CN109540983B - 一种用于检测α2,6唾液酸化聚糖的新型电化学生物传感器 - Google Patents
一种用于检测α2,6唾液酸化聚糖的新型电化学生物传感器 Download PDFInfo
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Abstract
本发明成功开发了基于新型金纳米棒‑链霉亲和素(AuNPs‑SA)复合材料和羧基化单壁碳纳米角‑硫掺杂铂(c‑SWCNHs/S‑PtNC)的特异性超敏夹心电化学免疫传感器,用于检测人血清中的α2,6唾液酸化聚糖(α2,6‑sial‑Gs)。金纳米棒‑链霉亲和素(AuNPs‑SA)不仅能增加电极导电性,增强电子间转移,同时通过链霉亲和素和生物素系统为固载生物素标记的黑接骨木素(bio‑SNA)提供活性位点。此外,c‑SWCNHs/S‑PtNC对H2O2的还原具有优异的催化性能,并且Pt‑NH2和‑NH‑CO‑可有效捕获三氨基苯硼酸(M‑APBA)。本发明的优点在于线性范围宽。灵敏度高,特异性强,检测迅速,以及良好的可重复使用性,并且该发明可用于测量人血清中α2,6‑sial‑Gs含量,有在临床检测中有着巨大潜力。
Description
技术领域:
本发明涉及一种在临床上定量检测α2,6唾液酸化聚糖的电化学免疫传感器的制备方法及应用,尤其是基于金纳米棒-链霉亲和素纳米粒子复合材料及羧基化单壁碳纳米角-硫掺杂铂纳米复合材料作为信号探针制备的生物传感器,用于检测α2,6唾液酸聚糖,属于电化学检测领域。
背景技术:
α2,6唾液酸化聚糖(α2,6-sial-Gs)是一种广谱肿瘤标志物,与肿瘤的增殖、转移、扩散及肿瘤的抗原性等恶性行为密切相关。在癌细胞凋亡过程中,α2,6-sial-Gs在糖苷酶的水解作用下脱落并释放进入血液循环,从而使血清中的α2,6-sial-Gs水平显著升高。因此,α2,6-sial-Gs的检测对于肿瘤的临床诊断、疗效观察和预后判断有着非常重要的临床价值。
用于α2,6-sial-Gs定量检测的常规方法包括气相色谱-质谱联用,高效液相色谱-质谱联用和毛细管电泳。然而这些方法需要昂贵的设备和材料,并且所检测样品必须经过复杂的前处理来消除其他的干扰物,除此之外,须由技术人员在专门的实验室进行分析。近年来,电化学免疫传感器技术作为新兴的检测技术,具有快速、灵敏度高、操作简单、稳定性好等特点,并且已广泛应用于生化分析、环境监测、临床研究和食品质量检测等领域,这种方法对α2,6-sial-Gs的检测提供了新的研究思路。
在电化学免疫传感器分析技术中,为了提高检测的灵敏度和缩短响应时间,满足临床上痕量物质的定量快速检测,目前多采用“三明治”型的传感器反应模式。其原理是基于构建负载生物素标记黑接骨木素 (bio-SNA)-被测目标(α2,6-sial-Gs)-三氨基苯硼酸(M-APBA)夹心免疫复合物的分析方法,其中bio-SNA 和M-APBA可以特异性识别α2,6-sial-Gs。如何简便、快速的实现对目标物质进行检测是将其推广应用的重要标准。为实现这一目的,电极修饰材料和纳米信标的选择显得尤为重要。近年来,由于金纳米棒(AuNR) 具有良好的导电性和优良的物理化学性能,在电化学生物传感技术中广泛应用。同时,为了增加抗体的固载量,本课题采用链霉亲和素(SA),它是四聚体蛋白,大小为66KDa。一分子链霉亲和素可以高度特异性地与四分子生物素结合。基于以上优点,本实验采用AuNRs-SA作为电极修饰材料增加导电性和抗体的固载量。同时,本课题拟采用首次合成的硫掺杂铂(S-PtNC)作为纳米信标,由于其具有良好的催化性能和大量的吸收/活性位点,可以放大电化学免疫传感器电信号,为了增加S-PtNC的固载量,采用了比表面积大的羧基化单壁碳纳米角(c-SWCNHs)结合S-PtNC形成c-SWCNHs/S-PtNC纳米复合材料,不仅增加了催化性,也可以与M-APBA通过金属-氨基和羧基-氨基配位结合,通过bio-SNA和M-APBA对α2,6-sial-Gs的特异性结合实现夹心型免疫传感器的构建。
该项目建立了一个简单、快速的检测方法实现了对α2,6-sial-Gs的特异、超灵敏检测。为肿瘤的临床诊断、疗效观察和预后判断提供依据。
发明内容:
1.本发明的目的是用于检测α2,6-sial-Gs的电化学免疫传感器的制备方法与应用,为肿瘤的临床诊断、疗效观察和预后判断提供依据,其特征包括以下步骤:
(1)金纳米棒-链霉亲和素(AuNPs-SA)纳米复合材料的制备;
(2)羧基化单壁碳纳米角-硫掺杂铂-三氨基苯硼酸(c-SWCNHs/S-PtNC/M-APBA)纳米信标的制备;
(3)建立电化学免疫传感器,检测α2,6-sial-Gs,绘制标准曲线。
2.本发明所述c-SWCNHs/S-PtNC纳米复合材料的制备过程具体包括以下步骤,其特征包括以下步骤:
(1)AuNRs-SA复合材料的制备:
首先将2.5mL 0.5mM的氯金酸(HAuCl4)溶液与2.5mL 0.2mM的十六烷基三甲基溴化铵(CTAB) 溶液混合;加入300mL 0.01mM硼氢化钠(NaBH4)并快速混合2分钟,合成金粒种子。然后,将5mL 0.2mM 的HAuCl4溶液加入到5mL 1mM的CTAB溶液中;将4mM 0.15mL的硝酸银(AgNO3)加入到溶液中并反应5分钟;再加入70μL 0.079mM的抗坏血酸(AA),使溶液反应2分钟。然后,将12mL制备的金粒种子加入该溶液中并剧烈搅拌20秒并使其在25℃下反应2小时。之后,通过以5000rpm离心30分钟收集AuNRs,洗涤3次并溶解在200μL超纯水中。然后,将40μL 1mg/mL-1链霉亲和素(SA)加入溶液中并摇动过夜。最后,洗涤3次后,将最终的沉淀物分散在200μL超纯水中进一步使用。
(2)c-SWCNHs/S-PtNC的制备:
将1mL 2.5mg mL-1羧基化单壁碳纳米角(c-SWCNHs)溶液超声处理5分钟;然后,加入5mL氯铂酸钾(K2PtCl4)和6mL 0.1mM亚甲蓝(MB)在90℃下加热下加热搅拌5小时。最后,将混合物以10000rpm离心5分钟,洗涤三次并溶解在1mL超纯水中,供下一步使用。
(3)c-SWCNHs/S-PtNC/M-APBA纳米信标的制备:
将1mL c-SWCNHs/S-PtNC复合物,50μL 50mg mL-1 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐 (EDC)和50μL 50mg mL-1 N-羟基琥珀酰亚胺(NHS)在4℃轻微混合30分钟。接下来,将50μL 50mg mL-1的三氨基苯硼酸(M-APBA)加入到混合物中并继续搅拌4小时;然后,将100μL 0.25wt% BSA溶液加入混合物中并在4℃下轻微混合1小时,封闭活性位点。随后,将所得溶液离心,彻底洗涤以除去未结合的M-APBA,再分散于1mL超纯水中得到纳米信标,然后在4℃下储存以供进一步使用。
3.根据权利要求1所述的建立电化学免疫传感器,检测α2,6-sial-Gs,绘制标准曲线,其特征在于包括以下步骤:
(1)分别用0.3和0.05μm的Al2O3粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
(2)将10μL电极修饰材料金纳米棒-链霉亲和素(AuNRs-SA)复合材料滴加在电极表面,在室温条件下干燥。
(3)将8μL的骨接黑木素(bio-SNA)第一抗体溶液(0.2mg mL-1)结合到干燥的电极表面,在 37℃孵育1.5h。
(4)用超纯水将孵育后的电极冲洗干净后滴加6μL,0.25%的BSA溶液室温孵育30min。
(5)用超纯水将电极冲洗干净后将不同浓度的α2,6-sial-Gs滴加在电极上并置于30℃孵育2.5h。
(6)在干燥后的电极上滴加10μLc-SWCNHs/S-PtNC/M-APBA置于37℃孵育2h。
(7)将孵育后的电极用超纯水冲洗干净后置于室温条件干燥。
(8)将电极置于5mL,0.1M PBS(0.1M Na2HPO4,0.1M KH2PO4,0.1M KCl)中进行表征,每隔 20s加入20μL,2mM H2O2,测量其计时电流变化电流值。
(9)根据所得电流变化值与α2,6-sial-Gs浓度呈线性关系,绘制工作曲线。
与现有技术相比,本发明的一种定量检测α2,6-sial-Gs的电化学免疫传感器的制备方法与应用,其突出的特点是:
(1)将AuNRs-SA作为电极修饰材料引入到电化学免疫传感器的制备中,提高了传感器的比表面积,以及导电性,加快电子传递,进而提高了电化学免疫传感器的灵敏度和生物相容性;同时引入生物素-链霉亲和素系统可以固载更多的bio-SNA,增加免疫反应效率,减少捕获抗体的时间,进一步提高传感器的灵敏度;
(2)首次合成的c-SWCNHs/S-PtNC作为纳米信标信号材料,良好的导电性、结合位点以及催化性,可以产生、放大信号;
(3)本方法制备的电化学免疫传感器可为肿瘤的临床诊断、疗效观察和预后判断提供有效信息
(4)本方法制备的电化学免疫传感器由于利用抗体抗原之间的特异性结合,具有良好的特异性,其制备过程简单、检测步骤较少,检测速度较快,便于实现商品化,有利于推进转化医学的发展。
附图说明:
图1为本发明的电化学免疫传感器的构建示意图。
图2为本发明的电极修饰材料和信号材料的不同合成步骤的场发射扫描电镜图、透射电镜图、 EDS图和XPS图。
图3为本发明的电化学免疫传感器在检测α2,6-sial-Gs时得到的计时电流变化电流与浓度的线性关系。
具体实施方式:
下面结合具体实施例对本发明进行进一步阐述,应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
步骤1.将1mL 2.5mg mL-1羧基化单壁碳纳米角(c-SWCNHs)溶液超声处理5分钟;然后,加入5mL氯铂酸钾(K2PtCl4)和6mL 0.1mM亚甲蓝(MB)在90℃下加热下加热搅拌5小时。最后,将混合物以10000rpm离心5分钟,洗涤三次并溶解在1mL超纯水中,最后置于真空干燥箱中干燥待用。
步骤2.分别用0.3和0.05μm的Al2O3粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
步骤3.将10μL电极修饰材料金纳米棒-链霉亲和素(AuNRs-SA)滴加在电极表面,室温干燥;
步骤4.将8μL的骨接黑木素(bio-SNA)第一抗体溶液(0.2mg mL-1)结合到干燥的电极表面,在37℃孵育1.5h;
步骤5.用超纯水将电极冲洗干净后将不同浓度的α2,6-sial-Gs滴加在电极上并置于30℃孵育 2.5h;
步骤6.在干燥后的电极上滴加10μLc-SWCNHs/S-PtNC/M-APBA置于37℃孵育2h;
步骤7.将孵育后的电极用超纯水冲洗干净后置于室温条件干燥;
步骤8.将电极置于5mL,0.1M PBS(0.1M Na2HPO4,0.1M KH2PO4,0.1M KCl)中进行表征,每隔 20s加入20μL,2mMH2O2,测量其计时电流变化电流值;
步骤9.将不同浓度的目标PCSK9滴加在电极上并置于37°孵育60min;
步骤10.在干燥后的电极上滴加8μLPt-PMB-Ab2纳米信标并置于37℃孵育60min;
步骤11.将孵育后的电极用清洗缓冲液冲洗干净后置于氮气中干燥;
步骤12.根据所得电流变化值与α2,6-sial-Gs浓度呈线性关系,绘制工作曲线;测定结果表明α2,6-sial-Gs浓度在100fg mL-1到100ng mL-1范围内成线性关系,线性相关系数为0.9995,检测限为 0.69fg mL-1
步骤13.将本发明上述传感器于4℃保存,间断检测传感器电流响应,储存28天后电流响应仍为初始电流的89.63%,表示传感器具有良好的稳定性;
步骤14.本发明取同一批次制备的免疫传感器5支,在相同条件下对10pg□mL-1的α2,6-sial-Gs 分别进行测定,每一支电极测定3次,结果响应电流的相对标准偏差为0.4796%,说明构建的传感器批内差异小,传感器重现性良好。
步骤15.将本发明上述传感器在血液中其他生物分子存在的条件下检测α2,6-sial-Gs,结果其他生物分子的存在不影响α2,6-sial-Gs电流的改变,说明传感器的特异性好,可以很好区分目标分子。
以上所述仅是本发明的优选实施方式,应当指出的是,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提条件下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种用于检测α2,6唾液酸聚糖的双响应电化学生物传感器制备方法,其特征在于包括以下步骤:
(1)金纳米棒-链霉亲和素纳米复合材料的制备;
(2)羧基化单壁碳纳米角-硫掺杂铂-三氨基苯硼酸纳米信标的制备;
(3)建立电化学免疫传感器,检测α2,6-sial-Gs,绘制标准曲线。
2.一种如权利要求1所述的双响应电化学生物传感器制备方法,其特征在于包括以下步骤:
(1)金纳米棒-链霉亲和素纳米复合材料的制备:
首先将2.5mL浓度为0.5mM的氯金酸溶液与2.5mL浓度为0.2mM的十六烷基三甲基溴化铵溶液混合,加入300mL浓度为0.01mM硼氢化钠并快速混合2分钟,合成金粒种子;然后,将5mL浓度为0.2mM的氯金酸溶液加入到5mL浓度为1mM的十六烷基三甲基溴化铵溶液中,将4mM浓度为0.15mL的硝酸银加入到溶液中并反应5分钟;再加入70μL浓度为0.079mM的抗坏血酸,反应2分钟;然后,将12mL制备的金粒种子加入上述溶液中并剧烈搅拌20秒且使其在25℃下反应2小时;之后,通过以5000rpm离心30分钟收集金纳米棒,洗涤3次并溶解在200μL超纯水中,然后,将40μL 1mg/mL-1链霉亲和素加入溶液中并摇动过夜,最后,洗涤3次后,将最终的沉淀物分散在200μL超纯水中以备进一步使用;
(2)羧基化单壁碳纳米角-硫掺杂铂-三氨基苯硼酸纳米信标的制备:
将1mL 2.5mg mL-1的羧基化单壁碳纳米角溶液超声处理5分钟,然后加入5mL氯铂酸钾和6mL浓度为0.1mM的亚甲蓝,在90℃下加热搅拌5小时,最后,将混合物以10000rpm离心5分钟,洗涤三次并溶解在1mL超纯水中,供下一步使用;
将1mL制备好的羧基化单壁碳纳米角-硫掺杂铂复合物与50μL 50mg mL-1 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和50μL 50mg mL-1 N-羟基丁二酰亚胺在4℃轻微混合30分钟,接下来,将50μL 50mg mL-1的三氨基苯硼酸加入到上述混合物中并继续搅拌4小时;然后,将100μL 0.25wt%的牛血清白蛋白溶液加入混合物中并在4℃下轻微混合1小时,封闭活性位点;随后,将所得溶液离心,彻底洗涤以除去未结合的三氨基苯硼酸,再重新分散于1mL超纯水中得到纳米信标,然后在4℃下储存以供进一步使用。
3.一种如权利要求1所述的电化学生物传感器检测方法,其特征在于包括以下步骤:
(1)分别用0.3和0.05μm的氧化铝粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
(2)将10μL电极修饰材料金纳米棒-链霉亲和素复合材料滴加在电极表面,在室温条件下干燥;
(3)将8μL 0.2mg mL-1的骨接黑木素第一抗体溶液结合到干燥的电极表面,在37℃孵育1.5h;
(4)用超纯水将孵育后的电极冲洗干净后滴加6μL,0.25wt%的牛血清白蛋白溶液室温孵育30min;
(5)用超纯水将电极冲洗干净后将不同浓度的α2,6-sial-Gs滴加在电极上并置于30℃孵育2.5h;
(6)在干燥后的电极上滴加10μL羧基化单壁碳纳米角-硫掺杂铂-三氨基苯硼酸纳米信标并置于37℃孵育2h;
(7)将孵育后的电极用超纯水冲洗干净后置于室温条件干燥;
(8)将电极置于用0.1M磷酸氢二钠,0.1M磷酸二氢钾,0.1M氯化钾配置而成的5mL浓度为0.1M的PBS溶液中进行表征,每隔20s加入20μL浓度为2mM过氧化氢溶液,测量其计时电流变化电流值;
(9)根据所得电流变化值与α2,6-sial-Gs浓度呈线性关系,绘制工作曲线。
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