Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.确定细胞内稀有蛋白质的定位通常需借助样本内扩增（on-sample amplification）技术。基于抗体识别与酶促扩增（enzymatic amplification）的传统方案，可在蛋白质表达位点生成大量可见标记物，但此类技术存在生物样本中非特异性反应的缺陷，且对标记物的空间调控能力欠佳。聚合扩增（polymerization based amplification）是近年新兴的荧光成像用样本内扩增替代技术，可规避内源性标记（endogenous labels）干扰与信号定位（signal localization）丢失的问题。本文基于聚合扩增技术，开发了一种稳定、可存档且具备比色检测能力的扩增模式，命名为聚合物染料标记（Polymer Dye Labeling）。其核心原理是在蛋白质表达位点原位生长界面聚合物（interfacial polymer），随后通过适配染料对该聚合物进行染色。研究人员最初在微阵列（microarray）实验中测试了伊文思蓝（Evans Blue）与曙红（eosin）的比色响应效果，二者均可特异性染色玻片上的聚合物薄膜。随后将该流程推广至人真皮成纤维细胞（human dermal fibroblast cells）内的蛋白质表达染色实验，结果显示聚合物染料标记仅在目标蛋白质表达的区域呈现特异性信号。该标记产物在室温环境下可稳定保存200天以上，且与现有主流封片介质（mounting medium）兼容。