Reverse enzyme-linked immunosorbent assay

Abstract

The present application discloses a reverse enzyme-linked immunosorbent assay (ELISA) method, which belongs to the field of biomedical detection technology. The method comprises the following steps: Step 1, centrifugation and gradient dilution of the biological sample, and simultaneously preparing uncoupled specific antibodies / antigens and double-enzyme-labeled secondary antibodies, and optimizing the reagent ratio to ensure optimal activity; Step 2, allowing the sample to be tested to compete with the specific antibody / antigen for binding, adding the double-enzyme-labeled secondary antibody to bind to the unoccupied antibody / antigen binding sites to form an enzyme-labeled complex; Step 3, adding nanomagnetic particles, and promoting the efficient capture of the complex by applying a pulsed magnetic field to form a complete immune complex; Step 4, using a gradient magnetic field to separate the immune complex, collecting the liquid phase of the unbound enzyme-labeled secondary antibody in the supernatant phase, and using time-resolved fluorescence-colorimetric dual mode to detect its enzyme activity; Step 5, accurately calculating the target antigen / antibody concentration in combination with the sample-specific correction factor; Step 6, generating a standardized test report.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, more specifically, to a reverse enzyme-linked immunosorbent assay method. BACKGROUND

[0002] Reverse enzyme-linked immunosorbent assay (rELISA) is a common immunological analysis method used for detecting and quantifying specific antigens or antibodies in samples. Unlike traditional enzyme-linked immunosorbent assay (ELISA), reverse enzyme-linked immunosorbent assay involves immobilizing target molecules (usually antigens or antibodies) on a solid support, and then adding enzyme-labeled antibodies that detect specific target molecules to generate a signal in the reaction. This method has high sensitivity and specificity, and is simple to operate, so it is widely used in medical diagnosis, food safety, environmental monitoring and other fields.

[0003] Currently, reverse enzyme-linked immunosorbent assay has achieved significant application results in the detection of biomarkers, disease diagnosis, and food allergen detection. Especially in early disease screening and pathogen detection, rELISA has become an important detection tool due to its high sensitivity. However, the existing reverse enzyme-linked immunosorbent assay method still faces some technical challenges, such as further improving the detection sensitivity, quickly and accurately analyzing the results, and automating and high-throughput detection in large-scale detection.

[0004] Although the application potential of rELISA technology is huge, in the actual application process, due to the complexity of the sample and the existence of interference factors, how to further improve its stability and accuracy and reduce human error in the operation process is still a bottleneck for technical development. In addition, the existing technology focuses more on small-scale testing under laboratory conditions, and is still insufficient in terms of large-scale on-site detection, integration of automated equipment, and optimization of data processing efficiency.

[0005] In summary, how to improve the sensitivity and stability of the reverse enzyme-linked immunosorbent assay method and solve the application problems in large-scale on-site detection has become a technical problem that needs to be solved. SUMMARY

[0006] In order to overcome the series of defects existing in the prior art, the purpose of the present application is to provide a reverse enzyme-linked immunosorbent assay method, which includes the following steps:

[0007] Step 1, centrifugal filtration and gradient dilution are performed on the biological sample, and un-coupled specific antibodies / antigens and double enzyme-labeled secondary antibodies are prepared, and the reagent ratio is optimized to ensure optimal activity;

[0008] Step 2, in the microfluidic reaction chamber, the sample to be tested competes with specific antibodies / antigens for binding, and then a double-enzyme-labeled secondary antibody is added to bind to the unoccupied antibody / antigen binding sites, forming an enzyme-labeled complex;

[0009] Step 3, add nanometer magnetic particles modified with specific antibodies / antigens, and apply a pulsed magnetic field to promote efficient capture of the complex, forming a complete immune complex;

[0010] Step 4, separate the immune complex using a gradient magnetic field, collect the liquid phase of the unbound enzyme-labeled secondary antibody in the supernatant, and detect its enzyme activity using time-resolved fluorescence-colorimetric dual-mode detection;

[0011] Step 5, based on the enzyme activity data, reverse calculate the amount of bound enzyme-labeled secondary antibody, and accurately calculate the target antigen / antibody concentration based on the sample-specific correction factor;

[0012] Step 6, perform internal quality control procedures to verify the accuracy of the detection results, and generate a standardized detection report.

[0013] Further, step 1 includes the following steps:

[0014] Centrifuge the collected biological sample at 3000 rpm for 10 minutes at 4°C, then filter it using a 0.22 μm filter to remove large particulate matter and cell debris;

[0015] Dilute the filtered sample with PBS buffer at pH 7.4, with dilution ratios including 1:2, 1:4, 1:8, 1:16, and 1:32, and store them in low-adsorption microcentrifuge tubes at 4°C for standby;

[0016] Prepare a solution of uncoupled specific antibodies / antigens, dilute the high-purity antibodies / antigens obtained by affinity chromatography purification to 5-10 μg / ml with 0.01M PBS buffer at pH 7.4, and store them at 4°C in the dark to maintain their optimal immunological activity;

[0017] Prepare a double-enzyme-labeled secondary antibody, select horseradish peroxidase and alkaline phosphatase to be labeled on the secondary antibody molecule by glutaraldehyde cross-linking, ensure that the active sites of the two enzymes do not interfere with each other, and then use gel filtration column to separate and purify, remove unbound enzyme molecules;

[0018] Determine the optimal concentration ratio of antigen / antibody to double-enzyme-labeled secondary antibody through orthogonal experiment design, test the activity under different ion strength and pH conditions, and select the condition with the highest signal-to-noise ratio as the final working parameter;

[0019] Prepare a stabilizer solution containing 1% BSA, 0.05% Tween-20 and 0.02% sodium azide, and add it to the antigen / antibody and double-enzyme-labeled secondary antibody solutions, respectively. After mixing, store at 4°C in the dark to ensure that the reagents remain stable and active during storage.

[0020] Further, step 2 includes the following steps:

[0021] Inject 50-100 μL of the biological sample into the reaction chamber of the microfluidic chip, and control the flow rate to be stable at 5-10 μL / min through capillary action and a micro-pump;

[0022] Inject the pre-optimized concentration of specific antibody / antigen solution, incubate at 37°C under constant temperature conditions for 20 minutes, and maintain oscillation at 60 rpm to promote the competitive binding of the target analyte in the sample to the specific antibody / antigen;

[0023] Precisely inject the double-enzyme-labeled secondary antibody solution through the independent channel of the microfluidic chip, control the flow rate to be 3-5 μL / min, ensure that it is fully mixed with the solution in the reaction chamber, and incubate at 25°C for 15 minutes to allow the double-enzyme-labeled secondary antibody to specifically bind to the antibody / antigen binding sites that are not occupied by the target analyte;

[0024] During the entire competitive binding process, the temperature and pH sensors built into the microfluidic chip monitor the reaction conditions in real time, and the feedback control system automatically adjusts the heating elements and micro-buffer injection to ensure that the reaction process is maintained at a pH of 7.2-7.4 and a temperature of 37±0.5°C;

[0025] After the completion of the competition reaction, switch the control micro-valve to introduce a 0.05% concentration of non-ionic surfactant washing solution, and perform gentle flushing at a flow rate of 3-5 μL / min to remove non-specific binding substances while retaining the specifically formed immune complexes;

[0026] In the mixing area of the microfluidic chip, the "H-type" micro-channel structure is used to enhance the collision probability between molecules through the laminar flow effect, and the built-in ultrasonic micro-vibrator is used for intermittent vibration with 5 seconds on and 10 seconds off to further promote the efficient binding of the double-enzyme-labeled secondary antibody to the unoccupied binding sites;

[0027] Real-time monitoring of the reaction process is performed through the built-in micro-spectrophotometric sensor in the reaction chamber, and when the signal intensity change rate decreases to below 0.5% / min, the reaction is automatically terminated and the next step is entered to ensure that the reaction reaches an equilibrium state and forms stable enzyme-labeled complexes.

[0028] Further, the microfluidic chip is made of polydimethylsiloxane, polymethyl methacrylate or epoxy resin material, the internal channel width is 100-200 μm, the depth is 40-100 μm, the total reaction volume is 10-100 μL, the chip surface is treated by plasma to enhance hydrophilicity, and is coated with an anti-adhesion layer to reduce non-specific adsorption.

[0029] Further, step 3 comprises the following steps:

[0030] The pre-prepared surface-modified nano-magnetic particles are injected into the microfluidic reaction chamber to covalently connect specific antibodies / antigens on the surface of the magnetic particles by EDC / NHS chemical cross-linking method, the concentration is controlled at 0.5-1 mg / mL, the injection rate is 2-3 μL / min, and the magnetic particles are uniformly dispersed in the reaction system to avoid aggregation phenomenon affecting the capture efficiency;

[0031] The electromagnetic coil array integrated at the bottom of the microfluidic chip is started to generate a pulsed magnetic field with a strength of 0.2-0.5 T, the pulse frequency is set to 1-5 Hz, the duty cycle is 30%, the magnetic field direction is perpendicular to the fluid flow direction, and the nano-magnetic particles produce controlled micro-movement in the reaction chamber to increase the collision probability with the immune complexes in the solution;

[0032] Under the action of the pulsed magnetic field, the specific antibodies / antigens on the surface of the nano-magnetic particles bind with the immune complexes formed in the solution with high affinity, and the unoccupied specific antibodies / antigens capture the target analytes in the solution to form a complete immune complex with a sandwich structure of "nano-magnetic particle-antigen / antibody-double enzyme-labeled secondary antibody";

[0033] As the capture reaction proceeds, the aggregation state and fluidity of the magnetic particles are monitored in real time by the built-in electron spin resonance micro-sensor in the microfluidic chip, and when the signal is stable, the pulsed magnetic field strength is gradually reduced to 0.1 T and the frequency is reduced to 0.5 Hz for a 2-minute low-intensity stabilization treatment;

[0034] After the capture reaction is completed, a PBS solution containing 0.1% BSA and 0.01% Tween-20 at pH 7.4 is injected at a flow rate of 8-10 μL / min, and a stable magnetic field of 0.05 T is maintained to ensure that the magnetic particles remain in the reaction chamber, while the non-specific binders and unbound components are eluted;

[0035] The magnetic immune complex in the reaction chamber is observed and recorded in real time using a 5-10-fold magnification, and the complex formation efficiency is evaluated by combining image analysis algorithms, and when the analysis parameters meet the preset standards, the next step is automatically entered to ensure that the quality and quantity of the immune complex meet the subsequent detection requirements.

[0036] Furthermore, step 4 includes the following steps:

[0037] Activate the quadrupole magnetic field generator in the microfluidic chip to generate a precise gradient magnetic field with a center strength of 0.8T and an edge strength of 0.2T. This causes the nanomagnetic immune complexes to migrate orderly along the magnetic field gradient within 3-5 minutes and accumulate in the bottom area of ​​the reaction chamber. At the same time, ensure that the liquid phase components are stably suspended in the upper solution, achieving preliminary solid-liquid phase separation.

[0038] While maintaining the gradient magnetic field, the flow control system of the microfluidic chip is activated to carefully extract the supernatant containing unbound enzyme-labeled secondary antibody from the upper part of the reaction chamber at a slow flow rate of 2-3 μL / min. The collected liquid is then precisely distributed into two parallel detection microcavities through a micro three-way valve, respectively, for subsequent fluorescence detection and colorimetric detection to ensure sample consistency.

[0039] An optimized fluorescent substrate solution was injected into the fluorescence detection microcavity, reacting with the HRP enzyme-labeled portion in the supernatant to generate a fluorescent signal. The signal was then collected using a time-resolved fluorescence detector at an excitation wavelength of 320 nm and an emission wavelength of 405 nm, with a 100 μs delay time and a 2 ms integration time. Data was recorded every 200 ms, continuously monitoring the reaction kinetics for 5 minutes.

[0040] At the same time, a chromogenic substrate solution was injected into the colorimetric detection microcavity to react with the AP enzyme-labeled portion in the supernatant to produce a yellow product. The absorbance change was measured every 15 seconds at a wavelength of 405 nm using a micro-spectrophotometer. The reaction curve was continuously recorded for 5 minutes to obtain the initial rate data of the enzyme-catalyzed reaction.

[0041] Automatically calculate the rate of change of fluorescence signal intensity and colorimetric absorbance, perform weighted fusion of the data from the two detection modes, eliminate potential matrix interference and nonspecific background, and generate a normalized comprehensive enzyme activity curve to directly reflect the concentration of unbound enzyme-labeled secondary antibody in the supernatant;

[0042] The real-time detection data were compared with the pre-established standard curve, and the Michaelis-Menten model based on the Hill equation was used to fit the enzyme reaction data to extract key parameters. The consistency coefficient of the two detection modes was evaluated as one of the internal quality control indicators.

[0043] Furthermore, the fluorescent substrate solution contains HPPA and hydrogen peroxide in a volume ratio of 1:1; the substrate used in the chromogenic substrate solution is 1 mg / mL 4-nitrophenyl phosphate disodium salt.

[0044] Furthermore, step 5 includes the following steps:

[0045] According to the supernatant enzyme activity comprehensive data obtained in step 4, the initial concentration value of the unbound enzyme label secondary antibody is calculated;

[0046] The total amount of the initial enzyme label secondary antibody added in step 1 is subtracted from the calculated amount of the unbound enzyme label secondary antibody, thereby obtaining the amount of the enzyme label secondary antibody captured by the immune complex;

[0047] The sample-specific correction factor matrix previously established for the specific target molecule to be detected is retrieved for eliminating the systematic errors caused by matrix interference and non-specific adsorption of different sample types;

[0048] The calculated initial concentration value is convoluted with the sample-specific correction factor, and a temperature compensation coefficient and an ion strength correction term are introduced, so as to comprehensively consider the slight differences between the actual detection conditions and the standard curve construction conditions, dynamically adjust the calculation parameters, and generate the target molecule concentration value after comprehensive correction;

[0049] By iteratively perturbing the key parameters 1000 times, a 95% confidence interval is constructed, and the coefficient of variation and the relative standard deviation are calculated. When the coefficient of variation is <10%, the data reliability is determined to be good. For the case where the coefficient of variation is in the range of 10%-15%, the correction weight is automatically increased to improve the accuracy;

[0050] The final calculation result is compared with the reference range database, different clinical significance levels are assigned according to the detection object and the clinical application scene, and a concentration result report is generated, which includes the core value, the reference range, the detection uncertainty and the concentration unit conversion table.

[0051] Further, the sample-specific correction factor matrix includes correction coefficients for different sample types, and three different concentration gradient correction values are set for each sample type, and the correction weight is dynamically adjusted according to the sample ion strength, pH value and protein content.

[0052] Further, step 6 includes the following steps:

[0053] Three concentration levels of quality control samples are automatically extracted from the built-in quality control sample storage module, and parallel analysis is performed using the same process as the detection sample, the quality control recovery rate and the linear correlation coefficient are calculated, and whether the slope and intercept of the calibration curve are within the preset allowable range is checked;

[0054] Repeated measurements are performed on the same sample for precision analysis, the within-batch coefficient of variation and the between-batch coefficient of variation are calculated, and Levey-Jennings plot analysis is performed; the current detection result is compared with the historical quality control data, Westgard multi-rule judgment method is applied to identify potential systematic errors and random errors, and it is ensured that all quality control parameters are within the acceptable range;

[0055] Parallel detection of the original sample after different degrees of dilution, analysis of the linear relationship and parallelism of the results before and after dilution, calculation of the dilution recovery rate and inspection of the linearity of the dilution curve, to verify whether there is a high dose hook effect or matrix interference in the sample;

[0056] Compare and analyze the current reverse ELISA detection results with the pre-stored reference method data, calculate the correlation coefficient, consistency coefficient and Bland-Altman bias chart between the two methods, and evaluate the accuracy and comparability of the system;

[0057] After completing all quality control verifications, automatically generate a detection report conforming to the laboratory information management system standard based on the XML structured template, and the report includes sample information, detection conditions, original detection data, result calculation process, final concentration value, reference range, quality control data, methodological explanation and result interpretation suggestion;

[0058] Retrospective evaluation of the key links of the entire detection process, including reaction condition stability, reagent performance, signal response curve characteristics and result reliability indicators, generate a system performance evaluation report as an attachment to the detection report; at the same time, save all original data and quality control records in an encrypted format to the cloud database, ensure long-term preservation and necessary audit traceability of the data, and complete the closed-loop management of the entire detection process.

[0059] Compared with the prior art, the present application has the following beneficial effects:

[0060] The present application accurately calculates the target antigen / antibody concentration by combining reverse enzyme-linked immunosorbent detection method with microfluidic chip, nanomagnetic particle, high-efficiency magnetic field capture, double-mode enzyme activity detection and sample-specific correction factor, and realizes efficient and accurate immunodetection. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 The flowchart of the reverse enzyme-linked immunosorbent detection method disclosed in the embodiments of the present application. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the embodiments of the present application will be described in more detail below in combination with the drawings of the embodiments of the present application. In the drawings, the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The described embodiments are part of the embodiments of the present application, not all of the embodiments.

[0063] Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.

[0064] The embodiments described below and the directional terms used are exemplary and are intended to be used in explaining the present application, and should not be construed as limiting the present application.

[0065] As shown in the figure, the reverse enzyme-linked immunosorbent assay method comprises the following steps: Figure 1

[0066] Step 1, centrifugal filtration and gradient dilution are performed on the biological sample, and uncoupled specific antibodies / antigens and double-enzyme-labeled secondary antibodies are prepared, and the reagent ratio is optimized to ensure the best activity;

[0067] Step 2, in the microfluidic reaction chamber, the sample to be tested is allowed to compete with the specific antibody / antigen for binding, and then the double-enzyme-labeled secondary antibody is added to bind to the unoccupied antibody / antigen binding site, forming an enzyme-labeled complex;

[0068] Step 3, nanometer magnetic particles modified with specific antibodies / antigens are added, and a pulsed magnetic field is applied to promote efficient capture of the complex, forming a complete immune complex;

[0069] Step 4, the immune complex is separated using a gradient magnetic field, the liquid phase of the unbound enzyme-labeled secondary antibody in the supernatant is collected, and the enzyme activity thereof is detected using time-resolved fluorescence-colorimetric dual-mode detection;

[0070] Step 5, based on the enzyme activity data, the amount of bound enzyme-labeled secondary antibody is reversely calculated, and the target antigen / antibody concentration is accurately calculated by combining the sample-specific correction factor;

[0071] Step 6, an internal quality control program is executed to verify the accuracy of the detection results, and a standardized detection report is generated.

[0072] ​In the present embodiment, step 1 is to carry out centrifugal filtration and gradient dilution to biological sample, can effectively remove impurities and interfering substances that may exist in sample, ensure that the reaction environment in the detection process is purer, and also make sample concentration reach suitable range, provide uniform and stable starting conditions for subsequent reaction. Centrifugal filtration technology can separate solid particles from liquid by high-speed rotation, further improve sample purity, thereby reducing the possibility of non-specific binding. Gradient dilution is to carry out multi-stage decrease according to the concentration characteristics of antigen or antibody in sample, make the detection signal be within reasonable dynamic range, both avoid the signal distortion caused by supersaturation, also ensure that low-abundance target molecule can be effectively captured and detected. At the same time, prepare uncoupled specific antibody / antigen and double enzyme labeled secondary antibody in this step, through accurate reagent ratio optimization, can not only ensure the high activity and high specificity of each reaction component, but also make subsequent competitive binding reaction and enzyme-labeled complex formation process smoother. Reasonable reagent ratio is the key to guaranteeing the reaction efficiency and sensitivity of whole detection system, it determines the equilibrium state of competitive reaction and the strength of signal, and then affects the quantitative accuracy of whole detection method. In this process, the quality and stability of the reagents are also very critical. Unconjugated antibodies / antigens often have higher activity and affinity, while double-enzyme-labeled secondary antibodies can provide signal amplification through special labeling technology. The optimization of reagents not only depends on strict quality control between batches, but also requires multiple preliminary experiments to determine the optimal conditions to achieve an organic combination of high sensitivity and high specificity. Overall, the implementation of step 1 provides a solid foundation for subsequent complex immune reactions, ensuring that each molecule can play its maximum role under strictly controlled conditions, and ultimately contributes to the stability and repeatability of the test results.

[0073] In this embodiment, the key technology of step 2 is to use a microfluidic reaction chamber to build a high-efficiency and precise reaction environment, so that the target molecules in the sample can effectively compete with the pre-added specific antibodies / antigens. Microfluidic technology can realize precise fluid operation in micro-scale space, with the characteristics of fast reaction speed, low sample consumption and controllable reaction conditions. In this step, the competition reaction between the sample and the specific antibody / antigen is the core of the entire detection system. Only under the condition of sufficient competition can a reliable inverse relationship between the signal and the target concentration be ensured. Then, a double-enzyme-labeled secondary antibody is added, which can react with the unoccupied antibody / antigen binding site to form an enzyme-labeled complex. The construction of this reaction system requires that the affinity and specificity of each component must be matched, and the reaction time and temperature conditions must be precisely controlled to ensure that all reactions are carried out in the best state. The fluid dynamics in the microfluidic chamber can ensure that the sample and reagents are fully mixed, thereby improving the reaction efficiency and signal consistency. In addition, microfluidics can greatly reduce the amount of sample and reagent, which has advantages in terms of economy and environmental protection. Through the precise design of the microchannel structure, the reaction rate can be effectively controlled, so that the competition binding process reaches equilibrium within a limited time, ensuring that the number of enzyme-labeled complexes formed has a stable quantitative relationship with the concentration of target molecules in the sample. Overall, using a microfluidic reaction chamber for competition binding and enzyme-labeled complex formation not only significantly improves the sensitivity and specificity of detection, but also lays a solid foundation for subsequent complex signal analysis.

[0074] In this embodiment, step 3 is a highlight of the detection method. Due to its small size and large specific surface area, nano-magnetic particles can specifically bind to enzyme-labeled complexes at the molecular level, greatly improving the sensitivity of detection. After special modification, the surface not only retains the recognition function of the antibody / antigen, but also ensures the movement response of the magnetic particles under an external magnetic field, so that the complexes can quickly gather in the microfluidic chamber. The application of pulsed magnetic field technology can dynamically control the movement trajectory and capture efficiency of magnetic particles. Pulsed magnetic field not only quickly attracts target complexes, but also effectively prevents particle aggregation, ensuring that each complex can be independently captured and further involved in subsequent detection reactions. In addition, this step also overcomes the interference problems caused by background noise and non-specific adsorption in traditional detection methods. Through magnetic separation technology, the signal-to-noise ratio of detection can be significantly improved, thereby realizing more accurate quantitative analysis. The selection and modification process of nano-magnetic particles is critical, requiring high affinity, good biocompatibility and chemical stability. Combined with the application of pulsed magnetic field, not only the rapid and centralized capture of target molecules is realized, but also the subsequent signal amplification is strongly supported, forming a complete and stable immune complex, ensuring the efficiency and reliability of the overall detection process.

[0075] In this embodiment, the core of step 4 is to separate the formed immune complex by using a gradient magnetic field, and to detect the unbound enzyme-labeled secondary antibody in the supernatant by time-resolved fluorescence-colorimetric dual-mode detection. The gradient magnetic field can effectively separate the magnetic particles in the immune complex according to their magnetic response characteristics, distinguish the magnetic particles bound to the complex from the unbound enzyme-labeled secondary antibody, reduce background signal interference, and ensure the accuracy of the detection data. With this technology, the target molecules can be effectively isolated from non-specific interferents, and the signals released by the enzyme-labeled secondary antibody can be concentrated for detection. The subsequent time-resolved fluorescence and colorimetric dual-mode detection method further enhances the reliability and sensitivity of the detection results. Time-resolved fluorescence detection utilizes the delay characteristics of fluorescence signals to effectively filter out short-lived background fluorescence noise, while colorimetric detection provides intuitive optical absorption changes. Through the complementary effects of the two detection modes, the sensitivity of the detection signal can be improved, and the accuracy of the data can be ensured through cross-validation. This method not only has high detection sensitivity for enzyme activity, but also can achieve rapid response and large-scale quantitative dynamic detection, meeting the needs of clinical rapid detection and laboratory precise analysis. The dual-mode detection scheme can significantly reduce the incidence of false positives and false negatives in practical applications, thereby improving the reliability of the detection results. Through the synergistic effect of the gradient magnetic field and the dual-mode detection, not only high resolution and high accuracy are achieved, but also excellent performance in detecting target molecules in complex biological samples is shown, providing strong technical support and data guarantee for the entire detection process, and promoting the development of immunodetection methods towards high sensitivity and high accuracy.

[0076] In this embodiment, step 5 is the key to data analysis in the entire detection process, and accurate enzyme activity data directly determines the accuracy of the final quantitative results. First, by establishing a standard curve between enzyme activity and enzyme-labeled secondary antibody binding amount in advance, the enzyme activity signal released during the detection process can be quantitatively analyzed. Due to the high catalytic amplification effect of enzyme reaction, the signal intensity and the target molecule concentration show a certain linear or quasi-linear relationship, but in actual operation, it will be disturbed by many factors, such as substrate concentration, temperature, pH value and reaction time, etc. Therefore, by introducing a sample-specific correction factor, the biological differences between different samples and the slight changes in reaction conditions can be adjusted to ensure the accuracy and repeatability of the quantitative results. The reverse calculation method not only depends on accurate mathematical models and data fitting algorithms, but also needs a strict quality control system as support to ensure that reliable data can be obtained under various interference conditions. This technology realizes efficient conversion from enzyme activity to target concentration, which not only makes full use of the signal amplification effect of enzyme catalytic reaction, but also avoids the limitations of directly detecting low-abundance molecules, so it has significant advantages in sensitivity and dynamic range. Through this reverse calculation method, not only the detection lower limit is greatly reduced, but also the accuracy of the detection data is improved, providing high-reliability quantitative basis for clinical diagnosis and scientific research experiments. Overall, the technical scheme of this part shows high level in data processing, mathematical modeling and correction algorithm, providing strong computational support for the detection method, ensuring that each experimental data can be converted into accurate target molecule concentration information, so as to realize high-precision quantitative detection.

[0077] In this embodiment, step 6 is crucial for ensuring the accuracy and reliability of the detection results. In the internal quality control program, multiple quality control samples and standards are usually set to judge the stability and repeatability by comparing the differences between the detection data and the expected results. The design of the quality control samples takes into account different concentration ranges and covers various interference factors that may exist in the samples, so as to monitor the reaction state and data fluctuation in real time throughout the detection process. Internal quality control not only involves the monitoring of physical and chemical parameters such as temperature, reaction time, batch-to-batch differences of reagents, etc., but also includes the accuracy verification of data processing software to ensure that each step of data acquisition and calculation process meets the standard operating procedures. The generated standardized detection report integrates all detection data, quality control indicators and correction parameters, providing a complete, intuitive and rigorously verified detection result to the detector through detailed charts, statistical data and trend analysis. Such a report not only quantitatively describes the detection results, but also explains the possible errors and biases, facilitating further clinical decision-making and scientific research. Through the implementation of the internal quality control process, remedial measures can be taken quickly in the event of abnormal conditions, effectively reducing the risk of errors and ensuring the stability and accuracy of the overall detection process in multiple repetitions. The standardized report provides a reliable basis for subsequent data comparison, clinical judgment and scientific research, reflecting the rigor and scientific nature of the method in quality management and data tracking. Overall, the implementation of internal quality control and the generation of standardized detection reports greatly enhance the credibility and application value of the entire detection method, enabling timely detection and correction of potential errors and providing sustained support and technical support for the long-term application of the detection process.

[0078] In summary, the reverse enzyme-linked immunosorbent assay method realizes sample pretreatment, reaction system construction, magnetic capture separation, dual-mode signal detection, reverse calculation quantification and strict internal quality control through the fine design of each step. Each step plays a unique technical advantage, and the steps are connected and complement each other to form a high-sensitivity, high-specificity, quantitative and accurate detection system, providing strong support for clinical diagnosis and scientific research applications.

[0079] Further, step 1 comprises the following steps:

[0080] The collected biological sample is centrifuged at 3000 rpm for 10 minutes at 4°C, and then filtered using a 0.22 μm filter to remove large particulate matter and cell debris;

[0081] The filtered sample is gradient diluted using PBS buffer with pH 7.4, with dilution ratios including 1:2, 1:4, 1:8, 1:16 and 1:32, respectively placed in low adsorption microcentrifuge tubes, and stored at 4°C for standby;

[0082] The uncoupled specific antibody / antigen solution is prepared by diluting the high-purity antibody / antigen obtained by affinity chromatography purification to 5-10 μg / ml with 0.01 M PBS buffer at pH 7.4 and stored at 4°C in the dark to maintain its optimal immunological activity;

[0083] The double-enzyme-labeled secondary antibody is prepared by cross-linking horseradish peroxidase and alkaline phosphatase to the secondary antibody molecules by glutaraldehyde cross-linking method, ensuring that the active sites of the two enzymes do not interfere with each other, and then purified by gel filtration column to remove unbound enzyme molecules;

[0084] The optimal concentration ratio of antigen / antibody to double-enzyme-labeled secondary antibody is determined by orthogonal experimental design, and the activity is tested under different ion strength and pH conditions, and the condition with the highest signal-to-noise ratio is selected as the final working parameter;

[0085] A stabilizer solution containing 1% BSA, 0.05% Tween-20 and 0.02% sodium azide is prepared and added to the antigen / antibody and double-enzyme-labeled secondary antibody solutions, respectively, and stored at 4°C in the dark after mixing, to ensure that the reagents remain stable and active during storage.

[0086] In summary, through a series of optimization processes, the purity of biological samples and the high activity of reagents are ensured, thereby improving the sensitivity and reliability of the reverse enzyme-linked immunosorbent assay method. First, low-temperature centrifugation and 0.22 μm filter filtration are used to effectively remove large particle impurities and cell debris, reducing non-specific interference. Subsequently, gradient dilution with PBS buffer is used to optimize detection sensitivity and ensure linear response in different concentration ranges. The uncoupled specific antibody / antigen solution is purified by high-purity affinity chromatography and stored in a suitable buffer system and in the dark to maintain its immunological activity. The preparation of double-enzyme-labeled secondary antibody uses glutaraldehyde cross-linking method to ensure the coexistence of horseradish peroxidase and alkaline phosphatase on the secondary antibody molecules without interfering with each other, while gel filtration purification is used to improve the specificity of the reagent. The ratio of antigen / antibody to double-enzyme-labeled secondary antibody is optimized by orthogonal experiment, and the signal-to-noise ratio is tested under different environmental parameters to determine the optimal reaction conditions. In addition, a stabilizer solution containing BSA, Tween-20 and sodium azide is used to improve the stability of the reagent during long-term storage and prevent activity reduction.

[0087] Further, the molar ratio of horseradish peroxidase to alkaline phosphatase is 1:1 to 1:2, the labeling density is 1-3 enzyme molecules per secondary antibody molecule, and the activity retention rate of both enzymes is greater than 80%.

[0088] Further, step 2 includes the following steps:

[0089] 50-100 μL of biological sample is injected into the reaction chamber of the microfluidic chip, and the flow rate is stabilized at 5-10 μL / min through capillary action and micro-pump control;

[0090] A pre-optimized concentration of specific antibody / antigen solution is injected, incubated at 37°C for 20 minutes, and kept at 60 rpm for shaking to promote the competitive binding of target analytes in the sample to specific antibodies / antigens;

[0091] The double-enzyme-labeled secondary antibody solution is precisely injected through the independent channel of the microfluidic chip, with a controlled flow rate of 3-5 μL / min, to ensure its full mixing with the solution in the reaction chamber, and incubated at 25°C for 15 minutes to allow the double-enzyme-labeled secondary antibody to specifically bind to the antibody / antigen binding sites not occupied by the target analyte;

[0092] During the entire competitive binding process, the temperature and pH sensors built into the microfluidic chip monitor the reaction conditions in real time, and the feedback control system automatically adjusts the heating elements and micro-buffer injection to ensure that the reaction process is maintained at pH 7.2-7.4 and temperature 37±0.5°C;

[0093] After the completion of the competition reaction, the 0.05% concentration of non-ionic surfactant washing solution is introduced by controlling the micro-valve switch, and is gently flushed at a flow rate of 3-5 μL / min to remove non-specific binding substances while retaining the specific immune complexes formed;

[0094] In the mixing area of the microfluidic chip, the "H-type" micro-channel structure is used to generate a laminar flow effect to enhance the collision probability between molecules, and the built-in ultrasonic micro-vibrator is used for intermittent vibration of 5 seconds on and 10 seconds off to further promote the efficient binding of the double-enzyme-labeled secondary antibody to the unoccupied binding sites;

[0095] The reaction progress is monitored in real time by the micro-spectrophotometric sensor built into the reaction chamber, and when the signal intensity change rate drops below 0.5% / min, the reaction is automatically terminated and proceeds to the next step to ensure that the reaction reaches an equilibrium state and forms stable enzyme-labeled complexes.

[0096] In summary, by precise control of the microfluidic chip, efficient and specific competitive binding reactions are achieved, thereby improving the sensitivity and repeatability of the reverse enzyme-linked immunosorbent assay method. First, the biological sample is injected into the reaction chamber of the microfluidic chip at a stable flow rate controlled by the micro pump, and competes with specific antibodies / antigens. Through 37°C constant temperature incubation and moderate oscillation, the binding efficiency of the target analyte and the antibody / antigen is enhanced. Subsequently, the double enzyme-labeled secondary antibody is injected under the condition of precise control of the flow rate and temperature, and specifically binds to the binding sites not occupied by the target analyte. During the entire reaction process, the temperature and pH sensors built-in the microfluidic chip monitor and automatically adjust the reaction conditions in real time, ensuring that the system maintains the optimal environmental parameters, thereby improving the stability and controllability of the reaction. In addition, non-ionic surfactant washing solution is introduced through the micro valve to remove non-specific binders in a gentle manner, thereby improving the specificity of the detection. The laminar flow effect of the H-type microchannel structure enhances the collision probability between molecules, and the intermittent vibration of the ultrasonic micro vibrator further promotes the efficient binding of the double enzyme-labeled secondary antibody, thereby improving the formation efficiency of the enzyme-labeled complex. Finally, the micro spectrophotometric sensor monitors the reaction process in real time and automatically terminates the reaction after the signal stabilizes, ensuring that the binding process reaches equilibrium and forms a stable enzyme-labeled complex.

[0097] Further, the microfluidic chip is made of polydimethylsiloxane, polymethyl methacrylate or epoxy resin material, the internal channel width is 100-200 μm, the depth is 40-100 μm, the total reaction volume is 10-100 μL, the chip surface is treated by plasma to enhance hydrophilicity, and is coated with an anti-adhesion layer to reduce non-specific adsorption.

[0098] Further, step 3 comprises the following steps:

[0099] The pre-prepared surface-modified nanomagnetic particles are injected into the microfluidic reaction chamber, so that the magnetic particles surface is covalently connected to the specific antibody / antigen by EDC / NHS chemical cross-linking method, the concentration is controlled at 0.5-1 mg / mL, the injection rate is 2-3 μL / min, and the magnetic particles are uniformly dispersed in the reaction system to avoid aggregation phenomenon affecting the capture efficiency;

[0100] The electromagnetic coil array integrated at the bottom of the microfluidic chip is started to generate a pulsed magnetic field with a strength of 0.2-0.5 T, a pulse frequency of 1-5 Hz and a duty cycle of 30%, the magnetic field direction is perpendicular to the fluid flow direction, so that the nanomagnetic particles produce controlled micro-movement in the reaction chamber, thereby increasing the collision probability of the nanomagnetic particles with the immune complexes in the solution;

[0101] Under the action of the pulsed magnetic field, the specific antibody / antigen on the surface of the nano-magnetic particles and the immune complex formed in the solution have high-affinity binding, and the unoccupied specific antibody / antigen sites capture the target analyte in the solution to form a complete immune complex of the "nano-magnetic particle-antigen / antibody-double enzyme-labeled secondary antibody" sandwich structure;

[0102] With the progress of the capture reaction, the aggregation state and fluidity change of the magnetic particles are monitored in real time by the electron spin resonance micro-sensor built in the microfluidic chip. When the signal is stable, the pulsed magnetic field strength is gradually reduced to 0.1 T, and the frequency is reduced to 0.5 Hz. A low-intensity stabilization treatment is performed for 2 minutes.

[0103] After the completion of the capture reaction, a PBS solution with pH 7.4, containing 0.1% BSA and 0.01% Tween-20, is injected at a flow rate of 8-10 μL / min, and a stable magnetic field of 0.05 T is maintained to ensure that the magnetic particles remain in the reaction chamber, while the non-specific binders and unbound components are eluted.

[0104] The magnetic immune complex in the reaction chamber is observed and recorded in real time using a 5-10-fold magnification, and the complex formation efficiency is evaluated by image analysis algorithm. When the analysis parameters meet the preset standards, the next step is automatically entered to ensure that the quality and quantity of the immune complex meet the subsequent detection requirements.

[0105] In summary, the above series of steps utilize surface-modified nano-magnetic particles combined with the precise control of the microfluidic chip to achieve efficient and stable immune complex capture and enrichment. By covalently linking specific antibodies / antigens through EDC / NHS chemical cross-linking, the magnetic particles are uniformly dispersed in the solution and aggregation is avoided, improving the immune capture efficiency. The controlled pulsed magnetic field generated by the integrated electromagnetic coil array causes the nano-magnetic particles to produce micro-movements in the reaction chamber, thereby increasing the collision probability with the immune complex and enhancing the binding efficiency. Under the real-time monitoring of the electron spin resonance micro-sensor, the magnetic field parameters are optimized to ensure stabilization treatment and improve the integrity of the immune complex. Subsequently, non-specific binders are eluted by PBS buffer, and the magnetic particles are fixed by a low-intensity magnetic field to ensure the enrichment and purification of the target complex. Finally, the complex formation efficiency is evaluated by magnification real-time observation and image analysis algorithm to ensure that the quality and quantity meet the subsequent detection requirements. Overall, this step integrates nano-magnetic capture technology, pulsed magnetic field control, and real-time monitoring to improve the enrichment efficiency, stability, and detection sensitivity of the immune complex.

[0106] Furthermore, the nano-magnetic particles have a particle size of 50-150 nm, and the surface is modified with carboxyl, amino, or epoxy groups. The saturation magnetization is 50-70 emu / g, the specific surface area is 80-100 m 2 / g.

[0107] Further, step 4 includes the following steps:

[0108] The quadrupole magnetic field generator in the microfluidic chip is activated to generate a precise gradient magnetic field with a central strength of 0.8T and an edge strength of 0.2T, so that the nano-magnetic immune complex migrates in order along the direction of the magnetic field gradient in 3-5 minutes and gathers in the bottom area of the reaction chamber, while ensuring that the liquid phase components are stably suspended in the upper solution, realizing the preliminary separation of solid-liquid phases;

[0109] Under the condition of maintaining the gradient magnetic field, the shunt control system of the microfluidic chip is started to carefully extract the supernatant containing unbound enzyme-labeled secondary antibody from the upper part of the reaction chamber at a slow flow rate of 2-3 μL / min, and the collected liquid is accurately distributed into two parallel detection microcavities through a micro three-way valve for subsequent fluorescence detection and colorimetric detection, respectively, to ensure sample consistency;

[0110] The optimized fluorescence substrate solution is injected into the fluorescence detection microcavity to react with the HRP enzyme-labeled part in the supernatant to generate a fluorescence signal, and then the time-resolved fluorescence detector is used to collect signals at an excitation wavelength of 320 nm and an emission wavelength of 405 nm with a 100 μs delay time and a 2 ms integration time, record data every 200 ms, and continuously monitor the reaction kinetics curve for 5 minutes;

[0111] At the same time, the color developing substrate solution is injected into the colorimetric detection microcavity to react with the AP enzyme-labeled part in the supernatant to generate a yellow product, and the micro spectrophotometer is used to measure the absorbance change every 15 seconds at a wavelength of 405 nm, continuously record the reaction curve for 5 minutes, and obtain the initial rate data of enzyme catalytic reaction;

[0112] The rate of change of fluorescence signal intensity and colorimetric absorbance is automatically calculated, the data of the two detection modes are weighted and fused, potential matrix interference and non-specific background are eliminated, and a normalized comprehensive enzyme activity curve is generated to directly reflect the concentration of unbound enzyme-labeled secondary antibody in the supernatant;

[0113] The real-time detection data is compared with the standard curve established in advance, the Michaelis-Menten model based on the Hill equation correction is applied to fit the enzyme reaction data, the key parameters are extracted, and the consistency coefficient of the two detection modes is evaluated as one of the internal quality control indicators.

[0114] In summary, the ordered migration of the nanomagnetic immunocomplexes is precisely controlled by the quadrupole gradient magnetic field, and the solid-liquid phase separation is efficiently achieved. The precise distribution of the supernatant is ensured by the microfluidic splitting. The fluorescence detection and the colorimetric detection are respectively realized by the high-sensitivity time-resolved fluorescence detector and the micro-spectrophotometer, and the enzyme-catalyzed reaction kinetics curve is monitored in real time to improve the sensitivity and stability of the detection. The data of the two detection modes are fused by weighting to eliminate the matrix interference and generate the normalized comprehensive enzyme activity curve to ensure the accuracy of the quantitative analysis. Finally, the Michaelis-Menten model modified by the Hill equation is used to fit the enzyme reaction data, the key parameters are extracted, and the consistency of the detection mode is evaluated to enhance the internal quality control capability. Overall, the step integrates the magnetic field-driven separation, dual-mode detection, and data fusion analysis to improve the sensitivity, accuracy, and repeatability of the immunoassay.

[0115] Further, the rates of change of the fluorescence signal intensity and the colorimetric absorbance are automatically calculated, the data of the two detection modes are fused by weighting to eliminate the potential matrix interference and non-specific background, and the normalized comprehensive enzyme activity curve is generated to directly reflect the concentration of the unbound enzyme-labeled secondary antibody in the supernatant, including the following steps:

[0116] The fluorescence signal intensity F at each time point in the experiment is obtained t and the initial fluorescence signal F0, the rate of change of the fluorescence signal is calculated by the formula: fluorescence rate of change = (F t -F0) / F0;

[0117] The absorbance value A at each time point is obtained t and the initial absorbance A0, the rate of change of the fluorescence signal is calculated by the formula: absorbance rate of change = (A t -A0) / A0;

[0118] The background signal B at each time point during the experiment is determined t , and the background signal is subtracted from the fluorescence signal and the absorbance signal to obtain the background-corrected fluorescence signal F' t and the absorbance signal A' t ;

[0119] Let a and β be the weighting coefficients of the fluorescence signal intensity and the absorbance, respectively, and require a + β = 1, use the formula The rates of change of the fluorescence signal and the absorbance signal are fused by weighting to obtain the comprehensive rate of change, wherein F'0 is the background-corrected initial fluorescence signal, indicating the fluorescence signal measured at the beginning of the experiment, which has removed any matrix interference or non-specific background; A'0 is the background-corrected initial absorbance signal, indicating the absorbance value measured at the beginning of the experiment, which has removed any matrix interference or non-specific background.

[0120] The integrated change rate R 综合 (t) is normalized to ensure comparability of results under different experimental conditions;

[0121] The integrated change rate R 标准化 (t) is normalized to ensure comparability of results under different experimental conditions;

[0122] Further, the fluorescence substrate solution contains HPPA and hydrogen peroxide in a volume ratio of 1:1; the substrate used in the chromogenic substrate solution is 1 mg / mL of 4-nitrophenyl phosphate disodium salt.

[0123] Further, step 5 includes the following steps:

[0124] According to the integrated data of supernatant enzyme activity obtained in step 4, the initial concentration value of unbound enzyme marker secondary antibody is calculated;

[0125] Subtract the calculated amount of unbound enzyme marker secondary antibody from the total amount of initial enzyme marker secondary antibody added in step 1 to obtain the amount of enzyme marker secondary antibody captured by immune complexes;

[0126] Retrieve the sample-specific correction factor matrix previously established for the specific target molecule being detected, to eliminate systematic errors caused by matrix interference and non-specific adsorption of different sample types;

[0127] Convolve the calculated initial concentration value with the sample-specific correction factor, and introduce a temperature compensation coefficient and an ionic strength correction term, to comprehensively consider the small differences between the actual detection conditions and the standard curve construction conditions, dynamically adjust the calculation parameters, and generate the target molecule concentration value after comprehensive correction;

[0128] By randomly perturbing key parameters 1000 times, a 95% confidence interval is constructed, and the coefficient of variation and relative standard deviation are calculated. When the coefficient of variation is <10%, the data is determined to be reliable; for cases where the coefficient of variation is in the range of 10%-15%, the correction weight is automatically increased to improve accuracy;

[0129] Compare the final calculation results with the reference range database, assign different clinical significance levels according to the detection object and clinical application scenarios, and generate a concentration result report containing core values, reference ranges, detection uncertainties, and concentration unit conversion tables.

[0130] In summary, the above steps calculate the initial concentration of unbound enzyme-labeled secondary antibody based on enzyme activity data, and determine the target molecular weight captured by immune complexes through subtraction operation. Combined with sample-specific correction factor matrix, matrix interference and system error are eliminated, and dynamic parameter adjustment is realized through temperature compensation and ion strength correction to improve measurement accuracy. Subsequently, 95% confidence interval is calculated through 1000 times of random disturbance iteration to ensure the reliability of data, and correction weight is dynamically adjusted according to the coefficient of variation. Finally, the corrected target molecule concentration is compared with the reference database to generate a detection report containing core numerical value, reference range, detection uncertainty and unit conversion table, thereby improving the accuracy and clinical applicability of the detection results.

[0131] Further, the sample-specific correction factor matrix includes correction coefficients for different sample types, and correction values of 3 different concentration gradients are set for each sample type, and the correction weight is dynamically adjusted according to the sample ion strength, pH value and protein content.

[0132] Further, the target molecule concentration value C final is expressed by the formula:

[0133]

[0134] wherein C0 is the initial concentration value; K s (t) is the sample-specific correction factor; is the convolution operator, representing the combination of the initial concentration C0 and the sample-specific correction factor K s (t); α(T) is the temperature compensation coefficient; β(I) is the ion strength correction factor; η1 and η2 are the adjustment coefficients of temperature and ion strength, respectively, used to correct the slight difference between the actual conditions and the standard conditions; T act and T std are the actual detection temperature and the standard temperature, respectively; I act and I std are the actual detection ion strength and the standard ion strength, respectively.

[0135] Further, step 6 includes the following steps:

[0136] Automatically extract three concentration levels of quality control from the built-in quality control storage module, use the same process as the detection sample for parallel analysis, calculate the quality control recovery rate and linear correlation coefficient, and check whether the slope and intercept of the calibration curve are within the preset allowable range;

[0137] The same sample was repeatedly measured for precision analysis, calculation of within-batch and between-batch coefficients of variation, and Levey-Jennings plot analysis; the current detection results were compared with historical quality control data, and Westgard multi-rule judgment method was used to identify potential systematic and random errors, to ensure that all quality control parameters were within the acceptable range;

[0138] Parallel detection was performed on the original sample after different degrees of dilution, the linear relationship and parallelism of the results before and after dilution were analyzed, the dilution recovery rate was calculated, and the linearity of the dilution curve was checked, to verify whether there was high-dose hook effect or matrix interference in the sample;

[0139] The current reverse ELISA detection results were compared and analyzed with the pre-stored reference method data, the correlation coefficient, consistency coefficient and Bland-Altman bias chart between the two methods were calculated, to evaluate the accuracy and comparability of the system;

[0140] After completing all quality control verifications, the detection report conforming to the laboratory information management system standard was automatically generated based on the XML structured template, including sample information, detection conditions, original detection data, result calculation process, final concentration value, reference range, quality control data, methodological explanation and result interpretation suggestion;

[0141] The key links of the entire detection process were retrospectively evaluated, including reaction condition stability, reagent performance, signal response curve characteristics and result reliability indicators, to generate a system performance evaluation report as an attachment to the detection report; at the same time, all original data and quality control records were saved in encrypted format to the cloud database, to ensure long-term preservation and necessary audit traceability of the data, and to complete the closed-loop management of the entire detection process.

[0142] In summary, step 6 ensures the accuracy, stability and reliability of the detection through automatic quality control verification, precision analysis, dilution linearity evaluation and methodological comparison. Potential errors are identified through Levey-Jennings plot analysis and Westgard multi-rule judgment method, and sample matrix interference is evaluated through dilution recovery rate and high-dose hook effect analysis. Combined with Bland-Altman bias chart calculation accuracy, a detection report conforming to the laboratory information management system standard is finally generated, with a performance evaluation report attached. At the same time, all original data and quality control records are encrypted and stored in the cloud, to realize closed-loop management, ensure long-term traceability and audit compliance of the data.

[0143] Further, the acceptable range of quality control recovery rate is 90-110%, the within-batch coefficient of variation is ≤5%, the between-batch coefficient of variation is ≤10%, the linear correlation coefficient r is ≥0.98, the acceptable range of dilution recovery rate is 85-115%, and the linearity deviation of the dilution curve is not more than ±8%.

[0144] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A reverse enzyme-linked immunosorbent assay method, characterized in that: The following steps are involved: Step 1: Centrifuge and filter the biological sample and perform gradient dilution. Prepare unconjugated specific antibodies / antigens and dual enzyme-labeled secondary antibodies at the same time, and optimize the reagent ratio to ensure optimal activity. Step 2: In a microfluidic reaction chamber, the sample to be tested competes with the specific antibody / antigen for binding, and then a dual-enzyme-labeled secondary antibody is added to bind to the unoccupied antibody / antigen binding sites to form an enzyme-labeled complex; Step 3: Add nanomagnetic particles modified with specific antibodies / antigens and apply a pulsed magnetic field to promote efficient capture of the complex to form a complete immune complex; Step 4: Separate the immune complex using a gradient magnetic field, collect the unbound enzyme-labeled secondary antibody in the supernatant, and detect its enzyme activity using a time-resolved fluorescence-colorimetric dual mode; Step 5: Reversely calculate the amount of bound enzyme-labeled secondary antibody based on the enzyme activity data, and accurately calculate the target antigen / antibody concentration in combination with the sample-specific correction factor; Step 6: Execute internal quality control procedures to verify the accuracy of the test results and generate a standardized test report; Step 3 includes the following steps: The pre-prepared surface-modified nanomagnetic particles were injected into the microfluidic reaction chamber to covalently link the specific antibodies / antigens to the surface of the magnetic particles through EDC / NHS chemical cross-linking. The concentration was controlled at 0.5-1 mg / mL and the injection rate was 2-3 μL / min to ensure that the magnetic particles were evenly dispersed in the reaction system to avoid aggregation that affects the capture efficiency. The electromagnetic coil array integrated at the bottom of the microfluidic chip is activated to generate a pulsed magnetic field with an intensity of 0.2-0.5 T. The pulse frequency is set to 1-5 Hz, with a duty cycle of 30%. The magnetic field is perpendicular to the direction of fluid flow, causing the nanomagnetic particles to produce controlled micro-motion within the reaction chamber, thereby increasing their collision probability with the immune complexes in the solution. Under the action of a pulsed magnetic field, the specific antibodies / antigens on the surface of the nanomagnetic particles bind with high affinity to the immune complexes already formed in the solution. At the same time, the unoccupied specific antibody / antigen sites capture the target analytes in the solution, forming a complete immune complex with a "nanomagnetic particle-antigen / antibody-dual enzyme-labeled secondary antibody" sandwich structure. As the capture reaction proceeds, the aggregation state and mobility changes of the magnetic particles are monitored in real time by the electron spin resonance microsensor built into the microfluidic chip. Once the signal stabilizes, the pulsed magnetic field intensity is gradually reduced to 0.1 T and the frequency is reduced to 0.5 Hz for a 2-minute low-intensity stabilization process. After the capture reaction is completed, a PBS solution containing 0.1% BSA and 0.01% Tween-20 at pH 7.4 is injected at a flow rate of 8-10 μL / min while maintaining a stable magnetic field of 0.05 T to ensure that the magnetic particles remain in the reaction chamber, while nonspecific binders and unbound components are eluted; The magnetic immune complexes in the reaction chamber are observed and recorded in real time using a 5-10x magnification, and the efficiency of complex formation is evaluated using an image analysis algorithm. When the analysis parameters reach the preset standards, the system automatically proceeds to the next step to ensure that the quality and quantity of the immune complexes meet the requirements of subsequent testing.

2. The reverse enzyme-linked immunosorbent assay method according to claim 1, wherein Step 1 includes the following steps: The collected biological samples were centrifuged at 3000 rpm for 10 min at 4°C and then filtered through a 0.22 μm filter membrane to remove large particulate matter and cell debris; The filtered samples were serially diluted with PBS buffer (pH 7.4) at ratios of 1:2, 1:4, 1:8, 1:16, and 1:32, placed in low-binding microcentrifuge tubes, and stored at 4°C until use. Prepare unconjugated specific antibody / antigen solution using high-purity antibody / antigen purified by affinity chromatography, dilute to 5-10 μg / ml with 0.01 M PBS buffer, pH 7.4, and store at 4°C in the dark to maintain optimal immunoreactivity; Prepare a dual-enzyme labeled secondary antibody. Horseradish peroxidase and alkaline phosphatase are simultaneously labeled on the secondary antibody molecule through glutaraldehyde cross-linking to ensure that the active sites of the two enzymes do not interfere with each other. Then, use a gel filtration column to separate and purify the secondary antibody to remove unbound enzyme molecules. The optimal concentration ratio of antigen / antibody to double-enzyme labeled secondary antibody was determined through orthogonal experimental design. Activity was tested under different ionic strength and pH conditions, and the condition with the highest signal-to-noise ratio was selected as the final working parameter. Prepare a stabilizer solution containing 1% BSA, 0.05% Tween-20, and 0.02% sodium azide, add it to the antigen / antibody and double-enzyme-labeled secondary antibody solutions, respectively, mix them, and store them at 4°C in the dark to ensure the stability and activity of the reagents during storage.

3. The reverse enzyme-linked immunosorbent assay method according to claim 1, wherein Step 2 includes the following steps: 50-100 μL of biological sample was injected into the reaction chamber of the microfluidic chip, and the flow rate was stabilized at 5-10 μL / min through capillary action and micropump control; Inject a specific antibody / antigen solution with a pre-optimized concentration and incubate at 37°C for 20 minutes with shaking at 60 rpm to promote competitive binding between the target analyte in the sample and the specific antibody / antigen. The dual-enzyme-labeled secondary antibody solution was precisely injected through independent channels of the microfluidic chip at a controlled flow rate of 3-5 μL / min to ensure that it was fully mixed with the solution in the reaction chamber. The solution was incubated at 25°C for 15 minutes to allow the dual-enzyme-labeled secondary antibody to specifically bind to the antibody / antigen binding sites not occupied by the target analyte. During the entire competitive binding process, the temperature and pH sensors built into the microfluidic chip monitor the reaction conditions in real time. The feedback control system automatically adjusts the heating element and microbuffer injection to ensure that the reaction process is maintained at pH 7.2-7.4 and temperature 37±0.5°C. After the competition reaction is completed, the microvalve is controlled to switch and introduce a 0.05% concentration of non-ionic surfactant washing solution, which is gently washed at a flow rate of 3-5 μL / min to remove non-specific binding substances while retaining the specifically formed immune complexes. In the mixing area of ​​the microfluidic chip, the laminar flow effect generated by the herringbone microchannel structure is used to enhance the probability of collisions between molecules. Intermittent vibrations, with a 5-second on and 10-second off cycle, are performed by the chip's built-in ultrasonic microvibrator, further promoting the efficient binding of the dual-enzyme-labeled secondary antibody to unoccupied binding sites. The reaction progress is monitored in real time by a micro-spectrophotometric sensor built into the reaction chamber. When the rate of change of signal intensity drops below 0.5% / min, the reaction is automatically terminated and the next step is entered to ensure that the reaction reaches equilibrium and forms a stable enzyme-labeled complex.

4. The reverse enzyme-linked immunosorbent assay method according to claim 3, characterized in that: The microfluidic chip is made of polydimethylsiloxane, polymethyl methacrylate or epoxy resin materials, with an internal channel width of 100-200μm, a depth of 40-100μm, and a total reaction volume of 10-100μL. The chip surface is plasma treated to enhance hydrophilicity and coated with an anti-adhesion layer to reduce nonspecific adsorption.

5. The reverse enzyme-linked immunosorbent assay method according to claim 1, wherein Step 4 includes the following steps: Activating the quadrupole magnetic field generator in the microfluidic chip generates a precise gradient magnetic field with a central intensity of 0.8 T and an edge intensity of 0.2 T. This causes the nanomagnetic immune complexes to migrate orderly along the magnetic field gradient within 3-5 minutes and accumulate in the bottom area of ​​the reaction chamber. At the same time, the liquid phase components are stably suspended in the upper solution, achieving preliminary solid-liquid phase separation. While maintaining the gradient magnetic field, the flow control system of the microfluidic chip is activated to carefully extract the supernatant containing unbound enzyme-labeled secondary antibody from the upper part of the reaction chamber at a slow flow rate of 2-3 μL / min. The collected liquid is then precisely distributed into two parallel detection microcavities through a micro three-way valve, respectively, for subsequent fluorescence detection and colorimetric detection to ensure sample consistency. An optimized fluorescent substrate solution was injected into the fluorescence detection microcavity, reacting with the HRP enzyme-labeled portion in the supernatant to generate a fluorescent signal. The signal was then collected using a time-resolved fluorescence detector at an excitation wavelength of 320 nm and an emission wavelength of 405 nm, with a 100 μs delay time and a 2 ms integration time. Data were recorded every 200 ms, and the reaction kinetics were continuously monitored for 5 minutes. At the same time, a chromogenic substrate solution was injected into the colorimetric detection microcavity to react with the AP enzyme-labeled portion in the supernatant to produce a yellow product. The absorbance change was measured every 15 seconds at a wavelength of 405 nm using a micro-spectrophotometer. The reaction curve was continuously recorded for 5 minutes to obtain the initial rate data of the enzyme-catalyzed reaction. Automatically calculate the rate of change of fluorescence signal intensity and colorimetric absorbance, perform weighted fusion of the data from the two detection modes, eliminate potential matrix interference and nonspecific background, and generate a normalized comprehensive enzyme activity curve to directly reflect the concentration of unbound enzyme-labeled secondary antibody in the supernatant; The real-time detection data were compared with the pre-established standard curve, and the Michaelis-Menten model based on the Hill equation was used to fit the enzyme reaction data to extract key parameters. The consistency coefficient of the two detection modes was evaluated as one of the internal quality control indicators.

6. The reverse enzyme-linked immunosorbent assay method according to claim 5, characterized in that: The fluorescent substrate solution contained HPPA and hydrogen peroxide in a volume ratio of 1:1; the substrate used in the chromogenic substrate solution was 1 mg / mL 4-nitrophenyl phosphate disodium salt.

7. The reverse enzyme-linked immunosorbent assay method according to claim 1, characterized in that: Step 5 includes the following steps: Based on the comprehensive data of supernatant enzyme activity obtained in step 4, calculate the initial concentration of unbound enzyme-labeled secondary antibody; Subtract the total amount of the initial enzyme-labeled secondary antibody added in step 1 from the calculated amount of unbound enzyme-labeled secondary antibody to obtain the amount of enzyme-labeled secondary antibody captured by the immune complex; Recall the sample-specific correction factor matrix pre-established for the specific target molecule currently being detected to eliminate systematic errors caused by matrix interference and nonspecific adsorption of different sample types; The calculated initial concentration value is convolved with the sample-specific correction factor, and a temperature compensation coefficient and ion strength correction term are introduced. The calculation parameters are dynamically adjusted to generate a fully corrected target molecule concentration value, taking into account the slight differences between the actual detection conditions and the standard curve construction conditions. By performing 1000 random perturbation iterations on key parameters, a 95% confidence interval is constructed, and the coefficient of variation and relative standard deviation are calculated. When the coefficient of variation is <10%, the data reliability is judged to be good. For coefficients of variation within the range of 10%-15%, the correction weight is automatically increased to improve accuracy. The final calculated results are compared with the reference range database, and different levels of clinical significance are assigned according to the test object and clinical application scenario. A concentration result report is generated, which includes the core value, reference range, detection uncertainty and concentration unit conversion table.

8. The reverse enzyme-linked immunosorbent assay method according to claim 7, characterized in that: The sample-specific correction factor matrix contains correction coefficients for different sample types. Three correction values ​​with different concentration gradients are set for each sample type, and the correction weights are dynamically adjusted according to the sample ionic strength, pH value, and protein content.

9. The reverse enzyme-linked immunosorbent assay method according to claim 1, wherein Step 6 includes the following steps: Automatically extract three concentration levels of quality control materials from the built-in quality control material storage module, perform parallel analysis using the same process as the test samples, calculate the quality control recovery rate and linear correlation coefficient, and check whether the slope and intercept of the calibration curve are within the preset allowable range; Repeated measurements of the same sample were performed, followed by precision analysis, calculation of intra-assay and inter-assay coefficients of variation, and Levey-Jennings plot analysis. Current test results were compared with historical quality control data, and the Westgard multi-rule judgment method was applied to identify potential systematic and random errors, ensuring that all quality control parameters were within acceptable ranges. Perform parallel tests on the original sample after different dilutions, analyze the linearity and parallelism of the results before and after dilution, calculate the dilution recovery rate, and check the linearity of the dilution curve to verify whether there is a high-dose hook effect or matrix interference in the sample; Compare and analyze the current reverse ELISA test results with the pre-stored reference method data, calculate the correlation coefficient, consistency coefficient and Bland-Altman deviation plot between the two methods, and evaluate the accuracy and comparability of the system; After all quality control verifications are completed, a test report that complies with laboratory information management system standards is automatically generated based on an XML structured template. The report includes sample information, test conditions, original test data, result calculation process, final concentration value, reference range, quality control data, methodological description, and result interpretation suggestions; A retrospective evaluation is conducted on the key links of the entire testing process, including the stability of reaction conditions, reagent performance, signal response curve characteristics, and result reliability indicators, and a system performance evaluation report is generated as an attachment to the test report. At the same time, all raw data and quality control records are saved in an encrypted format to a cloud database to ensure long-term data preservation and audit traceability when necessary, completing the closed-loop management of the entire testing process.

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