A microfluidic chip, its fabrication method, and a molecular measurement method using the same chip.

Abstract

This invention discloses a microfluidic chip, its fabrication method, and a molecular measurement method utilizing the same. The microfluidic chip includes an upper chip and a lower chip. The lower surface of the upper chip and the upper surface of the lower chip can be attached to each other, and the upper and lower chips can slide relative to each other along the attached surfaces. The lower surface of the upper chip has a first fluid channel structure for loading a first reactant and a second fluid channel structure for loading a second reactant. The upper surface of the lower chip has a microporous structure. When the upper and lower chips slide relative to each other, the vertical projection of the first or second fluid channel structure can be aligned with the microporous structure, allowing the first and second reactants to enter the microporous structure stepwise. This invention can increase the magnetic bead loading rate to approximately 90%, significantly improving detection performance. Furthermore, the stepwise loading scheme of this invention can significantly reduce background values, offering a more significant advantage in multiplex detection.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to a microfluidic chip, its fabrication method, and a molecular measurement method using the same. Background Technology

[0002] Nucleic acids and proteins are the basic molecules that make up life and participate in almost all physiological and pathological processes. Since the invention of polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in the 1980s, the role of nucleic acids and proteins as biomarkers in clinical diagnosis has been gradually gaining attention. Recent studies have shown that a large number of protein and nucleic acid molecules, though numerous, play a crucial role in the occurrence and development of diseases. These molecules are difficult to detect using traditional methods, thus limiting in-depth research and utilization.

[0003] Single-molecule array (SimoA) technology was first proposed in 2010. Walt et al. used antibody-modified magnetic microspheres to capture antigens in samples and form immune complexes on the surface of the magnetic microspheres. Then, a large number of magnetic beads were independently enclosed in a fly-ultra-high microreaction chamber for fluorescence catalysis. Due to the physical isolation between different microreaction chambers, the catalyzed fluorescent substrate aggregated in the fly-ultra-high droplets, preventing dilution due to diffusion and resulting in extremely high local substrate concentrations, thus enabling single-molecule signal amplification. Since the antigen distribution on the microsphere surface follows a Poisson distribution, each microsphere surface captures zero, one, or more antigen molecules. Finally, by statistically analyzing the ratio of positive to negative microspheres or the change in fluorescence intensity of positive microspheres, the absolute concentration of protein in the original system can be calculated. This technology has improved the detection sensitivity of traditional ELISA by nearly a thousand times and is now widely used in the detection of tumor markers, neurodegenerative diseases, and inflammatory and infectious diseases. While the above-mentioned digital protein detection technology significantly improves the sensitivity of protein detection, the following problems still exist:

[0004] 1. Microsphere loading efficiency is extremely low. Regardless of whether microcavity arrays or microdroplets are used as isolation modules, the loading efficiency of a single microsphere in a droplet is limited by the Poisson distribution, typically accounting for only about 10% of the total chambers. This results in a large number of microcavities or microdroplets becoming ineffective chambers, which not only wastes resources for subsequent data analysis, but more importantly, in order to achieve higher detection sensitivity or a wider linear range, it is necessary to increase the total number of chambers to meet the needs of practical applications. This leads to a significant increase in manufacturing difficulty, chip area, and a surge in the difficulty of matching optical detection systems.

[0005] 2. Difficulty in simultaneously detecting more than 10 proteins in a single reaction. Typically, digital detection methods requiring ultra-high sensitivity and a wide linear range need at least tens of thousands of effective chambers per target molecule (i.e., tens of thousands of isolation chambers loaded with individual microspheres). The more target molecules required, the greater the number of effective isolation microchambers needed. However, due to the Poisson distribution limitation of microsphere loading, achieving hundreds of thousands of effective isolation chambers necessitates millions or even tens of millions of total isolation chambers. This significantly increases redundant analytical resources and the difficulty of chip fabrication. Therefore, overcoming the limitations of the Poisson distribution in microsphere loading, achieving high microsphere loading within limited isolation chambers, and achieving ultra-sensitive digital detection with a wide linear range for the simultaneous detection of approximately 20 target molecules remains one of the biggest challenges for current digital detection technology.

[0006] 3. Existing digital isolation chips cannot achieve multi-step sample loading reactions. As a general-purpose digital protein detection tool platform, it not only needs to meet the application requirements of specific detection methodologies and raw materials, but also the versatility of the detection chip and its ability to be easily adjusted according to specific detection methodological needs; that is, the flexibility of the detection platform is also crucial. For example, some enzyme-catalyzed signal amplification reactions, due to their extremely fast reaction rates, require stepwise sample loading and precise control of reaction time to overcome the bottleneck problem of background signal enhancement caused by the enzyme contacting the substrate before microsphere loading, which leads to reduced detection sensitivity.

[0007] Since Vogelstein et al. proposed the concept of digital PCR in 1999 by using extreme dilution of the sample amount in each well of a 384-well plate and increasing the number of wells for detection, thus pointing out the development direction of digital PCR detection instruments, nucleic acid amplification methods have evolved from the relatively quantitative qPCR technology that relies on standard curves to the era of absolute quantitative detection that no longer relies on standard substances as quantitative references, and have developed rapidly in recent years. Currently, there are three main categories of digital detection platforms for nucleic acids: microchamber array technology, microdroplet technology, and barcode single-molecule counting analysis technology. Microchamber array technology is represented by LIFE Technologies' QuantStudio 3D digital PCR instrument. It prepares an array of 20,000 microwells with a diameter of 60 micrometers and a volume of 0.8 nL in a microfluidic chip. The sample to be tested is dispersed into the microwells to form several independent reaction units by segmentation. After sealing the microwells, amplification is performed. Finally, the "present" or "absent" signal of the amplification endpoint is read and counted, and the concentration of the sample to be tested is obtained by Poisson distribution statistics. Another type of technology currently dominant in applications is microdroplet technology, such as Bio-Rad's QX200, RainDance, and STILLA's Crystal digital PCR microdroplet digital PCR instruments, as well as BEAMing (Beads, Emulsion, Amplification, Magnetics) digital nucleic acid detection technology that combines droplets with magnetic bead capture amplification. Microdroplet digital PCR technology rapidly and consistently generates nanoliter to picoliter water-in-oil droplets using microfluidic chips, which serve as reaction chambers to isolate the nucleic acid molecules to be tested. Compared to microchamber array technology, its biggest advantage is that the number of droplets can be flexibly and continuously generated as needed, even reaching tens of millions (as in RainDance), thus significantly improving the sensitivity of nucleic acid detection. Furthermore, the manufacturing method of the microfluidic chips used for droplet generation is simpler and more controllable. The third type is fluorescent barcode nucleic acid single-molecule counting analysis systems, represented by Nanostring's nCounter. Its main principle is that the nucleic acid molecules to be tested hybridize with the capture probe and the reporter probe labeled with four-color fluorescent barcodes to form hybridization complexes. These complexes are then fixed on the detection chip and arranged upside down on the chip surface by an electric field. Finally, the type and number of barcodes are detected by a micro-fluorescence imaging system to achieve multiplex, absolute quantitative counting detection of the target nucleic acid molecules.Because the fluorescent barcode probe molecules are composed of four different fluorescent nucleic acid clusters arranged in a specific order, with six fluorescent clusters per barcode, 4096 barcode probes can be formed. Theoretically, this system has the capability to simultaneously detect over 4000 target nucleic acids, making it the system with the highest single-reaction detection multiplex for nucleic acid digital detection currently available. However, this system is complex to operate, the hybridization reaction to form the complex requires overnight incubation, and the multi-color reporter probes with multiple encodings are extremely expensive, hindering its application as a routine clinical testing method.

[0008] Digital PCR detectors, whether based on microchamber array technology or microdroplet technology, while unmatched by other nucleic acid detection technologies in achieving high sensitivity, still suffer from inherent drawbacks in their instrument design principles that are difficult to overcome.

[0009] (1) None of them have adequately addressed the need for multiplex (>10) nucleic acid detection in the era of precision medicine. Due to the limitations of the fluorescence detection channel, these digital detection systems have difficulty accommodating a large number of excitation light sources and fluorescence detection channels within the limited space of the optical detection module. Currently, the maximum number of detection channels in digital PCR is only 4 colors, meaning that a single tube can only detect 4 nucleic acid molecules simultaneously.

[0010] (2) The goal of equivalent amplification has not yet been achieved in multiplex nucleic acid amplification. Due to the limitations of traditional nucleic acid amplification methodologies, the more types of target nucleic acids that need to be detected in a single reaction (single tube), the more likely conventional multiplex primer-based amplification methods will suffer from primer interference and primer dimers. It is difficult to ensure that the amplification efficiency of different target nucleic acid molecules remains consistent, which greatly reduces the detection sensitivity and specificity. An even bigger problem is that the product concentration level may deviate from the original target nucleic acid molecule concentration level due to the inconsistency of amplification efficiency.

[0011] As analyzed in the preceding review, digital detection systems for low-abundance proteins and nucleic acids have brought unprecedented changes to the discovery and validation of novel biomarkers, early disease diagnosis, prognostic assessment, relapse monitoring, and medication guidance in the era of precision medicine. However, two major challenges remain:

[0012] (1) A breakthrough is urgently needed for a universal, simple and controllable digital detection system that can be applied to both proteins and nucleic acids. This may be because nucleic acids and proteins are two different target molecules, and their detection methodologies are also completely different, making it difficult to achieve the unification of two very different methodologies with a single detection system.

[0013] (2) Breakthroughs are urgently needed in single-reaction (single tube) multiplex (more than 10 target molecules) digital detection systems. Currently, neither digital PCR detection systems nor protein digital detection systems can achieve the simultaneous detection of more than 10 target molecules in a single tube reaction. Summary of the Invention

[0014] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art, such as low microsphere loading rate, difficulty in realizing multiple digital biological detection, and difficulty in realizing digital multi-index detection of multiple biomolecules, and to provide a microfluidic chip, its preparation method and molecular measurement method using it.

[0015] The present invention solves the above-mentioned technical problems through the following technical solution:

[0016] This invention provides a microfluidic chip, comprising an upper chip and a lower chip. The lower surface of the upper chip and the upper surface of the lower chip are able to adhere to each other, and the upper chip and the lower chip are able to slide relative to each other along the adhering surfaces. The lower surface of the upper chip is provided with a first fluid channel structure for loading a first reactant and a second fluid channel structure for loading a second reactant. The upper surface of the lower chip is provided with a microporous structure. When the upper chip and the lower chip slide relative to each other, the vertical projection of the first fluid channel structure or the second fluid channel structure can be made to coincide with the microporous structure, so that the first reactant and the second reactant enter the microporous structure stepwise.

[0017] This invention utilizes the relative sliding of upper and lower chips for fluid manipulation, eliminating the need for complex fluid manipulation structures and significantly reducing the dead volume used for fluid manipulation in the fluid channel structure to less than 40% of the total sample loading volume. Furthermore, by controlling the size of the micropores, this invention ensures that each micropore can only hold a single magnetic microsphere, with excess microspheres being flushed away by fluorocarbon compounds. This overcomes the limitations of Poisson distribution and improves the microsphere loading rate. Through these two methods, this invention increases the magnetic bead loading rate from 5-10% in traditional technologies to approximately 90%, significantly improving detection performance.

[0018] Furthermore, this invention employs a stepwise sample loading method, adding reactants in stages. This prevents the reaction from starting before sealing due to premature mixing of the first and second reactants, which could lead to increased background signals or even false positives. Therefore, the stepwise sample loading method of this invention can significantly reduce background values, offering a more significant advantage in multiplex detection.

[0019] Preferably, the microporous structure includes a plurality of micropores, and the first reactant includes magnetic microspheres bound to the analyte molecule, wherein the micropores are capable of accommodating a single magnetic microsphere.

[0020] Preferably, the diameter of the micropores is 1μm-100μm and the depth is 1μm-100μm; more preferably, the diameter is 5μm-10μm and the depth is 3μm-8μm, and the diameter of the magnetic microspheres is 3-6μm.

[0021] Preferably, the number of micropores is 10,000 to 1,000,000; more preferably, 400,000 to 800,000. This ensures that the number of effective chambers, i.e., the number of closed micropores loaded with magnetic microspheres, can meet the requirements for molecular detection up to twenty layers.

[0022] Preferably, the vertical projection contours of the first fluid channel structure and the second fluid channel structure can coincide with the contour of the microporous structure. This ensures that when the upper and lower chips are in the first and second sample loading positions, the first and second fluid channels can respectively coincide with the microporous array.

[0023] Preferably, the first reactant comprises magnetic microspheres incorporating the analyte molecule, the second reactant comprises a signal precursor capable of reacting with the magnetic microspheres to generate a detectable signal, the first fluid channel structure having a first depth, and the second fluid channel structure having a second depth less than the first depth.

[0024] By using a larger first depth, when the sample is loaded at the first loading position, the vertical projection density of the discrete magnetic microspheres injected into the first fluid channel structure is higher, which increases the probability of the magnetic microspheres falling into the microporous structure and improves the utilization rate of the micropores.

[0025] By employing a smaller second depth, the probability of oil seal damage during fluid manipulation and the likelihood of bubble formation can be reduced, thereby improving the efficiency of sealing micropores.

[0026] In some embodiments, the first depth is 10μm-200μm and the second depth is 10μm-200μm.

[0027] Preferably, the first depth is 50μm-175μm and the second depth is 50μm-175μm.

[0028] More preferably, the first depth is 175 μm and the second depth is 50 μm.

[0029] Preferably, the surfaces of the first fluid channel structure, the second fluid channel structure, and the microporous structure are provided with a hydrophobic layer.

[0030] Preferably, the hydrophobic layer is made of linear polydimethylsiloxane molecules.

[0031] Therefore, the chip materials, surface treatments, chip structure, and reagents used in this microfluidic chip are all compatible with digital protein and nucleic acid detection signal amplification methods. It can be used for protein detection with room temperature incubation and can also withstand PCR reactions requiring dozens of thermal cycles. This makes this microfluidic chip a universal detection device for proteins and nucleic acids.

[0032] This invention also provides a method for fabricating a microfluidic chip, used to fabricate the microfluidic chip as described above, the method comprising the following steps:

[0033] S10: Etch the first fluid channel structure and the second fluid channel structure on the upper chip, wherein the first fluid channel structure has a first depth, the second fluid channel structure has a second depth, and the first depth is greater than the second depth;

[0034] S20: Etch the microporous structure onto the lower chip;

[0035] Specifically, step S10 includes:

[0036] S11: A first etched region for removing the protective layer is formed on a chip covered with a protective layer, the first etched region corresponding to the outline of the first fluid channel structure.

[0037] S12: Etch the first etched area until the etch depth of the first etched area reaches the difference between the first depth and the second depth;

[0038] S13: A second etched region for removing the protective layer is formed on the upper chip covered with a protective layer, the second etched region corresponding to the outline of the second fluid channel structure;

[0039] S14: Further etch the first etched area and the second etched area until the etching depth of the first etched area reaches the first depth and the etching depth of the second etched area reaches the second depth.

[0040] Preferably, the method further includes the following after step S20:

[0041] S30: Prepare a hydrophobic layer on the upper and lower chips after etching.

[0042] The present invention also provides a molecular measurement method applicable to microfluidic chips as described above, for quantitative detection of molecules to be measured.

[0043] Preferably, the molecule to be tested is a protein or nucleic acid.

[0044] Preferably, when the analyte is a protein, the first reactant comprises magnetic microspheres bound to the analyte, and the second reactant comprises a signal precursor. The magnetic microspheres, upon binding to the signal precursor, can generate a detectable signal. The molecular measurement method includes the following steps:

[0045] S100: Assemble the upper chip and the lower chip, and set the lower surface of the upper chip to be in contact with the upper surface of the lower chip;

[0046] S200: The upper chip and the lower chip are slid relative to each other so that the vertical projection of the first fluid channel structure coincides with the microporous structure;

[0047] S300: Inject the magnetic microspheres into the first fluid channel structure, causing the magnetic microspheres to settle in the microporous structure;

[0048] S400: Inject a first fluorocarbon compound into the first fluid channel structure to seal the microporous structure;

[0049] S500: Inject a signal precursor into the second fluid channel structure, slide the upper chip and the lower chip relative to each other so that the second fluid channel structure coincides with the microporous structure, thereby allowing the signal precursor to enter the microporous structure and combine with the magnetic microspheres to generate a detectable signal;

[0050] S600: Inject a second fluorocarbon compound into the second fluid channel structure to seal the microporous structure a second time;

[0051] S700: Detect the signal emitted by the magnetic microspheres and calculate the concentration of the analyte molecule based on the signal.

[0052] Preferably, the fluorescent substrate is halogen-β-D-galactopyranoside, and the enzyme is β-galactosidase.

[0053] Preferably, the molecule to be tested binds to the magnetic microspheres modified with capture antibodies.

[0054] Preferably, the signal precursor includes a fluorescent substrate, and the magnetic microspheres, after binding to the analyte molecule, also bind an enzyme capable of cleaving the fluorescent substrate to generate a fluorescent signal.

[0055] Preferably, step S700 includes:

[0056] The fluorescence positive signal of the magnetic microspheres is read, and the concentration of the analyte is calculated based on the proportion of the fluorescence positive signal in the micropore structure, the Poisson distribution data of the analyte on the magnetic microspheres, and the amount of magnetic microspheres added.

[0057] Preferably, the first fluorocarbon compound is a mixture of FC-40 and Novec 7100, wherein the volume ratio of FC-40 to Novec 7100 is 1:4. This mixture has a high surface energy, which can effectively drive away excess magnetic microspheres, seal micropores, and not affect the magnetic microspheres and solution inside the micropores. At the same time, it can be completely removed from above the microporous structure as it slides, without remaining on the surface of the lower chip or leaking into the adjacent second fluid channel structure.

[0058] Preferably, the second fluorocarbon compound is composed of FC-40. This component has lower volatility than the first fluorocarbon compound, ensuring that the micropore array remains closed during signal amplification and that the micropores are not exposed due to the evaporation and drying of the fluorocarbon compound.

[0059] When the molecule to be tested is a nucleic acid, the molecular measurement method includes the following steps:

[0060] S1000: Assemble microfluidic chips so that the first fluid channel structure and the second fluid channel structure on the upper chip and the microporous structure on the lower chip are filled with liquid phase.

[0061] S2000: The upper chip and the lower chip are slid relative to each other so that the first fluid channel structure coincides with the microporous structure;

[0062] S3000: Inject PCR reaction solution into the first fluid channel structure, and then inject a first fluorocarbon compound into the first fluid channel structure to seal the micropores;

[0063] S4000: Injecting perfluoropolyether into the first fluid channel structure to replace the first fluorocarbon compound;

[0064] S5000: Perform PCR amplification on the microfluidic chip;

[0065] S6000: Calculate the nucleic acid concentration based on the fluorescence signal in the microporous structure.

[0066] The positive and progressive effects of this invention are as follows:

[0067] This invention successfully overcomes the limitations of Poisson distribution on microsphere loading, achieving a microsphere loading rate of nearly 90%, which is significantly higher than the traditional technology with a loading rate of only 5-10%. The greater number of microspheres makes the detection sensitivity of this technology superior to similar technologies, and when combined with fluorescently encoded microspheres, it can achieve more than 20-fold multiplex digital biomolecule detection.

[0068] This invention innovatively enables multi-step sample loading into hundreds of thousands of microwells, giving the chip greater versatility and making it applicable to various reaction principles. For example, in digital protein detection, enzymes and substrates can be added to the microwells stepwise, reducing the need for time control and improving reaction stability. Compared to traditional sample loading methods, this invention can significantly reduce false positive signals caused by premature contact between enzymes and substrates.

[0069] This invention enables digital multi-index detection of two biomolecules, nucleic acids and proteins, in a single chip. Attached Figure Description

[0070] Figure 1 This is a top view of the chip.

[0071] Figure 2 This is a top view of the lower chip;

[0072] Figure 3 This is a schematic diagram showing the overlap between the first fluid channel structure and the microporous structure.

[0073] Figure 4 A schematic diagram illustrating the injection of a first fluorocarbon compound to push away excess magnetic microspheres;

[0074] Figure 5 This is a schematic diagram showing the sedimentation of magnetic microspheres in a microporous structure.

[0075] Figure 6 A schematic diagram of injecting a signal precursor into the second fluid channel structure;

[0076] Figure 7 This is a schematic diagram of a microfluidic chip when it is stationary.

[0077] Figure 8 This is a schematic diagram of fluorescence signals emitted in a microporous structure.

[0078] Figure 9 A graph showing the relationship between the amount of magnetic beads used and the utilization rate of magnetic beads.

[0079] Figure 10 The background signal comparison diagram shows the results of single and double sample loading under different magnetic bead loading amounts, antigen loading amounts, and magnetic bead settling times.

[0080] Figure 11 A comparison chart showing the number of positive wells for DNA samples at different dilution ratios;

[0081] Figure 12 This is a graph showing the relationship between dilution factor and measured DNA concentration.

[0082] Figure 13 A view of signal values ​​for standard samples of interleukin-6 at different concentrations;

[0083] Explanation of reference numerals in the attached figures:

[0084] Chip 100

[0085] First fluid channel structure 110

[0086] Second fluid channel structure 120

[0087] Chip 200

[0088] Microporous structure 210

[0089] First reactant 300

[0090] Second reactant 400

[0091] Magnetic microspheres 500 Detailed Implementation

[0092] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments.

[0093] Example 1

[0094] like Figure 1 and Figure 2 This invention provides a microfluidic chip, which includes an upper chip 100 and a lower chip 200. The upper chip 100 and the lower chip 200 can be made of glass, plastic, metal, or composite materials. In this invention, both the upper chip 100 and the lower chip 200 are made of glass.

[0095] The lower surface of the upper chip 100 and the upper surface of the lower chip 200 can be attached to each other, and the attached upper chip 100 and lower chip 200 can slide relative to each other along their attachment surfaces.

[0096] The lower surface of the chip 100 is provided with a first fluid channel structure 110 for loading the first reactant 300 and a second fluid channel structure 120 for loading the second reactant 400. The first reactant 300 includes magnetic microspheres 500 with analyte molecules bound to them, and the second reactant 400 includes a signal precursor.

[0097] The upper surface of the lower chip 200 is provided with a microporous structure 210, which includes multiple micropores. Each micropore has a diameter of 5μm-10μm and a depth of 3μm-8μm. Correspondingly, the diameter of the magnetic microsphere 500 is 3-6μm, so that only one magnetic microsphere 500 can be accommodated in a single micropore.

[0098] The first fluid channel structure 110, the second fluid channel structure 120, and the microporous structure 210 can all be manufactured by wet etching, dry etching, injection molding, hot pressing, or machining.

[0099] like Figure 3 and Figure 6 When the upper chip 100 and the lower chip 200 slide relative to each other, the vertical projection of the first fluid channel structure 110 or the second fluid channel structure 120 can be made to coincide with the microporous structure 210.

[0100] The operation method of this microfluidic chip is as follows:

[0101] Combination Figure 8 The upper chip 100 and the lower chip 200 are assembled face-to-face, and the first fluid channel structure 110 in the upper chip 100 is aligned with the microporous structure 210 in the lower chip 200. At this time, the upper chip 100 and the lower chip 200 are in the first sample loading position. Then, magnetic microspheres 500 are injected into the first fluid channel structure 110. After the magnetic microspheres 500 settle into the microporous structure 210, fluorocarbon compounds are injected to push away excess magnetic microspheres 500 and close the micropores. Then, the second fluid channel structure 12 is installed. The signal precursor is injected into the upper chip 100. The upper and lower chips 200 are slid to align the second fluid channel structure 120 with the microporous structure 210. At this time, the upper chip 100 and the lower chip 200 are in the secondary sample loading position, and the signal precursor can be delivered into the micropores and mixed with the magnetic microspheres 500. After waiting for 5-30 seconds to complete the diffusion of the reactants, the fluorocarbon compound is injected again to seal the micropores. The sample loading operation is now complete. The analyte in the sample can be quantified by subsequent signal amplification (isothermal incubation catalysis or PCR amplification).

[0102] This invention utilizes the relative sliding of the upper and lower chips 200 for fluid manipulation, eliminating the need for complex fluid manipulation structures and significantly reducing the dead volume used for fluid manipulation in the fluid channel structure to less than 40% of the total sample loading volume. Furthermore, by controlling the size of the micropores, each micropore can only hold a single magnetic microsphere 500; excess microspheres can be flushed away by fluorocarbon compounds, thereby overcoming the limitations of Poisson distribution and improving the microsphere loading rate. Through these two methods, this invention increases the magnetic bead loading rate from 5-10% in traditional technologies to approximately 90%, significantly improving detection performance.

[0103] Furthermore, this invention employs a stepwise sample loading scheme, adding the reactants in stages. This prevents premature mixing of the first reactant 300 and the second reactant 400, which could lead to the reaction starting before sealing, resulting in increased background signal or even false positives. Therefore, the stepwise sample loading scheme of this invention can significantly reduce background values, offering a more significant advantage in multiplex detection.

[0104] In this embodiment, the number of micropores is preferably 400,000 to 800,000, so that the number of closed micropores in the effective chamber, i.e., the magnetic microspheres 500, can meet the requirements for molecular detection of up to twenty layers.

[0105] In this embodiment, the first fluid channel structure 110 includes a first flow channel and a first inlet communicating with the first flow channel, the second fluid channel structure 120 includes a second flow channel and a second inlet communicating with the second flow channel, and the multiple micropores in the microporous structure 210 are distributed in an array.

[0106] The contours of the first flow channel, the second flow channel, and the microporous array are consistent, so that when the upper chip 100 and the lower chip 200 are in the first and second sample loading positions, the first flow channel and the second flow channel can respectively coincide with the microporous array. Specifically, in this invention, the dimensions of the first flow channel, the second flow channel, and the microporous array are: length 15mm-22mm, width 1.5mm-3.5mm. Of course, the above dimensions are only specific embodiments of this invention, and different dimensions can be used as needed.

[0107] In this embodiment, the first fluid channel structure has a first depth, and the second fluid channel structure 120 has a second depth different from the first depth. Specifically, the first depth is 50μm-175μm, and the second depth is 50μm-175μm.

[0108] By employing a larger first depth, when the sample is loaded at the first loading position, the vertical projection density of the discrete magnetic microspheres 500 in the first fluid channel structure 110 is higher, increasing the probability that the magnetic microspheres 500 fall into the microporous structure 210 and improving the microporous utilization rate.

[0109] By employing a smaller second depth, the probability of oil seal damage during fluid manipulation and the likelihood of bubble formation can be reduced, thereby improving the efficiency of sealing micropores.

[0110] In this embodiment, a hydrophobic layer is provided on the surfaces of the first fluid channel structure 110, the second fluid channel structure 120, and the microporous structure 210. The hydrophobic layer is made of linear polydimethylsiloxane molecules. Therefore, the chip material, surface treatment, chip structure, and reagents used in this microfluidic chip are compatible with signal amplification methods for digital protein and nucleic acid detection, enabling it to be used for protein detection with room temperature incubation, and also to withstand PCR reactions requiring dozens of thermal cycles. This makes this microfluidic chip a universal detection device for proteins and nucleic acids.

[0111] Example 2

[0112] This invention also provides a method for fabricating a microfluidic chip, applicable to the microfluidic chip described above, comprising the following steps:

[0113] S10: A first fluid channel structure 110 and a second fluid channel structure 120 are etched on the upper chip 100. The first fluid channel structure 110 has a first depth of 50 μm and the second channel structure has a second depth of 10 μm, or the first fluid channel structure 110 has a first depth of 100 μm and the second channel structure has a second depth of 50 μm, or the first fluid channel structure 110 has a first depth of 200 μm and the second channel structure has a second depth of 175 μm.

[0114] S20: Micro-hole structure 210 is etched on the lower chip 200;

[0115] Specifically, step S10 includes:

[0116] S11: A first etched area for removing the protective layer is formed on the upper chip 100 covered with a protective layer. The first etched area corresponds to the outline of the first fluid channel structure 110 and the first etched area can be etched.

[0117] S12: Etch the first etched area until the etch depth of the first etched area reaches the difference between the first depth and the second depth;

[0118] S13: A second etched area for removing the protective layer is formed on the upper chip 100 covered with a protective layer. The second etched area corresponds to the outline of the second fluid channel structure 120 and can be etched.

[0119] S14: Etch the first etched area and the second etched area simultaneously until the etch depth of the first etched area and the second etched area reaches the second depth.

[0120] By using the above-described preparation method, a first fluid channel structure 110 and a second fluid channel structure 120 having a first depth and a second depth, respectively, can be fabricated on the upper chip 100.

[0121] In step S11, the protective layer includes a chromium layer and a photoresist layer sequentially deposited on the upper chip 100. The step of preparing the first etched area specifically includes:

[0122] The chip structure was designed using AutoCAD software, and a film mask corresponding to the contour of the first fluid channel structure 110 was fabricated. The mask was placed on top of the upper chip 100, which had been coated with a chromium layer and a photoresist layer, and exposed to parallel ultraviolet light in a photolithography machine.

[0123] The exposed glass was immersed in a 0.1 mol / L sodium hydroxide solution for 1 minute to remove the photoresist that reacts with ultraviolet light;

[0124] Subsequently, the glass was transferred to a chromium removal solution for 1 minute to remove the exposed chromium layer after the photoresist was removed. The chromium removal solution contained 0.6 mol / L perchloric acid and 0.365 mol / L cerium ammonium nitrate aqueous solution.

[0125] The treated glass is thoroughly rinsed with deionized water and air-dried to obtain the first etched area after the chromium layer and photoresist layer are removed.

[0126] In step S12, the step of etching the first etched area specifically includes:

[0127] The upper chip 100, which has undergone step S11, is placed in a constant temperature glass etching solution at 40°C for etching, and the shaker is kept shaking at a rate of 50 rpm. The glass etching solution contains 1 mol / L hydrofluoric acid, 0.5 mol / L ammonium fluoride and 0.75 mol / L nitric acid solution, and the etching rate is approximately 1.5 to 2 μm / min.

[0128] The depth of the flow channel is characterized by a step gauge and the etching time is adjusted. Once the etching depth reaches the difference between the first and second depths, the chip is removed.

[0129] In step S13, the fabrication process of the second etched region specifically includes:

[0130] A second mask with the outline of the second fluid channel structure 120 is placed above the upper chip 100, and the matching marks on the mask and the matching marks on the chrome plate are observed under a microscope.

[0131] Expose the glass again under parallel ultraviolet light, then immerse the exposed glass in a 0.1 mol / L sodium hydroxide solution for 1 minute to remove the photoresist that reacts with the ultraviolet light, temporarily retaining the chromium layer;

[0132] The treated glass is thoroughly rinsed with deionized water and then air-dried.

[0133] After step S12 is completed, the upper chip 100 is removed, cleaned with deionized water, and placed in a chromium removal solution to remove the chromium layer exposed by the second exposure, thereby obtaining the second etched area.

[0134] Specifically, step S14 includes:

[0135] The upper chip 100, which has undergone step S13, is then immersed in the glass etching solution for etching until the etching depth reaches the second depth.

[0136] Specifically, step S20 of fabricating the lower chip 200 includes:

[0137] The chip structure was designed using AutoCAD software, and a chromium mask corresponding to the micropore structure 210 was fabricated.

[0138] A 2-3µm thick photoresist is spin-coated onto the lower chip 200. Then, the pattern on the chromium mask is transferred onto the photoresist using ultraviolet lithography. The design of a single chip in the mask contains four parallel micro-hole arrays, with a total of 281,200 micro-holes in a single array. Each micro-hole has a diameter of 4.25µm and a center-to-center distance of 10.25µm between the micro-holes.

[0139] Inductively coupled reactive ion etching (ICD) is used to etch the photolithographically patterned substrate. The main etching gases used are CHF3 and Ar. The etching rate is about 350 nm / min, the etching time is about 10 min, and the etching depth is about 3.5 μm. A micro-hole array with a diameter of 4.25 μm and a depth of 3.5 μm is thus fabricated with a depth tolerance of < ±0.3 μm.

[0140] The residual photoresist on the lower chip 200 is removed by combining acetone wet stripping and plasma dry stripping. The substrate is then diced into unit chips using mechanical cutting equipment.

[0141] In this embodiment, the method further includes the following after step S20:

[0142] S30: Prepare a hydrophobic layer on the etched upper chip 100 and lower chip 200.

[0143] Specifically, step S30 includes:

[0144] Thoroughly clean the glass surface with deionized water and allow it to dry.

[0145] The chip was placed in a plasma cleaner for surface plasma cleaning and activation. The plasma cleaner used air as the generating gas, with a power of about 150w and a pressure of about 50Pa. The processing time was 2 minutes. Then the glass chip was quickly removed and placed in a plastic petri dish containing 50μL of dichlorodimethylsilane for vapor deposition. This step required a reaction at room temperature for 1 hour.

[0146] After the reaction is complete, rinse the chip surface with chloroform, acetone and ethanol, and let it air dry for later use.

[0147] Example 3

[0148] This invention also provides a method for measuring protein molecules, applicable to microfluidic chips as described above, wherein the first reactant 300 includes magnetic microspheres 500 bound to the analyte molecule, and the second reactant 400 includes a signal precursor. The magnetic microspheres 500, upon binding to the signal precursor, can generate a detectable signal. This molecular measurement method includes the following steps:

[0149] S100: Assemble the upper chip 100 and the lower chip 200, and set the lower surface of the upper chip 100 to be in contact with the upper surface of the lower chip 200;

[0150] Specifically, in this invention, the microfluidic chip is immersed in 0.05% Triton X-100 solution for cleaning, then transferred to ultrapure water, the chip is disassembled, the container is gently shaken to replace the liquid inside the chip with ultrapure water, and then the upper chip 100 and the lower chip 200 are assembled again in the aqueous phase. After the assembly is completed, the chip is removed, and the relative positions of the upper and lower chips 200 are fixed with dovetail clips to complete the chip assembly.

[0151] S200: Slide the upper chip 100 and the lower chip 200 relative to each other so that the vertical projection of the first fluid channel structure 110 coincides with the micropore structure 210;

[0152] S300: Combined Figure 3 Magnetic microspheres 500 are injected into the first fluid channel structure 110, causing the magnetic microspheres 500 to settle in the microporous structure 210.

[0153] Combination Figure 5 Specifically, in this invention, after injecting 500 magnetic microspheres, it is necessary to let it stand for 2 minutes to allow the microspheres to settle into the micropore array below;

[0154] S400: Combined Figure 4 A first fluorocarbon compound is injected into the first fluid channel structure 110 to seal the microporous structure 210.

[0155] S500: Combined Figure 6 A signal precursor is injected into the second fluid channel structure 120. The upper chip 100 and the lower chip 200 are slid relative to each other so that the second fluid channel structure 120 coincides with the microporous structure 210, thereby allowing the signal precursor to enter the microporous structure 210 and combine with the magnetic microspheres 500 to generate a detectable signal.

[0156] S600: Injecting a second fluorocarbon compound into the second fluid channel structure 120 to seal the microporous structure 210 for a second time;

[0157] Specifically, in this invention, after sealing the micropores, it is necessary to react at room temperature for 3 minutes to allow the signal precursor and magnetic microspheres 500 to react fully;

[0158] S700: Combined Figure 7 The signal emitted by the magnetic microspheres 500 is detected, and the concentration of the analyte is calculated based on the signal.

[0159] In this embodiment, the signal precursor includes a fluorescent substrate, and the magnetic microsphere 500, after binding to the analyte molecule, also binds to an enzyme capable of cleaving the fluorescent substrate to generate a fluorescent signal.

[0160] In this embodiment, the fluorescent substrate is halogen-β-D-galactopyranoside, and the enzyme is β-galactosidase.

[0161] In this embodiment, the molecule to be tested includes a protein molecule that binds to magnetic microspheres 500 modified with capture antibodies.

[0162] In this embodiment, the magnetic microspheres 500, which combine the target molecule and the enzyme, are prepared by the following method:

[0163] 1. Dilute the test samples to different concentrations using sample dilution buffer (10mM PBST + 1% sodium caseinate, pH = 7.4, 0.05% Tween-20), dilute the microspheres coated with the capture antibody to 2*10^7 / mL, dilute the biotinylated detection antibody to 0.25μg / mL, and dilute the streptavidin-β-galactosidase conjugate to 120pM;

[0164] 2. Shake the diluted coding microsphere working solution for 30 seconds to mix and sonicate for 2 minutes. Add 25 μL of the working solution containing microspheres to a 2 mL centrifuge tube, add 100 μL of the protein sample to be tested, and then add 20 μL of the detection antibody dilution solution. Mix well and place on a rotary reactor. Rotate the reactor at 37°C for 30 minutes.

[0165] 3. Place the centrifuge tube in a desktop centrifuge and centrifuge for 5 seconds. Centrifuge the liquid on the cap of the centrifuge tube to the bottom of the centrifuge tube, place it on a magnetic rack, and let it magnetically adhere for 2 minutes. Then remove the supernatant.

[0166] 4. Add 400 μL of washing buffer (10 mM PBST, pH 7.4, 0.05% Tween-20), vortex for 30 seconds to mix, centrifuge for 5 seconds, place on a magnetic rack, magnetically adsorb for 2 minutes, remove the supernatant, and repeat this operation 4 times.

[0167] 5. Add 100 μL of SβG diluent, mix well, and place on a rotary reactor. Rotate the reactor at 37°C for 10 minutes.

[0168] 6. Place the centrifuge tube in a desktop centrifuge and centrifuge for 5 seconds. Centrifuge the liquid on the cap of the centrifuge tube to the bottom of the centrifuge tube, place it on a magnetic rack, and let it magnetically adhere for 2 minutes. Then remove the supernatant.

[0169] 7. Add 400 μL of washing solution, vortex for 30 seconds to mix, centrifuge for 5 seconds, place on a magnetic rack, magnetically adsorb for 2 minutes, remove the supernatant, and repeat this operation 4 times;

[0170] 8. Add 5 μL of washing buffer, pipette 5 times, resuspend and mix the prepared immune complex microspheres.

[0171] In this embodiment, the detection method for the molecule to be tested in S700 specifically includes the following steps:

[0172] The fluorescence positive signal of the magnetic microspheres 500 is read, and the concentration of the analyte is calculated based on the proportion of the fluorescence positive signal in the micropore structure 210, the Poisson distribution data of the analyte on the magnetic microspheres 500, and the amount of magnetic microspheres 500 added.

[0173] In this embodiment, the first fluorocarbon compound is a mixture of FC-40 and Novec 7100, with a volume ratio of FC-40 to Novec 7100 of 1:4. This mixture has a high surface energy, which can effectively repel excess magnetic microspheres 500, seal micropores without affecting the magnetic microspheres 500 and the solution inside the micropores. Simultaneously, it can be completely removed from above the microporous structure 210 as the sliding process proceeds, without remaining on the surface of the lower chip 200 or leaking into the adjacent second fluid channel structure 120.

[0174] In this embodiment, the second fluorocarbon compound is FC-40. This compound has lower volatility than the first fluorocarbon compound, ensuring that the micropore array remains closed during signal amplification and preventing exposure of the micropores due to the evaporation and drying of the fluorocarbon compound.

[0175] Example 4

[0176] This embodiment provides a nucleic acid molecular detection method, including the following steps:

[0177] S1000: Assemble the microfluidic chip so that the first fluid channel structure 110 and the second fluid channel structure 120 on the upper chip 100 and the microporous structure 210 on the lower chip 200 are filled with liquid phase. Specifically, in this embodiment, the upper and lower chips are first immersed in a 0.5% wt Triton X-100 solution. The microstructure-containing parts of the upper and lower chips are joined together in the solution. Then, they are transferred to ultrapure water, the chips are disassembled, and the Triton in the chips is fully replaced. Then, the structure-containing parts of the chips are joined together again, and the chips are transferred to an 80% wt glycerol solution. The chips are disassembled, and the ultrapure water is fully replaced with glycerol solution. Then, the chips are joined together again.

[0178] S2000: The upper chip 100 and the lower chip 200 are slid relative to each other so that the first fluid channel structure 110 coincides with the microporous structure 210;

[0179] S3000: PCR reaction solution is injected into the first fluid channel structure 110, and then a first fluorocarbon compound is injected into the first fluid channel structure to seal the micropore. The first fluorocarbon compound is FC-40.

[0180] S4000: Inject a perfluoropolyether into the first fluid channel structure 110 to replace the first fluorocarbon compound. Specifically, the perfluoropolyether is polyperfluoromethyl isopropyl ether (CAS No.: 69991-67-9) with a molar mass of 1800g.

[0181] S5000: Place the microfluidic chip on a flat-panel PCR instrument for PCR amplification;

[0182] S6000: The nucleic acid concentration is calculated based on the fluorescence signal in the microporous structure 210. Specifically, in this embodiment, after amplification, a fluorescence microscope is used to perform microscopic imaging of the fluorescence signal of the FAM channel in the micropore. Since the distribution of nucleic acid molecules in the micropore conforms to the Poisson distribution, the nucleic acid in the original sample can be absolutely quantitatively analyzed by counting the number of positive micropores to negative micropores.

[0183] The sequence and primer information used for PCR amplification are shown in Table 1; the PCR reaction system is shown in Table 2; and the PCR reaction conditions are shown in Table 3. The DNA template was the BK virus genome.

[0184] Table 1 Primers and probes

[0185]

[0186] Table 2 PCR reaction system

[0187] Components Volume (total volume 100μL) ExTaq(Takara,RR001A) 2μL 10x ExTaq Buffer(Takara,RR001A) 10μL 2.5mM dNTP 8μL 10μM forward primer 10μL 10μM reverse primer 10μL 10μM hydrolysis probe 10μL 20 mg / mL BSA solution 5μL Ultrapure water 40μL template 5μL

[0188] The PCR reaction conditions are shown in the table below:

[0189]

[0190] Example 1

[0191] This invention characterizes the magnetic bead loading performance of a microporous array chip by analyzing the micropore utilization rate (number of micropores per magnetic bead / total number of micropores) under different magnetic bead loading conditions. After loading, the loading state of the magnetic beads is observed using a fluorescence microscope. The positioning of the magnetic beads can be achieved by detecting the green fluorescence signal emitted by FITC molecules modified on the surface of the magnetic beads. By statistically analyzing the number of magnetic beads loaded on the chip under different conditions, the magnetic bead loading performance of the sliding microporous array chip can be evaluated.

[0192] Combination Figure 9 In this invention, the amount of magnetic beads used in each reaction is 62,500, 125,000, 250,000, 500,000, and 1,000,000, respectively. It is evident that as the amount of magnetic beads increases, the utilization rate of the chip micropores significantly improves. When the amount of magnetic beads reaches 1,000,000, the micropore utilization rate reaches 90%, which is significantly better than similar technologies (5-10%).

[0193] Example 2

[0194] This invention analyzes the background signal intensity under two different sample loading methods: single loading and double loading, and systematically characterizes the effects of different loading methods. The tests include measuring the background signal intensity in the microporous structure under different amounts of magnetic beads, antigen, and magnetic bead settling times.

[0195] Test structure as follows Figure 10 As shown in the figure above, with the increase of magnetic beads and antigen input, the background signal of the single-sample loading group is significantly enhanced, significantly higher than that of the double-sample loading group. Under different magnetic bead settling times, the background signal of the single-sample loading group is significantly higher than that of the double-sample loading group. This result shows that the unique double-sample loading workflow of the sliding microporous array chip can suppress the influence of pre-catalysis on the reaction signal-to-noise ratio, and can become a potential technical solution to eliminate false positive signals in multi-index detection.

[0196] Example 3

[0197] The quantitative capability of digital PCR based on a microarray chip was evaluated by testing DNA samples at different dilutions. This experiment tested DNA samples diluted 1, 4, 16, and 64 times, which were then injected into the chip for PCR amplification. Figure 11 As shown, A and D represent the digital PCR results at different dilution factors. The number of positive wells decreases significantly with increasing dilution factor. Figure 12 As shown, there is a good linear relationship between the dilution factor and the measured DNA concentration.

[0198] Example 4

[0199] like Figure 13 This invention measured the signal values ​​of interleukin-6 standard samples at different concentrations. Specifically, interleukin-6 standard samples with concentrations of 0.05, 0.25, 1.25, 6.25, and 31.25 pg / mL, as well as a negative control group, were tested. Each concentration was replicated in triplicate. The limit of detection (LoD) and the limit of quantitation (LoQ) were the sum of the average background signal of the negative control group and 3 times and 10 times the standard deviation, respectively. Thus, the LoD of this system was calculated to be 5.3 pg / mL, and the LoQ was calculated to be 15.2 pg / mL.

[0200] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A microfluidic chip comprising an upper chip and a lower chip, a lower surface of the upper chip and an upper surface of the lower chip being capable of abutting, and the upper chip and the lower chip being capable of sliding relative to each other along the abutting surfaces, characterized in that: The lower surface of the upper chip is provided with a first fluid channel structure for loading a first reactant and a second fluid channel structure for loading a second reactant, and the upper surface of the lower chip is provided with a micropore structure, and the vertical projection profile of the first fluid channel structure and the second fluid channel structure can coincide with the profile of the micropore structure when the upper chip and the lower chip slide relative to each other. The micropore structure comprises a plurality of micropores, and the first reactant comprises magnetic microspheres combined with the to-be-detected molecules, and the micropores can be used to accommodate a single magnetic microsphere.

2. The microfluidic chip of claim 1, wherein: The micropore has a diameter of 1-100 μm and a depth of 1-100 μm, and / or the number of micropores is 10,000-1,000,000.

3. The microfluidic chip of claim 1, wherein: The first fluid channel structure has a first depth, and the second fluid channel structure has a second depth smaller than the first depth.

4. The microfluidic chip of claim 3, wherein: The first depth is 10-200 μm, and the second depth is 10-200 μm.

5. The microfluidic chip of claim 4, wherein: The first depth is 175 μm, and the second depth is 50 μm.

6. The microfluidic chip according to any one of claims 1 to 5, wherein: The surface of the first fluid channel structure, the second fluid channel structure and the micropore structure is provided with a hydrophobic layer.

7. The microfluidic chip of claim 6, wherein: The material of the hydrophobic layer is linear polydimethylsiloxane molecules.

8. A method for manufacturing a microfluidic chip according to any one of claims 1 to 7, characterized in that, The preparation method comprises the following steps: S10: etching the first fluid channel structure and the second fluid channel structure on the upper chip, wherein the first fluid channel structure has a first depth, and the second fluid channel structure has a second depth, and the first depth is greater than the second depth; S20: etching the micropore structure on the lower chip; S11: removing the first etching area of the protective layer on the chip covered with the protective layer, and the first etching area corresponds to the profile of the first fluid channel structure; S12: etching the first etching area until the etching depth of the first etching area reaches the difference between the first depth and the second depth; S13: removing the second etching area of the protective layer on the upper chip covered with the protective layer, and the second etching area corresponds to the profile of the second fluid channel structure; S14: further etching the first etching area and the second etching area until the etching depth of the first etching area reaches the first depth, and the etching depth of the second etching area reaches the second depth. After step S20, it further comprises:

9. The method for fabricating a microfluidic chip as described in claim 8, characterized in that, S30: preparing a hydrophobic layer on the etched upper chip and lower chip. The microfluidic chip as claimed in any one of claims 1-7 is used to quantitatively detect to-be-detected molecules.

10. A method of molecular measurement, characterized by, The to-be-detected molecules are proteins or nucleic acids.

11. The method of molecular measurement of claim 10, wherein, When the to-be-detected molecules are proteins, the first reactant comprises magnetic microspheres combined with the to-be-detected molecules, and the second reactant comprises a signal precursor, and the magnetic microspheres can generate a detectable signal after being combined with the signal precursor, and the molecular measurement method comprises the following steps:

12. The method of molecular measurement of claim 11, wherein, S100: assembling the upper chip and the lower chip, and arranging the lower surface of the upper chip to be attached to the upper surface of the lower chip; ​ S200: relatively sliding the upper chip and the lower chip to make the projection of the first fluid channel structure in the vertical direction coincide with the microwell structure; S300: injecting the magnetic microspheres into the first fluid channel structure to make the magnetic microspheres settle in the microwell structure; S400: injecting a first fluorocarbon compound into the first fluid channel structure to seal the microwell structure; S500: injecting a signal precursor into the second fluid channel structure, relatively sliding the upper chip and the lower chip to make the second fluid channel structure coincide with the microwell structure, so that the signal precursor enters the microwell structure, and combines with the magnetic microspheres to generate a detectable signal; S600: injecting a second fluorocarbon compound into the second fluid channel structure to seal the microwell structure again; S700: detecting the signal generated by the magnetic microspheres, and calculating the concentration of the to-be-detected molecules according to the signal.

13. The method of molecular measurement of claim 12, wherein: The signal precursor includes a fluorescent substrate, the magnetic microspheres further combine with an enzyme capable of cutting the fluorescent substrate to generate a fluorescent signal after combining with the to-be-detected molecules, and / or the to-be-detected molecules combine with the magnetic microspheres modified with a capture antibody.

14. The method of molecular measurement of claim 13, wherein: The fluorescent substrate is resorcinol-β-D-galactopyranoside, and the enzyme is β-galactosidase.

15. The method of molecular measurement of claim 12, wherein, Step S700 includes: reading the fluorescent positive signal of the magnetic microspheres, and calculating the concentration of the to-be-detected molecules according to the proportion of the microwells with fluorescent positive signals in the microwell structure, the Poisson distribution data of the to-be-detected molecules on the magnetic microspheres, and the amount of the magnetic microspheres.

16. The method of molecular measurement of claim 12, wherein: The first fluorocarbon compound is a mixture of FC-40 and Novec 7100, the volume ratio of FC-40 and Novec 7100 is 1:4, and the second fluorocarbon compound is FC-40.

17. The method of molecular measurement of claim 12, wherein: The second fluorocarbon compound is FC-40.

18. The method of molecular measurement of claim 12, wherein, When the to-be-detected molecules are nucleic acids, the following steps are included: S1000: assembling a microfluidic chip, so that the first fluid channel structure, the second fluid channel structure on the upper chip, and the microwell structure on the lower chip are filled with a liquid phase; S2000: relatively sliding the upper chip and the lower chip to make the first fluid channel structure coincide with the microwell structure; S3000: injecting a PCR reaction solution into the first fluid channel structure, and then injecting a first fluorocarbon compound into the first fluid channel structure to seal the microwells; S4000: injecting a perfluoropolyether into the first fluid channel structure to replace the first fluorocarbon compound; S5000: performing PCR amplification on the microfluidic chip; S6000: calculating the concentration of nucleic acids according to the fluorescent signal in the microwell structure.

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