Analytica Chimica Acta 676 (2010) 81–86 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay Hongwei Zhao a , Changguo Xue b , Tiegui Nan a , Guiyu Tan a , Zhaohu Li a , Qing X. Li c , Qingchuan Zhang b,∗∗ , Baomin Wang a,∗ a College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China b Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027, China c Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA article info Article history: Received 16 June 2010 Received in revised form 22 July 2010 Accepted 24 July 2010 Available online 3 August 2010 Keywords: Copper Detection Monoclonal antibody Enzyme-linked immunosorbent assay Microcantilever immunosensor abstract A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)–ethylenediamine–N,N,N ′ ,N ′ –tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)–EDTA–Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL -1 of Cu(II)–EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2–17 ng mL -1 , respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry. © 2010 Published by Elsevier B.V. 1. Introduction Copper, one of very important transition metals, is widely used in industrial and agricultural processes. It has resulted in serious environmental pollution due to the excessive exploitation and uti- lization. High concentrations of copper are frequently added to pig and poultry feeds to improve their growth [1]. Copper could accu- mulate in the livers of human body through the food chain [2]. Cu(II) is a potential mutagenic agent [3]. External applications of higher doses of Cu(II) could result in both clastogenic effects and spindle disturbances in mammalian cells [3]. Menkes’ and Wilson’s dis- eases are two kinds of human genetic maladies that closely relate with the disorder of copper metabolism [4]. China, U.S. Environ- mental Protection Agency (EPA) and World Health Organization (WHO) have set the copper limits of 1000, 1300 and 2000 ng mL -1 , respectively, in drinking water [5]. To monitor the copper content in the environment and study its toxicity, sensitive and specific analytical methods for copper are desirable. Current conventional methods for copper determination include atomic absorption spectroscopy (AAS) [6,7], inductively ∗ Corresponding author. Tel.: +86 10 62731305; fax: +86 10 62731145. ∗∗ Corresponding author. Tel.: +86 551 3607613; fax: +86 551 3607613. E-mail addresses: zhangqc@ustc.edu.cn (Q. Zhang), wbaomin@263.com, wbaomin@263.net (B. Wang). coupled plasma atomic emission spectroscopy (ICP-AES) [8], induc- tively coupled plasma-mass spectrometry (ICP-MS) [9,10], and stripping potentiometry [11]. Although these methods are well used to determine the trace metals, they are expensive, time- consuming and/or inconvenient for in situ monitoring. Other methods have also been developed for copper determination. For example, Gly-Gly-His tripeptide is highly sensitive and selective to Cu(II) and has been used to develop an electrochemical and a microcantilever biosensor for copper ions [12,13]. Chen et al. [14] developed a fluorescence sensor for the highly selective detection of Cu(II) with a limit of detection (LOD) of 3.6 nM based on the aggregation-induced fluorescence quenching of the highly fluo- rescent glutathione-capped gold nanoparticles. Summer et al. [15] described a wild type form of red fluorescent protein as a highly sensitive, selective, and reversible fluorescence-biosensor for both Cu(I) and Cu(II) and the LOD for Cu(II) was below 1 ppb. Zhou et al. [16] reported a colorimetric method for the detection of Cu(II) based on the color change of the colloid gold in aqueous solutions by using azide and terminal alkyne-functionalized gold nanoparticles, which the minimum concentration of Cu(II) detectable by naked eye was approximately 50 mM. These Cu(II) sensors are based on different principles and have provided alternative methods for the analysis of copper ions. Enzyme-linked immunosorbent assay (ELISA) is rapid, sensi- tive, selective and cost-effective. It has been one of the principal detection methods for environmental contaminants [17–19]. Since 0003-2670/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.aca.2010.07.041