CN114235925A

CN114235925A - Anti-interference electrochemical uric acid test paper and preparation method thereof

Info

Publication number: CN114235925A
Application number: CN202111548761.7A
Authority: CN
Inventors: 葛艳秋; 黄晶; 秦玉
Original assignee: Nanjing Jingjie Biotechnology Co ltd
Current assignee: Nanjing Jingjie Biotechnology Co ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-03-25

Abstract

Compared with the prior art, the anti-interference electrochemical uric acid test paper and the preparation method thereof disclosed by the application comprise the following steps: the anti-interference area is ingeniously designed at the inlet of the siphon pool of the reaction window, blood firstly flows through the anti-interference area containing ascorbic acid oxidase in the siphon pool through siphon action, ascorbic acid in blood samples passes through the anti-interference area to rapidly react with the ascorbic acid oxidase, interference of the ascorbic acid is eliminated, and then the blood flows into the reaction area to react with the urate oxidase and a water-soluble electronic mediator, so that the accuracy of determining different individual difference uric acid is improved. Meanwhile, the metal electrode is combined to accelerate the transmission of electrons, and the reduction current is carried out at a lower negative potential for detecting signals, so that the background current is further reduced, the sensitivity is improved, and the detection time can be reduced to be within 10 seconds. The interference enzyme removing layer is combined, the effect of removing the ascorbic acid is obvious, the sensitivity is high, and the accuracy is good.

Description

Anti-interference electrochemical uric acid test paper and preparation method thereof

Technical Field

The invention belongs to the technical field of medical equipment, and particularly relates to anti-interference electrochemical uric acid test paper and a preparation method thereof.

Background

Uric acid is the end product of purine metabolism and is present in the blood in vivo essentially in the free form of monosodium urate. Under normal purine diet and 2 continuous fasting tests on non-same day, male is more than 416 mu mol/L (7mg/dl), female is more than 357 mu mol/L (6mg/dl), and the hyperuricemia can be judged. The greater the likelihood of gout developing in the next 5 years of long-term hyperuricemia. Uric acid has a close relationship with kidney diseases, and can directly cause microangiopathy of glomerular arteriole, resulting in chronic kidney diseases.

Along with the improvement of living standard of people, the intake of high-purine diet is increased, and the blood uric acid is easily increased due to long-term high-fat and high-protein diet, and the addition of work tension, irregular work and rest, abnormal diet of three meals, night stay and the like. The disposable electrochemical uric acid test paper is used for detecting the concentration of uric acid in blood at home, so that the concentration of uric acid in the blood at that time can be obtained, the user is helped to know the physical condition of the user in time and adjust the diet structure of the user according to the physical condition, or the doctor is led to the hospital to adjust the dosage of the medicine according to daily monitoring data. It has been reported that strict diet control is the first choice for controlling hyperuricemia.

At present, the main methods for detecting uric acid in hospitals are a chemiluminescence method, a photochemical colorimetry, a high performance liquid chromatography and an electrochemical method. The chemiluminescence method can not test whole blood, the sample needs to be processed in the early stage, the test process is complicated, and the test time is long. The photochemical method needs large sample size, for example, the wound surface is large due to the detection of fingertip blood, the detection time is long, the detection is easily affected by reduction interference substances, and the result is not accurate enough. The electrochemical method has the advantages of high sensitivity, short detection time, simple and convenient operation and the like, and the preparation is easy for batch production and low in price. Because the concentration of the ascorbic acid and the uric acid in a normal human body is similar and the ascorbic acid and the uric acid have relatively similar oxidation potentials, the ascorbic acid can be directly subjected to electrooxidation or oxidized by an oxidation type electron mediator on the surface of an electrode by using an oxidation current as an electrochemical test paper for detecting a signal. In addition, other endogenous or exogenous reductive interference substances in blood also participate in the generation process of the current signal, so that the detection background is high, and the measurement result is inaccurate. For electrochemical test paper using reduction current as detection signal, ascorbic acid can react with hydrogen peroxide, resulting in negative deviation of the measurement result. Therefore, the test strip which is simple and convenient to operate, small-sized, portable, small in blood consumption, suitable for fingertip blood, low in price, capable of avoiding interference of ascorbic acid and interference of other interference substances in blood and capable of accurately measuring the concentration of uric acid has important significance. In order to reduce the interference of ascorbic acid on the detection result, an anti-interference layer is added on the conventional test paper structure in the market, for example, chinese patents CN102507670B, CN208239340U and CN205067415U, etc. use a water-insoluble redox electron mediator, mix with carbon powder, resin, solvent and auxiliary agent, print on an insulating substrate in a screen printing manner, and then drop-drop a reagent layer containing ascorbic acid oxidase on the surface. Since a water-insoluble redox electron mediator is used, a reagent layer containing ascorbic acid oxidase is dropped on the surface during production to form a water-soluble anti-interference enzyme layer and a water-insoluble reaction enzyme layer. Therefore, when the sample is tested, the sample firstly passes through the upper ascorbic acid oxidase layer to consume the ascorbic acid in the sample. Then when the reaction signal passes through the bottom layer, namely the working electrode, the reaction signal is the uric acid reaction signal. However, this solution not only has a complicated process, but also increases the thickness of the test strip, making it difficult to control the reproducibility in the quantitative production process. And the water-insoluble electronic mediator is mixed with the carbon slurry, so that the contact reaction process of uric acid in a blood sample reaching the surface of the electrode and the electronic mediator is slow, the reaction speed is slow, and the detection time is long and the reaction specificity is poor. In addition, the oxidation potential used is high, eliminating the interference of ascorbic acid, but not avoiding the interference of other substances.

It is also common to form mixed enzymes by mixing interfering enzymes with the test substance enzymes, as described in patent application CN 101349667A. Although the method can avoid the superposition of two interfaces of water solubility and water insolubility in the method, the problems of low reaction speed, long addition time and poor reaction specificity are avoided. However, the method has very poor anti-interference effect, and the interference substance and the substance to be detected react with the electron mediator at the same time, so that the anti-interference capability of the test strip on the ascorbic acid cannot be obviously improved.

Therefore, the development of a method for effectively reducing and eliminating interference of ascorbic acid on uric acid test has important significance in improving detection accuracy, sensitivity and shortening detection time.

Disclosure of Invention

In order to realize the rapid and accurate test of the content of uric acid in a whole blood sample, the invention aims to provide a uric acid test strip for resisting ascorbic acid which is a main interfering substance in blood, and the low-sensitivity high-accuracy rapid detection of uric acid in whole blood is realized. The invention also provides a preparation method of the anti-interference electrochemical uric acid test strip.

The invention provides an anti-interference electrochemical uric acid test strip which comprises an insulating substrate (1), an electrode layer (2) positioned on the substrate, and a hydrophilic film layer (7) formed by combining a double-sided adhesive and a hydrophilic film, wherein the hydrophilic film layer (7) is adhered to one side of the electrode layer (2), and the electrode layer (2) is close to the hydrophilic film layer (7) and covers a label layer (9) so that part of electrodes are exposed; the hydrophilic film layer (7) is provided with a reagent window (10), and an anti-interference area (12) of an anti-interference reagent reacting with an interference substance in the blood sample and a reaction area (11) of a reagent reacting with uric acid in the blood sample are covered in the reagent window (10); the anti-interference area (12) and the reaction area (11) are arranged in tandem, and are adjacent or connected in tandem, preferably adjacent in tandem. At the moment, a reaction area (11), an anti-interference area (12) and a hydrophilic film layer (7) with a reagent window (10) form a siphon pool (8) which can suck a blood sample.

In order to overcome the defects of the prior art, the anti-interference electrochemical uric acid test paper provided by the invention is provided with a section of water-soluble anti-interference area (12) before blood enters the water-soluble reaction area, and the anti-interference area (12) contains biological enzyme and reaction substances which react with ascorbic acid. And a reaction area (11) is arranged adjacent to or connected with the anti-interference area, and the reaction area (11) contains a biological enzyme, an electron mediator and a reaction substance which react with uric acid. Blood firstly flows through a reagent window (10) to form a siphon pool (8) through siphon action, an anti-interference enzyme layer in the siphon pool, and interfering substances in blood samples rapidly react with ascorbic acid oxidase, so that the interference of ascorbic acid is effectively eliminated. Because the anti-interference area (12) and the reaction area (11) are both water-soluble, the blood sample can easily flow from the anti-interference area (12) to the reaction area (11), and the reaction speed and the detection speed are further accelerated.

The anti-interference electrochemical uric acid test paper comprises an electrode layer (2) and a test strip, wherein the electrode layer at least comprises a working electrode (3) and a reference electrode (4); besides the working electrode and the reference electrode, a sample in-place detection electrode (5), a switch-on electrode (6) and the like can be designed according to needs; the present invention does not specifically require the position of each electrode.

In the invention, a layer of biological enzyme and non-reaction substances thereof which can rapidly react with ascorbic acid substances in blood samples are covered at the position of an anti-interference area, and the biological enzyme is preferably ascorbic acid oxidase; the content of the biological enzyme is preferably 10 wt% to 20 wt% based on 100 wt% of the total weight of the reagent layer dry film.

The reaction area is covered with one or more biological enzymes which react with the substance to be detected, and preferably, the biological enzymes which react with the substance to be detected are urate oxidase and peroxidase.

The reaction zone further contains an electron mediator, preferably the electron mediator is a reduced electron mediator or an oxidized electron mediator, preferably the electron mediator is a water-soluble reduced electron mediator or an oxidized electron mediator, preferably the water-soluble electron mediator is a metal or a noble metal complex, more preferably the electron mediator is potassium ferricyanide or potassium ferrocyanide; the preferred content range of the electron mediator is 1 wt% to 30 wt% based on 100% of the total weight of the dry film of the reagent layer; it is further preferred that the content of the electron mediator is in the range of 15 wt% to 25 wt%.

According to different requirements, the anti-interference electrochemical uric acid test paper provided by the invention can use an oxidized electron mediator or a reduced electron mediator. The electron mediator is preferably a water-soluble electron mediator of a metal or noble metal complex; more preferably, when a water-soluble oxidized electron mediator is used, the excitation potential applied is 200mV to 400mV, preferably 300mV, using the oxidation current as a detection signal.

The principle of the reaction of the oxidation type electron mediator is as follows: uric acid in a blood sample reacts with an oxidation-type electron mediator under the action of urate oxidase to generate allantoin and a reduction-state electron mediator, the potential applied on the surface of an electrode by the reduction-state electron mediator is oxidized to generate oxidation current, the current is transmitted to the other end of the electrode by electrode conduction on a working electrode and a reference electrode and is connected with a receiving device of an instrument, and a current signal is converted into the concentration of uric acid in the device.

When a reduced electron mediator is used, the reduction current is used as a detection signal, and the reduced electron mediator is preferably water-soluble potassium ferrocyanide.

The principle of the reduced electron mediator reaction is as follows: hydrogen peroxide generated after the action of urate oxidase and uric acid participates in the reaction under the action of peroxidase, and the reduction type electron mediator is changed from a reduction state to an oxidation state; applying an externally applied low negative voltage to the metal electrode causes the electron mediator to return from the oxidized state to the reduced state, thereby generating a reduced current signal, the current is conducted to the other end of the working electrode and the reference electrode through electrode conduction, the current signal is converted into the concentration of uric acid inside the device by connecting with a receiving device of the instrument, and the electron mediator is potassium ferrocyanide according to the preferred embodiment of the invention. Based on the combined action of the metal electrode and the electron mediator, the strong signal current can be generated at a low potential, the applied excitation potential is-50 mV to-150 mV, preferably-80 mV, and the reduction current is the excitation potential of the detection signal. The method for indirectly detecting the hydrogen peroxide generated in the uric acid reaction process by taking the reduction current as the detection signal under the low negative potential not only reduces the background current and further improves the sensitivity, but also can avoid other oxidizing interfering substances in the blood sample from being reduced to generate the interference current under the potential by applying the lower negative excitation potential, thereby avoiding the interference of the oxidizing substances and further improving the detection accuracy. The uric acid test paper can accurately detect the content of uric acid in a whole blood sample within 2-10 seconds, and is rapid, accurate, high in sensitivity and convenient to use, and provided for domestic and medical people to screen gout hidden dangers.

The anti-interference area (12) and the reaction area (11) can also contain non-reactive substances, wherein the non-reactive substances comprise biological buffers, thickening agents, protective agents and surfactants.

The biological buffer solution comprises one or more of Tris-HCl buffer solution, phosphate buffer solution, ACES buffer solution, sodium acetate buffer solution, MES buffer solution, Good's buffer solution and glycine buffer solution.

Preferably, the biological buffer is phosphate buffer, and the pH buffer range is 7.4-9.0.

The thickener comprises one or more of cellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and carboxymethyl cellulose.

Preferably, the content of the thickening agent is in the range of 20-35% based on 100% of the total weight of the dry film of the reagent layer; the thickener functions to make the enzyme layer more uniform during the coating and drying process.

The protective agent is one or more of bovine serum albumin, gelatin, trehalose and sucrose; the surfactant is TritonX-100, Tween 20, sodium dodecyl sulfate, etc.

The preferable content range of the protective agent is 1 wt% to 50 wt% based on 100% of the total weight of the reagent layer dry film; the addition of sugar or protein protective agents to the reagents can protect the enzyme from being active for a longer time under dry conditions; and the stability of the uric acid test strip in long-term storage is enhanced.

The polymer insulating substrate material of the present invention may include, but is not limited to, PET sheet, PVC sheet, PP sheet, etc.; preferably 1mm to 1.5mm thick.

The electrode is a metal thin layer or carbon paste attached to a high polymer matrix material, and the electrode with the same function is correspondingly realized through screen mask sputtering/electroplating or laser etching; the metallic material may preferably be gold or an alloy thereof. The thickness of the metal layer is preferably between 5nm and 20nm, more preferably 15 nm.

Therefore, the invention also provides a method for manufacturing the anti-interference electrochemical uric acid test paper, which comprises the steps of preparing an electrode by the method, and then dotting the liquid anti-interference enzyme in the anti-interference area (12) and the reaction enzyme in the reaction area (11) in a liquid-spotting way (as shown in figure 2); drying at constant temperature in a drying tunnel, preferably at 45-55 deg.C to obtain dried reagent layer with uniform thickness; and finally, adhering a middle interlayer to obtain the electrochemical biosensor.

On the other hand, the invention also provides a method for detecting uric acid by using the anti-interference electrochemical uric acid test paper.

The invention has the following advantages:

1. the design is ingenious: due to the unique design of the electrochemical uric acid test paper, an anti-interference area (12) and a reaction area (11) are respectively arranged in a reagent window (10) and are arranged in front of and behind each other. Therefore, blood firstly flows through an anti-interference area (12) containing ascorbic acid oxidase in the siphon pool under the siphon action of the siphon pool (8), and ascorbic acid in blood sample passes through the anti-interference area to rapidly react with the ascorbic acid oxidase so as to eliminate ascorbic acid interference; then the blood flows into the reaction area (11) to react with urate oxidase and a water-soluble electron mediator, so that the accuracy of uric acid determination of different individual differences is improved.

2. The detection is quick: by using urate oxidase and a water-soluble electron mediator, the dissolution speed of urate in a blood sample reaching the surface of an electrode is higher, the probability of collision between the electron mediator and a target substance and enzyme molecules is increased, and an enzymatic reaction can be rapidly carried out. In addition, because the anti-interference area (12) and the reaction area (11) both use water-soluble medium materials, the detection sample (blood) moves rapidly through the siphon action of the siphon pool (8), and the detection speed is effectively improved. On the other hand, the metal alloy electrode plate prepared by the sputtering process is preferably high in conductivity, the electron transfer between the substrate and the film is well promoted on the pure gold electrode, the electron transfer electron conduction rate between the electrode and the electroactive substance is further increased, and therefore the current signal is strong, the determination time is shortened, and the sensitivity is high. The invention can realize the high-accuracy test within 2-10 seconds. Avoiding printing carbon electrodes: the problems of low electron transfer rate, poor conductivity, high energy consumption, poor stability, relatively low sensitivity, large detection error, inaccurate measurement result and the like are caused.

3. Sensitivity is high, and the accuracy is high: according to the invention, the working electrode in the reaction zone specifically reacts with uric acid by using urate oxidase to generate allantoin and hydrogen peroxide, and the generated hydrogen peroxide is indirectly used for detecting uric acid, so that the background value is low, the detection value linearity is high, the sensitivity is high, and the lowest test concentration can be 50 mu mol/L or less. Because the applied negative potential is smaller (-80mV), the oxidation-reduction potential of most oxidation-reduction substances in the organism is avoided, the interference of ascorbic acid is eliminated, the interference of other substances is also avoided, and the interference of coexisting electroactive substances is avoided. Therefore, the product of the invention has high detection accuracy, the CV value is less than 4.0 percent, and the detection of the target substance with higher reliability can be realized.

Drawings

The attached drawings are used for further understanding and explaining the design, the structure and the performance effect of the electrochemical uric acid test paper, and are not used for limiting the invention.

FIG. 1 is a schematic structural decomposition diagram of an electrochemical uric acid test paper;

FIG. 2 is a schematic diagram of the positions of the electrochemical uric acid test paper reaction area and the anti-interference area in the invention;

FIG. 3 is a linear relationship graph of uric acid concentration and current during detection according to the present invention;

FIG. 4 is a diagram showing the effect of the anti-interference capability of the anti-interference test paper of the present invention;

FIG. 5 is a graph showing the effect of the anti-interference ability of the anti-interference free test paper of the present invention;

FIG. 6 is a linear relationship graph of current and concentration with oxidation current as a detection signal in the present invention;

FIG. 7 is a linear relationship between current and concentration, in which the reduction current is used as a detection signal in the present invention.

Detailed Description

The technical solutions of the present invention are further described in detail by the following specific examples, but it should be noted that the following examples are only illustrative of the contents of the present invention and do not limit the scope of the present invention.

Example 1 structure, composition, preparation and performance test of electrochemical uric acid test paper.

The structural decomposition diagram of the electrochemical uric acid test paper of the embodiment is shown in fig. 1.

The manufacturing method of the electrochemical uric acid test strip comprises the following steps:

preparing a metal electrode: a high-molecular insulating substrate (1) is taken, pure gold with the thickness of 20nm is sputtered in vacuum on the insulating substrate, and electrode systems such as a working electrode (3), a reference electrode (4), a sample in-place detection electrode (5) and a switch-on electrode (6) are engraved on the pure gold electrode through a laser engraving technology.

Preparing a basic buffer solution: preparing a buffer solution of ACES with the concentration of 0.2M and the pH value of 7.0, weighing 1% of trehalose, 1% of triton, 2% of bovine serum albumin and 4% of hydroxyethyl cellulose, and mixing until the trehalose, the triton, the bovine serum albumin and the hydroxyethyl cellulose are completely dissolved.

Preparation of a reaction enzyme solution: the enzyme solution uses the basic buffer solution as a solvent, 5 percent of urate oxidase, 5 percent of electron mediator potassium ferrocyanide, 1 percent of horseradish peroxidase and 1 percent of sodium chloride are weighed according to parts by weight and mixed and stirred for 20 minutes until being completely dissolved, and uniform enzyme solution is formed.

Preparing anti-interference enzyme solution: and taking the basic buffer solution as a solvent, weighing 2% of ascorbic acid oxidase by weight, and stirring for 20 minutes until the ascorbic acid oxidase is completely dissolved to form uniform enzyme solution.

The preparation of the uric acid test strip comprises the following steps: covering a layer of reaction enzyme liquid on a reaction area (11) of the electrode in a dispensing manner, covering anti-interference enzyme liquid on an anti-interference area (12) of the electrode in a dispensing manner, wherein the loading capacity of a reaction enzyme point is 1.0mg, the loading capacity of an anti-interference enzyme point is 2.0mg, and drying for 60 minutes by heat treatment at a drying tunnel section at 60 ℃. The septum layer is adhered to the surface of the dried fabric after drying, and comprises a single-sided adhesive and a hydrophilic film layer (7) covered on the single-sided adhesive. And finally, after pressing and cutting, storing the finished product of the uric acid electrochemical test strip in a closed plastic cylinder with a molecular sieve drying agent.

When the test strip is used, the bare and leaky conductive end of the test strip is inserted into the plug port of the detection equipment matched with the test strip, a blood sample is dripped from the inlet of the siphon pool, is firstly sucked into the anti-interference layer (12) in the siphon pool through the siphon action, and reacts with ascorbic acid in the process of passing through the anti-interference enzyme layer, in the process, the main interference substance ascorbic acid in the sample is gradually consumed and finished, then blood enters the reaction zone (11), at the moment, the uric acid instrument equipment matched with the test strip applies a low negative excitation potential-80 mV excitation voltage to generate a current value with the electrochemical reaction of enzyme liquid and an electronic mediator, and then the corresponding uric acid value is obtained through the conversion of software.

Now, the test performance of the electrochemical uric acid test strip under the experimental condition is researched, and the specific experimental steps are as follows: step 1: the method comprises the following steps of adding different amounts of uric acid solutions into normal human venous whole blood to prepare uric acid blood samples with different concentrations, wherein the concentrations measured by a biochemical analyzer are as follows: 750.0. mu. mol/L, 249.6. mu. mol/L, 348. mu. mol/L, 453.6. mu. mol/L, 586.8. mu. mol/L, 744. mu. mol/L, 826.8. mu. mol/L, 960. mu. mol/L, 1176. mu. mol/L. Step 2: the performance of the electrochemical uric acid test strip is tested under the voltage of minus 80mV, the current response of the uric acid test strip prepared in the case to uric acid samples with different concentrations is tested, the current value when the reaction time is 8 seconds is taken, and the concentration is plotted against the current to obtain a linear equation of the test strip. Testing the average value, the repeatability, the corresponding sensitivity and the linear range of the uric acid whole blood samples with different concentrations; the test results are shown in Table 1 and FIG. 3.

Table 1: electrochemical uric acid test paper with various concentration current values and repeatability of current values

As can be seen from the linear relationship graph of uric acid concentration and current in the attached figure 3 and the repetitive data of each concentration current value and current value of the electrochemical uric acid test paper in the graph 1, by measuring samples with different concentrations, the correlation coefficient of the uric acid concentration and current in a blood sample is greater than 0.99, the CV of the current value of each concentration section is below 4%, and the lowest concentration can be measured to 50 mu mol/L. The test was carried out by using 40 samples of whole blood with different concentrations, and the measured uric acid concentration of the blood sample was compared with the corresponding biochemical uric acid value, and the results are shown in tables 2 and 3.

Table 2: accuracy data chart for detecting uric acid by electrochemical test strip

TABLE 3 repeatability data chart for uric acid value detection according to the invention

The results on the data table show that the uric acid value of each concentration section tested by the test strip is very close to the biochemical value. The relative deviation is within 10%. The data in the above tables 1, 2, 3 and 3 show that the CV of the current of the quality control solution is less than 3%, the CV of the current of the whole blood sample is less than 4%, the correlation coefficient of the uric acid concentration and the current is 0.99, and the lowest concentration is 50 mu mol/L. The uric acid test paper prepared by the method has the advantages of high accuracy, good repeatability, high sensitivity and high reaction speed. Therefore, the test strip can accurately test the concentration of uric acid in whole blood.

Example 2: anti-interference capability comparison experiment of electrochemical uric acid test strip

This example compares the anti-interference ability of blood samples containing different concentrations of ascorbic acid in order to compare the presence of anti-interference regions. The preparation method of the anti-interference uric acid test paper and the non-anti-interference uric acid test paper comprises the following steps:

preparing a metal electrode: a high-molecular insulating substrate (1) and a pure gold electrode layer (2) with the thickness of 20nm based on the vacuum sputtering on the insulating substrate are taken, and electrode systems such as a working electrode (3), a reference electrode (4), a sample in-place detection electrode (5), a switch-on electrode (6) and the like are engraved on the pure gold electrode through a laser engraving technology.

Preparing an electrode plate: covering a layer of reaction enzyme liquid on a reaction area (11) of the electrode in a dispensing manner, wherein the loading capacity of a reaction enzyme point is 1.0mg, dispensing is not carried out in an anti-interference area (12) of the electrode, and the electrode plate subjected to dispensing is subjected to heat treatment and drying for 60 minutes in a drying tunnel section at 60 ℃. After drying, a hydrophilic film layer (7) is attached. And finally, after pressing and cutting, storing the finished product of the uric acid electrochemical test strip in a closed plastic cylinder with a molecular sieve drying agent.

The anti-interference performance of two kinds of test paper strips is tested respectively, the conductive end that bares leaks of test paper strip inserts the patch socket department of the check out test set who matches with it, and the blood sample drips into from the entrance of siphon pond, is at first inhaled anti-interference district (12) in the siphon pond through the siphon effect, has anti-interference enzyme district in-process and ascorbic acid reaction. In this process, the consumption of ascorbic acid, which is a main interfering substance in the sample, is gradually completed, and then the blood enters the reaction region (11). When the electrode test using the interference-free layer is used, the interference substance cannot be consumed when the blood passes through the interference-free region. After the blood reaches the reaction zone, the uric acid instrument and equipment matched with the blood applies a low negative excitation potential of negative potential, namely 80mV excitation voltage, and performs reduction electrochemical reaction with enzyme liquid and an electronic mediator to generate a reduction current value, and then the corresponding uric acid value is obtained through conversion of software.

Now, the anti-interference performance of the electrochemical uric acid test strip under the experimental condition is researched, and the specific experimental steps are as follows: step 1, preparing three uric acid whole blood samples with different concentration levels, namely low, medium and high, dividing the uric acid whole blood samples into 4 parts respectively, adding ascorbic acid with different concentrations to prepare interference samples with different concentrations, and preparing the indexes as shown in table 4.

Table 4: interference sample preparation table

Low value (300. mu. mol/L)	Median value (480. mu. mol/L)	High value (750. mu. mol/L)
			Ascorbic acid of normal human	Ascorbic acid of normal human	Ascorbic acid of normal human
+ 150. mu. mol/L ascorbic acid	+ 100. mu. mol/L ascorbic acid	+ 100. mu. mol/L ascorbic acid
			+ 200. mu. mol/L ascorbic acid	+ 200. mu. mol/L ascorbic acid	+ 200. mu. mol/L ascorbic acid
+ 300. mu. mol/L ascorbic acid	+ 300. mu. mol/L ascorbic acid	+ 300. mu. mol/L ascorbic acid

Step 2, adopting a uric acid tool with an excitation potential of-80 mV to test the test performance of the electrochemical uric acid test strip, and respectively testing the prepared interference samples with different concentrations by using an anti-interference test strip and an anti-interference-free test strip, wherein the results are shown in Table 4. The results of the concentration test using the prepared anti-interference test strip and the non-anti-interference test strip in Table 4 above are plotted against the amount of ascorbic acid added in FIGS. 4 and 5. The test result of the electrochemical uric acid test strip without interference is shown in fig. 5, and the measured uric acid concentration of the sample obviously changes along with the increase of the ascorbic acid concentration in the sample. The results of the anti-interference capability of the anti-interference test paper of the invention are shown in FIG. 4. The anti-interference electrochemical uric acid test strip has a relatively obvious effect of eliminating interference of ascorbic acid, and as can be seen from fig. 4, the concentration of uric acid to be tested is basically unchanged along with the increase of the concentration of ascorbic acid. The method can effectively reduce the influence of the ascorbic acid.

Example 3: comparison of Performance Using Oxidation Current as detection Signal and reduction Current as detection Signal

This example compares the performance of uric acid test strips using oxidation current as the detection signal and reduction current as the detection signal.

The preparation method of the uric acid test paper using the reduction current as the detection signal adopted in the embodiment is as follows:

preparing a metal electrode: a high-molecular insulating substrate (1) and a pure gold electrode layer (2) with the thickness of 20nm based on the vacuum sputtering on the insulating substrate are taken, and electrode systems such as a working electrode (3), a reference electrode (4), a sample in-place detection electrode (5), a switch-on electrode (6) and the like are engraved on the pure gold electrode through a laser engraving technology. Preparing a basic buffer solution: preparing a buffer solution of ACES with the concentration of 0.2M and the pH value of 7.0, weighing 1% of trehalose, 1% of triton, 2% of bovine serum albumin and 4% of hydroxyethyl cellulose, and mixing until the trehalose, the triton, the bovine serum albumin and the hydroxyethyl cellulose are completely dissolved.

Preparation of a reaction enzyme solution: the enzyme solution uses the basic buffer solution as a solvent, 5 percent of urate oxidase, 5 percent of electron mediator potassium ferrocyanide, 1 percent of horseradish peroxidase and 1 percent of sodium chloride are weighed according to parts by weight and mixed and stirred for 20 minutes until being completely dissolved, and uniform enzyme solution is formed. Covering a layer of reaction enzyme liquid on a reaction area (11) of the electrode in a coating or dispensing mode, wherein the loading amount of the reaction enzyme point is 1.0mg, and preparing the anti-interference enzyme liquid: and taking the basic buffer solution as a solvent, weighing 2% of ascorbic acid oxidase by weight, and stirring for 20 minutes until the ascorbic acid oxidase is completely dissolved to form uniform enzyme solution. The anti-interference area (12) of the electrode is covered with a layer of reaction enzyme liquid in a coating mode, and the loading amount of anti-interference enzyme points is 2.0 mg.

Drying and attaching: drying for 60 minutes by heat treatment in a drying tunnel section at 60 ℃. After drying, a hydrophilic film layer (7) is attached. And finally, after pressing and cutting, storing the finished product of the uric acid electrochemical test strip in a closed plastic cylinder with a molecular sieve drying agent.

The preparation method of the uric acid test paper using oxidation current as a detection signal adopted in the embodiment is as follows:

Preparing a basic buffer solution: preparing MES buffer solution with the concentration of 0.1M and the pH value of 7.5, weighing 1 percent of sucrose, 202 percent of Tween, 4 percent of bovine serum albumin and 4 percent of hydroxypropyl methyl cellulose, and mixing until the components are completely dissolved.

Preparation of a reaction enzyme solution: the enzyme solution uses the basic buffer solution as a solvent, and 3 weight parts of urate oxidase, 3 weight parts of electron mediator potassium ferricyanide and 1 weight percent of sodium chloride are mixed and stirred for 20 minutes until the urate oxidase, the electron mediator potassium ferricyanide and the sodium chloride are completely dissolved to form uniform enzyme solution.

Covering a layer of reaction enzyme liquid on a reaction area (11) of the electrode in a coating or dispensing mode, wherein the loading amount of the reaction enzyme point is 1.0mg, and preparing the anti-interference enzyme liquid: and taking the basic buffer solution as a solvent, weighing 2% of ascorbic acid oxidase by weight, and stirring for 20 minutes until the ascorbic acid oxidase is completely dissolved to form uniform enzyme solution. The anti-interference area (12) of the electrode is covered with a layer of reaction enzyme liquid in a coating mode, and the loading amount of anti-interference enzyme points is 2.0 mg.

Drying and attaching: drying for 60 minutes by heat treatment in a drying tunnel section at 60 ℃. After sticking, pressing and cutting, the finished product of the uric acid electrochemical test strip is stored in a closed plastic cylinder with a molecular sieve drying agent.

When the test strip is used, the naked and leaked conductive end of the test strip is inserted into a plug port of a detection device matched with the test strip, a blood sample is dripped from an inlet of a siphon pool, is firstly sucked into an anti-interference layer (12) in the siphon pool through siphon action and reacts with ascorbic acid in the process of passing through an anti-interference enzyme layer, in the process, the main interference substance ascorbic acid in the sample is gradually consumed and finished, then the blood enters a reaction area (11), the matched uric acid instrument and equipment applies a voltage of 300mV, and at the moment, enzyme liquid on the surface of an electrode and an electronic mediator electrochemically react with uric acid in the blood sample to generate an oxidation current value, and then the corresponding uric acid value is obtained through conversion of software.

Now, the performance of the two test strips under this experimental condition is investigated:

uric acid blood samples with uric acid concentrations in the range of 100-1200 mu mol/L at 5 different concentration levels.

The test paper with oxidation current as the detection signal prepared by the above example and the matched uric acid test instrument are used, the applied excitation potential is 300mV, the uric acid in 5 concentration blood samples is detected to obtain corresponding current signals, the current value when the reaction time is 10 seconds is taken, and the linear graph and data of the current versus the concentration are shown in FIG. 6 and Table 5, wherein the uric acid blood samples with 5 different concentration levels with the uric acid concentration in the range of 100-.

Table 5: current repeatability data table using oxidation current as detection signal

The test paper prepared by the above example and using the reduction current as the detection signal and the matched uric acid test instrument are used, the applied excitation potential is-80 mV, the uric acid in 5 concentration blood samples is detected by using a chronoamperometry to obtain corresponding current signals, the current value at the time of reaction is 10 seconds, and a linear graph of current versus concentration and data are shown in fig. 7 and table 6.

Table 6: current repeatability data table using reduction current as detection signal

The result on the data table shows that the CV of the test paper current with the reduction current as the detection signal is less than 4.5%, the correlation coefficient of the uric acid concentration and the current is 0.99, the lowest concentration is tested to 50 mu mol/L, the repeatability of the current of each concentration section of the test paper with the oxidation current as the detection signal is poor, the highest current value reaches 13%, the background current value is high, the detection sensitivity is low, and the correlation coefficient of the uric acid concentration and the current is 0.94. The experiment further shows that the method using the reduction current as the detection signal has low background value and high detection sensitivity, and can realize the detection of the target substance with higher reliability.

Claims

1. An anti-interference electrochemical uric acid test paper comprises an insulating substrate (1), an electrode layer (2) positioned on the substrate, a hydrophilic film layer (7) formed by combining a double-sided adhesive tape and a hydrophilic film, wherein the hydrophilic film layer (7) is adhered to one side of the electrode layer (2) to expose part of electrodes, a reagent window (10) is formed in the hydrophilic film layer (7), and an anti-interference area (12) of an anti-interference reagent reacting with an interference substance in a blood sample and a reaction area (11) of a reagent reacting with uric acid in the blood sample are respectively covered in the reagent window (10); the anti-interference area (12) and the reaction area (11) are arranged in tandem, and are adjacent or connected in tandem, preferably adjacent in tandem.

2. The electrochemical test strip according to claim 1, said electrode layer (2) comprising a working electrode (3) and a reference electrode (4).

3. The electrochemical strip according to claim 1, wherein said interference resistant region (12) contains one or more biological enzymes reacting with an interfering substance and a buffer, and said reaction region (11) contains one or more biological enzymes reacting with a substance to be measured and an oxidized or reduced electron mediator.

4. The electrochemical test strip of claim 3, wherein the biological enzyme of said interference-free zone is ascorbate oxidase; the biological enzyme in the reaction zone is selected from urate oxidase and/or peroxidase, and the oxidized or reduced electron mediator is selected from water-soluble electron mediators.

5. The electrochemical test strip according to claim 4, wherein the oxidized electron mediator is a water-soluble electron mediator of a metal or noble metal complex; preferably, the oxidized electron mediator is present in an amount ranging from 1 wt% to 30 wt%, based on 100 wt% of the total weight of the dry film of the reagent layer.

6. The electrochemical test strip according to claim 5, wherein the excitation potential using an oxidized electron mediator with an oxidation current as a detection signal is 200mV to 400 mV.

7. The electrochemical test strip according to claim 4, wherein said reduced electron mediator is a water-soluble potassium ferrocyanide, said electron mediator being present in an amount ranging from 1 wt% to 30 wt% based on 100% by weight of the total dry film of said reagent layer.

8. The electrochemical test strip according to claim 7, wherein the applied excitation potential using a reduced electron mediator with a reduction current as a detection signal is from-50 mV to-150 mV.

9. The electrochemical test strip according to one of claims 1 to 8, wherein said interference resistant zone (12), said reaction zone (11) and, with the hydrophilic membrane layer (7) therebetween, form a siphon (8) capable of aspirating a blood sample.

10. The electrochemical test strip according to one of claims 1 to 9, wherein the interference-resistant zone (12) and the reaction zone (11) may further comprise a non-reactive substance, respectively, said non-reactive substance comprising: biological buffer solution, thickening agent, protective agent and surfactant; wherein the biological buffer comprises one or more of Tris-HCl buffer, phosphate buffer, ACES buffer, sodium acetate buffer, MES buffer, Good's buffer and glycine buffer; the thickening agent comprises one or more of cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose and carboxymethyl cellulose; the protective agent is one or more of bovine serum albumin, gelatin, trehalose and sucrose; the surfactant is one or more of TritonX-100, Tween 20 and sodium dodecyl sulfate.

11. Electrochemical test strip according to one of claims 1 to 10, characterized in that said insulating substrate (1) is a polymer material, which can be selected from PET sheet, PVC sheet, or PP sheet, with a thickness of 1mm to 1.5 mm.

12. The electrochemical test paper according to one of claims 1 to 11, characterized in that said electrode layer (2) is a thin metal layer or printed carbon paste attached on a polymer insulating substrate, and a corresponding electrode with the same function is realized by screen mask sputtering/electroplating or laser etching.

13. The electrochemical test strip of claim 12, wherein the metal material comprises gold, platinum, palladium, nickel, titanium or an alloy thereof, preferably gold; the thickness of the metal layer is between 5nm and 20 nm.