Asrat Thomas M, Cho Whirang, Liu Favian A, Shapiro Sarah M, Bracht John R, Zestos Alexander G
Department of Chemistry, American University, Washington, D.C. 20016, United States.
Department of Biology, American University, Washington, D.C. 20016, United States.
ACS Omega. 2021 Mar 3;6(10):6571-6581. doi: 10.1021/acsomega.0c04845. eCollection 2021 Mar 16.
DNA and RNA have been measured with many techniques but often with relatively long analysis times. In this study, we utilize fast-scan cyclic voltammetry (FSCV) for the subsecond codetection of adenine, guanine, and cytosine, first as free nucleosides, and then within custom synthesized oligos, plasmid DNA, and RNA from the nematode . Previous studies have shown the detection of adenosine and guanosine with FSCV with high spatiotemporal resolution, while we have extended the assay to include cytidine and adenine, guanine, and cytosine in RNA and single- and double-stranded DNA (ssDNA and dSDNA). We find that FSCV testing has a higher sensitivity and yields higher peak oxidative currents when detecting shorter oligonucleotides and ssDNA samples at equivalent nucleobase concentrations. This is consistent with an electrostatic repulsion from negatively charged oxide groups on the surface of the carbon fiber microelectrode (CFME), the negative holding potential, and the negatively charged phosphate backbone. Moreover, as opposed to dsDNA, ssDNA nucleobases are not hydrogen-bonded to one another and thus are free to adsorb onto the surface of the carbon electrode. We also demonstrate that the simultaneous determination of nucleobases is not masked even in biologically complex serum samples. This is the first report demonstrating that FSCV, when used with CFMEs, is able to codetect nucleobases when polymerized into DNA or RNA and could potentially pave the way for future uses in clinical, diagnostic, or research applications.
人们已经使用多种技术来测量DNA和RNA，但分析时间往往相对较长。在本研究中，我们利用快速扫描循环伏安法（FSCV）在亚秒级对腺嘌呤、鸟嘌呤和胞嘧啶进行共检测，首先是作为游离核苷，然后是在定制合成的寡核苷酸、质粒DNA以及线虫的RNA中进行检测。先前的研究已经表明，利用FSCV能够以高时空分辨率检测腺苷和鸟苷，而我们将该检测方法进行了扩展，使其能够检测RNA以及单链和双链DNA（ssDNA和dsDNA）中的胞苷、腺嘌呤、鸟嘌呤和胞嘧啶。我们发现，在等效核碱基浓度下检测较短的寡核苷酸和ssDNA样品时，FSCV测试具有更高的灵敏度，并且能产生更高的氧化峰值电流。这与碳纤维微电极（CFME）表面带负电荷的氧化物基团、负保持电位以及带负电荷的磷酸骨架产生的静电排斥作用相一致。此外，与dsDNA不同，ssDNA核碱基彼此之间没有氢键结合，因此可以自由吸附到碳电极表面。我们还证明，即使在生物成分复杂的血清样本中，同时测定核碱基也不会受到干扰。这是第一份表明FSCV与CFME一起使用时能够在核碱基聚合成DNA或RNA时进行共检测的报告，这可能为未来在临床、诊断或研究应用中的使用铺平道路。
