Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling |
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Authors: | Dafeng Jiang Lei Wang Wei Jiang |
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Institution: | a School of Chemistry and Chemical Engineering, Shandong University, 27 Shanda Nanlu, 250100 Jinan, Shandong, PR China b School of Pharmacy, Shandong University, 250012 Jinan, PR China |
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Abstract: | 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. |
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Keywords: | Single-molecule counting Quantum dot Epi-fluorescence microscopy Surface modification Antibody |
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