CN115015342B - 金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法 - Google Patents
金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法 Download PDFInfo
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Classifications
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
本发明公开了金属离子掺杂硼纳米片M‑BNS复合物用作外泌体Exo比率电化学适体传感器的制备方法。在金属离子存在下,以硼粉末为原料采用超声辅助的液相剥离硼粉末途径制得M‑BNS。在玻碳电极GCE表面依次滴涂萘酚、M‑BNS、硫堇TH和二茂铁Fc标记的单链DNA适体,制得Fc‑DNA/TH/M‑BNS/GCE修饰电极。将含不同外泌体浓度CExo的液体样品滴涂在该修饰电极表面,在电解液中测定电化学方波伏安曲线。以硫堇TH为参比,二茂铁Fc为响应,以特征电位处电流峰强度比值IFc/ITH为信号输出,拟合IFc/ITH与样品中对应CExo之间的线性关系,构建比率电化学适体传感器用于外泌体检测。
Description
技术领域
本发明属于金属离子掺杂硼纳米片复合物和外泌体比率电化学适体传感器的制备技术领域,具体涉及金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法,该制备的比率电化学适体传感器可用于生物流体样品中外泌体的高灵敏性和高选择性定量检测。
背景技术
硼烯是后石墨烯时代科学家致力寻找的性能优良的极具代表性单元素二维纳米材料之一,硼烯是由单一硼元素构成的二维平面结构,具有独特的二维六角蜂窝状结构。理论计算结果表明,硼烯处于缺电子状态,在自由状态下其六角蜂窝状结构的存在形式是不稳定的,因此硼烯不是自然存在的,需通过人工合成获得。研究者早期采用了自下而上途径去合成硼烯,包括分子束外延生长法、化学气相沉积生长法、高真空原子溅射法等,但普遍存在成本高昂、条件苛刻、操作复杂、产量有限、产物转移困难等问题,阻碍了这些合成途径的推广应用。相比之下,采用自上而下的途径可有效合成出硼烯衍生物,例如在超声辅助下液相剥离硼粉末可获得独立式结构的薄层硼纳米片。密度泛函理论计算研究表明,在液相剥离过程中硼纳米片因暴露在氧环境中,硼容易与氧发生反应生成硼氧化物,进而使硼纳米片氧化和降解,结构稳定性和理化性能受到削弱。
硼原子核外仅三个电子,呈现电子缺失状态,硼sp2价电子易杂化,配位数大,共价半径短,易与其它元素形成强化学键。碱金属如钾,碱土金属如镁,过渡金属如铁、钴、镍、铜和银等的空轨道可与硼的孤对电子形成金属-硼配位,补偿硼原子和硼纳米片的电子缺陷,使硼的孤对电子被占据,不再与氧发生反应而使硼纳米片的结构遭到破坏。金属进入硼纳米片的层间结构促进了片层剥离,能防止金属原子聚集,也增强了硼纳米片电化学活性。因此,在金属离子存在下,采用自上而下的超声辅助液相剥离硼粉末的方法可制得结构稳定和性能优越的硼纳米片。
外泌体是一种新型的肿瘤生物标志物,能特异性识别特定肿瘤细胞的跨膜蛋白,用于特定肿瘤的早期精准筛查,是经历内溶体途径从多囊体中释放得到,为50~100纳米的细胞外囊泡,携带来自亲本的大量生物大分子,如跨膜蛋白、胞质蛋白、DNA、mRNA等。外泌体作为介导细胞间信息的信使,能反映肿瘤相关生理状态及其改变,而对肿瘤进行早期预判和诊断。外泌体检测方法主要包括流式细胞术、表面等离子共振、纳米颗粒追踪、比色、发光、电分析等。Zhang等报导了Ti3C2 MXenes纳米片催化的电致化学发光生物传感器检测外泌体(Huixin Zhang,Zonghua Wang,Qiuxia Zhang,Feng Wang,Yang Liu.Ti3C2MXenesnanosheets catalyzed highly efficient electrogenerated chemiluminescencebiosensor for the detection of exosomes.Biosensors and Bioelectronics,2019,124–125,184–190);Doldán等报导基于表面标志物介导信号放大的电化学三明治免疫传感器用于外泌体的检测(Ximena Doldán,Pablo Fagúndez,Alfonso Cayota,Justo Laíz,Juan Pablo Tosar,Electrochemical sandwich immunosensor for determination ofexosomes based on surface marker-mediated signal amplification,AnalyticalChemistry,2016,88,10466-10473)。金芳芳等公开了一种流式细胞仪检测外泌体的方法(金芳芳;王延博;徐学博;陈张朋;薛姜飞.一种流式细胞仪检测外泌体的方法.国家发明专利.公开号CN110702589A)。截止目前,尚未有关于金属离子掺杂硼纳米片复合物作为比率电化学适体传感器用于外泌体的高灵敏性和高选择性定量检测的国内外文献和专利报道。
发明内容
本发明之目的在于发展一种新颖和高效的基于金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法,该制备的比率电化学适体传感器可用于生物流体样品中外泌体的高灵敏性和高选择性定量检测。
为实现上述目的,本发明涉及的金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器,其制备方法具体包括以下步骤:
(1)在搅拌下,向150~200毫升N-甲基吡咯烷酮中加入120~150毫克硼粉末,以形成硼粉末分散液,然后在搅拌下逐滴加入金属盐,如氯化钾、氯化镁、氯化铁、氯化铜、硝酸钴、硝酸镍、硝酸银等的水溶液以形成混合液,调节硼浓度为0.6~1.0毫克/毫升,调节金属盐浓度为0.06~0.1毫克/毫升;
(2)采用探针超声在600~800瓦功率下处理上述混合液,首先超声5秒然后暂停10秒,重复超声-暂停的循环操作,整个过程维持在20~60分钟;
(3)量取5毫升二次蒸馏水,在搅拌下加入5~10毫克谷胱甘肽GSH以形成水溶液,将其逐滴加入到探针超声处理后的混合液中,然后采用浴池超声在1000~1100瓦功率下连续处理该混合液6~12小时,制得GSH修饰的金属离子掺杂薄层硼纳米片M-BNS分散液,其质量浓度调节为0.1~0.5毫克/毫升;
(4)取一根表面洁净的玻碳电极GCE,将0.5%萘酚的乙醇溶液5微升滴加在GCE表面,干燥后将制得的M-BNS分散液3~5滴滴涂在萘酚修饰的GCE表面,干燥后再滴加0.1%硫堇TH的乙醇溶液2~3滴,在上述逐滴添加过程中,每滴1滴后等GCE表面干燥后再滴下一滴,干燥后形成TH/M-BNS/GCE修饰电极;
(5)将1~10微摩尔/升的一端二茂铁Fc标记的单链DNA适体溶液在37℃下孵育30~120分钟,吸取3~5滴适体溶液滴涂在TH/M-BNS修饰的GCE表面,干燥后制得Fc-DNA/TH/M-BNS/GCE复合物修饰电极,干燥后在此复合物修饰电极的表面滴加含不同外泌体Exo浓度的液体样品,制得待测电极;
(6)将待测电极插入含10毫摩尔/升磷酸盐水缓冲液的电解池中,采用电化学工作站三电极系统测量电化学方波伏安曲线,以硫堇TH为参比,二茂铁Fc为响应,以特征电位处Fc(0.15V)与TH(-0.22V)电流峰强度比值IFc/ITH为输出,拟合IFc/ITH与样品中对应外泌体浓度CExo之间的线性关系,构建基于Fc-DNA/TH/M-BNS/GCE复合物修饰电极的外泌体比率电化学适体传感器,其外泌体浓度的线性检测范围为0.5×102~5×106个颗粒/微升,检测限为10~50个颗粒/微升。
本发明的效果是公开了一种基于金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法。以硼粉末为原料,在金属离子存在下采用探针超声辅助的液相剥离硼粉末途径制得金属离子掺杂的硼纳米片;以谷胱甘肽GSH为表面包覆剂,采用浴池超声辅助深度液相剥离硼纳米片,制得GSH修饰的金属离子掺杂薄层硼纳米片M-BNS。在萘酚修饰的玻碳电极GCE表面,滴涂M-BNS的分散液,干燥后滴加硫堇TH-乙醇溶液,干燥后再滴加一端二茂铁Fc标记的单链DNA适体溶液,干燥后制得Fc-DNA/TH/M-BNS/GCE修饰电极。将含不同外泌体浓度的液体样品依次滴涂在该修饰电极表面,干燥后插入含磷酸盐水缓冲液的电解池中,采用电化学工作站三电极系统测量电化学工作曲线。以硫堇TH为参比信号,二茂铁Fc为响应信号,以电化学方波伏安曲线上的特征电位处Fc(0.15V)与TH(-0.22V)电流峰强度的比值IFc/ITH为信号输出。
由于该单链Fc-DNA是特定设计的,其碱基序列为5’-Fc-(CH2)5-TTT TTT CAC CCCACC TCG CTC CCG TGA CAC TAA TGC TA-3′,可识别从乳腺癌MCF细胞中提取的外泌体Exo所高表达CD63蛋白而发生特异性结合,形成卷曲纠缠的复合物Exo/Fc-DNA,导致Fc随同DNA离开修饰电极表面,进而引起Fc电化学信号减弱,这个过程对附着在M-BNS表面的TH影响甚微,故Fc电化学信号基本不变。拟合IFc/ITH与样品中对应的外泌体浓度CExo之间的线性关系,进而构建基于金属离子掺杂硼纳米片复合物Fc-DNA/TH/M-BNS修饰电极的外泌体比率电化学适体传感器,用于生物流体样品中外泌体的高灵敏性和高选择性定量检测,便于执行特定肿瘤的早期筛查和精准诊断。
附图说明
图1.金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法及其检测外泌体的过程示意图。
具体实施方式
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1
本实施例涉及的金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法及其检测外泌体的过程如图1所示,具体制备步骤如下:
在搅拌下,向150毫升N-甲基吡咯烷酮中加入120毫克硼粉末,以形成硼粉末分散液,然后在搅拌下逐滴加入氯化铁的水溶液以形成混合液,调节硼浓度为0.7毫克/毫升,调节氯化铁浓度为0.07毫克/毫升。采用探针超声在650瓦功率下处理上述混合液,先超声5秒后暂停10秒,重复超声-暂停的循环操作,整个过程维持在30分钟。量取5毫升二次蒸馏水,在搅拌下加入5毫克谷胱甘肽GSH以形成水溶液,将其逐滴加入到探针超声处理后的混合液中,然后采用浴池超声在1050瓦功率下连续处理该混合液6小时,制得GSH修饰的金属离子掺杂薄层硼纳米片M-BNS分散液,其质量浓度调节为0.2毫克/毫升。取一根表面洁净的玻碳电极GCE,将0.5%萘酚的乙醇溶液5微升滴加在GCE表面,干燥后将制得的M-BNS分散液3滴滴涂在萘酚修饰的GCE表面,干燥后再滴加0.1%硫堇TH的乙醇溶液2滴,在上述逐滴添加过程中,每滴1滴后等GCE表面干燥后再滴下一滴,干燥后形成了修饰电极TH/M-BNS/GCE。
将2微摩尔/升的一端二茂铁Fc标记的单链DNA适体溶液在37℃下孵育30分钟,吸取3滴适体溶液滴涂在TH/M-BNS修饰的GCE表面,干燥后制得复合物修饰电极Fc-DNA/TH/M-BNS/GCE,干燥后在此复合物修饰电极的表面滴加含有不同外泌体Exo浓度的液体样品,制得待测电极。将待测电极插入含10毫摩尔/升磷酸盐水缓冲液的电解池中,采用电化学工作站三电极系统测量电化学方波伏安曲线,以硫堇TH为参比,二茂铁Fc为响应,以特征电位处Fc(0.15V)与TH(-0.22V)电流峰强度比值IFc/ITH为输出,拟合IFc/ITH与样品中对应外泌体浓度CExo之间的线性关系,构建基于Fc-DNA/TH/M-BNS/GCE复合物修饰电极的外泌体比率电化学适体传感器,其外泌体浓度的线性检测范围为1.0×102~5×106个颗粒/微升,检测限为50个颗粒/微升。
实施例2
本实施例涉及的金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法及其检测外泌体的过程如图1所示,具体制备步骤如下:
在搅拌下,向160毫升N-甲基吡咯烷酮中加入130毫克硼粉末,以形成硼粉末分散液,然后在搅拌下逐滴加入氯化铜的水溶液以形成混合液,调节硼浓度为0.8毫克/毫升,调节氯化铜浓度为0.08毫克/毫升。采用探针超声在700瓦功率下处理上述混合液,先超声5秒后暂停10秒,重复超声-暂停的循环操作,整个过程维持在40分钟。量取5毫升二次蒸馏水,在搅拌下加入6毫克谷胱甘肽GSH以形成水溶液,将其逐滴加入到探针超声处理后的混合液中,然后采用浴池超声在1060瓦功率下连续处理该混合液8小时,制得GSH修饰的金属离子掺杂薄层硼纳米片M-BNS分散液,其质量浓度调节为0.3毫克/毫升。取一根表面洁净的玻碳电极GCE,将0.5%萘酚的乙醇溶液5微升滴加在GCE表面,干燥后将制得的M-BNS分散液4滴滴涂在萘酚修饰的GCE表面,干燥后再滴加0.1%硫堇TH的乙醇溶液2滴,在上述逐滴添加过程中,每滴1滴后等GCE表面干燥后再滴下一滴,干燥后形成了修饰电极TH/M-BNS/GCE。
将5微摩尔/升的一端二茂铁Fc标记的单链DNA适体溶液在37℃下孵育60分钟,吸取4滴适体溶液滴涂在TH/M-BNS修饰的GCE表面,干燥后制得复合物修饰电极Fc-DNA/TH/M-BNS/GCE,干燥后在此复合物修饰电极的表面滴加了含不同外泌体Exo浓度的液体样品,制得待测电极。将待测电极插入含10毫摩尔/升磷酸盐水缓冲液的电解池中,采用电化学工作站三电极系统测量电化学方波伏安曲线,以硫堇TH为参比,二茂铁Fc为响应,以特征电位处Fc(0.15V)与TH(-0.22V)电流峰强度比值IFc/ITH为输出,拟合IFc/ITH与样品中对应外泌体浓度CExo之间的线性关系,构建基于Fc-DNA/TH/M-BNS/GCE复合物修饰电极的外泌体比率电化学适体传感器,其外泌体浓度的线性检测范围为0.5×102~1×106个颗粒/微升,检测限为25个颗粒/微升。
实施例3
本实施例涉及的金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法及其检测外泌体的过程如图1所示,具体制备步骤如下:
在搅拌下,向150~200毫升N-甲基吡咯烷酮中加入120~150毫克硼粉末,以形成硼粉末分散液,然后在搅拌下逐滴加入硝酸镍的水溶液以形成混合液,调节硼浓度为0.8毫克/毫升,调节硝酸镍浓度为0.08毫克/毫升。采用探针超声在780瓦功率下处理上述混合液,先超声5秒然暂停10秒,重复超声-暂停的循环操作,整个过程维持在60分钟。量取5毫升二次蒸馏水,在搅拌下加入8毫克谷胱甘肽GSH以形成水溶液,将其逐滴加入到探针超声处理后的混合液中,然后采用浴池超声在1080瓦功率下连续处理该混合液10小时,制得GSH修饰的金属离子掺杂薄层硼纳米片M-BNS分散液,其质量浓度调节为0.4毫克/毫升。取一根表面洁净的玻碳电极GCE,将0.5%萘酚的乙醇溶液5微升滴加在GCE表面,干燥后将制得的M-BNS分散液5滴滴涂在萘酚修饰的GCE表面,干燥后再滴加0.1%硫堇TH的乙醇溶液3滴,在上述逐滴添加过程中,每滴1滴后等GCE表面干燥后再滴下一滴,干燥后形成TH/M-BNS/GCE修饰电极。
将8微摩尔/升的一端二茂铁Fc标记的单链DNA适体溶液在37℃下孵育90分钟,吸取5滴适体溶液滴涂在TH/M-BNS修饰的GCE表面,干燥后制得复合物修饰电极Fc-DNA/TH/M-BNS/GCE,干燥后在此复合物修饰电极的表面滴加了含不同外泌体Exo浓度的液体样品,制得待测电极。将待测电极插入含10毫摩尔/升磷酸盐水缓冲液的电解池中,采用电化学工作站三电极系统测量电化学方波伏安曲线,以硫堇TH为参比,二茂铁Fc为响应,以特征电位处Fc(0.15V)与TH(-0.22V)电流峰强度比值IFc/ITH为输出,拟合IFc/ITH与样品中对应外泌体浓度CExo之间的线性关系,构建基于Fc-DNA/TH/M-BNS/GCE复合物修饰电极的外泌体比率电化学适体传感器,其外泌体浓度的线性检测范围为0.5×102~1×106个颗粒/微升,检测限为40个颗粒/微升。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,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 (1)
1.金属离子掺杂硼纳米片复合物用作外泌体比率电化学适体传感器的制备方法,其特征在于,该制备方法具体包括以下步骤:
(1)在搅拌下,向150~200毫升N-甲基吡咯烷酮中加入120~150毫克硼粉末,以形成硼粉末分散液,然后在搅拌下逐滴加入金属盐的水溶液以形成混合液,调节硼浓度为0.6~1.0毫克/毫升,调节金属盐浓度为0.06~0.1毫克/毫升;上述的金属盐为氯化铁、氯化铜或硝酸镍;
(2)采用探针超声在600~800瓦功率下处理上述混合液,首先超声5秒然后暂停10秒,重复超声-暂停的循环操作,整个过程维持在20~60分钟;
(3)量取5毫升二次蒸馏水,在搅拌下加入5~10毫克谷胱甘肽GSH以形成水溶液,将其逐滴加入到探针超声处理后的混合液中,然后采用浴池超声在1000~ 1100瓦功率下连续处理该混合液6~12小时,制得GSH修饰的金属离子掺杂薄层硼纳米片M-BNS分散液,其质量浓度调节为0.1~0.5毫克/毫升;
(4)取一根表面洁净的玻碳电极GCE,将0.5%萘酚的乙醇溶液5微升滴加在GCE表面,干燥后将制得的M-BNS分散液3~5滴滴涂在萘酚修饰的GCE表面,干燥后再滴加0.1%硫堇TH的乙醇溶液2~3滴,在上述逐滴添加过程中,每滴1滴后等GCE表面干燥后再滴下一滴,干燥后形成TH/M-BNS/GCE修饰电极;
(5)将1~10微摩尔/升的一端二茂铁Fc标记的单链DNA适体溶液在37°C下孵育30~120分钟,吸取3~5滴适体溶液滴涂在TH/M-BNS修饰的GCE表面,干燥后制得Fc-DNA/TH/M-BNS/GCE复合物修饰电极,干燥后在此复合物修饰电极的表面滴加含不同外泌体Exo浓度的液体样品,制得待测电极;其中,单链Fc-DNA的碱基序列为5’-Fc-(CH2)5-TTT TTT CAC CCC ACCTCG CTC CCG TGA CAC TAA TGC TA-3’,外泌体Exo是从乳腺癌MCF细胞中提取的,Fc-DNA通过特异性识别该外泌体Exo所高表达CD63蛋白而发生特异性结合;
(6)将待测电极插入含10毫摩尔/升磷酸盐水缓冲液的电解池中,采用电化学工作站三电极系统测量电化学方波伏安曲线,以硫堇TH为参比,二茂铁Fc为响应,Fc和TH的特征电位分别位于0.15 V和-0.22 V处,以特征电位处Fc与TH电流峰强度比值IFc/ITH为输出,拟合IFc/ITH与样品中对应外泌体浓度C Exo之间的线性关系,构建基于Fc-DNA/TH/M-BNS/GCE复合物修饰电极的外泌体比率电化学适体传感器;其外泌体浓度的线性检测范围为0.5×102~5×106个颗粒/微升,检测限为10~50个颗粒/微升。
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