Quantitative all-in-one high-performance thin-layer chromatography–planar start-zone enzyme assay applied for invertase activity

BACKGROUND: Conventional methods for assessing enzyme activity often rely on in vitro assays with spectrophotometric detection; however, they lack accuracy for complex substrates. The recently introduced nanoGIT concept demonstrated the potential of on-surface enzyme assays performed directly in the start zone, followed by high-performance thin-layer chromatography (HPTLC) of the formed products on the same adsorbent surface. For the first time, the potential of this all-in-one HPTLC concept was investigated for the quantification of enzyme (invertase) activity using a complex substrate. RESULTS: Enzymatic hydrolysis of sucrose or egg biscuit extract was carried out in the start zone under identical conditions for all studied samples. On the same adsorbent surface, the hydrolysis products were separated, detected via densitometric fluorescence measurement, and quantified. Even with complex substrates, the integrated separation provided accurate, in-depth information on enzyme characteristics from the individual products formed and their respective intensities. The method was validated and proved to be accurate and reliable in the nanomolar range, outperforming current spectrophotometric in vitro assays that operate in the millimolar range. The assay in the start zone exhibited remarkable sensitivity, requiring a 12,500-fold lower substrate quantity while using equivalent enzyme amounts. Limitations and benefits were discussed. SIGNIFICANCE: For the first time, a validated quantitative all-in-one HPTLC-start-zone enzyme assay-FLD method was successfully demonstrated for the determination of the enzyme activity. It enabled the quantification of even minor amounts of individual enzyme reaction products, providing improved sensitivity and more accurate insights into enzyme characteristics. Low enzyme levels can be analysed, extending current analytical capabilities.