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Langmuir 2008, 24, 4200-4204
Biotinylated Nanodiamond: Simple and Efficient Functionalization of Detonation Diamond Anke Krueger,* Jochen Stegk, Yuejiang Liang, Li Lu, and Gerald Jarre Otto-Diels-Institut fu¨r Organische Chemie, Christian-Albrechts-UniVersita¨t zu Kiel, Otto-Hahn-Platz 3, D-24098 Kiel, Germany ReceiVed NoVember 7, 2007. In Final Form: December 21, 2007 We have developed a simple and efficient method for the covalent functionalization of detonation nanodiamond. After homogenization of the surface by borane reduction, the surface was modified with (3-aminopropyl)trimethoxysilane. Subsequent grafting of biotin yielded covalently biotinylated nanodiamond, which was characterized by FTIR spectroscopy, X-ray powder diffractometry, thermogravimetry, and elemental analysis. The activity was tested with horseradish peroxidase-labeled streptavidin. The surface loading of biotin was found to be 1.45 mmol g-1. The new material opens the way to covalently bonded diamond bioconjugates for labeling, drug delivery, and other applications.
Introduction The development of new platforms for the immobilization of biologically active substances is a very active research area. Especially systems on the basis of silica, gold, and iron oxide particles, glass surfaces, and cadmium chalcogenide quantum dots are being investigated.1 Another candidate material in the form of films or particles is diamond. It shows only low cytotoxicity,2 and on a clean, hydrogen-terminated diamond surface nonspecific adsorption is very low.3 It is optically transparent, chemically inert, and commercially available in different forms and can be doped rather easily. Recently, the progress and modern developments in the field of nanodiamond particles and films and their potential applications have been recently reviewed.4 For the application of nanoparticles in biological systems it is in general necessary to modify the particle surface according to the specific requirements of the desired application. It is therefore important to find methods to functionalize the diamond surface with bioactive moieties to be able to assess the potential of this material in bioapplications. Various examples for the chemical modification of diamond films have been reported, which have been used as substrates, e.g., for biosensors and electrochemical applications.5 There have also been several reports on the noncovalent grafting of bioactive molecules onto the surface * To whom correspondence should be addressed. E-mail: akrueger@ oc.uni-kiel.de. Fax: (+)49(0)431 880 1558. (1) Niemeyer, C. M. Angew. Chem. 2001, 113, 4254-4287; Angew. Chem., Int. Ed. 2001, 40, 4128-4158. Katz, E.; Willner, I. Angew. Chem. 2004, 116, 6166-6235; Angew. Chem., Int. Ed. 2004, 43, 6042-6108. Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (2) Bakowicz, K.; Mitura, S. J. Wide Bandgap Mater. 2002, 9, 261-272. Mathieu, H. J. Surf. Interface Anal. 2001, 32, 3-9. Schrand, A. M.; Huang, H.; Carlson, C.; Schlager, J. J.; Osawa, E.; Hussain, S. M.; Dai, L. J. Phys. Chem. B 2007, 111, 2-7. (3) Granger, M. C.; Witek, M.; Xu, J.; Wang, J.; Hupert, M.; Hanks, A.; Koppang, M. D.; Butler, J. E.; Lucazeau, G.; Mermoux, M.; Strojek, J. W.; Swain, G. M. Anal. Chem. 2000, 72, 3793-3804. (4) Shenderova, O. A., Gruen, D. M., Eds. Ultrananocrystalline Diamond; William Andrew Publishing: Norwich, NY, 2006. Holt, K. B. Philos. Trans. R. Soc., A 2007, 365, 2845-2861. (5) Knickerbocker, T.; Strother, T.; Schwartz, M. P.; Russell, J. N., Jr.; Butler, J.; Smith, L. M.; Hamers, R. J. Langmuir 2003, 19, 1938-1942. Ha¨rtl, A.; Schmich, E.; Garrido, J. A.; Hernando, J.; Catharino, S. C. R.; Walter, S.; Feulner, P.; Kromka, A.; Steinmu¨ller, D.; Stutzmann, M. Nat. Mater. 2004, 3, 736-742. Delabouglise, D.; Marcus, B.; Mermoux, M.; Bouvier, P.; Chane-Tune, J.; Petit, J.-P.; Mailley, P.; Livache, T. Chem. Commun. 2003, 2698-2699.
of nanodiamond particles. These include the attachment of cytochrome c, apoobelin, protein lysozyme, and many others.6 However, there is little known about the covalent attachment of biological moieties on diamond particles. Nevertheless, they could, especially with low particle size, serve for targeted drug delivery and fluorescence labeling.7,8 Diamond particles have the advantage of inherent fluorescence from lattice defects such as N-V centers, which is nonbleaching and nonblinking and is visible in the red region of light.7 So far, in vitro labeling experiments have been carried out with noncovalently modified diamond particles, e.g., by adsorption of a biocompatible agent such as poly-L-lysine.9 Although quite stable in this case, noncovalently grafted moieties have in general a tendency to leach out from the label composite. Additionally, the production of conjugates with defined stoichiometry by noncovalent interaction is very challenging. It is hence desirable to produce covalently modified, biocompatible nanodiamond particles. Furthermore, it is important to develop a simple procedure with only a few steps to make the material accessible for routine applications. In general, the covalent attachment of functional groups on diamond particles has often been described for diamond materials with a crystallite size in the range of 500 nm.10 Additionally, there have been only a few reports on the grafting of biocompatible moieties onto diamond particles so far. For example, the oxidation of diamond particles with concentrated mineral acids at elevated temperatures leads to partially carboxylated particles. These have been coupled to thymidine and later modified with short DNA (6) Yu, S.-J.; Kang, M.-W.; Chang, H.-C.; Chen, K.-M.; Yu, Y.-C. J. Am. Chem. Soc. 2005, 127, 17604-17605. Chung, P.-H.; Perevedentseva, E.; Tu, J.-S.; Chang, C. C.; Cheng, C.-L. Diamond Relat. Mater. 2006, 15, 622-625. Nguyen, T.-T.-B.; Chang, H.-C.; Wu, V. W.-K. Diamond Relat. Mater. 2007, 16, 872-876. Huang, L.-C. L.; Chang, H.-C. Langmuir 2004, 20, 5879-5884. Bondar’, V. S.; Pozdnyakova, I. O.; Puzyr’, A. P. Phys. Solid State 2004, 46, 758-760. (7) The fluorescence originates from defect centers, here nitrogen defects. In addition to its long wave emission, it possesses a high stability (no bleaching). See: Gruber, A.; Dra¨benstedt, A.; Tietz, C.; Fleury, L.; Wrachtrup, J.;Borczyskowski, C. v. Science 1997, 276, 2012-2014. (8) Yu, S.-J.; Kang, M.-W.; Chang, H.-C.; Chen, K.-M.; Yu, Y.-C. J. Am. Chem. Soc. 2005, 127, 17604-17605. (9) Fu, C.-C.; Lee, H.-Y.; Chen, K.; Lim, T.-S.; Wu, H.-Y.; Lin, P.-K.; Wei, P.-K.; Tsao, P.-H.; Chang, H.-C.; Fann, W. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 727-732. (10) Tsubota, T.; Hirabayashi, O.; Ida, S.; Nagaoka, S.; Nagata, M.; Matsumoto, Y. Phys. Chem. Chem. Phys. 2002, 4, 806-811. Nakamura, T.; Ishihara, M.; Ohana, T.; Koga, Y. Chem. Commun. 2003, 900-901.
10.1021/la703482v CCC: $40.75 © 2008 American Chemical Society Published on Web 03/01/2008
Biotinylation of Nanodiamond
Langmuir, Vol. 24, No. 8, 2008 4201 Scheme 1. Biotinylation of Detonation Diamonda
a Conditions and reagents: (a) BH ‚THF, THF, reflux, 72 h; (b) (3-aminopropyl)trimethoxysilane, acetone, room temperature, 16 h; (c) 3 biotin, EDC, DMAP, CH2Cl2, 0 °C f room temperature, 65 h.
strands.11 The size of the crystallites was in the range of hundreds of nanometers though. Besides that, the reaction of so-called detonation diamond (vide infra) with fluorine in a hydrogen stream at ∼400 °C yielded a fluorinated material that has been reacted with glycine.12 The crystallite size in this case was very small (4-5 nm), and the resulting functionalized particles were in the range of 200 nm. Recently, detonation nanodiamond13 came into focus as a potentially useful material for bioapplications due to its unique properties.14 The pristine material carries various surface functional groups due to the production from explosives in a closed reactor15 and exhibits a highly oxidized, hydrophilic surface. Therefore, its application in biological systems is promising, but the surfaces of the particles need to be homogenized. Besides treatment with oxidizing acids16 and fluorination,12 we have described another versatile method for the initial surface homogenization consisting in the reduction of surface carbonyl groups on detonation nanodiamond with borane under mild conditions.17 The resulting hydroxyl functions are a good starting point for the further functionalization of the diamond surface. We have chosen to silanize the diamond surface by using (3-aminopropyl)trialkoxysilane to establish a simple method for further surface functionalization. From the modification of silica particles this simple technique is well-known and shows high efficiency and reproducibility.18 In a model reaction we have recently demonstrated the feasibility of this approach for nanodiamond.17 It is therefore reasonable to apply this technique for the immobilization of bioactive structures too. Here we report on the covalent attachment of a biocompatible moiety, namely biotin, onto the detonation diamond surface. Experimental Section Chemicals. Detonation diamond was purchased from Gansu Lingyun Corp., Lanzhou, China. All other chemicals have been purchased from Fluka and Sigma-Aldrich and used without further purification if not stated otherwise. Solvents were dried according to literature procedures. Biotin was purchased from Dako Chemicals Corp. (11) Ushizawa, K.; Sato, Y.; Mitsumori, T.; Machinami, T.; Ueda, T.; Ando, T. Chem. Phys. Lett. 2002, 351, 105-108. (12) Liu, Y.; Gu, Z.; Margrave, J. L.; Khabashesku, V. N. Chem. Mater. 2004, 16, 3924-393. (13) Yu Dolmatov, V. Russ. Chem. Bull. 2001, 70, 607-626. (14) Krueger, A. Chem.sEur. J. 2008, 14, 1382-1390. (15) Shenderova, O. A.; Zhirnov, V. V.; Brenner, D. W. Crit. ReV. Solid State Mater. Sci. 2002, 27, 227-356. Donnet, J.-B.; Lemoigne, C.; Wang, T. K.; Peng, C.-M.; Samirant, M.; Eckhardt, A. Bull. Soc. Chim. Fr. 1997, 134, 875-890. (16) Loktev, V. F.; Makal’ski, V. I.; Stoyanova, I. V.; Kalinkin, A. V.; Likholobov, V. A. Carbon 1990, 29, 817-819. (17) Krueger, A.; Liang, Y.; Jarre, G.; Stegk, J. J. Mater. Chem. 2006, 16, 2322-2328. (18) Wight, A. P.; Davis, M. E. Chem. ReV. 2002, 102, 3589-3614.
Instrumentation. The FTIR spectra were measured on a PerkinElmer Paragon 1000 spectrometer as KBr pellets. Thermogravimetry was carried out on a Netzsch ST2010, powder X-ray diffraction was measured on a Stoe Stadi P powder diffractometer using Cu R1 radiation. Elemental compositions were measured on a Euro EA 3000 series elemental analyzer. Measurements of the particle size were executed on a Photal Otsuka FPAR1000 fiber-optics particle analyzer. HRTEM images were obtained with a 200 kV Tecnai F20ST electron microscope equipped with a field emission gun. Synthesis of Biotinylated Detonation Nanodiamond. The reaction of nanodiamond with borane to 1 and the subsequent silanization with (3-aminopropyl)trimethoxysilane were carried out according to the procedure described in a recently published work (see also the Supporting Information).17 Synthesis of 3: 339 mg of silanized nanodiamond 2 was placed in a round-bottom flask under a nitrogen atmosphere, and a solution of 251 mg (1.02 mmol) of biotin and 61 mg (0.51 mmol) of (dimethylamino)pyridine in 100 mL of dry dichloromethane was added. Under ice cooling, 200 mg (1.02 mmol) of EDC (1-ethyl-3-[(3-dimethylamino)propyl]carbodiimide hydrochloride) was added, and the solution was stirred for 1 h at 0 °C and 65 h at room temperature. Centrifugation yielded a solid residue, which was washed with 250 mL of acetone in total. The solid was stirred in 200 mL of ethanol for 2.5 h. After another centrifugation step the material was washed with 250 mL of ethanol in total and with 50 mL of acetone. The sample was dried in vacuo. This powder was again stirred with 250 mL of ethanol for 1 h, centrifuged, and washed with 200 mL of ethanol until no biotin was detectable in the washing liquid anymore. After drying in vacuo, 455 mg of a light gray powder was obtained. IR (cm-1): 3360, 3306, 2932, 1704, 1688 (CdO), 1638, 1118 (Si-O-C), 1032 (SiO-Si), 650 (C-Si). Elemental analysis: C, 70.41; H, 3.266; N, 6.77; S, 4.66. Surface loading: 1.45 mmol g-1. Thermogravimetry (N2, 4 K min1): -21.93%. Binding Tests with Streptavidin. The test was carried out using horseradish peroxidase-labeled streptavidin in microtiter plates according to the established procedure (see the Supporting Information for details). The diamond was suspended, and the wells of a microtiter plate were coated with an aqueous suspension of 3. The plates were placed in a 37 °C incubator and dried overnight to form a thin layer of biotinylated nanodiamond on the bottom of the wells. Only plates with a sufficiently uniform and stable coating (no delamination) can be used for the tests. After several washing steps, streptavidin-horseradish peroxidase conjugate was added. The subsequent reaction with hydrogen peroxide solution and the addition of ABTS yielded the typical green coloring, which proves the existence of active biotin on the surface of the diamond particles (vide infra).19 (19) Matsuda, H.; Tanaka, H.; Blas, B. L.; Nosenas, J. S.; Tokawa, T.; Ohsawa, S. Jpn. J. Exp. Med. 1984, 54, 131-138. Gallati, V. H. J. Clin. Chem. Clin. Biochem. 1979, 17, 1-7. Porstmann, B.; Porstmann, T.; Nugel, E. J. Clin. Chem. Clin. Biochem. 1981, 19, 435-439.
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Figure 1. FTIR spectra of functionalized detonation diamond samples: (a) starting material (the arrow shows the carbonyl signal of the pristine detonation diamond, which disappears after reduction), (b) hydroxylated nanodiamond 1, (c) silanized nanodiamond 2, (d) biotinylated nanodiamond 3 (the underlying signals around 1635 and ∼3300 cm-1 in all spectra originate from adsorbed water, which is very difficult to remove).
Results and Discussion Here we report on the first covalent functionalization of detonation diamond with biotin. It serves as a model system for the biocompatible functionalization of this material. The inhomogeneously functionalized surface of the pristine detonation diamond is first reacted with borane (Scheme 1).17 Thus, carboxyl and carbonyl groups on the surface are reduced as can be seen from the IR spectra (Figure 1). The signal for CdO bonds at 1720 cm-1 (arrow) disappears after the reduction. From TPD-MS measurements the initial concentration of surface OH groups was calculated to be 0.05 mmol g-1; for the reduced sample 1 it increased almost 10 times to 0.49 mmol g-1. The resulting hydroxyl functions are then reacted with (3-aminopropyl)trimethoxysilane (APTMES), which covalently binds to the surface in a condensation reaction. The covalent nature can be established by the characteristic signal of the C-O-Si bond in the IR spectrum and the high decomposition temperature in thermogravimetric analysis. Nevertheless, it has to be pointed out that the trialkoxysilanes are prone to further react with additional trialkoxysilane molecules as well as to condense to larger structures by interparticle condensation, leading to an increased particle size (see below). In all, a high binding efficiency by several alkoxy groups and the tendency to form aggregates have to be balanced by the choice of the reagent and optimized reaction conditions.18,20 XRD measurements (not shown) prove that the surface modification did not affect the diamond core of the particles as shown by the existence of the diffraction signals for the diamond lattice. Additionally, we have obtained HRTEM images for the silanized and biotinylated samples (Figure 2). These images show unambiguously that the material consists of small and evenly sized diamond nanoparticles with little to no graphitic surface coverage. They exist in the form of small agglomerates (see also the size distribution below). The resulting silyl ether is stable except for significantly acidic conditions; e.g., in 0.1 N HCl the silyl groups are completely cleaved, and the diamond with a hydroxylated surface can be recovered. The material’s characterization is carried out using FTIR spectroscopy (Figure 1), thermogravimetry (Figure 3), and elemental analysis (Table 1). Using these methods, the covalent (20) Clark, J. H.; Macquarrie, D. J. Chem. Commun. 1998, 853-860.
Figure 2. HRTEM images of (a) the pristine detonation nanodiamond, (b) the (aminopropyl)silanized nanodiamond 2 (the inset shows a higher magnification of some particles), and (c) the biotinylated nanodiamond 3. The diamond lattice is clearly visible, and the primary nanoparticles exhibit a quite uniform size and shape distribution. The bright lines surrounding the particles result from the underfocus conditions, which were used to increase the contrast.
grafting of the silane was proven and a surface loading of 1.46 mmol g-1 was calculated for 2, indicating an efficient coating of the diamond surface. It has to be taken into account that the original diamond material already contains a certain amount of nitrogen, which stems from the explosives used in its synthesis.21 Therefore, it is necessary to deduct the inherent nitrogen content from the overall value for a correct calculation of the surface loading with the silane. Another important point is the fact that the surface loading with silane groups is roughly 3 times higher than the calculated concentration of OH groups on the diamond surface measured by TPD-MS. This indicates that further (21) Most of the nitrogen is most likely included as N2 in cavities between diamond particles or sootlike structures. Only a small fraction is incorporated into the lattice, forming substitutional defects; see ref 11.
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Figure 3. Thermogravimetric analysis of pristine and functionalized detonation diamond. The weight increase for pristine and hydroxylated detonation diamond 1 is most likely due to the reaction with the surrounding nitrogen atmosphere during TGA analysis and typical for this type of nanodiamond. This phenomenon is currently under investigation. Table 1. Elemental Composition (%) of Functionalized Diamond Materials and Surface Loading (mmol g-1) with Functional Groups substance
C
H
N
S
loading
starting material 1 2 3
88.46 88.65 81.21 70.41
0.49 0.46 1.54 3.27
2.24 2.28 3.96 6.77
0 0 0 4.66
a a 1.46 1.45
a Due to water adsorption the surface loading with OH groups could not be determined by elemental analysis or thermogravimetry.
condensation reactions of trialkoxysilanes onto already attached silanes play a major role in the surface coating. This is known for various kinds of trialkoxysilanes.18,20 In the next reaction step, the terminal amino groups of the silanized diamond 2 are reacted with biotin using EDC and DMAP for the coupling (Scheme 1).22 In the IR spectrum of 3 the characteristic signals for biotin as well as for the amide bond with the amino groups of the silane appear as strong peaks. An important issue is the removal of nonreacted biotin from the samples. This is done by thorough washing with ethanol until no biotin is detectable in the washing liquid anymore. After this procedure, the signal for the O-H valence vibration of free carboxylic groups (expected at ∼2500 cm-1) is almost not detectable, indicating that only negligible amounts of free biotin are present in the sample. The thermogravimetric analysis (Figure 3) shows the decomposition of the silanized sample 2 and the subsequently biotinylated sample 3 at temperatures above 250 °C. This is a further indication for the covalent nature of the grafting of the functional moieties onto the diamond surface. The elemental composition (Table 1) shows the increase in nitrogen content as well as the incorporation of sulfur after the biotinylation of the material. The surface loading for 3 calculated from these analytical data is 1.45 mmol g-1, which corresponds to 3 × 1014 biotin molecules/cm2 of the particle surface (the specific surface of 300 m2 g-1 was determined from the BET isotherm, not shown here). The obtained value indicates that the (22) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org. Chem. 1982, 47, 1962-1965. Han, S.-Y.; Kim, Y.-A. Tetrahedron 2004, 60, 2447-2467.
grafting of biotin proceeds virtually quantitatively on all free amino groups, underlining the efficiency of this process. For diamond film loadings of 1012-1015 cm-2 have been reported;23 hence, the result for the investigated diamond particles lies in the same range. Further improvement could be made by an increase in accessible diamond surface by deagglomeration during the silanization process. This work is currently under way. The particle size of the different diamond materials has been measured by dynamic light scattering. The pristine diamond material forms very strongly bound agglomerates with an average size of several micrometers. They are not easily destroyed due to remaining soot structures and interparticle covalent bonds.12,15,24 The reaction with borane, though, also results in the partial deagglomeration of the particles down to a size of ∼50 nm. The silanized diamond 2 shows an increased size of ∼200-250 nm, which is due to the thickness of the silane coating layer and condensation reactions with further free silane molecules (vide supra). The biotinylated diamond samples exhibit a size of ∼170 nm, indicating the existence of small (compared to the original) diamond agglomerates in the resulting material. Further deagglomeration down to very small agglomerates or even the primary particles should be achievable by deagglomeration during the silanization process (vide supra) or by the use of monoalkoxydimethylsilanes as they are not able to form interparticle bridges and hence do not promote the formation of aggregates. The resulting biotinylated material 3 was tested for its biological activity. To this end, a labeled streptavidin was used. The interaction of biotin with avidin or streptavidin occurs with extremely high selectivity and affinity. Therefore, the interaction of fluorescently or otherwise labeled derivatives of these proteins is often used to prove the grafting of biotin on a substrate.25 Here, horseradish peroxidase-labeled streptavidin was chosen for the simplicity of the test method and the easily detectable green coloring (Figure 3) of the ABTS (2,2′-azinobis(3ethylbenzothiazoline-6-sulfonic acid)) when reacted with the OH radicals from enzymatically decomposed hydrogen peroxide.19 This reaction is induced by the immobilized horseradish peroxidase, which is grafted to the bottom layer in the wells by the firm interaction between biotin moieties and the streptavidin. In blank tests with hydroxylated and silanized detonation diamond (1 and 2), a weak but significant nonspecific adsorption of the streptavidin conjugate on the hydrophilic diamond surface is observed. Such an adsorption of proteins on hydrophilic diamond surfaces is known for films as well as particles with oxygen-containing surface groups.26 In the present example the adsorption originates from the interaction of surface hydroxyl groups with the streptavidin conjugate. To inhibit this nonspecific interaction, the residual surface hydroxyl groups on the diamond particle surface are blocked with lactate before the reaction with streptavidin-horseradish peroxidase (HRP) conjugate. After this treatment, only biotinylated samples show the typical green coloring (Figure 4). The existence of covalently bound and bioactive biotin was hence unambiguously established with the (23) Lasseter Clare, T.; Clare, B. H.; Nichols, B. M.; Abbott, N. L.; Hamers, R. J. Langmuir 2005, 21, 6344-6355. Rezek, B.; Shin, D.; Nakamura, T.; Nebel, C. E. J. Am. Chem. Soc. 2006, 128, 3884-3885. Hamers, R. J.; Butler, J. E.; Lasseter, T.; Nichols, B. M.; Russel, J. N., Jr.; Tse, K.-Y.; Yang, W. Diamond Relat. Mater. 2005, 14, 661-668. Nichols, B. M.; Butler, J. E.; Russel, J. N., Jr.; Hamers, R. J. J. Phys. Chem. B 2005, 109, 20938. (24) Krueger, A.; Ozawa, M.; Kataoka, F.; Fujino, T.; Suzuki, Y.; Aleksenskii, A. E.; Ya Vul, A.; Osawa, E. Carbon 2005, 43, 1722-1730. (25) Smith, C. L.; Milea, J. S.; Nguyen, G. H. Top. Curr. Chem. 2006, 261, 63-90 and references therein. (26) Kong, X. L.; Huang, L. C. L.; Hsu, C.-M.; Chen, W.-H.; Han, C.-C.; Chang, H.-C. Anal. Chem. 2005, 77, 259-265. Guan, B.; Wu, L.; Ren, B.; Zhi, J. Carbon 2006, 44, 2849-2867. Huang, T. S.; Tzeng, Y.; Liu, Y. K.; Chen, Y. C.; Walker, K. R.; Guntupalli, R.; Liu, C. Diamond Relat. Mater. 2004, 13, 1098-1102. Huang, L.-C. L.; Chang, H.-C. Langmuir 2004, 20, 5879-5884.
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Figure 4. Activity test of biotinylated nanodiamond 3 with streptavidin-HRP conjugate (rows 2, 4, 6, and 8, blank test with ND-OH (1); rows 1, 3, 5, and 7, reaction of 3 with the following dilutions of the streptavidin-HRP reagent: 1, 2-1:10; 3, 4-1:100; 5, 6-1:1000; 7, 8-1:10000 (for details see the Supporting Information)).
test. Although a clear concentration-dependent coloration is visible in Figure 4, quantification of the interaction between the biotinylated diamond and HRP-labeled streptavidin proved difficult. The bottoms of the microtiter plate wells are covered with inhomogeneously thick layers of biotinylated diamond 3. During washing, partial delamination of the diamond layer at the well bottom took place in some cases too. Hence, a meaningful quantification by measuring the color intensity of the ABTS radical could not be accomplished as the amount of the biotinylated substrate 3 varies from well to well after sample preparation. To improve this situation, it would be desirable to use other coating techniques than simple film drying for the preparation of the deposits in the wells.
Conclusion In conclusion, we have shown here for the first time the covalent grafting of biotin, a biologically active moiety, on the surface
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of diamond nanoparticles with high surface loading under conservation of its activity. The covalent immobilization represents an important progress compared to adsorptive or noncovalent binding. It enables directed grafting onto the diamond surface and offers increased stability of the resulting conjugates. The versatile, simple, and efficient protocol enables its use in routine applications, not only for the immobilization of biotin but also for that of other bioactive compounds. In combination with the small particle size of detonation diamond and the inertness of the diamond lattice, it opens various opportunities for biological and medical applications of such diamond conjugates, especially as biocompatible fluorescence labels as well as for drug delivery devices. Future work will include the preparation of smaller diamond-bioconjugate particles, an increase in surface loading, or the preparation of adducts with defined stoichiometry. Acknowledgment. We thank the Deutsche Forschungsgemeinschaft and the European Commission (Contract LSHC-CT2005-019102) for financial support of the project, Prof. Dr. Th. K. Lindhorst and Dr. O. Sperling (Kiel University) for introduction to the activity tests and the permission to use their equipment, A. Puls and N. Stock (Kiel University) for powder XRD, C. Na¨ther and I. Jess (Kiel University) for access to the thermobalance, A. Thorel and M. Sennour (Ecole des Mines, Paris) for HRTEM images, and J. P. Boudou (UPMC, Paris) for TPD-MS measurements. A.K. is indebted to the Fonds der Chemischen Industrie for a Liebig fellowship. Supporting Information Available: Experimental procedure for the synthesis of 1 and 2 and detailed procedure for streptavidin testing. This information is available free of charge via the Internet at http://pubs.acs.org. LA703482V
