Electrochemical biosensor for rapid detection of fungal contamination in fuel systems.

There is an increased demand for real-time monitoring of biological and biochemical processes. While most sensor research focuses on physiological conditions, less has been done towards developing real-time biosensors that can operate in and survive exposure to extreme environments and harsh chemicals such as fuel. One interesting application is monitoring microbial load in fuel tanks to prevent both fuel spoilage and biocorrosion. We developed a comprehensive method to enable the first reagentless, real-time, microbial sensor platform that is also fuel resistant. We first identified an extracellular protein epitope conserved in fuel-degrading fungi then used this epitope to develop a suitable biorecognition element (BRE) through biopanning of a 7-mer phage displayed peptide library. After demonstrating the BRE's affinity to fungi using molecular and fluorescence assays, we incorporated the BRE into a reagentless, real-time electrochemical sensing platform based on a self-assembled monolayer of peptide BREs and redox reporters. Finally, we incorporated this real-time electrochemical sensing platform into a microfluidic device. We demonstrated detection of Yarrowia lipolytica as low as 1 × 104 CFU/mL in a bath cell, and demonstrate a microfluidic cell that functions even after exposure to jet fuel. In summary, this work describes development of a fuel-resistant biosensor for monitoring microbial growth in extreme environments.

FRET-based optical assay for selection of artificial riboswitches.

Artificial riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules and, therefore, can be useful tools to reprogram cellular behavior for different applications. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer with a randomized expression platform followed by in vivo selection and screening. Here, we describe an in vivo selection and screening technique to discover artificial riboswitches in E. coli cells that is based on TEV protease-FRET substrate reporter system.