Cerium/Gadolinium-Based Phosphatase-Like Nanozymes:Design,Ssynthesis,Catalytic Performance And Biological/Sensing Applications

Nanozymes have emerged as a new generation of artificial enzymes with remarkable properties such as high structural stability,controllable catalytic activity,functional diversity,recyclability,and scalability.They offer a promising solution to overcome the inherent limitations of traditional natural enzymes.Despite their wide-ranging applications in analytical detection,biomedicine,environmental remediation,and agriculture,most of the recent research efforts have been focused on oxidation and reduction type nanozymes,while hydrolytic nanozymes have received less attention.Phosphatase-like nanozymes,a type of enzyme-like material that can hydrolyze the phosphate ester bond,are expected to become an important branch of nanozyme research.However,the current research on phosphatase-like nanozymes is still in its infancy,and many issues remain unresolved,such as the limited variety of materials,low catalytic activity,insufficient understanding of the catalytic mechanisms,and simple application methods.Therefore,constructing new types of high-activity phosphatase-like nanozymes,exploring their catalytic mechanisms,and expanding their applications is crucial for enriching the development of nanozymes and deepening our understanding of them.This thesis aims to design and synthesize new phosphatase-like nanozymes,study surface/interface catalysis mechanisms,and develop biological and biosensor applications.The specific contents of the research are discussed below.1.This chapter presents an overview of the development of nanozymes,followed by a review of the current classification,activity regulation,and application status of phosphatase-like nanozymes.Moreover,this chapter critically analyzes the strengths and weaknesses of the current development of phosphatase-like nanozymes and provides a preliminary outlook on their potential future development.Based on this analysis,the chapter proposes a research theme for this thesis.2.Surface functional groups have a significant impact on the catalytic performance of phosphatase-like nanozymes,and a deeper understanding of their surface properties is helpful for the design and synthesis of high-performance phosphatase-like nanozymes.In this chapter,three porous hydroxide metal oxides,Zr OOH,Gd OOH,and Hf OOH,with significant phosphatase-like catalytic activity were synthesized at room temperature by using the metal-organic framework material M-BDC（M=Zr,Gd,Hf;BDC=terephthalic acid）as a template and replacing the BDC ligand with strong base hydroxide ions.The phosphatase-like catalytic activity of the synthesized Zr OOH,Gd OOH,and Hf OOH materials was studied and verified in depth.In particular,through isotope tracing,combined with high-resolution mass spectrometry and nuclear magnetic resonance technology experiments,it was found that a large number of surface hydroxyl groups of the synthesized hydroxide metal oxides can directly participate in the nucleophilic attack of the positively charged phosphorus atom in the phosphate ester,accelerating the cleavage of the phosphate ester bond at the end of the substrate molecule.In addition,it was found that Cr（III）ions,as"poison"inhibitors,can effectively inhibit the phosphatase-like catalytic activity of Gd OOH.Based on this effect,a Cr（III）ion colorimetric biosensing system was constructed.The results demonstrate the huge potential of Zr OOH,Gd OOH,and Hf OOH as phosphatase-like nanozymes and deepen our understanding of the role of surface hydroxyl groups in the catalytic dephosphorylation of phosphatase-like nanozymes.3.Using high-performance phosphatase-like nanozymes to control the ATP dephosphorylation metabolism of yeast cells has the potential to increase ethanol production.In this chapter,dextran and gadolinium salts were used as reaction materials to successfully synthesize dextran-coated nanogadolinia（DCNG）nanoparticles.The study showed that the prepared DCNG has good phosphatase-like catalytic activity,which can selectively catalyze the hydrolysis of the high-energy phosphate bond at the end of ATP under physiological conditions,thus achieving the catalytic reaction of ATP dephosphorylation.In addition,the special amorphous ultrafine structure of DCNG and the good biocompatibility of natural dextran make it easily absorbed by yeast cells through endocytosis.As a phosphatase-like nanozyme,DCNG accelerates the consumption of ATP in yeast cells through dephosphorylation hydrolysis,thus lowering the ATP level in yeast cells by artificial intervention and increasing the ethanol production of yeast cells.This research result is expected to provide new ideas for expanding the potential biological applications of nanozymes and to provide new directions for the deep application development of phosphatase-like nanozymes.4.Phosphatase-like nanozymes facilitate the hydrolysis of specific metabolic products in cells,enhancing the ferroptosis effect.In this chapter,we induced the synthesis of L-arginine（L-Arg）Ce O₂ ultrasmall nanoparticles with uniform morphology,high（111）crystal surface exposure,and good dispersion under solvothermal conditions.These nanoparticles exhibited excellent phosphatase-like activity.We studied the dephosphorylation and hydrolysis of physiologically active metabolites,nicotinamide adenine dinucleotide phosphate（NADP（H））and glucose-6-phosphate（G6P）,by L-Arg Ce O₂ phosphatase-like nanozyme.In vitro and cellular-level experiments showed that L-Arg Ce O₂ could lower the intracellular NADP（H）level through dephosphorylation,thereby inhibiting the regeneration synthesis of reduced glutathione（GSH）,reducing the consumption of reactive oxygen species（ROS）by GSH,enhancing the oxidative stress response,and ultimately promoting the ferroptosis of cells.This study explored the regulation of the intracellular NADP（H）level by Ce O₂ phosphatase-like nanozymes to affect cell ferroptosis from the perspective of cell phosphoric ester metabolism.It indicates that Ce O₂ phosphatase-like nanozyme can indirectly increase the intracellular ROS level by regulating specific metabolic products,rather than directly producing ROS through the catalysis of nanozymes themselves.The results of this study may provide new ideas and insights for the biological effects and related applications of Ce O₂ phosphatase-like nanozymes.5.Constructing a colorimetric sensing platform is an important application of phosphatase-like nanozymes,and non-redox colorimetric sensing is expected to overcome the interference of reducible substances in actual samples.In this chapter,L-Arg Ce O₂ nanoparticles with light color,uniform particle size,ultra-small structure,and good water dispersibility,synthesized in Chapter 4,were used to construct a colorimetric sensing method based on phosphatase-like nanozymes.Unlike previous research on colorimetric sensing based on Ce O₂ nanozymes,this study focused on the construction and application of a non-redox colorimetric sensing platform based on the phosphatase-like activity of Ce O₂.The obtained L-Arg Ce O₂ non-redox colorimetric sensing exhibits advantages such as low color of the nanozyme itself and resistance to interference from endogenous reducible coexisting substances.In particular,three simple,inexpensive,and highly sensitive hydrolytic enzyme-type colorimetric sensing platforms were constructed using L-Arg Ce O₂ phosphatase-like nanozyme.Among them,a colorimetric sensing method for fluoride ions was constructed by using the strong coordination between cerium and fluoride to inhibit the catalytic activity of L-Arg Ce O₂ phosphatase-like nanozymes;a colorimetric sensing method for zearalenone was constructed by utilizing the open-close characteristics of L-Arg Ce O₂ phosphatase-like nanozymes catalytic activity with a nucleic acid aptamer recognition unit;and a colorimetric sensing method for hydrogen peroxide was constructed by utilizing the property of L-Arg Ce O₂phosphatase-like nanozymes catalyzing the dephosphorylation of L-ascorbyl-2-phosphate（AAP）to produce strong reducing ascorbic acid and induce the formation of silver nanoparticles.The research results can enrich the non-redox colorimetric sensing detection methods based on nanozymes.6.Designing more efficient nanoceria phosphatase-like nanozymes holds promise for improving the detection sensitivity of colorimetric sensing methods.This chapter reports the design and synthesis of L-carnosine-modified nanoceria phosphatase-mimicking nanozymes（CMNC）,which are surface-functionalized with L-carnosine to simulate the microenvironment of the catalytic reaction center of natural phosphatases,thereby enhancing their catalytic activity towards phosphate removal.Furthermore,it was found that ultrasound irradiation could further promote the catalytic activity of CMNC,achieving a dual enhancement effect from both material surface structure optimization and specific external stimulation.Finally,based on the results obtained,an ultrasensitive nanoceria phosphatase-mimicking non-redox colorimetric immunoassay was constructed for colorimetric sensing analysis of C-reactive protein and prostate-specific antigen.On the one hand,ultrasound activation of phosphatase-like nanozymes can provide an innovative colorimetric signal amplification strategy for detection applications.On the other hand,the introduction of the terminal imidazole amino acid residue on the surface of the nanoparticles provides a new reference strategy for the rational design and synthesis of phosphatase-like nanozymes.