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

• 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
• 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

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.

基于鉴定的测定，如呼吸道感染的逆转录酶-聚合酶链反应（RT-PCR）， 分泌物常规用于检测病毒核酸。然而，RT-PCR检测具有许多 局限性如需要高纯度样品，基因分离和扩增，训练有素的人员， 用于样品处理的先进清洁设施，以及使用昂贵的实验室仪器。到 与正在进行的流行病作斗争，迫切需要可获得的、负担得起的和可视的分子 为地球仪资源有限地区提供服务的诊断。这种先进的技术也将 通过精确测量，促进早期疾病诊断，预后和发生后的条件， 特异性生物标志物靶点。与大多数DNA传感器相比，视觉比色传感器不需要昂贵的 仪器或实验室设置来诊断感染或病症。尽管有这些优势， 对比色传感器的有效使用的限制包括检测极限阈值差和依赖性 在一个靶分子上，从而导致变体的假阴性检测。因此，我们在此建议 本申请提供了一种多路传感器方法，该方法将通过一起靶向一个或多个传感器来固有地放大检测信号。 唾液和血清中的RNA标记物组，从而增加阳性鉴定的概率， 减少假阴性检测。我们的方法包括设计多寡核苷酸探针， 磁性可分离的铁氧化物核/金壳纳米颗粒，以选择性地捕获靶 唾液和血清中SARS-CoV-2野生型RNA和两种最常见的N501 Y和E484 K变体（步骤 1，10分钟）作为模型系统更广泛的应用到任何其他生物标志物的目标。第二 互补寡核苷酸（生物素化）杂交和磁性分离（步骤2，5 min），和 链霉亲和素-过氧化物酶分子随后选择性结合靶RNA， 寡核苷酸和磁性分离（第3步，5分钟）将允许目视和光谱仪颜色读数， 加入的过氧化氢和四甲基联苯胺底物的存在，如用本发明的载体所支持的， 新方法的初步结果。一个校准的颜色参考标度， 将设计一种类似pH试纸的图谱，用于确定基于RNA生物标志物的感染是否存在 在真实的样本中。采用标准核酸PCR技术验证比色传感器， 提出了我们的研究团队将主要由本科生组成。除了 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， 百名本科生，我们将进行课堂演示，在一般化学类，生物医学 工程，工艺和产品设计课程（由PI教授）。因此，所有本科生将 通过积极参与拟议的项目获得重要的生物医学研究经验， 加强俄克拉荷马州州立大学的研究环境。