This Small Business Innovation Research (SBIR) Phase I project proposes to construct a new microarray platform with high protein binding capacity that allows for enhanced fluorescence detection. Limitations of existing microarray surfaces include platform-based optical interferences and limited or ineffective binding capacity for biomolecules. These limit the ultimate sensitivity of binding reactions mounted on existing microarray surfaces. We will construct a composite or modified surface in two ways. First, by casting nitrocellulose (a polymer able to bind many different biomolecules essentially irreversibly) on an optically transparent porous track-etched membrane. We believe this new surface will maintain some properties of both starting materials. By varying the pore structure of the track-etched membrane, we will optimize the resulting membranes ability to capture complex protein mixtures and permit sensitive fluorescent detection. Secondly, we will directly modify the track-etched membrane with functional silanes to provide chemical groups permitting covalent coupling of proteins and nucleic acids. This approach should prove beneficial to maintain the optical compatibility of the original track-etched structure. This type of modified, optically transparent track-etched membrane may be optimal for antibody arrays where the capture molecule can be immobilized at a sufficient density to provide a sensitive assay surface.
The broader impact/commercial potential of this project will be to provide the basis for a new analytical tool that will allow establishment of antibody- and antigen-based assays of enhanced sensitivity. It also will provide an innovative surface to capture quantitatively the biochemical components of complex mixtures in such a way as to permit the detection of rare molecules. Microarrays play an increasingly important role in bioscience research, disease, and drug discovery processes as well as in human and animal diagnostics. They provide parallel processing tools required to extract multiple values from small amounts of precious clinical and research samples. Microarrays with enhanced sensitivity will permit the detection of rare biomolecules that may be involved in cellular regulation, cellular differentiation, and disease mechanisms. Reverse phase protein arrays (RPPA) are important for understanding cellular changes in a variety of disease states. In cancer, for example, lysates from small numbers of tumor cells can be spotted on a surface and then interrogated with many different antibodies to elucidate protein expression patterns in these tumor cell populations. The power of these techniques will be enhanced significantly by having a platform able to support the most sensitive assays.
This Small Business Innovation Research Phase I project demonstrated feasibility for creating thin film composite membranes with sufficient surface area and ability to bind bio-molecules (increased sensitivity) so they can participate in quantitative binding reactions (high binding capacity). These surfaces are appropriate for the immobilization of different cellular constituents for interrogation and are compatible with existing sensitive detection methods (fluorescence). The increased sensitivity and high binding capacity of this new surface will allow protein microarray technology to realize its full potential. It will enhance and support discovery of rare proteins that occur at levels too low for current technology to detect that are important in our understanding, diagnosis and treatment of human diseases. The surface will be incorporated into testing formats (such as protein microarrays) that will allow detection of many different proteins simultaneously at extremely low concentrations for use in determining and monitoring disease progress and response to treatment. The new surface also may be incorporated into standard analytical testing methods (immunoassays) to improve their sensitivity and expanding their use in the diagnosis and analysis of disease. In general, the new surface will significantly improve researchers’ ability to understand, detect and develop treatment strategies for complex diseases such as cancer. The broader impacts of this technology include its potential to enable personalized medicine, such as for cancer treatment. In one current method called "Reverse Phase Protein Microarrays," investigators will be able to use this new surface to detect very rare proteins in individual tumor cells. Measurement of protein expression is of extreme importance in understanding the type, metastatic state and potential drug response of individual tumors. This provides a key step in the diagnosis, prognosis and treatment of cancers at an individual level. In the future this may allow doctors to design individualized treatment schemes for cancer patients.
