有机磷农药胶体金免疫层析快速检测试纸条的研制

上海师范大学硕士学位论文有机磷农药胶体金免疫层析快速检测试纸条的研制姓名：刘莹申请学位级别：硕士专业：生物化学与分子生物学指导教师：魏新林20090401I14.32mg/ml7.6112pH8.024µg/ml3IgG90.25mg/kgIIAbstractOrganicphosphoruspesticides(OPs)arestillbeingwidelyusedallovertheworld,whichhaveareallyhighresiduedetectionandover-standardrateinfruits,vegetablesandtea.TheextremelyharmfultoxicityofOPsresiduesremainedinfoodhasposedagreatthreattoconsumers.Therefore,itissignificanttostrengthenOPssupervisionandtodeveloparapidresiduedetectionmethodtoensurefoodsafety.Inthisstudy,weusedColloidalGoldImmunochromatographicAssay(GICA)anddevelopedacolloidalgoldstripspecificallyforOPsresiduesdetectioninordertoprovideatheoreticbasisforcolloidalgoldstrippreparationforOPsresiduesrapiddetection.Themaincontentsandresultsofthestudyareasfollows:1AnOPsconjugateantigen(OP-BSA)wassynthesizedbydiazotization.TheproductwasidentifiedbyUVspectraandIRspectra.BothspectraindicatedthattheOPswereconjugatedtothecarrierproteinBSA.TheconcentrationofOP-BSAwasdeterminedto4.32mg/mlandthecouplingratiowas7.611.2AnantibodyimmuneprobeforOPswassynthesizedbycolloidalgoldlabeling.TheOPsmonoclonalantibodywaslabeledbycolloidalgoldatpH8.0withtheconcentrationof24µg/ml.Theantibodyprobewasidentifiedthroughdotgoldimmunofiltrationandimmunity-chromatographyassay.Theresultssuggestedthatthegoldprobehadanimmuneactivity.3AcolloidalgoldrapidteststripfordetectionofOPswaspreparedwithatestlinecoatedbyOP-BSAandacontrollinecoatedbygoatanti-mouseIgG.Eachcomponentofthestriphadbeenstudiedinthispaper,includingthemembranematerialsselection,thesolutionsystemscreeningandtheconcentrationdetermination.Atlasttheassembledstripwastestedinseveralaspects.Theresultsindicatedthatthestripwasabletodetectatleast9kindsofOPsresidueswithadetectionlimitamountof0.25-1.5mg/kg.Keywords:Organicphosphoruspesticides,residuedetection,GICA,teststripIIIOPsBSAGCOP-BSATEMGICANCTC11O157[1]720035005213[2]22.1(P=O)(P=S)[3]2R1R2X[4][5-6](AChE)-X-R()[7]Na+K+Mg2+Ca2+ATPaseROXRC-O-SH()-SH2.2(OPs)3195640[8]1982DDT[9]2032.8%50%~60%60%10~154[10]46%98%200161378[11]20005550129322%[12]200162.5%75%47.5%[13][14]200235413281737.6%131253%[15]200410~20054223175480%20056844247.4%31.97mg/kg5.9%[16]20056~200632848.1%12.32%3.68%[17]30070%1089.64%[18]3[19-20][21-24][25-27]3.1[28]0.18~10.0mg/kg3.2Giang[29]52530min2560minpH[30]0.04~10.0mg/L[31]0.3~3.5mg/kg0.05~5.0mg/kg15min30min80%3.3[32]3.4[33]-H2O20.05mol/l-H2O2pH=131mmol/l0.1mg/kgRoda[34](FIA)0.75µg/lMoris[35]6ng/l3.5Holger[36]2µg/kgKumaran[37]-pH1.310-5mol/l6.210-6mol/l[38]3min0.5~43.1µg/ml0.1~15.0µg/mlMulchandani[39]910-8710-8mol/lpH3.6()()Lueking[40]PVDF(poly-vinylidenedifluoride)(HRP)-3.7(RIA)(EIA)(FIA)(LIA)[41-44]RIAFIALIAEIA7(ELISA)[42,45](ELISA)0.1~10mg/lErcegovich[46]BSA0.1ppmClegg[47](r=0.99)[48](M1605)[49][50]NHS-DCCEDC[51]O,O-[52]()7RPYNCLGDYNT844.1colloidalgold1150nmHAuCl4[53-54]1971FaulkTaylor[55-56](GICA)2090Beggs[55](HCG)GICANCGICAGICAGICA5-10min4.21996Mika[57][58][59]2003BFNHBSA9BFNHIgG0.25mg/kg10[60][61]PDL-PDL[62]2,4-D2,4-D2,4-D-OVA2,4-D2,4-D2,4-D21µg/l1020034.3[63-68]ELISA1ng/mlUSEPA()10[69-70]HBsAgHCVAFP/CEA111-(1000D)[71][72-73]22.195%2.27000EppendorfpHMetllerToledoPL-203-IKARHR501-BSHZ-95BT6ALPHAI-612NBSAgilent6890n/973i400BrukerDMX400BrukerHERMLEZ-383K()AVATAR37033.1[74]BSA2.1NO2CH3OPOCH3OCH3SHClCH3OPOCH3OCH3SNH2NaNO2BSACH3OPOCH3OCH3SNNProtein2.1195%4g40ml1%Na2CO3320ml40ml-9:1V/V8g1h25ml2%pH1-210210%NaOHpH10-11CHCl3210ml2CHCl3240mg10ml1mmol100ml0.1mol/lNaNO2-30minHNO213BSA100mg20mLpH92h3[75]0.01mol/lpH7.4436h2l/3,500rpm15minOP-BSA-203.2121%3DMSO1H-NMRTMS400MHz3.33.3.1(1)OP-BSA-190-400nm14BSA-190-400nm-190-400nm(2)BSA3.3.2SDS-PAGE[76]3%10%BSA1mg/ml400µl100µl530min10µl80v120v1h3.3.3mlmgmg/ml(mg/mL)[77]15BSA123456BSA1.0mg/ml01.02.03.04.05.05.04.03.02.01.001A280A2803.3.4[78]OD2800.4-0.8OP0.05mg/mlBSA0.7mg/mlOP()BSAHεBSAε=-/4OVABSA164.14.1.12.22.24.1.21-2-2.34.1.32.42.5RT=21.01minRT=20.50min17m/z302.4.2182.54.1.41H-NMR2.6ppm=5.07NH2br:192.64.24.2.12.7[74][79]2.7204.2.21[75]2.81-2-BSA3-2.822.9BSAOP-BSA212.9BSA3300295916581541cm-1[80]BSAOP-BSAOP-BSABSA3300295916581541cm-1OP-BSA123411621070955859cm-1BSA123411621070cm-1P-O-C955859cm-1P=SOP-BSABSABSA4.2.31-Marker2-BSA3-2.102.10Marker674528KDBSAOP-BSABSA34.2.4BSAOD2800.4-0.84.32mg/ml22y=0.588x+0.0684R2=0.99300.10.20.30.40.50.60.70.800.511.5BSAConcentrationofBSAmg/mL)AbsA2802.114.2.5[78]=-/-A=LCL=A/CA/C)/A/C)7.611231colloidalgold1150nmHAuCl4[53-54]1971FaulkTaylor[55-56]17.5nmpH22.1sigma20000Tween-202.27,000EppendorfIKARHT6Z-383KHERMLEMilli-QSynthesisMilli-QHITACA6002433.13.1.172h100g,250ml1000ml24h373.1.218.20.22µm3.1.3lg1%411%10ml0.1g/l3.1.4G.Frens[81]0.1g/l100ml1001%1mL1.4mL1.6ml1.8ml300-300rpm200rpm15min100ml43.1.5-4ml1%(Na3C6H5O72H2O)0~5ml1%0~5ml25mmo/lK2CO320ml601ml1%HAuCl479ml60(0.5~1h)5min[82]253.1.60.1g/l100ml1ml0.5%[83]3.23.2.13.2.2400~700nm[84]3.2.3Formvar30050[84]3.33.43.50.1g/l100ml1.02.5ml1%263.63.6.110.01mol/lpH7.4PB3d6h212,000r/min430min3.6.2pH1.5ml1ml0.1mol/lK2CO30.1mol/lHClpH4.05.06.07.08.09.010.011.096pH200µl36µl1mg/ml5min40µl10%NaCl2hpH3.6.310ml0.1mol/lK2CO30.1mol/lHClpH96200µl3pH0.0