Fabrication and Characterization of Solid Mercury Amalgam

Mar 8, 2010 - Liquid mercury electrodes, with potential windows allowing detection of DNA and protein ..... Voltammetry of Os(VI)-Modified Polysacchar...
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Anal. Chem. 2010, 82, 2690–2695

Fabrication and Characterization of Solid Mercury Amalgam Electrodes for Protein Analysis Petra Juskova´,† Veronika Ostatna´,‡ Emil Palecˇek,*,‡ and Frantisˇek Foret*,† Institute of Analytical Chemistry of the ASCR, v. v. i., Veverˇ´ı 97, 60200 Brno, Czech Republic and Institute of Biophysics of the ASCR, v. v. i. Kra´lovopolska´ 135, 61265 Brno, Czech Republic Gold and carbon electrodes have been largely used as transducers in protein and DNA sensors and arrays. Liquid mercury electrodes, with potential windows allowing detection of DNA and protein reduction processes at highly negative potentials, were considered as useless in such arrays. Here, we show that solid amalgam electrode (SAE) arrays can be prepared as a substitution of liquid mercury in the analysis of the above biomacromolecules. Vacuum metal sputtering on a glass substrate, photolithography, and galvanic mercury amalgam formation were used for fabrication of an inexpensive disposable electrode array. The resulting ultrathin (less than 1 µm) amalgam microelectrodes were characterized with respect to influence of the electrode composition and size on the reproducibility and stability of electrochemical signals. Further characterization was performed using electron microscopy and the well-established ruthenium electrochemistry. Final, optimized, design was applied in protein analysis employing the recently described electrocatalytic chronopotentiometric peak H. Rapid, sensitive, selective, and reproducible analyses of biological samples command development of new analytical protocols and instrumentation. Growing demands in biotechnology and medicine require techniques amenable to automation and parallelization consuming very small amounts of biological material. While separations coupled to mass spectrometry are irreplaceable, especially in the discovery phases of research,1,2 smaller, selective, and much less expensive detection techniques are required for screening and diagnostic purposes. At present, this area is often addressed by technologies based on fluorescence detection.3 These methods can fulfill many detection requirements, but often require attachment of a suitable fluorescence tag, complicating the sample preparation. Additionally, while the new semiconductor based excitation sources (laser and LED) are much smaller and less power hungry than the previous generation of gas or metal ion lasers, these systems still require a significant amount of electric power, limiting their potential field applications. There * To whom correspondence should be addressed. E-mail: [email protected] (E.P.); [email protected] (F.F.). † Institute of Analytical Chemistry of the ASCR. ‡ Institute of Biophysics of the ASCR. (1) Ceglarek, U.; Leichtle, A.; Brugel, M.; Kortz, L.; Brauer, R.; Bresler, K.; Thiery, J.; Fiedler, G. M. Mol. Cell. Endocrinol. 2009, 301, 266–271. (2) Kolch, W.; Neususs, C.; Peizing, M.; Mischak, H. Mass Spectrom. Rev. 2005, 24, 959–977. (3) Myers, F. B.; Lee, L. P. Lab Chip 2008, 8, 2015–2031.

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are many cases when a simpler, less expensive, method is desirable. One of the options is the detection based on electrochemistry. A well-known example is the success of the blood glucose monitoring devices based on electrochemical measurement with multibillion markets worldwide. While this is an exceptional application, there are also many other important electrochemical applications in bioanalysis.4-8 Since the invention of polarography in 1922, the dropping mercury electrode dominated the electrochemical analysis for about half a century.9-11 Among specificities of Hg electrodes is the accessible potential window, which is shifted by about 1 V to more negative potentials as compared to nonmercury electrodes, such as gold and carbon. Accessibility of negative potentials at mercury electrodes was utilized in nucleic acid12 and protein11,13 and recently even in polysaccharide analysis.14 In addition, high affinity to sulfur compounds, high hydrophobicity, atomically smooth surface of liquid mercury, and other properties of mercury have played an important role in the analysis of proteins, nucleic acids, and their components.11,12 The current electrochemistry of proteins is oriented mainly at a relatively small group of conjugated proteins containing nonprotein electrochemically active centers (mainly metals);15 the potential for the electrochemical analysis of the majority of the proteins has not yet been explored in detail. Recently, we have shown that, using constant current chronopotentiometric stripping analysis (CPSA) in combination with bare solid amalgam and hanging mercury drop electrodes, (4) Bao, N.; Xu, J. J.; Dou, Y. H.; Cai, Y.; Chen, H. Y.; Xia, X. H. J. Chromatogr., A 2004, 1041, 245–248. (5) Tcherkas, Y. V.; Kartsova, L. A.; Krasnova, I. N. J. Chromatogr., A 2001, 913, 303–308. (6) Torimura, M.; Kano, K.; Ikeda, T.; Goto, M.; Ueda, T. J. Chromatogr., A 1997, 790, 1–8. (7) Xu, J. J.; Wang, A. J.; Chen, H. Y. TrAC, Trends Anal. Chem. 2007, 26, 125–132. (8) Palecek, E.; Fojta, M. Talanta 2007, 74, 276–290. (9) Heyrovsky, J.; Kuta, J. Principles of Polarography, 1st ed.; Czechoslovak Academy of Science: Prague, 1965. (10) Palecek, E.; Scheller, F.; Wang, J. Electrochemistry of nucleic acids and proteins; Elsevier: Amsterdam, 2005. (11) Palecek, E. In Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics; Palecek, E., Scheller, F., Wang, J., Eds.; Elsevier: Amsterdam, 2005, pp 690-750. (12) Palecek, E.; Jelen, F. In Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics.; Palecek, E., Scheller, F., Wang, J. , Eds.; Elsevier: Amsterdam, 2005, pp 74-174. (13) Palecek, E.; Ostatna, V. Electroanalysis 2007, 19, 2383–2403. (14) Strmecki, S.; Plavsic, M.; Cosovic, B.; Ostatna, V.; Palecek, E. Electrochem. Commun. 2009, in press. (15) Hammerich, O.; Ulstrup, J. Bioinorganic Electrochemistry; Springer: Dordrecht, Netherlands, 2008. 10.1021/ac902333s  2010 American Chemical Society Published on Web 03/08/2010

practically all proteins produce a well-defined chronopotentiometric peak H.13,16 The behavior of this peak suggested that it is due to the catalytic hydrogen evolution.17 This peak allows protein detection down to nanomolar and subnanomolar concentrations.13 Although the hanging mercury drop electrodes (HMDE) exhibit unique features, there are efforts focused on development of new analytical tools based on nontoxic, harmless, an environmentally-friendly materials retaining the electrochemical features of the mercury electrodes. One of the promising materials for replacing liquid mercury is nontoxic solid amalgam.18-21 The electrochemical stability of a solid amalgam electrode was found to be very good (several weeks without any maintenance), which is essential for online monitoring and/or application in a field apparatus. There are two basic types of amalgam electrodes: solid dental amalgam electrodes and mercury film or mercury layer electrodes on solid substrates.18,22,23 Silver and copper solid amalgam electrodes were applied in bioanalysis.19,20,24-27 With biological samples, it is often necessary to analyze small volume samples. Thus, electrode miniaturization is an important trend in bioelectroanalysis. Microlithography is the most common technique for reproducible fabrication of regular and well-defined structures.28 Once the fabrication protocol is optimized, a large number of identical structures can be replicated inexpensively, e.g., disposable electrode arrays for DNA29-31 or multiple bioagent analysis.32 Design (geometry, modifications) and fabrication techniques for microelectrode arrays depend on the particular application.29 While standard silicon or glass wafers can be used as a substrate for electrode fabrication, further improvement of electrochemical properties of the electrodes can be achieved by proper surface modification.33-36 In some studies, iridium was found as a suitable (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30)

(31) (32) (33) (34)

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Palecek, E.; Ostatna, V. Chem. Commun. 2009, 13, 1685–1687. Heyrovsky, M. Electroanalysis 2004, 16, 1067–1073. Mikkelsen, O.; Schroder, K. H. Electroanalysis 2003, 15, 679–687. Yosypchuk, B.; Fojta, M.; Havran, L.; Heyrovsky, M.; Palecek, E. Electroanalysis 2006, 18, 186–194. Yosypchuk, B.; Novotny, L. Talanta 2002, 56, 971–976. Coldrick, Z.; Steenson, P.; Millner, P.; Davies, M.; Nelson, A. Seville, Spain, Sep 07-12 2008; pp 4954-4962. Fadrna, R.; Cahova-Kucharikova, K.; Havran, L.; Yosypchuk, B.; Fojta, M. Electroanalysis 2005, 17, 452–459. Cahova-Kucharikova, K.; Fojta, M.; Mozga, T.; Palecek, E. Anal. Chem. 2005, 77, 2920–2927. Hason, S.; Vetterl, V. Talanta 2006, 69, 572–580. Barek, J.; Fischer, J.; Navratil, T.; Peckova, K.; Yosypchuk, B. Sensors 2006, 6, 445–452. Allison, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8–12. Selesovska-Fadrna, R.; Fojta, M.; Navratil, T.; Chylkova, J. Anal. Chim. Acta 2007, 582, 344–352. Madou, M. J. Fundamentals of microfabrication, 1st ed.; CRC press: New York, 1997. Huang, X. J.; O’Mahony, A. M.; Compton, R. G. Small 2009, 5, 776–788. Errachid, A.; Mills, C. A.; Pla-Roca, M.; Lopez, M. J.; Villanueva, G.; Bausells, J.; Crespo, E.; Teixidor, F.; Samitier, J. Mater. Sci. Eng., C 2008, 28, 777– 780. Liepold, P.; Wieder, H.; Hillebrandt, H.; Friebel, A.; Hartwich, G. Bioelectrochemistry 2005, 67, 143–150. Harper, J. C.; Polsky, R.; Wheeler, D. R.; Dirk, S. M.; Brozik, S. M. Langmuir 2007, 23, 8285–8287. Rehacek, V.; Shtereva, K.; Novotny, I.; Tvarozek, V.; Breternitz, V.; Spiess, L.; Knedlik, C. Vacuum 2005, 80, 132–136. Silva, P. R. M.; El Khakani, M. A.; Chaker, M.; Champagne, G. Y.; Chevalet, J.; Gastonguay, L.; Lacasse, R.; Ladouceur, M. Anal. Chim. Acta 1999, 385, 249–255. Reay, R. J.; Flannery, A. F.; Storment, C. W.; Kounaves, S. P.; Kovacs, G. T. A. Sens. Actuators, B: Chem. 1996, 34, 450–455.

base electrode material, because of its low solubility in mercury as well as physical and chemical stability.37 Silver, Pt, or Au dissolve in mercury, leading to amalgam film formation. Such electrodes were used for parallel detection of trace metals,38 iodate,39 and thiols.40 In the recent decade, a number of electrode arrays (using predominantly gold and carbon electrodes) for DNA and protein analyses have been developed.41 Platinum based mercury amalgam electrodes with phospholipid coating have recently been described for detection of biomembrane active drug molecules.21 In this work, we have explored vacuum metal sputtering for the deposition of ultrathin layers (100-800 nm) of silver and gold on glass substrate. Spin coated photoresist was used as the surface defining insulating material separating the individual metal spots of the array. Individual solid amalgam electrodes (SAEs) were then formed by precisely controlled electrodeposition of mercury on the photolithographically defined spots. Besides testing of their electrochemical properties, characteristics of resulting amalgam films were also examined by energy-dispersive X-ray microanalysis (EDS) and scanning electron microscope (SEM) analysis. EXPERIMENTAL SECTION Materials and Methods. Borosilicate glass disks (3 in. (∼75 mm) in diameter, 1.5 mm thick, used as the insulating substrate) were sputter coated with the layer of the desired base electrode material using a vacuum sputter coater (SCD 500, Bal-TEC AG, Lichtenstein). All the sputter targets were also obtained from BalTEC AG. Negative resist MaN-420 and developer ma -D 332S were from Micro resist technology GmbH, Berlin, Germany. Lithographic processes further included the use of a spin coater (WS400B-6NPP/LITE, Laurell Technologies, North Wales, PA) and a hot plate (PZ 28-2, H. Gestigkeit GmBH, Du¨sseldorf, Germany). Mercuric acetate (99.999%), perchloric acid (70%), and all other chemicals were of p.a. grade obtained from Sigma-Aldrich s. r. o., Prague, Czech Republic. Platinum wire (>99.99%) used as an electrode during electroplating was from Safina, a. s., Czech Republic. A fotolithographic mask was designed using ZW CAD 2007 Professional software (Techsoft s.r.o., Slovakia) and prepared on a plotter film HG NEW HPR-7S (Fujifilm, Japan), using a photoplotter (FP 8000, CADware s.r.o., Liberec, Czech Republic). A regulated constant current power supply was constructed locally. It allowed presetting the output current in the range of 1 nA to 10 mA with the stability better than 10 ppm and the maximum voltage limited to 5 V. Caution: Mercuric acetate (CAS # 1600-27-7) is extremely hazardous in case of ingestion, very hazardous in case of inhalation, hazardous in case of skin contact (irritant) and eye contact (irritant), and slightly hazardous in case of skin contact. (36) Kounaves, S. P.; Deng, W.; Hallock, P. R.; Kovacs, G. T. A.; Storment, C. W. Anal. Chem. 1994, 66, 418–423. (37) Nolan, M. A.; Kounaves, S. P. J. Electroanal. Chem. 1998, 453, 39–48. (38) Uhlig, A.; Paeschke, M.; Schnakenberg, U.; Hintsche, R.; Diederich, H. J.; Scholz, F. Sens. Actuators, B: Chem. 1995, 25, 899–903. (39) Lowinsohn, D.; Peres, H. E. M.; Kosminsky, L.; Paixao, T. R. L. C.; Ferreira, T. L.; Ramirez-Fernandez, F. J.; Bertotti, M. Sens. Actuators, B: Chem. 2006, 113, 80–87. (40) Batz, N. G.; Martin, R. S. Analyst 2009, 134, 372–379. (41) Wang, J. Analytical Electrochemistry, 3rd ed.; John Wiley&Sons, Inc: Hoboken, NJ, 2006.

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Oral (LD50): acute, 41 mg/kg [rat]; 24 mg/kg [mouse]; 65 mg/ kg [mammal]. Dermal (LD50): acute, 570 mg/kg [rat]. Morphological characterization and elemental analysis by the scanning electron microscopy (SEM) micrograph was performed on a MIRA II LMU (TESCAN s.r.o., Brno, Czech Republic) with qualitative and quantitative energy-dispersive X-ray microanalysis (EDS) system QUANTAX (Bruker AXS Microanalysis GmbH, Berlin, Germany). Silicon wafers (3 in. diameter, 〈100〉) used as substrates for electrodes during the SEM analysis were obtained from NESTEC (New Bedford, MA). Electrochemical measurements were performed with the AUTOLAB Analyzer (EcoChemie, The Netherlands). The standard cell with a three-electrode system with a Ag/AgCl/3 M KCl electrode as the reference electrode and a platinum wire as the auxiliary electrode were used. All experiments were carried out at room temperature under air. In our measurements, we applied cyclic voltammetry for determination of an electrochemically active surface of SAE and adsorptive chronopotentiometric stripping analysis (AdCPSA)13 for study of proteins. Adsorptive Stripping (AdS). The working electrode was immersed into a 20 µL drop of the protein solution (bovine serum albumin - BSA) in the background electrolyte for accumulation time, tA, at accumulation potential, EA of -0.1 V (if not stated otherwise), followed by chronopotentiogram recording. No stirring accompanied the accumulation. The initial potential, Ei of -0.1 V, the final potential, Ef of -1.81 V (-2 V), and the stripping current, Istr of -40 µA (-80 µA) for SAE (HMDE) were used. Adsorptive Transfer Stripping (AdTS). Protein modified amalgam working electrode was prepared as in AdS and then BSA modified SAE was washed by 200 µL of the background buffer, followed by placing 20 µL of the blank background electrolyte and recording of chronopotentiograms. Fabrication of Amalgam Electrodes. Electrode Deposition. The main goal of this work was preparation and characterization of a simple array of electrodes for analysis of small sample volumes. The selected ∼75 mm (3 in.) diameter glass substrate was first cleaned with piranha solution (4:1 mixture of concentrated sulfuric acid and 30% hydrogen peroxide), rinsed with deionized water, and dried on the hot plate at 80 °C. Next, it was placed in the vacuum chamber of the sputtering instrument, and the layer of selected metal was deposited by the magnetron in the argon plasma discharge. Since gold and silver are known to have lower adherence on glass, we have first tested application of a fine adhesion layer of chromium or titanium (∼10 nm) onto which the final electrode material (Ag or Au) was sputtered. After a number of experiments, we have realized that this procedure, commonly used in microfabrication practice,42 is not needed for this application since the electrodes are not mechanically stressed during the measurement. Omission of the adhesion layer simplified the preparation procedure and eliminated any danger of electrochemical interferences from the metal underlay. Final experiments have shown that both gold and silver suits well, and silver was selected as the preferred material. The preparation process begun with deposition of a uniform, 100-800 nm, silver layer on the whole surface of the glass wafer. Next, a ∼3.5 µm (42) Ling, T. G. I.; Beck, M.; Bunk, R.; Forsen, E.; Tegenfeldt, J. O.; Zakharov, A. A.; Montelius, L. Microelectron. Eng. 2003, 67-8, 887–892.

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Figure 1. (a) Cross section view of the electrode wells. Top, insulating photoresist layer defined the sample microwells. (b) Final electrode array positioned on the insulating pad with two gold contacts on both sides. Electrodes with 0.4 mm diameter were aligned concentrically with electric contact pads on the side.

photoresist layer was deposited on the silver surface using a spin coater (30 s, 1000 rpm) followed by “soft-baking” on the hot plate (5 min, 100 °C). The basis for the SAE array was designed as exposed circles in the electrically insulating photoresist layer with varying diameter (200 µm to 2 mm). This array was formed by UV exposition (15 min) through a previously prepared lithographic mask and removal of the unexposed photoresist. Depending on the sputtering instrument used, the sputtered silver layer thickness can vary. This could lead to variation of the spatial mercury/silver ratio during the mercury electrolysis in the next step. Indeed, with our sputtering instrument, the metal film thickness decreased from the center to the edge of the glass substrate by ∼20%. Thus, for obtaining a set of electrodes with identical thickness of the amalgam layer, it was best to arrange the electrodes aligned in concentric circles. In this arrangement, the distance of each electrode in the circle from the substrate center remained the same and so did the metal thickness. Better sputtering equipment can provide perfectly uniform metal films, yielding a reproducible mercury/metal ration across the whole substrate. While several thousand 200-400 µm spots can easily fit on the 3 in. diameter glass substrate, in this work, we have spaced the electrodes for easy access to the 20 µL sample drop. Thus, the array contained 60 electrodes arranged in concentric circles. The final metal spots were separated from one another by the insulating layer of the exposed photoresist and further stabilized by “hard-baking” on hot plate (30 min, 100 °C). The photoresist layer encloses metal spots, creating the small electrode wells shown in Figure 1a. The hydrophobic nature of the insulating photoresist material defining the electrode size also helped to confine the measured sample drop in the electrode area. Besides the design of the concentric array of spots, the lithographic mask also included two contact areas on both sides of the glass substrate for electric connection. Amalgam Formation. The exposed circular silver or gold spots on the glass surface described in the previous section were further modified by electrolytic mercury deposition. Each electrode was modified at 1 µA, from 20 µL of 5 mM Hg(CH3COO)2 in 0.1 M HClO4 solution43 using a spiral Pt wire electrode and the constant current power supply (in case of gold spots, electro-

plating was performed in presence of 20 µL of 1 M H2SO4). The amount of deposited mercury was controlled during the electrolysis step and calculated according the Faraday law. With the electrolysis times ranging from 10 to 700 s, the corresponding mercury layer thickness was 6-420 nm on the 400 µm diameter metal spots. The electrode surface was washed with deionized water and ready for measurement. The final array of amalgam electrodes with a detailed view of the one particular electrode can be seen in Figure 1. RESULTS AND DISCUSSION Morphological characterization and elemental analysis of the resulting film were examined using scanning electron microscope (SEM) with energy-dispersive X-ray microanalysis (EDS). Glass substrates were not suitable for these analyses, because of the surface charging in vacuum leading to the image distortion. For this reason, we have prepared electrodes, under the same conditions as described above, on the silicon substrates. It can be hypothesized that the amalgam forms instantly on the submicrometer thick silver layer during the electrolysis process.44 This can be further supported by the fact that the electrodes perform identically when freshly prepared or after a week of storage. Changes in electrode morphology during the amalgamation process were studied by SEM on the set of electrodes with increasing amount of deposited mercury (controlled by deposition times). The mass fraction of mercury in the final amalgam layer ranged from 2% to about 46%. Upon visual inspection (light microscope), even a small amount of mercury caused apparent changes on the silver surface. With an increasing amount of mercury, the electrode surface became rugged and small mercury droplets were formed. Later, isolated mercury islands propagated from the electrode periphery to its center. Finally, the islands merged at Hg content higher than 30%. At this concentration, the amalgam surface looked liquid like filling all the pores and gaps. This is shown in Figure 2. It is worth stressing that, for simplicity, we have operated only in the Hg content corresponding to the phase R in the amalgam phase diagram.44 Thus, one should expect only a homogeneous amalgam surface with uniform morphology. On the basis of the electron micrography, this was not always the case and formation of different phases was not excluded. However, unlike the experiments described by Rehacek et al.33 on mercury plated indium tin oxide (ITO) microelectrodes, the electrochemical properties of the present electrodes were related to the Ag-Hg amalgam formation. The electrochemical performance of the prepared electrodes was good and reproducible in the whole range of the compositions; however, at mercury content higher than 35%, the mechanical stability weakened. The EDS quantitative line scan analysis of identified elements as well as their distribution along the electrode diameter is in Figure 2e. Scan line was positioned to cover the whole electrode diameter, including a short photoresist border on both sides. From these results, it can be concluded that irrespective of the mercury concentration the electrode profile is regular with amalgam uniformly dispersed all over the silver surface. Similar observations were made also for the gold-mercury amalgam electrodes tested in this study. (43) Silva, P. R. M.; El Khakani, M. A.; Le Drogoff, B.; Chaker, M.; Vijh, A. K. Sens. Actuators, B: Chem. 1999, 60, 161–167. (44) Hudson, D. R. J. Phys. Chem. 1945, 49, 483–506.

Electrochemical Characterization. While electron microscopy measurements provide good characterization of the morphology of the amalgam surface and element distribution, the electrode performance was characterized using standard electrochemical measurements. The electrochemical measurements were facilitated by an insulating pad with two gold contacts on both sides, corresponding to contact circles on the electrode array. Each amalgam electrode served as a single working electrode. Although experiments with different analyte concentrations could be performed on a single electrode, the signals shown in Figure 2 were obtained from different electrodes with four different diameters. In another set of experiments, the signals obtained from different electrodes with the same diameter on the array were uniform within 15% rsd. Thus, individual electrodes in the array could also be operated as single measurement sensors. This might be important in cases when a sample cross-contamination is an issue. It is also worth noting that metal sputtering is a well-established technique, allowing preparation of practically identical metal layers from batch to batch. Similarly, the electroplating processes can be well controlled, and preparation of a large number of electrode arrays with practically identical performance can be easily achieved. Before measurement, sample (20 µL) was deposited on the electrode surface. Electrochemical measurement was carried out using a three-electrode configuration; a reference electrode and auxiliary platinum electrode were dipped into the analyte solution, and a cyclic voltammogram was recorded as shown in Figure 3. Detection of Proteins. Most negative electrochemical signals due to intrinsic electroactivity of DNA and RNA at mercury electrodes occur at about -1.4 V.12 Electrocatalytic signals of Oslabeled NAs45 and peptides/proteins46 appear at about -1.2 V and can be easily detected at SAE. Electrocatalytic peak H of proteins is at HMDE observed between -1.7 and -1.9 V. Measurements of this peak with SAE, thus, require high quality electrodes with the potential window not differing from that of HMDE. To test the quality of the new SAE described in this paper, we therefore choose the most difficult task, i.e., the measurements of peak H. Protein catalyzed hydrogen evolution17 responsible for peak H has not been, however, observed with gold, silver, or carbon electrode but solely with mercury-containing electrodes.11,13 Peak H of proteins was predominantly measured with HMDE.13,16,47 Here, we compared the electrocatalytic responses of BSA at HMDE with those obtained at SAE. Five micromolars of BSA was adsorbed either at HMDE or at single SAE (diameter 0.4 mm) in 0.2 M McIlvaine buffer, pH 7, for accumulation potential, tA, 1 min at accumulation potential, EA of -0.1 V. Well developed peak H was observed at both types of electrodes. Figure 4 shows a dependence of peak H on concentration of native BSA. At the HMDE, a large catalytic signal of BSA was observed in agreement with the previously published results16 showing a sigmoid curve with linear dependence on BSA concentration in a narrow range, followed by saturation level from about 350 nM. At SAE, peak H of BSA increased linearly with BSA concentration up to about 1 µM and then leveled off (Figure 4). (45) Fojta, M.; Kostecka, P.; Trefulka, M. R.; Havran, L.; Palecek, E. Anal. Chem. 2007, 79, 1022–1029. (46) Billova, S.; Kizek, R.; Palecek, E. Bioelectrochemistry 2002, 56, 63–66. (47) Dorcak, V.; Palecek, E. Anal. Chem. 2009, 81, 1543–1548.

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Figure 2. SEM images presenting changes in the metal morphology during electroplating. (a) Cross section view of SAE. Roughened metal surface with small amalgam droplets can be seen. The mass content of mercury in the electrodes was (b) 5% Hg, (c) 14% Hg, and (d) 24% Hg. The electrode surface with increasing amount of mercury became rough and porous (5-14% Hg), continuing amalgamation makes the surface more liquid like, with mercury filling all the pores and gaps (more than 30% Hg). The electrode with 24% Hg content still pertains to both surface types, rough in the center and liquid in the periphery as shown in (d). EDS quantitative line scan analysis of the amalgam electrode on the silicon substrate (e). The amalgam layer is equally distributed on the surface. Carbon content marks the photoresist border.

The sensitivity of determination of denatured BSA is very high;48 low concentration of native BSA can be detected at lower stripping currents (Istr of -10 µA) as documented in Figure 5b, showing a distinguished peak H of 5 nM BSA at tA 5 min. Using SAE, BSA was tested by conventional adsorptive stripping (AdS, in situ) or adsorptive transfer stripping (AdTS, ex situ) analysis.13 AdS chronopotentiograms were recorded with the HMDE immersed into the protein solution. On the other hand, in AdTS, the protein-modified HMDE was washed and transferred to the blank background electrolyte to record the chronopotentiogram. Using conventional AdS CPS analysis 5 µM BSA produced a well-developed peak H at -1.64 V, which differed only a little from peak H obtained by AdT CPS analysis (Figure 5a). A relatively small decrease of AdTS (ex situ) peak H (height) was (48) Ostatna, V.; Palecek, E. Electrochim. Acta 2008, 53, 4014–4021.

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probably due to the removal of loosely bound protein molecules during washing. The results observed with SAE, thus, did not significantly differ from those obtained earlier with HMDE.11,13,48 Our recent studies of the electrocatalytic peak H of different proteins at HMDE revealed that this chronopotentiometric peak is sensitive to local and global changes in protein structure.13,16,47-51 We showed that we can discriminate between native and denatured forms of BSA and other proteins at HMDE.16,48-50 Similar results were obtained at SAE with 300 nM native BSA and urea(49) Ostatna, V.; Dogan, B.; Uslu, B.; Ozkan, S.; Palecek, E. J. Electroanal. Chem. 2006, 593, 172–178. (50) Ostatna, V.; Kuralay, F.; Trnkova, L.; Palecek, E. Electroanalysis 2008, 20, 1406–1413. (51) Palecek, E.; Ostatna, V.; Masarik, M.; Bertoncini, C. W.; Jovin, T. M. Analyst 2008, 132, 76–84.

Figure 3. Cyclic voltammograms of 1 mM Ru[(NH)6]3+ in 50 mM Na-acetate, pH 5.1 on bare gold amalgam electrodes with different diameters. Black, 0.25 mm; red, 0.5 mm; blue, 1 mm; magenta, 2 mm. Scan rate: 100 mV/s.

Figure 4. Dependence of peak H height on concentration of native BSA on HMDE (red) and SAE (black). BSA was adsorbed at a working electrode for accumulation time, tA, 60 s at accumulation potential, EA of -0.1 V, followed by immediate chronopotentiogram recording in 200 mM McIlvaine, pH 7, using Istr of -40 µA (SAE) and -80 µA (HMDE).

denatured BSA in 50 mM McIlvaine buffer, pH 7 (Figure 5c). Denatured BSA produced 3 times higher peak H at more positive potentials (about 20 mV) than native BSA. CONCLUSIONS We have developed a simple, rapid, and inexpensive procedure for preparation of a solid silver-SAE array and characterized its material composition and electrochemical behavior. The protocol is based on deposition of a silver layer on an insulating material (glass) followed by electrolytic modification with mercury. While the present protocol uses a glass wafer as the array substrate, it can be easily substituted by many ceramic or plastic supporting materials for volume preparation, leading to cost-effective disposable electrodes. While the fabrication process allows preparation of electrodes with various geometries in an array or a single electrode arrangement, (52) Palecek, E.; Trefulka, M.; Fojta, M. Electrochem. Commun. 2009, 11, 359– 362.

Figure 5. (a) Peak H of 5 µM BSA at SAE using conventional adsorptive stripping, AdS (in situ) or adsorptive transfer stripping (ex situ) analysis. (b) AdS peak H of 5 nM native BSA (blue), electrolyte (black), tA of 5 min; Istr of -10 µA; (a,b) background electrolyte: 0.2 M McIlvaine buffer, pH 7. (c) Peak H of 300 nM native (black) and urea denatured (red) BSA in 50 mM Na-phosphate, pH 7; Istr of -30 µA. Higher buffer concentrations (e.g., 0.2 M) result in higher electrocatalytic peak H, while lower buffer concentrations should be used to detect changes in the protein structure.16 Denaturation of 14.4 µM BSA in 0.1 M Tris-HCl, pH 7.3, with 8 M urea was performed overnight at 4 °C. Other details as in Figure 4.

the composition of the mercury amalgam can be easily optimized by precise timing of the electrolysis step. The performance of individual electrodes is comparable to the standard mercury electrodes with the advantage of the batch-to-batch electrode reproducibility. Additionally, unlike pure mercury, the amalgam is not toxic and, as with every single use system, there is no danger of sample cross-contamination. Electrochemical measurements were performed on the electrodes prepared in various time intervals prior to analysis, ranging from hours to over a week. All measurements did not show significant changes in the electrochemical performance. We have also demonstrated that the electrodes can be applied for biomolecular detection. Further work is ongoing to incorporate the electrodes into a miniaturized bioanalytical system. Our results open the door for label-free, reagent-less parallel analysis of biomacromolecules yielding electrochemical signals at highly negative potentials, accessible at mercury-containing electrodes. In addition, DNA, RNA, and proteins can be easily specifically labeled to produce another type of electrocatalytic signals45,52 requiring these electrodes. Thus, a lot of knowledge in nucleic acid8,12 and protein electrochemistry13,16,47 acquired in the past with liquid mercury electrodes can now be utilized in fast developing science areas, such as genomics, proteomics, and biomedicine. ACKNOWLEDGMENT The authors wish to thank Jirˇ´ı Dluhosˇ and TESCAN, s.r.o. for valuable assistance in the electron microscopic work. This work was supported by the Czech Grants: GAAV-KAN400310651, M20004090, and GACR-202/07/P497. MEYS CR (LC06035 and ME 09038) and institutional research plans AV0Z 40310501, AV0Z50040507, and AV0Z50040702 are also acknowledged. Received for review October 15, 2009. Accepted February 22, 2010. AC902333S Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

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