Highly Integrated Nucleic-Acid Analysis Using Graphene Bioelectronics

项目总结 生物体液中循环中的microRNAs是各种疾病的理想生物标志物。护理点 对循环中的microRNA的分析需求永无止境，但典型的方法，如免疫分析和 MicroRNA检测以实验室为基础/集中化，价格昂贵(每项检测400-1000美元)，而且耗时(&gt；6小时)。我们 将开发一种高度集成的、全纳米生物电子平台技术，用于多路、高精度 50-μL血浆中循环microRNA的超高分辨分析 高灵敏度(亚调频)和高效率的时间(&lt；40分钟)和成本(&lt；美元/测试)，从而实现高- 性能循环--检测点的microRNA分析。该计划的新奇之处在于利用 基于石墨烯的生物电子学，集成循环microRNA分离、浓缩、扩增和 量化成一个自给自足的装置。为了证明这项技术的概念，该计划将 包括开发和验证两代基于石墨烯的分析平台Gap1和 GAP2.将追求具有可衡量里程碑的三个具体目标。(1)我们将展示多个 可以通过探针功能化石墨烯传感器上的杂交链式反应来扩增microRNA分析物 阵列和分析物浓度可以容易地被石墨烯传感器阵列询问并转化 转换成电信号。我们将开发Gap1来选择性地量化八个预先选择的目标microRNA (MDCis8)在5-μL缓冲液中添加。预计特定microRNAs的检测下限为FM级。(2) 我们将证明，通过将目标循环microRNA固定在一个 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 石墨烯-DNA电极与裸石墨烯之间施加偏压产生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，并用GAP2分析来自国家核科学研究院的50-μL血浆裂解样品中循环的MDCI_8 老鼠。预计GAP2将把microRNAs浓缩5倍，并提供亚FM级别的灵敏度。(3)我们 将展示将该平台技术用于诊断应用的可行性。我们将使用GAP2来 在50-μL中定量检测循环中mdcis8的表达水平，以指示浸润性前期乳腺癌 来自Mind小鼠模型的用户盲群的血浆样本。将对分析结果进行分析以 预测浸润性前乳腺癌的进展，其快速、廉价的诊断仍然是一个挑战。 GAP2的预测结果将与手术活检的结果相结合，以确定 进度预测技术。预期预测准确率为&gt；96%。如果成功，则 技术将为下一代护理点基因组诊断/预后微总体提供一条新的途径 分析系统将足够便宜和用户友好，足以在各种临床环境中使用。

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.