Biomolecule detection array plate and detection system for droplet PCR (Polymerase Chain Reaction)

Abstract

The utility model discloses a biomolecule detection array plate and a biomolecule detection system for liquid drop PCR (Polymerase Chain Reaction). The array plate comprises a substrate, the substrate is provided with a plurality of reaction surfaces which are distributed in an array mode and are separated from one another, and each reaction surface provides an area for liquid drop PCR reaction; the array plate further comprises a freeze-dried body of a specific probe 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 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] The utility model belongs to the field of biomolecule detection based on droplet PCR technology, and relates to a biomolecule detection array plate and system for droplet PCR reaction, in particular to a protein detection array plate or nucleic acid detection array plate based on droplet PCR technology. Background Art

[0002] The polymerase chain reaction (PCR) technology is one of the most important tools in modern biology, and it is widely used in medical diagnosis, personalized medicine, food inspection, genetically modified organism detection, pathogen identification, immunoassay, forensic science and other aspects. Droplet PCR technology is based on the oil-in-water droplet technology. Through the oil-in-water structure, a small amount of DNA molecules are encapsulated into separate micro-droplets, and the inertness of the oil is used to isolate DNA molecules from each other. The DNA molecules are restricted to amplify separately in their own micro-droplets, avoiding competition from other sequences. After the amplification of DNA molecules is completed under suitable temperature conditions, by recording the total number of micro-droplets and the number of micro-droplets that can detect fluorescence signals, the precise quantification of DNA copy number can be achieved using the Poisson distribution algorithm (droplet digital PCR). Each droplet can also be regarded as an independent reaction unit to observe its real-time fluorescence amplification process, so as to achieve quantification (droplet real-time quantitative PCR).

[0003] In the prior art, for droplet digital PCR detection systems and methods, samples and a PCR amplification system containing amplification enzymes, primers, and probes usually form droplets through a droplet generation system, and then the droplets are loaded into a chip for amplification reaction and detection (such as fluorescence detection) in a PCR instrument. Using this traditional method, specific probes are included in the droplets. If different targets (such as different proteins and different nucleic acid sites of nucleic acids) need to be specifically detected, different PCR amplification systems need to be constructed, the droplet generation system needs to be replaced, and droplets containing different specific probes are generated respectively, which cannot achieve true multiplex detection, the detection is not convenient enough, the operability is poor, the cost is high, and the detection time is long.

[0004] In addition, high-throughput mass spectrometry is mainly used in the prior art for protein metabolism-related detection research, which has the disadvantages of limited sensitivity, low specificity, limited detection dynamic range, low throughput, large sample consumption, high cost, etc., and has great limitations when detecting plasma and similar samples.

[0005] The above information disclosed in the background art section is only used to strengthen the understanding of the background of the present application, so it may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Utility Model

[0006] An object of the present utility model is to provide a biomolecule detection array plate for droplet PCR reaction, which can provide specific probes for droplet PCR detection, facilitate the realization of specific detection of different targets (such as different proteins, different nucleic acid sites), reduce the detection cost of multiplex detection, and shorten the detection time. Another object of the present utility model is to provide a biomolecule detection system for droplet PCR reaction.

[0007] The present utility model adopts the following technical solutions:

[0008] A biomolecule detection array plate for droplet PCR reaction, comprising a substrate, the substrate having a plurality of reaction surfaces which are arrayed and spaced apart from each other, each of the reaction surfaces respectively providing a region for droplet PCR reaction to proceed; the array plate further comprising a freeze-dried body of specific probes disposed on the reaction surfaces, the specific probes being used for specifically binding to a target biomolecule to be detected or a marker linked to the target biomolecule to be detected. Herein, for the convenience of description, the target biomolecule to be detected or the marker linked to the target biomolecule to be detected is collectively referred to as "analyte".

[0009] In a preferred embodiment, the reaction surface is a hydrophilic surface, and the array plate further comprises a hydrophobic layer, the hydrophobic layer being graphically disposed on the surface of the substrate to separate the plurality of reaction surfaces.

[0010] 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 - 0.5 mm.

[0011] In a more preferred embodiment, the upper surface of the substrate is a hydrophilic surface, and the hydrophobic layer is graphically coated on the upper surface to form the plurality of reaction surfaces separated and enclosed by the hydrophobic layer.

[0012] In a preferred embodiment, the surface of the substrate and the reaction surface are respectively flat surfaces.

[0013] In a preferred embodiment, a plurality of micropores are provided on the surface of the substrate, the pore walls of the micropores constituting the reaction surfaces; the hydrophobic layer is coated on the portion of the upper surface where the micropores are not opened.

[0014] In a further preferred embodiment, the depth of the micropores is less than or equal to 0.3 mm.

[0015] 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.

[0016] 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.

[0017] In a preferred embodiment, the array plate contains lyophilized bodies of one, two or more of the specific probes, and the lyophilized bodies of the specific probes provided on the plurality of reaction surfaces are the same, different or partially the same.

[0018] In a preferred embodiment, the reaction surface is planar, the width of the reaction surface is 0.1 to 0.4 mm, the length is 1 to 17 mm, and the area is less than or equal to 8 mm 2 . More preferably, the length of the reaction surface is 1 to 2 mm.

[0019] In a preferred embodiment, the sizes of the reaction surfaces are the same or different.

[0020] 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, a circular array distribution or a combination of the two.

[0021] In a preferred embodiment, the biomolecule detection array plate is a protein detection array plate or a nucleic acid detection array plate, and the array plate includes lyophilized bodies of 10 to 400 specific probes.

[0022] Another technical solution adopted by the present utility model is: a biomolecule detection system for droplet PCR reaction, including a droplet generation device, and the biomolecule detection system further includes the biomolecule detection array plate.

[0023] The droplet generation device of the present utility model is used to generate droplets containing the target biomolecule to be detected or markers linked with the target biomolecule to be detected and amplification primers and apply them to the respective reaction surfaces of the array plate.

[0024] In a preferred embodiment, the droplet generation device includes a variable-volume accommodation chamber, a control mechanism for controlling the volume of the accommodation chamber to change periodically, and a droplet generation tube having relatively distant first and second ports. The first port of the droplet generation tube communicates with the accommodation chamber. The droplet generation device further includes a driving fluid mechanism for introducing a driving fluid into the accommodation chamber. The inner diameter of the second port of the droplet generation tube is more than 0.1 mm, 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.

[0025] The above-mentioned droplet generation device can be adjusted to generate droplets with a desired and appropriate volume. According to a preferred aspect of the present invention, the droplet generation device is configured to generate droplets with a volume of 1 to 20 nL. Specifically, the droplet generation device is configured to generate droplets with a volume of 1 to 5 nL.

[0026] Preferably, the inner diameter of the second port is greater than 0.2 mm, specifically preferably 0.2 mm - 1 mm. More preferably, the inner diameter of the second port is 0.3 - 0.6 mm. Also preferably, the inner diameter of the first port is greater than that of the second port. The droplet generation tube includes a tapered tube portion, and the first port and the second port are respectively formed at both ends of the tapered tube portion, and the taper of the tapered tube portion is 0.05 - 0.2.

[0027] Furthermore, the volume of the droplet generation tube can be 10 - 200 microliters. Experiments show that the volume of the droplet generation tube has a relatively small impact on the droplet generation effect, which is also one of the advantages of the droplet generation device of the present invention.

[0028] According to a specific implementation and preferred aspect of the present invention, the periodic change is a reciprocating change of compression - recovery, or an expansion - recovery reciprocating change, or a compression - recovery - expansion - recovery reciprocating change.

[0029] According to a specific implementation and preferred aspect of the present invention, the driving fluid mechanism includes a pump and a fluid passage, the droplet generation device includes a base body, the base body provides a cylindrical hole and a fluid passage, and provides a connecting portion for connecting the liquid generation tube. On the same base body, the number of the cylindrical hole, the fluid passage, and the connecting portion is one or more. The cylindrical hole is a cylindrical shape with openings at both the upper and lower ends, and a diaphragm is covered on each cylindrical hole. The cylindrical hole and the diaphragm together form a receiving cavity.

[0030] Preferably, the receiving cavity, the droplet generation tube, and the droplet receiver are arranged in sequence from top to bottom. The first port of the droplet generation tube is communicated with the lower opening of the receiving cavity. The center line of the receiving cavity, the axis line of the droplet generation tube, the center line of the first port, and the center line of the second port coincide and all extend along the vertical direction.

[0031] Preferably, each diaphragm respectively includes a main body portion and a movable portion. The main body portion is fixedly connected to the base body, the movable portion is located directly above the cylindrical hole, and the movable portion is connected to the control mechanism through a connecting member.

[0032] Furthermore, the diaphragm can be a metal (such as stainless steel) or a non - metal diaphragm; the thickness of the diaphragm can be 5 microns - 2 mm.

[0033] Preferably, a sealing structure is provided between the diaphragm and the base body to keep the receiving cavity always in good sealing.

[0034] Preferably, the control mechanism is a vibration mechanism, and the vibration mechanism 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 present invention, the vibration mechanism preferably includes a piezoelectric ceramic.

[0035] Preferably, the vibration direction provided by the vibration mechanism is the up-and-down direction, and the vibration frequency provided is at least 50 - 600 Hz. During use, it can be specifically selected within this range, and the vibration amplitude provided is preferably at least 5 microns - 300 microns.

[0036] Preferably, the droplet generation tube is detachably connected to the connection portion. In this way, the droplet generation tube part can be independent of the entire system and can be disassembled and replaced when the droplet generation of a sample solution is completed. In some embodiments, the droplet generation tube can directly adopt commercially available nozzles of corresponding specifications. Of course, it is also possible to make the droplet generation tube and the connection portion into one body, which will not affect the droplet generation effect.

[0037] Preferably, on the same substrate, the number of each of the cylindrical holes, fluid passages, and connection portions is 2 - 20, preferably 4 - 10.

[0038] Preferably, there are multiple cylindrical holes, fluid passages, and connection portions. The two opposite side portions of the substrate are respectively higher than the middle portion between the two opposite side portions. The multiple cylindrical holes are independently distributed in the middle portion of the substrate and arranged in two rows. Each fluid passage includes a vertical passage formed on the two opposite side portions of the substrate and a horizontal passage that correspondingly communicates the vertical passage with the cylindrical hole. Adopting this structural design makes the overall device relatively compact and convenient for operation and control.

[0039] According to a specific implementation and preferred aspect of the present invention, a drainage portion is formed between the port of the horizontal passage and the inner peripheral side wall of the accommodation cavity, so that the liquid from the horizontal passage enters the accommodation cavity tangentially to the circumferential direction of the accommodation cavity.

[0040] According to a specific implementation and preferred aspect of the present invention, one end of the fluid passage communicates with the accommodation cavity of the substrate, and when the fluid is driven to enter the accommodation cavity from the fluid passage, the fluid forms a vortex that rotates circumferentially along the accommodation cavity and the droplet generation tube in the accommodation cavity and the droplet generation tube.

[0041] According to another specific implementation and preferred aspect of the present invention, one end of the fluid passage communicates with the accommodation cavity of the substrate, and the direction in which the fluid is discharged from the fluid passage deviates from the axis line of the accommodation cavity.

[0042] As described above, through the structural design of the fluid passage and the accommodation cavity, etc., as a whole, it is very convenient to discharge the air bubbles in the system cavity before droplet generation, avoiding the adverse effects of the presence of air bubbles on droplet generation.

[0043] According to the present utility model, the droplet receiver is a device having a receiving cavity, which can be in a sealed or non-sealed form, without any particular limitation.

[0044] The present utility model adopts the above solution and has the following advantages:

[0045] For the biomolecular detection array plate for droplet PCR reaction of the present utility model, the specific probe dry bodies are arranged on the reaction surface of the array plate. The same or different specific probe dry bodies can be arranged on different reaction surfaces. The probes are provided by the array plate and do not have to be included in the droplets. A general droplet and droplet generation device can be used, making the detection process relatively convenient; especially for multiplex detection of different targets (such as different proteins), it is very convenient.

[0046] In a further preferred solution, compared with the traditional PCR detection system using a microfluidic chip or a microwell plate as the bottom plate, the biomolecular detection array plate of the present utility model is easy to manufacture, and a high-density array design and an open structure can be adopted, which are beneficial to reducing the sample requirement and realizing highly sensitive, high-throughput and highly specific protein detection, and the detection is more accurate, reducing the cost of multiplex detection and shortening the time of multiplex detection. Description of the Drawings

[0047] In order to more clearly illustrate the technical solution of the present utility model, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present utility model. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0048] Figure 1 Schematic diagram of a biomolecular detection system according to an embodiment of the present utility model.

[0049] Figure 2 Top view of a biomolecular detection array plate according to an embodiment of the present utility model.

[0050] Figure 3 Partial cross-sectional view of a biomolecular detection array plate according to an embodiment of the present utility model. Detailed Embodiments

[0051] The following will elaborate on the preferred embodiments of the present utility model in conjunction with the accompanying drawings, so that the advantages and features of the present utility model can be more easily understood by those skilled in the art. It should be noted here that the description of these embodiments is for helping to understand the present utility model, but does not constitute a limitation to the present utility model. In addition, the technical features involved in the various embodiments of the present utility model described below can be combined with each other as long as they do not conflict with each other.

[0052] This embodiment provides a biomolecule detection system for droplet PCR reaction and consumables for this detection system. Figure 1 The biomolecule detection system is shown. Figures 2 to 3 The consumables are shown, specifically an array plate 2 for detecting biomolecules based on droplet PCR technology. The biomolecules typically include proteins, nucleic acids, etc.

[0053] As Figure 1 shown, the biomolecule detection system includes a droplet generation device 1 and the array plate 2. The array plate 2 is arranged below the droplet generation device 1 to receive the droplets generated by the droplet generation device. After the droplet addition is completed, the array plate 2 can be detected and observed. As the amplification reaction proceeds, the fluorescence change on the array plate 2 can be observed. The volume of the droplets is 1 - 20 nL, preferably 5 - 10 nL.

[0054] Droplet generation device 1:

[0055] The droplet generation device 1 includes a base body 10 and a droplet generation unit 11 provided on the base body 10. The droplet generation unit 11 includes a variable-volume accommodation chamber 110, a control mechanism 111 for controlling the volume of the accommodation chamber 110 to change periodically, a droplet generation tube 112 having relatively far-apart first port a1 and second port a2, and a driving fluid mechanism 113 for introducing a driving fluid into the accommodation chamber 110.

[0056] A vertically extending cylindrical hole is provided in the middle part of the base body 10, and a diaphragm 114 is covered on the cylindrical hole. The cylindrical hole and the corresponding diaphragm 114 together form the accommodation chamber 110. The control mechanism 111 is a vibration mechanism, which is installed above the cylindrical hole and connected to the diaphragm 114 through a connecting member 115, so as to control the movement of the diaphragm 114 to implement the volume change of the accommodation chamber 110. The droplet generation tube 112 is vertically arranged, the upper end of which is communicated with the connecting part 101, and the lower end forms a droplet outlet.

[0057] The central axis of the accommodation chamber 110, the axis of the droplet generation tube 112, the central axis of the first port a1, and the central axis of the second port a2 coincide and all extend along the vertical direction. The accommodation chamber 110 is an annular cavity with an inner diameter of about 5 mm, and the inner peripheral side wall extends along the vertical direction. The diaphragm 114 (made of stainless steel) forming the top of the accommodation chamber 110 includes a main body portion b1 and a movable portion b2 respectively. The main body portion b1 is fixedly connected to the base body 10, and the movable portion b2 is located directly above the cylindrical hole, and the movable portion b2 is connected to the control mechanism 111 through a connecting member 115. The control mechanism 111 specifically includes a piezoelectric ceramic, which can provide reciprocating vibration up and down. When it reciprocates, it will drive the movable portion b2 to move inward or outward relative to the accommodation chamber 110, thereby correspondingly changing the volume of the accommodation chamber 110 periodically. Correspondingly, the liquid located in the accommodation chamber 110 is disturbed due to this periodic volume change. Further, the periodic change can be a reciprocating change of compression - recovery, or an expansion - recovery reciprocating change, or a reciprocating change of compression - recovery - expansion - recovery. In this example, at least the periodic change that can be provided includes a reciprocating change of compression - recovery.

[0058] The droplet generation tube 112 includes a connecting tube portion c1 and a tapered tube portion c2. The tapered tube portion c2 has a first port a1 and a second port a2. The inner diameter of the tapered tube portion c2 gradually decreases from the first port a1 towards the second port a2, and the taper presented by the change in this inner diameter has an important influence on the droplet generation effect. Let the inner diameter of the first port a1 be R1, the inner diameter of the second port a be R2, and the distance between the first port a1 and the second port a2 (i.e., the length c1 of the tapered tube portion) be L, and the taper be (R1 - R2) / L. Then in this example, the taper of the tapered tube portion c2 adopted is 0.12, and the inner diameter R2 of the second port a2 is 0.5 ± 0.1 mm. The volume of the droplet generation tube 112 is about 10 microliters.

[0059] The connecting tube portion c1 intersects with the tapered tube portion c2 at the first port a1, and it is used to be detachably sleeved on the connecting portion 101. Its taper has no special requirement, but it is preferably greater than the taper of the tapered tube portion c2. A flow channel t communicating with the accommodation chamber 110 is formed in the middle of the connecting portion 101. After the droplet generation tube 112 is installed on the connecting portion 101, the accommodation chamber 110 is communicated with the droplet generation tube 112 through the flow channel t.

[0060] The driving fluid mechanism 113 includes a pump d1 and a fluid passage d2. The fluid passage d2 includes a vertical passage d21 formed on the left convex edge portion 10b of the base 10 and a horizontal passage d22 that corresponds to and communicates with the cylindrical hole 100. To facilitate the discharge of air bubbles in the system cavity, a drainage portion d3 is formed between the port of the horizontal passage d22 and the inner peripheral side wall of the accommodation cavity 110, so that the liquid from the horizontal passage d2 enters the accommodation cavity 110 tangentially to the circumferential direction of the accommodation cavity 110. In this way, a vortex that rotates along the circumferential direction of the accommodation cavity 110 and the droplet generation tube 112 is formed in the accommodation cavity 110 and the droplet generation tube 112, and thus the air bubbles can be easily discharged. A sealing ring 116 is also provided between the diaphragm 114 and the base 10 to improve the sealing performance of the accommodation cavity.

[0061] The array plate is located below the second port a2 and is used to accommodate the first liquid y1 and droplets. In the field of digital PCR, the first liquid y1 is usually a formulated oil, such as mineral oil, in which a surfactant is preferably added. The second liquid y2 is usually the aqueous phase of the biomolecule to be detected. The fluid y3 (driving oil) fills the inner cavities of the fluid passage d2, the accommodation cavity 110, and the droplet generation tube 112. Further, the fluid y3 and the first liquid y1 can be the same kind of mineral oil. Usually, when generating droplets, the liquid in the droplet generation tube 112 is sequentially divided into a driving fluid section and a second liquid section from top to bottom. Or, the liquid in the droplet generation tube 112 is sequentially 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 turned on to start generating droplets.

[0062] Array plate 2:

[0063] Referring to 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 have a hydrophilic surface. The substrate 100 has a plurality of reaction surfaces 10a, and each reaction surface 10a is used to provide a region for droplet PCR reaction to proceed. The plurality of reaction surfaces 10a are arrayed and spaced apart from each other, and each reaction surface 10a is a hydrophilic surface. The plurality of reaction surfaces 10a are preferably arranged in an array and can be divided into multiple groups according to the detection needs. For example, the plurality of reaction surfaces 10a in each row are a group, and multiple rows of reaction surfaces 10a are multiple groups of reaction surfaces 10a. The reaction surfaces 10a are arrayed, and the number of reaction surfaces 10a on each substrate 100 is 10 to 400.

[0064] The array plate 2 further includes a freeze-dried body 200 of a specific probe for droplet PCR detection. The specific probe refers to an oligonucleotide that can specifically bind to a target biomolecule to be detected or a marker linked to the target biomolecule to be detected, and has a specific nucleotide sequence. The specific probe is preferably a fluorescent probe, with a fluorescent reporter group labeled at the 5'-end and a fluorescent quenching group labeled at the 3'-end of the specific probe. During PCR amplification, the probe is cleaved and degraded by an enzyme (such as Taq enzyme), and the fluorescent reporter group and the fluorescent quenching group are separated, thereby emitting fluorescence. The fluorescent reporter groups linked to different specific probes can be the same, different, or partially the same. The freeze-dried body 200 of the specific probe is attached to the reaction surface 10a in the form of a freeze-dried body 200. Among them, the freeze-dried body 200 of the specific probe can be directly obtained by freeze-drying a known specific probe freeze-drying solution in the prior art. The specific probe freeze-drying solution and its freeze-drying process do not belong to the key points of the utility model of this application, and the known specific probe freeze-drying solution and freeze-drying process can be directly used, and will not be elaborated here.

[0065] The array plate 2 contains one, two, or more specific probes. The specific probes provided on multiple reaction surfaces 10a are the same, different, or partially the same. Preferably, the target biomolecule to be detected is a protein or nucleic acid, and the array plate 2 includes 10 to 400 specific probes.

[0066] The freeze-dried bodies 200 of the specific probes provided on several of the multiple reaction surfaces 10a are the same, different, or partially the same, and even the freeze-dried bodies 200 of the specific probes on each reaction surface 10a are all different. Specifically in this embodiment, as described above, the multiple reaction surfaces 10a are divided into multiple groups, and the freeze-dried bodies 200 of the specific probes on at least two groups of reaction surfaces 10a are different. The freeze-dried bodies 200 of the specific probes on each reaction surface 10a in each group can be the same or different. For example, Figure 2 the freeze-dried body 200 of the specific probe provided on the reaction surface 10a in the first row in [description] is the freeze-dried body 200 of the specific probe corresponding to the first protein to be detected; Figure 2 the freeze-dried body 200 of the specific probe provided on the reaction surface 10a in the first row in [description] is the freeze-dried body 200 of the specific probe corresponding to the second protein to be detected. Thus, specific detection of the first protein and the second protein can be simultaneously performed based on the same droplet system.

[0067] The array plate 2 further includes a hydrophobic layer 300, which is graphically disposed on the surface of the substrate 100 to separate the multiple reaction surfaces 10a. Among them, 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 to 0.5 mm. In this embodiment, as Figure 3As shown, a plurality of micro-holes 102 are provided on the upper surface of the substrate 100. The depth of the micro-holes 102 is less than or equal to 0.3 mm, and the pore walls of the micro-holes 102 constitute the reaction surface 10a. Specifically, the bottom wall of each micro-hole 102 forms a reaction surface 10a. The hydrophobic layer 300 is coated on the part of the upper surface where no micro-holes 102 are opened (that is, Figure 3 the upper surface of the convex part protruding upward in

[0068] The micro-holes 102 are arranged in an array, and the volume of the micro-holes 102 is less than or equal to 2.5 mL. The number of micro-holes 102 on each substrate is 10 to 400.

[0069] Each reaction surface 10a is a plane, and the area of the reaction surface 10a is (0.1 - 0.4) mm × (1 - 17) mm, and the area is less than 8 mm 2 . Among them, as Figure 2 shown, the length L of the reaction surface 10a is 1 - 2 mm, and the width W1 is 0.1 - 0.4 mm.

[0070] The array plate 2 can be obtained by the following preparation method:

[0071] Provide a substrate 100, on the upper surface of which a plurality of micro-holes 102 arranged in an array are opened. The bottom wall of each micro-hole 102 is a hydrophilic surface, that is, the reaction surface 10a;

[0072] Coat a hydrophobic material on the part of the upper surface of the substrate 100 where no micro-holes 102 are opened (the convex part), so as to form a patterned hydrophobic layer 300, so that each micro-hole 102 is surrounded by the hydrophobic layer 300 and separated into an independent PCR reaction space.

[0073] Among them, a specific probe freeze-dried body is fixed on each reaction surface 10a by a freeze-drying process.

[0074] The array plate 2 can also be obtained by the following preparation method:

[0075] Provide a substrate 100, the upper surface of which is a hydrophilic plane;

[0076] A hydrophobic material is graphically 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 reaction surfaces 10a spaced apart from each other. Among them, the periphery of each reaction surface 10a is surrounded by the hydrophobic layer 300, and is divided into an independent droplet PCR reaction space.

[0077] Among them, specific probe dry bodies are fixed on each reaction surface 10a through a lyophilization process. For example, one method is as follows: after adding a cryoprotectant to the specific probe reagent, it is dispensed onto the reaction surface of the substrate, frozen into a solid state, and then the water molecules are sublimated under vacuum conditions to leave a solid reagent with a stable structure, that is, the dry body. The sponge-like porous solid reagent can quickly recover when it encounters a dissolution solution. Another method is as follows: ① The specific probe reagent is quantitatively dropped into liquid nitrogen and quickly frozen into particles. Quick freezing better maintains the stability of its protein structure; ② Transfer it to a freeze dryer, and sublimate the water under vacuum. After the sublimation is completed, uniform solid freeze-dried microcore particles are formed; ③ Quantify the freeze-dried microcore particles and dispense them onto the reaction surface of the substrate according to the required quantity and variety.

[0078] This biomolecule detection system is typically used for protein detection and nucleic acid detection. The protein detection and nucleic acid detection methods are described in detail below.

[0079] Protein detection:

[0080] Traditional mass spectrometry-based protein detection techniques have the disadvantages of limited sensitivity, low specificity, limited detection dynamic range, low throughput, large sample consumption, and high cost. It has great limitations when detecting plasma and similar samples.

[0081] In this embodiment, based on the droplet PCR technology of macrofluidic microdroplet generation and microdroplet microarray technology, protein detection is combined with fluorescence PCR to achieve highly sensitive, high-throughput, and highly specific protein detection. The sample demand is extremely small, and only 6 μL of sample is required at least to detect 3000 proteins. When detecting each protein, a pair of specifically matched antibodies (or polyclonal antibodies are directly divided into two halves) are designed to ensure the specificity of the immunological reaction level. By adding paired nucleic acid single strands to a pair of antibodies respectively, the specificity of DNA pairing is used to avoid cross-talk at the immunological level, and then through a micro PCR reaction device, specific probes are used to quantitatively / semi-quantitatively detect the double strands of DNA pairing. Thus, ultrasensitive and unbiased targeted proteomics in the biological sense is achieved, which is especially suitable for blood samples that cannot be handled by traditional methods.

[0082] The protein detection method based on the droplet PCR technology, for non-diagnostic and non-therapeutic purposes, uses the above-mentioned biomolecule detection system to detect one protein or simultaneously detect multiple proteins. The protein detection method includes:

[0083] For each target protein to be detected, a corresponding specific antibody is designed and linked with a specific oligonucleotide. Then, the specific antibody linked with the specific oligonucleotide is mixed with the sample to be detected and incubated to enable antigen-antibody hybridization, obtaining a hybridization product.

[0084] The hybridization product, amplification primers, and polymerase are mixed to perform pre-amplification by PCR, obtaining a pre-amplified product.

[0085] The pre-amplified product is formed into droplets by the droplet generation device and applied to the reaction surface of the array plate or droplets are directly generated above each reaction surface. On the reaction surface, the specific probe dissolves and is mixed with the pre-amplified product to form an amplification system, where the specific probe specifically binds to the specific oligonucleotide.

[0086] An amplification reaction is carried out and then signal acquisition is performed.

[0087] The sample to be detected is typically a blood sample.

[0088] For each target protein to be detected, a pair of specific antibodies are designed. Single-stranded oligonucleotides are respectively linked to the pair of specific antibodies, and a section near one end of the two single-stranded oligonucleotides linked to the pair of specific antibodies can specifically bind to each other, and this specific binding section can specifically bind to the specific probe.

[0089] The length of the single-stranded oligonucleotide linked to the antibody is 60 - 80 bp. The length of the fragment capable of specific binding of the two single-stranded oligonucleotides is 10 - 15 bp.

[0090] The sequence of the other end of the single-stranded oligonucleotide is a universal sequence.

[0091] The method further includes: adding a fluorescent group to the amplification system and performing real-time fluorescence quantitative analysis on the amplification system.

[0092] Nucleic acid detection:

[0093] Nucleic acid site detection, typically such as next-generation sequencing (NGS), is playing an increasingly important role in the fields of tumor, genetic disease, and pathogenic microorganism detection. Traditional sequencing methods require steps such as extraction, library construction, pooling, enrichment, machine detection, and data analysis, with a relatively long detection time and a relatively high detection cost.

[0094] Using the above-mentioned biomolecular detection system, an improved nucleic acid detection method based on droplet PCR technology is proposed for non-diagnostic and therapeutic purposes. The nucleic acid detection method includes: forming droplets of a pre-amplification system containing a target nucleic acid to be detected, primers, and polymerase through the droplet generating device and applying them to the reaction surface of the array plate or directly generating droplets above each reaction surface. On the reaction surface, the specific probe dissolves and mixes with the pre-amplification system to form an amplification system, where the specific probe specifically binds to the target nucleic acid to be detected; performing an amplification reaction and then signal acquisition. For the detection of different nucleic acid sites, this method has the advantages of low cost and short time.

[0095] The method further includes performing real-time fluorescence quantitative analysis or digital PCR analysis on the amplification system.

[0096] The array plate includes a variety of specific probes, and the method is a multiplex detection for different sites of nucleic acids.

[0097] The test sample can be plasma.

[0098] Application Example 1

[0099] 1. Antigen-antibody hybridization and product pre-amplification

[0100] 1.1. According to the antigen to be detected (protein), design two antibodies, connect an oligonucleotide (single-stranded, preferably 60-80 bp long) to each antibody, and there are approximately 10-15 bp at the ends of the two oligonucleotides that can specifically bind. That is, if they bind, the total length is approximately: 2 x single-strand length - specific binding length (preferably about 150 bp). The sequences at both ends need to ensure universality, and the specific binding part is designed with different sequences for monitoring different antigens (for designing probes).

[0101] That is, the basic composition is as follows:

[0102] ① Strand: antibody - 5’--AATTCAGA---------------CGCGATGCA--3’

[0103] ② Strand: 3’--GCGCTAGCT------------------------TATAGCTTGC--5’--antibody

[0104] 1.2. Add a pair of designed antibodies to the antigen and incubate to allow antigen-antibody hybridization. After hybridization, the two strands are relatively close and can specifically bind (the CGCGATGCA and TATAGCTTGC parts).

[0105] 1.3. Design a universal primer to pre-amplify the combined product. It should be noted that the primers used for pre-amplification and subsequent PCR amplification must be the 5'-end parts (AATTCAGA, TATAGCTTGC) that are consistent with ② respectively, and must not be their complementary sequences. Otherwise, even if the ① and ② strands do not bind, during pre-amplification, the complementary strands of ① and ② (referred to as ①' strand and ②' strand) will be amplified separately. After re-raising the temperature, the complementary strands of the two single strands will dissociate. At this time, the ①' strand and ②' strand can also bind to each other, resulting in false positives).

[0106] To ensure relatively consistent amplification efficiency and relatively simplified experiments during the pre-amplification stage, a universal primer + specific probe must be designed, rather than a universal probe and specific primer. Add the universal primer to the product after antigen-antibody hybridization. For different samples, place them in different wells (the wells of the 96-well plate for antigen-antibody hybridization) or tubes (the PCR eight-strip tubes for antigen-antibody hybridization) respectively for PCR pre-amplification. The final product will obtain: 1 antibody + nucleic acid combination, and several double-stranded nucleic acids without protein linkage.

[0107] 1.4. Add the universal primer to the product after antigen-antibody hybridization. For different samples, place them in different wells (the wells of the 96-well plate for antigen-antibody hybridization) or tubes (the PCR eight-strip tubes for antigen-antibody hybridization) respectively for PCR pre-amplification. The final PCR pre-amplification product will obtain: 1 antibody + nucleic acid combination, and several double-stranded nucleic acids without protein linkage.

[0108] 2. Array plate design

[0109] Based on the macrofluidic digital droplet PCR technology, 48 samples (the above-mentioned PCR pre-amplification products) can be separately generated into a certain number of droplets with known sizes and quantities and enter the array plate 2. The ideal droplet size is 3 - 7 nanoliters, and the optimal is 5.6 nanoliters (diameter 210 micrometers). 200 - 300 micro-wells 102 can be arranged in the array plate 2. A specific probe dry body 200 is preset in each micro-well 102, and each micro-well 102 is a target to be measured. The ideal hydrophilic area is (0.2 - 0.4) mm × (1 - 2) mm, and the optimal is 0.3 × 1 mm.

[0110] For each experiment on each sample, first fill the consumables with the generated oil, and arrange the above-mentioned PCR pre-amplified products in each array plate 2 with a constant droplet size (macrofluidic microdroplet generation technology). The microarray in each array plate 2 can distribute several (3 - 5) small droplets with known droplet sizes. After the droplets absorb the freeze-dried probes, cover the lid and perform amplification, so as to fix each droplet in a determined microarray for amplification. Among them, 1. For the case of detecting one site for one sample, the above-mentioned microarray refers to a single micro-well or multiple micro-wells pre-embedded with the same probes. 2. For the case of detecting multiple sites for one sample, it is necessary to ensure that each micro-well has different probes, and the pre-amplified products of the same person are added to multiple micro-wells. That is, if one person detects 10 sites, the above-mentioned microarray can be understood as 10 micro-wells, each micro-well corresponding to one site, and the pre-amplified products of one person are added to 10 micro-wells.

[0111] 3. Monitoring of droplet PCR amplification in the microarray:

[0112] Design different probes according to the number of target sites to be detected, ensuring that the probes correspond one-to-one with the proteins to be detected, without conflict or repetition.

[0113] Generate droplets from the above-mentioned PCR pre-amplified products through a droplet generation device. The above-mentioned array plate 2 pre-embedded with specific probes receives the droplets, and fluorescence is monitored in real time on each reaction surface. Theoretically, the maximum throughput can reach 48 person-times × 200 - 300 target sites.

[0114] 4. Quality control design

[0115] 4.1. Designed an antibody directly connecting two oligonucleotides, that is, when antigen-antibody hybridization does not occur, nucleic acid complementary pairing can still occur, and subsequent pre-amplification, etc. can be carried out. For example: when other wells are negative and this well is positive, the detection is valid; when other wells are negative and this well is also negative, it proves that there is an abnormality in the amplification stage of the whole experiment, and the data of the whole plate is not credible.

[0116] 4.2. Set a negative control, that is, there is no antigen throughout the process, participate in the whole reaction, and monitor the LOB.

[0117] 4.3. Set a positive control, which participates in the reaction throughout the process, that is, a sample that will theoretically definitely be positive.

[0118] Application Example 2

[0119] 1. Pre-amplification: After measuring the concentration of the extracted DNA, take a partial volume according to a fixed concentration and add it to a PCR tube, add a mixed primer, and perform amplification in an ordinary PCR machine.

[0120] Remark: ①. For detecting the above-mentioned lung cancer ctDNA, about 100 pairs of primers + about 20 pairs of probes are required;

[0121] ②. In this step, 100 pairs of primers need to be pre-mixed, and it is necessary to verify in advance that all primer pairs can be amplified and the amplification efficiency is relatively consistent at a fixed ratio.

[0122] ③. In this step, it is necessary to pay attention to the quality control of the loading concentration and the number of amplification cycles, and control the final total number of loaded copies. When the copy number is too high, false positives may occur.

[0123] 2. Detection of the amplified product by loading it onto the instrument

[0124] At this time, there are two possible technical paths that need to be verified separately:

[0125] 2.1 Method 1:

[0126] ① The primers added in Step 1 need to be in appropriate excess, that is, it can ensure both Step 1 and the amount of primers in subsequent experiments.

[0127] ② Take the amplified product and re-add the probe method buffer (or the above buffer for trial use and its activity has not decreased).

[0128] ③ Place it in a digital PCR for reaction.

[0129] ④ In the microarray chip of the digital PCR, pre-embed the probe to be detected in advance. Observe the changes of several droplets (about 10) in each area, or it can be focused on observing, that is, fluorescence appears as positive; or continuous observation can be carried out.

[0130] 2.2 Method 2:

[0131] ① After the pre-amplification in Step 1 is completed, use magnetic beads for purification to remove the buffer and primers from the previous step and retain the amplified product.

[0132] ② Re-add the probe method buffer to the purified amplified product, and then follow Method 1 in 2.1.

[0133] 2.3 Method 3:

[0134] ① After the pre-amplification in Step 1 is completed, use magnetic beads for purification to remove the buffer and primers from the previous step and retain the amplified product.

[0135] Among them, in this Method 3, the primers used in Step 1 are specially treated and are different from the primers used in Step 1 in Method 1 and Method 2. The primers in Step 1 in Method 1 and Method 2 are fully paired multiplex primers. Taking one of the primers as an example, the following changes are made:

[0136] Primer design for Method 1 and Method 2:

[0137] Primer: 5’--AATTGCCGGT--3’

[0138] Template:..........................TTAACGGCCA..........................

[0139] The primer is completely complementary to the template

[0140] The primers used in Method 3 are as follows:

[0141] Primer: 5’--CGTCGGCA---AATTGCCGGT--3’

[0142] Template:............................TTAACGGCCA..........................

[0143] Modify the primer to the above form, that is, the AATTGCCGGT part is the original complementary part, and the CGTCGGCA part is the redesigned universal primer. It is necessary to ensure that the universal primer does not pair complementarily with the template to be tested;

[0144] When step 1 starts, amplify respectively starting from the AATTGCCGGT part, but each amplification product carries the CGTCGGCA part at the same time.

[0145] ② For the above special primer design in step 1, specifically design primer 2 for the PCR part, and this primer 2 is the universal primer.

[0146] ③ Add the purified and quantified pre-amplified PCR product to the universal primer, and generate droplets through a droplet generation device.

[0147] ④ The array plate receives the droplets. After the droplets absorb the probes freeze-dried on the reaction surface, PCR amplification is carried out on each reaction surface respectively.

[0148] Taking the detection of ctDNA in lung cancer using this method as an example, the size of human genomic DNA (Mb): 3000 Mb / copy x molecular weight of 1 bp: 1.096x10-21 g / bp x converting Mb to bp: 1x106 bp / Mb x converting g to ng: 1x109 ng / g = ng value per copy of DNA: 0.0033 ng / copy. Then, only the copy number concentration needs to be directly converted. If the copy number concentration is 3000 copies / μL, then the conversion using the following formula: 3000 copies / μL x 3.3 pg / copy (0.0033 ng / copy) = 9.9 ng / μL. That is, when the approximate loading amount is 10 ng, the total copy number is approximately 3000 copies. If the upper limit of the detectable mutant copy number is 3 in the experiment, then the lowest detection limit per locus at this time is approximately 0.1%. Therefore, for ctDNA with a relatively low concentration, to achieve the LOD, the most important thing is to load all the samples. Therefore, it is necessary to concentrate the initial DNA or increase the total reaction system. When the total volume corresponding to each well in this example is 20 μL, the loading volume is approximately 10 - 15 μL. Although samples may be wasted at this time, the LOD can barely be achieved. Therefore, in this example, before droplet splitting, pre-amplification is performed, and the method of re-splitting within the well is used to ensure that there are sufficient copy numbers in each minimum volume.

[0149] Comparative example: NGS detection small panel method

[0150] 3.1 Background introduction

[0151] Common NGS detections mainly include the semiconductor sequencing method of life and the reversible terminator method of illumina. Both methods need to go through: extraction, library construction, pooling, enrichment, on-machine detection, and data analysis. Among them, the ones closest to us in terms of throughput, time, and usage scenarios should be the S5 platform of life or the MiniSeq / NextSeq of illumina. Taking the S5 of life as an example, its main working process is introduced below.

[0152] 3.2 Practical introduction to the S5 platform

[0153] 1. Library construction:

[0154] Special amplicons and barcodes are added simultaneously, and a single ordinary PCR is used to achieve the purpose of the above three-stage process. After the amplification is completed, purification is carried out.

[0155] 2. Pooling

[0156] In library construction, the barcode is a label, that is, a known short sequence. During actual sequencing, according to the known short sequence and the initial number corresponding to the information of the sample to be tested, the one-to-one correspondence of all samples to be tested can be achieved.

[0157] For NGS, in each detection, multiple samples are mixed together and batch-sequenced on one chip, generating a total amount of data.

[0158] Therefore, each time the prepared library needs to be quantified first (quantified by the fluorescence quantitative PCR standard curve method or qubit quantification. Currently, to improve the speed, the latter is usually used for small panels, while the former is used only for ultra-high-throughput sequencing), because the quality (ng) during loading is in one-to-one correspondence with the data volume of a certain sample during sequencing. Therefore, according to the data volume requirements of the samples to be tested, preparing the required mass ratio. For the behavior of detecting the same panel of different samples, it is usually considered that all samples are mixed with equal mass.

[0159] Therefore, library mixing is the step of calculating the volume of library mixing based on equal mass and detection concentration, and then mixing different samples together according to the calculated volume.

[0160] The mixed library can obtain a theoretical concentration and an actual concentration. For example, the measured concentration after library mixing is 2.35 ng / μl, and it is also known that the maximum loading capacity of a single chip is 50 μl * 0.0066 ng / μl. Dilute the sample to 0.0066 ng / μl for standby.

[0161] 3. Enrichment

[0162] The complete enrichment is automatically completed inside the instrument, and the specific steps can include 3 steps.

[0163] ① OT2

[0164] On the special ISP beads, there are a sufficient number of upstream primers fixed, and this primer can be complementary paired with the adapter. By the instrument, a system like this is constructed: water-in-oil, where the water contains 1 ISP bead, a template to be tested, and a certain number of primers at the other end. Finally, in this water-in-oil system, 1 double-stranded ISP bead covered with the same sequence on the surface can be formed.

[0165] ② ES

[0166] In the end of the above steps, first use the demulsifier to open the water-in-oil system, and then remove the ISP beads that have not linked to DNA through magnetic beads, etc. And turn the double strand into a single strand. Finally, there will be left: ISP beads containing only 1 kind of sequence and those containing multiple kinds. In the final loading, the sequences output by those containing multiple kinds are disordered and will be discarded.

[0167] ③ Fabricate the loading chip

[0168] Add sequencing primers and enzymes to the above single-stranded ISP beads, and then inject them into the sequencing chip. The sequencing chip has multiple micro-pits, and each pit can accommodate 1 ISP bead.

[0169] 4. Sequencing

[0170] Place the prepared sequencing chip into the sequencer. Before using the sequencer, the sequencing reagent needs to be reloaded and the initialization completed. The sequencing time is about 2.5 - 3 hours. After that, the instrument will automatically analyze the data and generate a report. Usually, this step is selected for overnight analysis.

[0171] Comparative analysis:

[0172] Taking a small panel of about 10 genes in lung cancer as an example, the comparison of the experimental steps between Method 3 in Application Example 2 and the comparative example is shown in Table 1 below.

[0173] Table 1

[0174]

[0175] Method 3 in Application Example 2 has the advantages of low cost and short time.

[0176] For the above-mentioned biomolecular detection array plate 2 based on droplet PCR technology, the specific probe dry bodies 200 for droplet PCR detection are fixed on the reaction surface 10a of the substrate 100. The same or different specific probe dry bodies 200 can be fixed. The probes are provided by the array plate 2 and do not have to be included in the droplets, making the detection process more convenient; especially for the specific detection of different targets (such as different proteins) is very convenient. Compared with the traditional droplet PCR detection system using a microfluidic chip or a microwell 102 plate as the bottom plate, the above-mentioned array plate 2 is easy to manufacture, and can adopt a high-density array design and an open structure, which is conducive to reducing the sample requirement and realizing highly sensitive, high-throughput and highly specific protein detection. The minimum sample required for detecting 3000 grids of proteins is only 6 μL. For multiplex nucleic acid site detection, it has the advantages of low cost and short time.

[0177] Especially, the macrofluidic micro-droplet generation and droplet microarray technology based on droplet PCR. When detecting each protein, a pair of specifically matched antibodies (or polyclonal antibodies directly divided into two halves) are designed to ensure the specificity of the immunological reaction level. By adding a pair of nucleic acid single strands that are paired one by one to the pair of antibodies, the cross-talk at the immunological level is avoided by virtue of the specificity of DNA pairing. Then, through a micro-PCR detection device, the double-stranded DNA pairing is quantitatively / semi-quantitatively detected using specific probes. Thus, ultra-sensitive and unbiased targeted proteomics in the biological sense is achieved, which is especially suitable for blood samples that cannot be handled by traditional methods.

[0178] As shown in this specification and the claims, the terms "comprising" and "including" merely indicate the inclusion of the specifically identified steps and elements, and these steps and elements do not constitute an exclusive listing. The method or device may also include other steps or elements.

[0179] It can be further understood that in the present disclosure, "a plurality of" means two or more, and other quantifiers are similar thereto.

[0180] It can be further understood that the terms "first", "second", etc. are used to describe various information, but this information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other, and do not indicate a specific order or importance. In fact, the expressions "first", "second", etc. can be used interchangeably. For example, without departing from the scope of the present disclosure, the first information can also be referred to as the second information, and similarly, the second information can also be referred to as the first information.

[0181] The above embodiments are only for illustrating the technical concept and features of the present utility model, and are a preferred embodiment. The purpose is to enable those skilled in the art to understand the content of the present utility model and implement it accordingly, and it should not be used to limit the protection scope of the present utility model.

Claims

1. A biomolecule detection array plate for droplet PCR reaction, comprising a substrate, characterized in that: The substrate has a plurality of reaction surfaces distributed in an array and spaced apart from each other, each of the reaction surfaces providing an area for droplet PCR reaction; the array plate also includes a lyophilized body of a specific probe disposed on the reaction surface, the specific probe being used to specifically bind to a target biological molecule to be detected or a marker connected to a target biological molecule to be detected.

2. The biomolecule detection array plate according to claim 1, characterized in that: The reaction surface is a hydrophilic surface. 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.

3. The biomolecule detection array plate according to claim 2, characterized in that: The hydrophobic layer is coated on the surface of the substrate, and the width of the hydrophobic layer is 0.3-0.5 mm.

4. The biomolecule detection array plate according to claim 2, characterized in that: The upper surface of the substrate is a hydrophilic surface, and the hydrophobic layer is patternedly coated on the upper surface to form the plurality of reaction surfaces separated and enclosed by the hydrophobic layer.

5. The biomolecule detection array plate according to claim 2, characterized in that: The surface of the substrate and the reaction surface are respectively planes.

6. The biomolecule detection array plate according to claim 2, characterized in that: A plurality of micropores are arranged on the surface of the substrate, and the pore walls of the micropores constitute the reaction surface; the hydrophobic layer is coated on the portion of the surface where no micropores are arranged.

7. The biomolecule detection array plate according to claim 6, characterized in that: The depth of the micropores is less than or equal to 0.3 mm; and / or, the volume of the micropores is less than or equal to 2.5 mL; and / or, the micropores are distributed in an array; and / or, the number of micropores on each substrate is 10 to 400.

8. The biomolecule detection array plate according to claim 1, characterized in that: The array plate comprises one, two or more lyophilized bodies of the specific probes, and the lyophilized bodies of the specific probes arranged on the multiple reaction surfaces are the same or different or partially the same.

9. The biomolecule detection array plate according to claim 1, characterized in that: The reaction surface has a width of 0.1 to 0.4 mm, a length of 1 to 17 mm, and an area of ​​less than or equal to 8 mm. 2 ; and / or, the sizes of each of the reaction surfaces are the same or different.

10. The biomolecule detection array plate according to claim 1, characterized in that: The number of reaction surfaces on each substrate is 10 to 400; and / or, the array distribution is a square array distribution or a circular array distribution or a combination of the two.

11. The biomolecule detection array plate according to any one of claims 1 to 10, characterized in that: The biomolecule detection array plate is a protein detection array plate or a nucleic acid detection array plate, and the array plate comprises lyophilized bodies of 10 to 400 specific probes.

12. A biomolecule detection system for droplet PCR reaction, comprising a droplet generating device, characterized in that: The biomolecule detection system further comprises a biomolecule detection array plate as claimed in any one of claims 1 to 11.

13. The biomolecule detection system according to claim 12, characterized in that: The droplet generating device includes a accommodating chamber with a variable volume, a control mechanism for controlling the volume of the accommodating chamber to make it change periodically, and a droplet generating tube having a first port and a second port that are relatively far apart, the first port of the droplet generating tube is connected to the accommodating chamber, the droplet generating device also includes a driving fluid mechanism for introducing a driving fluid into the accommodating chamber, the inner diameter of the second port of the droplet generating tube is greater than 0.1 mm, 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 according to claim 13, characterized in that: The inner diameter of the second port is 0.1-0.2 mm or 0.3-0.6 mm.

15. The biomolecule detection system according to any one of claims 12 to 14, characterized in that: The droplet generating device is configured to generate droplets with a volume of 1 to 20 nL.

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