Single-molecule Detection Assays For Antibody Quantification Using Fluorescence Microscopy

In chapter one, significance of single-molecule detection (SMD) have been described. SMD, with its ability to detect single molecules, are powerful tools for investigation of dynamic and kinetics of single molecules. The related techniques and methods for SMD analysis have been described here. Corresponding to the mentioned techniques and methods, laser induced fluorescence microscopy (LIFM), quantum dots (QDs) and the strategies for solid-supported immobilization are reviewd. For a further step, a review of their applications for quantitative SMD analysis is provided here.In chapter two, we developed a sensitive method for quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy (TIRFM) with QDs labeling. Alexa Fluor labeled antibody molecules have been detected using TIRFM with adsorption equibrium in literature. The limit of detection (LOD) was only 5.4×10-11 mol L-1. We chose biotinylated monoclonal anti-human IgG molecules as the model antibody. First, antibody molecules were immobilized on the silanized glass substrate surface. By the strong biotin-streptavidin affinity, streptavidin-coated QDs were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots in the evanescent wave field were obtained by a high-sensitivity electron multiplying charge coupled device (EMCCD). Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted based on a single molecule counting approach, one by one. The linear range of 8.0×10-14 to 5.0×10"12 mol L-1 was obtained between the number of single molecules and the sample concentration. The lower limit of the linear range was 3 orders of magnitude lower than that reported in the literature.In chapter three, we characterized nonspecific adsorption of fluorescent dyes (dye-labeled antibody) on silanized substrate surfaces using single-molecule counting with epi-fluorescence microscopy (EFM). Nonspecific adsorption causes false positive events, decreasing the accuracy and sensitivity of the assays. The silanization of substrate surfaces is a widely used method to attach functional groups such as amino, aldehyde, epoxy, or thiol groups for cross-linking antibody molecules onto the glass surface. Since the silanized surfaces are often hydrophobic as a result of their hydrophobic chains, these surfaces are liable to cause protein adsorption through the hydrophobic interaction between them. At first, three different silanized glass substrates with differently terminated-functional groups were obtained. QDs and QDs-Antibody conjugates were selected as the model target analytes for characterizing nonspecific adsorption. Then EFM coupled with EMCCD was used to indentify and detect single adsorped molecules. Finally, the nonspecific adsorption of adsorped molecules is quantified based on the direct counting of individual fluorescent spots. The results demonstrate that a hydrophilic silanized surface has the lowest nonspecific adsorption and is highly suitable for bioassays.In chapter four, we described a hydrophilic substrate surface for antibody immobilization and presented a fluorescence single-molecule counting assays for antibody quantification using EFM. The covalent bonding of poly(vinyl alchol) (PVA) on a poly(dimethylsiloxane) (PDMS) surface provided a hydrophilic substrate surface in literature. Nonspecific adsorption of proteins was greatly reduced on the PVA-coated PDMS surface, and target molecules could be immolized with high loading. In our study, nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, QDs were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. EFM coupled with EMCCD was chosen as the tool for single-molecule fluorescence detection here. A linear range of 5.0×10-14 to 3.0×10-12 mol L-1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach. Compare with the literature, coverslip instead of PDMS as the substrate could reduce the background level; EFM instead of AFM as the detection tool could give a more widely application of the method mentioned here. In chapter five, we presented a platform of supported protein layers (SPLs) surface for oriented and specific antibody immobilization. Current methods for antibody immobilization are mainly based on the covalent bonding of them to substrate surfaces, which often results in steric problems and an inevitable loss in binding affinity. A common strategy for preventing nonspecific adsorption is to block the surfaces with bovine serum albumin (BSA), however, this step often hinder access of molecules of interest to binding sites. Here, our work is described as follows:1. Constructing the SPLs surface platform for antibody immobilization, which was achieved by attached BSA, anti-BSA, and protein G to carboxyl-terminated substrate surfaces by turns.2. Nonspecific adsorption of single molecules on SPLs surfaces was investigated. Two different kinds of dye-antibody conjugates were chosen for the characterization of nonspecific adsorption. The results indicated that SPLs had the ability to resit the nonspecific adsorption of antibody, which did not effecet by the kinds of labeled dyes.3. Quantifying the concentration of antibody binding to SPLs substrate. Protein immobilized on the BSA-blocked substrate surface has been detected using TIRFM with single-molecule counting in literature. The LOD was 1.0×10-10 mol L-1. We immobilized the target antibody molecules to the SPLs substrate surface. The oriented antibody immobilization with high affinity could reduce the steric problems and give a high binding capacity. QDs were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots were obtained using EFM coupled with EMCCD. The linear range of 1.0×10-14 to 3.0×10-12 mol L-1 was obtained via a single-molecule conting approach. The lower limit of the linear range was 4 orders of magnitude lower than that reported in the literature.