A fluorescent immunoprobe and its preparation method and application

Abstract

The present invention discloses a fluorescent immunoprobe and a preparation method and application thereof. The method for preparing a quantum dot fluorescent immunoprobe comprises: mixing a quantum dot-streptavidin conjugate, a biotinylated antibody and a borate buffer, and performing an oscillation reaction to obtain the quantum dot fluorescent immunoprobe. The preparation method of the present invention uses a reaction system of streptavidin and biotin, combined with a borate buffer and a high-speed oscillation reaction to prepare a quantum dot fluorescent immunoprobe, which can quickly prepare a highly sensitive quantum dot fluorescent immunoprobe, and effectively solves the problems of long preparation cycle and low sensitivity of the current quantum dot fluorescent immunoprobe.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a fluorescent immune probe and a preparation method and application thereof. Background Art

[0002] Immunolabeling technology refers to the use of color-developing luminescent substances as indicators, labeling antigens or antibodies through covalent or non-covalent bonds to conduct antigen-antibody reactions, and using instruments such as fluorescence analyzers, enzyme-linked immunosorbent analyzers, and grayscale detection instruments to qualitatively and quantitatively determine the experimental results. A major breakthrough has been made in trace analytical chemistry, combining radioisotope tracing with immune reactions to establish radioimmunoassay. Since then, immunoassay technology has continued to develop and break through, such as enzyme-linked immunosorbent assay, colloidal gold immunochromatography, fluorescent immunoassay, and chemiluminescent immunoassay.

[0003] In the synthesis process of fluorescent immunoprobe, it is usually necessary to couple the protein (antigen or antibody, etc.) and the labeling material. In order to make the coupling between the two more stable, it is usually necessary to react the two overnight, etc., which takes a long time, resulting in a long production cycle and high cost. In addition, if the labeling material is small in size, due to factors such as steric hindrance, only one or two molecules of antigen / antibody may be coupled to the surface of the material, which is not conducive to improving the detection sensitivity. For example, CN113671171A discloses a method for preparing a signal amplification quantum dot fluorescent immunoprobe, comprising the following steps: activating carboxyl quantum dot fluorescent microspheres with a condensing agent, adding avidin and casein to react to form a quantum dot fluorescent microsphere-avidin complex; connecting activated biotin and antibody to obtain a biotinylated antibody; mixing the biotinylated antibody and the quantum dot fluorescent microsphere-avidin complex for a primary reaction, adding the quantum dot fluorescent microsphere-avidin complex, and then adding the biotinylated antibody for a secondary reaction, and labeling for multiple cycles to obtain a signal amplification quantum dot fluorescent immunoprobe, but the preparation cycle is long.

[0004] In addition, during the synthesis of fluorescent immunoprobes, the pH value, temperature and reaction buffer type of the reaction system will affect the stability and reaction efficiency of the probe. Improper control may cause serious precipitation or low coupling efficiency. Therefore, it is necessary to fully consider the influence of various factors and accurately control the reaction process. How to achieve precise control, effectively improve the compatibility of the preparation method, and provide a method for quickly preparing highly sensitive fluorescent immunoprobes is one of the problems that urgently need to be solved in the field of fluorescence detection. Summary of the invention

[0005] In view of the deficiencies in the prior art and actual needs, the present invention provides a fluorescent immunoprobe and a preparation method and application thereof. The method can quickly prepare a highly sensitive quantum dot fluorescent immunoprobe, effectively solving the problems of long preparation cycle and low sensitivity of the current quantum dot fluorescent immunoprobe.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a quantum dot fluorescent immunoprobe, the method comprising:

[0008] The quantum dot-streptavidin conjugate, the biotinylated antibody and the borate buffer are mixed and shaken to react to obtain the quantum dot fluorescent immune probe.

[0009] In the present invention, a quantum dot fluorescent immunoprobe is prepared by using a reaction system of streptavidin and biotin in combination with a borate buffer and a high-speed oscillation reaction, which can effectively solve the problems of low reaction efficiency and easy aggregation during the reaction process, thereby significantly shortening the preparation cycle. It only takes 5 minutes to 1 hour for the quantum dot-streptavidin conjugate and the biotinylated antibody to complete the reaction, thereby preparing a highly sensitive quantum dot fluorescent immunoprobe.

[0010] Preferably, the molar ratio of the quantum dot-streptavidin conjugate to the biotinylated antibody is 1:(1-10), including but not limited to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0011] In the present invention, it is found that controlling the molar ratio of quantum dot-streptavidin conjugate to biotinylated antibody to 1:(1-10) can significantly improve the sensitivity and stability of the prepared quantum dot fluorescent immunoprobe.

[0012] Preferably, the borate buffer contains bovine serum albumin.

[0013] In the present invention, it is found that the use of a borate buffer containing bovine serum albumin can significantly improve the reaction efficiency, while improving the sensitivity and stability of the prepared quantum dot fluorescent immunoprobe and reducing the background signal.

[0014] Preferably, the mass percentage of bovine serum albumin in the borate buffer is 0.1% to 5%, including but not limited to 0.2%, 0.4%, 0.5%, 0.8%, 1%, 2%, 2.5%, 3%, 4%, 4.2%, 4.4%, 4.6%, 4.8% or 4.9%.

[0015] Preferably, the pH of the borate buffer is 6 to 9, including but not limited to 6.0, 6.4, 6.8, 7.2, 7.5, 8, 8.2, 8.5 or 8.9.

[0016] Preferably, the oscillating reaction is carried out in an oscillator, and the rotation speed of the oscillator is 200-500rpm, including but not limited to 201rpm, 202rpm, 203rpm, 204rpm, 205rpm, 206, 207rpm, 210rpm, 220rpm, 230rpm, 260rpm, 300, 350, 380, 400, 410, 420, 430, 440rpm, 450rpm, 460rpm, 470rpm, 480rpm or 490rpm.

[0017] Preferably, the temperature of the oscillating reaction is 20-30°C (for example, it can be 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C or 29°C), and the time is 5min to 1h (for example, it can be 6min, 7min, 8min, 10min, 12min, 15min, 20min, 0.6h, 0.7h, 0.8h, 0.9h or 1h).

[0018] Preferably, the method for preparing quantum dot fluorescent immunoprobe further comprises the step of preparing quantum dot-streptavidin conjugate.

[0019] Preferably, the method for preparing the quantum dot-streptavidin conjugate comprises:

[0020] The quantum dots are activated by using a condensing agent, and streptavidin is added to obtain a quantum dot-streptavidin conjugate.

[0021] Preferably, the method for preparing the quantum dot-streptavidin conjugate comprises the following steps:

[0022] (1-1) Add coupling liquid and condensing agent to the quantum dot solution, perform ultrasonic treatment, and centrifuge to remove the supernatant and retain the precipitate;

[0023] (1-2) Add coupling solution to the precipitate in (1-1), then add streptavidin and perform shaking reaction;

[0024] (1-3) adding a blocking solution to the solution obtained in (1-2) to carry out a reaction;

[0025] (1-4) The solution obtained in (1-3) is centrifuged to remove the supernatant and retain the precipitate, which is then re-dissolved in a storage solution to obtain a quantum dot-streptavidin conjugate.

[0026] Preferably, the condensing agent includes one or a combination of two or more of N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), NN'-carbonyldiimidazole, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride.

[0027] Preferably, the coupling solution comprises a borate solution containing Tween, and the pH of the coupling solution is 6.5-8 (eg, 6.6, 6.8, 6.9, 7, 7.2, 7.5, 7.8 or 7.9).

[0028] Preferably, the coupling solution comprises 1-5 mM (for example, 1.1, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.2 mM, 4.4 mM, 4.6 mM or 4.8 mM) borate solution (BSS) containing 0.01% to 0.1% Tween 20 (for example, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08% or 0.09%), and the pH is 6.5 to 8.

[0029] Preferably, the blocking solution comprises a borate solution containing casein and proclin 300, and the pH of the blocking solution is 6.5-8 (eg, 6.6, 6.8, 6.9, 7, 7.2, 7.5, 7.8 or 7.9).

[0030] Preferably, the blocking solution comprises 1-5 mM (for example, 1.1, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.2 mM, 4.4 mM, 4.6 mM or 4.8 mM) borate solution (BSS) containing 1% to 10% casein (for example, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%) and 0.01% to 0.1% proclin300 (for example, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08% or 0.09%), with a pH of 6.5 to 8.

[0031] Preferably, the preservation solution comprises a borate solution containing bovine serum albumin and proclin 300, and the pH of the preservation solution is 6.5-8 (eg, 6.6, 6.8, 6.9, 7, 7.2, 7.5, 7.8 or 7.9).

[0032] Preferably, the preservation solution comprises 1-5 mM borate solution (SBS) containing 0.1%-5% bovine serum albumin (BSA) (for example, it can be 0.2%, 0.3%, 0.5%, 0.8%, 1%, 2%, 3%, 3.2%, 3.4%, 3.8%, 4%, 4.2%, 4.3%, 4.5%, 4.6%, 4.8% or 4.9%) and 0.01%-0.1% proclin300 (for example, it can be 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08% or 0.09%), and the pH is 6.5-8.

[0033] Preferably, the method for preparing quantum dot fluorescent immunoprobe further comprises the step of preparing biotinylated antibodies.

[0034] Preferably, the method for preparing the biotinylated antibody comprises:

[0035] The antibody and biotin are mixed and reacted to obtain the biotinylated antibody.

[0036] Preferably, the method for preparing the biotinylated antibody comprises the following steps:

[0037] (2-1) adding biotin to the buffer containing the antibody and performing an oscillating reaction;

[0038] (2-2) centrifuging the solution obtained in (2-1), collecting the supernatant, and obtaining the biotinylated antibody;

[0039] Preferably, the molar ratio of the antibody to biotin is 1:(1-40), including but not limited to 1:2, 1:3, 1:4, 1:8, 1:10, 1:20, 1:25, 1:28, 1:30, 1:32, 1:33, 1:35, 1:37 or 1:39.

[0040] Preferably, the method for preparing quantum dot fluorescent immunoprobe further comprises a blocking step.

[0041] Preferably, the blocking solution used for blocking contains bovine serum albumin and biotin.

[0042] Preferably, the blocking comprises adding a blocking solution and blocking for 0.3 to 1 h, including but not limited to 0.4 h, 0.5 h, 0.6 h, 0.8 h or 0.9 h.

[0043] As a preferred technical solution, the method for preparing quantum dot fluorescent immunoprobe comprises the following steps:

[0044] (1) adding a coupling liquid and a condensing agent to a quantum dot solution, ultrasonically treating the solution, and centrifuging to remove the supernatant and retain the precipitate;

[0045] (2) adding coupling solution to the precipitate in step (1), then adding streptavidin, and performing an oscillating reaction;

[0046] (3) adding a blocking solution to the solution obtained in step (2) to carry out a reaction;

[0047] (4) centrifuging the solution obtained in step (3), removing the supernatant and retaining the precipitate, and re-dissolving it with a storage solution to obtain a quantum dot-streptavidin conjugate;

[0048] (5) adding biotin to the buffer containing the antibody and performing an oscillating reaction;

[0049] (6) centrifuging the solution obtained in step (5), collecting the supernatant, and obtaining the biotinylated antibody;

[0050] (7) mixing the quantum dot-streptavidin conjugate and the biotinylated antibody at a molar ratio of 1:(1-10), adding a borate buffer at 20-30° C., and shaking at 200-500 rpm for a reaction of 5 min-1 h;

[0051] (8) Adding a blocking solution to the solution obtained in step (7) for blocking to obtain the quantum dot fluorescent immunoprobe.

[0052] In a second aspect, the present invention provides a quantum dot fluorescent immunoprobe prepared by the method for preparing a quantum dot fluorescent immunoprobe as described in the first aspect.

[0053] In a third aspect, the present invention provides the use of the quantum dot fluorescent immunoprobe described in the second aspect in fluorescent immunoassay.

[0054] In a fourth aspect, the present invention provides a fluorescent immunoassay kit, which comprises the quantum dot fluorescent immunoassay probe described in the second aspect.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The present invention can effectively solve the problems of low reaction efficiency and easy aggregation during the reaction process, significantly shorten the preparation cycle, and the quantum dot-streptavidin conjugate and biotinylated antibody only need 5 minutes to 1 hour to complete the reaction, thereby preparing a highly sensitive quantum dot fluorescent immunoprobe;

[0057] (2) The present invention controls the molar ratio of quantum dot-streptavidin conjugate to biotinylated antibody to be 1:(1-10), which can significantly improve the sensitivity and stability of the prepared quantum dot fluorescent immunoprobe;

[0058] (3) The present invention has found that the use of a borate buffer containing bovine serum albumin can significantly improve the reaction efficiency, while improving the sensitivity and stability of the prepared quantum dot fluorescent immunoprobe and reducing the background signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1A The reaction system of Comparative Example 1 of the present invention;

[0060] Figure 1B This is a diagram showing the test results of the test paper prepared using the quantum dot fluorescent immunoprobe in Comparative Example 1;

[0061] Figure 2 This is a diagram showing the test results of the test paper prepared using the quantum dot fluorescent immunoprobe in Example 1;

[0062] Figure 3 This is a diagram showing the test results of the test paper prepared using the quantum dot fluorescent immunoprobe in Example 5;

[0063] Figure 4 This is a graph showing the detection results of the test paper prepared using the quantum dot fluorescent immunoprobe in Example 8. DETAILED DESCRIPTION

[0064] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0065] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0066] Example 1

[0067] This embodiment provides a quantum dot fluorescent immunoprobe, and the preparation method of the quantum dot fluorescent immunoprobe comprises the following steps:

[0068] (1) Add coupling solution, 50 μL 54 mg / mL LEDC and 50 μL 75.6 mg / mL NHS to 150 μL 5 mg / mL quantum dot solution, sonicate for 5 min, and then centrifuge to remove the supernatant and retain the precipitate;

[0069] (2) Add the coupling solution to the precipitate in step (1), then add 21 μL of 10 mg / mL streptavidin, and shake at 200 rpm for 0.7 h;

[0070] (3) Add blocking solution to the solution obtained in step (2) and react at 25°C for 0.5 h

[0071] (4) centrifuging the solution obtained in step (3), removing the supernatant and retaining the precipitate, and re-dissolving it with a storage solution to obtain a quantum dot-streptavidin conjugate;

[0072] (5) adding biotin to the buffer containing the cortisol antibody, with the molar ratio of biotin to antibody being 20:1, and shaking at 200 rpm for 1 h;

[0073] (6) adding the solution obtained in step (5) into a 30K concentration tube and centrifuging to remove unreacted biotin;

[0074] (7) The solution obtained in step (6) was fixed to volume with 1×PBS to obtain a biotinylated antibody;

[0075] (8) The quantum dot-streptavidin conjugate and the biotinylated antibody were mixed at a molar ratio of 1:5, and then twice the volume of 3 mM borate buffer containing 0.3% bovine serum albumin was added, and the reaction was rapidly shaken at 25° C. and 200 rpm for 0.5 h;

[0076] (9) adding 1 μL of 200 mM D-biotin and 1% bovine serum albumin (BSA) blocking solution to the solution obtained in step (8) and blocking for 0.3 h;

[0077] (10) The solution obtained in step (9) is centrifuged and washed three times, and then redissolved with a preservation solution to obtain the quantum dot fluorescent immunoprobe.

[0078] The coupling solution is a 3 mM borate solution (BSS) containing 0.05% Tween 20, with a pH of 7; the blocking solution in step (3) is a 3 mM borate solution (BSS) containing 5% casein and 0.5% proclin300, with a pH of 7; the preservation solution is a 3 mM borate solution (SBS) containing 0.3% bovine serum albumin (BSA) and 0.05% proclin300, with a pH of 7.

[0079] Example 2

[0080] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that the molar ratio of the quantum dot-streptavidin conjugate to the biotinylated antibody in step (8) is 1:1.

[0081] Example 3

[0082] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that the molar ratio of the quantum dot-streptavidin conjugate to the biotinylated antibody in step (8) is 1:10.

[0083] Example 4

[0084] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that the molar ratio of the quantum dot-streptavidin conjugate to the biotinylated antibody in step (8) is 1:0.5.

[0085] Example 5

[0086] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that the molar ratio of the quantum dot-streptavidin conjugate to the biotinylated antibody in step (8) is 1:12.

[0087] Example 6

[0088] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that in step (8), twice the volume of 3 mM borate buffer (without BSA) is added.

[0089] Example 7

[0090] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that the rotation speed of the oscillation reaction in step (8) is 150 rpm.

[0091] Example 8

[0092] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Embodiment 1, except that the oscillation reaction time in step (8) is 5 minutes.

[0093] Comparative Example 1

[0094] This embodiment provides a quantum dot fluorescent immunoprobe. The preparation method of the quantum dot fluorescent immunoprobe is different from that of Example 1, except that in step (8), an equal amount of PBS buffer is used to replace the 3 mM borate buffer containing 0.3% bovine serum albumin, and the reaction produces aggregation, as shown in FIG1 .

[0095] The reaction time of step (8) and whether aggregation occurs during the preparation of Examples 1-7 and Comparative Example 1 are shown in Table 1.

[0096] Table 1

[0097] Experimental Group Step (8) Reaction time Is aggregation occurring? Detection of fluorescence value Example 1 30min no 3653.345 Example 2 30min no 1572.377 Example 3 30min no 7908.281 Example 5 30min no 2553.981 Example 8 5min no 3291.989 Comparative Example 1 120min yes 476.9624

[0098] As can be seen from Table 1, the present invention can effectively solve the problems of low reaction efficiency and easy aggregation during the reaction process, significantly shorten the preparation cycle, and the quantum dot-streptavidin conjugate and biotinylated antibody only need 5 minutes to 1 hour to complete the reaction.

[0099] Application Examples

[0100] This application example uses the quantum dot fluorescent immunoprobes prepared in Examples 1-7 and Comparative Example 1 to prepare a fluorescent immunoassay kit and perform fluorescent immunoassay.

[0101] In the embodiment of the present invention, a quantum dot-coupled cortisol antibody is prepared as a probe, which can detect cortisol in a sample. The prepared quantum dot probe is sprayed on the conjugate pad of the test strip, and the probe concentration is 1 mg / mL; cortisol-BSA is sprayed on the detection line T line, and the concentration is 1 mg / mL; the quality control line is sprayed with sheep anti-streptavidin, and the concentration is 0.5 mg / mL.

[0102] The fluorescent immunoassay process is as follows:

[0103] During immunoassay, 20 μL of sample was mixed with 50 μL of loading buffer, mixed evenly and added to the test strip, incubated at 25°C for 10 min, and then the fluorescence signal values ​​of the T line and C line were measured.

[0104] The test results of the test paper prepared by using the quantum dot fluorescent immunoprobe in Example 1 are as follows: Figure 2 As shown, the results show that both the test line (T line) and the quality control line (C line) have strong fluorescence signal values, and there is no background signal in the area outside the T and C lines, and the signal-to-noise ratio is high; the test results of the test paper prepared by the quantum dot fluorescent immunoprobe in Example 5 are as follows Figure 3 As shown, the high proportion of biotinylated antibodies leads to unstable probes and aggregation, resulting in membrane blocking during chromatography on the test strip. The fluorescent signal of the probe can be clearly observed below the T line, and there is a strong background signal. The test results of the test strip prepared using the quantum dot fluorescent immunoprobe in Example 8 are shown in FIG. Figure 4 As shown, when the reaction time is 5 min, a biological probe with high sensitivity, high stability and high signal-to-noise ratio can be obtained; the test results of the test paper prepared by the quantum dot fluorescent immunoprobe in Comparative Example 1 are as follows Figure 1B As shown, the reaction efficiency of the quantum dot fluorescent probe is not high, and the signal intensity on the detection line is relatively weak.

[0105] In summary, the present invention can effectively solve the problems of low reaction efficiency and easy aggregation during the reaction process, significantly shorten the preparation cycle, and the quantum dot-streptavidin conjugate and biotinylated antibody only need 5 minutes to 1 hour to complete the reaction, thereby preparing a highly sensitive quantum dot fluorescent immunoprobe.

[0106] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a quantum dot fluorescent immunoprobe, characterized in that: The method for preparing quantum dot fluorescent immunoprobe comprises: The quantum dot-streptavidin conjugate, the biotinylated antibody and the borate buffer are mixed and shaken to react to obtain the quantum dot fluorescent immunoprobe; The molar ratio of the quantum dot-streptavidin conjugate to the biotinylated antibody is 1:(1-10); the borate buffer contains bovine serum albumin; and the oscillation reaction time is 5 minutes to 1 hour.

2. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The pH of the borate buffer is 6-9.

3. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The oscillating reaction is carried out in an oscillator, and the rotation speed of the oscillator is 200-500 rpm.

4. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The temperature of the shaking reaction is 20-30°C.

5. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The method for preparing quantum dot fluorescent immune probe also includes the step of preparing quantum dot-streptavidin conjugate.

6. The method for preparing quantum dot fluorescent immunoprobe according to claim 5, characterized in that: The preparation method of the quantum dot-streptavidin conjugate comprises: The quantum dots are activated by using a condensing agent, and streptavidin is added to obtain a quantum dot-streptavidin conjugate.

7. The method for preparing quantum dot fluorescent immunoprobe according to claim 6, characterized in that: The preparation method of the quantum dot-streptavidin conjugate comprises the following steps: (1-1) Add coupling liquid and condensing agent to the quantum dot solution, perform ultrasonic treatment, and centrifuge to remove the supernatant and retain the precipitate; (1-2) Add coupling solution to the precipitate in (1-1), then add streptavidin and perform shaking reaction; (1-3) adding a blocking solution to the solution obtained in (1-2) to carry out a reaction; (1-4) The solution obtained in (1-3) is centrifuged to remove the supernatant and retain the precipitate, which is then re-dissolved in a storage solution to obtain a quantum dot-streptavidin conjugate.

8. The method for preparing quantum dot fluorescent immunoprobe according to claim 7, characterized in that: The condensing agent includes one or a combination of two or more of N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-carbonyldiimidazole, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride.

9. The method for preparing quantum dot fluorescent immunoprobe according to claim 7, characterized in that: The coupling solution comprises a borate solution containing Tween, and the pH value of the coupling solution is 6.5-8.

10. The method for preparing quantum dot fluorescent immunoprobe according to claim 7, characterized in that: The blocking solution includes a borate solution containing casein and proclin 300, and the pH value of the blocking solution is 6.5-8.

11. The method for preparing quantum dot fluorescent immunoprobe according to claim 7, characterized in that: The storage solution comprises a borate solution containing bovine serum albumin and proclin 300, and the pH value of the storage solution is 6.5-8.

12. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The method for preparing quantum dot fluorescent immune probes also includes the step of preparing biotinylated antibodies.

13. The method for preparing quantum dot fluorescent immunoprobe according to claim 12, characterized in that: The method for preparing the biotinylated antibody comprises: The antibody and biotin are mixed and reacted to obtain the biotinylated antibody.

14. The method for preparing quantum dot fluorescent immunoprobe according to claim 13, characterized in that: The method for preparing the biotinylated antibody comprises the following steps: (2-1) adding biotin to the buffer containing the antibody and performing an oscillating reaction; (2-2) Centrifuge the solution obtained in (2-1), collect the supernatant, and obtain the biotinylated antibody.

15. The method for preparing quantum dot fluorescent immunoprobe according to claim 13 or 14, characterized in that: The molar ratio of the antibody to biotin is 1:(1-40).

16. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The method for preparing quantum dot fluorescent immune probe also includes a blocking step.

17. The method for preparing quantum dot fluorescent immunoprobe according to claim 16, characterized in that: The blocking solution used in the blocking process contains bovine serum albumin and biotin.

18. The method for preparing quantum dot fluorescent immunoprobe according to claim 1, characterized in that: The method for preparing quantum dot fluorescent immunoprobe comprises the following steps: (1) Add coupling liquid and condensing agent to the quantum dot solution, treat with ultrasound for 10 to 30 minutes, and centrifuge to remove the supernatant and retain the precipitate; (2) adding coupling solution to the precipitate in step (1), then adding streptavidin, and performing an oscillating reaction; (3) adding a blocking solution to the solution obtained in step (2) to carry out a reaction; (4) centrifuging the solution obtained in step (3), removing the supernatant and retaining the precipitate, and re-dissolving it with a storage solution to obtain a quantum dot-streptavidin conjugate; (5) adding biotin to the buffer containing the antibody and performing an oscillating reaction; (6) centrifuging the solution obtained in step (5), collecting the supernatant, and obtaining the biotinylated antibody; (7) mixing the quantum dot-streptavidin conjugate and the biotinylated antibody at a molar ratio of 1:(1-10), adding a borate buffer at 20-30° C., and shaking at 200-500 rpm for a reaction of 5 min-1 h; (8) Adding a blocking solution to the solution obtained in step (7) for blocking to obtain the quantum dot fluorescent immunoprobe.

19. A quantum dot fluorescent immunoprobe prepared by the method for preparing a quantum dot fluorescent immunoprobe as described in any one of claims 1 to 18.

20. Use of the quantum dot fluorescent immunoprobe according to claim 19 in fluorescent immunoassay.

21. A fluorescent immunoassay kit, characterized in that: The fluorescent immunoassay kit comprises the quantum dot fluorescent immunoprobe described in claim 19.

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