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Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay |
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| Authors: | Seungah Lee Young-Ho Ko Jung Dong Kim Jaebum Choo Seong Ho Kang |
| Institution: | a Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, 664-14, 1-Ga, Duckjin-Dong, Duckjin-Gu, Jeonju 561-756, South Korea b Department of Physical Education, Chonbuk National University, Jeonju 561-756, South Korea c Department of Biotechnology, Yonsei University, Seoul 120-749, South Korea d Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea e Department of Applied Chemistry, Hanyang University, Ansan 426-791, South Korea f Laboratory of Microbiology, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, South Korea |
| Abstract: | We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y = 456.35x + 80,382 (R = 0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time. |
| Keywords: | Human leptin Nanoarray protein chip Single-molecule sandwich immunoassay Total internal reflection fluorescence microscopy (TIRFM) |
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