LT3750B - Method for making of fermentation electrode for determinating of l-lactate - Google Patents

Abstract

Invention is used in the medical field for the analysis of biological solutions, for purposes of diagnostics and in the microbiology industry. For the production of a ferment electrode, a cytochrome b2 is adsorbed on the carbon electrode glass surface modified by the PM-75 soot. Before modification of the soot, nitric acid is oxidised and dispersed in an acetone suspension that has 2-5 percent of the soot. The invention ensures a simplification of the glass carbon electrode modification; reduction of the operational potential of the ferment electrode, increase selectivity of the same; increase it's sensitivity that enables it to use heavily diluted biological solutions.

Description

Field of the invention is biochemistry. Specifically, the present invention is directed to a method for the production of an enzyme electrode and can be used in enzymology for scientific research as well as in the medical or microbiological industry for electrochemical determination of L-lactate in biological media, e.g. blood, diagnostics of other media.

A known method for creating an L-lactate electrode using cytochrome b2 / MMMo6nnn3OBaHHbie φβρΜβΗΤΈΐ τ.2. ct. 169176 (1976)). In this method, the enzyme is immobilized on a gel to coat the electrode and a redox mediator is introduced into the test solution, transferring electrons from the active center of the enzyme to the electrode. The disadvantage of this technique is the need for a soluble mediator. Quinone toxic compounds, e.g. benzoquinone. Therefore, such electrodes cannot be used for in vivo assays as well as in microbiological reactors and for mass analysis of biological media.

A known enzyme electrode consists of a glass carbon working surface coated with cytochrome b ₂ and lactate dehydrogenase and covered with a dialysis membrane / USSR al. The disadvantage of this electrode is the use of an additional enzyme. In addition, a soluble mediator is also required to detect lactate.

A known method of creating an enzyme electrode for detecting L-lactate is based on cytochrome b ₂ based on adsorption of the enzyme onto a current-carrying matrix composed of organic cations (methylated phenazine derivatives, acridine, quinoline, pyridine derivatives) and 7,7,8,8-tetracyano-p. -quinodimethane anion-radical complexes / USSR a. I. No. 894534 /. The use of this electrode for analysis does not require the presence of a soluble mediator in the test solution. The disadvantage of the known method is the complexity of the process, which requires the additional synthesis of complexes and their components, such as the tetracyano-p-quinodimethane radical.

The object of the invention is to simplify the process.

SUMMARY OF THE INVENTION The present invention provides an electrode for adsorption of cytochrome b ₂ on a modified glass carbon electrode surface. The electrode is modified with soot PM-75, which is used as a dye in printing works or as a filler in the rubber industry. Prior to modification, the soot is oxidized with nitric acid and ultrasonically dispersed in a solution of fluoroplastic lacquer in acetone. The suspension used for the modification contains 2-5% fluoroplastic varnish and 12-20% soot.

The mechanism of action of the enzyme electrode is explained by the electrooxidation of reduced cytochrome b _{2 on} the surface of the modified electrode. L-lactate reduces cytochrome b ₂ .

Cit. b ₂ ( ₀ | _<s j + L- ^lactate- ^ ^itb 2 (red) ⁺ P ^iruvate ·

Oxidation of soot with nitric acid produces electroactive quinone groups (Q) on the surface that catalyze the electrooxidation of cytochrome b ₂ :

^Cit - ^b 2 (Ed.) + ^Q * ^{Cit b} 2 (Ox.) ^{+ QH} 2 'θ ^ 2 ~ 2θ ^_ 2Η Electrode * θ ·

According to the present invention, PM-75 carbon black is stored in 55% nitric acid at room temperature for 5 days prior to modification. They are then washed with water to a neutral reaction (pH 7.0) and dried. The mixture of prepared soot and fluoroplastic lacquer solution in acetone is dispersed by sonication (44 kHz) for 0.5-1 h. Immerse the glass carbon electrode in the resulting soot suspension for 3-5 seconds, then dry, wash with distilled water and buffer. Apply 10 ml of cytochrome b ₂ solution to the working plane of the soot modified electrode. The working plane is covered with a capron mesh and dialysis membrane, which are then secured with a rubber ring. Immerse the electrode in 0.1 M K-phosphatic citrate buffer (pH 7.0) and connect to a polarograph (operating potential -0.2V by Ag / AgCl). Introduce the test fluid into the electrode lattice and record the steady-state oxidation current dependence on the L-lactate concentration.

An example. Determination of L-lactate concentration in aqueous solution

The electrode is prepared according to the above procedure. Ultrasonic dispersion of soot takes up a suspension of acetone containing 2-5% fluoroplastic varnish and 12-20% soot. Set current to -0.2V according to h

Dependence of Ag / AgCI reference electrode on L-lactate concentration.

Table

L-lactate concentration, mM 0.1 0.2 0.3 0.5 0.8 1.0 Current at the electrode, mkA 0.55 1.25 1.80 3.13 4.5 5.0

According to the data in Table 1, the current is linearly dependent on the concentration of L-lactate up to 0.5 mM (Fig. 1).

Using the electrode produced in this way, L-lactate is oxidized to a sufficiently low potential, so that the signal is not affected by ascorbic acid (the electroactive component of biological fluids). From FIG. 1 shows that the electrode is sufficiently sensitive (5.3 mkA / mM L-lactate), which allows the use of more dilute biological solutions for analysis. Increasing the concentration of soot or varnish above the specified upper limit slows the electrochemical conversion of the surface carbon black groups. Lowering the concentration of the components below the lower limit will reduce the electrode stability.

According to Example 1, the electrooxidation of ascorbic acid is found to start at a potential of 0.1 V by Ag / AgCl, so its presence does not interfere with the determination of L-lactate at 0.2 V.

An example. Electrode stability study

The electrode is prepared according to the above procedure. Over a period of 12 days, the electrical current (potential -0.2 V by Ag / AgCl) is measured periodically at a concentration of 0.1-0.3 mM lactate. The data are shown in Table 2.

s

Table

Daily 1 2 3 4 5 8th 10th 1.1 12th Relative current,% 100 100 100 100 90 70 60 55 50

We see that the electrode retains its working capacity for at least 12 days. At least 40 measurements were made. Wash and measure the electrode for a maximum of 4 minutes.

An example. Determination of L-lactate concentration in biological solutions

The electrode is prepared according to the above procedure. L-lactate concentration was determined in blood plasma and culture medium for bovine leukosis virus according to Example 1. In parallel, L-lactate concentration is determined spectrophotometrically by ferricyanide reduction catalyzed by cytochrome b ₂ . Plasma or culture fluid samples are diluted 10-40 times when introduced into an electrochemical or spectrophotometric lattice. The data obtained are presented in Table 3.

Table

Test solution and method of determination L-lactate concentration, m M Blood plasma: (a) spectrophotometrically 0.125 0.185 0.43 0.53 b) according to the invention 0.10 0.16 0.39 0.485 Culture fluid: (a) spectrophotometrically 0.06 0.065 0.19 0.285 b) according to the invention 0.06 0.06 0.16 0.26

As can be seen from the data in Table 3, the concentration of L-lactate, determined spectrophotometrically and using an enzyme electrode, is virtually identical. Setting time - up to 4 minutes. Thus, the resulting electrode can be used to detect L-lactate in biological media.

Another advantage of the proposed method is the simplification of the modification of the glass carbon electrode.

Claims (1)

DEFINITION OF INVENTION A method for developing an enzyme electrode for detecting L-lactate using adsorption of cytochrome b _{2 in a} conductor layer, characterized in that the electrode is modified by soot oxidized in nitric acid and the soot is dispersed in an acetone suspension containing 2-5% fluoroplastic varnish and 12- 20% soot.