This Small Business Innovation Research (SBIR) Phase II project will provide an optimized composite polymer protein binding surface for proteomics applications. The new surface will be specifically designed for reverse phase protein microarrays to enable detection of rare molecules in complex biological mixtures. Discovery and quantification of rare molecules in complex mixtures is essential to improve the understanding of disease mechanism and progression, and responses to treatment regimes. Current surfaces used in these applications have properties that exhibit limited sensitivity of detection due to optical interferences, low protein binding and accumulation of nonspecific interactions. This project will optimize and introduce the application of a new track etched , nitrocellulose composite membrane for protein array applications. Manufacturing processes for the new composite will be developed to generate multiple forms of the composite to allow it to be incorporated into a variety of binding assay formats. This effort will also shed light on important properties for generating ultrasensitive binding surfaces. The result of this project will be an optimized composite membrane with characteristics and manufacturability suited for the most sensitive binding applications, such as reverse phase protein arrays. The platform initially will be optimized for fluorescent detection of rare molecules in complex cell lysates.
The broader impact/commercial potential of this project will be to provide a family of discovery and diagnostic tools that will expand the understanding, detection and treatment of human disease. The current focus in translational medicine for therapies in clinical trials is to identify expression patterns of proteins (biomarkers) in individual patients. These measurements allow the monitoring and understanding of individualized disease progression and responses to treatment. They will provide the data necessary to create targeted, personalized treatment regimens. Protein arrays have found utility over the past decade as research tools that provide multiplexed detection and quantification of protein expression. However, the full potential of these tools as diagnostic platforms that provide patient-specific information and guide drug treatment has not been realized due to insufficient binding capacity, limited dynamic range and poor sensitivity. This project defines a new composite surface that has a significant increase in both binding capacity and sensitivity when incorporated into multiplexed immunoassay systems. The composite can be included in a variety of platforms to enhance discovery and quantification of important markers on an individual scale as well as high throughput systems for broad diagnostic application.
This NSF Phase II SBIR grant funded the development of a new microporous material able to support highly sensitive detection of rare molecules in complex biological mixtures. We developed this material by creating a composite membrane which was a combination of polyethylene terephtalate track etched membrane (TEM) and nitrocellulose (NC). The composite, which we call composite reaction surface (CRS™), has a thin coating of NC on a TEM support. We successfully developed a method for coating NC onto TEM in a continuous roll format. Optimal thickness of coatings and porosity were determined to result in a product that can be used in numerous convenient product configurations. When used as a support for immunodetection of rare proteins in cell lysates, such as done with protein microarrays, CRS exhibits high protein binding capacity, good optical qualities and will support a more sensitive assay than existing nitrocellulose-coated surfaces. Prototype results indicate that CRS attached to glass slides supports highly sensitive protein binding and detection, with higher signal to noise ratios than nitrocellulose-coated slides commonly used for protein microarray analysis. The continuous roll format allows use of CRS in a variety of experimental and test devices. For instance we have heat sealed the material into multiwell plates, a common multiplexing research and test format. The flow-through capability provided by the porous nature of the TEM support will accommodate rapid test designs that require wash steps or reagent changes. CRS has been shared in roll format with several investigators for specific applications and optimization. We expect to launch CRS in roll format in the fall of 2014 so that it will be widely available for further applications development. We are currently working with a diagnostics company to incorporate CRS into their multiplexed test system. The sensitivity of detection on this surface and the flexibility of the material from a handling and manufacturability standpoint will make this, and other applications, successful. Advanced materials such as CRS will provide a new generation of immunoreactive surfaces that push forward the boundaries of sensitive detection of rare molecules in complex biological samples such as blood or tumor cells. CRS can be employed in such techniques as reverse phase protein arrays, a multiplexed technique that quanitates protein expression in cell lysates. With more sensitive detection comes the ability to detect molecules in smaller samples, an important consideration for many diagnostic tests including those for cancer, and affords development of quantitative analysis tools. CRS, with is flexibility in format and high sensitivity, will help proteomics advance our understanding of disease, response to treatment and eventually diagnose disease at the individual’s level.
