CN116626288A - 一种基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条及其制备方法 - Google Patents
一种基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条及其制备方法，所述免疫层析试纸条包括底板，样品垫，结合垫和硝酸纤维素膜，其制备方法为：将样品垫和结合垫进行预处理；通过柠檬酸三钠还原法制备金纳米颗粒，然后将其与癌胚抗原的抗体通过静电吸附偶联制备金纳米颗粒免疫探针，并将免疫探针固定在结合垫上；通过配体辅助再沉淀法合成钙钛矿纳米晶，并将其喷涂在硝酸纤维素膜上形成检测线和对照线，然后在检测线上单独喷涂BSA封闭的癌胚抗原；最后组装试纸条。本发明所述的试纸条具有检测快速、低成本、高灵敏度、低检测限、高选择性、无需大型仪器和专业操作人员等优点，在即时诊断中具有良好的应用前景。
Description
技术领域
本发明涉及一种免疫层析试纸条及其制备方法，尤其涉及一种基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条及其制备方法。
背景技术
近年来，癌症已经严重危害到人类的健康，癌症的早期筛查与诊断至关重要。患者血浆中的癌胚抗原是胰腺癌、结直肠癌、肺癌等癌症的临床诊断中常见的癌症标志物。血清中癌胚抗原的含量的异常升高，可能会导致患癌的风险的提高。
在之前的报道中通常采用荧光、化学发光、电化学、表面增强拉曼散射等方法来检测患者血清中的癌胚抗原含量，这些方法依赖于实验室基础设施并且现场操作起来很繁琐。因此，为了实现快速且方便的血清中癌胚抗原含量的监测，即时诊断(POCT)应运而生，POCT能够快速、灵敏、定量地检测癌胚抗原，对资源贫乏地区的诊断具有重要意义。目前亟待寻找一种能够快速、灵敏、定量检测癌胚抗原的测试方法。
发明内容
发明目的：本发明旨在提供一种能够实现癌胚抗原快速、灵敏检测的基于钙钛矿纳米晶的双读出信号免疫层析试纸条的制备方法。
技术方案：本发明所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条包括底板，样品垫，结合垫和硝酸纤维素膜，所述结合垫上固定有金纳米颗粒免疫探针，所述硝酸纤维素膜上涂有检测线和对照线，所述检测线由钙钛矿纳米晶溶液和牛血清白蛋白封闭的癌胚抗原喷涂得到，所述对照线由钙钛矿纳米晶溶液喷涂得到。
优选地，所述钙钛矿纳米晶溶液的喷涂浓度为1-3mg/mL，所述牛血清白蛋白封闭的癌胚抗原的喷涂浓度为2-20μg/mL。
优选地，所述检测线和对照线之间的距离为1-2cm，免疫层析试纸条上各部件的重叠部分的宽度为2-4mm。
所述基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，包括以下步骤：
(1)将样品垫浸泡在缓冲溶液中，然后取出放于真空烘箱中烘干备用；
(2)将结合垫浸泡在缓冲溶液中，然后取出放于真空烘箱中烘干备用；接着将金纳米颗粒免疫探针固定在上述预处理过的结合垫上，再放于真空烘箱中烘干备用；
(3)将钙钛矿纳米晶溶液和牛血清白蛋白(BSA)封闭的癌胚抗原喷涂在硝酸纤维素膜上形成检测线；并将等体积的钙钛矿纳米晶溶液喷涂在硝酸纤维素膜上形成对照线；然后，将这一硝酸纤维素膜放于真空烘箱中烘干备用；
(4)将上述预处理过的各部件和吸水纸依次组装到已切割成适宜宽度的PVC底板上，得到基于钙钛矿纳米晶的快速检测癌胚抗原双读出信号免疫层析试纸条。
优选地，步骤(2)中所述金纳米颗粒免疫探针的制备方法包括如下步骤：用碳酸钾调节金纳米颗粒溶液的pH，然后向溶液中加入癌胚抗原的抗体混合孵育，接着再加入牛血清白蛋白混合孵育以封闭多余的结合位点，最后将该溶液用磷酸缓冲溶液洗涤，再重新分散得到金纳米颗粒免疫探针溶液。
优选地，所述金纳米颗粒溶液的制备方法包括如下步骤：将氯金酸溶液和去离子水进行加热，然后迅速向溶液中加入柠檬酸三钠溶液，反应完全立即停止加热；待该溶液冷却至室温后进行洗涤，最后重新分散在蒸馏水中，得到金纳米颗粒溶液。
优选地，步骤(3)中所述钙钛矿纳米晶的制备方法包括如下步骤：将溴化铅和溴化铯用溶剂溶解并搅拌，加入辛胺接枝聚丙烯酸两亲性聚合物和油胺，搅拌至溶液由浑浊变澄清，即形成前驱体溶液；然后将上述前驱体溶液迅速注入甲苯中，反应完全后洗涤，烘干，得到钙钛矿纳米晶；将钙钛矿纳米晶分散在蒸馏水中，加入戊二醛活化，再加入牛血清白蛋白孵育，洗涤，再重新分散得到钙钛矿纳米晶溶液。
优选地，步骤(1)中所述的缓冲溶液为10-20mM PBS，pH＝7.2-7.6，1-2％BSA，0.2-0.5％吐温-20和4-5％蔗糖，步骤(2)中所述的缓冲溶液为10-20mM PBS，pH＝7.2-7.6，1-2％BSA和0.1-0.5％吐温-20。
优选地，步骤(1)和(2)中所述在缓冲溶液中的浸泡时间均为2-5h。
优选地，步骤(1)(2)(3)中所述烘干的温度均为35-37℃。
发明原理：首先，以辛胺接枝聚丙烯酸两亲性聚合物和油胺为配体，采用改进的配体辅助再沉淀法合成钙钛矿纳米晶，再通过戊二醛偶联法与BSA偶联形成BSA封闭的钙钛矿纳米晶。然后将其固定在硝酸纤维素膜的检测线和对照线上，再将BSA封闭的癌胚抗原固定于检测线上。金纳米颗粒与癌胚抗原的抗体偶联的免疫探针固定在免疫层析试纸条的结合垫上。在分析物中不含癌胚抗原的情况下，金纳米颗粒免疫探针在检测线上被相应抗原捕获，形成红色的金纳米颗粒线，并通过荧光内滤效应(IFE)淬灭检测线上固定的钙钛矿纳米晶的绿色荧光，而对照线上的荧光保持明亮，用于与检测线荧光进行对照。而当分析物中存在癌胚抗原时，样品中的癌胚抗原与检测线上的癌胚抗原竞争与金纳米颗粒免疫探针结合，检测线上的红色带逐渐“关闭”，而绿色荧光信号缓慢“打开”。由于金纳米颗粒的局部表面等离子体共振效应，用肉眼观察其色带。利用365nm紫外灯暗箱中观察钙钛矿荧光信号的变化，实现双模定量检测。
有益效果：与现有技术相比，本发明具有如下显著优点：
(1)所制备的基于钙钛矿纳米晶的快速检测癌胚抗原双读出信号免疫层析试纸条一方面能通过金纳米颗粒的比色信号进行定性分析，另一方面能通过钙钛矿纳米晶与金纳米颗粒之间的荧光内滤效应而产生的荧光信号进行定量分析。这一比色-荧光双读出信号免疫层析试纸条具有快速、低成本、高灵敏度、低检测限和高选择性等优点；
(2)所制备的基于钙钛矿纳米晶的快速检测癌胚抗原双读出信号免疫层析试纸条中在硝酸纤维素膜上喷涂的钙钛矿纳米晶具有优异的稳定性。这一试纸条在空气中保存40天后，检测线处的荧光强度可以保持初始强度96.69％，对照线处的荧光强度可以保持初始强度95.99％；
(3)所制备的基于钙钛矿纳米晶的快速检测癌胚抗原双读出信号免疫层析试纸条产生的比色-荧光信号在自然光和紫外光下均可肉眼定性评价测量效果。也可通过手机拍照记录，将照片采集检测结果通过Fjij软件进行比色和荧光强度的分析，从而实现对癌胚抗原的定量检测。也无需实验室基础设施和专业的操作人员，在POCT诊断中具有良好的应用前景。
附图说明
图1是双读出信号免疫层析试纸条的实物图；
图2是抗体的不同偶联量对双读出信号免疫层析试纸条检测效果的影响图；
图3是钙钛矿纳米晶在硝酸纤维素膜上喷涂40天的荧光强度变化图；
图4是基于钙钛矿纳米晶的快速检测癌胚抗原双读出信号免疫层析试纸条对癌胚抗原的检测结果图。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1
1、金纳米颗粒的制备
将0.5mL氯金酸溶液(1wt％)和50mL去离子水加入三颈烧瓶进行加热。当温度达到110℃时，迅速向溶液中加入2.5mL柠檬酸三钠溶液(1mg/mL)。待反应10分钟后停止加热，使溶液冷却至室温。最后用去离子水清洗3次，再分散于20mL去离子水中，4℃保存备用。
2、金纳米颗粒免疫探针的制备
在1mL金纳米颗粒溶液中滴加碳酸钾(0.1mol/L)，将癌胚抗原抗体(含0.5％ BSA)与上诉溶液混合，37℃孵育1h，再加入100μLBSA(10％)，再孵育30min，阻断溶液中未反应位点。孵育后的溶液以10000rpm离心10min，弃上清液，用200μL缓冲液(10mM PBS,pH＝7.4,1％BSA,5％蔗糖，0.5％吐温-20)重悬，4℃保存备用。
3、钙钛矿纳米晶的制备
将0.4mmol溴化铅和0.4mmol溴化铯加入到盛有10mL N,N-二甲基甲酰胺单颈烧瓶中搅拌，待瓶中固体完全溶解后加入2mg辛胺接枝聚丙烯酸(OPA)两亲性聚合物和0.5mL油胺，搅拌10min后溶液由浑浊变澄清，形成前驱体溶液。将0.5mL上述前驱体溶液迅速注入10mL甲苯中，溶液变绿。反应10min后以8000rpm离心5分钟，用甲苯洗涤三次。然后在60℃真空烘箱下干燥2h，得到钙钛矿纳米晶。将钙钛矿纳米晶分散在水溶液后加入10μL戊二醛(5％)活化30min，加入100μL BSA(100mg/mL)孵育2h。将孵育后的溶液以8000rpm离心5分钟，并用超纯水冲洗3次。最后将BSA封闭的钙钛矿纳米晶溶解于500μL超纯水中，4℃保存备用。
4、基于钙钛矿纳米晶的快速检测癌胚抗原双读出信号免疫层析试纸条的制备
(1)将样品垫浸泡在缓冲溶液(10mM PBS,pH＝7.4,1％BSA,0.2％吐温-20和4％蔗糖)中2h，然后取出放于37℃的真空烘箱中烘干备用；
(2)将结合垫浸泡在缓冲溶液(10mM PBS,pH＝7.4,1％BSA,0.1％吐温-20)中2h，然后取出放于37℃的真空烘箱中烘干备用；接着将金纳米颗粒免疫探针固定在上述预处理过的结合垫上，再放于37℃的真空烘箱中烘干备用；
(3)将钙钛矿纳米晶溶液和BSA封闭的癌胚抗原喷涂在硝酸纤维素膜上形成检测线；并将等体积的钙钛矿纳米晶溶液喷涂在硝酸纤维素膜上形成对照线；然后，将这一硝酸纤维素膜膜放于37℃的真空烘箱中烘干备用；
(4)将上述预处理过的各部件和吸水纸依次组装到已切割成5mm宽度的PVC底板上，得到基于钙钛矿纳米晶的快速检测癌胚抗原的双读出信号免疫层析试纸条，实物图见图1。
5、基于钙钛矿纳米晶的双读出信号免疫层析试纸条的癌胚抗原检测
将制备好的试纸条上样品垫的部分插入80μL一系列浓度含有癌胚抗原标准样品溶液中，反应10min。在自然光和紫外光下均可肉眼定性评价测量效果，并通过手机拍照记录。同时，将照片采集检测结果通过Fjij软件进行比色和荧光强度的分析，从而实现对癌胚抗原的定量检测。
实施例2
参照实施例1的制备方法，不同的是在制备金纳米颗粒免疫探针的过程中，调节与金纳米颗粒偶联的癌胚抗原抗体的量。
具体操作如下：分别将20μL、40μL、60μL、80μL、100μL的癌胚抗原抗体(100μg/mL)与等量的金纳米颗粒溶液混合，37℃孵育1h。
经对以上各实施例制得的基于钙钛矿纳米晶的双读出信号免疫层析试纸条进行癌胚抗原检测，如图4所示对不同浓度的癌胚抗原的检测结果图。
由图4可见，一方面，随着癌胚抗原浓度的增加，检测线上金纳米颗粒的红色带逐渐变浅直至消失，比色视觉模式下癌胚抗原的视觉检测极限(VLOD)为25ng/mL。另一方面，在紫外光照下，随着癌胚抗原浓度的增加，检测线线上钙钛矿纳米晶的绿色荧光信号“开启”，荧光强度逐渐增强，荧光视觉模式下癌胚抗原的VLOD为10ng/mL。
由图2可见，80μL的抗体(100μg/mL)为最佳剂量，此时金纳米颗粒免疫探针被捕获最多。
由图3可见，钙钛矿纳米晶喷涂在硝酸纤维素膜上后荧光仍具有良好的稳定性。试纸条在空气中保存40天后，对照线处的荧光强度可以保持初始强度96.69％，检测线处的荧光强度可以保持初始强度95.99％。
可见，本发明制备的基于钙钛矿纳米晶的双读出信号免疫层析试纸条具备较高的稳定性和较低的检测限。可望应用于人体血清中癌胚抗原含量的POCT诊断。
Claims (10)
1.一种基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条，其特征在于，包括底板，样品垫，结合垫和硝酸纤维素膜，所述结合垫上固定有金纳米颗粒免疫探针，所述硝酸纤维素膜上涂有检测线和对照线，所述检测线由钙钛矿纳米晶溶液和牛血清白蛋白封闭的癌胚抗原喷涂得到，所述对照线由钙钛矿纳米晶溶液喷涂得到。
2.根据权利要求1所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条，其特征在于，所述钙钛矿纳米晶溶液的喷涂浓度为1-3mg/mL，所述牛血清白蛋白封闭的癌胚抗原的喷涂浓度为2-20μg/mL。
3.根据权利要求1所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条，其特征在于，所述检测线和对照线之间的距离为1-2cm，免疫层析试纸条上各部件的重叠部分的宽度为2-4mm。
4.一种权利要求1-3任一所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，包括以下步骤：
(1)将样品垫浸泡在缓冲溶液中，然后烘干备用；
(2)将结合垫浸泡在缓冲溶液中，然后烘干备用；接着将金纳米颗粒免疫探针固定在上述预处理过的结合垫上，烘干备用；
(3)将钙钛矿纳米晶溶液和牛血清白蛋白封闭的癌胚抗原喷涂在硝酸纤维素膜上形成检测线；并将钙钛矿纳米晶溶液喷涂在硝酸纤维素膜上形成对照线，然后放于真空烘箱中烘干备用；
(4)将上述预处理过的各部件和吸水纸依次组装到底板上，得到所述免疫层析试纸条。
5.根据权利要求4所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，步骤(2)中所述金纳米颗粒免疫探针的制备方法包括如下步骤：用碳酸钾调节金纳米颗粒溶液的pH，然后向溶液中加入癌胚抗原的抗体混合孵育，接着再加入牛血清白蛋白混合孵育以封闭多余的结合位点，最后将该溶液用磷酸缓冲溶液洗涤，再重新分散得到金纳米颗粒免疫探针溶液。
6.根据权利要求5所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，所述金纳米颗粒溶液的制备方法包括如下步骤：将氯金酸溶液和去离子水进行加热，然后迅速向溶液中加入柠檬酸三钠溶液，反应完全立即停止加热；待该溶液冷却至室温后进行洗涤，最后重新分散在蒸馏水中，得到金纳米颗粒溶液。
7.根据权利要求4所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，步骤(3)中所述钙钛矿纳米晶的制备方法包括如下步骤：将溴化铅和溴化铯用溶剂溶解并搅拌，加入辛胺接枝聚丙烯酸两亲性聚合物和油胺，搅拌至溶液由浑浊变澄清，即形成前驱体溶液；然后将上述前驱体溶液迅速注入甲苯中，反应完全后洗涤，烘干，得到钙钛矿纳米晶；将钙钛矿纳米晶分散在蒸馏水中，加入戊二醛活化，再加入牛血清白蛋白孵育，洗涤，再重新分散得到钙钛矿纳米晶溶液。
8.根据权利要求4所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，步骤(1)中所述的缓冲溶液为10-20mM PBS，pH＝7.2-7.6，1-2％BSA，0.2-0.5％吐温-20和4-5％蔗糖，步骤(2)中所述的缓冲溶液为10-20mM PBS，pH＝7.2-7.6，1-2％BSA和0.1-0.5％吐温-20。
9.根据权利要求4所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，步骤(1)和(2)中所述在缓冲溶液中的浸泡时间均为2-5h。
10.根据权利要求4所述的基于钙钛矿纳米晶的快速检测癌胚抗原免疫层析试纸条的制备方法，其特征在于，步骤(1)(2)(3)中所述烘干的温度均为35-37℃。
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