CN115032258B - miRNA类肿瘤标志物检测试剂盒 - Google Patents
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Abstract
本发明提供了一种miRNA类肿瘤标志物检测试剂盒,所述检测试剂盒包括:电极、电极界面材料和捕获探针ssDNA;所述捕获探针、miRNA和功能化发光探针可通过碱基互补形成三明治结构;其中,所述电极为玻碳电极、所述电极界面材料为NiFe‑AuNPs;所述功能化发光探针TDN‑Ru是通过电荷吸附作用将发光分子负载在DNA四面体上组装制备而成;所述DNA四面体由四条DNA片段序列通过碱基配对自组装而成,其中一条DNA片段序列含有与miRNA部分互补配对的序列。
Description
技术领域
本发明涉及生物检测化学分析技术领域,特别是涉及miRNA类肿瘤标志物检测试剂盒。
背景技术
MicroRNAs(miRNA)是一类短的非编码的内源性单链RNA(18-25个碱基),在人类细胞网络中发挥着至关重要的作用。研究表明,miRNA参与癌基因和肿瘤抑制基因表达的调控,与肿瘤的发生和发展密切相关。因此,可以用作疾病诊断和筛查的生物标志物。目前,miRNA检测的金标准是定量逆转录聚合酶链式反应(qRT-PCR)。该技术灵敏度和通量都较高,但是需要采用昂贵的试剂盒、试剂和仪器。此外,还有一些技术比如荧光、比色、表面等离子体共振(SPR)等已被广泛应用于miRNA的检测。然而,昂贵的仪器、复杂的操作程序和熟练的技术是必需的,限制了其实用性。
电化学发光(electrochemiluminescence,ECL)是一种高灵敏的检测技术,通过对电极施加一定的电压使得电极界面产生电化学反应形成激发态物质,最终产生光信号。ECL结合了电化学和化学发光的优点,因其灵敏度高、检测范围宽、背景信号低等优点而备受关注,已被广泛用于临床检测、环境和食品安全等领域。由于生物样品中miRNA的尺寸小、丰度低,因此开发有效的ECL方法提高检测灵敏度至关重要。
中国发明专利申请CN113322305A公开了一种基于金纳米团簇/MnO2纳米花的电化学发光传感器的制备及对前列腺抗原(PSA)和Let-7a miRNA的检测应用。利用电化学发光共振能量转移(ECL-RET)技术,制备了电化学发光免疫传感器,通过“off-on”检测模式,实现了对前列腺抗原和Let-7a miRNA的灵敏分析。然而,该方法操作复杂,miRNA的检测是基于前期抗原检测实现的,且使用的试剂成本较高。
中国发明专利申请CN 112649484 B公开了一种基于CHA反应的光致电化学miRNA检测试剂盒的制备方法和产品,利用Au NCs与Mg-PTCAMOFs之间的共振能量转移,将目标物浓度与富集的次级目标物相关联,获得猝灭的PEC光电流信号,实现对miRNA21的超灵敏检测。但是,该方法操作复杂且耗时。
发明内容
针对miRNA检测的试剂和方法存在仪器昂贵、操作复杂和检测灵敏度低的技术问题,本发明提供了一种miRNA类肿瘤标志物检测试剂盒,所述检测试剂盒用于miRNA检测,是基于NiFe复合物较大的表面积,以及DNA四面体超电荷性质负载大量的信号探针,并利用碱基互补配对,实现miRNA的高灵敏检测。
本发明所采用的技术方案是:
第一方面,本发明提供一种miRNA类肿瘤标志物检测试剂盒,包括:电极、电极界面材料和捕获探针ssDNA;所述捕获探针、miRNA和功能化发光探针可通过碱基互补形成三明治结构;
其中,所述电极为玻碳电极、所述电极界面材料为NiFe-AuNPs;
其中,如图1所示,所述功能化发光探针TDN-Ru是通过电荷吸附作用将发光分子负载在DNA四面体(TDN)上组装制备而成;所述DNA四面体由四条DNA片段序列通过碱基配对自组装而成,其中一条DNA片段序列含有与miRNA部分互补配对的序列。进一步的,所述发光分子可选择Ru(bpy)3 2+,QDs中的任意一种;进一步优选的,所述发光分子为Ru(bpy)3 2+,此时,组装的功能化的发光探针TDN-Ru(bpy)3 2+的ECL发光信号相对标准偏差为1.42%,具有优良的ECL性能,可作为本发明优选的发光体;
进一步的,所述捕获探针ssDNA与NiFe-AuNPs通过自组装预先固定在电极上。
作为本发明的优选的实施方案,本发明的miRNA检测试剂盒包括:
第一试剂容器,该第一试剂容器包含所述玻碳电极;
第二试剂容器,该第二试剂容器包含NiFe-AuNPs复合物;
第三试剂容器,该第三试剂容器包含所述捕获探针ssDNA;
第四试剂容器,该第四试剂容器包含所述功能化发光探针;
第五试剂容器,该第五试剂容器包含磷酸盐缓冲液PBS;
第六试剂容器,该第六试剂容器包含磷酸盐缓冲液PBS、三丙胺TPrA;
第七试剂容器,该第七试剂容器包含TM缓冲溶液;
第八试剂容器,该第八实际容器包含封闭剂MCH。
其中,所述TM缓冲溶液的成分包括:10mM Tris-HCl,50mM MgCl2。
需要说明的是,上述检测试剂盒用于检测肿瘤标志物使用方法和原理为:首先,如图2所示,向所述第一试剂容器中的电极上滴加第二试剂容器,室温条件下反应,将NiFe-AuNPs复合物修饰在电极上;反应后采用第五试剂容器中的缓冲溶液清洗所述电极,然后滴加第三试剂容器中的ssDNA,将捕获探针ssDNA利用Au-S键作用修饰在电极上;反应一定时间后,滴加MCH封闭非特异性结合位点,构建ECL传感平台;优选的,所述第一试剂容器、第二试剂容器、第五试剂容器、第二试剂容器和第八试剂容器中的成分预先按照上述操作混合后预先构建ECL传感平台,采用一个试剂容器单独保存即可;然后,如图3所示,获取待测样品,置于第四试剂容器中,与第四试剂容器中的功能化发光探针在室温条件下孵育制备成miRNA-TDN-Ru(bpy)3 2+复合物,完成样品中检测对象miRNA的标记;然后,如图4所示采用第五试剂容器中的缓冲溶液清洗上述构建ECL传感平台,在ECL传感平台上滴加第四试剂容器中反应后形成的miRNA-TDN-Ru(bpy)3 2+复合物,反应后用PBS清洗,放置于第六试剂容器中含有TPrA的PBS溶液中,然后用于ECL检测。
有益效果
针对miRNA检测的试剂和方法存在仪器昂贵、操作复杂和检测灵敏度低的技术问题,本发明构建的miRNA检测方法具备如下优势:1.通过ECL技术进行检测,具备仪器设备简单、成本低廉的特点;2.该ECL传感平台构建方法简单,操作简便,检测速度快,易于实现多种miRNA标志物的检测;3.利用NiFe特殊的层状结构提高AuNPs和ssDNA负载量,利用AuNPs优良的催化性能提高发光效率,从而提高检测灵敏度。利用DNA四面体结构超电荷性质负载大量的信号分子,进而提高检测灵敏度,比起现有的传感平台灵敏度更高,检测限更低。
附图说明
图1为本发明所述功能化发光探针的制备过程;
图2为本发明所述电化学ECL传感平台的构建过程;
图3为本发明所述样品中miRNA的标记过程;
图4为本发明所述标记后的样本滴于ECL传感平台的反应和检测过程;
图5为本发明具体实施方式中所述所述NiFe-AuNPs复合物的制备过程;
图6为本发明具体实施方式中所述NiFe、NiFe-AuNPs的SEM表征图;
图7为本发明具体实施方式中所述NiFe-AuNPs复合物的TEM和EDS mapping表征图;
图8为本发明具体实施方式所述传感平台的ECL曲线表征图;
图9为本发明具体实施方式所述传感平台构建过程CV曲线表征图;
图10为本发明具体实施方式所述传感平台构建过程EIS曲线表征图;
图11为本发明具体实施方式所述传感平台条件优化;
图12为本发明具体实施方式中所述ssDNA与miRNA-TDN-Ru孵育时间对ECL强度影响;
图13为本发明具体实施方式中不同浓度miRNA的ECL曲线和线性曲线。
具体实施方式
下面结合附图,以胃癌标志物miRNA-27a为检测样本,采用一种较优的ECL传感平台,具体阐述本发明所述ECL传感平台构建过程和本发明所述检测试剂盒用于胃癌标志物miRNA-27a的检测过程。
以下具体制备中,涉及的试验材料及试验设备说明如下:
寡核苷酸序列由上海生工合成;Tris(2,2”-Bipyridine)Ruthenium Dichloride(Ru(bpy)3 2+)、HAuCl4·3H2O、购于Macklin Biochemical Co.,Ltd;Sodium borohydride(NaBH4)、6-Mercapto-1-hexanol(MCH)购于Sigma-Aldrich;6X Ficoll Gel LoadingBuffer、Acrylamide购于Sangon Biotech(Shanghai)Co.,Ltd;N,N,N',N'-Tetramethylethylenediamine(TEMED)、Tripropylamine(TPA)购于上海易恩化学技术有限公司;MgCl2·6H2O、Tris、Magnesium acetate、Ammonium peroxodisulphate(APS)、Orthoboric acid购于国药集团化学试剂有限公司;EDTA-2Na、Potassium ferricyanide(K3[Fe(CN)6])、Potassiumhexacyanoferrate(II)(K4[Fe(CN)6])、Potassium chloride(KCL)、Sodium Dihydrogen Phosphate(NaH2PO4)、Dibasic Sodium Phosphate(Na2HPO4)购于南京化学试剂有限公司,TM缓冲溶液成分为:10mM Tris-HCl,50mM MgCl2;MiRNA-27a序列如SEQ ID NO.5所示,具体为UUCACAGUGGCUAAGUUCCGC
超微弱发光测量仪BPCL购于微光科技有限公司,电化学工作站购于上海辰华。电化学发光检测实验中所施加的扫描电压范围为0~1.3V,扫描速度为0.1V/s,PMT高压为800V。
关于DNA四面体的制备
所述DNA四面体(TDN)由四条DNA片段自组织而成,形成的DNA四面体含有与目标miRNA碱基配对的序列。四条DNA片段在本实施例中如下SEQ ID NO.1、SEQ ID NO.2、SEQ IDNO.3和SEQ ID NO.4所示;这四条DNA片段可通过碱基配对形成四面体结构。
SEQ ID NO.1如下:
5’-CCACTGTGAATTCTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCGACGGTATTGGACCCTCGCATG-3’
SEQ ID NO.2如下:
5’-CGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCGACATGCGAGGGTCCAATACCG-3’
SEQ ID NO.3如下:
5’-CTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3’
SEQ ID NO.4如下:
5’-TTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3’
上述四条精确设计的首位互补的寡核苷酸链通过退火自组装形成,利用紫外分光光度计对每条DNA序列浓度进行定量,将4条序列以摩尔比1:1:1:1的比例混合,可保证大部分DNA都能自组装成DNA四面体,将DNA定量后,以SEQ ID NO.1:SEQ ID NO.2:SEQ ID NO.3:SEQ ID NO.4=1:1:1:1的比例将四条序列在TM缓冲液中混合均匀后,在95℃退火5min,然后降至4℃,形成DNA四面体结构。
功能化发光探针的制备
在380μL的TM缓冲液中加入10μL TDN(10μM)和10μL Ru(bpy)3 2+(10mM),4℃静置12h以保证Ru(bpy)3 2+充分与TDN结合。将上述溶液转移到超滤管中,以14,000×g的速度离心,以过滤多余的Ru(bpy)3 2+。将制备的TDN-Ru(bpy)3 2+储存在4℃备用。
将制备好的TDN-Ru(bpy)3 2+(50nm)与不同浓度的miRNA等体积混合,4℃下静置3h后组装成miRNA-TDN-Ru(bpy)3 2+。
NiFe-AuNPs的制备
根据文献Highly sensitive nonenzymatic H2O2 sensor based on NiFe-layered double hydroxides nanosheets grown on Ni foam.Surfaces and Interfaces12(2018)102-107报道的方法合成NiFe,具体的本实施例采用2mM Fe(NO3)3·9H2O、5mM Ni(NO3)2·6H2O和2.0mmol NH4F溶解在超纯水中并搅拌30min;将混合液转移到反应釜中,120℃反应6h;最后,离心、清洗、干燥后即得到NiFe产物;
如图5所示,将100μL 2mg/mL的NiFe分散在2mL H2O中以400rpm进行搅拌,在搅拌过程中依次加入50μLHAuCl4(1%)和40μL新鲜制备的NaBH4(3.8mg/mL),再以1200rpm充分搅拌30分钟,将得到的紫红色溶液离心分离收集,NiFe-AuNPs复合物,最后分散在1mL的超纯水中备用。
关于ECL传感平台的制备
将玻碳电极经打磨、超声、清洗得到干净的电极表面;将10μLNiFe-AuNPs修饰电极,自然晾干后滴加5μL ssDNA,室温反应1h,使得巯基修饰的ssDNA通过Au-S键结合在电极上。通过滴加MCH封闭材料的非特异性结合位点后,滴加5μL预先组装的miRNA-TDN-Ru(bpy)3 2+,反应2h后,经PBS清洗后制备得到传感平台。
如图6所示,其中,A图和B图分别是制备的NiFe低倍和高倍的SEM表征图,C图、D图中可以看出材料表面负载了密集的小颗粒(AuNPs),表明了NiFe-AuNPs的成功合成。通过高分辨TEM和EDS mapping分析上述制备的NiFe-AuNPs复合物,如图7中的A图和B图所示,AuNPs均匀的负载在NiFe层上,从元素分析结果可以看出(图7中的C图、D图、E图和F图),该复合物主要由Ni、Fe、Au元素组成,也进一步表明了NiFe-AuNPs复合物的成功制备。
分别利用ECL、CV和EIS对ECL传感平台的构建过程进行表征,图8为不同修饰过程的ECL曲线,图9为不同修饰过程的CV曲线,图10为不同修饰过程的EIS曲线。从ECL信号变化、CV曲线电流变化和EIS阻抗值变化表明了传感平台的成功制备。
将所制备的ECL传感平台为工作电极,Ag/AgCl电极作为参比电极,Pt丝作为辅助电极,使用循环伏安扫描电压,电压范围为0-1.3V,扫描速度为0.1V/s,光电倍增管高压为800V。
为了获得性能更好的传感器,对传感器的修饰过程进行了优化。首先是ssDNA的浓度优化,考察浓度分别为:0.1、0.5、1、5、10μM。如图11所示,随着ssDNA浓度的逐渐增加,传感器的ECL信号逐渐增强,在10μM时,信号达到最大。此外,对ssDNA和miRNA-TDN-Ru(bpy)3 2+的孵育时间进行了优化,考察时间为:15、30、60、120、180min。如图12所示,在120min时,发光强度达到了平台。因此,选择10μM ssDNA,120min为最佳条件。
人体血液中miRNA的浓度一般在1-1000pM,为了验证低丰度miRNA的灵敏检测,对不同浓度梯度的miRNA进行了检测,具体的,不同浓度的miRNA的浓度梯度设置为100aM,1fM,10fM,100fM,1pM,10pM,100pM和1nM)进行检测,图13所示,其中A图展示了传感器检测不同浓度miRNA的ECL曲线,B图为A图相应的线性关系,ECL强度与miRNA浓度在100aM-1nM之间具有良好的相关性,相关系数R2=0.9976,检测限为11.7aM(S/N=3)。
序列表
<110> 南京邮电大学
<120> miRNA类肿瘤标志物检测试剂盒
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 74
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccactgtgaa ttctcaactg cctggtgata cgaggatggg catgctcttc ccgacggtat 60
tggaccctcg catg 74
<210> 2
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgattacagc ttgctacacg attcagactt aggaatgttc gacatgcgag ggtccaatac 60
cg 62
<210> 3
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctactatggc gggtgataaa acgtgtagca agctgtaatc gacgggaaga gcatgcccat 60
cc 62
<210> 4
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tttatcaccc gccatagtag acgtatcacc aggcagttga gacgaacatt cctaagtctg 60
aa 62
<210> 5
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
uucacagugg cuaaguuccg c 21
Claims (5)
1.miRNA类肿瘤标志物检测试剂盒,其特征在于:所述检测试剂盒包括:电极、电极界面材料和捕获探针ssDNA;所述捕获探针、miRNA和功能化发光探针可通过碱基互补形成三明治结构;其中,所述电极为玻碳电极、所述电极界面材料为NiFe -AuNPs;所述功能化发光探针TDN-Ru是通过电荷吸附作用将发光分子负载在DNA四面体上组装制备而成;所述DNA四面体由四条DNA片段序列通过碱基配对自组装而成,其中一条DNA片段序列含有与miRNA部分互补配对的序列;所述DNA四面体的四条DNA片段序列通过碱基配对自组装时,所述四条DNA片段序列的数量比为1:1:1:1;
所述发光分子为Ru(bpy)3 2+;
所述捕获探针ssDNA与NiFe -AuNPs通过自组装预先固定在电极上;
所述miRNA为MiRNA-27a,所述MiRNA-27a序列如SEQ ID NO.5所示;所述DNA四面体的四条DNA片段序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示。
2. 根据权利要求1所述的miRNA类肿瘤标志物检测试剂盒,其特征在于:所述DNA四面体的四条DNA片段序列通过碱基配对自组装,是通过在TM缓冲液中将四条DNA片段序列混合均匀,在95 ℃退火5min,然后降至4 ℃,形成DNA四面体结构。
3.根据权利要求1所述的miRNA类肿瘤标志物检测试剂盒,其特征在于:所述检测试剂盒包括:
第一试剂容器,该第一试剂容器包含所述玻碳电极;
第二试剂容器,该第二试剂容器包含NiFe -AuNPs复合物;
第三试剂容器,该第三试剂容器包含所述捕获探针ssDNA;
第四试剂容器,该第四试剂容器包含所述功能化发光探针;
第五试剂容器,该第五试剂容器包含磷酸盐缓冲液PBS;
第六试剂容器,该第六试剂容器包含磷酸盐缓冲液PBS、三丙胺TPrA;
第七试剂容器,该第七试剂容器包含TM缓冲溶液;
第八试剂容器,该第八试剂容器包含封闭剂6-巯基-1-己醇MCH。
4.根据权利要求3所述的miRNA类肿瘤标志物检测试剂盒,其特征在于:所述第一试剂容器、第二试剂容器、第五试剂容器、第三试剂容器和第八试剂容器中的成分预先按照以下步骤混合,构建ECL传感平台,所述构建ECL传感平台采用一个试剂容器单独保存,具体顺序混合过程包括:向所述第一试剂容器中的电极上滴加第二试剂容器,室温条件下反应,将NiFe -AuNPs复合物修饰在电极上;反应后采用第五试剂容器中的缓冲溶液清洗所述电极,然后滴加第三试剂容器中的ssDNA,将捕获探针ssDNA利用Au-S键作用修饰在电极上;反应一定时间后,滴加MCH封闭非特异性结合位点,构建ECL传感平台。
5.根据权利要求4所述的miRNA类肿瘤标志物检测试剂盒,其特征在于:所述TM缓冲溶液的成分包括:10 mM Tris-HCl,50 mM MgCl2。
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