TY - JOUR
T1 - Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media
AU - Zanetti, Chiara
AU - Li, Liang
AU - Gaspar, Rafael Di Lazaro
AU - Santovito, Elisa
AU - Elisseeva, Sophia
AU - Collins, Stuart G.
AU - Maguire, Anita R.
AU - Papkovsky, Dmitri B.
N1 - Publisher Copyright:
© 2024 by the authors.
PY - 2024/1
Y1 - 2024/1
N2 - Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The ‘shielded’ probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.
AB - Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The ‘shielded’ probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.
KW - bacterial cell respiration
KW - optical oxygen respirometry
KW - quenched-phosphorescence oxygen sensing
KW - selective media
KW - water-soluble oxygen probes
UR - https://www.scopus.com/pages/publications/85181950155
U2 - 10.3390/s24010267
DO - 10.3390/s24010267
M3 - Article
C2 - 38203132
AN - SCOPUS:85181950155
VL - 24
JO - Sensors
JF - Sensors
IS - 1
M1 - 267
ER -