pubs.acs.org/Langmuir © 2009 American Chemical Society
Voltammetric Extraction of Heparin and Low-Molecular-Weight Heparin across 1,2-Dichloroethane/Water Interfaces Ping Jing, Yushin Kim, and Shigeru Amemiya* Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260 Received June 30, 2009. Revised Manuscript Received August 20, 2009 Heparin and low-molecular-weight heparin are voltammetrically extracted across 1,2-dichloroethane/water interfaces for the detection of these highly sulfated polysaccharides widely used as anticoagulants/antithrombotics in many medical procedures. A new heparin ionophore, 1-[4-(dioctadecylcarbamoyl)butyl]guanidinium, is the first to enable the voltammetric extraction of various polyanionic heparins with average molecular weights of up to ∼20 kDa including those in commercial preparations (i.e., Arixtra (1.5 kDa), Lovenox (4.5 kDa), and unfractionated heparin (15 kDa), as well as chromatographically fractionated heparins (7, 9, 15, and 20 kDa)). Facilitated Arixtra extraction is fully and quantitatively characterized by micropipet voltammetry to propose that cooperative effects from strong heparinbinding capability and high lipophilicity of this ionophore are required for the formation of an electrically neutral and highly lipophilic complex of a heparin molecule with multiple ionophore molecules to be extracted into the nonpolar organic phase. At the same time, the participation of multiple ionophore molecules in interfacial complexation with a heparin molecule slows down its extraction across the interface. This kinetic limitation is enhanced by fast mass transfer at a micropipet-supported interface to compromise thermodynamically favorable selectivity for heparin and an important contaminant, oversulfated chondroitin sulfate, thereby requiring a macroscopic interface for sensing applications. Another highly lipophilic guanidinium ionophore, N,N-dioctadecylguanidinium, cannot completely extract even Arixtra, which indicates the importance of elaborate ionophore design for heparin extraction.
Unfractionated heparin is widely used as an anticoagulant/ antithrombotic in many medical procedures despite its heterogeneous composition of a mixture of highly sulfated polysaccharides (average molecular weight ∼15 kDa and average charge ∼-75).1-3 Coagulation monitoring must be performed routinely to maintain narrow and low therapeutic concentrations of heparin for adequate anticoagulation without bleeding.4 Common commercial methods, however, are indirect, measuring the coagulation effect of heparin5 so that the calibration of each reagent lot and coagulometer is recommended to establish a concentration-derived therapeutic range.4 Importantly, these clot-based assays that have been used for quality control of unfractionated heparin6 cannot detect a serious anticoagulant contaminant, oversulfated chondroitin sulfate, which induces a rapid, acute allergic-type reaction that has recently caused a large number of patient deaths.7,8 Low-molecular-weight heparins (LMWHs) have a much better efficacy/safety ratio to replace unfractionated *To whom correspondence should be addressed. E-mail: amemiya@pitt. edu. (1) Garg, H. G., Linhardt, R. J., Hales, C. A., Eds. Chemistry and Biochemistry of Heparin and Heparan Sulfate; Elsevier: New York, 2005. (2) Linhardt, R. J. J. Med. Chem. 2003, 46, 2551–2564. (3) Lane, D. A.; Bjork, I.; Lindahl, U., Eds. Heparin and Related Polysaccharides; Plenum Press: New York, 1992. (4) Hirsh, J.; Raschke, R. Chest 2004, 126, 188S–203S. (5) Bates, S. M.; Weitz, J. I. Circulation 2005, 112, E53–E60. (6) U.S. Pharmacopeia web site. http://www.usp.org/pdf/EN/USPNF/revisionBullletinHeparinSodium.pdf (accessed on June 30, 2009). (7) Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; Al-Hakim, A.; Gunay, N. S.; Zhang, Z. Q.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R. J.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 669–675. (8) Kishimoto, T. K.; Viswanathan, K.; Ganguly, T.; Elankumaran, S.; Smith, S.; Pelzer, K.; Lansing, J. C.; Sriranganathan, N.; Zhao, G. L.; Galcheva-Gargova, Z.; Al-Hakim, A.; Bailey, G. S.; Fraser, B.; Roy, S.; Rogers-Cotrone, T.; Buhse, L.; Whary, M.; Fox, J.; Nasr, M.; Dal Pan, G. J.; Shriver, Z.; Langer, R. S.; Venkataraman, G.; Austen, K. F.; Woodcock, J.; Sasisekharan, R. N. Engl. J. Med. 2008, 358, 2457–2467.
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heparin gradually for many indications. LMWHs are derived from heparins by chemical or enzymatic depolymerization. Also, a synthetic pentasaccharide, Arixtra, was approved for clinical use as an anticoagulant drug.9 Assays for monitoring LMWHs are still in development or have not been clinically validated10 but are needed for patients with renal dysfunction, those at extremes of weight, infants, and pregnant women.4 During the past decade, ion-transfer voltammetry at the interface between two immiscible electrolyte solutions (ITIES)11-15 has found new analytical applications for the detection of biologically or biomedically important macromolecules including unfractionated heparin16-20 and Arixtra21 as well as proteins (protamines,22-26 cytochrome c,27,28 ribonuclease A,27 insulin,29 hemoglobin,30 (9) Petitou, M.; van Boeckel, C. A. A. Angew. Chem., Int. Ed. 2004, 43, 3118– 3133. (10) Walenga, J. M.; Hoppensteadt, D. A. Semin. Thromb. Hemostasis 2004, 30, 683–695. (11) Koryta, J. Electrochim. Acta 1979, 24, 293–300. (12) Girault, H. H.; Schiffrin, D. J. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1989; Vol. 15, pp 1-141. (13) Senda, M.; Kakiuchi, T.; Ohsakai, T. Electrochim. Acta 1991, 36, 253–262. (14) Samec, Z. Pure Appl. Chem. 2004, 76, 2147–2180. (15) Vanysek, P.; Ramirez, L. B. J. Chil. Chem. Soc. 2008, 53, 1455–1463. (16) Samec, Z.; Trojanek, A.; Langmaier, J.; Samcova, E. Electrochem. Commun. 2003, 5, 867–870. (17) Guo, J.; Yuan, Y.; Amemiya, S. Anal. Chem. 2005, 77, 5711–5719. (18) Guo, J.; Amemiya, S. Anal. Chem. 2006, 78, 6893–6902. (19) Langmaier, J.; Olsak, J.; Samcova, E.; Samec, Z.; Trojanek, A. Electroanalysis 2006, 18, 115–120. (20) Langmaier, J.; Olsak, J.; Samcova, E.; Samec, Z.; Trojanek, A. Electroanalysis 2006, 18, 1329–1338. (21) Rodgers, P. J.; Jing, P.; Kim, Y.; Amemiya, S. J. Am. Chem. Soc. 2008, 130, 7436–7442. (22) Amemiya, S.; Yang, X.; Wazenegger, T. L. J. Am. Chem. Soc. 2003, 125, 11832–11833. (23) Yuan, Y.; Wang, L.; Amemiya, S. Anal. Chem. 2004, 76, 5570–5578. (24) Yuan, Y.; Amemiya, S. Anal. Chem. 2004, 76, 6877–6886. (25) Rodgers, P. J.; Amemiya, S. Anal. Chem. 2007, 79, 9276–9285. (26) Trojanek, A.; Langmaier, J.; Samcova, E.; Samec, Z. J. Electroanal. Chem. 2007, 603, 235–242.
Published on Web 09/11/2009
DOI: 10.1021/la902336w
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R-chymotrypsin,31 and lysozyme32) and DNAs.33,34 This active voltammetric method offers several advantages over the passive potentiometric method, which has been successfully employed for polyion-sensitive membrane electrodes.35,36 In ion-transfer voltammetry, heparin molecules in an aqueous solution can be selectively transferred across the ITIES by externally controlling the phase boundary potential, where the heparin molecules form complexes with ionophores doped in the organic phase. The resulting ionic current is highly sensitive to the concentration of the heparin molecules, which carry multiple charges. Moreover, well-defined voltammograms thus obtained give useful insights into interactions of heparin with ionophores and the behavior of their complexes at the ITIES. Importantly, the voltammetric studies revealed that complexes of unfractionated heparin molecules with ionophores are adsorbed at the ITIES and are not significantly extracted into the bulk 1,2-DCE phase17 or poly(vinyl chloride) membranes plasticized with 1,6-dichlorohexane,16 2-nitrophenyl octyl ether,18-20 or bis(2ethylhexyl) sebacate.20 So far, Arixtra (1.5 kDa) is the only and largest heparin molecule that can be voltammetrically extracted into an organic phase.21 However, bulk extraction of unfractionated and low-molecular-weight heparins into plasticized polymer membranes has been considered to be a mechanism of super-Nernstian responses of potentiometric heparin-sensitive electrodes37,38 although such nonequilibrium responses can also be affected by adsorption.39,40 The main goal of this work is to develop an ionophore that enables the voltammetric extraction of heparin molecules into a water-immiscible nonpolar organic phase, which is required for their highly sensitive and selective detection by ion-transfer stripping voltammetry.17,18 Originally, exclusive adsorption of heparin-ionophore complexes at the ITIES was employed as a preconcentration step for adsorptive stripping voltammetry, where exhaustive desorption of heparin molecules from the interface results in an enhanced ionic current response.17,18 Heparin adsorption, however, not only readily causes saturation of the interface18 but also seriously competes with the adsorption of blood proteins such as serum albumin.17 Consequently, a limit of detection of unfractionated heparin is compromised to 0.13 unit/mL in sheep blood plasma (equivalent to ∼60 nM with an average molecular weight of ∼15 kDa),17 which is barely lower than therapeutic concentrations of unfractionated heparin (>0.2 unit/mL41). Such limitations of adsorptive preconcentration are potentially overcome by employing the extraction of an analyte ion into a thin organic phase supported on a solid electrode, which is thin enough for exhaustive stripping of the extracted analyte to give subnano(27) Shinshi, M.; Sugihara, T.; Osakai, T.; Goto, M. Langmuir 2006, 22, 5937– 5944. (28) Osakai, T.; Shinohara, A. Anal. Sci. 2008, 24, 901–906. (29) Kivlehan, F.; Lanyon, Y. H.; Arrigan, D. W. M. Langmuir 2008, 24, 9876– 9882. (30) Herzog, G.; Kam, V.; Arrigan, D. W. M. Electrochim. Acta 2008, 53, 7204– 7209. (31) Vagin, M. Y.; Trashin, S. A.; Karpachova, G. P.; Klyachko, N. L.; Karyakin, A. A. J. Electroanal. Chem. 2008, 623, 68–74. (32) Scanlon, M. D.; Jennings, E.; Arrigan, D. W. M. Phys. Chem. Chem. Phys. 2009, 11, 2272–2280. (33) Horrocks, B. R.; Mirkin, M. V. Anal. Chem. 1998, 70, 4653–4660. (34) Osakai, T.; Komatsu, H.; Goto, M. J. Phys.: Condens. Matter 2007, 19, 375103. (35) Ma, S. C.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1992, 64, 694–697. (36) Meyerhoff, M. E.; Fu, B.; Bakker, E.; Yun, J.-H.; Yang, V. C. Anal. Chem. 1996, 68, 168A–175A. (37) Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 2250–2259. (38) Fu, B.; Bakker, E.; Yang, V. C.; Meyerhoff, M. E. Macromolecules 1995, 28, 5834–5840. (39) Muslinkina, L.; Pretsch, E. Chem. Commun. 2004, 1218–1219. (40) Muslinkina, L.; Pretsch, E. Electroanalysis 2004, 16, 1569–1575. (41) Hyers, T. M.; Agnelli, G.; Hull, R. D.; Morris, T. A.; Samama, M.; Tapson, V.; Weg, J. G. Chest 2001, 119, 176S–193S.
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Figure 1. Molecular formulas of highly lipophilic guanidinium ionophores 1 and 2.
molar limits of detection for monovalent ions.42,43 An even lower limit of detection is expected for an ion with more charges because a current response based on exhaustive ion stripping is proportional to the square of the ionic charge.43 Voltammetric extraction of polyelectrolyte heparins into a nonpolar organic phase is a challenging chemical task because of their large negative charge density, which is the highest of any known biological macromolecule.44 A larger heparin molecule with a larger charge is not only more hydrophilic but also more strongly bound to cations in an aqueous electrolyte solution to screen ∼60%45,46 of the negative charges of a heparin molecule by counterion condensation.47 Thus, a large voltammetric response based on the extraction of a multiply charged heparin molecule requires its dissociation from the bound aqueous cations, which is driven by complexation with multiple molecules of a positively charged ionophore at the interface.17 Even Arixtra, however, cannot be extracted voltammetrically by an ionophore with strong heparin-binding sites such as octadecylammonium and octadecylguaninidium.21 Many more lipophilic quaternary ammoniums with much weaker heparin-binding capabilities such as methyltridodecylammonium and dimethyldioctadecylammonium allow for the voltammetric extraction of Arixtra21 but not unfractionated heparin.17,18 Here we report on a new finding that a highly lipophilic guanidinium ionophore, 1-[4-(dioctadecylcarbamoyl)butyl]guanidinium 1 (Figure 1), can voltammetrically extract heparin molecules with average molecular weights of up to ∼20 kDa into the 1,2dichloroethane phase, which include those in commercial preparations (i.e., Arixtra, Lovenox (4.5 kDa), and unfractionated heparin as well as chromatographically fractionated heparins (7, 9, 15, and 20 kDa)). Successful voltammetric extraction of various heparin molecules by ionophore 1 is ascribed to the formation of highly lipophilic heparin complexes based on cooperative effects of the high lipophilicity and strong heparin-binding capability of this ionophore. Another highly lipophilic guanidinium ionophore, N,Ndioctadecylguanidinium 2, cannot completely extract even Arixtra, thereby revealing the importance of an elaborate ionophore design for heparin extraction. Ion-transfer cyclic voltammetry at micropipet electrodes48,49 is employed as a powerful approach not only to discriminate between the adsorption and extraction of various heparin molecules but also to characterize Arixtra extraction facilitated by ionophore 1 quantitatively and fully.
Experimental Section Chemicals. Tetradodecylammonium (TDDA) bromide, methyltridodecylammonium iodide, octadecyltrimethylammonium bromide, 1,2-dichloroethane (g99.8%), chlorotrimethylsilane (99%), and tetraethylammonium hydroxide (20 wt % in water) (42) Kim, Y.; Amemiya, S. Anal. Chem. 2008, 80, 6056–6065. (43) Kim, Y.; Rodgers, P. J.; Ishimatsu, R.; Amemiya, S. Anal. Chem. 2009, 81, 7262-7270 (44) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 391–412. (45) Ascoli, F.; Botre, C.; Liquori, A. M. J. Phys. Chem. 1961, 65, 1991–1992. (46) Rabenstein, D. L.; Robert, J. M.; Peng, J. Carbohydr. Res. 1995, 278, 239– 256. (47) Manning, G. S. Acc. Chem. Res. 1979, 12, 443–449. (48) Taylor, G.; Girault, H. H. J. Electroanal. Chem. 1986, 208, 179–183. (49) Shao, Y.; Mirkin, M. V. Anal. Chem. 1998, 70, 3155–3161.
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were obtained from Aldrich (Milwaukee, WI). 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) was purchased from Sigma (St. Louis, MO). Dimethyldioctadecylammonium chloride was from Tokyo Kasei Kogyo (Tokyo, Japan). Potassium tetrakis(pentafluorophenyl)borate (KTFAB) was from Boulder Scientific Co. (Mead, CO). All reagents were used as received. Aqueous solutions of the following heparins and LMWHs were prepared with 18.3 MΩ 3 cm deionized water (Nanopure, Barnstead, Dubuque, IA). LMWHs investigated in this work are Arixtra (7.5 mg/0.6 mL, GlaxoSmithKline, Research Triangle Park, NC) and Lovenox (30 mg/0.3 mL, Sanofi-aventis U.S. LLC, Bridgewater, NJ). The original saline solution of Arixtra was dialyzed to remove NaCl.21 Unfractionated heparin from bovine intestinal mucosa (172 units/mg) was obtained from Sigma (St. Louis, MO). Sodium salts of unfractionated heparin with and without contamination of oversulfated chondroitin sulfate (>10 wt %) were obtained from U.S. Pharmacopeia (Rockville, MD). Heparins that are fractionated chromatographically and characterized by size-exclusion HPLC for the determination of a peak molecular weight (7, 9, 15, or 20 kDa) were obtained from Neoparin (Alameda, CA). A peak molecular weight that corresponds to the molecular weight value on the top of a chromatographic peak gives a good estimation of an average molecular weight.50 Preparation of Ionophore Salts. The TFAB salt of 1-[4-(dioctadecylcarbamoyl)butyl]guanidinium 1 or N,N-dioctadecylguanidinium 2 was prepared by metathesis of KTFAB in ethanol and the p-toluenesulfonate or lactate salt of the respective ionophores in dichloromethane as reported for preparation of TFAB salts of the other ionophores investigated in this work.21 The p-toluenesulfonate salts of ionophore 151 and octadecylguanidinium52 and the lactate salt of ionophore 253 were synthesized and characterized by 1H NMR as described in Supporting Information. A 1,2-DCE solution of Arixtra-ionophore 1 complexes was obtained by anion exchange between p-toluenesulfonate of the ionophore 1 salt in a 1,2-DCE solution and Arixtra in a dialyzed solution. Specifically, a 200 μL 1,2-DCE solution of 3 mM p-toluenesulfonate salt of ionophore 1 and 0.1 M TDDATFAB was washed three times with an 800 μL aqueous solution of 0.4 mM Arixtra and 1 mM HEPES at pH 7.0. The resulting 1,2DCE solution was directly used for cyclic voltammetric experiments. Electrochemical Measurements. A computer-controlled CHI 660B electrochemical workstation equipped with a CHI 200 picoampere booster and Faraday cage (CH instruments, Austin, TX) was used for all electrochemical measurements. The following electrochemical cells were employed: Agj0:5 mM TFAB salt of ionophore 1, 2, or octadecylguanidinium in 0:1 M TDDATFAB ð1, 2-DCEÞj1:2 mM Arixtra in 0:1 M Tris=H2 SO4 or 1 mM HEPES=NaOH at pH 7:0 ðaqueousÞjAgCljAg ðcell 1Þ Agj0:38 mM Arixtra-ionophore 1 complex in 0:1 M TDDATFAB ð1, 2DCEÞj1:0mM Arixtra in 1 mM HEPES=NaOH at pH 7:0 ðaqueousÞj AgCljAg
ðcell 2Þ
(50) Bertini, S.; Bisio, A.; Torri, G.; Bensi, D.; Terbojevich, M. Biomacromolecules 2005, 6, 168–173. (51) Kamino, A. K., H.; Ariga, K.; Kunitake, T Bull. Chem. Soc. Jpn. 1996, 69, 3619–3631. (52) Oishi, Y.; Torii, Y.; Kato, T.; Kuramori, M.; Suehiro, K.; Ariga, K.; Taguchi, K.; Kamino, A.; Koyano, H.; Kunitake, T. Langmuir 1997, 13, 519–524. (53) Fotsch, H. H., A.; Meyer, J.; Pascaly M. Use of Alkylguanidines as Cationic Emulsifiers. U.S. Patent 2006/134056 A1, June 22, 2006.
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Agj5 mM TFAB salt of an ionophore in 0:1 M TDDATFAB ð1, 2-DCEÞj Arixtra, Lovenox, unfractionated heparin, or fractionated heparins in 1 mM HEPES=NaOH at pH 7:0 ðaqueousÞ jAgCljAg
ðcell 3Þ
Ionophores and concentrations of heparins and LMWHs in cell 3 are given in the captions of the corresponding figures. A 1,2-DCE/water microinterface was formed at the tip of a micropipet electrode, which was made from a borosilicate glass capillary (o.d./i.d. = 1.0 mm/0.58 mm, 10 cm in length, Sutter Instrument Co., Novato, CA) by using a laser-based pipet puller (model P-2000, Sutter Instrument). The inner and outer tip radii, a and rg (= 1.3a), respectively, were determined as reported elsewhere.21 The inner or outer wall of each pipet was silanized with chlorotrimethylsilane49 so that an organic or an aqueous solution, respectively, was injected into the pipet from the back using a 10 μL syringe. The potential of a water phase was given with respect to a formal potential of tetraethylammonium transfer.25 A current carried by a negative charge from the aqueous phase to the organic phase was defined to be negative. All electrochemical experiments were performed at 22 ( 3 °C.
Results and Discussion Voltammetric Method for Discrimination between the Extraction and Adsorption of Heparin. Here we briefly introduce principles of cyclic voltammetry at micropipet electrodes to discriminate between the adsorption and extraction of heparin-ionophore complexes at the ITIES. Our experimental findings are discussed in the following chapters on the basis of the voltammetric principles. In general, heparin-ionophore complexes are adsorbed at the interface or further extracted into the bulk organic phase as defined by Az ðaqueous phaseÞ þ sL þ ðorganic phaseÞ hALs ðz þsÞ ðinterfaceÞhALs ðz þsÞ ðorganic phaseÞ
ð1Þ
where Az, L+, and ALs(z+s) represent anticoagulant heparin with charge z, ionophore, and their 1/s complexes, respectively. Discrimination between the adsorption and extraction of heparin-ionophore complexes is based on the voltammetry of their dissociation. On one hand, the resulting desorption of heparin from the interface gives a peak-shaped transient voltammogram. On the other hand, no current response based on the reverse extraction of heparin from the bulk organic phase is observed when one of three configurations in Figure 2 is employed (see below). Eventually, the configuration in Figure 2c is needed for discrimination between adsorption and extraction for samples of heterogeneous heparin whereas all three initial conditions are required for full characterization of Arixtra extraction facilitated by ionophore 1. It should be noted that the amplitude of a peak-shaped response based on heparin desorption is independent of a potential scan rate when the formation of heparin-ionophore complexes at the interface is controlled by steady-state diffusion of ionophore or heparin from the external solution to the microITIES (Figure 2a,c, respectively). The desorption peak current varies linearly not only with the scan rate but also with the number of heparin molecules adsorbed at the interface whereas this amount is inversely proportional to the scan rate. Overall, a potential scan rate does not affect either the steady-state forward DOI: 10.1021/la902336w
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Figure 2. Scheme of heparin adsorption and extraction at the 1,2-DCE/water interfaces formed at the tip of micropipet electrodes. The organic phases initially contain (a, c) free ionophore, L, (b) ionophore-heparin (or LMWHs) complexes, AL, and the aqueous phases (a, b) contain free heparin (or LMWHs), A. The arrows correspond to the direction of the forward reaction in eq 1.
wave or the following desorption wave, which contrasts with the cyclic voltammetry of heparin adsorption and desorption at a large ITIES.18 Adsorption and Extraction of Arixtra Facilitated by Guanidinium Ionophores. Highly lipophilic ionophores 1 and 2 with a guanidinium group as a strong heparin-binding site (Figure 1) were characterized voltammetrically by employing Arixtra. This synthetic heparin mimic with well-defined structure allows for the quantitative interpretation of voltammetric data.21 Cyclic voltammetry was performed with glass micropipet electrodes filled with an aqueous solution of an excess amount of Arixtra so that free ionophore molecules are depleted by complexation with Arixtra at the interface (Figure 2a). In fact, a CV with either ionophore 1 or 2 gives a sigmoidal negative response based on facilitated Arixtra transfer (Figure 3a,b, respectively), where mass transfer is controlled by the steady-state radial diffusion of an ionophore from the outer 1,2-DCE phase to the micrometer-sized interface. Importantly, the lack of a positive current response with ionophore 1 indicates that Arixtra-ionophore 1 complexes are extracted across the interface to diffuse radially into the outer 1,2DCE phase. In contrast, ionophore 2 gives a peak-shaped positive response based on the desorption of Arixtra from the interface, which indicates preceding adsorption of Arixtra-ionophore 2 complexes. Nevertheless, the positive peak current with ionophore 2 is smaller than that with octadecylguanidinium (Figure S1) so that a charge density of 8.7 10-4 C/cm2 under the desorption peak with ionophore 2 is smaller than the value of 2.6 10-3 C/cm2 with octadecylguanidinium. This result indicates that more lipophilic ionophore 2 can extract Arixtra more effectively although a certain fraction of Arixtra-ionophore 2 complexes are adsorbed at the interface. The strong binding capability of the guanidinium ionophores with Arixtra was demonstrated using more negative switching potentials in cyclic voltammetry at water-filled pipets. With a switching potential of ∼0.45 V, a sigmoidal negative response based on facilitated Arixtra transfer was followed by a peakshaped response based on the transfer of excess Arixtra without ionophore complexation (insets of Figures 3 and S1). The large separation between the half-wave potential of facilitated Arixtra transfer and the peak potential of simple Arixtra transfer (0.35, 0.30, and 0.38 V for ionophores 1, 2, and octadecylguanidinium, respectively) indicates that Arixtra forms highly stable complexes with the guanidinium ionophores. The high stability is due to strong binding between a guanidinium group of an ionophore molecule and oxoanion groups of an Arixtra molecule,21 which can be mediated by ion pairing and hydrogen bonding as found in (54) Linhardt, R. J.; Toida, T. Acc. Chem. Res. 2004, 37, 431–438.
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Figure 3. CVs of Arixtra with ionophores (a) 1 and (b) 2 at waterfilled pipets with tip diameters of 5.0 and 3.5 μm, respectively (cell 1). The aqueous buffer solution is 0.1 M Tris/H2SO4 at pH 7.0, and the scan rate is 20 mV/s. The insets show the corresponding CVs with more negative switching potentials.
recognition sites of heparin-binding proteins.44,54 Moreover, the participation of multiple ionophore molecules in complexation with an Arixtra molecule contributes to the high stability as confirmed more quantitatively later. It should also be noted that the more negative switching potentials do not significantly affect the amplitude of a peak-shaped positive response based on Langmuir 2009, 25(23), 13653–13660
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Figure 4. Steady-state CVs of (top) Arixtra-ionophore 1 complexes in cell 2 and (bottom) Arixtra with ionophore 1 in cell 1 at water-filled pipets with tip diameters of 5.6 and 6.8 μm, respectively. The aqueous buffer solution is 1 mM HEPES/NaOH at pH 7.0, and the scan rate is 20 mV/s. The open circles correspond to equations S1 and S4, respectively (Supporting Information). The dotted lines represent zero current.
Arixtra desorption with ionophore 2 or octadecylguanidinium (Figures 3b and S1, respectively). This result indicates that the adsorption of their Arixtra complexes at the interface reaches a saturated or steady state at the aforementioned charge densities. Slow Arixtra Transfer with Guanidinium Ionophores. CVs at water-filled pipets also revealed slow Arixtra transfer with guanidinium ionophores. The sigmoidal negative response with ionophore 2 or octadecylguanidinium is totally separated from the peak-shaped positive response (Figure 3b or S1, respectively), which corresponds to electrochemically irreversible adsorption and desorption of Arixtra, respectively. Moreover, electrochemically irreversible extraction is demonstrated by comparing CVs of forward and reverse Arixtra extraction with ionophore 1 (Figure 4). The reverse extraction was studied by dissolving Arixtra-ionophore 1 complexes in the outer 1,2-DCE solution to obtain a sigmoidal positive response (top of Figure 4), where mass transfer is controlled by steady-state radial diffusion of the complexes (Figure 2b). This result confirms the presence of Arixtra-ionophore 1 complexes in the 1,2-DCE phase as well as their dissociation at the interface. In addition, the lack of a peak-shaped wave indicates that the adsorption of Arixtra-ionophore 1 complexes is negligible. Noticeably, the sigmoidal positive response is completely separated from a negative response based on forward extraction as observed with an excess amount of Arixtra in the internal aqueous phase (bottom of Figure 4). The large separation of 0.24 V between half-wave potentials of the negative and positive responses corresponds to the electrochemical irreversibility of facilitated Arixtra extraction. The slow extraction of Arixtra with ionophore 1 was also confirmed by cyclic voltammetry at a micropipet filled with a 1,2DCE solution of excess ionophore (solid line in Figure 5). The facilitated extraction of Arixtra into the inner 1,2-DCE phase gives a major negative response at >0.03 V. The sigmoidal shape is due to the steady-state diffusion of Arixtra in the outer aqueous phase (Figure 2c). Preceding peak-shaped responses are minor and relevant to the adsorption of Arixtra, ionophore 1, or their complexes. The small negative prepeaks are mainly coupled with small and broad positive responses of around -0.15 V because the Langmuir 2009, 25(23), 13653–13660
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Figure 5. CVs of 18.5 μM Arixtra with ionophore 1 at an organicfilled pipet with a tip diameter of 8.5 μm (cell 3). The scan rate is 5 mV/s. The open circles correspond to a CV simulated with k0 = 3.5 10-7 cm/s by the finite element method (Supporting Information), where the CV was shifted to obtain the good fit for negative responses. The inset also shows CVs simulated with k0 = 3.5 10-5 (black), 3.5 10-6 (green), 3.5 10-7 (red), 3.5 10-8 (blue), and 3.5 10-9 cm/s (magenta) as plotted against potentials with respect to a formal potential. The other parameters are listed in Table 1.
feature of a voltammogram on the reverse potential sweep was similar when the direction of the potential sweep was switched prior to the major negative response (dotted line). The lack of a significant positive response that is coupled with the major negative response confirms that Arixtra-ionophore 1 complexes are mainly extracted into the 1,2-DCE phase. This result also indicates that the reverse extraction of Arixtra is too slow to give a significant positive response coupled with hindered diffusion of its complexes in the internal solution. At the same time, the slow extraction of Arixtra shifts the resulting negative response toward the negative limit of the potential window. Consequently, the application of very negative potentials in the presence of an excess amount of ionophores drives the facilitated transfer of other aqueous anions (e.g., HEPES anion) to increase the background current, thereby compromising Arixtra selectivity. The kinetically limited selectivity of ionophore 1 was also observed for the other heparins (see below). Interfacial Mechanism for Voltammetric Arixtra Extraction. Well-defined CVs of Arixtra extraction with ionophore 1 under three different configurations (Figures 4 and 5) were analyzed to reveal the importance of slow interfacial complexation, which controls the rates of the overall bulk extraction across the interface (Figure 6). Heterogeneous rate constants, kf and kb, for the formation and dissociation of complexes at the adsorption plane are given by eqs S10 and 11 with kinetic parameters k0, zeff, R, and β. Definitions of these parameters and their determination from CVs are detailed in Supporting Information. Two parameters of z and s in eq 1 were obtained from limiting currents (Table 1; see also Supporting Information) to demonstrate that a polyanionic Arixtra molecule forms an electrically neutral and highly lipophilic complex with multiple molecules of ionophore 1 to become extractable into the nonpolar 1,2-DCE phase. The charge of z = -7.8 ( 0.7 is carried by a polyanionic Arixtra molecule between the bulk phases whereas Arixtra possesses eight sulfate and two carboxyl groups. Importantly, the stoichiometry of s = 7.8 ( 0.8 indicates that ionophore DOI: 10.1021/la902336w
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Figure 6. Scheme of a multistep transfer mechanism for Arixtra extraction facilitated by ionophore 1.
molecules neutralize all charges on an Arixtra molecule to form a neutral complex surrounded by multiple octadecyl groups of the bound ionophore molecules. This result contrasts with complexation between Arixtra and octadecylguanidinium, where an Arixtra molecule is bound to only five ionophore molecules with one octadecyl group so that the resulting negatively charged and insufficiently lipophilic complexes are mainly adsorbed at the ITIES.21 All CVs of Arixtra extraction with ionophore 1 can be explained consistently using a multistep transfer mechanism with zeff, R, and β values listed in Table 1. This mechanism was proposed originally for protamine extraction facilitated by dinonylnaphthalene sulfonate (see Table 1 for the corresponding parameters).24,25 It is assumed in this model that Arixtra-ionophore complexation at an adsorption plane is the rate-determining step whereas the adsorption of free ionophores and Arixtra-ionophore complexes is fast enough to be always at equilibrium (Figure 6). This mechanism is supported by experimental β values of ∼0.80 for both Arixtra and protamine as a measure of the location of the adsorption plane.24 These β values agree with values for surfactant adsorption at 1,2-DCE/water interfaces determined by optical second-harmonic generation.55,56 The multistep transfer mechanism is also supported by the charges on an Arixtra molecule that effectively contribute to the transfer kinetics, zeff, which is close to the overall charge, z, carried by the molecule between two liquid phases. An effective transfer coefficient, R, for Arixtra extraction with ionophore 1 is larger than normal values in the range of 0.4-0.6, thereby suggesting a double-layer effect.57 The parameters thus determined above allow for the quantitative assessment of slow interfacial complexation between Arixtra and ionophore 1 as represented by a small heterogeneous standard rate constant, k0. The finite element method was employed to simulate CVs for Arixtra extraction using k0 values of 3.5 10-5-3.5 10-9 cm/s and experimentally determined parameters (Supporting Information).21,25,58 A peak-shaped positive response in a simulated CV becomes smaller and broader and shifts toward positive potentials as k0 becomes smaller (inset of Figure 5). The peak shape corresponds to the transient diffusion of Arixtra-ionophore complexes in the inner 1,2-DCE solution.21 Even(55) (56) (57) (58) 2296.
Higgins, D. A.; Corn, R. M. J. Phys. Chem. 1993, 97, 489–493. Piron, A.; Brevet, P. F.; Girault, H. H. J. Electroanal. Chem. 2000, 29–36. Marcus, R. A. J. Chem. Phys. 2000, 113, 1618–1629. Jing, P.; Rodgers, P. J.; Amemiya, S. J. Am. Chem. Soc. 2009, 131, 2290–
13658 DOI: 10.1021/la902336w
Jing et al.
tually, positive responses simulated with k0 e 3.5 10-7 cm/s (closed circles in Figure 5) overlap with or exceed the positive limit of the potential window, which is consistent with the experimental CV as obtained with an organic-filled pipet (solid line). This result indicates that a k0 value for Arixtra extraction with ionophore 1 is smaller than 3.5 10-7 cm/s. Arixtra extraction with ionophore 1 is slower than protamine extraction with DNNS (k0 = 3.5 10-5 cm/s), which is the slowest ion transfer at the ITIES reported so far.25 Noticeably, the electrochemical irreversibility of intrinsically slow Arixtra extraction with ionophore 1 is enhanced by fast mass transfer to a micropipet-supported ITIES. Extraction and Adsorption of Heparin in Commercial Preparations. Successful voltammetric extraction of Arixtra by ionophore 1 motivated us to examine larger heparin molecules in commercial preparations. Ionophore 1 can extract larger LMWH, Lovenox, with an average molecular weight, M, of 4.5 kDa and a molecular weight distribution mainly from 2 to 8 kDa.59 Lovenox extraction is confirmed by cyclic voltammetry at a micropipet electrode filled with a 1,2-DCE solution of excess ionophore 1 (Figure 7). The negative current response varies with the Lovenox concentration although the shape of these waves is distorted from an idealistic sigmoidal shape because of a background current around the negative limit of the potential window. The lack of a significant positive current response indicates electrochemically irreversible extraction of Lovenox into the 1,2-DCE phase. Lovenox is significantly adsorbed at the interface in the presence of highly lipophilic quaternary ammoniums (i.e., dimethyldioctadecylammonium and methyltridodecylammonium), as indicated by large desorption peaks on the reverse potential sweep (Figure S2). Moreover, the CVs with these weakly binding ionophores are observed at much more negative potentials than the CV with ionophore 1, which possesses a guanidinium group as a much stronger heparin-binding site.21 Overall, ionophore 1 with both strong binding capability and high lipophilicity is required for the extraction of Lovenox whereas smaller Arixtra can be extracted into the 1,2-DCE phase with either ionophore 1 or the quaternary ammoniums (data not shown). In contrast to the sole extraction of LMWHs, both extraction and adsorption were observed for unfractionated heparin with ionophore 1. The corresponding CV (solid line in Figure 8) shows a major negative response at
