Discovery of Surface-Induced Resonance Shift of 4-Nitrophenol Enabling Direct Monitoring of an Enzymatic Reaction

Chromogenic and fluorogenic substrates are important for the detection of enzymatic activity. Conjugates of 4-nitrophenol are among the earliest investigated classes of molecules and have been used in diagnostic applications, including those for type-2 diabetes. The detection of 4-nitrophenol under physiological conditions enables real-time monitoring of glucosidase activity. This becomes possible when the resonance structure of 4-nitrophenol is altered, making it distinguishable from its precursor chromogenic substrate. One of the surface-modified silica materials, comprising an acetyl-protected mannoside along with precipitated byproducts such as acyl-urea and urea formed during carbodiimide coupling, induces a unique resonance shift in 4-nitrophenol upon interaction. Raman microscopic analysis can distinguish the phenol-type band at 1330 cm⁻¹ and the quinone-type band at 860 cm⁻¹, thereby enabling monitoring of the α-glucosidase reaction.