Chiral Ligand Exchange Potentiometric Aspartic Acid Sensors with

Feb 2, 2009 - An enantioselective molecular sensor was fabricated by inserting a chiral ligand, N-carbobenzoxy-l-aspartic acid (N-CBZ-l-Asp) or ...
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Anal. Chem. 2009, 81, 1888–1892

Chiral Ligand Exchange Potentiometric Aspartic Acid Sensors with Polysiloxane Films Containing a Chiral Ligand N-Carbobenzoxy-Aspartic Acid Yanxiu Zhou,*,† Tsutomu Nagaoka,† Bin Yu,‡ and Kalle Levon‡ Department of Applied Chemistry, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan, and Polymer Research Institute, Polytechnic Institute of New York University, Six Metrotech Center, Brooklyn, New York 11201-3840 An enantioselective molecular sensor was fabricated by inserting a chiral ligand, N-carbobenzoxy-L-aspartic acid (N-CBZ-L-Asp) or N-CBZ-D-Asp, into an octadecylsiloxane (ODS) monolayer by polysiloxane film immobilization (PFI). The resulting system can recognize one enantiomer of aspartic acids (Asps) due to the chiral ligand exchange reaction at the N-CBZ-L-/D-Asp modified indium-tin oxide (ITO)-coated electrode. The enantioselective formation of diastereoisomeric complexes of Cu(II) with target enantiomers, in here L-/D-Asps, and N-CBZ-L-/D-Asp immobilized by PFI on the ITO electrode. Those diastereoisomeric complexes have different thermodynamic stabilities and Nernst factors and thus enable the sensors to convert the enantioselective recognition event into potential changes by detecting Asp enantiomers in a concentration range of (4.0 × 10-8)-(8.9 × 10-5) M without any pre- or postseparation process. The enantiomeric selectivity coefficients of the sensors for the counterisomers were in the range of (4.0 × 10-5)-(5.0 × 10-5). Stereochemistry is a crucial element in medicinal chemistry, drug design, food technology, biomedical research and diagnosis, agrochemicals, biotechnology, and other diverse areas.1-5 This is because chiral compounds have different kinetics, metabolism, disappearance, and stability behavior.6 For example, one of the enantiomers of a chiral drug demonstrates specific bioactivities while the counterisomer is probably causing serious side-effects or acts as an antagonist.7 Thus, many chiral drugs must be made * Corresponding author. E-mail: [email protected]. † Yamaguchi University. ‡ Polytechnic Institute of New York University. (1) Resolution of Optical Isomers; Osa, T., Ed.;Gakkai Shuppan Center: Tokyo, Japan, 1989. (2) Chiral Separations; Ahuja, S., Ed.; American Chemical Society: Washington, DC, 1997. (3) Davankov, V. A.; Navratil, J. D.; Walton H. F. In Ligand Exchange Chromatography; CRC Press: Boca Raton, FL, 1988. (4) Chirality in Drug Research; Francotte, E.; Linder, W. Wiley/VCH: Weinheim, Germany, 2006. (5) Chirality in Drug Design and Development; Reddy, I. K.; Mehvar, R., Eds.; Marcel Dekker, Inc.: New York, 2004. (6) Ali, I.; Aboul-Enein, H. Y. Chiral Pollutants, Distributions, Toxicity, and Analysis by Chromatography and Capillary Electrophoresis; Wiley: Chichester, U.K., 2004. (7) Eichelbaum, M.; Gross, A. S. Adv. Drug Res. 1996, 28, 1–64.

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with high enantiomeric purity. Therefore, the development of analytical methods for the quantitative analysis of chiral molecules and assessment of enantiomeric purity is an extremely important task. However, enantiomers are identical with respect to physiochemical properties, and the differences only arise when they are in the presence of other chiral molecules. The current chiral analysis uses chiral stationary phases in conjunction with chromatography and capillary electrophoresis (CE)8-10 as a way to separate and thereby individually quantitate the enantiomers of an enantiomeric pair. Following an enantiomeric separation process, detection modes such as UV-vis absorbance,11 mass spectroscopy,12 fluorescence,13,14 ICPMS,15 voltammetry,16,17 or chiral-selective detection mode, such as circular dichroism (CD)10 or polarimetry,10,18,19 can serve as an important means to enhance the quantitation of enantiomeric compounds. However, those techniques use large and expensive instruments and require sophisticated and extensive analysis procedures. The development of chemical and biological sensors is an extremely useful and exciting area of scientific research.20-28 The sensor is a small device that integrates molecular recognition and (8) Chiral Separation Techniques: A Practical Approach, Completely Revised and Updated, 3rd ed.; Subramanian, G., Ed; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2007. (9) Ahuja, S. Chiral Separations by Chromatography; Oxford University Press and American Chemical Society: Oxford, U.K. and Washington, DC, 2000. (10) Bobbitt, D. R.; Linder, S. W. Trends Anal. Chem. 2001, 20, 111–123. (11) Kaddoumi, A.; Nakashima, M. N.; Takahashi, M.; Nakashima, K. Anal. Sci. 2001, 17 (Suppl.), 1907–1909. (12) Richards, D. S.; Davidson, S. M.; Holt, R. M. J. Chromatogr., A 1996, 746, 9–15. (13) Rubio-Barroso, S.; Santos-Delgado, M. J.; Martı´n-Olivar, C.; Polo-Dı´ez, L. M. J. Dairy Sci. 2006, 89, 82–89. (14) Drenth, B. F. H.; Bosman, J.; Feitsma, K. G.; Nijhuis, A. Chromatographia 1988, 26, 281–284. (15) Sutton, K. L.; Ponce de Leon, C. A.; Ackley, K. L.; Sutton, R. M.; Stalcup, A. M.; Caruso, J. A. Analyst 2000, 125, 281–286. (16) Park, S.; McGrath, M.; Smyth, M.; Diamond, D.; Lunte, C. E. Anal. Chem. 1997, 69, 2994–3001. (17) Gerhardt, G.; Cassidy, R. M.; Baranski, A. J. Anal. Chem. 1998, 70, 2167– 2173. (18) Roussel, C.; Vanthuyne, N.; Jobert, J.-L.; Loas, A. I.; Tanase, A. E.; Gherase, D. Chirality 2007, 19, 497–502. (19) Roussel, C.; Hart, N.; Bonnet, B.; Suteu, C.; Hirtopeanu, A.; Kravtsov, V. C.; Luboradzki, R.; Vanthuyne, N. Chirality 2002, 14, 665–673. (20) Anderson, D. J. Anal. Chem. 1999, 71, 293R–372R. (21) Downstream Processing Biosurfactants/Carotenoids. In Advances in Biochemical Engineering/Biotechnology, Vol. 53; Fiechter, A., Ed.; Springer: New York, 1996. 10.1021/ac801751n CCC: $40.75  2009 American Chemical Society Published on Web 02/02/2009

signal transduction and is capable of carrying out analyte separation, preconcentration, and detection in a very effective way. The advantage of sensors over other analytical approaches is accuracy, real time, comparatively low cost, ease of use that has led to their widespread application in health care, environmental monitoring, the food industry, and control of industrial processes.1,20-29 Introduction of a chiral structure onto the electrodes30,31 with different functions such as catalytic nature has been the mark of the emergence of the chemically modified electrodes (CMEs) research field in 1975. However, development of enantioselective electrochemical sensors is still a very difficult task because electrochemical properties do not have any direct relationship with chirality. The enantio-recognition ability of sensors can only arise from the chiral recognition elements32-39 or surface imprinted chiral cavities left by the chiral template molecules on the electrode surfaces40-42 or from new chiral recognition approaches that bridge the chirality and electrochemical techniques that enable enantiomers to be stereoselectively recognized. As a part of our ongoing research to develop chiral electrochemical sensors,37,40-47 we have explored a chiral electrochemical method to enantio-recognize optical isomers, chiral ligand exchange potentiometry (CLEP).43 In CLEP, a chiral ligand, N-CBZ-L-Asp, preferentially recognizes D-Asp and undergoes ligand exchange with the enantiomeric labile coordination complexes of [Cu(II)(D-Asp)2] or [Cu(II)(L-Asp)2] to form a diastereoisomeric complex [(D-Asp)Cu(II)(N-CBZ-L-Asp)] or [(L-Asp)Cu(II)(22) Frontiers in Drug Design and Discovery, Vol. 2; Caldwell, G. W., Rahman, A. U., D’Andrea, M. R., Choudhary, M. I., Eds.; Bentham Science Publishers Ltd.: Hilversum, The Netherlands, 2006. (23) Biosensors for Environmental Monitoring; Bilitewski, U., Turner, A. P. F. , Eds.; Harwood Academic Publishers: Amsterdam, The Netherlands, 2000. (24) Principles of Chemical and Biological Sensors; Diamond, D., Ed.; John Wiley & Sons, Inc.: New York, 1998. (25) Wang, J. Anal. Chem. 1999, 71, 328R–332R. (26) Electroanalytical Methods for Biological Materials; Brajter-Toth, A., Chambers, J. Q., Eds.; Marcel Dekker, Inc.: New York, Basel, 2002. (27) Bakker, E.; Qin, Y. Anal. Chem. 2006, 78, 3965–3983. (28) Privett, B. J.; Shin, J. H.; Schoenfisch, M. H. Anal. Chem. 2008, 80, 4499– 4517. (29) Staff, G. K. Smart Biosensor Technology; CRC Press: Boca Raton, FL, 2007. (30) Moses, P. R.; Wier, L.; Murray, R. W. Anal. Chem. 1975, 47, 1882–1886. (31) Watkins, B. F.; Behling, J. R.; Kariv, E.; Miller, L. L. J. Am. Chem. Soc. 1975, 97, 3549–3550. (32) Aboul-Enein, H. Y.; Stefan, R.-I. Crit. Rev. Anal. Chem. 1998, 28 (3), 259– 266. (33) Zhang, S.; Ding, J.; Liu, Y.; Kong, J.; Hofstette, O. Anal. Chem. 2006, 78, 7592–7596. (34) Kataky, R.; Bates, P. S.; Parker, D. Analyst 1992, 117, 1313–1317. (35) Kieser, B.; Fietzek, C.; Schmidt, R.; Belge, G.; Weimar, U.; Schurig, V.; Gauglitz, G. Anal. Chem. 2002, 74, 3005–3012. (36) Inagaki, S.; Min, J. Z.; Toyo’oka, T. Anal. Chem. 2008, 80, 1824–1828. (37) Chen, Z.; Sakumoto, N.; Koyama, M.; Nakao, H.; Nagaoka, T. Electroanalysis 1999, 11, 1169–1171. (38) Trojanowicz, M.; Wcislo, M. Anal. Lett. 2005, 38, 523–547. (39) Weng, W.; Han, J. L.; Chen, Y. Z.; Huang, X. J. Prog. Chem. 2007, 19, 1820–1825. (40) Zhou, Y.; Yu, B.; Levon, K. Chem. Mater. 2003, 15, 2774–2779. (41) Zhou, Y.; Nagaoka, T. Chem. Sens. 1998, 14 (Suppl. B), 101–104. (42) Zhou, Y.; Yu, B.; Levon, K. In Immunoassay and Other Bioanalytical Techniques; Van Emon J. M., Ed.; CRC Press: New York, 2006; pp 167186. (43) Zhou, Y.; Yu, B.; Levon, K.; Nagaoka, T. Electroanalysis 2004, 16, 955– 960. (44) Zhou, Y.; Yu, B.; Zhu, G. Polymer 1997, 38, 5493–5495. (45) Deore, B.; Chen, Z.; Nagaoka, T. Anal. Chem. 2000, 72, 3989–3994. (46) Okuno, H.; Kitano, T.; Yakabe, H.; Kishimoto, M.; Deore, B. A.; Siigi, H.; Nagaoka, T. Anal. Chem. 2002, 74, 4184–4190. (47) Siigi, H.; Kishimoto, M.; Yakabe, H.; Deore, B. A.; Nagaoka, T. Anal. Sci. 2002, 18, 41–44.

(N-CBZ-L-Asp)]. Those complexes have different thermodynamic stabilities and net electrical charge, thus enabling chiral Asps to be distinguished by potentiometry. CLEP43 is the first enantioselective recognition approach that bridges chirality and electrochemical techniques. It makes electrochemical enantio-recognition of chiral compounds possible. However, there will be some practical issues to apply this technique directly to real samples. For example, the composition of a sample has to be roughly known. Otherwise, it is hard to decide what kind of ligand needed be added to the samples. It may contaminate samples or make the detection process tedious and complicated. Since the chiral ligand exchange reaction does not happen on the surface of the electrodes but in solution, it cannot be used to detect multianalytes as electrodes cannot distinguish the potentiometric output difference and will give an overall potentiometric response. However, when the chiral ligand was coated on the surface of an electrode as a CLEP sensor, above aforementioned problems would be overcome. Therefore, the objective of this article is to develop a chiral ligand exchange potentiometric sensor by immobilization of chiral ligands on the surface of an ITO-coated electrode glass coupled with CLEP detection for the enantioselective analysis of aspartic acid and investigate the performance of the resulting CLEP sensor as well. EXPERIMENTAL SECTION The ITO-coated glasses were pretreated as follows: 5 min of sonication in concentrated nitric acid, rinsed in doubly distilled water, 7 min of sonication in 0.02 M sodium hydroxide, individually washed in a jet of doubly distilled water, and finally dried in an oven. Then, the ITO-coated glass plates were soaked (effective surface area about (0.7 × 4 cm2)) in a CHCl3/CCl4 suspension (2:3 v/v) containing 0.8 mM C18H37SiCl3 octadecyltrichlorosilane (OTS)) and N-CBZ-L- or D-Asp ((1.9 × 10-2)-(3.8 × 10-2) M) for 2-4 min. After fully cured overnight at room temperature, these fabricated sensors were washed with distilled water to remove unimmobilized N-CBZ-L- or D-Asp. In an electrochemistry cell, 0.1 M KCl-phthalate buffer (pH 2.3) is used as a blank solution. Analytes are mixed with 6.9 × 10-5 M Cu(II) to form labile complexes before testing. The elemental analysis of the sensor surfaces was carried out using the Quantum 2000 (pH 1 Co) photoelectron spectroscopy. Other detailed experimental procedures were previously reported.43 RESULTS AND DISCUSSION The immobilization of chiral ligands or other chemical or biological recognition elements on the surface of substrates and maintaining their recognition ability are very difficult in sensor research. Adsorption, microencapsulation, entrapment, crosslinking, and covalent bonding are nowadays the five generally used methods for the immobilization of chemical or biological materials.48 More or less, those five methods have their own merits and demerits. For example, for the same sensors fabricated by different methods of immobilization, the longest lifetime is over months by covalent bonding, but this method needs a carefully designed bond between a functional group in the recognition (48) Eggins, B. R. Chemical Sensors and Biosensors; John Wiley & Sons Ltd: West Sussex, U.K., 2002; pp 98-104.

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Table 1. Elemental Analysis Resultsa element

C

O

In

Sn

N

Si

ITO glass plate CBZ-Asp/ODS/ITO

186 397.0

189 219.7

100 100

6.6 7.1

7.9 17.2

7.7 16.3

a

Normalized to indium.

element and the support matrix, otherwise the recognition elements will lose their recognition ability easily; while for the simple adsorption, the lifetime is around 1 day or so.48 Herein, the water soluble chiral ligand, N-CBZ-L- or D-Asp, was immobilized on the surface of an ITO-coated glass plate with polysiloxane film immobilization (PFI) by inserting it within the polysiloxane monolayer. The procedure of PFI is as follows: soaking the pretreated ITO plate in a L- or D-CBZ-Asp suspension dispersed in CHCl3/CCl4 containing OTS, and a hydrophobic octadecylsiloxane (ODS) monolayer40,49-53 was covalently bound onto the ITO surface around the N-CBZ-L-Asp or N-CBZ-D-Asp molecules. Hence, the amino acid was inserted into the polysiloxane macromolecular network via the hydrophobic interaction with the silanized ITO surface and immobilized onto it. The incorporation of the amino acid within the polysiloxane film was monitored by X-ray photoelectron spectroscopy, as shown in Table 1. The results show that carbon, silicon, and nitrogen intensity increased to 113, 112, and 118% over the initial values of the ITO electrode indicating that the amino acid was immobilized onto the surface of the ITO. A similar result was also documented by Kallury et al.,54 who used OTS and chlorodimethyloctadecylsilane (CDOS)-treated silicon wafers together with guest molecule hexadecane. They noticed that neither the original film nor its intercalated molecules within the silane network could be washed out by water, which is consistent with our results. Since the chiral ligands were only physically trapped within the ODS monolayer by hydrophobic interaction, it allowed N-CBZ-L- or D-Asp to keep their selective recognition ability to further exchange with chiral Asps in transient dibasic copper complexes [Cu(II)(D-Asp)2] or [Cu(II)(L-Asp)2]. The observation suggests that this simple surface modification technique could be utilized in immobilization of water soluble recognition elements. The optimization of conditions for sensor fabrication can be adjusted by changing the chiral ligand concentration in the deposition solution during the immobilization process and coadsorption time. The results depicted that the optimal concentration of CBZ-L-Asp in CHCl3/CCl4 solution for fabrication of the D-Asp sensor was (1.9 × 10-2)-(3.8 × 10-2) M. Meanwhile, the effect of the coadsorption time of CBZ-L-Asp in OTS-CHCl3/CCl4 solution for development of the D-Asp sensor demonstrated that the most efficient immersion time was between 2 to 4 min. The optimization of conditions for subsequent enantiodiscrimination has been studied. The concentration of the cupric ions should affect the sensors’ recognition of the preferred enantiomer coordination complexes. When the concentration of the D- or L-isomer of Asp was 6.88 × 10-6 M, as the concentration (49) (50) (51) (52) (53) (54)

Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92–98. Sagiv, J. Isr. J. Chem. 1979, 18, 346–353. Sagiv, J. Isr. J. Chem. 1979, 18, 339–345. Zhou, Y.; Yu, B.; Levon, K. Biosens. Bioelectron. 2005, 20, 1851–1855. Zhou, Y.; Yu, B.; Shiu, E.; Levon, K. Anal. Chem. 2004, 76, 2689–2693. Kallury, K. M. R.; Thompson, M. Langmuir 1992, 8, 947–954.

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Figure 1. The influence of Cu(II) concentration on potentiometric response of the D-Asp sensor to 6.88 × 10-6 M Asp in 0.1 M KCl-phthalate buffer solution (pH 2.3).

Figure 2. The effect of pH on the potentiometric response of the D-Asp sensor to 2.4 × 10-7 M Asp, [Cu(II)] ) 1.0 × 10-6 M.

of copper increased, the potential decreased. The potential of the sensor reached its maximum when the concentration of Cu2+ was at 1.0 × 10-5 M, as shown in Figure 1. Other metal cation ions such as Cr, Pb, Ni can also form complexes with aspartic acids. Moreover, the influence of solution pH on the recognition process of the sensor was studied intensively because of the three acid/base equilibia involved in both N-CBZ-Asp and Asp.40 The maximum potential difference between the two diastereoisomeric complexes was observed at pH 2.3, as indicated by the pH dependence of the output of the D-Asp sensor for 2.4 × 10-7 M Asp (Figure 2). Therefore, all subsequent experiments were performed in phthalate buffer with 0.1 M KCl at pH 2.3. In Figure 3, a sensor with a chiral ligand N-CBZ-L-Asp immobilized by PFI showed potential response in the presence of a labile complex [Cu(II)(D-Asp)2] (curve (9)). However, when the electrode was modified with a polysiloxane matrix but without N-CBZ-L-Asp, there is no potential change (curve (0)). This clearly depicted that the sensor enantio-discriminated the

Figure 3. Potentiometric response of D-Asp on the ODS/ITO electrode with (9) (sensor) and without (0) chiral ligand N-CBZ-LAsp (control). Experimental conditions: 0.1 M KCl-phthalate buffer (0.1 M, pH 2.3). D-Asp isomer after the labile complex ([Cu(II)(D-Asp)2]) exchanging with the ligand N-CBZ-L-Asp on the sensor and stereoselectively formed the ternary complex of [(N-CBZ-LAsp)Cu(II)(D-Asp)]. This result further confirmed that the N-CBZ-L-Asp chiral selector was immobilized within the adsorbed polysiloxane monolayer, and its enantio-recognition ability was retained as well. The differences between enantiomers of Asp will be distinguished only when they are in the presence of the chiral selector, N-CBZ-L-Asp. The chiral sensor allowed us to investigate the chirality between the D-and L-isomers of Asp by a potentiometric measurement. The chiral discrimination toward binding of the enantiomers of the labile coordination complexes, [Cu(II)(D-Asp)2] or [Cu(II)(LAsp)2], was investigated, and the results indicated that the chiral molecular sensors have preference for a particular isomer. The D-Asp sensor (chiral ligand, N-CBZ-L-Asp) was found to selectively identify the D-Asp isomer (Figure 4(A)) (s)). The potential response was proportional to the logarithmic concentration in a concentration range of (4.0 × 10-8)-(8.9 × 10-5) M with a slope of -29.6 mV dec-1, one order higher than that of chiral ligand in solution,43 while there was no potential alteration for the L-isomer complex (Figure 4(A)) (- - -)). The separate solution method55 was followed to establish the POT selectivity coefficients, KDL , a quantitative measurement of the sensor’s ability to discriminate the D-isomer against the interfering L-isomer:

0 ED ) ED + s log([(D-Asp)Cu(II)(N-CBZ-L-Asp)] + POT [(L-Asp)Cu(II)(N-CBZ-L-Asp])(1) KDL

where ED0 and ED are the potential of the standard electrode potential and the D-Asp sensor, respectively. s is the slope and POT POT K DL the selectivity coefficient. By simulation, the K DL value -5 of 4.0 × 10 has been obtained for the D-Asp sensor (Figure (55) Brett, A. C. M.; Brett, A. M. O. Electroanalysis; Oxford University Press: Oxford, U.K., 1998; p 39.

Figure 4. Potentiometric responses of the (A) D-Asp and (B) L-Asp sensor for (s) D-Asp and (- - -) L-Asp. (O) Added D-Asp from initial condition of 1.0 × 10-6 M L-Asp; (∇) added L-Asp from initial condition of 1.0 × 10-6 M D-Asp. Experimental conditions: 0.1 M KCl-phthalate buffer (0.1 M, pH 2.3). Table 2. Output of D-Asp Sensor for Racemic Mixtures D-Asp

sensor output (mV)

concn/Ma

100% D-Asp

50% D-Asp + 50% L-Asp

error (%)

× × × × × × ×

-30.2 -54.5 -64.2 -82.0 -89.1 -112.5 -116.9

-29.6 -53.8 -64.0 -83.3 -89.8 -106.6 -114.8

-2.0 -1.3 -0.3 1.6 0.8 -5.2 -1.8

1.00 6.25 1.30 5.00 8.50 4.98 6.95 a

10-7 10-7 10-6 10-6 10-6 10-5 10-5

Concentration of racemate is double of the pure enantiomer.

POT 4(A)) and the K LD value for the L-Asp sensor is 5.0 × 10-5 (Figure 4(B)). These values demonstrate that the sensitivities of these sensors for target enantiomers are 20 000 -25 000 times higher than those of for the counterisomers, which means that the present sensor possessed high enantioselectivity. Furthermore, this sensor not only owned selective recognition of one enantiomer in the presence of the counterisomer (see Figure 4(A) (O,3)) but also displayed specific molecular recognition ability toward one enantiomer in a racemic mixture as shown in Table 2. The potentials of the D-Asp sensor changed about only -1.17% in the presence of the equimolar amount of the L-Asp in comparison with pure D-isomer and even the total concentration of Asp in the racemic mixture is doubled. The fact that there was no substantial potential alteration observed with and without the presence of the L-isomer demonstrated high selectivity as shown from the potentiometric selectivity coefficients above. Since the enantiomeric selectivity measurements are usually performed in a solution of a mixture of amino acids, the selectivity of the D-Asp sensor to other amino acids was also investigated. In all the cases except for D-Asp, D-amino acids gave a slope of around 0 mV dec-1, which is similar to that of L-Asp. Even tridentate amino acids, such as D-glutamic acid (D-Glu) and D-histidine (D-His), can form [L-Cu-D] complexes with N-CBZL-Asp as D-Asp does. However, the free energies of those formed diastereoisomeric complexes are inadequate to permit potentiometric enantio-recognition even though the very weak intermolecular interactions can serve as a basis for a chro-

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L-Asp

Table 3. Potentiometric Selectivity Coefficients amino acids

D-Glu

POT K Dj (× 10-3)

0.11

D-

Pro

1.5

D-Phe

D-His

D-Val

D-Ala

D-Thr

5.4

1.1

1.1

5.4

4.8

matographic separation process.3 Therefore, the size matching between the chiral ligand and analytes also plays an important role in chiral ligand exchange potentiometry. The potentiometric selectivity coefficients were determined by the separate POT solution method,55 and KDj (D, D-Asp; j, interfering amino acids) values were shown in Table 3. The high preference of the D-Asp sensor for D-Asp is immediately evident. Hence, these chiral sensors not only demonstrate a selective recognition ability to discriminate between enantiomers of the same amino acid but also different amino acids. The response time, evaluated as the time required for a 95% signal response, was about 220 s for 8 × 10-7 M D-Asp. After the measurement was repeated more than 200 times, the potential response remains 92% of its initial magnitude, showing the long lifetime of the sensors. The relative standard deviation was 2.67% for 3.0 × 10-7 M (n ) 16). The mechanism of the Asp sensors distinguishing enantiomers is similar to that of the unmodified electrode in our previous work,43 except that the chiral ligand is immobilized on the surface of the ITO electrode. As described previously,43 CLEP is based on the enantioselective formation of different net electrical charged diastereoisomeric complexes with different isomers. In our experiment, Asp was mixed with Cu2+ first. Therefore, a transient dibasic copper complex [Cu(II)(D-Asp)2] or [Cu(II)(L-Asp)2] was formed. When they encountered the chiral ligand, N-CBZ-

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(for D-sensor, for example), which was on the surface of the electrode, a ligand exchange occurred between the L- or D-Asp in dibasic copper complexes and N-CBZ-L-Asp and different diastereoisomeric complexes [(D-Asp)Cu(II)(N-CBZL-Asp)] and/or [(L-Asp)Cu(II)(N-CBZ-L-Asp)] formed on the surface of sensor, which led to a potential change. CONCLUSION In summary, we presented a novel immobilization method (PFI) coupled with CLEP to develop chiral electrochemical amino acid sensors. The technique described gives rise to sensors with excellent selectivity and chemical stability and suggests a novel strategy for electrochemical distinction between enantiomers. It represents a significant improvement over existing CLEP asparatate detection and increases the selectivity by about 1 order of magnitude. Bridging of chiral recognition and electrochemical techniques makes the on-site monitoring and detection of chiral molecules and assessing of enantiomeric purity possible. Another important finding is that PFI could be used to immobilize a water soluble ligand on the surface of the substrate and retain its selective recognition ability. ACKNOWLEDGMENT Y.Z. gratefully acknowledges the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship (P. 97380) to accomplish part of this work. Received for review August 20, 2008. Accepted January 15, 2009. AC801751N