Electrochemical detection of specific DNA and respiratory activity of Escherichia coli

• Published in: Electrochimica acta Vol. 82; pp. 132 - 136
• Main Authors: Yamanaka, Keiichiro, Ikeuchi, Tomohiko, Saito, Masato, Nagatani, Naoki, Tamiya, Eiichi
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Kidlington Elsevier Ltd 01.11.2012; Elsevier
• Subjects: Biological and medical sciences; Chemistry; Electrochemistry; Escherichia coli; Exact sciences and technology; Food industries; Food safety; Fundamental and applied biological sciences. Psychology; General and physical chemistry; General aspects; Hygiene and safety; Polymerase chain reaction (PCR); Screen-printed electrode; USB-powered portable potentiostat; Screen-printed electrode; USB-powered portable potentiostat; Food safety; Polymerase chain reaction (PCR); Escherichia coli; Reaction product; Electrochemical detector; Polymerase chain reaction; Gene amplification; DNA; Food security; Potentiostat; Bacteria; Portable equipment; Enterobacteriaceae
• ISSN: 0013-4686; 1873-3859
• DOI: 10.1016/j.electacta.2012.03.032

We present two rapid and simplified detection methods for Escherichia coli involving the use of a hand-held potentiostat and a disposable screen-printed carbon electrode. E. coli is one of the indicator organisms used to access for food safety. Commonly, microbiological culture techniques take more than one day to yield results and therefore, a simple, cost-effective, in situ detection system is required for testing food safety. This report describes two complementary techniques for high- and low-sensitivity detection of E. coli. High-sensitivity detection relies upon quantification of DNA amplification by using polymerase chain reaction (PCR), while the simplified, low-sensitivity detection can be obtained through measurement of oxygen consumption due to respiration; importantly, both techniques utilize the same type of electrode. The former entails mixing the PCR mixture with Hoechst, an electro-active DNA intercalator, and then, measuring the oxidation current. Binding of Hoechst molecules to the amplified DNA causes the peak current to decrease because of the slow diffusion of the Hoechst-amplified DNA complex to the electrode surface. The results showed that the oxidation peak current of Hoechst decreased depending on the number of E. coli cells added to the PCR mixture as the template for amplification, and the sensitivity of the method was as low as a single bacterium. Oxygen consumption was detected by direct measurement of the cell-containing culture medium. This method required only 10μL to be applied on the screen-printed electrode, and the reduction in oxygen current was clearly observed within 30min when a minimum of 1×105cells were present. These results were obtained without purifying the culture, and the samples were applied onto the electrode without any surface modifications. The techniques describes in this report are versatile, because they require the same type of electrode, have simplistic nature, use a hand-held potentiostat, and have potential to provide an in situ system for assessing food safety.