Studies On The Design And Antibacterial Mechanism Of Reticular Chemistry Derived Nanozymes Targeting Microenvironment Of Foodborne Pathogens

Food contamination and food-borne diseases caused by food-borne pathogens pose serious challenges to the food industry.In the post-antibiotic era,traditional chemical antibacterials not only cause persistent ecological hazards and pollution problems to the environment and food chain,but also may greatly increase the risk of microbial resistance,thereby losing effective control over microbial safety.The rapid development of nanochemistry brings promise for more efficient,safe,green and sustainable antibacterial technologies.Inspired by the antibacterial activity of natural enzymes,"nanozymes"with enzyme-like properties can effectively inactivate drug-resistant bacteria through sustained biomimetic catalytic reactions,and thus are expected to be a class of candidate bactericidal agents with long-lasting and safer bacteriostatic efficacy.Reticular chemistry is a versatile toolbox for the biomimetic design of metal-organic frameworks（MOFs）nanozymes,which have been widely used in chemical sensing,biomedicine,and food safety.In this thesis,reticular chemistry is used as a design tool,and strategies such as rational design,derivatization,and biohybridization of MOFs are adopted to improve the biomimetic catalytic,targeted antibacterial,and biosafe properties of antibacterial nanozymes.Several smart-responsive MOFs-derived nanozymes have been designed for the construction of precise nano-antibacterial systems to target the microenvironment of food-borne pathogens and control food microbial safety.The main research contents are as follows:（1）By proposing a reticular chemistry derived“quasi-MOF”（Q-MOF）scheme to rationally design the reactivity and functional adaptability of MOFs derived POD nanozymes,a new mechanism for targeted control of foodborne pathogens was constructed based on a light-regulated p H-responsive nanozyme reaction.The results showed that by combining MOF crystal engineering with a ligand-controlled decarboxylation strategy,we have successfully synthesized Q-MOF_Ce0.5 nanosheets with a hierarchical zero-dimensional/two-dimensional（0D/2D）heterojunction-like interface,which enabled rational arrangement of the node-derived isolated Ce-O-Cu sites on the 2D decarboxylated MOF framework,thus accommodating oxygen-vacancy-coupled multivalent redox cycling and photoactive energy band configuration.With these structural advantages,Q-MOF_Ce0.5processed much higher peroxidase（POD）-mimicking activity and visible-light-responsive activity over corresponding MOFs or metal oxide derivatives.Through light-regulated POD-like reactions and adhesion to bacteria surface,abundant·OH and ¹O₂ free radicals were generated under slight acidic environment,inducing destruction of bacterial cell membrane and programmed death of bacteria.The bacteriostatic rates against Escherichia coli O157:H7（E.coli）and Staphylococcus aureus（S.aureus）reached 99.74%and 99.35%,respectively.（2）Through using the reticular chemistry-based ligand engineering strategy,the dual apyrase-like and oxidase-like（OXD）properties,cascade effects,and structure-activity relationships of the isomorphic Ce-UiO-66 MOFs were explored,enabling a new mechanism for targeted control of foodborne pathogenic biofilms based on an adenosine triphosphate（ATP）-responsive self-cascading nanozyme reaction.The results showed that the as-prepared isomorphic Ce-UiO-66-X MOFs（X=BDC,BDC-CH₃,BDC-OH,BDC-NH₂,BDC-NO₂,ADC,Fum）exhibited ligand-dependent enzymatic activities,and the ATP/ADP/AMP/Pi reactants related to apyrase-like reaction can effectively accelerate the OXD-like reaction of Ce-UiO-66-X,which could be further intensified by the progress of ATP hydrolysis.Ce-UiO-66-X exhibited a special hydrolase-oxidoreductase self-cascade reaction mechanism,which could simultaneously consume ATP molecules and strengthen the generation superoxide radicals（·O₂^-）.Further,by selecting Ce-BDC-NO₂,Ce-BDC and Ce-BDC-NH₂ as typical nanozyme isoforms and using methicillin-resistant S.aureus（MRSA）as model bacteria,we revealed that Ce-UiO-66-NO₂ can effectively target and interfere with the metabolic microenvironment of MRSA biofilm,due to the nanocatalytic mechanism of synergistic ATP consumption and ROS generation.As a result,Ce-UiO-66-NO₂ nanozyme significantly inhibited the adhesion and formation of MRSA biofilm on food-contact surface,and reduced the MRSA viability within biofilm to 3.64%.（3）By combining reticular chemistry derived single-atom catalysts and protein engineering strategies,a new mechanism for targeted control of foodborne pathogenic biofilms based on a glucose-activated bio-cascaded single-atom nanozyme reaction was constructed.Single-atom nanozymes（SAzymes）were prepared by derivatization of Cu-MOFs,and functionalized with glucose oxidase（GOX）and concanavalin A（Con A）to create protein-directed biocascade single-atom nanozymes（Bio SAzymes）.The results showed that the prepared SAzyme exhibited excellent horseradish peroxidase（HRP）,glutathione peroxidase（GPx）and glutathione oxidase（GSHOx）-like activities.Based on this,Bio SAzyme was found to catalyze the generation of H₂O₂ and gluconic acid from glucose,which regulated the p H microenvironment of reaction and effectively triggered its HRP/GPx/GSHOx-like activities,thereby consuming glucose and glutathione（GSH）molecules and generating large amounts of·OH radicals.Under this cascade reaction mechanism,using E.coli and MRSA biofilms as models,we found that Bio SAzyme not only utilizes the bioaffinity of Con A to glycocalyx compounds,but also utilizes the triggering effects of bacterial physiologically relevant glucose molecules on the cascade reaction,achieving active capture and efficient elimination of E.coli and MRSA biofilms.As a result,Bio SAzyme effectively inhibited the accumulation of biomass of E.coli and MRSA biofilm,and reduced the corresponding bacterial survival rates to 0.36%and 1.47%,respectively.（4）Through using the reticular chemistry based biomineralization strategy,a cellulose-based enzyme-mimicking spray packaging was constructed for the research of biomimetic catalytic preservation of fruits.Using carboxymethylcellulose nanofibers（CNF）and Ce-UiO-66-BDC nanozymes as active ingredients,Ce-UiO-66 was grown on CNF in situ by a biomineralization method,and the enzymatic properties and antibacterial activities of the obtained CNF@MOF was investigated.The results showed that CNF@MOF nanozymes exhibited superior OXD-like and apyrase-like activities as compared to crystalline Ce-UiO-66-BDC nanozyme due to the amorphous state of the grown Ce-MOF particles and the coexistence of Ce³⁺/Ce⁴⁺valence states.CNF@MOF nanozymes can simultaneously generates·O₂^-radicals and consume ATP biomolecules.The antibacterial effects of CNF@MOF nanozymes were evaluated using three typical food-borne pathogenic bacteria as model bacteria,namely E.coli,S.aureus,and MRSA.It was found that CNF@MOF could inhibit bacterial growth up to 90.46%,86.91%and 82.9%,respectively,and the biocompatibility of CNF@MOF was revealed by cell physiological experiments.The colloid solution of CNF@MOF nanozyme was utilized and investigated as protective active spray coating against food spoilage using banana and mango as model fruits.It was found that the CNF@MOF coating could significantly improve quality of the coated banana and mango with regard to the appearance,weight loss rate and firmness of the fruit samples.This study pioneered the development of biocompatible nanozymes for active packaging materials and practices.（5）The evidence about biosafety and in vivo anti-infection of the reticular chemistry derived nanozymes（Q-MOF,Ce-UiO-66-X and Bio SAzyme）developed in this thesis was preliminarily explained through in vitro cell physiological evaluation models and in vivo mouse models.Through cytotoxicity experiment with NIH 3T3 cells,hemolysis experiment with mouse red blood cells,mouse wound anti-infection model,and physiological and histological analysis of experimental mouse,we not only revealed that the use of Q-MOF,Ce-UiO-66-X or Bio SAzyme will possibly not affect the normal physiologies of animal cells and mammals,but also preliminarily confirmed the adaptability of the above nanozymes to complex microbial environments.The results provided references for application of reticular chemistry derived nanozymes in the food safety field.