CN109142486A - 一种用于microRNA检测的光电化学生物传感器的制备方法 - Google Patents
一种用于microRNA检测的光电化学生物传感器的制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测microRNA的环金属铱配合物生物传感器的制备方法。首先设计合成了一种可嵌插于DNA双链的环金属铱配合物作为光电材料。将捕获发夹DNA固定在修饰好的ITO电极上制得本传感器的工作电极。当靶microRNA存在时，与工作电极上固定的捕获发夹DNA杂交释放其颈部分，释放的发夹颈部分继而与另外两个发DNA相互杂交引发杂交链式反应，在工作电极表面形成长DNA双链聚合物，该聚合物能嵌入大量环金属铱配合物从而使信号放大，实现对microRNA的高灵敏检测。本发明具有灵敏度高、特异性好、稳定性好等优点。
Description
技术领域
本发明属于光电功能材料及生物分析领域，具体涉及一种用于检测microRNA的环金属铱配合物生物传感器的制备方法。
背景技术
MicroRNAs是一类内源性的非编码单链小分子RNA，长度通常为19～23个核苷酸，在细胞的增殖、分化、凋亡等生命过程中发挥着重要作用。研究表明，microRNAs的异常表达与多种疾病密切相关，如肿瘤、糖尿病、心脏病、神经性疾病等，已经成为一种新型的肿瘤特异性生物标志物。因此，实现对microRNA的高灵敏度检测对研究其生物功能及癌症的早期诊断具有重要意义。但是，由于microRNAs序列短，序列同源性高、表达水平低等特点，使得对microRNA的检测异常困难，亟需发展简单、快速、灵敏度高、特异性强的microRNA 检测新方法以满足科学研究及临床诊断的需要。
光电化学生物传感器融合了电化学和电化学发光检测方法的优点，具有灵敏度高、设备简单、成本低廉、易于微型化和集成化等特点，在生物分析领域备受瞩目。为实现对生物目标物(如microRNA)的高特异、高灵敏检测，设计合成转换效率高、光电性质稳定的光电功能材料成为关键。目前用于光电化学生物传感器的材料主要集中于无机半导体纳米材料及其复合物，主要包括TiO2、CdS、CdTe等。但该类材料电子-空穴复合率高、光稳定性差，另外镉离子的毒性，限制了其在生物体系中的应用。环金属铱配合物由于具有丰富且宽范围可调的光物性质和电化学性质近年来被广泛用于有机电致发光、电致化学发光、染料敏化太阳能电池等领域。而将该类物质用作光电材料才刚刚起步，但表现出非常大的潜力(Anal.Chem., 2015,87,4283-4291；Anal.Chem.,2017,87,4283-4291)。本发明设计合成了一种可见光激发的环金属铱配合物作为光电材料，构建了基于该材料的光电化学传感器，用于一种microRNA 的检测。
发明内容：
鉴于现有技术的不足，本发明旨在提供一种用于检测microRNA的环金属铱配合物生物传感器的制备方法，具有高灵敏度、高特异性的特点。
基于上述目的，本发明所涉及的技术方案如下：
本发明所采用的光电材料为离子型双配体环金属铱配合物，其结构式为 [(C6)2Ir(dppz)]+PF6 -，其中C6为环金属化配体香豆素-6，dppz为辅助配体二吡啶并[3,2-a:2’,3’-c] 吩嗪，其结构如下式所示：
本发明提供一种基于上述铱配合物光电材料[(C6)2Ir(dppz)]+PF6 -的生物传感器的制备方法，用于检测microRNA，包括以下步骤：
(1)将面积固定的ITO电极清洗干净并干燥后，将其浸入含30％H2O2,30％NH4OH和H2O(体积比为：1:1:5)混合溶液中在电极表面形成羟基化层。干燥后将其浸入3-氨丙基三甲氧基硅氧烷(APTMS)的乙醇溶液(5％)中在电极表面形成氨基自组装层，然后浸泡在胶体金(AuNPs)溶液中孵化12小时，得到胶体金修饰的ITO电极。与捕获发夹HP1的溶液孵化后通过Au-S键将其固定在电极上得到ITO工作电极。
所述胶体金溶液的制备方法为：将制备过程中所需的玻璃仪器、磁子及存放金纳米粒子的容器均用二次水洗净，用王水浸泡过夜，之后用大量超纯水冲洗至pH值为中性，烘干备用。氯金酸HAuCl4(1.0mmol/L,100mL)于洗净的单口烧瓶中边搅拌加热至沸腾，然后快速把柠檬酸三钠(38.8mmol/L,10mL)加入到上述溶液，继续反应10min溶液由淡黄色慢慢变为深酒红色，继续回流15min，停止加热，边搅拌边自然冷却至室温，4℃避光保存。
所述捕获发夹HP1的序列为：5’-AAAAAATCAAACACCATTGTCACACTCCAGCAATTTGGAGTGTGACAATGGT-3’(5’端修饰-SH)。
优选的，所述捕获发夹HP1的溶液为SPSC缓冲溶液(1M NaCl和50mMNa2HPO4,pH7.5)，捕获发夹HP1的浓度为2μM。
(2)向步骤(1)制得的ITO工作电极上滴加靶microRNA溶液，microRNA与固定在ITO电极上的捕获发夹HP1识别杂交使得发夹HP1颈部分释放出来，发夹HP1释放的部分与发夹HP2杂交，释放发夹HP2颈部分，释放的HP2颈部分继而与发夹HP3杂交，引发杂交链式反应(HCR)，如此循环往复，在ITO工作电极上形成长的DNA双链聚合物。
所述靶microRNA的序列为：UGGAGUGUGACAAUGGUGUUUGA
所述发夹HP2的序列为：TCAAACACCATTGTCACACTCCAGCAATTTGGAGTGTGAC AATGGT
所述发夹HP3的序列为：TGGAGTGTGACAATGGTGTTTGAACCATTGTCACACTCCAA ATTGC
优选的，所述靶microRNA及发夹HP2和HP3溶液均为SPSC缓冲溶液(1M NaCl和50mMNa2HPO4,pH 7.5)。
优选的，发夹HP2和HP3溶液的浓度均为1μM。
(3)将步骤(2)所制得的修饰有长DNA双链聚合物的ITO电极浸入铱配合物 [(C6)2Ir(dppz)]+PF6 -的溶液中孵化，配合物通过嵌插的方式负载在工作电极上从而制得了光电化学传感器。
优选的，所述铱配合物[(C6)2Ir(dppz)]+PF6 -溶液的溶剂为N，N-二甲基甲酰胺(DMF)： PBS(1:20)，浓度为0.01mM。
优选的，所述孵化时间为40min。
(4)将步骤(3)制备好的光电传感器浸入0.01M PBS缓冲溶液中，采用490nm可见光进行激发，每20s开关光源一次，偏置电压为-0.2–0.2V进行光电流检测，实现对不同浓度microRNA的信号响应。
优选的，所述PBS缓冲溶液的pH＝5.5。
优选的，所述偏置电压为-0.1V。
本发明的有益效果：
(1)本发明公开了一种环金属铱配合物，作为光电材料用于生物传感器的制备，该材料具有稳定性好，采用可见光进行激发，光电转换效率高的特点。
(2)采用公开的环金属铱配合物作为光电材料制备的生物传感器用于microRNA的检测，具有灵敏度高、特异性好的特点，对于靶microRNA，检测限低至0.23fM。
附图说明：
图1光电化学生物传感器制备示意图；
图2环金属配合物[(C6)2Ir(dppz)]+PF6 -的光电流响应示意图；
图3传感器对不同浓度靶microRNA的光电流响应示意图；
图4传感器对靶microRNA浓度的线性关系图。
具体实施方式
结合实施例对本发明作进一步的说明，应该说明的是，下述说明仅是为了解释本发明，并不对其内容进行限定。
实施例1
环金属铱配合物光电材料的合成与表征：
称取IrCl3·3H2O固体(0.34g,1mmol)溶于水(5mL)，搅拌使其充分溶解，加入香豆素-6 (0.77g,2.2mmol)和乙二醇乙醚(15mL)，反应混合液在氮气氛围下于120℃下反应24h，冷却至室温。过滤，用水、无水乙醇和丙酮洗涤，得氯桥联中间体。称取制得的氯桥联中间体(0.14 g,0.08mmol)，二吡啶并[3,2-a:2’,3’-c]吩嗪(0.06g,0.2mmol)，加入到乙二醇乙醚(10mL)中。氮气保护下120℃下反应15h，冷却至室温。减压蒸发去除溶剂，进行柱色谱提纯(200-300 目，洗脱剂：二氯甲烷/甲醇＝10/1)，得深红色固体(0.061g,0.046mmol,58％)。1H NMR (CDCl3,500MHz)δ:9.69(d,J＝9.0Hz,2H),9.03(d,J＝5.5Hz,2H),8.41(dd,J＝6.5,3.5Hz, 2H),8.30(dd,J＝8.0,5.5Hz,2H),8.04(dd,J＝7.5,3.5Hz,2H),7.72(d,J＝7.5Hz,2H),7.09(t, J＝7.5Hz,2H),6.82(t,J＝7.5Hz,2H),6.45(d,J＝2.5Hz,2H),6.08(d,J＝9.5Hz,2H),5.89(dd, J＝9.5,2.5Hz,2H),5.80(d,J＝9.0Hz,2H),3.38(q,J＝7.5Hz,8H),1.12(t,J＝7.5Hz,12H)。
实施例2
ITO工作电极的制备：
将ITO电极依次用丙酮、乙醇、超纯水进行超声清洗，然后在氮气氛围下充分干燥。将 ITO电极浸入含有1mL超纯水、0.2mLNH4OH(30％)、0.2mLH2O2(30％)的混合液中，浸泡10min后取出，用超纯水继续充分清洗，氮气氛围充分干燥，将上述处理好的ITO电极浸泡在3-氨丙基三甲氧基硅氧烷的乙醇溶液中(5％(V/V))，室温下12小时。用乙醇进行充分清洗，氮气氛围充分干燥，而后将其置于110℃烘箱中，保持1h。随后，将其浸泡于已制备好的胶体金溶液中组装12小时，用超纯水进行充分清洗，氮气下干燥。将捕获发夹HP1 溶解在SPSC缓冲溶液(1M NaCl，50mM Na2HPO4,10mM三(2-羧乙基)膦，pH 7.5)缓冲液中使其浓度为2μM。室温下活化1h，然后将其(20μL)滴到已经被金胶修饰好的ITO电极上，在室温下反应12h，然后将已孵化好的电极浸入10mM Tris-HCl(含有1mM 6-巯基-1- 己醇)缓冲液中，浸泡1h，随后Tris-HCl缓冲液进行充分洗涤，氮气氛围下干燥，制得ITO 工作电极。
实施例3
用于microRNA检测的光电化学生物传感器的制备：
将20μL不同浓度的microRNA-122b滴加到实施例2制备的ITO工作电极上，37℃孵化2h，用Tris-HCl(0.01M)缓冲液进行充分洗涤，氮气氛围下进行干燥。然后滴加20μL 发夹HP2和HP3的混合液(1μM)，37℃孵化2h，用Tris-HCl(0.01M)缓冲液进行充分洗涤后，滴加10μL实施例1中制得的环金属铱配合物溶液(溶剂为：DMF/PBS＝1:20,0.01 μM)孵化40分钟，再用缓冲溶液冲洗除去非特异性吸附的信号物质，用于光电信号的检测。
实施例4
将实施例3制备好的传感器浸入0.01M PBS缓冲溶液中(pH＝5.5)，采用饱和Ag/AgCl 为参比电极；铂丝为对电极；由光电化学分析仪记录和检测光电流。采用490nm可见光进行激发，每20s自动开关光源一次。光电流强度与凝血酶的浓度在1fM至1000fM范围内成线性关系，线性方程为I＝16.70lgc+18.73，R2＝0.998，其中I为光电流强度(nA)，c为microRNA-122b浓度(fM)。检测限为0.23fM。
序列表
<110> 王墨泽
青岛科技大学
<120> 一种用于microRNA检测的光电化学生物传感器的制备方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
uggaguguga caaugguguu uga 23
<210> 2
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaaaaatcaa acaccattgt cacactccag caatttggag tgtgacaatg gt 52
<210> 3
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcaaacacca ttgtcacact ccagcaattt ggagtgtgac aatggt 46
<210> 4
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tggagtgtga caatggtgtt tgaaccattg tcacactcca aattgc 46
Claims (7)
1.一种用于microRNA检测的光电化学生物传感器的制备方法，其特征在于，包括以下步骤：环金属铱配合光电材料的制备；修饰有捕获发夹HP1的ITO工作电极的制备；靶microRNA与修饰在工作电极上的捕获发夹DNA杂交引发杂交链式反应实现信号放大功能；光电材料在ITO工作电极上的负载及光电信号的检测。
2.根据权利要求1所述，其特征在于，所采用的光电材料为离子型双配体环金属铱配合物，其中环金属化配体为香豆素-6，辅助配体为二吡啶并[3,2-a:2’,3’-c]吩嗪，其结构式为：
3.根据权利要求1所述的方法，ITO工作电极的制备包括ITO电极的羟基化、氨基化、金胶的组装以及捕获发夹HP1的组装。
4.根据权利要求3所述的捕获发夹HP1的组装过程，其特征在于，HP1的缓冲溶液包含1MNaCl，50mMNa2HPO4,10mM三(2-羧乙基)膦，浓度为2μM。
5.根据权利要求1所述的方法，靶microRNA杂交引发的杂交链式反应借助发夹HP2和HP3实现，采用的浓度均为1μM。
6.根据权利要求1所述的方法，其特征在于光电材料在ITO工作电极上的负载过程，采用的环金属铱配合物的浓度为0.01mM,孵化时间为40min。
7.根据权利要求1所述的方法，其特征在于，光电化学生物传感器检测条件为：检测液为0.01M PBS缓冲溶液(pH＝5.5)，采用490nm光进行激发，偏置电压为-0.1V。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110376259A (zh) * | 2019-07-18 | 2019-10-25 | 济南大学 | 一种用于检测microRNA的纸基光电阴极生物传感器的制备方法 |
| CN111187806A (zh) * | 2020-01-09 | 2020-05-22 | 中国人民解放军陆军军医大学第一附属医院 | 一种基于3D DNA纳米网状结构双信号放大技术的microRNA检测方法 |
| CN113201580A (zh) * | 2021-04-28 | 2021-08-03 | 青岛科技大学 | 一种环金属铱配合物敏化NiO阴极光电化学生物传感器的制备方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110376259A (zh) * | 2019-07-18 | 2019-10-25 | 济南大学 | 一种用于检测microRNA的纸基光电阴极生物传感器的制备方法 |
| CN110376259B (zh) * | 2019-07-18 | 2021-10-26 | 济南大学 | 一种用于检测microRNA的纸基光电阴极生物传感器的制备方法 |
| CN111187806A (zh) * | 2020-01-09 | 2020-05-22 | 中国人民解放军陆军军医大学第一附属医院 | 一种基于3D DNA纳米网状结构双信号放大技术的microRNA检测方法 |
| CN111187806B (zh) * | 2020-01-09 | 2021-01-08 | 中国人民解放军陆军军医大学第一附属医院 | 一种基于3D DNA纳米网状结构双信号放大技术的microRNA检测方法 |
| CN113201580A (zh) * | 2021-04-28 | 2021-08-03 | 青岛科技大学 | 一种环金属铱配合物敏化NiO阴极光电化学生物传感器的制备方法 |
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