CN101694832A - 形成多长度尺度薄膜的方法和组合物 - Google Patents
形成多长度尺度薄膜的方法和组合物 Download PDFInfo
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Abstract
本发明涉及形成多长度尺度薄膜的方法及组合物。所述方法包括(a)制备包含病毒和与病毒相结合的肽的组合物,其中所述肽选择性地与具有不同组成或晶面的晶状表面结构结合;和(b)由上述组合物制备多长度尺度薄膜。所述组合物包含病毒和与病毒相结合的肽,其中所述肽选择性地与具有不同组成或晶面的晶状表面结构结合,且所述组合物以多长度尺度薄膜的形式存在。
Description
本申请是申请号为02819565.5、申请日为2002年10月2日、发明名称为“利用遗传工程的中尺度病毒对杂合材料进行纳米级的排序”的中国发明专利申请的分案申请。
技术领域
本发明涉及能够与无机材料结合的有机物,尤其涉及能够与半导体材料结合并形成整齐排序结构的噬菌体。
背景技术
本主题申请的研究部分由National Science Foundation基金支持,政府享有某些权力。
在生物学系统中,有机分子对诸如碳酸钙和硅石等无机材料的晶核形成以及矿石相位具有显著的控制作用,同时对生物功能所需的复杂结构中模块的组装也具有显著的作用。
生物方法制备出的材料通常很柔软,由分子模块(即,脂类、肽类、以及核酸)的非常简单的集合构成,而这些分子模块却具有非常复杂的排列结构。半导体工业需要依靠一系列光刻技术处理步骤从而在集成电路上构建最小的部件,而与此很不相同的是,活性生物体大部分情况下利用同时在许多分子成分上起作用的非共价力来实现其结构。而且,这些结构通常能够在两个或多个可用构型之间进行精细的重排,而不改变任何分子组成。
利用“生物”材料处理下一代微电子设备为解决传统的处理方法中所存在的问题提供了一种可能的解决方案。该处理方法中的关键性因素是确定生物--无机材料合适的兼容性以及结合性,以及恰当的模块的合成。
发明概述
本发明人设计了结构成分,并制备了生物材料,该生物材料能够介导并控制无机材料的装配从而形成受控的复杂结构。利用生物材料来制备和设计具有所需要的电学或光学特性的材料,这种应用可用于减小部件的尺寸并用来增加对材料的诸如光-电特性的控制。半导体材料通常是由硫化锌、砷化镓、磷酸铟、硫化镉、砷化铝、锑化铝(aluminum stibinide)以及硅来制备的。这些半导体材料通常被归为族II-族V以及族II-族VI的半导体材料。
有机-无机杂合材料为新材料以及装置提供了一种新的途径。本发明人使用有机-无机杂合来选择能够与半导体材料结合的肽类。尺寸控制的纳米结构为半导体材料提供了可调节的光学和电学特性。在本发明中,有机加成材料被用来对半导体材料的无机形态、相位、以及晶核形成方向等进行修饰。生物材料的单分散特性使得该系统对高度有序的近晶排列结构具有兼容性。
在纳米级上构建整齐排序且良好控制的二维和三维结构是构建下一代的光、电、磁学材料及装置的主要目标。许多研究人员都主要利用传统的材料制备途径来构建这种结构。如本文所公开,本发明人证实了柔软的材料可作为自组织者,在纳米水平上对无机材料进行组织。Alivisators和Mirkin利用DNA识别连接子来形成特异的纳米粒子组合结构。Stupp和Coworkers在溶致液晶介质中使ZnS和CdS成核来制备纳米导线和纳米结构。然而,上述两种方法在长度尺度上都很受限,并仅能提供限定种类的用来起作用的无机材料。因此,需要提供在纳米级水平制备良好排序结构的其它方法。
本发明是基于下述认识:具有各相异性形状的单分散性生物材料可以作为构建良好排序结构的手段。本发明包括使用生物选择性和自装配来构建具有良好排序的纳米颗粒层的方法。纳米颗粒层可由诸如CdS,FeS,和ZnS等族II-VI的半导体材料构成。
本发明的一种形式是使用诸如噬菌体等自装配生物分子的方法,所述的生物分子通过遗传工程与半导体材料相结合从而组织成良好的排序结构。这些结构可以是诸如纳米颗粒的纳米级排列。以噬菌体为例,可对自装配的生物材料进行选择,使之对特定半导体表面具有特异性的结合性质。因此,修饰的噬菌体以及本文所述的方法可用于构建选定材料的良好排序的结构。
本发明的另一种形式是构建具有特定排序特性的纳米颗粒的方法。该方法可采用诸如下述步骤来实现:构建具有特定结合特性的诸如M13噬菌体,利用聚合酶链式反应将噬菌体扩增至高浓度,然后对菌体进行重悬。
也可用同样的方法构建具有三种液晶相(liquid crystalline phases)的噬菌体,一种是在向列相(nemetic phase)的定向排列,一种在胆固醇液晶相的扭曲向列相结构(twisted nemetic structure),以及在近晶相定向和定位排列。本发明的一种技术方案涉及制备聚合物的方法,如薄膜,该方法包括下述步骤:将自装配的生物分子扩增至高浓度,其中所述的生物分子包含与特定半导体表面结合的部分,然后将一种或多种半导体材料前体与自装配的生物分子相接触,从而形成晶体,或者介导晶体的形成。
本发明的又一种技术方案是构建具有不同的胆固醇节距(cholestericpitches)的纳米颗粒的方法,该方法使用诸如M13噬菌体与半导体表面结合,并将菌体重悬至多种不同的浓度。本发明的再一种形式是利用排序的纳米颗粒制备铸型薄膜的方法,该方法使用诸如遗传工程的M13噬菌体并将噬菌体重悬。
附图简要说明
为了更完整地理解本发明的特征以及优点,现并结合附图对本发明进行详细描述,其中不同附图中的相应的数字代表相应的部分,其中:
图1描述了本发明的选择性随机氨基酸序列(selected random amino acidsequences);
图2描述了本发明的XPS结构光谱;
图3描述了本发明的噬菌体识别异质结构;
图4-8描述了本发明的特定氨基酸序列;
图9(a)和9(b)描述了本发明的M13菌体的近晶排列示意图;
图10(a)-10(f)是A7-ZnS悬液的图像:(a)和(b)POM图像,(c)AFM图像,(d)SEM图像,(e)TEM图像,以及(f)TEM图像(具有电子衍射嵌入);其中,(a):A7-ZnS悬液(127mg/ml)的近晶悬液的POM图像,(b):A7-ZnS悬液的胆固醇相,(c):25%近晶悬液铸型薄膜的AFM图像,(d):(a)悬液的SEM图像,(e):与纳米颗粒结合的单个噬菌体的TEM图像,以及(f):用2%乙酸双氧铀染色的0.1%稀释的A7-ZnS悬液的TEM图像以及ZnS wurtzite(插入)的电子衍射;以及
图11(a)-11(f)是M13噬菌体纳米颗粒生物薄膜的图像:(a)薄膜照片,(b)薄膜结构的示意图,(c)AFM图像,(d)SEM图像,(e)和(f)沿着x-z和z-y平面的TEM图像;其中,(a):M13噬菌体-ZnS纳米颗粒生物膜的照片,(b):膜结构的示意图,(c):膜的自由表面的AFM图像,(d):膜的SEM图像,以及(e,f):分别显示了x-z和y-z平面的纳米颗粒排序的TEM图像。
本发明详细描述
尽管本发明下面将对所使用的实施例进行详细讨论,应当了解本发明提供了许多使用的发明观念,这些观念可在多种特定环境下实施。本文所讨论的特定实施例仅仅阐述了制造和使用本发明的特定方式,而并非为了对本发明做出限制。
本发明人曾经证明肽类能够与半导体材料结合。这些肽类被进一步开发为纳米颗粒成核的手段,并介导这些颗粒的自装配。这类肽的主要特征是它们对具有表面特异性(face specificity)的重要材料的识别及结合能力、对尺寸受限的晶体半导体材料的成核能力、以及对有核的纳米颗粒的结晶相的控制能力。这类肽还能通过控制肽类的纵横比(aspect ratio)来控制光学特性。
简要地,生物系统在非常微小的尺度上装配及其复杂结构的装置极大地激发了发明人想要找出具有类似作用的非生物系统。能够发明出用在令发明人感兴趣的电子或光学特性材料的方法将尤其具有意义,但是天然进化并未选择出在生物分子和这类材料之间相互作用。
本发明所基于的认识是,生物系统能够有效、精确地将纳米级的构成模块装配为具有高度完整性、受控的尺寸以及复合的均一性的具有复杂功能的结构。
一种提供随机有机聚合物池(random organic polymer pool)的方法是使用噬菌体展示文库,它是基于与M13大肠杆菌噬菌体的pIII包衣蛋白融合的7至12个氨基酸的随机肽形成的组合库,提供了能够与晶状半导体结构相互作用的不同的肽类。在噬菌体颗粒一端具有pIII包衣蛋白的5个拷贝,它们在颗粒上为10-16nm。该噬菌体展示方法在多肽--底物相互作用和编码该相互作用的DNA之间提供了物理连接。本发明的实施例中,有5个不同的单晶半导体:GaAs(100),GaAs(111)A,GaAs(111)B,InP(100)和Si(100)。这些底物能够对肽类--底物相互反应进行系统评价,并确认本发明方法在不同晶型结构上的一般用途。
将与特定晶体成功结合的蛋白序列从晶体表面洗脱下来,扩增诸如百万倍,在更严格的条件下与底物反应。将这一过程重复5次,在库中选择具有最特异结合的噬菌体。在经过例如第三、第四和第五次噬菌体选择后,分离出晶体特异性的菌体,并对其DNA进行测序。鉴别对晶体组合物具有选择性的肽类结合(例如,与GaAs结合而不与Si结合)以及对晶面具有选择性的肽类结合(例如,与(100)GaAs结合,而不与(111)B GaAs结合)。
分析选自GaAs(100)的20个克隆,以确定针对GaAs晶面的表位结合域。图1所示为修饰的pIII或pVIII蛋白的部分肽序列,显示了与GaAs接触的肽中的类似氨基酸序列。随着与GaAs晶面接触的数量的增加,无电荷极性的和路易斯碱的官能团也增加。三个、四个和五个循环的噬菌体克隆序列分别平均包含30%、40%、和44%的极性官能团,而路易斯碱官能团的部分同时从41%增加至48%~55%。路易斯碱中观察到的增加仅占本发明文库的随机12-mer肽的官能团的34%,这说明肽上的路易斯碱和GaAs晶面的路易斯酸位点之间的相互反应可以介导由这些克隆提供的选择性结合。
选自文库中修饰的12-mers的预期结构可以为伸展的构象,这应该对小肽类更合适,该构象使得肽比GaAs晶胞(5.65A°)更长。因此,在肽类对GaAs晶体的识别中,仅需要很小的结合域。这些短肽结构域,如图1所示,除了包含诸如天冬酰氨和谷氨酸盐等胺类路易斯碱(amine Lewis bases)外,还包含富含丝氨酸和苏氨酸的区域。为了确定精确的结合序列,已经用更短的文库对晶面进行筛选,该文库包括7-met和二硫化限定的7-met。使用这些更短的文库能减小结合域的大小和柔性,能够允许更少的肽-晶面相互作用,使得在所选的代之间的相互作用力产生预期的增加。
采用20-nm胶体金粒子标记的噬菌体用于定量检测特异性结合,其中的胶体金粒子用抗生物素蛋白链菌素(streptavidin)标记,并通过M13包衣蛋白的生物素化的(biotinylated)抗体与菌体结合。进行X-射线光电子分光光谱(XPS)化学成分分析,通过金4f-电子信号强度监测噬菌体与底物之间的相互作用(图2a-c)。在G1-3噬菌体不存在的情况下,抗体和金-抗生物素蛋白链菌素(gold-streptavidin)不与GaAs(100)底物结合。因此,该金-抗生物素蛋白链菌素的结合对噬菌体具有特异性,并且还是噬菌体与底物结合的指示剂。利用XPS还发现从GaAs(100)分离的G1-3克隆与GaAs(100)而非Si(100)特异性结合(参考图2a)。在互补模式下,针对(100)Si晶面筛选的S1克隆几乎不与(100)GaAs晶面结合。
一些GaAs克隆也与另一种闪锌矿结构--InP(100)的晶面结合。选择性结合的基础是化学的、结构的还是电子的结合仍处于研究之中。此外,底物表面的天然氧化物的存在能够改变肽结合的选择性。
已经证实了在GaAs的(111)A(镓末端,gallium terminated)或者(111)B(砷末端,arsenic terminated)的晶面上,G1-3克隆与GaAs(100)优先结合(图2b,c)。在(100)晶面的G13克隆的表面浓度要比在富含镓的(111)A或者富含砷的(111)B晶面的浓度高,其中(100)晶面用于选择。已知这些不同的晶面表现出不同的化学反应性,因此噬菌体与多种晶面的结合具有选择性也就不足为奇了。尽管两个111晶面的大末端(bulk termination)具有相同的几何结构,当对表面改造进行比较时,在表面双分子层具有Ga或As原子外层之间的差别是很明显的。还认为多种GaAs晶面的氧化组合物也是不同的,这反过来会影响肽结合的特性。
针对底物结合能的Ga 2p电子强度如2c所示,其中该底物与G1-3噬菌体克隆相接触。如图2b结果所预测,在GaAs(100),(111)A和(111)B的晶面观察到的Ga 2p强度与金浓度呈反比。在具有更高的金--抗生物素蛋白链菌素浓度的晶面上Ga 2p强度的降低是由于菌体在晶面覆盖的增加造成的。XPS是一种表面技术,其取样深度约为30埃;因此,由于有机层厚度的增加,使得来自无机底物的信号降低。利用该观测可以确定,金--抗生物素蛋白链菌素的强度实际上是由于GaAs晶面上存在包含晶体特异性结合序列的噬菌体。进行了与XPS数据相关的结合研究,其中将等量的特异性噬菌体克隆与具有相同晶面面积的不同半导体底物相接触。野生型的克隆(没有随机的肽插入)不与GaAs结合(未检测到菌斑)。对于G1-3克隆来说,从GaAs(100)表面洗脱的噬菌体群要比从GaAs(111)A表面洗脱下的数高12倍。
利用原子力显微镜(AFM)对结合到GaAs(100)和InP(100)上的G1-3,G12-3和G7-4进行成像。尽管In-P结合比GaAs结合具有更大的离子特性,但InP晶体具有与GaAs晶体同构的闪锌矿结构。通过AFM观测到的10-nm宽、900-nm长的噬菌体与通过透射电镜(TEM)观测到的M13噬菌体的尺寸相匹配,并观察到与M13抗体结合的金球与噬菌体结合(数据未显示)。InP晶面具有高浓度的噬菌体。这些数据表明许多因素影响底物的识别,包括原子尺寸、电荷、极性以及晶体结构等。
在TEM图像中观察到G1-3克隆(负染)与GaAs晶片结合(未显示)。数据证实了该结合通过G1-3的修饰的pIII蛋白来介导,而不是通过与主要的包衣蛋白的非特异性相互作用介导。因此,本发明的肽可在装配纳米结构和异质结构中用来介导特定的肽-半导体的相互作用(图4e)。
用X-射线荧光显微镜来证明噬菌体与闪锌矿晶面的优选接触,其中闪锌矿晶面与不同的化学和结构组合物的表面密切接触。巢状的方块形(anested square pattern)被蚀刻成一GaAs晶片;该模块包含GaAs的1-μm线条,在每一线条之间有4μm的SiO2间隙(图3a,3b)。G12-3克隆与GaAs/SiO2形的底物相接触,洗涤以减少非特异性结合,用免疫荧光探针四甲基罗丹明(TMR)标记。发现标记的菌体为三条红线以及中央的圆点,相应于图3b中仅与GaAs结合的G12-3。该模块中SiO2区域未与噬菌体结合,为暗色区域。在未与菌体接触、但与一抗和TMR接触的对照组中没有观察到该结果(图3a)。利用未与菌体结合的G12-3肽得到相同的结果。
观察到GaAs克隆G12-3在AlGaAs上对GaAs具有底物特异性(图3c)。AlAs和GaAs在室温下具有基本相同的晶格属性(lattice constraints),分别为5.66A°和5.65A°,因此AlxGal-xAs的三重合金能够在GaAs底物上取向附生生长。GaAs和AlGaAs具有闪锌矿晶体结构,但是G12-3克隆仅对GaAs表现出结合特异性。采用包含GaAs和Al0.98Ga0.02As的交互层的多层底物。将底物材料进行裂解并随后与G12-3克隆相互反应。
G12-3克隆采用20-nm的金--抗生物素蛋白链菌素纳米颗粒进行标记。扫描电镜(SEM)的结果显示异质结构内GaAs和Al0.98Ga0.02As的交互层(图3c)。采用镓和铝的X-射线元素分析来绘制金--抗生物素蛋白链菌素颗粒的图谱,其中该颗粒仅针对异质结构的GaAs层,结果证实了对化学组成的高水平结合特异性。在图3d中,显示了一种用于半导体异质结构的噬菌体鉴别的模型,如荧光和SEM图像中所见到的(图3a-c)。
本发明阐述了采用噬菌体展示文库来对有机肽序列和无机半导体底物之间的结合进行鉴别、开发和放大。对无机晶体的这种肽识别和特异性已经延伸至其它底物,包括使用肽文库的GaN,ZnS,CdS,Fe3O4,Fe2O3,CdSe,ZnSe以及CaCO3。目前已经设计出具有两个成分识别的二价合成肽类(图4e);这类肽具有将纳米粒子定位在半导体结构特定位置上的能力。这些有机和无机对能够为下一代复杂的、综合性的电子结构加工提供有用的结构模块。
实施例1
肽的构建、分离、选择和定性
肽的选择。将噬菌体展示或肽文库与半导体或其它晶体在包含0.1%TWEEN-20的Tris-缓冲盐水(TBS)中相接触,以降低晶面上菌体-菌体之间的相互作用。在室温下摇动1小时后,用pH 7.5的Tris-缓冲盐水接触10次来洗涤晶面,该缓冲盐水中TWEEN-20的浓度从0.1%增加至0.5%(v/v)。噬菌体通过加入甘氨酸-HCl(pH 2.2)10分钟而从晶面上洗脱下来,转移至一新管中,然后用Tris-HCl(pH 9.1)中和。对洗脱下来的噬菌体进行浓度测定,比较结合能力。
将与底物接触三个循环后洗脱的噬菌体与其宿主Escherichia coliER2537混合,并置于LB XGal/IPTG平皿上。由于文库噬菌体来源于携带lacZα基因的M13mp19载体,因此当噬菌体置于包含Xgal(5-溴-氯-3-吲哚基-β-D-半乳糖苷)和IPTG(异丙基-β-D-硫代半乳糖苷)的介质中时噬菌体菌斑显示为蓝色。采用蓝色/白色筛选法来选择具有随机肽插入的噬菌体菌斑。从平皿上收集菌斑并进行DNA测序。
底物制备。通过X-射线衍射对底物进行定位,采用适当的化学特异性蚀刻技术除去天然氧化物。在GaAs和InP晶面上检验下述蚀刻:在蚀刻时间的1分钟和10分钟时NH4OH∶H2O 1∶10,HCl∶H2O 1∶10,H3PO4∶H2O2∶H2O 3∶1∶50。采用HCl∶H2O 1∶10蚀刻1分钟,然后用去离子水漂洗1分钟可使GaAs和InP蚀刻晶面具有最佳的元素比例和最少的氧化物形成(使用XPS)。然而,由于在文库的初始筛选中对GaAs使用了氢氧化铵蚀刻,因此在所有其它GaAs底物实施例中都使用了该蚀刻。Si(100)晶片采用下述方法蚀刻:在HF∶H2O 1∶40中蚀刻一分钟,然后用去离子水漂洗。所有的晶面直接从漂洗液中取出后立刻转入噬菌体文库中。对照组底物的晶面不与噬菌体接触,通过AFM和XPS对晶面蚀刻过程的效果以及形态学进行定性及绘制图谱。
GaAs和Al0.98Ga0.02As的多重底物通过分子束取向附生(molecular beamepitaxy)在(100)GaAs表面上生长。取向附生生长层为5×1017cm-3水平的掺杂Si-的生长层(Si-doped)(n-型)。
抗体和金标记。在XPS,SEM和AFM的实施例中,底物在Tris缓冲盐水中与噬菌体接触1小时,然后转入fd噬菌体pIII蛋白的抗-fd噬菌体-生物素偶联物抗体(1∶500于磷酸盐缓冲液中,Sigma)中30分钟,然后用磷酸盐缓冲液漂洗。通过生物素-抗生物素蛋白链菌素相互作用,将抗生物素蛋白链菌素/20-nm胶体金标记(1∶200于磷酸盐缓冲盐水(PBS)中,Sigma)与生物素偶联的噬菌体联结;将晶面与标记接触30分钟,然后用PBS漂洗几次。
X-射线光电子光谱学(XPS)。制备下述对照用于XPS实例,从而确定在XPS所见的金信号是源于与噬菌体结合的金而不是与GaAs晶面的非特异性抗体的相互反应。将制备的(100)GaAs晶面与下述材料接触:(1)抗体和抗生物素蛋白链菌素-金标记,但没有菌体,(2)G1-3菌体和抗生物素蛋白链菌素-金标记,但没有抗体,以及(3)抗生物素蛋白链菌素--金标记,但没有G1-3菌体或抗体。
所用的XPS仪器为Physical Electronics Phi ESCA 5700,该仪器带有可产生单频1,487-eV X射线的铝阳极。噬菌体用金标记(如上文所述)后,立即将所有样品转入小室中来降低GaAs晶面的氧化,然后在高度真空下抽吸过夜,从而降低XPS小室中样品的除气作用。
原子力显微镜(AFM)。所用的AFM为安装在Zeiss Axiovert 100s-2tv上的Digital Instruments Bioscope,用一个G扫描仪采用针尖扫描模式来运行。在空气中采用tapping模式输出图像。AFM探针是蚀刻的硅,其带有125-mm支架,在其共振频率200±400kHZ附近驱动的弹性常数为20±100Nm-1。扫描速率为1±5mms-1。利用一级水平面使图像水平,从而去除样品的倾斜。
透射电镜(TEM)。利用Philips EM208在60kV获得TEM图像。G1-3噬菌体(在TBS中1∶100稀释)与GaAs片段(500mm)温育30分钟,离心使未结合的噬菌体与颗粒分离,用TBS漂洗,然后用TBS重悬。样品用2%乙酸双氧铀染色。
扫描电镜(SEM)。G1-3噬菌体(在TBS中1∶100稀释)与新鲜裂解的异质结构晶面温育30分钟,然后用TBS漂洗。G12-3噬菌体用20-nm胶体金标记。在5kV下利用Hitachi 4700型场发射式扫描电镜的Norian检测系统收集SEM和元素绘制图像。
实施例II生物膜
本发明人已经发现有机-无机杂合材料能为新材料和装置提供新的途径。尺寸受控的纳米结构能为半导体材料提供光学和电学可调控的性质,并且有机加成物能对无机物的形态、相位以及晶核形成定向等进行修饰。生物材料的单分散性质使得该系统对高度有序的近晶排列结构具有兼容性。采用本发明的方法,利用遗传工程技术的、自装配的生物分子如对特定半导体晶面具有识别域(moiety)的M13噬菌体,创建了高度有序纳米级的以及多长度尺度排列的II-VI族半导体材料。
采用本发明的组合物和方法,使用本文所述的识别和自排序系统可完成纳米级的和多长度尺度排列的半导体材料。半导体材料的识别和自排序可用来提高电子设备的微加工,这种方法在性能上超过了目前使用的光蚀刻法。这类材料的应用包括:光电子设备,例如光发射显示装置、光学检测器以及激光;快速相互连接器(fast interconnects);以及纳米级的计算机组件和生物传感器。本发明所创建的生物膜的其它应用包括整齐排序的液晶显示材料以及有机-无机显示技术。
薄膜、光纤以及其它结构甚至还可包括用于检测包括生物毒素在内的小分子的高密度传感器。其它应用包括光学涂层以及光学开关。还可利用本文所公开的一种或多种材料,用在医用移植物的支架或甚至骨移植物上,这可以通过本领域技术人员公知的技术利用单层或多层或甚至以所述的任意的条纹或组合来进行构建。本发明的其它应用包括电和磁界面,或甚至用在高密度存储,如用于量子计算机的3D电子纳米结构的组成中。作为选择地,可采用本发明的薄膜或者基质来构建在药物应用中的高密度和稳定存储的病毒,例如具有生物兼容性的疫苗、以及佐剂和疫苗包含物等。基于量子点模式的信息存储可用于鉴别,例如在装甲或编码的织物中对防御助手或敌人进行鉴别。本发明的纳米光纤甚至还可用作对货币进行编码和鉴别。
开发下一代的光学、电子和磁力材料和设备的主要目标是在纳米级范围开发具有良好排序、可良好控制的二维和三维结构。目前制备特异性纳米颗粒的方法在长度和材料类型上都受到限制。本发明利用具有自组装性质的有机或生物分子或颗粒,诸如M13噬菌体来扩大纳米颗粒的排列、尺度和等级,并且扩大可使用半导体材料的范围。
本发明人已经发现具有各相异性形状的单分散型生物材料可作为构建整齐排序结构的替代途径。纳米级的和多长度尺度排列的II-VI族半导体材料是使用遗传工程的M13噬菌体来获得到,该菌体具有一针对特定半导体晶面的识别域(一肽类或氨基酸低聚体)。
Seth及其同事已经对具有位置和方向顺序的Fd病毒近晶排序结构进行了鉴别。Fd病毒的近晶结构可用在多尺度和纳米尺度排序的结构上,从而构建2维和3维排序的纳米颗粒。使用噬菌体M13是由于它能通过遗传修饰,已经成功选择出了与Fd病毒具有相同形状,并且对II-VI族半导体晶面具有特异性结合亲和力的噬菌体M13。因此,M13是近晶结构的一种理想来源,它能用于纳米颗粒的多尺度和纳米尺度的排序。
本发明人使用组合筛选方法来发现M13噬菌体,该噬菌体含有能与半导体晶面结合的肽插入片段。这些半导体晶面包括诸如硫化锌、硫化钙和硫化铁等材料。采用分子生物学技术,将能够与半导体材料和材料晶面特异性结合的噬菌体组合库克隆进行克隆,并扩增至能使液晶生成的足够高的浓度。
纤维状的噬菌体Fd为长杆状(长:880nm;直径:6.6nm),具有单分散性分子量(分子量:1.64x107)。这些性质使得在高浓度溶液下噬菌体具有溶致液晶行为。通过生物选择性和自装配性,利用噬菌体形状的各向异性作为构建整齐排序的纳米颗粒层的方法。通过常规的扩增方法来制备单分散性的噬菌体。在本发明中,对类似纤维状噬菌体M13进行遗传修饰,使之与诸如硫化锌、硫化钙和硫化铁等纳米颗粒相结合。
已经证实中尺度排列的噬菌体可形成纳米颗粒的纳米级排列。这些纳米颗粒可进一步被组织成微米结构域以及厘米级尺度。半导体纳米颗粒显示出量子阱效应,并能够在液晶内合成和排序。
制备含有特定肽插入物的噬菌体M13混悬液,并使用原子力显微镜(ATM)、透射电镜(TEM)和扫描电镜(SEM)其进行定性分析。样品中观察到均一的2D和3D排序的纳米颗粒。
原子力显微镜(ATM)。所用的ATM是装在Zeiss Axiovert 100s-2tv上的Digital Instruments Bioscope,利用G型扫描仪采用针尖扫描模式进行操作。利用tapping模式输出图像。AFM探针为蚀刻的硅,其带有125-mm支架,在其共振频率200±400kHZ附近驱动的弹性常数为20±100Nm-1。扫描速率为1±5mms-1。利用一级水平面使图像水平,从而去除样品的倾斜。图9(a)和9(b)为采用AFM观察到的M13菌体的近晶排序的示意图(未显示数据)。
透射电镜(TEM)。利用Philips EM208在60kV获取TEM图像。G1-3菌体(用TBS 1∶100稀释)与半导体材料温育30分钟,离心使未结合的噬菌体与颗粒分离,用TBS漂洗,然后用TBS重悬。样品用2%乙酸双氧铀染色。
扫描电镜(SEM)。菌体(用TBS 1∶100稀释)与新鲜裂解的异质结构晶面温育30分钟,然后用TBS漂洗。G12-3菌体用20-nm胶体金标记。在5kV下使用装在Hitachi 4700型场发射式扫描式电镜上的Norian检测系统收集SEM和元素绘制图像。
采用常规的分子生物学技术对遗传工程得到的M13噬菌体进行扩增和纯化,其中该噬菌体对半导体晶面具有特异的结合特性。为了进行大规模扩增(mass amplification),在400ml的LB介质中加入3.2ml的噬菌体混悬液(浓度:~107菌体/ul)和4ml的过夜培养物。扩增后,得到~30mg的沉淀物。在室温下向搀杂了ZnCl2的A7菌体混悬液中加入Na2S来制备混悬液。在约30mg的菌体沉淀中分别加入20ul的1mM ZnCl2和Na2S溶液来制备最高浓度的A7-菌体混悬液。利用269nm下3.84mg/ml的消光系数来计算浓度。
由于增加了各向同性混悬液的浓度,因此观察了具有定向序列的向列相(nemetic phase)、具有扭曲的向列相的胆固醇液晶相、以及具有定向和定位排列的近晶相。在没有纳米颗粒的Fd病毒中已观察到了这些相位。
偏振光显微镜:利用偏振光显微镜对M13菌体悬液进行定性分析。将每一悬液填充到直径为0.7mm的玻璃毛细管中。高浓度的悬液(127mg/ml)在平行偏振光下表现出多彩颜色[5],在垂直偏振光下表现出近晶的质地,如图10(a)所示。胆固醇节距(cholesteric pitches),如图10(b)可通过改变表1所示的悬液浓度来进行控制。测定节距长,并在样品制备24小时后拍摄显微照片。
表1.胆固醇节距和浓度之间的关系
浓度(mg/ml) | 节距长(um) |
76.30 | 31.9 |
71.22 | 51.6 |
56.38 | 84.8 |
50.52 | 101.9 |
43.16 | 163.7 |
37.04 | 176.1 |
27.54 | 259.7 |
原子力显微镜(AFM)观测:为进行AFM观测,取5ul的M13噬菌体悬液的M13悬液(浓度:30mg/ml)在8mmx8mm的云母基底上干燥24小时,该云母基底在干燥器中用3-氨基丙基三乙基硅烷进行硅烷化4小时。在空气中采用tapping模式摄取图像。由于M13噬菌体为长880nm、宽6.6nm的各向异性形状,因而观察到自装配的序列结构。图10(c)中M13菌体存在于照片的平面上,形成近晶序列。
扫描电镜(SEM)观测:为进行SEM观测,制备干燥噬菌体和ZnS纳米颗粒近晶悬液(噬菌体悬液浓度为127mg/ml)样品的临界点(critical point)。在图10(d)中,观察了富含纳米颗粒的区域和富含噬菌体的区域。纳米颗粒和噬菌体之间的分离长度与噬菌体的长度相关。用TEM利用近晶悬液的稀释样品通过电子衍射图证实了ZnS wurzite晶体结构。
生物膜的制备:用400ul Tris缓冲盐水(TBS,pH 7.5)和加入了1mMNa2S的200ul的1mM ZnCl2溶液对噬菌体沉淀进行重悬。在室温下摇动24小时后,将装在1ml eppendorff管中的悬液在干燥器中缓慢干燥1星期。在管内形成了~15um厚的半透膜。用镊子小心地将该膜收起,如图11(a),。
生物膜的SEM观测:利用SEM观测A7-ZnS膜的纳米级的噬菌体排列。为了进行SEM分析,切割薄膜,然后在氩保护气氛下用2nm的铬通过真空沉积来涂层。样品中观测到图11(d)所示的高度堆积结构(highly close-packed structures)。所测得的单个菌体的平均长度895nm与菌体长度880nm合理类似。薄膜表现出与近晶状A或C样的类薄层形态,其在纳米颗粒和噬菌体层之间具有周期性。周期的长度与噬菌体的长度相关。纳米颗粒的平均尺寸为~20nm,与单个颗粒的TEM观测类似。
生物膜的TEM观测:使用TEM研究ZnS纳米颗粒的排列。薄膜用环氧树脂(LR白)包埋1天,然后加入10ul促凝剂(accelerator)聚合。固化后,树脂用Leica Ultramicrotome切成薄片。将这些~50nm的切片漂浮于蒸馏水中,然后置于空白金格中。观察到平行排列的纳米颗粒在最低点(low),与示意图中x-z平面相对应,如图11(e)所示。由于每一噬菌体具有5个A7域的拷贝,因此每个A7识别一个纳米颗粒(2~3nm尺寸),排列为大约20nm宽,长度为超过2微米。2微米的颗粒被分成平行排列的20nm的条带,每个条带间隔~700nm。据Marvin小组报道,这种差异是源于在TEM观测中噬菌体层的倾斜的(tilted)近晶排列。还观察到了如图3(f)的y-z轴的纳米颗粒层平面。颗粒排列的SAED图显示ZnS颗粒具有wurzite六角形结构。
生物膜的AFM观测:使用AFM观测病毒薄膜的晶面定向。图11(c)显示噬菌体形成了平行排列的人字形,在大部分被命名为近晶O的晶面上,在临近的director normal(噬菌体轴)之间,这些平行排列的人字形几乎为直角。该薄膜显示出正常导向(normal director)的长范围排序,该正常导向具有几十微米。在两个结构域层彼此相遇的一些区域,两个或三个多重长度尺度的噬菌体平行排列并保持近晶C序列结构。
采用识别和自装配系统的纳米和多长度尺度排列的半导体材料能促进未来的电子设备的微加工。这些设备会具有超越目前的光蚀刻效能的潜力。这些材料的其它潜在的应用包括光电子设备,诸如光发射显示装置,光学检测器以及激光,快速相互连接器(fast interconnects),纳米级的计算机组件和生物传感器。
尽管使用多个实施例来详细讨论本发明,但可以知道,本发明提供的多种有实际意义的发明构思是不能囿于特定概念所限定的较宽的范围内的。本文所讨论的特定实施例仅仅是对制备和使用本发明进行特别阐述,而非对本发明的范围作出限制。
序列表
<110>得克萨斯州立大学董事会
<120>利用遗传工程的中尺度病毒对杂合材料进行纳米级的排序
<130>119927-1051
<140>10/157,775
<141>2002-05-29
<150>60/326,583
<151>2001-10-02
<160>95
<170>PatentIn version 3.1
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Claims (18)
1.形成多长度尺度薄膜的方法,其包括:
(a)制备包含病毒和与病毒相结合的肽的组合物,其中所述肽选择性地与具有不同组成或晶面的晶状表面结构结合;和
(b)由上述组合物制备多长度尺度薄膜。
2.形成多长度尺度薄膜的方法,其包括:
(i)制备包含病毒和与病毒相结合的肽的组合物以及纳米颗粒,其中所述肽选择性地与具有不同组成或晶面的晶状表面结构结合;和
(b)由上述组合物制备薄膜,其中所述纳米颗粒在薄膜内形成层。
3.根据权利要求1或2的方法,其进一步包括将组合物扩增至高浓度。
4.根据权利要求1或2的方法,其中所述薄膜包括液晶。
5.根据权利要求1或2的方法,其中所述肽是病毒的一部分。
6.根据权利要求1或2的方法,其中所述肽包括一个或多个肽结合序列。
7.根据权利要求1或2的方法,其中所述肽进一步成核并结合纳米晶体,所述肽与纳米晶体相结合。
8.根据权利要求1或2的方法,其中所述晶状表面结构包括单晶。
9.根据权利要求1或2的方法,其中所述晶体是无机晶体。
10.根据权利要求1或2的方法,其中所述晶体是半导体晶体。
11.根据权利要求2的方法,其中所述纳米颗粒包括硫化物。
12.一种组合物,其包含病毒和与病毒相结合的肽,其中所述肽选择性地与具有不同组成或晶面的晶状表面结构结合,且所述组合物以多长度尺度薄膜的形式存在。
13.根据权利要求12的组合物,其中所述肽进一步成核并结合纳米晶体,所述肽与纳米晶体相结合。
14.根据权利要求12的组合物,其中所述病毒是噬菌体。
15.根据权利要求12的组合物,其中所述晶状表面结构是单晶。
16.根据权利要求12的组合物,其中所述晶状表面结构是无机晶体。
17.根据权利要求12的组合物,其中所述晶状表面结构是半导体晶体。
18.根据权利要求12的组合物,其进一步包括含有晶状表面结构的组成的无机相。
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CNA028195655A Division CN1697884A (zh) | 2001-10-02 | 2002-10-02 | 利用遗传工程的中尺度病毒对杂合材料进行纳米级的排序 |
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US20030073104A1 (en) | 2003-04-17 |
US20080206838A1 (en) | 2008-08-28 |
WO2003029431A3 (en) | 2004-10-07 |
AU2008221594A1 (en) | 2008-10-16 |
JP4601292B2 (ja) | 2010-12-22 |
WO2003029431A2 (en) | 2003-04-10 |
CN1697884A (zh) | 2005-11-16 |
US20110300605A1 (en) | 2011-12-08 |
EP1488010A2 (en) | 2004-12-22 |
JP2005508163A (ja) | 2005-03-31 |
KR20040037230A (ko) | 2004-05-04 |
EP1488010A4 (en) | 2007-04-25 |
CA2462766A1 (en) | 2003-04-10 |
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