技术领域technical field
本发明涉及一种传感器的制备技术，尤其涉及一种利用纳米孔阵列构建人气味结合蛋白传感器的方法，以及利用纳米孔阵列的人气味结合蛋白传感器检测与疾病相关的脂肪酸(二十二碳六烯酸)分子和醛类分子(苯甲醛)的方法。The present invention relates to a preparation technology of a sensor, in particular to a method for constructing a human odor-binding protein sensor using a nanopore array, and a human odor-binding protein sensor using a nanopore array to detect disease-related fatty acids (docohexa enoic acid) molecules and aldehyde-like molecules (benzaldehyde).
背景技术Background technique
人气味结合蛋白属于脂质运载蛋白中的一种，是一种低分子量、可溶性蛋白，主要存在于人嗅觉神经细胞外侧的淋巴液中。其作用是与目标脂溶性分子可逆地结合，并运输此类疏水性小分子至嗅觉细胞上的嗅觉受体，进而促使产生嗅觉。人类能够检测并区分上亿种不同的气味刺激，其中人气味结合蛋白发挥了非常重要的作用。现阶段，基于气味结合蛋白的传感器已经受到了很多人的重视，而石英晶体微天平，表面等离子体共振，电化学阻抗等传感器的研究也已经开展，但是检测下限，灵敏度等仍不能满足要求。除此之外，研究所用气味结合蛋白主要来自于昆虫和其他哺乳动物，将人气味结合蛋白作为敏感元件构建传感器的很少。因此，利用纳米孔和人气味结合蛋白结合的纳米级别的优势，发展一种基于电化学阻抗分析的纳米孔阵列的人气味结合蛋白传感器，以期实现高灵敏的检测特异性配体分子，可以用于化学分子、疾病标志物检测及人气味结合蛋白特性研究。Human odorant binding protein is a kind of lipocalin, which is a low-molecular-weight, soluble protein, and mainly exists in the lymph fluid outside the human olfactory nerve cells. Its function is to reversibly combine with target fat-soluble molecules, and transport such small hydrophobic molecules to the olfactory receptors on olfactory cells, thereby promoting the generation of smell. Humans can detect and distinguish hundreds of millions of different odor stimuli, among which human odorant binding protein plays a very important role. At this stage, sensors based on odorant-binding proteins have attracted the attention of many people, and research on sensors such as quartz crystal microbalance, surface plasmon resonance, and electrochemical impedance has also been carried out, but the lower detection limit and sensitivity still cannot meet the requirements. In addition, the odorant-binding proteins used in the research mainly come from insects and other mammals, and there are very few sensors constructed using human odorant-binding proteins as sensitive elements. Therefore, taking advantage of the nanoscale advantages of combining nanopores and human odorant binding proteins, a human odorant binding protein sensor based on electrochemical impedance analysis nanopore arrays is developed in order to achieve highly sensitive detection of specific ligand molecules, which can be used It is used in the detection of chemical molecules, disease markers and the characteristics of human odor binding proteins.
发明内容Contents of the invention
本发明的目的在于对现有技术的不足，提供一种基于纳米孔阵列的人气味结合蛋白传感器的制备及应用。The purpose of the present invention is to provide a preparation and application of a human odor binding protein sensor based on a nanopore array to overcome the shortcomings of the prior art.
本发明的目的是通过以下技术方案来实现的：一种基于纳米孔阵列的人气味结合蛋白传感器的制备方法，包括以下步骤：The object of the present invention is achieved by the following technical solutions: a preparation method of a nanopore array-based human odor binding protein sensor, comprising the following steps:
(1)加工纳米孔阵列材料：采用标准的两步阳极氧化法制备。将纯度为99.99％以上的铝箔裁剪成长×宽为20mm×20mm的基片，进行机械抛光后依次在超纯水、丙酮溶液中用超声脱脂清洗10min，取出后用超纯水淋洗，用氮气吹干后以基片作为阳极，铜片作为阴极在0.25M的草酸中进行一次氧化3h，然后用超纯水淋洗后把基片放入质量百分比为4％的铬酸和质量百分比为8％的磷酸混合液中进行5min刻蚀，除去一次阳极氧化多孔氧化铝层，然后用超纯水淋洗后进行15min的二次氧化，条件与第一次氧化条件完全相同，第二次刻蚀溶液为质量百分比为8％的磷酸和0.1M的氯化铜混合溶液，时间为5min，取出基片，用超纯水淋洗后即得到纳米孔阵列材料；(1) Processing of nanopore array materials: prepared by a standard two-step anodic oxidation method. Cut the aluminum foil with a purity of more than 99.99% into a substrate with a length × width of 20 mm × 20 mm, perform mechanical polishing, and then use ultra-pure water and acetone solution to clean it with ultrasonic degreasing for 10 minutes. After taking it out, rinse it with ultra-pure water and use nitrogen gas. After drying, the substrate is used as the anode, and the copper sheet is used as the cathode, and is oxidized once in 0.25M oxalic acid for 3 hours, then rinsed with ultrapure water, and then put the substrate into chromic acid with a mass percentage of 4% and a mass percentage of 8 % phosphoric acid mixed solution for 5 minutes to remove the anodized porous aluminum oxide layer, and then rinse with ultrapure water for 15 minutes of secondary oxidation, the conditions are exactly the same as the first oxidation conditions, the second etching The solution is a mixed solution of phosphoric acid with a mass percentage of 8% and 0.1M copper chloride, and the time is 5 minutes. The substrate is taken out, and the nanopore array material is obtained after rinsing with ultrapure water;
(2)纳米孔阵列的人气味结合蛋白传感器的构建，该步骤通过以下子步骤来实现：(2) The construction of the human odor binding protein sensor of nanopore array, this step is realized by the following sub-steps:
(2.1)首先，将纳米孔阵列材料裁剪成3mm*3mm的纳米孔阵列，面积大于反应皿中间通道，然后将其固定于反应皿的中间，再向反应皿的两侧中加入超纯水，浸泡24h后，依次用无水乙醇和超纯水清洗上述的反应皿及纳米孔阵列，最后用氮气吹干；(2.1) First, cut the nanopore array material into a 3mm*3mm nanopore array with an area larger than the middle channel of the reaction vessel, then fix it in the middle of the reaction vessel, and then add ultrapure water to both sides of the reaction vessel, After soaking for 24 hours, wash the above-mentioned reaction vessel and nanopore array with absolute ethanol and ultrapure water in sequence, and finally blow dry with nitrogen;
(2.2)配制5mM的2-羧乙基磷酸溶液，溶剂为超纯水；将2-羧乙基磷酸溶液加入反应皿的两侧，2-羧乙基磷酸溶液体积为使得纳米孔阵列恰好完全浸泡在2-羧乙基磷酸溶液中。在室温下，避光静置72h，2-羧乙基磷酸中的磷酸根吸附于纳米孔阵列的表面，进而将2-羧乙基磷酸固定于纳米孔阵列上。之后再吸出反应皿中残留的2-羧乙基磷酸溶液，然后用超纯水清洗反应皿及纳米孔阵列，最后用氮气吹干；(2.2) Prepare 5mM 2-carboxyethyl phosphoric acid solution, the solvent is ultrapure water; 2-carboxyethyl phosphoric acid solution is added to both sides of the reaction vessel, the volume of 2-carboxyethyl phosphoric acid solution is such that the nanopore array is just completely Soak in 2-carboxyethyl phosphoric acid solution. At room temperature, it was left standing in the dark for 72 hours, and the phosphate radical in the 2-carboxyethyl phosphate was adsorbed on the surface of the nanopore array, thereby immobilizing the 2-carboxyethyl phosphate on the nanopore array. Then suck out the remaining 2-carboxyethyl phosphoric acid solution in the reaction vessel, then clean the reaction vessel and nanopore array with ultrapure water, and finally dry it with nitrogen;
(2.3)在反应皿的两侧分别加入8mg/ml的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)溶液和12mg/ml的NHS(N-羟基琥珀酰亚胺)溶液，EDC溶液和NHS溶液的体积均为2-羧乙基磷酸溶液体积的二分之一，EDC溶液的溶剂为0.1MMES缓冲液，NHS溶液的溶剂为0.1MPBS缓冲液，室温下静置15min后，再加入饱和NaHCO3溶液调节反应皿中溶液pH至7.2-7.4；(2.3) Add 8 mg/ml of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution and 12 mg/ml of NHS (N -hydroxysuccinimide) solution, the volume of EDC solution and NHS solution is 1/2nd of the volume of 2-carboxyethyl phosphoric acid solution, the solvent of EDC solution is 0.1MMES damping fluid, the solvent of NHS solution is 0.1MPBS buffering After standing still at room temperature for 15 minutes, add saturated NaHCO3 solution to adjust the pH of the solution in the reaction vessel to 7.2-7.4;
(2.4)在反应皿的两侧分别加入与EDC溶液等体积的20μg/ml人气味结合蛋白溶液，人气味结合蛋白溶液的溶质为人气味结合蛋白，溶剂为0.1MPBS缓冲液，内含质量百分比为5％的海藻糖和质量百分比为5％的甘露醇。将反应皿置于4℃条件下静置2h，使得人气味结合蛋白与2-羧乙基磷酸溶液中的2-羧乙基磷酸的羧基发生脱水缩合作用而形成稳定的酰胺键，进而形成酰胺键结构，然后吸出反应皿中残留的液体，再向反应皿两侧加满0.1MPBS缓冲液，静置5min后吸出PBS缓冲液，即可获得基于纳米孔阵列的人气味结合蛋白传感器，置于4℃条件下备用。(2.4) Add 20 μg/ml human odor binding protein solution equal to the volume of EDC solution on both sides of the reaction vessel, the solute of human odor binding protein solution is human odor binding protein, the solvent is 0.1MPBS buffer solution, and the contained mass percentage is 5% trehalose and 5% mannitol by mass. Place the reaction dish at 4°C for 2 hours, so that the human odor binding protein and the carboxyl group of 2-carboxyethyl phosphoric acid in the 2-carboxyethyl phosphoric acid solution undergo dehydration condensation to form a stable amide bond, and then form an amide bond structure, then suck out the remaining liquid in the reaction dish, and then fill the two sides of the reaction dish with 0.1MPBS buffer solution, and after standing for 5 minutes, suck out the PBS buffer solution to obtain a human odor-binding protein sensor based on a nanopore array. Reserve at 4°C.
应用上述基于纳米孔阵列的人气味结合蛋白传感器检测脂肪酸和醛类物质的方法，包括以下步骤：The method for detecting fatty acids and aldehydes by using the above-mentioned nanopore array-based human odor binding protein sensor comprises the following steps:
(1)配制苯甲醛和二十二碳六烯酸的标准样品溶液：采用分析纯为99％的苯甲醛和分析纯为99％的二十二碳六烯酸标准贮备溶液分别稀释配制5种不同浓度梯度的标准样品溶液，稀释液是分析纯为99％的甲醇溶液；(1) Preparation of standard sample solutions of benzaldehyde and docosahexaenoic acid: 5 kinds of benzaldehyde with analytical purity of 99% and docosahexaenoic acid standard stock solution with analytical purity of 99% were diluted respectively to prepare 5 kinds For standard sample solutions with different concentration gradients, the diluent is a methanol solution with an analytical purity of 99%;
(2)纳米孔阵列的人气味结合蛋白传感器构建的表征：首先是三电极检测系统的连接，先将三电极中金工作电极的一端连接电化学工作站的工作输入端，金工作电极的另一端插入反应皿中；银/氯化银参比电极的一端连接电化学工作站的参比输入端，铂丝对电极的一端连接电化学工作站的对电极输入端。银/氯化银参比电极和铂丝对电极的另外一端同时插入反应皿的另外一侧(与金工作电极分别位于反应皿的两侧)。当反应皿中间没有纳米孔阵列时，向反应皿的两侧中分别加入与2-羧乙基磷酸溶液等体积氧化还原对溶液，氧化还原对溶液中含有10mM铁氰化钾、10mM亚铁氰化钾和0.1M的KCl，溶剂为超纯水，再向反应皿的两侧中分别加入与氧化还原对溶液等体积的甲醇溶液，直接进行电化学阻抗图谱扫描，具体测试参数为初始电压为0.2V，交流电压幅度为5mV，扫频范围为1Hz～1MHz，最后得到无纳米孔阵列的电化学阻抗图谱。之后利用相同的测量方法，测量纳米孔阵列固定于反应皿中的电化学阻抗图谱，以及纳米孔阵列上固定人气味结合蛋白之后的电化学阻抗图谱，利用检测到的三条电化学阻抗图谱表征纳米孔阵列的人气味结合蛋白传感器的构建。(2) Characterization of the construction of the human odor binding protein sensor of the nanopore array: firstly, the connection of the three-electrode detection system, first connect one end of the gold working electrode among the three electrodes to the working input end of the electrochemical workstation, and the other end of the gold working electrode Insert it into the reaction vessel; connect one end of the silver/silver chloride reference electrode to the reference input of the electrochemical workstation, and one end of the platinum wire counter electrode to the counter electrode input of the electrochemical workstation. The silver/silver chloride reference electrode and the other end of the platinum wire counter electrode are simultaneously inserted into the other side of the reaction vessel (the gold working electrode is respectively located on both sides of the reaction vessel). When there is no nanopore array in the middle of the reaction vessel, add an equal volume redox pair solution to the two sides of the reaction vessel respectively. The redox pair solution contains 10 mM potassium ferricyanide and 10 mM ferrocyanide. Potassium chloride and 0.1M KCl, the solvent is ultrapure water, then add methanol solution equal to the redox solution to the two sides of the reaction vessel, and directly perform electrochemical impedance spectroscopy scanning. The specific test parameters are that the initial voltage is 0.2V, the AC voltage amplitude is 5mV, and the frequency sweep range is 1Hz-1MHz, and finally the electrochemical impedance spectrum without the nanohole array is obtained. Then use the same measurement method to measure the electrochemical impedance spectrum of the nanopore array immobilized in the reaction dish, and the electrochemical impedance spectrum of the human odor binding protein immobilized on the nanopore array, and use the three detected electrochemical impedance spectra to characterize the nanometer. Construction of a well-arrayed human odorant-binding protein sensor.
(3)检测苯甲醛标准样品溶液：三电极检测系统的连接及具体测试参数同步骤2，测试传感器是纳米孔阵列的人气味结合蛋白传感器，首先进行空白对照溶液电化学阻抗图谱的测量，向反应皿的两侧中分别加入与2-羧乙基磷酸溶液等体积的氧化还原对溶液，氧化还原对溶液中含有10mM铁氰化钾、10mM亚铁氰化钾和0.1M的KCl，溶剂为超纯水，再向反应皿的两侧中分别加入与氧化还原对溶液等体积的甲醇溶液，通过电化学工作站对纳米孔阵列的人气味结合蛋白传感器进行电化学阻抗图谱扫描，得到电化学阻抗图谱。测量结束后吸出反应皿两侧中残留的溶液，然后向反应皿的两侧均加满PBS缓冲液，静置5min后缓慢吸出PBS缓冲液，用于清洗纳米孔阵列，消除上次测量残留的铁氰化钾/亚铁氰化钾及PBS缓冲液的混合溶液影响。之后再进行苯甲醛标准样品溶液的检测，向反应皿的两侧分别加入与2-羧乙基磷酸溶液等体积的氧化还原对溶液和已知物质的量浓度的苯甲醛标准样品溶液，进行电化学阻抗图谱的测量，得到该物质的量浓度下的苯甲醛标准样品溶液对应的电化学阻抗图谱。测量结束后吸出反应皿两侧中残留的溶液，然后向反应皿的两侧均加满PBS缓冲液，静置5min后缓慢吸出PBS缓冲液，用于清洗纳米孔阵列。(3) Detect benzaldehyde standard sample solution: the connection of the three-electrode detection system and the specific test parameters are the same as step 2. The test sensor is the human odor binding protein sensor of the nanopore array. At first, the measurement of the electrochemical impedance spectrum of the blank control solution is carried out. Add redox pair solution equal to the volume of 2-carboxyethyl phosphoric acid solution to both sides of the reaction vessel, the redox pair solution contains 10mM potassium ferricyanide, 10mM potassium ferrocyanide and 0.1M KCl, and the solvent is Ultrapure water, then add methanol solution equal to the volume of the redox solution to both sides of the reaction vessel, scan the electrochemical impedance spectroscopy of the human odor binding protein sensor in the nanopore array through the electrochemical workstation, and obtain the electrochemical impedance Atlas. After the measurement, suck out the solution remaining on both sides of the reaction dish, then fill both sides of the reaction dish with PBS buffer, and slowly suck out the PBS buffer after standing for 5 minutes, which is used to clean the nanowell array and eliminate the residual solution from the previous measurement. Effect of the mixed solution of potassium ferricyanide/potassium ferrocyanide and PBS buffer. Carry out the detection of benzaldehyde standard sample solution again afterwards, add the oxidation-reduction pair solution and the benzaldehyde standard sample solution of the amount concentration of known substance respectively to the both sides of reaction vessel respectively with 2-carboxyethyl phosphoric acid solution, carry out electric The measurement of the chemical impedance spectrum obtains the corresponding electrochemical impedance spectrum of the benzaldehyde standard sample solution under the concentration of the substance. After the measurement, the remaining solution on both sides of the reaction dish was sucked out, and then both sides of the reaction dish were filled with PBS buffer solution, and after standing for 5 minutes, the PBS buffer solution was slowly sucked out to clean the nanopore array.
(4)重复步骤3中苯甲醛标准样品溶液的电化学阻抗图谱的测量过程，直至完成5种不同浓度梯度的苯甲醛标准样品溶液的测量，得到不同浓度下的电化学阻抗图谱。(4) Repeat the measurement process of the electrochemical impedance spectrum of benzaldehyde standard sample solution in step 3, until finishing the measurement of the benzaldehyde standard sample solution of 5 kinds of different concentration gradients, obtain the electrochemical impedance spectrum under different concentrations.
(5)建立纳米孔阵列的人气味结合蛋白传感器的等效电路模型：三电极(包含金工作电极，银/氯化银对电极，铂丝对电极)检测系统由经典Randles模型等效，包含溶液阻抗Rs,电子转移电阻Rct,Warburg阻抗Zw,及常相角元件CPE；纳米孔阵列等效电路为纳米孔电阻Rpore和纳米孔电容Cpore的并联电路；人气味结合蛋白的等效电路为人气味结合蛋白电阻Rp和人气味结合蛋白电容Cp的并联电路。其中，随着苯甲醛标准样品溶液的浓度增加，人气味结合蛋白修饰的纳米孔阵列阻抗R在增加，其中R＝(Rpore+Rp)/2；(5) Establish the equivalent circuit model of the human odor binding protein sensor of the nanopore array: the three-electrode (including gold working electrode, silver/silver chloride counter electrode, and platinum wire counter electrode) detection system is equivalent to the classic Randles model, including Solution impedance Rs , electron transfer resistance Rct , Warburg impedance Zw , and constant phase angle element CPE; nanopore array equivalent circuit is a parallel circuit of nanopore resistance Rpore and nanopore capacitance Cpore ; The equivalent circuit is a parallel circuit of the resistance Rp of the human odorant binding protein and the capacitance Cp of the human odorant binding protein. Wherein, as the concentration of the benzaldehyde standard sample solution increases, the impedance R of the nanopore array modified by the human odor binding protein increases, wherein R=(Rpore +Rp )/2;
(6)建立苯甲醛分子标准浓度-归一化阻抗曲线：利用步骤2-4中检测到的空白溶液和5种不同浓度梯度下的苯甲醛标准样品溶液的电化学阻抗图谱，和步骤5建立的等效电路模型计算人气味结合蛋白修饰的纳米孔阵列阻抗R，从而计算归一化阻抗，即标准样品溶液对应的人气味结合蛋白修饰的纳米孔阵列阻抗R1与空白对照溶液对应的人气味结合蛋白修饰的纳米孔阵列阻抗R0的差值相对于空白对照溶液对应的人气味结合蛋白修饰的纳米孔阵列阻抗R0的变化百分比(R1-R0)/R0，得到苯甲醛标准样品溶液浓度与归一化阻抗之间的关系曲线y＝ax+b，其中，x为苯甲醛标准样品溶液浓度，y为归一化阻抗，a和b为常数，实现对苯甲醛分子的检测。(6) Establish benzaldehyde molecular standard concentration-normalized impedance curve: use the electrochemical impedance spectra of the blank solution detected in steps 2-4 and the benzaldehyde standard sample solution under 5 different concentration gradients, and establish with step 5 The equivalent circuit model calculates the impedance R of the nanopore array modified by the human odorant binding protein, thereby calculating the normalized impedance, that is, the impedance Rof the nanopore array modified by the human odorant binding protein corresponding to the standard sample solution is equal to that of the blank control solution. The difference of the impedance R0 of the nanopore array modified by the odorant binding protein relative to the change percentage of the impedance R0 of the nanopore array modified by the human odorant binding protein corresponding to the blank control solution (R1 -R0 )/R0 , to obtain benzaldehyde The relationship curve y=ax+b between the standard sample solution concentration and the normalized impedance, wherein, x is the benzaldehyde standard sample solution concentration, y is the normalized impedance, a and b are constants, realize the parabenzaldehyde molecule detection.
(7)检测二十二碳六烯酸标准样品溶液，建立二十二碳六烯酸分子标准浓度-归一化阻抗曲线，方法同步骤2-6，最终得到5种不同浓度梯度下的二十二碳六烯酸标准样品溶液的电化学阻抗图谱与二十二碳六烯酸分子标准浓度-归一化阻抗曲线。(7) Detect the docosahexaenoic acid standard sample solution, and establish the docosahexaenoic acid molecular standard concentration-normalized impedance curve. Electrochemical impedance spectrum of docosahexaenoic acid standard sample solution and docosahexaenoic acid molecular standard concentration-normalized impedance curve.
(8)完成待测苯甲醛样品溶液和待测二十二碳六烯酸样品溶液的检测，测量得到待测苯甲醛样品溶液和待测二十二碳六烯酸样品溶液的电化学阻抗图谱及归一化阻抗，对应得出待测苯甲醛样品溶液和待测二十二碳六烯酸样品溶液的浓度。(8) Complete the detection of the benzaldehyde sample solution to be measured and the docosahexaenoic acid sample solution to be measured, and measure the electrochemical impedance spectrum of the benzaldehyde sample solution to be measured and the docosahexaenoic acid sample solution to be measured And the normalized impedance, corresponding to obtain the concentrations of the benzaldehyde sample solution to be measured and the docosahexaenoic acid sample solution to be measured.
本发明的有益效果是，本发明利用人气味结合蛋白固定在纳米孔阵列上，构建了纳米孔阵列的人气味结合蛋白传感器，可以用于苯甲醛分子和二十二碳六烯酸分子的检测。实验表明该方法构建的纳米孔阵列的人气味结合蛋白传感器可将人气味结合蛋白简便并稳定地固定在纳米孔阵列上，该纳米孔阵列的人气味结合蛋白传感器检测灵敏度高、检测下限低。The beneficial effect of the present invention is that the present invention uses the human odor binding protein to be immobilized on the nanopore array, and constructs a human odor binding protein sensor of the nanopore array, which can be used for the detection of benzaldehyde molecules and docosahexaenoic acid molecules . Experiments show that the human odorant-binding protein sensor constructed by this method can easily and stably immobilize human odorant-binding protein on the nanopore array, and the human odorant-binding protein sensor of the nanopore array has high detection sensitivity and low detection limit.
附图说明Description of drawings
图1为本发明纳米孔阵列的人气味结合蛋白传感器示意图；Fig. 1 is the schematic diagram of the human odor binding protein sensor of the nanopore array of the present invention;
图2为本发明人气味结合蛋白在纳米孔阵列上固定过程示意图；Figure 2 is a schematic diagram of the immobilization process of the human odorant binding protein on the nanopore array;
图3为本发明纳米孔阵列的人气味结合蛋白传感器制备过程中，无纳米孔阵列、电极之间固定纳米孔阵列及纳米孔阵列上固定人气味结合蛋白的电化学阻抗图谱；Fig. 3 is the electrochemical impedance spectrum of human odor binding protein sensor without nanopore array, fixed nanopore array between electrodes and fixed human odor binding protein on the nanopore array during the preparation process of the nanopore array sensor of the present invention;
图4为本发明纳米孔阵列的人气味结合蛋白传感器检测苯甲醛分子和二十二碳六烯酸分子溶液的电化学阻抗图谱；Fig. 4 is the electrochemical impedance spectrum of detection of benzaldehyde molecule and docosahexaenoic acid molecule solution by human odor binding protein sensor of nanopore array of the present invention;
图5为本发明纳米孔阵列的人气味结合蛋白传感器建立的等效电路模型；Fig. 5 is the equivalent circuit model established by the human odor binding protein sensor of the nanopore array of the present invention;
图6为本发明纳米孔阵列的人气味结合蛋白传感器器分别与不同浓度的苯甲醛分子溶液和二十二碳六烯酸分子溶液发生反应后归一化电子传递电阻与苯甲醛分子溶液和二十二碳六烯酸分子溶液浓度对数之间的关系图；Fig. 6 is the human odor binding protein sensor device of the nanopore array of the present invention reacted with different concentrations of benzaldehyde molecular solution and docosahexaenoic acid molecular solution respectively, and the normalized electron transfer resistance and benzaldehyde molecular solution and dicosahexaenoic acid molecular solution The relationship diagram between the concentration logarithm of docosahexaenoic acid molecular solution;
图中：反应皿1，金工作电极2，银/氯化银对电极3，铂丝对电极4，固定夹5，纳米孔阵列6，Randles模型7，纳米孔阵列等效电路8，人气味结合蛋白等效电路9，人气味结合蛋白修饰的纳米孔阵列阻抗R10，纳米孔阵列的人气味结合蛋白传感器的等效电路模型11。In the figure: reaction vessel 1, gold working electrode 2, silver/silver chloride counter electrode 3, platinum wire counter electrode 4, clamp 5, nanopore array 6, Randles model 7, nanopore array equivalent circuit 8, human odor Binding protein equivalent circuit 9, human odorant binding protein modified nanopore array impedance R10, equivalent circuit model of nanopore array human odorant binding protein sensor 11.
具体实施方式Detailed ways
以下结合附图及具体实施例对本发明作详细描述，但并不是限制本发明。The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited.
本发明基于纳米孔阵列的人气味结合蛋白传感器的制备方法，包括以下步骤：The preparation method of the human odor binding protein sensor based on the nanopore array of the present invention comprises the following steps:
1、纳米孔阵列的加工。1. Processing of nanohole arrays.
纳米孔阵列6的制备采用标准的两步阳极氧化法。首次按将高纯(99.99％以上)铝箔裁剪成尺寸合适的基片，用细绒布轻擦，进行机械抛光。制好的基片依次在超纯水，丙酮溶液中用超声脱脂清洗10min，取出后用超纯水淋洗干净，然后用含有质量百分比为5％的磷酸，质量百分比为5％的硫酸及0.2M的三氧化铬混合溶液对基片进行电化学抛光，其中电压为20V，时间15min,取出后用超纯水淋洗干净，用氮气吹干后以基片作为阳极，铜片作为阴极进行第一次阳极氧化。在物质的量浓度为0.25M的草酸中一次氧化3h以上，用超纯水淋洗后把基片放入含有质量百分比为4％的铬酸和质量百分比为8％的磷酸混合液中同时加热到60℃。用化学溶解的方法除去一次阳极氧化多孔氧化铝层，并在裸露的铝基底上再进行第二次阳极氧化，氧化时间为15min，且二次氧化时阳极氧化条件与第一次氧化条件完全相同，最后刻蚀溶液为含有质量百分比为8％的磷酸和0.1M氯化铜的混合溶液。The nanopore array 6 was fabricated using a standard two-step anodic oxidation method. For the first time, cut high-purity (above 99.99%) aluminum foil into a suitable size substrate, wipe it gently with a fine velvet cloth, and perform mechanical polishing. Prepared substrates were cleaned in ultrapure water and acetone solution by ultrasonic degreasing for 10 minutes, rinsed with ultrapure water after taking out, and then washed with phosphoric acid containing 5% by mass, sulfuric acid with 5% by mass and 0.2 The chromium trioxide mixed solution of M is used to electrochemically polish the substrate, wherein the voltage is 20V, and the time is 15min. After taking it out, it is rinsed with ultrapure water, dried with nitrogen, and the substrate is used as the anode, and the copper sheet is used as the cathode. Anodized once. Oxidize in oxalic acid with a substance concentration of 0.25M for more than 3 hours at a time, rinse with ultrapure water, put the substrate into a mixed solution containing 4% chromic acid and 8% phosphoric acid and heat it at the same time to 60°C. Remove the primary anodized porous aluminum oxide layer by chemical dissolution, and then conduct the second anodic oxidation on the bare aluminum substrate, the oxidation time is 15min, and the anodic oxidation conditions for the second oxidation are exactly the same as the first oxidation conditions , and finally the etching solution is a mixed solution containing 8% by mass of phosphoric acid and 0.1M copper chloride.
2、纳米孔阵列的人气味结合蛋白传感器的构建。2. Construction of human odor-binding protein sensor based on nanopore array.
首先，将纳米孔阵列6置于反应皿1的中间，用固定夹5固定后向反应皿2的两侧中加入等量的超纯水，浸泡24h后,依次用无水乙醇和超纯水清洗上述的反应皿1及纳米孔阵列6，最后用氮气吹干；配制物质的量浓度为5mM的2-羧乙基磷酸溶液，溶剂为超纯水；在反应皿1的两侧分别加入100μl的2-羧乙基磷酸溶液，使得纳米孔阵列6完全浸泡在2-羧乙基磷酸溶液中。在室温下，避光静置72h，2-羧乙基磷酸中的磷酸根能够吸附于纳米孔阵列6的表面，进而将2-羧乙基磷酸固定于纳米孔阵列6上。之后再吸出反应皿1中的残留的2-羧乙基磷酸溶液；随后分别向反应皿1的两侧均加入体积为2-羧乙基磷酸溶液一半的浓度为8mg/ml的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)溶液和浓度为12mg/ml的NHS(N-羟基琥珀酰亚胺)溶液，其中EDC溶液的溶剂为0.1MMES缓冲液,NHS溶液的溶剂为0.1MPBS缓冲液，15min后，再加入饱和NaHCO3溶液调节反应皿1两侧中溶液pH至7.2-7.4；然后在向反应皿1的两侧均加入50μl浓度为20μg/ml人气味结合蛋白(氨基酸序列如SEQIDNO.1所示)溶液，人气味结合蛋白溶液的溶剂为0.1MPBS缓冲液，内含质量百分比为5％的海藻糖和5％的甘露醇。将反应皿1置于4℃条件下静置2h，使得人气味结合蛋白与2-羧乙基磷酸溶液中的2-羧乙基磷酸的羧基发生脱水缩合作用而形成稳定的酰胺键，进而形成酰胺键结构，然后吸出反应皿1两侧的残留液体，再向反应皿1的两侧中加满0.1MPBS缓冲液，静置5min后吸出PBS缓冲液，用于清洗未被固定的人气味结合蛋白，重复用0.1MPBS缓冲液清洗3次后即可获得基于纳米孔阵列的人气味结合蛋白传感器，置于4℃条件下备用。人气味结合蛋白在纳米孔阵列上的固定过程如图2所示。First, place the nanopore array 6 in the middle of the reaction vessel 1, fix it with the fixing clip 5, add an equal amount of ultrapure water to both sides of the reaction vessel 2, soak it for 24 hours, and wash it with absolute ethanol and ultrapure water successively. Clean the above-mentioned reaction vessel 1 and the nanopore array 6, and finally dry them with nitrogen; prepare a 2-carboxyethyl phosphoric acid solution with a concentration of 5 mM, and use ultrapure water as the solvent; add 100 μl to both sides of the reaction vessel 1 2-carboxyethyl phosphoric acid solution, so that the nanopore array 6 is completely immersed in the 2-carboxyethyl phosphoric acid solution. At room temperature, after standing in the dark for 72 hours, the phosphate group in the 2-carboxyethyl phosphate can be adsorbed on the surface of the nanopore array 6 , and then the 2-carboxyethyl phosphate is fixed on the nanopore array 6 . Then suck out the remaining 2-carboxyethyl phosphoric acid solution in the reaction vessel 1; then add the EDC (1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution and the NHS (N-hydroxysuccinimide) solution with a concentration of 12mg/ml, wherein the solvent of the EDC solution is 0.1MMES Buffer solution, the solvent of NHS solution is 0.1MPBS buffer solution, after 15min, add saturated NaHCO3 solution to adjust the pH of the solution on both sides of reaction vessel 1 to 7.2-7.4; then add 50 μl concentration of 20 μg/ml human odor binding protein (amino acid sequence as shown in SEQ ID NO.1) solution, the solvent of the human odor binding protein solution is 0.1MPBS buffer, containing 5% trehalose and 5% mannitol by mass percentage. Place the reaction vessel 1 at 4°C for 2 hours, so that the human odor binding protein and the carboxyl group of 2-carboxyethyl phosphoric acid in the 2-carboxyethyl phosphoric acid solution undergo dehydration condensation to form a stable amide bond, and then form amide bond structure, then suck out the residual liquid on both sides of the reaction vessel 1, and then fill the two sides of the reaction vessel 1 with 0.1MPBS buffer solution, and after standing for 5min, suck out the PBS buffer solution to clean the unfixed human odor binding Protein, washed with 0.1MPBS buffer three times to obtain the human odor-binding protein sensor based on nanopore array, and placed at 4°C for standby. The immobilization process of human odorant binding protein on the nanopore array is shown in Figure 2.
本发明基于纳米孔阵列的人气味结合蛋白传感器可用于检测苯甲醛和二十二碳六烯酸。该应用具体如下：The human odor binding protein sensor based on the nanopore array of the present invention can be used to detect benzaldehyde and docosahexaenoic acid. The application is as follows:
(1)配制苯甲醛和二十二碳六烯酸的标准样品溶液：采用分析纯为99％的苯甲醛和二十二碳六烯酸标准贮备溶液稀释配制5种浓度分别为10-8mg/ml,10-7mg/ml,10-6mg/ml,10-5mg/ml,10-4mg/ml的相应苯甲醛和二十二碳六烯酸标准样品溶液，稀释液是分析纯为99％的甲醇溶液；(1) Preparation of standard sample solutions of benzaldehyde and docosahexaenoic acid: using standard stock solutions of benzaldehyde and docosahexaenoic acid with an analytical purity of 99% to dilute and prepare five concentrations of10-8 mg respectively /ml, 10-7 mg/ml, 10-6 mg/ml, 10-5 mg/ml, 10-4 mg/ml corresponding benzaldehyde and docosahexaenoic acid standard sample solution, the diluent is the analysis Pure 99% methanol solution;
(2)纳米孔阵列的人气味结合蛋白传感器构建的表征：首先是三电极检测系统的连接，如图1所示，先将三电极中金工作电极2的一端连接电化学工作站的工作输入端，金工作电极2的另一端插入反应皿1中；银/氯化银参比电极3的一端连接电化学工作站的参比输入端，铂丝对电极4的一端连接电化学工作站的对电极输入端。银/氯化银参比电极3及铂丝对电极4的另外一端同时插入反应皿1的另外一侧(与金工作电极2分别位于反应皿2的两侧)，电化学工作站可以采用上海辰华仪器有限公司CHI660型号的产品。当反应皿1中间没有纳米孔阵列6时，向反应皿1的两侧中分别加入与2-羧乙基磷酸溶液等体积的100μl氧化还原对溶液，氧化还原对溶液中含有10mM铁氰化钾、10mM亚铁氰化钾和0.1M的KCl，溶剂为超纯水，再向反应皿1的两侧中各加入与氧化还原对溶液等体积的100μl甲醇溶液，直接进行电化学阻抗图谱扫描，具体测试参数为初始电压为0.2V，交流电压幅度为5mV，扫频范围为1Hz～1MHz，最后得到无纳米孔阵列6条件下的电化学阻抗图谱。之后利用相同的测量方法，测量纳米孔阵列6固定于反应皿1中间的电化学阻抗图谱，以及纳米孔阵列6上固定了人气味结合蛋白的电化学阻抗图谱，3条电化学阻抗图谱如图3所示，表明成功构建了纳米孔阵列的人气味结合蛋白传感器。(2) Characterization of the construction of the human odor binding protein sensor of the nanopore array: first, the connection of the three-electrode detection system, as shown in Figure 1, first connect one end of the gold working electrode 2 in the three electrodes to the working input end of the electrochemical workstation , the other end of the gold working electrode 2 is inserted into the reaction vessel 1; one end of the silver/silver chloride reference electrode 3 is connected to the reference input end of the electrochemical workstation, and one end of the platinum wire counter electrode 4 is connected to the counter electrode input of the electrochemical workstation end. The silver/silver chloride reference electrode 3 and the other end of the platinum wire counter electrode 4 are inserted into the other side of the reaction vessel 1 at the same time (the gold working electrode 2 is located on both sides of the reaction vessel 2 respectively), and the electrochemical workstation can use Shanghai Chen China Instrument Co., Ltd. CHI660 model products. When there is no nanopore array 6 in the middle of the reaction vessel 1, add 100 μl redox pair solution equal to the volume of the 2-carboxyethyl phosphoric acid solution to both sides of the reaction vessel 1, and the redox pair solution contains 10 mM potassium ferricyanide , 10mM potassium ferrocyanide and 0.1M KCl, the solvent is ultrapure water, and then add 100 μl methanol solution equal to the redox solution to both sides of the reaction vessel 1, and directly perform electrochemical impedance spectroscopy scanning, The specific test parameters are as follows: an initial voltage of 0.2V, an AC voltage amplitude of 5mV, and a frequency sweep range of 1Hz-1MHz, and finally the electrochemical impedance spectrum without the nanopore array 6 is obtained. Then use the same measurement method to measure the electrochemical impedance spectrum of the nanopore array 6 fixed in the middle of the reaction vessel 1, and the electrochemical impedance spectrum of the human odor binding protein immobilized on the nanopore array 6. The three electrochemical impedance spectra are shown in the figure 3, indicating that the human odor-binding protein sensor of the nanopore array was successfully constructed.
(3)检测苯甲醛标准样品溶液：三电极检测系统的连接及具体测试参数同步骤2，测试传感器是纳米孔阵列的人气味结合蛋白传感器，首先进行空白对照溶液电化学阻抗图谱的测量，向反应皿1的两侧中分别加入与2-羧乙基磷酸溶液等体积的100μl氧化还原对溶液，氧化还原对溶液中含有10mM铁氰化钾、10mM亚铁氰化钾和0.1M的KCl，溶剂为超纯水，再向反应皿1的两侧中各加入与氧化还原对溶液等体积的100μl甲醇溶液，以电化学工作站作为仪器平台对的纳米孔阵列的人气味结合蛋白传感器进行电化学阻抗图谱扫描，最后得到电化学阻抗图谱。测量结束后吸出反应皿1的两侧中上次测量残留的溶液，然后反应皿1的两侧均加满0.1MPBS缓冲液，静置5min后缓慢吸出PBS缓冲液，用于清洗纳米孔阵列6，消除上次测量残留的铁氰化钾/亚铁氰化钾及PBS缓冲液的混合溶液影响。之后再进行苯甲醛标准样品溶液的检测，分别加入100μl氧化还原对溶液和100μl的10-8mg/ml的苯甲醛标准样品溶液至反应皿1的两侧中，进行电化学阻抗图谱的测量，得到10-8mg/ml的苯甲醛标准样品溶液对应的电化学阻抗图谱。一次测量结束后，先吸出反应皿1的两侧中上次测量残留的溶液，然后向反应皿1的两侧中各加满PBS缓冲液，静置5min后缓慢吸出PBS缓冲液，以消除上次测量残留的铁氰化钾/亚铁氰化钾及10-8mg/ml的苯甲醛标准样品溶液的影响。重复上述苯甲醛标准样品溶液的测量，直至完成10-7mg/ml,10-6mg/ml,10-5mg/ml,和10-4mg/ml浓度下苯甲醛标准样品溶液的测量。最终得到纳米孔阵列的人气味结合蛋白传感器与5种不同浓度(10-8mg/ml,10-7mg/ml,10-6mg/ml,10-5mg/ml,和10-4mg/ml)下的苯甲醛标准样品溶液相互作用的电化学阻抗图谱，如图4所示。(3) Detect benzaldehyde standard sample solution: the connection of the three-electrode detection system and the specific test parameters are the same as step 2. The test sensor is the human odor binding protein sensor of the nanopore array. At first, the measurement of the electrochemical impedance spectrum of the blank control solution is carried out. Add 100 μl redox pair solution equal to the volume of 2-carboxyethyl phosphoric acid solution to both sides of the reaction vessel 1, the redox pair solution contains 10 mM potassium ferricyanide, 10 mM potassium ferrocyanide and 0.1 M KCl, The solvent is ultrapure water, and 100 μl of methanol solution equal to the volume of the redox solution is added to both sides of the reaction vessel 1, and the electrochemical workstation is used as the instrument platform to conduct electrochemical experiments on the human odor binding protein sensor of the nanopore array. The impedance spectrum is scanned, and finally the electrochemical impedance spectrum is obtained. After the measurement, suck out the remaining solution from the previous measurement on both sides of the reaction vessel 1, then fill both sides of the reaction vessel 1 with 0.1MPBS buffer solution, and slowly suck out the PBS buffer solution after standing for 5 minutes to clean the nanopore array 6 , Eliminate the influence of the mixed solution of potassium ferricyanide/potassium ferrocyanide and PBS buffer in the last measurement. Then carry out the detection of the benzaldehyde standard sample solution, add 100 μl of redox solution and 100 μl of 10−8 mg/ml benzaldehyde standard sample solution to both sides of the reaction vessel 1 respectively, and measure the electrochemical impedance spectrum. The electrochemical impedance spectrum corresponding to the 10-8 mg/ml benzaldehyde standard sample solution was obtained. After one measurement, first suck out the solution remaining in the previous measurement on both sides of the reaction vessel 1, then fill the both sides of the reaction vessel 1 with PBS buffer solution, and slowly suck out the PBS buffer solution after standing for 5 minutes to eliminate the remaining solution on both sides of the reaction vessel 1. Measure the influence of residual potassium ferricyanide/potassium ferrocyanide and 10-8 mg/ml benzaldehyde standard sample solution. Repeat the measurement of the above-mentioned benzaldehyde standard sample solution until the measurement of the benzaldehyde standard sample solution at concentrations of 10-7 mg/ml, 10-6 mg/ml, 10-5 mg/ml, and 10-4 mg/ml is completed. Finally, human odorant-binding protein sensors with nanopore arrays were obtained with 5 different concentrations (10-8 mg/ml, 10-7 mg/ml, 10-6 mg/ml, 10-5 mg/ml, and 10-4 mg /ml) the electrochemical impedance spectrum of the interaction of the benzaldehyde standard sample solution, as shown in Figure 4.
(4)建立纳米孔阵列的人气味结合蛋白传感器的等效电路模型11：检测到纳米孔阵列的人气味结合蛋白传感器的电化学阻抗谱中包含2个半圆，不同浓度的苯甲醛标准样品溶液作用下，电化学阻抗谱中左侧的半圆基本没有发生变化，而右侧圆的直径随着检测苯甲醛标准样品溶液的浓度的增加而增大，因此建立纳米孔阵列的人气味结合蛋白传感器的等效电路模型11对电化学阻抗图普进行分析，如图5所示。其中三电极(包含金工作电极2，银/氯化银对电极3，铂丝对电极4)检测系统由经典Randles模型7等效，包含溶液阻抗Rs,电子转移电阻Rct,Warburg阻抗Zw,及常相角元件CPE；纳米孔阵列等效电路8为纳米孔电阻Rpore和纳米孔电容Cpore的并联电路；人气味结合蛋白的等效电路9包括人气味结合蛋白电阻Rp和人气味结合蛋白电容Cp。其中，右侧圆的直径代表了纳米孔阵列阻抗及人气味结合蛋白阻抗R的整体作用，其中R＝(Rpore+Rp)/2，随着苯甲醛标准样品溶液的浓度增加，电化学阻抗图谱右侧圆的直径逐渐增大，利用纳米孔阵列的人气味结合蛋白传感器的等效电路模型11进行分析，可以得到苯甲醛标准样品溶液作用下，人气味结合蛋白修饰的纳米孔阵列阻抗R10在增加。(4) Establishment of the equivalent circuit model 11 of the human odor-binding protein sensor of the nanopore array: the electrochemical impedance spectrum of the human odor-binding protein sensor of the nanopore array contains two semicircles, and different concentrations of benzaldehyde standard sample solutions Under the action, the semicircle on the left in the electrochemical impedance spectrum basically does not change, while the diameter of the circle on the right increases with the increase of the concentration of the detection benzaldehyde standard sample solution, so the human odor binding protein sensor of the nanopore array is established The equivalent circuit model 11 analyzes the electrochemical impedance map, as shown in FIG. 5 . The detection system of the three electrodes (including gold working electrode 2, silver/silver chloride counter electrode 3, and platinum wire counter electrode 4) is equivalent to the classic Randles model 7, including solution impedance Rs , electron transfer resistance Rct , and Warburg impedance Zw , and the constant phase angle element CPE; the nanopore array equivalent circuit 8 is a parallel circuit of the nanopore resistance Rpore and the nanopore capacitance Cpore ; the equivalent circuit 9 of the human odorant binding protein includes the human odorant binding protein resistance Rp and Human odorant binding protein capacitance Cp . Among them, the diameter of the circle on the right represents the overall effect of the nanopore array impedance and the human odor binding protein impedance R, where R=(Rpore +Rp )/2, as the concentration of the benzaldehyde standard sample solution increases, the electrochemical The diameter of the circle on the right side of the impedance spectrum increases gradually. Using the equivalent circuit model 11 of the human odor binding protein sensor of the nanopore array to analyze, the impedance of the nanopore array modified by the human odor binding protein under the action of the benzaldehyde standard sample solution can be obtained. R10 is increasing.
(5)建立苯甲醛分子溶液标准浓度-归一化阻抗曲线：利用步骤4中建立的纳米孔阵列的人气味结合蛋白传感器的等效电路模型11，选择步骤2中检测到的人气味结合蛋白修饰的纳米孔阵列阻抗R10进行分析，利用5种不同浓度(10-8mg/ml,10-7mg/ml,10-6mg/ml,10-5mg/ml,和10-4mg/ml)苯甲醛标准样品溶液作用下的人气味结合蛋白修饰的纳米孔阵列阻抗R10，计算归一化阻抗，即标准样品溶液对应的人气味结合蛋白修饰的纳米孔阵列阻抗R1与空白对照溶液对应的人气味结合蛋白修饰的纳米孔阵列阻抗R0的差值相对于空白对照溶液对应的人气味结合蛋白修饰的纳米孔阵列阻抗R0的变化百分比(R1-R0)/R0，得到苯甲醛标准样品溶液浓度与归一化阻抗之间的关系曲线y＝ax+b，其中，x为苯甲醛标准样品溶液浓度，y为归一化阻抗，a和b为常数，实现对苯甲醛标准样品溶液的检测，如图6所示；(5) Establish the standard concentration-normalized impedance curve of benzaldehyde molecular solution: Utilize the equivalent circuit model 11 of the human odor binding protein sensor of the nanopore array established in step 4, select the human odor binding protein detected in step 2 The modified nanopore array impedance R10 was analyzed using 5 different concentrations (10-8 mg/ml, 10-7 mg/ml, 10-6 mg/ml, 10-5 mg/ml, and 10-4 mg/ ml) the human odor binding protein modified nanopore array impedance R10 under the actionof benzaldehyde standard sample solution, calculate the normalized impedance, that is, the human odor binding protein modified nanopore array impedance R1 corresponding to the standard sample solution and the blank control solution The difference of the impedance R0 of the nanopore array modified by the corresponding human odorant binding protein relative to the change percentage of the impedance R0 of the nanopore array modified by the human odorant binding protein corresponding to the blank control solution (R1 -R0 )/R0 , Obtain the relationship curve y=ax+b between benzaldehyde standard sample solution concentration and normalized impedance, wherein, x is the benzaldehyde standard sample solution concentration, y is normalized impedance, and a and b are constants, realize the paraben The detection of formaldehyde standard sample solution, as shown in Figure 6;
(6)检测二十二碳六烯酸标准样品溶液，建立二十二碳六烯酸分子溶液标准浓度-归一化阻抗曲线：方法同步骤3，最终得到纳米孔阵列的人气味结合蛋白传感器与5种不同浓度(10-8mg/ml,10-7mg/ml,10-6mg/ml,10-5mg/ml,和10-4mg/ml)下的二十二碳六烯酸标准样品溶液相互作用的电化学阻抗图谱，如图4所示；方法同上述步骤4和5，最终得到纳米孔阵列的人气味结合蛋白传感器与5种不同浓度(10-8mg/ml,10-7mg/ml,10-6mg/ml,10-5mg/ml,和10-4mg/ml)下的二十二碳六烯酸标准样品溶液与归一化阻抗之间的关系曲线，如图6所示。(6) Detect the docosahexaenoic acid standard sample solution, and establish the standard concentration-normalized impedance curve of the docosahexaenoic acid molecular solution: the method is the same as step 3, and finally the human odor binding protein sensor of the nanopore array is obtained Docosahexaene at 5 different concentrations (10-8 mg/ml, 10-7 mg/ml, 10-6 mg/ml, 10-5 mg/ml, and 10-4 mg/ml) The electrochemical impedance spectrum of acid standard sample solution interaction is as shown in Figure 4; method is the same as above-mentioned steps 4 and 5, finally obtains the human odor binding protein sensor of nanopore array and 5 kinds of different concentrations (10-8 mg/ml, 10-7 mg/ml, 10-6 mg/ml, 10-5 mg/ml, and 10-4 mg/ml) the relationship between docosahexaenoic acid standard sample solution and normalized impedance Curve, as shown in Figure 6.
(7)完成待测苯甲醛样品溶液和待测二十二碳六烯酸样品溶液的检测，测量得到待测苯甲醛样品溶液和待测二十二碳六烯酸样品溶液的电化学阻抗图谱及归一化阻抗，对应得出待测苯甲醛样品溶液和待测二十二碳六烯酸样品溶液的浓度。(7) Complete the detection of the benzaldehyde sample solution to be measured and the docosahexaenoic acid sample solution to be measured, and measure the electrochemical impedance spectrum of the benzaldehyde sample solution to be measured and the docosahexaenoic acid sample solution to be measured And the normalized impedance, corresponding to obtain the concentrations of the benzaldehyde sample solution to be measured and the docosahexaenoic acid sample solution to be measured.
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