WO2021233186A1 - 基于虚拟分割技术的生物靶标数字化定量检测方法和系统 - Google Patents

基于虚拟分割技术的生物靶标数字化定量检测方法和系统 Download PDF

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WO2021233186A1
WO2021233186A1 PCT/CN2021/093464 CN2021093464W WO2021233186A1 WO 2021233186 A1 WO2021233186 A1 WO 2021233186A1 CN 2021093464 W CN2021093464 W CN 2021093464W WO 2021233186 A1 WO2021233186 A1 WO 2021233186A1
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reaction
magnetic beads
biological
tested
digital
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PCT/CN2021/093464
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French (fr)
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荆高山
王栋
许俊泉
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格物致和生物科技(北京)有限公司
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Priority claimed from CN202010416301.8A external-priority patent/CN113687062B/zh
Priority claimed from CN202010416295.6A external-priority patent/CN113687061B/zh
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    • 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

Definitions

  • the invention relates to the field of biological detection, in particular to a method and system for digital quantitative detection of biological targets based on virtual segmentation technology.
  • In Vitro Diagnosis is a technology that obtains clinical diagnosis information by performing sample processing, biochemical reaction and result detection on human samples (blood, body fluids, tissues, etc.) outside the human body.
  • the detection object of in vitro diagnostic technology is liquid, and the conventional detection volume is 1-100ml.
  • the biological and chemical substances in the liquid are mainly nucleic acid molecules (DNA/RNA) and protein molecules.
  • the main human sample for in vitro diagnostic testing is blood. Since the concentration of normal human biological and chemical substances in the blood is relatively constant, changes in the concentration of specific biological and chemical substances can indicate whether the human body is in a healthy state.
  • the in vitro diagnostic process can be divided into the following three stages.
  • Human samples especially blood, contain a variety of biological and chemical substances, such as DNA/protein target molecules.
  • the sample needs to be processed, and the target molecule to be detected is enriched and purified, so as to reduce the interference of the remaining substances in the human sample on the biochemical reaction and result detection.
  • the target molecules that have been processed and captured are generally at a low concentration. It needs to undergo a ligand amplification reaction to increase the mass of the target molecule or characterize the mass of the target molecule.
  • the commonly used DNA target molecule ligand amplification reaction is the PCR reaction, which increases the total amount of substance in the DNA molecule to be tested;
  • the commonly used protein target molecule ligand amplification reaction is ELISA (enzyme-linked stimulating reaction), which is generated by the reaction
  • a large number of chemiluminescent molecules are used to characterize protein target molecules and increase the detection signal of protein target molecules.
  • the strategy is: evenly distribute a sample to be tested into a large number of tiny reaction units; then, these tiny reaction units perform PCR amplification reactions at the same time to achieve single copy or multiple copies PCR amplification of target sequence molecules; after amplification, a threshold value is set for the fluorescent signal detected by each reaction unit, when the fluorescent signal is higher than the threshold value, the reaction unit of the fluorescent signal is interpreted as 1 ("positive"), when the fluorescent signal is lower than the threshold value The reaction unit is interpreted as 0 ("negative").
  • target sequence molecules DNA templates
  • the strategy is to capture the target protein to be tested in the sample through magnetic beads.
  • the magnetic beads that have captured the protein are allocated to a micropit array close to their size.
  • Each micropit can only contain one magnetic bead, and each micropit is individually isolated by fluorinated oil.
  • each micropit is subjected to an ELISA reaction.
  • a threshold value is set for the luminescence signal detected by each reaction unit. When the threshold value is higher than the threshold value, the reaction unit of the luminescence signal is interpreted as 1 ("positive"), and when the threshold value is lower than the threshold value, the reaction unit of the luminescence signal is interpreted as 0 (" feminine").
  • the reaction units are independent of each other.
  • the biochemical reactions in each reaction unit do not "crosstalk” with the biochemical reactions of other reaction units.
  • the PCR reactions in the two reaction units cannot "crosstalk” each other;
  • the digital ELISA technology for protein detection as an example, the ELISA reactions in the two reaction units cannot "crosstalk” each other.
  • the reaction units are not independent of each other, the space size of the reaction units is not uniform, and the number of reaction units is too low, which will cause errors in the downstream result detection.
  • Absolute quantification The absolute number of target molecules can be directly calculated, and accurate absolute quantitative detection can be performed without relying on control standard samples and standard curves.
  • microfluidic chips related to digital detection technology require the design and processing of micron-scale high-precision microfluidic chips to perform uniform physical segmentation of DNA/protein target molecules to be tested.
  • the "water-in-oil" digital PCR technology (Bole, Raindance) requires the design and processing of high-precision micro-channels with a scale of tens to hundreds of micrometers, and the use of oil and water immiscibility characteristics, so as to form a uniform size independent Reaction unit (“microdroplets").
  • the micro-pit type digital PCR chip (Thermofei chip) needs to process a uniform micro-pit array with a scale of tens of microns on a silicon substrate.
  • micro-pit The upper layer of the micro-pit is covered with fluorinated oil to physically isolate the sample to form an independent reaction unit of uniform size.
  • Micro-pit type digital ELISA chip (Quanterix), it is necessary to process a high-density micro-pit array of several microns on the polymer surface. A single magnetic bead is distributed into the micro-pit, and the upper layer is covered with fluorinated oil to achieve physical isolation of the sample.
  • Independent reaction units (“micropits") of uniform size.
  • the present invention provides a digital quantitative chip detection method for biological targets based on virtual segmentation technology.
  • the method includes: Step 1: Use magnetic beads to process, enrich and capture biological targets to be measured. The surface is modified with ligand molecules specifically connected to the biological target to be tested, and the liquid containing the biological target to be tested is concentrated and enriched; Driven into the microfluidic chip, in the microfluidic chip, the biological target to be tested connected to the magnetic beads is combined with the intermediate ligand, and the intermediate ligand functions to catalyze the liquid-solid phase In-situ luminescence reaction; Step 3: Randomly tile and fix the magnetic beads connected to the intermediary ligand on the plane of the reaction zone of the microfluidic chip; Step 4: Perform on the plane of the reaction zone of the microfluidic chip The liquid phase-solid phase in-situ luminescence reaction, the reaction is optically amplified for the biological target to be tested, and a solid-phase luminescent area is formed around the magnetic beads containing
  • the biological target is a DNA and/or protein molecule.
  • step 1 includes: modifying the surface of the magnetic beads with ligand molecules specifically connected to the biological target to be tested; the modified magnetic beads capture the biological target to be tested; using magnetic force to capture the biological target to be tested The target is cleaned and purified; then the purified biological target to be tested is evenly distributed in the liquid.
  • a magnet is applied to the reaction zone of the microfluidic chip, so that the magnetic beads that capture the biological target to be measured are adsorbed to the bottom of the reaction zone of the chip.
  • the magnetic beads that capture the biological target to be tested are adsorbed to the bottom of the reaction zone of the chip and/or the biological target to be tested and the intermediary ligand are placed in the chip.
  • the reaction also includes a step of washing with a washing liquid.
  • the magnetic beads are magnetic beads with a diameter of micrometers and nanometers, preferably magnetic beads with a diameter of 10 nanometers to 100 micrometers.
  • a magnet and an ultrasonic device are alternately used at the bottom of the chip, so that the magnetic beads are randomly tiled and fixed on the plane of the reaction zone of the microfluidic chip.
  • the intermediary ligand is horseradish peroxidase
  • the surface of the magnetic beads or the surface of the substrate is modified with a group capable of reacting with horseradish peroxidase, preferably an aromatic group, and more Preferably, it is a toluene group; the luminescent molecule generated by the catalyzed reaction of horseradish peroxidase is connected to the plane-modified group of the reaction zone of the chip.
  • a magnetic force is applied to the reaction area of the chip to keep the magnetic beads fixed; after the reaction is completed, a cleaning solution is added for elution, leaving the reacted luminescent molecules on the reaction area of the chip.
  • the virtual segmentation method in step 5 includes: uniformly segmenting the digital picture into a plurality of uniform virtual reaction units, each virtual reaction unit includes a light-emitting molecular region formed around each magnetic bead, After division, the luminescent molecule area formed around a single magnetic bead cannot be in the two reaction units; a threshold value is set for the luminescence signal detected by the "virtual reaction unit", and the luminescence signal reaction unit is judged as positive when the threshold value is higher , The reaction unit of the luminescence signal is judged as negative when it is lower than the threshold; and the absolute number of the biological target to be tested is determined by digital analysis.
  • the present invention provides a digital quantitative detection system for biological targets based on virtual segmentation technology
  • the system includes: a sample processing and enrichment device, the sample processing and enrichment device uses magnetic beads to perform the biological target For processing, enrichment and capture, the surface of the magnetic beads is modified with ligand molecules specifically connected to the biological target to be tested, and the liquid containing the biological target to be tested is obtained by concentration and enrichment; microfluidic detection chip, the biological target to be tested The liquid and the intermediate ligand reaction liquid are driven by pressure into the microfluidic chip respectively.
  • the biological target to be tested connected to the magnetic beads is combined with the intermediate ligand, so
  • the role of the intermediary ligand is to catalyze the liquid-solid phase in-situ luminescence reaction;
  • the magnetic beads connected to the intermediary ligand are randomly tiled and fixed on the plane of the reaction zone of the microfluidic chip;
  • the liquid phase-solid phase in-situ luminescence reaction is performed on the plane of the chip reaction zone, and the reaction is optically amplified for the biological target to be tested, and a solid phase luminescent area is formed around the magnetic bead containing the biological target to be tested; and the surface or the surface of the magnetic bead
  • the surface of the reaction zone of the microfluidic chip is modified in advance with functional groups that bind to the luminescent molecules generated by the liquid-solid phase in-situ luminescence reaction, so that the luminescent molecules generated by the reaction are covalently connected to the plane of the reaction zone;
  • a detection device which is used to obtain
  • the sample processing and enrichment device is a device used to capture multiple DNA and/or protein molecular biological targets from one biological sample, or to capture DNA and/or protein molecules from multiple biological samples. / Or a device for protein molecular biological targets.
  • the surface of the magnetic beads is modified with ligand molecules that are specifically connected to the biological target to be tested; the modified magnetic beads capture the biological target to be tested; the captured biological target is cleaned by magnetic force. Purification; then the purified biological target to be tested is evenly distributed in the liquid.
  • a magnet is applied to the reaction zone of the microfluidic chip, so that the magnetic beads that capture the biological target to be tested are adsorbed to the bottom of the reaction zone of the chip.
  • washing is further included. Liquid for cleaning.
  • the magnetic beads are magnetic beads with a diameter of micrometers and nanometers, preferably magnetic beads with a diameter of 10 nanometers to 100 micrometers.
  • a magnet and an ultrasonic device are alternately used at the bottom of the chip, so that the magnetic beads are randomly laid out and fixed on the plane of the reaction zone of the microfluidic chip.
  • the intermediate ligand is horseradish peroxidase
  • the surface of the magnetic beads or the substrate is modified with a group capable of reacting with horseradish peroxidase, preferably an aromatic group, and more Preferably, it is a toluene group; the luminescent molecule generated by the catalyzed reaction of horseradish peroxidase is connected to the plane-modified group of the reaction zone of the chip.
  • a magnetic force is applied to the reaction area of the chip to keep the magnetic beads fixed; after the reaction is completed, a cleaning solution is added for elution, leaving the reacted luminescent molecules on the reaction area of the chip.
  • the virtual segmentation method includes uniformly segmenting the digital picture into a plurality of uniform virtual reaction units, each virtual reaction unit includes a light-emitting molecular region formed around each magnetic bead, and after the division, a single The luminescent molecule area formed around the magnetic beads cannot be located in the two reaction units; a threshold value is set for the luminescence signal detected by the "virtual reaction unit", when the luminescence signal is higher than the threshold value, the luminescence signal reaction unit is judged as positive, which is lower than the threshold value When the reaction unit of the luminescent signal is judged as negative; and the absolute number of the biological target to be tested is determined through digital analysis.
  • the method of the present invention is a pioneering invention in the field of biodigital detection.
  • the present invention proposes virtual segmentation based on the resultant digital image of the biological target to be tested to realize the digital quantitative detection of the biological target to be tested.
  • the advantages of the method of the present invention are: (1) For conventional substrates, the target molecule to be tested in the detection result image is uniformly divided by the "virtual segmentation" technology, so as to realize high-precision, high-accuracy, and low-cost digital detection. Avoid using the complicated, high-precision, and high-cost microfluidic chip design in the existing digital detection technology. (2) Using conventional microscopic image detection technology to achieve high-throughput, fast and low-cost digital detection. Avoid using existing dedicated detectors for digital detection.
  • the present invention proposes for the first time a digital quantitative detection system for biological targets based on virtual segmentation technology to realize digital quantitative chip detection of biological targets to be tested.
  • the advantages of the system of the present invention are: (1) The target molecule to be tested in the detection result image is uniformly divided by the "virtual segmentation" technology, so as to realize high-precision, high-accuracy, and low-cost digital detection. Avoid using the complicated, high-precision, and high-cost microfluidic chip design in the existing digital detection technology. (2) Using conventional microscopic image detection technology to achieve high-throughput, fast and low-cost digital detection. Avoid using existing dedicated detectors for digital detection.
  • liquid manipulation, elution, connection, and liquid-phase solid-phase in-situ luminescence reactions are all completed in the microfluidic chip. There are few manual operations, high elution and reaction efficiency, low background noise, and reliable, sensitive, fast, and inexpensive digital detection can be realized.
  • the microfluidic chip can design multiple parallel flow channels, and simultaneously detect multiple indicators of a single sample or multiple samples in parallel.
  • the detection system required by the entire method of the present invention is greatly simplified, the cost of detection consumables and the detection system is greatly reduced, and the application of digital quantitative technology is greatly expanded. Based on the method of the present invention, reliable, sensitive, fast, and inexpensive digital detection can be realized.
  • the whole detection system of the present invention is greatly simplified, the cost of detection consumables and the detection system is greatly reduced, and the application of digital quantitative technology is greatly expanded. Based on the system of the present invention, reliable, sensitive, fast, and inexpensive digital detection can be realized.
  • the system of the present invention can realize multiple samples and multiple detection for each sample.
  • the entire detection process is completed by the instrument, including enrichment, reaction, and detection of trace DNA/protein target molecules from biological samples (blood, body fluid, tissue).
  • the operation process is automated by an instrument with a pre-set program. The highest detection sensitivity can reach the single-molecule level.
  • Figure 1 is a schematic diagram of the principle of a digital detection method for trace DNA/protein based on virtual segmentation technology
  • Figure 2 is a schematic diagram of the principle of capturing multiple DNA/protein target molecules from a biological sample
  • Figure 3 is a schematic diagram of the principle of capturing DNA/protein target molecules from multiple biological samples
  • Figure 4 is a schematic diagram of the structure of a microfluidic chip based on the "virtual segmentation technology" of trace DNA/protein digital detection technology.
  • Fig. 5 is a schematic diagram of the process of image imaging detection, virtual segmentation, and digital detection.
  • Example 1 Digital quantitative detection of biological targets based on virtual segmentation technology
  • the liquid sample (blood, body fluid, tissue, etc.) of the human body is processed and enriched using micro-nano magnetic beads, and the target DNA/protein molecule to be tested is captured.
  • the surface of the magnetic beads is modified with specific ligands (nucleic acids, proteins) connected to the target DNA/protein molecules to be tested.
  • the magnetic beads are fully mixed with the biological and chemical substances (nucleic acid, protein) to be tested in the sample tube (1-100ml) to capture the target DNA/protein molecules to be tested.
  • the magnetic beads that capture the DNA/protein target molecules to be tested are adsorbed on the tube wall to remove the suspension waste.
  • an intermediate ligand is connected to the captured DNA/protein target molecule through a specific ligand reaction.
  • the role of the intermediary ligand is to catalyze the liquid-solid phase in-situ luminescence reaction, such as horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the magnetic beads connected to the intermediary ligand are adsorbed on the tube wall to remove the suspended waste liquid. Remove the magnet and add cleaning solution to elute the biological and chemical substances (nucleic acid, protein) that are not characteristically adsorbed on the surface of the magnetic beads. Then, using a magnet, the magnetic beads connected to the intermediate ligand are adsorbed on the tube wall to remove the cleaning waste liquid. If necessary, after multiple washings, the magnetic beads connected to the intermediary ligand are concentrated and enriched in a 1-100 ⁇ l liquid system.
  • the number of magnetic beads is much higher than the number of DNA/protein target molecules to be tested.
  • the number of target molecules ranges from 1 molecule to 10,000 molecules, and the number of magnetic beads is greater than 50,000.
  • the typical ratio of the largest target molecule to the magnetic beads is 1:10. The larger the number of magnetic beads, the better the quantification effect. The result is that one target molecule is captured on the surface of most of the magnetic beads.
  • the magnetic beads connected to the intermediary ligand are randomly tiled and fixed to a flat substrate (such as a glass slide). Then drop onto a flat substrate (such as a glass slide). A magnet is used at the bottom of the substrate to fix the magnetic beads to the flat substrate. At this time, the magnetic beads are randomly distributed on the surface of the substrate.
  • the liquid phase-solid phase in-situ luminescence reaction solution is dropped on the flat substrate.
  • the light-reflecting molecules generated by the reaction are deposited on the area near the magnetic beads of the planar substrate; the surface of the planar substrate is modified in advance with functional groups combined with the light-emitting reaction molecules, so that the light-emitting molecules generated by the reaction are covalently connected to the surface of the planar substrate.
  • the surface of the planar substrate is modified with aromatic groups (for example, toluene groups) in advance, and the luminescent molecules generated by the HRP catalyzed reaction are connected to the toluene groups.
  • a magnet is applied to the bottom of the flat substrate to keep the magnetic beads fixed.
  • the solid-phase luminescent molecule area formed around each magnetic bead is several square micrometers to hundreds of square micrometers.
  • the planar substrate is imaged under a conventional fluorescence microscope to obtain high-definition digital pictures.
  • the "virtual segmentation” algorithm is adopted to realize digital detection, and the detection sensitivity can reach the single-molecule level.
  • the “virtual segmentation” calculation method is divided into several parts:
  • the high-definition digital picture is composed of pixels.
  • the solid-phase light-emitting molecule area formed around each magnetic bead is several micrometers to several hundred micrometers.
  • the high-definition picture is evenly divided into multiple uniform "virtual reaction units", each " The "virtual reaction unit” includes a light-emitting molecular region formed around each magnetic bead. Once fixed, the number of "virtual reaction units” is determined.
  • the pixel area of the virtual unit needs to be based on the light-emitting molecular area formed around each magnetic bead.
  • the area of the light-emitting molecule area formed around each magnetic bead is smaller than the area of the dummy cell. For example, the area of the light-emitting molecule formed around each magnetic bead is 100 square microns, and the area of the dummy cell is greater than 100 square microns. After the division, there will be two situations:
  • the Poisson distribution can be used for digital analysis.
  • the pixels of a picture are 1920 x 1280.
  • the maximum area of the luminescent molecule area formed around each magnetic bead is 100 square microns.
  • the largest pixel in the light-emitting molecular area surrounding a single magnetic bead is 4 x 4, so the number of pixels in a single "virtual reaction unit" is 16.
  • the total number of "virtual reaction units" N 0 is 153,600.
  • a threshold value is set for the luminescence signal detected by each "virtual reaction unit". When the threshold value is higher than the threshold value, the luminescence signal reaction unit is interpreted as 1 ("positive"), and when the threshold value is lower than the threshold value, the luminescence signal reaction unit is interpreted as 0 (" feminine").
  • the number of positive units M is 5000
  • the total number of virtual units N 0 is 153,600
  • the absolute number of positive molecules is calculated by the following formula:
  • the absolute number of molecules is 5083.
  • Embodiment 2 Biological target digital quantitative detection system based on virtual segmentation technology
  • the digital quantitative detection system for biological targets based on virtual segmentation technology is shown in Figure 1, and includes the following devices:
  • Figure 2 is a schematic diagram of the principle of capturing multiple DNA/protein target molecules from a biological sample
  • Figure 3 is a schematic diagram of the principle of capturing DNA/protein target molecules from multiple biological samples.
  • Micro-nano magnetic beads are used to process and enrich human liquid samples (blood, body fluids, tissues, etc.), and capture target DNA/protein molecules to be tested.
  • the surface of the magnetic beads is modified with specific ligands (nucleic acids, proteins) connected to the target DNA/protein molecules to be tested.
  • the magnetic beads are fully mixed with the biological and chemical substances (nucleic acid, protein) to be tested in the sample tube (1-100ml) to capture the target DNA/protein molecules to be tested.
  • the magnetic beads that capture the DNA/protein target molecules to be tested are adsorbed on the tube wall to remove the suspension waste.
  • the microfluidic detection chip can perform multiple detection reactions in parallel: it can detect multiple DNA/protein target molecules in a biological sample at the same time, and it can also detect multiple biological samples at the same time.
  • the biological sample needs to be cyclically processed, and the magnetic beads that capture different DNA/protein target molecules are concentrated and enriched in 1-100 ⁇ l liquid system. .
  • the number of magnetic beads is much higher than the number of DNA/protein target molecules to be tested.
  • the number of target molecules ranges from 1 molecule to 10,000 molecules, and the number of magnetic beads is greater than 50,000.
  • the captured DNA/protein target molecule is connected with an intermediary ligand.
  • the role of the intermediary ligand is to catalyze the liquid-solid phase in-situ luminescence reaction, such as horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • an intermediate ligand is connected to the captured DNA/protein target molecule.
  • the role of the intermediary ligand is to catalyze the liquid-solid phase in-situ luminescence reaction, such as horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • valve 1A (1) Sample injection. Open valve 1A, valve 1B, and close the remaining valves. Driven by air pressure or hydraulic pressure, sample 1 enters the chip from the sample 1 port. A magnet is applied in the reaction zone to adsorb the magnetic beads that capture the DNA/protein target molecules to be tested to the bottom of the chip reaction zone. After the injection is complete, close valve 1A and valve 1B.
  • the magnetic beads connected to the intermediary ligand are randomly tiled and fixed in the plane of the reaction zone of the chip.
  • the key to this operation is that the magnetic beads do not agglomerate.
  • the necessary measures include alternate use of magnets and ultrasonic equipment at the bottom of the chip, and finally the magnetic beads are randomly distributed in the plane of the chip's reaction zone.
  • the liquid-solid phase in-situ luminescence reaction is carried out in the microfluidic chip.
  • the light-reflecting molecules generated by the reaction are deposited on the area near the magnetic beads of the chip substrate; the surface of the chip substrate is modified in advance with functional groups combined with the light-emitting reaction molecules, so that the light-emitting molecules generated by the reaction are covalently connected to the surface of the chip substrate.
  • the surface of the flat substrate is modified with toluene groups in advance, and the luminescent molecules generated by the HRP catalyzed reaction are connected with the toluene groups.
  • a magnet is applied to the bottom of the flat substrate to keep the magnetic beads fixed.
  • the solid-phase luminescent molecule area formed around each magnetic bead is several square micrometers to hundreds of square micrometers.
  • the detection device obtains high-definition digital pictures from the surface of the microfluidic chip, which can be imaged under a conventional fluorescent microscope, for example.
  • the "virtual segmentation” algorithm is adopted to realize digital detection, and the detection sensitivity can reach the single-molecule level.
  • the “virtual segmentation” calculation method is divided into several parts:
  • the high-definition digital picture is composed of pixels.
  • the solid-phase light-emitting molecule area formed around each magnetic bead is several micrometers to several hundred micrometers.
  • the high-definition picture is evenly divided into multiple uniform "virtual reaction units", each " The "virtual reaction unit” includes a light-emitting molecular region formed around each magnetic bead. Once fixed, the number of "virtual reaction units” is determined.
  • the pixel area of the virtual unit needs to be based on the light-emitting molecular area formed around each magnetic bead.
  • the area of the light-emitting molecule area formed around each magnetic bead is smaller than the area of the dummy cell. For example, the area of the light-emitting molecule formed around each magnetic bead is 100 square microns, and the area of the dummy cell is greater than 100 square microns. After the division, there will be two situations:
  • the Poisson distribution can be used for digital analysis.
  • the pixels of a picture are 1920 x 1280.
  • the maximum area of the luminescent molecule area formed around each magnetic bead is 100 square microns.
  • the largest pixel in the light-emitting molecular area surrounding a single magnetic bead is 4 x 4, so the number of pixels in a single "virtual reaction unit" is 16.
  • the total number of "virtual reaction units" N 0 is 153,600.
  • a threshold value is set for the luminescence signal detected by each "virtual reaction unit". When the threshold value is higher than the threshold value, the luminescence signal reaction unit is interpreted as 1 ("positive"), and when the threshold value is lower than the threshold value, the luminescence signal reaction unit is interpreted as 0 (" feminine").
  • each magnetic bead to capture DNA/protein target molecules there are three possibilities for each magnetic bead to capture DNA/protein target molecules: zero molecules, single molecules or multiple molecules.
  • the number of magnetic beads is large enough, most of the magnetic beads only capture one molecule or zero molecules; in the end, most "virtual reaction units” contain only one molecule or zero molecules inside, and finally only one solid-phase luminescent molecular region or zero.
  • a solid-phase light-emitting molecular region so as to achieve single-molecule optical signal amplification. Even if a single "virtual reaction unit" contains more than two solid-phase luminescent molecular regions, the proportion and number of reaction units with two signal types, positive and negative, can be counted, and Poisson statistical analysis can be performed to finally calculate the original sample to be tested.
  • the number of DNA/protein target molecules is the number of DNA/protein target molecules.
  • the number of positive units M is 5000
  • the total number of virtual units N 0 is 153,600
  • the absolute number of positive molecules is calculated by the following formula:
  • the absolute number of molecules is 5083.

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Abstract

基于虚拟分割技术的生物靶标数字化定量检测方法和系统，该方法包括：（一）用磁珠对待测生物靶标进行处理、富集和捕获；（二）连接在磁珠上的待测生物靶标与中介配体结合；（三）将连接中介配体的磁珠随机平铺并固定到平面基板上；（四）在平面基板上进行液相-固相原位发光反应，并且平面基板表面事先修饰与液相-固相原位发光反应生成的发光分子结合的功能基团；和（五）获得反应后平面基板的数字图片，然后采用虚拟分割方法，实现待测生物靶标的数字化定量检测。整个方法和系统所要求的检测系统简化，检测耗材和检测系统成本降低，拓宽了数字定量技术应用。可以实现可靠、灵敏、快速并且价格低廉的数字化检测。

Description

基于虚拟分割技术的生物靶标数字化定量检测方法和系统 技术领域
本发明涉及生物检测领域，具体涉及基于虚拟分割技术的生物靶标数字化定量检测方法和系统。
背景技术
体外诊断技术(In Vitro Diagnosis，IVD)是在人体之外，通过对人体的样品(血液、体液、组织等)进行样品处理、生化反应以及结果检测，从而获得临床诊断信息的技术。体外诊断技术的检测对象是液体，常规检测体积1～100ml。液体中的生物、化学物质主要是核酸分子(DNA/RNA)，蛋白分子。体外诊断检测的主要人体样品是血液。由于正常人体生物、化学物质在血液中的浓度相对恒定，特定生物、化学物质的浓度变化，可以表征人体是否处于健康状态。体外诊断过程可以分为以下三个阶段。
(一)样品处理
人体样品，尤其是血液，含有多种生物、化学物质，例如DNA/蛋白靶分子。需要对样品进行处理，对待检测的靶分子进行富集、纯化，从而减少人体样品中其余物质对生化反应和结果检测的干扰。
(二)生化反应
体外诊断中，经过处理和捕获的靶分子一般浓度较低。需要经过配体放大反应，增大靶分子物质量或者表征靶分子的物质量。例如，常用的DNA靶分子配体放大反应是PCR反应，通过反应增大待测DNA分子的物质总量；常用的蛋白靶分子配体放大反应是ELISA(酶联促发反应)，通过反应生成大量化学发光分子用于表征蛋白靶分子，增大蛋白靶分子的检测信号。
(三)结果检测
常规生物、化学检测技术基于光学检测。经过配体放大反应后，较高浓度的核酸、蛋白光学标记物(例如荧光基团、化学发光物质)通过光学器件检测，例如光电倍增管(PMT)或者CCD/CMOS成像光学器件。
针对人体样品(尤其是血液)中痕量靶分子的可靠、灵敏、快速检测，是目前精准医学的重大需求。其中，数字化检测技术是目前的重点研发技术。数字化检测的核心过程是将待测样品均匀分配到大量的反应单元中，这些反应单元同时进行生化反应并进行结果检测。以针对核酸分子检测的数字PCR技术为例，策略是：将一个待测样品均匀分配到大量微小的反应单元中；然后，这些微小的反应单元同时进行PCR扩增反应，实现单拷贝或者多拷贝靶序列分子PCR扩增；扩增后，对每个反应单元检测到的荧光信号设定一个阈值，高于阈值时荧光信号的反应单元判读为1(“阳性”)，低于阈值时荧光信号的反应单元判读为0(“阴性”)。理论上讲，每个反应单元中靶序列分子(DNA模板)的分配存在三种可能性：零拷贝、一个拷贝或多个拷贝。当反应单元的数目足够大，大部分反应单元的内部只含有一个拷贝或者零拷贝靶序列分子(近似于泊松分布)，从而实现单拷贝靶序列分子PCR扩增。最后，通过统计阳性和阴性两种信号类型的反应单元比例和数目，并进行泊松统计学分析，最终计算出原始待测样本中的靶序列拷贝数。
以针对蛋白检测的数字ELISA技术为例，策略是：通过磁珠在样品中捕获待测蛋白靶分子。捕获到蛋白的磁珠被分配到与其尺寸接近的微坑阵列中，每个微坑只能容纳一个磁珠，每个微坑被氟化油单独隔离。然后，每个微坑进行ELISA反应。反应后，对每个反应单元检测到的发光信号设定一个阈值，高于阈值时发光信号的反应单元判读为1(“阳性”)，低于阈值时发光信号的反应单元判读为0(“阴性”)。理论上讲，每个磁珠捕获蛋白靶分子存在三种可能性：零分子、单分子或者多分子。当磁珠的数目足够大，大部分磁珠只捕获一个蛋白靶分子或者零蛋白靶分子；最终，大部分反应单元的内部只含有一个分子或者零分子，从而实现单分子光学信号放大。最后，通过统计阳性和阴性两种信号类型的反应单元比例和数目，并进行泊松统计学分析，最终计算出原始待测样本中的蛋白靶分子数目。
数字化检测的核心概念是：
(1)反应单元之间相互独立。每个反应单元内的生化反应不与其他反应单元的生化反应“串扰”。以针对核酸检测的数字PCR技术为例，两个反应单元内的PCR反应不能相互“串扰”；以针对蛋白检测的数字ELISA技术为例，两个反应单元内的ELISA反应不能相互“串扰”。
(2)反应单元空间尺寸均一，分布随机性。待测样品分布到每个反应单 元的概率一样，为结果检测的精准分析奠定基础。
(3)反应单元数目远远高于待检测的DNA/蛋白靶分子。从而，低浓度靶分子进入反应单元符合泊松分布，为结果检测的数据分析奠定理论基础。
反应单元不相互独立、反应单元空间尺寸不均一以及反应单元数目过低，都会对下游的结果检测产生误差。
数字化检测技术的优点是：
(1)绝对定量。可以直接计算靶分子的绝对数目，无需依赖于对照标准样品和标准曲线就可以进行精确的绝对定量检测。
(2)灵敏度高。可在物理层面实现单分子级检测。对每个反应单元的反应结果判读仅判断有/无两种状态。高于阈值时荧光信号的反应单元判读为1(“阳性”)，低于阈值时荧光信号的反应单元判读为0(“阴性”)。
(3)准确度高。待测样品的反应体系分配过程可以极大降低与靶分子有竞争性作用的背景物质浓度，对生化反应抑制物的耐受能力也大大提高，因此数字化检测技术非常适合在复杂背景中检测痕量DNA/蛋白靶分子。
现有数字化检测技术的不足是：
(1)数字化检测技术相关的微流体芯片设计、加工要求高。现有数字化检测技术需要设计、加工微米量级高精密微流体芯片，对待测DNA/蛋白靶分子进行均匀物理分割。例如“油包水”数字PCR技术(伯乐、Raindance)，需要设计、加工几十～上百微米尺度的高精度微流道，并且利用油和水不相溶的特性，从而形成尺寸均一的独立反应单元(“微液滴”)。微坑式数字PCR芯片(赛默飞芯片)，需要在硅基上加工尺度是几十微米的均匀微坑阵列，微坑上层覆盖氟化油，对样品物理隔离，形成尺寸均一的独立反应单元(“微坑”)。微坑式数字ELISA芯片(Quanterix公司)，需要在聚合物表面加工几微米量级的高密度微坑阵列，单个磁珠被分配到微坑中，上层覆盖氟化油，实现样品物理隔离，形成尺寸均一的独立反应单元(“微坑”)。
(2)数字化检测技术对于检测器的要求高。物理分割后的单元经过生化反应(PCR、ELISA)，需要通过流式检测或者高清成像技术进行检测和分析。
目前体外诊断，急需可靠、灵敏、快速，并且价格低廉的数字化检测方法，实现数字化精准诊断，对疾病做到早诊、早治、早预防。
发明内容
为了解决上述问题，本发明提供一种基于虚拟分割技术的生物靶标数字化定量芯片检测方法，所述方法包括：步骤1：用磁珠对待测生物靶标进行处理、富集和捕获，所述磁珠表面修饰有与待测生物靶标特异连接的配体分子，浓缩富集得到含有所述待测生物靶标的液体；步骤2：将所述待测生物靶标的液体和中介配体反应液分别在压力驱动下进入微流控芯片内，在所述微流控芯片内，连接在所述磁珠上的待测生物靶标与中介配体结合，所述中介配体的作用是催化液相-固相原位发光反应；步骤3：将连接所述中介配体的磁珠随机平铺并固定到所述微流控芯片反应区平面上；步骤4：在所述微流控芯片反应区平面上进行所述液相-固相原位发光反应，该反应对待测生物靶标进行光学放大，在含有待测生物靶标的磁珠周边形成固相发光区域；并且所述微流控芯片反应区平面上事先修饰与所述液相-固相原位发光反应生成的发光分子结合的功能基团，使得反应生成的发光分子共价连接到所述反应区平面上；和步骤5：获得反应后所述微流控芯片反应区平面上的数字图片，然后采用虚拟分割方法，实现所述待测生物靶标的数字化定量检测。
在一种实施方式中个，所述生物靶标是DNA和/或蛋白质分子。
在一种实施方式中，步骤1包括：在所述磁珠表面修饰与待测生物靶标特异连接的配体分子；修饰后的磁珠捕获待测生物靶标；利用磁力对所述捕获待测生物靶标进行清洗和纯化；然后将纯化后的待测生物靶标在液体中均匀分布。
在一种实施方式中，在所述步骤2中，在所述微流控芯片反应区内施加磁铁，使得捕获待测生物靶标的磁珠吸附到所述芯片反应区底部。
在一种实施方式中，在所述步骤2中，在所述捕获待测生物靶标的磁珠吸附到所述芯片反应区底部和/或将所述待测生物靶标和中介配体在芯片内反应后，还包括用清洗液进行清洗的步骤。
在一种实施方式中，所述磁珠是直径为微米级和纳米级别的磁珠，优选直径为10纳米-100微米的磁珠。
在一种实施方式中，在所述步骤3中，在所述芯片底部交替使用磁铁和超声设备，使得所述磁珠随机平铺并固定到所述微流控芯片反应区平面上。
在一种实施方式中，所述中介配体是辣根过氧化物酶，磁珠表面或者和所述基板表面修饰能够与辣根过氧化物酶反应的基团，优选为芳香基团，更优选为甲苯基团；经过辣根过氧化物酶催化反应生成的发光分子与所述芯片 反应区平面修饰的基团连接。
在一种实施方式中，步骤4反应期间，给所述芯片反应区施加磁力，保持磁珠固定；反应结束后，加入清洗液洗脱，所述芯片反应区上留下反应的发光分子。
在一种实施方式中，步骤5中所述虚拟分割方法包括：将所述数字图片均匀分割划分为多个均匀虚拟反应单元，每个虚拟反应单元包括每个磁珠周边形成的发光分子区域，划分后，单个磁珠周边形成的发光分子区域不能处于两个所述反应单元内；对“虚拟反应单元”检测到的发光信号设定一个阈值，高于阈值时发光信号的反应单元判读为阳性，低于阈值时发光信号的反应单元判读为阴性；和通过数字化分析确定待测生物靶标的绝对数量。
在一种实施方式中，本发明提供基于虚拟分割技术的生物靶标数字化定量检测系统，所述系统包括：样品处理与富集装置，所述样品处理与富集装置用磁珠对待测生物靶标进行处理、富集和捕获，所述磁珠表面修饰有与待测生物靶标特异连接的配体分子，浓缩富集得到含有所述待测生物靶标的液体；微流控检测芯片，待测生物靶标的液体和中介配体反应液分别在压力驱动下进入所述微流控芯片内，在所述微流控芯片内，连接在所述磁珠上的待测生物靶标与中介配体结合，所述中介配体的作用是催化液相-固相原位发光反应；连接所述中介配体的磁珠随机平铺并固定到所述微流控芯片反应区平面上；在所述微流控芯片反应区平面上进行所述液相-固相原位发光反应，该反应对待测生物靶标进行光学放大，在含有待测生物靶标的磁珠周边形成固相发光区域；并且磁珠表面或者所述微流控芯片反应区平面上事先修饰与所述液相-固相原位发光反应生成的发光分子结合的功能基团，使得反应生成的发光分子共价连接到所述反应区平面上；和检测装置，所述检测装置用于获得反应后所述微流控芯片反应区平面上的数字图片，然后采用虚拟分割方法，实现所述待测生物靶标的数字化定量检测。
在一种实施方式中，所述样品处理与富集装置是用于从一个生物样品中分别捕获多个DNA和/或蛋白质分子生物靶标的装置，或者是从多个生物样品中分别捕获DNA和/或蛋白质分子生物靶标的装置。
在一种实施方式中，在所述磁珠表面修饰与待测生物靶标特异连接的配体分子；修饰后的磁珠捕获待测生物靶标；利用磁力对所述捕获待测生物靶标进行清洗和纯化；然后将纯化后的待测生物靶标在液体中均匀分布。
在一种实施方式中，在所述微流控芯片反应区内施加磁铁，使得捕获待测生物靶标的磁珠吸附到所述芯片反应区底部。
在一种实施方式中，在所述捕获待测生物靶标的磁珠吸附到所述芯片反应区底部和/或将所述待测生物靶标和中介配体在芯片内反应后，还包括用清洗液进行清洗。
在一种实施方式中，所述磁珠是直径为微米级和纳米级别的磁珠，优选直径为10纳米-100微米的磁珠。
在一种实施方式中，在所述芯片底部交替使用磁铁和超声设备，使得所述磁珠随机平铺并固定到所述微流控芯片反应区平面上。
在一种实施方式中，所述中介配体是辣根过氧化物酶，和磁珠表面或者所述基板表面修饰能够与辣根过氧化物酶反应的基团，优选为芳香基团，更优选为甲苯基团；经过辣根过氧化物酶催化反应生成的发光分子与所述芯片反应区平面修饰的基团连接。
在一种实施方式中，给所述芯片反应区施加磁力，保持磁珠固定；反应结束后，加入清洗液洗脱，所述芯片反应区上留下反应的发光分子。
在一种实施方式中，所述虚拟分割方法包括将所述数字图片均匀分割划分为多个均匀虚拟反应单元，每个虚拟反应单元包括每个磁珠周边形成的发光分子区域，划分后，单个磁珠周边形成的发光分子区域不能处于两个所述反应单元内；对“虚拟反应单元”检测到的发光信号设定一个阈值，高于阈值时发光信号的反应单元判读为阳性，低于阈值时发光信号的反应单元判读为阴性；和通过数字化分析确定待测生物靶标的绝对数量。
本发明的方法是生物数字检测领域的开拓性发明，本发明第一次提出了基于对于待测生物靶标的结果数字化图像进行虚拟分割，实现待测生物靶标的数字化定量检测。本发明方法的优点是：(1)对常规基片，通过“虚拟分割”技术对检测结果图像中待测靶分子进行均匀分割，实现高精密、高准确、低成本的数字化检测。避免采用现有数字化检测技术中复杂、高精度、高成本的微流控芯片设计。(2)采用常规显微图像检测技术，实现高通量、快速、低成本的数字化检测。避免使用现有数字化检测的专用检测器。
本发明第一次提出了基于虚拟分割技术的生物靶标数字化定量检测系统，实现待测生物靶标的数字化定量芯片检测。本发明系统的优点是：(1)通过“虚拟分割”技术对检测结果图像中待测靶分子进行均匀分割，实现高 精密、高准确、低成本的数字化检测。避免采用现有数字化检测技术中复杂、高精度、高成本的微流控芯片设计。(2)采用常规显微图像检测技术，实现高通量、快速、低成本的数字化检测。避免使用现有数字化检测的专用检测器。(3)在本发明中，液体操控、洗脱、连接、液相固相原位发光反应均在微流控芯片内完成。手动操作少，洗脱和反应效率高，背景噪声低，可以实现可靠、灵敏、快速，并且价格低廉的数字化检测。(4)在本发明中，通过实现设计的芯片结构，微流控芯片可以设计多个的并行流道，同时对单个样品的多种指标、或者多个样品进行并行检测。
本发明整个方法所要求的检测系统大大简化，检测耗材和检测系统成本大大降低，大大拓宽了数字定量技术应用。基于本发明方法，可以实现可靠、灵敏、快速，并且价格低廉的数字化检测。本发明整个检测系统大大简化，检测耗材和检测系统成本大大降低，大大拓宽了数字定量技术应用。基于本发明系统，可以实现可靠、灵敏、快速，并且价格低廉的数字化检测。本发明的系统，可以实现多个样品、每个样品多重检测。整个检测流程都由仪器完成，包括从生物样品(血液、体液、组织)中富集、反应、检测痕量DNA/蛋白靶分子，该操作流程经事先设定好程序的仪器自动化完成。检测灵敏度最高可以达到单分子级别。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1是基于虚拟分割技术的痕量DNA/蛋白数字化检测方法原理示意图；
图2是从一个生物样品中分别捕获多个DNA/蛋白靶分子原理示意图；
图3是从多个生物样品中分别捕获DNA/蛋白靶分子原理示意图；
图4是基于“虚拟分割技术”的痕量DNA/蛋白数字化检测技术的微流控芯片的结构示意图；和
图5是图成像检测、虚拟分割、数字化检测流程示意图。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案，下面将结 合下面结合实施例对本发明作进一步说明，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都应当属于本申请保护的范围。
实施例一基于虚拟分割技术的生物靶标数字化定量检测
基于虚拟分割技术的痕量DNA/蛋白数字化检测技术完整技术方案如图1所示，包括以下五个步骤：
(一)痕量样品处理与富集
在此步骤，利用微纳磁珠对人体的液体样品(血液、体液、组织等)进行处理、富集，捕获待测DNA/蛋白靶分子。首先，磁珠表面修饰有与待测DNA/蛋白靶分子相连接的特异配体(核酸、蛋白)。磁珠在样品管内(1～100ml)和待测生物、化学物质(核酸、蛋白)充分混合，捕获待测DNA/蛋白靶分子。然后，利用磁体，将捕获待测DNA/蛋白靶分子的磁珠吸附到管壁上，去除悬浮废液。接着，除去磁体，加入清洗液，洗脱磁珠表面非特性吸附的生物、化学物质(核酸、蛋白)；然后利用磁体，将捕获待测DNA/蛋白靶分子的磁珠吸附到管壁上，去除清洗废液。有必要的话，经过多次清洗，将捕获待测DNA/蛋白靶分子的磁珠浓缩富集、纯化在1～100μl液体体系中。
(二)中介配体连接
在此步骤，通过特异性配体反应，给捕获的待测DNA/蛋白靶分子连接中介配体。中介配体的作用是催化液相-固相原位发光反应，例如辣根过氧化物酶(Horseradish peroxidase，HRP)。利用磁体，将捕获待测DNA/蛋白靶分子的磁珠吸附到管壁上，去除悬浮废液。接着，除去磁体，加入中介配体反应液，将催化液相-固相原位发光反应的中介配体与待测DNA/蛋白靶分子连接。利用磁体，将连接中介配体的磁珠吸附到管壁上，去除悬浮废液。除去磁体，加入清洗液，洗脱磁珠表面非特性吸附的生物、化学物质(核酸、蛋白)。然后利用磁体，将连接中介配体的磁珠吸附到管壁上，去除清洗废液。有必要的话，经过多次清洗，将连接中介配体的磁珠浓缩富集在1～100μl液体体系中。
需要注意的是，磁珠的数量要远远高于待测的DNA/蛋白靶分子数量。例如，靶分子的数量范围为：1个分子～1万个分子，磁珠的数量大于5万。 典型的最大靶分子和磁珠的比例为1：10。磁珠的数量越大，量化效果越好。其结果是：大部分磁珠表面捕捉到1个靶分子。
(三)随机分布：磁珠随机分布、固定到平面基板
在此步骤，将连接中介配体的磁珠随机平铺并固定到一个平面基板(例如载玻片)。然后滴加到一个平面基板(例如载玻片)。基板底部使用磁铁，将磁珠固定到平面基板，这时磁珠随机分布在基板表面。
(四)液相-固相原位发光反应
在此步骤，在平面基板上滴加液相-固相原位发光反应液。该反应生成的反光分子沉积到平面基板磁珠附近区域；平面基板表面事先修饰与发光反应分子结合的功能基团，使得反应生成的发光分子共价连接到平面基板表面。例如，平面基板表面事先修饰芳香基团(例如：甲苯基团)，经过HRP催化反应生成的发光分子与甲苯基团连接。反应期间，平面基板底板施加磁铁，保持磁珠固定。反应结束后，加入清洗液，洗脱磁珠，平面基板仅留下反应的发光分子。每个磁珠周边形成的固相发光分子区域在几个平方微米～几百平方微米。
(五)成像检测、虚拟分割、数字化分析
在此步骤，平面基板在常规荧光显微镜下成像，获得高清数字图片。之后，采用“虚拟分割”算法，实现数字化检测，检测灵敏度最高可以达到单分子级别。“虚拟分割”计算方法分为几个部分：
(1)设定单位“虚拟反应单元”的区域大小
高清数字图片由像素点构成，每个磁珠周边形成的固相发光分子区域在几个微米～几百微米，通过算法，将高清图片均匀分割为多个均匀“虚拟反应单元”，每个“虚拟反应单元”包括每个磁珠周边形成的发光分子区域。一旦固定，“虚拟反应单元”的数目确定。虚拟单元的像素面积，需要根据每个磁珠周边形成的发光分子区域。每个磁珠周边形成的发光分子区域面积小于虚拟单元的面积。例如，每个磁珠周边形成的发光分子区域面积100平方微米，虚拟单元的面积大于100平方微米。划分后，会出现两种情形：
a.如果两个磁珠周边形成的发光分子区域没有交集。“虚拟反应单元”划分时，每个磁珠周边形成的发光分子区域处于各自反应单元内。
b.如果两个磁珠周边形成的荧光发光区域有交集，就需要扩大反应单元分割面积，使得一个反应单元内可能容纳两个以上的发光分子区域。
这两种情形，都可以通过泊松分布进行数字化分析。
例如，一张图片的像素是1920 x 1280。通过实验，每个磁珠周边形成的发光分子区域最大面积100平方微米。此时，对应的单个磁珠周边发光分子区域最大像素为4 x 4，因此单个“虚拟反应单元”的像素数目是16。“虚拟反应单元”的总数N 0为15.36万。
(2)确定阳性信号阈值
对每个“虚拟反应单元”检测到的发光信号设定一个阈值，高于阈值时发光信号的反应单元判读为1(“阳性”)，低于阈值时发光信号的反应单元判读为0(“阴性”)。
(3)数字化分析–泊松分析
理论上讲，每个磁珠捕获DNA/蛋白靶分子存在三种可能性：零分子、单分子或者多分子。当磁珠的数目足够大，大部分磁珠只捕获一个分子或者零分子；最终，大部分“虚拟反应单元”的内部只含有一个分子或者零分子，最终只含有一个固相发光分子区域或者零个固相发光分子区域，从而实现单分子光学信号放大。即使单个“虚拟反应单元”含有两个以上固相发光分子区域，可以通过统计阳性和阴性两种信号类型的反应单元比例和数目，并进行泊松统计学分析，最终计算出原始待测样本中的DNA/蛋白靶分子数目。
例如：经过检测，阳性单元的数目M为5000，虚拟单元的总数N 0为15.36万，阳性分子的绝对数目由以下公式计算：
Figure PCTCN2021093464-appb-000001
绝对分子数量为5083。
实施例二基于虚拟分割技术的生物靶标数字化定量检测系统
基于虚拟分割技术的生物靶标数字化定量检测系统如图1所示，包括以下装置：
一、样品处理与富集装置
如图2和图3所示，图2是从一个生物样品中分别捕获多个DNA/蛋白靶分子原理示意图；和图3是从多个生物样品中分别捕获DNA/蛋白靶分子原理示意图。利用微纳磁珠对人体的液体样品(血液、体液、组织等)进行处理、富集，捕获待测DNA/蛋白靶分子。首先，磁珠表面修饰有与待测DNA/ 蛋白靶分子相连接的特异配体(核酸、蛋白)。磁珠在样品管内(1～100ml)和待测生物、化学物质(核酸、蛋白)充分混合，捕获待测DNA/蛋白靶分子。然后，利用磁体，将捕获待测DNA/蛋白靶分子的磁珠吸附到管壁上，去除悬浮废液。接着，除去磁体，加入清洗液，洗脱磁珠表面非特性吸附的生物、化学物质(核酸、蛋白)；然后利用磁体，将捕获待测DNA/蛋白靶分子的磁珠吸附到管壁上，去除清洗废液。有必要的话，经过多次清洗，将捕获待测DNA/蛋白靶分子的磁珠浓缩富集、纯化在1～100μl液体体系中。
二、微流控检测芯片
微流控检测芯片可以并行进行多个检测反应：可以同时检测一个生物样品中的多个DNA/蛋白靶分子，也可以同时检测多个生物样品。对于检测可以同时检测一个生物样品中的多个DNA/蛋白靶分子，需要对该生物样品进行循环处理，将捕获到不同DNA/蛋白靶分子的磁珠分别浓缩富集在1～100μl液体体系中。
如图4所示，如果同时检测多个生物样品检测，需要对多个生物样品并行处理，将捕获到DNA/蛋白靶分子的磁珠分别浓缩富集在1～100μl液体体系中。
需要注意的是，磁珠的数量要远远高于待测的DNA/蛋白靶分子数量。例如，靶分子的数量范围为：1个分子～1万个分子，磁珠的数量大于5万。磁珠的数量越大，量化效果越好。其结果是：大部分磁珠表面捕捉到1个靶分子。
在微流控芯片内通过特异性配体反应，给捕获的待测DNA/蛋白靶分子连接中介配体。中介配体的作用是催化液相-固相原位发光反应，例如辣根过氧化物酶(Horseradish peroxidase，HRP)。通过特异性配体反应，给捕获的待测DNA/蛋白靶分子连接中介配体。中介配体的作用是催化液相-固相原位发光反应，例如辣根过氧化物酶(Horseradish peroxidase，HRP)。对于样本1的流程顺序如下：
(1)进样。打开阀1A，阀1B，关闭其余阀。气压或者液压驱动下，样本1自进样1口进入芯片。反应区内施加磁铁，将捕获待测DNA/蛋白靶分子的磁珠吸附到芯片反应区底部。进样完成后，关闭阀1A，阀1B。
(2)清洗。打开缓冲阀、连接阀1、连接阀2、连接阀3、阀1B，关闭其余阀。气压或者液压驱动下，缓冲液自缓冲液进样口进入芯片洗脱磁珠表 面非特性吸附的生物、化学物质(核酸、蛋白)。清洗完成后，关闭缓冲阀、连接阀1、连接阀2、连接阀3、阀1B。
(3)中介配体反应。打开反应阀2、连接阀1、连接阀2、连接阀3、阀1B，关闭其余阀。气压或者液压驱动下，中介配体反应液自中介配体反应液进样口进入芯片，将催化液相-固相原位发光反应的中介配体与待测DNA/蛋白靶分子连接。中介配体反应完成后，关闭反应阀2、连接阀1、连接阀2、连接阀3、阀1B。
(4)清洗。打开缓冲阀、连接阀1、连接阀2、连接阀3、阀1B，关闭其余阀。气压或者液压驱动下，缓冲液自缓冲液进样口进入芯片洗脱磁珠表面非特性吸附的生物、化学物质(核酸、蛋白)。有必要的话，经过多次清洗，将连接中介配体的磁珠浓缩富集在1～100μl液体体系中。清洗完成后，关闭缓冲阀、连接阀1、连接阀2、连接阀3、阀1B。
将连接中介配体的磁珠随机平铺并固定到芯片反应区平面内。这个操作的关键是磁珠不发生团聚。必要的措施包括，芯片底部交替使用磁铁和超声设备，最终磁珠随机分布在芯片反应区平面内。
在微流控芯片内进行液相-固相原位发光反应。该反应生成的反光分子沉积到芯片基板磁珠附近区域；芯片基板表面事先修饰与发光反应分子结合的功能基团，使得反应生成的发光分子共价连接到芯片基板表面。例如，平面基板表面事先修饰甲苯基团，经过HRP催化反应生成的发光分子与甲苯基团连接。反应期间，平面基板底板施加磁铁，保持磁珠固定。反应结束后，加入清洗液，磁珠表面或者平面基板仅留下反应的发光分子。每个磁珠周边形成的固相发光分子区域在几个平方微米～几百平方微米。
参照图4，对于样本1的流程顺序如下：
(1)液相-固相反应液进样、反应。反应阀2、连接阀1、连接阀2、连接阀3、阀1B，关闭其余阀。气压或者液压驱动下，液相-固相反应液自液相-固相反应液进样口进入芯片，在中介配体(例如HRP)的催化下，进行液相-固相反应，将催化液相-固相原位发光反应的中介配体与待测DNA/蛋白靶分子连接。每个磁珠周边形成的固相发光分子区域在几个平方微米～几百平方微米。反应完成后，关闭反应阀2、连接阀1、连接阀2、连接阀3、阀1B。
(2)清洗。打开缓冲阀、连接阀1、连接阀2、连接阀3、阀1B，关闭其余阀。气压或者液压驱动下，缓冲液自缓冲液进样口进入芯片洗脱磁珠表 面非特性吸附的生物、化学物质(核酸、蛋白)。加入清洗液，洗脱磁珠，微流控芯片表面仅留下反应的发光分子。每个磁珠周边形成的发光分子区域在几个微米～上百微米。清洗完成后，关闭缓冲阀、连接阀1、连接阀2、连接阀3、阀1B。
三、检测装置
如图5所示，检测装置从微流控芯片表面获得高清数字图片，例如可以使用常规荧光显微镜下成像。之后，采用“虚拟分割”算法，实现数字化检测，检测灵敏度最高可以达到单分子级别。“虚拟分割”计算方法分为几个部分：
(1)设定单位“虚拟反应单元”的区域大小
高清数字图片由像素点构成，每个磁珠周边形成的固相发光分子区域在几个微米～几百微米，通过算法，将高清图片均匀分割为多个均匀“虚拟反应单元”，每个“虚拟反应单元”包括每个磁珠周边形成的发光分子区域。一旦固定，“虚拟反应单元”的数目确定。虚拟单元的像素面积，需要根据每个磁珠周边形成的发光分子区域。每个磁珠周边形成的发光分子区域面积小于虚拟单元的面积。例如，每个磁珠周边形成的发光分子区域面积100平方微米，虚拟单元的面积大于100平方微米。划分后，会出现两种情形：
a.如果两个磁珠周边形成的发光分子区域没有交集。“虚拟反应单元”划分时，每个磁珠周边形成的发光分子区域处于各自反应单元内。
b.如果两个磁珠周边形成的荧光发光区域有交集，就需要扩大反应单元分割面积，使得一个反应单元内可能容纳两个以上的发光分子区域。
这两种情形，都可以通过泊松分布进行数字化分析。
例如，一张图片的像素是1920 x 1280。通过实验，每个磁珠周边形成的发光分子区域最大面积100平方微米。此时，对应的单个磁珠周边发光分子区域最大像素为4 x 4，因此单个“虚拟反应单元”的像素数目是16。“虚拟反应单元”的总数N 0为15.36万。
(2)确定阳性信号阈值
对每个“虚拟反应单元”检测到的发光信号设定一个阈值，高于阈值时发光信号的反应单元判读为1(“阳性”)，低于阈值时发光信号的反应单元判读为0(“阴性”)。
(3)数字化分析–泊松分析
理论上讲，每个磁珠捕获DNA/蛋白靶分子存在三种可能性：零分子、单分子或者多分子。当磁珠的数目足够大，大部分磁珠只捕获一个分子或者零分子；最终，大部分“虚拟反应单元”的内部只含有一个分子或者零分子，最终只含有一个固相发光分子区域或者零个固相发光分子区域，从而实现单分子光学信号放大。即使单个“虚拟反应单元”含有两个以上固相发光分子区域，可以通过统计阳性和阴性两种信号类型的反应单元比例和数目，并进行泊松统计学分析，最终计算出原始待测样本中的DNA/蛋白靶分子数目。
例如：经过检测，阳性单元的数目M为5000，虚拟单元的总数N 0为15.36万，阳性分子的绝对数目由以下公式计算：
Figure PCTCN2021093464-appb-000002
绝对分子数量为5083。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质，因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的，而不是意欲限制本发明的范围，本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到，或者能够确认使用不超过常规实验，在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。

Claims (16)

  1. 基于虚拟分割技术的生物靶标数字化定量检测方法，其特征在于，所述方法包括：
    步骤1：用磁珠对待测生物靶标进行处理、富集和捕获，所述磁珠表面修饰有与待测生物靶标特异连接的配体分子；
    步骤2：连接在所述磁珠上的待测生物靶标与中介配体结合，所述中介配体的作用是催化液相-固相原位发光反应；
    步骤3：将连接所述中介配体的磁珠随机平铺并固定到平面基板上；
    步骤4：在所述平面基板上进行所述液相-固相原位发光反应，该反应对待测生物靶标进行光学放大，在含有待测生物靶标的磁珠周边形成固相发光区域；并且所述平面基板表面事先修饰与所述液相-固相原位发光反应生成的发光分子结合的功能基团，使得反应生成的发光分子共价连接到磁珠表面或者所述平面基板表面上；和
    步骤5：获得反应后所述平面基板的数字图片，然后采用虚拟分割方法，实现所述待测生物靶标的数字化定量检测。
  2. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，所述生物靶标是DNA和/或蛋白质分子。
  3. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，所述磁珠是直径为微米级和纳米级别的磁珠，优选直径为10纳米-100微米的磁珠。
  4. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，所述步骤1包括：在所述磁珠表面修饰与待测生物靶标特异连接的配体分子；修饰后的磁珠捕获待测生物靶标；利用磁力对所述捕获待测生物靶标进行清洗和纯化；然后将纯化后的待测生物靶标在液体中均匀分布。
  5. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，所述步骤2包括：将催化液相-固相原位发光反应的中介配体在液相中与待测生物靶标连接后，利用磁力对连接中介配体的磁珠进行清洗，清洗后的连接中介配体的磁珠浓缩富集在液相体系中。
  6. 根据权利要求5所述的生物靶标数字化定量检测方法，其特征在于，催化液相-固相原位发光反应的中介配体在液相中与待测生物靶标的数量之比为1:1-1:100000，优选地为1:10-1:10000；和更优选地为1:100-1:1000。
  7. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，在步骤3中随机平铺到所述基板上的磁珠通过磁力固定于所述基板上。
  8. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，所述中介配体是辣根过氧化物酶，和所述基板表面修饰能够与辣根过氧化物酶反应的基团，优选为芳香基团，更优选为甲苯基团；经过辣根过氧化物酶催化反应生成的发光分子与磁珠表面或者所述基板表面修饰的基团连接。
  9. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，步骤4反应期间，给所述基板施加磁力，保持磁珠固定；反应结束后，加入清洗液洗脱，磁珠表面或者所述基板上留下反应的发光分子。
  10. 根据权利要求1所述的生物靶标数字化定量检测方法，其特征在于，步骤5中所述虚拟分割方法包括：将所述数字图片均匀分割划分为多个均匀虚拟反应单元，每个虚拟反应单元包括每个磁珠周边形成的发光分子区域，划分后，单个磁珠周边形成的发光分子区域不能处于两个所述反应单元内；对“虚拟反应单元”检测到的发光信号设定一个阈值，高于阈值时发光信号的反应单元判读为阳性，低于阈值时发光信号的反应单元判读为阴性；和通过数字化分析确定待测生物靶标的绝对数量。
  11. 基于虚拟分割技术的生物靶标数字化定量检测系统，其特征在于，所述系统包括：
    样品处理与富集装置，所述样品处理与富集装置用磁珠对待测生物靶标进行处理、富集和捕获，所述磁珠表面修饰有与待测生物靶标特异连接的配体分子，浓缩富集得到含有所述待测生物靶标的液体；
    微流控检测芯片，待测生物靶标的液体和中介配体反应液分别在压力驱动下进入所述微流控芯片内，在所述微流控芯片内，连接在所述磁珠上的待测生物靶标与中介配体结合，所述中介配体的作用是催化液相-固相原位发光反应；连接所述中介配体的磁珠随机平铺并固定到所述微流控芯片反应区平面上；在所述微流控芯片反应区平面上进行所述液相-固相原位发光反应，该反应对待测生物靶标进行光学放大，在含有待测生物靶标的磁珠周边形成 固相发光区域；并且磁珠表面或者所述微流控芯片反应区平面上事先修饰与所述液相-固相原位发光反应生成的发光分子结合的功能基团，使得反应生成的发光分子共价连接到所述反应区平面上；和
    检测装置，所述检测装置用于获得反应后所述微流控芯片反应区平面上的数字图片，然后采用虚拟分割方法，实现所述待测生物靶标的数字化定量检测。
  12. 根据权利要求11所述的基于虚拟分割技术的生物靶标数字化定量检测系统，其特征在于，所述样品处理与富集装置是用于从一个生物样品中分别捕获多个DNA和/或蛋白质分子生物靶标的装置，或者是从多个生物样品中分别捕获DNA和/或蛋白质分子生物靶标的装置。
  13. 根据权利要求11所述的基于虚拟分割技术的生物靶标数字化定量检测系统，其特征在于，在所述磁珠表面修饰与待测生物靶标特异连接的配体分子；修饰后的磁珠捕获待测生物靶标；利用磁力对所述捕获待测生物靶标进行清洗和纯化；然后将纯化后的待测生物靶标在液体中均匀分布。
  14. 根据权利要求1所述的基于虚拟分割技术的生物靶标数字化定量检测系统，其特征在于，在所述微流控芯片反应区内施加磁铁，使得捕获待测生物靶标的磁珠吸附到所述芯片反应区底部。
  15. 根据权利要求11所述的基于虚拟分割技术的生物靶标数字化定量检测系统，其特征在于，在所述芯片底部交替使用磁铁和超声设备，使得所述磁珠随机平铺并固定到所述微流控芯片反应区平面上。
  16. 根据权利要求11所述的基于虚拟分割技术的生物靶标数字化定量检测系统，其特征在于，给所述芯片反应区施加磁力，保持磁珠固定；反应结束后，加入清洗液洗脱，所述磁珠表面或者芯片反应区上留下反应的发光分子。
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