A ratiometric fluorescence biosensor based on upconversion nanoprobes for ultra-sensitive detection of plasmin activity.

Plasmin regulates fibrinolysis and haemostasis, and its abnormal fibrinolytic activity is closely associated with thrombotic and bleeding disorders. Many plasmin-like drugs are used for the treatment of thrombotic diseases. Therefore, it is crucial to evaluate the fibrinolytic activity of such drugs. Herein, a ratiometric fluorescence biosensor based on upconversion nanoprobes was constructed for quantitative analysis of fibrinolytic activity. Upconversion nanoprobes were functionalized with DSPE-PEG2000 and conjugated to a fibrinolytic-responsive peptide labeled with Cy5. Under 980 nm excitation, the fluorescence emission of upconversion nanoprobes at 654 nm was quenched by Cy5 based on fluorescence resonance energy transfer while the fluorescence emission at 541 nm was not affected. In the presence of plasmin, Cy5 was released due to enzymatic cleavage of the peptide, restoring the fluorescence of upconversion nanoprobes and producing a ratiometric fluorescence signal that correlated with the fibrinolytic activity. The assays demonstrated a linear response over 10-7 to 10-1 U mL-1 and a limit of detection of 2.04 × 10-8 U mL-1 with high specificity for the target enzyme and strong anti-interference performance in the presence of common biological matrix components. Moreover, this biosensor was used to evaluate the fibrinolytic activity of the active components in leech extract, providing an ultra-sensitive method for the activity evaluation of plasmin-like ingredients of Chinese medicine.