CN113203781A - 一种基于RGO-CS-Hemin@Pt NPs纳米材料和适配体检测GPC3的方法 - Google Patents
一种基于RGO-CS-Hemin@Pt NPs纳米材料和适配体检测GPC3的方法 Download PDFInfo
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Abstract
一种基于RGO‑CS‑Hemin@Pt NPs纳米材料和适配体检测GPC3的方法,采用电沉积技术将Au NPs@rGO修饰在SPCE表面,通过静电吸附作用将GPC3AptI负载在Au NPs@rGO表面,分别将GPC3和RGO‑CS‑Hemin@Pt NPs‑AptII信号探针孵育在电极表面,构建了RGO‑CS‑Hemin@Pt NPs‑AptII/GPC3/GPC3AptI/Au NPs@rGO/SPCE夹心型电化学纳米适配体传感器。借助于RGO‑CS‑Hemin@Pt NPs的过氧化物酶作用,催化分解支持液中的H2O2和HQ,采用电化学工作站的DPV进行扫描,记录其峰电流,实现对GPC3的检测。
Description
技术领域
本发明属于生物检测领域,具体涉及一种基于纳米复合材料和适配体检测GPC3的方法。
背景技术
磷脂酰肌醇蛋白聚糖3 ( glypican-3 , GPC3)是一种肝癌标志物。目前GPC3检测方法主要有流式免疫分析法、电化学免疫传感器、压电免疫传感器、酶联免疫吸附法(ELISA)、放射免疫分析法等。公开号CN 111909902 A的发明专利,涉及一种杂交瘤细胞株、单克隆抗体及其制备方法和应用、制备致敏胶乳原液的方法以及试剂盒,但该方法对操作环境和技术要求较高。公开号CN 111751534 A的发明专利,涉及一种磷脂酰肌醇蛋白聚糖3检测试剂盒及其应用,但该方法所用试剂昂贵且技术要求高,因此,需要建立一种快速、灵敏、便携检测GPC3的方法。
发明内容
本发明所要解决的技术问题是提供一种基于还原性氧化石墨烯-壳聚糖-血红素@纳米铂(RGO-CS-Hemin@Pt NPs)的纳米材料,构建夹心型适配体传感器,实现GPC3检测,最低检测限为0.0647 ng/mL。
为解决该技术问题,利用RGO-CS-Hemin@Pt NPs纳米材料为载体,制备了RGO-CS-Hemin@Pt NPs-AptII信号探针;采用电沉积技术将Au NPs@rGO修饰在丝网印刷电极表面;通过静电吸附作用将GPC3适配体(GPC3AptI)负载在修饰Au NPs@rGO的电极表面,适配体以单链形式形成不稳定的空间结构而存在电极表面;分别将GPC3和RGO-CS-Hemin@Pt NPs-AptII信号探针孵育在上述电极表面,由于GPC3能够与GPC3适配体特异性结合,形成稳定的空间结构,有序排列在电极表面,构建了RGO-CS-Hemin@Pt NPs-AptII /GPC3/GPC3AptI/AuNPs@rGO/SPCE夹心型电化学纳米适配体传感器。借助于RGO-CS-Hemin@Pt NPs纳米材料的过氧化物酶作用,催化支持液中的H2O2和氢醌(HQ)并将HQ还原为苯醌(BQ)沉积在电极表面,采用电化学工作站的微分脉冲伏安法 (DPV),记录其峰电流,从而实现对GPC3的检测。
本发明按照以下步骤进行:
步骤1:RGO-CS-Hemin@Pt NPs-AptII信号探针的制备
(1)还原性氧化石墨烯(RGO)的制备
氧化石墨烯(GO)溶液倒入蒸馏水中,超声破碎,制成GO水溶液,再加入抗坏血酸(AA)还原,得RGO溶液;
(2)还原性氧化石墨烯-壳聚糖(RGO-CS)的制备
在RGO溶液加入壳聚糖(CS),超声破碎,得到均匀的RGO-CS分散液;
(3)还原性氧化石墨烯-壳聚糖-血红素(RGO-CS-Hemin)的制备
将血红素(Hemin)加入氨水中溶解,得血红素溶液。再加RGO-CS溶液中,搅拌,得到RGO-CS-Hemin分散液;
(4)RGO-CS-Hemin@Pt NPs复合材料的制备
在RGO-CS-Hemin分散液中加入氯铂酸钠和抗坏血酸,放置在磁力搅拌器上搅拌12h后取出。把分散液放入离心机进行分离。离心后取沉淀溶于超纯水,得到 RGO-CS-Hemin@Pt NPs溶液;
(5)RGO-CS-Hemin@Pt NPs-AptII信号探针的制备
将GPC3适配体(GPC3-AptII)和RGO-CS-Hemin@PtNPs溶液混合,孵育,离心洗涤,去除游离的适配体,即得RGO-CS-Hemin@Pt NPs-AptII溶液。
步骤2:电极的修饰与生物传感界面的构建
(1)将丝网印刷电极(SPCE)置于H2SO4溶液中活化;
(2)将活化后的丝网印刷电极置入含有氯金酸(HAuCl4)和RGO的混合液中,进行恒电位沉积,得Au NPs@rGO/SPCE电极;
(3)将GPC3AptI滴加在Au NPs@rGO/SPCE的表面,孵育,洗涤晾干,得GPC3AptI /AuNPs@rGO/SPCE;
(4)将GPC3标准液滴加到GPC3AptI /Au NPs@rGO/SPCE表面,孵育一段时间后,清洗晾干,得到GPC3/GPC3AptI /Au NPs@rGO/SPCE;
(5)在GPC3/GPC3AptI /Au NPs@rGO/SPCE上滴加RGO-CS-Hemin@Pt NPs-AptII溶液,孵育一定时间,清洗晾干,得到工作电极(RGO-CS-Hemin@Pt NPs-AptII/GPC3/GPC3AptI/Au NPs@rGO/SPCE),备用。
步骤3:GPC3工作曲线的绘制
(1)将步骤2得到的工作电极浸入到含有H2O2-HQ的PBS溶液,采用电化学工作站的差分脉冲伏安法(DPV)进行扫描,记录传感器的响应电流值;
(2)分别对不同浓度的GPC3进行检测,记录峰电流;根据传感器的电流响应值与GPC3浓度的关系,绘制GPC3的工作曲线,计算出该方法的最低检测限。
步骤4:实际血清样本中GPC3的检测
(1)用待测实际血清样品制备的工作电极浸入到含有H2O2-HQ的PBS溶液里面,采用电化学工作站的DPV进行扫描,记录传感器的响应电流值;
(2)根据步骤3所得到的工作曲线,计算得到所述待测实际样品中GPC3的浓度。
作为优选:
步骤1中所述RGO-CS-Hemin@Pt NPs溶液浓度为1.0 mg/mL;
步骤1中所述RGO-CS-Hemin@Pt NPs-AptII溶液浓度为10.0 μmol/L;
步骤2中所述H2SO4溶液浓度为0.5 mol/L;所述扫描电压为-0.4 V ~ 1.2 V,扫描圈数为20;
步骤2中所述用于沉积溶液的HAuCl4溶液为0.01%,RGO溶液浓度为1.0 mg/mL,沉积电位为-0.5 V,沉积时间120 s;
步骤3和步骤4中所述电极的孵育温度为15°C,孵育时间为1h;
步骤3和步骤4中所述H2O2-HQ溶液中H2O2和HQ浓度均为1 mmol/L;
步骤3和步骤4中所述的扫描范围为-0.18V ~ 0.24V,扫描速率为0.01V/s。
其中,步骤1制备一种独特的RGO-CS-Hemin@Pt NPs纳米材料,为GPC3-AptII的固定提供了良好的载体,形成了具有大比表面积和高的电子转移效率的RGO-CS-Hemin@PtNPs-AptII纳米信号探针。步骤2构成特异性识别GPC3的生物传感界面,利用GPC3适配体和GPC3蛋白的特异性结合以及RGO-CS-Hemin@Pt NPs纳米材料兼备类过氧化酶的性质,实现良好的协同效应及催化作用,并有利于电子的传递。步骤2中生物传感界面的构建为步骤3和步骤4中GPC3的电化学检测中必不可少的关键步骤。可见步骤1-4相互支撑,共同作用,才能利用RGO-CS-Hemin@Pt NPs-AptII纳米信号探针实现GPC3的检测。
本发明与现有技术相比具有如下优点:
1.目前一般采用抗体免疫法测定血清GPC3水平,因其试剂昂贵,操作复杂而难以实现,而电化学适配体传感器具有优异的灵敏度,快速响应,从而实现了GPC3的灵敏检测。
2.RGO-CS-Hemin@Pt NPs-AptII纳米信号探针具有比表面积大,吸附能力强,导电性强等特点,可有效的提高检测速率;且RGO-CS-Hemin@Pt NPs纳米材料兼具高效的类过氧化酶的催化性质,可将H2O2催化分解为H2O和O2,HQ催化还原为BQ,从而产生较强的电流信号,实现对GPC3蛋白的灵敏检测,该方法的最低检测限为0.0647 ng/mL。
附图说明
图1 基于RGO-CS-Hemin@Pt NPs纳米材料和适配体的夹心型电化学适配体传感器检测GPC3的原理图;
图2 RGO-CS-Hemin@Pt NPs复合纳米材料的透射电镜图(TEM);
图3 电极表面不同修饰过程的扫描电镜图(SEM);
图4 GPC3的工作曲线。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
一种基于RGO-CS-Hemin@Pt NPs纳米材料和适配体的夹心型电化学适配体传感器检测GPC3的原理图见图1。首先,制备RGO-CS-Hemin@Pt NPs纳米复合材料,应用RGO-CS-Hemin@Pt NPs纳米复合材料固定GPC3-AptII形成RGO-CS-Hemin@Pt NPs-AptII信号探针。采用电沉积技术将Au NPs@rGO修饰在丝网印刷电极表面。通过静电吸附作用将GPC3AptI负载在修饰Au NPs@rGO的电极表面,适配体以单链形式形成不稳定的空间结构而存在电极表面;分别将GPC3和RGO-CS-Hemin@Pt NPs-AptII信号探针孵育在上述电极表面,由于GPC3能够与GPC3适配体特异性结合,形成稳定的空间结构,有序排列在电极表面,构建了RGO-CS-Hemin@Pt NPs-AptII /GPC3/GPC3AptI/Au NPs@rGO/SPCE夹心型电化学纳米适配体传感器。借助于RGO-CS-Hemin@Pt NPs纳米材料的过氧化物酶作用,催化分解支持液中的H2O2和氢醌(HQ)并将HQ还原为苯醌(BQ)沉积在电极表面,采用电化学工作站的微分脉冲伏安法(DPV),记录其检测GPC3前后的电化学电流信号,描绘出该电流与GPC3浓度的关系曲线,从而实现对GPC3的检测。
实施步骤如下:
1.RGO-CS-Hemin@Pt NPs-AptII信号探针的制备
(1)称取35mg GO,并将GO倒入于35 mL的蒸馏水中,使用超声细胞破碎仪超声1h使其充分溶解均匀,制成 1.0 mg/mL的GO水溶液,加入10mg AA,持续搅拌12h,即得RGO。图2a为RGO的透射电镜图(TEM),呈黑色褶皱薄膜结构。
(2)将1 mg CS加入100 mL 1%的乙酸溶液中,搅拌均匀,观察到溶液中无气泡,得到1.0 mg/mL CS溶液。将20 mg Hemin加入8μL氨水至20 mL纯水中溶解,得血红素溶液。取Hemin溶液加入到RGO-CS溶液中,搅拌反应,得到还浓度为1.0 mg/mL的RGO-CS-Hemin复合材料。图2b为RGO-CS-Hemin的TEM,黑色片状结构呈褶皱膜状结构,表明RGO-CS-Hemin材料制备成功。
(3)在RGO-CS-Hemin溶液中加入5mL 1.0 mg/mL氯铂酸钠,加入抗坏血酸10mg,搅拌12后取出,离心,转速设置为10000r/min,转速时间设置为10min,离心后取沉淀溶于超纯水,可得到浓度为1.0 mg/mL RGO-CS-Hemin@Pt NPs溶液。图2c为RGO-CS-Hemin@Pt NPs的TEM图,褶皱膜状结构上明显分散着纳米颗粒,证明Pt NPs已成功附着在RGO-CS-Hemin表面,表明RGO-CS-Hemin@Pt NPs材料构建成功。
(4)将GPC3-AptII和RGO-CS-Hemin@Pt NPs溶液混合,孵育,产物经离心洗涤后,去除游离的适配体,即得RGO-CS-Hemin@Pt NPs-AptII溶液。
电极的修饰与生物传感界面的构建
(1)将电极置于0.5 mol/L H2SO4中进行循环伏安扫描20段,电压范围为0.4 V ~1.0 V。将活化后的SPCE电极放入5mL 含有0.01% HAuCl4溶液和1.0 mg/mL RGO溶液的混合液溶液,在-0.5 V恒电位沉积120 s,沉积完成后用纯水洗涤3次,吹干得到Au NPs@rGO/SPCE电极。
(2) 将3μL巯基化的GPC3适配体溶液(GPC3AptI,5'-TAACGCTGACCTTAGCATGGCTTTACATGTTCCA-SH-3')滴加在Au NPs@rGO/SPCE的表面,孵育1h,洗涤晾干,得到GPC3AptI /AuNPs@rGO/SPCE。
(3)滴加3μL 0.2%的MCH溶液进行封闭,自然晾干。将3μL 不同浓度的GPC3标准液(或者待测血清样品)滴加到GPC3AptI /Au NPs@rGO/SPCE表面,孵育1h后,清洗晾干后得到GPC3/GPC3AptI /Au NPs@rGO/SPCE。
(4)在步骤(3)制备的传感界面上滴加3μL 10.0 μmol/L RGO-CS-Hemin@Pt NPs-AptII溶液,孵育30min,清洗晾干得到工作电极(RGO-CS-Hemin@Pt NPs-AptII/GPC3/GPC3AptI /Au NPs@rGO/SPCE)。采用扫描电镜(SEM)对电极表面不同修饰过程进行表征,如图3所示。图3A为SPCE的SEM图,电极表面因其固有的碳颗粒而呈现出排列均匀的颗粒;图3B为Au NPs@rGO /SPCE的SEM图,可以看见在碳颗粒的表层覆盖一层黑色薄膜并且均匀分布着许多明亮白色的球形微粒,说明Au NPs@rGO成功沉积到了电极表面;图3C 为GPC3AptI /Au NPs@rGO/SPCE的 SEM图,可以看到表面覆盖了一层薄膜,可知GPC3AptI成功固定在电极表面。图3D中可以看到薄膜表面较为平滑,归因于GPC3与GPC3AptI之间反应形成稳定的结构,可知GPC3成功吸附在电极表面。图3E为RGO-CS-Hemin@Pt NPs-AptII/GPC3/GPC3AptI /Au NPs@rGO/SPCE,表面呈现典型的褶皱结构,且包裹着许多球形纳米颗粒,证明RGO-CS-Hemin@Pt NPs-AptII已均匀的修饰在电极表面。
工作曲线的绘制
(1)在步骤2构建的GPC3电化学生物传感界面滴加3μL GPC3标准溶液,放到25℃孵育箱中孵育1h,得到GPC3的工作电极(RGO-CS-Hemin@Pt NPs-AptII/GPC3/GPC3AptI /AuNPs@rGO/SPCE)。
(2)将上述所得的工作电极放到含有H2O2-HQ的PBS溶液(0.2 mol/L,pH6.0,1:1)中,采用电化学工作站的DPV进行扫描,记录其峰电流。不同GPC3浓度的DPV曲线图见图4。GPC3浓度在3.0~60.0 µg/mL内,传感器电流响应值(Y)与GPC3浓度(X)之间的关系呈线性,其线性方程为Y=2.6301+0.0298X,相关系数为0.9908。将空白对照的三倍标准差定义为检测下限,通过公式CLOD=3Sb/b计算得到该方法的最低检测限为0.0647 ng/mL。
实际血清样本中GPC3的检测
通过加标法在最佳条件下检测人血清样本中的GPC3水平。将正常人血清样本以1:1的比例分别与10.0 μg/mL,20.0 μg/mL,40.0 μg/mL的GPC3标准溶液充分混合,制成混合液。在步骤2构建的GPC3电化学生物传感界面滴加3μL混合液,放到25℃孵育箱中孵育1h,得到GPC3的工作电极。按照步骤3所述,将工作电极置于含有H2O2-HQ的PBS溶液(0.2 mol/L,pH6.0,1:1)中进行DPV扫描,记录电流值。通过步骤3所得到的GPC3工作曲线,计算人血清样品中GPC3的浓度,结果见表1所示,其回收率在99.95-104.06%范围内,RSD值为1.31-5.22%。这些结果表明,所开发的GPC3适配体传感器具有良好的应用前景。
表1 实际血清样本中GPC3的检测结果
(注:血清样本由中国人民解放军联勤保障部队第九二四医院提供)。
Claims (8)
1.一种基于RGO-CS-Hemin@Pt NPs纳米材料和适配体检测GPC3的方法,其特征在于,按以下步骤进行:
步骤1:RGO-CS-Hemin@Pt NPs-AptII信号探针的制备
RGO的制备
GO倒入蒸馏水中,破碎均匀,加入AA还原,得RGO溶液;
RGO-CS的制备
在RGO溶液加入CS,超声破碎,获得RGO-CS溶液;
RGO-CS-Hemin的制备
将Hemin加入氨水中溶解,得血红素溶液;
取Hemin溶液加入到RGO-CS溶液中,搅拌反应,得到RGO-CS-Hemin溶液;
RGO-CS-Hemin@PtNPs复合材料制备
在RGO-CS-Hemin溶液中加入氯铂酸钠和抗坏血酸,搅拌反应,离心分离,得到 RGO-CS-Hemin@Pt NPs溶液;
(5) RGO-CS-Hemin@Pt NPs-AptII信号探针的制备
将GPC3-AptII和RGO-CS-Hemin@PtNPs溶液混合,孵育,离心去除上清液,即得RGO-CS-Hemin@Pt NPs-AptII溶液;
步骤2:电极的修饰与生物传感界面的构建
(1) 将SPCE置于H2SO4溶液活化;
(2) 将活化后的丝网印刷电极置入含有HAuCl4和RGO溶液的混合液中,进行恒电位沉积,得到Au NPs@rGO/SPCE;
(3) 将GPC3AptI滴加在Au NPs@rGO/SPCE的表面,孵育,洗涤干燥,得到GPC3AptI /AuNPs@rGO/SPCE;
(4) 将GPC3标准液或者待测样品滴加到GPC3AptI /Au NPs@rGO/SPCE表面,孵育,清洗干燥,得到GPC3/GPC3AptI /Au NPs@rGO/SPCE;
(5) 在GPC3/GPC3AptI/Au NPs@rGO/SPCE上滴加RGO-CS-Hemin@Pt NPs-AptII溶液,孵育,清洗干燥,得到工作电极(RGO-CS-Hemin@Pt NPs-AptII/GPC3/GPC3AptI /Au NPs@rGO/SPCE);
步骤3:GPC3工作曲线的绘制
(1) 将步骤2得到的工作电极浸入到含有H2O2-HQ的PBS溶液,采用DPV进行扫描,记录传感器的响应电流值;
(2) 分别对不同浓度的GPC3进行检测,记录峰电流;根据传感器的电流响应值与GPC3浓度的关系,绘制GPC3的工作曲线,计算出该方法的最低检测限;
步骤4:实际血清样本中GPC3的检测
(1) 用待测实际血清样本制备的工作电极浸入到含有H2O2-HQ的PBS溶液里面,采用电化学工作站的DPV进行扫描,记录传感器的响应电流值;
(2) 根据步骤3所得到的工作曲线,计算得到所述待测实际样品中GPC3的浓度。
2.按照权利要求1所述检测GPC3的方法,其特征在于:步骤1中所述RGO-CS-Hemin@PtNPs溶液浓度为1.0 mg/mL。
3.按照权利要求1所述检测GPC3的方法,其特征在于:步骤1中所述RGO-CS-Hemin@PtNPs-AptII溶液浓度为10.0 μmol/L。
4.按照权利要求1所述检测GPC3的方法,其特征在于:步骤2中所述H2SO4溶液浓度为0.5mol/L;所述扫描电压为-0.4 V ~ 1.2 V,扫描圈数为20。
5.按照权利要求1所述检测GPC3的方法,其特征在于步骤2中所述用于沉积溶液的HAuCl4溶液为0.01%,RGO溶液浓度为1.0 mg/mL,沉积电位为-0.5 V,沉积时间120 s。
6.按照权利要求1所述检测GPC3的方法,步骤3和步骤4中所述电极的孵育温度为15°C,孵育时间为1h。
7.按照权利要求1所述检测GPC3的方法,其特征在于:步骤3和步骤4中所述H2O2-HQ溶液中H2O2和HQ浓度均为1 mmol/L。
8.按照权利要求1所述检测GPC3的方法,其特征在于:步骤3和步骤4中所述的扫描范围为-0.18V ~ 0.24V,扫描速率为0.01V/s。
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Application publication date: 20210803 Assignee: Guangxi Silizhao Biotechnology Co.,Ltd. Assignor: GUILIN University OF ELECTRONIC TECHNOLOGY Contract record no.: X2023980045668 Denomination of invention: A non diagnostic purpose based on RGO-CS-Hemin@Pt Method for Detecting GPC3 with NPs Nanomaterials and Adapters Granted publication date: 20220531 License type: Common License Record date: 20231102 |