Article pubs.acs.org/ac
Polypyrrolic Bipyridine Bis(phenantrolinequinone) Ru(II) Complex/ Carbon Nanotube Composites for NAD-Dependent Enzyme Immobilization and Wiring Bertrand Reuillard, Alan Le Goff, and Serge Cosnier* Université Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, France CNRS, DCM UMR 5250, F-38000 Grenoble, France ABSTRACT: We report the synthesis and electrochemical characterization of a novel electropolymerizable Ru(II) complex containing two phenanthrolinequinone ligands, RuII(PhQ)2(bpy-pyrrole)(PF6)2. This complex was electropolymerized on glassy carbon (GC) and multiwalled carbon nanotube (MWCNT) electrodes. Higher apparent surface concentrations (80 nmol cm−2) were obtained on MWCNTs than on GC electrodes and correspond to ∼1000 equivalent compact monolayers of Ru complex. Moreover, the nanostructured metallopolymer exhibits efficient electrocatalytic properties toward oxidation of NADH. This metallopolymer can be electrogenerated in water from the adsorbed Ru(II) monomer. This property was applied to the immobilization of enzymes by coadsorption of Ru complex and enzyme and then electropolymerization of coatings. This two-step procedure leads to the entrapment of 70%−90% of the deposited amount of enzyme in poly-RuII(PhQ)2(bpy-pyrrole) films. Glucose dehydrogenase (GDH) was thus efficiently immobilized in the electrogenerated polymer matrix. In presence of NAD+ (10 mM), the resulting enzyme electrode exhibits high current densities for glucose oxidation of 1.04 mA cm−2 at low overpotentials (−0.1 V) with a detection limit of 1 μM of glucose.
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polyvinyl polymers.8,9 However, in all cases, the immobilization of enzymes was ensured using multilayer constructs, using a cross-linking agent or a chemically generated polymer. Although the surfaces are conductive, the immobilization of dehydrogenases was not performed electrochemically and, therefore, without precise spatial resolution. In this context, here, we report the synthesis and electrochemical characterization of an electropolymerizable phenanthrolinequinone-Ru(II) complex: RuII(PhQ)2(bpy-pyrrole)(PF6)2. This compound is functionalized by a pyrrole group that can be electropolymerized in its adsorbed state in water. Thanks to this original property, this Ru complex was applied to the electrochemical entrapment of enzymes via the adsorption of an enzyme-Ru complex mixture on an electrode surface and the electropolymerization of the adsorbed monomers. Moreover, the electrogenerated metallopolymer film presents efficient electrocatalytic properties toward the oxidation of NADH. To further improve electron transfer in these types of polymers and increase the surface concentrations of wired enzymes, the electrodeposition at carbon nanotube (CNT)based electrodes has been investigated. CNTs have excellent physicochemical properties toward efficient electropolymeriza-
he use of NAD-dependent enzymes in biosensing or biofuel cell applications relies on their large diversity, which makes them suitable for the oxidation of many relevant substrates, such as glucose, lactate, aldehyde, or alcohols. In particular, their use in enzymatic biofuel cells is envisioned to supply power from biofuels such as glucose,1 alcohols2 or Llactate.3 Another advantage is their possible use in enzyme cascade systems, where different dehydrogenases employ NADH/NAD+ as the single cofactor for the complete oxidation of substrates.2 In particular, high-power biofuel cells rely on the oxidation of glucose by glucose dehydrogenase (GDH) immobilized in polymer matrices.1 Beyond the intrinsic enzyme activity, the efficiency of the electrocatalysis mostly depends on efficient oxidation of the NADH cofactor. Furthermore, if the enzyme electrode must be integrated into a biofuel cell setup, the NADH oxidation must be achieved at low overpotential to maximize the output potential of the biofuel cell. While the redox potential of NADH is set at −0.56 V vs SCE, its oxidation at the electrode surface often occurs at high overpotentials. In this context, the electropolymerization of organic dyes has been extensively employed to oxidize NADH at low overpotentials.4−6 Among redox molecules that lower the NADH oxidation overpotential, Abruna et al.7 proposed a ruthenium complex bearing phenanthrolinequinone groups that achieves electrocatalytic NADH oxidation at −0.1 V vs SCE. Then, several attempts have been focused on the immobilization of NAD-dependent enzymes at Ru(II)-functionalized © 2014 American Chemical Society
Received: January 21, 2014 Accepted: April 10, 2014 Published: April 10, 2014 4409
dx.doi.org/10.1021/ac500272v | Anal. Chem. 2014, 86, 4409−4415
Analytical Chemistry
Article
were synthesized according to previously described procedures.7,15,16 Synthesis of [bis(Phendion) (4-methyl-4′-butylpyrrole-2,2′b i p y r i d i ne ) Ru t he n i u m ( I I ) H e x afl uo ro ph o s ph a t e (RuII(PhQ)2(bpy-pyrrole)). The synthesis of [bis(Phendion) (4-methyl-4′-butylpyrrole-2,2′-bipyridine) ruthenium(II) hexafluorophosphate (RuII(PhQ)2(bpy-pyrrole)) proceeded as follows. A solution of 4-methyl-4′-butylpyrrole-2,2′-bipyridine (21 mg) and [Ru(Phendion)2Cl2] (40 mg) in ethylene glycol (2.5 mL) was refluxed for 40 min under nitrogen. After cooling to room temperature, an aqueous solution of NH4PF6 was added, allowing the formed product to precipitate. The orange brown precipitate was then filtrated, washed with water, and dried with Et2O yielding to 58 mg of product (78%). 1H NMR: δH ppm (400 MHz, CD3CN): 1.57 (m, 2H), 1.70 (m, 2H), 2.74 (m, 2H), 3.39 (s, 3H), 3.87 (m, 2H), 5.93 (s, 2H), 6.68 (s, 2H), 7.21 (m, 2H), 7.46 (m, 2H), 7.58 (m, 2H), 7.71 (m, 2H), 7.80 (m, 2H), 7.98 (m, 1H), 8.03 (m, 1H), 8.66 (m, 3H), 8.73 (m, 3H). MS (ESI+): 406.7 (M-2PF62+), 958.1 (M-PF6+). UVvis (DMF): λmax/nm (ε/M−1 cm−1) = 295 (29 200), 360 (11 200), 444 (12 400). Biosensor Fabrication. Commercial-grade thin MWCNTs (9.5 nm diameter, purity >95%,) were obtained from Nanocyl. MWCNT films were prepared using a modified procedure from Wu et al.17 MWCNTs (10 mg) were dispersed in pure water (250 mL) and sonicated during 30 min. The solution was carefully decanted overnight and the remaining transparent supernatant (100 mL) was then filtered over cellulose nitrate filter (Sartorius, 0.45 μm pore size, overall diameter of ⌀ = 3.5 cm), resulting in the deposition of MWCNTs. The obtained membrane was deposited on a glassy carbon (GC) electrode (surface area of 0.07 cm−2) and carefully dissolved by several washings with acetone. Electropolymerization was performed in MeCN or water. In MeCN, MWCNT electrodes were functionalized by electropolymerization between 0 and 0.9 V (40 scans) of 1 mM RuII(PhQ)2(bpy-pyrrole). The electrodes were then successively rinsed with MeCN and water. In water, 20 μL of an aqueous solution containing enzyme (1 mg mL−1) and pyrrole monomer (1 mM) were spread and dried under vacuum for 15 min, yielding adsorbed coatings. Then, the resulting electrodes were transferred to a cell containing 0.1 M LiClO4 (aqueous) and the adsorbed monomers were electropolymerized at 0.8 V vs SCE for 2 min. All the bioelectrodes were then kept in a pH 7 phosphate buffer solution.
tion procedures, including the immobilization of high amounts of enzymes and efficient electron transfer properties for molecular electrocatalysis.10,11 Furthermore, carbon nanomaterials such as carbon nanofibers have also demonstrated direct oxidation of NADH for the detection of dehydrogenase substrates.12 Furthermore, we showed the beneficial effects of combining electrogenerated metallopolymers, in terms of improved surface concentrations and electron transfer properties.13,14 For these reasons, CNTs are one of the privileged nanomaterials that have been developed for use as the electrodes of enzymatic biofuel cells. We show that the electrogenerated poly-[RuII(PhQ)2(bpy-pyrrole)] metallopolymers, when nanostructured on multiwalled carbon nanotube (MWCNT) surfaces, strongly immobilizes high amounts of dehydrogenase enzyme while achieving efficient NADH oxidation at low overpotentials. Thus, the combination of CNT electrodes with coordination complexes leads to functional hybrids with enhanced surface concentrations of immobilized coordination complexes and improved diffusion of substrates to the enzymes. We especially underline the facile electrochemical entrapment process without need of additional cross-linkers and the flexibility of the poly-[RuII(PhQ)2(bpypyrrole)] to immobilize enzymes such as GDH. These nanostructured nanocomposite bioelectrodes are envisioned for electrochemical biosensors and bioanodes in biofuel cells.
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EXPERIMENTAL SECTION Materials and Reagents. Acetonitrile (HPLC grade), which was used for electrochemistry measurements, was obtained from Rathburn and used without further modification. Tetrabutylammonium perchlorate ([Bu4N]ClO4, TBAP, Fluka) was used as supporting electrolyte in organic media. All chemical products purchased from Aldrich were of reagentgrade quality and used as received, unless noted otherwise. NMR spectra were recorded on a Bruker AVANCE 400 system that was operating at 400.0 MHz for 1H. Electron spin ionization (ESI) mass spectra were recorded with a Bruker APEX-Qe ESI FT-ICR mass spectrometer. Ultraviolet−visible (UV-vis) spectra were recorded with a Varian Cary 1 spectrophotometer with a quartz cuvette (1 cm depth). Electrochemistry. The electrochemical experiments performed in MeCN were carried out in a three-electrode electrochemical cell under a dry argon atmosphere and in a glovebox ([O2]
