A Microsystem Compatible Strategy For Viable E.coli Detection

There are a significant number of pathogenic microorganisms in the environment (air, water, soil, food, etc.). In order to protect the public health, routine environmental assessment is essential, especially the pathogen viability detection since only the living cells are harmful. Practical challenges for timely and effective viability detection include speed and portability. Conventional method for viable pathogen detection typically relies on the culture-based assays, which is generally time-consuming and replies on laboratory facilities, In recent years, with the dramatic development of micromachining technologies, more and more biological analyses have been successfully demonstrated in miniaturized analysis systems (lab on a chip or micro total analysis system, μTAS). Biodetection in a microscale format has many inherent advantages including small sample volume and less reagent consumption. In addition, the ability to integrate various processing modules on the same device and the hope for a truly portable analyzer has attracted a large academic community to this research endeavor. However, the available strategies for microsystem compatible detection of pathogen viability have been seldom reported till now.This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e., mRNAs of a common Escherichia coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 °C) for 20 minutes. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au based electrochemical detection is done after the amplification of mRNAs by reverse-transcription polymerase chain reaction(RT-PCR).Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle based mRNA extraction technique were tackled. A sensitive Ag/Au based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 102 cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis.The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.