UNS: Translation of nano-biorecognition into macroscopic counting for attomolar detection of biomolecules

UNS：将纳米生物识别转化为用于生物分子阿摩尔检测的宏观计数

• 批准号: 1512664
• 负责人: Chuanbin Mao
• 金额: $ 30万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1512664&HistoricalAwards=false
• 关键词: UNS; Translation; nano; biorecognition; into

1512664 Mao, Chuanbin There is growing evidence that microRNAs, a type of a biomolecule in body fluids can be used as non-invasive biomarkers diseases. At present, detection and quantification of them is by current methods yields poor reproducibility and low accuracy when the concentration is very low. This research will develop a new novel approach called plaque counting (PC) method, which is likely to yield superior results. MicroRNAs (miRNAs) in body fluids are a class of non-invasive Alzheimer's disease (AD) biomarkers. At present their detection and quantification is by quantitative real-time polymerase chain reaction (qRT-PCR), which has inherent limitations such as poor reproducibility. In the proposed study, a plaque counting (PC) approach is proposed, which uses T7 phage to establish a one-to-one correspondence among target miRNA, T7 phages, and phage-developed viral plaques. A gold NP (AuNP)-probe dually functionalized with a recombinant AuNP-binding fluorescent T7 phage and an oligonucleotide (ONT) capable of hybridizing with one segment of the target miRNA and a magnetic microparticle (MMP)-probe functionalized with another ONT capable of hybridizing with a segment of the same miRNA co-capture the target miRNA to form a sandwich complex. T7 phages are then released from the complex by competitive binding of a reported gold-binding peptide and plated to develop fluorescent plaques. The number of fluorescent T7 phages is equal to not only that of the target miRNAs but also to that of the developed fluorescent plaques. Two aims are: Aim 1: Understand how the solution and probe conditions influence the accuracy and sensitivity of the PC method for quantifying single and multiple AD-associated miRNAs. The PC method will be used to quantify AD biomarker miRNAs (miR-9, miR-29a, and miR-137) with a series of dilutions in water to identify the detection limit of the method. The PI will then examine how the background RNAs, the sizes of MMPs and AuNPs, and the concentrations of saline influence both single and multiplexed quantifications of the three target miRNAs. Aim 2: Validate the use of the PC strategy for quantifying single and multiple miRNAs in human serum. MMPs capable of capturing target miRNAs will be first used to magnetically remove the pre-existing target miRNAs from the commercial human serum, which is then used to make serum samples with known concentrations of target miRNAs, followed by the quantification tests described in Aim 1. Intellectual Merit : This project will develop and optimize a new facile method for quantifying circulating miRNAs with high sensitivity, accuracy, and reproducibility. It overcomes some of the disadvantages of qRT-PCR technique and thus advances the fields of miRNA-based diagnosis. Broader Impacts : The results of this study is likely to improve detection limit of microRNA in body fluids; and therefore, disease diagnosis. Broader impact activities include: (1) A curriculum on bionanotechnology (BNT) will be strengthened to educate a new generation of scientists; (2) community college and high school students will be motivated to learn hands-on skills in BNT and choose to pursue higher education; (3) A Native American Nanotechnology Outreach (NANO) program will be solidified in Oklahoma to help Native Americans become interested in scientific careers and be more competitive on job markets; (4) Public awareness of BNT will be increased in Oklahoma statewide.

1512664毛传斌越来越多的证据表明，microRNA是体液中的一种生物分子，可用作非侵入性疾病的生物标志物。目前，当浓度很低时，通过现有方法对其进行检测和定量产生差的重现性和低准确度。这项研究将开发一种新的方法称为斑块计数（PC）方法，这是可能产生上级的结果。 体液中的微小RNA（miRNAs）是一类非侵入性阿尔茨海默病（AD）生物标志物。目前，它们的检测和定量是通过定量实时聚合酶链反应（qRT-PCR），其具有固有的局限性，如重现性差。在拟议的研究中，提出了一种空斑计数（PC）方法，该方法使用T7噬菌体建立靶miRNA、T7噬菌体和噬菌体产生的病毒空斑之间的一一对应关系。用重组AuNP结合荧光T7噬菌体和能够与靶miRNA的一个区段杂交的寡核苷酸（ONT）双重功能化的金NP（AuNP）-探针和用能够与相同miRNA的区段杂交的另一个ONT功能化的磁性微粒（MMP）-探针共捕获靶miRNA以形成夹心复合物。然后，通过竞争性结合报道的金结合肽从复合物中释放T7 β，并铺板以形成荧光斑块。荧光T7标记的数量不仅与靶miRNA的数量相等，而且与所形成的荧光斑块的数量相等。两个目标是：目标1：了解溶液和探针条件如何影响PC方法定量单个和多个AD相关miRNA的准确性和灵敏度。PC方法将用于定量AD生物标志物miRNA（miR-9、miR-29 a和miR-137），并在水中进行一系列稀释，以确定该方法的检测限。然后PI将检查背景RNA、MMP和AuNP的大小以及盐水浓度如何影响三种靶miRNA的单一和多重定量。目的2：验证PC策略在定量人血清中单个和多个miRNAs中的应用。能够捕获靶miRNA的MMP将首先用于从商业人血清中磁性去除预先存在的靶miRNA，然后用于制备具有已知浓度的靶miRNA的血清样品，然后进行目标1中描述的定量测试。智力优势：该项目将开发和优化一种新的简便方法，用于定量循环miRNA，具有高灵敏度，准确性和重现性。它克服了qRT-PCR技术的一些缺点，从而推进了基于miRNA的诊断领域。更广泛的影响：这项研究的结果可能会提高体液中microRNA的检测限;因此，疾病诊断。 影响更广泛的活动包括：（1）加强生物纳米技术课程，以教育新一代科学家;（2）鼓励社区大学和高中学生学习生物纳米技术的实践技能，并选择接受高等教育;（3）美国本土纳米技术推广（NANO）该计划将在俄克拉荷马州得到巩固，以帮助美洲原住民对科学事业感兴趣，并在就业市场上更具竞争力;（4）将在俄克拉荷马州全州范围内提高公众对BNT的认识。