CN116297756A - 一种检测黑色素瘤标志物s100b蛋白的电化学传感器及其制备方法 - Google Patents
一种检测黑色素瘤标志物s100b蛋白的电化学传感器及其制备方法 Download PDFInfo
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Abstract
本发明属于电化学传感器技术领域,公开了一种检测黑色素瘤标志物S100B蛋白的电化学传感器及其制备方法。将捕获探针通过金硫自组装固定到金电极表面,用MCH封闭后,得到有捕获探针修饰的金电极。然后通过捕获探针与S100B蛋白特异性结合修饰检测物S100B蛋白,最后通过在水中由于亲疏水作用发生自组装的信号放大探针与S100B蛋白特异性结合,即得到检测S100B蛋白的电化学生物传感器。本发明基于多肽中的序列KRLRRSAHARKETEFLRLKRTRLGLE与S100B蛋白之间的2:1的特异性识别构建了一种检测S100B蛋白的电化学生物传感器,采用CV和SWV对传感器的性能和特性进行表征,准确捕捉电化学信号的变化。该传感器具有灵敏度高、特异性强、分析速度快等特点。
Description
技术领域
本发明属于电化学传感器技术领域,具体涉及一种检测黑色素瘤标志物S100B蛋白的电化学传感器及其制备方法。
背景技术
S100B蛋白是一种21kDa二聚体蛋白,黑色素瘤的标志物中,乳酸脱氢酶是临床常规使用效果的最突出的标志物,而S100B蛋白效果甚至优于乳酸脱氢酶。因此,S100B蛋白作为一种新的标志物,在黑素瘤的早期检测和预防中起着至关重要的作用。目前检测S100B蛋白的主要方法有:酶联免疫吸附试验(ELISA)和使用抗体的免疫组织化学,酶联免疫吸附法特异性和灵敏性较差,操作过程繁琐,检测时间较长;使用抗体的免疫组织化学灵敏性差,抗原质量要求高,很难获得各种市售抗原抗体。这里我们建议使用多肽作为抗体的简化等价物,用电化学而不是光谱学测量。一方面,众所周知,电化学技术简单、快速且具有成本效益。另一方面,多肽的几个优点,如合成可接近性和易于修饰,使它们成为蛋白质测定的靶向配体的一个令人信服的选择。目前,利用多肽和电化学结合的方法还未见相关报道。
发明内容
有鉴于此,本发明的目的在于提供一种检测黑色素瘤标志物S100B蛋白的电化学传感器及其制备方法。能对S100B蛋白进行即时检测,灵敏度高、特异性强且简单廉价。为达到上述目的,本发明采取如下技术手段:
本发明提供一种检测黑色素瘤标志物S100B蛋白的电化学生物传感器的制备方法,包括如下步骤:
(1)制备S100B蛋白溶液:
将S100B蛋白冻干粉溶于含三(2-氯乙基)磷酸酯TCEP的Tris-HCl溶液中,得到S100B蛋白溶液;
(2)制备捕获探针溶液:
将捕获探针多肽A冻干粉溶于含TCEP的去离子水中,得到捕获探针溶液;
(3)制备信号放大探针溶液:
将信号放大探针多肽B冻干粉溶于去离子水中,经过自组装后得到信号放大探针溶液;
(4)电化学生物传感器的组装:
步骤4.1,将预处理后的金电极放入步骤(2)所得的捕获探针溶液中进行自组装修饰,然后用乙醇和水清洗;
步骤4.2,将步骤4.1中修饰和清洗完的金电极放入6-巯基-1-己醇MCH溶液中,封闭电极表面未被捕获探针占据的空白位点,封闭后用乙醇和去离子水冲洗电极表面;
步骤4.3,将步骤4.2中处理完的金电极放入步骤(1)所得的S100B蛋白溶液中,通过金电极表面已修饰上的捕获探针与S100B蛋白的特异性结合,得到修饰上S100B蛋白的金电极表面;
步骤4.4,将步骤4.3中修饰上S100B蛋白的金电极放入步骤(3)所得的信号放大探针溶液中,通过S100B蛋白与信号放大探针之间的特异性结合,形成多肽-蛋白-多肽的“三明治”夹心结构,即为检测S100B蛋白的电化学生物传感器。
进一步地,
步骤(1)中,含三(2-氯乙基)磷酸酯TCEP的Tris-HCl溶液中,TCEP的浓度为1mM;Tris-HCl的浓度为20mM;S100B蛋白溶液的浓度为0.2nM-12.8nM。
步骤(2)中,含TCEP的去离子水中,TCEP的浓度为1mM;捕获探针溶液的浓度为30μM;多肽A的氨基酸序列为:CP4-KRLRRSAHARKETEFLRLKRTRLGLE。
步骤(3)中,信号放大探针溶液的浓度为30μM;多肽B的氨基酸体序列为:C16-GGG-KRLRRSAHARKETEFLRLKRTRLGLE-Fc。
步骤4.1中,自组装修饰的条件为:在黑暗环境中于4度环境下反应12-16h。
步骤4.2中,MCH溶液的浓度为1mM,封闭时间为0.5-1h。
步骤4.3中,S100B蛋白的修饰时间至少2.5h。
步骤4.4中,信号放大探针的修饰时间为2h。
步骤4.1中,金电极的预处理步骤包括:将金电极浸泡在食人鱼溶液中去除有机物后用去离子水冲洗,将电极表面抛光;抛光后的金电极放入硫酸溶液中以-0.4mV到1.6mV的电压扫描CV 20圈;再将金电极放入铁氰化钾/亚铁氰化钾溶液中以-0.2到0.6V的电压扫描CV,预处理后的金电极在铁氰化钾/亚铁氰化钾溶液中ΔEP≤90mV。
另外,本发明还提供了一种黑色素瘤标志物S100B蛋白的检测的方法,步骤为:以检测S100B蛋白的电化学生物传感器为工作电极,以Ag/AgCl参比电极,铂丝作为对电极;将工作电极放入PBS电解液的电解杯中,用方波伏安法进行电化学测试,通过电化学信号状态表征S100B蛋白浓度。
进一步地,所述方波伏安法的参数为:扫描电压从0.2V~0.8V,电位增量5mV,振幅25mV;PBS电解液中还添加有NaClO4,NaClO4的浓度为0.1M。
与现有技术相比,本发明的有益效果是:
本发明中我们设计的捕获探针是一种多肽,多肽的具体氨基酸序列是CP4-KRLRRSAHARKETEFLRLKRTRLGLE,我们设计的多肽序列不仅具有与S100B特异性结合的识别序列KRLRRSAHARKETEFLRLKRTRLGLE,还有与金电极结合的CP4序列,这是因为捕获探针上的CP4序列带有巯基,可通过Au-S自组装将捕获探针固定在金电极表面。
在此发明中,我们提出了一种新的基于原位多肽自组装的信号放大方法来灵敏地检测S100B蛋白。有趣的是,在我们的生物传感平台中,多肽同时发挥了多种作用。特别是,设计的多肽具有与S100B结合的识别区和自组装区域,在温和的条件下形成多肽纳米球,以增加信号标签Fc的负载量。这两个反应同时进行,简化了操作,同时提高了性能。信号放大探针C16-GGG-KRLRRSAHARKETEFLRLKRTRLGLE-FC的特异性结合能力不受自组装的影响,这是因为C16尾部包装在疏水核心中,并将识别片段KRLRRSAHARKETEFLRLKRTRLGLE和Fc标签暴露在自组装纳米球上。由于可控的多肽自组装可提高电活性标记的负载量,因此这种多功能多肽为制备具有理想灵敏度的电化学生物传感器提供了一种通用的手段。采用CV和SWV对传感器的性能和特性进行表征,准确捕捉电化学信号的变化。具有特异性强、灵敏度高的优点。使用本发明所提供的电化学传感器对黑色素瘤的早期检测发展具有一定的促进作用。
附图说明
图1为电化学传感器的原理图;
图2为电化学传感器构建过程表征的循环伏安曲线图;
图3为不同浓度S100B蛋白对应的方波伏安法图;
图4为SWV信号值与S100B蛋白浓度的线性关系图;
图5为电化学生物传感器检测S100B蛋白的选择性结果图。
具体实施方式
本发明公开了一种检测黑色素瘤标志物S100B蛋白的电化学传感器及其制备方法。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
以下实施案例中的方法、设备、材料,如果未进行特别说明,均为本领域常规方法、设备和材料,均可由市场购得。
实施例1:电化学传感器的制备
(1)金电极的预处理:
将金电极浸泡在食人鱼溶液(浓硫酸与过氧化氢的体积比为7/3的混合溶液)中30分钟,将电极取出用去离子水将电极上残留的食人鱼溶液冲洗干净。用白色尼龙抛光布和鹿皮抛光布加0.3μm和0.05μm的氧化铝抛光粉进行抛光,将电极表面抛光如镜面;随后,将电极放入0.5M的硫酸溶液中以-0.4到1.6mV的电压扫描CV 20圈,使曲线峰值稳定在89mV以下。之后将电极放入5mM铁氰化钾/亚铁氰化钾溶液中以-0.2到0.6V的电压扫描CV,使ΔEP≤90mV;完成金电极的预处理。
(2)金电极的修饰:
将预处理后的金电极放入300μL的1mM三(2-氯乙基)磷酸酯(TCEP)和30μM捕获探针的去离子水溶液中,4℃黑暗环境下反应至少12个小时,之后残留的溶液用乙醇和水洗掉;
然后将电极浸入到1mM的MCH的乙醇溶液中来淬灭金电极上剩余的活性位点,反应在通风橱中于室温进行1h;
封闭之后的金电极插入300μL浓度分别为0.2nM、0.4nM、0.8nM、1.6nM、3.2nM、6.4nM、12.8nM的S100B蛋白待测液,室温下反应至少2.5h;
最后把修饰上S100B蛋白的金电极插入300μL的30μM信号放大探针的去离子水溶液中,修饰至少2h,即完成电化学传感器中金电极的全部修饰过程。
图1是电化学传感器的原理图;如图1可见,在预处理完的金电极表面依次修饰捕获探针、MCH、S100B蛋白、信号放大探针,形成多肽-蛋白-多肽的“三明治”夹心结构,即为检测S100B蛋白的电化学生物传感器。本发明中信号放大的原理是通过最后一步修饰过程中的信号放大探针在水中由于亲疏水作用发生自组装,从而增加信号分子的负载量,达到信号放大的效果,实现对S100B蛋白的超灵敏检测。
图2是电化学传感器构建过程表征的循环伏安曲线图。循环伏安法的参数为:扫描电压从-0.2V~0.6V,扫速50mV;循环伏安法的测量在5mM K3[Fe(CN)6]/K4[Fe(CN)6]SWV溶液中进行;
如图2所示,裸金电极的电流最大,氧化还原峰之间的电压差ΔV在90mV左右。当修饰捕获探针后,电压差值明显增大且电流变小。说明捕获探针不具备导电特性,阻碍了电子传递。随后依次修饰MCH和S100B蛋白后电流仍出现不同程度的降低,说明了MCH和S100B蛋白成功修饰到了电极上。而当信号放大探针特异性吸附到电极上后电流值表现出回升,可能是由于二茂铁分子和铁氰化钾之间的静电作用促进了电子转移。图2主要证明了我们构建的电化学传感器是逐层修饰的,间接证明了多肽-蛋白-多肽的“三明治”夹心结构的形成。
实施例2:电化学传感器检测S100B蛋白
在本实施例中使用三电极体系对S100B蛋白进行测试:
以实施例1制备的电化学传感器为工作电极,以Ag/AgCl参比电极,铂丝作为对电极。将工作电极放入加有0.1M的NaClO4的PBS电解液的玻璃电解杯(CHI222)中,根据实验需求检测不同浓度的S100B蛋白。利用方波伏安法(SWV)来表征传感器的性能和特性,SWV的参数为:扫描电压从0.2V到0.8V,电位增量5mV,振幅25mV。电化学测试均在辰华电化学工作站(CHI660E)上完成。SWV在0.1M的NaClO4的PBS(pH=7.4)溶液中进行。
根据测试结果发现,SWV信号值Ip在S100B蛋白浓度为0.2nM-12.8nM范围内呈线性关系Ip=1.172+1.042log C(Ip单位:μA C单位:nM/L R2=0.995),不同浓度的S100B蛋白对应的SWV信号值如图3所示,SWV信号值与S100B蛋白浓度之间的线性关系图如图4所示。
通过循环伏安法(CV)对电化学传感器构建过程进行监测,使用方波伏安法(SWV)对S00B蛋白浓度进行定量检测;由实施例2可知,SWV信号值与S100B蛋白浓度在0.2nM-12.8nM浓度范围内呈线性关系,因此,根据检测限计算公式3σ/S,经计算实施例2制备得到的检测S100B蛋白的电化学生物传感器检测S100B蛋白的检测限可达0.027nM,这说明本发明的电化学生物传感器的灵敏度很高。
实施例3:特异性测试
以上述实施例1制备得到的检测S100B蛋白的电化学生物传感器为工作电极,实验条件与上述具体实施例2相同,检测0.2nM S100B蛋白以及20nM常见干扰蛋白:BSA、Thrombin、CEA、溶菌酶,结果如图5所示。结果表明:除了目标物S100B蛋白外,其余干扰蛋白与电化学生物传感器相互作用后,SWV信号值变化均很小。这说明100倍的常见的干扰蛋白不影响检测,本发明的生物传感器具有较好的特异性。这主要是因为生物传感器与S100B蛋白相互作用后,S100B蛋白对多肽序列KRLRRSAHARKETEFLRLKRTRLGLE进行特异性识别,使得含信号分子Fc的信号放大探针吸附在电极表面,从而使得SWV信号上升,基于此实现对S100B蛋白高特异性和高灵敏度的检测。
本发明扩展了黑色素瘤标志物S100B蛋白的检测方法,相较基于ThT的荧光分析,本发明通过电化学技术实现对S100B蛋白进行简单、快捷、低成本的实施监测,为医学工作者开发早期检测黑色素瘤提供了参考。本发明在预处理后的金电极上修饰捕获探针,主要通过捕获探针上的CP4序列带有的巯基,可通过Au-S自组装将捕获探针固定在金电极表面。随后用6-巯基-1-己醇(MCH)封闭电极表面上未被捕获探针占据的空白位点,然后分别修饰S100B蛋白、信号放大探针,主要基于多肽(KRLRRSAHARKETEFLRLKRTRLGLE)与S100B蛋白之间的2:1的特异性识别,构建了一种多肽-蛋白-多肽的“三明治”夹心结构的检测黑色素瘤标志物S100B蛋白的电化学传感器。以Fc作为探针检测传感器和S100B蛋白待测液相互作用后传感器界面上SWV信号值的变化。S100B蛋白浓度在0.2nM-12.8nM范围内与SWV信号值呈现良好的线性关系,检出限为0.027nM。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.一种检测黑色素瘤标志物S100B蛋白的电化学传感器的制备方法,其特征在于,包括如下步骤:
(1)制备S100B蛋白溶液:
将S100B蛋白冻干粉溶于含三(2-氯乙基)磷酸酯TCEP的Tris-HCl溶液中,得到S100B蛋白溶液;
(2)制备捕获探针溶液:
将捕获探针多肽A冻干粉溶于含TCEP的去离子水中,得到捕获探针溶液;
(3)制备信号放大探针溶液:
将信号放大探针多肽B冻干粉溶于去离子水中,经过自组装后得到信号放大探针溶液;
(4)电化学生物传感器的组装:
步骤4.1,将预处理后的金电极放入步骤(2)所得的捕获探针溶液中进行自组装修饰,然后用乙醇和水清洗;
步骤4.2,将步骤4.1中修饰和清洗完的金电极放入6-巯基-1-己醇MCH溶液中,封闭电极表面未被捕获探针占据的空白位点,封闭后用乙醇和去离子水冲洗电极表面;
步骤4.3,将步骤4.2中处理完的金电极放入步骤(1)所得的S100B蛋白溶液中,通过金电极表面已修饰上的捕获探针与S100B蛋白的特异性结合,得到修饰上S100B蛋白的金电极表面;
步骤4.4,将步骤4.3中修饰上S100B蛋白的金电极放入步骤(3)所得的信号放大探针溶液中,通过S100B蛋白与信号放大探针之间的特异性结合,形成多肽-蛋白-多肽的“三明治”夹心结构,即为检测S100B蛋白的电化学生物传感器。
2.如权利要求1所述的制备方法,其特征在于:步骤(1)中,含三(2-氯乙基)磷酸酯TCEP的Tris-HCl溶液中,TCEP的浓度为1mM;Tris-HCl的浓度为20mM;S100B蛋白溶液的浓度为0.2nM-12.8nM。
3.如权利要求1所述的制备方法,其特征在于:步骤(2)中,含TCEP的去离子水中,TCEP的浓度为1mM;捕获探针溶液的浓度为30μM;多肽A的氨基酸序列为:CP4-KRLRRSAHARKETEFLRLKRTRLGLE。
4.如权利要求1所述的制备方法,其特征在于:步骤(3)中,信号放大探针溶液的浓度为30μM;多肽B的氨基酸体序列为:C16-GGG-KRLRRSAHARKETEFLRLKRTRLGLE-Fc。
5.如权利要求1所述的制备方法,其特征在于:步骤4.1中,自组装修饰的条件为:在黑暗环境中于4度环境下反应12-16h。
6.如权利要求1所述的制备方法,其特征在于:步骤4.2中,MCH溶液的浓度为1mM,封闭时间为0.5-1h。
7.如权利要求1所述的制备方法,其特征在于:步骤4.3中,S100B蛋白的修饰时间至少2.5h;步骤4.4中,信号放大探针的修饰时间为2h。
8.将权利要求1-7任一项所述制备方法制得的电化学传感器用于检测黑色素瘤标志物S100B蛋白用途。
9.如权利要求8所述的用途,其特征在于:检测步骤为:
以检测S100B蛋白的电化学生物传感器为工作电极,Ag/AgCl参比电极,铂丝作为对电极;将工作电极放入PBS电解液的电解杯中,用方波伏安法进行电化学测试,通过电化学信号状态表征S100B蛋白浓度。
10.如权利要求9所述的用途,其特征在于:所述方波伏安法的参数为:扫描电压从0.2V~0.8V,电位增量5mV,振幅25mV;PBS电解液中还添加有NaClO4,NaClO4的浓度为0.1M。
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