Polymer-Based Modular Systems with Nanosensors for DNA/RNA Sequencing

具有用于 DNA/RNA 测序的纳米传感器的基于聚合物的模块化系统

基本信息

批准号：
8179098
负责人：
Steven Allan Soper
金额：
$ 31.34万
依托单位：
LOUISIANA STATE UNIV A&M COL BATON ROUGE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8179098
关键词：
2&apos -deoxyguanosine 5&apos -phosphate Affect Aluminum Oxide Applications Grants Area Automation Bioreactors Caliber Characteristics Chemicals Clinic DNA DNA Sequence Data Deposition Detection Diagnostic Dimensions Drops Electric Conductivity Electrodes Elements Enzymes Evolution Exonuclease Feeds Foundations Genome Goals In Vitro Indium Individual Length Lifting Literature Masks Measurement Measures Mechanics Metals Methods Minor Modality Modification Molecular Nanostructures Nucleotides Output Pattern Phase Plastics Plexiglas Plexiglass Polymers Positioning Attribute Process Production Property RNA Sequences Reaction Reading Reporter Sampling Scheme Secure Side Signal Transduction Solid Solutions Stretching Structure Surface System Techniques Technology Time Transducers Travel Variant Width base conditioning cost ds-DNA fighting genome sequencing improved innovation instrument molecular assembly/self assembly nano nanochannel nanofluidic nanoimprint lithography nanometer nanopore nanoscale nanosensors nanowire new technology next generation novel response sensor single molecule two-dimensional

项目摘要

DESCRIPTION (provided by applicant): While the cost of DNA sequencing has dropped significantly over the last few years due to the evolution of next-generation sequencing instruments, there still exists the need to produce new technologies that can significantly reduce sequencing cost and time and improve the level of automation to realize the ability of transitioning DNA sequencing into currently inaccessible areas, such as the clinic for in vitro diagnostics. In fact, reaching the goals mandated by the $1,000 Genome Project will provide the ability to use DNA sequencing as a de facto standard for looking at any sequence variation over the entire genome. The long term goal of this project is to generate a novel DNA sequencing platform that can substantially reduce the cost, labor and time associated with acquiring DNA sequencing information using a fully automated platform. The strategy uses nano-scale sensors that read the identity of mononucleotide bases from their characteristic flight- time through a 2-dimensional (2D) nanochannel (<10 nm in width and depth; >5 ¿m in length) fabricated in a thermoplastic, such as Plexiglas, via low-cost nanoimprint lithography and other replication-based techniques. The mononucleotide bases are generated from an intact DNA fragment (~50,000 bp) using a processive exonuclease, which is covalently anchored to a support contained within a bioreactor that feeds the mononucleotides into the 2D nanochannel. The identity of the mononucleotide is deduced from a molecular- dependent flight-time through the 2D nanochannel. The major focus of this R21 application is to develop a transduction modality that can measure the flight-time of mononucleotides through a 2D polymer nanochannel without requiring a reporter molecule covalently attached to the mononucleotide. The transducer to be investigated consists of 2 pairs of nanoelectrodes poised at each end of the 2D nanochannel with the signal resulting from perturbations in the conductivity induced by the mononucleotide. The sensing platform is produced from nanowires built using templating methods from anodized aluminum oxide materials and then, electrochemically thinned to the desired diameter (~10 nm). The wires are strategically placed on a nanofluidic chip using chemical patterns made via nanoimprint lithography with the required gap (<10 nm) generated via mechanical or chemical steps. The nanosensor chips are produced on a plastic module that can be integrated via novel interconnect technologies to other DNA processing modules to provide complete automation of the DNA sample processing pipeline. The envisioned DNA sequencing platform will produce ~1 x 106 nucleotide base reads s-1 when configured in an arrayed format, process an entire sample in a fully automated fashion with the cost of the modular fluidic system <$200. The low-cost of the fluidic system results not only from the use of replication technologies to produce the fluidic network spanning over multiple size scales, but also the simple and highly parallel strategies used to produce the nano-scale components required for this chip. PUBLIC HEALTH RELEVANCE: A novel single-molecule DNA sequencing system is envisioned that utilizes a modular 3D approach to process input DNA with each module spanning several size domains (mm ? nm). One module is a nanosensor chip, which is comprised of 2D nanochannels used to identify individual mononucleotides through their molecular- dependent flight-time through the nanochannel. The flight-time is transduced using single-molecule conductivity measurements, which is measured using nano-electrodes poised at the input and output ends of the nanochannel. In this R21 application, the feasibility of measuring the conductivity response of single mononucleotides will be demonstrated.

描述（由适用提供）：虽然由于下一代测序仪器的发展，在过去的几年中，DNA测序的成本已大大下降，但仍然存在需要生产新技术，这些新技术可以显着降低测序成本和时间，并提高自动化水平，以实现当前无效诊断的能力，例如，在climccessiencecessimcessible climccessiment clinist上，例如，在无效的领域中进行了临床。实际上，达到1,000美元基因组项目规定的目标将提供使用DNA测序作为事实上的标准，以查看整个基因组上的任何序列变化。该项目的长期目标是生成一个新颖的DNA测序平台，该平台可以大大降低与使用全自动平台获取DNA测序信息相关的成本，人工和时间。该策略使用纳米尺度传感器，这些传感器从其特征飞行时间读取单核苷酸基碱基的身份 - 通过二维（2D）纳米渠道（宽度<10 nm的宽度和深度<10 nm；> 5 nm；长度为5»m的长度），该热塑料通过plexiglas，例如通过低表含量的纳米含量nanoimprint section和其他复杂来制造的热塑料。单核苷酸碱基是通过完美的外核酸酶从完整的DNA片段（〜50,000 bp）产生的，该核酸酶将其共价固定在生物反应器中的支撑物中，该辅导剂将单核苷酸喂入2D纳米胆道中。单核苷酸的身份是从通过2D纳米渠道的分子依赖飞行时间推导的。该R21应用的主要重点是开发一种翻译方式，该模态可以通过2D聚合物纳米渠道测量单核苷酸的飞行时间，而无需将记者分子共同连接到单核苷酸上。要研究的传感器由2对在2D纳米通道的两端中毒的2对纳米电极组成，并由单核苷酸引起的电导率扰动引起的信号。传感平台是由使用氧化铝材料的模板方法构建的纳米线产生的，然后在电化学上稀释到所需的直径（〜10 nm）。通过纳米印刷岩石学制造的化学模式，将电线策略性地放置在纳米流体芯片上，并具有通过机械或化学步骤产生的所需间隙（<10 nm）。纳米传感器芯片是在塑料模块上生产的，可以通过新型的互连技术集成到其他DNA加工模块，以提供DNA样品处理管道的完全自动化。设想的DNA测序平台将在以阵列格式配置时产生〜1 x 106核苷酸底座读取S-1，以完全自动化的方式处理整个样品，而模块化流体系统的成本<$ 200。流体系统的低成本不仅是由于使用复制技术在多个尺寸尺度上产生流体网络而产生的，而且还导致了用于生产此芯片所需的纳米级组件的简单且高度平行的策略。公共卫生相关性：设想一种新型的单分子DNA测序系统，它利用模块化3D方法处理输入DNA，每个模块跨越多个尺寸域（mm？nm）。一个模块是一种纳米传感器芯片，其中包括2D纳米渠道，用于通过纳米渠道通过其分子依赖的飞行时间来鉴定单个单核苷酸。使用单分子电导率测量值对飞行时间进行翻译，该测量是使用在纳米渠道的输入和输出末端中毒的纳米电极进行测量的。在此R21应用中，将证明测量单单核苷酸的电导率响应的可行性。