Electrochemical Study of Insulin at the Polarized Liquid−Liquid

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Electrochemical Study of Insulin at the Polarized Liquid-Liquid Interface Francine Kivlehan, Yvonne H. Lanyon, and Damien W. M. Arrigan* Tyndall National Institute, Lee Maltings, UniVersity College, Cork, Ireland ReceiVed March 17, 2008. ReVised Manuscript ReceiVed May 28, 2008 This paper reports on the electrochemical behavior of bovine insulin at the interface between two immiscible electrolyte solutions (ITIES). The voltammetric ion-transfer response obtained in the presence of insulin was dependent on the aqueous phase pH conditions and on the nature of the organic phase electrolyte employed in experiments. Optimal detection was obtained at acidic pH below the isoelectric point of insulin where it was positively charged. A shift in transfer potentials to lower potential values was observed with decreasing hydrophobicity of the anion of the organic phase electrolyte. No ion-transfer response was observed at pH values of the aqueous phase above the isoelectric point, where insulin was negatively charged. These results suggest that the voltammetric response is due to ion-pairing interactions at the ITIES between positively charged insulin and the hydrophobic anion of the organic phase electrolyte, together with adsorption of the ion-pair at the interface. The voltammetric response was obtained for insulin at concentrations down to 1 µM. These results show that electrochemistry is useful in studying the behavior of this important protein molecule at the polarized water-1,2-DCE interface and provides an alternative detection mode for bioanalytical applications.

Introduction Insulin is a small protein (RMM ∼5700) composed of two polypeptide chains A and B and containing three disulphide bonds, two of which are interchain between A and B, while the third is an intrachain bond within chain A.1,2 Insulin regulates blood glucose by signaling when high levels of blood glucose are present in the body.3 It stimulates the uptake of glucose by muscle tissue and works together with another hormone, glucagon, to maintain a controlled level of blood glucose. Diabetes mellitus is caused by a deficiency in the secretion (type 1 diabetes) or action (type 2 diabetes) of insulin. According to one study, the prevalence of diabetes for all age groups worldwide is estimated at 2.8% for 2000 rising to 4.4% in 2030.4 The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. Furthermore, the American Diabetes Association (ADA) estimated the national cost of diabetes in the U.S. for 2002 to be $132 billion, increasing to $192 billion in 2020,5 so clearly insulin monitoring is an important subject for medical and physiological studies of this disease. Present analytical methods for the detection of insulin include bioassays, immunoassays and chromatography. The development of bioassays6–9 and immunoassays10–13 for the detection of insulin * To whom correspondence should be addressed. Phone: 353-21-4904079. Fax: 353-21-4270271. E-mail: [email protected]. (1) Ryle, A. P.; Sanger, F.; Smith, L. F.; Kitai, R. Biochem. J. 1955, 60, 1541. (2) Stankovich, M. T.; Bard, A. J. J. Electroanal. Chem. 1977, 85, 173. (3) Lehninger, A. L.; Nelson, D. L.; Cox, M. M., Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, 1993. (4) Wild, S.; Roglic, G.; Green, A.; Sicree, R.; King, H. Diabetes Care 2004, 27, 1047. (5) Hogan, P.; Dall, T.; Nikolov, P. Diabetes Care 2003, 26, 917. (6) Kowarski, C. R.; Bado, B.; Shah, S.; Kowarski, D.; Kowarski, A. A. J. Pharm. Sci. 1983, 72, 692. (7) van Zoelen, E. J. J. Prog. Growth Factor Res. 1990, 2, 131. (8) Trethewey, J. J. Pharm. Biomed. Anal. 1989, 7, 189. (9) Vu, L.; Pralong, W. F.; Cerini, F.; Gjinovci, A.; Stocklin, R.; Rose, K.; Offord, R. E.; Kippen, A. D. Anal. Biochem. 1998, 262, 17. (10) Wherry, F. E.; Miller, R. E.; Mason, J. W. Metabolism 1966, 15, 163. (11) Taylor, K. W.; Howell, S. L.; Montague, W.; Edwards, J. C. Clin. Chim. Acta 1968, 22, 71. (12) Kimura, H. J. Immunol. Methods 1980, 38, 353. (13) Roper, M. G.; Shackman, J. G.; Dahlgren, G. M.; Kennedy, R. T. Anal. Chem. 2003, 75, 4711.

has been well reported in the literature. Of these, radioimmunoassays (RIA)14–17 and enzyme-linked immunosorbent assays (ELISA)18–22 are the most commonly used methods. A number of high performance liquid chromatography (HPLC) methods coupled with UV absorbance,23 mass spectrometry,24 photolytic electrochemical detection25 and fluorescence26 have been developed for the direct detection of insulin. These methods can detect insulin down to sub-nM and pM concentrations, but are limited in their use as they can be time-consuming and slow, and frequently require isotope- or fluorophore-labeled insulin to increase the sensitivity and selectivity. Furthermore, much of the instrumentation required is laboratory-based, and measurements can involve multistep procedures. The direct detection of insulin by electrochemical means offers an excellent opportunity for the development of portable systems. This has already been achieved to a considerable extent using solid electrodes such as the glassy carbon (GC) electrode. This approach however has its drawbacks such as slow oxidation kinetics and surface fouling, which are overcome by complex surface modification27–30 and/or the use of mediators.31–35 (14) Feldman, J. M.; Chapman, B. A. Clin. Chem. 1973, 19, 1250. (15) Kagan, A. Semin. Nucl. Med. 1975, 5, 183. (16) Banks, W. A.; Jaspan, J. B.; Kastin, A. J. Peptides 1997, 18, 1257. (17) Murayama, H.; Matsuura, N.; Kawamura, T.; Maruyama, T.; Kikuchi, N.; Kobayashi, T.; Nishibe, F.; Nagata, A. J. Autoimmun. 2006, 26, 127. (18) Burgi, W.; Briner, M.; Franken, N.; Kessler, A. C. Clin. Biochem. 1988, 21, 311. (19) MacDonald, M. J.; Gapinski, J. P. Metab., Clin. Exp. 1989, 38, 450. (20) Storch, M. J.; Alexopoulos, A.; Kerp, L. J. Immunol. Methods 1989, 119, 53. (21) Webster, H. V.; Bone, A. J.; Webster, K. A.; Wilkin, T. J. J. Immunol. Methods 1990, 134, 95. (22) Kumada, Y.; Katoh, S.; Imanaka, H.; Imamura, K.; Nakanishi, K. J. Biotechnol. 2007, 127, 288. (23) Ohkubo, T. Biomed. Chromatogr. 1994, 8, 301. (24) Sun, Y. P.; Smith, D. L.; Shoup, R. E. Anal. Biochem. 1991, 197, 69. (25) Dou, L.; Krull, I. S. Anal. Chem. 1990, 62, 2599. (26) Toriumi, C.; Imai, K. Anal. Chem. 2002, 74, 2321. (27) Zhang, M. G.; Mullens, C.; Gorski, W. Anal. Chem. 2005, 77, 6396. (28) Wang, J.; Musameh, M. Anal. Chim. Acta 2004, 511, 33. (29) Wang, J.; Musameh, M. Anal. Chim. Acta 2005, 539, 209. (30) Wang, J.; Tangkuaram, T.; Loyprasert, S.; Vazquez-Alvarez, T.; Veerasai, W.; Kanatharana, P.; Thavarungkul, P. Anal. Chim. Acta 2007, 581, 1. (31) Cox, J. A.; Gray, T. J. Anal. Chem. 1989, 61, 2462.

10.1021/la800842f CCC: $40.75  2008 American Chemical Society Published on Web 07/31/2008

Electrochemical Study of Insulin

Detection limits within the sub-µM to nM range have been achieved by such methods, while pM detection has been reported most recently using modified carbon composite electrodes with flow injection amperometry.36 Insulin’s main role in the body is the regulation of blood glucose levels, which it does by binding to transmembrane receptors of muscle and adipose cells in order to stimulate a complex cell signaling pathway for the transport of glucose across the cell membrane.37 Insulin interacts with these receptors as a monomer but also has the ability to form different association states such as dimers, tetramers and hexamers. This allows the body to store large quantities of insulin in the pancreatic β-cells because hexamer formation ensures efficient packing of the protein and helps with conversion of inactive proinsulin to insulin.38 Insulin exists as an equilibrium mixture of these different associations depending on the experimental factors involved. For example, while the main storage form of insulin is a zinccoordinated hexamer, zinc-free insulin exists as a dimer at low protein concentrations over a pH range of 2 to 8, but shifts to a tetramer at protein concentrations above 1.5 mg mL-1 (2.6 × 10-4 M).39 pH conditions have been shown to determine the preferred associations of the insulin molecule. Nielsen et al. demonstrated that at pH 7.4, insulin is hexameric, and dissociates on going to lower pH, existing as a tetramer at pH 3 and as a dimer/monomer at pH 1.6 or 2.0.40 The nature of the acid used at very acidic pH also dictates the association state of insulin, as it was reported that insulin is dimeric at pH 1.6 in HCl, and monomeric at pH 2 in acetic acid, which may be a result of the strong hydrogenbonding properties of carboxylic acids.40 Insulin is one of about 20 known proteins that are classed as amyloidogenic, meaning that it can undergo an alternative folding pathway leading to the formation of fibrils consisting of a crossβ-sheet structure.41 These amyloid structures have the same physical characteristics as those responsible for neurodegenerative diseases such as Alzheimer’s disease.42,43 The biologically active monomeric form of insulin is prone to fibrillation when it is much less stable, as opposed to the hexamer form where the burial of hydrophobic surfaces prevents the insulin molecule from undergoing such random aggregation interactions. Details of the underlying mechanism of insulin aggregation remain unclear, but studies on insulin fibers have shown that experimental factors which bring about monomerization of the molecule, such as low pH, heat, organic solvents and agitation, promote fibril formation.44,45 Insulin aggregation may also be promoted by (32) Gorski, W.; Aspinwall, C. A.; Lakey, J. R. T.; Kennedy, R. T. J. Electroanal. Chem. 1997, 425, 191. (33) Pikulski, M.; Gorski, W. Anal. Chem. 2000, 72, 2696. (34) Cheng, L.; Pacey, G. E.; Cox, J. A. Anal. Chem. 2001, 73, 5607. (35) Salimi, A.; Pourbeyram, S.; Haddadzadeh, H. J. Electroanal. Chem. 2003, 542, 39. (36) Salimi, A.; Roushani, M.; Soltanian, S.; Hallaj, R. Anal. Chem. 2007, 79, 7431. (37) Blundell, T. L.; Dodson, G. G.; Hodgkin, D. M.; Merola, D. AdV. Protein Chem. 1972, 26, 279. (38) Brange, J.; Skelbaek-Perdersen, B.; Langkjaer, L.; Damgaard, U.; Ege, H.; Havelung, S.; Heding, L. G.; Jorgensen, K. H.; Lykkeberg, J.; Markussen, J.; Pingel, M.; Rasmussen, E. Galenics of Insulin. The physicochemical and pharmaceutical aspects of insulin and insulin preparations; Springer-Verlag: Berlin, 1987. (39) Bryant, C.; Spencer, D. B.; Miller, A.; Bakaysa, D. L.; McCune, K. S.; Maple, S. R.; Pekar, A. H.; Brems, D. N. Biochemistry 1993, 32, 8075. (40) Nielsen, L.; Frokjaer, S.; Brange, J.; Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 8397. (41) Kraineva, J.; Smirnovas, V.; Winter, R. Langmuir 2007, 23, 7118. (42) Sunde, M.; Blake, C. C. F. Q. ReV. Biophys. 1998, 31, 1. (43) Serpell, L. C.; Smith, J. M. J. Mol. Biol. 2000, 299, 225. (44) du Vigneaud, V.; Sifferd, R. H.; Sealock, R. R. J. Biol. Chem. 1933, 102, 521. (45) Waugh, D. F. J. Am. Chem. Soc. 1946, 68, 247.

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interactions with hydrophobic interfaces, for example, air-water or lipid-water interfaces,46,47 as well as hydrophobic surfaces such as polytetrafluoroethylene and polyvinylchloride.46,48 This paper reports on the electrochemical behavior of insulin at the interface between two immiscible electrolyte solutions (ITIES) as a step toward the detection of insulin. This approach may be used to overcome the associated drawbacks of its detection at solid electrodes, while retaining its potential for the development of out-of-laboratory detection systems. Although there are currently no reports on the behavior of insulin at the liquid-liquid interface, other proteins have been studied, e.g., protamine.49,50 The ITIES has been employed for evaluating counterion binding to protamine with various hydrophobic anions present in the organic phase,51 as well as for the study of ionized macromolecules such as dendrimers.52 The electrochemical extraction of protamine and cytrochrome c has been achieved at a polarized liquid-liquid interface by reverse micelle formation,53 while the study and detection of heparin, a sulfonated polysaccharide, at a plasticized polyvinylchloride membrane|solution interface has also been reported.54,55 Furthermore, potentiometric membrane electrodes have been developed for the sensing of heparin and protamine, where the response mechanism relies on the phase boundary potential at the sample solution-membrane interface.56–58 The use of electrochemistry at the liquid-liquid interface as a means for the electrochemical study of insulin offers a number of advantages. It is label-free in that insulin can be detected without the need for its modification, the sensor signal is directly related to the presence of insulin and it can, in principle, be implemented with portable electrochemical instrumentation. This offers scope for the development of this system to suit applications such as point-of-care diagnostics and the real-time monitoring of pharmaceutical insulin preparations. We report here the results of studies of the electrochemical behavior of insulin at the ITIES. The results show that insulin may be detected but that its dependence on solution phase chemistry in both phases suggests that careful optimization of the conditions for detection will be necessary. The detection mechanism involves ion-transfer accompanied by adsorption, whereby the cationic insulin forms an ion-pair with organic phase electrolyte anion at the ITIES.

Experimental Section Reagents. All solutions were prepared in ultrapure water (g18 MΩ cm purity). Sodium hydroxide, potassium dihydrogen phosphate, Tween80, bis(triphenylphosphoranylidene) ammonium chloride (BTPPACl) and 1.0 M HCl were purchased commercially (SigmaAldrich GmbH), as was 1,2-dichloroethane (1,2-DCE) (Merck KGaA, Darmstadt, Germany), and potassium tetrakis(4-chlorophenyl)borate (KTPClB), lithium chloride and tetraethylammonium (46) Sluzky, V.; Tamada, J. A.; Klibanov, A. M.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9377. (47) Sharp, J. S.; Forrest, J. A.; Jones, R. A. L. Biochemistry 2002, 41, 15810. (48) Feingold, V.; Jenkins, A. B.; Kraegen, E. W. Diabetologia 1984, 27, 373. (49) Amemiya, S.; Yang, X. T.; Wazenegger, T. L. J. Am. Chem. Soc. 2003, 125, 11832. (50) Yuan, Y.; Amemiya, S. Anal. Chem. 2004, 76, 6877. (51) Troja´nek, A.; Langmaier, J.; Samcova´, E.; Samec, Z. J. Electroanal. Chem. 2007, 603, 235. (52) Berduque, A.; Scanlon, M. D.; Collins, C. J.; Arrigan, D. W. M. Langmuir 2007, 23, 7356. (53) Shinshi, M.; Sugihara, T.; Osakai, T.; Goto, M. Langmuir 2006, 22, 5937. (54) Samec, Z.; Troja´nek, A.; Langmaier, J.; Samcova´, E. Electrochem. Commun. 2003, 5, 867. (55) Langmaier, J.; Olsa´k, J.; Samcova´, E.; Samec, Z.; Troja´nek, A. Electroanalysis 2006, 18, 1329. (56) Ma, S. C.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1992, 64, 694. (57) Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 2250. (58) Yun, J. H.; Meyerhoff, M. E.; Yang, V. C. Anal. Biochem. 1995, 224, 212.

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Figure 1. CV response of bovine insulin at the ITIES. (Aq): 10 mM LiCl + (a) 2.5 µM; (b) 5 µM; (c) 10 µM; (d) 20 µM; (e) 40 µM; (f) 60 µM; (g) 80 µM insulin. pH ) 2.85. (O): 10 mM BTPPATPClB. Scan rate, 5 mV s-1.

chloride (Fluka Chemie GmbH). Insulin (from Bovine pancreas; g 27 USP units mg-1) was also purchased from SigmaAldrich. Preparation of Insulin Solutions. A stock solution of 1 mM insulin was prepared by dissolving powdered bovine insulin in ultrapure water containing 0.02% (v/v) Tween80. Tween80 surfactant was used to minimize adsorption of the insulin onto the glass walls of the cell.28,31 The stock solution contained 0.02 mM HCl, in order to fully dissolve the insulin. Subsequent dilutions were made in the aqueous phase electrolyte solution and the pH of all solutions were adjusted to required values using 1.0 M NaOH or 1.0 M HCl. pH measurements were made with a combination glass pH electrode and pH meter (Orion model 520A). Solutions were prepared fresh for each day of experimentation. Electrochemical Cell and Measurements. The experimental system used for electrochemical measurements at the liquid-liquid interface was a four-electrode system, as described in the literature.59–61 A typical cell setup used for this study was as follows.

Ag|AgCl|10mM BTPPACl + 10mM LiCl|10mM BTPPATPClB in 1,2-DCE ||x mM Insulin + 10 mM LiCl|AgCl|Ag 10 mM LiCl served as the aqueous phase to which insulin was added, while the organic phase was 10 mM bis(triphenylphosphoranylidene) ammonium tetrakis(4-chlorophenyl)borate (BTPPATPClB) in 1,2-DCE. The supporting electrolyte for the organic phase, BTPPATPClB, was prepared by metathesis of KTPClB and BTPPACl.62 The counter electrodes were platinum mesh wires and Ag/AgCl wires served as the reference electrodes in each phase. The stem of the organic phase counter electrode was shrouded in a glass capillary to avoid any contact with the external organic or aqueous solutions. The organic phase reference solution was made up of 10 mM LiCl and 10 mM BTPPACl. All figures presenting data display the potential axis relative to the experimentally used reference electrodes. Transposition to the Galvani potential scale are presented in the discussions where relevant. The experimentally determined potential values obtained can be transposed to the tetraphenylarsonium tetraphenylborate Galvani scale by comparing the experimentally observed transfer potentials to the reversible transfer potential for tetraethylammonium (TEA+; 0.430 V), a model cation well characterized in the literature and of known transfer potential on the Galvani scale (0.049 V).61 Electrochemical measurements were made with CHI620B, CHI660B (CH Instruments, Austin, Texas), or Autolab PGSTAT30 (59) Samec, Z.; Marecek, V.; Koryta, J.; Khalil, M. W. J. Electroanal. Chem. 1977, 83, 393. (60) Wickens, J.; Dryfe, R. A. W.; Mair, F. S.; Pritchard, R. G.; Hayes, R.; Arrigan, D. W. M.; New, J. Chem. 2000, 24, 149. (61) Samec, Z. Pure Appl. Chem. 2004, 76, 2147. (62) Qian, Q. S.; Wilson, G. S.; Bowman-James, K.; Girault, H. H. Anal. Chem. 2001, 73, 497.

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Figure 2. CV response of control experiments testing the response of (I) Blank 10 mM LiCl (aq) phase, and (II) additions of insulin-free solution prepared in 10 mM LiCl. Scan rate, 5 mV s-1. Solution additions to create curve (II) are equivalent to those used to produce insulin concentrations of 50 µM in insulin-containing solutions.

Figure 3. CV response, (Aq): 10 mM LiCl + 0.05 mM insulin pH 2.06. (O): 10 mM BTPPATPClB. Scan rates: 5 mV s-1; 10 mV s-1; 25 mV s-1; 50 mV s-1; 75 mV s-1; 100 mV s-1. Inset (A): current response to (V)1/2 for peak (2); Inset (B): current response to (V)1/2 for peak (3); Inset (C): current response to V for wave (1).

(EcoChemie BV, Utrecht, The Netherlands) potentiostats. All electrochemical experiments were performed at room temperature, ∼21-22 °C. Cyclic voltammetry (CV) and square wave stripping voltammetry (SWSV) at the polarized aqueous-1,2-DCE interface were employed in the study of insulin behavior at the ITIES and are reported in the following sections. For CV, the potential was scanned from lower to higher potentials on the forward scan. For SWSV the scan direction was from higher to lower potentials.

Results and Discussion Cyclic Voltammetry. Figure 1 illustrates CVs of insulin at increasing concentrations in the aqueous phase (pH 2.85) in the ITIES cell. At this pH, insulin is cationic. A forward scan transfer peak at a potential of 0.70 V and a reverse scan transfer peak at 0.54 V were present, with the magnitude of both forward and reverse peak currents being dependent on the concentration of insulin in the aqueous phase. The separation between the peak potentials of the forward and reverse peaks (∆Ep) was 163 mV and the midpoint half-wave transfer potential (E1/2) was 0.62 V. The striking feature of these CVs is that they are markedly different from that for a reversible diffusion-controlled ion-transfer process.61 Such a process would exhibit a peak-peak separation value of 59/zi mV. Insulin is capable of possessing a net positive charge of 6+ at acidic pH,38 therefore a ∆Ep value of ∼9 mV would be expected if reversible transfer of the fully protonated molecule was occurring. Based on the large ∆Ep value and peak

Electrochemical Study of Insulin

Figure 4. Effect of change in pH on forward and reverse transfer peaks for 0.05 mM insulin. Scan rate, 5 mV s-1. A diffusion coefficient of 1 × 10-6 cm2 s-1 68 was used to calculate the predicted current response.

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the transfer of ionic species from the organic to the aqueous phase, which is facilitated by ion-pair formation with insulin and subsequent adsorption of this complex at the interface. Considering the acidic pH conditions employed for this experiment, where insulin is known to be positively charged,38 it may act as a facilitator for the transfer of the organic phase anion across the interface via the formation of an ion-pair. As the concentration of insulin in the aqueous phase is lower than the concentration of anionic species in the organic phase, facilitated transfer of the anion across the ITIES is controlled by the diffusion of insulin in the aqueous phase toward the interface. The currents of peaks (2) and (3) both had a linear dependence on the square root of the scan rate (V)1/2 (inset A and B of Figure 3), in accordance with a diffusion-limited transfer process at a large interface and in agreement with the Randles-Sevcik model (eq 1).

Ip ) 2.807 × 105zi3 ⁄ 2AciD1 ⁄ 2V1 ⁄ 2

(1)

shapes observed, the transfer process occurring across the ITIES is not a simple reversible ion-transfer reaction. Nevertheless, a linear correlation between the transfer peak current and the concentration of insulin was obtained, (Figure 1, inset). The coefficient of variation at each concentration was no greater than 10%, based on triplicate measurements. The CVs of Figure 1 may be due to a process such as insulin-facilitated transfer of the organic phase electrolyte anion, accompanied by some conformational rearrangement of the insulin at the interface, yielding the markedly different peak shapes on forward and reverse scans. In effect, this means that two different substances are responsible for the forward and reverse peaks: insulin and a polypeptide of identical chemical composition but different conformation, respectively. The data shown in Figure 1 were obtained from a series of experiments where the insulin solution analyzed was prepared by dilution of the stock solution in 10 mM LiCl, which serves as the aqueous electrolyte. Control experiments on the aqueous phase constituents were carried out in the absence of insulin. Figure 2 illustrates the results of these control experiments for solutions prepared by dilution in 10 mM LiCl (pH 3.0). A small signal was observed at a potential of ∼0.75-0.80 V, which is most likely due to the presence of the Tween80 surfactant. On further investigation of the influence of Tween80, it was found that small transfer currents were obtained in the presence of Tween80, but these were miniscule in comparison to the peaks obtained on subsequent spiking of insulin into the aqueous phase. This indicated that the Tween80 employed at 0.02% was not responsible for the voltammetric peaks observed. Scan Rate Studies. Figure 3 illustrates the influence of the CV scan rate on the response to insulin at the ITIES, at pH 2.06. Two processes were observed on the forward scan, with wave (1) appearing at the foot of peak (2) with increasing scan rate. One process was observed on the reverse sweep, peak (3). The development of wave (1) at the foot of peak (2) suggests the occurrence of an adsorption process, supported by a linear correlation observed between the current for this wave and scan rate (V) (see inset (C), Figure 3). The occurrence of a prewave such as this indicates adsorption of the product resulting from the ion-transfer process.63,64 This may be explained by considering

Although peak (3) exhibits some aspects of the shape of an adsorption/desorption process (i.e., the absence of diffusionaltailing), it reflects a diffusion-controlled transfer process based on its (V)1/2 dependency, which exhibited a greater linear correlation coefficient than the corresponding peak current-scan rate (V). Thus the experimental data for insulin electrochemistry at the ITIES indicate a diffusion-controlled ion-transfer process, accompanied by adsorption of the resulting ion-pair product formed between insulin and the organic phase anion at the interface. The results shown in Figure 3 are similar to those reported by Ulmeanu et al. on the transfer and adsorption characteristics of polyethylenimine (a positively charged polyelectrolyte, PEI+) at the ITIES,65 where adsorption was strong enough at the interface to form an adsorbed layer visible as a “cloudy” interface. Amemiya et al. reported the transfer and adsorption of protamine at the micro-ITIES using micropipets, where CV studies confirmed protamine transfer during the forward scan followed by adsorption near the interface rather than diffusion into the bulk organic phase.49 Effect of pH. The influence of pH on transfer peaks was studied by varying the pH of the aqueous phase electrolyte at constant concentration of insulin (50 µM). Insulin has an isoelectric point (pI) of ∼5.5.66,67 Below its pI, it is positively charged and above it, it is negatively charged. Insulin contains many ionizable groups, with six amino acid residues capable of achieving a net charge of up to 6+ below the pI, and 10 amino acid residues capable of achieving a net charge of up to 10above the pI.38 Figure 4 illustrates the effect of pH on the forward and reverse transfer peaks obtained in the presence of insulin. A general decrease in the peak height was observed for both peaks on approaching the pI from lower pH solutions and past it toward more basic conditions where no transfer peaks were obtained in the presence of insulin. The dashed line in Figure 4 represents the predicted current response to insulin as a function of the aqueous phase pH. This was calculated by use of the Randles-Sevcik equation for ion-transfer reactions at the liquid-liquid interface (equation 1). The predicted current response (Ip) was calculated for a range of pH values between 2 and 12. Net charge values (zi) were estimated for each pH value, based on the number of ionizable groups in an insulin

(63) Pletcher, D.; Greef, R.; Peat, R.; Peter, L. M.; Robinson, J. Instrumental Methods in Electrochemistry; Horwood Publishing Limited: Chichester, U.K., 2001. (64) Bard, A. J.; Faulkner, L. R., Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley and Sons Inc.: New York, 2001.

(65) Ulmeanu, S.; Lee, H. J.; Girault, H. H. Electrochem. Commun. 2001, 3, 539. (66) Gao, J.; Mrksich, M.; Gomez, F. A.; Whitesides, G. M. Anal. Chem. 1995, 67, 3093. (67) Tanford, C.; Epstein, J. J. Am. Chem. Soc. 1954, 76, 2163.

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Figure 5. Photograph of the (aq)|(o) interface, at pH 6.0-7.0.

monomer that are capable of protonation or deprotonation.38 A diffusion coefficient of 1 × 10-6 cm2 s-1 was used, in accord with values reported for the diffusion of insulin in aqueous solution.68 The diffusion of insulin toward the interface is the controlling factor, since the organic phase electrolyte anion is in excess over the insulin concentration. For this reason the calculations use the characteristics of insulin, both diffusion coefficient and charge number. It is clear from Figure 4 that the experimental data obtained at acidic pH for the forward peak follows the predicted response quite well on approaching the region near the pI. The experimental data do not follow the predicted response at pH values above the isoelectric point, demonstrating that the voltammetric response is directly related to the interaction of cationic insulin at the ITIES. As previously discussed, both pH and concentration can dictate the preferred association state of insulin. The hexameric form of insulin found at pH 7.4 dissociates on going to acidic pH values, ultimately leading to dimeric/monomeric forms at pH 1.6/2.0.40 Low concentrations in solution also favor the presence of monomeric insulin.38 Optimal detection of insulin was observed in acidic solutions in the range pH 1.0-2.0, using HCl to adjust the solution pH, indicating a preferred tendency for insulin to interact at the polarized interface in monomeric and/or dimeric form. The low concentrations employed further suggest that insulin behavior at the ITIES is dominated by fully protonated monomeric/dimeric species. The fact that optimal detection was obtained at acidic pH values, under which the insulin molecule is positively charged, supports the view that the transfer mechanism is insulin interaction with the organic phase anion, tetrakis(4-chlorophenyl)borate, via the formation of an ion-pair that drives the transfer of the anion from the organic to aqueous phase. It was observed on approaching the pI (pH of 5.0-6.0) that it was difficult to measure the transfer peaks around this pH region, causing deviations from the general decreasing trend shown in Figure 4. The pI is also the pH at which solubility of a protein is minimal and may therefore precipitate. A low solubility and subsequent precipitation of insulin between pH values of 4 and 7 has been reported67 in conditions very similar to those of the present work. A pictorial example of this is shown in Figure 5. Precipitation of insulin in the aqueous phase resulting in a cloudy appearance of the electrolyte was observed at pH 5.07.0, which collected mainly in the vicinity of the interface during (68) Hosoya, O.; Chono, S.; Saso, Y.; Juni, K.; Morimoto, K.; Seki, T. J. Pharm. Pharmacol. 2004, 56, 1501.

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Figure 6. Effect of pH on the voltammetric response. (Aq): 10 mM LiCl + 50 µM insulin + 0.2 mM TEA+. (O): 10 mM BTPPATPClB. Scan rate, 5 mV s-1. Table 1. Evaluation of TEA+ Transfer under Various pH Conditions, in the Presence of Insulin pH

∆Ep, mV

1 2.6 4.17 5.2 7.4 9.7

66 73 118 90 62 68

experiments upon polarization and forming what appears to be an adsorbed layer at the interface. As well as a decrease in peak current, a shift to more positive potentials with increasing pH was observed for the transfer peaks, as shown in Figure 6. For example, peak potentials (when adjusted to the Galvani scale) shifted from 0.334 V (forward) and 0.221 V (reverse) at a pH of 1.0, to 0.427 V (forward) and 0.257 V (reverse) at a pH of 5.2. These data show that the transfer process is easiest to achieve at lower pH conditions when insulin is protonated. Simple ion transfer of TEA+ 61,69–71 was also studied in the presence of insulin at different pH values of the aqueous phase. While the addition of TEA+ had no effect on the voltammetric response to insulin, the opposite was not the case. Table 1 summarizes the influence of insulin on the TEA+ electrochemistry at different pH values of the aqueous phase. Reversible TEA+ transfer occurred within the range of pH 1-2, but peak separation ∆E p increased (>60 mV) at higher pH values. Within the pH range 7-11 where there is no detection of transfer peaks related to insulin, TEA+ transfer was again reversible. This suggests that when insulin is least soluble, close to its pI, it has an adverse effect on the reversible transfer of TEA+, perhaps by creating a partially blocked ITIES by buildup of precipitate at the interface. Effect of the Supporting Organic Phase Electrolyte. Shinshi et al. have reported on the electrochemical extraction of proteins such as cytochrome c and protamine by complexation with an anionic surfactant, bis(2-ethylhexyl) sulfosuccinate (AOT), at the polarized water-1,2-DCE interface.53 In the absence of AOT in the organic phase, the voltammetric response for cytochrome c was quite similar to that obtained for insulin in our experiments (although at lower potentials). It was suggested however that the (69) Abraham, M. H.; Namor, A. F. D. d. J. Chem. Soc., Faraday Trans. 1976, 72, 955. (70) Wandlowski, T.; Marecek, V.; Samec, Z. Electrochim. Acta 1990, 35, 1173. (71) Samec, Z.; Langmaier, J.; Trojanek, A. J. Electroanal. Chem. 1996, 409, 1.

Electrochemical Study of Insulin

response was not due to the transfer of cytochrome c, but to the facilitated transfer of the tetraphenylborate anion of the supporting organic electrolyte (TPB-) via adsorption of cytochrome c at the aqueous side of the interface. The effect of the nature of the membrane or organic phase ion has also been seen in studies of other biological polyions, such as protamine and heparin.56–58 Potentiometric studies have shown that lipophilic quaternary ammonium species, (i.e., tridodecylmethylammonium; TDMA+) can bind to heparin, a large polyanion with RMM ∼15 000 g mol-1. A similar potentiometric sensor for protamine, which has a net charge of 20+ at physiological pH or lower,72 was developed by incorporating tetrakis(4-chlorophenyl)borate (TPClB-) into the polymer membrane to serve as the protein carrier. Based on the data presented in this paper, one of the possible processes taking place is that the insulin at the ITIES serves as a facilitator for the transfer across the interface of anionic electrolyte species from the organic phase. Experiments were performed to assess the effect of the organic electrolyte on the voltammetric response to insulin. Bis(triphenylphosphoranylidene) ammonium tetrakis(4-fluorophenyl)borate (BTPPATPFB) and bis(triphenylphosphoranylidene) ammonium tetraphenylborate (BTPPATPB) were investigated as organic electrolytes, with the results compared to that obtained for BTPPATPClB. The voltammetric response to insulin in the case of each anion present in the organic phase is shown in Figure 7. Measurements were all recorded under similar pH conditions, within the range pH 3.11-3.17. Both the forward and reverse peaks obtained upon addition of insulin to the aqueous phase show a dependency on the nature of the hydrophobic anion present in the organic supporting electrolyte. The peak potentials shifted to lower values for both forward and reverse peaks, following the trend ETPFB- > ETPClB> ETPB- (where E refers to a transfer peak potential in the presence of the indicated anion in the organic phase). This suggests that the voltammetric response is due to the interaction between the hydrophobic anion in the organic phase and cationic insulin in the aqueous phase. The transfer potentials of forward and reverse peaks decreased with decreasing hydrophobicity of the anion, which is consistent with the formation of a complex between cationic insulin and the respective anion. The net charge (Q) measured under the reverse peaks were quite similar in the case of TPFB- and TPClB- (8.64 × 10-5 C and 8.86 × 10-5 C, respectively). In the case of TPB-, however, Q was 2.24 × 10-5 C. This shows that insulin does not interact with the less hydrophobic TPB- anion as much as with TPFB- and TPClB-. These data illustrate that as the organic anion becomes more hydrophobic, the transfer process requires more energy but that the extent of interaction (i.e., the amount of anion associating with the insulin) is greater, perhaps due to insulin’s hydrophobic sites being occupied to a greater extent by the more hydrophobic anion. These results clearly show that the voltammetric response for insulin is very much dependent on the nature of the organic phase electrolyte. The data presented in Figure 7 is similar to that reported by Troja´nek et al. for the study of counterion binding to protamine at the polarized aqueous-1,2-DCE interface.51 A decrease in the stability of the protamine-organic anion ionpair with increasing anion size was reported in the order TPB> TPClB- > TPFB-, with which our experimental data agrees. Interfacial tension measurements reported by Troja´nek et al.51 showed that the adsorption of protamine at the liquid-liquid interface was electrostaticsa direct result of the attraction between (72) Ando, T.; Yamasaki, M.; Suzuki, K., Protamines: Isolation, Characterization, Structure and Function; Springer-Verlag: New York, 1973.

Langmuir, Vol. 24, No. 17, 2008 9881

Figure 7. CV response, (Aq): 10 mM LiCl + 50 µM insulin pH 3.11-3.17. (O): (a) 10 mM BTPPTPFB, (b) 10 mM BTPPATPClB and (c) 10 mM BTPPATPB in 1,2-DCE. Scan rate, 5 mV s-1.

positively charged protamine molecules and negatively charged ions in the organic phase. The shift in transfer potential with different organic electrolytes observed in this work demonstrates that the voltammetric response is due to ion-pair interactions between positively charged insulin and the organic anion at the polarized liquid-liquid interface. As discussed in the scan rate studies above, peak (2) on the forward scan (Figure 3) can be explained by the facilitated transfer of TPClB- by insulin from the organic phase, via the formation of an ion-pair complex. Peak (3) is due to subsequent stripping of organic phase anion from the ion-pair complex at the interface. Detection Range. A working concentration range of 10-50 µM insulin was easily established using CV. Along with CV experiments, stripping voltammetry was employed in order to obtain a voltammetric response at lower concentrations of insulin. Figure 1 shows CV results obtained at pH 2.85 for increasing concentrations of insulin. A voltammetric response was observed

9882 Langmuir, Vol. 24, No. 17, 2008

Figure 8. (A) SWSV response, (Aq): (a) 10 mM LiCl (0.02% Tween80) + (b) 1 µM; (c) 2 µM; (d) 3 µM; (e) 5 µM insulin pH ) 1.67. (O): 10 mM BTPPATPClB. Deposition at 0.7 V for 180 s. Square wave parameters: Potential increment: 0.004 V; Amplitude: 0.01 V; Frequency: 5 Hz. (B) SWSV response with background extraction performed, (Aq): (a) 10 mM LiCl (0.02% Tween80) + (b) 1 µM; (c) 2 µM; (d) 3 µM; (e) 5 µM insulin pH ) 1.67. (O): 10 mM BTPPATPClB. Deposition at 0.7 V for 180 s. Square wave parameters: Potential increment: 0.004 V; Amplitude: 0.01 V; Frequency: 5 Hz.

for insulin concentrations as low as 2.5 µM. A linear correlation between transfer current and concentration of insulin was also observed, (inset of Figure 1). Squarewave stripping voltammetry (SWSV) experiments resulted in slightly lower detection of transfer peaks down to a concentration of 2 µM insulin. These experiments were performed with a constant background concentration of Tween80 surfactant, with results shown in Figure 8. Additions of insulin result in the appearance of a peak at ∼0.5 V from as low a concentration of 2 µM, which correlates well with the sharp bell-shaped peak previously observed on the reverse of scan of CV experiments. Figure 8b shows SWSV results obtained after background subtraction was performed, which served to lower the detection capability down to 1 µM of insulin. Further optimization of the waveform parameters should yield an improvement on this detection limit.

Conclusions The voltammetric study of insulin at the polarized water-1,2DCE interface has been presented in this paper. The effect of pH on the voltammetric response to insulin was in agreement with its pI and acid-base chemistry. Optimal detection was observed

KiVlehan et al.

under acidic conditions, with the transfer current decreasing to eventually no transfer under basic conditions. These results together with the already known effect pH has on the charge of insulin show that the voltammetric response obtained is largely dependent on the protein’s interaction at the polarized interface when positively charged. Scan rate studies indicate a diffusioncontrolled transfer process with an adsorption prewave on the forward scan, indicative of the adsorption of the product of the electrochemical process. Investigation of the effect of the organic supporting electrolyte showed that the voltammetric response was dependent on the nature of the hydrophobic anion employed in the organic phase. A shift to more negative transfer potentials was observed with decreasing hydrophobicity of the organic anion. It is therefore proposed that the voltammetric response is due to insulin-facilitated anion transfer and ion-pairing interactions between positively charged insulin and the hydrophobic anion of the organic phase. The formation of an adsorbed layer at the aqueous side of the polarized interface was generally observed during the course of this work such as that shown in Figure 5, where the adsorption process is readily seen due to a low solubility of insulin within a pH range of 5.0-7.0. This cloudiness close to the interface may be associated with the proximity of the organic phase, which is known to promote fibril formation.46,48 A detection limit of 2.5 µM was obtained using cyclic voltammetry and improved to 1 µM using stripping voltammetry. Although insulin is not the transferring species, it can be detected by this approach when the other reagent (organic anion in this case) is in excess. The fact that insulin interacts with organic anions as the basis of its detection at the ITIES may be reliant on insulin’s wellknown hydrophobic regions and ability to assemble into supramolecular complexes. Low pH and low concentrations in solution are known to favor insulin present in monomeric or dimeric forms in which the hydrophobic regions of the molecule are not buried and thus open to interaction with the interface and the organic anions located there. At higher pH values, insulin is known to associate into e.g. hexameric forms, with the hydrophobic regions buried and not accessible. The ability to detect insulin using the ITIES approach to electrochemistry offers new schemes for detection in bioanalytical applications, although the inability to detect insulin at a physiological pH creates problems for application of this electrochemical behavior to the direct detection of insulin in blood samples. Detection limits achieved with the present experimental system imply that an optimization of all experimental parameters is required in order to detect physiological levels of insulin, which are usually in the nM-pM concentration range. Improved performance can be achieved using microinterfaces as well as configuring it for use in flow-analytical or liquid chromatographic systems. Overall, use of this electrochemistry has proved to be successful in uncovering the behavior of this important protein molecule at liquid-liquid interfaces and may provide the basis for further advances in its detection. Acknowledgment. This work was supported by Science Foundation Ireland (Grants No. 02/IN.1/B84 and 07/IN.1/B967) and the Irish Research Council for Science Engineering and Technology (Grant No. RS/2004/74). LA800842F