Spore-Based Biosensing Systems: A Stabilized Dormant-Active Approach to Whole-Cell Biosensors

There is great interest in the development of biosensing systems that can be applied effectively to field analysis. In that regard, whole-cell-based biosensors are ideal in bioanalysis as well as in environmental monitoring in that they allow for the sensitive, selective, quantitative and rapid detection of the analytes of interest. Moreover, they provide valuable information on the bioavailability and activity of the target compounds. However, the issues of the shelf-life and transportability of whole cell biosensing systems, and of their adaptation to miniaturized systems for field analysis have been scarcely addressed. To that end, the goal of this research is to develop a method based on the use of bacterial spores for the longterm maintenance of the viability and activity of bacterial sensing cells, and to integrate such preserved sensor cells into portable systems for on-field sensing. Specifically, the objectives of the proposed research are: (1) Development of genetically engineered whole-cell sensing systems based on sporeforming bacteria and production of spores as means of preservation, storage and transport of the sensing cells. Spores are dormant forms of life that exhibit long-term resistance towards extreme environmental conditions, and are able to return to a vegetative state and resume full metabolic activity in the presence of appropriate stimuli. (2) Evaluation of different luminescent reporter genes in the construction of the whole-cell sensing systems, in order to identify those more appropriate for long-term storage and field studies. (3) Development of whole-cell sensing systems for different target analytes, representatives of different classes of compounds, such as oxoanions, metals, and sugars. Specifically, arsenite, zinc, and L-arabinose will be used as models of the broad applicability of the spore-based sensing systems in different fields of analysis. (4) Incorporation of these dormant sensors into miniaturized microfluidic systems, for example into a CD microcentrifuge microfluidic platform, which can be transported to the field, where the spores can be germinated to vegetative cells and employed for sensing. (5) Use of the sensing spores in easy, ready-to-use configurations, based, for example, on the immobilization of the spores on paper strips by employing different technologies. These sensing strips should be easy to use, even by non-trained personnel. Intellectual Merit: The proposed research addresses several key challenges in the field of biosensing. In particular, those related to the stability of whole-cell biosensing systems for prolonged periods of time and in unfavorable conditions, and to the packaging of the sensors and their integration into analytical devices in such a way that improves their shelf-life and allows their use in different environments in the field. The proposed approach is general in that it can be applied to the detection of any analyte, provided that specific genetic constructs can be prepared. It is envisioned that this new strategy can expand the use of whole-cell biosensors for on-field analysis, in environments and applications in which they have not been able to be employed before. These include developing countries with unfavorable climate conditions and inadequate distribution and storage facilities, extreme environments, such as deserts, polar regions and space, and the remote sensing of chemical warfare agents used for bioterrorism. Furthermore, the proposed set-up is amenable to the incorporation of multiple sensing systems in one device and provides with a portable solution to multiplexed, high-throughput field-analysis.