| In chapter one, fluorescence methods in single-molecule detection (SMD) for biomolecules and fluorescent probes were reviewed briefly. The main fluorescence methods in SMD are confocal fluorescence microscopy (CFM), total internal reflection fluorescence microscopy (TIRFM), epi-fluorescence microscopy (EFM) and two-photon or multi-photon laser scanning fluorescence microscopy (TPFM, MPFM). The principles of these techniques and methods and their applications for quantification of biomolecules were reviewed. The fluorescent probes including organic dye, nanoparticles, fluorescent protein, rare-earth-metal ions were also described.In chapter two, we developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6-CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope cover slip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40×40μm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge coupled device. One hundred images of the fluorescent molecules accumulated on the cover slip were taken consecutively, one by one, by moving the cover slip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5×10-15 to 5×10-12 mol/L for R6G,3×10-15 to 2×10-12 mol/L for IgG(H+L)-488, and 3×10-15 to 2×10-12 mol/L for DNA-R6G.In chapter three, a novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of protein was developed using total internal reflection fluorescence microscopy coupled with fluorescent quantum dot (QD)-labeling. In life sciences, many protein microarray assays are required to detect proteins from small sample volumes and to describe low abundance levels in absolute terms (numbers or molar amounts). Therefore, in protein microarray assays, ultra-sensitive detection methods with ultra-small volumes have become increasingly important. SMD is rarely used in microarray assays, because the conventional scanners used in microarray assays cannot acquire images of single fluorescent molecules due to low sensitivity and resolution, and when SMD is used in microarray assays, the biggest difficulty is acquiring the whole microarray image. It is because the size of the images taken using SMD techniques is much less than that of a microspot on a microarray. In almost all reports concerning SMD-based microarray assays, only a part of each microspot was acquired. The readout system or method is crucial for the SMD-based microarray assays. In the SMCMA, a single-molecule microarray reader was used to acquire the whole images of microarrays at single-molecule level. Owing to the remarkably high photostability, QDs as labels are much better than fluorescent dyes and can withstand numerous illumination cycles without photoquenching during laser scanning the microarrays when acquiring single-molecule images. These perfect photochemical characteristics of QDs improved the signal and increased the sensitivity in the SMCMA. Using the present SMCMA, an amount as low as 1.8×10-21 mole (1080 molecules) for proteins in 1.8-nL samples could be detected. The SMCMA with 1.8-nL sample volume makes dynamic detection of protein expression for the same alive cells possible. Here, the SMCMA was applied to dynamically measure osteopontin (OPN) expression of decidual stromal cells (DSCs).In chapter four, A novel ultra-sensitive and high-selective single-molecule-counting microarray assay (SMCMA) of DNA for single-cell multi-gene expression was developed using a single-molecule microarray reader coupled with fluorescent quantum dot (QD)-labeling. In the SMCMA, a microarray fabricated on a silanized glass coverslip consists of 10 subarrays with 9 spots for each subarray. Each spot with a diameter of-300μm is modified with different capture DNAs (DNA1). The cDNAs corresponding to mRNAs in a single cell are captured to the complementary capture DNAls at different spots of a subarray. After the cDNAs are labeled with QDs using QD-labeled detection DNAs, the image of the microassay is acquired using a single-molecule microarray reader. The amounts of the cDNAs are quantified by counting the bright dots corresponding to single cDNA molecules on the microassy. Using the SMCMA,2×10-16 mol/L DNA in 3 μL sample or as low as 360 molecules of mRNAs in a single cell can be detected. For a microarray, nine different genes in ten different cells can be quantified in parallel. Since quantification relies on the number of bright dots corresponding to single DNA molecules rather than their signal size, the reproducibility of the detected signal intensity becomes irrelevant, thus guaranteeing reliability of the results and reducing detection error. To our knowledge, this is the first report on PCR amplification-independent quantification of multi-gene expression profiling in single cells.In chapter five, a novel visible SMD method for DNA analysis using conventional biological microscope was provided. In the method,300-nm-diameter magnet microbeads (MBs) as labels were bound to target DNA molecules immobilized on microarrays. Target DNA could be quantified based on counting the number of MBs corresponded to single target DNA molecules on the microarrays under a conventional microscope. Using the method, DNA as low as 1.2×10-21 mol (720 molecules) could be detected and multi-gene expression in single cell could be quantified. The method is simple without need of expensive instruments.In chapter six, multicolor optical coding for DNA single-molecule-detection has been achieved by binding dye-DNAs to magnetic microbeads at different ratios. The AMCA, FAM and Cy5 as dyes were used. About one thousand of dyes could be binded to a bead. The use of different ratios of dyes could make multicolor besds. Using these encoded besds, single-molecule-detection for DNA was achieved. This coding technology is expected to open new opportunities in gene expression studies, highthroughput screening, and medical diagnosticsIn chapter seven, the principal of fluorescence resonance energy transfer (FRET) and its biological application were reviewed briefly. The FRET as a powerful technique has been applied in measuring conformational change of biomolecules, immunnoassay and DNA analysis. Luminescent quantum dots (QDs) have been used in FRET, bioluminescence resonance energy transfer (BRET) and chemiluminescence resonance energy transfer (CRET) as acceptors or donors. The application of QDs in resonance energy transfer is also described.In chapter eight, we developed a novel resonance energy transfer, electrochemiluminescence resonance energy transfer (ECRET). In the ECRET technique, the emitters of N-(4-aminobutyl)-N-ethylisoluminol/H2O2 system generated at an electrode through electrochemical reactions act as electrochemiluminescent donors to emit light with a maximum emission of 460 nm and the red fluorescent luminescent semiconductor nanocrystals (quantum dots, QDs), having a maximum emission at 655 nm, serve as acceptors. When a potential is applied to the electrode, the electrochemiluminescent donors transfer energy to the proximal ground-state QD acceptors, producing efficient ECRET. As a result, the QD acceptors emit a light with a longer wavelength of 655 nm than that of the electrochemiluminescent donors (460 nm). From the ECRET spectra consisting of ECL spectra of the electrochemiluminescent donors and emission spectra of the QD acceptors via ECRET, many biological events can be evaluated. We report the ECRET between the luminol molecules and the QDs in luminol-DNA-DNA-QD, luminol-protein-protein-QD and lumino-protein-QD conjugates immobilized on the Au electrode. The ECRET technique could be applied to the investigation of interactions between nucleic acids or proteins and conformational changes of DNA and protein.In chapter nine, the ECRET between the QDs as donors and the Cy5 as acceptors was studied. The new ECRET system was applied to investigate the conformational changes of protein. The ECRET technique based on QDs-Cy5 system could provide a powerful tool to the study in chemistry and biology. |
