多功能酶标仪原理及应用

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1、酶标仪检测原理及应用July-11-2013李军辉©2009PerkinElmer酶标仪的应用AgglutinationGeneexpressionPCRproduct(quantitativeApoptosisEnvironmentaltoxinsandqualitative)BacterialadhesionFIApHindicatorsBacterialidentificationPhagocytosisBluefluorescentproteinGeneexpressionPhospholipidassayBRET2™Gree

2、nfluorescentPKUCelladhesionproteinGFPProteaseactivityCellcountingHTSProteinassaysCellexpressionHomogeneousTRFquantificationCellmembraneintegrity(LANCE™)ReceptorbindingstudiesandlysisImmunoassaysReportergeneCellproliferationIntracellularCa2+ReversetranscriptaseCellvia

3、bilityandmeasurement,Fura-2,RNAhybridizationcytotoxicitydual-labelratiometricRNAquantificationChemotaxisandkineticSignaltransductionChlorophyllKinaseactivitySNP(SingleNucleotideColorimetricassaysLANCEPolymorphisms)CytokineanalysisMacrophageactivitySteroidhormonesCytoto

4、xicityMetabolicactivityToxicologyDELFIA,preferablyMIC(minimuminhibitoryUraniumbRDUassayconcentration)DNAhybridizationMitochondrialmembraneDNAquantificationpotential(fluorescenceandUVMultiprobeassaysabsorbance)NADH,NADPHELISA/FELISANa+/H+exchangeEndocrinehormonesNKcel

5、lactivityEnzymeactivityNucleicacidquantificationEnzymaticcleavageOxidativeburstEnzymekineticsOxidationreactions2技术原理光吸收化学发光荧光强度荧光偏振时间分辨荧光Alpha技术Label-free技术3光吸收物质对光的吸收比耳定律A=KCLK：吸收系数；C：物质的浓度；L：溶液光径长度物质浓度测定DNA：260nmProtein：280nmBCA：562nmElisa：450nmMTT：490nmNADH：340nmChlorophyll

6、:K=105cm-1M-1Bacteria:600nmBSA:6.67cm-1M-1Plate：Clear4IgG:14cm-1M-1荧光能量由光辐射提供激发:物理激发E=hν=hc/λh：普朗克常量ν：频率c：光速λ：波长E发射λ激发荧光素λ发射=535nm＞荧光素λ激发=485nmPlate:BlackGuess?NielsBohrMaxPlank5荧光应用Ca2+测定：Fura-2，Fluo-3DNA定量：PicoGreenRNA定量：RiboGreenGUS报告基因：细胞增殖：AlamarBlu

7、eAc-DEVD——AMC细胞凋亡：Caspase-36化学发光能量由化学反应提供激发：化学激发A+B=C*C+light常消耗高键能物质如ATP闪光性（flash）、辉光型（glow）Plate：White7化学发光应用:Chemistry8化学发光应用：Luciferase9化学发光应用：ATP-Cellviability10化学发光应用：ATP-Kinaseassay11化学发光应用：Luciferin-P450(ADME)12时间分辨荧光：“剩”者为王镧系元素：Eu、Sm、Tb、DyEu320615DoubleLSm320642Longha