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

摘要 心脏病是美国的主要死亡原因，每年夺走60多万人的生命。这个 以血液为基础的生物标志物的测量在缩小患者的鉴别诊断和分诊范围方面起着至关重要的作用 在急慢性心血管疾病的管理方面。这个R01项目将开发一个手持点- 护理测试(POCT)，使用低成本、一次性分析卡来测量心脏多路复用板 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方面的主要进展。 集成在薄膜晶体管(CNT-TFT)中的碳纳米管(CNT-TFT)和用于检测蛋白质的分析平台(称为D4 从一滴血中获得类似于酶联免疫吸附试验的分析结果。CNT-TFT和D4都是由 打印，使其易于定制和低成本。CNT-TFT提供了极其灵敏的纳米管 与以前的基于碳纳米管的生物传感器不同，碳纳米管-TFT提供了可重复性 性能和高产量。D4是一种基于荧光的检测方法，它使用打印到 聚合物刷涂(“POEGMA”)，消除背景噪音，并允许高度敏感 直接在复杂的生物环境中进行测量。将这两种创新技术融合在一起，就产生了“D4- TFT“，它结合了碳纳米管-TFT的电子灵敏度和D4的无污染选择性，以产生 一种无需漂洗即可通过电子手段快速检测全血中生物标志物浓度的分析方法 转导。广泛的初步数据表明，D4-TFT的完全印刷制造，保存了 晶体管操作，以及从血清和血液中成功检测目标分析物。这样做的具体目的是 该项目包括开发一个手持POCT单元，带有所有需要的控制电子设备和简单的用户 与显示器接口，并使D4-TFT适应插入手持设备的兼容盒式磁带 用于操作的单元。D4-TFT检测的分析性能将最大化，并与两个 心脏生物标志物面板：(1)包括肌酸激酶(CK)在内的一组有效的心脏生物标志物， 肌钙蛋白同工酶(CKMB)、肌钙蛋白(TN)和N末端b型利钠肽(BNP)，对诊断和治疗有重要意义。 管理一系列心血管疾病，包括急性胸痛、心肌梗死、 和心力衰竭(HF)；以及(2)两个正在研究的心脏生物标志物，血管生成素2(ANG2)和肿瘤坏死 α因子(肿瘤坏死因子α)，因其与晚期出血事件的潜在关联而备受关注。 植入左心室辅助装置(LVAD)的心衰患者(这是该人群中最常见的并发症)。 这两个不同的心脏生物标志物小组将展示这种POCT的多功能性和价值，两者都用于 使用经过验证的生物标记物的诊断潜力以及对研究生物标记物的快速临床筛选。 实现这些目标将导致一种新的心脏生物标志物的POCT，它可以很容易地放大。它完全是 打印的性质使D4-TFT高度可定制，并广泛适用于任何其他基于血液的测试 生物标记物与免疫分析试剂。

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.