阅读说明：本技术 信号放大量子点荧光免疫探针及其制备方法和应用 (Signal amplification quantum dot fluorescence immunoassay probe and preparation method and application thereof ) 是由 陈竹 吴才桂 陈一凡 陈永晨 张海龙 于 2021-07-06 设计创作，主要内容包括：本发明公开了一种信号放大量子点荧光免疫探针的制备方法,包括以下步骤：利用缩合剂将羧基量子点荧光微球活化,加入亲和素、酪蛋白进行反应,形成量子点荧光微球-亲和素复合物；连接活化生物素和抗体,得到生物素化抗体；将生物素化抗体和量子点荧光微球-亲和素复合物混合进行一次反应,加入量子点荧光微球-亲和素复合物,再加入生物素化抗体进行二次反应,多次循环标记,制得信号放大的量子点荧光免疫探针。还公开了该制备方法制得的信号放大量子点荧光免疫探针及其在荧光免疫检测中的应用。通过上述方法制得的探针,荧光标记物和抗体充足,结构稳定,用于免疫检测具有捕获能力强、灵敏度高和线性宽等特点。(The invention discloses a preparation method of a signal amplification quantum dot fluorescence immunoassay probe, which comprises the following steps: activating the carboxyl quantum dot fluorescent microspheres by using a condensing agent, adding avidin and casein to react to form a quantum dot fluorescent microsphere-avidin compound; connecting the activated biotin and the antibody to obtain a biotinylated antibody; mixing the biotinylated antibody and the quantum dot fluorescent microsphere-avidin compound for primary reaction, adding the quantum dot fluorescent microsphere-avidin compound, adding the biotinylated antibody for secondary reaction, and circularly labeling for multiple times to obtain the quantum dot fluorescent immune probe with amplified signals. Also discloses a signal amplification quantum dot fluorescence immunoassay probe prepared by the preparation method and application thereof in fluorescence immunoassay. The probe prepared by the method has sufficient fluorescent markers and antibodies, has stable structure, and has the characteristics of strong capture capacity, high sensitivity, wide linearity and the like when being used for immunodetection.)

1. A preparation method of a signal amplification quantum dot fluorescence immunoassay probe is characterized by comprising the following steps:

(1) activating the carboxyl quantum dot fluorescent microspheres by using a condensing agent, adding avidin and casein to react to form a quantum dot fluorescent microsphere-avidin compound;

(2) connecting the activated biotin and the antibody to obtain a biotinylated antibody;

(3) mixing the biotinylated antibody and the quantum dot fluorescent microsphere-avidin compound for primary reaction;

(4) and adding the quantum dot fluorescent microsphere-avidin compound into the solution after the primary reaction, adding the biotinylated antibody to perform secondary reaction, and circularly marking for multiple times to prepare the quantum dot fluorescent immune probe with amplified signals.

2. The method for preparing the signal amplification quantum dot fluorescent immune probe according to claim 1, wherein the specific steps of the step (1) comprise:

(101) adding a condensing agent into the quantum dot solution for activation, uniformly mixing, ultrasonically dispersing for 5-20 s, and carrying out oscillation reaction for 10-30 min;

(102) adding avidin into the solution obtained in the step (101), and performing coupling reaction for 0.5-3 h;

(103) adding confining liquid into the solution obtained in the step (102), and reacting for 0.5-2 h at normal temperature;

(104) and (4) redissolving the precipitate obtained after the centrifugation in the step (103) by using a preservation solution to obtain the quantum dot-avidin compound.

3. The method for preparing a signal amplification quantum dot fluorescence immunoassay probe according to claim 2, wherein the condensing agent is one or a mixture of two or more of N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, NN' -carbonyldiimidazole, and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride.

4. The method for preparing a signal amplification quantum dot fluorescence immunoassay probe according to claim 2, wherein the blocking solution is 5-200 mM PBS containing 0.5-2% casein and 0.01-1% Tween 20 by mass, and has a pH of 6-8.

5. The method for preparing a signal amplification quantum dot fluorescence immunoassay probe according to claim 2, wherein the preservation solution is 10-200 mM PBS containing 0.5-5% bovine serum albumin, 5-30% trehalose, 0.5-5% polyvinylpyrrolidone K30 and 0.1-5% Tween 20, with pH 6.5-7.8.

6. The method for preparing the signal amplification quantum dot fluorescent immune probe according to claim 1, wherein the specific steps of the step (2) are as follows:

adding an antibody into a buffer solution containing activated biotin, wherein the molar mass ratio of the biotin to the antibody is 20: 1-1: 1, reacting in a dialysis bag for 6-12 h to obtain the biotinylated antibody.

7. The method for preparing the signal amplification quantum dot fluorescent immune probe according to claim 1, wherein the specific steps of the step (3) are as follows:

mixing the biotinylated antibody and the quantum dot fluorescent microsphere-avidin compound according to the proportion of 1: 0.1-1: 2, and reacting for 1-8 h.

8. A signal amplification quantum dot fluorescent immunoprobe prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the signal amplifying quantum dot fluorescent immunoprobe of claim 8 in fluoroimmunoassay.

Technical Field

The invention relates to the technical field of immune rapid analysis, in particular to a signal amplification quantum dot fluorescence immunoassay probe and a preparation method and application thereof.

Background

The immunolabeling technology is that a substance capable of developing color and emitting light is used as an indicator, an antigen or an antibody is labeled by covalent or non-covalent bonds to perform antigen-antibody reaction, and the experimental result is qualitatively and quantitatively determined by means of instruments such as a fluorescence analyzer, an enzyme-linked immunoassay analyzer, a gray detection instrument and the like. The trace analysis chemistry makes a major breakthrough, and the radioimmunoassay is established by combining the radioactive isotope labeling with the immune reaction. Thereafter, immunoassay techniques such as enzyme-linked immunosorbent assay, colloidal gold immunochromatography, fluorescence immunoassay, and chemiluminescence immunoassay have been developed and developed.

The different marking techniques described above have their own drawbacks due to the different principles. For example, the colloidal gold protein labeling process is a passive adsorption process, and the generated probe is unstable and has low sensitivity; enzyme labeling has high requirements on environment, and the enzyme is easy to degrade or inactivate; the radioactive isotope labeling has potential safety hazard and can cause harm to organisms and environment.

At present, fluorescent labeling probes such as phycoerythrin, CY5 and fluorescent microspheres are widely used and have the characteristics of high sensitivity, high quantification and the like, but the method also has the problem of difficulty in detection when the method is used for trace substances. Therefore, the market has further demands for the development of an immunolabeling method and a detection technology, which have high biosecurity, strong sensitivity and stable signal. The biotin-avidin system is a reaction amplification system which is more applied, and the immune labeling and tracing analysis technology is more sensitive due to the ultrahigh affinity and the multi-stage amplification effect of the reaction amplification system.

The patent with publication number CN112326953A provides a method for preparing biotinylated antibody, which comprises the steps of directionally crosslinking an aldehyde-based antibody and biotin, improving the effective binding rate, and achieving the purpose of signal amplification. However, the technology only realizes one-time amplification of the signal, and the signal strength is improved to a limited extent.

The signal amplification fluorescent probe described in patent publication No. CN102565383A is [ fluorescence-biotinylated antibody ] -streptavidin- [ fluorescence-polylysine-biotin ], and the preparation method thereof is relatively complicated in steps and requires two substances labeled with biotin.

Therefore, it is highly desirable to provide a novel signal amplification quantum dot fluorescent immunoprobe to solve the above problems.

Disclosure of Invention

The invention aims to solve the technical problem of providing a signal amplification quantum dot fluorescence immunoassay probe and a preparation method and application thereof, wherein the prepared probe fluorescence label and antibody are sufficient, the structure is stable, and the probe fluorescence label and antibody used for immunoassay have the characteristics of strong capture capability, high sensitivity, wide linearity and the like.

In order to solve the technical problems, the first technical scheme adopted by the invention is as follows: the preparation method of the signal amplification quantum dot fluorescence immunoassay probe comprises the following steps:

(1) activating the carboxyl quantum dot fluorescent microspheres by using a condensing agent, adding avidin and casein to react to form a quantum dot fluorescent microsphere-avidin compound;

(2) connecting the activated biotin and the antibody to obtain a biotinylated antibody;

(3) mixing the biotinylated antibody and the quantum dot fluorescent microsphere-avidin compound for primary reaction;

(4) and adding the quantum dot fluorescent microsphere-avidin compound into the solution after the primary reaction, adding the biotinylated antibody to perform secondary reaction, and circularly marking for multiple times to prepare the quantum dot fluorescent immune probe with amplified signals.

In a preferred embodiment of the present invention, the step (1) comprises the following steps:

(101) adding a condensing agent into the quantum dot solution for activation, uniformly mixing, ultrasonically dispersing for 5-20 s, and carrying out oscillation reaction for 10-30 min;

(102) adding avidin into the solution obtained in the step (101), and performing coupling reaction for 0.5-3 h;

(103) adding confining liquid into the solution obtained in the step (102), and reacting for 0.5-2 h at normal temperature;

(104) and (4) redissolving the precipitate obtained after the centrifugation in the step (103) by using a preservation solution to obtain the quantum dot-avidin compound.

Further, the condensing agent is one or a mixture of more than two of N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, NN' -carbonyldiimidazole and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride.

Further, the blocking solution is 5-200 mM PBS with pH 6-8 and containing 0.5-2% casein and 0.01-1% Tween 20 by mass fraction.

Further, the preservation solution is 10 to 200mM PBS containing 0.5 to 5% of bovine serum albumin, 5 to 30% of trehalose, 0.5 to 5% of polyvinylpyrrolidone K30, and 0.1 to 5% of Tween 20, with pH of 6.5 to 7.8.

In a preferred embodiment of the present invention, the step (2) comprises the following specific steps:

adding an antibody into a buffer solution containing activated biotin, wherein the molar mass ratio of the biotin to the antibody is 20: 1-1: 1, reacting in a dialysis bag for 6-12 h to obtain the biotinylated antibody.

In a preferred embodiment of the present invention, the step (3) comprises the following specific steps:

mixing the biotinylated antibody and the quantum dot fluorescent microsphere-avidin compound according to the proportion of 1: 0.1-1: 2, and reacting for 1-8 h.

In order to solve the above technical problems, the second technical solution adopted by the present invention is: provides the signal amplification quantum dot fluorescence immunoassay probe prepared by the preparation method.

In the preparation process of the common quantum dot fluorescent probe, the quantum dot and the antibody are only marked once; the invention is based on the fact that biotin and avidin can be combined with high affinity: the fluorescent quantum dot is marked with avidin and biotin is marked with antibody, a quantum dot-avidin-biotin-antibody composite structure is formed in the first step, and quantum dot-avidin and biotin-antibody are sequentially added in the second step to form a (quantum dot-avidin-biotin-antibody) - (quantum dot-avidin-biotin-antibody) composite, so that the fluorescent probe signal is circularly marked to achieve the purpose of enhancing the fluorescent probe signal.

In order to solve the above technical problems, the third technical solution adopted by the present invention is: provides the application of the signal amplification quantum dot fluorescence immunoassay probe prepared by the preparation method in fluorescence immunoassay.

The invention has the beneficial effects that:

(1) on the basis that biotin and avidin can be combined with high affinity, the biotin and the avidin are combined in a cascade overlapping manner, so that the signal intensity of the probe can be improved, the capture capacity of the probe is improved, the sensitivity and the linearity are greatly improved, and the probe prepared by the method has the characteristics of sufficient fluorescent markers and antibodies, stable structure, strong capture capacity, high sensitivity, wide linearity and the like when used for immunodetection;

(2) the invention utilizes the high affinity characteristic of biotin and avidin to realize the signal amplification of the fluorescent immune probe by multiple-cycle amplification and marking, and can develop a new-mode immune diagnostic reagent by combining a full-automatic immunoassay instrument based on the invention.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention, and to clearly and clearly define the scope of the present invention.

Example 1: the preparation method of the gastrin 17(G-17) quantum dot immunofluorescence chromatography kit comprises the following steps:

(1) preparation of G-17 fluorescent probe:

a. adding 1ml of morpholine ethanesulfonic acid buffer solution (0.05M pH 5.5) into 10mg of quantum dot microspheres, adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to the final concentration of 5mM, and oscillating for 30min at room temperature in a dark place to obtain activated quantum dot microspheres;

b. washing the activated quantum dot microspheres with 50mM phosphate buffer solution with pH 6.5, taking 3mg avidin and fully mixing the avidin, oscillating the mixture for 2 hours at room temperature in a dark place, adding casein with the final concentration of 1% and 0.05% Tween 20 after the reaction is finished, sealing the rest active sites, oscillating the mixture for 1 hour at room temperature in a dark place, washing the mixture with 0.02M PBS buffer solution with pH 7.4 containing 1% BSA after the reaction is finished, and resuspending to obtain 3mg/ml quantum dot-avidin compound for later use at 4 ℃;

c. dialyzing 3mg of the gastrin 17 detection antibody against 0.1M sodium bicarbonate buffer pH 8.0 in a dialysis bag for 12 h; adding N-hydroxysuccinimide biotin into the dialyzed gastrin 17 detection antibody to a final concentration of 0.12mg/ml, stirring at room temperature for 6h, adding 9.6ul of 1M ammonium chloride, and stirring for 15 min; dialyzing with PBS at 4 deg.C for 12h, removing free biotin, collecting dialyzed liquid to obtain 1.5mg/ml biotinylated gastrin 17 antibody at 4 deg.C;

d. mixing 0.1ml of the solution obtained in the step b with 0.2ml of the solution obtained in the step c, and stirring at room temperature for reaction for 3 hours to obtain a fluorescent probe;

e. adding 0.1ml of the solution obtained in the step (b) into the solution obtained in the step (d), adding 0.2ml of the solution obtained in the step (c) for secondary labeling, and stirring and reacting at room temperature for 3 hours to obtain a fluorescent probe;

f. and (4) adding 0.1ml of the solution obtained in the step (b) into the solution obtained in the step (e), adding 0.2ml of the solution obtained in the step (c), carrying out labeling for three times, and stirring and reacting at room temperature for 3 hours to obtain the signal-enhanced G-17 fluorescent probe.

(2) Preparing a marking pad:

diluting the signal-enhanced fluorescent probe by 10 times with 0.02M PBS buffer solution with pH 7.4 and containing 1% BSA, spraying the diluted fluorescent probe on a glass fiber membrane according to the parameter of 5ul/cm, and drying in a constant-temperature drying oven at 37 ℃ in a dark place for 6 hours;

(3) preparation of stationary phase:

diluting the gastrin 17 capture antibody to 1.5mg/ml by using a coating buffer solution, diluting the quality control antibody to 1.0mg/ml, coating the antibody in an amount of 1ul/cm by using XYZ3060 of BIODOT company, and drying the antibody in a constant-temperature drying oven at 37 ℃ for 6 hours in a dark place; wherein the coating buffer consists of: 0.02M phosphate buffer + 2% trehalose.

(4) Sample pad preparation:

soaking the glass fiber membrane in a treatment buffer solution for 0.5h, and drying in a constant-temperature drying oven at 37 ℃ in a dark place for 6h, wherein the treatment buffer solution is 0.02M PBS (pH 7.4) containing 1% BSA and 0.2% Tween 20;

(5) assembling the test strip:

the marking pad, the stationary phase, the sample pad, the water absorption pad and the backing plate are matched and assembled in a clean and dry room with the grade of 10 ten thousand, cut into the width of 4mm by a cutting machine and put into a card shell for standby.

Comparative example 1:

(1) preparation of G-17 fluorescent probe:

a. adding 1ml of morpholine ethanesulfonic acid buffer solution (0.05M pH 5.5) into 10mg of quantum dot microspheres, adding N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to the final concentration of 5mM, and oscillating for 30min at room temperature in a dark place to obtain activated quantum dot microspheres;

b. washing the activated quantum dot microspheres with 50mM phosphate buffer solution with pH 6.5, taking 3mg of gastrin 17 detection antibody, fully and uniformly mixing the detection antibody with the activated quantum dot microspheres, oscillating the mixture for 2 hours at room temperature in a dark place, adding casein with the final concentration of 1% and 0.05% of tween-20 after the reaction is finished, sealing the rest active sites, oscillating the mixture for 1 hour at room temperature in a dark place, washing the mixture with 0.02M PBS buffer solution with pH 7.4 containing 1% BSA, and resuspending the mixture to obtain 3mg/ml of G-17 quantum dot fluorescent probe for later use at 4 ℃; (ii) a

(2) Preparing a marking pad:

diluting the signal-enhanced fluorescent probe by 10 times with 0.02M PBS buffer solution with pH 7.4 and containing 1% BSA, spraying the diluted fluorescent probe on a glass fiber membrane according to the parameter of 5ul/cm, and drying in a constant-temperature drying oven at 37 ℃ in a dark place for 6 hours;

(3) preparation of stationary phase:

diluting the gastrin 17 capture antibody to 1.5mg/ml by using a coating buffer solution, diluting the quality control antibody to 1.0mg/ml, coating the antibody in an amount of 1ul/cm by using XYZ3060 of BIODOT company, and drying the antibody in a constant-temperature drying oven at 37 ℃ for 6 hours in a dark place; wherein the coating buffer consists of: 0.02M phosphate buffer + 2% trehalose.

(4) Sample pad preparation:

soaking the glass fiber membrane in a treatment buffer solution for 0.5h, and drying in a constant-temperature drying oven at 37 ℃ in a dark place for 6h, wherein the treatment buffer solution is 0.02M PBS (pH 7.4) containing 1% BSA and 0.2% Tween 20;

(5) assembling the test strip:

the marking pad, the stationary phase, the sample pad, the water absorption pad and the backing plate are matched and assembled in a clean and dry room with the grade of 10 ten thousand, cut into the width of 4mm by a cutting machine and put into a card shell for standby.

Comparative example 1 differs from example 1 in that the labeling step is different, comparative example 1 does not employ this signal amplification step, and only the antibody to gastrin 17 is covalently labeled with quantum dot microspheres; example 1 and comparative example 1 were tested with a calibrator for gastrin 17 and the test data are shown in table 1.

TABLE 1 comparison of fluorescence signal values for comparative example 1 and example 1

As can be seen from the data in table 1, the sensitivity of example 1 of the present invention is significantly improved, and the better linearity is y-445.99 x +1696.9, R2-0.995, and the linear equations of the comparative example are y-139.67 x +1088, and R2-0.9338.

And (3) repeatability experiment: measuring 3 positive samples for 20 times in the same time period, and calculating the variation coefficient (within batch); and (3) continuously manufacturing 3 batches of fluorescent probes, respectively measuring 3 positive samples for 10 times, and calculating the variation coefficient (between batches), wherein the variation coefficient in each batch and between batches is less than 10% according to the result, which shows that the fluorescent probes have good repeatability when being applied to the immunochromatography reagent.

TABLE 2 repeatability in batches

Stability test: the reagent (example 1) is stored in an oven at 37 ℃ in a sealed manner for 14 days, 3 positive samples and 1 negative sample are detected in parallel with the reagent placed at normal temperature, and the result shows that no change is found in the detection result, which indicates that the fluorescence stability of the probe is good.

Table 3 example 1 stability experiment

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.