Rapid Electrochemical Detection and Identification of Microbiological and Chemical Contaminants for Manned Spaceflight Project

<p>A great deal of effort has gone into the development of point-of-use methods to meet the challenge of rapid bacterial identification for both environmental monitoring and clinical applications.&nbsp; Unfortunately, most of the methods developed rely on Preliminary Chain Reaction (PCR) and face inherent limitations because of the requirement for enzymatic components and thermal control.&nbsp; Other methods based on surface plasmon resonance, quartz crystal microbalance, and fluorescence has been reported with good detection limits, but, these methods are immunological and cannot provide genetic-level information.&nbsp; Further, they require labeled markers, complicated fluid handling systems, and sensitive optics that drive up cost and complexity and preclude them from outside the laboratory.&nbsp; Recent work by a group at the University of Toronto has focused on developing an electrochemical platform that combines ultrasensitive detection, straightforward sample processing, and inexpensive components to create a cost-effective, user-friendly device for detection and identification of microorganisms.&nbsp; The platform combines an electrical cell lysis chamber, and electrochemical reporter system, and nanostructured microelectrodes (NMEs) to detect specific nucleic acid sequences.&nbsp; The nucleic acid sequences are unique to a given type of microorganism and can be used to identify the microorganisms present in a sample.</p><p>From the perspective of the anticipated prototype device &nbsp;(Lam, et al. 2012. <em>Polymerase Chain Reaction-Free, Sample-to-Answer Bacterial Detection in 30 Minutes with Integrated Cell Lysis</em>. Anal. Chem. <strong>84(1)</strong>: 21-5), detection of microbial contaminants will begin with a lysis chamber designed to release DNA and RNA from microorganisms present in the sample using ultrasonic or electrochemical technology.&nbsp; The DNA and RNA mixture is then passed into an analysis chamber containing an array of nanostructured microelectrodes (NMEs).&nbsp; The surface of the NMEs will be functionalized with probe molecules for DNA or RNA sequences specific to the bacteria being targeted.&nbsp; Binding of the DNA or RNA to the appropriate detection probe on the NME surface in the presence of an electrochemical reporter system will change the electrochemical properties of the NMEs.&nbsp; A potentiostat is used to measure the current at each individual electrode before and after addition of the DNA and RNA mixture.&nbsp; The difference in current before and after addition of the mixture to the NMEs is compared against a pre-determined threshold to check for the presence of target bacteria in the sample.&nbsp; The process for detection of chemical contaminants is very similar.&nbsp; The lysis chamber would be bypassed and the sample would flow directly into the analysis chamber.&nbsp; The NMEs will be functionalized with molecules to selectively bind the desired targets (analytes) and the change in the electrochemical response of each NME can again be used to detect and quantify the contaminants.&nbsp; Depending on the analyte of interest, it may be possible to directly measure analyte binding on the surface of the NMEs without the use of an electrochemical reporter system. The overall project will focus on optimization of the individual aspects of the detection platform in preparation for construction of a prototype for a flight experiment.&nbsp; The scope of the work in this proposal is limited to characterization and optimization of the lysis step/sample preparation, probe selection, and NME structure.&nbsp; Lysis conditions will be optimized by evaluating parameters associated with the oscillation frequency and lysis time for ultrasonic techniques and applied voltage for the electrochemical techniques.&nbsp; Cell viability, as determined by fluorescent detection of DNA or R