Biosensor for detecting GADA and application thereof

Abstract

The invention discloses a biosensor for detecting GADA and application of the biosensor. A ruthenium-labeled immune complex is fixed on a flexible polyethylene glycol (PEG) chain on the surface of an electrode, a loose structure of an immune complex assembly layer is provided, and a larger free space is provided for collision ECL reaction of the ruthenium complex on the electrode, so that current and ECL emission are enhanced. The designed biosensor is high in detection specificity, high in sensitivity, good in accuracy and flexible in construction method, a flexible PEG chain with proper length and concentration can be selected according to detection requirements, and an effective strategy is provided for diagnosis of early-stage type 1 diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to a biosensor for detecting GADA and application thereof, and in particular to a biosensor for detecting GADA with high sensitivity and application thereof. Background Art

[0002] Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease caused by a combination of susceptibility genes and environmental factors, leading to pancreatic β-cell damage. The disease is usually diagnosed in early adulthood and adolescence. The classic markers for diagnosing T1DM currently include glutamic acid decarboxylase antibodies (GADA), protein tyrosine phosphatase antibodies (IA-2A), insulin antibodies (IAA), islet cell antibodies (ICA), and transporter 8 antibodies (ZnT8A). Among these antibodies, GADA has the highest sensitivity and specificity and is an important immune indicator for diagnosing T1DM. GADA is involved in the pathogenesis of immune-mediated (type 1) diabetes. Compared with type 2 diabetes patients with similar diabetes severity, GADA-positive diabetic patients have a higher risk of cognitive decline. GADA may mediate cognitive dysfunction by disrupting the production of γ-aminobutyric acid (GABA) and may lead to dementia in diabetic patients. Radioligand Binding Assay (RBA) has become an internationally recognized standardized method for GADA detection due to its high detection sensitivity. However, this technology faces challenges in future applications due to its high cost, potential radioactive contamination, and complex and time-consuming process.

[0003] Electrochemiluminescence (ECL) technology is a combination of electrochemical reaction and chemiluminescence, in which the electrochemical reaction produces active intermediates and excited luminescent substances. Subsequently, the excited luminescent substances return to the ground state and emit light. Since there is no light source in the whole process, the ECL method overcomes the interference of background signals and has the advantages of high sensitivity and fast response time. Therefore, ECL-based detection has been widely used in biosensing and clinical testing. Although significant achievements have been made in the development of ECL-based immunosensors, in order to better detect and analyze low-abundance proteins in serum, there is still a need to further enhance ECL emission and improve sensor performance. Summary of the invention

[0004] Purpose of the invention: The present invention aims to provide a biosensor suitable for detecting extremely low concentrations of GADA and convenient for identifying signals, and its application in the field of type 1 diabetes diagnosis.

[0005] Technical solution: The biosensor for detecting GADA described in the present invention is prepared by the following method:

[0006] (1) Carboxylation of the gold substrate electrode with HS-PEG-COOH to obtain Au / HS-PEG-COOH;

[0007] (2) coupling the GAD65 antigen (Ag) to the carboxylated electrode obtained in step (1) to obtain Au / HS-PEG-COOH / Ag bioconjugate;

[0008] (3) coupling the GADA antibody to the electrode obtained in step (2) to obtain Au / HS-PEG-COOH / Ag / GADA;

[0009] (4) coupling the ruthenium-labeled GAD65 antigen (Ru-Ag) to the electrode obtained in step (3) to obtain the biosensor Au / HS-PEG-COOH / Ag / GADA / Ru-Ag;

[0010] Among them, the molecular weight of PEG is 2000~10000Da.

[0011] Different from the traditional ECL immunosensor strategy that focuses on improving the electron transfer rate, the present invention fixes the ruthenium-labeled immune complex on a flexible polyethylene glycol (PEG) chain to induce more free collisions of ruthenium complexes on the electrode, thereby enhancing the ECL detection signal.

[0012] Specifically, PEG with a long chain structure was first self-assembled on a gold electrode, which served as a recognition layer for connecting and fixing glutamate decarboxylase 65 (GAD65). Then, through the specific binding interaction between the GAD65 antigen and the antibody, GADA was introduced as the target analyte to form a sandwich complex. Then, Ru(bpy)3 2+ Labeled GAD65 captures the target antibody to form an immune complex, Ru(bpy)3 2+ As a luminophore, under certain voltage conditions, Ru(bpy)3 2+ Excited Ru(bpy)3 2+* The structure constructed with long-chain PEG linkers will cause the sandwich structure to collapse, resulting in more collisions between the ruthenium complex and the electrode surface, promoting Ru(bpy) 2+ / 3+ The direct electron transfer between them can enhance the ECL emission, providing a new strategy for the construction of sensitive immunosensors.

[0013] Preferably, the molecular weight of PEG is 3400 to 10000 Da.

[0014] More preferably, the molecular weight of PEG is 3400 Da.

[0015] Preferably, the concentration of HS-PEG-COOH in step (1) is 1 μM to 10 mM.

[0016] More preferably, the concentration of the HS-PEG-COOH is 5 to 50 μM.

[0017] More preferably, the concentration of the HS-PEG-COOH is 10 μM.

[0018] Specifically, in step (1), HS-PEG-COOH is added to the cleaned Au substrate electrode and reacted at 4° C. to prepare a carboxylated electrode surface, and then the electrode is washed to remove excess HS-PEG-COOH.

[0019] In step (2), a mixture of EDC / NHS (1:1) was added to the Au electrode obtained in step (1), activated at 4°C, and the electrode was rinsed. Then, GAD65 antigen (Ag) was added to the Au electrode and incubated at 4°C. In order to reduce nonspecific adsorption, the Au / HS-PEG-COOH / Ag bioconjugate was incubated in a BSA solution at 37°C. Finally, the modified electrode was washed to remove excess BSA and stored in a humid environment at 4°C for future use.

[0020] In step (3), the GADA solution is added to the Au electrode and incubated at 37° C. to obtain Au / HS-PEG-COOH / Ag / GADA.

[0021] In step (4), Ru(bpy)3 2+ Stir with streptavidin at 4°C, and purify the mixed solution by ultrafiltration using an ultrafiltration membrane. Then concentrate the streptavidin-modified Ru(bpy)3 2+ The complex was diluted with PBS (pH 7.4) and incubated with biotinylated GAD65 antigen at 37°C to obtain Ru(bpy)3 2+ The product of labeled GAD65 antigen (Ru-Ag). Finally, the diluted Ru-Ag solution was added to the electrode obtained in step (3) under dark conditions at 37°C, and the electrode was washed to remove excess Ru-Ag to obtain the sandwich immunosensor Au / HS-PEG(n)-COOH / Ag / GADA / Ru-Ag, which was stored in a humid environment at 4°C for further ECL detection.

[0022] Preferably, when the concentration of the GADA to be detected is 10 pg / mL to 10 ng / mL, the ECL intensity of the biosensor is linearly correlated with the concentration of the GADA to be detected.

[0023] More preferably, the linear correlation equation between the ECL intensity of the biosensor and the GADA concentration to be measured is y=488.34x+281.51.

[0024] Preferably, the detection limit of the biosensor for the GADA to be detected is 1 pg / mL.

[0025] Preferably, the biosensor has a peak current increase value of 1V or more.

[0026] Further preferably, the biosensor has a peak current increase value of at least 1.2V.

[0027] Application of the biosensor of the present invention in preparing a diagnostic reagent for type 1 diabetes.

[0028] Preferably, the biosensor is used in the preparation of a diagnostic reagent for early stage type 1 diabetes.

[0029] Preferably, when the biosensor is detecting, tripropylamine is added to react with the biosensor.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0031] The present invention uses a flexible polyethylene glycol (PEG) chain to fix the ruthenium-labeled immune complex on the substrate electrode, which can induce more ruthenium complexes to collide freely, thereby enhancing the ECL detection signal. The designed biosensor has strong detection specificity, high sensitivity, good accuracy, and low sample consumption. The construction method is flexible, and the flexible PEG chain of appropriate length and concentration can be selected according to the detection needs, providing an effective strategy for the diagnosis of early type 1 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the construction process of the immunosensor;

[0033] Figure 2 The electrochemical characterization and ECL performance evaluation results of the immunosensor prepared in Example 1;

[0034] Figure 3 The electrochemical characterization and ECL performance evaluation results of immunosensors with different PEG chain lengths are shown;

[0035] Figure 4 The immunosensor prepared in Example 1 was used to detect serum GADA. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with embodiments.

[0037] Example 1: Preparation of immunosensor

[0038] The Au electrode was cleaned three times in ethanol and aqueous solution using ultrasound. A volume of 10 μL of 10 μM HS-PEG (MW 3400)-COOH (10 μL, diluted with ultrapure water) was dropped on the Au electrode and reacted overnight at 4°C to prepare a carboxylated electrode surface. Then, the electrode was washed with deionized water to remove excess HS-PEG (MW 3400)-COOH. EDC (N-ethyl-N'-(3-(dimethylamino)propyl) carbodiimide) and NHS (N-hydroxysuccinimide) were dissolved in MES (2-(N-morpholino)ethanesulfonic acid) buffer to obtain a 0.1 M solution.

[0039] Then, a 10 μL volume of EDC / NHS (1:1) mixture was added to the Au electrode and activated at 4°C for 30 min, and then the electrode was rinsed with 0.1 M phosphate buffered saline (PBS) containing 0.05% Tween 20. Finally, 5 μL of 10 μg / mL GAD65 antigen (Ag) was added to the Au electrode and incubated at 4°C for 2 h. In order to reduce nonspecific adsorption, the Au / HS-PEG (MW 3400)-COOH / Ag bioconjugate was incubated in 5 μL of 3% BSA solution at 37°C for 30 min. Finally, the modified electrode was washed with 0.1 M PBS (pH 7.4) to remove excess BSA and stored in a humid environment at 4°C for future use.

[0040] Ru(bpy)3 2+ The preparation method of labeled antigen is as follows: 1mg / mL Ru(bpy)3 2+ The mixture was stirred with 0.1 mg / mL streptavidin at 4 °C for 2 h, and the mixed solution was purified by ultrafiltration using an ultrafiltration membrane with a molecular weight of 10K. 2+ The complex was diluted to 20 μg / mL with PBS (pH 7.4) and incubated with biotinylated GAD65 antigen at 37°C for 30 min to obtain Ru(bpy)3 2+ The product of labeled GAD65 antigen (Ru-Ag). 5 μL of sample solution with different concentrations of GADA was dropped on the electrode and incubated at 37°C for 1 h to obtain Au / HS-PEG (MW 3400)COOH / Ag / GADA. Then, 5 μL of the diluted Ru-Ag solution was dropped on the electrode surface at 37°C in the dark for 1 h, and the excess Ru-Ag was removed with a washing solution.

[0041] Finally, the sandwich immunosensor (Au / HS-PEG (MW 3400)-COOH / Ag / GADA / Ru-Ag) was stored in a humid condition at 4 °С for further ECL detection.

[0042] Other immunosensors (Au / HS-PEG(n)-COOH / Ag / GADA / Ru-Ag) constructed using HS-PEG(MW 2000)-COOH, HS-PEG(MW 5000)-COOH, and HS-PEG(MW 10000)-COOH were prepared according to the above method.

[0043] Other immunosensors (Au / HS-PEG (MW 3400)-COOH / Ag / GADA / Ru-Ag) constructed with other concentrations of HS-PEG (MW 3400)-COOH (1 μM, 5 μM, 50 μM, 100 μM, 1 mM, 10 mM) were prepared according to the above method.

[0044] Example 2: Electrochemical Characterization of Immunosensor

[0045] 1. Characterization methods

[0046] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements of the immunosensor were performed using an electrochemical workstation. Au electrode, Ag / AgCl wire and Pt wire were used as working electrode, reference electrode and counter electrode, respectively. CV measurements were performed between -0.2 and 0.8 V with a scan rate of 50 mV / s. EIS measurements were performed in the frequency range of 100 KHz to 0.01 Hz. The electrolyte solution used for EIS measurements was 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] (1:1), PBS (pH 7.4). To ensure operation in the linear current-voltage range, the AC potential amplitude was adjusted to ±5 mV relative to the open circuit potential. In all measurements, the initial open circuit potential remained stable at 399 ± 1 mV, and the minimum fluctuation observed in the long-term Faraday EIS measurement was less than 5 mV. EIS measurements were performed at room temperature.

[0047] 2. Characterization results

[0048] The immunosensor was prepared using a self-assembly strategy. The thiol groups of HS-PEG with different lengths were reacted with the Au surface for initial assembly. Then, the electrode surface was exposed to EDC / NHS to activate the carboxylic acid groups. The amine reaction intermediates formed, such as succinyl ester groups, can further react with the amino groups on the GAD65 antigen, which bind to the corresponding GADA and Ru-Ag to form an immune complex. The entire assembly process is as follows: Figure 1 To characterize the assembly process, an electrochemical probe [Fe(CN)6] 3- / 4-The electrochemical behavior of Au electrodes with different numbers of layers is shown in Figure 2. The continuous addition of PEG, GAD65 antigen, GADA, and Ru-Ag will continuously introduce an insulating layer on the Au electrode, thereby inhibiting the electrochemical behavior of [Fe(CN)6] 3- / 4- Penetration into the electrode surface creates a greater barrier. When the antigen and antibody are fixed on the Au electrode in sequence, Figure 2 The redox peak currents in (A) decrease sequentially because the electron transfer rate is hindered by the poorly conductive protein (CV of the immunosensor prepared in 0.1 M PBS (pH = 7.4) over the potential range of -0.2 to 0.8 V). Figure 2 The EIS characterization in (B) further shows the gradual increase of the semicircle in the spectrum, more insulating layers at the supporting electrodes and the resulting impedance increase (EIS characterization of the prepared immunosensor in 5.0 mM K3Fe(CN)6 / K4Fe(CN)6, frequency range 0.01 Hz to 10 KHz, black line: Au / HS-PEG(MW 3400)-COOH; red line: Au / HS-PEG(MW 3400)-COOH / GAD65 antigen; green line: Au / HS-PEG(MW 3400)-COOH / GAD65 antigen / BSA; blue line: Au / HS-PEG(MW 3400)-COOH / GAD65 antigen / BSA / GADA; purple line: Au / HS-PEG(MW 3400)-COOH / GAD65 antigen / BSA / GADA / Ru-GAD65).

[0049] Example 3: ECL performance evaluation

[0050] 1. Evaluation method

[0051] The prepared electrode was placed in phosphate buffer (PB, pH 7.4) containing 0.1 M tripropylamine (TrPA) as a co-reactant. The ECL response was measured with a photomultiplier tube (PMT). The voltage of the PMT was 700 V. The scanning potential was 0-1.5 V, and the scanning rate was 500 mV / s. The detection of GADA in serum, the source of samples and ethics were provided by the Run Run Shaw Hospital of Nanjing Medical University (2022-SRS038), and the experimental process was in full compliance with ethical approval. The serum samples were diluted 100 times with phosphate buffer (pH 7.4), and the concentration of GADA in serum was determined by the ECL standard addition method.

[0052] 2. Evaluation results

[0053] After forming the immunosensor on the Au electrode, TPrA was added to the solution as a co-reactant to react with the ruthenium complex to produce an ECL signal. In order to clearly demonstrate the role of the PEG chain in the ECL process, TGA of a short-chain rigid structure was selected to make another immunosensor. Compared with the CV trajectory of the TGA immunosensor, the PEG immunosensor Figure 2 The peak current increase at 1.2 V is given in (C) (CV intensity of the immunosensor with 100 mM TPrA in 0.1 M PBS with TGA (black line) and HS-PEG (MW 3400)-COOH (red line) as linker). It is worth noting that the long chain PEG increases the diffusion distance of TPrA radicals from the electrode surface to the Ru complex, so if there is only an “access pathway”, the observed current is smaller. Therefore, Ru(bpy) 2+ / 3+ Direct electron transfer must occur between TPrA and Ru(bpy), which is attributed to the flexible PEG chain. In the presence of such long and flexible PEG chains, the immune complex cannot be positioned vertically on the electrode surface. Some collapse of the immune layer occurs, resulting in direct contact of the ruthenium complex on the electrode. Therefore, TPrA and Ru(bpy) 2+ Electron losses at this point begin to contribute more current.

[0054] At the same time, the ECL signals of PEG and TGA immunosensors were recorded, as Figure 2 (D) shows the ECL intensity of the immunosensor with TGA (black line) and HS-PEG (MW 3400)-COOH (red line) as linker and 100 mM TPrA in 0.1 M PBS. A 34-fold increase in the ECL intensity was observed, confirming the acceleration of the ECL process. More importantly, the ECL enhancement observed on the PEG immunosensor facilitates the measurement and may improve the detection sensitivity.

[0055] Example 4: Optimization of electrodes

[0056] 1. Preparation of immunosensor

[0057] Different immunosensors were prepared according to the method of Example 1.

[0058] 2. Optimize results

[0059] The length of PEG affects the collision probability of the ruthenium complex at the electrode and needs to be optimized to obtain the best conditions for the ECL reaction. Therefore, polyethylene glycol with different molecular weights (molecular weight of 2000, 3400, 5000 and 10000Da) was used to make immunosensors. In principle, short PEG chains tend to form a loose stacking layer on the electrode surface, which limits the transfer of electrons and is not conducive to the ECL process. When the chain length increases, the polyethylene glycol chain tends to form a mushroom configuration on the electrode surface, which is beneficial to increase the electron transfer rate. Figure 3 (A) and Figure 3 As shown in (B), when the molecular weight increases from 2000 to 3400Da, the current increases and the impedance decreases. Longer PEG chains lead to the formation of a thicker layer on the electrode surface, which increases the tunneling distance of electron transfer and reduces the electron transfer rate (black line: 2000Da; red line: 3400Da, green line: 5000Da, blue line: 10000Da).

[0060] Therefore, when the molecular weight of PEG increases from 3400 to 5000 and 10000Da, the current decreases as the impedance increases. Consistent with the changes in current and impedance at the electrode, the ECL intensity of the PEG-modified electrode with a molecular weight of 3400Da is the strongest, as shown in Figure 2. Figure 3 (C) This result confirms that the thickness of the PEG layer plays an important role in regulating the emission of ECL.

[0061] At the same time, the density of PEG chains at the electrode affects the collapse of the chains and the subsequent Ru(bpy) 2+ In particular, a low density of PEG chains introduced fewer immune complexes onto the electrode, whereas a high density of PEG chains induced a crowded environment that did not allow Ru(bpy) 2+ Complete collision. To evaluate this effect, different concentrations of PEG (MW 3400Da) were modified on the electrode. ECL measurements showed that the emission was strongest at a PEG concentration of 10 μM, as shown in Figure 3 (D) Under these conditions, it is expected that Ru(bpy) can be achieved by optimizing the distance between each PEG chain. 2+ Highest collision efficiency at the electrodes.

[0062] Example 5: Detection of serum GADA

[0063] 1. Detection method

[0064] Under the optimal preparation conditions, the Au electrode was modified with HS-PEG (MW3400)-COOH at an optimal concentration of 10uM and a molecular weight of 3400Da; the carboxyl group was activated by EDC / NHS, and then the electrode surface was modified with GAD65 antigen; the target antibody and Ru-labeled antigen were modified to the electrode surface using the antigen-antibody binding principle to prepare the immunosensor. The immunosensor was exposed to a 100mM co-reactant TrPA (pH 7.4, PBS) solution, and the ECL signal was detected by an electrochemiluminescence analyzer, and the ECL signal intensity of the formed immune complex was recorded.

[0065] 2. Test results

[0066] Figure 4 (A) shows that the ECL intensity gradually increases with the increase of GADA, indicating that the GADA detection sensitivity is high. In order to investigate the specificity of the sensor, interfering antibodies, including protein tyrosine phosphatase antibody (IA-2A), insulin antibody (IAA) or transporter 8 antibody (ZnT8A), were added to the solution for detection. The ECL intensity of GADA is significantly higher than that of other antibodies, such as Figure 4 As shown in (B), the ECL immunosensor has good selectivity for GADA. This good property should also be attributed to the presence of PEG chains on the electrode, which are reported to minimize the adsorption of nonspecific proteins. The PEG layer is a non-toxic polymer that has the ability to increase the affinity of the material with water and can produce a microenvironment that is conducive to protein stabilization and improved biomolecular interactions. By properly adjusting the chain length, it can facilitate the interaction between proteins and substrates and prevent the diffusion of nonspecific proteins. Therefore, the immunosensor prepared using polyethylene glycol chains can improve the selectivity of immunoassays, which is crucial for subsequent serum testing.

[0067] Figure 4(C) shows the relationship between ECL intensity and GADA concentration from 10 pg / mL to 10 ng / mL. The near-linear correlation confirms the good response of the sensor to GADA. The calculated detection limit was 1 pg / mL, which is 100 times lower than the result of the classical enzyme-linked immunosorbent assay (ELISA). ELISA is a commonly used GADA detection method in hospitals, and its lowest detection limit is still about 5 IU / mL (equivalent to 1 ng / mL). However, this amount is significantly lower in the early stages of autoimmune diabetes. Compared with the ELISA method, the advantages of immunosensors include smaller sample volume, speed, equivalent or better sensitivity, larger dynamic range, and multiplexing capabilities. In addition, ECL immunoassay (5 μL) allows the serum volume required for reference standards and test samples to be reduced by ≥20 times compared with ELISA (100 μL). In contrast, TGA as a rigid chain has also been used to make immunosensors for the detection of GADA, with the lowest concentration measured being 100 pg / mL ( Figure 4 (D)), which is less sensitive than the sensor using flexible PEG chains. Although ECL detection using either modification layer can sensitively detect GADA, the ECL detection using flexible PEG chains showed a higher signal, lower detection limit, and higher sensitivity.

[0068] Next, the established ECL immunosensor was used to perform the test on clinical serum samples.

[0069] The results showed that the concentration of GAD65 Ab in serum was 11.99 ng / mL, which was consistent with the results of the Meso Scale Discovery (MSD) ECL method. The RSD of the ECL intensity measured 4 times was 8.69%. The recovery rate of GADA was 92%. Based on these evaluations, the developed ECL immunosensor is capable of detecting GADA in human serum samples.

[0070] In summary, the present invention constructs a new ECL immunosensor by introducing a flexible PEG chain on the electrode to fix the immune complex, and the sensor has a high detection sensitivity for GADA. 2+ The movement on the complex provides greater freedom, making it easier to enter the electrode for initial ECL reaction. Compared with the rigid chain, the flexible chain supports the successfully designed sensor with higher ECL intensity and detection sensitivity. The detection limit of this method is nearly 2 orders of magnitude lower than that of the classic ELISA kit, providing a new strategy for the early diagnosis of T1DM based on sensitive detection of GADA.

Claims

1. A biosensor for detecting GADA, characterized in that: Prepared by the following method: (1) Carboxylation of the gold substrate electrode with HS-PEG-COOH to obtain Au / HS-PEG-COOH; (2) coupling the GAD65 antigen (Ag) to the carboxylated electrode obtained in step (1) to obtain Au / HS-PEG-COOH / Ag bioconjugate; (3) coupling the GADA antibody to the electrode obtained in step (2) to obtain Au / HS-PEG-COOH / Ag / GADA; (4) coupling the ruthenium-labeled GAD65 antigen (Ru-Ag) to the electrode obtained in step (3) to obtain the biosensor Au / HS-PEG-COOH / Ag / GADA / Ru-Ag; Among them, the molecular weight of PEG is 2000~10000Da.

2. The biosensor according to claim 1, characterized in that The molecular weight of PEG is 3400~10000Da.

3. The biosensor according to claim 1, characterized in that The concentration of HS-PEG-COOH in step (1) is 1 μM to 10 mM.

4. The biosensor according to claim 3, characterized in that The concentration of the HS-PEG-COOH is 5 to 50 μM.

5. The biosensor according to claim 1, characterized in that When the concentration of the GADA to be detected is 10 pg / mL to 10 ng / mL, the ECL intensity of the biosensor is linearly correlated with the concentration of the GADA to be detected.

6. The biosensor according to claim 5, characterized in that The linear correlation equation between the ECL intensity of the biosensor and the GADA concentration to be measured is y=488.34x+281.

51.

7. The biosensor according to claim 1, characterized in that The detection limit of GADA was 1pg / mL.

8. The biosensor according to claim 1, characterized in that It has a peak current increase value of more than 1V.

9. Use of the biosensor according to claim 1 in preparing a diagnostic reagent for type 1 diabetes.

10. The use according to claim 9, characterized in that: When performing detection, tripropylamine is added to co-react with the biosensor.

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