The long term objective of this research is to perfect fiber optic biosensors as a rapid, highly reliable and specific method for detection and quantitation of drugs and/or metabolites in urine and blood. Biosensors shall be used to analyze urines of patients who participate in the Methadone Maintenance Program of the Drug Treatment Center of School Medicine, Univ. of Maryland at Baltimore. They visit the clinic six days a week for methadone and counseling, are closely monitored and if their urine test positive are dismissed from the program. The most common drugs they abuse are heroin and cocaine. In a double blind study, the biosensor data will be compared with results of the routinely performed immune assays on the same urine samples and if discrepancies occur GC-MS will be used. This research utilizes a multi-disciplinary approach, (molecular biological, synthetic and analytical chemical, pharmacological, biophysical and clinical) to improve the sensitivity, specificity, selectivity and reproducibility of the biosensor. Technological innovations to be employed include the utilization of recombinant cloning to provide a stable reliable source of highly specific monoclonal antibodies (mAbs) against benzoylecgonine (BE) and morphine-3-beta-glucuronide (M3G), the major metabolites of cocaine and heroin in urine, respectively. These mAbs will be used as biological sensing elements in the biosensors, which will hopefully eliminate false positive and false negative results. The fiber optic instrument will be modified to be more selective and amenable to miniaturization. The outstanding features of biosensors are their quantitative capabilities, user-friendly nature, quick on-site analysis, low cost and improved sensitivity and specificity. Not only would such biosensors be valuable for detection and quantitation of cocaine and heroin abuse but they will also be very important in emergency rooms where rapid identification of the drug is important for initiating treatment. There are five specific aims: 1- Clone BE and M3G mAbs in. 2- Synthesize fluorescent, 125I-labeled and protein conjugates of BE and M3G. 3- Select mAbs with high specificity and selectivity for BE and M3G. 4- Enhance evanescent excitation of the fiber optic biosensor. 5- Validate the reliability and specificity of biosensor analysis of BE and M3G in patients' urine. The following several hypotheses will be tested: Biosensors can be highly sensitive, [specific, easy to operate, do not require sample pretreatment and provide rapid quantitation.] The sensitivity of the fiber optic biosensor will be improved by use of LEDs, red fluorescent dye and polystyrene fibers. Recombinant gene cloning technology provides a large variety of mAbs from which to select rapidly the most sensitive and specific ones against BE and M3G.
|Eldefrawi, M E; Azer, N L; Nath, N et al. (2000) A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine. Appl Biochem Biotechnol 87:25-35|
|O'Connell, K P; Valdes, J J; Azer, N L et al. (1999) Assessment of an automated solid phase competitive fluoroimmunoassay for benzoylecgonine in untreated urine. J Immunol Methods 225:157-69|
|Nath, N; Eldefrawi, M; Wright, J et al. (1999) A rapid reusable fiber optic biosensor for detecting cocaine metabolites in urine. J Anal Toxicol 23:460-7|