Multiplex Hybridization Colorimetric Sensor for Wild Type and Variant RNA Biomarkers

用于野生型和变异 RNA 生物标志物的多重杂交比色传感器

基本信息

批准号：
10580293
负责人：
Sadagopan Krishnan
金额：
$ 43.89万
依托单位：
OKLAHOMA STATE UNIVERSITY STILLWATER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-11 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10580293
关键词：
2019-nCoV Amides Amines Base Pairing Binding Biological Assay Biological Markers Biological Models Biomedical Engineering Biomedical Research Biotin Biotinylation COVID-19 COVID-19 detection Calibration Carbodiimides Carboxylic Acids Chemicals Chemistry Color Complex DNA Detection Diagnosis Disease Educational process of instructing Environment Exposure to Genes Glucose Goals Gold Hormones Human Resources Hydrogen Peroxide Immobilization Infection Ions Label Laboratories Lateral Magnetism Measurement Measures Metals Methods Molecular Mutation Nucleic Acids Nucleotides Oklahoma Oligonucleotide Probes Oligonucleotides Outcome Output Paper Pathogenicity Peroxidases Polymerase Chain Reaction Population Probability Process Prognosis RNA RNA marker ROC Curve Reagent Research Resource-limited setting Reverse Transcriptase Polymerase Chain Reaction Saliva Sampling Schools Sensitivity and Specificity Serum Signal Transduction Streptavidin Sulfhydryl Compounds Surface Techniques Technology Testing Training Translations Universities Validation Variant Viral Load result Virus Virus Diseases Visual amplification detection combat coronavirus disease cost design detection limit detection sensitivity detector disease diagnosis experience innovation instrument iron oxide minimally invasive molecular diagnostics nano nanoGold nanomaterials nanoparticle nanoshell novel strategies pandemic disease plasmonics public health relevance respiratory sensor specific biomarkers targeted biomarker undergraduate student variant detection viral RNA viral detection

项目摘要

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.

基于实验室的检测，例如呼吸系统的逆转录酶聚合酶链反应 (RT-PCR) 分泌物通常用于检测病毒核酸。然而，RT-PCR 检测有许多优点诸如需要高纯度样品、基因分离和扩增、训练有素的人员等限制，用于样品处理的先进清洁设施，以及昂贵的实验室仪器。到与当前的大流行作斗争，迫切需要可获取、负担得起的、可视化的分子为全球资源有限的地区提供服务的诊断。如此先进的技术也将通过精确测量，促进早期疾病诊断、预后和发病后状况特定的生物标志物目标。与大多数 DNA 传感器相比，视觉比色传感器不需要昂贵的用于诊断感染或疾病的仪器或实验室环境。尽管有这些优点，现有的有效使用比色传感器的限制包括较差的检测极限阈值和可靠性一个目标分子上，从而导致变体检测的假阴性。因此，我们在此建议应用多重传感器方法，通过将多个传感器一起瞄准来本质上放大检测信号唾液和血清中的 RNA 标记组，从而增加阳性识别和减少误报检测。我们的方法涉及设计携带可磁分离的防污氧化铁核/金壳纳米颗粒，可选择性捕获目标 SARS-CoV-2 野生型 RNA 以及唾液和血清中两种最常见的 N501Y 和 E484K 变体（步骤 1、10 分钟）作为模型系统，可更广泛地应用于任何其他生物标志物目标。一秒钟互补寡核苷酸（生物素化）杂交和磁力分离（步骤 2，5 分钟），以及随后链霉亲和素-过氧化物酶分子选择性结合靶 RNA 杂交生物素寡核苷酸和磁性分离（步骤 3，5 分钟）将允许目视和光谱仪颜色读数存在添加的过氧化氢和四甲基联苯胺底物，如所提出的那样拟议新方法的初步结果。具有颜色强度的校准颜色参考标度将设计像 pH 试纸这样的配置文件，用于对基于 RNA 生物标志物的感染进行是/否识别在实际样品中。使用标准核酸 PCR 技术验证比色传感器建议的。我们的研究团队将主要由本科生组成。此外在我校建立R15本科生俱乐部，为多名学生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、工艺和产品设计课程（由 PI 教授）。所有本科生将因此通过积极参与拟议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。