway 3345 ion meter) by adding NaOH (0.1 M) and nitrogen was bubbled through the solution for few minutes prior to titration. Another aqueous solution containing Cd(II)
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Semiconductor nanoparticles exhibit a blue shift in the absorption spectra as the size is reduced below the characteristic Bohr exciton diameter of the bulk material.31 The quantitative relationship between absorption spectra and particle size is now well understood.32 The band gap (Eg) was calculated from absorption onset (lonset) in the UV-Vis absorption spectra of each nanoparticle solution using the relation Eg = hc/lonset, where h is the Planck’s constant and c is the speed of light. The average size of nanoparticles (d) was obtained using the correlation of band gap shift (DEg = Eg(nanocrystal) Eg(bulk)) and particle size deduced by tight-binding approximation32 (eqn (1)). DEg = a1e
d/b1
+ a2e
d/b2
(1)
The values of the parameters for CdS nanoparticles are a1 = 2.83, b1 = 8.22, a2 = 1.96, b2 = 18.07. The relative percentage
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distribution of particle sizes within nanocrystal solution was determined from the absorption spectra by following the method reported earlier.33,34 It was demonstrated earlier that the particle size determined by this optical method was found to be in good agreement with the size obtained from TEM measurements.
Results and discussion In the studied model system producing the CdS nanocrystals, various synthetic parameters, the nature of the reaction medium, and the interplay between them affect the nanocrystal formation. In a synthetic batch, thermodynamic equilibriums can exist between the reactant monomers and the CdS nanocrystals produced. The concentration of the reactant monomers together with temperature may affect both the chemical reaction kinetics and the thermodynamic equilibriums. Also, the reaction medium affects the reactivity of the reactant precursors leading to the formation of the reactant monomers, as well as the thermodynamic properties, such as solubility and chemical stability, of nanocrystals produced. The affinity is indeed the change in free energy with its sign changed. Hence a high affinity means an important decrease in the free energy upon binding. Anyway, the condition for the spontaneous evolution of a system is that DG o 0. In order to understand the thermodynamics of the evolution of biofunctional semiconductor nanoparticles, we have measured the change in heat associated with the growth of CdS nanoparticles in different conditions by carrying out ITC measurements. The heat change associated with growth is exothermic in all the conditions studied. Similar to the evolution of Au nanoparticles,35 the observed heat change as a function of time indicating the growth process in semiconductor nanocrystals appears to be sigmoidal in nature. The sigmoidal growth model suggests that there are three stages of the total process, nucleation, growth and saturation. Interestingly, the heat change pattern with the growth of the semiconductor particles is reverse to what was observed in
metal nanoclusters. In the case of Au nanoparticles, heat change decreases with the growth of the particles,35 while we observe that heat change increases with growing particles and reaches almost zero in semiconductor nanocrystals. In the present study, the experimental data (corrected with blank titration of S2 ions) determined under all reaction conditions employed satisfactorily fit into the typical single site binding model of commercially available software (ORIGIN, Microcal, Northampton, MA) suggesting that one type of interaction is involved in the formation of semiconductor nanocrystals, presumably, electrostatic or non-covalent in nature.36,37 Thus, this fit can provide an overall estimate of the thermodynamic parameters. In order to determine the heat of dilution, sulfide at the same concentration as in the formation reaction was titrated into water. The interaction of Cys-Cd(II) or BSA-Cd(II) with S2 ions resulted in a relatively large exothermic enthalpy change. The obtained parameter values from the fitting process were only marginally changed with the subtraction of the blank titration. Since the heat of dilution of S2 was very small, the values of the overall heat change in the Cys-CdS or BSA-CdS formation reactions were reported without subtraction. It is evident from the calorimetric measurements that the reaction of S2 ions with Cys-Cd(II) or BSA-Cd(II) involves one site binding between the two entities. The binding constants (K), reaction stoichiometry (n), and enthalpy changes (DH) were determined from the curve fitting analyses. The Gibbs free energy changes (DG) and entropy changes (DS) were calculated using the standard thermodynamic equations DG = RT ln K and DG = DH TDS.38 Fig. 1 illustrates the calorimetric profile of the binding of Cys-Cd(II) with sulfide. In order to investigate the effect of temperature on nanoparticle formation, the ITC was done at different temperatures: 25, 35, 55 and 75 1C (Fig. 1). Fig. 1: A1, B1, C1 and D1 show raw ITC curves resulting from the injections of Na2S solution into Cys-Cd(II) solution. Each negative peak shown in the heat signal curves from Cys-Cd(II)–sulfide (Fig. 1: A1, B1, C1 and D1) represents an exothermic process, which
Fig. 1 ITC analyses for the complexation of Cys-Cd(II) with sulfide at different temperatures A1, A2 at 25 1C; B1, B2 at 35 1C; C1, C2 at 55 1C and D1, D2 at 75 1C. A1, B1, C1, D1 are ITC profiles for Cys-Cd(II) binding of sulfide and A2, B2, C2, D2 are plots of heat evolved (mcal) against the molar ratio of sulfide to Cd(II) corresponding to the ITC.
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denotes the heat released in one injection of sulfide into the Cd(II) solution as a function of time. During the initial injection cycles, the exothermic peak heights are roughly similar, indicating complete complexation between sulfide ensuing into the sample cell and cysteine-Cd(II) during these cycles. As the injections progress, the exothermicity of the peaks decreases because of depleting available binding sites. Toward the end of the injection cycles, it is observed that little binding takes place and that the cysteine-Cd(II)–sulfide complex has already formed. Fig. 1: A2, B2, C2 and D2 are plots of the integrated heat response obtained from the raw data plotted as a function of the ratio of [sulfide]/[Cd(II)]. Fig. 1: A2, B2, C2 and D2 would thus correspond to binding isotherms of CdS nanoparticle formation. The data points reflect the experimental injection heats, while the solid line reflects the calculated fit of the data with a model for a single set of identical sites.39 The absorption and luminescence spectra of the end product of the titration are shown in Fig. 2. It is observed that UV-Vis and PL spectra represent the characteristic spectral bands of CdS nanocrystals which are similar to those observed in earlier work.7 To illustrate, typical UV-Vis and PL spectra of cysteine capped CdS NPs synthesized following the usual procedure are given in the ESI† (Fig. S1). These results suggest that the observed thermogram actually represents heat changes involved in the evolution of CdS nanocrystals. The average particle size of CdS NPs synthesized at different temperatures was determined from absorption onset using the correlation with bandgap as described in the Characterization section. It is shown that as the temperature is increased, the absorption peak and the onset of the nanocrystals shift to a higher
Fig. 2
Table 1
wavelength. With the increase in synthesis temperature from 25 1C to 75 1C, the average size of CdS nanoparticles increased from 3 to 5 nm and size distribution increased from 8% to 20%. The thermodynamic parameters obtained from our ITC experiments at different temperatures are summarized in Table 1. Inspection of these thermodynamic data reveals that the value of K decreases with the increase in synthesis temperature which corresponds to the rule that the higher temperature goes against the exothermic reactions. The temperature dependence of thermodynamic parameters is similar to what was observed in the formation of bare CdS nanocrystals at two temperatures by Jiang et al.23 However, the difference between their reported values of thermodynamic parameters and our observed values signifies the role of surface capping groups in the nanocrystal formation process. The value of DS increases (less negative) as temperature increases. It was shown earlier that growth of cysteine capped CdS NPs is governed by the ‘Ostwald ripening’ mechanism.7,40 The smaller particles are thermodynamically less stable because of their higher surface free energy (Es). Thus, there exists a stability gradient and, as the solution is heated, there is a slow diffusion of materials from smaller particles to the surface of larger particles. As a result, the particle size increases continuously during growth. As particle size increases with increasing temperature, entropy becomes more positive indicating an increase in randomness. This suggests more randomness at higher temperature, which may be due to the de-focusing in the particle size distribution. It has been shown by D. Alloyeau et al.41 that for small NPs, the surface energy (Es) is not negligible and the overall energy should be treated as G = H TS + Es. Since the increase in
Absorbance (a) and luminescence (b) spectra showing the formation of cysteine capped CdS nanoparticles at different temperatures.
Thermodynamic parameters determined from calorimetric measurements of temperature dependent formation of cysteine-CdS nanoparticles
Temperature (1C)
n
K (M 1)
25 35 55 75
0.9 1.10 1.08 1.06
(8.09 (1.59 (799 (302
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DH (kJ mol 1) 0.05) 103 0.11) 103 72.6) 11.3)
50.04 44.00 41.81 34.65
1.04 1.18 1.04 1.83
DS (kJ mol 0.09 0.08 0.07 0.05
1
deg 1)
DG (kJ mol 1) 22.29 19.00 18.22 16.52
TDS (kJ mol 1) 27.75 25.00 23.60 18.13
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Fig. 3
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(a) Absorption and (b) luminescence spectra of cysteine capped CdS NPs during the progress of calorimetric titration.
synthesis temperature led to an increase in particle size, it was expected that DG should be more negative for a given enthalpy and entropy. However, the observed less negative values of DG in the formation of nanocrystals in question with the rise in temperature suggest that the entropy and enthalpy contributions outweigh the decrease in the surface energy of the nanoparticles formed at higher temperature. The n value of 1 indicates that each sulfide ion is able to bind on a single site of the cadmium thiolate complex, presumably with the Cd(II) site, leading to the formation of CdS nanoparticles in water. In order to investigate how the properties of nanoparticles evolve at a given temperature (55 1C) during calorimetric titration, UV-Vis and PL spectra were recorded at different time intervals. From Fig. 3a, the shift of the absorption peak and onset of the nanocrystals to a higher wavelength is proof for particle growth. The average particle size, calculated by tight binding approximation as described earlier, increases with the time of progress of reactions (from 3.4 to 4.6 nm). The size distribution of the nanocrystals was also determined as described in the Characterization section. It was observed that size distribution increases from 10% to 15% with the progress of reaction. Fig. 3b shows the PL spectra at different time intervals. The determination of FWHM in PL spectra also shows that the distribution of particles increases with the progress of reaction. Studies on the thermodynamics of nanomaterial formation in the BSA matrix lead to significant advances in our understanding of its growth mechanism, as well as the correlation between thermodynamic data and structure. The characteristic absorption and luminescence spectra of the as-synthesized BSA encapsulated CdS nanocrystals are shown in ESI† (Fig. S2a). Fig. 4 illustrates the raw data and fitted curves for binding of sulfide to BSA-Cd(II) solution. The binding isotherm indicates that the binding process is also exothermic in nature. Excellent fit of the data is achieved for a model with single site binding. The thermodynamic parameters obtained from our ITC experiments are n = 1.26, K = 3.31 104 M 1, DH = 95.23 kJ mol 1, DS = 0.23 kJ mol 1 deg 1, DG = 25.78 kJ mol 1 and TDS = 69.43 kJ mol 1. The evolution of particle growth as a function of protein concentration was followed. The variation of thermodynamic
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Fig. 4
ITC analysis for the reaction of BSA-Cd(II) with sulfide at 25 1C.
parameters for formation of CdS nanoparticles synthesized at different concentrations of BSA (2, 3 and 4 mg ml 1) at pH 6 is given in Table 2. It is observed that the value of DS increases (becomes less negative) and the value of DG decreases (becomes more negative) with the increase of BSA concentration. In our earlier work,30 it was shown that the size of CdS nanoparticles decreased with increasing concentration of BSA from 2 mg ml 1 to 4 mg ml 1. As the size of the NPs decreases, randomness of particles, i.e. the DS value, increases with increasing BSA concentration. This was further supported by the fact that the size of gold particles decreased with increasing concentration of capping thiol groups.42 It can be argued that in order to stabilize nanocrystals in solution, this energy per capping group must be sufficiently negative. This signifies that since it is energetically unfavorable for metal or semiconductor particles to be brought to the interface, the energy of bringing the capping group to the interface must overcompensate. BSA has reversible conformational isomerization in different pH conditions. BSA has two conformers, such as N (normal) and F (fast) at pH 6 and 4 respectively (Fig. 5).43 When the pH
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Table 2
PCCP Thermodynamic parameters determined from calorimetric measurements of formation of BSA-CdS nanoparticles at different concentrations of BSA
BSA concentration (mg ml 1)
n
K (M 1)
4 3 2
1.34 1.40 1.18
(4.72 0.24) 104 (3.15 0.12) 104 (2.48 0.06) 104
DH (kJ mol 1) 92.46 1.56 96.23 1.15 100.83 2.30
DS (kJ mol 0.21 0.23 0.25
1
deg 1)
DG (kJ mol 1) 27.00 26.00 25.07
TDS (kJ mol 1) 65.46 70.23 75.76
Fig. 5 The two conformers of BSA, which are N (normal) and F (fast) at pH 6 and 4, respectively.
value is lower than 4.7, the normal conformer (N) of BSA (at pH 6) undergoes an expansion leading to the formation of relatively open structured F conformer. In our earlier work,30 it was demonstrated that these conformational variations result in the formation of different sizes of semiconductor nanoparticles with varying quality. To investigate the effect of protein conformation on the thermodynamics of CdS nanoparticle formation in the BSA matrix, the titration of sulfide in BSA-Cd(II) solution was done at two different pHs (pH 6 and 4) of BSA (Fig. 6). The characteristic absorption and luminescence spectra of the as-synthesized CdS nanocrystals are shown in Fig. 7. It is observed that the average particle size varies with the pH of BSA solution (3 nm at pH 6 and 4.5 nm at pH 4). Thermodynamic parameters obtained from the ITC study are given in Table 3. The binding constant K and DS values increase but DG and n decrease with the increasing pH of BSA. Since the observed data fit into the single independent site
Fig. 6
Fig. 7 Absorption and luminescence spectra (inset) of BSA encapsulated CdS NPs at two different pH of BSA solution.
model, it is apparent that the binding of sulfide ions on the Cd-BSA complex occurs at the cationic Cd(II) site. However, the observed n value of 4 at pH 4 suggests that there has been an increase in the available binding sites for sulfide ions due to the conformational change in BSA from N to F form. As CdS formation plays the dominant role, the DH value does not change significantly. Further, we had observed earlier that NPs bound with the N form of BSA (at pH 6) have a smaller size and particles bound with the F form (at pH 4) have a larger size due to the expanded conformation of BSA at the latter pH.
ITC analyses for the reaction of BSA-Cd(II) with sulfide at different pH of BSA, A = pH 6, B = pH 4.
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Table 3
Paper Thermodynamic parameters determined from calorimetric measurements of pH dependent formation of BSA-CdS nanoparticles
pH
n
K (M 1)
6 4
1.26 3.81
(3.31 0.09) 104 (1.13 0.22) 103
DH (kJ mol 1) 95.23 2.40 108.07 4.24
As the size of NPs at pH 6 is smaller than the particle size at pH 4, randomness is greater at pH 6 than that at pH 4. This results in a greater DS value at pH 6 than at pH 4. A closer examination of the thermodynamic quantities determined in the present investigation reveals that the formation of cysteine or BSA mediated CdS nanoparticles characterizes a favorable enthalpy change (DH o 0), which is offset partially by unfavorable entropy loss (DS o 0), contributing overall negative free energy changes (DG). The thermodynamics of complexation depend on two simultaneous processes featuring noncovalent interaction (including electrostatic, hydrophobic, hydrogen bonding, and Van der Waal’s interactions) and solvent reorganization. From an enthalpic consideration, the noncovalent interaction is exothermic (DHintrinsic o 0) while the solvent reorganization is endothermic (DHdesolv > 0).19 The observed negative enthalpy suggests that the intrinsic bond formation between Cd(II) associated with BSA or cysteine and inorganic sulfide plays a predominant role in the complex formation. Water molecules at interfaces can sometimes enhance the complementarity of the interacting surfaces; however, the negative entropy changes do not necessarily indicate that the hydration of the CysCdS or BSA-CdS interface remains unchanged or increases in comparison with that of the free Cys-Cd(II) or BSA-Cd(II) and sulfide. In addition, an unfavorable contribution to the entropy change to a greater extent in BSA-CdS formation in comparison with that in cysteine-CdS may arise from the conformational restriction of the surface groups in BSA. When the entropy increase due to desolvation is not large enough to compensate the entropy loss owing to solute freedom reduction, overall unfavorable entropy changes are observed for the binding of Cys-Cd(II) or BSA-Cd(II) with sulfide. However, in the present case, a dominating contribution to the unfavorable entropy is that the nanocluster formation follows the nucleation process induced by initial monomer formation. In all the cases of formation of Cys-CdS or BSA-CdS, it is observed that the contribution of DH to the free energy DG is greater than the one in TDS. Therefore, we may infer that the interaction between Cys-Cd(II) or BSA-Cd(II) and sulfide is an enthalpy driven process. However, if we compare the formation processes of CdS nanocrystals with cysteine and BSA functional groups under their optimum synthesis conditions, greater negative free energy and higher binding affinity suggest more efficient stabilization of nanoparticles by BSA. It is apparent from the tables that favorable enthalpy changes in Cys-Cd(II)–sulfide interactions are always balanced by entropic loss, i.e., enthalpy–entropy compensation.44–49 Although no explicit relationship between the enthalpic and the entropic terms can be deduced from fundamental thermodynamics, the compensation effect has been observed
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DS (kJ mol
1
deg 1)
DG (kJ mol 1)
0.23 0.30
25.78 17.42
TDS (kJ mol 1) 69.45 90.65
universally in host–guest complexation.50 Inoue et al. have carried out quantitative correlation analyses of compensatory enthalpy–entropy relationships using a wide variety of molecular recognition systems.49,51 To analyze the compensation, if any, the TDS value is linearly correlated with the DH value to give eqn (2). When eqn (3) is incorporated into the Gibbs–Helmholtz equation followed by the differential, eqn (3) is obtained TDS = aDH + TDS0
(2)
a) jDH
(3)
jDG = (1
According to eqn (3), the slope (a) of DH TDS plots reflects the contribution of enthalpic gains (jDH) induced by alterations in host, guest, and/or solvent to the free energy change (jDG), as some enthalpy has been cancelled by the accompanying entropic loss (jDS).43 The intercept (TDS0) represents the inherent complex stability (DG) obtained at DH = 0. By employing this correlation approach, the entropy changes (TDS) are plotted (Fig. 7) against corresponding enthalpy changes (DH) for the formation of BSA-CdS and cysteine-CdS nanomaterials studied. As shown in Fig. 8, an excellent linear relationship is obtained in the case of BSA-CdS or Cys-CdS for these thermodynamic quantities with a correlation coefficient of 0.99, slope a = 0.96, and intercept TDS0 = 17.69 kJ mol 1. Since the intercept value is not as high as in the case of protein–protein interaction, it may be assumed that the contribution of the desolvation effect is minimal. On the other hand, the low intercept value is consistent with earlier reported results on interactions of protein–nonpeptide ligand or macromolecule– small organic molecule.19
Fig. 8 Plot of entropy (TDS) versus enthalpy (DH) for the formation of biofunctionalized CdS nanoparticles.
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Conclusions We have determined the different thermodynamic parameters and showed how these can influence the evolution of semiconductor nanocrystals. It is also demonstrated that enthalpy predominantly contributes to the formation processes and protein conformation can play a significant role in nanostructure formation and stabilization. A whole protein molecule can be a more effective capping group compared to an amino acid, even if it contains metal affinic thiol groups. It is observed that the evolution process is not favored by entropy, but favorable enthalpy compensates entropy loss leading to favorable Gibbs free energy. It is established from thermodynamic consideration that lower synthesis temperature can help in the formation of smaller particles. This investigation is possibly the first endeavor to shed light on the basic energetics of semiconductor nanocrystal formation, which may facilitate, in future, the development of a strategy for designing biofunctional nanostructured materials with tunable and optimal properties.
Acknowledgements One of the authors (D. Ghosh) is grateful to CSIR, Govt of India, for the award of senior Research Fellowship. Both authors (S. Mondal and C. N. Roy) are thankful to the University Grants Commission for NET fellowship. We sincerely appreciate the valuable suggestions of one of the reviewers.
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