A novel immunochromatographic assay based on a time-resolved chemiluminescence strategy for the multiplexed detection of ractopamine and clenbuterol.

A novel multiplexed immunochromatographic assay (ICA) based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of β-agonists, by utilizing ractopamine (RAC) and clenbuterol (CLE) as the models. Different from conventional multiplexed ICA methods which usually require two or more test lines, this strategy was developed for detection of two β-agonists by using only one test line on the nitrocellulose membrane. In this study, horseradish peroxidase and alkaline phosphatase were used as the signal probes to label RAC antibody and CLE antibody, respectively. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by injecting the coreactants, then the signals for RAC and CLE detections were recorded at 3 s and 300 s after coreactants injection, respectively. Owing to the utilization of CL detection, this protocol showed ideal sensitivity for quantitation. Under the optimal conditions, the detection limits for RAC and CLE were 0.17 ng mL(-1) and 0.067 ng mL(-1) (S/N = 3), respectively. The whole assay process can be accomplished within 20 min without complicated sample pretreatment. The proposed method was successfully applied for the detection of RAC and CLE in spiked swine urine. It opens up a new pathway for designing a low cost, time-efficiency and multiplexed strategy for rapid screening and field assay.

[1]  A. Berlina,et al.  'Traffic light' immunochromatographic test based on multicolor quantum dots for the simultaneous detection of several antibiotics in milk. , 2015, Biosensors & bioelectronics.

[2]  Suherman,et al.  Highly selective and sensitive detection of β-agonists using a surface plasmon resonance sensor based on an alkanethiol monolayer functionalized on a Au surface. , 2015, Biosensors & bioelectronics.

[3]  Xiaoyun Liu,et al.  Determination of ractopamine in pig hair using liquid chromatography with tandem mass spectrometric detection. , 2014, Journal of pharmacological and toxicological methods.

[4]  Huan Yu,et al.  Application of ionic liquid as additive in determination of three β‐agonists by capillary electrophoresis with amperometric detection , 2013, Electrophoresis.

[5]  Eshaq Isaac Shishani,et al.  Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry , 2003 .

[6]  Guodong Liu,et al.  Multiplex electrochemical immunoassay using gold nanoparticle probes and immunochromatographic strips , 2008 .

[7]  M. Himmelsbach,et al.  A sensitive non-aqueous capillary electrophoresis-mass spectrometric method for multiresidue analyses of beta-agonists in pork. , 2009, Biomedical chromatography : BMC.

[8]  Yahong Liu,et al.  Determination of ractopamine and clenbuterol in feeds by gas chromatography–mass spectrometry , 2007 .

[9]  Yan Zhang,et al.  Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution. , 2010, Journal of chromatography. A.

[10]  Shijia Yang,et al.  Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol. , 2014, Analytica chimica acta.

[11]  Ting Xu,et al.  Strip-based immunoassay for the simultaneous detection of the neonicotinoid insecticides imidacloprid and thiamethoxam in agricultural products. , 2012, Talanta.

[12]  Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane , 2011, Analytical and bioanalytical chemistry.

[13]  Fanggui Ye,et al.  Magnetic bead-sensing-platform-based chemiluminescence resonance energy transfer and its immunoassay application. , 2012, Analytical chemistry.

[14]  P. Carrola,et al.  Clenbuterol food poisoning diagnosis by gas chromatography–mass spectrometric serum analysis , 2003 .

[15]  Xianbo Qiu,et al.  A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids. , 2009, Lab on a chip.

[16]  Wei Zhang,et al.  Separation and determination of abused drugs clenbuterol and salbutamol from complex extractants in swine feed by capillary zone electrophoresis with simple pretreatment. , 2008, Talanta.

[17]  H. Kuiper,et al.  Illegal use of beta-adrenergic agonists: European Community. , 1998, Journal of animal science.

[18]  G A Mitchell,et al.  Illegal use of beta-adrenergic agonists in the United States. , 1998, Journal of animal science.

[19]  Parin Chaivisuthangkura,et al.  Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples. , 2013, Biosensors & bioelectronics.

[20]  Fan Zhang,et al.  Lanthanide-labeled immunochromatographic strips for the rapid detection of Pantoea stewartii subsp. stewartii. , 2014, Biosensors & bioelectronics.

[21]  A. Abad‐Somovilla,et al.  Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron. , 2013, Biosensors & bioelectronics.

[22]  H. Ouyang,et al.  Multiplexed detection of two proteins by a reaction kinetics-resolved chemiluminescence immunoassay strategy. , 2015, The Analyst.

[23]  Y. Gan,et al.  Determination of salbutamol in human plasma and urine by high-performance liquid chromatography with a coulometric electrode array system. , 2004, Journal of chromatographic science.

[24]  Yihua Zhang,et al.  Determination of salbutamol using R-phycoerythrin immobilized on eggshell membrane surface as a fluorescence probe , 2010, Analytical and bioanalytical chemistry.

[25]  Peilong Wang,et al.  A sensitive and quantitative fluorescent multi-component immuno-chromatographic sensor for β-agonist residues. , 2015, Biosensors & bioelectronics.

[26]  Zuanguang Chen,et al.  A sensitive electrochemical sensor with sulfonated graphene sheets/oxygen-functionalized multi-walled carbon nanotubes modified electrode for the detection of clenbuterol , 2015 .

[27]  A. Molina-Díaz,et al.  Combined data mining strategy for the systematic identification of sport drug metabolites in urine by liquid chromatography time-of-flight mass spectrometry. , 2013, Analytica chimica acta.

[28]  Shen-Ming Chen,et al.  Simultaneous determination for toxic ractopamine and salbutamol in pork sample using hybrid carbon nanotubes , 2013 .

[29]  Mingyuan Gao,et al.  Lateral flow immunochromatographic assay for sensitive pesticide detection by using Fe3O4 nanoparticle aggregates as color reagents. , 2011, Analytical chemistry.