CN1314873A - 阵列印刷用基片 - Google Patents

阵列印刷用基片 Download PDF

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CN1314873A
CN1314873A CN98813145A CN98813145A CN1314873A CN 1314873 A CN1314873 A CN 1314873A CN 98813145 A CN98813145 A CN 98813145A CN 98813145 A CN98813145 A CN 98813145A CN 1314873 A CN1314873 A CN 1314873A
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substrate
glass
polar silanes
polar
mean roughness
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A·卡雷
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Corning Inc
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Abstract

一种用于支承高密度生物或化学阵列的基片,它由硼硅酸盐或硼铝硅酸盐玻璃制成。已经表明,当用于固定寡核苷酸的涂层施涂在低氧化钠含量的玻璃材料构成的载玻片时,在暴露于环境作用后仍会保留其功能性。

Description

阵列印刷用基片
发明领域
本发明涉及高密度的生物和化学阵列，更具体地涉及一种阵列可沉积在其上面的改进的基片材料。
发明背景
寡核苷酸杂交广泛用于确定在核酸中是否存在与寡核苷酸探针互补的序列。在许多情况下，这种方法提供了一种可的替代常规测序的简便、快速、价廉方法。杂交并不需要核酸克隆和纯化，不需要进行碱基特异性反应，也不需要繁琐的电泳分离。核酸探针的杂交已成功地应用于各种用途，例如分析遗传多态性、诊断遗传疾病、癌症诊断、检测病毒和微生物病原体、筛选克隆、基因组定位和片段文库的有序化。
寡核苷酸阵列是由以规则方式固定在固相载体表面上的许多单独的寡核苷酸种类所构成的，每种寡核苷酸位于一个不同区域，使得各寡核苷酸的位置是已知的。阵列可含有一群选定的寡核苷酸，例如对所有已知的临床上重要病原体特异的探针，或者对遗传病所有已知序列标记特异的探针。这样的阵列可满足诊断实验室的需要。或者，一个阵列可含有给定长度n的所有可能的寡核苷酸。核酸与这种综合性阵列的杂交，可导致列出所有其构成性的n-聚物，这些n-聚物可用来明确地识别基因(例如在法医研究中)、用来确定未知的基因变异体和突变(包括一旦知道其中之一的序列，就可对相关基因组进行测序)、用来重迭克隆、以及用来检查用常规方法确定的序列。最后，通过与综合性阵列的杂交而调查n-聚物，可以提供足够信息用来确定完全未知核酸的序列。
寡核苷酸阵列的制备，可采用固相化学合成法结合定点掩模(如美国专利5,510,270所述)，直接在载体上平行地合成全部寡核苷酸。使用有效的光刻技术，已得到在1平方厘米面积上含105种寡核苷酸的微型阵列。
另一种制造寡核苷酸阵列的技术，涉及采用如美国专利5,474,796所述的压电泵进行精确的点滴沉积。这种压电泵能将微量液体逐滴输送到基片表面上。泵的设计与喷墨打印中所用的泵非常相似。这种微微泵(picopump)能够以3000Hz之内的频率输送50微米尺寸和65微微升体积的液滴，并且能够准确喷击在250微米的目标上。当泵工作时，这种细微液滴就从泵射出并沉积在阵列板上的功能化结合位点。
其他形成阵列的方法是将基片表面与内装液滴的印刷针反复接触，并用喷墨印刷技术来排列阵列矩阵。
在选择基片用作附着寡核苷酸的载体时，必须考虑几个特性。首先，基片表面必须与杂交检测方法相容。因为光谱、化学发光和荧光检测技术是用来研究涉及高密度阵列DNA的检测技术，为了能使用这些技术，需要基片是透光的。第二个重要特性是次末寡核苷酸与基片表面的连接应具有化学稳定性，该稳定性至少与DNA中聚磷酸骨架的稳定性相同。
支持阵列的基片通常是1×3英寸的钠石灰玻璃载玻片，其上面涂有一层极性硅烷，该硅烷含有例如适用于锚定固相寡核苷酸合成并且特别适合于连接DNA分子的氨基。通过光致抗蚀或掩模技术，可以在表面上产生一定的图案。用这种方式，可以在原本非湿化表面上形成图案湿化位点，并且在原本非功能化表面上形成图案形式的功能化位点。
常规使用钠石灰玻璃作为高密度阵列载体基质的一个问题，是会存在颗粒污染，这在生产这种低等级玻璃过程是常见的。每个载玻片有10,000个靶位点如此精细分布样品，对其处理时颗粒污染就特别会有不利的影响。此外，在钠石灰玻璃中所含的钠会容易迁移离开玻璃。结果产生的雾化会对玻璃透明度产生不利影响，因而会干扰前述的检测技术。最后，在目前常规使用的载玻片表面上形成均匀功能化的涂层(如具有氨基官能团的硅烷涂层)是不容易的。没有均匀的涂层，寡核苷酸的附着就不均匀，会使检测结果波动和不可靠。
发明概述
本发明公开了一种用于印刷或合成生物和化学阵列的改进基片。该基片是用硼硅酸盐或硼铝硅酸盐玻璃制成的基本平坦的载体。
发明详述
在具有例如氨基官能化胺的玻璃基片表面上的功能化涂层，是高密度阵列制品的骨架。如上所述，需要基本上均匀的功能涂料的涂层。已经发现，可采用已知方法制造获得一定平滑度的已知玻璃，用作生物基片的重要用途。
基片是本发明的主题，它宜采用1英寸×3英寸载玻片的形式，是用硼硅酸盐或硼铝硅酸盐玻璃制成的，更佳地是用Corning Incorporated 1737LCD玻璃制成的。以摩尔百分数表示，这种玻璃基本上由下列成分构成：
 SiO2     67.6  BaO     4.31
 Al2O3     11.4  MgO     1.31
 B2O3     8.53  SrO     1.29
 CaO     5.2  As2O3     0.39
载玻片可由玻璃板切割而成，该玻璃板宜用美国专利3,338,696和3,682,609(这些专利参考结合于此)所公开的熔拉法(fusion draw process)制造。该方法可由高液相线粘度玻璃(例如硼硅酸盐或硼铝硅酸盐玻璃)制造具有极高平滑度的玻璃板。尽管硼硅酸盐或硼铝硅酸盐玻璃板还可以用其他方法制造，随后再抛光，然而熔拉法更好，因为在制造过程中采用抛光步骤会导致基片表面上形成颗粒污染。在另一优选实施方案中，载玻片是用Corning Incorporated 7059 LCD玻璃制造的。无论如何，载玻片基片的优选玻璃组成具有重量百分数低于15％的氧化钠或其他碱金属氧化物。数种合适的玻璃组成列于共同转让的美国专利5,374,595中。
更优选的是，载玻片应具有均匀的表面平滑度，使得用原子力显微镜(atomicforce microscope,AFM)在20微米×20微米范围扫描测量时，所测上表面的平均粗糙度(Ra)小于10纳米，较佳地小于10埃米。上表面是载玻片上结合实体阵列优选被合成、沉积或附着的那个部分。其平均粗糙度小于5埃米更佳。当用于生产LCD1737玻璃时，用美国专利3,338,696和3,682,609所述方法形成的熔拉平板玻璃，所提供的表面就具有低于5埃米的较佳平均粗糙度。这种较佳的平均粗糙度也可以通过抛光实现。表面的平滑度有助于施涂均匀的表面涂层。
较佳施涂在硼硅酸盐或硼铝硅酸盐基片上用来固定寡核苷酸的涂层是极性硅烷，它含有例如适用于锚定固相寡核苷酸合成并且特别适合于连接DNA分子的氨基。此极性硅烷可在水解后含有羟基(在水解前，该基团宜为烷氧基)。合适的涂层包括功能化的烷氧基硅烷或氯代硅烷，该硅烷具有1-3个烷氧基或氯基团。此外，通过例如光致抗蚀或掩模技术，可以使其上表面产生一定的图案。
采用硼硅酸盐或硼铝硅酸盐玻璃作为基片并不局限于使用用于寡核苷酸阵列载体的胺功能化涂层。基片可以用作各种不同结合实体的固相载体，这些结合实体包括通过共价键或非共价键对另一种分子具有特异亲和力的任何生物的或合成的分子。较佳的是这些结合实体天然地或通过修饰而含有功能化学基团(伯胺、巯基、醛、羧基、丙烯酸基等)、共同序列(核酸)、表位(抗体)、半抗原或配体，它们使结合实体可与基片表面上的共同功能基团共价地反应或非共价地结合。具体的结合实体包括(但并不限于)：脱氧核糖核酸(DNA)、核糖核酸(RNA)、人工合成的寡核苷酸、抗体、蛋白质、肽、凝集素、修饰的多糖、合成的复合大分子、功能化的纳米结构物(nanostructures)、合成的聚合物、修饰的/受保护的(blocked)核苷酸/核苷、修饰的/受保护的氨基酸、荧光团、生色团、配体、螯合物和半抗原。
实施例
进行了比较研究，确定涂有相同涂层，但由3种不同玻璃(钠石灰玻璃、硼硅酸盐玻璃和硼铝硅酸盐玻璃)制成的1″×3″载玻片的耐久性。对每种待测试的载玻片，施涂上γ-氨基丙基三乙氧基硅烷涂层。然后将载玻片浸入沸水0.5-5小时。若胺化的涂层在暴露于某些环境作用(在本例中为沸水)后仍保留在载玻片表面上，就称表面的功能性是保留了其功能性，耐久性测试的结果就是阳性的。
胺化的涂层的耐久性，用基于Au/Ag生长法的着色试验法进行测试。该方法可揭示基片表面上胺功能的存在与否。当发生Au/Ag的生长时，对于胺功能存在与否的测试结果就是阳性的。此时肉眼观察到一层浓密而均匀的金属灰色薄层，就表示是阳性的测试结果。没有胺功能的基片不会着色，仍保持透明。
着色法试验如下进行：将载玻片浸入AURODYE FORTE RPN 490(AmershamLife Science,Amersham International)1小时。用纯水漂洗载玻片2次。再用氮气对载玻片进行干燥。接着将载玻片浸入INTENSE BL SILVER ENHANCEMENTSOLUTION RPN492(Amersham Life Science,Amersham International)5分钟。再次用纯水漂洗载玻片，再用氮气干燥。通过肉眼观察确定金属灰色薄层的存在与否。
如上所述，测试了由3种不同材料即钠石灰玻璃、硼硅酸盐玻璃和硼铝硅酸盐玻璃制成的基片。表1显示了为了使涂有γ-氨基丙基三乙氧基硅烷的载玻片丧失其胺功能，暴露于沸水所需的时间长短(即，使着色试验结果为阴性的时间)。表1
    玻璃基片     涂层耐久性(小时)
    钠石灰玻璃     0.5
    硼硅酸盐玻璃     2.0
硼铝硅酸盐玻璃(1737 LCD玻璃)     4.0
表1所示结果表明，在硼硅酸盐或硼铝硅酸盐玻璃上的γ-氨基丙基三乙氧基硅烷涂层的耐久性，远远优于钠石灰玻璃上相同涂层的耐久性。
尽管不准备进行什么理论解释，但是可以认为，在硼硅酸盐和硼铝硅酸盐玻璃样品中，氧化钠水平较低或根本不存在氧化钠提供了测试结果所示的有利耐久性能。用于高密度分析基片的玻璃材料的氧化钠含量低于12摩尔％，较佳低于8摩尔％，更佳是甚至不含氧化钠。由于这个理由，可以设想使用任何具有这种关键氧化钠含量的玻璃(例如包括铝硅酸盐玻璃)来代替上述硼硅酸盐或硼铝硅酸盐玻璃。
在表2中给出了钠石灰玻璃的组成。表3中给出了该实施例中所用的硼硅酸盐玻璃的组成。在本实施例中所用的硼铝硅酸盐玻璃1737LCD玻璃的组成已在上面给出。
表2
    组分     摩尔百分数(％)
    SiO2     71.5
    Na2O     13.3
    K2O     0.3
    CaO     8
    MgO     4.1
    Al2O3     1.5
    SO3     0.37
    TiO2     0.06
    Fe2O3     0.07
    As2O3     0.015
表3
    组分     摩尔百分数(％)
    SiO2     65
    Na2O     6.4
    K2O     6.6
    Al2O3     4.1
    TiO2     4.2
    B2O3     8.1
    ZnO     5.6
    Sb2O3     0.2
尽管为了说明起见详细描述了本发明，然而应该理解，这些说明细节仅用于这个目的。在不背离权利要求所限定的本发明精神和本发明范围的情况下，本领域的技术人员可以进行各种改动。

Claims (27)

1．一种用于支承高密度生物或化学阵列的基片，其特征在于，它是由氧化钠含量低于12摩尔％的硼硅酸盐或硼铝硅酸盐玻璃构成，基本平坦的1英寸×3英寸载玻片。
2．如权利要求1所述的基片，其特征在于，它还具有平均粗糙度小于10纳米的上表面。
3．如权利要求1所述的基片，其特征在于，它还具有平均粗糙度小于5埃米的上表面。
4．如权利要求1所述的基片，其特征在于，它在表面上具有至少局部的极性硅烷涂层。
5．如权利要求4所述的基片，其特征在于，该极性硅烷含有胺基。
6．如权利要求4所述的基片，其特征在于，该极性硅烷含有至少一个羟基。
7．如权利要求4所述的基片，其特征在于，该极性硅烷含有至少一个烷氧基。
8．如权利要求4所述的基片，其特征在于，该极性硅烷含有至少一个氯原子基团。
9．如权利要求4所述的基片，其特征在于，该极性硅烷是氨基丙基三乙氧基硅烷。
10．如权利要求1所述的基片，其特征在于，该硼铝硅酸盐玻璃是1737LCD玻璃。
11．如权利要求1所述的基片，其特征在于，该硼铝硅酸盐玻璃是7059LCD玻璃。
12．一种将基片用于高密度生物或化学阵列的方法，其特征在于，包括如下步骤：
(a)提供由氧化钠含量低于12摩尔％的玻璃材料构成，基本平坦的载玻片；
(b)将结合实体阵列附着于该载玻片。
13．如权利要求12所述的方法，其特征在于，还包括步骤：在该附着步骤之前，将功能化的极性硅烷涂料施涂在该材料的至少一部分表面上。
14．如权利要求12所述的方法，其特征在于，该载玻片具有平均粗糙度小于10纳米的上表面。
15．如权利要求13所述的方法，其特征在于，该极性硅烷含有胺基。
16．如权利要求13所述的方法，其特征在于，该极性氨基是氨基丙基三乙氧基硅烷。
17．如权利要求12所述的方法，其特征在于，该玻璃材料是硼铝硅酸盐玻璃。
18．如权利要求12所述的方法，其特征在于，该玻璃材料是硼硅酸盐玻璃。
19．一种用于支承高密度生物或化学阵列的基片，其特征在于，它包括氧化钠含量低于12摩尔％并且上表面平均粗糙度小于10纳米的玻璃材料。
20．一种用于支承高密度生物或化学阵列的基片，其特征在于，它包括
氧化钠含量低于12摩尔％并且其上表面平均粗糙度小于10纳米的玻璃材料；
覆盖该材料的至少一部分表面的极性硅烷的功能化涂层；
并且该基片的特性是，在浸入沸水超过1小时后该涂层的功能性仍保留。
21．如权利要求20所述的基片，其特征在于，该材料的平均粗糙度小于5埃米。
22．如权利要求20所述的基片，其特征在于，所述的氧化钠含量低于8摩尔％。
23．如权利要求20所述的基片，其特征在于，该极性硅烷含有胺基。
24．如权利要求20所述的基片，其特征在于，该极性硅烷含有至少一个羟基。
25．如权利要求20所述的基片，其特征在于，该极性硅烷含有至少一个烷氧基。
26．如权利要求20所述的基片，其特征在于，该极性硅烷含有至少一个氯原子基团。
27．如权利要求20所述的基片，其特征在于，该极性硅烷是氨基丙基三乙氧基硅烷。
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