Colorimetric Detection of Salivary α-Amylase using Maltose as Inhibitor for Polysaccharide Cleavage.

This paper describes an approach for colorimetric detection of salivary α-amylase, one of the potential biomarkers of autonomic nervous system (ANS) activity, for enabling assessment of fatigue. The ability of α-amylase to cleave α-bonds of polysaccharides is utilized for developing a colorimetric assay. In the proposed approach, 2-chloro-4-nitrophenyl-α-D-maltotrioside as substrate releases a colored by-product upon cleavage by salivary α-amylase. Introduction of maltose as a non-competitive inhibitor yields desirable linear responses in the physiologically relevant concentration range (20-500 μg/mL) with a limit of detection (LOD) of 8 μg/mL (in aqueous solution). The concentrations of substrate and non-competitive inhibitor are subsequently optimized for colorimetric detection of salivary α-amylase. A facile paper-based "strip" assay is proposed for analysis of human saliva samples with marginal interference from saliva components. The proposed assay is rapid, specific and easy-to-implement for colorimetric detection of salivary α-amylase between 20-500 μg/mL. Complementary RGB (red, green, blue components) analysis offers quantitative detection with a LOD of 11 μg/mL. The two assay formats are benchmarked against the Phadebas test, a state of art method for spectrophotometric detection of α-amylase. The reported paper-based methodology possesses a high potential for estimation of altered ANS responses towards stressors that possibly could find applications in assessment of fatigue and for monitoring onset of fatigue.