Manufacturing process of portable 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 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

Technical Field

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

[0002] During the oxygen metabolism process in the human body, harmful oxygen or free radicals called reactive oxygen species are produced as by-products. 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 continuously occurs in cell membranes, which causes oxidative damage to unsaturated lipids.

[0003] In the prior art, malondialdehyde is one of the measurement methods for determining the degree of lipid peroxidation. The methods mainly use the HLPC method and the TBARS method. The HLPC method requires the use of high-cost equipment to achieve, and the TBARS method requires relatively complex pretreatment of samples, 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 of a portable oxygen free radical detector, so that the obtained portable oxygen free radical detector can detect human oxygen free radicals conveniently, efficiently, in real time and at low cost.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: A manufacturing process of a portable oxygen free radical detector, comprising the following steps: S11: Mix superoxide dismutase, potassium ferricyanide, isothiazolinone preservative, hexammine ruthenium chloride, polyethylene glycol octyl phenyl ether, trehalose and bovine serum albumin, and stir evenly to obtain an enzyme preparation; S12: Mix the enzyme preparation with a polymer material solution to form a reagent layer slurry with viscosity; S13: Uniformly coat the reagent layer slurry on a substrate material and dry it; S14: Print electrode materials on the substrate material to form a working electrode and a counter electrode; S15: Place a silver electrode in an electrolyte containing chloride ions, apply a potential to cause a chlorination reaction on the silver surface to form a silver / silver chloride electrode; S16: Cover the reagent layer above the electrode, and make it tightly combined by pasting or hot pressing, and then cut it to obtain a test strip; S21: Prepare a detection instrument, which includes an electromotive force signal receiving unit, an electromotive force signal amplifying unit, an A / D conversion unit, a central processor and a display module that are 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 amplification unit is used to amplify the tiny electromotive force signal. The A / D conversion unit is used to convert the obtained electromotive force signal into a digital signal. The central processor is used to calculate the corresponding oxygen free radical concentration value according to the electromotive force digital signal. The display module is used to display the oxygen free radical concentration value.

[0006] Further, in the manufacturing process of the above 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 preservative in the total mass of the enzyme preparation is 0.1 - 0.3%; the mass percentage of hexammine ruthenium chloride in the total mass of the enzyme preparation is 16%; the mass percentage of polyethylene glycol octyl phenyl ether in the total mass of the enzyme preparation is 2%; the mass percentage of trehalose in the total mass of the enzyme preparation is 1.5%.

[0007] Further, in the manufacturing process of the above portable oxygen free radical detector, in S11, the stirring and mixing is specifically carried out by magnetic stirring, the stirring speed is controlled at 100 - 300 rpm, and the stirring time is 1 - 2 hours.

[0008] Further, in the manufacturing process of the above portable oxygen free radical detector, in S12, the mass ratio of the enzyme preparation to the polymer material solution is 70 - 90∶10 - 30.

[0009] Further, in the manufacturing process of the above 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.

[0010] Further, in the manufacturing process of the above portable oxygen free radical detector, in S14, the electrode material is selected from carbon electrode materials. The carbon paste is composed of carbon powder and binder. The printing is carried out by screen printing. The mesh number of the screen printing screen is 100 - 300 meshes, and the printing thickness is 50 μm.

[0011] Further, in the manufacturing process of the above portable oxygen free radical detector, in S15, the chloride ion-containing electrolyte is selected from sodium chloride solution or hydrochloric acid solution, the applied potential value is 0.2 - 0.3 V; the chlorination reaction time is 10 - 30 minutes.

[0012] Further, in the manufacturing process of the above portable oxygen free radical detector, in S16, it is tightly combined by hot pressing. The hot pressing temperature is 80 - 120 °C, the pressure is 0.1 - 0.5 MPa, and the hot pressing time is 1 - 3 seconds.

[0013] Further, in the manufacturing process of the above-mentioned portable oxygen free radical detector, in S12, the polymer material is selected from sodium carboxymethyl cellulose.

[0014] The present invention also protects the portable oxygen free radical detector obtained by the manufacturing process of the above-mentioned portable oxygen free radical detector.

[0015] The beneficial effects of the present invention are as follows: In the manufacturing process of the portable oxygen free radical instant detector involved in the present invention, the ability of superoxide dismutase to scavenge oxygen free radicals is utilized. It can catalyze the disproportionation reaction of oxygen free radicals to generate oxygen and hydrogen peroxide. The hydrogen peroxide generates an electrochemical reaction under the action of the working electrode, resulting in electron transfer, thereby generating a current signal. By detecting the strength of the generated current signal, the concentration of oxygen free radicals in the detection object can be judged. By using an electron mediator, the oxygen free radical test strip can achieve more stable and accurate monitoring of oxygen free radicals, reducing the influence of external factors on the measurement results, enabling the prepared portable oxygen free radical detector to detect human oxygen free radicals conveniently, efficiently, in real time and at low cost. This detector is not only easy to carry, but also can monitor in real time. It is a POCT device suitable for home use, capable of detecting anytime and anywhere and giving rapid results immediately. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic structural diagram of a test strip involved in the manufacturing process of a portable oxygen free radical real-time detector according to a specific embodiment of the present invention; Figure 2 It is a schematic structural diagram of a portable oxygen free radical real-time detector according to a specific embodiment of the present invention; Figure 3 It is a regression equation curve graph of the test data of a portable oxygen free radical real-time detector according to a specific embodiment of the present invention and an oxidative stress free radical detection analyzer (model FRAS5) of an Italian company for different blood samples; Label Description: 1. Test strip; 11. Substrate material layer; 12. Reagent layer; 13. Working electrode; 14. Counter electrode; 2. Detection instrument; 21. Screen. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] To describe in detail the technical content, achieved objectives and effects of the present invention, the following is described in conjunction with the embodiments and with reference to the accompanying drawings.

[0018] The present invention relates to a manufacturing process of a portable oxygen free radical detector, including the following steps: S11: Mix 5 mg / mL of superoxide dismutase (the activity of superoxide dismutase ≥ 60000 U / mg, purity ≥ 99%), 0.05 mg / mL of potassium ferricyanide, 0.03%wt of isothiazolinone preservative (PC - 300), 16%wt of hexammine ruthenium chloride, 2.0%wt of polyethylene glycol octyl phenyl ether, 1.5%wt of trehalose, and 5%wt of bovine serum albumin, and stir and mix evenly with a magnetic stirrer at a stirring speed of 200 rpm for 1.5 hours to fully dissolve and mix various reagents evenly to obtain an enzyme preparation; Among the components of the above enzyme preparation, polyethylene glycol octyl phenyl ether is a non - ionic surfactant and is a non - reactive component of the test strip, which helps to 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.

[0019] Among the components of the above enzyme preparation, the preservative PC - 300 (isothiazolinone preservative) is mainly a mixture of methylchloroisothiazolinone (CMIT) and methylisothiazolinone (MIT).

[0020] Among the components of the above enzyme preparation, potassium ferricyanide [K3Fe(CN)6] is an electron mediator. Superoxide dismutase (SOD) catalyzes the disproportionation reaction of oxygen free radicals (O 2- ·) to generate oxygen and hydrogen peroxide, while releasing electrons. These electrons are transferred to the electrode through the electron mediator to generate a current signal. By measuring the magnitude of the current, the concentration of oxygen free radicals can be calculated. By using an electron mediator, the oxygen free radical test strip can achieve more stable and accurate monitoring of oxygen free radicals, reducing the influence of external factors on the measurement results.

[0021] S12: Mix 80% of the enzyme preparation with 20% of the polymer material solution to form a reagent layer slurry with viscosity; The polymer material is selected from sodium carboxymethyl cellulose.

[0022] S13: Use a roll - coating device to evenly coat the reagent layer 12 slurry on the substrate material layer 11 (PET film) with a thickness of 0.1 mm, and dry it for 3 hours at 45 °C and a relative humidity of 45%; The 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 is required to be smooth and flat to ensure uniform reagent coating; S14: For the carbon electrode material, print the carbon paste (composed of carbon powder and binder) on the substrate material by screen printing to form a working electrode 13 and a counter electrode 14. The mesh number of the screen printing screen is 200 meshes, and the printing thickness is 50 μm; The working electrode uses a carbon electrode, which has good electrical conductivity and chemical stability; The counter electrode uses a carbon electrode, which is used in conjunction with the working electrode to provide a path for electron transfer; S15: Place the silver electrode in an electrolyte containing chloride ions (such as sodium chloride or hydrochloric acid solution), apply a potential of 0.3 V, and allow the silver surface to undergo a chlorination reaction for 20 minutes to form a silver / silver chloride electrode; S16: Cover the reagent layer 12 above the electrode and make it closely combined by hot pressing. The hot pressing temperature is 100 °C, the pressure is 0.3 MPa, and the hot pressing time is 2 seconds to form good contact between the reagent layer and the electrode, preventing the appearance of bubbles or delamination. After cutting, a test strip is prepared. The length of the test strip 1 is 27 - 33 mm, the width is 6 mm, and the error is ±0.1 mm to ensure that the test strip can be accurately inserted into the detector for detection; The structure of the test strip 1 refers to Figure 1 ; It should be noted that the manufacturing of the test strip is based on the following principle: During the electron transfer of the enzymatic reaction, the oxidation-reduction reaction process, or under the action of light and heat, the covalent bond of the compound molecule undergoes homolytic cleavage to form atoms or groups with unpaired electrons, becoming free radicals. Superoxide dismutase (SOD) has the ability to scavenge oxygen free radicals (O 2- ·), and it can catalyze the disproportionation reaction of oxygen free radicals (O 2- ·) to generate oxygen and hydrogen peroxide. Hydrogen peroxide undergoes an electrochemical reaction under the action of the working electrode, resulting in electron transfer, and thus generating a current signal. By detecting the current, the purpose of detecting the concentration of oxygen free radicals is achieved. The specific reaction equation is as follows: S21: Prepare the detection instrument 2. The detection instrument includes an electromotive force signal receiving unit, an electromotive force signal amplification unit, an A / D conversion unit, a central processor, and a display module that are electrically connected to each other; The structure of the detection instrument refers to Figure 2 ; The electromotive force signal receiving unit is used to receive the tiny electromotive force generated by the electrochemical reaction on the test strip. 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 obtained electromotive force signal into a digital signal. The central processor is used to calculate the corresponding oxygen free radical concentration value according to the electromotive force digital signal. The display module (screen 21) is used to display the oxygen free radical concentration value.

[0023] The detection instrument also includes a temperature acquisition unit and a regulated power supply unit, which are used to balance the interference of the power supply and environmental temperature on the test accuracy.

[0024] Instructions for using the detection instrument: Auto-sleep and wake-up function: After the portable tester is installed with a battery, when it is not in use, the screen turns off and it enters the sleep state 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 the standby state, the screen lights up, and a 100s countdown is started. If there is still no detection when the countdown time reaches, the screen turns off again and enters the sleep state.

[0025] Detection process: After blood collection, when the test strip is close to the blood, the blood will automatically enter the detection reaction area of the test strip due to the siphon phenomenon. After the system detects a weak electrical signal, it emits a "beep" sound again and enters the oxygen free radical detection state. The screen switches to a 10s countdown. During this short period, the central processing unit receives the digital signal from the A / D conversion module and processes the data according to the temperature signal collected by the temperature acquisition unit to obtain the concentration of oxygen free radicals in the blood. After the 10s countdown ends, the calculated data is stored in the storage unit and displayed on the screen.

[0026] Test strip discard: After the detection is completed, since the test strip is contaminated with blood, if it is pulled out by hand, it is easy to cause infection. Therefore, a pushing device is made in the test strip slot. By pushing the push rod, the test strip is pushed out of the slot by the push rod.

[0027] Control software: Through the control software and the buttons on the instrument, the date, time can be adjusted, and the data of the most recent 1 test, the average value of 10 tests, and the average value of 30 tests can be displayed in sequence.

[0028] Verification of test data: To verify the reliability of the data, different blood samples were detected using a portable oxygen free radical detector (referred to as portable) and an oxidative stress free radical detection analyzer (model FRAS5) from an Italian company. The experimental data is shown in the following table: The test results show that there is a large correlation between the two. Linear regression analysis is performed on the data, and the fitted linear regression equation is: y = 0.0307x - 0.2025, where x is the detected value of blood oxygen free radicals (u.carr) by the FRAS5 analyzer, and y is the detected value of blood oxygen free radicals (nmol / L) by the portable oxygen free radical detector involved in the present invention. Refer to Figure 3 , the discrete detection value relationship between y and x for each sample is very close to the regression equation curve, indicating that the detected value of the portable oxygen free radical detector involved in the present invention has high reliability.

[0029] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent transformation made using the content of the specification and drawings of the present invention, or directly or indirectly applied in the relevant technical fields, shall be included in the patent protection scope of the present invention by the same token.

Claims

1. The manufacturing process of a portable oxygen free radical detector, characterized in that, It includes the following steps: S11: Mix superoxide dismutase, potassium ferricyanide, isothiazolinone preservative, hexammine ruthenium chloride, polyethylene glycol octyl phenyl ether, trehalose and bovine serum albumin, stir and mix evenly to obtain an enzyme preparation; S12: Mix the enzyme preparation with a polymer material solution to form a reagent layer slurry with viscosity; S13: Uniformly coat the reagent layer slurry on a substrate material and dry it; S14: Print electrode materials on the substrate material to form a working electrode and a counter electrode; S15: Place a silver electrode in an electrolyte containing chloride ions, apply a potential to cause a chlorination reaction on the silver surface to form a silver / silver chloride electrode; S16: Cover the reagent layer above the electrode, make it tightly combined by pasting or hot pressing, and then cut it to obtain a test strip; S21: Prepare a detection instrument, which includes an electromotive force signal receiving unit, an electromotive force signal amplifying unit, an A / D conversion unit, a central processor and a display module that are 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 strip, 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 obtained electromotive force signal into a digital signal, the central processor is used to calculate 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 ≥ 20000 U / mg, and the purity ≥ 98%; the concentration of potassium ferricyanide is 0.01 - 0.1 mol / L, the mass percentage of the isothiazolinone preservative in the total mass of the enzyme preparation is 0.1 - 0.3%; the mass percentage of hexammine ruthenium chloride in the total mass of the enzyme preparation is 16%; the mass percentage of polyethylene glycol octyl phenyl ether in the total mass of the enzyme preparation is 2%; 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 specific operation of stirring and mixing evenly is to use magnetic stirring, the stirring speed is controlled at 100 - 300 rpm, and the stirring time is 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 materials, the carbon paste is composed of carbon powder and a binder, the printing is carried out by screen printing, the mesh number of the screen printing screen 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, the applied potential value is 0.2 - 0.3 V; 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, make it tightly combined by hot pressing, the hot pressing temperature is 80 - 120 °C, the pressure is 0.1 - 0.5 MPa, and the hot pressing time is 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 prepared by the manufacturing process of the portable oxygen free radical detector according to any one of claims 1 to 9.

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