Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids
Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids
复制标题
DOI：
10.1021/acs.analchem.6b04609
复制
发表时间：
2017-02-07
影响因子：
7.4
通讯作者：
Yang, Haesik
中科院分区：
文献类型：
--
作者：
Fang, Chiew San;Kim, Kwang-sun;Yang, Haesik
高灵敏度和高特异性对于检测已成为重要临床生物标志物的miRNA至关重要。Just Another Zinc finger proteins（JAZ，ZNF 346）与单链RNA、单链DNA和双链DNA相比，更优选（但非序列特异性地）与DNA RNA杂交体结合。我们提出了一种用于miRNA-21检测的超灵敏度和高度特异性的电化学方法，该方法基于JAZ与DNA捕获探针和靶miRNA-21之间形成的DNA RNA杂交体的选择性结合。这使我们能够使用化学稳定的DNA作为捕获探针，而不是RNA，以及应用标准的三明治型检测格式的miRNA检测。高信号放大通过（i）碱性磷酸酶（ALP）的酶促放大与（ii）涉及ALP产物（氢醌）的电化学化学（ECC）氧化还原循环偶联获得。低非特异性吸附ALP共轭JAZ获得使用聚合物自组装单层修饰和酪蛋白处理的氧化铟锡电极。该检测方法可以区分靶miRNA-21和非靶核酸（DNA DNA杂合体、单链DNA、miRNA-125 b、miRNA-155、单碱基错配的miRNA和三碱基错配的miRNA）。缓冲液和10倍稀释血清中miRNA-21的检测限分别约为2和30 fM，表明该检测方法是超灵敏的。这种检测方法可以很容易地扩展到多重检测的miRNA，由于其非序列特异性结合特性，只有一个ALP共轭JAZ探针。我们还相信，该方法可以为体液中miRNA的即时检测提供一种有前途的解决方案。
Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA RNA hybrids over single stranded RNAs, single stranded DNAs, and double stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical chemical chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein treated indium tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA DNA hybrid, single stranded DNA, miRNA-125b, miRNA-155, single base mismatched miRNA, and three base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10 fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point of care testing of miRNAs in body fluids.