Peptide-Based Fluorescent Biosensing for Rapid Detection of Fuel Biocontamination

摘要

To reduce the impact of biodeterioration in fuel systems, effective methods for early detection and monitoring of microbial growth in the fuel are required. This study presents the development of broad-range peptide biorecognition elements (BREs) that target cell surface determinants produced by hydrocarbon-degrading microorganisms during growth in fuel. BREs were used to biofunctionalize fluorescent semiconductor particles (quantum dots, QDs) to produce a sensitive quatititative fluorescence-based assay for detection of microbial growth in fuel. A biopanning method in the presence of fuel was used to select phage-displayed heptameric peptide BREs against epitopes conserved in fuel-degrading Gram-negative bacteria including Pseudomonas spp. and Acinetobacter spp. Fluorescence microscopy analysis and fluorescence signal measurements relative to colony-forming units (CFUs) demonstrated the binding and specificity of the BRE-QDs for fuel-degrading Gram-negative bacteria. Cross-reactivity with Gram-positive bacteria Arthrobacter and Lysinibacillus was not observed. The assay was shown to be specific for detection of Gram-negative-bacteria. Jet fuel samples amended with different concentrations of fuel-degrading bacteria were used-so determine the sensitivity and limit of detection (LOD) of the assay; an LOD of 5 X 10(4) colony forming units (CFUs) with detection levels as low as 5 X 10(3) CFUs was established for the best performing BRE-QD conjugate. The peptide BRE-QD chemistry effectively detected biocontaminated fuel samples from fuel tanks. The peptide BREs may serve to biofunctionalize various fluorescent, chemiluminescent, and colorimetric molecules as well as optical and electrical transducers to developed effective biosensors for detection of microbial contamination in fuel.