A magnetic force assisted centrifugal loading device and detection method for improving the sensitivity of digital ELISA detection

Abstract

The application provides a magnetic force auxiliary centrifugal sample loading device, comprising a sample injection device, a multi-channel chip rack, a chip card slot, a magnetic attraction device, a rotating motor and a base. The application also provides a magnetic force auxiliary centrifugal sample loading detection method. The combination of magnetic force and centrifugal sample loading makes the magnetic force auxiliary centrifugal sample loading device simple and efficient, and the device does not need to be equipped with an electric field, a pressure or an electrophoresis device. The device can be used for multi-channel parallel sample loading, accelerates the sample loading speed, improves the work efficiency, increases the pit entry rate of magnetic beads, and thus can improve the detection sensitivity of digital ELISA.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, and particularly to a magnetic-assisted centrifugal loading device and detection method for improving the sensitivity of digital ELISA detection. BACKGROUND

[0002] Digital ELISA (enzymelinkedimmunosorbentassay) is a method for precise detection of low-abundance proteins. Compared with traditional ELISA, it has great advantages in sensitivity and accuracy of low-concentration samples. Compared with conventional immunoassay techniques (such as traditional ELISA, chemiluminescence immunoassay (CLIA), etc.), digital ELISA technology can improve the sensitivity by 1000 times, and can be applied to the early detection, companion diagnosis, drug research and development of major diseases such as tumors, neurological diseases, infectious diseases, and immune inflammation. Among them, the representative products are SIMOA (Single-moleculeArray) detection system of Quanterix company. TM

[0003] A core parameter of digital ELISA technology is the loading rate of the sample dispensing unit. In the SIMOA reaction system, magnetic beads are dropped into microwells by natural sedimentation, and then the magnetic beads not falling into the microwells are washed away with substrate solution by sealing oil. This method makes the loading rate of the sample dispensing unit (magnetic beads) low, which affects the sensitivity and accuracy of the detection. In addition, the loading device should be simple, fast and efficient to operate. The loading device of the SIMOA reaction system is complex, and its working mode is to mix magnetic beads with substrate solution, then drive fluid movement by negative pressure to spread the magnetic bead solution on the surface of the microwell array. The pressure source provided by the conventional pump can only be operated individually in each channel, or a very complex flow path interface is needed to realize parallel operation.

[0004] Kristopher D. Barbee et al. reported a method of assisting the magnetic bead group to enter the microwell chip by external magnet. This method uses 3-5 times of reciprocating manual magnetic attraction operation to drag the magnetic beads to the surface of the microarray. The disadvantage of this manual operation method is that it is difficult to ensure the consistency of the operation. In addition, the author also reported a method of increasing the loading rate of magnetic beads by applying an electric field. This method requires a multi-step processing process and a complex electric field generation process.

[0005] ​A method for increasing the loading rate on magnetic beads by the combined action of an applied force field (magnetic or electric) and the meniscus tension of the fluid is reported in WO2021 / 211754A2. This scheme uses a traditional way of sample injection, and the sample injection effect is not good, and the efficiency is low. And because the magnetic bead solution needs to be reciprocated on the surface of the micro-pit by pressure or electrophoresis device, the action force in two directions needs to be accurately controlled, which increases the complexity and controllability of the system. SUMMARY

[0006] In view of the defects of the prior art, the present application provides a magnetic force assisted centrifugal sample loading device and detection method for improving the sensitivity of digital ELISA, which is used to speed up the sample loading speed, improve the work efficiency, increase the pit rate of magnetic beads, and thus improve the detection sensitivity of digital ELISA.

[0007] According to one aspect of the present application, a magnetic force assisted centrifugal sample loading device is provided, comprising: a sample injection device, a multi-channel chip rack, a chip card slot, a magnetic attraction device, a rotary motor, a base,

[0008] A plurality of chip card slots are designed on the multi-channel chip rack for placing a plurality of microfluidic chips;

[0009] The rotary motor is placed below the multi-channel chip rack for controlling the angle, speed and time of the rotation of the multi-channel chip rack;

[0010] The magnetic attraction device includes a magnet and a bracket for placing the magnet;

[0011] The magnet is movably fixed on the base through the magnet bracket, so that the magnet is located below or away from the lower part of the multi-channel chip rack;

[0012] The rotary motor is fixed on the base.

[0013] According to another aspect of the present application, a magnetic force assisted centrifugal sample loading detection method applied to the magnetic force assisted centrifugal sample loading device is provided, and the method comprises the following steps:

[0014] S1. Magnetic bead solution sample injection: the sample injection device adds the magnetic bead microspheres connected with β-galactosidase and the buffer solution without fluorescent substrate into the microfluidic chip through the sample injection port;

[0015] S2. Add magnet: place the magnet below the chip rack, rotate the chip rack to rotate the microfluidic chip to the upper part of the magnet, and stand for a first time;

[0016] S3. Centrifugation: rotate the chip rack according to the second centrifugation condition;

[0017] S4. Reaction substrate solution sample injection: the sample injection device adds the solution containing the reaction substrate into the microfluidic chip through the sample injection port;

[0018] S5. Centrifugation: rotate the chip rack according to the third centrifugal condition;

[0019] S6. Partition oil injection: inject the partition oil into the microfluidic chip through the injection port by the injection device;

[0020] S7. Enzymatic reaction: wait for the second duration;

[0021] S8. Imaging detection and digital analysis: capture the image by the CCD / CMOS camera and perform the image detection; perform the Poisson statistical analysis to calculate the number of protein molecules in the original sample to be detected.

[0022] The magnetic force assisted centrifugal sample loading device is simple and efficient, does not need to be configured with an electric field, pressure or electrophoresis device, can perform multi-channel parallel sample loading, accelerates the sample loading speed, improves the work efficiency, can increase the pit entry rate of the magnetic beads, and thus can improve the detection sensitivity of the digital ELISA.

[0023] The features and advantages of the present application will become apparent from the following detailed description of the application, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 a and 1b are respectively the top view and the sectional view of the magnetic force assisted centrifugal sample loading device of the present application.

[0025] Figure 2 The structural schematic diagram of the microfluidic chip is shown.

[0026] Figure 3 The flowchart of the magnetic force assisted centrifugal sample loading and detection method of the present application is shown.

[0027] Figure 4 The comparison diagram of the pit entry number of the magnetic beads loaded according to two ways is shown.

[0028] Figure 5 The reaction result linear diagram according to two ways of sample loading is shown. DETAILED DESCRIPTION

[0029] In order to make the technical scheme of the present application more clear and explicit, further detailed description will be made below in conjunction with the drawings, and it should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0030] Pictures 1a and 1b are respectively the top view and the sectional view of the magnetic force assisted centrifugal sample loading device of the present application.As shown in pictures 1a and 1b, the magnetic force assisted centrifugal sample loading device comprises: an injection device (not shown in the figure), a multi-channel chip rack 31, a chip card slot 33, a magnetic attraction device 20, a rotating motor 32, and a base 33. Figure 1

[0031] As shown in the figure, a plurality of chip card slots 33 are designed on the multi-channel chip rack 31 for placing a plurality of microfluidic chips 10. Preferably, the number of chip card slots 33 is eight. The rotating motor 32 is placed below the multi-channel chip rack 31 and controls the angle, speed and time of rotation of the multi-channel chip rack 32 under the action of the control system (not shown in the figure). The magnetic attraction device 20 includes a magnet 21 and a magnet holder 22. The magnet 21 is movably fixed on the base 33 through the magnet holder 22 (for example, the magnet holder 22 is detachably or hingedly fixed on the base 33), so that the magnet 21 is located below or away from the multi-channel chip rack 31. When the magnet 21 is located below the multi-channel chip rack 31, the distance from the magnet 21 to the center axis of the rotating motor is equal to the distance from the reaction chamber of the microfluidic chip to the center axis of the rotating motor. By controlling the multi-channel chip rack 31 to rotate to a specific angle through the rotating motor 32, the reaction chamber of the microfluidic chip 10 can be rotated to directly above the magnet 21, and the magnet 21 can exert a force on the magnetic beads in the reaction chamber. When the microfluidic chip 10 rotates out of the direct above of the magnet 21, the magnetic field force weakens or disappears. Although only one magnetic attraction device is shown in the figure, in fact, the number of magnetic attraction devices can be multiple, and the number of magnetic attraction devices is the same as the number of chip card slots, each magnetic attraction device corresponds to a microfluidic chip, so that parallel magnetic force assisted sample loading of multiple channels can be realized. Similarly, the number of sample injection devices can be multiple, and the number of sample injection devices is the same as the number of chip card slots, each sample injection device corresponds to a microfluidic chip, so that parallel sample injection of multiple channels can be realized. The rotating motor 32 is fixed on the base 33 to avoid displacement and shaking. In addition, the magnetic force assisted centrifugal device is fixed on the precision displacement table 40 through the base 33. The precision displacement table 40 can move accurately in the horizontal direction of the XY axis, adjust the position of the microfluidic chip detection window and the signal collection lens (not shown in the figure), so as to facilitate signal collection of multiple regions of the same microfluidic chip.

[0032] The magnetic force assisted centrifugal sample loading device of the present application integrates a centrifugal device and a magnetic attraction device. The advantage of the centrifugal device is that one power source can handle multiple fluid channels in parallel, and multiple channel parallel sample loading is realized through centrifugation, which simplifies the complexity of the instrument equipment and improves the sample loading rate. The advantage of the magnetic attraction device is that it can realize parallel magnetic force assisted sample loading of multiple channels, which accelerates the rate of magnetic beads into the pit. Therefore, under the same standing time, the magnetic force assisted centrifugal sample loading device of the present application can increase the rate of magnetic beads into the pit without configuring a pressure or electrophoresis device, thereby improving the detection sensitivity of digital ELISA.

[0033] Figure 2The structural schematic diagram of the microfluidic chip is shown. The microfluidic chip is composed of three parts: an upper shell 15, a lower shell 16, and a microwell array chip 17. The upper shell and the lower shell are bonded to form a sample injection cavity 18, a biochemical reaction cavity 13, and a waste liquid cavity 14.

[0034] The sample injection cavity 18 is connected with an external sample injection device through a sample injection port 19. The sample injection cavity 18 is connected with the biochemical reaction cavity 13 through a sample injection flow guide structure.

[0035] A detection window 20 is formed on the top cover of the biochemical reaction cavity 13 for optical detection. A lower concave cavity is formed at the bottom of the biochemical reaction cavity 13, and a micrometer-scale microwell array chip 17 is integrated in the lower concave cavity for performing a digital ELISA reaction. A micropit 12 is formed on the microwell array chip 17. The height of the biochemical reaction cavity 13 is adjusted according to the type of biochemical reaction. The biochemical reaction cavity 13 is connected with the waste liquid cavity 14 through a waste liquid flow guide structure.

[0036] An exhaust hole or a gas permeable film 21 is formed on the top cover of the waste liquid cavity 14. The waste liquid cavity 14 serves to store waste liquid and release the air pressure in the cavity.

[0037] Figure 3 The flowchart of the magnetic force assisted centrifugal sample loading detection method of the application is shown. The magnetic force assisted centrifugal detection method comprises the following steps:

[0038] S1. Magnetic bead solution sample injection

[0039] First, a plurality of microfluidic chips are installed in the chip card slots of the multi-channel chip rack. Then, a sample injection device (such as a sample injection needle) is used to inject a specific volume (such as 20 μL) of magnetic bead microspheres connected with β-galactosidase and buffer solution without fluorescent substrate into the microfluidic chip through the sample injection port. Multiple sample injection devices can be used to perform parallel sample injection on multiple microfluidic chips. The magnetic bead solution enters the biochemical reaction cavity through self-suction by the sample injection flow guide structure. If necessary, the chip rack is rotated, and the magnetic bead solution is assisted to enter the biochemical reaction cavity under the action of the centrifugal force generated by rotation, which can improve the efficiency of sample injection.

[0040] For example, under a specific centrifugal condition (such as a rotation speed of 200 rpm and a duration of 10 seconds), the centrifugal force sends the magnetic beads and the buffer solution without fluorescent substrate into the biochemical reaction cavity. Due to the constraint of the flow guide structure, the fluid is kept in the biochemical reaction cavity.

[0041] S2. Add magnet

[0042] The magnet is located below the chip holder, and the chip holder is rotated to rotate the microfluidic chip to the directly above the magnet, and then it is left for several seconds to several minutes (e.g., 1 min). Under the dual action of gravity and magnetic field, the magnetic bead solution is settled into the microwell array of the microwell array chip in the biochemical reaction chamber. By using the magnetic attraction of the magnetic field, the speed and quantity of the magnetic beads falling into the microwell array can be increased.

[0043] S3. Centrifugation

[0044] The chip holder is set to rotate at a specific centrifugal speed (e.g., 1000 rpm for 10 seconds). By centrifugal force, the excess magnetic bead solution in the biochemical reaction chamber is thrown out, while the magnetic bead microspheres in the microwell remain inside. The excess magnetic bead solution that does not enter the microwell is guided by the centrifugal force through the waste liquid guide structure into the waste liquid chamber. During the centrifugation process, the remaining volume of the waste liquid chamber decreases, and the gas pressure increases; the gas pressure is discharged outside the chip through the exhaust hole or the gas permeable membrane.

[0045] S4. Reaction substrate solution injection

[0046] The injection device adds a specific volume (e.g., 20 μL) of solution containing the reaction substrate (resorcinol-β-galactoside, RGP) into the microfluidic chip through the injection port. Multiple injection devices can simultaneously perform parallel injection on multiple microfluidic chips. The reaction substrate solution enters the biochemical reaction chamber through the self-suction effect of the injection guide structure. If necessary, the chip holder is rotated, and the reaction substrate solution is injected under the action of the centrifugal force generated by the rotation.

[0047] For example, under a specific centrifugal condition (e.g., 200 rpm for 10 seconds), the centrifugal force sends the reaction substrate solution into the biochemical reaction chamber. Due to the constraint of the guide structure, the fluid remains in the biochemical reaction chamber. The reaction substrate entering the biochemical reaction chamber exchanges substances with the magnetic bead solution in the microwell through liquid exchange or molecular diffusion, etc.

[0048] S5. Centrifugation

[0049] The chip holder is set to rotate at a specific centrifugal speed (e.g., 1000 rpm for 10 seconds). By centrifugal force, the excess magnetic bead solution in the biochemical reaction chamber is thrown out, while the magnetic bead microspheres in the microwell remain inside. The excess magnetic bead solution that does not enter the microwell is guided by the centrifugal force through the waste liquid guide structure into the waste liquid chamber. During the centrifugation process, the remaining volume of the waste liquid chamber decreases, and the gas pressure increases; the gas pressure is discharged outside the chip through the exhaust hole or the gas permeable membrane.

[0050] S6. Partition oil injection

[0051] The sample injection device injects a specific volume of high viscosity partition oil (e.g. 40 μL of fluorinert or silicon oil) into the microfluidic chip through the injection port. Multiple sample injection devices can be used to inject multiple microfluidic chips in parallel. The partition oil enters the reaction chamber through the injection channel by capillary action, separating the microwells into individual reaction units. If necessary, the chip holder can be rotated to assist the injection of the partition oil under the centrifugal force generated by the rotation.

[0052] For example, under certain centrifugal conditions (e.g. 200 rpm for 10 seconds), the centrifugal force drives the partition oil into the reaction chamber. This process removes the residual magnetic beads on the surface of the microwell array chip that do not fall into the microwells, and separates the solutions in each reaction unit.

[0053] S7. Enzymatic reaction

[0054] The sample is incubated for a certain period of time for the enzymatic reaction at room temperature. For example, 1 minute. In this example, the β-galactosidase attached to the magnetic beads via the double antibody sandwich reaction can catalyze the hydrolysis of resorufin-β-galactoside (RGP), which does not emit fluorescence, to generate resorufin molecules that emit fluorescence.

[0055] S8. Imaging detection and digital analysis

[0056] The images are captured by a CCD / CMOS camera and analyzed. The microfluidic chip is mounted on a precision translation stage. The microfluidic chip is moved by the precision translation stage to align the lens with different positions on the chip, and the fluorescent images (577 nm excitation, 620 nm emission, exposure time 600 ms) and brightfield images (mercury lamp light source, exposure time 50 ms) of multiple regions are taken. The brightfield images are used to identify the brightfield signals of the microwells containing magnetic beads (referred to as effective microwells), and the number of microwells containing magnetic beads (N) is counted. The fluorescent images are used to identify the fluorescent signals in the microwells containing magnetic beads, and the number of microwells with positive enzymatic reaction (referred to as positive microwells) is counted. After the enzymatic reaction, the microwells with fluorescent signals higher than the threshold value are judged to be positive, and the microwells with fluorescent signals lower than the threshold value are judged to be negative.

[0057] Finally, Poisson statistical analysis is performed to calculate the number of protein target molecules in the original sample to be tested.

[0058] In theory, there are three possibilities for each magnetic bead to capture a protein target molecule: zero molecule, single molecule or multiple molecules. When the number of magnetic beads is large enough, most of the magnetic beads only capture one molecule or zero molecule; finally, most of the reaction detection units only contain one molecule or zero molecule in the interior, and finally only contain one solid-phase luminescent molecule region or zero solid-phase luminescent molecule region, so as to realize single-molecule optical signal amplification. Even if a single reaction detection unit contains more than two solid-phase luminescent molecule regions, the proportion and number of reaction detection units of the two signal types of positive and negative can be counted, and Poisson statistical analysis can be performed, and finally the number of protein target molecules in the original sample to be measured can be calculated.

[0059] For example: the measured protein molecule is interleukin-6 (IL-6), and the molecular weight is 21Kda. The initial sample volume is 100 μL, the total number of sample distribution units (magnetic beads) (N0) is 753,600, the total number of sample detection units (micropits) is 188,000, the number of positive reaction detection units (M) is 5,000, the number of reaction detection units (N) is 121,000, and the probability p that the protein molecules to be measured in the sample are captured and further connected with signal molecules is 80%.

[0060] The absolute number of positive molecules is calculated by the following formula:

[0061]

[0062] It is calculated that the absolute number of positive molecules is 39,753, and the concentration of sample protein molecules is 13.9 fg / ml.

[0063] The beneficial effects of the present application will be illustrated by comparing the loading rates of magnetic beads in the magnetic force assisted centrifugal loading scheme and the non-magnetic force assisted loading scheme.

[0064] Take 20 μL of magnetic bead solution, which contains 400,000, 800,000, 1,200,000 magnetic beads respectively for loading test. The reaction conditions are that the magnetic beads connected with streptavidin are reacted with different concentrations of biotin-β-galactosidase (BβG), the reaction buffer solution is 1×PBS, and the reaction time is 30 min. After the reaction is completed, 1×PBST is used for cleaning five times. The loading is carried out according to the magnetic force assisted centrifugal loading operation process and the non-magnetic force assisted loading operation process of the present application. The difference between the non-magnetic force assisted loading operation process is that in steps S1, S4, S6, only the sample is loaded by fluid self-suction, in steps S3, S5, the waste liquid is discharged by negative pressure driving, and in step S2, the process of standing for 1 min under the action of magnetic field is changed to not adding magnetic field, standing for 2 min, and the magnetic beads are naturally settled into the pits under the action of gravity.

[0065] The number of magnetic beads in each micro-pit of the chip and the loading rate were counted according to two different loading methods, wherein the loading rate was the number of magnetic beads in the micro-pit / the number of loaded magnetic beads. The results are shown in Table 1.

[0066] Table 1. Comparison of the number of magnetic beads and the loading rate of magnetic beads loaded according to the magnetic force assisted centrifugal loading and the non-magnetic force assisted loading of different numbers of magnetic beads

[0067]

[0068] Figure 4 A comparison chart of the number of magnetic beads loaded in the micro-pit of the microfluidic chip according to the magnetic force assisted centrifugal loading and the non-magnetic force assisted loading is shown, wherein the number of magnetic beads loaded by the magnetic force assisted centrifugal loading is obviously more.

[0069] The number of positive magnetic beads, the total number of magnetic beads, the AEB (the ratio value of the number of positive magnetic beads to the total number of magnetic beads (AEB, Average Enzyme per Bead)) and the LOD (detection sensitivity) of each chip were counted according to two different loading methods. The detailed data are shown in Table 2. After the magnetic field is increased, the number of positive magnetic beads, the total number of magnetic beads and the AEB are greatly increased, and the detection sensitivity is also increased from 4.0 aM to 2.1 aM.

[0070] Table 2. Comparison of the reaction results of the magnetic force assisted centrifugal loading and the non-magnetic force assisted loading

[0071]

[0072] Figure 5 A linear chart of the reaction results according to the magnetic force assisted centrifugal loading and the non-magnetic force assisted loading is shown, wherein the AEB value of the magnetic force assisted centrifugal loading is obviously higher.

[0073] In summary, the combination of the magnetic force and the centrifugal loading makes the magnetic force assisted centrifugal loading device simple and efficient, does not need to configure an electric field, a pressure or an electrophoresis device, can be loaded in multiple channels in parallel, speeds up the loading speed, improves the working efficiency, increases the loading rate of the magnetic beads into the pit, and further improves the detection sensitivity of the digital ELISA.

[0074] The above only describes the preferred embodiments of the present application, and does not limit the patent scope of the present application. Any equivalent structural transformation made according to the content of the specification and the drawings of the present application, or direct / indirect application in other related technical fields is included in the patent protection scope of the present application.

Claims

1. A magnetic-assisted centrifugal loading detection method, applied to a magnetic-assisted centrifugal loading device, the magnetic-assisted centrifugal loading device comprising: Sample injection device, multi-channel chip rack, chip card slot, magnetic device, rotating motor, base; The multi-channel chip rack is designed with multiple chip slots for placing multiple microfluidic chips; The rotary motor is placed under the multi-channel chip rack to control the speed and time of the multi-channel chip rack rotation; the magnetic device includes a magnet and a bracket for placing the magnet; the bracket and the rotary motor are fixed to the base; the method is characterized in that the number of magnetic devices and the number of sample injection devices are the same as the number of chip card slots, each magnetic device corresponds to a microfluidic chip, and each sample injection device corresponds to a microfluidic chip; the method comprises the following steps: S1. Magnetic bead solution injection: The injection device introduces β-galactosidase-linked magnetic microspheres and a buffer solution without fluorescent substrate into the microfluidic chip through the injection port; S2. Magnetization: Place a magnet below the chip holder, rotate the chip holder to position the microfluidic chip directly above the magnet, and allow the chip to rest for a first period of time. S3. Centrifugation: Rotate the chip rack according to the second centrifugation condition; S4. Injection of reaction substrate solution: The injection device adds the solution containing the reaction substrate to the microfluidic chip through the injection port; S5. Centrifugation: Rotate the chip rack according to the third centrifugation condition; S6. Split oil injection: The injection device adds split oil to the microfluidic chip through the injection port; S7. Enzymatic reaction: wait for the second time period; S8. Imaging detection and digital analysis: Capture images with a CCD camera and perform image detection; perform Poisson statistical analysis to calculate the number of protein molecules in the original sample to be tested; In steps S1, S4, and S6, multiple sample injection devices simultaneously inject samples into multiple microfluidic chips in parallel.

2. The magnetic-assisted centrifugal sample loading detection method according to claim 1, wherein in steps S1, S4, and S6, the chip holder is rotated according to a first centrifugation condition; the first centrifugation condition is a rotation speed of 200 rpm and a duration of 10 seconds.

3. The magnetic-assisted centrifugal sample loading detection method according to claim 1, wherein the second centrifugation condition is a rotation speed of 1000 rpm and a duration of 10 seconds.

4. The magnetic-assisted centrifugal sample loading detection method according to claim 1, wherein the third centrifugation condition is a rotation speed of 300 rpm and a duration of 10 seconds. The magnetic-assisted centrifugal sample loading detection method according to claim 1 , wherein the reaction substrate is resorufin-β-galactoside.

6. The magnetic-assisted centrifugal sample loading detection method according to claim 1, wherein before step S1, a plurality of microfluidic chips are installed in the chip card slots of the multi-channel chip rack.

7. The magnetic-assisted centrifugal sample loading detection method according to any one of claims 1 to 6, wherein in step S8, the microfluidic chip is moved by a precision translation stage so that the lens of a CCD camera is aligned with different positions of the microfluidic chip to capture fluorescence images and bright field images of multiple areas; in, Bright field images were used to count the number of effective micropits containing magnetic beads; fluorescence images were used to count the number of positive micropits that were positive for enzymatic reactions.

8. The magnetic-assisted centrifugal sample loading detection method according to any one of claims 1 to 6, wherein: The absolute number of protein molecules M0 is calculated by the following formula: ， N0 is the total number of magnetic beads, M is the number of positive micropits, N is the number of effective micropits, and the probability that the protein molecules of the sample to be tested are captured and further connected to the signal molecules is p.

9. The magnetic-assisted centrifugal sample loading detection method according to claim 1, wherein the base is fixed on a precision translation stage, and the precision translation stage can perform precise horizontal movement.

10. The magnetic-assisted centrifugal loading detection method according to claim 1 or 9, wherein the magnet is movably fixed to the base by a bracket, so that the magnet is located below the multi-channel chip rack or away from the bottom of the multi-channel chip rack. When the magnet is located below the multi-channel chip rack, the distance from the magnet to the central axis of the rotating motor is equal to the distance from the reaction chamber of the microfluidic chip to the central axis of the rotating motor.

11. The magnetic-assisted centrifugal sample loading detection method according to claim 1, wherein the microfluidic chip is composed of three parts: The upper shell, the lower shell and the micropore array chip are bonded to form a sample injection cavity, a biochemical reaction cavity and a waste liquid cavity.

12. The magnetic-assisted centrifugal sample loading detection method according to claim 11, wherein the sample injection cavity is connected to an external sample injection device via a sample injection port, and the sample injection cavity is connected to a biochemical reaction cavity via a sample injection guide structure.

13. The magnetic-assisted centrifugal loading detection method according to claim 11 or 12, wherein a detection window is provided on the top cover of the biochemical reaction chamber for optical detection, a concave cavity is formed at the bottom of the biochemical reaction chamber, and a micron-scale microwell array chip is integrated in the concave cavity for performing digital ELISA reactions, and the biochemical reaction chamber is connected to the waste liquid chamber via a waste liquid diversion structure.

14. The magnetic-assisted centrifugal sample loading detection method according to claim 11 or 12, wherein an exhaust hole or a breathable membrane is provided on the top cover of the waste liquid chamber.

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