In this study, a novel method for the fast, sensitive and selective detection of Cu 2+ using gold nanoparticles (AuNPs) was developed and used in immunoassays. In the presence of L-cysteine, L-cysteine can bind to the surface of citrate-stabilized AuNPs through Au-S bonds. As a result, aggregation of AuNPs occurs through electrostatic interactions between the cysteinebound AuNPs. In contrast, in the presence of Cu 2+ , Cu 2+ can catalyze O 2 oxidation of cysteine, leading to the quick formation of disulfide cystine. An increase in the concentration of Cu 2+ decreased L-cysteine-induced AuNPs aggregation by decreasing the number of free cysteine thiol groups, and the solution color changed from purple to red. Therefore, the concentration of Cu 2+ can be detected with the naked eye or with ultraviolet-visible spectroscopy, and the detection limits of Cu 2+ were 20 nM and 10 nM, respectively. This sensitivity was approximately three orders of magnitude higher than that of traditional AuNPs-based colorimetric Cu 2+ detection methods. Because of the high sensitivity of the proposed method, we further used it with a labeled antibody in colorimetric immunoassays. The detection limit of the cancer biomarker a-fetoprotein was 2 ng ml À1 , which is comparable to the detection limit of the enzyme-linked immunosorbent assay method.
INTRODUCTIONCopper is an essential metal ion for biological functions. It is a cofactor and/or a structural component of numerous enzymes and other proteins needed in metabolic processes. 1 However, the accumulation of Cu 2+ in humans leads to many serious conditions, including neurodegenerative diseases and prion diseases. 2,3 As such, the detection and measurement of Cu 2+ has become increasingly important. To date, there are many technologies that have been developed to detect Cu 2+ , including inductively coupled plasma 4 detectors, electrochemical sensors, 5 surface-plasmon resonance 6 detectors, fluorescenceanisotropy assays, 7 quantum-dot-based assays 8 and Cu 2+ -specificDNAzyme-based fluorescence sensors. 9 Although these technologies can detect Cu 2+ sensitively and selectively, the need for sophisticated instrumentation and highly trained operators limited their application in routine detection.Recently, gold-nanoparticles (AuNPs)-based colorimetric methods, which without the aid of advanced instrumentation, have attracted attention for their use in many applications because they can be easily monitored with the naked eye or with low-cost portable instruments. AuNPs possess intrinsically strong surface-plasmon resonance absorptions and high extinction coefficients (approximately four orders of magnitude greater than typical organic dyes). 10 The color of the AuNPs solution can change from red to purple, in response to the surface-plasmon resonance absorption of dispersed and aggregated