Highly Integrated Nucleic-Acid Analysis Using Graphene Bioelectronics

使用石墨烯生物电子学进行高度集成的核酸分析

• 批准号: 10584520
• 负责人: Jinglei Ping
• 金额: $ 18.99万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10584520
• 关键词: Affinity; Animal Model; Biological Assay; Biological Markers; Biopsy; Biopsy Specimen; Blinded; Buffers; Clinical; Clinical Trials; Complex; Consumption; Core Facility; DNA; DNA Probes; Development; Devices; Diagnosis; Diagnostic; Disease; Disease Progression; Electrodes; Electronics; Generations; Genomics; Goals; Hospitals; Hour; Immunoassay; Intervention; Investigation; Measurable; Measures; MicroRNAs; Monitor; Mus; Noninfiltrating Intraductal Carcinoma; Nucleic Acids; Oligonucleotides; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Performance; Plasma; Population; Prognosis; Progressive Disease; Property; Public Health; Reaction; Recurrence; Research; Risk Assessment; Sampling; Signal Pathway; Signal Transduction; Technology; Testing; Therapeutic; Time; Transducers; Transistors; Translating; Translational Research; Validation; Visit; Xenograft Model; bioelectronics; breast cancer progression; circulating microRNA; clinical care; clinical translation; cohort; cost; detection limit; diagnostic technologies; disease diagnosis; early screening; genomic predictors; graphene; human subject; improved; individualized medicine; liquid biopsy; malignant breast neoplasm; micro-total analysis system; mouse model; new therapeutic target; next generation; outcome prediction; overtreatment; pH gradient; point of care; point-of-care diagnosis; pre-clinical; prevent; prognostic; programs; response; screening; sensor; targeted treatment; technology platform; technology validation; treatment response; treatment strategy; user-friendly; voltage

PROJECT SUMMARY The circulating population of microRNAs in biofluids are ideal biomarkers for various diseases. Point-of-care profiling of circulating microRNAs is in insatiable demand, but typical approaches, e.g., immunoassays and microRNA assays are lab-based/centralized, expensive ($400–1,000/test), and time-consuming (>6 hours). We will develop a highly integrated, all-nanobioelectronic platform technology for multiplex, high-accuracy circulating-microRNA analysis that is capable of profiling circulating microRNAs in a 50-μL plasma with ultra- high sensitivity (sub-fM) and efficiencies in time (<40 minutes) and cost (<$10/test), thereby enabling high- performance circulating-microRNA analysis at the point of test. The novelty of the program is to harness graphene-based bioelectronics to integrate circulating microRNA isolation, concentration, amplification, and quantification into a self-contained device. In order to proof the concept of this technology, the program will include the development and validation of two generations of graphene-based analytical platforms, GAP1 and GAP2. Three specific aims with measurable milestones will be pursued. (1) We will demonstrate that multiple microRNA analytes can be amplified via hybridization chain reaction on a probe-functionalized graphene sensor array and the analyte concentrations can be readily interrogated by the graphene sensor array and translated into electrical signals. We will develop GAP1 to selectively quantify eight pre-selected target microRNAs (MDCIS8) spiked in 5-μL buffer. The detection limit of the specific microRNAs is expected to be at fM level. (2) We will demonstrate that target circulating microRNAs can be isolated from plasma by immobilizing them on a DNA-functionalized graphene electrode and releasing them into a small-volume simple cargo solution upon the generation of pH gradient by applying voltage bias between the graphene-DNA electrode with a bare graphene electrode. We will develop a graphene-based circulating-microRNA isolation module, combine the module with GAP1 to form GAP2, and use GAP2 to profile circulating MDCIS8 in lysed samples of 50-μL plasma from NSG mice. The GAP2 is expected to concentrate the microRNAs by >5× and deliver sub-fM level sensitivity. (3) We will demonstrate the feasibility of using this platform technology for diagnostic applications. We will use GAP2 to quantify circulating MDCIS8, whose expression levels are indicative to pre-invasive breast cancer, in 50-μL plasma samples from a user blinded cohort of the MIND murine model. The profiling result will be analyzed to predict the progression of pre-invasive breast cancer whose rapid, inexpensive diagnosis remains a challenge. The GAP2 prediction outcome will be combined with that based on surgical biopsy to establish the accuracy of the technology for progression prediction. The expected prediction accuracy is >96%. If successful, the technology will offer a new pathway to next-generation point-of-care genomic diagnostic/prognostic micro total analysis systems that would be cheap enough and user friendly enough to be used in various clinical settings.

项目概要 生物体液中循环的 microRNA 群体是各种疾病的理想生物标志物。现场护理 对循环 microRNA 的分析有着无法满足的需求，但典型的方法，例如免疫分析和 microRNA 测定是基于实验室/集中化的、昂贵的（每次测试 400-1,000 美元）且耗时（> 6 小时）。我们 将开发高度集成的全纳米生物电子平台技术，用于多重、高精度 循环 microRNA 分析，能够分析 50 µL 血浆中的循环 microRNA 高灵敏度（sub-fM）以及时间效率（<40 分钟）和成本（<10 美元/测试），从而实现高 测试时进行循环 microRNA 分析。该计划的新颖之处在于利用 基于石墨烯的生物电子学，集成循环 microRNA 分离、浓缩、扩增和 量化到一个独立的设备中。为了证明这项技术的概念，该程序将 包括两代基于石墨烯的分析平台 GAP1 和 差距2。将追求三个具有可衡量里程碑的具体目标。 (1) 我们将证明多个 microRNA 分析物可以通过探针功能化石墨烯传感器上的杂交链式反应进行扩增 阵列和分析物浓度可以很容易地通过石墨烯传感器阵列询问并转换 转化为电信号。我们将开发 GAP1 来选择性地量化八个预选的目标 microRNA (MDCIS8) 加标于 5 µL 缓冲液中。特定 microRNA 的检测限预计为 fM 水平。 (2) 我们将证明，通过将目标循环 microRNA 固定在血浆上，可以将其从血浆中分离出来。 DNA 功能化的石墨烯电极，并将其释放到小体积的简单货物溶液中 通过在石墨烯-DNA 电极与裸石墨烯之间施加偏压来产生 pH 梯度 电极。我们将开发一种基于石墨烯的循环microRNA分离模块，将该模块与 GAP1 形成 GAP2，并使用 GAP2 分析来自 NSG 的 50 µL 血浆裂解样品中的循环 MDCIS8 老鼠。 GAP2 预计将 microRNA 浓缩 >5 倍，并提供亚 fM 水平的灵敏度。 (3) 我们 将展示使用该平台技术进行诊断应用的可行性。我们将使用 GAP2 来 定量 50 µL 循环 MDCIS8，其表达水平指示浸润前乳腺癌 血浆样本来自 MIND 小鼠模型的用户盲态队列。分析结果将被分析为 预测浸润前乳腺癌的进展，其快速、廉价的诊断仍然是一个挑战。 GAP2 预测结果将与基于手术活检的结果相结合，以确定 GAP2 预测结果的准确性 进展预测技术。预期预测准确度>96%。如果成功的话， 技术将为下一代护理点基因组诊断/预后微总数提供新途径 分析系统足够便宜且用户友好，可用于各种临床环境。