CN115639364A - Absolute quantitative digital ELISA detection method - Google Patents
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- CN115639364A CN115639364A CN202211183184.0A CN202211183184A CN115639364A CN 115639364 A CN115639364 A CN 115639364A CN 202211183184 A CN202211183184 A CN 202211183184A CN 115639364 A CN115639364 A CN 115639364A
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Abstract
The invention particularly relates to an absolute quantitative digital ELISA detection method, which comprises the following steps: preparing a sample containing target molecules to be detected, capturing the target molecules to be detected by using magnetic beads, and forming a compound of magnetic beads, the target molecules to be detected and enzyme through an affinity reaction; transferring the sample formed with the complex and the fluorescent substrate to a microwell array chip; adding a spacer fluid to the microporous array chip; waiting for an enzymatic reaction of an enzyme with a fluorogenic substrate to produce a fluorescent molecule; determining the number of microwells containing magnetic beads in the microwell array and the number of microwells with fluorescence signals higher than a threshold value in the microwell array; and determining the number of the target molecules to be detected in the sample based on the two numbers and the number of the magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further combined with the enzyme. The invention does not need to rely on a standard sample and a standard curve, and the dynamic detection range is accurate, controllable, sensitive and accurate.
Description
Technical Field
The invention relates to biological detection, in particular to an absolute quantitative digital ELISA detection method.
Background
Currently, a variety of detection techniques exist on the market for the quantitative detection of low concentrations of analytes.
A representative product is SMC of Merck company TM (Single Molecule Counting) system, however, the light path structure of this system is complicated, expensive, and detection speed is slow, carries out serial detection to the microballon, and detection time is also very long.
Another class uses amplification techniques to increase the number of target molecules to be detected to provide a sufficient number of signal molecules, typically from Chimera
Systems, however, have complex assay methods and are prone to false positive signals.
Simoa of Quanterix corporation TM The Single-molecule Array system adopts digital ELISA (Enzyme Linked Immunosorbent Assay) technology similar to digital PCR (Polymerase Chain Reaction), however, the system is a relatively quantitative digital ELISA detection method, in order to determine the number of target molecules to be detected, standard target molecules with known concentration are required to be calibrated to make a standard curve, and the number of target molecules to be detected and the concentration thereof are reversely deduced through a fitted standard curve formula. In addition, depending on the target to be measuredThe system uses two analysis algorithms of digitalization and simulation for quantification, so that the dynamic detection range of digital analysis is not fully utilized, and the resolution and robustness are reduced.
Therefore, there is a need for an absolute quantitative digital ELISA method that does not rely on standard samples and standard curves, has a precisely controllable dynamic detection range, high sensitivity, and high accuracy.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an absolute quantitative digital ELISA detection method, which does not depend on a standard sample and a standard curve, has a precise and controllable dynamic detection range, high sensitivity and high accuracy.
The invention provides an absolute quantitative digital ELISA detection method, which comprises the following steps:
step 2, transferring the sample with the complex and the fluorescent substrate into a microwell array chip, wherein the microwell array chip comprises a microwell array and a fluid chamber for accommodating the microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead;
step 3, adding a spacer fluid into the micropore array chip to separate all micropores in the micropore array from one another;
step 4, waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate the fluorescent molecules, wherein the fluorescent signals of the micropores where the magnetic beads capturing the single target molecules to be detected are located are higher than a threshold value, and the fluorescent signals of the micropores where the magnetic beads capturing zero target molecules to be detected are located are lower than the threshold value;
step 5, determining the number of micropores containing the magnetic beads in the micropore array, and determining the number of micropores with fluorescence signals higher than a threshold value in the micropore array;
and 6, determining the number of the target molecules to be detected in the sample based on the number of the micropores containing the magnetic beads in the micropore array and the number of the micropores with the fluorescence signals higher than a threshold value in the micropore array, and based on the number of the magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further combined with the enzyme.
In one embodiment of the invention, the number of magnetic beads in the sample is determined based on the counting mode of a particle counter, flow cytometry or a cell counting plate.
In an embodiment of the present invention, a ratio of the number of the magnetic beads in the sample to the number of the target molecules to be detected in the sample is greater than or equal to 5.
In one embodiment of the present invention, in the step 5, one or more bright field images and fluorescence images of the field of view are taken of the microwell array, wherein the number of microwells containing the magnetic beads in the microwell array is determined based on the bright field image of the one or more field of view, and the number of microwells whose fluorescence signal is higher than a threshold value in the microwell array is determined based on the fluorescence image of the one or more field of view.
In one embodiment of the present invention, in step 6, the following formula is used to determine the number of the target molecules to be detected in the sample:
wherein, M 0 Is the number of target molecules to be detected in the sample, N 0 Is the number of the magnetic beads in the sample, M is the number of microwells in the microwell array for which the fluorescence signal is above a threshold, N is the number of microwells in the microwell array containing the magnetic beads, and p is the probability that the target molecule to be detected is captured by the magnetic beads and further binds to the enzyme.
In one embodiment of the present invention, the ratio of the number of all microwells in the microwell array to the number of the magnetic beads in the sample is in the range of 0.1 to 10.
In one embodiment of the present invention, in the step 2, the sample with the complex formed and the fluorescent substrate are transferred to the fluid chamber of the microwell array chip and the microwells of the microwell array by self-suction, centrifugation or pressure injection, and the redundant magnetic beads and fluorescent substrate in the fluid chamber are removed and the magnetic beads and fluorescent substrate in the microwells are retained by centrifugation.
In one embodiment of the present invention, in the step 3, the isolation solution is added to the fluid chamber of the microwell array chip by self-priming, centrifugation or pressure sample injection, and the magnetic beads and the fluorescent substrate in the microwells of the microwell array are sealed and isolated.
In one embodiment of the present invention, the target molecule to be detected is a protein molecule to be detected.
In one embodiment of the present invention, the isolation fluid is fluorine oil or silicone oil.
As described above, the present invention has the following advantageous effects:
the present invention can absolutely quantify the target molecules to be detected in the sample by the number of sample distribution units clearly determined by counting means such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields, the number of positive reaction detection units determined by fluorescence images of one or more fields, and the probability that the target molecules to be detected are captured by magnetic beads and further bound to the enzyme as a constant.
In the present invention, the number of magnetic beads in the sample determines the upper limit of the dynamic detection range, and by changing the number of magnetic beads in the sample, the dynamic detection range can be accurately controlled. Theoretically, the upper limit of the dynamic detection range is less than 5 times the number of magnetic beads.
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FIG. 1 is a schematic diagram of the principle of an absolute quantitative digital ELISA detection method according to one embodiment of the present invention;
FIG. 2 is a schematic flow diagram of an absolute quantitative digital ELISA detection method according to one embodiment of the present invention.
Detailed Description
Embodiments of the present invention are described below with reference to the drawings.
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.
The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. The word "comprising" or "comprises", and the like, means that the element or item listed as preceding "comprising" or "includes" covers the element or item listed as following "comprising" or "includes" and its equivalents, and does not exclude other elements or items.
Fig. 1 is a schematic view of the principle of an absolute quantitative digital ELISA detection method according to one embodiment of the present invention, and fig. 2 is a schematic view of the flow of an absolute quantitative digital ELISA detection method according to one embodiment of the present invention.
As shown in fig. 1 and 2, the digital ELISA method of absolute quantification includes:
at step 1 (S1), a sample is prepared, the sample contains a target molecule to be detected, the target molecule to be detected in the sample is captured by using a magnetic bead, and a complex of the magnetic bead-the target molecule to be detected-an enzyme is formed through an affinity reaction, and the enzyme can perform an enzymatic reaction with a fluorescent substrate to generate a fluorescent molecule.
Specifically, the target molecule to be detected may be a protein molecule to be detected, and the protein molecule to be detected may be derived from a liquid sample (blood, body fluid, tissue, etc.) of a human body. More specifically, the test protein molecule may be derived from serum, plasma, tissue homogenate, or supernatant from a cell extract. Based on the method, the protein molecules with ultralow abundance, which are difficult to detect by a conventional method in normal people and disease patients, can be accurately quantified, and brand-new application is developed in the fields of early detection, accompanying diagnosis, medicament research and development and the like of serious diseases such as tumors, neurological diseases, infectious diseases, immunoinflammation and the like.
In particular, the magnetic beads may have a diameter in the micrometer scale. As a sample distribution unit, the surface of the magnetic bead is modified with a capture antibody capable of specifically connecting with a target molecule to be detected, for example, the capture antibody reacts with an antibody antigen generated by a protein molecule to be detected, so as to capture the protein molecule to be detected.
The number of magnetic beads in the sample can be unambiguously determined based on counting means such as particle counting instruments, flow cytometry or cell counting plates. In addition, the homogeneity of the magnetic beads in the sample can also be precisely controlled based on the above techniques. Based on this, the present invention can absolutely quantify the target molecules to be detected in the sample based on at least the number of magnetic beads in the sample.
In addition, the number of magnetic beads in the sample should be much larger than the number of target molecules to be detected in the sample. Preferably, the ratio of the number of the magnetic beads in the sample to the number of the target molecules to be detected in the sample is greater than or equal to 5, so that the statistical distribution of the target molecules to be detected captured to the magnetic beads conforms to the poisson distribution.
Theoretically, there are three possibilities for the number of target molecules to be detected captured per magnetic bead: capture zero target molecules to be detected, capture a single target molecule to be detected, or capture multiple target molecules to be detected, and when the number of magnetic beads is sufficiently large (e.g., at least 5 times or more the number of target molecules to be detected), most magnetic beads capture only zero target molecules to be detected or only a single target molecule to be detected, thereby achieving single-molecule fluorescence signal amplification as will be described below.
In the present invention, the number of magnetic beads in the sample determines the upper limit of the dynamic detection range, and by changing the number of magnetic beads in the sample, the dynamic detection range can be precisely controlled. Theoretically, the upper limit of the dynamic detection range is less than 5 times the number of magnetic beads. For example, the number of magnetic beads in the sample is in the range of 10 to 1000 ten thousand, so that the upper limit of the dynamic detection range is in the range of 50 to 5000 ten thousand.
As an example, the capture antibody modified on the surface of the magnetic bead specifically captures the target molecule to be detected in the sample, and further links the detection antibody and the enzyme, finally forming an immune complex of "magnetic bead-capture antibody-target molecule to be detected-detection antibody-enzyme", and the enzyme can perform an enzymatic reaction with a fluorescent substrate to generate a fluorescent molecule. For example, the magnetic beads may have β -galactosidase attached thereto via the double antibody sandwich reaction described above, and the fluorescent substrate may be non-fluorescent resorufin- β -galactoside (RGP), which catalyzes the hydrolysis of the non-fluorescent resorufin- β -galactoside (RGP) to produce a resorufin molecule capable of emitting fluorescence.
At step 2 (S2), the sample with the complexes formed and the fluorescent substrate are transferred into a microwell array chip, wherein the microwell array chip comprises a microwell array and a fluid chamber containing the microwell array, each microwell of the microwell array being configured to be capable of containing only one magnetic bead.
In particular, the microwell array comprises a plurality of microwells, each microwell of which may be slightly larger in size than a magnetic bead, and thus configured to be able to accommodate only one magnetic bead. Wherein the ratio of the number of all microwells in the microwell array to the number of magnetic beads in the sample is in the range of 0.1 to 10, such that as many magnetic beads as possible fall into a microwell. If the magnetic beads fall into a microwell, as shown in FIG. 1, the microwell can be referred to as an active microwell or a reaction detection unit. The number of reaction detection units (i.e. effective microwells) is not larger than the number of sample distribution units (i.e. magnetic beads), and theoretically the accuracy and resolution of digital detection is higher the closer the number of reaction detection units is to the number of sample distribution units.
For example, the micropore array comprises 188000 micropores, the micropores are circular micropores, the diameter of the micropore is 4 μm, the depth of the micropore is 4 μm, and the center-to-center distance between the micropores is 8 μm.
Specifically, the sample with the complex formed and the fluorogenic substrate are transferred to the microwell array chip under a first centrifugation condition, for example, 200rpm for 10 seconds, and the centrifugation force feeds the sample and the fluorogenic substrate into the fluid chamber of the microwell array chip. Then, left to stand for a certain time (e.g., 2 minutes), the sample and the fluorescent substrate are allowed to settle into the microwells of the microwell array. The excess magnetic beads and fluorescent substrate in the fluid chamber are then removed and the magnetic beads and fluorescent substrate in the microwells are retained under a second centrifugation condition, such as 600rpm for 10 seconds, at which speed the excess magnetic beads and fluorescent substrate in the fluid chamber are lifted out while the magnetic beads and fluorescent substrate in the microwells are retained. It is understood that the sample and the fluorescent substrate formed with the complex can be transferred to the microwell array chip by self-priming or pressure injection.
At step 3 (S3), a spacer fluid is added to the microwell array chip to isolate all microwells in the microwell array from each other.
Specifically, the spacer fluid may be fluorine oil or silicone oil having a high viscosity coefficient. Under a third centrifugal condition, such as 200rpm holding for 10 seconds, a spacer fluid is added to the fluid chamber of the microwell array chip and the magnetic beads and the fluorescent substrate in the microwell array are sealed and isolated, the spacer fluid is fed into the fluid chamber of the microwell array chip by a centrifugal force, the hydrophobic spacer fluid can sufficiently wet the surface of the microwell array, the magnetic beads which do not fall into the microwell are further removed while isolating all the microwells from each other, and the fluorescent molecules generated subsequently are difficult to diffuse, and the thermal stability of the individual microwells is excellent. It is understood that the isolation liquid can also be added to the fluid chamber of the microwell array chip by self-suction or pressure injection.
At step 4 (S4), waiting for an enzymatic reaction of an enzyme with a fluorogenic substrate to generate a fluorescent molecule, wherein the fluorescent signal of the microwell in which the magnetic bead with a single target molecule to be detected is located is above a threshold value, and the fluorescent signal of the microwell in which the magnetic bead with zero target molecules to be detected is located is below the threshold value.
For example, waiting for 1 minute at room temperature for e.g.beta-galactosidase to catalyse the hydrolysis of non-fluorescent resorufin-beta-galactoside (RGP) to generate a resorufin molecule capable of emitting fluorescence. As already described above, in the case that the target molecules to be detected are captured to obtain a statistical distribution of magnetic beads conforming to a poisson distribution, most of the magnetic beads capture only zero target molecules to be detected or only a single target molecule to be detected, so as to achieve single-molecule fluorescent signal amplification, that is, as shown in fig. 1, the fluorescent signal of the microwell where the magnetic bead capturing the single target molecule to be detected is located is higher than a threshold, and the fluorescent signal of the microwell where the magnetic bead capturing zero target molecules to be detected is located is lower than the threshold. Microwells with a fluorescence signal above the threshold value in the microwell array can be interpreted as 1 ("positive"), and microwells with a fluorescence signal below the threshold value in the microwell array can be interpreted as 0 ("negative").
At step 5 (S5), the number of microwells containing magnetic beads in the microwell array is determined, and the number of microwells whose fluorescence signal is higher than a threshold value in the microwell array is determined.
In other words, step 5 determines the number of reaction detection units and the number of positive reaction detection units, respectively. Specifically, a bright field image (e.g., mercury lamp light source, and exposure time 50 ms) and a fluorescent image (e.g., 577nm excitation, 620nm emission, and exposure time 600 ms) of one or more fields of view (e.g., 35 fields of view) are taken of the microwell array, wherein, as shown in fig. 1, the number of microwells containing magnetic beads in the microwell array is determined based on the bright field image of the one or more fields of view, and the number of microwells whose fluorescent signal is above a threshold value in the microwell array is determined based on the fluorescent image of the one or more fields of view.
At step 6 (S6), the number of target molecules to be detected in the sample is determined based on the number of microwells having magnetic beads in the microwell array and the number of microwells having fluorescence signals above a threshold value in the microwell array, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bind to the enzyme.
Specifically, the following formula is used to determine the number of target molecules to be detected in the sample:
wherein, M 0 Is the number of target molecules to be detected in the sample, N 0 Is the number of magnetic beads in the sample, M is the number of microwells in the microwell array for which the fluorescence signal is above the threshold, N is the number of microwells in the microwell array containing magnetic beads, and p is the probability that the target molecule to be detected is captured by a magnetic bead and further binds to the enzyme (if the operating homogeneity is high, probability p is a constant less than 100%).
In other words, the present invention can absolutely quantify the target molecules to be detected in the sample by the number of sample distribution units clearly determined by counting means such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields, the number of positive reaction detection units determined by fluorescence images of one or more fields, and the probability that the target molecules to be detected are captured by magnetic beads and further bound to the enzyme as a constant.
More specifically, the following formula is used to determine the concentration of the target molecule to be detected in the sample: c = M 0 M/V, where c is the concentration of the target molecule to be detected in the sample, M 0 Is the number of target molecules to be detected in the sample, m is the mass of a single target molecule to be detected in the sample, and V is the volume of the sample.
For example, the target molecule to be detected in the sample is interleukin-6 (IL-6), the molecular weight of which is 21kDa, and the volume V of the sample is 100. Mu.L. Number N of magnetic beads (sample distribution units) in a sample determined based on flow cytometry 0 75.36, the number of all microwells in a microwell array is 18.8 ten thousand, the number N of microwells containing magnetic beads (reaction detection units) in a microwell array determined based on bright field images of one or more fields of view is 12.1 ten thousand, based on oneThe number M of microwells (positive reaction detection units) with fluorescence signals higher than a threshold value in the microwell array determined by the fluorescence image of the one or more fields of view is 5000, the probability p that the target molecules to be detected are captured by the magnetic beads and further combined with the enzyme is 80%, and then the number M of the target molecules to be detected in the sample is 5000 0 Using the following formula
Is calculated as 39753, and the concentration c of the target molecule to be detected adopts the following formula c = M 0 The m/V was calculated to be 13.9fg/ml.
While the invention has been shown and described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A method of digital ELISA detection of absolute quantification, comprising:
step 1, preparing a sample, wherein the sample contains target molecules to be detected, magnetic beads are used for capturing the target molecules to be detected in the sample, a compound of the magnetic beads, the target molecules to be detected and enzyme is formed through an affinity reaction, and the enzyme can perform an enzymatic reaction with a fluorescent substrate to generate fluorescent molecules;
step 2, transferring the sample with the complex and the fluorescent substrate into a microwell array chip, wherein the microwell array chip comprises a microwell array and a fluid chamber for accommodating the microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead;
step 3, adding a spacer fluid into the micropore array chip to separate all micropores in the micropore array from one another;
step 4, waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate the fluorescent molecules, wherein the fluorescent signals of the micropores where the magnetic beads capturing the single target molecules to be detected are located are higher than a threshold value, and the fluorescent signals of the micropores where the magnetic beads capturing zero target molecules to be detected are located are lower than the threshold value;
step 5, determining the number of micropores containing the magnetic beads in the micropore array, and determining the number of micropores with fluorescence signals higher than a threshold value in the micropore array;
and 6, determining the number of the target molecules to be detected in the sample based on the number of the micropores containing the magnetic beads in the micropore array and the number of the micropores with the fluorescence signals higher than a threshold value in the micropore array, and based on the number of the magnetic beads in the sample and the probability of the target molecules to be detected being captured by the magnetic beads and further being combined with the enzyme.
2. The method of claim 1, wherein the number of magnetic beads in the sample is determined based on a counting regime of a particle counter, flow cytometry, or a cell counting plate.
3. The method of claim 1, wherein the ratio of the number of magnetic beads in the sample to the number of target molecules to be detected in the sample is greater than or equal to 5.
4. The method of claim 1, wherein in step 5, one or more bright field images and fluorescence images of the microwell array are taken, wherein the number of microwells in the microwell array containing the magnetic beads is determined based on the bright field image of the one or more fields, and the number of microwells in the microwell array having a fluorescence signal above a threshold value is determined based on the fluorescence image of the one or more fields.
5. The method of claim 1, wherein in step 6, the number of target molecules to be detected in the sample is determined using the following formula:
wherein, M 0 Is the number, N, of the target molecules to be detected in the sample 0 Is the number of the magnetic beads in the sample, M is the number of microwells in the microwell array for which the fluorescence signal is above a threshold, N is the number of microwells in the microwell array containing the magnetic beads, and p is the probability that the target molecule to be detected is captured by the magnetic beads and further binds to the enzyme.
6. The method of claim 1, wherein the ratio of the number of all microwells in the microwell array to the number of magnetic beads in the sample is in the range of 0.1 to 10.
7. The method of claim 1, wherein in the step 2, the sample with the formed complex and the fluorescent substrate are transferred to the fluid chamber of the microwell array chip and the microwells of the microwell array chip by self-suction, centrifugation or pressure injection, and redundant magnetic beads and fluorescent substrate in the fluid chamber are removed and the magnetic beads and fluorescent substrate in the microwells are retained by centrifugation.
8. The method of claim 1, wherein in step 3, the isolation solution is added to the fluid chamber of the microwell array chip by self-priming, centrifugation, or pressure injection, and the magnetic beads and the fluorescent substrate in the microwells of the microwell array are sealed and isolated.
9. The method of claim 1, wherein the test target molecule is a test protein molecule.
10. The method of claim 1, wherein the spacer fluid is a fluorine oil or a silicone oil.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116735460A (en) * | 2023-06-16 | 2023-09-12 | 深圳大学 | Quick magnetic bead counting device and manufacturing and using method thereof |
| WO2024067667A1 (en) * | 2022-09-27 | 2024-04-04 | 格物致和生物科技(北京)有限公司 | Microfluidic chip, operating method therefor, digital elisa detection method, and use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024067667A1 (en) * | 2022-09-27 | 2024-04-04 | 格物致和生物科技(北京)有限公司 | Microfluidic chip, operating method therefor, digital elisa detection method, and use |
| CN116735460A (en) * | 2023-06-16 | 2023-09-12 | 深圳大学 | Quick magnetic bead counting device and manufacturing and using method thereof |
| CN116735460B (en) * | 2023-06-16 | 2024-03-29 | 深圳大学 | Quick magnetic bead counting device and manufacturing and using method thereof |
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