Fabrication of Nanomechanical Biosensors to Map 3D Surface Strain in Live Cells and Tissue
Fabrication of Nanomechanical Biosensors to Map 3D Surface Strain in Live Cells and Tissue
复制标题
制造纳米机械生物传感器来绘制活细胞和组织中的 3D 表面应变图
DOI：
10.21203/rs.3.pex-1169/v1
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
A. Feinberg
中科院分区：
文献类型：
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作者：
Daniel J. Shiwarski;J. Tashman;A. Tsamis;J. Bliley;Malachi A. Blundon;E. Aranda;Q. Jallerat;John M. Szymanski;B. McCartney;A. Feinberg
本协议详细介绍了一步一步的制造过程，通过适应表面启动组装（SIA）程序，创建一个基于细胞外基质（ECM）蛋白质的纳米机械生物传感器（NMBS）。NMBS是一种荧光标记的超薄纤维连接蛋白晶格网，通过调整晶格的宽度和间距从2-100 μ m定制空间分辨率。通过将NMBS应用于细胞和组织表面，可以直接测量从亚细胞到组织长度尺度的变形。这里概述的过程涵盖了NMBS构建的所有方面，从图案设计、模具制作的光刻、聚二甲基硅氧烷（PDMS）硅胶印章的铸造、荧光染料与纤维连接蛋白的结合、纤维连接蛋白的微接触印刷、明胶载体的创建、将NMBS转移到明胶载体上，以及使用明胶载体将NMBS应用到细胞和组织上。该方案可分为三个阶段：材料制备（2天），NMBS图案（4小时）和转移到细胞或组织（1小时）。材料准备和NMBS图案化可以提前进行，图案化的NMBS可以在使用前储存长达1年。
This protocol details the step-by-step fabrication process to create an extracellular matrix (ECM) protein-based nanomechanical biosensor (NMBS) via an adaptation of the surface-initiated assembly (SIA) procedure1. The NMBS is a fluorescently labeled, ultra-thin fibronectin lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2-100 µm. By applying the NMBS to the surface of cells and tissues one can directly measure deformation from subcellular to tissue length-scales. The procedure outlined here covers all aspects of NMBS construction from pattern design, photolithography for mold creation, casting of polydimethylsiloxane (PDMS) silicone stamps, conjugation of fluorescent dye to fibronectin, microcontact printing of fibronectin, creation of gelatin carriers2, transfer of the NMBS to gelatin carriers, and the use of gelatin carriers for application of the NMBS onto cells and tissue. The protocol can be broken down into three phases: material preparation (2 days), NMBS patterning (4 hours), and transfer to cell or tissue (1 hour). Material preparation and NMBS patterning can be performed ahead of time and the patterned NMBS can be stored for up to 1 year prior to use.