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

早期诊断提供了重大和前所未有的好处，因为在疾病的早期阶段诊断的患者通常有很好的机会治愈和获得功能结果。此外，快速检测对于抗击这种大流行至关重要，正在进行的新冠肺炎大流行就是例证。该项目旨在设计一种基于直接聚类规则间隔短回文重复序列(CRISPR)的护理点诊断系统，而不需要预先扩增病毒基因组。它将使许多疾病(例如心血管疾病、癌症和传染病)在体液(即尿液、血液、唾液)中的病毒基因组浓度仍然非常低、不足以被现有技术检测到的早期阶段被超低和超灵敏地检测出来。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护理点诊断方面的知名专家合作实现这一目标的机会。该项目的成功完成将为疾病的早期检测、治疗结果评估带来非侵入性的、廉价的、可批量生产的系统，极大地改善患者的发病率并降低医疗成本，这对内华达州尤其重要，该州在12种主要死亡原因中的较高发病率一直接近垫底。该项目的目标是(1)将纳米天线与超疏水表面相集成，以增强CRISPR/Cas12a检测灵敏度，而无需预扩增；(2)将设计的增强型CRISPR/Cas12a荧光检测模块与微流控技术集成，用于血液样本中的病毒检测。尽管基于CRISPR的护理点诊断系统因其简单、灵活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，如对复杂生物样本的稳定性不高。一个有希望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激元。然而，纳米天线技术在生物医学领域的常规应用还远远不够，这是因为一个主要的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靠扩散来捕获目标分子，这使得检测时间太长。该项目将纳米天线与超疏水表面相结合，以解决这一扩散限制。超疏水表面上的液滴在蒸发过程中保持准球形，不会弄湿表面。因此，液滴蒸发取代了扩散，将分子集中到纳米天线的敏感区域，成为主要的传质机制。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。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智力优势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.