ARTICLE pubs.acs.org/ac
Experimental Platform to Study Heavy Metal Ion-Enzyme Interactions and Amperometric Inhibitive Assay of Agþ Based on Solution State and Immobilized Glucose Oxidase Chao Chen,† Qingji Xie,*,† Lihua Wang,† Cong Qin,† Fangyun Xie,† Shouzhuo Yao,*,†,‡ and Jinhua Chen‡ †
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China ‡ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China
bS Supporting Information ABSTRACT: The heavy metal (HM) ion-enzyme interaction is an important research topic in many areas. Using glucose oxidase (GOx) as an example, a comprehensive experimental platform based on quartz crystal microbalance and electroanalysis techniques is developed here to quantitatively study the HM ion-enzyme interactions and amperometric inhibitive assays of HM ions. The effects of some common HM ions on the bioactivities of solution-state GOx (GOxs), electrode surface-adsorbed GOx (GOxads), and polymerentrapped GOx (GOxe) are comparatively examined on the basis of anodic amperometric detection of enzymatically generated H2O2. Agþ shows the strongest inhibition effect among the HM ions examined, and the inhibitive assays of Agþ based on GOxs, GOxads, and GOxe entrapped in poly(L-noradrenalin) (PNA) give limits of detection (LOD) of 2.0, 8.0, and 5.0 nM (S/N = 3), respectively. Inhibition effects of Hg2þ, Cu2þ, and Co2þ are detectable only at 15 μM or higher concentrations, and the other HM ions show undetectable inhibition even at 1.0 mM. The developed experimental platform allows one to quantify the number of the bound HM ions per GOxads molecule at various inhibition percentages. In addition, the electrosynthesized PNA matrix to entrap GOx for an inhibitive assay of Agþ shows the lowest competitive affinity to HM ions and gives the highest sensitivity, as compared with several other polymer matrixes commonly used for the inhibitive assay. The suggested experimental platform is recommended for wide applications in enzymatic inhibitive assays and quantitative studies of the inhibition effects of HM ions on many other redox-event-relevant enzymes.
E
nvironmental contamination from toxic heavy metal (HM) ions is a worldwide concern.1-3 General consensus holds that the toxicity of HM ions mainly results from their interactions with proteins (especially various enzymes) or with nucleic acids.4 HM ions alter prooxidant/antioxidant balance and bind to sulphydryl groups or other ligands in life-relevant species, resulting in inhibition of glutathione metabolism and functions of numerous enzymes and hormones. In addition, HM ions are antagonistic to essential trace elements and usually compete with nutrient elements for binding sites on transport and storage proteins, receptors, and metalloenzymes, which may result in marked aberrations in the metabolism of carbohydrates, proteins, neurotransmitters, and hormones. The in-depth understanding of the inhibition effects of various HM ions on various enzymes is of obvious biomedical and environmental interests,5 including acquisition of the quantitative information on the relationship of the number of the bound HM ions per enzyme molecule with the inhibition percentage of enzymatic activity. r 2011 American Chemical Society
The strongest interactions of HM ions with enzymes usually take place in the cases of Hg2þ, Cu2þ, Pb2þ, Cd2þ, and Agþ.6,7 Many immobilized enzymes have been used for inhibitive assays of Hg2þ and Cu2þ, but fewer reports are on Agþ.6-12 Glucose oxidase (GOx) of low cost, good stability, and high specific activity has been widely used for glucose biosensing,13 and the inhibitive assays of several HM ions based on polymer-entrapped GOx (GOxe) have also been investigated.2,10,14-16 However, it seems that the reported inhibition effects of HM ions on GOxe are rather scattering and even controversial. For instance, GOxe entrapped in poly(o-phenylenediamine) (PoPD) was reported to be sensitive to Hg2þ and Cu2þ,14 while it was also used for quantification of the total amount of HM ions in water samples.16 GOxe entrapped in polyvinylpyridine or polyaniline was reported Received: December 1, 2010 Accepted: February 20, 2011 Published: March 10, 2011 2660
dx.doi.org/10.1021/ac1031435 | Anal. Chem. 2011, 83, 2660–2666
Analytical Chemistry to be effective in determining Hg2þ free of interferences.2,17 GOxe entrapped in poly(neutral red) was sensitive to Ca2þ, Cu2þ, Pb2þ, and Zn2þ.10 In our opinion, it is highly likely that the competitive coordination of the GOx-immobilization matrixes with HM ions are different from one another, which may affect the GOxe-based inhibitive assay but has been paid insufficient attention to date. The presence of Agþ in wastewater is an issue of environmental and economic interests. The annual release of silver to the environment is estimated to be as high as 2500 tons.18 The wide utilization of silver and its compounds in industries (e.g., electronics, photography, and mirrors) and the relatively high toxicity of Agþ highly call for the sensitive determination of Agþ.18-21 It is regulated by WHO that the standard safe concentration of Agþ for human is
