RU117011U1 - DEVICE FOR DETERMINING THE GLUCOSE CONTENT ON THE BASIS OF PENICILLIUM ADAMETZII GLUCOSOO-OXIDASE AND OXYGEN CLARK TYPE ELECTRODE - Google Patents

Abstract

A device for determining glucose content in food, fermentation processes and in physiological fluids, containing a measuring cuvette with a magnetic stirrer, a biosensor consisting of a Clark electrode with a bioreceptor placed on it and made with the possibility of placing it in a measuring cuvette and connecting to a data processing recording unit on the basis of a computer, glucose oxidase immobilized on a carrier, synthesized by the Penicillium adametzii F-3298 D. strain, was used as a bioreceptor.

Description

The utility model relates to biosensor analytical devices with a scope in biotechnology, food industry, agriculture and medicine. The biosensor can be used to determine the glucose content in food products, fermentation processes, as well as in physiological fluids.

The use of glucose oxidase (YEAR) synthesized by Penicillium adametzii F-3298 D strain as the biological basis of the biosensor for glucose determination is shown.

To immobilize the YEAR, the method of immobilizing enzymes in the DEAE-dextran layer covalently linked to the nitrocellulose membrane by benzoquinone (Zaitsev M.G., Ashin V.V., Reshetilov A.N., A new method for immobilizing methylotrophic cells and alcohol oxidase isolated from them for biosensor detection of lower alcohols. Abstracts of the III International Youth School-Conference "Actual Aspects of Modern Microbiology". Moscow; 2007. P.35).

In the proposed, utility model, immobilized is used as a biologically sensitive element. YEAR, a Clark type oxygen electrode is used as a converter.

The following are analogues of the proposed biosensor based on an oxygen electrode.

An implantable glucose biosensor for continuous monitoring of blood glucose in patients with diabetes is presented in (Gamburzev S., Atanasov P. et al. Performance of glucose biosensor based on oxygen electrode in physiological fluids and at body temperature // Sensors and Actuators B: Chemical . 1996. V.30. No. 3. P. 179-183). YEAR (isolated from Aspergillus niger) was immobilized on carbon powder using a carbodiimide crosslinking agent. The linear range was 0-2.4 mg / ml. The responses of the sensor were stable for 2 weeks of continuous operation of the sensor in blood plasma.

Joint immobilization of YEAR (isolated from Aspergillus niger) and glucoamylase was used to create biosensors for starch detection (Renneberg R., Scheller F., Rieldel K., Litschko E., Richter M. Development of anti-interference enzyme layer for a-amylase measurements in glucose containing samples // Anal. Lett. 1983. V. 16 (B 12). P. 877-890.). Enzymes were immobilized on a silk membrane by crosslinking with glutaraldehyde. The upper limit of the linear relationship was 5 mm glucose. The response time was 10 s.

The prototype used an amperometric biosensor for glucose detection, presented in (Wu B., Zhang G. et al. Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane // Talanta. 2004. V. 64. No. 2. P. 546-553). YEAR (isolated from Aspergillus niger) was immobilized on the shell of an egg shell using glutaraldehyde. The response time of the sensor was 100 s, and the storage stability for 4 months was 85% of the initial response level. The linear detection range was 1 × 10 ⁻⁵ −1 × 10 ⁻³ M.

In contrast to the above prototype, the proposed model uses the enzyme glucose oxidase synthesized by the strain Penicillium adametzii F-3298 D, not previously used for these purposes. In addition, the difference between the proposed model and the closest analogue is the response time of the sensor, which is two times less than the response in the prototype model. In the proposed type of biosensor also used a new method of immobilization of the enzyme, providing stability and detection limits comparable with the parameters of the prototype.

The problem to which the claimed utility model is directed is to create a receptor element for a biosensor for the determination of glucose and applications in such areas as the food industry, biotechnology, medicine and agriculture.

The technical result that can be obtained using the proposed utility model is that the proposed receptor element allows you to study the kinetic parameters of the enzyme and has a short response time, two times less than the prototype model, high sensitivity and stability, allowing you to study the kinetic parameters enzyme.

The essence of the utility model lies in the fact that a bioreceptor containing an enzyme immobilized on a carrier, glucose oxidase, is coupled to a Clark type electrode.

Figure 1 presents a diagram of a device.

The proposed device includes the following elements: a biosensor, consisting of a converter - Clark electrode (1), on which a bioreceptor (2) is placed, which is an enzyme immobilized on a carrier, as well as a measuring cell (3) and a magnetic stirrer (4). The device also contains a computer-based data recording and processing unit.

The main analytical parameter of the biosensor is the calibration dependence. For its construction, various concentrations of glucose were added to the measuring cell. Figure 2 shows the calibration dependence of the sensor based on the YEAR synthesized by the strain Penicillium adametzii F-3298 D. The range of determined glucose concentrations was 0.05-5 mM. The sensor responses were recorded in a 30 mM potassium phosphate buffer solution with pH 6. The sensitivity in the linear range was 1.6 (nA / s) / mM. The response time was 50 s.

The calibration dependence was approximated by Hill's three-parameter equation. The apparent kinetic constants are shown in Table 1. For comparison, the commercial enzyme glucose oxidase Aspergillus niger from Sigma was investigated. The immobilized YEAR Penicillium adametzii F-3298 D has a higher V _max value compared to a commercial enzyme, which indicates a higher sensor response. Moreover, the YEAR of Aspergillus niger has a lower value of K _m , compared to the YEAR of Penicillium adametzii.

Table 1. Kinetic constants YEAR. Strain Seemingly kinetic constants Maximum reaction rate (V _max ) Michaelis Constant (K _M ) Hill parameter (h) Penicillium adametzii F-3298 D 4.47 ± 0.30 1.77 ± 0.23 1.30 ± 0.10 Aspergillus niger (Sigma) 2.54 ± 0.11 0.98 ± 0.08 1.88 ± 0.21

As a result of the studies, the basic properties of Penicillium adametzii F-3298 D glucose oxidase were established, a biosensor for glucose determination was developed, which provides a quick determination of glucose content in a sample without the use of complex and expensive equipment. The formation of a bioreceptor reduces the analysis time.

Claims (1)

A device for determining the glucose content in food products, fermentation processes and physiological fluids, containing a measuring cell with a magnetic stirrer, a biosensor consisting of a Clark electrode with a bioreceptor placed on it and made with the possibility of placing it in a measuring cell and connected to a data processing recording unit based on a computer, glucose oxidase immobilized on a carrier synthesized by Penicillium adametzii F-3298 D strain was used as a bioreceptor.