Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Amperometric enzyme-based biosensors for application in food and beverage industry
4. Selected practical examples
4.1. REDOX HYDROGEL INTEGRATED PEROXIDASE BASED HYDROGEN
PEROXIDE BIOSENSORS
The importance of finding new and more efficient peroxidases, with improved
bioelectrochemical characteristics, has been outlined in section 3.2. In this example,
development of hydrogen peroxide sensors has been targeted, using two newly purified
peroxidases extracted from tobacco (TOP) and sweet potato (SPP), comparing their
characteristics to the ones obtained for similarly constructed electrodes based on the
generally used HW. Electrodes were prepared according to a previously published
protocol [2 1]. Briefly: the enzymes were cross-linked to poly(vinylimidazole)
complexed with Os(4,4’dimethylbipyridine) 2C1 (PV17dmeOs) using poly
(ethyleneglycol) diglycidyl ether (PEGDGE) as the cross-linker. Premixed hydrogels
were made using stock solutions of peroxidases (4.78 mg ml-1 of TOP in 0.15 M Tris-
HCI, pH 6.0; 5 mg ml -1 of HRP in 0.1 M phosphate buffer pH 7.0; 1.86 mg ml-1 of SPP
in 0.005 M Tris-HC1, pH 8.1), PVI 7 dmeOs (3.3 mg ml -1 ) and PEGDGE (2.5 mg ml -1 ).
Defined amounts of hydrogels (film thickness) were applied on spectrographic graphite
rods and the electrodes were cured at room temperature for 20 h in desiccated
conditions. The electron-transfer pathway is outlined in scheme 4.
Scheme 4. Electron-transfer pathway in peroxidase containing redox hydrogels.
The comparative study concerning the bioelectrochemical characteristics of the
developed biosensors showed that, irrespective of studied peroxidase, the biosensors’
sensitivity was strongly influenced by the composition of the redox hydrogel, curing
procedure, film thickness and applied potential. Therefore, all hydrogels were
optimised with regard to these parameters. The optimal enzyme and cross-linker
content was found to be of 15 - 40 % POD and 10 - 25 % PEGDGE, respectively. A
loading of 22 µg per electrode was yielding the best results (adhesion, stability, and
sensitivity). The current response increased with the applied potential and have shown a
levelling off tendency at 0, -50 and around -100 mV vs. Ag/AgCl, KC1 0.1 M for TOP,
SPP and HRP, respectively. Calibrations curves obtained using the optimal electrodes
in a single manifold flow-injection system (see figure 5) with a flow-through wall-jet
electrochemical cell [34] are presented in figure 6. The optimised SPP biosensor (48 %
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