ARTICLE pubs.acs.org/Langmuir
Nanomolar Hydrogen Peroxide Detection Using Horseradish Peroxidase Covalently Linked to Undoped Nanocrystalline Diamond Surfaces Qi Wang,†,‡ Alexander Kromka,§ Jana Houdkova,§ Oleg Babchenko,§ Bohuslav Rezek,§ Musen Li,‡ Rabah Boukherroub,† and Sabine Szunerits*,† †
Institut de Recherche Interdisciplinaire (IRI, USR 3078), Universite de Lille1, Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France ‡ School of Materials Science and Engineering, Shandong University, 73 Jingshi Road, Jinan, Shandong Province, PR China § Institute of Physcis ASCR, Cukrovamicka 10, 16000 Prag, Czech Republic ABSTRACT: In this article, we report on the low-level detection of hydrogen peroxide, a key player in the redox signaling pathway and a toxic product in the cellular system, using a colorimetric solution assay. Amine-terminated undoped nanocrystalline diamond thin films were grown on glass using a linear-antenna microwave plasma CVD process. The diamond surface consists mainly of NH2 termination. The aminated diamond surface was decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HRP immobilization was confirmed by X-ray photoelectron spectroscopy (XPS). The enzymatic activity of immobilized HRP was determined with a colorimetric test based on the HRP-catalyzed oxidation of 2,20 -azino-bis(3-ethylbenzothiazoline-6-sufonic acid (ABTS) in the presence of hydrogen peroxide. The surface coverage of active HRP was estimated to be Γ = 7.3 1013 molecules cm2. The use of the functionalized diamond surface as an optical sensor for the detection of hydrogen peroxide with a detection limit of 35 nM was demonstrated.
1. INTRODUCTION During the past decade, diamond has proven its capability of being a promising material for biochemical applications. This is mainly due to its physicochemical stability and its biocompatibility.14 Although its high cost has limited the use of the material, nowadays the development of diamond growth by chemical vapor deposition (CVD) has enabled the preparation of large-area synthetic diamond films on different substrates at a reasonable cost.5,6 Depending on the growth parameters (gas mixture, temperature, substrate seeding, etc.), different kinds of diamond films that are generally classified according to the crystal grain size as polycrystalline (1 μm), nanocrystalline (100 nm), and ultrananocrystalline (below 10 nm) can be produced.7 The electronic and optical properties of these films strongly depend on the presence of sp2-hybridized carbon atoms as well as embedded impurities. The most widely used form of diamond for biosensing is boron-doped diamond (BDD).815 The intentional introduction of boron increases the conductivity of diamond sufficiently to make electrodes with distinct electrochemical properties. Intrinsic (nominally undoped) diamond films can exhibit conductivity when H-terminated.16,17 However, the covalent grafting of organic moieties typically replaces hydrogen surface atoms and thus the diamond becomes highly resistive and unusable as an electrode material, yet undoped diamond films can be used for optical-based sensing applications. The development of such sensors has lagged behind electrochemical-based detection schemes. r 2011 American Chemical Society
Until now, intrinsic diamond substrates were mainly used in combination with fluorescence-based detection schemes.1820 Undoped single-crystalline (100) diamond has been used by Rezek and Nebel for the construction of DNA arrays where the success of the hybridization event was detected using fluorescence microsopy.18 Hamers et al. investigated the nonspecific adsorption of proteins such as avidin, casein, and fibrinogen on triethylene glycol-modified diamond interfaces.19 Beside these examples, undoped ultrananocrystalline diamond thin films were mainly used as coatings for biological implants.21 In this work, we show that undoped nanocrystalline diamond interfaces can be used for the sensitive detection of hydrogen peroxide using a colorimetric solution assay. The technique represents an interesting alternative to electrochemicalbased detection schemes reported on boron-doped diamond electrodes.8,10,22,23 In this study, horseradish peroxidase HRP catalyzes the reduction of hydrogen peroxide. The oxidized enzyme requires two electrons to be reduced back to its initial state, which can be provided by a mediator present in solution or by direct electron transfer from the diamond electrode to the active center of the enzyme. In the case of direct electron transfer, the active center of the enzyme has to be in an appropriate orientation and an appropriate distance away from the electrical Received: July 29, 2011 Revised: October 26, 2011 Published: November 08, 2011 587
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interface.24 In the case of the colorimetric test presented here, the enzyme is reduced back by 2,20 -azino-bis(3-ethylbenzthiazoline6-sulfonic acid) (ABTS), a peroxidase substrate that has been shown to be suitable for use in ELISA procedures. The reduction process produces a soluble end product that is green in color and can be readily observed spectrophotometrically at 405 nm. This optical H2O2 sensor can be seen as an alternative detection principle compared to the widely used electrochemically based ones. The choice of transparent aminated diamond as an immobilization interface beyond the range of transparent materials (e.g., indium tin oxide, doped SnO2) was motivated by the fact that diamond has shown the strongest binding stability to biomolecules such as DNA.14,20,25,26 Additionally, Rubio-Retama et al. have reported on the interest in synthetic nanocrystalline diamond as a third-generation biosensor support.8 The proximity of the HRP heme groups to boron-doped nanocrystalline diamond electrodes allowed direct electron transfer between them and a calculation of the amount of immobilized HRP. The HRP-modified interface was used as a biosensor for hydrogen peroxide determination with a linear response in the millimolar range. Herein, we will show that one of the advantages of the optical detection scheme is a nanomolar detection limit together with a very simple read-off scheme.
Figure 1. Scheme of HRP immobilization on hydrogenated diamond surfaces. in a protocol similar to that described by H€artl and Stutzmann.10,22 It is based on an aminolysis reaction between the surface NH2 groups and the carboxylic acid groups of the four lysine residues on HRP in the presence of EDC, as shown in Figure 1. The protocol for HRP attachment was achieved by immersing amine-terminated NCD in a 100 μg/L HRP solution in PBS buffer (pH 7.4) for 24 h at room temperature containing NHS (0.1 M) and EDC (2 mM). The interface was then washed with PBS-tween (0.1%) for 5 min and repeatedly rinsed with water to remove any nonspecifically adsorbed enzyme. The interface was stored in PBS buffer at 4 °C before use. 2.3. Assay for the Detection of Hydrogen Peroxide. HRP, a prototypical hemoprotein peroxidase, catalyzes the oxidation of a number of substrates in the presence of hydrogen peroxide. The reaction of HRP with H2O2 gives compound I, where the heme center is its pentavalent redox state.29 Compound I is reduced back to the trivalent ferric redox state of the native enzyme resting state by peroxidase substrates. 2,20 -Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) is such a peroxidase substrate and has been shown to be suitable for use in ELISA procedures. The reduction process produces a soluble end product that is green in color and can be readily detected spectrophotometrically at 405 nm. This colorimetric reaction was used to explore the sensitivity of HRP-terminated NCD to different hydrogen peroxide concentrations. The HRP-terminated interface was immersed into a UV/vis cuvette containing 2 mL of PBS solution mixed with 3.6 mM 2,20 -azino-bis(3-ethylbenzothiazoline-6-sufonic acid) diammonium salt (ABTS). The UV/vis spectra were recorded after a reaction time of 20 min. Increasing concentrations of hydrogen peroxide were injected, and the UV/vis spectra of the resulting solutions were measured. 2.4. Surface Characterization. 2.4.1. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) was used to evaluate the composition and chemical bonding of the diamond surfaces before and after surface modifications. The near-surface composition of NCD films was studied by XPS using an ADES 400 angular-resolved photoelectron spectrometer (VG Scientific, U.K.) equipped with a twin anode X-ray source with the standard Al/Mg anodes and a hemispherical analyzer. XPS spectra were recorded using a Mg Kα source operated at a power of 200 W at a constant pass energy of 100 or 20 eV. Peak areas were determined following the Shirley inelastic background subtraction method. Static sample charging of the spectra was corrected with respect to the C 1s peak at 285.0 eV. 2.4.2. Atomic Force Microscopy. The surface topography of NCD samples was investigated by atomic force microscopy (AFM) in tapping mode (AFM Microscope Dimension 3100, Veeco). Silicon AFM cantilevers were used with a typical tip radius of 10 nm and a resonance frequency of 70 kHz. 2.4.3. Contact Angle Measurements. Water contact angles were measured using deionized water. We used a remote-computer-controlled goniometer system (DIGIDROP by GBX, France) to measure the contact angles. The accuracy was (2°. All measurements were made under ambient conditions at room temperature. 2.4.4. UV/Vis Measurements. Absorption and transmission spectra were recorded using a Perkin-Elmer Lambda UV/vis 950 spectrophotometer in polystyrene cuvettes with an optical path of 10 mm. The wavelength range was 400800 nm.
2. EXPERIMENTAL SECTION 2.1. Materials. Horseradish peroxidase (HRP, EC 1.11.1.7, type II), N-hydroxysuccinimide (NHS), N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC), hydrogen peroxide (H2O2), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sufonic acid (ABTS), phosphate buffered saline (PBS), and tween were purchased from Sigma-Aldrich and used as received unless otherwise specified. 2.2. Undoped Nanocrystalline Diamond Films. Undoped nanocrystalline diamond (NCD) thin films were grown on glass substrates (Schott AF 45, 1 1 cm2). The substrates were first nucleated by ultrasonic pretreatment in a suspension of deionized water and ultradispersed detonation diamond powder (Φ ≈ 510 nm, New Metals and Chemicals Corp. Ltd., Kyobashi).27 The NCD films were grown with a large-area linear antenna microwave plasma-enhanced CVD deposition system (AK 400 modified system, Roth and Rau, AG), which employs two cooper antennas located in the quartz tube and two microwave generators (2.45 GHz, MX4000D, Muegge) on each side of the linear conductor.6 The microwave power used was 2.5 kW in pulsed mode (frequency 111 Hz with a cycle ON:OFF = 2:1), the substrate temperature was 750 °C, the total gas pressure was 2 mbar, and the hydrogen-rich gas mixture consisted of 2.5% methane and 10% carbon dioxide. Under these experimental conditions, the diamond film thickness was 200 nm. The NCD surface was inherently hydrogen-terminated by the deposition process as confirmed by a static water contact angle of close to 90°, in accordance with values reported in the literature for hydrogen-terminated diamond surfaces.28 2.2.1. Diamond Amination. Amine-termination of NCD films was achieved by NH3-plasma treatment in radio frequency plasma (13.56 MHz) using an rf power of 4 W, ammonia flow of 50 sccm, and a total gas pressure of 25 Pa. All NCD samples were treated for 10 min at room temperature without preheating. The technique is easy to carry out and allows the introduction of NH2 groups onto the diamond surface under mild conditions. 2.2.2. HRP Immobilization. Anchoring amine terminal functional groups on the NCD surface represents a significant advance for the incorporation of molecules bearing COOH groups. The aminated diamond surface was hence used for the covalent linking of horseradish peroxidase (HRP). HRP was linked to the amine-terminated interfaces 588
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Figure 3. (A) Wide-range XPS survey spectra of (a) hydrogenated, (b) aminated, and (c) HRP-modified diamond surfaces. (B) Highresolution C 1s and (C) N 1s XPS spectra of (a) hydrogenated, (b) aminated, and (c) HRP-modified diamond surfaces.
3.2. Biosensor Preparation. The hydrogenated diamond interface was used in this section for the covalent linking of horseradish peroxidase (HRP) in a two-step process as depicted in Figure 1. X-ray photoelectron spectroscopy was used to evaluate the chemical composition of the diamond surfaces before and after NH3 plasma treatment and the nature of the chemical bonding associated with transformations that occurred on the surface after HRP linking. Figure 3A displays a wide range of XPS survey spectra of the hydrogenated, aminated, and HRPmodified diamond surfaces. The atomic percentage of oxygen present on the surface of the different diamond interfaces increased from 4.9% on NCDH to 6.3% on NCDNH2 and 19.6% on NCDNHHRP. The detected oxygen on the hydrogenated NCD is most likely due to the adsorption of air moisture and other hydrocarbon impurities during sample transfer from CVD to XPS. The effect is even more pronounced on the aminated NCD surface, which is known to be very hygroscopic. This would explain the apparent increase in oxygen content on aminated diamond films. The atomic concentration of nitrogen reached 4.5% in the case of NCDNH2. This shows that the developed plasma technique is easy to carry out and allows us to introduce nitrogen functions onto the surface under mild conditions. The linking of HRP resulted in a significant increase in the N 1s peaks (7.7 atom %). In addition, a peak centered at 170.6 eV attributed to sulfur S 2p (0.7 atom %) was detected on the NCDNHHRP sample. The HRP monomer contains 308 amino acids with 12 sulfur atoms and carbon
Figure 2. (A) AFM topography, (B) SEM image, and (C) Raman spectrum of an undoped nanocrystalline diamond film prepared by a linear antenna CVD process.
3. RESULTS AND DISCUSSION 3.1. Surface Morphology of Undoped NCD. Figure 2A shows an AFM image in tapping mode of the undoped nanocrystalline diamond (NCD) surface investigated in this study. The diamond film displays an average grain size of 27 ( 3 nm. The rms surface roughness is 6 ( 1 nm, as calculated from a scan area of 1 1 μm2. The SEM-determined surface morphology of undoped NCD reveals nanosized surface features of less than 50 nm (Figure 2B). Figure 2C shows a typical Raman spectrum of the employed NCD films. The spectrum is dominated by D and G bands at 1340 and 1520 cm1, respectively.30 Two weaker and broad bands centered at around 1148 and 1485 cm1 are also observed. They can be attributed to short transpolyacetylene segments most probably localized at diamond grain boundaries.31 However, these bands are also attributed to NCD films. 32 The characteristic diamond peak centered at 1332 cm 1 is well resolved.33 589
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hydrates attached around each HRP.34 The XPS thereby confirms the presence of HRP on the diamond surface. Figure 3B displays the different high-resolution XPS spectra of the C 1s peak. The FWHM of the C 1s peak increased from 1.1 eV (NCDH) to 1.4 eV (NCDNH2) and finally to 1.6 eV for the NCDNHHRP surface. This indicates the increasing complexity of carbon bonding states. In the case of NCDH, the C 1s band was deconvoluted into three components: 285.1 eV (CC/CH) and 286.5 eV (CO) due to carbon impurities adsorbed on the surface. In addition, a weak band at 284.1 eV due to sp2 carbon was observed. The NCDNH2 interface showed bands at 284.1 (C sp2), 285.1 (CC/CH), 285.8 (CN), and 286.6 eV (CO). The surface of the diamond was partially oxidized during the amination process. In the case of NCDNHHRP, the C 1s band was deconvoluted into bands at 285.1 (CC/CH), 286.1 (CN), 286.6 (CO), and 288.1 eV (CdO). The peak-fitting results are in agreement with the literature data.35 Figure 3C displays high-resolution N 1s spectra of the two nitrogen-containing samples, NCDNH2 and NCDNH HRP, with peaks at 399.4 and 400.2 eV, respectively. In the case of NCDNH2, peak fitting resulted in a dominant primary amine contribution at 399.4 eV (NH2) together with an imide and/or secondary amine contribution at 398.4 eV (CdN, CNC) and other contributions at 400.8 eV. On the basis of the diamond structure, it is expected that the sp3 CH bonds on the (111) facets will be terminated with NH2 groups whereas the CH2 bonds on the (100) facets will be transformed to imine (CdN) and (CNC) functional groups.36 The most complex high-resolution N 1s XPS spectrum was measured for an HRP-modified diamond sample. Peak fitting of the N 1s spectrum of NCDNHHRP revealed an amide spectral signal at 399.4 eV, a negligible imide contribution at 397.9 eV, and a dominant signal from other contributions at 400.4 eV. The latter spectral feature can be assigned to positively charged nitrogen, such as N+
