Protein-Functionalized Hairy Diamond Nanoparticles - Langmuir (ACS

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Protein-Functionalized Hairy Diamond Nanoparticles Si Amar Dahoumane,† Minh Ngoc Nguyen,† Alain Thorel,‡ Jean-Paul Boudou,*,§ Mohamed M. Chehimi,*,† and Claire Mangeney*,† †

ITODYS, Universit
e Paris Diderot and CNRS (UMR 7086), 15 rue Jean de Baif, 75013 Paris, France, ‡ Centre des Mat
eriaux, Mines-ParisTech, BP 87, 91000 Evry, France, and §Structure and Activity of Normal and Pathological Biomolecules - INSERM/UEVE U829, Universit
e d’Evry-Val d’Essonne, 91025 Evry, France Received March 18, 2009. Revised Manuscript Received July 17, 2009

Diazonium salt chemistry and atom transfer radical polymerization (ATRP) were combined in view of preparing new bioactive hairy diamond nanoparticles containing, or potentially containing, nitrogen-vacancy (NV) fluorescent centers (fluorescent nanodiamonds, or fNDs). fNDs were modified by ATRP initiators using the electroless reduction of the diazonium salt BF4-,þN2-C6H4-CH(CH3)-Br. The strongly bound aryl groups -C6H4-CH(CH3)-Br efficiently initiated the ATRP of tert-butyl methacrylate (tBMA) at the surface of the nanodiamonds, which resulted in obtaining ND-PtBMA hybrids. The grafted chain thickness, estimated from X-ray photoelectron spectroscopy (XPS), was found to increase linearly with respect to time before reaching a plateau value of ca. 2 nm. These nanoobjects were further hydrolyzed into ND-PMAA (where PMAA is the poly(methacrylic acid) graft) and further decorated by bovine serum albumin through the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling procedure.

Introduction Fluorescent nanodiamonds (fNDs) have recently attracted a growing interest for applications in physics, chemistry and biology because of intrinsic fluorescence related to optically active nitrogen-vacancy (NV) defects.1 They are prepared by diamond irradiation and subsequent annealing, performed before or after milling of high-pressure, high-temperature diamond grit, which converts isolated substitutional nitrogen atoms (C centers) into fluorescent NV centers. fNDs show very intense near-infrared fluorescence with no photobleaching, sufficient for single-particle tracking within a cell. After preliminary investigations, fNDs display a low cytotoxicity. Yu et al.2 found that cell viability only diminished slightly upon the addition of relatively large quantities of fNDs. In biology, fNDs, with exceptional photostable NV centers and low potential cytotoxicity and human cytotoxicity, open up many applications in single molecule imaging and tracking2,3 and for quantitative aspects of biochemistry and living processes. In this prospect, colloidal fND bioconjugation is the first step toward several biological applications (biolabeling, biosensor, diagnostic, biochemical analysis, etc.), as for quantum dot (QD) labels,4 first reported by Alivisatos’ group.5 As the light emission results from defect sites within the core of the fND, the surface of the particle can be modified and bioconjugation *Corresponding author. (M.M.C.) Phone: þ33 1 57 27 68 63; e-mail: [email protected]. (C.M.) Phone: þ33 157 27 68 76; e-mail: [email protected]. (J.P.B.) Phone: þ33 1 47 09 31 34; e-mail: [email protected]. (1) Gruber, A.; Dr€abenstedt, A.; Tietz, C.; Fleury, L.; Wrachtrup, J.; von Borczyskowski, C. Science 1997, 276, 2012. (2) Yu, S.; Kang, M.; Chang, H.; Chen, K.; Yu, Y. J. Am. Chem. Soc. 2005, 21, 17604. (3) Neugart, F.; Zappe, A.; Jelezko, F.; Tietz, C.; Boudou, J. P.; Krueger, A.; Wrachtrup, J. Nano Lett. 2007, 7, 3588. (4) Smith, A. M.; Duan, H.; Mohs, A. M.; Nie, S. Adv. Drug Delivery. Rev. 2008, 60, 1226. (5) Bruchez, M. Jr; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013.

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performed without altering the fluorescence signal. Previous fND bioconjugations have been performed via - physical adsorption;6,7 - covalent immobilization, which relies upon the functional groups introduced by acid oxidation with carboxylic group 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction;8,9 - fND encapsulation with hydroxyl or amino group bearing polymers, which give a stable suspension during peptide grafting either via hydroxyl group silanization or amine group reaction with a maleimido function.10 These previously reported methods present drawbacks such as weak and uncontrolled linkage and increase of the particle size by agglomeration. Furthermore, classical methods are time-consuming and operate under harsh conditions.11 Although plasma chemistry has proved to be fast and effective for modifying diamond prior to protein immobilization,12 dry plasma treatment is hardly applicable to individual diamond nanocrystals piled up as a thin layer or a bed, hence the importance to find a new route to modify the surface of spatially isolated nanodiamonds well dispersed in a liquid media. In the present work, diazonium salts were used for the robust and well controlled grafting of surface functional groups on fNDs dispersed in water media. (6) Kong, X. L.; Huang, L. C. L.; Hsu, C. M.; Chen, W. H.; Han, C. C.; Chang, H. C. Anal. Chem. 2005, 77, 259. (7) Chung, P. H.; Perevedentseva, E.; Tu, J. S.; Chang, C. C.; Cheng, C. L. Diamond Relat. Mater. 2006, 15, 622. (8) Fu, C.; Lee, H.; Chen, K.; Lim, T.; Wu, H.; Lin, P.; Wei, P.; Tsao, P.; Chang, H.; Fann, W. Proc. Natl Acad. Sci. U.S.A. 2007, 104, 727. (9) Chang, C.-K.; Wu, C.-C.; Wang, Y.-S.; Chang, H.-C. Anal. Chem. 2008, 80, 3791. (10) Vial, S.; Mansuy, C.; Sagan, S.; Irinopoulou, T.; Burlina, F.; Boudou, J. P.; Chassaing, G.; Lavielle, S. ChemBioChem 2008, 9, 2113. (11) Li, L.; Davidson, J. L.; Lukehart, C. M. Carbon 2006, 44, 2308. (12) Coffinier, Y.; Szunerits, S.; Jama, C.; Desmet, R.; Melnyk, O.; Marcus, B.; Gengembre, L.; Payen, E.; Delabouglise, D.; Boukherroub, R. Langmuir 2007, 23, 4494.

Published on Web 07/27/2009

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Figure 1. Surface modification of fNDs (gray sphere) by electroless grafting of a bromoethyl aryl group derived from BF4-,þN2-C6H4CH(CH3)-Br diazonium salt (step 1) followed by surface-confined ATRP of tBMA (step 2). Preparation of fND-PMAA hybrids by hydrolysis of fND-PtBMA (step 3). Covalent attachment of BSA to fND-PMAA via the NHS/EDC activation (steps 4 and 5).

The advantage of such a procedure is that it ensures a soft and rapid route toward grafting atom transfer radical polymerization (ATRP) initiators for the controlled growth of polymer at the nanodiamond surface. Since fND dispersions flocculate by addition of salts at an ionic strength as low as 50 mM, the grafting protocol has been carefully adapted to avoid any flocculation while fNDs dispersed in water are modified under ultrasound by slow introduction of diazonium salt. As shown in Figure 1, and in a similar approach that we have devised for obtaining nitrophenyl-functionalized diamond nanoparticles,13 fNDs were modified, in very mild conditions, by the electroless attachment of brominated aryl groups derived from the parent 4-(1-bromoethyl)benzene diazonium salt: BF4-,þN2C6H4-CH(CH3)-Br (BrEB diazonium salt). It is likely that, because of the existence of surface sp2 carbon atom types as judged by X-ray photoelectron spectroscopy (XPS),13 the aryl groups attach spontaneously and form, according to Toupin and B
elanger,14 the following interfacial molecular joints: -sp2C-C6 H4 -CHðCH3 ÞBr and -sp2C-N¼ N-C6 H4 -CHðCH3 ÞBr The modified fND particles (hereafter fND-Br) served as macroinitiators for ATRP of tert-butyl methacrylate (tBMA). The resulting hairy fND-PtBMA nanoparticles were further hydrolyzed to obtain the carboxylated nanoparticles fND-PMAA (poly(methacrylic acid)-functionalized fNDs), which served for binding bovine serum albumine via the N-hydroxysuccinimide/ 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC) coupling procedure. The rationale for preparing hairy nanodiamond platforms for the immobilization of proteins is that the introduction of polymers directly onto nanoparticles ensures a (13) Mangeney, C.; Qin, Z.; Dahoumane, S. A.; Adenier, A.; Herbst, F.; Boudou, J. P.; Pinson, J.; Chehimi, M. M. Diamond Relat. Mater. 2008, 17, 1881. (14) Toupin, M.; B
elanger, D. J. Phys. Chem. C 2007, 111, 5394. (15) Tomczaka, N.; Ja
nczewski, D.; Hana, M.; Julius Vancso, G. Prog. Polym. Sci. 2009, 34, 393.

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better stability for the nanosupports.15 Additionally, by using polymeric spacers, multiple and diverse chemical functionalities can be introduced at the surface. Moreover, and as far as the nanomedicine field is concerned, most of the efforts were put on CdS QDs for the preparation of QD/polymer hybrids15 and quasi nothing, to the best of our knowledge, on nanodiamonds. Therefore, the aim of our actual preliminary piece of work is to fill this gap and bring hairy nanodiamonds to the field of nanomedicine via a fundamental and exploratory approach. The hairy nanodiamonds prepared and postmodified so far, together with their precursors, were characterized by transmission electron microscopy (TEM) under lattice imaging contrast conditions to distinguish the amorphous grafted polymer shell from the underlying crystalline nanoparticles; by XPS to monitor surface chemical modifications by initiator and polymeric species; and by Fourier transform infrared (FT-IR) to give evidence of an effective reduction of BrEB diazonium salt on fND, growth of PtBMA, and its modification by hydrolysis and protein binding.

Experimental Section Materials. A nanodiamond dispersion in pure water (van Moppes SYP 0-0.05 GAF) contained 81.42 carats/kg (i.e., 16.284 mg/mL). SYP 0-0.05 nanodiamonds contain ∼ 200 ppm of nitrogen and have a nominal size of 50 nm. The suspension was sonicated for 5 min before use. BF4- þN2-C6H4-CH(CH3)Br diazonium salt was synthesized as described by Matrab et al.16,17

Electroless Grafting of Aryl Layer: Preparation of fNDBr. Electroless grafting of BrEB to fNDs was achieved in the as-received fND-water suspension (at pH 5.5). BrEB diazonium salt was added to 2 mL of fND aqueous suspension (20 mg/mL) so that its initial concentration was 5.10-2 M. The mixture was sonicated for 15 min then stirred for 1 h at room temperature. The BrEB-modified fNDs (hereafter fND-Br’s) were cleaned in distilled water by five centrifugation-redispersion cycles. (16) Matrab, T.; Chehimi, M. M.; Perruchot, C.; Adenier, A.; Guillez, V.; Save, M.; Charleux, B.; Cabet-Deliry, E.; Pinson, J. Langmuir 2005, 21, 4686. (17) Matrab, T.; Save, M.; Charleux, B.; Pinson, J.; Cabet-Deliry, E.; Adenier, A.; Chehimi, M. M.; Delamar, M. Surf. Sci. 2007, 601, 2357.

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Surface-Confined ATRP: Preparation of fND-PtBMA. The fND-Br macroinitiator was used to surface-initiate ATRP of tBMA at 90 °C in toluene under a stream of argon in order to obtain fND-PtBMA hybrids. The mass of macroinitiators was 30 mg, and the monomer, N,N,N0 ,N00 ,N00 -pentamethyldiethylenetriamine (PMDETA) ligand, and CuCl catalyst were in the relative concentrations 100:1:1 (tBMA quantity was 61.5 mmol). ATRP was conducted for a period ranging from 0.5 to 20 h.

Preparation of ND-PMAAs and Their Modification by Bovine Serum Albumin (BSA). Hydrolysis of fND-PtBMA was conducted with 170 mg of fND-PtBMA in a chloroform (24 mL)/CF3COOH (8 mL) mixture at 25 °C under argon, for 24 h. The activation of fND-PMAA by NHS/EDC was conducted in a phosphate buffer saline (PBS) solution for 2 h using a suspension of fND-PMAA of 10 mg/mL. The concentrations of NHS and EDC were 0.2 and 0.1 M, respectively. The N-succinimidyl ester-functionalized products were cleaned in PBS and 50 mg of these nanocomposites were further incubated in 5 mL of 400 μg/mL solution of BSA. Instrumentation. Infrared absorption spectra were recorded on a Magna-860 FT-IR spectrometer (Nicolet Instrument Corp., Madison, WI-USA), at 4 cm-1 spectral resolution. Untreated and treated fNDs were dried and compressed to KBr pellets. XPS spectra were recorded using a Thermo VG Scientific ESCALAB 250 system fitted with a microfocused, monochromatic Al KR X-ray source (hν = 1486.6 eV; spot size = 650 μm; power = 15 kV  200 W). The pass energy was set at 150 and 40 eV for the survey and the narrow regions, respectively. An electron flood gun was used, under a 2.10-8 mBar partial pressure of argon, for static charge compensation. These conditions resulted in negative but uniform static charge. Spectral calibration was determined by setting the main C1s component at 285 eV. The surface composition was determined using the integrated peak areas and the corresponding Scofield sensitivity factors corrected for the analyzer transmission function. TEM analyses were performed on a Tecnai F-20ST (FEI) field emission gun transmission microscope equipped with supertwin polar pieces and a Gatan Imaging Filter/Electron Energy Loss Spectrometer, and operated at 200 kV. The point-to-point resolution is 0.28 nm, which can be experimentally extended to less than 0.15 nm, thanks to the coherency of the field emission gun. The samples, prepared by ultrasonic dispersion, were deposited on a copper grid coated with holey carbon. The deposited suspensions were thereafter dried in air prior to TEM observations. To avoid any electron irradiation damage that could induce allotropic transformations of carbon materials, reduced beam intensity was used.

Results and Discussion Transmission Electron Microscopy. The morphology and structure of the fNDs, before and after growth of polymer chains, were examined. Many grains were observed close to the Scherzer defocus, but no precise zone axis orientations were found and hence no atomic structure was recorded. For the purpose of the present study, lattice imaging conditions were proved to be relevant enough, in particular for imaging {111} planes (Figure 2). We systematically observed that fND {111} planes were visible from the core to the edge of pure fND grains (Figure 2a) while the surface of fND-PtBMA hybrids appeared to be systematically covered with an amorphous 1-2 nm thick layer (Figure 2b). A statistical comparison of fNDs and fND-PtBMA lattice imaging bright-field images taken above and below the minimum contrast condition indicates clearly that this edge structure does not correspond to Fresnel fringes, particularly numerous in images recorded via a FEG TEM, but to a very low Z coating, compatible with a polymer layer. This is consistent with additional electron energy loss spectrometry (EELS) analyses that Langmuir 2009, 25(17), 9633–9638

Figure 2. Lattice imaging TEM images of the bare fND (a) and of the fND-PtBMA hybrid (b). The inset is an enlargement showing (a) the individual fND lattice planes, which are clearly visible and not affected from the core to the very edge of the grain, and (b) the non crystalline low Z edge layer, which is consistent with a 2 ( 1 nm (5 to 15 d111) shell of fND-PtBMA. On the left-hand side of panel b, two diamond nanoparticles are aggregated (presence of Moir
e fringes). This aggregation would result from either the drying after suspension deposition on the TEM grid or pre-existing polymer chain bridging between the two particles.

showed the presence of carbon and oxygen, which are present in PtBMA. However, if these TEM observations strongly suggest the presence of a polymer layer on the surface of nanodiamonds, they are not appropriate to determine the exact chemical nature of the polymer itself. X-ray Photoelectron Spectroscopy. The main peaks detected in XPS analysis of untreated and modified fNDs are C1s, O1s and Br3d, centered at 285, 533, and 71 eV, respectively. Br3d testifies for the grafting of the aryl groups onto fNDs and also for the living character of the polymer chains. The C1s regions were peak-fitted in order to determine the contribution of the ester carbon species (Cester) to the total C1s peak areas. Figure 3a displays peak-fitted high-resolution C1s regions of fND-PtBMA hybrids prepared after 2 and 4 h of polymerization time. The spectra give evidence of the screening of fNDs by the polymer chains, with the disappearance of the complex structure observed elsewhere for bare fNDs.13 The spectra are fitted with four components centered at ∼285, 286.5, 288, and 289 eV corresponding to C-C/C-H, C-O, CdO, and O-CdO chemical environments, respectively. For a short polymerization time, a fifth component was added at 291.5 eV in order to account for the so-called πfπ* , shakeup . satellite, which is characteristic of aromatic aryl groups derived from the parent diazonium salt.16-18 It is important to note the increase of the relative peak area of the C1s component from the ester groups (Cester), which is characteristic of polymethacrylates. Figure 3b displays plots of O/C and Cester/Br atomic ratios versus polymerization time. It shows that upon ATRP, O/C atomic ratios increase from 0.17 to 0.20 due to the addition of methacrylate repeat units, which are oxygen-rich compared to the underlying fNDs. However, the ratio still remains below the theoretical value of 0.25 calculated for pure PtBMA, suggesting that the underlying fNDs particles are detected by XPS through the organic polymer top layer. The plot of Cester/Br atomic ratio versus polymerization time shows a similar behavior with a progressive increase until a plateau is reached after approximately 4 h of polymerization. Considering the Cester/Br ratio as a rough degree of polymerization (DP) together with molecular weight of tBMA repeat units (MtBMA = 142 g/mol), the mean molecular weight of the chains (18) Matrab, T.; Chehimi, M. M.; Boudou, J. P.; Benedic, F.; Wang, J.; Naguib, N. N.; Carlisle, J. A. Diamond Relat. Mater. 2006, 15, 639.

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Figure 3. (a, left) Peak-fitted high resolution C1s regions of fND-PtBMA after 2 h and 4 h of ATRP. (b, right) Atomic O/C and Cester/Br ratios for fND-PtBMA vs polymerization time.

tethered to fND nanoparticles can be estimated as Mn = DP  MtBMA.17 An Mn value of ca. 4000 g 3 mol-1 is calculated after 4 h polymerization. The thickness of the polymer grafts could also be assessed by XPS using the approach of Strohmeier19 as described in the Supporting Information SI1. The PtBMA layer growth is linear with respect to time up to approximately 4 h. At longer reaction times, the increase in layer thickness seems to slow down. A similar behavior is observed when plotting the polymer shell thickness versus the molecular weight of the polymer. A linear relationship between thickness and molecular weight is observed at polymerization times less than 4 h, as shown in Figure 4. From the molecular weight (Mn) of the surface attached polymer chains and the film thickness (d), the graft density Γ of the chains could be obtained using Γ ¼ N A 3 d 3 F 3 100=M n For this calculation the unknown material density of the polymer shell around fND was assumed to be close to the bulk density of the same material (1.022 g/cm3, Aldrich source). These calculations yield an average grafting density of 0.25 chains/nm2 where the area in square nanometers is that of the underlying fNDs. This value was constant throughout the polymerization except at longer polymerization times (20 h) where significant deviations were observed. The polymer shell thickness and molecular weight increase linearly as a function of time, while the graft density remains constant until 4 h of polymerization, a situation similar to that reported by Jones et al.20 in their study of the growth of polymer brushes without any use of the deactivator Cu2þ. This indicates (19) Strohmeier, B. R. J. Vac. Sci. Technol. A 1989, 7, 3238. (20) Jones, D. M.; Brown, A. A.; Huck, W. T. S. Langmuir 2002, 18, 1265.

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Figure 4. Average thickness of PtBMA, estimated by XPS (as described in the Supporting Information SI1) as a function of the Mn of the tethered polymer chains (r2 = 0.998).

that the rate of exchange between the active and dormant chain was sufficiently fast that all chains grew slowly and at the same rate with the number of chains remaining constant. From the grafting density, and assuming that the consumed monomers form the polymer chains, one can estimate at each time the residual monomers in the ATRP medium and hence conversion. Again, Figure 5 shows that conversion varies linearly with time until 4 h (Figure 5a). In addition, the molecular weight of PtBMA varies linearly with conversion, until 4 h (Figure 5b). This XPS analysis suggests continued polymer growth at least during the first 4 h polymerization period. However, for extended time range, the kinetic data are indicative of a loss of active chain ends, probably, by termination reactions. The loss of some Langmuir 2009, 25(17), 9633–9638

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Figure 5. Growth of PtBMA on nanodiamond surface: (a) conversion versus time (h) and (b) Mn versus conversion.

Figure 6. FT-IR spectra of modified fNDs and the reference materials (fNDs and the BrEB diazonium salt).

control in this polymerization system is compensated by the simplicity of the protocol and the possibility of growing polymer grafts from NDs. Fourier Transform Infrared Spectroscopy. Figure 6 compares the IR spectra of BrEB diazonium salt and untreated and modified fND particles. In the 4000-1400 cm-1 range, the IR spectrum of the free diazonium salt exhibits several well-resolved Langmuir 2009, 25(17), 9633–9638

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peaks, including four characteristic bands at 2940, 2850, 2260, and 1580 cm-1. The two medium intensity bands at 2940 and 2850 cm-1 correspond to the asymmetric (νas(C-H)) and symmetric (νs(C-H)) C-H stretching vibrations, respectively. The sharp one at 2260 cm-1 is assigned to the NtN stretching vibration, whereas at 1580 cm-1, one can observe the phenyl cycle vibration. The fNDs spectrum shows a different pattern with two broad bands at 1623 and 1790 cm-1. The former is attributed to the O-H bending of physically adsorbed water molecules and the latter to the CdO stretching vibrations of anhydride carboxylic acid from the surface of untreated fNDs. The spectrum of fND-Br displays a combined pattern of the pure fNDs spectrum (with bands at 1623 and 1790 cm-1) and the attached C6H4-CH(CH3)-Br moieties (with bands at 2925, 2850, and 1580 cm-1). Furthermore, the absence of the 2260 cm-1 band (previously assigned to the NtN stretching vibration) in both product spectra indicates the loss of the diazonium group which suggests the covalent attachment of aryl initiators to fNDs. In the case of fND-PtBMA, one can notice strong modifications of the IR spectra with the appearance of a sharp CdO ester band at 1720 cm-1 together with a significant increase of the band intensities in the aliphatic region. These features prove that the polymerization process was successful on fND-Br. The hairy fND-PtBMA platforms were further tested for specific biomedical applications. As an example, we evaluated their capabilities to carry proteins, BSA being chosen as a model biomacromolecule. For such application, it is more interesting to have PMAA chains covering the fNDs particles, the pendant carboxylic acid groups being easily activated using NHS in the presence of EDC. As this polymer is difficult to grow using ATRP because of the possible complexation of the copper catalyst by the carboxylate groups, it is usually obtained by hydrolysis of a preformed PtBMA polymer. The fND-PtBMA hybrids were therefore hydrolyzed in order to obtain functional PMAA grafts which in turn were activated using NHS/EDC in order to attach BSA. The hydrolysis of ND-PtBMA was successful as evidenced by the spectra of the generic fND-PMAA. Indeed, the intensity of the sharp ester band (at 1720 cm-1) from fND-PtBMA has strongly decreased (compared to those of the fNDs bands) while a new band at 1700 cm-1, assigned to CdO stretching vibrations in COOH groups, has appeared. Furthermore, the bands corresponding to the stretching vibrations of C-H bonds (at 2940 and 2850 cm-1) are much less intense, and the elongation band of the C-O bonds (1255 cm-1) has vanished. Concerning the attachment of BSA to fND-PMAA by the NHS/EDC coupling procedure, it can be characterized by the disappearance of the CdO stretching vibrations due to COOH groups, and the concomitant appearance of two new bands centered at 1539 and 1650 cm-1, assigned to the amide groups from the protein. It is also worth noting the presence of a pronounced shoulder at ca. 3250 cm-1, due to N-H vibrations, from protein amine and amide groups. Therefore, this IR analysis permits to monitor the chemical changes that occur at the surface of fNDs as a result of ATRP initiator grafting, attachment of PtBMA, their hydrolyis into PMAA chains and finally the functionalization of the hairy particles by BSA.

Conclusions In this work we evaluated the potentialities offered by the diazonium salt chemistry to provide new ATRP initiators for modifying the surface of a nanodiamond. The diazonium salt DOI: 10.1021/la9009509

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BF4- þN2-C6H4-CH(CH3)Br was reduced at the surface of nanodiamonds via the spontaneous, electroless chemical method, which was conducted in a very friendly and simple way. The as-modified fNDs (fND-Br) were further used as macroinitiators for the surface-confined ATRP of tBMA. In order to further functionalize the hairy fND particles (fND-PtBMA) by peptides, these hybrids were hydrolyzed to obtain first fND-PMAA nanoparticles, and in turn were activated using NHS/EDC for the covalent bonding of BSA. The present work firmly highlights the interest of using aryl diazonium salts to modify nanodiamonds in a very simple and friendly manner. Given the wide panel of functional groups that can be attached to diazonium salts, clearly one can design carbon surfaces on demand, suitable for a variety of domains such as nanomedicine, electrochemistry, and aerospace. The newly

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prepared protein-functionalized hairy nanodiamonds pave the way toward the utilization of nanodiamonds as bioactive platforms and hence enlarge the panel of potential applications of these carbon nanomaterials in the field of biotechnology. Acknowledgment. The authors wish to thank Mr. C. Bangerter (van Moppes, Switzerland) for the gift of a sample of SYP 0-0.05 GAF. J.P.B. and A.T. gratefully acknowledge the support of the European project Nano4Drugs under FP6 Contract LSHCCT-2005-019102. Prof. P. Curmi (INSERM/UEVE U829, Evry, France) is acknowledged for helpful discussion. Supporting Information Available: XPS estimation of PtBMA thickness on fND. This material is available free of charge via the Internet at http://pubs.acs.org.

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