Electrochemical flow-through biosensors based on microfiber enzymatic filter discs placed at printed electrodes.

A new type of electrochemical biosensors in a flow injection system with printed electrodes were developed and tested. A filter disc (7 mm diameter) with immobilized enzyme was placed at the printed electrode. This conception combines the advantages of biosensors with a bioreceptor at the electrode surface and systems with spatially separated enzymatic and detection parts. Filters of different composition (glass, quartz, and cellulose), thickness, porosity, and ways of binding enzyme to their surface were tested. Only covalent bonds throughout a filter-aminosilane-glutaraldehyde-enzyme chain ensured a long-time and reproducible biosensor response. The developed method of biosensor preparation has been successfully applied to enzymes glucose oxidase, laccase and choline oxidase. The dependences of peak current on detection potential, flow rate, injection volume, analyte concentration as well as biosensor lifetime and reproducibility were investigated for glucose oxidase biosensor. The sensitivity of measurements was two or more times higher than that of biosensor with a mini-reactor filled by powder with immobilized enzyme. The developed biosensor with laccase was tested by determining dopamine in the pharmaceutical infusion product Tensamin®. Results of the analysis (40.0 ± 0.7 mg mL-1, SD = 0.8 mg mL-1, RSD = 1.85 %, N = 11) show a good agreement with the manufacturer's declared value.

Influence of different covalent immobilization protocols on electroanalytical performance of laccase-based biosensors.

This paper presents a simple and effective flow-through electrochemical biosensor, consisting of Trametes versicolor laccase (Lac)-based mini-reactor and a tubular detector of silver solid amalgam (TD-AgSA), capable of rapid and selective detection of phenolic compounds. Amperometric detection relies on the reduction of the quinone molecule (formed during the enzymatic reaction in a mini-reactor) on TD-AgSA at -50 mV vs SCE. Since different enzyme immobilization techniques may contribute to differing biosensor performances, four covalent strategies for Lac attachment were compared: (i) through glutaraldehyde to supports -NH2, (ii) via disuccinimidyl suberate to supports -NH2, (iii) using EDC/NHS for Lac coupling by its -COOH groups to supports -NH2, and (iv) using EDC/NHS to supports -COOH. Additionally, five supports (mesoporous silica (SBA-15, MCM-41), cellulose, carbon-based (glassy carbon, graphite) powders) were investigated. It was found that different methods of immobilization, as well as different types of supports, significantly affect the amount of immobilized Lac and, in turn, the analytical characteristics of the obtained biosensors. Thus, TD-AgSA with enzymatic mini-reactor based on Lac covalently attached via glutaraldehyde to aminated MCM-41 proved to be the most promising biosensor, providing the best detection limit (18.3 µmol L-1) and the long-term stability (47.9 % of the initial response/4 months/100 measurements).