納米金及熒光成像

納米金生物合成及其在生命科學(xué)中的應(yīng)用學(xué)生：張晶璞

主要內(nèi)容1納米金的生物合成1.1光合細(xì)菌1.2光合細(xì)菌合成納米金2納米金在生命科學(xué)中的應(yīng)用2.1納米金性質(zhì)2.2基因檢測2.3蛋白質(zhì)和氨基酸檢測2.4細(xì)胞成像和光熱治療3熒光光譜與成像(Fluorescencespectrumandimaging)3.1Introduction3.2GNPs-Based“Turn-On〞FluorescentProbe3.3金納米棒與熒光1納米金的生物合成光合細(xì)菌(PhotosyntheticBacteria,PSB)不放氧光合異養(yǎng)生長：厭氧光照條件，有機(jī)物作電子供體兼碳源或二氧化碳為碳源；好氧異養(yǎng)生長：好氧黑暗條件，有機(jī)物作碳源。分布土壤、泥潭沼澤、海水、淡水、地下水污泥、水生植物根系等區(qū)域分類藍(lán)細(xì)菌(門)紅螺菌(目)〔不放氧光合作用〕紅螺菌科紅假單孢菌屬沼澤紅假單胞菌(Rhodopseudomonaspalustris)(菌種形態(tài)短桿狀或卵圓形)莢膜紅假單胞菌(Rhodopseudomonascapsulata)著色菌科綠菌科功能固氮、固碳、放氫等1.1光合細(xì)菌1.2光合細(xì)菌合成納米金1.2.1納米金生物合成現(xiàn)狀1概述納米技術(shù)一個重要的開展方向開展新的實(shí)驗(yàn)方法以重復(fù)合成尺寸、多分散性、化學(xué)組成和形狀均可控制的納米材料。納米材料的生物合成清潔、無毒、綠色化學(xué)、環(huán)境友好金納米金顆粒的生物合成植物：檸檬香草、蘆薈、海藻、香樟樹葉真菌：端孢菌放線菌細(xì)菌：莢膜紅假單胞菌、芽孢桿菌2

BiologicalsynthesisofGNPswithRhodopseudomonascapsulata(1)

CFE(10mL)+HAuCl4(pH=6,30℃,48h)①c(HAuCl4

)=2.5×10-4Mcolor:vividrubyred;UV-visspectra:asinglebutstrongSPRbandabsorptionpeakcenteredatabout530nm;TEM:sphericalwithparticlesizerangingfrom10to20nm.Figure1.UV-visiblespectraofgoldnanoparticlespreparedbyCFEat2.5×10-4MHAuCl4.Figure2.(A)RepresentativeTEMimageofgoldnanoparticlesproducedbyCFEat2.5×10-4MHAuCl4.(B)SAEDpatternfromgoldnanoparticlescorrespondingtoA.ShiyingHe,NingGu.

Biotechnol.Prog,2008,24:476-480.②c(HAuCl4

)=5×10-4Mcolor:

grayblue;UV-visspectra:a

boardpeak

observedfrom500to900nm;TEM:goldnanowireswiththediameterofapproximately50-60nm.Figure3.UV-visiblespectraofgoldnanowiresspreparedbyCFEat5.0×10-4MHAuCl4.Figure4.(A)RepresentativeTEMimageofgoldnanoparticlesproducedbyCFEat5×10-4MHAuCl4.InsetistheSAEDpatternfromgoldnanoparticlescorrespondingtoA.(B)HighermagnificationTEMimageofnanowiresShiyingHe,NingGu.

Biotechnol.Prog,2008,24:476-480.(2)R.capsulata(1gwetweight)+HAuCl4(c(HAuCl4)=1.0×10-3M,20mL,30℃,48h)Figure5.(A)TEMimageofthegoldnanoparticlesproducedbythereactionof10?3MaqueousHAuCl4solutionwithbacteriaR.capsulatabiomassatpH7and(B)atpH4.TheinsetshowstheirEDpatterns.(C)UV–VisabsorptionspectraofgoldnanoparticlesproducedatpH7(curve1)andpH4(curve2).①pH=7UV-visspectra:540nm;TEM:sphericalwithasizerangeof10–50nm.②pH=4UV-visspectra:540nm(TSPR,bandI);900nm(LSPR,bandII).TEM:triangulargoldnanoplatesinthesizeof50–400nm.S.Heetal.Materialsletters,2007,61:3984–3987.Detectingtheviabilityofthebacteriaonagarplates:resistanttothegoldionsandgoldNPs.producinggoldnanoparticles

extracellularly.2

BiologicalsynthesisofGNPswithFungus,Trichotheciumsp.(1)ExtracellularGoldNanoparticleSynthesisunderstationaryconditionsreleaseofreducingproteins/enzymesinsolutionFigure6.(A)UV-visspectrarecordedasafunctionoftimeofreactionofa10?3MaqueoussolutionofHAuCl4withTrichotheciumsp.biomassgrowninstationaryphase.Theinsetshowsatesttubeofthegoldnanoparticlesolutionformedattheendofthereaction(48h).color:purpleUV-visspectra:～535nm;steadilyincreasesinintensityasafunctionoftimeofreaction;stablewithlittlesignsofaggregation.acappingagentlikelytobeproteinssecretedbythefungalmat.periodicsamplingofaliquots(2ml)oftheaqueouscomponent.AbsarAhmadetal.J.Biomed.Nanotech,2005(1):47–53.avarietyofshapes:

goldnanoparticlescrystallizeintheformofpolygons(mainly

hexagonalandtriangularshapes);

size:5-200nm;

asolution-castfilm:depositedonacarbon-coatedcopperTEMgrid.Figure7.Fig.2.(A–E)RepresentativeTEMmicrographsrecordedfromdrop-castfilmsofthegoldnanoparticlessolutionformedbythereactionofchloroauricacidsolutionwithTrichotheciumsp.biomassgrowninstationaryphasefor48hatdifferentmagnifications.Arrowspointtosomeplaceswheretwoormoreflatparticlesoverlap.(F)Selectedareadiffractionpatternrecordedfromasinglegoldtriangle.TEM:AbsarAhmadetal.J.Biomed.Nanotech,2005(1):47–53.(2)IntracellularGoldNanoparticleSynthesisundershakingconditionsNoreleaseofreducingproteins/enzymesinsolutionUV-visspectra:Aunano-Trichotheciumcells:

～540nm,thebroadeningoftheresonancescatteringfromtheroughbiomasssurface;noabsorption:theaqueousHAuCl4solution;theas-harvestedfungalcells.Filmsofthefungalcells:solution-castingthewashedfungalcellsontoquartzandSi(111)wafersandthoroughlydryingthefilminflowingN2;operatinginthereflectionmodeataresolutionof2nm;thefiltrateofthereactionmixtureinthetransmissionmode.Figure8.UV-visspectrarecordedfrombiofilmsoftheTrichotheciumsp.biomassgrownundershakingconditionbefore(curve1)andafterexposureto10?3MaqueousHAuCl4solutionfor48h(curve2).TheinsetshowsaconicalflaskofTrichotheciumsp.biomassgrownundershakingconditionafterexposureto10?3MaqueousHAuCl4solutionfor72h.AbsarAhmadetal.J.Biomed.Nanotech,2005(1):47–53.TEM:Figure9.(A–C)RepresentativeTEMmicrographsrecordedatdifferentmagnificationsfromthinsectionsofstainedTrichotheciumsp.cellsafterreactionwithAuCl4?ionsfor72h.size:thenanoparticlesrangedfrom10–25nm.locationon:thecellwall(outerboundary)onthecytoplasmicmembrane(innerboundary)thinsectionsoftheAunano-fungalcell:60kV,40μmmeshcoppergrids

preparation,stainingAbsarAhmadetal.J.Biomed.Nanotech,2005(1):47–53.用光合細(xì)菌(沼澤紅假單胞菌)制取納米金顆粒的反響條件及機(jī)理初探1實(shí)驗(yàn)方案主要內(nèi)容2

預(yù)實(shí)驗(yàn)結(jié)果時間:實(shí)驗(yàn)條件：細(xì)菌細(xì)胞〔1g濕重〕+HAuCl4〔1×10﹣3M、pH=7〕+搖床200rmp，30℃實(shí)驗(yàn)現(xiàn)象：2天時混合液無明顯變化，即仍為淺黃色；4-5天后混合液中心懸浮有紫色絮狀物，水溶液仍為淡黃色。TEM結(jié)果未經(jīng)前處理而直接作電子顯微所得照片如下：1SAED結(jié)果

圖1混合液TEM圖像及相應(yīng)區(qū)域的ED圖像納米金顆粒合成位置：胞內(nèi)〔細(xì)胞膜及細(xì)胞壁〕胞外納米金顆粒濃度較低2細(xì)胞內(nèi)合成情況

細(xì)胞內(nèi)，量較少，粒徑較小圖2混合液TEM圖像團(tuán)聚，外包蛋白質(zhì)等有機(jī)物質(zhì)；單個分散體粒徑10-20nm.圖3混合液TEM圖像3細(xì)胞外合成情況細(xì)胞內(nèi)或外，無法判別；團(tuán)聚或重疊，外包蛋白質(zhì)等有機(jī)物質(zhì)4其它絮狀黑影可能是蛋白質(zhì)等有機(jī)物，在高電壓下不斷變化；泡狀結(jié)構(gòu)為細(xì)胞，由于未經(jīng)切片、染色等處理，故無法區(qū)分其結(jié)構(gòu)。3下一步改進(jìn)措施1細(xì)菌細(xì)胞+搖床UV-visspectra:細(xì)胞外合成：濾除細(xì)胞后直接測定濾液細(xì)胞內(nèi)合成：取混合液滴加于石英圓片上，后于流動N2中枯燥TEM:〔注意其電壓、網(wǎng)規(guī)格、膜性質(zhì)等〕細(xì)胞內(nèi)合成：經(jīng)切片、染色等前處理2細(xì)菌細(xì)胞+靜相細(xì)胞外合成3無細(xì)胞提取液細(xì)胞外合成2納米金在生命科學(xué)領(lǐng)域的應(yīng)用納米金顆粒(goldnanoparticals,GNPs)至少在一個維度上尺寸<100nm的金顆粒。

獨(dú)特的物理性質(zhì)和化學(xué)性質(zhì)：(l)易于制備，穩(wěn)定性好；(2)具有納米顆粒所共有的效應(yīng)；(3)具有獨(dú)特的電學(xué)效應(yīng)、光學(xué)效應(yīng)(光吸收和光散射)、磁學(xué)效應(yīng)、催化效應(yīng)和特殊的生物親和效應(yīng)。2.1納米金性質(zhì)突出的外表等離子共振性質(zhì)：球形納米金顆粒表現(xiàn)為單一的SPR峰。5nm、24.5nm、41nm、71.5nm、80(含>80)nm：橙、橙紅、紅、紫灰、金色。棒狀納米金顆粒具有橫向(TSPR)和縱向(LSPR)兩個SPR峰。LSPR峰隨金納米棒的長短軸比率增加而紅移。圖1球形納米金粒徑與SPR峰的關(guān)系[1]圖2不同長短軸比率的金納米棒的SPR譜圖[4]

2.2基因檢測納米金新型基因芯片1目視化基因芯片圖3目視化基因芯片檢測基因突變的原理[2]檢測機(jī)制：“三明治〞式反響〔1〕捕獲探針的固定〔2〕與靶DNA雜交〔3〕雜交信號放大特點(diǎn)：解鏈溫度(meltingtemperature，Tm)

高，半峰寬小。需在小范圍內(nèi)調(diào)節(jié)溫度。2微電極芯片圖4微電極芯片檢測基因突變的原理[2]根本原理：DNA雜交可以“牽引〞納米金顆粒至兩個微金電極之間，銀染后形成完整的銀層從而接通整個電路系統(tǒng)特點(diǎn)：不需嚴(yán)格控溫，室溫下即可到達(dá)檢測目的；微型化，高通量、高自動化，方便攜帶，快速檢測。2.2.2生物傳感器

石英晶體微天平(quartzcrystalmicrobalance,QCM)：質(zhì)量敏感的檢測傳導(dǎo)裝置1直接標(biāo)記堿基互補(bǔ)配對“三明治〞式雜交體系：巰基化的單鏈寡核苷酸探針固定在金膜外表作為捕獲探針金顆粒沉積金電極質(zhì)量增加探針振蕩頻率下降檢測靈敏度：納米金粒徑：靈敏度與納米金的粒徑大小成正比電極外表所占空間比較大，比較重pH和鹽濃度2間接標(biāo)記圖5利用QCM二級信號放大檢測基因突變的原理[3]生物素-鏈酶親和素原理：雜交信號一級放大：納米金與靶基因相連，檢測限為100fmol/L；雜交信號二級放大：納米金與生物素標(biāo)記的polyA和polyT結(jié)合，PolyT一端的生物素再次與納米金結(jié)合形成，檢測限為10fmol/L。特點(diǎn)：靈敏度要顯著高于直接標(biāo)記法。2.2.3交聯(lián)式檢測

圖6利用DNA連接酶檢測基因突變的原理[2]1三維網(wǎng)狀結(jié)構(gòu)〔“斑點(diǎn)識別法〞〕以DNA雜交體為紐帶的多個金顆粒構(gòu)成的三維網(wǎng)狀結(jié)構(gòu)原理：兩種探針：兩種不同的、分別與靶基因的兩端互補(bǔ)的巰基化寡核苷酸與納米金通過“Au-S〞共價鍵結(jié)合。DNA連接酶檢測限為74pmol/L。2二維聚集檢測體系：基底層：聚乙烯比色皿的底部脂質(zhì)雙分子層：兩面均帶正電荷金探針層：外表帶負(fù)電荷

2.2.4非交聯(lián)式檢測

根本原理：納米金分散單體呈紅色；納米金聚集體呈藍(lán)紫色。方法：特殊的能“變形〞的DNA探針(aptamer)單鏈DNA和雙鏈DNA對納米金外表不同的作用效果突變型引物對靶序列進(jìn)行等位特異性擴(kuò)增觀測信號：吸收光譜和顏色方面差異2.3蛋白質(zhì)和氨基酸檢測2.3.1蛋白質(zhì)檢測圖7用于三靶標(biāo)免疫檢測的金納米棒探針[4]

1免疫檢測〔1〕三靶標(biāo)免疫檢測3種不同比率(AR=2.1、4.5、6.5)的金納米棒偶聯(lián)3種不同的抗體分子(goatanti-humanIgGFab、rabbitanti-mouseIgGFab、rabbitanti-sheepIgG(H+L))金納米棒的LSPR產(chǎn)生紅移十六烷基三甲基溴化銨(CTAB)，11-巰基十一酸(11-mercaptoundecanoicacid,MUA)

〔2〕h-IgG檢測圖8基于沉積在石英片上的Aurod@SiO2復(fù)合顆粒膜對蛋白質(zhì)分子的比色檢測[4]

PVP:聚4-乙烯吡啶，APTMS:(3-氨基丙基)三甲基硅烷，GA:戊二醛

根本原理：抗原-抗體特異性識別作用LSPR的變化圖9通過抗體-抗原識別誘導(dǎo)金納米棒自組裝檢測IgG示意圖[4]PSS:聚苯乙烯磺酸鈉自組裝聚集

圖10SiO2@Aurod@ATP@SiO2復(fù)合顆粒的制備及其檢測h-IgG分子示意圖。APS:(3-氨基丙基)三甲氧基硅烷；GA:戊二醛；PEI:聚乙烯亞胺；PVP:聚乙烯吡咯烷酮；ATP:對巰基苯胺[4]拉曼信號的增強(qiáng)作用：多拉曼信號分子(ATP)吸附產(chǎn)生的多分子增強(qiáng)效應(yīng)金納米棒顆粒的金基底增強(qiáng)效應(yīng)2檢測鏈霉親和素(streptavidin)的蛋白傳感器

圖8金納米棒傳感器的制備示意圖[4]

三乙氧基巰醇(EG3SH)，巰基十六酸(MHA)

在磷酸緩沖液中的蛋白檢出限為94pM,在血清(serum)中的檢出限為19nM。

2.3.2氨基酸檢測圖9金納米棒的自組裝機(jī)理[4]

半胱氨酸和谷胱甘肽選擇性檢測：微摩爾量級圖11金納米棒(0.12nM)分別參加半胱氨酸(A)(a)0,(b)1.75,(c)2.0,(d)2.25,(e)2.5,(f)3μM和谷胱甘肽(B)(a)0,(b)7,(c)9,(d)11,(e)13,(f)14μM的吸收光譜圖[4]圖10半胱氨酸存在(a)和不存在(b,c)情況下的金納米棒的電鏡照片[4]

金納米棒鏈：端點(diǎn)到端點(diǎn)(end-to-end)的方式2.4細(xì)胞成像和光熱治療2.4.1細(xì)胞成像2.4.2光熱治療圖12anti-EGFR/Au納米球(A)和anti-EGFR/Au(B)納米棒,分別與HaCaT細(xì)胞、HSC細(xì)胞和HOC細(xì)胞在室溫下孵育30min后的光散射照片[4]

球形金納米顆粒：對可見光的強(qiáng)吸收特性使得光能轉(zhuǎn)換為熱能皮膚癌治療或者診斷金納米棒顆粒：對與其LSPR峰波長相近的近紅外光的強(qiáng)吸收使得光能轉(zhuǎn)換為熱能皮下組織癌癥的治療或者診斷3熒光光譜與成像(Fluorescencespectrumandimaging)3.1IntroductionPrinciplesandcorrelatedconcepts

PrinciplesoffluorecenceexcitationFluorophors

QDs(Quantumdots)RSFPs(Reversiblyswitchablefluorescentproteins)LAMPs(Localactivationofmolecularfluorescentprobes)GNPs(Goldnanoparticles)HJ(Hollidayjunction)Fluorescenceimagingtechnologyandtheir

applications

inlifesciencesmFRET(Single-moleculefluorescenceresonanceenergytrasfer)SCFM(Scanningconfocalfluorescencemicroscope)2PLSM(Twophoton-excitedfluorescencelaser-scanningmicroscopy)FLIM(Fluorescencelifetimeimaging)3.2GNPs-Based“Turn-On〞FluorescentProbeHaoWang,RonghuaYangetal.Anal.Chem.2008,80(23):9021–9028.1Mechanism

Figure1(A)SchematicdescriptionofcolorimetricandfluorescentsensingofHg2+basedonthemodification-freeAu-NPs.(B)StructuresofFAM,theFAM-labeledssDNA,1and2,theperfectedcDNA3,and11-merT-TmismatchedbasesDNA4of2.OligorepresentsthepositionofattachmentoftetheredthecolorofgoldcolloidandtheaggregationsofAu-NPs(pink-red/blue-gray);ssDNAadsorbsonnegativelychargedAu-NPs,whiledsDNAdoesnot;theexceptionalquenchingcapabilityofAu-NPstotheproximatefluorescentdye.3.2.1GNPs—GoldNanoparticle-BasedColorimetricand“Turn-On〞FluorescentProbeforMercury(II)IonsinAqueousSolution2ExperimentalresultsFigure2(A)EffectofHg2+ontheabsorptionspectraof1stabilizedAu-NPssolution.(a)AbsorptionspectrumofthemixturesolutionofAu-NPsand1(pH7.4,0.1MNaCl)intheabsenceofHg2+;(b)absorptionspectrumofthemixturesolutioninthepresenceof6.0μMHg2+.(B)Thecorrespondingcolorchangeof1stabilizedAu-NPsintheabsence(a)andthepresenceofHg2+(b).Figure3TEMimagesof1/Au-NPsin0.1MNaClsolutionintheabsence(A)andthepresenceof6.0μMHg2+(B).HaoWang,RonghuaYangetal.Anal.Chem.2008,80(23):9021–9028.Figure4Fluorescenceemissionspectraof1atdifferentexperimentconditions:(a)40nM1

inTris-HClbuffer(0.1MNaCl);(b)a+4.0μMHg2+;(c)a+0.8nMAu-NPs;(d)a+0.8nMAu-NPs+4.0μMHg2+.Excitationwavelengthwasat480nm.Figure5

Changesinthefluorescenceemissionspectraof1/Au-NPsintheTris-HClbuffer(0.1MNaCl)uponadditionofdifferentconcentrationsofHg2+.ThearrowsindicatethesignalchangesasincreasesinHg2+concentrations(0,0.08,0.2,0.4,0.8,1.6,2.4,4.0,and6.0μM).Excitationwavelengthwasat480nm.Inset:Responseparameter(R)asafunctionoflogarithmofHg2+concentration(M)atpH7.4.HaoWang,RonghuaYangetal.Anal.Chem.2008,80(23):9021–9028.3.2.2Awavelength-shiftingmo