Electrochemical detection method based on screen-printed electrodes

Abstract

The present invention relates to an electrochemical detection method based on a screen-printed electrode, which specifically comprises the following steps: S1 preparation of protein binding groups: treating the working electrode on the screen-printed electrode substrate, and adhering the protein binding groups; S2 basic protein reaction: adding basic protein, and the active groups on the basic protein react with the protein binding groups in the step S1; S3 blocking basic protein: using a blocking liquid to block the basic protein after the reaction in the step S2, and obtaining the screen-printed electrode to be detected; S4 electrochemical detection: adding a detection reagent containing a target protein, and the basic protein reacts specifically with the target protein, and collecting data from the working electrode after the reaction for electrochemical analysis to obtain the detection result. The collected data is electrochemically analyzed, and the results of different detection items can be obtained according to different basic proteins and target proteins, with high sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical detection, and in particular to an electrochemical detection method based on screen-printed electrodes. Background Art

[0002] Electrochemical detection is a method that uses the redox properties of a substance to determine the characteristics of the substance being tested by measuring changes in electrochemical parameters in a solution.

[0003] In Chinese patent document CN109490399A, an electrochemical detection device and an electrochemical detection method are disclosed, including causing the voltage scanning unit to generate a plurality of voltage scanning signals of different waveforms according to the scanning waveform control signal and applying the voltage scanning signal to the electrode unit; causing the electrode unit to participate in an electrochemical reaction driven by the voltage scanning signal to generate a detection current signal; and causing the detection unit to obtain the detection current signal from the electrode unit. When performing electrochemical detection, an electric signal with a certain current and potential strength is generally applied to the solution through an electrode system as an excitation signal to stimulate an oxidation-reduction reaction in the solution, causing the electrochemical parameters of the solution to change accordingly. By measuring the changes in the electrochemical parameters in the solution, the electrochemical detection device can accurately measure the characteristic information of the substance to be tested in the solution.

[0004] However, in the above technical solution, there is no detailed description on the detection of different projects or even multiple projects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an electrochemical detection method based on screen-printed electrodes, which can carry out different detection items in complex system samples with high sensitivity.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: the electrochemical detection method based on screen-printed electrodes specifically comprises the following steps:

[0007] Preparation of S1 protein binding groups: the working electrode on the screen-printed electrode substrate is treated to adhere the protein binding groups;

[0008] S2 basic protein reaction: adding basic protein, and the active groups on the basic protein react with the protein binding groups in step S1;

[0009] S3: blocking the basic protein: using a blocking solution to block the basic protein after the reaction in step S2 to obtain a screen-printed electrode to be detected;

[0010] S4 electrochemical detection: adding a detection reagent containing the target protein, the base protein reacts specifically with the target protein, and the data collected by the working electrode after the reaction is electrochemically analyzed to obtain the detection result.

[0011] The protein binding group is adhered and fixed on the surface of the working electrode. When it reacts and binds with the active groups on the basic protein, the original membrane charge density will change, eventually leading to a change in the membrane potential. This can characterize or define how much basic protein is involved in the reaction and binding with the protein binding group. The role of the blocking solution is to react with the active groups on the basic protein that have not reacted, and then add a permeable protective membrane to make the "active groups on the basic protein and the protein binding groups bind" more firmly. During electrochemical detection, the basic protein reacts specifically with the target protein, and the collected data includes the collection of electrode + electrical signals, especially the impedance or voltage difference between the working electrode and the reference electrode of the screen-printed electrode, and electrochemical analysis is performed. According to different basic proteins and target proteins, the results of different detection items can be obtained with high sensitivity.

[0012] Preferably, in step S1, the surface of the working electrode is pretreated, and the specific method of the pretreatment is:

[0013] S11 Clean the electrode surface: mechanical grinding and polishing to mirror surface;

[0014] S12 identification of electrode surface cleanliness: within 1×10 -3 mol / L K3Fe(CN)6 phosphate buffer solution until reversible cathode and anode peaks appear, that is, the surface is cleaned, otherwise return to step S11.

[0015] It should be noted that when performing step S11 polishing, grinding is performed in the order of decreasing particle size, and after polishing, the particles are moved into an ultrasonic water bath for cleaning until they are clean.

[0016] Preferably, in step S1, the protein binding group is an aldehyde group, a carboxyl group or an acyl group.

[0017] The aldehyde groups can be exposed as protein binding groups by treating the surface of the working electrode substrate with 25% (molar concentration) glutaraldehyde.

[0018] Preferably, the base protein in step S2 and the target protein in step S4 correspond to each other and are used for different detection items.

[0019] Preferably, the basic protein in step S2 is troponin cTnI antibody; and the target protein in step S4 is troponin cTnI, which is used for troponin cTnI detection project.

[0020] Troponin is composed of three subunits: troponin I, T, and C. Together with tropomyosin, it regulates the interaction between actin and myosin through the activity of calcium ions on striated actin ATPase. When the myocardium is damaged, the cardiac troponin complex is released into the blood. After 4-6 hours, it begins to increase in the blood. The elevated troponin I can remain in the blood for a long time, 6-10 days. Troponin I has high myocardial specificity and sensitivity, so troponin I has become the most ideal marker for myocardial infarction.

[0021] Preferably, the basic protein in step S2 is a myoglobin MYO-specific monoclonal antibody; the target protein in step S4 is myoglobin MYO; and it is used for the myoglobin MYO detection project.

[0022] The MYO method is a two-step enzyme immunoassay based on the "sandwich" principle, where the sample is incubated with chromium dioxide particles coated with myoglobin-specific monoclonal antibodies to form a particle / MYO complex. The particles are separated by magnetization and the supernatant is removed. In the second step, the particle / MYO complex is incubated with a conjugate reagent (myoglobin-specific monoclonal antibody labeled with β-galactosidase) to form a particle / MYO / conjugate sandwich. Unbound conjugate and analyte are separated by magnetization and removed by washing. The sandwich-bound β-galactosidase binds to the chromophore substrate chlorophenol red-β-d-pyranoside (CPRG) and catalyzes the hydrolysis of the substrate to chlorophenol red (CPR). The CPR formation changes the absorbance at 577 nm that is proportional to the concentration of MYO present in the patient sample.

[0023] Preferably, the basic protein in step S2 is creatine kinase isoenzyme CK-MB antibody; the target protein in step S4 is creatine kinase isoenzyme CK-MB; used for creatine kinase isoenzyme CK-MB detection project.

[0024] Creatine kinase isoenzyme CK-MB detection items: Creatine kinase is a dimeric enzyme, and its subunits are of two types: M type (muscle type) and B type (brain type). It is composed of different combinations of three isoenzymes, namely CK-MM, CK-MB, and CK-BB. CK-MM (CK3) is mainly present in striated muscle, and a small amount is present in myocardium. CK-BB (CK1) is mainly present in brain tissue, and its content in other major organs such as liver, kidney, lung, spleen and red blood cells is extremely small, and it is not easy to detect under normal conditions. CK-MB (CK2) is mainly present in myocardium and almost does not exist in other tissues. In normal serum, there are mainly CK-MM and a small amount of CK-MB, and CK-BB is almost non-existent. A significant increase in CK-MB in the blood indicates myocardial infarction. It can better judge myocardial damage than the total CK activity assay, and has higher specificity and sensitivity.

[0025] There is almost no CK-BB in normal serum. Antiserum against the M subunit can form an antigen-antibody complex with the M subunit in CK-MM and CK-MB, thereby completely inhibiting the enzyme activity of the M subunit. Multiplying the measured CK activity value by 2 represents the activity of CK-MB. The detection principle of creatine kinase (CK) activity is: CK catalyzes the reaction of creatine phosphate and adenosine diphosphate (ADP) to produce adenosine triphosphate (ATP) and creatine; coupled hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) catalytic reaction. HK catalyzes the reaction of glucose and ATP to form glucose-6-phosphate, and G6PDH catalyzes the oxidation of glucose-6-phosphate to form 6-phosphogluconate and NADPH. The rate of NADPH generation represents CK activity. The reaction mechanism is as follows:

[0026]

[0027]

[0028]

[0029] Preferably, the basic protein in step S2 is N-terminal pro-brain natriuretic peptide NT-proBNP antibody; the target protein in step S4 is N-terminal pro-brain natriuretic peptide NT-proBNP; used for N-terminal pro-brain natriuretic peptide NT-proBNP detection project.

[0030] ProBNP, BNP, and NT-proBNP coexist in the blood circulation. NT-proBNP is an inactive amino acid fragment, and no clear biological function has been found so far. Left ventricular stretch and ventricular wall tension regulate the release of NT-proBNP. In normal people, NT-proBNP and BNP are always secreted in equal moles, and their plasma concentrations are basically the same. However, in heart failure, the concentration of plasma NT-proBNP is 2-10 times higher than that of BNP, and the half-life is 60-120 minutes, which is longer than BNP (20 minutes).

[0031] The N-terminal pro-brain natriuretic peptide NT-proBNP test project is to detect the concentration of NT-proBNP in human blood. The diagnostic values ​​of NT-proBNP values ​​according to age stratification are shown in Table 1 below:

[0032] Table 1 Diagnostic cutoffs of NT-proBNP according to age stratification

[0033]

[0034] BNP (type B natriuretic peptide / brain natriuretic peptide) was originally isolated from pig brain tissue and was called brain natriuretic peptide, but its synthesis and secretion are mainly in ventricular myocardial cells. The gene of BNP exists at the end of the short arm of chromosome 1, including 3 exons and 2 introns. It is transcribed into BNP precursor (proBNP) composed of 108 amino acids through mRNA and exists in some endocrine granules of ventricular myocardium. When the ventricular volume load or pressure load increases, causing the ventricular myocardium to be stretched or the ventricular wall pressure to increase, the stored proBNP is secreted and released, and quickly decomposed into inactive amino-terminal proBNP (NT-proBNP) composed of 76 amino acids and BNP composed of 32 amino acids with endocrine activity. The effects of BNP include diuresis, sodium excretion, vasodilation, inhibition of the renin-angiotensin-aldosterone system, inhibition of the release of adrenocorticotropic hormone (ACTH) and excessive sympathetic nerve response, and participation in the regulation of blood pressure, blood volume and salt balance. Recent studies have shown that BNP can also inhibit myocardial fibrosis, vascular smooth muscle cell proliferation, and anti-coronary artery spasm.

[0035] Preferably, the basic protein in step S2 is serum amyloid A antibody and C-reactive protein antibody; the target protein in step S4 is serum amyloid A and C-reactive protein; and it is used for serum amyloid A / C-reactive protein detection project.

[0036] SAA: Human serum amyloid A, the precursor of tissue amyloid A, is an acute phase reaction protein. Under normal circumstances, its content in plasma is extremely small. When the body is stimulated by antigens such as bacteria, viruses, mycoplasma, and chlamydia, liver cells synthesize and secrete a large amount of SAA into the blood, which rapidly increases by about 1000 times within 5-6 hours.

[0037] CRP: C-reactive protein, an acute phase protein synthesized by the liver under stress. Its physiological functions include infection defense and phagocytosis and regulation during inflammation. Under normal circumstances, its content in plasma is low. When the body is infected or traumatized, it rises rapidly within 5-8 hours and reaches a peak within 24 hours.

[0038] During viral infection, SAA changes are more sensitive than CRP changes, so SAA levels can be used as a sensitive and reliable monitoring indicator for infection. Joint monitoring of SAA and CRP can make up for the disadvantage that CRP levels do not change significantly during viral infection.

[0039] Preferably, the basic protein in step S2 is procalcitonin (PCT) antibody; the target protein in step S4 is procalcitonin (PCT); and it is used for the procalcitonin (PCT) detection project.

[0040] PCT, also known as serum calcitonin, is a polypeptide hormone extracted from thyroid tumor cell culture fluid. It is a tumor marker for thyroid tumor serum. Therefore, the PCT level index of normal people will be low, and the detection rate for diagnosing infectious diseases is high. IL-6 is mainly an interleukin-6, which is a lymphokine for activated T cells and fibroblasts. It can effectively make the bone marrow cells in the patient's body gradually grow and differentiate, thereby enhancing the lysis of natural killer cells. Therefore, when the patient is infected, the IL-6 index will gradually increase. Relevant data have shown that PCT is a calcitonin propeptide substance with no hormonal activity. When the PCT value gradually increases, it indicates that the patient has an infectious disease. It is a relatively sensitive indicator for distinguishing infectious diseases.

[0041] Procalcitonin (PCT) is a protein synthesized and secreted by the thyroid gland. It can be produced in various tissues under the influence of endotoxins, TNF-α and other cytokines during infection. The level of PCT in plasma increases in severe bacteria, sepsis, and multiple organ failure, but does not increase in autoimmune, allergic and viral infections. PCT reflects the activity of systemic inflammatory response and is a new specific indicator for early diagnosis, differential diagnosis and treatment monitoring of bacterial infections.

[0042] Preferably, in step S3, the blocking solution is BSA bovine serum albumin or casein or a protein-free compound.

[0043] Preferably, in step S4, the collected data includes the impedance or voltage difference between the working electrode and the reference electrode in the screen-printed electrode.

[0044] The technical solution of the present invention is based on the electrochemical detection method of screen-printed electrodes, and detects reagents by detecting electrical signals through electrochemical technology. The reagent reaction is detected by (electro)chemical reaction / interaction and converted into a detectable analytical signal. Specifically: the sample is fixed on the electrode surface, and then the target molecule is captured on the electrode surface through the specific recognition effect between biological molecules. The base electrode converts the concentration signal into current, and the measurable electrical signal is used as the corresponding signal, thereby realizing quantitative or qualitative analysis of the target analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a flow chart of the electrochemical detection method based on screen-printed electrodes of the present invention. DETAILED DESCRIPTION

[0046] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0047] The electrochemical detection method based on screen-printed electrodes of this embodiment is as follows: Figure 1 As shown, the specific steps include:

[0048] Preparation of S1 protein binding groups: the working electrode on the screen-printed electrode substrate is treated to adhere the protein binding groups;

[0049] S2 basic protein reaction: adding basic protein, and the active groups on the basic protein react with the protein binding groups in step S1;

[0050] S3: blocking the basic protein: using a blocking solution to block the basic protein after the reaction in step S2 to obtain a screen-printed electrode to be detected;

[0051] The blocking solution is BSA bovine serum albumin or casein or a protein-free compound;

[0052] S4 electrochemical detection: adding a detection reagent containing the target protein, the base protein reacts specifically with the target protein, and performing electrochemical analysis on the data collected by the working electrode after the reaction to obtain the detection result;

[0053] The collected data includes the impedance or voltage difference between the working electrode and the reference electrode in the screen-printed electrode.

[0054] The basic protein in step S2 and the target protein in step S4 correspond to each other and are used for different detection items. Different detection items have different target proteins corresponding to different basic proteins. For example, (1) the basic protein in step S2 is troponin cTnI antibody; the target protein in step S4 is troponin cTnI, which is used for troponin cTnI detection item; (2) the basic protein in step S2 is myoglobin MYO-specific monoclonal antibody; the target protein in step S4 is myoglobin MYO; which is used for myoglobin MYO detection item; (3) the basic protein in step S2 is creatine kinase isoenzyme CK-MB antibody; the target protein in step S4 is creatine kinase isoenzyme C K-MB; used for the creatine kinase isoenzyme CK-MB detection project; (4) the basic protein in the step S2 is the N-terminal pro-brain natriuretic peptide NT-proBNP antibody; the target protein in the step S4 is the N-terminal pro-brain natriuretic peptide NT-proBNP; used for the N-terminal pro-brain natriuretic peptide NT-proBNP detection project; (5) the basic protein in the step S2 is the serum amyloid A antibody and the C-reactive protein antibody; the target protein in the step S4 is the serum amyloid A and the C-reactive protein; used for the serum amyloid A / C-reactive protein detection project; (6) the basic protein in the step S2 is the procalcitonin PCT antibody; the target protein in the step S4 is the procalcitonin PCT; used for the procalcitonin PCT detection project.

[0055] In the step S1, the surface of the working electrode is pretreated, and the specific method of the pretreatment is:

[0056] S11 Clean the electrode surface: mechanical grinding and polishing to mirror surface;

[0057] S12 identification of electrode surface cleanliness: within 1×10 -3 mol / L K3Fe(CN)6 phosphate buffer solution until reversible cathode and anode peaks appear, that is, the surface is cleaned, otherwise return to step S11.

[0058] In the step S1, the protein binding group is an aldehyde group, a carboxyl group or an acyl group; the aldehyde group can be used as the exposed protein binding group by treating the surface of the working electrode substrate with 25% (molar concentration) glutaraldehyde.

[0059] For ordinary technicians in this field, the specific embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, such as replacing the basic protein and the target protein for other detection items, they are all within the protection scope of the present invention.

Claims

1. An electrochemical detection method based on screen-printed electrodes, characterized in that: The specific steps include: Preparation of S1 protein binding group: pre-treating the working electrode on the screen-printed electrode substrate, adhering the protein binding group; the protein binding group is an aldehyde group, and the aldehyde group is treated with 25% molar concentration of glutaraldehyde on the surface of the working electrode substrate; The specific method of the pretreatment is: S11 Clean the electrode surface: mechanical grinding and polishing to mirror surface; S12 identification of electrode surface cleanliness: within 1×10 -3 Scanning in a phosphate buffer solution of 10 mol / L K3Fe(CN)6 until reversible cathode and anode peaks appear, that is, the surface is cleaned, otherwise returning to step S11; S2 basic protein reaction: adding basic protein, and the active group on the basic protein reacts with the protein binding group in step S1; the basic protein is any one of troponin cTnI antibody, myoglobin MYO specific monoclonal antibody, creatine kinase isoenzyme CK-MB antibody, N-terminal pro-brain natriuretic peptide NT-proBNP antibody, serum amyloid A antibody, C-reactive protein antibody or procalcitonin PCT antibody; S3: blocking the basic protein: using a blocking solution to block the basic protein after the reaction in step S2 to obtain a screen-printed electrode to be detected; S4 electrochemical detection: adding a detection reagent containing a target protein corresponding to the basic protein, wherein the target protein is any one of troponin cTnI, myoglobin MYO, creatine kinase isoenzyme CK-MB, N-terminal pro-brain natriuretic peptide NT-proBNP, serum amyloid A, C-reactive protein or procalcitonin PCT; the basic protein reacts specifically with the target protein, and electrochemically analyzes the data collected by the working electrode after the reaction, wherein the collected data includes the impedance or voltage difference between the working electrode and the reference electrode in the screen-printed electrode, and obtains the detection result.

2. The electrochemical detection method based on screen-printed electrodes according to claim 1, characterized in that: In step S3, the blocking solution is BSA bovine serum albumin or casein or a protein-free compound.

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