Non-enzymatic electrochemical assay of N-acetyl-d-neuraminic acid through competitive chemoreceptor binding with (thiophen-3-yl)boronic acid†

Electroanalytical methods which can aid in the selective quantitation of saccharides such as the sialic acid N-acetyl-D-neuraminic acid (Neu5Ac) are very attractive due to their significance in a wealth of human diseases and food/nutritional products. Using cyclic voltammetry, boronic acid–diol recognition based on a redox indicator displacement assay (RIDA) strategy was exploited for non-enzymatic comparative electroanalysis of Neu5Ac vs. fructose using the redox active reporter Alizarin Red S (ARS). The concept has its foundation in the classical competition between an analyte and an indicator (ARS) for the same binding site on a host (boronic acid) molecule. The pH dependent assay employed first-time use of (thiophen-3-yl)boronic acid (TBA) as heterocyclic chemoreceptor. Electrochemistry of ARS in equilibrium with TBA resulted in proton coupled redox processes at −0.48 V (free ARS), −0.29 V (ARS–TBA boronate ester) and +0.51 V vs. Ag|AgCl (free ARS) correlating with ARS concentration in the TBA–ARS equilibrium or in competition equilibrium with a sugar species. Saccharide driven boronic acid displacement resulted in the reinstatement of the free ARS redox processes, forming the basis for the analytical signal. Voltammetry and optical investigations established the optimum conditions for Neu5Ac measurement relative to competing species such as fructose, enabling pH tunable ratiometric quantitation over the range 1–10 mM Neu5Ac (0.1 M sodium acetate buffer pH 5.6) with sensitivity 0.119 ± 0.009 μA mM⁻¹ and LOD 0.63 mM (using differential pulse voltammetry). The homogeneous studies paved the way for film formation and preliminary displacement testing when ARS was surface confined within a chitosan biopolymer layer on a glassy carbon electrode.