Data from: Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.在单分子荧光研究中，标记底物产生的背景发射通常会将其浓度限制在非生理的纳摩尔量级。解决这一难题的一种方案是采用零模波导（zero-mode waveguides, ZMWs）——即金属薄膜上的纳米级孔洞，可在物理与光学层面限制观测体积，从而允许使用浓度更高的荧光底物。ZMW的常规制备工艺依赖耗时且成本高昂的电子束纳米光刻技术。本文中，研究人员采用聚苯乙烯微球自组装掩模制备ZMW，无需精密设备即可并行加工数千个ZMW。直径1微米的聚苯乙烯微球可在载玻片上自组装形成六边形阵列，作为模板用于在微球间隙中沉积金属柱。通过在聚苯乙烯的玻璃化转变温度下对微球进行部分熔融，可将间隙（以及后续形成的金属柱）的宽度调控在100~300纳米范围内。随后将微球在甲苯中溶解，在金属柱周围沉积铝或金镀层，再移除金属柱，最终得到ZMW阵列。本文还对工艺参数优化及ZMW的性能进行了展示。借助胶体自组装技术，普通实验室即可采用亚波长ZMW技术，无需配备精密洁净室环境与设备，也无需承担相关高昂成本。