Label-Free Evaluation of Advanced Glycation End Products by a Comprehensive Biophysical Analysis

Nonenzymatic glycation is a post-translational modification of proteins, leading to the formation of advanced glycation end products (AGEs) implicated in diabetes, neurodegenerative disorders (NDDs), and aging-related complications. The sensitive and precise detection of protein-bound AGEs is essential for comprehending their pathological implications. Conventional detection methods for AGEs have several drawbacks, such as being expensive and time-consuming, complex sample preparation steps, etc. Further, AGEs' structural complexity and heterogeneity affect their accurate determination. Therefore, we introduce an autofluorescence-based methodology (LED-induced autofluorescence) for detecting and tracking the formation of AGEs on proteins using a low-power fiber-coupled near-UV LED (340 nm) and a blue LED (430 nm) as excitation sources covering the heterogeneous excitation maxima of multiple AGEs that form on proteins. The device captures the wide range of AGEs-specific autofluorescence signatures without the interference of protein autofluorescence, revealing their heterogeneous composition on proteins. The device demonstrated high sensitivity in detecting standard pentosidine (3.66 pg/μL) at 340 nm excitation, while enabling effective monitoring of the formation and progression of clinically relevant AGEs such as pentosidine, argpyrimidine, vesperlysine (A, B, and C), etc., generated on various structurally distinct glycated proteins in vitro. While noninvasive diabetes risk assessment and diagnosis based on AGEs fluorescence is gaining growing attention, the use of low-power, cost-effective light-emitting diodes (LEDs) as excitation sources offers significant advantages. Their stable power output ensures high reproducibility, enabling a rapid and reliable approach to assessing protein glycation, facilitating the routine monitoring of accumulation of AGEs, and advancing research addressing glycation-related complications in diabetes, aging, and NDDs.