Adsorption and Immobilization of Cytochrome c on Nanodiamonds

Jun 4, 2004 - in Γ between these two adsorption cases (i.e., 8 × 1012 versus 3 × 1012 molecules/cm2) is ... Yu.; Titov, V. M. Chem. Phys. Lett. 199...
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Langmuir 2004, 20, 5879-5884

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Adsorption and Immobilization of Cytochrome c on Nanodiamonds L.-C. Lora Huang and Huan-Cheng Chang* Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei, Taiwan 106, Republic of China Received February 18, 2004. In Final Form: April 14, 2004 Methods have been developed to immobilize proteins onto the surfaces of nanodiamonds with an average size of 5 ( 1 nm. The immobilization started with carboxylation/oxidization of diamonds with strong acids, followed by coating the surfaces with poly-L-lysine (PL) for covalent attachment of proteins using heterobifunctional cross-linkers. The feasibility of this approach is proven with fluorescent labeling of the PL-coated diamonds by Alexa Fluor 488 and subsequent detection of the emission using a confocal fluorescence microscope. Immobilization of proteins onto the surfaces is also demonstrated with yeast cytochrome c, which possesses a free SH group for linkage and a characteristic Soret absorption band for observation.

Introduction Nanobiotechnology is an emerging interdisciplinary field, which combines nanotechnology with a number of areas in life sciences.1 The technology provides not only a tool for the study of interactions between proteins and small molecules but also an opportunity to explore the functionality of proteins on genome wide scales. Protein immobilization is a crucial step in nanobiotechnology as well as biochip developments.2-4 Many methods are currently under development to immobilize proteins on solid supports and to preserve their activity and native conformation.3 Amine-terminated diamonds,5 in particular, have the potential to serve as a useful intermediate for further functionalization and as a substrate for selfassembly of biomolecules. The standard synthetic techniques utilized in organic chemistry can be fruitfully applied,6 including ammonium salt formation, alkylation, and amide generation. Diamond, because of its optical transparency, chemical stability, biological compatibility,7,8 and the ability of being doped and deposited in very thin films on a variety of substrates,9 is gaining increasing attention in biosensor and biochip applications. Hamers and collaborators,10 for example, employed nanocrystalline diamond films for DNA hybridization via photochemical modification of diamond surfaces. A comparison of DNA hybridization on * To whom correspondence may be addressed. E-mail: [email protected]. (1) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (2) Lee, Y.-S.; Mrksich, M. Trends Biotechnol. 2002, 20, S14. (3) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55. (4) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192. (5) Miller, J. B.; Brown, D. W. Langmuir 1996, 12, 5809. (b) Miller, J. B. Surf. Sci. 1999, 439, 21. (6) Buriak, J. M. Angew. Chem., Int. Ed. 2001, 40, 532. (7) Tang, L.; Tsai, C.; Gerberich, W. W.; Kruckeberg, L.; Kania, D. R. Biomaterials 1995, 16, 483. (8) Hauert, R. Diamond Relat. Mater. 2003, 12, 583. (9) Davis, R. F. Diamond Films and Coatings. Development, Properties, and Applications; Noyes: Park Ridge, NJ, 1993. (10) Yang, W.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N., Jr.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253. (b) Strother, T.; Knickerbocker, T.; Russell, J. N.; Butler, J. E.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 968. (c) Knickerbocker, T.; Strother, T.; Schwartz, M. P.; Russell, J. N., Jr.; Butler, J.; Smith, L. M.; Hamers, R. J. Langmuir 2003, 19, 1938.

diamond and crystalline silicon indicates that the former exhibits superior chemical stability over the latter under the conditions for denaturation of the surface-bound DNA. One origin for this difference is that the DNA molecules are covalently immobilized on diamond, which offers stronger, sturdier, and also closer attachment of the adsorbate to the substrate. On account of sensor durability, covalent immobilization is perhaps the best approach. However, the surface functionalization of diamonds in aqueous solution leading to covalent immobilization of biomolecules is often a time-consuming process.11 Furthermore, the process does not appear to be very effective, as revealed by infrared spectroscopy probing the surface ester11 and amide12 linkages. Noncovalent functionalization is a method widely applied to immobilization of biomolecules on the surfaces of metal oxides,13 silicon single crystals,14 and carbon nanotubes.15-17 On nanoparticles that are stabilized by anionic ligands such as citrate or lipoic acid, biomolecules can be coupled to them through noncovalent electrostatic interactions. The method is particularly applicable to diamond, whose surfaces can be easily terminated with carboxylate or other CO-containing groups18 for noncovalent interactions with the amino groups of proteins or DNA. We have studied in our laboratory hydrogenterminated diamond surfaces of single crystals and (11) Ushizawa, K.; Sato, Y.; Mitsumori, T.; Machinami, T.; Ueda, T.; Ando, T. Chem. Phys. Lett. 2002, 351, 105. (12) The surface amide linkage was formed by reaction of carboxylated/oxidized diamonds with neat thionyl chloride at 50 °C for 1 day, followed by reaction with ethylenediamine in anhydrous pyridine under reflux for more than 24 h. Only a very weak feature that can be assigned to the amide I band was found at ∼1633 cm-1. Huang, L. C. L.; Chang, H.-C. Unpublished results. (13) Rezania, A.; Johnson, R.; Lefkow, A. R.; Healy K. E. Langmuir 1999, 15, 6931. (14) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205. (15) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838. (b) Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, Y.; Kim, W.; Utz, P. J.; Dai, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4984. (16) Baker, S. E.; Cai, W.; Lasseter, T. L.; Weidkamp, K. P.; Hamers, R. J. Nano Lett. 2002, 2, 1413. (17) Sun, Y.-P.; Fu, K.; Lin, Y.; Huang, W. Acc. Chem. Res. 2002, 35, 1096. (18) John, P.; Polwart, N.; Troupe, C. E.; Wilson, J. I. B. J. Am. Chem. Soc. 2003, 125, 6600.

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powders using infrared spectroscopy for several years.19 The associated hydrophobic interactions between the surface CH groups and small molecules such as C2H2 were investigated in detail at low temperatures under high vacuum.19c In this work, we extend the investigation to hydrophilic diamond powders (sizes of 5 and 100 nm) prepared by oxidization and carboxylation in strong acids using various spectroscopic methods. We examine closely the surface structures and properties of the hydrophilic diamonds, followed by protein immobilization beginning with poly-L-lysine for noncovalent surface amination.14 Yeast cytochrome c was chosen as the protein for immobilization. The protein absorbs strongly at 409 nm (the Soret band) and contains a single free SH group (cysteine 102) for covalent linkage. The Soret band, with an exceptionally large molar absorptivity, has been frequently employed as a probe for the conformational change(s) of the protein in solution20,21 and on surface.22 The cysteine residue, on the other hand, is reactive to the maleimide moieties of heterobifunctional cross-linkers commonly used in immunological assays, making regiospecific immobilization of the protein on diamond possible.23 As will be demonstrated in this work, the method we propose is facile and general and is expected to be applicable to immobilization of other cysteinecontaining proteins on nanocrystalline diamonds as well. Experiments Materials and Chemicals. Synthetic diamond powders of sizes in the range of 100 and 5 nm were obtained from Kay Industrial Diamond (KDM, USA) and Toron (ultraFine Diamond, Russia), respectively. Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) was from Pierce, and polyL-lysine (PL, MW ∼30 000), horse cytochrome c (HCC), and yeast cytochrome c (YCC) were all from Sigma and used without further purification. Deionized water was purified with a Milli Q plus system (Millipore), having a resistivity >18 MΩ/cm. Surface Functionalization. Diamond powders were carboxylated and oxidized following the procedures of Ushizawa et al.11 The sample (0.5 g) was first heated in a 9:1 (v/v) mixture of concentrated H2SO4 and HNO3 at 75 °C for 3 days, subsequently in 0.1 M NaOH aqueous solution at 90 °C for 2 h, and then in 0.1 M HCl aqueous solution at 90 °C for 2 h. The resulting carboxylated/oxidized diamonds were extensively rinsed with deionized water and separated by sedimentation with a Kubota 3700 centrifuge at 12 000 rpm. To conduct surface amination, the acid-treated diamonds (0.07 g) and PL (0.03 g) were mixed together in boric acid (10 mL, pH adjusted by NaOH aqueous solution to 8.5) for 30 min.24 The amine-terminated diamonds were thoroughly washed with deionized water before use. IR and UV-vis Measurements. Prior to infrared spectroscopic measurements, both the carboxylated/oxidized diamonds and the amine-terminated diamonds were made to water suspensions, deposited on a single-polished Ge(111) wafer, and dried in air to form thin films. The film-containing wafer was then mounted on a Mo sample holder and positioned in a small high-vacuum chamber (base pressure < 10-3 Torr)19 to eliminate water in the atmosphere and on the film. A N2-purged Fourier transform infrared (FTIR) spectrometer (Bomem MB154), (19) Chang, H.-C.; Lin, J.-C.; Wu, J.-Y.; Chen, K.-H. J. Phys. Chem. 1995, 99, 11081. (b) Cheng, C.-L.; Lin, J.-C.; Chang, H.-C.; Wang, J.-K. J. Chem. Phys. 1996, 105, 8977. (c) Chang, H.-C.; Lin, J.-C. J. Phys. Chem. 1996, 100, 7018. (d) Cheng, C.-L.; Lin, J.-C.; Chang, H.-C. J. Chem. Phys. 1997, 106, 7411. (e) Chen, Y.-R.; Chang, H.-C.; Cheng, C.-L.; Wang, C.-C.; Jiang, J. C. J. Chem. Phys. 2003, 119, 10626. (20) Kamatari, Y. O.; Konno, T.; Kataoka, M.; Akasaka, K. J. Mol. Biol. 1996, 259, 512. (21) Bhuyan, A. K.; Udgaonkar, J. B. J. Mol. Biol. 2001, 312, 1135. (22) Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C. J. Phys. Chem. A 2003, 107, 10687. (23) Wood, L. L.; Cheng, S.-S.; Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 571. (24) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642.

Huang and Chang equipped with a liquid nitrogen cooled MCT detector, acquired the spectra at room temperature. Absorbance spectra were obtained by ratioing the sample spectra with the background spectra taken for pure Ge(111) wafers. The spectra were typically acquired at 100 scans and an instrumental resolution of 4 cm-1. Adsorption isotherms were obtained for HCC on carboxylated/ oxidized diamonds in phosphate buffers at pH 6.5 using a UVvis spectrophotometer (Hitachi U-3200). The amount of proteins adsorbed (mg/g) was determined from the difference in protein concentration before and after addition of the diamond samples into the solution. A molar absorptivity, b ) 1.06 × 105 M-1 cm-1 at λmax ) 409 nm and pH 7,25 served to calibrate the protein concentration based on the measured absorbance at the Soret band maximum. To ensure equilibration of the adsorption, the proteins and the diamond suspension were thoroughly mixed together with a shaker (ELMI RM-2L) for more than 30 min, after which the mixture was centrifuged. The final equilibrium concentration of the proteins in solution was determined from the spectra acquired with a quartz cell (Hellma) of 1 mm path length at a spectrometric resolution of 2 nm. No correction was made for scattering losses by the remaining suspending particles. Dye Labeling and Protein Immobilization. The amineterminated diamonds were fluorescently labeled with an Alexa Fluor 488 dye-labeling kit (Molecular Probes), following the protocols of the manufacturer. The fluorescently labeled nanocrystals, after separation by centrifugation and dispersion on a glass slide, were inspected using a confocal laser scanning fluorescence microscope (Nikon c1). The microscope operated at 488 nm with the Alexa Fluor 488 dye molecule excited by an Ar ion laser. A spectral bandwidth of 5 nm was used for both excitation and collection of the emission at ∼520 nm.26 Protein immobilization was conducted by first activating the amine-terminated diamonds (0.07 g) in 10 mL of phosphate buffer saline (PBS) at pH 8.5 to react with the heterobifunctional linker SSMCC (2.2 mg) for 1 h. The excess bifunctional linker was later removed by centrifugation, and the sedimentary diamonds were washed once with deionized water. The SSMCC-anchored particles were then mixed with 26 µM phosphate-buffered Saccharomyces cerevisiae iso-1 cytochrome c (1.6 mg in 5 mL of PBS at pH 6.5), and the mixture was left in a centrifuge tube for 1 h. The resulting protein-diamond mixture went through several circles of washing with deionized water until the supernatant fraction of the sample consisted of a clear and transparent solution after centrifugation, showing negligible absorption at 409 nm. In detection of the surface-bound proteins with the UV-vis spectrophotometer, adjustment of the suspension concentration to a proper value is critical, because light scattering from the diamond particles severely interferes with the measurements at this wavelength region.

Results and Discussion Characterization of Diamond Surfaces. Figure 1a shows the FTIR spectrum of the 100 nm diamonds after acid treatment. Similar to the results reported previously,27a several distinct features appear at 3560, 1824, 1623, and 1277 cm-1 in the spectral region. The first feature is most likely to derive from the O-H stretching vibration, which has been similarly observed at 3540 cm-1 by Pehrsson and Mercer28 in thermal oxidation of diamond C(100) single-crystal surfaces. We assign it here to the O-H stretch of the surface carboxyl group. The assignment is supported by the observation that the intensity of this band diminishes after surface reaction. Further confirmation of the assignment comes from a comparison of (25) Babul, J.; Stellwagen, E. Biochemistry 1972, 11, 1195. (26) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes: Eugene, OR, 1996. (27) Ando, T.; Inoue, S.; Ishii, M.; Kamo, M.; Sato, Y.; Yamada, O.; Nakano, T. J. Chem. Soc., Faraday Trans. 1993, 89, 749. (b) Ando, T.; Yamamoto, K.; Ishii, M.; Kamo, M.; Sato, Y. J. Chem. Soc., Faraday Trans. 1993, 89, 3635. (28) Pehrsson, P. E.; Mercer, T. W. Surf. Sci. 2000, 460, 49. (b) Pehrsson, P. E.; Mercer, T. W. Surf. Sci. 2000, 460, 74.

Cytochrome c Adsorbed on Nanodiamonds

Figure 1. Infrared spectra of 100 nm diamonds after (a) treatment with strong acids, (b) physisorption of horse cytochrome c, and (c) coating with poly-L-lysines. Polynomial fits are used for baseline subtraction of each spectrum. Inset: SEM image of a single 100 nm diamond particle. The scale bar is 40 nm.

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Figure 2. (a) Typical UV-vis absorption spectra of horse cytochrome c solutions before (solid curves) and after (dash dot curves) exposure to 100 nm diamonds at two different protein concentrations. (b) Adsorption isotherms of horse cytochrome c on diamonds with sizes of 100 and 5 nm in phosphate buffers at pH 6.5. Solid curves are the best fits of the experimental data to the Langmuir adsorption model (eq 1 in text).

this spectrum with that of benzoic acid,29 which possesses an intramolecular H-bond within the -COOH group, resulting in a large red shift (∼100 cm-1) of the O-H stretching frequency to 3567 cm-1. In accord with this assignment, the corresponding CdO stretching vibration of the carboxyl group can be identified at 1824 cm-1, which is the most prominent feature of all (Figure 1a). We rule out the possibility for assigning the 1623 cm-1 band to the CdO stretching of the carboxylate anion, because when the spectrum was acquired under high vacuum (∼10-5 Torr), it disappeared nearly completely. Hence, the most plausible assignment of this band is the O-H bending of physically adsorbed water. Similarly, the much weaker feature appearing as a shoulder at ∼3704 cm-1 should be assigned to the free-OH stretches of physisorbed water and the associated hydrogen-bonded-OH stretches are responsible to the broad absorption bands observed at 3000-3600 cm-1.30 The lowest-frequency feature in the spectral region of interest (the 1277 cm-1 band) has been ascribed by Ando et al.27b to ether-like groups on the diamond powders. The strong acid pretreatment described herein is effective in removing metallic and graphitic carbon impurities from the diamond nanocrystal surfaces. However, no significant microstructural or morphological changes were observed by scanning electron microscopy (SEM) for the particles. These acid-resulted changes are

also too small to be revealed by Raman spectroscopy, presumably because of the high quality of the diamond powders used in this work.19a The inset in Figure 1 shows the typical SEM image of a single particle of 100 nm diamonds, which are rough and irregularly shaped but are crystalline and optically transparent. Physical Adsorption. Carboxylated/oxidized diamonds are good adsorbents for proteins or polypeptides. The adsorption is established through electrostatic attraction between the surface-terminating anionic groups (-COO-) and the positively charged amino groups (-NH3+) of the biomolecules. Aside from the charge-charge interactions, ionic hydrogen bonds can also form between -NH3+ and any CO-containing surface groups such as ether, carbonyl, peroxide, etc. Evidence for the existence of these interactions is abundant in the literature for gasphase clusters.31 The typical binding energies for such ionic H-bonds are in the range of 10-30 kcal/mol. Hence, the heme-containing cytochrome c, which comprises 19 lysines,32 is highly attractive to the acid-treated diamond nanocrystals. Figure 2a shows the UV-vis absorption spectra for HCC-containing solutions (2.5 mL) before and after exposure to the 100 nm diamonds (50 mg). At low protein concentration, the removal of the proteins from the solution is so dramatic that the absorbance essentially decreases to zero soon after addition of the powers into the solution. Only after saturation of the diamond surfaces with the

(29) Stepanian, S. G.; Reva, I. D.; Radchenko, E. D.; Sheina, G. G. Vib. Spectrosc. 1996, 11, 123. (30) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic: Boston, MA, 1991.

(31) Meot-Ner, M. J. Am. Chem. Soc. 1984, 106, 1257. (b) Meot-Ner, M.; Sieck, L. W.; Liebman, J. F.; Scheiner, S. J. Phys. Chem. 1996, 100, 6445. (32) Moore, G. R.; Pettigrew, G. W. Cytochrome c; Springer-Verlag: Heidelberg, 1990.

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protein molecules can noticeable absorbance be detected for the solution. Figure 2b shows the corresponding adsorption isotherm of HCC on 100 nm diamonds. The isotherm is seen to reach rapidly to its maximum with a very steep slope, denoting high affinity of the protein to the diamond surface. The affinity is so high that HCC remains attached to the surfaces even after washing the diamond particles thoroughly with deionized water. This striking behavior is evidenced from FTIR measurements for the protein-diamond film (cf. Figure 1b), where the bands arising from the vibrations of the peptide backbones of the physisorbed proteins can be readily identified at 3316, 1665, and 1531 cm-1 for the N-H stretch, the CdO stretch (amide I), and the N-H deformation (amide II), respectively.33 The adsorption of HCC to 100 nm diamonds saturates at 97 mg/g (Figure 2b). According to the manufacturer, the specific surface area of the 100 nm diamond powders is ∼60 m2/g. Combining these two numbers gives a density of 1.6 mg/m2 for HCC physisorbed on the diamond surface at saturation. On the basis of the isotope-averaged molecular mass (12360 Da)32 of the protein, this transforms to a packing density of Γ ) 8 × 1012 molecules/cm2. Cytochrome c is a globular protein with a crystallographic dimension of 2.5 × 2.5 × 3.7 nm.34 Assuming that the conformational change is insignificant upon the adsorption, the maximum packing density of the protein should range from 1.0 × 1013 to 1.6 × 1013 molecules/cm2, depending on the protein’s orientation.22,23 Our observation of the Γ for HCC is in close agreement with these estimates, strongly suggesting that a protein film consisting of closely packed cytochrome c forms on the surface. In contrast to the steplike isotherm of HCC on 100 nm diamonds, the adsorption of the same protein to 5 nm diamonds exhibits a much gradual progress and saturates at 195 mg/g (Figure 2b). From a fit of the experimental data to the Langmuir adsorption model

Θ)

KaC 1 + KaC

(1)

where Θ is the ratio of the occupied adsorption sites to the total sites at saturation, Ka is the adsorption constant, and C is the equilibrium protein concentration, we obtained Ka ) 2.4 × 105 and 3.4 × 106 M-1 for the 5 and 100 nm diamonds, respectively. Note that the two equilibrium constants differ by more than 1 order of magnitude, revealing an interesting size effect. On the premise that the 5 nm diamonds are all spheroids weighing ∼2 × 10-19 g per particle, this amount of HCC adsorbed at saturation (i.e., 195 mg/g or 9.42 × 1018 molecules/g) means that each nanodiamond can carry up to two protein molecules. The associated packing density is Γ ) 3 × 1012 molecules/cm2, given a specific surface area of ∼300 m2/g from the manufacturer for the nanodiamonds. Elements that affect protein adsorption to diamond include hydrophilic35 and hydrophobic36,37 interactions. The present experiment was executed in phosphate buffers at pH 6.5, and hence, the diamond surface is mainly negatively charged (due to the presence of the carboxylate (33) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry; Freeman: San Francisco, CA, 1980; Part II, Chapter X. (34) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (35) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344. (36) Rivas, L.; Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2002, 106, 4823. (37) Vinu, A.; Streb, C.; Murugesan, V.; Hartmann, M. J. Phys. Chem. B 2003, 107, 8297.

groups) and the protein (with an isoelectric point of pI ) 10.6)38 is positively charged. While the carbon species on diamond are very hydrophobic and are attractive to the nonpolar side chains of the amino acid residues on the HCC surface, the affinity of the protein to the hydrophilic adsorption matrix should still be dominated by electrostatic forces between -NH3+ and -COO- on both 5 and 100 nm diamonds. In this context, the marked difference in Γ between these two adsorption cases (i.e., 8 × 1012 versus 3 × 1012 molecules/cm2) is thus likely to be a result that the 5 nm diamonds are rounded in shape,39,40 which in effect reduces the feasibility of multiple hydrogen bond formation between the folded proteins and the curved surfaces. To increase the density as well as the strength of the adsorption of the proteins on nanodiamonds, a method for covalent immobilization is developed. Protein Immobilization. Poly-L-lysine was selected as the polycation for surface amination and subsequent protein immobilization. Previous experiments14,24 have shown that functionalization of a surface with PL is useful for chemical sensor applications via the free lysine residues. At neutral pH, the PL is highly positively charged and it is expected to entrap the surface CO moieties via multiple H-bonding in an extended configuration, forming a firm hydrophilic layer on the diamond surface. The adsorption should proceed facilely, irrespective of the size and curvature of the particles. Figure 1c shows the FTIR spectrum of the 100 nm PL-coated diamonds. Two absorption bands at 1672 and 1502 cm-1 are attributed to the amide I and amide II modes of the polypeptide backbone, respectively.30,33 However, with reference to the 1809 cm-1 band of the surface CdO groups, which are derived from the diamond substrate only, they both are 3-fold lower in intensity as compared to the corresponding features in Figure 1b for HCC. It means that the adsorbed PL layer has a lower density than the adsorbed HCC layer on the same surface. Since HCC is adsorbed on the diamond with a folded configuration, the present observations (Figure 1b,c) corroborate the suggestion that PL is attached to the surface in an extended configuration. The attachment is so robust that the PL layer is resistant to several circles of washing with deionized water, even on 5 nm diamonds. The ability of lysine to be modified via the free -NH3+ moieties is a crucial property in implementation of the PL layer for practical chemical sensor applications. While PL is adsorbed to diamond in an extended configuration, some of the lysine residues may not be bound to the surface but remain available for interaction or reaction with molecules from solution. As a test for the availability of the free lysine residues for protein immobilization, the PL-coated 5 nm diamonds were exposed to a solution of Alexa Fluor 488 reactive dye (Scheme 1). This dye molecule was chosen for this study because it contains a succinimidyl ester moiety that reacts efficiently with the primary amines of proteins or polypeptides in solution.26 Moreover, it has absorption and emission maxima of 497 and 518 nm, respectively, and the emission is insensitive to the solution pH between 4 and 10. Indeed, by utilizing a confocal laser scanning fluorescence microscope, we have been able to perform the fluorescence imaging of the nanodiamond particles. Given in Figure 3 is the result of (38) Lehninger, A. L. Biochemistry: The Molecular Basis of Cell Structure and Function, 2nd ed.; Worth Publishers: New York, 1975; Chapter 7. (39) Kuznetsov, V. L.; Chuvilin, A. L.; Butenko, Yu. V.; Mal’kov, I. Yu.; Titov, V. M. Chem. Phys. Lett. 1994, 222, 343. (40) Lin, K.-W.; Cheng, C.-L.; Chang, H.-C. Chem. Mater. 1998, 10, 1735.

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Scheme 1. Reactions for Surface Amination, Dye-Labeling, and Protein Immobilization on Nanodiamonds

Figure 4. Infrared spectra of (a) 100 nm and (b) 5 nm diamonds after coating with poly-L-lysines (dash dot curves) and immobilization of yeast cytochrome c (solid curves) on the surfaces. Polynomial fits are used for baseline subtraction of all the spectra, which are normalized separately with respect to the absorption bands of the surface CdO stretches at ∼1800 cm-1 in each series.

Figure 3. Fluorescence image of 5 nm diamonds labeled with Alexa Fluor 488 after surface amination with poly-L-lysines.

the measurement, showing the images of the 5 nm diamonds (i.e., the bright spots), which are clearly visible as individual particles or as clusters thanks to the fluorescent labeling.41 Such dye-labeled nanodiamonds potentially may be utilized as a probe to detect and sense the states of single living cells.42 The heterobifunctional cross-linker presently used for covalent protein immobilization is SSMCC. One end of the linker reacts with the amino groups of the PL-coated surfaces, whereas the other end reacts specifically with (41) Hazani, M.; Naaman, R.; Hennrich, F.; Kappes, M. M. Nano Lett. 2003, 3, 153. (42) Bruchez, M. Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos A. P. Sci. 1998, 281, 2013. (b) Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47.

the sulfhydryl group of the cysteine-containing protein (Scheme 1). Figure 4a shows the FTIR spectrum of YCC immobilized on the 100 nm diamond surfaces in the presence of PL and SSMCC. Both PL and YCC contribute to the observation of the amide I and II bands in the spectrum. The contribution of the latter, however, can be deduced semiquantitatively by proper normalization of the spectrum (solid curve in Figure 4a) with respect to the surface CdO absorption bands at ∼1800 cm-1, followed by subtracting the spectrum of PL (dash dot curve in Figure 4a) in the amide vibration region. Similar analysis applies to YCC on 5 nm diamonds (cf. Figure 4b). On the basis of the intensity-normalized spectra, we compare in Figure 5 the amide band intensities of the covalently immobilized proteins (part b) to those of the physisorbed ones (part a). The former are nearly twice as high as the latter, implying an increase in the protein adsorption density Γ with the aid of the cross-linkers. We conducted a stability test for the protein films composed of (a) YCC only and (b) PL/SSMCC/YCC on

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Figure 5. Stability tests of yeast cytochrome c films on 5 nm diamonds, prepared by (a) physisorption and (b) covalent immobilization. The spectra, all normalized with respect to the absorption bands at ∼1780 cm-1, were acquired for freshly prepared samples (solid curves) and samples stored in a refrigerator for 5 months (dash dot curves).

nanodiamonds (cf. Figure 5). Samples employed here were subjected to extensive washing with deionized water. Both of the protein films were so stable that the spectra remain essentially unchanged after 10 cycles of washes. Even after storage of the sample suspensions in a refrigerator (4 °C) for 5 months, the YCC film shows only slight decreases in intensity of both the amide bands (Figure 5a). In contrast, the PL/SSMCC/YCC film gives a spectrum essentially identical to that of the freshly prepared one (Figure 5b), depicting exceptionally high stability of the substance. The results demonstrate not only that the protein-immobilized diamond surface is very stable but also that diamond is an excellent material for preserving protein stability.7,8 A surface-bound protein may change its conformation upon adsorption to a substrate. For YCC in solution, the information can be deduced from the peak position of the Soret band, which has been observed to shift distinctly from 409 nm (pH 7) to 395 nm (pH 2) when the protein unfolds due to acid denaturation.43 We compare in parts a and b of Figure 6 the respective spectra of the covalently immobilized YCC and the corresponding protein in solution. While there is a shift of the baseline from 600 to 350 nm in Figure 6a because of severe light scattering from the suspending nanodiamond particles and their aggregates, the Soret band can be readily identified at λmax ) 409 nm at pH 6.5. The observation of the Soret bands appearing at the same position for both surfacebound YCC and free YCC is consistent with the picture that the protein changes its conformation insignificantly upon immobilization on the diamond surface.22 (43) Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124, 14840.

Huang and Chang

Figure 6. UV-vis absorption spectra of yeast cytochrome c (a) covalently immobilized on 5 nm diamonds and (b) in solution at pH 6.5. The rise of the background from 600 to 300 nm in (a) is due to light scattering from the nanodiamonds and their agglomerates, and the glitch appearing at ∼390 nm is due to the change of the grating of the spectrometer.

Conclusion We have developed a method that allows facile functionalization of nanocrystalline diamond surfaces with amino groups for protein immobilization as well as fluorescent labeling. Compared to the method developed previously for covalent immobilization of DNA on diamond microcrystals,11 this approach is faster, more effective, easier to operate, and independent of the origin and size of the diamond sample used in the preparation. Since the diamond surfaces thus prepared are stable and biocompatible, the surface-modified nanocrystalline diamonds with an average size of 5 nm are potentially useful in biomedical applications; for example, they may be applicable as an immobilizer for biologically active substances or low-molecular-weight compounds44 to be probed by optical microscopy and mass spectrometry. The present work establishes a foundation for future applications in this direction. Acknowledgment. The research was supported by the Academia Sinica and the National Science Council (Grant No. NSC 92-3112-B-001-012-Y under the National Research Program for Genomic Medicine) of Taiwan, Republic of China. We thank C.-M. Hsu for technical assistance and W. H. Chen for taking the fluorescence microscope images for us. LA0495736 (44) Liotta, L. A.; Ferrari, M.; Petricoin, E. Nature 2003, 425, 905.