Lecture
Identification of bioactive substances using Thin Layer Chromatography coupled bioassays - A Practical Journey in Industrial PhD Research
- at -
- ICM Saal 4b
- Type: Lecture
Lecture description
CC Clappier Ludwigshafen/DE, MS Silber Ludwigshafen/DE, KS Schneider Ludwigshafen/DE, GB Baier Ludwigshafen/DE, BV Vacano Ludwigshafen/DE, SK Kolle Ludwigshafen/DE, GF Fricker Heidelberg/DE
This study aims to identify bioactive substances by combining thin-layer chromatography (TLC) with suspect-targeted bioassays for genotoxic, endocrine-active, and antimicrobial activity. Bioactive substances that persist in industrial products—e.g., recycling streams
or fermentation outputs—can pose health risks. Detecting them is challenging because recycling streams typically contain complex mixtures of polymers, additives and nonintentionally added substances (NIAS), and recycling processes can chemically transform constituents, further complicating sample composition and analysis. Consequently, it is often unclear whether bioactive substances are present and, if so, which compounds are responsible. To address these challenges, we established a TLCcoupled SOS-UmuC assay for genotoxicity screening. The assay reliably detected reference genotoxins such as 4-nitroquinoline-1-oxide (4-NQO) and aflatoxin B1 at nanomolar to picomolar concentrations. Metabolic activation is simulated in the assay using rat liver S9 mix, allowing detection of metabolites with genotoxic potential. For endocrine activity we evaluated genetically engineered yeast reporter strains, and for antimicrobial activity we implemented a TLC-coupled MTT assay.
TLC-coupled bioassays are well suited to non-targeted screening because chromatographic separation reduces mixture complexity and preserves spatial separation of components on the plate. Applying a biological test directly to the plate enables simultaneous bioactivity screening of all separated components. Bioactive zones can then be targeted for solvent elution and follow-up structure elucidation by LC-MS and GC-MS, enabling identification of the compounds responsible for the biological effects.
Limit-of-detection experiments indicate that the fluorescence-based TLC-SOS-UmuC assay is more sensitive for many genotoxins than conventional colorimetric bioassays performed in microwell plates. However, when applied to samples from a simulated mechanical recycling process, the TLC-SOS-UmuC assay did not detect genotoxic substances directly on the plates. Notably, a 4-NQO spike in the output material was identified by LC-MS/MS after solvent elution of the corresponding genotoxic band, demonstrating that the overall workflow—TLC separation, bioassay localization, fraction elution and instrumental analysis—works in principle, even if direct on-plate detection has not yet been achieved for complex recycling samples.
This study aims to identify bioactive substances by combining thin-layer chromatography (TLC) with suspect-targeted bioassays for genotoxic, endocrine-active, and antimicrobial activity. Bioactive substances that persist in industrial products—e.g., recycling streams
or fermentation outputs—can pose health risks. Detecting them is challenging because recycling streams typically contain complex mixtures of polymers, additives and nonintentionally added substances (NIAS), and recycling processes can chemically transform constituents, further complicating sample composition and analysis. Consequently, it is often unclear whether bioactive substances are present and, if so, which compounds are responsible. To address these challenges, we established a TLCcoupled SOS-UmuC assay for genotoxicity screening. The assay reliably detected reference genotoxins such as 4-nitroquinoline-1-oxide (4-NQO) and aflatoxin B1 at nanomolar to picomolar concentrations. Metabolic activation is simulated in the assay using rat liver S9 mix, allowing detection of metabolites with genotoxic potential. For endocrine activity we evaluated genetically engineered yeast reporter strains, and for antimicrobial activity we implemented a TLC-coupled MTT assay.
TLC-coupled bioassays are well suited to non-targeted screening because chromatographic separation reduces mixture complexity and preserves spatial separation of components on the plate. Applying a biological test directly to the plate enables simultaneous bioactivity screening of all separated components. Bioactive zones can then be targeted for solvent elution and follow-up structure elucidation by LC-MS and GC-MS, enabling identification of the compounds responsible for the biological effects.
Limit-of-detection experiments indicate that the fluorescence-based TLC-SOS-UmuC assay is more sensitive for many genotoxins than conventional colorimetric bioassays performed in microwell plates. However, when applied to samples from a simulated mechanical recycling process, the TLC-SOS-UmuC assay did not detect genotoxic substances directly on the plates. Notably, a 4-NQO spike in the output material was identified by LC-MS/MS after solvent elution of the corresponding genotoxic band, demonstrating that the overall workflow—TLC separation, bioassay localization, fraction elution and instrumental analysis—works in principle, even if direct on-plate detection has not yet been achieved for complex recycling samples.
