Electrochemical test paper for detecting uric acid and preparation method thereof

Abstract

The electrochemical test paper comprises a substrate, a conductive layer, an enzyme reaction layer and an anti-interference layer, the conductive layer is sprayed on the substrate, the enzyme reaction layer is coated on the conductive layer, and the anti-interference layer is located on the enzyme reaction layer; the substrate is a PET (Polyethylene Terephthalate) substrate; the conductive layer is made of a graphene and nanogold composite material, and the mass ratio of graphene to nanogold is 3: 1; the enzyme reaction layer comprises 0.5-1.5 U / mm < 2 > of urate oxidase, 0.3-0.8 U / mm < 2 > of horse radish peroxidase, an electronic mediator potassium ferrocyanide and a chitosan cross-linking agent; and the anti-interference layer is used for selectively filtering ascorbic acid. The invention has the following beneficial effects: the graphene and nanogold composite conductive layer improves the electron transmission efficiency, and the lower limit of detection reaches 0.1 [mu] M; the molecularly imprinted membrane inhibits interference of more than 90% of ascorbic acid; after 6 months of preservation at 4 DEG C, the enzyme activity retention rate is gt; 95%.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and particularly to a uric acid electrochemical test strip and a preparation method thereof. Background Art

[0002] Uric acid is the end product of purine metabolism in the human body, and abnormal concentrations thereof are closely related to diseases such as gout and renal dysfunction. Existing detection methods (such as chromatography and chemical colorimetry) have problems such as complex operation, high cost, or poor anti-interference ability. The electrochemical test strip method has the advantages of rapidity and portability, but the existing test strips have the following problems:

[0003] 1. The enzyme immobilization efficiency is low, resulting in insufficient sensitivity;

[0004] 2. Coexisting substances (such as ascorbic acid and dopamine) easily interfere with the detection results;

[0005] 3. The stability of the test strip is poor, and the performance deteriorates after long-term storage. Summary of the Invention

[0006] In summary, in order to overcome the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a uric acid electrochemical test strip with high sensitivity, strong anti-interference ability and excellent stability, and a preparation method thereof.

[0007] The technical solution of the present invention for solving the above technical problems is as follows: A uric acid electrochemical test strip includes a substrate, a conductive layer, an enzyme reaction layer, and an anti-interference layer; the conductive layer is sprayed on the substrate, the enzyme reaction layer is coated on the conductive layer, and the anti-interference layer is located on the enzyme reaction layer;

[0008] The substrate is a PET substrate;

[0009] The conductive layer is a composite material of graphene and nano gold, and the mass ratio of graphene to nano gold is 3:1;

[0010] The enzyme reaction layer includes 0.5 - 1.5 U / mm of uricase 2 , 0.3 - 0.8 U / mm of horseradish peroxidase 2 , an electron mediator potassium ferrocyanide and a chitosan crosslinking agent;

[0011] The anti-interference layer is a selective filter for ascorbic acid.

[0012] Based on the above technical solutions, the present invention can be further improved as follows.

[0013] Further, the conductive layer is a composite material of graphene and nano gold, and the mass ratio of graphene to nano gold is 3:1.

[0014] Further, the enzyme reaction layer comprises uricase at 0.5 - 1.5 U / mm 2 , horseradish peroxidase at 0.3 - 0.8 U / mm 2 .

[0015] The preparation method of the above-mentioned uric acid electrochemical test strip comprises the following steps:

[0016] Step 1, substrate pretreatment: cleaning the PET substrate and performing plasma treatment;

[0017] Step 2, preparation of the conductive layer: spraying a graphene and nano-gold dispersion liquid on the substrate and then drying;

[0018] Step 3, coating of the enzyme reaction layer: mixing uricase, horseradish peroxidase, potassium ferrocyanide and a 0.5% chitosan solution, and screen-printing to form a film and coating it on the conductive layer;

[0019] Step 4, construction of the anti-interference layer: depositing a dopamine-ascorbic acid molecularly imprinted membrane on the surface of the enzyme reaction layer;

[0020] Step 5, encapsulation of the insulating layer: encapsulating the semi-finished product obtained in Step 4 with an insulating layer and laser-cutting to form a detection window to obtain the finished product.

[0021] Further, the plasma treatment in Step 1 is carried out for 10 - 15 min.

[0022] Further, the solvent of the dispersion liquid in Step 2 is NMP.

[0023] Further, drying is carried out at 60 °C in Step 2.

[0024] Further, the dopamine-ascorbic acid molecularly imprinted membrane is deposited on the surface of the enzyme reaction layer by an electro-polymerization method in Step 4.

[0025] Further, the diameter of the detection window in Step 5 is 3 mm.

[0026] The beneficial effects of the present invention are as follows: the graphene and nano-gold composite conductive layer improves the electron transfer efficiency, and the detection lower limit reaches 0.1 μM; the molecularly imprinted membrane inhibits more than 90% of the interference of ascorbic acid; the enzyme activity retention rate is > 95% after storage at 4 °C for 6 months. Specific Embodiments

[0027] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0028] An electrochemical test strip for detecting uric acid, comprising a substrate, a conductive layer, an enzyme reaction layer, and an anti-interference layer; the conductive layer is sprayed on the substrate, the enzyme reaction layer is coated on the conductive layer, and the anti-interference layer is located on the enzyme reaction layer. The substrate is a PET substrate. The conductive layer is a composite material of graphene and nano-gold, and the mass ratio of graphene to nano-gold is 3:1. The enzyme reaction layer includes 0.5 - 1.5 U / mm of uricase 2 , 0.3 - 0.8 U / mm of horseradish peroxidase 2 , an electron mediator potassium ferrocyanide and a chitosan cross-linking agent. The anti-interference layer is a selective filter for ascorbic acid.

[0029] The preparation method of the above-mentioned electrochemical test strip for detecting uric acid includes the following steps:

[0030] Step 1, substrate pretreatment: Clean the PET substrate and perform plasma treatment for 10 - 15 minutes;

[0031] Step 2, preparation of the conductive layer: Spray a dispersion of graphene and nano-gold on the substrate, the solvent of the dispersion is NMP, and then dry at 60 °C;

[0032] Step 3, coating of the enzyme reaction layer: Mix uricase, horseradish peroxidase, potassium ferrocyanide with a 0.5% chitosan solution, and screen-print and coat on the conductive layer;

[0033] Step 4, construction of the anti-interference layer: Deposit a dopamine-ascorbic acid molecularly imprinted membrane on the surface of the enzyme reaction layer by electro-polymerization;

[0034] Step 5, encapsulation of the insulating layer: Encapsulate the insulating layer on the semi-finished product obtained in Step 4 and laser-cut to form a detection window with a diameter of 3 mm to obtain the finished product.

[0035] I. Sensitivity and linear range

[0036] 1. Detection limit and linear response

[0037] In the optimized test strip formula of the present invention (the conductive layer is a composite material of graphene and nano-gold, and the enzyme reaction layer contains 1.0 U / mm of uricase 2 ), the detection limit of the test strip for uric acid reaches 0.1 μM, which is significantly better than the detection limit (4.083 μM) of the existing enzyme-free carbon cloth electrode.

[0038] The linear range covers 0.5 - 500 μM (R 2 = 0.998). The test strip of the present invention is superior to the 10 - 400 μM range of traditional electrochemical test strips and is suitable for the accurate detection of clinical hyperuricemia (>420 μM) and low-concentration samples.

[0039] 2. Verification of the performance of the conductive layer

[0040] Through cyclic voltammetry (CV) testing, the electron transfer efficiency of the graphene and nano-gold composite material of the test strip of the present invention is increased by 2.3 times compared with that of the pure graphene electrode, and the oxidation peak current intensity has a significant positive correlation with the uric acid concentration.

[0041] The introduction of nano-gold particles (particle size 20 nm) in the test strip of the present invention increases the effective surface area of the electrode by 45%, improving the enzyme immobilization efficiency.

[0042] II. Anti-interference ability

[0043] 1. Inhibition efficiency of ascorbic acid

[0044] The inhibition efficiency of the polydopamine molecularly imprinted membrane of the test strip of the present invention against the common interferent ascorbic acid reaches 93% (compared with the test strip without the anti-interference layer, the error is reduced from ±15% to ±3%).

[0045] Verification by differential pulse voltammetry (DPV) of the test strip of the present invention shows that in a simulated body fluid containing 100 μM ascorbic acid, the deviation of the uric acid detection signal is <5%, meeting the clinical requirements.

[0046] 2. Influence of other interferents

[0047] The cross-reactivity rates of the test strip of the present invention with substances such as dopamine (50 μM) and glucose (10 mM) are all <2%, indicating that the test strip has high selectivity.

[0048] III. Stability and repeatability

[0049] 1. Long-term storage performance

[0050] After the test strip of the present invention is stored at 4°C for 6 months, the enzyme activity retention rate is >95% (the enzyme activity of the control test strip without the anti-interference layer drops to 60%), which is attributed to the stabilizing effect of the chitosan cross-linking agent.

[0051] The accelerated aging experiment (37°C / 70% humidity, 30 days) shows that the response current decay rate of the test strip of the present invention is <8%, meeting the stability standard of medical devices.

[0052] 2. Repeatability between batches and within batches

[0053] When testing 100 μM uric acid samples with test strips of the same batch, the relative standard deviation (RSD) of the test strip of the present invention is 1.2%; the RSD of test strips of different batches is 2.8%, which is better than the industry requirement of 5%.

[0054] IV. Clinical verification data

[0055] 1. Recovery rate test

[0056] In the serum samples added with 50 μM and 200 μM uric acid, the recovery rates of the test strips of the present invention were 98.2%-102.5% and 97.6%-103.8% respectively, showing high consistency with the results of laboratory mass spectrometry (correlation coefficient 0.992).

[0057] 2. Detection of actual samples

[0058] The detection of 50 clinical blood samples showed that the average deviation between the results of the test strips of the present invention and those of the hospital biochemical analyzer (enzymatic colorimetry) was 3.1%, meeting the ISO 15197:2013 standard (deviation < ±15%).

[0059] V. Comparative experiments

[0060] 1. Comparison with enzyme-free test strips

[0061] The traditional enzyme-free carbon cloth test strips showed signal saturation when the uric acid concentration > 200 μM, while the test strips of the present invention still maintained a linear response at 500 μM.

[0062] The sensitivity of the enzyme reaction test strips (0.1 μM) was significantly higher than that of the enzyme-free test strips (4.083 μM).

[0063] 2. Comparison with commercially available products

[0064] The inhibition efficiency of the Sannuo UA-1 test strips (anti-interference enzyme layer design) against ascorbic acid was 85%, while the test strips of the present invention increased the inhibition rate to 93% through the molecularly imprinted membrane.

[0065] VI. Detailed description of the experimental method

[0066] 1. Electrochemical test conditions

[0067] A three-electrode system was adopted (working electrode: conductive layer of the test strip; reference electrode: Ag / AgCl; counter electrode: platinum wire), with a scanning rate of 50 mV / s and a potential range of -0.2 - 0.6 V.

[0068] 2. Interference test scheme

[0069] In the PBS buffer containing 10% fetal bovine serum, interference substances such as ascorbic acid and dopamine were added respectively, and the change in the oxidation peak current was recorded by the DPV method.

[0070] Conclusion

[0071] The above data comprehensively show that the test strips of the present invention are superior to the prior art in terms of sensitivity, anti-interference ability and stability, and are particularly suitable for home self-testing and primary medical scenarios.

[0072] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. An electrochemical test paper for detecting uric acid, characterized in that: It comprises a substrate, a conductive layer, an enzyme reaction layer, and an anti-interference layer; the conductive layer is sprayed on the substrate, the enzyme reaction layer is coated on the conductive layer, and the anti-interference layer is on the enzyme reaction layer; The substrate is a PET substrate; The conductive layer is a composite material of graphene and nano-gold, and the mass ratio of graphene to nano-gold is 3:1; The enzyme reaction layer includes urate oxidase 0.5-1.5U / mm 2 , horseradish peroxidase 0.3-0.8U / mm 2 , electron mediator potassium ferrocyanide and chitosan cross-linking agent; The anti-interference layer selectively filters ascorbic acid.

2. The electrochemical test paper for detecting uric acid according to claim 1, characterized in that: The conductive layer is a composite material of graphene and nano-gold, and the mass ratio of graphene to nano-gold is 3:

1.

3. The electrochemical test paper for detecting uric acid according to claim 1, characterized in that: The enzyme reaction layer includes urate oxidase 0.5-1.5U / mm 2 , horseradish peroxidase 0.3-0.8U / mm 2 .

4. A method for preparing an electrochemical test paper for detecting uric acid, characterized in that: The steps include: Step 1, substrate pretreatment: cleaning the PET substrate and plasma treatment; Step 2, preparation of the conductive layer: spraying graphene and nano-gold dispersion on the substrate, and then drying; Step 3, coating the enzyme reaction layer: mixing urate oxidase, horseradish peroxidase, potassium ferrocyanide and 0.5% chitosan solution, and coating the mixture on the conductive layer by screen printing; Step 4, anti-interference layer construction: depositing a dopamine-ascorbic acid molecular imprinting film on the surface of the enzyme reaction layer; Step 5, insulation layer packaging: the semi-finished product obtained in step 4 is packaged with an insulation layer, and laser cutting is performed to form a detection window to obtain a finished product.

5. The method for preparing an electrochemical test paper for detecting uric acid according to claim 4, characterized in that: In step 1, plasma treatment is performed for 10-15 minutes.

6. The method for preparing an electrochemical test paper for detecting uric acid according to claim 4, characterized in that: The solvent of the dispersion in step 2 is NMP.

7. The method for preparing an electrochemical test paper for detecting uric acid according to claim 4, characterized in that: In step 2, the mixture is dried at 60°C.

8. The method for preparing an electrochemical test paper for detecting uric acid according to claim 4, characterized in that: In step 4, the dopamine-ascorbic acid molecular imprinting film is deposited on the surface of the enzyme reaction layer by electropolymerization.

9. The method for preparing an electrochemical test paper for detecting uric acid according to claim 4, characterized in that: The diameter of the detection window in step 5 is 3 mm.