Flow-Chamber Hybridization Rate Enhancement for Distributed Viral Load Measuremen

描述（由申请人提供）：在资源有限的环境中，病毒载量测量是一个持续需要的领域。在紧凑、易于使用、每次测试成本低的仪器方面的最新进展，有望开发用于HIV RNA定量的高度便携式测定仪（HPAs）。由于缺乏热循环和对污染的最低敏感性，支链DNA (bDNA) HIV RNA夹心核酸杂交/信号扩增试验是资源有限环境下病毒载量测量的良好候选。然而，成功开发HPA-bDNA方法和仪器需要加速HIV RNA与固相捕获探针的杂交。在目前的bDNA方案中，杂交发生在一夜之间；这样一个冗长的杂交步骤是不切实际的HPA病毒载量测试在资源有限的设置。这项可行性研究的中心假设是，可以利用微尺度流体力学过程和非线性扩散效应来显著加速流室HIV RNA杂交——在不到20分钟的时间内，样品中足够大的病毒RNA总量的一部分就能与固相结合。本文将研究一种利用微尺度流体流动层流性和扩散效应非线性标度的新方法——序贯分层。初步建模表明，顺序分层增强流室可以实现接近侧流膜的分析物捕获效率（在100微升样品中杂交超过30%的HIV RNA，初级杂交步骤持续时间仅为10分钟），同时保持完全主动流体运输的操作参数控制。为了确定可行性并为HPA-bDNA系统的开发奠定基础，将制作原型流室并使用bDNA探针和参考分析物进行测试，将使用有限元建模进行设计优化和性能参数估计，并建立系统需求。血浆病毒载量，由复杂的血液测试确定，是艾滋病毒/艾滋病患者病情严重程度以及他或她对治疗反应如何的指标。定期检测病毒载量对照顾艾滋病毒/艾滋病患者很重要，但目前并非人人都能进行这些检测。该项目探索了新的方法，使病毒载量测量更便宜，更容易为远离先进医疗设施的医生和患者提供。

DESCRIPTION (provided by applicant): Viral load measurement in resource-limited settings is a persistent area of need. Recent advances in compact, easy-to-use instrumentation with low per-test costs is promising for the development of highly portable assayers (HPAs) for HIV RNA quantitation. Lacking thermal cycling and minimally susceptible to contamination, the branched DNA (bDNA) HIV RNA sandwich nucleic acid hybridization/signal amplification assay is a good candidate for viral load measurement in resource-limited settings. However, successful development of HPA-bDNA methods and instruments requires speeding hybridization of HIV RNA to solid- phase capture probes. In the current bDNA protocol, hybridization takes place overnight; such a lengthy hybridization step is impractical for HPA viral load testing in resource-limited settings. The central hypothesis of this feasibility study is that microscale fluid mechanical processes and nonlinear diffusion effects can be leveraged to significantly accelerate flow-chamber HIV RNA hybridization-to the extent that a sufficiently large fraction of total viral RNA in a sample binds to the solid phase in less than twenty minutes. A novel method referred to as sequential delamination, which takes advantage of the microscale fluid flow laminarity and nonlinear scaling of diffusive effects, will be studied. Preliminary modeling indicates that sequential delamination-enhanced flow chambers can achieve analyte capture efficiencies approaching those of lateral flow membranes (hybridization of over 30% of HIV RNA in a 100 microliter sample with a primary hybridization step only ten minutes in duration) while maintaining the operational parameter control of fully active fluid transport. To determine feasibility and lay the groundwork for HPA-bDNA system development, prototype flow chambers will be fabricated and tested using bDNA probes and reference analytes, finite element modeling will be used for design optimization and performance parameter estimation, and system requirements will be established. Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project explores new ways of making viral load measurements less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities.