Biological molecule detection system, array plate and method based on liquid drop PCR (Polymerase Chain Reaction) technology

Abstract

The invention discloses a biomolecule detection system, array plate and method based on a droplet PCR (Polymerase Chain Reaction) technology. The biomolecule detection system comprises a liquid drop generation device and an array plate, a substrate of the array plate is provided with a plurality of reaction surfaces which are distributed in an array and are separated from one another, and each reaction surface provides an area for a liquid drop PCR (Polymerase Chain Reaction) reaction; a specific probe is arranged on the reaction surface, and the specific probe is used for being specifically combined with a to-be-detected target biomolecule or a marker connected with the to-be-detected target biomolecule; the liquid drop generation device is used for generating liquid drops containing to-be-detected target biomolecules or connected with markers and amplification primers of the to-be-detected target biomolecules and applying the liquid drops to the reaction surfaces of the array plate. The array plate of the biomolecule detection system can provide a specific probe for droplet PCR detection, specific detection of different targets (such as different proteins and different nucleic acid sites) can be conveniently realized, the detection cost of multiple detection is reduced, and the detection time is shortened.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection based on droplet PCR technology, and relates to a biomolecular detection array plate and system based on droplet PCR technology, as well as a protein detection method and a nucleic acid detection method based on droplet PCR technology. Background Technology

[0002] Polymerase chain reaction (PCR) technology is one of the most important tools in modern biology, widely used in medical diagnostics, personalized medicine, food inspection, transgenic detection, pathogen identification, immunoassay, and forensic science. Droplet PCR is based on water-in-oil droplet technology. It encapsulates a small number of DNA molecules into individual microdroplets using a water-in-oil structure. The inertness of the oil isolates the DNA molecules, confining them to their own droplets for amplification, avoiding competition from other sequences. After DNA amplification under suitable temperature conditions, the total number of droplets and the number of droplets with detectable fluorescence signals can be recorded. Using the Poisson distribution algorithm, precise quantification of DNA copy number can be achieved (droplet digital PCR). Alternatively, each droplet can be observed as an independent reaction unit to monitor its real-time fluorescence amplification process, thus achieving quantification (droplet real-time quantitative PCR).

[0003] Existing droplet digital PCR detection systems and methods typically involve forming droplets from a sample and a PCR amplification system containing amplifying enzymes, primers, and probes using a droplet generation system. These droplets are then loaded onto a chip, and amplification and detection (e.g., fluorescence detection) are performed in a PCR instrument. Using this traditional method, specific probes are contained within the droplets. To specifically detect different targets (such as different proteins or different nucleic acid sites), different PCR amplification systems need to be constructed, and the droplet generation system changed to generate droplets containing different specific probes. This approach fails to achieve true multiplexing, is inconvenient, has poor operability, is costly, and has a long detection time.

[0004] In addition, existing technologies mainly use high-throughput mass spectrometry for protein metabolism-related detection research, which has shortcomings such as limited sensitivity, low specificity, limited detection dynamic range, low throughput, large sample consumption, and high cost. It also has great limitations when detecting plasma and similar samples.

[0005] The information disclosed in the background section is only intended to enhance the understanding of 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

[0006] One objective of this invention is to provide a biomolecular detection system based on droplet PCR technology, wherein the array plate can provide specific probes for droplet PCR detection, facilitating the specific detection of different targets (such as different proteins or different nucleic acid sites), reducing the detection cost of multiplex detection, and shortening the detection time. Another objective of this invention is to provide a biomolecular detection array plate based on droplet PCR technology.

[0007] Another objective of this invention is to provide a protein detection method based on droplet PCR technology, which can achieve highly sensitive, high-throughput, and highly specific protein detection with minimal sample requirements, convenient multiplex detection, and applicability to the detection of various trace proteins in blood samples.

[0008] Another objective of this invention is to provide a nucleic acid detection method based on droplet PCR technology, which can conveniently perform multiplex detection on multiple nucleic acid sites, and the multiplex detection is low in cost and short in time.

[0009] One aspect of the present invention provides a biomolecule detection system based on droplet PCR technology, including a droplet generation device. The biomolecule detection system further includes an array plate, the array plate including a substrate, the substrate having a plurality of reaction surfaces arranged in an array and spaced apart from each other, each of the reaction surfaces providing an area for droplet PCR reaction to be performed; the array plate further includes specific probes disposed on the reaction surfaces, the specific probes being used to specifically bind to a target biomolecule to be tested or a marker attached to the target biomolecule to be tested (for ease of description, both are collectively referred to as "analyte");

[0010] The droplet generating device is used to generate droplets containing the target biomolecule or a marker or amplification primer connected to the target biomolecule and apply them to each of the reaction surfaces of the array plate.

[0011] In a preferred embodiment, the specific probe is attached to the reaction surface in the form of a lyophilized body.

[0012] In a preferred embodiment, the reaction surface is a hydrophilic surface, and the array plate further includes a hydrophobic layer patterned on the surface of the substrate to space the plurality of reaction surfaces apart.

[0013] In a more preferred embodiment, the hydrophobic layer is coated on the surface of the substrate, and the width of the hydrophobic layer is 0.3 to 0.5 mm.

[0014] In a more preferred embodiment, the upper surface of the substrate is a hydrophilic surface, and the hydrophobic layer is patterned on the upper surface to form the plurality of reactive surfaces separated and enclosed by the hydrophobic layer.

[0015] In a preferred embodiment, the surface of the substrate and the reaction surface are both planar.

[0016] In a preferred embodiment, the surface of the substrate is provided with a plurality of micropores, the walls of the micropores forming the reaction surface; the hydrophobic layer is coated on the portion of the upper surface where the micropores are not formed.

[0017] In a further preferred embodiment, the depth of the micropore is less than or equal to 0.3 mm.

[0018] In a further preferred embodiment, the volume of the micropores is less than 2.5 mL. More preferably, the volume of the micropores is less than 2.1 mL.

[0019] In a further preferred embodiment, the micropores are arranged in an array; and / or, the number of micropores on each substrate is 10 to 400.

[0020] In a preferred embodiment, the array plate includes one, two or more of the aforementioned specific probes, and the specific probes disposed on the plurality of reaction surfaces are the same, different, or partially the same.

[0021] In a preferred embodiment, the reaction surface is planar, with a width of 0.1–0.4 mm, a length of 1–17 mm, and an area of ​​less than or equal to 8 mm². 2 More preferably, the length of the reaction surface is 1 to 2 mm.

[0022] In a preferred embodiment, the dimensions of each of the reaction planes may be the same or different.

[0023] In a preferred embodiment, the number of reaction surfaces on each substrate is 10 to 400; and / or the array distribution is a square array distribution or a ring array distribution or a combination of both.

[0024] In a preferred embodiment, the target biomolecule to be tested is a protein or nucleic acid, and the array plate includes 10 to 400 specific probes.

[0025] In a preferred embodiment, the biomolecule includes a protein, and at least a few of the plurality of reaction surfaces are provided with different specific probes to detect multiple proteins; the analyte contained in the droplet is a PCR pre-amplification product of an antigen-antibody hybridization product of the sample to be tested, the primer contained in the droplet is a universal primer, and the volume of the droplet is 1-20 nL, preferably 5-10 nL.

[0026] In a preferred embodiment, the biomolecule includes nucleic acid, and at least a few of the plurality of reaction surfaces are provided with different specific probes to detect multiple nucleic acid sites; the analyte contained in the droplet is a PCR pre-amplification product of the sample to be tested, the primer contained in the droplet is a universal primer, and the volume of the droplet is 1 to 20 nL, preferably 5 to 10 nL.

[0027] In a preferred embodiment, the droplet generating device includes a variable-volume receiving cavity, a control mechanism for controlling the volume of the receiving cavity to change periodically, and a droplet generating tube having a first port and a second port relatively far apart. The first port of the droplet generating tube communicates with the receiving cavity. The droplet generating device further includes a driving fluid mechanism for introducing driving fluid into the receiving cavity. The inner diameter of the second port of the droplet generating tube is 0.1 mm or more. The periodic change is a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery. The volume of the generated droplet is 1–20 nL, preferably 5–10 nL.

[0028] Preferably, the inner diameter of the second port is greater than 0.2 mm, specifically 0.2 mm to 1 mm. More preferably, the inner diameter of the second port is 0.3 to 0.6 mm. Even more preferably, the inner diameter of the first port is greater than the inner diameter of the second port. The droplet generating tube includes a tapered tube section, with the first port and the second port formed at both ends of the tapered tube section, respectively, and the taper of the tapered tube section is 0.05 to 0.2.

[0029] Furthermore, the volume of the droplet generating tube can be 10-200 microliters. Experiments show that the volume of the droplet generating tube has little impact on the droplet generation effect, which is one of the advantages of the droplet generation method of this invention.

[0030] According to a specific embodiment and preferred aspect of the invention, the periodic change is a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery.

[0031] According to a specific embodiment and preferred aspect of the present invention, the driving fluid mechanism includes a pump and a fluid passage, and the droplet generating device includes a substrate, the substrate providing cylindrical holes and fluid passages, and providing a connecting portion for connecting the liquid generating tube. On the same substrate, the number of cylindrical holes, fluid passages and connecting portions is one or more, the cylindrical holes are cylindrical with openings at both the top and bottom, and a diaphragm is provided on each cylindrical hole, the cylindrical holes and the diaphragm together forming a receiving cavity.

[0032] Preferably, the receiving cavity, the droplet generating tube, and the droplet receiver are arranged sequentially from top to bottom. The first port of the droplet generating tube is connected to the lower opening of the receiving cavity. The centerline of the receiving cavity, the axis of the droplet generating tube, the centerline of the first port, and the centerline of the second port coincide and all extend in the vertical direction.

[0033] Preferably, each diaphragm includes a main body and a movable part. The main body is fixedly connected to the substrate, the movable part is located directly above the cylindrical hole, and the movable part is connected to the control mechanism through a connector.

[0034] Furthermore, the diaphragm can be a metal (e.g., stainless steel) or a non-metallic diaphragm; the thickness of the diaphragm can be 5 micrometers to 2 millimeters.

[0035] Preferably, a sealing structure is provided between the diaphragm and the substrate to ensure that the cavity is always well sealed.

[0036] Preferably, the control mechanism is a vibration mechanism, which includes one or more of a galvanometer motor, a piezoelectric ceramic, and a voice coil motor, without particular limitation. According to a specific preferred aspect of the invention, the vibration mechanism preferably includes a piezoelectric ceramic.

[0037] Preferably, the vibration direction provided by the vibration mechanism is up and down, and the vibration frequency provided is at least 50-600Hz. In use, it can be specifically selected within this range, and the vibration amplitude provided is preferably at least 5 micrometers-300 micrometers.

[0038] Preferably, the droplet generating tube is detachably connected to the connecting part. This allows the droplet generating tube to be independent of the entire system and can be disassembled and replaced after each droplet generation of a sample solution. In some embodiments, the droplet generating tube can directly use a commercially available nozzle of the corresponding specification. Of course, it is also possible to integrate the droplet generating tube and the connecting part as one piece without affecting the droplet generation effect.

[0039] Preferably, on the same substrate, the number of each of the cylindrical holes, fluid passages, and connecting parts is 2 to 20, and more preferably 4 to 10.

[0040] Preferably, there are multiple cylindrical holes, fluid passages, and connecting parts. The two opposite sides of the base are respectively higher than the middle part between the two opposite sides. The multiple cylindrical holes are independently distributed in the middle part of the base and arranged in two rows. Each fluid passage includes a vertical passage formed on the two opposite sides of the base and a horizontal passage connecting the vertical passage to the corresponding cylindrical hole. This structural design makes the device more compact and convenient to operate and control.

[0041] According to a specific embodiment and preferred aspect of the invention, a drainage portion is formed between the port of the horizontal passage and the inner peripheral sidewall of the receiving cavity, so that liquid from the horizontal passage enters the receiving cavity tangentially to the circumference of the receiving cavity.

[0042] According to a specific embodiment and preferred aspect of the invention, one end of the fluid passage is in communication with the receiving cavity of the substrate, and when the fluid is driven from the fluid passage into the receiving cavity, the fluid forms a vortex that rotates circumferentially within the receiving cavity and the droplet generating tube.

[0043] According to another specific embodiment and preferred aspect of the invention, one end of the fluid passage is in communication with the receiving cavity of the substrate, and the direction in which the fluid is discharged from the fluid passage deviates from the axis of the receiving cavity.

[0044] Through the structural design of fluid pathways and containment cavities, it is possible to easily remove air bubbles from the system cavity before droplet generation, thus avoiding the adverse effects of air bubbles on droplet generation.

[0045] According to the present invention, the droplet receiver is a device having a receiving cavity, which can be hermetically sealed or non-hermetically sealed, without particular limitation.

[0046] Another aspect of the present invention provides a protein detection method based on droplet PCR technology for non-diagnostic and therapeutic purposes, wherein the biomolecular detection system is used to detect one protein or simultaneously detect multiple proteins, and the protein detection method includes:

[0047] For each target protein to be tested, a corresponding specific antibody is designed and linked to a specific oligonucleotide. Then, the specific antibody linked to the specific oligonucleotide is mixed with the sample to be tested and incubated to allow the antigen and antibody to hybridize and obtain the hybridization product. The oligonucleotide is a nucleotide with an amino acid length of less than 100 bp.

[0048] The hybridization product, amplification primers, and polymerase were mixed and subjected to PCR pre-amplification to obtain the pre-amplified product.

[0049] The pre-amplification product is formed into droplets by the droplet generation device and applied to the reaction surface of the array plate or droplets are generated directly on each of the reaction surfaces. On the reaction surface, the specific probe dissolves and mixes with the pre-amplification product to form an amplification system, wherein the specific probe specifically binds to the specific oligonucleotide.

[0050] The amplification reaction is performed, followed by signal acquisition.

[0051] In a preferred embodiment, a pair of specific antibodies is designed for each target protein to be tested. Each pair of specific antibodies is linked to a single-stranded oligonucleotide, and a segment of the two single-stranded oligonucleotides linked to the pair of specific antibodies can specifically bind to one end of each other. This specifically binding segment can specifically bind to the specific probe.

[0052] In a preferred embodiment, the length of the single-chain oligonucleotide linked to the antibody is 60-80 bp; and / or, the length of the fragments that the two single-chain oligonucleotides can specifically bind to is 10-15 bp.

[0053] In a preferred embodiment, the sequence at the other end of the single-stranded oligonucleotide is a universal sequence.

[0054] In a preferred embodiment, the method further includes: adding a fluorescent group to the amplification system and performing real-time quantitative fluorescence analysis on the amplification system.

[0055] In a preferred embodiment, the sample to be tested includes a blood sample.

[0056] Another aspect of the present invention provides a nucleic acid detection method based on droplet PCR technology for non-diagnostic and therapeutic purposes, employing the aforementioned biomolecular detection system. The nucleic acid detection method includes: forming droplets from a pre-amplification system containing a target nucleic acid, primers, and polymerase using the droplet generation device and applying them to the reaction surface of the array plate or directly generating droplets on each of the reaction surfaces; on the reaction surface, dissolving the specific probe and mixing it with the pre-amplification system to form an amplification system, wherein the specific probe specifically binds to the target nucleic acid; performing an amplification reaction; and subsequently acquiring a signal.

[0057] In a preferred embodiment, the method further includes performing real-time quantitative fluorescence analysis or digital PCR analysis on the amplification system.

[0058] In a preferred embodiment, the array plate includes multiple specific probes, and the method is for multiplex detection of different sites of nucleic acids.

[0059] Another aspect of the present invention provides an array plate as described above for the detection of biomolecular droplets by PCR.

[0060] The array plate includes a substrate having a plurality of reaction surfaces arranged in an array and spaced apart from each other, each reaction surface providing a region for droplet PCR reaction; the array plate also includes specific probes disposed on the reaction surfaces, the specific probes being used to specifically bind to the target biomolecule or a marker connected to the target biomolecule.

[0061] In a preferred embodiment, the specific probe is attached to the reaction surface in the form of a lyophilized body.

[0062] In a preferred embodiment, the reaction surface is a hydrophilic surface, and the biomolecule detection array plate further includes a hydrophobic layer, which is patterned on the surface of the substrate to space the plurality of reaction surfaces apart.

[0063] In a more preferred embodiment, the hydrophobic layer is coated on the surface of the substrate, and the width of the hydrophobic layer is 0.3 to 0.5 mm.

[0064] In a more preferred embodiment, the upper surface of the substrate is a hydrophilic surface, and the hydrophobic layer is patterned on the upper surface to form the plurality of reactive surfaces separated and enclosed by the hydrophobic layer.

[0065] In a preferred embodiment, the surface of the substrate and the reaction surface are both planar.

[0066] In a specific and preferred embodiment, the substrate has a plurality of micropores on its surface, the walls of the micropores forming the reaction surface; the hydrophobic layer is coated on the portion of the upper surface where no micropores are formed.

[0067] More preferably, the depth of the micropore is less than or equal to 0.3 mm.

[0068] In a preferred embodiment, the volume of the micropores is less than 2.5 mL. More preferably, the volume of the micropores is less than 2.1 mL.

[0069] In a preferred embodiment, the micropores are arranged in an array; and / or, the number of reaction surfaces on each substrate is 10 to 400.

[0070] In a further preferred embodiment, the reactive surface is a hydrophilic surface.

[0071] In a further preferred embodiment, the reaction surface is planar, with a width of 0.1–0.4 mm, a length of 1–17 mm, and an area of ​​less than or equal to 8 mm². 2 More preferably, the length of the reaction surface is 1 to 2 mm. The dimensions of each of the reaction planes may be the same or different.

[0072] In a further preferred embodiment, the array plate includes one, two or more of the aforementioned specific probes, wherein the specific probes disposed on the plurality of reaction surfaces are the same or different or partially the same.

[0073] The present invention adopts the above solution and has the following advantages:

[0074] The biomolecular detection system based on droplet PCR technology of the present invention places specific probes on the reaction surface of an array plate. The same or different specific probes can be placed on different reaction surfaces. The probes are provided by the array plate and do not need to be contained in droplets. Universal droplets and droplet generation devices can be used, making the detection process more convenient. In particular, it is very convenient for multiple detection of different targets (e.g., different proteins).

[0075] The protein detection method based on droplet PCR technology of the present invention pre-embeds a protein-specific probe on an array plate. The array plate receives droplets containing the analyte and universal primers, and the droplet PCR reaction is performed on the reaction surface of the array plate. This method can achieve highly sensitive, high-throughput, and highly specific protein detection with very small sample requirements. Multiplex detection is also convenient and applicable to the detection of various trace proteins in blood samples.

[0076] The nucleic acid detection method based on droplet PCR technology of the present invention pre-embeds specific probes for different nucleic acid sites on an array plate. The array plate receives droplets containing the analyte and universal primers, and performs droplet PCR reaction on the reaction surface of the array plate. This method can conveniently perform multiplex detection of multiple nucleic acid sites, and the multiplex detection is low in cost and short in time.

[0077] In a further preferred embodiment, compared with traditional PCR detection systems that use microfluidic chips or microplates as substrates, the biomolecular detection array plate of the present invention is easy to manufacture and can adopt a high-density array design and an open structure, which helps to reduce the amount of samples required and achieve high-sensitivity, high-throughput and high-specificity protein detection, and the detection is more accurate, reducing the cost of multiplex detection and shortening the time of multiplex detection. Attached Figure Description

[0078] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0079] Figure 1 This is a schematic diagram of a biomolecular detection system according to an embodiment of the present invention.

[0080] Figure 2 This is a top view of a biomolecular detection array plate according to an embodiment of the present invention.

[0081] Figure 3 This is a partial cross-sectional view of a biomolecular detection array plate according to an embodiment of the present invention. Detailed Implementation

[0082] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is for the purpose of aiding understanding the present invention, but does not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0083] This embodiment provides a biomolecular detection system based on droplet PCR technology and the consumables for the detection system. Figure 1 The biomolecule detection system is shown. Figures 2 to 3 The consumable is shown, specifically an array plate 2 for detecting biomolecules using droplet-based PCR technology. These biomolecules typically include proteins, nucleic acids, etc.

[0084] like Figure 1 As shown, the biomolecular detection system includes a droplet generating device 1 and an array plate 2. The array plate 2 is positioned below the droplet generating device 1 to receive droplets generated by the device. After the droplet addition is complete, the array plate 2 can be detected and observed. As the amplification reaction proceeds, fluorescence changes on the array plate 2 can be observed. The droplet volume is 1–20 nL, preferably 5–10 nL.

[0085] Droplet generating device 1:

[0086] The droplet generating device 1 includes a substrate 10 and a droplet generating unit 11 disposed on the substrate 10. The droplet generating unit 11 includes a variable volume receiving cavity 110, a control mechanism 111 for controlling the volume of the receiving cavity 110 to change periodically, a droplet generating tube 112 having a first port a1 and a second port a2 that are relatively far apart, and a driving fluid mechanism 113 for introducing driving fluid into the receiving cavity 110.

[0087] A cylindrical hole extending vertically is provided in the middle of the substrate 10, and a diaphragm 114 is covered on the cylindrical hole. The cylindrical hole and the corresponding diaphragm 114 together form a receiving cavity 110. The control mechanism 111 is a vibration mechanism, which is installed above the cylindrical hole and connected to the diaphragm 114 through a connector 115, thereby controlling the movement of the diaphragm 114 to implement the volume change of the receiving cavity 110. The droplet generating tube 112 is vertically arranged, with its upper end communicating with the connecting part 101 and its lower end forming a droplet outlet.

[0088] The centerline of the receiving cavity 110, the axis of the droplet generating tube 112, the centerline of the first port a1, and the centerline of the second port a2 coincide and all extend vertically. The receiving cavity 110 is an annular cavity with an inner diameter of approximately 5 mm, and its inner peripheral sidewalls extend vertically. The diaphragm 114 (made of stainless steel) constituting the top of the receiving cavity 110 includes a main body b1 and a movable part b2. The main body b1 is fixedly connected to the base 10, and the movable part b2 is located directly above the cylindrical hole and is connected to the control mechanism 111 via a connector 115. The control mechanism 111 specifically includes a piezoelectric ceramic that can provide reciprocating vibration. When it reciprocates, it drives the movable part b2 to move inward or outward relative to the receiving cavity 110, thereby periodically changing the volume of the receiving cavity 110. Correspondingly, the liquid in the receiving cavity 110 is disturbed due to this periodic volume change. Furthermore, the periodic change can be a compression-recovery reciprocating change, or an expansion-recovery reciprocating change, or a compression-recovery-expansion-recovery reciprocating change. In this example, at least the periodic change that can be provided includes a compression-recovery reciprocating change.

[0089] The droplet generating tube 112 includes a connecting tube section c1 and a tapered tube section c2. The tapered tube section c2 has a first port a1 and a second port a2. The inner diameter of the tapered tube section c2 gradually decreases from the first port a1 to the second port a2, and the taper of this inner diameter has a significant impact on the droplet generating effect. Let the inner diameter of the first port a1 be R1, the inner diameter of the second port a1 be R2, the distance between the first port a1 and the second port a2 (i.e., the length c1 of the tapered tube section) be L, and the taper be R1-R2 / L. In this example, the taper of the tapered tube section c2 is 0.12, and the inner diameter R2 of the second port a2 is 0.5±0.1 mm. The volume of the droplet generating tube 112 is approximately 10 microliters.

[0090] The connecting tube portion c1 and the tapered tube portion c2 intersect at the first port a1. The connecting tube portion c1 is detachably fitted onto the connecting portion 101. Its taper is not particularly required, but is preferably greater than that of the tapered tube portion c2. A flow channel t is formed in the middle of the connecting portion 101, communicating with the receiving cavity 110. After the droplet generating tube 112 is installed on the connecting portion 101, the receiving cavity 110 communicates with the droplet generating tube 112 through the flow channel t.

[0091] The driving fluid mechanism 113 includes a pump d1 and a fluid passage d2, wherein the fluid passage d2 includes a vertical passage d21 formed on the left protruding edge portion 10b of the base 10 and a horizontal passage d22 correspondingly communicating the vertical passage d21 with the cylindrical hole 100. To facilitate the removal of air bubbles from the system cavity, a drainage portion d3 is formed between the port of the horizontal passage d22 and the inner peripheral sidewall of the receiving cavity 110, allowing liquid from the horizontal passage d2 to enter the receiving cavity 110 tangentially in the circumferential direction. Thus, the fluid forms a vortex rotating circumferentially within the receiving cavity 110 and the droplet generating tube 112, thereby easily removing air bubbles. A sealing ring 116 is also provided between the diaphragm 114 and the base 10 to improve the sealing of the receiving cavity.

[0092] The array plate is located below the second port a2 and is used to contain the first liquid y1 and droplets. In the field of PCR, the first liquid y1 is usually a formulated oil, such as mineral oil, preferably with added surfactants. The second liquid y2 is usually an aqueous phase containing the biomolecule to be detected. Fluid y3 (driving oil) fills the fluid passage d2, the receiving cavity 110, and the inner cavity of the droplet generation tube 112. Further, fluid y3 and the first liquid y1 can use the same mineral oil. Typically, during droplet generation, the liquid in the droplet generation tube 112 is divided into a driving fluid section and a second liquid section from top to bottom. Alternatively, the liquid in the droplet generation tube 112 is divided into a driving fluid section, a second liquid section, and a first liquid section from top to bottom; then, the driving mechanism and the fluid driving mechanism are activated to begin droplet generation.

[0093] Array board 2:

[0094] Reference Figure 2 and Figure 3 As shown, the array plate 2 for detecting biomolecules includes a substrate 100. The substrate 100 is entirely made of a hydrophilic material; or the surface of the substrate 100 is treated to make it hydrophilic. The substrate 100 has multiple reaction surfaces 10a, each reaction surface 10a providing an area for droplet PCR reaction. The multiple reaction surfaces 10a are arranged in an array and spaced apart from each other, and each reaction surface 10a is a hydrophilic surface. The multiple reaction surfaces 10a are preferably arranged in an array and can be divided into multiple groups according to detection needs. For example, multiple reaction surfaces 10a in each row constitute a group, and multiple rows of reaction surfaces 10a constitute multiple groups of reaction surfaces 10a. The reaction surfaces 10a are distributed in an array, and the number of reaction surfaces 10a on each substrate 100 is 10 to 400.

[0095] The array plate 2 also includes lyophilized versions 200 of specific probes for droplet PCR detection. Specific probes are oligonucleotides that can specifically bind to target biomolecules or markers linked to target biomolecules, and have a specific nucleotide sequence. These specific probes are preferably fluorescent probes, with a fluorescent reporter group labeled at the 5' end and a fluorescent quencher group labeled at the 3' end. During PCR amplification, the probe is cleaved and degraded by an enzyme (e.g., Taq polymerase), separating the fluorescent reporter group and the fluorescent quencher group, thereby emitting fluorescence. The fluorescent reporter groups attached to different specific probes can be the same, different, or partially the same. The specific probes are attached to the reaction surface 10a in the form of lyophilized versions 200. The lyophilized versions 200 of the specific probes can be obtained directly by lyophilizing known specific probe lyophilization solutions. The specific probe lyophilization solutions and their lyophilization processes are not part of the inventive point of this application; known specific probe lyophilization solutions and processes can be directly utilized and will not be elaborated upon here.

[0096] The array plate 2 contains one, two, or more specific probes, and the specific probes disposed on multiple reaction surfaces 10a are the same, different, or partially the same. Preferably, the target biomolecule to be tested is a protein or nucleic acid, and the array plate 2 includes 10 to 400 specific probes.

[0097] The specific probe lyophilized bodies 200 disposed on several of the multiple reaction surfaces 10a may be the same, different, or partially the same; even, the specific probe lyophilized bodies 200 on each reaction surface 10a may be different. Specifically, in this embodiment, as described above, the multiple reaction surfaces 10a are divided into multiple groups, and the specific probe lyophilized bodies 200 on at least two groups of reaction surfaces 10a are different. The specific probe lyophilized bodies 200 on each reaction surface 10a within each group may be the same or different. For example, Figure 2 The specific probe lyophilized body 200 disposed on the reaction surface 10a in the first row is a specific probe lyophilized body 200 corresponding to the first test protein; Figure 2 The specific probe lyophilized body 200 disposed on the reaction surface 10a in the first row is a specific probe lyophilized body 200 corresponding to the second analyte protein. Thus, the first and second proteins can be specifically detected simultaneously based on the same droplet system.

[0098] The array plate 2 further includes a hydrophobic layer 300, which is patterned on the surface of the substrate 100 to space the plurality of reaction surfaces 10a. The hydrophobic layer 300 is coated on the upper surface of the substrate 100, and the width W2 of the hydrophobic layer 300 is 0.3–0.5 mm. In this embodiment, as... Figure 3As shown, a plurality of micropores 102 are provided on the upper surface of the substrate 100. The depth of the micropores 102 is less than or equal to 0.3 mm, and the walls of the micropores 102 constitute a reaction surface 10a. Specifically, the bottom wall of each micropore 102 forms a reaction surface 10a. A hydrophobic layer 300 is coated on the portion of the upper surface where no micropores 102 are formed (i.e., Figure 3 On the upper surface of the upward-protruding portion. In other embodiments, the upper surface of the substrate 100 is planar and entirely hydrophilic, without micropores 102, and a hydrophobic layer 300 is patternedly coated on the upper surface to form multiple reactive surfaces 10a separated and enclosed by the hydrophobic layer 300. This greatly simplifies the fabrication process of the array plate 2, making the array plate 2 easy to manufacture.

[0099] The micropores 102 are arranged in an array, and the volume of each micropore 102 is less than or equal to 2.5 mL. The number of micropores 102 on each substrate is 10 to 400.

[0100] Each reaction surface 10a is planar, and the area of ​​reaction surface 10a is (0.1~0.4)mm×(1~17)mm, with an area less than 8mm². 2 Among them, such as Figure 2 As shown, the length L of the reaction surface 10a is 1-2 mm and the width W1 is 0.1-0.4 mm.

[0101] Array plate 2 can be obtained by the following preparation method:

[0102] A substrate 100 is provided, and a plurality of micropores 102 arranged in an array are formed on the upper surface of the substrate 100. The bottom wall of each micropore 102 is a hydrophilic surface, i.e., a reaction surface 10a.

[0103] A hydrophobic material is coated on the portion (protrusion portion) of the upper surface of the substrate 100 where the micropores 102 are not formed, thereby forming a patterned hydrophobic layer 300, such that each micropore 102 is surrounded by the hydrophobic layer 300, separating it into an independent PCR reaction space.

[0104] Each reaction surface 10a has a specific probe freeze-dried body fixed on it by a freeze-drying process.

[0105] Array plate 2 can also be obtained by the following preparation method:

[0106] A substrate 100 is provided, the upper surface of which is a hydrophilic plane;

[0107] A hydrophobic material is patternedly coated on the upper surface of the substrate 100 to form a patterned hydrophobic layer 300. The upper surface of the substrate 100 is divided into a plurality of spaced-apart reaction surfaces 10a. Each reaction surface 10a is surrounded by the hydrophobic layer 300, forming an independent droplet PCR reaction space.

[0108] Each reaction surface 10a contains a specific probe lyophilized body fixed via a freeze-drying process. For example, one method involves: after adding a protective agent, the specific probe reagent is dispensed onto the reaction surface of the substrate, frozen into a solid state, and then sublimated under vacuum, leaving a structurally stable solid reagent, i.e., the lyophilized body. The sponge-like porous solid reagent can be rapidly thawed upon contact with a dissolving solution. Another method involves: ① quantitatively adding the specific probe reagent to liquid nitrogen for rapid freezing to form particles; rapid freezing better maintains the stability of its protein structure; ② transferring it to a freeze dryer, where water is sublimated under vacuum, resulting in uniform solid lyophilized microparticles; ③ quantitatively dispensing the lyophilized microparticles, according to the required quantity and type, onto the reaction surface of the substrate.

[0109] This biomolecular detection system is typically used for protein and nucleic acid detection. The methods for protein and nucleic acid detection are described in detail below.

[0110] Protein detection:

[0111] Traditional mass spectrometry-based protein detection techniques suffer from drawbacks such as limited sensitivity, low specificity, limited dynamic range, low throughput, high sample consumption, and high cost. They are particularly limited when detecting plasma and similar samples.

[0112] This embodiment utilizes macrofluidic microdroplet generation and microdroplet microarray technology based on droplet PCR to combine protein detection with fluorescent PCR, achieving highly sensitive, high-throughput, and highly specific protein detection with minimal sample requirements—only 6 µL of sample is needed to detect 3000 protein samples. For each protein detection, a pair of specifically matched antibodies (or polyclonal antibodies split in half) is designed to ensure the specificity of the immunological reaction. By adding a pair of paired nucleic acid single strands to each antibody, crosstalk at the immunological level is avoided by leveraging the specificity of DNA pairing. Then, a micro-PCR reaction device is used to quantitatively / semi-quantitatively detect the paired DNA double strands using specific probes. This achieves biologically ultrasensitive, unbiased targeted proteomics, particularly suitable for blood samples where traditional methods are inadequate.

[0113] This embodiment of the protein detection method based on droplet PCR technology is for non-diagnostic and non-therapeutic purposes. It employs the aforementioned biomolecular detection system to detect one protein or simultaneously detect multiple proteins. The protein detection method includes:

[0114] For each target protein to be tested, a corresponding specific antibody is designed and linked to a specific oligonucleotide. Then, the specific antibody linked to the specific oligonucleotide is mixed with the sample to be tested and incubated to allow the antigen and antibody to hybridize and obtain the hybridization product.

[0115] The hybridization product, amplification primers, and polymerase were mixed and subjected to PCR pre-amplification to obtain the pre-amplified product.

[0116] The pre-amplification product is formed into droplets by the droplet generation device and applied to the reaction surface of the array plate or droplets are generated directly on each of the reaction surfaces. On the reaction surface, the specific probe dissolves and mixes with the pre-amplification product to form an amplification system, wherein the specific probe specifically binds to the specific oligonucleotide.

[0117] The amplification reaction is performed, followed by signal acquisition.

[0118] The sample to be tested is typically a blood sample.

[0119] For each target protein to be tested, a pair of specific antibodies are designed. Each pair of specific antibodies is linked to a single-stranded oligonucleotide, and a segment near one end of the two single-stranded oligonucleotides linked to the pair of specific antibodies can specifically bind to each other, and this specifically binding segment can specifically bind to the specific probe.

[0120] The single-stranded oligonucleotide linked to the antibody is 60–80 bp in length. The fragments of the two single-stranded oligonucleotides that can specifically bind are 10–15 bp in length.

[0121] The sequence at the other end of the single-stranded oligonucleotide is a universal sequence.

[0122] The method further includes: adding a fluorescent group to the amplification system and performing real-time quantitative fluorescence analysis on the amplification system.

[0123] Nucleic acid amplification testing:

[0124] Nucleic acid site detection, typically using next-generation sequencing (NGS), plays an increasingly important role in the detection of tumors, genetic diseases, and pathogenic microorganisms. Traditional sequencing methods require steps such as extraction, library construction, library mixing, enrichment, sequencing, and data analysis, which are time-consuming and relatively costly.

[0125] This embodiment proposes an improved nucleic acid detection method based on droplet PCR technology for non-diagnostic and therapeutic purposes, employing the aforementioned biomolecular detection system. The nucleic acid detection method includes: forming droplets from a pre-amplification system containing the target nucleic acid, primers, and polymerase using the droplet generation device and applying them to the reaction surface of the array plate, or directly generating droplets on each of the reaction surfaces; on the reaction surface, the specific probe dissolves and mixes with the pre-amplification system to form an amplification system, wherein the specific probe specifically binds to the target nucleic acid; performing the amplification reaction; and subsequently acquiring the signal. This method offers advantages such as low cost and short detection time for different nucleic acid sites.

[0126] The method also includes performing real-time quantitative fluorescence analysis or digital PCR analysis on the amplification system.

[0127] The array plate includes multiple specific probes, and the method is a multiplex detection of different sites of nucleic acid.

[0128] The test sample can be plasma.

[0129] Application Example 1

[0130] 1. Antigen-antibody hybridization and product pre-amplification

[0131] 1.1 Based on the antigen (protein) to be tested, design two antibodies, each with a single-chain oligonucleotide (preferably 60-80 bp) attached. Approximately 10-15 bp at the ends of both oligonucleotides should specifically bind. That is, if binding occurs, the total length is approximately: 2 x single-chain length - specific binding length (preferably approximately 150 bp). The sequences at both ends must ensure universality, and the specific binding portion should be designed with different sequences (for probe design) for monitoring different antigens.

[0132] The basic structure is as follows:

[0133] ① Chain: Antibody—5'--AATTCAGA---------------CGCGATGCA--3'

[0134] ② Chain: 3'--GCGCTAGCT------------------------TATAGCTTGC--5'--antibody

[0135] 1.2. The designed pair of antibodies is added to the antigen and incubated to allow antigen-antibody hybridization. After hybridization, the two chains are close together and can undergo specific binding (CGCGATGCA, TATAGCTTGC parts).

[0136] 1.3 Design a universal primer to pre-amplify the bound product. It is important to note that the primers used for pre-amplification and subsequent PCR amplification must have the same 5' end portion as ② (AATTCAGA, TATAGCTTGC), and must not be complementary sequences. Otherwise, even if strands ① and ② do not bind, complementary strands of ① and ② will be amplified during pre-amplification (referred to as ①' and ②' strands). After reheating, the complementary strands of the two single strands will become free. At this point, ①' and ②' strands can also bind to each other, resulting in false positives.

[0137] To ensure consistent amplification efficiency and simplify the experiment during the pre-amplification stage, a combination of universal primers and a specific probe must be designed, rather than a combination of a universal probe and a specific primer. The universal primers are added to the antigen-antibody hybridization product, and different samples are placed in separate wells for PCR pre-amplification. The final product will consist of: one antibody-nucleic acid combination and several protein-free double-stranded nucleic acids.

[0138] 1.4. Add universal primers to the antigen-antibody hybridization product. For different samples, place them in different wells (wells of a 96-well plate for antigen-antibody hybridization) or tubes (8-tube PCR tubes for antigen-antibody hybridization) for PCR pre-amplification. The final PCR pre-amplification product will yield: one antibody-nucleic acid combination and several protein-free double-stranded nucleic acids.

[0139] 2. Array board design

[0140] Based on macrofluidic digital droplet PCR technology, 48 samples (the aforementioned PCR pre-amplification products) can be generated into droplets of a known size and quantity, which are then introduced into array plate 2. The ideal droplet size is 3–7 nanoliters, with an optimal size of 5.6 nanoliters (210 micrometers in diameter). Array plate 2 can contain 200–300 microwells 102, each containing a pre-prepared lyophilized specific probe 200, representing a single target site. The ideal hydrophilic area is (0.2–0.4) mm × (1–2) mm, with an optimal area of ​​0.3 × 1 mm.

[0141] For each experiment on each sample, the consumables are first filled with the generated oil. The PCR pre-amplification products described above are then arranged in each array plate 2 at a constant droplet size (macrofluidic droplet generation technology). Each array plate 2 can be allocated several (3-5) small droplets of known droplet size to the microarray. After the droplets absorb the lyophilized probe, the plate is capped and amplification is performed, thus fixing each droplet within the defined microarray for amplification. Specifically: 1. For a sample testing one site, the microarray refers to a single microwell or multiple microwells pre-embedded with the same probe. 2. For a sample testing multiple sites, it is necessary to ensure that each microwell contains a different probe, and that the pre-amplification products of the same individual are added to multiple microwells. That is, if one individual tests 10 sites, the microarray can be understood as 10 microwells, each microwell corresponding to one site, with the individual's pre-amplification products added to the 10 microwells.

[0142] 3. Monitoring of droplet PCR amplification within the microarray:

[0143] Different probes are designed according to the number of target sites to be tested, ensuring that the probes correspond one-to-one with the target proteins, without conflict or duplication.

[0144] The PCR pre-amplified product is generated into droplets using a droplet generation device. The array plate 2 with the pre-embedded specific probe receives the droplets, and fluorescence is monitored in real time on each reaction surface. Theoretically, the maximum throughput can reach 48 samples × 200 to 300 target sites.

[0145] 4. Quality control design

[0146] 4.1 An antibody was designed to directly link two oligonucleotides, allowing for complementary pairing of nucleic acids and subsequent pre-amplification even without antigen-antibody hybridization. For example, if all other wells are negative and this well is positive, the detection is valid; if all other wells are negative and this well is also negative, it indicates an abnormality in the amplification phase of the experiment, rendering the entire dataset unreliable.

[0147] 4.2 Set up a negative control, i.e., no antigen is present throughout the entire reaction, and LOB is monitored.

[0148] 4.3 Set up a positive control, which participates in the entire reaction process, meaning that it is a sample that will theoretically be positive.

[0149] Application Example 2

[0150] 1. Pre-amplification: After determining the concentration of the extracted DNA, take a portion of the volume according to the fixed concentration and add it to a PCR tube. Add the mixed primers and perform pre-amplification in a regular PCR machine.

[0151] Note: ① Approximately 100 pairs of primers and 20 pairs of probes are needed to detect the above-mentioned lung cancer ctDNA.

[0152] ② This step requires mixing 100 primer pairs in advance and verifying beforehand that all primer pairs can be amplified under a fixed ratio, and that the amplification efficiency is relatively consistent.

[0153] ③ In this step, it is essential to pay attention to the quality control of the sample concentration and the number of amplification cycles to control the final total copy number. If the copy number is too high, false positives may occur.

[0154] 2. Detection of amplified products

[0155] At this point, there are two possible technical paths that need to be verified separately:

[0156] 2.1 Method 1:

[0157] ① The primers added in step 1 should be in appropriate excess, that is, enough to ensure the amount of primers in both step 1 and subsequent experiments;

[0158] ② Take the amplification product and add it back to the probe buffer (or try the above buffer again, provided that the activity has not decreased);

[0159] ③ Place it in a digital PCR reactor for reaction;

[0160] ④ In the digital PCR microarray chip, the probe to be tested is pre-embedded. Observe the changes of a number of droplets (about 10) in each region, or focus on observing the key droplets, i.e., the appearance of fluorescence indicates a positive result; or continue to observe.

[0161] 2.2 Method Two:

[0162] ①After step 1 - pre-amplification is completed, use magnetic beads to purify the product, remove the buffer and primers from the previous step, and retain the amplification product.

[0163] ② Add the purified amplification product back into the probe buffer, and proceed as in Method 1 of 2.1.

[0164] 2.3 Method Three:

[0165] ①After step 1 - pre-amplification is completed, use magnetic beads to purify the product, remove the buffer and primers from the previous step, and retain the amplification product.

[0166] In method three, the primers used in step 1 have undergone special processing and are different from those used in methods one and two. In methods one and two, the primers in step 1 are fully paired multiple primers. Taking one primer as an example, the following changes are made:

[0167] Primer design for Method 1 and Method 2:

[0168] Primer: 5'--AATTGCCGGT--3'

[0169] Template: ........... ....TTAACGGCCA...................

[0170] Primer and template are a perfect match

[0171] The primers used in Method 3 are as follows:

[0172] Primer: 5'--CGTCGGCA---AATTGCCGGT--3'

[0173] Template: ................TTAACGGCCA...................

[0174] The primers are changed to the above form, that is, the AATTTGCCGGT part is the original complementary part, and the CGTCGGCA part is a redesigned universal primer. It is necessary to ensure that the universal primer and the template to be tested do not pair complementarily.

[0175] When step 1 begins, amplification is performed starting from the AATTTGCCGGT portion, but each amplification product also carries the CGTCGGCA portion.

[0176] ② For the special primer design in step 1 mentioned above, primer 2 is specifically designed for the PCR part, and this primer 2 is the universal primer.

[0177] ③ Add the purified and quantified PCR pre-amplification product to universal primers and generate droplets using a droplet generation device.

[0178] ④ The array plate receives droplets. After the droplets absorb the lyophilized probes on the reaction surface, PCR amplification is performed on each reaction surface.

[0179] Taking the detection of ctDNA in lung cancer using this method as an example, the human genomic DNA size (Mb) is 3000Mb / copy. The molecular weight of 1bp is 1.096 x 10⁻²¹ g / bp. Converting Mb to bp yields 1 x 10⁶ bp / Mb. Converting g to ng yields 1 x 10⁹ ng / g. This equals 0.0033 ng / copy per copy of DNA. Then, the copy number concentration can be directly converted. If the copy number concentration is 3000 copies / μL, the formula is: 3000 copies / μL x 3.3 pg / copy (0.0033 ng / copy) = 9.9 ng / μL. That is, with a loading amount of approximately 10 ng, the total copy number is approximately 3000 copies. If we take the copy number of the mutation (3) as the upper limit detectable in the experiment, the limit of detection per unit spot is approximately 0.1%. Therefore, for relatively low concentrations of ctDNA, achieving the limit of detection (LOD) requires loading the entire sample. Therefore, the initial DNA needs to be concentrated, or the total reaction volume needs to be increased. In this example, when the total volume corresponding to each well is 20 μL, the loading volume is approximately 10-15 μL. Although this may waste sample, it barely achieves the required level of density (LOD). Therefore, in this example, pre-amplification is performed before droplet splitting, using an in-well splitting method to ensure sufficient copy number within each minimum volume.

[0180] Comparative Example: NGS Detection of Small Panel Method

[0181] 3.1 Background Introduction

[0182] Common NGS assays mainly include Life's semiconductor sequencing method and Illumina's reversible termination method. Both methods require the following steps: extraction, library construction, library mixing, enrichment, assay, and data analysis. Among them, Life's S5 platform or Illumina's MiniSeq / NextSeq are the closest to our needs in terms of throughput, time, and application scenarios. Taking Life's S5 as an example, its main working process is described below.

[0183] 3.2 S5 Platform Practical Introduction

[0184] 1. Database creation:

[0185] Simultaneously, a special amplicon and barcode are added to achieve the above three-stage purpose with a single ordinary PCR, followed by purification after amplification.

[0186] 2. Mixed storage

[0187] In library construction, the barcode is a tag, i.e. a known short sequence. During actual sequencing, the information of the known short sequence and the initial number corresponding to the sample to be tested can be used to achieve a one-to-one correspondence between all the samples to be tested.

[0188] For NGS, each test involves batch sequencing of multiple samples together on a single chip, resulting in a total amount of data.

[0189] Therefore, each time the prepared library needs to be quantified (either by quantitative real-time PCR standard curve method or qubit quantification; currently, to improve speed, small panels usually use the latter, while ultra-high-throughput sequencing uses the former), because the quality (ng) at the time of sequencing corresponds one-to-one with the amount of data of a certain sample. Therefore, according to the data requirements of the sample to be tested, the required quality ratio is prepared. For the behavior of detecting different samples in the same panel, it can usually be considered that all samples are mixed with equal quality.

[0190] Therefore, pooling involves calculating the pool volume based on equal mass and concentration, and then mixing different samples together according to the calculated volume.

[0191] The mixed libraries can yield a theoretical concentration and an actual concentration. For example, if the measured concentration after mixing the libraries is 2.35 ng / ul, and the maximum capacity of a single chip is known to be 50ul * 0.0066 ng / ul, the sample can be diluted to 0.0066 ng / ul for later use.

[0192] 3. Enrichment

[0193] The complete enrichment is completed automatically within the instrument, and the specific steps may include three steps.

[0194] ①OT2

[0195] A sufficient number of upstream primers are fixed onto specially designed ISP beads, which can complementaryly pair with the adapter. An instrument is used to construct a water-in-oil system containing one ISP bead, a template to be tested, and a certain number of primers at the other end. Ultimately, a double-stranded ISP bead with the same sequence growing on its surface can be formed within this water-in-oil system.

[0196] ②ES

[0197] The above steps culminate in the following process: first, an emulsion-breaking agent is used to open the water-in-oil system; then, magnetic beads are used to remove ISP beads that have not been linked to DNA. Double strands are then converted to single strands. Ultimately, this will leave two types of ISP beads: those containing only one sequence and those containing multiple sequences. In the final output, the sequences containing multiple sequences are scrambled and will be discarded.

[0198] ③ Fabrication of the onboard chip

[0199] Sequencing primers and enzymes are added to the single-stranded ISP beads described above, and then the beads are injected into a sequencing chip. The sequencing chip consists of multiple micro-pits, each of which can hold one ISP bead.

[0200] 4. Sequencing

[0201] Place the prepared sequencing chip into the sequencer. Before use, the sequencer needs to be refilled with sequencing reagents and initialized. The sequencing time is approximately 2.5-3 hours. Afterward, the instrument will automatically analyze the data and generate a report; this step is usually performed overnight.

[0202] Comparative analysis:

[0203] Taking a small panel of about 10 genes in lung cancer as an example, the experimental steps of Method 3 in Example 2 and the comparative example are compared in Table 1 below.

[0204] Table 1

[0205]

[0206]

[0207] Method 3 in Application Example 2 has the advantages of low cost and short time.

[0208] The aforementioned biomolecular detection array plate 2 based on droplet PCR technology uses lyophilized specific probes 200 fixed on the reaction surface 10a of the substrate 100. The same or different lyophilized specific probes 200 can be fixed, and the probes are provided by the array plate 2 and do not need to be contained within the droplets, making the detection process more convenient. This is particularly convenient for the specific detection of different targets (e.g., different proteins). Compared to traditional droplet PCR detection systems using microfluidic chips or microwell 102 plates as the substrate, the array plate 2 is easier to manufacture and can employ a high-density array design and an open structure, which helps reduce sample requirements and achieve highly sensitive, high-throughput, and high-specificity protein detection. Detection of 3000 protein samples requires as little as 6 μL of sample. For multiplex nucleic acid site detection, it offers advantages of low cost and short detection time.

[0209] In particular, macrofluidic microdroplet generation and droplet microarray technology based on droplet PCR are employed. For each protein detection, a pair of specifically matched antibodies (or polyclonal antibodies split in half) are designed to ensure the specificity of the immunological reaction. By adding a pair of paired nucleic acid single strands to each antibody, crosstalk at the immunological level is avoided by leveraging the specificity of DNA pairing. Then, a miniature PCR detection device uses specific probes to quantitatively / semi-quantitatively detect the paired DNA double strands. This achieves biologically ultrasensitive, unbiased targeted proteomics, especially suitable for blood samples where traditional methods are inadequate.

[0210] As indicated in this specification and claims, the terms "comprising" and "including" only indicate the inclusion of explicitly identified steps and elements, and these steps and elements do not constitute an exclusive list, as the method or apparatus may also include other steps or elements.

[0211] It can be further understood that in this disclosure, "multiple" refers to two or more, and other quantifiers are similar.

[0212] It is further understood that the terms "first," "second," etc., are used to describe various types of information, but this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another, and do not indicate a specific order or degree of importance. In fact, expressions such as "first," "second," etc., are completely interchangeable. For example, without departing from the scope of this disclosure, first information can also be referred to as second information, and similarly, second information can also be referred to as first information.

[0213] The above embodiments are only for illustrating the technical concept and features of the present invention, and are preferred embodiments. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the scope of protection of the present invention.

Claims

1. A biomolecule detection system based on a droplet PCR technique, comprising a droplet generation device, characterized by, The biomolecule detection system further comprises an array plate; The array plate comprises a substrate having a plurality of reaction surfaces arranged in an array and spaced apart from each other, each of the reaction surfaces provides an area for a droplet PCR reaction; the array plate further comprises specific probes arranged on the reaction surfaces, the specific probes are used for specific binding with target biomolecules or markers connected with target biomolecules; The droplet generating device is used for generating droplets containing target biomolecules or markers connected with target biomolecules, amplification primers and applying to each of the reaction surfaces of the array plate.

2. The biomolecule detection system according to claim 1, wherein The specific probes are attached to the reaction surfaces in a lyophilized form.

3. The biomolecule detection system of claim 1, wherein The reaction surfaces are hydrophilic surfaces, the array plate further comprises a hydrophobic layer, the hydrophobic layer is patterned on the surface of the substrate to separate the plurality of reaction surfaces.

4. The biomolecule detection system according to claim 3, wherein The hydrophobic layer is coated on the surface of the substrate, and the width of the hydrophobic layer is 0.3-0.5mm.

5. The biomolecule detection system according to claim 3, wherein The upper surface of the substrate is a hydrophilic surface, and the hydrophobic layer is patterned on the upper surface to form the plurality of reaction surfaces separated and enclosed by the hydrophobic layer.

6. The biomolecule detection system according to claim 3, wherein The surface of the substrate and the reaction surfaces are respectively planes.

7. The biomolecule detection system according to claim 3, wherein The surface of the substrate is provided with a plurality of micropores, and the pore walls of the micropores constitute the reaction surfaces; the hydrophobic layer is coated on the part of the surface which is not provided with micropores.

8. The biomolecule detection system of claim 7, wherein The depth of the micropores is less than or equal to 0.3mm; and / or, the volume of the micropores is less than or equal to 2.5mL; and / or, the micropores are arranged in an array; and / or, the number of micropores on each substrate is 10-400.

9. The biomolecule detection system of claim 1, wherein, The array plate comprises one, two or more specific probes, and the specific probes arranged on the plurality of reaction surfaces are the same or different or partially the same.

10. The biomolecule detection system of claim 1, wherein, The width of the reaction surface is 0.1-0.4 mm, the length is 1-17 mm, and the area is less than or equal to 8 mm 2 ; and / or the dimensions of each reaction plane are the same or different.

11. The biomolecule detection system of claim 1, wherein The number of reaction surfaces on each substrate is 10-400; and / or, the array distribution is a square array distribution or a circular array distribution or a combination of the two.

12. The biomolecule detection system according to any one of claims 1 to 11, wherein, The biomolecules are proteins or nucleic acids, and the array plate comprises 10-400 specific probes; and / or, the droplet generating device is used for generating droplets with a volume of 1-20nL.

13. The biomolecule detection system according to any one of claims 1 to 11, wherein The droplet generating device comprises a volume-variable accommodating cavity, a control mechanism for controlling the volume of the accommodating cavity to be periodically changed, and a droplet generating tube having a first port and a second port relatively far away from each other, the first port of the droplet generating tube communicates with the accommodating cavity, the droplet generating device further comprises a driving fluid mechanism for introducing a driving fluid into the accommodating cavity, the inner diameter of the second port of the droplet generating tube is greater than 0.1mm, and the periodic change is a reciprocating change of compression-recovery, or a reciprocating change of expansion-recovery, or a reciprocating change of compression-recovery-expansion-recovery.

14. The biomolecule detection system of claim 13, wherein, The inner diameter of the second port is 0.1-0.2mm or 0.3-0.6mm; and / or, the droplet generating device is used for generating droplets with a volume of 1-20nL.

15. An array plate for biomolecule droplet PCR detection, characterized in that, The array plate is as claimed in any one of claims 1-14.

16. A method for protein detection based on droplet PCR technology for non-diagnostic and therapeutic purposes, characterized by, The biomolecule detection system according to any one of claims 1 to 14 is used for detecting a protein or simultaneously detecting multiple proteins, and the protein detection method comprises the following steps: For each target protein to be detected, a corresponding specific antibody is designed and connected with a specific oligonucleotide, and then the specific antibody connected with the specific oligonucleotide is mixed with a sample to be detected, and incubation is performed to allow antigen-antibody hybridization, so as to obtain a hybridization product, wherein the oligonucleotide is a nucleotide with an amino acid length of less than 100 bp; The hybridization product, an amplification primer and a polymerase are mixed to perform PCR pre-amplification, so as to obtain a pre-amplification product; The pre-amplification product is formed into droplets by the droplet generation device and is applied to reaction surfaces of the array plate or droplets are directly generated above the reaction surfaces, and on the reaction surfaces, the specific probe is dissolved and mixed with the pre-amplification product to form an amplification system, wherein the specific probe is specifically combined with the specific oligonucleotide. An amplification reaction is performed, and then signal acquisition is performed.

17. The protein detection method based on the droplet PCR technique according to claim 16, wherein, For each target protein to be detected, a pair of specific antibodies is designed, the specific antibodies are connected with single-stranded oligonucleotides, and a specific combination can be formed between a segment close to one end of the two single-stranded oligonucleotides connected with the specific antibodies, and the specific combination can be specifically combined with the specific probe.

18. The protein detection method based on the droplet PCR technique according to claim 17, wherein, The length of the single-stranded oligonucleotide connected with the antibody is 60-80 bp; and / or the length of the segment capable of being specifically combined between the two single-stranded oligonucleotides is 10-15 bp.

19. The protein detection method based on the droplet PCR technique according to claim 17, characterized by, The sequence of the other end of the single-stranded oligonucleotide is a universal sequence.

20. The protein detection method according to claim 17, wherein, The method further comprises adding a fluorescent group to the amplification system and performing real-time fluorescent quantitative analysis on the amplification system.

21. The protein detection method according to claim 17, wherein The sample to be detected comprises a blood sample.

22. A method for nucleic acid detection based on droplet PCR technology for non-diagnostic and therapeutic purposes, characterized by, The biomolecule detection system according to any one of claims 1 to 14 is used, and the nucleic acid detection method comprises the following steps: a pre-amplification system containing a target nucleic acid to be detected, a primer and a polymerase is formed into droplets by the droplet generation device and is applied to reaction surfaces of the array plate or droplets are directly generated above the reaction surfaces, and on the reaction surfaces, the specific probe is dissolved and mixed with the pre-amplification system to form an amplification system, wherein the specific probe is specifically combined with the target nucleic acid to be detected; an amplification reaction is performed, and then signal acquisition is performed.

23. The nucleic acid detection method of claim 22, wherein, The method further comprises performing real-time fluorescent quantitative analysis or digital PCR analysis on the amplification system.

24. The method of claim 22, wherein the nucleic acid is DNA. 25 The array plate comprises multiple specific probes, and the method is a multiplex detection performed on different sites of a nucleic acid.

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