ИСТИНА

Being true dielectrics, the enzymes must be properly oriented on the electrode surfaces to maintain direct bioelectrocatalysis [1]. To improve efficiency of bioelectrocatalysis we proposed to use conductive polymers, containing analogues of PQQ-Glucose Dehydrogenase (PQQ-GDH) substrates as substituents. On the one hand, displaying affinity to the active site, these polymers are expected to anchor the enzyme and facilitate electron transfer. On the other hand, rather low redox potentials of polyazines would allow to avoid interfering of reductants. Moreover, compared to biosensors based on freely diffusing azine dyes with inherently low operation stability [2], the use of polymer would provide advanced operational stability of the corresponding biosensors. In order to check orientation effect of the polyazines on the PQQ-GDH we electropolymerized Methylene Green, Methylene Blue, Toluidine Blue, Azure A and Coomassie Brilliant Blue in cyclic voltammetry mode [3]. PQQ-GDH was immobilized on the polyazines modified electrodes by adsorption. In the present study, characteristics of the developed biosensors were investigated in in chronoamperometric regime. Chronoamperometric response remain more than 75% of the initial response after 250 injections of 0.1 mM glucose solutions. Thus, allowing long-term monitory without sensitivity loss. The steady-state current-potential curves of bioelectrocatalytic glucose oxidation for PQQ-GDH were investigated. The half-wave potential of curve for Methylene Blue is 80±10 mV. This value is close to the half-wave potential of that for PQQ-GDH adsorbed on blank electrode. Though redox potentials of the electropolymerized Methylene Green are higher, in addition to a similar anodic wave of 120±20 mV, the cathodic one with the half-wave potential of –80±7 mV appears. A difference between anodic and cathodic waves reflects a thermodynamic stability of the intermediate state of PQQ, a semiquinone radical, as it is related to semiquinone formation constant. This value implies that poly(Methylene Green) stabilizes PQQ semiquinone radical. The use of polymer of azine dyes increases the operational stability. The half-wave potentials for poly(Methylene Green) are in a good agreement with those registered for PQQ-glucose dehydrogenase covalently bound to carbon nanotubes. Efficient of direct bioelectroOn an unmodified graphite electrode the catalytic currents were 0.2 μA•cm-2, that is more than 70 times lower than on biosensor based on poly(Methylene blue). At the same time for poly(Methylene green) electrocatalytic currents were 170 μA•cm-2 already at 0.0 V vs. Ag/AgCl, that is more than 10 times higher than for poly(methylene blue) and 700 times higher than on unmodified biosensor. In the presence of both glucose and the freely diffusing mediator phenazine metasulfate the observed current values over an entire potential range are rather similar indicating similar amount of active PQQ-glucose dehydrogenase on all types of electrodes. Accordingly, these studies allow us to conclude that poly(Methylene green) exhibits an orienting effect in relation to the enzyme protein. Wherein, the maximal current for the biosensor based on poly(methylene green) – up to 220 μA•cm-2 - is only 5 times lower, than that in the presence of diffusing mediator phenazine methosulphate. SPE|polyMG|PQQ-GDH electrode has been tested for sweat analysis. Pearson correlation coefficient is of 0.88 pointing to good agreement with reference method. A successful validation of the biosensor confirms a possibility of using this sensors for non-invasive diagnostics.