Insulin affinity determination device based on cell membrane directional reconstruction technology, detection method and application

Abstract

The invention belongs to the technical field of electrochemical biosensors, and discloses an insulin affinity determination device based on a cell membrane directional reconstruction technology, a detection method and application of the insulin affinity determination device based on the cell membrane directional reconstruction technology. The measuring electrode comprises a measuring layer made of a conductive material, a directional connecting layer formed by chemically modifying the measuring layer, and a cell membrane layer formed by spreading a cell membrane from bottom to top. The measuring layer transmits an electric signal to external signal recording equipment, the directional connection layer is combined with specific molecules distributed on the inner side of a cell membrane through chemical bonds, the cell membrane layer is directionally fixed to the surface of the measuring electrode, insulin receptor protein on the cell membrane is exposed to the outer side of the cell membrane layer, and insulin molecules are captured; the measuring cavity comprises a sample feeding pipeline and a sample discharging pipeline and is used for placing a measuring electrode during measurement. The device is simple in structure, the detection method is convenient, and the device can be applied to screening of diabetes-related compounds and research of pathological mechanisms of diabetes and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical biosensors, and in particular relates to an insulin affinity determination device, a detection method and an application thereof based on a cell membrane directional reconstruction technology. Background Art

[0002] Insulin receptor is a key target related to diabetes, and it is mainly present on the cell membrane of liver cells, muscle cells and fat cells. The main function of insulin receptor is to mediate insulin signal, thereby regulating the uptake, utilization and storage of glucose. In the process of diabetes drug screening, the activity and functional status of insulin receptor is an important evaluation indicator. Effective diabetes drugs should be able to enhance the sensitivity of insulin receptors, improve insulin signal transduction, and thus lower blood sugar levels. Therefore, affinity testing for insulin plays an important role in target verification, efficacy testing, safety assessment, and personalized medication. At present, the test of insulin affinity is mainly carried out in model animals or cell experiments, which has a long cycle, high throughput and low cost, and is not suitable for specific analysis of individual cells.

[0003] The present invention proposes a method of combining a cell membrane with an analytical electrode to achieve in vitro insulin affinity testing. At the same time, the insulin receptor is a transmembrane protein, and its extracellular and intracellular parts have different functions. Therefore, when constructing a cell membrane on an electrode, its directionality needs to be controlled to ensure that the extracellular side faces the liquid environment to be tested. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide an insulin affinity determination device, detection method and application based on cell membrane directional reconstruction technology. The present invention introduces a directional connection layer on the surface of the analysis electrode to connect with specific molecules mainly distributed on the inner side of the cell membrane, thereby achieving directional reconstruction of the cell membrane on the surface of the measurement electrode to meet its directional requirements.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides an insulin affinity determination device based on cell membrane directional reconstruction technology, comprising at least a measuring electrode and a measuring cavity, wherein the measuring electrode comprises, from bottom to top, a measuring layer composed of a conductive material, a directional connection layer formed by chemically modifying the measuring layer, and a cell membrane layer formed by depositing and spreading a cell membrane on the directional connection layer, wherein: The measuring layer is used to conduct the electrical signal to an external signal recording device. The directional connection layer is combined with specific molecules distributed on the inner side of the cell membrane through chemical bonds, and the cell membrane layer is fixed to the surface of the measuring electrode in a directional manner, so that the insulin receptor protein on the cell membrane is exposed to the outer side of the cell membrane layer, thereby capturing the insulin molecules during measurement. The measuring cavity comprises a sample inlet pipeline and a sample outlet pipeline, which are used to place the measuring electrode in a sealed environment constructed by the measuring cavity during measurement, and to implement continuous sample addition operation through the sample inlet pipeline and the sample outlet pipeline in a preset sequence.

[0006] Preferably, the conductive material of the measuring layer includes gold, carbon or conductive polymer.

[0007] Preferably, the specific molecules distributed on the inner side of the cell membrane include one or a combination of phosphatidylethanolamine, a phosphate group, a liposome-linked protein with a carboxyl group, a fatty acid chain, and phosphatidylinositol.

[0008] Preferably, the substance used for chemically modifying the measurement layer to form the directional connection layer includes one or a combination of mercapto, enol compounds, and tetrabutylammonium fluoride.

[0009] Preferably, the cell membrane constituting the cell membrane layer is collected from muscle cells, fat cells or liver cells having insulin receptor proteins on their surfaces.

[0010] The second aspect of the present invention provides a method for detecting cell insulin affinity, which uses the above-mentioned insulin affinity determination device for detection, comprising the following steps: S1, placing the measuring electrode prepared with the cells to be tested in a clean measuring cavity, injecting the electrode reaction liquid from the sample inlet pipeline, testing the response current of the measuring electrode at this time, and recording the current I1 through the connected signal recording device; then injecting the cleaning liquid, so that the electrode reaction liquid is emptied through the sample outlet pipeline; S2, injecting a measuring solution containing insulin molecules into the measuring cavity through the injection pipeline and incubating the solution, so that the insulin molecules fully bind to the insulin receptor protein on the cell membrane layer; S3, continue to inject cleaning solution from the sample inlet pipeline, so that the measuring solution containing insulin molecules is emptied through the sample outlet pipeline, and use the cleaning solution to clean and remove unbound insulin molecules; S4, injecting the electrode reaction solution into the measuring cavity again, and testing the response current of the measuring electrode at this time, and recording the current I2 through the connected signal recording device; S5. Determine the insulin affinity of the cells to be tested by the current change amplitude (I1-I2).

[0011] Preferably, the concentration range of insulin molecules in the measuring solution is 10 pM-10 nM, and the incubation time is 5-10 minutes.

[0012] Preferably, the electrode reaction liquid is a mixed solution of potassium ferrocyanide and potassium ferrocyanide, wherein the concentrations of potassium ferrocyanide and potassium ferrocyanide are both 2.5 mM; and the cleaning liquid is deionized water.

[0013] The third aspect of the present invention provides an application of the above-mentioned insulin affinity determination device in the screening of diabetes-related compounds and / or the study of the pathological mechanism of diabetes.

[0014] Preferably, the diabetes-related compounds include compounds that cause cells to be resistant to insulin and / or compounds that promote cells to be sensitized to insulin.

[0015] The present invention has the following beneficial effects: (1) Insulin affinity measurement is of great significance for diabetes-related drug analysis and personalized medication. For example, in pathological research, by measuring the affinity of insulin receptors, researchers can better understand the mechanism of insulin resistance in type II diabetes; such as high-throughput screening, measuring the affinity of a large number of compounds to insulin receptors, and quickly screening out potential candidate drugs; such as treatment monitoring, evaluating changes in receptor affinity during insulin treatment, monitoring treatment effects and adjusting medication strategies. However, the current test for insulin affinity is mainly carried out in model animals or cell experiments, which has a long cycle, high throughput and low cost, and is not suitable for specific analysis of individual cells. The insulin affinity determination device provided by the present invention has a simple structure and is easy to operate. It realizes the measurement of in vitro insulin affinity and can replace the expensive and long-cycle in vivo insulin affinity test.

[0016] (2) The cell membrane is a directional cell component that can be collected by cell lysis, ultracentrifugation and other methods. The cell membrane is composed of a phospholipid bimolecular skeleton to form the basic membrane morphology. The distribution of proteins, glycosyl groups, receptors and other molecules on the cell membrane facing the inside and outside of the cell is very different. Therefore, if the cell membrane directionality is not controlled during the in vitro reconstruction process, the obtained cell membrane orientation is random, and the functionality of the membrane protein cannot be effectively analyzed. The insulin affinity determination device of the present invention is prepared based on the cell membrane directional reconstruction technology. The directional reconstruction maintains the natural conformation of the membrane protein, allowing the insulin molecule to effectively bind to it, thereby ensuring the accuracy and reliability of the in vitro insulin affinity measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the measuring electrode structure of the insulin affinity determination device based on the cell membrane oriented reconstruction technology according to Example 1 of the present invention.

[0018] Figure 2 This is a schematic diagram of detecting the change in current response before and after the insulin-resistant cell membrane binds to the insulin molecule by the insulin affinity determination device of Example 2 of the present invention.

[0019] Figure 3 This is a schematic diagram of detecting the change in current response before and after the insulin sensitizing drug-treated cell membrane binds to the insulin molecule by the insulin affinity determination device of Example 3 of the present invention.

[0020] In the figure: 1. Measurement layer; 2. Directed connection layer; 3. Cell membrane layer; 31. Insulin receptor protein; 32. Phospholipid bilayer skeleton; 33. Specific molecules. DETAILED DESCRIPTION

[0021] In order to make the purpose and technical solution of the present invention clearer and more complete, the present invention is further described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which all belong to the scope of protection of the present invention.

[0022] Unless otherwise specified, the reagents and materials involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0023] Example 1 In this embodiment, the insulin affinity determination device based on the cell membrane directional reconstruction technology includes a measuring electrode and a measuring cavity, such as Figure 1 As shown, the measuring electrode includes, from bottom to top, a measuring layer 1 made of a conductive material, a directional connection layer 2 formed by chemical modification on the measuring layer 1, and a cell membrane layer 3 formed by cell membrane deposition and spreading. It is worth noting that Figure 1 As shown, the cell membrane is a directional cell component, which is composed of a phospholipid bilayer skeleton 32 to form the basic membrane morphology. The insulin receptor protein 31 is a transmembrane protein, and its extracellular and intracellular parts have different functions. It is the extracellular part of the insulin receptor protein 31 that is responsible for binding to ligands such as insulin molecules. Therefore, when constructing the cell membrane layer 3 on the measuring electrode, it is necessary to control its directionality to ensure that the extracellular part of the insulin receptor protein 31 is facing the outside of the cell membrane layer 3 so that it can effectively capture insulin molecules during measurement.

[0024] Specifically, the measuring layer 1 is mainly used to convert the biological signal of the insulin receptor protein 31 of the cell membrane layer 3 and the insulin molecule into an electrical signal and transmit it to an external signal recording device. The conductive material constituting the measuring layer 1 includes gold, carbon or conductive polymer. Chemical modification is performed on the measuring layer 1 to form a directional connection layer 2, which is mainly used to directional fix the cell membrane layer 3 on the surface of the measuring electrode. Preferably, the substances used to chemically modify the measuring layer 1 to form the directional connection layer 2 include thiol, enol compound, tetrabutylammonium fluoride, etc., or a combination of one or more of them. Since the distribution of molecules such as proteins, glycosyls, receptors, etc. on the cell membrane facing the inside of the cell and the outside is very different, the directional connection layer 2 is formed by combining with specific molecules 33 mainly distributed on the inside of the cell membrane to form a chemical bond to achieve directional fixation of the cell membrane layer 3. It should be noted that the specific molecules 33 selected to be located on the inner side of the cell membrane include one or more combinations of phosphatidylethanolamine, phosphate groups, liposomes with carboxyl groups, fatty acid chains and phosphatidylinositol, because the distribution content of the above molecular groups on the inner side of the cell membrane is much higher than their distribution content on the outer side of the cell membrane.

[0025] Furthermore, the cell membrane layer 3 is mainly constructed by extracting the cell membrane by ultracentrifugation after lysis of living cells, and then depositing and spreading the cell membrane on the surface of the measuring electrode. Figure 1 As shown, the extracellular part of the insulin receptor protein 31 on the cell membrane that binds to the insulin molecule is oriented toward the outside of the cell membrane layer 3. It should be understood that the cell membrane constituting the cell membrane layer 3 includes, but is not limited to, living cells collected from in vitro culture and having abundant insulin receptor protein 31 on the surface, such as muscle cells, fat cells or liver cells. In the field of drug development and drug screening, various model cells of the above cells can be used to regulate and edit related genes by adding dexamethasone, rosiglitazone and other compounds or plasmid transfection in in vitro culture, so as to achieve directional transformation of the degree of cell insulin resistance.

[0026] It should be noted that the measuring cavity includes a sample inlet pipeline and a sample outlet pipeline, which are used to construct a sealed environment. During measurement, the measuring electrode is placed in the measuring cavity, and the sample is added to the measuring cavity through the sample inlet pipeline and the sample outlet pipeline in a set order, so that the measuring liquid containing insulin molecules reacts with the measuring electrode, and realizes continuous operation during the insulin affinity determination process. It is a common component in the prior art and is not shown here.

[0027] Furthermore, this embodiment also provides a method for detecting the insulin affinity of cells using the above-mentioned insulin affinity determination device, comprising the following steps: S1. Place the measuring electrode prepared with the cells to be tested in a measuring cavity that has been rinsed with a cleaning solution, such as deionized water, and inject an electrode reaction solution consisting of a mixed solution of potassium ferrocyanide and potassium ferrocyanide from the injection line. The concentrations of potassium ferrocyanide and potassium ferrocyanide in the electrode reaction solution are both 2.5 mM. Test the response current of the measuring electrode at this time, and record the current I1 through the connected signal recording device; then inject deionized water from the injection line to drain the electrode reaction solution through the sample outlet line, and rinse the measuring cavity again with deionized water; S2, injecting a measuring solution containing insulin molecules into the measuring cavity through the injection pipeline, and incubating for 5-10 minutes to allow the insulin molecules to fully bind to the insulin receptor protein 31 on the cell membrane layer 3, wherein the concentration range of the insulin molecules in the measuring solution is 10pM-10nM; S3, continue to inject deionized water from the sample inlet line, so that the measuring liquid containing insulin molecules is emptied through the sample outlet line, and the unbound insulin molecules are washed with deionized water; S4, injecting the electrode reaction solution composed of the mixed solution of potassium ferrocyanide and potassium ferrocyanide into the measuring cavity again, testing the response current of the measuring electrode at this time, and recording the current I2 through the connected signal recording device; S5. Due to the changes in the steric hindrance and charge effect after the specific binding of insulin molecules to the insulin receptor protein 31, the electron transfer of potassium ferrocyanide and potassium ferrocyanide on the measuring layer 1 is affected. That is, when the affinity of the insulin molecules to the insulin receptor protein 31 is high, the number of insulin molecules bound to the surface of the cell membrane layer 3 increases, which reduces the chance of potassium ferrocyanide and potassium ferrocyanide approaching the surface of the measuring layer 1, reduces the number of electron transfers, and reduces the current. Therefore, the affinity of the insulin receptor protein 31 to the insulin molecule can be judged by the current change amplitude (I1- I2) generated by the two electron transfers.

[0028] Example 2 In the insulin affinity determination device based on the cell membrane directional reconstruction technology of this embodiment, the measuring layer 1 uses gold as the conductive material, and the gold electrode layer is accurately deposited by magnetron sputtering. The sulfhydryl group is modified on the surface of the gold electrode to form a directional connection layer 2, and the directional connection layer 2 forms a covalent bond with the amino group on the phosphatidylethanolamine on the inner side of the cell membrane through the sulfhydryl group. The cell source of the cell membrane layer 3 is L6 muscle cells, which are cultured and differentiated into L6 insulin-resistant muscle cells by adding 1 μM dexamethasone to the cell culture medium. The cells cultured without dexamethasone are L6 normal muscle cells. After that, the L6 insulin-resistant muscle cells and the L6 normal muscle cells as the control are subjected to cell lysis and ultracentrifugation respectively to obtain cell membrane fragments, and then the above two cell membrane fragments are spread on the directional connection layer 2 of the measuring electrode respectively to prepare the corresponding two measuring electrodes. Three replicate samples of each type of measuring electrode are prepared in the same batch to ensure the reliability of the experiment. The sulfhydryl groups on the directional connection layer 2 and the amino groups on the phosphatidylethanolamine on the inner side of the cell membrane combine to form a covalent bond, thereby anchoring the cell membrane, and the unanchored cell membrane is washed away during the cleaning process. Repeat three times to ensure that a complete cell membrane covering layer is formed on the surface of the directional connection layer 2, meeting the requirements of the insulin affinity test.

[0029] During the measurement process, deionized water was first injected through the sample inlet of the measuring chamber to flush the pipe and the chamber. Then, the measuring electrode was placed in the measuring chamber containing a mixed solution of 2.5 mM potassium ferrocyanide and potassium ferrocyanide, and the response current of the measuring electrode was tested. The current I corresponding to the L6 normal muscle cell was recorded by the connected signal recording device. 1a The current I corresponding to L6 insulin-resistant myocytes 1b After the mixed solution of potassium ferrocyanide and potassium ferrocyanide was discharged, the measurement liquid containing insulin molecules was injected into the measurement chamber. The concentration of insulin molecules was 10 nM. After the measurement liquid was allowed to stand for 5 minutes, deionized water was injected for cleaning to flush away the free insulin molecules that were not bound to the insulin receptor protein 31. Then, a mixed solution of potassium ferrocyanide and potassium ferrocyanide with a concentration of 2.5 mM was injected again, and the response current of the measuring electrode was tested at this time. The current I corresponding to the normal muscle cells of L6 was recorded by the connected signal recording device. 2a The current I corresponding to L6 insulin-resistant myocytes 2b .like Figure 2 As shown, normal muscle cells produced an average current change of 2.94nA (I 1a -I 2a ), insulin-resistant muscle cells produced an average current change of 1.46nA when faced with the same concentration of insulin molecules (I 1b -I 2b). Since the specific binding of insulin receptor protein 31 to insulin molecules causes steric hindrance changes, it will reduce the chances of potassium ferrocyanide and potassium ferrocyanide approaching the surface of the measuring layer 1, reducing the number of electron transfers. Therefore, the current change amplitude of normal muscle cells is greater than that of insulin-resistant muscle cells. That is, from the perspective of current increment, it can be explained that the binding ability of insulin receptor protein 31 on the cell membrane surface of insulin-resistant muscle cells to insulin molecules has decreased.

[0030] Example 3 In the insulin affinity determination device based on the cell membrane directional reconstruction technology of this embodiment, the measuring layer 1 uses gold as the conductive material, and the gold electrode layer is accurately deposited by magnetron sputtering. The sulfhydryl group is modified on the surface of the gold electrode to form a directional connection layer 2, and the directional connection layer 2 is combined with the carboxyl group of the fatty acid chain on the inner side of the cell membrane through the sulfhydryl group to form an amide bond. The cell source of the cell membrane layer 3 is L02 hepatocytes, which are cultured and differentiated into L02 insulin-sensitized hepatocytes by adding 1 μM rosiglitazone to the cell culture medium, and the cells cultured without rosiglitazone are L02 normal hepatocytes. After that, the L02 insulin-sensitized hepatocytes and the L02 normal hepatocytes as the control are subjected to cell lysis and ultracentrifugation respectively to obtain cell membrane fragments, and then the above two cell membrane fragments are spread on the directional connection layer 2 of the measuring electrode respectively to prepare two corresponding measuring electrodes, and three replicate samples of each type of measuring electrode are prepared in the same batch to ensure the reliability of the experiment. The sulfhydryl groups on the directional connection layer 2 and the carboxyl groups of the fatty acid chains on the inner side of the cell membrane combine to form an amide bond, thereby anchoring the cell membrane, and the unanchored cell membrane is washed away during the cleaning process. Repeat three times to ensure that a complete cell membrane covering layer is formed on the surface of the directional connection layer 2 to meet the requirements of the insulin affinity test.

[0031] During the measurement process, deionized water was first injected through the sample inlet of the measuring chamber to flush the pipe and the chamber. Then, the measuring electrode was placed in the measuring chamber containing a mixed solution of 2.5 mM potassium ferrocyanide and potassium ferrocyanide, and the response current of the measuring electrode was tested. The current I corresponding to the normal liver cells of L02 was recorded by the connected signal recording device. 3a The current I corresponding to L02 insulin-sensitized hepatocytes 3b After the mixed solution of potassium ferrocyanide and potassium ferrocyanide was discharged, the measuring solution containing insulin molecules was injected into the measuring chamber. The concentration of insulin molecules was 8 nM. After the measuring solution was allowed to stand for 5 minutes, deionized water was injected for cleaning to flush away the free insulin molecules that were not bound to the insulin receptor protein 31. Then, a mixed solution of potassium ferrocyanide and potassium ferrocyanide with a concentration of 2.5 mM was injected again, and the response current of the measuring electrode was tested at this time. The current I corresponding to the normal liver cells of L02 was recorded by the connected signal recording device. 4a The current I corresponding to L02 insulin-sensitized hepatocytes4b .like Figure 3 As shown, normal liver cells produced an average current change of 2.98nA (I 3a -I 4a ), insulin-sensitized hepatocytes produced an average current change of 3.79nA when faced with the same concentration of insulin molecules (I 3b -I 4b ). Since the specific binding of insulin receptor protein 31 to insulin molecules causes steric hindrance changes, it will reduce the chances of potassium ferrocyanide and potassium ferrocyanide approaching the surface of the measuring layer, reducing the number of electron transfers. Therefore, the current change amplitude of normal liver cells is smaller than that of insulin-sensitized liver cells. That is, from the perspective of current increment, it can be explained that the insulin receptor protein 31 on the cell membrane surface of insulin-sensitized liver cells has enhanced its binding ability to insulin molecules under the action of sensitizing drugs.

Claims

1. An insulin affinity determination device based on cell membrane directional reconstruction technology, characterized in that: The method comprises at least a measuring electrode and a measuring cavity, wherein the measuring electrode comprises, from bottom to top, a measuring layer (1) made of a conductive material, a directional connection layer (2) formed by chemically modifying the measuring layer (1), and a cell membrane layer (3) formed by depositing and spreading a cell membrane on the directional connection layer (2), wherein: The measuring layer (1) is used to conduct the electrical signal to an external signal recording device, and the directional connection layer (2) is combined with a specific molecule (33) distributed on the inner side of the cell membrane through a chemical bond, so as to directionally fix the cell membrane layer (3) to the surface of the measuring electrode, so that the insulin receptor protein (31) on the cell membrane is exposed to the outer side of the cell membrane layer (3), thereby achieving the capture of insulin molecules during measurement; The measuring cavity comprises a sample inlet pipeline and a sample outlet pipeline, which are used to place the measuring electrode in a sealed environment constructed by the measuring cavity during measurement, and to implement continuous sample addition operation through the sample inlet pipeline and the sample outlet pipeline in a preset sequence.

2. The insulin affinity measuring device according to claim 1, characterized in that: The conductive material of the measuring layer (1) includes gold, carbon or conductive polymer.

3. The insulin affinity measuring device according to claim 1, characterized in that: The specific molecules (33) distributed on the inner side of the cell membrane include one or a combination of phosphatidylethanolamine, phosphate groups, liposomes with carboxyl groups, fatty acid chains, and phosphatidylinositol.

4. The insulin affinity measuring device according to claim 3, characterized in that: The substance used for chemically modifying the measurement layer (1) to form the directional connection layer (2) comprises one or a combination of mercapto, enol compounds, and tetrabutylammonium fluoride.

5. The insulin affinity measuring device according to claim 1, characterized in that: The cell membranes constituting the cell membrane layer (3) are collected from muscle cells, fat cells or liver cells having insulin receptor proteins on their surfaces.

6. A method for detecting cell insulin affinity, characterized in that: The method of using the insulin affinity determination device as described in any one of claims 1 to 5 for detection comprises the following steps: S1, placing the measuring electrode prepared with the cells to be tested in a clean measuring cavity, injecting the electrode reaction liquid from the sample inlet pipeline, testing the response current of the measuring electrode at this time, and recording the current I1 through the connected signal recording device; then injecting the cleaning liquid, so that the electrode reaction liquid is emptied through the sample outlet pipeline; S2, injecting a measuring solution containing insulin molecules into the measuring chamber through the injection pipeline and incubating the solution, so that the insulin molecules fully bind to the insulin receptor protein (31) on the cell membrane layer (3); S3, continue to inject cleaning solution from the sample inlet pipeline, so that the measuring solution containing insulin molecules is emptied through the sample outlet pipeline, and use the cleaning solution to clean and remove unbound insulin molecules; S4, injecting the electrode reaction solution into the measuring cavity again, and testing the response current of the measuring electrode at this time, and recording the current I2 through the connected signal recording device; S5. Determine the insulin affinity of the cells to be tested by the current change amplitude (I1-I2).

7. The detection method according to claim 6, characterized in that: The concentration range of insulin molecules in the measuring solution is 10 pM-10 nM, and the incubation time is 5-10 minutes.

8. The detection method according to claim 6, characterized in that: The electrode reaction solution is a mixed solution of potassium ferrocyanide and potassium ferrocyanide, wherein the concentrations of potassium ferrocyanide and potassium ferrocyanide are both 2.5 mM; the cleaning solution is deionized water.

9. Use of the insulin affinity determination device according to any one of claims 1 to 5 in screening diabetes-related compounds and / or studying the pathological mechanism of diabetes.

10. The use according to claim 9, characterized in that: The diabetes-related compounds include compounds that cause cells to be resistant to insulin and / or compounds that promote cells to be sensitized to insulin.

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