Antibody detection method based on organic electrochemical transistor

Abstract

The application discloses an antibody detection method based on an organic electrochemical transistor and belongs to the technical field of biosensing and antibody detection. The application prepares a novel organic electrochemical transistor, conducts antibody detection research, and the main content and conclusion are as follows: the preparation of the organic electrochemical transistor is completed through steps such as spin coating, annealing and evaporation, and then the schistosome antigen is fixed on the surface of the sensor. Functionalization characterization is conducted through a semiconductor parameter analyzer, a scanning electron microscope and an atomic force microscope. In PBS and serum, as the antibody is continuously combined with the modified antigen, the current change amount in the organic electrochemical transistor gradually increases, and thus a standard curve can be drawn to quantitatively detect the schistosome infection. The experimental results show that the device can detect schistosome antibodies as low as 0.001 mu g / mL, and the sensitivity is high. Compared with the commercially available test strip, the sensor has great improvement in sensitivity and specificity.

Description

TECHNICAL FIELD

[0001] The application relates to an antibody detection method based on an organic electrochemical transistor, and belongs to the technical field of biosensing and antibody detection. BACKGROUND

[0002] The detection method of schistosomiasis is a complex and delicate process, involving multiple aspects to ensure the accuracy and reliability of the diagnosis. In the etiological diagnosis, the direct smear method is suitable for areas with severe infection, and the eggs of the patient's stool or mucous blood of the acute schistosomiasis patient are observed under a microscope, but this method is limited by the quality of the sample and the uniformity of the egg distribution, so the detection rate of the eggs is relatively low. In order to improve the positive detection rate, the miracidium hatching method is widely used, which hatches the eggs under suitable conditions to make the miracidia hatch out, so that they are easier to be detected. However, this method requires a certain time, usually several hours to several days, so it is not suitable for urgent diagnosis. The quantitative transparency method provides a more accurate egg counting method, which can quantitatively evaluate the degree of infection by using a microscope to observe and count. For chronic and advanced schistosomiasis patients, due to the thickening of the intestinal wall tissue, the egg discharge is blocked, so rectal mucosa biopsy is very important, which can more accurately judge the disease condition by observing the intestinal mucosa lesions through a rectoscope.

[0003] In the immunological diagnosis, intradermal test is a commonly used screening method, which observes the skin reaction by injecting antigen, and has a high coincidence rate with the positive rate of fecal egg examination, but false positive or false negative reactions may occur, so it needs to be combined with other examination results for comprehensive judgment.

[0004] Due to high sensitivity and specificity, purified antigen and recombinant antigen can be used for the diagnosis of schistosomiasis. The detection of antibodies is a more sensitive and specific method, which can indirectly infer the infection by detecting specific antibodies such as IgM, IgG, IgE, etc. in the serum. If the specific antibodies of the subject are positive without pathogen treatment, it is more meaningful for the diagnosis. Circulating antigen enzyme immunoassay is another important immunological examination method, which provides a strong basis for the diagnosis of schistosomiasis by detecting circulating antigens. Studies by Wang Bolan et al. show that the detection rates of TRFIA and DDIA are higher than ELISA in detecting advanced, acute and chronic schistosomiasis. Studies by Yang Hui et al. show that the sensitivity can be increased to 99.3% by detecting ELISA together with DDIA and DIGFA. In addition to etiological and immunological diagnosis, other examination methods also play an auxiliary role in the diagnosis of schistosomiasis. The use of probes to detect schistosome antibodies provides another angle for the detection of schistosomes. Hemogram examination can reflect the characteristics of the changes of peripheral blood in acute schistosomiasis patients, such as significant increase of eosinophils, etc.

[0005] Liver function tests can assess liver damage, acute patients with increased serum globulin, serum ALT, AST slightly increased, and late patients with decreased albumin and increased globulin. Imaging examinations such as B-mode ultrasound and CT scans can directly show the lesion site, range and degree, providing important reference information for doctors.

[0006] In summary, there are various detection methods for schistosomiasis, and doctors will choose appropriate examination methods for diagnosis according to the specific situation and clinical manifestations of patients. At the same time, with the continuous progress of medical technology, new detection methods are also emerging, providing a broader space for the diagnosis and treatment of schistosomiasis.

[0007] Organic electrochemical transistors have made great progress in antigen-antibody research. Studies have shown that using pH-sensitive organic electrochemical transistors, it is possible to achieve in-situ application of wearable electronic devices in physiological monitoring, showing broad application prospects. Liu Jian's flexible organic electrochemical transistor improves the detection capability of H2O2, and Chen Lizhen et al. use organic electrochemical transistors to realize qualitative measurement of cell surface glycan. Feng Xiaojian and his team of organic electrochemical transistor biosensors have made a detailed exposition in the field of biological detection of glucose, DNA, antibody antigen, cells, dopamine, etc., focusing on their sensing mechanism and detection performance. OECT has shown broad application prospects in the fields of biosensing, environmental monitoring and medical diagnosis, and is expected to play an important role in emerging fields such as flexible electronic skin and smart wearable devices.

[0008] In summary, the research on OECT is continuously making new progress, and its application prospects in the field of sensing are broad. SUMMARY

[0009] (1) Based on the study of organic electrochemical transistors at home and abroad, find the appropriate method to modify the organic electrochemical transistor, and then prepare for the subsequent experiment.

[0010] (2) Probe modification, antibodies can bind to antigen epitopes through non-covalent interaction. Adopting the strategy of surface grafting, schistosome egg antigens are combined to the surface of the detection area, and the modification effect is evaluated by current change, Raman, scanning electron microscopy, etc.

[0011] (3) The data obtained are used to draw standard curves, and the relationship between current response and concentration of the measured substance is studied by detecting different concentrations of schistosome antibodies in phosphate buffer. According to the results, the conditions such as binding temperature and incubation time are optimized to explore the detection performance of the sensor. According to the results of multiple experiments, a standard curve is established, and key indicators such as detection limit and sensitivity are calculated.

[0012] (4) Serum testing was performed by preparing serum samples containing different concentrations of antibodies and detecting them using the antigen-modified sensor. The actual performance of the sensor was analyzed and compared with the standard method.

[0013] (5) Specificity analysis was performed, and control experiments were conducted using liver fluke and lung fluke antibodies as interferences to verify the specificity of the sensor and compare it with the standard method.

[0014] This research uses Schistosoma japonicum antibodies as a model, modifying the surface of a semiconductor sensor with worm egg antigens. This method enables quantitative detection of antibodies by measuring changes in current. This method requires no labeling and offers a direct detection mode. Other biomolecules can be detected by replacing the probe, thus offering potential applications and potentially providing new insights into food contaminant detection.

[0015] The present invention provides an antibody sensor based on an organic electrochemical transistor. The device comprises, from bottom to top, a substrate layer, an organic semiconductor layer, a solid ion gel electrolyte layer, an organic semiconductor doping layer, and a gate. The organic semiconductor layer and the organic semiconductor doping layer are both P-type organic semiconductors, and the organic semiconductor doping layer is a P-type organic semiconductor doped with a P-type dopant.

[0016] After the gate surface is cleaned with ethanol and water, cysteamine and soluble insect egg antigen SEA are used as modifiers in sequence to modify the gate surface of the device to form a self-assembled molecular film.

[0017] In one embodiment of the present invention, the substrate layer is: a glass sheet or a polyimide substrate of the source and drain electrodes;

[0018] In one embodiment of the present invention, the solid ion gel electrolyte layer is formed by spin coating PVDF-HFP solution doped with ionic liquid;

[0019] In one embodiment of the present invention, the ionic liquid is EMIM TFSI, EMIM BF4 or a mixture thereof.

[0020] In one embodiment of the present invention, the method for modifying the gate self-assembled molecular film includes:

[0021] (1) Wash with water and ethanol respectively and then blow dry;

[0022] (2) After drying, add cysteamine to the gate and react in the dark for 6 to 8 hours. After washing with water and drying, add glutaraldehyde and react for 1 to 2 hours.

[0023] (3) After the reaction in step (2), the grid is rinsed with water and blown dry, and soluble egg antigen SEA is added and reacted again for 1 to 2 hours;

[0024] (4) After the reaction in step (3) is completed, rinse and blow dry, add bovine serum albumin and react for 1 to 2 hours to passivate the aldehyde groups that have not reacted with SEA, and then wash and dry.

[0025] In one embodiment of the present invention, in step (2), the concentration of cysteamine is 0.4-0.5 mM.

[0026] In one embodiment of the present invention, the volume fraction of glutaraldehyde is: 2 to 2.5%;

[0027] In one embodiment of the present invention, the concentration of the soluble egg antigen SEA in step (3) is: 45-50 μg / mL;

[0028] In one embodiment of the present invention, the mass fraction of bovine serum albumin in step (4) is 1 to 1.5%.

[0029] The present invention also provides a method for preparing an antibody sensor based on an organic electrochemical transistor, the method comprising:

[0030] Step 1): spin-coating a layer of organic semiconductor solution on the substrate with the active and drain electrodes formed thereon, and annealing to form an organic semiconductor layer;

[0031] Step 2): spin-coating an ionic gel electrolyte solution on the organic semiconductor layer, and heating and curing the solution to form a solid ionic gel electrolyte layer;

[0032] Step 3): spin-coating a mixed solution of an organic semiconductor and a dopant on the solid ion gel electrolyte layer, and annealing to form an organic semiconductor doping layer;

[0033] Step 4): evaporating a gate electrode on the organic semiconductor doping layer;

[0034] Step 5): After the gate surface is cleaned with ethanol and water, cysteamine and soluble insect egg antigen SEA are used as modifiers in sequence to modify the gate surface of the device to form a self-assembled molecular film.

[0035] In one embodiment of the present invention, the organic semiconductor in step 1) and the organic semiconductor in step 3) are P-type organic semiconductors.

[0036] In one embodiment of the present invention, the ionic gel electrolyte solution is a PVDF-HFP solution doped with an ionic liquid.

[0037] In one embodiment of the present invention, the ionic liquid is EMIM TFSI, EMIM BF4 or a mixture thereof.

[0038] In one embodiment of the present invention, the dopant in step 3) is a P-type dopant;

[0039] In one embodiment of the present invention, the method for modifying the gate self-assembled molecular film includes:

[0040] (1) Wash with water and ethanol respectively and then blow dry;

[0041] (2) After drying, add cysteamine to the gate and react in the dark for 6 to 8 hours. After washing with water and drying, add glutaraldehyde and react for 1 to 2 hours.

[0042] (3) After the reaction in step (2), the grid is rinsed with water and blown dry, and soluble egg antigen SEA is added and reacted again for 1 to 2 hours;

[0043] (4) After the reaction in step (3) is completed, rinse and blow dry, add bovine serum albumin and react for 1 to 2 hours to passivate the aldehyde groups that have not reacted with SEA, and then wash and dry.

[0044] The present invention also provides a method for detecting SEA antibodies in a sample. The method is not for the purpose of disease diagnosis and treatment. The method is to add the sample to the gate of the above-mentioned organic electrochemical transistor-based antibody sensor for reaction, and then detect whether the sample contains SEA antibodies through the current-voltage curve, and at the same time detect the content of SEA antibodies in the sample.

[0045] In one embodiment of the present invention, the sample is a body fluid sample.

[0046] In one embodiment of the present invention, the body fluid is selected from any one of saliva, tissue fluid, blood, PBS buffer, and sweat.

[0047] In one embodiment of the present invention, after the sample is added to the gate, the voltage between the source and the drain is adjusted to 1V, and the gate voltage is adjusted to -3V to +3V.

[0048] In one embodiment of the present invention, when the gate voltage is fixed at -3 V and the sample is a buffer solution, the content of SEA antibodies in the sample is detected by the following formula: y = -0.0085x4 + 0.0978x3 - 0.3255x2 + 0.4524x - 0.0417;

[0049] In one embodiment of the present invention, the gate voltage is fixed at -3 V, and when the sample is serum, the content of SEA antibodies in the sample is detected by the following formula: y = -0.0016x4 + 0.0205x3 - 0.0918x2 + 0.2061x - 0.0592.

[0050] The application provides a product containing the above-mentioned antibody sensor based on an organic electrochemical transistor; the product is a biochip for detecting SEA antibodies in a sample, or the product is a device for detecting SEA antibodies in a sample, or the product is a kit for detecting SEA antibodies in a sample.

[0051] In an embodiment of the application, the product is used for detecting SEA antibodies in a sample; the detection method is as above.

[0052] The application also provides use of the above-mentioned antibody sensor in preparation of a product for detecting SEA antibodies in a sample, the product being a biochip for detecting SEA antibodies in a sample, or the product being a device for detecting SEA antibodies in a sample, or the product being a kit for detecting SEA antibodies in a sample.

[0053] Beneficial effects

[0054] 1. A surface-functionalized organic electrochemical transistor (OECT) device was prepared by design. By adjusting the voltages of the source, drain, and gate electrodes, the current-voltage curve was measured, which proved the reliability and stability of the device.

[0055] 2. Using phosphate buffered saline (PBS) and rabbit serum as antibody solvents, a standard detection curve was drawn to achieve quantitative detection of SEA antibodies. This result shows that the OECT device is not only suitable for antibody detection under different solvent conditions, but also has high accuracy and repeatability.

[0056] 3. Compared with commercially available schistosome infection detection test strips, the OECT device designed in this experiment showed extremely high sensitivity and specificity, and it could detect as low as 0.001 μg / mL of schistosome antibodies, and the current change was significantly higher than the antibodies (liver fluke and lung fluke antibodies) that the other two test strips could not distinguish. This feature makes the OECT device have a significant advantage in the field of antibody detection, especially in the rapid detection scenarios that require to distinguish different types of antibodies or detect low concentration of antibodies.

[0057] 4. Based on the high sensitivity and specificity of the OECT device in antibody detection, we can foresee its broad application prospects in the fields of clinical diagnosis, biological research, food safety monitoring, etc. For example, in the medical field, the OECT device can be used to quickly and accurately detect various pathogen antibodies, providing strong support for early diagnosis and prevention and control of diseases; in the field of biological research, it can be used to study the interaction between biological molecules and the signal transduction mechanism; in the field of food safety monitoring, it can be used to detect harmful substances and pollutants in food. Although the current OECT device has shown good detection performance, we can still further improve its performance by optimizing the detection conditions, optimizing the device structure, improving the material selection, etc.

[0058] 5. The OECT device designed in this experiment showed extremely high sensitivity and specificity. It can detect schistosome antibodies as low as 0.001 μg / mL, which can achieve early detection of schistosome infection (can be diagnosed within 10 days). However, the specificity analysis of existing schistosome detection kits on the market shows that it cannot be detected below 2 mg / mL. As a result, patients often have to wait for about one month after infection for the number of antibodies in their bodies to reach a certain concentration before being diagnosed, delaying the optimal period for diagnosis and treatment. It can be seen that the detection method of the present invention shortens the patient's infection time. The current detectable infection time has been advanced from one month (test strip) to within 10 days.

[0059] Furthermore, the test strips currently used cannot distinguish between liver flukes and lung flukes, while the sensor of the present invention can achieve quantitative and qualitative distinction between liver flukes and lung flukes. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : Structural diagram and physical picture of organic electrochemical transistor.

[0061] Figure 2 : Stability detection of OECT devices; (a) is the unidirectional scanning IV curve of the OECT device, and (b) is the transfer characteristic curve.

[0062] Figure 3 : Diagram of the surface functionalization process of OECT devices.

[0063] Figure 4 : Electron microscopy observation of the modified area of ​​the organic electrochemical transistor before and after SEA modification; a) electron microscopy image before modification, (b) electron microscopy image after modification.

[0064] Figure 5 : Atomic force microscopy (AFM) observation results of the modified area of ​​the organic electrochemical transistor before and after SEA modification, (a) AFM image 3D characterization before modification and (b) 3D characterization after modification.

[0065] Figure 6 :(a) Bidirectional scanning IV curve before and after modification and (b) current change bar graph before and after modification.

[0066] Figure 7 : (a) Current-voltage curve and (b) current-time curve.

[0067] Figure 8 : Standard curve of current change and anti-SEA concentration in PBS system.

[0068] Figure 9 :Standard curve of current change and anti-SEA concentration in rabbit serum system

[0069] Figure 10 : Current changes of three antibodies at concentrations of (a) 0.1 μg / mL, (b) 1 μg / mL, and (c) 10 μg / mL, respectively.

[0070] Figure 11 :Test results of the kit: (a) Schistosoma japonicum antibody, (b) Liver fluke antibody, (c) Lung fluke antibody. DETAILED DESCRIPTION

[0071] In the present invention, the gate is attached Figure 1 The modifiers in the formula are different expressions of the same substance.

[0072] The ethanol involved in the following examples was purchased from Sinopharm Chemical Reagent Co., Ltd., phosphate buffered saline (PBS) was purchased from Aladdin Chemical Co., Ltd., Shanghai, China, cysteamine (Cys) was purchased from Aladdin Chemical Co., Ltd., Shanghai, China, glutaraldehyde was purchased from China Pharmaceutical Chemical Reagent Co., Ltd., soluble egg antigen SEA, schistosome antibody anti-(SEA), rabbit serum, liver fluke antibody, and lung fluke antibody were provided by the Jiangsu Schistosoma Control Institute, bovine serum albumin (BSA) was purchased from Aladdin Chemical Co., Ltd., Shanghai, China, and the BCA kit was purchased from Anhui White Shark Biotechnology Co., Ltd.

[0073] The detection methods involved in the following embodiments

[0074] Determination of antigen-antibody concentration

[0075] In this experiment, Schistosoma japonicum antigen-antibody, liver fluke antigen-antibody, and lung fluke antigen-antibody were provided by Jiangsu Schistosoma japonicum Control Institute. The antigen-antibody samples were used to prepare a standard curve using the White Shark BCA kit, and the concentration was measured by comparing with the standard curve. The samples were then diluted to the required concentration in preparation for subsequent experiments.

[0076] Measuring characterization of OECT devices

[0077] It is mainly carried out using electron microscope, atomic force microscope and kickstart2 semiconductor analyzer.

[0078] The device surface morphology was observed using a scanning electron microscope (SEM) to characterize the device's surface morphology and electrode structure. The accelerating voltage and probe current were adjusted to obtain high-resolution surface morphology images. Next, a transmission electron microscope was used to examine the device's internal structure, exploring the microstructure and interface characteristics of the organic semiconductor layer.

[0079] The OECT device is characterized using an atomic force microscope (AFM) to obtain the morphology of the device surface and nanoscale surface features. By adjusting the position and vibration frequency of the probe, high-resolution surface topography images and surface roughness information can be obtained.

[0080] The electrical performance of the OECT device is measured and analyzed using a kickstart2 semiconductor parameter analyzer.

[0081] The device is placed in a test fixture, the probe is placed on the specified source, gate, and drain electrodes, and connected to a computer for analysis. Then, by setting appropriate test parameters such as voltage range, scan rate, scan point number, and scan time, etc., the electrical performance test is carried out, including output characteristics, current-voltage curve, current-time curve, etc.

[0082] The application provides a low operating voltage electrochemical transistor, which is an electrochemical transistor with Cr / Au:5nm / 50nm electrodes as source and drain electrodes, P3HT as an organic semiconductor layer, PVDF-HFP mixed EMIM TFSI ionic gel as a solid ionic gel electrolyte layer, a TrTPFB and PBBT-2T mixed solution as an organic semiconductor doping layer, and gold as a gate.

[0083] The application provides an antibody sensor based on an organic electrochemical transistor, which comprises, from bottom to top, a substrate layer, a semiconductor layer, an ionic gel layer, a doping layer, and a gate; after the surface of the gate is cleaned with ethanol and water, cysteamine and soluble egg antigen SEA are used as modifiers to modify the surface of the gate of the device to form a self-assembled molecular film.

[0084] Example 1: Preparation of OECT device

[0085] (1) The glass substrate with Cr / Au (5nm / 50nm) as source and drain electrodes is ultrasonically cleaned with deionized water, acetone, and isopropanol for 2 minutes, and then dried with an argon gun after cleaning.

[0086] (2) The source and drain electrode glass substrate cleaned in step (1) is transferred into an ozone treatment device and treated for 15 minutes to change the wettability of the substrate surface, which is beneficial to subsequent film deposition;

[0087] (3) Preparation of semiconductor P3HT solution: In an argon-filled glove box, P3HT is dissolved in chlorobenzene at a concentration of 5g / L, heated at 50℃ for 1h, and filtered with a PTFE filter with a pore size of 0.45μm to remove excess impurities.

[0088] (4) Spin-coat the prepared semiconductor P3HT solution on the glass substrate treated in step (2). Set the spin-coating speed to 1500 rpm, the time to 30 s, and the annealing process to heat at 130° C. for 5 min to form an organic semiconductor layer.

[0089] (5) Preparation of ionic gel electrolyte layer solution: PVDF-HFP was dissolved in 2-butanone at a concentration of 100 g / L. Then, ionic liquid (EMIM TFSI) was added to the PVDF-HFP solution at a volume ratio of 1%. The solution was then heated on a hot plate at 60°C and stirred at 200 rpm overnight. A uniform ionic gel electrolyte solution was obtained after the solution was fully dissolved.

[0090] The prepared gel electrolyte layer solution is spin-coated on the organic semiconductor layer obtained in step (4). Before spin coating, it needs to be preheated at 60°C and stirred at 200 rpm for 10 minutes to prevent solute separation. The spin coating speed is 2000 rpm and the time is 30 seconds. After the spin coating is completed, the organic semiconductor layer is immediately transferred to a hot plate for heating at 90°C for 2 hours to form a dense and highly elastic solid ion gel layer.

[0091] (6) Preparing an organic semiconductor solution and a dopant solution: In an argon-filled glove box, PBBT-2T and TrTPFB were dissolved in chlorobenzene at a concentration of 5 g / l, respectively, and heated at 50°C for 1 h to dissolve. Excess impurities were removed using a PTFE filter with a pore size of 0.45 μm to prepare an organic semiconductor solution and a dopant solution, respectively.

[0092] Preparation of a mixed solution of organic semiconductor and dopant: The dopant solution and the organic semiconductor solution are mixed in a molar ratio of TrTPFB to PBBT-2T monomers of 6:10. The mixture is ready for use without heating or filtering.

[0093] The prepared mixed solution of organic semiconductor and dopant is spin-coated on the solid ion gel electrolyte layer at a rotation speed of 1500 rpm for 30 seconds. The annealing process is heating at 90° C. for 20 minutes to form an organic semiconductor doping layer.

[0094] (7) 50 nm Au was deposited on the organic semiconductor doping layer through a mask as a gate ( Figure 1 A low operating voltage electrochemical transistor device can be obtained.

[0095] Preferably, Figure 1 The right side of the picture is the actual picture.

[0096] The results show:

[0097] Figure 1The left side of the middle is the structure diagram of the OECT device. Through the structure diagram, the arrangement and connection mode between each layer inside the device can be seen. In particular, the distribution and configuration of the doped layer, ion glue and P3HT- and other materials are key factors affecting the performance of the OECT device. The doped layer is used to control the current flow, and the selection of its structure and material has an important influence on the conductive performance of the device. The ion glue acts as an adhesive to fix and connect each layer, and its quality and stability are directly related to the reliability and life of the device. The P3HT- and other organic materials may bring specific electrical or optical properties to the device.

[0098] Figure 1 The right side of the middle is the physical diagram of the OECT device. As can be observed from the diagram, the device has a clear source, drain and gate, which are the basic components of the OECT operation. The channel part between the source and the drain is the key area of the organic electrochemical transistor, and its form and structure have an important influence on the performance of the device. In addition, there are multiple metal contact points on the OECT device, which are used to connect with the external circuit to realize the input and output of current. The position and layout of these contact points are reasonable, which not only ensures the smooth transmission of current, but also avoids the possible short circuit or leakage problem.

[0099] Example 2: Characterization of OECT device

[0100] The electrical performance of the OECT device was measured and analyzed using the kickstart2 semiconductor parameter analyzer:

[0101] The device was placed in the test fixture, the probe was placed on the specified source, gate and drain electrodes, and connected to the computer for analysis. Then, by setting appropriate test parameters such as voltage range, scan rate, scan point number and scan time, etc., the electrical performance test was carried out, including output characteristics, current-voltage curve, current-time curve, etc.

[0102] In this example, the source voltage was set to 1V, the drain voltage was set to 1V, and the gate voltage was set to -3V, -2V, -1V, 0V, 1V, 2V or 3V. The current-voltage curve of the device was measured. The test data was processed and analyzed using origin, and the key device parameters of the device were obtained, such as the change in current before and after modification, etc.

[0103] The results show that:

[0104] (1) In order to prove the stability of the OECT device, we tested the I-V curve of the device when the voltage between the source and the drain electrodes was 1V and the gate voltage varied from -3V to +3V, and the result is shown in Figure 2 (a);

[0105] (2) Figure 2(b) in the figure is the transfer characteristic curve of the OECT device. For the prepared organic electrochemical transistor, under the condition of controlling the scanning voltage between the source and the drain to be 0~1V, by changing the gate voltage to -3V, -2V, -1V, 0V, 1V, 2V, 3V, the current change curve of the device over time was measured, and it was observed that as the gate voltage continued to increase, the current continued to decrease.

[0106] The phenomenon that the current of OECT decreases with increasing gate voltage may be due to the regulatory effect of gate voltage on channel conductivity and source-drain current, as well as the combined effect of factors such as interface effect and charge accumulation.

[0107] Example 3: OECT probe modification

[0108] The present invention performs probe modification on the prepared OECT, wherein: Figure 3 This is a schematic diagram of the modification process. Specifically, the cysteamine, with a thiol group (-SH) at one end, reacts with a hydroxyl group on the device surface under strong interaction, binding to the gate surface. The amino group (-NH2) at the other end of the cysteamine reacts with the aldehyde group (-CHO) of glutaraldehyde to form a Schiff base reaction, attaching the glutaraldehyde to the device via a carbon-nitrogen bond. Subsequently, the amino group of SEA undergoes the same reaction with the other end of glutaraldehyde, binding to it. Finally, the unbound aldehyde groups are passivated with BSA. The resulting modified OECT device is then used for assays with anti-SEA.

[0109] The specific steps are as follows:

[0110] (1) The OECT device prepared in Example 1 was taken out, washed with distilled water and anhydrous ethanol for 5-10 minutes respectively, and dried in a fume hood.

[0111] (2) After drying, add 20 μL of 0.5 mM cysteamine to the gate ( Figure 1 The modified grid was placed in a refrigerator in the dark for 6.5 hours. Unreacted cysteamine was then rinsed with ultrapure water and dried. 10 μL of 2.5% glutaraldehyde was added and allowed to react for 2 hours.

[0112] (3) After the reaction in step (2), rinse the gate with ultrapure water ( Figure 1 The modified grid was removed and blown dry, and 10 μL of 50 μg / mL soluble egg antigen SEA was added and reacted again for 2 h.

[0113] (4) After the reaction in step (3) is completed, rinse with PBS solution 2-3 times to prevent unreacted SEA from remaining. After drying the electrode, add 10 μL of 1% (v / v) bovine serum albumin (BSA) to passivate the aldehyde groups that did not react with SEA. After 1 hour, rinse with ultrapure water and dry the electrode. Then, seal it with a vacuum sealer and store it in a -4°C refrigerator.

[0114] Example 4: OECT probe characterization

[0115] After device modification, SEA antigen was bound to the surface of the OECT device, which was again characterized and observed using electron microscopy, atomic force microscopy, and kickstart2 semiconductor analyzer.

[0116] The device surface morphology was observed using scanning electron microscopy (SEM), with particular attention paid to the morphology and distribution of the antigen-binding region. Parameters were adjusted to obtain high-resolution surface images, and the differences before and after modification were compared. Atomic force microscope (AFM) was used to observe the device surface topology and the nanoscale features of the antigen-binding region. The electrical performance of the modified OECT device was further analyzed using a Kickstart2 semiconductor parameter analyzer. Electrical performance testing was performed by connecting the device to a computer and setting appropriate test parameters, such as voltage range and scan rate.

[0117] The results show:

[0118] (1) Electron microscopy observation of the modified area of ​​the organic electrochemical transistor before and after SEA antigen modification revealed obvious contrast results, such as Figure 4 As shown. Before modification, the surface of OECT showed a typical gold nanoparticle morphology, ( Figure 4 (a)); After SEA antigen modification, the electron micrograph shows that the modified area has undergone significant changes. The modified layer successfully adheres to the OECT surface, forming a uniform and dense covering layer ( Figure 4 (b)) shows that its morphological features have changed significantly compared to those before modification, proving that SEA modification is successful.

[0119] (2) Atomic force microscopy (AFM) observation results of the modified area of ​​the organic electrochemical transistor before and after SEA antigen modification are as follows Figure 5 As shown. Before modification, the surface atomic arrangement of OECT showed relatively flat and orderly characteristics ( Figure 5 (a)), Ra = 33.8 nm, Rq = 28.3 nm; after SEA antigen modification of OECT, the surface showed obvious morphological changes ( Figure 5In (b), the atomic arrangement becomes more compact, and parameters such as surface roughness and surface roughness increase to Ra = 109 nm and Rq = 86.3 nm, which directly reflects the successful coverage and improvement of the SEA antigen modification layer on the OECT surface. After modification, obvious protrusions caused by SEA antigens appear on the surface.

[0120] (3) In order to prove the stability of the OECT device, we tested the IV curves of the device before and after modification when the voltage between the source and drain electrodes was 1V and the gate voltage varied from -3V to +3V; that is, the modified OECT probe prepared in Example 2 was placed in the test fixture, and the probe was placed on the designated source, gate, and drain electrodes respectively, and the source voltage was set to 1V, the drain voltage was set to 1V, and the gate voltage was set to -3V, 2V, -1V, 0V, 1V, 2V, or 3V;

[0121] The results are as follows Figure 6 ,in Figure 6 (a) is the IV line of a single experiment. Figure 6 (b) is the average result of multiple experiments. It can be seen from the figure that the current of the modified OECT device is smaller than that before modification.

[0122] Example 5: Detection of SEA Antibody Concentration in Buffer System after OECT Device Surface Modification

[0123] After the OECT device surface was modified, a series of different concentrations of SEA antibodies were used, and the known concentration of SEA antibodies was diluted to 0.001μg / mL, 0.01μg / mL, 0.1μg / mL, 1μg / mL, 10μg / mL and 100μg / mL using phosphate buffered saline (PBS) as a solvent to construct a set of concentration gradients. This step obtained different concentrations of SEA antibodies, and these antibodies had different binding conditions with the modified device, resulting in different changes in the current in the analyzer.

[0124] The specific steps are as follows:

[0125] (1) 10 μL of SEA antibody with different concentration gradients (0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL and 100 μg / mL) were added to the gate electrode of the modified OECT device prepared in Example 2 ( Figure 1 The device was then incubated at 25°C for 1 hour. This allowed the antibody to fully bind and interact with the modified layer on the device surface, forming a stable complex.

[0126] (2) After the reaction is completed, use PBS to rinse and remove the SEA antibody that is not bound to the device surface to ensure the accuracy and reliability of the experimental results.

[0127] (3) The modified device was tested for electrical properties using a Kickstart2 semiconductor analyzer. The specific steps are as follows: the modified OECT device prepared in Example 2 was placed in a test fixture, and the probes were placed on the designated source, gate, and drain electrodes, respectively. The source voltage was set to 1V, the drain voltage was set to 1V, and the gate voltage was set to -3V, 2V, -1V, 0V, 1V, 2V, or 3V. By adjusting the instrument parameters, such as the voltage range and scan rate, we were able to obtain key data such as the current-voltage curve and the current-time curve. These curves reflect the changes in electrical properties after different concentrations of antibodies bind to the device surface, providing a quantitative analysis and evaluation of the antigen binding effect.

[0128] The results show:

[0129] (1) SEA antibody was diluted with PBS to obtain SEA antibody with concentrations of 0.001 μg / mL, 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively. The SEA antibody was reacted with the modified OECT device for 1 h, and the voltage between the source and drain was adjusted to 1 V, and the gate voltage was -3 V to +3 V. Figure 7 In (a), it can be seen that as the concentration of bound SEA antibody continues to increase, the current gradually increases.

[0130] (2) Since the current is most stable at -3V voltage, when other conditions remain unchanged, the gate voltage is selected as -3V, and the current time variation curve is obtained as follows: Figure 7 As shown in (b), the change in current over time after combination is very small and can be ignored, which further improves the stability of subsequent measurements.

[0131] (3) Take IV curves of different concentration gradients, fix the gate voltage at -3V, and subtract the measured current from the unbound current after modification to obtain the current change. Plot it against the concentration gradient to obtain a standard curve. The results are as follows: Figure 8 The standard curve can not only detect the presence of SEA antibodies by measuring the change in current, but also quantitatively detect the concentration of SEA antibodies.

[0132] The content of SEA antibodies in the sample was detected by the following formula: y = -0.0085x4 + 0.0978x3 - 0.3255x2 + 0.4524x - 0.0417.

[0133] Example 6: Detection of SEA Antibody Concentration in Serum System after OECT Device Surface Modification

[0134] High quality rabbit serum was obtained from Jiangsu Institute of Parasitic Diseases, which was used as the diluent for the SEA antibody. Then, the known concentration of SEA antibody was gradually diluted to six different concentration gradients: 0.001 pg / mL, 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL and 100 pg / mL, according to the predetermined ratio. During the dilution process, each step was strictly controlled to ensure the accuracy of each concentration gradient.

[0135] The specific steps are as follows:

[0136] (1) The modified OECT device prepared in Example 2 was prepared respectively, and different concentrations (0.001 pg / mL, 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL and 100 pg / mL) of anti-SEA were added to the modified gate (modified gate) in the modified OECT device. The device was allowed to react at 25°C for 1 h, so that the antibody had enough time to interact with the molecules on the surface of the device. Figure 1

[0137] (2) After the reaction, the device was rinsed with PBS to remove unbound anti-SEA, in order to reduce experimental errors and improve measurement accuracy.

[0138] (3) The kickstart2 semiconductor analyzer was used to measure the current-voltage of the OECT device combined with different concentrations of antibodies. The settings of the analyzer were adjusted to capture the weak electrical signal changes produced after the antibody combined with the device. By recording and analyzing these curves, the current-voltage change curves of the antibody interacting with the OECT device under different concentration gradients were obtained.

[0139] The results showed that:

[0140] The above experiment proved that the OECT device can play a good detection role in the PBS solvent system. In order to make detection more convenient, we replaced the PBS solvent with rabbit serum for repeated experiments, and the standard curve graph obtained is shown in Figure 9 Compared with the PBS solvent system curve, the change in device current in serum is reduced, which may be due to the fact that compared with the PBS system, the serum system has more complex components, including non-specific antibodies, fatty acids, steroids, serum proteins and other substances, which have a certain impact on the entire system. However, the overall trend remains the same and can be used to quantitatively measure the concentration of SEA antibody in serum.

[0141] The content of SEA antibody in the sample was detected by the following formula: y = -0.0016x4+0.0205x3-0.0918x2+0.2061x-0.0592.​

[0142] Example 7: Specificity analysis of SEA antibody detection in serum system after surface modification of OECT device

[0143] High-quality rabbit serum was obtained from the Jiangsu Provincial Schistosoma Control Institute and used as a solvent for diluting the SEA antibody. Liver fluke and lung fluke antibodies were prepared as interfering substances. The concentrations of the liver fluke and lung fluke antibodies provided by the Jiangsu Provincial Schistosoma Control Institute were measured using a White Shark BCA kit to ensure that their concentrations were comparable to those of the SEA antibody used in the experiment. These two interfering substances were used in control experiments to verify the OECT device's specific recognition of the SEA antibody. The liver fluke and lung fluke antibodies provided by the Jiangsu Provincial Schistosoma Control Institute were at concentrations of 30 mg / mL and 35 mg / mL, respectively. These solutions were diluted to obtain concentrations of 0.1 μg / mL, 1 μg / mL, and 10 μg / mL, respectively, for the schistosoma japonicum, liver fluke, and lung fluke antibody solutions.

[0144] The specific steps are as follows:

[0145] (1) Take the modified OECT device prepared in Example 2, and then select 10 μL of SEA antibody (concentrations are 0.1 μg / mL, 1 μg / mL, 10 μg / mL), liver fluke antibody (concentrations are 0.1 μg / mL, 1 μg / mL, 10 μg / mL) and lung fluke antibody (concentrations are 0.1 μg / mL, 1 μg / mL, 10 μg / mL), and add them to the gate of different modified OECT devices ( Figure 1 in the modified grid).

[0146] The device was allowed to react at 25°C for 1 hour to allow the antibody to fully interact with the molecules on the device surface.

[0147] (2) After the reaction is completed, the device is rinsed with PBS to remove unbound antibodies.

[0148] (3) The current-voltage and current-time curves of OECT devices bound to different antibodies were measured using a kickstart2 semiconductor analyzer. The source voltage was set to 1V, the drain voltage was 1V, and the gate voltage was -3V; the changes in the electrical signals generated after each antibody bound to the device were recorded and analyzed. By comparing the changes in the electrical signals after different antibodies bound to the OECT device, the specific recognition ability of the OECT device for SEA antibodies was analyzed. If the electrical signal changes generated by the SEA antibody were significantly higher than those of the liver fluke antibody and the lung fluke antibody, it indicated that the OECT device had good specificity for the SEA antibody. The specific recognition results of the OECT device were then compared with the standard test strip method to verify its accuracy and reliability.

[0149] (4) Prepare commercially available SEA test strips and operate according to the instructions. Use the test strips to detect schistosome antibodies (4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.1 mg / mL), liver fluke antibodies (4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.1 mg / mL), and lung fluke antibodies (4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.1 mg / mL). Observe the color development of the test strips at high and low concentrations. Based on the experimental results of specificity analysis and sensitivity comparison, compare the test strips with the OECT device to find the performance advantages of the OECT device in SEA antibody detection.

[0150] The results show:

[0151] (1) According to the measurement, when the source-drain voltage is 1V and the gate voltage is -3V, the current change of the schistosoma japonicum antibody under the three concentration conditions is much higher than that of the other two antibodies, such as Figure 10 As shown, it is proved that the OECT device is specific for the detection of Schistosoma japonicum antibodies; Figure 10 Current changes of three antibodies at concentrations of (a) 0.1 μg / mL, (b) 1 μg / mL, and (c) 10 μg / mL, respectively.

[0152] (2) The specificity of the existing schistosoma detection kits on the market was analyzed. The concentrations of the five test strips from left to right were 4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, and 0.1 mg / mL. The results showed that regardless of the schistosoma antibody ( Figure 11 (a)), liver fluke antibodies ( Figure 11 (b)) or Paragonimus antibodies ( Figure 11 (c)) At concentrations above 2 mg / mL, a color reaction can occur, which cannot clearly distinguish the three antibodies and has no specificity; and below 2 mg / mL, it cannot be detected;

[0153] In comparison, even at very low antibody concentrations (0.1 μg / mL), the OECT device can detect significant current changes, indicating that the OECT device has high sensitivity and can meet the needs of actual detection of schistosome antibodies.

[0154] (3) The results show that:

[0155] Compared to commercially available schistosoma japonicum infection detection test strips, the OECT device designed in this experiment demonstrated extremely high sensitivity and specificity. It was able to detect schistosoma japonicum antibodies as low as 0.001 μg / mL (while commercially available schistosoma japonicum test strips could not detect antibodies below 2 mg / mL), and the current change was significantly higher than that of antibodies that could not be distinguished by the other two test strips (liver fluke and lung fluke antibodies). This characteristic gives OECT devices a significant advantage in the field of antibody detection, especially in rapid testing scenarios where different types of antibodies need to be distinguished or low concentrations of antibodies need to be detected.

[0156] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An antibody sensor based on an organic electrochemical transistor, characterized in that: The antibody sensor comprises, from bottom to top, a substrate layer, an organic semiconductor layer, a solid ion gel electrolyte layer, an organic semiconductor doping layer, and a gate; the organic semiconductor layer and the organic semiconductor doping layer are both P-type organic semiconductors, and the organic semiconductor doping layer is a P-type organic semiconductor doped with a P-type dopant; after the gate surface is cleaned with ethanol and deionized water, cysteamine and soluble insect egg antigen SEA are used as modifiers to modify the gate surface to form a self-assembled molecular film; The substrate layer is a glass sheet of source and drain electrodes, and the solid ion gel electrolyte layer is formed by spin coating after doping ionic liquid in PVDF-HFP solution, and the ionic liquid is EMIM TFSI, EMIM BF4 or a mixture of the two; The method for modifying the gate self-assembled molecular film comprises: (1) Wash with deionized water and ethanol respectively and then blow dry; (2) After drying, add cysteamine to the gate and react in the dark for 6-8 hours. After washing and drying, add glutaraldehyde and react for 1-2 hours. (3) After the reaction in step (2), rinse the grid with water and blow dry, then add soluble egg antigen SEA and react again for 1-2 hours; (4) After the reaction in step (3) is completed, rinse and blow dry, add bovine serum albumin and react for 1-2 hours to passivate the aldehyde groups that have not reacted with SEA, and then wash and dry; In step (2), the concentration of cysteamine is 0.4-0.5 mM, and the volume fraction of glutaraldehyde is 2-2.5%; in step (3), the concentration of soluble egg antigen SEA is 45-50 μg / mL; and in step (4), the mass fraction of bovine serum albumin is 1-1.5%.

2. A method for preparing an antibody sensor based on an organic electrochemical transistor according to claim 1, characterized in that: The method comprises: Step 1): Spin-coat a layer of organic semiconductor solution on the substrate with the active and drain electrodes, and anneal to form an organic semiconductor layer; Step 2): Spin-coating an ionic gel electrolyte solution on the organic semiconductor layer and heating and curing the solution to form a solid ionic gel electrolyte layer; Step 3): Spin-coating a mixed solution of an organic semiconductor and a dopant on the solid ion gel electrolyte layer and annealing to form an organic semiconductor doping layer; Step 4): evaporating a gate electrode on the organic semiconductor doping layer; Step 5): After the gate surface is washed with ethanol and water, cysteamine and soluble egg antigen SEA are used as modifiers in sequence to modify the gate surface to form a self-assembled molecular film.

3. The preparation method according to claim 2, characterized in that The organic semiconductor in step 1) and the organic semiconductor in step 3) are P-type organic semiconductors; the ionic gel electrolyte solution is an ionic liquid doped in a PVDF-HFP solution, and the ionic liquid is EMIM TFSI, EMIM BF4 or a mixture of the two; the dopant in step 3) is a P-type dopant; The method for modifying the gate self-assembled molecular film comprises: (1) Wash with water and ethanol respectively and then blow dry; (2) After drying, add cysteamine to the gate and react in the dark for 6-8 hours. After washing and drying, add glutaraldehyde and react for 1-2 hours. (3) After the reaction in step (2), rinse the grid with water and blow dry, then add soluble egg antigen SEA and react again for 1-2 hours; (4) After the reaction in step (3) is completed, rinse and blow dry, add bovine serum albumin and react for 1 to 2 hours to passivate the aldehyde groups that have not reacted with SEA, and then wash and dry.

4. A product, characterized in that The product contains the organic electrochemical transistor-based antibody sensor according to claim 1; the product is a biochip for detecting SEA antibodies in a sample, or the product is a device for detecting SEA antibodies in a sample, or the product is a kit for detecting SEA antibodies in a sample.

5. Use of the antibody sensor according to claim 1 in preparing a product for detecting SEA antibodies in a sample, characterized in that: The product is a biochip for detecting SEA antibodies in a sample, or the product is a device for detecting SEA antibodies in a sample, or the product is a kit for detecting SEA antibodies in a sample.

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