Integrated Nano-Opto-Fluidic System on Sapphire towards Single-Molecule Protein Sequencing

蓝宝石上的集成纳米光流控系统用于单分子蛋白质测序

• 批准号: 10473301
• 负责人: Chao Wang
• 金额: $ 133.95万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10473301
• 关键词: Address; Amino Acid Sequence; Amino Acids; Biology; Cells; Classification; Complex; DNA-Protein Interaction; Detection; Development; Device Designs; Devices; Diagnostic; Electric Capacitance; Face; Future; Genes; Genome Mappings; Genomics; Genotype; Heterogeneity; Individual; Knowledge; Liquid substance; Masks; Mass Spectrum Analysis; Measurement; Medicine; Methods; Microscopy; Nanostructures; Noise; Nucleic acid sequencing; Optics; Peptide Sequence Determination; Peptides; Polymerase Chain Reaction; Protein Fingerprints; Proteins; Proteomics; Research; Sapphire; Signal Transduction; Speed; Structure; System; Technology; Therapeutic; base; clinical application; cost; deep learning algorithm; design; fluorescence microscope; improved; integrated circuit; method development; multiple omics; nano; nanofabrication; nanophotonic; nanopore; next generation; portability; sensor; single molecule; transcriptomics; waveguide

PROJECT SUMMARY/ ABSTRACT Enabled by the development of high-throughput, low-cost nucleic acids sequencing technologies, there have been accelerated development in genomics and transcriptomics in the past two decades, profoundly reshaping our knowledge in biology and medicine. However, similar technologies have yet to emerge for rapid identification and quantification of proteins. This is attributed to more complex structures of proteins, lack of polymerase chain reaction-like amplification methods, cellular heterogeneity, etc. Conventional protein sequencing methods, such as Edman degradation and mass spectrometry, are slow, expensive, and not suitable for detecting low- abundance proteins. Such ensemble measurements also can mask our fundamental understandings on how cells of a particular genotype function and respond to therapeutics. Single-molecule protein sequencing (SMPS) is an emerging research direction that directly reads amino acids sequence from individual protein or peptide molecules. Yet, a promising strategy using fluorosequencing still relies on long Edman degradation cycles and bulky fluorescent microscopes, not ideal for fast and low-cost readout. Electronic SMPS technologies using tunneling or nanopore sensors are emerging methods for development of portable and inexpensive sequencing systems. However, they still face challenges in precise nanofabrication, structural instability, high electronic noise, and inability in detection of all amino acids. To address the multi-faceted challenges in next-generation SMPS, we will design an on-chip integrated, electronic system that incorporates nano-opto-fluidic structures to transduce protein fingerprints into electronic signals at a high speed, a low cost and a small system foot-print. Our platform features an all-sapphire nanopore (AlSaPore) fluidic device that has a small capacitance and a greatly improved structural stability, and accordingly suited for high-speed, low-noise, high-throughput, electronic detection. Further, an ultrathin nitride-based metasurface-integrated circuit (MIC) structure is created on the AlSaPore to optically interrogate the fluorescently tagged single amino acids passing through the nanopore without conventional fluorescent microscopy. Subsequently, the fluorescent tag signals are transmitted through the waveguide and collected by on-chip integrated photodetectors. The optoelectronic channel (IA for amino acids tags) and ionic current channel (IP for protein primary structure) will be synchronously recorded, classified by deep learning algorithms, and used in combination to improve the protein sequencing accuracy. Supported by well-established nitride-on-sapphire device design and manufacturing technology, our MIC-AlSaPore is a scalable and compact platform that achieves single-molecule sensitivity with a potential to read out all 20 amino acids. The development of MIC-AlSaPore platform will have far-reaching impact in biomedicine beyond protein sequencing. It may be used for studying DNA-protein interactions at single-molecule levels, classification of specific genes, genome mapping and de novo assembly. Additionally, it may inspire future multi-omic (genomic, transcriptomic, and proteomic) diagnostic solutions with potential clinical applications.

项目概要/摘要 随着高通量、低成本核酸测序技术的发展， 过去二十年基因组学和转录组学加速发展，深刻重塑 我们在生物学和医学方面的知识。然而，类似的快速识别技术尚未出现 和蛋白质的定量。这是由于蛋白质结构更复杂，缺乏聚合酶链 类反应扩增方法、细胞异质性等。传统的蛋白质测序方法，如 埃德曼降解法和质谱法速度慢、成本高，并且不适合检测低浓度 丰富的蛋白质。这种集合测量也可能掩盖我们对如何进行的基本理解 特定基因型的细胞发挥功能并对治疗做出反应。单分子蛋白质测序 (SMPS) 是一个新兴的研究方向，直接读取单个蛋白质或肽的氨基酸序列 分子。然而，使用荧光测序的有前途的策略仍然依赖于长埃德曼降解周期和 笨重的荧光显微镜，不适合快速和低成本的读出。电子 SMPS 技术使用 隧道或纳米孔传感器是开发便携式且廉价测序的新兴方法 系统。然而，他们仍然面临着精密纳米加工、结构不稳定性、高电子性等方面的挑战。 噪音，并且无法检测所有氨基酸。应对下一代的多方面挑战 SMPS，我们将设计一个片上集成电子系统，该系统结合了纳米光流控结构 以高速、低成本和小系统占用空间将蛋白质指纹转换为电子信号。 我们的平台采用全蓝宝石纳米孔 (AlSaPore) 流体装置，该装置具有小电容和 大大提高了结构稳定性，适合高速、低噪声、高通量、电子 检测。此外，在超薄氮化物超表面集成电路（MIC）结构上创建了 AlSaPore 以光学方式询问穿过纳米孔的荧光标记的单个氨基酸 无需传统的荧光显微镜。随后，荧光标签信号通过 波导并由片上集成光电探测器收集。光电通道（氨基酸IA 标签）和离子电流通道（蛋白质一级结构的IP）将被同步记录，分类为 深度学习算法，并结合使用，提高蛋白质测序的准确性。支持者 我们的 MIC-AlSaPore 拥有完善的蓝宝石氮化物器件设计和制造技术 可扩展且紧凑的平台，可实现单分子灵敏度，并有可能读出所有 20 个氨基 酸。 MIC-AlSaPore平台的开发将对蛋白质以外的生物医药产生深远影响 测序。它可用于研究单分子水平上的 DNA-蛋白质相互作用、分类 特定基因、基因组作图和从头组装。此外，它可能会激发未来的多组学（基因组、 具有潜在临床应用的转录组学和蛋白质组学）诊断解决方案。