Peptide-gold Nanozyme Construction And Its Applications In Dual-signal Rapid Detection Of Harmful Residues

Biomimetic inorganic nanozymes are a class of inorganic nanomaterials with enzyme-like catalytic activity.Compared with natural enzymes,nanoenzymes have the advantages of simple preparation,low cost,good stability,easy long-term storage.Due to their special shell-core structure,size effect,and surface effect,gold nanozymes not only exhibit excellent enzyme-like activity,but also have unique photophysical and chemical properties,such as electron leaping process of fluorescence emission due to discrete quantumization energy levels,thus showing strong photoluminescence（PL）.Therefore,the fluorescence emission spectra of gold nanozymes with different sizes and surface ligands can be tuned from the visible to near-infrared light range.Among them,rational ligand design is one of the effective ways to regulate the structure and performance of gold nanozymes.Given the the diversity of amino acid sequences,structures,and biological properties of peptides,the peptide-protected gold nanoenzymes are highly flexible in design and highly similar to natural enzymes in overall conformation,composition and chemical microenvironment,thus exhibiting excellent catalytic and optical properties,which may find applications in various practical detection,including heavy metals,biological substances,pesticides,drugs,etc.Herein,this thesis successfully synthesized gold nanozymes with both fluorescent and enzymatic properties,termed as CK-Au NE,using the designed peptide as a ligand template via a green synthesis method.By establishing a multi-enzyme cascade hydrogel system and combining the effects of the enzyme inhibitor method on the dual properties of CK-Au NE,OPs and NSAIDs can be detected by this dual-signal sensor to meet the requirements of rapid,quantitative,visualization and accurate analysis for real samples.The content mainly includes the following three parts:1.CK-Au NE with both highly efficient fluorescence and enzyme-like activity were synthesized by a green method using the designed peptide（CCYGGYRFKYRFK）as the ligand for the stabilization and reduction of Au ions with cysteine and tyrosine,respectively.Firstly,the size,shape,and valence states of CK-Au NE were characterized using transmission electron microscopy,dynamic light scattering,X-ray photoelectron spectroscopy;Secondly,the optimal excitation and emission wavelengths and fluorescence lifetime were tested by fluorescence spectrometer and steady-transient fluorescence spectrometer;Finally,the peroxidase-like activity was evaluated by3,3’,5,5’-tetramethylbenzidine（TMB）-H₂O₂ colorimetric method and the parameters of V_max and K_m for enzymatic reaction were also calculated.Meanwhile,Fe²⁺-mediated Fenton reaction was investigated to confirm that the generation of·OH by H₂O₂ cause the fluorescence quenching of CK-Au NE.2.Colorimetric and fluorometric dual-signal detection method was established based on the cascade reaction of acetylcholinesterase（ACh E）/choline oxidase（Ch Ox）.The detailed mechanism is that the inhibition of ACh E by OPs is able to affect the end product（H₂O₂）of ACh E/Ch Ox reaction,which in turn regulate the color development of CK-Au NE-catalyzed TMB reaction and quench the fluorescence of CK-Au NE by·OH via Fe²⁺-catalyzed Fenton reaction.The linear detection range,minimum detection limit,and anti-interference ability of the method were evaluated for different kinds of OPs（e.g.chlorpyrifos,dimethoate,methyl paraoxon）.In addition,CK-Au NE can be further immobilized in sodium alginate hydrogel films to construct a multi-enzyme cascade dual-signal sensor based on the above-mentioned detection principle,and its"naked eye"detection range and minimum detection limit were determined by smartphone-assisted digital colorimetry,which can be applied to the rapid trace detection of OPs in fruits and vegetables.The accuracy of this detection method was verified by High Performance Liquid Chromatography-Mass Spectrometry.3.A dual-signal detection method based on the cascade reaction of lactate dehydrogenase/glutathione reductase/glutathione peroxidase（LDH/GR/GPx）was developed to regulate the enzymatic and fluorescence properties of CK-Au NE through the inhibition of LDH by NSAIDs（e.g.aspirin,ibuprofen,nimesulide,and indomethacin）,which affects the end product（H₂O₂）of LDH/GR/GPx cascade reaction.The linear detection range,minimum detection limit,selectivity and anti-interference ability of this detection method were determined respectively.Using sodium alginate as the solid phase carrier and CK-Au NE as the catalytic element,a dual-signal sensor was constructed by enzyme immobilization to realize the highly sensitive and rapid“naked eye”detection of aspirin via the combination of smartphones and Image J software to analyze the gray value,which can be used in trace detection of NSAIDs in milk samples.