Applications Of Spectroscopy In Biomolecules Detection

Biomolecules including vitamins,amino acids,and enzymes have important biological and pharmacological activities,which play essential roles in varieties of physiological processes.For example,ascorbic acid(vitamin C)has antioxidant,cancer prevention,free radical scavenging functions.Histidine can control the metal transfer process in living organisms.Trypsin participates in the regulation of pancreatic exocrine function and is a marker for the diagnosis of pancreatic diseases.Curcumin possesses some pharmacological activities such as antioxidative,anticoagulant,hypolipidemic,and anticancer effects.Therefore,biomolecules are important analysis factors for disease diagnosis and clinical research,which are closely related to human health.Accurate detection of biomolecules in real samples is essential for biological analysis and clinical research.This thesis focuses on the development of several simple,sensitive,fast and accurate new spectroscopic analysis methods,and explores the applications of these methods in sensitive detections for ascorbic acid,histidine,trypsin,and curcumin.The main research contents are outlined as follows:1.A rapid and sensitive colorimetric strategy for the determination of ascorbic acid(AA)is presented,using amino-functionalized copper metal-organic frameworks nanoparticles(Cu-BDC-NH2 NPs)with effective intrinsic peroxidase-like properties.In our design,Cu-BDC-NH2 NPs catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine(TMB)generating blue-colored oxidized TMB(ox-TMB)with a significant absorbance at 655 nm in UV-vis spectrum.AA can reduce the ox-TMB,inducing a dramatic blue color fading and a decrease in absorbance.Under the optimal experimental conditions,this proposed system presents a good linear relationship(0.5-60 ?M)and the detection limit is 0.15?M.Repeatability,high selectivity,and practicability are also well-studied,proving that the sensing system based on Cu-BDC-NH2 NPs can be successfully applied for colorimetric detection of AA in human serum,food,and pharmaceutical samples.2.A fluorometric assay for histidine(His)is described.It is based on the inhibitory effect of His on nanocubes consisting of cobalt-containing Prussian blue analog(CoFe NCbs),which have a strong oxidation effect on thiamine(THI)in the presence of NaOH.THI is nonfluorescent but the oxidized form(thiochrome;ThC)has strong blue fluorescence,with excitation/emission maxima at 370/445 nm.His inhibits the oxidation effect of the CoFe NCbs due to the strong interaction between its imidazole side chain and the amino groups of the CoFe NCbs.As a result,the production of ThC decreased,and the fluorescence intensity at 445 nm gradually decreased with the increase of His concentration.The lower detection limit is 14.3 nM of His,and the linear range extends from 0.05 to 2.5 ?M.And the method was successfully employed to quantify His in spiked serum samples.3.A fluorometry assay for trypsin-sensitive determination has been presented.The fluorescence of the system at 370/445 um is derived from thiochrome(ThC)obtained by in-situ oxidation of thiamine.Based on the inner filter effect,cytochrome C(Cyt C)can quench the fluorescence at 445 nm effectively.Cyt C is specifically hydrolyzed by trypsin through an enzymatic reaction,giving rise to the recovery of the fluorescence intensity.The change value of fluorescence intensity is proportional to trypsin concentration,which is successfully used for trypsin quantitative detection.This method exhibits good repeatability and selectivity with a detection limit of 0.15?g mL-1 and a quantification limit of 0.50 ?g mL-1 for trypsin sensing.Moreover,it is applied to detect trypsin in practical serum and urine samples with accurate results.4.A novel fluorometric strategy is proposed for detecting curcumin by polyvinyl pyrrolidone-templated Cu NCs(PVP-Cu NCs)as a fluorescent probe that exhibits excitation/emission peaks at 380/510 nm.The fluorescent excitation and emission spectra of PVP-Cu NCs have a striking overlap with the UV-vis spectrum of curcumin,and the fluorescence lifetime of PVP-Cu NCs decreases after the addition of curcumin.Curcumin leads to fluorescence quenching based on fluorescence resonance energy transfer.This method allows for the determination of curcumin in the range of 0.1-10 ?g mL-1 and the detection limit is 21 ng mL-1.Furthermore,the method displays good selectivity and is successfully applied for real serum and food samples analysis.