DOI: 10.1021/la9009509 9633Langmuir 2009, 25(17), 9633–9638 Published on Web 07/27/2009

pubs.acs.org/Langmuir

© 2009 American Chemical Society

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 ofNormal 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 newbioactive 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 thediazonium salt BF4

-,þN2-C6H4-CH(CH3)-Br. The strongly bound aryl groups -C6H4-CH(CH3)-Br efficientlyinitiated the ATRP of tert-butyl methacrylate (tBMA) at the surface of the nanodiamonds, which resulted in obtainingND-PtBMA hybrids. The grafted chain thickness, estimated from X-ray photoelectron spectroscopy (XPS), was foundto increase linearly with respect to time before reaching a plateau value of ca. 2 nm. These nanoobjects were furtherhydrolyzed into ND-PMAA (where PMAA is the poly(methacrylic acid) graft) and further decorated by bovine serumalbumin through the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) couplingprocedure.

Introduction

Fluorescent nanodiamonds (fNDs) have recently attracteda growing interest for applications in physics, chemistry andbiology because of intrinsic fluorescence related to opticallyactive nitrogen-vacancy (NV) defects.1 They are prepared bydiamond irradiation and subsequent annealing, performedbefore or after milling of high-pressure, high-temperaturediamond grit, which converts isolated substitutional nitrogenatoms (C centers) into fluorescent NV centers. fNDs showvery intense near-infrared fluorescence with no photobleach-ing, sufficient for single-particle tracking within a cell. Afterpreliminary investigations, fNDs display a low cytotoxicity.Yu et al.2 found that cell viability only diminished slightly uponthe addition of relatively large quantities of fNDs. In biology,fNDs, with exceptional photostable NV centers and lowpotential cytotoxicity and human cytotoxicity, open up manyapplications in single molecule imaging and tracking2,3 and forquantitative aspects of biochemistry and living processes. Inthis prospect, colloidal fND bioconjugation is the first steptoward several biological applications (biolabeling, biosensor,diagnostic, biochemical analysis, etc.), as for quantum dot(QD) labels,4 first reported by Alivisatos’ group.5 As the lightemission results from defect sites within the core of the fND,the surface of the particle can be modified and bioconjugation

performed without altering the fluorescence signal. PreviousfND bioconjugations have been performed via

- physical adsorption;6,7

- covalent immobilization, which relies upon the func-tional groups introduced by acid oxidation withcarboxylic group 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated coupling reaction;8,9

- fND encapsulation with hydroxyl or amino groupbearing polymers, which give a stable suspensionduring peptide grafting either via hydroxyl groupsilanization or amine group reaction with amaleimidofunction.10

These previously reported methods present drawbacks such asweak and uncontrolled linkage and increase of the particle size byagglomeration. Furthermore, classical methods are time-consum-ing and operate under harsh conditions.11 Although plasmachemistry has proved to be fast and effective for modifyingdiamond prior to protein immobilization,12 dry plasma treatmentis hardly applicable to individual diamond nanocrystals piledup as a thin layer or a bed, hence the importance to find a newroute to modify the surface of spatially isolated nanodiamondswell dispersed in a liquid media. In the present work, diazoniumsalts were used for the robust and well controlled graftingof surface functional groups on fNDs dispersed in water media.

*Corresponding author. (M.M.C.) Phone: þ33 1 57 27 68 63; e-mail:chehimi@univ-paris-diderot.fr. (C.M.) Phone: þ33 157 27 68 76; e-mail:mangeney@univ-paris-diderot.fr. (J.P.B.) Phone: þ33 1 47 09 31 34; e-mail:jpb.cnrs@free.fr.(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. DrugDelivery. Rev. 2008,

60, 1226.(5) Bruchez, M. Jr; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science

1998, 281, 2013.

(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.

9634 DOI: 10.1021/la9009509 Langmuir 2009, 25(17), 9633–9638

Letter Dahoumane et al.

The advantage of such a procedure is that it ensures a soft andrapid route toward grafting atom transfer radical polymerization(ATRP) initiators for the controlled growth of polymer at thenanodiamond surface.

Since fNDdispersions flocculate by addition of salts at an ionicstrength as low as 50mM, the grafting protocol has been carefullyadapted to avoid any flocculation while fNDs dispersed in wateraremodified under ultrasoundby slow introduction of diazoniumsalt. As shown in Figure 1, and in a similar approach that we havedevised for obtaining nitrophenyl-functionalized diamond nano-particles,13 fNDs were modified, in very mild conditions, by theelectroless attachment of brominated aryl groups derived fromthe parent 4-(1-bromoethyl)benzene diazonium salt: BF4

-,þN2-C6H4-CH(CH3)-Br (BrEB diazonium salt). It is likely that,because of the existence of surface sp2 carbon atom types asjudged by X-ray photoelectron spectroscopy (XPS),13 the arylgroups attach spontaneously and form, according to Toupin andB�elanger,14 the following interfacial molecular joints:

-sp2C-C6H4-CHðCH3ÞBr and

-sp2C-N¼ N-C6H4-CHðCH3ÞBrThe modified fND particles (hereafter fND-Br) served as

macroinitiators for ATRP of tert-butyl methacrylate (tBMA).The resulting hairy fND-PtBMA nanoparticles were furtherhydrolyzed to obtain the carboxylated nanoparticles fND-PMAA(poly(methacrylic acid)-functionalized fNDs), which served forbinding bovine serum albumine via the N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC)coupling procedure. The rationale for preparing hairy nanodia-mond platforms for the immobilization of proteins is that theintroduction of polymers directly onto nanoparticles ensures a

better stability for the nanosupports.15 Additionally, by usingpolymeric spacers, multiple and diverse chemical functionalitiescan be introduced at the surface. Moreover, and as far as thenanomedicine field is concerned, most of the efforts were put onCdSQDs for the preparation of QD/polymer hybrids15 and quasinothing, to the best of our knowledge, on nanodiamonds. There-fore, the aim of our actual preliminary piece of work is to fill thisgap and bring hairy nanodiamonds to the field of nanomedicinevia a fundamental and exploratory approach.

The hairy nanodiamonds prepared and postmodified so far,togetherwith their precursors, were characterized by transmissionelectron microscopy (TEM) under lattice imaging contrast con-ditions to distinguish the amorphous grafted polymer shell fromthe underlying crystalline nanoparticles; by XPS to monitorsurface chemicalmodifications by initiator and polymeric species;and by Fourier transform infrared (FT-IR) to give evidence of aneffective reduction of BrEB diazonium salt on fND, growth ofPtBMA, 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.,16.284mg/mL). SYP 0-0.05 nanodiamonds contain∼ 200 ppmofnitrogen and have a nominal size of 50 nm. The suspension wassonicated for 5 min before use. BF4

- þN2-C6H4-CH(CH3)Brdiazonium salt was synthesized as described by Matrab et al.16,17

Electroless Grafting of Aryl Layer: Preparation of fND-

Br. Electroless grafting of BrEB to fNDs was achieved in theas-received fND-water suspension (at pH 5.5). BrEB diazoniumsalt was added to 2 mL of fND aqueous suspension (20 mg/mL)so that its initial concentration was 5.10-2 M. The mixturewas sonicated for 15min then stirred for 1 h at room temperature.The BrEB-modified fNDs (hereafter fND-Br’s) were cleaned indistilled water by five centrifugation-redispersion cycles.

Figure 1. Surface modification of fNDs (gray sphere) by electroless grafting of a bromoethyl aryl group derived from BF4-,þN2-C6H4-

CH(CH3)-Br diazonium salt (step 1) followed by surface-confined ATRP of tBMA (step 2). Preparation of fND-PMAA hybrids byhydrolysis of fND-PtBMA (step 3). Covalent attachment of BSA to fND-PMAA via the NHS/EDC activation (steps 4 and 5).

(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.

(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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Dahoumane et al. Letter

Surface-Confined ATRP: Preparation of fND-PtBMA.The fND-Br macroinitiator was used to surface-initiate ATRPof tBMA at 90 �C in toluene under a stream of argon in orderto obtain fND-PtBMA hybrids. The mass of macroinitiators was30mg, and themonomer,N,N,N0,N0 0,N0 0-pentamethyldiethylene-triamine (PMDETA) ligand, and CuCl catalyst were in therelative 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-PtBMAwas conducted with 170 mg of fND-PtBMA in a chloroform(24 mL)/CF3COOH (8 mL) mixture at 25 �C under argon, for24h.Theactivationof fND-PMAAbyNHS/EDCwas conductedin a phosphate buffer saline (PBS) solution for 2 h using asuspension of fND-PMAA of 10 mg/mL. The concentrations ofNHS and EDC were 0.2 and 0.1 M, respectively. The N-succini-midyl ester-functionalized products were cleaned in PBS and50 mg of these nanocomposites were further incubated in 5 mLof 400 μg/mL solution of BSA.

Instrumentation. Infrared absorption spectra were recordedon aMagna-860 FT-IR spectrometer (Nicolet Instrument Corp.,Madison,WI-USA), at 4 cm-1 spectral resolution.Untreated andtreated fNDs were dried and compressed to KBr pellets.

XPS spectra were recorded using a Thermo VG ScientificESCALAB 250 system fitted with a microfocused, monochro-matic Al KRX-ray source (hν= 1486.6 eV; spot size = 650 μm;power = 15 kV � 200 W). The pass energy was set at 150 and40 eV for the survey and the narrow regions, respectively. Anelectron flood gunwas used, under a 2.10-8mBar partial pressureof argon, for static charge compensation. These conditionsresulted innegative but uniform static charge. Spectral calibrationwasdeterminedby setting themainC1s component at 285 eV.Thesurface composition was determined using the integrated peakareas and the corresponding Scofield sensitivity factors correctedfor the analyzer transmission function.

TEM analyses were performed on a Tecnai F-20ST (FEI) fieldemission gun transmission microscope equipped with supertwinpolar pieces and a Gatan Imaging Filter/Electron Energy LossSpectrometer, and operated at 200 kV. The point-to-point resolu-tion is 0.28 nm, which can be experimentally extended to less than0.15 nm, thanks to the coherency of the field emission gun. Thesamples, prepared by ultrasonic dispersion, were deposited on acopper grid coated with holey carbon. The deposited suspensionswere thereafter dried in air prior to TEM observations. To avoidany electron irradiation damage that could induce allotropictransformations of carbon materials, reduced beam intensitywas used.

Results and Discussion

Transmission Electron Microscopy. The morphology andstructure of the fNDs, before and after growth of polymer chains,were examined. Many grains were observed close to the Scherzerdefocus, but no precise zone axis orientations were found andhence no atomic structure was recorded. For the purpose of thepresent study, lattice imaging conditions were proved to berelevant enough, in particular for imaging {111} planes (Figure 2).

We systematically observed that fND {111} planes were visiblefrom the core to the edge of pure fND grains (Figure 2a) while thesurface of fND-PtBMA hybrids appeared to be systematicallycovered with an amorphous 1-2 nm thick layer (Figure 2b).A statistical comparison of fNDs and fND-PtBMA latticeimaging bright-field images taken above and below the minimumcontrast condition indicates clearly that this edge structure doesnot correspond to Fresnel fringes, particularly numerous inimages recorded via a FEG TEM, but to a very low Z coating,compatible with a polymer layer. This is consistent with addi-tional electron energy loss spectrometry (EELS) analyses that

showed the presence of carbon and oxygen, which are present inPtBMA. However, if these TEM observations strongly suggestthe presence of a polymer layer on the surface of nanodiamonds,they are not appropriate to determine the exact chemical nature ofthe polymer itself.X-ray Photoelectron Spectroscopy. The main peaks de-

tected in XPS analysis of untreated and modified fNDs are C1s,O1s and Br3d, centered at 285, 533, and 71 eV, respectively. Br3dtestifies for the grafting of the aryl groups onto fNDs and also forthe living character of the polymer chains.

The C1s regions were peak-fitted in order to determine thecontribution of the ester carbon species (Cester) to the total C1speak areas. Figure 3a displays peak-fitted high-resolution C1sregions of fND-PtBMA hybrids prepared after 2 and 4 h ofpolymerization time. The spectra give evidence of the screening offNDs by the polymer chains, with the disappearance of thecomplex structure observed elsewhere for bare fNDs.13 Thespectra are fitted with four components centered at ∼285, 286.5,288, and 289 eV corresponding to C-C/C-H, C-O, CdO, andO-CdO chemical environments, respectively. For a short polym-erization time, a fifth componentwas added at 291.5 eV in order toaccount for the so-called πfπ* , shakeup . satellite, which ischaracteristic of aromatic aryl groups derived from the parentdiazonium salt.16-18 It is important to note the increase of therelative 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 ratiosversus polymerization time. It shows that upon ATRP, O/Catomic ratios increase from 0.17 to 0.20 due to the addition ofmethacrylate repeat units, which are oxygen-rich compared to theunderlying fNDs. However, the ratio still remains below thetheoretical value of 0.25 calculated for pure PtBMA, suggestingthat the underlying fNDs particles are detected by XPS throughthe organic polymer top layer.

The plot of Cester/Br atomic ratio versus polymerization timeshows a similar behavior with a progressive increase until aplateau is reached after approximately 4 h of polymerization.Considering the Cester/Br ratio as a rough degree of polymeriza-tion (DP) together with molecular weight of tBMA repeat units(MtBMA = 142 g/mol), the mean molecular weight of the chains

Figure 2. Lattice imaging TEM images of the bare fND (a) and ofthe fND-PtBMA hybrid (b). The inset is an enlargement showing(a) the individual fND lattice planes, which are clearly visible andnot affected from the core to the very edge of the grain, and (b) thenon crystalline lowZedge layer,which is consistentwith a 2( 1 nm(5 to15d111) shell of fND-PtBMA.On the left-hand sideofpanel b,two diamond nanoparticles are aggregated (presence of Moir�efringes). This aggregation would result from either the drying aftersuspension deposition on the TEM grid or pre-existing polymerchain bridging between the two particles.

(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.

9636 DOI: 10.1021/la9009509 Langmuir 2009, 25(17), 9633–9638

Letter Dahoumane et al.

tethered to fND nanoparticles can be estimated as Mn = DP �MtBMA.

17 AnMn value of ca. 4000 g 3mol-1 is calculated after 4 hpolymerization. The thickness of the polymer grafts could also beassessed by XPS using the approach of Strohmeier19 as describedin the Supporting Information SI1.

The PtBMA layer growth is linear with respect to time up toapproximately 4 h. At longer reaction times, the increase in layerthickness seems to slow down.

A similar behavior is observed when plotting the polymer shellthickness versus the molecular weight of the polymer. A linearrelationship between thickness and molecular weight is observedat polymerization times less than 4 h, as shown in Figure 4.

From the molecular weight (Mn) of the surface attachedpolymer chains and the film thickness (d), the graft density Γ ofthe chains could be obtained using

Γ ¼ NA 3 d 3 F 3 100=Mn

For this calculation the unknown material density of thepolymer shell around fND was assumed to be close to the bulkdensity of the same material (1.022 g/cm3, Aldrich source). Thesecalculations yield an average grafting density of 0.25 chains/nm2

where the area in square nanometers is that of the underlyingfNDs. This value was constant throughout the polymerizationexcept at longer polymerization times (20 h) where significantdeviations were observed.

The polymer shell thickness and molecular weight increaselinearly as a function of time, while the graft density remainsconstant until 4 h of polymerization, a situation similar to thatreported by Jones et al.20 in their study of the growth of polymerbrushes without any use of the deactivator Cu2þ. This indicates

that the rate of exchange between the active and dormant chainwas sufficiently fast that all chains grew slowly and at the samerate with the number of chains remaining constant.

From the grafting density, and assuming that the consumedmonomers form the polymer chains, one can estimate at each timethe residual monomers in the ATRP medium and hence conver-sion. Again, Figure 5 shows that conversion varies linearly withtime until 4 h (Figure 5a). In addition, the molecular weight ofPtBMA varies linearly with conversion, until 4 h (Figure 5b).

This XPS analysis suggests continued polymer growth at leastduring the first 4 h polymerization period. However, for extendedtime range, the kinetic data are indicative of a loss of active chainends, probably, by termination reactions. The loss of some

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/Brratios for fND-PtBMA vs polymerization time.

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

(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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Dahoumane et al. Letter

control in this polymerization system is compensated by thesimplicity of the protocol and the possibility of growing polymergrafts from NDs.Fourier Transform Infrared Spectroscopy. Figure 6 com-

pares the IR spectra of BrEB diazonium salt and untreated andmodified fND particles. In the 4000-1400 cm-1 range, the IRspectrum of the free diazonium salt exhibits several well-resolved

peaks, including four characteristic bands at 2940, 2850, 2260,and 1580 cm-1. The two medium intensity bands at 2940 and2850 cm-1 correspond to the asymmetric (νas(C-H)) and sym-metric (νs(C-H)) C-H stretching vibrations, respectively. Thesharp one at 2260 cm-1 is assigned to the NtN stretchingvibration, whereas at 1580 cm-1, one can observe the phenylcycle vibration.

The fNDs spectrum shows a different pattern with two broadbands at 1623 and 1790 cm-1. The former is attributed to theO-H bending of physically adsorbed water molecules and thelatter to the CdO stretching vibrations of anhydride carboxylicacid from the surface of untreated fNDs.

The spectrum of fND-Br displays a combined pattern of thepure fNDs spectrum (with bands at 1623 and 1790 cm-1) andthe 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) inboth product spectra indicates the loss of the diazonium groupwhich suggests the covalent attachment of aryl initiators to fNDs.

In the case of fND-PtBMA, one can notice strong modifica-tions of the IR spectra with the appearance of a sharp CdO esterband at 1720 cm-1 togetherwith a significant increase of the bandintensities in the aliphatic region. These features prove that thepolymerization process was successful on fND-Br.

The hairy fND-PtBMA platforms were further tested forspecific biomedical applications. As an example, we evaluatedtheir capabilities to carry proteins, BSA being chosen as a modelbiomacromolecule. For such application, it is more interesting tohave PMAA chains covering the fNDs particles, the pendantcarboxylic acid groups being easily activated using NHS inthe presence of EDC. As this polymer is difficult to grow usingATRP because of the possible complexation of the coppercatalyst by the carboxylate groups, it is usually obtained byhydrolysis of a preformed PtBMA polymer. The fND-PtBMAhybrids were therefore hydrolyzed in order to obtain functionalPMAA grafts which in turn were activated using NHS/EDC inorder to attach BSA.

The hydrolysis of ND-PtBMA was successful as evidenced bythe spectra of the generic fND-PMAA. Indeed, the intensity of thesharp ester band (at 1720 cm-1) from fND-PtBMA has stronglydecreased (compared to those of the fNDs bands) while a newband at 1700 cm-1, assigned to CdO stretching vibrations inCOOH groups, has appeared. Furthermore, the bands corre-sponding to the stretching vibrations of C-H bonds (at 2940 and2850 cm-1) are much less intense, and the elongation band of theC-O bonds (1255 cm-1) has vanished.

Concerning the attachment of BSA to fND-PMAA by theNHS/EDC coupling procedure, it can be characterized by thedisappearance of the CdO stretching vibrations due to COOHgroups, and the concomitant appearance of two new bandscentered at 1539 and 1650 cm-1, assigned to the amide groupsfrom the protein. It is also worth noting the presence of apronounced shoulder at ca. 3250 cm-1, due to N-H vibrations,fromprotein amine and amide groups. Therefore, this IR analysispermits to monitor the chemical changes that occur at the surfaceof fNDs as a result of ATRP initiator grafting, attachment ofPtBMA, their hydrolyis into PMAA chains and finally thefunctionalization of the hairy particles by BSA.

Conclusions

In this work we evaluated the potentialities offered by thediazonium salt chemistry to provide new ATRP initiators formodifying the surface of a nanodiamond. The diazonium salt

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

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

9638 DOI: 10.1021/la9009509 Langmuir 2009, 25(17), 9633–9638

Letter Dahoumane et al.

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. Theas-modified fNDs (fND-Br) were further used as macroinitiatorsfor the surface-confined ATRP of tBMA. In order to furtherfunctionalize the hairy fND particles (fND-PtBMA) by peptides,these hybrids were hydrolyzed to obtain first fND-PMAA nano-particles, and in turn were activated using NHS/EDC for thecovalent bonding of BSA.

The present work firmly highlights the interest of using aryldiazonium salts to modify nanodiamonds in a very simple andfriendly manner. Given the wide panel of functional groups thatcan be attached to diazonium salts, clearly one can design carbonsurfaces on demand, suitable for a variety of domains suchas nanomedicine, electrochemistry, and aerospace. The newly

prepared protein-functionalized hairy nanodiamonds pave theway toward the utilization of nanodiamonds as bioactive plat-forms and hence enlarge the panel of potential applications ofthese carbon nanomaterials in the field of biotechnology.

Acknowledgment.Theauthorswish to thankMr.C.Bangerter(vanMoppes, Switzerland) for the gift of a sample of SYP 0-0.05GAF. J.P.B. and A.T. gratefully acknowledge the support ofthe European project Nano4Drugs under FP6 Contract LSHC-CT-2005-019102. Prof. P. Curmi (INSERM/UEVE U829, Evry,France) is acknowledged for helpful discussion.

Supporting Information Available: XPS estimation ofPtBMA thickness on fND. This material is available freeof charge via the Internet at http://pubs.acs.org.