Paramagnetic antibody-modified microparticles coupled with voltammetry as a tool for isolation and detection of metallothionen as a bioindicator of metal pollution Libuse Trnkova, a Ivo Fabrik, b Dalibor Huska, b Helena Skutkova, c Miroslava Beklova, d Jaromir Hubalek, e Vojtech Adam, bc Ivo Provaznik c and Rene Kizek * b Received 7th April 2011, Accepted 11th July 2011 DOI: 10.1039/c0em00306a Low-molecular mass proteins rich in cysteines called metallothioneins (MT) can be considered as markers for the pollution of the environment by metals. Here, we report on suggestion for an automated procedure for the isolation of MT followed by voltammetric analysis. Primarily, we optimized the automated detection of MT using an electrochemical analyser. It was found that the most sensitive and repeatable analyses are obtained at a temperature of 4  C for the supporting electrolyte. Further, we optimized experimental conditions for the isolation of MT by using antibody-linked paramagnetic microparticles. Under the optimal conditions (4 h long interaction between the microparticles and MT), the microparticles were tested on isolation of various amounts of MT. The lowest isolated amount of MT by antibody-linked paramagnetic microparticles was 5 mg ml 1 of MT (50 ng). The automated procedure of MT isolation was further tested on isolation of MT from guppy fish (Poecilia reticulata) treated with silver(I) ions (50 mM AgNO 3 ). The whole process lasted less than five hours and was fully automated. We attempted to correlate these results with the standard method for MT isolation. The correlation coefficient is 0.9901, which confirms that results are in good agreement. Moreover, the concentration of silver ions in tissues of fish treated with Ag(I) ions was determined by high performance liquid chromatography with electrochemical detection. 1. Introduction Pollution of the environment with toxic metals due to anthro- pogenic activities poses a threat to plants as well as animals. Mining, fertilizing, using fossil fuels and others belong to the main sources of metal-pollution. 1 Prior to the development and use of silver, the majority of silver occurring in surface waters originated from natural leaching, mining and the photographic industry, 2 with measured concentrations in natural and contaminated waters in the ng l 1 and higher range (Sanudo- Willhelmy and Flegal, 1992). Since the 1970s, concentrations of silver have decreased in surface waters due to the restrictions of using Ag in the photographic industry and more stringent a Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic b Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic. E-mail: kizek@sci.muni.cz; Fax: +420-5-4521-2044; Tel: +420-5-4513-3350 c Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Kolejni 4, CZ-612 00 Brno, Czech Republic d Department of Veterinary Ecology and Environmental Protection, Faculty of Veterinary Hygiene, Ecology, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, CZ-612 42 Brno, Czech Republic e Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of Technology, Udolni 53, CZ-602 00 Brno, Czech Republic Environmental impact An easy-to-use and automated procedure for the isolation of metallothioneins (MT) as markers of metal pollution in the envi- ronment is missing. In this study, we optimize experimental conditions for the isolation of MT by using antibody-linked para- magnetic microparticles and also for the automated electrochemical determination of the isolated proteins. The automated procedure of MT isolation and determination was tested on isolation of MT from guppy fish (Poecilia reticulata) treated with silver (I) ions (50 mM AgNO 3 ). Moreover, we attempted to correlate the MT levels determined using standard procedures with the optimized one based on antibody-linked paramagnetic microparticles. We attempted to correlate these results with standard method for MT isolation. The correlation coefficient is 0.9901, which confirms that results are in good agreement. This journal is ª The Royal Society of Chemistry 2011 J. Environ. Monit., 2011, 13, 2763–2769 | 2763 Dynamic Article Links C < Journal of Environmental Monitoring Cite this: J. Environ. Monit., 2011, 13, 2763 www.rsc.org/jem PAPER