Fluorescence Method For Simultaneous Detection Of Two Apoptosis-associated Proteases

The function Proteases are a large group of enzymes that selectively hydrolyze peptide bonds within proteins and polypeptides.The human genomes encode approximately 600different proteases which regulate virtually all biological pathways and networks.Furthermore,protease activities are associated with a variety of diseases and even cancers.Therefore,it is of great significance to develop highly sensitive and selective protease assays.Conventional methods for proteases assay include enzyme-linked immune sorbent assay（ELISA）,western blot,flow cytometry,and mass spectrometry.They usually involve multi-step and time-consuming processes,and extensive pre-treatment of samples.Recently,a variety of new methods have been developed for in vitro and in vivo detection of proteases,including electrochemical,colorimetric and fluorescence measurements.Most of these methods rely on either antibodies or peptide substrates.The main limitation of antibody-based assays is the requirement for high quality antibodies.Alternatively,peptide substrates are more compelling choice for protease assay with distinct advantages of accessibility,simplicity,cost-effectiveness,and chemical definition.In the peptide substrate-based assays,peptide substrates are often labelled with fluorophores and the fluorescence signals are directly measured after protease digestion,resulting in the limited sensitivities.The development of new methods for sensitive protease assay is highly desirable.Herein,we developed a sensitive nanosensor based on the integration of exonuclease Ⅲ（Exo Ⅲ）-powered three-dimensional（3-dimension,3D）DNA walker with single-molecule detection for simultaneous measurement of proteases using initiator caspase-8 and caspase-9 as model targets.Extrinsic apoptotic pathway and intrinsic apoptotic pathway are the only two pathways of apoptosis,and caspase-8 and caspase-9 are critical participants in the extrinsic and intrinsic apoptotic,respectively.The simultaneous detection of multiple initiator caspases is essential to apoptosis mechanism study and disease therapy.This assay involves two peptide–DNA detection probe-conjugated magnetic beads and two signal probe-conjugated gold nanoparticles（signal probes@AuNPs）.The presence of caspase-8 and caspase-9 can induce the peptide cleavage in two peptide–DNA detection probes,releasing two trigger DNAs from the magnetic beads,respectively.The two trigger DNAs can serve as the walker DNAs,respectively,to walk on the surface of signal probes@AuNPs powered by Exo Ⅲ digestion,liberating numerous Cy5 and Texas Red fluorophores which can be quantified by single-molecule detection,with Cy5 indicating caspase-8 and Texas Red indicating caspase-9.Notably,the introduction of magnetic separation into AuNPs-based 3D DNA walker greatly reduces the background signal,and the introduction of single-molecule detection further improves the detection sensitivity.This nanosensor is very sensitive with a detection limit of 2.08×10^-6 U/μL for caspase-8 and 1.71×10^-6U/μL for caspase-9,and it can be used for simultaneous screening of caspase inhibitors and measurement of endogenous caspase activity in various cell lines at the single-cell level.Moreover,this nanosensor can be extended to detect various proteases by simply changing the peptide sequences of detection probes.