Biotechnology Resource Center of Biomodular Multi scale Systems CBM2 for Precision Molecular Diagnostics

摘要 目前制造纳米级设备的路线需要高端纳米制造技术，如聚焦 离子束研磨或电子束光刻与无机衬底的使用相结合。尽管他们 独特的操作特性，纳米流体设备很难用于一次性应用，因为 体外诊断所需的。构思了新的制造策略，将允许产生 由热塑性塑料制成的纳米流体器件采用大规模生产模式，以低成本获得器件- 成本和严格的合规性，适合一次性使用的应用。设想的设备将采用 单分子鉴定和/或定量利用固相分子分析，可以 使用同一平台查询DNA和RNA分子中的各种序列变异 配置。制造策略将采用高通量纳米压印光刻(NIL)，用于 与其他微加工技术相结合，制造出混合尺度结构。利用先进的 专门为基于热塑性塑料的纳米流体平台、封闭式设备量身定做的组装/结合工艺 具有较高的收益率可以实现。利用这些纳米制造技术，一种新型的传感平台将被 探索可以利用单分子数字计数来保护精致的定量数据， 使用非光学读出方式。该传感器由具有锥形2D的纳米通道飞行管组成 合成孔(开放10 nm)，允许通过它们的特征来识别分子实体 通过聚合物纳米通道的飞行时间使用瞬时电流阻塞事件确定 需要建立平面内和纳米间隙电极。传感器还将由微尺度结构组成，以允许 对于能够生成序列变化的独特分子签名的固相分子反应 从循环标记中获得的DNA和RNA，如生物细胞、游离DNA和 外显体。深入了解聚合物纳米通道特有的单分子行为和 聚合物固相反应器是关键，将通过实验和 模拟。该传感器可以在4英寸晶片上形成图案，以提供进行高吞吐量处理的能力 寻找罕见的分子事件，并且具有高度的特异性和敏感性。晶片规模的生产 传感器将允许在许多有趣的生物医学应用中使用这些引人注目的设备 例如在从循环肿瘤细胞中分离基因组DNA中寻找点突变、诊断中风 通过它们的mRNA片段的表达差异或确定其甲基化状态来从外体中分离出来 从癌症患者体内提取的游离DNA。

Abstract Current routes for producing nanoscale devices require high-end nanofabrication techniques, such as focused ion beam milling or electron beam lithography coupled with the use of inorganic substrates. In spite for their unique operational characteristics, nanofluidic devices are difficult to be utilized for single-use applications as required for in vitro diagnostics. Novel fabrication strategies are conceived that will allow for the generation of nanofluidic devices made from thermoplastics using high-scale production modalities that yield devices at low- cost and with tight compliance, appropriate for single-use applications. The devices envisioned will employ single-molecule identification and/or quantification taking advantage of solid-phase molecular assays that can query for a variety of sequence variations in both DNA and RNA molecules using the same platform configuration. The fabrication strategy will employ high throughput nanoimprint lithography (NIL) used in combination with other micromachining techniques to produce mixed-scale structures. Utilizing an advanced assembly/bonding process specifically tailored for thermoplastic-based nanofluidic platforms, enclosed devices with high yield rates can be achieved. Using these nanofabrication techniques, a novel sensing platform will be explored that can take advantage of single-molecule digital counting to secure exquisite quantitative data that uses a non-optical readout modality. The sensor consists of a nanochannel flight tube with tapered 2D synthetic pores (opening <10 nm), which allows for identification of molecular entities via their characteristic flight time through a polymer nanochannel determined using transient current blockage events without the need to build in-plane and nano-gap electrodes. The sensor will also consist of microscale structures to allow for solid-phase molecular reactions that can generate unique molecular signatures of sequence variations in DNA and RNA that have been harvested from circulating markers such as biological cells, cell free DNA and exosomes. An in-depth understanding of single molecule behavior specific to polymer nanochannels and polymer solid-phase reactors is critical and will be extensively evaluated through experimentation and simulation. The sensor can be patterned over 4” wafers to provide the ability to do high throughput processing to search for rare molecular events and do so with high specificity and sensitivity. Wafer-scale production of the sensors will allow for using these compelling devices in a number of interesting biomedical applications such as searching for point mutations in genomic DNA isolated from circulating tumor cells, diagnosing stroke from exosomes through expression differences in their mRNA cargo or determining the methylation status of cell free DNA isolated from cancer patients.