Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University , Busan 46241, Korea.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST) , Daejeon 34141, Korea.
Anal Chem. 2017 Feb 7;89(3):2024-2031. doi: 10.1021/acs.analchem.6b04609. Epub 2017 Jan 6.
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.
高灵敏度和高特异性对于检测 miRNA 至关重要，miRNA 已成为重要的临床生物标志物。另一种锌指蛋白（JAZ，ZNF346）优先（但非特异性）结合 DNA-RNA 杂交体，而不是单链 RNA、单链 DNA 和双链 DNA。我们提出了一种基于 JAZ 与 DNA 捕获探针和靶 miRNA-21 之间形成的 DNA-RNA 杂交体选择性结合的超灵敏和高度特异性电化学方法来检测 miRNA-21。这使我们能够使用化学稳定的 DNA 作为捕获探针，而不是 RNA，并且可以将标准夹心型测定格式应用于 miRNA 检测。通过（i）碱性磷酸酶（ALP）偶联的酶促扩增和（ii）涉及 ALP 产物（对苯二酚）的电化学-化学-化学（ECC）循环实现高信号放大。使用聚合自组装单分子层修饰和酪蛋白处理的铟锡氧化物电极，可以获得低的 ALP 偶联 JAZ 的非特异性吸附。该检测方法可以区分靶 miRNA-21 和非靶核酸（DNA-DNA 杂交体、单链 DNA、miRNA-125b、miRNA-155、单碱基错配 miRNA 和三碱基错配 miRNA）。在缓冲液和 10 倍稀释血清中的 miRNA-21 的检测限分别约为 2 和 30 fM，表明该检测方法具有超灵敏性。由于其非特异性结合特性，该检测方法可以很容易地扩展到仅使用一个 ALP 偶联 JAZ 探针对 miRNA 进行多重检测。我们还认为，该方法可以为体液中 miRNA 的即时护理测试提供有前途的解决方案。
