Development Of FӦrster Resonance Energy Transfer Based Homogeneous Immunoassays For Determination Of (R)-Methadone
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Date
2016-08
Authors
Ooi, May Yui
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Abstract
Drug analysis is commonly performed using an accurate and specific chromatographic method, such as GC-MS and LC-MS. These methods are known to be highly sensitive and specific. However, they require sophisticated equipment and also skilled manpower for the analysis. For analysis of large number of samples, less descending methods like immunoassays are used prior to confirmation by GC-MS or LC-MS. Thus, high throughput screening is essential in increasing the efficiency of drug testing. In this study, the fluorescence properties from various types of fluorescence materials, such as organic dyes, quantum dot and lanthanide chelates were investigated and employed. As a consequence, new FRET and TR-FRET based homogeneous competitive immunoassays were successfully designed and developed for detection of (R)-methadone in serum sample. Then, the concentrations of (R)-methadone in the 50 MMT patient’s serums were tested. Consequently, the accuracy and sensitivity of the developed assays were studied and validated by using LC-MS/MS method. Generally, comparison of the LC-MS/MS method with other three immunoassays methods showed adequate correlation. The regression analysis of FRET based immunoassay, point of care - FRET based immunoassay and TR-FRET based immunoassay are R2 = 0.898, 0.797 and 0.910, respectively. In this study, the developed FRET based assays were also compared with conventional ELISA method in terms of assay sensitivity, simplicity of handling and assay time. Overall results suggest that the reported homogeneous immunoassays advantageous over ELISA as
it required shorter assay time with easy handling. Nevertheless, comparison among these immunoassays, TR-FRET immunoassay is the best analytical method due to its higher accuracy and sufficient sensitivity. These results demonstrated the potential of the developed immunoassays as alternatives in high throughput screening method in drug testing. Besides, through this study, it was also found that quantum dots are not suitable to be used in small drug study as there are interactions between quantum dot and small drug molecules either after conjugation or via passive adsorption. Therefore, further studies are needed to deepen the understanding of these interactions.
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Homogeneous competitive immunoassays were successfully designed and developed , for detection of (R)-methadone in serum sample.