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Chem. Mater. 2003, 15, 2774-2779
Potentiometric Sensing of Chiral Amino Acids Yanxiu Zhou, Bin Yu, and Kalle Levon* Department of Chemistry, Chemical Engineering and Materials Science, Polytechnic University, Six Metrotech Center, Brooklyn, New York 11201 Received January 30, 2003. Revised Manuscript Received May 5, 2003
Recognition of enantiomers of amino acids was achieved with potentiometric measurements using chiral sensors constructed by a surface imprinting technique. The sensors exhibited recognition properties toward one isomer of racemic N-carbobenzoxy-aspartic acids (N-CBZAsp) without any preseparation processes. The sensors translated the enantioselective recognition event into a potential change by detecting optically active N-CBZ-Asp in a concentration range of 5.0 × 10-6-1.2 × 10-2 M. The enantiomeric selectivity coefficients of the sensors for the counter isomers were in the range of 4.0 × 10-3-9.0 × 10-3. The sensitivity of these sensors is assigned to the specific surface modification of the electrodes resulting in chiral recognition and to the coupled transduction activity of the sensor.
Introduction An interesting challenge in the design of electrochemical sensors is to achieve electrochemical discrimination between enantiomers. The detection of chiral amino acids is of importance as they are essential substances in protein metabolism as well as in pharmaceutical and food products, for example.1 The present enantioselective detection systems without separation process requirements are electrochemical enzyme sensors and enantioselective membrane electrodes.2-4 The specific selectivity of the enzymes is the strong basis for the high reliability of these sensors,5-7 but because of the presence of the biological components, biosensors do have problems with long-term stability, irreversible deactivation at high temperatures or under harsh chemical environments, and operation in organic phases.8-10 As for nonenzymatic sensors, enantioselective membranes2 or column electrodes3,4 provide more stable alternatives, although low selectivity and poor reproducibility always remain as a problematic issue. One successful example, a cross-linked poly(methacrylic acid) with a chiral cavity prepared by molecular imprinting against L-phenylalanine anilide, has been applied for the construction of a chiral column electrode.4 Although the preparation of such a modified electrode can be tedious and the * Corresponding author e-mail:
[email protected]. (1) Bruckner, H.; Wittner, R.; Hausch, M.; Godel, H. Fresenius Z. Anal. Chem. 1989, 333, 775. (2) Horva´th, V.; Taka´cs, T.; Horvai, G.; Huszthy, P.; Bradshaw, J. S.; Izatt, M. Anal. Lett. 1997, 30, 1591. (3) Chen, Z.; Sakumoto, N.; Koyama, M.; Nakao, H.; Nagaoka T. Electroanalysis 1999, 11 (16), 1169. (4) Andersson, L. I.; Miyabayashi, A.; O’Shannessy D. J.; Mosbach, K. J. Chromatogr. 1990, 516, 323. (5) Alvarez-Crespo, S. L.; Lobo-Castanˇo´n, M. J.; Miranda-Ordieres, A. J.; Tunˇo´n-Blanco, P. Biosens. Bioelectron. 1997, 12, 739. (6) Va´rdi, M.; Ada´nyi, N.; Szabo´, E. E.; Trummer, N. Biosens. Bioelectron. 1999, 14, 335. (7) Olschewski, H.; Erlenkotter, A.; Zaborosch, C.; Chemnitius, G.C. Enzyme Microb. Technol. 2000, 26, 537. (8) Griffiths, D.; Hall, G. Trends Biotechnol. 1993, 11, 122. (9) Scheller, F.; Schubert, F. Biosensors; Elsevier: Amsterdam, 1992. (10) Khan, G. F.; Wernet W. Anal. Chem. 1997, 69, 2682.
detection results may still not be satisfactory, the molecular imprinting technique seems to be effective for chiral separation and could potentially be used for sensor application.11 In this paper, we report the fabrication of a chemical sensor and its use for the potentiometric measurement of chiral amino acids. The chiral recognition ability was imparted to an indium-tin oxide (ITO) glass plate modified in a manner similar to the previously reported surface imprinting technology.12-22 An octadecylsiloxane (ODS) layer was covalently bound onto the ITO surface in the presence of the chiral N-CBZ-Asp molecules. After the removal via washing out the chiral component, the sensor showed chiral recognition ability toward chiral N-CBZ-Asp. Experimental Section Chemicals and Apparatus. Chloroform and carbon tetrachloride were distilled over CaH2. Other chemicals were used without further purification. The ITO glass plates as indicator electrodes were obtained from Kinoene Optical Industry Co. Ltd., Japan. All aqueous solutions were prepared from water purified using a Millipore system (resistivity: 18.2 MΩ‚cm). The two-electrode system consisted of an Ag/AgCl (saturated KCl) as reference electrode and the ODS/ITO sensor as working electrode. The electrochemical cell was a 25-mL beaker. The working electrode clip was directly connected to (11) Sellergren, B., Ed. Molecularly imprinted polymers, man-made mimics of antibodies and their application in analytical chemistry; Elsevier: New York, 2001 (12) Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92. (13) Sagiv, J. Isr. J. Chem. 1979, 18, 346. (14) Sagiv, J. Isr. J. Chem. 1979, 18, 339. (15) Polymeropoulos, E. E.; Sagiv, J. J. Chem.Phys. 1978, 69 (5), 1836. (16) Cohen, S. R.; Naaman, R.; Sagiv, J. J. Phys. Chem. 1986, 90, 3045. (17) Neizer, L.; Sagiv, J. J. Am. Chem. Soc. 1983, 105, 674. (18) Maoz R.; Sagiv, J. J. Colloid Interface Sci. 1984, 100 (2), 465. (19) Gun, J.; Iscovici, R.; Sagiv, J. J. Colloid Interface Sci. 1984, 101 (1), 201. (20) Iscovici, R.; Sagiv, J. Thin Solid Films 1983, 99, 235. (21) Iscovici, R.; Sagiv, J. Thin Solid Films 1983, 100, 67. (22) Untereker, D. F.; Lennox, J. C.; Wier, L. M,; Moses, P. R.; Murray, R. W. J. Electroanal. Chem. 1977, 81, 309.
10.1021/cm030060e CCC: $25.00 © 2003 American Chemical Society Published on Web 06/13/2003
Potentiometric Sensing of Chiral Amino Acids
Chem. Mater., Vol. 15, No. 14, 2003 2775
Table 1. Elemental Analysis Results element
C
O
Ina Sn
N
Si
ITO glass plate 186 189 100 6.6 7.9 7.7 adsorbed CBZ-Asp and ODS 397.0 219.7 100 7.1 17.2 16.3 on ITO glass plate rinsed 351.3 191.3 100 7.9 6.1 10.1 *Normalized to Indium.
the ITO glass plate. The potentials of the ODS/ITO indicator electrodes were measured against the Ag/AgCl reference electrode with Orion 920A potentiometer. Thereafter, the potential response of the sensor was defined as the difference between the electrode potential with and without N-CBZ-LAsp or N-CBZ-D-Asp in solution (i.e., ∆E ) E1 - E0, where E0 and E1 are the electrode potentials before and after the amino acid addition, respectively). The dissociation constants (pKa) of N-CBZ-Asp were obtained by titration using a pH meter. The elemental analysis of the sensors surface was carried out using the Quantum 2000 (pH 1 Co) photoelectron spectroscopy. The potential responses of the sensors as a function of pH were detected in the same two-system described above but with an additional pH electrode in the solution, which was used to record the pH change. Sensor Fabrication. ITO glass plates were pretreated by a method described by Sagiv.12 N-CBZ-L-Asp or N-CBZ-D-Asp (chiral template) and octadecyltrichlorosilane (OTS) (silylating agent) were co-adsorbed on the polar solid surface of the ITO glass plate (effective surface area about 1 × 4 cm2) from the CHCl3/CCl4 solution (2:3 v/v) at 20 ( 1°C for 3 min. The electrode was rinsed with CHCl3 (30 × 1 mL) to remove the embedded chiral amino acid molecules and followed by drying at room temperature for 12 h. ODS-modified electrodes without the N-CBZ-L-Asp and N-CBZ-D-Asp cavity were also prepared as a control.
Results and Discussion The chiral compound (N-CBZ-L-Asp or N-CBZ-D-Asp) and the silylating agent (OTS) were co-adsorbed on the polar solid surface of the ITO glass plate. A hydrophobic layer consisting of the polymerized organosiloxane groups was formed in this way in the presence of the N-CBZ-L-Asp or N-CBZ-D-Asp molecules.12-23 The films were then repeatedly washed with chloroform to remove the physically adsorbed N-CBZ-L-Asp or N-CBZ-D-Asp molecules. This possible removal of the adsorbed NCBZ-L-Asp or N-CBZ-D-Asp by the repeated extractions is assumed to create chiral cavities in a network of silane molecules, as the ODS layers are stable in solvents of low polarity (e.g., CHCl3).13 The incorporation of amino acid into polysiloxane film and removal from it during templating process were confirmed by X-ray photoelectron spectroscopy (the nitrogen and oxygen concentrations were restored to the initial values (
