A reagentless signal-on architecture for electronic, real-time copper sensors based on self-cleavage of DNAzymes† Lidong Li, * Long Luo, Xiaojiao Mu, Tianyu Sun and Lin Guo * Received 18th March 2010, Accepted 17th April 2010 First published as an Advance Article on the web 28th April 2010 DOI: 10.1039/c0ay00176g Herein, a rapid electrochemical biosensor based on highly specific, metal-induced self-cleaving DNAzymes was designed. This proposed sensing protocol offers reasonable selectivity, amazingly fast speed and operational convenience for copper assays on-line. The development of specific ion sensors is linked to pressing needs for the rapid detection of toxic metals, such as Cu 2+ . Currently, flame and graphite furnace atomic absorption spectrometry and spectropho- tometric methods are known as the most common techniques used for copper determinations. 1 These techniques suffer from their insufficient sensitivity to very low concentration of the metal in many environmental and biological samples. Very recently, scanning elec- trochemical microscopy (SECM), combined with surface plasmon resonance (SPR), SECM-SPR, was applied for real-time detection of the incorporation of copper ion, 2 which requires expensive robotic facilities, and complicated operation process. In recent years, fluo- rescent and optical fiber sensors have been developed to achieve parts-per-billion detection limits. 3 These optical methods, however, suffer from possible drawbacks including unstable chemical and florescence properties, potential false signals arising from contami- nating colorants, fluorophores and quenchers, and, frequently, a reliance on cumbersome optical equipment. 4 Electrochemical methods, in contrast, benefit from the impressive miniaturization of modern microelectronics, the relative paucity of electroactive contaminants, and the relative stability and environmental insensi- tivity of electroactive labels and thus are less likely to suffer from these potential drawbacks. Nevertheless, the classical polarographic tech- niques, which employ a dropping mercury electrode (DME) with a glass capillary, lead to irreproducible dropping characteristics and inaccuracy because of the etching of the glass capillary itself. 5 The electrochemical copper detection methods reported to date require complex, multistep protocols involving the reductive deposition of metallic copper followed by anodic stripping voltammetry, and the relatively high detection limit suffers as well. 6 Aptamers, short oligonucleotides selected in vitro for specific, high- affinity binding to a broad range of molecular targets, are considered as promising recognition elements for biosensor applications. 7 Recently, electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform. 8 E-AB signal generation occurs when a binding-induced conformational change significantly alters electron tunneling from a redox-tagged, electrode-bound aptamer to the sensing electrode. E-AB sensors have been widely applied to biological systems by Plaxco and Fan’s group. 9 Herein, based on E-AB sensing mechanisms, we propose a simple electrochemical copper detection approach based on the highly specific, metal-induced self-cleaving DNAzymes. Generally, biological catalysis is dominated by enzymes made of protein and, to a lesser extent, by enzymes made of RNA. 10 DNA is considerably more resistant than RNA to degradation in aqueous solutions and hence is well-suited to serve as a reservoir for biological information. However, in vitro selection for catalytic polynucleotieds, has been used recently to create several classes of DNA enzymes, including DNAs that cleave RNA, ligate chemically activated DNAs and promote porphyrin metallation. It was found that DNA is more susceptible to scission via depurination followed by elimination or via oxidative mechanisms than by hydrolysis. 11 Based on in vitro selection of self-cleaving DNAs, Breaker et al. reported that single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism, suggesting that efficient DNA enzyme might be made to cleave DNA in a biological context. 12 In their findings, an optimized single stranded DNA displays significant cleavage when only Cu 2+ is added. Our copper sensing protocol is based on the copper-dependent and highly site- specific cleavage of single stranded DNA. The copper-dependent self-cleaving DNAzyme (DNA(1)) we have employed is totally composed of 87 nucleotides. Previous studies indicate that the most frequent sites of cleavage are located at two different regions: from 9 to 14 nucleotides and near position 72 of the sequence domain. 12 To construct a signal-on architecture for a self- cleaving DNAzyme sensor, a short ferrocene(Fc)-tagged 26 base oligonucleotide DNA(2) was introduced. The DNA(2) is predesigned by partially complementary to DNA(1) from 1 to 8 nucleotides, and School of Chemistry & Environment, Beijing University of Aeronautics & Astronautics, Beijing, 100191, China. E-mail: lilidong@buaa.edu.cn; guolin@buaa.edu.cn; Tel: +086-010-82316498 † Electronic supplementary information (ESI) available: Materials and methods, supplementary Fig. S1–S7 and Scheme S1. See DOI: 10.1039/c0ay00176g This journal is ª The Royal Society of Chemistry 2010 Anal. Methods, 2010, 2, 627–630 | 627 COMMUNICATION www.rsc.org/methods | Analytical Methods Published on 28 April 2010. Downloaded on 22/03/2015 16:56:06. 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