ADVANCED MATERIALS FOR ELECTRODE MODIFICATION IN TRACE ELECTROANALYSIS
Creators: name: PIFFERI, VALENTINA
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: Trace analysis [1] (i.e. the analysis of analytes in concentration low enough to cause difficulty, generally under 1 ppm) albeit very challenging, in the last years has shown a tremendous growth, prompted by the urgent need of many International Organizations (US Environmental Protection Agency EPA, U.S. Food and Drug Administration FDA, European Food Safety Authority EFSA, World Health Organization WHO) looking for new analytical techniques for the detection of different molecules in different and increasingly more complex matrixes. Trace analysis is therefore a basic and fundamental technique in many scientific and technological areas, from the environmental monitoring, the food safety and the clinical diagnosis to the national security and the forensic investigation.
The determination of trace analytes requires reliable and robust analytical methods characterized by high level of sensitivity, precision, accuracy, selectivity and specificity. Among different analytical techniques suitable for this purpose, such as mass spectrometry, which is characterized by high accuracy and sensitivity and low limits of detection, electroanalytical techniques and particularly those based on pulsed voltammetry, seem to be a promising independent alternative in terms of very high precision, accuracy and sensitivity, simplicity of use, portability, easy automation and possibility of on-line and on-site monitoring without sample pre-treatments and low costs. These methods are no more confined to the detection of inorganic species and have been already and successfully employed for the determination of organic compounds and environmental carcinogens [2, 3, 4], as the Jir� Barek UNESCO Laboratory of Environmental Electrochemistry and the Trace Element Satellite Centre haves amply demonstrated in the last decades.
In this context, two quite recent technological developments have enhanced the chances of progress and growth of electroanalitycal methodologies for trace analysis:
? the screen-printing microfabrication technology [5, 6], which offers the possibility of large-scale mass production of extremely inexpensive, disposable and reproducible electrochemical sensors increasing the potentialities of the voltammetric techniques, since it allows to work with small amounts of samples, considerably reducing the analytical costs and facilitating on-line and on-site monitoring; 
? the use of nanosized and/or nanostructured materials sometimes combined with the use of polymeric materials for the modification of electrodes, with the aim of increasing the affinity for the analyte, increasing sensitivity, lowering the limits of detection and minimizing or completely avoiding interferences.
This PhD thesis has sought to provide a contribution in this framework, trying to enhance the technological potentialities of electroanalytical methodologies in the field of inorganic and organic trace analysis, with the use of screen-printed electrodes and electrodes modified by nanomaterials and/or polymeric membranes. 
SCREEN-PRINTED electrodes
Different types of screen-printed electrodes (SPEs) were employed for the determination of organic and inorganic carcinogenic hazardous compounds, included in the Priority Pollutants List of many countries. In particular, the following analytes have been the subject of the study: furan, benzidines, chromium and arsenic. 
Furan, a volatile oxygen-containing heterocyclic compound, was classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC) in 1995, with the liver as primary target organ. It is unintentionally produced, together with dioxin, during most forms of combustion. For this reason, it falls into the Organic Persistent Pollutants list of Stockholm Convention. It is also formed during the thermal degradation of carbohydrates in foods [7], such as jarred baby foods, coffee, canned meat and toasted bread. The standard analytical procedure [8] for the detection of furan is based on GC/MS, which reaches the very low detection limits required by the analytical problem, but it is affected by results overestimation since furan can be produced during the heating required by the technique. 
A new electroanalytical method based on Square Wave Voltammetry (frequency of 100-200 Hz) at Pt disk and Pt-SPE covered by Nafion was studied. Furan shows an oxidation peak in acetonitrile at 1.85-1.95 V (SCE) for Pt disk and 1.95 V (SCE) for Pt-SPE. Both electrodes display a good linear correlation in the dynamic range between 1.02 ppm and 68.07 ppm. Pt disk presents a problem of saturation for higher concentration. The detection limits are quite good (0.11 ppm for Pt disk and 0.52 ppm for Pt-SPE), while apparent recovery factors (in both cases &gt; 95%) are better than those determined for the conventional GC/MS method. The applicability of the new method in a real matrix was tested using Pt-SPE covered with Nafion membrane for experiments in coffee, spiked with known quantities of furan. Good calibration plot (R2 = 0.997) and apparent recovery factor (102 %) were obtained also in this case. 
Benzidine is an organic synthetic compound which exists as an odourless, white or slightly reddish crystalline solid and that evaporates slowly, especially from water and soil. Benzidine and its derivatives are employed in many fields but the main use remains the synthesis of azo-dyes, by coupling benzidine with phenols and amines [9], which are widely employed in textile, printing, leather, paper making, drug and food industries and can be released in the effluents and wastewaters. Benzidine was identified as a carcinogenic agent [10] for human urinary bladder by IARC because its oxidation by human enzymes can permit its binding with DNA. Furthermore, its derivatives generate benzidine through reduction by intestinal and environmental microorganisms. Though production and use of azo-dyes were forbidden in many countries since 1970s, their use in companies of emerging countries is still increasing, thus affecting the work place of many environments. 
Electrochemical techniques for the detection of benzidine were tested at standard electrodes (Platinum and GC) with promising results [11, 12]. Benzidine and its derivatives (o-tolidine, o-toluidine, tetrametylbenzidine) were detected by Differential Pulse Voltammetry (DPV) using Carbon-based Screen-Printed Electrodes (C-SPE) in comparison with Glassy Carbon electrodes. Cyclic voltammograms of these molecules show that the reaction is a bielectronic chemical and electrochemical reversible one for benzidine, o-tolidine and tetrametylbenzidine, while it is monoelectronic and irreversible for o-toluidine. The four molecules display different peak potential position, due to the presence or absence of electrodonating groups, tetrametylbenzidine characterized by the lower peak potential, followed by o-tolidine, benzidine and finally o-toluidine. In the case of the detection with DPV and C-SPE, all molecules show excellent linearity in the linear dynamic range 2 ppb-18 ppb (R2 &gt; 0.9), high accuracy (with apparent recovery factors very close to 100%) and very low detection limits (0.33 ppb for benzidine, 1.45 ppb for tetrametylbenzidine, 0.43 ppb for o-tolidine and 123 ppb for o-toluidine). Since the innovative technique seems to be very reliable and each molecule presents a different peak position, an interesting research development was the study of the behaviour of the mixture of the four molecules. Cyclic voltammograms show that they can be revealed at the same time and that their response remains linear and with a good correlation (R2 &gt; 0.99), also when they are present contemporaneously. Preliminary results using DPV display four observable peaks. 
Chromium, particularly its hexavalent species, is a carcinogenic and mutagenic pollutant and it is located in the Priority Pollutants List of many countries. It is employed in several industrial processes (metal plating, leather tanning, paint making) and it can be especially found in waste waters. Therefore, in recent years, many efforts have been made to develop efficient and accurate techniques for its determination [13]. For this purpose mercury electrodes are widely used in association with voltammetric stripping techniques preceded by cathodic or anodic preconcentration steps, especially in the presence of chromium complexing agents. Nowadays, mercury tends to be replaced by other less toxic materials [14]. A promising alternative seems to be the environmentally friendly bismuth electrode [15, 16], since it presents an electrochemical behaviour very similar to mercury, in particular in the wide cathodic potential window. 
Commercially available bismuth Screen-Printed Electrodes (Bi-SPE) were employed using Square Wave Voltammetry (SWV) to develop a new technique for the detection of Cr(VI), using pyrocatechol violet (PCV) as Cr(VI) complexing agent and electroactive probe and HEDTA as Cr(III) complexing agent to remove possible Cr(III) interferences. This innovative method was compared with the traditional procedure based on Differential Pulse Adsorptive Stripping Voltammetry (DPAdSV) at Hanging-Mercury Drop Electrode (HMDE). Many differences can be envisaged: first of all, PCV displays at Bi-SPE an intensive reduction peak at -1.18 V, which increases for consecutive additions of Cr(VI), in contrast with the decrease observed in the case of HMDE in the same conditions. This behaviour can be explained assuming that the complex Cr(VI)-PCV is electroactive at Bi-SPE and non-electroactive at HMDE. Secondly, PCV at Bi-SPE can be revealed without stripping, which is instead a necessary step required when adopting HMDE. The new method shows a very good linearity range (R2 = 0.996) and accuracy (Apparent Recovery Factors around 102%) and its limit of detection is an order of magnitude lower than that using HMDE (0.28 ppb against 2.8 ppb). The applicability of the new optimized procedure was tested analyzing samples coming from Cr(VI) photocatalysis in liquid phase to follow the photoreduction of Cr(VI) from a concentration of 2.8 ppm to the complete disappearance. The analysis was performed with an analyte addition method (three addition of the sample) after a calibration plot built with 8 standard additions. This method allows distinguishing the performances of different types of photocatalysts. The same test was also performed at HMDE for comparison: the new technique displays better results since it is less affected by interferences of the complex matrix. 
 Arsenic is a hazardous, dangerous and toxic compound, especially in its trivalent form, and arsenic contamination is widely recognized as a global health problem, ascribing it in the first places of the Priority Pollutants List. High levels of As can be found in soil, groundwater and drinking water, since arsenic derivatives are mainly used in agricultural poisons, such as fungicides, insecticides, pesticides. Chronic arsenic exposure can cause a lot of health diseases, such as skin lesions, cancers, cardio-vascular system problems. For these reasons, many methods characterized by pros and cons are present in the Literature for As detection [17].
In this work, Gold-based screen-printed electrodes (Au-SPE) were used for the determination of As by Linear Sweep Voltammetry with a preconcentration step and a cleaning procedure. Citric acid was employed as supporting electrolyte instead of hydrochloric acid, which caused electrode damaging, and good calibration plots were obtained in the range 4.9-59 ppb, in particular for gold nanoparticles-based screen-printed electrodes. The optimized method was applied to As detection during its photocatalytic oxidation by titanium dioxide, allowing to discriminate among different types of photocatalysts.
ELECTRODES modified by NANOMATERIALS
An important advantage of electroanalytical techniques is the possibility to modify the working electrode with different types of advanced materials to increase the affinity for the analyte, to lower the limits of detection and to avoid interferences. In particular, during the last years, nanomaterials appeared to be very promising for application in the field of sensors and biosensors. Among different types of nanomaterials, carbon nanotubes, metal and semiconductive nanoparticles, show very interesting properties and features for electrochemical performances and were chosen for the modification of electrodes to be used in selected trace electroanalytical applications.
All the new modified electrodes were firstly characterized and studied by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS), in the presence or in the absence of a model probe molecule, in order to obtain important information about electrochemical properties and the behaviour of the electrode in solution and with the redox probe. After the characterization study, some electrodes were used as sensors for the determination of relevant compounds or pollutants at trace level.
Carbon nanotubes (CNT) [18, 19] are extensively employed in the electroanalytical field, due to their large surface area, electrocatalytic activity, fast electron transfer rate and easy functionalization. Since the procedure of purification with acids plays an important role for electrode performance, initially, a detailed study on different purification procedures was performed. 24 h sulfonitric mixture treatment appeared to be the best procedure for our CNTs yielding to materials characterized by an high metal nanoparticles removal, high amount of covalent acidity (responsible of CNTs activity), high surface area and mesoporosity. Moreover, the final removal of amorphous carbon by NaOH treatment highly improved the reversibility of the final electrodic device and favoured the diffusion mechanism of the process. The best type of purified and activated CNTs was employed for applications in electroanalysis, in particular in the determination of some previously studied pollutants: o-toluidine, benzidine and furan. 
o-toluidine was detected using Linear Sweep Voltammetry in the range 1.5-7 ppm with good correlation, obtaining a limit of detection of 0.16 ppm and excellent apparent recovery factors and repeatability, in comparison with carbon based-screen printed electrodes, which presented problems of fouling, probably due to polymerization products. This new method was used for the determination of o-toluidine during its photodegradation mediated by ZnO photocatalyst, showing better performances than C-SPE and comparable with HPLC. Moreover, the methodology was also employed to monitor o-toluidine absorption by cyclodextrine nanosponges based on hydrogel polyamidoamines (PAA), allowing to discriminate among various types of resins and to obtain absorption kinetic parameters. 
Benzidine was determined by using Square Wave Voltammetry in the range 0.05-2.2 ppm, obtaining good correlation and good limits of detection at ppb level, with better apparent recovery factors and repeatability in comparison with the previously optimized technique based on C-SPE. 
In the case of furan, preliminary promising results were obtained with deposition of Pt nanoparticles on carbon nanotubes by cyclic voltammetry, but optimization of nanoparticles deposition procedure and application of other voltammetric techniques are still needed and are currently under investigation.
Metal and semiconductor nanoparticles [20] present unique peculiar properties, dependent on their size and shape, very different from bulk materials, such as high active surface area, high surface-to-volume ratio, selectivity, easy functionalization and electrocatalysis, and for these reasons they are extensively employed in electroanalysis. In this work, gold, silver and titanium dioxide nanoparticles were studied and characterized. 
Gold nanoparticles, synthesized by colloidal procedure with or without a protective polymer on carbon nanotubes as support, showed in comparison with CNTs, an increase in the peak current and capacitance, followed by the decrease of charge transfer resistance. The polymer, if the content of gold is low, is detrimental for the electrochemical behaviour, probably because it isolates too much the gold nanoparticles. The best results were obtained with 1% Au or 5% Au-Polymer. The optimized electrode was tested for the determination of glycerol obtaining really promising preliminary results using cyclic voltammetry. 
Silver nanoparticles were synthesized via colloidal method using two different supports: Nafion membrane and carbon nanotubes. In the case of Nafion, Ag nanoparticles show higher current intensity than bare electrode, probably due to higher surface area, a change in the diffusion mechanism from planar to convergent and small double layer formation. This electrode was tested for the determination of halothane and dichloromethane, showing promising preliminary results. In the case of CNTs, silver nanoparticles allowed the extension of the potential range towards negative values and peak currents were higher than the previous case with Nafion, showing the important contribute played by CNTs. Moreover, the use of a protective polymer (PVA) caused the decrease of the electrode activity, probably due to less available Ag nanoparticles. 
Titanium dioxide nanorods were studied in combination with single-walled carbon nanotubes, in the dark or under UV illumination, considering the photoactivity of titania. The best electrochemical performances were obtained for SWCNTs, since titania, as semiconductor brings a more resistive behaviour. Differences between dark and irradiation appeared only in the presence of titania, as expected. UV irradiation caused a change in the model probe molecule diffusion through the nanomaterials, probably ascribable to excited electrons of the titanium dioxide.
ELECTRODES modified by POLYMERIC MEMBRANES
Conducting polymers are polymeric systems characterized by ionic conductivity. They can be divided into two classes: electron and proton conducting polymers. Electron conducting polymers present a conjugated chain structure, with an extended ?-bond system, leading to the formation of broad valence and conduction bands. Among all the types of electron conducting polymers, electroactive polymers seem to have the best qualities for the construction of sensors [21, 22, 23]. Indeed, they can act as electron donors/acceptors, adding to the high conductivity, an electrocatalytic effect and a possibility of redox-mediation, showing both electronic and ionic conductivity in contact with the electrolyte solution. 
Brilliant Green (BG), belonging to the triphenylmethane family, was chosen as electroactive polymer for the production of modified electrodes, in combination with CNTs and PEDOT [24], another non-redox electron conducting polymer. CNTs in combination with PEDOT gave the best electrochemical performance in terms of capacitance and low resistance, but when the determination of hydrogen peroxide was considered, electrode with CNTs and polyBG gave the best results for the presence of the redox centre (LoD around 30 ppb). This electrode was also tested as biosensor for glucose and ethanol, immobilizing on the electrode glucose oxidase (GOx) and alcohol oxidase (AlOx), respectively and showing very good results in comparison with the biosensors of the Literature, with limit of detections of 2 ppm for glucose and 1 ppm for ethanol. Moreover, the influence of oxygen was studied, obtaining better results in its presence for glucose detection and in its absence for ethanol determination, probably due to the aerobic or anaerobic character of the enzyme bacterium. 
Proton conducting polymers show a cation/proton conductivity along the polymer backbone thanks to the presence of carboxylated or sulfonated groups with a cationic counter ion, whose mobility can be increased by water swelling. For this peculiarity, they present low electrical resistance (obtained increasing ion exchange capacity and water content and decreasing membrane thickness), high permeoselectivity for anions and non-ionized molecules, good mechanical and chemical stability over long periods. Their properties depend on many factors, such as the chemical nature of the polymer backbone, the polymer molecular weight and molecular weight distribution, the nature of the solvent used for casting and the possible presence of residual solvent in the polymeric film. 
Poly(aryl ether sulfone) (PES) was studied as a new material for the production of modified electrodes in comparison with Nafion. For its characterization, different parameters have been studied: the quantity and the form of the polymer, its storage, its method of drying and the casting solvent. In particular, 1 % linear PES in the acidic form, dried at 25 �C in oven, dissolved in N-Methylpyrrolidone, showed the best performance, superior to Nafion. These polymers presented a very interesting behaviour, since without the redox probe, capacitance was comparable to glassy carbon, while when the redox probe was present, capacitance increased of two orders of magnitude and diffusion of the probe changed, probably due to variation of diffusion mechanism in the polymeric structure.
Future developments will consider the applications of these new interesting systems for the detection of various important analytes or pollutants. Furthermore, new types of sensors and biosensors based on different types of the advanced materials studied and their combination will be considered.
[1] D.T. Pierce, J.X. Zhao, Trace Analysis with Nanomaterials, Wiley-VCH, Weinheim (Germany), (2010) [2] J. Zima, I. Svancara, J. Barek, K. Vytras, Crit. Rev. Anal. Chem. 2009, 39, 204-227 [3] J. Barek, K. Peckova, V. Vyskocil, Current Anal. Chem. 2008, 4, 242-249 [4] J. Barek, J. Cvacka, A. Muck, V. Quaiserov�, J. Zima, Fres. J. Anal. Chem. 2001, 369, 556-562 [5] M. Alvarez-Icaza, U. Bilitewski, Anal. Chem. 1993, 65, 525A-533A [6] J. Wang, B. Tian, V.B. Nascimento, L. 2000, 12, 1293-129 [7] J.A. Maga, CRC Crit. Rev. Food Sci. and Nutrition 1979, 11, 355-400 [8] J. Vranov�, Z. Ciesarov�, Czech J. Food Sci. 2009, 27, 1-10 [9] K.-T. Chung, S.-C. Chen, L. D. Claxton, Mutation Research 2006, 612, 58?76 [10] T.J. Haley, Clin. Toxicol. 1975, 8, 13?42 [11] J. Barek, A. Berka, Z. Tocksteinov, J. Zima, Talanta 1986, 33, 811-815 [12] J. Barek, J. Cvacka, A. Muck, V. Quaiserov�, J. Zima, Electroanalysis 2001, 13, 799-803 [13] V. Gomez, M.P. Callao, TrAC, Trends Anal. Chem. 2006, 25, 1006?1015 [14] Directive 2008/51/EC [15] I. Svancara, C. Prior, S.B. Hoc�var, J. Wang, Electroanalysis 2010, 22, 1405?1420 [16] J. Barek, K. Peckova, V. Vyskocil, Curr. Anal. Chem. 2008, 42, 42?249 [17] V.K. Sharma, M. Sohn, Environ. Int. 2009, 35, 743 [18] C.N.R. Rao, B.C. Satishkumar, A. Govindaraj, M. Nath, ChemPhysChem, 2001, 2, 78-105 [19] J. J. Gooding, Electrochimica Acta, 2005, 50, 3049-3060 [20] L. Rassaei, M. Amiri, C.M. Cirtiu, M. Sillanpaa, F. Marken, M. Sillanpaa, Trends in Analytical Chemistry, 2011, 30 (11), 1705-1715 [21] M.E. Ghica, C.M.A. Brett, Electroanalysis, 2006, 18, No. 8, 748-756 [22] M.M. Barsan, E.M. Pinto, C.M.A. Brett, Electrochimica Acta, 2008, 53, 3973-3982 [23] M.E. Ghica, C.M.A. Brett, Journal of Electroanalytical Chemistry, 2009, 629, 35-42 [24] X. Crispin, F.L.E. Jakobsson, A. Crispin, P.C.M. Grim, P. Andersson, A. Volodin, C. Van Haesendonck, M. Van der Auweraer, W.R. Salaneck, M. Berggren, Chem. Mater., 2006, 18, 4353-4360