IMPEDIMETRIC Au-NTA SENSOR FOR LEAD DETERMINATION

Marina Palcic1, Irena Kerkovic2, S. Milardovic2 and Zorana Grabaric1

1University of Zagreb, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10 000 Zagreb, Croatia

2University of Zagreb, Faculty of Chemical Engineering and Technology, Marulicev trg 19, 10 000 Zagreb, Croatia

Summary A self assembled monolayer (SAM) of cysteamine was prepared on the surface of gold disc electrode and further modified with nitrilotriacetic acid (NTA). Sensor’s surface characterization was performed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of potassium hexacyanoferrate (II)/(III) redox pair. Complexation of NTA with Pb2+ was tested by EIS measurements using the same redox pair as in the surface characterization. Electrode response (log ∆Rct) was linearly proportional to log c(Pb2+) with correlation coefficient R2 = 0.9865 for measurements in buffer pH = 4.60 and R2 = 0.9931 for pH = 7.06 in the concentration range of 0.06-66 µmol/L.

Keywords: impedimetric sensor, lead, NTA, self assembled monolayers

Introduction

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Thiol monolayers are frequently used for modification of metal electrodes (gold, silver, copper and platinum) as stable anchor for surface functionalization. Cysteamine monolayer attached to surface of metal electrode makes a platform containing large number of free surface amino groups convenient for further modifications in order to enlarge the number of other functional groups. Using metal coordination chemistry, –COOH, –OH-, –PO4

3-,–NH2 terminal groups were so far used to attach large number of different metal ions (Cu2+, Ca2+, Ni2+, Pb2+, Zr4+) as reported in the review by Cao et al. (2009). SAMs can be applied for sensor preparation for detection of biologically important molecules such as glucose, dopamine, proteins, cholesterol, antibodies and antigens and DNA (Davis et al., 2005). SAM’s terminated with nitrilotriacetic acid complexed with Me2+ are used for selective immobilization of His-tagged proteins (Sigal et al., 1996), too.The main purpose of this work was to investigate and characterize gold electrode modified with cysteamine self-assembled monolayer (Au/CA) functionalized with nitrilotriacetic acid (Au/CA/NTA) and the possibility of application in lead determination.

Materials and Methods

Materials and reagents

All chemicals used were of analytical grade and all solutions were prepared with water from Millipore-MilliQ system (USA).Cysteamine (HSCH2CH2NH2, CA), Fluka, was used for building SAM platform. Other reagents used for electrode surface modification, such as 1-ethyl-3(3-(dimethylamino)propyl)carboimide (EDC), nitrilotriacetic acid (NTA), N-hydroxysuccinimide (NHS) were from Sigma-Aldrich. Reagents used for preparation of 0.1 M acetic, phosphate and boric buffer solutions were obtained from Kemika. Standard solution of lead was prepared from lead nitrate (Merck). Sodium perchlorate as supporting electrolyte, potassium hexacyanoferrate(II) trihidrate and potassium hexa-cyanoferrate(III) used as redox probe, were obtained from Merck, too.

Instrumentation

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Potentiostat 264 A (Princeton Applied Research, USA) connected to a computer via EG&G-PowerSuite software for data collecting and analysis was used for CV measurements. Lock-in amplifier 5210 (PAR, USA) was connected to Potentiostat 264 A for EIS measurements.

Cyclic voltammetry and electrochemical impedance spectroscopy

Both CV and EIS were used for electrode surface characterization, whereas EIS alone for lead determination. All electrochemical measurements were carried out in a three-electrode cell. Gold disc electrode was used as working electrode, Hg|Hg2Cl2| KCl saturated (SCE) electrode and platinum wire as reference and counter electrodes, respectively. The applied potential scan rate in cyclic voltammetry was 50 mV s-1. The frequency range for impedance measurements was between 100 mHz and 100 kHz. Small sinusoidal AC signal of 5 mV was applied to potential of +200 mV vs. SCE. Equivalent circuit parameters were calculated by fitting the EIS data using appropriate circuit by ZsimpWin software provided by EG&G. All solutions were deaerated with nitrogen gas for 10 minutes before measurements and all measurements were performed at room temperature.

Electrode preparation

Prior to modification of gold disc electrode (1 mm in diameter) with thiols, surface was chemically cleaned by soaking for 3 minutes in „piranha“ solution (1:3 (by volume) mixture of 30 % H2O2 and 98 % H2SO4). Mirror like surface was then obtained by polishing electrode on a flat pad with SiC powder of different mesh (240, 800 and 1200) and finally by Al2O3 powder with particle size of 1 and 0.25 µm. After each polishing, adsorbed particles were removed by immersing electrode in water and ethanol solutions using ultrasonic bath. Final step in surface preparation was electrochemical cleaning performed by cycling the electrode potential in 0.1 M HClO4 at 50 mV/s from 0 to +1.5 V vs. SCE, until the reproducible voltammograms were obtained. Electrode surface modification is summarised in Fig.1. First step was formation of stable SAM containing free amino groups by adsorption of cysteamine (18 mM ethanol solution) for 24 h in dark at room temperature. For further modification of Au/CA electrode the solution of 0.002 M EDC, 0.005 M NHS and 0.01 M NTA prepared in phosphate buffer pH = 5 was kept in dark for 3 h in order to activate NTA carboxyl

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groups. To obtain Au/CA/NTA modified surface electrode was immersed in this solution for 24 h in dark at room temperature.

Determination of lead

Lead binding capacity was studied by EIS. After 10 minute of Pb2+

accumulation from standard solutions (c = 0.01 to 66 µM) the concentration of lead was measured. Two sets of measurements were performed in order to investigate the influence of pH. One set in acidic media (acetic buffer pH = 4.60), and the other in neutral media (boric buffer pH = 7.06). Both solutions contained 0.1 M NaClO4 as supporting electrolyte and 1 mM K4[Fe(CN)6]/K3[Fe(CN)6] as redox probe.

Results and Discussion

Characterization of Au/CA/NTA electrode

Formation of self-assembled monolayer creates additional barrier on the electrode, generally leading to a decrease in both, peak current (Ip) and apparent rate constant, while peak-to-peak separation (∆Ep) increases. However pH changes in measurement conditions can influence SAM’s apparent behaviour (Sabatini et al ., 1987) . Introducing negatively charged redox probe into the measurement system enables detection of changes in surface charge.Au/CA monolayer containing neutrally charged amino groups at pH = 9 caused decrease in Ip more evident in cathodic part of a voltammogram and increase in ∆Ep of approximately 10 mV (Fig. 2a). The same surface layer is highly protonated in acetic media at pH = 3, enabling electrostatic attraction to negatively charged redox couple facilitating approach of redox probe to electrode surface that is evident in slight Ip increase and ∆Ep diminishing (Fig. 2b). Further modification of the surface with NTA and formation of Au/CA/NTA SAM led to another decrease in Ip and broadening of ∆Ep

which is apparent in both acidic and alkaline media. These changes are more evident in alkaline media on account of NTA carboxyl groups being highly deprotonated at pH = 9, causing prevalence of negative charge at electrode surface inhibiting approach of the negative redox couple.

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When studying influence of electrode surface modification on kinetics of electrochemical reaction, EIS performed in frequency range between 100 mHz and 100 kHz is the method of choice, mainly because it is a non-invasive technique (Shervedani et al., 2006). Measured data were processed according to altered Randles’ model shown in Fig.3., where double layer capacitance is replaced by constant phase element (CPE). Parameter values of the model are shown in Table 1. with respect to unit electrode surface (1 cm2).

Electrochemical process under study can be influenced by two major effects, thickening of barrier between redox probe and electrode and facilitation or blocking approach of redox pair to electrode surface trough electrostatic interactions. Later effect is dominant in investigated system, because cysteamine SAM covered with NTA monolayer isn’t thick enough to act as insulating barrier. Nyquist plots obtained by EIS measurements were used to characterize effects that SAMs formed on top of gold electrode have on electrochemical reaction in question. Formation of CA monolayer that has neutrally charged terminal amino groups at pH = 9 caused increase in Rct as shown in Nyquist plot in Fig.4a., whereas the same surface layer is highly protonated in acetic media at pH = 3 (Fig.4b.), enabling electrostatic attraction to negatively charged redox couple significantly facilitating approach of redox probe to electrode surface that is in agreement to Rct decrease. Further modification of surface with NTA and formation of Au/CA/NTA SAM led to another increase in Rct in both acidic and alkaline media. These changes are more evident in alkaline media on account of NTA carboxyl groups being deprotonated at pH = 9, causing prevalence of negative charge at electrode surface to significantly inhibit approach of the negative redox couple. In addition to presented results, decrease in double layer capacitance, which is dependent on gold electrode surface coverage, indicates formation of stabile film on top of electroactive metal surface. Increase of CPE parameter (n) also confirms layer formation causing thus increase in surface homogeneity.

Determination of lead

NTA demonstrates high binding capacity towards metal ions from solutions because of coordination ability of three carboxyl groups and nitrogen to metal ions. When NTA is attached to cysteamine one carboxyl group is engaged, thus the binding of metal is somewhat weaker. The

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binding of metal is influenced by pH of sample solution used for lead preconcentration, too. In alkaline media the electrode surface charge becomes more negative, but at the same time the precipitation of lead as Pb(OH)2 occures. That is why Pb2+ accumulation was performed in the solution pH=7. In order to determine appropriate conditions of EIS measurements, experiments were performed in two different media, acidic (Fig.5a) and neutral (Fig.5b). Comparing the two Nyquist plots, change of Rct

contributed to Pb2+ attachment on the top of electrode surface is visible in both cases. When immersed in neutral boric buffer solution NTA is deprotonated, causing electrostatic rejection of redox probe. As surface concentration of Pb2+ increases with each accumulation step, Rct decreases due to the decrease of negative surface charge.When the same process is measured in acidic media, unexpected increase in Rct occurs as surface concentration of Pb2+ increases with each accumulation step. One of the possible explanation relies on the fact that at pH = 4.60 NTA surface becomes partially protonated and some of Pb2+

is released in the solution in the vicinity of the electrode surface thus reacting with OH- and forming Pb(OH)3

- that would correlate to Rct

increase, since EIS technique is a type of static measurement. Linear relation between c(Pb2+)/M in sample, ranging from 60 nM to 66 µM, and measured ΔRct/kΩ is described with Eq.1. with R2 = 0.9865.

log ΔRct/kΩ = (1.30 ± 0.06) + (0.15 ± 0.01) ∙ log c(Pb2+)/M (Eq.1.)

The same linear relation described with Eq.2. for c(Pb2+)/M in sample, ranging from 60 nM to 66 µM, was confirmed for measurements in boric buffer with R2 = 0.9931.

log ΔRct/kΩ = (2.24 ± 0.01) + (0.15 ± 0.03) ∙ log c(Pb2+)/M (Eq.2.)

Conclusions

The self assembled layer of cysteamine on gold disc electrode, further functionalised by NTA, was tested for determination of lead. The binding of Pb2+ onto the modified electrode was successfully done for a wide range of tested concentrations using EIS in two different media. Electrode response (log ∆Rct/kΩ) was linearly proportional to log c(Pb2+)/M with correlation coefficient R2 = 0.9865 for measurements in

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acetic buffer pH = 4.6 and R2 = 0.9931 for measurements in boric buffer pH = 7.06 tested on water solutions with lead content from 0.06 to 66 μM.Further optimization regarding accumulation time, preconcentration conditions (buffer composition and pH) and interferences with other metal ions is required in order to use proposed modified electrode for detection of lead in various samples.

References

Cao, R. Jr., Diaz-Garcia, A.M., Cao, R. (2009): Coordination compounds built on metal surfaces, Coordin Chem Rev 253, 1262–1275.

Davis, F., Higson, S.P.J. (2005): Structured thin films as functional components within biosensors, Biosens Bioelectron 21, 1–20.

Sabatani, E., Rubinstein, I., Maoz, R., Sagiv, J. (1987): Organized self-assembling monolayers on electrodes. 1. Octadecyl derivatives on gold, J. Electroanal. Chem. 219, 365-371.

Sigal, G.B., Bamdad, C., Barberis, A., Strominger, J., Whitesides, G.M., (1996): A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance, Anal. Chem. 68, 490-497.

Shervedani, R.K., Mozaffari, S.A. (2006): Impedimetric sensing based on phosphate functionalised cysteamine self-assembled monolayers, Anal. Chim. Acta. 562 (2006) 223-228.

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Fig.1. NTA coupling to Au/CA surface.

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Fig.2. Cyclic voltammograms of Au, Au/CA and Au/CA/NTA electrodes in buffered solution containing 0.1 M NaClO4 and 1 mM K4[Fe(CN)6]/K3[Fe(CN)6]; a) 0.1 M phosphate buffer pH=9b) 0.1 M acetic buffer pH=3

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Fig.3. Randles’ model equivalent circuit, where double layer capacitance is replaced by CPE.

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Fig.4. Nyquist plots of Au (solid line), Au/CA (dashed line) and Au/CA/NTA electrodes before (dotted line). Symbols represent measured values, whereas lines are values obtained by modeling. Measurements are performed in buffered solutions containing 0.1 M NaClO4 and 1 mM K4[Fe(CN)6]/K3[Fe(CN)6]; a) 0.1 M phosphate buffer pH=9

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b) 0.1 M acetic buffer pH=3

Fig.5. Nyquist plots of Au/CA/NTA electrodes before (dashed line) and after 10 minutes of lead accumulation (solid lines). Dots represent measured values, whereas lines are values obtained by modeling. Measurements are performed in buffered solutions containing 0.1 M NaClO4 and 1 mM K4[Fe(CN)6]/K3[Fe(CN)6]; a) 0.1 M acetic buffer pH=4.6 b) 0.1 M boric buffer pH=7

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Table 1. Parameters of Randles’ model based on EIS measurements of bare gold electrode (Au), gold electrode after formation of CA SAM (Au/CA) and after formation of NTA SAM (Au/CA/NTA) taken in alkaline and acidic media. Parameters of the model are solution resistance (Rs), charge transfer resistance (Rct), Warburg diffusion parameter (W), double layer capacitance defined trough constant phase element (CPE) and CPE parameter (n). χ2 represents error in fitting model to measured EIS data.pH electrode Rs / k Rct / k Cdl / F n W / ms0.5 χ2 9 Au 0.18 ± 0.01 1.64 ± 0.02 1.33 ± 0.07 0.85 ± 0.01 0.05 ± 0.01 2.45∙10-4

Au/CA 0.19 ± 0.01 3.46 ± 0.02 0.61 ± 0.07 0.90 ± 0.01 0.05 ± 0.01 6.56∙10-4 Au/CA/NTA 0.17 ± 0.02 11.02 ± 0.02 0.23 ± 0.05 0.89 ± 0.01 0.04 ± 0.02 1.14∙10-4

3 Au 0.35 ± 0.01 3.88 ± 0.03 1.95 ± 0.06 0.87 ± 0.01 0.07 ± 0.01 3.65∙10-4 Au/CA 0.35 ± 0.01 0.79 ± 0.01 0.56 ± 0.09 0.76 ± 0.02 0.04 ± 0.02 7.89∙10-4

Au/CA/NTA 0.34 ± 0.01 1.79 ± 0.04 0.24 ± 0.10 0.89 ± 0.01 0.04 ± 0.01 7.68∙10-4

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