High-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology

Abstract

The invention provides a high-sensitivity multi-index protein detection instrument based on an OECT and ELISA combined technology, belongs to the technical field of biomedical detection, and is characterized in that the high-sensitivity multi-index protein detection instrument comprises: an integrated carrier plate, which comprises a plurality of units, and each unit is provided with a detection unit and an immunoreaction unit in an integrated manner; the immunoreaction unit is used for generating small molecules related to the target protein concentration; the trapping agents coated by the at least two immunoreaction units are different; the detection unit comprises an organic electrochemical transistor, and the organic electrochemical transistor generates changing electric signals; the hardware circuit system is used for collecting electric signals; and the upper computer is in communication connection with the hardware circuit system and is used for receiving the electric signal and displaying a detection result related to the target protein concentration based on the electric signal. According to the invention, a miniaturized instrument integrating immune reaction and immunoassay functions and capable of performing multi-index protein high-sensitivity detection is realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a highly sensitive multi-index protein detection instrument based on the combined use of OECT and ELISA technology. Background Technology

[0002] Early tumor marker screening is crucial for improving cancer cure rates. Currently, widely used clinical detection technologies include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), and electrochemiluminescence immunoassay (ECIA). While these technologies are mature, they still have the following significant limitations: First, most commercially available testing kits target a single protein marker, failing to enable combined detection of multiple markers and thus hindering a comprehensive assessment of a patient's health. Combining multiple single-marker kits can lead to signal crosstalk due to inconsistencies in protein probes and chromogenic substrates. Second, existing immunoassay devices, such as ELISA readers and fully automated chemiluminescence immunoassay analyzers, separate the immunoreaction process within the kit from the immunoassay process performed in the dedicated instrument, making integration impossible. Finally, these dedicated testing devices are bulky, complex to operate, require professional personnel, and have high maintenance costs, hindering their widespread adoption in primary healthcare, home, or on-site testing. Therefore, there is an urgent need for a portable protein detection instrument that integrates immunoreaction and immunoassay, enabling highly sensitive multi-marker detection. Summary of the Invention

[0003] This invention provides a highly sensitive multi-index protein detection instrument based on OECT and ELISA combined technology, realizing a miniaturized instrument that integrates immune reaction and immunoassay functions and can perform highly sensitive detection of multiple index proteins.

[0004] This invention provides a highly sensitive multi-index protein detection instrument based on OECT and ELISA combined technology, comprising: An integrated carrier plate includes multiple units, each of which integrates a detection unit and an immune response unit; The immune reaction unit is used to perform an immune reaction based on the principle of enzyme-linked immunosorbent assay (ELISA) and generate small molecules related to the concentration of the target protein; wherein at least two of the immune reaction units are coated with different capture agents, and the capture agents are antibodies, antigens or nucleic acids; The detection unit includes an organic electrochemical transistor, which generates a changing electrical signal in response to the concentration of the small molecule. A hardware circuit system, which is electrically connected to the organic electrochemical transistor, is used to provide a working voltage signal to the organic electrochemical transistor and to acquire the electrical signal; A host computer, which is communicatively connected to the hardware circuit system, is used to receive the electrical signal and display the detection results related to the concentration of the target protein based on the electrical signal.

[0005] In some embodiments, the detection unit includes a substrate and an organic electrochemical transistor microelectrode disposed on the substrate; The immune response unit includes a reaction substrate and a signal amplification unit. The capture agent is coated on the reaction substrate. The signal amplification unit includes a biotin-labeled secondary antibody and an enzyme-labeled streptavidin for an enzymatic reaction.

[0006] In some embodiments, the organic electrochemical transistor microelectrode includes a source, a drain, and a gate, with a channel between the source and the drain, the gate being made of a hydrogen peroxide-sensitive material, and the channel being made of an organic semiconductor material.

[0007] In some embodiments, the hydrogen peroxide-sensitive material includes at least one of noble metals, noble metal alloys, transition metals, transition metal oxides, transition metal hydroxides, carbon-based materials, composite materials, composite nanoenzyme materials, or organic polymer materials, and the organic semiconductor material includes polyethylene dioxythiophene material.

[0008] In some embodiments, the hydrogen peroxide-sensitive material is platinum or polybenzimidazole-benzophenanthridine, and the organic semiconductor material is a mixture of monolayer polyethylenedioxythiophene and polystyrene sulfonate, or a stacked structure composed of a first mixture and a second mixture; wherein the first mixture is a mixture of polyethylenedioxythiophene and bromothymol blue, and the second mixture is a mixture of polyethylenedioxythiophene and polystyrene sulfonate.

[0009] In some embodiments, the plurality of units are arranged in a unit array; The immune response units described in the same series are coated with the same capture agent, and the corresponding detection circuits described in the same series are used to collect the electrical signals corresponding to different concentrations of the same target protein; The immune response units described in the same column are coated with different types of capture agents, and the corresponding detection circuits described in the same column are used to collect the electrical signals corresponding to the same concentration of different target proteins.

[0010] In some embodiments, the hardware circuit system includes a wireless communication module, a microcontroller unit, a digital-to-analog converter module, and an analog-to-digital converter module; The digital-to-analog converter module is used to provide the operating voltage signal to the organic electrochemical transistor, and the analog-to-digital converter module is used to acquire the electrical signal output by the organic electrochemical transistor; The microcontroller unit is connected to the wireless communication module and is used to process the electrical signal and transmit the processed signal to the host computer through the wireless communication module.

[0011] In some embodiments, the hardware circuit system is disposed below each of the units; The integrated substrate has inter-row apertures corresponding to the unit array formed by the arrangement of the multiple units. The digital-to-analog converter module includes three output ports. The conductive leads connected to the three output ports are led out through the inter-row apertures and respectively connected to the source, drain and gate of the organic electrochemical transistor.

[0012] In some embodiments, the wireless communication module is a Bluetooth module, and all the wireless communication modules are of the same model and signal synchronization is achieved through a timing calibration algorithm.

[0013] In some embodiments, the integrated carrier plate has a length and width ranging from 15 mm to 25 cm, and a thickness ranging from 3 mm to 5 cm; The length, width, and depth of the unit all range from 1 millimeter to 2 centimeters. The length and width ranges of the detection unit and the immune response unit are both 0.5 mm to 1 cm.

[0014] This invention integrates the immunoreaction unit and the detection unit into each unit, enabling the immunoreaction and immunoassay processes to be completed on the same device. This overcomes the drawbacks of separating the two processes in traditional technologies and simplifies the operation process. Furthermore, by coating different immunoreaction units with different capture agents, a single instrument can simultaneously detect multiple target proteins, achieving multi-index detection and overcoming the limitations of single-index detection. Simultaneously, utilizing the high sensitivity of organic electrochemical transistors to hydrogen peroxide and their inherent signal amplification capabilities, weak biochemical signals are converted into strong electrical signals, laying the foundation for highly sensitive detection. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 This is a top view schematic diagram of a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology provided in an embodiment of the present invention.

[0017] Figure 2 This is a three-dimensional structural diagram of a unit provided in an embodiment of the present invention.

[0018] Figure 3 This is a three-dimensional structural schematic diagram of another unit provided in an embodiment of the present invention.

[0019] Figure 4 This is a schematic diagram of a scanning electron microscope image of a single-layer PEDOT:PSS provided in an embodiment of the present invention.

[0020] Figure 5 This is a schematic scanning electron microscope image of a stacked PEDOT:PSS and PEDOT:BTB provided in an embodiment of the present invention.

[0021] Figure 6 This is a schematic diagram of a hardware circuit system provided in an embodiment of the present invention.

[0022] Figure 7 This is a three-dimensional exploded structure diagram of a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology provided in an embodiment of the present invention.

[0023] Figure 8 This is a schematic diagram of the electrical connection between the hardware circuit system and the OECT microelectrode of the detection unit.

[0024] Figure 9 This is a schematic cross-sectional view of the OECT microelectrode fabrication process in a detection unit provided by an embodiment of the present invention, showing the sequential formation of each step.

[0025] Figure 10 This is a schematic diagram of the cross-sectional structure formed sequentially by each step in the preparation process of the channel laminate material in a detection unit provided by an embodiment of the present invention.

[0026] Figure 11 This is a flowchart illustrating the specific operation steps of a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology, as provided in this embodiment of the invention.

[0027] Figure 12 This is a top view schematic diagram of a CEA single-index protein detection instrument based on OECT and ELISA combined technology provided in an embodiment of the present invention.

[0028] Figure 13 This is a schematic diagram of the relationship between CEA antigen concentration and normalized current sensitivity provided in an embodiment of the present invention.

[0029] Figure 14 This is a top view schematic diagram of a single-index protein detection instrument for SA based on OECT and ELISA combined technology provided in an embodiment of the present invention.

[0030] Figure 15 This is a schematic diagram of the relationship between SA antigen concentration and normalized current sensitivity provided in the embodiments of the present invention.

[0031] Figure 16 This is a schematic diagram of the recovery rate curve of serum samples from human lung cancer patients provided in an embodiment of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Air pollution from industrial waste gas and vehicle exhaust, increased psychological stress and irregular work-rest schedules caused by competition across various industries, inadequate medical conditions in remote areas, and rising cancer risks due to an aging population all pose serious threats to people's health and well-being. Although the five-year survival rate for cancer patients in my country has improved, it remains relatively low, with significant room for improvement. Early detection and treatment could significantly increase the five-year survival rate. Cancer biomarkers, as objectively measurable and assessable macromolecules, serve as indicators of normal or disease processes and pharmacological responses to treatment interventions. Therefore, the development of highly sensitive early tumor biomarker detection equipment is currently a hot topic in the market.

[0034] Currently, early tumor marker screening mainly relies on enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and electrochemiluminescence assay (ECL). ELISA is a classic immunological detection technique based on the specific binding of antigen and antibody, directly reacting with an enzyme-labeled antibody or a biotin-labeled antibody and an enzyme-labeled streptavidin-catalyzed chromogenic substrate. Because clinical quantitative or qualitative detection typically uses inexpensive commercial kits paired with ELISA readers, it is widely used for early screening of tumor markers, with detection limits usually reaching the ng / mL to pg / mL level. CLIA is a technique based on the specific binding of enzyme-labeled antibodies or antigens to a target substance, which then reacts with a specific luminescent substrate of a catalytic enzyme to produce light emission. A photosensitive device captures the photon signal, converting the light signal into an electrical signal for quantitative or qualitative analysis of the target substance. Clinical quantitative or qualitative testing typically employs highly sensitive commercial reagent kits paired with fully automated electrochemiluminescence immunoassay analyzers, making it widely used in populations requiring real-time dynamic monitoring and high-precision detection, as well as those with complex clinicopathological conditions. Its detection limits typically reach the pg / mL level. Electrochemiluminescence immunoassay (ECL) is a technique combining electrochemistry and CLIA, where antibody-antigen specific binding occurs on a solid phase (i.e., fixed on the surface of magnetic beads), followed by binding to a luminescent reagent-labeled detection antibody. The resulting electrochemical reaction on the electrode surface excites the substance to emit light. Clinical quantitative or qualitative testing typically employs highly sensitive commercial reagent kits paired with fully automated electrochemiluminescence immunoassay analyzers, making it widely used in populations requiring real-time dynamic monitoring and high-precision detection, as well as those with complex clinicopathological conditions. Its detection limits typically reach the pg / mL to fg / mL level.

[0035] Immunoassay techniques for early cancer biomarkers, such as ELISA, CLIA, and ECL, vary depending on their market positioning. CLIA and ECL technologies hold a significant advantage in customized precision medicine and high-precision, wide-range quantitative detection, but they rely on bulky, dedicated electrical or chemiluminescence detection instruments, which are expensive, have high maintenance costs, are complex to operate, and are prone to cross-contamination due to residual samples. ELISA technology excels in large-scale screening and qualitative detection, but suffers from a limited detection range, low sensitivity, slow detection speed, and a high risk of subjective error. Therefore, there is an urgent market need for an early tumor biomarker detection device that integrates high sensitivity, low cost, and high portability to meet the needs of primary healthcare, smart healthcare, and home healthcare. Furthermore, the common defects and shortcomings of immunoassay technologies are as follows: First, in order to ensure the consistency of protein probes and chromogenic substrates and to unify their immune reaction systems, commercially available ELISA, CLIA, and ECL kits tend to be kits with a single protein indicator rather than kits with multiple protein indicators.

[0036] In clinical practice, tumor markers are commonly used as indicators of the occurrence, presence, or progression of pathological conditions in cancer patients. This requires measuring low concentrations of proteins in complex samples. Clinically, most tumor markers are identified in the plasma supernatant (serum) of early-stage cancer patients, including embryonic antigens, carbohydrate antigens, enzymes and proteins, hormones, and others—approximately 15 to 20 in total. Commercially available kits often only detect a single protein marker. Customized multi-marker protein marker kits often use different protein probes and chromogenic substrates, which may lead to signal crosstalk. Currently, single-marker protein kits are the most popular on the market, but they fail to comprehensively assess the health status of cancer patients.

[0037] Secondly, current immunoassay technologies for early cancer biomarkers have not yet integrated immune responses with immunoassay measurements.

[0038] Currently available immunoassay devices mainly include ELISA readers, CLIA fully automated chemiluminescence immunoassay analyzers, and ECL fully automated electrochemiluminescence immunoassay analyzers. All of these require the immunoassay to be completed first using commercially available reagent kits, followed by the immunoassay assay using their respective dedicated instruments. It is impossible to integrate the immunoassay and immunoassay into a single device.

[0039] Finally, commercially available detection devices for early cancer biomarkers do not meet the requirements for miniaturization.

[0040] Currently available immunoassay devices, such as ELISA readers, CLIA fully automated chemiluminescence immunoassay analyzers, and ECL fully automated electrochemiluminescence immunoassay analyzers, all feature three main modules: sample introduction, detection, and display. These devices are relatively large and have not achieved miniaturization of these modules. Furthermore, fully automated electrochemiluminescence or chemiluminescence immunoassay analyzers for CLIA and ECL technologies generally require professional operation and present a certain level of difficulty.

[0041] To address the aforementioned problems, embodiments of the present invention provide a highly sensitive multi-index protein detection instrument based on OECT and ELISA combined technology. Figure 1 This is a top view schematic diagram of a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology provided in an embodiment of the present invention. Figure 2 This is a three-dimensional structural diagram of a unit provided in an embodiment of the present invention. (In conjunction with...) Figure 1 and Figure 2 The high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology includes an integrated carrier plate 1, a hardware circuit system 12, and a host computer. The integrated carrier plate 1 includes multiple units 13, each unit 13 integrating a detection unit 131 and an immune reaction unit 132. The immune reaction unit 132 is used to perform an immune reaction based on the principle of enzyme-linked immunosorbent assay and generate small molecules related to the concentration of the target protein. Among them, at least two immune reaction units 132 are coated with different capture agents, which are antibodies, antigens, or nucleic acids. The nucleic acid can be, for example, an aptamer. That is, the different ELISA immune reaction units 132 in each unit 13 can be coated with the same or multiple antibodies 1322, antigens 1323, or nucleic acids. The detection unit 131 includes an organic electrochemical transistor (OECT), which generates a changing electrical signal in response to the concentration of small molecules, such as hydrogen peroxide. The hardware circuit system 12 is electrically connected to the organic electrochemical transistor to provide a working voltage signal to the organic electrochemical transistor and to acquire the electrical signal. The host computer is communicatively connected to the hardware circuit system 12 to receive the electrical signal and display the detection results related to the target protein concentration based on the electrical signal.

[0042] Specifically, the integrated carrier plate 1 refers to the overall plate-like structure that carries multiple functional units 13, the unit 13 refers to the smallest independent area on the integrated carrier plate 1 that has complete detection function, the immune reaction unit 132 refers to the area that performs a specific binding reaction of specific biomolecules, such as antigens and antibodies, the coating refers to the process of fixing biomolecules, such as antibodies or antigens, onto the surface of a solid carrier, and the capture agent refers to a molecule that can specifically bind to the target protein, such as antibodies, antigens, or nucleic acids.

[0043] Organic electrochemical transistors (OETTs) are three-terminal devices that utilize organic semiconductor materials as the channel 1316 and control the current in the channel 1316 by adjusting the gate potential. Specifically, an OETT is a three-terminal device, mainly consisting of a source 1313, a drain 1314, and a gate 1315. A high-gain current signal is output by adjusting the channel 1316 between the source and drain through a gate voltage of, for example, less than or equal to 1V. OETTs offer advantages such as simple structure, low power consumption, high gain, and good biocompatibility.

[0044] Hydrogen peroxide is a signal molecule produced by enzyme catalysis of substrate in enzyme-linked immunosorbent assay (ELISA). Hardware circuit system 12 refers to the collection of circuit modules that provide working conditions for electronic devices and process electrical signals. Host computer refers to a computer or intelligent device, such as a personal computer, mobile phone or tablet computer, that receives, processes and displays data. Electrical signal refers to the current or voltage signal output by organic electrochemical transistors whose magnitude varies with the concentration of hydrogen peroxide.

[0045] The immunoreaction unit 132 operates based on the mature principle of enzyme-linked immunosorbent assay (ELISA). When a sample containing the target protein is added to the immunoreaction unit 132, the target protein specifically binds to a capture agent pre-coated on the reaction substrate 1321. Subsequently, a signal amplification structure is formed by introducing biotin-labeled secondary antibody and glucosidase-labeled streptavidin. Finally, a glucose solution is added, and glucosidase catalyzes the oxidation of glucose to generate hydrogen peroxide proportional to the concentration of the target protein. The gate 1315 of the organic electrochemical transistor in the detection unit 131 is directly exposed to the electrolyte containing hydrogen peroxide. The hydrogen peroxide undergoes an electrochemical reaction on the surface of the gate 1315, causing an electrical signal at the gate 1315, such as a change in potential. This small change in potential is amplified by the organic electrochemical transistor, modulating the current in the channel 1316 between its source 1313 and drain 1314, thereby outputting a significantly changed and easily measurable electrical signal. The electrical signal is acquired by the hardware circuit system 12 and transmitted to the host computer. The host computer converts the electrical signal value into the concentration of the target protein using a preset algorithm and displays it to the user.

[0046] Therefore, by integrating the immunoreaction unit 132 and the detection unit 131 onto each unit 13, this embodiment of the invention enables the immunoreaction and immunoassay processes to be completed on the same device, overcoming the drawbacks of separating the two processes in traditional technologies and simplifying the operation process. Furthermore, by coating different immunoreaction units 132 with different capture agents, a single instrument can simultaneously detect multiple target proteins, achieving multi-index detection functionality and overcoming the limitations of single-index detection. Simultaneously, by utilizing the high sensitivity of organic electrochemical transistors to hydrogen peroxide and their inherent signal amplification capabilities, weak biochemical signals are converted into strong electrical signals, laying the foundation for highly sensitive detection.

[0047] In some embodiments, combined with Figure 1 and Figure 2 The detection unit 131 includes a substrate 1312 and an organic electrochemical transistor microelectrode 1311 disposed on the substrate 1312; the immunoreaction unit 132 includes a reaction substrate 1321 and a signal amplification unit 133, wherein a capture agent is coated on the reaction substrate 1321, and the signal amplification unit 133 includes a biotin-labeled secondary antibody and an enzyme-labeled streptavidin for an enzymatic reaction. Exemplarily, the enzyme-labeled streptavidin for the enzymatic reaction may include glucose-labeled streptavidin, lactase-labeled streptavidin, or uricase-labeled streptavidin, etc., substances involved in enzymatic reactions.

[0048] Specifically, substrate 1312 refers to the base material supporting the organic electrochemical transistor microelectrode 1311, such as silicon, glass, or polymer. Organic electrochemical transistor microelectrode 1311 refers to the microelectrode structure fabricated on substrate 1312 and constituting an organic electrochemical transistor. Reaction substrate 1321 refers to the solid surface on which the immune response depends, such as a polystyrene plate. Signal amplification unit 133 refers to the collection of components that convert a single biomolecule binding event into a large number of signal molecules through a cascade reaction. Biotin-labeled secondary antibody refers to an antibody bound to a primary antibody or target protein and linked with a biotin molecule. Glucosease-labeled streptavidin refers to glucose oxidase covalently bound to a streptavidin molecule, such as, but not limited to, glucose oxidase.

[0049] The detection unit 131 and the ELISA immunoreaction unit 132 are integrated on the same unit 13. The detection unit 131 consists of a substrate 1312 and an OECT microelectrode 1311. The substrate 1312 of the detection unit 131 provides mechanical support and electrical insulation for the organic electrochemical transistor microelectrode 1311. The reaction substrate 1321 of the immunoreaction unit 132 provides a fixation site for the capture agent. The signal amplification unit 133 works on the principle that biotin and streptavidin have extremely high affinity and can form a stable complex. One streptavidin molecule can bind four biotin molecules, which allows multiple glucose molecule to be introduced in each immunobinding event. When glucose molecule-labeled streptavidin is immobilized on the immunocomplex through the biotin-streptavidin system, each enzyme molecule can continuously catalyze the production of hydrogen peroxide molecules from glucose, thereby achieving cascade amplification of the signal.

[0050] Figure 2 The structure shown corresponds to the immune response unit 132 of the double antibody sandwich structure. The immune response unit 132 includes a substrate 1321, a coated antibody 1322, an antigen 1323, and a signal amplification unit 133. The signal amplification unit 133 includes a biotin-labeled secondary antibody 1332 and a glucose-labeled streptavidin 1333. Figure 3 This is a three-dimensional structural schematic diagram of another unit provided in an embodiment of the present invention. Figure 3 The structure shown corresponds to the immune response unit 132 of the immune competition structure. The immune response unit 132 includes a substrate 1321, a coated antigen 1323, and a signal amplification unit 133. The signal amplification unit 133 includes a biotin-labeled secondary antibody 1332 and a glucosidase-labeled streptavidin 1333.

[0051] Therefore, this embodiment of the invention constructs a highly efficient signal amplification mechanism by combining the biotin-streptavidin system with enzyme-catalyzed reactions, enabling a single antigen-antibody binding event to generate tens of thousands of hydrogen peroxide molecules, greatly improving detection sensitivity. Simultaneously, the detection unit 131 and the immune reaction unit 132 are structurally clearly separated yet tightly integrated, allowing the biological reaction section and the electrical sensing section to be optimized separately before integration, improving the instrument's performance and reliability.

[0052] In some embodiments, combined with Figures 1 to 3 The organic electrochemical transistor microelectrode 1311 includes a source 1313, a drain 1314 and a gate 1315. A channel 1316 is provided between the source 1313 and the drain 1314. The gate 1315 is made of a material that is sensitive to small molecules, such as hydrogen peroxide. The channel 1316 is made of an organic semiconductor material.

[0053] Specifically, the channel 1316 is an organic semiconductor region connecting the source 1313 and the drain 1314, and its conductivity is regulated by the potential of the gate 1315. Materials sensitive to hydrogen peroxide refer to materials that can undergo electrochemical reactions and generate electrical signals in the presence of hydrogen peroxide, such as materials with potential changes. Organic semiconductor materials refer to organic polymers or small molecule materials with semiconductor properties.

[0054] When a voltage is applied between the source 1313 and drain 1314, current flows through the channel 1316. When the gate 1315 is immersed in an electrolyte containing hydrogen peroxide, the hydrogen peroxide undergoes a redox reaction on the surface of the gate 1315, which is made of a hydrogen peroxide-sensitive material. This reaction alters the double-layer structure at the interface between the gate 1315 and the electrolyte, causing a drift in the potential of the gate 1315. According to the transport characteristics of organic electrochemical transistors, a small change in the potential of the gate 1315 effectively modulates the carrier density in the channel 1316, resulting in a significant change in the current between the source 1313 and drain 1314. This current change is the output electrical signal.

[0055] Therefore, this embodiment of the invention utilizes the transistor amplification effect of organic electrochemical transistors to amplify the weak chemical potential signal on the gate 1315 into a significant change in source-drain current, enabling the instrument to detect extremely low concentrations of hydrogen peroxide, thereby indirectly achieving the detection of extremely low concentrations of target proteins. Simultaneously, the functional separation design of the gate 1315 and channel 1316 materials allows for the selection of optimal materials for sensing and signal amplification respectively, thereby improving the overall performance of the device. Sensing corresponds to the gate 1315, and signal amplification corresponds to the channel 1316.

[0056] In some embodiments, combined with Figures 1 to 3Materials sensitive to hydrogen peroxide include at least one of noble metals, noble metal alloys, transition metals, transition metal oxides, transition metal hydroxides, carbon-based materials, composite materials, composite nanozyme materials, or organic polymer materials. Organic semiconductor materials include, for example, polyethylene dioxythiophene materials.

[0057] Specifically, precious metals refer to metallic elements such as ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, and gold. Precious metal alloys refer to alloys formed between or with other metals, such as platinum-palladium alloys. Transition metal oxides and hydroxides refer to oxides or hydroxides formed from transition metals, such as manganese dioxide, cobalt hydroxide, and iron oxide. Carbon-based materials refer to functional materials mainly composed of carbon, such as graphite glassy carbon, carbon nanotubes, and graphene. Composite materials refer to materials composed of two or more different materials, such as platinum-carbon composites and manganese dioxide-carbon nanotube composites. Composite nanoenzyme materials refer to nanocomposite materials with enzyme-like catalytic activity, such as PtO / PtO2-C3N4, i.e., platinum oxide / platinum dioxide-carbon nitride composites. Organic polymer materials refer to polymers with specific electrochemical activities, such as polybenzimidazole-benzophenanthridine, i.e., poly BBL. Polyethylene dioxythiophene materials refer to conductive polymer materials with poly(3,4-ethylenedioxythiophene) as the main component.

[0058] Different types of hydrogen peroxide-sensitive materials exhibit varying mechanisms for catalyzing hydrogen peroxide decomposition. Noble metals, such as platinum, primarily lower the reaction energy barrier for hydrogen peroxide decomposition by providing surface catalytically active sites, while certain metal oxides, such as manganese dioxide or nanozyme materials, catalyze hydrogen peroxide decomposition through a valence-changing process. Regardless of the mechanism, both ultimately lead to a change in the gate 1315 potential. Polyethylene dioxythiophene, as a typical organic semiconductor, exhibits excellent ion-electron mixing conductivity in aqueous electrolytes. When the gate 1315 potential changes, ions in the electrolyte are injected into or extracted from the channel 1316 material, thereby significantly altering its conductivity.

[0059] Therefore, the embodiments of the present invention provide a wide range of gate 1315 materials, allowing for flexible selection of the most suitable material based on different detection requirements, such as cost, sensitivity, and stability. Meanwhile, the polyethylene dioxythiophene material exhibits good biocompatibility and high transconductance, ensuring stable operation of the organic electrochemical transistor under physiological conditions and enabling it to generate high-gain output signals.

[0060] In some embodiments, combined with Figures 1 to 3The material sensitive to hydrogen peroxide is platinum or polybenzimidazole and benzo[a]phenanthridine, and the organic semiconductor material is a mixture of monolayer polyethylene dioxythiophene and polystyrene sulfonate, or a stacked structure composed of a first mixture and a second mixture; wherein the first mixture is a mixture of polyethylene dioxythiophene and bromothymol blue, and the second mixture is a mixture of polyethylene dioxythiophene and polystyrene sulfonate.

[0061] Specifically, platinum is a noble metal element and a highly efficient hydrogen peroxide catalyst. Polybenzimidazole-benzophenanthridine is a conductive organic polymer material with a ladder-like structure that exhibits electrochemical responsiveness to hydrogen peroxide. Organic semiconductor materials can be monolayer mixtures of polyethylene dioxythiophene and polystyrene sulfonate, referring to thin films composed of a single conductive layer of polyethylene dioxythiophene and polystyrene sulfonate, such as monolayer mixtures of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate (PEDOT:PSS).

[0062] Organic semiconductor materials can also be multilayer structures composed of a first mixture and a second mixture; wherein the first mixture is a mixture of polyethylene dioxythiophene and bromothymol blue, and the second mixture is a mixture of polyethylene dioxythiophene and polystyrene sulfonate. Exemplarily, a layer of a mixture of polyethylene dioxythiophene and polystyrene sulfonate is first prepared, and then a layer of a mixture of polyethylene dioxythiophene and bromothymol blue is prepared on top thereon, forming a bilayer composite structure. The mixture of polyethylene dioxythiophene and bromothymol blue can be a mixture of poly(3,4-ethylenedioxythiophene) and bromothymol blue (PEDOT: BTB), and the mixture of polyethylene dioxythiophene and polystyrene sulfonate can be a mixture of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate (PEDOT: PSS).

[0063] The platinum gate generates protons and electrons through the catalytic decomposition of hydrogen peroxide, causing a change in interfacial potential. Polybenzimidazole-benzophenanthridine, as an organic electrocatalytic material, also efficiently catalyzes the decomposition of hydrogen peroxide through its active sites in its molecular structure. Regarding the channel 1316 material, the stacked structure utilizes the synergistic effect of different materials: the lower layer, a mixture of polyethylene dioxythiophene and polystyrene sulfonate, provides an excellent conductive substrate and ion implantation capability; the upper layer, a mixture of polyethylene dioxythiophene and bromothymol blue, contains bromothymol blue molecules that react with the protons generated by the gate 1315, such as H+. + It is highly sensitive, and its structural changes will further modulate the electrical performance of the channel 1316, thereby achieving dual signal response and amplification.

[0064] A preferred combination for achieving ultra-low detection limits may be a platinum-based gate 1315 material and a multilayer structure consisting of a first mixture and a second mixture for the channel 1316 material; wherein the first mixture is a mixture of polyethylene dioxythiophene and bromothymol blue, and the second mixture is a mixture of polyethylene dioxythiophene and polystyrene sulfonate. Another preferred combination may be a polybenzimidazole-benzophenanthridine BBL-based gate 1315 material and a single-layer mixture of polyethylene dioxythiophene and polystyrene sulfonate, PEDOT:PSS. Yet another preferred combination may be a polybenzimidazole-benzophenanthridine BBL-based gate 1315 material and a multilayer structure consisting of a first mixture and a second mixture for the channel 1316 material; wherein the first mixture is a mixture of polyethylene dioxythiophene and bromothymol blue, and the second mixture is a mixture of polyethylene dioxythiophene and polystyrene sulfonate.

[0065] Using one of the aforementioned preferred combinations, the gate 1315 material of the OECT microelectrode 1311 is made of platinum, and the channel 1316 material is a multilayer material, with a lower layer of PEDOT:PSS and an upper layer of PEDOT:BTB. The PEDOT:PSS material for the channel 1316 is produced using a spin-coating process, and the PEDOT:BTB material for the channel 1316 is produced using an electrodeposition process. The specific operating steps are as follows: First, pipette 3 to 5 μL of the prepared PEDOT:PSS mixture and add it dropwise to channel 1316. Then, spin coat the mixture on a spin coater for 60 to 90 seconds at a speed of 2000 to 4000 rpm. After spin coating, place the mixture in a drying oven and dry at 130°C to 150°C for 10 to 15 minutes to complete the preparation of the PEDOT:PSS layer. Next, the polymer solution for PEDOT:BTB electrodeposition consists of 10 mM 3,4-ethylenedioxythiophene (EDOT), 1 mM BTB, 1 mM phosphate-buffered saline (PBS), and 0.1 M potassium nitrate aqueous solution. After stirring the polymer solution continuously for 15 to 20 minutes, purge with nitrogen for 5 to 10 minutes. Then, PEDOT:BTB electrodeposition was performed in a three-electrode cell using cyclic voltammetry, with a saturated calomel electrode as the reference electrode, a platinum wire as the counter electrode, and a semiconductor channel 1316 as the working electrode. After immersing the working electrode in the polymer solution, the electrode was voltammetrically deposited at 0.1 V / s. -1 A scanning rate of 0 to 1 V relative to a saturated calomel electrode was applied to the working electrode for 15 to 20 cycles. After electrodeposition, the electrodeposit was rinsed with deionized water and dried at room temperature, thus completing the preparation of the PEDOT:BTB layer.

[0066] Therefore, the platinum and polybenzimidazole-benzophenanthridine used in the embodiments of the present invention are proven high-performance gate 1315 materials, which can ensure high sensitivity and low detection limit. At the same time, the use of multilayer channel 1316 material, especially the introduction of bromothymol blue, creates a dual response mechanism to hydrogen peroxide and its reaction byproducts, resulting in a synergistic amplification effect, enabling the detection limit to reach the femtogram per milliliter level. Figure 4 This is a schematic diagram of a scanning electron microscope image of a single-layer PEDOT:PSS provided in an embodiment of the present invention. Figure 5 This is a schematic scanning electron microscope image of a stacked PEDOT:PSS and PEDOT:BTB provided in an embodiment of the present invention.

[0067] In some embodiments, combined with Figures 1 to 3 Multiple units 13 are arranged into a unit array 14; the same row of immune reaction units 132 are coated with the same capture agent, and the corresponding row detection circuit is used to collect the electrical signals corresponding to different concentrations of the same target protein; the same column of immune reaction units 132 are coated with different capture agents, and the corresponding column detection circuit is used to collect the electrical signals corresponding to the same concentration of different target proteins.

[0068] Specifically, the cell array 14 refers to a matrix formed by arranging multiple cells 13 according to row and column rules, i.e., a single-multi-index cell array 14. In the cell array 14, immunoreaction cells 132 coated with the same capture agent, such as all cells coated with carcinoembryonic antigen (CEA) antibodies, are arranged in the same row. When a series of standard solutions of the same target protein, i.e., CEA, at different concentrations are added to each cell 13 in that row, each cell 13 will generate an electrical signal corresponding to that concentration. By plotting the electrical signal-to-concentration curve of all cells 13 in that row, the quantitative detection calibration of that protein can be completed. Simultaneously, immunoreaction cells 132 coated with different capture agents are arranged in the same column; for example, the first column is coated with CEA antibodies, and the second column is coated with prostate-specific antigen (PSA) antibodies. When a mixed sample containing multiple target proteins is added, different cells 13 in the same column will simultaneously respond to their respective target proteins, thereby completing the qualitative or quantitative analysis of multiple proteins in a single detection.

[0069] For example, each row of ELISA immunoreaction units 132, such as those corresponding to units 1A to 1H, can be configured with the same coating antibody 1322. Each column of ELISA immunoreaction units 132, such as those corresponding to units A1 to A5, can have different coating antibodies 1322 or antigens, such as carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), prostate-specific antigen (PSA), epididymal protein (HE4), and sialic acid (SA). Each row of detection units 131, such as those corresponding to units 1A to 1H, collects electrical signals representing different concentrations of the same antigen 1323. Each column of detection units 131, such as those corresponding to units A1 to A5, collects electrical signals representing different types of antigens 1323, such as carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), prostate-specific antigen (PSA), epididymal protein (HE4), and sialic acid (SA) at the same concentration.

[0070] Therefore, this embodiment of the invention, through array design, enables simultaneous single-index quantitative calibration and multi-index synchronous detection on a single carrier board, greatly improving detection efficiency and information volume. Simultaneously, the clear and rational layout facilitates signal addressing and data parsing by the hardware circuit system 12, simplifying the software algorithm of the host computer.

[0071] Figure 6 This is a schematic diagram of a hardware circuit system provided in an embodiment of the present invention. (In conjunction with...) Figures 1 to 6 The hardware circuit system 12 includes a wireless communication module 122, a microcontroller unit 123, a digital-to-analog converter (DAC) module 124, and an analog-to-digital converter (ADC) module 125. The DAC module 124 provides a working voltage signal to the organic electrochemical transistor, and the ADC module 125 acquires the electrical signals output by the organic electrochemical transistor. The microcontroller unit 123 is connected to the wireless communication module 122 and processes the electrical signals, transmitting the processed signals to a host computer via the wireless communication module 122. It may also include a power supply module 121 to power all devices. For example, Figure 6 The digital-to-analog converter module 124 is shown to include DA1 and DA2, corresponding to the output gate voltage V. G and drain voltage V D The source 1313 can be grounded, for example. The analog-to-digital converter module 125 includes an AD converter, which acquires the source-drain current I via a current-to-voltage converter. DS .

[0072] Specifically, the wireless communication module 122 is an electronic module that realizes wireless data transmission functions, such as a Bluetooth module or a WiFi module. The microcontroller unit 123 is a chip that integrates a processor, memory, and peripheral interfaces, and is the control core of the hardware circuit system 12. The digital-to-analog converter module 124 is a circuit that converts digital signals into analog voltage signals, and the analog-to-digital converter module 125 is a circuit that converts analog electrical signals into digital signals, realizing high-precision data sampling.

[0073] The microcontroller unit 123 issues digital commands, which, through the analog-to-digital converter module 124, generate precise and stable analog voltage signals, i.e., operating voltage signals, and apply them to the source 1313, drain 1314, and gate 1315 of the organic electrochemical transistor to provide the necessary operating bias. The analog current or voltage signals generated by the organic electrochemical transistor in response to hydrogen peroxide are acquired by the analog-to-digital converter module 125 and converted into digital signals. The microcontroller unit 123 performs preliminary processing on these digital signals, such as filtering and averaging, and then transmits the processed digital signals to a host computer via the wireless communication module 122. For example, the data processing, transmission, and storage modules are integrated into the microcontroller unit 123 equipped with a Bluetooth system, and the data is transmitted in real time to a host computer, such as a mobile phone, via Bluetooth for data visualization.

[0074] Therefore, this invention enables precise control of the operating state of organic electrochemical transistors and high-precision signal acquisition, ensuring the accuracy and repeatability of detection results. Wireless transmission eliminates the constraints of cables, enhancing the portability and ease of use of the instrument, and facilitating remote data viewing and sharing.

[0075] Figure 7 This is a three-dimensional exploded structure diagram of a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology provided in an embodiment of the present invention. Figure 8 This is a schematic diagram of the electrical connection between the hardware circuit system and the OECT microelectrode of the detection unit. (Combined with...) Figures 1 to 8 In the OECT-ELISA immunoassay system, a hardware circuit system 12 is configured below each unit 13; the unit array 14 formed by multiple units 13 on the integrated carrier board 1 is provided with inter-row apertures 15; the digital-to-analog conversion module 124 includes three output ports, and the conductive leads connected to the three output ports are led out through the inter-row apertures 15 and respectively connected to the source 1313, drain 1314 and gate 1315 of the organic electrochemical transistor; the three output ports are located in the vertical projection area of ​​the inter-row apertures 15.

[0076] Specifically, the inter-row aperture 15 is a gap or channel located between rows of cell 13 on the integrated carrier board 1, used for laying conductive leads; it can also be replaced by an inter-column aperture. The output port is the physical interface or pad on the analog-to-digital converter module 124 used to output analog voltage signals, and the conductive leads are metal wires, such as gold wires or copper wires, used to connect the circuit and the electrodes.

[0077] Each unit 13 is independently configured with a hardware circuit system 12, enabling parallel signal acquisition and processing and improving detection speed. The inter-row aperture 15 provides a physical channel for the electrical connection from the lower hardware circuit system 12 to the upper detection unit 131. The three output ports of the digital-to-analog converter module 124 are precisely bonded to the source 1313, drain 1314, and gate 1315 of the organic electrochemical transistor microelectrode 1311 via very fine conductive leads through the inter-row aperture 15, completing the electrical connection. For example, all three output ports can be configured to use ultra-fine gold wires led out from the inter-row aperture 15 with a lead diameter adapted to the wire diameter, and connected to the source 1313, drain 1314, and gate 1315 of the OECT microelectrode 1311, respectively, completing the electrical connection and providing a stable operating voltage. Alternatively, an organic electrochemical transistor chip with microelectrodes 1311 can be attached to the lower half of unit 13, and then a programmable automatic wire bonding machine can be used to connect the pins of the organic electrochemical transistor chip to the pins of the three output ports of the digital-to-analog converter module 124 in the hardware circuit system 12.

[0078] Therefore, this embodiment of the invention adopts a distributed design where unit 13 corresponds one-to-one with the hardware circuit, avoiding signal crosstalk and delay caused by multiplexing, and ensuring the independence and signal quality of each detection channel. The structural design of the inter-row aperture 15 makes high-density electrical connections possible, which is a key structure for realizing the miniaturization and integration of the instrument.

[0079] In some embodiments, combined with Figures 1 to 8 The wireless communication module 122 is a Bluetooth module. All wireless communication modules 122 have the same model and achieve signal synchronization through a timing calibration algorithm.

[0080] Specifically, the timing calibration algorithm is a software program running on the microcontroller unit 123, used to coordinate the communication timing between multiple modules, ensuring that the data they send is synchronized in time and avoiding conflicts. Each unit 13's hardware circuit system 12 independently sends data to the host computer via, for example, a Bluetooth module. To ensure that the host computer can correctly receive and distinguish data from a large number of units 13, each microcontroller unit 123 runs a timing calibration algorithm. This algorithm allocates a specific communication time slice to each Bluetooth module, or uses a master-slave negotiation mechanism to ensure that all modules send data sequentially in a predetermined order, thereby achieving synchronized and orderly transmission of data packets.

[0081] Therefore, in this embodiment of the invention, the wireless communication module 122 of each hardware circuit system 12 is of the same model, and signal delay differences are eliminated through a preset timing calibration algorithm, enabling the display of data from multiple modules on a host computer, such as a mobile phone. Simultaneously, timing calibration solves the data collision problem of multiple transmitting modules under limited wireless channels, ensuring the stability and reliability of large-scale array data transmission.

[0082] In some embodiments, combined with Figures 1 to 8 The integrated carrier plate 1 has a length and width range of 15 mm to 25 cm and a thickness range of 3 mm to 5 cm; the unit 13 has a length, width and depth range of 1 mm to 2 cm; the detection unit 131 has a length and width range of 0.5 mm to 1 cm and the immune reaction unit 132 has a length and width range of 0.5 mm to 1 cm.

[0083] Specifically, the aforementioned size range can be determined by comprehensively optimizing the performance of organic electrochemical transistors, the liquid volume required for the immune reaction, the hardware circuit integration density, and ergonomics. Smaller unit sizes allow for the integration of more detection units 131 on a limited substrate area, achieving high-throughput detection, while the overall size of the integrated substrate 1 is limited to a range suitable for handheld or portable use. For example, as... Figure 7 As shown, the width of the three electrodes can be set to be 1 mm to 2 mm, the length of the channel 1316 to be 100 μm to 200 μm, the length of the hardware circuit system 12 to be 1 cm to 2 cm, the width to be 1 cm to 2 cm, and the thickness to be 1 cm to 2 cm; the length of the inter-row aperture 15 to be 14 cm to 24 cm, the width to be 0.5 cm to 1 cm, and the depth to be 1 cm to 2 cm.

[0084] Therefore, the specific size limitation adopted in the embodiments of the present invention ensures the miniaturization and portability of the entire instrument, which is much smaller than that of traditional ELISA readers or chemiluminescence analyzers. This size design achieves an optimal balance between portability and functional integrity, ensuring a sufficient number of detection units 131 while making the instrument compact and lightweight overall.

[0085] In summary, on the one hand, this invention employs OECT coupled with ELISA technology to develop a highly sensitive multi-index protein detection instrument based on OECT and ELISA combined technology. This system integrates the immunoreaction unit 132 and the detection unit 131 onto a single-index multi-index OECT / ELISA plate, effectively solving the drawbacks of traditional immunoassay techniques such as ELISA, CLIA, and ECL where the immunoreaction unit 132 and detection unit 131 are separate. Compared to traditional immunoassay techniques, this system has a wider detection range, a lower detection limit, and multi-index immunoassay measurement capabilities. On the other hand, compared to the detection equipment configured for traditional immunoassay techniques such as ELISA, CLIA, and ECL, the system developed in this invention has a smaller footprint, a simpler operation method, and adds portability and wireless communication capabilities. Finally, the channel 1316 material of the OECT microelectrode 1311 used in this invention can be an organic semiconductor stack of PEDOT:BTB plus PEDOT:PSS, and the gate 1315 material can be platinum or polybenzimidazole-benzophenanthridine. The synergistic effect between the Nernst potential generated by the platinum or polybenzimidazole-benzophenanthridine gate electrode catalyzing hydrogen peroxide in the ELISA reaction termination solution and the Nernst potential generated by the interaction of BTB molecules with hydrogen ions, a byproduct of hydrogen peroxide catalysis, greatly reduces the detection limit of the target antigen. Compared with traditional immunoassay techniques, the detection limit is reduced by two to four orders of magnitude.

[0086] It should be noted that the embodiments of the present invention are not limited to the CEA index, but are applicable to all protein biomarkers; they are not limited to use in conjunction with ELISA technology, but also include related immunoassay technologies such as CLIA and ECL; they are not limited to double antibody sandwich structures, but also cover immune competitive structures and other related extended structures; they are not limited to the size of single or multiple index plates of OECT plus ELISA, but also cover designs of any shape, size, and structure.

[0087] The following three specific embodiments illustrate in detail the solutions provided by the embodiments of the present invention: Example 1 Combination Figure 1 and Figure 8 The high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology has the following positional relationship from bottom to top: substrate 11, hardware circuit system 12, and unit 13. Unit 13 consists of two parts: detection unit 131 and ELISA immunoreaction unit 132. The detection unit 131 can be located in the upper half of unit 13 and the ELISA immunoreaction unit 132 can be located in the lower half of unit 13, or the detection unit 131 can be located in the lower half of unit 13 and the ELISA immunoreaction unit 132 can be located in the upper half of unit 13. Unit 13 constitutes a single-multi-index unit array 14.

[0088] The substrate 1312 of the detection unit 131 is made of titanium or gold. The OECT microelectrode 1311 includes a source 1313, a drain 1314, and a gate 1315. The channel 1316 is located in the region between the source 1313 and the drain 1314. The gate 1315 is made of platinum. The reaction substrate 1321 of the ELISA immunoreaction unit 132 is made of polystyrene. A large number of antibodies 1322 or antigens 1323 are coated on the reaction substrate 1321 of the ELISA immunoreaction unit 132 due to hydrophobic interactions and van der Waals forces.

[0089] The single / multi-index unit array 14 is composed of units 13, wherein each unit 13 contains at least one coated antibody 1322 or antigen 1323. In the single / multi-index unit array 14, each row of ELISA immunoreaction units 132, such as 1A to 1H, has the same coated antibody 1322, while each column of ELISA immunoreaction units 132, such as A1 to A5, has different coated antibodies 1322, such as embryonic antigen (CEA), human chorionic gonadotropin (HCG), prostate-specific antigen (PSA), epididymal protein (HE4), and sialic acid (SA), respectively. Each row of detection units 131, such as 1A to 1H, collects electrical signals representing the electrical signals of the same antigen 1323 at different concentrations; each column of detection units 131, such as A1 to A5, collects electrical signals representing the electrical signals of different types of antigens 1323, such as carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), prostate-specific antigen (PSA), epididymal protein (HE4), and sialic acid (SA) at the same concentration.

[0090] The specific steps for manufacturing a high-sensitivity, multi-index protein detection instrument based on OECT and ELISA combined technology are as follows: Figure 9 This is a schematic cross-sectional view of the sequentially formed steps in the OECT microelectrode fabrication process of a detection unit provided in an embodiment of the present invention. Figure 10 This is a schematic cross-sectional view of the sequentially formed steps in the preparation process of the channel laminate material in a detection unit provided by an embodiment of the present invention. For example... Figure 9 As shown, the preparation and detection unit includes: The substrate was cleaned, and a 500 nm thick SiO2 insulating layer 22 was thermally grown on the Si substrate 21. The substrate was then ultrasonically treated with acetone, alcohol and deionized water for 5 min each, and then dried with nitrogen.

[0091] Photoresist was applied by spin coating a layer of negative photoresist onto an alumina wafer using a spin coater. The process was repeated three times, with the following parameters: First spin coating: 500 rpm, 25 s, 100 rpm acceleration; Second spin coating: 3000 rpm, 54 s, 200 rpm acceleration; Third spin coating: 4000 rpm, 6 s, 1000 rpm acceleration.

[0092] Photoresist annealing: Fix the silicon oxide wafer with photoresist on the back of the mask using high-temperature tape, and place it on a heating platform. Heat it continuously at a constant temperature of 110°C for 90 seconds.

[0093] Exposure: The photomask with the silicon oxide wafer fixed is placed flat in the photolithography area of ​​the photolithography machine, and photolithography patterning is performed under the photolithography conditions of photovoltage 0.65V and time 6.5s.

[0094] Development: The silicon oxide wafer with completed photolithographic patterning is placed in the developing solution for 60 seconds, then removed and cleaned with deionized water, and then dried with nitrogen.

[0095] Magnetron sputtering: A 20 nm Ti layer 23 and a 100 nm Au layer 24 were sequentially sputtered onto the developed silicon oxide wafer using a magnetron sputtering machine at a power of 50 W for 600 s. The Ti layer was used to increase the adhesion of the Au electrode and the SiO2 substrate. Residual photoresist was then removed with acetone. Finally, the fabricated microelectrode chip was rinsed with deionized water and dried with nitrogen. The microelectrode chip includes a source structure S, a drain structure D, and a gate structure G.

[0096] Gate electrode fabrication: Parylene C powder was poured into the deposition machine's storage hopper. Two Parylene C films were deposited on the microelectrode chip using a molecular layer deposition method with controllable and more uniform film thickness. During the deposition of the first Parylene C film 251, a silane coupling agent was added to enhance the adhesion of the first layer and prevent peeling. A cleaning agent diluted ten times was applied between the two Parylene C films to facilitate the peeling of the second Parylene C film 252. Then, a reactive ion etching machine was used to simultaneously pattern the two Parylene C layers to a set area, etching the channel region and the gate region. Using a magnetron sputtering process, before sputtering the platinum layer in the gate region, the channel region was shielded with PET (polyethylene terephthalate) or polyethylene terephthalate tape 26. The sputtering parameters were: sputtering power 45W and sputtering time 30 minutes, finally fabricating the platinum gate electrode, i.e., gate 1315.

[0097] like Figure 10As shown, an organic semiconductor channel was prepared. After removing the PET tape 26 from the channel region and treating the channel region with oxygen plasma, the gate electrode region was shielded with PET tape 26. 3 μL of PEDOT:PSS solution was spin-coated onto the electrode chip at a rotation speed of 4300 rpm, an acceleration of 200 rpm, and a spin-coating time of 60 s. The spin-coated chip was then dried in a 130°C oven for 15 min to form a PEDOT:PSS film 27, and then the PET tape 26 was removed. The polymer solution for PEDOT:BTB electrodeposition was continuously stirred for 15 to 20 minutes, followed by purging with nitrogen for 5 to 10 minutes. Then, PEDOT:BTB electrodeposition was performed in a three-electrode cell using cyclic voltammetry, with a saturated calomel electrode as the reference electrode, a platinum wire as the counter electrode, and the semiconductor channel as the working electrode. The working electrode was immersed in the polymer solution for PEDOT:BTB electrodeposition at 0.1 V∙s⁻¹. -1 A potential of 0 to 1 V relative to a saturated calomel electrode was applied to the working electrode at a scan rate. After electrodeposition, the electrodeposit was rinsed with deionized water and dried at room temperature, thus completing the preparation of the PEDOT:BTB film 28. The second layer, Parylene C film 252, i.e., the upper Parylene C film, was carefully peeled off with tweezers and placed in a drying oven at 130°C for 10 min.

[0098] The preparation process of ELISA immunoreaction unit 132 is as follows: The silicon wafer is ultrasonically cleaned with deionized water and then air-dried. The surface is then treated with a gas lamp for further cleaning. Polystyrene is then dissolved in toluene to prepare a 0.1 wt% polystyrene solution. This solution is dropped onto the silicon wafer surface and allowed to self-assemble at room temperature for approximately 2 hours. Finally, the wafer is cleaned with deionized water and air-dried. This process forms a porous, rough polystyrene ultrathin film on the silicon wafer surface, increasing its specific surface area. Plasma treatment further optimizes the hydrophobic-hydrophilic balance.

[0099] When 100 μL of a 100 ng / mL CEA antibody (carcinoembryonic antigen antibody) or SA antigen (sialic acid antigen) solution is contacted with a polystyrene ultrafilm, the hydrophobic groups of the antibody-antigen solution spontaneously adsorb onto the film surface, forming a hydrophobic anchor. Van der Waals forces further stabilize this binding, causing biomolecules to be directionally immobilized on the surface. The solution is then discarded, and 200 to 300 μL of washing buffer (equal to the amount of the solution) is added. The mixture is allowed to stand for 1 to 2 minutes, the washing buffer is discarded, and the washing process is repeated 3 to 5 times.

[0100] Add 100 μL of 0.5% to 1% BSA solution (bovine serum albumin solution) to block unbound sites and reduce nonspecific adsorption. Discard the solution, add 200 to 300 μL of washing buffer, let stand for 1 to 2 minutes, discard the washing buffer, and repeat the washing 3 to 5 times.

[0101] The prepared carrier plate coated with antibody 1322 or antigen 1323 is attached to the lower half of unit 13 and closely attached to detection unit 131.

[0102] The connection process of the three output ports of the digital-to-analog converter module 124 is as follows: Figure 8 As shown, a programmable automatic wire bonding machine is used. After setting the height of the three output port pins of the digital-to-analog converter module 124 of the hardware circuit system 12 from the chip pins, the spacing between each corresponding pin, and the spacing between each unit 13, the wires are automatically bonded through the interrow holes 15.

[0103] For the overall packaging design: The prepared detection unit 131 and ELISA immunoreaction unit 132 are pasted together, and then pasted onto unit 13.

[0104] Units 13 are pasted together in an array to form a single-multiple indicator unit array 14.

[0105] The hardware circuit system 12 is installed below each unit 13, and the digital-to-analog conversion module 124 of the hardware circuit system 12 is aligned with the interrow hole 15 with three output ports and located directly below the interrow hole 15.

[0106] The hardware circuit system 12 is installed under each unit 13 of the single-multi-index unit array 14 and then mounted on the OECT-ELISA immunoassay system integrated with the OECT multi-index ELISA carrier plate 1 to complete the entire encapsulation.

[0107] The operating steps for a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology are as follows: Add 50-100 μL of target antigen 1323 solution to unit 13.

[0108] For the double-antibody sandwich structure, after sealing the plate with the sealing film, incubate at 30 to 40°C for 50 to 70 minutes. Remove the ELISA plate, discard the liquid, and without washing, add 50 to 100 μL of biotin-1331-labeled secondary antibody 1332 solution. After sealing the plate with the sealing film, incubate at 30 to 40°C for 40 to 60 minutes. For the immune competition structure, immediately add 50 to 100 μL of biotin-1331-labeled secondary antibody 1332 solution. After sealing the plate with the sealing film, incubate at 30 to 40°C for 40 to 60 minutes.

[0109] Discard the solution, add 200 to 300 μL of washing buffer, let stand for 1 to 2 minutes, discard the washing buffer, and repeat the washing process 3 to 5 times.

[0110] Add 50 to 100 μL of glucosidase-labeled streptavidin 1333, cover with a sealing film, and incubate at 30 to 40°C for 20 to 30 minutes.

[0111] Discard the solution, add 200 to 300 μL of washing buffer, let stand for 1 to 2 minutes, discard the washing buffer, and repeat the washing process 3 to 5 times.

[0112] Add 50 to 100 μL of a 10 to 56 μM high-concentration glucose solution, cover with a sealing film, and incubate at 30 to 40 °C for 5 to 20 min. The final unit 13 contains the ELISA reaction termination solution and serves as the electrolyte for the detection unit 131.

[0113] The OECT microelectrode 1311 in the detection unit 131 is provided with a gate voltage of -0.6V for the gate 1315, a source voltage of -0.6V for the source 1313, and a drain voltage of -0.6V for the drain 1314 through the hardware circuit system 12.

[0114] After the test is completed, the hardware circuit system 12 outputs an electrical signal and collects the electrical signal to be transmitted to the host computer indefinitely.

[0115] Figure 11 This is a flowchart illustrating the specific operating steps of a high-sensitivity multi-index protein detection instrument based on OECT and ELISA combined technology, as provided in this embodiment of the invention. Figure 11 As shown, the operation steps include: Taking the CEA indicator as an example, in a single-multiple indicator unit array 14, such as... Figure 1 100 μL of CEA antigen 1323 solution with concentrations of 0.125 fg / mL, 1.25 fg / mL, 12.5 fg / mL, 125 fg / mL, 1.25 pg / mL, 12.5 pg / mL, 125 fg / mL, and 1250 pg / mL, respectively, was added to units 131A to 1I, respectively. A blank group was set up, which was CEA antigen 1323 solution with a concentration of 0 pg / mL. Figure 12 This is a top view schematic diagram of a CEA single-index protein detection instrument based on OECT and ELISA combined technology provided in an embodiment of the present invention. For example, it can be used in... Figure 12 In the single-multi-index unit array 14 shown in unit 13, three sets of parallel experiments are conducted by selecting well positions 1A to 1I, 2A to 2I, and 3A to 3I. The detection unit 13 is not limited to rows A to I and columns 1 to 5. The number of units 13 can be increased according to the requirements.

[0116] After sealing the plate with the membrane, incubate at 37°C for 60 min. Remove the ELISA plate, discard the liquid, and without washing, add 100 μL of biotin-1331-labeled CEA antibody 1332 solution directly to the entire structure. After sealing the plate with the membrane, incubate at 37°C for 60 min.

[0117] Discard the solution, add 300 μL of washing buffer, let stand for 1 min, discard the washing buffer, and repeat the washing process 3 times.

[0118] Add 100 μL of glucosidase-labeled streptavidin 1333, cover with a sealing film, and incubate at 37°C for 30 min.

[0119] Discard the solution, add 300 μL of washing buffer, let stand for 1 min, discard the washing buffer, and repeat the washing process 5 times.

[0120] Add 100 μL of 10 μM high-concentration glucose solution, cover with sealing film, and incubate at 37°C for 10 min. The final unit 13 contains ELISA reaction termination solution and serves as the electrolyte for detection unit 131.

[0121] The OECT microelectrode 1311 in the detection unit 131 is provided with a gate voltage of 0.6 V, a source voltage of 1313 grounded, and a drain voltage of 0.6 V through the hardware circuit system 12.

[0122] After the test is completed, the hardware circuit system 12 outputs an electrical signal and collects the electrical signal to send it to the host computer indefinitely. The source and leakage current values ​​that change over time are read and an Excel data table is generated.

[0123] Import the Excel data table into the corresponding software, obtain the stable sensing current at different concentrations, and plot the sensitivity curve of normalized current versus concentration. The formula for calculating the normalized current is: ΔI / I = (Istandard - Iblank) / Iblank. Figure 13 This is a schematic diagram of the relationship between CEA antigen concentration and normalized current sensitivity provided in an embodiment of the present invention. Figure 13 The horizontal axis represents the CEA antigen concentration C. CEA The logarithm is expressed in pg / mL, and the ordinate represents ΔI / I0, where I0 is the blank group. Figure 13 The lowest point of the fitted curve was found, and the detection limit of CEA antigen was 0.125 fg / mL.

[0124] Example 2 Based on Example 1, by selecting cells 135A to 5H of the single-multi-index cell array 14, simultaneous detection of CEA and SA samples can be achieved. Specific experimental steps are as follows: Figure 11 As shown, it includes: In single-multi-indicator unit array 14, such as Figure 1 100 μL of CEA antigen 1323 solution with concentrations of 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.2 pg / mL, 78.1 pg / mL, 39.06 pg / mL, and 19.53 pg / mL, respectively, were added to units 135A to 5H, respectively. A blank group, namely SA antigen 1323 solution with a concentration of 0 pg / mL, was also set up. Figure 14This is a top view schematic diagram of a single-index protein detection instrument for SA based on OECT and ELISA combined technology provided in an embodiment of the present invention. For example, it can be used in... Figure 14 Three sets of parallel experiments were conducted on the well positions 131A to 1H, 2A to 2H, and 3A to 3H in the unit 13 of the single index unit array 14 shown. The detection unit 13 is not limited to rows A to H and columns 1 to 5. The number of units 13 can be increased as needed.

[0125] After sealing the plate with the membrane, incubate at 37°C for 60 min. Remove the ELISA plate, discard the liquid, and without washing, add 100 μL of biotin-1331-labeled CEA antibody 1332 solution directly to all units. Immediately add 50 to 100 μL of biotin-1331-labeled antibody SA1332 solution. After sealing the plate with the membrane, incubate at 37°C for 60 min.

[0126] Discard the solution, add 300 μL of washing buffer, let stand for 1 min, discard the washing buffer, and repeat the washing process 3 times.

[0127] Add 100 μL of glucosidase-labeled streptavidin 1333, cover with a sealing film, and incubate at 37°C for 30 min.

[0128] Discard the solution, add 300 μL of washing buffer, let stand for 1 min, discard the washing buffer, and repeat the washing process 5 times.

[0129] Add 100 μL of 10 μM high-concentration glucose solution, cover with sealing film, and incubate at 37°C for 10 min. The final unit 13 contains ELISA reaction termination solution and serves as the electrolyte for detection unit 131.

[0130] The OECT microelectrode 1311 in the detection unit 131 is provided with a gate voltage of -0.6V for the gate 1315, a source voltage of -0.6V for the source 1313, and a drain voltage of -0.6V for the drain 1314 through the hardware circuit system 12.

[0131] After the test is completed, the hardware circuit system 12 outputs an electrical signal and collects the electrical signal to send it to the host computer indefinitely. The source and leakage current values ​​that change over time are read and an Excel data table is generated.

[0132] Import the Excel data table into the corresponding software, obtain the stable sensing current at different concentrations, and plot the sensitivity curve of normalized current versus concentration. The formula for calculating the normalized current is: ΔI / I = (Istandard - Iblank) / Iblank. Figure 15 This is a schematic diagram of the relationship between SA antigen concentration and normalized current sensitivity provided in the embodiments of the present invention. Figure 15 The horizontal axis represents the SA antigen concentration C. SAThe logarithm is expressed in pg / mL, and the ordinate represents ΔI / I0, where I0 is the blank group. Figure 15 The lowest point of the fitted curve was found, and the detection limit of SA antigen was 0.0928 fg / mL.

[0133] Example 3 Based on Example 1, the standard antigen 1323 solution used for measurement was replaced with real serum samples from human lung cancer patients. Specific experimental procedures are as follows: Figure 11 As shown, it includes: In the single-index unit array 14, such as Figure 12 In units 131A to 1G, 100 μL of CEA antigen 1323 solution with concentrations of 12.5 fg / mL, 125 fg / mL, 1.25 pg / mL, 12.5 pg / mL, 125 pg / mL, and 1250 pg / mL, respectively, was added, along with a blank control group containing 0 pg / mL of CEA antigen 1323 solution. In units 131H to 1J, 2H to 2J, and 3H to 3J of the single-index unit array 14, serum samples from human lung cancer patients with concentrations of 0.48 pg / mL, 3.17 pg / mL, and 122.8 to 0.48 pg / mL, respectively, were added, and three parallel experiments were designed.

[0134] After covering with the sealing film, incubate at 37°C for 60 min. Remove the ELISA plate, discard the liquid, and without washing, add 100 μL of biotin-1331-labeled CEA antibody 1332 solution. Cover with the sealing film and incubate at 37°C for 60 min.

[0135] Discard the solution, add 300 μL of washing buffer, let stand for 1 min, discard the washing buffer, and repeat the washing process 3 times.

[0136] Add 100 μL of glucosidase-labeled streptavidin 1333, cover with a sealing film, and incubate at 37°C for 30 min.

[0137] Discard the solution, add 300 μL of washing buffer, let stand for 1 min, discard the washing buffer, and repeat the washing process 5 times.

[0138] Add 100 μL of 10 μM high-concentration glucose solution, cover with sealing film, and incubate at 37°C for 10 min. The final unit 13 contains ELISA reaction termination solution and serves as the electrolyte for detection unit 131.

[0139] The OECT microelectrode 1311 in the detection unit 131 is provided with a gate voltage of -0.6V for the gate 1315, a source voltage of -0.6V for the source 1313, and a drain voltage of -0.6V for the drain 1314 through the hardware circuit system 12.

[0140] After the test is completed, the hardware circuit system 12 outputs an electrical signal and collects the electrical signal to send it to the host computer indefinitely. The source and leakage current values ​​that change over time are read and an Excel data table is generated.

[0141] Import the Excel data table into the corresponding software, obtain stable sensing currents at different concentrations, and plot the normalized current versus concentration sensitivity curve as a calibration curve. Then, test real serum samples from human lung cancer patients. The formula for calculating the normalized current is: ΔI / I = (Istandard - Iblank) / Iblank. Figure 16 This is a schematic diagram of the recovery rate curve of serum samples from human lung cancer patients provided in an embodiment of the present invention. Figure 16 The horizontal axis represents the CEA antigen concentration C. CEA The logarithm is expressed in pg / mL, and the ordinate represents ΔI / I0, where I0 is the blank group. Figure 15 The difference between the ordinate of the real sample a shown in the figure and the ordinate of the corresponding point of the fitted curve shown by the dashed line indicates that the recovery rate of real serum samples from human lung cancer patients is 97.5% to 101.7%.

[0142] In summary, this invention utilizes the mature immunoassay system of ELISA and the portability of OECT devices to develop a highly sensitive multi-index protein detection instrument based on the combination of OECT and ELISA, which has the following advantages: First, this detection instrument integrates a multi-index carrier plate with different coated antibodies or antigens, enabling the detection of multiple cancer biomarkers. The instrument employs a highly consistent immune reaction system: coated antibody / antigen-antigen-biotin-labeled antibody-glucosidase-labeled streptavidin. Indirect detection of the target sample is achieved by detecting hydrogen peroxide, a common intermediate product generated from the enzymatic reaction of glucose and glucosidase. For multi-index protein detection, there is no need to change protein probes and chromogenic substrates, thus avoiding signal crosstalk to some extent. The developed multi-index carrier plate has a detection unit array. This array can use the same coated antibody or antigen in the same row or column, and different coated antibodies or antigens in different rows or columns to achieve single or multiple index detection. The same row or column of the detection unit array can quantitatively detect a single cancer biomarker, while different rows or columns can qualitatively detect multiple cancer biomarkers.

[0143] Secondly, this detection instrument integrates OECT and ELISA technologies, achieving a combination of immune reaction and immunoassay. Its design integrates a detection unit with both OECT and ELISA functions, peripheral hardware circuitry, and a host computer, thus achieving system miniaturization. The difference between OECT-coupled ELISA technology and OECT-based immunosensing technology lies in the fact that the latter's immune modification and immune reaction occur at the OECT gate, often requiring significant resources to explore modification methods and characterize modification stability. This invention utilizes the mature immune reaction system of ELISA and the portability of OECT devices to develop a highly sensitive multi-index protein detection instrument based on OECT-ELISA combined technology. This sensing and testing system eliminates the need for immune modification at the OECT gate; it only requires integrating the mature ELISA immune reaction process and the OECT immunoassay process into a single unit, along with peripheral hardware circuitry and a host computer, thus achieving system miniaturization.

[0144] Finally, this detection instrument features a wide detection range and ultra-low detection limit for tumor markers. The OECT-ELISA combination leverages the biocompatibility and signal amplification capabilities of OECT, along with the strong affinity binding of the capture antibody-target antigen-biotin-labeled antibody sandwich structure in ELISA to enzyme-labeled streptavidin. The ratio of enzyme-labeled streptavidin to biotin-labeled antibody is 1:4. This constructed biosignal amplification architecture enables synergistic signal amplification, thereby achieving ultra-low detection limits for cancer markers.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology, characterized in that, The application relates to a protein concentration detection device. The device comprises an integrated carrier plate, which comprises a plurality of units, each of which is integrally provided with a detection unit and an immune reaction unit. The immune reaction unit is used for immune reaction based on the enzyme-linked immunosorbent assay principle and generates small molecules related to the concentration of target proteins; at least two immune reaction units are coated with different capture agents, and the capture agents are antibodies, antigens or nucleic acids. The detection unit comprises an organic electrochemical transistor, which is used for generating an electric signal that changes in response to the concentration of the small molecules. Hardware circuit system, which is electrically connected with the organic electrochemical transistor, is used for providing a working voltage signal for the organic electrochemical transistor and collecting the electric signal. A host computer is in communication connection with the hardware circuit system, which is used for receiving the electric signal and displaying a detection result related to the concentration of the target proteins based on the electric signal.

2. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 1, characterized in that, The detection unit comprises a substrate and an organic electrochemical transistor microelectrode arranged on the substrate. The immune reaction unit comprises a reaction substrate and a signal amplification unit, the capture agent is coated on the reaction substrate, and the signal amplification unit comprises a biotin-labeled secondary antibody and an enzyme-labeled streptavidin for enzyme reaction.

3. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 2, characterized in that, The organic electrochemical transistor microelectrode comprises a source electrode, a drain electrode and a gate electrode, a channel is arranged between the source electrode and the drain electrode, the gate electrode is made of a material sensitive to hydrogen peroxide, and the channel is made of an organic semiconductor material.

4. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 3, characterized in that, The material sensitive to hydrogen peroxide comprises at least one of a noble metal, a noble metal alloy, a transition metal, a transition metal oxide, a transition metal hydroxide, a carbon-based material, a composite material, a composite nano-enzyme material or an organic polymer material, and the organic semiconductor material comprises a polyethylene dioxythiophene material.

5. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 4, characterized in that, The material sensitive to hydrogen peroxide is platinum or polybenzimidazobenzophenanthroline, the organic semiconductor material is a mixture of a single-layer polyethylene dioxythiophene and polystyrene sulfonate, or a laminated structure composed of a first mixture and a second mixture; the first mixture is a mixture of polyethylene dioxythiophene and bromothymol blue, and the second mixture is a mixture of polyethylene dioxythiophene and polystyrene sulfonate.

6. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 1, characterized in that, The plurality of units are arranged into a unit array. The same row of immune reaction units is coated with the same capture agent, and the corresponding detection circuit in the same row is used for collecting the corresponding electric signal under different concentrations of the same target protein. The same column of immune reaction units is coated with different capture agents, and the corresponding detection circuit in the same column is used for collecting the corresponding electric signal under the same concentration of different target proteins.

7. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to any one of claims 1-6, characterized in that, The hardware circuit system comprises a wireless communication module, a micro control unit, a digital-to-analog conversion module and an analog-to-digital conversion module. The digital-to-analog conversion module is used for providing the working voltage signal for the organic electrochemical transistor, and the analog-to-digital conversion module is used for collecting the electric signal output by the organic electrochemical transistor. The micro control unit is connected with the wireless communication module and is used for processing the electric signal and transmitting the processed signal to the host computer through the wireless communication module.

8. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 7, characterized in that, Each of the units is configured with the hardware circuit system below; The unit array formed by the arrangement of the plurality of units on the integrated carrier plate is provided with an inter-row aperture, the digital-to-analog conversion module includes three output ports, the conductive leads connected to the three output ports are led out through the inter-row aperture, and are respectively connected to the source, drain and gate of the organic electrochemical transistor.

9. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to claim 7, characterized in that, The wireless communication module is a Bluetooth module, the models of the wireless communication modules are the same, and signal synchronization is achieved through a timing calibration algorithm.

10. The high-sensitivity multi-index protein detection instrument based on the combination of OECT and ELISA technology according to any one of claims 1-6, characterized in that, The length range and the width range of the integrated carrier plate are both 15 mm to 25 cm, and the thickness range is 3 mm to 5 cm; The length range, the width range and the depth range of the unit are all 1 mm to 2 cm; The length range and the width range of the detection unit, and the length range and the width range of the immune reaction unit are all 0.5 mm to 1 cm.

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