中科大高分子课件(五)聚合物在生物高分子分离中的应用2

电渗是CE的基本现象之一，它可以控制组分的迁移速度和方向，进而影响CE的分离效率和重现性．如何控制电渗流？理论：能影响电渗的因素都有可能用于电渗的控制实际：能理想地调节电渗而又不影响分离过程的控制方法目前不多见．对于DNA分离及测序来说，pH-8.3，常用管壁涂层（涂覆)技术.化学涂覆如聚丙烯酰胺、聚乙烯基吡咯烷酮涂层可在pH9-pH6以内基本消除电渗优点•抑制熔硅毛细管表面Si－OH解离，修饰表面Zeta电势．•聚合物或硅烷化试剂的取代基覆盖已离解的Si－OH．•增加了内壁表面层附近溶液的粘度，涂层的厚度和密度都将影响到表面层附近溶液的粘度，从而影响涂层的涂覆效果。•涂层寿命长，稳定性好，分离效率高。缺点•化学键合的过程中涉及毛细管硅烷化和引发单体聚合两步反应，一方面受两步反应的转化率影响，Si－OH很难被完全屏蔽；另一方面，毛细管内的聚合反应很难控制，容易造成涂层的不均一性、不规整性，甚至阻塞毛细管。•价格昂贵。物理涂覆•涂层制备过程简单易操作，涂覆过程可一步完成，•涂层可再生。这种涂层可以是中性的、带正电荷的、也可以是吸附在带正电的聚合物涂层表面转而带负电荷的。优点缺点物理吸附涂层稳定性不及化学键合涂层．用于DNA分离的聚合物交联聚合物:交联聚丙烯酰胺、琼脂糖非交联聚合物均聚物poly-N-hydroxyethylacrylamide（PHEA)存在的问题：共聚物a.无规共聚物理想的筛分介质：高筛分能力、低粘度、自涂覆功能poly(DEA-co-DMA)聚(N,N-二乙基丙烯酰胺-co-N,N-二甲基丙烯酰胺）poly(AM-co-DMA)聚(丙烯酰胺-co-N,N-二甲基丙烯酰胺)poly(AA-co-AG)聚(丙烯酰胺-co-β-吡喃型葡萄糖)poly(AM-co-AAG)聚(丙烯酰胺-co-葡糖酰丙胺)poly(NEEA-co-NMEA)聚(乙氧基乙基丙烯酰胺-co-乙氧基甲基丙烯酰胺)等b.嵌段共聚物PEG-(CnF2n+1)2末端带有碳氟化合物的聚乙二醇PEO-PPO-PEO低分子量的聚环氧乙烷和聚环氧丙烷的三嵌段共聚物BEB/EBE聚环氧乙烷和聚环氧丁烷的三嵌段共聚物等C.接枝共聚物PAM-g-PNIPAM（聚丙烯酰胺-g-聚N-异丙基丙烯酰胺)PNIPAM-g-PEO(聚N-异丙基丙烯酰胺-g-聚环氧乙烷)PAM-g-PDMA（聚丙烯酰胺-g-聚N，N-二甲基丙烯酰胺）PDMA-g-PMMA（聚N，N-二甲基丙烯酰胺-g-聚甲基丙烯酸甲酯）等存在的问题：共混聚合物不同分子量的PEO分离ds和ssDNAHEC，LPA等不同分子量的混合物也都能成功地对DNA分离或排序存在的问题：准互穿网络聚合物YanmeiWang,DehaiLiang,JingchengHao,DufeiFang,BenjaminChu,Electrophoresis2002,23,1460-1466MMRMMMMMMMMMMRMRMMMMMMMMMMechanismofparticleformationbyinversemicroemulsionpolymerizationofAM/AIBNsystem.Mismonomeracrylamide;Iisinitiator;Risfreeradical.18192021222302x1064x1062502001501005024252627282902x1064x10640035030030313233343502x1065505004503637383940414201x1062x10675070065060043444546474801x1062x106850800migrationtime,minElectrophoreticseparationofDNAfragmentsgeneratedonBigDyeTMTerminatorCycleSequencingStandardwithAmpliTaqFSunderoptimumexperimentalconditions:2.5%IPNs,200V/cmandroomtemperature.A-trackred,T-trackblue,G-trackgreen,andC-trackblack.Electrophoreticconditionswereasfollows:effectivelength36cm,totallength41cm,75-m-i.d.,365-m-o.d.anodebuffer1TTE;cathodebuffer1TTE/7Murea.Thesamplewasinjectedataconstantelectricfieldof73V/cmfor40s.YanmeiWang,DehaiLiang,Q.Ying,B.Chu，Electrophoresis2005,26,126-136quasi-IPNGNPsquasi-IPN/GNPsDMAinitiatorquasi-IPNLPAPreparationofquasi-IPN/GNPscompositematricesquasi-IPN/GNPs复合介质TEMimagesof(A)GNPs20(~20nm),(B)GNPs40(~40nm),and(C)GNPs60(~60nm)inwater,and(D)GNPs40(~40nm)inquasi-IPN3polymersolution.DuringthepreparationofAucolloids,citrateactedasbothareducingagentandacappingagenttoavoidaggregationofGNPs.TheparticlesizesofpreparedGNPscouldbecontrolledbytheamountoftrisodiumcitrate.Thefullfour-color(baseC,T,A,G)electropherogramsofBigdyeTerminatorV3.1sequencingstandardDNAsamplebyCEusing2.5%w/vquasi-IPN3/GNPs40-1at50℃.Sequencingconditions:effective/totallengthofbarefused-silicacapillaries,50/61cm;id/od,75/365μm;sequencingelectricfieldstrength,150V/cm;DNAelectrokineticinjection,41V/cmfor30s;anodebuffer,1×TTE;cathodebuffer,1×TTE/7Murea.30405060708090200030004000500060007000304050607080902000300040005000600070003040506070809020003000400050006000700030405060708090200030004000500060007000Blue,GGreen,AYellow,TFluorescenceintensityMigrationtime(min)Red,CDNAsequencingbyCEanddataanalysis1214161820222426283032200040006000121416182022242628303220004000600012141618202224262830322000400060001214161820222426283032200040006000365218Migrationtime(min)Blue,G358210Green,A301Yellow,T356207FluorescenceintensityRed,CThepartialfour-color(baseC,T,A,G)electropherogramsofstandardDNAsampleusing2.5%w/vquasi-IPN3/GNPs40-1byCEat50℃.Electrophoresis,2007,28,1072-1080.Electrophoresis,2007,28,2998-3007中国发明专利：申请号：200610114099.3.公开号：CN1966719A1)HNO3COOHCOOH2)SOCl23)HOCH2CH2OHBrBrO4)COOCH2CH2OOCCBrCH3CH3COOCH2CH2OOCCBrCH3CH3COOCH2CH2OOCCCH3CH3CH2CHBrmC=ONH3CCH3COOCH2CH2OOCCCH3CH3CH2CHBrmC=ONH3CCH3DMACuBrMe6[14]aneN4MWNTMWNT-COOHMWNT-BrMWNT-PDMAElectrophoresis,2008,29,4637-4645.+MWNT-PDMAquasi-IPNquasi-IPN/MWNT-PDMAdouble-networkmatrixquasi-IPN/functionalizedmulti-walledcarbonnanotubes020040060080010000.40.60.81.01.21.4ResolutionBasenumberquasi-IPN(2.5%)quasi-IPN/MWNT-PDMA2(2.0%+0.038mg/mL)quasi-IPN/MWNT-PDMA2(2.5%+0.038mg/mL)quasi-IPN-H(2.5%)POP-6SchematicrepresentationofpreparationofMWNT-COOH,MWNT-BrandMWNT-PDMAviaATRPSchematicrepresentationoftheformationofquasi-IPN/MWNT-PDMAdouble-networkcompositesievingmatrix.TEMimagesof(A)crudeMWNTsinwaterand(B)MWNT-PDMAinquasi-IPNsolution.Resolutionvs.basenumberinDNAsequencingbyCEusingquasi-IPNOHHydroxyethylcelluloseHydroxyethylcelluloseHydroxyethylcelluloseOHOHOHOOBrOOONH2BrOOONH2OHOOBrBrOBrBracrylamideligand,cat.OHHydroxyethylcelluloseHydroxyethylcelluloseHydroxyethylcelluloseOHOHOHOOBrOOONH2BrOOONH2OHOOBrBrOBrBrnnTEA,DMAP■HEC○HEC-g-PAM-1▲HEC-g-PAM-2◇HEC-g-PAM-3▼HEC-g-PAM-41015202530354045505560020406080100120140Temperature(oC)Viscosity(mPa/s)02004006008001000120014000.000.050.100.150.200.250.300.35ResolutionBasepair(bp)HECHEC-g-PAM-1HEC-g-PAM-2HEC-g-PAM-3HEC-g-PAM-4PDMAElectrophoresis,2007,28,3223-3231.HEC-g-PAMFormationofHEC-g-PAMViscosityversustemperaturefordifferentHEC-g-PAMcopolymers.SchematicrepresentationofthestructureofHEC-g-PAMcopolymerandofitsadsorptiontoacapillary