Microfluidic multi-target detection system based on fluidized bed magnetic enrichment and cds / qds@zif-8 physical amplification

Abstract

The application discloses a microfluidic multi-target detection system based on fluidized bed magnetic enrichment and CDs / QDs@ZIF-8 physical amplification. The system comprises capture magnetic particles, a fluidized bed magnetic enrichment microfluidic chip, CDs / QDs@ZIF-8 biological tracers and a dissolution release agent. The capture magnetic particles can be used for synchronously separating and enriching multi-targets in a large-volume sample to be detected in the fluidized bed magnetic enrichment microfluidic chip. The CDs / QDs@ZIF-8 biological tracers can be used for identifying the enriched multi-targets by loading different carbon dot or quantum dot fluorescent signals, and the dissolution release agent can be used for enhancing the signals of the identified multi-targets, so that the multi-targets in the sample to be detected can be detected with high sensitivity, and the system has the characteristics of high integration and high automation.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, specifically to a microfluidic multi-target biosensing hypersensitive immunoassay system and its detection method based on fluidized bed magnetic enrichment and physical amplification of carbon dots / quantum dots@zeolitic imidazolate framework nanomaterials (CDs / QDs@ZIF-8). Background Technology

[0002] Foodborne pathogens easily cause food safety problems and seriously endanger human health. Therefore, rapid screening for foodborne pathogens is an important means of ensuring food safety. Foodborne pathogens in food have significant characteristics such as the ability to proliferate, complex substrates, and diverse species, which places clear performance requirements on detection technologies, including high sensitivity, resistance to interference, and multi-target capabilities. Conventional detection methods for foodborne pathogens, such as plate counting, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), cannot meet practical needs.

[0003] To achieve the required high sensitivity and interference resistance for detecting foodborne pathogens, immunomagnetic separation technology based on antigen-antibody immune reactions and electrochemical detection technology have been widely used in the isolation, enrichment, and detection of bacteria. For example, patent document CN103217529A (hereinafter referred to as Document 1) discloses a rapid detection method for foodborne pathogens based on indirect enrichment immunomagnetic separation using γ-Fe2O3 nanoparticles. It uses immunomagnetic beads coupled with specific monoclonal antibodies to enrich specific pathogens in liquid / liquefied test samples, thereby achieving interference resistance and high sensitivity detection. However, in the method disclosed in Document 1, the amount of test sample that can be processed is small (≤1mL), the mixing / capture / enrichment steps rely on manual operation, it cannot be automated, it cannot process large volumes of test samples, and it can only detect a single target. On the other hand, the method in Document 1 also requires pretreatment such as filtration and enrichment activation of the test sample, so the operation is cumbersome. Zhong Miao (Highly Sensitive Electrochemical Detection of Pathogenic Bacteria and Uric Acid, Hunan Normal University, Master's Thesis, June 2018, hereinafter referred to as Reference 2) synthesized a CdS@ZIF-8 nanocapsule and constructed a sandwich-type electrochemical immunosensor using it as a signal probe for the quantitative detection of E. coli O157:H7. The results showed that compared with the traditional analytical method using CdS nanoparticles as markers, using CdS@ZIF-8 nanocapsules as markers significantly amplified the electrochemical signal, with a sensitivity 16 times that of the traditional method. However, Reference 2 detects Cd released into solution by CdS@ZIF-8 nanocapsules. 2+This amplifies the electrochemical signal, thus preventing simultaneous detection of multiple targets. Summary of the Invention

[0004] To address one or more problems existing in the prior art, one aspect of this application provides a microfluidic multi-target detection system based on fluidized bed magnetic enrichment and physical amplification of CDs / QDs@ZIF-8, comprising a magnetic particle capture device, a fluidized bed magnetic enrichment microfluidic chip, CDs / QDs@ZIF-8 biotracers for each target, and a dissolution and release agent; wherein:

[0005] The surface of the captured magnetic particles is modified with antibodies targeting one or more of multiple targets, which can specifically bind to their corresponding targets.

[0006] The fluidized bed magnetic enrichment microfluidic chip includes a substrate, multiple inlets and outlets disposed on the surface of the substrate, and a fluidized bed chamber, a fluorescence detection cell, and a microfluidic channel disposed within the substrate. The multiple inlets include a CDs / QDs@ZIF-8 biological tracer inlet, a multi-target inlet for injecting the sample to be tested, and a dissolving and releasing agent inlet. The multiple outlets include a waste liquid outlet and a fluorescence signal molecule collection outlet. Each inlet is connected to the chamber inlet of the fluidized bed chamber via the microfluidic channel. The chamber outlet of the fluidized bed chamber is connected to the waste liquid outlet and the inlet of the fluorescence detection cell via the microfluidic channel, respectively. The outlet of the fluorescence detection cell is connected to the fluorescence signal molecule collection outlet via the microfluidic channel.

[0007] The CDs / QDs@ZIF-8 biological tracer includes a ZIF-8 framework and a fluorescent signal molecule loaded on the ZIF-8 framework. The surface of the CDs / QDs@ZIF-8 biological tracer is modified with an antibody that can specifically bind to the corresponding target. The fluorescent signal molecule is selected from carbon dots and quantum dots.

[0008] The dissolving and releasing agent is used to dissolve ZIF-8 in the CDs / QDs@ZIF-8 biotracer.

[0009] In some embodiments, in the fluidized bed magnetic enrichment microfluidic chip, the cross-section of the fluidized bed chamber is spindle-shaped, with a total length of 20-100 mm, a width at its widest point of 2-20 mm, a chamber height of 100-800 μm (optionally 400-600 μm), a chamber inlet angle of 10-30°, a chamber outlet angle of 40-75°, and a diameter of 1-1.5 mm for both the chamber inlet and outlet.

[0010] In some embodiments, in the fluidized bed magnetic enrichment microfluidic chip, the height of the microfluidic channel is the same as the height of the fluidized bed chamber.

[0011] In some embodiments, the diameter of the plurality of liquid inlets and the plurality of liquid outlets is 1-1.5 mm.

[0012] In some embodiments, the length of the fluorescence detection cell is 5-20 mm, the width is 2-10 mm, and the height is the same as that of the fluidized bed chamber.

[0013] In some embodiments, the microfluidic multi-target detection system further includes an external magnetic field; optionally, the magnetic field strength of the external magnetic field is 2-10 Tesla (T); further optionally, the external magnetic field is generated by an external magnet, which is located on the outer side of the substrate at a distance of 1-5 mm from the chamber inlet of the fluidized bed chamber.

[0014] In some embodiments, the CDs / QDs@ZIF-8 biological tracers are prepared by the following steps:

[0015] (1) Zinc nitrate hexahydrate Zn(NO3)2·6H2O and dimethylimidazole were mixed and reacted to obtain ZIF-8 nanocarriers;

[0016] (2) The fluorescent signal molecules are mixed and reacted with the ZIF-8 nanocarrier prepared in step (1) above, so that the fluorescent signal molecules are loaded on the ZIF-8 nanocarrier to obtain the CDs / QDs@ZIF-8 complex; and

[0017] (3) Modify the surface of the CDs / QDs@ZIF-8 complex obtained in step (2) above so that it can specifically bind to the corresponding target to obtain CDs / QDs@ZIF-8 biotracer.

[0018] In some embodiments, the capturing magnetic particles are prepared by the following steps: mixing and incubating magnetic particles modified with streptavidin with an antibody modified with biotin targeting a specific target.

[0019] In another aspect, the present invention provides the application of the aforementioned microfluidic multi-target detection system in the preparation of reagent kits for detecting foodborne pathogens.

[0020] In another aspect, the present invention provides an operational method for the aforementioned microfluidic multi-target detection system to detect multiple targets for non-disease diagnostic purposes, comprising the following steps:

[0021] S1) Block the fluorescent signal molecule collection outlet and open the waste liquid outlet;

[0022] S2) The liquid containing the captured magnetic particles is injected into the fluidized bed chamber from the multi-target inlet, and the captured magnetic particles are made to circulate and reciprocate in the fluidized bed chamber;

[0023] S3) The sample to be tested containing multiple targets is injected into the fluidized bed chamber through the multi-target inlet. In the fluidized bed chamber, the capturing magnetic particles specifically bind to the targets in the sample to form a magnetic particle-antibody-target complex. Optionally, the sample to be tested is injected into the fluidized bed chamber at a flow rate of 0.4-0.6 mL / min, preferably 0.4 mL / min.

[0024] S4) The CDs / QDs@ZIF-8 biological tracer is injected into the fluidized bed chamber through the CDs / QDs@ZIF-8 biological tracer inlet, and it specifically binds to the magnetic particle-antibody-target complex to form a double antibody sandwich complex.

[0025] S5) Block the waste liquid outlet, open the fluorescent signal molecule collection outlet, and inject the dissolving and releasing agent into the fluidized bed chamber from the dissolving and releasing agent inlet, so as to dissolve the ZIF-8 framework in the dual-antibiotic sandwich complex and release the fluorescent signal molecules therein; and

[0026] S6) After the fluorescent signal molecules released in step S5) enter the fluorescence detection cell, the fluorescent signal molecules in the fluorescence detection cell are detected, and the multi-target information in the sample to be tested is determined based on the detected fluorescent signal molecule information.

[0027] Another aspect of the present invention provides a fluidized bed magnetic enrichment microfluidic chip, comprising a substrate, a plurality of liquid inlets and a plurality of liquid outlets disposed on the surface of the substrate, and a fluidized bed chamber, a fluorescence detection cell, and a microfluidic channel disposed within the substrate. The plurality of liquid inlets include a CDs / QDs@ZIF-8 biotracer inlet, a multi-target inlet for injecting a sample to be tested, and a dissolving and releasing agent inlet. The plurality of liquid outlets include a waste liquid outlet and a fluorescence signal molecule collection outlet. Each of the plurality of liquid inlets is connected to the chamber inlet of the fluidized bed chamber via the microfluidic channel. The chamber outlet of the fluidized bed chamber is connected to the waste liquid outlet and the inlet of the fluorescence detection cell via the microfluidic channel, respectively. The outlet of the fluorescence detection cell is connected to the fluorescence signal molecule collection outlet via the microfluidic channel.

[0028] The fluidized bed chamber has a spindle-shaped cross-section, with a total length of 20-100 mm, a maximum width of 2-20 mm, a chamber height of 100-800 μm (optionally 400-600 μm), a chamber inlet angle of 10-30°, a chamber outlet angle of 40-75°, and a diameter of 1-1.5 mm for both the chamber inlet and outlet.

[0029] The height of the microfluidic channel is the same as the height of the fluidized bed chamber;

[0030] The diameter of the plurality of liquid inlets and the plurality of liquid outlets is 1-1.5 mm;

[0031] The length of the fluorescence detection cell is 5-20 mm, the width is 2-10 mm, and the height is the same as that of the fluidized bed chamber.

[0032] In another aspect, the present invention provides a CDs / QDs@ZIF-8 biotracer for use in the aforementioned microfluidic multi-target detection system. The CDs / QDs@ZIF-8 biotracer includes a ZIF-8 framework and a fluorescent signal molecule loaded on the ZIF-8 framework. The surface of the CDs / QDs@ZIF-8 biotracer is modified with an antibody that can specifically bind to a target. The fluorescent signal molecule is selected from carbon dots and quantum dots.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) In the microfluidic multi-target detection system provided by the present invention, the fluidized bed magnetic enrichment microfluidic chip organically integrates the liquid circulation control and immunomagnetic separation technology of microfluidic fluidized bed (a reactor that uses gas or liquid to make solid particles in a suspended state through a particulate solid layer and carry out gas-solid phase reaction process or liquid-solid phase reaction process). The captured magnetic particles can maintain dynamic equilibrium and continuously perform dynamic reciprocating motion in the fluidized bed chamber of the fluidized bed magnetic enrichment microfluidic chip. On the one hand, this allows the system of the present invention to process large volume (e.g., 10 ml) of test samples. On the other hand, it can significantly improve the contact rate between the captured magnetic particles and the target in the test sample, thereby improving the separation and enrichment efficiency of the target in the test sample, which is beneficial to improving the detection sensitivity. Furthermore, there is no need to perform pretreatment such as filtration, enrichment and activation on the test sample.

[0035] (2) In the microfluidic multi-target detection system provided by the present invention, the fluidized bed magnetic enrichment microfluidic chip integrates the separation, enrichment and detection of multiple targets on a single microfluidic chip. Therefore, the system has the characteristics of high integration and automation, which can reduce manual operation, thereby effectively shortening the detection time and improving detection efficiency and accuracy.

[0036] (3) In the microfluidic multi-target detection system provided by the present invention, CDs / QDs@ZIF-8 biological tracers can identify enriched multi-targets by loading different carbon dot or quantum dot fluorescence signals, and use dissolving and releasing agents to enhance the fluorescence signals of each identification, thereby enabling simultaneous detection of multi-targets with low concentrations in the sample, and having high detection sensitivity. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the microfluidic chip designed in an embodiment of the present invention;

[0038] Figure 2 This is a simulation diagram of the magnetic field of the fluidized bed magnetic enrichment microfluidic chip using COMSOL software in an embodiment of the present invention;

[0039] Figure 3 This is a diagram showing the optimized channel height and flow rate in the microfluidic chip in an embodiment of the present invention.

[0040] Figure 4 This is a diagram showing the optimized results of the amount of magnetic particles used in the fluidized bed magnetic enrichment microfluidic chip in this embodiment of the invention.

[0041] Figure 5 This illustrates the dissolution status of the ZIF-8 framework by the dissolution-releasing agent Na2EDTA in this embodiment of the invention.

[0042] Figure 6 This illustrates the stability of fluorescent signal molecules in Na2EDTA in embodiments of the present invention.

[0043] Figure 7 This is the CDs / QDs@ZIF-8 physical amplification system designed in this embodiment of the invention;

[0044] Figure 8a The image shows the fluorescence spectral scanning results of different concentrations of Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B in the embodiments of the present invention.

[0045] Figure 8b This is a standard curve diagram of the microfluidic multi-target biosensor system for three target bacteria in an embodiment of the present invention. Detailed Implementation

[0046] To address the problems of existing methods or devices for detecting foodborne pathogens, such as small sample volume, low automation, single-target detection (unique tracer signal), and / or cumbersome pretreatment like filtration and enrichment, this invention aims to provide a system for the simultaneous separation, enrichment, and ultrasensitive detection of multiple foodborne pathogens in large volumes of test samples. This system includes magnetic capture particles for capturing one or more targets, a fluidized bed magnetic enrichment microfluidic chip, CDs / QDs@ZIF-8 biotracers, and a dissolution-releasing agent. The magnetic capture particles specifically capture and enrich multiple foodborne pathogens in large volumes (e.g., 10 mL) of test samples within the fluidized bed magnetic enrichment microfluidic chip. Furthermore, the captured and enriched multiple foodborne pathogens are separated from each other in the sample to be tested, as well as from other interfering impurities. The CDs / QDs@ZIF-8 biological tracers can identify the enriched multiple foodborne pathogens by loading different carbon dots or quantum dots with fluorescence signals, and the dissolving and releasing agents are used to enhance the signals of each identification. The organic combination of the above processes enables qualitative and quantitative detection of multiple foodborne pathogens in large volumes of samples to be tested, with high detection sensitivity. At the same time, the fluidized bed magnetic enrichment microfluidic chip in the system integrates the separation, enrichment and detection of multiple foodborne pathogens on a single microfluidic chip. Therefore, the system also features a high degree of integration and automation, reducing manual operation, effectively shortening the detection time, and improving detection efficiency and accuracy.

[0047] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0048] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0049] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0050] Example 1: Design and fabrication of a fluidized bed magnetic enrichment microfluidic chip based on a fluidized bed magnetic enrichment chamber as the main structure.

[0051] A schematic diagram of the structure of the fluidized bed magnetic enrichment microfluidic chip designed and fabricated in this embodiment, based on a fluidized bed magnetic enrichment chamber as the main structure, is shown below. Figure 1As shown, the microfluidic chip includes a substrate (which can be made of a transparent or semi-transparent material, such as a rigid resin material), with three inlets on the substrate surface (serving as the inlet for CDs / QDs@ZIF-8 biological tracers, the inlet for multi-target injection of the sample to be tested, and the inlet for Na2EDTA, respectively) and two outlets (serving as the outlet for waste liquid and the outlet for collecting fluorescent signal molecules, respectively). Inside the substrate, there is a fluidized bed chamber, a fluorescence detection cell, and microfluidic channels connecting the aforementioned inlets, outlets, chamber, and detection cell. The fluidized bed chamber has a spindle-shaped cross-section. Its inlet is connected to three liquid inlets, with an inlet angle α of 10-30°. The outlet is connected to a waste liquid outlet and a fluorescence detection cell via microfluidic channels. The outlet angle β is greater than the inlet angle α, for example, β can be 40-75°. The total length of the fluidized bed chamber is 20-100 mm, the width at its widest point is 2-20 mm, and the height is 100-800 μm, optionally 400-600 μm. The fluorescence detection cell can be a square chamber with a length of 5-20 mm, a width of 2-10 mm, and a height designed to be the same as the fluidized bed chamber, 100-800 μm, optionally 400-600 μm, connected to the fluorescence signal molecule collection outlet. The height of the microfluidic channel is designed to be the same as the height of the fluidized bed chamber, ranging from 100-800 μm, with a selectable height of 400-600 μm. It connects the inlet, fluidized bed chamber, waste outlet, fluorescence detection cell, and fluorescence signal molecule collection outlet. The diameters of the inlet (CDs / QDs@ZIF-8 biological tracer inlet, multi-target inlet, Na2EDTA inlet) and outlet (waste outlet and fluorescence signal molecule collection outlet), as well as the fluidized bed chamber inlet and outlet, can all be 1-1.5 mm. By controlling the above parameters and coupling an external magnetic field (e.g., placing a magnet with a magnetic field strength of 2-10 Tesla (T) on the outside of the substrate 1-5 mm away from the inlet of the spindle-shaped chamber), the captured magnetic particles injected into the fluidized bed chamber through the multi-target inlet can continuously reciprocate within the fluidized bed chamber, thereby improving the capture (enrichment) efficiency of the target in the sample to be tested added from the multi-target inlet and shortening the sample processing time (detailed below).

[0052] The design and fabrication method of the microfluidic chip provided in this embodiment is as follows:

[0053] (1) A microfluidic chip was designed and drawn using Solidworks software, including a substrate, and three inlets (CDs / QDs@ZIF-8 biological tracer inlet, multi-target inlet, and Na2EDTA inlet) and outlets (waste outlet and fluorescent signal molecule collection outlet) on the surface of the substrate, as well as a fluidized bed chamber, a fluorescence detection cell, and microfluidic channels arranged within the substrate, as described above. Various microfluidic chips with different design parameters were fabricated (exemplary structures are shown below). Figure 1 As shown below, three of them (microfluidic chips 1-3) are used in the experiments described later:

[0054] Microfluidic chip 1: The diameter of each inlet (three liquid inlets and fluidized bed chamber inlet) and each outlet (fluidized bed chamber outlet, waste liquid outlet, and fluorescent signal molecule collection outlet) is 1 mm; the fluidized bed chamber is 50 mm long, 10 mm wide at its widest point, and 400 μm high; the chamber inlet angle α is 13°, and the chamber outlet angle β is 60°; the fluorescence detection cell is 15 mm long, 5 mm wide, and 400 μm high; the microfluidic channel is 400 μm high.

[0055] Microfluidic chip 2: The diameter of each inlet and outlet is 1 mm; the fluidized bed chamber is 50 mm long, 10 mm wide at its widest point, and 500 μm high; the chamber inlet angle α is 13°, and the chamber outlet angle β is 60°; the fluorescence detection cell is 15 mm long, 5 mm wide, and 500 μm high; the microfluidic channel is 500 μm high.

[0056] Microfluidic chip 3: The diameter of each inlet and outlet is 1 mm; the fluidized bed chamber is 50 mm long, 10 mm wide at its widest point, and 600 μm high; the chamber inlet angle α is 13°, and the chamber outlet angle β is 60°; the fluorescence detection cell is 15 mm long, 5 mm wide, and 600 μm high; the microfluidic channel is 600 μm high.

[0057] (2) Use 3D printing technology to print and fabricate the microfluidic chips 1-3 pre-designed in step (1) above. Then clean with anhydrous ethanol to remove residual rigid resin material. Finally, irradiate under ultraviolet light for 2-3 minutes to complete ultraviolet curing, thereby completing the fabrication of the microfluidic chip.

[0058] Example 2: How to use a microfluidic chip to detect samples containing multiple targets

[0059] This embodiment utilizes the microfluidic chip (e.g., microfluidic chip 1-3) fabricated in Embodiment 1 above to specifically illustrate its usage method in detecting samples containing multiple targets, including the following steps:

[0060] S1) Before starting the experiment, seal the fluorescent signal molecule collection outlet of the microfluidic chip with tape, and only open the waste liquid outlet;

[0061] S2) A liquid containing the captured magnetic particles (detailed in Example 3 below) (e.g., a liquid obtained by dispersing the captured magnetic particles in ultrapure water or PBS solution) is injected into the multi-target inlet of the microfluidic chip. To facilitate the flow of the liquid containing the captured magnetic particles into the fluidized bed chamber, the captured magnetic particles can be slowly introduced into the fluidized bed chamber using a magnet at the bottom of the microfluidic chip. After the captured magnetic particles enter the fluidized bed chamber, a magnet (e.g., a magnetic field strength of 2-10 Tesla (T)) is placed on the outside of the substrate 1-5 mm away from the inlet of the fluidized bed chamber. Due to the placement of the magnet, an external magnetic field (e.g., a magnetic field strength of 2-10 Tesla (T)) can be generated around the fluidized bed chamber. Figure 2 As shown, an external magnetic field simulated using COMSOL simulation software is illustrated (where lines represent magnetic field lines of a magnet and arrows indicate the direction of the magnetic field generated by the magnet). This external magnetic force is applied to the captured magnetic particles. Meanwhile, fluid (e.g., ultrapure water or PBS solution) is continuously and slowly injected from the multi-target inlet. The fluid eventually flows out from the waste liquid outlet. The injection of fluid will generate an additional fluid drag force on the captured magnetic particles. Therefore, the captured magnetic particles located in the fluidized bed chamber are simultaneously subjected to the dual forces of the magnetic force of the external magnetic field and the drag force of the injected fluid. This allows the captured magnetic particles to continuously perform dynamic reciprocating motion in the fluidized bed chamber. This reciprocating motion can significantly improve the contact rate between the captured magnetic particles and the targets in the sample to be tested, thereby significantly improving the capture and enrichment efficiency of the captured magnetic particles on the targets in the sample to be tested.

[0062] S3) The sample to be tested containing multiple targets is slowly injected through the multi-target inlet. The sample to be tested enters the fluidized bed chamber through the fluidized bed chamber inlet. The capture magnetic particles located in the fluidized bed chamber will specifically capture and enrich the target bacteria in the sample to be tested, forming a complex of capture magnetic particles and target bacteria (also referred to as magnetic particle-antibody-target complex in this article), thereby separating the target bacteria from the sample to be tested and realizing the separation of multiple target bacteria.

[0063] S4) The CDs / QDs@ZIF-8 biotracer (detailed in Example 4 below) is injected into the microfluidic chip through the CDs / QDs@ZIF-8 biotracer inlet and flows continuously for a period of time. The CDs / QDs@ZIF-8 biotracer will further form a double antibody sandwich complex (also referred to in this article as magnetic particle-antibody-target-antibody-(CDs / QDs@ZIF-8) complex) with the complex that captures magnetic particles and target bacteria in the fluidized bed chamber. Then, a washing solution (e.g., ultrapure water or PBS solution) is injected through the CDs / QDs@ZIF-8 biotracer inlet to wash away the unbound CDs / QDs@ZIF-8 biotracer. The waste liquid flows out from the waste liquid outlet.

[0064] S5) Seal the waste liquid outlet with tape and open the fluorescent signal molecule collection outlet. Then, inject Na2EDTA solution into the microfluidic chip through the Na2EDTA inlet. The injected Na2EDTA solution will dissolve the ZIF-8 framework in the double-antibody sandwich complex formed in step S4) and release fluorescent signal molecules (e.g., CD515, QD570, and QD620) into the fluorescence detection cell; and

[0065] S6) Detect fluorescent signal molecules in the fluorescence detection cell of the microfluidic chip, and determine the multi-target information in the sample to be tested based on the detected fluorescent signal molecule information.

[0066] Example 3: Detection of the capture efficiency of magnetic particle-antibody complexes (also known as capturing magnetic particles) for multiple targets in a fluidized bed magnetic enrichment microfluidic chip with a fluidized bed magnetic enrichment chamber as the main structure.

[0067] This embodiment utilizes the microfluidic chips 1-3 fabricated in Embodiment 1 above, and follows the usage method of Embodiment 2 above to test the capture efficiency of magnetic particles in the fluidized bed chamber for multiple targets (multiple bacteria). The multiple targets in the test sample are taken as three foodborne pathogens: *Escherichia coli* O157:H7, *Salmonella paratyphi A*, and *Salmonella paratyphi B*. The magnetic particles used to capture these multiple targets in the test sample are magnetic particles modified with monoclonal antibodies against these three bacteria. The specific detection of capture efficiency includes the following operations:

[0068] 1) Preparation of magnetic particles for trapping: Magnetic particles (MNPs, 3 μm in diameter) modified with streptavidin at different masses (100 μg, 150 μg, 200 μg, 250 μg, 300 μg) were respectively mixed with monoclonal antibodies that specifically bind to *Escherichia coli* O157:H7, *Salmonella paratyphi A*, and *Salmonella paratyphi B* (the three monoclonal antibodies were mixed in equal masses; all three monoclonal antibodies were modified with biotin, which was purchased from Zhengzhou Saitukang Biotechnology Co., Ltd., and targeted each...). Bacterial monoclonal antibodies (biotin, 60 μg) were mixed and incubated for 45 min. Magnetic particle-antibody complexes with different amounts of magnetic particles were prepared by centrifugation and washing. These were the captured magnetic particles and named 100-MNPs-Abs, 150-MNPs-Abs, 200-MNPs-Abs, 250-MNPs-Abs, and 300-MNPs-Abs, respectively. The prepared magnetic particle-antibody complexes were resuspended in PBS solution for later use.

[0069] 2) Following steps S1) and S2) in Example 2 above, the magnetic capture particles (taking 200-MNPs-Abs as an example) prepared in step 1) above are injected into the fluidized bed chamber of microfluidic chip 1-3, and the magnetic capture particles are continuously subjected to dynamic reciprocating motion in the fluidized bed chamber. The specific parameters of the magnet used are: NdFeB, N50, length, width and height: 30*22*20mm, and it is placed on the outside of the substrate 2mm away from the inlet of the fluidized bed chamber.

[0070] 3) Following step S3) of Example 2 above, inject the test sample (10 mL) containing a mixture of Escherichia coli O157:H7, Salmonella paratyphi A and Salmonella paratyphi B) into the microfluidic chips (microfluidic chips 1-3) of each group at a flow rate of 0.4 mL / min, 0.5 mL / min, or 0.6 mL / min respectively. After the test sample flows into the fluidized bed chamber, the target bacteria in it will be specifically captured and enriched by the capturing magnetic particles that circulate in the fluidized bed chamber, and a complex of capturing magnetic particles and target bacteria will be formed through antigen-antibody reaction.

[0071] 4) After all the samples to be tested have been injected into the microfluidic chip, ultrapure water or PBS solution is continuously injected from the multi-target inlet to ensure that all the samples to be tested flow out from the waste liquid outlet, and all the waste liquid flowing out is collected for later use; then the magnet is removed, and PBS solution is continuously injected from the multi-target inlet, so that the complex of the captured magnetic particles and target bacteria formed in step 3) flows out from the waste liquid outlet under the drive of the flowing PBS solution, and is collected for later use.

[0072] All the waste liquid collected in step 4) (used to count the number of uncaptured bacterial colonies) and the collected complex of the capturing magnetic particles and target bacteria (used to count the number of captured bacterial colonies) were separately spread on LB agar plates and incubated at 37°C for 18-24 hours. The number of colonies was then counted, and the capture efficiency of the capturing magnetic particles in the fluidized bed chamber was calculated as (number of captured bacterial colonies / (number of captured bacterial colonies + number of uncaptured bacterial colonies)). The results are shown below. Figure 3 As shown, the capture efficiency of the magnetic capture particles for *E. coli* O157:H7 is only exemplified. It can be seen that the magnetic capture particles in the fluidized bed chambers of the microfluidic chips 1-3 prepared in Example 1 can capture target bacteria in the test samples at flow rates of 0.4 mL / min, 0.5 mL / min, or 0.6 mL / min, respectively. The magnetic capture particles in the fluidized bed chamber of microfluidic chip 1 exhibit the highest capture efficiency for target bacteria in the test sample at a flow rate of 0.4 mL / min. It is evident that the height of the microfluidic chip's tubing (including the fluidized bed chamber, fluorescence detection cell, and microfluidic channel) and the flow rate of the test sample are factors that may affect the capture efficiency of the magnetic capture particles in the fluidized bed chamber for target bacteria in the test sample. When the height of the microfluidic chip's tubing is 400 μm and / or the sample flow rate is 0.4 mL / min, it is more conducive to the capture of multiple target bacteria in the test sample by the magnetic capture particles in the fluidized bed chamber.

[0073] 5) The magnetic capture particles (100-MNPs-Abs, 150-MNPs-Abs, 200-MNPs-Abs, 250-MNPs-Abs, or 300-MNPs-Abs) prepared in PBS and dissolved in step 1) above are slowly injected into the fluidized bed chamber of the microfluidic chip 1 prepared in Example 1 above through the multi-target inlet according to steps S1) and S2) in Example 2 above. Then, Escherichia coli O157:H7 and type A bacteria are injected into the microfluidic chip of each group at a flow rate of 0.4 mL / min through the multi-target inlet. The test samples of Salmonella paratyphi and Salmonella paratyphi B (10 mL of test sample injected) were specifically captured by the capture magnetic particles located in the fluidized bed chamber. A complex of the capture magnetic particles and target bacteria was formed through an antigen-antibody reaction. Following step 4) above, all waste liquid and the complex of the capture magnetic particles and target bacteria were collected from the waste liquid outlet and spread evenly on LB agar plates. After incubation at 37°C for 18-24 h, the number of colonies was counted, and the capture efficiency of the capture magnetic particles for the target bacteria in the fluidized bed chamber was calculated. The results are as follows: Figure 4As shown, the average capture efficiency of the magnetic capture particles for three types of bacteria is illustrated. It can be seen that the capture efficiency of the various magnetic capture particles used for the target bacteria in the sample to be tested can reach more than 35%. Among them, 200-MNPs-Abs has the highest capture efficiency for the target bacteria, reaching more than 50%, followed by 150-MNPs-Abs and 250-MNPs-Abs, both of which can reach more than 40%.

[0074] Example 4: Construction of a physically amplified fluorescence signal system based on CDs / QDs@ZIF-8 biotracers and releasing multiple fluorescent signal molecules using Na2EDTA.

[0075] This embodiment constructs a physically amplified fluorescence signal system based on CDs / QDs@ZIF-8 biological tracers and releases multiple fluorescent signal molecules using Na2EDTA, specifically including the following operations:

[0076] (1) Zn(NO3)2·6H2O (0.1M, 5mL) was mixed with 2-methylimidazole (0.8M, 5mL) and stirred at 15 rpm for 2 min. One carbon dot (CD515, emission wavelength 515nm, 400μg) and one of two quantum dots (CdSe / ZnS, QD570 and QD620, emission wavelengths 570nm and 620nm respectively, where QD570: 5mg; QD620: 4mg) were added to the mixture. The mixture was stirred on a magnetic stirrer at room temperature for 1 h. Finally, the three CDs / QDs@ZIF-8 nanomaterials loaded with CD515, QD570 and QD620 respectively (named CD515@ZIF-8, QD570@ZIF-8 and QD620@ZIF-8 respectively) were collected by centrifugation and resuspending in ultrapure water.

[0077] (2) BSA (100 μL of BSA (10%)) was adsorbed onto the surface of the three CDs / QDs@ZIF-8 nanomaterials (1 mL) prepared above by electrostatic adsorption. Using the amino groups provided by BSA, antibodies that can specifically bind to the target were modified onto the surface of the CDs / QDs@ZIF-8 nanomaterials by covalent binding of amino and carboxyl groups, respectively, to obtain three CDs / QDs@ZIF-8 antibody complexes, which were named as CD515@ZIF-8 antibody complex, QD570@ZIF-8 antibody complex and QD620@ZIF-8 antibody complex, which are CDs / QDs@ZIF-8 biotracers;

[0078] (3) The dissolving and releasing agent Na2EDTA was mixed with ZIF-8 and fluorescent signal molecules (CD515, QD570, and QD620) respectively to verify the solubility of ZIF-8 and the effect of Na2EDTA on the fluorescence of the fluorescent signal molecules. The results are as follows: Figure 5 and Figure 6 As shown. By Figure 5 As shown, the dissolution and release agent Na2EDTA can significantly promote the dissolution of ZIF-8, and thus can be used to release CDs / QDs in the ZIF-8 biotraceable. Figure 6 As shown, the dissolving and releasing agent Na2EDTA has no significant effect on the fluorescence of the fluorescent signal molecules under various wavelength conditions.

[0079] Example 5: A microfluidic multi-target detection system based on fluidized bed magnetic enrichment and physical amplification of CDs / QDs@ZIF-8 and its multi-target ultrasensitive detection of three foodborne pathogens: Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B.

[0080] This embodiment uses Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B as detection models. These foodborne pathogens can easily cause foodborne illnesses, with clinical symptoms mainly including fever, nausea, and vomiting. In severe cases, they can even cause meningitis and death. Moreover, their concentration in the test sample is usually low, and they often exist in the same test sample. Therefore, when testing such test samples, it is often necessary to use a large volume of test sample, and there is a need for simultaneous isolation, enrichment, and ultrasensitive detection of multiple foodborne pathogens in the test sample.

[0081] This embodiment, based on Embodiments 1-4 above, provides a system for the simultaneous separation, enrichment, and ultrasensitive detection of multiple foodborne pathogens in large-volume samples. The system includes magnetic particles for capture (e.g., the magnetic particle-antibody complex prepared in Embodiment 3), a fluidized bed magnetic enrichment microfluidic chip (e.g., microfluidic chips 1-3 designed and fabricated in Embodiment 1), CDs / QDs@ZIF-8 biotracers for each target (e.g., CD515@ZIF-8-antibody complex, QD570@ZIF-8-antibody complex, and QD620@ZIF-8-antibody complex prepared in Embodiment 4), and a dissolving and releasing agent (e.g., Na2EDTA). The application of this system in the multi-target ultrasensitive detection of three foodborne pathogens—Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B—is described below.

[0082] Specifically, the following steps are included:

[0083] (1) In accordance with steps S1) and S2) of Example 2 above, the magnetic particle-antibody complex (capture magnetic particles) 200-MNPs-Abs prepared in Example 3 and dissolved in PBS is slowly injected from the multi-target inlet into the fluidized bed chamber of the microfluidic chip 1 prepared in Example 1 above, and the magnetic particle-antibody complex 200-MNPs-Abs continuously circulates in the fluidized bed chamber;

[0084] (2) Following step S3 in Example 2 above, inject cells containing gradient bacterial concentrations (10) into each group of microfluidic chips at a flow rate of 0.4 mL / min from the multi-target inlet. 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 The test samples or negative controls for *Escherichia coli* O157:H7 (CFU / mL), *Salmonella paratyphi A*, and *Salmonella paratyphi B*; such as Figure 7 As shown, these different target bacteria are specifically captured and enriched by the magnetic particle-antibody complex located in the fluidized bed chamber, and form magnetic particle-antibody-target complex through antigen-antibody reaction;

[0085] (3) Following step S4) in Example 2 above, a mixture of the three CDs / QDs@ZIF-8 antibody complexes (CD515@ZIF-8 antibody complex, QD570@ZIF-8 antibody complex, and QD620@ZIF-8 antibody complex) prepared in Example 4 above was injected into the microfluidic chip at a flow rate of 0.4 mL / min through the CDs / QDs@ZIF-8 biotracer inlet, and the flow continued for 45 min; Figure 7 As shown, these CDs / QDs@ZIF-8 antibody complexes will react with the corresponding magnetic particle-antibody-target complex in the fluidized bed chamber to form a magnetic particle-antibody-target-antibody-(CDs / QDs@ZIF-8) complex (i.e., a double antibody sandwich complex).

[0086] (4) Following step S5) in Example 2 above, inject 1 mL of Na2EDTA (100 mM, pH = 7.0) solution into the microfluidic chip through the Na2EDTA inlet. Figure 7As shown, due to the metal chelating effect of Na2EDTA, it can chelate zinc ions in the ZIF-8 framework, causing the ZIF-8 framework to dissolve and thus releasing three fluorescent signal molecules (CD515, QD570 and QD620) loaded on the magnetic particle-antibody-target-antibody-(CDs / QDs@ZIF-8) complex. The released fluorescent signal molecules enter the fluorescence detection cell.

[0087] (5) The fluorescence emission wavelength and fluorescence intensity of three fluorescent signal molecules in the fluorescence detection cell were measured using a fluorescence spectrophotometer F-7000 to qualitatively and quantitatively detect *Escherichia coli* O157:H7, *Salmonella paratyphi A*, and *Salmonella paratyphi B*, thereby achieving ultrasensitive detection of multiple foodborne pathogens. Results are as follows: Figure 8a and Figure 8b As shown, where Figure 8a The microfluidic multi-target detection system of the present invention can detect different concentrations (10) 1 -10 6 Fluorescence spectral scanning results of *Escherichia coli* O157:H7 (CD515), *Salmonella paratyphi A* (QD570), and *Salmonella paratyphi B* (QD620) at CFU / mL. Figure 8b The standard curves for three types of bacteria at different concentrations are provided by the microfluidic multi-target biosensor system of this invention.

[0088] Depend on Figure 8a and Figure 8b The results show that as the bacterial concentration increases, the collected fluorescence intensity also increases accordingly, with all three foodborne pathogens detectable down to a minimum intensity of 10. 1 CFU / mL, establish a standard curve between the fluorescence intensity at the characteristic emission wavelengths of different fluorescent molecules in the fluorescence spectra of three bacteria and the bacterial concentration (e.g., CFU / mL). Figure 8b As shown in the figure, a good linear correlation was found between fluorescence intensity and bacterial concentration. This indicates that the microfluidic multi-target detection system provided by this invention can achieve simultaneous isolation, enrichment, and ultrasensitive detection of multiple foodborne pathogens in large-volume samples. Furthermore, the above detection process clearly demonstrates that the microfluidic multi-target detection system provided by this invention integrates the simultaneous isolation, enrichment, and ultrasensitive detection steps of multiple foodborne pathogens in a sample onto a single microfluidic chip. Therefore, compared to existing technologies, it has a higher degree of integration and automation, is relatively simple to operate, and does not require pretreatment operations such as filtration or enrichment activation of the sample.

[0089] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be considered part of the present invention.

Claims

1. A microfluidic multi-target detection system based on fluidized bed magnetic enrichment and physical amplification of CDs / QDs@ZIF-8, characterized in that, The microfluidic multi-target detection system includes a magnetic particle capture device, a fluidized bed magnetic enrichment microfluidic chip, CDs / QDs@ZIF-8 biotracers for each target, a dissolution and release agent, and an external magnetic field; wherein: The surface of the capturing magnetic particles is modified with monoclonal antibodies that can specifically bind to Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B; the mass of the capturing magnetic particles is 100~250 μg. The fluidized bed magnetic enrichment microfluidic chip includes a substrate, multiple inlets and outlets disposed on the surface of the substrate, and a fluidized bed chamber, a fluorescence detection cell, and a microfluidic channel disposed within the substrate. The multiple inlets include a CDs / QDs@ZIF-8 biological tracer inlet, a multi-target inlet for injecting the sample to be tested, and a dissolving and releasing agent inlet. The multiple outlets include a waste liquid outlet and a fluorescence signal molecule collection outlet. Each inlet is connected to the chamber inlet of the fluidized bed chamber via the microfluidic channel. The chamber outlet of the fluidized bed chamber is connected to the waste liquid outlet and the inlet of the fluorescence detection cell via the microfluidic channel, respectively. The outlet of the fluorescence detection cell is connected to the fluorescence signal molecule collection outlet via the microfluidic channel. The height of the microfluidic channel is 400–600 μm. The CDs / QDs@ZIF-8 biotracers include a ZIF-8 framework and a fluorescent signal molecule loaded on the ZIF-8 framework. The surface of the CDs / QDs@ZIF-8 biotracers is modified with monoclonal antibodies that can specifically bind to Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B. The fluorescent signal molecule is a carbon dot or a quantum dot. The dissolving and releasing agent is Na2EDTA, which is used to dissolve ZIF-8 in the CDs / QDs@ZIF-8 biological tracer. The external magnetic field is generated by an external magnet, which is located on the outside of the substrate at a distance of 1-5 mm from the inlet of the fluidized bed chamber, so that the captured magnetic particles continuously perform dynamic reciprocating motion in the fluidized bed chamber.

2. The microfluidic multi-target detection system according to claim 1, characterized in that, In the fluidized bed magnetic enrichment microfluidic chip, the cross-section of the fluidized bed chamber is spindle-shaped, with a total length of 20-100 mm, a width at the widest point of 2-20 mm, a chamber height of 100-800 μm, a chamber inlet angle of 10-30°, a chamber outlet angle of 40-75°, and a diameter of 1-1.5 mm for both the chamber inlet and outlet.

3. The microfluidic multi-target detection system according to claim 2, characterized in that, In the fluidized bed magnetic enrichment microfluidic chip, the height of the fluidized bed chamber is 400-600 μm.

4. The microfluidic multi-target detection system according to claim 1, characterized in that, In the fluidized bed magnetic enrichment microfluidic chip, the height of the microfluidic channel is the same as the height of the fluidized bed chamber; and / or The diameters of the plurality of liquid inlets and the plurality of liquid outlets are all 1-1.5 mm; and / or The length of the fluorescence detection cell is 5-20 mm, the width is 2-10 mm, and the height is the same as that of the fluidized bed chamber.

5. The microfluidic multi-target detection system according to any one of claims 1-4, characterized in that, The magnetic field strength of the applied magnetic field is 2-10 Tesla.

6. The microfluidic multi-target detection system according to any one of claims 1-4, characterized in that, The CDs / QDs@ZIF-8 biological tracers were prepared through the following steps: (1) Zinc nitrate hexahydrate Zn(NO3)2·6H2O and dimethylimidazole were mixed and reacted to obtain ZIF-8 nanocarriers; (2) The fluorescent signal molecules are mixed and reacted with the ZIF-8 nanocarrier prepared in step (1) above, so that the fluorescent signal molecules are loaded on the ZIF-8 nanocarrier to obtain the CDs / QDs@ZIF-8 complex; and (3) Modify the surface of the CDs / QDs@ZIF-8 complex obtained in step (2) above so that it can specifically bind to the corresponding target to obtain CDs / QDs@ZIF-8 biotracer.

7. The microfluidic multi-target detection system according to any one of claims 1-4, characterized in that, The captured magnetic particles are prepared by the following steps: mixing and incubating magnetic particles modified with streptavidin with an antibody modified with biotin targeting a specific target.

8. The microfluidic multi-target detection system according to any one of claims 1-4, characterized in that, The mass of the captured magnetic particles is 200 μg; the height of the microfluidic channel is 400 μm.

9. The use of the microfluidic multi-target detection system according to any one of claims 1-4 in the preparation of a kit for detecting foodborne pathogens.

10. A method of operating the microfluidic multi-target detection system according to any one of claims 1-9 for non-disease diagnostic purposes, characterized in that, The operation method includes the following steps: S1) Block the fluorescent signal molecule collection outlet and open the waste liquid outlet; S2) The liquid containing the captured magnetic particles is injected into the fluidized bed chamber from the multi-target inlet, and the captured magnetic particles are made to circulate and reciprocate in the fluidized bed chamber; S3) The sample to be tested containing multiple targets is injected into the fluidized bed chamber from the multi-target inlet at a flow rate of 0.4-0.6 mL / min. In the fluidized bed chamber, the captured magnetic particles specifically bind to the targets in the sample to be tested to form a magnetic particle-antibody-target complex. S4) The CDs / QDs@ZIF-8 biological tracer is injected into the fluidized bed chamber through the CDs / QDs@ZIF-8 biological tracer inlet, and it specifically binds to the magnetic particle-antibody-target complex to form a double antibody sandwich complex. S5) Block the waste liquid outlet, open the fluorescent signal molecule collection outlet, and inject the dissolving and releasing agent into the fluidized bed chamber from the dissolving and releasing agent inlet, so as to dissolve the ZIF-8 framework in the dual-antibiotic sandwich complex and release the fluorescent signal molecules therein; and S6) After the fluorescent signal molecules released in step S5) enter the fluorescence detection cell, the fluorescent signal molecules in the fluorescence detection cell are detected, and the multi-target information in the sample to be tested is determined based on the detected fluorescent signal molecule information.

11. The operating method according to claim 10, characterized in that, The sample to be tested was injected into the fluidized bed chamber at a flow rate of 0.4 mL / min.

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