Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
Lab Chip. 2013 Oct 7;13(19):3910-20. doi: 10.1039/c3lc50229e.
Confirmatory diagnostics offer high clinical sensitivity and specificity typically by assaying multiple disease biomarkers. Employed in clinical laboratory settings, such assays confirm a positive screening diagnostic result. These important multiplexed confirmatory assays require hours to complete. To address this performance gap, we introduce a simple 'single inlet, single outlet' microchannel architecture with multiplexed analyte detection capability. A streptavidin-functionalized, channel-filling polyacrylamide gel in a straight glass microchannel operates as a 3D scaffold for a purely electrophoretic yet heterogeneous immunoassay. Biotin and biotinylated capture reagents are patterned in discrete regions along the axis of the microchannel resulting in a barcode-like pattern of reagents and spacers. To characterize barcode fabrication, an empirical study of patterning behaviour was conducted across a range of electromigration and binding reaction timescales. We apply the heterogeneous barcode immunoassay to detection of human antibodies against hepatitis C virus and human immunodeficiency virus antigens. Serum was electrophoresed through the barcode patterned gel, allowing capture of antibody targets. We assess assay performance across a range of Damkohler numbers. Compared to clinical immunoblots that require 4-10 h long sample incubation steps with concomitant 8-20 h total assay durations; directed electromigration and reaction in the microfluidic barcode assay leads to a 10 min sample incubation step and a 30 min total assay duration. Further, the barcode assay reports clinically relevant sensitivity (25 ng ml(-1) in 2% human sera) comparable to standard HCV confirmatory diagnostics. Given the low voltage, low power and automated operation, we see the streamlined microfluidic barcode assay as a step towards rapid confirmatory diagnostics for a low-resource clinical laboratory setting.
确证诊断通常通过检测多个疾病生物标志物来提供高临床灵敏度和特异性。在临床实验室环境中使用时，这些检测可确认阳性筛查诊断结果。这些重要的多重确证检测需要数小时才能完成。为了解决这一性能差距，我们引入了一种简单的“单入口、单出口”微通道架构，具有多重分析物检测能力。在直玻璃微通道中，经链霉亲和素功能化的填充通道的聚丙烯酰胺凝胶作为纯电泳但异质免疫测定的 3D 支架。生物素和生物素化捕获试剂沿微通道的轴以离散区域图案化，导致试剂和间隔物形成条码样图案。为了表征条码的制造，在一系列电迁移和结合反应时间尺度上进行了图案化行为的实证研究。我们将异质条码免疫测定法应用于检测丙型肝炎病毒和人类免疫缺陷病毒抗原的人类抗体。血清通过条码图案化凝胶电泳，允许捕获抗体靶标。我们评估了在一系列 Damkohler 数范围内的测定性能。与需要 4-10 小时的样品孵育步骤和 8-20 小时的总测定时间的临床免疫印迹相比；在微流控条码测定中定向电迁移和反应导致 10 分钟的样品孵育步骤和 30 分钟的总测定时间。此外，条码测定报告了与临床相关的灵敏度（2%人血清中的 25ngml(-1)），与标准 HCV 确证诊断相当。鉴于低电压、低功率和自动化操作，我们认为简化的微流控条码测定法是朝着在资源有限的临床实验室环境中进行快速确证诊断迈出的一步。
