Universidade de São Paulo, Instituto de Química, Av. Prof. Lineu Prestes, 748, Cidade Universitária, 05508-000 São Paulo, SP, Brazil.
An Acad Bras Cienc. 2022 Aug 1;94(3):e20210917. doi: 10.1590/0001-3765202220210917. eCollection 2022.
Molecular machines, as exemplified by the kinesin and microtubule system, are responsible for molecular transport in cells. The monitoring of the cellular machinery has attracted much attention in recent years, requiring sophisticated techniques such as optical tweezers, and dark field hyperspectral and fluorescence microscopies. It also demands suitable procedures for immobilization and labeling with functional agents such as dyes, plasmonic nanoparticles and quantum dots. In this work, microtubules were co-polymerized by incubating a tubulin mix consisting of 7 biotinylated tubulin to 3 rhodamine tubulin. Rhodamine provided the fluorescent tag, while biotin was the anchoring group for receiving streptavidin containing species. To control the microtubule alignment and consequently, the molecular gliding directions, functionalized iron oxide nanoparticles were employed in the presence of an external magnet field. Such iron oxide nanoparticles, (MagNPs) were previously coated with silica and (3-aminopro-pyl)triethoxysilane (APTS) and then modified with streptavidin (SA) for linking to the biotin-functionalized microtubules. In this way, the binding has been successfully performed, and the magnetic alignment probed by Inverted Fluorescence Microscopy. The proposed strategy has proved promising, as tested with one of the most important biological structures of the cellular machinery.
分子机器，以驱动蛋白和微管系统为例，负责细胞内的分子运输。近年来，对细胞机器的监测引起了广泛关注，需要使用光学镊子、暗场高光谱和荧光显微镜等复杂技术。它还需要合适的固定和标记程序，如用染料、等离子体纳米粒子和量子点等功能试剂进行标记。在这项工作中，通过孵育含有 7 个生物素化微管蛋白和 3 个罗丹明微管蛋白的微管混合液，共同聚合微管。罗丹明提供荧光标记，而生物素是接收含有链霉亲和素的物质的锚固基团。为了控制微管的取向，从而控制分子滑行方向，在外部磁场的存在下使用功能化的氧化铁纳米粒子。这些氧化铁纳米粒子（MagNPs）之前用硅烷和（3-氨丙基）三乙氧基硅烷（APTS）进行了涂层，然后用链霉亲和素（SA）进行了修饰，以与生物素化微管结合。通过荧光倒置显微镜探测到了这种结合。该策略已被成功应用于细胞机器的最重要的生物结构之一，证明了其具有广阔的应用前景。
