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

越来越多的证据表明，体液中的一类生物分子microrna可以作为非侵入性疾病的生物标志物。目前，用现有的方法对其进行检测和定量，在浓度很低的情况下，重现性差，准确度低。这项研究将开发一种新的方法，称为斑块计数（PC）方法，这可能会产生更好的结果。体液中的MicroRNAs （miRNAs）是一类非侵入性阿尔茨海默病（AD）的生物标志物。目前对其进行检测和定量的方法是实时定量聚合酶链反应（qRT-PCR），这种方法存在固有的局限性，如重复性差。本研究提出了一种斑块计数（PC）方法，该方法利用T7噬菌体在靶miRNA、T7噬菌体和噬菌体产生的病毒斑块之间建立一一对应关系。一个金NP （AuNP）探针与重组AuNP结合的荧光T7噬菌体和一个能够与目标miRNA的一个片段杂交的寡核苷酸（ONT）双功能化，一个磁性微粒（MMP）探针与另一个能够与同一miRNA的一个片段杂交的ONT功能化，共同捕获目标miRNA形成三明治复合物。然后，T7噬菌体通过一种报道的金结合肽的竞争性结合从复合物中释放出来，并被镀以形成荧光斑块。荧光T7噬菌体的数量不仅与靶mirna的数量相等，而且与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。将使用PC方法定量AD生物标志物mirna (miR-9， miR-29a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、MMPs和AuNPs的大小以及盐水浓度如何影响三种目标mirna的单次和多次定量。目的2：验证PC策略用于定量人血清中单个和多个mirna的使用。能够捕获目标mirna的MMPs将首先用于从商业人血清中磁性去除预先存在的目标mirna，然后用于制作具有已知浓度的目标mirna的血清样本，然后进行Aim 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，准确性和可重复性。它克服了qRT-PCR技术的一些缺点，从而推动了基于mirna的诊断领域的发展。更广泛的影响：本研究结果可能提高体液中microRNA的检测限；因此，疾病诊断。更广泛的影响活动包括：(1)将加强生物纳米技术课程，以培养新一代科学家；(2)社区大学和高中的学生将有动力在BNT学习动手技能，并选择接受高等教育；(3)俄克拉何马州将巩固一项美洲原住民纳米技术推广（NANO）计划，以帮助美洲原住民对科学事业产生兴趣，并在就业市场上更具竞争力；(4)俄克拉何马州将提高公众对BNT的认识。

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.