Development of a rapid method for the detection of biological threats in water

摘要

In this paper, progress towards the development of real-time sensing of chemical and biological threats in liquid samples will be presented. This overall goal of this work is to combine the selective, molecular recognition of nucleic acid aptamers with a rapid signal transduction using fluorescence resonance energy transfer (FRET) for a single step identify and detect approach. Of particular interest is the application to whole-cell target recognition of biologicals, such as environmental pathogens (e.g., Campylobacter jejuni), without requiring cell lysis or other complex protocols to access biochemical species internal to the organism. An aptamer staining protocol for whole cell targets is developed and applied to the investigation of aptamers against Campylobacter jejuni cells. A comparison of aptamer binding using this method with and without the primer regions utilized in the aptamer selection process is presented and the primer regions were found to have little impact on binding performance. C. jejuni aptamers exhibited strong binding as evidenced through the fluorescence images acquired and little to no background fluorescence was observed from non-specific binding of the streptavidin-dye conjugate used in the staining method. A thrombin targeted molecular aptamer beacon was also studied and a rapid analysis was demonstrated. A 10 nM sample of thrombin was distinguishable from the fluorescence baseline of the probe alone, when using a 40 nM aptamer probe concentration. The fluorescence intensity was found to increase until saturation of the aptamer probe was achieved. These results show promise for the development of single-step identification of whole-cell targets using an aptamer bioreceptor and fluorescence resonance energy transfer transduction signaling scheme.