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3(,110004)HRPH2O2BPRH2O2,,,,2001204220;2001208210(No.2000031)1,(FI2RST,flowinjectionrenewablesurfacetechnique)1,FI-RST,27,,H2O2H2O28(bromopyrogallolred,BPR)(horseradishperoxidase,HRP)H2O2H2O2,(glucoseoxidase,GOD),22.1(E.C.1.11.1.7,268U/mg,RZ3.1,Sigma)(115U/mg,ToyoboCo.,Ltd.,Japan)(GLUKit,)()PolysorbACT21C218(Transgenomic,Omaha,USA),2060m,2.2(SI)ASD210(Bodenseewerk,Perkin2Elmer,Uberlingen,Germany)10(Valco,Houston,TX,USA),5mL20mm,30W,XMT2(CHNTGroupCorporation,China)1.0,722()LM142164()(9),556nm3,,0.75mm500m1.8mm,20m556nm,SI25cm0.7mmi.d.HC0.96mm,150cm,1100LWHC0.96mm,56cm,400L0.7mm,20cm3020023(FENXIHUAXUE)ChineseJournalofAnalyticalChemistry33073111Fig.1Flowinjectionrenewablesurfaceassaysystema.SI2RST(schematicdiagramofthesequentialinjection2renewablesurfacetechnique(SI2RST)system)C.(H2Ocarrier);SP.(syringepump);HC.(holdingcoil);Heater.(heatingrod);MPV.(multipositionvalve);VSP.(syringevalve);FC.(microchipflowcell);REC.(recorder);L.(light);D.(detector);WHC.(wasteholdingcoil);Bead.(2.4g/Lbeadsuspension)b.(schematicdiagramofthesequenceofzonesinHCwhenstep12ofTable1)Pho.(phosphatebuffer);BHP.(mixtureofbromopyrogallolred(BPR),horseradishperoxidase(HRP)andbuffer)0.5mm452.32.3.1HRP40U/mL,GOD11.5U/mL,BPR210-4mol/L,4H2O2KMnO4,H2O2:34.0gKH2PO4500mLH2O,90mL0.5mol/LKOHpH6.2C218:,2.410-3g/mL2.3.2SI2RSTSI2RST1aSI,SISI1HC1b22BPR1,6222,BPR1SITable1SequenceofoperationofthesequentialinjectionsystemSequenceNo.MPVMPVpositionVSPVSPpositionSyringesteps3Volume3(L)Time(s)Function11Left2404003H2Oaspiration27Right1202002Phosphatebufferaspiration34Right2103502Beadsuspensionaspiration48Right1202001H20aspiration55Right-390-65010Flowcellwashing,beadloading61Right601001Airaspiration72Right18301BPR+HRPaspiration83Right18301H2O2aspiration92Right12201BPR+HRPaspiration103Right12201H2O2aspiration111Right601001Airaspiration128Right1202002H20aspiration136Right-120-2002H20pushing142196Right263Repeat1213for3times206Right1202005H20aspiration216Right-174-2905H20andairwaste225Right-70-1162Phosphatebufferloading235Right-120-20012Sampleandairloading245Right3005003Wastebeadback2suction256Right-536-8935Wasteandholdingcoilwashing3,(+foraspiratinginHC,-forpushingoutHC)8033033.1SI2RST9SI2RST2FI2RST2Fig.2Absorbance2timecurveforFI2RSTassayA0.A0(absorbance2timecurvefordeterminationofA0);A.A(absorbance2timecurvefordeterminationofA)a.114(curveforstep14ofTable1);b.15(curveforstep5ofTable1);c.1622(curveforstep622ofTable1);d.123(curveforstep23ofTable1);e.12425(curveforstep2425ofTable1)H2O2HC3.23.2.19,500m,2L40m,30000(0.8mg)3.2.2BPRC218BPR510-6mol/L,0.51.5mL/min,,1.5mL/min,:BPR,,BPR,,BPR,,BPR,,BPR,,,1.0mL/min3.2.3BPRC218BPR,BPR1.010-5mol/L,1.0mL/min,BPR,V2A3,150L,,200L100L27%100L,1.0mL/minBPR510-8510-6mol/L,A=0.00323+1.70104C,r=0.9998(n=5),RSD=1.1%(n=11),(3)=2.510-8mol/LBPR,BPR8.010-61.010-5mol/L3.2.4pHpH4.86.5,pH6.180.030.3mol/L,0.04mol/L3.2.5HRPHRP0.05U/mL,1050,HRP0.04U/mL,BPR1.010-5mol/L,H2O25.510-6mol/L,1050,74minHRP0.1U/mL,,HRPA2t4,,,,,HRP,HRP,9033:37,HRP0.40.2U/mL,3060s3BPRFig.3InfluenceofBPRvolume(1.010-5mol/L)onsensitivity4HRP2H2O22BPRFig.4InfluenceofreactiontimeofHRP2H2O22BPRsystemonsensitivityBPR1.010-5mol/L,H2O25.010-6mol/L,(reactiontemperature)37,(totalvolumeofsample)100L,(flowrateofsamplezone)1.0mL/min,pH6.18a.HRP014U/mL;b.HRP0.2U/mL;c.HRP0.16U/mL;d.HRP0.1U/mL3.2.60.01mol/LEDTA,BPR8.010-6mol/L,H2O27.010-6mol/L,HRP0.4U/mL,37,30s,100L,1.0mL/min,25%2Table2Toleranceofforeignionsinthedeterminationof7.010-6mol/LH2O2Cations(mg/L)Tolerance(mg/L)Anions(mg/L)Tolerance(mg/L)CationsTolerance(mg/L)Fe3+,CrG0.5S2-,S2O2-30.050.1Al3+,Co2+,Mn2+1.0Cu2+,Cr3+,Pb2+2.0Cl-,SO2-4,PO3-4,NO-35000Cd2+,Ni2+4.0SeG,Ca2+10I-0.2Mg2+,Zn2+K+,Na+20003.3H2O2,H2O2,38,4,10,3.4,GOD0.2U/mL,3710min,14500,EDTA242(Phenol242AAP)Phenol242AAP5,3H2O2Table3AnalyticalperformancefordeterminationofH2O2Reactiontime(s)30r0.9997(n=6)BPR(mol/L)8.010-6Linearrange5.010-85.010-6HRP(U/mL)0.4Detectionlimit(3)(mol/L)3.010-8Volumeofsample(L)140Samplingfrequency(h-1)45Flowrate(mL/min)1.0RSD(n=11)3.5%LinearregressionequationA=4.910-3+1.97104C013304Table4AnalyticalperformancefordeterminationforglucoseReactiontime(s)30Flowrate(mL/min)1.0BPR(mol/L)8.010-6LinearregressionequationA=3.910-3+1.02104CHRP(U/mL)0.4r0.9998(n=6)Volumeofsample(L)140Linearrange1.010-71.010-55Table5AnalyticalresultsforglucoseinhumanserumSampleNo.Thismethod(mol/L)242Phenol242AAPmethod3(mol/L)Differencebetweentwomethods(%)14.610-34.510-3+2.225.110-35.210-3-1.933.910-33.810-3+2.6Phenol242APP:phenol242aminoantipyrineReferences1RuzickaJ,ScampaviaL.Anal.Chem.,1999,71:257263A2PollemaCH,RuzickaJ,ChristianGD,LernmarkA.Anal.Chem.,1992,64:135613603RuzickaJ,PollemaCH,ScudderKM.Anal.Chem.,1993,65:356635714HolmanDA,ChristianGD,RuzickaJ.Anal.Chem.,1997,69:176317655MayerM,RuzickaJ.Anal.Chem.,1996,68:380838146OleschukRD,Shultz2LockyearLL,NingY,HarrisonDJ.Anal.Chem.,2000,72:5855907AnderssonH,AhmadianA,WijngaartWVD.MicroTotalAnalysisSystem2000.Netherlands:KluwerAcademicPublishers,2000:4734768GuoZX,LiL,ShenHX.Anal.Chim.Acta,1999,379:63689WangJianya(),FangZhaolun().Spectro