A method for detecting intermolecular interactions based on micro-magnetic drive

Abstract

The present invention relates to the technical field of detecting intermolecular interactions, and mainly discloses a method for detecting intermolecular interactions based on micro-magnetic drive. By utilizing the characteristics of nano-magnetic beads, group coating treatment is carried out. The group can undergo a specific coupling reaction with sample A. The nano-magnetic beads or sample A are labeled, and the label can be detected by a photoelectric detector. The nano-magnetic beads and sample A are mixed and incubated, and then incubated one by one with sample B at gradient concentrations to form multiple mixed solutions B. The signal intensity of the set detection area of the multiple mixed solutions B is detected in real time by a photoelectric detector, and the increase and decrease changes of the signal are collected without magnetic force and within a certain time after the magnetic force is applied. Through algorithm fitting, an affinity fitting curve graph is obtained, and finally the affinity K is obtained. D , through the above detection method, common nano-magnetic beads can be used to coat probes, with a wide range of biological groups and a wide range of applicable molecular types. The sample consumption is extremely low, the detection sensitivity is higher, and the detection time is shorter.

Description

Technical Field

[0001] The present invention relates to the technical field of detecting intermolecular interactions, and in particular to a method for detecting intermolecular interactions based on micro-magnetic drive. Background Art

[0002] There are mainly many existing technologies for performing molecular interactions. Optical detection means mainly include surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), biolayer interferometry (BLI), microscale thermophoresis (MST), etc. Generally speaking, traditional methods for detecting the affinity between biomolecules include equilibrium dialysis, spectroscopic measurement, calorimetry, co-immunoprecipitation method, etc. Most methods require at least one molecule to be fixed on a chip or other carrier, another molecule to flow through, or at least one molecule to be fluorescently labeled when obtaining quantitative affinity data, giving a thermal driving force to the complex molecules after interaction, so as to obtain the signal change caused by the interaction, and obtaining the affinity (constant K D ). K D : Affinity constant, also known as dissociation equilibrium constant, is a constant parameter used to evaluate the strength of a reversible reaction. When entering the equilibrium state, it is the constant related to the relationship among free A (receptor), free B (ligand) and AB (receptor-ligand complex) in the reaction system.

[0003] Chinese Patent CN103698278A discloses a method for measuring intermolecular interactions. The specific steps are as follows: (1) Connect one molecule A in the interaction system to be measured to the surface of nanoparticles; (2) Fix the nanoparticles in a microfluidic chip and record the local surface plasmon resonance (LSPR) spectrum of a single nanoparticle; (3) Pass the other molecule B in the interaction system through the nanoparticles at different concentration gradients respectively, and record the LSPR spectrum of a single nanoparticle at the same time; (4) Plot the concentration of molecule B as the abscissa and the LSPR peak value as the ordinate, and fit the graph according to the algorithm formula to obtain the binding constant, the number of binding sites, and the maximum LSPR peak shift of the interaction system between molecule A and molecule B. This method needs to fix one molecule on the chip through nanoparticles, and cannot be directly and effectively docked with the upstream and downstream experiments such as sample purification and enrichment of the sample after interaction, resulting in a certain amount of sample waste and process increase. In addition, it has high requirements for the purity of the sample and the skills of the operator, and the overall equipment and consumable costs are higher.

[0004] The prior art document "Determination of intermolecular interactions in natural biological solutions using MST" discloses a method for determining intermolecular interactions by microscale thermophoresis. The experimental setup is such that an infrared laser is coupled into the fluorescence excitation / emission channel through an infrared dichroic mirror and finally imaging is completed (Fig. 1a). The laser is focused on the sample through an objective lens (which is also used for fluorescence detection). In this way, thermophoresis can be observed in various microfluidic sample compartments, such as capillaries or microfluidic channels. The MST technique requires at least one sample to be fluorescently labeled. Since fluorescent molecules are generally large in size, and fluorescent molecules directly bound to the sample are more likely to form steric hindrance effects, thus affecting the accuracy of the binding results. In addition, heating is not suitable for temperature-sensitive samples, and it cannot be directly and effectively docked with upstream and downstream experiments such as sample purification and enrichment of samples after interaction, resulting in a certain amount of sample waste and process increase.

[0005] In summary, the above-mentioned means, including existing typical techniques such as ITC (isothermal titration calorimetry), all have their own deficiencies, such as high requirements for sample purity, large consumption of sample molecules, long measurement time, influence of heating on molecules, insufficient sensitivity, data accuracy, etc. In addition, observing and analyzing changes in images or signals caused by the movement of magnetic beads has been applied, but the method of using the characteristics of magnetic beads to determine intermolecular interactions has not been applied. Summary of the Invention

[0006] The object of the present invention is to address the above-mentioned problems. The present invention provides a method for detecting intermolecular interactions based on micro-magnetic drive. By virtue of the characteristics of superparamagnetism, small particle size, easy coating with other groups to couple with target molecules, and wide application in purification experiments of nanomagnetic beads, which are mature in technology and application, it is particularly suitable for qualitative determination and rapid, highly sensitive, and direct quantitative measurement of the interaction between any biological macromolecules and small molecules, and can further facilitate the specific enrichment, purification, and analysis of interacting molecules. The acquisition of specific interacting molecules is a further application expansion of traditional molecular interaction methods.

[0007] Glossary:

[0008] Nanomagnetic beads refer to small magnetic particles whose sizes are suitable to be measured in nanometers, generally ranging from 1 to 1000 nanometers. Such magnetic beads have superparamagnetic properties, that is, they have strong magnetic responsiveness in an external magnetic field, and the magnetism of the magnetic particles disappears immediately and there is no residual magnetism after the magnetic field is removed, and they are evenly dispersed in the solution again. Nanomagnetic beads coated with biological or other groups, such as functional groups like amino, hydroxyl, carboxyl, epoxy groups, etc., also possess excellent suspension in liquid, high surface area, high affinity, and high specificity. By specifically binding these groups to the target molecules and then collecting the magnetic beads by magnetic force, the substances needed can be separated, and controllable magnetic force is generated by the magnetic field to manipulate particles, molecules, and even single molecules.

[0009] A method for detecting intermolecular interactions based on micro-magnetic drive uses nanomagnetic beads as carriers for molecular labeling and tracing and / or molecular movement, a highly sensitive spectral detection element as a signal recorder, an external and / or internal magnetic force generating element of a sample carrier as a driving source, the adjustment of the distance between the magnetic force generating element and the detection area or / and the magnitude of the magnetic force as a driving force adjustment means, and the signal (or self-luminescence signal) of one of the nanomagnetic beads or / and any interacting molecules as a probe as the detection object, and measures the affinity (equilibrium dissociation constant) of the intermolecular interaction system in a single or multiple detection areas (the geometric structure of the area is not limited, such as circular, square, or any area that remains unchanged):

[0010] S1. Respectively prepare solution A uniformly dispersed with sample A, solution B uniformly dispersed with sample B, and solution C uniformly dispersed with nanomagnetic beads, and the sample A can interact with the sample B;

[0011] S2. Perform group coating treatment on the nanomagnetic beads in the solution C, and the group can specifically couple with the sample A, and the group is a biological group or other organic group or inorganic group;

[0012] S3. Perform probe labeling and / or self-luminescence treatment on the nanomagnetic beads in the solution C described in S2 and / or the sample A in the solution A, and the probe labeling and / or self-luminescence can be detected by a photoelectric detector;

[0013] S4. Mix and incubate the solution C described in S2 and the solution A described in S3 to form mixture A. The nanomagnetic beads coated with biological groups in mixture A specifically couple with the sample A to form complex A. Among them, there will be more nanomagnetic beads than those required for coupling. The excess nanomagnetic beads are removed by filtration means. For the sample A with probe labeling, it may not be filtered. Mixture A is allowed to react fully and maintain uniform dispersion for a certain period of time;

[0014] S5. Dilute the solution B described in S1 successively by multiples, with the multiple range being 2 - 10 times and the number of dilution gradients ranging from 5 - 50, preferably 10 - 20, to form multiple series of solutions B1, B2... Bn with gradient concentrations. Mix equal volumes of the multiple series of solutions B with the mixture A described in S4 one by one and incubate them to form multiple mixtures B. The concentration of mixture A in the mixtures B is the same. In the multiple mixtures B, part of complex A and part of sample B interact to form complex B. The mixtures B are allowed to react fully for a certain period of time while maintaining uniform dispersion;

[0015] S6. Use multiple carriers to hold equal volumes of the multiple mixtures B. A detection area is provided inside the carrier. Apply a magnetic field to one end and / or other areas of the carrier. Applying a magnetic field to one end of the carrier can be an attractive or repulsive force; Use a photoelectric detector to detect in real time the change in the signal intensity over time in the detection area before and after the magnetic field is turned on. This intensity reflects the probe or self-luminescence intensity bound by complex A and / or complex B, that is, the signal intensity is the signal intensity of the probe-labeled signal and / or self-luminescence signal passing through the detection area; Because different proportions of sample A bind to form complex B, it causes differences in the signal intensity in the detection area at the same time point. The regular differences generated by a series of gradients are calculated through formulas to obtain the affinity K of the binding force between sample A and sample B D , and the data indication obtained is called the equilibrium dissociation constant;

[0016] S7.1. Obtain the data of the signal intensity change over time of multiple mixtures B before the magnetic field is turned on. Use the signal intensity S of the multiple mixtures B detected in real time in S6 1 as the ordinate and the time T 1 as the abscissa to obtain the curve graph of the signal intensity S 1 and the time T 1 ;

[0017] S7.2. Obtain the data of the signal intensity change over time of multiple mixtures B after the magnetic field is turned on under the same magnetic field intensity. Use the signal intensity S of the multiple mixtures B detected in real time in S6 2 as the ordinate and the time T under the same intensity magnetic field 2 as the abscissa to obtain the curve graph of the signal intensity S 2 and the time T 2 ;

[0018] S7.3. Obtain the affinity fitting curve graph. Use the ratio S of the signal intensity within the set time range of multiple mixtures B after the magnetic field is turned on to the signal intensity within the set time range of multiple mixtures B before the magnetic field is turned on at the same set time point as the ordinate (i.e., normalization processing to eliminate systematic or operational errors), S = S 1 / S2 Using the respective concentrations (gradient concentration series) of Sample B in Mixture B as the abscissa, plot a graph to obtain the curve of signal intensity S versus concentration B C The abscissa value corresponding to the inflection point in this curve graph is the affinity K between Sample A and Sample B D ;

[0019] A further technical solution of the present invention is that: the range of the gradient concentration series is 5 - 50, preferably, the range of the gradient concentration series is 10 - 20.

[0020] A further technical solution of the present invention is that: the shape of the nanomagnetic beads is spherical, rod-shaped, fusiform, triangular, star-shaped, polygonal, and irregular shapes.

[0021] A further technical solution of the present invention is that: the diameter of the nanomagnetic beads is 1 nm - 10000 nm, preferably, the diameter of the nanomagnetic beads is 1 nm - 1000 nm, and most preferably, the diameter of the nanomagnetic beads is 2 nm - 100 nm.

[0022] A further technical solution of the present invention is that: the temperatures of Solution A, Solution B, Solution C, Mixture A, and Mixture B during the detection of the interaction process are 0°C - 100°C, preferably in the range of 4°C - 60°C, more preferably in the range of 15°C - 50°C, and most preferably in the range of 20°C - 45°C; during the detection process, the temperature is controlled constantly, and the preferred range of the temperature change amplitude is within ±0.5°C, and the most preferred range is within ±0.05°C.

[0023] A further technical solution of the present invention is that: the carrier is a hollow capillary, a porous plate or a centrifuge tube made of glass or plastic, a flat plate or a plate with grooves, and their combination forms.

[0024] A further technical solution of the present invention is that: the combination forms of Sample A and Sample B at least include any combination of the following types: antibody - antigen, nucleic acid - protein, protein - small molecule, protein - protein, nucleic acid - small molecule, virus particle - protein, virus particle - small molecule, carbohydrate - protein, carbohydrate - small molecule, carbohydrate - ion, liposome - protein, liposome - nucleic acid, liposome - small molecule, liposome - ion, organelle - ion, organelle - small molecule, organelle - nucleic acid, organelle - protein, enzyme - enzyme, enzyme - antagonist, polypeptide - protein, polypeptide - nucleic acid, polypeptide - small molecule, polypeptide - ion, nanoparticle - protein, nanoparticle - nucleic acid, nanoparticle - small molecule.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This method can be applied to the measurement of weakly to strongly interacting molecules at a constant temperature in solution. Common nanobeads can be coated with probes, have a wide range of biological groups, and are applicable to a wide range of molecular species. Using a microfluidic flow cell as a carrier, the sample consumption is extremely low. Changes in the molecular weight, conformation, or surface characteristics during molecular binding can easily cause differences in velocity, resulting in higher detection sensitivity. The generation and intensity of the magnetic field are controllable, so the detection time is shorter. High-throughput detection can be easily carried out using standard 96- or 384-well plates or multi-channel flow cells as carriers. Based on nanobeads, during the pretreatment experiment, the sample can be purified, and the purified sample can be directly used for interaction detection. After the interaction, the sample can be directly enriched and the reaction molecules can be collected. Since this method detects the change in migration velocity in the bound or unbound state of the sample, the amount of binding and the affinity of intermolecular interaction can be reflected. Therefore, the target molecules in the mixed sample can be labeled with characteristic markers (such as fluorescent probes) to distinguish them from other molecules, or the nanobeads with characteristic signals can specifically react and couple with the target molecules, thus avoiding the need to label the sample molecules. Due to the detection signal intensity, both the light flux and the signal intensity of the characteristic marker can be used as detection means, thus avoiding the disadvantages of traditional interaction methods, such as the need for molecular purification, mandatory fluorescent labeling, large molecular consumption, and heating the sample, which affects the binding accuracy. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0028] Figure 1 It is a graph showing the change of signal intensity with time after the magnetic field is turned on in Embodiment 1 of the present invention.

[0029] Figure 2 It is a curve fitting graph of the affinity in Embodiment 1 of the present invention.

[0030] Figure 3 It is a reference diagram of the working principle of the present invention.

[0031] Figure 4 It is a schematic diagram of the magnetic field generating device and the detection area of the present invention.

[0032] In the figure: 1. Photoelectric detector, 2. Carrier, 3. Light source, 4. Recorder, 5. Magnetic field generating device, 6. Detection area, 7. Magnetic field shielding device. Detailed Embodiments

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] As Figure 1 shown, a method for detecting intermolecular interactions based on micro-magnetic drive includes the following steps:

[0035] S1. Respectively set solution A uniformly dispersed with sample A, solution B uniformly dispersed with sample B, and solution C uniformly dispersed with nano-magnetic beads, where sample A can interact with sample B;

[0036] S2. Perform group coating treatment on the nano-magnetic beads in solution C, and the group can undergo a specific coupling reaction with sample A;

[0037] S3. Perform probe labeling and / or self-luminescence treatment on the nano-magnetic beads in solution C described in S2 and / or sample A in solution A, and the probe labeling and / or self-luminescence can be detected by a photoelectric detector;

[0038] S4. Mix and incubate solution C described in S2 and solution A described in S3 to form mixture A. The nano-magnetic beads coated with biological groups in mixture A and sample A undergo a specific coupling reaction to form complex A. Among them, there will be more nano-magnetic beads than those required for coupling. The excess nano-magnetic beads can be removed by filtration means, or not filtered. Mixture A is allowed to react fully and maintain uniform dispersion for a certain period of time;

[0039] S5. Dilute solution B described in S1 successively by a factor of 2 - 10 times, with the number of dilution gradients ranging from 5 - 50, preferably 10 - 20, to form multiple gradient concentration series of solutions B1, B2... Bn. Mix and incubate multiple gradient concentration series of solutions B with mixture A described in S4 one by one in equal volume to form multiple mixtures B. The concentration of mixture A in mixtures B is the same. In multiple mixtures B, part of complex A and part of sample B interact to form complex B. Mixtures B are allowed to react fully and maintain uniform dispersion for a certain period of time;

[0040] S6. Use multiple carriers to hold equal volumes of the multiple mixtures B. A detection area is provided inside the carrier. Apply a magnetic field to one end and / or other areas of the carrier. The magnetic field applied to one end of the carrier is an attractive force or a repulsive force. Use a photoelectric detector to detect in real time the change in the signal intensity of the detection area over time before and after the magnetic field is turned on. This intensity reflects the probe or self-luminescence intensity bound by complex A and / or complex B, that is, the signal intensity is the signal intensity of the probe-labeled signal and / or self-luminescence signal passing through the detection area. Because different proportions of sample A bind (to form complex B), it causes differences in the signal intensity of the detection area at the same time point. The regular differences generated by a series of gradients are obtained through formula calculation to get the affinity of the binding force between sample A and sample B, and the data indication obtained is called the equilibrium dissociation constant K D 。

[0041] S7.1. Obtain the data of the signal intensity change over time of multiple mixtures B before the magnetic field is turned on. Use the signal intensity S of the multiple mixtures B detected in real time in S6 1 as the ordinate, and use time T 1 as the abscissa to obtain the curve graph of signal intensity S 1 versus time T 1 ;

[0042] S7.2. Obtain the data of the signal intensity change over time of multiple mixtures B after the magnetic field is turned on under the same magnetic field intensity. Use the signal intensity S of the multiple mixtures B detected in real time in S6 2 as the ordinate and use the time T under the same intensity magnetic field 2 as the abscissa to obtain the curve graph of signal intensity S 2 versus time T 2 ;

[0043] S7.3. Obtain the affinity fitting curve graph. Use the ratio S of the signal intensity of multiple mixtures B within the set time range after the magnetic field is turned on to the signal intensity of multiple mixtures B within the set time range before the magnetic field is turned on at the same set time point as the ordinate (i.e., normalization processing to eliminate system or operation errors), S = S 1 / S 2 , use the respective concentrations (gradient concentration series) of sample B in mixture B as the abscissa, and plot a graph to obtain the curve graph of signal intensity S versus concentration B C . The abscissa value corresponding to the inflection point in this curve graph is the affinity K of sample A and sample B D ;

[0044] A further technical solution of the present invention also lies in that: the range of the gradient concentration series is 5 - 50, preferably, the range of the gradient concentration series is 10 - 20.

[0045] A further technical solution of the present invention also lies in that: the shape of the nano magnetic beads is spherical, rod-shaped, fusiform, triangular, star-shaped, polygonal and irregular shapes.

[0046] A further technical solution of the present invention also lies in that: the diameter of the nano magnetic beads is 1 nm to 10,000 nm, preferably, the diameter of the nano magnetic beads is 1 nm to 1,000 nm, and most preferably, the diameter of the nano magnetic beads is 5 nm to 100 nm.

[0047] A further technical solution of the present invention also lies in that: the temperatures of solution A, solution B, solution C, mixture A and mixture B during the detection of the interaction process are 0 °C to 110 °C, preferably in the range of 4 °C to 60 °C, more preferably in the range of 15 °C to 50 °C, and most preferably in the range of 20 °C to 45 °C; during the detection process, the temperature is controlled constantly, and the preferred range of the temperature change is within ±0.5 °C, and the most preferred range is within ±0.05 °C.

[0048] A further technical solution of the present invention also lies in that: the carrier is a hollow capillary, a porous plate or a centrifuge tube made of glass or plastic, a flat plate or a plate with grooves and their combination modes.

[0049] A further technical solution of the present invention also lies in that: the combination forms of sample A and sample B at least include any combination of the following types: antibody - antigen, nucleic acid - protein, protein - small molecule, protein - protein, nucleic acid - small molecule, virus particle - protein, virus particle - small molecule, carbohydrate - protein, carbohydrate - small molecule, carbohydrate - ion, liposome - protein, liposome - nucleic acid, liposome - small molecule, liposome - ion, organelle - ion, organelle - small molecule, organelle - nucleic acid, organelle - protein, enzyme - enzyme, enzyme - antagonist, polypeptide - protein, polypeptide - nucleic acid, polypeptide - small molecule, polypeptide - ion, nanoparticle - protein, nanoparticle - nucleic acid, nanoparticle - small molecule.

[0050] Example 1

[0051] Take 250 μl of a carboxylated (group-coated treatment) nano magnetic bead solution with a diameter of 100 nm (the particle density range is 5X10 12 / ml), and mixed with 250 μl of bovine serum albumin BSA solution (solution A) with a final concentration of 500 nM at a ratio of 1:1 and incubated for 10 - 15 minutes to obtain mixture A. 420 μl of a solution (solution B) with a concentration of 4000 μM was prepared by dissolving analytical pure tauroursodeoxycholic acid sodium with a molecular weight of approximately 520. The 4000 μM tauroursodeoxycholic acid sodium was diluted 2 times respectively to obtain 14 concentration gradients of 30 μl each with final concentrations of 4000 μM, 2000 μM, 1000 μM......0.4883 μM (i.e., B1 - B14). 30 μl of each of mixture A was mixed with multiple concentration gradients of tauroursodeoxycholic acid sodium solution (solution B1 - B14) at a ratio of 1:1 and incubated for 10 - 15 minutes to obtain mixture B. The above steps are preferably carried out in a PCR tube. The PCR tubes (mixture B1 - B14) carrying the mixture B samples were respectively detected for the fluorescence intensity at a wavelength of 340 ± 5 nm before and after the magnetic field was turned on (BSA protein emits fluorescence at approximately 340 nm when excited at approximately 280 nm). To avoid interference before and after the sample moves, the recording of mixture B starts 10 seconds after the detection begins, and a series of curves with the fluorescence intensity of mixture B1 - B14 as the ordinate and time as the abscissa are obtained ( Figure 1 ), the ratio of the average fluorescence intensity value of each 65S - 70S to the average fluorescence intensity value of the corresponding 10S - 15S (normalized) to obtain S1 - S14 values; taking the S1 - S14 values as the ordinate and the corresponding concentration of tauroursodeoxycholic acid sodium in the mixture B1 - B14 solutions as the abscissa, and fitting according to the algorithm, the affinity KD = 18.75 μM is obtained.

[0052] Note: Sample A = bovine serum albumin BSA, Sample B = tauroursodeoxycholic acid sodium.

[0053] As Figure 3-4 shown, the mixture B is placed in a carrier. Here, the carrier is selected as a PCR centrifuge tube, and a detection area is set in the area near one side wall above the centrifuge tube. A magnetic field shielding device and a magnetic field generating device are placed obliquely under the PCR tube. The magnetic field generating device uses a permanent magnet, and the magnetic field shielding device uses some magnetic shielding materials. The principle of the photodetector is as shown in the figure. The signal intensity of the detection area is detected through a series of lenses, objective lenses, mirrors, and gratings inside the light source and the photodetector, and the data of the signal intensity is obtained through a recorder; after the test is started, by adding / removing the magnetic field shielding device between the magnet and the sample, the magnetic field of the environment where the nanobeads are located is made to be absent / present, and the N or S pole of the permanent magnet faces the bead area.

[0054] Obtain the data of the signal intensity change of multiple mixture B over time before the magnetic field is turned on. Using the signal intensity S of multiple mixture B detected in real - time in S6 1 as the ordinate, and time T 1Using [ [ ID = 0 ] ] as the abscissa, the signal intensity S is obtained. 1 and time T 1 to obtain a curve graph;

[0055] Obtain the data of the signal intensity change with time of multiple mixtures B after the magnetic field is turned on at the same magnetic field intensity. Using the signal intensity S of multiple mixtures B detected in real time in S6 2 as the ordinate and the time T under the magnetic field of the same intensity 2 as the abscissa, the signal intensity S is obtained. 2 and time T 2 to obtain a curve graph;

[0056] Table 1 shows the data of the signal intensity change with time of multiple mixtures B before and after the magnetic field is turned on at the same magnetic field intensity:

[0057]

[0058] Table 1

[0059] Plot a curve graph for the data in Table 1. As Figure 1 shown, after the magnetic field is turned on for each mixture B, the detected signal intensity decreases with the extension of time. Because the nanomagnetic beads will move towards the magnetic poles in the magnetic field (magnetotaxis, note that when the magnetic beads are in the middle of the N and S poles, they cancel each other out, and the magnetic beads can move towards either the N or S pole, and the magnetic beads do not pick the magnetic poles), so the samples driven by the magnetic beads (the samples have fluorescence probe-labeled signals) move from the detection area to the outside of the detection area, so the signal intensity decreases with time; the combination causes changes in properties such as the size and conformation (i.e., the shape in the solution) of the molecules, and such changes change / or affect the moving speed.

[0060] Obtain an affinity fitting curve graph. Using the ratio S of the signal intensity within the set time range of multiple mixtures B after the magnetic field is turned on to the signal intensity within the set time range of multiple mixtures B before the magnetic field is turned on at the same set time point as the ordinate (i.e., normalization processing to eliminate system or operation errors), S = S 1 / S 2 , and using the respective concentrations (gradient concentration series) of sample B in mixture B as the abscissa, plot a graph to obtain the curve graph of signal intensity S and concentration B C . The abscissa value corresponding to the inflection point in this curve graph is the affinity K of sample A and sample B D ,

[0061] Table 2 shows the data of the ratio S1 - S14 of the signal intensity and the respective concentrations of sample B in mixture B.

[0062]

[0063] Table 2

[0064] Plot the graph of signal intensity S versus concentration B C , as shown in Figure 2 . According to the affinity fitting curve, the affinity K of sample A and sample B is obtained D . K D = 18.75 uM

[0065] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims involved

Claims

1. A method for detecting intermolecular interactions based on micro-magnetic drive, characterized in that, it comprises the following steps: S1. Respectively prepare solution A uniformly dispersed with sample A, solution B uniformly dispersed with sample B, and solution C uniformly dispersed with nano-magnetic beads, and the sample A can interact with the sample B; S2. Perform group coating treatment on the nano-magnetic beads in the solution C, and the group can undergo a specific coupling reaction with the sample A; S3. Perform probe labeling and / or self-luminescence treatment on the nano-magnetic beads in the solution C described in S2 and / or the sample A in the solution A, and the probe labeling and / or self-luminescence can be detected by a photoelectric detector; S4. Mix and incubate the solution C described in S2 and the solution A described in S3 to form a mixture A, and the group-coated nano-magnetic beads and the sample A in the mixture A undergo a specific coupling reaction to form complex A; S5. Dilute the solution B described in S1 in multiples in sequence to form multiple gradient concentration series of solutions B1, B2... Bn, and mix and incubate the multiple gradient concentration series of solutions B with the mixture A described in S4 one by one in equal volume to form multiple mixtures B. A part of complex A and a part of sample B in the multiple mixtures B interact to form complex B, and the mixtures B are kept for a certain time for sufficient reaction and uniform dispersion; S6. Use multiple carriers to hold equal volumes of the multiple mixtures B, a detection area is provided in the carrier, a magnetic field is applied to one end and / or other areas of the carrier, and the change of the signal intensity in the detection area before and after the magnetic field is turned on is detected in real time by a photoelectric detector; S7.

1. Obtain the data of the signal intensity change of multiple mixtures B over time before the magnetic field is turned on. Take the signal intensity S1 of the multiple mixtures B detected in real time in S6 as the ordinate and the time T1 as the abscissa to obtain a curve graph of the signal intensity S1 and the time T1; S7.

2. Obtain the data of the signal intensity change of multiple mixtures B over time under the same magnetic field intensity after the magnetic field is turned on. Take the signal intensity S2 of the multiple mixtures B detected in real time in S6 as the ordinate and the time T2 under the same intensity magnetic field as the abscissa to obtain a curve graph of the signal intensity S2 and the time T2; S7.

3. Obtain an affinity fitting curve graph. Take the ratio S of the signal intensity within the set time range of multiple mixtures B after the magnetic field is turned on to the signal intensity within the set time range of multiple mixtures B before the magnetic field is turned on at the same set time point as the ordinate, and the respective concentrations of sample B in the mixture B as the abscissa, and plot a curve graph of the signal intensity S and the concentration BC. The abscissa value corresponding to the inflection point in this curve graph is the affinity KD of sample A and sample B.

2. The method for detecting intermolecular interactions based on micro-magnetic drive according to claim 1, characterized in that: In S5, the range of the multiple is 2 - 10 times, and the range of the number of dilution gradients is 5 - 50.

3. The method for detecting intermolecular interactions based on micro-magnetic drive according to claim 2, characterized in that: The shapes of the nanomagnetic beads are spherical, rod-shaped, fusiform, triangular, star-shaped, polygonal, and irregular shapes.

4. A method for detecting intermolecular interactions based on micro-magnetic force drive according to claim 3, characterized in that: the diameter of the nanomagnetic beads is 1 nm to 10,000 nm.

5. A method for detecting intermolecular interactions based on micro-magnetic force drive according to claim 4, characterized in that: In S1-S7, the temperatures of solution A, solution B, solution C, mixture A, and mixture B are 0 to 100 °C, and the temperature is controlled to be constant during the detection process, and the temperature change range is within ±0.5 °C.

6. A method for detecting intermolecular interactions based on micro-magnetic force drive according to claim 5, characterized in that: The carrier is a hollow capillary, a porous plate or a centrifuge tube made of glass or plastic, a flat plate, or a plate with grooves, and their combination modes.

7. A method for detecting intermolecular interactions based on micro-magnetic force drive according to any one of claims 1-6, characterized in that: The combination forms of sample A and sample B at least include any combination of the following types: antibody-antigen, nucleic acid-protein, protein-small molecule, protein-protein, nucleic acid-small molecule, virus particle-protein, virus particle-small molecule, carbohydrate-protein, carbohydrate-small molecule, carbohydrate-ion, liposome-protein, liposome-nucleic acid, liposome-small molecule, liposome-ion, organelle-ion, organelle-small molecule, organelle-nucleic acid, organelle-protein, enzyme-enzyme, enzyme-antagonist, polypeptide-protein, polypeptide-nucleic acid, polypeptide-small molecule, polypeptide-ion, nanoparticle-protein, nanoparticle-nucleic acid, nanoparticle-small molecule.

8. A method for detecting intermolecular interactions based on micro-magnetic force drive according to claim 6, characterized in that: In S5, the range of the multiple is 2 times, the number range of dilution gradients is 10-20, the diameter of the nanomagnetic beads is 5 nm to 100 nm, the temperature is 20 °C to 45 °C, and the carrier is a PCR centrifuge tube.

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