A respiratory multi-pathogen micro-quick in-vitro detection system based on particle transfer immunoassay

Abstract

The application discloses a respiratory tract multi-pathogen micro rapid in-vitro detection system based on particle transfer immune analysis, which comprises a disposable multi-chamber detection chip, a magnetic control transfer mechanism, an optical detection module and a quantitative analysis module. The chip is sequentially provided with a sample loading chamber, an immune reaction chamber, a magnetic bead cleaning chamber and a signal detection chamber along the transfer direction of the magnetic microspheres, and a plurality of groups of coded magnetic microspheres are pre-packaged in the immune reaction chamber. The magnetic control transfer mechanism drives the physical transfer of the magnetic microspheres between the chambers through a magnetic field, replacing the pump valve liquid path. A low-residue transfer channel is arranged between adjacent chambers, comprising an inlet contraction section, a narrow-diameter transfer section and an outlet diffusion section, which realizes liquid film shear separation in cooperation with a liquid-repellent coating and a liquid film stripping step. The magnetic control transfer mechanism drives the magnetic microspheres according to a four-stage magnetic field motion program. The quantitative analysis module eliminates multi-pathogen joint detection signal crosstalk through a cross-correction matrix.

Description

Technical Field

[0001] This invention relates to the fields of in vitro diagnostics (IVD) and immunoassay, specifically to the fields of immunoassay, microfluidic chips, magnetic separation technology and point-of-care testing (POCT), and particularly to a rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay. Background Technology

[0002] Respiratory tract infections (RTIs) are among the most prevalent and burdensome infectious diseases worldwide. Various respiratory pathogens, including influenza A virus, influenza B virus, respiratory syncytial virus (RSV), adenovirus, and Mycoplasma pneumoniae (MP), can alternate in epidemics and cause co-infections, posing a significant clinical burden on children and the elderly.

[0003] The clinical diagnosis and treatment of respiratory infections faces the following contradictions: First, the clinical symptoms of different respiratory pathogens are highly homogeneous, making it impossible to distinguish them based on physical signs alone, resulting in a high rate of misdiagnosis; Second, children and elderly patients have poor cooperation in sampling, and traditional tests require sample volumes of 100μL to 200μL, making micro-sampling difficult; Third, there is a mismatch between existing testing capabilities and clinical needs.

[0004] Currently, respiratory point-of-care testing (POCT) has formed three main technical routes. Multiplex nucleic acid detection technology has high sensitivity but expensive equipment, requires professional personnel to operate, and is not suitable for large-scale deployment at the grassroots level. Rapid immunochromatography technology is fast but mainly qualitative or semi-quantitative, with relatively low sensitivity. Miniaturized chemiluminescence immunoassay can provide quantitative results but is mostly designed for single items and requires large sample volumes.

[0005] In the prior art, several technical solutions have been proposed for magnetic bead immunoassay. Patent document CN202420674034.8 discloses a magnetic bead transfer device that uses a magnetic field to attract magnetic beads and transfer them between reaction containers. However, this solution lacks a specific design for the inter-chamber transfer structure, leading to a significant amount of pre-fluid being carried into the magnetic beads during transfer, and it does not consider scenarios involving trace samples or simultaneous detection of multiple pathogens. Patent document CN202420677342.6 discloses an immunoassay analyzer, including an analysis module and a reaction chamber, which automates the immunoassay process. However, this solution still relies on peristaltic pumps and valves for fluid path drive, resulting in a complex structure and high cost. Patent documents CN202420697962.6 and CN202410397892.7 disclose chemiluminescence immunoassay analyzers, primarily addressing the acquisition and analysis of luminescence signals, but do not address low-residue control for the physical transfer of magnetic beads between multiple chambers or anti-crosstalk processing for multiple pathogen signals. Patent document CN202420678309.5 discloses a heating device for immunoassay, which only addresses the temperature control problem in the immunoassay process.

[0006] A comprehensive analysis of the existing technologies reveals that current solutions generally employ a working mode where peristaltic pumps, multi-position valves, and tubing, among other sophisticated liquid path components, drive reagent flow through a solid-phase carrier. Alternatively, magnetic bead transfer solutions often lack structural design for controlling liquid film residue across chambers. This working mode suffers from the following technical drawbacks: high cost, complex structure, and susceptibility to clogging of liquid path components; multiple pumping and valve switching steps during multi-step cleaning, leading to reagent residue and cross-contamination; dead zones in the liquid path volume limit further reduction in sample volume; liquid path switching between multiple chambers increases the structural complexity of multi-pathogen detection; the lack of a dedicated liquid film stripping structure during magnetic bead cross-chamber transfer makes it difficult to control the amount of preceding liquid carried over; and the lack of an effective mathematical correction mechanism for signal crosstalk between coded magnetic beads in multi-pathogen detection. Therefore, a novel in vitro detection technology solution is urgently needed that can overcome the technical bottlenecks of the aforementioned liquid path-driven modes and meet the requirements of multi-pathogen detection for micro-samples. Summary of the Invention

[0007] The purpose of this invention is to solve the technical problems of existing immunoassay POCT products, which are limited by the liquid-driven mode, insufficient control of magnetic bead transfer residues across chambers, and difficulty in correcting crosstalk signals for multi-pathogen detection. The invention proposes a rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay.

[0008] To achieve the above objectives, this invention proposes a rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay, comprising a disposable multi-chamber detection chip, a magnetically controlled transfer mechanism, an optical detection module, and a quantitative analysis module. The disposable multi-chamber detection chip is sequentially configured along the magnetic microsphere transfer direction with a sample loading chamber, an immunoreaction chamber, at least one magnetic bead washing chamber, and a signal detection chamber. The magnetically controlled transfer mechanism drives the magnetic microspheres to physically transfer sequentially between the chambers by controlling the spatial position and trajectory of the magnetic field, thereby replacing the pump-valve liquid circuit drive method to achieve continuous processing of immunoreaction, washing separation, and signal detection. The optical detection module acquires the optical signals in the signal detection chamber. The quantitative analysis module converts the optical signals into quantitative concentration values ​​for each respiratory pathogen based on a preset standard curve model.

[0009] Preferably, the multiple sets of magnetic microspheres are multiple sets of coded magnetic microspheres, and the different sets of coded magnetic microspheres are distinguished by at least one of particle size coding, fluorescence ratio coding or surface optical reflectance coding; the respiratory pathogens targeted by the multiple sets of coded magnetic microspheres include at least influenza A virus, influenza B virus, respiratory syncytial virus and mycoplasma pneumoniae; the volume of the sample to be tested received by the sample loading chamber is 10 μL to 30 μL.

[0010] Preferably, the at least one magnetic bead cleaning chamber includes a first cleaning chamber and a second cleaning chamber arranged in series; the optical detection module includes a photomultiplier tube or a silicon photomultiplier tube, a darkroom light-shielding structure, and a signal amplification circuit; the quantitative analysis module uses a four-parameter logistic regression model to perform standard curve fitting and concentration back calculation.

[0011] Preferably, a low-residue transfer channel is provided between adjacent chambers, and the low-residue transfer channel includes, in sequence along the transfer direction of the magnetic microspheres, an inlet constriction section, a narrow-diameter transfer section, and an outlet diffusion section; the inner wall of the narrow-diameter transfer section is at least partially provided with a hydrophobic coating with a water contact angle of not less than 110°; at least one of the inlet and outlet of the low-residue transfer channel is provided with a liquid film peeling step or a liquid-limiting lip; the length, width, or hydrophobic coating contact angle of the low-residue transfer channel between the first cleaning chamber and the second cleaning chamber is different from that of the low-residue transfer channel between the immune reaction chamber and the first cleaning chamber.

[0012] Preferably, the magnetic transfer mechanism drives the magnetic microspheres to move according to a preset four-stage magnetic field motion program, including a aggregation-dispersion mixing trajectory in the immune reaction stage, a continuous traction trajectory in the cross-chamber transfer stage, a release-reaggregation trajectory in the cleaning stage, and a fixed-point enrichment trajectory in the signal detection stage; the magnet moving speed in the continuous traction trajectory is less than the aggregation-dispersion moving speed in the aggregation-dispersion mixing trajectory.

[0013] Preferably, the multiple sets of coded magnetic microspheres are packaged according to a preset ratio; the quantitative analysis module establishes an independent standard curve for each set of coded magnetic microspheres and pre-stores a cross-correction matrix. The original signal vector is corrected by using the inverse matrix of the cross-correction matrix before the concentration is calculated.

[0014] Preferably, the sample loading cavity includes a micro-sample volume control area, an overflow buffer, and a sample introduction microchannel that are interconnected; the inlet of the sample introduction microchannel is provided with at least one filtration structure selected from microfiltration membrane, fiber barrier layer, or lateral separation microcolumn.

[0015] The beneficial effects of this invention are as follows: Through the synergistic effect of the three-segment structure of the low-residue transfer channel, the hydrophobic coating, and the liquid film peeling steps, this invention achieves multiple shearing and peeling of the pre-stage liquid film during the cross-chamber transfer of magnetic microspheres, effectively suppressing the residual introduction of pre-stage liquid. The four-stage magnetic field motion program enables the magnetic microspheres to execute differentiated motion trajectories at each detection stage, improving the antigen-antibody binding efficiency and cleaning effect under micro-sample conditions. Furthermore, the combined application of the preset ratio of encoded magnetic microspheres and the cross-calibration matrix eliminates signal crosstalk in multi-pathogen joint detection, improving the accuracy of quantitative results for each pathogen. Attached Figure Description

[0016] The above and other features, properties and advantages of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings and embodiments, in which the same reference numerals always denote the same features, wherein: Figure 1 This is a schematic diagram of the overall architecture of a rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay according to the present invention; wherein, 1 is the overall system, 11 is a disposable multi-chamber detection chip, 12 is a magnetically controlled transfer mechanism, 13 is an optical detection module, 14 is a quantitative analysis module / display terminal, 111 is a sample loading chamber, 112 is an immune reaction chamber, 113 is a primary cleaning chamber, 114 is a secondary cleaning chamber, 115 is a signal detection chamber, 121 is a stepper motor, 122 is a linear guide rail, 123 is a magnet mounting base, and 124 is a magnet assembly.

[0017] Figure 2 This is a schematic diagram of the structure of the disposable multi-chamber detection chip of the present invention; wherein, 2 is the chip, 20 is the sample loading chamber, 21 is the sample inlet, 22 is the micro-sampling and metering area, 23 is the overflow buffer, 24 is the sample introduction microchannel, 25 is the filter / mucus blocking microstructure, 26 is the immune reaction chamber, 27 is the first magnetic bead cleaning chamber, 28 is the second magnetic bead cleaning chamber, 29 is the signal detection chamber, 210 is the transparent optical detection window, and 211 is the low-residue transmission channel.

[0018] Figure 3This is a schematic diagram of the three-section structure and liquid film stripping mechanism of the low-residue transfer channel of the present invention; wherein, 31 is the front chamber, 32 is the rear chamber, 33 is the inlet constriction section, 34 is the narrow-state transmission section, 35 is the outlet diffusion section, 36 is the magnetic bead cluster, 37 is the hydrophobic coating, 38 is the liquid film stripping step, 39 is the liquid limiting lip, and 310 is the stripping liquid film / reflux.

[0019] Figure 4 This is a timing diagram of the four-stage magnetic field motion program of the magnetic transfer mechanism of the present invention; wherein, 41 is the first stage aggregation-dispersion trajectory, 42 is the second stage continuous traction trajectory, 43 is the third stage release-reaggregation trajectory, 44 is the fourth stage fixed-point enrichment trajectory, 45 is the magnet, 46 is the magnetic bead cluster, 47 is the reaction chamber, 48 is the cleaning chamber, and 49 is the detection chamber / detection window.

[0020] Figure 5 This is a schematic diagram of the principle of multi-channel cross-correction of coded magnetic beads in this invention; wherein, 51 is the coded magnetic bead group, 52 is the optical detection module, 53 is the original signal vector S_raw, 54 is the cross-correction matrix M / inverse matrix M^(-1), 55 is the corrected signal vector S_corrected, 56 is the independent standard curve, and 57 is the quantitative result output.

[0021] Figure 6 This is a schematic flowchart of the in vitro detection method based on particle transfer immunoassay of the present invention; wherein, 61 is sample loading, 62 is immune reaction, 63 is cross-compartment transfer and low residual liquid membrane peeling, 64 is two-stage washing, 65 is signal excitation and site enrichment, 66 is encoding recognition and signal acquisition, 67 is cross-correction and quantitative back calculation, 68 is result output, and 69 is residual quality control.

[0022] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0023] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] The core technical concepts involved in the embodiments of this invention will be explained below. Particle transfer immunoassay is the core technical route of the embodiments of this invention. Its core principle is as follows: using magnetic microspheres with specific antibodies modified on their surface as a solid-phase carrier, the magnetic microspheres are driven by an external controllable magnetic field to physically transfer between multiple independent chambers pre-encapsulated with different reagents, thereby completing steps such as immune capture reaction, washing and separation of non-specific conjugates, and excitation and detection of signal substrates. Unlike the traditional immunoassay mode in which liquid flows through a solid-phase carrier, in this technical route, the solid-phase carrier (magnetic microspheres) is actively moving, while the liquid (reagents) is statically pre-encapsulated in each chamber. This reversal of the technical route brings several structural advantages: no need for precision liquid circuit components such as pumps and valves; each chamber is pre-encapsulated with reagents and is not interconnected, eliminating the risk of cross-contamination; the magnetic microspheres enrich and carry the target analyte, adapting to trace samples. Encoded magnetic microspheres refer to magnetic microspheres identified by means of particle size, fluorescent dye doping ratio, or surface optical reflectivity. Different codes correspond to different types of capture antibodies, ensuring that when multiple sets of magnetic microspheres with different codes are placed in the same reaction system, each set of microspheres captures only its corresponding pathogen antigen. During detection, the signals of each pathogen are distinguished by recognizing the microsphere codes, enabling simultaneous detection of multiple pathogens. Chemiluminescence immunoassay (CLIA) is an immunoassay technique based on chemiluminescence reactions. Enzymes labeled on the detection antibody (such as horseradish peroxidase HRP or alkaline phosphatase ALP) catalyze the luminescent substrate (such as luminol, acridinium ester, etc.) to generate photons; the number of photons is directly proportional to the concentration of the analyte. The sensitivity of chemiluminescence can reach the pg / mL level.

[0025] See Figures 1 to 6 This invention discloses a rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay, comprising four core components: a disposable multi-chamber detection chip 11, a magnetic transfer mechanism 12, an optical detection module 13, and a quantitative analysis module 14. Each component is described in detail below.

[0026] Disposable multi-chamber detection chip: such as Figure 2 As shown, the disposable multi-chamber detection chip 2 is the core consumable of this invention, and is molded in one piece using injection molding. The chip is generally elongated or card-shaped, and along the magnetic microsphere transfer direction, it is sequentially arranged with a sample loading chamber 20, an immune reaction chamber 26, a first magnetic bead cleaning chamber 27, a second magnetic bead cleaning chamber 28, and a signal detection chamber 29. Adjacent chambers are connected by a low-residue transmission channel 211.

[0027] (1) Sample loading chamber 20. The sample loading chamber is located at the beginning of the chip and has a sample inlet 21 for receiving micro-volume samples of 10 μL to 30 μL. The sample loading chamber includes a micro-sampling measurement area 22, an overflow buffer 23, and a sample introduction microchannel 24 that are interconnected. The volume of the micro-sampling measurement area 22 is precisely designed to be 20 μL, and its structural parameters are 5 mm in length, 2 mm in width, and 2 mm in depth. When the volume of the injected sample exceeds 20 μL, the excess sample flows into the overflow buffer 23 through the lateral overflow port, thereby precisely limiting the volume of sample entering the immunoreaction chamber 26 to 20 μL, eliminating the influence of fluctuations in the sample volume added by medical staff or patients on the quantitative accuracy of the test results.

[0028] A filtration / mucus-blocking microstructure 25 is provided at the entrance of the sample introduction microchannel 24. In one embodiment, the filtration structure is a polyvinylidene fluoride microfiltration membrane with a pore size of 5 μm, used to filter blood cells in capillary blood samples; in another embodiment, the mucus-blocking structure is an array of staggered lateral separation microcolumns with a diameter of 0.2 mm and a spacing of 0.15 mm, used to separate mucus clumps and epithelial cell debris in the swab eluent.

[0029] The beneficial effects of the sample loading chamber structure in this embodiment are as follows: by combining the design of the micro-sample volume control area and the overflow buffer, the sample volume entering the immunoreaction chamber is precisely limited to 20 μL, eliminating the impact of volume fluctuations during manual sample loading on quantitative accuracy; by setting up the filter structure, large particles in the sample are effectively blocked from entering the immunoreaction chamber, avoiding sample matrix interference and improving the stability and repeatability of the detection results.

[0030] (2) Immunoreaction Chamber 26. The immunoreaction chamber is located downstream of the sample loading chamber and is the core chamber for the antigen-antibody immunobinding reaction. The volume of this chamber is 50 μL to 100 μL, and it is pre-encapsulated with multiple sets of encoded magnetic microspheres, a mixture of chemiluminescently labeled detection antibodies, and reaction buffer. In a preferred embodiment, four sets of encoded magnetic microspheres are provided, corresponding to influenza A virus nucleoprotein antigen, influenza B virus nucleoprotein antigen, respiratory syncytial virus F protein antigen, and Mycoplasma pneumoniae P1 protein antigen, respectively. The particle size of each set of encoded magnetic microspheres is 2 μm to 5 μm, and the surface is modified with monoclonal antibodies corresponding to the pathogen by chemical coupling. Each set of microspheres contains approximately 5,000 to 10,000 microspheres. The multiple sets of encoded magnetic microspheres are encapsulated according to a preset ratio. The principle for determining the preset ratio is: for pathogens with a high clinical concentration range but strong antibody affinity, the number of corresponding magnetic microspheres is relatively small; for pathogens with a low clinical concentration range or weak antibody affinity, the number of corresponding magnetic microspheres is relatively large. This differentiated ratio can keep the signal response range of each pathogen detection channel relatively balanced.

[0031] (3) Magnetic bead cleaning chambers 27 and 28. A first cleaning chamber 27 and a second cleaning chamber 28 are set up for two-stage series cleaning. The volume of each cleaning chamber is 200 μL to 500 μL, and it is pre-encapsulated with phosphate buffered saline (PBS-T) containing Tween-20 surfactant as the cleaning solution.

[0032] (4) Signal detection cavity 29. The signal detection cavity is located downstream of the cleaning cavity and is pre-encapsulated with a chemiluminescent substrate solution. A transparent optical detection window 210 is provided at the bottom of the signal detection cavity for the optical detection module 13 to collect photon signals.

[0033] (5) Low-residue transfer pathway 211. For example... Figure 3 As shown, the low-residue transfer channel is one of the core structural innovations of this invention. Along the transfer direction of the magnetic microspheres, the low-residue transfer channel comprises three structural segments: an inlet contraction section 33, a narrow-state transport section 34, and an outlet diffusion section 35.

[0034] The function of the inlet contraction section 33 is to compress the thickness of the liquid film attached to the outer periphery of the magnetic bead cluster 36 when it enters the low-residue transfer channel. A liquid film peeling step 38 is provided at the inlet of the inlet contraction section 33. When the magnetic bead cluster 36 passes through the liquid film peeling step 38 under magnetic traction, the liquid film attached to the top and periphery of the magnetic bead cluster is subjected to the shearing action of the step, and a part of the liquid film is peeled off and flows back to the previous chamber 310.

[0035] The narrow-section transfer segment 34 is the core functional segment of the low-residue transfer channel, and its structural parameters are precisely designed as follows: width 0.5 mm to 1.5 mm, depth 0.3 mm to 0.8 mm, and length 3 mm to 8 mm. The inner wall of the narrow-section transfer segment 34 is provided with a hydrophobic coating 37. In a preferred embodiment, the hydrophobic coating 37 is made of perfluoropolyether or fluorosilane, with a water contact angle of not less than 110° (preferably 120° to 140°). The hydrophobic coating 37 makes it difficult for aqueous cleaning fluid to continuously creep along the inner wall of the channel.

[0036] The outlet diffusion section 35 is used to rapidly disperse the magnetic bead cluster 36 after it enters the subsequent chamber 32. A liquid-limiting lip 39 is provided at the outlet of the outlet diffusion section 35. The liquid-limiting lip 39 forms a local height abrupt change with the bottom wall of the channel, which further reduces the volume of liquid carried by the magnetic bead cluster from the previous chamber into the subsequent chamber.

[0037] The liquid residue carryover rate in low-residue transfer channels can be estimated using the following formula: R_carry=V_film / V_chamber×100% In the above formula, R_carry is the liquid residue carryover rate, V_film is the volume of liquid film ultimately attached and carried by the magnetic bead cluster when passing through the low-residue transfer channel, and V_chamber is the total liquid volume of the previous chamber. In this embodiment, with the synergistic effect of the three-stage structure, liquid film stripping step 38, and hydrophobic coating 37, V_film is controlled between 1 μL and 3 μL, while the liquid volume of the previous chamber, V_chamber, is typically between 200 μL and 500 μL. Therefore, the single-stage R_carry is typically less than 2%.

[0038] Furthermore, in this embodiment, the narrow diameter of the low-residue transfer channel between the immune reaction chamber 26 and the first cleaning chamber 27 is 5 mm, and the contact angle of the hydrophobic coating is 120°; while the narrow diameter of the low-residue transfer channel between the first cleaning chamber 27 and the second cleaning chamber 28 is 7 mm, and the contact angle of the hydrophobic coating is 130°. Through the cumulative cleaning effect of the two-stage tandem low-residue transfer channels, the total removal rate of nonspecific contaminants is greater than 99.9%.

[0039] The beneficial effects of the low-residue transfer channel structure in this embodiment are as follows: through the synergistic effect of the three-section structure of the inlet contraction section, the narrow-state transmission section, and the outlet diffusion section, along with the hydrophobic coating, the liquid film peeling step, or the liquid-limiting lip, the magnetic bead cluster is subjected to triple liquid film peeling action during the cross-chamber transfer process, including physical compression at the inlet, liquid-repellent obstruction on the inner wall, and shearing peeling at the step or lip. This controls the single-stage liquid residue carry-in rate to below 2%, fundamentally overcoming the technical problem of high background signal and decreased sensitivity caused by the large amount of liquid carried in the previous stage in traditional magnetic bead immunoassay in the case of micro-sample scenarios.

[0040] Magnetic transfer mechanism and four-stage magnetic field motion program: such as Figure 1 and Figure 4 As shown, the magnetic transfer mechanism 12 is located below the multi-chamber detection chip and includes a stepper motor 121, a linear guide rail 122, a magnet mounting base 123, and a magnet assembly 124.

[0041] The magnetic transfer mechanism of this invention employs a four-stage magnetic field motion program for precise control, such as... Figure 4 As shown: Phase 1, Stage 41: Aggregation-Dispersion Trajectory in the Immune Response Phase. Magnet 45, located at the bottom of reaction chamber 47, performs small-amplitude periodic displacement movements at a preset frequency, causing the magnetic bead clusters 46 to periodically aggregate and disperse. The displacement amplitude is 2mm to 5mm, and the movement period is 10 to 20 seconds. This periodic aggregation and dispersion is equivalent to microscale mixing of the sample, reducing the immune response time from the traditional 30 to 60 minutes of static reactions to 5 to 10 minutes.

[0042] Phase 2, Section 42: Continuous Traction Trajectory in the Inter-Cavity Transfer Stage. Magnet 45 moves continuously at a constant speed from the bottom of the current chamber to the bottom of the adjacent chamber, continuously pulling the magnetic bead cluster 46 through the low-residue transfer channel. The magnet's moving speed is 0.5 mm / s to 2 mm / s, much slower than the coalescing / dispersing speed in Phase 1. This slower continuous traction speed ensures that the inlet contraction section, hydrophobic coating, and liquid film stripping step fully utilize their liquid film stripping capabilities in sequence.

[0043] Phase 3 (43): Release-Reaggregation Trajectory in the Cleaning Stage. Magnet 45 performs a combination of lateral reciprocating motion and vertical approach-remote movement at the bottom of the cleaning chamber 48. The specific sequence of actions is as follows: First, the magnet briefly moves away from the bottom of the chip (3mm to 5mm away, lasting 1 to 2 seconds), allowing the magnetic bead clusters 46 to disperse freely in the cleaning solution; then, the magnet approaches the bottom again to reaggregate the magnetic beads; next, the magnet performs lateral reciprocating motion 3 to 5 times; finally, the magnet moves away and approaches once more. The frequency of each action in Phase 3 is higher than the aggregation and dispersion frequency in Phase 1 to enhance the scouring and shearing effect on the non-specific adsorbates on the surface of the magnetic microspheres.

[0044] Phase 44: Fixed-point enrichment trajectory during signal detection. The magnet 45 is fixed at a preset position below the detection window at the bottom of the detection chamber 49 and remains stationary for 20 to 30 seconds, allowing the magnetic bead cluster 46 to stably accumulate in the detection window area to form a uniformly thick enrichment layer.

[0045] The beneficial effects of the four-stage magnetic field motion procedure in this embodiment are as follows: the periodic aggregation and dispersion motion in the immune reaction stage shortens the immune reaction time; the slow, continuous traction in the cross-chamber transfer stage ensures the liquid film peeling effect; the release-reaggregation action combination in the cleaning stage enhances the cleaning effect; and the fixed-point enrichment in the signal detection stage ensures the consistency and reproducibility of optical detection. The coordinated operation of the differentiated motion trajectories in each stage compresses the entire detection process to within 30 minutes.

[0046] Optical detection module: such as Figure 1 As shown, the optical detection module 13 is located at the detection position of the signal detection cavity 29, and includes a photodetector (PMT / PD), an optical lens, a filter, and an excitation source. When using coded magnetic microspheres for multi-pathogen detection, the optical detection module first identifies the code on the magnetic microspheres through the excitation source, and then turns off the source to collect the chemiluminescence signal intensity.

[0047] Quantitative analysis module and cross-correction: such as Figure 5 As shown, the quantitative analysis foundation of module 14 is the standard curve fitting and inverse calculation of four-parameter logistic regression (4PL). The mathematical expression of the 4PL model is: y = D + (AD) / (1 + (x / C)^B) In the above formula, y represents the optical signal intensity, x represents the analyte concentration, A represents the lower asymptote of the curve, D represents the upper asymptote of the curve, C represents the half-effective concentration EC50, and B represents the curve slope factor. The inverse function is: x = C × ((AD) / (yD) - 1)^(1 / B) like Figure 5 As shown in Figure 54, this invention introduces a cross-correction matrix M. The dimension of the cross-correction matrix M is n×n, where n is the number of pathogen channels. The element M_ij in the i-th row and j-th column of matrix M represents the cross-reactivity coefficient of the pure signal of the j-th pathogen to the detection channel of the i-th pathogen.

[0048] Signal correction process as follows Figure 5 As shown in Figures 53 to 55: First, acquire the original signal vector S_raw; second, perform matrix correction using the inverse of the cross-correction matrix M. S_corrected=M^(-1)×S_raw The third step, as Figure 5 As shown in Figure 56, by substituting each component of the corrected signal vector S_corrected into the independent 4PL standard curves of the corresponding pathogens, the quantitative concentration values ​​of each pathogen are calculated. Figure 5 As shown in Figure 57.

[0049] For example, taking a 4-way cross-checking test, assume the cross-checking matrix is ​​as follows: M=[[1.00,0.05,0.02,0.01], [0.04, 1.00, 0.03, 0.02], [0.02,0.03,1.00,0.04], [0.01,0.02,0.03,1.00]] Suppose the original signal vector of a sample is S_raw=[100,50,80,30]^T, then the corrected signal vector is approximately S_corrected=[95.4,44.9,76.3,26.6]^T, and the pure signal of each pathogen is reduced by 4% to 11%.

[0050] The beneficial effect of the quantitative analysis module in this embodiment is that by combining the application of the preset ratio of coded magnetic microspheres with the cross-correction matrix, signal crosstalk in multi-pathogen joint detection is suppressed from both the source and post-processing dimensions, so that the quantitative accuracy of each pathogen in multiplex detection is close to the level of single independent detection.

[0051] Detection methods based on particle transfer immunoassay: such as Figure 6As shown, the method includes the following steps: Step 61, Sample loading. A micro-sample of 10 μL to 30 μL is dropped into the sample loading cavity 20 of the chip. The sample is precisely limited to a preset volume through the micro-sampling metering area 22. Excess sample flows into the overflow buffer 23, and the filter structure 25 blocks particulate impurities.

[0052] Step 62, Immunoreaction. The sample automatically flows into the immunoreaction chamber 26 and mixes with pre-encapsulated multiple groups of encoded magnetic microspheres and labeled antibodies. The magnetron transfer mechanism executes the first-stage aggregation-dispersion trajectory 41 for 5 to 10 minutes.

[0053] Step 63, cross-chamber transfer and low residual liquid film stripping. The magnetic transfer mechanism executes the second-stage continuous traction trajectory 42, and the magnetic bead cluster passes sequentially through the inlet contraction section 33, the narrow transmission section 34 (the hydrophobic coating 37 hinders the liquid film crawling), the liquid film stripping step 38 (the liquid film is sheared and stripped back 310), and the outlet diffusion section 35.

[0054] Step 64, two-stage cleaning. The magnetic transfer mechanism executes the third-stage release-re-aggregation trajectory 43, completing two-stage cleaning in the first cleaning chamber 27 and the second cleaning chamber 28. The cumulative liquid residue rate after two-stage cleaning is less than 0.04%. Optionally, residue quality control 69 is performed.

[0055] Step 65: Signal excitation and site-specific enrichment. The magnetron transfer mechanism executes the fourth-stage site-specific enrichment trajectory 44, and the magnetic bead clusters are stably enriched below the detection window. The HRP enzyme catalyzes the luminescent substrate to produce a chemiluminescent reaction.

[0056] Step 66, Encoding Recognition and Signal Acquisition. The optical detection module 13 first identifies the encoding information of each magnetic microsphere by excitation light source, and then turns off the light source to acquire the intensity of chemiluminescence signal, forming the original signal vector S_raw.

[0057] Step 67, cross-correction and quantitative back-calculation. For example... Figure 5 As shown in Figures 54 to 56, the quantitative analysis module uses the inverse of the cross-correction matrix M to correct S_raw to obtain S_corrected, and then substitutes it into the independent 4PL standard curves of each pathogen to calculate the concentration.

[0058] Step 68, Results Output. A test report is generated by combining the quantitative concentration values ​​of each pathogen. The entire process takes no more than 30 minutes.

[0059] Residual quality control methods: such as Figure 6As shown in Figure 69, the detection method of the present invention further includes a residue control step. Negative control encoded magnetic microspheres or blank encoded magnetic microspheres, whose surfaces are not modified with any pathogen-specific capture antibodies, are additionally added to the immunoreaction chamber. When abnormal residues are present in the low-residue transfer channel or during the washing process, the free labeled antibody generates a detectable background signal through non-specific adsorption. When the background optical signal exceeds a preset background threshold, it is determined that there is a residue abnormality in this detection, and a retest prompt or abnormal report is output.

[0060] The beneficial effect of the residue quality control steps in this embodiment is that the residue control level is converted into a quantifiable background signal by the built-in negative control magnetic microspheres, which can realize automated real-time residue monitoring without additional instruments and ensure the reliability of the quantitative results of the detection system.

[0061] VII. Examples of Electronic Devices In some embodiments, an electronic device includes at least one processor and a memory communicatively connected to the processor. The memory stores instructions executable by the processor to perform the code recognition, cross-correction, and quantitative inverse calculation steps in the detection method provided by the present invention. The electronic device is integrated into a portable detection device.

[0062] This invention discloses a rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay. During operation, magnetic microspheres, acting as solid-phase carriers, are physically transferred between chambers, replacing the traditional pump-valve liquid-driven mode. The three-segment structure of the low-residue transfer channel enables multiple shearing and stripping of the pre-stage liquid membrane. A four-stage magnetic field motion program executes differentiated motion trajectories in each detection stage. The preset ratio of coded magnetic beads and the cross-correction matrix eliminate signal crosstalk in the joint detection of multiple pathogens.

[0063] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A rapid in vitro detection system for multiple respiratory pathogens based on particle transfer immunoassay, characterized in that, The system includes: A disposable multi-chamber detection chip, comprising a sample loading chamber, an immune reaction chamber, at least one magnetic bead washing chamber, and a signal detection chamber arranged sequentially along the magnetic microsphere transfer direction; the immune reaction chamber is pre-encapsulated with multiple sets of magnetic microspheres and labels, each set of magnetic microspheres having a surface modified with specific capture antibodies against different respiratory pathogen antigens; the magnetic bead washing chamber is pre-encapsulated with a washing solution; and the signal detection chamber is pre-encapsulated with a signal excitation substrate. A magnetically controlled transfer mechanism is located below or to the side of the disposable multi-chamber detection chip. It includes a movable permanent magnet assembly or an electromagnet assembly. By controlling the spatial position and movement trajectory of the magnetic field, the magnetic microspheres are driven to physically transfer sequentially between the immune reaction chamber, the magnetic bead cleaning chamber, and the signal detection chamber. This replaces the pump valve liquid circuit drive method with the spatial transfer of the magnetic microspheres, thereby realizing continuous processing of immune reaction, cleaning separation, and signal detection. An optical detection module is disposed at the detection position of the signal detection cavity and is used to collect the optical signal generated by the immune reaction in the signal detection cavity; The quantitative analysis module is communicatively connected to the optical detection module and is used to receive the optical signal and convert the optical signal into quantitative concentration values ​​of each respiratory pathogen based on a preset standard curve model.

2. The system according to claim 1, characterized in that, The multiple sets of magnetic microspheres are multiple sets of coded magnetic microspheres, and different sets of coded magnetic microspheres are distinguished by at least one of particle size coding, fluorescence ratio coding, or surface optical reflectance coding; the respiratory pathogens targeted by the multiple sets of coded magnetic microspheres include at least influenza A virus, influenza B virus, respiratory syncytial virus, and mycoplasma pneumoniae; the volume of the sample to be tested received in the sample loading chamber is 10 μL to 30 μL, and the sample to be tested includes one or more of nasopharyngeal swab eluent, pharyngeal swab eluent, and peripheral blood.

3. The system according to claim 1, characterized in that, The at least one magnetic bead cleaning chamber includes a first cleaning chamber and a second cleaning chamber arranged in series. The magnetic microspheres are sequentially cleaned through the first cleaning chamber and the second cleaning chamber under the drive of the magnetically controlled transfer mechanism. The optical detection module includes a photomultiplier tube or a silicon photomultiplier tube, a darkroom light-shielding structure, and a signal amplification circuit for collecting photon signals from the chemiluminescence reaction. The quantitative analysis module uses a four-parameter logistic regression model to perform standard curve fitting and concentration back-calculation on the optical signal. The four-parameter logistic regression model is y=D+(AD) / (1+(x / C)^B), where y is the optical signal intensity, x is the concentration of the analyte, A is the lower asymptote of the curve, D is the upper asymptote of the curve, C is the half-effective concentration, and B is the curve slope factor.

4. The system according to any one of claims 1 to 3, characterized in that, A low-residue transfer channel is provided between adjacent chambers. The low-residue transfer channel, along the transfer direction of the magnetic microspheres, sequentially includes an inlet constriction section, a narrow-diameter transfer section, and an outlet diffusion section. The inlet constriction section is used to compress the thickness of the liquid film adhering to the outer periphery of the magnetic microspheres when they enter the low-residue transfer channel. The narrow-diameter transfer section has a width of 0.5 mm to 1.5 mm, a depth of 0.3 mm to 0.8 mm, and a length of 3 mm to 8 mm. At least a portion of the inner wall of the narrow-diameter transfer section is provided with a hydrophobic coating, and the water contact angle of the hydrophobic coating is not less than 110°. The outlet diffusion section is used for… To enable the magnetic microspheres to disperse rapidly after entering the next chamber; at least one of the inlet and outlet of the low-residue transfer channel is provided with a liquid film peeling step or a liquid-limiting lip, and a local height abrupt change is formed between the liquid film peeling step or liquid-limiting lip and the bottom wall of the low-residue transfer channel, so as to shear and peel off the liquid film attached to the periphery of the magnetic microspheres when the magnetic microspheres pass through; wherein, the length, width or hydrophobic coating contact angle of the low-residue transfer channel between the first cleaning chamber and the second cleaning chamber is different from that of the low-residue transfer channel between the immune reaction chamber and the first cleaning chamber.

5. The system according to any one of claims 1 to 3, characterized in that, The magnetically controlled transfer mechanism drives the magnetic microspheres to move within and between the chambers according to a preset four-stage magnetic field motion program. The four-stage magnetic field motion program includes: a first magnetic field motion trajectory executed during the immune reaction phase, causing the magnetic microspheres to periodically aggregate and disperse at a preset frequency within the immune reaction chamber; a second magnetic field motion trajectory executed during the inter-chamber transfer phase, causing the magnetic microspheres carrying immune complexes to pass through the low-residue transfer channel in a continuous traction manner; a third magnetic field motion trajectory executed during the washing phase, including at least one combination of lateral reciprocating or vertical approaching and moving away within the washing chamber, causing the magnetic microspheres to be released, dispersed, and re-aggregated in the washing solution; and a fourth magnetic field motion trajectory executed during the signal detection phase, causing the magnetic microspheres to be fixedly enriched in the region below the detection window of the signal detection chamber to form a stable enrichment layer; wherein, the magnet moving speed in the second magnetic field motion trajectory is less than the aggregation and dispersion moving speed in the first magnetic field motion trajectory.

6. The system according to claim 2, characterized in that, The multiple sets of encoded magnetic microspheres are encapsulated in the immune reaction chamber according to a preset ratio. The preset ratio is determined based on the clinical concentration range of the target antigen of each respiratory pathogen, the affinity of the corresponding antibody, and the signal response range of each detection channel, so as to prevent the signal of high-abundance pathogens from masking the signal of low-abundance pathogens. The quantitative analysis module establishes an independent standard curve for each set of encoded magnetic microspheres and pre-stores a cross-correction matrix for correcting signal crosstalk between different encoded magnetic microspheres. The signal processing procedure of the quantitative analysis module includes: acquiring the original optical signals of the detection channels corresponding to each coded magnetic microsphere to form an original signal vector S_raw; performing matrix correction on the original signal vector using the inverse matrix of the cross-correction matrix M to obtain a corrected signal vector S_corrected=M^(-1)×S_raw; and then substituting each component in the corrected signal vector into the independent standard curve of the corresponding respiratory pathogen to calculate the quantitative concentration value of each respiratory pathogen. The element M_ij in the i-th row and j-th column of the cross-correction matrix M represents the cross-response coefficient of the j-th pathogen signal to the i-th pathogen detection channel, which is determined through factory calibration or user calibration.

7. The system according to any one of claims 1 to 3, characterized in that, The sample loading chamber includes an interconnected micro-sample volume control area, an overflow buffer, and a sample introduction microchannel. The volume of the micro-sample volume control area is configured to be 10 μL to 30 μL. When the volume of the injected sample exceeds the volume of the micro-sample volume control area, the excess sample flows into the overflow buffer to limit the volume of sample entering the immunoreaction chamber. The inlet of the sample introduction microchannel is provided with a sample filtration structure or a mucus barrier structure, which includes at least one of a microfiltration membrane, a fiber barrier layer, or a lateral separation microcolumn, to reduce particulate impurities, mucus clumps, or cellular components in the swab eluent or capillary blood sample from entering the immunoreaction chamber.

8. A rapid in vitro detection method for multiple respiratory pathogens based on particle transfer immunoassay, characterized in that, The method of using the system of any one of claims 1 to 7 comprises: A small amount of test sample with a volume of 10 μL to 30 μL is added to the sample loading chamber of the disposable multi-chamber detection chip, and the small amount of test sample is then introduced into the immune reaction chamber. Within the immune reaction chamber, the trace sample to be tested undergoes an immune reaction with the multiple sets of magnetic microspheres and the label, forming an immune complex of magnetic microsphere-antigen-labeled antibody; the magnetically controlled transfer mechanism executes the first magnetic field motion trajectory during the immune reaction, causing the magnetic microspheres to periodically aggregate and disperse at a preset frequency; The magnetically controlled transfer mechanism executes the second magnetic field motion trajectory, causing the magnetic microspheres carrying the immune complex to be transferred through the low-residue transfer channel to the magnetic bead washing chamber in a continuous traction manner; within the low-residue transfer channel, the liquid film attached to the periphery of the magnetic microspheres is peeled off by the combination of the inlet contraction section, the hydrophobic coating, and the liquid film peeling step or the liquid-limiting lip. The magnetic transfer mechanism executes the third magnetic field motion trajectory, causing the magnetic microspheres to complete a two-stage release-re-aggregation cleaning process sequentially in the first cleaning chamber and the second cleaning chamber. The magnetic transfer mechanism executes the fourth magnetic field motion trajectory to transfer the cleaned magnetic microspheres to a fixed point below the detection window of the signal detection cavity for enrichment. The optical detection module identifies the coding information of the coded magnetic microspheres and acquires the corresponding optical signals. The quantitative analysis module corrects the original signal vector based on the cross-correction matrix and then calculates the quantitative concentration value of each respiratory pathogen based on the independent standard curve corresponding to each pathogen. The entire process, from the addition of the trace sample to the output of the quantitative concentration value, takes no more than 30 minutes.

9. The method according to claim 8, characterized in that, The method further includes a residue quality control step, which includes: placing negative control coded magnetic microspheres or blank coded magnetic microspheres in the signal detection cavity; collecting the background optical signal of the negative control coded magnetic microspheres or the blank coded magnetic microspheres; determining whether the residue control of the low-residue transfer channel and the multi-stage cleaning process meets the preset residue threshold requirements based on the background optical signal; when the background optical signal exceeds the preset background threshold, determining that there is a residue abnormality in this detection, and outputting a retest prompt or abnormal report; wherein, the execution frequency of the aggregation and dispersion action of the first magnetic field movement trajectory in the immune reaction stage is lower than the execution frequency of the release-reaggregation action of the third magnetic field movement trajectory in the cleaning stage.

10. An electronic device, characterized in that, The electronic device includes a processor and a memory communicatively connected to the processor. The memory stores a computer program executable by the processor. When the computer program is executed by the processor, it implements the encoding recognition, cross-correction, and quantitative inverse calculation steps in the method of claim 8 or 9.

Images