CN103293293B - 用于无标记癌胚抗原检测的电化学免疫传感器的制备方法 - Google Patents
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Abstract
本发明公开了一种用于无标记癌胚抗原检测的电化学免疫传感器的制备方法,所述传感器为采用标准的MEMS工艺加工而成的平面金电极阵列。制备时先依次使用己二硫醇和N-羟基琥珀酰亚胺-聚乙二醇-马来酰亚胺交联剂处理金电极,然后组装上癌胚抗原捕获抗体,再用质量浓度为1%牛血清蛋白的PBS缓冲液封闭,获得所述电化学免疫传感器。本发明将癌胚抗原捕获抗体固定在金电极上,用于癌胚抗原的检测。实验表明该方法构建的电化学免疫传感器可将抗体稳定、简便的固定在金电极表面,传感器检测灵敏度高,检测下限低,特异性强。
Description
技术领域
本发明涉及一种传感器件的制备技术,尤其涉及一种用于无标记癌胚抗原检测的高通量电化学阻抗免疫传感器的制备方法。
背景技术
免疫学检测主要是利用抗原和抗体的特异性反应进行检测的一种手段,由于其可以利用同位素、酶和化学发光物质对检测信号进行放大和显示,因此常被用于蛋白质、激素等微量物质的检测。而电化学免疫分析方法由于其灵敏度高,无标记,分析时间短等优势受到人们的重视。电化学免疫传感器的重点在于检测物所对应的捕获抗体在电极表面的固定。常用的方法是利用自组装单分子层将抗体固定在电极上。如先在金电极上固定上一层硫醇自组装层,再通过EDC/NHS处理活化硫醇末端的羧基,最后通过NHS-ester基团与蛋白氨基的共价结合将抗体固定在金电极上。然而EDC/NHS对反应条件要求高,如溶液的酸碱性、反应时间和顺序等,而且EDC活化羧基后易水解。
发明内容
本发明的目的在于针对现有技术的不足,提供一种用于无标记癌胚抗原(carcino-embryonic antigen,CEA)检测的高通量电化学阻抗免疫传感器的制备方法。
本发明的目的是通过以下技术方案来实现的:一种用于无标记癌胚抗原检测的电化学免疫传感器的制备方法,该方法包括以下步骤:
(1)加工金电极阵列阻抗传感器:以厚度为0.5mm、直径为4英寸的玻璃为基底,在玻璃上磁控溅射20nm厚的钛化钨(TiW)薄膜作为粘附层,接着磁控溅射300nm厚的金(Au)薄膜作为电极层;用AZ光刻胶光刻出电极图形后,采用干法刻蚀将非电极区域刻蚀掉;一个芯片单元上共有6个金电极,每个金电极的直径为7mm;划片后使用金属夹一头夹住电极引线引出的接触盘,金属夹的另一头焊在PCB板上形成金电极与PCB板的电气连接,最后将有机玻璃材料制作而成的腔体用环氧树脂胶封在芯片上;大腔的高度为2cm,底面积为8cm2,每个大腔中包含六个小腔,小腔的高度为大腔的一半,每个小腔对应一个电极通道,每个小腔内的有效电极面积为12.56mm2;电化学阻抗谱扫描时,一个芯片上的所有金电极共用一对外置Ag/AgCl参考电极和铂丝对电极;
(2)清洗金电极:依次用1M的HCl水溶液和1M的NaOH水溶液浸泡电极5-10min,用于除去电极表面的无机污染物;然后将金电极作为工作电极,铂电极为对电极,Ag/AgCl电极作为参考电极,以0.5M的H2SO4溶液为背景液,在电压0V-1.5V下进行伏安循环扫描30min,除去芯片表面的氧化物,最后依次使用无水乙醇和超纯水超声清洗金电极各5min;
(3)制备电化学免疫传感器:首先将金电极浸泡在体积百分比为0.5%的HDT溶液(溶剂由无水乙醇和纯水按体积比1:1混合组成)中,室温下浸泡8-12h后取出,无水乙醇超声清洗5min,再用超纯水反复清洗,用氮气吹干;接着将电极置于10mM SM(PEG)6溶液(溶剂为DMSO)中,室温浸泡30min,取出后用超纯水反复清洗,用氮气吹干;将处理后的金电极浸泡在200μg/ml 的 anti-CEA 抗体溶液(溶剂为PBS缓冲液)中,室温下浸泡2h,取出后用PBS缓冲液反复清洗,洗去电极表面的非特异性吸附的anti-CEA 抗体,用氮气吹干;最后再将结合抗体的金电极浸泡在质量百分比为1%的牛血清白蛋白溶液(溶剂为PBS缓冲液)中,室温下浸泡1h对抗体的非活性位点进行封闭,取出后用PBS缓冲液反复清洗,用氮气吹干,获得所述的用于CEA检测的电化学免疫传感器,在4℃下保存备用。
本发明的有益效果是,本发明将CEA捕获抗体固定在金电极上,用于CEA的检测。实验表明该方法构建的电化学免疫传感器可将抗体稳定、简便的固定在金电极表面,传感器检测灵敏度高,检测下限低,特异性强。
附图说明
图1为金电极阵列的电极排布图;
图2为本发明电化学免疫传感器腔体图;
图3为本发明电化学免疫传感器的制备图;
图4为本发明电化学免疫传感器制备过程中每步处理后的电化学阻抗谱曲线;
图5为本发明电化学免疫传感器检测不同浓度CEA的电化学阻抗谱曲线;
图6为本发明电化学免疫传感器分别与不同浓度的CEA发生免疫反应后;电子传递阻抗与CEA浓度的对数之间的线性关系图;
图7为电化学免疫传感器与浓度为10-15 g/ml的HCG反应前后的电化学阻抗谱曲线。
具体实施方式
本发明利用金与己二硫醇(HDT)的一个硫键形成的稳定的Au-S键作用,HDT的另一个硫键又与N-羟基琥珀酰亚胺-聚乙二醇-马来酰亚胺交联剂(NHS-(PEG)n-Maleimide Crosslinkers,优选SM(PEG)6)的马来酰亚胺(maleimide)基团共价结合,紧接着SM(PEG)n的N-羟基琥珀酰亚胺酯(NHS-ester)基团与抗体上的氨基形成稳定的酰胺键。与传统的EDC/NHS活化硫醇末端羧基反应相比,SM(PEG)n与HDT的反应时间快,且SM(PEG)n不易水解,反应在室温中性环境下进行,反应成功率高,因此固定抗体的效率也会变高。本发明所述的电化学免疫传感器灵敏度下限为0.1fg/ml。通过固定不同类型的捕获抗体,可用于不同抗原的检测。
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。
1、金电极阵列阻抗传感器的加工。传感器的加工采用标准的MEMS 工艺。传感器以厚度为0.5mm,直径为4英寸的玻璃为基底,首先在玻璃上磁控溅射20nm厚的钛化钨(TiW)薄膜作为粘附层,接着磁控溅射300nm厚的金(Au)薄膜作为电极层;用AZ光刻胶光刻出电极图形后,采用干法刻蚀将非电极区域刻蚀掉。芯片电极图形如图1所示,一个芯片单元上共有6个金电极1,每个金电极的直径为7mm。划片后使用金属夹5一头夹住电极引线2引出的接触盘3,金属夹的另一头焊在PCB板4上形成金电极与PCB板的电气连接,最后将有机玻璃材料制作而成的腔体用环氧树脂胶封在芯片上。腔体如图2所示。大腔的高度为2cm,底面积为8cm2。每个大腔中包含六个小腔1,小腔的高度为大腔的一半,每个小腔对应一个电极通道,每个小腔内的有效电极面积为12.56mm2。电化学阻抗谱扫描时,一个芯片上的所有金电极共用一对外置Ag/AgCl参考电极和铂丝对电极。
2、金电极的清洗:依次用1M HCl水溶液和1M NaOH水溶液浸泡电极5-10min(优选10min),用于除去电极表面的无机污染物。然后将金电极作为工作电极,铂电极为对电极,Ag/AgCl电极作为参考电极,以0.5M H2SO4溶液为背景液,在电压0V-1.5V下进行伏安循环扫描30min,用于除去芯片表面的氧化物,最后依次使用无水乙醇和超纯水超声清洗金电极各5min。
3、电化学免疫传感器制备过程如图3所示:首先将金电极浸泡在体积百分比为0.5%的HDT溶液(溶剂由无水乙醇和纯水按体积比1:1混合组成)中,室温下浸泡8-12h后取出,无水乙醇超声清洗5min,再用超纯水反复清洗,用氮气吹干。接着将电极置于10mM SM(PEG)6溶液(溶剂为DMSO)中,室温浸泡30min,取出后用超纯水反复清洗,用氮气吹干。将处理后的金电极浸泡在200μg/ml 的 anti-CEA 抗体溶液(溶剂为0.1MPBS缓冲液,pH=7.2,0.1M指的是PBS缓冲液中磷酸盐的摩尔浓度,本专利中使用的PBS缓冲液均是指0.1M,pH=7.2的PBS缓冲液)中,室温下浸泡2h,取出后用PBS缓冲液反复清洗,洗去电极表面的非特异性吸附的anti-CEA 抗体,用氮气吹干。最后再将结合抗体的金电极浸泡在质量百分比为1%的牛血清白蛋白溶液(BSA,溶剂为PBS缓冲液)中,室温下浸泡1h对抗体的非活性位点进行封闭,取出后用PBS缓冲液反复清洗,用氮气吹干,获得所述的用于CEA检测的电化学免疫传感器,放在4℃条件下备用。
以上每一步处理完毕后,都需要对每个金电极进行电化学阻抗谱扫描用于对电极表面进行电化学表征和对处理过程进行验证。将每一步处理后的金电极分别作为工作电极,以铂丝作为对电极,以Ag/AgCl电极作为参考电极,芯片腔内加入铁氰化钾/亚铁氰化钾的混合溶液(1:1,5mM,底液为0.1M的KCl水溶液) 作为电解液,以电化学工作站作为仪器平台对每个通道的金电极进行电化学阻抗谱扫描。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0.01Hz~100KHz。每步处理后得到的阻抗谱曲线如图4所示。从图中可以看出,金电极修饰己二硫醇后,阻抗显著增加,原因在于己二硫醇在很大程度上阻碍了电子的转移。SM(PEG)6处理后,电极阻抗降低,是因为SM(PEG)6末端的NHS-ester基团促进了电子的传递。而抗体固定上之后,导致阻抗进一步增加,源于氧化还原对透过单分子层的难度增加。因此在金电极上已成功的修饰了相应的化学物质。
用本发明的方法制备的电化学免疫传感器可用于无标记癌胚抗原检测:电化学免疫传感器制备好之后,在芯片的每个小腔中加入不同浓度的CEA溶液(浓度范围为10-16~10-12g/ml,溶剂为PBS缓冲液),37℃下处理2h,之后用PBS缓冲液反复清洗,洗去非特异性吸附在抗体上的CEA,获得结合抗原的电化学传感器。另外预留一个小腔加入10-15 g/ml的HCG(人绒毛膜促性腺激素,一种肿瘤标志物,溶剂为PBS缓冲液)溶液作为阴性对照,同样37℃下处理2h,之后用PBS缓冲液反复清洗,洗去非特异性吸附在电极上的HCG。
以上述结合不同浓度抗原的金电极作为工作电极,以铂丝作为对电极,以Ag/AgCl电极作为参考电极,芯片腔内加入铁氰化钾/亚铁氰化钾的混合溶液(1:1,5mM,底液为0.1M的KCl水溶液) 作为电解液,以电化学工作站作为仪器平台对每个通道的金电极进行电化学阻抗谱扫描。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0.01Hz~100KHz。得到电化学阻抗扫频曲线,电路拟合后得到电极的电子传递电阻Rct。不同浓度抗原处理后的金电极的阻抗谱曲线如图5所示。以传递电阻Rct的变化值((Rct_CEA-Rct_BSA)/ Rct_BSA)为纵坐标,CEA抗原浓度的对数值为横坐标,制作线性曲线,如图6所示。线性关系公式为 △Rct=11.96lg[C]+0.714,线性度为0.97。同时,阴性对照组处理前后的阻抗曲线没有明显的变化,如图7所示,说明本发明电化学免疫传感器特异性强。
Claims (1)
1.一种用于无标记癌胚抗原检测的电化学免疫传感器的制备方法,其特征在于,该方法包括以下步骤:
(1)加工金电极阵列阻抗传感器:以厚度为0.5mm、直径为4英寸的玻璃为基底,在玻璃上磁控溅射20nm厚的钛化钨薄膜作为粘附层,接着磁控溅射300nm厚的金薄膜作为电极层;用AZ光刻胶光刻出电极图形后,采用干法刻蚀将非电极区域刻蚀掉;一个芯片单元上共有6个金电极,每个金电极的直径为7mm;划片后使用金属夹一头夹住电极引线引出的接触盘,金属夹的另一头焊在PCB板上形成金电极与PCB板的电气连接,最后将有机玻璃材料制作而成的腔体用环氧树脂胶封在芯片上;大腔的高度为2cm,底面积为8cm2,每个大腔中包含六个小腔,小腔的高度为大腔的一半,每个小腔对应一个电极通道,每个小腔内的有效电极面积为12.56mm2;电化学阻抗谱扫描时,一个芯片上的所有金电极共用一对外置Ag/AgCl参考电极和铂丝对电极;
(2)清洗金电极:依次用1M的HCl水溶液和1M的NaOH水溶液浸泡电极5-10min,用于除去电极表面的无机污染物;然后将金电极作为工作电极,铂电极为对电极,Ag/AgCl电极作为参考电极,以0.5M的H2SO4溶液为背景液,在电压0V-1.5V下进行伏安循环扫描30min,除去芯片表面的氧化物,最后依次使用无水乙醇和超纯水超声清洗金电极各5min;
(3)制备电化学免疫传感器:首先将金电极浸泡在体积百分比为0.5%的HDT溶液中,所述HDT溶液的溶剂由无水乙醇和纯水按体积比1:1混合组成,室温下浸泡8-12h后取出,无水乙醇超声清洗5min,再用超纯水反复清洗,用氮气吹干;接着将电极置于10mM SM(PEG)6溶液中,所述SM(PEG)6溶液的溶剂为DMSO,室温浸泡30min,取出后用超纯水反复清洗,用氮气吹干;将处理后的金电极浸泡在200μg/ml 的 anti-CEA 抗体溶液中,所述anti-CEA 抗体溶液的溶剂为PBS缓冲液,室温下浸泡2h,取出后用PBS缓冲液反复清洗,洗去电极表面的非特异性吸附的anti-CEA 抗体,用氮气吹干;最后再将结合抗体的金电极浸泡在质量百分比为1%的牛血清白蛋白溶液中,所述牛血清白蛋白溶液的溶剂为PBS缓冲,室温下浸泡1h对抗体的非活性位点进行封闭,取出后用PBS缓冲液反复清洗,用氮气吹干,获得所述的用于CEA检测的电化学免疫传感器,在4℃下保存备用。
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