The modified screen-printed electrodes based on glucose oxidase immobilized by different methods

Kamanin S. S., Arlyapov V. A.

Intensive development of biosensor technology makes new demands on the quality of analysis. In this situation, one of the most promising ways to improve the technology is the development of biosensors based on screen-printed electrodes. Screen-printed electrodes are already being used in commercial glucometers – biosensors for the determination of glucose in the blood, which are now virtually dominate the biosensors. Most available biosensors used for the determination of glucose based on the electrochemical determining of hydrogen peroxide generated by glucose oxidase (GO) by the equation:

glucose + O₂ → gluconic acid + H₂O₂

Oxidation or reduction of hydrogen peroxide usually requires the application of a high potential for non-modified electrodes. To reduce the operating voltage measurements and increase the sensitivity of glucose sensors electron transfer mediator can be applied. In commercial glucometers potassium ferrocyanide is used as a mediator (for example, blood glucose meters Accu-Chek). A derivative of this compound – Prussian Blue – is an inorganic catalyst for the decomposition of hydrogen peroxide, which allows to use it as a mediator in biosensors based on glucose oxidase.

Along with the mediator screen-printed electrodes’ properties are determined by the method of biological material immobilization. One of the most modern and advanced methods of immobilizing enzymes is including in polymeric sol-gel matrix. The method of immobilization in a sol-gel layer has a number of advantages: high speed, and ease of performance, non-toxicity, availability of precursors and high mechanical strength matrices. Another promising method for immobilizing enzymes on the printed electrodes is the inclusion of biological material in polymer gels. An example of such is immobilized in a gel including a crosslinked bovine serum albumin (BSA). Obtained after cross-linking with glutaraldehyde molecules BSA matrix provides a good diffusion of substrates and metabolites and forms a favorable sites for immobilized protein environment.

In this paper, a comparative analysis of methods for immobilizing the enzyme on the surface of graphite screen-printed electrodes modified with Prussian blue was carried out, and the results of the development of screen-printed mediator electrodes modified with Prussian blue, immobilized GO for determination of low concentrations of glucose are submitted.

To develop a biosensor circuit electrodes, characterized by stability and high sensitivity, it was necessary to choose a method of immobilization in which the enzyme will function longer period of time without losing its activity. The most promising methods of immobilization have been voted GO incapsulation in agar gel, gel cross-linked BSA and hybrid composition sol-gel/PVA, further characterization of the modified electrode was carried out using these methods of immobilization.

The modified electrode based on GO immobilized in BSA has higher sensitivity and a wider range of detectable concentrations although its detection limit is much higher than the electrodes made using other methods of immobilization. At the same time, electrodes based on GO immobilized in hybrid composition sol-gel/PVA and in agar gel and gives a higher standard deviation. Thus, the modified electrodes fabricated by immobilizing a hybrid composition and cross-linked BSA outperforms electrodes with an enzyme immobilized in agar gel.

Importantly, despite a relatively low sensitivity coefficient obtained modified electrodes have a lower or equal to the lower limit of detectable concentrations compared to their counterparts. Compared with the method of high performance liquid chromatography analysis using the modified electrodes has developed a number of advantages: low detection limit concentration, rapidity of analysis, its simplicity and low cost of the hardware.

To test the developed electrodes real samples were analyzed with them. Electrodes modified with GO immobilized in a hybrid composition sol-gel/PVA and BSA gel were selected for the tests, as in the work it was found that they are superior to the electrodes with GO immobilized agar gel on a number of parameters. Testing of electrodes on the basis of GO immobilized in a hybrid composition sol-gel/PVA carried out on samples of wine and fermenting mass sampled at different times of the fermentation process (164 hours and 185 hours). Electrodes based on GO immobilized in BSA tested on samples of juice products: orange juice and apple juice. Statistical methods demonstrated that the glucose concentration is determined using the modified electrode insignificantly different from the results obtained by the reference method. The obtained data demonstrate the ability to use the developed screen-printed electrodes modified as prototypes for mass use.

The work was supported by the Federal Target Program "Research and scientific-pedagogical personnel of innovative Russia" for 2009-2013 ", the state contract № 16.740.11.0766.