SBIR, Phase I (R43): Ultrasensitive Lateral Flow Assays Enabled by Fluorescent Nanodiamond Labels

摘要提供医学上重要的分析物的快速现场检测的侧流测定（LFA）可以 对于最大限度地减少经济损失所必需的高容量、高速人群筛查而言， 以及流行病爆发的社会影响。然而，LFA性能主要局限于比色法， 二元诊断，主要为高浓度的临床相关分析物提供是/否测试 浓度（µM-nM），无法满足可靠病毒检测（fM-aM）的灵敏度要求， 目前仅使用实验室技术（ELISA、PCR）实现。虽然信号放大通过 荧光已经稳定地获得牵引力，以将灵敏度提高1-2个数量级， 标准比色试验中，用于构建LFA测试条的材料的固有自发荧光干扰 测试性能并引入可变性。通过实现快速的阿摩尔灵敏度， LFA可以促进即时检测分析能力的革命性转变， 可采取行动的数据，以告知卫生当局及时采取应对措施。这种发展的途径包括 荧光检测系统的进步和报告分子的材料特性。这项建议旨在 通过推进最近出现的LFA平台，从战略上解决这两个方向， 荧光纳米金刚石（FND）报告基因。FND具有LFA报告子的理想特征，例如明亮的非荧光性。 漂白荧光和无与伦比的坚固性。使用FND作为新型LFA报告子的原型是 Debina Diagnostics最近在皮克水平上检测埃博拉病毒糖蛋白， 定制的光电读取器（Axxin，Inc.）Adámas生产的200 nm FND 纳米技术。FND最显著的特性是设想将基于FND的LFA灵敏度提高2-3 数量级来自粒子的独特耦合磁光性质，其中氮- 具有电子自旋的空位（NV）色心允许FND荧光的强度被电子自旋调制。 外部磁场基于这种独特的性质，FND相关信号可以从 背景自发荧光在频域通过锁相分析，这是广泛使用的信号， 处理以提取低于噪声水平的小周期信号。FND的锁定信号处理可以 允许高达100倍的灵敏度增加，如在生物成像中所证明的，包括LFA的最新结果。 Adámas最近开发了一种方法，将磁调制对比度进一步提高了3-5倍。在这 建议，Adámas和Debina加入他们的努力，以定制FND材料属性，以消除非特定 通过改变颗粒的物理（形状、尺寸）和化学（涂层）性质，将颗粒保留在LFA带上， 同时优化亮度和磁光特性（目标1）。实施和优化后， 锁定分析，包括接口设置与光电阅读器（目标2），我们的目的是证明 埃博拉病毒抗原LOD提高> 300倍，SARS-CoV-2抗原检测LOD <1 pg（目标3）。

Summary. Lateral flow assays (LFAs) providing rapid on-site detection of medically significant analytes could be extremely valuable for high-volume, high-speed population screening necessary to minimize the economic and societal impact of epidemic outbreaks. However, LFA performance has been mostly limited to colorimetric binary diagnostics, primarily providing yes/no testing for clinically relevant analytes present at high concentrations (µM-nM) and cannot satisfy sensitivity requirements for reliable virus detection (fM-aM) which is presently achieved only using laboratory techniques (ELISA, PCR). Though signal amplification through fluorescence has steadily gained traction to improve sensitivity by 1-2 orders of magnitude in comparison with standard colorimetric tests, the inherent autofluorescence of materials used to construct LFA test strips interferes with test performance and introduces variability. By enabling rapid, attomolar sensitivity, fluorescence-based LFAs could facilitate a revolutionary shift in the analytical capability of point-of-care tests to provide quantitative actionable data to inform health authorities to launch timely responses. The pathway to this development involves advancement of the fluorescence detection system and material properties of the reporters. This proposal aims to strategically address both of these directions by advancing a recently emerged LFA platform based on fluorescent nanodiamond (FND) reporters. FNDs possess ideal features for an LFA reporter such as bright non- bleaching fluorescence and unsurpassed ruggedness. A prototype using FND as a novel LFA reporter was recently demonstrated by Debina Diagnostics in detecting Ebola virus glycoprotein at the picograms level, using a custom-made optoelectronic reader (Axxin, Inc.) and off-the-shelf 200 nm FND produced by Adámas Nanotechnologies. The most striking attribute of FND envisioned to improve FND-based LFA sensitivity by 2-3 orders of magnitude arises from the uniquely coupled magneto-optical properties of the particles, where nitrogen- vacancy (NV) color centers with an electron spin allow the intensity FND fluorescence to be modulated by an external magnetic field. Based on this unique property, the FND-related signal can be separated from background autofluorescence in the frequency domain through lock-in analysis which is widely used in signal processing to extract small periodic signals present below noise levels. Lock-in signal processing of FND can allow up to 100x increase in sensitivity, as was demonstrated in bioimaging including recent results in LFA. Adámas recently developed a method further increasing magnetic modulation contrast by 3-5 folds. In this proposal, Adámas and Debina join their efforts to tailor FND material properties in order to eliminate non-specific retention of particles on the LFA strip by modifying their physical (shape, size) and chemical (coating) properties, while optimizing brightness and magneto-optical properties (aim 1). After implementation and optimization of the lock-in analysis including interfacing the set-up with the opto-electronic reader (aim 2), we aim to demonstrate >300x improvement in LOD of Ebola virus antigen and <1pg LOD for detection of SARS-CoV-2 antigen (aim 3).