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Direct Conjugation of Semiconductor Nanoparticles with Proteins Mohammed J. Meziani, Pankaj Pathak, Barbara A. Harruff, Razvan Hurezeanu, and Ya-Ping Sun* Department of Chemistry, Howard L. Hunter Chemistry Laboratory, Clemson University, Clemson, South Carolina 29634-0973 Received August 27, 2004. In Final Form: December 8, 2004 Nanocrystalline CdS particles directly conjugated with bovine serum albumin (BSA) protein were prepared by applying the supercritical fluid processing technique, rapid expansion of a supercritical solution into a liquid solvent. The direct conjugation takes advantage of the unique features of the process for nanoparticle formation. The BSA-conjugated CdS nanoparticles in stable aqueous suspension or in the solid state were characterized by using microscopy, X-ray diffraction, and optical spectroscopy methods. The results show that well-dispersed CdS nanoparticles are coated with BSA in a core-shell-like arrangement and that the protein species associated with the nanoparticles remain functional according to the modified Lowry assay. These BSA-conjugated CdS nanoparticles are also strongly luminescent, with the luminescence spectrum contributed to primarily by the exciton emission.
Introduction The availability of colloidal semiconductor nanoparticles with controlled optical and electrical properties has sparked widespread interest in their biologically relevant applications such as luminescence tagging and imaging, medical diagnostics, drug delivery, and implantable nanoelectronics.1 For example, ZnS- and CdS-capped CdSe nanocrystals were developed as luminescent probes in biological labeling for their unique advantages including the tunable color and the exceptional photochemical stability.2,3 Recent investigations to impart biocompatibility and bioactive functionalities to semiconductor nanoparticles have concentrated on the particle surface modification with DNA,4 peptides,5 and proteins.6-9 For proteins, in particular, there has been significant progress in the understanding of their specificity and binding capabilities toward nanoscale semiconductor particles. Among the widely used proteins is the globular protein bovine serum albumin (BSA). For example, BSA was used to form (1) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 113, 4128; 2003, 42, 5796. (2) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (3) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (4) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607. (b) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078. (c) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849. (d) Mirkin, C. A. Inorg. Chem. 2000, 39, 2258. (5) (a) Dameron, C. T.; Reese, R. N.; Mehra, R. K.; Kortan, A. R.; Carroll, P. J.; Steigerwald, M. L.; Brus, L. E.; Winge, D. R. Nature 1989, 338, 596. (b) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (6) (a) Kreuter, J. In Microcapsules and Nanoparticules in Medicine and Pharmacy; Donbrow, M., Ed.; CRC Press: Boca Raton, 1992. (b) Hermanson, G. T. In Bioconjugate Techniques; Academic Press: New York, 1996. (7) (a) Douglas, T.; Young, M. Nature 1998, 393, 152. (b) Gestwicki, J. E.; Strong, L. E.; Kiessling, L. L. Angew. Chem., Int. Ed. 2000, 39, 4567. (c) Mamedova, N. N.; Kotov, N. A.; Rogach, A. L.; Studer, J. Nano Lett. 2001, 1, 281. (d) Wang, S. P.; Mamedova, N. N.; Kotov, N. A.; Chen, W.; Studer, J. Nano Lett. 2002, 2, 817. (e) Ishii, D.; Kinbara, K.; Ishida, Y.; Ishii, N.; Okochi, M.; Yohda, M.; Aida, T. Nature 2003, 423, 628. (8) Meziani, M. J.; Rollins, H. W.; Allard, L. F.; Sun, Y.-P. J. Phys. Chem. B 2002, 106, 11178. (9) Meziani, M. J.; Sun, Y.-P. J. Am. Chem. Soc. 2003, 125, 8015.
bioconjugate with luminescent CdTe nanoparticles that are capped with L-cysteine.7c Such conjugation was reported to increase the luminescence intensities of the CdTe nanoparticles, and the luminescence enhancement was attributed to possibly the presence of resonance energy transfer from the tryptophan moieties in BSA to the CdTe nanoparticles.7c However, the preparation of bioconjugates of inorganic nanoparticles is by no means a straightforward process. Typically, the nanoparticle surface has to be modified first by chemical functionalization with a linker, which recognizes the biomolecules and also protects the nanoparticles from uncontrolled growth or agglomeration, before the desired bioconjugation.1 There have only been a few investigations on the direct conjugation of biological species to inorganic nanoparticles despite some obvious advantages. Recently, Sun and co-workers used a supercritical fluid processing technique known as RESOLV (rapid expansion of a supercritical solution into a liquid solvent) to produce Ag2S nanoparticles directly conjugated with BSA.9 The direct conjugation took advantage of the unique feature of the RESOLV method that the formation of nanoparticles in an aqueous solution requires no nanoscale templating agents such as surfactants because the templating effect is provided by the supercritical fluid rapid expansion process.10,11 RESOLV differs from the traditional RESS (rapid expansion of supercritical solution) process by expanding the supercritical solution into a liquid solvent or solution instead of air or vacuum.12,13 Mechanistically, the liquid at the receiving end of the rapid expansion in RESOLV (10) Sun, Y.-P.; Rollins, H. W.; Jayasundera, B.; Meziani, M. J.; Bunker, C. E. In Supercritical Fluid Technology in Materials Science and Engineering: Synthesis, Properties, and Applications; Sun, Y.-P., Ed.; Marcel Dekker: New York, 2002; p 491. (11) (a) Sun, Y.-P.; Rollins, H. W. Chem. Phys. Lett. 1998, 288, 585. (b) Sun, Y.-P.; Guduru, R.; Lin, F.; Whiteside, T. Ind. Eng. Chem. Res. 2000, 39, 4663. (c) Sun, Y.-P.; Rollins, H. W.; Guduru, R. Chem. Mater. 1999, 11, 7. (d) Sun, Y.-P.; Riggs, J. E.; Rollins, H. W.; Guduru, R. J. Phys. Chem. B 1999, 103, 77. (e) Sun, Y.-P.; Atorngitjawat, P.; Meziani, M. J. Langmuir 2001, 17, 5707. (12) (a) Matson, D. W.; Fulton, J. L.; Petersen, R. C.; Smith, R. C. Ind. Eng. Chem. Res. 1987, 26, 2298. (b) Lele, A. K.; Shine, A. D. AIChE J. 1992, 38, 742. (13) Debenedetti, P. G. Supercritical Fluids: Fundamentals for Application; Kluwer Academic Publishers: Boston, 1994; p 719.
10.1021/la0478550 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/01/2005
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Figure 1. Experimental setup of RESOLV for the preparation of BSA-conjugated CdS nanoparticles.
likely inhibits or disrupts the condensation and coagulation in the expansion jet, thus, effectively quenching the rapid particle growth processes found in the traditional RESS.14 It has been shown that the RESOLV process produces exclusively nanoscale particles.10,11,15 In addition, the use of an aqueous receiving solution under ambient conditions provides accommodation for protein species, which may conjugate directly with the inorganic nanoparticles as they form in the RESOLV process. In the reported work, we applied this technique to the preparation of BSA-conjugated CdS nanoparticles without the need for any linkers. The bioconjugates thus obtained were characterized by several instrumental techniques including X-ray powder diffraction, transmission electron microscopy (TEM), and atomic force microscopy (AFM). The optical properties of the BSA-conjugated CdS nanoparticles were also investigated. Experimental Section Materials. Cadmium nitrate [Cd(NO3)2, >99.9%] and sodium sulfide (Na2S, >99.9%) were purchased from Aldrich, and the BSA protein sample was obtained from Sigma. Anhydrous ammonia (>99.9999%) was supplied by Air Products. Water was deionized and purified by being passed through a Labconco WaterPros water purification system. The membrane tubing for dialysis was purchased from Spectrum. Measurement. UV/vis absorption spectra were recorded on a Shimadzu UV-3100 spectrophotometer. X-ray powder diffraction measurements were carried out on a Scintag XDS-2000 powder diffraction system. TEM images were obtained on Hitachi H-7000 and HD-2000 STEM systems. AFM analysis was conducted in the acoustic AC mode on a Molecular Imaging PicoPlus system equipped with a multipurpose scanner for a maximum imaging area of 10 µm ×10 µm and a NanoWorld Pointprobe NCH sensor (125 µm length). CdS Nanoparticles. The RESOLV apparatus for the preparation of nanoparticles is illustrated in Figure 1.10 It consists of a syringe pump for pressure generation and pressure maintenance during the rapid expansion and a gauge for monitoring the system pressure. The heating unit is a cylindrical solid copper block of high heat capacity in a tube furnace. The copper block is wrapped with stainless steel tubing to ensure a close contact between the tubing coil and the copper block for efficient heat transfer. The copper block/tubing coil assembly is preheated to a set temperature before each rapid expansion experiment. The expansion nozzle is a fused silica capillary hosted in a stainless steel tubing, which is inserted into a chamber containing the room-temperature receiving solution. In a typical experiment, a solution of Cd(NO3)2 in methanol (40 mM, 0.5 mL) was added to the syringe pump, followed by the evaporation of the solvent methanol. The syringe pump was then filled with liquid ammonia. When pumped through the heating (14) Weber, M.; Thies, M. C. In Supercritical Fluid Technology in Materials Science and Engineering: Synthesis, Properties, and Applications; Sun, Y.-P., Ed.; Marcel Dekker: New York, 2002; p 387. (15) Meziani, M. J.; Pathak, P.; Hurezeanu, R.; Thies, M. C.; Enick, R. M.; Sun, Y.-P. Angew. Chem., Int. Ed. 2004, 43, 704.
Figure 2. Optical absorption spectrum of the BSA-conjugated CdS nanoparticles in a stable aqueous suspension. unit, the ammonia solution of Cd(NO3)2 was heated and equilibrated at 160 °C before reaching the expansion nozzle. The supercritical solution was rapidly expanded via a 50-µm fused silica capillary nozzle into an ambient aqueous Na2S solution (0.15 mg/mL). The system pressure was maintained at 4000 psia during the rapid expansion. The aqueous receiving solution also contained BSA protein (2 mg/mL) for the direct conjugation with the CdS nanoparticles as they form in the rapid expansion process. Upon the completion of the rapid expansion, the ambient suspension of nanoparticles was transferred to a membrane tubing (cutoff molecular weight 100 000) and dialyzed against fresh deionized water to remove excess BSA and other salts and reagents. Protein Assay. The modified Lowry procedure16 was used to determine the total protein content in the BSA-nanoparticle conjugate sample. In a typical experiment, a dilute sample solution in sodium chloride solution (∼1 mg/mL) was prepared. To a small aliquot of the solution (0.2 mL) was added Biuret reagent (2.2 mL). After the solution was allowed to stand for 10 min at room temperature, Folin-Ciocalteu’s phenol reagent (0.1 mL) was added. The resulting solution was kept at room temperature for another 30 min, and then the visible absorption spectrum of the solution was recorded. The same experimental procedure was applied to the pristine BSA to generate the standard curve for calculating the protein content in the conjugate sample.
Results and Discussion The rapid expansion of the supercritical ammonia solution of Cd(NO3)2 into an ambient solution of Na2S results in the precipitation of CdS to form nanoscale particles.11a The nanoparticles generally require protection from agglomeration to remain in a stable suspension. In this work, BSA protein was used as a stabilization agent in the aqueous suspension. The protein species protect the CdS nanoparticles via their direct conjugation. The aqueous suspension remains stable in the dialysis for the removal of free BSA and other residual reagents, which suggests that the interactions between BSA protein species and CdS nanoparticles are relatively strong to sustain the conjugation. This is similar to what was observed in the previously reported direct conjugation of BSA with Ag2S nanoparticles.9 The stable aqueous suspension of BSA-conjugated CdS nanoparticles appears as a homogeneous solution with a yellowish color. The UV/vis absorption spectrum of the suspension consists of a well-defined peak at 320 nm (Figure 2). These optical absorption features typically correspond to small semiconductor nanoparticles of a (16) (a) Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (b) Ohnishi, S. T.; Barr, J. K. Anal. Biochem. 1978, 86, 193. (b) Peterson, G. L. Anal. Biochem. 1979, 100, 201.
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Figure 4. X-ray powder diffraction pattern of the BSAconjugated CdS nanoparticles in the solid state. The cubic and hexagonal patterns from the JCPDS library are also shown as references.
Figure 3. Typical TEM image of the BSA-conjugated CdS nanoparticles prepared via RESOLV (top). The image at high resolution shows the single-crystal structure for one of the nanoparticles (bottom).
relatively narrow size distribution.17 According to the relationship between the average CdS particle size and the absorption band position already in the literature,18 these BSA-conjugated CdS nanoparticles should have a size on the order of 2-3 nm in diameter. A more precise determination of the average particle size and size distribution was performed via TEM analysis. The specimen for TEM imaging was prepared by depositing a few drops of the nanoparticle suspension after dilution onto a carbon-coated copper grid. The purpose was to maintain the characteristics and distribution of the CdS nanoparticles as they were originally prepared and suspended. A typical TEM image of the specimen is shown in Figure 3. According to a statistical analysis of the TEM results, the CdS nanoparticles have an average particle size of 2.9 nm and a size distribution standard deviation of 0.7 nm. The TEM imaging at high resolution also suggests that these BSA-conjugated CdS nanoparticles are mostly single crystals (Figure 3). The identification of the CdS nanoparticles was based on powder X-ray diffraction analysis. The solid sample was obtained by removing water from the suspension in evaporation under a vacuum. A typical X-ray powder diffraction pattern of the BSA-conjugated CdS nanoparticles is shown in Figure 4. The broad diffraction peaks are typical of the particles being nanoscale. Because of the broadness, however, it is difficult to determine an exact match of the observed diffraction pattern with that of the cubic or hexagonal CdS in the JCPDS library. It seems that the nanoparticle sample is probably a mixture of cubic and hexagonal CdS. The TEM results are consistent with the fact that the electron density of the CdS nanoparticles is significantly higher than that of the conjugated BSA; namely, the protein species are not easily visible in the images. Indirectly, however, the presence of BSA on the CdS nanoparticle surface is made evident by the affinity of the conjugate to the surface of gold. For example, when colloidal gold particles (∼20 nm in diameter) were added to the aqueous suspension of the BSA-conjugated CdS (17) (a) Brus, L. J. Phys. Chem. 1986, 90, 2555. (b) Henglein, A. Chem. Rev. 1989, 89, 1861. (18) Wang, Y. Adv. Photochem. 1995, 19, 179.
Figure 5. Typical STEM (Z-contrast mode) image for the coating of colloidal gold by the BSA-conjugated CdS nanoparticles (left). The marked regions are enlarged (right).
nanoparticles,19 the relatively large gold particles were each coated with the CdS nanoparticles, as illustrated by the scanning TEM (STEM) images in Figure 5. It is wellknown that BSA is affinitive to nanoscale gold surface,6a,20 and such affinity is likely responsible for the observed coating shown in Figure 5. The visualization of BSA protein species conjugated to the CdS nanoparticle surface was achieved via AFM imaging. The sample for AFM measurements was deposited on a mica substrate. Shown in Figure 6 are typical height and phase images of the BSA-conjugated CdS nanoparticles. These images are dominated by circular features of around 20 nm in size, which are consistent with well-dispersed individual CdS nanoparticles that are each coated with BSA protein species. The phase image provides a better contrasting between CdS and BSA, and the features of white dots each immersed in a pot of soft materials may be considered as more visual evidence that the CdS nanoparticles are associated with BSA in a coreshell-like arrangement (Figure 6).21 Because AFM is a scanning technique, the observed feature size in the image is larger than the actual particle by a factor.21,22 In a rough approximation, the BSA-conjugated particle diameter should be (π/2)-1 of what the AFM image shows, or about 12 nm. This length scale corresponds to BSA (∼4 nm)CdS (∼3 nm)-BSA. Thus, the BSA coverage on a CdS nanoparticle is likely close to a monolayer. After being conjugated to the CdS nanoparticles, the BSA protein species are still intact according to the (19) A colloidal gold suspension (0.1 mL) was added to the BSA-CdS suspension (0.5 mL), and the specimen for imaging was prepared after 30 min. (20) Quaroni, L.; Chumanov, G. J. Am. Chem. Soc. 1999, 121, 10642. (21) The apparently larger particle sizes in AFM images than those determined by TEM may be attributed to the tip convolution effect, which is a well-established scanning probe microscopy phenomenon.22 (22) Waner, M. J.; Gilchrist, M.; Schindler, M.; Dantus, M. J. Phys. Chem. B 1998, 102, 1649 and references therein.
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Figure 7. Luminescence spectrum of the BSA-conjugated CdS nanoparticles in a stable aqueous suspension (solid line) compared with that of the similarly sized CdS nanoparticles prepared and encapsulated in AOT micelles (dashed line).26
Figure 6. Height (left) and phase (right) images from the AFM analysis of the BSA-conjugated CdS nanoparticles on the mica substrate.
modified Lowry assay for total protein analysis, which measures the tryptophan and tyrosine contents in the analyte.16 Thus, the BSA conjugation serves the dual purposes of stabilizing the CdS nanoparticles to prevent agglomeration in aqueous suspension and introducing biocompatible functionalities into these nanoparticles for further biological interactions or couplings (antibody attachment, for example). Mechanistically, the relatively strong interactions between BSA and CdS (strong enough to remain stable in dialysis) are worth noting. BSA is rich in amino moieties and also contains several disulfide bonds with one free thiol in the cysteine residues.23 These functionalities are probably responsible for the conjugation to CdS nanoparticles via thiolate linkages and/or weak covalent bonds with alkylamines.24 The aqueous suspension of BSAconjugated CdS nanoparticles was stable without precipitation for an extended period of time (at least months). There were also little changes in the properties of the nanoparticles according to UV/vis absorption and microscopy characterization results. Therefore, it is safe to conclude that the BSA species remain conjugated with the nanoparticles over time and the conjugated nanoparticles do not agglomerate via protein-protein interactions in the absence of any significant pH increases.9 The luminescence properties of the BSA-conjugated CdS nanoparticles in the aqueous suspension were evaluated. As shown in Figure 7, the luminescence spectrum has a relatively narrow bandwidth and is close to the absorption onset. This is primarily the exciton emission associated (23) Foster, J. F. In Albumin Structure, Function and Uses; Rosenoer, V. M., Oratz, M., Rothschild, M. A., Eds.; Pergamon: Oxford, U.K., 1977; p 53. (24) (a) Sasaki, Y. C.; Yasuda, K.; Suzuki, Y.; Ishibashi, T.; Satoh, I.; Fujiki, Y.; Ishiwata, S. Biophys. J. 1997, 72, 1842. (b) Brelle, M. C.; Zhang, J. Z.; Nguyen, L.; Mehra, R. K. J. Phys. Chem. A 1999, 103, 10194. (c) Leff, D. V.; Brandt, L.; Heath, J. R. Langmuir 1996, 12, 4723.
with the band gap excitation.18 The observed luminescence quantum yield is high, ∼20% in reference to the fluorescence standard 9,10-diphenylanthracene, which is also consistent with substantial contributions from the exciton emission.18 Sun and co-workers reported previously that, in the production of CdS nanoparticles via RESOLV, the rapid expansion of the supercritical ammonia solution may have a special passivation effect on the nanoparticle surface.10 The passivation suppresses the surface defect luminescence and enhances the exciton emission from the CdS nanoparticles.10,11,18,25 Apparently, the same luminescence properties are largely preserved in the BSAconjugated CdS nanoparticles produced via RESOLV with the supercritical ammonia solution. For comparison, the luminescence spectrum of the CdS nanoparticles prepared and encapsulated in the micelles of sodium dioctyl sulfosuccinate (AOT)26 is also shown in Figure 7. The broad emission from those CdS nanoparticles is due primarily to energy trapping states associated with the surface defects, as already discussed in the literature.18,25,26 In summary, small CdS nanoparticles of narrow size distribution can be produced via the supercritical fluid processing technique RESOLV. Because the nanoparticle formation involves no templating agents in an aqueous environment, the method enables direct conjugation of the CdS nanoparticles to the protein BSA. These proteinconjugated nanoparticles are stable (without agglomeration and precipitation) in an aqueous suspension, and the protein species associated with the nanoparticles remain intact, amenable to further biofunctionalizations (such as conjugation with antibodies or other biological species). The strongly luminescent features of these CdS nanoparticles may be valuable to biosensor-related applications. Acknowledgment. Financial support from DOE (DEFG02-00ER45859) and the Center for Advanced Engineering Fibers and Films (NSF-ERC at Clemson University) is gratefully acknowledged. R.H. was a participant of the Summer Undergraduate Research Program sponsored jointly by NSF and Clemson University. LA0478550 (25) Wang, Y.; Suna, A.; McHugh, J.; Hilinski, E. F.; Lucas, P. A.; Johnson, R. D. J. Chem. Phys. 1990, 92, 6927. (26) Harruff, B. A.; Bunker, C. E. Langmuir 2003, 19, 893.
