Ratiometric fluorometry on microfluidic paper-based analytical device for simultaneous glucose and cholesterol detection using MnFe-layered double hydroxides as peroxidase mimic

A 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 (K_M) 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.