Detection method of glycoprotein hormone

Abstract

The invention provides an electrochemical detection method for glycoprotein hormones, which comprises the following steps: identifying and capturing glycoprotein hormones by using a specific receptor, oxidizing ortho-hydroxyl on glycoprotein hormone glycan chains into aldehyde groups by using periodate, further reducing a large amount of Ag < + > into Ag particles by using the generated aldehyde groups, and depositing the Ag particles on the surface of an electrode, and then solid volt-ampere analysis is carried out on the Ag particles deposited on the surface of the electrode, so that high-sensitivity and high-specificity detection on the glycoprotein hormone is realized. In addition, the electrochemical detection method of the glycoprotein hormone also has the advantages of simplicity, convenience, rapidness, low cost and the like, and has a very good application prospect in in-vitro diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of electroanalytical chemistry, and in particular relates to a method for detecting glycoprotein hormones. Background Art

[0002] Glycoprotein hormones (GPH), such as chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH), have been widely used in early pregnancy screening and other aspects. At present, the gold standard method for glycoprotein hormone detection is colloidal gold immunochromatography, and its basic principle is: when the sample to be tested (such as urine) is siphoned through the colloidal gold labeled antibody, an antigen-antibody-colloidal gold complex is formed, and the complex continues to crawl and passes through the anti-glycoprotein hormone monoclonal antibody coated at the test line (T line), forming a double antibody sandwich colloidal gold complex, showing a color band at the T line, and the excess colloidal gold antibody continues to crawl and combines with the antibody at the control line (C line) to form a colloidal gold immune complex, showing a color band at the C line. Therefore, when clear color bands appear at both the T line and the C line, it indicates that glycoprotein hormones are present in the sample to be tested. Although this method has the advantages of simple operation, rapid response, and strong specificity, it has disadvantages such as low sensitivity, poor stability, and easy to produce false positive results. Summary of the invention

[0003] The purpose of the present invention is to provide a method for detecting glycoprotein hormones, in particular, an electrochemical detection method for glycoprotein hormones; the method uses a specific receptor to recognize and capture the glycoprotein hormone, and then uses periodate to oxidize the ortho-hydroxyl groups on the polysaccharide chain of the glycoprotein hormone into aldehyde groups, and the generated aldehyde groups further bind a large amount of Ag. + The Ag particles are reduced to Ag particles and deposited on the electrode surface, and then the Ag particles deposited on the electrode surface are subjected to solid-state voltammetry analysis to achieve high-sensitivity detection of glycoprotein hormones.

[0004] The first aspect of the present invention provides a method for detecting a glycoprotein hormone, which specifically comprises the following steps:

[0005] 1) incubating the electrode with surface modified specific receptors with the sample to be tested;

[0006] 2) incubating the electrode obtained in step 1) with a periodate solution;

[0007] 3) incubating the electrode obtained in step 2) with a silver ion solution;

[0008] 4) Performing solid-state voltammetry analysis on the electrode obtained in step 3).

[0009] In some embodiments of the invention, the specific receptor does not contain an ortho-hydroxyl group.

[0010] In some embodiments of the present invention, the specific receptors include polypeptides, nucleic acid aptamers, nanobodies and single-chain antibodies.

[0011] In some embodiments of the present invention, the sample to be tested includes glycoprotein hormones, and the glycoprotein hormones include chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH).

[0012] In the present invention, the specific receptor refers to a receptor that can specifically recognize a specific glycoprotein hormone, such as a receptor that specifically recognizes CG, a receptor that specifically recognizes LH, a receptor that specifically recognizes FSH, or a receptor that specifically recognizes TSH.

[0013] In the present invention, the specific receptor only specifically recognizes one glycoprotein hormone, and the specific receptor cannot recognize more than one glycoprotein hormone at the same time; for example, the receptor that specifically recognizes CG only recognizes CG, but cannot recognize LH, FSH or TSH; similarly, the receptor that specifically recognizes LH cannot recognize CG, FSH or TSH, the receptor that specifically recognizes FSH cannot recognize CG, LH or TSH, and the receptor that specifically recognizes TSH cannot recognize CG, LH or FSH.

[0014] In the present invention, specific detection of glycoprotein hormones is achieved through specific recognition of specific receptors, that is, the receptor that specifically recognizes CG can achieve specific detection of CG, the receptor that specifically recognizes LH can achieve specific detection of LH, the receptor that specifically recognizes FSH can achieve specific detection of FSH, and the receptor that specifically recognizes TSH can achieve specific detection of TSH.

[0015] In some embodiments of the present invention, the sample to be tested includes a glycoprotein hormone, the glycoprotein hormone includes chorionic gonadotropin (CG), and the specific receptor includes a receptor that recognizes CG.

[0016] In some embodiments of the present invention, the sample to be tested includes a glycoprotein hormone, the glycoprotein hormone includes human chorionic gonadotropin (hCG), and the specific receptor includes a receptor that recognizes hCG.

[0017] In some embodiments of the present invention, the glycoprotein hormone is human chorionic gonadotropin (hCG), and the specific receptor is a receptor that recognizes hCG.

[0018] In some embodiments of the present invention, the specific receptor comprises the sequence shown in SEQ ID NO:1.

[0019] In some embodiments of the present invention, the specific receptor sequence is shown as SEQ ID NO:1.

[0020] In some embodiments of the present invention, the periodate includes NaIO4, KIO4, LiIO4 and NH4IO4.

[0021] In some embodiments of the invention, the electrode comprises a gold electrode.

[0022] In some embodiments of the present invention, the electrolyte solution for solid-state voltammetry analysis includes KCl, NaCl, NH4Cl, KBr, NaBr and NH4Br.

[0023] The detection method of the glycoprotein hormone of the present invention shows high sensitivity and high specificity for the detection of hCG. In the range of 1.0mIU / mL to 25mIU / mL, there is a good linear relationship between the dissolution peak current and the hCG concentration (R 2 =0.996), the detection limit of hCG was as low as 0.45mIU / mL.

[0024] In some embodiments of the present invention, the silver ion solution comprises a silver ammonia solution ([Ag(NH3)2] + ) and AgNO3 solution.

[0025] In some embodiments of the present invention, the concentration of periodate in step 2) is 0.0001-0.1M.

[0026] In some embodiments of the present invention, the concentration of silver ions in step 3) is 0.0001-0.1M.

[0027] In some embodiments of the present invention, the electrode with surface modified with specific receptors in step 1) is blocked with a blocking solution.

[0028] In some embodiments of the invention, the blocking solution comprises mercaptohexanol (MCH).

[0029] The second aspect of the present invention provides a product for detecting glycoprotein hormones, which comprises an electrode with a surface modified with a specific receptor, periodate and silver ions.

[0030] In some embodiments of the present invention, the product further comprises a blocking solution.

[0031] In some embodiments of the present invention, the product includes a detection reagent or a detection kit.

[0032] The third aspect of the present invention provides the use of the product of the second aspect of the present invention in the preparation of a detection product for glycoprotein hormone detection.

[0033] Using the product of the second aspect of the present invention, the test sample including the glycoprotein hormone is tested according to the detection method of the first aspect of the present invention. After electrochemical analysis, if a dissolution peak is observed in the potential range of -0.1 to 0.1 V, it means that the test sample contains the target glycoprotein hormone to be tested; if not, it does not contain the target glycoprotein hormone to be tested.

[0034] Furthermore, in order to quantitatively detect the concentration of glycoprotein hormone in the sample to be tested, a standard curve of the change in dissolution peak current with the concentration of the glycoprotein hormone standard is first drawn, and then the dissolution peak current measured for the sample to be tested is substituted into the standard curve to obtain the concentration of the glycoprotein hormone in the sample to be tested.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides an electrochemical detection method for glycoprotein hormones, wherein the method uses a specific receptor to recognize and capture the glycoprotein hormones, and then uses periodate to oxidize the ortho-hydroxyl groups on the polysaccharide chains of the glycoprotein hormones into aldehyde groups, and the generated aldehyde groups further bind a large amount of Ag. + The Ag particles are reduced to Ag particles and deposited on the electrode surface, and then the Ag particles deposited on the electrode surface are subjected to solid-state voltammetric analysis, thereby achieving high-sensitivity and high-specificity detection of glycoprotein hormones. In addition, the electrochemical detection method of glycoprotein hormones of the present invention also has the advantages of simplicity, rapidity, low cost, etc., and has a good application prospect in in vitro diagnosis.

[0037] The detection method of the glycoprotein hormone of the present invention shows high sensitivity and high specificity for the detection of hCG. In the range of 1.0mIU / mL to 25mIU / mL, there is a good linear relationship between the dissolution peak current and the hCG concentration (R 2 =0.996), the detection limit of hCG was as low as 0.45mIU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The principle of the highly sensitive electrochemical detection method for glycoprotein hormones.

[0039] Figure 2 This is the feasibility study result of human chorionic gonadotropin (hCG) detection in Example 1.

[0040] Figure 3 This is the SEM image of the electrode surface in Example 1.

[0041] Figure 4 This is the result of the sensitivity study of hCG detection in Example 2.

[0042] Figure 5 This is the result of the specificity study of hCG detection in Example 3.

[0043] Figure 6 This is the result of the practical study of hCG detection in Example 4. DETAILED DESCRIPTION

[0044] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0045] Figure 1 The principle of the highly sensitive electrochemical detection method of glycoprotein hormones is shown as follows:

[0046] Glycoprotein hormones usually carry a certain number of polysaccharide chains, and most of the monosaccharide residues that make up the polysaccharide chains have ortho-hydroxyl groups. - ) is oxidized to aldehyde groups, which can convert a large amount of Ag + The Ag particles are reduced to Ag particles and deposited on the electrode surface, and then the Ag particles deposited on the electrode surface are subjected to solid-state voltammetric dissolution analysis to achieve highly sensitive detection of glycoprotein hormones.

[0047] Example 1: Feasibility study of hCG detection

[0048] A 0.8 mM (7.5 μL) solution of a polypeptide terminally modified with a Cys residue (whose sequence is CGGSSPPLRINRHILTR (SEQ ID NO: 1)) was dripped onto the gold electrode surface and incubated for 15 min. Then, the remaining active sites on the electrode surface were blocked with a 2.5 mM mercaptohexanol (MCH) solution for 15 min. Subsequently, the obtained electrode surface was incubated with a 25 mIU / mL hCG solution for 30 min and immersed in a 10 mM NaIO4 solution for 15 min. Next, the obtained electrode surface was incubated with a 20 mM silver ammonia solution ([Ag(NH3)2] + ) and incubate for 30 min; finally, the obtained electrode was inserted into a 0.5 M potassium chloride solution, and saturated calomel electrode (SCE) and platinum wire were used as the reference electrode and counter electrode, respectively, and detection was performed by square wave voltammetry (SWV) (initial potential: -0.2 V; potential increment: 2 mV; termination potential: 0.2 V).

[0049] from Figure 2It can be seen that for the fully modified gold electrode, a strong dissolution peak can be observed at 0.03V; if no polypeptide solution, hCG solution, NaIO4 solution or silver ammonia solution is added during the modification of the electrode surface, no obvious dissolution peak can be observed. It can be seen that the obtained electrochemical signal can be used for the detection of hCG. Figure 3 The scanning electron microscope (SEM) image shown shows that a large amount of silver nanoparticles are deposited on the electrode surface.

[0050] Example 2: Sensitivity study of hCG detection

[0051] Under the conditions described in Example 1, the sensitivity of the method was studied. Compared with the experimental conditions in Example 1, this example explored the detection results when the hCG concentration range was 1.0-25mIU / mL, and the specific concentrations were 1.0mIU / mL, 2.5mIU / mL, 5mIU / mL, 10mIU / mL, 15mIU / mL, 20mIU / mL and 25mIU / mL. Figure 4 It can be seen that the dissolution peak current increases with the increase of hCG concentration. Moreover, in the range of 1.0mIU / mL to 25mIU / mL, there is a good linear relationship between the dissolution peak current and the hCG concentration. The linear regression equation can be expressed as y=0.021x+0.004 (where y is the dissolution peak current, in mA; x is the hCG concentration, in mIU / mL), and the correlation coefficient R 2 =0.996, and the lower limit of detection is calculated to be 0.45mIU / mL. Therefore, the method of the present invention can be used for high-sensitivity detection of hCG.

[0052] Example 3: Study on the specificity of hCG detection

[0053] To evaluate the specificity of the method, the dissolution peak currents corresponding to 25mIU / mL hCG, 15ng / mL LH, 15ng / mL FSH, 15ng / mL CA15-3, 15ng / mL carcinoembryonic antigen (CEA), 15ng / mL glycosylated albumin (GA), 15ng / mL thrombin, 15ng / mL human serum albumin (HSA) and 15ng / mL glucose were collected. Other experimental conditions and steps were the same as those in Example 1. Figure 5 It can be seen that only hCG can produce a strong dissolution peak current, while the dissolution peak currents produced by other interfering components are the same or similar to the blank control without hCG. It can be seen that the method of the present invention can be used for high-specific detection of hCG.

[0054] Example 4: Practical study of hCG detection

[0055] In order to evaluate the practicality of the method of the present invention, 10-fold diluted urine of a 24-year-old healthy male was used as a matrix to prepare three hCG solutions with concentrations of 25mIU / mL, 10mIU / mL and 1.0mIU / mL. A 0.8mM (7.5μL) solution of a polypeptide with a terminal modified with a Cys residue (whose sequence is CGGSSPPLRINRHILTR (SEQ ID NO: 1)) was dropped onto the surface of the gold electrode and incubated for 15 minutes; then, the remaining active sites on the electrode surface were blocked with a 2.5mM mercaptohexanol (MCH) solution for 15 minutes; subsequently, the resulting electrode surface was incubated with the three hCG solutions for 30 minutes and soaked in a 10mM NaIO4 solution for 15 minutes; then, the resulting electrode surface was incubated with a 20mM silver ammonia solution for 30 minutes; finally, the resulting electrode was inserted into a 0.5M potassium chloride solution, and SCE and platinum wire were used as reference electrodes and counter electrodes, respectively, and SWV was used for detection. The results are as follows. Figure 6 As shown, there is almost no difference between the peak current signal of the dissolution from the urine sample of a healthy male and the peak current signal of the dissolution from the sample of 10 mmol / L phosphate buffer (PBS, pH 7.4). It can be seen that the method of the present invention can be used for quantitative analysis of hCG concentration in actual samples.

[0056] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for detecting a glycoprotein hormone, characterized in that: The following steps are involved: 1) incubating the electrode with surface modified specific receptors with the sample to be tested; 2) incubating the electrode obtained in step 1) with a periodate solution; 3) incubating the electrode obtained in step 2) with a silver ion solution; 4) Performing solid-state voltammetry analysis on the electrode obtained in step 3).

2. The method according to claim 1, characterized in that The specific receptor contains no vicinal hydroxyl groups.

3. The method according to claim 1, characterized in that The glycoprotein hormones include chorionic gonadotropin (CG), luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH).

4. The method according to claim 1, characterized in that: The specific receptors include polypeptides, nucleic acid aptamers, nanobodies and single-chain antibodies.

5. The method according to claim 4, characterized in that The specific receptor comprises the sequence shown in SEQ ID NO:

1.

6. The method according to claim 1, characterized in that The periodates include NaIO4, KIO4, LiIO4 and NH4IO4.

7. The method according to claim 1, characterized in that The electrolyte solution for the solid-state voltammetry analysis includes KCl, NaCl, NH4Cl, KBr, NaBr and NH4Br.

8. A product for the detection of glycoprotein hormones, characterized in that: The product comprises an electrode with a surface modified with a specific receptor, periodate and Ag + .

9. The product according to claim 8, characterized in that The products include detection reagents and detection kits.

10. Use of the product according to any one of claims 8 to 9 in the preparation of a test product for glycoprotein hormone detection.

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