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的关键组成部分 治疗是减少母婴传播艾滋病毒发病率的工具，也是常规工作的必要内容 诊断性检测，使人们了解自己的艾滋病毒状况。尽管巨大的研究和产品 开发工作已被应用于护理点病毒负载测试，这是目前核酸测试的范例 而抗原分析继续显示出它们固有的复杂性所产生的根本局限性。 以及缺乏健壮性，这反过来又影响了它们在资源有限的情况下采用的成本和实用性。 我们寻求通过三种技术的组合来解决现有技术能力中的一个重要差距 创新以产生集成、快速、简单、超灵敏、高度选择性、健壮和廉价的系统 用于病毒载量的定量测量。首先，我们利用微流控技术从全血中分离病毒粒子， 在10分钟内从20-100微米的L全血中提取10-50微米的L血浆样本，并进行95%的病毒提取 效率。其次，我们将实现对合成血清中完整的HIV病毒粒子的超选择性识别 设计者DNA纳米结构，其形式是一个大分子“网”，其顶点是一个精确的 与HIV外壳上显示的尖峰gp120蛋白矩阵的间距和位置的机械匹配 浮出水面。DNA网络顶点结合了核酸适体探针，这些探针已经被选择性地选择用于 以HIV gp120为靶点，导致多个部位的高亲和力附着，从而可将该“网”用作 一种当共价连接到光子晶体生物传感器表面时的有效捕获探针。最后，我们会 利用一种新发明的名为光子谐振器干涉散射的生物传感器显微镜 光子晶体表面放大完整捕获的激光散射的显微镜(PRISM) 病毒粒子，使每一个都能以高信噪比进行计数。因为PRISM不需要标签 或者酶扩增，我们的方法实现了对捕获的病毒的动态、实时计数 精确度和超高灵敏度。在拟议的项目中，我们将把病毒分离和光子晶体 将生物传感器集成到塑料墨盒中，并开发一种快速工作流程，该工作流程将简单快速地与 护理点设置，目标是在&lt；30分钟的样本到答案时间内得出结果。我们的目标包括 开发PRISM工具的护理点版本，并统计稳健地描述 检测限、重复性和稳健性。我们的研究将以该系统在临床上的验证为结束 并直接与金标实验室RT-PCR分析进行比较。

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.