Full-liquid-phase escherichia coli immunoassay method, system, device and medium based on alternating current magnetization response of superparamagnetic nanoparticles

Abstract

The invention provides a full-liquid-phase escherichia coli immunoassay method, system, device and medium based on alternating current magnetization response of superparamagnetic nanoparticles. The method comprises the following steps: pre-dissolving a detection reagent in a reagent buffer solution; when escherichia coli detection is carried out, a detection reagent is taken into a detection reagent bottle, the magnetization response signal intensity of the reagent detected by detection equipment is used as an initial signal to be stored, a detected reagent is added into the initial signal, incubation is carried out after uniform mixing, and the incubation time is determined according to the binding force and affinity of an antigen and an antibody and the antigen and antibody detection time; and after the reaction is completed, taking out the reagent, measuring the magnetization response intensity of the reagent again by using the detection equipment, storing the magnetization response intensity as a signal after the reaction, calculating the change value of the corresponding emigration to complete the detection of the concentration or quantity of the escherichia coli, and finally carrying out rapid quantitative detection on the escherichia coli in the detected liquid. The anti-interference capability is strong, and the detection reagent is not interfered by other microorganisms in the environment after being treated.

Description

Technical Field

[0001] The invention relates to the technical field of Escherichia coli immunity, and in particular to a full liquid phase Escherichia coli immunoassay method, system, device and medium based on the alternating current magnetization response of superparamagnetic nanoparticles. Background Art

[0002] The existing traditional methods for detecting Escherichia coli mainly include: multiple tube fermentation method, membrane filter method and plate colony counting method.

[0003] (1) The multiple tube fermentation method is a traditional method for detecting Escherichia coli in water. This method is to continuously culture on a medium containing a fluorescent substrate at 44.5°C for 24 hours, and then produce glucuronidase, which decomposes the fluorescent substrate, thereby releasing fluorescent products, and then the culture medium produces characteristic fluorescence under ultraviolet light. This method can be used to make a statistical estimate of the number of colonies in the original sample. Generally, it involves the following experiments: first, fermentation in a single or double lactose bile salt fermentation tube; then, separation and culture test, separation using eosin methylene blue culture dishes; followed by secondary fermentation in a lactose fermentation tube, and finally completion through spore staining, Gram staining, microscopic observation, etc.

[0004] Advantages and disadvantages of the multiple-tube fermentation method: it does not require high test conditions and has low cost, but the detection time is long and it is easily interfered by other conditions, which in turn affects the accuracy of the measurement results.

[0005] (2) The basic steps of the membrane filter method are: first, add about 10 mL of sterile dilution water to the filter, then add an appropriate amount of the aqueous solution of the food to be tested that has been fully mixed to the filter, and perform suction filtration. Before the filtration is completed, add a small amount of sterile dilution water to rinse the inner wall of the filter so that all the bacteria in the aqueous solution are concentrated on the filter membrane; use sterile tweezers to clamp the edge of the filter membrane and transfer it to the M-FC culture medium, with the bacteria-retaining side of the filter membrane facing up, and the filter membrane and the culture medium completely close to each other without bubbles. After the culture dish is tightly covered, place it in a constant temperature incubator that can accurately maintain a constant temperature of 44.5±0.5℃. After 24±2h of incubation, if the fecal coliform bacteria appear blue or blue-green, count the number of such colonies. For difficult colonies, inoculate the suspicious colonies into EC culture medium, incubate at 44.5±0.5℃ for 24±2h, and observe whether gas is produced to determine whether such suspicious colonies are fecal coliform bacteria. Then calculate the approximate number of fecal coliforms per liter of water sample and finally convert it into the number of E. coli contained in the food.

[0006] Advantages and disadvantages of the membrane filtration method: The advantage of the membrane filtration method is that it is very convenient to detect large quantities of low turbidity water samples, and is particularly suitable for routine monitoring in water plants. The disadvantage is that it is not suitable for water samples with more impurities and easy to clog the filter pores. The specificity is not high, and the results are easily affected by other bacteria in the water samples and misjudged. Further confirmation tests are required to finally determine the results, but confirmation of the results takes at least 2 to 3 days, which is not conducive to the prevention and response to public health emergencies.

[0007] (3) Plate method: Aseptic operation: Use a sterile pipette to draw 1 mL of sample solution with the same dilution as the lactose bile salt fermentation method, transfer it into a sterile culture dish, inject about 10 mL of COLIID selective colorimetric medium cooled to 45°C into the culture dish, and quickly rotate the culture dish to mix evenly. After solidification, inject about 5 mL of medium cooled to 45°C into the culture dish again, and quickly shake it to cover the surface of the plate. After solidification, turn the culture dish over and incubate at 37°C for 24 hours, observe the color and morphology of the colonies, and count them. Make 2 culture dishes in parallel for each dilution. This method is to dilute the sample at a certain ratio, disperse the microorganisms in it into single cells after dilution, and then culture them under certain conditions until they grow into colonies that can be observed with the naked eye. Finally, the number of bacteria in the unit sample is calculated based on the sample quantity and dilution.

[0008] Advantages and disadvantages of plate colony counting method: Plate colony counting method usually performs gradient dilution, so the linear range of counting is large, and the number of live bacteria in the sample can be measured. It is intuitive, can read directly, has sensitive detection, and has good parallelism. In addition, since the bacterial suspension is poured or spread, it is relatively uniform and can better reflect the density of the colony. However, the operation of the plate method is more cumbersome, and the measured value is often affected by various factors. A single colony may also come from 2 to 3 or more cells in the sample. Therefore, the results of plate colony counting are often low, and the inspection time is long.

[0009] The new technical methods for Escherichia coli detection mainly include: gas chromatography (GC) and high performance liquid chromatography (HPLC), ATP bioluminescence technology, PCR detection technology and immunomagnetic bead technology (IMS).

[0010] (1) Gas chromatography (GC) and high performance liquid chromatography (HPLC). Gas chromatography mainly provides as many chemical components as possible for subsequent analysis and research after a series of derivatization pretreatments of Escherichia coli. The degree of contamination of Escherichia coli can be analyzed by headspace gas chromatography, which is based on the use of CO2, a microbial metabolite at the top of the food sealing system, to analyze the microorganisms. This method is of great significance for food quality and safety testing. Compared with the above-mentioned traditional technologies, this method has the advantages of fast detection speed and high sensitivity. High performance liquid chromatography (HPLC) mainly analyzes nucleic acids based on the principle of ion pairing reverse phase chromatography. HPLC is mainly used for the separation of DNA fragments. Long-chain DNA carries more phosphate groups, so more TEAA will fuse with it, and its residence time in the column will increase. Because acetonitrile is hydrophilic, DNA can be separated by it. At a certain denaturation temperature, obvious absorption peaks can be shown through the spectrum. This technology has the advantages of high accuracy, high sensitivity, and low cost, which can greatly improve work efficiency.

[0011] (2) ATP bioluminescence technology. ATP bioluminescence technology is a rapid microbial detection method that has developed rapidly in recent years. ATP is the most common energy metabolite in living cells, providing cells with energy required for various physiological activities, and its content in organisms is maintained within a certain range. One method for detecting ATP content is fluorescence spectrophotometry. Bioluminescence is the fluorescence emitted by living cells under the catalysis of luciferase. The luciferase currently used is mainly derived from fireflies in North America. It is a protein with a relative molecular mass of 62,000. It can catalyze the oxidation reaction of luciferin. The end product of this reaction is unstable and quickly decomposes to produce fluorescence. Compared with traditional detection methods, the difference is that the test results can be obtained within a few minutes, and the fluorescence spectrophotometer is portable and very convenient to use. It is suitable for on-site detection. This technology can be used to detect the level of tiny pollutants and the cleanliness of food processing equipment and its surface.

[0012] (3) PCR detection technology. The polymerase chain reaction technology, also known as PCR (polymerase chain reaction), was first proposed by Kleppe et al. in 1971 and was not recognized as a detection method until 1985. PCR detection technology is a polymerase chain reaction that uses three steps of denaturation-annealing-extension to enable DNA genes to unwind and unwind in vitro, similar to a form of in vitro increase and replication. Theoretically, a DNA molecule can be amplified to 106-109 times within 2 hours, and agarose gel electrophoresis is used to detect the fluorescent bands of PCR amplification products.

[0013] At present, PCR technology is widely used in many disciplines of life sciences. In the field of food microbial detection, PCR can be used to quickly detect the content of microorganisms in food. Commonly used methods include immune PCR, real-time fluorescence quantitative PCR, multiplex PCR, reverse transcription PCR and real-time quantitative PCR. Shi Yun et al. established a double PCR detection method for Escherichia coli in meat and meat products, but this method requires a long time for sample purification and requires high professional knowledge of the operator.

[0014] (4) Immunomagnetic bead technology (IMS). Immunomagnetic bead technology is a technology for separating Escherichia coli. The principle is to use magnetic beads as antibody carriers, bind to Escherichia coli, and use magnetic force to achieve mechanical movement to separate Escherichia coli. Compared with other bacterial separation methods, immunomagnetic bead technology has greatly improved the detection efficiency of pathogenic Vibrio parahaemolyticus in samples. Immunomagnetic bead technology can quickly process different microorganisms in samples of different strains, and this method can greatly improve detection efficiency.

[0015] In general, the existing methods for detecting E. coli, whether traditional or new detection technologies, are too cumbersome to detect, requiring the E. coli in the test sample to be cultured, filtered, and processed, which consumes a lot of time. In addition, the detection equipment cannot be portable, and some detection methods have strict requirements on the source, type, and state of the sample. New technologies have high portability and fast detection speed, but the detection markers in this method are relatively common metabolites in microorganisms and cannot accurately detect E. coli.

[0016] Therefore, there is a need for a new technology for accurate detection of Escherichia coli, and it is necessary to meet the requirements of high portability of the equipment, high detection sensitivity (can realize the detection of a single bacterium or a bacterial group) and low requirements for the detection sample (such as liquid, colloid, etc.). The present invention uses the magnetization response characteristics and targeting characteristics of biofunctionalized superparamagnetic nanoparticles to develop a new Escherichia coli detection technology, which has high specificity and can directly bind to the Escherichia coli in the object to be detected without being interfered by other microorganisms; high portability, small size of the detection equipment, and convenient to carry; fast detection speed, using the change of the magnetization response signal of the biofunctionalized superparamagnetic nanoparticles before and after the reaction with Escherichia coli to determine the number of Escherichia coli, only need to wait for 3 to 5 minutes of reaction time; strong anti-interference ability, by closing and modifying the biofunctionalized superparamagnetic nanoparticles, the anti-interference force will be improved and the targeted reaction rate will be improved; high sensitivity, can realize the detection of a single bacterium; low requirements for the detection sample, whether the Escherichia coli in the solution or the surface of the object can be detected; low cost, the cost of a single detection reagent is only 2 to 3 yuan. Summary of the invention

[0017] The present invention aims to solve at least one of the technical problems existing in the prior art or related art. Therefore, there is a need for a new technology for accurately detecting Escherichia coli, which needs to meet the requirements of high portability, high detection sensitivity (capable of detecting a single bacterium or a bacterial group), and low requirements for the detection sample (such as liquid, colloid, etc.). The invention develops a new Escherichia coli detection technology by utilizing the magnetization response characteristics and targeting characteristics of biofunctionalized superparamagnetic nanoparticles, which has high specificity and can directly specifically combine with a specific type of Escherichia coli in a north side sample; it has high portability, and the detection device is small in size and can be conveniently carried; it has high detection speed, and the overall detection process is simple to operate, and it is not necessary to culture the Escherichia coli, and the number of the Escherichia coli can be determined by only utilizing the change in the magnetization response signal of the biofunctionalized superparamagnetic nanoparticles before and after the reaction with the Escherichia coli, and it only needs to wait for a reaction time of 3 to 5 minutes; it has strong anti-interference ability, and the anti-interference ability and the targeting reaction rate can be improved by sealing and modifying the biofunctionalized superparamagnetic nanoparticles; it has high sensitivity and can realize the detection of a single bacterium; it has low requirements on the detection sample, and both the Escherichia coli in the solution and the surface of the object can be detected; it has low cost, and the cost of a single detection reagent is only 2 to 3 yuan.

[0018] To this end, the first object of the present invention is to provide a full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles.

[0019] The second objective of the present invention is to provide a full liquid phase Escherichia coli immunoassay system based on the AC magnetization response of superparamagnetic nanoparticles.

[0020] A third objective of the present invention is to provide a computer device.

[0021] A fourth objective of the present invention is to provide a computer-readable storage medium.

[0022] In order to achieve the above-mentioned object, the technical solution of the first aspect of the present invention provides a full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles, characterized in that it comprises:

[0023] Reagent buffer system: The detection reagent is pre-dissolved in the reagent buffer, and the reagent buffer can be selected from various commonly used buffer systems that will not interfere with the antigen-antibody reaction;

[0024] Reagent reaction system: When performing E. coli detection, first take the detection reagent into the detection reagent bottle, use the detection equipment to detect the magnetization response signal intensity of the reagent as the initial signal for preservation, then add the detected reagent (which can be a solution or colloid) to it, mix well and incubate, the incubation time is determined according to the binding force, affinity and antigen-antibody detection time of the antigen and antibody, and can usually be set to 3 to 5 minutes. After the reaction is completed, take out and use the detection equipment to measure the magnetization response intensity of the reagent again as the post-reaction signal for preservation, and finally calculate the change value of the corresponding migration to complete the detection of the concentration or number of E. coli. Since the detection reagent contains a superparamagnetic nano-magnetic core affected by the Brownian relaxation time, after the antibody on its surface reacts with the protein on the surface of E. coli to form a large aggregate, its superparamagnetic response will change significantly, and then the E. coli in the detected liquid can be quickly and quantitatively detected.

[0025] Preferably, the detection reagent comprises biofunctionalized superparamagnetic nanoparticles, and specific antibodies or proteins are combined with the superparamagnetic nanoparticles by covalent bonds or non-covalent coupling to react specifically with the object to be detected; the buffer may contain an appropriate amount of preservatives, surfactants, blocking agents and other necessary substances to improve the stability and shelf life of the reagent.

[0026] Preferably, the selection of weak magnetic sensors in the detection system: This technology detects the concentration of the detected object based on the changes in the AC magnetization response of superparamagnetic nanoparticles before and after the reaction, and the magnetization of superparamagnetic nanoparticles and the collection of magnetization response signals use weak magnetic detection coils. The weak magnetic detection coil consists of two parts: an excitation coil and a detection coil; the highly sensitive harmonic signal measurement is achieved through the independently developed high-sensitivity weak magnetic detection system with a balance fine-tuning function and low thermal noise. To ensure the accuracy of continuous signal acquisition, a linear bipolar power supply system is used, and the input signal is generated by a function waveform generator, which is connected to the excitation coil after amplification by a power amplifier. In order to increase the excitation intensity, the excitation coil is connected in series with a power capacitor to form a resonance, reducing the AC impedance of the excitation circuit. The function waveform generator synchronously provides a reference signal for the phase-locked amplifier, and the detection coil collects the sample signal. After filtering and extracting the harmonic signal by the phase-locked amplifier, the system detection sensitivity can reach 100nV.

[0027] Preferably, the selection of other components such as a phase-locked amplifier, a power supply, a signal generator (whether to use it can be determined based on the performance of the power supply), a shielding device, a display device, a signal amplifier, a filter, etc., needs to be further selected according to specific sensitivity requirements. The important components such as the phase-locked amplifier and the power supply are explained in the embodiments.

[0028] The technical solution of the second aspect of the present invention also provides a full liquid phase E. coli immunoassay system based on the AC magnetization response of superparamagnetic nanoparticles, including: a reagent buffer system; the detection reagent is pre-dissolved in a reagent buffer, and the reagent buffer can be selected from various commonly used buffer systems that do not interfere with the antigen-antibody reaction; a reagent reaction system; when performing E. coli detection, firstly, the detection reagent is taken into the detection reagent bottle, and the magnetization response signal intensity of the detection reagent is detected by the detection device as the initial signal for preservation, and then the detected reagent (which can be a solution or a colloid) is added thereto, mixed and incubated, and the incubation time is determined according to the binding force, affinity and antigen-antibody detection time of the antigen and antibody, and can usually be set to 3 to 5 minutes. After the reaction is completed, the magnetization response intensity of the reagent is taken out and measured again by the detection device as a post-reaction signal for preservation, and finally the detection of the concentration or quantity of E. coli can be completed by calculating the change value of the corresponding migration. Since the detection reagent contains superparamagnetic nano-cores that are affected by the Brownian relaxation time, after the antibodies on its surface react with the proteins on the surface of Escherichia coli to form large aggregates, its superparamagnetic response will change significantly, thereby enabling rapid quantitative detection of Escherichia coli in the liquid being tested.

[0029] Preferably, the detection reagent comprises biofunctionalized superparamagnetic nanoparticles, and specific antibodies or proteins are combined with the superparamagnetic nanoparticles by covalent bonds or non-covalent coupling to react specifically with the object to be detected; the buffer may contain an appropriate amount of preservatives, surfactants, blocking agents and other necessary substances to improve the stability and shelf life of the reagent.

[0030] Preferably, the selection of weak magnetic sensors in the detection system: This technology detects the concentration of the detected object based on the changes in the AC magnetization response of superparamagnetic nanoparticles before and after the reaction, and the magnetization of superparamagnetic nanoparticles and the collection of magnetization response signals use weak magnetic detection coils. The weak magnetic detection coil consists of two parts: an excitation coil and a detection coil; the highly sensitive harmonic signal measurement is achieved through the independently developed high-sensitivity weak magnetic detection system with a balance fine-tuning function and low thermal noise. To ensure the accuracy of continuous signal acquisition, a linear bipolar power supply system is used, and the input signal is generated by a function waveform generator, which is connected to the excitation coil after amplification by a power amplifier. In order to increase the excitation intensity, the excitation coil is connected in series with a power capacitor to form a resonance, reducing the AC impedance of the excitation circuit; the function waveform generator synchronously provides a reference signal for the phase-locked amplifier, and the detection coil collects the sample signal. After filtering and extracting the harmonic signal by the phase-locked amplifier, the system detection sensitivity can reach 100nV.

[0031] Preferably, the selection of other components such as a phase-locked amplifier, a power supply, a signal generator (whether to use it can be determined based on the performance of the power supply), a shielding device, a display device, a signal amplifier, a filter, etc., needs to be further selected based on specific sensitivity requirements.

[0032] The technical solution of the third aspect of the present invention also provides a computer device, which includes a memory, a processor, and a computer program stored in the memory and executable on the processor. When the processor executes the computer program, the steps of the full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles as in any of the above technical solutions are implemented.

[0033] The technical solution of the fourth aspect of the present invention also provides a computer-readable storage medium on which a computer program is stored. When the computer program is executed by a processor, the steps of the full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles as in any of the above technical solutions are implemented.

[0034] Beneficial effects of the present invention:

[0035] (1) The present invention provides a full liquid phase E. coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles. It proposes for the first time to use the relaxation time characteristics and magnetization response characteristics of superparamagnetic nanoparticles to detect E. coli. E. coli is a relatively common bacterium, a single-cell organism, with a volume of several microns. The present invention utilizes the targeting effect of antibodies that have the function of specifically recognizing E. coli surface proteins, modifies the antibodies on the surface of superparamagnetic nanoparticles, utilizes the change in the volume of superparamagnetic nanoparticles caused by the binding of antibodies to bacteria, and then reduces the magnetization response intensity. The concentration or number of E. coli is determined based on the change in the magnetization response intensity.

[0036] (2) The full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles provided by the present invention has a simple detection process and a fast detection speed. It does not require operations such as culturing Escherichia coli, and only requires waiting for the reaction time between the antibody and the Escherichia coli surface protein.

[0037] (3) The full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles provided by the present invention has high specificity. The use of superparamagnetic nanoparticles with biological functionalization can specifically identify the type of Escherichia coli to be detected in the detected object.

[0038] (4) Strong anti-interference ability. After treatment, the detection reagent will not be interfered by other microorganisms in the environment.

[0039] (5) The detection equipment is highly portable.

[0040] (6) High detection sensitivity.

[0041] (7) Low testing cost.

[0042] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 A schematic diagram showing single-stage structure detection in a full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0044] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0045] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0046] Among them, the signal detection principle is:

[0047] Among them, single-level structure detection:

[0048] Figure 1 (b): taking out the detection reagent and adding it to a reagent bottle, adding a buffer solution to the reagent bottle, and detecting the magnetization response signal of the reagent by the sensor as the initial signal value;

[0049] Figure 1 (c): After adding blood to the sample bottle, the object to be detected in the blood will specifically bind to the targeted magnetic beads to form aggregates, and the particle size will increase. There will also be some aggregates formed by non-specific adsorption. The particle size of the aggregates is large, so the magnetization response signal value of the reagent is reduced. The sensor detects the magnetization response signal at this time as the reflected signal value, and the concentration value of the object to be detected can be obtained by calculating the signal change.

[0050] The structure and specific implementation of the excitation coil and the detection coil refer to patents "202210091820.0" and "202210091551.8". The important components such as the phase-locked amplifier and the power supply are described in the embodiment.

[0051] Among them, the new technology for Escherichia coli detection: applying magnetic sensitive immunoassay technology to the rapid detection of Escherichia coli can achieve fast and accurate detection, and can adjust the detection reagent type according to different Escherichia coli types. It has fast detection speed, strong specificity, high sensitivity, low cost, strong anti-interference ability, and can also achieve the separation and purification of Escherichia coli and reagents.

[0052] Among them, the detection device: according to the Brownian relaxation time characteristics of superparamagnetic nanoparticles, when the volume of magnetic nanoparticles increases several times or even dozens of times, its magnetization response signal will decrease. Utilizing this characteristic of magnetic nanoparticles, an alternating magnetic field is used to magnetize superparamagnetic nanoparticles to generate a magnetization response signal, and a weak magnetic sensor is used to collect weak magnetic signals, such as a weak magnetic detection coil, a TMR sensor, etc.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A full liquid phase Escherichia coli immunoassay method based on the alternating current magnetization response of superparamagnetic nanoparticles, characterized in that: include: Reagent buffer system; The detection reagent is pre-dissolved in a reagent buffer, and the reagent buffer can be any commonly used buffer system that does not interfere with the antigen-antibody reaction; Reagent reaction system; when performing E. coli detection, first take the detection reagent into the detection reagent bottle, use the detection equipment to detect the magnetization response signal intensity of the reagent as the initial signal for preservation, then add the detected reagent (which can be a solution or colloid) into it, mix well and incubate, the incubation time is determined according to the binding force, affinity and antigen-antibody detection time of the antigen and antibody, and can usually be set to 3 to 5 minutes; after the reaction is completed, take out and use the detection equipment to measure the magnetization response intensity of the reagent again as the post-reaction signal for preservation, and finally calculate the change value of the corresponding shift to complete the detection of the concentration or number of E. coli. Since the detection reagent contains superparamagnetic nano-magnetic cores affected by Brownian relaxation time, after the antibodies on its surface react with the proteins on the surface of E. coli to form large aggregates, its superparamagnetic response will change significantly, and then the E. coli in the detected liquid can be quickly and quantitatively detected.

2. The full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: The detection reagent comprises biologically functionalized superparamagnetic nanoparticles, which are combined with specific antibodies or proteins by means of covalent bonds or non-covalent coupling to react specifically with the detected object; The buffer may contain appropriate amounts of preservatives, surfactants, blocking agents and other necessary substances to improve the stability and shelf life of the reagents.

3. The full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: Selection of weak magnetic sensors in the detection system: This technology detects the concentration of the detected object based on the changes in the AC magnetization response of superparamagnetic nanoparticles before and after the reaction. The magnetization of superparamagnetic nanoparticles and the collection of magnetization response signals use weak magnetic detection coils; the weak magnetic detection coils include an excitation coil and a detection coil. High-sensitivity harmonic signal measurement is achieved through the independently developed high-sensitivity weak magnetic detection system with balance fine-tuning function and low thermal noise. In order to ensure the accuracy of continuous signal acquisition, a linear bipolar power supply system is adopted. The input signal is generated by the function waveform generator, and then connected to the excitation coil after amplification by the power amplifier; in order to increase the excitation intensity, the excitation coil and the power capacitor are connected in series to form resonance, reducing the AC impedance of the excitation circuit; The function waveform generator synchronously provides the reference signal for the phase-locked amplifier. The detection coil collects the sample signal. After filtering and the phase-locked amplifier extracts the harmonic signal, the system detection sensitivity can reach 100nV level.

4. The full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: The selection of other components such as phase-locked amplifier, power supply, signal generator (the use of which may be determined based on the performance of the power supply), shielding device, display device, signal amplifier, filter, etc., needs to be further selected based on specific sensitivity requirements.

5. A full liquid phase Escherichia coli immunoassay system based on the alternating current magnetization response of superparamagnetic nanoparticles, characterized in that: include: Reagent buffer system; The detection reagent is pre-dissolved in a reagent buffer, and the reagent buffer can be any commonly used buffer system that does not interfere with the antigen-antibody reaction; Reagent reaction system: When performing E. coli detection, first take the detection reagent into the detection reagent bottle, use the detection equipment to detect the magnetization response signal intensity of the reagent as the initial signal for preservation, then add the detected reagent (which can be a solution or colloid) to it, mix well and incubate, the incubation time is determined according to the binding force, affinity and antigen-antibody detection time of the antigen and antibody, and can usually be set to 3 to 5 minutes. After the reaction is completed, take out and use the detection equipment to measure the magnetization response intensity of the reagent again as the post-reaction signal for preservation, and finally calculate the change value of the corresponding migration to complete the detection of the concentration or number of E. coli. Since the detection reagent contains a superparamagnetic nano-magnetic core affected by the Brownian relaxation time, after the antibody on its surface reacts with the protein on the surface of E. coli to form a large aggregate, its superparamagnetic response will change significantly, and then the E. coli in the detected liquid can be quickly and quantitatively detected.

6. The all-liquid phase Escherichia coli immunoassay system based on superparamagnetic nanoparticles AC magnetization response according to claim 5, characterized in that: The detection reagent comprises biologically functionalized superparamagnetic nanoparticles, which are combined with specific antibodies or proteins by means of covalent bonds or non-covalent coupling to react specifically with the detected object; The buffer may contain appropriate amounts of preservatives, surfactants, blocking agents and other necessary substances to improve the stability and shelf life of the reagents.

7. The all-liquid phase Escherichia coli immunoassay system based on superparamagnetic nanoparticles AC magnetization response according to claim 5, characterized in that: Selection of weak magnetic sensors in the detection system: This technology detects the concentration of the detected object based on the changes in the AC magnetization response of superparamagnetic nanoparticles before and after the reaction. The magnetization of superparamagnetic nanoparticles and the collection of magnetization response signals use weak magnetic detection coils. The weak magnetic detection coils include an excitation coil and a detection coil. High-sensitivity harmonic signal measurement is achieved through the independently developed high-sensitivity weak magnetic detection system with balance fine-tuning function and low thermal noise. To ensure the accuracy of continuous signal acquisition, a linear bipolar power supply system is adopted. The input signal is generated by the function waveform generator, and the excitation coil is connected after amplification by the power amplifier. To increase the excitation intensity, the excitation coil and the power capacitor are connected in series to form resonance and reduce the AC impedance of the excitation circuit. The function waveform generator synchronously provides the reference signal of the phase-locked amplifier, and the detection coil collects the sample signal. After filtering and extracting the harmonic signal by the phase-locked amplifier, the system detection sensitivity can reach 100nV level.

8. The all-liquid phase Escherichia coli immunoassay system based on superparamagnetic nanoparticle AC magnetization response according to claim 5, characterized in that: The selection of other components such as phase-locked amplifier, power supply, signal generator (the use of which may be determined based on the performance of the power supply), shielding device, display device, signal amplifier, filter, etc., needs to be further selected based on specific sensitivity requirements.

9. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein: When the processor executes the computer program, the steps of the full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles are implemented as described in any one of claims 1 to 4.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the full liquid phase Escherichia coli immunoassay method based on the AC magnetization response of superparamagnetic nanoparticles as claimed in any one of claims 1 to 4 are implemented.

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