CN103954775B - A kind of method detecting biomacromolecule or microbial body - Google Patents

Abstract

The invention relates to a method for detecting biological macromolecules or microorganisms. The method comprises the steps of: using biomacromolecule or microbial antibody-coated monodisperse superparamagnetic nano-magnetic beads to contact with the sample to be detected for incubation reaction to generate multi-particle superparamagnetic nano-magnetic bead clusters; Aggregates are obtained through magnetic separation, and the concentration of biological macromolecules or microorganisms in the sample to be detected is determined according to the degree of aggregation of the aggregates. The method of the present invention integrates immunomagnetic enrichment and visual detection, is simple to operate, takes a short time for the entire detection process, does not require special equipment, and the method of the present invention does not require high purity of antibodies, which can greatly reduce Testing costs.

Description

A method for detecting biological macromolecules or microorganisms

technical field

The invention relates to the technical field of immunoassay analysis, in particular to a fast, sensitive and low-cost method for visually detecting biological macromolecules or microorganisms.

Background technique

Rapid, sensitive, and low-cost detection and diagnosis of infectious animal and plant pathogenic bacteria, viruses, and proteins in clinical samples are of great significance to the protection of human health and life safety, the protection of the environment and national security, and the maintenance of social stability . At present, relatively mature detection methods mainly include methods such as isolation and culture detection, immunological detection and molecular biology. The separation and culture method is simple and easy to implement, but due to its low detection sensitivity and time-consuming, it cannot meet the on-site and rapid requirements. Immunological methods mainly include ELISA and immunochromatographic test strips. ELISA has the advantages of simplicity, high sensitivity, and low cost. It is suitable for large-scale sample screening, but it requires multi-step washing. It is time-consuming and labor-intensive, so its use is also subject to certain restrictions. Colloidal gold immunochromatographic test strips have the advantages of quickness, simplicity, and low cost, but their sensitivity is relatively low and quantitative detection cannot be achieved. Molecular biology methods are mainly PCR-based analysis methods, which have the advantages of high detection sensitivity and good specificity, but require expensive instruments and highly skilled personnel to operate, and their application is limited to a certain extent. Therefore, it is of great significance to construct an analytical method suitable for on-site, rapid, highly sensitive and personalized diagnosis to realize the detection of pathogenic bacteria, viruses and proteins in environmental, food and biological samples.

Superparamagnetic nano-magnetic beads have excellent magnetic and optical properties such as strong saturation magnetism, good monodispersity, controllable particle size and multiple colors, so the immunomagnetic separation technology based on superparamagnetic nano-magnetic beads has a high specificity of immune response It is the most widely used separation and enrichment technology due to its fast, simple and efficient advantages of magnetic separation. In recent years, it has been widely used in biological macromolecules or microorganisms such as pathogenic bacteria, viruses, proteins, and tumor cells. separation and enrichment. In the immunomagnetic enrichment reaction, the recognition of the immunomagnetic beads and the target is carried out in a suspension solution, which is a homogeneous immune reaction mode, which is conducive to the full reaction of the antibody-antigen and can speed up the reaction. Therefore, It can be combined with many other technologies to construct a series of new analysis methods, such as magnetic separation-fluorescence immunoassay, magnetic separation-chemiluminescence analysis, enrichment-microfluidic chip technology, magnetic separation-surface plasmon resonance biosensor , Magnetic enrichment-quartz crystal microbalance biosensor and other methods. Due to the development of immunomagnetic separation technology, the development of related analysis technology has been greatly promoted, and its application potential is constantly being explored.

Contents of the invention

Aiming at the deficiencies of the existing technology, we conducted an in-depth study on the detection of immunomagnetic beads, and found that the aggregation state of multi-particle superparamagnetic nanomagnetic bead clusters formed by immune reaction is different from that of monodisperse superparamagnetic nanomagnetic beads in a magnetic field, and The degree of aggregation of the immunosupercisive nanomagnetic beads is positively correlated with the content of the target in the sample.

Therefore, the object of the present invention is to provide a kind of fast, sensitive and low-cost method for visually detecting biomacromolecules or microorganisms, in the described method, immunosuperparamagnetic nano-magnetic beads are both used as magnetic separation and enrichment carriers; It has color, and multi-particle superparamagnetic nanomagnetic bead clusters are different from monodisperse superparamagnetic nanomagnetic beads in the magnetic field. It can be used as a visual signal reporting mark, and the magnetic separation, enrichment and detection in the immune reaction It is completed in one step to realize the rapid and sensitive detection of the target in the sample.

For realizing the purpose of the present invention, the present invention provides following technical scheme:

A method for detecting biomacromolecules or microorganisms, comprising the following steps: using monodisperse superparamagnetic nano-magnetic beads coated with antibodies of biomacromolecules or microorganisms to contact with a sample to be detected for incubation and reaction to generate multi-particle superparamagnetic Nano-magnetic bead clusters; magnetic separation is performed after the reaction to obtain aggregates, and the concentration of biomacromolecules or microorganisms in the sample to be detected is determined according to the degree of aggregation of the aggregates.

Antibodies of corresponding biomacromolecules or microorganisms are coupled on the surface of superparamagnetic nano-magnetic beads to prepare immune superparamagnetic nano-magnetic beads. In the immune response, biological macromolecules or microorganisms have multiple antigenic determinants, and through the specific recognition between antibodies and antigens, multiple monodisperse immune superparamagnetic nanomagnetic beads can be connected. The monodisperse immune superparamagnetic nano-magnetic beads become clustered multi-particle superparamagnetic nano-magnetic bead clusters through the "bridge" connection of the antigen. Then under the action of an external magnetic field, it becomes an aggregated multi-particle superparametric nanomagnetic bead cluster (aggregation), due to the dispersion of the monodisperse superparamagnetic nanomagnetic beads and the aggregated multiparticle superparamagnetic nanomagnetic beads in the magnetic field Different states lead to different degrees of aggregation, and the degree of aggregation of monodisperse immunosuperparamagnetic nano-magnetic beads is positively correlated with the content of target objects (biological macromolecules or microorganisms) in the sample. Based on this, the degree of aggregation of aggregates can be Determine the concentration of biomacromolecules or microorganisms in the sample to be detected, so as to construct a visual analysis method for detecting biomacromolecules or microorganisms.

As a preferred solution of the present invention, the aggregation degree of the aggregate is positively correlated with the concentration of the biomacromolecule or microorganism. The higher the concentration of the biomacromolecule or microorganism, the darker the color of the aggregate; therefore, standard samples can be used in advance to construct a curve relationship between the concentration of the biomacromolecule or microorganism and the degree of aggregation of the aggregate According to the graph, the concentration of biomacromolecules or microorganisms corresponding to a specific color depth can be determined, and the quantitative determination of the concentration of biomacromolecules or microorganisms in the sample can be realized.

As a preferred solution of the present invention, the biomacromolecule includes protein, nucleic acid and polysaccharide, preferably protein. Since proteins, nucleic acids and polysaccharides may have corresponding epitopes for the binding of corresponding antibodies to realize the generation of multi-particle superparamagnetic nano-magnetic bead clusters, they can all be used as the detection objects of biological macromolecules in the present invention.

Preferably, said proteins include lipoproteins, glycoproteins, nucleoproteins and biomarkers.

As a preferred solution of the present invention, the microorganisms include bacteria, fungi and viruses. Since the proteins on the surface of bacteria, fungi and viruses have antigenic epitopes, which can be combined with corresponding antibodies to realize the generation of multi-particle superparamagnetic nano-magnetic bead clusters, they can all be used as the detection objects of microorganisms in the present invention. The aforementioned microorganisms may be pathogenic bacteria or non-pathogenic bacteria, but the present invention excludes the case of disease diagnosis.

As a preferred solution of the present invention, the sample to be detected is a sample of environmental, biological or food origin. The samples from the environment can be, for example, samples from polluted water bodies, which contain biological macromolecules or microorganisms to be detected. By using the method of the present invention, these substances can be quantified to realize the assessment of the pollution status. Samples derived from food can be, for example, samples derived from artificially processed cooked food or packaged food, which contain biomacromolecules or microorganisms to be detected. By using the method of the present invention, these substances are quantified to achieve food safety assessment.

As a preferred solution of the present invention, the antibody is a monoclonal antibody and/or a polyclonal antibody, preferably a polyclonal antibody. Since there may be multiple antigenic determinants on the surface of biological macromolecules or microorganisms in the sample to be detected, the use of monoclonal antibodies can also achieve "bridge" connection in the case of the same or similar antigenic determinants; polyclonal antibodies have The binding sites of various antigenic determinants can easily realize the role of "bridge" connection. Undoubtedly, the present invention can also use a mixture of monoclonal antibodies and polyclonal antibodies, and the present invention does not require high purity of monoclonal antibodies, even if it contains impurities, the impact on the present invention can be ignored.

As a preferred solution of the present invention, the biomacromolecule or microorganism has multiple antigenic determinants. Multiple antigenic determinants of the same biomacromolecule or microorganism are combined with antibodies on the surface of different immune superparamagnetic nano-magnetic beads to realize the "bridge" connection and generate superparamagnetic nano-magnetic bead clusters.

As a preferred version of the present invention, the incubation reaction time is 5-60min, such as 5min, 6min, 10min, 12min, 15min, 20min, 25min, 30min, 40min, 45min, 50min, 55min, 58min, 10-40min, 10- 30min, 5-20min, 15-30min, preferably 15min.

As a preferred solution of the present invention, the magnetic separation time is 0.5-5 min, such as 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 4.5 min, 4.8 min, preferably 1 min.

As a preferred solution of the present invention, the concentration of biological macromolecules or microorganisms in the sample to be detected is determined according to the degree of aggregation of the aggregates, specifically:

A photo of the aggregate is obtained, and the concentration of the biomacromolecule or microorganism in the sample to be detected is determined by quantifying the gray value of the photo.

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

The method for detecting biological macromolecules or microorganisms of the present invention is based on the excellent magnetic and optical properties of superparamagnetic nano-magnetic beads, integrates immunomagnetic enrichment and visual detection, is simple to operate, and takes a short time for the entire detection process. No special equipment is needed, only one type of antibody is needed, which avoids the need for two types of antibodies (capture antibody and detection antibody) in traditional immunoassays, and the method of the present invention does not require high purity of antibodies, which can greatly reduce the detection time. cost. In addition, the method of the present invention has universal applicability, can detect bacteria, fungi, viruses, proteins, etc., is suitable for on-site detection, and has great application prospects.

Description of drawings

Fig. 1 is a schematic diagram of the reaction principle of the detection of biological macromolecules or microorganisms by the visualization method based on the aggregation of immunosupercisive nano-magnetic beads of the present invention. Among them, (1) represents the immune reaction between monodisperse immunosupercisive nano magnetic beads and biomacromolecules or microorganisms in the sample; Magnetic bead cluster; (3) the immunosuperparamagnetic nanomagnetic bead cluster that expresses multiparticle is under the effect of applied magnetic field, forms the immunosuperparamagnetic nanomagnetic bead aggregate of aggregation; Target (biological macromolecule or microbial body ) more, the greater the degree of aggregation of the immunosupercisive nano-magnetic beads, and the darker its color.

Fig. 2 is a mechanism diagram of the magnetic aggregation of the immunosuperparamagnetic nano magnetic beads of the present invention. Among them, (a) shows that the immune superparamagnetic nano-magnetic beads are mixed with the sample, and after reacting for 15 minutes, there is no state diagram under an external magnetic field; The state diagram under the magnetic field; (c) represents that the external magnetic field is removed, the immunosuperparamagnetic nano-magnetic beads in the aggregated state are resuspended, and then placed on a test tube rack without an external magnetic field, and the immune superparamagnetic nano-magnetic beads are under gravity A state diagram for a single action. Numbers indicate the concentration (cfu/mL) of biomacromolecules or microorganisms.

Fig. 3 is a graph showing the results of detection of Escherichia coli by the method of the present invention. Among them, (a) represents the state diagram of immune superparamagnetic nano-magnetic beads mixed with Escherichia coli sample after the reaction under a magnetic field; The greater the degree of aggregation, the darker its color; (b) represents the gray value of the photo of the aggregate and the concentration gradient of E. coli in the sample (1-6 in the figure respectively represent the concentration of E. coli 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 0cfu/mL); the higher the concentration of Escherichia coli, the larger the gray value.

Fig. 4 is a graph showing the results of detection of fetal oncoprotein (CEA) by the method of the present invention. Wherein, (a) represents the immunosuperparamagnetic nano-magnetic beads mixed with the CEA sample, after the reaction, the state diagram under the magnetic field; The bigger it is, the darker its color is; (b) shows the relationship between the gray value of aggregate photos and the concentration of CEA in the sample (ng/mL); the higher the concentration of CEA, the larger the gray value.

Fig. 5 is a graph showing the results of detection of alpha-fetoprotein (AFP) by the method of the present invention. Among them, 1-6 respectively represent the state diagram under the magnetic field after the samples with AFP concentration of 40, 20, 10, 5, 2.5 and 0ng/mL are mixed with the immunosuperparamagnetic nano-magnetic beads; the concentration of AFP in the sample The higher the (ng/mL), the greater the degree of aggregation of the immunosuperparamagnetic nano-magnetic beads, and the darker its color.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only preferred examples of the present invention, so as to better understand the present invention, and thus should not be considered as limiting the scope of the present invention.

The experimental methods in the following examples, unless otherwise specified, are conventional methods; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

The schematic diagram of the reaction principle of the present invention is as shown in Figure 1, (1) represents the immune reaction between the monodisperse immune super-parallel nano-magnetic beads and the biological macromolecules or microorganisms in the sample; (2) represents the "bridge" through the antigen The multi-particle immunosuperparamagnetic nano-magnetic bead cluster formed by the interaction; (3) means that the multi-particle immunosuperparamagnetic nano-magnetic bead cluster forms an aggregated immunosuperparamagnetic nano-magnetic bead aggregate under the action of an external magnetic field; the sample The more targets (biological macromolecules or microorganisms) in the medium, the greater the aggregation degree of the immunosuperparamagnetic nano-magnetic beads, and the darker the color.

The sources of reagents, instruments and equipment used in the following examples are as follows: Escherichia coli was purchased from American Culture Collection Center, Escherichia coli antibody (polyclonal antibody) was purchased from Genway, USA; alpha-fetoprotein, fetal oncoprotein and related antibodies were purchased from Beijing Rejing Biotechnology Co., Ltd.; the magnetic separation frame was purchased from Shanghai Aorun Micro-Nano New Material Co., Ltd.; the eddy current oscillator was purchased from IKA, Germany.

Example 1

The preparation of immunosuperparamagnetic nano-magnetic beads and the visual immunodetection of magnetic aggregation, the specific steps are as follows:

(1) Preparation of immunosupercisive nano-magnetic beads:

Disperse a certain amount of superparamagnetic nano-magnetic beads (0.2-0.5mg) modified with carboxyl groups on the surface in a certain volume of MES buffer solution (pH=5.6, 0.01M), and then add a certain amount of carbodiimide (EDC, 0.01-0.1mg) and N-hydroxysuccinimide (NHS, 0.01-0.1mg), gently shake the reaction on a vortex shaker for 0.5h, then magnetically separate, and resuspend with PBS buffer at pH=7.4 , then add a certain amount of antibody (0.1-0.5mg), react for 2h, then add 5% BSA (100-500μL) for 0.5h, finally magnetically separate, remove the supernatant, then add a certain amount of PBST washing solution to resuspend The superparamagnetic nano-magnetic beads were then magnetically separated, the supernatant was removed, and the wash was repeated 3 times. The finally obtained immune superparamagnetic nano-magnetic beads were resuspended in PBS buffer and stored in a refrigerator at 4°C.

(2) Magnetic aggregation visualization immunoassay steps:

Mix a certain amount of immunosupercisive nanomagnetic beads with a series of concentrations of biomacromolecules (such as proteins) or microorganisms (such as bacteria, fungi or viruses), react on a shaker for 10-20min, and then put the The test tube of the immunosuperparamagnetic nano-magnetic beads is placed on the magnetic separation rack, and the aggregation state of the immuno-superparamagnetic nano-magnetic beads is observed after 1 min.

The results after the above steps are shown in Figure 2, (a) shows that the immunosuperparamagnetic nano-magnetic beads are mixed with the sample, and after reacting for 15 minutes, there is no state diagram under an external magnetic field; (b) shows that the immunosuperparamagnetic nano-magnetic beads and The samples are mixed together and reacted for 15 minutes, the state diagram under the magnetic field; (c) shows that the external magnetic field is removed, the immunosuperparamagnetic nano-magnetic beads in the aggregated state are resuspended, and then placed on the test tube rack without the external magnetic field. The state diagram of the above-mentioned immunosuperparamagnetic nano-magnetic beads under the action of gravity alone. It can be seen that the more target substances (such as Escherichia coli) in the sample, the greater the degree of aggregation of the immunosupercisive nano-magnetic beads, and the darker their color; after resuspension, the greater the sedimentation speed and degree under the action of gravity alone.

Example 2

To detect Escherichia coli in water samples, the specific experimental steps are as follows:

(1) Mix a series of different concentrations of Escherichia coli (10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ and 0 cfu/mL) each in 900 μL and a certain amount of superparamagnetic nano-magnetic beads (100 μL), Shake the reaction on a vortex shaker for 15 minutes;

(2) Place the above-mentioned reacted immune mixture on a magnetic separation rack, and magnetically separate for 1 min;

(3) Observing the state of the superparamagnetic nano-magnetic beads with naked eyes, it can be seen that the higher the concentration of Escherichia coli (cfu/mL) in the sample, the greater the aggregation degree of the immune superparamagnetic nano-magnetic beads, and the darker its color (Figure 3a); Take photos with a camera, and use relevant graphics processing software to quantify the gray value. The results show that the higher the concentration of E. coli, the larger the gray value (Fig. 3b).

Example 3

To detect Newcastle disease virus in chicken embryo sac fluid, the specific experimental steps are as follows:

(1) Mix 900 μL each of a series of different concentrations of Newcastle disease virus (10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ and 0 copy/mL) and a certain amount of superparamagnetic nano-magnetic beads (100 μL), Shake the reaction on a vortex shaker for 15 minutes;

(2) Place the above-mentioned reacted immune mixture on a magnetic separation rack, and magnetically separate for 1 min;

(3) Observe the state of the superparamagnetic nano-magnetic beads with naked eyes, take pictures with a camera, and quantify its gray value with relevant graphics processing software. The result is similar to Figure 3.

Example 4

Detect the content of fetal cancer protein (CEA) in the urine, the specific experimental steps are as follows:

(1) Mix a series of different concentrations of CEA (20, 10, 5, 2, 1, 0 ng/mL) each 900 μL and a certain amount of superparamagnetic nano-magnetic beads (100 μL), and shake the reaction on a vortex shaker 15min;

(2) Place the above-mentioned reacted immune mixture on a magnetic separation rack, and magnetically separate for 1 min;

(3) Observing the state of the superparamagnetic nano-magnetic beads with the naked eye, it can be seen that the higher the concentration of CEA (ng/mL) in the sample, the greater the aggregation degree of the immune superparamagnetic nano-magnetic beads, and the darker the color (Fig. 4a); The camera took pictures, and the gray value was quantified with relevant graphics processing software. The results showed that the higher the concentration of CEA, the larger the gray value (Fig. 4b).

Example 5

To detect the content of alpha-fetoprotein (AFP) in urine, the specific experimental steps are as follows:

(1) Mix a series of different concentrations of AFP (40, 20, 10, 5, 2.5, and 0 ng/mL) with 900 μL each and a certain amount of superparamagnetic nano-magnetic beads (100 μL), and shake the reaction on a vortex shaker 15min;

(2) Place the above-mentioned reacted immune mixture on a magnetic separation rack, and magnetically separate for 1 min;

(3) Observe the state of the superparamagnetic nano-magnetic beads with the naked eye. The higher the AFP concentration (ng/mL) in the sample, the greater the aggregation degree of the immuno-superparamagnetic nano-magnetic beads, and the darker the color (Figure 5). Take pictures with a camera, and use related graphics processing software to quantify the gray value, which shows that the gray value is positively correlated with the AFP concentration.

The applicant states that the present invention illustrates the detailed features and detailed methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed features and detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed features and detailed methods. implement. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of selected components of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (14)

1. one kind is detected the method for biomacromolecule or microbial body, comprise the steps: that using the super suitable nanometer magnetic bead of single dispersing of the antibody bag quilt of biomacromolecule or microbial body to contact with detected sample carries out incubation reaction, generates granose super suitable nanometer magnetic bead cluster; Carry out Magneto separate 0.5-5min after reaction and obtain aggregation, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation.

2. method according to claim 1, is characterized in that, the aggregation extent of described aggregation and the concentration of described biomacromolecule or microbial body are proportionate.

3. method according to claim 1 and 2, is characterized in that, described biomacromolecule is protein, nucleic acid or polysaccharide.

4. method according to claim 3, is characterized in that, described biomacromolecule is protein.

5. method according to claim 4, is characterized in that, described protein is lipoprotein, glycoprotein, nucleoprotein or biomarker.

6. method according to claim 1, is characterized in that, described microbial body is bacterium, fungi or virus.

7. method according to claim 1, is characterized in that, described detected sample is the sample of environment, biology or food sources.

8. method according to claim 1, is characterized in that, described antibody is monoclonal antibody and/or polyclonal antibody.

9. method according to claim 8, is characterized in that, described antibody is polyclonal antibody.

10. method according to claim 1, is characterized in that, described biomacromolecule or microbial body have multiple antigenic determinant.

11. methods according to claim 1, is characterized in that, the described incubation reaction time is 5-60min.

12. methods according to claim 11, is characterized in that, the described incubation reaction time is 15min.

13. methods according to claim 1, is characterized in that, the time of described Magneto separate is 1min.

14. methods according to claim 1, is characterized in that, according to the concentration of biomacromolecule or microbial body in the aggregation extent determination detected sample of described aggregation, are specially:

Obtain the photo of described aggregation, quantitatively determined the concentration of biomacromolecule or microbial body in detected sample by comparison film gray-scale value.