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

使用设计的 DNA 纳米结构捕获探针和光子谐振器干涉散射显微镜进行超灵敏 HIV 病毒载量定量

• 批准号: 10196015
• 负责人: Brian T. Cunningham
• 金额: $ 74.21万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-21 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10196015
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Address; Adoption; Affinity; Antibodies; Antigens; Avidity; Awareness; Base Sequence; Binding; Biological Assay; Biosensor; Blood; Buffers; Calibration; Chemistry; Clinical; Collection; Complex; Crystallization; Custom; Cytolysis; DNA; Detection; Development; Device Designs; Elements; Engineering; Environment; Epidemic; Epitopes; Exclusion; Exposure to; Filtration; Glycoproteins; Goals; Gold; HIV; HIV Envelope Protein gp120; HIV-1; Immobilization; Incidence; Individual; Label; Laboratories; Lasers; Measures; Mechanics; Microfluidic Microchips; Microfluidics; Microscopy; Monitor; Mother-to-child HIV transmission; Nanostructures; Noise; Nucleic Acid Amplification Tests; Nucleic Acids; Pathogen detection; Patients; Pattern; Performance; Plasma; Positioning Attribute; Proteins; Protocols documentation; Reagent; Reporting; Reproducibility; Resolution; Resources; Reverse Transcriptase Polymerase Chain Reaction; Routine Diagnostic Tests; Sampling; Serum; Signal Transduction; Site; Specificity; Specimen; Stains; Surface; Surface Plasmon Resonance; System; Technology; Temperature; Testing; Time; Validation; Vertical Disease Transmission; Viral; Viral Genome; Viral Load result; Viral load measurement; Virion; Virus; Whole Blood; antiretroviral therapy; aptamer; base; clinically relevant; cost; design; detection limit; digital; experimental study; innovation; instrument; light scattering; nanoparticle; particle; photonics; point of care; point of care testing; product development; research and development; sample collection; sensor; service utilization; stem; tool; transmission process

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.

抽象的 频繁，准确且高度敏感的HIV-1病毒载荷监测是AIDS抗逆转录病毒的关键成分 治疗，一种减少母亲艾滋病毒传播事件的工具，以及常规的必需要素 诊断测试使人们意识到自己的艾滋病毒状况。虽然庞大的研究和产品 开发工作已应用于护理点病毒负荷测试，即当前的核酸测试范式 抗原测定继续证明从继承复杂性得出的基本限制 并且缺乏鲁棒性，这反过来影响了他们在资源有限的环境中采用的成本和实用性。 我们试图通过三个组合来解决现有技术能力的重要差距 创新以产生综合，快速，简单，超敏感，高度选择性，健壮且廉价的系统 用于定量病毒负荷测量。首先，我们利用病毒与全血的微流体分离， 在<10分钟内从20-100 µL全血中产生10-50 µL血浆样品，> 95％的病毒提取 效率。其次，我们将通过使用的血清实现对完整的HIV病毒的超选择性识别 设计师DNA纳米结构采用大分子“网”的形式，其顶点是精确的 机械匹配与艾滋病毒外显示的尖峰GP120蛋白基质的间距和定位 表面。 DNA NET顶点纳入了已选择为选择性的核酸APATMER问题 针对HIV GP120，导致多个高亲和力附件，因此可以将“网”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，我们会的 利用一种新发明的生物传感器显微镜，称为光子谐振器干扰散射 显微镜（棱镜），其中光子晶体表面放大器激光散射从捕获的完整 病毒体，使每个人都能以高信噪比进行计数。因为棱镜不需要标签 或酶扩增，我们的方法可以使被捕获的病毒的动态，实时计数使用数字计数 精度和超敏。在拟议的项目中，我们将整合病毒分离和光子晶体 生物传感器进入塑料墨盒，并发展出快速的工作流程，该工作流将简单而快速，以兼容与 护理点设置，目的是在<30分钟的样本到撤离器中产生结果。我们的目标包括 开发Prism工具的即将点版本，并在统计学上稳健地表征 检测极限，可重复性和鲁棒性。我们的研究将包括使用临床验证系统的验证 标本和直接比较与金标准实验室RT-PCR分析。