CN109001164B - 一种基于锰卟啉淬灭CdSe量子点的光电生物传感器及其制法和应用 - Google Patents
一种基于锰卟啉淬灭CdSe量子点的光电生物传感器及其制法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于DNA酶的放大反应以及链式杂交掺杂锰卟啉淬灭CdSe量子点的光电生物传感器及其制法和应用。本发明的技术方案是首先合成了形貌较好的TiO2修饰材料和光电信号很强的CdSe QDs，然后利用了DNA酶的循环放大反应以及线性链式反应掺杂锰卟啉淬灭CdSe QDs构建光电生物传感器，对凝血酶进行检测。DNA S1和S2与凝血酶特异性结合，形成了“钳子”结构，选择性和灵敏度高。在链式杂交反应中掺杂锰卟啉，可以使大量的锰卟啉通过链式DNA连接到电极上，阻碍电子的传递，使光电信号明显降低，显示了很高的灵敏度。该PEC传感分析方法，在生物分子的微量生物分析和临床医学有很大的应用前景。
Description
技术领域：
本发明涉及一种基于DNA酶的放大反应以及链式杂交掺杂锰卟啉淬灭CdSe量子点的光电生物传感器；以及所述生物传感器的制备方法及其检测凝血酶的分析应用。
背景技术：
光电分析(PEC)不仅具有光学分析方法和电化学分析方法的优点[Li,L.L.,Ge,P.,Selvin,P.R.,et.al.Analytical Chemistry,2012,84(18):7852–7856.]，也具备比传统方法更可取的优势，比如灵敏度高，背景信号较低，操作简单和更低的成本。光电分析方法由于其优越性已经广泛应用于生物标志物检测。量子点(QDs)独特的电化学和光物理性质在一系列领域中发现了越来越多的应用，特别是在生物分析领域，QDs的生物相容性非常好，在生物检测方面有独特的优势。如功能化的QDs可以标记在各种生物分子上，可以为PEC检测生物分子提供光电信号。基于QDs的PEC生物分析由于它们的独特的传感方式，在生化分析领域得到广泛的应用，同时临床诊断和工业分析的长期需求又继续推动QDs在PEC传感分析中的发展。
凝血酶是血液中的一种丝氨酸蛋白酶，能将可溶性纤维蛋白原转化为不溶性物质。纤维蛋白，它在各种生命过程中起着关键的作用，并与许多疾病有关，如心血管疾病、炎症反应、血栓栓塞性疾病和抗凝血治疗[Xu,W.,Xue,S.,Yi,H.,et.al.ChemicalCommunications,2015,51(8):1472–1474.]。因此定量检测凝血酶在临床研究与诊断过程中十分重要。
适体(aptamer)是用配体指数富集法系统演化(SELEX)技术从人工体外合成的随机寡核苷酸序列库中反复筛选得到的能以极高的亲和力和特异性与靶分子结合的一段寡核苷酸序列[Abnous K,Danesh N M,Alibolandi M,et al.Microchimica Acta,2017,184(4):1151–1159.]。适体与靶分子的结合和抗原-抗体作用相似，适体具有明显优于抗体的许多特性。由于其优越性已被广泛应用于各种分析方法，如比色法[Chang,C.C.,Wei,S.C.,Wu,T.H.,et.al.Biosensors&Bioelectronics,2013,42(1):119–123.]、荧光[Chang,H.,Tang,L.,Wang,Y.,et.al.Analytical Chemistry,2010,82(6):2341–2346.]和电化学[Radi,A.E.,Acero,Sánchez,J.L.,Baldrich,E.,et.al.Journal of the AmericanChemical Society,2006,128(1):117–124.]。为了进一步提高灵敏度，降低检测限，信号放大的方法包括聚合酶链反应[Wang,X.L.,Li,F.,Su,Y.H.,et.al.Analytical Chemistry,2004,76(19):5605–5610.]、纳米材料标记[Sun,A.,Qi,Q.,Wang,X.,et.al.Biosensors&Bioelectronics,2014,57(10):16–21.]、滚环放大[He,P.,Liu,L.,Qiao,W.,et.al.Chemical Communications,2014,50(12):1481–1484.]和酶辅助循环放大[Peng,K.,Zhao,H.,Yuan,Y.,et.al.Biosensors&Bioelectronics,2014,55(55C):366–371已被广泛纳入基于适体的凝血酶检测。事实上，这些新方法可以显著放大信号，实现高灵敏的凝血酶检测。
到目前为止，光电检测中的信号淬灭大部分都是量子点与金纳米颗粒之间能量共振转移。在本实验中我们选用了一种新的淬灭剂-锰卟啉。锰卟啉(MnPP)是一种锰与卟啉的金属络合产物，具有多种优点，如生产成本低，化学性质稳定，催化性能高和优越的生物相容性。卟啉衍生物是一类具有一定尺寸、相对缺电子的平面结构化合物。由于其特殊的结构特点曾作为双链DNA结合剂[Balagurumoorthy,P.,Brahmachari,S.K.,Mohanty,D.,et.al.Nucleic Acids Research,1992,20(15):4061–4067.][Sari,M.A.,Battioni,J.P.,Dupre,D.,et.al.Biochemistry,1990,29(17):4205–4215.]。MnPP作为本工作中的有效淬灭剂，可通过π-π共轭作用和静电相互作用掺杂在双链DNA中，同时利用杂交链式反应增加MnPP的量。在正常情况下，CdSe QDs的PEC信号显著降低，淬灭率达到82％左右，表明MnPP可作为CdSe QDs的有效淬灭剂。
在本实验中我们分别采用了DNA酶辅助循环放大和链式杂交反应。首先利用适体与凝血酶特异性结合，释放出DNA S3与目标建立关系，再通过DNA酶循环放大，产出产物链I，然后DNA I充当桥梁使S4/S5链式反应能够连接到电极上，同时将淬灭剂锰卟啉(MnPP)掺杂到链式结构中去，使信号降低，实现对凝血酶的高灵敏检测。
发明内容：
本发明的目的之一提供一种光电性能较好的CdSe量子点作为探针，利用TiO2修饰CdSe QDs，从而检测光电信号。具体包括以下步骤：
步骤1.NaHSe的制备：将0.0950g的NaBH4放入10mL离心管中，在离心管中加入6mL去离子水，加入磁子搅拌溶解，通氮气除氧。然后称取0.0947g硒粉快速加入到体系中，在常温下搅拌至无色透明溶液，即硒粉完全溶解。
步骤2.MPA-CdSe的制备：将0.280g CdCl2加入到盛有25mL去离子水的50mL的三口烧瓶中，移取30μL巯基乙酸加入到上述溶液中，然后用0.1M的NaOH溶液调节pH＝8，溶液变得澄清透明，通N2除O2 20min。然后将制备的NaHSe前驱体加入到三口烧瓶中，加热回流2h，得到淡黄色溶液。冷却至室温，保存到4℃待用。
步骤3.制备TiO2：精确称取0.1465g草酸钛钾于烧杯中，加入8mL去离子水，然后将8mL H2O2加入到上述溶液中，磁力搅拌30min，用HCl调节pH＝4，溶液由亮黄色变为暗红色。然后将溶液转移到聚四氟乙烯高压反应釜中，于真空烘箱中加热到150℃反应1h。取出冷却至室温，加入2mL去离子水分散待用。
本发明的目的之二是提供一种基于DNA酶的放大反应以及链式杂交掺杂锰卟啉淬灭CdSe量子点的光电生物传感器，以及利用该生物传感器检测凝血酶的分析应用。它由以下步骤组成：
步骤1.DNA和凝血酶的预处理：配制pH＝7.4的TE缓冲溶液(10mM EDTA，1.0mMTris-HCl和12.5mM的MgCl2)作为DNA的稀释液。将干粉DNA在使用前，用离心机10000rpm下离心1min，使DNA收集至管底，然后按照具体要求将配置好的TE缓冲溶液加入到离心管中配制成浓度均为100μM(即1.0×10-4M)溶液，然后将DNA稀释成5μM，于4℃下保存备用。然后将目标凝血酶配置成不同浓度待用。
步骤2.目标的循环放大：将S1(5μL，5μM)与S3(5μL，5μM)混合反应2h，形成双链S1/S3。之后将S2(5μL，5μM)和不同浓度的凝血酶5μL与S1/S3混合，室温条件下摇床中反应30min，释放出S3。然后将上述反应溶液中加入HP(10μL，5μM)，使S3和HP杂交，然后反应2h。之后将10μL 0.01M的MgSO4加入到反应体系中，在放入摇床中培养2h，就会得到循环产物DNA I。
步骤3.CdSe QDs-C-DNA探针的组装：取100μL CdSe QDs，然后加适量乙醇，离心纯化再分散于等体积的二次水中。之后将10μL 0.1M EDC和10μL 0.025M NHS加入到量子点中，于摇床中室温下活化1h。最后将10μL 5μM的C-DNA加入到活化好的量子点中反应6h，待用。
步骤4.基于DNA酶的循环放大反应以及链式杂交掺杂锰卟啉淬灭CdSe量子点检测凝血酶：首先将ITO电极用稀盐酸、稀乙醇和二次水分别浸泡超声15min，然后烘干待用。将TiO210μL滴在电极表面，室温下自然晾干，然后将10μL CdSe QDs-C-DNA滴到TiO2表面，再将10μL目标循环放大产物DNA I，10μL S4/S5，锰卟啉取滴到相同的位置，湿润条件下反应4h，自然晾干之后用PBS冲洗，将未杂交的产物连冲洗掉，自然晾干。之后进行光电信号检测。
该方法的检测是在PBS中室温条件下进行的，采用三电极系统：ITO为工作电极，饱和甘汞电极为参比电极，铂丝电极为对电极，施加的电压是0.1V，激发光是蓝光。
本发明合成了一种新颖的CdSe量子点，并以该量子点作为光电材料。基于CdSe量子点探针和DNA酶的循环放大技术，构建了一种线性链式杂交放大反应，锰卟啉(MnPP)掺杂在双链DNA中，阻碍电子的传递，使信号降低，实现了对凝血酶的灵敏检测。
本发明与现有技术相比，主要优点在于，本发明利用制备的CdSe量子点作为光电信号探针，具有显著的光电性能，产生较强的光电信号，极大的提高了检测的灵敏度；利用DNA酶的循环放大反应以及链式杂交掺杂锰卟啉淬灭CdSe量子点光电信号，提高了检测灵敏度，实现了对凝血酶的准确检测。
本发明的光电生物传感器表现出优良的准确性、高灵敏性、高选择性，稳定性与重现性，检测迅速、方便，在生物医学分析检测和早期临床诊断中具有巨大的应用潜力。
附图说明：
图1透射电子显微镜(TEM)图：(A)CdSe QDs，(B)TiO2。
图2基于DNA酶的循环放大反应以及链式杂交掺杂锰卟啉淬灭CdSe量子点检测凝血酶的原理图。
图3电泳表征：(1)5μM S1,(2)5μM S2,(3)5μM S3,(4)将5μM S1和5μM S3杂交产物,(5)将5μM S1、5μM S3、5μM S2和凝血酶杂交产物，(6)替换下来的S3和HP,再加入Mg2+的产物，(7)5μM HP，(8)S4与S5链式反应杂交产物，(9)mark。
图4各阶段的光电信号响应：(a)ITO,(b)ITO/TiO2,(c)ITO/TiO2/CdSe QDs/c-DNA/I/链式杂交产物，(d)ITO/TiO2/CdSe QDs/c-DNA/I，(e)ITO/TiO2/CdSe QDs/c-DNA(f)ITO/TiO2/CdSe。
图5基于锰卟啉淬灭量子点检测不同浓度凝血酶(pM)的光电响应：(a)0，(b)1.0×10-3，(c)1.0×10-2，(d)0.1，(e)1.0，(f)10，(g)1.0×102，(h)1.0×103，(i)1.0×104，(j)1.0×105
图6基于锰卟啉淬灭CdSe QDs检测凝血酶的选择性：免疫球蛋白G，溶菌酶，牛血清蛋白，凝血酶。凝血酶和其他干扰物浓度为1.0nM
具体实施方式：
实施例1.信号探针的制备及对凝血酶的检测
目标的循环放大过程：将S1(5μl，5μM)与S3(5μl，5μM)混合反应2h，形成双链S1/S3。之后将S2(5μl，5μM)和不同浓度的凝血酶5μl与S1/S3混合，室温条件下摇床中反应30min，释放出S3。然后将上述反应溶液中加入HP(10μl，5μM)，使S3和HP杂交，然后反应2h。之后将10μl 0.01M的MgSO4加入到反应体系中，在放入摇床中培养2h，就会得到循环产物DNA I。
制备信号探针过程:取100μL CdSe QDs，然后加适量乙醇，离心纯化再分散于等体积的二次水中。之后将10μL 0.1M EDC和10μL 0.025M NHS加入到量子点中，于摇床中室温下活化1h。最后将10μL 5μM的C-DNA加入到活化好的量子点中反应6h，待用。
电极的预处理:首先将ITO电极用稀盐酸、稀乙醇和二次水分别浸泡超声15min，然后烘干待用。将TiO2 10μL滴在电极表面，每次滴加的位置应相同，然后室温下自然晾干，然后将10μL CdSe QDs-C-DNA滴到TiO2表面，然后再将10μL目标循环放大产物DNA I，10μLS4/S5，锰卟啉取滴到相同的位置，湿润条件下反应4h，自然晾干之后用PBS冲洗，将未杂交的产物连冲洗掉，自然晾干。之后进行光电信号检测。
实施例2.电致化学发光生物传感器的制备及对凝血酶的检测
将“首先将S1(5μl，5μM)与S3(5μl，5μM)混合反应2h，形成双链S1/S3。之后将S2(5μl，5μM)和不同浓度的凝血酶5μl与S1/S3混合，室温条件下摇床中反应30min”改为“将S1(5μl，5μM)与S3(5μl，5μM)混合反应2h，形成双链S1/S3。之后将S2(5μl，5μM)和不同浓度的凝血酶5μl与S1/S3混合，室温条件下摇床中反应50min。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对凝血酶检测的结果同实施例1。
实施例3.电致化学发光生物传感器的制备及对凝血酶的检测
将“然后将上述反应溶液中加入HP(10μl，5μM)，使S3和HP杂交，然后反应2h”改为“然后将上述反应溶液中加入HP(10μl，5μM)，使S3和HP杂交，然后反应3h”,。制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对凝血酶检测的结果同实施例1。
实施例4.电致化学发光生物传感器的制备及对凝血酶的检测
将“最后将10μL 5μM的C-DNA加入到活化好的量子点中反应6h，待用”改为“最后将10μL 6μM的C-DNA加入到活化好的量子点中反应6h，待用”。制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对凝血酶检测的结果同实施例1。
Claims (1)
1.一种基于锰卟啉淬灭CdSe量子点的光电生物传感方法，其特征是：利用DNA酶辅助循环放大目标产物，释放出DNA I与目标凝血酶建立关系；在电极上通过TiO2修饰CdSe QDs量子点，利用DNA I建立链式反应，将淬灭剂锰卟啉(MnPP)掺杂到链式结构中去，使光电信号降低，构建检测凝血酶的光电生物传感器；
其中，所述基于锰卟啉淬灭CdSe量子点的光电生物传感方法，具体步骤组成：
步骤1.目标的循环放大：将5 μL，5 μM的S1与5 μL，5 μM的S3混合反应2 h，形成双链S1/S3；之后将5 μL，5 μM的S2和目标凝血酶5 μL与S1/S3混合，室温条件下摇床中反应30min，释放出S3；然后将上述反应溶液中加入10 μL，5 μM的HP，使S3和HP杂交，然后反应2 h；之后将10 μL 0.01 M的MgSO4加入到反应体系中，在放入摇床中培养2 h，就会得到循环产物DNA I；步骤2.生物传感器的制备：首先将ITO电极用稀盐酸、稀乙醇和二次水分别浸泡超声15 min，然后烘干待用；将TiO2 10 μL滴在电极表面，每次滴加的位置应相同，然后室温下自然晾干，然后将10 μL CdSe QDs-C-DNA 滴到TiO2表面，然后再将10 μL目标循环放大产物DNA I，10 μL S4/S5，锰卟啉取滴到相同的位置，湿润条件下反应4 h，自然晾干之后用PBS冲洗，将未杂交的产物链 冲洗掉，自然晾干；之后进行光电信号检测；光电信号检测是在PBS中室温条件下进行的，采用三电极系统：ITO为工作电极，饱和甘汞电极为参比电极，铂丝电极为对电极，施加的电压是0.1 V，激发光是蓝光。
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