ABSTRACT
Many families of enzymes such as oxidases, dehydrogenases, phosphatases, kinases and ureases have been used for heavy metals detection. The detection is based on activation or inhibition of enzyme activity. Detected heavy metal ion activates the enzyme in case the ion is a part of the enzyme structure, or inhibits it when the ion is able to bond to the active centre of employed enzyme and, thus, inactivates it (Verma and Singh 2005). The biosensor for zinc (II) ions detection based on phosphatases alkali activation has been developed because this ion is a component of the enzyme active centre. The whole system was implemented in micro-injected fl uid analysis coupled with calorimetric sensor. Enzyme was covalently immobilized and the authors declare that they were able to determine zinc (II) ions within the range from micromolar to milimolar concentration. The response time was 3 min, which is very important for measurements of larger set of samples. Furthermore, the authors achieved the notable stability of the biosensor, which is very important for analysis of samples without losing the sensitivity and selectivity for a period of longer than 2 mon (Satoh 1991). Biosensors for heavy metal ions determination based on inhibition of enzyme activity use more than one enzyme compared to those based on activation. Oxidases and dehydrogenases belong to the most commonly used enzymes in these types of biosensors. These enzymes are immobilized due to reticulate gelatinous fi lm or affi nity interaction with a special type of membrane. For mercury (II) ions determination, L-glycerol phosphate oxidase coupled with Clarc electrode was used; the detection limit 20 µM was achieved. The biosensor was able to regenerate using ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (Gayet et al. 1993). The same authors used pyruvate monoamino oxidase to compare it with the previous enzymes and achieved three times lower detection limit (50 nM mercury (II) ions). It was also possible to regenerate the biosensor by the same chemicals (Gayet et al. 1993). Furthermore, the biosensor consisting of enzymatic system including L-lactate dehydrogenase and L-lactate oxidase as the
substance non-sensitive for heavy metals-was developed and proved to be a good choice for various metal ions detection. The detection limits were as follows: 1.0 µM HgCl2, 0.1 µM AgNO3, 10 µM CdCl2, 10 µM ZnCl2, 50 µM Pb (CH3COO)2 and 250 µM CuSO4 (Gayet et al. 1993; Verma and Singh 2005). This system was also applied for detection of Hg(II), Ag(I), Pb(II), Cu(II) and Zn(II) ions, where Fennouh et al. obtained twice the lower limit detection as compared with the previous study (Fennouh et al. 1998). It was possible to regenerate the biosensor in mixture of EDTA, KCN and dithiothreitol. In addition, inhibitive infl uence of chromium (III) ions to L-lactate dehydrogenase, hexokinase and pyruvate kinase was utilized for chromium (III) sensitive biosensors. Moreover, various interferences including other metal ions were used in this study and the results were evaluated using artifi cial neural networks (Cowell et al. 1995). The specifi c inhibition of peroxidase by mercury (II) ions was observed after enzyme immobilization in chitosan. The attained concentration interval was from 0.02 to 1000 µM Hg(II) (Shekhovtsova et al. 1997). Other large groups of enzymatic biosensors are based on urease. Optical biosensor based on urease immobilized on glass pores was developed for mercury (II) ions determination. However, the concentration interval, in which the biosensor operates, was only from one to 10 µM (Andres and Narayanaswamy 1995). One of the one-shot approaches using urease was established on combination with ammonia sensitive optode and ammonium ions sensitive optode. Limits of detection were as follows: Ag (I) 0.18 µM, Hg(II) 0.35 µM, and Cu(II) 3.94 µM. The study also showed that the above mentioned metals exhibited synergic effects to inhibition, which was confi rmed by the highest inhibition of metal ions mixture in comparison with single metals (Preininger and Wolfbeis 1996). Ion-sensitive fi eld-effect transistors (ISFETs) in combination with urease were used besides optodes. Based on the suggested system the detection limits for Ag(I), Hg(II) and Cu(II) were down to units of µM. Furthermore, the authors succeeded in fi nding a way to modify the specifi c biosensor to be sensitive to Hg(II) only. The suggested method employs NaI as a masking agent for Ag(I) and EDTA for Cu(II) (Volotovsky et al. 1997). Urease inhibition by mercury (II) ions was also studied using the potentiometric biosensor (Krawczyk et al. 2000). Interaction between urease and nickel (II) ions is another promising way to modify the biosensor for heavy metals ion detection (Verma and Singh 2006). Recently, it was shown that urease system and glutamate dehydrogenase may be employed for mercury (II), copper (II), cadmium (II) and zinc (II) ions detection. Amperometric determination was used for the suggested method (Rodriguez et al. 2004).
