Oxidases biosensors

F.N. Kok and V. Hasirci, Determination of binary pesticide mixtures by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19, 661-665 (2004). 

Some applications of amperometric biosensing strategies for pesticide detection in real or spiked food samples have been recently reported. Most of the applications have been developed for vegetable matrices. Different formats of biosensors have been used disposable screen-printed choline oxidase biosensors [23] using AChE in solution were utilized to detect pesticides in real samples of fruit and vegetables. 

To perform electrochemical analysis with the choline oxidase biosensor, dichlorvos needed to be transferred to PBS solution, thus avoiding any electrochemical interference by organic solvents. 

Determination of methyl mercury in fish tissue using electrochemical glucose oxidase biosensors based on invertase inhibition... 

Scheme 20.1. Glucose oxidase biosensor combined with the enzyme invertase in solution.

GLUCOSE OXIDASE BIOSENSOR PREPARATION Enzyme immobilisation was carried out, at room temperature. 

After sucrose hydrolysis by the invertase enzyme in solution, perform all measurements at a constant applied potential of +0.60 Y in order to oxidase the hydrogen peroxide produced by the enzyme reaction of glucose oxidase biosensors (Scheme 20.1). 

After current stabilisation, add a defined volume of concentrated stock solution of substrate (sucrose) to the electrochemical cell containing the glucose oxidase biosensor and measure the steady-state current, corresponding to H202 oxidation (/,). 

It should be noted that the activity of the glucose oxidase biosensor remained stable during the whole period of the experiments (more than 4 weeks). 

Fig. 20.1. Dependence of glucose oxidase biosensors and invertase on pH values for 20 mM of sucrose addition and 5 min as reaction time. Experimental conditions Invertase (4pg/mL) acetate buffer ( ) and phosphate buffer ( ) Eapp = + 0.60 V vs. Ag/AgCl.

The effect of the inhibitor in glucose oxidase biosensor response was investigated. The biosensor was incubated for 10 min in the presence of different concentrations of methyl mercury. It was seen that after exposure to the high concentration equal to 1 ppm of methyl mercury the response of glucose remained constant (results not shown). 

Fig. 20.2. Amperometric study of the percentage of inhibition as function of the concentration of the enzyme in presence of 50 ppb of methyl mercury using phosphate buffer/toluene mixture and glucose oxidase biosensor. Phosphate buffer pH 6.0 Eapp — 0.60 V vs. Ag/AgCl reaction time = 5 min and incubation time = 10 min.

Fig. 20.4. Calibration curve for inhibition of invertase (0.05 pg/mL) by methyl mercury after 10-min incubation using biphasic system (phosphate buffer/ toluene mixture) and glucose oxidase biosensor. Eapp — +0.60 V vs. Ag/AgCl and reaction time = 5 min.

Knowing that the inorganic mercury is biomagnified in the aquatic food chain through bacterial conversion to methyl mercury and then accumulated primarily in fish, this part is focused to the determination of methyl mercury in fish samples. The developed method described above using the combination of biphasic system and glucose oxidase biosensor was used. 

Determination of methyl mercury using biphasic system (aqueous solution/ solvent) in the presence of fish matrix. The method is based on enzyme inhibition combined with glucose oxidase biosensor... 

The principle of combination of electrochemical glucose oxidase biosensor with the clean-up method for direct extraction and determination of methyl mercury has been successfully demonstrated. The extraction of methyl mercury from the organic solvent has been based on invertase enzyme inhibition. The combination of very low concentration of invertase enzyme and 10 min of incubation time allows the detection of methyl mercury at 5 ppb level. Our method permits the detection of this inhibitor below the legal limit given by the European Union with good recoveries when fish samples were measured. 

The choline oxidase biosensor was constructed using disposable SPEs. The working electrode was modified with Prussian blue (PB) catalyst and with the biorecognition element (choline oxidase). 

The inhibitory effect of dichlorvos on both eeAChE and rAChE was evaluated comparing the decrease in the current produced by the choline oxidase biosensor. 

After 2 min, 100 pi was placed on the choline oxidase biosensor surface and the steady-state current recorded for 2 min. 

We successfully applied an AChE inhibition assay to the detection of dichlorvos in durum wheat samples using a simplified extraction procedure. The total assay time, including the extraction step, was 30 min. Considering that several extractions and assay steps can be run simultaneously, the throughput for one operator is 12 determinations per hour. It is also important to stress that the choline oxidase biosensor used in this work showed an excellent functioning stability after 20 days from preparation, the blank measurement lost only 10% of the signal intensity. The method allowed the accurate analysis of dichlorvos in wheat samples at the MRL, 2 mg/kg, and below that value. The mean recovery was 75%, and neither false nor positive samples were detected. Finally, the portable electrochemical instrumentation combined with the simple extraction procedure was quite well suited for in situ analysis of dichlorvos in durum wheat. 

Saurina, J. Hernandez-Cassou, S. Alegret, S. Fabregas, E. Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites. Biosens. Bioelectron. 1999,14, 211-220. 

Glucose Oxidase Biosensor Based on Colloidal Gold-Carbon Nanotubes... 

Welzel, H.-R, G. Kossmehl, G. Engelmann, B. Neumann, U. Wollenberger, F. Scheller, and W. Schroder. 1996. Reactive groups on polymer covered electrodes. Lactate-oxidase-biosensor based on electrodes modified by polythiophene. Macromol Chem Phys 197 3355-3363. 

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