Ultrasensitive HIV viral load quantitation using designer DNA nanostructure capture probes and photonic resonator interference scattering microscopy

摘要 频繁、准确和高度敏感的HIV-1病毒载量监测是艾滋病抗逆转录病毒治疗的关键组成部分 艾滋病毒/艾滋病治疗是减少母婴传播艾滋病毒发生率的一种手段，也是日常工作的一个必要组成部分。 诊断检测，使人们了解他们的艾滋病毒状况。尽管大量的研究和产品 开发工作已应用于即时病毒载量检测，即当前核酸检测的范例 并且抗原测定继续显示出源自其固有复杂性的基本局限性 和缺乏鲁棒性，这反过来又影响了它们在资源有限的环境中采用的成本和实用性。 我们寻求通过以下三个方面的结合来解决现有技术能力中的一个重要差距 创新，以产生一个集成的，快速的，简单的，超灵敏的，高选择性的，强大的和廉价的系统 用于定量病毒载量测量。首先，我们利用微流体从全血中分离病毒体， 在<10分钟内从20-100 µl全血中获得10-50 µl血浆样本，病毒提取率>95% 效率第二，我们将实现从所得血清中超选择性识别完整的HIV病毒体， 设计的DNA纳米结构，采取大分子“网”的形式，其顶点是精确的 与HIV外膜上显示的刺突gp 120蛋白基质的间距和定位机械匹配 面DNA网顶点掺入已经选择性地用于DNA扩增的核酸适体探针。 靶向HIV gp 120，导致多个高亲和力连接位点，因此“网”可用作 当共价连接到光子晶体生物传感器表面时有效的捕获探针。最后我们将 利用一种新发明的生物传感器显微镜形式，称为光子共振干涉散射 光子晶体表面放大激光散射的显微镜（PRISM）， 病毒粒子，使每一个都能以高信噪比计数。因为PRISM不需要标签 或酶促扩增，我们的方法能够动态，实时计数捕获的病毒与数字 精确度和超灵敏度。在本计画中，我们将整合病毒分离与光子晶体 将生物传感器装入塑料盒中，并开发一种快速的工作流程，该工作流程将简单快速， 床旁环境，目标是在<30分钟的采样到回答时间内得出结果。我们的目标包括 PRISM仪器的即时版本的开发，以及 检测限、重复性和耐用性。我们的研究将结束与验证系统使用临床 标本和直接比较金标准实验室RT-PCR分析。

Abstract Frequent, accurate, and highly sensitive HIV-1 viral load monitoring is a critical component of AIDS antiretroviral therapy, a tool for reducing the incidence of mother-to-child HIV transmission, and a required element of routine diagnostic testing to make people aware of their HIV status. Although enormous research and product development effort has been applied to point-of-care viral load testing, the current paradigm of nucleic acid tests and antigen assays continues to demonstrate fundamental limitations that derive from their inherent complexity and lack of robustness, which in turn impact their costs and practicality for adoption in resource-limited settings. We seek to address an important gap in the capabilities of existing technologies through a combination of three innovations to yield an integrated, rapid, simple, ultrasensitive, highly selective, robust, and inexpensive system for quantitative viral load measurement. First, we utilize microfluidic separation of virions from whole blood, yielding a 10-50 µl plasma sample from 20-100 µl of whole blood in <10 min, with >95% virus extraction efficiency. Second, we will achieve ultraselective recognition of intact HIV virions from the resulting serum using designer DNA nanostructures that take the form of a macromolecular “net” whose vertices are a precise mechanical match to the spacing and positioning of the spike gp120 protein matrix displayed on the HIV outer surface. The DNA net vertices incorporate nucleic acid aptamer probes that have been selected for selectively targeting the HIV gp120, resulting in multiple sites of high affinity attachment, and thus the “net” can be used as an effective capture probe when covalently attached to a photonic crystal biosensor surface. Finally, we will utilize a newly-invented form of biosensor microscopy called Photonic Resonator Interference Scattering Microscopy (PRISM) in which the photonic crystal surface amplifies laser light scattering from captured intact virions, enabling each one to be counted with high signal-to-noise ratio. Because PRISM does not require labels or enzymatic amplification, our approach enables dynamic, real-time counting of captured virus with digital precision and ultrasensitivity. In the proposed project, we will integrate viral separation and the photonic crystal biosensor into a plastic cartridge and develop a rapid workflow that will be simple and rapid for compatibility with point-of-care settings, with the goal of yielding a result in <30 minutes sample-to-answer. Our Aims include development of a point-of-care version of the PRISM instrument, and statistically robust characterization of detection limits, repeatability, and robustness. Our study will conclude with validation of the system using clinical specimens and direct comparison against gold-standard laboratory RT-PCR analysis.