Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay

Seungah Lee, Shinae Lee, Young Ho Ko, Hyungil Jung, Jung Dong Kim, Joon Myong Song, Jaebum Choo, Seong Kug Eo, Seong Ho Kang

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)608-612
Number of pages5
Issue number2
Publication statusPublished - 2009 Apr 30

Bibliographical note

Funding Information:
The authors thank KBSI for supporting the AFM experiments. This work was supported by a grant from the Korea Science & Engineering Foundation (R01-2007-000-20238-0).

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry


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