Exploiting bioluminescence to enhance the analytical performance of whole-cell and cell-free biosensors for environmental and point-of-care applications

The routine health monitoring of living organisms and environment has become one of the major concerns of public interest. Therefore, there has been an increasing demand for fast and easy to perform monitoring technologies. The current available analytical techniques generally offer accurate and precise results; however, they often require clean samples and sophisticated equipment. Thus, they are not suitable for on site, real-time, cost-effective routine monitoring. To this end, biosensors represent suitable analytical alternative tools. Biosensors are analytical devices integrating a biological recognition element (i.e. antibody, receptor, cell) and a transducer able to convert the biological response into an easily measurable analytical signal. These tools can easily quantify an analyte or a class of analytes of interest even in a complex matrix, like clinical or environmental samples, thanks to the specificity of the biological components. Whole-cell biosensors among others offer unique features such as low cost of production and provide comprehensive functional information (i.e. detection of unclassified compounds and synergistic effects, information about the bioavailable concentration). During this PhD, several bioengineered whole-cell biosensors have been developed and optimized for environmental and point-of-care applications. Analytical performance of biosensors have been improved (i.e. low limit of detection, faster response time and wider dynamic range) thanks to synthetic biology and genetic engineering tools. Bacterial, yeast and 3D cell cultures of mammalian cell lines have been tailored at the molecular level to improve robustness and predictivity. Several reporter genes, i.e. colorimetric, fluorescent and bioluminescent proteins, have been also profiled for finding the best candidate for each point-of-need application. Furthermore, spectral resolution of different optical reporter proteins has been exploited and multiplex detection has been achieved. The inclusion of viability control strains provided a suitable tool for assessing non-specific effects on cell viability, correcting the analytical signal and increasing the analytical performance of ready-to-use cartridges.