Hydrogen peroxide (H2O2) is associated with diseases and food safety. Thus, it is essential to achieve sensitive and efficient detection of H2O2. Herein, a ratiometric luminescence composite nanoprobe was designed for single particle imaging sensing of H2O2 by combining NaYbF4:Er@NaYbF4:Tm@NaGdF4:Yb upconversion nanoparticles (UCNPs) with cyanine dye IR-628. High brightness NaYbF4:Er@NaYbF4:Tm@NaGdF4:Yb UCNPs with double-peak emission (665 and 812 nm) were synthesised. Cyanine dye IR-628 with an absorption peak at 628 nm was synthesised and served as a recognition unit. More importantly, on the basis of the upconversion luminescence total internal reflection imaging technique, we developed a ratiometric single particle imaging quantitative analysis for sensing H2O2 with a limit of quantitation of 5 nM. This ratiometric single particle imaging method not only greatly eliminates the influence of the probe concentration and instrumental and environmental factors, but also reduces the dosage of the reagent used and improves the sensitivity of detection.
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