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A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Pseudomonas aeruginosa Siderophore Pyochelin
ACS Sensors ( IF 8.2 ) Pub Date : 2021-09-03 , DOI: 10.1021/acssensors.1c01023 Daniela Visaggio 1, 2 , Mattia Pirolo 1 , Emanuela Frangipani 3 , Massimiliano Lucidi 1 , Raffaella Sorrentino 4 , Emma Mitidieri 4 , Francesca Ungaro 5 , Andrea Luraghi 6 , Francesco Peri 6 , Paolo Visca 1, 2
ACS Sensors ( IF 8.2 ) Pub Date : 2021-09-03 , DOI: 10.1021/acssensors.1c01023 Daniela Visaggio 1, 2 , Mattia Pirolo 1 , Emanuela Frangipani 3 , Massimiliano Lucidi 1 , Raffaella Sorrentino 4 , Emma Mitidieri 4 , Francesca Ungaro 5 , Andrea Luraghi 6 , Francesco Peri 6 , Paolo Visca 1, 2
Affiliation
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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.
中文翻译：
用于铜绿假单胞菌铁载体绿脓杆菌微量检测的高灵敏度发光生物传感器
由病原菌铜绿假单胞菌产生的绿脓杆菌 (PCH) 铁载体是一种重要的毒力因子，在感染过程中充当生长促进剂。虽然体内PCH 生产存在强有力的证据，但由于分析的复杂性，生物样品中的 PCH 量化存在问题，需要从大量提取和耗时的纯化步骤。在这里，报告了一种基于生物发光全细胞的生物传感器的构建，该传感器允许在生物样品中进行快速、灵敏和单步 PCH 量化。该生物传感器通过熔合PCH生物合成基因的启动子工程化PCHE到luxCDABE操纵子，并将得到的构建体插入到 Δ pvdA Δ pchD Δ fpvA铁载体无效铜绿假单胞菌突变体的染色体中。在 96 孔微孔板格式中设置了生物测定，能够在几个小时内对多个样品进行现代筛选。观察到高达 40 nM PCH 的线性响应，检测下限为 1.64 ± 0.26 nM PCH。考虑了不同的参数来校准生物传感器，并建立了详细的分步操作协议，包括解决样品制备过程中可能出现的特定问题，以实现铜绿假单胞菌的快速、灵敏和特定的 PCH 定量培养上清液和生物样品。该生物传感器被用作筛选工具，以检测固体培养基上产生 PCH 的铜绿假单胞菌菌株。
更新日期：2021-09-24
中文翻译：
用于铜绿假单胞菌铁载体绿脓杆菌微量检测的高灵敏度发光生物传感器
由病原菌铜绿假单胞菌产生的绿脓杆菌 (PCH) 铁载体是一种重要的毒力因子，在感染过程中充当生长促进剂。虽然体内PCH 生产存在强有力的证据，但由于分析的复杂性，生物样品中的 PCH 量化存在问题，需要从大量提取和耗时的纯化步骤。在这里，报告了一种基于生物发光全细胞的生物传感器的构建，该传感器允许在生物样品中进行快速、灵敏和单步 PCH 量化。该生物传感器通过熔合PCH生物合成基因的启动子工程化PCHE到luxCDABE操纵子，并将得到的构建体插入到 Δ pvdA Δ pchD Δ fpvA铁载体无效铜绿假单胞菌突变体的染色体中。在 96 孔微孔板格式中设置了生物测定，能够在几个小时内对多个样品进行现代筛选。观察到高达 40 nM PCH 的线性响应，检测下限为 1.64 ± 0.26 nM PCH。考虑了不同的参数来校准生物传感器，并建立了详细的分步操作协议，包括解决样品制备过程中可能出现的特定问题，以实现铜绿假单胞菌的快速、灵敏和特定的 PCH 定量培养上清液和生物样品。该生物传感器被用作筛选工具，以检测固体培养基上产生 PCH 的铜绿假单胞菌菌株。
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