Multistage full liquid phase immunoassay method based on direct current magnetization response of superparamagnetic nanoparticles

Abstract

The invention relates to a multistage full-liquid-phase immunoassay method based on direct current magnetization response of superparamagnetic nanoparticles, and provides a brand-new three-stage superparamagnetic sensitive detection structure, interference caused by non-specific factors such as whole blood can be eliminated through reaction of a first-stage reagent and a second-stage reagent, and full-liquid-phase superparamagnetic sensitive signals are amplified and detected through reaction of the second-stage reagent and the third-stage reagent. According to the technical effects, the traditional advantages of a full-liquid-phase superparamagnetic sensitive detection technology are reserved, interference caused by most of whole blood is eliminated, signal amplification is carried out through a biotin avidin system, the detection sensitivity is improved, the clinical application level can be achieved, and the method is suitable for popularization and application. As a brand-new immunodetection protein technology which is completely put forward in China, the method has extremely important industrial and social values.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to a multi-stage full liquid phase immunoassay method based on direct current magnetization response of superparamagnetic nanoparticles. Background Art

[0002] The existing magnetic sensitive detection technology that supports full liquid phase detection is the single-stage structure and two-stage structure developed by our team in the early stage. The single-stage structure is mainly used for the detection of multimeric proteins, such as C-reactive protein, or large-volume objects to be detected, such as fungi and cells. The single-stage detection structure mainly uses superparamagnetic nanoparticles modified with specific targeting proteins as detection reagents. Since the objects to be detected are multimeric proteins or large-volume objects to be detected, when the reagent undergoes a specific binding reaction with the object to be detected, the volume will increase tenfold or even more, and the Brownian relaxation time will increase, causing the magnetization response signal value of the reagent to decrease. The concentration of the object to be detected can be analyzed by detecting the change in the magnetization response signal before and after the reaction.

[0003] The two-stage structure is mainly used for the detection of monomeric proteins, such as cardiac troponin, or small molecules, such as exosomes. The two-stage detection structure mainly uses two superparamagnetic nanoparticles modified with different target proteins as detection reagents. These two target proteins can react with the target substance at the same time. The target positions are located at different positions of the target substance and will not interfere with each other. Two different detection steps can be selected for detection: First, reagent 1 is mixed with the object to be detected, and reagent 2 is added for sandwich reaction after sufficient reaction. By measuring the initial magnetization response signal before the addition of the antigen and the magnetization response signal after sufficient reaction with reagent 2, the signal change value is obtained, and then the concentration value of the object to be detected is analyzed. In this detection method, since reagent 1 and reagent 2 are added separately, the selection of reagent 2 requires the selection of microspheres without magnetic signals or magnetic spheres with a larger inner diameter, which can effectively reduce interference; second, before detection, reagent 1 and reagent 2 are mixed, and the object to be detected is added after mixing, and the reaction is sufficient. The concentration of the object to be detected can be analyzed by detecting the change value of the magnetization response signal before and after the addition of the object to be detected. In this way, both reagent 1 and reagent 2 can be selected to be superparamagnetic nanoparticles with magnetization response signals, and the materials of reagent 1 and reagent 2 described in "1" can also be the same.

[0004] The detection speed of both the single-stage detection structure and the two-stage detection structure can achieve 10 minutes of detection, but they are extremely susceptible to nonspecific interference in the blood. That is, the change value of the magnetic response signal after adding blood includes the signal change caused by nonspecific aggregation, and the change value of this change in different detection items is uncontrollable, so it is necessary to eliminate the influence of nonspecific aggregation to the greatest extent. Therefore, based on these two structures, a three-stage detection structure is proposed to eliminate nonspecific aggregation interference and improve detection accuracy. Summary of the invention

[0005] The technical scheme adopted by the present invention is as follows: the multi-level full liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles comprises a tertiary structure, and the present technology develops a brand-new immunoassay technology with a tertiary structure taking superparamagnetic nanoparticles as the core; the tertiary structure comprises reagent 1, which is a superparamagnetic nanoparticle with biological functionalization, and a specific antibody or protein is combined with the superparamagnetic nanoparticles by means of covalent bonds or non-covalent coupling, so as to react specifically with the detected object; reagent 2, which is a functionally modified specific antibody, which is another specific antibody that can react specifically with the detected object and is paired with the antibody in reagent 1, and is specially functionalized on the surface so that it can react specifically with reagent 3 with high intensity; and reagent 3, which is a biofunctionalized particle carrier modified with avidin, and the carrier itself can be a colloidal microsphere, a magnetic sphere or a protein polymer, etc., which is a material that can be uniformly suspended in a liquid phase, and the particle size of which is much larger than that of reagent 1, and the surface of which is modified with a substance that can react specifically with reagent 2 with high intensity.

[0006] The detection device, the full liquid phase immunoassay device can detect extremely low concentrations of molecules with an accuracy of up to 10 -19 mol level, far exceeding the semi-liquid phase equipment; and the device has strong anti-interference and easy operation, suitable for complex application environments such as vehicles and battlefields; the device is a pure magnetic signal measurement, hemolysis, jaundice and other blood color changes do not affect the detection accuracy, support whole blood, serum, cell lysis fluid and other types of samples.

[0007] The three-level structure described in the present invention can be modified according to the specific detection category, and a two-level structure detection can also be selected; the detection equipment is not limited to the detection device used in the present invention, and a branch home device can also be selected for detection.

[0008] The secondary and tertiary levels use high-affinity substances, avidin, and anti-avidin antibodies; high-affinity avidins are usually designed to bind specifically to target molecules. They are able to bind tightly to target molecules, while showing low or no binding ability to other non-specific molecules; this high selectivity makes them very useful in purifying and enriching target molecules; anti-avidin antibodies have high affinity and can bind tightly to avidin tags; this high affinity ensures high specificity and high sensitivity of the tagged protein, making it a powerful tool for detecting and locating target proteins.

[0009] Paired antibodies, proteins, and genes can also be used for gene testing, etc.; the detection range of this technology is not limited to antigen proteins, but can also be used for the detection of genes, enzymes, drugs, biological targeted small molecules (such as RGD peptides, folic acid, galactose, glucose, etc.), etc. The detection process only requires replacing the modified antibody with the detected substance to achieve detection, and the application prospects are broad.

[0010] Beneficial effects of the present invention: The new three-stage superparamagnetic sensitive detection structure proposed by the present invention can eliminate the interference caused by non-specific factors such as whole blood through the reaction of the first and second stage reagents, and amplify and detect the full liquid phase superparamagnetic sensitive signal through the reaction of the second and third stage reagents. In terms of technical effects, while retaining the traditional advantages of the full liquid phase superparamagnetic sensitive detection technology (completely separation-free, fast detection speed, and simple use), it eliminates the interference caused by most whole blood, and amplifies the signal through the biotin avidin system, improving the detection sensitivity, so that it can reach the level of clinical application. As a new immunodetection protein technology completely proposed by my country, it has extremely important industrial and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram of the process of combining to form agglomerates according to the present invention.

[0012] Figure 2 It is a block diagram of the detection system of the present invention.

[0013] Figure 3 It is a structural diagram of the experimental equipment of the present invention.

[0014] Figure 4 It is a flowchart of the operation process of the experimental equipment of the present invention.

[0015] Figure 5 It is a reaction flow chart of the immunoassay method of the present invention.

[0016] Figure 6 is a polymer diagram of the reaction of the present invention. DETAILED DESCRIPTION

[0017] Existing pure liquid phase immunoassay technology can usually only detect serum plasma or a small amount of whole blood. The main reason is that there are a large number of biological components in whole blood, such as cells, exosomes or other pathogens, which are easy to agglomerate with magnetic nanotracers due to positive and negative charges, hydrophobic effects, etc., which greatly affect the nonspecificity of the detection signal and form background noise interference; at the same time, homogeneous detection usually requires magnetic or colloidal particles to maintain stable dispersion in a liquid environment, and when specific antigen-antibody binding occurs, the force can cause the particles to aggregate, but the interaction force between different antigens and antibodies (usually expressed as a dissociation constant) is different, so the time required for specific aggregation and the final detection sensitivity are quite different, which also makes it difficult to further improve the sensitivity of pure liquid phase wash-free magnetic sensitive immunoassay.

[0018] The magnetic beads are mainly subjected to the following forces in the solution: electrostatic repulsion Fs, residual magnetic attraction Fm, hydrophobic binding force, hydrogen bonding force and antigen-antibody reaction force. There are two states in the reagent bottle. First, before the test reaction, there is no binding reaction force. The magnetic ball reagent must remain stable and evenly dispersed in the solution, so Fs>>Fm+Fb must be satisfied. If this condition is met, the magnetic beads can be evenly dispersed in the solution; second, when adding antigens for binding reaction tests, in order to better react to form large aggregates, the binding reaction force Fk must meet Fk>>Fs to achieve the requirement of binding to form aggregates.

[0019] For example, the secondary structure superparamagnetic magnetic sensitive immunoassay technology proposed by the team of Professor Keiji Enfuku of Kyushu University in Japan uses the secondary structure of reagent 1 and reagent 2 to detect antigen concentration. Reagent 1 is a superparamagnetic nanoparticle modified with a specific antibody, reagent 2 is a particle size much larger than reagent 1 and modified with another specific antibody that can be paired with the antibody of reagent 1, and the base material of reagent 2 is polystyrene microspheres. After adding the antigen, reagents 1 and 2 form a polymer through the antigen, causing the magnetization response signal of the superparamagnetic particles to change, and the concentration of the detected object is detected by the change of the magnetization response signal before and after the specific reaction. This detection method has a very fast detection speed, is easy to use, and is not affected by blood color, but it is greatly affected by the non-specific effects of blood, especially anions and cations or larger cells in whole blood. At the same time, the efficiency of the secondary structure reaction is affected by the affinity of antigen and antibody, and the detection sensitivity still needs to be further improved in clinical applications.

[0020] This multi-stage all-liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles is the first in the world to propose the use of a tertiary structure to achieve superparamagnetic sensitive protein immunoassay. Compared with the existing technology, its main feature is to remove the nonspecific effects of whole blood while performing specific reactions through primary and secondary reagents, and then achieve signal amplification and detection through the reaction between secondary and tertiary reagents.

[0021] The specific implementation plan is:

[0022] 1. Reagents

[0023] Reagent 1: Superparamagnetic nanoparticles with biological functionality, which are used to combine specific antibodies or proteins with superparamagnetic nanoparticles by covalent bonds or non-covalent coupling to react specifically with the object to be detected.

[0024] Reagent 2: Functionally modified specific antibody, another specific antibody that can react specifically with the object to be detected and is paired with the antibody in reagent 1, and a special functional modification is performed on its surface so that it can bind specifically with reagent 3 with high intensity. For example, in the embodiment of the present invention, biotin modification is performed on the surface of reagent 2 so that it can bind specifically with avidin on the surface of reagent 3 with high intensity, and the dissociation constant between biotin and avidin reaches *** (the dissociation constant (Kd) between biotin and avidin describes the equilibrium property of their binding and dissociation. Since the binding affinity between biotin and avidin is high, the dissociation constant is usually low. The dissociation constant between biotin and avidin depends on the specific biotin and avidin. Different biotin and avidin pairs may have different binding affinities and dissociation constants), which is one of the specific reactions with the strongest binding force currently known in nature.

[0025] Reagent 3: Biofunctionalized particle carrier. The carrier itself can be made of colloidal microspheres, magnetic spheres or protein polymers, etc., which are materials that can be evenly suspended in the liquid phase. Their particle size is much larger than that of reagent 1, and their surface is modified with substances that can undergo high-intensity specific binding with reagent 2. For example, in the example of this study, 3.3um polystyrene microspheres modified with avidin are used as the material of reagent 3.

[0026] Reagent buffer system: Reagent 1, reagent 2 and reagent 3 are pre-dissolved in reagent buffer. The reagent buffer can be selected from various commonly used buffer systems that will not interfere with the antigen-antibody reaction. The buffer may contain an appropriate amount of preservatives, surfactants, blocking agents and other necessary substances to improve the stability and shelf life of the reagents.

[0027] Reagent reaction system: When performing protein detection, first mix reagent 1 and reagent 2 evenly, add the liquid to be tested (which can be whole blood, serum, cell lysate, plasma, etc.), and incubate with reagent 1 and reagent 2. The incubation time is determined by the binding force, affinity and antigen-antibody detection time of the antigen and antibody, and can usually be set to 3 to 15 minutes. The liquid to be tested can react with reagent 1 first and then add reagent 2, or reagent 1 and reagent 2 can be pre-mixed and then directly added to the liquid to be tested. After reagent 1 and reagent 2 react, add reagent 3. Since the surface of reagent 3 is modified with a substance that can specifically bind to reagent 2 with high intensity, the mixture of reagent 1 and reagent 2 will quickly form a large volume polymer with reagent 3. Since reagent 1 contains superparamagnetic nano-magnetic cores affected by Brownian relaxation time, after it is affected by reagent 1 and reagent 2 to form a large aggregate, its superparamagnetic response will change significantly, thereby quickly and quantitatively detecting the substance in the liquid to be tested.

[0028] 2. Detection method

[0029] 1. Detection system:

[0030] The purpose of the invention is to provide a multi-stage full liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles. Different from the existing immunoassay method, the present invention eliminates the interference caused by non-specific factors such as whole blood through a three-stage superparamagnetic sensitive detection structure, and has a fast detection speed.

[0031] First, the magnetic marker is magnetized by a permanent magnet. When the external magnetic field is removed, a weak magnetism will remain on the magnetic marker, and the HMC2003 magnetoresistive sensor is used to detect the weak magnetic signal of the magnetic marker. This method can detect the concentration of the measured object with its simple and fast operation and detection characteristics without cleaning steps.

[0032] 2. Experimental equipment

[0033] (1) Motor control component: responsible for controlling the start, stop and switching of the motor operation mode. The motor controller of the device is a DC motor controller. 2. Control circuit board: mainly controls the start, stop and operation of the components in the device. 3. PAD: the pad and protective gear inside the device shell, mainly to protect the inner surface of the device shell from damage. 4. Lifting and swinging mechanism (lifting and swinging mechanism motor): the swinging motor controls the start, operation and stop of the swinging mechanism. The swinging mechanism lifts the pipette up and down and moves it left and right appropriately, so that the pipette can accurately transfer the test solution into the sample bottle. 5. Pipette: quantitative transfer of test reagents required. 6. 10-equal division mechanism (equal division mechanism motor): the equal division mechanism motor controls the start, operation and stop of the equal division mechanism. The 10-equal division mechanism is driven by the motor to rotate the workbench to divide the sample bottle into 10 equal parts, and at the same time realizes the positioning and braking of the workbench.

[0034] (2) Reagent reaction process:

[0035] like Figure 5 Shown is a reaction flow diagram for this immunoassay method, with the magnetic signal represented by the color of the bar below.

[0036] First, we added biofunctionalized superparamagnetic nanoparticles into the sample tube. Since the magnetic nanoparticles are superparamagnetic, they have no hysteresis and thus no magnetism. Therefore, when no magnetic field is applied, the sensor does not detect any magnetic signal, and the magnetic signal is zero at this time.

[0037] Next, we add blood (the liquid to be tested) to the sample tube. Since the blood contains antigens (other interfering substances), the antigens will target and bind to the magnetic nanoparticles to form magnetic particle polymers, and other interfering substances in the blood will not target and bind to the magnetic nanoparticles; then add reagent 2 (functionally modified specific antibodies). After a period of time, the specific antibodies will bind to the already targeted antigens, and continue to increase the magnetic particle polymers. After the reaction is completed, a magnetic field is applied to the polymers, and the magnetization response signal at this time is measured. The magnetization response signal of the reagent at this time is taken as the initial signal; continue to add reagent 3 (biofunctionalized particle carrier) with a surface modified with a substance that can specifically bind to reagent 2 with high intensity. After a period of time, the carrier will combine the magnetic nanoparticle polymers after the previous step of reaction, further increase the size of the magnetic nanoparticle polymers, and form a large volume polymer. At this time, a magnetic field is applied to the substances in the liquid to be tested. Since the superparamagnetic response of the nanoparticles will change significantly after a series of reactions, the magnetic signal at this time will be larger than the initial signal. By comparing the changes in the magnetization response signals before and after the reaction, and using data analysis methods such as the four-parameter fitting method, the concentration value of the object to be tested is obtained. Rapid quantitative detection of substances in the liquid being tested.

[0038] During the detection process, reagent 1 and reagent 2 are stored in a sample reaction solution in advance, and a liquid to be detected, such as whole blood, is added to allow detection reagents 1 and 2 to react specifically in the sample to be detected. Due to the action of antibodies, a sandwich structure of primary reagent-antigen-secondary reagent can be formed. At the same time, there are factors affecting nonspecific aggregation in whole blood, which also combine with reagent 1 and reagent 2. After reagent 1 and reagent 2 fully react with the solution to be detected, the magnetization response signal of the reagents after the reaction is used as the initial signal, and reagent 3 is added. Since reagent 3 does not show a nonlinear magnetization signal and has a large particle size, its nonspecific binding with whole blood will not interfere with the detection signal. At the same time, the avidin modified on reagent 3 and the biotin modified on reagent 2 have extremely strong affinity, which can make reagent 2 and the polymerized structure formed by reagent 2 and reagent 1 aggregate on the surface of reagent 3 in a very short time, obtain a specific superparamagnetic response change signal that is significantly different from the initial signal, and obtain the concentration value of the object to be detected through data analysis methods such as the four-parameter fitting method.

[0039] In addition, there are a few things to note:

[0040] 1. In order to achieve a sufficient and rapid reaction, the amount of reagent 1 must be greater than the amount of the object to be detected, the amount of reagent 2 must be greater than the amount of reagent 1, and the biotin in the secondary reagent must be modified in large quantities;

[0041] 2. Reagent 1 and reagent 2 can be directly mixed and stored, and the object to be detected can be directly put into them during the test. Alternatively, reagent 1 and the object to be detected can be mixed and reacted first, and then reagent 2 can be added for mixing and reaction;

[0042] 3. The amount of avidin modified on reagent 3 must be much greater than the amount of reagent 1 and reagent 2, and if magnetic balls are used as the material of reagent 3, the magnetic signal and residual magnetic characteristics need to be considered;

[0043] 4. The biotin modified on reagent 2 and the avidin used on reagent 3 can also be replaced by other substances with high affinity reaction.

Claims

1. A multi-stage all-liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles, characterized in that: Including a tertiary structure, this technology has developed a brand-new immunoassay technology with a tertiary structure based on superparamagnetic nanoparticles; the tertiary structure includes reagent 1 - superparamagnetic nanoparticles with biological functionality, which are combined with specific antibodies or proteins with superparamagnetic nanoparticles by covalent bonds or non-covalent coupling to react specifically with the object to be detected; reagent 2 - functionally modified specific antibody, which is another specific antibody that can react specifically with the object to be detected and is paired with the antibody in reagent 1, and has special functional modification on its surface so that it can have high-intensity specific binding with reagent 3; reagent 3 - a biofunctionalized particle carrier modified with avidin, the carrier itself can be made of colloidal microspheres, magnetic spheres or protein polymers, etc., which can be uniformly suspended in the liquid phase, and its particle size is much larger than reagent 1, and the surface is modified with a substance that can have high-intensity specific binding with reagent 2.

2. The multi-stage all-liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: The detection device, the full liquid phase immunoassay device can detect extremely low concentrations of molecules with an accuracy of up to 10 -19 mol level, far exceeding the semi-liquid phase equipment; and the device has strong anti-interference and easy operation, suitable for complex application environments such as vehicles and battlefields; the device is a pure magnetic signal measurement, hemolysis, jaundice and other blood color changes do not affect the detection accuracy, support whole blood, serum, cell lysis fluid and other types of samples.

3. The multi-stage all-liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: The three-level structure described can be modified according to the specific detection category, and a two-level structure detection can also be selected; the detection equipment is not limited to the detection device used in the present invention, and a branch home device can also be selected for detection.

4. The multi-stage all-liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: The secondary and tertiary levels use high-affinity substances, avidin, and anti-avidin antibodies; high-affinity avidins are usually designed to bind specifically to target molecules. They are able to bind tightly to target molecules, while showing low or no binding ability to other non-specific molecules; this high selectivity makes them very useful in purifying and enriching target molecules; anti-avidin antibodies have high affinity and can bind tightly to avidin tags; this high affinity ensures high specificity and high sensitivity of the tagged protein, making it a powerful tool for detecting and locating target proteins.

5. The multi-stage all-liquid phase immunoassay method based on the DC magnetization response of superparamagnetic nanoparticles according to claim 1, characterized in that: Paired antibodies, proteins, and genes can also be used for gene testing, etc. The detection range of this technology is not limited to antigen proteins, but can also be used for the detection of genes, enzymes, drugs, and biological targeted small molecules (such as RGD peptides, folic acid, galactose, glucose, etc.). The detection process only requires replacing the modified antibody with the substance to be detected to achieve detection.

Images