Novel microbiosensors prepared utilizing biomimetic silicification method

We demonstrate fabrication of microbiosensors utilizing a simple, rapid biomimetic silicification method catalyzed by poly-L-lysine at ambient temperature to provide a mild and efficient method for entrapment of the enzymes required for a range of analytes. To obtain a robust poly-L-lysine layer for precipitating silica onto the Pt surface, a Pt microelectrode was first functionalized with abundant carboxyl groups by electrochemical deposition of poly(pyrrole-1-propanoic acid). By means of zero length cross-linking reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (NHSS), poly-L-lysine was covalently immobilized onto microelectrode surface. Under mild chemical conditions, three enzymes including adenosine deaminase (AD, EC 3.5.4.4), nucleoside phosphorylase (NP, EC 2.4.2.1) and xanthine oxidase (XO, EC 1.1.3.22) could then be simultaneously entrapped into a continuous silicate layer formed on top of Pt microelectrode from a mixture of enzymes and hydrolyzed silanes in Tris buffer (0.1M, pH 7.2) via the catalytic action of the attached poly-L-lysine. The fabricated adenosine biosensors exhibited good analytical performance with a sensitivity of 153.0+/-2.4 microA mM(-1)cm(-2) (n=4, R.S.D.=2.1%), a lower detection limit of 40 nM and a favourable response time (estimated as 10-90% response rise time) of 25+/-2s (n=4). The good selectivity of the adenosine microbiosensor against coexisting interfering substances such as ascorbic acid, urate and 5-HT was achieved through formation of a screening barrier from electrodeposited poly(diaminobenzene) following the biomimetic deposition process. We found that our methods were adaptable for other enzymes and analytes allowing fabrication of l-glutamate and lactate biosensors.