Rapid Identification of Pathogenic Bacteria Based on Long-range SERS Microarray Biosensors

Yu 1159609开发用于快速鉴定具有高灵敏度和特异性的病原菌的生物传感器对于环境监测、生物医学诊断和国土安全是高度期望的。目前的检测方法，如细菌学培养、血清学和PCR测试，要么耗时，要么需要使用高端仪器和物种特异性抗体，这大大增加了它们向真实的世界应用的工程过渡的复杂性。基于表面增强拉曼光谱（Sers）技术的生物传感器与现有的检测方法相比具有许多优点，如无需试剂、多路复用以及在线和现场应用的潜力。然而，传统Sers是一种近场效应，严重限制了细菌的检测。智力优势：在这项工作中，PI挑战目前的病原菌检测方法，提出开发一种新的生物传感器平台，基于长距离Sers（LRSERS）的敏感检测和快速鉴定病原菌。LR-SERS的概念得到了基础物理学的支持，因为表面等离子体共振（SPR）可以通过金属薄膜的金属/电介质界面处的SPR的耦合而延伸到离金属表面很长的距离。本工作的目的是利用电磁场理论计算合理设计新型LR-SERS基底，并在此基础上开发微阵列生物传感器。首先将进行电磁时域有限差分（FDTD）计算，以便合理设计等离子体纳米结构，其具有远离金属表面至约50 nm的最强局部电场，该长度覆盖细菌细胞壁。随后，LR-SERS基底将通过电子束光刻（EBL）制造用于检测细菌。本项目开发的LRSRS微阵列生物传感器主要用于快速鉴定任何致病菌，由于海洋致病性副溶血弧菌是世界上引起海产品细菌性疾病的主要原因，因此我们将使用弧菌的分离株进行检测。收集并分析来自不同系统发生群和来源（环境和临床）的副溶血性弧菌分离株的LR-SERS光谱，以确定LR-SERS光谱特征峰与致病菌株编码外膜和细胞壁蛋白的基因的相关性。利用LR-SERS条形码技术和主成分分析技术，建立了一种快速鉴定混合菌群中致病性副溶血性弧菌的方法。最后，LR-SERS微阵列生物传感器将与微流体系统集成，用于原位、灵敏、高通量检测和鉴定病原菌。更广泛的影响：本项目开发的LR-SERS微阵列生物传感器提供了一种新的方法策略，例如，可以导致开发部署在海岸外的健康预警系统的生物传感器网络。该项目的成果将对分析化学、生物医学、药理学、法医学、食品安全、农业、生物燃料研究、环境监测和生物防御等相关领域产生重大影响。研究生、本科生和社区学院的学生将接受培训并参与这一高度跨学科的研究项目，特别是来自女性等代表性不足群体的学生。国际交换生的参与将促进国际合作。从这项工作中获得的知识将通过PI每年为课程提供的讲座以及通过UW纳米技术中心和西雅图太平洋科学中心建立的外展计划进行传播。

Yu1159609The development of biosensors for the rapid identification of pathogenic bacteria with high sensitivity and specificity is highly desired for environmental monitoring, biomedical diagnostics, and homeland security. Current detection methods, such as bacteriologic culture, serological, and PCR tests, are either time consuming or require the use of high-end instruments and species-specific antibodies, greatly complicating their engineering transition to real world applications. Biosensors based on surface-enhanced Raman spectroscopy (SERS) for direct detection and discrimination of bacteria offers many advantages over the current detection methods such as being reagent-less, multiplexing and the potential for on-line and infield applications. However, conventional SERS is a near-field effect which severely limits the detection of bacteria.Intellectual Merit: In this work, the PI challenge the current pathogenic bacteria detection methods by proposing to develop a new biosensor platform based on long-rang SERS (LRSERS) for the sensitive detection and rapid identification of pathogenic bacteria. The LR-SERS concept is supported by the fundamental physics in that the surface plasmon resonance (SPR) can be extended to a long distance from the metal surface by the coupling of SPRs at both metal/dielectric interfaces of a metal thin film. This work is aimed to rationally design fundamentally new LR-SERS substrates using theoretical electromagnetic calculations and to develop a microarray biosensor based on the LR-SERS platform. Electromagnetic finite-difference time-domain (FDTD) calculations will be performed first in order to rationally design the plasmonic nanostructures with the strongest local electric field away from the metal surface to ~ 50 nm, the length covering bacterial cell walls. Subsequently, the LR-SERS substrates will be fabricated via electron beam lithography (EBL) for the detection of bacteria. While the LRSERS microarray biosensor developed in this project is targeted for rapid identification of anypathogenic bacteria, we will use the isolates of vibrio species for testing because the pathogenicmarine bacterium V. parahaemolyticus is the leading cause of seafood-borne bacterial illness in the word. The LR-SERS spectra of isolates of V. parahaemolyticus from different phylogenetic groups and sources (environment and clinical) will be collected and analyzed to determine the correlation of LR-SERS spectral characteristic peaks to the gene encoding outer membrane andcell wall proteins of pathogenic strains. A method for the quick identification of pathogenic V.parahaemolyticus from a mixture of bacteria strains will be established using LR-SERS barcode and principle component analysis (PCA). And finally, LR-SERS microarray biosensors will be integrated with a microfluidic system for the in-situ, sensitive, and high-throughput detection and identification of pathogenic bacteria.Broader Impact: The LR-SERS microarray biosensors developed in this project provide a new approach strategy that can lead to, for example, the development of biosensor networks deployed off-coast for health early warning systems. Results from this project will make significant impact on the related fields such as analytical chemistry, biomedicine, pharmacology, forensics, food safety, agriculture, bio-fuel research, environmental monitoring, and bio-defense.Graduate, undergraduate, and community college students will receive training and participate in this highly interdisciplinary research project, especially students from underrepresented groups such as female. The participation of international exchange students will promote the international collaborations. The knowledge gained from this work will be disseminated through the lectures the PI provides for the courses every year and via outreach programs established by the UW Center for Nanotechnology and the Seattle Pacific Science Center.