CN101044401A - 采用水凝胶的微阵列 - Google Patents
采用水凝胶的微阵列 Download PDFInfo
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- CN101044401A CN101044401A CNA2005800284159A CN200580028415A CN101044401A CN 101044401 A CN101044401 A CN 101044401A CN A2005800284159 A CNA2005800284159 A CN A2005800284159A CN 200580028415 A CN200580028415 A CN 200580028415A CN 101044401 A CN101044401 A CN 101044401A
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Abstract
一种通过用含有均匀分散于其中的锚定分子的可聚合水凝胶层涂布平板基材来制造微阵列的方法。固化之后在基材的上表面安全附加贯穿其微阵列区域的厚度均匀的连续层。然后可通过将探针连接到固化的水凝胶内的锚定分子从而固定多种不同探针以便在该板层表面的不连续空间位置形成微斑点。这种锚定分子可采用例如能用铜或一些其它金属活化的有机螯合剂以及诸如亲和素-生物素互补对的连接系统。
Description
发明领域
本发明一般涉及制造微阵列的方法和所得产品,更具体涉及通过用可聚合的水凝胶层基本均匀地涂布合适基材区域而在该基材或芯片上制造微阵列的方法。
发明背景
微阵列被用于生物测定以检测生物样品中靶材料的存在情况和/或含量;它们构成了一个正在成长的有时称为表面试验的领域,其中的靶材料或代表性靶材料分子被捕获在固体支持物上然后检测。由于这种DNA微阵列能够同时监测大量基因,因此现在已经被广泛接受用于研究基因表达和其它基因型功能。例如,大量不同探针序列可结合于微阵列表面的不连续空间位置或微斑点(microspot),每个这种微斑点可各含有不同的探针。当这种微阵列与含有标记的样品材料的溶液杂交时,若存在靶物质,它就会与阵列各种不同微斑点上的互补DNA链杂交。洗涤除去未结合的材料之后,可利用特征标记物,例如荧光,然后被用来确定各微斑点上标记材料的强度;这种成像能够为样品中存在的特定靶物质含量提供量度。
已经制造了可提供其它分子的这种阵列,如用来结合靶材料的蛋白质,包括抗体、半抗原或适体。这些基于表面的试验也可用于ELISA。总之,这种微量试验芯片已经代替凝胶电泳成为生物试验的备选方法,据信,随着蛋白质组学的进一步发展,这种倾向很可能继续下去。
为使微量试验芯片可有效用于这种生物试验,它应该能够固定要从有关样品分离的令人满意量的分析物或靶材料;这样在随后阅读芯片时才能提供满意数量级的信号。当然,芯片还应该能够高度均匀地制造以便在试验之间产生可重现的结果。
过去十年间已经开发了许多微阵列芯片,其中,探针被固定在修饰的玻璃基材、硅基材等的不连续空间位置上,以形成代表大量不同探针的阵列。最初开发的微阵列是二维形式,其中的探针直接结合在基材表面。最近已经用水凝胶材料开发出了三维微阵列,其中的微斑点可以类似于微小的半球,其多孔结构提供了三维框架或基质。这种类型的微阵列描述于美国专利No.6,174,683和已公开的国际申请WO02/059372。
美国公开申请2003/0124371公开了使用水可溶胀的亲水性水凝胶,认为这种水凝胶特别适用于将多肽分析物固定到吸收层上,所述吸收层是通过改变水凝胶中亲水部分和疏水部分的比例而构造的。构成水凝胶的亲水和疏水单体交联形成所需聚合物。一个例子是,用二氧化硅涂布铝基材,然后用烷基硅烷处理,之后将单体施加到多个可寻址位置(微斑点),然后通过辐射照射交联。使用结合缓冲剂将探针加到芯片的各微斑点上,并将加载好的芯片孵育30分钟。然后洗涤并将芯片用于试验。
美国公开申请2003/0138649传授了制造特别适合结合蛋白质作为探针或捕获剂、采用明胶基材的微阵列的方法。用IV型明胶溶液涂布诸如玻璃、硅或照相纸等的基材;例如,将明胶涂布到反射照相纸上然后冷冻(chill-set)并干燥。然后在整体用明胶涂布的平板上点微斑点以结合双官能化合物,例如山羊抗小鼠抗体IgG,该化合物的一个基团将结合明胶,第二个基团能够以高特异性与蛋白质反应。在相关美国公开申请No.2003/0170474中,硅胶片或玻璃板首先用烷基硅烷处理,再浸入明胶溶液中。然后将明胶涂布的基材浸入聚乙烯亚胺(PEI)溶液中。据报道,这种表面对蛋白质具有相对低的非特异性结合容量,并可用作微阵列基材,将蛋白质捕获剂固定在表面相间隔的微斑点上。
美国公开申请2003/0096257传授了通过用氨基烷基硅烷涂布玻璃载玻片然后通过结合到氨基将乙烯基磺酰基附加到整个表面来制造DNA芯片的方法。然后将具有接头的寡核苷酸点到反应性平板上并适当孵育以确保连接,从而制成可用于杂交分析的DNA芯片。
尽管这些制造生物芯片等的方法可能有许多优点,但它们都有缺点。因此将继续研究以得到制造这种微阵列的更好的方法,而重点通常集中于在这种微阵列中使用水凝胶。
发明概述
我们现在发现,通过提供上表面被有机分子官能化的基材并用可聚合的水凝胶层涂布该表面可制造微阵列,所述水凝胶层含有均匀分布于其中的锚定分子从而覆盖将作为微阵列的连续表面区域。固化涂布的基材以聚合所述涂布的水凝胶层,通过将探针连接到存在于所述固化的水凝胶层内的锚定分子从而将多种不同探针固定到表面的不连续空间位置以形成微斑点。需要的话可遮盖微斑点周围区域。然而,当所述水凝胶基于PEG或PPG(或其共聚物)和聚异氰酸酯交联接头,以及当所用锚定分子是有机螯合剂时,非特异性结合应该不高因此无需遮盖。
在一具体方面,本发明提供了一种制造微阵列的方法,该方法包括以下步骤:提供上表面用有机分子官能化的基材;用可聚合水凝胶层涂布所述表面以便完全覆盖所述表面的阵列区域,所述水凝胶层包含均匀分散于其中的锚定分子;固化所述涂布的基材以聚合所述水凝胶层;和然后通过将所述探针连接到存在于所述固化的水凝胶层内的锚定分子从而将多种不同探针固定到所述涂布表面的不连续空间位置上。
在另一具体方面,本发明提供了一种微阵列,其包括:上表面用有机分子官能化的基材;完全覆盖所述表面阵列区域的聚合的水凝胶层,所述水凝胶层包含均匀分散于其中的锚定分子;和固定在所述水凝胶涂层所述表面的不连续空间位置上的多种不同探针,所述探针连接到所述聚合的水凝胶层内的所述锚定分子,且所述不连续位置周围的所述涂层区域基本上不含探针。
优选实施方案详述
用来形成平板的基材可由化学实验室通常使用的任何合适材料制成;其例子包括玻璃、石英、硅、二氧化硅、不锈钢和惰性聚合物,如聚乙烯、聚丙烯、聚丙烯酸、聚碳酸酯等,这些都是本领域熟知的。平板可任选用反射层涂布,这也是本领域熟知的。反射层应优选覆盖该基材基本全部将固定探针的的表面区域,即阵列区域;然而,为便于制造,反射涂层通常覆盖基材的整个上表面。反射层可以是反光金属,例如铝、银、金、铑等,它可提供镜面层。反光金属是指能反射至少90%感兴趣波长区域内的入射光(通常是可见光(400-800nm))并可能包括近红外区域的较长波长(如800-1100nm)和极少(或接近0%)折射如介质的光的金属。可用常规的蒸汽涂布法或本领域熟知的提供这种镜面涂层的涂布方法来提供这种薄金属层。这种层的厚度并不特别重要,只要连续即可,但包括这种层时其厚度通常约为0.01-15微米。
当用该基材来产生用于微量试验的微阵列时,采用能自发光或对激发光反应而发光的标记或标签,例如荧光、发光标记或磷光标记,这些不同标记发射的光将具有已知波长。2003年9月16日提交的待批美国专利序列号10/664,248教导到,在反光金属层上施加能够基本上除去特定波长的绝缘层可以非常充分地降低微斑点位置或其它此类探针位置的背景伪像(artifact)发射。可采用由诸如一氧化硅和/或二氧化硅等材料制成的透明绝缘层,其厚度通常约为0.1-5微米。如果基材的上表面被均匀涂布上这种金属镜面薄层,则整个上表面宜涂布上这种均匀的绝缘层。从蒸汽沉积这种非常薄的二氧化硅、氧化铝、氟化镁等的绝缘膜是本领域熟知的。
一旦施加了这种薄的绝缘层,则优选用化学方法处理该绝缘层的上表面以促进水凝胶的强附着而提供探针或捕获剂阵列。绝缘表面优选用诸如PEI、聚赖氨酸或氨基烷基硅烷等合适试剂衍生,这些覆盖整个表面的试剂具有悬垂(pendent)的氨基,如本领域所熟知的,氨基然后可用于牢固结合反应性分子。这种硅烷偶联剂的例子包括氨基丙基三乙氧基硅烷、N-β-(氨基乙基)-α-氨基丙基三甲氧基硅烷和N-β-(氨基乙基)-α-氨基丙基甲基二甲氧基硅烷。
用水凝胶三维微斑点作为探针或捕获剂固定点的微阵列,描述于美国专利No.6,174,683和已经公开的题为“三维形式生物芯片(Three Dimensional Format Biochips)”的国际申请WO 09/059372和题为“捕获活细胞和有机分子的方法和凝胶组合物(Methods and Gel Compositions For Encapsulating Living Cells and Organic Molecules)”的WO 02/081662中。
精选的可聚合的水凝胶涂层优选是用异氰酸酯官能化的前聚合物(prepolymer)制备的,这种前聚合物是将聚氧化烯二醇或多元醇与双官能和/或多官能(即三官能或多官能)异氰酸酯化合物反应制备的。优选的前聚合物是从聚氧化烯二醇或多元醇制备的,其中包括环氧乙烷单元的均聚物或含有环氧乙烷单元和环氧丙烷或环氧丁烷单元的混合物的嵌段或随机共聚物。就这种嵌段或随机共聚物而言,至少75%的单元优选是环氧乙烷单元。这种聚氧化烯二醇或多元醇的分子量优选约500-10,000道尔顿,更优选约1,000-6,000道尔顿,有时最优选的分子量是5,000-6,000道尔顿。合适的前聚合物可将所选聚氧化烯二醇或多元醇与聚异氰酸酯反应制备,这样可使几乎所有的羟基被聚异氰酸酯加帽,如下文更加详细描述的。通常优选聚乙二醇(PEG)、聚丙二醇(PPG)或其共聚物。异氰酸酯官能化前聚合物优选含有一定量的活性异氰酸酯,所述含量约为0.1-1meq/g,更优选约为0.2-0.8meq/g。如果使用分子量较低的前聚合物,例如小于2,000道尔顿,优选它们具有相对高的异氰酸酯含量(约1meq/g或更高)。然而,可能需要更加精确地控制这种较小前聚合物的聚合速度以避免过于迅速的聚合。此外,具有较高异氰酸酯含量的前聚合物在聚合之后可能具有相对高含量的游离胺,中性pH时,这种胺官能度上的正电荷可能会增加负电荷生物分子的非特异性结合,从而可能导致较高水平不需要的背景信号。因此,含有相对低异氰酸酯含量的分子量较高的前聚合物通常是优选的。
这种高分子量前聚合物通常用两种常用方法中的任何一种制备,但也可采用本领域已知的其它方法:(1)将分子量至少2000道尔顿的多元醇(三元醇或更高)与聚异氰酸酯如异氟尔酮二异氰酸酯反应,或(2)将分子量至少2000道尔顿的二元醇与聚异氰酸酯和交联剂反应,所述交联剂诸如甘油、三羟甲基丙烷、三羟甲基乙烷、三乙醇胺或有机三胺反应。
可使用芳族、脂族或环脂族聚异氰酸酯;所用聚异氰酸酯包括双和多(官能)异氰酸酯。高分子量脂族异氰酸酯加帽的前聚合物通常在约20-90分钟内胶凝成水合聚合物状态,而用芳族聚异氰酸酯加帽的前聚合物更加迅速地胶凝。合适的双官能或多官能异氰酸酯的例子如下:4,4’-亚甲基双-(苯基(phyenyl)异氰酸酯)(MDI)、甲苯-2,4-二异氰酸酯、甲苯-2,6-二异氰酸酯(其同分异构体的混合物作为TDI出售)、异氟尔酮二异氰酸酯(IPDI)、乙烯基二异氰酸酯、亚乙基二异氰酸酯、丙烯-1,2-二异氰酸酯、亚环己基(cyclobexylene)-1,2-二异氰酸酯、亚环己基-1,4-二异氰酸酯、苯二异氰酸酯、3,3”-二苯基-4,4”-二苯二异氰酸酯、1,6-六亚甲基二异氰酸酯、1,4-四亚甲基二异氰酸酯、1,10-十亚甲基二异氰酸酯、枯烯-2,4-二异氰酸酯、1,5-萘二异氰酸酯、亚甲基二环己基二异氰酸酯、1,4-亚环己基二异氰酸酯、对-四甲基苯二甲基二异氰酸酯、对-苯二异氰酸酯、4-甲氧基-1,3-苯二异氰酸酯、4-氯-1,3-苯二异氰酸酯、4-溴-1,3-苯二异氰酸酯、4-乙氧基-1,3-苯二异氰酸酯、2,4-二甲基-1,3-苯二异氰酸酯、2,4-二甲基-1,3-苯二异氰酸酯、5,6-二甲基-1,3-苯二异氰酸酯、1,4-二异氰酸根合二苯基醚、4,4’-二异氰酸根合二-苯基醚、联苯胺二异氰酸酯、4,6-二甲基-1,3-苯二异氰酸酯、9,10-蒽二异氰酸酯、4,4’-二异氰酸根合二-苯基、3,3’-二甲基-4,4’-二异氰酸根合二苯基甲烷、1,6-二甲基-4,4’-二异氰酸根合二苯基、2,4-二异氰酸根合芪(stibene)、3,3’-二甲氧基-4,4’-二异氰酸根合二苯基、1,4-蒽(antracene)二异氰酸酯、2,5-荧光酮二异氰酸酯、1,8-萘二异氰酸酯、2,6-二异氰酸根合苯并呋喃(benzluran)、2,4,6-甲苯三异氰酸酯、p,p’,p”-三苯基甲烷三异氰酸酯、异氟尔酮二异氰酸酯的三官能三聚体(异氰脲酸酯)、六亚甲基二异氰酸酯的三官能缩二脲、六亚甲基二异氰酸酯的三官能三聚体(异氰脲酸酯)、聚合4,4’-二苯基甲烷二异氰酸酯、苯二甲基二异氰酸酯和间-四甲基苯二甲基二异氰酸酯。
采用化学计量量的反应物可有效地用聚异氰酸酯加帽选择的二醇或多元醇形成前聚合物。如本领域已知的,异氰酸酯与羟基的比例是可变的,但优选约为1-3,更优选约为1.2-2.2。加帽反应可采用任何合适条件进行,如在约20℃-150℃在干燥氮气下反应约2小时至约14天,优选不使用催化剂。优选的温度是约60-100℃,当异氰酸酯浓度接近理论值时终止反应。
优选的前聚合物包含末端用甲苯二异氰酸酯加帽的聚乙二醇;环氧乙烷和环氧丙烷(任选含有三羟甲基丙烷)以及甲苯二异氰酸酯的共聚物;甲苯二异氰酸酯-聚乙二醇-三羟甲基丙烷(trimethylopropane),亚甲基二异氰酸酯-亚甲基均聚物;聚合的亚甲基二异氰酸酯-聚乙二醇;环氧乙烷-环氧丙烷三羟甲基丙烷和异氟尔酮二异氰酸酯的聚合物,以及聚乙二醇三乳酸盐和甲苯二异氰酸酯。合适的上述类型的前聚合物购自Dow Chemical Company,名为HYPOL PreMAG-50、HYPOL2000、HYPOL3000、HYPOL4000和HYPOL5000;这些制剂通常包含聚氧化乙烯和少量分子量约6,000道尔顿的聚氧化丙烯的共聚物。其它可以商品名Urepol购自EnviroChemTechnologies,也可从市售原料制备类似前聚合物。
总的看来,水凝胶聚合物的主链优选由聚乙二醇、聚丙二醇或者聚乙二醇和聚丙二醇的共聚物构成。聚乙二醇和聚丙二醇水凝胶的非离子亲水特性能够使分析物与水凝胶低水平非特异性结合,并能够为要固定的生物分子提供良好相容性,从而可维持其天然构象和生物反应性。这种常规类型的基于聚氨基甲酸酯(polyurethane)的异氰酸酯官能化水凝胶描述于美国专利Nos.3,939,123(Mathews等)、4,110,286(Vandegaer等)和4,098,645(Hartdegan等)。
在一优选实施方案中,微阵列的基材是用异氰酸酯官能化水凝胶制造的,所述水凝胶基于高分子量聚氧化乙烯、聚氧化丙烯或者聚氧化乙烯和聚氧化丙烯共聚物的二元醇或三元醇,用水活化二异氰酸酯加帽,并任选用合适的交联剂轻微交联。如上所述,前聚合物中存在的活性异氰酸酯的量优选约为0.1-1meq/g。通常优选的二异氰酸酯包括芳族二异氰酸酯:甲苯二异氰酸酯(TDI)和亚甲基二苯基-异氰酸酯(MDI),以及脂族二异氰酸酯:异氟尔酮二异氰酸酯。前聚合物中约0.01-15%的反应性异氰酸酯通常被用来连接涂层和基材,这为固定锚定的分子(entity)留下了充足够位点。前聚合物可在可与水混溶的有机溶剂中预先配制,且聚合通常通过简单加水形成脲键而发生。
所施加的水凝胶涂层含有均匀分散于其中的锚定分子,该部分被用来直接或间接锚定探针作为微阵列的一部分。它们可被溶于水溶液并与前聚合物混合以启动聚合反应。合适的锚定分子的例子包括有机螯合剂和有机接头,所述有机接头可以是一对互补接头(如链霉亲和素和生物素)的一半,而另一半然后可连接到感兴趣的探针上。当将有机螯合剂作为可聚合层的一个组分混合时(因此可均匀分散在固化层中),在其固化之后用水溶性金属离子的水溶液处理该涂层从而金属阳离子可结合到有机螯合剂,通常在3或4个位点结合。当采用该系统时,也可将探针连接到将与这些金属离子络合的有机分子上,而这些金属离子已经活化固化涂层内的螯合剂。
固化的水凝胶层或板的厚度可以约为1-1000微米;优选至少约5微米厚。例如,当使用三配位或四配位有机螯合剂(如亚氨基二乙酸(IDA)或次氮基三乙酸(NTA))时,它们在涂层组合物中的浓度可以约为50mM-1mM,这样的量足以使这些锚定分子均匀分布在整个涂层中从而覆盖阵列区域。
一旦施加了水凝胶层或板就可以固化直到该制备物基本上完全交联。由于施加和固化可以是封闭条件下进行,因此可以基本排除溶剂蒸发的问题(不同于常规方法中在平板上施加微斑点);因此可以采用许多溶剂和水凝胶制备物而不会遇到环境难题或其它制造难题。同时,由于铸造/模压/涂布步骤可以非常一致,且可以一次涂布大量平板,因此涂布平板之间的差异将会很小,同时可避免机械打点的许多固有限制。
一旦成功固化载玻片,即已经基本完成交联,便可洗涤载玻片以除去任何未与水凝胶结合的有机螯合剂或其它有机接头,以及除去配制和/或施加涂层组合物期间使用的溶剂。当锚定分子是有机螯合剂时,可将涂布的基材与诸如硝酸铜或硝酸镍的溶液一起孵育。其结果是,这种溶液中的铜或镍离子与螯合剂结合,这一过程在该领域被称为螯合剂的活化。然后可任选干燥涂布的基材并储存不定时间。如果进行了这种干燥,宜在形成微阵列之前或者形成微阵列时进行所有涂层或者至少微斑点位于区域的再水合。
被用作捕获剂或探针的生物材料可以是本领域熟知的各种类型。它们可涉及从DNA序列和肽到许多大分子,如抗体;甚至可采用合适的互补接头将活细胞固定到多孔水凝胶的不连续空间位置上。除这里提到的螯合剂和生物素-亲和素外,许多其它此类结合对描述于上述专利和公开的国际申请中。
在常规试验中,微阵列在杂交/结合条件下接触含有生物材料样品的溶液,通常是水溶液;溶液中含有已经用报告物质或信号材料或用随后可与报告材料螯合的接头添加尾部或标记的潜在靶物质,和培育。标记或尾部用来指可连接到靶物质(如核酸序列)以使其能被检测和/或定量的取代基。其例子包括放射性标记,如32P、33P和35S;比色指示剂,如荧光化合物、化学发光化合物或有色化合物;配体,如生物素;以及可通过质谱或其它光谱性能进行鉴别的化学基团。更加具体的合适标记的例子包括黄嘌呤染料、罗丹明染料、萘胺(naphtylamine)、苯并二唑(benzoxadiazole)、芪、芘、吖啶、花青3(Cy-3)和花青5(Cy-5)。当靶是核酸时,优选将标记作为引物的一部分而直接掺入分析物核酸中;然而,可通过化学反应或酶反应,或者也可将标记与中间配体杂交或退火来间接加入。为了简便和有效,优选在杂交步骤中简单采用在分析物样品中的荧光、发光或磷光等发光标记。尽管结合对如亲和素-生物素可用来随后添加标记,但特别优选用这些物质来产生试验所用的微阵列,该阵列中的标签已经与培育步骤所用样品溶液中的靶生物材料共价结合。荧光标记如Cy-3和Cy-5是这种常用标记的例子。
将微阵列与样品溶液孵育适当时间后进行洗涤,通常重复洗涤,这是本领域熟知的和常用的;然后通常将微阵列干燥,之后再进行成像。当采用发射(无论是自发的或是受激的)光的标记时,各种市售光度计可用于成像。例如,美国专利No.5,672,880公开了一种荧光成像系统,该系统用激光束激发荧光,聚集发出的荧光使聚集光照射光电检测器,而产生信号。公布的美国专利2002/0109841公开了一种用于高通量荧光检测的扫描分光光度计,其中,光线被收集并发送到光电倍增管(PMT),再将信号反馈到处理单元。
以下实施例提供了目前已知的执行本发明的最佳模式;然而,这些实施例只是为了阐述而不是要以任何方式限制本发明的范围,本发明的范围由说明书随后出现的权利要求书限定。
实施例1
采用在反射铝层之上涂有氧化硅薄层的标准实验室玻璃载玻片,载玻片涂布有一层连续的其中分布锚定分子的水凝胶前聚合物溶液。将0.075克Hypol PreMaG-50(Dow Chemical Company)和0.225克乙腈和0.225克N-甲基-2-吡咯烷酮溶液混合制备溶液A。溶液B为50mM硼酸盐缓冲水溶液(pH 8.2)中的0.5μM衍生的NTA螯合剂。该螯合剂的伯胺共价结合在交联的水凝胶中。然后将200μL溶液A与50μL溶液B混合使聚合开始,所得组合物涂布在胺处理过的玻璃载玻片上。在室温和约95%RH的湿度室中小心聚合含有螯合剂的水凝胶涂层以避免脱水。该制备物在室温下在约2小时内聚合。固化后的水凝胶板厚约50微米,它是物理稳定牢固附着于玻璃载玻片。固化的载玻片然后用浓度约50μM的硝酸铜水溶液在室温处理2小时以使铜阳离子被均匀分散在多孔水凝胶板中的螯合剂分子螯合。用这种方式活化螯合剂之后,载玻片用含0.1M KNO3(pH 4)的50μM乙酸溶液洗涤1小时,然后用去离子水冲洗。然后将其干燥用于微打点(microspotting)以形成测试微阵列。
用5微升玻璃微毛细管进行微打点以在活化的水凝胶板表面形成规则图案的微斑点。出于测试目的,每管都含有绿色荧光蛋白以模拟探针,探针尾部是能与铜复合的富含组氨酸肽段。微打点的表面然后在室温孵育约2小时以使这些探针依照微斑点的规则图案有效连接到固化的水凝胶板表面。
洗涤微阵列以除去任何过量探针,然后用电荷耦合照相机扫描。图象显示均匀发光的微斑点以规则图案贯穿阵列表面,微斑点之间的区域没有发出显著荧光;这说明与水凝胶板的非特异性结合不是问题,且这种基材非常适合制造高灵敏微阵列。
实施例2
用类似的异氰酸酯加帽的聚氨基甲酸酯前聚合物重复实施例1所述的过程。然而此时,溶液B为50mM硼酸盐缓冲水溶液(pH 8.2)中的0.5mM链霉亲和素溶液。如上所述,再将约200μL溶液A与50μL溶液B混合,所得组合物涂布在胺处理过的玻璃载玻片上作为连续层并使其聚合。
固化后,再用5微升玻璃微毛细管进行微打点以在水凝胶板表面形成规则图案的微斑点,从而形成微阵列。该测试中,各管含有前列腺特异性抗原(PSA)的生物素化的抗体以用作探针,这是本领域熟知的。将微打点的表面在25℃孵育约60分钟以使生物素-亲和素以规则图案有效连接到固化的水凝胶板表面上。
洗涤微阵列以除去任何过量探针,然后与PSA水溶液杂交,PSA用Cy-3标记作为测试靶材料;标记的PSA选择性结合探针。在25℃孵育约60分钟后洗涤阵列,然后用激光扫描仪(ScanArrayLite,Perkin Elmer)成像以获得微阵列表面的荧光图象。图象显示均匀发光的微斑点以规则图案贯穿阵列表面,微斑点之间的区域没有发出显著荧光;这说明与水凝胶板的非特异性结合不是问题,且这种基材非常适合制造高灵敏微阵列。
由此可见,采用具有这种连续水凝胶板的基材可大大提高效率,从而可用均匀分散于其中的锚定分子制造微阵列。
尽管已经就某些优选实施方案描述了本发明,但应理解,本领域的一般技术人员显然可作出各种变化和改进而不超出本发明的范围,本发明的范围在附带的权利要求中列出。
Claims (18)
1.一种制造微阵列的方法,所述方法包括以下步骤:
提供上表面用有机分子官能化的基材,
用可聚合水凝胶层涂布所述表面以完全覆盖所述表面的阵列区域,所述水凝胶层包含均匀分散于其中的锚定分子,
固化所述涂布的基材以聚合所述水凝胶层,和
然后通过将所述探针连接到存在于所述固化的水凝胶层内的锚定分子从而将多种不同探针固定到所述涂布表面的不连续空间位置上。
2.如权利要求1所述的方法,其特征在于,所述锚定分子是与所述可聚合水凝胶层组分混合的有机螯合剂。
3.如权利要求2所述的方法,其特征在于,所述固化步骤之后,用水溶性金属离子的水溶液处理所述螯合剂以活化所述螯合剂。
4.如权利要求3所述的方法,其特征在于,所述探针是已用强烈结合所述金属离子的化合物衍生的分子。
5.如权利要求3所述的方法,其特征在于,所述探针用富含组氨酸的肽衍生。
6.如权利要求1所述的方法,其特征在于,所述探针用在所述附加步骤中直接结合所述锚定分子的互补接头衍生。
7.如权利要求6所述的方法,其特征在于,所述接头和所述锚定分子分别为生物素和链霉亲和素。
8.如权利要求1-7中任一项所述的方法,其特征在于,所述可聚合水凝胶包含PEG或PPG或其共聚物。
9.如权利要求8所述的方法,其特征在于,所述可聚合水凝胶包含聚异氰酸酯交联剂。
10.如权利要求9所述的方法,其特征在于,所述交联剂包括脂族或芳族聚异氰酸酯。
11.如权利要求9所述的方法,其特征在于,所述涂布的基材在所述固化后被干燥,然后在连接所述探针的位置上再水合。
12.一种微阵列,其包括:
上表面用有机分子官能化的基材,
完全覆盖所述表面阵列区域的聚合的水凝胶层,所述水凝胶层包含均匀分散于其中的锚定分子,和
连接在所述水凝胶涂层所述表面的不连续空间位置上的多种不同探针,所述探针连接于所述聚合的水凝胶层内的所述锚定分子,以及所述不连续位置周围的所述涂层区域基本上不含探针。
13.如权利要求12所述的微阵列,其特征在于,所述锚定分子是用金属离子活化的有机螯合剂。
14.如权利要求13所述的微阵列,其特征在于,所述探针用富含组氨酸的肽衍生。
15.如权利要求12所述的微阵列,其特征在于,所述探针用直接结合所述锚定分子的互补接头衍生。
16.如权利要求15所述的微阵列,其特征在于,所述探针是生物素化探针,所述锚定分子是链霉亲和素。
17.如权利要求12-16中任一项所述的微阵列,其特征在于,所述聚合的水凝胶包含PEG或PPG或其共聚物的反应产物。
18.如权利要求12-16中任一项所述的微阵列,其特征在于,所述聚合的水凝胶包含(a)分子量约为1,000-6,000道尔顿的PEG或PPG或其共聚物和(b)多官能异氰酸酯的反应产物。
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