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

摘要 目前生产纳米级器件的途径需要高端纳米制造技术，例如聚焦纳米技术。 离子束研磨或电子束光刻结合无机衬底的使用。尽管他们 由于独特的操作特性，纳米流体装置难以用于一次性应用， 用于体外诊断。构思了新颖的制造策略，其将允许产生 使用大规模生产方式由热塑性塑料制成的纳米流体装置， 成本和严格的合规性，适用于一次性应用。所设想的装置将采用 利用固相分子测定进行单分子鉴定和/或定量， 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）， 与其他微机械加工技术相结合，以产生混合规模的结构。利用先进的 专门为基于热塑性塑料的纳米流体平台、封闭设备定制的组装/粘合工艺 可以获得高产率。利用这些纳米纤维技术，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获得精确的定量数据， 使用非光学读出模式。该传感器由具有锥形2D的纳米通道飞行管组成 合成孔（开口<10 nm），允许通过其特征识别分子实体 通过聚合物纳米通道的飞行时间使用瞬时电流阻断事件确定，而不使用 需要构建面内和纳米间隙电极。该传感器还将包括微尺度结构， 对于固相分子反应，可以产生序列变异的独特分子特征， 已经从循环标记物如生物细胞、无细胞DNA和DNA中收获的DNA和RNA， 外来体深入了解聚合物纳米通道特定的单分子行为， 聚合物固相反应器至关重要，将通过实验进行广泛评估， 仿真传感器可以在4”晶片上形成图案，以提供进行高通量处理的能力 以高特异性和灵敏度搜索罕见的分子事件。晶圆级生产 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的基因组DNA中寻找点突变， 通过其mRNA货物的表达差异或确定外泌体的甲基化状态， 从癌症患者身上分离的游离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.