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

描述（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已经显著下降，但仍然需要产生能够显著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，例如用于体外诊断的诊所。事实上，达到1,000美元基因组计划规定的目标将提供使用DNA测序作为观察整个基因组中任何序列变异的事实标准的能力。该项目的长期目标是产生一种新型的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、劳动力和时间。该策略使用纳米级传感器，通过低成本的纳米压印光刻和其他基于复制的技术，通过在热塑性塑料（如有机玻璃）中制造的二维（2D）纳米通道（宽度和深度<10 nm;长度>5 μ m），从它们的特征飞行时间读取单核苷酸肽碱基的身份。使用进行性核酸外切酶从完整的DNA片段（~ 50，000 bp）产生单核苷酸肽碱基，所述进行性核酸外切酶共价锚定至包含在生物反应器内的支持物，所述生物反应器将单核苷酸肽进料至2D纳米通道中。从通过2D纳米通道的分子依赖性飞行时间推导出monopoltide的身份。该R21应用的主要焦点是开发一种转导方式，其可以通过2D聚合物纳米通道测量monoclutide的飞行时间，而不需要共价连接到monoclutide的报告分子。待研究的传感器由2对纳米电极组成，所述纳米电极位于2D纳米通道的每一端，所述纳米通道具有由单核苷酸引起的电导率扰动产生的信号。传感平台由纳米线制成，纳米线使用阳极氧化铝材料的模板方法构建，然后电化学减薄到所需的直径（约10 nm）。使用通过纳米压印光刻法制成的化学图案将线策略性地放置在纳米流体芯片上，其中通过机械或化学步骤产生所需的间隙（<10 nm）。纳米传感器芯片在塑料模块上生产，该塑料模块可以通过新颖的互连技术集成到其他DNA处理模块，以提供DNA样品处理管道的完全自动化。设想的DNA测序平台在以阵列形式配置时将产生约1 × 106个核苷酸碱基读数s-1，以完全自动化的方式处理整个样品，模块化流体系统的成本<200美元。流体系统的低成本不仅来自于使用复制技术来生产跨越多个尺寸尺度的流体网络，而且还来自于用于生产该芯片所需的纳米级组件的简单且高度并行的策略。 公共卫生相关性：设想了一种新型的单分子DNA测序系统，其利用模块化3D方法来处理输入DNA，其中每个模块跨越几个尺寸域（mm？nm）。一个模块是纳米传感器芯片，其由2D纳米通道组成，用于通过它们通过纳米通道的分子依赖性飞行时间来识别单个单核苷酸。使用单分子电导率测量来转换飞行时间，所述单分子电导率测量使用在纳米通道的输入端和输出端处保持平衡的纳米电极来测量。在此R21应用中，将证明测量单个单核苷酸的电导率响应的可行性。

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.