(189a) Genetically Diverse Biosensors and Bionanoanalytical Methods | AIChE

(189a) Genetically Diverse Biosensors and Bionanoanalytical Methods

Authors 

Daunert, S. - Presenter, University of Kentucky


The design of instruments and techniques capable of detection, quantification, and delivery of small amounts of biomolecules is essential for the development of new bioengineering methods and devices. Microfabrication and microfluidics have been instrumental in the advancement of this field. In order to detect target molecules in small volumes and microfabricated structures, it is necessary to prepare bioreagents that provide enough sensitivity for their detection. In our laboratory, we engineer proteins and cells to design a diverse array of biosensing systems. Specifically, the photoprotein aequorin has been genetically engineered by preparing mutant and fusion proteins, and by designing molecular switches. We have incorporated various chromophore analogues into the newly produced aequorin variants in order to shift the emission maxima and alter the bioluminescent decay kinetics. Moreover, we have genetically encoded aequorin with non-natural amino acids to create newly ?colored' aequorin variants, which have found application in simultaneous multianalyte detection. Additionally, we have prepared a bioluminescent molecular switch for glucose by dissecting the gene of the aequorin molecule into two halves and inserting in between the gene of the glucose binding protein to produce a protein molecular switch capable of glucose detection. In the presence of glucose, the glucose binding protein undergoes a conformational change bringing the two ?halves' of the aequorin molecule and allowing for the emission of bioluminescence in a manner proportional to the concentration of glucose present. In another strategy, a protein immobilized within a hydrogel acts as a recognition element for drugs. The integrated molecular recognition within the hydrogel allows for simultaneous sensing and actuating, thus providing with a novel approach to responsive drug delivery systems. Finally, we have taken advantage of the bioluminescence produced from bacterial luciferase in order to study quorum sensing and the mechanism of action of quorum sensing molecules (QSMs) in relation to gastrointestinal (GI) disorders. To that end, we developed and employed genetically engineered bioluminescent whole-cell-based sensing systems for the detection of QSMs in physiological samples, both in subjects with various GI disorders and healthy volunteers. Finally, non-invasive diagnostics/management tools for these diseases have been developed by preparing colorimetric paper strips incorporating these QSM biosensors.