Tomimuro Kosuke, Tenda Keisuke, Ni Yan, Hiruta Yuki, Merkx Maarten, Citterio Daniel
Department of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, 223-8522 Yokohama, Japan.
Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
ACS Sens. 2020 Jun 26;5(6):1786-1794. doi: 10.1021/acssensors.0c00564. Epub 2020 Jun 5.
Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.
抗体除了越来越多地用于治疗目的外，还是临床诊断中的重要生物标志物。尽管存在多种检测和定量抗体的方法，但它们主要需要由专业人员操作的台式仪器。为了能够在护理点（POC）检测抗体，开发独立于实验室设备的简单快速检测方法具有高度相关性。在本研究中，我们展示了基于微流控线的分析装置（μTADs）作为一种通过生物发光共振能量转移（BRET）切换传感器蛋白检测抗体的新平台。这些装置由垂直组装的层组成，包括血液分离膜和带有缝入棉线的塑料薄膜，BRET传感器蛋白与底物呋喃嗪已预先沉积在该薄膜上。与基于强度的信号传导不同，BRET机制能够在暗室中用数码相机或带有3D打印镜头适配器的智能手机相机对生物发光信号进行与时间无关的比率读出。该装置设计允许在单根缝线上空间分离地沉积多种生物发光蛋白，从而能够在5分钟内对5μL全血中的多种抗体进行定量。生物发光响应在5 - 15μL范围内与所加样品体积无关。因此，μTADs与基于BRET的传感器蛋白相结合，代表了一种用户友好的分析工具，可在手指刺破（5μL）全血时无需任何实验室设备即可对抗体进行POC定量。
