Fluorescence Analysis Method Based On Cupric Oxide Nanoparticles

Nanoparticle shows many unique physical and chemical properties due to its surface effect,small size effect and macroscopic quantum tunneling effect.In recent years,the enzyme mimetic activity of nanomaterials has been widely concerned and employed for bioanalysis and biotechnological applications.Cupric oxide nanoparticles（CuO NPs）,which are considerably stable and possess almost unchanged catalytic activity over a wide range of pH and temperatures,have been found to possess intrinsic enzyme mimetic activity similar to that of natural peroxidase.In this study,we have investigated the alkaline peroxidase-like activity of CuO NPs by using 3-（4-hydroxyphenyl）propionic acid（HPPA）as fluorescent substrate and demonstrated that the nanozyme function can be modulated by ammonia.Based on these findings,a“turn-off”fluorescent sensor for urea and urease is developed and successfully applied to the real sample detection.The fluorescence analysis method of alkaline phosphatase was successfully established by using the ascorbic acid oxidase-like activity of CuO NPs and coupling the alkaline phosphatase enzymatic reaction.In addition,we encapsulated CuO NPs in liposomes,the prepared CuO NPs-liposome complex can be quantitatively detected by the methionine gold cluster,which is expected to be used as a novel marker.This paper was divided into three parts:Chapter I:Alkaline peroxidase activity of cupric oxide nanoparticles and ammonia modulate effectIn this chapter,CuO nanoparticles were discovered to possess alkaline peroxidase-like activity by using 3-（4-hydroxyphenyl）propionic acid as a substrate.Based on this finding,a fluorometric method was developed for H₂O₂ determination in alkaline condition with a low detection limit of 0.81μM.Notablely,ammonia was found to inhibit the alkaline peroxidase-like activity of CuO nanoparticle.Therefore,a novel sensing platform for the determination of urea and urease has been successfully constructed.The limit of detection for urea and urease was 27μM and 2.6 U/L,respectively.The real samples,such as urea in human urine and urease in soil were detected with satisfied results.These studies not only extend the catalytic reaction condition of peroxidase and its mimetics from acidic and neutral to alkaline regions,but also provide a novel method to the analysis of urea and urease.As well as the developed assay has the features of easy handling,low-cost,more convenient,and high selectivity and sensitivity,it is accounting for the further insight of enzyme nano-mimics and their potential applications.Chapter II:Dual enzyme-like activities of CuO nanoparticles and construction of alkaline phosphatase biosensorIn this chapter,alkaline phosphatase can selectively hydrolyze sodium ascorbate to produce ascorbic acid.CuO NPs as an ascorbate oxidase mimetic which can be used to catalyze the oxidation of ascorbic acid（AA）to hydrogen peroxide.Then,CuO NPs as a peroxidase oxidized terephthalic acid（TA）to produce strong fluorescence.Base on that,an alkaline phosphatase sensing protocol was successfully established.The linear range of alkaline phosphatase sensing was 1.2¹4.4 U/L,and the detection limit was 0.058 U/L.It has the advantages of good selectivity,high sensitivity,less sample requirement,good reproducibility and low cost.In addition,alkaline phosphatase inhibitor was explored in this study,in which the IC50 of L-phenylalanine was 0.11 mM.We had successfully applied the method to the biological sample analysis,and the results of serum sample in recovery experiments were satisfactory.Chapter III:Preparation of CuO nanoparticles-liposome complex and construction of fluorescence quenching detection systemIn this chapter,homogeneous and uniformly distributed CuO NPs-liposome complexes were prepared.All CuO NPs were monodisperse particles（about 6 nm in diameter）and were tightly wrapped in spherical liposomes.The prepared CuO NPs-liposomal complex can be detected by methionine gold nanocluster（Met-AuNCs）.The fluorescence of Met-AuNCs was quenched by Cu²⁺which were produced through the reaction of CuO and HCl.The results were consistent with ICP-MS and BCO colorimetric method.The limit of detection for CuO NPs-liposome in proposal method and BCO colorimetric method were 0.054μg/mL and 0.088μg/mL,respectively.The actual determination of CuO in CuO NPs-liposome complex was also demonstrated.Compared with BCO colorimetric method,the proposal method has the features of easy handling,more convenient,time consuming.