Development Of Methods For Determination Of Physiological Active Substances Based On Fluorescent Silicon Nanoparticles

Physiological active substances existing in human blood play key roles in maintenance and regulation of physiological activities of human body.And certainly,activity or concentration variations of these substances can indicate the health status of human body.Therefore,rapid determination and real-time monitoring of physiological active substances in human serum are vital for bioanalysis and clinical diagnosis.Fluorescent probes have been widely used in the fields of bioanalysis and clinical diagnosis in recent years.Analytical performance and applicability of fluorescent probes are generally determined by the features of fluorescent materials,the mechanisms for sensing,the strategies for spectroscopic measurements and the performance of instruments.Fluorescent silicon nanoparticles（SiNPs）usually refer to the zero-dimensional nanoparticles containing silicon.SiNPs possess intrinsic properties（e.g.,strong fluorescence and robust photostability）and distinct dominances（e.g.,low cost,excellent biocompatibility and good biodegradability）,which facilitate their applications in bioanalysis and clinical diagnosis.Inner filter effect（IFE）is a common effect in fluorescence spectroscopy,which are mainly induced by the overlap between the absorption band of a certain substance and the excitation or emission bands of a fluorophore,and will result in the quenching of fluorescence emitted from the fluorophore.Based on IFE,the substance which absorbs incident radiation or fluorescence emission of a fluorophore can be detected as a routine quencher.In this dissertation,three types of SiNPs were prepared for the determination of alkaline phosphatase（ALP）activity,glucose and xanthine in human serum based on IFE.Common fluorometry,fluorescent and colorimetric dual-mode assay and ratiometric fluorometry were employed for the development of corresponding analytical methods.1.A simple and sensitive fluorescence assay was established for the effective monitoring of ALP activity in serum based on IFE.The SiNPs as the fluorescent indicator of this sensing system,were prepared via a one-pot strategy using N-[3-（tri-methoxysiyl）propyl]-ethylenediamine（DAMO）and catechol as the silicon source and reductant,respectively.The zymolyte of this assay,4-nitrophenyl phosphate（PNPP）,would be hydrolyzed with the catalyzation of ALP,resulting in the production of p-nitrophenol（PNP）.PNP would induce a significant IFE on the fluorescence emitting from SiNPs due to the obvious overlap of absorption spectrum of PNP and fluorescence excitation spectrum of SiNPs,which was employed to evaluate the activity of ALP.The mechanism of this assay was further investigated and confirmed by using fluorescent lifetime measurements as well as UV-Vis absorption and fluorescence spectroscopy.The limit of detection（LOD）and linear range for the present assay are 0.0027 U/L and0.01–5.0 U/L,respectively.Inorganic ions,enzymes and other biological compounds commonly coexisting in human serum exhibited little influence on the present assay.Good correlation（r=0.97）between the testing results of real serum samples obtained by the present assay and a commercial reagent kit demonstrates the reliability of this assay.By introducing the enzyme-catalyzed hydrolytic reaction for the generation of energy absorber of fluorescence excitation light,the specific and sensitive activity detection of target enzyme was realized in this work.Excellent analytical performance of the present assay exhibits broad potential of the facile-prepared fluorescent SiNPs for bioanalysis and clinical diagnosis.Moreover,the present assay can also be applied to the determination of ALP inhibitors.2.A novel colorimetric and fluorescent dual-mode approach based on fluorescent organosilicon nanodots（SiNPs）with¹00%photoluminescence quantum yield was established for sensing glucose in serum.SiNPs were facilely-synthesized by a one-pot hydrothermal strategy using trimethoxysilylpropyldiethylenetriamine（AEEA）and rose bengal（RB）as the silicon source and the reductant respectively,and were employed as the fluorescence indicator in this work.The product of the oxidation reaction between p-phenylenediamine（PPD）and hydrogen peroxide（H₂O₂）,2,5-diamino-N,N’-bis（p-aminophenyl）-l,4-benzoquinone di-imine（PPDox）,would exhibit IFE on the fluorescence of SiNPs due to the overlap of the its absorption band and the fluorescence excitation band of SiNPs.By measuring the fluorescence changes of SiNPs or absorption（reflection）changes induced by PPDox,an ultrasensitive fluorescent and colorimetric dual-mode assay of glucose in human serum was realized,since glucose would participate in the H₂O₂-generation reaction catalyzed by glucose oxidase（GOx）.The limits of detection（LODs）and linear dynamic range of the present assay are 0.36μmol/L and 0.5-500μmol/L for colorimetric mode,and 0.06μmol/L and 0.1-200μmol/L for fluorescent mode,respectively.Inorganic ions,enzymes and other biomolecules commonly coexisting in human serum exhibited little interference on the assay.Good correlations between the testing results obtained by the present assay and a commercial glucometer indicate the good reliability of the present assay.The excellent analytical performance of the present assay makes it suitable for further clinical diagnosis and H₂O₂ screening.3.A new type of fluorescent silicon nanoparticles（SiNPs）was prepared via a facile one-pot hydrothermal method by using N-[3-（trimethoxysiyl）propyl]-ethylenediamine（DAMO）and glucose as reagents,and was subsequently applied to constructing a ratiometric fluorescent probe for sensitive and rapid sensing of xanthine in human serum.With the one-step addition of xanthine oxidase（XOD）,o-phenylenediamine（OPD）,horseradish peroxidase（HRP）and SiNPs as testing reagents into a serum sample,a series of catalytic oxidation reactions would be triggered and accordingly lead to the fluorescence signal changes of the testing system.Hydrogen peroxide（H₂O₂）,a product of the oxidation of xanthine under the catalyzation of XOD,would oxidize o-phenylenediamine（OPD）to fluorescent 2,3-diaminophenazine（DAP）with the catalysis of HRP.By measuring the yellow fluorescence emission of DAP and the inner filter effect（IFE）of DAP on the blue fluorescence emitted from SiNPs,a ratiometric fluorescence assay for xanthine was established with a detection limit（LOD）of 0.12μmol/L.Furthermore,the as-prepared SiNPs were applied for pH sensing in the range of 4.5-10.5.The strategies proposed in this work for both preparation and application of fluorescent SiNPs can be easily employed in future fabrications of novel fluorescent probes,especially for the sensing of biological metabolites involved in H₂O₂ generation or consumption reactions.