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簡(jiǎn)介：中文中文2830字出處出處WANGJ,GUODONGLIUAULTRASENSITIVEELECTRICALBIOSENSINGOFPROTEINSANDDNA鈥CARBONNANOTUBEDERIVEDAMPLIFICATIONOFTHERECOGNITIONANDTRANSDUCTIONEVENTSJJOURNALOFTHEAMERICANCHEMICALSOCIETY,2004,1261030101超靈敏免疫和超靈敏免疫和DNADNA電化學(xué)生物分析電化學(xué)生物分析應(yīng)用碳納米管放大分子識(shí)別和傳導(dǎo)過(guò)程應(yīng)用碳納米管放大分子識(shí)別和傳導(dǎo)過(guò)程蛋白質(zhì)和DNA檢測(cè)技術(shù)在基因疾病的診斷和治療，傳染性疾病的檢測(cè)，藥物的開(kāi)發(fā)，生物戰(zhàn)爭(zhēng)預(yù)警中起著非常重要的作用。這些生物檢測(cè)通常依賴DNA雜交或抗原抗體相互作用，可達(dá)到超靈敏度的檢測(cè)。由于電化學(xué)傳感器具有靈敏度高、簡(jiǎn)單、可微型化，低成本和高需求的特點(diǎn)，非常適用于生物檢測(cè)。酶標(biāo)記在蛋白質(zhì)和DNA超靈敏電化學(xué)生物親和性檢測(cè)中起著很大的作用。HLELLER，小組5，6通過(guò)DNA上連接HRP酶標(biāo)記物和采用一種可加快電子傳遞的氧化聚合物可實(shí)現(xiàn)DNA的高靈敏度的電化學(xué)檢測(cè)（低至5ZMOL）。WILLNER,S小組7～8通過(guò)生物催化沉積酶反應(yīng)產(chǎn)物可獲得信號(hào)的多重放大，從而實(shí)現(xiàn)極低的檢測(cè)限（25AMOL），酶聯(lián)電化學(xué)蛋白質(zhì)檢測(cè)信號(hào)可通過(guò)雙酶底物體系底物循環(huán)或者酶產(chǎn)物的離子交換富集產(chǎn)物來(lái)進(jìn)行放大。然而，在電化學(xué)生物檢測(cè)中，放大生物識(shí)別傳導(dǎo)信號(hào)仍是一個(gè)重大的挑戰(zhàn)。為了滿足蛋白質(zhì)和核酸電化學(xué)檢測(cè)高靈敏度的要求，我們需要新的方法，通過(guò)酶生物催化反應(yīng)來(lái)放大信號(hào)。在本文中，我們利用碳納米管（CNTS）顯著放大蛋白質(zhì)和DNA的識(shí)別和電化學(xué)傳導(dǎo)信號(hào)。CNTS所特有的電學(xué)性能、化學(xué)性能和機(jī)械性質(zhì)使其非常適用于電化學(xué)傳感器1，2。大多數(shù)CNT傳感器主要是利用CNTS特有的表面性質(zhì)來(lái)促進(jìn)在生物催化裝置中電子轉(zhuǎn)移反應(yīng)，在我們新的生物親和性檢測(cè)中（圖1），CNTS起著放大識(shí)別和傳導(dǎo)信號(hào)的雙重作用，也就是CNTS上載有大量酶及積累了大量酶反應(yīng)的產(chǎn)物。這些新奇的方法和CNTS預(yù)富集的功能反映了CNTS具有很大的比表面積，并可用ALP酶標(biāo)來(lái)證實(shí)。通過(guò)采用CNTS的放大處理從而降低檢測(cè)限的一些方法已被報(bào)道過(guò)，因此很適用于電化學(xué)DNA檢測(cè)。圖，這些微觀圖片是HITACHIH7000儀器在工作電壓為75KV下拍下的。從圖3DNA分子雜交（A）和抗原抗體生物檢測(cè)中可以看到由于CNT的雙重放大作用引起了傳感信號(hào)的明顯增強(qiáng)?；趩蚊笜?biāo)記物和一個(gè)玻碳電極的傳統(tǒng)檢測(cè)方法既不能對(duì)10PGML1的目標(biāo)DNA（A，A）也不能對(duì)80PGML1的IGG（B，B）產(chǎn)生響應(yīng)?；谳d有ALP酶的CNTS（B）的第一放大步驟為這些分析物的低濃度檢測(cè)提供了方便。單ALP酶檢測(cè)即使在分析物濃度較高（1000倍）的條件下仍顯示一個(gè)較低的信號(hào)（圖中未標(biāo)出）。改進(jìn)方法后的檢測(cè)靈敏度達(dá)到將近104，正好與每個(gè)CNT上載有的ALP酶估計(jì)量相一致。用涂覆上鏈霉親合素的聚苯乙烯代替CNT來(lái)裝載粒子獲得的靈敏度增強(qiáng)約僅為單酶檢測(cè)的50倍。在信號(hào)第二放大途徑，即用CNT修飾傳感器可獲得更強(qiáng)的DNA和蛋白質(zhì)檢測(cè)信號(hào)（約是用鏈霉親合素修飾聚苯乙烯檢測(cè)的30倍）（C）。后者反映了在CNT層強(qiáng)烈地吸附著大量游離的Α奈酚。在CNT上富集產(chǎn)物的現(xiàn)象可用由沉積時(shí)間的不同而引起的Α奈酚信號(hào)的突增來(lái)描述（與裸電極上產(chǎn)生的時(shí)間信號(hào)關(guān)系相比較；見(jiàn)圖2的支持信息）。圖310PGML1目標(biāo)寡核苷酸（A）和80PGML1IGG（B）分別在玻碳電極（A）單ALP酶標(biāo)（B）和載有大量ALP酶標(biāo)的CNT上用計(jì)時(shí)電勢(shì)分析法進(jìn)行檢測(cè)產(chǎn)生的信號(hào)。在檢測(cè)中（C）除了使用CNT修飾的玻碳電極外與（B）均相同。磁性粒子量，50UG；分別進(jìn)行20和30分鐘的DNA雜交和抗原/抗體免疫反應(yīng)；樣品體積，50UL。檢測(cè)，往樣品中加入50ULΑ奈基磷酸鹽（50MM）溶液進(jìn)行酶反應(yīng)20分鐘。產(chǎn)物Α奈酚的測(cè)量在裸或者是已用

簡(jiǎn)介：MICROSATELLITEINSTABILITYAND/ORLOSSOFHETEROZYGOSITYINYOUNGGASTRICCANCERPATIENTSINITALYYIHHORNGSHIAO1,DANIELABOVO2,MARIAGUIDO2,CARLOCAPELLA3,MAUROCASSARO2,GRAZIELLABUSATTO2,VALENTINARUSSO2,4,ANGELOSIDONI5,ANNARPARENTI2ANDMASSIMORUGGE21LABORATORYOFCOMPARATIVECARCINOGENESIS,NCIFCRDC,NATIONALINSTITUTESOFHEALTH,FREDERICK,MD,USA2DEPARTMENTOFONCOLOGICALANDSURGICALSCIENCES,CATTEDRADIISTOCHIMICAEIMMUNOHISTOCHIMICAPATOLOGICA,IIICATTEDRADIANATOMIAPATOLOGICA,UNIVERSITA′DEGLISTUDI,PADOVA,ITALY3DEPARTMENTOFCLINICALANDBIOLOGICALSCIENCE,UNIVERSITYOFPAVIA,VARESE,ITALY4DEPARTMENTOFPATHOLOGY,UNIVERSITYOFCATANIA,CATANIA,ITALY5DEPARTMENTOFPATHOLOGY,UNIVERSITYOFPERUGIA,PERUGIA,ITALYGASTRICCANCERSARERARELYDIAGNOSEDBEFORETHEAGEOF40YEARSANDTHEINCIDENCEREACHESAPEAKDURINGTHE7THDECADEINTHEGENERALPOPULATIONAMOLECULARMECHANISMOFEARLYTUMORONSETMAYBEDETERMINEDBYCOMPARINGMICROSATELLITEINSTABILITYMSI,INDICATIVEOFERRORPRONEMISMATCHREPAIR,ANDLOSSOFHETEROZYGOSITYLOHBETWEENGASTRICCANCERSINPATIENTSI40YEARSOFAGEANDTHOSEOFOLDERAGESTHREETO5CHROMOSOMALLOCI,WHEREMSIAND/ORLOHARECOMMONLYFOUNDINGASTRICCANCERSINTHEGENERALPOPULATION,WEREEXAMINEDINFORMALINFIXED,PARAFFINEMBEDDEDSAMPLESFROM102PATIENTSI40YEARSOFAGEUSINGAPOLYMERASECHAINREACTIONBASEDNONRADIOACTIVESCREENINGMETHODMSIAND/ORLOHATAMINIMUMOF1LOCUSWEREDETECTEDIN11/102PATIENTSTHEFREQUENCYOFMSIAND/ORLOHATTHED11S904LOCUSWASSIGNIFICANTLYHIGHERTHANTHATATTHED2S119,D2S123,D5S409ANDIFNAREGIONSNOPREFERENTIALGENETICCHANGESATTHED11S904LOCUSWEREOBSERVEDINELDERLYPATIENTSAMONGSEVERALCLINICOPATHOLOGICALVARIABLES,ASTATISTICALLYSIGNIFICANTASSOCIATIONWITHMSIAND/ORLOHWASOBSERVEDONLYFORTUMORSLOCATEDATTHECARDIA,COMPAREDWITHTUMORSATTHEANTRUMANDTHECORPUSOURFINDINGSSUGGESTTHATAUNIQUEMECHANISMMAYBEINVOLVEDININCREASINGTHESUSCEPTIBILITYOFTHED11S904LOCUSFOREITHERMSIORLOH,ESPECIALLYFORCARDIATUMORSINYOUNGPATIENTSEARLYONSETOFGASTRICCANCERSINPATIENTSI40YEARSOFAGEISASSOCIATEDWITHGENETICCHANGESATPREFERENTIALCHROMOSOMALLOCI,INCLUDINGD11S904INTJCANCER8259–62,1999?1999WILEYLISS,INCALTHOUGHGASTRICCANCERINCIDENCEHASBEENDECLINING,ITWASSTILLRANKEDASTHE2NDMOSTCOMMONCANCERANDTHE2NDLEADINGCAUSEOFCANCERDEATHINTHEWORLDINTHE1990SPARKINETAL,1993BECAUSEOFTHEAGGRESSIVENATUREOFGASTRICCANCERS,THEOVERALL5YEARSURVIVALRATEISLESSTHAN20BREAUXETAL,1990GASTRICCANCERSUSUALLYOCCURINPATIENTSOLDERTHAN50YEARSOFAGEONLYABOUT5OFGASTRICCANCERPATIENTSAREYOUNGERTHAN40YEARSNEUGUTETAL,1996GASTRICCANCERSINPATIENTS?40YEARSOFAGEAREMOREAGGRESSIVETHANTHOSEINELDERLYPATIENTSFUJIMOTOETAL,1994COMPARISONOFGENETICALTERATIONSINGASTRICCANCERSBETWEENPATIENTS?40YEARSOFAGEANDOLDERPATIENTSMAYIDENTIFYASPECIFICMOLECULARMECHANISMASSOCIATEDWITHANEARLYONSETOFTUMORMICROSATELLITESARESHORTTANDEMLYREPEATEDDNASEQUENCESANDPRESENTTHROUGHOUTMAMMALIANGENOMESREPETITIVESEQUENCESOFDI,TRIANDTETRANUCLEOTIDEREPEATSAREPRESENTINMORETHAN50OFTHEHUMANGENOMEBECKMANANDWEBER,1992THETANDEMREPEATSOFDCDANAREPARTICULARLYABUNDANTALTERATIONSINTHENUMBEROFREPEATSPERSITE,KNOWNASMICROSATELLITEINSTABILITYMSI,HAVEBEENIMPLICATEDINVARIOUSHUMANDISEASES,INCLUDINGNEOPLASMSSPEICHER,1995MICROSATELLITESEQUENCESAREALSOHIGHLYPOLYMORPHICPOLYMORPHISMDUETODIFFERENTNUMBEROFREPEATSISVERYUSEFULTODETERMINEGENETICALTERATIONS,SUCHASLOSSOFHETEROZYGOSITYLOHINTHISSTUDY,WEEXAMINED5CHROMOSOMALLOCI,WHEREMSIAND/ORLOHARECOMMONLYFOUNDINGASTRICCANCERSINTHEGENERALPOPULATIONRHYUETAL,1994CHONGETAL,1994NAGELETAL,1995TAMURAETAL,1995LINETAL,1995SERUCAETAL,1995BUONSANTIETAL,1997,INGASTRICCANCERPATIENTS?40YEARSOFAGETHEFREQUENCYOFMSIAND/ORLOHANDTHEIRASSOCIATIONWITHOTHERCLINICOPATHOLOGICALDATAAREDISCUSSEDMATERIALANDMETHODSPATIENTSFORMALINFIXED,PARAFFINEMBEDDEDTISSUEBLOCKSFROM102PATIENTS?40YEARSOFAGEMEANAGE35YEARSRANGE16–40YEARSWERERETRIEVEDFROMTHEARCHIVESOFMANYITALIANHOSPITALSWITHCOMPARABLEGASTRICCANCERINCIDENCEDEMOGRAPHICANDPATHOLOGICALDATA,INCLUDINGAGE,GENDER,SITEOFTUMORANDTUMORSTAGEUSINGTHETNMSYSTEMBEAHRSANDMYERS,1983,WEREOBTAINEDFROMACOLLABORATIVEMULTICENTERSTUDYTHESITEOFTUMORWASCATEGORIZEDINTOTHEANTRUM,CORPUSORCARDIAOFSTOMACHNOCANCEREXTENDEDBEYONDTHEGASTRICESOPHAGEALJUNCTIONACCORDINGTOTHELAURE′N(xiāo)SYSTEM1965,GASTRICCANCERSWERECLASSIFIEDASEITHERINTESTINALORDIFFUSETYPEWHENBOTHPHENOTYPESCOEXISTED,CLASSIFICATIONOFTHETUMORTYPEWASBASEDONTHEMOSTREPRESENTATIVEHISTOLOGYGASTRITISNONATROPHICVSATROPHIC/METAPLASTICWASCLASSIFIEDUSINGTHEHOUSTONUPDATEDSYDNEYSYSTEMDIXONETAL,1996ALLCASESWEREJOINTLYASSESSEDBY2AUTHORSMCANDMRDNAEXTRACTIONNEOPLASTICANDADJACENTNONNEOPLASTICAREASWEREMICRODISSECTEDSEPARATELYFROMUNSTAINEDFORMALINFIXED,PARAFFINEMBEDDEDSECTIONSFORDEPARAFFINIZATION,PROTEINASEKDIGESTIONANDDNAPURIFICATION,ASDESCRIBEDPREVIOUSLYSHIAOETAL,1994MICRODISSECTEDNEOPLASTICAREASCONTAINEDATLEAST50TUMORCELLSWHENPOSSIBLE,LYMPHOCYTESWEREUSEDASANONNEOPLASTICCELLCONTROLPOLYMERASECHAINREACTIONPCRFIVECHROMOSOMALLOCIWITHCANDINUCLEOTIDEREPEATSD2S119,D2S123,D5S409,IFNAANDD11S904WERESELECTEDFORPCRPRIMERSD2S123AFM093XH3AANDAFM093XH3MD5S409AFM184YB6AANDAFM184YB6MIFNAIFNAPCR21ANDIFNAPCR22D11S904AFM081ZA5AANDAFM081ZA5MWEREOBTAINEDFROMTHEGENOMEDATABASEWEBSITEWWWGDBORGFORD2S119,UPPER5?CCAGT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