Biosensors & Bioelectronics 16 (2001) 9 – 16 A methylene blue-mediated enzyme electrode for the determination of trace mercury(II), mercury(I), methylmercury, and mercury – glutathione complex Shubo Han a , Min Zhu a , Zhuobin Yuan a, *, Xin Li b a Department of Chemistry, Graduate School, Uniersity of Science and Technology of China, Chinese Academy of Sciences, Yuquan Road, 19A Box 3908, Beijing 100039, People’s Republic of China b Department of Enironmental Science, Hebei Uniersity of Science and Technology, Shijiazhuang 050018, People’s Republic of China Received 1 September 1999; received in revised form 10 August 2000; accepted 29 August 2000 Abstract A methylene blue-mediated enzyme biosensor has been developed for the detection of inhibitors including mercury(II), mercury(I), methylmercury, and mercury – glutathione complex. The inhibition to horseradish peroxidase was apparently reversible and noncompetitive in the presence of HgCl 2 in less than 8 s and irreversibly inactivated when incubated with different concentrations of HgCl 2 for 1–8 min. The binding site of horseradish peroxidase with HgCl 2 probably was a cysteine residue SH. Mercury compounds can be assayed amperometrically with the detection limits 0.1 ng ml -1 Hg for HgCl 2 and methylmercury, 0.2 ng ml -1 Hg for Hg 2 (NO 3 ) 2 and 1.7 ng ml -1 Hg for mercury – glutathione complex. Inactivation of the immobilized horseradish peroxidase was displayed in the AFM images of the enzyme membranes. © 2001 Published by Elsevier Science B.V. Keywords: Mercury compounds; Peroxidase inhibition; Methylene blue; Mediated biosensor www.elsevier.com/locate/bios 1. Introduction Rapid and sensitive measurements of mercury(II) and related compounds are required in various fields such as environment, food industry and medicine. Clas- sical methods such as atomic absorption spectroscopy, inductively coupled plasma optical emission spectrome- try, inductively coupled plasma mass spectrometry and their combination with chromatographic techniques are in wide use (Yuan et al., 1999a). These methods need sophisticated instrumentation, skilled personnel, com- plicated sample pretreatment and a long measuring period (Evtugyn et al., 1998). Therefore, electrochemi- cal methods such as ion selective electrodes, polarogra- phy, and other voltammetry are also widely used due to their less complex instrumentation and shorter measur- ing period. However, more sensitive methods are ur- gently needed in the analysis of environmental and clinical samples to measure trace amounts of mercury compounds usually in the range of ng ml -1 or lower therein. Biosensors provide rapid measurements without time-consuming purification or fractionation proce- dures for the analysis of heavy metal compounds. Sev- eral configurations have been described in the past including enzyme biosensors (Mattiasson et al., 1978; O  gren and Johansson, 1978; Danielsson et al., 1981; Gayet et al., 1993; Shekhovtsova and Chernetskaya, 1994; Amine et al., 1995; Preininger and Wolfbeis, 1996; Wong et al., 1997; Fennouth et al., 1998), whole cell biosensors (Evtugyn et al., 1998; Bontidean et al., 1998) and genetically modified biosensors (Watton et al., 1990; Salifonova et al., 1993; Tescione and Belfort, 1993; Virta et al., 1995; Klein et al., 1997; Evtugyn et al., 1998) for the determination of mercury(II) and related compounds. Among them, an inhibitor biosen- sor is far more sensitive than a substrate biosensor (Uo et al., 1992). The detection limit is much lower than the * Corresponding author. Tel.: +86-10-68224596; fax: +86-10- 68210501. E-mail address: yuanzhuobin@263.net (Z. Yuan). 0956-5663/01/$ - see front matter © 2001 Published by Elsevier Science B.V. PII: S0956-5663(00)00114-7