Synthesis Of Peptide-functionalized MXene Nanoprobes And Their Applications In Biosensing

Two-dimensional nanomaterials are a class of materials,in which electrons can only move freely on two dimensional nanoscales（1-100 nm）.Graphene,molybdenum disulfide,carbon nitride nanosheets and other two-dimensional nanomaterials have been gradually discovered by researchers in the past decade,showing great application prospects in the field of biosensing and biomedicine due to their unique physical and chemical properties.In recent years,transition metal carbon/nitride（MXenes）have been prepared by etching and stripping MAX phase precursors with ceramic properties.As a representative MXene,titanium carbide（Ti₃C₂ MXene）is one of the most interesting multifunctional nanomaterials.The thickness of monolayer Ti₃C₂MXene is generally less than 1 nm,and its lateral size ranges from nanometers to micronmeters.It has large surface area,strong light absorption from ultraviolet to near-infrared regions,and its mass extinction coefficient is more than twice that of GO in the wavelength range of 400-1000 nm.Additionally,unlike graphene,molybdenum disulfide and C₃N₄ nanosheets and other hydrophobic two-dimensional materials,Ti₃C₂MXene has a large number of hydrophilic groups on the surface,such as-OH and-F,which contribute to a good dispersity in aqueous solution.These characteristics render Ti₃C₂ MXene with great advantages in the construction of fluorescent biosensors.Peptides are chain polymers of amino acids linked by amide bonds,which have the advantages of high stability,easy chemical synthesis and modification.By reasonably designing the amino acid sequence and chemical modification of peptides,they can specifically bind to target molecules or serve as the substrates of enzymes.In this study,based on the excellent physicochemical properties of Ti₃C₂ MXene,including broad and strong fluorescence quenching ability,high specific surface area,good hydrophilicity and biocompatibility,we attempted to construct peptide-functionalized Ti₃C₂ MXene nanoprobes,and explored its application potentials in biosensing and imaging.The specific research contents are as follows:1.Construction of peptide-functionalized Ti₃C₂ MXene based on a noncovalent strategy.In this section,monolayer Ti₃C₂ MXene was prepared by organic base etching,and its surface was modified with lecithin to enhance the stability of Ti₃C₂ MXene in complex physiological environments.We designed chimeric peptide probes consisting of a functional sequence and a membrane insertion peptide（MIP）sequence.The MIP sequence at the end of the probe binds the phospholipid bilayer to form?helix,and self-assembles on the surface of titanium carbide MXenes to form Ti₃C₂ MXene（Pep-Ti₃C₂）.The noncovalent peptide functionalization strategy developed in this section has the advantages of simplicity and speediness,and avoides complex multi-step covalent modification,which also provids a good biocompatible interface.2.Construction of Pep-Ti₃C₂ nanoprobes for the detection of post-translational modification enzyme activities.Post-translational modification（PTM）of proteins can fine-regulate the structure and function of proteins and play a crucial role in the life process.The abnormal activity of PTM enzymes is closely related to a series of serious diseases such as immune deficiency,neurodegeneration and cancer.Therefore,the development of analytical methods for PTM enzymes and inhibitor screening is of great significance in basic scientific research and clinical diagnosis.In this section,we designed chimeric peptides Pep1 and Pep3 responding to different PTM enzymes,which were assembled on the phospholipid modified Ti₃C₂ MXene surface through membrane insertion to form Pep-Ti₃C₂ nanoprobes.In these probes,the fluorescent groups modified on the polypeptides were efficiently quenched by Ti₃C₂.In the presence of PTM enzymes,they catalyze the removal of the modification groups;and then carboxypeptidase（CPY）hydrolyzes the polypeptides and releases the labeled fluorophores,leading to the recovery of their fluorescence,thus realizing the detection of the activity of PTM enzymes.Under the optimized experimental conditions,Pep-Ti₃C₂ nanoprobes could sensitively respond to histone deacetylase and protein phosphatase with detection limit of 0.1 n M and 2.3 n M,respectively.In addition,the nanoprobes can also be used for simultaneous analysis of the activities of these two PTM enzymes and their inhibitors in different cell lysates,providing a new method for highly sensitive detection of PTM enzymes in complex biological samples.3.Construction of nucleic acid/peptide functionalized Ti₃C₂MXene nanoprobes（DP-Ti₃C₂）for the simultaneous detection and imaging of Hepatitis C virus（HCV）RNA and protease in host cells.HCV is a typical plus-strand RNA virus of the flaviviridae,its replication and maturation in host cells depend on genomic RNA and NS3-4A serine protease.Therefore,simultaneous detection of HCV RNA genome and NS3-4A protease activity not only provides spatio-temporal information on genome replication and functional protein expression,but also offers a powerful tool for the development of antiviral drug.In this section,polyacrylic acid was used to modify Ti₃C₂ MXene,and DNA and peptide probes responding to HCV virus RNA and NS3-4A protease were simultaneously coupled on the surface of Ti₃C₂ MXene to form the DP-Ti₃C₂ nanoprobes.The results showed that DP-Ti₃C₂ probes could be used for simultaneous detection of HCV RNA and NS3-4A,as well as imaging these two viral biomarkers in host cells.Additionally,DP-Ti₃C₂ probes were employed to evaluate the inhibitory effect of VX-950 on HCV virus protease NS3-4A.In conclusion,the DP-Ti₃C₂ nanoprobes constructed in this section provide an effective tool for studying the virus infection process at the living cell level.