Deconvolution and Correction of the Inner Filter Effect in a Tri-Enzymatic Fluorescent Cascade for Ultra-Extended Cholesterol Quantification.

The sensing architecture leveraging a tri-component fluorescent bio probe (Cholesterol Oxidase-Horse Reddish Peroxidase-Ampliflu Red) drives a highly efficient enzymatic cascade for the acutely sensitive and selective spectrophotometric quantification of cholesterol. The reaction culminates in the HRP catalyzed oxidation of Ampliflu Red, generating an intense, quantifiable emission centered at λem = 585 nm and excitation at λex = 570 nm, whose intensity is stoichiometrically correlated with cholesterol concentration (c). Crucially, the inner filter effect, which typically imposes nonlinearity at high analyte concentrations, is mathematically deconvoluted and corrected. This computational approach drastically expands the linear dynamic range from 0.2 mg/dL to unprecedented 975 mg/dL, demonstrating a paradigm shift in overcoming fundamental limitations (encompassing concentrations beyond the typical > 375 mg/dL inflection point) and extending the analytical applicability of fluorescence biosensors into previously inaccessible high-concentration regimes.