Printed Electronic Biosensors for Point-of-Care Testing of Cardiovascular Biomarkers

摘要 心脏病是美国的主要死因，每年夺去60多万人的生命。的 测量血液中的生物标志物，以缩小鉴别诊断和分诊病人起着关键作用， 急性和慢性心血管疾病的管理。这个R 01项目将开发一个手持点- 使用低成本一次性检测盒测量多组心脏 直接从一滴全血中提取生物标志物。该分析汇集了碳纳米管的主要进展 本发明涉及集成到薄膜晶体管（CNT-TFT）中的碳纳米管（CNT）和用于检测蛋白质的测定平台（称为“D4”）。 分析物与ELISA一样的性能从一滴血。CNT-TFT和D4都是通过以下方式制造的： 印刷，使它们易于定制和低成本。CNT-TFT提供极其敏感的纳米管， 并且，与现有的基于CNT的生物传感器不同，CNT-TFT提供了可重复的电转导。 性能和高产量。D4是一种基于荧光的检测方法，其使用打印到荧光显微镜上的抗体（Ab）对。 聚合物刷涂层（“POEGMA”），其消除背景噪声并允许高灵敏度的 在复杂的生物环境中直接测量。将这两项创新技术结合起来，就可以实现“D4- TFT”，其将CNT-TFT的电子灵敏度与D4的不结垢选择性相结合，以产生 一种可以在不冲洗的情况下通过电化学分析从全血中快速检测生物标志物浓度的测定法， 转导大量的初步数据表明，D4-TFT的全印刷制造，保存， 晶体管操作，并成功地从血清和血液中检测目标分析物。具体目标是 该项目包括一个手持POCT单元的开发，所有需要的控制电子和一个简单的用户 与显示器的接口，以及D4-TFT与插入手持设备的兼容卡匣的适配 单位进行操作。D4-TFT检测试剂盒的分析性能将最大化，并针对两种 一组心脏生物标志物：（1）一组经验证的心脏生物标志物，包括肌酸激酶（CK）， CKMB、肌钙蛋白（Tn）和N-末端前B型利钠肽（BNP），这些是诊断的核心， 管理广泛的心血管疾病，包括急性胸痛，心肌梗死， 和心力衰竭（HF）;（2）两种研究性心脏生物标志物，血管生成素2（ANG 2）和肿瘤坏死因子（TNF） α因子（TNFα），由于其与晚期出血事件的潜在相关性而受到关注。 植入左心室辅助装置（LVAD）的HF患者（该人群中最常见的并发症）。 这两组不同的心脏生物标志物将证明这种POCT的多功能性和价值， 使用经验证的生物标志物的诊断潜力以及用于研究性生物标志物的快速临床筛选。 实现这些目标将导致一种新的POCT心脏生物标志物，可以很容易地扩大规模。其完全 打印的性质使得D4-TFT高度可定制，并广泛适用于任何其他基于血液的测试 生物标志物与免疫测定试剂。

ABSTRACT Heart disease is the leading cause of death in the United States, claiming over 600,000 lives annually. The measurement of blood-based biomarkers to narrow differential diagnoses and triage patients plays a critical role in the management of acute and chronic cardiovascular disease. This R01 project will develop a handheld point- of-care test (POCT) that uses low-cost, disposable assay cassettes to measure multiplexed panels of cardiac biomarkers directly from a drop of whole blood. The assay brings together major advances in carbon nanotubes (CNTs) integrated into thin-film transistors (CNT-TFTs) and an assay platform (called “D4”) for detecting protein analytes with ELISA-like performance from a single drop of blood. Both the CNT-TFTs and D4 are fabricated by printing, making them readily customizable and low cost. CNT-TFTs offer extremely sensitive nanotubes for electrical transduction in an assay and, unlike prior CNT-based biosensors, the CNT-TFTs provide reproducible performance and high yield. The D4 is a fluorescence-based assay that uses antibody (Ab) pairs printed into a polymer brush coating (“POEGMA”) that eliminates background noise and permits highly sensitive measurements directly in complex biological milieu. Merging these two innovative technologies leads to the “D4- TFT”, which combines the electronic sensitivity of the CNT-TFTs with the nonfouling selectivity of the D4 to yield an assay that can rapidly detect biomarker concentration from whole blood without rinsing, via electrical transduction. Extensive preliminary data demonstrate the D4-TFT’s fully printed fabrication, preservation of transistor operation, and successful detection of target analytes from serum and blood. The specific aims of this project include the development of a handheld POCT unit, with all needed control electronics and a simple user interface with display, and adaptation of the D4-TFT to compatible cassettes that are inserted into the handheld unit for operation. Analytical performance of the D4-TFT assay will be maximized and multiplexed against two panels of cardiac biomarkers: (1) A panel of validated cardiac biomarkers that includes creatine kinase (CK), CKMB, troponin (Tn), and N-terminal pro b-type natriuretic peptide (BNP), which are central to the diagnosis and management of a wide spectrum of cardiovascular conditions, including acute chest pain, myocardial infarction, and heart failure (HF); and (2) two investigative cardiac biomarkers, angiopoietin 2 (ANG2) and tumor necrosis factor alpha (TNFα), which are of major interest for their potential association with bleeding events in advanced HF patients with left ventricular assist device (LVAD) implants (the most common complication in this population). These two diverse panels of cardiac biomarkers will demonstrate the versatility and value of this POCT, both for diagnostic potential using validated biomarkers as well as for rapid clinical screening of investigative biomarkers. Accomplishing these aims will lead to a novel POCT for cardiac biomarkers that can be scaled up easily. Its fully printed nature makes the D4-TFT highly customizable and broadly applicable to other blood-based tests for any biomarker with immunoassay reagents.