Multiplex Hybridization Colorimetric Sensor for Wild Type and Variant RNA Biomarkers

呼吸道逆转录聚合酶链式反应(RT-PCR)等实验室检测 分泌物通常被用来检测病毒核酸。然而，RT-PCR法有许多 限制因素，如需要高纯度样本、基因分离和扩增、训练有素的人员、 用于样品处理的先进的清洁设施，以及昂贵的实验室仪器。至 抗击持续的大流行迫切需要可获得的、负担得起的和可视的分子 为全球资源有限地区提供服务的诊断服务。如此先进的技术也将 通过精确测量以下指标，促进疾病早期诊断、预后和发病后状况 特定的生物标志物靶标。与大多数DNA传感器不同，视觉比色传感器不需要昂贵的 诊断感染或疾病的仪器或实验室环境。尽管有这些优势，现有的 有效使用比色传感器的限制包括检测下限差和依赖 在一个靶分子上，从而导致对变体的假阴性检测。因此，我们在此提出 应用一种多路传感器方法，通过将目标集中在一起来固有地放大检测信号 唾液和血清中的一组RNA标志物，从而增加阳性识别和 减少假阴性检测。我们的方法涉及多个寡核苷酸探针的设计 磁性可分离、防污染的氧化铁核/金壳纳米颗粒选择性地捕获目标 SARS-CoV-2野生型RNA以及唾液和血清中最常见的两个N501Y和E484K变异(STEP 1、10分钟)作为更广泛应用于任何其他生物标志物靶标的模型系统。一秒钟 互补寡核苷酸(生物素化)杂交和磁分离(步骤2，5分钟)，以及 链霉亲和素-过氧化物酶分子与生物素杂交靶RNA的选择性结合 寡核苷酸和磁分离(步骤3，5分钟)将允许视觉和光谱分析颜色读数 添加的过氧化氢和四甲基联苯胺底物的存在 拟议的新方法的初步结果。以色彩强度为特征的校准颜色参考标尺 将设计像pH指示器纸这样的配置文件，用于对基于RNA生物标记物的感染进行是/否识别 在真实的样品中。使用标准的基于核酸的聚合酶链式反应技术对比色传感器的验证是 建议。我们的研究团队将主要由本科生组成。除了……之外 在我们学校建立R15本科生俱乐部，为几个人提供病毒传感器研究机会 一百名本科生，我们将在普通化学课、生物医学课上进行课堂演示 工程学、工艺和产品设计课程(由PI授课)。所有本科生都将因此 通过积极参与提议的项目获得重要的生物医学研究经验，从而 改善俄克拉荷马州立大学的研究环境。

Laboratory-based assays such as reverse transcriptase-polymerase chain reaction (RT-PCR) of respiratory secretions are being used routinely to detect viral nucleic acids. The RT-PCR assay, however, has many limitations such as the need for high purity samples, gene isolation and amplification, highly trained personnel, sophisticated clean facilities for sample processing, and access to expensive laboratory instruments. To combat the ongoing pandemic there is an urgent need for accessible, affordable, and visual molecular diagnostics that serve resource-limited regions across the globe. Such advanced technology would also facilitate early disease diagnosis, prognosis, and post-occurrence conditions through precise measurements of specific biomarker targets. In contrast to most DNA sensors, visual colorimetric sensors do not require costly instruments or a laboratory setting to diagnose an infection or a disorder. Despite these advantages, existing limitations on the effective use of colorimetric sensors include poor detection limit thresholds and the reliance on one target molecule, thus resulting in false-negative detection of variants. Hence, we propose in this application a multiplex sensor approach that will inherently amplify detection signals by targeting together a panel of RNA markers in saliva and serum and thus increase the probability of positive identification and mitigate false negative detection. Our approach involves the design of multi-oligonucleotide probes carrying magnetically-separable, antifouling iron oxide core/gold shell nanoparticles to selectively capture the target SARS-CoV-2 wild type RNA and the two most common N501Y and E484K variants in saliva and serum (Step 1, 10 min) as the model system for broader applications to any other biomarkers targets. A second complementary oligonucleotide (biotinylated) hybridization and magnetic separation (Step 2, 5 min), and subsequent selective binding of streptavidin-peroxidase molecules to the target RNAs hybridized biotin oligonucleotides and magnetic separation (Step 3, 5 min) will allow visual and spectrometer color readouts in the presence of added hydrogen peroxide and tetramethylbenzidine substrate as supported with the presented preliminary results from the proposed new approach. A calibrated color reference scale featuring color intensity profiles like a pH indicator paper will be devised for a yes/no identification of RNA biomarkers-based infections in real samples. Validation of the colorimetric sensor with the standard nucleic acid-based PCR techniques is proposed. Our research teams will be composed primarily of undergraduate students. In addition to establishing an R15 undergraduate club at our school, to offer virus sensor research exposure for several hundred undergraduates, we will conduct in-class demonstrations in general chemistry classes, Biomedical Engineering, and Process and Product Design classes (taught by the PIs). All undergraduate students will thus gain significant biomedical research experience through active involvement in the proposed project and thus enhance the research environment of Oklahoma State University.