Talanta 78 (2009) 608–612 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay Seungah Lee a , Shinae Lee b , Young-Ho Ko b , Hyungil Jung c , Jung Dong Kim c , Joon Myong Song d , Jaebum Choo e , Seong Kug Eo f , Seong Ho Kang a,∗ 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 article info Article history: Received 20 October 2008 Received in revised form 6 December 2008 Accepted 8 December 2008 Available online 24 December 2008 Keywords: Human leptin Nanoarray protein chip Single-molecule sandwich immunoassay Total internal reflection fluorescence microscopy (TIRFM) abstract We report a method for the quantitative analysis of human serum leptin, which is a protein hormone asso- ciated 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 150nm on a self-assembled mono- layer 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 flu- orescence 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 stan- dards 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. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Leptin is a protein hormone associated with obesity, appetite regulation, energy expenditure, and reproduction in animals and humans. Noticeably higher leptin levels have been observed in obese humans than in non-obese humans [1–3]. Leptin is thought to contribute to body weight regulation by controlling food intake and energy expenditure at the hypothalamic level [4]. It has been suggested that abnormalities in its level increase the propen- sity to obesity. In addition to its role in metabolic disorders and obesity, leptin also has an important regulatory effect on bodily hormonal [5,6] and gonadal [7] functions. So far, the capillary elec- trophoresis [8–10], immunofunctional assay [11], enzyme-linked immunosorbent assay (ELISA) [12,13], radioimmunoassay [14–18] and Western blotting [19–21] techniques have been convention- ally used to determine human leptin. Although they are reliable, these methods are relatively expensive and are restricted to the determination of single target specificity. ∗ Corresponding author. Tel.: +82 63 270 3421; fax: +82 63 270 3408. E-mail address: shkang@chonbuk.ac.kr (S.H. Kang). The possible application of microarray biochips to the analy- sis of various cytokines, including leptin, in serum samples, was demonstrated by Du et al. [22]. The ability to fabricate highly robust microarrays in which a thousand samples are immobilized enables the generation of massive amounts of biologically relevant data using low sample quantities. Recently, nanoarray technology has been suggested as a means of overcoming the problems of microar- ray technology, such as the relatively large sample volumes and long incubation times that are required, and the high limits of detection (LOD). A new generation nanoarray biochip has also been described which is capable of supporting high-throughput and multiplexed ELISA. Despite its advantages, the nanoarray assay is not able to achieve the femtomolar (fM, ×10 -15 M) LOD of ELISA, which limits its applications in immunoassays [23,24]. However, the detection of proteins in the zeptomolar concentration range (zM, ×10 -21 M) has recently been demonstrated using a mass spectrometer or gold nanoparticles conjugated with antibodies [25,26]. Proteins in the zM concentration range exhibit almost no detectable, nonspecific binding to the passivated portions of nanoarrays, even when they are present in the form of complex mixtures, and therefore provide the opportunity to study a variety of surface-mediated, biological recognition processes. 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.12.018