Amalgam electrodes as tool for study of environmental important compounds and for detection of DNA damages

Transcription

1 Amalgam electrodes as tool for study of environmental important compounds and for detection of DNA damages Bogdan Yosypchuk, Miroslav Fojta, and Jiří Barek Abstract This paper will deal with recent results regarding voltammetric and amperometric determinations of micromolar and submicromolar concentrations of various environmentally important biologically active inorganic and organic substances using solid and paste amalgam electrodes, which can be environmentally friendly alternatives to mercury electrodes. Attention will be paid to amalgam electrodes either in batch analysis or in flow liquid systems (especially HPLC or flow-injection analysis with electrochemical detection). Silver amalgam electrodes can be used in voltammetric and amperometric analysis as an alternative to mercury electrodes. Other solid amalgam electrodes (e.g., CuSAE, AuSAE) are convenient for specific purposes, where properties of the metal, of which the solid amalgam consists, are employed. Different types of amalgam electrodes have been used as highly sensitive tools for the detection of DNA strand breaks, as sensors for DNA cleaving substances, for the detection of enzymatic or chemical DNA cleavage in solution or at the electrode surface, as a sensor for DNA nicking substances and for the detection of DNA strand breaks induced by ionizing radiation. Keywords Amalgam electrodes, DNA, Electrochemistry, Environmental analysis. M I. INTRODUCTION ONITORING of detrimental compounds in the environment is one of the most important tasks of modern analytical chemistry. Electrochemical methods are especially suitable for large scale environmental monitoring of electrochemically active pollutants because they are inexpensive, extremely sensitive and they present an independent alternative to so far prevalent spectrometric and separation techniques [1]. Development of sufficiently sensitive and selective voltammetric and amperometric methods for determination of various environmentally Manuscript received September 29, This work was supported by the GA AV ČR (project IAA ) and by the Ministry of Education, Youth and Sports of the ČR (project LC06063). B. Yosypchuk is with the Department of Biophysical Chemistry, J.Heyrovský Institute of Physical Chemistry of AS CR, v.v.i., Dolejskova 3, Prague 8, Czech Republic (corresponding author to provide phone: ; josypcuk@jh-inst.cas.cz). M. Fojta is with the Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics of AS CR, v.v.i., Kralovopolska 135, Brno, Czech Republic ( fojta@bpi.cz). J. Barek is with the Charles University in Prague, Faculty of Science, Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry, Albertov 6, Prague 2, Czech Republic ( barek@natur.cuni.cz). important substances is the main task of different laboratories. We pay attention to new electrode materials characterized by broader potential window, higher signal-to-noise ratio, mechanical stability enabling their application in flowing systems, and resistance toward passivation [2]. The last requirement is especially important because electrode fouling is probably the biggest obstacle to more frequent applications of electroanalytical methods in environmental analysis. Last but not least we search for nontoxic electrode material friendly toward the environment and thus compatible with the concept of so called green analytical chemistry. Amalgam electrodes (AE) are environmentally friendly alternatives to mercury electrodes suitable both for batch analysis and for HPLC with electrochemical detection (HPLC-ED) or for flow injection analysis with electrochemical detection (FIA-ED). They can be easily prepared in any laboratory and their simple electrochemical pretreatment in many cases eliminates problems with their passivation [2]-[4]. Development of electrochemical sensors responding to DNA damage - together with sensors for DNA hybridization and those for DNA-drug interactions - belong to the main present challenges addressed to researchers dealing with nucleic acid electrochemistry [5], [6]. Sensitive determination of DNA damage can be used as an important diagnostic criterion giving evidence about the exposure of an organism to various toxicants. In addition, DNA biosensors may be used as tools for the detection of genotoxic agents in biological samples, in food or in the environment [5], [7]. II. DEVELOPMENT OF NEW TYPES OF WORKING ELECTRODES Mercury is the best electrode material because of easily renewable and atomically smooth surface and large potential window. However, there is a tendency to avoid the use of mercury because of fears of its toxicity and because of its low mechanical stability complicating the use of mercury electrodes in flowing systems and in portable devices. Development of new solid electrode materials is the result of compromise between achieving the desired characteristics and keeping the measurement reproducibility on a required level. Modern trends in analytical applications (flow analysis, automatic systems, application of monitoring devices, electrochemical detection in HPLC, etc.) require introduction of solid electrodes and practically exclude the possibility of ISBN:

2 mechanical polishing or chemical surface regeneration of the working electrode. Qualitatively close alternative to liquid mercury electrodes should be solid electrode with a wide interval of working potential, comparable with mercury electrodes, which can be regenerated electrochemically with sufficient effectiveness. The use of modern, computer controlled devices, enables the realization of automatic electrochemical regeneration before each measurement in a chosen way. Our efforts in development and applications of solid [2], [8]-[10] and paste [2], [11] amalgam electrodes (AE) have been motivated by the need of new analytical tools based on nontoxic, harmless, environment-friendly materials retaining the electrochemical features of mercury electrodes. III. WORKING AMALGAM ELECTRODES FOR ENVIRONMENTAL ANALYSIS Amalgams can be liquid, paste or solid in dependence on the mercury/metal ratio. Solid and paste amalgam working electrodes (WE) can be classified according to the state of their surface as follows: polished solid amalgam electrode not containing liquid mercury (p-mesae) the surface of which was mechanically polished; film modified polished MeSAE covered by mercury film (MF-MeSAE); meniscus modified polished MeSAE covered by mercury meniscus (m-mesae); composite WE based on fine amalgam powder dispersed in a solid polymer (MeSA-CE); paste with pasting liquid WE based on fine amalgam powder dispersed in a suitable pasting liquid (MeSA-PE); paste without pasting liquid WE based on paste amalgam with relatively low content of a metal (MeA-PE). MeSAE can be used directly after surface polishing (p-mesae). The potential window of p-agsae is for the electrodes, which do not contain any liquid mercury extremely wide, often comparable with mercury electrodes. This fact enables to apply p-agsae for determination of very electronegative compounds (Zn 2+, Mn 2+, IO 3, etc.), for determination of electrochemical processes in region of very negative potentials (catalytic hydrogen evolution, adsorptiondesorption of organic compounds, etc.), and in such cases when the use of mercury containing electrodes is unsuitable. The solid surface of p-mesae can be modified in various ways, which substantially extends its applicability. Very important is the fact that it is possible to prepare the electrodes relatively easily from solid amalgams of various metals and subsequently to utilize specific interaction of these metals with the investigated compound. From the analytical point of view, AEs modified by mercury proved to be the best, namely modified by mercury meniscus [2], [8] or by mercury film [10]. Their liquid surface is ideally smooth and homogeneous, which solves the principal problem of all solid electrodes the mechanical regeneration of their surfaces and with this complication often connected insufficient reproducibility of repeated measurements. Electrochemical regeneration of the surface of meniscus or film electrode before each measurement can be carried out by means of a computer controlled analyzer (where it is possible to build-in this operation relatively easily into controlling software). It enables to reach relative standard deviation (RSD) of repeated determinations below 2 3 %. A. Determination of inorganic compounds The amalgam electrodes can find very wide field of application for determination of cations, namely of almost all heavy metals. Determination of metallic ions is essential part of environmental monitoring, tracing occurrence of harmful substances and of biogenous elements in drinking water, beverages, body fluids, in control of technological processes, etc. Hanging mercury drop electrode (HMDE), electrodes from noble metals eventually modified by mercury film, by mercury meniscus or by biologically active substances, composite electrodes and electrodes from various types of carbon are most frequently used in voltammetry for these purposes. We have tested the possibility of determination of As(III), Cd(II), Co(II), Cr(III), Cu(II), Fe(III), In(III), Mn(II), Ni(II), Pb(II), Sn(II), Tl(I), Zn(II), etc. using various AEs (e.g., [2], [4]). For determination of anions realized with AEs it is possible to utilize various electrochemical processes, such as reduction (IO 3, Nb(V)), catalytic effects (NO 3 ), chemisorption in combination with follow-up cathodic scan (Cl, Br, I, S 2, CNS etc.), or adsorption (Cr(VI)). Determinations of some compounds (mostly in different types of water) are described below. Some of these determinations were published in detail in our papers [2]-[4]. Determination of Cu, Pb, Cd, Zn and Tl. AgSAE enables, similarly as mercury electrodes, to determine copper, lead, cadmium and zinc in one potential scan. Concentration of these ions in drinking water can vary from µg l -1 level for Cd and Tl to a few mg l -1 for Cu and Zn. Anodic stripping voltammetry is usually used for these determinations the determined metals are electrolytically deposited (accumulated) on the working electrode surface in the first step and they are stripped out in the second step, which process is connected with registration of the passing currents. The time of accumulation depends on analyte concentration. It usually varies from a few minutes (for µg l -1 levels) to a few seconds (for mg l -1 levels). If the concentration of determined ions in the analyzed solution is about 0.5 mg l -1 and higher, it is possible to use direct cathodic scan and to leave out the accumulation process. For analytical purposes differential pulse voltammetry is most frequently used, which exhibits higher sensitivity and yields well developed and symmetric, easily evaluable peaks. Determination of Ni. Adsorption stripping voltammetry belongs to one of the most sensitive methods, enabling to determine concentrations of, e.g., chromium, uranium, vanadium, aluminium on the level of mol l -1. Selection of the proper complexing compound can essentially affect the analytical selectivity. Determination of nickel in ISBN:

3 ammonium buffer in the presence of dimethylglyoxime represents a classical example of the adsorptive stripping voltammetry. Nickel forms sufficiently selectively a complex with dimethylglyoxime, which is adsorbed on the electrode surface. During the cathodic scan nickel is reduced in the adsorbed complex and it yields a well developed peak. The best linearity was achieved in nickel concentration interval from 10 to 100 µg l -1. The low limits of detection (0.9 µg l -1 ) and RSD 3.0 % enable reliable determination of current nickel concentration in drinking water. Even at lower nickel concentrations (1-10 µg l -1 ) the registered current is well measurable, however, the linearity of concentration dependences is somewhat worse (R = ). Determination of Fe(III). The well developed reduction wave corresponding to the process Fe(III) Fe(II), recorded on mercury electrodes from triethanolamine complex in alkaline solutions is mostly used for analytical purposes. Due to the fact that accumulation of iron is not possible on the surface of mercury working electrode, the sensitivity of such determinations is not too high. With respect to low iron toxicity, this process is sufficient for its determination at limiting value concentrations (according to the valid standards) in drinking water. Alkaline supporting electrolyte containing triethanolamine was chosen as the most suitable for iron determination with m-agsae. To eliminate the interfering effect of zinc, and manganese eventually, which can occur in waters in concentrations comparable with iron concentration, complexing agent Na 2 EDTA was added to this solution. Peak potentials of zinc and manganese in EDTA-complex were thus shifted to very negative values and these ions cannot affect the peak of Fe(III). The achieved acceptable repeatability (RSD = 2.3%) and sufficient sensitivity (0.2 mg l -1 ) confirm the possibility of the use of m-agsae for determination of Fe(III) in drinking water. The dependence of heights of the well developed peaks of iron on its concentration, exhibits linear course (R = ) in a wide concentration range of Fe(III). Determination of NO 3 -. Since the nitrate ions are not reduced on mercury electrodes directly, the catalytic reoxidation in the presence of multivalent elements is utilized. The cerium solution, which proved suitable with mercury electrodes, was applied for testing the possibilities of determination of nitrates with m-agsae. The linearity of the current response in dependence on concentration (in range mg l -1 ) seems very good (R = ) and it attests the application of m-agsae for nitrates analysis as possible. The linear part of the concentration dependence includes the limiting values of concentration of nitrates, which are defined in standards for different types of waters. Nevertheless, if the concentration of NO 3 - ions exceeds the mentioned linear part, the analyzed sample can be easily diluted by distilled water. Determination of IO 3 -. The reduction of iodates is carried out at relatively highly negative potential range and therefore it was mostly accomplished on the classic mercury electrodes. It was proved that it is possible to carry out the determination of iodates using p-agsae containing no liquid mercury. The suggested fast and very simple method offers a very suitable possibility of iodate determination in real samples (waters or table salt). It was found by testing of various amalgam electrodes that AgSAE is the best electrode for the determination of iodates. The potential window of m-agsae, p-agsae and mercury electrode in the used 0.1M NaOH solution is almost identical. It is possible to note for both electrodes that the linearity of achieved calibration curves and the repeatability of measurements is excellent (R = and RSD = 0.5 % for m-agsae, and R = and RSD = 1.2 % for p-agsae). B. Determination of organic compounds Various modes of voltammetry can be used for the determination of organic compounds in dependence on their electrochemical properties. Most of compounds which we studied are reduced during their scan and exhibit linear dependences of the peak height on their concentration in ranges of 2 3 concentration orders. Limits of detection of these methods are about 10-6 mol l -1. Therefore, it is possible to determine various biologically active organic compounds, pesticides, agrochemicals, drugs or derivatives of these substances with amalgam electrodes using their direct reduction [2], [4]. Usually some functional group is reduced (e.g., nitro group, azo group, single bond (Cl-Cl, C-S, etc.), double or triple bond). Therefore, the selectivity of such determinations is not too high. Selectivity and sensitivity of these measurements can be substantially improved by proper sample pretreatment, by combination of voltammetry with preseparation using HPLC or by separation and preconcentration using solid phase extraction (SPE) [12]. SPE enables determination of nanomolar concentrations of the tested substances. Cathodic stripping voltammetry (CSV) is a very sensitive method for determination of organic compounds. CSV is based on the principle that the material of the working electrode is dissolved during fixed period at defined potential at first, the resulting cations (Ag(I) and/or Hg(I) in case of AgSAE; Cu(I) in case of CuSAE, etc.) form compounds with the determined analyte (adenine, guanine, cysteine, phytochelatins, etc), which are adsorbed at the electrode. The reduction of such metal ions from the adsorbed component is observable as a voltammetric peak. This procedure is applicable for voltammetric analysis of such substances, which can react with metal cations, released from the working electrode (e.g., compounds containing SH or SS- groups). To the advantages of amalgam electrodes belongs the fact that it is possible to prepare the electrode material directly with the metal, which interacts with the analyzed compound and by this procedure to increase the selectivity of the discussed electrochemical measurements. Since the analyte is accumulated at the working electrode in case of CSV, the limit of detection of thus realized determinations is commonly in the level of mol l -1 [2]. Amalgam electrodes, similarly as mercury electrodes, do not possess great potentiality for oxidation processes due to dissolution of electrode material at relatively low positive ISBN:

4 potentials. Suitable compound for such measurements is ascorbic acid in beverages and waters. On AgA-PE, the response was a linear function of concentration of ascorbic acid in the region mol l -1 (R = ). Appropriate repeatability was provided by electrochemical regeneration, no exchange of active surface was necessary. Some substances are strongly adsorbed at the surface of the working electrodes and they can be reduced, oxidized or desorbed in such adsorbed state. Each of these processes is accompanied by change of the registered signals. On favorable terms is the current change proportional to the amount of the analyte at the electrode surface. The adsorption at the surface of the discussed amalgam electrodes is so much stable in case of some biological macromolecules that it is possible to talk about electrodes modified by the corresponding macromolecule. The electrode with strongly adsorbed molecules can be rinsed and transferred into the cell with clean supporting electrolyte. Using proper electrochemical method, it is possible to record the electrochemical signal. This procedure is usually called adsorptive transfer stripping voltammetry (AdTSV) [5], [13]. One of the most pronounced advantages of the use of the solid amalgam electrodes in AdTSV consists in fact that in some special cases 1 2 µl of the sample is the sufficient amount for analysis. This small volume of the substance is put on directly at the active working surface of the electrode by a micropipette (for the traditional analysis at least 1 ml of the sample is necessary) [14]. One of the ways to substantially increase the sensitivity of the measurement is the adsorption of a compound, which is catalytically active. The catalytic currents are by several orders of magnitude higher than those controlled by diffusion, and it results in sensitivity increase. For example, the detection limit of DNA modified by complex of osmium tetroxide with 2,2 -bipyridine on mercury electrode was 0.1 ng ml -1 (RSD = 2.3 %, N = 11), and on m-agsae it was 0.2 ng ml -1 (RSD = 3.1 %, N = 11) [14]. Determination of dinitronaphthalenes. Dinitro-naphthalenes (DNN) belong to the group of genotoxic nitrated polycyclic aromatic hydrocarbons. Environment is contaminated with these compounds first of all due to combustion processes of fossil fuels and by photochemical reactions of polycyclic aromatic hydrocarbons with nitrogen oxides in the atmosphere. In consequence of the presence of electrochemically reducible nitro groups it is possible to apply modern electroanalytical methods for DNN analysis. Optimized determinations of 1,3- and 1,8-dinitronaphthalenes with m-agsae were used for their measurements in drinking water as model matrix. Under optimal conditions, voltammograms of 1,3-DNN a 1,8-DNN in dependence on concentration were recorded and plotted in corresponding calibration graphs (R 1,3-DNN = and R 1,8- DNN = ). Limits of detections of 1,3-DNN and of 1,8- DNN are 1 µm l -1 and 0.5 µm l -1, respectively. Determination of 2-nitrophenol. Nitrophenols the coming from pesticide degradation products, car exhausts, and industrial wastes are listed as priority pollutants by the US Environmental Protection Agency. They are potential carcinogens, teratogens, and mutagens. Because of their toxicity and vast scale distribution in the environment, their determinations have become one of the important goals of environmental analysis. The practical applicability of these methods after model experiments with deionized water was confirmed by determination of 2-nitrophenol (2-NP) in drinking water as a simple environmental matrix. To improve limit of determination, preconcentration by solid phase extraction (SPE) from 100 ml and 1000 ml samples was used. It was shown that in combination m-agsae with a preliminary separation and preconcentration by SPE it is possible to determine concentration of 2-NP down to 20 nmol l -1. For methods without pre-concentration, limits of determination are similar as for mercury electrode (2 µmol l -1 ). Electrochemical detection with flow-injection system. A combination of electrochemical detection with flow-injection system vastly improves the productivity by substantially decreasing the time per one analysis. An important issue concerning electrochemical detection at solid electrodes is electrode fouling due to irreversible adsorption of reaction products and intermediates, which results in a need for electrode pretreatment. Moreover, the electrode itself should be mechanically stable which complicates the use of mercury electrodes in flow techniques. Electrode materials based on non-toxic solid amalgam were successfully tested for FIA-ED and HPLC-ED determination of trace amounts of electrochemically reducible environmental active organic compounds in water samples. It was shown that mechanically stable AgSAE can be used for determination of organic water pollutants in flowing systems. Wall-jet arrangement with AgSAE was used for determination of model samples of electrochemically active pesticides (2-methyl-4,6- dinitrophenol), grow stimulators (4-nitrophenol) and carcinogens (5-nitroquinoline). The main advantage of the use of AgSAE in flowing system is its mechanical stability and good reproducibility of measurements. The effect of passivation of this electrode is very low. Detecting DNA damage. Amalgam electrodes were successfully used for the investigations of natural DNAs, various DNA bases, oligonucleotides (ODNs), as well as chemically modified nucleic acids. The adsorption of DNA on the surface of amalgam working electrodes is sufficiently strong to allow preparation of a DNA-modified electrode resisting washing. The m-agsae was used for the detection of enzymatic or chemical DNA cleavage in solution or at the electrode surface. AgSAE modified with supercoiled DNA was utilized as a sensor for DNA nicking substances. Specific voltammetric peak appears only when supercoiled DNA is damaged [15]. Similarly as with mercury electrodes and m-agsae, measurements on MF-AgSAE and p-agsae were successfully applied for the detection of DNA strand breaks induced by ionizing radiation [16]. IV. CONCLUSION Environmentally friendly solid and paste amalgam electrodes are suitable both for batch analysis and for HPLC or ISBN:

5 flow injection analysis with electro-chemical detection of electrochemically active substances with limit of quantitation about mol l -1. They can be easily prepared in any laboratory and their simple electrochemical pretreatment in many cases ensure a good reproducibility of measurements. Application of AEs presents a suitable alternative to traditionally used mercury electrodes for determination of different analytes in practically all types of water, drinking water including, as well as in relatively complicated plant matrixes [17]. The achieved results are comparable with those achieved with hanging mercury drop electrode or using atomic absorption spectrometry method in the case of trace metals determination. Amalgam electrodes have been introduced in DNA biosensor development, DNA electroanalysis including detecting DNA damage. Solid and paste amalgam electrodes are being considered as analytical tools exhibiting similar features as the classical mercury drop electrodes (e.g., broad potential window, simple regeneration, good reproducibility) and possessing some advantages typical for solid electrodes (simplicity, mechanical properties, environmental safety). REFERENCES [1] J. Wang, Analytical Electrochemistry. New York: Wiley- VCH, 2000, ch. 1. [2] B. Yosypchuk and J. Barek, Analytical Applications of Solid and Paste Amalgam Electrodes, Crit. Rev. Anal. Chem., vol. 39, no. 3, pp , [3] J. Barek, J. Fischer, T. Navratil, K. Peckova, B. Yosypchuk, and J. Zima, Nontraditional Electrode Materials in Environmental Analysis of Biologically Active Organic Compounds, Electroanalysis, vol. 19, no , pp , [4] B. Yosypchuk, T. Navratil, J. Barek, K. Peckova, and J. Fischer, Amalgam Electrodes as Sensors in the Analysis of Aquatic Systems, in Progress on Drinking Water Research, M. H. Lefebvre and M. M. Roux, Eds. New York: Nova Science Publishers, 2008, pp [5] M. Fojta, Electrochemical Sensors for DNA Interactions and Damage, Electroanalysis, vol. 14, no. 21, pp , [6] M. Fojta, Mercury Electrodes in Nucleic Acid Electrochemistry: Sensitive Analytical Tools and Probes of DNA Structure, Collect. Czech. Chem. Commun., vol. 69, no. 4, pp , [7] J. Wang, Electrochemical nucleic acid biosensors, Anal. Chim. Acta, vol. 469, no. 1, pp , [8] B. Yosypchuk and L. Novotný, Nontoxic Electrodes of Solid Amalgams, Crit. Rev. Anal. Chem., vol. 32, no. 2, pp , [9] B. Yosypchuk and L. Novotný, Copper solid amalgam electrodes, Electroanalysis, vol. 15, no. 2, pp , [10] B. Yosypchuk, M. Fojta and J. Barek, Preparation and Properties of Mercury Film Electrodes on Solid Amalgam Surface, Electroanalysis, vol. 22, no , pp , [11] B. Yosypchuk and I. Sestakova, Working Electrodes from Amalgam Paste for Electrochemical Measurements, Electroanalysis, vol. 20, no. 4, pp , [12] J. Fischer, L. Vanourková, A. Danhel, V. Vyskocil, K. Cizek, J. Barek, K. Peckova, B. Yosypchuk, and T. Navratil, Voltammetric Determination of Nitrophenols at a Solid Silver Amalgam Electrode, International Journal of Electrochemical Science, vol. 2, no. 3, pp , [13] E. Palecek and I. Postbieglova, Adsorptive Stripping Voltammetry of Biomacromolecules with Transfer of the Adsorbed Layer, J. Electroanal.Chem., vol. 214, no. 1-2, pp , [14] B. Yosypchuk, M. Fojta, L. Havran, M. Heyrovsky, and E. Palecek, Voltammetric Behavior of Osmium-labeled DNA at Mercury Meniscus-modified Solid Amalgam Electrodes. Detecting DNA Hybridization, Electroanalysis, vol. 18, no. 2, pp , [15] K. Kuchaříková, L. Novotný, B. Yosypchuk, and M. Fojta, Detecting DNA Damage with a Silver Solid Amalgam Electrode, Electroanalysis, vol. 16, no. 5, pp , [16] R. Fadrná, K. Cahová-Kuchaříková, L. Havran, B. Yosypchuk, and M. Fojta, Use of Polished and Mercury Film-Modified Silver Solid Amalgam Electrodes in Electrochemical Analysis of DNA, Electroanalysis, vol. 17, no. 5-6, pp , [17] P. Cizkova, T. Navratil, I. Sestakova, and B. Yosypchuk, Verification of applicability of mercury meniscus modified silver solid amalgam electrode for determination of heavy metals in plant matrices, Electroanalysis, vol. 19, no. 2-3, pp , ISBN: