Rapid, Binary-Mode Nucleic Acid Sequence Detection

技术摘要特定核酸(NA)序列的检测在传染病诊断、遗传病基因筛查、个性化医学、犯罪学、亲子鉴定和动植物育种等许多应用中都是至关重要的。尽管聚合酶链式反应(PCR)技术与寡核苷酸阵列芯片相结合，能够以极低的拷贝数对样本中存在的多个序列进行特异性鉴定，但这种方法需要额外的标记和试剂来检测目标NA。这些标签和检测它们所需的硬件(例如，光学设备)显著增加了用于检测特定NA序列的设备的分析时间、成本和占用空间。作为目前方法的替代，该项目将开发能够以单分子分辨率检测致病核酸的新型纳米孔。这些纳米孔将在硅中制造，并将被改造成含有标记有互补NA序列的珠子。靶向寡核苷酸与微球的结合将导致凝胶性质的改变，并将导致纳米孔被微球遮挡。对于目标NA的二进制是/否读数，将以电化学方式检测该事件。非技术摘要需要开发简单且廉价的方法来检测医疗或食品安全应用中的病原体。这项提议将开发一种基于含有珠子的小孔的新方法。这些珠子会与病原体生物标志物发生反应，堵塞毛孔，并导致电流的变化。这种方法被认为比目前的方法具有更高的灵敏度和更简单的方法。

Technical abstractDetection of specific nucleic acid (NA) sequences is crucial to infectious diseasediagnostics, genetic screening for hereditary diseases, personalized medicine, criminology,paternity disputes, and animal and plant breeding, among many other applications. Althoughpolymerase chain reaction (PCR) technology, coupled with oligonucleotide array chips, hasenabled specific identification of multiple sequences present in a sample at very low copynumber, this method requires added labels and reagents to detect the target NA. These labelsand the hardware (e.g., optics) required to sense them significantly increase the analysis time,cost, and footprint of devices for detecting specific NA sequences. As an alternative to current approaches, this project will develop novel nanopores capable of detecting pathogenic nucleic acids with single molecule resolution. These nanopores will be fabricated in silicon and will be engineered to contain beads labeled with complementary NA sequences. Binding of target oligonucleotides to the beads will result in the change of electrophoretic properties and will lead to nanopores becoming occluded by the beads. This event will be detected electrochemically for binary yes/no readout of the target NA. Non-technical abstractThere is a need to develop simple and inexpensive methods for detecting pathogens for medical or food safety applications. This proposal will develop a new approach based on small pores containing beads. The beads will react to pathogen biomarkers, will clog the pores and will result in the change of electrical current. This method is envisioned to have improved sensitivity and be simpler than the current approaches.