Application Of MnO₂ Nanosheets As Resonance Light Scattering Probes For Biochemical Analysis

Resonance light scattering?RLS?technology is widely used in the analysis of inorganic ions,drugs,food additives,biomolecules and other substances because of its simple operation,high sensitivity and short analysis time.At present,most RLS analysis methods are based on the determination of the enhanced scattering signal?Turn-on?caused by the interaction of analyte and RLS probe,such as association,aggregation,electrostatic interaction and so on.However,these simple interactions are easily interfered by non-target analytes,making RLS analysis less selective.In addition,there are many factors leading to the formation of aggregates in the liquid-phase sensing system,which will generate false-positive scattering signals.Therefore,we can solve the problems in the above RLS analysis methods through the following two efforts:First,because certain conditions are needed for the chemical reaction,the change of the scattering signal of the system caused by the chemical reaction between the probe and the analyte can improve the selectivity of RLS analysis method to a certain extent;second,since there are few factors that lead to the destruction of the probe in the liquid-phase sensing system,the false positive scattering signal can be effectively avoided by destroying the scatterer and decreasing the the system scattering signal.MnO₂ nanosheets is a new-type two-dimensional nanomaterial,which is widely used in fluorescence sensing system for its large specific surface area,high chemical stability,good biocompatibility,easy synthesis,easy reduction and other excellent properties.It is found that MnO₂ nanosheets have strong RLS signal.Reducing substances can reduce MnO₂ nanosheets to Mn²⁺,resulting in the decrease or disappearance of their scattering signal and the concentration of reducing substances is directly proportional to the decrease of scattering signal within a certain concentration range.On this basis,MnO₂ nanosheets are used as self-sacrificing RLS nanoprobe and combined with the high specificity of the enzyme-catalyzed reaction in the paper,an RLS analysis method based on the"Turn-off"mode is established and successfully applied to enzyme activity evaluation,enzyme inhibitor screening and enzyme substrate analysis.This dissertation can be divided into four chapters:Chapter 1:A brief introduction about the development of resonance light scattering technology is made,in which the principle and quantitative basis of resonance light scattering technology are emphasized,and the related applications of resonance light scattering technology are comprehensively reviewed.In addition,the application of MnO₂ nanosheets in fluorescence sensing system is summarized.Chapter 2:MnO₂ nanosheets are used as RLS probe for enzyme activity evaluation.Acid phosphatase?ACP?,an important biomarker of metastatic prostate cancer,is selected as the target analyte.The ACP catalytic product of ascorbic acid-2-phosphate?AAP?is ascorbic acid?AA?,which can reduce MnO₂ nanosheets,then result in the decrease of resonance light scattering signal of the system.The quantitative analysis of ACP is realized according to the decrease value of scattering signal??I?.Under the optimum conditions,the concentration of ACP and?I have a good linearity?R²=0.992?in the range of 0.05-0.40 mU/mL,and the detection limit is 0.015 mU/mL.This method is not only successfully applied to the determination of ACP in human serum samples,but also to the screening of ACP inhibitors,showing great potential for clinical diagnosis and biomedical research.Chapter 3:MnO₂ nanosheets are used as RLS probes for enzyme inhibitor screening.Xanthine oxidase?XO?is a drug target for the treatment of gout.It is of great significance to establish a simple and rapid XO inhibitor screening system for the research and development of related drugs.Therefore,in this chapter,XO catalyzes the substrate xanthine to produce uric acid?UA?and H₂O₂ to reduce MnO₂nanosheets,resulting in the decrease of resonance light scattering signal of the system.When the XO activity is inhibited,the generation efficiency of enzymatic hydrolysis product will reduce,further affects the decrease value of the scattering signal of the system,and accordingly,the screening of XO inhibitors can be achieved.Febuxostat,allopurinol and apigenin were selected as inhibitors in the experiment,and the IC₅₀were determined to be 1.85 nM,1.66?M and 2.00?M,respectively,which is consistent with the literature reports,indicating that the inhibitor screening system established in this chapter is expected to be used for the in vitro screening of XO inhibitors.Chapter 4:MnO₂ nanosheets are used as RLS probes for determination of enzyme substrate.Select sarcosine?SO?as the target analyte,and use sarcosine oxidase?SOx?to catalyze SO to generate H₂O₂,which can reduce MnO₂ nanosheets to decrease its RLS signal.The results show that the RLS signal variation value??I?and SO concentration have good linearity?R²=0.991?in the range of 0.075-7.5?M and the detection limit is 39.3 nM under the optimum conditions.The determination method is not interfered by isomer alanine and other coexistent substances.It is expected to be used for the selective determination of SO in complex matrices,and it has great application potential in the study of biomarkers related to SO.