分光光度法快速检测细菌-李冬阳

DOI10.3724/SP.J.1096.2010.00573*310029H2O2。E.coliDH5αH2O2H2O2H2O2H2O2。H2O2240nm。4.2×10－13L/s·cell5.7×106～5.7×107cfu/mL5.7×106cfu/mL。5～10min。2009-08-102009-12-02No.NCET-07-0725、No.2008C14077No.2009ZX08012-004B*[e-mail]1。CDC760052001。1999～2000E.coliOl57∶H72。。、。、。PCR、ELISA34。PCR5。ELISA。。ATP67。H2O2Catalase。H2O2H2O2H2O2O2H2O2。H2O28、9、`10、11、1213。。。Stop-watchH2O2。H2O2。22.12550ⅡESCOSOVALLSPX-250SK3300HPBS224SartoriusOrigin。3820104FENXIHUAXUEChineseJournalofAnalyticalChemistry4573～576LB10mmol/LPBSpH7.430%H2O2Millipore。2.22.2.1E.coliDH5α。LB37℃24h4℃、8000g15minPBS5min。0.1mL。。E.coliE000.5E00.25E00.125E06E-1E-2E-3E-4E-5E-6E0。2.2.2H2O2H2O20.01～1mol/L200～350nm240nmH2O2。2.2.3H2O2H2O210mmol/L103cfu/mL1。1H2O2Table1CompositionofblankandsamplewhenbacteriaofdifferentconcentrationsactwithH2O2PipettesuccesivelyintothecuvetteBlankSampleConcentrationinassaymixturePBS1.00mL－PBS10mmol/LSamplebacteriasolution2.00mL2.00mL0.67V/VH2O230mmol/L－1.00mLH2O210mmol/LH2O20.5300sStartthereactionbyadditionofH2O2followthedecreaseinabsorbancewitharecorderfor300s。33.1H2O2H2O2-H2O2O2H2OH2O2H2O2。0.01～1.0mol/LH2O2350～200nm1H2O2。Beers240nmH2O2-H2O21H2O2Fig.1Wavelength-scanspectrogramofH2O2withdifferentconcentrationsH2O213Beers、200nm240nmH2O210mmol/LH2O214。H2O21。3.2H2O2240nm10mmol/LH2O2240nm10min0.0040～1±0.002～0.004H2O2240nm。3.3H2O2－dc/dt=kc1475381c=c0exp－kt2215。lnctk。Lambert-BeerA=εbc2A=A0exp－kt3H2O2H2O2pH6.8～7.514。pH7.4PBS。210mmol/LH2O2E05.7×107cfu/mL。k10。y=y0+A0exp－kx16。2E0y=0.0307+0.35781exp－0.01629xR2=0.97565。IntactcellH2O2H2O2。kR22k3kR2=0.98573。K′k/CSpecificactivity。K′。K′=4.2×10－13L/scell。210mmol/LH2O2240nmFig.2KineticcurvesofE.coliwithdifferentconcentra-tionreactingwithH2O23kFig.3RelationshipbetweenE.coliconcentrationsandcorrespondingrateconstantk4ΔAFig.4RelationshipbetweenE.coliconcentrationsandcorrespondingΔA2H2O2Table2BacteriaconcentrationCvaluesandcorrespondingcatalasekvaluesfittedConcentration5.7×107cfu/mLkR210.016340.975650.50.008460.997750.250.005740.996630.1250.002420.993480.10.001410.99800.050.001590.99470.0250.0001910.99610.01250.0004960.8604H2O214。H2O20～5minΔΑA－A0ΔA4。ΔAR2=0.95163。5754。。。5min。10min10min。。References1MeadPSSlutskerL.EmergInfectDis199955607～6252LIDu-JuanWANGJian-PingYINGYi-BinLIYan-Bin.ChineseJournalofBiochemistryandMolecularBiology2007233194～1993LIDu-JuanWANGJian-PingGELinYINGYi-Bin.ChineseJournalofSensorsandActuators2008215709～7144LUOJin-PingTIANQingYUEWei-WeiHEBao-ShanCAIXin-Xia.ChineseJ.Anal.Chem.2009372306～3105LazckaODelCampoFJMunozFX.Biosen.Bioelectron20072271205～12176QiuJZhouYChenHLinJM.Talanta2009793787～7957TomasLALOrdonezJAdeFernandoGG.Meat.Sci.2006722222～2288BonnichsenRK.MethodsEnzymol.195527819CohenGDembiecDMarcusJ.Anal.Biochem.197034130～3810MuellerSRiedelHDStremmelW.Anal.Biochem.1997245155～6011WuMLinZWolfbeisO.Anal.Biochem.20033201129～13512LiuXPZweierJL.FreeRadic.Biol.Med.2001317894～90113BeersRFSizerIW.J.Biol.Chem.19521951133～14014AebiH.MethodsEnzymol.1984105121～12615WANGJing-YanZHUShen-GenXUChang-Fa.Biochemistry.3rdEd.BeijingHigherEducationPress200235316DavisonAJKettleAJFaturDJ.J.Biol.Chem.198626131193～1200SpectrophotometricMethodforRapidDetectionofBacteriaLIDong-YangRUShi-PingWUJian*YINGYi-BinCollegeofBiosystemEngineeringandFoodScienceZhejiangUniversityHangzhou310029AbstractThecatalaseactivityofintactcellswasinvestigatedwiththeUVspectrophotometrytorealizetherapiddetectionforbacteriaE.coliDH5αasamodel.Withtheadditionofhydrogenperoxidetothebacteriasolutionthecatalaseofthebacteriawoulddecomposethehydrogenperoxidewhichfollowedthefirst-orderkinetic.Thefirst-orderrateconstantthatreflectedthebacteriaconcentrationwascalculatedaccordingtotheabsorbancechangeofhydrogenperoxideat240nm.TheresultsindicatedthattherewasalinearrelationshipbetweentherateconstantandtheE.coliconcentrationintherangeof5.7×106－5.7×107cfu/mLthecata-laseactivityofintactE.coliwas4.2×10－13L/s·cell.Thismethodcouldbeusedtodetectthebacteriaof5.7×106cfu/mLwithin5－10min.KeywordsBacteriadetectionCatalaseEnzymeactivitySpectrophotometryBiosensorReceived10August2009accepted2December200967538