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

精准医疗是治疗人类疾病的新兴方法，通过针对个体患者疾病的潜在基础进行靶向治疗。虽然癌症和其他慢性病的检测和治疗计划需要数天至数周的时间来制定，但对于急性发作的患者来说，可能挽救生命的治疗必须在几分钟至几小时内提供，这需要患者附近的快速诊断技术。这项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的耗时要求，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），促进西班牙裔，墨西哥裔和美洲原住民科学（SACNAS）协会和女性工程师协会（SWE）。激发少数族裔学生对工程和纳米技术生物传感器设备开发的兴趣。本研究的目的是开发一种床旁（POC）数字免疫测定生物传感器平台，以满足对严重危及生命疾病的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测定技术上，该技术包括（i）将抗体缀合的磁珠限制在微流体芯片上的飞升大小的微孔阵列中，每个微孔形成当靶分析物结合时激活的荧光“像素”，和（ii）从用于预处理的整个微孔阵列的“快照”图像中计数荧光信号激活像素的数目，平衡的分析物结合事件。 凭借其高性能和鲁棒性，我们新的微流体数字生物传感技术可以实现近实时，近患者的循环血液细胞因子生物标志物的同时定量。使我们的方法具有创新性和独特性的是前所未有的能力，可以同时实现速度（总共10分钟，孵育30秒），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重性（多达24个样品）。本研究将涉及一个多物理场模型的发展，提供了一个理论基础和优化的指导方针，建议的生物传感器，建设一个独立的实验室上的一个磁盘系统的便携式，半自动微阵列生物传感的细胞因子生物标志物，并在重症监护室设置下的系统性能的验证。该技术中使用的受体类型可以进一步扩展，以采用多种抗体、肽和寡核苷酸。这将使这项技术扩展到其他生物检测领域，包括受体-配体检测、酶检测和DNA检测。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。

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.