An integrated impedance detection device and method for isolation-detection of pathogenic bacteria

Abstract

The application discloses a kind of separation-detection integrated impedance detection device and method for pathogenic bacteria.The device includes nano-channel electrode chip INCE, pressurizing separation module, impedance analysis module, control module, power module and shell;The method includes fixing INCE in electrode fixed part;A series of solutions containing pathogenic bacteria are added dropwise in detection area respectively, and pressure is controlled;Electrolyte solution is added dropwise in detection area, and impedance analysis module is controlled to detect impedance change rate;Obtain the standard curve of pathogenic bacteria concentration and impedance change rate;The sample solution to be measured is added dropwise in detection area, and impedance change rate is obtained, and the concentration of pathogenic bacteria is obtained by comparing the standard curve, and the impedance detection of pathogenic bacteria is completed.The method of the application realizes quantitative detection of pathogenic bacteria by detecting electrode impedance change rate.The device of the application integrates pressurizing separation module and impedance analysis module, is used for rapid detection of pathogenic bacteria, and has the advantages of small volume, portability, simple operation, rapid detection.

Description

Technical Field

[0001] This invention relates to an impedance detection method, specifically to an integrated impedance detection device and method for the isolation and detection of pathogenic bacteria. Background Technology

[0002] Pathogenic contamination has become a serious public health problem, significantly impacting human health worldwide and imposing a heavy economic burden. To monitor microbial contamination in real time, providing a basis for public health policy and intervention measures; to directly and rapidly diagnose pathogenic infections and reduce related economic losses; and to prevent food safety issues caused by pathogens and safeguard public health, it is essential to develop accurate and convenient new detection methods and devices.

[0003] The main methods for detecting pathogenic bacteria include traditional culture methods, routine rapid detection methods, and biosensor methods. Among these, traditional culture and colony counting are currently the most reliable and accurate bacterial detection methods, capable of qualitative and quantitative detection of a variety of pathogenic bacteria. However, despite the improved sensitivity and specificity of current technologies and techniques, the long time required and complex operation of traditional culture methods remain unresolved. Therefore, to enable sensitive and rapid bacterial detection in livestock and poultry, many rapid detection methods have been developed, including immunoassay techniques such as enzyme-linked immunosorbent assay (ELISA), immunomagnetic bead separation technology (IMBS), and immunochromatographic assay (ICA), as well as molecular diagnostic methods such as polymerase chain reaction (PCR), quantitative real-time PCR, gene chip technology, and loop-mediated isothermal amplification (LAMP). However, these routine rapid detection methods require expensive equipment, and the cumbersome procedures demand a high level of expertise from operators. Some also suffer from poor stability and low sensitivity. Biosensor methods are a novel and rapid detection method that can quantitatively or semi-quantitatively analyze the concentration of a target substance by measuring data corresponding to the concentration of the target substance. It has the advantages of high specificity, high sensitivity, low detection limit, and simple operation, and can provide real-time measurement results and perform multi-target detection.

[0004] Solid-state nanochannels are artificial nanochannels developed based on biological nanochannels. Due to their advantages such as structural stability, tunable pore size, and ease of modification, they have attracted widespread research interest in fields such as DNA sequencing, biosensing, and energy conversion. Nanochannels can act as "nanosieves" to achieve rapid sorting of molecules of different sizes. Therefore, constructing electrochemical sensing and detection systems based on solid-state nanochannels and using electrochemical methods to monitor the transmembrane electrical signals of nanochannels offers higher sensitivity and wider applications.

[0005] Existing electrochemical analysis methods mainly rely on electrochemical workstations. With the continuous development of technology, although commercial electrochemical workstations can perform various basic electrochemical characterization methods, they have drawbacks such as large size and high cost. At the same time, they all require additional sample processing devices for the detection process, resulting in low integration of the entire detection device and inconvenience for moving the device to a new location and conducting on-site detection. Summary of the Invention

[0006] To address the problems existing in the background art, this invention provides an integrated impedance detection device and method for the separation and detection of pathogenic bacteria. The method of this invention utilizes solid-state nanochannels with electrodes introduced on their surface. Nanoparticles and nanoparticle-bacterial conjugates are separated by filtration, and changes in electrode impedance are detected to achieve quantitative detection of pathogenic bacteria. A portable device integrating a pressurized separation system and an impedance analysis system is provided for rapid detection of pathogenic bacteria. This method is convenient, sensitive, highly integrated, and easy to operate.

[0007] The technical solution adopted in this invention is:

[0008] I. An integrated impedance detection device for the isolation and detection of pathogenic bacteria:

[0009] The device includes a nanochannel electrode chip (INCE) for adding a sample solution and generating an impedance change; a pressure separation module for separating target pathogens from the sample solution, with the INCE mounted on and electrically connected to the pressure separation module; an impedance analysis module for receiving and transmitting the impedance change measurement results generated by the INCE, electrically connected to the pressure separation module; a control module for receiving signals from a host computer to control the impedance analysis module and the pressure separation module, and for receiving and analyzing the impedance change rate from the impedance measurement module before transmitting it to the host computer, electrically connected to the host computer, the impedance analysis module, and the pressure separation module; a power supply module for supplying power to the control module, the pressure separation module, and the impedance analysis module, electrically connected to the control module; and a housing for mounting the power supply module, control module, pressure separation module, and impedance analysis module, all of which are housed inside the housing.

[0010] The housing has an upper cover and a front cover. When the upper cover is open, it is used to install the nanochannel electrode chip INCE, add the sample solution to be tested, and observe the surface state of the nanochannel electrode chip INCE. When the front cover is open, it is used to observe and disassemble the waste liquid collection section.

[0011] The pressurized separation module includes an electrode fixing part, a waste liquid collection part, and a pressurization part. The electrode fixing part is installed at the top of the waste liquid collection part, the waste liquid collection part is connected to the pressurization part, and the pressurization part is electrically connected to the control module.

[0012] The electrode fixing part includes a fixing box and a fixing plate. The fixing plate is horizontally mounted on the fixing box, and an adjusting screw is vertically mounted on the top of the fixing box, located directly above the fixing plate. The fixing plate includes an upper flip cover, a base, and a connecting circuit board. One side of the upper flip cover is hinged to one side of the base. A groove is formed on the top surface of the base away from the hinge side, and the connecting circuit board is installed in the groove. A first through hole penetrating the surface of the base is formed on the side of the base near the hinge side. The nanochannel electrode chip INCE is horizontally placed on the top surface of the base and the connecting circuit board and electrically connected. The circuit board has an upper flip cover that rotates around the hinge edge to completely cover the top surface of the nanochannel electrode chip INCE. The upper flip cover has a second through hole with the same shape as the first through hole that penetrates its own surface on the side near the hinge edge. After the upper flip cover is closed, the first through hole and the second through hole are arranged opposite each other. The adjusting screw presses down on the top surface of the upper flip cover so that the upper flip cover clamps the nanochannel electrode chip INCE. The nanochannel electrode chip INCE has a detection area. The upper and lower sides of the detection area of ​​the nanochannel electrode chip INCE are directly opposite the second through hole and the first through hole of the electrode fixing part.

[0013] The electrode fixing section is used to flatten and fix the nanochannel electrode chip, guide the filtered waste liquid into the waste liquid collection section, and connect the two electrodes of the electrode chip to the impedance analysis module. The pressurization section is used to create negative pressure under the nanochannel electrode chip to promote the filtration of solution from the nanochannels.

[0014] The impedance analysis module includes a microcontroller and an impedance analysis chip. The microcontroller is electrically connected to the impedance analysis chip and the control module, and the impedance analysis chip is electrically connected to the connection circuit board of the pressure separation module.

[0015] II. A method for preparing a nanochannel electrode chip (INCE) for an integrated impedance detection device combining separation and detection: The method includes the following steps:

[0016] 1) A gold layer is uniformly sputtered on both sides of the porous anodic aluminum oxide (AAO) film. Two electrodes are used to connect the two gold layers respectively, and then the film is fixed and sealed with tape, exposing only the detection area on the surface of the gold layers on both sides, thus obtaining a sealed electrode.

[0017] 2) After cleaning the sealed electrode with H2SO4 solution and deionized water and drying it, immerse it in bovine serum albumin (BSA) solution. Finally, rinse it with deionized water to remove excess BSA solution and dry it to obtain the nanochannel electrode chip INCE.

[0018] In step 1), the nanochannel electrode chip INCE is electrically connected to the connection circuit board of the pressure separation module through two electrodes; the shape of the detection area on both sides of the gold layer surface of the nanochannel electrode chip INCE is the same as the shape of the first through hole on the fixing plate of the electrode fixing part; when the nanochannel electrode chip INCE is fixed on the electrode fixing part, the upper and lower sides where the detection area of ​​the nanochannel electrode chip INCE is located are directly opposite the second through hole and the first through hole of the electrode fixing part.

[0019] In step 2), the sealed electrode is washed and dried sequentially with H2SO4 solution and deionized water, and then immersed in bovine serum albumin (BSA) solution. Specifically, the sealed electrode is washed and dried sequentially with 1 μm H2SO4 solution and deionized water, and then immersed in 0.2%-1% m / v bovine serum albumin (BSA) solution for 30-60 min.

[0020] III. A method for detecting the impedance of pathogenic bacteria using an integrated impedance detection device for separation and detection:

[0021] The method includes the following steps:

[0022] 1) Fix the nanochannel electrode chip INCE to the electrode fixing part, so that the detection area of ​​the nanochannel electrode chip INCE is directly opposite the second through hole and the first through hole of the electrode fixing part.

[0023] 2) A series of solutions containing pathogenic bacteria of the same volume with preset gradient concentrations are dropped onto the detection area of ​​the nanochannel electrode chip INCE through the second through hole of the electrode fixing part. The pressurization part of the pressurization separation module is controlled by the control module to draw air from the waste liquid collection part below the detection area of ​​the nanochannel electrode chip INCE to form a negative pressure, which promotes the solution to be tested to pass through the nanochannel, so that the target is trapped on the surface and inside of the nanochannel electrode chip INCE.

[0024] 3) The electrolyte solution is dripped from the second through-hole of the electrode fixing part onto the detection area of ​​the INCE nanochannel electrode chip. The impedance measurement program is run by the control module to control the impedance analysis module to detect the impedance change of the two electrodes of the INCE nanochannel electrode chip and transmit the measurement results to the control module. First, the microcontroller runs and initializes all serial ports and the impedance analysis chip. It determines whether it is the first detection. If so, it calibrates the impedance analysis chip. Then, the frequency scanning conditions are written into the registers of the impedance analysis chip, including the starting frequency, frequency increment, and increment number. The impedance analysis chip runs, and the microcontroller reads the real value register data R and the imaginary value register data I of the impedance analysis chip and calculates the impedance. The status register content is used to determine whether the frequency scan is complete. The measurement continues until the last frequency point, after which the impedance analysis chip is turned off.

[0025] 4) The control module obtains a standard curve of pathogen concentration and impedance change rate based on the preset gradient concentration of each solution containing pathogens and the measurement results of their impedance changes.

[0026] 5) Drop the sample solution to be tested onto the detection area of ​​the nanochannel electrode chip INCE through the second through hole of the electrode fixing part, and then continue the operation of step 3) to obtain the measurement result of the impedance change of the sample solution to be tested. Compare with the standard curve obtained in step 4) to obtain the concentration of pathogenic bacteria in the sample solution to be tested, and complete the impedance detection of pathogenic bacteria.

[0027] In step 5), the sample solution to be tested is prepared using the following method:

[0028] The inactivated pathogenic bacteria solution was diluted with sterile phosphate-buffered saline (PBS) or chicken extract. The diluted pathogenic bacteria solution was then mixed with antibody-modified nanoparticles and incubated to capture the nanoparticle-pathogen conjugate formed by the target pathogenic bacteria. After magnetic separation, the supernatant was discarded to obtain the precipitate. The precipitate was washed with PBS and then resuspended in PBS to obtain the enriched and purified sample solution of the target pathogenic bacteria.

[0029] In the preparation of the test sample solution, the inactivated pathogenic bacteria solution is diluted to 10 μL using sterile phosphate-buffered saline (PBS) or chicken extract. 2 -10 6 CFU / mL, take 1 mL of diluted pathogenic bacterial solution and mix with antibody-modified nanoparticles at a volume ratio of 10:1 and incubate for 30-60 min to capture the nanoparticle-pathogenic bacterial conjugate 11 formed by the target pathogenic bacteria. After magnetic separation, discard the supernatant to obtain the precipitate. Wash the precipitate with phosphate-buffered saline (PBS) solution and resuspend it in 50-100 μL of PBS solution to obtain the test sample solution enriched and purified from the target pathogenic bacteria.

[0030] The negative pressure formed by the pressurization part in step 2) is 50-100 kPa.

[0031] The target material is a nanoparticle-pathogenic bacteria conjugate.

[0032] This invention is based on a nanochannel electrode chip. An internal pressurized separation system allows small nanoparticles to pass through the nanochannels, while large nanoparticle-bacterial conjugates are trapped on the surface and inside the nanochannels. By trapping the nanoparticle-bacterial conjugates, this invention hinders the passage of electrochemical probe molecules through the nanochannels, causing a change in electrode impedance. An impedance measurement module is used to measure this impedance change to achieve bacterial detection. This invention obtains the impedance values ​​and impedance changes at different frequencies by setting the initial frequency, frequency increment, and number of increments.

[0033] The beneficial effects of this invention are:

[0034] 1) This invention is based on a miniaturized nanochannel electrode chip and integrates a separation sensing and detection module, which can realize the integrated separation and detection of bacteria.

[0035] 2) This invention can be applied to the detection of pathogenic bacteria. It is simple and efficient to operate, and the equipment is portable and inexpensive, avoiding the use of a variety of expensive large instruments.

[0036] 3) This invention integrates the sample separation and detection steps into a single device, eliminating the need for multiple electrode movements and improving detection stability.

[0037] In summary, the method of this invention achieves quantitative detection of pathogenic bacteria by detecting changes in electrode impedance. The device of this invention integrates a pressure separation module and an impedance analysis module for rapid detection of pathogenic bacteria, and has the advantages of small size, portability, simple operation, and rapid detection. Based on a nanochannel electrode chip, this invention highly integrates sample processing and detection modules, achieving both rapid bacterial separation and highly sensitive detection, realizing integrated rapid detection within a small device. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the overall structure of the device of the present invention;

[0039] Figure 2 This is a schematic diagram of the pressurization separation module structure of the present invention;

[0040] Figure 3 This is a schematic diagram of the electrode fixing part of the present invention;

[0041] Figure 4 This is a schematic diagram of the separation-detection process of the present invention;

[0042] Figure 5 This is a schematic diagram of the module connection of the present invention;

[0043] Figure 6 This is a flowchart of the impedance measurement process of the present invention;

[0044] Figure 7 This is a flowchart of the method of the present invention;

[0045] Figure 8 This is a standard curve diagram showing the relationship between the rate of change of impedance and the concentration of pathogenic bacteria, obtained in this invention.

[0046] In the diagram: 1. Electrode fixing part, 2. Waste liquid collection part, 3. Pressurization part, 4. Shell, 5. Top cover, 6. Front cover, 7. Top flip cover, 8. Base, 9. Connecting circuit board, 10. Nanoparticles, 11. Nanoparticle-bacterial complex, 12. Nanochannel, 13. Electrode. Detailed Implementation

[0047] To enable those skilled in the art to better understand the technical solution of the present invention, the method provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0048] The present invention relates to an integrated impedance detection system for the separation and detection of pathogenic bacteria, comprising: a nanochannel electrode chip (INCE) for adding a sample solution to generate an impedance change; a pressure separation module for separating the target pathogenic bacteria from the sample solution, wherein the INCE is mounted on and electrically connected to the pressure separation module; an impedance analysis module for receiving the measurement results of the impedance change generated by the INCE and transmitting the data, wherein the impedance analysis module is electrically connected to the pressure separation module; a control module for receiving signals from a host computer to control the impedance analysis module and the pressure separation module, and for receiving the measurement results transmitted from the impedance measurement module, performing data analysis to obtain the impedance change rate, and transmitting the data to the host computer; the control module is electrically connected to the host computer, the impedance analysis module, and the pressure separation module; a power supply module for supplying power to the control module, the pressure separation module, and the impedance analysis module, wherein the power supply module is electrically connected to the control module; and a housing 4 for mounting the power supply module, the control module, the pressure separation module, and the impedance analysis module, all of which are installed inside the housing 4. The housing 4 has an upper cover 5 and a front cover 6. When the upper cover 5 is open, it is used to install the nanochannel electrode chip INCE, add the sample solution to be tested, and observe the surface state of the nanochannel electrode chip INCE. When the front cover 6 is open, it is used to observe and disassemble the waste liquid collection section 2. The impedance analysis module includes a microcontroller and an impedance analysis chip. The microcontroller is electrically connected to the impedance analysis chip and the control module. The impedance analysis chip is electrically connected to the connection circuit board 9 of the pressure separation module.

[0049] The pressurized separation module includes an electrode fixing part 1, a waste liquid collection part 2, and a pressurization part 3. The electrode fixing part 1 is installed at the top of the waste liquid collection part 2, which is connected to the pressurization part 3. The pressurization part 3 is electrically connected to the control module. The electrode fixing part 1 includes a fixing box and a fixing plate. The fixing plate is horizontally installed on the fixing box, and an adjusting screw is vertically installed on the top of the fixing box, located directly above the fixing plate. The fixing plate includes an upper flip cover 7, a base 8, and a connecting circuit board 9. One side of the upper flip cover 7 is hinged to one side of the base 8. A groove is formed on the top surface of the base 8 away from the hinge side, and the connecting circuit board 9 is installed in the groove. A first through-hole is formed on the side of the base 8 near the hinge side. The nanochannel electrode chip INCE is horizontally placed on the top surface of the base 8 and the connecting circuit board 9 and electrically connected to the connecting circuit board 9. The upper cover 7 rotates around the hinge edge to completely cover the top surface of the nanochannel electrode chip INCE. A second through-hole, identical in shape to the first through-hole, is formed on the side of the upper cover 7 near the hinge edge, penetrating its own surface. After the upper cover 7 is closed, the first and second through-holes are aligned. The adjusting screw presses down on the top surface of the upper cover 7, clamping the nanochannel electrode chip INCE. The nanochannel electrode chip INCE has a detection area, with its upper and lower sides aligned with the second and first through-holes of the electrode fixing part 1. The electrode fixing part 1 is used to flatten and fix the nanochannel electrode chip, guide the filtered waste liquid into the waste liquid collection part 2, and connect the two electrodes of the electrode chip to the impedance analysis module. The pressurizing part 3 is used to create negative pressure below the nanochannel electrode chip, promoting the filtration of the solution from the nanochannel 12.

[0050] Specific embodiments of the present invention are as follows:

[0051] The waste liquid collection section 2 of the pressurization separation module is specifically a collection tank for collecting waste liquid, and the pressurization section 3 is specifically a miniature negative pressure pump; the impedance analysis chip of the impedance analysis module is specifically an AD5933 chip, etc.; the control module is specifically an STM32, etc.; and the host computer is specifically a computer.

[0052] Example 1:

[0053] Fabrication of nanochannel electrode chips INCE:

[0054] Gold layers were uniformly sputtered onto both sides of a porous anodic aluminum oxide (AAO) film with a pore size of approximately 300 nm, a thickness of 60 μm, and a diameter of 4 mm. Two electrodes 13 were used to connect the two gold layers respectively, and then the films were fixed and sealed with tape, exposing only the detection areas on the gold layer surfaces on both sides to obtain a sealed electrode. The sealed electrode was then washed sequentially with 1 μm H2SO4 solution and deionized water and dried. It was then immersed in 0.2% m / v bovine serum albumin (BSA) solution for 30 min. Finally, it was rinsed with deionized water to remove excess BSA solution and dried to obtain the nanochannel electrode chip INCE.

[0055] Preparation of the sample solution for testing using the immunomagnetic bead method:

[0056] Dilute the inactivated pathogenic bacteria solution to 10 using sterile phosphate-buffered saline (PBS) or chicken extract. 6 CFU / mL, take 1 mL of diluted pathogenic bacterial solution and mix with immunomagnetic beads MNPs-Ab at a volume ratio of 10:1 and incubate for 45 min to capture the MNPs-Ab-bacterial conjugate formed by the target pathogenic bacteria. After magnetic separation, discard the supernatant to obtain the precipitate. Wash the precipitate with phosphate-buffered saline (PBS) and resuspend it in 50 μL of PBS to obtain the test sample solution enriched and purified from the target pathogenic bacteria.

[0057] Using the pathogenic bacteria impedance detection device and method of the present invention, immunomagnetic bead MNPs-Ab and MNPs-Ab-bacterial conjugates were separated by filtration:

[0058] Take 10 μL of the above solution and drop it onto the detection area on the upper surface of INCE through the second through hole of the electrode fixing part. Apply 100 kPa pressure through the internal pressure separation module of the device to filter the solution through the nanochannel and rinse it thoroughly with deionized water.

[0059] Impedance detection was performed using the pathogenic bacteria impedance detection device and method of the present invention:

[0060] 10 μL of electrolyte was added to the surface of INCE. The electrolyte was connected through nanochannels to form a microelectrolysis cell. The impedance modulus Z at this frequency was measured, and the rate of change of impedance modulus ΔZ% was calculated. Finally, as shown... Figure 8 The standard curve showing the relationship between impedance change rate and pathogen concentration yields the concentration of pathogens in the sample solution.

[0061] This invention proposes an integrated impedance device and method for pathogen detection, combining separation and detection. Based on an integrated nanochannel electrode, it completes the separation and detection steps within a single device. Pressure is applied to filter the solution through the nanochannels, separating free antibody-modified magnetic beads (MNPs-Ab) and MNPs-Ab-bacterial conjugates. Simultaneously, quantitative detection is achieved through electrode impedance measurement, and the results can be transmitted to a computer. This device is convenient, sensitive, highly integrated, and easy to operate, showing promising prospects for rapid on-site detection.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the method of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An integrated impedance detection device for the isolation and detection of pathogenic bacteria, characterized in that: This includes the INCE nanochannel electrode chip, which is used to add the sample solution to be tested and generate an impedance change; It includes a pressurized separation module for separating target pathogens from the sample solution to be tested, and a nanochannel electrode chip INCE is mounted on the pressurized separation module and electrically connected to the pressurized separation module; It includes an impedance analysis module, which receives the measurement results of the impedance change generated by the nanochannel electrode chip INCE and transmits the data. The impedance analysis module is electrically connected to the pressure separation module. It includes a control module, which receives signals from the host computer to control the impedance analysis module and the pressure separation module, and receives the measurement results transmitted by the impedance measurement module, performs data analysis to obtain the impedance change rate, and then transmits it to the host computer; the control module is electrically connected to the host computer, the impedance analysis module, and the pressure separation module. Includes a power supply module for powering the control module, the pressure separation module, and the impedance analysis module; the power supply module is electrically connected to the control module. Includes a housing (4) for mounting the power module, control module, pressure separation module and impedance analysis module, all of which are mounted inside the housing (4); The pressurized separation module includes an electrode fixing part (1), a waste liquid collection part (2) and a pressurizing part (3). The electrode fixing part (1) is installed at the top of the waste liquid collection part (2). The waste liquid collection part (2) is connected to the pressurizing part (3). The pressurizing part (3) is electrically connected to the control module. The electrode fixing part (1) includes a fixing box and a fixing plate. The fixing plate is horizontally installed on the fixing box. An adjusting screw is vertically installed on the top of the fixing box. The adjusting screw is located directly above the fixing plate. The fixing plate includes an upper flip cover (7), a base (8), and a connecting circuit board (9). One side of the upper flip cover (7) is hinged to one side of the base (8). A groove is opened on the top surface of the base (8) away from the hinge side. The connecting circuit board (9) is installed in the groove. A first through hole is opened on the side of the base (8) near the hinge side, penetrating the surface of the plate. The nanochannel electrode chip INCE is horizontally placed on the top surface of the base (8) and the connecting circuit board (9) and Electrically connect the circuit board (9). After the upper cover (7) rotates around the hinge edge, it completely covers the top surface of the nanochannel electrode chip INCE. The upper cover (7) has a second through hole with the same shape as the first through hole on the side close to the hinge edge. After the upper cover (7) is covered, the first through hole and the second through hole are arranged opposite each other. The adjusting screw presses down on the top surface of the upper cover (7) so that the upper cover (7) clamps the nanochannel electrode chip INCE. The nanochannel electrode chip INCE has a detection area. The upper and lower sides of the detection area of ​​the nanochannel electrode chip INCE are directly opposite the second through hole and the first through hole of the electrode fixing part (1).

2. The integrated impedance detection device for the isolation and detection of pathogenic bacteria according to claim 1, characterized in that: The impedance analysis module includes a microcontroller and an impedance analysis chip. The microcontroller is electrically connected to the impedance analysis chip and the control module. The impedance analysis chip is electrically connected to the connection circuit board (9) of the pressure separation module.

3. The method for preparing the nanochannel electrode chip INCE of the integrated impedance detection device for separation and detection according to any one of claims 1-2, characterized in that: The method includes the following steps: 1) Gold layers are uniformly sputtered on both sides of the porous anodic aluminum oxide (AAO) film. Two electrodes (13) are used to connect the two gold layers respectively. Then, tape is used to fix and seal the film, exposing only the detection area on the surface of the gold layers on both sides to obtain a sealed electrode. 2) After cleaning the sealed electrode with H2SO4 solution and deionized water and drying it, it was immersed in bovine serum albumin (BSA) solution. Finally, it was rinsed with deionized water to remove excess BSA solution and dried to obtain the nanochannel electrode chip INCE. In step 1), the nanochannel electrode chip INCE is electrically connected to the connecting circuit board (9) of the pressure separation module through two electrodes (13); the shape of the detection area on both sides of the gold layer surface of the nanochannel electrode chip INCE is the same as the shape of the first through hole on the fixing plate of the electrode fixing part (1). When the nanochannel electrode chip INCE is fixed on the electrode fixing part (1), the upper and lower sides where the detection area of ​​the nanochannel electrode chip INCE is located are directly opposite the second through hole and the first through hole of the electrode fixing part (1).

4. The method for preparing the nanochannel electrode chip INCE of the integrated impedance detection device for separation and detection according to claim 3, characterized in that: In step 2), the sealed electrode is washed and dried sequentially with H2SO4 solution and deionized water, and then immersed in bovine serum albumin (BSA) solution. Specifically, the sealed electrode is washed and dried sequentially with 1 μm H2SO4 solution and deionized water, and then immersed in 0.2%-1% m / v bovine serum albumin (BSA) solution for 30-60 min.

5. The method for detecting the impedance of pathogenic bacteria using the integrated impedance detection device for separation and detection according to any one of claims 1-2, characterized in that: The method includes the following steps: 1) Fix the nanochannel electrode chip INCE to the electrode fixing part (1) so that the detection area of ​​the nanochannel electrode chip INCE is directly opposite the second through hole and the first through hole of the electrode fixing part (1). 2) A series of solutions containing pathogenic bacteria of the same volume with preset gradient concentrations are dripped from the second through hole of the electrode fixing part (1) onto the detection area of ​​the nanochannel electrode chip INCE. The pressurizing part (3) of the pressurizing separation module is controlled by the control module to draw air from the waste liquid collection part (2) below the detection area of ​​the nanochannel electrode chip INCE to form a negative pressure, so that the target is trapped on the surface and inside of the nanochannel electrode chip INCE. 3) The electrolyte solution is dropped from the second through hole of the electrode fixing part (1) onto the detection area of ​​the nanochannel electrode chip INCE. The impedance analysis module is controlled by the control module to detect the impedance change of the two electrodes (13) of the nanochannel electrode chip INCE and the measurement results are transmitted to the control module. 4) The control module obtains a standard curve of pathogen concentration and impedance change rate based on the preset gradient concentration of each solution containing pathogens and the measurement results of their impedance changes. 5) The sample solution to be tested is dropped from the second through hole of the electrode fixing part (1) onto the detection area of ​​the nanochannel electrode chip INCE, and then the operation of step 3) is continued to obtain the measurement result of the impedance change of the sample solution to be tested. The concentration of pathogenic bacteria in the sample solution to be tested is obtained by referring to the standard curve obtained in step 4), and the impedance detection of pathogenic bacteria is completed.

6. The method for detecting the impedance of pathogenic bacteria using the integrated impedance detection device for separation and detection according to claim 5, characterized in that: In step 5), the sample solution to be tested is prepared using the following method: The inactivated pathogenic bacteria solution was diluted with sterile phosphate-buffered saline (PBS) or chicken extract. The diluted pathogenic bacteria solution was mixed with antibody-modified nanoparticles (10) and incubated to capture the nanoparticle-pathogenic bacteria conjugate (11) formed by the target pathogenic bacteria. After magnetic separation, the supernatant was discarded to obtain the precipitate. The precipitate was washed with phosphate-buffered saline (PBS) and then resuspended in phosphate-buffered saline (PBS) to obtain the test sample solution.

7. The method for detecting the impedance of pathogenic bacteria using the integrated impedance detection device for separation and detection according to claim 5, characterized in that: In the preparation of the test sample solution, the inactivated pathogenic bacteria solution is diluted to 10 μL using sterile phosphate-buffered saline (PBS) or chicken extract. 2 -10 6 CFU / mL, take 1 mL of diluted pathogenic bacteria solution and mix with antibody-modified nanoparticles (10) at a volume ratio of 10:1 and incubate for 30-60 min to capture the nanoparticle-pathogenic bacteria conjugate (11) formed by the target pathogenic bacteria. Then, after magnetic separation, discard the supernatant to obtain the precipitate. Wash the precipitate with phosphate-buffered saline (PBS) solution and resuspend it in 50-100 μL of PBS solution to obtain the sample solution to be tested.

8. The method for detecting the impedance of pathogenic bacteria using the integrated impedance detection device for separation and detection according to claim 5, characterized in that: The negative pressure formed by the pressurization part (3) in step 2) is 50-100 kPa; The target material is the nanoparticle-pathogenic bacteria conjugate (11).

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