CN115825419A - FRET (fluorescence resonance energy transfer) immunological probe as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a FRET (fluorescence resonance energy transfer) immune probe as well as a preparation method and application thereof, belongs to the technical field of analysis and detection, and solves the problem of how to improve the detection sensitivity and selectivity. The probe is prepared from protein G-coated carboxyl magnetic beads and an antigen-antibody coupling compound marked by fluorescein and a quencher. The preparation method comprises the following steps: activating the carboxylic acid groups with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; after being coated by protein G, goat anti-human immunoglobulin G marked by a fluorescence quenching agent is added, and after incubation reaction at room temperature, magnetic separation is carried out to obtain a nano magnetic bead compound loaded with a fluorescence quenching agent marked antibody; and mixing with human immunoglobulin G labeled by fluorescein diacetate, performing coupling reaction at 37 ℃, and performing magnetic separation to obtain the final product. The probe of the invention is applied to the detection of human immunoglobulin G. The probe of the invention is convenient to prepare, does not relate to a complex chemical process, and has high specificity and sensitivity to a detection target object.

Description

A kind of FRET immune probe and its preparation method and application

technical field

The invention belongs to the technical field of analysis and detection, and in particular relates to a FRET immune probe and its preparation method and application.

Background technique

Immunoglobulin G (IgG) is the antibody with the highest content in human serum. It is produced by B lymphocytes in the immune response and accounts for 70% to 80% of the immunoglobulin in serum. It has the characteristics of antibacterial, antiviral and antitoxin. Play an important role in the body's immune defense. Since IgG is a specific clinical indicator for the diagnosis of chronic infection, chronic liver disease, and immune disease, the determination of IgG content in human serum can provide a basis for the early diagnosis of many diseases.

Taking advantage of the exceptional affinity and specificity of biological recognition, including antibody-antigen, receptor-protein, and DNA-protein interactions, a series of sensitive and highly selective biosensors have been developed. At present, the main method for detecting IgG is enzyme-linked immunoassay (ELISA). However, the labeled enzyme needs special storage, and the sensitivity and accuracy need to be further improved. The fluorescent immunosensor is based on the immune reaction of antigen-antibody binding, and uses the fluorescence of the substance as the output signal to achieve quantitative detection. The system has both the specificity of the immune response and the high sensitivity of the fluorescence response. Among the many fluorescence detection systems, fluorescence resonance energy transfer (FRET) has attracted much attention, which is extremely sensitive to the separation distance between the donor and the acceptor, where the luminescent donor interacts through non-radiative dipole-dipole Transferring energy to a fluorescent or non-fluorescent acceptor, FRET occurs between two fluorophores when a bioaffinity reaction brings them into proximity. However, these methods often require expensive instruments, complex structure design and preparation processes, and skilled operators, and the detection sensitivity and selectivity also need to be improved, which greatly limits the practical application of these methods.

Contents of the invention

The purpose of the present invention is to provide a FRET immune probe to solve the problem of how to improve detection sensitivity and selectivity.

Another object of the present invention is to provide a preparation method of FRET immunoprobe.

The third object of the present invention is to provide an application of FRET immune probe.

The technical solution of the present invention is: a FRET (fluorescence resonance energy transfer) immunoprobe, which is prepared from carboxyl magnetic beads coated with protein G and antigen-antibody coupling complexes labeled with fluorescein and quencher. The FRET immunoprobe uses carboxyl magnetic beads as the carrier material, and uses protein G as the connecting protein for the binding of the antibody to the carboxyl magnetic beads. hatched.

Further, the carboxyl magnetic beads are polyacrylic acid (PAA) modified magnetic beads, and the fluorescent quencher-labeled antibody is 4-[4-(dimethylamino)phenylazo]benzoic acid-labeled goat anti-human immunoglobulin G ( DABCYL-anti-IgG), the fluorescein-labeled antigen is fluorescein diacetate-labeled human immunoglobulin G (FAM-IgG).

Further, the magnetic beads are composed of ferric oxide nanoclusters.

A preparation method of FRET immune probe, comprising the following steps:

A. Carboxyl magnetic beads were prepared by microwave-assisted polyol method, and carboxylic acid groups were activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC);

B. After the carboxyl magnetic beads activated in step A are coated with protein G, goat anti-human immunoglobulin G (DABCYL-anti-IgG) labeled with a fluorescent quencher is added, incubated at room temperature and then magnetically separated to obtain A nano-magnetic bead complex loaded with a fluorescent quencher-labeled antibody;

C. Mix the nano-magnetic bead complex obtained in step B with fluorescein diacetate-labeled human immunoglobulin G (FAM-IgG), conduct a coupling reaction at 37°C and then magnetically separate, and the obtained magnetic beads are complexed The object is the FRET immunoprobe.

Further, in step A, the concentration of carboxyl magnetic beads is 5-10 mg/mL.

Further, in step B, the concentration of the fluorescent quencher-labeled goat anti-human immunoglobulin G solution is 0.5-1 mg/mL,

Further, in step C, the antigen-antibody coupling reaction time is 1-3 h.

Application of a FRET immunoprobe in the detection of human immunoglobulin G (IgG).

A kind of FRET immune probe is detecting the application of human immunoglobulin G, comprising the following steps:

(1) Add the FRET immunoprobe (Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG) into the PBS buffer solution, and measure its initial fluorescence with a fluorescence spectrometer;

(2) Divide the FRET immunoprobe into multiple equal parts, add different concentrations of human immunoglobulin G, and incubate at 37 °C for a period of time to react;

(3) The multiple reaction solutions treated in step (2) were separated by magnetics, the supernatants were poured into the sample pools, and the spectra were recorded with a fluorescence spectrometer, thereby quantitatively determining human immunoglobulin G.

Further, in step (1), the pH value of the PBS buffer solution is 6-8, the concentration of the FRET immunoprobe is 5-8 mg/mL, and the reaction time is 1-3 h.

Due to the unique structure and properties of nanomaterials, new nanoprobes constructed by coupling them with biomolecules can greatly improve the performance of biosensors. Biocompatible magnetic beads can effectively couple biomolecules on their surface, providing a convenient method for the detection of biomolecules. Protein G is a cell wall protein isolated from type G Streptococcus. It can reversibly bind to various IgGs by binding to IgG Fc fragments (Fragment crystallization crystallization fragments), especially for human, rabbit and mouse IgG. affinity. Since protein G can recognize the Fc end of IgG, it will mediate the immobilization of an ordered Fc site-specific antibody on magnetic beads, resulting in targeted Fab (Fagment antigen-binding antigen-binding fragment) expression. Combining carboxylated magnetic beads with protein G is used for smart immobilization of antibodies, so that all antibodies are presented in the same direction on the magnetic beads, and the active Fab part is oriented to the optimal configuration for antigen binding, which will improve the targeting efficiency of antibodies .

Fluorescence resonance energy transfer (FRET) occurs when fluorescein-labeled human IgG is conjugated to fluorescence quencher-labeled goat anti-human IgG, and the fluorescence of fluorescein-labeled human IgG is quenched. When the target detection substance IgG is added, IgG and fluorescein-labeled human IgG will undergo a competitive immune reaction, and fluorescein will detach from the probe surface into the solution, and the fluorescence signal of the supernatant of the reaction system will increase after magnetic separation; with the addition of IgG As the amount increases, the immune competition reaction continues to occur, and the fluorescent signal continues to increase. IgG is proportional to the fluorescent signal response. Finally, the detection of IgG concentration can be realized by detecting the fluorescence intensity of the supernatant. The immunoprobe has the advantages of simple preparation, no complex chemical process involved, high specificity and sensitivity to target objects, etc., and can detect human immunoglobulin G sensitively.

The present invention connects protein G to Fe ₃ O ₄ magnetic nanoclusters modified by polyacrylic acid (PAA), thereby directionally connecting antibodies on its surface, and utilizes the distance-dependent optical properties of the fluorophore/quencher pair to design separately labeled Antigen-antibody coupling complexes of fluorophores and quenchers were used to prepare FRET fluorescent/magnetic nano-immunoprobes with simple structures, and to establish a new sensitive and rapid detection of IgG by using the probes to compete with the target IgG immunoreaction. method. The carboxyl group in PAA can be connected to the amino group of protein G, which is equivalent to building a "molecular bridge" between the interface of protein G and magnetic nanoclusters, which improves the connection efficiency and strength; the carboxylated magnetic nanoclusters and protein The composite material of G is used for the intelligent immobilization of antibodies, so that all antibodies are presented in the same direction on the nanoclusters, and the active Fab part is oriented to the optimal configuration for antigen binding, which greatly improves the targeting efficiency of antibodies; small Nano-clusters with large size can amplify the signal, and the above preparation characteristics can improve the detection sensitivity. In addition, antigen-antibody conjugation, competing immunoreactions, and magnetic separation manipulations can enhance selectivity.

Compared with the prior art, the present invention has the following advantages:

1. The FRET immunoprobe (Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG) of the present invention can be magnetically manipulated and separated magnetically, is easy to prepare, does not involve complex chemical processes, and is highly effective for detecting target substances Has high specificity and sensitivity.

2. The structure design of the FRET immunoprobe (Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG) of the present invention is ingenious. The magnetic beads in the probe are composed of nano-clusters with strong magnetic response and active sites There are many points; the carboxyl group in PAA can be connected to the amino group of protein G, which is equivalent to building a "molecular bridge" between protein G and the interface of magnetic nanoclusters, which improves the connection efficiency and strength; protein G can be oriented to connect antibodies, improving Antigen load.

3. The FRET immune probe of the present invention can design other nano-composite immune probes by changing the corresponding antigen and corresponding antibody, which has certain versatility.

4. The preparation method of the FRET immunoprobe of the present invention is simple, does not require high requirements for operators, can be magnetically manipulated, and does not involve complicated chemical processes.

5. By using the FRET immunoprobe of the present invention, the method of competitive immune reaction and magnetic separation detection supernatant can be organically combined, and human immunoglobulin G can be detected efficiently and sensitively. The detection method of the present invention has the advantages of simple operation, high sensitivity, and strong anti-interference ability. The linear range for detecting human immunoglobulin G is 0.067 nM to 146 nM, and the detection limit is 0.022 nM. The detection method of the invention opens up a new path for fluorescence immunoassay, and has good popularization and application value in the fields of biological science, pharmaceutical research, clinical diagnosis and the like.

Description of drawings

Fig. 1 is the preparation flowchart of FRET immune probe of the present invention;

Fig. 2 is the schematic diagram of detection of human immunoglobulin G by FRET immunoprobe of the present invention;

Fig. 3 is the X-ray diffraction pattern of carboxyl magnetic beads in the first embodiment of the present invention;

Fig. 4 is the infrared spectrogram of carboxyl magnetic beads in the first embodiment of the present invention;

Figure 5 is a Zeta potential diagram of the composite immune probe in the first embodiment of the present invention;

Fig. 6 is a comparison diagram of fluorescence microscopy before and after fluorescence resonance energy transfer of the composite immune probe in the first embodiment of the present invention;

Fig. 7 is a fluorescent spectral response diagram of immunocompetitive reaction between IgG and FAM-IgG in the seventh embodiment of the present invention;

Fig. 8 is a graph showing changes in the fluorescence spectrum of the detection system after adding different concentrations of human immunoglobulin G in the eighth embodiment of the present invention;

Fig. 9 is the linear curve of the human immunoglobulin G response of the detection system after adding different concentrations of human immunoglobulin G in the eighth embodiment of the present invention;

Figure 10 is the anti-interference diagram of human immunoglobulin G.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Embodiment 1,

A kind of preparation method of FRET immune probe, as shown in Figure 1, comprises the following steps:

A. Take 500μL of 10mg/mL carboxyl magnetic bead dispersion into a 5mL centrifuge tube for magnetic separation, wash with 0.05M 2-morpholineethanesulfonic acid (MES) buffer (pH=6.0, 3×500μL) for 3 times, Add 500 μL of 10 mg/mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and incubate at room temperature for 50 min to activate the carboxylic acid group;

B. Add 200 μL of 1 mg/mL protein G to the activated carboxyl magnetic beads, incubate at room temperature for 3 hours, discard unlinked protein G by magnetic separation, wash with MES buffer, and redisperse in 500 μL PBS containing 1.0 % BSA Incubate at room temperature for 2 hours to eliminate the risk of non-specific binding, and magnetically separate to remove excess BSA;

After washing, 500 μL of 0.16 mg/mL fluorescent quencher-labeled goat anti-human immunoglobulin G (DABCYL-anti-IgG) was added, and incubated at room temperature for 24 hours to obtain Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG, After washing, disperse again in 500 μL PBS containing 1.0% BSA, incubate at room temperature for 2 h to eliminate the risk of non-specific binding, and perform magnetic separation, and finally wash 3 times with PBS buffer (3×500 μL), at 4 Store at ℃ for subsequent use;

C. Take 12 μL of 2.5 mg/mL FAM-IgG and 488 μL of PBS, add it into the sample pool to measure the fluorescence intensity, and set the excitation and emission wavelengths to 490nm and 517nm respectively; recover the FAM-IgG solution and combine it with the Fe ₃ O Mix ₄ NCs-protein G-DABCYL-anti-IgG, incubate at 37 ℃ for 1 hour to obtain Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG immune composite probe, wash and magnetically separate the obtained magnetic The bead complex is the FRET immunoprobe.

In this embodiment, the X-ray diffraction pattern of the carboxyl magnetic beads used is shown in FIG. 3 , and the infrared spectrum of the carboxyl magnetic beads is shown in FIG. 4 . It can be seen from Figure 3 that it matches the standard diffraction spectrum of inverse spinel Fe ₃ O ₄ (JCPDS. 19-0629). In addition, an amorphous polymer-coated peak appears at the position where the diffraction angle is about 20.00°, indicating that there is polymer coating on the surface of the magnetic beads. From Figure 4, the peak at 602cm ^-1 is the stretching vibration peak of Fe-O bond, which is the characteristic peak of Fe ₃ O ₄ . The broad peak at 3400 cm ^-1 is the stretching vibration peak of (-OH), and the peak at 1663 cm ^-1 is the stretching vibration peak of (C=O). The appearance of these two peaks indicates the presence of carboxyl groups in the sample. The peak at 2948 cm ^-1 is the antisymmetric stretching vibration peak of (-CH ₂ -) on the main chain of polyacrylic acid, further confirming the existence of polyacrylic acid (PAA).

The prepared composite immunoprobe was characterized by the spectrum. Fig. 5 is a Zeta potential diagram of the composite immunoprobe, and Fig. 6 is a comparison diagram of fluorescence microscopy before and after fluorescence resonance energy transfer of the composite immunoprobe. In Fig. 5, curve a is the Zeta potential curve of Fe ₃ O ₄ NCs, curve b is the Zeta potential curve of Fe ₃ O ₄ NCs-protein G, and curve c is Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG The Zeta potential curve of , with the layer-by-layer connection of protein G and DABCYL-anti-IgG on the surface of magnetic beads, the Zeta potential has been moving to the negative direction, indicating that there are more and more negatively charged active groups, which is consistent with the connection results. Fig. 6 is a fluorescence microscope image of FAM-IgG and Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG. As shown in Figure A, FAM-IgG has fluorescence, indicating that the fluorescein dye has been successfully labeled on human IgG; when FAM-IgG and Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG undergo fluorescence resonance energy transfer , the fluorescence was significantly quenched (Panel B). It shows that the magnetic/fluorescent composite immunoprobe Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG has been prepared successfully.

Embodiment 2,

The difference between this example and Example 1 is that the concentration of carboxyl magnetic beads is 10 mg/mL, the concentration of goat anti-human immunoglobulin G solution labeled with fluorescent quencher is 0.5 mg/mL, and the antigen-antibody coupling reaction time is 1 h .

Embodiment 3,

The difference between this example and Example 1 is that the concentration of carboxyl magnetic beads is 5 mg/mL, the concentration of goat anti-human immunoglobulin G solution labeled with fluorescent quencher is 0.5 mg/mL, and the antigen-antibody coupling reaction time is 1 h .

Embodiment 4,

The difference between this example and Example 1 is that the concentration of carboxyl magnetic beads is 10 mg/mL, the concentration of goat anti-human immunoglobulin G solution labeled with fluorescent quencher is 0.5 mg/mL, and the antigen-antibody coupling reaction time is 3 h .

Embodiment 5,

The difference between this example and Example 1 is that the concentration of carboxyl magnetic beads is 10 mg/mL, the concentration of goat anti-human immunoglobulin G solution labeled with fluorescent quencher is 1 mg/mL, and the antigen-antibody coupling reaction time is 3 h .

Embodiment 6,

The difference between this example and Example 1 is that the concentration of carboxyl magnetic beads is 5 mg/mL, the concentration of goat anti-human immunoglobulin G solution labeled with fluorescent quencher is 0.5 mg/mL, and the antigen-antibody coupling reaction time is 3 h .

By characterizing the composite immune probes prepared in Examples 2-6, it can be found that composite immune probes with good performance can be prepared under the above raw material concentrations and reaction times.

Embodiment 7,

The principle of the method for detecting human immunoglobulin G by the FRET immunoprobe of the present invention is shown in Figure 2. When the human IgG labeled with fluorescein is coupled with the goat anti-human immunoglobulin G labeled with a fluorescent quencher, fluorescence resonance will occur Fluorescence of fluorescein-labeled human IgG is quenched by energy transfer (FRET). When the target detection substance IgG is added, IgG and fluorescein-labeled human IgG will undergo a competitive immune reaction, and fluorescein will detach from the probe surface into the solution, and the fluorescence signal of the supernatant of the reaction system will increase after magnetic separation; with the addition of IgG As the amount increases, the competitive immune response continues to occur, and the fluorescent signal continues to increase, and the IgG concentration is proportional to the fluorescent signal response.

In Fig. 7, curve a is the fluorescence spectrum curve of Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG mixed with FAM-IgG after fluorescence resonance energy transfer occurs, the fluorescence is quenched, and there is almost no fluorescence intensity. When the target detection substance IgG is added, IgG and FAM-IgG will undergo a competitive immune reaction, so that FAM-IgG reappears in the solution, and the fluorescence signal is enhanced, as shown in curves b, c, and d. increased, the competitive immune response continued to occur, and the fluorescent signal continued to increase. Figure 7 effectively demonstrates the design principle of the FRET immunoprobe to detect IgG.

Embodiment 8,

The method for detecting human immunoglobulin G (IgG) using the FRET immunoprobe prepared in Example 1 may further comprise the steps:

(1) Add 8 mg/mL FRET immunoprobe (Fe ₃ O ₄ NCs-protein G-DABCYL-anti-IgG-FAM-IgG) to PBS buffer solution with pH=7.4, and measure its initial fluorescence with a fluorescence spectrometer;

(2) Divide the immunoprobe into 5 equal parts, add IgG with different concentration gradients, and incubate at 37 °C for 3 h to react;

(3) The multiple reaction solutions treated in step (2) were separated by magnetics, the supernatants were poured into the sample pools, and the spectra were recorded with a fluorescence spectrometer to quantitatively measure human IgG.

Figure 8 is the fluorescence spectrum change diagram of the detection system after adding different concentrations of human immunoglobulin G in this example, curves a to e respectively represent the concentrations of human immunoglobulin G in the detection system are 0.067 nM, 0.2 nM, 5.4 nM, 48.6 nM and 146 nM. Fig. 9 is a linear curve of the human immunoglobulin G response of the detection system after adding different concentrations of human immunoglobulin G in this example. From the data in Figure 8, it can be seen that when the concentration of human immunoglobulin G (IgG) gradually increases in the range of 0.067 nM to 146 nM, the fluorescence intensity of the detection system also increases, which indicates that the probe has a certain effect on the concentration of IgG. Changes are very responsive. From the data in Figure 9, it can be seen that there is a good linear relationship between the concentration of IgG and the fluorescence intensity of the detection system. The linear range of IgG detection is 0.067 nM to 146 nM, the detection limit is 0.022 nM, and the correlation coefficient is 0.9979.

Embodiment 9,

This example is to verify the anti-interference ability of the FRET immunoprobe prepared in Example 1 to detect human immunoglobulin G (IgG). Immunoglobulin (Ig) is a group of proteins with antibody activity, mainly present in the blood, tissue fluid and exocrine fluid of organisms, and is an important indicator for examining the body's humoral immune function. The three items of immunoglobulins refer to serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM). We used IgA and IgM to test the selectivity of the immunosensor and evaluate its anti-interference ability. The detection steps are similar to those in Example 8, with the difference that: the target IgG to be tested in the detection system is replaced by the interfering substances IgM and IgA that may exist in the actual sample in turn, and the fluorescence intensity of the supernatant of the reaction system is detected. The results are shown in Figure 10. Show.

It can be seen from Figure 10 that when there are anti-interfering substances that may coexist with IgG in the detection system, only IgG can obviously cause changes in fluorescence intensity, and IgA and IgM have no obvious interference. The results showed that the method had good selectivity and anti-interference ability for IgG detection, and was able to distinguish IgG from its analogues in complex samples, which was attributed to the high specificity and magnetic separation and purification of antigen-antibody immune reaction.

In summary, the present invention proposes to use polyacrylic acid (PAA) to modify magnetic beads, and then combine with protein G, so as to connect antibodies on the surface of magnetic beads in a directional manner, and utilize the distance-dependent optical properties of fluorophore/quencher pairs to design Antigen-antibody conjugated complexes of fluorophores and quenchers were labeled separately to prepare FRET immunoprobes, and a new method for simple, sensitive and rapid detection of IgG was established by using the competitive immunoreaction between this probe and the target IgG. The preparation method of the FRET immune probe of the present invention is simple, has high specificity and sensitivity to detection targets, has good response to human immunoglobulin G, and can be used for detection of human immunoglobulin G. The preparation and detection method of this probe has opened up a new path for the construction of a fluorescent immunoassay method, and is expected to promote its application in the fields of biological science, pharmaceutical research, and clinical diagnosis.

Claims (10)

1. A FRET immune probe, characterized in that: the FRET immune probe is prepared from carboxyl magnetic beads coated by protein G and an antigen-antibody coupling compound labeled by fluorescein and a quencher, wherein the FRET immune probe is prepared by taking the carboxyl magnetic beads as a carrier material, directionally connecting the carboxyl magnetic beads and an antibody labeled by the fluorescence quencher by using the protein G as a connecting protein for combining the antibody and the carboxyl magnetic beads, and incubating the antibody and the antigen labeled by the fluorescein.

2. A FRET immunological probe as claimed in claim 1, wherein: the carboxyl magnetic beads are polyacrylic acid modified magnetic beads, the antibody marked by the fluorescence quencher is 4- [4- (dimethylamino) phenylazo ] benzoic acid marked goat anti-human immunoglobulin G, and the antigen marked by the fluorescein is fluorescein diacetate marked human immunoglobulin G.

3. A FRET immunoprobe according to claim 1 or 2, wherein: the magnetic beads are composed of ferroferric oxide nanoclusters.

4. A method for preparing a FRET immune probe is characterized by comprising the following steps:

A. preparing carboxyl magnetic beads by using a microwave-assisted polyol method, and activating carboxylic groups by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;

B. coating the activated carboxyl magnetic beads in the step A with protein G, adding goat anti-human immunoglobulin G marked by a fluorescence quenching agent, incubating at room temperature, and carrying out magnetic separation to obtain a nano magnetic bead compound loaded with a fluorescence quenching agent marked antibody;

C. and C, mixing the nano magnetic bead compound obtained in the step B with human immunoglobulin G marked by fluorescein diacetate, performing coupling reaction at 37 ℃, and performing magnetic separation to obtain the magnetic bead compound, namely the FRET immune probe.

5. The method of claim 4 for preparing a FRET immunoprobe, wherein: in the step A, the concentration of the carboxyl magnetic beads is 5-10 mg/mL.

6. The method of claim 4 or 5, wherein the FRET immune probe is prepared by: in step B, the concentration of the goat anti-human immunoglobulin G solution marked by the fluorescence quenching agent is 0.5-1 mg/mL.

7. The method of claim 4, wherein the FRET immune probe is prepared by: in step C, the coupling reaction time is 1-3 h.

8. Use of a FRET immuno probe according to any one of claims 1-3 for the detection of human igg.

9. Use of a FRET immuno probe according to claim 8 for the detection of human igg, comprising the steps of:

(1) Adding the FRET immune probe into a PBS buffer solution, and measuring the initial fluorescence of the FRET immune probe by using a fluorescence spectrometer;

(2) Dividing the FRET immune probe into a plurality of equal parts, adding human immunoglobulin G with different concentrations, and incubating for a period of time at 37 ℃ for reaction;

(3) And (3) carrying out magnetic separation on the multiple reaction solutions treated in the step (2), respectively pouring the supernate into sample cells, and recording the spectrum by using a fluorescence spectrometer, thereby quantitatively determining the human immunoglobulin G.

10. Use of a FRET immuno probe according to claim 9 for detection of human igg, wherein: in step (1), the pH value of the PBS buffer solution is 6-8, the concentration of the FRET immune probe is 5-8 mg/mL, and the reaction time is 1-3 h.

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