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。0854979 AdvinculaProteomics有助于了解基本生物现象和疾病的检测和治疗的进步。蛋白质微阵列使大量的数据和定量研究的蛋白质组学。然而，这些并不是最佳的研究基本和复杂的性质的蛋白质在真实的时间。这与微阵列在酶表征、抗体特异性、理解基因功能和药物开发中的越来越多的使用特别相关。 在一种变革性的方法中，该提案将专注于一系列新的分子，这些分子基于用于蛋白质捕获的电可聚合聚乙二醇线性树枝化层，通过自组装Langmuir-Blodgett技术排列成一层，并在表面上形成图案。通过将三种分析技术：电化学，表面等离子体共振和原子力显微镜放在一个单一的仪器设置中，可以为程序化蛋白质组学研究提供实时和实时的分析方法，从而更好地了解蛋白质中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，这将使前所未有的控制蛋白质组学阵列研究从微米到纳米级。用于多种蛋白质生物标志物的最广泛使用的方法包括质谱法、蛋白质印迹法、凝胶电泳法、脂质双层法和免疫学测定法。然而，这些技术中没有一种非常适合于分析大量样品或同时检测单个样品中的许多靶标。这在很大程度上是通过使用微阵列来解决的，微阵列能够在疾病诊断和药物发现等领域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，一些挑战仍然存在的蛋白质性质，同时分析技术的验证，以及在蛋白质构象-功能研究中缺乏变量控制。荧光成像，和无标记的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势控制和电化学方法尚未与这些方法相结合。通过使用单一平台的仪器设置，将有可能利用高数据阵列与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化基元，将有可能限制吸附、控制束缚位点、引导取向以及探针位点或蛋白质的聚集而不抑制稳定性。广泛的影响。研究和筛选具有药理学意义的常见蛋白质、酶、激素、食物蛋白质和其他肽与基础科学和医学有关。虽然已经报道了商业阵列和生物测定，但最先进的蛋白质组学方法仍然面临挑战。如果成功，该项目将为改善阵列平台的研究和设备的最终商业化带来重大进展。这将提高以更快的速度检测和治疗疾病的能力。一个更重要的广泛影响是在材料合成，表面分析，仪器开发和生物工程专业知识的学生和研究人员的同时培训。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在材料和生物工程相结合的项目中直接接受首席研究员的培训和指导。结果将通过出版物、研讨会、会议介绍和合作进行宣传。将与医学博士的研究人员合作。安德森癌症中心、贝勒医学院、休斯顿大学和几家公司利用设备开发和蛋白质组学应用。最后，调查员在过去15年里致力于向代表性不足的少数民族学生和女学生开展外联活动，包括高中辅导，这是本项目将继续开展的一个优先事项。

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.