Development Of Enzymatic Cleavage-responsive Fluorescent Probes For DNA Methytransferase And Beta-amyloid Cleaving Enzyme-1 Assays

The bioprobe-based methods have the advantages such as good specificity,excellent accuracy and rapid response,and play an important role in human disease research.Due to their good water solubility,thermal stability,and ease of synthesis and modification,fluorescent probes have attracted more and more attention recently.Fluorescent probe-based sensors have been developed for use in a range of fields,including medical diagnostics,biochemical analysis,food safety,and environmental monitoring because of their high sensitivity and good selectivity.In the field of biochemical analysis,the detection of biomarkers is a powerful tool for understanding and treating various diseases,and is of great value for epidemiological investigation and analysis,disease screening,clinical diagnosis and prognosis.Therefore,we developed two biosensors based on enzyme cleavage responsive fluorescent probes using DNA methyltransferase（MTase）andβ-site amyloid precursor protein-cleaving enzyme 1（BACE-1）as models.Main research contents are as follows:（1）We developed a signal amplification method to detect DNA methylase based on probe CRISPR/Cas12a-mediated cleavage of double-labeled nucleic acid fluorescent.We designed a double-labeled nucleic acid fluorescent probe that can be non-specifically digested by double-stranded DNA（ds DNA）activated CRISPR/Cas12a effector.In the presence of DNA methyltransferase,the ds DNA substrate is methylated and it cannot be digested by Bss HII endonuclease.Subsequently,the ds DNA substrate can bind with g RNA to activate the Cas12a,resulting in the cleavage of fluorescent probes and the recovery of Cy5 fluorescence which can be accurately quantified by single-molecule imaging.The length of the double-labeled fluorescent probe is 5 nt,which ensures its high quenching efficiency and low background signal.The CRISPR/Cas12a effector can convert biometric recognition event into a detectable fluorescent signal,enabling the easy amplification of output signal.This assay exhibits good specificity and high sensitivity with a detection limit of 4.61×10^-4 U/m L,and it can be further applied for inhibitor screening,enzyme kinetic analysis,and the measurement of DNA MTase activity in human serum,holding great potential in clinical diagnosis,drug development,and drug evaluation.（2）We developed a fluorescent probes-modified gold nanoparticle（Au NP）nanosensors for in vitro detection and in vivo imaging of BACE-1.We designed two fluorescent probes,including a Cy5-modified peptide probe and a dark quencher 2（BHQ2）-labeled DNA probe.Through the self-assembly of both fluorophore-labeled peptide probes and quencher-labeled assistant DNA probes on the surface of a single Au NP,a superquenched Au NP nanoprobe is obtained with a near-zero background fluorescence.The presence of target BACE-1 induces a distinct fluorescence signal as a result of the BACE-1-catalyzed cleavage of peptide probe and the subsequent release of abundant fluorophore moieties from the Au NP nanoprobe.The fluorescence signal can be directly quantified by single-molecule counting.This nanosensor involves only a single nanoprobe for the one-step homogeneous detection of the BACE-1 activity without the requirements of any antibodies and separation steps,and it possesses good selectivity and high sensitivity with a low detection limit of 26.48 p M.Moreover,it can be employed to screen BACE-1 inhibitors and analyze kinetic parameters.Especially,this nanoprobe possesses good stability and can be easily transferred into live cells for the real-time imaging of cellular BACE-1 activity,providing a new platform for BACE-1 associated research and early diagnosis of Alzheimer′s disease.