Near-Infrared Fluorescence Hydrogen Peroxide Assay for Versatile Metabolite Biosensing in Whole Blood

Abstract

In emergency medicine, blood lactate levels are commonly measured to assess the severity and response to treatment of hypoperfusion-related diseases (e.g., sepsis, trauma, cardiac arrest). Clinical blood lactate testing is conducted with laboratory analyzers, leading to a delay of three hours between triage and lactate result. Here we describe a fluorescence-based blood lactate assay, which could be utilized for bedside testing, based on measuring the hydrogen peroxide generated by the enzymatic oxidation of lactate. To establish a hydrogen peroxide assay, near-infrared cyanine derivatives were screened and sulfo-cyanine 7 was identified as a new HRP substrate, which loses its fluorescence in presence of HRP and hydrogen peroxide. As hydrogen peroxide is rapidly cleared by erythrocytic catalase and glutathione peroxidase, sulfo-cyanine 7, HRP, and lactate oxidase were encapsulated in a liposomal reaction compartment. In lactate-spiked bovine whole blood, the newly developed lactate assay exhibited a linear response in a clinically relevant range after 10 min. Substituting lactate oxidase with glucose and alcohol oxidase allowed for blood glucose, ethanol, and methanol biosensing, respectively. This easy-to-use, rapid, and versatile assay may be useful for the quantification of a variety of enzymatically oxidizable metabolites, drugs, and toxic substances in blood and potentially other biological fluids.