A Pre-Equilibrium Single-Molecule Digital Counting Biosensor for Near-Patient Precision Medicine of Life-Threatening Illnesses

精准医学是一种新兴的治疗人类疾病的方法，通过针对个体患者疾病的潜在基础进行靶向治疗。癌症和其他慢性疾病的检测和治疗计划需要数天到数周的时间才能制定出来，而对于急性发作的病人来说，一种可能挽救生命的治疗方法必须在几分钟到几小时内交付，这需要病人身边的快速诊断技术。本研究旨在通过开发一种创新的诊断生物传感器平台来满足这一需求，该平台克服了传统生物标志物分析技术耗时的要求，具有挽救器官衰竭等危机患者生命的潜力。该平台将同时实现更高的速度、更高的灵敏度和更低的仪器成本，同时允许在患者床边同时分析多种血液生物标志物蛋白。这项研究中开发的技术可能使未来生物标志物引导的危及生命的疾病的精确治疗成为可能。此外，该计划将允许研究团队参加由科学-技术-工程-数学（STEM）协会组织的会议，如西班牙裔专业工程师协会（SHPE），推进西班牙裔，奇卡诺人和美国原住民科学协会（SACNAS）协会和女性工程师协会（SWE），以激发未被充分代表的少数民族学生对工程和基于纳米技术的生物传感器设备开发的兴趣。本研究的目的是开发一个点护理（POC）数字免疫测定生物传感器平台，以解决严重危及生命疾病的精准医学对非常规诊断方法的迫切需求。所提出的平台建立在创新的免疫分析技术的基础上，其中包括(i)将抗体共轭磁珠限制在微流控芯片上的飞升大小的微孔阵列中，每个微孔形成一个荧光“像素”，当目标分析物被结合时激活，以及（ii）从整个微孔阵列的“快照”图像中计算荧光信号激活的像素的数量，用于预平衡分析物结合事件。凭借其高性能和鲁棒性，我们的新型微流控数字生物传感技术可以实现近实时，近患者同步量化循环血液细胞因子生物标志物。使我们的方法创新和独特的是前所未有的同时实现速度（总共10分钟，30秒孵育）的能力，灵敏度导致检测限（LOD）为0.1 - 1 pg/mL，从而实现临床阈值，大线性动态范围和多样性（多达24个样品）在同一个平台上一起。本研究将包括建立一个多物理场模型，为所提出的生物传感器提供理论基础和优化指导，构建一个独立的磁盘实验室系统，用于便携式，半自动微阵列细胞因子生物标志物的生物传感，并在重症监护病房环境下验证系统的性能。在该技术中使用的受体类型可以进一步扩展到使用各种各样的抗体、肽和寡核苷酸。这可以将该技术扩展到其他生物检测，包括受体配体检测、酶检测和DNA检测。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。

Precision medicine is the emerging approach to treat human diseases by targeting therapy towards the underlying basis of the disease for individual patients. While testing and therapeutic plans for cancers and other chronic diseases are formulated over days to weeks, a potentially life-saving treatment for patients suffering an immediate acute attack must be delivered in minutes to hours, requiring fast diagnostic technologies near the patient. This research aims to meet this need by developing an innovative diagnostic biosensor platform that overcomes the time-consuming requirements of conventional biomarker analysis technologies, with the potential of saving patient lives undergoing crises such as organ failure. This proposed platform will simultaneously achieve higher speed, higher sensitivity, and lower instrumentation cost, while permitting concurrent analyses of multiple blood biomarker proteins at the patient's bedside. The technology developed in this research may enable future biomarker-guided precision treatment of life-threatening illnesses. Additionally, this program will allow the research team to participate in conferences organized by Science-Technology-Engineering-Mathematics (STEM) societies, such as the Society of Hispanic Professional Engineers (SHPE), Advancing Hispanics, Chicanos, and Native Americans in Science (SACNAS) Society, and Society of Women Engineers (SWE), to stimulate the interests of underrepresented minority students in engineering and nanotechnology-based biological sensor device development. The objective of this study is to develop a point-of-care (POC) digital immunoassay biosensor platform to address urgent needs for unconventional diagnostic approaches to precision medicine of severe life-threatening illnesses. The proposed platform is built upon innovative immunoassay technology, which involves (i) confining antibody-conjugated magnetic beads into an array of femtoliter-sized microwells on a microfluidic chip, each forming a fluorescent 'pixel' activated when the target analyte is bound, and (ii) counting the number of fluorescence signal-activated pixels from a 'snapshot' image of the entire microwell array taken for pre-equilibrated analyte binding events. With its high performance and robustness, our new microfluidic digital biosensing technology may enable near-real-time, near-the-patient concurrent quantification of circulating blood cytokine biomarkers. What makes our approach innovative and unique is the unprecedented ability to simultaneously achieve speed ( 10 minutes in total with 30seconds of incubation), sensitivity leading to limit of detection (LOD) of 0.1 - 1 pg/mL, which achieves clinical thresholds, a large linear dynamic range, and multiplexity (up to 24 samples) all together in the same platform. This research will involve the development of a multiphysics model to provide a theoretical foundation and optimization guideline for the proposed biosensor, construction of a self-contained lab-on-a-disk system for portable, semi-automated microarray biosensing of cytokine biomarkers, and validation of the system performance under an intensive care unit setting. The receptor types used in this technology may be further extended to employ a wide variety of antibodies, peptides, and oligonucleotides. This could allow an expansion of this technology for other biological assays including receptor-ligand assays, enzyme assays, and DNA assays.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.