www.afm-journal.de

FULL PAPER

Stabilization of PbS Nanocrystals by Bovine Serum Albumin in its Native and Denatured States By Mandeep Singh Bakshi,* Pankaj Thakur, Gurinder Kaur, Harpreet Kaur, Tarlok Singh Banipal, Fred Possmayer, and Nils O. Petersen* delivery, biological labeling, and others.[1] Construction of a suitable bioconjugate PbS nanocrystals (NCs) are synthesized in aqueous phase within a temperature nanomaterial for a specific application range of 40–80 8C in the presence of native and denatured states of bovine requires appropriate synergism between serum albumen (BSA) as the capping/stabilizing agent. The NCs are the biological molecules and the nanoparcharacterized with the help of field-emission scanning electron microscopy, ticle surface at the nanoscale level.[1f,2] Lead high-resolution transmission electron microscopy, X-ray diffraction, and sulfide (PbS) is an important semiconducting material with a narrow band gap energy energy-dispersive X-ray analysis. At 40 8C, large ball-shaped NCs (145  37 nm) 4 1 and a large Bohr radius of 18 nm. Several with small surface protrusions are formed when 1  10 g mL BSA is used. studies have reported various shape conAs the reaction temperature is increased towards 80 8C, the size of NCs trolled morphologies of PbS nanocrystals decreases and they acquire somewhat cubic geometries (49.1  7.0 nm) due to (NCs).[3] Surfactant-assisted hydrothermal a change in the capping behavior of BSA between its native and denatured methods have primarily been employed for such synthesis. An appropriate use of such states. The native and denatured states of BSA are simultaneously studied by NCs for biological labeling requires the fluorescence spectroscopy using tryptophan emission, and pH measurements coating of biological receptor molecules on with respect to time and temperature. Gel electrophoresis is used to determine the NC surface. This can be accomplished the polarity of the BSA capped NCs. Only the small sized NCs conjugated with if the synthesis is carried out in the relatively larger amounts of BSA show a displacement towards the positively presence of appropriate biomolecules charged electrode in comparison to larger NCs with lower amounts of BSA which have a strong affinity for the NC surface. With surfactant assisted synthesis, capping. It was concluded that the denatured state of BSA is more effective in surfactants are best suited for charge and controlling the crystal growth of PbS than its native state especially in the low steric-stabilizations to attain colloidal staconcentration range. bility. The same stability can also be achieved when, instead of surfactants, amphiphillic biomolecules are used for the synthesis of 1. Introduction bioconjugate nanomaterials.[1f,2] The main advantage of this method is that the NCs thus obtained at the end of the reaction Bioconjugate semiconducting nanomaterials comprise a highly can directly be used for specific bioactive functionalities. important class of materials which can serve in diverse There are other issues which need to be addressed such as the biomedical applications such as luminescence tagging, drug native or denatured state of the biomolecules, for example, proteins. What would be the fate of a protein if it were used as a receptor molecule on a synthesized bioconjugate nanomaterial, [*] Dr. M. S. Bakshi and would it be possible to preserve its functionalities? These Department of Chemistry, Acadia University 6 University Ave., Elliot Hall, Wolfville, NS B4P 2R6 (Canada) questions can be answered if one synthesizes such materials in E-mail: [email protected] the presence of the native and denatured states of a protein. To Prof. N. O. Petersen address this issue we have undertaken a study in which we took National Institute for Nanotechnology, Edmonton, AL, (Canada) bovine serum albumen (BSA) a low molecular weight, water E-mail: [email protected] soluble, and highly important carrier protein as a capping/ P. Thakur, H. Kaur, Prof. T. S. Banipal stabilizing agent to synthesize BSA-PbS bioconjugate nanomaDepartment of Applied Chemistry, Guru Nanak Dev University terials. Amritsar, 143005 (India) BSA, an important blood protein with a molecular weight of Dr. G. Kaur Nanotechnology Research Lab., College of North Atlantic 66 500 Da, is composed of 580 amino acid residues.[4] BSA is a Labrador City, NL A2V 2K7 (Canada) versatile carrier protein with wide hydrophobic, hydrophilic, Prof. F. Possmayer anionic, and cationic properties. The overall shape of BSA is Department of Biochemistry, University of Western Ontario oblate and it contains three domains, I, II, and III, which are 339 Windermere Rd, London, ON N6A 5A5 (Canada) stabilized by an internal network of disulfide bonds. BSA has previously been used as a capping/stabilizing agent for the DOI: 10.1002/adfm.200801212

Adv. Funct. Mater. 2009, 19, 1451–1458

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1451

FULL PAPER

www.afm-journal.de

synthesis of various noble metal and semiconductor nanomaterials.[4a,b,5] This protein is weakly reducing and can act as a shape directing agent to promote anisotropic growth.[4a] A number of studies have reported the heat induced denaturation of BSA.[6] BSA remains stable up to 40 8C; between 40–50 8C, the conformational changes of BSA are reversible, while unfolding of a-helices is irreversible up to 60 8C. Above approximately 60 8C, unfolding of BSA progresses and baggregation begins. Beyond 70 8C, gel formation takes place. The overall denaturation temperature is usually reported to be close to 60 8C depending on different methodologies and detection techniques. In the present study, we have used BSA as a capping as well as a stabilizing agent for the synthesis of PbS NCs. The reactions were carried out in aqueous phase at different temperatures from 40–80 8C. Since heat induces drastic conformational changes in the overall morphology of BSA the capping/stabilizing ability should also vary accordingly. We expected a drastic change in the morphology of PbS NC as we conducted the reaction in the temperature range of 40–80 8C due to collapse of the short segment chains connecting BSA a-helical segments around 60 8C, and a change in the tertiary structure of BSA by intermolecular b-sheet formation above 60 8C. This allowed us to examine differences between the capping ability of the native and denatured states of BSA on the synthesis of PbS NCs. This report is the first of its kind in this direction and the results will help us to understand the complex capping behavior of similar proteins in order to design and synthesize different bio-nanomaterials.

Figure 1. a) FESEM of PbS NCs prepared with BSA ¼ 1  104 g mL1 at 40 8C. b) A close up image showing surface petrustions of approximately 25 nm in size. Size distribution histogram of NCs shown in (a0 ) and (a00 ) represents the XRD patterns of same sample. c) FESEM image of PbS NCs entangled with denatured protein thread at 60 8C. d) A close up image shows that the NCs are completely enveloped by denatured protein. c0 ) Size distribution histogram of NCs of (c). e) FESEM of PbS NCs prepared at 80 8C. Note the interconnected NCs with smooth surfaces. e0 ) Size distribution histogram of NCs and e00 ) their corresponding XRD patterns.

2. Results and Discussion Figure 1 shows various micrographs of PbS NCs synthesized at 40, 60, and 80 8C in the presence of 1  104 g mL1 of BSA. Figure 1a shows a number of individual spherical PbS NCs of 145  37 nm (Fig. 1a0 ) with rough surfaces. A closer inspection reveals that the surface of each NC is covered by protrusions of roughly 25 nm (Fig. 1b). The spherical NCs appear to be monodispersed but to have a slight tendency to clump. The X-ray diffraction (XRD) patterns of these PbS NCs are shown in Figure 1a00 . All peaks are very prominent and are consistent with the cubic rock salt structure of crystalline PbS. The intensity of the (200) peak is much higher than that of (111), which suggests a higher growth rate on {100} facets in comparison to {111}. At 60 8C (Fig. 1c), the morphology of NCs, although a bit less regular, remains more or less the same (shown by the filled arrow), but the size decreases to 83.5  20 nm (Fig. 1c0 ). The NCs also appear to

1452

be entangled with denatured protein threads (indicated by the empty arrow). It appears that some of the denatured protein has completely enveloped the NCs as evidenced from the magnified image in Figure 1d. At 80 8C (Fig. 1e), the NCs acquire more nearly spherical shapes but prefer to exist in a fused state (Fig. 1f) and their size further decreases to 49.1  7.0 nm (Fig. 1e0 ). Interestingly, the intensity of the (200) peak has risen significantly (Fig. 1e00 ) in comparison to rest of the peaks suggesting a markedly increased growth at {100} crystal planes from 40 to 80 8C. As we increase the amount of BSA ¼ 5  104 g mL1 with each reaction, instead of large spherical NCs, groups of dendritic type NCs are formed at 40 8C (Fig. 2a) with a fine crystalline nature (Fig. 2a0 ). At 60 8C, they are attached to a background film of denatured BSA indicated by the empty arrow (Fig. 2b). A further increase in the temperature to 80 8C almost completely eliminates the dendritic nature of the NCs and they acquire rather

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2009, 19, 1451–1458

www.afm-journal.de

smooth morphologies (Fig. 2c), again apparently because of the predominant growth at {100} crystal planes (Fig. 2c0 ). A similar sequence of morphological changes was demonstrated by the NCs when BSA ¼ 10  104 g ml1 is used (Fig. 3a–c). However, here a reaction temperature of 80 8C produced fine fused double or triple cubic NCs (Fig. 3c) of 27.6  6.6 nm (Fig. 3c0 ), which were not observed with any of the previous cases. High-resolution transmission electron microscopy (HRTEM) imaging suggests the presence of a protein shell around each NC (indicated by the empty arrow in Fig. 3d). The NCs are crystalline with predominant growth at {100} crystal planes (Fig. 3c00 ) and lattice-resolved imaging (Fig. 3d) clearly indicates the presence of {100} crystal planes with lattice spacing 3 A˚. Energy dispersive X-ray (EDX) analysis confirms the presence of Pb and S (Fig. 3e). The protein coating can be further identified with the help of high-angle annular dark field (HAADF) imaging (Fig. 4). Figure 4a shows the bright field while the Figure 4b shows the dark field image of the same sample with bright spots as small PbS NCs, identified by the corresponding EDX spectrum (Fig. 4c). Such a thick coating is probably due to the use of 10  104 g mL1 of BSA as the capping/stabilizing agent for this sample. Performing the reactions at fixed pH ¼ 7 provided essentially similar results. Some of the TEM images from these samples were compared with those synthesized in pure water in the presence of 5  104 g mL1 BSA (Figs. 2 and 3). Figure 2c and d show field-

Adv. Funct. Mater. 2009, 19, 1451–1458

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER

Figure 2. a–c) FESEM images of PbS NCs prepared at 40 8C, 60 8C, and 80 8C, respectively, with BSA ¼ 5  104 g mL1. a0 and c0 ) XRD patterns of samples (a) and (c). d) TEM image of the same sample prepared at pH ¼ 7.

emission scanning electron microscope (FESEM) and TEM images of two different samples prepared in pure water and at pH ¼ 7, respectively, under identical conditions. Similar morphologies were obtained in both cases. Similarly, Figure 3c and f represent FESEM and TEM images, respectively, of another two samples prepared under identical conditions in the presence of 10  104 g mL1 BSA. Here too identical morphologies on inter connected PbS NCs are evident from the TEM image. Thus, the pH effect is considered to be minimal because BSA in the presence of salts seems to have a buffering effect. UV-Vis measurements were used to further investigate these results. Figure 5a and b illustrates the profiles of NCs of Figure 1e (1  104 g ml1 of BSA, 80 8C) and Figure 3c (10  104 g mL1 of BSA, 80 8C), respectively, with respect to time from 30 min to 48 hrs. One can observe a clear absorbance shift with a broad maximum in the former case, while this is no longer so in the latter case. It is expected that the fused NCs in Figure 1e would evolve from the larger monodisperse ones as seen in Figure 1a within 48 h. A similar transition is, however, not observed for the NCs of Figure 3c even though their size is comparable to NCs of Figure 1e. The evolution of a relatively broad peak at higher wavelength (Fig. 5a) of the NCs in Figure 1e further supports their self-associating nature in comparison to those of Figure 3c. This indicates that in both the cases, the NCs have ultimately attained smaller morphologies even though the difference in the amount of BSA used is ten-fold. It is known that some of the molecular regions of BSA become accessible to new intermolecular interactions due to conformational changes through disulfide and non-covalent interactions as the temperature of aqueous BSA solutions increases above room temperature.[6a,7] In addition, BSA has two tryptophans embedded in two different domains.[8] Try-314 is located close to the protein surface but is buried in a hydrophobic pocket of domain I, while Try-214 is located in an internal part of domain II. Our results show that tryptophan emission decreases both with respect to time (Fig. 5c) as well as temperature (Fig. 5d) due to denaturation of the protein. The emission becomes almost negligible within 5 hours of reaction time (see inset in Fig. 5c), apparently due to a rapid increase in fluorescence quenching and a decrease in quantum yield with the increase in temperature as tryptophan residues become exposed to aqueous solvent.[9] The influence of denaturation of the protein on its capping ability is clearly visible in Figure 5a because 1  104 g mL1 is barely sufficient to effectively control the PbS crystal growth during the start of the reaction to attain smaller morphologies. But at later stages the denatured state of BSA provided more effective capping and consequently led to much smaller geometries. On the other hand, the use of 10  104 g mL1 BSA is fully capable of controlling the crystal growth even at the beginning of the reaction and hence the peak shift is observed early in Figure 5b. To understand how 10  104 g mL1 BSA controls the crystal growth in the latter case, we carried out a protein assay by employing the Bradford method to determine the amount of BSA bound to NCs during the growth of the NCs of Figure 3c.[10] Equivalent aliquots were drawn from the reaction mixtures at fixed intervals of time and then each sample was used to determine the amount of BSA associated with the isolated NCs (see Experimental Section). Figure 5e shows a plot of the BSA bound by capping with respect to time. There is a sharp increase in the amount of BSA trapped by capping during the initial

1453

FULL PAPER

www.afm-journal.de

Figure 3. a–c) FESEM images of PbS NCs synthesized at 40 8C, 60 8C, and 80 8C, respectively, with BSA ¼ 10  104 g mL1. b0 and c00 ) XRD patterns of NCs of sample (b) and (c). c0 ) The size distribution histogram of (c). d) HRTEM of single NCs capped with a thick protein layer indicated by an empty arrow. e) EDX of NC shown in (d). Note the clear emissions due to Pb and S. f) TEM image of the same sample prepared at pH ¼ 7.

10 hours of reaction time which subsequently tends to become constant for up to 48 hours. The initial increase in the BSA bound is complementary to the decrease in the tryptophan emission (Fig. 5c, inset) which suggests the denaturation of the protein proceeds with and presumably facilitates the capping mechanism of PbS NCs. Gel electrophoresis assists in determining the polarity of BSA capped PbS NCs. Figure 6a shows the displacement of NCs with some of the samples. Samples F.3c (Fig. 3c) and F.2b (Fig. 2b) show a displacement towards the positively charged electrode under the applied potential suggesting that the surface of BSA capped NCs is predominantly negatively charged. The displacement of sample F.3c can readily be understood from its small size as well as relatively less self-associated nature (Fig. 3c), while that of F.2b cannot be properly understood on these grounds. One possible explanation could be an appropriate denaturation temperature (i.e., 6 8C) which might deposit

1454

relatively greater amounts of BSA on the PbS NC surface. The amount of protein bound evaluated from the Bradford method is listed below each well in Figure 6a. Interestingly, samples F.3c and F.2b show maximum amounts of protein bound among all of the samples. We have further evaluated these results by measuring the pH of BSA þ water, BSA þ aqueous acetic acid, and BSA þ all of the reaction components, with respect to temperature (Fig. 6b and c, Scheme 1). With different amounts of BSA in pure water the pH initially decreased slightly up to 40 8C from a value close to 7, but then it started increasing up to 80 8C, where it slightly exceeded a pH of 7. Thus the overall pH varied about half a unit within 20– 90 8C for a range of 1–10  104 g mL1 of BSA. A clear minimum was observed in each case which indicated the onset of denaturation temperature around 40 8C, beyond which it is was not very sensitive to different conformational changes. On the contrary, in aqueous acetic acid medium (Fig. 6c), the overall

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2009, 19, 1451–1458

www.afm-journal.de

FULL PAPER

Figure 4. a and b) Bright field and dark field (HADAF) HRTEM images. The respective dark and bright spots in left and right panels represent to small PbS NCs surrounded by a layer of protein coating. c) EDX spectrum further confirms the PbS NCs.

Figure 5. UV–Vis spectra of PbS NCs of colloidal suspensions of samples Figure 1e (a) and Figure 4c (b) with respect to time, i.e., 30 min, 1 h, 2 h, 4 h, 16 h, and 48 h (from bottom to top scan). Tryptophan emission spectra with respect to time (c) and temperature (d) for a reaction solution containing BSA ¼ 5  104 g mL1. e) Plot of the amounts of BSA bound to NCs with respect to reaction time. Inset in (c) shows a variation of lmax with time. See text for details.

Adv. Funct. Mater. 2009, 19, 1451–1458

Figure 6. a) Gel electrophoresis of some of the PbS NCs suspensions. The symbols above each of the gel well represent the corresponding FESEM figure of NCs. (b) and (c) demonstrate the pH versus temperature profiles of aqueous BSA and BSA in different media, respectively. See the text for details.

trend is reversed with little initial variation in pH but the values show a continuous fall starting around 40–50 8C, again possibly due to the onset of denaturation. Interestingly, the pH profile of an actual reaction mixture with all components remains identical to that of corresponding BSA þ acetic acid, with the exception that the values (pH  5) are higher than the previous case, presumably due to salt buffering. In acidic medium (pH  5), amino acids exposed to aqueous phase become protonated, thereby acquire a zwitterionic character. This will allow BSA to have an extended form which would have a greater impact on its capping ability than its globular form.[11] In addition, thermal denaturation of BSA brings most of the changes in the ß-sheet structure of BSA around 60 8C and that brings a drastic change from globular to fibrous nature of BSA.[6a,d] The zwitterionic nature of surface amino acids would further facilitate their interfacial adsorption on the NC surface driven by short range electrostatic interactions. The ammonium groups just like that of quaternary ammonium surfactants show favorable interactions in comparison to acidic groups, thus water exposed acidic groups of BSA might provide predominantly negatively charged to BSA capped NCs.[3f ] Thus, all of the results indicate that there is a significant difference between the capping ability of BSA at 40 and 80 8C regarding the shape and structure of PbS NCs. These are summarized in Scheme 2. The NCs are either large and spherical (BSA ¼ 1  104 g mL1) or dendritic shaped (BSA > 1  104 g mL1) at 40 8C. But the NCs ultimately attain much smaller cubic shapes at 80 8C. Such a significant change in the shape of the NCs is clearly evident from XRD analysis. A comparison between the XRD patterns of Figure 1a00 with e00 , and that of Figure 3b0 with c00 , indicates a clear shift in the

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1455

www.afm-journal.de

FULL PAPER

Therefore, during the transition at 60 8C, the predominant growth will give dendritic geometries (Scheme 1). The current studies demonstrate that PbS NCs with distinct morphology can readily be generated using BSA as a reducing agent. Depending on the conditions, the NCs can be coated with BSA to varying extents. Such NCs have potential utility in biological applications including contrast agents for enhanced imaging, with magnetic resonance imaging (MRI), and X-ray analysis. Furthermore, NCs have proven useful for targeted drug delivery and molecular diagnostics.[12] Such applicability will obviously be subject to biocompatibility, in particular given the toxic nature of Pb.[13] Superparamagnetic iron oxide NPs can be coated with a number of biocompatible substances, including complex sugars such as dextran and starch and by various polyvinyl alcohols. Such coated NCs are not toxic to brain cells in culture nor do they appear to trigger an inflammatory response,[14] and they are tolerated well by animals. Other studies have demonstrated that the toxic effects of certain NCs can be gainfully exploited. Therapeutic drugs which distribute to throughout the body will have poor treatment potential. Directing such drugs to specifically defined sites has theoretical and practical significance. Blood bound particles tend to be taken up by phagocytic macrophages, including hepatic von Kupfer cells. A number of chemical modifications to proteins or other chemicals coating these particles reduces macrophage uptake. Increasing the surface positive charge tends to assist such discrimination. Finally, including specific recognition molecules, for example targeted to receptors that are taken up by clathrincoated or caveolar uptake mechanisms, can be used to direct toxic substances to cancer cells, leucocytes, and to transport across the blood-brain barrier.[15] These previous studies provide rationale for the present investigations. Scheme 1. Schematic representation of a proposed reaction.

crystal growth from {111} crystal planes to {100}. The {111} crystal planes possess the highest atomic density for any fcc crystal geometry (Scheme 2b). This will provide maximum charge on {111} planes, thereby making them more vulnerable to interaction with charged species. At 40 8C, a relatively less of the surface amino acids of BSA globular form will acquire zwitterionic character in comparison to those of fibrous form at 80 8C (Scheme 2c) and hence may not able to completely cap the {111} planes at 40 8C; this might result in the spherical geometry of PbS NCs. However, unfolded protonated amino acids at 80 8C are expected to be in a better position to effectively interact with {111} crystal planes and hence this would effectively reduce the nucleation on these planes. Such a situation would obviously shift the crystal growth from highest atomic density {111} planes to low atomic density {100} crystal planes at 80 8C as evident from Figures 1e00 and 3c00 . This is because the low atomic density {100} crystal planes are expected to be poorly capped by protein due to weak electrostatic interactions. Therefore, a predominant growth at {100} planes at 80 8C will convert the roughly spherical shaped NCs at 40 8C into first dendritic shaped at 60 8C, and then ultimately cubic geometry bound with {100} planes at 80 8C. The appearance of a dendritic shape is thought to be an intermediate shape between the spheres and cubes, and the crystal growth seems to proceed from {111} to {100} via {110} planes at 60 8C.

1456

3. Conclusions The native and denatured states of BSA have been used in the synthesis of PbS NCs to design bio-nanomaterials whose shape and structure depend on the nature of protein biomacromolecules. Due to the water soluble nature of BSA, it acts as a capping as well as stabilizing agent for colloidal PbS NCs, while thermal denaturation of BSA effectively alters this property. A significant dependence of NC morphology on the native to denatured states of BSA has been observed where latter state proves to be quite effective in controlling the NC size. This process has been studied by carrying out the reaction at different temperatures in the range of 40–80 8C. At 40 8C, large spherical PbS NCs are obtained; their size decrease as temperature is increased to 80 8C. This effect is more prominent when small amounts of BSA (1  104 g mL1) are used, but becomes less significant as the amount of BSA is increased above this. With larger amounts of BSA, smaller PbS NCs are always formed and these are entangled with threads of denatured BSA, especially at 60 8C, the temperature where denaturation of the protein initiates. Between 60–80 8C, the protein is already in a denatured state, although the degree of unfolded form increases as the temperature approaches towards 80 8C. The unfolded denatured protein proved to be a better capping agent in comparison to globular folded form possibly due to easier access of the zwitterionic amino acids to the

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2009, 19, 1451–1458

www.afm-journal.de

NC surface. This is a first report which clearly indicates that crystal growth depends on the native as opposed to the denatured state of proteins and helps to understand the complex capping behavior of proteins during the fabrication of shape controlled bioconjugate nanomaterials.

FULL PAPER

Scheme 2. A flow diagram showing a change in the morphology of PbS nanocrystals with respect to temperature.

UV-Vis and Fluorescence Measurements: UV-Vis measurements of dilute colloidal PbS NC suspensions were carried out using a Shimadzu 4500 spectrophotometer in the wavelength range of 200–900 nm to determine the shape dependent absorbances. Interaction between BSA and PbS NCs with respect to the reaction time as well as temperature was measured by using a Hitachi Spectrophotometer F-2500. When excited at 295 nm, tryptophan residue emission around 340 nm helps to evaluate such interactions that change with respect to reaction time as well as temperature due to a change in the native to denatured states of BSA. Gel Electrophoresis: The polarity of the BSA-capped NCs was determined by gel electrophoresis by using tris-HCl buffer as a gel running medium with pH ¼ 7. For this purpose, 1% aqueous agarose solution was first brought to the boil in a microwave and left in the gel plate to harden. Then, 20 ml of colloidal PbS aqueous suspension was loaded in the gel wells and a direct voltage of 90 V was applied for 30 minutes to monitor the movement of NCs. No staining agent was used because the PbS NC suspension in each case was colored black. Protein Assay: The total protein content of the BSA-nanoparticle conjugate suspensions was determined by the Bradford method [10]. First of all, standard BSA (reference) solutions of concentrations 0, 2, 4, 6, 8, and 10 mg mL1 were prepared in 100-mL pure water. 10 mL of each of these solutions was taken in triplicate in different wells of a UV plate. The purified BSA-PbS conjugate suspensions were also placed in the same plate in another series of wells in duplicate. Extra care was taken not to lose sample during the assay procedure. 20 ml of pure water and 170 ml of the Bradford reagent were mixed in all the wells so as to achieve a total volume of 200 ml. The absorbance of each solution was measured and from the absorbance values the amount of BSA conjugated to PbS NCs in each sample was determined.

Acknowledgements This work was supported by Grants MOP 66406 and FRN 15462 from the Canadian Institutes of Health Research.

4. Experimental Materials: Lead acetate, thioacetamide, acetic acid, and bovine serum albumin (BSA), all 99% or better, were purchased from Aldrich. Water was used after purification through double distillation. Preparation of PbS Nanoparticles: In a typical procedure, 10 mL of aqueous BSA (1, 5, and 10  104 g mL1) was taken in a round bottom glass flask. Under constant stirring, 1 mL of 0.5 M aqueous acetic acid was added. This was followed by the addition of 0.5 mL of 0.1 M aqueous lead acetate and 0.5 mL of aqueous 0.1 M thioacetamide. After mixing all the components at room temperature, the reaction mixture was kept in a water thermostated bath (Julabo F 25) under precise temperature control for 48 hours under static conditions. In the presence but not the absence of BSA, the color of the solution changed from colorless to dull yellow, then to a pinkish black, and finally it attained a black color within 16 hours and remained the same till 48 hours. A schematic representation (Scheme 2) explains the complete reaction. The colloidal black solution thus obtained indicated the presence of PbS NCs. The colloidal PbS NCs were collected by centrifuging at 10 000 rpm for 5 minutes and washing 2–3 times with distilled water. Similar reactions were carried out at pH 7 using phosphate buffer. The overall reaction observations were essentially similar to those observed in the above case. The reaction color immediately changed from colorless to light yellow. Within fifteen minutes it turned brown black and then predominantly black in 2 hours. It remained so up to 48 hours. Methods: FESEM, TEM, and XRD Measurements: The characterization of PbS NCs was carried out by using a Hitachi S-4800 FESEM. A diluted suspension of each sample was placed on a piece of silicon wafer and dried in air. It was then coated with 4-nm chromium film (Gatan-PECs-682 ion beam sputtering) and observed at an accelerating voltage of 5 KV. Lowresolution TEM images were obtained by using a Phillips CM 10 TEM operating at 100 KV; HRTEM and HAADF images were determined by using a CM 20 model. The diffraction and energy EDX analysis were also performed with the same instrument. The XRD patterns were characterized by using Bruker-AXS D8-GADDS with Tsec ¼ 480.

Adv. Funct. Mater. 2009, 19, 1451–1458

Received: August 17, 2008 Revised: December 13, 2008 Published online: March 13, 2009

[1] a) K. T. Yong, J. Qian, I. Roy, H. H. Lee, E. J. Bergey, K. M. Tramposch, S. He, M. T. Swihart, A. Maitra, P. N. Prasad, Nano Lett. 2007, 7, 761. b) K. Eggenberger, A. Merkulov, M. Darbandi, T. Nann, P. Nick, Bioconjugate Chem. 2007, 18, 1879. c) Y. Wang, Z. Tang, S. Tan, N. A. Kotov, Nano Lett. 2005, 5, 243. d) S. S. Narayanan, R. Sarkar, S. K. Pal, J. Phys. Chem. C 2007, 111, 11539. e) Q. Wang, Y. Kuo, Y. Wang, G. Shin, C. Ruengruglikit, Q. Huang, J. Phys. Chem. B 2006, 110, 16860. f) D. Chen, G. Wang, J. Li, J. Phys. Chem. C 2007, 111, 2351. [2] a) M. S. Bakshi, F. Possmayer, N. O. Petersen, J. Phys. Chem. C. 2007, 111, 14113. b) M. S. Bakshi, G. Kaur, P. Thakur, T. S. Banipal, F. Possmayer, N. O. Petersen, J. Phys. Chem. C 2007, 111, 5932. c) M. S. Bakshi, F. Possmayer, N. O. Petersen, Chem. Mater. 2007, 19, 1257. [3] a) L. Cademartiri, J. Bertolotti, R. Sapienza, D. S. Wiersma, G. von Freymann, G. A. Ozin, J. Phys. Chem. B 2006, 110, 671. b) D. V. Talapin, H. Yu, E. V. Shevchenko, A. Lobo, C. B. Murray, J. Phys. Chem. C 2007, 111, 14049. c) Z. Zhang, S. H. Lee, J. J. Vittal, W. S. Chin, J. Phys. Chem. B 2006, 110, 6649. d) K. A. Abel, J. Shan, J.-C. Boyer, F. Harris, F. C. J. M. van Veggel, ¨, Z. Xiu, S. Wang, H. Zhang, Chem. Mater. 2008, 20, 3794. e) G. Zhou, M. Lu Y. Zhou, S. Wang, J. Phys. Chem. B 2006, 110, 6543. f) C. Zhang, Z. Kang, E. Shen, E. Wang, L. Gao, F. Luo, C. Tian, C. Wang, Y. Lan, J. Li, X. Cao, J. Phys. Chem. B 2006, 110, 184. g) M. S. Bakshi, P. Thakur, S. Sachar, G. Kaur, T. S. Banipal, F. Possmayer, N. O. Petersen, J. Phys. Chem. C 2007, 111, 18087. h) J. W. Stouwdam, J. Shan, F. C. J. M. van Veggel, A. G. Pattantyus-

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1457

FULL PAPER

www.afm-journal.de

1458

Abraham, J. F. Young, M. Raudsepp, J. Phys. Chem. C 2007, 111, 1086. i) F. Zuo, S. Yan, B. Zhang, Y. Zhao, Y. Xie, J. Phys. Chem. C 2008, 112, 2831. j) M. S. Bakshi, G. Kaur, F. Possmayer, N. O. Petersen, J. Phys. Chem. C 2008, 112, 4948. [4] a) J. Xie, J. Y. Lee, D. I. C. Wang, J. Phys. Chem. C 2007, 111, 10226. b) Li. Shang, Y. Wang, J. Jiang, S. Dong, Langmuir 2007, 23, 2714. c) J. L. Burt, C. Gutierrez-Wing, M. Miki-Yoshida, M. Jose-Yacaman, Langmuir 2004, 20, 11778. d) D. C. Carter, J. X. Ho, J. Adv. Protein Chem. 1994, 45, 153. e) K. Kaibera, T. Okazaki, H. B. Bohidar, P. L. Dubin, Biomacromolecules 2000, 1, 100. f) A. Papadopoulou, R. J. Green, R. A. Frazier, J. Agric. Food Chem. 2005, 53, 158. [5] a) M. J. Meziani, P. Pathak, B. A. Harruff, R. Hurezeanu, Y.-P. Sun, Langmuir 2005, 21, 2008. b) M. J. Meziani, H. W. Rollins, L. F. Allard, Y.-P. Sun, J. Phys. Chem. B 2002, 106, 11178. c) S. H. Brewer, W. R. Glomm, M. C. Johnson, M. K. Knag, S. Franzen, Langmuir 2005, 21, 9303. d) M. Mikhaylova, D. K. Kim, C. C. Berry, A. Zagorodni, M. Toprak, A. S. G. Curtis, M. Muhammed, Chem. Mater. 2004, 16, 2344. [6] a) K. Murayama, M. Tomida, Biochemistry 2004, 43, 11526. b) S. Baier, D. J. McClements, J. Agric. Food Chem. 2001, 49, 2600. c) Y. Moriyama, K.

[7] [8] [9]

[10] [11]

[12] [13] [14] [15]

Takeda, Langmuir 2005, 21, 5524. d) V. Militello, V. Vetri, M. Leone, Biophys. Chem. 2003, 105, 133. a) C. Honda, H. Kamizono, T. Samejima, K. Endo, Chem. Pharm. Bull. 2000, 48, 464. b) W. Wang, Int. J. Pharm. 1999, 185, 129. Y. Moriyama, D. Ohta, K. Hachiya, Y. Mitsui, K. Takeda, J. Protein Chem. 1996, 15, 265. a) E. Bismuto, I. Sirangelo, G. Irace, E. Gratton, Biochemistry 1996, 35, 1173. b) M. Lasanga, E. Gratton, D. M. Jameson, J. E. Brunet, Biophys. J. 1999, 76, 443. c) C. Sun, J. Yang, X. Wu, X. Huang, F. Wang, S. Liu, Biophys. J. 2005, 88, 3518. M. M. Bradford, Anal. Biochem. 1976, 72, 248. a) T. Nakamura, S. Utsumi, T. Mori, Agric. Biol. Chem. 1986, 50, 2429. b) N. Kitabatake, Y. Tani, E. Doi, J. Food Sci. 1989, 54, 1632. c) J. F. Foster, in Albumin Structure, Function and Uses (Eds: V. M. Rosenoer, M. Oratz, M. A. Rothschild), Pergamon, Oxford 1977, p. 53. M. N Helmus,, Nat. Nanotechnol. 2007, 2, 333. M. T. Crisp, N. A. Kotov, Nano Lett. 2003, 3, 173. W. Lu, Q. Sun, J. Wan, Z. She, X.-G. Jiang, Cancer Res. 2006, 66, 11878. L. Juillerat-Jeanneret, F. Schmitt, Med. Res. Rev. 2007, 27, 574.

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2009, 19, 1451–1458

Stabilization of PbS Nanocrystals by Bovine Serum ...

Mar 13, 2009 - www.afm-journal.de. [*] Dr. M. S. Bakshi. Department of Chemistry ..... plate in another series of wells in duplicate. Extra care was taken not to.

846KB Sizes 1 Downloads 189 Views

Recommend Documents

quinolizine with bovine serum albumin
Available online 14 July 2004. Abstract. Interaction of ... protein has three domains, each consisting of a large double ... connecting segment to the next domain.

quinolizine with bovine serum albumin
Available online 14 July 2004. Abstract ... The binding constant and free energy change. (DG0) for the ... +91 332483 4133; fax: +91 332414. 6266. E-mail ...

Sirivat Voravetvuthikun - PBS
SIRIVAT VORAVETVUTHIKUN: Before I had a big, big dream: I wanted to be a .... chance to become successful in life, because big companies would employ ...

RELATIONSHIP OF SERUM MESOTHELIN AND MIDKINE LEVELS ...
RELATIONSHIP OF SERUM MESOTHELIN AND MIDK ... PATIENTS WITH MALIGNANT MESOTHELIOMA.pdf. RELATIONSHIP OF SERUM MESOTHELIN AND ...

Epidemiological Modeling of Bovine Brucellosis in India
limitations in surveillance data, this study illustrates the comparative ... Brucellosis represents a significant threat to the future of public health given evidence of ...

02 Dimensional stabilization of wood by chemical modification using ...
... Center, Indian Institute of Science, Bengaluru. 85. Page 3 of 9. 02 Dimensional stabilization of wood by chemical modification using isopropenyl acetate.pdf.

serum wajah.pdf
Loading… Whoops! There was a problem loading more pages. Whoops! There was a problem previewing this document. Retrying... Download. Connect more apps... Try one of the apps below to open or edit this item. serum wajah.pdf. serum wajah.pdf. Open. E

Formation and Stabilization of Anisotropic ... - CSIRO Publishing
Sep 23, 2008 - mission electron microscopy. Rapid microwave heating resulted in 'star-shaped' palladium nanoparticles, but platinum nanoparticles were ...

Epidemiological Modeling of Bovine Brucellosis in India
by which cell invasion and immune system evasion occur [5]. Brucellosis represents a significant threat to the future of public health given evidence of highly ...

Serum osteocalcin as a specific marker of bone ...
Abstract. The field of bone turnover markers has developed considerably in the past decade. Biochemical monitoring of bone metabolism depends upon measurement of enzymes and proteins released during bone formation and of degradation products produced

Serum albumin metabolism.pdf
There was a problem previewing this document. Retrying... Download. Connect more apps... Try one of the apps below to open or edit this item. Serum albumin ...

Curvature effects on optical response of Si nanocrystals ...
Aug 26, 2009 - At planar interfaces (or surfaces) the electronic charge density is shown to ... method for measuring interface effects due to Si suboxides in Si NC in SiO2 is also ...... 8 O. Madelung, Semiconductors: Data Handbook (Springer-.

Evaluation of quinolones residues in bovine meat in ...
antimicrobial therapy in human medicine. Public health risks comming from Salmonellas and. Cmpylobacter resistance strains increased morbidity and mortality ...

Stabilization Wedges
Aug 14, 2008 - ... August 14, 2008 ):. The following resources related to this article are available online at ... sion, space-based solar electricity, and artificial photosynthesis (2). ... panied by 2% growth in primary energy con- sumption and 3% 

PBS Rock Roll [.pdf
Loading… Page 1. Whoops! There was a problem loading more pages. PBS Rock Roll [.pdf. PBS Rock Roll [.pdf. Open. Extract. Open with. Sign In. Main menu.

Kinetic Stabilization of Biopolymers in Single-Crystal ...
Department of Chemistry, Box 351700. UniVersity of Washington ... lactose solution and allowed to stand for 3-4 days at room temperature in a 24-well ... band (λex ) 470 nm) to prevent photobleaching.9 The data are comparable to those from ...

Video Stabilization of Atmospheric Turbulence Distortion
May 20, 2012 - blurry image frames ⇒ sharpen individual frame temporal oscillations ⇒ stabilize temporal direction. We propose the following PDE model for video stabilization: ut (x,y,k) = S[u(x,y,k)] + ต△k u where S[·] denotes the Sobolev s

PBS Home Matrix Blank HP.pdf
There was a problem previewing this document. Retrying... Download. Connect more apps... Try one of the apps below to open or edit this item. PBS Home Matrix Blank HP.pdf. PBS Home Matrix Blank HP.pdf. Open. Extract. Open with. Sign In. Main menu.

PBS Academy for England FINAL.pdf
There was a problem previewing this document. Retrying... Download. Connect more apps... Try one of the apps below to open or edit this item. PBS Academy ...

SOALAN LAZIM PBS 2014.pdf
Loading… Page 1. Whoops! There was a problem loading more pages. Retrying... SOALAN LAZIM PBS 2014.pdf. SOALAN LAZIM PBS 2014.pdf. Open. Extract.

Pekeliling PBS Bil1.2014.pdf
There was a problem previewing this document. Retrying... Download. Connect more apps... Pekeliling PBS Bil1.2014.pdf. Pekeliling PBS Bil1.2014.pdf. Open.