CN110398585B - 一种测定甲胎蛋白抗原的免疫生物传感器及其制备方法与应用 - Google Patents
一种测定甲胎蛋白抗原的免疫生物传感器及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种测定甲胎蛋白抗原的免疫生物传感器及其制备方法与应用,所述的测定甲胎蛋白抗原的免疫生物传感器,其基底电极表面依次经过羧基化多壁碳纳米管、乙烯基二茂铁‑N‑羟基琥珀酰亚胺丙烯酸酯电聚合和甲胎蛋白抗原抗体修饰即得。与现有技术相比,本发明具有如下优点:(1)MWCNTs具有比表面积大、吸附力强、导电性能好等优点;(2)制备的电化学免疫传感器具有灵敏度高,反应时间短,组装简单,信号强,线性范围宽等优点,并实现了对肿瘤标志物AFP的痕量检测;(3)该传构建方法也可用于其他免疫蛋白或致病抗原物质的测定,在临床方面有广泛的应用价值;(4)本发明的免疫生物传感器操作简单,成本低。
Description
技术领域
本发明属于肿瘤标志物快速检测技术领域,具体涉及一种测定甲胎蛋白抗原的免疫生物传感器及其制备方法。
背景技术
甲胎蛋白(AFP)是目前发现最重要且最具说服力的肿瘤标志物。有研究表明血清甲胎蛋白测定结果大于500μg/L以上,或含量有不断增高者,更应高度警惕,肝癌患者血清甲胎蛋白含量变化的速率和程度与肿瘤组织分化程度高低有一定相关性,分化程度较高的肿瘤甲胎蛋白含量常大于200μg/L。甲胎蛋白的动态变化与病情有一定的关系。甲胎蛋白含量上升提示病情恶化,其动态变化是显示治疗效果的一项敏感指标。原发性肝癌患者在检测甲胎蛋白时出现阴性或假阴性可能与测定方法、癌肿类型、原发部位等因素有关。肝细胞肝癌血清甲胎蛋白的水平高低与肝细胞分化程度有关,I级肝癌细胞分化接近正常细胞,故甲胎蛋白量尚不足以测出,IV级由于分化极差,已失去类似胚胎细胞的功能,也不能从血清中检出甲胎蛋白,Ⅱ、Ⅲ级癌细胞认为是和胚胎肝细胞相似的幼稚细胞能产生甲胎蛋白,阳性率较高,因此,研究快速准确高精度的甲胎蛋白的检测方法具有重要的意义。免疫蛋白的固定是制备免疫传感器的关键步骤之一。
多壁碳纳米管(MWCNTs)作为一种功能型的材料,具有比表面积大、吸附力强、导电性能好,生物相容性好等优点。碳纳米管上碳原子的P电子形成大范围的离域π键,由于共轭效应显著,碳纳米管具有一些特殊的电学性质,碳纳米管具有良好的导电性能,由于碳纳米管的结构与石墨的片层结构相同,所以具有很好的电学性能当管径小于6nm时,碳纳米管可以制作成具有良好导电性能的一维量子导线。因此,电化学免疫传感器是一种将化学反应转化为电信号利用计算机等媒介进行处理检测的方法。其中其具有导电性高(提高信号响应)和比表面积大(负载更多的抗体)的优势,MWCNTs常被用于增强免疫传感器的检测灵敏度。
发明内容
本发明所要解决的问题是为解决现有技术存在的缺陷,提供一种测定甲胎蛋白抗原的免疫生物传感器。
本发明还要解决的技术问题是提供上述免疫生物传感器的制备方法。
本发明最后要解决的技术问题是提供上述免疫生物传感器的应用。
为解决上述技术问题,本发明采用的技术方案如下:
一种测定甲胎蛋白抗原的免疫生物传感器的制备方法,它包括如下步骤:基底电极表面依次经过羧基化多壁碳纳米管(c-MWCNT)、乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯(FC/NSA)电聚合和甲胎蛋白(AFP)抗原抗体修饰即得。
具体的基底电极由c-MWCNT修饰,FC/NSA结合和c-MWCNT,AFP抗体修饰结合到FC/NSA,其中AFP抗体通过酰胺键共价连接NSA,甲胎蛋白(AFP)是一种糖蛋白,它属于白蛋白家族,主要由胎儿肝细胞及卵黄囊合成,检测中AFP成为AFP抗原。
上述测定甲胎蛋白抗原的免疫生物传感器的制备方法,包括如下步骤:
(1)电极预处理:将基底电极进行打磨、抛光、超声清洗,用氮气吹干燥;
(2)将c-MWCNT加入到水中,超声25~60分钟直至其分散均匀,取2~8μL,优选5μL滴加到步骤(1)处理后的基底电极表面,18~40℃避光干燥1~3h;
(3)FC/NSA的电极组装:将FC/NSA溶液电聚合到步骤(2)处理后的基底电极的表面,其通过打开C=C使FC/NSA与c-MWCNT紧密交联结合;
(4)共价连接AFP抗体:将步骤(3)已经电聚合后的工作电极取出并用PBS清洗,滴加甲胎蛋白包被抗体(cAnti-AFP)于清洗后的基底电极表面,1~10℃,避光放置6~18h,待cAnti-AFP与FC/NSA以酰胺键形式相结合,用PBS缓冲液清洗,室温干燥;
(5)封闭非特异性位点:滴加BSA溶液于步骤(4)处理后的基底电极表面,室温放置1~15h,优选2h,封闭非特异性结合位点,用PBS缓冲液清洗,室温干燥;
(6)接AFP抗原:将甲胎蛋白抗原滴加于步骤(5)处理后的基底电极表面,30~40℃,保温箱中避光0.5~2h干燥,待cAnti-AFP与AFP抗原复合物结合,用PBS缓冲液清洗。
步骤(1)中,所述的抛光为用0.05和0.03μm Al2O3粉末抛光,然后分别用无水乙醇、超纯水超声清洗5min;
步骤(2)中,所述的羧基化多壁碳纳米管的浓度为0.2~4mg/mL,优选1mg/mL,所述的羧基化多壁碳纳米管的体积为2~8μL,优选5μL。
步骤(3)中,所述的电聚合为连续循环伏安法,其扫描范围为(-2.4)~(-1)V,扫描速率为0.1~0.5V/s,优选0.1V/s,扫描圈数为10~17圈,优选15圈;乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液的用量为1~5mL。
步骤(4)中,所述的甲胎蛋白包被抗体的浓度为12~20μg/mL,滴加量为1~10μL,在4℃冰箱,所述的PBS缓冲液的浓度为0.1M,pH=7.4;
步骤(5)中,BSA溶液的质量分数为0.2~2%,优选1%,滴加量为1~10μL,优选5μL;
步骤(6)中,所述的AFP抗原的浓度为10ng/mL~10μg/mL,滴加量为5~10μL,优选5μL。
其中,所述基底电极为玻碳电极。
其中,所述的羧基化多壁碳纳米管的制备方法:
(Ⅰ)将多壁碳纳米管粉末加入到混合酸中,于65~90℃超声1~3.5h,再加热搅拌1~5h,离心分离,得到沉淀物;其中,超声温度优选为80℃,超声时间优选为2h,搅拌时间优选为5h;
(Ⅱ)将步骤(Ⅰ)得到的沉淀物水洗至中性,或加入稀氢氧化钠洗涤至中性,室温下干燥,即得羧基化多壁碳纳米管(c-MWCNT)。
步骤(Ⅰ)中,所述的混合酸为浓硫酸与浓硝酸的混合物,其质量分数分别为98%和65%,体积比为3:1~1:3;控制混酸的添加量,使多壁碳纳米管的浓度为3~7mg/mL,优选5mg/mL;所述的加热指的是加热至温度为24~26℃。
其中,所述的乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯的制备方法如下:
取乙烯基二茂铁、N-羟基琥珀酰亚胺丙烯酸酯和室温离子液体于玛瑙研钵中,均匀研磨5~8分钟直至溶液中无清晰可见颗粒物并转移到小烧杯中,得到乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液。
其中,乙烯基二茂铁和N-羟基琥珀酰亚胺丙烯酸酯的摩尔质量比为1:10~10:1,优选2:1,控制室温离子液体的体积,使乙烯基二茂铁浓度为1~15mg/mL,优选7.069mg/mL;N-羟基琥珀酰亚胺丙烯酸酯的浓度为1~5mg/mL,优选2.835mg/mL;所述的室温离子液体优选1-丁基-3-甲基咪唑六氟磷酸盐,其添加量为2mL。
本发明进一步的目的是提供上述的制备方法制备得到的免疫生物传感器。
本发明更进一步的目的是提供上述免疫生物传感器在定量检测AFP中的应用。
本文采用滴涂法将c-MWCNT固定到基底电极(GCE)表面,将FC与NSA以室温离子液体(RTILs)作为电解质和溶剂制成FC-NSA混合物,通过电聚合打开碳碳双键连接到c-MWCNT表面。然后利用FC-NSA复合物与cAnti-AFP以形成酰胺键的方式相结合,通过抗原与抗体特异的结合制备电化学免疫传感器,利用AFP浓度与电流变化呈一定相关关系对其浓度进行定量分析。由于MWCNTs本身优越的导电性能和高比表面积,可以聚合大量FC-NSA复合物,在三电极体系,电化学催化的条件下,FC作为信号分子。NSA作为一种高效组装的氨基供体试剂,既有提供氨基与大分子蛋白形成酰胺键的能力,又可以作为一种高效交联剂,可以在电催化的条件下打开C=C双键与MWNCTs形成复合材料。RTILs是一种十分优良的电解质,由于其特有的分散的性质,可以将碳管解缠,而且可以通过其粘性将室温不宜组装的FC与NSA通过粘性作用研磨混合在一起,进而在用电化学催化的方式促进其结合。结果表明,该传感器具有制备方法简单、响应迅速、灵敏度高、稳定性好、线性范围宽的优势,将其用于临床样品检测,可以得到满意结果。
本发明中技术术语的缩写如下:
甲胎蛋白:AFP
N-羟基琥珀酰亚胺丙烯酸酯:NSA;
乙烯基二茂铁:FC;
室温离子液体:RTILs(1-丁基-3-甲基咪唑六氟磷酸盐);
多壁碳纳米管:MWCNTs;
羧基化多壁碳纳米管:c-MWCNT;
甲胎蛋白包被抗体:cAnti-AFP
磷酸盐缓冲液:PBS
牛血清白蛋白:BSA
有益效果:与现有技术相比,本发明具有如下优点:
(1)MWCNTs具有比表面积大、吸附力强、导电性能好、相容性好等优点;
(2)制备的电化学免疫传感器具有灵敏度高,反应时间短,组装简单,信号强度大,线性范围宽等优点,并实现了对肿瘤标志物AFP的痕量检测。;
(3)该传感器构建方法也可用于其他免疫蛋白或致病抗原物质的测定,在临床方面有广泛的应用价值;
(4)本发明的免疫生物传感器操作简单,成本低。
附图说明
图1为本发明测定AFP的免疫传感器的制备示意图。
图2为本发明测定AFP的免疫生物传感器循环伏安图。
图3为本发明测定AFP的免疫生物传感器峰电流强度与抗原浓度之间的线性关系。
图4为本发明测定AFP的免疫生物传感器稳定性与选择性研究。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1 c-MWCNT的制备
将0.1g多壁碳纳米管粉末(MWCNTs)加入到20mL混酸(浓稍酸:浓稍酸体积比为3:1)中80℃下超声2h,接着加热至25℃在6000rpm下搅拌5h,离心并用超纯水洗涤产物至中性,温室下干燥24h,得到羧基化多壁碳纳米管浓度为5mg/mL(c-MWCNT)。
实施例2免疫传感器的构建
(1)电极预处理:将玻碳电极进行打磨、抛光和超声清洗;
(2)将5μL c-MWCNT(1mg/mL)滴加到步骤(1)处理后的电极表面,37℃保温箱中避光干燥2h;
(3)FC-NSA在电极表面电聚合
取14.138mg FC、5.368mg NSA及2mL RTILs(1-丁基-3-甲基咪唑六氟磷酸盐),FC与NSA呈现2:1的最适摩尔比例,将已经称量好的颗粒置于玛瑙研钵中,均匀研磨5~8分钟直至溶液中无清晰可见颗粒物,得到乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液。采用连续循环伏安法将乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液电聚合到c-MWCNT表面,连续扫描15圈。电化学参数如下,扫描范围:(-2.4)V~(-1)V;扫速:0.1V/s。
(4)免疫复合物的组装
将步骤(3)已经电聚合后的工作电极取出并用PBS清洗,在该玻碳电极表面滴加5μL cAnti-AFP(15μg/mL),然后置于4℃冰箱中避光放置12h,待cAnti-AFP与FC/NSA复合物以酰胺键形式紧密结合,用PBS缓冲液清洗,室温干燥。
(5)免疫复合物的封闭
滴加5μL 1%BSA于上述孵育抗体后的玻碳电极上以封闭包被抗体上非特异性结合位点,室温下避光放置2h,PBS清洗,室温干燥。
(6)免疫传感器的抗原修饰
滴加5μL AFP溶液于上述修饰的玻碳电极上,在37℃恒温箱中避光反应(请补充具体的时间:0.5~2h),PBS清洗。
实施例3:说明免疫传感器的建设(图1)
免疫生物传感器的制备方法包括如下步骤:基底电极表面依次经过羧基化多壁碳纳米管(c-MWCNT)修饰、乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯(FC/NSA)电聚合和甲胎蛋白(AFP)抗原抗体修饰即得。
具体的基底电极由MWCNT修饰,FC/NSA电聚合到c-MWCNT,AFP抗体修饰结合到FC/NSA,其中AFP抗体通过酰胺键共价连接NSA,滴加BSA为免疫复合物的封闭。然后免疫生物传感器面对AFP抗原检测。
实施例4:说明NSA/FC在MWCNT上的电聚合(图2)
将FC与NSA以室温离子液体(RTILs)作为电解质和溶剂制成FC-NSA混合物,通过第一圈循环伏安出来在(-1.8)V上有峰(第一圈),电聚合打开碳碳双键连接到c-MWCNT表面后,峰消失了(第十五圈)。说明FC-NSA混合物被c-MWCNT表面上成功地聚合。
实施例5:免疫传感器对甲胎蛋白抗原的定量分析
在抗原组装前后,均在0.1M PBS缓冲液中进行示差脉冲伏安法(DPV)测定,每个免疫传感器用DPV连续测试直至稳定,取最后扫描结果,获取其电流峰值。利用检测大电流峰值减去小电流峰值的差值与未组装AFP抗原前的电流峰值作比,得到最终的峰电流变化率来衡量不同浓度AFP抗原与不同峰电流变化率的定量关系。本实验测定了10μg/mL、1000ng/mL、100ng/mL和10ng/mL AFP抗原浓度与上述峰电流变化率的定量关系如图3。
实施例6:免疫传感器的选择性
考察AFP的检测时其他干扰蛋白如cTnT、CEA和TNF-α对甲胎蛋白(100ng/mL)检测的影响。免疫生物传感器的制备方法同实施例3,不同的是抗原不同,结果见图4。相同浓度下即100ng/mL的AFP测定信号分别是所测cTnT,CEA信号TNF-α的7.3倍、80倍和13.36倍,说明本发明所述的免疫传感器的具有较高的特异性性能,并且对AFP的测定具有很高的选择性。
Claims (10)
1.一种测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,基底电极表面依次经过羧基化多壁碳纳米管、乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯电聚合和甲胎蛋白抗体与抗原修饰即得;其中,乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯电聚合是将乙烯基二茂铁与N-羟基琥珀酰亚胺丙烯酸酯以室温离子液体作为电解质和溶剂制成乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯混合物,通过电聚合打开碳碳双键连接到羧基化多壁碳纳米管表面;所述室温离子液体为1-丁基-3-甲基咪唑六氟磷酸盐。
2.根据权利要求1所述测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,包括如下步骤:
(1)将基底电极进行打磨、抛光、清洗;
(2)将羧基化多壁碳纳米管加入到水中,超声至分散均匀,再滴加到步骤(1)处理后的基底电极表面,18~40 °C避光干燥1~3 h;
(3)将乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液电聚合到步骤(2)处理后的基底电极的表面;
(4)将步骤(3)已经电聚合后的工作电极取出,并用PBS清洗;并滴加甲胎蛋白包被抗体于清洗后的基底电极表面,1~10 ℃ 放置6~18 h,用PBS清洗,室温干燥;
(5)滴加牛血清白蛋白溶液于步骤(4)处理后的基底电极表面,室温放置1~15 h,用PBS清洗,室温干燥;
(6)将甲胎蛋白抗原滴加于步骤(5)处理后的基底电极表面,30~40 ℃,避光保存0.5~2h,干燥,用PBS缓冲液清洗。
3.根据权利要求1或2所述的测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,所述基底电极为玻碳电极。
4.根据权利要求1或2所述的测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,所述的羧基化多壁碳纳米管的制备方法如下:
(Ⅰ)将多壁碳纳米管加入到混合酸中,于 65~90 ℃ 超声1~3.5 h,再加热搅拌1~5 h,离心分离,得到沉淀物;
(Ⅱ)将步骤(Ⅰ)得到的沉淀物水洗至中性,或加入稀氢氧化钠洗涤至中性,干燥,即得羧基化多壁碳纳米管。
5.根据权利要求4所述的测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,步骤(Ⅰ)中,所述的混合酸为浓硫酸与浓硝酸的混合物,体积比为 3:1~1:3;控制混酸的添加量,使多壁碳纳米管的浓度为3~7 mg/mL;所述的加热指的是加热至温度为24~26 °C。
6.根据权利要求1或2所述的测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,所述的乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯的制备方法如下:
取乙烯基二茂铁、N-羟基琥珀酰亚胺丙烯酸酯和室温离子液体于玛瑙研钵中,均匀研磨5~8分钟直至溶液中无清晰可见颗粒物,得到乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液。
7.根据权利要求6所述的测定甲胎蛋白抗原的免疫生物传感器的制备方法,其特征在于,乙烯基二茂铁和N-羟基琥珀酰亚胺丙烯酸酯的摩尔质量比为1:10~10:1,控制室温离子液体的添加量,使乙烯基二茂铁浓度为1~15 mg/mL,N-羟基琥珀酰亚胺丙烯酸酯的浓度为1~5 mg/mL。
8.根据权利要求2所述的制备方法,其特征在于,步骤(2)中,所述的羧基化多壁碳纳米管的浓度为0.2~4 mg/mL,所述的羧基化多壁碳纳米管的体积为2~8 µL;步骤(3)中,所述的电聚合为连续循环伏安法,其扫描范围为-2.4~-1V,扫描速率为0.1~0.5 V/s,扫描圈数为10~17圈,乙烯基二茂铁-N-羟基琥珀酰亚胺丙烯酸酯溶液的用量为1~5 mL;步骤(4)中,所述的甲胎蛋白包被抗体的浓度为12~20 μg/mL,滴加量为1~10 µL;步骤(5)中,所述的牛血清白蛋白溶液的质量分数为0.2~2 %,滴加量为1~10 µL;步骤(6)中所述的甲胎蛋白抗原的浓度为10 ng/mL~10 µg/mL,滴加量为5~10 µL。
9.权利要求1~8任意一项所述的制备方法制备得到的免疫生物传感器。
10.权利要求9所述的免疫生物传感器在制备定量检测甲胎蛋白抗原诊断试剂中的应用。
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