Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe
Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe
复制标题
DOI：
10.1039/c2an36153a
复制
发表时间：
2013-01-01
期刊：
影响因子：
4.2
通讯作者：
Zourob, Mohammed
中科院分区：
文献类型：
--
作者：
Ahmed, Minhaz Uddin;Nahar, Sharifun;Zourob, Mohammed
静电氧化还原探针相互作用已广泛用于DNA定量。我们已经通过使用钌六胺分子[Ru（NH3）（6）]（3+）建立了原理证明。我们已经应用了这种方法的实时电化学监测的环介导等温扩增（LAMP）扩增的大肠杆菌和金黄色葡萄球菌的靶基因的方波伏安法（SWV）。钌六胺与溶液中的游离DNA相互作用而不被固定到生物芯片表面上，使我们能够放弃DNA定量中耗时的过夜探针固定步骤。我们已经测量了阴极电流信号的变化，使用丝网印刷的低成本的生物芯片在存在和不存在的LAMP扩增子的目标DNA在溶液相中。利用这种新型探针，我们成功地实现了实时等温扩增和检测，在不到30分钟的S。aureus和E.大肠杆菌的敏感性分别为30拷贝μ L ~（-1）和20拷贝μ L ~（-1）。电流的阴极峰高与扩增子形成的程度和引入的模板基因组DNA的量有关。重要的是，由于不需要费力的探针固定，并且体外扩增和实时监测都是使用单个生物芯片在单个聚丙烯管中进行的，因此这种新方法可以避免整个过程中所有潜在的交叉污染。
Electrostatic redox probes interaction has been widely rendered for DNA quantification. We have established a proof-of-principle by using the ruthenium hexaamine molecule [Ru(NH3)(6)](3+). We have applied this method for real-time electrochemical monitoring of a loop mediated isothermal amplification (LAMP) amplicon of target genes of Escherichia coli and Staphylococcus aureus by square wave voltammetry (SWV). Ruthenium hexaamine interaction with free DNAs in solution without being immobilized onto the biochip surface enabled us to discard the time-consuming overnight probe immobilization step in DNA quantification. We have measured the changes in the cathodic current signals using screen printed low-cost biochips both in the presence and the absence of LAMP amplicons of target DNAs in the solution-phase. By using this novel probe, we successfully carried out the real-time isothermal amplification and detection in less than 30 min for S. aureus and E. coli with a sensitivity up to 30 copies mu L-1 and 20 copies mu L-1, respectively. The cathode peak height of the current was related to the extent of amplicon formation and the amount of introduced template genomic DNA. Importantly, since laborious probe immobilization is not necessary at all, and both the in vitro amplification and real-time monitoring are performed in a single polypropylene tube using a single biochip, this novel approach could avoid all potential cross-contamination in the whole procedure.