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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Enzyme Assays

Background:

• Conventional enzyme activity assays often use spectrophotometric detection, which struggles with accuracy for complex substrates.
• The nanoGIT concept pioneered on-surface enzyme assays in the start zone, followed by HPTLC separation on the same surface.
• This study explores the all-in-one HPTLC concept for quantifying enzyme activity with complex substrates.

Purpose of the Study:

• To investigate the potential of an all-in-one HPTLC concept for enzyme activity quantification.
• To develop a method for analyzing enzyme activity using complex substrates like sucrose or egg biscuit extract.
• To validate a novel assay for enzyme activity determination.

Main Methods:

• Enzymatic hydrolysis was performed directly in the start zone of an HPTLC plate.
• Hydrolysis products were separated, detected using densitometric fluorescence measurement, and quantified on the same adsorbent surface.
• The method involved high-performance thin-layer chromatography (HPTLC) coupled with fluorescence detection (FLD).

Main Results:

• The all-in-one HPTLC assay accurately quantified enzyme (invertase) activity with complex substrates.
• The method demonstrated high sensitivity, operating in the nanomolar range and outperforming millimolar range spectrophotometric assays.
• A 12,500-fold lower substrate quantity was required compared to conventional methods, with equivalent enzyme amounts.

Conclusions:

• A validated quantitative all-in-one HPTLC-start-zone enzyme assay-FLD method was successfully developed.
• The assay enables precise quantification of individual enzyme reaction products, offering improved sensitivity and enzyme characteristic insights.
• This method extends analytical capabilities for analyzing low enzyme levels and complex biological samples.