CN114113258B - 同时检测多种猪腹泻类冠状病毒的高通量比率型芯片式传感器的构建方法 - Google Patents
同时检测多种猪腹泻类冠状病毒的高通量比率型芯片式传感器的构建方法 Download PDFInfo
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Abstract
本发明提供了同时检测多种猪腹泻类冠状病毒的高通量比率型芯片式传感器的构建方法,高通量比率型电极基底与普鲁士蓝颜色变化的改变可以直接读出信号,实现对三种猪腹泻类冠状病毒的同时检测。步骤为:步骤1、制备ZnIn2S4/3DNG;步骤2、构建同时检测PEDV、TGEV和PDCoV的高通量比率型芯片式免疫传感器。本发明构建的高通量比率型芯片式免疫传感器,制备三个检测区域和相应的信号输出区域,增加作为参比的空白区域提高精确度,引入抗体作为识别元件构建免疫传感平台,加上3D打印的模具,可以将检测物与信号输出区域隔开,除此之外,隔开每个检测区域,减少干扰,可以实现同时特异性地检测三种检测物,提高了检测效率。
Description
技术领域
本发明属于光电化学生物传感技术领域,主要涉及一种基于光电化学免疫传感器件的构建方法,具体为同时检测多种猪腹泻类冠状病毒的高通量比率型芯片式传感器的构建方法。
背景技术
光电致变色可视化传感器是一种将光电致变色器件与光电化学传感器相结合的新型传感技术,它利用光敏材料能够在光激发下产生光电流的特点,将其应用在电致变色的电信号响应光学性能的变化上,电致变色材料能够在不同电流信号下产生不同变色效果的特点,达到目视定量分析检测物的目的,这一技术成功的将光电化学传感技术的信号输出部分由电化学工作站简化为颜色变化。光电致变色可视化传感器具有操作步骤简单、检测结果可以直接肉眼观察以及无需外接仪器检测易于便携等优点,这让其在环境检测,食品检测领域具有广泛的应用。
猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)是仔猪常见的重要病毒性腹泻病原,临床上均以呕吐、厌食、腹泻、脱水为主要特征,仔猪的发病率和死亡率较高,给养猪业造成重大经济损失。尤其是PEDV变异株,是近年来仔猪腹泻的主要病原。由于临床症状极其相似,难以区分,只能借助实验室手段加以鉴别诊断。建立PEDV、TGEV和PDCoV快速鉴别检测方法,对仔猪病毒性腹泻的诊断和防控具有重要意义。
目前关于猪冠状病毒检测方法的研究多集中于血清学和分子生物学检测方法。其中ELISA、免疫层析等血清学检测方法成本低、操作简单快速,可满足大批量样本检测,在基层兽医临床诊断和血清抗体监测中得到广泛应用,但也存在特异性、敏感性相对较低的缺点。而传统分子生物学检测方法如PCR、多重PCR、荧光定量PCR等技术在特异性和灵敏性上有了大幅度的提高,实现了对病原定性到定量的分析,单一病原到多重病原的鉴别检测,提升了检测效率及准确率,可快速鉴别多种肠道病原以及快速区分不同基因型,但仍需要较高的试验条件和成本而不适用于大规模的临床样本和基层兽医诊断。
发明内容
本发明旨在提供集快速、简单、微型化、使用模式灵活等优点为一体的便携式高通量比率型光电致变色免疫传感器应用于同时对PEDV、TGEV和PDCoV的检测,在FTO上设计三个检测区域和一个空白对照区域,实现基于普鲁士蓝颜色变化的光电致变色可视化比率型检测。
本发明同时检测多种猪腹泻类冠状病毒的高通量比率型芯片式传感器的构建方法,包括如下步骤:
步骤1、制备光电材料硫铟锌三维氮杂石墨烯(ZnIn2S4/3DNG):
首先,将氧化石墨烯分散液与甘氨酸加入乙二醇中,搅拌形成均匀的溶液。然后,将无水醋酸锌、四水合氯化铟和硫代乙酰胺加入上述溶液中并搅拌均匀。最后将溶液转移到高压反应釜并在烘箱中进行溶剂热反应,至此便成功制备出了ZnIn2S4/3DNG纳米复合材料。最后,待其自然冷却至室温后经过多次水、乙醇交替离心清洗,随后真空干燥,从而制备了固体产物ZnIn2S4/3DNG。
步骤2、制备高通量比率型电极基底
将FTO依次在甲苯、丙酮、乙醇和水超声清洗下,以去除表面可能吸附的杂质与有机污染物。用激光刻蚀技术将设计的图案呈现在干净的FTO上,刻蚀出若干个电致变色区域和若干个电子注入区域,建立高通量比率型检测的基础。
步骤3、制备电致变色区域:
在FTO的电致变色区域电沉积普鲁士蓝,称取FeCl3·6H2O和K3Fe(CN)6倒入烧杯中,向烧杯中加入去离子水搅拌使其完全溶解,随后加入HCl,搅拌至混合均匀。采用恒电压法在信号输出区域电沉积普鲁士蓝,用Ag/AgCl电极,铂丝电极和FTO构建三电极体系,电沉积参数设置为施加电压为0.3V,时间为75s。电沉积结束后用纯水缓慢冲洗电极,干燥一夜以备用。
步骤4、制备电子注入区域:
将步骤1中得到的ZnIn2S4/3DNG分散于N,N-二甲基甲酰胺(DMF)中,得到ZnIn2S4/3DNG分散液,将ZnIn2S4/3DNG分散液滴涂于电子注入区域,置于红外灯下烘干,得到以ZnIn2S4/3DNG为光敏材料的电子注入区域。
步骤5、构建检测PEDV、TGEV和PDCoV的高通量比率型传感器
首先,在电子注入区域ZnIn2S4/3DNG/FTO上滴涂壳聚糖(CHIT)溶液,置于红外灯下烘干。接着,将戊二醛(GA)溶液滴于电极表面,并置于室温下反应,反应完毕后,用PBS淋洗,除去电极表面多余的GA。用PBS为溶剂配制PEDV、TGEV和PDCoV抗体溶液,将三种抗体溶液分别滴加在相应的电子注入区域,反应一段时间后,用PBS淋洗以除去过量未结合的抗体,然后滴加牛血清蛋白(BSA)溶液以封闭非特异性活性位点。3D打印的模具盖在电极上。最终得到抗体修饰的电子注入区域(Ab/ZnIn2S4/3DNG/FTO),与电致变色区域构成光电致变色免疫传感器件。
步骤1中,
所述的溶液中,氧化石墨烯分散液、甘氨酸、乙二醇、无水醋酸锌、四水合氯化铟和硫代乙酰胺的用量比例为3.525mL:0.0976g:10mL:0.0732g:0.1953g:0.2g,其中氧化石墨烯分散液浓度为2.0mg/mL。
所述溶剂热反应的温度为160℃~200℃,反应时间为14~17h。
步骤2中,
所述电极基底面积为2.2cm×5.3cm;每个电子注入区域的面积为1cm×1cm;每个电致变色区域的面积为0.5cm×0.5cm;所用刻蚀仪器为激光刻蚀仪,刻蚀所用功率是50%,刻蚀速度为1000mm/s。
步骤3中,
所述溶液中,FeCl3·6H2O,K3Fe(CN)6,HCl浓度均为0.005mol/L,超纯水的用量为90~110ml;
恒电压法电沉积的参数为电压为0.3V,时间为75s;
步骤4中,
所述ZnIn2S4/3DNG分散液的浓度为2mg/mL,滴加量20μL。
步骤5中,
所述CHIT的质量百分浓度为0.1%,滴加量20μL;
所述GA的体积百分浓度为2.5%,滴加量20μL;CHIT与GA反应时间为1~2h;
PEDV、TGEV和PDCoV抗体溶液浓度分别为2μM、2μM和4μM,滴加量均为20~40μL,反应时间10~14h;
BSA的质量百分浓度为3%,滴加量20μL。
所述3D打印的模具的尺寸为530mm×220mm×8mm,四个电子注入区域的尺寸为11mm×12mm×8mm,电致变色区域尺寸为500×4mm×8mm。
将本发明制备的高通量比率型芯片式传感器件用于同时检测PEDV、TGEV和PDCoV的用途,具体步骤为:
(1)将不同浓度的PEDV、TGEV和PDCoV的病毒溶液分别滴到Ab/ZnIn2S4/3DNG/FTO电子注入区域上,并在室温下孵育一段时间;
(2)将PBS溶液等体积的分别注入到四个电子注入区域所在的槽子中,再将KCl溶液滴加到电致变色区域所在的槽子中,氙灯光源垂直照射该芯片式传感器上,以电致变色区域中的普鲁士蓝颜色变化作为输出信号;将变色区域普鲁士蓝灰度值与空白区域的普鲁士蓝的比值与PEDV、TGEV和PDCoV浓度做标准曲线;
步骤(1)中,PEDV、TGEV和PDCoV浓度范围分别为102~105TCID50/mL、103~107.5TCID50/mL和5×103~107TCID50/mL,具体为102、7×102、103、104、5×104、105TCID50/mL与103、5×103、5×105、5×106、107、107.5TCID50/mL和5×103、7×103、104、5×104、5×106、107TCID50/mL,滴加量为10~30μL;
步骤(2)中,PBS量为0.5~1.0mL,KCl溶液的用量为0.5~1.0mL,浓度为0.1mol/L;氙灯光源的强度为25%~100%。
本发明的有益效果为:
本发明以带有三个检测区域和作为参照的一个空白区域FTO为基底,其中检测区域与空白区域均由电致变色区域与电子注入区域组成。以修饰ZnIn2S4/3DNG纳米颗粒与抗体的三个区域作为电子注入区域,电沉积普鲁士蓝的四个区域作为电致变色区域,成功建立了高通量比率型免疫传感器,实现同时对PEDV、TGEV和PDCoV毒素的分析检测,其特色和优点表述如下:
(1)本发明制备ZnIn2S4/3DNG纳米颗粒作为电子注入区域,电化学沉积的普鲁士蓝作为电致变色区域来构建高通量比率型免疫传感器,变色信号明显,增加参比检测更准确。
(2)本发明制备三个检测区域,能同时检测三种检测物,提高了检测效率,增加作为参比的空白区域提高精确度,引入抗体作为识别元件,能实现特异性检测。
(3)本发明所提出的高通量比率型免疫传感器实现了同时对PEDV、TGEV和PDCoV的灵敏检测。在102~105TCID50/mL浓度区间内,PEDV浓度的对数值(lgCPEDV)与对于电致变色区域普鲁士蓝灰度值与空白区域对应变色区域普鲁士蓝灰度值的比值呈现良好的线性关系,检测限可达0.33×102TCID50/mL。同理,TGEV和PDCoV分别在103~107.5TCID50/mL和5×103~107TCID50/mL浓度区间内,TGEV和PDCoV浓度的对数值(lgCTGEV和lgCPDCoV)与对于电致变色区域普鲁士蓝灰度值与空白区域对应变色区域普鲁士蓝灰度值的比值呈现良好的线性关系,检测限可达0.33×103TCID50/mL和1.67×103TCID50/mL。
(4)本发明构建的高通量比率型免疫传感器,加上3D打印的模具,可以将检测物与信号输出区域隔开,除此之外,隔开每个检测区域,减少干扰,提高精确度。
附图说明
图1为高通量比率型电极区域示意图;
图2为构建的高通量比率型免疫传感器的机理图;
图3(A)、(B)、(C)为制备的ZnIn2S4/3DNG的扫描电子显微镜图、透射电镜图、X-射线衍射图;
图4(A)、(B)、(C)为PEDV、TGEV、PDCoV浓度与普鲁士蓝灰度值的比值关系图。
具体实施方式
以下结合实例对本发明进行详细描述,但本发明不局限于这些实施例。
图2为构建的高通量比率型免疫传感器的机理图。
实施例1:
(1)ZnIn2S4/3DNG的制备
首先,将3.525mL氧化石墨烯分散液与0.0976g甘氨酸加入到10mL乙二醇中,搅拌形成均匀的溶液。然后,将0.0612g无水醋酸锌、0.1953g四水合氯化铟和0.4g硫代乙酰胺加入上述溶液中并搅拌均匀。最后将溶液转移到高压反应釜并在烘箱中进行溶剂热反应,至此便成功制备出了ZnIn2S4/3DNG纳米复合材料。最后,待其自然冷却至室温后经过多次水、乙醇交替离心清洗,随后真空干燥,从而制备了固体产物ZnIn2S4/3DNG。
至此便成功制备出了ZnIn2S4/3DNG纳米复合材料,材料复合结构如图3所示。
(2)修饰高通量比率型电极的制造
在制备电极之前,先对FTO进行预处理。将FTO电极置于1M氢氧化钠溶液中煮沸30分钟,再依次用丙酮、蒸馏水和乙醇超声清洗,氮气吹干备用。用激光刻蚀将设计的图案呈现在干净的FTO上,刻蚀出若干个电致变色区域和若干个电子注入区域,如图1所示,建立高通量比率型检测的基础;
将电致变色区域置于普鲁士蓝溶液中,用恒电压法电沉积,得到电致变色区域。
用1cm×1cm的正方形茶色耐高温胶带将电子注入区域固定,称取2mg ZnIn2S4/3DNG分散于1mL DMF中,得到浓度为2mg/mL的ZnIn2S4/3DNG分散液,分别移取20μL ZnIn2S4/3DNG分散液分别均匀滴涂于四个电子注入区域,置于红外灯下烘干,得到ZnIn2S4/3DNG/FTO。
将PBS溶液与KCl溶液分别滴在电子注入区域所在的四个槽子与电致变色区域所在的槽子中,用氙灯垂直照射在电极上,用肉眼观看光电致变色区域普鲁士蓝的变色情况,完成光电致变色检测。其中,PBS浓度为0.1mol/L,pH=7.4,KCl浓度为0.1mol/L。
(3)光电/可视化适配体传感器件的构建
首先,在工作电极ZnIn2S4/3DNG/FTO上滴涂20μL壳聚糖(CHIT)溶液,置于红外灯下烘干。接着,将20μL 2.5%戊二醛(GA)溶液滴于工作电极表面,并置于室温下反应1h,反应完毕后,用PBS(pH=7.4,0.1mol/L)淋洗,除去电极表面多余的GA。用PBS(pH=7.4,0.1mol/L)为溶剂配制浓度分别为2μM、2μM和4μM的PEDV、TGEV和PDCoV抗体溶液。将体积为20μL的抗体溶液滴加在电子注入区域,置于4℃冰箱中反应12h,用PBS淋洗以除去过量的未结合的抗体,然后滴加20μL 3%牛血清蛋白(BSA)溶液以封闭非特异性活性位点,盖上3D打印模具,最终得到抗体修饰的电子注入区域(Ab/ZnIn2S4/3DNG/FTO),与电致变色区域构成高通量比率型免疫传感器件。
高通量比率型免疫传感器件同时检测PEDV、TGEV和PDCoV
此后,将20μL浓度为102、7×102、103、104、5×104、105TCID50/mL与103、5×103、5×105、5×106、107、107.5TCID50/mL和5×103、7×103、104、5×104、5×106、107TCID50/mL的PEDV、TGEV和PDCoV分别滴到三个对应Ab/ZnIn2S4/3DNG/FTO电极上,并在室温下孵育一段时间。最后,将PBS溶液等体积的分别注入到四个电子注入区域所在的槽子中,再将KCl溶液滴加到电致变色区域所在的槽子中,氙灯光源垂直照射该芯片式传感器上进行电化学分析。
检测结果如图4:
图4中(A)、(B)、(C)为PEDV、TGEV、PDCoV浓度与普鲁士蓝灰度值的比值关系图,从图中可以看出随着PEDV、TGEV、PDCoV浓度的增大,电致变色区域普鲁士蓝灰度值与空白区域对应变色区域普鲁士蓝灰度值的比值减小。PEDV、TGEV、PDCoV浓度区间分别为102~105TCID50/mL、103~107.5TCID50/mL和5×103~107TCID50/mL内,电致变色区域普鲁士蓝灰度值与空白区域对应变色区域普鲁士蓝灰度值的比值与PEDV、TGEV、PDCoV浓度之间呈现良好的线性关系,检测限分别为0.33×102TCID50/mL、0.33×103TCID50/mL和1.67×103TCID50/mL。
Claims (8)
1.同时检测多种猪腹泻类冠状病毒的高通量比率型芯片式传感器的构建方法,其特征在于,包括如下步骤:
步骤1、制备光电材料硫铟锌三维氮杂石墨烯ZnIn2S4/3DNG,备用;
将氧化石墨烯分散液与甘氨酸加入乙二醇中,搅拌形成均匀的溶液,然后,将无水醋酸锌、四水合氯化铟和硫代乙酰胺加入上述溶液中并搅拌均匀,最后将溶液转移到高压反应釜并在烘箱中进行溶剂热反应160~200℃下反应14~17h;待其自然冷却至室温后经清洗,真空干燥,得到固体产物ZnIn2S4/3DNG;
其中,氧化石墨烯分散液、甘氨酸、乙二醇、无水醋酸锌、四水合氯化铟和硫代乙酰胺的用量比例为3.525mL:0.0976g:10mL:0.0732g:0.1953g:0.2g,氧化石墨烯分散液浓度为2.0mg/mL;
步骤2、制备高通量比率型电极基底:
将FTO依次用甲苯、丙酮、乙醇和水超声清洗后,用激光刻蚀技术将设计的图案呈现在干净的FTO上,刻蚀出若干个电致变色区域和若干个电子注入区域,建立高通量比率型检测的基础;
步骤3、制备电致变色区域:
在步骤2中FTO的电致变色区域电沉积普鲁士蓝,形成带有电致变色材料的电致变色区域;
步骤4、制备电子注入区域:
将步骤1中得到的ZnIn2S4/3DNG分散于N,N-二甲基甲酰胺DMF中,得到ZnIn2S4/3DNG分散液,将ZnIn2S4/3DNG分散液滴涂于电子注入区域,置于红外灯下烘干,得到以ZnIn2S4/3DNG为光敏材料的电子注入区域;
步骤5、构建检测PEDV、TGEV和PDCoV的高通量比率型传感器:
首先,在电子注入区域ZnIn2S4/3DNG/FTO上滴涂壳聚糖CHIT溶液,置于红外灯下烘干;接着,将戊二醛GA溶液滴于电极表面,并置于室温下反应,反应完毕后,用PBS淋洗,除去电极表面多余的GA;
用PBS为溶剂配制PEDV、TGEV和PDCoV抗体溶液,将三种抗体溶液分别滴加在相应的电子注入区域,反应一段时间后,用PBS淋洗以除去过量未结合的抗体,然后滴加牛血清蛋白BSA溶液以封闭非特异性活性位点;3D打印的模具盖在电极上,最终得到抗体修饰的电子注入区域Ab/ZnIn2S4/3DNG/FTO,与电致变色区域构成光电致变色免疫传感器件;
利用分别添加了PEDV、TGEV和PDCoV抗体的三个检测区域和一个空白对照区域,实现基于普鲁士蓝颜色变化的光电致变色可视化比率型检测。
2.如权利要求1所述的构建方法,其特征在于,步骤2中,所述电极基底面积为2.2cm×5.3cm;每个电子注入区域的面积为1cm×1cm;每个电致变色区域的面积为0.5cm×0.5cm;所用刻蚀仪器为激光刻蚀仪,刻蚀所用功率是50%,刻蚀速度为1000mm/s。
3.如权利要求1所述的构建方法,其特征在于,步骤3中,电沉积普鲁士蓝的具体步骤为:采用三电极体系:工作电极为FTO中的电致变色区域、辅助电极为铂丝、参比电极为Ag/AgCl电极,在以HCl、K3[Fe(CN)6]和FeCl3·6H2O组成的沉积液中用循环伏安法电沉积普鲁士蓝,将制备完成的电极置于温度为60℃的烘箱中过夜;
其中,电沉积参数设置为施加电压为0.3V,时间为75s;电沉积结束后用纯水缓慢冲洗电极,干燥一夜以备用;
所述沉积液中,K3[Fe(CN)6]、FeCl3·6H2O和HCl的浓度均为0.005mol/L,超纯水的用量为90~110ml。
4.如权利要求1所述的构建方法,其特征在于,步骤4中,所述ZnIn2S4/3DNG分散液的浓度为2mg/mL,滴加量20μL。
5.如权利要求1所述的构建方法,其特征在于,步骤5中,
所述CHIT的质量百分浓度为0.1%,滴加量20μL;
所述GA的体积百分浓度为2.5%,滴加量20μL;CHIT与GA反应时间为1~2h;
PEDV、TGEV和PDCoV抗体溶液浓度分别为2μM、2μM和4μM,滴加量均为20~40μL,反应时间10~14h;
BSA的质量百分浓度为3%,滴加量20μL;
所述3D打印的模具的尺寸为530mm×220mm×8mm,四个电子注入区域的尺寸为11mm×12mm×8mm,电致变色区域尺寸为500mm×4mm×8mm。
6.将权利要求1~5任一项所述构建方法构建的高通量比率型芯片式传感器件用于同时检测PEDV、TGEV和PDCoV的用途。
7.如权利要求6所述的用途,其特征在于,检测的具体步骤为:
(1)将不同浓度的PEDV、TGEV和PDCoV的病毒溶液分别滴到Ab/ZnIn2S4/3DNG/FTO电子注入区域上,并在室温下孵育一段时间;
(2)将PBS溶液等体积的分别注入到四个电子注入区域所在的槽子中,再将KCl溶液滴加到电致变色区域所在的槽子中,氙灯光源垂直照射该芯片式传感器上,以电致变色区域中的普鲁士蓝颜色变化作为输出信号;将变色区域普鲁士蓝灰度值与空白区域的普鲁士蓝的比值分别与PEDV、TGEV和PDCoV浓度做标准曲线。
8.如权利要求7所述的用途,其特征在于,
步骤(1)中,PEDV、TGEV和PDCoV浓度范围分别为102~105TCID50/mL、103~107.5TCID50/mL和5×103~107TCID50/mL,滴加量为10~30μL;
步骤(2)中,PBS量为0.5~1.0mL,KCl溶液的用量为0.5~1.0mL,浓度为0.1mol/L;氙灯光源的强度为25%~100%。
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