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。蛋白质微阵列使蛋白质组学的大量数据和定量研究成为可能。然而，对于实时研究蛋白质的基本和复杂性质来说，这些并不是最优化的。这与越来越多的微阵列用于酶特性、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，这项提议将专注于一系列新的分子，这些分子基于可电聚合的聚乙二醇线性树枝状分子层，用于捕获蛋白质，通过自组装朗缪尔-布洛杰特技术排列为一层，并在表面图案化。通过将三种分析技术：电化学、表面等离子体共振和原子力显微镜结合在一起，有可能拥有实时的分析方法来进行程序化蛋白质组研究，从而更好地了解蛋白质中生化识别事件的物理、化学和电势性质。再加上多仪器模式研究和浸笔纳米级光刻的结合，这将使蛋白质组阵列研究从微米到纳米级的研究获得前所未有的控制。最广泛使用的多个蛋白质生物标志物的方法包括质谱学、蛋白质印迹、凝胶电泳法、脂双层和免疫学分析。然而，这些技术都不能很好地适用于大量样本的分析或单个样本中多个目标的同时检测。这在很大程度上是通过使用微阵列来解决的，它使得在疾病诊断和药物发现等领域能够进行大量的蛋白质组数据筛选。虽然蛋白质微阵列已被用于检测各种临床上感兴趣的蛋白质，但在蛋白质性质、同时分析技术的验证以及蛋白质构象-功能研究中缺乏变量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，将有可能利用高数据排列和高定量表面分析技术。通过使用自组装的电活性聚乙二醇线性树枝状分子，可以限制吸附，控制系链位置，指导探针或蛋白质的定向和聚集，而不会抑制稳定性。广泛的影响。研究和筛选常见的蛋白质、酶、激素、食品蛋白和其他具有药理意义的多肽，涉及基础科学和医学。虽然已经报道了商业阵列和生物检测，但最先进的蛋白质组学方法仍然面临挑战。如果成功，该项目将在改进阵列平台的研究和最终实现设备商业化方面取得重大进展。这将提高以更快的速度发现和治愈疾病的能力。更重要的广泛影响是同时培养学生和具有材料合成、表面分析、仪器开发和生物工程专业知识的研究人员。每个项目方面都带来了研究问题的独特视角，并加强了批判性思维和技能的发展。为此，两名研究生和一名本科生将直接接受联合材料和生物工程专业首席研究员的培训和指导。结果将通过出版物、研讨会、在会议上的陈述和合作来公布。将与M.D.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，以利用蛋白质组学中的设备开发和应用。最后，调查员在过去15年中一直致力于接触代表不足的少数群体和女学生，包括高中辅导，这是本项目将追求的一个优先事项。

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.