A magnetic bead array partition chip and system for digital PCR

Abstract

This invention discloses a magnetic bead array partitioning chip and system for digital PCR. The chip features a high-density micro-pit array with a micro-pit diameter to magnetic bead diameter ratio of 1.0–2.0. A micromagnetic structure can be optionally installed at the bottom of the micro-pits for bead positioning in conjunction with a programmable magnetic field. The system includes the aforementioned chip, a programmable magnetic field generator, a controller, a temperature control module, an optical detection module, and a main controller. The system utilizes the programmable magnetic field generator to produce controlled-movement magnetic field peaks, driving functionalized magnetic beads one by one into the micro-pits to form a monodisperse partition of "one bead per pit." It integrates temperature-controlled amplification and fluorescence imaging functions, achieving absolute nucleic acid quantification and supporting automated magnetic field-driven recovery of positive magnetic beads after amplification. This invention solves the problems of high cost, complex operation, and inability to actively locate and recover templates in existing digital PCR systems, offering advantages such as low cost, high throughput, ease of operation, support for positive partition recovery, and scalability.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics and digital nucleic acid detection technology, specifically relating to a magnetic bead array partition chip and system for digital PCR, which is particularly suitable for applications such as absolute nucleic acid quantification, low copy number detection, rare mutation screening, multiplex target analysis and positive partition recovery. Background Technology

[0002] Digital polymerase chain reaction (dPCR) disperses nucleic acid templates into a large number of independent partitions, each containing 0 or 1 template molecule. After amplification, absolute quantification is achieved by statistically analyzing the proportion of positive partitions, eliminating the need for a standard curve. Current digital PCR technologies are mainly divided into droplet-based (ddPCR) and chip-based (cdPCR). Droplet-based PCR uses water-in-oil droplets, resulting in a large number of partitions, but requires complex equipment such as droplet generators and droplet readers, making operation cumbersome and costly in terms of consumables. Chip-based PCR fabricates microwells or microcavities on microfluidic chips, offering good partition stability, but chip manufacturing costs are high, the number of partitions is limited by chip area and micro / nano fabrication precision, and sample loading typically relies on passive diffusion.

[0003] Furthermore, both methods require random distribution of nucleic acid templates into droplets or microwells, making active template localization or enrichment impossible, and detection sensitivity is limited by template loading efficiency. Simultaneously, positive regions after amplification are difficult to recover for downstream sequencing, resulting in the loss of traceability information for nucleic acid samples. Regarding the integration of magnetic beads with microwell arrays, while studies have reported schemes using magnetic fields to position magnetic beads into microwell arrays, these schemes generally employ permanent magnets as the magnetic field source. The magnetic bead assembly process relies on passive random adsorption, repeated fluid sweeping, or capillary assistance, making time- and region-specific programmable control of the magnetic field impossible. Some schemes rely on complex digital microfluidic electrowetting platforms, resulting in complex system architectures and high costs. Moreover, existing schemes are either used for DNA array preparation rather than digital PCR absolute quantification, or for immunoassay rather than PCR amplification, failing to organically integrate programmable magnetic positioning with PCR amplification, fluorescence quantification, and positive sample recovery.

[0004] It should be noted that the information disclosed in the background section above is only for understanding the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The main objective of this invention is to overcome the deficiencies in the aforementioned background technology and provide a magnetic bead array partitioning chip and system for digital PCR. This system uses a programmable magnetic field generator to drive functionalized magnetic beads one by one into a micro-pit array, forming a monodisperse partition of "one bead per pit." It integrates temperature control and optical detection modules to achieve absolute nucleic acid quantification and supports the recovery of positive magnetic beads after amplification.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In the first aspect of the present invention, a magnetic bead array partitioning chip for digital PCR is characterized in that it comprises: a chip substrate (1) having a micro-pit array (2) composed of a plurality of regularly arranged micro-pits; the diameter of the micro-pits is 5 to 100 μm, the depth is 5 to 50 μm, and the center-to-center distance between adjacent micro-pits is 10 to 100 μm; the ratio of the diameter of the micro-pit to the particle size of the magnetic bead (9) to be positioned is 1.0 to 2.0; and a sealing cover (3) covering the chip substrate (1) for sealing the micro-pit array (2).

[0007] In a second aspect of the present invention, a magnetic bead array partitioning system for digital PCR is characterized in that it comprises: a chip as described above; a programmable magnetic field generator disposed below the chip for generating a spatially movable magnetic field peak; a controller (12) electrically connected to the programmable magnetic field generator, wherein the controller (12) independently controls multiple magnetic field sources of the programmable magnetic field generator to move the magnetic field peak along a preset path, thereby driving the magnetic beads (9) in the chip to sequentially enter each micro-pit; a temperature control module thermally coupled to the chip for providing the temperature conditions required for PCR; an optical detection module disposed above the chip for acquiring fluorescence images of the micro-pit array (2); and a main controller (20) electrically connected to the controller (12), the temperature control module, and the optical detection module of the programmable magnetic field generator, respectively, for coordinating the workflow of each module.

[0008] Furthermore, the programmable magnetic field generating device is a flexible electromagnetic coil array (11), including a flexible substrate (10) and a plurality of planar spiral microcoils disposed thereon, wherein the spacing of the microcoils matches or is an integer multiple of the spacing of the micropit array (2). In a preferred embodiment, the programmable magnetic field generating device may be the device described in the invention patent application "An Artificial Intelligence Programmable Flexible Electromagnetic Coil Microarray Magnetic Control System" filed on the same day. The specific structure, driving circuit and control method of the device have been fully disclosed in that application, the entire contents of which are incorporated herein by reference.

[0009] Furthermore, the controller (12) synthesizes a magnetic field peak through a virtual magnetic field center algorithm, and moves the magnetic field peak along at least one of the following paths: row-by-row scanning, column-by-column scanning, or point-by-point addressing.

[0010] Furthermore, the virtual magnetic field center algorithm is an inverse distance weighting method, which allocates current according to the distance from each magnetic field source to the virtual center, so that the center of the synthesized magnetic field falls on the virtual center position.

[0011] Furthermore, each micro-pit of the micro-pit array (2) is provided with a micro-magnet structure at the bottom of the micro-pit. The micro-magnet is a ferromagnetic thin film, namely a NiFe thin film (8), deposited at the bottom of the micro-pit, with a thickness of 50-500 nm, which is used to enhance the trapping force of the magnetic beads (9) and prevent the magnetic beads (9) from shifting due to thermal convection during the amplification process.

[0012] Furthermore, the chip also includes at least one sample inlet (4), one sample outlet (5), and one collection chamber (6). The sample outlet (5) is connected to the collection chamber (6) via a microchannel (7) and is used to push out the magnetic beads (9) in the positive micropits and transport them to the collection chamber (6) after amplification by the programmable magnetic field generator.

[0013] Furthermore, the electromagnetic coil array (11) is driven by a matrix addressing drive circuit (13), which adopts a row and column scanning architecture. Each micro coil is connected in series with an isolation diode. The sum of the number of row drive lines and column drive lines of the controller (12) is equal to the sum of the number of rows and columns of the electromagnetic coil array (11).

[0014] Furthermore, the chip substrate (1) is made of a transparent polymer material selected from cyclic olefin polymer (COP), polycarbonate (PC) or polymethyl methacrylate (PMMA), and the micro-pit array (2) is formed on the chip substrate (1) by hot stamping, injection molding or photolithography.

[0015] Furthermore, the number of micropits in the micropit array (2) is 1,000 to 1,000,000.

[0016] Furthermore, the temperature control module includes a PI film heater (14) and a fan (15), with a heating and cooling rate of not less than 3°C / s.

[0017] Furthermore, the optical detection module includes a multi-channel LED excitation light source (17) and corresponding emission filters (18) and excitation filters (19), and acquires images through a CMOS camera (16) to simultaneously detect amplification products of at least two different fluorescent labels.

[0018] Furthermore, the main controller (20) runs system control software (21) and is connected to each module via communication bus (22). It is also programmed to identify positive micro-pits after amplification and generate a repulsive magnetic field by applying a reverse pulse current to the positive micro-pit location through the programmable magnetic field generator, thereby pushing out the magnetic beads (9) in the positive micro-pit and transporting them to the chip's collection cavity (6).

[0019] In a third aspect of the present invention, a digital PCR absolute quantification method based on the above system is characterized by comprising the following steps: (a) introducing functionalized magnetic beads (9) carrying nucleic acid templates into the surface of the micro-pit array (2) of the chip; (b) activating the controller (12) to drive the magnetic beads (9) one by one into the micro-pits through a programmable magnetic field to form a monodisperse distribution; (c) adding PCR reaction solution and sealing the chip; (d) activating the temperature control module to perform PCR amplification; (e) activating the optical detection module to acquire fluorescence images of the micro-pit array (2) and counting the number of positive micro-pits; (f) calculating the initial copy number of the template according to the Poisson distribution formula.

[0020] Furthermore, after step (b) and before step (c), the process includes imaging the micro-pit array (2) to identify and mark micro-pits containing multiple magnetic beads (9) for removal in subsequent counting; after step (e), the process includes recovering magnetic beads (9) from positive micro-pits using the programmable magnetic field generator.

[0021] The present invention has the following beneficial effects: (1) Low cost and easy to mass-produce: Micro-pit array chips can be mass-produced through hot stamping or injection molding processes. The cost per chip is much lower than that of traditional photolithography chips and micro-droplet consumables.

[0022] (2) Programmable active partitioning: Unlike the passive loading method using permanent magnets in the existing technology, this system achieves orderly and controllable positioning of magnetic beads through a programmable magnetic field generator, resulting in high loading efficiency and good partitioning consistency.

[0023] (3) High integration: The micro-pit array chip, programmable magnetic field generator, temperature control module and optical detection module are integrated into an integrated system to realize a fully automatic process from sample loading to result output.

[0024] (4) Support positive partition recovery: Through the combination of programmable magnetic field reverse drive and micromagnet (NiFe thin film (8)) assisted capture, the automatic non-contact recovery of positive magnetic beads is realized, and the recovered nucleic acid can be directly used for downstream sequencing.

[0025] (5) High scalability: The density of the micro-pit array can be flexibly adjusted according to the detection requirements, and can be realized from thousands to millions of partitions; the coil density of the magnetic field generator is matched with the micro-pit array, and can be scaled proportionally to adapt to different throughput requirements. Attached Figure Description

[0026] Figure 1This is a schematic diagram of the magnetic bead array partitioned chip structure of the present invention (top view, partial magnification). In the figure: 1-chip substrate, 2-micro-pit array, 3-sealing cover, 4-sample inlet, 5-sample outlet, 6-collection chamber, 7-microchannel, AA is the position of the cross-sectional line.

[0027] Figure 2 for Figure 1 A schematic diagram of the AA cross-section. In the diagram: 1-chip substrate, 2-micro-pit, 3-sealing cover, 8-NiFe thin film, 9-magnetic bead.

[0028] Figure 3 This is a schematic diagram of the overall system structure of the present invention (including a programmable magnetic field generator, temperature control, and optical module). In the figure: 1-chip substrate, 2-micro-pit array chip, 10-flexible substrate, 11-electromagnetic coil array, 12-FPGA+MCU controller, 13-matrix drive circuit, 14-PI film heater, 15-fan, 16-CMOS camera, 17-LED excitation light source, 18-emission filter, 19-excitation filter, 20-main controller, 21-software, 22-communication bus.

[0029] Figure 4 A schematic diagram illustrating the principle of driving magnetic beads into micro-pits by scanning line by line to the virtual magnetic field center. In the figure: (a) before scanning, (b) during scanning, (c) after scanning; 2-micro-pit array, 9-magnetic beads, 11-electromagnetic coil array.

[0030] Figure 5 This is a schematic diagram of fluorescence imaging of a single magnetic bead within a micro-pit. In the figure: (a) bright-field view, (b) positive fluorescence signal, (c) negative fluorescence signal; 2-micro-pit, 9-magnetic bead.

[0031] Figure 6 This is a flowchart of the system operation of the present invention.

[0032] Figure 7 This is a schematic diagram illustrating the principle of programmable magnetic field-driven positive magnetic bead recovery. In the diagram: (a) before recovery, (b) reverse pulse ejection, (c) gradient-guided transmission, (d) recovery completed; 1-chip substrate, 2-micro-pit array, 6-collection cavity, 7-microchannel, 8-NiFe thin film, 9-magnetic bead, 11-electromagnetic coil array. Detailed Implementation

[0033] The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary and not intended to limit the scope and application of the present invention.

[0034] It should be noted that when a component is referred to as "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as "connected to" another component, it can be directly connected to or indirectly connected to that other component. Furthermore, a connection can be used for fixing, coupling, or communication.

[0035] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the present invention.

[0036] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

[0037] General Description: The "programmable magnetic field generator" described in this invention refers to any device capable of generating and moving magnetic field peaks in space through electronic control. In a preferred embodiment, the device is the system described in the invention patent application "An Artificial Intelligence Programmable Flexible Electromagnetic Coil Microarray Magnetic Control System" filed on the same day. The specific structure, driving circuit, control algorithm, and manufacturing process of this system have been disclosed in detail in the aforementioned application, and all of its contents are incorporated herein by reference. Alternatively, the programmable magnetic field generator can also be composed of multiple independent electromagnets disposed below the chip (each electromagnet is grouped by row / column), or can be implemented by a movable single electromagnet in conjunction with a positioning platform. As long as a controlled moving magnetic field peak can be generated to drive the magnetic beads (9) to enter the pit sequentially, it falls within the protection scope of this invention.

[0038] Application Area Description: The chip and system provided by this invention are a general technical solution. The above embodiments are only used to illustrate some applications of this invention and are not intended to limit the scope of application of this invention. Those skilled in the art should understand that, based on the principles of this invention, this system can be widely applied to any scenario requiring absolute quantification by digital PCR, including but not limited to: viral load monitoring, tumor liquid biopsy, non-invasive prenatal testing, pathogen identification, quantitative gene expression analysis, gene copy number variation analysis, quantitative detection of transgenic components, and food safety microbial detection. All these applications fall within the protection scope of this invention.

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0040] Example 1: Magnetic bead array chip and system for absolute quantification of HBV DNA 1. Chip Structure Chip substrate (1): Made of COP material (cyclic olefin polymer), with dimensions of 20mm×20mm and a thickness of 1mm.

[0041] Micro-pit array (2): 100×100 micro-pits (a total of 10,000) are formed by hot stamping process. The micro-pits have a diameter of 20μm, a depth of 20μm, and a center-to-center spacing of 40μm. The ratio of the micro-pit diameter to the particle size (10μm) of the magnetic beads (9) used is 2.0. A 50nm thick nickel-iron alloy (NiFe) thin film (8) is deposited at the bottom of the micro-pit as a micro-magnet structure, which is deposited by magnetron sputtering after hot stamping.

[0042] Sealing cover (3): A COP film with a thickness of 0.2 mm, which is sealed to the chip substrate (1) by thermal bonding process. The chip is provided with a sample inlet (4) and a sample outlet (5), and the sample outlet (5) is connected to the collection chamber (6) through a microchannel (7).

[0043] 2. System Composition Programmable magnetic field generator: The system described in the invention patent application "An Artificial Intelligence Programmable Flexible Electromagnetic Coil Microarray Magnetic Control System" filed on the same day is adopted (8×8 flexible electromagnetic coil array (11), the coil is a planar spiral structure with an outer diameter of 5mm, an inner diameter of 0.5mm, 15 turns, FPC process, and the flexible substrate (10) is a 50μm polyimide film). The coil spacing is about 5mm, and a virtual magnetic field center is synthesized by the inverse distance weighting method, which covers the entire micro-pit array (2) in a row-by-row activation manner.

[0044] Controller (12): FPGA+MCU (STM32F407), which realizes multi-channel independent current control through matrix drive circuit (13) and can programmatically generate a virtual magnetic field center for line-by-line scanning.

[0045] Temperature control module: PI membrane heater (14) works with fan (15) to dissipate heat and realize PCR thermal cycling (95°C→60°C→72°C), with a heating and cooling rate ≥3°C / s.

[0046] Optical detection module: CMOS camera (16) (5 million pixels) equipped with LED excitation light source (17) (wavelength 480nm / 530nm / 630nm three channels) and corresponding emission filter (18) and excitation filter (19), which can capture multi-channel fluorescence images of the entire micro-pit array (2).

[0047] Main controller (20): Industrial computer or embedded ARM processor, running system control software (21), coordinating the working timing of magnetic field generation, temperature control and imaging modules through communication bus (22).

[0048] 3. Workflow (a): Magnetic bead preparation. Streptavidin magnetic beads (9) with a diameter of 10 μm were incubated with biotin-labeled HBV-specific capture probes to attach the probes to the surface of the magnetic beads (9). The magnetic beads (9) were mixed with the sample to be tested and incubated for 30 minutes to capture the target nucleic acid. The concentration of the magnetic beads (9) was controlled by dilution to ensure that each magnetic bead (9) carried an average of approximately 0.1 templates.

[0049] (b): Magnetic bead loading. A suspension of magnetic beads (9) is injected into the chip through the injection port (4), distributing the magnetic beads (9) on the surface of the micro-pit array (2). The main controller (20) activates the programmable magnetic field generator, calculates the current of each coil using the inverse distance weighting method, and moves the center of the synthesized virtual magnetic field row by row from row 1 to row 100 (with a dwell time of 0.5 seconds per row), driving the magnetic beads (9) to fall sequentially into the micro-pits of the corresponding rows, forming a monodisperse distribution. The filling rate is approximately 95%.

[0050] (c): Imaging rejection. Bright-field imaging is performed on the micro-pit array (2), and micro-pits containing multiple magnetic beads (9) are identified and marked by image analysis software (21) and rejected in subsequent counting.

[0051] (d): PCR amplification. Add PCR premix containing TaqMan probes and seal the chip. Start the temperature control module (PI membrane heater 14, fan 15) for thermal cycling: 95°C for 3 min; [95°C for 15 s, 60°C for 30 s] × 45 cycles.

[0052] (e): Fluorescence imaging. Start the optical detection module (LED excitation light source 17, CMOS camera 16, emission filter 18, excitation filter 19) to capture fluorescence images of the amplified micro-pit array (2), and automatically identify and count the number of positive micro-pits.

[0053] (f): Absolute quantification. The main controller (20) automatically calculates the initial copy number of the template and the original sample concentration according to the Poisson distribution formula.

[0054] (g) (Optional): Positive magnetic bead recovery. For micropits marked as positive, the controller (12) activates the corresponding coil and applies a reverse pulse current to push the magnetic bead (9) out of the micropit and transport it to the collection chamber (6) through the guiding magnetic field via the microchannel (7).

[0055] 4. Effect Comparison The HBV DNA standard (expected concentration 14000 copies / μL) was detected using this system, and compared with the traditional droplet dPCR (Bio-Rad QX200). The results are shown in the table below: This invention system 14000 copies / μL 14140 copies / μL <1% Droplet dPCR 14000 copies / μL 13950 copies / μL <1% Quantitative results: The concentration measured by the system of this invention was 14140 copies / μL, with a relative error of <1% compared with the expected value of the standard, and highly consistent with the result of droplet dPCR (13950 copies / μL).

[0056] Example 2: High-density array (100,000-level micro-pits) By reducing the diameter of the micropits to 8 μm, the depth to 8 μm, and the center-to-center spacing to 16 μm, approximately 100,000 micropits can be integrated within an area of ​​1 cm². A 30 nm thick NiFe film (8) is deposited at the bottom of the micropits as a micromagnet. The electromagnetic coil array (11) of the programmable magnetic field generator is adjusted accordingly to adjust the coil array density. The chip substrate (1) still uses COP material, and the micropits are formed by injection molding with higher precision. The structures such as the sealing cover (3), the sample inlet (4), the sample outlet (5), the microchannel (7), and the collection chamber (6) are adjusted proportionally. The remaining system components (controller 12, PI film heater 14 and fan 15 of the temperature control module, CMOS camera 16 and LED excitation light source 17 and filters 18 / 19 of the optical detection module, and main controller 20) are similar to those in Example 1. This embodiment is suitable for detection scenarios that require a higher number of partitions and a wider dynamic range.

[0057] Example 3: Automated Recycling of Positive Magnetic Beads After amplification imaging in Example 1, the main controller (20) reads the list of positive micro-pit coordinates. For each positive micro-pit, the controller (12) performs the retrieval process: (a) activating the coil corresponding to the micro-pit and applying a reverse pulse current (100ms, 300mA) to generate a repulsive magnetic field that pushes the magnetic bead (9) out from above the NiFe film (8); (b) simultaneously activating 2-3 adjacent coils in the same row to generate a guiding magnetic field gradient that transports the magnetic bead (9) through the microchannel (7) to the collection chamber (6). The NiFe film (8) at the bottom of the micro-pit assists in fixing the magnetic bead (9) during normal amplification. During reverse pulse excitation, due to its low remanence (NiFe is a soft magnetic material), it does not hinder the release of the magnetic bead (9). The total retrieval time for 850 positive magnetic beads (9) is about 7 minutes. The retrieved magnetic beads (9) are directly used for Sanger sequencing verification with good sequence fidelity.

[0058] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. A magnetic bead array partitioning chip for digital PCR, characterized in that, include: A chip substrate with a micro-pit array consisting of multiple regularly arranged micro-pits; The diameter of the micro-pit is 5–100 μm, the depth is 5–50 μm, and the center-to-center distance between adjacent micro-pits is 10–100 μm; The ratio of the diameter of the micro-pit to the particle size of the magnetic bead to be positioned is 1.0 to 2.0; A sealing cover is placed on the chip substrate to seal the micro-pit array.

2. The chip according to claim 1, characterized in that, The chip substrate is made of a transparent polymer material selected from cyclic olefin polymers (COP), polycarbonate (PC), or polymethyl methacrylate (PMMA).

3. The chip according to claim 1, characterized in that, Each micro-pit in the micro-pit array is further provided with a micro-magnet structure at the bottom. The micro-magnet is a ferromagnetic thin film deposited at the bottom of the micro-pit with a thickness of 50 to 500 nm.

4. The chip according to claim 1, characterized in that, It also includes at least one inlet, one outlet, and one collection chamber, wherein the outlet is connected to the collection chamber via a microchannel.

5. A magnetic bead array partitioning system for digital PCR, characterized in that, include: The chip according to any one of claims 1 to 4; A programmable magnetic field generator is disposed below the chip to generate a spatially movable magnetic field peak. A controller is electrically connected to the programmable magnetic field generator. The controller independently controls multiple magnetic field sources of the programmable magnetic field generator, causing the magnetic field peak to move along a preset path, thereby driving the magnetic beads in the chip to enter each micro-pit sequentially. A temperature control module, thermally coupled to the chip, is used to provide the temperature conditions required for PCR; An optical detection module is disposed above the chip and is used to acquire fluorescence images of the micro-pit array; A main controller is electrically connected to the controller, temperature control module, and optical detection module of the programmable magnetic field generator.

6. The system according to claim 5, characterized in that, The programmable magnetic field generator is a flexible electromagnetic coil array, including a flexible substrate and a plurality of planar spiral microcoils disposed thereon. The spacing between the microcoils is matched with or is an integer multiple of the spacing between the micro-pit array.

7. The system according to claim 5, characterized in that, The controller synthesizes a magnetic field peak using a virtual magnetic field center algorithm, and moves the magnetic field peak along at least one of the following paths: row-by-row scanning, column-by-column scanning, or point-by-point addressing.

8. The system according to claim 7, characterized in that, The virtual magnetic field center algorithm is an inverse distance weighting method, which allocates current according to the distance from each magnetic field source to the virtual center, so that the center of the synthesized magnetic field falls on the virtual center position.

9. The system according to claim 6, characterized in that, The electromagnetic coil array is driven by a matrix addressing drive circuit, which adopts a row and column scanning architecture. Each micro coil is connected in series with an isolation diode. The sum of the number of row drive lines and column drive lines of the controller is equal to the sum of the number of rows and columns of the electromagnetic coil array.

10. The system according to claim 5, characterized in that, The number of micro-pits in the micro-pit array is 1,000 to 1,000,000, and the micro-pit array is formed on the chip substrate by hot stamping, injection molding or photolithography.

11. The system according to claim 5, characterized in that, The main controller is also programmed to identify positive micro-pits after amplification and to generate a repulsive magnetic field by applying a reverse pulse current to the location of the positive micro-pit through the programmable magnetic field generator, thereby pushing out the magnetic beads in the positive micro-pit and transporting them to the chip's collection cavity.

12. The system according to claim 5, characterized in that, The temperature control module includes a thin-film heater and a fan, with a heating and cooling rate of not less than 3°C / s.

13. The system according to claim 5, characterized in that, The optical detection module includes a multi-channel excitation light source and a corresponding emission filter, used to simultaneously detect amplification products of at least two different fluorescent labels.

14. A digital PCR absolute quantification method based on the system of claim 5, characterized in that, Includes the following steps: (a) Introducing functionalized magnetic beads carrying nucleic acid templates onto the surface of the micro-pit array of the chip; (b) The controller is activated, and the magnetic beads are driven one by one into the micro-pits by a programmable magnetic field to form a monodisperse distribution; (c) Add PCR reaction solution and seal the chip; (d) Start the temperature control module to perform PCR amplification; (e) Activate the optical detection module to acquire fluorescence images of the micro-pit array and count the number of positive micro-pits; (f) Calculate the initial copy number of the template according to the Poisson distribution formula.

15. The method according to claim 14, characterized in that, After step (b) and before step (c), the process further includes imaging the micro-pit array to identify and mark micro-pits containing multiple magnetic beads, which are then discarded in subsequent counting; after step (e), the process further includes recovering magnetic beads from positive micro-pits using the programmable magnetic field generator.

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