Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

• Published in: Analytica chimica acta Vol. 590; no. 1; pp. 104 - 109
• Main Authors: Wang, Lei, Xu, Guang, Shi, Zhikun, Jiang, Wei, Jin, Wenrui
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 02.05.2007; Elsevier
• Subjects: Adsorption; Analytical chemistry; Chemistry; Exact sciences and technology; Immunoglobulin Heavy Chains - analysis; Microscopy, Fluorescence - methods; Protein; Proteins - analysis; Single-molecule detection; Spectrometric and optical methods; Total internal reflection fluorescence microscopy; Total internal reflection fluorescence microscopy; Protein; Single-molecule detection; Glass; Charge coupled device; IgG; Fluorescence spectrometry; Chemical enrichment; Total internal reflection; Adsorption; Imaging; Evanescent wave; Fluorescence microscopy; Quantitative analysis
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2007.03.018

We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 × 10 −11 to 8.1 × 10 −10 mol L −1.