Label-Free Protein Arrays Based on Linear Dendron Macromolecular Layers and In-Situ Real Time EC-SPR-AFM Methods

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。[854979]蛋白质组学对理解基本生物现象以及疾病的检测和治疗作出了贡献。蛋白质微阵列为蛋白质组学的大量数据和定量研究提供了可能。然而，这些并没有优化用于实时研究蛋白质的基本和复杂特性。这与微阵列越来越多地用于酶表征、抗体特异性、了解基因功能和药物开发有关。在一种变革性的方法中，该提案将重点关注一系列新的分子，这些分子基于可电聚合的聚乙二醇线性树突层，用于蛋白质捕获，通过自组装Langmuir-Blodgett技术排列成一层，并在表面上形成图案。通过将电化学、表面等离子体共振和原子力显微镜这三种分析技术结合在一起，就有可能为程序化蛋白质组学研究提供实时和实时的分析方法，从而更好地理解蛋白质生化识别事件的物理、化学和电势特性。结合多仪器模式研究和蘸笔纳米级光刻技术，这将使蛋白质组学阵列研究从微米级到纳米级的前所未有的控制成为可能。知识价值。多种蛋白质生物标志物最广泛使用的方法包括质谱法、western blotting、凝胶电泳、脂质双层和免疫学分析。然而，这些技术都不适合分析大量样品或同时检测单个样品中的许多目标。微阵列的使用在很大程度上解决了这一问题，微阵列能够在疾病诊断和药物发现等领域进行大量蛋白质组学数据筛选。尽管蛋白质微阵列已被用于检测各种临床感兴趣的蛋白质，但在蛋白质特性、同步分析技术验证以及蛋白质构象功能研究中缺乏可变控制方面仍存在许多挑战。荧光成像、无标记表面等离子体共振成像和原子力显微镜方法已被用于这些研究。另一方面，用于探测蛋白质结构和活性的静电电位控制和电化学方法尚未与这些方法相结合。通过使用单一平台仪器设置，可以利用高定量表面分析技术的高数据阵列。通过使用自组装的电活性聚乙二醇线性树突，可以在不抑制稳定性的情况下限制吸附，控制系聚位点，指导取向和探针位点或蛋白质的聚类。广泛的影响。研究和筛选具有药理意义的常见蛋白质、酶、激素、食物蛋白和其他多肽，涉及基础科学和医学。虽然商业阵列和生物分析已经被报道，但最先进的蛋白质组学方法仍然存在挑战。如果成功，该项目将为阵列平台的改进研究和最终的设备商业化提供重大进展。这将提高以更快的速度检测和治疗疾病的能力。更重要的广泛影响是同时培养学生和研究人员在材料合成、表面分析、仪器开发和生物工程方面的专业知识。每个项目方面都为研究问题带来了独特的视角，并增强了批判性思维和技能发展。为此，两名研究生和一名本科生将由联合材料和生物工程项目的首席研究员直接培训和指导。研究结果将通过出版物、研讨会、会议报告和合作来公布。将与md安德森癌症中心、贝勒医学院、休斯顿大学和几家公司的研究人员合作，利用蛋白质组学的设备开发和应用。最后，在过去的15年里，研究者一直致力于向代表性不足的少数民族和女性学生提供服务，包括高中辅导，这是本项目将追求的优先事项。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).0854979AdvinculaProteomics has contributed to advances in understanding fundamental biological phenomena and the detection and curing of diseases. Protein microarrays enable large amounts of data and quantitative studies for proteomics. However, these are not optimized for investigating fundamental and complex properties of proteins in real time. This is especially relevant with the increasing use of microarrays for enzyme characterization, anti-body specificity, understanding gene function, and drug development. In a transformative approach, this proposal will focus on a new series of molecules based on electropolymerizable polyethyelene glycol linear dendron layers for protein capture arranged as a layer by self-assembly Langmuir-Blodgett techniques and patterned on a surface. By putting together three analytical techniques: electrochemistry, surface plasmon resonance, and atomic force microscopy in a single instrumental set-up, it is possible to have live and real-time analysis methods for programmed proteomic studies, giving greater understanding to the physical, chemical, and electrical potential properties of biochemical recognition events in proteins. Together with the combined multi-instrument mode studies and dip pen nanoscale lithography, this will enable unprecedented control for proteomics array studies from micron- to nano-scale.Intellectual Merit. The most widely used methods for multiple protein biomarkers include mass spectrometry, western blotting, gel electrophoresis, lipid bilayers, and immunological assays. However, none of these techniques are well-suited for the analysis of a large number of samples or the simultaneous detection of many targets within an individual sample. This has been largely addressed by the use of microarrays which enable high amounts of proteomic data screening in areas such as disease diagnosis and drug discovery. Although protein microarrays have been used to detect a variety of clinically interesting proteins, a number of challenges remain in terms of protein properties, verification by simultaneous analytical techniques, and the absence of variable control in protein conformation-function studies. Fluorescence imaging, and label-free surface plasmon resonance imaging and atomic force microscopy methods have been used for these studies. On the other hand, electrostatic potential control and electrochemical methods for probing protein structure and activity have not been combined with these methods. By using a single platform instrumental set-up, it will be possible to take advantage of the high data arraying with highly quantitative surface analytical techniques. By employing the self-assembled electroactive polyethyelene glycol linear dendrons, it will be possible to limit adsorption, control tethering sites, direct the orientation, and clustering of probe sites or proteins without inhibiting stability. Broad Impact. The study and screening of common proteins, enzymes, hormones, food proteins, and other peptides with pharmacological significance are relevant to fundamental science and medicine. While commercial arrays and bioassays have been reported, the challenges for state-of-the-art proteomics methods remain. If successful, this project will allow significant advances for improved research in array platforms and eventual commercialization of devices. This will improve the ability to detect and cure diseases at a faster rate. A more important broad impact is in the simultaneous training of students and researchers with expertise in materials synthesis, surface analysis, instrument development, and bioengineering. Each project aspect brings a unique perspective to research problems and enhances critical thinking and skills development. To this effect, two graduate students and an undergraduate student will be directly trained and mentored by the principal investigator in the combined materials and bioengineering program. Results will be made know through publications, seminars, presentations to conferences, and collaborations. Collaborations will be made with researchers at the M.D. Anderson Cancer Center, Baylor College of Medicine, University of Houston and several companies to take advantage of device developments and applications in proteomics. Lastly, the investigator has shown commitment for the last 15 years on outreach towards under-represented minority and women students including high school mentoring, a priority that will be pursued in this project.