The high sensitivity detection system, which detects the smaller amounts of chemicals and biomolecules, is a prospective technology and can be applied not only for medical application but for the preparations and responses in case of emergency in various fields; environmental technology, water poisoning, safety of food and drug supply, and public health security such as preparation against bioterrorism. However, a lot of processes are required to detect these smaller amounts of harmful substances. Hence, there is a problem to require much time in detection of biomolecules.The immunological diagnostic methods were widely performed in the medical field. In these cases, almost all of the samples are provided as blood, tissue, exudation fluid, or excrement, and the antigen-antibody reaction was commonly performed as the detection method in addition to the colorimetric methods. In this article we developed a high sensitive and quick antigen detecting system using the fluorescence nanocrystal quantum dots (QDs) about a substance detrimental to the biomolecules, such as a bacterial toxin that caused food poisoning or bioterrorism. In order to detect bacterial toxins sensitively and quickly, we adapted the fluorescence-ELISA using QDs to the origin of evanescent microscopy. In this system, the fluorescence of the QDconjugated antibody, which detects bacterial toxins, is placed on the evanescent filed, resulted in detecting bacterial toxins countable by single molecules. Imaging Abstract: Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.
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