Number Of Single Molecules Research Articles

Solid phase single-molecule counting of antibody binding to supported protein layers surface with low nonspecific adsorption

On the basis of the supported protein layers (SPLs) substrate, the study presented an ultrasensitive and highly specific platform for single-molecule fluorescence detection of antibody using quantum dots (QDs) as probes. To construct the SPLs surface platform for antibody immobilization, bovine serum albumin (BSA), anti-BSA, and protein G were firstly attached to carboxyl-terminated substrate surfaces by turns. Then nonspecific adsorption of single antibody molecules on SPLs surfaces was investigated. Through the irreversible interaction between streptavidin and biotin, streptavidin-QD conjugates were employed to conjugate with biotinylated antibody, producing QD-antibody conjugates for generating fluorescent signals in fluorescent imaging. Epi-fluorescence microscopy equipped with an electron multiplying charge-coupled device was chosen as the tool for single-molecule fluorescence detection here. The concentration of antibody is quantified based on the direct counting of individual fluorescent spots, one by one. The generated fluorescent signals increased with the increasing concentration of immobilized antibody and were found to be proportional to antibody concentrations. The better brightness and photostability of QDs, and slower increase in the number of counted molecules make a large linear dynamic range of 1.0 × 10 −14 to 3.0 × 10 −12 mol L −1 between the number of single molecules and antibody concentrations, which is comparable to the previously reported surface-based SMD analysis.

Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 × 10 −14–3.0 × 10 −12 mol L −1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

Quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling

We presented a sensitive method to quantify antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling. In this method, the biotinylated monoclonal anti-human IgG molecules were immobilized on the silanized glass substrate surface. By the strong biotin–streptavidin affinity, streptavidin-coated quantum dots 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. Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted, 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.