Fluorescence Biosensor Based On Functional Biomolecules And Functional Nanomaterials

The covalent modification of histones by the post-translational modification(PTM)enzyme plays an important role in a variety of physiological processes,such as transcriptional activation/inactivation,chromosome packaging,and DNA damage/repair.Therefore,the analysis of PTM enzyme activity and high-throughput screening of its inhibitors are very important for clinical diagnosis,drug development and drug research.Glucose is the main source of energy in the organism,as well as important intermediate product in the metabolic process.Thus,it is important to develop a rapid and reliable biosensor for detecting glucose content in the process of disease prevention,clinical medicine and other processes.Functional biomolecules G-quadruplexes and supercharged green fluorescent proteins(ScGFPs)are widely used as a versatile molecular tool for highly sensitive detection of multiple analytes.Functional nano-materials gold clusters(AuNCs)have become a new class of nano-probes for the development of new sensing methods due to their ultra-small size,excellent light stability,strong luminous efficiency and good biocompatibility.In this paper,we have carried out a series of studies based on functional biomolecules G-quadruplexes,ScGFP and functional nanomaterials AuNCs,and then developed different fluorescent biosensors for the detection of PTM enzymes and glucose.The details are described below:1.To simulate the interactions between DNA and histone proteins,we propose a novel method for detecting acetylation-related peptides.The proposed method is based on the electrostatic interaction between G-quadruplexes(G4s)and acetylation-related peptides,which results in marked change of fluorescent intensity of G4/Thioflavin T(ThT)complexes.We selected a peptide derived from H4 N-terminal histone tail and thrombin-binding aptamer(TBA)as the study object.The average particle size of TBA/acetylation-related peptide complex was 195 ± 69 nm,characterized by dynamic light scattering.Competitive experiments by Heparin demonstrated that the main interaction between G4 and acetylated peptides was the electrostatic interaction.Then the feasibility of the proposed method was verified by hydrogel imaging,fluorescence detection and circular dichroism spectroscopy.These results show that the proposed bioassay is simple and convenient,and it is expected to be used as a novel biosensing platform for the detection of acetylation-related enzymes and screening their inhibitors.2.A label-free fluorescent method has been established for the homogeneous bioassay of histone acetyltransferases(HATs)and histone deacetylases(HDACs)activity and respective inhibitors.The proposed approach is primarily based on the electrostatic interaction between G-quadruplexes(G4s)and acetylation-related peptides,which results in marked change of fluorescent intensity of G4/Thioflavin T(ThT)complexes.This HAT(p300)activity assay is exceedingly sensitive and selective,with a linear range from 0.1 to 120 nM and a detection limit of 0.05 nM.Moreover,this biosensor is feasible to detect the HDAC(Sirtl)activity with a linear range from 1 to 450 nM and a detection limit of 1 nM.The potency of this assay is further demonstrated by detecting HAT/HDAC activity in cell lysates and evaluating HAT and HDAC-targeted inhibitors,C464 and EX 527,respectively.The proposed assay is convenient,label-free and cost-efficient,which is promising for HAT/HDAC-targeted epigenetic research and pharmaceutical development.3.We developed a novel fluorescent biosensor for sensitive and specific detecting the content of H2O2 and glucose,which is primarily based on the interaction between AuNCs and ScGFP.To the best of our knowledge,this work demonstrated for the first time that the AuNCs synthesized by CoA as ligands can effectively quench the fluorescence of ScGFP,and the hydroxyl radicals produced by Fenton reactions can effectively oxidize the synthesized AuNCs.Based on this principle,we proposed a novel fluorescent biosensor for detecting H2O2 content.In addition,glucose in the glucose oxidase catalysis,can produce H2O2.Then,we use this phenomenon to achieve the detection of glucose.The low detection limits of 0.1 mM and 0.05 mM were achieved for glucose and H2O2.The linear ranges of glucose and H2O2 were 0.1-5.0 mM and 0.1-2.0 mM,respectively.In addition,we tested the potential of our bio-sensors in practical samples by testing in 5%of human serum samples.The biosensors we propose are convenient,lable-free and highly sensitive,and are promising to be applied in biomedical analysis and clinical testing.