IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa , Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Anal Chem. 2014 May 6;86(9):4340-7. doi: 10.1021/ac5001288. Epub 2014 Apr 21.
Microfluidic paper-based analytical devices (μPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from noncomplementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-labeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via the CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed μPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step toward the development of easy to use and affordable molecular diagnostic tests.
基于蜡印技术的微流控纸基分析器件（μPADs）是开发用于传染病的简单且经济实惠的分子诊断检测方法的合适平台，尤其是在资源有限的环境中。可以通过向测试区域添加适当的试剂来修改纸基器件以进行生物检测。为此，亲和固定化策略的使用可以为生物活性纸的制造提供很好的解决方案。本文描述了一种通过将抗体与融合蛋白结合来捕获标记的 DNA 链和杂交物的方法，该融合蛋白结合了来自热纤维梭菌的家族 3 碳水化合物结合模块（CBM），对纤维素具有高亲和力，以及葡萄球菌蛋白 A 的 ZZ 片段，该片段通过其 Fc 部分识别 IgG 抗体。通过 CBM-ZZ 固定的抗体能够捕获适当标记的（生物素、荧光素）DNA 链和 DNA 杂交物。在斑点和微通道检测中，均证明了针对生物素的抗体识别互补和非互补的、生物素标记的靶标的能力。通过荧光素标记的 DNA 探针的荧光发射检测杂交。与没有其他分子中介的情况下将抗体简单地点在纸上的物理吸附方法相比，通过 CBM-ZZ 构建将标记的 DNA 固定在纸上的抗体的捕获效率明显更高。本研究中提出的在室温下用 CBM-ZZ 功能化的蜡印 μPADs 进行 DNA 检测的实验概念验证是朝着开发易于使用且经济实惠的分子诊断测试迈出的重要一步。
