Manufacturing process of portable and reliable oxygen free radical detector

Abstract

The invention relates to the technical field of oxygen free radical detection, in particular to a manufacturing process of a portable and reliable oxygen free radical detector. Comprising the following steps: mixing superoxide dismutase, potassium ferricyanide, an isothiazolinone preservative, hexammine ruthenium chloride, polyethylene glycol octylphenol ether, trehalose and bovine serum albumin, and uniformly stirring to prepare an enzyme preparation; mixing an enzyme preparation with the high polymer material solution to form reagent layer slurry with viscosity; uniformly coating the reagent layer slurry on a substrate material, and drying; printing an electrode material on the substrate material to form a working electrode and a counter electrode; the silver electrode is placed in an electrolyte containing chloride ions, potential is applied, the silver surface is subjected to a chlorination reaction, and a silver / silver chloride electrode is formed; covering the reagent layer above the electrode, tightly combining the reagent layer with the electrode in a sticking or hot pressing manner, and cutting to obtain the test paper; the prepared portable oxygen free radical detector can be used for conveniently and efficiently detecting oxygen free radicals of a human body at low cost.

Description

[0001] This case is a divisional application of the patent application with application number 202510929704.5, application date 2025-07-07, entitled "Manufacturing process of portable oxygen free radical detector". Technical Field

[0002] This invention relates to the field of oxygen free radical detection technology, and in particular to the manufacturing process of a portable and reliable oxygen free radical detector. Background Technology

[0003] During the process of oxygen metabolism in the human body, harmful oxygen or free radicals, also known as reactive oxygen species, are produced as byproducts. These oxygen free radicals cause various adverse effects on the human body. For example, they produce lipid peroxides in human tissues. Lipid peroxidation is a physiological process that occurs continuously in cell membranes, leading to oxidative damage to unsaturated lipids.

[0004] In the existing technology, malondialdehyde is one of the methods that can determine the degree of lipid peroxidation. The main methods used are HLPC and TBARS. HLPC requires expensive equipment, while TBARS requires complex sample pretreatment, resulting in poor convenience and low efficiency of detection. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a manufacturing process for a portable and reliable oxygen free radical detector, so that the resulting portable oxygen free radical detector can conveniently, efficiently, in real time and at low cost detect oxygen free radicals in the human body.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] The manufacturing process of a portable oxygen free radical detector includes the following steps:

[0008] S11: Superoxide dismutase, potassium ferricyanide, isothiazolinone preservatives, hexaammineruthenium chloride, polyethylene glycol octylphenyl ether, trehalose and bovine serum albumin are mixed and stirred to prepare an enzyme preparation.

[0009] S12: Mix the enzyme preparation with the polymer solution to form a viscous reagent slurry;

[0010] S13: Apply the reagent layer slurry evenly onto the substrate material and allow it to dry;

[0011] S14: Printing electrode material onto substrate material to form working electrode and counter electrode;

[0012] S15: Place the silver electrode in an electrolyte containing chloride ions, apply a potential, and cause a chlorination reaction on the silver surface to form a silver / silver chloride electrode.

[0013] S16: The reagent layer is placed over the electrode and bonded tightly by adhesive or hot pressing. After cutting, the test strip is obtained.

[0014] S21: Prepare a detection instrument, which includes an electromotive force signal receiving unit, an electromotive force signal amplification unit, an A / D conversion unit, a central processing unit, and a display module that are electrically connected to each other;

[0015] 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 amplification 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 calculate the corresponding oxygen free radical concentration value based on the electromotive force digital signal. The display module is used to display the oxygen free radical concentration value.

[0016] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in S11, the concentration of superoxide dismutase is 1-10 mg / mL, the activity of superoxide dismutase is ≥60000 U / mg, and the purity is ≥99%; the concentration of potassium ferricyanide is 0.01-0.1 mol / L; the mass percentage of isothiazolinone preservatives in the total enzyme preparation is 0.1-0.3%; the mass percentage of hexaammineruthenium chloride in the total enzyme preparation is 16%; the mass percentage of polyethylene glycol octylphenyl ether in the total enzyme preparation is 2%; and the mass percentage of trehalose in the total enzyme preparation is 1.5%.

[0017] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in step S11, the stirring and mixing is specifically carried out by magnetic stirring, with the stirring speed controlled at 100-300 rpm and the stirring time at 1-2 hours.

[0018] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in S12, the mass ratio of enzyme preparation to polymer material solution is 70-90:10-30.

[0019] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in step S13, the drying temperature is 30-60℃, the relative humidity is 30%-60%, and the drying time is 1-5 hours.

[0020] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in S14, the electrode material is selected from carbon electrode material, the carbon paste is composed of carbon powder and binder, and the printing is done by screen printing, with a screen mesh number of 100-300 mesh and a printing thickness of 50μm.

[0021] Furthermore, in the manufacturing process of the aforementioned 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; the chlorination reaction time is 10-30 minutes.

[0022] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in S16, the components are tightly bonded by hot pressing. The hot pressing temperature is 80-120℃, the pressure is 0.1-0.5MPa, and the hot pressing time is 1-3 seconds.

[0023] Furthermore, in the manufacturing process of the aforementioned portable oxygen free radical detector, in step S12, the polymer material is selected from sodium carboxymethyl cellulose.

[0024] This invention also protects the portable oxygen free radical detector manufactured using the above-mentioned manufacturing process.

[0025] The beneficial effects of this invention are as follows: The manufacturing process of the portable oxygen free radical instantaneous detector involved in this invention utilizes the ability of superoxide dismutase to scavenge oxygen free radicals. Superoxide dismutase catalyzes the dismutation reaction of oxygen free radicals, generating oxygen and hydrogen peroxide. Hydrogen peroxide undergoes an electrochemical reaction under the action of the working electrode, resulting in electron transfer and the generation of a current signal. The concentration of oxygen free radicals in the target object is determined by detecting the strength of the generated current signal. By using an electronic medium, the oxygen free radical test strip can achieve more stable and accurate oxygen free radical monitoring, reducing the influence of external factors on the measurement results. This allows the resulting portable oxygen free radical detector to conveniently, efficiently, in real-time, and at low cost detect oxygen free radicals in the human body. This detector is not only portable but also provides real-time monitoring, making it a POCT device suitable for home use, allowing for testing anytime, anywhere, and providing immediate and rapid results. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the 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.

[0027] Figure 2 This is a schematic diagram of the structure of a portable real-time oxygen free radical detector according to a specific embodiment of the present invention;

[0028] Figure 3 The regression equation curves of experimental data from different blood samples were obtained using a portable real-time oxygen free radical detector and an oxidative stress free radical detection and analysis instrument (model FRAS5) from an Italian company, as a specific embodiment of the present invention.

[0029] Label Explanation:

[0030] 1. Test strip; 11. Substrate layer; 12. Reagent layer; 13. Working electrode; 14. Counter electrode;

[0031] 2. Testing instruments; 21. Screen. Detailed Implementation

[0032] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0033] This invention relates to a manufacturing process for a portable oxygen free radical detector, comprising the following steps:

[0034] S11: Mix 5 mg / mL of superoxide dismutase (superoxide dismutase activity ≥60000 U / mg, purity ≥99%), 0.05 mg / mL of potassium ferricyanide, 0.03% wt of isothiazolinone preservative (PC-300), 16% wt of hexaammineruthenium chloride, 2.0% wt of polyethylene glycol octylphenyl ether, 1.5% wt of trehalose, and 5% wt of bovine serum albumin. Stir with a magnetic stirrer at 200 rpm for 1.5 hours to ensure that all reagents are fully dissolved and mixed evenly to obtain the enzyme preparation.

[0035] Among the enzyme preparations mentioned above, polyethylene glycol octylphenyl ether is a nonionic surfactant. As a non-reactive component of the test strip, it helps improve the wettability and dispersibility of the test strip, ensuring that the blood sample can be evenly distributed on the test strip, thereby improving the accuracy of the test.

[0036] Among the enzyme preparations mentioned above, the preservative PC-300 (isothiazolinone preservative) is mainly a mixture of methylchloroisothiazolinone (CMIT) and methylisothiazolinone (MIT).

[0037] Among the enzyme preparations mentioned above, potassium ferricyanide [K3Fe(CN)6] serves as an electron mediator, and superoxide dismutase (SOD) catalyzes the oxidation of oxygen free radicals (O2). 2- A disproportionation reaction occurs, producing oxygen and hydrogen peroxide, while releasing electrons. These electrons are transferred to the electrodes 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 can achieve more stable and accurate monitoring of oxygen free radicals, reducing the influence of external factors on the measurement results.

[0038] S12: Mix 80% of the enzyme preparation with 20% of the polymer solution to form a viscous reagent slurry;

[0039] The polymer material is selected from sodium carboxymethyl cellulose.

[0040] S13: Use a roller coating equipment to uniformly coat the reagent layer 12 slurry onto the base material layer 11 (PET film) with a thickness of 0.1 mm, and dry it for 3 hours at 45°C and 45% relative humidity.

[0041] PET film has good mechanical properties, chemical stability and dimensional stability, which can provide stable support for the reagent layer of the test strip. The surface is required to be smooth and flat to ensure uniform reagent coating.

[0042] S14: Carbon electrode material is printed onto a substrate material by screen printing carbon paste (composed of carbon powder and binder) to form working electrode 13 and counter electrode 14. The screen printing mesh number is 200 mesh and the printing thickness is 50μm.

[0043] The working electrode is a carbon electrode, which has good electrical conductivity and chemical stability;

[0044] The counter electrode is a carbon electrode, used in conjunction with the working electrode to provide a pathway for electron transfer;

[0045] S15: Place the silver electrode in an electrolyte containing chloride ions (such as sodium chloride or hydrochloric acid solution), apply a potential of 0.3V, and allow the silver surface to undergo a chlorination reaction for 20 minutes to form a silver / silver chloride electrode.

[0046] S16: Cover the electrode with reagent layer 12 and bond it tightly by hot pressing. The hot pressing temperature is 100℃, the pressure is 0.3MPa, and the hot pressing time is 2 seconds. This ensures good contact between the reagent layer and the electrode, preventing air bubbles or delamination. After cutting, the test strip 1 is obtained. The length of the test strip 1 is 27-33mm, the width is 6mm, and the error is ±0.1mm, ensuring that the test strip can be accurately inserted into the detector for testing. The structure of test strip 1 is as follows: Figure 1 ;

[0047] It should be noted that the test strips are manufactured based on the following principles:

[0048] During enzymatic reactions involving electron transfer, redox reactions, or under photothermal conditions, covalent bonds in compound molecules undergo homolytic cleavage, forming atoms or groups with unpaired electrons, which become free radicals. Superoxide dismutase (SOD) has the ability to scavenge oxygen free radicals (O2). 2- Its ability to catalyze oxygen free radicals (O2) 2- • A disproportionation reaction occurs, producing oxygen and hydrogen peroxide. Hydrogen peroxide undergoes an electrochemical reaction under the influence of the working electrode, resulting in electron transfer and the generation of a current signal. By detecting this current, the concentration of oxygen free radicals can be detected. The specific reaction equation is as follows:

[0049]

[0050] H₂O₂→O₂+2H₂ + +2e -

[0051] S21: Prepare detection instrument 2, which includes an electromotive force signal receiving unit, an electromotive force signal amplification unit, an A / D conversion unit, a central processing unit, and a display module that are electrically connected to each other; the structure of the detection instrument is as follows. Figure 2 ;

[0052] 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 amplification 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 calculate the corresponding oxygen free radical concentration value based on the electromotive force digital signal. The display module (screen 21) is used to display the oxygen free radical concentration value.

[0053] The testing instrument also includes a temperature acquisition unit and a regulated power supply unit, which are used to balance the interference of power supply and ambient temperature on the test accuracy.

[0054] Instructions for use of the testing instrument:

[0055] Automatic sleep and wake-up function: After the portable tester is equipped with batteries, the screen will turn off when it is not in use, and it will be in sleep mode to save power. When the oxygen free radical detection test strip is inserted into the test strip slot, the system will beep and enter standby mode. The screen will light up and start a 100-second countdown. If no detection is detected after the countdown time, the screen will turn off and enter sleep mode again.

[0056] Detection Process: After blood is collected, the test strip is brought close to the blood. Due to the siphon effect, the blood automatically enters the detection reaction area of ​​the test strip. After the system detects a weak electrical signal, it emits a "beep" sound, entering the oxygen free radical detection state. 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 conversion module and processes the data based on the temperature signal collected by the temperature acquisition unit to determine the oxygen free radical concentration in the blood. After the 10-second countdown ends, the calculated data is stored in the storage unit and displayed on the screen.

[0057] Test strip disposal: After the test is completed, since the test strip contains blood, it is easy to cause infection if you pull it out by hand. Therefore, a push-out device is made in the paper strip slot. Push the push rod and the test strip is pushed out of the slot by the push rod.

[0058] Control software: The date and time can be adjusted by using the control software and buttons on the instrument, and the data of the most recent test, the average of 10 tests, and the average of 30 tests can be displayed in sequence.

[0059] Test data verification: To verify the reliability of the data, different blood samples were tested using a portable oxygen free radical detector (hereinafter referred to as portable) and an oxidative stress free radical detection and analysis instrument (model FRAS5) from an Italian company. The experimental data are shown in the table below:

[0060]

[0061] Test results show that the two are highly correlated. Linear regression analysis was performed on the data, and the fitted linear regression equation is as follows:

[0062] y = 0.0307x - 0.2025, where x is the blood oxygen free radical detection value (u.carr) measured by the FRAS5 analyzer, and y is the blood oxygen free radical detection value (nmol / L) measured by the portable oxygen free radical detector involved in this invention. (Refer to...) Figure 3 The discrete detection values ​​of y and x for each sample are very close to the regression equation curve, indicating that the detection values ​​of the portable oxygen free radical detector involved in this invention have high reliability.

[0063] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A manufacturing process for a portable and reliable oxygen free radical detector, characterized in that, Includes the following steps: S11: Superoxide dismutase, potassium ferricyanide, isothiazolinone preservatives, hexaammineruthenium chloride, polyethylene glycol octylphenyl ether, trehalose and bovine serum albumin are mixed and stirred to prepare an enzyme preparation. S12: Mix the enzyme preparation with the polymer solution to form a viscous reagent slurry; S13: Apply the reagent layer slurry evenly onto the substrate material and allow it to dry; S14: Printing electrode material onto substrate material to form working electrode and counter electrode; S15: Place the silver electrode in an electrolyte containing chloride ions, apply a potential, and cause a chlorination reaction on the silver surface to form a silver / silver chloride electrode. S16: Cover the working electrode with the reagent layer, and then bond it tightly by adhesive or hot pressing. After cutting, the test strip is obtained. S21: Prepare a detection instrument, which includes an electromotive force signal receiving unit, an electromotive force signal amplification unit, an A / D conversion unit, a central processing unit, and a display module that are electrically connected to each other; the display module is a screen. The electromotive force (EMF) signal receiving unit receives the minute EMF generated by the electrochemical reaction on the test strip. The EMF signal amplification unit amplifies the minute EMF signal. The A / D conversion unit converts the acquired EMF signal into a digital signal. The central processing unit calculates the corresponding oxygen free radical concentration value based on the digital EMF signal. The display module displays the oxygen free radical concentration value. The instrument integrates automatic sleep and wake-up functions: when the portable tester is equipped with batteries, the screen is turned off when not in use, and it is in sleep mode to save power. When the oxygen free radical test strip is inserted into the test strip slot, the system emits a "beep" sound, the system enters standby mode, the screen lights up, and a 100-second countdown begins. If no detection is performed after the countdown, the screen turns off again and the system enters sleep mode.

2. The manufacturing process of the portable and reliable 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 ≥20000 U / mg, and the purity is ≥98%; the concentration of potassium ferricyanide is 0.01-0.1 mol / L; isothiazolinone preservatives account for 0.1-0.3% of the total mass of the enzyme preparation; hexaammineruthenium chloride accounts for 16% of the total mass of the enzyme preparation; 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.

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

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

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

6. The manufacturing process of the portable and reliable 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 binder, and the printing is screen printing with a screen mesh number of 100-300 mesh and a printing thickness of 50μm.

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

9. The manufacturing process of the portable and reliable 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 radical detector manufactured using the manufacturing process of any one of claims 1 to 9.

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