Manufacturing process of portable oxygen free radical detector

Abstract

The invention relates to the technical field of oxygen free radical detection, and in particular to a manufacturing process of a portable oxygen free radical detector. The method comprises the following steps: mixing superoxide dismutase, potassium ferricyanide, an isothiazolinone preservative, hexaammineruthenium chloride, polyethylene glycol octylphenyl ether, trehalose and bovine serum albumin, stirring and mixing to obtain an enzyme preparation; mixing the enzyme preparation with a polymer material solution to form a reagent layer slurry with viscosity; uniformly coating the reagent layer slurry on a base material, and drying; printing an electrode material on the base material to form a working electrode and a counter electrode; placing a silver electrode in an electrolyte containing chloride ions, applying a potential to cause a chlorination reaction on the silver surface, and forming a silver / silver chloride electrode; covering the electrode with a reagent layer, bonding them tightly by gluing or hot pressing, and then cutting to obtain a test paper. The portable oxygen free radical detector thus obtained can detect oxygen free radicals in the human body conveniently, efficiently and at low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of oxygen free radical detection, in particular to a manufacturing process of a portable oxygen free radical detector. Background Art

[0002] During oxygen metabolism in the human body, harmful oxygen or free radicals called reactive oxygen species are produced as byproducts. These oxygen free radicals have various adverse effects on the human body. For example, they produce lipid peroxides inside human tissues. Lipid peroxidation is a physiological process that occurs continuously in cell membranes, which leads to oxidative damage to unsaturated lipids.

[0003] In the existing technology, malondialdehyde is one of the measurement methods that can determine the degree of lipid peroxidation. The method mainly uses the HLPC method and the TBARS method. The HLPC method requires the use of expensive equipment to implement, and the TBARS method requires more complicated pre-treatment of the sample, resulting in poor detection convenience and low efficiency. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a manufacturing process for a portable oxygen free radical detector, so that the manufactured portable oxygen free radical detector can detect oxygen free radicals in the human body conveniently, efficiently, in real time and at low cost.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] The manufacturing process of the portable oxygen free radical detector includes the following steps:

[0007] S11: mixing superoxide dismutase, potassium ferricyanide, an isothiazolinone preservative, hexaammineruthenium chloride, polyethylene glycol octylphenyl ether, trehalose, and bovine serum albumin, and stirring to obtain an enzyme preparation;

[0008] S12: mixing the enzyme preparation and the polymer material solution to form a reagent layer slurry with viscosity;

[0009] S13: evenly coating the reagent layer slurry on the base material and drying;

[0010] S14: printing the electrode material on the substrate material to form a working electrode and a counter electrode;

[0011] S15: placing the silver electrode in an electrolyte containing chloride ions, applying a potential to cause a chlorination reaction on the silver surface to form a silver / silver chloride electrode;

[0012] S16: Covering the reagent layer on the electrode, bonding them tightly by gluing or hot pressing, and then cutting to prepare a test paper;

[0013] S21: preparing a detection instrument, the detection instrument including an electromotive force signal receiving unit, an electromotive force signal amplifying unit, an A / D conversion unit, a central processing unit, and a display module electrically connected to each other;

[0014] The electromotive force signal receiving unit is used to receive the tiny electromotive force generated by the electrochemical reaction on the test paper, the electromotive force signal amplifying unit is used to amplify the tiny electromotive force signal, the A / D conversion unit is used to convert the acquired electromotive force signal into a digital signal, the central processing unit is used to convert the corresponding oxygen free radical concentration value according to the electromotive force digital signal, and the display module is used to display the oxygen free radical concentration value.

[0015] Furthermore, in the manufacturing process of the above-mentioned portable oxygen free radical detector, in S11, the concentration of superoxide dismutase is 1-10 mg / mL, the activity of superoxide dismutase is ≥60,000 U / mg, and the purity is ≥99%; the concentration of potassium ferrocyanide is 0.01-0.1 mol / L, the mass percentage of isothiazolinone preservatives in the total mass of the enzyme preparation is 0.1-0.3%; the mass percentage of hexaammineruthenium chloride in the total mass of the enzyme preparation is 16%; polyethylene glycol octylphenyl ether accounts for 2% of the total mass of the enzyme preparation; and trehalose accounts for 1.5% of the total mass of the enzyme preparation.

[0016] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S11, the stirring and mixing is specifically performed by magnetic stirring, the stirring speed is controlled at 100-300 rpm, and the stirring time is 1-2 hours.

[0017] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S12, the mass ratio of the enzyme preparation to the polymer material solution is 70-90:10-30.

[0018] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S13, the drying temperature is 30-60° C., the relative humidity is 30%-60%, and the drying time is 1-5 hours.

[0019] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S14, the electrode material is selected from a carbon electrode material, the carbon paste is composed of carbon powder and a binder, and the printing is performed by screen printing, the mesh size of the screen printing is 100-300 meshes, and the printing thickness is 50 μm.

[0020] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S15, the electrolyte containing chloride ions is selected from sodium chloride solution or hydrochloric acid solution, and the applied potential value is 0.2-0.3V; and the chlorination reaction time is 10-30 minutes.

[0021] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S16, the components are tightly combined by hot pressing, with a hot pressing temperature of 80-120° C., a pressure of 0.1-0.5 MPa, and a hot pressing time of 1-3 seconds.

[0022] Furthermore, in the manufacturing process of the portable oxygen free radical detector, in S12, the polymer material is selected from sodium carboxymethyl cellulose.

[0023] The present invention also protects the portable oxygen free radical detector manufactured by the manufacturing process of the portable oxygen free radical detector.

[0024] The present invention provides the following beneficial effects: The manufacturing process for the portable instant oxygen free radical detector utilizes the ability of superoxide dismutase to scavenge oxygen free radicals. It catalyzes the dismutation reaction of oxygen free radicals to generate oxygen and hydrogen peroxide. The hydrogen peroxide, under the action of the working electrode, undergoes an electrochemical reaction, resulting in electron transfer and a current signal. The strength of the generated current signal is then used to determine the concentration of oxygen free radicals in the test subject. By using an electron mediator, the oxygen free radical detection test strip achieves more stable and accurate oxygen free radical monitoring, reducing the influence of external factors on the measurement results. This portable oxygen free radical detector enables convenient, efficient, real-time, and low-cost detection of human oxygen free radicals. The detector is not only portable but also capable of real-time monitoring, making it a point-of-care (POCT) device suitable for home use, allowing for on-the-go testing and rapid, immediate results. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic structural diagram of a test strip involved in the manufacturing process of a portable real-time oxygen free radical detector according to a specific embodiment of the present invention;

[0026] Figure 2 This is a structural schematic diagram of a portable real-time oxygen free radical detector according to a specific embodiment of the present invention;

[0027] Figure 3 This is a regression equation graph of experimental data of different blood samples detected by a portable real-time oxygen free radical detector according to a specific embodiment of the present invention and an oxidative stress free radical detection analyzer (model FRAS5) from an Italian company;

[0028] Description of labels:

[0029] 1. Test strip; 11. Base material layer; 12. Reagent layer; 13. Working electrode; 14. Counter electrode;

[0030] 2. Testing instrument; 21. Screen. DETAILED DESCRIPTION

[0031] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following description is given in conjunction with the embodiments and accompanying drawings.

[0032] The present invention relates to a manufacturing process of a portable oxygen free radical detector, comprising the following steps:

[0033] S11: 5 mg / mL superoxide dismutase (superoxide dismutase activity ≥ 60,000 U / mg, purity ≥ 99%), 0.05 mg / mL potassium ferricyanide, 0.03% wt isothiazolinone preservative (PC-300), 16% wt hexaammineruthenium chloride, 2.0% wt polyethylene glycol octylphenyl ether, 1.5% wt trehalose, and 5% wt bovine serum albumin were mixed, and the mixture was stirred with a magnetic stirrer at a speed of 200 rpm for 1.5 hours to fully dissolve and mix the various reagents, thereby preparing an enzyme preparation;

[0034] Among the above enzyme preparation ingredients, polyethylene glycol octylphenyl ether is a non-ionic surfactant. As a non-reactive component of the test paper, it helps to improve the wettability and dispersibility of the test paper, ensuring that the blood sample can be evenly distributed on the test paper, thereby improving the accuracy of the test.

[0035] Among the above enzyme preparation ingredients, the preservative PC-300 (isothiazolinone preservative) is mainly composed of a mixture of methylchloroisothiazolinone (CMIT) and methylisothiazolinone (MIT).

[0036] Among the above enzyme preparations, potassium ferrocyanide [K3Fe(CN)6] is an electron mediator, and superoxide dismutase (SOD) catalyzes the oxidation of oxygen free radicals (O 2- ·) undergoes a disproportionation reaction, generating oxygen and hydrogen peroxide while releasing electrons. These electrons are transferred to the electrode via an electron mediator, generating a current signal. By measuring the magnitude of this current, the concentration of oxygen free radicals can be calculated. By using an electron mediator, oxygen free radical detection test strips enable more stable and accurate oxygen free radical monitoring, reducing the influence of external factors on measurement results.

[0037] S12: mixing 80% of the enzyme preparation with 20% of the polymer material solution to form a reagent layer slurry having viscosity;

[0038] The polymer material is selected from sodium carboxymethyl cellulose.

[0039] S13: Use a roller coating device to evenly coat the reagent layer 12 slurry on the base material layer 11 (PET film) with a thickness of 0.1 mm, and dry it at 45°C and a relative humidity of 45% for 3 hours;

[0040] PET film has good mechanical properties, chemical stability and dimensional stability, and can provide stable support for the reagent layer of the test strip. The surface must be smooth and flat to ensure uniform coating of the reagent.

[0041] S14: Printing a carbon electrode material by screen printing a carbon paste (composed of carbon powder and a binder) on a substrate material to form a working electrode 13 and a counter electrode 14, wherein the screen printing screen has a mesh size of 200 and a printing thickness of 50 μm;

[0042] The working electrode adopts carbon electrode, which has good conductivity and chemical stability;

[0043] The counter electrode is a carbon electrode, which is used in conjunction with the working electrode to provide a pathway for electron transfer.

[0044] S15: placing the silver electrode in an electrolyte containing chloride ions (such as sodium chloride or hydrochloric acid solution), applying a potential of 0.3 V, and causing a chlorination reaction on the silver surface for 20 minutes to form a silver / silver chloride electrode;

[0045] S16: Cover the reagent layer 12 on the electrode and make it tightly bonded by hot pressing. The hot pressing temperature is 100°C, the pressure is 0.3MPa, and the hot pressing time is 2 seconds. Make good contact between the reagent layer and the electrode to prevent bubbles or delamination. After cutting, the test paper is made. The length of the test paper strip 1 is 27-33mm, the width is 6mm, and the error is ±0.1mm. Ensure that the test strip can be accurately inserted into the detector and tested; the structure of the test strip 1 refers to Figure 1 ;

[0046] It should be noted that the manufacture of test strips is based on the following principles:

[0047] During the electron transfer of enzymatic reactions, redox reactions, or under the action of light and heat, the covalent bonds of compound molecules are split to form atoms or groups with unpaired electrons, which become free radicals. Superoxide dismutase (SOD) has the function of scavenging oxygen free radicals (O 2- ·) ability, which can catalyze oxygen free radicals (O 2- ·) undergoes a disproportionation reaction, generating oxygen and hydrogen peroxide. Hydrogen peroxide undergoes an electrochemical reaction at the working electrode, causing electron transfer and generating a current signal. By detecting the current, the concentration of oxygen free radicals can be measured. The specific reaction equation is as follows:

[0048]

[0049] S21: Prepare a detection instrument 2, which includes an electromotive force signal receiving unit, an electromotive force signal amplifying unit, an A / D conversion unit, a central processing unit, and a display module electrically connected to each other; the structure of the detection instrument refers to Figure 2 ;

[0050] The electromotive force signal receiving unit is used to receive the tiny electromotive force generated by the electrochemical reaction on the test paper, the electromotive force signal amplifying unit is used to amplify the tiny electromotive force signal, the A / D conversion unit is used to convert the acquired electromotive force signal into a digital signal, the central processing unit is used to convert the corresponding oxygen free radical concentration value based on the electromotive force digital signal, and the display module (screen 21) is used to display the oxygen free radical concentration value.

[0051] The testing instrument also includes a temperature acquisition unit and a voltage-stabilized power supply unit, which are used to balance the interference of power supply and ambient temperature on the test accuracy.

[0052] Instructions for use of the testing instrument:

[0053] Automatic sleep and wake-up function: After the portable tester is equipped with batteries, the screen will be turned off when not in use, and it will be in sleep mode to save energy. When the oxygen free radical detection test strip is inserted into the test strip slot, the system will emit a "beep" sound, the system will be in standby mode, the screen will be lit, and a 100s countdown will be counted. If there is still no detection when the timer expires, the screen will be turned off and it will enter sleep mode.

[0054] Testing process: After blood is drawn, the test strip is placed near the blood. Siphoning will automatically draw blood into the test strip's detection reaction area. Once the system detects a weak electrical signal, it beeps again, signaling the start of oxygen free radical detection. The screen then switches to a 10-second countdown. During this brief period, the central processing unit receives the digital signal from the A / D converter module and processes the data based on the temperature signal collected by the temperature acquisition unit to determine the concentration of oxygen free radicals in the blood. At the end of the 10-second countdown, the calculated data is stored in the storage unit and displayed on the screen.

[0055] Discarding the test strip: After the test is completed, since the test strip contains blood, it is easy to cause infection if it is pulled out by hand. Therefore, a push-out device is made in the paper strip slot. Push the push rod and the edge of the test strip will be pushed out of the slot by the push rod.

[0056] Control software: Through the control software and the buttons on the instrument, the date and time can be adjusted, and the data of the most recent test, the average value of 10 times, and the average value of 30 times can be displayed in sequence.

[0057] Test data verification: To verify the reliability of the data, different blood samples were tested using a portable oxygen free radical detector (referred to as portable) and an Italian company's oxidative stress free radical detection analyzer (model FRAS5). The experimental data are shown in the following table:

[0058]

[0059] The test results show that there is a large correlation between the two. The linear regression analysis of the data is performed, and the fitted linear regression equation is:

[0060] y = 0.0307x -0.2025, where x is the blood oxygen free radical detection value (u.carr) detected by the FRAS5 analyzer, and y is the blood oxygen free radical detection value (nmol / L) detected by the portable oxygen free radical detector of the present invention. Figure 3 The relationship between the discrete detection values ​​of y and x for each sample is very close to the regression equation curve, indicating that the detection values ​​of the portable oxygen free radical detector involved in the present invention have high reliability.

[0061] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's description and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A manufacturing process for a portable oxygen free radical detector, characterized in that: The following steps are involved: S11: mixing superoxide dismutase, potassium ferricyanide, an isothiazolinone preservative, hexaammineruthenium chloride, polyethylene glycol octylphenyl ether, trehalose, and bovine serum albumin, and stirring to obtain an enzyme preparation; S12: mixing the enzyme preparation and the polymer material solution to form a reagent layer slurry with viscosity; S13: evenly coating the reagent layer slurry on the base material and drying; S14: printing the electrode material on the substrate material to form a working electrode and a counter electrode; S15: placing the silver electrode in an electrolyte containing chloride ions, applying a potential to cause a chlorination reaction on the silver surface to form a silver / silver chloride electrode; S16: Covering the reagent layer on the working electrode, bonding them tightly by gluing or hot pressing, and then cutting to prepare a test paper; S21: preparing a detection instrument, the detection instrument including an electromotive force signal receiving unit, an electromotive force signal amplifying unit, an A / D conversion unit, a central processing unit, and a display module electrically connected to each other; The electromotive force signal receiving unit is used to receive the tiny electromotive force generated by the electrochemical reaction on the test paper, the electromotive force signal amplifying unit is used to amplify the tiny electromotive force signal, the A / D conversion unit is used to convert the acquired electromotive force signal into a digital signal, the central processing unit is used to convert the corresponding oxygen free radical concentration value according to the electromotive force digital signal, and the display module is used to display the oxygen free radical concentration value.

2. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S11, the concentration of superoxide dismutase is 1-10 mg / mL, the activity of superoxide dismutase is ≥20,000 U / mg, and the purity is ≥98%; the concentration of potassium ferrocyanide is 0.01-0.1 mol / L, the mass percentage of isothiazolinone preservatives in the total mass of the enzyme preparation is 0.1-0.3%; the mass percentage of hexaammineruthenium chloride in the total mass of the enzyme preparation is 16%; the mass percentage of polyethylene glycol octylphenyl ether in the total mass of the enzyme preparation is 2%; and the mass percentage of trehalose in the total mass of the enzyme preparation is 1.5%.

3. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S11, the stirring and mixing is specifically performed by magnetic stirring, with the stirring speed controlled at 100-300 rpm and the stirring time being 1-2 hours.

4. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S12, the mass ratio of the enzyme preparation to the polymer material solution is 70-90:10-30.

5. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S13, the drying temperature is 30-60° C., the relative humidity is 30%-60%, and the drying time is 1-5 hours.

6. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S14, the electrode material is selected from carbon electrode material, the carbon paste is composed of carbon powder and a binder, and the printing is performed by screen printing, the mesh number of the screen printing is 100-300 meshes, and the printing thickness is 50 μm.

7. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S15, the electrolyte containing chloride ions is selected from sodium chloride solution or hydrochloric acid solution, and the applied potential value is 0.2-0.3V; the chlorination reaction time is 10-30 minutes.

8. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S16, the components are tightly bonded by hot pressing, with a hot pressing temperature of 80-120° C., a pressure of 0.1-0.5 MPa, and a hot pressing time of 1-3 seconds.

9. The manufacturing process of the portable oxygen free radical detector according to claim 1, characterized in that: In S12, the polymer material is selected from sodium carboxymethyl cellulose.

10. A portable oxygen free radical detector manufactured by the manufacturing process of the portable oxygen free radical detector according to any one of claims 1 to 9.

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