A ratiometric electrochemical strategy for sensitive determination of Furin activity based on dual signal amplification and antifouling nanosurfaces

Developing a sensitive and accurate method for Furin activity is still the bottleneck for understanding the role played by Furin in cell-surface systems and even in Alzheimer's disease. In this work, a ratiometric electrochemical biosensor was developed for sensitive and accurate determination of Furin activity in the cell based on dual signal amplification stemming from a peptide with multiple response sites and the antifouling gold nano-bellflowers (GBFs). A new peptide, HS-CMRVRR↓YKDFDFG (P3), was designed for the first time to be selectively cleaved by Furin at site↓. More importantly, this peptide P3 constitutes three amino acid residues with the -COOH group subsequently used to bind with the response molecule of ferrocene, and can remarkably improve the determination sensitivity by about 2.3 fold. Meanwhile, GBFs stabilized by PEG were taken as a second element to magnify the signal of the ferrocene group via a large ratio surface area and good conductivity, as well as an antibiofouling nanosurface to reduce the biofouling of the electrode surface in cells. This double amplification strategy can greatly enhance the sensitivity of Furin detection by 6.5-fold, which is favorable for detection of low amounts of Furin. In addition, 5'-MB-GGCGCGA(T)13-SH-3' was co-assembled as an inner reference to provide a built-in element to correct the determination error resulting from a complicated analysis environment. Finally, this sensitive and accurate Furin biosensor was successfully applied to detect Furin activity in Furin overexpressed U251 and MDA-MB-468 cells. As far as we know, this is the first report to mention an electrochemical strategy to detect Furin activity in cells.