CN113687062B

CN113687062B - Biological target digital quantitative chip detection method based on virtual segmentation method

Info

Publication number: CN113687062B
Application number: CN202010416301.8A
Authority: CN
Inventors: 荆高山; 王栋; 许俊泉
Original assignee: Gewu Zhihe Biotechnology Beijing Co ltd
Current assignee: Gewu Zhihe Biotechnology Beijing Co ltd
Priority date: 2020-05-17
Filing date: 2020-05-17
Publication date: 2023-06-06
Anticipated expiration: 2040-05-17
Also published as: CN113687062A

Abstract

The invention provides a biological target digital quantitative chip detection method based on a virtual segmentation method, which comprises the steps of processing, enriching and capturing a biological target to be detected by using magnetic beads, enabling liquid of the biological target to be detected and intermediate ligand reaction liquid to enter a microfluidic chip under the driving of pressure, randomly tiling and fixing the magnetic beads connected with the intermediate ligand on a micro-fluidic chip reaction area plane, carrying out liquid-solid in-situ luminescence reaction on the micro-fluidic chip reaction area plane to obtain a digital picture on the micro-fluidic chip reaction area plane after the reaction, and then adopting the virtual segmentation method to realize digital quantitative detection of the biological target to be detected. The detection system required by the whole method is greatly simplified, the detection consumable and the detection system cost are greatly reduced, and the application of the digital quantitative technology is greatly widened.

Description

Biological target digital quantitative chip detection method based on virtual segmentation method

Technical Field

The invention relates to the field of biological detection, in particular to a biological target digital quantitative chip detection method based on a virtual segmentation method.

Background

The in vitro diagnostic technique (In Vitro Diagnosis, IVD) is a technique for obtaining clinical diagnostic information by performing sample processing, biochemical reaction, and result detection on a sample (blood, body fluid, tissue, etc.) of a human body, outside the human body. The detection object of the in vitro diagnosis technology is liquid, and the conventional detection volume is 1-100 ml. 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 tests is blood. Because the concentration of normal human body biological and chemical substances in blood is relatively constant, the concentration change of specific biological and chemical substances can be used for representing whether the human body is in a health state or not. The in vitro diagnostic process can be divided into the following three phases.

Sample processing

Human samples, especially blood, contain a variety of biological, chemical substances, such as DNA/protein target molecules. The sample is required to be processed, and the target molecules to be detected are enriched and purified, so that the interference of other substances in the human body sample on biochemical reaction and result detection is reduced.

(II) Biochemical reaction

In vitro diagnostics, the concentration of the treated and captured target molecules is generally low. It is necessary to increase the target molecule mass or characterize the target molecule mass through ligand amplification reactions. For example, a commonly used DNA target molecule ligand amplification reaction is a PCR reaction, by which the total amount of substance of the DNA molecule to be detected is increased; a commonly used protein target ligand amplification reaction is ELISA (enzyme linked immunosorbent assay) which generates a large number of quantitative luminescent molecules for characterizing the protein target molecules and increasing the detection signal of the protein target molecules.

(III) detection of results

Conventional biological, chemical detection techniques are based on optical detection. After ligand amplification reactions, higher concentrations of nucleic acid, protein optical labels (e.g., fluorophores, chemiluminescent substances) are detected by an optical device, such as a photomultiplier tube (PMT) or CCD/CMOS imaging optics.

The reliable, sensitive and rapid detection of trace target molecules in human body samples (especially blood) is a great demand of current accurate medicine. Among them, the digital detection technology is the key research and development technology at present. The core process of digital detection is to uniformly distribute the sample to be detected into a large number of reaction units, and the reaction units simultaneously perform biochemical reaction and result detection. Taking digital PCR technology for nucleic acid molecule detection as an example, the strategies are: uniformly distributing a sample to be tested into a large number of tiny reaction units; then, the tiny reaction units simultaneously carry out PCR amplification reaction to realize single-copy or multi-copy target sequence molecule PCR amplification; after amplification, a threshold value is set for each fluorescent signal detected by each reaction cell, and the reaction cell of the fluorescent signal above the threshold value interprets as 1 ("positive") and the reaction cell of the fluorescent signal below the threshold value interprets as 0 ("negative"). Theoretically, there are three possibilities for the partitioning of the target sequence molecule (DNA template) in each reaction unit: zero copy, one copy, or multiple copies. When the number of reaction units is large enough, most of the reaction units contain only one copy or zero copy of the target sequence molecule (approximately poisson distribution) inside, thereby achieving single copy target sequence molecule PCR amplification. Finally, calculating the copy number of the target sequence in the original sample to be detected by counting the proportion and the number of the reaction units of the positive signal type and the negative signal type and carrying out poisson statistical analysis.

Taking the digital ELISA technique for protein detection as an example, the strategies are: the target molecules of the protein to be detected are captured in the sample by magnetic beads. The beads that capture the protein are distributed into an array of micro-pits of similar size, each capable of holding only one bead, each isolated by fluorinated oil. Then, each dimple was subjected to ELISA reaction. After the reaction, a threshold value is set for the luminescence signal detected by each reaction unit, and the reaction unit of the luminescence signal judges that the luminescence signal is 1 (positive) when the luminescence signal is higher than the threshold value, and the reaction unit of the luminescence signal judges that the luminescence signal is 0 (negative) when the luminescence signal is lower than the threshold value. Theoretically, there are three possibilities for capturing protein target molecules per bead: zero molecule, single molecule or multiple molecules. When the number of magnetic beads is large enough, most magnetic beads capture only one protein target molecule or zero protein target molecule; finally, most of the reaction units contain only one molecule or zero molecule inside, thereby realizing single-molecule optical signal amplification. Finally, counting the proportion and the number of the reaction units of the positive signal type and the negative signal type, carrying out poisson statistical analysis, and finally calculating the number of protein target molecules in the original sample to be detected.

The core concept of digital detection is:

(1) The reaction units are mutually independent. The biochemical reactions within each reaction unit do not "cross-talk" with the biochemical reactions of the other reaction units. Taking a digital PCR technique for nucleic acid detection as an example, the PCR reactions in the two reaction units cannot "cross-talk" to each other; taking the digital ELISA technique for protein detection as an example, ELISA reactions in two reaction units cannot "cross-talk" to each other.

(2) The space size of the reaction unit is uniform, and the distribution is random. The probability that the sample to be detected is distributed to each reaction unit is the same, and a foundation is laid for accurate analysis of result detection.

(3) The number of reaction units is much higher than the DNA/protein target molecule to be detected. Therefore, the low-concentration target molecules enter the reaction unit to accord with poisson distribution, and a theoretical basis is laid for data analysis of result detection.

The reaction units are not independent of each other, the space size of the reaction units is not uniform, and the number of the reaction units is too low, so that errors can be generated on downstream result detection.

The digital detection technology has the advantages that:

(1) Absolute quantification. The absolute number of target molecules can be directly calculated, and accurate absolute quantitative detection can be performed without depending on a control standard sample and a standard curve.

(2) The sensitivity is high. Single molecular level detection can be achieved at the physical level. The reaction result of each reaction unit is interpreted to judge only the presence/absence of two states. The reaction units with fluorescence signals above the threshold interpret as 1 ("positive") and the reaction units with fluorescence signals below the threshold interpret as 0 ("negative").

(3) The accuracy is high. The distribution process of the reaction system of the sample to be detected can greatly reduce the concentration of background substances having competitive action with target molecules and greatly improve the tolerance capability to biochemical reaction inhibitors, so that the digital detection technology is very suitable for detecting trace DNA/protein target molecules in complex backgrounds.

The defects of the existing digital detection technology are as follows:

(1) The design and processing requirements of the microfluidic chip related to the digital detection technology are high. The existing digital detection technology needs to design and process a micron-sized high-precision microfluidic chip, and performs uniform physical segmentation on the DNA/protein target molecules to be detected. For example, "water-in-oil" digital PCR technology (bure, rain) requires the design, fabrication of high precision microchannels on the order of tens to hundreds of microns, and the use of the property of oil and water insolubility to form individual reaction units ("microdroplets") of uniform size. Micro-pit type digital PCR chip (Siemens flight chip) needs to process uniform micro-pit array with the size of tens micrometers on silicon substrate, and the upper layer of micro-pits is covered with fluorinated oil to physically isolate the sample so as to form independent reaction units ('micro-pits') with uniform size. Micropit digital ELISA chip (Quantix Co.) requires processing high density micropit array on the order of several microns on the polymer surface, single magnetic beads are distributed into micropits, upper layer is covered with fluorinated oil to realize physical isolation of sample, and independent reaction unit ("micropits") with uniform size is formed.

(2) Digital detection techniques are demanding on the detector. The physically separated units undergo biochemical reactions (PCR, ELISA) requiring detection and analysis by flow detection or high-definition imaging techniques.

At present, a digital detection method which is reliable, sensitive, quick and low in cost is urgently needed for in vitro diagnosis, digital accurate diagnosis is realized, and early diagnosis, early treatment and early prevention of diseases are realized.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for detecting a biological target digitized quantitative chip based on a virtual segmentation method, which is characterized in that the method comprises: step 1: treating, enriching and capturing a biological target to be detected by using magnetic beads, wherein ligand molecules specifically connected with the biological target to be detected are modified on the surfaces of the magnetic beads, and concentrating and enriching to obtain liquid containing the biological target to be detected; step 2: respectively enabling the liquid of the biological target to be detected and the intermediate ligand reaction liquid to enter a microfluidic chip under the driving of pressure, combining the biological target to be detected connected to the magnetic beads with the intermediate ligand in the microfluidic chip, wherein the intermediate ligand has the function of catalyzing liquid phase-solid phase in-situ luminescence reaction; step 3: randomly tiling and fixing the magnetic beads connected with the intermediate ligands on the plane of the reaction area of the microfluidic chip; step 4: carrying out liquid-solid phase in-situ luminescence reaction on the plane of the reaction zone of the microfluidic chip, wherein the reaction optically amplifies a biological target to be detected, and a solid phase luminescence zone is formed at the periphery of a magnetic bead containing the biological target to be detected; functional groups combined with luminescent molecules generated by the liquid-solid phase in-situ luminescence reaction are modified on the plane of the reaction zone of the microfluidic chip in advance, so that the luminescent molecules generated by the reaction are covalently connected to the plane of the reaction zone; and step 5: and obtaining a digital picture on the plane of the reaction area of the microfluidic chip after the reaction, and then adopting a virtual segmentation method to realize the digital quantitative detection of the biological target to be detected.

In one embodiment, the biological target is a DNA and/or protein molecule.

In one embodiment, step 1 comprises: modifying ligand molecules which are specifically connected with biological targets to be detected on the surfaces of the magnetic beads; capturing a biological target to be detected by the modified magnetic beads; washing and purifying the biological target to be detected by utilizing magnetic force; and then uniformly distributing the purified biological target to be detected in the liquid.

In one embodiment, in the step 2, a magnet is applied in the reaction region of the microfluidic chip, so that magnetic beads capturing biological targets to be detected are adsorbed to the bottom of the reaction region of the chip.

In one embodiment, in the step 2, after the magnetic beads capturing the biological target to be tested are adsorbed to the bottom of the reaction area of the chip and/or the biological target to be tested and the intermediate ligand are reacted in the chip, the method further comprises a step of washing with a washing liquid.

In one embodiment, the magnetic beads are micron-sized and nanoscale in diameter, preferably 10 nm-100 microns in diameter.

In one embodiment, in the step 3, magnets and ultrasonic devices are alternately used at the bottom of the chip, so that the magnetic beads are randomly tiled and fixed on the reaction area plane of the microfluidic chip.

In one embodiment, the mediator ligand is horseradish peroxidase, and the substrate surface is modified with a group capable of reacting with horseradish peroxidase, preferably an aromatic group, more preferably a toluene group; and the luminescent molecules generated by horseradish peroxidase catalytic reaction are connected with the groups modified on the plane of the chip reaction area.

In one embodiment, during the step 4 reaction, magnetic force is applied to the chip reaction zone, keeping the magnetic beads stationary; after the reaction is finished, the magnetic force is removed, a cleaning solution is added for eluting, and the reacted luminescent molecules are left on the chip reaction area.

In one embodiment, the virtual segmentation method in step 5 includes: dividing the digital picture into a plurality of uniform virtual reaction units, wherein each virtual reaction unit comprises a luminescent molecular region formed around each magnetic bead, and after division, the luminescent molecular region formed around a single magnetic bead cannot be positioned in two reaction units; setting a threshold value for the luminous signals detected by the virtual reaction unit, judging positive by the reaction unit of the luminous signals when the luminous signals are higher than the threshold value, and judging negative by the reaction unit of the luminous signals when the luminous signals are lower than the threshold value; and determining the absolute number of biological targets to be detected by digital analysis.

The method is a pioneering invention in the field of biological digital detection, and the invention provides a method for realizing digital quantitative chip detection of a biological target to be detected based on virtual segmentation of a result digital image of the biological target to be detected for the first time. The invention has the advantages that: (1) The target molecules to be detected in the detection result image are uniformly segmented by a virtual segmentation technology, so that high-precision, high-accuracy and low-cost digital detection is realized. The design of a complex, high-precision and high-cost micro-fluidic chip in the existing digital detection technology is avoided. (2) The conventional microscopic image detection technology is adopted to realize high-flux, rapid and low-cost digital detection. Avoiding the use of dedicated detectors for existing digital detection. (3) In the method, liquid control, elution, connection and liquid-solid phase in-situ luminescence reaction are all completed in the microfluidic chip. The method has the advantages of less manual operation, high elution and reaction efficiency and low background noise, and can realize reliable, sensitive and quick digital detection with low price. (4) In the method, the micro-fluidic chip can design a plurality of parallel flow channels by realizing the designed chip structure, and simultaneously detect a plurality of indexes of a single sample or a plurality of samples in parallel.

The detection system required by the whole method is greatly simplified, the detection consumable and the detection system cost are greatly reduced, and the application of the digital quantitative technology is greatly widened. Based on the method of the invention, the digital detection with reliability, sensitivity, rapidness and low price can be realized.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a trace DNA/protein digital detection method based on a virtual segmentation method;

FIG. 2 is a schematic diagram of the principle of capturing a plurality of DNA/protein target molecules from a biological sample, respectively;

FIG. 3 is a schematic diagram of the principle of capturing DNA/protein target molecules from a plurality of biological samples, respectively;

FIG. 4 is a schematic structural diagram of a microfluidic chip based on the "virtual segmentation technique" of trace DNA/protein digital detection technique; and

fig. 5 is a schematic diagram of the imaging detection, virtual segmentation, and digital detection flow.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to the following examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

Embodiment biological target digital quantitative chip detection method based on virtual segmentation method

The complete technical scheme of trace DNA/protein digital detection technology based on the virtual segmentation method is shown in figure 1, and comprises the following five steps:

trace sample treatment and enrichment

FIG. 2 is a schematic diagram showing the principle of capturing a plurality of DNA/protein target molecules from a biological sample, respectively, as shown in FIGS. 2 and 3; and FIG. 3 is a schematic diagram of the principle of capturing DNA/protein target molecules from a plurality of biological samples, respectively. In the step, the micro-nano magnetic beads are utilized to process and enrich liquid samples (blood, body fluid, tissues and the like) of human bodies, and capture DNA/protein target molecules to be detected. First, the surface of the magnetic beads is modified with specific ligands (nucleic acids, proteins) that are linked to the DNA/protein target molecules to be tested. The magnetic beads are fully mixed with biological and chemical substances (nucleic acid and protein) to be detected in a sample tube (1-100 ml) to capture target molecules of the DNA/protein to be detected. Then, using a magnet to adsorb the magnetic beads for capturing the DNA/protein target molecules to be detected on the tube wall, and removing the suspension waste liquid. Then, removing the magnet, adding a cleaning solution, and eluting biological and chemical substances (nucleic acid and protein) which are not adsorbed by the surface of the magnetic beads; and then, using a magnet to adsorb the magnetic beads for capturing the DNA/protein target molecules to be detected on the tube wall, and removing the cleaning waste liquid. If necessary, the magnetic beads for capturing the DNA/protein target molecules to be detected are concentrated, enriched and purified in a liquid system of 1-100 mu l through multiple times of cleaning.

(II) on-chip intermediate ligand ligation

At this step, the chip may perform multiple detection reactions in parallel: multiple DNA/protein target molecules in one biological sample may be detected simultaneously, or multiple biological samples may be detected simultaneously. For detection, a plurality of DNA/protein target molecules in a biological sample can be detected simultaneously, the biological sample needs to be circularly treated, and magnetic beads which capture different DNA/protein target molecules are respectively concentrated and enriched in a liquid system of 1-100 mu l.

As shown in FIG. 4, if a plurality of biological samples are detected simultaneously, the plurality of biological samples are required to be processed in parallel, and the magnetic beads capturing DNA/protein target molecules are respectively concentrated and enriched in 1-100 mu l of liquid system.

It should be noted that the number of magnetic beads is far higher than the number of DNA/protein target molecules to be detected. For example, the number of target molecules ranges from: 1 molecule to 1 ten thousand molecules, and the number of the magnetic beads is more than 5 ten thousand. The larger the number of magnetic beads, the better the quantization effect. The result is that: most of the magnetic bead surface captured 1 target molecule.

In this step, intermediate ligands are attached to the captured DNA/protein target molecules to be tested by specific ligand reactions within the microfluidic chip. The mediator ligand serves to catalyze a liquid-solid phase in situ luminescence reaction, such as horseradish peroxidase (Horseradish peroxidase, HRP). The captured target DNA/protein molecules to be tested are linked to an intermediate ligand by a specific ligand reaction. The mediator ligand serves to catalyze a liquid-solid phase in situ luminescence reaction, such as horseradish peroxidase (Horseradish peroxidase, HRP).

The flow sequence for sample 1 is as follows:

(1) And (5) sample injection. Valve 1A, valve 1B, and the remaining valves are opened. Under the pneumatic or hydraulic drive, the sample 1 enters the chip from the sample inlet 1. And applying a magnet in the reaction area to adsorb the magnetic beads for capturing the DNA/protein target molecules to be detected to the bottom of the chip reaction area. After the sample injection is completed, the valve 1A and the valve 1B are closed.

(2) And (5) cleaning. The buffer valve, the connection valve 1, the connection valve 2, the connection valve 3, the valve 1B are opened, and the remaining valves are closed. Under the driving of air pressure or hydraulic pressure, the buffer solution enters the chip from the buffer solution sample inlet to elute biological and chemical substances (nucleic acid and protein) which are not adsorbed by the surface of the magnetic beads. After the cleaning is completed, the buffer valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.

(3) Intermediate ligand reactions. The reaction valve 2, the connection valve 1, the connection valve 2, the connection valve 3, the valve 1B are opened, and the remaining valves are closed. Under the driving of air pressure or hydraulic pressure, the intermediate ligand reaction liquid enters the chip from the intermediate ligand reaction liquid injection port, and the intermediate ligand for catalyzing the liquid-solid phase in-situ luminescence reaction is connected with the DNA/protein target molecules to be detected. After the intermediate ligand reaction is completed, the reaction valve 2, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.

(4) And (5) cleaning. The buffer valve, the connection valve 1, the connection valve 2, the connection valve 3, the valve 1B are opened, and the remaining valves are closed. Under the driving of air pressure or hydraulic pressure, the buffer solution enters the chip from the buffer solution sample inlet to elute biological and chemical substances (nucleic acid and protein) which are not adsorbed by the surface of the magnetic beads. If necessary, the beads to which the mediator ligands are attached are concentrated and enriched in 1 to 100. Mu.l of a liquid system by washing several times. After the cleaning is completed, the buffer valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.

(III) magnetic beads are randomly distributed in the chip

In this step, the beads attached to the mediator ligands are randomly tiled and immobilized into the plane of the chip reaction area. The key to this step is that the beads do not agglomerate. The necessary measures include that the bottom of the chip is alternately provided with a magnet and ultrasonic equipment, and finally, the magnetic beads are randomly distributed in the plane of the reaction area of the chip.

(IV) liquid phase-solid phase in-situ luminescence reaction is carried out in the chip

In the step, liquid phase-solid phase in-situ luminescence reaction is carried out in the microfluidic chip. The reflective molecules generated by the reaction are deposited in the area near the magnetic beads of the chip substrate; the functional group combined with the luminescence reaction molecule is modified on the surface of the chip substrate in advance, so that the luminescence molecule generated by the reaction is covalently connected to the surface of the chip substrate. For example, the surface of the planar substrate is modified with a toluene group in advance, and a luminescent molecule generated by HRP catalytic reaction is connected with the toluene group. During the reaction, a magnet is applied to the planar substrate base plate to keep the magnetic beads fixed. After the reaction is finished, the magnet is removed, a cleaning solution is added, the magnetic beads are eluted, and only the reacted luminescent molecules are left on the planar substrate. The solid-phase luminescent molecular area formed around each magnetic bead is between several square micrometers and several hundred square micrometers.

Referring to fig. 4, the flow sequence for sample 1 is as follows:

(1) And (3) liquid phase-solid phase reaction liquid sample injection and reaction. The reaction valve 2, the connection valve 1, the connection valve 2, the connection valve 3, the valve 1B, and the remaining valves are closed. Under the drive of air pressure or hydraulic pressure, liquid-solid phase reaction liquid enters the chip from a liquid-solid phase reaction liquid inlet, liquid-solid phase reaction is carried out under the catalysis of an intermediate ligand (such as HRP), and the intermediate ligand catalyzing the liquid-solid phase in-situ luminescence reaction is connected with the DNA/protein target molecules to be detected. The solid-phase luminescent molecular area formed around each magnetic bead is between several square micrometers and several hundred square micrometers. After the completion of the reaction, the reaction valve 2, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.

(2) And (5) cleaning. The buffer valve, the connection valve 1, the connection valve 2, the connection valve 3, the valve 1B are opened, and the remaining valves are closed. Under the driving of air pressure or hydraulic pressure, the buffer solution enters the chip from the buffer solution sample inlet to elute biological and chemical substances (nucleic acid and protein) which are not adsorbed by the surface of the magnetic beads. Removing the magnet, adding the cleaning solution, eluting the magnetic beads, and leaving only the reacted luminescent molecules on the surface of the microfluidic chip. The luminescent molecular area formed around each magnetic bead is several micrometers to hundreds of micrometers. After the cleaning is completed, the buffer valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.

Fifth, imaging detection, virtual segmentation and digital analysis

As shown in fig. 5, in this step, the surface of the microfluidic chip was imaged under a conventional fluorescence microscope, and a high-definition digital picture was obtained. And then, a virtual segmentation algorithm is adopted to realize digital detection, and the highest detection sensitivity can reach a single molecular level. The "virtual segmentation" calculation method is divided into several parts:

(1) Setting the area size of the unit "virtual reaction unit

The high-definition digital picture consists of pixel points, wherein the solid-phase luminescent molecular area formed at the periphery of each magnetic bead is between a few micrometers and hundreds of micrometers, and the high-definition picture is uniformly divided into a plurality of uniform virtual reaction units through an algorithm, and each virtual reaction unit comprises the luminescent molecular area formed at the periphery of each magnetic bead. Once fixed, the number of "virtual reaction units" is determined. The pixel area of the dummy cell needs to be formed according to the luminescent molecular region formed around each magnetic bead. The area of the luminescent molecular area formed around each magnetic bead is smaller than that of the virtual unit. For example, the area of the luminescent molecular region formed around each magnetic bead is 100 square micrometers, and the area of the virtual cell is greater than 100 square micrometers. After division, two situations can occur:

a. if the luminescent molecular regions formed around the two beads do not intersect. When the virtual reaction units are divided, luminescent molecular regions formed around each magnetic bead are positioned in the respective reaction units.

b. If the fluorescent light-emitting regions formed around the two magnetic beads intersect, it is necessary to enlarge the divided area of the reaction cell so that more than two light-emitting molecular regions may be accommodated in one reaction cell.

In both cases, the digitized analysis can be performed by poisson distribution.

For example, the pixel of a picture is 1920x 1280. By experiment, each magnetic bead was formed aroundThe maximum area of the luminescent molecular area is 100 square micrometers. At this time, the maximum pixel of the corresponding single magnetic bead peripheral light-emitting molecular region is 4×4, and thus the number of pixels of a single "virtual reaction unit" is 16. Total number N of "virtual reaction units ₀ 15.36 ten thousand.

(2) Determining a threshold for a positive signal

A threshold value is set for each of the luminescence signals detected by the "virtual reaction units", and the reaction unit of the luminescence signal above the threshold value interprets as 1 ("positive") and the reaction unit of the luminescence signal below the threshold value interprets as 0 ("negative").

(3) Digital analysis-poisson analysis

Theoretically, there are three possibilities for capturing DNA/protein target molecules per magnetic bead: zero molecule, single molecule or multiple molecules. When the number of magnetic beads is large enough, most magnetic beads capture only one molecule or zero molecules; finally, most of the virtual reaction units contain only one molecule or zero molecule, and finally only one solid-phase luminescent molecule region or zero solid-phase luminescent molecule region, so that single-molecule optical signal amplification is realized. Even if a single 'virtual reaction unit' contains more than two solid-phase luminescent molecular regions, the number of DNA/protein target molecules in an original sample to be detected can be finally calculated by counting the proportion and the number of the reaction units of positive and negative signal types and carrying out poisson statistical analysis.

For example: through detection, the number M of positive units is 5000, and the total number N of virtual units ₀ The absolute number of positive molecules was 15.36 ten thousand, calculated by the following formula:

the absolute molecular number was 5083.

It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Claims

1. The biological target digital quantitative chip detection method based on the virtual segmentation method is characterized by comprising the following steps of:

step 1: treating, enriching and capturing a biological target to be detected by using magnetic beads, wherein ligand molecules specifically connected with the biological target to be detected are modified on the surfaces of the magnetic beads, and concentrating and enriching to obtain liquid containing the biological target to be detected;

step 2: respectively enabling the liquid of the biological target to be detected and the intermediate ligand reaction liquid to enter a microfluidic chip under the driving of pressure, combining the biological target to be detected connected to the magnetic beads with the intermediate ligand in the microfluidic chip, wherein the intermediate ligand has the function of catalyzing liquid phase-solid phase in-situ luminescence reaction;

step 3: randomly tiling and fixing the magnetic beads connected with the intermediate ligands on the plane of the reaction area of the microfluidic chip;

step 4: carrying out liquid-solid phase in-situ luminescence reaction on the plane of the reaction zone of the microfluidic chip, wherein the reaction optically amplifies a biological target to be detected, and a solid phase luminescence zone is formed at the periphery of a magnetic bead containing the biological target to be detected; functional groups combined with luminescent molecules generated by the liquid-solid phase in-situ luminescence reaction are modified on the reaction zone plane of the microfluidic chip in advance, and tolyl groups are modified on the reaction zone plane so that the luminescent molecules generated by the reaction are covalently connected to the reaction zone plane; during the reaction of the step 4, magnetic force is applied to the chip reaction area to keep the magnetic beads fixed; after the reaction is finished, removing the magnetic force, adding a cleaning solution for eluting, and leaving reacted luminescent molecules on the chip reaction area; and

step 5: obtaining a digital picture on the plane of the reaction area of the microfluidic chip after the reaction, and then adopting a virtual segmentation method to realize the digital quantitative detection of the biological target to be detected;

the virtual segmentation method in step 5 comprises the following steps: dividing the digital picture into a plurality of uniform virtual reaction units, wherein each virtual reaction unit comprises a luminescent molecular region formed around each magnetic bead, and after division, the luminescent molecular region formed around a single magnetic bead cannot be positioned in two reaction units; setting a threshold value for the luminous signals detected by the virtual reaction units, judging the luminous signals to be positive by the reaction units higher than the threshold value, and judging the luminous signals to be negative by the reaction units lower than the threshold value; and determining the absolute number of biological targets to be detected by digital analysis.

2. The method of claim 1, wherein the biological target is a DNA and/or protein molecule.

3. The method for detecting biological targets by using the digital quantitative chip according to claim 1, wherein the step 1 comprises: modifying ligand molecules which are specifically connected with biological targets to be detected on the surfaces of the magnetic beads; capturing a biological target to be detected by the modified magnetic beads; washing and purifying the biological target to be detected by utilizing magnetic force; and then uniformly distributing the purified biological target to be detected in the liquid.

4. The method according to claim 1, wherein in the step 2, a magnet is applied in the reaction region of the microfluidic chip, so that magnetic beads capturing biological targets to be detected are adsorbed to the bottom of the reaction region of the chip.

5. The method according to claim 4, wherein in the step 2, after the magnetic beads capturing the biological target to be detected are adsorbed to the bottom of the reaction area of the chip and/or the biological target to be detected and the intermediate ligand are reacted in the chip, the method further comprises a step of washing with a washing liquid.

6. The method for detecting biological targets by using the digital quantitative chip according to claim 1, wherein the magnetic beads are magnetic beads with the diameter of micrometer scale and nanometer scale.

7. The method for detecting biological targets by using the digital quantitative chip according to claim 1, wherein the magnetic beads are magnetic beads with diameters of 10 nanometers to 100 micrometers.

8. The method according to claim 1, wherein in the step 3, magnets and ultrasonic devices are alternately used at the bottom of the chip, so that the magnetic beads are randomly tiled and fixed on the reaction area plane of the microfluidic chip.

9. The method for detecting the biological target digital quantitative chip according to claim 1, wherein the intermediate ligand is horseradish peroxidase, and a luminescent molecule generated by horseradish peroxidase catalytic reaction is connected with a group modified by the plane of the chip reaction area.