FRET-Based Nanoprobe with Adaptive Background Suppression for Reliable Detection of ONOO/ClO in Whole Blood: Facilitating Monitoring of Sepsis Progression and Hemolytic Disorders.

Abnormal fluctuations in blood biomarker levels serve as critical indicators of the disease. However, detecting endogenous substances in whole blood using fluorescent probes is challenging due to its complex composition. This challenge primarily arises from two factors: the high autofluorescence of whole blood and the intrinsic fluorescence of the probe, both contributing to significant background fluorescence in the detection system. To overcome these obstacles, we introduced a donor-acceptor "one-to-many" FRET-based sensing strategy integrated with blood autofluorescence suppression to design a multifunctional fluorescent nanoprobe. The donor effectively suppresses blood autofluorescence through the inner filter effect and efficiently quenches donor fluorescence by adjusting the acceptor-to-donor ratio, achieving a "zero" background in whole blood detection. Leveraging this excellent background fluorescence quenching effect, we successfully detected endogenous ONOO and ClO levels in whole blood samples from mice with sepsis or hemolytic diseases. Furthermore, we monitored the changes in the ONOO and ClO levels throughout the disease course, revealing a positive correlation between the ONOO and ClO concentrations and disease severity. This innovative sensing strategy for achieving a "zero" background in whole blood detection provides valuable insights for designing fluorescent probes to directly detect endogenous substances in whole blood.