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Shradha V. Prabhulkar, Adam de la Zerda, Sanjiv Gambhir, Richard Awdeh; Development Of Single Step Nano-Optical Immunoassay As A Replacement For ELISA For The Measurement of Protein Biomarkers Upregulated in Disease State. Invest. Ophthalmol. Vis. Sci. 2012;53(14):332.
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This study focuses on the development of a rapid single-step, highly sensitive immunoassay for the measurement of protein biomarkers with a wide detection range. Gold nanorods (GNRs) were used as the signaling labels, as they are not affected by reagent chemistry or photobleaching, and exhibit unique optical properties such as tunable enhanced scattering/absorption of light and biocompatibility. Optical coherence tomography (OCT) was chosen as the tool to detect the GNR labels, utilizing their high scattering properties.
GNRs [size:15X60nm, SPR:840nm] were covalently attached to Glut-1 specific monoclonal antibodies via carbodiimide chemistry. Glut-1 protein (model analyte) was directly attached to the surface of micto-titter plates by overnight incubation at 4oC. The Glut-1 monoclonal antibody functionalized GNRs were then allowed to incubate within the wells for 60 minutes at room temperature. The wells were then twice-rinsed with 1X PBS buffer to remove any unbound GNRs. 100µl of pH = 14, 1X PBS buffer was then infused into each well and the plates were sonicated for 45 minutes. The sonication and alkaline buffer causes the protein and gold nanorod conjugate to break up and float freely in the buffer solution. OCT imaging of the sonicated immunoassay plates containing functionalized GNRs + bound analyte dispersed in PBS solution was conducted using the Bioptigen Spectral Domain Ophthalmic Imaging System (SDOIS). A calibration curve was obtained by plotting the concentration of the Glut-1 protein initially incubated in each well versus the average signal intensity obtained from normalized OCT images.
Our detection range using this nano-optical immunoassay was wide, ranging from 10ng/ml to 1ug/ml. The minimal detectable concentration based on the intercept of our calibration curve of Glut-1 was 2.8ng/ml.
This novel nano-optical immunoassay holds promise to act as a simple, selective, sensitive strategy for effective disease diagnosis, and offers advantages over traditional ELISA techniques, such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development required. Our future goal is to incorporate this strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.
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