RU2731411C1 - Biosensor with high sensitivity factor - Google Patents

Abstract

FIELD: electroanalytical analysis systems.

SUBSTANCE: invention relates to electroanalytical analysis systems and can be used to determine concentration of glucose in diluted blood, as well as serum and blood plasma. Planar biosensor for determining glucose in a liquid sample includes silver chloride comparison electrode, working and auxiliary electrodes placed on a substrate, a Prussian blue film is applied on the working electrode, and a membrane comprising as an enzyme glucose oxidase immobilized inside the membrane layer is made of poly-y-aminopropyltriethoxysilane, wherein the comparison electrode is located at distance of 3–5 mm from the working electrode with possibility of setting a constant zero potential on the working electrode.

EFFECT: invention provides high sensitivity of analysis and enables to determine glucose, as well as provides high accuracy of blood analysis using the disclosed biosensor as part of the electroanalytical system, by eliminating systematic error.

8 cl, 1 tbl, 3 ex, 7 dwg

Description

Technology area

The invention relates to electroanalytical analysis systems and can be used in medical diagnostic centers or ambulances to determine the concentration of glucose in diluted blood, as well as serum and plasma.

State of the art

From the prior art, technical solutions are known that provide the electrochemical enzymatic determination of glucose. In particular, the first approach to such an analysis was the solution [L.C.J. Clark, C. Lyons. Annals of the New York Academy of Sciences 102 (1962) 29], which consists in placing the enzyme glucose oxidase, which catalyzes the oxidation of glucose by oxygen, near the membrane of an amperometric oxygen platinum sensor, which records a local decrease in oxygen concentration. Subsequently, a modification of this solution was proposed [S.J. Updike, G.P. Hicks. Nature 214 (1967) 986], which consists in the immobilization of the enzyme on the surface of the membrane of the oxygen sensor.

However, this solution has the following disadvantages:

i) in equilibrium with air, the oxygen concentration in aqueous solutions is quite high (0.2 mM), which complicates the analysis of low concentrations of the analyte;

ii) in real objects, the equilibrium oxygen concentration may change; and

iii) the hydrogen peroxide formed during the enzymatic reaction can be electrochemically reduced under the same conditions, which leads to errors in the analysis.

Also known solution [G.G. Guilbault, G.J. Lubrano. Anal. Chim. Acta 64 (3) (1973) 439], in which the determination of hydrogen peroxide released during the enzymatic oxidation of glucose is used for glucose analysis.

This method also has several disadvantages. The use of the oxidation reaction of hydrogen peroxide at 0.6 V leads to the appearance of a systematic error in the analysis of blood due to the presence of a number of reducing agents (urate, ascorbate, paracetamol, and others), which are also oxidized at this potential. In addition, a suboptimal method of enzyme immobilization is used - covalent binding to the electrode surface - which can lead to a partial loss of enzyme activity due to disruption of its structure.

It is also known the decision [Karyakin AA, Karyakina EE, Mokrushina AV, Andreev EA. METHOD FOR MANUFACTURING A MICROBIOSENSOR FOR DETERMINING GLUCOSE OR LACTATE, RU 2580288], aimed at creating a microbiosensor using a two-electrode microsystem, Prussian blue (BL) and immobilized enzymes of glucose oxidase and lactate oxidase.

However, in this source of information, Nafion is used for the immobilization of glucose oxidase, and γ-aminopropyltriethoxysilane is used only for the immobilization of lactate oxidase. At the same time, despite the similar name and belonging to the class of oxidoreductases, glucose oxidase and lactate oxidase are fundamentally different enzymes in terms of the structure of the protein sequence (from primary to quaternary), the structure of the active center and size (lactate oxidase (LOD) is several times larger than glucose oxidase (GOD )), in addition, each enzyme requires a separate selection of a matrix for immobilization and immobilization conditions.

For example, immobilization of lactate oxidase into the Nafion matrix does not lead to the production of any sensitive biosensors. In addition, the substrates of these enzymes belong to different classes of compounds - glucose (for glucose oxidase) - sugars and lactate (for lactate oxidase) - hydroxy acids. The two-electrode microsensors mentioned in the source can be used for implantation and determination of glucose or lactate directly in blood, but not in laboratory flow-injection installations for analyzing a large number of diluted blood samples, in which the claimed biosensors are easily applicable. In addition, the mass production of microbiosensors is difficult, and their use requires special, more sensitive devices for measuring low currents and special insulation from external electromagnetic noise, since, due to the small area of the electrodes (<2 mm, the entire system, including external insulation) measurement of very small (10 ^-9 - 10 ^-12 A) currents.

The closest to the claimed is the solution presented in the article [A. Karyakin, E.A. Kotel'nikova et. al. Anal. Chem. 74 (2002) 1597], which allows with high sensitivity and precision to determine the concentration of glucose in various objects. To create biosensors for glucose, glassy carbon electrodes are used, electrochemically modified with a film of Prussian blue (BL), on top of which an enzyme-containing membrane based on Nafion is applied. A flow-injection system based on a biosensor has the following characteristics: a sensitivity coefficient of 50 mA / Mcm ² , a lower limit of detectable concentrations of 0.1 μM. The system allows the determination of glucose in diluted blood with minimal sample preparation. However, the use of glassy carbon electrodes is not suitable for the industrial production of biosensors due to the high costs of producing the electrode and the high cost of materials. In addition, each electrode is made individually by the master, which is unsuitable for creating replaceable units for a device - a glucose analyzer, which are mainly used as biosensors. The electrochemical method for the synthesis of BL is also inconvenient for the mass production of biosensors due to the high time required for production and the impossibility of automating this process, since each sensor is manufactured individually, in several stages, including the use of the device, which excludes the parallel production of several sensors.

Disclosure of invention

The technical problem of the claimed invention is the need to create an express, accurate, highly sensitive and selective biosensor, providing the ability to conduct a blood test (determination of glucose concentration) in laboratory conditions.

The technical result achieved with the use of the invention is to increase the sensitivity of the proposed biosensor.

The advantage of the proposed biosensor can also be attributed to the provision of the possibility of small-scale production of biosensors for glucose by using the technology of printing electrodes on a sheet of polyethylene terephthalate (about 350-400 electrodes per batch, production cycle 6 hours) and the technology of chemical deposition of BL (up to 60 sensors per batch , production cycle 2.5 hours).

The technical result is achieved through the use of polyalkoxysilane gel as a membrane-forming component, which improves the analytical characteristics of the biosensor. The technical result is also achieved by increasing the content of the enzyme in the enzyme-containing membrane, which became possible due to the replacement of the membrane-forming component with polyalkoxysilane gel.

The problem is solved by the fact that a planar biosensor for determining glucose in a liquid sample includes a silver chloride reference electrode, a working and auxiliary electrodes placed on a substrate, while a Prussian blue film and a membrane made of polyalkoxysilane containing glucose oxidase as an enzyme are applied to the working electrode. immobilized inside the membrane layer, while the reference electrode is located at a distance of 3-5 mm from the working electrode with the possibility of setting a constant zero potential on the working electrode. The area of the auxiliary electrode of the claimed biosensor is larger than the area of the working electrode, while all electrodes of the biosensor are made with the ability to connect to a potentiostat. The membrane containing the enzyme can be obtained by hydrolysis followed by polycondensation from a monomer selected from methyltrimethoxysilane, vinyltrimethoxysilane, γ-mercaptopropylmethyltrimethoxysilane, phenyltrimethoxysilane, benzyltriethoxysilane, γ -triethoxypropyl-glythoxysyl The membrane can also be obtained by incorporating the enzyme into the polyalkoxysilane gel directly upon its formation from the monomer. Such a membrane contains - 2-12 μg of glucose oxidase and is obtained from 0.5-2.5 _vol. % solution of γ-aminopropyltriethoxysilane in isopropyl alcohol with a water content of 15 _vol. %. The working electrode can be made with a radius of 0.8-1 mm and is located at a distance from the reference electrode no more than 5 mm. The biosensor can be screen printed and can have a thickness of 0.22-0.35 mm.

Brief Description of Drawings

The invention is illustrated by drawings, where

in fig. 1 shows a diagram of a planar electrode - the basis for the manufacture of a biosensor for glucose,

in fig. 2 shows a diagram of the action of the sensor material,

in fig. 3 shows a diagram of the sensitivity of glucose determination for a working electrode with a film containing YEAR and various membrane-forming components (concentration 0.5 _vol. % In the membrane-forming mixture),

in fig. 4 - a diagram of the sensitivity of glucose determination for a working electrode with a film containing GOD and various concentrations of γ-aminopropyltriethoxysilane and GOD in a membrane-forming mixture (3D),

in fig. 5 - a diagram of the sensitivity of glucose determination for a working electrode with a film containing GOD and various concentrations of γ-aminopropyltriethoxysilane and GOD in the membrane-forming mixture (2D),

in fig. 6 - an example of a calibration dependence for the claimed biosensor is presented,

and in FIG. 7 shows a graph of checking the correctness of biosensor readings by comparison with an accredited device (Eco-Basic glucose analyzer).

Positions in the figures indicate:

1 - substrate,

2 - auxiliary electrode,

3 - working electrode,

4 - reference electrode,

5 - photocurable insulator separating the electrodes,

6 - contacts,

7 - membrane based on polyalkoxysilane gel containing the enzyme YEAR,

8 - film of Prussian blue (BL),

9 - surface of the working electrode,

10 - Methyltrimethoxysilane,

11 - Vinyltrimethoxysilane,

12 - Phenyltrimethoxysilane,

13 - (γ-mercaptopropyl) trimethoxysilane,

14 - Benzyltriethoxysilane,

15 - Tolyltriethoxysilane,

16 - (γ-aminopropyl) triethoxysilane,

17 - (γ-glycidyloxypropyl) triethoxysilane

18 - nafion.

Implementation of the invention

The inventive biosensor includes a reference electrode, an auxiliary electrode and a working electrode modified with a sensor coating that allows selective determination of the target analyte - glucose (a diagram of a three-electrode printed structure on the basis of which the biosensor is created is shown in Fig. 1). The sensor coating includes a film of an electrocatalyst for the reduction of hydrogen peroxide (BL) and a biorecognition element (a polyalkoxysilane gel membrane containing an enzyme - YEAR) (the scheme of the sensor coating and its principle of operation are shown in Fig. 2).

A BL film and a membrane based on polyalkoxysilane gel, containing an enzyme - YEAR, are sequentially applied to the working electrode to obtain a sensor coating for glucose determination. Continuous homogeneous BL film 60-80 nm thick was applied according to the method [Borisova AV, Karyakina EE, Cosnier S., Karyakin AA // Electroanalysis. 2009. Vol. 21 P. 409]. The membrane containing the enzyme - YEAR, is obtained from a membrane-forming mixture with a concentration of γ-aminopropyltriethoxysilane of 0.5-2.5 _vol. % (Fig. 5, 6), or a number of other alkoxysilanes (methyltrimethoxysilane, vinyltrimethoxysilane, γ-mercaptopropylmethyltrimethoxysilane, phenyltrimethoxysilane, benzyltriethoxysilane, tolyltriethoxysilane, γ-glycidyloxypropyl _. % (Fig. 3) YEAR - 1-6 mg / ml and water - 15 _vol. % in isopropyl alcohol (Fig. 5, 6), according to the technique, which consists in mixing a solution of alkoxysilane monomer in isopropyl alcohol and an aqueous solution of the enzyme, followed by application to the electrode surface and drying at 4 ° C for 12 hours. the enzyme-catalyzed reaction of hydrolysis and polycondensation of the alkoxysilane monomer and a polyalkoxysilane gel is formed with the enzyme molecules incorporated inside it and retained in the film. The effect of increasing the sensitivity of the biosensor when using polyalkoxysilane gel can be achieved by creating a more favorable environment for the enzyme in such a matrix, due to which it exhibits greater activity.

The reference electrode is located at a distance of no more than 5 mm from the working electrode with the provision of the possibility of setting a constant zero potential on the working electrode.

The auxiliary electrode has an area larger than the area of the working electrode in order to reduce the influence of the processes taking place on the auxiliary electrode on the measured signal.

All electrodes are designed to be connected to a potentiostat.

The biosensor thickness is 0.22-0.35 mm. This thickness provides sufficient mechanical strength of the obtained sensors without leading to their excessive bulkiness; at the same time, the substrate material is not brittle and it is impossible to split it (in contrast to the ceramics used in some analogs).

To determine glucose, a biosensor based on a three-electrode printing system (TPN) is used (Fig. 1). TPR is made by screen printing on a plastic substrate made of polyethylene terephthalate (PET). Manufacturing of the printing system includes several stages: a layer of silver-containing paste is applied to the plastic substrate, which serves as conductive contacts, as well as the material of the reference electrode; then a layer of carbon paste is applied, which serves as the material of the auxiliary and working electrodes, then a layer of photocurable insulator is applied, which separates the working electrode from the reference electrode and the auxiliary electrode and closes the current-carrying contacts from contact with the solution in the cell. A method for manufacturing a biosensor sensor coating includes chemical modification of the surface of the working electrode with a BL film followed by immobilization of the GOD enzyme into a membrane on the surface of the modified electrode. The membrane consists of a polyalkoxysilane gel with an enzyme included in it - YEAR. The principle of operation of the sensor coating is based on the selective electrocatalytic reduction of hydrogen peroxide, released during the enzyme-catalyzed reaction, on the surface of the electrode with the applied BL film (Fig. 2). The membrane containing the enzyme - YEAR, is obtained by applying a drop of a membrane-forming mixture containing alkoxysilane with a concentration of 0.5-2.5 _vol. %, YEAR - 1-6 mg / ml and water - 15 _vol. % in isopropyl alcohol (Fig. 4), on the surface of the working electrode, followed by drying at 4 ° C for 1 hour.

The analytical signal of the biosensor is recorded by the amperometry method when a potential of 0 V is applied (relative to the silver chloride reference electrode, hereinafter referred to as CSE) to increase the current in the presence of the analyte - glucose.

Determination of glucose concentrations by the claimed biosensor is carried out as follows:

• At the first stage, a background buffer solution (50 mM KH ₂ PO ₄ / K ₂ NRO ₄ , 0.1 M KCl, pH 6.0) is passed through the electroanalytical system, which includes the proposed biosensor, while simultaneously measuring the current at the working electrode at a potential of 0 mV relative to internal silver chloride reference electrode. This stage ends with the establishment of a constant background current at the working electrode.

• At the second stage, the analyzed solution is injected into the injector loop (in the closed position) (the volume of the injector loop is 50 µl, the sample volume is 300 µl, the excess sample is poured out through the loop drain). After that, the injector is set in the open position, and a part of the injected sample from the loop (50 μL) enters the flow-injection cell through the hose system. In this case, the potentiostat measures the change in the cathodic current at the working electrode, which is expressed in the appearance of peaks on the experimental curve of the current versus time. The height of the peaks is proportional to the concentration of the analyte in the test solution. After measurement, the sample is washed out of the cell with the background buffer solution through the cell drain.

• Steps 1 and 2 are repeated for a number of model solutions with analyte concentrations of 1 μM - 1 mM. Each concentration must be determined at least three times. Based on the measurement results, a calibration dependence is plotted. Then, blood samples (diluted 100 times with the background buffer solution) are used as the analyzed solution. The concentration of glucose in blood samples is found from the previously constructed calibration dependence.

Sensor materials and a biosensor based on them make it possible to selectively distinguish the signal caused by the presence of glucose in a blood sample against the background of all other components of the sample. The sensitivity coefficient of the biosensor is more than 200 mA⋅M ^-1 ⋅cm ^-2 (Fig. 6) when determining glucose; the range of determined contents (DOS) is from 5 to 1000 μM. The biosensor has high reproducibility (systematic error does not exceed 3%), operational stability (over 50 measurements without the need for recalibration when determining glucose concentration). The DOS of the biosensor completely overlaps the range of physiological concentrations of glucose in the blood both in normal conditions and in pathology. The volume of a blood sample required for measurements is only 10 μl, which is 2 times less than for analogs. The analysis time for a biosensor does not exceed one minute.

Example 1. The creation of enzyme-containing membranes based on various polyalkoxysilanes was carried out by mixing a solution of polyalkoxysilane in isopropyl alcohol (0.5 _vol % in the final mixture) and an aqueous solution of YEAR (1.5 mg / ml in the final mixture), the water content in the final mixture was 15 _vol. %, since such a water content in water-organic mixtures provides the best retention of activity YEAR. The quality of the obtained membranes was judged by the value of the sensitivity coefficient of biosensors based on such membranes, measured as the slope of the dependence of the biosensor response (in mA cm ^-2 ) on the concentration of the model glucose solution to which this response was obtained (in M). Experimental data for biosensors with membranes based on polymethyltrimethoxysilane, polyvinyltrimethoxysilane, poly-γ-mercaptopropylmethyltrimethoxysilane, poly-γ-glycidyloxypropyltriethoxysilane, polyphenyltrimethoxysilane, polybenzyltriethoxysilane, are presented on fig. 3, for comparison, the data obtained for a biosensor with a membrane based on Nafion (prototype) under similar conditions are shown. The best sensitivity was possessed by biosensors with a membrane based on poly-γ-aminopropyltriethoxysilane, for which an additional selection of the content of various membrane components was carried out (Fig. 4, 5) and the possibility of a further increase in sensitivity with an increase in the amount of YEAR in the membrane was shown, which is possible due to the larger membrane capacity based on polyalkoxysilane compared to Nafion (for which an increase in the amount of enzyme in the membrane is impossible). An example of the dependence of the response of a biosensor with a membrane based on poly-γ-aminopropyltriethoxysilane on the concentration of model glucose solutions (calibration graph) is shown in Fig. 6. The sensitivity of such a biosensor exceeds 200 mA⋅M ^-1 ⋅cm ^-2 , which is much higher than the prototype (40-50 mA⋅M ^-1 ⋅cm ^-2 ).

Example 2. The correct operation of a biosensor based on a membrane containing 1.5 vol.% Γ-aminopropyltriethoxysilane and 4 mg / ml YEAR in a membrane-forming mixture was checked by analyzing standardized blood serum samples with a known concentration of glucose with normal (Spintpol H Normal) and pathological (Spintrol H Pathologic) content produced by Spinreact (Spain).

After measuring a series of responses to the samples, the response to the model solutions of the analyte was measured, while no change in the responses to the model solutions after the measurement of the samples was noticed. Data on certain concentrations of glucose and lactate are presented in table. 1. They did not differ from the data presented in the passport of samples. Thus, the developed biosensor can be used to analyze diluted blood samples.

The data obtained using the proposed biosensor (presented in the second column) coincide both with the data obtained using the Eco-Basic glucose analyzer (Care Diagnostica, Germany), which is widely used in clinical laboratories for blood analysis (presented in the second column), as well as and with data on the content of the determined component given in the certificate of reference materials.

Example 3. Using a biosensor, the concentration of glucose in diluted blood samples from donors was determined (Fig. 7). The sample preparation procedure was reduced to diluting blood samples by a factor of 100 with the background buffer solution.

The results obtained are in good agreement with the results obtained by the comparison method (Eco-Basic glucose analyzer), the Pearson correlation coefficient was 0.95.

Thus, the claimed technical result of the invention, which consists in ensuring an increase in the sensitivity of the biosensor, is achieved (in particular, the sensitivity coefficient is increased by a factor of 4.5 relative to the prototype), and, as a consequence, an increase in the accuracy of blood analysis is achieved using the proposed biosensor as part of an electroanalytical system , due to the elimination of systematic error, which, as a rule, is present when measuring by known analyzers. In addition, it becomes possible to use small-scale production of biosensors for glucose.

Claims (8)

1. A planar biosensor for determining glucose in a liquid sample, including a silver chloride reference electrode placed on a substrate, a working and auxiliary electrodes, characterized in that a film of Prussian blue is applied to the working electrode, and a membrane including glucose oxidase immobilized inside the membrane layer as an enzyme is made from poly-y-aminopropyltriethoxysilane, while the reference electrode is located at a distance of 3-5 mm from the working electrode with the possibility of setting a constant zero potential on the working electrode. 2. The biosensor according to claim 1, characterized in that the area of the auxiliary electrode is greater than the area of the working electrode, and all the electrodes of the biosensor are made with the possibility of being connected to a potentiostat. 3. Biosensor according to claim 1, characterized in that the membrane is obtained by incorporating the enzyme into the polyalkoxysilane gel directly during its formation from the monomer by hydrolysis with subsequent polycondensation. 4. Biosensor under item 1, characterized in that the membrane contains 2-12 μg of glucose oxidase and is obtained from 0.5-2.5 vol. % solution of y-aminopropyltriethoxysilane in isopropyl alcohol with a water content of 15 vol. %. 5. Biosensor according to claim 1, characterized in that the working electrode is made with a radius of 0.8-1 mm. 6. Biosensor according to claim 1, characterized in that the working electrode is located at a distance from the reference electrode of no more than 5 mm. 7. Biosensor according to claim 1, characterized in that it is made by screen printing. 8. Biosensor according to claim 1, characterized in that it has a thickness of 0.28-0.35 mm.

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