CN110967386B - 基于核酸适配体的镉离子电化学传感方法 - Google Patents
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Abstract
一种基于核酸适配体的镉离子电化学传感方法，将六水硝酸钴的水溶液和钛酸四丁基酯的乙醇溶液混合并搅拌，收集滴入氨水后的沉淀并煅烧制备得到钛修饰的四氧化三钴；然后将钛修饰的四氧化三钴纳米材料悬浊液滴加至丝网印刷碳电极上，与涂有镉离子核酸适配体层的工作电极组成电化学传感器；最后通过检测核酸适配体特异性捕捉作为靶标的镉离子并产生的电流信号，实现镉离子的定量检测。本发明检测限为0.027ng/mL，线性范围为0.01‑0.1，0.1‑15ng/mL，同时具有很好的重现性和稳定性。
Description
技术领域
本发明涉及的是一种传感分析领域的技术，具体是一种基于核酸适配体的镉离子电化学传感方法。
背景技术
自通过SELEX首次筛选所得以来，核酸适配体aptamer在近三十年受到了广泛关注。一般而言，核酸适配体是一段核苷酸数量小于100的寡核苷酸序列，具有灵敏识别靶标的功能。因其特异性强，生物兼容性好，无毒，易于合成和修饰而被广泛应用于检测各类靶标。
纳米材料受益于分子尺寸效应，在理化性质上的表现出超出一般材质(如具有更高的比表面积，更强的催化能力，更好的导电性等)，其在生物传感器领域的应用，能显著提高检测性能。
发明内容
本发明针对现有技术存在的上述不足，提出一种基于核酸适配体的镉离子电化学传感方法，能精确且灵敏的检测镉离子的含量，并绘制了相应的工作曲线。用该传感器检测水溶液中的镉离子，检测限为0.027ng/mL，线性范围为0.01-0.1，0.1-15ng/mL。该方法还具有很好的重现性和稳定性。通过实际水样加标回收率实验，进一步验证了该方法在检测镉离子的实际应用中具有可行性。
本发明是通过以下技术方案实现的：
本发明将六水硝酸钴的水溶液和钛酸四丁基酯的乙醇溶液混合并搅拌，收集滴入氨水后的沉淀并煅烧制备得到钛修饰的四氧化三钴；然后将钛修饰的四氧化三钴纳米材料悬浊液滴加至丝网印刷碳电极上，与涂有镉离子核酸适配体层的工作电极组成电化学传感器；最后通过检测核酸适配体特异性捕捉作为靶标的镉离子并产生的电流信号，实现镉离子的定量检测。
所述的混合是指：钴：钛质量比为2:1的六水硝酸钴的水溶液与钛酸四丁基酯的乙醇溶液混合，并持续搅拌3h。
所述的煅烧制备，具体包括：将沉淀洗净、抽虑并后以90℃干燥10h，再以500℃煅烧3h，即可得到钛修饰四氧化三钴纳米材料。
所述的悬浊液，通过将钛修饰的四氧化三钴纳米材料加入得到超纯水然后超声处理形成，其浓度为1mg/mL。
所述的工作电极是指：在丝网印刷碳材质的工作电极上均匀地涂覆2.5μL 2.0μmol/L镉离子核酸适配体孵育1.5h后用超纯水冲洗该工作电极，洗去未结合的核酸适配体得到。
所述的检测，通过预制循环伏安法曲线后，再对待测样品溶液进行检测，比照得到的峰值电流的变化程度得到重金属离子浓度，具体为：通过设置扫描电位范围为-0.65～0V，扫描速度为0.1V/S，对已知浓度的重金属离子进行检测，扫描后根据重金属离子添加前后还原峰值电流的变化程度与重金属离子浓度的关系，绘制工作曲线。
技术效果
与现有技术相比，本发明整体所解决的技术问题是：对低浓度(ng/mL)重金属镉离子的高灵敏高特异性的原位快速检测；由此产生的技术效果包括：基于修饰有导电材料的电极所建立的电化学传感器，灵敏度高，检出限低，且操作简单，重现性好，在原位监测环境样品重金属方面具有一定应用价值。
附图说明
图1为本发明中传感器的层层组装及检测Cd2+示意图；
图2A为钛修饰四氧化三钴纳米材料的扫描电镜图
图2B为钛修饰四氧化三钴纳米材料的透射电镜图；
图3为循环伏安法(CV)测定的电极修饰过程示意图；
图中：(a)裸电极，(b)纳米材料修饰电极，(c)纳米材料/核酸适配体修饰电极，(d)纳米材料/核酸适配体/镉离子修饰电极；
图4A为循环伏安法测定不同浓度的镉离子与CV关系示意图；
图4B为循环伏安法测定不同浓度的镉离子与电流差值关系示意图；
图中：(a)0.01,(b)0.02,(c)0.03,(d)0.05,(e)0.1,(f)0.2,(g)0.5,(h)1.0,(i)2.0,(j)3.0,(k)5.0,(l)7.5,(m)10,(n)12.5,(o)15.0ng/mL。
具体实施方式
如图1所示，为本实施例涉及一种基于钛修饰四氧化三钴纳米材料的镉离子检测方法，包括以下步骤：
①将钴：钛质量比为2:1的六水硝酸钴的水溶液与钛酸四丁基酯的乙醇溶液混合，并持续搅拌3h后，逐滴加入10mL氨水并再持续搅拌2h。收集所得沉淀，用超纯水洗净并抽滤分离，再90℃干燥10h，最后500℃煅烧3h，即可得到如图2所示的钛修饰四氧化三钴纳米材料。
②基于核酸适配体的电化学传感：
2.1)称量5mg钛修饰四氧化三钴纳米材料加入到5mL超纯水中，超声10分钟使其形成均匀悬浊液。然后取2.5μL该悬浊液均匀涂到丝网印刷碳电极的工作电极表面，最后在室温下干燥2h；
2.2)在工作电极上均匀的涂覆2.5μL 2.0μmol/L镉离子核酸适配体，其核苷酸序列如Seq ID No.1所示，具体为5’-GGACTGTTGTGGTATTATTTTTGGTTGTGCAGTCC-3’，后静置在室温下孵育1.5h后用超纯水冲洗修饰电极，洗脱未结合的核酸适配体，即制得电化学传感器，最后放置于
冰箱中保存。如图3所示，在电极修饰的每一步中，都用电化学循环伏安法(CV)进行了扫描电位范围为-0.65～0V，扫描速度为0.1V/S的信号检测。用钛修饰四氧化三钴纳米材料修饰电极后，其电流信号显著增强，为电化学传感器的高灵敏度奠定了坚实基础。
③镉离子含量的检测：
3.1)用电化学工作站(上海辰华仪器公司，CHI1030A)的循环伏安法(CV)测定电流，扫描电位范围为-0.65～0V，扫描速度为0.1V/S，记录数据I。
3.2)将2.5μL 2.0μmol/L镉离子核酸适配体均匀涂覆到工作电极后孵育30min，再次用循环伏安法(CV)测定电流，扫描电位范围为-0.65～0V，扫描速度为0.1V/S，记录数据I’。
3.3)根据电流的差值ΔI(I与I’的差值)绘制工作曲线。其检测限为0.027ng/mL，线性范围为0.01-0.1，0.1-15ng/mL，表明基于该生物传感器的电化学方法检测性能优良，在检测重金属镉离子方面行之有效。
经过具体实际实验，应用上述电化学传感方法检测实际环境样品(河水/自来水)中外源加标的镉离子(ng/mL)，回收率可达94.89％-107.21％，相对误差小于5％。
与现有技术相比，本方法灵敏准确，检出限低，相比于常规大型仪器设备(如ICP，AAS)，操作简便灵活，更适用于原位快速检测。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。
序列表
<110> 上海交通大学
<120> 基于核酸适配体的镉离子电化学传感方法
<130> fnb848e
<141> 2019-12-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列()
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ggactgttgt ggtattattt ttggttgtgc agtcc 35
Claims (4)
1.一种基于核酸适配体的镉离子电化学传感方法，其特征在于，将六水硝酸钴的水溶液和钛酸四丁基酯的乙醇溶液混合并搅拌，收集滴入氨水后的沉淀并煅烧制备得到钛修饰的四氧化三钴；然后将钛修饰的四氧化三钴纳米材料悬浊液滴加至丝网印刷碳电极上，与涂有镉离子核酸适配体层的工作电极组成电化学传感器；最后通过检测核酸适配体特异性捕捉作为靶标的镉离子并产生的电流信号，实现镉离子的定量检测；
所述的工作电极是指：在丝网印刷碳材质的工作电极上均匀地涂覆2.5μL2.0μmol/L镉离子核酸适配体孵育1.5h后用超纯水冲洗该工作电极，洗去未结合的核酸适配体得到；
所述的镉离子核酸适配体，其核苷酸序列如Seq ID No.1所示，具体为5’-GGACTGTTGTGGTATTATTTTTGGTTGTGCAGTCC-3’；
所述的检测，通过预制循环伏安法曲线后，再对待测样品溶液进行检测，比照得到的峰值电流的变化程度得到重金属离子浓度，具体为：通过设置扫描电位范围为-0.65～0V，扫描速度为0.1V/S，对已知浓度的重金属离子进行检测，扫描后根据重金属离子添加前后还原峰值电流的变化程度与重金属离子浓度的关系，绘制工作曲线。
2.根据权利要求1所述的基于核酸适配体的镉离子电化学传感方法，其特征是，所述的混合是指：钴：钛质量比为2:1的六水硝酸钴的水溶液与钛酸四丁基酯的乙醇溶液混合，并持续搅拌3h。
3.根据权利要求1所述的基于核酸适配体的镉离子电化学传感方法，其特征是，所述的煅烧制备，具体包括：将沉淀洗净、抽虑并后以90℃干燥10h，再以500℃煅烧3h，即可得到钛修饰四氧化三钴纳米材料。
4.根据权利要求1所述的基于核酸适配体的镉离子电化学传感方法，其特征是，所述的悬浊液，通过将钛修饰的四氧化三钴纳米材料加入到超纯水然后超声处理形成，其浓度为1mg/mL。
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