Systematic Evaluation of a Two-Layer LF1/pH-Indicator Paper Assay for Serum Cholinesterase: Influence of Assay Conditions and Sample Background

Paper-based colorimetric devices could support decentralized screening of cholinesterase inhibition after organophosphate (OP) exposure. We systematically evaluated how substrate identity, concentration and volume, reagent hydration, reading time, glycerol addition, and serum volume affect color development in a two-layer platform comprising a glass-fiber (LF1) reaction layer and a pH-indicator detection layer. Acetylcholine chloride (AChCl), butyrylcholine chloride (BChCl), and butyrylcholine iodide were first screened in solution; AChCl and BChCl were then tested on paper with commercial serum and six individual human sera, and color was quantified as blue-channel intensity from scanned images of the pH-indicator. A working configuration using 8 µL of 60 mM substrate, with readings taken after 20 min, was selected based on the observed colorimetric response. AChCl and BChCl did not differ significantly under these conditions, and larger serum volumes gave no consistent improvement. The selected configuration was 4 µL of serum and 8 µL of 60 mM AChCl containing 5% (v/v) glycerol. Commercial serum-only controls without added substrate shifted the indicator from yellow to green, showing a sample-related contribution to the basal indicator color that may influence the final RGB signal. In a separate exploratory comparison using BChCl, blue-channel intensity was not significantly correlated with pre-treatment reference cholinesterase activity in the six human sera. These results provide a working configuration for further microdevice development and highlight the need to account for sample background.