Direct bioelectronic detection of SARS-CoV-2 from saliva using single-molecule field-effect transistor array

使用单分子场效应晶体管阵列直接生物电子检测唾液中的 SARS-CoV-2

• 批准号: 10266395
• 负责人: Kenneth L Shepard
• 金额: $ 81.73万
• 依托单位: QUICKSILVER BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-21 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266395
• 关键词: 2019-nCoV; Address; Binding Proteins; Biological Assay; Biological Sciences; COVID test; COVID-19 detection; COVID-19 diagnostic; COVID-19 pandemic; COVID-19 testing; Carbon Nanotubes; Clinical; Communicable Diseases; Complex; Defect; Detection; Development; Devices; Diagnosis; Diagnostic tests; Differential Diagnosis; Electrolytes; Gold; Hour; ImProv; Immobilization; Immunoassay; Individual; Infection; Infectious Agent; Influenza; Kinetics; Laboratories; Llama; Membrane Proteins; Nanotubes; New York; Nucleic Acid Amplification Tests; Nucleocapsid Proteins; Optics; Phase; Polymerase Chain Reaction; Population Surveillance; Preparation; Process; Proteins; Public Health; Quantitative Reverse Transcriptase PCR; Reader; Reagent; Recombinants; Research Personnel; Reverse Transcription; SARS-CoV-2 transmission; Saliva; Sampling; Semiconductors; Sensitivity and Specificity; Site; Small Business Innovation Research Grant; Specificity; Specimen; Structural Protein; Symptoms; Technology; Testing; Time; Training; Transistors; Universities; Vaccines; Viral; Viral Load result; Viral Proteins; Virus; antibody detection; antigen detection; antigen test; base; biobank; cost; design; diagnostic assay; env Gene Products; experimental study; improved; innovation; metal oxide; molecular array; molecular diagnostics; multiplex detection; nanobodies; particle; pathogen; point of care; point of care testing; programs; protein structure; saliva sample; sensor; single molecule; temporal measurement; tool; transmission process; viral RNA; viral detection

Direct bioelectronic detection of SARS-CoV-2 from saliva using single-molecule field-effect transistor array Nucleic acid tests have become the gold-standard for diagnostic testing for COVID-19, usually performed in specialized laboratories. Most are based on reverse-transcription quantitative polymerase chain reaction (qRT-PCR). The time required for specimen transport and processing results in a turnaround time that is typi- cally several days. The few rapid (<1 hour) point-of-care (POC) tests are more expensive, still require sample preparation and specialized reagents, and do not have the throughput needed for population surveillance. Di- rect testing for the virus, which also reduces requirements for multiple reagents, is a necessary step to improv- ing diagnostic testing. While four such antigen tests have been approved for detection of SARS-CoV-2 based on immunoassays to the N protein, sensitivity is limited and no quantitation of viral load is possible. We will address this gap by using DiagnostikosTM, an in-development rapid POC platform for direct, real- time, multiplexed, quantitative bioelectronic detection of biomolecules that employs an all-electronic detection device that functions at the single-molecule level. These single-molecule field-effect transistors (smFETs) are arrayed on a complementary metal-oxide-semiconductor (CMOS) integrated circuit chip. Chips will interface with an envisioned USB-stick-form-factor reader device. Robust single-domain antibodies, known as nanobod- ies and immobilized on these devices, are used for sensitive detection of viral particles and viral debris. The use of multiple nanobodies for a single protein and nanobodies for different proteins in a single assay allows for significant improvements in specificity. Nanobodies will be specific for one or more of the four major struc- tural proteins in SARS-CoV-2; the nucleocapsid (N) protein engulfing the viral RNA, the spike (S) protein, the membrane (M) protein and the envelope (E) protein. No sample preparation or specialized reagents are re- quired for detection, and the device will be designed to operate with saliva, which has very recently been shown to be a reliable medium for detecting SARS-CoV-2. Individual sensor chips can be manufactured at a cost of $35. With the addition of other nanobodies, these large dense arrays can also allow detection of many pathogens in a single test. In this Direct-To-Phase-2 SBIR program we will pursue several key innovations that are required to make such a platform possible, including isolation of nanobodies for key structure proteins of SARS-CoV-2 (Specific Aim 1), development of the smFET platform for antigen detection (Specific Aim 2), development of large CMOS arrays of these smFET devices (Specific Aim 3), and verification of detection in increasingly complex samples up to and including clinical samples (Specific Aim 4). This project is a partnership between university researchers who developed the smFET technology and a venture-based start-up venture, Quicksilver Biosci- ences, spun out to commercialize smFET technology and develop smFET/CMOS arrays for molecular diag- nostic applications.

从唾液中直接生物电子检测 SARS-CoV-2 使用单分子场效应晶体管阵列 核酸检测已成为 COVID-19 诊断检测的黄金标准，通常进行 在专门的实验室。大多数基于逆转录定量聚合酶链反应 （qRT-PCR）。样本运输和处理所需的时间导致典型的周转时间 好几天。少数快速（<1 小时）即时护理 (POC) 测试更昂贵，仍然需要样本 制剂和专用试剂，并且不具备群体监测所需的通量。迪- 对病毒进行正确的检测，这也减少了对多种试剂的需求，是改进病毒检测的必要步骤。 荷兰国际集团的诊断测试。虽然四种此类抗原测试已被批准用于检测基于 SARS-CoV-2 的 N 蛋白的免疫测定的灵敏度有限，并且无法定量病毒载量。 我们将通过使用 DiagnostikosTM 来解决这一差距，这是一个正在开发的快速 POC 平台，可用于直接、真实的 采用全电子检测的生物分子的时间、多重、定量生物电子检测 在单分子水平上发挥作用的装置。这些单分子场效应晶体管 (smFET) 排列在互补金属氧化物半导体（CMOS）集成电路芯片上。芯片将接口 与设想的 USB 棒形状因素读取器设备。强大的单域抗体，称为纳米抗体 病毒颗粒和病毒碎片固定在这些设备上，用于灵敏检测病毒颗粒和病毒碎片。这 在一次测定中使用多个纳米抗体检测单个蛋白质，并使用纳米抗体检测不同的蛋白质 显着提高特异性。纳米抗体将特异性针对四种主要结构中的一种或多种 SARS-CoV-2 中的天然蛋白；吞噬病毒 RNA 的核衣壳 (N) 蛋白、刺突 (S) 蛋白、 膜（M）蛋白和包膜（E）蛋白。无需重新制备样品或专用试剂 检测所需，该设备将设计为利用唾液进行操作，最近已被 被证明是检测 SARS-CoV-2 的可靠介质。单独的传感器芯片可以在 费用为 35 美元。通过添加其他纳米抗体，这些大型密集阵列还可以检测许多 一次测试中的病原体。 在这个 Direct-To-Phase-2 SBIR 计划中，我们将追求几项关键创新，以实现 这样一个平台是可能的，包括分离 SARS-CoV-2 关键结构蛋白的纳米抗体（具体 目标1），开发用于抗原检测的smFET平台（具体目标2），开发大型 这些 smFET 器件的 CMOS 阵列（具体目标 3），以及日益复杂的检测验证 样本直至并包括临床样本（具体目标 4）。该项目是大学之间的合作伙伴关系 开发 smFET 技术的研究人员和一家基于风险投资的初创企业 Quicksilver Biosci- ences，分拆出来将 smFET 技术商业化，并开发用于分子诊断的 smFET/CMOS 阵列 诺斯提应用程序。