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).

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder.

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

Flow electrochemical biosensors based on enzymatic porous reactor and tubular detector of silver solid amalgam.

A flow amperometric enzymatic biosensor for the determination of glucose was constructed. The biosensor consists of a flow reactor based on porous silver solid amalgam (AgSA) and a flow tubular detector based on compact AgSA. The preparation of the sensor and the determination of glucose occurred in three steps. First, a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) was formed at the porous surface of the reactor. Second, enzyme glucose oxidase (GOx) was covalently immobilized at MUA-layer using N-ethyl-N'-(3-dimethylaminopropyl) carboimide and N-hydroxysuccinimide chemistry. Finally, a decrease of oxygen concentration (directly proportional to the concentration of glucose) during enzymatic reaction was amperometrically measured on the tubular detector under flow injection conditions. The following parameters of glucose determination were optimized with respect to amperometric response: composition of the mobile phase, its concentration, the potential of detection and the flow rate. The calibration curve of glucose was linear in the concentration range of 0.02-0.80 mmol L(-1) with detection limit of 0.01 mmol L(-1). The content of glucose in the sample of honey was determined as 35.5±1.0 mass % (number of the repeated measurements n=7; standard deviation SD=1.2%; relative standard deviation RSD=3.2%) which corresponds well with the declared values. The tested biosensor proved good long-term stability (77% of the current response of glucose was retained after 35 days).