Ertürk Gizem, Lood Rolf
Department of Clinical Sciences Lund, Division of Infection Medicine, Lund University.
Department of Clinical Sciences Lund, Division of Infection Medicine, Lund University;
J Vis Exp. 2018 Feb 16(132):57208. doi: 10.3791/57208.
The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.
在复杂溶液中检测和定量生物分子的能力一直是自然科学领域备受追捧的；它被用于检测生物标志物、污染物和其他感兴趣的分子。用于此目的的一种常用技术是酶联免疫吸附测定（ELISA），通常一种抗体针对特定的靶分子，第二种标记抗体用于检测一抗，从而实现对所研究生物分子的绝对定量。然而，使用抗体作为识别元件限制了该方法的稳健性；使用标记分子的需求也是如此。为了克服这些限制，人们采用了分子印迹技术，创建与模板分子互补的人工识别位点，从而不再需要使用抗体进行初始结合。此外，为了获得更高的灵敏度，可以用依赖电容来定量靶分子的生物传感器取代二级标记抗体。在本方案中，我们描述了一种快速且无需标记地检测和定量复杂样品中低丰度生物分子（蛋白质和病毒）的方法，其灵敏度明显优于常用的检测系统，如ELISA。这一切都是通过分子印迹与电容生物传感器相结合来实现的。
