CN107109141A - 在防污支撑件上印刷粘附图案 - Google Patents
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Abstract
一种在聚合物刷子上印刷粘附图案的方法,其中所述聚合物刷子在支撑件(1)表面处延伸而形成纳米防污层(2),该方法包括以下步骤:‑将所述层(2)与含有二苯甲酮的第一水溶液(4)接触;‑然后根据图案且根据表面能量,用波长在二苯甲酮的吸收光谱内的辐射(3)照射所述层。
Description
技术领域
本发明涉及根据光学限定的图案将蛋白质接枝到基底上的领域。
背景技术
国际公布号为WO 2013/135844(以下称为“STUDER”或“公布”)的国际申请公开了一种用于将蛋白质微结构接枝到基底上的装置或光化学印刷装置。在该公布中,水溶液中的二苯甲酮(BP)和蛋白质的混合物在根据基底顶部图案的位置处被照射,并且在照射位置处获得蛋白质的持久转移,以实现印刷。然而,该公布中所描述的方法是在BP存在下、并在被照射的同时将蛋白质转移到基底上的。
该装置涉及根据基底上图案的图像进行照射的照射装置和能够输送同时含有蛋白质和BP的水溶液的微流体装置在相同位置处的组合。这导致印刷系统体积庞大的问题,并且还存在由二苯甲酮和来自照射装置的光的组合作用损坏蛋白质的风险。理想的情况下,借助于照射系统,在不粘附蛋白质的情况下,将对蛋白质仅有粘附性的图案印刷到基底上是非常有用的。随后在与蛋白质(例如荧光蛋白)水溶液接触的情况下,实际图案会在基底上显影,其中,蛋白质优先附着在粘附图案中的被照射的部分以形成实际图案。然而,对于蛋白质,这种溶液取决于能够产生潜在的或随后显影的粘附图案的方法的有效性,其中,所述粘附图案印刷在由蛋白质防污层覆盖的支撑件(support)上。蛋白质防污层应理解为是指由在进行所要实现的印刷的时间尺度内蛋白质不会附着到所述层的材料制成的层。
被其防污层覆盖的这种基底或防污基底可以特别地由支撑件形成,例如硬支撑件,其一个实例是具有对于来自照射系统的光呈透明的光学性能的玻璃,或例如软支撑件,其一个实例是PDMS(聚二甲基硅氧烷),其中,玻璃或PDMS由聚合物刷子材料、或通过分子链附着到支撑件上而作为刷子的聚合物(例如PEG(聚乙二醇)和聚NIPAM(N-异丙基丙烯酰胺))覆盖。对于这种类型的防污基底,聚合物链在其一端连接到支撑件并且在另一端自由,类似于刷子的刷毛。
其它技术例如在防污基底上进行防污材料的图案的激光烧蚀的、通过掩模应用于蛋白质防污基底的光刻技术在现有技术中可以获得具有图案的防污支撑件,该图案允许随后根据已去除聚合物刷子或防污材料的基底的照射区域,通过光能将蛋白质选择性接枝到基底上。
材料的烧蚀被认为是由基底的照射引起的,并且所产生的程度差使得随后的实际图像的反影图像成为可能。当通过仅对光路敏感的光学相位对比技术观察这些差异时,粘附图案可能以同样的方式归因于防污材料的烧蚀或材料性质的变化,防污材料的烧蚀或材料性质的变化改变其光学指数并随后使蛋白质优先粘附到已经照射的聚合物链区域。能够观察潜像的其它技术(特别是原子力显微镜、椭偏仪、X射线分析等)能够在某些情况下证实潜像是由用于这些技术的PEG层的完全烧蚀产生的。因此,这种烧蚀技术不能产生密度梯度,PEG或防污层的烧蚀是预先完成的。
最终需要有一种用于生产防污或聚合物刷子支撑件的方法,其按照图案而具有随着刷子被照射而成比例或连续变化的粘附性,而分子不必在被照射的同时粘附到刷子。相反,需要这些分子以延迟的方式粘附到刷子上。
发明内容
以下定义适用于本申请:
“粘附图案”:表示表面图案,根据所述表面图案,某些分子特别是蛋白质(并尤其是抗体)、纳米壳、DNA(脱氧核糖核酸)链或RNA链或细菌以时间稳定的方式分布在由所述粘附图案之外的防粘附或防污或聚合物刷子层覆盖的支撑件上。由于图案被限定在防粘附或防污区域或者一组防粘附或防污区域之外,粘附图案也可以在基底上被限定为一组区域或图案,其与基底上的该组区域的补充表面相比,对于感兴趣的分子更粘附。对于聚合物刷子,至少可通过以下两种已在现有技术中存在的技术预测无需与分子的水溶液接触的图案的存在所需的粘附效果的差异:
-原子力显微镜,其可以显示刷子的聚合物链长度的减小,这种减少随后导致防粘附效果的降低或这些区域中的粘附效果的增加。
-相差显微镜,其可以显示在大多数粘附区域中通过刷子的光路变化,这种变化随后与粘附效果变化相关联。
“聚合物刷子”:表示纳米层(即其厚度在纳米级,即通常在1nm和100nm之间),其具有防污性,特别是对于蛋白质、纳米壳、DNA链和细菌有防污作用,这种纳米层存在于支撑件的表面以形成防污基底。据估计,在本申请的申请日时,这种刷子由一组接枝到支撑件表面上的聚合物链组成,该组聚合物链在支撑件表面处的区域中延伸,对于PEG,其厚度在1nm和20nm之间,而对于聚NIPAM,其厚度在1nm和30nm之间。据估计,在1nm和20nm之间,这种刷子具有防粘附或防污性质,特别是对于蛋白质、纳米壳、DNA链或细菌。聚乙二醇或称“PEG”层、或聚(N-异丙基丙烯酰胺)或称聚NIPAM层是聚合物刷子的实例。
对于聚合物刷子,“厚度”表示从形成刷子的聚合物链的自由端到支撑件的距离。例如,对于PEG,层的厚度取决于PEG链的长度,也即构成这些链的乙二醇单体的数量。这些链可以特别地相对于基底倾斜,或者以类似于刷子刷毛的作用的任何方式被压缩或修改,以便在刷子的自由表面上印刷浮凸或改变厚度。
在本文中,本发明涉及在聚合物刷子上印刷粘附图案的方法,其中所述聚合物刷子在支撑件的表面处延伸而形成纳米防污层,该方法包括以下步骤:
-将所述层与含有二苯甲酮的第一水溶液接触,
-然后根据图案且根据表面能量,用波长在二苯甲酮的吸收光谱范围内的辐射照射该层。
在方法的变体中:
-所述层的厚度在1nm和20nm之间;
-所述波长在300nm和400nm之间选择;
-所述聚合物为聚乙二醇(PEG);
-所述聚合物为聚NIPAM;
-所述支撑件为玻璃;
-所述支撑件为聚二甲基硅氧烷(PDMS);
-传递到PEG层的照射的表面能量在10mJ/mm2和1000mJ/mm2之间;
-传递到聚NIPAM层的照射的表面能量在100mJ/mm2和10000mJ/mm2之间;
–PDMS支撑件的杨氏模量小于15kPa。
本发明还涉及一种用于在聚合物刷子上印刷蛋白质图案的上述方法,其包括以下附加步骤:
-冲洗以消除所述层和所述第一溶液之间的接触,
-然后将所述层与含所述有蛋白质的第二水溶液接触。
本发明还涉及一种用于在聚合物刷子上印刷纳米壳图案的上述方法,其包括以下附加步骤:
-冲洗以消除所述层和所述第一溶液之间的接触,
-然后将所述层与含有所述纳米壳的第二溶液接触。
本发明还涉及一种用于在聚合物刷子上印刷DNA链图案的上述方法,其包括以下附加步骤:
-冲洗以消除所述层和所述第一溶液之间的接触,
-然后将所述层与含有所述DNA链的第二溶液接触。
本发明还涉及一种用于印刷粘附图案的方法的应用,其应用于通过表面能量的空间变化来制备在支撑件的表面处具有粘附力梯度的粘附图案。
附图说明
结合下面的附图将更好地理解本发明,其中:
图1表示由玻璃支撑件和防污材料层组成的防污基底的横截面,特别是对蛋白质、纳米壳或DNA链有防污性,也就是说,聚合物刷子接枝或附着到支撑件上。在所有或一些聚合物刷子上用含有二苯甲酮的水溶液的液滴覆盖基底。覆盖有二苯甲酮层的区域AB用包括二苯甲酮吸收光谱内的波长的辐射(即在300nm与400nm之间的辐射)透过支撑件(透过液滴也是可行的)照射。
图2表示图1中的液滴被冲洗且具有由区域AB处防污材料表面中的中空示意性地表示的潜像的基底。这种材料能够根据与区域AB表面相对应的图案用于印刷图案,尤其是蛋白质、纳米壳或者DNA或RNA链的图案。具体地,在二苯甲酮存在下,对聚合物刷子的照射有可能使聚合物刷子在区域AB中具有粘附性,并且随后通过使聚合物刷子层分别与特别是蛋白质溶液、纳米壳或DNA链的溶液接触,随后能够使特别是蛋白质、纳米壳或DNA链粘附。因此,根据如AB的一组区域所进行的照射能够产生粘附图案或聚合物刷子上的分子的粘附图案,用于对于刷子通常不呈现粘附性的分子。
具体实施方式
在第一实施例中,参考图1的括号中的附图标记,公开了一种由玻璃支撑件(1)和聚合物刷子材料的层(2)组成的防污基底,在该第一实施例中,所述聚合物刷子材料为PEG或聚NIPAM。
在该第一实施例中,辐射(3)在此透过支撑件(1)照射区域AB(AB)上的层(2),其中,所选择的支撑件(1)对所使用的辐射透明,且二苯甲酮水溶液的液滴(4)沉积在覆盖区域AB(AB)的层(2)上。以相同的方式,可以透过液滴(4)照射相同的区域AB上的层。
所使用的辐射包括在二苯甲酮的吸收光谱内的至少一个波长,该光谱实际上可有利地在300nm和400nm之间延伸。优选地,在该范围内使用波长小于390nm的辐射,在这种情况下,对层进行辐射的时间将最小化。
在所选波长下二苯甲酮的吸收越低,光源的功率将必须越大或者对所照射区域进行辐射的时间必须越长,所接收的辐射剂量等于照射功率和照射时间的乘积,所接收的辐射剂量是控制本发明所获得的效果的参数。
由于溶液中没有待接枝的蛋白质,根据需要,辐射可具有与对随后待接枝的蛋白质产生破坏的功率相比更高的功率,并且仅受限于由上述层所接受的光能的表面密度,而不会产生降解。然而,对于PEG,在二苯甲酮的存在下,可以使用比烧蚀或掩模技术低10至100倍的光学功率。
因此可以使用10mJ/mm2和1000mJ/mm2之间的能量密度来获得PEG上的粘附图案的出现。因此,对于来自半导体激光器的在372nm处的紫外线波长产生具有400微米边长的正方形范围的2mW照射的光源可以满足本发明。对于PDMS支撑件上的聚NIPAM,可用的能量密度在100mJ/mm2到10000mJ/mm2之间。可以通过简单地对PEG的照射时间乘以10来再次使用相同的半导体激光源。
在本实施方式的第一步中,将防污基底与二苯甲酮水溶液的液滴接触,然后在第二步中,用紫外线光源照射基底的防污层的区域AB。
可以使用能够使光源的能量同时聚焦在区域AB或一组区域上的任何光学系统,并且这些系统是现有技术中已知的。因此,可以设想具有微镜阵列的显微镜用于产生该实施例的照射系统。类似地,可以用二苯甲酮水溶液的膜替代液滴以与上述层接触,然后用已知的微流体装置在照射后冲洗。
图2示意性地表示形成为纳米层且通过冲洗二苯甲酮溶液的液滴而提供的聚合物刷子,其中,在区域AB中具有本身相对于其深度而言也为纳米级别的中空潜在图案。为了显影,需要随后使该中空或潜在图案与能够在聚合物刷子中的该中空处粘附到支撑件上的分子或分子组件(蛋白质、纳米壳、DNA链、细菌等)接触,随后在水溶液中存在这些分子的情况下,这些分子根据潜在图案在二苯甲酮存在下已照射的层(这里为AB)的区域处发生粘附。分子的粘附在不提供光能的条件下发生。分子在潜在图案或粘附图案处直接被吸附在聚合物刷子上。因此,在刷子上形成分子的实际图案。特别地,如果分子是荧光的,则可以通过现有技术中已知的技术作出其图像,以展现粘附的结果。
然而,即使不与水溶液(例如蛋白质溶液)接触,也可以在刷子的日照之后通过在照射之后使用原子力显微镜(AFM)在照射位置处测量刷子中是否存在纳米深度的中空,或者通过利用相差显微镜光学地观察在这些相同位置处刷子中是否存在光路变化,与随后的实际图案的产生无关地预测是否将获得本发明的效果。因此,可以在没有其它实验的情况下选择适用于本发明方法的聚合物刷子,特别是用于在二苯甲酮存在下照射后观察到刷子的聚合物链的长度减小的那些。
在本发明的第二实施方式中,使图2的装置与蛋白质水溶液或纳米壳水溶液接触。从能够粘附到支撑件上的蛋白质和纳米壳体中选择蛋白质和纳米壳体的类型,以获得尽可能最持久的实际图像。
因此,通过该第二实施方式的方法,可以例如通过使用荧光蛋白获得区域AB的实际图像,但是更一般地,使用蛋白质防污基底上的蛋白质图案。此外,即使区域AB对应于光学照明系统的分辨率极限,防污基底在照射条件下的特性使得可以产生具有随该区域所接收的照射或光辐射剂量而连续变化的值的荧光,更一般地,产生随着该区域中的照射而连续变化的蛋白质、纳米壳或DNA链的浓度,而不需要求助于二相点的密度来模拟可变的蛋白质密度。
因此,可以将本发明应用于例如蛋白质、纳米壳或DNA链沿着基底或防污层的表面在密度方向产生粘附梯度,通过将类型AB的几个区域端对端对齐,并且通过改变传递到这些区域的表面能量,例如通过在二苯甲酮的存在下照射聚合物刷子或者印刷潜像或粘附图案的步骤期间,以可变(表面能,以J/m2计)的表面区域照射它们。
例如,对于在粘附区域外具有估计为5nm的厚度的PEG聚合物刷子,通过在二苯甲酮存在下在PEG刷子上的可变剂量照射,使得厚度减少量在0nm(无粘附或图案外部区域)和2nm(最大粘附)之间,从而获得蛋白质的连续可变粘附效果。
在所提出的实施方式中,以毫摩尔二苯甲酮/升水溶液(mmol/l)计,可以使用5mmol/l至50mmol/l的浓度范围。
本发明在工业上适用于用于在聚合物刷子上印刷蛋白质的粘附图案的基底生产领域。
Claims (14)
1.一种在聚合物刷子上印刷粘附图案的方法,其中所述聚合物刷子在支撑件(1)的表面处延伸而形成纳米防污层(2),所述方法包括以下步骤:
-将所述层(2)与含有二苯甲酮的第一水溶液(4)接触;以及
-然后根据图案且根据表面能量,用波长在所述二苯甲酮的吸收光谱内的辐射(3)照射所述纳米防污层。
2.根据权利要求1所述的方法,其中,所述层(2)的厚度在1nm和20nm之间。
3.根据权利要求2所述的方法,其中,所述波长在300nm和400nm之间选择。
4.根据权利要求3所述的方法,其中,所述聚合物为聚乙二醇(PEG)。
5.根据权利要求3所述的方法,其中,所述聚合物为聚NIPAM。
6.根据权利要求3所述的方法,其中,所述支撑件(1)为玻璃。
7.根据权利要求3所述的方法,其中,所述支撑件(1)为聚二甲基硅氧烷(PDMS)。
8.根据权利要求4所述的方法,其中,所述照射传递到PEG层的表面能量在10mJ/mm2和1000mJ/mm2之间。
9.根据权利要求5所述的方法,其中,所述照射传递到聚NIPAM层的表面能量在100mJ/mm2和10000mJ/mm2之间。
10.根据权利要求7所述的方法,其中,所述PDMS的杨氏模量小于15kPa。
11.根据权利要求1所述的方法,所述方法用于在所述聚合物刷子上印刷蛋白质图案,所述方法还包括以下步骤:
-冲洗以消除所述层(2)和所述第一水溶液(4)之间的接触;以及
-将所述层(2)与含有蛋白质的第二水溶液接触。
12.根据权利要求1所述的方法,所述方法用于在所述聚合物刷子上印刷纳米壳图案,所述方法还包括以下步骤:
-冲洗以消除所述层(2)和所述第一水溶液(4)之间的接触;以及
-将所述层(2)与含有纳米壳的第二溶液接触。
13.根据权利要求1所述的方法,所述方法用于在所述聚合物刷子上印刷DNA链图案,所述方法还包括以下步骤:
-冲洗以消除所述层和所述第一水溶液(4)之间的接触;以及
-将所述层(2)与含有DNA链的第二溶液接触。
14.根据权利要求1所述方法的应用,所述应用用于通过表面能量的空间变化来制备在所述支撑件(1)的表面处具有粘性梯度的粘附图案。
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