The goal of this project is to exploit the high reflection density of integrated optical waveguides (IOWs) to develop evanescent wave biosensors for applications in high throughput genetic screening. In particular, the applicants plan to develop nucleic acid hybridization assays (also know as """"""""molecular diagnostics"""""""" or """"""""MDx"""""""" assays) for use in screening and diagnosis of hereditary cardiovascular disease. Although there are several potential disease targets, they intend to focus on long-QT syndrome (LQTS) because there is a compelling need in this disease for rapid and inexpensive methods for genetic screening of family members of affected individuals. Traditional diagnostic methods (e.g., ECG) are equivocal in about 40% of LQTS cases because the affected individual exhibits either a normal or borderline prolonged QT interval. Such individuals often go undiagnosed, resulting in 3000-4000 sudden deaths per year in the United States. LQTS has been linked to genetic polymorphisms in four genes (KVLQT1,HERG, SCN5A & KCNE1) that encode for cardiac ion channels. Present-day methodology for assessing genetic polymorphism involves isolating genes from afflicted individuals using polymerase chain reaction (PCR), sequencing them, and then cataloging the observed mutations. However, this procedure is too expensive and time-consuming for use in routine patient screening, which has lead to development of so-called """"""""DNA chips"""""""" that contain hundreds to thousands of oligonucleotides immobilized to a single substrate in a microarray. Patient screening with a DNA chip involves isolating the gene of interest from the patient's DNA using PCR and them allowing the PCR product to hybridize to the chip. Hybridization is detected using either an epifluorescence or confocal microscope. The detection process is time-consuming because each array element is imaged sequentially for a few seconds or more. Moreover, the instrumentation required to read the chips is very expensive, costing between $100,000 and $200,000 for a typical setup. Thus, assay time and cost are limiting factors in the application of MDx technology to routine patient screening and diagnosis. The approach taken in this application to this problem is to use an integrated optical waveguide sensor as the immobilization support for a DNA chip. By doing so the applicants can dramatically reduce assay time (to 5 minutes, or less) and instrumentation cost (our prototype analyzer cost about $3,000 to produce, production versions would probably retail for about $10,000) because the entire oligonucleotide array is monitored in real time by charge-coupled device (CCD) camera. Moreover, data acquisition in real time enables us monitoring hybridization kinetics, which is essential for identifying point mutations.
| Throne, R; Wilber, D; Olshansky, B et al. (1993) Autoregressive modeling of epicardial electrograms during ventricular fibrillation. IEEE Trans Biomed Eng 40:379-86 |
