Detection of Heavy Metal Ions at Femtomolar Levels Using Protein

Milan Sasmal , Tapas Kumar Maiti , Tarun Kanti Bhattacharyya .... A Simple Colorimetric Sensor with High Selectivity for Mercury Cation in Aqueous Sol...
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Anal. Chem. 1998, 70, 4162-4169

Detection of Heavy Metal Ions at Femtomolar Levels Using Protein-Based Biosensors Ibolya Bontidean,†,⊥ Christine Berggren,‡ Gillis Johansson,‡ Elisabeth Cso 1 regi,† Bo Mattiasson,*,† § § § Jonathan R. Lloyd, Kenneth J. Jakeman, and Nigel L. Brown

Departments of Biotechnology and Analytical Chemistry, Chemical Center, P.O. Box 124, Lund University, S-221 00 Lund, Sweden, and School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.

Sensors based on proteins (GST-SmtA and MerR) with distinct binding sites for heavy metal ions were developed and characterized. A capacitive signal transducer was used to measure the conformational change following binding. The proteins were overexpressed in Escherichia coli, purified, and immobilized in different ways to a self-assembled thiol layer on a gold electrode placed as the working electrode in a potentiostatic arrangement in a flow analysis system. The selectivity and the sensitivity of the two protein-based biosensors were measured and compared for copper, cadmium, mercury, and zinc ions. The GST-SmtA electrodes displayed a broader selectivity (sensing all four heavy metal ions) compared with the MerR-based ones, which showed an accentuated selectivity for mercury ions. Metal ions could be detected with both electrode types down to femtomolar concentration. The upper measuring limits, presumably due to near saturation of the proteins’ binding sites, were around 10-10 M. Control electrodes similarly constructed but based on bovine serum albumin or urease did not yield any signals. The electrodes could be regenerated with EDTA and used for more than 2 weeks with about 40% reduction in sensitivity. Heavy metals are ubiquitous in nature, thus constituting a serious environmental problem in many countries. Due to their toxicity, there is an obvious need to determine them at trace levels. There is also a need to measure metal ion concentrations in medicine, food, and other products. Various classical methods such as atomic absorption spectroscopy,1 inductively coupled plasma optical emission spectrometry,1 and inductively coupled plasma mass spectrometry (ICPMS)1,2 are in wide use. These methods require sophisticated instrumentation and skilled personnel, and there is, therefore, a need for simpler methods. Electrochemical methods require less complex instrumentation, and the techniques for metal ion determination include the use of ion-

selective electrodes, polarography, and other voltammetric methods.3 The electrochemical methods are generally able to selectively detect bioavailable heavy metal ions. Biosensors are promising analytical devices, and several different configurations have been described in the past for heavy metal detection. Whole cell biosensors can be based on bacteria, yeasts, fungi, lichens, mosses, and water plants as the recognition element.4 Another approach is to use enzymes for detection.5 So far, the use of apoenzymes has been the most successful.6 However, these sensors are characterized by limited selectivity and fairly low sensitivity. Design and development of new sensor types using specific proteins as the biorecognition elements to meet the high selectivity and sensitivity requirements is strongly desirable for environmental protection. Metal-binding proteins are synthesized by many cell types in response to the presence of specific metals and play important roles in metal homeostasis and detoxification mechanisms.7 Due to a wide spectrum of selectivity, metal-binding proteins could be used as the basis of biosensors of varying specificity, depending on the required application. Recent advances in molecular biology and genetics have facilitated rapid progress in the study of these proteins.7,8 The characteristics of two classes of metal-binding proteins which could be used in heavy metal biosensors are described below. The metallothioneins are low-molecular-weight, cysteine-rich proteins which bind heavy metal ions nonspecifically in metalthiolate clusters.9 They are ubiquitous in higher eukaryotes and are synthesized in response to elevated concentrations of heavy metal ions (e.g., silver, bismuth, cadmium, cobalt, copper, mercury, nickel, or zinc). Metallothioneins have also been identified in the cyanobacterium Synechococcus sp.10 The synechococcal metallothionein SmtA has been studied in detail.11 Synthesis of

Department of Biotechnology, Lund University. Department of Analytical Chemistry, Lund University. § The University of Birmingham. ⊥ On leave from Faculty of Chemistry, Department of Physical Chemistry, The Babes-Bolyai University, Str. Arany Ja´nos No. 11, Ro-3400 Cluj-Napoca, Romania. (1) Jackson, K. W.; Chen, G. Anal. Chem. 1996, 68, 231R-256R. (2) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R651R.

(3) Anderson, J. L.; Bowden, E. F.; Pickup, P. G. Anal. Chem. 1996, 68, 379R444R. (4) Wittman, C.; Riedel, K.; Schmid, R. D. In Handbook of Biosensors and Electronic Noses; Kress-Rogers, E., Ed.; CRC: Boca Raton, FL, 1997; pp 299332. (5) O ¨ gren, L.; Johansson, G. Anal. Chim. Acta 1978, 96, 1-11. (6) Mattiasson, B.; Nilsson, H.; Olsson, B. J. Appl. Biochem. 1979, 1, 377384. (7) Beveridge, T. J.; Hughes, M. N.; Lee, H.; Leung, K. T.; Poole, R. K.; Savvaidis, I.; Silver, S.; Trevors, J. T. Adv. Microb. Physiol. 1997, 38, 177-243. (8) Silver, S. Gene 1996, 179, 9-19. (9) Stillman, M. J. Coord. Chem. Rev. 1995, 144, 461-511. (10) Olafson, R. W. Int. J. Pept. Protein Res. 1984, 24, 303-308. (11) Turner, J. S.; Robinson, N. J. J. Ind. Microbiol. 1995, 14, 119-125.

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S0003-2700(98)00363-1 CCC: $15.00

† ‡

© 1998 American Chemical Society Published on Web 09/04/1998

SmtA is increased in response to elevated concentrations of Cd2+ or Zn2+. Mutants unable to synthesize the protein are more sensitive to these metals.12 The protein has been expressed as a recombinant fusion protein, and its metal-binding characteristics have been determined in vitro. The protein has a greater affinity for Zn2+ than equine metallothionein but a reduced affinity for Cd2+ and Cu2+.13 SmtA also binds Hg2+. The mercury resistance proteins encoded by the mer operon of Tn501 in Pseudomonas aeruginosa offer increased selectivity and could possibly be used in biosensors specific for Hg2+. One protein of particular interest is the regulatory protein MerR, which controls the expression of itself and the other mer gene products required for mercury detoxification.14 Binding of Hg2+ to MerR results in a conformational change, aligning the protein with contacts at the promotor region of the operon-activating transcription of the mer genes.15 The proteins encoded by these genes could also be used as the recognition component of a biosensor. These include not only MerR but also MerP which sequesters Hg2+ in the periplasm (the outer compartment of the cell), protecting other proteins in this location from damage by reactive Hg2+ ions, and MerA, which is a cytoplasmic protein that catalyzes the reduction of Hg2+ to relatively nontoxic Hg(0), which can diffuse out of the cell as mercury vapor.16 Two metal-binding proteins were overexpressed, purified to homogeneity, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the protein visualized by Coomassie blue stain, and used in prototype biosensors. The broadspecificity metallothionein SmtA was overexpressed in Escherichia coli as a carboxy-terminal extension of glutathione-S-transferase. The mercuric ion-binding MerR protein was overexpressed in E. coli as the native protein. It is believed that a large conformational change takes place when heavy metal ions bind to these proteins. A suitable transducer should be able to detect this change directly. A capacitive transducer with immobilized antibodies has recently shown high selectivity and detection limits down to 10-15 M.17 The coupling of this type of transducer with the proteins mentioned above is the subject of the present work. EXPERIMENTAL SECTION Protein Production and Purification. Expression and Purification of the GST-SmtA Fusion Protein. The cyanobacterial metallothionein SmtA was overexpressed in E. coli as a carboxyterminal extension of glutathione-S-transferase (GST) and purified using glutathione Sepharose 4B (Pharmacia Biotechnology, Uppsala, Sweden) affinity chromatography. Plasmid pGEX3X containing the smtA gene was supplied by Prof. N. J. Robinson (University of Newcastle) and transformed into E. coli using standard techniques. The construction of the plasmid, which encodes the GST-SmtA fusion protein, was described by Shi et al.13 A single colony from an agar plate was used to inoculate a 500-mL flask (12) Turner, J. S.; Morby, A. P.; Whitton, B. A.; Gupta, A.; Robinson, N. J. J. Biol. Chem. 1993, 268, 4494-4498. (13) Shi, J.; Lindsay, W. P.; Huckle, J. W.; Morby, A. P.; Robinson, N. J. FEBS Lett. 1992, 303, 159-163. (14) Foster, T. J.; Brown, N. L. J. Bacteriol. 1985, 163, 1153-1157. (15) O’Halloran, T. V.; Frantz, B.; Shin, M. K.; Ralston, D. M.; Wright, J. G. Cell 1989, 56, 119-129. (16) Hobman, J. L.; Brown, N. L. Metal Ions Biol. Syst. 1997, 34, 527-568. (17) Berggren, C.; Johansson, G. Anal. Chem. 1997, 69, 3651-3657.

containing 100 mL of Luria broth supplemented with tetracycline (15 µg/mL) and ticarcillin (200 µg/mL). After 16 h of incubation at 37 °C with constant agitation (orbital shaker, 150 rpm), an 80mL aliquot was used to inoculate 800 mL of Luria broth supplemented with ampicillin (100 µg/mL) in a 2-L conical flask. The culture was incubated at 37 °C on an orbital shaker, and after 45 min, Zn2+ (as ZnSO4) was added to a final concentration of 500 µM. The production of the recombinant fusion protein was induced after a further 15 min by the addition of isopropyl β-Dthiogalactoside (IPTG) to a final concentration of 1 mM. Cells were then grown for 4 h, after which they were collected by centrifugation (5000g for 15 min). The pellet was resuspended in 1% of the original volume of ice-cold 1% (v/v) Triton X-100, 150 mM NaCl, 16 mM NaH2PO4, and 4 mM Na2HPO4 (pH 7.3), and the cells were lysed by gentle sonication at 4 °C. The centrifugation step was repeated to remove the cellular debris, and the soluble GST-SmtA fusion protein was purified from the supernatant by single-step affinity chromatography using glutathione Sepharose 4B. After three wash steps with phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.3), the fusion protein was eluted with 3 bed volumes of 5 mM glutathione in 50 mM Tris-HCl, pH 8.0. Finally, the eluant was passed through a column of Sephadex G25 (Pharmacia Biotechnology) to remove the glutathione. GST-SmtA was stored at -20 °C in PBS containing 50% (v/v) glycerol until use. All chemicals used were supplied by Sigma-Aldrich (Poole, UK). Expression and Purification of MerR. The mercuric ion-binding protein MerR was also overexpressed in E. coli and purified using sonication, salt extraction, and HPLC affinity chromatography using a method modified from that of Parkhill et al.18 Plasmid pKK containing the merR gene was transformed into E. coli TG2. A 10-mL starter culture was grown from a single colony on an agar plate for 16 h in Luria broth supplemented with tetracycline and ticarcillin. This culture was then added to 800 mL of Luria broth supplemented with glucose (0.2% w/v) in a 2-L conical flask. The cells were grown at 37 °C on an orbital shaker, and MerR production was induced by the addition of IPTG to a final concentration of 50 µM, when the optical density (OD600 nm) reached 0.7. Cells were grown for 1 h and collected by centrifugation at 5000g for 15 min. The cell pellet was resuspended in Tris buffer (100 mM Tris, 10 mM MgCl2, 10% glycerol, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol, 0.01 mg/mL soybean trypsin inhibitor, 0.02 mg/mL L-1-tosylamide 2-phenylethyl chloromethyl ketone, 0.02 mg/mL N-p-tosyl-L-lysine chloromethyl ketone, 0.01 mg/mL leupeptin, 0.01 mg/mL aprotonin; pH 8.0) to a final concentration of 1 g of biomass (wet weight) per 2 mL of buffer. After gentle sonication, the cell debris was removed by centrifugation (14000g, 30 min) and resuspended in Tris buffer (20 mM Tris, 1 M NaCl, 10 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA; pH 7.4). The cell debris was removed again by centrifugation, and 0.2% streptomycin sulfate added to the MerR-containing supernatant. A further centrifugation step preceded the addition of ammonium sulfate to the supernatant (to 50% saturation). After 30 min, the precipitated protein was collected by centrifugation (14000g for 30 min). (18) Parkhill, J.; Ansari, A. Z.; Wright, J. G.; Brown, N. L.; O’Halloran, T. V. EMBO J. 1993, 12, 413-421.

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Final purification was done using a Biocompatible HPLC system (Bio-Rad, Watford, UK). The protein pellet was dissolved in Tris buffer (20 mM Tris, 0.1 M NaCl, 10 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA; pH 7.4; 100 mL of buffer per 800 mL of bacterial culture used), and 200 mL was loaded onto a heparin column (Bio-Rad). The MerR was eluted with a salt gradient from 0.1 to 0.5 M NaCl in a mobile phase of 20 mM phosphate buffer (pH 6.0) containing 2 mM dithiothreitol, 0.1 mM EDTA, and glycerol (10% v/v). Fractions containing MerR were identified by SDS-PAGE electrophoresis and dialyzed overnight against a 100-fold excess of 0.1 M NaCl, 1 mM dithiothreitol, and 20 mM phosphate buffer (pH 7.0). The sample was then loaded onto a Poros SP/M cation-exchange column (Boehringer Mannheim UK, Lewes, UK) and eluted with a 0.11.0 M NaCl gradient. MerR-containing fractions were identified using SDS-PAGE and stored under nitrogen at -20 °C in buffer supplemented with 2 mM dithiothreitol and glycerol (50%, v/v). All buffers were purged with nitrogen and degassed prior to use. All chemicals used were supplied by Sigma-Aldrich, except dithiothreitol, which was obtained from Melford Laboratories (Ipswich, UK). Chemicals. The fusion proteins GST-SmtA and MerR were dissolved in phosphate-buffered saline (70 mM NaCl, 1.3 mM KCl, 5 mM Na2HPO4, 0.9 mM KH2PO4; pH 7.3) containing 50% (v/v) glycerol to a final concentration of 1 mg/mL protein. Thioctic acid, glutaraldehyde (GA), and bovine serum albumin (BSA, initial fractionation by heat shock) were purchased from Sigma (St. Louis, MO), and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) was obtained from Fluka AG (Buchs, Switzerland). Gold rods, 99.99%, used as electrode material (Catalog No. 26,583-7, 3 mm in diameter) and 1-dodecanethiol were from Aldrich Chemicals (Milwaukee, WI). The heavy metal salts CuCl2‚2H2O, ZnCl2, HgCl2, and Cd(NO3)2‚4H2O were all from Merck (Darmstadt, Germany). Polyethyleneglycol diglycidyl ether (PEGDGE) was from Polysciences Inc. ( Warrington, PA). All reagents were of analytical grade. Solutions were, if not otherwise specified, prepared with water obtained from a Milli-Q system, preceded by a reverse osmosis step, both from Millipore (Bedford, MA). To remove heavy metal traces, all glassware was treated with 3 M HNO3 for 3 days, followed by soaking in Millipore water, before use. Biosensor Design. Biosensors were prepared by immobilizing the fusion proteins on the gold surface by (i) EDC coupling, (ii) PEGDGE entrapment, and (iii) glutaraldehyde coupling. In all cases, 20 µL of the protein was diluted with 480 µL of 100 mM borate buffer, pH 8.75, and the solution was ultrafiltered on a microfilter (Amicon, Beverly, MA) with a molecular weight cutoff of 3000. After ultrafiltration, the protein was recovered from the filter. The protein concentration was adjusted to approximately 0.04 mg/mL with borate buffer. Gold electrodes were cleaned and pretreated with thioctic acid, as described earlier,17 or with cysteamine. As the MerR protein is sensitive to oxidation in air, the preparation of these electrodes was performed under a nitrogen atmosphere. The activation was performed at room temperature if not otherwise specified. (i) EDC Coupling. Thioctic acid self-assembled electrodes were thoroughly washed with ethanol, dried, and thereafter activated in a 1% solution of EDC in dried acetonitrile for 5 h. 4164 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

Figure 1. Schematic drawing of the experimental setup.

After being washed with 100 mM borate buffer, pH 8.75, the electrodes were dipped into the protein solution at 4 °C for 24 h. Next, each electrode was thoroughly washed with borate buffer and immersed in a solution of 1-dodecanethiol for 20 min. (ii) PEGDGE Entrapment. The thioctic acid-activated electrodes were covered with 1.5 µL of a 0.04 mg/mL protein solution in 100 mM borate buffer also containing 10 or 30% (w/w) PEGDGE, and the electrodes were cured at 45 °C for 15 min. Next, the electrodes were immersed into 1-dodecanethiol for 20 min, washed with borate buffer, and placed in the measuring cell. PEGDGE solutions were freshly prepared and used within 15 min. (iii) Glutaraldehyde Coupling. Prior to glutaraldehyde immobilization, the electrodes were modified with cysteamine, instead of thioctic acid. The dried electrodes were dipped into a solution containing 12.5% (w/v) glutaraldehyde in coupling buffer (0.1 M sodium phosphate, 0.15 M NaCl; pH 7) and 6 g/L NaCNBH3 for 4 h. Thereafter, the electrodes were washed thoroughly with the same buffer, dipped into the protein solution (0.04 mg/mL) with 6 g/L NaCNBH3 for 4 h, washed with buffer again, and immersed in 1-dodecanethiol for 20 min. Final washing with buffer completed the electrode preparation. It has been observed during work on another biomolecule that there were no capacitance changes if the 1-dodecanthiol treatment was extended to 2 h. Displacement by the thiol is thus negligible. Capacitance Measurements. The biosensor was inserted as the working electrode in a specially constructed three (four)electrode flow cell with a dead volume of 10 µL, as shown in Figure 1. The electrodes were connected to a fast potentiostat described separately.19 A platinum foil and a platinum wire served as auxiliary and reference electrodes, respectively. An extra reference electrode (Ag/AgCl) was placed in the outlet stream, as described below. The buffer solution was pumped by a Minipulse 3 peristaltic pump (Gilson Medical Electronics, Villiers-le-Bel, France) with a flow rate of 0.5 mL/min. Samples were injected into the flow via a 250-µL sample loop. The carrier buffer (10 mM borate; pH 8.75) was filtered through a 0.22 µm Millipore filter and degassed before use. (19) Berggren, C.; Bjarnason, B.; Johansson, G. Instrum. Sci. Technol., in press.

The working electrode had a rest potential of 0 mV vs the Ag/AgCl reference electrode. Measurements were made by applying a potential pulse of 50 mV and recording the current transients following the potential step according to eq 1, where

i(t) ) u/Rs exp(-t/RsC1)

(1)

i(t) is the current at time t, u is the amplitude of the potential pulse applied, Rs is the resistance between the gold and the reference electrodes, C1 is the total capacitance over the immobilized layer, and t is the time elapsed after the potential pulse was applied. It is known that, for thiols, the values of resistance and capacitance are independent of the pulse amplitude. The current values were collected with a frequency of 50 kHz, and the first 10 values were used for the evaluation of the capacitance. An equal current transient but with opposite sign was obtained when the potential was stepped back to the rest value. Ten measurements were made with an interval of 1 s, and the capacitance was calculated from the mean. The platinum reference electrode controls the working electrode potential, but it does not have a well-defined potential. However, such an electrode is necessary to obtain a sharp response in a small dead volume cell. The platinum and Ag/AgCl reference electrodes were, therefore, compared potentiometrically just before a step was applied. The computer adjusted the working electrode potential so that the potentiostat behaved as if it had a Ag/AgCl reference controlling the working electrode. A more detailed description of the measurements has been given before,17 as well as a thorough description of the potentiostat.19 Cyclic Voltammetry Measurements. Cyclic voltammograms were recorded in 5 mM K3[Fe(CN)6], 0.1 M KCl in a batch cell, with the unmodified/modified gold electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum flag as the auxiliary electrode, at a scan rate of 100 mV/s. The electrodes were connected to a Princeton Applied 274 A potentiostat (Princeton Applied Research, Princeton, NJ), connected to a computer. Spectrometry. The heavy metal content in the test buffers was determined with ICPMS (VG PQ2+, Fisons Elemental, Winsford, Cheshire, UK) for Cu, Zn, and Cd and with cold vapor atomic fluorescence spectrometry (Merlin Plus System, P. S. Analytical Ltd., Orpington, Kent, UK) for Hg. RESULTS AND DISCUSSION Well-established and easy methods based on the self-assembly of thiol, sulfide, or disulfide compounds onto gold were used to immobilize different proteins on the surface of the electrode. Selfassembly is a spontaneous process producing well-ordered monolayers of molecules.20 Furthermore, the binding between sulfur and gold is rather strong,21 an important aspect in biosensor design, where leaking of the recognition element will result in loss of activity and, hence, decreased stability. Figure 2 shows the relevant amino acid sequences of both the GST-SmtA and MerR proteins, with the cysteine residues that are required for (20) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (21) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335.

Figure 2. Amino acid sequences of (a) GST-SmtA (b) and MerR. The cysteine residues required for heavy metal binding are marked (/). The GST-SmtA and MerR protein sequences of the heavy metal ion binding domain are denoted with a line.

Figure 3. Protein-based biosensor concept for measuring conformational change upon binding of heavy metal ions.

heavy metal binding marked with asterisks. The protein is bound to the self-assembled molecules through one or more amino acid residues. The detection principle used in this work is based on measuring the conformational change of the immobilized protein when a heavy metal ion is bound to it (see Figure 3). Using capacitance measurements, the interface between a conducting electrode and the sample solution can be studied. The interface consists of a metallic conductor which is covered by nonconducting organic molecules, outside of which ions in an aqueous conducting liquid form a space-charge. The total capacitance is composed of a series of capacitances representing the self-assembled molecules, the protein, and the space-charge of ions. Since the inverse total capacitance is the sum of the inverse capacitances of each layer, it is important to make each capacitance as large as possible so that the changes caused by recognition dominate as much as possible. Most studies on selfassembly have been made on surfaces covered with long-chain alkanethiols.22 Such layers result in small capacitances which, as discussed below, are undesirable. We have, therefore, used the thinner and much more polar thioctic acid or cysteamine. The metal ion binding will cause the space-charge in the aqueous solution to move closer to the electrode because of changes in the protein conformation. The importance of a good insulation of the electrode surface has earlier been demonstrated.17,19 If an aqueous conducting solution can penetrate the layer, it will increase its capacitance due to the greater polarity of water. The conductivity of the water will, furthermore, be equivalent to a resistor in parallel with the (22) Anderson, J. L.; Bowden, E. F.; Pickup, P. G. Anal. Chem. 1996, 68, 410R412R.

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Figure 5. Chemical structure of the immobilizing agents used: (a) EDC, (b) PEGDGE, and (c) GA.

Figure 4. Cyclic voltammograms recorded for (a) a clean gold electrode, (b) gold modified with thioctic acid, (c) gold modified with thioctic acid and EDC-immobilized GST-SmtA protein, and (d) as in (c) but with additional 1-dodecanethiol treatment. Measurements were performed in 5 mM K3[Fe(CN)6], 0.1 M KCl, with a scan rate of 100 mV/s. A SCE was used as a reference electrode.

capacitance, thereby partially shortcircuiting it. Well-ordered surface structures, as those which can be obtained by selfassembly, are thus crucial. Treatment with dodecanethiol will reduce the capacitance further and prevent shortcircuiting of the hydrophobic layer. Blocking of Faradaic reactions at the metal surface will prevent interferences from redox couples in solution, and it will also reduce the background current by orders of magnitude compared to an untreated metal electrode. The degree of insulation can be studied with cyclic voltammetry, by having a small, permeable redox couple such as, e.g., K3[Fe(CN)6], in the solution. In Figure 4, it is shown how the blocking increases for each additional layer immobilized on the electrode surface. On a clean gold surface, the redox couple is oxidized and reduced at the metal surface during cycling. A surface covered with selfassembled thioctic acid reduces the access to the surface to a certain degree. Immobilization of the protein further insulates the surface, but it is not until the treatment in 1-dodecanethiol that the oxidation/reduction peaks totally disappear. All electrodes in the following experiments were, therefore, treated for 20 min with 1-dodecanethiol. The straight-chain dodecanethiol will penetrate through the space between protein molecules and bind to the gold surface. Impurities, e.g., copper, or defect sites in the monolayer might not be covered by thioctic acid or cysteamine and thus might be accessible to ions from the solution. Long-chain dodecanethiol molecules will bind to nearby gold atoms, after which the alkyl chains might cover the metal through fold-over. Protein Immobilization. Since the measuring principle is highly dependent on the electrode surface configuration, the protein immobilization procedure is of key importance. Three different immobilization methods were, therefore, considered in this work, to produce a suitable surface for capacitance measurements: (i) EDC-based coupling, (ii) PEGDGE entrapment, and (iii) glutaraldehyde coupling. In all cases, a short compound containing one or more sulfur groups was first self-assembled onto the gold surface. (i) The EDC-based coupling produces a monolayer of protein molecules on the surface without any cross-linking between 4166 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

Figure 6. Capacitance changes for Cu2+ measured with the GSTSmtA electrode: (a) EDC immobilization, (b) 10% (w/w) PEGDGE, and (c) 30% (w/w) PEGDGE. Measurements were performed in 10 mM borate buffer, pH 8.75, flow rate 0.5 mL/min, sample injection volume 250 µL.

individual molecules. This method has been previously shown to be successful for immobilization of antibodies onto gold electrodes with self-assembled thioctic acid on the surface.17,19 (ii) PEGDGE (see structure in Figure 5) is widely used for the construction of “wired” enzyme electrodes, when various enzymes are cross-linked to redox polymeric cations, resulting in highly permeable redox hydrogels and stable biosensor designs.23-25 Calibration curves obtained for EDC-coupled and PEGDGE-entrapped protein-based electrodes using Cu2+ as the substrate are shown in Figure 6. A thorough investigation of the effect of PEGDGE is in progress. As seen, the EDC-activated electrodes displayed sensitivities twice as high as those prepared by protein entrapment in PEGDGE when the concentration of the polymer was 30% (w/w). Previous work using this polymer to cross-link various enzymes to polycationic redox networks23-25 showed that a rigid network will result in a decreased rate of electron transfer and, thus, decreased current signals. Similarly, it can be assumed that, if cross-linking of the proteins is too tight, a certain degree of conformational change, when binding the heavy metal ions, will be prohibited, thus resulting in lower signals. Therefore, new batches of electrodes were prepared using only 10% (w/w) of the cross-linker. These electrodes displayed 50% higher signals than those prepared with 30% PEGDGE, but the recorded signals were still 50% lower than those obtained for EDC-activated electrodes (see Figure 6). However, (23) Heller, A. J. Phys. Chem. 1992, 96, 3579-3587. (24) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65, 3512-3517. (25) Cso¨regi, E.; Schmidtke, D. W.; Heller, A. Anal. Chem. 1995, 67, 12401244.

Figure 7. (A) Calibration curves obtained for an EDC-modified GST-SmtA-based electrode for Hg2+, Cu2+, Cd2+, and Zn2+. The lowest plot represents a control which shows the Cu2+ response for a BSA-based electrode. (B) Capacitance changes vs logarithmic heavy metal ion concentration for an EDC-modified MerR electrode for Hg2+, Cu2+, Zn2+, and Cd2+. Other experimental conditions are as in Figure 6.

another reason for the lower sensitivity with PEGDGE coupling might be that the protein is partially denatured during coupling. Current experiments are expected to elucidate the observed phenomena. (iii) GA (see structure in Figure 5) is also widely used and, in many applications, has been shown to be an effective crosslinker26-28 which requires, however, the presence of amino groups at both the protein and the electrode surface. For glutaraldehydecoupled electrodes, the cysteamine-covered gold rods were first activated with glutaraldehyde before the protein was added, to avoid cross-linking between protein molecules. However, no signals could be observed with this immobilization method. This might be because the coupling procedure could damage the protein or because this type of cross-linking resulted in a too rigid structure, prohibiting conformational changes. Accordingly, only the EDC-coupling method was used in all following experiments, and the results presented are the means of four separate equivalent electrode preparations. The relative standard deviation between electrodes varied depending on the metal ion and concentration. The lowest values, 1.6-13%, were obtained for Cu2+ and Cd2+, while Zn2+ and Hg2+ gave the highest values, 9-42%. The results were obtained with freshly prepared electrodes, each with a new protein coating, and the standard deviation would probably have been much lower if the electrodes had been treated with EDTA first. GST-SmtA Electrodes. The affinity of the GST-SmtA protein was studied for Cu2+, Cd2+, Hg2+, and Zn2+ (see Figure 7A). The electrodes responded for all four metal ions with decreasing sensitivity in the order Cu2+ > Cd2+ > Zn2+, well in agreement with previously reported dissociation results obtained at higher metal concentrations with gel chromatography of this protein.13 The Hg2+ response seems to be steeper, displaying a higher sensitivity than the other metal ions at 10-10 M. No leveling off tendency could be observed at 10-10 M for Hg2+, in contrast to (26) Bradley, C. R.; Rechnitz, G. A. Anal. Chem. 1985, 57, 1401-1404. (27) Verdyuyn, C.; Van Dijken, J. P.; Scheffers, W. A. Biotechnol. Bioenerg. 1983, 25, 329-340. (28) Mattiasson, B.; Danielsson, B. Carbohydr. Res. 1982, 102, 273-282.

the behavior of the other studied ions. The working range is from the femtomolar range to the nanomolar range for each of the studied heavy metal ions. Control electrodes prepared by using BSA instead of GST-SmtA did not give any usable signals for Cu2+ (see Figure 7A), even though BSA binds Cu in vitro,29 nor did it give any signals with the other ions. Similar results were obtained when using another protein, urease, known to bind mercury ions.5 In the latter case, the absence of any detectable signal was assumed to be due to lack of conformational changes upon exposure to heavy metal ions. The activity of a GST-SmtA electrode was studied for an extended concentration range. Samples of 10-15-0.1 M Cu2+ were injected, and the capacitance changes were recorded. A capacitance decrease was obtained up to approximately 10-10 M, where saturation seemed to occur. The capacitance remained thereafter constant up to 10-5 M, where the signal decreased again to approximately 10-2 M (see Figure 8). It is believed that the decrease in the first part of the curve is due to the reversible binding of heavy metal ions and that the second one probably is due to a major alteration in the structure of the protein on the electrode surface. The electrode could not be regenerated after injection of 1 mM Cu2+, supporting this assumption. In vivo metallothionein is responsible for sequestering excess metal,11 and a conformational change as the protein binds a complete complement of metal ions might be expected above 10-5 M. The stability of the GST-SmtA-based biosensor was studied over 15 days. A calibration curve between 10-15 and 10-10 M Cu2+ was recorded, and the accumulated capacitance change for each calibration was plotted vs time in Figure 9. The sensor was stored at 4 °C in 100 mM borate buffer, pH 8.75, between measurements, and it was regenerated with EDTA just before a new measurement was started. A loss of activity was observed after 10 days, and the sensitivity had decreased by approximately 40% after 15 days. The surface of the biosensor could, however, be regenerated by injecting 250 µL of 1 mM EDTA. It was found that, if the (29) McArdle, H. J.; Gross, S. M.; Danks, D. M.; Wedd, A. G. Am. J. Physiol. 1990, 258, 988-991.

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Table 1. Highest Permitted Metal Ion Concentration To Avoid Metal Hydroxide Precipitation at pH 8.75 (Second Row) and Free Metal Ion Concentration Given as a Percentage of Total (Fourth Row) or Given as Concentration at Me2+tot ) 10-10 (Fifth Row) Cu2+ pKsob limit of pp, M log βnc Me2+, free, % Me2+, M free at Me2+tot ) 10-10 M

Figure 8. Cu2+ response for EDC-coupled GST-SmtA electrodes in the concentration range of 10-15-0.1 M. Other experimental conditions are as in Figure 6. The line in the inset was an automatic nonlinear least-squares fit made in the Graph program.

Figure 9. Storage stability of an EDC-modified GST-SmtA electrode. Accumulated capacity changes between 10-15 and 10-11 M were recorded on different days. Between measurements, the sensor was stored at 4 °C in 100 mM borate buffer at pH 8.75.

biosensor was stored in regenerated condition, it lost the total activity overnight, but if the electrode was stored (straight after measuring Cu2+ concentrations up to 10-10 M) in 100 mM borate buffer, and the regeneration procedure took place immediately before a new measurement, no such additional activity loss was observed. The activity loss could be due to the fact that the metalbinding cysteine residues are more prone to oxidation in the uncoordinated state. Alternatively, the protein might have a more open structure when the heavy metal ion is absent, and, hence, this structure is more easily denatured. Regeneration with EDTA after injection of a sample containing 1 mM Cu2+ was unsuccessful, which could be attributed to an irreversible conformational change or denaturation by the high metal ion concentration. The capacitance increased from 2600 to 3500 nF/cm2, with a variation of 2% between the highest and lowest values obtained for different electrodes the first time an electrode was treated with EDTA, indicating that the complexing agent removed metal ions bound to the protein during the preparation. All results in this study, except those shown in Figure 9, were obtained using freshly prepared electrodes not treated with EDTA. Calibration curves thus show the accumulated changes for a particular electrode, after which it is provided with a new coating. 4168 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

19.1 2.51 × 10-9 log β1, 7.13 log β2, 12.45 log β3, 15.17 2.1 × 10-6 2.1 × 10-18

Cd2+

Zn2+

Hg2+ a

14.6 16.42 25.44 7.94 × 10-5 1.20 × 10-6 1.15 × 10-15 log β4, 10.6 log β4, 11.8 46 4.6 × 10-11

5.2 5.2 × 10-12

a No complex constants were available for Hg2+. b pK so is the solubility product of the OH- complex. c βn is the overall stability constant.

MerR Electrodes. Using the described EDC-based design, similar biosensors were constructed with the second protein, MerR, and signals were recorded for Cu2+, Cd2+, Hg2+, and Zn2+ (see Figure 7B). This protein has been reported to be highly specific toward Hg2+.30 The MerR-based electrodes displayed high selectivity for Hg2+, and this electrode showed higher selectivity than the GST-SmtA-based electrodes (see Figure 7). The starting capacitance was around 1800 nF/cm2, as compared to 2600 nF/ cm2 for the GST-SmtA electrode. As discussed in a separate paper,31 several factors besides size influence the value of the capacitance. The capacitance changes on binding will be related to the conformation change rather than to protein size. The resistances were approximately 280 Ωcm2 for both the MerR and GST-SmtA electrodes. We have presently no explanation for the different curve shapes obtained with the different metal ions. However, the concentration range across which data are obtained may be less than that showing specific binding in vivo.32 Solution Equilibria. When working with metal ions in alkaline media, it is important to calculate conditions under which precipitation can occur. Table 1 shows the solubility products33 in the first row and the metal ion concentration at which precipitation might be expected at pH 8.75 in the second row. In cases where several values were available in the literature, we have selected the most unfavorable. A pKw of 14.0 was used for the calculations.34 Borate forms complexes with metal ions according to eq 2,

M + nL ) MLn, βn ) [ML]n/[M][L]n

(2)

where βn is the overall stability constant of the metal complex MLn. Stability constants for borate complexes were available for (30) Frantz, B.; O’Halloran, T. V. Biochemistry 1990, 29, 4747-4751. (31) Berggren, C.; Bjarnason, B.; Johansson, G. Biosens. Bioelectron. 1998, in press. (32) Ralston, D. M.; O’Halloran, T. V. O. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3846-3850. (33) Ho ¨gfeldt, E. IUPACsStability Constants of Metal-Ion Complexes. Part A: Inorganic Ligands; Pergamon Press Ltd.: Oxford, 1982. (34) Bates, R. G. Determination of pH, Theory and Practice; John Wiley & Sons: New York, 1973.

Table 2. Free Mercury Concentrations at pH 8.75 and in the Presence of 1 mM Chelating Agent chelating agent log βn

a

pKnb

Hg2+, free, % Hg2+, M free at Hg2+tot ) 10-10 M

picolinic acid

glycine

EDTA

log β1, 7.70 log β2, 15.55 pK1, 5.39

log β1, 10.3 log β2, 19.2 pK1, 2.35 pK2, 9.87

log β1, 21.7

2.8 × 10-12 2.8 × 10-24

6.3 × 10-8 6.3 × 10-20

pK1, 2.0 pK2, 2.66 pK3, 6.32 pK4, 11.01 2.0 × 10-17 2.0 × 10-29

a β is the overall stability constant of the metal-ligand. b pK is n n the negative logarithm of the protonation constant.

all metal ions except mercury,35 and they are given in the third row of Table 1. Boric acid is present in a very large excess, and with a pK of 9.24,35 it can be assumed that all the borate is present as B(OH)4-. The percentage of free metal ions can now be calculated, and the obtained values are given in the fourth row of Table 1. The last row indicates the free metal ion concentration at 10-10 M. It can be seen that there should not be any problems with precipitation, except, possibly, for mercury. It can also be seen that buffers without chelating properties could have been used for Cd2+ and Zn2+, since the solubility products were fairly low. The strength of the binding between the protein and the metal ion was studied by allowing the electrode to compete for metal ions with soluble complexes. We selected Hg2+ as an example since a suitable complex could be added to the buffers in order to make it even less likely that mercury ion precipitation would affect the results. Table 2 gives the relevant constants for glycine and picolinic acid as well as those for EDTA.35 However, no constants were available for phthalic acid. EDTA could be used to regenerate the electrode, and the protein is thus obviously a weaker complex-former than EDTA. Runs with glycine added to the carrier showed that the capacitance slowly increased from 2600 to around 2800 nF cm-2 during an initial pumping period of about 1 h. Treatment with EDTA resulted in a capacitance value of about 3500 nF cm-2. The glycine ligand thus seems to remove metal ions from the protein, although more slowly and to a lesser degree than EDTA. Injections of 10-15-10-11 M Hg2+ in 1 mM glycine, 1 mM picolinic acid, and 1 µM phthalic acid (all in 10 mM borate buffer) did not give significant changes in the capacitances. To test whether the metal ion uptake from complexes was slow, a solution with 10-11 M Hg2+ and 1 µM phthalic acid was run through the cell for 1 h. No decrease in capacitance was observed. Our conclusion was, therefore, that the protein was not able to take up mercury ions, even from a complex as weak as that formed with picolinic acid. The data obtained thus give an indication of the binding strength of Hg2+ to the protein. Furthermore, Hg2+ binding at concentrations of 10-15-10-11 M Hg2+ was found to be reversible, whereas Shi et al.13 reported that Hg2+ binding at millimolar concentrations was irreversible, even when the pH is reduced. (35) Kotrly, S.; Sucha, L. In Handbook of Chemical Equilibria in Analytical Chemistry; Chalmers, R. A., Masson, M., Eds.; Ellis Horwood Limited: Chichester, 1985.

Buffer Contamination. Metal ion contamination of buffers represents a great problem in all methods for trace metal determinations, and the validity of the low levels reported here should, whenever possible, be checked by independent methods. The sensitivity of the capacitive sensors is so high, however, that a comparison with other methods over the whole range is impossible. Hg2+ was studied using cold vapor atomic fluorescence spectrometry, and it was found that a reagent blank corresponded to 5 × 10-12 M Hg2+. Measurements on the borate buffer gave 4 × 10-12 M, i.e., less than the blank. An upper limit for mercury contamination as given by this method should be around 10-12 M. Considerable difficulties were encountered with ICPMS for Cu2+, Zn2+, and Cd2+. Responses were obtained even on the introduction of argon into the plasma. The recordings showed peaks at the respective mass numbers, but it is unlikely that the responses represent real metal contamination, because different levels were obtained at the different mass numbers, particularly for Cu2+. Readings with Millipore water as a sample were in some cases lower than those with argon alone. Measurements on the borate buffer resulted in slightly higher values than those in the Millipore water. The significance of the low-level ICPMS measurements is questionable, and no relevant conclusions can be drawn regarding Cu2+ and Zn2+ contamination. The corrected readings for Cd2+ were 10-10 and 6 × 10-11 M at mass numbers 111 and 114, respectively. The subtracted argon blank was 3 × 10-10 and 2 × 10-10 M, respectively. Although the values indicate absence of gross contamination, we do not regard them as sufficiently reliable to set an upper limit of contamination. An observation indicating extremely low contamination in the buffer was that a constant capacitance reading was obtained when the buffer was pumped through the cell for 1 h at 0.5 mL/min. Free metal ions in the buffer, if present, should have been picked up by the protein, thus decreasing the capacitance. ACKNOWLEDGMENT The authors thank the following organizations for financial support: (B.M. and N.L.B.) the European Commission, project no. ENV4-CT95-0141, (E.C.) the Swedish Natural Research Council (NFR) and the Swedish Council for High Education (Ho¨gskoleverket), (B.M.) MISTRA (Coldren project), (N.L.B. and K.J.J.) the UK Medical Research Council, grant no. G.9309093, (I.B.) the Swedish Institute and Soros Foundation, and (C.B. and G.J.) the Swedish Natural Research Council (NFR). ICPMS and cold vapor measurements were made by Mr. Anders Ekstro¨m and Dr. Andrejs Schu¨tz at the Department of Occupational and Environmental Medicine, Lund. N.L.B. and J.R.L. also thank Dr. Jon Hobman and Dr. Jon Wilson for valuable discussions and Prof. N. Robinson for supplying the pGEX3X plasmid encoding GSTSmtA.

Received for review March 31, 1998. Accepted July 4, 1998. AC9803636 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

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