Triple-color emission ratiometric fluorescence in biochemical sensing analysis; principles, probe fabrication and applications

Triple-color emission ratiometric fluorescence sensing has emerged as a powerful tool in biochemical analysis, enabling a shift from semi-quantitative to fully quantitative and highly precise measurements. This review presents a comprehensive overview of the design principles, fabrication strategies, and practical applications of triple-color emission probes excited under single-wavelength excitation. By incorporating three distinct emission channels, these probes offer self-calibrating, interference-resistant, and sensitive detection with enhanced color discrimination. We categorize the fabrication strategies into one-, two-, and three-emitter designs, discussing the mode of action for each and how they ensure spectral stability, tunability, and robustness in multiplexed sensing. Special attention is given to how these systems outperform conventional binary systems in terms of sensitivity, dynamic range, and color hue variation. Recent advancements span metal ion detection, pH sensing, enzyme assays, antibiotic and biomarker analysis, explosives, and others. This review provides key insights and guidance for advancing next-generation quantitative biochemical sensing technologies.