Kitchawengkul N.Prakobkij A.Saenmuangchin R.Citterio D.Nacapricha D.Jarujamrus P.Mahidol University2025-04-022025-04-022025-07-15Sensors and Actuators B: Chemical Vol.435 (2025)09254005https://repository.li.mahidol.ac.th/handle/20.500.14594/108597A highly sensitive ratiometric fluorescence sensing system was developed for simultaneous glucose and total cholesterol (TC) detection in whole blood using MnFe-layered double hydroxides (MnFe-LDHs) as a peroxidase mimic, combined with an o-phenylenediamine (OPD) substrate and nitrogen-doped graphene quantum dots (N-GQDs). The detection platform, an X-shaped laminated microfluidic paper-based analytical device (XL-μPAD), was fabricated via laser printing and cutting. The MnFe-LDHs' large surface area and layered structure provide a high affinity for OPD, with a Michaelis–Menten constant (KM) of 0.0127 mmol L−1. Upon placing a drop of blood on the XL-μPAD sample pad, the enzymatic reactions of glucose and TC produce H2O2, which MnFe-LDHs convert to hydroxyl radicals (•OH). These radicals oxidize OPD into fluorescent 2,3-diamino phenazine (DAP) with emission at 560 nm. Meanwhile, the N-GQDs emit fluorescence at 415 nm, which is quenched by DAP through the inner filter effect (IFE) and dynamic quenching, enabling ratiometric sensing via the intensity ratio (I560/I415). As H2O2 levels increase, a visible green emission appears, correlating with glucose and TC levels. This XL-μPAD system demonstrates promising potential as a portable device for multiplex biomarker detection and diagnostic applications.Materials SciencePhysics and AstronomyEngineeringArticleSCOPUS10.1016/j.snb.2025.1376712-s2.0-105000988941