A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Siderophore Pyochelin.

The pyochelin (PCH) siderophore produced by the pathogenic bacterium is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production , PCH quantification in biological samples is problematic due to analytical complexity, requiring extraction from large volumes and time-consuming purification steps. Here, the construction of a bioluminescent whole cell-based biosensor, which allows rapid, sensitive, and single-step PCH quantification in biological samples, is reported. The biosensor was engineered by fusing the promoter of the PCH biosynthetic gene to the operon, and the resulting construct was inserted into the chromosome of the ΔΔΔ siderophore-null mutant. A bioassay was setup in a 96-well microplate format, enabling the contemporary screening of several samples in a few hours. A linear response was observed for up to 40 nM PCH, with a lower detection limit of 1.64 ± 0.26 nM PCH. Different parameters were considered to calibrate the biosensor, and a detailed step-by-step operation protocol, including troubleshooting specific problems that can arise during sample preparation, was established to achieve rapid, sensitive, and specific PCH quantification in both culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing strains on a solid medium.