Application of carbon paste containing potassium ferricyanide in preparation of electrochemical test paper, electrochemical test paper for creatinine detection, device and method

Abstract

The invention provides application of carbon paste containing potassium ferricyanide in preparation of electrochemical test paper, the electrochemical test paper for creatinine detection, a device and a method, and relates to the technical field of biological detection. The research of the inventor finds that when the carbon paste containing potassium ferricyanide is used for preparing the electrode, the reaction activity of the conductive material carbon paste can be improved, and the sensitivity and the test linear range of the creatinine sensor are remarkably improved. The electrochemical test paper provided by the invention is low in cost, the electrode is prepared from the carbon paste containing potassium ferricyanide, and the electrochemical test paper is good in detection sensitivity and accuracy. The creatinine detection method provided by the invention is simple to operate, the sensor is activated in a pulse excitation mode and then detected, and the detection consistency is good.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an application of carbon slurry containing potassium ferrocyanide in the preparation of electrochemical test paper, and an electrochemical test paper, device and method for creatinine detection. Background Art

[0002] Creatinine (Cr) is a low molecular weight nitrogen compound in the human body and is a product of muscle metabolism in the human body. Serum creatinine comes from both exogenous and endogenous sources. Exogenous creatinine is the product of meat food metabolism in the body; endogenous creatinine is the product of muscle tissue metabolism in the body. When the exogenous creatinine intake is stable and the in vivo production is constant, the blood creatinine concentration mainly depends on the glomerular filtration function. When the glomerular filtration rate drops to 1 / 3 of that of a normal person, blood creatinine will increase significantly. Therefore, when blood creatinine is significantly higher than normal, it often indicates that renal function has been severely damaged. In the case of impaired renal function, blood creatinine is the most reliable indicator of glomerular function. Generally speaking, the reference range of blood creatinine is: 57-97umol / L for men (20-59 years old), 57-111umol / L for men (60-79 years old), 41-73umol / L for women (20-59 years old), and 41-81umol / L for women (60-79 years old).

[0003] Traditional methods for detecting creatinine mainly include the Jaffe reaction method and the enzymatic method. The Jaffe reaction is fast and economical, but it is easily affected by interfering chromogens such as ketones and glucose in the serum, making it sometimes inaccurate. Therefore, the enzymatic method is considered to be a better method. However, for patients who are not convenient to go to the hospital but need to check their renal function regularly, the two existing creatinine detection methods are not easy to implement.

[0004] Biosensors are an electrochemical detection technology that converts the concentration of biological materials into electrical signals. Compared with traditional measurement methods, biosensors have the advantages of short measurement time, simple operation, and good selectivity, and are a promising alternative method for creatinine detection. However, the current use of biosensors to detect creatinine still has problems such as insufficient electrical signal precision (CV) and low accuracy.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first object of the present invention is to provide the use of carbon slurry containing potassium ferrocyanide in preparing electrochemical test paper.

[0007] The second object of the present invention is to provide an electrochemical test paper for creatinine detection to solve the above technical problems.

[0008] The third object of the present invention is to provide a device for creatinine detection.

[0009] The fourth object of the present invention is to provide a method for detecting creatinine.

[0010] In order to achieve the above objectives, the following technical solutions are adopted:

[0011] In a first aspect, the present invention provides the use of a carbon slurry containing potassium ferrocyanide in the preparation of an electrochemical test paper, wherein the carbon slurry is used as a raw material for preparing an electrochemical test paper electrode;

[0012] In the carbon slurry, the mass proportion of potassium ferrocyanide is 0.1%-5%.

[0013] In a second aspect, the present invention provides an electrochemical test paper for creatinine detection, comprising: an electrode substrate, an electrode system, a first patch, a second patch and a hydrophilic membrane;

[0014] The electrode system is arranged on the electrode substrate to form a hematocrit working electrode, a hematocrit auxiliary electrode, a creatine working electrode, a creatinine auxiliary electrode, and a creatinine working electrode which are separated and not connected; the electrode system is mainly prepared from a carbon slurry containing potassium ferrocyanide;

[0015] One end of the hematocrit working electrode and the hematocrit auxiliary electrode are located in the reaction area A, and the other end is located in the connection area, for transmission of electrical signals;

[0016] One end of the creatine working electrode is located in the reaction area B, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0017] One end of the creatinine auxiliary electrode is located in the reaction area C, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0018] One end of the creatinine working electrode is located in the reaction area D, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0019] From the sampling end to the other end of the electrochemical test paper, the reaction area A, the reaction area B, the reaction area C and the reaction area D are arranged in sequence;

[0020] The reaction zone B is loaded with creatine hydrolase and sarcosine oxidase; the reaction zone D is loaded with creatine hydrolase, sarcosine oxidase and creatinine hydrolase;

[0021] The first patch covers the electrode area except the connection area, and has windows in the reaction area A, the reaction area B, the reaction area C and the reaction area D;

[0022] The second patch covers the first patch, and has windows in the reaction area A, the reaction area B, the reaction area C and the reaction area D and the areas therebetween, the windows extending to the sampling end of the electrochemical test paper to form a sampling port;

[0023] The hydrophilic membrane covers the window of the second patch.

[0024] As a further technical solution, the electrode substrate includes a PET (polyethylene terephthalate) substrate, a PMMA (polymethyl methacrylate) substrate, a PVC (polyvinyl chloride) substrate or a PCB (circuit board).

[0025] As a further technical solution, the electrode system is prepared by screen printing technology.

[0026] As a further technical solution, the step of loading the reaction zone B with creatine hydrolase and sarcosine oxidase comprises: dispensing solution B on the reaction zone B by using a dispensing process;

[0027] The solution B is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP (polyvinyl pyrrolidone) or PEG (polyethylene glycol), 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant, 1% to 5% potassium ferrocyanide, 800 to 1200 U / ml creatine hydrolase and 800 to 1200 U / ml sarcosine oxidase.

[0028] As a further technical solution, the step of loading the reaction zone D with creatine hydrolase, sarcosine oxidase and creatinine hydrolase comprises: dispensing the solution C on the reaction zone D by using a dispensing process;

[0029] The solution C is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant, 1% to 5% potassium ferrocyanide, 800 to 1200 U / ml creatine hydrolase, 800 to 1200 U / ml sarcosine oxidase and 1000 to 2000 U / ml creatinine hydrolase.

[0030] As a further technical solution, the reaction zone C is loaded with non-reactive substances, and the loading step includes: dispensing solution A on the reaction zone C by using a dispensing process;

[0031] The solution A is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant and 1% to 5% potassium ferrocyanide.

[0032] As a further technical solution, the buffer solution is a PBS buffer solution;

[0033] The surfactant includes triton x-100;

[0034] The molecular weight of the PVP is 10k to 100k;

[0035] The molecular weight of the PEG is 1k to 10k;

[0036] The viscosity of the hydroxyethyl cellulose is 50-300cp;

[0037] The viscosity of the methyl cellulose is 25-100 cp.

[0038] In a third aspect, the present invention provides a device for creatinine detection, comprising the above-mentioned electrochemical test paper, a connector, a processor and a display;

[0039] The connector is used to transmit the electrical signal from the electrochemical test paper to the processor;

[0040] The processor and the display are used to convert the received electrical signal into creatinine concentration and display it.

[0041] In a fourth aspect, the present invention provides a method for detecting creatinine, using the above-mentioned electrochemical test paper for detection, comprising the following steps:

[0042] a. Pulse excitation: applying pulse cycles to the creatine working electrode and the creatinine auxiliary electrode for pulse excitation;

[0043] b. Detection: The creatine working electrode and the creatinine auxiliary electrode form a test circuit, and the current measured by the constant potential method is I1; the creatinine working electrode and the creatinine auxiliary electrode form a test circuit, and the current measured by the constant potential method is I2; then the creatinine reaction current is I2-I1.

[0044] As a further technical solution, the pulse cycle is: first apply 0.1 to 0.6 mv voltage excitation for 0.01 to 0.1 s, and then apply -0.1 to -0.6 mv voltage excitation for 0.01 to 0.1 s.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The inventors have found that preparing electrodes with carbon slurry containing potassium ferrocyanide can improve the reactivity of the conductive material carbon slurry and significantly improve the sensitivity and test linear range of the creatinine sensor.

[0047] The electrochemical test paper provided by the invention has low cost, good detection sensitivity and good accuracy.

[0048] The creatinine detection method provided by the present invention is simple to operate, and the sensor is activated by pulse excitation before detection, and the detection consistency is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 The structure of the electrochemical test paper provided by the embodiment of the present invention;

[0051] Figure 2 This is the result curve of the detection of scheme 1;

[0052] Figure 3 This is the result curve of the detection of scheme 2;

[0053] Figure 4 This is the result curve of the detection of scheme 3;

[0054] Figure 5 This is the result curve of the detection of Scheme 4.

[0055] Icon: 1-electrode base; 2-hematocrit working electrode; 3-creatine working electrode; 4-creatinine working electrode; 5-creatinine auxiliary electrode; 6-hematocrit auxiliary electrode; 7-reaction area A; 8-reaction area B; 9-reaction area C; 10-reaction area D; 11-first patch; 12-second patch; 13-hydrophilic membrane. DETAILED DESCRIPTION

[0056] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0057] In a first aspect, the present invention provides the use of a carbon slurry containing potassium ferrocyanide in the preparation of an electrochemical test paper, wherein the carbon slurry is used as a raw material for preparing an electrochemical test paper electrode;

[0058] In the carbon slurry, the mass proportion of potassium ferrocyanide may be, for example, but not limited to, 0.1%, 1% or 5%, preferably 1%.

[0059] The inventors have found that preparing electrodes with carbon slurry containing potassium ferrocyanide can improve the reactivity of the conductive material carbon slurry and significantly improve the sensitivity and test linear range of the electrochemical sensor.

[0060] In a second aspect, the present invention provides an electrochemical test paper for creatinine detection, comprising: an electrode substrate 1, an electrode system, a first patch 11, a second patch 12 and a hydrophilic membrane 13;

[0061] The electrode system is arranged on an electrode substrate 1, forming a hematocrit working electrode 2, a hematocrit auxiliary electrode 6, a creatine working electrode 3, a creatinine auxiliary electrode 5, and a creatinine working electrode 4 that are separated and not connected; the electrode system is mainly prepared from a carbon slurry containing potassium ferrocyanide, wherein the mass concentration of potassium ferrocyanide in the carbon slurry can be, for example, but not limited to, 1%, 3% or 5%;

[0062] One end of the hematocrit working electrode 2 and the hematocrit auxiliary electrode 6 are located in the reaction area A7, and the other end is located in the connection area, for transmission of electrical signals;

[0063] One end of the creatine working electrode 3 is located in the reaction area B8, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0064] One end of the creatinine auxiliary electrode 5 is located in the reaction area C9, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0065] One end of the creatinine working electrode 4 is located in the reaction area D10, and the other end is located in the connection area for transmission of electrical signals;

[0066] From the sampling end to the other end of the electrochemical test paper, the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10 are arranged in sequence;

[0067] The reaction zone B8 is loaded with creatine hydrolase and sarcosine oxidase; the reaction zone D10 is loaded with creatine hydrolase, sarcosine oxidase and creatinine hydrolase;

[0068] The first patch 11 covers the electrode area except the connection area, and has windows at the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10 to leak out the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10;

[0069] The second patch 12 covers the first patch 11, and has windows in the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10 and the areas therebetween, the windows extending to the sampling end of the electrochemical test paper to form a sampling port;

[0070] The hydrophilic membrane 13 covers the window of the second patch 12. Since the hydrophilic membrane 13 is hydrophilic and there is a gap between the hydrophilic membrane 13 and the substrate or the first patch 11, the sample will be sucked into the reaction area along the gap during sampling, thereby realizing the detection of the sample to be tested.

[0071] The inventors have found that preparing electrodes with carbon slurry containing potassium ferrocyanide can improve the reactivity of the conductive material carbon slurry and significantly improve the sensitivity and test linear range of the creatinine sensor.

[0072] In some optional embodiments, the test sample of the electrochemical test paper includes blood.

[0073] In some optional embodiments, reaction zone A7, reaction zone B8, reaction zone C9 and reaction zone D10 do not intersect with each other.

[0074] In some optional embodiments, the electrode substrate includes but is not limited to a PET substrate, a PMMA substrate, a PVC substrate or a PCB, or other substrates known to those skilled in the art.

[0075] The first patch 11 and the second patch 12 are each independently a double-sided adhesive patch.

[0076] In some optional embodiments, the electrode system is prepared by screen printing technology.

[0077] In some optional embodiments, the step of loading the reaction zone B8 with creatine hydrolase and sarcosine oxidase comprises: dispensing solution B on the reaction zone B8 using a dispensing process; the amount of solution B used is 0.5 to 1.5 uL, preferably 0.8 uL.

[0078] The solution B is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant, 1% to 5% potassium ferrocyanide, 800 to 1200 U / ml creatine hydrolase and 800 to 1200 U / ml sarcosine oxidase.

[0079] In some optional embodiments, the step of loading the reaction zone D10 with creatine hydrolase, sarcosine oxidase and creatinine hydrolase comprises: dispensing solution C on the reaction zone D10 using a dispensing process; the amount of solution C used is 0.5 to 1.5 uL, preferably 0.8 uL.

[0080] The solution C is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant, 1% to 5% potassium ferrocyanide, 800 to 1200 U / ml creatine hydrolase, 800 to 1200 U / ml sarcosine oxidase and 1000 to 2000 U / ml creatinine hydrolase.

[0081] In some optional embodiments, the reaction zone C9 is loaded with a non-reactive substance, and the loading step includes: dispensing solution A on the reaction zone C9 using a dispensing process; the amount of solution A used is 0.5 to 1.5 uL, preferably 0.8 uL.

[0082] The solution A is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant and 1% to 5% potassium ferrocyanide.

[0083] The electrode where solution A is located is used as the counter electrode (auxiliary electrode) and reference electrode of the circulating circuit at the same time. The auxiliary electrode functions to form the negative electrode of the circuit, and the reference electrode functions to correct the voltage applied to the working electrode. Therefore, solution A plays the role of electrode conduction on the one hand, and serves as a reference to the working electrode on the other hand. Solution A is solution C without the three enzymes, and is also solution B without the two enzymes, to ensure that the applied voltage of the working electrode (solution B or C) relative to the auxiliary electrode (solution A) is fixed.

[0084] In solution A, PVP and surfactant play the role of protecting the enzyme, and hydroxyethyl cellulose is a binder, providing the viscosity and mechanical properties of the reagent.

[0085] In some optional embodiments, the buffer solution is a PBS buffer solution;

[0086] The surfactant includes but is not limited to triton x-100, or other surfactants known to those skilled in the art;

[0087] The molecular weight of the PVP may be, for example, but not limited to, 10k, 20k, 50k or 100k, preferably 20k;

[0088] The molecular weight of the PEG may be, for example, but not limited to, 1k, 5k or 10k;

[0089] The viscosity of the hydroxyethyl cellulose may be, for example, but not limited to, 50 cp, 100 cp, 200 cp or 300 cp, preferably 100 cp;

[0090] The viscosity of the methyl cellulose may be, for example, but not limited to, 25 cp, 50 cp, 75 cp or 100 cp.

[0091] In some optional embodiments, the diameters of the creatine working electrode 3, the creatinine auxiliary electrode 5 and the creatinine working electrode 4 in the reaction zone B8, the reaction zone C9 and the reaction zone D10 may be, for example, but not limited to, 0.6 mm, 0.8 mm, 1 mm, 1.2 mm or 1.4 mm, preferably 1 mm; the sensor area where the chemical reaction can occur may be 1 mm 2 , 1.3mm 2 , 1.6mm 2 or 2mm 2 , preferably 1.6 mm 2 .

[0092] In a second aspect, the present invention provides a device for creatinine detection, comprising the above-mentioned electrochemical test paper, a connector, a processor and a display;

[0093] The connector is used to transmit the electrical signal from the electrochemical test paper to the processor;

[0094] The processor and the display are used to convert the received electrical signal into creatinine concentration and display it.

[0095] The device provided by the invention is simple and has high sensitivity, and can accurately detect creatinine.

[0096] In a third aspect, the present invention provides a method for detecting creatinine, using the above-mentioned electrochemical test paper for detection, comprising the following steps:

[0097] a. Pulse excitation: applying a pulse cycle to the creatine working electrode 3 and the creatinine auxiliary electrode 5 for pulse excitation;

[0098] b. Detection: The creatine working electrode 3 and the creatinine auxiliary electrode 5 form a test circuit, and the current I1 is measured by constant potential method; the reaction formula of the creatine working electrode 3 is:

[0099]

[0100] The creatinine working electrode 4 and the creatinine auxiliary electrode 5 form a test circuit, and the current I2 is measured by constant potential method; the reaction formula of the creatinine working electrode 4 is:

[0101]

[0102] Among them, I1 is the test current of creatine, I2 is the test current of creatine and creatinine, and the creatinine reaction current is I2-I1.

[0103] In addition, the creatinine concentration is also affected by the hematocrit in the sample. The electrochemical impedance spectroscopy method is used to test the hematocrit of the sample for correction of the creatinine concentration.

[0104] The creatinine detection method provided by the present invention is simple to operate, and the sensor is activated by pulse excitation before detection, and the detection consistency is good.

[0105] In some optional embodiments, the pulse cycle is: first apply 0.1 to 0.6 mv voltage excitation for 0.01 to 0.1 s, and then apply -0.1 to -0.6 mv voltage excitation for 0.01 to 0.1 s.

[0106] In some optional embodiments, the number of pulse cycles may be, for example, 5 times.

[0107] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0108] Example 1

[0109] An electrochemical test paper for creatinine detection, such as Figure 1 As shown, it includes: an electrode substrate 1, an electrode system, a first patch 11, a second patch 12 and a hydrophilic membrane 13;

[0110] The substrate is a PET substrate; the first patch 11 and the second patch 12 are double-sided adhesive patches;

[0111] The electrode system is arranged on the electrode substrate 1, forming a separated and unconnected hematocrit working electrode 2, a hematocrit auxiliary electrode 6, a creatine working electrode 3, a creatinine auxiliary electrode 5, and a creatinine working electrode 4; the electrode system is mainly obtained by screen printing of a carbon paste containing potassium ferrocyanide;

[0112] One end of the hematocrit working electrode 2 and the hematocrit auxiliary electrode 6 are located in the reaction area A7, and the other end is located in the connection area for transmission of electrical signals;

[0113] One end of the creatine working electrode 3 is located in the reaction area B8, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0114] One end of the creatinine auxiliary electrode 5 is located in the reaction area C9, and the other end is located in the connection area, which is used for the transmission of electrical signals;

[0115] One end of the creatinine working electrode 4 is located in the reaction area D10, and the other end is located in the connection area for transmission of electrical signals;

[0116] Reaction area A7, reaction area B8, reaction area C9 and reaction area D10 do not intersect each other, and from the sampling end to the other end of the electrochemical test paper, reaction area A7, reaction area B8, reaction area C9 and reaction area D10 are arranged in sequence;

[0117] The reaction zone B8 is loaded with solution B. Solution B is dispensed on the reaction zone B8 by a dispensing process. The amount is 0.8uL. The loading is completed after drying at 45 degrees Celsius for 30 minutes. The reaction zone C9 is loaded with solution A. Solution A is dispensed on the reaction zone C9 by a dispensing process. The amount is 0.8uL. The loading is completed after drying at 45 degrees Celsius for 30 minutes. The reaction zone D10 is loaded with solution C. Solution C is dispensed on the reaction zone D10 by a dispensing process. The amount is 0.8uL. The loading is completed after drying at 45 degrees Celsius for 30 minutes.

[0118] The first patch 11 covers the electrode area except the connection area, and has windows at the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10 to leak out the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10;

[0119] The second patch 12 covers the first patch 11, and has windows in the reaction area A7, the reaction area B8, the reaction area C9 and the reaction area D10 and the areas therebetween, the windows extending to the sampling end of the electrochemical test paper to form a sampling port;

[0120] The hydrophilic film 13 covers the window of the second patch 12 .

[0121] The carbon slurry containing potassium ferrocyanide is prepared by dissolving 20% ​​potassium ferrocyanide in a diluent, N-methylpyrrolidone, to obtain a diluent D. 5% of the diluent D is added to a carbon slurry (purchased from Japan Jujo Chemical Co., Ltd., model CH-8), and mechanically stirring at room temperature for 1 hour to obtain a carbon slurry containing potassium ferrocyanide.

[0122] Solution A is a PBS solution with a pH of 7.4, containing 1% PVP (molecular weight 20k), 3% high molecular weight polymer hydroxyethyl cellulose (viscosity 100 cp), 1% triton x-100 and 3% potassium ferrocyanide;

[0123] Solution B is a PBS solution with a pH of 7.4 and containing 1% PVP (molecular weight 20k), 3% high molecular weight polymer hydroxyethyl cellulose (viscosity 100cp), 1% triton x-100, 3% potassium ferrocyanide, 960U / ml creatine hydrolase and 980U / ml sarcosine oxidase;

[0124] Solution C is a PBS solution with a pH of 7.4 and contains 1% PVP (molecular weight 20k), 3% high molecular weight polymer hydroxyethyl cellulose (viscosity 100cp), 1% triton x-100, 3% potassium ferrocyanide, 960U / ml creatine hydrolase, 980U / ml sarcosine oxidase and 1550U / ml creatinine hydrolase.

[0125] The diameters of the creatine working electrode 3, the creatinine auxiliary electrode 5 and the creatinine working electrode 4 in the reaction zone B8, the reaction zone C9 and the reaction zone D10 are 1 mm respectively, and the sensor area where the chemical reaction can occur is 1.6 mm respectively. 2 .

[0126] Example 2

[0127] An electrochemical test paper for creatinine detection, which differs from Example 1 in that:

[0128] The carbon slurry containing potassium ferrocyanide is prepared by dissolving 20% ​​of potassium ferrocyanide in a diluent, N-methylpyrrolidone, to obtain a diluent D. Then, 3% of the diluent D is added to the carbon slurry, and the mixture is mechanically stirred at room temperature for 1 hour to obtain a carbon slurry containing potassium ferrocyanide.

[0129] Solution A is a PBS solution with a pH of 7 and containing 0.5% PVP (molecular weight of 100k), 1% high molecular weight polymer hydroxyethyl cellulose (viscosity of 300cp), 0.5% triton x-100 and 1% potassium ferrocyanide;

[0130] Solution B is a PBS solution with a pH of 7 and containing 0.5% PVP (molecular weight of 100k), 1% high molecular weight polymer hydroxyethyl cellulose (viscosity of 300cp), 0.5% triton x-100, 1% potassium ferrocyanide, 800U / ml creatine hydrolase and 800U / ml sarcosine oxidase;

[0131] Solution C is a PBS solution with a pH of 7 and contains 0.5% PVP (molecular weight 100k), 1% high molecular weight polymer hydroxyethyl cellulose (viscosity 300cp), 0.5% triton x-100, 1% potassium ferrocyanide, 800U / ml creatine hydrolase, 800U / ml sarcosine oxidase and 1000U / ml creatinine hydrolase.

[0132] Example 3

[0133] An electrochemical test paper for creatinine detection, which differs from Example 1 in that:

[0134] The carbon slurry containing potassium ferrocyanide is prepared by dissolving 20% ​​of potassium ferrocyanide in a diluent, N-methylpyrrolidone, to obtain a diluent D. Then, 10% of the diluent D is added to the carbon slurry, and mechanically stirred at room temperature for 1 hour to obtain a carbon slurry containing potassium ferrocyanide.

[0135] Solution A is a PBS solution with a pH of 8 and containing 2% PVP (molecular weight of 10k), 5% high molecular weight polymer hydroxyethyl cellulose (viscosity of 50cp), 2% triton x-100 and 5% potassium ferrocyanide;

[0136] Solution B is a PBS solution with a pH of 8 and containing 2% PVP (molecular weight of 10k), 5% high molecular weight polymer hydroxyethyl cellulose (viscosity of 50cp), 2% triton x-100, 5% potassium ferrocyanide, 1200U / ml creatine hydrolase and 1200U / ml sarcosine oxidase;

[0137] Solution C is a PBS solution with a pH of 8 and contains 2% PVP (molecular weight 10k), 5% high molecular weight polymer hydroxyethyl cellulose (viscosity 50cp), 2% triton x-100, 5% potassium ferrocyanide, 1200U / ml creatine hydrolase, 1200U / ml sarcosine oxidase and 2000U / ml creatinine hydrolase.

[0138] Comparative Example 1

[0139] The difference from Example 1 is that the carbon slurry does not contain potassium ferrocyanide.

[0140] Comparative Example 2

[0141] The difference from Example 1 is that the carbon slurry does not contain potassium ferrocyanide, and after the electrode is obtained by screen printing, a layer of potassium ferrocyanide is electrodeposited on the surface of the electrode.

[0142] Test Example 1

[0143] Human whole blood containing creatinine (75, 160, 270, 550, 815, 1235 mmol / L) was used as a sample to test the electrochemical test papers provided in Example 1 and Comparative Examples 1-2, and the groups were as follows:

[0144] Scheme 1: The electrochemical test paper provided in Comparative Example 1 is used for detection, and no pulse potential is used for excitation during detection;

[0145] Solution 2: Using the electrochemical test paper provided in Example 1 for detection, without using pulse potential for excitation during detection;

[0146] Scheme 3: The electrochemical test paper provided in Example 1 is used for detection. During the detection, pulse potential is first used for excitation. The pulse potential excitation method is: pulse excitation is performed by applying a pulse cycle to the creatine working electrode 3 and the creatinine auxiliary electrode 5. The pulse cycle is: first applying a 0.5 mv voltage for 0.1 s, and then applying a -0.5 mv voltage for 0.1 s;

[0147] Scheme 4: Using the electrochemical test paper provided in Comparative Example 2 for detection, without using pulse potential for excitation during detection;

[0148] Scheme 5: The electrochemical test paper provided in Example 2 is used for detection. During the detection, pulse potential is first used for excitation. The pulse potential excitation method is: pulse excitation is performed by applying a pulse cycle to the creatine working electrode 3 and the creatinine auxiliary electrode 5. The pulse cycle is: first applying a 0.1 mv voltage for 0.01 s, and then applying a -0.1 mv voltage for 0.01 s;

[0149] Scheme 6: The electrochemical test paper provided in Example 3 is used for detection. During detection, pulse potential is first used for excitation. The pulse potential excitation method is: pulse excitation is performed by applying a pulse cycle to the creatine working electrode 3 and the creatinine auxiliary electrode 5. The pulse cycle is: first apply a 0.6mv voltage for 0.05s, and then apply a -0.6mv voltage for 0.05s.

[0150] Detection: The creatine working electrode 3 and the creatinine auxiliary electrode 5 form a test circuit, the applied voltage is 0.15V, and the current I1 is measured by the constant potential method;

[0151] The creatinine working electrode 4 and the creatinine auxiliary electrode 5 form a test circuit, the applied voltage is 0.15V, and the current is I2 measured by the constant potential method;

[0152] The hematocrit working electrode 2 and the hematocrit auxiliary electrode 6 form a test circuit, and the hematocrit of the sample is tested by AC impedance method, with an amplitude of 0.15V and a frequency of 400kHz;

[0153] Then the creatinine reaction current is I2-I1.

[0154] Since creatinine concentration is also affected by the hematocrit in the sample, the electrochemical impedance spectroscopy method was used to test the hematocrit of the sample for correction of creatinine concentration.

[0155] The test results are shown in Table 1 and Figure 2-Figure 5 shown.

[0156] Table 1 Creatinine test current under different schemes

[0157]

[0158] From the experimental data, it can be seen that the linear range of creatinine test in scheme 1 is narrow, and the fitting coefficient R2 Less than 0.99, and the CV (coefficient of variation) is relatively high. The linear range of creatinine test current in scheme 2 is relatively wide, but the CV is relatively high. The creatinine test in schemes 3-5 is not only linearly wider, but also has a relatively low CV. Among them, scheme 3 has the best performance.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of carbon slurry containing potassium ferrocyanide in the preparation of electrochemical test paper, characterized in that: The carbon slurry is used as a raw material for preparing electrochemical test paper electrodes; In the carbon slurry, the mass proportion of potassium ferrocyanide is 0.1%-5%.

2. An electrochemical test paper for creatinine detection, characterized in that: include: An electrode substrate, an electrode system, a first patch, a second patch and a hydrophilic membrane; The electrode system is arranged on the electrode substrate to form a hematocrit working electrode, a hematocrit auxiliary electrode, a creatine working electrode, a creatinine auxiliary electrode, and a creatinine working electrode which are separated and not connected; the electrode system is mainly prepared from a carbon slurry containing potassium ferrocyanide; One end of the hematocrit working electrode and the hematocrit auxiliary electrode are located in the reaction area A, and the other end is located in the connection area, for transmission of electrical signals; One end of the creatine working electrode is located in the reaction area B, and the other end is located in the connection area, which is used for the transmission of electrical signals; One end of the creatinine auxiliary electrode is located in the reaction area C, and the other end is located in the connection area, which is used for the transmission of electrical signals; One end of the creatinine working electrode is located in the reaction area D, and the other end is located in the connection area, which is used for the transmission of electrical signals; From the sampling end to the other end of the electrochemical test paper, the reaction area A, the reaction area B, the reaction area C and the reaction area D are arranged in sequence; The reaction zone B is loaded with creatine hydrolase and sarcosine oxidase; the reaction zone D is loaded with creatine hydrolase, sarcosine oxidase and creatinine hydrolase; The first patch covers the electrode area except the connection area, and has windows in the reaction area A, the reaction area B, the reaction area C and the reaction area D; The second patch covers the first patch, and has windows in the reaction area A, the reaction area B, the reaction area C and the reaction area D and the areas therebetween, the windows extending to the sampling end of the electrochemical test paper to form a sampling port; The hydrophilic membrane covers the window of the second patch.

3. The electrochemical test paper according to claim 2, characterized in that: The electrode substrate includes a PET substrate, a PMMA substrate, a PVC substrate or a PCB; And / or, the electrode system is prepared by screen printing technology.

4. The electrochemical test paper according to claim 2, characterized in that: The step of loading the reaction zone B with creatine hydrolase and sarcosine oxidase comprises: dispensing solution B on the reaction zone B by using a dispensing process; The solution B is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant, 1% to 5% potassium ferrocyanide, 800 to 1200 U / ml creatine hydrolase and 800 to 1200 U / ml sarcosine oxidase.

5. The electrochemical test paper according to claim 2, characterized in that: The step of loading the reaction zone D with creatine hydrolase, sarcosine oxidase and creatinine hydrolase comprises: dispensing the solution C on the reaction zone D by using a dispensing process; The solution C is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant, 1% to 5% potassium ferrocyanide, 800 to 1200 U / ml creatine hydrolase, 800 to 1200 U / ml sarcosine oxidase and 1000 to 2000 U / ml creatinine hydrolase.

6. The electrochemical test paper according to claim 2, characterized in that: The reaction zone C is loaded with non-reactive substances, and the loading step includes: dispensing solution A on the reaction zone C by using a dispensing process; The solution A is a buffer solution with a pH value of 7 to 8, containing 0.5% to 2% PVP or PEG, 1% to 5% hydroxyethyl cellulose or methyl cellulose, 0.5% to 2% surfactant and 1% to 5% potassium ferrocyanide.

7. The electrochemical test paper according to any one of claims 4 to 6, characterized in that: The buffer solution is a PBS buffer solution; The surfactant includes triton x-100; The molecular weight of the PVP is 10k to 100k; The molecular weight of the PEG is 1k to 10k; The viscosity of the hydroxyethyl cellulose is 50-300cp; The viscosity of the methyl cellulose is 25-100 cp.

8. A device for creatinine detection, characterized in that: The invention comprises the electrochemical test paper, connector, processor and display according to any one of claims 2 to 7; The connector is used to transmit the electrical signal from the electrochemical test paper to the processor; The processor and the display are used to convert the received electrical signal into creatinine concentration and display it.

9. A method for detecting creatinine, characterized in that: The electrochemical test paper according to any one of claims 2 to 7 is used for detection, comprising the following steps: a. Pulse excitation: applying pulse cycles to the creatine working electrode and the creatinine auxiliary electrode for pulse excitation; b. Detection: The creatine working electrode and the creatinine auxiliary electrode form a test circuit, and the current measured by the constant potential method is I1; the creatinine working electrode and the creatinine auxiliary electrode form a test circuit, and the current measured by the constant potential method is I2; then the creatinine reaction current is I2-I1.

10. The detection method according to claim 9, characterized in that: The pulse cycle is: firstly apply 0.1-0.6mv voltage excitation for 0.01-0.1s, and then apply -0.1-0.6mv voltage excitation for 0.01-0.1s.

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