| 项目编号: | 1512664
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| 项目名称: | UNS: Translation of nano-biorecognition into macroscopic counting for attomolar detection of biomolecules |
| 作者: | Chuanbin Mao
|
| 承担单位: | University of Oklahoma Norman Campus
|
| 批准年: | 2014
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| 开始日期: | 2015-09-01
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| 结束日期: | 2018-08-31
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| 资助金额: | USD300000
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| 资助来源: | US-NSF
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| 项目类别: | Standard Grant
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| 国家: | US
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| 语种: | 英语
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| 特色学科分类: | Engineering - Chemical, Bioengineering, Environmental, and Transport Systems
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| 英文关键词: | target mirna
; t7 phage
; detection
; detection limit
; aim
; plaque counting
; body fluid
; qrt-pcr
; bnt
; native american nanotechnology outreach
|
| 英文摘要: | 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. |
| 资源类型: | 项目
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| 标识符: | http://119.78.100.158/handle/2HF3EXSE/93604
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| Appears in Collections: | 影响、适应和脆弱性
气候减缓与适应
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| Recommended Citation: | |
Chuanbin Mao. UNS: Translation of nano-biorecognition into macroscopic counting for attomolar detection of biomolecules. 2014-01-01.
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