Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, USA.
Anal Chem. 2010 Jul 1;82(13):5591-7. doi: 10.1021/ac101269u.
In this paper, we report the development of an on-chip aptamer-based fluorescence assay for protein detection and quantification based on sandwich ELISA principles. Thrombin was selected as a model analyte to validate the assay design, which involves two DNA thrombin aptamers recognizing two different epitopes of the protein. Aptamer-functionalized magnetic beads were utilized to capture the target analyte, while a second aptamer, functionalized with quantum dots, was employed for on-chip detection. The binding of thrombin to the two aptamers via sandwich assay was monitored by fluorescence microscopy. The sandwich assay was performed on disposable microfluidic devices, fabricated on double-sided tapes and polymeric materials using a laser cutting approach. The approach enabled rapid thrombin detection with high specificity. Experimental conditions, such as reagent consumption and incubation time, were optimized in the microchip platform for the lowest limit of detection, highest specificity, and shortest assay time. The analytical performance of the microchip based assay was compared to that in the well plate format (generally utilized for ELISA-based methodologies). The results show that microfluidic chip proved to be a rapid and efficient system for aptamer-based thrombin assays, requiring only minimal (microliter) reagent use. This work demonstrated the successful application of on-chip aptamer-based sandwich assays for detection of target proteins of biomedical importance.
在本文中，我们报告了一种基于芯片的适体荧光分析方法的开发，用于基于夹心 ELISA 原理的蛋白质检测和定量。选择凝血酶作为模型分析物来验证该分析设计，该设计涉及两种 DNA 凝血酶适体，它们识别蛋白质的两个不同表位。适体功能化的磁性珠用于捕获目标分析物，而第二个适体则与量子点功能化，用于芯片上检测。通过荧光显微镜监测凝血酶与两个适体通过夹心测定的结合。夹心测定在一次性微流控设备上进行，该设备使用双面胶带和聚合物材料通过激光切割方法制造。该方法能够以高特异性快速检测凝血酶。在微芯片平台上优化了实验条件，如试剂消耗和孵育时间，以获得最低检测限、最高特异性和最短测定时间。基于微芯片的测定法的分析性能与微孔板格式（通常用于基于 ELISA 的方法）进行了比较。结果表明，微流控芯片证明是一种快速有效的基于适体的凝血酶测定法的系统，仅需要最小（微升）试剂用量。这项工作证明了基于芯片的适体夹心测定法在检测生物医学重要靶蛋白方面的成功应用。
