阅读说明：本技术 一种适配体包覆鲁米诺的中空多孔二氧化硅材料的制备及其应用 (Preparation and application of aptamer-coated luminol hollow porous silicon dioxide material ) 是由 罗川南 孙元玲 王雪莹 代玉雪 刘浩 朱晓冬 高丹丹 韩蕊 王鹏飞 张少华 王喜 于 2019-09-23 设计创作，主要内容包括：本发明公开了一种适配体包覆鲁米诺的中空多孔二氧化硅材料的制备及其在化学发光分析中的应用技术。主要技术特征是：用三步法制得中空多孔二氧化硅,并用适配体将鲁米诺分子包覆在中空多孔二氧化硅的内部空腔中,得到对PSA有高特异性识别能力、可释放化学发光试剂分子鲁米诺的中空多孔二氧化硅材料；本发明同时提供了一种将适配体包覆鲁米诺分子的中空多孔二氧化硅材料应用于化学发光检测PSA的方法,该方法检测PSA表现出优异的选择性、稳定性和重现性,同时具有高的灵敏度,并且成功用于血清中PSA的检测,为该传感器进一步应用于临床PSA的检测和监控提供了理论支撑。(The invention discloses a preparation method of an aptamer-coated luminol hollow porous silicon dioxide material and an application technology of the aptamer-coated luminol hollow porous silicon dioxide material in chemiluminescence analysis. The main technical characteristics are as follows: preparing hollow porous silica by a three-step method, and coating luminol molecules in an inner cavity of the hollow porous silica by using an adapter to obtain a hollow porous silica material which has high specificity recognition capability on PSA and can release chemiluminescent reagent molecules luminol; the invention also provides a method for applying the aptamer-coated luminol molecule hollow porous silica material to chemiluminescence detection of PSA, the method has excellent selectivity, stability and reproducibility in PSA detection, has high sensitivity, is successfully used for detection of PSA in serum, and provides theoretical support for further application of the sensor to detection and monitoring of clinical PSA.)

1. A preparation method of a hollow porous silica material with luminol molecules coated by aptamers is characterized by comprising the following process steps:

(1) preparing hollow porous silica by a three-step method, namely firstly decomposing ethyl orthosilicate under weak base (PH = 8.0 ~ 9.0.0) to prepare solid silica, then etching under strong base (PH = 12.5 ~ 13.0.0) to obtain porous silica, finally coating polyelectrolyte polydiene dimethyl ammonium chloride on the surface layer of the porous silica, centrifugally collecting the porous silica, dispersing the porous silica in 100 mL of 1.0 ~ 2.0 moL/L ammonia solution, carrying out stirring reaction at 60 ~ 80 ℃ for 60 ~ 100 min and carrying out centrifugal separation, and then calcining the product in a 500 ~ 600 ℃ muffle furnace for 110 ~ 150 min to obtain the hollow porous silica;

(2) the preparation of the aptamer-coated luminol molecule hollow porous silica material comprises the steps of preparing 100 mL of 0.05 ~ 0.15.15 g/L hollow porous silica dispersion with PBS (phosphate buffer solution) with the pH = 7.0 ~ 7.4.4, simultaneously placing 5 mL of the dispersion and 50 uL0.01 ~ 0.1.1 mol/L luminol solution in a centrifuge tube, oscillating the centrifuge tube at room temperature for 2 ~ 4 h, then adding 2.0 ~ 4.0.0 nmol of aptamer into the centrifuge tube, oscillating the centrifuge tube at room temperature for 2 ~ 4 h, centrifugally separating the centrifuge tube after oscillation is completed to remove redundant aptamer and uncoated luminol molecules, and placing the centrifuged product in a 50 mL volumetric flask with PBS and in a 4C refrigerator for later use.

2. The method for preparing the aptamer luminol molecule-coated hollow porous silica material according to claim 1, wherein the method comprises the following steps: the hollow porous silicon dioxide material is synthesized in the step (1) through a three-step synthesis method, and the hollow porous silicon dioxide material is strong in condition controllability and simple to operate.

3. The method for preparing the aptamer luminol molecule-coated hollow porous silica material according to claim 1, wherein the method comprises the following steps: the synthetic material has high specificity recognition capability on PSA and can release chemiluminescent reagent molecule luminol.

4. The method for detecting PSA by chemiluminescence by using the hollow porous silica material with luminol molecules coated by the aptamer is characterized by adopting a chemiluminescence method for detection, and the process for detecting PSA by chemiluminescence is as follows:

starting a main pump and an auxiliary pump, respectively flowing sodium hydroxide, hydrogen peroxide, horseradish peroxidase solution and sample solution into a solution mixing pool from four sample inlet pipes, mixing and flowing into a detection pool after passing through the mixing pool, and generating chemiluminescenceIThe chemiluminescence intensity corresponding to the PSA with a certain concentration; when PSA exists, PSA and aptamer specificity identification on the surface of the aptamer-coated luminol molecule hollow porous silica material are combined together to form aptamer @ PSA, and simultaneously, luminol molecules coated in the hollow porous silica material are released, and the released luminol molecules, hydrogen peroxide and sodium hydroxide react under the catalytic action of horseradish peroxidase to generate strong chemiluminescent signals.

Technical Field

The invention relates to a preparation method of an aptamer-coated luminol hollow porous silica material and an application technology thereof in chemiluminescence analysis, belongs to the field of chemiluminescence sensing, and particularly relates to a preparation method of an aptamer-coated luminol hollow porous silica material and an application thereof in chemiluminescence detection of prostate specific antigen.

Background

Nano silicon dioxide (SiO)₂) The SiO has small particle size, large specific surface area and good biocompatibility, and has the advantages of surface interface effect, small size effect, quantum size effect and the like of nano materials, so that the SiO has the advantages of high strength, high toughness and high toughness₂Can be widely applied to the fields of microelectronics, biomedical engineering, composite materials, adsorption materials and the like. The hollow porous silica material has the characteristics of specific pore structure, hollowness, small density and large specific surface area, so that the hollow porous silica material has better permeability, adsorptivity, molecular sieving capacity and optical performance. Therefore, the material becomes a hotspot material with wide application prospect. At present, a plurality of methods for synthesizing the hollow porous silicon dioxide are available, such as a surface deposition method, a layer-by-layer assembly method, an atom transfer radical polymerization method, a spraying method, a microemulsion method and the like. In the patent with application number 2014107036911, a rapid one-step hydrothermal method for preparing hollow porous silica and further using hydrazine hydrate for one-step reduction to prepare hollow porous silica is disclosedA method of silicon/silver nanoparticles. In the patent with application number 2015100603893, a preparation method of a microorganism and nanoparticle composite system for degrading phenol is disclosed, wherein a temperature-sensitive polymer is introduced into the hollow cavity of a hollow porous silica nanoparticle, so that the adsorption capacity of the nanoparticle is increased, and the adsorption and desorption of phenol on the nanoparticle can be realized at different temperatures, thereby providing a wider choice for sewage treatment agents.

Luminol (Luminol) is a chemiluminescent molecule that can be converted to excited aminophthalic acid in the presence of hydrogen peroxide molecules, which emits strong fluorescence. Hydrogen peroxide is a product of many biological oxidation reactions and it is therefore easy to link these biological oxidation reactions with light detection by introducing luminol. Therefore, the luminol-hydrogen peroxide chemiluminescence system is widely applied to analysis methods such as fluorescence, chemiluminescence, electrochemiluminescence and the like. Many substances can catalyze the luminol-hydrogen peroxide reaction, such as metal particles (e.g., gold, silver nanoparticles, etc.), metal ions (e.g., iron ions, chromium ions, etc.), metal oxides (e.g., cerium dioxide), quantum dots (e.g., cadmium telluride quantum dots), biological enzymes (e.g., horseradish peroxidase, catalase, etc.), and mimic enzymes (e.g., G-quadruplex mimic enzyme).

Prostate disease is a common disease in the male population, in which the influence of prostate cancer on the patient's life health is self-evident, and the high-grade male malignant tumor ranking list is shown at the 6 th place. The research finds that a Prostate Specific Antigen (PSA) exists in the seminal plasma of a male, the PSA is a main component of the seminal plasma and is a substance capable of specifically reflecting the prostate, the prostate specific antigen of a normal person mainly exists in the prostate, the content of the prostate specific antigen in serum is extremely low, and the prostate specific antigen in the serum is obviously increased when the prostate is greatly cancerized or diseased, so that the detection of the level of the prostate specific antigen in the serum has higher application value in the clinical diagnosis of the prostate cancer. In the traditional detection method, mainly a radioimmunoassay is used, but the method needs a radioactive reagent for detection, and a container for storing the reagent cannot keep the effect of the reagent for a long time. Compared with the traditional method, the detection method combined with the chemiluminescence technology has the advantages of good repeatability, short time for testing samples and the like, and particularly has strong advantages in the aspects of specificity and sensitivity of clinical diagnosis. Therefore, the chemiluminescence-based detection method for PSA has extremely important value, can effectively improve the early diagnosis rate of the prostate cancer, provides reliable basis for clinical diagnosis of the prostate cancer, and has wide application prospect.

The invention aims to prepare a hollow porous silica material with an aptamer coated with luminol molecules, luminol is used as a chemiluminescent reagent and encapsulated inside the hollow porous silica by the aptamer, and the aptamer is simultaneously used as a specific recognition material and can specifically recognize a target detection object, so that the obtained hollow porous silica material with the aptamer coated with the luminol molecules can encapsulate the chemiluminescent reagent molecules and has high specific recognition capability; the hollow porous silica material with the aptamer coated with luminol molecules is used for constructing a chemiluminescence sensor, so that high-sensitivity, high-selectivity, accurate and rapid detection of PSA is realized, and a novel method for detecting PSA is invented.

Disclosure of Invention

The invention aims to provide a preparation method of an aptamer-coated luminol molecule hollow porous silica material, which is mainly used for preparing hollow porous silica, and luminol molecules are coated in an internal cavity of the hollow porous silica by using an aptamer to obtain the hollow porous silica material which has high specificity recognition capability on PSA and can release chemiluminescent reagent molecules luminol.

The purpose of the invention is realized by the following technical scheme:

(1) preparing hollow porous silica by a three-step method, namely, firstly decomposing ethyl orthosilicate under weak base (PH = 8.0 ~ 9.0.0) to prepare solid silica, then etching under strong base (PH = 12.5 ~ 13.0.0) to obtain porous silica, finally coating polyelectrolyte polydiene dimethyl ammonium chloride on the surface layer of the porous silica, centrifugally collecting the porous silica, dispersing the porous silica in 100 mL of 1.0 ~ 2.0 moL/L ammonia solution, carrying out stirring reaction at 60 ~ 80 ℃ for 60 ~ 100 and 100 min, and after centrifugal separation, calcining the product in a 500 ~ 600 ℃ muffle furnace for 110 ~ 150 min to obtain the hollow porous silica;

(2) the preparation of the aptamer-coated luminol molecule hollow porous silica material comprises the steps of preparing 100 mL of 0.05 ~ 0.15.15 g/L hollow porous silica dispersion with PBS (phosphate buffer solution) with the pH = 7.0 ~ 7.4.4, simultaneously placing 5 mL of the dispersion and 50 uL0.01 ~ 0.1.1 mol/L luminol solution in a centrifuge tube, oscillating the centrifuge tube at room temperature for 2 ~ 4 h, then adding 2.0 ~ 4.0.0 nmol of aptamer into the centrifuge tube, oscillating the centrifuge tube at room temperature for 2 ~ 4 h, centrifugally separating the centrifuge tube after oscillation is completed to remove redundant aptamer and uncoated luminol molecules, and placing the centrifuged product in a 50 mL volumetric flask with PBS and in a 4C refrigerator for later use.

Another object of the present invention is to apply the hollow porous silica material coated with luminol molecule by aptamer to detect PSA by chemiluminescence. The prepared hollow porous silica with the aptamer coated with the luminol molecule is placed in a PSA sample tube, when PSA exists, PSA and the aptamer are specifically identified and combined, so that the aptamer falls off from the surface of the hollow porous silica, the coated luminol molecule is indirectly released, the released luminol molecule initiates a chemiluminescence reaction of luminol-hydrogen peroxide, and the chemiluminescence reaction is catalyzed by catalytic peroxidase under the action of horseradish peroxidase, so that detection of PSA is realized. The chemiluminescence detection PSA is characterized in that: good selectivity, high sensitivity and convenient operation. In the construction of the chemiluminescence chemical sensor, the release response of luminol under the stimulation of PSA is researched, chemiluminescence conditions are selected, a working curve is drawn, the selectivity, reproducibility and stability of the sensor are researched, and the chemiluminescence chemical sensor is finally used for detecting PSA in serum.

The invention has the advantages and effects that:

(1) the hollow porous silica is prepared by the method, and the solid silica, the porous silica and the hollow porous silica are synthesized in sequence by a three-step synthesis method, so that the method has strong condition controllability and simple operation. The prepared hollow porous silicon dioxide has the advantages of hollow porosity, ordered pore size distribution, large specific surface area and the like, and provides support for coating luminol molecules;

(2) the invention prepares the hollow porous silicon dioxide with the aptamer coated with the luminol molecule, and the synthetic material has high specificity recognition capability on PSA and can release the luminol molecule of the chemiluminescent reagent;

(3) the hollow porous silica with the aptamer coated with the luminol molecule, prepared by the invention, is used for detecting PSA through chemiluminescence, and the chemiluminescence sensor has excellent selectivity, stability and reproducibility in PSA detection, high sensitivity, accuracy and precision, is successfully used for detecting PSA in serum, and provides theoretical support for further application of the sensor to detection and monitoring of clinical PSA.

Detailed Description