CN110455896A - 金属有机骨架复合物比率电化学miR3123适体传感器的制备方法 - Google Patents
金属有机骨架复合物比率电化学miR3123适体传感器的制备方法 Download PDFInfo
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Abstract
本发明公开了基于黑磷纳米片BPNSs和硫堇TH掺杂的铜基金属有机骨架Cu‑MOF复合物的比率电化学miR3123适体传感器的制备方法。将TH与Cu‑MOF前驱体反应制备TH/Cu‑MOF，滴涂BPNSs制备BPNSs/TH/Cu‑MOF，将其滴涂在电极上。二茂铁Fc标记的DNA单链适体吸附在BPNSs上，制得适体‑BPNSs/TH/Cu‑MOF。靶分子miR3123与DNA单链适体Fc‑DNA结合，使Fc‑DNA脱离BPNSs，引起Fc电化学信号减弱，而对TH信号影响甚微。拟合电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建比率电化学miR3123适体传感器。
Description
技术领域：
本发明属于金属有机骨架复合材料和比率电化学适体传感器的制备技术领域，具体涉及一种基于黑磷纳米片和硫堇共掺杂的铜基金属有机骨架纳米复合物的比率电化学miR3123适体传感器的制备方法，其制备的传感器可用于生物医学样品中miR3123的高灵敏和高选择性定量检测。
背景技术：
MicroRNAs简写为miRNAs，是一类非编码的单链RNA分子，由长度约为22个核苷酸的内源性基因编码。在细胞分化和生物发育过程中，参与调节动植物转录后的基因表达。miRNAs因其显著的功能，引起了科技工作者广泛的研究兴趣。随着研究工作的不断深入，研究者发现小RNA基因调控在肿瘤、心脏病和神经系统疾病等的发展中起着重要作用，小RNA因此被认为是一种新的生物标记物，用于疾病诊断研究。目前已知检测小RNA的方法，主要包括荧光猝灭、指数等温扩增、电化学发光和实时聚合酶链式反应平台。miR3123是一种含17核苷酸的微小RNA，它在早期胃癌中表现出明显的表达变化，可作为胃癌早期诊断的潜在生物标志物。对人体中miR3123的定量检测，有助于胃癌的早期诊断和治疗。
Daneshpour等设计了一种双信号标记的电化学纳米生物传感器，用于胃癌相关标志物miR-106a的检测(Maryam Daneshpour,Behzad Karimi,Kobra Omidfar.Simultaneousdetection of gastric cancer-involved miR-106a and let-7a through a dual-signal-marked electrochemical nanobiosensor.Biosensors and Bioelectronics,2018,109,197-205)。Ki等发展了酶辅助靶循环的局域表面等离子体共振传感器，用于胃癌相关标志物miR-10b的检测(Jisun Ki,Hyo young Lee,Hye Young Son,Yong-Min Huh,Seungjoo Haam.Sensitive plasmonic detection of miR-10b in biological samplesusing enzyme-assisted target recycling and developed LSPR probe.ACSAppl.Mater.Interfaces,2019,11,18923-18929)。刘寒梢等公开了一种对胃癌生物标志物miR-378进行反转录和实时定量PCR反应，以U6snRNA作为内参计算miR-378的PCR相对定量值的方法(刘寒梢；孟宪欣；张伟；张春秀；肖华胜.miR-378生物标志物在胃癌检测、诊断中的应用.国家发明专利.公开号CN102605042A)。王振宁等公开了一种胃癌疾病进展分子标志物miR-1258的试剂盒，用于胃癌术后早期进行预后治疗(王振宁；宋永喜；周欣；黄选章；高鹏；孙景旭；陈晓婉.胃癌进展及预后相关分子标志物miR-1258.国家发明专利.公开号CN104531856A)。
尽管有关胃癌疾病相关标志物小RNA检测的工作已有国内外文献和专利报道，但迄今为止，尚未检索到有关miR3123定量检测的国内外文献和专利报道。基于此，本发明公开了一种胃癌疾病相关标志物miR3123定量检测的方法，报道了一种基于黑磷纳米片BPNSs和硫堇TH共掺杂的铜基金属有机骨架Cu-MOF纳米复合物的比率电化学miR3123适体传感器的制备方法。将TH与Cu-MOF前驱体一起反应制备TH/Cu-MOF复合物，将BPNSs分散液滴涂在复合物上制备BPNSs/TH/Cu-MOF复合物，将此复合物滴涂在裸玻碳电极表面，二茂铁Fc标记的DNA单链适体吸附在BPNSs上，制得适体-BPNSs/TH/Cu-MOF纳米复合物。靶分子miR3123与其DNA单链适体特异性结合，使Fc-DNA脱离BPNSs而远离电极表面，引起Fc电化学信号减弱，此过程对TH的电化学信号影响甚微。由此，以TH为参比，Fc为信号响应单元，拟合氧化还原电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建比率电化学miR3123适体传感器。截止目前，尚未有基于黑磷纳米片和硫堇共掺杂的铜基金属有机骨架纳米复合物，以及比率电化学适体传感器用于miR3123定量检测的国内外文献和专利的报道。
发明内容：
本发明的目的在于克服上述现有技术存在的不足，设计一种方法简便、成本低、灵敏高和选择性好的一种基于黑磷纳米片和硫堇共掺杂铜基金属有机骨架纳米复合物的比率电化学miR3123适体传感器的制备方法。
为实现上述目的，本发明涉及的一种基于黑磷纳米片和硫堇共掺杂铜基金属有机骨架纳米复合物的比率电化学miR3123适体传感器的制备工艺包括以下步骤：
1.金属有机骨架复合物比率电化学miR3123适体传感器的制备方法，其特征在于，该方法具体包括以下步骤：
(1)TH/Cu-MOF复合物的制备：称取4mg三水合硝酸铜、10μL三氟乙酸和10mg聚乙烯吡咯烷酮分别加入12mL混合溶剂中，含有9mL N,N-二甲基甲酰胺和3mL乙醇，搅拌均匀形成混合液A。称取4mg的4,4’,4”,4”’-(卟吩-5,10,15,20-四烷基)四苯甲酸和4mg硫堇分别加入含有3mL N,N-二甲基甲酰胺和1mL乙醇的混合溶剂中，搅拌均匀形成混合液B。将混合液A逐滴加入混合液B中，搅拌和超声作用10min后，将A-B混合液加热至80℃反应3h。反应产物溶液在8000rpm转速下离心10min，用乙醇和蒸馏水洗涤、干燥后得到TH/Cu-MOF复合物，将此复合物分散在乙醇中备用。
(2)BPNSs/TH/Cu-MOF复合物的制备：称取15mg黑磷块状晶体加入30mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中处理6h后，转入探头式超声波发生器中处理4h，超声处理后的分散液在10000rpm转速下离心20min去除尺寸较大的产物，取上层分散液在3500rpm转速下离心20min，得到BPNSs分散液。将其逐滴加入TH/Cu-MOF复合物的分散液中，保持搅拌和超声处理1h，然后在8000rpm转速下离心15min，制得BPNSs/TH/Cu-MOF复合物分散液备用。
(3)适体-BPNSs/TH/Cu-MOF纳米复合物的制备：在抛光打磨处理后的裸玻碳电极表面滴加交联剂Nafion，然后滴涂BPNSs/TH/Cu-MOF复合物分散液，形成该复合物改性的玻碳电极。将此改性电极浸没在含有1～10μM的miR3123对应DNA单链适体Fc-DNA的磷酸盐水缓冲液中，在37℃下孵育2h，然后取出电极，自然干燥后在电极表面制得适体-BPNSs/TH/Cu-MOF纳米复合物。
(4)以适体-BPNSs/TH/Cu-MOF纳米复合物改性的玻碳电极为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，向其中加入一定量miR3123，测定不同miR3123浓度下的电化学方波伏安曲线，以TH和Fc的氧化还原电流峰强度为参比信号和响应信号，拟合两个电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建用于miR3123定量检测的比率电化学适体传感器。其中，miR3123的线性检测浓度范围为1nM～10μM，检测限为1～5nM。
本发明的效果是：报道了一种基于BPNSs和TH共掺杂的Cu-MOF纳米复合物的比率电化学miR3123适体传感器的制备方法。首先将TH与Cu-MOF前驱体共同反应制备TH/Cu-MOF复合物，将新鲜制备的BPNSs分散液滴涂在TH/Cu-MOF上制备BPNSs/TH/Cu-MOF复合物，将此复合物滴涂在裸玻碳电极表面，构建该复合物改性的玻碳电极。Fc标记的DNA单链适体通过吸附富集在BPNSs上，制得适体-BPNSs/TH/Cu-MOF纳米复合物。靶分子miR3123与其DNA单链适体Fc-DNA特异性结合，使Fc-DNA脱离BPNSs，即Fc远离电极表面，引起Fc电化学信号减弱，此过程对TH的电化学信号影响甚微。基于此，以TH为参比，Fc为信号响应单元，拟合氧化还原电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建比率电化学miR3123适体传感器。与现有技术相比，本发明方法操作简便，比率电化学信号抗干扰能力强，检测灵敏度高和选择性好，可作为一种新颖的比率电化学传感器用于生物医学样品中miR3123的高灵敏和高选择性定量检测。
附图说明：
图1为基于黑磷纳米片和硫堇共掺杂的铜基金属有机骨架纳米复合物的比率电化学适体传感器的制备与miR3123检测的原理示意图；
图2(a)为测定不同miR3123浓度存在下该比率电化学适体传感器体系对应的电化学方波伏安曲线；
图2(b)为不同miR3123浓度对应的二茂铁和硫堇的氧化还原电流峰强度比率IFc/ITH，拟合不同IFc/ITH比率值与miR3123浓度之间的线性关系。
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：
本实施例涉及的基于黑磷纳米片和硫堇共掺杂的铜基金属有机骨架纳米复合物的比率电化学miR3123适体传感器的制备方法，其制备工艺和miR3123检测的原理示意图如图1所示，具体工艺步骤如下：
TH/Cu-MOF复合物的制备：称取4mg三水合硝酸铜、10μL三氟乙酸和10mg聚乙烯吡咯烷酮分别加入12mL混合溶剂中，含有9mL N,N-二甲基甲酰胺和3mL乙醇，搅拌均匀形成混合液A。称取4mg的4,4’,4”,4”’-(卟吩-5,10,15,20-四烷基)四苯甲酸和4mg硫堇分别加入含有3mL N,N-二甲基甲酰胺和1mL乙醇的混合溶剂中，搅拌均匀形成混合液B。将混合液A逐滴加入混合液B中，搅拌和超声作用10min后，将A-B混合液加热至80℃反应3h。反应产物溶液在8000rpm转速下离心10min，用乙醇和蒸馏水洗涤、干燥后得到TH/Cu-MOF复合物，将此复合物分散在乙醇中备用。
BPNSs/TH/Cu-MOF复合物的制备：称取15mg黑磷块状晶体加入30mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中处理6h后，转入探头式超声波发生器中处理4h，超声处理后的分散液在10000rpm转速下离心20min去除尺寸较大的产物，取上层分散液在3500rpm转速下离心20min，得到BPNSs分散液。将其逐滴加入TH/Cu-MOF复合物的分散液中，保持搅拌和超声处理1h，然后在8000rpm转速下离心15min，制得BPNSs/TH/Cu-MOF复合物分散液备用。
适体-BPNSs/TH/Cu-MOF纳米复合物的制备：在抛光打磨处理后的裸玻碳电极表面滴加交联剂Nafion，然后滴涂BPNSs/TH/Cu-MOF复合物分散液，形成该复合物改性的玻碳电极。将此改性电极浸没在含有2μM的miR3123对应DNA单链适体Fc-DNA的磷酸盐水缓冲液中，在37℃下孵育2h，然后取出电极，自然干燥后在电极表面制得适体-BPNSs/TH/Cu-MOF纳米复合物。
以适体-BPNSs/TH/Cu-MOF纳米复合物改性的玻碳电极为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，向其中加入一定量miR3123，测定不同miR3123浓度下的电化学方波伏安曲线(如图2(a)所示)，以TH和Fc的氧化还原电流峰强度为参比信号和响应信号，拟合两个电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系(如图2(b)所示)，构建用于miR3123定量检测的比率电化学适体传感器。miR3123的线性检测浓度范围为10nM～2μM，检测限为2nM。
实施例2：本实施例涉及的基于黑磷纳米片和硫堇共掺杂的铜基金属有机骨架纳米复合物的比率电化学适体传感器，其制备工艺和miR3123检测的原理示意图，TH/Cu-MOF和BPNSs/TH/Cu-MOF复合物制备的工艺步骤同实施例1。
其它具体工艺步骤如下：
在抛光打磨处理后的裸玻碳电极表面滴加交联剂Nafion，再滴涂BPNSs/TH/Cu-MOF复合物分散液，形成该复合物改性的玻碳电极。将此改性电极浸没在含有5μM的miR3123对应DNA单链适体Fc-DNA的磷酸盐水缓冲液中，在37℃下孵育2h，然后取出电极，自然干燥后在电极表面制得适体-BPNSs/TH/Cu-MOF纳米复合物。
以适体-BPNSs/TH/Cu-MOF纳米复合物改性的玻碳电极为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，向其中加入一定量miR3123，测定不同miR3123浓度下的电化学方波伏安曲线，以TH和Fc的氧化还原电流峰强度为参比信号和响应信号，拟合两个电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建用于miR3123定量检测的比率电化学适体传感器。miR3123的线性检测浓度范围为10nM～10μM，检测限为5nM。
实施例3：本实施例涉及的基于黑磷纳米片和硫堇共掺杂的铜基金属有机骨架纳米复合物的比率电化学适体传感器，其制备工艺和miR3123检测的原理示意图，TH/Cu-MOF和BPNSs/TH/Cu-MOF复合物制备的工艺步骤同实施例1。
其它具体工艺步骤如下：
在抛光打磨处理后的裸玻碳电极表面滴加交联剂Nafion，再滴涂BPNSs/TH/Cu-MOF复合物分散液，形成该复合物改性的玻碳电极。将此改性电极浸没在含有8μM的miR3123对应DNA单链适体Fc-DNA的磷酸盐水缓冲液中，在37℃下孵育2h，然后取出电极，自然干燥后在电极表面制得适体-BPNSs/TH/Cu-MOF纳米复合物。
以适体-BPNSs/TH/Cu-MOF纳米复合物改性的玻碳电极为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，向其中加入一定量miR3123，测定不同miR3123浓度下的电化学方波伏安曲线，以TH和Fc的氧化还原电流峰强度为参比信号和响应信号，拟合两个电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建用于miR3123定量检测的比率电化学适体传感器。miR3123的线性检测浓度范围为1nM～1μM，检测限为1nM。
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.金属有机骨架复合物比率电化学miR3123适体传感器的制备方法，其特征在于，该方法具体包括以下步骤：
(1)TH/Cu-MOF复合物的制备：称取4mg三水合硝酸铜、10μL三氟乙酸和10mg聚乙烯吡咯烷酮分别加入12mL混合溶剂中，含有9mL N,N-二甲基甲酰胺和3mL乙醇，搅拌均匀形成混合液A，称取4mg的4,4’,4”,4”’-(卟吩-5,10,15,20-四烷基)四苯甲酸和4mg硫堇分别加入含有3mL N,N-二甲基甲酰胺和1mL乙醇的混合溶剂中，搅拌均匀形成混合液B，将混合液A逐滴加入混合液B中，搅拌和超声作用10min后，将A-B混合液加热至80℃反应3h，反应产物溶液在8000rpm转速下离心10min，用乙醇和蒸馏水洗涤、干燥后得到TH/Cu-MOF复合物，将此复合物分散在乙醇中备用；
(2)BPNSs/TH/Cu-MOF复合物的制备：称取15mg黑磷块状晶体加入30mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中处理6h后，转入探头式超声波发生器中处理4h，超声处理后的分散液在10000rpm转速下离心20min去除尺寸较大的产物，取上层分散液在3500rpm转速下离心20min，得到BPNSs分散液，将其逐滴加入TH/Cu-MOF复合物的分散液中，保持搅拌和超声处理1h，然后在8000rpm转速下离心15min，制得BPNSs/TH/Cu-MOF复合物分散液备用；
(3)适体-BPNSs/TH/Cu-MOF纳米复合物的制备：在抛光打磨处理后的裸玻碳电极表面滴加交联剂Nafion，然后滴涂BPNSs/TH/Cu-MOF复合物分散液，形成该复合物改性的玻碳电极，将此改性电极浸没在含有1～10μM的miR3123对应DNA单链适体Fc-DNA的磷酸盐水缓冲液中，在37℃下孵育2h，然后取出电极，自然干燥后在电极表面制得适体-BPNSs/TH/Cu-MOF纳米复合物；
(4)以适体-BPNSs/TH/Cu-MOF纳米复合物改性的玻碳电极为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，向其中加入一定量miR3123，测定不同miR3123浓度下的电化学方波伏安曲线，以TH和Fc的氧化还原电流峰强度为参比信号和响应信号，拟合两个电流峰强度比率IFc/ITH与miR3123浓度之间的线性关系，构建用于miR3123定量检测的比率电化学适体传感器，其中，miR3123的线性检测浓度范围为1nM～10μM，检测限为1～5nM。
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| WO2023206075A1 (en) * | 2022-04-26 | 2023-11-02 | Micropoint Biotechnologies, Co., Ltd. | Electrochemical dual-signal detection method and device for detecting biomarkers |
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| US11099150B1 (en) | 2021-08-24 |
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