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

University Of Houston, Houston TX

Investigators

Abstract

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). 0854979 Advincula Proteomics 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.