An ultrasensitive FRET-based fluorescent low molecular weight heparin nanoprobe for quantifying heparanase activity

Heparanase (HPA) is a multifaceted endo-β-glucuronidase, and its dysregulation facilitates cancer metastasis. Developing techniques for fast and sensitively monitoring HPA enzymatic activity is crucial for searching for molecular therapies targeting HPA. Herein, we developed a novel fluorescence resonance energy transfer (FRET)-based nanoprobe AuNCs-LMWH-AuNRs, with AuNCs@GSH-cys and AuNRs/end-NH₂/side-SiO₂ attached to the non-reducing terminus and reducing terminus of low molecular weight heparin (LMWH), respectively. AuNCs@GSH-cys exhibited an absolute quantum yield of 1.1%. The absorption spectra of AuNRs/end-NH₂/side-SiO₂ (825 nm for maximum longitudinal absorption) and the emission spectra of AuNCs@GSH-cys (824 nm for maximum emission) were precisely overlapping, further enhancing the efficiency of FRET. In the presence of HPA, the LMWH nanoprobe exhibited an ultrasensitive response with excitation/emission wavelength (lambda (ex) = 560 nm, lambda (em) = 824 nm). The probe presented a wide linear dynamic detection range (LDR) of 0.125 ng/μL - 0.01 μg/μL in vitro with a limit of detection (LODs) of 82.15 pM (0.43 pg/μL). The excellent selectivity and good fluorescence turn-on efficiency of the probe made it possible for one-step detection of cellular heparanase activity. High throughput screening of HPA inhibitors also can be accomplished using the highly efficient LMWH nanoprobe.