In Situ and Point of Sale Quantification of Human Pathogens Associated with Aquaculture and Shellfish Farming Using Novel Surface Enhanced Raman Spectroscopy

There is a dire need for technology to rapidly and accurately detect and quantify Escherichia coli serotypes 0157:H7 and K12 and Vibrio parahaemolyticus and total fecal coliforms. In Phase I we developed a novel approach to Surface Enhanced Raman Spectroscopy (SERS) for detecting human pathogens in cultured bacteria, mixtures of organisms and in oyster tissue. Our approach uses a higher energy laser wavelength (405nm) compared with the lower energy of more the common lasers at 633 and 785nm, a novel mixture and preparation of Ag-coated nano-particles passed through a microfluidic mixing platform, and classification of Raman spectra with a novel 1D Convolutional Neural Network (CNN). Through serial dilutions of cultured bacteria, the detection limit of the SERS platform was evaluated at a dilution of 1:100, which was equivalent to ~100 CFU/mL and a MPN of 44.8 /mL. In Phase II we will further refine the approach and build several units for testing by managers in the field. The microfluidic instrument will allow in situ identification and quantification of pathogen species. Real-time detection of E. coli and other fecal coliforms from buoys or the end of docks would allow for immediate mapping of contamination sources and early response by environmental managers.