Development of aptamer nanosensors for detection of Staphylococcus aureus

基于纳米颗粒的检测方法在体外和体内病原体检测中越来越受到关注，因为它们在高灵敏度和快速的现场监测中具有潜在的实用性。近年来，核酸适体寡核苷酸链以高亲和力和选择性结合生物靶标，已被提出作为生物分子、药物和全细胞的替代识别元件。尽管适体功能化纳米材料在检测这些和其他分析物方面显示出巨大的效用，但使用适体功能化纳米材料检测完整病原体的报道很少。本项目将制备适配体功能化的金纳米粒子Apt AuNPs，用于金黄色葡萄球菌作为一种备受关注的新型环境病原体的定量分析。PI的假设是，将适体赋予的特异性与通过表面等离子体促进信号转导实现的灵敏度相结合，将产生强大的传感器平台，并易于转化为现场应用。为此，他们提出了一种Apt-AuNP构建体的设计，使其敏感性和特异性最大化。值得注意的是，过去的研究表明，适体功能化纳米颗粒在传感器应用中的潜在基本适用性，并不一定产生完全保留适体特异性的纳米颗粒。特别是，PI认为适体结合密度通常不被考虑，即使已经确定适体构象必须改变以响应识别事件。他们提出了一种系统的方法来评估表面密度对适体特异性的作用。本文确定了四个研究任务：任务1：产生并表征具有不同适配体身份和表面密度的Apt-AuNP。任务2：将使用比色筛选法评估Apt-AuNP的特异性。任务3：使用表面增强拉曼光谱法测定Apt-AuNP的灵敏度。任务4：将使用便携式拉曼分光光度计评估Apt-AuNP的现场能力。这个项目是新颖的，因为它将是第一个开发适合体功能化纳米粒子用于病原体检测。在此之前，纳米材料的病原体生物传感器几乎完全依赖于抗体来提供检测特异性。PI设计Apt-AuNP颗粒的策略采用了一种基本方法，该方法将系统地考虑Apt-AuNP结构的不同成分如何影响适体敏感性。研究人员认为，这种基于系统工程科学的方法将被其他人在设计适体功能化纳米粒子时所模仿，因此具有重大的变革潜力。耐甲氧西林金黄色葡萄球菌MRSA是在美国以及世界范围内越来越多的致命疾病爆发的病原体。虽然这种生物历来与医院有关，但最近，MRSA的环境爆发以及在废水中检测到的MRSA使MRSA成为一种令人关注的新兴环境病原体。不幸的是，现有的MRSA和其他金黄色葡萄球菌菌株检测方案是缓慢的，不容易转化为现场应用。提出的生物传感器将解决全球对改进金黄色葡萄球菌检测的需求，同时为未来开发其他基于适体的探针提供框架。除了对纳米技术环境健康和安全方面的迫切研究需求作出贡献之外，该项目还将建立在现有的教育推广组成部分——纳米技术教育和环境推广NEEO计划——的基础上，该计划旨在培训下一代环境专业人员了解和量化新兴技术对人类和环境健康的影响。在这个项目中，弗吉尼亚理工大学环境生物纳米技术实验室研究团队的成员正在与西弗吉尼亚公共教育联盟合作，开发一系列外部资助的实习机会，使中学教育工作者和学生能够跟随研究团队，学习观察生物和环境界面发生的纳米级现象的新方法。这些实习旨在为教育工作者提供开发新技术课程的机会，并为学生提供培训和专业准备。

PI:VikeslandProposal Number:1133746Nanoparticle based assays are of growing interest for the in vitro and in vivo detection of pathogens because of their potential utility in highly sensitive and rapid field monitoring. Recently aptamers oligonucleotide strands that bind to biological targets with high affinity and selectivity, have been proposed as alternative recognition elements for biomolecules, pharmaceuticals, and whole cells. Although aptamer functionalized nanomaterials have shown great utility for detection of these and other analytes there are few reports of the use of aptamer-functionalized nanoparticles for intact pathogen detection. This project will produce aptamer functionalized gold nanoparticles Apt AuNPs for quantification of Staphylococcus aureus as a model emerging environmental pathogen of concern. It is the PI's hypothesis that coupling of the specificity imparted by an aptamer with the sensitivity achieved via surface plasmon facilitated signal transduction will produce sensor platforms that will be robust and readily translatable to field applications. To this end, they have proposed a design for Apt-AuNP constructs such that their sensitivity and specificity is maximized. It is noted that past studies illustrating the underlying fundamental applicability of aptamer functionalized nanoparticles for sensor applications have not necessarily produced nanoparticles that fully retain the specificity of the aptamer. In particular the PI contends that aptamer binding density is often not considered, even though it is well established that aptamer conformation must change in response to a recognition event. They propose a systematic approach to evaluate the role of surface density on aptamer specificity. Four research tasks have been identified: Task 1: Apt-AuNP of varying aptamer identity and surface density will be produced and characterized. Task 2: Apt-AuNP specificity will be assessed using a colorimetric screening assay. Task 3: Apt-AuNP sensitivity will be determined using a surface enhanced Raman spectroscopic assay. Task 4: The field capabilities of Apt-AuNP will be evaluated using a portable Raman spectrophotometer. This project is novel in that it will be the first to develop an aptamer functionalized nanoparticle for pathogen detection. Prior to this effort, nanomaterial enabled biosensors for pathogens have relied almost exclusively on antibodies to provide assay specificity. The PI's strategy for the design of the Apt-AuNP particles utilizes a fundamental approach that will systematically consider how the different components of the Apt-AuNP construct affect aptamer sensitivity. The researchers believe this systematic engineering-science based approach will be emulated by others when they undertake the design of aptamer functionalized nanoparticles and as such has significant transformative potential.Methicillin resistant S. aureus MRSA is the causative agent for a growing number of deadly disease outbreaks both within the United States as well as worldwide. Although this organism is historically associated with hospitals, recently, environmental outbreaks of MRSA as well as its detection in wastewater effluent have inaugurated MRSA as an emerging environmental pathogen of concern. Unfortunately existing protocols for detection of MRSA and other S. aureus strains are slow and not easily translatable to field applications. The proposed biosensor will address the global need for improved S. aureus detection specifically while providing a framework for the development of other aptamer based probes in the future. Beyond a contribution to pressing research needs in nanotechnology environmental health andsafety, the project will build upon an existing educational outreach component, the Nanotechnology Educational and Environmental Outreach NEEO Program, that has been designed to train the next generation of environmental professionals in understanding and quantifying the effects of emerging technologies on human and environmental health. In this program, members of the Virginia Tech Environmental BioNanotechnology Laboratory research team are working in partnership with the Western Virginia Public Education Consortium to develop a series of extramurally funded internships that enable middle school educators and students to shadow the research team and learn about new methods for observing nanoscale phenomena occurring at biological and environmental interfaces. These internships are intended to provide opportunities for the development of new technology inspired curricula for educators as well as training and professional preparation for students.