阅读说明：本技术 一种丝素蛋白光电免疫传感器的制备方法 (Preparation method of silk fibroin photoelectric immunosensor ) 是由 王秉 陈博逸 马维维 邓博之 万军民 彭志勤 于 2019-09-08 设计创作，主要内容包括：本发明涉及文物保护技术领域,公开了一种丝素蛋白光电免疫传感器的制备方法,包括聚多巴胺的制备；纳米二氧化钛的制备；玻碳电极预处理；聚多巴胺/二氧化钛GCE电极的制备；丝素蛋白光电免疫传感器的制备；本发明以聚多巴胺/二氧化钛复合材料作为光电免疫传感器的敏感材料,该材料在405nm LED光源激发下,具有强而稳定的光电流,能够实现对丝素蛋白的高灵敏检测。本发明将纳米二氧化钛引入光电化学传感体系,该颗粒生物相容性好、具有较强稳定性。本发明的光电免疫传感器测定丝素蛋白的操作过程简便,不需要特殊实验条件,仪器要求简单,在文物鉴定领域具有较好的应用前景。(The invention relates to the technical field of cultural relic protection, and discloses a preparation method of a silk fibroin photoelectric immunosensor, which comprises the steps of preparing polydopamine; preparing nano titanium dioxide; pretreating a glassy carbon electrode; preparing a polydopamine/titanium dioxide GCE electrode; preparing a silk fibroin photoelectric immunosensor; the poly-dopamine/titanium dioxide composite material is used as a sensitive material of the photoelectric immunosensor, and has strong and stable photocurrent under the excitation of a 405nm LED light source, so that the high-sensitivity detection of the silk fibroin can be realized. The invention introduces the nano titanium dioxide into a photoelectrochemical sensing system, and the particles have good biocompatibility and stronger stability. The photoelectric immunosensor provided by the invention has the advantages of simple operation process for determining silk fibroin, no need of special experimental conditions, simple instrument requirements and good application prospect in the field of cultural relic identification.)

1. A preparation method of a silk fibroin photoelectric immunosensor is characterized by comprising the following steps of:

1) Preparation of polydopamine: adding 8-10 ml of absolute ethyl alcohol and 18-20 ml of deionized water into a container, and then adding 500-; stirring the obtained mixed solution for 20-40min, and slowly adding 1.5-2.5ml dopamine solution of 45-55 mg/ml; stirring at normal temperature for reaction, centrifuging the obtained solution, and taking the solid to obtain polydopamine;

2) Preparing nano titanium dioxide: taking 20-30ml of absolute ethyl alcohol, sequentially adding 0.8-1.2ml of n-butyl alcohol, 0.8-1.2ml of polyethylene glycol, 0.35-0.40ml of hydrofluoric acid, 0.8-1.2ml of ethylene glycol and 0.4-0.6ml of deionized water, and stirring for 15-20 min to prepare a solution A; taking 2.5-3.5ml of tetrabutyl titanate, adding 8-12ml of absolute ethyl alcohol, and stirring for 15-20 min to prepare a solution B; injecting the solution B into the solution A at an injection speed of 3.5-4.5ml/h, stirring the solution A vigorously, and continuing to stir for 3-5 h after the injection is finished; stirring the stirred solution at the temperature of 140 ℃ and 160 ℃ for 3.5-4 h, respectively centrifugally washing with deionized water, n-butyl alcohol and absolute ethyl alcohol until the reaction is finished, drying the product obtained after cleaning, grinding and calcining to obtain nano titanium dioxide;

3) Pretreating a glassy carbon electrode: polishing the working glassy carbon electrode on the chamois by using nano-grade aluminum oxide and deionized water; then soaking the substrate in absolute ethyl alcohol for ultrasonic cleaning, and finally performing ultrasonic cleaning by using deionized water;

4) dripping 2-4ul of polydopamine solution with the concentration of 8-12ug/L on the surface of the electrode, drying, washing with PBS buffer solution, continuously dripping 2-4ul of 8-12ug/L rice titanium dioxide solution on the surface of the electrode, and drying to obtain the polydopamine/titanium dioxide/GCE electrode;

5) Cleaning and drying the electrode, dripping 8-12ul of 0.04-0.06M 3-hydroxypropionic acid aqueous solution, and drying and reacting at 35-39 ℃ for 0.5-1.5 h; incubating the electrode modified with 3-hydroxypropionic acid in a buffer solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride containing 0.04-0.06M EDC/0.02-0.04M NHS for 0.5-1.5h to convert the terminal carboxyl group of 3-hydroxypropionic acid into active NHS ester; dripping 4-6ul of 8-12ug/mL quantum dot-labeled silk fibroin antibody solution onto the surface of the electrode, drying at room temperature, and washing the unfixed antibody with PBS buffer solution to obtain an antibody/polydopamine/titanium dioxide/GCE modified electrode; and then, blocking the non-specific binding sites possibly existing on the surface of the electrode by using BSA solution for 20-40min, taking out, washing by using PBS buffer solution, and airing to obtain the silk fibroin photoelectric immunosensor.

2. The preparation method as claimed in claim 1, wherein in step 1), the stirring time after adding dopamine solution is 25-35h, the stirring is protected from light, and the centrifugation conditions are 9000-.

3. The method as claimed in claim 1, wherein in step 2), the product obtained by washing is dried at 55-65 ℃, ground for 1-2 h, and then calcined at 440 ℃ for 20-40min to obtain the nano titanium dioxide.

4. The preparation method according to claim 1, wherein in the step 3), the mixture is soaked in absolute ethyl alcohol and cleaned by ultrasonic wave for 8-12 min; and finally, ultrasonically cleaning the glass substrate for 8-12min by using deionized water.

5. the method of claim 1, wherein in step 4), the drying is performed at room temperature.

6. The method according to claim 1, wherein the PBS buffer has a pH of 7.4 in steps 4) to 6).

7. The method of claim 1, wherein the concentration of the BSA solution in step 5) is 0.8-1.2 wt%.

Technical Field

The invention relates to the technical field of cultural relic protection, in particular to a preparation method of a silk fibroin photoelectric immunosensor.

Background

china is one of the earliest countries in the world that produce textiles. As early as in the original society, people master a simple textile technology, and wild hemp and the like are made into clothes by rubbing, weaving and knitting to replace animal fur for preventing cold, keeping warm and covering bodies. With the development of agriculture, silkworm industry and animal husbandry, a Chinese ancient textile system taking hemp, silk and wool as main textile raw materials is formed. In a long history river, the textile cultural relics show social alternation, economic development and cultural blend in China, and are precious historical materials for researching the ancient social, economic, technical and cultural levels in China. At present, protein textiles account for the majority of the unearthed textiles. The textile is easy to age and decompose under the influence of moisture, temperature, pH value, microorganisms and the like in the burial environment, so that macromolecular chains are broken, peptide segments are degraded, and fragments and micro traces are formed when the textile is unearthed. Therefore, it is necessary to establish a scientific, effective and sensitive detection method for the identification of cultural relics, and the photoelectrochemistry immunosensing sensitivity is higher and the detection limit can be further reduced. In addition, because of using the electric signal response, compared with the traditional immunization, the PEC detection instrument has the advantages of simplicity, low price, easy miniaturization and the like. The photoelectrochemistry immunosensor has a strong application prospect in the field of cultural relic detection.

₂The principle of the basic method of photoelectrochemical immunoassay is that under the condition of illumination, immunoreaction is converted into a photoelectric signal of a photoelectric active substance, so that qualitative and quantitative detection of an object to be detected is realized.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a silk fibroin photoelectric immunosensor.

The specific technical scheme of the invention is as follows: a preparation method of a silk fibroin photoelectric immunosensor comprises the following steps of:

1) Preparation of polydopamine: adding 8-10 ml of absolute ethyl alcohol and 18-20 ml of deionized water into a container, and then adding 500-; stirring the obtained mixed solution for 20-40min, and slowly adding 1.5-2.5ml dopamine solution of 45-55 mg/ml; stirring at normal temperature for reaction, centrifuging the obtained solution, and taking the solid to obtain the polydopamine.

2) Preparing nano titanium dioxide: taking 20-30ml of absolute ethyl alcohol, sequentially adding 0.8-1.2ml of n-butyl alcohol, 0.8-1.2ml of polyethylene glycol, 0.35-0.40ml of hydrofluoric acid, 0.8-1.2ml of ethylene glycol and 0.4-0.6ml of deionized water, and stirring for 15-20 min to prepare a solution A; taking 2.5-3.5ml of tetrabutyl titanate, adding 8-12ml of absolute ethyl alcohol, and stirring for 15-20 min to prepare a solution B; injecting the solution B into the solution A at an injection speed of 3.5-4.5ml/h, stirring the solution A vigorously, and continuing to stir for 3-5 h after the injection is finished; and (3) stirring the stirred solution at the temperature of 140 ℃ and 160 ℃ for 3.5-4 h, respectively centrifugally washing with deionized water, n-butyl alcohol and absolute ethyl alcohol until the reaction is finished, drying the product obtained by cleaning, grinding and calcining to obtain the nano titanium dioxide.

3) Pretreating a glassy carbon electrode: polishing the working glassy carbon electrode on the chamois by using nano-grade aluminum oxide and deionized water; then soaking the mixture in absolute ethyl alcohol for ultrasonic cleaning, and finally performing ultrasonic cleaning by using deionized water.

4) Dripping 2-4ul of prepared polydopamine solution (10ug/L), drying, washing with PBS buffer solution, continuously dripping 2-4ul of prepared nano titanium dioxide solution (10ug/L) on the surface of the electrode, and drying to obtain polydopamine/titanium dioxide/GCE electrode;

5) Cleaning and drying the electrode, dripping 8-12ul of 0.04-0.06M 3-hydroxypropionic acid aqueous solution, and drying and reacting at 35-39 ℃ for 0.5-1.5 h; incubating the electrode modified with 3-hydroxypropionic acid in a buffer solution of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride containing 0.04-0.06M EDC/0.02-0.04M NHS (N-hydroxysuccinimide) for 0.5-1.5h to convert the terminal carboxyl group of 3-hydroxypropionic acid into active NHS ester; dripping 4-6ul of 8-12ug/mL quantum dot-labeled silk fibroin antibody solution onto the surface of the electrode, drying at room temperature, and washing the unfixed antibody with PBS buffer solution to obtain an antibody/polydopamine/titanium dioxide/GCE modified electrode; and then, blocking the non-specific binding sites possibly existing on the surface of the electrode by using BSA solution for 20-40min, taking out, washing by using PBS buffer solution, and airing to obtain the silk fibroin photoelectric immunosensor.

Preferably, in the step 1), the stirring time after the dopamine solution is added is 25-35h, the stirring is carried out in a dark place, and the centrifugation condition is 9000-.

Preferably, in the step 2), the product obtained by cleaning is dried at 55-65 ℃, ground for 1-2 h and then calcined at 420-440 ℃ for 20-40min to obtain the nano titanium dioxide.

Preferably, in the step 3), soaking and ultrasonic cleaning are carried out for 8-12min by using absolute ethyl alcohol; and finally, ultrasonically cleaning the glass substrate for 8-12min by using deionized water.

Preferably, in step 4), drying is performed at room temperature.

Preferably, in steps 4) to 6), the pH of the PBS buffer is 7.4.

Preferably, in step 5), the concentration of the BSA solution is 0.8-1.2 wt%.

Compared with the prior art, the invention has the beneficial effects that:

In the step 4), the poly-dopamine and nano-titanium dioxide are attached to the surface of the electrode twice, so that the conduction of electrochemical signals can be greatly enhanced.

In the step 5), the terminal carboxyl of the 3-hydroxypropionic acid is converted into active NHS ester, the combination with the antibody can be realized by converting the group, and the quantum dot labeled antibody is introduced into the system.

The poly-dopamine/titanium dioxide composite material is used as a sensitive material of the photoelectric immunosensor, and has strong and stable photocurrent under the excitation of a 405nm LED light source, so that the high-sensitivity detection of the silk fibroin can be realized.

The invention introduces the nano titanium dioxide into a photoelectrochemical sensing system, and the particles have good biocompatibility and stronger stability.

The photoelectric immunosensor provided by the invention has the advantages of simple operation process for determining silk fibroin, no need of special experimental conditions, simple instrument requirements and good application prospect in the field of cultural relic identification.

Detailed Description

The present invention will be further described with reference to the following examples.