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重要组成部分 治疗是减少艾滋病毒母婴传播发生率的工具，也是常规治疗的必要组成部分 诊断测试使人们了解自己的艾滋病毒状况，尽管有大量的研究和产品。 开发工作已应用于即时病毒载量测试，这是当前核酸测试的范例 和抗原测定继续证明其固有的复杂性所带来的根本局限性 以及缺乏稳健性，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。 我们寻求通过结合以下三种技术来解决现有技术能力的重要差距 创新产生集成、快速、简单、超灵敏、高选择性、稳健且廉价的系统 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 全血中提取 10-50 µl 血浆样本，病毒提取率 >95% 其次，我们将使用所得血清实现对完整 HIV 病毒体的超选择性识别。 设计者 DNA 纳米结构采用大分子“网”的形式，其顶点是精确的 与 HIV 外部显示的刺突 gp120 蛋白矩阵的间距和定位进行机械匹配 DNA 网顶点包含经过选择性选择的核酸适体探针。 靶向HIV gp120，产生多个高亲和力附着位点，因此“网”可以用作 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，我们将。 利用一种新发明的生物传感器显微镜，称为光子谐振器干涉散射 显微镜 (PRISM)，其中光子晶体表面放大捕获的完整激光散射 病毒粒子，使每个病毒粒子都能以高信噪比进行计数，因为 PRISM 不需要标签。 或酶扩增，我们的方法可以通过数字技术对捕获的病毒进行动态、实时计数 在拟议的项目中，我们将整合病毒分离和光子晶体。 生物传感器装入塑料盒中，并开发一个快速的工作流程，该工作流程将简单快速地兼容 护理点设置，目标是在 30 分钟内获得结果，我们的目标包括。 开发 PRISM 仪器的护理点版本，并进行统计上稳健的表征 我们的研究将通过临床验证系统来结束。 样本并与金标准实验室 RT-PCR 分析进行直接比较。