Biosensors and Bioelectronics 24 (2009) 2858–2863 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Automated and ultrasensitive detection of methyl-3-quinoxaline-2-carboxylic acid by using gold nanoparticles probes SIA-rt-PCR Wei Chen a , Yuan Jiang b , Baoqing Ji a , Changqin Zhu b , Liqiang Liu a , Chifang Peng a , M. Kim Jin a , Ruirui Qiao c , Zhengyu Jin a , Libing Wang a , Shuifang Zhu d,∗ , Chuanlai Xu a,∗ a School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, PR China b Food Laboratory, Jiangsu Import and Export Inspection and Quarantine Bureau, Nanjing, Jiangsu 210000, PR China c Institute of Chemistry, CAS, Zhong Guan Cun, Bei Yi Jie 2, Beijing 10008, PR China d Chinese Academy of Inspection and Quarantine, Beijing 10008, PR China article info Article history: Received 29 December 2008 Received in revised form 12 February 2009 Accepted 13 February 2009 Available online 27 February 2009 Keywords: MQCA Gold Superparamagnetic nanoparticles Sequential injection analysis rt-PCR abstract An ultrasensitive and rapid sequential injection analysis (SIA) based on real-time PCR (SIA-rt-PCR) assay was developed by using different nanoparticles for the detection of small molecule chemicals residues. Gold (Au) nanoparticle, conjugated with goat anti-rabbit IgG and duplex strand DNA (dsDNA), was used as a substitute for chemiluminescent probes in an SIA system. By indirect competitive immunoreactions in the SIA system, the gold nanoparticles were attached to antigens which were immobilized by super- paramagnetic nanoparticles (SMNPs). The dsDNA on the gold nanoparticles was dehybridized and then the single-stranded DNA (ssDNA) was collected and quantified with rt-PCR, which showed a rather low linearity range from 2.5 atto mol L -1 (aM) to 250 femto mol L -1 (fM) and the LOD was 1.4 aM. This method, which is rapid, automated and capable of high-throughput, was used to detect methyl-3-quinoxaline-2- carboxylic acid (MQCA) residues in real samples. The analytical results had a coefficient of variation of less than 15% and the recovery was 89–108%. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. 1. Introduction Recently, a great deal of attention has been focused on the application of metal nanoparticles conjugated with oligonu- cleotide probes, which are called nanoparticle probes (Marshall and Hodgson, 1998; Seeman, 2003; Mirkin, 2000; Mirkin et al., 1996). Mirkin and his co-workers first functionalized the gold nanoparticles with both DNA and antibody to establish a semiquan- titative method for diagnosing disease (Nam et al., 2003; Jin et al., 2003; Keating, 2005; Dubertret et al., 2001). However, the lack of automation and the complicated separation steps make the method unsuitable for routine sample analysis. Since the sequential injection analysis (SIA) system, also called the second generation of flow injection analysis (FIA), was described by Ruzicka and Marshall in 1990 (Ruzicka and Marshall, 1990), it has been widely applied in bioanalytical science due to its tremendous advantages, which include suitability for automation and minia- turizing fluidic operation, sensitive, and short periods of incubation and microbeads trapping (Lenghor et al., 2003; Guzman et al., 1993). The beads injection (BI) technique, also developed by Ruzicka, can ∗ Corresponding author. Tel.: +86 510 85329076; fax: +86 510 85329076. E-mail addresses: zhushf@netchina.com.cn (S. Zhu), xcl@jiangnan.edu.cn (C. Xu). be combined with the SIA system, allowing immunoreactions to proceed on the microbeads with a quite unique “jet ring cell” (Chandler and Brockman, 2000; Ruzicka and Ivaska, 1997; Hartwell et al., 2004). Chemiluminescence (CL) is one of the most sensitive techniques for trace analysis and has been applied successfully with the SIA system in an immunoassay format (Xu et al., 2006; Zhang et al., 2007; Luo and Yang, 2007). Unfortunately, the sensitivity of this technique is limited by the following drawbacks: (a) it is difficult to select an optimum pH value for both enzymatic and chemilumi- nescent reaction with a desirable flow rate; (b) incomplete contact between enzyme and substrate; (c) the light scattering phenomena induced by the reactor cell decreases the flux and reproducibility; and (d) the chemiluminescent signal declined with the detection time. According to our previously reported paper (Xu et al., 2006), the limit of detection of clenbuterol is 0.01 ng mL -1 , which is much higher than the LOD of the constructed method in this paper. Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is the last major remaining detectable biomarker of olaquindox (OLA), an antibacterial agent that has been banned in the EU since January 1st 1999 due to the health concerns over possible carcinogenic, muta- genic and photoallergenic effects of the drug and its metabolites. It was banned in 2002, however there are many cases of illegal use in animal production in China (FAO/WHO, 1995; Hurtaud-Pessel et al., 0956-5663/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.02.015