CN113687061A - Biological target digital quantitative detection system based on virtual segmentation method - Google Patents
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Abstract
The invention provides a biological target digital quantitative detection system based on a virtual segmentation method, which comprises the following steps: the sample processing and enriching device is used for processing, enriching and capturing the biological target to be detected by using the magnetic beads; the liquid of the biological target to be detected and the intermediary ligand reaction liquid respectively enter the micro-fluidic chip under the pressure drive, and the biological target to be detected connected on the magnetic beads is combined with the intermediary ligand in the micro-fluidic chip; and the detection device is used for obtaining a digital picture on the reaction area plane of the microfluidic chip after reaction, and then realizing digital quantitative detection of the biological target to be detected by adopting a virtual segmentation method. The whole detection system is greatly simplified, the detection material consumption and the detection system cost are greatly reduced, and the application of the digital quantitative technology is greatly widened.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a biological target digital quantitative detection system based on a virtual segmentation method.
Background
In Vitro Diagnostic (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 testing is blood. Because the concentration of the biological and chemical substances in the blood of a normal human body is relatively constant, and the concentration change of the specific biological and chemical substances can represent whether the human body is in a healthy state or not. The in vitro diagnostic procedure can be divided into the following three phases.
(I) sample treatment
Human samples, especially blood, contain a variety of biological, chemical substances, such as DNA/protein target molecules. The sample needs 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 mass of the target molecule or characterize the mass of the target molecule by a ligand amplification reaction. For example, the commonly used DNA target molecule ligand amplification reaction is a PCR reaction, which increases the total amount of substance of the DNA molecule to be detected; the commonly used protein target molecule ligand amplification reaction is ELISA (enzyme-linked immunosorbent assay), and a large number of chemiluminescent molecules are generated through the reaction and used for characterizing the protein target molecules, so that the detection signals of the protein target molecules are increased.
(III) detection of the results
Conventional biological, chemical detection techniques are based on optical detection. After the ligand amplification reaction, higher concentrations of nucleic acids, protein optical labels (e.g., fluorophores, chemiluminescent species) are detected by optical devices, such as photomultiplier tubes (PMTs) 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 precise medicine at present. Among them, the digital detection technology is the currently important research and development technology. The core process of digital detection is to uniformly distribute samples to be detected into a large number of reaction units, and the reaction units carry out biochemical reaction and result detection at the same time. Taking the digital PCR technology for nucleic acid molecule detection as an example, the strategy is: uniformly distributing a sample to be detected into a large number of tiny reaction units; then, the micro 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 the fluorescence signal detected in each reaction unit, above which the reaction unit for the fluorescence signal is interpreted as 1 ("positive") and below which the reaction unit for the fluorescence signal is interpreted as 0 ("negative"). Theoretically, there are three possibilities for the assignment of target molecules (DNA templates) per reaction unit: zero copies, one copy, or multiple copies. When the number of reaction units is large enough, most reaction units contain only one copy or zero copy of target sequence molecules inside (similar to poisson distribution), thereby realizing PCR amplification of single copy target sequence molecules. And 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 strategy is: and capturing the protein target molecules to be detected in the sample through the magnetic beads. The protein-captured magnetic beads are distributed in an array of micro-pits of close size, each micro-pit containing only one magnetic bead, each micro-pit being individually isolated by a fluorinated oil. Then, each micro-well was subjected to ELISA reaction. After the reaction, a threshold value is set for the luminescence signal detected by each reaction unit, and above the threshold value, the reaction unit of the luminescence signal is judged as 1 ("positive"), and below the threshold value, the reaction unit of the luminescence signal is judged as 0 ("negative"). Theoretically, there are three possibilities for capturing protein target molecules per magnetic bead: zero molecules, single molecules or multiple molecules. When the number of the magnetic beads is large enough, most of the magnetic beads capture only one protein target molecule or zero protein target molecule; finally, most reaction units only contain one molecule or zero molecules inside, so that monomolecular optical signal amplification is realized. And finally, 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 to finally calculate the number of the protein target molecules in the original sample to be detected.
The core concept of digital detection is:
(1) the reaction units are independent from each other. The biochemical reaction in each reaction cell does not "cross-talk" with the biochemical reactions of the other reaction cells. Taking the digital PCR technology for nucleic acid detection as an example, the PCR reactions in the two reaction units cannot "cross talk" with each other; taking the digital ELISA technique for protein detection as an example, the ELISA reactions in the two reaction units cannot "cross-talk" each other.
(2) The reaction units are uniform in spatial size and random in distribution. The probability that the samples to be detected are 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 molecules 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 non-independence of the reaction units, the non-uniform spatial dimension of the reaction units, and the low number of reaction units all cause errors in downstream result detection.
The advantages of the digital detection technology are that:
(1) absolute quantification. The absolute number of target molecules can be directly calculated, and accurate absolute quantitative detection can be carried out without depending on a control standard sample and a standard curve.
(2) The sensitivity is high. The single molecule level detection can be realized at the physical level. The reaction result of each reaction unit is interpreted to judge only the presence/absence of two states. Reaction units with a fluorescence signal above the threshold read 1 ("positive") and reaction units with a fluorescence signal below the threshold read 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 of biochemical reaction inhibitors, so that the digital detection technology is very suitable for detecting trace DNA/protein target molecules in a complex background.
The existing digital detection technology has the following defects:
(1) the design and processing requirements of the microfluid chip related to the digital detection technology are high. The existing digital detection technology needs to design and process a micro-scale high-precision micro-fluidic chip to perform uniform physical segmentation on DNA/protein target molecules to be detected. For example, "water-in-oil" digital PCR technology (berle, raidance) requires the design, processing of high-precision microchannels on the scale of tens to hundreds of microns, and the use of the property of oil and water incompatibility to form individual reaction cells ("micro-droplets") of uniform size. A micro-pit type digital PCR chip (a Saimei flying chip) needs to process a uniform micro-pit array with the size of tens of microns on a silicon substrate, the upper layer of the micro-pit is covered with fluorinated oil, and a sample is physically isolated to form an independent reaction unit (a micro-pit) with uniform size. A crater type digital ELISA chip (Quanterix company) needs to process a high-density crater array with the magnitude of a few microns on the surface of a polymer, single magnetic beads are distributed in the craters, and the upper layer is covered with fluorinated oil to realize physical isolation of samples and form independent reaction units (craters) with uniform size.
(2) Digital detection techniques are highly demanding for detectors. The physically segmented units undergo biochemical reactions (PCR, ELISA) and need to be detected and analyzed by flow detection or high-definition imaging technology.
At present, a digital detection method which is reliable, sensitive, rapid and low in price is urgently needed for in vitro diagnosis, so that 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 digital quantitative detection system for biological targets based on a virtual segmentation method, the system comprising: the sample processing and enriching device is used for processing, enriching and capturing a biological target to be detected by using magnetic beads, ligand molecules specifically connected with the biological target to be detected are modified on the surfaces of the magnetic beads, and liquid containing the biological target to be detected is obtained by concentration and enrichment; the liquid of the biological target to be detected and the intermediary ligand reaction liquid respectively enter the micro-fluidic chip under the pressure drive, the biological target to be detected connected on the magnetic beads is combined with the intermediary ligand in the micro-fluidic chip, and the intermediary ligand is used for catalyzing liquid phase-solid phase in-situ luminescence reaction; magnetic beads connected with the intermediary ligand are randomly tiled and fixed on the reaction area plane of the microfluidic chip; performing the liquid-solid phase in-situ luminescence reaction on the plane of the reaction area of the microfluidic chip, wherein the reaction optically amplifies the biological target to be detected and forms a solid-phase luminescence area around the magnetic bead containing the biological target to be detected; and the plane of the reaction area of the microfluidic chip is modified with a functional group which is combined with luminescent molecules generated by the liquid-solid phase in-situ luminescent reaction in advance, so that the luminescent molecules generated by the reaction are covalently connected to the plane of the reaction area; and the detection device is used for obtaining a digital picture on the reaction area plane of the microfluidic chip after reaction, and then realizing digital quantitative detection of the biological target to be detected by adopting a virtual segmentation method.
In one embodiment, the sample processing and enriching means is a means for capturing a plurality of DNA and/or protein molecule biological targets from one biological sample, respectively, or a means for capturing a plurality of DNA and/or protein molecule biological targets from a plurality of biological samples, respectively.
In one embodiment, the surface of the magnetic bead is modified with a ligand molecule specifically linked to a biological target to be detected; capturing a biological target to be detected by the modified magnetic beads; cleaning and purifying the captured biological target to be detected by using magnetic force; and then uniformly distributing the purified biological target to be detected in the liquid.
In one embodiment, a magnet is applied to the reaction area of the microfluidic chip, so that the magnetic beads for capturing the biological target to be detected are adsorbed to the bottom of the reaction area of the chip.
In one embodiment, after the magnetic beads capturing the biological target to be detected are adsorbed to the bottom of the reaction region of the chip and/or the biological target to be detected and the intermediary ligand are reacted in the chip, the method further comprises washing with a washing solution.
In one embodiment, the magnetic beads are micro-sized and nano-sized in diameter, preferably between 10 nanometers and 100 micrometers in diameter.
In one embodiment, the magnetic beads are randomly tiled and fixed on the reaction area plane of the microfluidic chip by using a magnet and an ultrasonic device alternately at the bottom of the chip.
In one embodiment, the intermediary ligand is horseradish peroxidase and the substrate surface is modified with groups capable of reacting with horseradish peroxidase, preferably aromatic groups, more preferably toluene groups; luminescent molecules generated by the catalytic reaction of horseradish peroxidase are connected with the groups modified on the plane of the reaction area of the chip.
In one embodiment, a magnetic force is applied to the reaction region of the chip to keep the magnetic beads immobilized; and after the reaction is finished, removing the magnetic force, adding a cleaning solution for elution, and leaving the reacted luminescent molecules on the reaction area of the chip.
In one embodiment, the virtual segmentation method includes uniformly segmenting the digital picture into a plurality of uniform virtual reaction units, each virtual reaction unit includes a luminescent molecule region formed around each magnetic bead, and after segmentation, the luminescent molecule region formed around a single magnetic bead cannot be located in two reaction units; setting a threshold value for the luminescence signal detected by the virtual reaction unit, wherein the reaction unit of the luminescence signal judges as positive when the threshold value is higher than the threshold value, and the reaction unit of the luminescence signal judges as negative when the threshold value is lower than the threshold value; and determining the absolute number of the biological target to be detected by digital analysis.
The invention provides a biological target digital quantitative detection system based on a virtual segmentation method for the first time, and realizes digital quantitative chip detection of a biological target to be detected. 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 complicated, high-precision and high-cost micro-fluidic chip design in the existing digital detection technology is avoided. (2) The conventional microscopic image detection technology is adopted to realize high-throughput, rapid and low-cost digital detection. The use of existing dedicated detectors for digital detection is avoided. (3) In the invention, liquid control, elution, connection and liquid-phase solid-phase in-situ luminescence reaction are all completed in the microfluidic chip. The method has the advantages of few manual operations, high elution and reaction efficiency, low background noise, and capability of realizing reliable, sensitive and rapid digital detection with low price. (4) In the invention, by realizing the designed chip structure, the microfluidic chip can design a plurality of parallel flow channels and simultaneously carry out parallel detection on a plurality of indexes of a single sample or a plurality of samples.
The whole detection system is greatly simplified, the detection material consumption and the detection system cost are greatly reduced, and the application of the digital quantitative technology is greatly widened. Based on the system of the invention, the digital detection with reliability, sensitivity, rapidness and low price can be realized. The system of the invention can realize multiple samples and multiple detection of each sample. The whole detection process is completed by an instrument, and comprises the steps of enriching, reacting and detecting trace DNA/protein target molecules from biological samples (blood, body fluid and tissues), and the operation process is automatically completed by the instrument with a preset program. The detection sensitivity can reach single molecule level.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used 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 it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of the principle of a trace amount DNA/protein digital detection method based on a virtual segmentation method;
FIG. 2 is a schematic diagram of the principle of capturing multiple DNA/protein target molecules from a biological sample;
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 a "virtual segmentation technology" for trace DNA/protein digital detection technology; and
fig. 5 is a schematic view of the flow of imaging detection, virtual segmentation and digital detection.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Biological target digital quantitative detection system based on virtual segmentation method
The biological target digital quantitative detection system based on the virtual segmentation method is shown in fig. 1, and comprises the following devices:
first, sample treatment and enrichment device
FIG. 2 is a schematic diagram illustrating the principle of capturing multiple DNA/protein target molecules from a biological sample; and FIG. 3 is a schematic diagram of the principle of capturing DNA/protein target molecules from multiple biological samples, respectively. The micro-nano magnetic beads are utilized to process and enrich liquid samples (blood, body fluid, tissues and the like) of a human body, and DNA/protein target molecules to be detected are captured. Firstly, the surface of the magnetic bead is modified with a specific ligand (nucleic acid, protein) connected with a DNA/protein target molecule to be detected. And (3) fully mixing the magnetic beads with biological and chemical substances (nucleic acid and protein) to be detected in a sample tube (1-100 ml) to capture DNA/protein target molecules to be detected. Then, a magnet is used for adsorbing the magnetic beads for capturing the DNA/protein target molecules to be detected to the tube wall, and the suspension waste liquid is removed. Then, removing the magnet, adding cleaning solution, and eluting the biological and chemical substances (nucleic acid and protein) adsorbed on the surfaces of the magnetic beads in the non-characteristic way; and then, adsorbing the magnetic beads for capturing the DNA/protein target molecules to be detected onto the tube wall by using a magnet, and removing the cleaning waste liquid. If necessary, after multiple times of washing, the magnetic beads for capturing the DNA/protein target molecules to be detected are concentrated, enriched and purified in a 1-100 mu l liquid system.
Second, micro-fluidic detection chip
The microfluidic detection chip can perform a plurality of detection reactions in parallel: can simultaneously detect a plurality of DNA/protein target molecules in one biological sample, and can also simultaneously detect a plurality of biological samples. For the detection, a plurality of DNA/protein target molecules in a biological sample can be detected simultaneously, the biological sample needs to be subjected to circulating treatment, and magnetic beads capturing different DNA/protein target molecules are concentrated and enriched in a liquid system of 1-100 mu l respectively.
As shown in FIG. 4, if a plurality of biological samples are detected simultaneously, the plurality of biological samples need to be processed in parallel, and the magnetic beads capturing DNA/protein target molecules are concentrated and enriched in a liquid system of 1-100 μ l respectively.
It should be noted that the number of magnetic beads is much 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 magnetic beads is more than 5 ten thousand. The larger the number of magnetic beads, the better the quantification effect. The result is: most magnetic bead surfaces capture 1 target molecule.
And connecting the captured DNA/protein target molecules to be detected with an intermediary ligand through a specific ligand reaction in the microfluidic chip. The role of the intermediary ligand is to catalyze liquid-solid phase in situ luminescent reactions, such as Horseradish peroxidase (HRP). The captured test DNA/protein target molecules are linked to an intermediary ligand by a specific ligand reaction. The role of the intermediary ligand is to catalyze liquid-solid phase in situ luminescent reactions, such as Horseradish peroxidase (HRP). The flow sequence for sample 1 is as follows:
(1) and (6) sample injection. Valve 1A, valve 1B is opened and the remaining valves are closed. Under the drive of air pressure or hydraulic pressure, the sample 1 enters the chip from the sample introduction 1 port. And applying a magnet in the reaction area, and adsorbing the magnetic beads for capturing the DNA/protein target molecules to be detected to the bottom of the reaction area of the chip. After the sample injection is completed, the valves 1A and 1B are closed.
(2) And (5) cleaning. The cushion valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are opened, and the remaining valves are closed. Under the drive of air pressure or hydraulic pressure, the buffer solution enters the chip from the buffer solution sample inlet to elute the biological and chemical substances (nucleic acid and protein) nonspecifically adsorbed on the surfaces of the magnetic beads. After the completion of the cleaning, the cushion valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.
(3) And (3) intermediate ligand reaction. The reaction valve 2, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B were opened, and the remaining valves were closed. Under the drive of air pressure or hydraulic pressure, the intermediary ligand reaction liquid enters the chip from the intermediary ligand reaction liquid sample inlet, and the intermediary ligand for catalyzing the liquid-solid phase in-situ luminescent reaction is connected with the DNA/protein target molecule to be detected. After the intermediary 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 cushion valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are opened, and the remaining valves are closed. Under the drive of air pressure or hydraulic pressure, the buffer solution enters the chip from the buffer solution sample inlet to elute the biological and chemical substances (nucleic acid and protein) nonspecifically adsorbed on the surfaces of the magnetic beads. If necessary, the magnetic beads connected with the intermediate ligand are concentrated and enriched in a liquid system of 1-100 mu l after a plurality of times of washing. After the completion of the cleaning, the cushion valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.
The magnetic beads connected with the intermediary ligand are randomly tiled and fixed in the plane of the reaction area of the chip. The key to this operation is that the beads do not agglomerate. The necessary measures include that the magnet and the ultrasonic device are alternately used at the bottom of the chip, and finally, the magnetic beads are randomly distributed in the plane of the reaction area of the chip.
And carrying out liquid phase-solid phase in-situ luminescent reaction in the microfluidic chip. Reflecting molecules generated by the reaction are deposited in the area near the magnetic beads on the substrate of the chip; the surface of the chip substrate is modified with a functional group combined with the luminescent reaction molecule in advance, so that the luminescent molecule generated by the reaction is covalently connected to the surface of the chip substrate. For example, a toluene group is modified in advance on the surface of the planar substrate, and a luminescent molecule generated by HRP-catalyzed reaction is bonded to the toluene group. During the reaction, a magnet was applied to the bottom plate of the planar substrate to keep the beads immobilized. After the reaction is finished, the magnet is removed, cleaning solution is added, the magnetic beads are eluted, and only the reacted luminescent molecules are left on the planar substrate. The solid phase luminous molecule area formed around each magnetic bead is several square microns to several hundred square microns.
Referring to fig. 4, the flow sequence for sample 1 is as follows:
(1) and (4) injecting a liquid phase-solid phase reaction solution and reacting. Reaction valve 2, connecting valve 1, connecting valve 2, connecting valve 3, valve 1B, the remaining valves were 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 sample inlet, liquid-solid phase reaction is carried out under the catalysis of an intermediary ligand (such as HRP), and the intermediary ligand for catalyzing the liquid-solid phase in-situ luminescence reaction is connected with the DNA/protein target molecule to be detected. The solid phase luminous molecule area formed around each magnetic bead is several square microns to several hundred square microns. After the reaction, the reaction valve 2, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B were closed.
(2) And (5) cleaning. The cushion valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are opened, and the remaining valves are closed. Under the drive of air pressure or hydraulic pressure, the buffer solution enters the chip from the buffer solution sample inlet to elute the biological and chemical substances (nucleic acid and protein) nonspecifically adsorbed on the surfaces of the magnetic beads. And removing the magnet, adding a cleaning solution, eluting the magnetic beads, and only leaving the reacted luminescent molecules on the surface of the microfluidic chip. The luminous molecule area formed on the periphery of each magnetic bead is several micrometers to hundreds of micrometers. After the completion of the cleaning, the cushion valve, the connection valve 1, the connection valve 2, the connection valve 3, and the valve 1B are closed.
Third, the detection device
As shown in fig. 5, the detection device obtains a high-definition digital picture from the surface of the microfluidic chip, for example, it can be imaged using a conventional fluorescence microscope. And then, a virtual segmentation algorithm is adopted to realize digital detection, and the highest detection sensitivity can reach a single molecule level. The calculation method of the virtual division is divided into several parts:
(1) setting the size of the region of the unit "virtual reaction cell
The high-definition digital picture is composed of pixel points, a solid-phase luminescent molecule area formed on the periphery of each magnetic bead is several microns to several hundred microns, the high-definition picture is uniformly divided into a plurality of uniform virtual reaction units through an algorithm, and each virtual reaction unit comprises a luminescent molecule area formed on the periphery of each magnetic bead. Once fixed, the number of "virtual reaction units" is determined. The pixel area of the dummy unit needs to be determined by the light-emitting molecule region formed around each magnetic bead. The area of the luminous molecule area formed on the periphery of each magnetic bead is smaller than that of the virtual unit. For example, the area of the luminescent molecule region formed around each magnetic bead is 100 square microns, and the area of the dummy unit is larger than 100 square microns. After partitioning, two situations can occur:
a. if the areas of luminescent molecules formed by the peripheries of the two magnetic beads do not intersect. When the "virtual reaction unit" is divided, the luminescent molecule region formed around each magnetic bead is located in the respective reaction unit.
b. If the fluorescent light-emitting regions formed around two magnetic beads intersect, the divided areas of the reaction units need to be enlarged, so that more than two light-emitting regions can be accommodated in one reaction unit.
In both cases, the analysis can be digitally carried out by Poisson distribution.
For example, the pixel of one picture is 1920x 1280. Through experiments, the maximum area of the luminous molecule region formed on the periphery of each magnetic bead is 100 square microns. At this time, the maximum pixel of the light-emitting molecule region around the corresponding single magnetic bead is 4 × 4, and thus the number of pixels of a single "virtual reaction cell" is 16. Total number N of "virtual reaction units0Is 15.36 ten thousand.
(2) Determining a positive signal threshold
A threshold value is set for the luminescence signal detected by each "virtual reaction unit", and above the threshold value, the reaction unit of the luminescence signal is judged as 1 ("positive"), and below the threshold value, the reaction unit of the luminescence signal is judged as 0 ("negative").
(3) Digital analysis-Poisson analysis
Theoretically, there are three possibilities for capturing DNA/protein target molecules per magnetic bead: zero molecules, single molecules or multiple molecules. When the number of the magnetic beads is large enough, most of the magnetic beads capture only one molecule or zero molecules; finally, the interior of most of the virtual reaction units only contains one molecule or zero molecule, and finally only contains one solid-phase luminescent molecule area or zero solid-phase luminescent molecule area, so that the single-molecule optical signal amplification is realized. Even if a single 'virtual reaction unit' contains more than two solid-phase luminous molecule 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 reaction units with positive and negative signal types and carrying out Poisson statistical analysis.
For example: the number M of the positive units is 5000 and the total number N of the virtual units is detected0At 15.36 million, the absolute number of positive molecules was calculated by the following equation:
the absolute molecular number was 5083.
It is to be understood that the invention disclosed 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 intended to be encompassed by the following claims.
Claims (10)
1. The biological target digital quantitative detection system based on the virtual segmentation method is characterized by comprising the following steps:
the sample processing and enriching device is used for processing, enriching and capturing a biological target to be detected by using magnetic beads, ligand molecules specifically connected with the biological target to be detected are modified on the surfaces of the magnetic beads, and liquid containing the biological target to be detected is obtained by concentration and enrichment;
the liquid of the biological target to be detected and the intermediary ligand reaction liquid respectively enter the micro-fluidic chip under the pressure drive, the biological target to be detected connected on the magnetic beads is combined with the intermediary ligand in the micro-fluidic chip, and the intermediary ligand is used for catalyzing liquid phase-solid phase in-situ luminescence reaction; magnetic beads connected with the intermediary ligand are randomly tiled and fixed on the reaction area plane of the microfluidic chip; performing the liquid-solid phase in-situ luminescence reaction on the plane of the reaction area of the microfluidic chip, wherein the reaction optically amplifies the biological target to be detected and forms a solid-phase luminescence area around the magnetic bead containing the biological target to be detected; and the plane of the reaction area of the microfluidic chip is modified with a functional group which is combined with luminescent molecules generated by the liquid-solid phase in-situ luminescent reaction in advance, so that the luminescent molecules generated by the reaction are covalently connected to the plane of the reaction area; and
and the detection device is used for obtaining a digital picture on the reaction area plane of the microfluidic chip after reaction, and then realizing digital quantitative detection of the biological target to be detected by adopting a virtual segmentation method.
2. The digital quantitative detection system for biological targets based on virtual segmentation method as claimed in claim 1, wherein the sample processing and enriching device is a device for capturing multiple biological targets of DNA and/or protein molecules from one biological sample or a device for capturing biological targets of DNA and/or protein molecules from multiple biological samples.
3. The digital quantitative detection system for biological targets based on virtual segmentation method as claimed in claim 1, wherein the surface of the magnetic bead is modified with ligand molecules specifically linked to the biological targets to be detected; capturing a biological target to be detected by the modified magnetic beads; cleaning and purifying the captured biological target to be detected by using magnetic force; and then uniformly distributing the purified biological target to be detected in the liquid.
4. The digital quantitative detection system for biological targets based on virtual segmentation method as claimed in claim 1, wherein a magnet is applied in the reaction area of the microfluidic chip, so that the magnetic beads for capturing the biological targets to be detected are adsorbed to the bottom of the reaction area of the chip.
5. The digital quantitative detection system for biological targets based on virtual segmentation method as claimed in claim 4, further comprising washing with a washing solution after the magnetic beads for capturing biological targets to be detected are adsorbed to the bottom of the reaction region of the chip and/or the biological targets to be detected and the intermediary ligands are reacted in the chip.
6. The digital quantitative detection system for biological targets based on the virtual segmentation method as claimed in claim 1, wherein the magnetic beads are micro-sized and nano-sized magnetic beads, preferably 10 nm to 100 μm in diameter.
7. The digital quantitative detection system of biological target based on virtual segmentation method as claimed in claim 1, wherein the magnetic beads are randomly tiled and fixed on the reaction area plane of the microfluidic chip by using the magnet and the ultrasonic device alternatively at the bottom of the chip.
8. The digital quantitative detection system for biological targets based on virtual segmentation method as claimed in claim 1, wherein the intermediate ligand is horseradish peroxidase, and the substrate surface is modified with groups capable of reacting with horseradish peroxidase, preferably aromatic groups, more preferably toluene groups; luminescent molecules generated by the catalytic reaction of horseradish peroxidase are connected with the groups modified on the plane of the reaction area of the chip.
9. The digital quantitative detection system for biological targets based on virtual segmentation method as claimed in claim 1, wherein magnetic force is applied to the reaction region of the chip to keep the magnetic beads fixed; and after the reaction is finished, removing the magnetic force, adding a cleaning solution for elution, and leaving the reacted luminescent molecules on the reaction area of the chip.
10. The digital quantitative detection system of biological target based on virtual segmentation method as claimed in claim 1, wherein the virtual segmentation method comprises: uniformly dividing the digital picture into a plurality of uniform virtual reaction units, wherein each virtual reaction unit comprises a luminescent molecule area formed around each magnetic bead, and the luminescent molecule area formed around a single magnetic bead cannot be positioned in two reaction units after division; setting a threshold value for the luminescence signal detected by the virtual reaction unit, wherein the reaction unit of the luminescence signal judges as positive when the threshold value is higher than the threshold value, and the reaction unit of the luminescence signal judges as negative when the threshold value is lower than the threshold value; and determining the absolute number of the biological target to be detected by digital analysis.
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Denomination of invention: A Digital Quantitative Detection System for Biological Targets Based on Virtual Segmentation Method Effective date of registration: 20231124 Granted publication date: 20230620 Pledgee: Haidian Beijing science and technology enterprise financing Company limited by guarantee Pledgor: Gewu Zhihe Biotechnology (Beijing) Co.,Ltd. Registration number: Y2023110000490 |