技术领域technical field
本发明涉及一种用于检测雌二醇的传感器的制备方法，属于新型纳米功能材料与电化学生物传感分析技术领域。The invention relates to a preparation method of a sensor for detecting estradiol, and belongs to the technical field of novel nanometer functional materials and electrochemical biosensing analysis.
背景技术Background technique
环境雌激素是指一类进入机体后，具有干扰体内正常内分泌物质的合成、释放、运输、结合、代谢等过程，激活或抑制内分泌系统的功能，从而破坏维持机体稳定性和调控作用的化合物，环境雌激素种类繁多，包括人工合成化合物及植物天然雌激素，广泛分布于自然界中。雌二醇便是一种类固醇类合成雌激素。目前，检测雌二醇的方法主要有色谱法、质谱法等。此类方法仪器贵重、操作复杂，化验人员需要专业培训后才能进行检测。因此，研发成本低、检测快、灵敏度高、特异性强的雌二醇传感器具有重要意义。Environmental estrogen refers to a class of compounds that interfere with the synthesis, release, transport, binding, and metabolism of normal endocrine substances in the body after entering the body, activate or inhibit the function of the endocrine system, and thereby destroy the stability and regulation of the body. There are many kinds of environmental estrogens, including synthetic compounds and plant natural estrogens, which are widely distributed in nature. Estradiol is a synthetic estrogen that is a steroid. At present, the methods for detecting estradiol mainly include chromatography and mass spectrometry. Such methods require expensive instruments and complex operations, and laboratory personnel need professional training before they can perform detection. Therefore, it is of great significance to develop estradiol sensors with low cost, fast detection, high sensitivity and strong specificity.
电化学生物传感分析技术由于操作简便、检测速度快等优势，日益得到人们的重视。目前，用于检测雌二醇的电化学生物传感分析技术按照检测手段来分主要有电化学传感器、电致化学发光传感器和光电化学传感器三种。其中，电致化学发光传感器和光电化学传感器相对于电化学传感器，具有背景信号噪音少、灵敏度高、检测成本低等特点，近几年被越来越多的研究者所关注。Due to the advantages of simple operation and fast detection speed, electrochemical biosensing analysis technology has been paid more and more attention by people. At present, the electrochemical biosensing analysis technologies used to detect estradiol are mainly divided into three types: electrochemical sensors, electrochemiluminescence sensors and photoelectrochemical sensors according to the detection methods. Among them, compared with electrochemical sensors, electrochemiluminescence sensors and photoelectrochemical sensors have the characteristics of less background signal noise, high sensitivity, and low detection cost. In recent years, more and more researchers have paid attention to them.
电致化学发光也称为电化学发光，是指通过电化学方法在电极表面产生一些特殊的物质，这些物质之间或与体系中其他组分之间通过电子传递形成激发态，由激发态返回到基态产生发光现象。电致化学发光传感器即通过改变电极表面的修饰材料，与分析物产生电化学发光，在最优条件下，根据分析物浓度与电化学发光强度的相关变化实现对分析物的定性定量分析。Electrochemiluminescence, also known as electrochemiluminescence, refers to the generation of some special substances on the surface of electrodes by electrochemical methods, and these substances form excited states through electron transfer between them or with other components in the system, and return from excited states to The ground state produces luminescence. The electrochemiluminescence sensor generates electrochemiluminescence with the analyte by changing the modified material on the electrode surface. Under optimal conditions, the qualitative and quantitative analysis of the analyte is realized according to the correlation between the concentration of the analyte and the intensity of the electrochemiluminescence.
光电化学传感器是基于外加光源激发光电敏感材料导致电子-空穴对进行分离，在合适的偏电位条件下，实现电子在电极、半导体及修饰物和分析物上的快速传递，并形成光电流。在最优条件下，分析物浓度的变化会直接影响光电流的大小，可以根据光电流的变化实现对分析物的定性定量分析。The photoelectrochemical sensor is based on the excitation of photosensitive materials by an external light source, resulting in the separation of electron-hole pairs. Under appropriate bias conditions, the rapid transfer of electrons on electrodes, semiconductors, modifiers, and analytes is realized, and photocurrents are formed. Under optimal conditions, the change of analyte concentration will directly affect the size of the photocurrent, and the qualitative and quantitative analysis of the analyte can be realized according to the change of photocurrent.
但是，由于电致化学发光传感器需要外置光信号捕捉设备如光电二极管等，而光电化学传感器需要外设光源来激发光电敏感材料，这在一定程度上影响了二者操作的便捷性，限制了他们在实际生产、生活中更为广泛的应用。因此，设计、制备更为简便、快捷的检测雌二醇的电化学生物传感分析技术具有十分重要的实用价值。However, since electrochemiluminescence sensors require external light signal capture devices such as photodiodes, while photoelectrochemical sensors require external light sources to excite photosensitive materials, this affects the convenience of both operations to a certain extent and limits They are more widely used in actual production and life. Therefore, it is of great practical value to design and prepare a simpler and faster electrochemical biosensing analysis technology for the detection of estradiol.
发明内容Contents of the invention
本发明的目的在于提供一种操作简单、携带方便、检测快、成本低的雌二醇传感器的制备方法，所制备的传感器，可用于日常生产、生活等领域的对雌二醇的快速、灵敏检测。基于此目的，本发明在同一电解池中，采用四电极系统，即两个工作电极、一个对电极和一个参比电极，其中工作电极1采用磷酸镍铵微纳材料NH4NiPO4和电聚合的鲁米诺进行修饰，作为电致化学发光工作电极W1，也作为自发光光源，工作电极2采用二氧化钛纳米粒子溶胶TiO2NPs和雌二醇抗体共同进行修饰，作为光电化学工作电极W2。进行检测时，在电解池中加入固定浓度过氧化氢后，在W1上施加阶跃电压，由于NH4NiPO4的稳定发光的作用，鲁米诺与过氧化氢反应，产生电致化学发光，这便相当于“开灯”，当阶跃电压为0时，电致化学发光消失，这便相当于“关灯”，与此同时在W2上一直施加恒定电压，由于TiO2NPs会因为电致化学发光产生的发光激发导致电子-空穴对进行分离，标记在雌二醇二抗上的辣根过氧化物酶HRP催化过氧化氢产生氧气，使过氧化氢成为空穴“给体”，从而在W2上得到光电流，当电致化学发光消失，即“关灯”时，光电流随即消失。由于在固定过氧化氢浓度的前提下，光电流大小与HRP浓度正相关，当被测物中雌二醇浓度越大，当与一抗免疫结合到W2上时，再免疫结合标记有HRP的雌二醇二抗的浓度就会越大，产生的光电流也就越大，因此通过记录光电流的大小即可实现对雌二醇的检测。The purpose of the present invention is to provide a method for preparing an estradiol sensor that is simple to operate, easy to carry, fast to detect, and low in cost. The prepared sensor can be used for rapid and sensitive detection of estradiol in the fields of daily production and life detection. Based on this purpose, the present invention adopts a four-electrode system in the same electrolytic cell, i.e. two working electrodes, a counter electrode and a reference electrode, wherein the working electrode 1 adopts nickel ammonium phosphate micro-nano material NH4 NiPO4 and electropolymerization The luminol was modified as the electrochemiluminescent working electrode W1 and also as the self-luminous light source. The working electrode 2 was modified with titanium dioxide nanoparticle sol TiO2 NPs and estradiol antibody as the photoelectrochemical working electrode W2. When performing detection, after adding a fixed concentration of hydrogen peroxide into the electrolytic cell, a step voltage is applied to W1. Due to the stable luminescence ofNH4NiPO4 , luminol reacts with hydrogen peroxide to produce electrochemiluminescence. This is equivalent to "turning on the light". When the step voltage is 0, the electrochemiluminescence disappears, which is equivalent to "turning off the light". At the same time, a constant voltage is always applied to W2, because TiO2 NPs will The luminescent excitation generated by chemiluminescence leads to the separation of electron-hole pairs, and the horseradish peroxidase HRP labeled on the estradiol secondary antibody catalyzes hydrogen peroxide to generate oxygen, making hydrogen peroxide a hole "donor" , so that the photocurrent is obtained on W2, and when the electrochemiluminescence disappears, that is, when the light is turned off, the photocurrent disappears immediately. Since the magnitude of the photocurrent is positively correlated with the concentration of HRP under the premise of a fixed concentration of hydrogen peroxide, when the concentration of estradiol in the analyte is higher, when the primary antibody is immunocombined with W2, the immunocombination with HRP will be more effective. The greater the concentration of the estradiol secondary antibody, the greater the photocurrent generated, so the detection of estradiol can be realized by recording the magnitude of the photocurrent.
基于以上发明原理，本发明采用的技术方案如下：Based on above invention principle, the technical scheme that the present invention adopts is as follows:
1.一种基于自发光激发的雌二醇光电化学传感器的制备方法，其特征在于，制备步骤为：1. a kind of preparation method based on the estradiol photoelectrochemical sensor excited by self-luminescence, it is characterized in that, preparation step is:
（1）电致化学发光工作电极W1的制备：(1) Preparation of electrochemiluminescence working electrode W1:
1）以ITO导电玻璃为工作电极，在电极表面滴涂磷酸镍铵微纳材料NH4NiPO4溶液，覆盖面积为1cm×1cm，室温下晾干；1) Using ITO conductive glass as the working electrode, drop-coat nickel ammonium phosphate micro-nano material NH4 NiPO4 solution on the surface of the electrode, covering an area of 1cm×1cm, and dry it at room temperature;
2）将1）得到的工作电极，浸入电解液中，浸入面积为NH4NiPO4所覆盖的面积，利用三电极系统对工作电极进行电化学沉积，沉积后取出工作电极，使用超纯水清洗，4℃下避光干燥，制得电致化学发光工作电极W1；2) Immerse the working electrode obtained in 1) into the electrolyte, and the immersion area is the area covered by NH4 NiPO4 , use the three-electrode system to electrochemically deposit the working electrode, take out the working electrode after deposition, and clean it with ultrapure water , and dried at 4°C in the dark to prepare the electrochemiluminescence working electrode W1;
所述的NH4NiPO4溶液为磷酸镍铵微纳材料水溶液，所述磷酸镍铵微纳材料的制备步骤为：在40mL水中加入2.0~4.0g铵盐和0.15~0.25g磷酸盐，完全溶解后，加入0.15~0.25g二氯化镍，在30~45℃下搅拌10~14h，离心分离，将产品置于50℃下干燥，即得到NH4NiPO4；The NH4 NiPO4 solution is an aqueous solution of nickel ammonium phosphate micro-nano material, and the preparation steps of the nickel ammonium phosphate micro-nano material are: adding 2.0-4.0 g of ammonium salt and 0.15-0.25 g of phosphate in 40 mL of water, completely dissolving Finally, add 0.15~0.25g of nickel dichloride, stir at 30~45°C for 10~14h, centrifuge and dry the product at 50°C to obtain NH4 NiPO4 ;
所述的铵盐选自下列之一：氯化铵、溴化铵、磷酸铵、磷酸二氢铵、磷酸氢二铵；The ammonium salt is selected from one of the following: ammonium chloride, ammonium bromide, ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate;
所述的磷酸盐选自下列之一：磷酸铵、磷酸二氢铵、磷酸氢二铵；The phosphate is selected from one of the following: ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate;
所述的电解液为含有鲁米诺的硫酸溶液，所述的电解液中鲁米诺的浓度为1~10mmol/L，硫酸浓度为0.1~1.0mol/L；Described electrolytic solution is the sulfuric acid solution containing luminol, and the concentration of luminol in described electrolytic solution is 1～10mmol/L, and sulfuric acid concentration is 0.1～1.0mol/L;
所述的三电极系统，包括工作电极、参比电极和对电极，所述的参比电极为饱和甘汞电极，所述的对电极为铂丝电极；The three-electrode system includes a working electrode, a reference electrode and a counter electrode, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode;
所述的电化学沉积过程，采用的电化学方法为循环伏安法，起始电压为-0.2V，终止电压为1.5V，扫速为100mv/s，循环20~30圈；In the electrochemical deposition process, the electrochemical method adopted is cyclic voltammetry, the initial voltage is -0.2V, the final voltage is 1.5V, the scan rate is 100mv/s, and the cycle is 20~30 cycles;
（2）光电化学工作电极W2的制备：(2) Preparation of photoelectrochemical working electrode W2:
1）以ITO导电玻璃为工作电极，在电极表面滴涂8~12μL二氧化钛纳米粒子溶胶TiO2NPs，室温下晾干；1) Use ITO conductive glass as the working electrode, drop-coat 8~12 μL titanium dioxide nanoparticle sol TiO2 NPs on the surface of the electrode, and dry it at room temperature;
2）将1）中得到的工作电极放入马弗炉中，在450℃下进行退火处理，处理后冷却至室温；2) Put the working electrode obtained in 1) into a muffle furnace, perform annealing treatment at 450°C, and cool to room temperature after treatment;
3）将2）中得到的工作电极表面滴涂8~12μL雌二醇抗体溶液，4℃下干燥，干燥后用超纯水清洗，4℃下干燥，制得光电化学工作电极W2；3) Apply 8-12 μL of the estradiol antibody solution on the surface of the working electrode obtained in 2), dry at 4°C, wash with ultrapure water after drying, and dry at 4°C to prepare the photoelectrochemical working electrode W2;
所述的TiO2NPs为1mg/mL的二氧化钛纳米粒子水溶液；The TiO2 NPs is a 1 mg/mL aqueous solution of titanium dioxide nanoparticles;
所述的雌二醇抗体溶液的浓度为300μg/mL；The concentration of the estradiol antibody solution is 300 μg/mL;
（3）基于自发光激发的雌二醇光电化学传感器的制备方法：(3) Preparation method of estradiol photoelectrochemical sensor based on self-luminescence excitation:
1）将W1和W2面面相对插入电解池中，W1与W2间距为0.5cm~1.5cm；1) Insert W1 and W2 face to face into the electrolytic cell, the distance between W1 and W2 is 0.5cm~1.5cm;
2）以Ag/AgCl为参比电极RE、铂丝电极为对电极CE，插入电解池中，与W1和W2共同组成四电极系统；2) Take Ag/AgCl as the reference electrode RE and platinum wire electrode as the counter electrode CE, insert it into the electrolytic cell, and form a four-electrode system together with W1 and W2;
3）在电解池中加入10mLpH值为11~13的NaOH溶液和0.2mL浓度为1mmol/L的过氧化氢溶液；3) Add 10mL NaOH solution with a pH value of 11~13 and 0.2mL hydrogen peroxide solution with a concentration of 1mmol/L in the electrolytic cell;
4）将1）~3）所制得四电极系统以及电解池置于暗盒中，即制得基于自发光激发的雌二醇光电化学传感器。4) Put the four-electrode system and the electrolytic cell prepared in 1) to 3) in a cassette to prepare a photoelectrochemical sensor for estradiol based on self-luminescence excitation.
2.根据技术方案1所述的制备方法所制备的传感器，应用于雌二醇的检测，其检测步骤为：2. The sensor prepared according to the preparation method described in technical scheme 1 is applied to the detection of estradiol, and its detection steps are:
（1）在W2上滴加待测雌二醇溶液，在待测雌二醇溶液中的雌二醇与W2上的雌二醇抗体免疫结合后，冲洗掉其余物质，再滴加辣根过氧化物酶HRP标记的雌二醇二抗进行免疫结合，结合后冲洗掉多余的HRP标记的雌二醇二抗，制得待测W2；(1) Add the estradiol solution to be tested dropwise on W2. After the estradiol in the estradiol solution to be tested is immunocombined with the estradiol antibody on W2, wash off the remaining substances, and then add horseradish dropwise for a long time. The oxidase HRP-labeled estradiol secondary antibody was immunocombined, and after binding, the excess HRP-labeled estradiol secondary antibody was washed away to prepare W2 to be tested;
（2）利用电化学工作站，在含有固定浓度过氧化氢的电解液中，在W1上采用阶跃电压的方法对W1施加阶跃电压，初始电压为0v，阶跃电压为0.7~0.9v，阶跃时间为10~30s；同时，在待测W2上采用时间-电流方法对待测W2施加恒定电压，电压为0~0.6v；待测W2上的电流会随着待测雌二醇溶液中雌二醇的浓度增大而相应增大，根据所得电流增大值得出待测雌二醇溶液中雌二醇的浓度；(2) Using an electrochemical workstation, in an electrolyte containing a fixed concentration of hydrogen peroxide, apply a step voltage to W1 by using a step voltage method on W1. The initial voltage is 0v, and the step voltage is 0.7~0.9v. The step time is 10~30s; at the same time, a constant voltage is applied to the W2 to be tested by using the time-current method, and the voltage is 0~0.6v; the current on the W2 to be tested will follow the estradiol solution The concentration of estradiol increases and correspondingly increases, and the concentration of estradiol in the estradiol solution to be measured is obtained according to the obtained current increase value;
所述的HRP标记的雌二醇二抗的浓度为300μg/mL。The concentration of the HRP-labeled estradiol secondary antibody is 300 μg/mL.
本发明的有益成果Beneficial results of the present invention
（1）本发明所述的雌二醇传感器制备简单，操作方便，无需外部辅助设备，利用检测设备的微型化、便携化，并实现了对雌二醇的快速、灵敏、高选择性检测，具有广阔的市场发展前景；(1) The estradiol sensor of the present invention is simple to prepare, easy to operate, does not require external auxiliary equipment, utilizes the miniaturization and portability of detection equipment, and realizes rapid, sensitive and highly selective detection of estradiol, Has broad market development prospects;
（2）本发明首次在同一电解池中采用四电极系统检测雌二醇，并实现了电致化学发光与光电化学双功能信号放大策略。在电解池中随着雌二醇浓度的增加，修饰在W2上的HRP标记的雌二醇二抗就会增加，也就意味着HRP对过氧化氢催化的提高。如此以来，一方面，使得电致化学发光强度线性增加，所激发的光电流线形增大；另一方面，过氧化氢作为电子给体，使得光电化学反应中光电流线形增大。因此，电致化学发光和光电化学两种方法在同一电解池中、同一电化学工作站下共同反应、相互作用，实现了对雌二醇检测电信号的双重放大，极大地提高了检测灵敏度和检出限，具有重要的科学意义和应用价值。(2) The present invention uses a four-electrode system to detect estradiol in the same electrolytic cell for the first time, and realizes a dual-function signal amplification strategy of electrochemiluminescence and photoelectrochemistry. As the concentration of estradiol increases in the electrolytic cell, the number of HRP-labeled estradiol secondary antibodies modified on W2 will increase, which means that HRP catalyzes hydrogen peroxide. In this way, on the one hand, the intensity of electrochemiluminescence increases linearly, and the excited photocurrent increases linearly; on the other hand, hydrogen peroxide acts as an electron donor, which makes the photocurrent linearly increase in the photoelectrochemical reaction. Therefore, the two methods of electrochemiluminescence and photoelectrochemistry react and interact together in the same electrolytic cell and under the same electrochemical workstation, realizing the double amplification of the electrical signal of estradiol detection, which greatly improves the detection sensitivity and detection efficiency. It has important scientific significance and application value.
具体实施方式detailed description
实施例1一种基于自发光激发的雌二醇光电化学传感器Embodiment 1 A kind of estradiol photoelectrochemical sensor based on self-luminous excitation
一种基于自发光激发的雌二醇光电化学传感器的制备方法，具体的制备步骤为：A method for preparing an estradiol photoelectrochemical sensor based on self-luminescence excitation, the specific preparation steps are:
（1）电致化学发光工作电极W1的制备：(1) Preparation of electrochemiluminescent working electrode W1:
1）以ITO导电玻璃为工作电极，在电极表面滴涂NH4NiPO4，覆盖面积为1cm×1cm，室温下晾干；1) Use ITO conductive glass as the working electrode, drop-coat NH4 NiPO4 on the surface of the electrode, covering an area of 1cm×1cm, and dry it at room temperature;
2）将1）得到的工作电极，浸入电解液中，浸入面积为NH4NiPO4所覆盖的面积，利用三电极系统对工作电极进行电化学沉积，沉积后取出工作电极，使用超纯水清洗，4℃下避光干燥，制得电致化学发光工作电极W1；2) Immerse the working electrode obtained in 1) into the electrolyte, and the immersion area is the area covered by NH4 NiPO4 , use the three-electrode system to electrochemically deposit the working electrode, take out the working electrode after deposition, and clean it with ultrapure water , and dried at 4°C in the dark to prepare the electrochemiluminescence working electrode W1;
所述的NH4NiPO4溶液为磷酸镍铵微纳材料水溶液，所述磷酸镍铵微纳材料的制备步骤为：在40mL水中加入2.0~4.0g铵盐和0.15~0.25g磷酸盐，完全溶解后，加入0.15~0.25g二氯化镍，在30~45℃下搅拌10~14h，离心分离，将产品置于50℃下干燥，即得到NH4NiPO4；The NH4 NiPO4 solution is an aqueous solution of nickel ammonium phosphate micro-nano material, and the preparation steps of the nickel ammonium phosphate micro-nano material are: adding 2.0-4.0 g of ammonium salt and 0.15-0.25 g of phosphate in 40 mL of water, completely dissolving Finally, add 0.15~0.25g of nickel dichloride, stir at 30~45°C for 10~14h, centrifuge and dry the product at 50°C to obtain NH4 NiPO4 ;
所述的铵盐选自下列之一：氯化铵、溴化铵、磷酸铵、磷酸二氢铵、磷酸氢二铵；The ammonium salt is selected from one of the following: ammonium chloride, ammonium bromide, ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate;
所述的磷酸盐选自下列之一：磷酸铵、磷酸二氢铵、磷酸氢二铵；The phosphate is selected from one of the following: ammonium phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate;
所述的电解液为含有鲁米诺的硫酸溶液，所述的电解液中鲁米诺的浓度为1mmol/L，硫酸浓度为0.1mol/L；Described electrolytic solution is the sulfuric acid solution containing luminol, and the concentration of luminol in described electrolytic solution is 1mmol/L, and sulfuric acid concentration is 0.1mol/L;
所述的三电极系统，包括工作电极、参比电极和对电极，所述的参比电极为饱和甘汞电极，所述的对电极为铂丝电极；The three-electrode system includes a working electrode, a reference electrode and a counter electrode, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode;
所述的电化学沉积过程，采用的电化学方法为循环伏安法，起始电压为-0.2V，终止电压为1.5V，扫速为100mv/s，循环20圈。In the electrochemical deposition process, the electrochemical method adopted is cyclic voltammetry, the initial voltage is -0.2V, the final voltage is 1.5V, the scan rate is 100mv/s, and the cycle is 20 cycles.
（2）光电化学工作电极W2的制备：(2) Preparation of photoelectrochemical working electrode W2:
1）以ITO导电玻璃为工作电极，在电极表面滴涂8μL二氧化钛纳米粒子溶胶TiO2NPs，室温下晾干；1) Using ITO conductive glass as the working electrode, drop-coat 8 μL titanium dioxide nanoparticle sol TiO2 NPs on the surface of the electrode, and dry it at room temperature;
2）将1）中得到的工作电极放入马弗炉中，在450℃下进行退火处理，处理后冷却至室温；2) Put the working electrode obtained in 1) into a muffle furnace, perform annealing treatment at 450°C, and cool to room temperature after treatment;
3）将2）中得到的工作电极表面滴涂8μL雌二醇抗体溶液，4℃下干燥，干燥后用超纯水清洗，4℃下干燥，制得光电化学工作电极W2；3) Apply 8 μL of the estradiol antibody solution on the surface of the working electrode obtained in 2), dry at 4°C, wash with ultrapure water after drying, and dry at 4°C to prepare the photoelectrochemical working electrode W2;
所述的TiO2NPs为1mg/mL的二氧化钛纳米粒子水溶液；The TiO2 NPs is a 1 mg/mL aqueous solution of titanium dioxide nanoparticles;
所述的雌二醇抗体溶液的浓度为300μg/mL；The concentration of the estradiol antibody solution is 300 μg/mL;
（3）基于自发光激发的雌二醇光电化学传感器的制备方法：(3) Preparation method of estradiol photoelectrochemical sensor based on self-luminescence excitation:
1）将（1）中制备的W1和（2）中制备的W2面面相对插入电解池中，W1与W2间距为0.5cm；1) Insert W1 prepared in (1) and W2 prepared in (2) face to face into the electrolytic cell, and the distance between W1 and W2 is 0.5cm;
2）以Ag/AgCl为参比电极RE、铂丝电极为对电极CE，插入电解池中，与W1和W2共同组成四电极系统；2) Take Ag/AgCl as the reference electrode RE and platinum wire electrode as the counter electrode CE, insert it into the electrolytic cell, and form a four-electrode system together with W1 and W2;
3）在电解池中加入10mLpH值为11的NaOH溶液和0.2mL浓度为1mmol/L的过氧化氢溶液；3) Add 10 mL of NaOH solution with a pH value of 11 and 0.2 mL of a hydrogen peroxide solution with a concentration of 1 mmol/L into the electrolytic cell;
4）将1）~3）所制得四电极系统以及电解池置于暗盒中，即制得基于自发光激发的雌二醇光电化学传感器。4) Put the four-electrode system and the electrolytic cell prepared in 1) to 3) in a cassette to prepare a photoelectrochemical sensor for estradiol based on self-luminescence excitation.
实施例2一种基于自发光激发的雌二醇光电化学传感器Embodiment 2 A kind of estradiol photoelectrochemical sensor based on self-luminous excitation
一种基于自发光激发的雌二醇光电化学传感器的制备方法，具体的制备步骤为：A method for preparing an estradiol photoelectrochemical sensor based on self-luminescence excitation, the specific preparation steps are:
（1）电致化学发光工作电极W1的制备：(1) Preparation of electrochemiluminescence working electrode W1:
制备步骤同实施例1中W1的制备步骤，不同之处为：磷酸镍铵微纳材料的制备步骤中铵盐加入量为0.3g，磷酸盐加入量为0.20g，二氯化镍加入量为0.20g，搅拌温度和时间分别为38℃和12h；电解液中鲁米诺的浓度为5mmol/L，硫酸浓度为0.5mol/L；循环伏安法进行电化学沉积时，循环25圈。The preparation steps are the same as the preparation steps of W1 in Example 1, the difference is: the ammonium salt addition is 0.3g in the preparation step of the nickel ammonium phosphate micro-nano material, the phosphate addition is 0.20g, and the nickel dichloride addition is 0.20g, the stirring temperature and time are 38°C and 12h respectively; the concentration of luminol in the electrolyte is 5mmol/L, and the concentration of sulfuric acid is 0.5mol/L; when electrochemical deposition is carried out by cyclic voltammetry, cycle 25 times.
（2）光电化学工作电极W2的制备：(2) Preparation of photoelectrochemical working electrode W2:
1）以ITO导电玻璃为工作电极，在电极表面滴涂10μL二氧化钛纳米粒子溶胶TiO2NPs，室温下晾干；1) Using ITO conductive glass as the working electrode, drop-coat 10 μL titanium dioxide nanoparticle sol TiO2 NPs on the surface of the electrode, and dry it at room temperature;
2）将1）中得到的工作电极放入马弗炉中，在450℃下进行退火处理，处理后冷却至室温；2) Put the working electrode obtained in 1) into a muffle furnace, perform annealing treatment at 450°C, and cool to room temperature after treatment;
3）将2）中得到的工作电极表面滴涂10μL雌二醇抗体溶液，4℃下干燥，干燥后用超纯水清洗，4℃下干燥，制得光电化学工作电极W2；3) Apply 10 μL of the estradiol antibody solution on the surface of the working electrode obtained in 2), dry at 4°C, wash with ultrapure water after drying, and dry at 4°C to prepare the photoelectrochemical working electrode W2;
其余同实施例1中W2的制备步骤。The rest are the same as the preparation steps of W2 in Example 1.
（3）基于自发光激发的雌二醇光电化学传感器的制备方法：(3) Preparation method of estradiol photoelectrochemical sensor based on self-luminescence excitation:
制备步骤同实施例1，不同之处为W1与W2间距为1.0cm，在电解池中加入的NaOH溶液的pH值为12。The preparation steps are the same as in Example 1, except that the distance between W1 and W2 is 1.0 cm, and the pH value of the NaOH solution added to the electrolytic cell is 12.
实施例3一种基于自发光激发的雌二醇光电化学传感器Embodiment 3 A kind of estradiol photoelectrochemical sensor based on self-luminous excitation
一种基于自发光激发的雌二醇光电化学传感器的制备方法，具体的制备步骤为：A method for preparing an estradiol photoelectrochemical sensor based on self-luminescence excitation, the specific preparation steps are:
（1）电致化学发光工作电极W1的制备：(1) Preparation of electrochemiluminescence working electrode W1:
制备步骤同实施例1中W1的制备步骤，不同之处为：磷酸镍铵微纳材料的制备步骤中铵盐加入量为0.4g，磷酸盐加入量为0.25g，二氯化镍加入量为0.25g，搅拌温度和时间分别为45℃和14h；电解液中鲁米诺的浓度为10mmol/L，硫酸浓度为1.0mol/L；循环伏安法进行电化学沉积时，循环30圈。The preparation steps are the same as the preparation steps of W1 in Example 1, the difference is: the ammonium salt addition is 0.4g in the preparation step of the nickel ammonium phosphate micro-nano material, the phosphate addition is 0.25g, and the nickel dichloride addition is 0.25g, the stirring temperature and time are 45°C and 14h respectively; the concentration of luminol in the electrolyte is 10mmol/L, and the concentration of sulfuric acid is 1.0mol/L; when electrochemical deposition is carried out by cyclic voltammetry, cycle 30 times.
（2）光电化学工作电极W2的制备：(2) Preparation of photoelectrochemical working electrode W2:
1）以ITO导电玻璃为工作电极，在电极表面滴涂12μL二氧化钛纳米粒子溶胶TiO2NPs，室温下晾干；1) Using ITO conductive glass as the working electrode, drop-coat 12 μL titanium dioxide nanoparticle sol TiO2 NPs on the surface of the electrode, and dry it at room temperature;
2）将1）中得到的工作电极放入马弗炉中，在450℃下进行退火处理，处理后冷却至室温；2) Put the working electrode obtained in 1) into a muffle furnace, perform annealing treatment at 450°C, and cool to room temperature after treatment;
3）将2）中得到的工作电极表面滴涂12μL雌二醇抗体溶液，4℃下干燥，干燥后用超纯水清洗，4℃下干燥，制得光电化学工作电极W2；3) Apply 12 μL of the estradiol antibody solution on the surface of the working electrode obtained in 2), dry at 4°C, wash with ultrapure water after drying, and dry at 4°C to prepare the photoelectrochemical working electrode W2;
其余同实施例1中W2的制备步骤。The rest are the same as the preparation steps of W2 in Example 1.
（3）基于自发光激发的雌二醇光电化学传感器的制备方法：(3) Preparation method of estradiol photoelectrochemical sensor based on self-luminescence excitation:
制备步骤同实施例1，不同之处为W1与W2间距为1.5cm，在电解池中加入的NaOH溶液的pH值为13。The preparation steps are the same as in Example 1, except that the distance between W1 and W2 is 1.5 cm, and the pH value of the NaOH solution added to the electrolytic cell is 13.
实施例4一种基于自发光激发的雌二醇光电化学传感器的应用Example 4 Application of an Estradiol Photoelectrochemical Sensor Based on Spontaneous Luminescence Excitation
实施例1制备的一种基于自发光激发的雌二醇光电化学传感器应用于雌二醇的检测，其检测步骤为：A kind of estradiol photoelectrochemical sensor based on self-luminous excitation prepared in Example 1 is applied to the detection of estradiol, and its detection steps are:
（1）在W2上滴加10μL不同浓度的雌二醇标准溶液，孵化30min后，雌二醇与W2上的雌二醇抗体进行免疫结合，冲洗后，再滴加10μL辣根过氧化物酶HRP标记的雌二醇二抗，孵化30min后，HRP标记的雌二醇二抗与雌二醇进行免疫结合，冲洗后，制得待测W2；(1) Add 10 μL of estradiol standard solution of different concentrations on W2 dropwise, after incubation for 30 minutes, estradiol and the estradiol antibody on W2 are immunocombined, after washing, add 10 μL of horseradish peroxidase dropwise HRP-labeled estradiol secondary antibody, after incubation for 30min, the HRP-labeled estradiol secondary antibody was immunocombined with estradiol, and after washing, the W2 to be tested was prepared;
（2）利用电化学工作站，在W1上采用阶跃电压的方法对W1施加阶跃电压，初始电压为0v，阶跃电压为0.7v，阶跃时间为10s；同时，在待测W2上采用时间-电流方法对待测W2施加恒定电压，电压为0v；待测W2上的电流会随着雌二醇浓度的增大而相应增大，根据所得电流增大值与雌二醇浓度之间的关系，绘制工作曲线；(2) Use the electrochemical workstation to apply a step voltage to W1 by using a step voltage method. The initial voltage is 0v, the step voltage is 0.7v, and the step time is 10s; at the same time, use The time-current method applies a constant voltage to W2 to be tested, and the voltage is 0v; the current on W2 to be tested will increase correspondingly with the increase of estradiol concentration, according to the difference between the obtained current increase value and the estradiol concentration relationship, draw the working curve;
（3）将待测雌二醇溶液代替雌二醇的标准溶液，按照（1）和（2）所述的雌二醇检测方法进行检测，根据所得到的电流增大值与所绘制的工作曲线得出待测雌二醇溶液的浓度；(3) Replace the standard solution of estradiol with the estradiol solution to be tested, and perform detection according to the estradiol detection method described in (1) and (2). The curve draws the concentration of the estradiol solution to be tested;
所述的HRP标记的雌二醇二抗的浓度为300μg/mL。The concentration of the HRP-labeled estradiol secondary antibody is 300 μg/mL.
实施例5一种基于自发光激发的雌二醇光电化学传感器的应用Example 5 Application of an estradiol photoelectrochemical sensor based on self-luminescence excitation
实施例2制备的一种基于自发光激发的雌二醇光电化学传感器应用于雌二醇的检测，其检测步骤除以下步骤外，其余步骤同实施例4：A kind of estradiol photoelectrochemical sensor based on self-luminous excitation prepared in Example 2 is applied to the detection of estradiol, and the detection steps are the same as in Example 4 except for the following steps:
步骤（2）利用电化学工作站，在W1上采用阶跃电压的方法对W1施加阶跃电压，初始电压为0v，阶跃电压为0.8v，阶跃时间为20s；同时，在待测W2上采用时间-电流方法对待测W2施加恒定电压，电压为0.3v；待测W2上的电流会随着雌二醇浓度的增大而相应增大，根据所得电流增大值与雌二醇浓度之间的关系，绘制工作曲线。Step (2) Use the electrochemical workstation to apply a step voltage to W1 by using a step voltage method. The initial voltage is 0v, the step voltage is 0.8v, and the step time is 20s; at the same time, on W2 to be tested Using the time-current method to apply a constant voltage to W2 to be tested, the voltage is 0.3v; The relationship between, draw the working curve.
实施例6一种基于自发光激发的雌二醇光电化学传感器的应用Example 6 Application of an estradiol photoelectrochemical sensor based on self-luminescence excitation
实施例3制备的一种基于自发光激发的雌二醇光电化学传感器应用于雌二醇的检测，其检测步骤除以下步骤外，其余步骤同实施例4：A kind of estradiol photoelectrochemical sensor based on self-luminous excitation prepared in Example 3 is applied to the detection of estradiol, and the detection steps are the same as in Example 4 except for the following steps:
步骤（2）利用电化学工作站，在W1上采用阶跃电压的方法对W1施加阶跃电压，初始电压为0v，阶跃电压为0.9v，阶跃时间为30s；同时，在待测W2上采用时间-电流方法对待测W2施加恒定电压，电压为0.6v；待测W2上的电流会随着雌二醇浓度的增大而相应增大，根据所得电流增大值与雌二醇浓度之间的关系，绘制工作曲线。Step (2) Use the electrochemical workstation to apply a step voltage to W1 by using a step voltage method. The initial voltage is 0v, the step voltage is 0.9v, and the step time is 30s; at the same time, on W2 to be tested Using the time-current method to apply a constant voltage to W2 to be tested, the voltage is 0.6v; The relationship between, draw the working curve.
实施例7实施例1-3所制备的雌二醇传感器，按照实施例4-6的检测步骤应用于雌二醇的检测，具有优良的检测效果，检测限为12pmol/L。Example 7 The estradiol sensor prepared in Examples 1-3 was applied to the detection of estradiol according to the detection steps of Examples 4-6, and had excellent detection effect, with a detection limit of 12 pmol/L.
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