Multiplexed Detection Of Lung Cancer Biomarkers Based On Fluorescent Quantum Dot Probes In Suspension Assay

Developing a rapid and accurate method to detect disease related biomarkers is important for early detection of lung cancer to reduce the mortality effectively. Immunoassay is a powerful technique that utilizes labeled antibodies to detect specific target antigens. It is used widely in protein quantitation due to its excellence performance. The emergence of nanotechnology bring up many opportunities for the progress of immunoassay. A magnetic immunoassay that involves fluorescent quantum dot (QD) probes has the potential to provide an alternative to improve the protein detection simultaneously. In this study, we prepared and developed an immune fluorescent assay to detect low concentration of biomarkers simultaneously. The topics of this dissertation are summarized as follows:1. Quantum dot probes and magnetic bead (MB) capture probes were prepared. QDs are outstanding fluorescent label attribute to the unique optical property, like high brightness or narrow and size-tunable fluorescence spectra. QDs can be conjugated with biomolecules due to the advantage of nanoparticle, such as the large surface area-to-volume ratio and the biocompatibility. The detect monoclonal antibody was tagged with QDs by using SMCC chemistry successfully. Antibody labels on MBs are capable of effective reactions with analytes, and the fluorescent signal of QDs would be confined to the MB surface which can be easily collected. The MBs are functionalized with monoclonal antibody using EDC/NHS chemistry to capture the target antigens in sample.2. Based on the unique properties of QDs and MBs, an immune fluorescent assay to detect low concentration carcinoembryonic antigen (CEA) was built. Target proteins form a fluorescent sandwich between the magnetic beads and the QD probes through specific antibody-antigen interactions. The fluorescent intensities produced by the QDs, which were confined to the surface of the beads, could be read efficiently to quantify the concentration of the CEA. The limit of detection was 38 pg/mL, which was approximately 40-fold more sensitive than the conventional enzyme-linked immunosorbent assay (ELISA).3. Multivariate tests are becoming increasingly important to diagnose lung cancer precisely. We have develop a multiplexed detect method for multiple lung cancer biomarkers based on the multicolor QDs and MBs. The capture antibodies were conjugated to the surface of the MBs, while the secondary antibodies specific to neuron specific enolase (NSE), CEA and cytokeratin-19 fragments (CYRFA 21-1) were conjugated to QDs with emission maxima at 525 nm,585 nm and 625 nm, respectively, for each antigen. By identifying and quantifying the fluorescent signal generated by the QDs on the beads, we were able to simultaneously detect NSE, CEA and CYRFA 21-1 using very low sample volumes. The 3-plex assay was able to offer clinically relevant detection limits, and with a broad linear working range for all three markers. Additional advantages of the presented method include ease of operation, low cost and very low sample volume requirement, give this method significant potential for clinical use.