Shahrokhtash Ali, Sutherland Duncan S
Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000Aarhus C, Denmark.
The Centre for Cellular Signal Patterns (CellPAT), Gustav Wieds Vej 14, 8000 Aarhus C ,Denmark.
ACS Appl Mater Interfaces. 2024 May 1;16(17):21534-21545. doi: 10.1021/acsami.4c00831. Epub 2024 Apr 18.
Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.
纳米级生物分子的布局对于推进细胞信号传导、传感器技术和分子相互作用研究至关重要。尽管如此，目前的方法在实现多种生物分子的大面积纳米图案化同时最小化非特异性相互作用方面仍存在不足。通过在亚微米尺度上使用生物正交标签，我们引入了一种新颖的孔掩膜胶体光刻方法，用于在大面积、完全钝化的表面上排列多达三种不同的蛋白质、DNA或肽。这些表面与单分子荧光显微镜和微孔板格式兼容，便于在细胞和单分子检测中进行多种应用。我们使用完全钝化且不含金属和纳米拓扑特征的透明基板，以确保精确图案化并最小化非特异性相互作用。表面图案化是通过生物正交的反式环辛烯 - 四嗪（逆电子需求狄尔斯 - 阿尔德反应，IEDDA）连接、二苯并环辛炔 - 叠氮化物（应变促进的叠氮化物 - 炔烃环加成反应，SPAAC）点击化学以及生物素 - 抗生物素蛋白相互作用来实现的。这些反应被安排在通过选择性和逐步去除牺牲性金属和聚合物层而制备的致密聚（乙二醇）（PEG）刷钝化的表面上，从而能够以纳米级精度定向附着生物特异性标签。在一个概念验证实验中，与纳米簇中的cRGD（精氨酰甘氨酰天冬氨酸）偶联的DNA张力计系链（TGT）力传感器测量了成纤维细胞整合素张力。这种新颖的应用能够量化皮牛顿范围内的力，该力被限制在纳米图案化的簇内。该平台用于研究整合素和表皮生长因子（EGF）近端信号传导的第二个演示揭示了清晰的机械转导和细胞形态变化。这些发现表明该平台作为一种强大工具的潜力，可用于探究涉及以纳米精度排列的多个分子和纳米级细胞相互作用的复杂生化途径。
