阅读说明：本技术 一种检测α-乳白蛋白的光电化学生物传感器的制备方法及应用 (Preparation method and application of photoelectrochemical biosensor for detecting alpha-lactalbumin ) 是由 陈志伟 孔玲 李灿国 朱琪颖 姜蒙 常慧琴 刘红亮 张秀珍 于 2021-02-18 设计创作，主要内容包括：本发明涉及一种检测乳制品致敏原α-乳白蛋白的光电化学免疫传感器的制备方法,属于功能纳米材料,食品分析以及光电化学生物传感技术领域。将CdWO-4-CdS异质结复合材料固定在ITO电极表面,应用聚多巴胺作连接剂固定致敏原α-乳白蛋白抗体,有效提高光电流响应及其灵敏度,通过层层组装方法,利用CdWO-4-CdS异质结良好的光电活性以及α-乳白蛋白与α-乳白蛋白抗体间的特异性结合,实现α-乳白蛋白的超灵敏检测,对于乳品中α-乳白蛋白的分析检测应用具有重要的意义。(The invention relates to a preparation method of a photoelectrochemical immunosensor for detecting dairy product allergen alpha-lactalbumin, belonging to the technical field of functional nano materials, food analysis and photoelectrochemical biosensing. Mixing CdWO 4 the-CdS heterojunction composite material is fixed on the surface of an ITO electrode, polydopamine is used as a connecting agent to fix an allergen alpha-lactalbumin antibody, the photocurrent response and the sensitivity of the photocurrent response are effectively improved, and the layer-by-layer assembly method is adopted and utilizedCdWO 4 The good photoelectric activity of the CdS heterojunction and the specific combination between the alpha-lactalbumin and the alpha-lactalbumin antibody realize the ultra-sensitive detection of the alpha-lactalbumin, and have important significance for the analysis and detection application of the alpha-lactalbumin in the dairy product.)

1. A preparation method and application of a photoelectrochemical biosensor for detecting alpha-lactalbumin are characterized by comprising the following steps:

(1) ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;

(2) adding CdWO of 8-12 μ L and 3-7 mg/mL₄Dropping the CdS suspension on an ITO electrode, naturally airing at room temperature, and calcining at 300 ℃ for 30 minutes;

(3) dripping 3-5 mu L of Tris-HCl solution with the pH of 8.5 and containing 1 mg/mL of dopamine hydrochloride into CdWO₄Reacting the electrode surface modified by CdS for 50-70 minutes at room temperature, and washing the electrode surface by using ultrapure water;

(4) dropwise adding 3-5 mu L of 10 mu g/mL alpha-lactalbumin antibody solution, reacting for 20-40 minutes, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;

(5) continuously dropwise adding 3-5 mu L of BSA solution with the mass fraction of 1% to seal the non-specific active sites on the surface of the electrode, reacting for 20-40 minutes, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;

(6) continuously dropwise adding 3-5 mu L of 0.001-10 ng/mL alpha-lactalbumin, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the CdWO-based electrode₄A non-standard photoelectrochemical biosensor of alpha-lactalbumin of CdS, stored at 4 ℃ in a refrigerator for future use.

2. The preparation method and application of the photoelectrochemical biosensor for detecting alpha-lactalbumin of claim 1, wherein the CdWO is₄-preparation of a CdS electrode, characterized by the following steps:

(1) taking 0.5-1.5 mmol of CH₄N₂S and 0.5-1.5 mmol of Na₂WO₄·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution A; 0.5-1.5 mmol Cd(CH₃COO)₂·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution B; dropwise adding the solution A into the solution B under magnetic stirring, stirring for 2 hours, transferring the white suspension into a 50 ml polytetrafluoroethylene reaction kettle, reacting for 24 hours at 160 ℃, centrifugally washing the obtained mixture with deionized water and absolute ethyl alcohol for 3 times respectively, and then carrying out vacuum drying at 50 ℃;

(2) adding CdWO of 8-12 μ L and 3-7 mg/mL₄dripping-CdS suspension on an ITO electrode, naturally airing at room temperature, calcining at 300 ℃ for 30 minutes, and naturally cooling to room temperature to obtain CdWO₄-a CdS electrode.

3. The preparation method and application of the photoelectrochemical biosensor for detecting alpha-lactalbumin as claimed in claim I, wherein the photoelectrochemical biosensor for detecting alpha-lactalbumin comprises the following steps:

(1) performing signal measurement by using a three-electrode system of an electrochemical workstation, wherein a test solution is 10-15 mL of 0.1 mol/L ascorbic acid phosphate buffer solution with the pH value of 5.0-8.0;

(2) detecting standard product alpha-lactalbumin with different concentrations by an i-t method, setting the voltage to be 0V, the running time to be 50 s, recording the change of current by using an LED as an excitation light source, and drawing a working curve;

(3) and (3) diluting the sample to be detected, and then replacing the standard substance in the step (2) for detection, and obtaining the content of the alpha-lactalbumin in the sample to be detected according to the photocurrent response intensity and the working curve.

Technical Field

The invention belongs to the technical field of bioanalysis chemistry, nano materials, immunoassay and photoelectrochemical biosensor, and provides a preparation method and application of a photoelectrochemical biosensor for detecting alpha-lactalbumin. In particular to a hydrothermal method for synthesizing CdWO₄The CdS heterojunction is used as an antibody capture substrate, polydopamine is used as a connecting agent to fix an allergen alpha-lactalbumin antibody, and a non-standard photoelectrochemical biosensor for detecting alpha-lactalbumin is prepared.

Background

Milk allergy is one of the most important allergic reactions, and milk is also one of the most common allergens in food products, characterized by a strong IgE response to milk proteins. The prevalence of CMA in children under 2 years of age is approximately 2.5%. CMA is a serious problem for children because milk is an important source of protein for their supplementation, and it is not feasible to strictly avoid using milk, nor is it practical to replace it. In addition, severe allergy may persist into adulthood, thus drawing the attention of allergic consumers. The allergen in milk, alpha-lactalbumin, is one of the major allergens in milk.

Alpha-lactalbumin is a Ca²⁺The binding protein has more than 72 percent of sequence homology between human and cattle, and has important physiological action and nutritional significance. Alpha-lactalbumin is rich in essential amino acids of the human body, especially tryptophan and cysteine, which are precursors of serotonin. It also has a number of branched chain amino acids, such as isoleucine and leucine, which are involved in protein synthesis in muscle. However, alpha-lactalbumin can cause about 30-35% of IgE-mediated milk allergy. At present, no food allergy treatment method exists, so that accurate detection of the content of alpha-lactalbumin in milk is very important for quality control of milk and prevention of milk allergy.

Currently, high performance liquid chromatography is a common method for detecting whey protein in the dairy industry, and liquid chromatography-mass spectrometry is used for analyzing milk protein due to its high resolution and short analysis time. However, these methods require pretreatment for separating the target protein, and development of alternative methods with high response speed, good selectivity, high sensitivity and low cost is urgently required. The photoelectrochemistry immunosensor has received wide attention in the field of bioanalysis due to the advantages of good selectivity and sensitivity, high analysis speed, small background influence and the like.

For the photoelectrochemical immunosensor, sensitivity is an index for evaluating the performance thereof. Methods for improving sensitivity are various, and increasing the initial signal is one of the most effective methods. CdWO₄Is an important photoelectric active material, but the photoelectric activity is limited by poor separation capability of electron-hole pairs, small specific surface area and weak surface adsorption capacity due to stability, proper band gap and lower cost. Therefore, there is a need to further optimize CdWO₄The photoelectric activity of (1). In the invention, CdS is adopted for sensitization to synthesize CdWO₄The CdS heterojunction is more favorable for charge separation and transfer, and the photoelectric response of the CdS heterojunction is enhanced.

The invention utilizes photoelectrochemical analysis method to perform sensitization by CdS to synthesize CdWO₄the-CdS heterojunction enhances the visible light absorption of the-CdS heterojunction to obtain a composite material with remarkably improved photoelectric activity, and polydopamine, alpha-lactalbumin antibody, bovine serum albumin and alpha-lactalbumin antigen are assembled into CdWO (cadmium telluride phosphate) by a layer-by-layer self-assembly method₄CdWO is utilized on-CdS heterojunction composite material₄Good photoelectric activity of a CdS heterojunction and specific combination between alpha-lactalbumin and an alpha-lactalbumin antibody to construct a CdWO-based antibody₄-alpha-lactalbumin photoelectrochemical sensor of CdS. The sensor has excellent photoelectrochemical activity, has the advantages of high sensitivity, wide linear range, low detection limit, quick detection, relatively simple preparation process and the like, realizes the ultra-sensitive analysis on the alpha-lactalbumin, and has important significance for the analysis and detection application of the alpha-lactalbumin in dairy products.

Disclosure of Invention

The invention provides a preparation method and application of a photoelectrochemical biosensor for detecting alpha-lactalbumin, which realize the ultrasensitive detection of the alpha-lactalbumin. One of the purposes of the invention is to provide a preparation method of a photoelectrochemical biosensor for detecting alpha-lactalbumin. The invention also aims to realize the ultra-sensitive detection of the alpha-lactalbumin by the prepared photoelectrochemistry immunosensor.

The technical scheme of the invention comprises the following steps:

(1) preparation of CdWO₄-a CdS heterojunction;

(2) preparing a working curve of the photoelectrochemical biosensor for detecting the alpha-lactalbumin.

Wherein the step (1) is used for preparing CdWO₄-the CdS heterojunction comprises:

taking 0.5-1.5 mmol of CH₄N₂S and 0.5-1.5 mmol of Na₂WO₄·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution A; 0.5-1.5 mmol Cd (CH)₃COO)₂·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution B; dropwise adding the solution A into the solution B under magnetic stirring, stirring for 2 hours, transferring the white suspension into a 50 ml polytetrafluoroethylene reaction kettle, reacting for 24 hours at 160 ℃, centrifugally washing the obtained mixture with deionized water and absolute ethyl alcohol for 3 times respectively, and then carrying out vacuum drying at 50 ℃;

wherein the working curve of the photoelectrochemical biosensor for detecting the alpha-lactalbumin in the step (2) without the standard comprises the following steps:

firstly, ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;

② CdWO 8-12 mu L and 3-7 mg/mL₄Dropping the CdS suspension on an ITO electrode, naturally airing at room temperature, and calcining at 300 ℃ for 30 minutes;

thirdly, 3-5 mu L of Tris-HCl solution with the pH of 8.5 and containing 1 mg/mL of dopamine hydrochloride is dripped into CdWO₄Reacting the electrode surface modified by CdS for 50-70 minutes at room temperature, and washing the electrode surface by using ultrapure water;

dripping 3-5 mu L of 10 mu g/mL alpha-lactalbumin antibody solution, reacting for 20-40 minutes, washing the surface of the electrode by ultrapure water, and airing in a refrigerator at 4 ℃;

continuously dropwise adding 3-5 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water after reacting for 20-40 minutes, and drying in a refrigerator at 4 ℃;

sixthly, continuously dropwise adding 3-5 mu L of 0.001-10 ng/mL alpha-lactalbumin, washing the surface of the electrode by using ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the CdWO-based nano-composite material₄-a non-standard photoelectrochemical biosensor of alpha-lactalbumin of CdS, stored in a refrigerator at 4 ℃ for later use;

the raw materials used in the present invention are all available from chemical or biopharmaceutical companies.

Advantageous results of the invention

(1) The invention is based on CdWO₄The CdS heterojunction constructs a novel photoelectrochemical immunosensor for detecting the allergen alpha-lactalbumin in the dairy product. Sensitization of CdWO with CdS₄A method for growing a heterojunction in situ.

(2) The immunosensor has high detection sensitivity on alpha-lactalbumin, the linear range is from 0.001 ng/mL to 10 ng/mL, the detection limit is low and is 0.17 pg/mL^-1. The prepared immunosensor has high stability and good reproducibility.

(3) The invention provides a novel and feasible detection method for the early detection of the alpha-lactalbumin, has simple operation and quick detection, and can be used for detecting actual samples.

Detailed Description

The invention will now be further illustrated by, but not limited to, the following specific embodiments

EXAMPLE 1 preparation of CdWO₄-a CdS heterojunction, the steps being as follows:

0.5 mmol of CH is taken₄N₂S and 0.5 mmol Na₂WO₄·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution A; 0.5 mmol of Cd (CH)₃COO)₂·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution B; dropwise adding the solution A into the solution B under magnetic stirring, and stirring 2After an hour, the white suspension was transferred to a 50 ml teflon reaction kettle and reacted at 160 ℃ for 24 hours, and the resulting mixture was centrifuged to wash deionized water, absolute ethanol 3 times each, and then dried under vacuum at 50 ℃.

EXAMPLE 2 preparation of CdWO₄-a CdS heterojunction, the steps being as follows:

1.0 mmol of CH is taken₄N₂S and 1.0 mmol Na₂WO₄·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution A; 1.0 mmol of Cd (CH)₃COO)₂·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution B; dropwise adding the solution A into the solution B under magnetic stirring, stirring for 2 hours, transferring the white suspension into a 50 ml polytetrafluoroethylene reaction kettle, reacting for 24 hours at 160 ℃, centrifugally washing the obtained mixture with deionized water and absolute ethyl alcohol for 3 times respectively, and then drying in vacuum at 50 ℃.

EXAMPLE 3 preparation of CdWO₄-a CdS heterojunction, the steps being as follows:

1.5 mmol of CH is taken₄N₂S and 1.5 mmol Na₂WO₄·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution A; 1.5 mmol of Cd (CH)₃COO)₂·2H₂Dissolving O in 20 mL of deionized water, and marking as a solution B; dropwise adding the solution A into the solution B under magnetic stirring, stirring for 2 hours, transferring the white suspension into a 50 ml polytetrafluoroethylene reaction kettle, reacting for 24 hours at 160 ℃, centrifugally washing the obtained mixture with deionized water and absolute ethyl alcohol for 3 times respectively, and then drying in vacuum at 50 ℃.

Example 4 a working curve of a photoelectrochemical biosensor for the detection of alpha-lactalbumin without a standard was prepared, with the following steps:

firstly, ultrasonically cleaning 2.5 cm of ITO conductive glass by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;

② adding 8 mu L of 3 mg/mL CdWO₄Dropping the CdS suspension on an ITO electrode, naturally airing at room temperature, and calcining at 300 ℃ for 30 minutes;

③ 3 mu L of dopamine hydrochloride with 1 mg/mL of pH8.5 Tris-HCl solution drop wise to CdWO₄The electrode surface modified by CdS reacts for 50 minutes at room temperature, and the electrode surface is washed by ultrapure water;

dripping 3 mu L of 10 mu g/mL alpha-lactalbumin antibody solution, washing the surface of the electrode by using ultrapure water after reacting for 20 minutes, and airing in a refrigerator at 4 ℃;

continuously dropwise adding 3 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water after reacting for 20 min, and airing in a refrigerator at 4 ℃;

sixthly, continuously dropwise adding 3 mu L of alpha-lactalbumin of 0.001-10 ng/mL, washing the surface of the electrode by using ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the CdWO-based nano-composite material₄A non-standard photoelectrochemical biosensor of alpha-lactalbumin of CdS, stored at 4 ℃ in a refrigerator for future use.

Example 5 a working curve of a photoelectrochemical biosensor for the detection of alpha-lactalbumin without a standard was prepared, with the following steps:

firstly, ultrasonically cleaning 2.5 cm of ITO conductive glass by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;

② adding 10 mu L of 5 mg/mL CdWO₄Dropping the CdS suspension on an ITO electrode, naturally airing at room temperature, and calcining at 300 ℃ for 30 minutes;

③ 4 mu L of Tris-HCl solution with pH of 8.5 and containing 1 mg/mL of dopamine hydrochloride is dripped into CdWO₄The electrode surface modified by CdS reacts for 60 minutes at room temperature, and the electrode surface is washed by ultrapure water;

dripping 4 mu L of 10 mu g/mL alpha-lactalbumin antibody solution, washing the surface of the electrode by ultrapure water after reacting for 30 minutes, and drying in a refrigerator at 4 ℃;

continuously dropwise adding 4 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water after reacting for 30 min, and airing in a refrigerator at 4 ℃;

sixthly, continuously dropwise adding 4 mu L of 0.001-10 ng/mL alpha-lactalbumin, washing the surface of the electrode by using ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the CdWO-based nano-composite material₄Non-standard photoelectrochemical biosensor of alpha-lactalbumin of CdS, ice at 4 ℃And storing in the box for later use.

Example 6 a working curve of a photoelectrochemical biosensor for the detection of alpha-lactalbumin without a standard was prepared, with the following steps:

firstly, ultrasonically cleaning 2.5 cm of ITO conductive glass by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;

② adding 12 mu L of 7 mg/mL CdWO₄Dropping the CdS suspension on an ITO electrode, naturally airing at room temperature, and calcining at 300 ℃ for 30 minutes;

③ 5 mu L of Tris-HCl solution with pH of 8.5 and containing 1 mg/mL of dopamine hydrochloride is dripped into CdWO₄The electrode surface modified by CdS reacts for 70 minutes at room temperature, and the electrode surface is washed by ultrapure water;

dripping 5 mu L of 10 mu g/mL alpha-lactalbumin antibody solution, washing the surface of the electrode by ultrapure water after reacting for 40 minutes, and drying in a refrigerator at 4 ℃;

continuously dropwise adding 5 mu L of BSA solution with the mass fraction of 1% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water after reacting for 40 min, and airing in a refrigerator at 4 ℃;

sixthly, continuously dropwise adding 5 mu L of 0.001-10 ng/mL alpha-lactalbumin, washing the surface of the electrode by using ultrapure water, and airing in a refrigerator at 4 ℃ to obtain the CdWO-based nano-composite material₄A non-standard photoelectrochemical biosensor of alpha-lactalbumin of CdS, stored at 4 ℃ in a refrigerator for future use.

Example 7A method for preparing a non-standard photoelectrochemical biosensor for detecting alpha-lactalbumin as claimed in claim l and its use, wherein the method is used for detecting alpha-lactalbumin and the detection steps are as follows:

firstly, a three-electrode system of an electrochemical workstation is used for signal measurement, and a test solution is 10-15 mL of 0.1 mol/L ascorbic acid phosphate buffer solution with the pH value of 5.0-8.0;

detecting the standard product alpha-lactalbumin with different concentrations by adopting an i-t method, setting the voltage to be 0V, the running time to be 50 s, using an LED as an excitation light source, recording the change of current, and drawing a working curve;

and thirdly, diluting the sample to be detected, replacing the standard product in the step II, and detecting to obtain the content of the alpha-lactalbumin in the sample to be detected according to the photocurrent response intensity and the working curve.