Tools and Methods for Producing High Quality Functional Protein Microarrays for Biomarker Discovery

High-density protein arrays are critical to identifying biomarkers for the early detection of disease, confirming specificity of commercial antibodies, identifying new protein-protein interactions, and more. Yet, access to these arrays is still hindered by high cost, largely as a result of the high cost of individual proteins that need to be pre- purified for printing. To facilitate access of protein arrays to the research community, INanoBio developed a functioning semi-automated protein array production instrument that utilizes lower cost DNA arrays to express proteins in situ, which are immediately covalently captured on a corresponding slide. The end product is a protein array without DNA or expression reagents that is immediately ready for assay. In Phase I, we met all milestones aimed at demonstrating system functionality, discrete protein capture, and functionality of captured proteins; we also identified sources of variation that will be addressed in this proposal. The ProCap system was proven to produce its capacity of four protein arrays simultaneously with over 10,000 discrete proteins per array. The proteins captured on the array were shown to maintain known protein-protein interactions, enzymatic activity, and substrate activity. In this proposal, we aim to develop a market-ready, user-friendly benchtop version of ProCap called Genetein (genes to proteins). This instrument will be more compact, operate by intuitive software, and most importantly will come pre-loaded with automation protocols for processing arrays on- instrument to add post-translational modifications and/or probe with samples for biomarker discovery and other assays. A key feature added to ProCap outside of the Phase I proposal was the ability to use different capture slide substrates, adding gold biosensor slides as an option for capture and ultimately enabling the user to investigate proteins in high throughput via surface plasmon resonance imaging. This feature will be included as part of Genetein . Ultimately, INanoBio and external partners will build, test, field-test, improve, and validate Genetein to ensure functionality, usability, and reproducibility. The outcome of the proposed work will be a ready-to-manufacture Genetein instrument for sale to customers, customized and off- the-shelf protein arrays produced on Genetein by INanoBio for shipping to customers, and a full-service option to allow customers to ship their samples to INanoBio for processing on Genetein arrays.