Lambert Christopher J, Clarke Eamonn, Patel Dhruv, Laurentius Lars B, Gale Bruce K, Sant Himanshu J, Porter Marc D
Departments of Mechanical Engineering, University of Utah, Salt Lake City, UT, 84112, USA.
Chemistry, University of Utah, Salt Lake City, UT, 84112, USA.
Anal Methods. 2024 Aug 8;16(31):5475-5481. doi: 10.1039/d4ay00772g.
Tuberculosis (TB) represents a major public health threat, with millions of new cases reported worldwide each year. A major hurdle to curtailing the spread of this disease is the need for low-cost, point-of-care (PoC) diagnostics. Mannose-capped lipoarabinomannan, a significant component of the bacillus, has been heavily studied as a biomarker for TB, but with little success due to its complexation with endogenous components of body fluids in a manner that sterically interferes with its detection by ELISA and other immunoassays. Recent work by our group and others has shown that complexation can be disrupted with protein-denaturing protocols. By way of followup, we recently described an enzymatic digestion (Proteinase K) sample pretreatment that enables quantitative recovery of ManLAM spiked into healthy human control serum. Herein, we report on the transfer of our benchtop sample pretreatment methodology to an automated microfluidic platform. We show that this platform can be configured to: (1) carry out the pretreatment process with very little user interaction and, (2) yield recoveries for ManLAm spiked into control serum which are statistically indistinguishable from those achieved by the benchtop process. Plans to integrate this device with a portable sample reader as a possible basis for a PoC TB diagnostic system and analyze patient samples are briefly discussed.
结核病是一项重大的公共卫生威胁，全球每年报告数百万新病例。控制这种疾病传播的一个主要障碍是需要低成本的即时检测（PoC）诊断方法。甘露糖封端的脂阿拉伯甘露聚糖是结核杆菌的一个重要成分，作为结核病的生物标志物已得到大量研究，但由于它与体液内源性成分络合，在空间上干扰了酶联免疫吸附测定（ELISA）和其他免疫测定对它的检测，因此成效甚微。我们团队及其他团队最近的研究表明，采用蛋白质变性方案可破坏这种络合。作为后续工作，我们最近描述了一种酶消化（蛋白酶K）样品预处理方法，该方法能够定量回收添加到健康人对照血清中的甘露糖脂阿拉伯甘露聚糖（ManLAM）。在此，我们报告将我们的台式样品预处理方法转移到自动化微流控平台的情况。我们表明，该平台可以配置为：（1）在用户极少干预的情况下进行预处理过程，以及（2）对于添加到对照血清中的ManLAm的回收率，在统计学上与台式方法所获得的回收率没有差异。本文简要讨论了将该设备与便携式样品读数器集成作为PoC结核病诊断系统的可能基础并分析患者样品的计划。
