Packaging Sensing Cells in Spores for Long-Term Preservation of Sensors: A Tool for Biomedical and Environmental Analysis Amol Date, Patrizia Pasini, Abhishek Sangal, and Sylvia Daunert* Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055 Whole-cell sensing systems have successfully been em- ployed for detection of various biologically and environ- mentally important analytes. A limitation to their use for on-field analysis is the paucity of preservation methods for long-term storage and transport. For that, we have previously developed spore-based genetically engineered whole-cell sensing systems that are able not only to maintain the activity of the sensing cells but also to preserve it for long periods of time in normal and extreme environmental conditions. Herein, we have employed these spore-based sensing systems for analysis of real samples, such as blood serum and freshwater. Spores were able to germinate in the presence of the sample matrix, and the minimum time required for the spores to germinate and generate vegetative sensing cells able to elicit a measurable response to target analytes resulted to be around 2 h. Of the two spore-based sensing systems selected to detect model analytes in real samples, one was able to detect arsenic concentrations as low as 1 × 10 -7 M in freshwater and serum samples, and the other one could sense down to 1 × 10 -6 M of zinc in serum. The analysis of human serum samples from healthy sub- jects for their zinc content proved the viability of spore- based sensing systems. The complete assays, including spore germination and analyte detection, were per- formed in 2.5 h or less for arsenic and zinc. Further- more, the assay is inexpensive and simple to carry out and offers unique advantages for the incorporation of the spore-based sensing systems into portable analyti- cal platforms, such as microfluidic devices, to be employed for on-site analysis. Genetically engineered bacterial whole-cell sensing systems are based on coupling of a specific binding event between a regulatory protein and its ligand analyte with the expression of a reporter protein within intact cells. Such systems are constructed by inserting into host cells the gene sequences of a regulatory protein, an operator/promoter (O/P) region, and a reporter gene whose transcription is under the transcriptional control of the regulatory protein and the O/P. These genes are usually carried by a DNA plasmid, which is transformed into the bacterial cells; alternatively, they can be inserted in the bacterial chromosome. An analyte permeates through the cell membrane and binds to the recognition/regulatory protein to activate the transcription of the reporter gene. Subsequent translation of the reporter mRNA produces a protein, which generates a measurable signal that is directly related to the amount of analyte. With the choice of the appropriate regulatory and reporter proteins, these sensing sys- tems can yield quantitative information about the analyte of interest in in vitro as well as in vivo settings. Additionally, since the target analyte needs to be uptaken by the sensing cells to induce a response, only the bioavailable amount of analyte is measured. This feature is unique to whole-cell sensing systems, which contributed to the appeal that these analytical tools gained in several fields of bioanalysis, such as environmental monitoring, drug screening, and clinical analysis. Bacterial whole-cell biosens- ing systems have been developed for the detection of sugars, drugs, quorum sensing molecules, various toxic metals, such as mercury, arsenic, cadmium, and several organic pollutants. 1-7 These bacterial sensing systems can selectively, sensitively, and rapidly detect very low levels of analytes. Furthermore, the potential of these sensors to be integrated into portable devices, including attachment onto optic fiber tips, incorporation into miniaturized microfluidics platforms, and immobilization on paper strips, among others, makes them attractive for on-field analysis. 2,8,9 However, limitations to the on-site use of these sensing systems are posed by their short shelf life and the requirement of specific growth and storage conditions. 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