生物传感器-11-2

纳米生物传感器—分子信标分子信标作为具有高灵敏度和选择性的核酸探针，能方便的运用于核酸与蛋白质的研究，能够为生物大分子的功能和作用机理的研究提供丰富的实时信息，成为一种极具发展潜力的新型核酸探针。分子信标是Tyagi和Kramer在1996年首次合成的，它的设计非常巧妙。分子信标将核酸的序列结构信息转变为荧光信号，具有很高的灵敏度与选择性。分子信标是一段呈发夹结构的合成的短链DNA，其环状部分是一段含有20个左右碱基的与目标核酸互补的核酸序列，发夹的两臂是序列互补的5～8个碱基对并在两端分别固定有荧光基团和荧光熄灭基团。基于荧光共振能量转移原理，分子信标类似于一个开关，当荧光基团和熄灭集团靠得很近时不发荧光，即处于关闭状态。但是当分子信标与互补的DNA发生相互作用时，由于分子间的相互作用力使发夹状的结构打开，此时就能观察到较强的荧光，即处于开启状态。QFQFDNA分子信标与目标DNA作用的原理图SSB+MBFQ分子信标与蛋白质反应的原理图原理分子信标的设计分子信标一般包括三部分：1.识别探针部分（即发夹结构的环部）此部分与目标核酸分子杂交，这一部分长度为15-30个碱基，序列与目标核酸的序列完全互补。在杂交双链中若有一个错配的碱基，其热稳定性就会显著下降。如果以19个碱基的DNA链中，设只有一个位点A发生点突变，则杂交物在50C时的相对稳定性有很大差别：A-T（完全配对）1.0G-T（稳定错配）0.18A-A（不稳定错配）0.005因此，分子信标可用于检测点突变。发夹结构的环部2.信标柄部是指信标分子两端长度为5-8个碱基相互互补的部分，往往由GC含量较高的碱基组成以提高结合能力并减少长度。柄部结合能的选择要与分子信标和目标核酸杂交结合能相匹配。柄部的存在使信标分子的杂交特异性明显高于相应的线性探针。3.荧光基团和荧光熄灭基团以及连接臂最常用的荧光熄灭基团二甲氨基偶氮苯甲酰（DABSYL）基，采用DABSYL作猝灭剂是由于它对多种荧光素都有很强的荧光猝灭效率。荧光剂和猝灭剂与核苷酸的连接要引入长度为7-9个原子的连接臂。分子信标的柄部分子信标的性质在室温处于自由状态时分子信标的荧光信号微弱，随着溶液温度升高荧光逐渐增强，说明分子信标的柄部受温度影响而打开了。分子信标的熔点温度取决于柄部长度、柄部中GC含量以及溶液中盐的浓度。二价阳离子尤其是Mg2+对分子信标的柄部杂交有稳定作用。例如，13bp的柄部，在Mg2+不存在时熔点为27C，而在1mmol/LMgCl2为56C。5-813bp的柄部，在Mg2+不存在时不能形成稳定的杂交物，而在1mmol/LMgCl2中形成稳定的发夹结构，所以分子信标杂交实验均在含有MgCl2的缓冲溶液中进行。优点•高灵敏,信噪比高•无须分离多余的探针分子,直接在均相溶液里测定•高特异性,可以识别单碱基突变•形成分子信标阵列•连接不同的荧光素，可以形成多核酸检测Real-timemonitoringofpolymerasechainreaction(PCR)在PCR扩增时管内加入与扩增靶标序列互补的分子信标，用荧光计监测就可以不需分离多余的信标而动态地实时定量测定扩增靶标的量。FIG.2.Fluorescenceofanallele-specificmolecularbeaconasafunctionoftemperatureintheabsenceoftarget(bottomcurve),inthepresenceofperfectlycomplementarytarget(topcurve),andinthepresenceofatargetcontainingamismatchednucleotideatthesameposition(middlecurve).Duetotheirhigherfluorescenceperfectlycomplementaryhybridscanbedistinguishedfrommismatchedhybridsattemperaturesinthe“windowofdiscrimination.”FIG.3.Phasetransitionsofmolecularbeacons.Whenannealed,themolecularbeacon–targethybridisfluorescent.Asthetemperatureisraised,thefluorescentprobe–targethybriddenaturesandanonfluorescentmolecularbeaconinaclosedformationisformed.Onfurtherriseintemperature,thehairpinstemdenaturesandthemolecularbeaconformsafluorescentrandomcoil.Peptide-linkedmolecularbeaconsforefficientdeliveryandrapidmRNAdetectioninlivingcellsRNAdetectioninlivingcellsDualFRETmolecularbeaconsformRNAdetectioninlivingcellsHybridizationofdonorandacceptormolecularbeaconstoadjacentregionsonthesamemRNAtargetresultsinFRETbetweendonorandacceptorfluorophoresupondonorexcitation.(AandC)Fluorescencesignalofsinglerandomsequencemolecularbeaconsin(A)normally-growingand(C)stimulatedHDFcells,respectively,representingthebackgroundduetobeacondegradationandnon-specificinteractions.(BandD)FluorescencesignalduetosingleK-rastargetingdonorbeaconsin(B)normallygrowingand(D)stimulatedHDFcellsunderCy3excitation(545nm)andemissiondetection(570nm).(E)normallygrowingand(G)stimulatedHDFcells,respectively,underCy3excitation(545nm)andFRETdetection(665nm),representingthebackgroundindualFRETbeaconassays.(FandH)FluorescencesignalduetoK-rastargetingdualFRETmolecularbeaconsin(F)normallygrowingand(H)stimulatedHDFcellsusingFREToptics(i.e.545nmexcitationand665nmemissiondetection).ThedualFRETmolecularbeaconsgaveamuchbettersignal-to-backgroundratio,andamorequantitativemeasureofmRNAexpressionlevel(I).ProteinstudiesMolecularbeaconaptamer¯uorescesinthepresenceofTatproteinofHIV-1GenestoCells(2000)5,389-396RatioofenhancementoffluorescenceofoligomerF-BA1-DbytheTat-derivedpeptides.Real-timemonitoringofnucleicacidligationinhomogenoussolutionsusingmolecularbeaconsNucleicAcidsResearch,2003,Vol.31,No.23e148Real-timefluorescencescansandcorrespondinggelelectrophoresis.Lanes1and2areforsampleD;3and4forsampleC;5and6forsampleB;and7and8forsampleA.Lanes1,3,5and7representsamplesD,C,BandAbeforetheadditionofT4DNAligase,whilelanes2,4,6and8representcorrespondingsamplesobtainedat360saftertheadditionofligase.SpectroscopicFeaturesofDualFluorescence/LuminescenceResonanceEnergy-TransferMolecularBeaconsAnal.Chem.2003,75,3697-3703GeneticanalysisTripartitemolecularbeaconsNucleicAcidsResearch,2002,Vol.30No.18e94ReversibleRatiometricProbeforQuantitativeDNAMeasurementsAnal.Chem.2004,76,947-952MBbiosensorMolecular-beacon-basedarrayforsensitiveDNAanalysisGangYaoandWeihongTan*AnalyticalBiochemistry331(2004)216–223Hybridization-BasedUnquenchingofDNAHairpinsonAuSurfaces:Prototypical“MolecularBeacon”BiosensorsHuiDu,‡MatthewD.Disney,‡BenjaminL.Miller,*,†andToddD.Krauss*,‡DepartmentofDermatology,DepartmentofChemistry,andTheCenterforFutureHealth,UniVersityofRochester,J.AM.CHEM.SOC.2003,125,4012-4013Electrochemicalinterrogationofconformationalchangesasareagentlessmethodforthesequence-specificdetectionofDNAChunhaiFan*,KevinW.Plaxco*†‡§,andAlanJ.Heeger*‡¶UniversityofCalifornia,SantaBarbara,CA93106PNASAugust5,2003vol.100no.16,9134–9137PhotoluminescenceandelectrochemiluminescenceofaRu(II)(bpy)3-quencherdual-labeledoligonucleotideprobeCHEM.COMMUN.,20032710–2711应用其它方法研究分子信标与目标核酸（或目标蛋白）的相互作用TetheredDNAhairpinsfacilitateelectrochemicaldetectionofDNAligationAnalyst,2005,130,345–349Structure:AG-richnucleicacidsequencebindsheminandyieldsabiocatalyticcomple