Development of Quantum dot Reporters for Immunoassay Applications
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DEVELOPMENT OF QUANTUM DOT REPORTERS FOR IMMUNOASSAY APPLICATIONS D.M. Speckman, T.L. Jennings, S.D. LaLumondiere, and S.C. Moss The Aerospace Corporation, P.O. Box 92957, Los Angeles, CA 90009 ABSTRACT Multi-pathogen biosensors that take advantage of sandwich immunoassay detection schemes usually rely on spatial resolution patterns of capture antibodies on a detector plate for pathogen recognition, and typically utilize fluorescent, organic dyes as optical labels to identify captured pathogens. An immunoassay-based, fiber optic detector that utilizes varying sized fluorescent semiconductor quantum dots (QDs) as the reporter labels for different antibodies would have the ability to detect multiple pathogens within a single fiber, using a single excitation source. Such a detector requires that QDs be attached to antibody proteins, such that the specificity of the antibody is maintained. We have been involved in efforts to develop a reproducible method for attaching QDs to antibodies for use in such a biosensor. We synthesized CdSe/ZnS core-shell QDs of differing size, functionalized their surfaces with several types of organic groups for water solubility, and covalently attached these functionalized QDs to rabbit anti-ovalbumin antibody protein. We also demonstrated that these labeled antibodies exhibit selective binding to ovalbumin antigen. We characterized the QDs at each step in the overall synthesis by UV-VIS absorption spectroscopy and by picosecond (psec) transient photoluminescence (TPL) spectroscopy. TPL spectroscopy measurements indicate that QD lifetime depends on the size of the QD, the intensity of their optical excitation, and whether or not they are functionalized and conjugated to antibody. We describe details of these experiments and discuss the impact of our results on our biosensor program. INTRODUCTION The use of semiconductor QDs in place of organic dyes as fluorescent reporters in biological systems has become a topic of great interest recently, due to the potential advantages that QDs appear to offer over organic dyes. Organic dye molecules have traditionally been used as sensitive, non-isotopic indicators or “tags” for the detection of specific species in biological systems such as DNA conjugates in DNA sequencing schemes and antigen-antibody complexes in biological assays. However, these dyes have some inherent properties that limit their effectiveness, such as the tendency to photobleach, the phenomenon of red-tailing, and narrow excitation bands. Semiconductor QDs are nanocrystals with optical and electronic properties that arise from the wave mechanics of a “particle in a sphere”, and fluorescent QDs can be tailored to emit light at discrete wavelengths depending on the size and composition of the nanocrystals. Due to their unique luminescent properties, QDs do not suffer the same limitations as organic dyes. QDs exhibit little photobleaching or red-tailing, their emission spectra can be tailored to a variety of different wavelengths by varying the size of the QDs, and they have a broad ex
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