Protein Films of Bovine Serum Albumen Conjugated Gold Nanoparticles

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Protein Films of Bovine Serum Albumen Conjugated Gold Nanoparticles: A Synthetic Route from Bioconjugated Nanoparticles to Biodegradable Protein Films Mandeep Singh Bakshi,*,† Harpreet Kaur,§ Poonam Khullar,^ Tarlok Singh Banipal,§ Gurinder Kaur,‡ and Narpinder Singh|| †

Department of Chemistry, Wilfrid Laurier University, Science Building, 75 University Avenue West, Waterloo ON N2L 3C5, Canada Nanotechnology Research Laboratory, College of North Atlantic, Labrador City NL A2 V 2K7, Canada § Department of Chemistry and Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, Punjab, India ^ Department of Chemistry, BBK DAV College for Women, Amritsar 143005, Punjab, India )

‡

bS Supporting Information ABSTRACT: Bovine serum albumen (BSA) conjugated gold (Au) nanoparticles (NPs) were directly synthesized by using BSA as a weak reducing agent against HAuCl4 in aqueous phase. A systematic variation in Au/BSA mole ratio showed a dramatic change in the size and shape of NPs which was very much dependent on the physical state of BSA. The nature of both colloidal NPs (due to surface plasmon resonance) and BSA (due to tryptophan residues) was monitored simultaneously by UV-visible measurements during the course of the reaction. A systematic variation in the reaction temperature from 20 to 70 C demonstrated a clear denaturation process of BSA and how it influenced the synthesis of Au NPs. A predominantly native state of BSA that existed up to 40 C proved to be a very mild reducing agent to convert Au(III) into Au(0). However, the reducing potential increased with unfolding of BSA beyond 40 C and became maximum in the denaturation temperature range (i.e., 52-58 C). Unfolded BSA conjugated NPs thus produced then started a seeding process with other similar NPs or free BSA to produce self-assembled colloidal assemblies in the form of soft film of BSA bearing NPs. SEM, TEM, and AFM studies were used to characterize the BSA conjugated NPs in the form of soft film. The soft film was used with water insoluble zein protein to produce very robust biodegradable protein films suitable for various food and pharmaceutical applications. Tensile strength and strain at failure measurements of zein protein films demonstrated that the film made with BSA conjugated NPs existed in the form of a soft film was much stronger and flexible in comparison to that made with nonaggregated NPs.

’ INTRODUCTION Synthesis of functional inorganic materials by organisms is a unique natural process where biomolecules are directly involved in their synthesis, and this process is known as biomineralization. For instance, the gravity sensor crystals of calcite that are produced in the inner ear are sensitive to any change in the acceleration. Similar functions are performed by the crystals of magnetite in a variety of magnetotactic bacteria. Biomineralization1-12 is a rapidly developing area to synthesize biofunctional nanomaterials by directly using biomolecules in their shape control synthesis13-15 with several biological applications.16-21 Biomolecules such as amino acids and phospholipids also act as fine capping/stabilizing agents22-25 to achieve appropriate geometries. They occupy low energy crystal planes selectively13-15 and hence reduce their participation in the overall nucleation process that directs the overall crystal growth at uncapped or poorly capped high energy crystal planes to achieve desired morphologies. For instance, in the face centered cubic (fcc) geometry, low energy {100}/{110} planes are preferentially r 2011 American Chemical Society

occupied by surface active molecules, which can direct crystal growth at high energy {111} planes to produce rods or wires from spheres.13,15 But not all biomolecules effectively control the shape.14 Better interfacial adsorption leads to better shape controlled effects, and thus it is the deciding criterion to control the overall shape of nanomaterials. Zwitterionic amino acids with low hydrophobicity cannot effectively control the crystal growth, and it is also true for zwitterionic phospholipids.14 Likewise, low molecular weight water-soluble globular proteins are less surface active than fibrous proteins due to their relatively less interfacial adsorption.26,27 On the other hand, unfolded proteins are always more surface active than folded ones.28,29 Unfolded protein with its large surface area can also provide soft-template effects30-32 for growing nucleating centers. We have tried to evaluate all these aspects by choosing a simple ternary mixture of BSA þ HAuCl4 þ water. In this Received: October 23, 2010 Revised: January 6, 2011 Published: February 2, 2011 2982

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The Journal of Physical Chemistry C

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system, BSA initiates33-36 the biomineralization of Au NPs simply by following the reduction of Au(III) into Au(0). Once atoms are produced, they undergo a nucleation process which is simultaneously controlled by the capping/stabilizing behavior of BSA. Here, the physical state of BSA that is folded or unfolded plays a governing role in configuring the overall shape and size of Au NPs. BSA is composed of 580 amino acid residues with 17 interchain disulfide bonds.37-39 The overall shape of BSA is oblate, and it consists of three domains I, II, and III, which are divided into nine loops. Each domain is made up of a sequence of large-small-large loops forming a triplet. BSA remains in native state up to 40 C; between 40 and 50 C, the reversible changes in its conformation occur, whereas they become irreversible between 50 and 60 C. Above 60 C, unfolding of BSA with βaggregation begins which ultimately leads to a gel formation around 70 C.39 The unfolded gel form of BSA is expected to act as a soft template and is the most ideal candidate to study the protein film formation in its soft state. Thus, temperature is the main contributing factor to achieve the gel state, but it also speeds up the reduction and nucleation of NPs. Therefore, an appropriate balance between the temperature and concentration parameters of a biomineralization process is required to achieve an appropriate protein film formation. These films may not be directly used for various applications (such as food or pharmaceutical packaging applications, disposable medical products such as gloves, gowns, etc., and biodegradable agricultural mulches)40-44 due to their liquid crystalline gel state, but they can be converted into more robust forms by using water insoluble zein protein. Pure zein films have little use due to their highly brittle and least flexible nature. Therefore, appropriate blending is to be done by incorporating conventional plasticizers such as glycerol, poly(ethylene glycol), fatty acids, and monoglycerides to explore their best applications.42,43 Although plasticizers play a significant role in achieving the flexibility, they also cause a decrease in tensile strength. We have tried to address this issue by blending zein films with BSA conjugated NPs to improve their mechanical properties. BSA conjugated NPs are first produced through biomineralization of gold salt by BSA and then used in the protein film formation to achieve better mechanical properties.

’ EXPERIMENTAL SECTION Materials. Chloroauric acid (HAuCl4), bovine serum albumin (BSA), and zein protein were purchased from Aldrich. Double distilled water was used for all preparations. Biomineralization of Au NPs. Aqueous mixtures (total 10 mL) of BSA (0.0015-0.015 mM) and HAuCl4 (0.25-1.0 mM) were taken in screw-capped glass bottles. After the components were mixed at room temperature, the reaction mixtures were kept in a water thermostat bath (Julabo F25) at precise 40, 60, or 80 ( 0.1 C for 6 h under static conditions. The color of the solution changed from colorless to pink-purple and finally purple within half an hour and remained the same thereafter in most of the cases. The following overall reaction (eq 1) was considered to take place where BSA acts as a weak reducing agent due to the presence of several reducing amino acids like cysteine. The reduction progresses as more and more cysteine residues come in contact with aqueous phase during the unfolding of BSA due to temperature or pH effects.

Au3þ ðaqÞ þ BSA ðaqÞ þ 3e- f Auo ðsÞ

ð1Þ

After 6 h, the samples were cooled to room temperature and kept overnight. They were purified from pure water at least two

times in order to remove unreacted BSA. Purification was done by collecting the Au NPs at 10000-14000 rpm for 5 min after washing each time with distilled water. Methods. UV-visible spectra of as prepared gold colloidal suspensions were recorded by UV spectrophotometer (Multiskan Spectrum, model no. 1500) in the wavelength range of 200-900 nm to determine the absorbance due to surface plasmon resonance (SPR). In addition, time dependent scans of some selective reactions at 40, 60, and 80 ( 0.1 C at regular intervals were also carried out to understand the growth kinetics of Au NPs. Reactions were also monitored over a wide temperature range from 20 to 70 C in order to determine the influence denaturation of BSA on the synthesis of Au NPs. Scanning electron microscopy (SEM) analysis was carried out on a Zeiss NVision 40 Dual Beam FIB/SEM instrument. Photomicrographs were obtained in bright field scanning/imaging mode, using a spot size of ∼1 nm and 12 cm of a camera length. Energy dispersive X-ray spectroscopy (EDS) microanalysis was carried out using an Oxford-INCA Atmospheric Ultrathin Window (UTW), and the data were processed using Oxford INCA Microanalysis Suite Version 4.04. Transmission electron microscopy (TEM) measurements were carried out by mounting a drop of a sample on a carbon coated Cu grid and allowed to dry in air. They were observed with the help of a Philips CM10 Transmission Electron Microscope operating at 100 kV. Atomic force microscopic (AFM) measurements were carried out by model Veeco diCaliber at room temperature. Twenty-five microliters of purified aqueous suspension of gold colloids was first spin coated (MTI corporation, PC100) at 500 rpm for 2 min on ultracleaned glass coverslip and was left to dry in a drybox. It was then scanned with silicon nitride tips in contact mode to get amplitude and height images. Survey scanned images were processed and analyzed by using SPM graphic software to obtain three-dimensional topography of BSA β-sheet bearing Au NPs. X-ray diffraction (XRD) patterns were recorded by using Bruker-AXS D8-GADDS with Tsec = 480. Samples were prepared on glass slides by placing a concentrated drop of aqueous suspension and then dried in vacuum desiccator. The pH of some of the reactions was measured by using a pH meter. Each reaction solution was taken in a glass flask with double walled jacket, and the temperature was precisely controlled by externally circulating thermostatted water (Julabo F25). pH measurements were carried out at regular intervals over a period of 6 h. Total protein contents were determined by the Bradford method as mentioned elsewhere.45 Zein protein film formation with BSA conjugated NPs was carried out as follows. First of all, a clear solution of zein (10% w/v) in aqueous ethanol (90% v/v) along with glycerol (30% on zein weight basis) as plasticizer was prepared. Then, a freshly produced BSA conjugated NPs suspension (10% v/v) was added into this solution. Five grams of this filmogenic solution was placed in a 9 cm diameter plastic Petri dish and gently swirled to coat the bottom of the dish. It was placed without a lid on a level surface (checked with a spirit level) in an oven at 40 C for 24 h. Dehydration of this filmogenic solution led to a protein film formation with average thickness of 0.05 mm which was easily peeled off. Color coordinate data of the films were determined using a Hunter colorimeter model D 25 optical sensor (Hunter Associates). The Hunter L, a, and b color space is a threedimensional rectangular space based on the opponent color theory. The L* is a measure of brightness, while a* and b* are 2983

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Figure 1. (a) UV-visible scans of a reaction with [Au/BSA] = 167 at 40 C at different time intervals over a time span of 6 h. Prominent peaks at 220 and 295 nm belong to AuCl3- ions and BSA, respectively. The intensity of both peaks decreases from top to bottom with time. Relative to these peaks, the absorbance due to SPR of Au NPs close to 520 nm is insignificant. The inset shows plots of normalized intensity versus time for the three peaks (see details in text). (b) The same reaction carried out at 80 C now shows prominent peaks at 550 nm due to SPR of Au NPs. (c) Plots of intensity (at 550 nm) versus time for the reactions carried out at 40, 60, and 80 C, demonstrating the effect of temperature on the synthesis of Au NPs. (d) Reactions of different mole ratios carried out with temperature ranging from 20 to 70 C showing the denaturation process of BSA. (e) Plots similar to (c) at 80 C indicating the effect of BSA concentration on the synthesis of Au NPs. (f) A variation in the reaction pH at different mole ratios. See details in text.

the color coordinates that range from -90 to þ90 in the color space “circle”. For a*, -90 = green and þ90 = red, and for b*, -90 = blue and þ90 = yellow. To perform the color tolerance, a standard sample was measured and saved for subsequent comparisons. The mechanical properties of the films containing different samples of BSA conjugated Au NPs were measured using a Texture Analyzer (TAXT2, Stable Microsystems, Godalming, U.K.) with a 5 kg load cell as mentioned elsewhere.43,46

’ RESULTS UV-Visible Studies. Temperature Effect. Each reaction is monitored simultaneously by UV-visible measurements over a

period of 6 h. Figure 1a and b show typical scans of a reaction with Au/BSA mole ratio =167 at 40 and 80 C, respectively. The peak close to 220 nm belongs to AuCl4- ions, while that at 295 nm is due to aromatic tryptophan residues of BSA. Colloidal Au NPs usually show absorbance due to SPR around 520 nm. At 40 C (Figure 1a), peaks at 220 and 295 nm are quite prominent, and their intensity decreases with time, whereas no peak is visible around 520 nm. Normalized intensity plots (Figure 1a, inset) indicate that the absorbance due to tryptophan diminishes within 90 min of the reaction, whereas that for AuCl4- ions takes about 250 min. The AuCl4- ions peak takes more than double the time to disappear and demonstrates the weak reducing behavior of BSA. The same reaction at 80 C (Figure 1b) produces a prominent peak at 550 nm due to Au NPs whose intensity 2984


The Journal of Physical Chemistry C increases with time. Note a much broader tryptophan peak at 295 nm, which vanishes as the peak at 550 nm achieves maximum intensity and the reaction equilibrates. The disappearance of the tryptophan peak can be related to the simultaneous adsorption of BSA on the freshly produced NPs. As more and more NPs are produced by simultaneous conversion of AuCl4- ions into the nucleating centers, more BSA is associated with the NP surface. Such an adsorption leads to a partial unfolding of BSA due to the breaking of disulfide bridges in view of the covalent interactions of cysteine residues with NP surface47 and causes conformational changes.35,48,49 Tryptophan has been found to be deeply embedded in the hydrophobic environment thus produced, with the result of which its absorbance decreases.47 The progress of a reaction can be studied by plotting the intensity of the 550 nm peak versus reaction time (Figure 1c) because it can be related to the number density of Au NPs.50,51 Figure 1c indicates that a negligible amount of NPs is produced at 40 C, but the amount increases at 60 C and becomes maximum at 80 C. At 80 C, as soon as the reaction is placed in the thermostat bath, the intensity of this peak increases exponentially and the reaction equilibrates within an hour. Obviously, the low temperature of the reaction (i.e., 40 C) provides much weaker reducing ability of BSA that increases as the temperature increases to 60 C and becomes maximum at 80 C. The 80 C further accelerates it due to the unfolding of BSA, which provides even better reducing ability due to a breakdown of a maximum number of disulfide bridges. Thus, two factors, temperature and unfolding of BSA, simultaneously control the reduction of Au(III) into Au(0) and can be best studied by monitoring the reaction with respect to temperature. Denaturation of BSA. A systematic change in the reaction temperature from 20 to 70 C not only indicates the formation of NPs but also simultaneously demonstrates the denaturation of BSA (see UV-visible scans with temperature in the Supporting Information, Figure S1). A variation in the intensity of the Au NPs peak at 550 nm for the reactions at different Au/BSA mole ratios is illustrated in Figure 1d. It is quite similar in all cases but much different from that of Figure 1c. Note that Figure 1c represents the intensity variation with time at a constant temperature where BSA exists in a particular physical state. But this is not the case with Figure 1d, where reaction temperature increases successively from 20 to 70 C, which causes a complete change in the BSA structure from native to unfolded state. Since BSA is a reducing agent and its physical state significantly influences the reduction process, the variation in 550 nm peak intensity should reflect the BSA denaturation behavior. Thus, Figure 1d can be explained in terms of a phase diagram with distinct regions. Region I refers to a temperature variation from 20 to 42 C with a little change in the intensity because no significant amount of NPs is produced in this region as observed in Figure 1c (at 40 C), although intensity values for mole ratio 68 are slightly higher than those of the lower mole ratios due to a greater amount of gold salt. This region represents predominantly the native state of BSA where BSA exists in the globular form with most of its disulfide bridges intact. It is to be noted that BSA consists of 17 disulfide bridges; therefore, breaking of a few disulfide bridges due to its association with a few small Au NPs will not significantly influence the tertiary structure of BSA, and hence the 550 nm peak intensity does not show any marked change within this region. In region II from 42 to 52 C, marked increase in the intensity is observed for the mole ratio of 68 again because of the greater

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amount of Au, but the variation is not so significant for the lower mole ratios except for a slight discontinuity around 42 C, suggesting some visible conformational changes have taken place in the BSA tertiary structure. Region II represents the temperature range where reversible conformational changes39 are taking place in the BSA structure, but a dramatic increase in the intensity especially with mole ratio 68 suggests much more than this. In fact, no reversible changes are expected in the presence of Au NPs because the partially unfolded domains will speed up the rate of the reduction process which will in turn produce a greater number of nucleating centers. This will allow a greater amount of BSA to be adsorbed on the NPs surface leading to a partial breakdown of the disulfide bridges. Freshly exposed cysteine residues further participate in the reaction, and the reaction turns into an autocatalytic process. This effect will obviously be a minimum with lower mole ratios. Further increase in the temperature under such circumstances leads us to region III from 52 to 58 C, where the most dramatic and identical changes are taking place for all the mole ratios. This temperature range brings irreversible changes to BSA tertiary structure by breaking all disulfide bridges and unfolding of R-helices.39 The reduction process thus passes through a maximum at 55 C when all cysteine residues fully participate in the reaction.52 Thereafter, the system relaxes at 58 C and progresses toward β-aggregation and gel formation in region IV. Of interest, region IV indicates that intensity continues to rise for the mole ratio 68, whereas it almost vanishes for 34 and 17. It means that the gel phase thus created at lower mole ratios where the amount of BSA is sufficiently higher than that of Au encloses all NPs in such a way that their SPR completely screens. The concentration effect will make this point more clear. BSA Concentration Effect. An increase in the amount of BSA at fixed HAuCl4 (Figure 1e) especially at 80 C (as shown in region IV of Figure 1d) reduces the absorbance of Au NPs due to entrapment of NPs by the gel phase. The origin of this behavior can be explained on the basis of simultaneous measurement of reaction pH (Figure S2). pH remains fairly constant and acidic after a slight initial fall within 15 min in each reaction. Acidic pH is generated by the dissociation of HAuCl4 to release Hþ ions, which in turn allows electrostatic interactions between R-NH3þ groups of BSA and AuCl4- ions (eq 2). Figure 1f demonstrates the variation in pH with a Au/BSA mole ratio at 40 C and at 80 C. In both cases, pH decreases with a steep slope up to Au/BSA = 350 and then tends to level off. The pH values at 80 C are lower than at 40 C. It means that greater dissociation of HAuCl4 takes place at 80 C and is obviously understood from the greater unfolding of BSA at 80 C rather than at 40 C because more aqueous exposed amino acids will attract more AuCl4- ions. Thus, high temperature, high BSA contents, and low pH facilitate the reduction and simultaneous entrapment of Au NPs in unfolded BSA. That is why practically no SPR is observed for Au/BSA = 17 and 34 in comparison to 68 especially in region IV of Figure 1d and is further explained by the following mechanism.

A lower pH than isoelectric point, pH = 4.7, is primarily responsible for the denaturation of BSA53,54 and is expected to enhance its reducing ability with more pronounced effect at 80 C 2985


The Journal of Physical Chemistry C rather than at 40 C. This is all related to the fact that albumins consist of a high content of cysteine and the charged amino acids with 17 disulfide bridges. The disulfide bridges are located almost exclusively between helical segments. None of the disulfide bonds is accessible to solvent in the pH range 5-7, they but become progressively available as the pH falls below the isoelectric point. As unfolding progresses due to low pH or high temperature of 80 C, more reducing amino acids like cysteine are exposed to the aqueous phase and hence participate in the reduction of AuCl4- ions to a much greater extent. The denatured BSA thus produced instantaneously adsorbs on the NP surface55-57 due to covalent interactions of cysteine residues with gold surface.47 Such assemblies then act as seeds to further interact with BSA conjugated NPs or free BSA in either the unfolded or partially folded states,58,59 leading to the formation of large aggregated assemblies. Under such circumstances, the reaction temperature decides if the conjugated BSA should exist in the form of a sol or gel state.60,61 If the reaction temperature lies in region I of Figure 1d, then obviously BSA conjugated NPs will exist in the form of sol, and if it is in region IV, it will exist in the form of gel. Thus, Au NPs present in sol show more SPR than those embedded deep in the gel phase. Furthermore, protein analysis based on the Bradford method allows us to quantitatively evaluate the amount of BSA associated with NPs (BSAc) (Figure S3). It is lowest at 40 C where BSA is considered to be mainly in its folded state, whereas a maximum at 80 C where BSA acquires a complete unfolded form shows that the latter form has maximum affinity for Au NPs. The gel phase thus created also acts as a soft template30-32 and provides a kind of semisolid support for the growing nucleating centers. It separates out from the aqueous phase in the form of small black flakes upon aging. Figure S4 shows a picture of such fragments deposited on a glass coverslip with a marked difference from a drop cast of a sol. The above results not only indicate the facilitation of the reduction process at 80 C and at high BSA contents, but the latter conditions are best suited for a soft protein film formation with entrapped Au NPs. Kinetic parameters evaluated from the similar plots following BSA absorption at 295 nm of Figure 1a indicate that reduction process primarily depends on the amount of BSA and facilitates at 80 C (rate constant = 1.03 10-5 s-1) rather than 40 C (rate constant = 6.18 10-5 s-1). With respect to the amount of BSA, the rate constant (i.e., 4.82, 4.41, and 4.31 10-5 s-1) at Au/BSA = 17, 34, and 167, respectively, decreases with the decrease in the amount of BSA. It means that the greater amount of BSA in its unfolded state is required for the facilitation of the reduction process which allows a maximum number of reducing amino acids to come in contact with AuCl4ions to initiate the reduction. Hence, unless BSA is not in its unfolded state, appropriate reduction cannot be achieved even if we have a greater amount of AuCl4- in the solution. Microscopic Studies. BSA Concentration Effect. SEM and TEM studies not only help us to evaluate the shape and size of Au NPs but also provide direct evidence of the presence of NPs in the gel phase. Figure 2a and b show SEM and TEM micrographs, respectively, of denatured BSA bearing Au NPs of 7.5 ( 3.5 nm (size distribution histogram from TEM image, Figure S5) of a sample prepared with a Au/BSA mole ratio of 167 at 80 C. Bright and dark field SEM images (Figure 2c and d, respectively) further confirm the presence of BSA as a gel support for the NPs. EDS analysis (Figure 2e) performed on a large NP (shown in dotted circle) shows emission only due to gold, while XRD

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patterns (Figure 2f) can be indexed to fcc geometry of the bulk gold. Surface topography of a BSA film with NPs can be best studied with the help of AFM measurements in contact mode. Figure 2g, h shows height images of adsorbed NPs as bright spots, whereas Figure 2i demonstrates the surface topography. Line analysis (Figure 2h) computes the average size of NPs as 32.1 ( 10.6 nm, which is much larger than 7.5 ( 3.5 nm determined from the TEM analysis (Figure 2b) because it is quite difficult to differentiate between the actual NP boundary and BSA coating. On the other hand, no typical BSA film with NPs is observed for the same sample prepared at 40 C (Figure S6a) due to the presence of predominantly globular nature of BSA. Instead, deformed shapes with larger size are obtained, which were indeed expected at 40 C rather than 80 C due to a weaker capping ability and reducing potential of globular BSA than that of an unfolded BSA at 80 C (see corresponding intensity plots in Figure 1c). A stronger reducing potential produces many folds of higher number of nucleating centers62 than a weaker reducing potential, and hence a relatively lower number of Auo atoms are accommodated per center in comparison to a fewer nucleating centers63 against a constant amount of HAuCl4. That is why the former produces many more small NPs (Figure 2a, b) than the latter. An increase in the amount of BSA (from Au/BSA mole ratio of 167 to 17) at 80 C produces even much more aggregated BSA with entrapped NPs (Figure S6b) as indicated by Figure 1d in its region IV where practically no SPR is observed for mole ratio 17. But it also produces much larger well-defined platelike geometries (Figure 2j) as observed previously by other authors,33 demonstrating a clear correlation between Figure 1d and Figure 1e. As nucleation is a time dependent process (in the presence of a weak reducing agent like BSA), such morphologies evolve with time (see a slow increase in the intensity of Au NPs absorbance in 6 h in Figure 1e for mole ratio 17). Gold Salt Concentration Effect. Figure 3a shows a SEM image of a sample prepared with Au/BSA mole ratio of 334 at 80 C. A fine interconnected treelike arrangement of NPs is evident but without the presence of any BSA gel support contrary to the sample made with a mole ratio of 167 (Figure 2a). A closeup image (inset, Figure 3a) indicates the presence of fused NPs in the form of small nanowires (NWs). An AFM image provides an even better picture (Figure 3b) where treelike branches are formed due to an oriented attachment (Figure 3c) of large NPs (194 ( 38.6 nm, see line analysis) with clear facets (Figure 3d). It is usually explained on the basis of Ostwald ripening process64-66 where poorly capped NPs undergo interparticle fusions to produce bigger geometries. Further increase in the amount of gold salt from 334 to 668 mol ratio converts the NPs with facets into even much larger triangular platelike morphologies (Figure 3e) of 217 ( 158 nm (size distribution histogram, Figure S7a) and smaller icosahedral NPs (Figure 3f) of 21 ( 8 nm (Figure S7b). It demonstrates that a systematic increase in the amount of HAuCl4 in the following order of mole ratios— 167 (Figure 2a) > 334 (Figure 3a) > 668 (Figure 3e, f)— dramatically changes the shape and size of NPs as well as their association with BSA. In fact, the appearance of triangular plates bound with {111} facets is due to a preferential adsorption of unfolded BSA on {111} crystal planes33 that prevents any crystal growth along the Æ111æ directions as the amount of HAuCl4 increases. It first converts the small polyhedral NPs (Figure 2a) into large NPs with clear facets (Figure 3d), which ultimately acquire platelike geometries (Figure 3e) as growth proceeds on 2986



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Figure 2. (a) FESEM (scale bar = 200 nm) and (b) TEM micrographs of Au NPs as bright spots on unfolded BSA as a soft template of a sample with Au/BSA mole ratio 167 synthesized at 80 C (scale bar = 100 nm). (c) and (d) Bright field (scale bar = 250 nm) and dark field images (scale bar = 250 nm), respectively, differentiating between Au NPs and unfolded BSA. (e) and (f) EDS spectrum and XRD patterns of Au NPs, respectively. (g) AFM height image of unfolded BSA as a soft template bearing Au NPs (scale bar = 870 nm). (h) Close-up image showing the line analysis of various Au NPs (scale bar = 100 nm). (i) Topography of the BSA film bearing Au NPs showing peaks and valleys. (j) TEM micrograph of large platelike NPs of a sample with Au/BSA mole ratio 17 prepared at 80 C (scale bar = 100 nm). See details in text.

{110} crystal planes as depicted in Figure 3g. Thus, increasing size and acquiring platelike geometries in fact facilitate the interfacial adsorption of unfolded BSA67 rather than the film

formation. Hence, low amounts of HAuCl4 with small NPs favors their adsorption on the BSA film, while high amounts result in the formation of large platelike geometries. 2987



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Figure 3. (a) FESEM image showing treelike branches of Au NWs formed through oriented attachment of Au NPs of sample with Au/BSA mole ratio 334 prepared at 80 C (scale bar 1 μm). Inset, close-up of (a) (scale bar = 200 nm). (b) Low resolution AFM height image of this sample showing the presence of treelike long NWs of Au NPs similar to those shown in the FESEM image (a) (scale bar = 10 μm). (c) Close-up image of NWs shows that they are made through the oriented attachment of prismlike Au NPs (scale bar = 2.7 μm). (d) AFM height image of independent NPs with clear facets (scale bar = 1.9 μm). (e) (scale bar = 1 μm) and (f) (scale bar = 60 nm) represent TEM images of a sample with Au/BSA mole ratio of 668 prepared at 80 C. (g) Schematic representation of the evolution of NPs morphology with the increase in the amount of gold salt. See details in text.

Protein Film and Its Properties. From the above results, it is clear that BSA conjugated NPs with Au/BSA mole ratio = 167 exist in the form of a clear protein film, whereas further increase in this ratio only increases the size with platelike morphologies of NPs without any clear film formation. This aqueous insoluble film precipitates out on aging in the form of small flakes (Figure S4) which could have limited applications. However, it can be converted into a large robust continuous biodegradable sheet by combining it with water insoluble zein protein (see Experimental Section). Figure 4a and b show such protein films made from samples of mole ratios 167 and 668, respectively. Visually both films do not show any practical difference, but color coordinates and mechanical properties demonstrate a significant difference between the two because the first one is made from a sample whose images are shown in Figure 2a, b while the second one is made from the sample of Figure 3e, f.

Color coordinates help us to determine the isotropic nature of the protein film. A variation in L*-, a*-, and b*-values (see Experimental Section) of zein films without NPs (i.e., zein þ BSA) and with BSA conjugated Au NPs (of mole ratios 167, 334, and 668) is shown in Figure 4c. A film containing bioconjugate NPs of mole ratio 167 has lower L*-values and almost equal a*value to a film without NPs. A lower L*-value indicates that the film is less bright or more dark and is attributed to a uniform distribution of small NPs throughout the film. But further increase in the mole ratio (to 334 and 668) increases L* due to an uneven distribution of large NPs arranged in NWs (Figure 3a-d) or platelike morphologies (Figure 3e). The variation in b* is considered to be entirely related to the protein structure because it refers to the blue and yellow parts of the electromagnetic spectrum. The positive b* value is due to the light yellow color of the protein film which represents the dried state of unfolded self-aggregated protein. The presence of NPs is 2988



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Figure 4. Photos of biodegradable protein films made with BSA conjugated NPs with Au/BSA mole ratios of (a) 167 and (b) 668. See Experimental Section for the composition of different components. (c) Histogram of color coordinates for the films made with different mole ratios. (d) and (e) Histograms of tensile strength and strain at failure versus different mole ratios, respectively. See details in the text.

expected to decrease the b*-value due to the incorporation of NPs in the cohesive arrangement of unfolded protein. Figure 4d and e show the variation in the tensile strength (TS) and strain at failure (E), respectively, for the above-mentioned films. Interestingly, film made with Au/BSA mole ratio of 167 (or with a sample of Figure 2a, b) shows a large increase in both properties in comparison to that in the absence of NPs (i.e., zein þ BSA), but further increase in the mole ratio to 334 and 668 regularly decreases these properties. Generally, addition of plasticizer decreases the TS and increases E (also known as elongation), which makes the film more flexible and less strong than in the absence of plasticizer. However, in our case, mole

ratio 167 produces a remarkably stronger film with even better flexibility than in the absence of BSA conjugated NPs, which is of course not maintained with mole ratios 334 and 668 because these samples do not contain BSA conjugated NPs in the form of a self-assembled film (Figure 3).

’ DISCUSSION A collective comparison of all results indicates that a robust biodegradable protein film with better mechanical properties can only be obtained when self-assembled biocojugated NPs (SA NPs) are used along with zein (Figure 5). Our results show that it 2989



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Figure 5. Schematic representation of the synthesis of BSA conjugated NPs under different experimental conditions and their use in the zein film formation. See details in the text.

happens with Au/BSA mole ratio of 167 where one can find the formation of SA NPs in the form of soft film following the seeding method (Figure 5a-c) as discussed in the Results section. Simple calculations suggest that there are about 3.4 sites available on a single BSA macromolecule per AuCl4- ion if BSA is considered to be in a fully unfolded state which is usually not the case because even at 80 C some of the hydrophobic domains are away from the aqueous phase. This perception is based on the fact that we do not observe any independent Au NPs in this sample (Figure 2a, b). But on the contrary, as the mole ratio increases to 334 or 668, there are plenty of independent NPs which exist as NWs (Figure 3a-c) or large platelike geometries (Figure 3e, f). Likewise, neither does a decrease in the mole ratio from 167 to 17 help us to achieve the film formation (Figure 2j) because an excessive amount of BSA promotes its self-aggregation without any clear film formation (Figure 5). Moreover, it is not only the bioconjugated NPs which initiate the seeding for self-aggregation,46,47 but desorption of denatured protein from the NP surface also triggers the seeding with free protein to promote the protein-protein aggregation.58 Thus, an appropriate balance between the amounts of HAuCl4 and BSA is required to obtain bioconjugated NPs which entirely exist in the form of self-assembled soft films (Figure 5). The following mechanism explains how SA NPs promote the mechanical properties of zein film rather than large nanoplates. In fact, the seeding process which is responsible for the synthesis of SA NPs is also expected to work with zein. Zein is a highly hydrophobic protein; therefore, it will have preferential hydrophobic interactions with SA NPs because SA NPs are also

predominantly hydrophobic due to the charge neutralization in the course of BSA adsorption on the NP surface. This is not the case with NPs existing in the form of NWs or large plates. Therefore, an appropriate BSA coating around NP in its selfassembled state (Figure 5c) favors the BSA-zein interactions for better assimilation of SA NPs in zein film (Figure 5d) rather than the interactions between poorly capped nanoplates and zein protein (only {111} crystal planes are mainly occupied by unfolded BSA, Figure 3g). In addition, lateral alignment of unfolded BSA with zein (Figure 5d) in producing a liquid crystalline arrangement also matters a lot in deciding the cohesive strength of the resulting protein film. Again, such kind of association is predominantly due to hydrophobic interactions between the nonpolar domains of both proteins and re-establishment of hydrogen bonding68 between the respective charged amino acids. Colloidal SA NPs in the form of soft films thus provide better orientations for unfolded zein to interact rather than independent platelike morphologies to produce strong and flexible protein films (Figure 5e).

’ CONCLUSION The results conclude that one can synthesize bioconjugated nanomaterials by following a simple reduction of metal ions by biomolecules. These biomaterials can then be used for biodegradable protein film formation. We have carried out this study by following a direct reduction of gold ions into NPs by BSA and have further used them along with zein protein to form biodegradable protein films. The reduction of gold ions into atoms 2990


The Journal of Physical Chemistry C depends on various factors which include the concentration, pH, temperature, and physical state of BSA. All factors significantly change the shape, structure, and morphology of BSA conjugated NPs which are the governing criteria for a versatile biodegradable protein film formation with better tensile strength and flexibility. We have found that a narrow Au/BSA mole ratio of 167 only produces small NPs adsorbed on the unfolded BSA in the form of a soft protein film in colloidal state. Any decrease or increase in this mole ratio produces large platelike triangular shapes of Au NPs with no sign of soft protein film formation or much aggregated BSA entrapping NPs, respectively. We have used these different morphologies of BSA conjugated NPs along with zein to produce more robust biodegradable protein films appropriate for various applications. Zein film produced with BSA conjugated NPs (especially in their colloidal state of soft film) has been found to have better tensile strength and flexibility than the one produced with large platelike NPs. Thus, the present results conclude that an appropriate biodegradable film with better strength and flexibility can be achieved by choosing appropriate morphologies of bioconjugated nanomaterials in their selfaggregated state.

’ ASSOCIATED CONTENT

bS

Supporting Information. UV-visible spectra and histograms. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail [email protected].

’ ACKNOWLEDGMENT These studies were partially supported by financial assistance from CSIR [ref. no. 01(2220)/08/EMR-II] and New Delhi [ref no. 01(2219)/09/EMR-II]. ’ REFERENCES (1) So, C. R.; Kulp, J. L.; Oren, E. E.; Zareie, H.; Tamerler, C.; Evans, J. S.; Sarikaya, M. ACS Nano 2009, 3, 1525. (2) Barnard, A. S.; Russo, S. P. Nanotechnology 2009, 20, 115702. (3) Wang, X.; Muller, W. E. Trends Biotechnol. 2009, 27, 375. (4) Muthukumar, M. J. Chem. Phys. 2009, 130, 161101. (5) Villarreal-Ramirez, E.; Moreno, A.; Mas-Oliva, J.; Chavez-Pacheco, J. L.; Narayanan, A. S.; Gil-Chavarria, I.; Zeichner-David, M.; Arzate, H. Biochem. Biophys. Res. Commun. 2009, 384, 49. (6) Nuraje, N.; Mohammed, S.; Yang, L.; Matsui, H. Angew. Chem., Int. Ed. Engl. 2009, 48, 2546. (7) Kunz, W.; Kellermeier, M. Science 2009, 323, 344. (8) Masica, D. L.; Schrier, S. B.; Specht, E. A.; Gray, J. J. J. Am. Chem. Soc. 2010, 132, 12252. (9) Marin-Garcia, L.; Frontana-Uribe, B. A.; Juan, P. R.-G.; Stojanoff, V.; Serrano-Posada, H. J.; Moreno, A. Cryst. Growth Des. 2008, 8, 1340. (10) Faivre, D.; Sch€uler, D. Chem. Rev. 2008, 108, 4875. (11) Prozorov, T.; Palo, P.; Wang, L.; Nilsen-Hamilton, M.; Jones, D.; Orr, D.; Mallapragada, S. K.; Narasimhan, B.; Canfield, P. C.; Prozorov, R. ACS Nano 2007, 1, 228. (12) Meldrum, F. C.; C€olfen, H. Chem. Rev. 2008, 108, 4332. (13) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. Chem. Mater. 2007, 19, 1257. (14) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2007, 111, 14113.

ARTICLE

(15) Bakshi, M. S.; Kaur, G.; Thakur, P.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2007, 111, 5932. (16) Wang, K. G.; Niu, H. Methods Mol. Biol. 2009, 544, 17. (17) Murphy, J. N.; Cheng, A. K.; Yu, H. Z.; Bizzotto, D. J. Am. Chem. Soc. 2009, 131, 4042. (18) Feng, W. Y.; Chiu, N. F.; Lu, H. H.; Shih, H. C.; Yang, D.; Lin, C. W. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2008, 2008, 5757. (19) Bardin, F.; Bellemain, A.; Roger, G.; Canva, M. Biosens. Bioelectron. 2009, 24, 2100. (20) Peng, L.; Eltgroth, M. L.; LaTempa, T. J.; Grimes, C. A.; Desai, T. A. Biomaterials 2009, 30, 1268. (21) Fischer, K. E.; Aleman, B. J.; Tao, S. L.; Hugh, Daniels, R.; Li, E. M.; Bunger, M. D.; Nagaraj, G.; Singh, P.; Zettl, A.; Desai, T. A. Nano Lett. 2009, 9, 716. (22) Shang, L.; Qin, C.; Wang, T.; Wang, M.; Wang, L.; Dong, S. J. Phys. Chem. C 2007, 111, 13414. (23) Zhang, H.; Cui, H. Langmuir 2009, 25, 2604. (24) He, P.; Urban, M. W. Biomacromolecules 2005, 6, 1224. (25) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21, 9303. (26) Neurath, H.; Bull, H. B. Chem. Rev. 1938, 23, 391. (27) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 1392. (28) Bakshi, M. S.; Thakur, P.; Kaur, G.; Kaur, H.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. Adv. Funct. Mater. 2009, 19, 1451. (29) Bakshi, M. S.; Jaswal, V. S.; Kaur, G.; Simpson, T. W.; Banipal, P. K.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2009, 113, 9121. (30) Hua, Z.; Chen, Z.; Li, Y.; Zhao, M. Langmuir 2008, 24, 5773. (31) Bull, S. R.; Palmer, L. C.; Fry, N. J.; Greenfield, M. A.; Messmore, B. W.; Meade, T. J.; Stupp, S. I. J. Am. Chem. Soc. 2008, 130, 2742. (32) Holmstr€om, S. C.; King, P. J. S.; Ryadnov, M. G.; Butler, M. F.; Mann, S.; Woolfson, D. N. Langmuir 2008, 24, 11778. (33) Xie, J.; Lee, J. Y.; Wang, D. I. C. J. Phys. Chem. C 2007, 111, 10226. (34) Meziani, M. J.; Rollins, H. W.; Allard, L. F.; Sun, Y. P. J. Phys. Chem. B 2002, 106, 11178. (35) Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, 2714. (36) Peters, T., Jr. All about Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (37) Carter, D. C.; Ho, X. J. Adv. Protein Chem. 1994, 45, 153. (38) Papedopoulou, A.; Green, R. J.; Frazier, R. A. J. Agric. Food Chem. 2005, 53, 158. (39) Murayama, K.; Tomida, K. Biochemistry 2004, 43, 11526. (40) Protein Based Films and Coatings; Gennadios, A., Ed.; CRC Press: Boca Raton, FL, 2002. (41) Buffo, R. A.; Weller, C. L.; Gennadios, A. Cereal Chem. 1997, 74, 473. (42) Bai, J.; Alleyne, V.; Hagenmeier, R. D.; Mattheis, J. P.; Baldwin, E. A. Postharvest Biol. Technol. 2003, 28, 259. (43) Singh, N.; Georget, D. M. R.; Belton, P. S.; Barker, S. A. J. Agric. Food Chem. 2009, 57, 4334. (44) Gao, C.; Stading, M.; Wellner, N.; Parker, M. L.; Noel, T. R.; Mills, E. N. C.; Belton, P. S. J. Agric. Food Chem. 2006, 54, 4611. (45) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (46) Bakshi, M. S.; Kaur, H.; Banipal, T. S.; Singh, N.; Kaur, G. Langmuir 2010, 26, 13535. (47) Zhang, D; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J. Nano Lett. 2009, 9, 666. (48) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404. (49) Chan, S.; Hammond, M. R.; Zare, R. N. Chem. Biol. 2005, 12, 323. (50) Bakshi, M. S.; Sachar, S.; Kaur, G.; Bhandari, P.; Kaur, G.; Biesinger, M. C.; Possmayer, F.; Petersen, N. O. Cryst. Growth Des. 2008, 8, 1713. 2991



ARTICLE

(51) Bakshi, M. S.; Sharma, P.; Banipal, T. S.; Kaur, G.; Torigoe, K.; Petersen, N. O.; Possmayer, F. J. Nanosci. Nanotechnol. 2007, 7, 916. (52) Militello, V.; Vetri, V.; Leone, M. Biophys. Chem. 2003, 105, 133. (53) Chakraborty, T.; Chakraborty, I.; Moulik, S. P.; Ghosh, S. Langmuir 2009, 25, 3062. (54) Donato, L.; Garnier, C.; Doublier, J. L.; Nicolai, T. Biomacromolecules 2005, 6, 2157. (55) Roberts, J. R.; Xiao, J.; Schliesman, B.; Parsons, D. J.; Shaw, C. J., III. Inorg. Chem. 1996, 35, 424. (56) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (57) Lewis, S. D.; Misra, D. C.; Shafer, J. A. Biochemistry 1980, 19, 6129. (58) McMasters, M. J.; Hammer, R. P.; McCarley, R. L. Langmuir 2005, 21, 4464. (59) Jarrett, J. T.; Lansbury, P. T. Biochemistry 1992, 31, 12345. (60) Clark, A. H. In Functional Properties of Food mMacromolecules, 2nd ed.; Hill, S. E., Ledward, D. A., Mitchell, J. R., Eds.; Aspen Publishers: Gaithersburg, MD, 1998; p77. (61) Gosal, W.; Murphy, S. B. Curr. Opin. Colloid Interface Sci. 2000, 5, 188. (62) Murphy, C. J.; San, T. K.; Gole, A. M.; Orendorff, C. J.; Gao, J. X.; Gou, L.; Hunyadi, S. E.; Li, T. J. Phys. Chem. B 2005, 109, 13857. (63) Jana, N. R.; Gearheart, L.; Murphy, C. J. Langmuir 2001, 17, 6782. (64) Redmond, P. L.; Hallock, A. J.; Brus, L. E. Nano Lett. 2005, 5, 131. (65) Li, R.; Luo, Z.; Papadimitrakopoulos, F. J. Am. Chem. Soc. 2006, 128, 6280. (66) Lilly, G. D.; Lee, J.; Sun, K.; Tang, Z.; Kim, K. S.; Kotov, N. A. J. Phys. Chem. C 2008, 112, 370. (67) Bakshi, M. S.; Thakur, P.; Sachar, S.; Kaur, G.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. J Phys. Chem. C 2007, 111, 18087. (68) Oncley, J. L.; Ellenbogen, E.; Gitlin, D.; Gurd, F. R. N. J. Phys. Chem. 1952, 56, 85.

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Protein Films of Bovine Serum Albumen Conjugated Gold Nanoparticles

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