Enzyme based bacterial biosensor

The objective of the project is to provide colorimetric bacteria detection. Creating colorimetric biosensors gives us an opportunity to get more precise data. Biosensors have advantages over traditional ones such as low cost, short time of analysis, high selectivity and simplicity. Our analyte was chosen as -gal. This enzyme is present in lactose fermented bacteria and it is a great fecal bacteria indicator. Bacteria use glucose as their carbon sources but in some cases, like in environments where glucose is not found, bacteria use lactose. In order to metabolize lactose bacteria needs -gal enzyme. To detect -gal presence and to create colorimetric change a chromogenic substrate was used. O-Nitrophenyl-β-D-galactopyranoside (ONPG) was used as a substrate due to structural similarities with lactose. ONPG is colorless. Hydrolysis of ONPG with -gal causes to produce galactose and 2-Nitrophenol. 2-nitrophenol is the compound which is yellow and provides visual evidence of -gal presence and activity. Besides colorimetric reading, our data is supported with SERS. During SERS 2-nitrophenol is chosen as the target molecule and 2-Nitrophenol signals are detected. 2-Nitrophenol has characteristic Raman signal at the wavelength 1380 cm-1. Calibration graph (R2 = 0,9103) is drawn focusing on that point. The system produced was applied to real samples. Cheese was chosen as a real sample and E. coli was spiked with cheese. Total time of analysis completed less than 2 hours. The results that were obtained from both spiked and not-spiked samples show promising results where the developed biosensor can detect even the lowest concentrations of E. coli.