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

总结。侧向流动分析(LFA)提供了对医学上有重要意义的分析物的现场快速检测 对于高容量、高速的人口筛查非常有价值，这是将经济损失降至最低所必需的 以及疫情暴发的社会影响。然而，LFA的性能主要局限于色度学 二元诊断，主要为临床相关分析物提供是/否测试 浓度(µM-NM)，不能满足可靠病毒检测(FM-AM)的灵敏度要求 目前仅使用实验室技术(ELISA法、PCR法)实现。通过对信号的放大 荧光稳步获得牵引力，将灵敏度提高了1-2个数量级 标准比色测试，用于构建LFA测试条的材料的固有自发荧光干扰 具有测试性能并引入了可变性。通过实现快速、全分子敏感性、基于荧光的 LFA可以促进护理点检测分析能力的革命性转变，以提供定量 可采取行动的数据，以告知卫生当局及时采取应对措施。实现这一发展的途径包括 荧光检测系统的进展和记者的材料性质。这项提议旨在 通过推进最近出现的LFA平台来战略性地解决这两个方向，该平台基于 荧光纳米钻石(FND)记者。FND具有LFA记者的理想功能，如明亮的非 漂白的荧光和无与伦比的坚固性。使用FND作为新颖的LFA报告程序的原型是 Debina Diagnostics最近演示了在皮克级检测埃博拉病毒糖蛋白，使用 定制光电阅读器(安信，Inc.)和阿达马斯生产的现成200 nm FND 纳米技术。FND最显著的特性是将基于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)色心允许强度FND荧光被一个 外部磁场。基于这一独特的属性，与FND相关的信号可以从 背景锁定分析在信号频域中的应用 提取低于噪声电平的小周期信号的处理。FND罐头的锁定信号处理 可将灵敏度提高高达100倍，正如生物成像(包括最近的LFA结果)所显示的那样。 阿达马斯最近开发了一种方法，将磁调制对比度进一步提高3-5倍。在这 Proposal、Adáas和Debina共同努力定制FND材料属性，以消除非特定 通过改变颗粒的物理(形状、大小)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性能(目标1)。在实施和优化了 锁定分析，包括将设置与光电阅读器接口(目标2)，我们的目标是演示 将埃博拉病毒抗原的LOD提高300倍，并将检测SARS-CoV-2抗原的&lt；1PG LOD提高300倍(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).