CN102735653B - Biological detection method by utilization of surface plasma resonance biosensor - Google Patents

Abstract

The invention provides a biological detection method using a surface plasmon resonance biosensor; the detection method comprises the following steps: 1) labeling the sample to be tested with a catalyst, the catalyst corresponding to the target nucleic acid or target protein to be identified; The labeled target nucleic acid or target protein is combined with the probe immobilized on the surface of the SPR; 3) the substrate corresponding to the catalyst is passed into the sample pool on the surface of the SPR, and the substrate corresponding to the catalyst is capable of interacting with the 4) Detecting changes in SPR characteristic parameters amplified by bubbles. The detection method of the invention has high specificity and sensitivity; it is suitable for rapid detection; and the chip can be regenerated and used.

Description

A biological detection method using a surface plasmon resonance biosensor

technical field

The invention relates to the technical field of surface plasmon resonance sensing, in particular, the invention relates to a biological detection method using a surface plasmon resonance biosensor.

Background technique

Surface plasmon resonance (Surface Plasmon Resonances, SPR) sensing technology is a biochemical optical sensing technology developed in the 1980s. The principle is based on the fact that p-polarized light is incident on the surface of the noble metal through a prism or grating at a specific wavelength and angle, and the evanescent wave formed resonates with the plasmonic vibration on the surface of the noble metal, resulting in the disappearance of the light wave. Because the conditions for surface plasmon resonance to occur are very sensitive to the refractive index of the noble metal surface, and biochemical effects can cause significant changes in the surface refractive index, SPR can be used for label-free detection of macromolecules and for the kinetics of intermolecular reactions. Constants are checked. Since the successful commercialization of SPR in the 1990s, SPR biosensors have developed rapidly and been widely used due to their outstanding label-free, rapid sensitivity, and real-time detection. At present, SPR biosensors have been applied to protein interaction, nucleic acid interaction, interaction between macromolecules and macromolecules, interaction between small molecules and macromolecules, food safety, pesticide residues, environmental safety and drug screening, rapid and sensitive infectious diseases detection and other fields.

Furthermore, people have developed Surface Plasmon Resonance Imaging (SPRI) technology on the basis of traditional SPR detection technology. SPRI technology is the real-time monitoring conversion of traditional SPR detection technology for changes in wavelength, angle or phase. For the real-time observation of the intensity of light, the traditional simultaneous detection of one or several substances can be transformed into a high-throughput detection technology that can be combined with biochip technology. SPRI technology has the advantages of label-free, rapid and real-time detection, and high throughput. It will have a wide range of applications for high-throughput detection of proteomics and genomic infectious diseases, drug screening, epitope profiling, and cancer research.

However, all current SPR biosensors, regardless of angle, wavelength, phase, or intensity detection methods, suffer from insufficient sensitivity for direct detection. In the SPR direct detection method, the signal obtained by the combination of the reactant is very small, and the non-specific effect of the sample without the reactant as a control will also produce a certain obvious signal, so even if the SPR resolution is very high, it cannot be distinguished. Concentrations of reactants combined. Therefore, most current SPR detection methods need to use various signal amplification methods to amplify the signal of reactant binding.

In the prior art, there are methods of using nanoparticles such as gold nanoparticles to amplify signals and using enzyme labels to amplify signals for generating immune substrates.

Among them, for the signal amplification method of nanoparticles, gold nanoparticles are taken as an example, and the SPR technology using immune reaction to detect antibodies is taken as an example to illustrate. First, the antigen is immobilized on the matrix where SPR occurs, and then it is incubated with the antibody solution, and the secondary antibody labeled with gold nanoparticles is used for detection. Due to the large mass and density of the gold nanoparticles, the change in the refractive index caused by only incubating the antibody is larger than that The change in the refractive index of the secondary antibody labeled with gold nanoparticles incubated with gold nanoparticles is about an order of magnitude smaller, and the plasma of gold nanoparticles can resonate with SPR under certain circumstances to increase the response of the signal, thereby achieving signal amplification. Its defect is that the amplification efficiency is still insufficient. Generally, the amplification efficiency can only reach 5-20 times. Also, chip regeneration is flawed due to the tendency of nanoparticles to aggregate after incubation. In addition, in the above signal amplification method, the non-specific signal caused by nanoparticles will be enhanced again, so this signal amplification method is not suitable for the detection of complex systems.

For the signal amplification method of directly using the enzyme label to generate the immune substrate, the SPR technology using the immune reaction to detect the antibody is taken as an example to illustrate. First, the antigen is immobilized on the matrix where SPR occurs, and then it is incubated with the antibody solution, and the enzyme-labeled secondary antibody is used for detection. Due to the large mass and density of the precipitate produced by the enzyme catalysis, the change in the refractive index caused by only incubating the antibody is larger than that The change in the refractive index of the precipitate catalyzed by incubation with the enzyme-labeled secondary antibody is about an order of magnitude smaller, thereby achieving signal amplification. However, due to the inability to regenerate the enzyme-catalyzed substrate, the use efficiency of the SPR chip is reduced.

In summary, the existing SPR signal amplification methods have problems such as insufficient amplification efficiency and inability to regenerate, which lead to low throughput and low sensitivity of SPR biological detection, and it is difficult to detect in real time. Therefore, it is urgent to provide a A high-throughput, high-sensitivity, real-time detection method for SPR.

Contents of the invention

The purpose of the present invention is to establish a high-throughput, high-sensitivity, real-time detection SPR detection method using gas catalyzed by catalysts such as nanoparticles or enzymes as a reporter molecule.

In order to achieve the above-mentioned purpose of the invention, the present invention provides a biological detection method utilizing a surface plasmon resonance biosensor, the surface plasmon resonance biosensor includes an SPR surface and a sample pool made on one side of the SPR surface; the detection The method includes the following steps:

1) labeling the sample to be tested with a catalyst corresponding to the target nucleic acid or target protein to be identified;

2) combining the labeled target nucleic acid or target protein with the probe immobilized on the surface of the SPR;

3) In the sample pool on the surface of the SPR, pass through the substrate corresponding to the catalyst, the substrate corresponding to the catalyst is a liquid that can combine with the catalyst and generate gas;

4) Detecting changes in SPR characteristic parameters of the SPR surface amplified by the bubbles, the SPR characteristic parameters include but not limited to SPR resonance angle, wavelength, intensity or phase. Qualitative or quantitative judgments can be made on the sample to be tested by detecting the change of SPR characteristic parameters. For example, it is judged whether the detected target nucleic acid or target protein exists in the test sample.

Wherein, in above-mentioned step 1), the method for catalyst labeling nucleic acid comprises:

1.1) Realize the labeling of nucleic acid through oligonucleotide linker;

1.2) or achieve the labeling of the product nucleic acid through primers;

1.3) or labeling directly to nucleic acids.

Wherein, the step of realizing the labeling method of nucleic acid through the oligonucleotide linker in the above-mentioned step 1.1) comprises:

1.1.1) preparing catalyst solution;

1.1.2) mixing the catalyst solution with the oligonucleotide in a ratio of 1-100:1, and reacting to obtain an oligonucleotide linker containing the catalyst;

1.1.3) Extract the DNA that needs to be labeled, and remove the phosphate group at the 5' end of the DNA by dephosphorylation to prevent its own connection;

1.1.4) Under the action of ligase, combine the catalyst-labeled oligonucleotide linker to obtain catalyst-labeled DNA.

Wherein, in the above-mentioned step 1.2) in the method for labeling nucleic acid by primers, the steps of using the PCR reaction primers for labeling the product DNA include:

1.2.1) preparing catalyst solution;

1.2.2) mixing the catalyst solution with the PCR primer in a ratio of 1-100:1, and reacting to obtain the PCR primer containing the catalyst;

1.2.3) Using the labeled primers to perform a PCR reaction to obtain catalyst-labeled DNA.

Wherein, in the above-mentioned step 1.2) in the method for labeling nucleic acid by primers, the steps of using primers for RT-PCR reaction to label the product DNA include:

1.2.1) preparing catalyst solution;

1.2.2) mixing the catalyst solution with the RT-PCR primers in a ratio of 1-100:1, and reacting to obtain RT-PCR primers containing the catalyst;

1.2.3) Use the labeled primers to carry out RT-PCR reaction to obtain catalyst-labeled DNA.

Wherein, the method for directly labeling the nucleic acid in the above step 1.3) is: by reacting with the terminal group of the nucleic acid, the terminal of the nucleic acid is modified with amino, carboxyl, sulfur-containing or imide-containing compounds, such compounds It can interact with the enzyme in the catalyst or nanoparticles, so as to realize the labeling of the catalyst.

Wherein, the method step of catalyst labeling protein in above-mentioned step 1.3) is:

1.3.1) preparing catalyst solution;

1.3.2) Mixing the catalyst solution with the reduced protein in a ratio of not less than 5:1, and reacting to obtain the protein containing the catalyst;

Wherein, the method step of combining the labeled nucleic acid with the probe immobilized on the SPR surface in the above step 2) is:

2.1) Denaturing the nucleic acid modified by the catalyst;

2.2) adding the DNA modified by the denatured catalyst to the surface of the SPR chip, and performing a hybridization reaction;

2.3) washing to remove unhybridized catalyst-labeled DNA;

2.4) Dried for later use.

Wherein, in above-mentioned step 2), the method step of binding the labeled protein to the probe immobilized on the SPR surface is:

2.1) PBS processing biochip;

2.2) adding the catalyst-modified protein to the surface of the SPR chip for binding reaction;

2.3) washing to remove unbound catalyst-labeled protein;

2.4) Dried for later use.

Wherein, in the above step 3), the reaction of the corresponding catalyst substrate and the labeled catalyst is used to generate gas, and the generated bubbles are used to amplify the signal of the resonance angle, wavelength and intensity of the SPR light. Its method steps are:

2.1) Add the substrate solution of the catalyst to the surface of the SPR chip, and incubate to generate bubbles;

2.2) Sampling and observing the position where the bubbles are generated.

Compared with the prior art, the present invention has the following technical effects:

1. The specificity and sensitivity of the present invention are very high.

2. The reaction speed of the present invention is very fast, which is very suitable for rapid detection.

3. Compared with the traditional method of generating precipitation, the chip of the present invention can be regenerated and used, which greatly improves the use efficiency of the chip.

Description of drawings

Fig. 1 shows the SPR detection system and measurement method schematic diagram thereof that one embodiment of the present invention uses;

Fig. 2 shows a schematic diagram of the measured SPR peak excursion in one embodiment of the present invention;

Fig. 3 shows the schematic diagram of the SPRI detection system used by another embodiment of the present invention;

Figure 4 shows the antigen-antibody binding curve and signal amplification curve measured in an embodiment of the present invention; wherein RU=10 ^-6 RIU, RIU is the International Refractive Index Unit;

Fig. 5 shows the antigen-antibody binding curve and signal amplification curve measured in another embodiment of the present invention.

Detailed ways

Hereinafter, the present invention will be described in detail with reference to the drawings and embodiments.

According to one embodiment of the present invention, a biological detection method using a surface plasmon resonance biosensor is provided.

The surface plasmon resonance biosensor is the SPR biosensor, which is a sensitive component of the SPR device for measurement. SPR devices include SPR devices based on angle detection, SPR devices based on wavelength detection, SPR devices based on intensity detection, SPR devices based on phase detection, and also include SPR devices based on microfluidic and automatic sample injection systems. It also includes the remaining SPR devices for detection based on SPR properties, and also includes local SPR devices, long-range SPR devices, waveguide mode SPR devices, and the like. All of the above-mentioned SPR devices can be called SPR biosensors. Generally speaking, an SPR biosensor includes an SPR implementation device for realizing SPR. A sample cell is arranged on the surface of the SPR to accommodate a liquid containing a biological sample, and the sample cell has a liquid inlet and an outlet for continuous flow of liquid. The SPR implementation device is used to introduce the incident light and detect the signal of the reflected light. When the SPR (surface plasmon resonance) condition is satisfied, the intensity of the reflected light decreases sharply. According to this property, the change of the detected reflected light can be obtained close to the SPR The change in the refractive index of the surface is used to determine whether the biological sample contains molecules that interact with the material on the surface.

The following example illustrates the above-mentioned SPR implementing device. FIG. 1 shows an SPR implementation device (ie, an SPR detection system) based on the Kretschmann structure of angle detection. As shown in Figure 1, the prism 3 is bonded to the glass side of a glass chip plated with precious metal on one side; a sealed flow cell is bonded to the side plated with precious metal, with openings at both ends of the flow cell and an external pump The valve carries the liquid through the sample cell, the area where the fluid is sampled. Among them, the noble metal plated on the glass chip forms the SPR surface 1, and the p-polarized light of different incident angles is incident on the prism 3, and part of the incident light at continuous angles is plasmon-coupled with the SPR surface 1 to form surface plasmon resonance in different degrees. The device 5 (which can use a charge-coupled element sensor) detects the change of the intensity of reflected light at different angles, and the relationship between the angle and intensity of reflected light can be obtained. By reading the SPR angle (the angle with the lowest reflected light intensity) in real time, the obtained For the refractive index change close to the SPR surface 1 (typically less than 200nm) on the side of the sample cell. When the refractive index close to the SPR surface 1 changes with time due to various reasons, real-time detection data can be obtained. When a reaction occurs between the biomolecules 4 and the substances on the surface, it will cause a change in the refractive index close to the SPR surface 1. Therefore, the real-time detection data of the detector 5 can be used to judge whether the biomolecules 4 and the substances on the surface react in turn. Reaction to obtain biological detection results. For example, when nucleic acid is bound, it will cause a change in the refractive index on the surface of the SPR, and the SPR resonance angle will change from the position of the thin line in Figure 1 to the position of the broad line, so that the resulting binding and dissociation reactions can be observed in real time. Furthermore, if the signal processing is performed on the resonance angle in real time, the kinetic curve of biomolecular binding can be obtained. Through the detection system, the change of the refractive index of the surface of the SPR metal layer caused by the combination and dissociation can be observed in real time, and then the change of the resonance angle or the resonance wavelength caused by it. The SPR detection system consists of two parts, one is the optical path system, and the other is the fluid sampling pool. In the optical path system, the incident light of different angles is incident on the gold-plated substrate (SPR chip) through the prism, and the SPR resonance of different situations occurs on the SPR chip, and the reflected light with a wide angle is incident on the charge-coupled device (CCD) through the prism, etc. The detector is connected to the computer for real-time recording and analysis of the resonance angle.

In this embodiment, the corresponding catalyst and substrate are selected so that when the nucleic acid or protein modified by the catalyst is captured by a substance that can react with it on the surface, the catalyst can catalyze the substrate and generate gas at the captured position. At this time, the SPR peak at this position (the position where the catalyst is trapped) is changed from the SPR peak of the aqueous solution to the SPR peak of the gas. The refractive indices of air and aqueous solution are 1.0 and 1.33, respectively, so the SPR peaks generated in the place where there are bubbles (that is, the place where gas is generated) and the place where the aqueous solution flows are obviously different. In Fig. 1, the solid line shows the SPR peak in the case of air, and the dotted line shows the SPR peak in the case of aqueous solution. According to the principle of SPR, since the position difference of the resonance angle between the SPR peak of the air and the SPR peak produced by the aqueous solution is more than 30 times that of the conventional detectable signal, the position of the resonance angle will be drastically reduced, so that the surface captures the catalyst label Small changes caused by nucleic acids or proteins that translate into gas bubbles can cause dramatic signal enhancements.

More specifically, in one embodiment, the biological detection method utilizing surface plasmon resonance biosensor comprises the following steps 1) to 4):

Step 1) label the sample to be tested with a catalyst.

The catalyst corresponds to the target nucleic acid or target protein to be identified. In a preferred embodiment, the tested biological sample can be nucleic acid or protein. Such nucleic acids include DNA, RNA, cDNA and peptide nucleic acids. The protein includes various antigens, antibodies and polypeptides. Catalysts for labeling nucleic acids or proteins include nanoparticles and enzymes. The nanoparticles are nanoparticles with catalytic properties with a diameter of 0.1-1000nm, examples of which include gold-platinum alloy\platinum shell nanoparticles, etc. Nanoparticles of molecules, peptides or proteins. In the present invention, gold-core platinum-shell nanoparticles of 100 nm may be preferred. The enzyme is an enzyme or ribozyme that can catalyze the generation of gas from a substrate, examples of which include catalase, etc., and also include peptides or mutants with catalase activity. Bovine liver catalase may be preferred in the present invention. It should be pointed out that the use of ELISA for detection (mainly referring to traditional horseradish peroxidase and alkaline phosphatase) is a routine detection method in the biological field, and the catalytic activity of nanoparticles is used instead of the role of enzymes for labeling It has also been reported a lot. For example, it was reported in Nature Nanotechnology magazine in 2008 that Fe ₃ O ₄ nanoparticles have peroxidase activity, which is similar to that of horseradish peroxidase, and the nanoparticles also have the advantage of stable storage, which can Makes a very good substitute for horseradish peroxidase.

Above-mentioned step 1) in, the method for catalyst labeling nucleic acid comprises:

1.1) Realize the labeling of nucleic acid through oligonucleotide linker;

1.2) or achieve the labeling of the product nucleic acid through primers;

1.3) or labeling directly to nucleic acids.

Furthermore, in a preferred embodiment, the steps in the above-mentioned 1.1) to implement the nucleic acid labeling method through the oligonucleotide linker include:

1.1.1) preparing catalyst solution;

1.1.2) mixing the catalyst solution with the oligonucleotide in a ratio of 1-100:1, and reacting to obtain an oligonucleotide linker containing the catalyst;

1.1.3) Extract the DNA that needs to be labeled, and remove the phosphate group at the 5' end of the DNA by dephosphorylation to prevent its own connection;

1.1.4) Under the action of ligase, combine the catalyst-labeled oligonucleotide linker to obtain catalyst-labeled DNA.

In a preferred embodiment, in the above-mentioned 1.2) in the method for labeling nucleic acids by primers, the steps of using the PCR reaction primers for labeling the product DNA include:

1.2.1) preparing catalyst solution;

1.2.2) mixing the catalyst solution with the PCR primer in a ratio of 1-100:1, and reacting to obtain the PCR primer containing the catalyst;

1.2.3) Using the labeled primers to perform a PCR reaction to obtain catalyst-labeled DNA.

In another preferred embodiment, in the above-mentioned 1.2) in the method for labeling nucleic acids by primers, the steps of using primers for RT-PCR reactions to label product DNA include:

1.2.1) preparing catalyst solution;

1.2.2) mixing the catalyst solution with the RT-PCR primers in a ratio of 1-100:1, and reacting to obtain RT-PCR primers containing the catalyst;

1.2.3) Use the labeled primers to carry out RT-PCR reaction to obtain catalyst-labeled DNA.

In a preferred embodiment, the method of directly labeling the nucleic acid in the above 1.3) is: by reacting with the terminal group of the nucleic acid, the end of the nucleic acid is modified with amino, carboxyl, sulfur-containing or imide-containing compounds, etc. , this type of compound can interact with the enzyme in the catalyst or nanoparticles, so as to realize the labeling of the catalyst.

In another preferred embodiment, the method step of catalyst labeling protein in above-mentioned 1.3) is:

1.3.1) preparing catalyst solution;

1.3.2) Mix the catalyst solution with the reduced protein in a ratio of not less than 5:1, and react to obtain the protein containing the catalyst.

The academic term "oligonucleotide linker" used in the present invention refers to a nucleic acid substance consisting of 1-2000 bases, including nucleic acid substances modified with groups such as sulfhydryl, amino, and acyl groups.

The definitions of the academic terms "primer", "PCR" and "RT-PCR" used in the present invention refer to: "PCR Technical Experiment Guide", C.W, Dieffenbach, G.S. Dewexler. Science Press, first edition in August 1998.

The academic term "product nucleic acid" used in the present invention refers to the nucleic acid material generated by PCR or RT-PCR reaction, such as DNA, cDNA.

Step 2) Binding the labeled target nucleic acid or target protein to the probe immobilized on the metal-coated side of the SPR surface.

In a preferred embodiment, the method for immobilizing probes on the surface includes immobilization methods based on physical adsorption, chemical covalent interaction, electrostatic adsorption, hydrogen bonding or other weak forces or any combination of the above forces. Further, in another preferred embodiment, the SPR surface includes surfaces of various metals and alloys, and surfaces of inorganic materials such as various glasses and quartz coated with various noble metals, and also includes surfaces coated with various noble metals. Surfaces of polymers such as polydimethylsiloxane and polystyrene. At the same time, it also includes rough surfaces coated with various precious metals, including surfaces coated with various precious metals of molecules that have undergone micro-nano treatment and modification, such as the surface of electrospun non-woven fabrics coated with various precious metals of different materials.

In a preferred embodiment, the method steps of combining the labeled nucleic acid with the probe immobilized on the surface of the SPR in the above step 2) are:

2.1) Denaturing the nucleic acid modified by the catalyst;

2.2) adding the DNA modified by the denatured catalyst to the surface of the SPR chip, and performing a hybridization reaction;

2.3) washing to remove unhybridized catalyst-labeled DNA;

2.4) Dried for later use.

In another preferred embodiment, the method steps of combining the labeled protein with the probe immobilized on the surface of the SPR in the above step 2) are:

2.1) PBS processing biochip;

2.2) adding the catalyst-modified protein to the surface of the SPR chip for binding reaction;

2.3) washing to remove unbound catalyst-labeled protein;

2.4) Dried for later use.

3) Pass the substrate corresponding to the catalyst into the sample cell on the surface of the SPR.

The substrate corresponding to the catalyst is a liquid capable of binding to the catalyst and generating a gas.

In a preferred embodiment, the substrate of the catalyst includes a reagent comprising a catalyst substrate, such as catalase substrate H ₂ O ₂ can be in pure water, in phosphate buffered saline (PBS), carbonate buffer, etc. Various concentrations of hydrogen peroxide are preferred in the present invention.

In a preferred embodiment, in step 3) above, the substrate of the corresponding catalyst reacts with the labeled catalyst to generate gas, and the generated bubbles are used to amplify the signal of the resonance angle, wavelength and intensity of the SPR light. Its method steps are:

3.1) Add the substrate solution of the catalyst to the surface of the SPR chip, and incubate to generate bubbles;

3.2) Sampling and observing the position where the bubbles are generated.

4) Detecting changes in the SPR characteristic parameters amplified by the bubbles, and judging whether the detected target nucleic acid or target protein exists in the sample to be tested. SPR characteristic parameters include but are not limited to SPR resonance angle, wavelength, intensity or phase.

When the target nucleic acid or target protein exists in the sample to be tested, the catalyst will catalyze the substrate and generate gas (that is, generate bubbles) at the position where it is captured, resulting in an SPR peak at the corresponding position on the SPR surface (the position where the catalyst is captured) The SPR peak of the aqueous solution is changed to the SPR peak of the gas, that is, the SPR peak is significantly shifted, so that when the SPR realization device detects that the SPR peak is significantly shifted, it can be conversely inferred that there is a target nucleic acid in the sample to be tested or target protein. The SPR implementation device can detect the shift of the SPR peak through the change of the SPR resonance angle, wavelength, intensity or phase, which is well known to those skilled in the art, so the more specific detection process will not be repeated here.

Next, through four more specific comparison experiments, the recognition of the target nucleic acid or target protein by the biological detection method using the surface plasmon resonance biosensor of the present invention will be demonstrated. Combined with the following comparative experiments, step 4) can be better understood.

Comparative experiment 1

In this experiment, the SPR resonance angle signal was amplified by modifying proteins with platinum nanoparticles. The experimental device used is shown in Figure 1, and its principle has been introduced above, so it will not be repeated here.

The specific steps of this comparative experiment are as follows:

1. Immobilize 1mg/ml human immunoglobulin G on the surface of the SPR chip, and immobilize 1mg/ml rabbit serum albumin on the reference tube as a reference signal.

2. Fix the chip on the SPR device, select the fixed position of human immunoglobulin G and rabbit serum albumin through the program to observe the resonance angle, and pass the surface into phosphate buffer for baseline scanning, as shown in Figure 4 baseline , where the dotted line represents rabbit serum albumin, and the solid line represents human immunoglobulin G.

3. Preparation of platinum nanoparticles solution.

The platinum nanoparticle solution is mixed with the reduced protein G at a ratio of 2:1, and the protein containing the platinum nanoparticle label is obtained through reaction.

4. Inject the protein labeled with platinum nanoparticles and incubate for 10 minutes, as shown in the binding curve in Figure 4.

5. Wash with phosphate buffer until the baseline is stable, as shown in the dissociation curve in Figure 4.

6. Pass through 1% H ₂ O ₂ solution, and observe the change of the resonance angle at the positions of human immunoglobulin G and rabbit serum albumin. From this, it can be obtained that there is basically no change in the resonance angle at the position of rabbit serum albumin, but there is a very large change in the resonance angle at the position of human immunoglobulin G. In this experiment, SPRI was carried out by modifying the protein with platinum nanoparticles. Amplification of the signal.

Comparative experiment 2

This experiment also uses platinum nanoparticles to modify proteins to amplify SPR resonance wavelength signals. The principle is the same as that of comparative experiment 1, and will not be repeated here.

The specific steps of this experiment are as follows:

1. Immobilize 1mg/ml human immunoglobulin G on the surface of the SPR chip, and immobilize 1mg/ml rabbit serum albumin on the reference tube as a reference signal.

2. Fix the chip on the SPR device, select the fixed position of human immunoglobulin G and rabbit serum albumin through the program to observe the resonance wavelength, and pass the surface into the phosphate buffer for baseline scanning, as shown in Figure 4 baseline , where the dotted line represents rabbit serum albumin, and the solid line represents human immunoglobulin G.

3. Preparation of platinum nanoparticles solution;

The platinum nanoparticle solution is mixed with the reduced protein G in a ratio of 2:1, and the protein containing the platinum nanoparticle label is obtained by reaction;

4. Inject the protein labeled with platinum nanoparticles and incubate for 10 minutes. The binding curve is shown in FIG. 4 .

5. Wash with phosphate buffer until the baseline is stable, as shown in the dissociation curve in Figure 4.

6. Pass through 1% H ₂ O ₂ solution, and observe the change of the resonance angle at the positions of human immunoglobulin G and rabbit serum albumin. From this, it can be obtained that there is basically no change in resonance wavelength at the position of rabbit serum albumin, but there is a very large change in resonance wavelength at the position of human immunoglobulin G. In this experiment, SPRI was carried out by modifying protein with platinum nanoparticles. Amplification of the signal.

Comparative experiment 3

In this experiment, the SPRI signal was amplified by modifying nucleic acid with platinum nanoparticles. The principle is as follows:

The SPRI device is shown in Figure 3. Through this device, the change of the light intensity caused by the change of the refractive index on the surface of the gold sheet can be observed. The SPRI device includes an optical path part and a fluid sampling pool 37 .

The light path part uses a light emitting diode (LED) light source or a laser as the original light source 31, and the light beam provided by the original light source 31 passes through the collimating lens 32 and the optical filter 33 to generate monochromatic light, and enters the gold-plated substrate (SPR chip 36) through the prism 34 On the SPR chip 36, different situations of SPR resonance occur at different positions, and the reflected light of different intensities is incident on the charge-coupled device 35 (CCD) through the prism, and then connected to the computer for recording and processing the intensity image.

As shown in Figure 2, the refractive indices of air and aqueous solution are 1.0 and 1.33, respectively, so the SPR peaks generated at the place where there are air bubbles and where the aqueous solution flows are obviously different. The solid line shows the SPR peak in the case of air, and the dashed line shows the SPR peak in the case of aqueous solution. According to the principle of SPRI, when detecting the change of surface refraction, the incident angle position shown by the vertical dotted line is selected to observe the intensity change. Under normal circumstances, since the SPRI device is used to observe the change of the refractive index caused by the aqueous solution on the surface of the gold sheet, the selected observation position is the intersection of the horizontal dotted line and the vertical dotted line in the diagram. When the catalyst-modified nucleic acid or protein is captured by a substance that can react with it on the surface, it will catalyze the substrate to generate gas, which is generated at the captured position. Therefore, the SPR peak at this position changes from aqueous solution to gas In the case of the SPR peak, since the position of the observation is unchanged, the intensity value of the reflected light at this time is transformed to be close to 100%, thus, the slight change caused by the nucleic acid or protein tagged with the catalyst captured on the surface is transformed into the generation of air bubbles The situation can cause great signal enhancement.

The specific steps of this experiment are as follows:

1. Immobilize 1 μM 40-base DNA sequence-1 and sequence-2 on the surface of the SPRI chip, sequence-1 and sequence-2 are sequences that are not identical and have no matching relationship.

2. Fix the chip on the SPRI device, select the fixed positions of DNA sequence-1 and sequence-2 through the program to observe the intensity, and pass the surface into phosphate buffer for baseline scanning, as shown in Figure 5 baseline.

3. Preparation of platinum nanoparticles solution;

Mix it with the PCR primer of the complementary sequence of DNA sequence-1, the ratio is 1:1, and react to obtain the PCR primer containing platinum nanoparticles;

The labeled primers are used to carry out PCR reaction to obtain the complementary sequence of the platinum nanoparticle-labeled DNA sequence-1.

4. Pass in the complementary sequence of the platinum-modified DNA sequence-1 and incubate for 10 minutes, as shown in the binding curve in FIG. 5 .

5. Wash with phosphate buffer until the baseline is stable, as shown in the dissociation curve in Figure 5.

6. Pass through 1% H ₂ O ₂ solution, and observe the change of the intensity at the position of sequence-1 and sequence-2. It can be obtained that there is basically no intensity change at the position of sequence-2, but there is a very large intensity change at the position of sequence-1. Therefore, this experiment uses platinum nanoparticles to modify nucleic acids to amplify the SPRI signal. In Fig. 5, the dotted line represents sequence-2, and the solid line represents sequence-1.

Comparative experiment 4

In this experiment, the protein was modified by catalase to amplify the SPRI signal. The principle is the same as that of Comparative Experiment 3, and will not be repeated here.

The specific steps of this experiment are as follows:

1. Immobilize 1 mg/ml human immunoglobulin G on the surface of the SPRI chip, and use 1 mg/ml rabbit serum albumin as a reference signal.

2. Fix the chip on the SPRI device, select the fixed positions of human immunoglobulin G and rabbit serum albumin through the program to observe the intensity, and pass the surface into the phosphate buffer for baseline scanning, as shown in Figure 5 baseline.

3. Prepare 10mg/ml catalase solution;

Mix the 10 mg/ml catalase solution with the reduced protein G at a ratio of 2:1, and react to obtain a protein labeled with catalase;

4. Add catalase-labeled protein and incubate for 10 minutes, as shown in the binding curve in Figure 5.

5. Wash with phosphate buffer until the baseline is stable, as shown in the dissociation curve in Figure 5.

6. Pass through 1% H ₂ O ₂ solution, and observe the changes in the intensity of the positions of human immunoglobulin G and rabbit serum albumin. It can be obtained that there is basically no change in intensity at the position of rabbit serum albumin, but there is a very large intensity change at the position of human immunoglobulin G. Therefore, this experiment uses catalase to modify the protein to perform SPRI signal zoom in. In Fig. 5, the dotted line represents rabbit serum albumin, and the solid line represents human immunoglobulin G.

The above-mentioned embodiment is only an exemplary description of the present invention, and those skilled in the art can easily understand that the method of generating bubbles can be used to amplify the resonance angle, wavelength and intensity signal of the SPR light, as well as the signal of the SPR light. Resonant phase signal.

In the present invention, the signal amplification method of SPR by generating bubbles through the catalyst makes the SPR biological detection method not only have the advantages of label-free and real-time testing, but also have high sensitivity, good specificity, and rapid detection. It can be applied to rapid and accurate detection of infectious diseases, high-throughput screening of protein-protein interactions, interactions between drugs and macromolecules, and other fields. Due to the simple conditions achieved by the method and the high sensitivity of the generated bubbles, it is also very suitable for rapid testing in ordinary laboratories and in the field. It will produce better economic and social benefits.

Finally, the above-mentioned embodiments are only used to illustrate the present invention, and it should not be construed as any limitation to the protection scope of the present invention. Moreover, those skilled in the art can understand that without departing from the spirit and principle of the above-mentioned embodiments, various equivalent changes, modifications and various improvements not described in the above-mentioned embodiments are protected by this patent. within range.

Claims (9)

1. A biological detection method utilizing a surface plasmon resonance biosensor, comprising the following steps: 1) labeling the sample to be tested with a catalyst corresponding to the target nucleic acid or target protein to be identified; 2) Combine the sample to be tested labeled with the corresponding catalyst with the probe immobilized on the surface of SPR; 3) Passing a substrate corresponding to the catalyst into the sample cell on the surface of the SPR, and the substrate corresponding to the catalyst is a liquid capable of combining with the catalyst and generating gas; 4) Detecting changes in SPR characteristic parameters of the SPR surface amplified by air bubbles. 2. The biological detection method according to claim 1, characterized in that, in the above step 1), the sample to be tested is nucleic acid, and the method for catalyst-labeled nucleic acid comprises: 1.1) Labeling of nucleic acids through oligonucleotide adapters; 1.2) Or label the product nucleic acid through primers; 1.3) or labeling directly to nucleic acids. 3. The biological detection method according to claim 2, characterized in that the step of realizing the nucleic acid labeling method through the oligonucleotide linker in the above step 1.1) comprises: 1.1.1) Prepare catalyst solution; 1.1.2) Mix the catalyst solution with the oligonucleotide at a ratio of 1-100:1, and react to obtain the oligonucleotide linker containing the catalyst; 1.1.3) Extract the DNA that needs to be labeled, and remove the phosphate group at the 5' end of the DNA by dephosphorylation to prevent its own connection; 1.1.4) Under the action of ligase, combine the catalyst-labeled oligonucleotide linker to obtain catalyst-labeled DNA. 4. The biological detection method according to claim 2, characterized in that, in the above-mentioned step 1.2) in the method for marking nucleic acid by primers, the steps of using primers for PCR to mark the product DNA include: 1.2.1) Preparation of catalyst solution; 1.2.2) Mix the catalyst solution with the PCR primer at a ratio of 1-100:1, and react to obtain the PCR primer containing the catalyst; 1.2.3) Use the labeled primers to carry out PCR reaction to obtain catalyst-labeled DNA. 5. The biological detection method according to claim 2, characterized in that, in the above-mentioned step 1.2) in the method for labeling nucleic acid by primers, the step of using primers for RT-PCR reaction to label the product DNA includes: : 1.2.1) Preparation of catalyst solution; 1.2.2) Mix the catalyst solution with the RT-PCR primer at a ratio of 1-100:1, and react to obtain the RT-PCR primer containing the catalyst; 1.2.3) Use the labeled primers to perform RT-PCR reaction to obtain catalyst-labeled DNA. 6. The biological detection method according to claim 2, characterized in that, in the above step 1.3), the method of directly labeling the nucleic acid is: by reacting with the terminal group of the nucleic acid, the terminal of the nucleic acid is modified with an amino group, a carboxyl group, Compounds containing sulfur or imides can interact with enzymes or nanoparticles in the catalyst to achieve labeling of the catalyst. 7. The biological detection method according to claim 2, characterized in that the step of combining the labeled nucleic acid with the probe immobilized on the surface of the SPR in the above step 2) is: 2.1) Denaturing the nucleic acid modified by the catalyst; 2.2) Add the denatured catalyst-modified DNA to the surface of the SPR chip for hybridization reaction; 2.3) Wash to remove unhybridized catalyst-labeled DNA; 2.4) Dry for later use. 8. The biological detection method according to claim 2, characterized in that, in the above-mentioned step 1.3), the sample to be tested is protein, and the method steps of catalyst labeling protein are: 1.3.1) Preparation of catalyst solution; 1.3.2) Mix the catalyst solution with the reduced protein at a ratio of not less than 5:1, and react to obtain the protein containing the catalyst. 9. The biological detection method according to claim 8, characterized in that, the step of combining the labeled protein with the probe immobilized on the surface of the SPR in the above step 2) is as follows: 2.1) PBS processing biochip; 2.2) Add the catalyst-modified protein to the surface of the SPR chip for binding reaction; 2.3) Wash to remove unbound catalyst-labeled protein; 2.4) Dry for later use.