Anal. Chem. 1998, 70, 2949-2956
Gel-Integrated Microelectrode Arrays for Direct Voltammetric Measurements of Heavy Metals in Natural Waters and Other Complex Media C. Belmont-He´bert, M. L. Tercier,* and J. Buffle
CABE, Department of Inorganic, Analytical and Applied Chemistry, Sciences II, 30 quai Ansermet, CH 1211 Geneva 4, Switzerland
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G. C. Fiaccabrino, N. F. de Rooij, and M. Koudelka-Hep
Institute of Microtechnology, University of Neuchaˆ tel, Jaquet-Droz 1, 2007 Neuchaˆ tel, Switzerland
A voltammetric sensor developed for in situ trace metal analysis in natural waters is presented. It consists of an array of 100 mercury-plated, iridium-based microdisk electrodes, coated with a 300-600-µm-thick 1.5% agarose gel membrane. This membrane acts as a dialysis membrane by allowing the diffusion of metal ions and complexes and by hindering the diffusion of colloids and macromolecules. Chronoamperometry and square wave anodic stripping voltammetry (SWASV) have been used to characterize the diffusion of hexacyanoferrate(III), lead, and cadmium in the agarose gel. For these species, the diffusion coefficients have been found to be half of the diffusion coefficient in free solution, and the time necessary for complete equilibration with the test solution varied with the gel thickness in accordance with the theory and can be lowered to 5 min for a gel thickness of 300 µm. The same techniques have been used to demonstrate the efficiency of the membrane against fouling and convection. Pressures in the range 1-600 bar have been found to have no effect on the sensor response. In contrast, variations in temperature in the range 4-22 °C considerably affected diffusion and charge-transfer kinetics, the resulting currents obeying a simple Arrhenius equation. These results confirm the suitability of the voltammetric sensor for in situ analysis of heavy metals in natural waters. In situ, real-time continuous measurement and speciation of trace elements in natural waters is strongly needed for quality monitoring purposes as well as to minimize artifacts such as contaminations, losses by adsorption, or speciation changes which often occur during sampling, sample handling, and storage. Voltammetric techniques coupled to small microsensors are well designed for this purpose.1 Voltammetric microsensors are selective only to the mobile fraction of metals, i.e., free ions and small complexes which diffuse quickly enough to be measured in the time scale of the voltammetric technique used, not to the total concentrations as with most other techniques. The concen(1) Tercier, M.-L.; Buffle, J. Electroanalysis 1993, 5, 187. S0003-2700(97)01194-3 CCC: $15.00 Published on Web 06/17/1998
© 1998 American Chemical Society
tration of these metal species, which is the most difficult to measure unambiguously with classical techniques, is important, as it is more closely related to metal bioavailability and processes linked to transport through biological membrane than total metal concentrations.2 This makes such sensors also useful for other purposes, such as clinical analysis or industrial applications. However, their applications for direct measurements in complex media are frequently limited by the well-known fouling problem.3-6 To overcome this, studies have been done using different types of thin protective membranes based on size exclusion or electric charge repulsion, or both.7-12 Unfortunately, none of these membranes is totally efficient for direct measurements in natural water samples, and, in addition, most of them are not totally inert toward the target elements. Recently, it has been shown that, thanks to the properties of microelectrodes (i.e., low iR drop and spherical diffusion which allows trace metal measurements by stripping techniques in quiescent solution),13 these thin membranes can be replaced by relatively thick gel membranes.6 The gel membrane acts as a dialysis membrane, i.e., allows diffusion of small metal ions and complexes but retains colloids and macromolecules. The voltammetric microsensor measures the test compounds inside the gel after equilibration. Systematic studies have made it possible to select an agarose gel which is totally inert toward trace elements and can be prepared in a (2) Campbell, P. G. C. In Metal Speciation and Bioavailability in Aquatic Systems; Tessier, A., Turner, D. R., Eds.; IUPAC Series on Analytical and Physical Chemistry of Environmental Systems; Wiley: New York, 1995; Vol. 3, Chapter 2, p 45. (3) Buffle, J.; Vuilleumier, J. J.; Tercier, M.-L; Parthasarathy, N Sci. Tot. Environ. 1987, 60, 75. (4) Brezonik, P. L.; Brauner, P. A.; Stumm, W. W. Water Res. 1976, 10, 605. (5) Sagberg, P.; Lund, W. Talanta 1982, 29, 457. (6) Tercier, M.-L.; Buffle, J. Anal. Chem. 1996, 68, 3670. (7) Aldstadt, J. H.; Dewald, H. D. Anal. Chem. 1993, 65, 922. (8) Hoyer, B.; Florence, T. M. Anal. Chem. 1987, 59, 2839. (9) Morrison, G. M. P.; Florence, T. M. Electroanalysis 1990, 2, 383. (10) Dam, M. E. R.; Thomsen, K. N.; Pickup, P. G.; Schroder, K. H. Electroanalysis 1995, 7, 70. (11) Wang, J.; Taha, Z. Electroanalysis 1990, 2, 383. (12) Wang, J.; Galal, A.; Zimmer, H.; Mark, H. B. Electroanalysis 1992, 4, 77. (13) Montenegro, M. I.; Queiros, M. A.; Daschbar, J. L. Microelectrodes: Theory and Applications; NATO ASI Series, 497; Kluwer: Dordrecht, The Netherlands, 1991.
Analytical Chemistry, Vol. 70, No. 14, July 15, 1998 2949
reproducible way.6 This agarose gel was used to cover a Hgplated, Ir-based single microelectrode developed specifically to allow reproducible and reliable measurements of trace metals in low ionic strength freshwater.14 Applications of the agarose membrane-covered Hg-plated, Ir-based single microelectrode (µAMMIE) for direct analysis of Pb(II) and Cd(II) in samples containing 10-31 mg/L fulvic compounds (MW 1000-2000) and in raw river waters containing high amounts of suspended matters (e78 mg/L) have demonstrated the efficiency of the agarose gel membrane against fouling.6 In particular, it has been shown15 that the major fouling compounds of natural waters (fulvic, colloids, etc.) do not penetrate the gel, and thus no cleaning of the gel is necessary between analyses. It has also been shown6,15 that the renewal of the Hg film through the protective agarose gel is reproducible and that the µ-AMMIE can be used for long-term measurements (a few days) using the same Hg semidrop. The last point is particularly important to allow continuous in situ measurements using a submersible voltammetric probe.15 Despite all its advantages, the limitation of this µ--AMMIE is linked to its fabrication steps, which can only be done manually and thus are time-consuming and costly. An alternative is to use photolithographic techniques. Indeed, it has been demonstrated16 that, using this technique, interconnected Hg-plated, Ir-based microsensor arrays with various geometry, showing reproducibility and reliability similar to those of the single microelectrode, can be mass produced at low cost. To allow its applications for direct, long-term measurements in complex media, the microsensor’s surface also has to be protected with the agarose gel membrane. To the best of our knowledge, this is the first report concerning voltammetric gel-integrated microsensor arrays. The aim of this paper is to define the optimum conditions for the gel coating and to characterize the gel-integrated microsensor arrays obtained in the absence and in the presence of different fouling materials. In addition, the effects of convection,17,18 pressure,19-21 and temperature22-24 on the voltammetric signals, which are to be considered with great care when performing in situ measurements, are also studied. EXPERIMENTAL SECTION Fabrication and Preparation of the Gel-Integrated Microelectrode Arrays. Two types of microelectrode arrays were fabricated. The first one (type I, Figure 1), previously described without the gel,16 consisted of 10 × 10 Ir microdisk electrodes of 5 µm diameter and a center-to-center spacing of 150 µm. The microelectrode arrays were prepared on a silicon wafer covered with successive layers of Si3N4, Ir, and Si3N4 with thicknesses of 2000 Å. The Si3N4 top layer was patterned by photolithography to define the microelectrodes and the bonding pads. The individual devices were mounted on a printed circuit board, wire (14) Tercier, M.-L.; Parthasarathy, N.; Buffle, J. Electroanalysis 1995, 7, 55. (15) Tercier, M.-L.; Buffle, J.; Graziottin, F. Electroanalysis, in press. (16) Belmont, C.; Tercier, M.-L.; Buffle, J.; Fiaccabrino, G. C.; Koudelka-Hep, M. Anal. Chim. Acta 1996,329, 203. (17) Kubiak, W. W.; Strozik, N. M. J. Electroanal. Chem. 1996, 417, 95. (18) Amatore, C. A.; Fosset, B. J. Electroanal. Chem. 1992, 328, 21. (19) Whitfield, M. Electrochim. Acta 1970, 15, 83. (20) Jerman, R.; Tercier, M.-L.; Buffle, J. Anal. Chim. Acta 1992, 269, 49. (21) Mu ¨ ller, B.; Hauser, P. C. Anal. Chim. Acta 1996, 320, 69. (22) Chen, H. Y.; Neeb, R. Fresenius’ Z. Anal. Chem. 1984, 319, 240. (23) Grundler, P.; Kirbs, A.; Zerihun, T. Analyst 1996, 121, 1805. (24) Zhou, H.; Dong, S. Electrochim. Acta 1996, 41, 2395.
2950 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
Figure 1. Schematic figure of type I and type II gel-integrated microelectrode arrays.
bonded and encapsulated with epoxy resin. The tip of the sensors, together with a flat cellulose spacer, was introduced and sealed in a 5-mm heat-shrinkable tubing. A 4 × 4 mm2 window in the tubing was then cut with a scalpel above the silicon chip. The spacer was removed, and the volume left by the spacer was filled with a 1.5% agarose solution heated at 80 °C. More details on the gel characteristics and preparation are given elsewhere.6 A Teflon tape was used to seal the end of the tubing. The thickness of the gel could be varied from 400 to 600 µm by changing the thickness of the spacer. The second type of microelectrode arrays (type II, Figure 1) was constructed in a similar way. It consisted of 5 × 20 Ir microdisk electrodes with the same characteristics. But, in addition, a 300-µm-thick Epon SU8 ring was deposited and patterned around the array of microelectrodes, forming a containment ring, allowing good attachment of the agarose gel and control of the gel thickness. The 1.8- × 4-mm individual devices were mounted and wire bonded on PCB prior to encapsulation with epoxy resin. The gel layer was formed on the microelectrode arrays by simply dipping the array in the heated 1.5% agarose solution.
In both cases, the agarose gel was left to harden in the air for approximately 1 min and then was kept constantly hydrated. Electrochemical deposition and reoxidation of the mercury semidrops were performed through the gel membrane (see below). Instrumentation and Experimental Conditions. The electrochemical measurements were performed with a computercontrolled AMEL 433 A polarograph and a homemade preamplifier25 set at a value of 100. Unless specified, a three-electrode system was used, with an Ag/AgCl/KCl saturated reference electrode protected with a 0.1 M NaNO3 bridge. The counter electrode was a platinum rod. Plexiglas cells were used to minimize metal adsorption and contaminations with the usual conditioning method.16 An optical inverted microscope (Leica-DMIL) was used to check that no impurities are present at the Ir substrate surfaces before covering them with the agarose gel. The gel thickness was also measured with this microscope once the gel had been removed and side cut. This implied the destruction of the sensors and was performed only when required, and after use. Mercury semidrops were deposited by maintaining the Ir potential at -400 mV in deoxygenated solution of 5 mM Hg(CH3COO)2 and 0.1 M HClO4. The Hg semidrops were removed by scanning the electrode potential from -300 to 300 mV at 5 mV‚s-1 in a deoxygenated solution of 1 M potassium thiocyanate. In both cases, the currents were recorded, and, from the electric charge involved, the diameters of the mercury semidrops were determined by assuming they were portions of spheres. The diffusion properties of the agarose gel were first investigated by running chronoamperometric reduction of 6 mM potassium hexacyanoferrate(III) at 0 V in deoxygenated 1 M NaNO3 solutions. The concentrations of Pb(II) and Cd(II) in deoxygenated 0.1 M NaNO3 were measured by square wave anodic stripping voltammetry (SWASV). After a cleaning phase at -100 mV for 1 min, metals were preconcentrated at -1100 mV for a given time (typically 5 min for concentrations in the range 1-10 nM), and the potential was then increased from -1100 to -100 mV with a pulse amplitude of 25 mV, a step amplitude of 8 mV, and a frequency of 50 Hz. The same conditions were used for the analysis of Pb(II) in a natural water with high organic content (137 ppm) and for the analysis of Zn(II) in a 2 × 10-2 M HEPES and 10-2 M KNO3 solution in which bacteria (Rhodococcus opacus Corynebacterium, Gram positive) had been incubated for 0-4 h. (Bacteria were eliminated by filtration on a 0.2-µm membrane before voltammetry.) The concentration of Zn was also measured by atomic adsorption spectroscopy (AAS, Pye-Unicam SP9). The concentration of Mn(II) in the anoxic part of Lake Bret (Switzerland) was measured with the same technique. The mercury microelectrodes were first electrochemically cleaned at -800 mV for 30 s, Mn(II) was then preconcentrated at -1600 mV for 20 s, and the potential was then increased from -1600 to -1300 mV with a pulse amplitude of 25 mV, a step amplitude of 1 mV, and a frequency of 6.7 Hz. For comparison, the concentration of Mn was also measured by ICP-AES (Perkin-Elmer, Plasma 1000), at an emission wavelength of 258 nm, in acidified raw samples, in samples acidified after filtration on 0.2-µm pore size (25) Huang, H. J.; He, P.; Faulkner, L. R. Anal. Chem. 1986, 58, 2889.
Figure 2. Pressure chamber and Plexiglas cell for measurements under high pressure.
membranes, and in ultracentrifuged samples (30 000 rpm for 15 h) (Beckman L7-55). The pressure cell used for the pressure tests is represented in Figure 2. It was built in a Plexiglas rod with diameter and height of 70 mm, in which an internal cylindrical hole of 40 mm diameter was extruded. It included a platinum wire sealed at the bottom, used simultaneously as an auxiliary electrode and a reference electrode. In this case, the applied potentials were adjusted by comparing the voltammograms obtained in the pressure cell with those obtained in the classical cell. The pressure was applied with nitrogen up to 10 bar. For higher pressures, this cell was placed in an aluminum homemade pressure chamber with wall thicknesses of 40 mm (Figure 2). All the air was replaced by paraffin oil, and the pressure was applied mechanically by pushing the lead into the bottom part of the chamber. The pressure inside the chamber was measured with an integrated manometer and could be increased up to 600 bar. A Kemlon connector (3-pin Kemtite) was used for the electrical connections. For the measurements made at controlled temperature, a thermostated glass cell was used with a Frigomix thermostated bath. The precision on the temperature measured was in the order of (1 °C. All reagents, except potassium hexacyanoferrate, Hg(CH3COO)2, and KSCN, were suprapur grade, and all the solutions were prepared before use with Milli-Q water. The LGL agarose (maximum sulfur, 0.03%; gel strength at 1.5%, 2000 g‚cm-2) was purchased from Biofinex (Switzerland). RESULTS AND DISCUSSION Diffusion Coefficients in the Agarose Gel. The diffusion process through the agarose gel was first studied by measuring the concentration change of potassium hexacyanoferrate(III) at Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
2951
Table 1. Diffusion Parameters Obtained with Five Different Arrays for the Diffusion of Hexacyanoferrate through a 1.5% Agarose Gel
array
gel thickness (µm) (error, (25 µm)
II I-a I-b I-c I-d
275 375 450 550 600
equilibration time (min) t90 t95 3.5 6.1 8.4 11.7 14.2
5.3 8.3 10.1 14.8 17.7
D (10-10m2‚s-1) 4.3 ( 0.3 4.2 ( 0.2 4.1 ( 0.2 4.4 ( 0.2 4.0 ( 0.2
Fe(III) was observed. The influence of the gel thickness on the diffusion profiles is shown in Figure 3-II. The average values of the diffusion coefficients of hexacyanoferrate(III) in 1.5% agarose gel with various thickness, reported in Table 1, were determined with eq 1,26 based on rigorous numerical calculations of diffusion through a membrane of thickness l under the limiting condition (0.2Csol + 0.8Cm) e C e (0.9Csol + 0.1Cm):
ln
Figure 3. Diffusion profiles of hexacyanoferrate(III). (I) Repeated measurements of diffusion in 1.5% agarose gel (a) toward the microelectrode surface and (b) from the microelectrode surface, with an array of type I and a gel thickness of 375 ( 25 µm. (c) Measurement at the microelectrode array without gel. (II) Diffusion of hexacyanoferrate(III) toward the microelectrode surface, in 1.5% agarose gel; measurements with a series of arrays of type I (O, ], 4, 3) and II (0) with different gel thicknesses. Full line curves: theoretical curves based on eq 1 for D ) 4.2 × 10-10 m2‚s-1. Concentrations of the hexacyanoferrate(III) solution: 6 mM in degassed 1 M NaNO3. Applied potential: 0 V. (III) Influence of the gel thickness on the equilibration time t90. Comparison of the experimental data (9) with the theoretical response from eq 1 (full line), with D ) 4.2 × 10-10 m2‚s-1.
the Ir substrate surfaces using chronoamperometry. The gelintegrated Ir-based microelectrode arrays (type I and II) were first immersed in a degassed solution of 1 M NaNO3. The solution was then replaced by a degassed solution containing 6 mM Fe(III) and 1 M NaNO3, and the dc reduction currents at 0 V were recorded as a function of time. The diffusion of Fe(III) out of the gel in a solution of 1 M NaNO3 was also monitored. These operations were repeated several times. As shown in Figure 3-I, the diffusion profiles were very reproducible, and no retention of 2952 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
[
]
Csol - C Dt ) 0.2306 - 2.452 2 Csol - Cm l
(1)
where Csol is the solution concentration, Cm is the initial uniform concentration in the gel, and C is the concentration at time t and x ) 0 (i.e., at the microelectrode surface). Diffusion coefficients were found to be independent of the gel thickness (Table 1), and the average value calculated from the different diffusion profiles was (4.2 ( 0.2) × 10-10 m2‚s-1. This value corresponds to 55% of the value measured in free electrolytes.27 Excellent fitting between experimental (Figure 3-II, symbols) and theoretical (Figure 3-II, full line) curves was obtained for the various gel membrane thicknesses. The time required for the equilibration of the agarose gel with the hexacyanoferrate(III) solution was evaluated from the values of t95 and t90, i.e., the times needed for the hexacyanoferrate(III) concentration at the microelectrode surface to be respectively 95% or 90% of the concentration outside the gel (i.e., i/imax ) 95% or 90%, respectively). Figure 3-III reports the values of t90 as a function of the gel thickness and compares the experimental values with the theoretical response calculated with eq 1, using D ) 4.2 × 10-10 m2‚s-1. For practical use, gel thicknesses of 200-300 µm are preferable since equilibration times of 3-5 min are then usable. In that respect, the microelectrode arrays of type II have a significant advantage over type I arrays or over the single microelectrode,6 as thinner thicknesses can be produced under well-defined conditions due to the containment ring. The diffusion of Pb(II) and Cd(II) through the agarose gel was studied in the same way as for hexacyanoferrate(III) but using micromolar concentrations and SWASV with gel-integrated Hgplated, Ir-based microelectrode arrays. Figure 4 shows the diffusion of these two ions toward the microelectrode surface. Equation 1 was found again to fit well the experimental data. Using (26) Jost, W. Diffusion in solids, liquids, gases, 3rd ed.; Academic Press: New York, 1960. (27) Heyrovsky, J.; Kuta, J. Principles of Polarography; Czechoslovak academy of Sciences: Prague, Czechoslovakia, 1965; p 106.
Figure 4. Diffusion of Pb(II) and Cd(II) through a 600-µm-thick agarose gel, toward the microelectrode surface, measured by SWASV with degassed solution of 9.65 µM Pb(II) and 8.89 µM Cd(II) in 0.1 M NaNO3. Hg diameter, 6.6 µm; deposition potential, -1.1 V; deposition time, 5 s; final potential, -0.1 V; pulse amplitude, 25 mV; step amplitude, 8 mV; and frequency, 50 Hz. (I) Repeated measurements for t ) 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 30, and 60 min. (II) Corresponding diffusion profiles of Pb(II) and Cd(II).
this equation and knowing the thickness of the agarose gel, the diffusion coefficients of both species were found to be 51% of the values measured in free solution: (4.2 ( 0.2) × 10-10 and (3.5 ( 0.2) × 10-10 m2‚s-1 instead of 8.25 × 10-10 and 6.90 × 10-10 m2‚s-1 for Pb(II) and Cd(II), respectively.27 These values were found to be independent of the gel thickness for the range investigated, and no shift of potential was observed with and without gel. It can thus be concluded that the gel is chemically inert vis-a´-vis these ions. All the above results are analogous to those obtained previously with the µ-AMMIE6 and thus confirm the reproducibility of the agarose gel obtained under controlled preparation mode. The only difference observed between single and array sensors was that the absolute value of the reduction current for a given analyte concentration with either the Ir or the Hg gelintegrated microelectrode array does not differ from that measured with a microelectrode array without gel, as shown for the example in Figure 3-I (curve c). This is assumed to be due to the existence of a thin aqueous layer between the gel and the array surface. Diffusion in this aqueous layer would then be the controlling factor during the time scale of the reduction process, whereas diffusion in the gel is the controlling factor for equilibration. Since, at the time scale of this latter process, diffusion is controlled by the gel even for the smallest gel thickness used (275 µm), the thickness of the aqueous layer at the electrode surface should be much smaller (i.e.,
