CAREER: Smartphone-Based CRISPR Biosensor for Point-of-Care HIV Viral Load Testing

艾滋病毒/艾滋病的一种治疗方法是抗逆转录病毒治疗（ART）。抗逆转录病毒疗法有助于艾滋病毒感染者活得更长、更健康，并降低艾滋病毒传播的风险。临床试验数据表明，与标准的每日口服方案相比，每月一次的长效注射抑制HIV。为了准确监测治疗进展并确保患者对长期治疗的信心，一种能够检测艾滋病毒某些生物标志物的技术是必不可少的。该职业提案旨在研究CRISPR技术在超灵敏分子诊断中的应用的基本特性，并设计和开发一种基于芯片的、智能手机可读的CRISPR生物传感器，用于监测HIV治疗的治疗进展。该项目的成功将大大提高长效艾滋病毒治疗的质量和效果。通过整合研究和教育，该项目将通过吸引、留住和培训工程和生物技术跨学科领域的学生，增加多样化的STEM劳动力。本次CAREER proposal的研究目标是研究CRISPR反式核酸酶的独特特性，将基因组编辑CRISPR- cas平台转化为下一代、快速、超灵敏的生物传感器。为了实现这一目标，我们将系统地研究CRISPR Cas蛋白的酶活性和动力学，并提出一种新的一锅式CRISPR诊断反应动力学模型。该项目还将探索将基于溶液的CRISPR检测转变为基于芯片的生物传感器，可以通过智能手机荧光显微镜进行光学检测，以确保亚原子水平的高检测灵敏度。这种芯片上的新传感机制将消除对样品预扩增的需要，从而大大简化和缩短当前CRISPR诊断的步骤。通过将病毒动态模型集成到定制开发的智能手机应用程序界面中，智能手机集成的艾滋病毒检测平台将允许病毒载量测量和计算预测。该项目将革新目前用于POC艾滋病毒载量检测的工具箱，这是阻止艾滋病毒流行和加强治疗的主要障碍之一。本项目的研究结果将为深入了解CRISPR - Cas蛋白反式切割的详细机制提供新的见解。这种分布式、联网的POC传感器的成功开发，将在新一波长效注射和植入式HIV药物的浪潮中非常及时。我们期望开发的感官方法也可以广泛应用于未来在资源有限的环境中检测许多其他疾病。该奖项反映了美国国家科学基金会的法定使命，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。

One treatment of HIV/AIDS is antiretroviral therapy (ART). Antiretroviral therapy helps people with HIV live longer, healthier lives and reduces the risk of HIV transmission. Clinical trial data have shown that once-a-month long-acting injectables suppress HIV comparably to the standard daily oral regimens. To accurately monitor the therapeutic progress and to assure patients’ confidence in the long-period treatment, a technology that is capable of testing certain biomarkers for HIV is essential. This CAREER proposal seeks to study the fundamental properties of the CRISPR technology for applications in ultrasensitive molecular diagnostics and to design and develop a chip-based, smartphone-readable CRISPR biosensor for monitoring the therapeutic progress of HIV treatments. The success of the project will significantly improve the quality and outcome of long-acting HIV treatment. By integrating research and education, this project will increase a diverse STEM workforce by attracting, retaining, and training students in the interdisciplinary field of engineering and biotechnology. The research objective of this CAREER proposal is to study the unique characteristics of CRISPR trans-nuclease and convert the genome-editing CRISPR-Cas platform into next-generation, rapid, and ultrasensitive biosensors. In pursuit of this goal, the enzymatic activity and kinetics of CRISPR Cas proteins will be systematically studied, and a new kinetic model for the one-pot CRISPR diagnostic reaction will be proposed. This project will also explore the transformation of the solution-based CRISPR assay into a chip-based biosensor that can be optically detected by a smartphone fluorescence microscope to ensure high detection sensitivity to the sub-attomolar level. This new sensing mechanism on a chip will eliminate the need for pre-amplifications of the samples to significantly simplify and shorten the steps of current CRISPR diagnostics. The smartphone-integrated HIV testing platform will allow for both viral load measurement and computational predication, through the incorporation of a viral dynamic model into a custom-developed smartphone application interface. The project will innovate the current toolbox for POC HIV viral load testing, which is one of the major roadblocks in halting the HIV epidemic and enhancing treatment. The results of this project will provide new insights into the detailed mechanism of trans-cleavage of CRISPR Cas proteins. The successful development of such distributed and connected POC sensors will be very timely in the new wave of long-acting injectable and implantable HIV drugs. We expect the developed sensory methodology can also be broadly applied to the detection of many other diseases in resource-limited settings in the future.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.