WO2024067667A1 - 微流体芯片及其操作方法、数字elisa检测方法和用途 - Google Patents

微流体芯片及其操作方法、数字elisa检测方法和用途 Download PDF

Info

Publication number
WO2024067667A1
WO2024067667A1 PCT/CN2023/121903 CN2023121903W WO2024067667A1 WO 2024067667 A1 WO2024067667 A1 WO 2024067667A1 CN 2023121903 W CN2023121903 W CN 2023121903W WO 2024067667 A1 WO2024067667 A1 WO 2024067667A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
magnetic beads
microfluidic chip
detected
microwells
Prior art date
Application number
PCT/CN2023/121903
Other languages
English (en)
French (fr)
Inventor
许俊泉
刘燕
李芳�
朱家君
褚衍桥
蔡志刚
吴浩扬
Original Assignee
格物致和生物科技(北京)有限公司
格物智造科技(成都)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202211183184.0A external-priority patent/CN115639364A/zh
Priority claimed from CN202211183084.8A external-priority patent/CN115624991A/zh
Priority claimed from CN202211183268.4A external-priority patent/CN116008523A/zh
Application filed by 格物致和生物科技(北京)有限公司, 格物智造科技(成都)有限公司 filed Critical 格物致和生物科技(北京)有限公司
Publication of WO2024067667A1 publication Critical patent/WO2024067667A1/zh

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the present invention relates to biological detection, and in particular to a microfluidic chip and an operation method thereof, a digital ELISA detection method and application thereof.
  • microfluidic chips currently used in biological testing generally do not include fluid control structures such as liquid storage tanks and waste liquid tanks. This means that when adding samples, the speed of liquid addition by the sampling needle must be coordinated with the speed of liquid extraction by the negative pressure pump. On the other hand, when discharging waste liquid, a waste liquid bucket and connecting joints and fluid pipelines are required, which increases the complexity. At the same time, the waste liquid is stored in the waste liquid bucket, which increases the risk of environmental pollution.
  • microfluidic chips generally adopt a multi-channel detection channel chip structure design, which ensures the detection throughput but increases the cost of a single detection and reduces flexibility.
  • One type uses highly sensitive optical instruments to detect single molecule signals.
  • Representative products include Merck's SMC TM (Single Molecule Counting) system.
  • SMC TM Single Molecule Counting
  • the optical path structure of this system is complex and expensive, the detection speed is slow, and the detection time is also very long for serial detection of microspheres.
  • Another type uses amplification technology to increase the number of target molecules to be detected in order to provide enough signal molecules.
  • Representative products include Chimera's
  • the detection method of this system is complicated and prone to false positive signals.
  • Quanterix's Simoa TM Single-molecule Array
  • digital ELISA Enzyme Linked Immunosorbent Assay
  • PCR Polymerase Chain Reaction
  • this system is a relatively quantitative digital ELISA detection method.
  • standard target molecules of known concentrations are required for calibration to make a standard curve.
  • concentration of target molecules to be detected are inferred by fitting the standard curve formula.
  • the system uses two analysis algorithms, digital and analog, for quantification, which neither fully utilizes the dynamic detection range of digital analysis nor reduces resolution and robustness.
  • the background signal of a single reaction detection unit in some current systems is high, and the fluorescent substance generated in a single reaction detection unit will diffuse to adjacent reaction detection units, thereby generating fluorescent signal crosstalk, affecting the accuracy of distinguishing negative and positive signals.
  • the present invention provides a microfluidic chip and its operation method and use, which has anti-pollution function, reliable, sensitive and rapid detection, simple structure and low price.
  • the present invention also provides an absolute quantitative digital ELISA detection method, which does not need to rely on standard samples and standard curves, has a precise and controllable dynamic detection range, high sensitivity and high accuracy.
  • the present invention also provides a digital ELISA detection method for preventing fluorescence signal crosstalk, reducing the fluorescence signal background of the reaction system, and reducing the diffusion of fluorescence signals in the reaction detection unit, thereby improving the accuracy of distinguishing negative signals and positive signals.
  • the present invention provides a microfluidic chip, the microfluidic chip comprising:
  • a lower shell comprising a reaction pool, a guide groove and a waste liquid pool formed on the inner surface of the lower shell and connected to each other;
  • a chip body wherein the chip body is arranged in a chip area of the reaction pool
  • the upper shell comprises an injection hole penetrating the upper shell and corresponding to the injection end of the reaction pool, and an air vent penetrating the upper shell and corresponding to the waste liquid pool.
  • the microfluidic chip further comprises a sealing gasket, and the sealing gasket is disposed on the injection hole.
  • the microfluidic chip further comprises a gas permeable membrane, and the gas permeable membrane is disposed on the gas permeable hole.
  • the chip body is a micropore array chip body including a micropore array
  • the micropore array includes 5,000 to 10 million micropores
  • the diameter of the micropores is 1 ⁇ m to 120 ⁇ m
  • the depth of the micropores is 1 ⁇ m to 120 ⁇ m
  • the center distance between the micropores is 3 ⁇ m to 180 ⁇ m.
  • the micropore array includes 188,000 micropores, the diameter of the micropores is 4 ⁇ m, the depth of the micropores is 4 ⁇ m, and the center distance between the micropores is 8 ⁇ m.
  • the micropore array includes 8800 micropores, the diameter of the micropores is 70 ⁇ m, the depth of the micropores is 70 ⁇ m, and the center distance between the micropores is 105 ⁇ m.
  • one or more positioning holes are arranged on the inner surface of the lower shell, and one or more positioning columns matched with the one or more positioning holes are arranged on the inner surface of the upper shell.
  • an overflow trough surrounding the reaction pool, the guide trough and the waste liquid pool is provided on the inner surface of the lower shell, and a welding line surrounding the injection hole and the air vent and matching the overflow trough is provided on the inner surface of the upper shell.
  • the upper shell further comprises an exhaust groove formed on the outer surface of the upper shell and connected to the exhaust hole, and one end of the exhaust groove away from the exhaust hole extends on the outer surface of the upper shell, so that the upper shell extends to form a hand-held portion.
  • the present invention further provides an operating method of the microfluidic chip described above, the operating method comprising:
  • Step 1 adding the sample into the injection hole once or in batches
  • Step 2 the sample added each time enters the reaction pool and the chip body in the chip area by self-absorption, centrifugation or pressure injection;
  • Step 3 centrifuging the microfluidic chip so that the sample enters the waste liquid pool from the reaction pool, while the sample in the chip body remains therein;
  • Step 4 adding the isolation liquid into the injection hole
  • Step 5 the isolation liquid enters the reaction pool by self-absorption, centrifugation or pressure injection to isolate the sample in the chip body;
  • Step 6 waiting for the sample in the chip body to undergo a biochemical reaction
  • Step 7 Perform imaging detection and digital analysis on the chip body.
  • the present invention further provides a use of the microfluidic chip described above in digital ELISA detection.
  • the present invention further provides a use of the microfluidic chip described above in digital PCR detection.
  • the present invention further provides an absolute quantitative digital ELISA detection method implemented using the microfluidic chip described above, the method comprising:
  • Step 1 preparing a sample, wherein the sample contains a target molecule to be detected, capturing the target molecule to be detected in the sample using magnetic beads, and forming a complex of "magnetic beads-target molecule to be detected-enzyme" through affinity reaction, wherein the enzyme can undergo enzymatic reaction with a fluorescent substrate to generate a fluorescent molecule;
  • Step 2 transferring the sample and the fluorescent substrate formed with the complex into the microfluidic chip, wherein the chip body is a microwell array chip body including a microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead;
  • Step 3 adding an isolation liquid into the microfluidic chip to isolate all microwells in the microwell array from each other;
  • Step 4 waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate the fluorescent molecule, wherein the fluorescence signal of the microwell where the magnetic beads that capture a single target molecule to be detected are located is higher than a threshold value, and the fluorescence signal of the microwell where the magnetic beads that capture zero target molecules to be detected are located is lower than a threshold value;
  • Step 5 determining the number of microwells in the microwell array containing the magnetic beads, and determining the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value;
  • Step 6 Determine the number of target molecules to be detected in the sample based on the number of microwells in the microwell array containing the magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.
  • the number of the magnetic beads in the sample is determined based on counting using a particle counter, flow cytometry or a cell counting plate.
  • the ratio of the number of the target molecules to be detected in the sample to the number of the magnetic beads in the sample is less than or equal to 100.
  • the ratio of the number of the target molecules to be detected in the sample to the number of the magnetic beads in the sample is less than or equal to 5.
  • step 5 bright field images and fluorescence images of one or more fields of view are taken of the microwell array, wherein the number of microwells in the microwell array containing the magnetic beads is determined based on the bright field images of the one or more fields of view, and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold is determined based on the fluorescence images of the one or more fields of view.
  • step 6 the following formula is used to determine the number of the target molecules to be detected in the sample: Wherein, M0 is the number of the target molecules to be detected in the sample, N0 is the number of the magnetic beads in the sample, M is the number of microwells in the microwell array whose fluorescence signals are higher than the threshold, N is the number of microwells in the microwell array containing the magnetic beads, and p is the probability of the target molecules to be detected being captured by the magnetic beads and further binding to the enzyme, or the capture probability.
  • the ratio of the number of all microwells in the microwell array to the number of the magnetic beads in the sample is in the range of 0.1 to 10.
  • step 2 the sample and the fluorescent substrate forming the complex are transferred to the reaction pool of the microfluidic chip and the microwells of the microwell array by self-absorption, centrifugation or pressure injection, and the excess magnetic beads and fluorescent substrate in the reaction pool are removed by centrifugation, and the magnetic beads and fluorescent substrate in the microwells are retained.
  • step 3 the isolation liquid is added to the reaction pool of the microfluidic chip by self-absorption, centrifugation or pressure injection, and the magnetic beads and fluorescent substrate in the microwells of the microwell array are sealed and isolated.
  • the target molecule to be detected is a protein molecule to be detected.
  • the isolation liquid is fluorine oil or silicone oil.
  • the present invention further provides a digital ELISA detection method for preventing fluorescence signal crosstalk implemented by the microfluidic chip described above, the method comprising:
  • Step 1 prepare a sample, wherein the sample contains a target molecule to be detected, capture the target molecule to be detected in the sample using magnetic beads, and form a complex of "magnetic beads-target molecule to be detected-enzyme" through affinity reaction;
  • Step 2 transferring the sample formed with the complex into the microfluidic chip, wherein the chip body is a microwell array chip body including a microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead;
  • Step 3 transferring the fluorescent substrate into the microfluidic chip, wherein the enzyme can perform an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules;
  • Step 4 adding an isolation liquid into the microfluidic chip to isolate all microwells in the microwell array from each other;
  • Step 5 waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate the fluorescent molecule, wherein the fluorescence signal of the microwell where the magnetic beads that capture a single target molecule to be detected are located is higher than a threshold value, and the fluorescence signal of the microwell where the magnetic beads that capture zero target molecules to be detected are located is lower than a threshold value;
  • Step 6 determining the number of microwells in the microwell array containing the magnetic beads, and determining the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value;
  • Step 7 Determine the number of target molecules to be detected in the sample based on the number of microwells in the microwell array containing the magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.
  • the isolation fluid is fluorine oil, and the viscosity coefficient of the fluorine oil at 25° C. is in the range of 0.1 cSt to 12500 cSt.
  • the viscosity coefficient of the fluorine oil at 25° C. is in the range of 1 cSt to 5000 cSt.
  • the viscosity coefficient of the fluorine oil at 25° C. is 11.4 cSt, 451 cSt or 1366 cSt.
  • the isolation fluid is silicone oil
  • the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 12500 cSt.
  • the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 5000 cSt.
  • the viscosity coefficient of the silicone oil at 25° C. is 350 cSt.
  • the number of the magnetic beads in the sample is determined based on counting using a particle counter, flow cytometry or a cell counting plate.
  • the ratio of the number of the target molecules to be detected in the sample to the number of the magnetic beads in the sample is less than or equal to 100.
  • the ratio of the number of the target molecules to be detected in the sample to the number of the magnetic beads in the sample is less than or equal to 5.
  • step 6 bright field images and fluorescence images of one or more fields of view are taken of the microwell array, wherein the number of microwells containing the magnetic beads in the microwell array is determined based on the bright field images of the one or more fields of view, and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value is determined based on the fluorescence images of the one or more fields of view.
  • step 7 the following formula is used to determine the number of the target molecules to be detected in the sample:
  • M0 is the number of the target molecules to be detected in the sample
  • N0 is the number of the magnetic beads in the sample
  • M is the number of microwells in the microwell array whose fluorescence signals are higher than the threshold
  • N is the number of microwells in the microwell array containing the magnetic beads
  • p is the probability of the target molecules to be detected being captured by the magnetic beads and further binding to the enzyme, or the capture probability.
  • the ratio of the number of all microwells in the microwell array to the number of the magnetic beads in the sample is in the range of 0.1 to 10.
  • step 2 the sample formed with the complex is transferred to the reaction pool of the microfluidic chip and the microwells of the microwell array by self-aspiration, centrifugation or pressure injection, and the excess magnetic beads in the reaction pool are removed by centrifugation, and the magnetic beads in the microwells are retained.
  • step 3 the fluorescent substrate is transferred to the reaction pool of the microfluidic chip and the microwells of the microwell array by self-absorption, centrifugation or pressure injection, and the excess fluorescent substrate in the reaction pool is removed by centrifugation, and the fluorescent substrate in the microwell is retained.
  • step 4 the isolation liquid is added to the reaction pool of the microfluidic chip by self-absorption, centrifugation or pressure injection, and the magnetic beads and fluorescent substrate in the microwells of the microwell array are sealed and isolated.
  • the target molecule to be detected is a protein molecule to be detected.
  • the present invention has the following beneficial effects:
  • the microfluidic chip integrates the injection chamber, the reaction chamber and the waste liquid chamber, and has a simple and compact structure, low cost, and does not require an additional negative pressure pump and a waste liquid bucket as well as connecting joints and fluid pipelines.
  • the microfluidic chip can be a disposable consumable. After the reaction is completed, the chip containing the waste liquid is stored in a specific area for unified treatment to prevent contamination.
  • each microfluidic chip is an independent detection channel for the detection of a sample. A single chip and multiple chips can be selected to work simultaneously, and the detection is reliable, sensitive and fast.
  • the microfluidic chip by selecting a microfluidic chip including a suitable chip body and by adding a suitable sample, the microfluidic chip can be operated to achieve different detections, such as digital ELISA detection and digital PCR detection.
  • the target molecules to be detected in the sample can be absolutely quantified by the number of sample distribution units clearly determined by counting methods such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields of view, the number of positive reaction detection units determined by fluorescent images of one or more fields of view, and the probability of the target molecules to be detected being captured by magnetic beads and further binding to the enzyme as a constant.
  • the number of magnetic beads in the sample determines the upper limit of the dynamic detection range.
  • the dynamic detection range can be precisely controlled. Theoretically, when the capture probability is 100%, the number of target molecules to be detected in the sample is less than or equal to 5 times the number of magnetic beads. When the capture probability is 5%, the number of target molecules to be detected in the sample is less than or equal to 100 times the number of magnetic beads.
  • the fluorescence signal background of the reaction system is greatly reduced by the two-step sample addition method, and the diffusion of the fluorescence signal in the reaction detection unit is greatly reduced by preferably using an isolation liquid with a high viscosity coefficient, thereby improving the accuracy of distinguishing negative signals and positive signals.
  • FIG1 is a schematic diagram of the overall structure of a microfluidic chip according to an embodiment of the present invention.
  • FIG2 is an exploded schematic diagram of a microfluidic chip according to an embodiment of the present invention.
  • FIG3 is a schematic structural diagram of a lower housing of a microfluidic chip according to an embodiment of the present invention.
  • FIG4 is a schematic structural diagram of an upper housing of a microfluidic chip according to an embodiment of the present invention.
  • FIG5 is a side view of a microfluidic chip according to an embodiment of the present invention.
  • 6A and 6B are scanning electron microscope images of a microwell array chip body of a microfluidic chip according to an embodiment of the present invention
  • FIG7 is a schematic flow chart of a method for operating a microfluidic chip according to an embodiment of the present invention.
  • FIG8 is a schematic diagram of the principle of an absolute quantitative digital ELISA detection method implemented using a microfluidic chip according to an embodiment of the present invention.
  • FIG9 is a schematic flow diagram of an absolute quantitative digital ELISA detection method implemented using a microfluidic chip according to an embodiment of the present invention.
  • FIG10 is a schematic diagram of the principle of a digital ELISA detection method for preventing fluorescence signal crosstalk using a microfluidic chip according to an embodiment of the present invention
  • FIG11 is a schematic flow diagram of a digital ELISA detection method for preventing fluorescence signal crosstalk using a microfluidic chip according to an embodiment of the present invention
  • FIG. 12A is a fluorescence image obtained using a two-step sample addition method
  • FIG. 12B is a fluorescence image obtained using a one-step sample addition method
  • FIG. 13A is a fluorescence signal of a certain column of microwells extracted and calculated from the fluorescence image shown in FIG. 12A
  • FIG. 13B is a fluorescence signal of a certain column of microwells extracted and calculated from the fluorescence image shown in FIG. 12B ;
  • FIG. 14A1-FIG. 14D2 are fluorescence images after 1 minute and 5 minutes of isolation using different isolation solutions
  • FIG. 15A1-FIG. 15B2 are schematic diagrams showing changes in the fluorescence values of the positive micropores and the crosstalk micropores over time after isolation using different isolation solutions.
  • FIG. 1 is a schematic diagram of the overall structure of a microfluidic chip according to an embodiment of the present invention
  • FIG. 2 is an exploded schematic diagram of the microfluidic chip according to an embodiment of the present invention.
  • the microfluidic chip 10 includes a lower housing 11, and the lower housing 11 can be made of one or more plastic materials such as polycarbonate (PC), polystyrene (PS), polymethyl methacrylate (PMMA), cyclo olefin copolymer (COC), and cyclo olefin polymer (COP) by conventional injection molding process, and the minimum processing accuracy is above 20 ⁇ m.
  • the material of the lower housing 11 is PC material.
  • the lower shell 11 may be in an elongated shape, and may have a first direction, a second direction, and a third direction perpendicular to each other, wherein the first direction is a length direction, the second direction is a width direction, and the third direction is a depth direction. It is understood that, depending on actual needs, the lower shell 11 may also be in any other shape, which is not limited here.
  • the lower housing 11 may have a first surface and a second surface opposite to each other, wherein the first surface located inside the microfluidic chip 10 is an inner surface, and the second surface located outside the microfluidic chip 10 is an outer surface.
  • FIG. 3 is a schematic structural diagram of a lower housing of a microfluidic chip according to an embodiment of the present invention.
  • the lower shell 11 includes a reaction pool (also referred to as a fluid chamber) 111, a guide groove 112 and a waste liquid pool 113 formed on the inner surface and interconnected with each other.
  • the reaction pool 111, the guide groove 112 and the waste liquid pool 113 can be arranged in sequence in the length direction (such as from right to left).
  • the reaction pool 111 includes an injection end, a chip area 111b and a sample outlet end, which can be arranged sequentially in the length direction (such as from right to left) so that the sample can pass through the injection end, the chip area 111b and the sample outlet end sequentially.
  • the injection end of the reaction pool 111 corresponds to the injection hole 131 formed on the outer surface of the upper shell 13 to be described below.
  • the sample will enter the injection end of the reaction pool 111 from the injection hole 131 and pass through the chip area 111b and the sample outlet end in sequence.
  • the chip region 111 b of the reaction pool 111 may be a through hole penetrating the lower housing 11 , and the shape and size of the through hole are adapted to the shape and size of the chip body 12 to be described below.
  • the sample outlet of the reaction pool 111 is connected to the guide groove 112, and the width of the guide groove 112 can be set to be relatively small, so as to avoid the sample from flowing into the waste liquid pool 113 before sufficient biochemical reaction.
  • the guide groove 112 can adopt a slope flow channel structure, so that the fluid can easily flow into the waste liquid pool 113 and is not easy to flow back from the waste liquid pool 113.
  • the guide groove 112 is connected to the waste liquid pool 113, so that the sample after sufficient reaction can flow into the waste liquid pool 113, thereby preserving the waste liquid.
  • the microfluidic chip 10 further includes a chip body 12.
  • the chip body 12 and the chip area 111b of the reaction pool 111 are engaged in the chip area 111b by size matching. It is understandable that the chip body 12 can be further fixed by a sealant. After the sample passes through the injection end, it will contact the chip body 12 in the chip area 111b, and a biochemical reaction will be performed at the chip body 12.
  • the chip body 12 can be made of one or more plastic materials such as polycarbonate (PC), polystyrene (PS), polymethyl methacrylate (PMMA), cyclo olefin copolymer (COC), and cyclo olefin polymer (COP), etc., using a CD injection molding process, and the minimum processing accuracy is 100nm-50 ⁇ m.
  • the chip body 12 can also be made of one or more semiconductor materials such as silicon and glass using a semiconductor processing process, and the minimum processing accuracy is 100nm-50 ⁇ m.
  • the microfluidic chip 10 further includes an upper shell 13, and the upper shell 13 can be made of one or more plastic materials selected from polycarbonate (PC), polystyrene (PS), polymethyl methacrylate (PMMA), cyclo olefin copolymer (COC), and cyclo olefin polymer (COP) by conventional injection molding process, and the minimum processing accuracy is above 20 ⁇ m.
  • the upper shell 13 is made of PC material.
  • the upper shell 13 may be in an elongated shape, and may have a first direction, a second direction, and a third direction perpendicular to each other, wherein the first direction is a length direction, the second direction is a width direction, and the third direction is a depth direction. It is understood that, depending on actual needs, the upper shell 13 may also be in any other shape, which is not limited here.
  • the upper housing 13 may have a first surface and a second surface opposite to each other, wherein the first surface located inside the microfluidic chip 10 is an inner surface, and the second surface located outside the microfluidic chip 10 is an outer surface.
  • FIG. 4 is a schematic structural diagram of an upper housing of a microfluidic chip according to an embodiment of the present invention.
  • the upper shell 13 includes an injection hole 131 penetrating the upper shell 13 and corresponding to the injection end of the reaction pool 111 , and an air vent 132 penetrating the upper shell 13 and corresponding to the waste liquid pool 113 .
  • the injection hole 131 corresponds to the injection end of the reaction cell 111 , and is connected to an external sample injection device, so that the sample enters the injection end of the reaction cell 111 through the injection hole 131 .
  • the vent hole 132 corresponds to the waste liquid pool 113 and can release the air pressure in the microfluidic chip 10 .
  • the upper shell 13 further includes an exhaust groove 133 formed on the outer surface of the upper shell 13 and connected to the air vent 132.
  • the injection hole 131, the air vent 132 and the exhaust groove 133 may be arranged sequentially in the length direction (such as from right to left).
  • the exhaust groove 133 is connected to the air hole 132 to further release the air pressure in the microfluidic chip 10 .
  • FIG. 5 is a side view of a microfluidic chip according to one embodiment of the present invention.
  • the lower shell 11 and the upper shell 13 are assembled together so that the inner surface of the lower shell 11 is opposite to the inner surface of the upper shell 13, and the side surfaces extending toward each other on the two inner surfaces are jointly surrounded to form an injection chamber 51, a reaction chamber 52 and a waste liquid chamber 53.
  • the depths of the injection chamber 51, the reaction chamber 52, and the waste chamber 53 can be reasonably set to limit the volumes of these chambers respectively.
  • the depths of the injection chamber 51 and the reaction chamber 52 are both 500 ⁇ m, and the total volume of the two chambers is 26 ⁇ L.
  • the depth of the waste chamber 53 can be set larger, so that the volume of the waste chamber 53 is also larger to accommodate all samples and isolation fluid (also referred to as separation oil).
  • the chip body 12 is embedded in the chip area 111b, and the upper surface of the chip body 12 is flush with the lower surface of the injection chamber 51 and the lower surface of the guide groove 112 before the waste liquid chamber 53, so that the lower surface of the injection chamber 51, the lower surface of the reaction chamber 52 and the lower surface of the guide groove 112 are flush with each other, which is convenient for the flow of the reaction fluid and the isolation liquid. It can be understood that when the guide groove 112 adopts a ramp flow channel structure, the lower surface of the injection chamber 51 and the lower surface of the reaction chamber 52 are flush with each other.
  • the microfluidic chip further includes a sealing gasket 14, which is disposed on the injection hole 131, thereby isolating the injection hole 131 from contact with the outside during non-sampling, and preventing foreign matter from entering the microfluidic chip 10 and contaminating the microfluidic chip 10. It is understandable that the sealing gasket 14 can be further fixed by a sealant.
  • the sealing pad 14 can be a silicone pad, and the size of the sealing pad 14 is adapted to the injection hole 131.
  • the outer diameter of the sealing pad 14 is 5 mm, the height is 1.5 mm, and the inner diameter is a cross-blade structure to achieve a better sealing effect.
  • the microfluidic chip further includes a breathable membrane 15, which is disposed on the breathable hole 132, thereby isolating the breathable hole 132 from contact with the outside world, and preventing foreign objects from entering the microfluidic chip 10 and contaminating the microfluidic chip 10. It is understandable that the breathable membrane 15 can be further fixed by a sealant.
  • the breathable membrane 15 may be a nitrocellulose membrane, and the size of the breathable membrane 15 is adapted to the breathable holes 132 .
  • the upper housing 13 may be made of a transparent material or a translucent material. Additionally or alternatively, as shown in FIG. 5 , a detection window 134 may be provided in a portion of the upper housing 13 corresponding to the chip area 111 b of the reaction pool 111. By so configuring, the biochemical reaction can be observed from the upper housing 13, and the chip body 12 can be imaged and detected.
  • 6A and 6B are scanning electron microscope images of a microwell array chip body of a microfluidic chip according to an embodiment of the present invention.
  • the chip body 12 is a micropore array chip body including a micropore array, and the micropore array chip body can have different shapes and sizes depending on the detection needs.
  • the micropore array chip body is a rectangular chip body of 3mm ⁇ 4mm.
  • the micropore array chip body is a square chip body of 10mm ⁇ 10mm.
  • the micropore array includes 5,000 to 10 million micropores 61 , the diameter of the micropore 61 is 1 ⁇ m to 120 ⁇ m, the depth of the micropore 61 is 1 ⁇ m to 120 ⁇ m, and the center distance between the micropores 61 is 3 ⁇ m to 180 ⁇ m.
  • the shape of the micropore 61 may be circular, and depending on the detection requirements, the micropore 61 may have different shapes, such as a hexagon.
  • the diameter of the micropore 61 may refer to the diameter of the circumscribed circle of the micropore 61.
  • the microwell array chip body is a rectangular chip body of 3 mm ⁇ 4 mm, and the microwell array includes 188,000 circular microwells 61, the diameter of the microwell 61 is 4 ⁇ m, the depth of the microwell 61 is 4 ⁇ m, and the center distance between the microwells 61 is 8 ⁇ m.
  • This microwell array chip body is particularly suitable for digital ELISA detection.
  • the microwell array chip body is a 10 mm ⁇ 10 mm square chip body, and the microwell array includes 8800 hexagonal microwells 61, the circumscribed circle diameter of the microwell 61 is 70 ⁇ m, the depth of the microwell 61 is 70 ⁇ m, and the center distance between the microwells 61 is 105 ⁇ m.
  • This microwell array chip body is particularly suitable for digital PCR detection.
  • one or more positioning holes 114 are provided on the inner surface of the lower shell 11, and one or more positioning posts 135 are provided on the inner surface of the upper shell 13 to match the one or more positioning holes 114.
  • the positioning holes 114 and the positioning posts 135 Through the cooperation of the positioning holes 114 and the positioning posts 135, the lower shell 11 and the upper shell 13 can be positioned relative to each other, so that the injection hole 131 corresponds to the injection end of the reaction pool 111, and the air vent 132 corresponds to the waste liquid pool 113, so as to avoid the injection chamber 51, the reaction chamber 52 and the waste liquid chamber 53 no longer being closed due to misalignment.
  • two positioning holes 114 are provided on the inner surface of the lower shell 11, and at both ends in the length direction.
  • two positioning columns 135 matching the two positioning holes 114 are provided on the inner surface of the upper shell 13, and also at both ends in the length direction.
  • an overflow groove 115 is provided on the inner surface of the lower shell 11 surrounding the reaction pool 111, the guide groove 112 and the waste liquid pool 113, and a welding line 136 is provided on the inner surface of the upper shell 13 surrounding the injection hole 131 and the vent hole 132 and matching the overflow groove 115.
  • the lower shell 11 and the upper shell 13 can be welded together by ultrasonic welding or laser welding the welding line 136, and the overflow generated by welding flows into the overflow groove 115, avoiding flowing into the reaction pool 111, the guide groove 112 and the waste liquid pool 113.
  • the overflow groove 115 and the welding line 136 can be reasonably arranged to fix the welding position and the height of the package.
  • one end of the exhaust groove 133 away from the air vent 132 extends on the outer surface of the upper housing 13, so that the upper housing 13 extends to form a hand-held portion 137.
  • the exhaust groove 133 extends outward in the length direction, and the gas in the microfluidic chip 10 can be released to the outside, and the hand-held portion 137 formed is convenient for the user to operate the microfluidic chip 10.
  • the microfluidic chip integrates the injection chamber, the reaction chamber and the waste liquid chamber, and has a simple and compact structure, low cost, and does not require an additional negative pressure pump and a waste liquid bucket as well as connecting joints and fluid pipelines.
  • the microfluidic chip can be a disposable consumable. After the reaction is completed, the chip containing the waste liquid is stored in a specific area for unified treatment to prevent contamination.
  • each microfluidic chip is an independent detection channel for the detection of a sample. A single chip and multiple chips can be selected to work simultaneously, and the detection is reliable, sensitive and fast.
  • FIG. 7 is a schematic flow chart of a method for operating a microfluidic chip according to an embodiment of the present invention.
  • the sample is added to the injection hole once or in batches.
  • the sample may be different.
  • the sample may include protein sample molecules to be tested and magnetic beads or microspheres
  • the sample may include nucleic acid sample molecules to be tested and PCR reaction reagents.
  • the sample may also include a fluorescent substrate, which may be added to the injection hole together with the protein sample molecules to be tested and magnetic beads or microspheres once, or may be added to the injection hole in batches, for example, the protein sample molecules to be tested and magnetic beads or microspheres are added first, and then the fluorescent substrate is added.
  • the microfluidic chip may also be centrifuged during the addition of the sample, and the centrifugal force generated is converted into liquid shear force to improve the efficiency of the addition of the sample.
  • each added sample enters the reaction pool and the chip body in the chip area by self-absorption, centrifugation or pressure injection.
  • the microfluidic chip is centrifuged by centrifugal injection so that each added sample enters the reaction pool and the chip body in the chip area.
  • specific centrifugal conditions for example, 200rpm for 10 seconds
  • the centrifugal force sends the sample into the reaction pool. Due to the constraints of the flow guide groove, the fluid remains in the reaction pool. Stand for a specific time (for example, 2 minutes) until the sample settles into the micropores of the chip body.
  • microfluidic chip can also be centrifuged during the period of standing and waiting for sedimentation (for example, 200rpm for 20 seconds), and the centrifugal force generated is converted into liquid shear force to improve the efficiency of sample addition. It is understandable that self-absorption or pressure injection can also be used to make the sample added each time enter the reaction pool and the chip body in the chip area.
  • the microfluidic chip is centrifuged so that the sample enters the waste liquid pool from the reaction pool, while the sample in the chip body is retained therein.
  • centrifugation is applied again (for example, 600rpm for 10 seconds).
  • the excess sample in the reaction pool enters the waste liquid pool through the guide groove of the ramp flow channel structure, while the sample in the micropores of the chip body is retained.
  • the volume of the waste liquid cavity decreases, the gas pressure increases, and the gas pressure is released to the outside of the microfluidic chip through the air vents and the air permeable membrane.
  • a spacer liquid is added to the injection hole.
  • the spacer liquid can be different.
  • the spacer liquid can include fluorine oil or silicone oil with a high viscosity coefficient.
  • the isolation liquid enters the reaction pool by self-absorption, centrifugation or pressure injection to isolate the samples in the chip body.
  • the microfluidic chip is centrifuged by centrifugal injection to allow the isolation liquid to enter the reaction pool to isolate the samples in the chip body.
  • the centrifugal force sends the isolation liquid into the reaction pool.
  • the hydrophobic isolation liquid can fully infiltrate the chip surface, further remove the samples that have not fallen into the microwells, and at the same time isolate the samples in the microwells from each other, and the fluorescent products are difficult to diffuse, and the thermal stability of a single microwell is excellent. It can be understood that self-absorption or pressure injection can also be used to allow the isolation liquid to enter the reaction pool.
  • step 6 wait for the sample in the chip body to undergo a biochemical reaction.
  • the waiting time and the required biochemical reaction temperature are different. For example, for digital ELISA testing, you can wait for 1 minute at room temperature, and for digital PCR testing, you can wait for 15 seconds at 95°C, wait for 50 seconds at 60°C, and repeat 35 times.
  • the chip body is imaged and digitally analyzed.
  • An imaging system is used to capture the image of the chip body, and the imaging system may include components such as a mercury lamp light source, a filter, an objective lens, and a CCD camera.
  • the microfluidic chip can be fixed on a mobile platform, and the mobile platform moves the microfluidic chip so that the lens is aligned with different positions of the chip body to capture images of multiple areas.
  • a precise autofocus platform can adjust the distance between the lens and the micropore to ensure the capture of the clearest image.
  • a 20x objective lens can be used, and each microfluidic chip can capture images of multiple fields of view (for example, for digital ELISA detection, 35 fields of view are captured, and for digital PCR detection, 256 fields of view are captured).
  • a fluorescent image for example, 577nm excitation, 620nm emission, and exposure time 600ms
  • a bright field image for example, a mercury lamp light source, and exposure time 50ms
  • Digital analysis is performed based on the fluorescent image and the bright field image.
  • the microfluidic chip can be a disposable consumable. After the reaction is completed, the chip containing the waste liquid is stored in a specific area for unified treatment to prevent contamination. It is understandable that a cleaning liquid can be added to the injection hole, and the microfluidic chip is centrifuged so that the sample after the reaction enters the waste liquid pool, thereby achieving the cleaning of the microfluidic chip and the harmless treatment of the waste liquid.
  • the microfluidic chip by selecting a microfluidic chip including a suitable chip body and by adding a suitable sample, the microfluidic chip can be operated to achieve different detections, such as digital ELISA detection and digital PCR detection.
  • the microfluidic chip is used in a digital ELISA assay, and according to one embodiment of the present invention, the microfluidic chip is used in a digital PCR assay.
  • FIG8 is a schematic diagram of the principle of an absolute quantitative digital ELISA detection method implemented using a microfluidic chip according to an embodiment of the present invention
  • FIG9 is a schematic diagram of the process of an absolute quantitative digital ELISA detection method implemented using a microfluidic chip according to an embodiment of the present invention.
  • the absolute quantitative digital ELISA detection method implemented by the microfluidic chip includes:
  • step 1 a sample is prepared, the sample contains the target molecule to be detected, the target molecule to be detected in the sample is captured using magnetic beads, and a "magnetic bead-target molecule to be detected-enzyme" complex is formed through an affinity reaction, and the enzyme can undergo an enzymatic reaction with a fluorescent substrate to generate a fluorescent molecule.
  • the target molecule to be detected can be a protein molecule to be detected, and the protein molecule to be detected can be derived from a liquid sample of the human body (blood, body fluid, tissue, etc.). More specifically, the protein molecule to be detected can be from the supernatant of serum, plasma, tissue homogenate or cell extract. Based on this, the present invention can accurately quantify the ultra-low abundance protein molecules in normal people and disease patients that are difficult to detect by conventional methods, and develop new applications in the fields of early detection, companion diagnosis, and drug development of major diseases such as tumors, neurological diseases, infectious diseases, and immune inflammation.
  • major diseases such as tumors, neurological diseases, infectious diseases, and immune inflammation.
  • the magnetic beads may have a diameter of micrometer scale.
  • the surface of the magnetic beads is modified with a capture antibody that can specifically bind to the target molecule to be detected, for example, to produce an antibody-antigen reaction with the protein molecule to be detected, thereby capturing the protein molecule to be detected.
  • the number of magnetic beads in the sample can be clearly determined based on counting methods such as a particle counter, flow cytometry or a cell counting plate.
  • the uniformity of the magnetic beads in the sample can also be accurately controlled based on the above technology. Based on this, the present invention can absolutely quantify the target molecule to be detected in the sample based at least on the number of magnetic beads in the sample.
  • the number of magnetic beads in the sample should be much larger than the number of target molecules to be detected captured on the magnetic beads, so that the statistical distribution of the target molecules to be detected captured on the magnetic beads conforms to the Poisson distribution.
  • the number of magnetic beads in the sample may be less than or equal to the number of target molecules to be detected captured on the magnetic beads.
  • the ratio of the number of target molecules to be detected captured on the magnetic beads to the number of magnetic beads in the sample is less than or equal to 5 (i.e., each magnetic bead captures a maximum of 5 target molecules to be detected on average)
  • the statistical distribution of the target molecules to be detected captured on the magnetic beads still conforms to the Poisson distribution, and single-molecule fluorescence signal amplification can still be achieved.
  • the number of magnetic beads in the sample determines the upper limit of the dynamic detection range.
  • the dynamic detection range can be precisely controlled. Theoretically, when the capture probability is 100%, the number of target molecules to be detected in the sample is less than or equal to 5 times the number of magnetic beads. For example, the number of magnetic beads in the sample is in the range of 100,000 to 10 million, so the upper limit of the dynamic detection range is in the range of 500,000 to 50 million. It is understandable that, considering the capture probability factor, the ratio of the number of target molecules to be detected in the sample to the number of magnetic beads in the sample can also be greater than or equal to 5. When the capture probability is 5%, the number of target molecules to be detected in the sample is less than or equal to 100 times the number of magnetic beads.
  • the capture antibody modified on the surface of the magnetic beads specifically captures the target molecule to be detected in the sample, and further connects the detection antibody and the enzyme, and finally forms an immune complex of "magnetic beads-capture antibody-target molecule to be detected-detection antibody-enzyme", and the enzyme can react enzymatically with the fluorescent substrate to generate fluorescent molecules.
  • the magnetic beads can be connected with ⁇ -galactosidase through the above-mentioned double antibody sandwich reaction
  • the fluorescent substrate can be non-fluorescent resorufin- ⁇ -galactoside (RGP)
  • ⁇ -galactosidase can catalyze the hydrolysis of non-fluorescent resorufin- ⁇ -galactoside (RGP) to generate resorufin molecules that can emit fluorescence.
  • the sample and the fluorescent substrate formed with the complex are transferred to a microfluidic chip, wherein the chip body is a microwell array chip body including a microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead. As described above, the microwell array and its chip body are accommodated in the chip area of the reaction pool.
  • the micropore array includes a plurality of micropores, and the size of each micropore can be slightly larger than the size of the magnetic bead, so as to be configured to accommodate only one magnetic bead.
  • the ratio of the number of all micropores in the micropore array to the number of magnetic beads in the sample is in the range of 0.1 to 10, so that as many magnetic beads as possible fall into the micropores.
  • the micropores can be called effective micropores or reaction detection units.
  • the number of reaction detection units i.e., effective micropores
  • the number of reaction detection units is not greater than the number of sample distribution units (i.e., magnetic beads). In theory, the closer the number of reaction detection units is to the number of sample distribution units, the higher the accuracy and resolution of digital detection.
  • the microwell array includes 188,000 microwells, the microwells are circular microwells, the diameter of the microwells is 4 ⁇ m, the depth of the microwells is 4 ⁇ m, and the center distance between the microwells is 8 ⁇ m.
  • the sample and fluorescent substrate formed with the complex are transferred to the microfluidic chip, and the first centrifugal condition is, for example, 200rpm for 10 seconds, and the centrifugal force sends the sample and the fluorescent substrate into the reaction pool of the microfluidic chip. Then, stand for a specific time (for example, 2 minutes) until the sample and the fluorescent substrate are settled in the micropores of the micropore array. Then, under the second centrifugal condition, the excess magnetic beads and fluorescent substrate in the reaction pool are removed, and the magnetic beads and fluorescent substrate in the micropores are retained.
  • the second centrifugal condition is, for example, 600rpm for 10 seconds.
  • sample and fluorescent substrate formed with the complex can also be transferred to the microfluidic chip by means of self-absorption or pressure injection.
  • a spacer liquid is added to the microfluidic chip to isolate all microwells in the microwell array from each other.
  • the isolation liquid can be fluorine oil or silicone oil with a high viscosity coefficient.
  • the isolation liquid is added to the reaction pool of the microfluidic chip, and the magnetic beads and fluorescent substrates in the micropores of the micropore array are sealed and isolated.
  • the third centrifugal condition is, for example, 200rpm for 10 seconds.
  • the centrifugal force sends the isolation liquid into the reaction pool of the microfluidic chip.
  • the hydrophobic isolation liquid can fully infiltrate the surface of the micropore array, further remove the magnetic beads that have not fallen into the micropores, and isolate all the micropores from each other.
  • the fluorescent molecules subsequently generated are difficult to diffuse, and the thermal stability of a single micropore is excellent. It is understandable that the isolation liquid can also be added to the reaction pool of the microfluidic chip by self-absorption or pressure injection.
  • step 4 wait for the enzyme to undergo an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules, wherein the fluorescence signal of the microwell where the magnetic beads capturing a single target molecule to be detected are located is higher than a threshold, and the fluorescence signal of the microwell where the magnetic beads capturing zero target molecules to be detected are located is lower than a threshold.
  • ⁇ -galactosidase catalyzes the hydrolysis of non-fluorescent resorufin- ⁇ -galactoside (RGP), generating resorufin molecules capable of emitting fluorescence.
  • RGP non-fluorescent resorufin- ⁇ -galactoside
  • the fluorescence signal of the micropore where the magnetic beads with a single target molecule to be measured are located is higher than the threshold
  • the fluorescence signal of the micropore where the magnetic beads with zero target molecules to be measured are located is lower than the threshold.
  • the micropores with a fluorescence signal higher than the threshold in the micropore array can be read as 1 ("positive")
  • the micropores with a fluorescence signal lower than the threshold in the micropore array can be read as 0 ("negative").
  • step 5 (S95) the number of microwells in the microwell array containing magnetic beads is determined, and the number of microwells in the microwell array having a fluorescence signal higher than a threshold value is determined.
  • step 5 determines the number of reaction detection units and the number of positive reaction detection units, respectively.
  • bright field images e.g., mercury lamp light source, and exposure time 50ms
  • fluorescent images e.g., 577nm excitation, 620nm emission, and exposure time 600ms
  • the number of microwells containing magnetic beads in the microwell array is determined based on the bright field images of one or more fields of view, and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value is determined based on the fluorescent images of one or more fields of view.
  • the number of target molecules to be detected in the sample is determined based on the number of microwells in the microwell array containing magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than the threshold, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.
  • M0 is the number of target molecules to be detected in the sample
  • N0 is the number of magnetic beads in the sample
  • M is the number of microwells in the microwell array whose fluorescence signals are above the threshold
  • N is the number of microwells in the microwell array containing magnetic beads
  • p is the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme (if the operation uniformity is high, the probability p is a constant less than 100%).
  • the target molecules to be detected in the sample can be absolutely quantified by the number of sample distribution units clearly determined by counting methods such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields of view, the number of positive reaction detection units determined by fluorescent images of one or more fields of view, and the probability of the target molecules to be detected being captured by magnetic beads and further binding to the enzyme as a constant.
  • c M 0 m/V
  • M 0 the number of target molecules to be detected in the sample
  • m the mass of a single target molecule to be detected in the sample
  • V the volume of the sample
  • the target molecule to be detected in the sample is interleukin-6 (IL-6), whose molecular weight is 21 kDa, and the volume V of the sample is 100 ⁇ L.
  • the number N0 of magnetic beads (sample distribution units) in the sample determined by flow cytometry is 753,600, the number of all microwells in the microwell array is 188,000, the number N of microwells (reaction detection units) containing magnetic beads in the microwell array determined based on the bright field image of one or more fields of view is 121,000, the number M of microwells (positive reaction detection units) in the microwell array with fluorescence signals higher than the threshold value determined based on the fluorescence image of one or more fields of view is 5000, and the probability p that the target molecule to be detected is captured by the magnetic beads and further bound to the enzyme is 80%, then the number M0 of the target molecule to be detected in the sample is calculated using the following formula: The calculated value is 39753, and the concentration c of the target molecule to be detected is
  • microfluidic chip used in the absolute quantitative digital ELISA detection method belongs to the same concept as the corresponding microfluidic chip embodiment described above.
  • the specific implementation process is detailed in the corresponding microfluidic chip embodiment and will not be repeated here.
  • Figure 10 is a schematic diagram of the principle of a digital ELISA detection method for preventing fluorescence signal crosstalk using a microfluidic chip according to an embodiment of the present invention
  • Figure 11 is a schematic diagram of the process of a digital ELISA detection method for preventing fluorescence signal crosstalk using a microfluidic chip according to an embodiment of the present invention.
  • the digital ELISA detection method for preventing fluorescence signal crosstalk implemented by a microfluidic chip includes:
  • step 1 a sample is prepared, the sample contains a target molecule to be detected, the target molecule to be detected in the sample is captured by magnetic beads, and a complex of "magnetic beads-target molecule to be detected-enzyme" is formed through an affinity reaction.
  • the target molecule to be detected can be a protein molecule to be detected, and the protein molecule to be detected can be derived from a liquid sample of the human body (blood, body fluid, tissue, etc.). More specifically, the protein molecule to be detected can be from the supernatant of serum, plasma, tissue homogenate or cell extract. Based on this, the present invention can accurately quantify the ultra-low abundance protein molecules in normal people and disease patients that are difficult to detect by conventional methods, and develop new applications in the fields of early detection, companion diagnosis, and drug development of major diseases such as tumors, neurological diseases, infectious diseases, and immune inflammation.
  • major diseases such as tumors, neurological diseases, infectious diseases, and immune inflammation.
  • the magnetic beads may have a diameter of micrometer scale.
  • the surface of the magnetic beads is modified with a capture antibody that can specifically bind to the target molecule to be detected, for example, to produce an antibody-antigen reaction with the protein molecule to be detected, thereby capturing the protein molecule to be detected.
  • the number of magnetic beads in the sample can be clearly determined based on counting methods such as a particle counter, flow cytometry or a cell counting plate.
  • the uniformity of the magnetic beads in the sample can also be accurately controlled based on the above technology. Based on this, the present invention can absolutely quantify the target molecule to be detected in the sample based at least on the number of magnetic beads in the sample.
  • the number of magnetic beads in the sample should be much larger than the number of target molecules to be detected captured on the magnetic beads, so that the statistical distribution of the target molecules to be detected captured on the magnetic beads conforms to the Poisson distribution.
  • the number of magnetic beads in the sample may be less than or equal to the number of target molecules to be detected captured on the magnetic beads.
  • the ratio of the number of target molecules to be detected captured on the magnetic beads to the number of magnetic beads in the sample is less than or equal to 5 (i.e., each magnetic bead captures a maximum of 5 target molecules to be detected on average)
  • the statistical distribution of the target molecules to be detected captured on the magnetic beads still conforms to the Poisson distribution, and single-molecule fluorescence signal amplification can still be achieved.
  • the number of magnetic beads in the sample determines the upper limit of the dynamic detection range.
  • the dynamic detection range can be precisely controlled. Theoretically, when the capture probability is 100%, the number of target molecules to be detected in the sample is less than or equal to 5 times the number of magnetic beads. For example, the number of magnetic beads in the sample is in the range of 100,000 to 10 million, so the upper limit of the dynamic detection range is in the range of 500,000 to 50 million. It is understandable that, considering the capture probability factor, the ratio of the number of target molecules to be detected in the sample to the number of magnetic beads in the sample can also be greater than or equal to 5. When the capture probability is 5%, the number of target molecules to be detected in the sample is less than or equal to 100 times the number of magnetic beads.
  • the capture antibody modified on the surface of the magnetic bead specifically captures the target molecule to be detected in the sample, and further connects the detection antibody and the enzyme, finally forming an immune complex of "magnetic bead-capture antibody-target molecule to be detected-detection antibody-enzyme", and the enzyme can react enzymatically with the fluorescent substrate to generate fluorescent molecules.
  • the magnetic bead can be connected with ⁇ -galactosidase through the above-mentioned double antibody sandwich reaction
  • the fluorescent substrate can be non-fluorescent resorufin- ⁇ -galactoside (RGP)
  • ⁇ -galactosidase can catalyze the hydrolysis of non-fluorescent resorufin- ⁇ -galactoside (RGP) to generate resorufin molecules that can emit fluorescence.
  • the sample formed with the complex is transferred to a microfluidic chip, wherein the chip body is a microwell array chip body including a microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead. As described above, the microwell array and its chip body are accommodated in the chip area of the reaction pool.
  • the micropore array includes a plurality of micropores, and the size of each micropore can be slightly larger than the size of the magnetic bead, so as to be configured to accommodate only one magnetic bead.
  • the ratio of the number of all micropores in the micropore array to the number of magnetic beads in the sample is in the range of 0.1 to 10, so that as many magnetic beads as possible fall into the micropores.
  • the micropores can be called effective micropores or reaction detection units.
  • the number of reaction detection units i.e., effective micropores
  • the number of reaction detection units is not greater than the number of sample distribution units (i.e., magnetic beads). In theory, the closer the number of reaction detection units is to the number of sample distribution units, the higher the accuracy and resolution of digital detection.
  • the microwell array includes 188,000 microwells, the microwells are circular microwells, the diameter of the microwells is 4 ⁇ m, the depth of the microwells is 4 ⁇ m, and the center distance between the microwells is 8 ⁇ m.
  • the sample formed with the complex is transferred to the microfluidic chip, and the first centrifugal condition is, for example, 200rpm for 10 seconds, and the centrifugal force sends the sample into the reaction pool of the microfluidic chip. Then, stand for a specific time (for example, 2 minutes) until the sample settles in the micropores of the micropore array. Then, under the second centrifugal condition, the excess magnetic beads in the reaction pool are removed, and the magnetic beads in the micropores are retained.
  • the second centrifugal condition is, for example, 600rpm for 10 seconds. At this speed, the excess magnetic beads in the reaction pool are separated, and the magnetic beads in the micropores are retained. It is understandable that the sample formed with the complex can also be transferred to the microfluidic chip by self-absorption or pressure injection.
  • the fluorescent substrate is transferred into the microfluidic chip.
  • the fluorescent substrate is transferred to the microfluidic chip, and the third centrifugal condition is, for example, 200rpm for 10 seconds, and centrifugal force sends the fluorescent substrate into the reaction pool of the microfluidic chip.
  • the fluorescent substrate enters the micropore by means of fluid shear force or molecular diffusion.
  • the fourth centrifugal condition the excess fluorescent substrate in the reaction pool is removed, and the fluorescent substrate in the micropore is retained, and the fourth centrifugal condition is, for example, 600rpm for 10 seconds. At this speed, the excess fluorescent substrate in the reaction pool is separated, and the fluorescent substrate in the micropore is retained.
  • the fluorescent substrate can also be transferred to the microfluidic chip by means of self-absorption or pressure injection.
  • a spacer liquid is added into the microfluidic chip to isolate all microwells in the microwell array from each other.
  • the isolation liquid can be fluorine oil or silicone oil with a high viscosity coefficient.
  • the fifth centrifugal condition is, for example, 200rpm for 10 seconds. The centrifugal force sends the isolation liquid into the reaction pool of the microfluidic chip.
  • the hydrophobic isolation liquid can fully infiltrate the surface of the micropore array, further remove the magnetic beads that have not fallen into the micropores, and isolate all the micropores from each other.
  • the fluorescent molecules subsequently generated are difficult to diffuse, and the thermal stability of a single micropore is excellent. It is understandable that the isolation liquid can also be added to the reaction pool of the microfluidic chip by self-priming or pressure injection.
  • step 5 wait for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules, wherein the fluorescence signal of the microwell where the magnetic beads that have captured a single target molecule to be detected are located is higher than the threshold, and the fluorescence signal of the microwell where the magnetic beads that have captured zero target molecules to be detected are located is lower than the threshold.
  • ⁇ -galactosidase catalyzes the hydrolysis of non-fluorescent resorufin- ⁇ -galactoside (RGP), generating resorufin molecules capable of emitting fluorescence.
  • RGP non-fluorescent resorufin- ⁇ -galactoside
  • the fluorescence signal of the micropore where the magnetic beads with a single target molecule to be measured are located is higher than the threshold
  • the fluorescence signal of the micropore where the magnetic beads with zero target molecules to be measured are located is lower than the threshold.
  • the micropores with a fluorescence signal higher than the threshold in the micropore array can be read as 1 ("positive")
  • the micropores with a fluorescence signal lower than the threshold in the micropore array can be read as 0 ("negative").
  • step 6 the number of microwells in the microwell array containing magnetic beads is determined, and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value is determined.
  • step 6 determines the number of reaction detection units and the number of positive reaction detection units, respectively.
  • bright field images e.g., mercury lamp light source, and exposure time 50ms
  • fluorescence images e.g., 577nm excitation, 620nm emission, and exposure time 600ms
  • the number of microwells containing magnetic beads in the microwell array is determined based on the bright field images of one or more fields of view, and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value is determined based on the fluorescence images of one or more fields of view.
  • the number of target molecules to be detected in the sample is determined based on the number of microwells in the microwell array containing magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than the threshold, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.
  • M0 is the number of target molecules to be detected in the sample
  • N0 is the number of magnetic beads in the sample
  • M is the number of microwells in the microwell array whose fluorescence signals are above the threshold
  • N is the number of microwells in the microwell array containing magnetic beads
  • p is the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme (if the operation uniformity is high, the probability p is a constant less than 100%).
  • the target molecules to be detected in the sample can be absolutely quantified by the number of sample distribution units clearly determined by counting methods such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields of view, the number of positive reaction detection units determined by fluorescent images of one or more fields of view, and the probability of the target molecules to be detected being captured by magnetic beads and further binding to the enzyme as a constant.
  • c M 0 m/V
  • M 0 the number of target molecules to be detected in the sample
  • m the mass of a single target molecule to be detected in the sample
  • V the volume of the sample
  • the target molecule to be detected in the sample is interleukin-6 (IL-6), whose molecular weight is 21 kDa, and the volume V of the sample is 100 ⁇ L.
  • the number N0 of magnetic beads (sample distribution units) in the sample determined by flow cytometry is 753,600, the number of all microwells in the microwell array is 188,000, the number N of microwells (reaction detection units) containing magnetic beads in the microwell array determined based on the bright field image of one or more fields of view is 121,000, the number M of microwells (positive reaction detection units) in the microwell array with fluorescence signals higher than the threshold value determined based on the fluorescence image of one or more fields of view is 5000, and the probability p that the target molecule to be detected is captured by the magnetic beads and further bound to the enzyme is 80%, then the number M0 of the target molecule to be detected in the sample is calculated using the following formula: The calculated value is 39753, and the concentration c of the target molecule to be detected is
  • Example 1 Comparison of microwell signals between two-step sample addition method and one-step sample addition method
  • the sample with a complex and the fluorescent substrate are transferred to the microfluidic chip in different steps, and more specifically, the sample with a complex and the fluorescent substrate are centrifuged and plated in batches, and this loading method is called a two-step loading method.
  • this loading method is called a one-step loading method.
  • the magnetic beads and fluorescent substrate are pre-mixed and then added to the microfluidic chip.
  • the chips are left to stand and wait for the magnetic beads to settle into the microwells of the microwell array, and then isolation fluid is added to isolate the microwells.
  • the enzymatic reaction begins while the magnetic beads and fluorescent substrate are mixed, and the generated fluorescent signal will diffuse throughout the reaction solution.
  • the isolation fluid isolates the magnetic beads and the fluorescent substrate
  • the enzyme in the positive reaction detection unit further catalyzes the fluorescent substrate to produce a stronger fluorescent signal
  • the fluorescent signal in the negative reaction detection unit is the background signal generated by the enzyme-catalyzed reaction from the mixing of the enzyme and the fluorescent substrate to the isolation of the isolation fluid.
  • the more enzyme molecules are connected to the magnetic beads, the stronger the background signal.
  • the reaction time for the enzyme to react with the fluorescent substrate to produce the background signal is greatly shortened.
  • the reaction time is about 3 minutes, while in the two-step addition method, the reaction time is about 20 seconds, thereby greatly reducing the fluorescent signal background of the reaction system.
  • biotin-linked magnetic beads (1,000,000) were reacted with a specific volume (100 ⁇ L) of streptavidin- ⁇ -galactosidase (100 fM, 1 pM, 10 pM) outside the microfluidic chip.
  • the reaction buffer solution was 1 ⁇ PBS, and the reaction time was 30 minutes.
  • the chip was washed five times with 1 ⁇ PBST washing solution, and then the operation inside the microfluidic chip was performed according to the technical schemes of the two-step sample addition method and the one-step sample addition method, respectively.
  • the negative micropore signal i.e., background signal or background
  • the negative micropore signal in the one-step addition method was significantly increased, with an average value of 678 for the negative micropore signal at a S ⁇ G concentration of 100 fM, an average value of 1172 for the negative micropore signal at a S ⁇ G concentration of 1 pM, and an average value of 6410 for the negative micropore signal at a S ⁇ G concentration of 100 pM, and the high background value leads to a low degree of differentiation between the signal and the background.
  • the background increase in the two-step addition method was much lower, with an average value of 561 for the negative micropore signal at a S ⁇ G concentration of 100 fM, an average value of 1046 for the negative micropore signal at a S ⁇ G concentration of 1 pM, and an average value of 1662 for the negative micropore signal at a S ⁇ G concentration of 100 pM.
  • FIG12A is a fluorescence image obtained by a two-step sample addition method for magnetic beads after a 10 pM S ⁇ G concentration reaction
  • FIG12B is a fluorescence image obtained by a one-step sample addition method for magnetic beads after a 10 pM S ⁇ G concentration reaction
  • FIG13A is a fluorescence signal of a certain column of microwells extracted and calculated from the fluorescence image shown in FIG12A using ImageJ software
  • FIG13B is a fluorescence signal of a certain column of microwells extracted and calculated from the fluorescence image shown in FIG12B using ImageJ software.
  • the background of the one-step addition method is significantly increased, and the distinction between the signal and the background is reduced.
  • Example 2 As a simplified system for evaluating enzymatic reactions, in Example 2, biotin-linked magnetic beads (1,000,000) were reacted with 100 fM streptavidin- ⁇ -galactosidase outside the microfluidic chip.
  • the reaction buffer solution was 1 ⁇ PBS and the reaction time was 30 minutes. After the reaction was completed, it was washed five times with 1 ⁇ PBST washing solution, and then the operation in the microfluidic chip was carried out according to the technical scheme of the two-step sample addition method, and isolation was performed using isolation fluids with different viscosity coefficients.
  • the low viscosity coefficient fluoro oil No. 1 (viscosity coefficient at 25°C is 0.77 cSt) showed obvious dye diffusion phenomenon
  • the high viscosity coefficient fluoro oil No. 2 (viscosity coefficient at 25°C is 11.4 cSt)
  • No. 3 viscosity coefficient at 25°C is 451 cSt
  • No. 4 viscosity coefficient at 25°C is 1366 cSt fluoro oils did not show obvious dye diffusion phenomenon.
  • the low viscosity coefficient silicone oil No. _#5 (viscosity coefficient at 25°C is 2.3 cSt) and No. _#6 (viscosity coefficient at 25°C is 6.6 cSt) both showed obvious dye diffusion phenomenon, while the high viscosity coefficient silicone oil No. _#7 (viscosity coefficient at 25°C is 350 cSt) did not show obvious dye diffusion phenomenon.
  • the viscosity coefficient of the fluorine oil at 25° C. is in the range of 0.1 cSt to 12500 cSt, preferably in the range of 1 cSt to 5000 cSt, and more preferably in the range of 10 cSt to 1500 cSt.
  • the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 12500 cSt, preferably in the range of 10 cSt to 5000 cSt, and more preferably in the range of 100 cSt to 500 cSt.
  • Figures 14A1, 14B1, 14C1 and 14D1 are fluorescence images taken 1 minute after isolation using isolation fluids No. 1, No. 2, No. 3 and No. 4, respectively, and Figures 14A2, 14B2, 14C2 and 14D2 are fluorescence images taken 5 minutes after isolation using isolation fluids No. 1, No. 2, No. 3 and No. 4, respectively.
  • Figures 15A1 and 15A2 are schematic diagrams of the changes in the fluorescence value of the positive micropores and the crosstalk micropore fluorescence value over time after isolation using the isolation liquid No. 1
  • Figures 15B1 and 15B2 are schematic diagrams of the changes in the fluorescence value of the positive micropores and the crosstalk micropore fluorescence value over time after isolation using the isolation liquid No. 3.
  • microfluidic chip used in the digital ELISA detection method for preventing fluorescence signal crosstalk belongs to the same concept as the corresponding microfluidic chip embodiment described above.
  • the specific implementation process is detailed in the corresponding microfluidic chip embodiment and will not be repeated here.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Clinical Laboratory Science (AREA)
  • Sustainable Development (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

本发明涉及生物检测，特别涉及微流体芯片及其操作方法、数字ELISA检测方法和用途。本发明的微流体芯片包括：下壳体，所述下壳体包括形成在所述下壳体的内表面上且互相连通的反应池、导流槽以及废液池；芯片本体，所述芯片本体设置在所述反应池的芯片区中；上壳体，所述上壳体包括贯穿所述上壳体且与所述反应池的进样端对应的进样孔以及贯穿所述上壳体且与所述废液池对应的透气孔。本发明的微流体芯片具备防污染功能，检测可靠、灵敏、快速，结构简单，并且价格低廉。

Description

微流体芯片及其操作方法、数字ELISA检测方法和用途 技术领域
本发明涉及生物检测，特别涉及微流体芯片及其操作方法、数字ELISA检测方法和用途。
背景技术
目前在生物检测中使用的微流体芯片一般不包括储液池、废液池等流体控制结构，这就导致一方面加样时，需要加样针加液的速度与负压泵抽液的速度相配合，另一方面排废液时，需要有废液桶以及连接的接头及流体管路，这增加了复杂性，同时废液被存储在废液桶中增加了环境污染的风险。
另外，微流体芯片一般采用多路检测通道芯片的结构设计，这虽然保证检测的通量，但是提高了单次检测的成本，降低了灵活性。
因此，亟需一种微流体芯片，具备防污染功能，检测可靠、灵敏、快速，结构简单，并且价格低廉。
此外，目前，市场上已有多种定量检测低浓度分析物的检测技术。
一类采用高灵敏度的光学仪器来检测单分子信号，代表性的产品有默克公司的SMCTM(Single Molecule Counting)系统，然而该系统的光路结构复杂、昂贵，检测速度慢，针对微球进行串行检测，检测时间也很长。
另一类采用扩增技术来增加待测靶标分子的数量，以提供足够多的信号分子，代表性的产品有Chimera公司的系统，然而该系统的测定方法复杂且容易产生假阳性信号。
Quanterix公司的SimoaTM(Single-molecule Array)系统则采用类似数字PCR(Polymerase Chain Reaction，聚合酶链式反应)的数字ELISA(Enzyme Linked Immunosorbent Assay，酶联反应免疫分析)技术，然而该系统是一种相对定量的数字ELISA检测方法，为了确定待测靶标分子的数量，需要已知浓度的标准靶标分子进行标定以制作标准曲线，通过拟合的标准曲线公式，反推出待测靶标分子的数量及其浓度。另外，取决于待测靶标分子的浓度，该系统使用数字化和模拟化两种分析算法进行定量，既没有充分利用数字化分析的动态检测范围，又降低了分辨率和鲁棒性。
因此，亟需一种绝对定量的数字ELISA检测方法，无需依赖于标准样本和标准曲线，动态检测范围精确可控，灵敏度高，并且准确度高。
进一步，目前某些系统中的单个反应检测单元的本底信号高，并且单个反应检测单元内生成的荧光物质会扩散到邻近的反应检测单元，从而产生荧光信号串扰，影响阴性信号和阳性信号的判别准确度。
因此，亟需一种防荧光信号串扰的数字ELISA检测方法，降低反应体系的荧光信号背景，并且降低反应检测单元内的荧光信号扩散，从而提高阴性信号和阳性信号的判别准确度。
发明内容
为了解决现有技术中的上述问题，本发明提供了微流体芯片及其操作方法和用途，具备防污染功能，检测可靠、灵敏、快速，结构简单，并且价格低廉。本发明还提供了一种绝对定量的数字ELISA检测方法，无需依赖于标准样本和标准曲线，动态检测范围精确可控，灵敏度高，并且准确度高。本发明又提供了一种防荧光信号串扰的数字ELISA检测方法，降低反应体系的荧光信号背景，并且降低反应检测单元内的荧光信号扩散，从而提高阴性信号和阳性信号的判别准确度。
本发明提供了一种微流体芯片，所述微流体芯片包括：
下壳体，所述下壳体包括形成在所述下壳体的内表面上且互相连通的反应池、导流槽以及废液池；
芯片本体，所述芯片本体设置在所述反应池的芯片区中；
上壳体，所述上壳体包括贯穿所述上壳体且与所述反应池的进样端对应的进样孔以及贯穿所述上壳体且与所述废液池对应的透气孔。
本发明的一个实施例中，所述微流体芯片还包括密封垫，所述密封垫设置在所述进样孔上。
本发明的一个实施例中，所述微流体芯片还包括透气膜，所述透气膜设置在所述透气孔上。
本发明的一个实施例中，所述芯片本体为包括微孔阵列的微孔阵列芯片本体，所述微孔阵列包括5000个-1000万个微孔，所述微孔的直径为1μm-120μm，所述微孔的深度为1μm-120μm，所述微孔之间的中心距为3μm-180μm。
本发明的一个实施例中，所述微孔阵列包括188000个微孔，所述微孔的直径为4μm，所述微孔的深度为4μm，所述微孔之间的中心距为8μm。
本发明的一个实施例中，所述微孔阵列包括8800个微孔，所述微孔的直径为70μm，所述微孔的深度为70μm，所述微孔之间的中心距为105μm。
本发明的一个实施例中，所述下壳体的内表面上设置一个或多个定位孔，所述上壳体的内表面上设置与所述一个或多个定位孔相适配的一个或多个定位柱。
本发明的一个实施例中，所述下壳体的内表面上设置围绕所述反应池、所述导流槽以及所述废液池的溢料槽，所述上壳体的内表面上设置围绕所述进样孔以及所述透气孔且与所述溢料槽相适配的焊接线。
本发明的一个实施例中，所述上壳体还包括形成在所述上壳体的外表面上且与所述透气孔连通的排气槽，所述排气槽的远离所述透气孔的一端在所述上壳体的外表面上延伸，使得所述上壳体延伸形成手持部。
本发明进一步提供了一种根据上面描述的微流体芯片的操作方法，所述操作方法包括：
步骤1、单次或分批地将样本加入到所述进样孔中；
步骤2、每次加入的所述样本通过自吸、离心或压力进样的方式进入所述反应池以及所述芯片区中的所述芯片本体；
步骤3、对所述微流体芯片进行离心，使得所述样本由所述反应池进入所述废液池，同时所述芯片本体中的样本保留在其中；
步骤4、将隔离液加入到所述进样孔中；
步骤5、所述隔离液通过自吸、离心或压力进样的方式进入所述反应池，以隔离所述芯片本体中的所述样本；
步骤6、等待所述芯片本体中的所述样本进行生化反应；
步骤7、对所述芯片本体进行成像检测和数字化分析。
本发明进一步提供了一种上面描述的微流体芯片在数字ELISA检测中的用途。
本发明进一步提供了一种上面描述的微流体芯片在数字PCR检测中的用途。
本发明进一步提供了一种利用上面描述的微流体芯片实施的绝对定量的数字ELISA检测方法，所述方法包括：
步骤1、制备样本，所述样本含有待测靶标分子，利用磁珠捕获所述样本中的所述待测靶标分子，并且通过亲和反应形成“磁珠-待测靶标分子-酶”的复合物，所述酶能够与荧光底物进行酶促反应以生成荧光分子；
步骤2、将形成有所述复合物的所述样本和所述荧光底物转移到所述微流体芯片中，其中，所述芯片本体为包括微孔阵列的微孔阵列芯片本体，所述微孔阵列中的每个微孔被配置为仅能够容纳一个磁珠；
步骤3、将隔离液加入到所述微流体芯片中，以将所述微孔阵列中的所有微孔彼此隔离；
步骤4、等待所述酶与所述荧光底物进行酶促反应以生成所述荧光分子，其中，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值；
步骤5、确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且确定所述微孔阵列中的荧光信号高于阈值的微孔的数量；
步骤6、基于所述微孔阵列中的含有所述磁珠的微孔的数量以及所述微孔阵列中的荧光信号高于阈值的微孔的数量，并且基于所述样本中的所述磁珠的数量以及所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率，确定所述样本中的所述待测靶标分子的数量。
本发明的一个实施例中，基于粒子计数仪、流式细胞术或细胞计数板的计数方式来确定所述样本中的所述磁珠的数量。
本发明的一个实施例中，所述样本中的所述待测靶标分子的数量与所述样本中的所述磁珠的数量的比值小于等于100。
本发明的一个实施例中，所述样本中的所述待测靶标分子的数量与所述样本中的所述磁珠的数量的比值小于等于5。
本发明的一个实施例中，在所述步骤5中，对所述微孔阵列拍摄一个或多个视野的明场图像和荧光图像，其中，基于所述一个或多个视野的明场图像来确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且基于所述一个或多个视野的荧光图像来确定所述微孔阵列中的荧光信号高于阈值的微孔的数量。
本发明的一个实施例中，在所述步骤6中，采用以下公式来确定所述样本中的所述待测靶标分子的数量：其中，M0是所述样本中的所述待测靶标分子的数量，N0是所述样本中的所述磁珠的数量，M是所述微孔阵列中的荧光信号高于阈值的微孔的数量，N是所述微孔阵列中的含有所述磁珠的微孔的数量，并且p是所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率，或称为捕获几率。
本发明的一个实施例中，所述微孔阵列中的所有微孔的数量与所述样本中的所述磁珠的数量的比值在0.1至10的范围内。
本发明的一个实施例中，在所述步骤2中，通过自吸、离心或压力进样的方式，将形成有所述复合物的所述样本和所述荧光底物转移到所述微流体芯片的所述反应池以及所述微孔阵列的所述微孔中，并且通过离心的方式，移除所述反应池中的多余磁珠和荧光底物，并且保留所述微孔中的磁珠和荧光底物。
本发明的一个实施例中，在所述步骤3中，通过自吸、离心或压力进样的方式，将所述隔离液加入到所述微流体芯片的所述反应池中，并且对所述微孔阵列的所述微孔中的磁珠和荧光底物进行密封和隔离。
本发明的一个实施例中，所述待测靶标分子为待测蛋白分子。
本发明的一个实施例中，所述隔离液为氟油或者硅油。
本发明进一步提供了一种利用上面描述的微流体芯片实施的防荧光信号串扰的数字ELISA检测方法，所述方法包括：
步骤1、制备样本，所述样本含有待测靶标分子，利用磁珠捕获所述样本中的所述待测靶标分子，并且通过亲和反应形成“磁珠-待测靶标分子-酶”的复合物；
步骤2、将形成有所述复合物的所述样本转移到所述微流体芯片中，其中，所述芯片本体为包括微孔阵列的微孔阵列芯片本体，所述微孔阵列中的每个微孔被配置为仅能够容纳一个磁珠；
步骤3、将荧光底物转移到所述微流体芯片中，所述酶能够与所述荧光底物进行酶促反应以生成荧光分子；
步骤4、将隔离液加入到所述微流体芯片中，以将所述微孔阵列中的所有微孔彼此隔离；
步骤5、等待所述酶与所述荧光底物进行酶促反应以生成所述荧光分子，其中，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值；
步骤6、确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且确定所述微孔阵列中的荧光信号高于阈值的微孔的数量；
步骤7、基于所述微孔阵列中的含有所述磁珠的微孔的数量以及所述微孔阵列中的荧光信号高于阈值的微孔的数量，并且基于所述样本中的所述磁珠的数量以及所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率，确定所述样本中的所述待测靶标分子的数量。
本发明的一个实施例中，所述隔离液为氟油，所述氟油在25℃下的粘滞系数在0.1cSt至12500cSt的范围内。
本发明的一个实施例中，所述氟油在25℃下的粘滞系数在1cSt至5000cSt的范围内。
本发明的一个实施例中，所述氟油在25℃下的粘滞系数为11.4cSt、451cSt或1366cSt。
本发明的一个实施例中，所述隔离液为硅油，所述硅油在25℃下的粘滞系数在10cSt至12500cSt的范围内。
本发明的一个实施例中，所述硅油在25℃下的粘滞系数在10cSt至5000cSt的范围内。
本发明的一个实施例中，所述硅油在25℃下的粘滞系数为350cSt。
本发明的一个实施例中，基于粒子计数仪、流式细胞术或细胞计数板的计数方式来确定所述样本中的所述磁珠的数量。
本发明的一个实施例中，所述样本中的所述待测靶标分子的数量与所述样本中的所述磁珠的数量的比值小于等于100。
本发明的一个实施例中，所述样本中的所述待测靶标分子的数量与所述样本中的所述磁珠的数量的比值小于等于5。
本发明的一个实施例中，在所述步骤6中，对所述微孔阵列拍摄一个或多个视野的明场图像和荧光图像，其中，基于所述一个或多个视野的明场图像来确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且基于所述一个或多个视野的荧光图像来确定所述微孔阵列中的荧光信号高于阈值的微孔的数量。
本发明的一个实施例中，在所述步骤7中，采用以下公式来确定所述样本中的所述待测靶标分子的数量：其中，M0是所述样本中的所述待测靶标分子的数量，N0是所述样本中的所述磁珠的数量，M是所述微孔阵列中的荧光信号高于阈值的微孔的数量，N是所述微孔阵列中的含有所述磁珠的微孔的数量，并且p是所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率，或称为捕获几率。
本发明的一个实施例中，所述微孔阵列中的所有微孔的数量与所述样本中的所述磁珠的数量的比值在0.1至10的范围内。
本发明的一个实施例中，在所述步骤2中，通过自吸、离心或压力进样的方式，将形成有所述复合物的所述样本转移到所述微流体芯片的所述反应池以及所述微孔阵列的所述微孔中，并且通过离心的方式，移除所述反应池中的多余磁珠，并且保留所述微孔中的磁珠。
本发明的一个实施例中，在所述步骤3中，通过自吸、离心或压力进样的方式，将所述荧光底物转移到所述微流体芯片的所述反应池以及所述微孔阵列的所述微孔中，并且通过离心的方式，移除所述反应池中的多余荧光底物，并且保留所述微孔中的荧光底物。
本发明的一个实施例中，在所述步骤4中，通过自吸、离心或压力进样的方式，将所述隔离液加入到所述微流体芯片的所述反应池中，并且对所述微孔阵列的所述微孔中的磁珠和荧光底物进行密封和隔离。
本发明的一个实施例中，所述待测靶标分子为待测蛋白分子。
如上所述，本发明具有以下有益效果：
在本发明的实施例中，微流体芯片将进样腔体、反应腔体和废液腔体集成在一起，结构简单紧凑，成本低，不需要额外的负压泵和废液桶以及连接的接头及流体管路。其次，微流体芯片可以为一次性耗材，反应结束后，含有废液的芯片被存放到特定区域统一处理，防止污染。另外，每个微流体芯片是一个独立的检测通道，用于一个样本的检测，可以选择单个芯片和多个芯片同时工作，检测可靠、灵敏、快速。
在本发明的实施例中，通过选择包括合适的芯片本体的微流体芯片，并且通过加入合适的样本，可以操作微流体芯片以实现不同的检测，例如数字ELISA检测和数字PCR检测。
在本发明的实施例中，可以通过由粒子计数仪、流式细胞术或细胞计数板等计数方式所明确确定的样本分配单元的数量、由一个或多个视野的明场图像所确定的反应检测单元的数量、由一个或多个视野的荧光图像所确定的阳性反应检测单元的数量以及作为常数的待测靶标分子被磁珠捕获并进一步与酶结合的几率，来绝对定量样本中的待测靶标分子。
在本发明的实施例中，样本中的磁珠的数量决定动态检测范围的上限，通过改变样本中的磁珠的数量，可以精确控制动态检测范围。理论上，当捕获几率为100％时，样本中的待测靶标分子的数量小于等于5倍的磁珠数量。当捕获几率为5％时，样本中的待测靶标分子的数量小于等于100倍的磁珠数量。
在本发明的实施例中，通过两步加样法，大大降低了反应体系的荧光信号背景，并且通过优选高粘滞系数的隔离液，大大降低了反应检测单元内的荧光信号扩散，从而提高了阴性信号和阳性信号的判别准确度。
附图说明
图1是根据本发明的一个实施例的微流体芯片的整体结构示意图；
图2是根据本发明的一个实施例的微流体芯片的爆炸示意图；
图3是根据本发明的一个实施例的微流体芯片的下壳体的结构示意图；
图4是根据本发明的一个实施例的微流体芯片的上壳体的结构示意图；
图5是根据本发明的一个实施例的微流体芯片的侧视图；
图6A和图6B分别是根据本发明的一个实施例的微流体芯片的微孔阵列芯片本体的扫描电镜图；
图7是根据本发明的一个实施例的微流体芯片的操作方法的流程示意图；
图8是根据本发明的一个实施例的利用微流体芯片实施的绝对定量的数字ELISA检测方法的原理示意图；
图9是根据本发明的一个实施例的利用微流体芯片实施的绝对定量的数字ELISA检测方法的流程示意图；
图10是根据本发明的一个实施例的利用微流体芯片实施的防荧光信号串扰的数字ELISA检测方法的原理示意图；
图11是根据本发明的一个实施例的利用微流体芯片实施的防荧光信号串扰的数字ELISA检测方法的流程示意图；
图12A是采用两步加样法得到的荧光图像，并且图12B是采用一步加样法得到的荧光图像；
图13A是从图12A示出的荧光图像中提取并计算出的某一列微孔的荧光信号，并且图13B是从图12B示出的荧光图像中提取并计算出的某一列微孔的荧光信号；
图14A1-图14D2是采用不同隔离液隔离后1分钟和5分钟的荧光图像；
图15A1-图15B2是采用不同隔离液隔离后阳性微孔荧光值以及串扰微孔荧光值随时间的变化示意图。
具体实施方式
以下根据附图对本发明的实施例进行说明。
这里将详细地对示例性实施例进行说明，其示例表示在附图中。下面的描述涉及附图时，除非另有表示，不同附图中的相同数字表示相同或相似的要素。以下示例性实施例中所描述的实施方式并不代表与本申请相一致的所有实施方式。相反，它们仅是与如所附权利要求书中所详述的、本申请的一些方面相一致的装置和方法的例子。
在本申请中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。在本申请和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。还应当理解，本文中使用的术语“和/或”是指并包含一个或多个相关联的列出项目的任何或所有可能组合。“包括”或者“包含”等类似词语意指出现在“包括”或者“包含”前面的元件或者物件涵盖出现在“包括”或者“包含”后面列举的元件或者物件及其等同，并不排除其他元件或者物件。
图1是根据本发明的一个实施例的微流体芯片的整体结构示意图，并且图2是根据本发明的一个实施例的微流体芯片的爆炸示意图。
如图1和图2所示，微流体芯片10包括下壳体11，并且下壳体11可以采用常规注塑工艺，材质为聚碳酸酯(PC，polycarbonate)、聚苯乙烯(PS，polystyrene)、聚甲基丙烯酸甲酯(PMMA，polymethyl methacrylat)、环状烯烃共聚物(COC，cyclo olefin copolymer)和环状烯烃聚合物(COP，cyclo olefin polymer)等中的一个或多个塑料材料，最小加工精度在20μm以上。优选地，下壳体11的材质为PC材料。
下壳体11可以为长条形，并且下壳体11可以具有互相垂直的第一方向、第二方向和第三方向，其中，第一方向为长度方向，第二方向为宽度方向，并且第三方向为深度方向。可以理解的是，取决于实际需要，下壳体11也可以为其它任意形状，在此不受限制。
在深度方向上，下壳体11可以具有彼此相对的第一表面和第二表面，其中，位于微流体芯片10内部的第一表面为内表面，并且位于微流体芯片10外部的第二表面为外表面。
图3是根据本发明的一个实施例的微流体芯片的下壳体的结构示意图。
如图3所示，并且参考图1和图2，下壳体11包括形成在内表面上且互相连通的反应池(也可以被称为流体腔室)111、导流槽112以及废液池113，反应池111、导流槽112以及废液池113可以在长度方向上(诸如从右到左)依次设置。
反应池111包括进样端、芯片区111b和出样端，进样端、芯片区111b和出样端可以在长度方向上(诸如从右到左)依次设置，使得样本可以依次通过进样端、芯片区111b和出样端。
反应池111的进样端与下面将要描述的形成在上壳体13的外表面上的进样孔131对应，样本将从进样孔131进入反应池111的进样端，并且依次通过芯片区111b和出样端。
反应池111的芯片区111b可以是贯穿下壳体11的通孔，通孔的形状和尺寸与下面将要描述的芯片本体12的形状和尺寸适配。
反应池111的出样端与导流槽112连通，导流槽112的宽度可以设置得比较小，从而避免在样本进行充分的生化反应之前流入废液池113。另外，导流槽112可以采用斜坡流道结构，使得流体容易流入废液池113而不容易从废液池113回流。
导流槽112与废液池113连通，使得充分反应后的样本可以流入废液池113，从而保存废液。
返回到图1和图2，微流体芯片10还包括芯片本体12。参考图3，芯片本体12与反应池111的芯片区111b通过尺寸配合嵌合在芯片区111b。可以理解的是，芯片本体12还可以进一步通过密封胶进行固定。样本在通过进样端之后，将在芯片区111b与芯片本体12接触，并且在芯片本体12处进行生化反应。
芯片本体12可以采用光盘注塑工艺，材质为聚碳酸酯(PC，polycarbonate)、聚苯乙烯(PS，polystyrene)、聚甲基丙烯酸甲酯(PMMA，polymethyl methacrylat)、环状烯烃共聚物(COC，cyclo olefin copolymer)和环状烯烃聚合物(COP，cyclo olefin polymer)等中的一个或多个塑料材料，最小加工精度在100nm-50μm。芯片本体12也可以采用半导体加工工艺，材质为硅和玻璃等中的一个或多个半导体材料，最小加工精度在100nm-50μm。
返回到图1和图2，微流体芯片10还包括上壳体13，并且上壳体13可以采用常规注塑工艺，材质为聚碳酸酯(PC，polycarbonate)、聚苯乙烯(PS，polystyrene)、聚甲基丙烯酸甲酯(PMMA，polymethyl methacrylat)、环状烯烃共聚物(COC，cyclo olefin copolymer)和环状烯烃聚合物(COP，cyclo olefin polymer)等中的一个或多个塑料材料，最小加工精度在20μm以上。优选地，上壳体13的材质为PC材料。
上壳体13可以为长条形，并且上壳体13可以具有互相垂直的第一方向、第二方向和第三方向，其中，第一方向为长度方向，第二方向为宽度方向，并且第三方向为深度方向。可以理解的是，取决于实际需要，上壳体13也可以为其它任意形状，在此不受限制。
在深度方向上，上壳体13可以具有彼此相对的第一表面和第二表面，其中，位于微流体芯片10内部的第一表面为内表面，并且位于微流体芯片10外部的第二表面为外表面。
图4是根据本发明的一个实施例的微流体芯片的上壳体的结构示意图。
如图4所示，并且参考图1和图2，上壳体13包括贯穿上壳体13且与反应池111的进样端对应的进样孔131以及贯穿上壳体13且与废液池113对应的透气孔132。
进样孔131与反应池111的进样端对应，进样孔131与外界的注样器件连接，使得样本通过进样孔131进入反应池111的进样端。
透气孔132与废液池113对应，可以释放微流体芯片10内的气压。
上壳体13还包括形成在上壳体13的外表面上且与透气孔132连通的排气槽133，进样孔131、透气孔132以及排气槽133可以在长度方向上(诸如从右到左)依次设置。
排气槽133与透气孔132连通，可以进一步释放微流体芯片10内的气压。
图5是根据本发明的一个实施例的微流体芯片的侧视图。
如图5所示，下壳体11和上壳体13组装在一起，使得下壳体11的内表面与上壳体13的内表面相对，并且分别在两内表面上朝向彼此延伸的侧面共同包围以形成进样腔体51、反应腔体52和废液腔体53。
可以合理设置进样腔体51、反应腔体52和废液腔体53的深度，以分别限定这些腔体的体积。优选地，进样腔体51和反应腔体52的深度均为500μm，并且两个腔体的总体积为26μL。优选地，相比于进样腔体51和反应腔体52，废液腔体53的深度可以设置得比较大，从而废液腔体53的体积也比较大，以容纳所有的样本和隔离液(也可以被称为分割油)。
芯片本体12嵌合在芯片区111b，并且芯片本体12的上表面与进样腔体51的下表面以及废液腔体53之前的导流槽112的下表面齐平，从而进样腔体51的下表面、反应腔体52的下表面以及导流槽112的下表面互相齐平，便于反应流体和隔离液的流动。可以理解的是，在导流槽112采用斜坡流道结构的情况下，进样腔体51的下表面以及反应腔体52的下表面彼此齐平。
返回到图1和图2，微流体芯片还包括密封垫14，密封垫14设置在进样孔131上，从而在非加样期间隔绝进样孔131与外界的接触，避免外来物进入微流体芯片10内部而污染微流体芯片10。可以理解的是，密封垫14还可以进一步通过密封胶进行固定。
密封垫14可以为硅胶垫，并且密封垫14的尺寸与进样孔131相适配。优选地，密封垫14的外径为5mm，高度为1.5mm，并且内径为十字刀口结构，以实现较好的密封效果。
继续参考图1和图2，微流体芯片还包括透气膜15，透气膜15设置在透气孔132上，从而隔绝透气孔132与外界的接触，避免外来物进入微流体芯片10内部而污染微流体芯片10。可以理解的是，透气膜15还可以进一步通过密封胶进行固定。
透气膜15可以为硝酸纤维素膜，并且透气膜15的尺寸与透气孔132相适配。
继续参考图1和图2，并且参考图3和图4，上壳体13可以采用透明材料或半透明材料。另外地或替代地，如图5所示，上壳体13的与反应池111的芯片区111b对应的部分可以开设检测窗口134。通过如此设置，可以从上壳体13观测生化反应，并且对芯片本体12进行成像检测。
图6A和图6B分别是根据本发明的一个实施例的微流体芯片的微孔阵列芯片本体的扫描电镜图。
如图6A和图6B所示，并且参考图5，芯片本体12为包括微孔阵列的微孔阵列芯片本体，并且取决于检测需要，微孔阵列芯片本体可以具有不同的形状和尺寸。优选地，微孔阵列芯片本体为3mm×4mm的长方形芯片本体。优选地，微孔阵列芯片本体为10mm×10mm的正方形芯片本体。
微孔阵列包括5000个-1000万个微孔61，微孔61的直径为1μm-120μm，微孔61的深度为1μm-120μm，微孔61之间的中心距为3μm-180μm。
微孔61的形状可以为圆形，并且取决于检测需要，微孔61可以具有不同的形状，例如六边形。在微孔61为非圆形形状的情况下，微孔61的直径可以指微孔61的外接圆的直径。
如图6A所示，微孔阵列芯片本体为3mm×4mm的长方形芯片本体，并且微孔阵列包括188000个圆形微孔61，微孔61的直径为4μm，微孔61的深度为4μm，微孔61之间的中心距为8μm。这种微孔阵列芯片本体特别适用于数字ELISA检测。
如图6B所示，微孔阵列芯片本体为10mm×10mm的正方形芯片本体，并且微孔阵列包括8800个六边形微孔61，微孔61的外接圆直径为70μm，微孔61的深度为70μm，微孔61之间的中心距为105μm。这种微孔阵列芯片本体特别适用于数字PCR检测。
返回到图3和图4，下壳体11的内表面上设置一个或多个定位孔114，上壳体13的内表面上设置与一个或多个定位孔114相适配的一个或多个定位柱135。通过定位孔114和定位柱135的配合，下壳体11和上壳体13可以彼此定位，从而进样孔131与反应池111的进样端对应，并且透气孔132与废液池113对应，避免因错位而导致进样腔体51、反应腔体52和废液腔体53不再闭合。
优选地，下壳体11的内表面上设置两个定位孔114，并且在长度方向上的两端。相应地，上壳体13的内表面上设置与两个定位孔114相适配的两个定位柱135，并且也在长度方向上的两端。
继续参考图3和图4，下壳体11的内表面上设置围绕反应池111、导流槽112以及废液池113的溢料槽115，上壳体13的内表面上设置围绕进样孔131以及透气孔132且与溢料槽115相适配的焊接线136。通过对焊接线136进行超声焊接或激光焊接，可以将下壳体11和上壳体13焊接在一起，焊接产生的溢料流入溢料槽115，避免流入反应池111、导流槽112以及废液池113。可以合理布置溢料槽115和焊接线136，从而固定焊接的位置和封装的高度。
返回到图1和图2，并且参考图4，排气槽133的远离透气孔132的一端在上壳体13的外表面上延伸，使得上壳体13延伸形成手持部137。排气槽133在长度方向上向外延伸，可以将微流体芯片10内的气体释放到外界，并且形成的手持部137便于使用者操作微流体芯片10。
在本发明的实施例中，微流体芯片将进样腔体、反应腔体和废液腔体集成在一起，结构简单紧凑，成本低，不需要额外的负压泵和废液桶以及连接的接头及流体管路。其次，微流体芯片可以为一次性耗材，反应结束后，含有废液的芯片被存放到特定区域统一处理，防止污染。另外，每个微流体芯片是一个独立的检测通道，用于一个样本的检测，可以选择单个芯片和多个芯片同时工作，检测可靠、灵敏、快速。
图7是根据本发明的一个实施例的微流体芯片的操作方法的流程示意图。
如图7所示，在步骤1(S71)处，单次或分批地将样本加入到进样孔中。取决于检测的类型和内容，样本可以不同。例如，对于数字ELISA检测，样本可以包括待测蛋白样品分子以及磁珠或微球，对于数字PCR检测，样本可以包括待测核酸样品分子和PCR反应试剂。另外，对于数字ELISA检测，样本还可以包括荧光底物，荧光底物可以与待测蛋白样品分子以及磁珠或微球单次地一起加入到进样孔中，也可以分批地加入到进样孔中，例如先加入待测蛋白样品分子以及磁珠或微球，再加入荧光底物。可以理解的是，在加样期间也可以对微流体芯片进行离心，产生的离心力转换为液体剪切力，提高加样效率。
在步骤2(S72)处，每次加入的样本通过自吸、离心或压力进样的方式进入反应池以及芯片区中的芯片本体。作为示例，采用离心进样的方式，对微流体芯片进行离心，使得每次加入的样本进入反应池以及芯片区中的芯片本体。在特定的离心条件下(例如，200rpm保持10秒)，离心力将样本送入反应池。由于导流槽的约束，流体保持在反应池内。静置特定时间(例如，2分钟)，待样本沉降到芯片本体的微孔中。可以理解的是，在静置等候沉降期间也可以对微流体芯片进行离心(例如，200rpm保持20秒)，产生的离心力转换为液体剪切力，提高加样效率。可以理解的是，也可以采用自吸或压力进样等方式，使得每次加入的样本进入反应池以及芯片区中的芯片本体。
在步骤3(S73)处，对微流体芯片进行离心，使得样本由反应池进入废液池，同时芯片本体中的样本保留在其中。继续上面的示例，再次施加离心(例如，600rpm保持10秒)，在该转速下，反应池内多余的样本经过斜坡流道结构的导流槽进入废液池，同时芯片本体的微孔内的样本保留。离心过程中，废液腔体体积减小，气体压力增大，气压通过透气孔和透气膜释放到微流体芯片外部。
在步骤4(S74)处，将隔离液加入到进样孔中。取决于检测的类型和内容，隔离液可以不同。例如，对于数字ELISA检测和数字PCR检测，隔离液可以包括粘滞系数高的氟油或者硅油。
在步骤5(S75)处，隔离液通过自吸、离心或压力进样的方式进入反应池，以隔离芯片本体中的样本。继续上面的示例，采用离心进样的方式，对微流体芯片进行离心，使得隔离液进入反应池，以隔离芯片本体中的样本。在特定的离心条件下(例如，200rpm保持10秒)，离心力将隔离液送入反应池。疏水性的隔离液可以充分浸润芯片表面，进一步去除未落入微孔的样本，同时将微孔内的样本互相隔离，并且荧光产物难以扩散，单个微孔的热稳定优良。可以理解的是，也可以采用自吸或压力进样等方式，使得隔离液进入反应池。
在步骤6(S76)处，等待芯片本体中的样本进行生化反应。取决于检测的类型和内容，等待的时间和所需的生化反应温度不同。例如，对于数字ELISA检测，可以在室温下等待1分钟，并且对于数字PCR检测，可以在95℃下等待15秒，在60℃下等待50秒，并且重复35次。
在步骤7(S77)处，对芯片本体进行成像检测和数字化分析。利用成像系统来捕获芯片本体的图像，成像系统可以包括汞灯光源、滤光片、物镜和CCD相机等组件。微流体芯片可以固定在移动平台上，移动平台移动微流体芯片，使镜头对准芯片本体的不同位置，拍摄多个区域的图像。在图像拍摄过程中，精确的自动对焦平台可以调整镜头与微孔之间的距离，保证捕获最清晰的图像。可以使用20倍物镜镜头，每张微流体芯片可以拍摄多个视野的图像(例如，对于数字ELISA检测，拍摄35个视野，并且对于数字PCR检测，拍摄256个视野)。对于每个视野的图像，分别获取荧光图像(例如，577nm激发，620nm发射，并且曝光时间600ms)和明场图像(例如，汞灯光源，并且曝光时间50ms)。根据荧光图像和明场图像，进行数字化分析。
微流体芯片可以为一次性耗材，反应结束后，含有废液的芯片被存放到特定区域统一处理，防止污染。可以理解的是，可以将清洗液加入到进样孔中，并且对微流体芯片进行离心，使得反应后的样本进入废液池，从而实现对微流体芯片的清洗以及对废液的无害化处理。
在本发明的实施例中，通过选择包括合适的芯片本体的微流体芯片，并且通过加入合适的样本，可以操作微流体芯片以实现不同的检测，例如数字ELISA检测和数字PCR检测。
根据本发明的一个实施例，微流体芯片用在数字ELISA检测中，并且根据本发明的一个实施例，微流体芯片用在数字PCR检测中。
图8是根据本发明的一个实施例的利用微流体芯片实施的绝对定量的数字ELISA检测方法的原理示意图，并且图9是根据本发明的一个实施例的利用微流体芯片实施的绝对定量的数字ELISA检测方法的流程示意图。
如图8和图9所示，利用微流体芯片实施的绝对定量的数字ELISA检测方法包括：
在步骤1(S91)处，制备样本，样本含有待测靶标分子，利用磁珠捕获样本中的待测靶标分子，并且通过亲和反应形成“磁珠-待测靶标分子-酶”的复合物，酶能够与荧光底物进行酶促反应以生成荧光分子。
具体地，待测靶标分子可以为待测蛋白分子，并且该待测蛋白分子可以源自人体的液体样本(血液、体液、组织等)。更具体地，该待测蛋白分子可以来自血清、血浆、组织匀浆或细胞提取液的上清液。基于此，本发明能够精确定量正常人以及疾病患者体内用常规方法难以检测到的超低丰度的蛋白分子，为肿瘤、神经性疾病、感染性疾病、免疫炎症等重大疾病的早期检测、伴随诊断、药物研发等领域开展崭新的应用。
具体地，磁珠可以具有微米尺度的直径。作为样本分配单元，磁珠表面修饰有能够与待测靶标分子特异连接的捕获抗体，例如与待测蛋白分子产生抗体抗原反应，从而捕获待测蛋白分子。
样本中的磁珠的数量可以基于粒子计数仪、流式细胞术或细胞计数板等计数方式来明确确定。另外，样本中的磁珠的均一性也可以基于以上技术来精确控制。基于此，本发明能够至少基于样本中的磁珠的数量来绝对定量样本中的待测靶标分子。
另外，样本中的磁珠的数量应该远大于被捕获到磁珠上的待测靶标分子的数量，从而使得待测靶标分子被捕获到磁珠的统计分布符合泊松分布。
理论上，每个磁珠捕获的待测靶标分子数量存在三种可能性：捕获零个待测靶标分子、捕获单个待测靶标分子或者捕获多个待测靶标分子，而当磁珠的数量足够大时，大部分磁珠只捕获零个待测靶标分子或者单个待测靶标分子，从而实现如下面将要描述的单分子荧光信号放大。
但是，在本发明的实施例中，样本中的磁珠的数量也可以小于等于被捕获到磁珠上的待测靶标分子的数量。作为示例，在被捕获到磁珠上的待测靶标分子的数量与样本中的磁珠的数量的比值小于等于5(即，平均每个磁珠最多捕获5个待测靶标分子)的情况下，待测靶标分子被捕获到磁珠的统计分布依然符合泊松分布，并且依然能够实现单分子荧光信号放大。
样本中的磁珠的数量决定动态检测范围的上限，通过改变样本中的磁珠的数量，可以精确控制动态检测范围。理论上，当捕获几率为100％时，样本中的待测靶标分子的数量小于等于5倍的磁珠数量。例如，样本中的磁珠的数量在10万至1000万的范围内，从而动态检测范围的上限就在50万至5000万的范围内。可以理解的是，考虑到捕获几率的因素，样本中的待测靶标分子的数量与样本中的磁珠的数量的比值也可以大于等于5。当捕获几率为5％时，样本中的待测靶标分子的数量小于等于100倍的磁珠数量。
作为示例，磁珠表面修饰的捕获抗体特异性地捕获样本中的待测靶标分子，并且进一步连接检测抗体和酶，最终形成“磁珠-捕获抗体-待测靶标分子-检测抗体-酶”的免疫复合物，并且该酶能够与荧光底物进行酶促反应以生成荧光分子。例如，磁珠可以通过上述双抗夹心反应连接有β-半乳糖苷酶，并且荧光底物可以为不发射荧光的试卤灵-β-半乳糖苷(RGP)，β-半乳糖苷酶能够催化不发射荧光的试卤灵-β-半乳糖苷(RGP)的水解，生成能够发射荧光的试卤灵分子。
在步骤2(S92)处，将形成有复合物的样本和荧光底物转移到微流体芯片中，其中，芯片本体为包括微孔阵列的微孔阵列芯片本体，微孔阵列中的每个微孔被配置为仅能够容纳一个磁珠。如上面已经描述的，微孔阵列及其芯片本体被容纳在反应池的芯片区中。
具体地，微孔阵列包括多个微孔，每个微孔的尺寸可以比磁珠的尺寸略大，从而被配置为仅能够容纳一个磁珠。其中，微孔阵列中的所有微孔的数量与样本中的磁珠的数量的比值在0.1至10的范围内，以使得尽可能多的磁珠落入微孔。如图8所示，如果磁珠落入微孔，则该微孔就可以被称为有效微孔或反应检测单元。反应检测单元(即，有效微孔)的数量不大于样本分配单元(即，磁珠)的数量，理论上，反应检测单元的数量越接近样本分配单元的数量，数字检测的精度和分辨率越高。
例如，微孔阵列包括188000个微孔，微孔为圆形微孔，微孔的直径为4μm，微孔的深度为4μm，微孔之间的中心距为8μm。
具体地，在第一离心条件下，将形成有复合物的样本和荧光底物转移到微流体芯片中，该第一离心条件例如是200rpm保持10秒，离心力将样本和荧光底物送入微流体芯片的反应池中。然后，静置特定时间(例如，2分钟)，待样本和荧光底物沉降到微孔阵列的微孔中。然后，在第二离心条件下，移除反应池中的多余磁珠和荧光底物，并且保留微孔中的磁珠和荧光底物，该第二离心条件例如是600rpm保持10秒，在该转速下，反应池中的多余磁珠和荧光底物离出，同时微孔中的磁珠和荧光底物得以保留。可以理解的是，也可以采用自吸或压力进样等方式，将形成有复合物的样本和荧光底物转移到微流体芯片中。
在步骤3(S93)处，将隔离液加入到微流体芯片中，以将微孔阵列中的所有微孔彼此隔离。
具体地，隔离液可以为粘滞系数高的氟油或者硅油。在第三离心条件下，将隔离液加入到微流体芯片的反应池中，并且对微孔阵列的微孔中的磁珠和荧光底物进行密封和隔离，该第三离心条件例如是200rpm保持10秒，离心力将隔离液送入微流体芯片的反应池中，疏水性的隔离液可以充分浸润微孔阵列表面，进一步移除未落入微孔的磁珠，同时将所有微孔彼此隔离，并且随后产生的荧光分子难以扩散，单个微孔的热稳定优良。可以理解的是，也可以采用自吸或压力进样等方式，将隔离液加入到微流体芯片的反应池中。
在步骤4(S94)处，等待酶与荧光底物进行酶促反应以生成荧光分子，其中，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值。
例如，在室温下等待1分钟，以用于例如β-半乳糖苷酶催化不发射荧光的试卤灵-β-半乳糖苷(RGP)的水解，生成能够发射荧光的试卤灵分子。如上面已经描述的，在待测靶标分子被捕获到磁珠的统计分布符合泊松分布的情况下，大部分磁珠只捕获零个待测靶标分子或者单个待测靶标分子，从而实现单分子荧光信号放大，即，如图8所示，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值。可以将微孔阵列中的荧光信号高于阈值的微孔判读为1(“阳性”)，并且可以将微孔阵列中的荧光信号低于阈值的微孔判读为0(“阴性”)。
在步骤5(S95)处，确定微孔阵列中的含有磁珠的微孔的数量，并且确定微孔阵列中的荧光信号高于阈值的微孔的数量。
换句话说，步骤5分别确定反应检测单元的数量和阳性反应检测单元的数量。具体地，对微孔阵列拍摄一个或多个视野(例如，35个视野)的明场图像(例如，汞灯光源，并且曝光时间50ms)和荧光图像(例如，577nm激发，620nm发射，并且曝光时间600ms)，其中，如图8所示，基于一个或多个视野的明场图像来确定微孔阵列中的含有磁珠的微孔的数量，并且基于一个或多个视野的荧光图像来确定微孔阵列中的荧光信号高于阈值的微孔的数量。
在步骤6(S96)处，基于微孔阵列中的含有磁珠的微孔的数量以及微孔阵列中的荧光信号高于阈值的微孔的数量，并且基于样本中的磁珠的数量以及待测靶标分子被磁珠捕获并进一步与酶结合的几率，确定样本中的待测靶标分子的数量。
具体地，采用以下公式来确定样本中的待测靶标分子的数量：其中，M0是样本中的待测靶标分子的数量，N0是样本中的磁珠的数量，M是微孔阵列中的荧光信号高于阈值的微孔的数量，N是微孔阵列中的含有磁珠的微孔的数量，并且p是待测靶标分子被磁珠捕获并进一步与酶结合的几率(如果操作均一性高，则几率p是一个小于100％的常数)。
换句话说，在本发明的实施例中，可以通过由粒子计数仪、流式细胞术或细胞计数板等计数方式所明确确定的样本分配单元的数量、由一个或多个视野的明场图像所确定的反应检测单元的数量、由一个或多个视野的荧光图像所确定的阳性反应检测单元的数量以及作为常数的待测靶标分子被磁珠捕获并进一步与酶结合的几率，来绝对定量样本中的待测靶标分子。
更具体，采用以下公式来确定样本中的待测靶标分子的浓度：c＝M0m/V，其中，c是样本中的待测靶标分子的浓度，M0是样本中的待测靶标分子的数量，m是样本中的单个待测靶标分子的质量，并且V是样本的体积。
例如，样本中的待测靶标分子为白介素-6(IL-6)，其分子量为21kDa，样本的体积V为100μL。基于流式细胞术所确定的样本中的磁珠(样本分配单元)的数量N0为75.36万，微孔阵列中的所有微孔的数量为18.8万，基于一个或多个视野的明场图像所确定的微孔阵列中的含有磁珠的微孔(反应检测单元)的数量N为12.1万，基于一个或多个视野的荧光图像所确定的微孔阵列中的荧光信号高于阈值的微孔(阳性反应检测单元)的数量M为5000，并且待测靶标分子被磁珠捕获并进一步与酶结合的几率p为80％，则样本中的待测靶标分子的数量M0采用以下公式计算为39753，并且待测靶标分子的浓度c采用以下公式c＝M0m/V计算为13.9fg/ml。
可以理解的是，在绝对定量的数字ELISA检测方法中利用的微流体芯片与如上面已经描述的相应微流体芯片实施例属于同一构思，其具体实现过程详见相应微流体芯片实施例，这里不再赘述。
图10是根据本发明的一个实施例的利用微流体芯片实施的防荧光信号串扰的数字ELISA检测方法的原理示意图，并且图11是根据本发明的一个实施例的利用微流体芯片实施的防荧光信号串扰的数字ELISA检测方法的流程示意图。
如图10和图11所示，利用微流体芯片实施的防荧光信号串扰的数字ELISA检测方法包括：
在步骤1(S111)处，制备样本，样本含有待测靶标分子，利用磁珠捕获样本中的待测靶标分子，并且通过亲和反应形成“磁珠-待测靶标分子-酶”的复合物。
具体地，待测靶标分子可以为待测蛋白分子，并且该待测蛋白分子可以源自人体的液体样本(血液、体液、组织等)。更具体地，该待测蛋白分子可以来自血清、血浆、组织匀浆或细胞提取液的上清液。基于此，本发明能够精确定量正常人以及疾病患者体内用常规方法难以检测到的超低丰度的蛋白分子，为肿瘤、神经性疾病、感染性疾病、免疫炎症等重大疾病的早期检测、伴随诊断、药物研发等领域开展崭新的应用。
具体地，磁珠可以具有微米尺度的直径。作为样本分配单元，磁珠表面修饰有能够与待测靶标分子特异连接的捕获抗体，例如与待测蛋白分子产生抗体抗原反应，从而捕获待测蛋白分子。
样本中的磁珠的数量可以基于粒子计数仪、流式细胞术或细胞计数板等计数方式来明确确定。另外，样本中的磁珠的均一性也可以基于以上技术来精确控制。基于此，本发明能够至少基于样本中的磁珠的数量来绝对定量样本中的待测靶标分子。
另外，样本中的磁珠的数量应该远大于被捕获到磁珠上的待测靶标分子的数量，从而使得待测靶标分子被捕获到磁珠的统计分布符合泊松分布。
理论上，每个磁珠捕获的待测靶标分子数量存在三种可能性：捕获零个待测靶标分子、捕获单个待测靶标分子或者捕获多个待测靶标分子，而当磁珠的数量足够大时，大部分磁珠只捕获零个待测靶标分子或者单个待测靶标分子，从而实现如下面将要描述的单分子荧光信号放大。
但是，在本发明的实施例中，样本中的磁珠的数量也可以小于等于被捕获到磁珠上的待测靶标分子的数量。作为示例，在被捕获到磁珠上的待测靶标分子的数量与样本中的磁珠的数量的比值小于等于5(即，平均每个磁珠最多捕获5个待测靶标分子)的情况下，待测靶标分子被捕获到磁珠的统计分布依然符合泊松分布，并且依然能够实现单分子荧光信号放大。
样本中的磁珠的数量决定动态检测范围的上限，通过改变样本中的磁珠的数量，可以精确控制动态检测范围。理论上，当捕获几率为100％时，样本中的待测靶标分子的数量小于等于5倍的磁珠数量。例如，样本中的磁珠的数量在10万至1000万的范围内，从而动态检测范围的上限就在50万至5000万的范围内。可以理解的是，考虑到捕获几率的因素，样本中的待测靶标分子的数量与样本中的磁珠的数量的比值也可以大于等于5。当捕获几率为5％时，样本中的待测靶标分子的数量小于等于100倍的磁珠数量。
作为示例，磁珠表面修饰的捕获抗体特异性地捕获样本中的待测靶标分子，并且进一步连接检测抗体和酶，最终形成“磁珠-捕获抗体-待测靶标分子-检测抗体-酶”的免疫复合物，并且该酶能够与荧光底物进行酶促反应以生成荧光分子。例如，磁珠可以通过上述双抗夹心反应连接有β-半乳糖苷酶，并且荧光底物可以为不发射荧光的试卤灵-β-半乳糖苷(RGP)，β-半乳糖苷酶能够催化不发射荧光的试卤灵-β-半乳糖苷(RGP)的水解，生成能够发射荧光的试卤灵分子。
在步骤2(S112)处，将形成有复合物的样本转移到微流体芯片中，其中，芯片本体为包括微孔阵列的微孔阵列芯片本体，微孔阵列中的每个微孔被配置为仅能够容纳一个磁珠。如上面已经描述的，微孔阵列及其芯片本体被容纳在反应池的芯片区中。
具体地，微孔阵列包括多个微孔，每个微孔的尺寸可以比磁珠的尺寸略大，从而被配置为仅能够容纳一个磁珠。其中，微孔阵列中的所有微孔的数量与样本中的磁珠的数量的比值在0.1至10的范围内，以使得尽可能多的磁珠落入微孔。如图10所示，如果磁珠落入微孔，则该微孔就可以被称为有效微孔或反应检测单元。反应检测单元(即，有效微孔)的数量不大于样本分配单元(即，磁珠)的数量，理论上，反应检测单元的数量越接近样本分配单元的数量，数字检测的精度和分辨率越高。
例如，微孔阵列包括188000个微孔，微孔为圆形微孔，微孔的直径为4μm，微孔的深度为4μm，微孔之间的中心距为8μm。
具体地，在第一离心条件下，将形成有复合物的样本转移到微流体芯片中，该第一离心条件例如是200rpm保持10秒，离心力将样本送入微流体芯片的反应池中。然后，静置特定时间(例如，2分钟)，待样本沉降到微孔阵列的微孔中。然后，在第二离心条件下，移除反应池中的多余磁珠，并且保留微孔中的磁珠，该第二离心条件例如是600rpm保持10秒，在该转速下，反应池中的多余磁珠离出，同时微孔中的磁珠得以保留。可以理解的是，也可以采用自吸或压力进样等方式，将形成有复合物的样本转移到微流体芯片中。
在步骤3(S113)处，将荧光底物转移到微流体芯片中。
具体地，在第三离心条件下，将荧光底物转移到微流体芯片中，该第三离心条件例如是200rpm保持10秒，离心力将荧光底物送入微流体芯片的反应池中。荧光底物通过流体剪切力或分子扩散等方式进入微孔中。然后，在第四离心条件下，移除反应池中的多余荧光底物，并且保留微孔中的荧光底物，该第四离心条件例如是600rpm保持10秒，在该转速下，反应池中的多余荧光底物离出，同时微孔中的荧光底物得以保留。可以理解的是，也可以采用自吸或压力进样等方式，将荧光底物转移到微流体芯片中。
在步骤4(S114)处，将隔离液加入到微流体芯片中，以将微孔阵列中的所有微孔彼此隔离。
具体地，隔离液可以为粘滞系数高的氟油或者硅油。在第五离心条件下，将隔离液加入到微流体芯片的反应池中，并且对微孔阵列的微孔中的磁珠和荧光底物进行密封和隔离，该第五离心条件例如是200rpm保持10秒，离心力将隔离液送入微流体芯片的反应池中，疏水性的隔离液可以充分浸润微孔阵列表面，进一步移除未落入微孔的磁珠，同时将所有微孔彼此隔离，并且随后产生的荧光分子难以扩散，单个微孔的热稳定优良。可以理解的是，也可以采用自吸或压力进样等方式，将隔离液加入到微流体芯片的反应池中。
在步骤5(S115)处，等待酶与荧光底物进行酶促反应以生成荧光分子，其中，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值。
例如，在室温下等待1分钟，以用于例如β-半乳糖苷酶催化不发射荧光的试卤灵-β-半乳糖苷(RGP)的水解，生成能够发射荧光的试卤灵分子。如上面已经描述的，在待测靶标分子被捕获到磁珠的统计分布符合泊松分布的情况下，大部分磁珠只捕获零个待测靶标分子或者单个待测靶标分子，从而实现单分子荧光信号放大，即，如图10所示，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值。可以将微孔阵列中的荧光信号高于阈值的微孔判读为1(“阳性”)，并且可以将微孔阵列中的荧光信号低于阈值的微孔判读为0(“阴性”)。
在步骤6(S116)处，确定微孔阵列中的含有磁珠的微孔的数量，并且确定微孔阵列中的荧光信号高于阈值的微孔的数量。
换句话说，步骤6分别确定反应检测单元的数量和阳性反应检测单元的数量。具体地，对微孔阵列拍摄一个或多个视野(例如，35个视野)的明场图像(例如，汞灯光源，并且曝光时间50ms)和荧光图像(例如，577nm激发，620nm发射，并且曝光时间600ms)，其中，如图10所示，基于一个或多个视野的明场图像来确定微孔阵列中的含有磁珠的微孔的数量，并且基于一个或多个视野的荧光图像来确定微孔阵列中的荧光信号高于阈值的微孔的数量。
在步骤7(S117)处，基于微孔阵列中的含有磁珠的微孔的数量以及微孔阵列中的荧光信号高于阈值的微孔的数量，并且基于样本中的磁珠的数量以及待测靶标分子被磁珠捕获并进一步与酶结合的几率，确定样本中的待测靶标分子的数量。
具体地，采用以下公式来确定样本中的待测靶标分子的数量：其中，M0是样本中的待测靶标分子的数量，N0是样本中的磁珠的数量，M是微孔阵列中的荧光信号高于阈值的微孔的数量，N是微孔阵列中的含有磁珠的微孔的数量，并且p是待测靶标分子被磁珠捕获并进一步与酶结合的几率(如果操作均一性高，则几率p是一个小于100％的常数)。
换句话说，在本发明的实施例中，可以通过由粒子计数仪、流式细胞术或细胞计数板等计数方式所明确确定的样本分配单元的数量、由一个或多个视野的明场图像所确定的反应检测单元的数量、由一个或多个视野的荧光图像所确定的阳性反应检测单元的数量以及作为常数的待测靶标分子被磁珠捕获并进一步与酶结合的几率，来绝对定量样本中的待测靶标分子。
更具体，采用以下公式来确定样本中的待测靶标分子的浓度：c＝M0m/V，其中，c是样本中的待测靶标分子的浓度，M0是样本中的待测靶标分子的数量，m是样本中的单个待测靶标分子的质量，并且V是样本的体积。
例如，样本中的待测靶标分子为白介素-6(IL-6)，其分子量为21kDa，样本的体积V为100μL。基于流式细胞术所确定的样本中的磁珠(样本分配单元)的数量N0为75.36万，微孔阵列中的所有微孔的数量为18.8万，基于一个或多个视野的明场图像所确定的微孔阵列中的含有磁珠的微孔(反应检测单元)的数量N为12.1万，基于一个或多个视野的荧光图像所确定的微孔阵列中的荧光信号高于阈值的微孔(阳性反应检测单元)的数量M为5000，并且待测靶标分子被磁珠捕获并进一步与酶结合的几率p为80％，则样本中的待测靶标分子的数量M0采用以下公式计算为39753，并且待测靶标分子的浓度c采用以下公式c＝M0m/V计算为13.9fg/ml。
实施例一、两步加样法和一步加样法的微孔信号对比
根据上面的描述，在根据本发明的一个实施例的利用微流体芯片实施的防荧光信号串扰的数字ELISA检测方法中，形成有复合物的样本与荧光底物是在不同的步骤中分别转移到微流体芯片中的，并且更具体地，形成有复合物的样本与荧光底物分次离心进样铺板，这种加样法被称为两步加样法。作为对比，如果形成有复合物的样本与荧光底物是在同一步骤中一起转移到微流体芯片中的，并且更具体地，形成有复合物的样本与荧光底物单次离心进样铺板，则这种加样法被称为一步加样法。
在一步加样法中，磁珠和荧光底物预先混合后再加入微流体芯片，静置等待磁珠沉降到微孔阵列的微孔中，再加入隔离液将微孔隔离。该过程中，在磁珠与荧光底物混合的同时，酶促反应即开始进行，产生的荧光信号会扩散在整个反应溶液中。待隔离液将磁珠和荧光底物进行隔离时，阳性反应检测单元内的酶进一步催化荧光底物以产生更强的荧光信号，阴性反应检测单元内的荧光信号则为从酶与荧光底物混合至隔离液隔离期间酶催化反应产生的背景信号。另外，磁珠上连接酶分子个数越多，背景信号越强。
而在两步加样法中，由于磁珠先加入微流体芯片并静置等待沉降，再加入荧光底物，而后立即加入隔离液隔离，因此酶与荧光底物反应以产生背景信号的反应时间大大缩短。在一步加样法中该反应时间约为3分钟，而在两步加样法中该反应时间约为20秒，从而大大降低了反应体系的荧光信号背景。
作为用于评测酶促反应的简化系统，实施例一在微流体芯片外将连接有生物素的磁珠(1,000,000个)与特定体积(100μL)链霉亲和素-β-半乳糖苷酶(100fM,1pM,10pM)进行反应，反应缓冲溶液为1×PBS，反应时间为30分钟，反应完成后用1×PBST洗液清洗五次，再分别按照两步加样法和一步加样法的技术方案进行微流体芯片内的操作。
如下面的表1所示，一步加样法中的阴性微孔信号(即，背景信号或背景)抬升明显，SβG浓度为100fM时阴性微孔信号的平均值为678，SβG浓度为1pM时阴性微孔信号的平均值为1172，并且SβG浓度为100pM时阴性微孔信号的平均值为6410，高背景值会导致信号与背景间的区分度变低。而两步加样法中的背景抬升幅度则降低很多，SβG浓度为100fM时阴性微孔信号的平均值为561，SβG浓度为1pM时阴性微孔信号的平均值为1046，并且SβG浓度为100pM时阴性微孔信号的平均值为1662。
表1.两步加样法和一步加样法的微孔信号对比
图12A是10pM SβG浓度反应后的磁珠采用两步加样法得到的荧光图像，并且图12B是10pM SβG浓度反应后的磁珠采用一步加样法得到的荧光图像。另外，图13A是采用ImageJ软件从图12A示出的荧光图像中提取并计算出的某一列微孔的荧光信号，并且图13B是采用ImageJ软件从图12B示出的荧光图像中提取并计算出的某一列微孔的荧光信号。
从图12A-图13B可以看出，一步加样法的背景抬升明显，并且降低了信号与背景间的区分度。
实施例二、不同粘滞系数的隔离液的隔离效果对比
作为用于评测酶促反应的简化系统，实施例二在微流体芯片外将连接有生物素的磁珠(1,000,000个)与100fM链霉亲和素-β-半乳糖苷酶进行反应，反应缓冲溶液为1×PBS，反应时间为30分钟，反应完成后用1×PBST洗液清洗五次，再按照两步加样法的技术方案进行微流体芯片内的操作，并采用不同粘滞系数的隔离液进行隔离。
如下面的表2所示，低粘滞系数的编号_#1氟油(在25℃下的粘滞系数为0.77cSt)出现了明显的染料扩散现象，而高粘滞系数的编号_#2氟油(在25℃下的粘滞系数为11.4cSt)、编号_#3氟油(在25℃下的粘滞系数为451cSt)和编号_#4氟油(在25℃下的粘滞系数为1366cSt)氟油均未出现明显的染料扩散现象。
同样，低粘滞系数的编号_#5硅油(在25℃下的粘滞系数为2.3cSt)、编号_#6硅油(在25℃下的粘滞系数为6.6cSt)均出现了明显的染料扩散现象，而高粘滞系数的编号_#7硅油(在25℃下的粘滞系数为350cSt)未出现明显的染料扩散现象。
表2.不同粘滞系数的隔离液的隔离效果对比
更具体地，当隔离液为氟油时，氟油在25℃下的粘滞系数在0.1cSt至12500cSt的范围内，优选地在1cSt至5000cSt的范围内，并且更优选地在10cSt至1500cSt的范围内。而当隔离液为硅油时，硅油在25℃下的粘滞系数在10cSt至12500cSt的范围内，优选地在10cSt至5000cSt的范围内，并且更优选地在100cSt至500cSt的范围内。
图14A1、图14B1、图14C1和图14D1是分别采用编号_#1、编号_#2、编号_#3和编号_#4的隔离液隔离后1分钟的荧光图像，并且图14A2、图14B2、图14C2和图14D2是分别采用编号_#1、编号_#2、编号_#3和编号_#4的隔离液隔离后5分钟的荧光图像。
从图14A1和图14A2可以看出，在采用低粘滞系数的编号_#1隔离液隔离后，阳性反应检测单元周边的临近反应检测单元有很强的荧光信号，严重影响阳性微孔和阴性微孔的判断，同时随着反应时间的增加，部分阳性反应检测单元的信号降低(由于染料扩散的速度大于酶促反应产生新的荧光分子的速度)，而被串扰的阴性反应检测单元的信号持续增强。而从图14B1至图14D2可以看出，在采用高粘滞系数的编号_#2、编号#3和编号#4隔离液隔离后，阳性反应检测单元周边无荧光信号增强现象。
另外，图15A1和图15A2是采用编号_#1的隔离液隔离后阳性微孔荧光值以及串扰微孔荧光值随时间的变化示意图，并且图15B1和图15B2是采用编号_#3的隔离液隔离后阳性微孔荧光值以及串扰微孔荧光值随时间的变化示意图。
从图15A1和图15A2可以看出，在采用低粘滞系数的编号_#1隔离液隔离后，串扰微孔荧光值上升很快。而从图15B1和图15B2可以看出，在采用高粘滞系数的编号_#3隔离液隔离后，串扰微孔荧光值无明显上升。
可以理解的是，在防荧光信号串扰的数字ELISA检测方法中利用的微流体芯片与如上面已经描述的相应微流体芯片实施例属于同一构思，其具体实现过程详见相应微流体芯片实施例，这里不再赘述。
虽然通过参照某些优选实施例，已经对本发明进行了图示和描述，但本领域的普通技术人员应该明白，可以在形式上和细节上对其作各种改变，而不偏离本发明的精神和范围。

Claims (34)

  1. 一种微流体芯片，其特征在于，所述微流体芯片包括：
    下壳体，所述下壳体包括形成在所述下壳体的内表面上且互相连通的反应池、导流槽以及废液池；
    芯片本体，所述芯片本体设置在所述反应池的芯片区中；
    上壳体，所述上壳体包括贯穿所述上壳体且与所述反应池的进样端对应的进样孔以及贯穿所述上壳体且与所述废液池对应的透气孔。
  2. 根据权利要求1所述的微流体芯片，其特征在于，所述微流体芯片还包括密封垫，所述密封垫设置在所述进样孔上。
  3. 根据权利要求1所述的微流体芯片，其特征在于，所述微流体芯片还包括透气膜，所述透气膜设置在所述透气孔上。
  4. 根据权利要求1所述的微流体芯片，其特征在于，所述芯片本体为包括微孔阵列的微孔阵列芯片本体，所述微孔阵列包括5000个-1000万个微孔，所述微孔的直径为1μm-120μm，所述微孔的深度为1μm-120μm，所述微孔之间的中心距为3μm-180μm。
  5. 根据权利要求4所述的微流体芯片，其特征在于，所述微孔阵列包括188000个微孔，所述微孔的直径为4μm，所述微孔的深度为4μm，所述微孔之间的中心距为8μm。
  6. 根据权利要求4所述的微流体芯片，其特征在于，所述微孔阵列包括8800个微孔，所述微孔的直径为70μm，所述微孔的深度为70μm，所述微孔之间的中心距为105μm。
  7. 根据权利要求1所述的微流体芯片，其特征在于，所述下壳体的内表面上设置一个或多个定位孔，所述上壳体的内表面上设置与所述一个或多个定位孔相适配的一个或多个定位柱。
  8. 根据权利要求1所述的微流体芯片，其特征在于，所述下壳体的内表面上设置围绕所述反应池、所述导流槽以及所述废液池的溢料槽，所述上壳体的内表面上设置围绕所述进样孔以及所述透气孔且与所述溢料槽相适配的焊接线。
  9. 根据权利要求1所述的微流体芯片，其特征在于，所述上壳体还包括形成在所述上壳体的外表面上且与所述透气孔连通的排气槽，所述排气槽的远离所述透气孔的一端在所述上壳体的外表面上延伸，使得所述上壳体延伸形成手持部。
  10. 一种根据权利要求1-9中任一项所述的微流体芯片的操作方法，其特征在于，所述操作方法包括：
    步骤1、单次或分批地将样本加入到所述进样孔中；
    步骤2、每次加入的所述样本通过自吸、离心或压力进样的方式进入所述反应池以及所述芯片区中的所述芯片本体；
    步骤3、对所述微流体芯片进行离心，使得所述样本由所述反应池进入所述废液池，同时所述芯片本体中的样本保留在其中；
    步骤4、将隔离液加入到所述进样孔中；
    步骤5、所述隔离液通过自吸、离心或压力进样的方式进入所述反应池，以隔离所述芯片本体中的所述样本；
    步骤6、等待所述芯片本体中的所述样本进行生化反应；
    步骤7、对所述芯片本体进行成像检测和数字化分析。
  11. 一种根据权利要求1-9中任一项所述的微流体芯片在数字ELISA检测中的用途。
  12. 一种根据权利要求1-9中任一项所述的微流体芯片在数字PCR检测中的用途。
  13. 一种利用根据权利要求1-9中任一项所述的微流体芯片实施的绝对定量的数字ELISA检测方法，其特征在于，所述方法包括：
    步骤1、制备样本，所述样本含有待测靶标分子，利用磁珠捕获所述样本中的所述待测靶标分子，并且通过亲和反应形成“磁珠-待测靶标分子-酶”的复合物，所述酶能够与荧光底物进行酶促反应以生成荧光分子；
    步骤2、将形成有所述复合物的所述样本和所述荧光底物转移到所述微流体芯片中，其中，所述芯片本体为包括微孔阵列的微孔阵列芯片本体，所述微孔阵列中的每个微孔被配置为仅能够容纳一个磁珠；
    步骤3、将隔离液加入到所述微流体芯片中，以将所述微孔阵列中的所有微孔彼此隔离；
    步骤4、等待所述酶与所述荧光底物进行酶促反应以生成所述荧光分子，其中，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值；
    步骤5、确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且确定所述微孔阵列中的荧光信号高于阈值的微孔的数量；
    步骤6、基于所述微孔阵列中的含有所述磁珠的微孔的数量以及所述微孔阵列中的荧光信号高于阈值的微孔的数量，并且基于所述样本中的所述磁珠的数量以及所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率，确定所述样本中的所述待测靶标分子的数量。
  14. 根据权利要求13所述的方法，其特征在于，基于粒子计数仪、流式细胞术或细胞计数板的计数方式来确定所述样本中的所述磁珠的数量。
  15. 根据权利要求13所述的方法，其特征在于，所述样本中的所述待测靶标分子的数量与所述样本中的所述磁珠的数量的比值小于等于100。
  16. 根据权利要求13所述的方法，其特征在于，所述样本中的所述待测靶标分子的数量与所述样本中的所述磁珠的数量的比值小于等于5。
  17. 根据权利要求13所述的方法，其特征在于，在所述步骤5中，对所述微孔阵列拍摄一个或多个视野的明场图像和荧光图像，其中，基于所述一个或多个视野的明场图像来确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且基于所述一个或多个视野的荧光图像来确定所述微孔阵列中的荧光信号高于阈值的微孔的数量。
  18. 根据权利要求13所述的方法，其特征在于，在所述步骤6中，采用以下公式来确定所述样本中的所述待测靶标分子的数量：其中，M0是所述样本中的所述待测靶标分子的数量，N0是所述样本中的所述磁珠的数量，M是所述微孔阵列中的荧光信号高于阈值的微孔的数量，N是所述微孔阵列中的含有所述磁珠的微孔的数量，并且p是所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率。
  19. 根据权利要求13所述的方法，其特征在于，所述微孔阵列中的所有微孔的数量与所述样本中的所述磁珠的数量的比值在0.1至10的范围内。
  20. 根据权利要求13所述的方法，其特征在于，在所述步骤2中，通过自吸、离心或压力进样的方式，将形成有所述复合物的所述样本和所述荧光底物转移到所述微流体芯片的所述反应池以及所述微孔阵列的所述微孔中，并且通过离心的方式，移除所述反应池中的多余磁珠和荧光底物，并且保留所述微孔中的磁珠和荧光底物。
  21. 根据权利要求13所述的方法，其特征在于，在所述步骤3中，通过自吸、离心或压力进样的方式，将所述隔离液加入到所述微流体芯片的所述反应池中，并且对所述微孔阵列的所述微孔中的磁珠和荧光底物进行密封和隔离。
  22. 根据权利要求13所述的方法，其特征在于，所述待测靶标分子为待测蛋白分子。
  23. 根据权利要求13所述的方法，其特征在于，所述隔离液为氟油或者硅油。
  24. 一种利用根据权利要求1-9中任一项所述的微流体芯片实施的防荧光信号串扰的数字ELISA检测方法，其特征在于，所述方法包括：
    步骤1、制备样本，所述样本含有待测靶标分子，利用磁珠捕获所述样本中的所述待测靶标分子，并且通过亲和反应形成“磁珠-待测靶标分子-酶”的复合物；
    步骤2、将形成有所述复合物的所述样本转移到所述微流体芯片中，其中，所述芯片本体为包括微孔阵列的微孔阵列芯片本体，所述微孔阵列中的每个微孔被配置为仅能够容纳一个磁珠；
    步骤3、将荧光底物转移到所述微流体芯片中，所述酶能够与所述荧光底物进行酶促反应以生成荧光分子；
    步骤4、将隔离液加入到所述微流体芯片中，以将所述微孔阵列中的所有微孔彼此隔离；
    步骤5、等待所述酶与所述荧光底物进行酶促反应以生成所述荧光分子，其中，捕获有单个待测靶标分子的磁珠所处的微孔的荧光信号高于阈值，并且捕获有零个待测靶标分子的磁珠所处的微孔的荧光信号低于阈值；
    步骤6、确定所述微孔阵列中的含有所述磁珠的微孔的数量，并且确定所述微孔阵列中的荧光信号高于阈值的微孔的数量；
    步骤7、基于所述微孔阵列中的含有所述磁珠的微孔的数量以及所述微孔阵列中的荧光信号高于阈值的微孔的数量，并且基于所述样本中的所述磁珠的数量以及所述待测靶标分子被所述磁珠捕获并进一步与所述酶结合的几率，确定所述样本中的所述待测靶标分子的数量。
  25. 根据权利要求24所述的方法，其特征在于，所述隔离液为氟油，所述氟油在25℃下的粘滞系数在0.1cSt至12500cSt的范围内。
  26. 根据权利要求25所述的方法，其特征在于，所述氟油在25℃下的粘滞系数在1cSt至5000cSt的范围内。
  27. 根据权利要求26所述的方法，其特征在于，所述氟油在25℃下的粘滞系数为11.4cSt、451cSt或1366cSt。
  28. 根据权利要求24所述的方法，其特征在于，所述隔离液为硅油，所述硅油在25℃下的粘滞系数在10cSt至12500cSt的范围内。
  29. 根据权利要求28所述的方法，其特征在于，所述硅油在25℃下的粘滞系数在10cSt至5000cSt的范围内。
  30. 根据权利要求29所述的方法，其特征在于，所述硅油在25℃下的粘滞系数为350cSt。
  31. 根据权利要求24所述的方法，其特征在于，在所述步骤2中，通过自吸、离心或压力进样的方式，将形成有所述复合物的所述样本转移到所述微流体芯片的所述反应池以及所述微孔阵列的所述微孔中，并且通过离心的方式，移除所述反应池中的多余磁珠，并且保留所述微孔中的磁珠。
  32. 根据权利要求24所述的方法，其特征在于，在所述步骤3中，通过自吸、离心或压力进样的方式，将所述荧光底物转移到所述微流体芯片的所述反应池以及所述微孔阵列的所述微孔中，并且通过离心的方式，移除所述反应池中的多余荧光底物，并且保留所述微孔中的荧光底物。
  33. 根据权利要求24所述的方法，其特征在于，在所述步骤4中，通过自吸、离心或压力进样的方式，将所述隔离液加入到所述微流体芯片的所述反应池中，并且对所述微孔阵列的所述微孔中的磁珠和荧光底物进行密封和隔离。
  34. 根据权利要求24所述的方法，其特征在于，所述待测靶标分子为待测蛋白分子。
PCT/CN2023/121903 2022-09-27 2023-09-27 微流体芯片及其操作方法、数字elisa检测方法和用途 WO2024067667A1 (zh)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN202211183084.8 2022-09-27
CN202211183184.0A CN115639364A (zh) 2022-09-27 2022-09-27 绝对定量的数字elisa检测方法
CN202211183084.8A CN115624991A (zh) 2022-09-27 2022-09-27 微流体芯片及其操作方法和用途
CN202211183268.4A CN116008523A (zh) 2022-09-27 2022-09-27 防荧光信号串扰的数字elisa检测方法
CN202211183184.0 2022-09-27
CN202211183268.4 2022-09-27

Publications (1)

Publication Number Publication Date
WO2024067667A1 true WO2024067667A1 (zh) 2024-04-04

Family

ID=90476374

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/121903 WO2024067667A1 (zh) 2022-09-27 2023-09-27 微流体芯片及其操作方法、数字elisa检测方法和用途

Country Status (1)

Country Link
WO (1) WO2024067667A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130204076A1 (en) * 2010-06-25 2013-08-08 Tsinghua University Integrated microfluidic device for single oocyte trapping
CN105543064A (zh) * 2015-12-29 2016-05-04 西安交通大学 一种数字pcr芯片及其使用方法
CN112322453A (zh) * 2020-12-03 2021-02-05 中国计量科学研究院 一种用于核酸提取、扩增及检测的微流控芯片
CN114308163A (zh) * 2021-12-31 2022-04-12 北京京东方技术开发有限公司 微流控芯片检测卡盒
CN115624991A (zh) * 2022-09-27 2023-01-20 格物致和生物科技(北京)有限公司 微流体芯片及其操作方法和用途
CN115639364A (zh) * 2022-09-27 2023-01-24 格物致和生物科技(北京)有限公司 绝对定量的数字elisa检测方法
CN116008523A (zh) * 2022-09-27 2023-04-25 格物致和生物科技(北京)有限公司 防荧光信号串扰的数字elisa检测方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130204076A1 (en) * 2010-06-25 2013-08-08 Tsinghua University Integrated microfluidic device for single oocyte trapping
CN105543064A (zh) * 2015-12-29 2016-05-04 西安交通大学 一种数字pcr芯片及其使用方法
CN112322453A (zh) * 2020-12-03 2021-02-05 中国计量科学研究院 一种用于核酸提取、扩增及检测的微流控芯片
CN114308163A (zh) * 2021-12-31 2022-04-12 北京京东方技术开发有限公司 微流控芯片检测卡盒
CN115624991A (zh) * 2022-09-27 2023-01-20 格物致和生物科技(北京)有限公司 微流体芯片及其操作方法和用途
CN115639364A (zh) * 2022-09-27 2023-01-24 格物致和生物科技(北京)有限公司 绝对定量的数字elisa检测方法
CN116008523A (zh) * 2022-09-27 2023-04-25 格物致和生物科技(北京)有限公司 防荧光信号串扰的数字elisa检测方法

Similar Documents

Publication Publication Date Title
JP6400630B2 (ja) 試料使用の極大化のためのシステム及び方法
US10365276B2 (en) Device and method for the detection of particles
US9212994B2 (en) Fluorescence detector, filter device and related methods
KR101722548B1 (ko) 원심력기반의 미세유동장치 및 이를 이용한 유체시료 내 분석대상물질 검출방법
CA1310566C (en) Element and method for performing biological assays accurately, rapidlyand simply
JPWO2004036194A1 (ja) 分析チップおよび分析装置
CN106290279B (zh) 一种单细胞蛋白检测系统及其应用
US20060228256A1 (en) Multi-shell microspheres with integrated chomatographic and detection layers for use in array sensors
CN1926424A (zh) 靶标物质的识别芯片、其检测方法和设备
EP1735618A2 (en) System and method for integrating fluids and reagents in self-contained cartridges containing particle and membrane sensor elements
US11904313B2 (en) Liquid quantifying device and application thereof
US20070148759A1 (en) Biological sample discrimination device, biological sample discriminating method, and biological sample discriminating plate
CN113607704A (zh) 一种基于磁珠混匀的微流控芯片检测方法
JP2007101221A (ja) 超高速で生体分子反応を測定する方法
EP1682891A1 (en) Method and apparatus for detecting analyte with filter
WO2024067667A1 (zh) 微流体芯片及其操作方法、数字elisa检测方法和用途
JP2008268198A (ja) 分離チップおよび分離方法
CN115624991A (zh) 微流体芯片及其操作方法和用途
CN115283026A (zh) 集成式滑动芯片
CN110531065B (zh) 一种基于水凝胶的微量全血分离和血浆检测一体化微流控芯片
CN117288960A (zh) 一种对抗体抗原反应进行蛋白芯片检测的方法
CN111504907A (zh) 一种微通道内发光信号的检测方法
CN116727008A (zh) 一种基于sers的微流控芯片及制备方法及应用
NZ706352B2 (en) Systems and methods for sample use maximization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23870894

Country of ref document: EP

Kind code of ref document: A1