RII Track-4: The Integration of Plasmonic Nanoantenna and Super-hydrophobic Surface for Ultrasensitive Fluorescence CRISPR Biosensing

早期诊断提供了显著和前所未有的益处，因为在疾病的早期阶段诊断的患者通常有很好的治愈和功能结果的机会。此外，快速检测对于抗击这一流行病至关重要，正在发生的COVID-19大流行就是例证。该项目旨在设计一种基于CRISPR（Regularly Interspaced Short Palindromic Repeats）的床旁诊断系统，而无需预扩增病毒基因组。它将允许对许多疾病进行超低和超灵敏的检测（例如，心血管疾病、癌症和传染病）的早期阶段，此时体液中病毒基因组的浓度（即，尿液、血液、唾液）仍然非常低，不足以被现有技术检测到。这个NSF EPSCoR RII Track-4奖学金提供了与康涅狄格大学生物医学工程系传染病即时诊断领域的知名专家合作的机会，以实现这一目标。该项目的成功完成将导致非侵入性，廉价，大规模生产的系统，用于早期检测，疾病的治疗结果评估，大大改善患者发病率和降低医疗成本，特别是对内华达州，在12个主要死因中，纳米天线的高发病率一直排在最后。该项目的目标是（1）将纳米天线与超级天线相结合，（2）将设计的增强型CRISPR/Cas 12 a荧光检测模块与微流体集成，用于血液样品中的病毒检测。尽管基于CRISPR的床旁诊断系统由于其简单性和灵活性而成为一种流行的技术和快速筛查的有力工具，但它仍然存在许多局限性，例如在复杂生物样品中的稳定性低。一个有前途的解决方案是探索纳米天线技术来触发增强的局域表面等离子体。然而，纳米天线技术仍然远未在生物医学领域常规实施，这是由于主要障碍不是来自等离子体而是来自质量传输：大多数纳米天线通常依赖于扩散来捕获目标分子，这使得检测时间不切实际地长。该项目将纳米天线与超疏水表面相结合，以解决这种扩散限制。超疏水表面上的液滴在蒸发过程中保持准球体，并且不润湿表面。因此，液滴蒸发取代扩散并将分子集中到纳米天线的敏感区域上，成为传质的主要机制。液滴蒸发时间不仅比扩散时间短得多，而且可以主动控制，这是一个额外的好处。等离子体和超疏水表面的结合为上述关键挑战提供了独特的解决方案，并为CRISPR实现的超低和超灵敏生物传感平台带来了希望。该RII Track-4奖学金提供的培训和研究经验将使PI成功地从材料科学和工程背景过渡到生物医学研究人员。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Early diagnosis provides significant and unprecedented benefits since patients diagnosed at an early stage of diseases often have a good chance for cure and functional outcomes. In addition, rapid testing is crucial to combat the pandemic as exemplified by the ongoing COVID-19 pandemic. This project aims to design a direct Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based point of care diagnostic system without pre-amplification of viral genomes. It will allow ultralow and ultrasensitive detection of many diseases (e.g., cardiovascular diseases, cancer, and infectious diseases) at an early stage when the concentration of viral genomes in body fluids (i.e., urine, blood, saliva) is still very low and not sufficient to be detected by existing technologies. This NSF EPSCoR RII Track-4 fellowship provides the opportunity to collaborate with a renowned expert in point-of-care diagnostics for infectious diseases in the Department of Biomedical Engineering at the University of Connecticut to achieve this goal. The successful completion of this project will lead to noninvasive, inexpensive, mass-producible systems for early detection, treatment outcome evaluation of diseases, greatly improving patient morbidity and reducing healthcare cost, particularly important to Nevada, which consistently ranks near the bottom in terms of higher rates of the 12 leading causes of death.The objectives of the project are to (1) integrate nanoantenna with super-hydrophobic surfaces for enhancing the CRISPR/Cas12a detection sensitivity without pre-amplification; (2) integrate the designed enhanced CRISPR/Cas12a fluorescence detection module with microfluidics for viral detection in the blood sample. Although CRISPR based point of care diagnostic system has emerged as a popular technology and a powerful tool for rapid screening due to its simplicity and flexibility, it still has many limitations such as low stability in complex biological samples. One promising solution is to explore the nanoantenna technique to trigger the enhanced Localized surface plasmon. However, the nanoantenna technique is still far from being routinely implemented in biomedical fields due to a major obstacle not from plasmonics but from the mass transport: Most nanoantennas typically rely on diffusion to capture target molecules, which makes the detection time impractically long. This project integrates the nanoantenna with the superhydrophobic surface to address this diffusion limit. Droplets over super-hydrophobic surfaces maintain quasi spheres during evaporation and do not wet the surface. Therefore, the droplet evaporation replaces the diffusion and concentrates molecules onto the sensitive regions of the nanoantenna, becoming the dominant mechanism of mass transfer. The droplet evaporation time is not only much shorter than the diffusion time but also can be actively controlled, which is an additional benefit. The combination of plasmonics and super-hydrophobic surfaces offers a unique solution to the aforementioned key challenge and holds the promising for ultralow and ultrasensitive biosensing platforms enabled by CRISPR. The training and research experience provided by this RII Track-4 fellowship will allow the PI to successfully transition from the background of material science and engineering to a biomedical researcher.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.