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

描述（由申请人提供）：虽然由于下一代测序仪器的发展，DNA测序的成本在过去几年中显着下降，但仍然需要产生能够显着降低测序成本和时间并提高自动化水平的新技术，以实现将DNA测序过渡到目前无法进入的领域的能力，例如临床体外诊断。事实上，达到1000美元基因组计划所规定的目标，将提供使用DNA测序作为实际标准的能力，用于观察整个基因组的任何序列变化。该项目的长期目标是生成一个新的DNA测序平台，该平台可以大大降低使用全自动平台获取DNA测序信息的成本，劳动力和时间。该策略使用纳米级传感器，通过在热塑性塑料（如有机玻璃）中通过低成本纳米压印光刻和其他基于复制的技术制造的二维（2D）纳米通道（宽度和深度<10 nm；长度为50 μ m），从其特征飞行时间读取单核苷酸碱基的身份。单核苷酸碱基由完整的DNA片段（~50,000 bp）使用过程外切酶生成，该过程外切酶共价锚定在生物反应器内的载体上，将单核苷酸送入二维纳米通道。单核苷酸的身份是从分子依赖的飞行时间通过二维纳米通道推断出来的。该R21应用程序的主要重点是开发一种转导模式，可以测量单核苷酸通过二维聚合物纳米通道的飞行时间，而不需要报告分子共价附着在单核苷酸上。所研究的换能器由两对纳米电极组成，分别位于二维纳米通道的两端，其信号由单核苷酸引起的电导率扰动引起。该传感平台是由阳极氧化铝材料采用模板法构建的纳米线制成的，然后通过电化学稀释到所需的直径（~10 nm）。这些金属线被策略性地放置在纳米流控芯片上，使用通过纳米压印光刻技术制作的化学图案，通过机械或化学步骤产生所需的间隙（<10纳米）。纳米传感器芯片是在塑料模块上生产的，可以通过新颖的互连技术集成到其他DNA处理模块上，以提供DNA样品处理管道的完全自动化。设想的DNA测序平台将产生~1 × 106个核苷酸碱基读数s-1，当配置为阵列格式时，以全自动方式处理整个样品，模块化流体系统的成本< 200美元。流体系统的低成本不仅是因为使用了复制技术来生产跨越多个尺寸尺度的流体网络，而且还因为使用了简单和高度并行的策略来生产该芯片所需的纳米级组件。

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.