HPTLC-coupled bioassays

Test organism

Detectable effects (impact)

  • Several endpoints can be combined with high performance thin-layer chromatography (HPTLC). The Ecotox Centre routinely evaluates inhibition of bacterial luminescence, activation of estrogen receptor (yeast estrogen screen), activation of DNA repair pathways (umuC SOS response test)
  • The effects of multiple separated chemicals are detectable simultaneously, linking to individual chemicals or giving a profile of bioactivity

Test principle

  • Chemicals in a sample are first chromatographically separated from each other on a HPTLC plate.
  • Microorganisms of a desired test system are applied to the plate containing the separated chemicals.
  • The plate is sprayed with the substrate, which, in the bioassay, leads to a color change or luminescence.
  • The test organisms respond to bioactive chemicals by reduced luminescence or induction of a color change, thereby indicating the location of the chemicals on the HPTLC plate.
  • The chromatographic behavior of chemicals can be compared between samples, and to standard chemicals.
  • The bioactive bands are extracted from the plate and used for further biological or chemical analyses. For example, the identity of the bioactive substances can be clarified using high-resolution mass spectrometry (LC-HRMS/MS).

Test duration

  • 1-3 d (exposure time: 0.5 - 3 h)

Relevance

  • Can detect multiple, separated, chemicals simultaneously.
  • Can reduce impact of matrix on the test’s specific effect (for example, masking from cytotoxicity).
  • HPTLC assays are usually more sensitive than the microtiter based counterparts.

Guidelines and literature

  • Bergmann, A. J., Simon, E., Schifferli, A., Schönborn, A., & Vermeirssen, E. L. M. (2020). Estrogenic activity of food contact materials - evaluation of 20 chemicals using a yeast estrogen screen on HPTLC or 96-well plates. Analytical and Bioanalytical Chemistry, 412, 4527-4536. doi.org/10.1007/s00216-020-02701-w 
  • Bergmann, A. J., Breitenbach, M., Muñoz, C., Simon, E., McCombie, G., Biedermann, M., Schönborn, A., Vermeirssen, E. L. M. (2023). Towards detecting genotoxic chemicals in food packaging at thresholds of toxicological concern using bioassays with high-performance thin-layer chromatography. Food Packaging and Shelf Life, 36, 101052 (11 pp.). doi.org/10.1016/j.fpsl.2023.101052 
  • Bergmann, A. J., Masset, T., Breider, F., Dudefoi, W., Schirmer, K., Ferrari, B. J. D., & Vermeirssen, E. L. M. (2024). Estrogenic, genotoxic, and antibacterial effects of chemicals from cryogenically milled tire tread. Environmental Toxicology and Chemistry. doi.org/10.1002/etc.5934