A Facile HPLC-UV-Based Method for Determining the Concentration of the Bacterial Universal Signal Autoinducer-2 in Environmental Samples

As a universal quorum sensing (QS) signal, autoinducer-2 (AI-2) is utilized by both Gram-negative and Gram-positive bacteria to coordinate several group behaviors, such as biofilm formation, virulence, and motility, when the bacterial cell density exceeds the thresholds. The determination of the AI-2 level is essential to understand the physiological and biochemical processes involved in bacterial communication. However, the current methods for AI-2 determination are complicated, time-consuming, and require costly equipment, such as a mass spectrometer (MS) or fluorescence detector (FLD). In this study, we present a new and easily applicable method for AI-2 determination. This method, based on the primary derivatization of AI-2 with 2,3-diaminonaphthalene (DAN), uses an affordable high-performance liquid chromatography (HPLC) instrument with a UV detector. Under optimized conditions, our method showed a good linearity (r2 = 0.999) and demonstrated the effective detection of AI-2 levels in various environmental samples, as follows: 0.38 (±0.05) μM for E. coli K12, 0.48 (±0.05) μM for Aeromonas sp. YB-2, 0.32 (±0.06) μM for the Enterobacter sp. YB-3, and 0.28 (±0.16) μM for activated sludge.