CN108627559B - 基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法 - Google Patents
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Abstract
本发明公开了一种基于铝‑普鲁士蓝自供能电化学传感器构建的免疫分析方法，其是先制备普鲁士蓝修饰电极，再将其和铝电极、数字万用表、反应池串联构建成所述自供能电化学传感器，再将葡萄糖氧化酶和抗体共同负载在纳米金颗粒上形成酶标纳米探针，再在酶标板上将其与待测样品形成免疫夹心结构，利用该免疫夹心结构使葡萄糖催化生成大量过氧化氢，再将过氧化氢注入自供能电化学传感器中，通过其对普鲁士蓝的氧化能够显著改变普鲁士蓝的颜色及传感器的电流信号，从而通过读取产生的电流值，或观察普鲁士蓝的颜色变化来测定目标物的浓度。本发明方法设备简单，便于携带，并可用于多种抗体的检测，为便携式检测提供了一种简单、灵敏且稳定的方法。
Description
技术领域
本发明属于生物化学分析技术领域，具体涉及一种基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法。
背景技术
随着社会的发展，环境监测、食品安全和疾病诊断越来越受到人们的重视，因此发展快速、简便、灵敏的检测方法仍然是现在研究的热点之一。在现代分析检测体系中，大部分的标准检测方法是基于光学、色谱、质谱等建立的，这些检测方法具有准确、灵敏、稳定等优点。但是，昂贵的设备、复杂的操作等限制了其在快速检测及现场分析等方面的进一步应用，尤其是在偏远地区。因此，设计一种设备简单、易于操作、稳定实用的检测方法具有重要意义。
自供能电化学传感器作为一种便携式的传感器，其主要由两根修饰的电极组成一个原电池结构。该传感器自身能够产生能量输出，无需外接其他能源，并且其能量输出和检测液中目标物的浓度有紧密关联。在这种传感器被提出后，已经进行了许多研究。通过在电极上修饰不同的材料，成功实现了对葡萄糖、乳酸、胆固醇等物质的检测。但在这些传统自供能电化学传感器中，其检测信号往往是输出功率，检测装置往往也是专业的电化学工作站，不利于其普及和便携化。另一方面，大部分自供能电化学传感器通常被分成阴极和阳极两个部分来进行检测，这也不利于其进一步的一体化。因此，设计更加简单、小型的自供能电化学传感器，并将其应用于快速、便携的免疫分析领域具有重要研究意义。
发明内容
基于以上背景，本发明的目的在于提供一种基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法。
为实现上述目的，本发明采用如下技术方案：
一种基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法，其具体包括如下步骤：
（1）普鲁士蓝修饰电极的制备：将洗净的FTO玻璃电极、铂丝电极和饱和甘汞电极分别作为工作电极、对电极和参比电极，置于含有0.01-0.5 mol/L HCl、0.01-0.5 mol/LKCl、0.1-10 mmol/L K3[Fe(CN6)]、0.1-10 mmol/L FeCl3的电解质中，在0.1-0.6 V的恒电压下电聚合300-400秒，然后用水洗涤、干燥，制得普鲁士蓝修饰电极；
（2）铝-普鲁士蓝自供能电化学传感器的构建：将步骤（1）制得的普鲁士蓝修饰电极、铝电极、数字万用表串联及两电极间的一小型检测池串联，制得所述铝-普鲁士蓝自供能电化学传感器；
（3）葡萄糖氧化酶和抗体共同负载的纳米探针的制备：将葡萄糖氧化酶和抗体依靠静电吸附作用负载在纳米金颗粒上，制得所述酶标纳米探针；
（4）抗体-抗原-抗体夹心型免疫复合物的形成：在高亲和力的96孔聚苯乙烯微孔板中加入30-80 μL抗体，过夜封闭，再依次加入30-80 μL目标物、30-80μL酶标纳米探针形成抗体-抗原-抗体夹心型免疫复合物；
（5）采用铝-普鲁士蓝自供能电化学传感器进行检测：将所得夹心型免疫复合物用磷酸缓冲溶液洗涤后，加入50-150 μL含50 mmol/L葡萄糖的磷酸缓冲液（pH=6）进行反应，然后将反应液注入到小型检测池中，25 s后读取数字万用表上的电流信号，或者直接观察普鲁士蓝电极的颜色变化来测定目标物的浓度。
步骤（1）中所述FTO玻璃电极的大小为1×5 cm2；所述铝电极是由铝箔粘贴在FTO玻璃电极上组成铝箔电极；
所得普鲁士蓝修饰电极上普鲁士蓝薄膜为直径6 mm的圆形，呈现蓝色，紫外可见光吸收峰在约700 nm处。
步骤（2）中所述小型检测池由聚二甲基硅氧烷（PDMS）制备，横截面为直径6 mm的圆形，体积为40 μL。
步骤（3）中所述纳米金颗粒的粒径为16 nm。
本发明以铝-普鲁士蓝双电极构建的单室原电池，结合抗原抗体免疫反应，构建自供能电化学免疫传感器。其技术原理是通过在免疫探针上引入葡萄糖氧化酶来催化葡萄糖分解生成过氧化氢，并将过氧化氢注入检测池中。当没有目标物存在时，普鲁士蓝被还原为无色的普鲁士白，铝-普鲁士蓝原电池放出较低的电流信号；当目标物存在时，由于过氧化氢的存在，普鲁士白能够被过氧化氢重新氧化生成普鲁士蓝，并使检测的电流信号显著增强，其中，电流信号的变化及普鲁士蓝的颜色均与目标物浓度在一定范围内成正相关，故可以通过数字万用表来定量检测，也可以根据普鲁士蓝颜色变化来定性检测。
本发明的优点如下：
（1）本发明所需要的材料原料易得，制备工艺简单，成本低廉。
（2）和传统检测装置相比，本发明可使用数字万用表作为信号读取装置进行定量检测或以普鲁士蓝颜色变化进行定性检测，而无需外部能源接入，极大简化了检测装置，其操作简单，适于便携式检测。
（3）本发明通过引入葡萄糖氧化酶催化葡萄糖分解生成过氧化氢来使信号增强，可用于多种抗体目标物的检测，其通用性强，具有广泛的应用前景。
（4）本发明检测方法具有灵敏度高、稳定性强、选择性好、线性范围广等特点，并可用于多种抗体的检测。
附图说明
图1为本发明基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法的示意图；
图2为用于构建本发明自供能电化学传感器的部件实物图；
图3为实施例1中对甲胎蛋白所做的标准工作曲线；
图4为实例例1中不同浓度的标准样品对应的普鲁士蓝电极颜色。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
实施例1
1. 普鲁士蓝修饰电极的制备
将洗净的FTO玻璃电极、铂丝电极和饱和甘汞电极分别作为工作电极、对电极和参比电极，置于含有0.1 mol/L HCl、0.1 mol/L KCl、5 mmol/L K3[Fe(CN6)]、5 mmol/L FeCl3的电解质中，在0.4 V的恒电压下电聚合360秒，然后用水洗涤、干燥，制得普鲁士蓝修饰电极。
2. 自供能电化学传感器构建
将制备好的普鲁士蓝修饰电极、铝电极、数字万用表串联及两电极间的一小型检测池串联，制得所述铝-普鲁士蓝自供能电化学传感器。
3. 酶标抗体纳米探针的制备
首先，将5 mL纳米金溶液用碳酸钠调节pH到8.5，再加入50 μL、0.5 mg/mL的甲胎蛋白抗体和200 μL、0.5 mg/mL的葡萄糖氧化酶，4 ℃孵化过夜。然后将上述溶液在4 ℃、14000 rmp条件下离心20分钟，将离心得到的沉淀重新分散在2 mL含有1.0 wt % BSA和0.1wt % 叠氮化钠的PBS(pH=7.4)中，储存于4 ℃备用。
4. 夹心型免疫复合物的制备
首先，在96孔高亲和板中加入50 μL、10 μg/mL的甲胎蛋白抗体，4 ℃下隔夜孵化，再加入300 µL、1.0 wt %的BSA封闭1小时。用PBS溶液洗涤后，分别依次加入50 µL甲胎蛋白特异性抗原的系列浓度标准样品和50 µL的酶标纳米探针，分别反应30分钟，除去上清液。
5. 对甲胎蛋白特异性抗原的检测
在所得夹心型免疫复合物中加入100 µL含有50 mM葡萄糖的PBS缓冲液（pH=6），反应30分钟。随后，取40 µL反应液注入传感器的检测池中，反应25秒，读取数字万用表上的电流值或观察普鲁士蓝电极颜色的变化。其所得电流对甲胎蛋白特异性抗原的标准工作曲线如图3所示，相对应的普鲁士蓝电极颜色变化如图4所示。
由图3可见，其线性范围为5-200 ng/mL，检测限为1.67 ng/mL。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (3)
1.一种基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法，其特征在于：所述方法包括如下步骤：
（1）普鲁士蓝修饰电极的制备：将洗净的FTO玻璃电极、铂丝电极和饱和甘汞电极分别作为工作电极、对电极和参比电极，置于含有0.01-0.5 mol/L HCl、0.01-0.5 mol/L KCl、0.1-10 mmol/L K3[Fe(CN6)]、0.1-10 mmol/L FeCl3的电解质中，在0.1-0.6 V的恒电压下电聚合300-400秒，然后用水洗涤、干燥，制得普鲁士蓝修饰电极；
（2）铝-普鲁士蓝自供能电化学传感器的构建：将步骤（1）制得的普鲁士蓝修饰电极、铝电极、数字万用表串联及两电极间的一小型检测池串联，制得所述铝-普鲁士蓝自供能电化学传感器；
（3）葡萄糖氧化酶和抗体共同负载的纳米探针的制备：将葡萄糖氧化酶和抗体依靠静电吸附作用负载在纳米金颗粒上，制得酶标纳米探针；
（4）抗体-抗原-抗体夹心型免疫复合物的形成：在高亲和力的96孔聚苯乙烯微孔板中加入30-80 μL抗体，过夜封闭，再依次加入30-80 μL目标物、30-80μL酶标纳米探针形成抗体-抗原-抗体夹心型免疫复合物；
（5）采用铝-普鲁士蓝自供能电化学传感器进行检测：将所得抗体-抗原-抗体夹心型免疫复合物用磷酸缓冲溶液洗涤后，加入50-150 μL、pH=6的含50 mmol/L葡萄糖的磷酸缓冲液进行反应，然后将反应液注入到铝-普鲁士蓝自供能电化学传感器中，25 s后读取数字万用表上的电流信号，或者直接观察普鲁士蓝电极的颜色变化来测定目标物的浓度。
2. 根据权利要求1所述的基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法，其特征在于：步骤（1）中所得普鲁士蓝薄膜为直径6 mm的圆形。
3. 根据权利要求1所述的基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法，其特征在于：步骤（3）中所述纳米金颗粒的粒径为16 nm。
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