Simultaneous Multicolor Detection System of the Single‐Molecular Microbial Antigen with Total Internal Reflection Fluorescence Microscopy

Abstract

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.

Label-Free, Single-Molecule Detection with Optical Microcavities

“…In the current report, they provide a double benefit through the thermo-optic detection mechanism which is responsible for the observed single-molecule detection sensitivity. Like other detection techniques, such as Surface Plasmon Resonance (SPR) (9,10) or total internal reflection fluorescence microscopy (TIRF), (11,12) the interaction which leads to detection occurs at the surface of the resonant cavity. As molecules bind to the surface of the microresonator, they interact directly with the evanescent tail of the whispering gallery mode (Fig.…”

Semiconductor Quantum Dots for Bioanalysis

“…In another example, multiplexed immunoassay formats based on antibody-functionalized QDs were used for the simultaneous detection of Escherichia coli O157:H7 and Salmonella typhimurium [68] and for the discrimination between the diphtheria toxin and the tetanus toxin proteins. [69] This was achieved by the modification of QDs having two different emission wavelengths with the respective antibodies.…”

Quantum Dots for Sensing

“…They also open the way to multiplexed protein detection due to their wide absorption band (allows only one light source excitation), their narrow emission peak (allow signal multiplexing), their size control (allows color tunability for signal multiplexing), and their surface engineering (allows multiple-target-specific reactions). This multiple-target detection has been successfully proved by several groups using modified ELISA techniques [71,72].…”

“…There are several factors that make QDs suitable for genomic applications; their high photostability, their narrow and tunable emission peak (allows multiplexing), their great bio-functionaliztion potential, and their high fluorescence quantum yield that provides higher signal-to-noise ratios with lower analyte concentrations. This high fluorescence emission allows a better spatial definition, as far as almost single QD detection can be achieved with advanced fluorescence microscopy techniques [72,75,76,77,78]. This spatial resolution is a basic requirement of some DNA detection techniques such as fluorescence in situ hybridization (FISH).…”