Micro-fluidic chip sensor, and device and method for detecting microorganisms or extracellular vesicles

Abstract

The invention provides a micro-fluidic chip sensor and a microorganism or extracellular vesicle detection device and method, and belongs to the field of biological detection.The sensor comprises a cover plate, a channel layer, an electrode layer and a substrate; a liquid inlet and a sample inlet are formed in a certain position, corresponding to a micro-channel structure, of the cover plate; a discharge port is formed in a position corresponding to the tail end of the micro-channel structure; one or more electrodes for enrichment and sensor detection are arranged on one surface, facing the channel layer, of the electrode layer; the enrichment unit is positioned below the cover plate and on the electrode layer and is positioned in the channel of the channel layer; or the device further comprises a plurality of enrichment units which are all located below the cover plate and on the electrode layer and located in the channel of the channel layer; after the cover plate, the channel layer, the electrode layer, the substrate and other units are bonded and packaged, the detection electrode and the enrichment unit are both located in the micro-channel structure. According to the invention, high-sensitivity and high-selectivity detection of microorganisms and extracellular vesicles can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a microfluidic chip sensor, a device and method for enriching, separating and detecting microorganisms or extracellular vesicles by a sensor. Background Art

[0002] Microbial detection is of great significance in many fields. (1) By detecting the types and quantities of microorganisms in aqueous solutions, the safety, pollution degree and self-purification ability of water quality can be evaluated; (2) Microorganisms in aerosols are an important part of air pollutants. By detecting the types and quantities of microorganisms in aerosols, the air quality can be evaluated. Microorganisms in aerosols may enter the human body through the respiratory tract and cause respiratory diseases. In recent decades, respiratory infections have become the deadliest infectious diseases in the world. Some fatal infections, including coronavirus disease, severe acute respiratory syndrome (SARS) and influenza A virus (IAV), are transmitted through bioaerosols, posing a huge threat to public health, resulting in huge economic losses and casualties. Therefore, timely and on-line detection of bioaerosols is crucial for the early prediction and real-time warning of airborne infectious diseases, and thus for timely controlling the outbreak of airborne infectious diseases; (3) By detecting the types and quantities of microorganisms in food, it can be determined whether the food is contaminated, whether there are pathogenic bacteria or spoilage bacteria. This helps to ensure food safety and prevent food poisoning and the occurrence of infectious diseases. In addition, during the production, processing, storage and sales of food, microbial detection can monitor the hygiene conditions of each link to ensure that the food meets the hygiene standards; (4) By detecting the microorganisms in the environment in crowded places such as hospitals and schools, potential sources of infection can be discovered in a timely manner, providing support for the prevention and control of diseases and helping to prevent cross-infection and the occurrence of infectious diseases. At the same time, the results of microbial detection can provide a scientific basis for public health intervention. For example, disinfection measures can be formulated according to the detection results, and environmental hygiene management can be strengthened to reduce the risk of disease transmission; (5) The distribution and quantity changes of microorganisms in environments such as soil and water bodies can reflect the health status of the ecosystem and provide guidance for environmental protection and ecological restoration. And microorganisms are sensitive and indicative to environmental pollution. By detecting the changes in the types and quantities of microorganisms in the environment, the pollution sources and pollution degrees can be discovered in a timely manner, providing a scientific basis for pollution control. Aerosols usually refer to bioaerosols with a diameter within 100 um, mainly referring to bacteria, viruses, fungi and some microbial fragments suspended in the air.

[0003] The classic method for microbial detection is the cultivation method. The cultivation method is simple, accurate, and effective, but it also has certain limitations, including: (1) time-consuming (more than 24 hours); (2) not applicable to non-cultivable pathogens; (3) unable to detect in real time and not suitable for on-site monitoring. Molecular biology methods based on polymerase chain reaction (PCR) and other techniques have developed rapidly in recent years. Compared with the microbial cultivation method, some new detection techniques, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, are used to identify and quantify microorganisms in samples. Molecular biology detection methods have advantages such as high sensitivity and good specificity. However, they also have some limitations, including complex nucleic acid extraction and amplification techniques that require relatively high requirements for personnel, complex procedures, easy to have false positives, a detection time of 3 - 5 hours, and being not conducive to automation. Therefore, they are not suitable for point-of-care testing [Li M, Wang L, Qi W, et al. Challenges and Perspectives for Biosensing of Bioaerosol Containing Pathogenic Microorganisms[J]. Micromachines, 2021, 12(7): 798]. Moreover, in some cases, the concentration of microorganisms is very low. For example, in the detection of aerosol microorganisms, the concentration of pathogenic microorganisms in bioaerosols is usually very low and cannot reach the detection limit of microbial detection. Therefore, microbial enrichment and concentration are required before detection. For example, for microorganisms in aerosols, current classic enrichment methods include the natural sedimentation method and instrument sampling methods based on principles such as centrifugation, filtration, and impaction. Subsequently, biological analysis is performed on the enriched microbial samples. Currently, the collection, enrichment, and detection of most bioaerosols are still two independent parts. It is necessary to collect bioaerosols containing pathogenic microorganisms and then manually transfer them to a suitable medium for downstream detection. This not only leads to an increase in detection errors and risks of biosafety but also low efficiency and low automation. Therefore, the integration of microbial collection and detection is a good way to solve this problem. There is an urgent need for a continuous and automated bioaerosol monitoring system, including collection, detection, and early warning, to achieve automatic and continuous online monitoring.

[0004] Extracellular vesicles (EVs) are an important form of cell secretions, which are tiny vesicles wrapped by cell membranes or organelle membranes and contain specific substances, including exosomes, apoptotic bodies, and microvesicles, etc. These vesicles are formed inside the cell and then transported to the vicinity of the cell membrane, releasing their contents into the extracellular or other intracellular regions through fusion. Extracellular vesicles have some important biological functions. They are not only responsible for the transport of substances inside and outside the cell and cell-to-cell communication but also participate in regulating physiological processes such as cell growth, differentiation, and apoptosis. In addition, the vesicles secreted by cells also play an important role in the occurrence, development, and treatment of diseases. For example, extracellular vesicles such as exosomes can be used as biomarkers for disease diagnosis, novel drug delivery systems, etc. Traditional EV isolation methods, such as ultracentrifugation, density gradient centrifugation, ultrafiltration, etc., although they can isolate EVs, often have problems such as low isolation efficiency and low purity. Emerging isolation techniques, such as size exclusion chromatography, immunoaffinity capture, etc., although they have improved the isolation efficiency and purity to a certain extent, still have problems such as high cost and complex operation. Current EV detection methods mainly include flow cytometry, enzyme-linked immunosorbent assay, nanoparticle tracking analysis, etc., and there are still some technical bottlenecks, such as insufficient sensitivity, complex operation, and high cost.

[0005] The principle of biological separation and detection lies in utilizing the differences in the physical and biochemical properties of biological objects to achieve the effective separation and accurate detection of target substances through specific technical means. Extracellular vesicles (represented by exosomes) play important roles in aspects such as intercellular communication and material exchange, as well as in aspects such as the metabolism, growth, and reproduction of microorganisms. Although extracellular vesicles and microbial cells have different origins and biological functions, they have similarities in structure and molecular composition. For example, the intracellular components of both extracellular vesicles and microbial cells contain various bioactive molecules such as proteins, lipids, and nucleic acids (DNA, RNA, miRNA, etc.). Secondly, the surfaces of both are cell membranes composed of a phospholipid bilayer structure, surface proteins, and polysaccharides. The vesicles secreted by cells, especially exosomes, usually have diameters between 30 and 150 nanometers and belong to nanoscale particles. Microbial cells, especially prokaryotic microorganisms, also have relatively small sizes. The length of most prokaryotic microorganisms is between 0.5 and 10 micrometers, and the diameter of the smallest prokaryotic microorganism is only 0.2 micrometers. Both belong to nanoscale structures. Therefore, based on the similarities in structure and surface composition between extracellular vesicles (EVs) and microbial cells, there are commonalities in their separation and detection methods. Therefore, during the separation process, technologies that can accurately distinguish different particle sizes and characteristics, such as ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and immunoisolation techniques, are often required to achieve effective separation and purification. At the same time, in terms of detection, since they may both be present in complex biological samples and at low concentrations, highly sensitive and specific detection methods, such as flow cytometry, fluorescence microscopy, or antibody-based detection techniques, are often required to accurately identify and quantify these two types of objects.However, existing separation and detection methods are cumbersome to operate, inefficient, and costly, etc., and there are still certain limitations and challenges [Mi F, Hu C, Wang Y, et al. Recent advancements in microfluidic chip biosensor detection of foodborne pathogenic bacteria: a review [J]. Analytical and Bioanalytical Chemistry, 2022, 414(9): 2883-2902.][Zhang G, Huang X, Liu S, et al. Demystifying EV heterogeneity: emerging microfluidic technologies for isolation and multiplexed profiling of extracellular vesicles [J]. Lab on a Chip, 2025.][Wang Y K, Bao Y R, Liang Y X, et al. Current progress and prospect of microfluidic-based exosome investigation [J]. TrAC Trends in Analytical Chemistry, 2023, 168: 117310.][Kant K, Shahbazi M A, Dave V P, et al. Microfluidic devices for sample preparation and rapid detection of foodborne pathogens [J]. Biotechnology advances, 2018, 36(4): 1003-1024.].

[0006] It is necessary to find a collection, separation, enrichment and detection system that can be integrated and connected to quickly, portable, accurate and sensitive detection methods to automatically identify common pathogenic organisms in public health and biological warfare agents without human supervision, or to achieve rapid detection of tumor-derived exosomes in clinical samples. Microfluidic chips have shown obvious advantages over traditional methods. With their miniaturization, integration and multifunctionality, they have been widely used in many fields such as medical diagnosis and food safety. Microfluidic chips have been used as laboratory-on-a-chip platforms, showing excellent capabilities in integrated particle collection and enrichment, sample processing, and target molecule analysis and detection, overcoming the limitations of time and space, thus highlighting their potential in simpler, cheaper, and more portable on-site bioaerosol monitoring [Lee I, Jeon E, Lee J. On-site bioaerosol sampling and detection in microfluidic platforms[J]. TrAC Trends in Analytical Chemistry, 2023, 158: 116880.] and clinical sample testing [Wang YK, Bao YR, Liang YX, et al. Current progress and prospect of microfluidic-based exosome investigation[J]. TrAC Trends in Analytical Chemistry, 2023, 168: 117310.]. In the microbial collection and enrichment unit, microfluidic chip technology can be combined with traditional bioaerosol collection methods in an independent separation mode or an overall combination mode. Different structures of microfluidic chips can also be used, such as fishbone-shaped staggered herringbone shapes to generate chaotic vortices, which are used to generate curved, circular or spiral channels for centrifugal force [Wang L, Qi W, Liu Y, et al. Recent advances on bioaerosol collection and detection in microfluidic chips [J]. Analytical Chemistry, 2021, 93 (26): 9013-9022]. However, the enrichment efficiency is limited when the microstructure is made by microelectromechanical processing technology alone, and there are few reports on the integration of microbial and extracellular vesicle microfluidic chip enrichment and sensor detection, which needs to be further improved.

[0007] In the detection unit of the microfluidic chip, leveraging the integration advantages of the microfluidic chip, the lysis, nucleic acid extraction, and amplification of microorganisms or extracellular vesicles can be completed on the chip. However, there are still problems such as long detection time, complex operation, high cost, and difficulty in meeting the requirements of rapid and real-time detection. There is a need for sensors and biosensor technologies that can directly achieve the detection of whole microorganisms or extracellular vesicles. For example, based on a microfluidic integrated platform, by efficiently collecting and concentrating microorganisms from a large amount of air samples and further integrating with a biosensing strategy, highly selective, sensitive, real-time, and continuous detection of bioaerosols can be achieved, enabling a rapid response to sudden risks.

[0008] Sensors have the advantages of low cost, fast response speed, small size, and easy integration, and are increasingly attracting people's attention and are considered effective tools for biological detection. In the sensor detection unit, there are optical and electrical sensors. Optical sensors require an additional optical signal acquisition system, and electrochemical biosensors have received particular attention. Their key advantages are high sensitivity, good selectivity, point-of-care testing, easy miniaturization, and low cost. Different from conventional aerosols, biological recognition elements (such as genes, antibodies, nucleic acid aptamers, etc.) are key components of electrochemical biosensors and are immobilized on the electrode surface by physical or chemical methods. When the analyte interacts with the recognition element on the surface of the biosensor, it will cause a change in the electrical signal, thereby achieving detection. However, electrochemical detection still requires expensive reagents such as antibodies, peptides, and aptamers [Batra J S, Chi T Y, Huang M F, et al. Wearable Biosensor with Molecularly Imprinted Conductive Polymer Structure to Detect Lentivirus in Aerosol[J]. Biosensors, 2023, 13(9):861]. However, the poor tolerance of antibodies makes them unsuitable for on-site applications, and there is an urgent need for suitable materials to replace antibodies as the sensitive layer of electrochemical sensors. Summary of the Invention

[0009] To solve the above problems, the present invention provides a microfluidic chip and a preparation method thereof. Specifically, it relates to an integrated microfluidic chip capable of realizing on-line enrichment and integrated sensor detection of microorganisms in samples such as aerosols or extracellular vesicles in liquid samples. It can not only greatly improve the detection sensitivity and selectivity of the existing technology, but also improve portability, shorten the detection time, and is expected to realize on-line continuous detection. Biological particles refer to tiny particles with biological activity, including cells, cell debris, viruses, protein aggregates, etc. These particles are widely present in the internal or external environment of organisms and participate in various biological processes and interactions. A detection device provided by the present invention can be applied to the analysis and detection of gas and aqueous solution samples, including but not limited to the detection of microorganisms such as bacteria and viruses, and biological particles such as cells or exosomes. Further, it also includes a detection method for microorganisms and EVs, which uses the detection device proposed in this application to analyze and detect biological particles such as microorganisms, cells, and extracellular vesicles in gas and solution samples through electrical signal acquisition and with the help of data analysis.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A microfluidic chip sensor includes a cover glass, a channel layer, an electrode layer, and a substrate arranged in sequence;

[0012] There is a microchannel structure in the channel layer. The cover glass is provided with a liquid inlet and a sampling port at positions corresponding to the microchannel structure, and a discharge port at a position corresponding to the end of the microchannel structure;

[0013] On the side of the electrode layer facing the channel layer, one or more enrichment or detection electrodes are provided;

[0014] It further includes an enrichment unit, which is located under the cover plate and on the electrode layer, within the channel of the channel layer; or it further includes a plurality of enrichment units, and all the plurality of enrichment units are located under the cover plate and on the electrode layer, within the microchannels of the channel layer;

[0015] Both the channel layer and the electrode layer include single-layer or multi-layer structural units.

[0016] After the cover plate, the channel layer, the electrode layer, and the substrate are bonded and encapsulated, the detection electrodes and the enrichment units are all located within the microchannel structure.

[0017] Preferably, the enrichment unit is at least one of a biological enrichment unit, a chemical enrichment unit, a hydrodynamic enrichment unit, a surface acoustic wave enrichment unit, or a dielectrophoretic physical enrichment unit based on interdigital electrodes.

[0018] Preferably, the hydrodynamic enrichment unit includes a channel body and any one of microstructures, nanostructures or micro-nanostructures provided inside the channel body.

[0019] Preferably, the dielectrophoresis physical enrichment unit based on interdigital electrodes includes a channel body and an electrode structure provided inside the channel body.

[0020] Preferably, the surface acoustic wave enrichment unit includes a piezoelectric substrate and interdigital electrodes, and the detection electrode is in the form of interdigital electrodes, double electrodes or triple electrodes.

[0021] Preferably, chemical modification or coating treatment, antibody immobilization, DNA immobilization, RNA immobilization, enzyme immobilization, cell immobilization or molecular imprinting polymer or any combination thereof is performed on the surface of a local area inside the channel body or on the micro-nanostructure of the hydrodynamic enrichment unit or on the electrode structure of the enrichment unit based on interdigital electrodes.

[0022] Preferably, the surface of the detection electrode has a sensitive recognition layer, and the sensitive recognition layer is prepared using any one or more of nanomaterials, antibodies, DNA, RNA, enzymes, cells or molecular imprinting polymers. The polymerization preparation method of the molecular imprinting polymer is electro-polymerization, photo-initiated polymerization, self-assembly, electrospinning or thermal polymerization.

[0023] The present invention also provides a microbial or extracellular vesicle detection device, including a liquid inlet pipeline, a sampling pipeline, a discharge pipeline, an infusion device, a signal generator and the microfluidic chip sensor as claimed in the claims, wherein the positions of the sampling pipeline and the discharge pipeline are not fixed and are arranged according to actual requirements;

[0024] The liquid inlet pipeline, the sampling pipeline and the discharge pipeline are respectively communicated with the liquid inlet, the sampling port and the discharge port of the cover plate of the microfluidic chip sensor. Delivery pumps are connected to both the sampling pipeline and the liquid inlet pipeline; the sampling pipeline is used to pump a sample to be detected from the outside into the microchannel structure and perform enrichment in the enrichment unit; the liquid inlet pipeline is used to pump a liquid to elute and release the microorganisms or extracellular vesicles in the enrichment unit into the sensor area for detection after the microorganisms or extracellular vesicles in the sample are enriched in the enrichment unit area; the discharge pipeline is used to discharge the liquid or gas in the microchannel structure;

[0025] When the enrichment unit is a dielectrophoresis enrichment unit based on interdigital electrodes, it further includes an interdigital electrode control unit of the signal generator. The interdigital electrode control unit is connected to the interdigital electrodes of the enrichment unit and is used to apply an alternating current signal to the interdigital electrodes for enriching microorganisms or extracellular vesicles;

[0026] When the enrichment unit is a surface acoustic wave enrichment unit, it further includes a radio frequency generator unit. The radio frequency generator is connected to the interdigital electrodes of the enrichment unit. By setting appropriate frequency and power, a radio frequency signal is applied to the interdigital electrodes to excite surface acoustic waves for the enrichment of microorganisms or extracellular vesicles.

[0027] The signal acquisition and processing unit is electrically connected to the detection electrodes of the electrode layer. The electrical signals of the detection electrodes are collected by the signal acquisition and processing unit. When the target microorganisms or cell vesicles bind to the surface of the detection electrodes, the electrical signals collected by the signal acquisition unit connected to the detection electrodes change. The collected signals are converted and processed by the signal processing unit.

[0028] Preferably, the signal acquisition and processing unit adopts any one of the signal excitation methods of constant current, constant potential, differential pulse voltammetry, cyclic voltammetry or impedance.

[0029] The present invention also provides a detection method for a detection device of microorganisms or extracellular vesicles, including the following steps:

[0030] The sample to be detected from the outside is transported to the microchannel structure through the injection pipeline.

[0031] The enrichment unit of the microfluidic chip sensor is continuously used to enrich microorganisms in aerosol samples and microorganisms or extracellular vesicles in solutions.

[0032] After a period of time, the enrichment is stopped, and liquid is transported into the microchannel structure through the liquid inlet pipeline to elute and release the microorganisms or extracellular vesicles in the enrichment unit. For the microorganisms or extracellular vesicles enriched based on dielectrophoresis of interdigital electrodes, the enriched microorganisms or extracellular vesicles can also be released by applying electricity.

[0033] The released microorganisms or extracellular vesicles enter the sensor area. After incubation and enrichment for a period of time, the signal acquisition and processing unit connected to the sensor electrodes performs electrical signal acquisition and processing.

[0034] Based on the change in the electrical signal value before and after the sensor binds to the target microorganisms or extracellular vesicles, and the linear relationship between the degree of change in the electrical signal and the concentration of the target microorganisms or extracellular vesicles, the detection and quantification of the target microorganisms or extracellular vesicles are achieved.

[0035] The microfluidic chip provided by the present invention has the following beneficial effects:

[0036] The present invention is provided with an enrichment unit on the channel layer, and one or more detection electrodes are provided on one side of the electrode layer facing the channel layer. After bonding and encapsulating each layer, both the detection electrodes and the enrichment unit are located within the microchannel structure, forming a microbial or extracellular vesicle sensor that integrates physical hydrodynamic enrichment and / or electroenrichment and / or chemical and / or biological enrichment. Combining with the molecular imprinted artificial antibody technology, it can achieve highly sensitive and highly selective detection of microorganisms or extracellular vesicles for real-time online monitoring, and at the same time has the advantages of low cost, stability, and rapidity. The method provided by the present invention can be used for the detection of microorganisms or extracellular vesicles in aerosol and aqueous solution samples. Description of the Drawings

[0037] In order to more clearly illustrate the embodiments of the present invention and its design solutions, the following will briefly introduce the drawings required for this embodiment. The drawings in the following description are only partial embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0038] Figure 1 It is a schematic structural diagram of the encapsulated microfluidic chip sensor provided in Embodiment 1 of the present invention;

[0039] Figure 2 It is an unencapsulated structural diagram when the enrichment unit is located on the side of the cover plate close to the channel layer;

[0040] Figure 3 It is an unencapsulated structural diagram when the enrichment unit is located on the side of the electrode layer close to the channel layer;

[0041] Figure 4 It is a schematic structural diagram of the detection electrode; where (a) is an interdigitated electrode and (b) is a three-electrode;

[0042] Figure 5 It is a schematic structural diagram of the microcolumn array.

[0043] Explanation of the Reference Numerals in the Drawings:

[0044] Cover plate 1, channel layer 2, electrode layer 3, substrate 4, microchannel structure 5, detection electrode 6, enrichment unit 7, liquid inlet pipeline 8, sampling pipeline 9, discharge pipeline 10. Detailed Embodiments

[0045] In order to enable those skilled in the art to better understand the technical solutions of the present invention and be able to implement them, the present invention will be described in detail below with reference to the drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0046] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the technical solutions of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0047] In addition, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. In the description of the present invention, it should be noted that unless otherwise clearly specified or limited, the terms "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more, which will not be elaborated here.

[0048] The present invention integrates aerosol microfluidic chip enrichment and electrochemical sensing for the first time, that is, a dual-mode enrichment strategy based on the integration of hydrodynamic enrichment and electrokinetic enrichment technologies is adopted to achieve highly sensitive and rapid detection in a low-cost manner and improve the enrichment efficiency; the artificial antibody technology is adopted for the first time to realize the detection of microorganisms or extracellular vesicles in the microfluidic chip, which has high selectivity, low cost, stability, can be reused, and is more conducive to on-site real-time and continuous detection.

[0049] Example 1

[0050] This example provides a microfluidic chip sensor, specifically an integrated microfluidic chip sensor capable of realizing aerosol microorganism enrichment and on-line electrochemical sensing detection, as Figure 1 shown, the sensor includes a cover plate 1, a channel layer 2, an electrode layer 3, and a substrate 4 arranged in sequence.

[0051] Specifically, a microchannel structure 5 is processed on the channel layer 2. The cover plate 1 is provided with a liquid inlet and a sampling port at a position corresponding to the front end of the microchannel structure 5, and a discharge port at a position corresponding to the end of the microchannel structure 5; one or more detection electrodes 6 are arranged on the side of the electrode layer 3 facing the channel layer 2. In this example, as Figure 4As shown, the detection electrode 6 is in the form of an interdigital electrode, a double electrode or a triple electrode. The surface of the detection electrode 6 has a sensitive recognition layer, and the sensitive recognition layer is prepared using any one or more of antibodies, DNA, RNA, enzymes, cells or molecularly imprinted polymers. The sensor provided in this embodiment is an electrochemical sensor combined with an artificial antibody molecularly imprinted recognition layer, and the polymerization method is electro-polymerization, photo-initiated polymerization, self-assembly, electrospinning or thermal polymerization.

[0052] Meanwhile, the sensor further includes an enrichment unit 7. The enrichment unit 7 is located on the lower surface of the cover plate 1 or the upper surface of the electrode layer 3, facing the channel layer 2. This embodiment may also include a plurality of enrichment units 7, and the plurality of enrichment units 7 are all located on the lower surface of the cover plate 1 and the upper surface of the electrode layer 3, and are in the channels of the channel layer 2. As Figure 3 shown, the enrichment unit 7 may also be located on the side of the electrode layer 3 close to the channel layer 2, that is, on the same layer as the detection electrode 6.

[0053] Both the detection electrode 6 and the enrichment unit 7 are opposite to the microchannel structure 5 in position. After the cover plate 1, the channel layer 2, the electrode layer 3 and the substrate 4 are bonded and encapsulated, both the detection electrode 6 and the enrichment unit 7 are located within the microchannel structure 5

[0054] Further, in this embodiment, the enrichment unit 7 is at least one of a biological enrichment unit, a chemical enrichment unit, a hydrodynamic enrichment unit, a surface acoustic wave enrichment unit or a dielectrophoresis physical enrichment unit based on an interdigital electrode.

[0055] Specifically, the hydrodynamic enrichment unit includes a channel body and any one of a micro-structure, a nano-structure or a Wiener structure provided inside the channel body. The enrichment unit based on an interdigital electrode includes a channel body and an electrode structure provided inside the channel body. The surface acoustic wave enrichment unit includes a piezoelectric substrate and an interdigital electrode.

[0056] This embodiment includes two enrichment units 7, wherein the microchannel structure 5 adopts a serpentine channel structure. The first enrichment unit is a hydrodynamic enrichment unit, as Figure 1 shown by 7 on the left side, there is a micro-column array in the channel, and the micro-column array structure is as Figure 5 shown. The micro-column array structure can also be of different shapes and array structures. The second enrichment unit adopts dielectrophoresis enrichment based on an interdigital electrode, as Figure 1 shown by 7 on the right side.

[0057] In order to improve the quality and efficiency of enrichment, the surfaces of local regions inside the channel body, the micro-nano structures of the hydrodynamic enrichment unit, the electrodes of the enrichment unit based on interdigitated electrodes, etc. are subjected to chemical modification or coating treatment, antibody immobilization, DNA immobilization, RNA immobilization, enzyme immobilization, cell immobilization, or any one or more of molecularly imprinted polymers, which can exert chemical effects or the specific affinity of biomolecules to highly selectively capture target microorganisms.

[0058] Among them, the preparation method of the sensitive recognition layer on the surface of the detection electrode 6 is as follows:

[0059] First, purchase commercially available flexible electrodes or glass electrodes (the sensor electrode patterns on the flexible matrix or glass surface are interdigitated electrode or three-electrode patterns), as Figure 4 shown, rinse the indium tin oxide electrode with organic solvents such as isopropanol, acetone, and ethanol respectively, finally rinse with distilled water, and dry with nitrogen for standby. Preparation of the sensor recognition sensitive layer: Place the indium tin oxide electrode as the working electrode in a solution containing target microorganisms, pyrrole, thionine, and phosphate buffer solution, and at the same time put in an Ag / AgCl reference electrode and a platinum wire counter electrode. Connect the working electrode on the ITO surface, as well as the reference and counter electrodes to an electrochemical workstation respectively, and electro-polymerize to prepare a molecularly imprinted polymer on the electrode surface in a cyclic voltammetry mode. The parameters considered during this process are the polymerization scan rate, the number of scan cycles, the pyrrole concentration, the polymerization voltage range, and the template microorganism concentration. Wash the electrode with sodium chloride solution (or sodium dodecyl sulfate-acetic acid mixture), and dry with nitrogen for standby to obtain an electrode modified with a selective recognition membrane (including but not limited to the above-mentioned preparation method of the sensitive layer). Among them, the thionine added in the system for synthesizing the sensitive layer can also be replaced by or simultaneously added with redox-active substances such as Prussian blue, methylene blue, ferrocene, or potassium ferricyanide. After the detection electrode selectively recognizes and binds to the target microorganism, there will be a change in the electrical signal.

[0060] During use, multiple sensors can be used in parallel, and each sensor can detect different types of microorganisms respectively to achieve simultaneous detection of multiple targets. The detection objects are not limited to microbial cells, and can be biological particles such as small molecules, large molecule proteins and nucleic acids, as well as animal and plant cells.

[0061] The preparation method of the microfluidic chip sensor provided in this embodiment is as follows:

[0062] Fabrication of the channel layer: Fabricate a mask, and then use photolithography technology to transfer the pattern on the mask plate to a silicon wafer with a certain thickness of SU-8 photoresist; after the positive mold of the chip is fabricated, pour PDMS on the mold and cure it, and then peel it off to obtain a hollow PDMS microchannel layer;

[0063] Microcolumn array layer: After making a mask, a silicon wafer with a microcrystal array is fabricated by deep silicon etching. Using this silicon wafer as a mold, a polydimethylsiloxane (PDMS) prepolymer solution is poured, and after polymerization and curing, the PDMS is peeled off to obtain a PDMS chip layer with a microcolumn array on its surface.

[0064] The PDMS cover sheet without microcolumns is made by pouring ordinary PDMS into a thin film, and after curing, it is cut into the required size. Holes are drilled at both ends of the channels in the corresponding channel layer as the liquid inlet and outlet.

[0065] As Figure 2 and Figure 3 shown, the microfluidic chip assembly can be carried out in different ways, including but not limited to, different enrichment units being located on the same layer, or on different layers respectively.

[0066] Specifically, two chip assembly methods can be adopted:

[0067] First: Align and stack the PDMS cover sheet, the PDMS channel layer, the flexible interdigital electrode with a recognition sensitive layer on its surface, the microcolumn array layer, and the glass substrate from top to bottom in sequence, and finally bond the different layers by plasma treatment or using double-sided tape to encapsulate and form a complete microfluidic chip sensor.

[0068] Second: Align and stack the PDMS layer with the microcolumn array facing downwards, the PDMS channel layer, and the glass electrode with a recognition sensitive layer on its surface from top to bottom in sequence, and finally bond the different layers by plasma treatment or using double-sided tape to encapsulate and form a complete microfluidic chip sensor.

[0069] Including but not limited to the above assembly and matching methods to achieve the integration of aerosol enrichment and bioelectrochemical sensors.

[0070] Through the precise design of the microfluidic chip sensor and using advanced microelectromechanical processing technology, micro-nano structures can be introduced into the microchannels of the microfluidic chip sensor. By using hydrodynamic, electroenrichment, and biological and chemical enrichment technologies, target microorganisms can be selectively captured and enriched from a large amount of air or liquid. Therefore, it can sensitively detect trace pathogens and other microorganisms. And based on the integrated advantages of the microfluidic chip, the enrichment and sensor detection units can be directly integrated and flexibly combined, allowing real-time and continuous monitoring of aerosol and liquid samples on-site, and enabling a rapid response to sudden risks.

[0071] Example 2

[0072] Based on the same inventive concept, this embodiment also provides a microbial detection device, which includes a liquid inlet pipeline 8, a sampling pipeline 9, a discharge pipeline 10, a signal acquisition and processing unit, and a microfluidic chip sensor provided in Embodiment 1. The microfluidic chip sensor serves as the signal acquisition and processing unit. The positions of the sampling pipeline 9 and the discharge pipeline 10 are not fixed and can be flexibly arranged according to actual requirements.

[0073] Specifically, the sampling pipeline 9, the liquid inlet pipeline 8, and the discharge pipeline 10 are respectively connected to the liquid inlet, the sampling port, and the discharge port of the cover plate 1 of the microfluidic chip sensor. A delivery pump is connected to both the sampling pipeline 9 and the liquid inlet pipeline 8. The sampling pipeline 9 is used to pump the aerosol to be detected in the external environment into the microchannel structure 5 and perform enrichment in the enrichment unit 7. The liquid inlet pipeline 8 is used to pump in liquid to elute and release the microorganisms in the enrichment unit 7 into the sensor area when the microorganisms in the aerosol are enriched in the area of the enrichment unit 7. The discharge pipeline 10 is used to discharge the liquid or gas in the microchannel structure 5. Specifically, the delivery pump is a micro peristaltic pump or a micro air pump, and both are connected to the liquid inlet and the sampling port of the microchannel structure 5 through hoses.

[0074] The signal acquisition and processing unit is connected to the detection electrode 6 of the electrode layer 3. When the target microorganism binds to the surface of the detection electrode 6, it causes a change in the electrical signal of the detection electrode 6. The signal acquisition and processing unit collects the electrical signal of the detection electrode 6 and converts and processes the signal.

[0075] Specifically, the signal acquisition and processing unit includes a main control module, an analog-to-digital conversion module, and a potentiostat. The analog-to-digital conversion module is in bidirectional communication connection with both the main control module and the potentiostat. The main control module is used to control the analog-to-digital converter to generate a corresponding excitation voltage according to the received control instruction. The potentiostat is used to apply the excitation voltage to the detection electrode and at the same time convert the electrical signal generated on the detection electrode into a voltage signal that meets the amplitude range. The analog-to-digital conversion module is also used to convert the voltage signal converted by the potentiostat into a digital signal and send the digital signal to the main control module. The potentiostat includes a feedback amplifier and a high-gain amplifier. The high-gain amplifier maintains an ultra-low input impedance and at the same time converts signals such as current from the sensing unit into voltage signals.

[0076] In order to display the detected data in real time, this embodiment also includes a power management unit and a data display unit. The power management unit adopts self-powered methods such as ordinary lithium batteries, photovoltaic cells, and friction. The power management unit is used to provide power to the sensing unit and the signal acquisition and processing unit. The data display unit is connected to the signal acquisition and processing unit and is used to display the detection results. The signal acquisition and processing unit also includes a wireless communication module. The wireless communication module connects the main control module to a mobile terminal through a Bluetooth chip and is used to complete wireless reception and transmission of data.

[0077] The main control module is used to control the digital-to-analog converter to generate a corresponding excitation voltage according to the control instruction sent by the mobile terminal, and is also used to send the digital signal converted by the analog-to-digital conversion module to the mobile terminal through the Bluetooth communication module.

[0078] In this embodiment, the signal acquisition and processing unit adopts any one of the signal excitation methods such as constant current, constant potential, differential pulse voltammetry, cyclic voltammetry or impedance. That is to say, when the excitation signal method is different, for example, a dynamically changing excitation signal is given, and the current and voltage of the electrode are dynamically collected, the detection result may be more sensitive. When the enrichment unit 7 is an enrichment unit based on interdigitated electrodes, it further includes an interdigitated electrode control unit, which is connected to the interdigitated electrodes of the enrichment unit and is used to apply an alternating current signal to the interdigitated electrodes for microbial enrichment.

[0079] During the detection process, the enrichment time of the enrichment unit and the sensor incubation time can be optimized and adjusted according to different conditions.

[0080] First, the constant potential (IT) mode is used to collect the current change of the sensor before and after binding the target microorganism. Later, the quantitative detection of the target microorganism will be realized based on the linear relationship between the current change and the microorganism concentration.

[0081] Second, the cyclic voltammetry (DPV) mode is used to collect the peak current change of the sensor before and after binding the target microorganism. Later, the quantitative detection of the target microorganism will be realized based on the linear relationship between the peak current change and the microorganism concentration.

[0082] Third, the impedance mode is used to collect the peak current change of the sensor before and after binding the target microorganism. Later, the quantitative detection of the target microorganism will be realized based on the linear relationship between the impedance value change and the microorganism concentration.

[0083] The signal acquisition and processing unit can be powered in different ways: including but not limited to: external power supply, ordinary lithium battery, photovoltaic cell, friction and other self-power supply methods, as well as Bluetooth wireless power supply, etc.

[0084] Embodiment 3

[0085] Step 1. Based on the microbial detection device provided in Embodiment 2, this embodiment further provides an aerosol microbial detection method, including the following steps:

[0086] Step 1. Start the delivery pump and at the same time start the enrichment unit, and transport the samples such as aerosol to be detected from the outside to the microchannel structure 5 through the sampling pipeline 9.

[0087] Step 2. Continuously use the enrichment unit 7 of the microfluidic chip sensor to enrich the microorganisms in the aerosol.

[0088] Step 3: After a period of enrichment is completed, stop the enrichment, start the liquid pump, and deliver liquid into the microchannel structure 5 through the liquid inlet pipeline 8 to elute and release the microorganisms in the enrichment unit 7.

[0089] Step 4: The released microorganisms enter the electrode layer 3. After incubation and enrichment for a period of time, the signal acquisition and processing unit acquires and processes the electrical signals of the detection electrode 6.

[0090] Step 5: Based on the change in the electrical signal value and the linear relationship between the change degree and the concentration of the target microorganism, the detection and quantification of the target microorganism are achieved.

[0091] Example 4

[0092] Based on the same inventive concept, this example also provides an exosome detection device, including a liquid inlet pipeline 8, a sampling pipeline 9, a discharge pipeline 10, a signal acquisition and processing unit, and a microfluidic chip sensor provided in Example 1.

[0093] Specifically, the sampling pipeline 9, the liquid inlet pipeline 8, and the discharge pipeline 10 are respectively connected to the liquid inlet, the sampling port, and the discharge port of the cover plate 1 of the microfluidic chip sensor. Delivery pumps are connected to both the sampling pipeline 9 and the liquid inlet pipeline 8; the sampling pipeline 9 is used to pump the sample to be detected outside into the microchannel structure 5 for enrichment in the enrichment unit 7; the liquid inlet pipeline 8 is used to pump in liquid to elute and release the exosomes in the enrichment unit 7 into the sensor area when the exosomes are enriched in the area of the enrichment unit 7; the discharge pipeline 10 is used to discharge the liquid in the microchannel structure 5. Specifically, the delivery pump is a micro peristaltic pump or a micro air pump, and both are connected to the liquid inlet and the sampling port of the microchannel structure 5 through hoses.

[0094] The signal acquisition and processing unit is connected to the detection electrode 6 of the electrode layer 3; when the target exosome binds to the surface of the detection electrode 6, it causes a change in the electrical signal of the detection electrode 6. The signal acquisition and processing unit acquires the electrical signal of the detection electrode 6 and converts and processes the signal.

[0095] Specifically, the signal acquisition and processing unit includes a main control module, an analog-to-digital conversion module, and a potentiostat. The analog-to-digital conversion module is in bidirectional communication connection with both the main control module and the potentiostat. The main control module is used to control the analog-to-digital converter to generate a corresponding excitation voltage according to the received control instruction. The potentiostat is used to apply the excitation voltage to the detection electrode and at the same time convert the electrical signal generated on the detection electrode into a voltage signal that meets the amplitude range. The analog-to-digital conversion module is also used to convert the voltage signal converted by the potentiostat into a digital signal and send the digital signal to the main control module. The potentiostat includes a feedback amplifier and a high-gain amplifier. The high-gain amplifier maintains an ultra-low input impedance and at the same time converts signals such as current from the sensing unit into voltage signals.

[0096] To display the detected data in real time, this embodiment further includes a power management unit and a data display unit. The power management unit adopts self-powered methods such as ordinary lithium batteries, photovoltaic cells, and friction. The power management unit is used to supply power to the sensing unit and the signal acquisition and processing unit. The data display unit is connected to the signal acquisition and processing unit and is used to display the detection results. The signal acquisition and processing unit further includes a wireless communication module. The wireless communication module connects the main control module to the mobile terminal through a Bluetooth chip and is used to complete wireless reception and transmission of data.

[0097] The main control module is used to control the digital-to-analog converter to generate a corresponding excitation voltage according to the control instruction sent by the mobile terminal, and is also used to send the digital signal converted by the analog-to-digital conversion module to the mobile terminal through the Bluetooth communication module.

[0098] In this embodiment, the signal acquisition and processing unit adopts any one of signal excitation methods such as constant current, constant potential, differential pulse voltammetry, cyclic voltammetry, or impedance. That is to say, when the excitation signal method is different, for example, a dynamic excitation signal is given, and the current and voltage of the electrode are dynamically collected, the detection result may be more sensitive. When the enrichment unit 7 is a surface acoustic wave enrichment unit based on interdigital electrodes, it further includes a radio frequency generator unit. The radio frequency generator unit is connected to the interdigital electrodes of the enrichment unit and is used to apply a radio frequency signal to the interdigital electrodes to trigger surface acoustic waves for exosome enrichment.

[0099] During the detection process, the enrichment time of the enrichment unit and the sensor incubation time can be optimized and adjusted according to different conditions.

[0100] First, the constant potential (IT) mode is used to collect the current change of the sensor before and after binding the target exosomes. Later, the quantitative detection of the target exosomes will be realized based on the linear relationship between the current change and the exosome concentration.

[0101] Second, the cyclic voltammetry (DPV) mode is used to collect the peak current change of the sensor before and after binding the target exosomes. Later, the quantitative detection of the target exosomes will be realized based on the linear relationship between the peak current change and the exosome concentration.

[0102] Third, the impedance mode is used to collect the peak current change of the sensor before and after binding the target exosomes. Later, the quantitative detection of the target exosomes will be realized based on the linear relationship between the impedance value change and the exosome concentration.

[0103] The signal acquisition and processing unit can be powered in different ways, including but not limited to: external power supply, self-powered methods such as ordinary lithium batteries, photovoltaic cells, and friction, as well as Bluetooth wireless power supply, etc.

[0104] Embodiment 5

[0105] Based on the detection device provided in Example 2, this embodiment also provides a method for detecting extracellular vesicles and exosomes, comprising the following steps:

[0106] Step 1: Start the delivery pump and the enrichment unit at the same time, and deliver the aerosol sample to be detected from the outside to the microchannel structure 5 through the sample inlet pipeline 9.

[0107] Step 2: Continue to use the enrichment unit 7 of the microfluidic chip sensor to enrich the exosomes in the solution.

[0108] Step 3: After a period of enrichment, the enrichment is stopped, and the liquid pump is started to transport liquid into the microchannel structure 5 through the liquid inlet pipeline 8 to elute and release the exosomes in the enrichment unit 7.

[0109] Step 4: The released exosomes enter the electrode layer 3 , and after incubation and enrichment for a period of time, the signal acquisition and processing unit acquires and processes the electrical signal of the detection electrode 6 .

[0110] Step 5: Detection and quantification of target exosomes are achieved based on the change in the value of the electrical signal and the linear relationship between the degree of change and the concentration of target exosomes.

[0111] It should be pointed out that the specific implementation methods described above can enable those skilled in the art to understand the invention more comprehensively, but do not limit the invention in any way. Therefore, although the invention has been described in detail in this specification and embodiments, those skilled in the art should understand that the invention can still be modified or replaced by equivalents; and all technical solutions and improvements that do not deviate from the spirit and scope of the invention are included in the protection scope of the patent for the invention. Any figure mark in the claims should not be regarded as limiting the claims involved. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the field within the technical scope disclosed in the present invention belongs to the protection scope of the present invention.

Claims

1. A microfluidic chip sensor, characterized in that, It includes a cover plate (1), a channel layer (2), an electrode layer (3) and a substrate (4) arranged in sequence; There is a microchannel structure (5) in the channel layer (2). The cover plate (1) is provided with a liquid inlet and a sampling port at positions corresponding to the microchannel structure (5), and a discharge port at a position corresponding to the end of the microchannel structure (5); One or more enrichment or detection electrodes (6) are arranged on one side of the electrode layer (3) facing the channel layer (2); It further includes an enrichment unit (7). The enrichment unit (7) is located under the cover plate (1) and on the electrode layer (3), and is in the channel of the channel layer (2); or it further includes a plurality of enrichment units (7), and the plurality of enrichment units (7) are all located under the cover plate (1) and on the electrode layer (3), and are in the microchannel (5) of the channel layer (2); Both the channel layer (2) and the electrode layer include single-layer or multi-layer structural units. After the cover plate (1), the channel layer (2), the electrode layer (3) and the substrate (4) are bonded and encapsulated, the detection electrode (6) and the enrichment unit (7) are both located in the microchannel structure (5).

2. The microfluidic chip sensor according to claim 1, characterized in that, The enrichment unit (7) is at least one of a biological enrichment unit, a chemical enrichment unit, a hydrodynamic enrichment unit, a surface acoustic wave enrichment unit or a dielectrophoresis physical enrichment unit based on interdigital electrodes.

3. The microfluidic chip sensor according to claim 2, characterized in that, The hydrodynamic enrichment unit includes a channel body and any one of a microstructure, a nanostructure or a micro-nanostructure arranged inside the channel body.

4. The microfluidic chip sensor according to claim 2, characterized in that, The dielectrophoresis physical enrichment unit based on interdigital electrodes includes a channel body and an electrode structure arranged inside the channel body.

5. The microfluidic chip sensor according to claim 2, characterized in that, The surface acoustic wave enrichment unit includes a piezoelectric substrate and interdigital electrodes. The detection electrode (6) is in the form of interdigital electrodes, double electrodes or triple electrodes.

6. The microfluidic chip sensor according to claim 3 or 4, characterized in that, Chemical modification or coating treatment, antibody immobilization, DNA immobilization, RNA immobilization, enzyme immobilization, cell immobilization or molecular imprinting polymer, or any one or more of these methods are carried out on the surface of a local area inside the channel body, the micro-nanostructure of the hydrodynamic enrichment unit or the electrode structure of the enrichment unit based on interdigital electrodes.

7. The microfluidic chip sensor according to claim 1, wherein The surface of the detection electrode (6) has a sensitive recognition layer. The sensitive recognition layer is prepared using any one material or multiple materials among nanomaterials, antibodies, DNA, RNA, enzymes, cells or molecular imprinting polymers. The polymerization preparation method of the molecular imprinting polymer is electro-polymerization, photoinitiated polymerization, self-assembly, electrospinning or thermal polymerization.

8. A microbial or extracellular vesicle detection device, characterized in that It includes a liquid inlet pipeline (8), a sampling pipeline (9), a discharge pipeline (10), an infusion device, a signal generator, a radio frequency generator and the microfluidic chip sensor as claimed in claim 1, wherein the positions of the sampling pipeline (9) and the discharge pipeline (10) are not fixed and are arranged according to actual requirements; The sample injection pipeline (9), the liquid inlet pipeline (8), and the discharge pipeline (10) are respectively connected to the liquid inlet, the sample injection port, and the discharge port of the cover plate (1) of the microfluidic chip sensor. A delivery pump is connected to both the sample injection pipeline (9) and the liquid inlet pipeline (8). The sample injection pipeline (9) is used to pump the sample to be detected from the outside into the microchannel structure (5) and perform enrichment in the enrichment unit. The liquid inlet pipeline (8) is used to pump liquid to elute and release the microorganisms or extracellular vesicles in the enrichment unit into the sensor area for detection after the microorganisms or extracellular vesicles in the sample are enriched in the enrichment unit area. The discharge pipeline (10) is used to discharge the liquid or gas in the microchannel structure (5). When the enrichment unit (7) is a dielectrophoresis enrichment unit based on interdigitated electrodes, it further includes a signal generator and an interdigitated electrode control unit. The interdigitated electrode control unit is connected to the interdigitated electrodes of the enrichment unit and is used to apply an alternating current signal to the interdigitated electrodes for enriching microorganisms or extracellular vesicles. When the enrichment unit (7) is a surface acoustic wave enrichment unit, it further includes a radio frequency generator unit. The radio frequency generator is connected to the interdigitated electrodes of the enrichment unit. By setting appropriate frequency and power, a radio frequency signal is applied to the interdigitated electrodes to excite surface acoustic waves for enriching microorganisms or extracellular vesicles. The signal acquisition and processing unit is electrically connected to the detection electrode (6) of the electrode layer (3). The electrical signals of the detection electrode (6) are acquired by the signal acquisition and processing unit. When the target microorganism or cell vesicle binds to the surface of the detection electrode (6), the electrical signals acquired by the signal acquisition unit connected to the detection electrode (6) change. The acquired signals are converted and processed by the signal processing unit.

9. The microbial or extracellular vesicle detection device according to claim 7, wherein, The signal acquisition and processing unit adopts any one of the signal excitation methods of constant current, constant potential, differential pulse voltammetry, cyclic voltammetry, or impedance.

10. A detection method for the microorganism or extracellular vesicle detection device according to claim 8, characterized in that, It includes the following steps: Transport the sample to be detected from the outside to the microchannel structure (5) through the sample injection pipeline (9). Continuously use the enrichment unit of the microfluidic chip sensor to enrich microorganisms in the aerosol sample and microorganisms or extracellular vesicles in the solution. After a period of time, stop the enrichment. Pump liquid into the microchannel structure (5) through the liquid inlet pipeline (8) to elute and release the microorganisms or extracellular vesicles in the enrichment unit. For microorganisms or extracellular vesicles enriched by dielectrophoresis based on interdigitated electrodes, the enriched microorganisms or extracellular vesicles can also be released by applying an electric current. The released microorganisms or extracellular vesicles enter the sensor area. After incubation and enrichment for a period of time, the signal acquisition and processing unit connected to the sensor electrode (6) performs electrical signal acquisition and processing. Based on the change in the electrical signal value before and after the sensor binds to the target microorganism or extracellular vesicle, and the linear relationship between the degree of change in the electrical signal and the concentration of the target microorganism or extracellular vesicle, the detection and quantification of the target microorganism or extracellular vesicle are achieved.

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