SBIR-STTR Award

Homocysteine is a sulfur-containing amino acid and a metabolite of methionine metabolism. Recent clinical studies have strongly suggested that an elevated level of homocysteine in blood is a risk factor for cerebrovascular, peripheral vascular and coronary heart diseases. However, the exact role that homocysteine plays in these diseases. is not well understood. In addition the prognostic value of plasma homocysteine concentrations for cardiovascular diseases has not yet been validated. In order to expedite the ongoing effort to better understand the role that homocysteine and other endogenous sulfhydryls and disulfides play in cardiovascular diseases, the development of a simple, reliable, high throughput and cost effective assay for the determination of homocysteine and other biological important thiols in plasma is essential. The objective of the proposed work is the development of a fast, sensitive and selective clinical assay for the determination of homocysteine and other endogenous thiols and disulfides in plasma. This will be accomplished using capillary electrophoresis with electrochemical detection. This assay should offer attractive features such as simpler sample processing (since native thiols are detected), high sensitivity, specificity, portability and the possibility of high sample throughput. The resulting methodology will provide clinical scientists with a simple and reliable assay that can be used to evaluate the role of homocysteine in the management of heart disease and can eventually be used for routine clinical testing in doctor's offices. PROPOSED COMMERCIAL APPLICATIONS: As the outcome from this research, we will develop two marketable devices for the rapid determination of homocysteine in plasma. The first will be a hand held portable analyzer suitable for use in the clinic or hospital. The second device, automated high throughput analyzer, will consist of a multichannel device compatible with existing 96-well microtiter plate formats. Automation by means of robotics should increase sample throughput by 8-fold compared to liquid chromatography based methods

The objective of the proposed work is fast, sensitive integrated clinical analyzers for the diagnosis of two metabolism disorders, homocysteinuria and phenylketonuria (PKU). The assays are based on microchip capillary electrophoresis with electrochemical detection. This format has several advantages, including small sample requirements, fast analysis times, and inexpensive manufacture of multiple chips. Electrochemical detectors are ideally suited to microchip CE because they can be easily miniaturized and integrated into the analysis system. Monitoring phenylanlanine concentration in plasma samples of newborns is required by law. The current assay has a relatively high rate of failure (1:70) and requires instrumentation frequently not found in-house, especially in small hospitals. High levels of homocysteine in plasma has been associated with cerebrovascular, peripheral vascular, and coronary heart diseases. The assay for homocysteine involves online reduction of the protein-bound homocysteine with tris-(2-carboxyethyl)phosphine, separation by electrophoresis, and electrochemical detection at a gold/mercury electrode. Phenylalanine will be determined in plasma in a similar manner using a copper electrode. The chips will be used with a portable analysis system based on a battery-powered high voltage power supply, miniaturized potentiostat, and integrated data analysis system. The chips we develop will generate a foundation for other clinical assays based on electrochemical detection. PROPOSED COMMERCIAL APPLICATION: The current trend in clinical analysis is away form large centralized labs and toward point of care testing. Thus, a compact device for performance of assays for homocysteine and other clinically relevant animo acids in the clinic or physician's office has large commercial potential. Recently, the American Heart Association recommended homocysteine screening for patients at risk for cardiovascular disease. This should lead to a large demand for accurate and cost-effective assays for homocysteine similar to the current cholesterol screening assays. The development of a small, fast and reliable assay for phenylalanine in blood will make it possible to test infants quickly and accurately for PKU. The electrochemical based clinical analyzers developed here will provide a format that can be used for the development of a number of other assays for clinically important analytes. What is demonstrated with homocysteine and phenylalanine is just the beginning of a large array of clinical analyzers based on microchip CEEC.