A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Pseudomonas aeruginosa Siderophore Pyochelin
| Autor: | Francesca Ungaro, Raffaella Sorrentino, Mattia Pirolo, Daniela Visaggio, Emma Mitidieri, Andrea Luraghi, Massimiliano Lucidi, Paolo Visca, Francesco Peri, Emanuela Frangipani |
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| Přispěvatelé: | Visaggio, D, Pirolo, M, Frangipani, E, Lucidi, M, Sorrentino, R, Mitidieri, E, Ungaro, F, Luraghi, A, Peri, F, Visca, P, Visaggio, D., Pirolo, M., Frangipani, E., Lucidi, M., Sorrentino, R., Mitidieri, E., Ungaro, F., Luraghi, A., Peri, F., Visca, P. |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Siderophore
siderophore Operon Mutant Siderophores Bioengineering Biosensing Techniques medicine.disease_cause biosensor Article Biosensing Technique Phenols medicine Bioassay Bioluminescence Instrumentation Fluid Flow and Transfer Processes Phenol Pseudomonas aeruginosa Chemistry Process Chemistry and Technology luciferase bioluminescence pyochelin Thiazoles Biochemistry Bioreporter Thiazole Biosensor |
| Zdroj: | ACS Sensors |
| Popis: | The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production in vivo, 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 pchE to the luxCDABE operon, and the resulting construct was inserted into the chromosome of the ΔpvdAΔpchDΔfpvA siderophore-null P. aeruginosa 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 P. aeruginosa culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing P. aeruginosa strains on a solid medium. |
| Databáze: | OpenAIRE |
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