A single molecule immunoassay detection method

Abstract

The application discloses a novel single-molecule immunoassay detection method, which is based on a single-molecule immunoassay method for mild dissociation and enrichment under specific enzyme treatment conditions. The detection method comprises a series of operation steps such as activation of the inner wall of a container, preparation of non-magnetic microspheres-capture antibodies, preparation of detection antibodies-magnetic microbeads, preparation of magnetic microbeads-target-non-magnetic microspheres, nucleic acid double-strand dissociation and enrichment under mild conditions, collection of reporters, signal detection and the like. Compared with existing detection methods, the method has a series of advantages such as reduction of statistical errors, improvement of detection sensitivity, shortening of reaction time, reduction of detection limit and the like, and improves the stability and operation repeatability of the detection system.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay and relates to a single-molecule immunoassay detection method, specifically a new technology that combines a digital enzyme-linked immunosorbent assay (dELISA) method with gentle unwinding separation of DNA double strands. Background Technology

[0002] As early as 2010, Rissin et al. proposed a digital ELISA method (Rissin et al., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnology, 28:595-599, 2010). This method involves binding target-specific capture antibodies to the surface of microbeads with a particle size of approximately 1-100 μm. After specifically recognizing the capture target, a biotinylated detection antibody specifically recognizes the target protein and binds specifically to the target capture antibody complex. Subsequently, a pre-linked avidin-dependent reporter molecule (enzyme molecule) is bound to the detection antibody, ultimately forming a double-antibody sandwich immune complex. These microbeads are loaded into an array of micro-pits with a specific diameter and depth, ensuring that each micro-pit contains ≤1 microbead based on the size relationship between the microbeads and the pits. Then, an enzyme substrate is added, and the micro-pits are sealed. The substrate is catalyzed by the enzyme molecule to generate a fluorescent product, which emits fluorescence under excitation light and is detected by an optical system. The volume of a micro-pit is approximately tens of femtoliters. This extremely small volume ensures a high concentration of fluorescent molecules in a localized area, greatly reducing interference from background light.

[0003] For a single-molecule immunoreactivity system (SIR) to achieve single-target binding of a bead to only one target (Bm) and form an enzyme-linked immunosorbent assay (ELISA), it is essential to ensure that each bead binds to only one target. According to the Poisson distribution theory, to achieve this, an excessive number of beads are needed to bind to the target. Therefore, each bead either has only one opportunity to bind to the target or none at all. Consequently, only a small percentage (1.5%) of the beads can form an ELISA, thus qualifying as effective beads. The vast majority (98.5%) of the beads do not bind to the target and therefore cannot form an ELISA, classifying them as ineffective beads.

[0004] With the development of single-molecule technology, how to efficiently separate effective microbeads that capture targets from other ineffective beads; how to improve the loading effect of magnetic beads that capture targets when developing single-molecule detection chips; and how to improve the utilization rate of counting chips are all important research objectives in the field of single-molecule immunology. Summary of the Invention

[0005] The purpose of this invention is to provide a single-molecule immunoassay detection method, and a single-molecule immunoassay method for mild dissociation and enrichment under specific enzyme treatment conditions. Therefore, the following will describe in detail how the mild dissociation of immunomagnetic beads is achieved.

[0006] The invention introduces a novel method for capturing and separating targets using a dual-bead immunomagnetic array. This method utilizes a mild enzymatic treatment to dissociate the immunomagnetic bead complex, and then, based on the 1:1 ratio of the dissociated immunomagnetic beads to the target, it enables the counting of fluorescent substances in single-molecule immunotherapy.

[0007] This invention provides a single-molecule immunoassay detection method, comprising the following steps:

[0008] (1) Coat the inner wall of the activated container with PL1, and then use a passivating agent to block the remaining activation sites on the inner wall of the container.

[0009] (2) The surface of the activated non-magnetic microspheres is coated with capture antibody and PL2. The remaining activation sites on the surface of the non-magnetic microspheres are blocked with a passivating agent. The excess capture antibody and PL2 are washed away to prepare the CPL complex.

[0010] (3) Add the CPL complex into the container obtained in step (1) so that the CPL complex reacts with PL1 on the inner surface of the container and binds the CPL complex to the inner surface of the container; wash away the unreacted CPL complex.

[0011] (4) The detection antibody is reacted with PL3 in a ratio of 1:1 to 1:5, and unreacted PL3 is removed by washing and centrifugation to obtain the PD complex.

[0012] (5) React PL4 with magnetic microbeads, coat the activated magnetic microbeads with fluorescent material with PL4, and use a passivating agent to block the unactivated sites on the surface of the magnetic microbeads to obtain PM reporter; the amount of PL4 added is 100-1000 times that of the magnetic beads.

[0013] (6) The PD complex and PM reporter obtained in steps (4) and (5) are fixed and bound together in a complementary base sequence manner. The unbound and fixed PD complex is then removed by a magnetic cleaning step to obtain the DM complex.

[0014] (7) Add the target to react with the CPL complex and DM complex bound to the inner wall of the container, incubate and wash to obtain DM-Bm-CPL double antibody sandwich complex; wash away unreacted DM complex.

[0015] (8) Add nucleotide dissociation enzyme to the DM-Bm-CPL double antibody sandwich complex to dissociate PL4 and PL3 to obtain PM reporter;

[0016] (9) Load the dissociated PM report substance obtained in step (8) into the detection chip and measure the PM report substance value;

[0017] PL1, PL2, PL3, and PL4 are oligonucleotides;

[0018] The base sequences of PL1 and PL2 have complementary regions;

[0019] The base sequences of PL3 and PL4 have complementary regions.

[0020] Both the detection antibody and the capture antibody can bind to the target;

[0021] The magnetic microspheres are fluorescent magnetic microspheres.

[0022] Further restrictions can be imposed on the methods described above:

[0023] In step (1), PL1 is coated by chemical embedding. The container used is usually a multi-well plate, typically with 96 wells, with each well corresponding to one container.

[0024] In step (2), the ratio of the number of the capture antibody and PL2 to the number of non-magnetic microspheres is 500:1 to 10000:1.

[0025] In step (4), the ratio of the detection antibody to PL3 can reach 1:1 to 1:10.

[0026] The diameter of the magnetic microspheres is 0.1–50 μm.

[0027] The ratio of PM reporter to Mb-PL4 complex was 1:1 to 1:3.

[0028] Because the complementary base sequences of PL3-PL4 contain specific restriction endonuclease recognition sites, while PL1-PL2 lack similar sites, a specific restriction endonuclease is selected for processing and dissociation of the DM-Bm-CPL double antibody sandwich complex. In the DM-Bm-CPL immune complex, the non-magnetic microspheres, target, and magnetic microbeads are in a 1:1:1 ratio. Therefore, the Mb free reporter (PM reporter) coated with PL4 obtained from the immobilized container Ct can accurately reflect the target content, enabling precise detection, identification, and quantitative analysis.

[0029] PM reporter substances are dispersed and resuspended, collected by magnetic attraction, and then concentrated after washing and rinsing. The PM reporter substances are then loaded onto a counting chip, and the final PM fluorescence signal is the desired target signal value. In this application, the fluorescence of the PM reporter substances is excited by light with a wavelength of 530 nm.

[0030] As a preferred option:

[0031] The nucleic acid sequence of PL1 is shown in SEQ ID NO:1;

[0032] The nucleic acid sequence of PL2 is shown in SEQ ID NO:2;

[0033] The nucleic acid sequence of PL3 is shown in SEQ ID NO:3;

[0034] The nucleic acid sequence of PL4 is shown in SEQ ID NO:4;

[0035] The nucleotide dissociation enzyme used was EcoR-I.

[0036] The magnetic microbeads were purchased from Beaver Biotechnology, model number 22316-1.

[0037] Preferably, the ratio of the inner surface area of ​​the container to the cross-sectional area of ​​the non-magnetic microspheres is not less than 6.02 × 10⁻⁶. 9 .

[0038] Preferably, the number of capture antibodies in step (2) is not less than 100 times the number of targets.

[0039] Preferably, the number of detection antibodies in step (3) is not less than 100 times the number of targets.

[0040] Preferably, the ratio of the number of detection antibodies to the number of PL3 reactions in step (4) is no greater than 3.

[0041] Preferably, the material of the container and the material of the non-magnetic microspheres include, but are not limited to, polystyrene, polyethylene, polypropylene, and glass; the passivating agent includes, but is not limited to, bovine serum albumin.

[0042] Preferably, the reaction order of the reactants in step (7) is one of the following:

[0043] a) The target first reacts with the CPL complex, and then with the DM complex;

[0044] b) The target reacts first with the DM complex, and then with the CPL complex;

[0045] c) The target reacts simultaneously with the CPL complex and the DM complex.

[0046] In another aspect, the present invention provides a method for detecting the concentration of a target to be tested, comprising the following steps:

[0047] The above method was used to measure the PM reporter values ​​of several groups of targets with known concentrations; then a standard curve was established based on the target concentration and the number of PM reporter microspheres; the above method was used to measure the PM reporter values ​​of the target to be tested to further determine the target concentration.

[0048] As a preferred method, the above-mentioned method for detecting the concentration of the target to be tested is applicable to diseases such as tumors and infectious diseases, as well as food and environmental testing.

[0049] In some further cases, the above detection method specifically refers to:

[0050] (1) Preparation process of embedding Ct on the inner surface of the container using oligonucleotide 1 (PL1):

[0051] The inner wall surface of a selected container made of a certain material is repeatedly cleaned. Based on its physicochemical properties, the inner wall is activated using appropriate chemical reagents. Excess activating reagent that has not reacted is then washed away with a cleaning buffer. The 5' amino terminus of PL1 is specially chemically modified, allowing PL1 to connect to the activation sites on the inner surface of the container Ct via covalent or non-covalent bonds, thus immobilizing PL1 at the activation sites on the container Ct surface. Excess unreacted nucleic acid sequences are then washed away with a cleaning buffer, and the remaining activation sites are blocked with a passivating agent. Excess passivating agent is then washed away with a cleaning buffer.

[0052] (2) Activation coating process of non-magnetic microspheres PS with oligonucleotide 2 (PL2) and capture antibody Cp that specifically recognizes the target:

[0053] Non-magnetic microspheres (PS) were reacted with capture antibody Cp or oligonucleotide 2PL2 at a ratio of 1:500 to 1:10000 using conventional biochemical coupling methods, resulting in a uniform coating of the PS surface with both. The reaction was carried out in excess of oligonucleotide and capture antibody, and an inactivating agent was used to block the remaining PS surface activation sites. Afterward, multiple low-speed centrifugations were performed to remove unreacted excess PL2 and Cp, followed by high-speed centrifugation to remove excess inactivating agent, yielding a highly purified Cp-PS-PL2 complex (CPL complex). This complex was collected and transferred to 4°C for later use.

[0054] (3) Preparation of CPL composite coating on the inner wall of the container. The specific preparation process is as follows:

[0055] Add a certain concentration of CPL complex to the container with Ct-PL1 surface treatment obtained in the above implementation step (1) and incubate it. The CPL complex will bind and fix to the surface of Ct-PL1 of the container in the form of complementary base sequence. Then wash away the unbound and fixed CPL complex with washing buffer to obtain the desired surface Ct-CPL complex. After cleaning and drying at room temperature, seal the container and transfer it to 4°C for later use.

[0056] (4) The detection antibody Dp was conjugated with oligonucleotide 3 (PL3):

[0057] The detection antibody Dp and PL3 sequence were added to a test tube in a reaction ratio of 1:1 to 1:5. A specific chemical coupling agent was added and mixed thoroughly before incubation. After repeated washing and centrifugation at a specific speed, unbound PL3 was removed, yielding the desired simply purified PL3-Dp complex (i.e., the PD complex).

[0058] (5) Coating of magnetic microbeads Mb with fluorescent material encapsulation using the oligonucleotide sequence PL4:

[0059] Using conventional biochemical coating methods, PL4 and Mb were uniformly added to the reaction tube at a specific ratio and mixed thoroughly. The entire reaction ensured that the amount of PL4 was significantly greater than that of the Mb magnetic beads (100:1–1000:1). Due to the magnetic properties of the beads, excess PL4 was washed away with a washing buffer after magnetization, and an inactive site on the Mb surface was blocked using a passivating agent. Excess passivating agent was then washed away again using the same magnetic properties. Thus, PL4 was coated onto the surface of fluorescently coated magnetic microbeads (Mb), and after magnetization, the purified PL4-Mb complex (i.e., the PM reporter) was obtained.

[0060] (6) Preparation process of Dp-PL3-PL4-Mb complex (DM complex preparation):

[0061] The PM reporter and PD complex generated in specific implementation steps (4) and (5) are added to a new test tube at a certain concentration ratio and mixed evenly, and then incubated at a specific temperature. The PM reporter and PD complex will be fixed and bound in a complementary base sequence. Then, the magnetic adsorption properties of the magnetic beads are used to collect the magnetic material. The unbound PD complex is washed away with washing buffer to obtain the desired DM complex. The purified DM complex suspension is transferred to 4°C for later use.

[0062] (7) Based on the principle of single-molecule immune Poisson distribution, a preparation process for the DM-Bm-CPL double antibody sandwich complex was established:

[0063] According to the Poisson distribution principle, the reaction is carried out using a pre-defined feeding method for the immune response, P(k) = k / ! +e -λ (k=0,1,2…), using this formula as the preferred condition, wherein the number of captured objects in step (1) is not less than 100 times the number of targets.

[0064] The DM complex and target Bm generated from the reaction are then added to a container containing the Ct-CPL complex, and all three are incubated simultaneously. Because the inner wall surface of the container is specially treated, based on the principle of the double antibody sandwich method, the complexes will couple in a specific binding manner, forming the DM-Bm-CPL double antibody sandwich complex.

[0065] (8) Enzymatic mild dissociation process for the DM-Bm-CPL double antibody sandwich complex:

[0066] The obtained DM-Bm-CPL double antibody sandwich complex was dissociated using a mild enzymatic treatment. Based on a clever design in nucleic acid sequence synthesis, the oligonucleotide sequences PL3-PL4 were first specially treated. Specifically, their complementary base sequences contain specific recognition sites for restriction endonucleases; however, the complementary base sequences of PL3-PL4 do not contain similar restriction sites. Therefore, the dissociation of the DM-Bm-CPL double antibody sandwich complex requires the use of a specific restriction endonuclease.

[0067] The immunoglobulin complex was digested with a specific restriction endonuclease and incubated for 0.5–2 hours. The dissociated PM reporter beads were then washed and enriched using magnetic collection.

[0068] In the DM-Bm-CPL complex, PS, target Bm, and Mb are in equal proportions of 1:1:1. Therefore, the PM reporter obtained from the container Ct theoretically has a one-to-one quantitative relationship with the content of target Bm. By counting the number of PM particles on the magnetic beads, the concentration of the target can be reflected.

[0069] (9) Preparation of single-molecule immunoassay chip samples and magnetic bead-loaded microscopic imaging;

[0070] The magnetic bead suspension Mb obtained by dissociation is eluted and extracted from the container. After multiple washing and magnetic collection operations, the free PM reporter substance after concentration and purification is loaded into the detection chip to obtain the required digital enzyme-linked immunosorbent assay (ELISA) sample. After oil sealing, the obtained digital ELISA sample is subjected to microscopic scanning and software counting, and the data is analyzed.

[0071] More specifically, during the detection process, the fluorescent material coating the surface of the magnetic microspheres is excited with a wavelength of 530nm, causing the magnetic microspheres to emit fluorescence.

[0072] In certain specific operational methods, the above steps can be performed and implemented in the following ways:

[0073] Prepare a reaction vessel Ct with the oligonucleotide sequence PL1 labeled on its inner surface;

[0074] ① First, the inner surface of the container is activated so that the container surface can carry active groups that can specifically covalently bind to the PL1 sequence. Then, the inner wall of the container is cleaned with a cleaning buffer.

[0075] ② Add the PL1 sequence to the activated container, seal the outer wall of the reaction container with a sealing strip, and incubate at 50°C with shaking for 2-4 hours to allow the nucleotide sequence PL1 to fully bind to the active groups on the inner surface of the container, thereby fixing PL1 on the inner surface of the container. Then, wash the container with a washing buffer.

[0076] ③ Add a passivating agent solution to the container to block the active sites on the inner surface of the container that have not reacted with the PL1 sequence, so as to avoid binding of other proteins or impurities or non-specific adsorption of Ct. Then clean the container with a cleaning buffer.

[0077] ④ Place the container coated with the nucleotide sequence PL1 into a sealed polyethylene bag and store it at 4°C or lower for later use; this completes the preparation of the inner wall of the PL1-Ct container with a treated surface.

[0078] The following method is used to prepare a specially treated container containing an inner surface of a CPL composite:

[0079] ① In another new container, non-magnetic microspheres PS with a special group on their surface are washed and surface activated to fully disperse the PS treated with added surfactant; and excess surfactant is washed away multiple times using a cleaning buffer.

[0080] ② Add a certain concentration of capture antibody Cp and oligonucleotide sequence PL2 to the activated PS spheres, and vortex to mix. Ensure that the amount of nucleotides and capture antibody added far exceeds the amount of PS spheres throughout the reaction, and incubate the reaction at room temperature for 2 hours. Then, centrifuge at an appropriate speed to remove unbound excess PL2 and capture antibody Cp.

[0081] ③ Add a passivating agent to the container to block the remaining activation sites on the PS surface. Then, centrifuge at a certain speed to remove excess PL2 and Cp that have not undergone the binding reaction. Then, centrifuge at high speed multiple times to remove excess passivating agent, and obtain a highly purified Cp-PS-PL2 complex (CPL complex).

[0082] ④ The highly purified CPL complex was added to the surface of a specially labeled reaction vessel (Ct) containing the oligonucleotide sequence PL1 at a specific ratio. The vessel was then shaken on a horizontal shaker for 30 minutes at room temperature. The nucleotide sequence PL2 on the CPL complex pairs complementaryly with the nucleotide sequence PL1 on the plate wall, forming a Ct-CPL surface. The inner wall of the vessel was then washed with washing buffer, dried at room temperature, sealed, and transferred to 4°C for later use.

[0083] The DM complex was prepared using the following method:

[0084] ① First, prepare the detection antibody Dp-oligonucleotide sequence PL3. Take a certain amount of the detection antibody Dp, dissolve it in PBS buffer, mix and vortex. Then add a certain amount of activator to activate Dp.

[0085] ② Use a cleaning and washing solution to repeatedly wash away excess activator.

[0086] ③ Add the oligonucleotide sequence PL3 to the washed Dp antibody tube and incubate for 1 hour (the reaction ratio of Dp antibody to PL3 sequence is 1:2 to 1:5). Select a specific high-speed rotation to wash away excess unreacted oligonucleotide sequence PL3. The purified Dp-PL3 complex is obtained.

[0087] ④ Next, prepare free oligonucleotide 4-magnetic bead complex (PL4-Mb).

[0088] A certain amount of magnetic bead solution containing SA treatment was taken, and the magnetic bead stock solution was washed with PBS buffer. Excess supernatant was removed by magnetic agglutination with the assistance of a magnetic rack. Based on the high affinity between streptavidin and biotin, biotin labeling was introduced at the 5' end in advance during the synthesis of sequence PL4, so the magnetic beads can have a high affinity for sequence PL4.

[0089] ⑤ Add an excess of oligonucleotide sequence PL4 to the washed magnetic bead test tube, incubate for 0.5-2 hours, and then use a magnetic rack to collect the magnets to remove the excess unreacted oligonucleotide sequence PL4.

[0090] ⑥ The prepared PL4-Mb complex is mixed and reacted with the Dp-PL3 complex, and then magnetically cleaned with the assistance of a magnetic rack to obtain the desired Dp-PL3-PL4-Mb, i.e., the DM complex.

[0091] The preparation method for the DM-Bm-CPL double antibody sandwich complex is as follows:

[0092] ① Take the CPL complex-containing surface container prepared in step (2), and add the DM complex and the target protein Bm into it; the ratio of the two added is based on the Poisson distribution principle.

[0093] ② The prerequisite for single-molecule immunocounting is that each microbead binds to only one target Bm and forms an enzyme-linked immunosorbent assay (ELISA) complex. According to the theory of Poisson distribution, to achieve this, an excess of microbeads (PS or Mb) is needed to bind to the target Bm. In this way, the target Bm will be completely bound by both PS and Mb microbeads. Washing away the excess unbound complex yields a purified DM-Bm-CPL double antibody sandwich complex. The ratio of Bm content to Mb content in this complex is 1:1.

[0094] The DM-Bm-CPL double antibody sandwich complex was dissociated using a gentle enzymatic unwinding process to obtain the dissociated PM complex. Then, effective microbeads were eluted and a counting chip was loaded, as follows:

[0095] ① For the DM-Bm-CPL double antibody sandwich complex obtained in step (4), a mild enzymatic dissociation method was adopted. This is because the oligonucleotide sequences PL3-PL4 have specific nuclease cleavage sites due to their complementary base sequences.

[0096] ② Add a certain volume of enzyme digestion reaction buffer to the container, wash 2-3 times, then quickly add the enzyme digestion solution and react for 1-2 hours. Extract the reaction supernatant and use a magnetic device to concentrate the magnetic beads. The number of magnetic beads obtained is in a 1:1 ratio to the concentration of Bm.

[0097] ③ Load the collected magnetic beads onto the counting chip in this experiment for image acquisition and signal output. Achieve equimolar conversion between Bm and PM.

[0098] Due to the principles of intermolecular reaction mechanics and the numerous cleaning steps and container changes involved in each reaction before loading, some signal loss of the PM reporter will occur. Therefore, the conversion of the Bm molecule number above contains some error, and the actual signal quantity obtained is less than the Bm quantity in the sample. Therefore, correction using a calibration curve is necessary to accurately reflect the actual Bm concentration in the sample. Establishment of the standard curve and conversion of detection results: Dilute the standard with known concentration and plot the standard curve. Based on the standard curve, convert the detection result of the sample to the actual target concentration.

[0099] Prepare at least five known Bm standard samples with concentration gradients from pM to aM. Obtain the samples using the sample preparation method described above, and then measure the final signal response values ​​of the test samples at different concentration gradients using the optical detection method described above. Each sample is repeated at least three times, and the mean and standard deviation (SD) of the final signal response for each sample are calculated. Plot a scatter plot with sample concentration on the x-axis and the mean of the final signal response on the y-axis. Perform linear fitting on the scatter plot to establish a standard curve, and calculate the goodness of fit r. 2 As is customary, the undetectable limit (LoD) is determined by adding three times the SD of the background signal to the background signal.

[0100] This invention provides a single-molecule immunoassay method that combines DNA polymerase unwinding multiplexing technology with a novel reporter, namely fluorescently coated magnetic microbeads. This invention offers significant advantages in improving the detection rate of target proteins, the detection rate of final products, and detection efficiency, possessing the following benefits:

[0101] (1) The use of DNA double-strand ligation and enzymatic dissociation significantly reduces PM reporter signal loss during the washing process, ultimately reducing statistical error and lowering the detection limit. Furthermore, the entire reaction is conducted under mild conditions, with a short reaction time and high specificity recognition efficiency. In addition to the initial reagent sample preparation, the subsequent analyte detection efficiency is extremely high, greatly shortening the reaction time. Furthermore, the fact that the final reporter detection step is wash-free further reduces the detection time for a single sample to 5 minutes.

[0102] (2) The use of fluorescently coated magnetic microbeads to replace the original enzyme-substrate fluorescence excitation method improves the stability and reproducibility of the test samples and extends the storage time of the test samples. Attached Figure Description

[0103] Figure 1 This is a flowchart illustrating the specific operation of the dELISA and gentle dissociation method of the present invention;

[0104] Figure 2 This is a schematic diagram illustrating the preparation of PM reporter substances that reflect the content of the target Bm in this invention;

[0105] Figure 3 This is the standard curve detection graph from Example 1.

[0106] Figure 4 This is the standard curve detection graph from Example 2. Detailed Implementation

[0107] The following examples will illustrate the implementation of this application in detail, so that the process of how this application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0108] Unless otherwise specified, all raw materials and equipment used in this application are commonly used in the field and are derived from commercially available products. Unless otherwise specified, all methods used in this application are conventional methods in the field.

[0109] There are many other feasible technical solutions in this application, which will not be listed here. All technical solutions claimed in the claims of this application are feasible.

[0110] The terms "comprising" or "including" are intended to indicate that a composition (e.g., a medium) and a method include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting substantially of" means excluding other elements that are of any significance to the combination for the stated purpose. Therefore, a composition consisting substantially of the elements defined herein does not exclude other materials or steps that do not materially affect the essential and novel features of the claimed application. "Constitutes" means excluding trace elements and substantial method steps that are other components. Embodiments defined by each of these transitional terms are within the scope of this application.

[0111] The invention will now be further described with reference to the accompanying drawings.

[0112] The embodiments described above use different systems to prepare novel dELISA samples according to this invention patent, and then test them to verify the universality of this method.

[0113] Example 1

[0114] This embodiment provides a method for detecting the signal of human IL6 target, which is converted to PM reporter after the formation of a bispherical complex. The specific operation flowchart of the embodiment is as follows. Figure 1 As shown, Figure 2 This is an illustration of the preparation of the PM report complex. The specific implementation process is as follows:

[0115] Step 1, the process of preparing the inner surface (Ct) of the container embedded with oligonucleotide 1 (PL1):

[0116] (1) Surface activation treatment of polystyrene container interior (actually using 96-well plates, each well is equivalent to one container):

[0117] The container was rinsed twice with PBST (0.01M phosphate-buffered saline containing 0.1% Tween-20); then 1 nmol PL1 and 100 μL of EDC (i.e., 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, concentration 10 mg / mL, solvent using MES (morpholinoethanesulfonic acid) buffer, pH 5.5) were added to the container. The reaction well was sealed with the connecting strip and incubated at 50°C with shaking for 4 h. The sequence of oligonucleotide PL1 is shown in SEQ ID NO:1.

[0118] (2) At room temperature, the reaction wells of the container were cleaned with 100 μL of cleaning buffer (containing 100 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20 pH ~ 7.5) three times, each time soaking for 5 min. Finally, the liquid in the reaction wells of the container was emptied. The empty reaction container was then cleaned again with 100 μL of deionized water, three times, each time soaking for 5 min. Finally, the liquid in the reaction wells was emptied. At this point, PL1 had been coated on the Ct surface inside the container.

[0119] (3) After sealing the prepared container with PL1 on the inner surface, put it into a sealed polyethylene plastic self-sealing bag and store it at 4°C or lower for later use.

[0120] Step 2, Coating preparation process of IL6 capture antibody (CP)-nonmagnetic microspheres (PS)-oligonucleotide 2 (PL2) complex (i.e., CPL complex):

[0121] (1) Take 10 μL of non-magnetic microspheres, i.e., PS spheres (approximately 3 × 10⁻⁶). 10 Add 25 μL of EDC and 25 μL of NHS (N-hydroxysuccinimide, 10 mg / mL) to 500 μL of 0.05 M MES buffer and vortex to mix.

[0122] (2) Shake for 1 hour at room temperature on a vertical mixer. Centrifuge at 12000g for 15 minutes to remove the supernatant, leaving a white precipitate, thus removing excess activator.

[0123] (3) Add borate buffer (0.05M, pH 8.0), 1 μL of IL6 capture antibody (0.5 mg / mL, 20 times the number of molecules of PS), and 0.3 μL of oligonucleotide 2, i.e., PL2 (200 times the number of molecules of PS). The sequence of oligonucleotide PL2 is shown in SEQ ID NO:2.

[0124] (4) Place the mixture on a vertical mixer and shake it. React at room temperature for 1 hour. Then centrifuge at 6000g for 15 minutes, remove the supernatant and leave a white precipitate. Then add 300μL of PBS to reconstitute the mixture, disperse it, centrifuge at 12000g for 15 minutes, remove the supernatant and leave a white precipitate to obtain the CPL complex.

[0125] (5) Add 300 μL of PBS to the container containing the CPL complex to reconstitute and disperse it, then set aside for later use (if the white CPL complex adheres to the tube wall, it can be briefly sonicated). This yields 3 × 10⁻⁶ CPL complex. 10 A CPL complex.

[0126] Step 3: The Ct coated with PL1 binds to the CPL complex:

[0127] (1) Remove the container obtained in step one from the low temperature environment and place it at room temperature for 0.5 h. Then add 100 μL of Buffer1 to the container. (Buffer1 is also called nucleotide buffer solution, which is composed of 0.01M Tris-HCl, pH=7.5, 1mM EDTA, and 0.1M NaCl).

[0128] (2) Take 6 μL of the CPL complex (approximately 6 × 10⁻⁶) 8 Add (one) to the container mentioned above.

[0129] (3) At room temperature, the container was placed on a horizontal shaker and shaken for 30 min. The CPL complex was bound to the inner surface of the Ct container by means of the principle of complementary sequence bases (i.e., PL1 and PL2 are complementary).

[0130] Step 4: Prepare the PD complex by labeling the IL6 detection antibody (Dp) with oligonucleotide 3 (i.e., PL3) (taking 0.5 mg / mL IL6 detection antibody as an example):

[0131] (1) Take 1 μL of IL6 detection antibody, add it to 300 μL of PBS, mix and shake to disperse.

[0132] (2) Add 5 μL of EDC (10 mg / mL) and 5 μL of NHS (10 mg / mL), followed immediately by 1.25 μL of PL3. The ratio of detection antibody to PL3 is 1:3.

[0133] (3) Place the mixture on a vertical suspension apparatus and shake at room temperature for 3 hours.

[0134] (4) Centrifuge at 12000g for 20min to remove 270μL of supernatant and leave about 30μL of residual liquid at the bottom.

[0135] (5) Add 280 μL of PBS to the remaining liquid and shake to reconstitute. Set aside for later use. The final quantity is 1.5 × 10⁻⁶. 13 PD complex.

[0136] Step 5: Oligonucleotide 4 (PL4) is coated onto the surface-activated magnetic microbeads (Mb) with fluorescent material to obtain the PM complex.

[0137] (1) 100 μL Mb (approximately 1.8 × 10⁻⁶ mb) 7 Each Mb sample was coated with a fluorescent material (purchased from Suzhou Beaver Biotechnology Co., Ltd., model number 22316-1). The sample was added to 200 μL of PBS for washing, and after magnetic collection, the supernatant was discarded. The sample was then washed three times with 300 μL of PBST (this magnetic collection refers to the process of using magnetism to attract Mb).

[0138] (2) Add 1.2 μL of PL4 (biotinylated oligonucleotide sequence 4, number of Mb 10). 5 The PM complex was obtained by mixing the PM with water at room temperature on a vertical vortex mixer for 1 hour.

[0139] (3) Collect magnetic separation, extract the supernatant and transfer the PM complex to a new EP tube.

[0140] (4) Wash the PM complex three times with 300 μL of PBST.

[0141] (5) Finally, add 300 μL of PBS, resuspend and disperse for later use, yielding approximately 1.8 × 10⁻⁶ ppm. 7 A PM complex.

[0142] Step 6: Prepare the free DM complex:

[0143] (1) Take 50 μL of the PD complex obtained in step four into a new test tube;

[0144] (2) Add 100 μL of the PM reporter from step five;

[0145] (3) At room temperature, the DM complex was obtained by oscillating the reaction for 4 hours using the complementary base pairing of PL3 and PL4.

[0146] Step 7: Prepare the DM-Bm-CPL double antibody sandwich complex and perform mild enzymatic dissociation to obtain an effective PM reporter:

[0147] (1) Take the prepared container (96-well plate, each well equals one container) containing the CPL complex, and add 100 μL of IL-6 target to each well. The target concentrations in the experimental groups are 100 fg / mL, 50 fg / mL, 20 fg / mL, 10 fg / mL, and 0.1 fg / mL, respectively, while the control group is 0 fg / mL. Each group has 3 wells. Place the plate on a horizontal shaker and shake for 30 min. Then immediately add 3 μL (1×10⁻⁶) of the target to each well. 6 (One DM complex) was placed on a horizontal shaker and shaken for 60 min to obtain the DM-Bm-CPL double antibody sandwich complex.

[0148] (2) After shaking and mixing, collect the supernatant of the reaction by magnetic attraction; add 200 μL Buffer1 to each reaction well and wash 3 times to remove the DM complex that has not bound to the CPL complex.

[0149] (3) Add EcoR-I enzyme and 200 μL of buffer solution to each well, react for 1 h, and dissociate to obtain effective PM reporter.

[0150] (4) Remove the reaction supernatant, concentrate the sample magnetically to obtain effective PM reporter materials, and load the PM reporter materials onto a counting chip (each micrometer pit on the counting chip contains one PM reporter material), and count them under a 20× objective lens of a microscope. The specific counting method is as follows: after excitation with 530nm fluorescence, record the number of Mb emitted (i.e., the number of luminescent micrometer pits).

[0151] Step 8: Establish the standard curve:

[0152] Based on the counting results from step seven, calculate the average value and standard deviation (SD) of the final signal response for each concentration. Plot a scatter plot with target concentration on the x-axis and the average value of the final target signal response on the y-axis. Perform linear fitting on the scatter plot to establish a standard curve and calculate the goodness of fit r. 2 The detection limit (LoD) was calculated using extrapolation. LoD equals the background signal plus three times the SD. Based on the standard curve, the achievable LoD in this embodiment is 2.5 fg / mL. Example 1: Standard curve detection... Figure 3 As shown.

[0153] The sequence of PL1 is SEQ ID NO: 1: TTTTTTTTTTTTTTTTTTTTTTTTTTCGTCGCCGTCCAGCTCGACCAGTCG;

[0154] The sequence of PL2 is SEQ ID NO: 2: TTTTTTTTTTTTTTTTTTTTTTTTTTTCGACTGGTCGAGCTGGACGGCGACG;

[0155] The sequence of PL3 is SEQ ID NO: 3: TTTTTTTTTTTTTTTTTTTTTTTTTTTTCCAAAGAATTCAACTTTAAGCCGG;

[0156] The sequence of PL4 is SEQ ID NO: 4: TTTTTTTTTTTTTTTTTTTTTTTTTTTCCGGCTTAAAGTTGAATTCTTTGGA;

[0157] The IL6 protein target was purchased from Pujian Biotechnology, catalog number ATMP00015HU. The IL6 capture antibody and IL6 detection antibody are based on patent CN202011200143.9.

[0158] Example 2

[0159] This embodiment provides a COVID-19N protein target, which is converted into a reporter substance after the formation of a biglobulin complex. The operation method is the same as that in Example 1, except that the IL6 target, IL6 capture antibody, and IL6 detection antibody used in Example 1 are replaced with the COVID-19N protein target, COVID-19N capture antibody, and COVID-19N detection antibody.

[0160] The COVID-19N protein target, COVID-19 capture antibody, and COVID-19 detection antibody were purchased from Phytobio, with the models being Cov19-MAb-15 (batch number: 20220524) and Cov19-MAb-17 (batch number: 20220222), respectively.

[0161] Example 2 Test standard curve detection as follows Figure 4 As shown. According to the standard curve, the actual achievable LoD in this embodiment is 1.9 fg / mL.

[0162] Comparative Example 1:

[0163] Step 1:

[0164] (1) Activation treatment of the inner surface of polystyrene container (actually using 96-well plate, each well is equivalent to 1 container): Activation treatment of the inner surface of polystyrene container: Wash the inner wall of the container twice with PBST (0.01M phosphate buffer containing 0.1% Tween 20); dilute 25% glutaraldehyde solution with PBS (phosphate buffer) to 8% (v / v) glutaraldehyde solution, add 350μL of 8% glutaraldehyde solution to the container, shake at room temperature for 5h, and repeat the washing of the inner surface of the container twice with PBST.

[0165] (2) Immobilize Cp (IL6 capture antibody) on the inner surface of the container: Add 350 μL of 8% glutaraldehyde solution to the container and allow the antibody to equilibrate at room temperature for at least 15 minutes. Add 1.75 μg of Cp to the container and shake the reaction overnight at room temperature to fix Cp on the inner surface of the container. Then wash the inner surface of the container twice with PBST solution.

[0166] (3) Passivation treatment of unreacted active sites on the inner surface of the container: Pour out the remaining liquid, add 0.2M ethanolamine, and incubate at room temperature for 30 min to inactivate the unreacted glutaraldehyde sites on the inner surface of the container. Use 10% bovine serum albumin (BSA) solution as a blocking buffer and incubate the inner wall of the container at room temperature for 30 min to ensure the passivation of inactive protein regions on the inner surface of the container. Wash the inner surface of the container three times with PBST, and the last time with deionized water.

[0167] (4) Specific binding of Cp to Bm: Add Bm (IL6 antigen) to the container and shake at room temperature for 5 hours to form Cp-Bm. The amount of Bm should be less than the amount of Cp (1 < number of Bm molecules < number of Cp molecules) to ensure that all Bm binds to Cp.

[0168] Step 2: Immobilize detection antibody (Dp) and enzyme molecule (EZ) as reporter on the surface of biotin-coated magnetic microbeads (MMS):

[0169] (1) Dispersion and activation of MMS: Take an appropriate amount of MMS with a diameter of 3.5 μm and place it in a new test tube. Place it at room temperature for 10 min and vortex for 20 s to allow the MMS to diffuse fully. Centrifuge at 8000g for 5 min at room temperature and remove the supernatant (MMS diffuses in PBS buffer at pH 7.4. After MMS diffuses into the buffer, it forms a suspension. After standing for a period of time, MMS will precipitate). Wash the MMS twice with PBST and remove the supernatant. Dilute the 25% glutaraldehyde solution with PBS to 8% (v / v) glutaraldehyde solution. Add 350 μL of 8% glutaraldehyde solution to the MMS and mix by shaking at room temperature for 5 h. Centrifuge at 8000g for 5 min at room temperature and remove the supernatant. Wash the MMS twice with PBST and discard the supernatant.

[0170] (2) Add CaptAvidin (avidin variant, AM) and avidin-labeled β-galactosidase (EZ) in a molar ratio of 1:1 to the container, shake and mix at room temperature for 5 h. After sufficient incubation, the surface of MMS is coated with EZ and AM. Wash the MMS three times with PBST, and separate the MMS and free AM and EZ using a magnetic rack. After separation, the test tube contains almost no free AM and EZ.

[0171] (3) Add biotin-labeled Dp (IL6 detection antibody) to the test tube, shake and incubate at room temperature for 5 hours to allow Dp to bind to the free sites on the AM on the MMS surface. Due to the large surface area of ​​MMS, multiple Dp and EZ can be linked to one MMS.

[0172] (4) Separate MMS and free Dp using a magnetic rack, centrifuge at 8000g at room temperature for 5 min, and discard the supernatant; wash three times with PBST, discard the supernatant, and only the (Dp)n-MMS-(EZ)m complex remains in the container.

[0173] Step 3, specific binding of (Dp)n-MMS-(EZ)m to Bm:

[0174] The (Dp)n-MMS-(EZ)m complex prepared in step two is added to the container from step one (containing the Cp-Bm complex). The mixture is shaken and incubated at room temperature for 2 hours to allow Dp and Bm to specifically bind, forming the Cp-Bm-(Dp)n-MMS-(EZ)m immune complex. In this step, the amount of (Dp)n-MMS-(EZ)m complex should be greater than the amount of Cp-Bm to ensure that each Bm molecule binds to one (Dp)n-MMS-(EZ)m complex, thus forming the Cp-Bm-(Dp)n-MMS-(EZ)m immune complex.

[0175] Step 4, enrichment of the effective MMS reporter complex:

[0176] The MMS in the above steps exists in two forms: immobilized Cp-Bm-(Dp)n-MMS-(EZ)m immune complexes (effective microbeads) and unbound free (Dp)n-MMS-(EZ)m complexes (ineffective microbeads). The solution in the container is poured out and washed three times with PBST. Washing removes the ineffective microbeads, leaving only effective microbeads in the container.

[0177] Step 5, Elution of effective microbeads:

[0178] After the previous step is completed, a reaction buffer solution (pH 10) is added to the reaction vessel to weaken the binding force between AM and Dp and MMS, thereby separating them and allowing MMS to return to the solution, obtaining the free MMS reporter complex and realizing the conversion of the marker with the microbeads.

[0179] Step 6, Optical detection of the MMS-reported complex:

[0180] (1) The free reporter complex generated in step five was subjected to fluorescence detection for quantification. The free MMS reporter complex in the container was loaded into a pre-prepared array of micro-pits. Specifically, the MMS reporter complex was added through the sample inlet of the microfluidic chamber containing the array of micro-pits. A magnet was used to move back and forth several times under the substrate of the micro-pits to load the MMS reporter complex into the micro-pits by magnetic force. The diameter of the micro-pit can only accommodate one MMS reporter complex, and the number of micro-pits is much greater than the number of MMS reporter complexes. Therefore, it is basically guaranteed that all MMS reporter complexes can be loaded into the micro-pits.

[0181] (2) After adding the substrate (halogen-β-D-galactopyranoside), seal the container in oil. After enzyme catalysis for a certain period, observe and record the fluorescence reaction under a fluorescence microscope (excitation wavelength 558 nm; emission wavelength 577 nm). Each bright spot indicates a positive molecule. Theoretically, the sum of all bright spots is approximately equal to the number of Bm molecules to be measured.

[0182] Step 7, establish the standard curve:

[0183] Prepare Bm (IL6 antigen) samples at concentrations of 100 fg / mL, 50 fg / mL, 20 fg / mL, 10 fg / mL, and 0.1 fg / mL. Measure the final signal response values ​​of the Bm samples at different concentration gradients using steps one through six. Each sample was measured at least three times. The mean and standard deviation (SD) of the final signal response for each sample were calculated. A scatter plot was plotted with sample concentration on the x-axis and the mean of the final signal response on the y-axis. A standard curve was established by linearly fitting the scatter plot, and the goodness of fit (r) was calculated. 2 The limit of detection (LoD) was calculated by extrapolation. LoD equals the background signal plus three times the SD. Based on the standard curve, the actual achievable LoD for this comparative example is 24.8 fg / mL.

[0184] The main difference between Comparative Example 1 and Example 1 is:

[0185] 1. The magnetic microbeads are linked to the detection antibody via biotin and avidin, rather than through DNA double strands;

[0186] 2. Comparative Example 1 requires the addition of a substrate to the detection substrate, and the reporter enzyme molecule dissociates the substrate to emit fluorescence.

[0187] Therefore, Example 1 has the following advantages compared to Comparative Example 1: 1. It uses DNA double-strand ligation, which is more stable and the dissociation conditions are mild and efficient; 2. The sample can be stored for a longer time, which can reduce measurement errors. Table 1 shows the number of fluorescent spots on the detection plates prepared from the 10 fg / mL samples in Example 1 and Comparative Example 1 after 0.5 h, 8 h, and 24 h.

[0188] Table 1

[0189] 0.5h 8h 24h Example 1 4546 4251 4089 Comparative Example 1 2239 1532 524

[0190] As shown in Table 1, even at 0.5 h, the number of fluorescent spots detected for the same sample varied considerably. This is mainly because biotin and avidin variants are linked by reversible chemical bonds, and the reporter is easily broken and eluted during multi-step elution, resulting in a lower overall detection rate and increased detection error.

[0191] The contents not described in detail in this application specification are common knowledge to those skilled in the art.

[0192] As used throughout the specification and claims, the term "comprising" is an open-ended term and should therefore be interpreted as "comprising but not limited to". "Approximately" means that within an acceptable margin of error, those skilled in the art can solve the technical problem and substantially achieve the technical effect within a certain margin of error.

[0193] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a product or system comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a product or system. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the product or system that includes said element.

[0194] The foregoing description illustrates and describes several preferred embodiments of this application. However, as previously stated, it should be understood that this application is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this application should be protected within the scope of the appended claims.

Claims

1. A single-molecule immunoassay method for non-disease diagnostic purposes, characterized in that... And includes the following steps: (1) Coat the inner wall of the activated container with PL1, and then use a passivating agent to block the remaining activation sites on the inner wall of the container; (2) The surface of the activated non-magnetic microspheres is coated with capture antibody and PL2. The remaining activation sites on the surface of the non-magnetic microspheres are blocked with a passivating agent. The excess capture antibody and PL2 are washed away to prepare the CPL complex. (3) Add the CPL complex into the container obtained in step (1) so that the CPL complex reacts with PL1 on the inner surface of the container and binds the CPL complex to the inner surface of the container; wash away the unreacted CPL complex. (4) The detection antibody is added to PL3 in a ratio of 1:1 to 1:5 to react and bind. Unreacted PL3 is removed by washing and centrifugation to obtain the PD complex. (5) React PL4 with magnetic microbeads, coat the activated magnetic microbeads with fluorescent material with PL4, and use a passivating agent to block the unactivated sites on the surface of the magnetic microbeads to obtain PM reporter; the amount of PL4 added is 100-1000 times that of the magnetic beads. (6) The PD complex and PM reporter obtained in steps (4) and (5) are fixed and bound together in a complementary base sequence manner. The unbound PD complex is then removed by a magnetic cleaning step to obtain the DM complex. (7) Add the target to react with the CPL complex and DM complex bound to the inner wall of the container, incubate and wash to obtain DM-Bm-CPL double antibody sandwich complex; wash away unreacted DM complex. (8) Add nucleotide dissociation enzyme to the DM-Bm-CPL double antibody sandwich complex to dissociate PL4 and PL3 to obtain PM reporter; (9) Load the dissociated PM report obtained in step (8) into the detection chip and measure the PM report value; PL1, PL2, PL3 and PL4 are oligonucleotides; The base sequences of PL1 and PL2 have complementary regions; The base sequences of PL3 and PL4 have complementary regions. Both the detection antibody and the capture antibody can bind to the target; The magnetic microspheres are fluorescent magnetic microspheres; The number of capture antibodies mentioned in step (2) shall not be less than 100 times the number of targets; The number of antibodies detected in step (3) shall not be less than 100 times the number of targets.

2. The single-molecule immunoassay detection method according to claim 1, characterized in that, The nucleic acid sequence of PL1 is shown in SEQ ID NO:1; The nucleic acid sequence of PL2 is shown in SEQ ID NO:2; The nucleic acid sequence of PL3 is shown in SEQ ID NO:3; The nucleic acid sequence of PL4 is shown in SEQ ID NO:4; The nucleotide dissociation enzyme used was EcoR-I. The magnetic microbeads were purchased from Beaver Biotechnology, model number 22316-1.

3. The single-molecule immunoassay detection method according to claim 1, characterized in that, The ratio of the inner surface area of ​​the container to the cross-sectional area of ​​the non-magnetic microspheres is not less than 6.02 × 10⁻⁶. 9 .

4. The single-molecule immunoassay detection method according to claim 1, characterized in that, The ratio of the number of detection antibodies to the number of PL3 reactions in step (4) shall not exceed 3.

5. The single-molecule immunoassay detection method according to claim 1, characterized in that, The materials of the container and the non-magnetic microspheres include polystyrene, polyethylene, polypropylene, and glass; the passivating agent includes bovine serum albumin.

6. The single-molecule immunoassay detection method according to claim 1, characterized in that, The reaction order of the reactants in step (7) is one of the following: a) The target reacts first with the CPL complex, and then with the DM complex; b) The target reacts first with the DM complex, and then with the CPL complex; c) The target reacts simultaneously with both the CPL complex and the DM complex.

7. A method for detecting the concentration of a target analyte for non-disease diagnostic purposes, characterized in that, Includes the following steps: The PM reporter values ​​of several groups of targets with known concentrations are measured using the method described in claim 1; a standard curve is then established based on the target concentration and the number of PM reporter microspheres; the PM reporter values ​​of the target to be tested are measured using the method described in claim 1 to further determine the target concentration.

8. The method according to claim 7, characterized in that, Suitable for food and environmental testing.

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