Coupling of Amine-Containing Osmium Complexes and Glucose Oxidase with Carboxylic Acid Polymer and Carbon Nanotube Matrix to Provide Enzyme Electrodes for Glucose Oxidation

Abstract

The immobilization methodology of enzyme and redox complex on electrode surfaces can have an impact on the magnitude and stability of amperometric current response, with implications for application as biosensor and fuel cell enzyme electrodes. Here we report on an investigation of carboxymethyl dextran (CMD) and polyacrylic acid polymers, bearing carboxylic functional groups, as chemical supports for immobilization of amine-containing osmium redox complexes and enzymes at electrode surfaces. Crosscoupling using carbodiimide reagent of the CMD polymer support, [Os (2,2 -bipyridine) 2 (4-aminomethyl pyridine)Cl].PF 6 redox complex, glucose oxidase (GOx) and multiwall carbon nanotubes (MWCNT), provides a 3-dimensional matrix for catalytic electrooxidation of glucose yielding current density of 1.0 ± 0.2 mA cm −2 and 4.5 ± 1.0 mA cm −2 at 0.45 V vs. Ag/AgCl, in 50 mM phosphate buffer saline (pH 7.4, 37 • C) containing 5 mM and saturated glucose amounts, respectively. Similar enzyme electrodes, but instead using [Os (4,4 -dimethoxy-2,2 -bipyridine) 2 (4-aminomethyl pyridine)Cl].PF 6 of lower redox potential, produce current densities of 0.83 ± 0.21 mA cm −2 in 5 mM glucose and 3.4 ± 0.7 mA cm −2 in saturated glucose solution at 0.2 V vs. Ag/AgCl thus showing promise for application as low potential glucose oxidizing biosensors and as anodes for in-vivo enzymatic fuel cells for power generation. Enzyme electrodes for glucose oxidation are of increasing interest due to their potential applications as biosensors and as anodes in membrane-less fuel cells operating on sugar as a fuel. Such enzymatic fuel cells (EFC) can use enzymes as specific catalysts to oxidize glucose at the anode and reduce oxygen at the cathode, that when combined as a fuel cell convert chemical energy into electrical power.1-3 The advantages to the use of enzyme-based catalysts are substrate specificity, which can eliminate the need for casings and ion exchange membranes in assembled fuel cells, and of being capable of operating under moderate ambient conditions, compared to metal catalysts. 1,2A substantial body of research exists on approaches for maximising catalytic current capture as a result of enzyme redox reactions in enzyme electrodes through the co-immobilization of enzymes and electron-shuttling mediators within redox-conducting hydrogels on solid electrodes. For example, over the past two decades, Heller and co-workers 3 have pioneered use of epoxy cross-linking of electrostatic adducts of redox enzyme and osmium redox polymers, using polyvinylimidazole (PVI) as the polymer backbone, at electrode surfaces to provide "wired" enzyme electrodes capable of producing glucose oxidation current. Mediated electron transfer by electron-hopping selfexchange within these hydrogels allows connection between redox active sites of enzymes and electrode surfaces thus generating bioelectrocatalytic current.4-6 As mediators, osmium based polypyridyl complexes are broadly explored, 6-8 as these complexes possess advantages over iron and ruthenium based ...

A glucose anode for enzymatic fuel cells optimized for current production under physiological conditions using a design of experiment approach

“…All prepared enzyme electrodes, in the absence of glucose substrate, exhibit oxidation and reduction peaks at 0.07 ± 0.01 V (vs. Ag/AgCl), Fig. 1, which is similar to the redox potential observed for the osmium complexes in solution and also close to that previously reported for the complex immobilized within CMD films or directly coupled to a carbon electrode [16,18]. Redox peak currents vary linearly with scan rate for scan rates less than 20 mV s −1 , indicative of a surface-controlled response [22].…”

“…At higher scan rates, the peak currents scale linearly with the square root of scan rate, indicative of semiinfinite diffusion control of the response, as expected for multi-layered films on electrodes [24,25]. Osmium complex surface coverage (Г Os ), calculated by integration of the charged passed under the redox complex oxidation peak using slow scan rate voltammetry in PBS solution [25], is comparable to values obtained previously [18] and approximately one thousand-fold that expected for monolayer coverage of osmium polypyridyl complexes [26,27]. Upon addition of glucose substrate to the PBS electrolyte, a sigmoidal-shaped slow-scan rate cyclic voltammogram characteristic of electrocatalytic oxidation of glucose is obtained for all enzyme electrodes, Fig.…”

“…Substantial glucose oxidation current densities are obtained under pseudo-physiological conditions, with a current density of 1.2 ± 0.1 mA cm −2 at 0.2 V vs. Ag/AgCl in 5 mM glucose for the DoE optimized enzyme electrodes, compared to 0.83 mA cm −2 obtained previously for Os(dmobpy) 2 4AMP based enzyme electrodes under similar conditions, optimized using a one-factor-at-a-time approach [18]. For comparison to other enzyme electrodes, a glucose-oxidizing current density of 0.9 mA cm −2 was reported for enzyme electrodes based on co-immobilization of a [Os(4,4´-dimethyl-2,2´-bipyridine) 2 (poly-vinylimidazole) 10 Cl] + redox polymer and MWCNTs, operating in 5 mM glucose [14].…”

“…Similarly amine functionalized osmium complexes and enzymes have been coupled to a polyallylamine support using a di-epoxide reagent, to produce enzyme electrodes [17]. More recently, co-immobilization of amine functionalized osmium complexes and enzymes to a CMD polymer and MWCNTs on graphite electrodes using a carbodiimide coupling approach has been used to prepare enzyme electrodes [18]. Given the number of different components, and the range of amounts and methods used to prepare these enzyme electrodes, a comprehensive study is required on optimization of each component used in enzyme electrode preparation to identify the interaction or dependency of these components on the performance of enzyme electrodes.…”

“…The enzyme electrodes are prepared by co-immobilizing [Os(4,4′-dimethoxy-2,2′-bipyridine) 2 (4-aminomethylpyridine)Cl].PF 6 , GOx, MWCNTs and CMD using carbodiimide coupling, as described previously [17]. The DoE-optimized enzyme electrodes display a 32% improved glucose oxidation current density compared to previously reported values for the system optimized by variation of one factor at a time [18].…”