Whole Cell and Regulatory Protein-Based Sensors in Bioanalysis

PROJECT SUMMARY In our previous work, we developed a series of highly specific and sensitive whole cell bacteria-based sensing systems for environmental contaminants and other important analytes. We also demonstrated the use of bioluminescent, chemiluminescent, and fluorescent reporters in the development of these sensing systems, and we employed them in the determination of the levels of a target analyte in real samples. We now postulate that the utility of the whole cell-sensing systems developed in our laboratory could be further enhanced by (1) packaging them in an effective manner so they can be used in the field. (2) integrating the bacterial sensors into micrototal analytical systems (j..ITAS). In addition and to expand the application of whole cell sensing systems in bioanalytical chemistry, we propose the development of sensing systems for quorum sensing. One of the intellectual merits of this proposal lies in the design of cell-based sensing systems that are specific, sensitive, easy to store, and portable and can be employed in environmental analysis. There are certain requirements that need to be met for a sensing system to be effective in field analysis. We will evaluate several strategies for facilitating the long-term stability of these bacteria-based sensing systems in a manner that they can be integrated in j..ITAS devices for field use. First, in order to make our whole cell sensing systems amenable for packaging at room temperature, we propose to lyophilize the bacterial cells. Cells that respond to arsenite/antimonite will be used as a model system. We plan to employ different lyophilization techniques for this purpose. Our second approach involves the use of spores. Spores of bacteria can maintain their viability under harsh environmental conditions for decades. Since these spores can be revived when desired, we propose to transform our sensing cells into spores. This will allow for transport, storage and use of the bacterial sensing systems upon revival when needed. The second goal of our work is the integration of our cell-based sensing systems into a j..ITAS platform. For that, we propose to incorporate these dry forms of cells into a portable miniaturized system, namely a centrifuge (CD) microfluidics platform. The dried cells or spores will be stored directly on the microfabricated structures of the CD platform and will be reconstituted when analysis is desired. These optimized cell-based sensors on the CD platform should extend their applications in field studies. Part of the intellectual merit of this proposal concerns the goal to develop methods for detection of signaling molecules involved in bacterial cell-cell communication termed as "quorum sensing". Quorum sensing is the key factor in many processes occurring in bacteria, such as antibiotic production, competence, release of bioluminescence, and virulence. Signaling molecules involved in this process include acyl homoserine lactones (AHL), furanones, autoinducer-2, and certain peptides. Intervention in bacterial communication has been proposed as an alternative to traditional antibiotic therapy. There is a need for detection of these signaling molecules and we propose to design highly sensitive whole cell and protein-based methods for sensing of signaling molecules. Cell-based methods will be developed by employing reporter gene technologies and the same principles as those previously prepared in our laboratory for other analytes. In another approach, specific regulatory proteins that bind signaling molecules and undergo a conformational change will be used in the design of protein-based sensors. An environment-sensitive fluorophore will be conjugated to the regulatory protein at a site that is allosterically linked to the binding site for the signaling molecule. Upon addition of signaling molecule, the change in the conformation of the protein will result in a concomitant change in fluorescence intensity of the fluorophore, which will be correlated with the amount of the signaling molecule present. The broader impact of this research lies in that the systems developed will not only find applications in environmental analysis, but in biomedicine as well. Gaining insight in cell-cell communications and understanding the levels at which the signaling molecules trigger aggregation of cells should allow for the design of new drugs that inhibit quorum sensing. Thus, it potentially can aid in the development of new therapies for diseases where quorum sensing plays or is suspected to playa role (i.e., cystic fibrosis, Crohn's disease, etc.). Moreover, the multidisciplinary nature of the work will allow students to participate and gain knowledge in different areas of Science including analytical chemistry, molecular biology, microfabrication, mechanical engineering, biomedicine, nanotechnology, etc.