Dodecylbenzenesulfonate

3 Jun 2005 - Different aspects of mixtures of decanoic acid and sodium decanoate were investigated in aqueous solution up to a total concentration of ...
51 downloads 0 Views 638KB Size
Download PDF
6210

Langmuir 2005, 21, 6210-6219

From Decanoate Micelles to Decanoic Acid/ Dodecylbenzenesulfonate Vesicles Trishool Namani and Peter Walde* Department of Materials, ETH-Ho¨ nggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zu¨ rich, Switzerland Received December 3, 2004. In Final Form: April 27, 2005 Different aspects of mixtures of decanoic acid and sodium decanoate were investigated in aqueous solution up to a total concentration of 300 mM. Depending on the ratio of ionized to nonionized decanoic acid, micelles or vesicles form above the critical concentrations of micelle (cmc) or the critical concentration for vesicle formation (cvc). The micelles and the vesicles are always present together with nonmicellized or nonvesiculized decanoate. The latter was determined for different total concentrations. On the basis of titration curves, by application of the Gibbs phase rule, and on the basis of differential scanning calorimetry measurements and an electron microscopy analysis, the pH region within which vesicles exist was identified (pH 6.8-7.8). At pH 7.0, the concentration of nonvesiculized decanoate is ∼20 mM. Decanoic acid/decanoate vesicles can be sized down by the extrusion technique to form stable and mainly unilamellar vesicles with a mean diameter of less than 100 nm. By coaddition of an equimolar amount of sodium dodecylbenzenesulfonate (SDBS) to decanoic acid, vesicles also formed below pH 6.8. These mixed vesicles were investigated as potential templates for the peroxidase-catalyzed polymerization of aniline at pH 4.3. Furthermore, decanaote micelles (at pH 11.0) were applied as reaction modifiers for the simultaneous competitive alkaline hydrolysis of p-nitrophenylacetate and fluorescein diacetate. While the rate of hydrolysis of fluorescein diacetate is slowed considerably in the presence of the micelles in comparison with the micelle-free system, the rate of hydrolysis of p-nitrophenylacetate remains almost unaffected.

Introduction From a chemical point of view, decanoic acid, H3C(CH2)8-COOH, is a simple amphiphilic molecule with a polar headgroup that can be in its protonated (-COOH) or deprotonated (-COO-) state, depending on the experimental conditions. Aqueous mixtures of decanoic acid and potassium (or sodium) decanoate show a complex aggregation behavior. Depending on the relative amounts of decanoic acid, decanoate, and water, different phases form at thermodynamic equilibrium, just like in the case of the more intensively studied octanoic acid/sodium octanoate/water system.1,2 In dilute alkaline solution and at room temperature, decanoate micelles start to form at the critical micellization concentration (cmc).3 At intermediate pH, the decanoate and decanoic acid molecules self-organize into bilayers (vesicles)4-6 above the critical concentration for bilayer (vesicle) formation (abbreviated as cbc4b or cvc6). Bilayers form if about half of the molecules are present in anionic form and half of the molecules are present in protonated, nonionic form.4-6 * To whom correspondence should be addressed. Phone: +41(0)44-63 20473. Fax: +41-(0)44-63 212 65. E-mail: peter.walde@ mat.ethz.ch. (1) (a) Ekwall, P.; Holmberg, P. Acta Chem. Scand. 1965, 19, 455468. (b) Ekwall, P.; Mandell, L.; Fontell, K. Acta Chem. Scand. 1968, 22, 697-699. (c) Ekwall, P.; Mandell, L. Kolloid Z. Z. Polym. 1969, 233, 938-944. (d) Fontell, K.; Mandell, L. Colloid Polym. Sci. 1993, 271, 974-991. (2) Laughlin, R. G. The Aqueous Phase Behavior of Surfactants; Academic Press: London, 1994. (3) McBain, J. W.; Salmon, C. S. J. Am. Chem. Soc. 1920, 42, 426460. (4) (a) Hargreaves, D.; Deamer, D. W. Biochemistry 1978, 17, 37593768. (b) Monnard, P.-A.; Deamer, D. W. Methods Enzymol. 2003, 372, 133-151. (c) Apel, C. L.; Deamer, D. W.; Mautner, M. N. Biochim. Biophys. Acta 2002, 1559, 1-9. (5) (a) Cistola, D. P.; Atkinson, D.; Hamilton, J. A.; Small, D. M. Biochemistry 1986, 25, 2804-2812. (b) Cistola, D. P.; Hamilton, J. A.; Jackson, D.; Small, D. M. Biochemistry 1988, 27, 1881-1888. (6) Morigaki, K.; Walde, P.; Misran, M.; Robinson, B. H. Colloids Surf., A. 2003, 213, 37-44.

The present paper first reports on detailed investigations of dilute decanoic acid/decanoate systems, focusing particularly on the concentration dependency of the bilayer formation and on the possible application of micelles and decanoic acid based vesicles as submicrometer- and micrometer-sized reaction systems. In the case of vesicles, we were particularly interested in finding conditions for vesicle formation that allow us to apply these vesicles for enzmye-catalyzed reactions. There are some drawbacks in using decanoic acid/ decanoate vesicles as reaction systems, e.g., the narrow pH range for vesicle formation (pH 6.4-7.8) and the relatively high concentration of nonaggregated decanoate molecules (∼20 mM at pH 7). To extend the pH range for vesicle formation to a lower pH, mixed decanoic acid/ sodium dodecylbenzenesulfonate (SDBS) vesicles were also investigated. Preliminary results on the use of these novel vesicle systems as templates for the enzymecatalyzed polymerization of aniline are presented at the end of the paper. Experimental Section Materials. Decanoic acid (capric acid, 99%), pyrene (>99%), merocyanine 540, arsenazo III, aniline (99.5%), hydrogen peroxide (30%, puriss.), p-nitrophenylacetete (puriss.), p-nitrophenyloctanoate (>97%), fluorescein diacetate (>98%), sodium dodecyl sulfate (SDS, g99%), 1,6-diphenylhexatriene (DPH), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), sodium 1-decanesulfonate (g99%), and horseradish peroxidase (560 U/mg) were purchased from Fluka (Switzerland). Pinacyanol chloride and sodium decanoate (>99%) were from Sigma (Switzerland). Sodium dodecylbenzenesulfonate (hard type, >95%, SDBS) was from TCI Europe (Zwijndrecht, Belgium). POPC (1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine) was from Avanti Polar Lipids (Alabaster, AL). The cmc Determinations. Four different spectroscopic methods for the determination of the cmc were used, basically following the description in the appropriate literature. The

10.1021/la047028z CCC: $30.25 © 2005 American Chemical Society Published on Web 06/03/2005

Decanoic Acid Micelles and Vesicles procedures used were either 13C NMR measurements7 or they were based on using pinacyanol chloride,8,9 pyrene,10 or DPH,11 as probe molecules. In the case of pinacyanol chloride, the dye concentration was kept constant at 3 µM (added as a 0.35 mM solution in methanol) and the absorbance measurements were carried out at 605 nm. For the measurements with pyrene, a concentration of 2 µM was used (added as a 4 mM solution in DMSO). The excitation wavelength was 332 nm, and the emission was recorded at 373 and 384 nm. In the case of DPH, the concentration was 5 µM (added as 10 mM solution in THF). The excitation was at 358 nm, and the emission was at 430 nm. The cvc Determinations. (a) Based on Sample Turbidity. The cvc was determined by measuring the optical density at 350 nm of a number of samples prepared separately at fixed pH in a buffered solution (100 mM sodium phosphate buffer) and at various total decanoic acid + decanoate concentrations. The cvc is defined as the lowest concentration at which turbidity could be measured. (b) Based on the Spectrophotometric Properties of Merocyanine 540. A series of decanoic acid/sodium decanoate samples (each 1 mL) were prepared in 100 mM sodium phosphate buffer (pH 7). After addition of 1 µL of a 10 mM merocyanine 540 solution prepared in ethanol, the visible absorption spectrum was recorded and the ratio of optical density (OD) at 570 nm (monomer) over optical density at 530 nm (dimer) was determined. A discontinuity in the OD570/OD530 vs [decanoic acid + decanoate] was taken as indication of the formation of vesicles at the discontinuity point. Ultraviolet/Visible, Near-Infrared, Fluorescence, and 13C NMR Measurements. Absorption measurements in the ultraviolet and visible ranges were carried out with a Cary 1E spectrophotometer from Varian (Australia), using quartz cells with a path length of 1 cm. For measurements in the near-infrared (NIR) and in the visible region, a Lambda 19 spectrometer from Perkin-Elmer was used. Fluorescence measurements were performed with a Spex Fluorolog 2 from Jobin Yvon (U.K.) with 1 cm quartz cells. The 13C NMR spectra were recorded on an AVANCE 300 MHz spectrometer from Bruker. Ester Hydrolysis Reactions in the Absence and Presence of Micelles. The alkaline hydrolysis of the three substrates p-nitrophenylacetate, p-nitrophenyloctanoate, and fluorescein diacetate were followed at 25 °C spectrophotometrically at 400 nm (release of p-nitrophenolate) and 490 nm (formation of fluorescein), respectively. The reactions were initiated by adding 0.1 mL of a DMSO substrate solution (either 0.8 mM pnitrophenylacetate or 0.8 mM p-nitrophenyloctanoate or 0.2 mM fluorescein diacetate) to 1.9 mL of 120 mM decanoate solution (pH 11, prepared in 50 mM phosphate). Control measurements were carried out in the absence of decanoate. For the competitive reaction experiments p-nitrophenylacetate and fluorescein diacetate were added simultaneously. In the case of p-nitrophenyloctanoate, the total decanoate concentration was varied between 0 and 180 mM in order to elaborate an alternative estimation of the cmc. Titration Experiments. Titration curves were obtained by first dissolving 1 M decanoic acid in water in the presence of a 10 mol % excess of NaOH. This stock solution was then diluted to the desired concentration, and a number of samples were prepared that contained a fixed decanoic acid + decanoate concentration and a defined amount of added 1 N HCl. After equilibration for 3 days at room temperature, the pH was measured with an InLab 423 pH electrode from Mettler Toledo (Switzerland).12 In the case of acetic acid as control, the same procedure was carried out. (7) Drakenberg, T.; Lindman, B. J. Colloid Interface Sci. 1973, 44, 184-186. (8) Mukerjee, P.; Mysels, K. J. J. Am. Chem. Soc. 1955, 77, 29372943. (9) Menger, F. M.; Portnoy, C. E. J. Am. Chem. Soc. 1967, 89, 46984703. (10) Ananthapadmanabhan, K. P.; Goddard, E. D.; Turro, N. J.; Kuo, P. L. Langmuir 1985, 1, 352-355. (11) Chattopadhyay, A.; London, E. Anal. Biochem. 1984, 139, 408412. (12) The use of electrodes in surfactant solutions to measure the pH (with pH electrodes) or the monomeric surfactant concentration (with surfactant-specific electrodes) may be influenced by interactions of the surfactants with the electrodes. Bloor, D.; Gray, J.; Hughes, J.; Tiddy, G. J. T. Langmuir 2001, 17, 6127-6131.

Langmuir, Vol. 21, No. 14, 2005 6211 Differential Scanning Calorimetry (DSC) Measurements. DSC measurements were performed with a Perkin-Elmer DSC 7 instrument in the range of -10 to 50 °C. The aluminum pan was filled with 20 µL sample. After equilibration at -10 °C for 30 min, the sample pan was heated at a heating rate of 5 °C/min. Empty pans were used as reference. Ultrafiltration Experiments. For the determination of the concentration of monomeric decanoate coexisting with the vesicles, 1 mL of the vesicle suspensions prepared for the titration experiments were treated by ultrafiltration using Centricon-10 microconcentrators (Amicon, Beverly, MA) that had membrane filters with a molecular weight cutoff of 10 000 Da. The samples were centrifuged for 1 h at 2000 rpm in an Hermle Z-320K centrifuge. The concentration of decanoate in the ultrafiltrate was analyzed by FTIR as described before.6 Vesicle Extrusion and Sonication. Large unilamellar vesicles (LUV) were prepared by the extrusion technique (ET) using The Extruder from Lipex Biomembranes (Vancouver, Canada) and Nucleopore polycarbonate membranes from Sterico (Dietikon, Switzerland).13 The vesicle suspensions were first passed 10 times through membranes with 400 nm pores, followed by extrusion 10 times through 200 nm and finally 100 nm membranes. The resulting vesicle suspension is abbreviated as LUVET100. Probe sonications of the vesicle suspensions were performed at room temperature by using a Branson Sonifier 250. The sonication time was 1-2 min. Dynamic Light Scattering and Electron Microscopy. Dynamic light scattering measurements were carried out on a Zetasizer 5000 from Malvern Instruments (U.K.) at three different scattering angles (60°, 90°, 120°). Because of the high concentration of nonassociated surfactant molecules, dilution of vesicle suspensions for dynamic light scattering measurements were avoided to prevent possible dilution-induced vesicle disintegrations. Freeze-fracture electron microscopy analysis of the vesicle suspensions were carried out by Dr. Martin Mu¨ller and Dr. Ernst Wehrli at the Institute of Applied Physics, ETH Zu¨rich, as described before.14 Dye Entrapment Experiments. For the entrapment of the water-soluble dye arsenazo III inside the vesicles, the dye was present at a concentration of 5 mM during vesicle formation. After extrusion (or sonication), nonentrapped dye molecules were separated from the vesicles by sepharose 4B (from Amersham Biosciences, Switzerland). The column length was 25 cm, and the diameter was 1.2 cm. Enzyme-Catalyzed Polyaniline Synthesis and Product Characterization. Aniline was polymerized at pH 4.3 (100 mM phosphate buffer) as follows:15 To 10 mL of buffer solution containing decanoic acid/SDBS vesicles (1:1, molar ratio, total concentration of 10 mM) were added 0.5 mL of 100 mM aniline (in phosphate buffer) and 0.05 mL of a 5 mg/mL peroxidase solution (prepared in phosphate buffer). After the sample was mixed, the reaction was started by adding 0.05 mL of a 100 mM hydrogen peroxide solution (prepared in phosphate buffer). The visible/NIR spectrum was recorded after 20 min using a 1 cm quartz cell. For comparison, the reaction was also performed in the absence of vesicles.

Results and Discussion Determination of the cmc of Decanoate Micelles. The cmc for decanoate was determined at room temperature (22-25 °C) by using different methods: (a) by 13C NMR spectrometry, (b) by using pinacyanol chloride, and (c) by using pyrene as probe molecules. (d) Furthermore, the cmc was evaluated from the decanoate concentration (13) (a) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. (b) Mui, B.; Chow, L.; Hope, M. J. Methods Enzymol. 2003, 367, 3-14. (c) Walde, P. In Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S., Ed.; American Scientific Publishers: Stevenson Ranch, CA, 2004; Vol. 9, pp 43-79. (14) (a) Mu¨ller, M.; Meister, M.; Moor, H. Mikrosopie 1980, 36, 129140. (b) Egelhaaf, S. U.; Wehrli, E.; Mu¨ller, M.; Adrian, M.; Schurtenberger, P. J. Microsc. 1996, 184, 214-228. (15) (a) Liu, W.; Kumar, J.; Tripathy, S.; Samuelson, L. A. Langmuir 2002, 18, 9696-9704. (b) Liu, W.; Kumar, J.; Tripathy, S.; Senecal, K. J.; Samuelson, L. J. Am. Chem. Soc. 1999, 121, 71-78.

6212

Langmuir, Vol. 21, No. 14, 2005

Namani and Walde

Figure 2. Schematic drawing of two competitive hydrolysis reactions occurring in an alkaline micellar solution. Because of a different partitioning between the bulk aqueous medium and the decanoate micelles, the reaction rates for the two substrates S1 and S2 are influenced differently. S1 has a lower affinity for the micelles than S2 has. The reaction products are P1 and P2 for S1 and P3 and P4 for S2: S1, p-nitrophenylacetate; S2, fluorescein diacetate; P1, acetate; P2, p-nitrophenolate; P3, acetate; P4, fluorescein.

Figure 1. The cmc determination of sodium decanoate by 13C NMR measurements (a) and by using pinacyanol chloride as probe molecule (b). In (a), the chemical shifts of the carbonyl carbon (C1, O) and of the terminal carbon (C10, b) are plotted. In (b), the absorbance of 3 µM pinacyanol chloride at 605 nm is plotted. The dotted lines indicate the cmc as estimated from these measurements. The samples were prepared in 50 mM sodium phosphate, pH 11.5.

dependency of the rate of hydrolysis of p-nitrophenyloctanoate (see below). All these methods indicate that sodium decanoate micelle formation occurs below 100 mM, independent of whether the measurements were done in the presence of 50 mM phosphate at pH 11.5 or just in water. Figure 1 shows experimental data obtained by the 13 C NMR analysis (Figure 1a) and by using pinacyanol chloride (Figure 1b). Both types of measurements indicate that micelle formation starts at about 50 mM. For a comparison, earlier literature values are listed in Table 1, together with the results obtained in the present study. With DPH as a neutral probe molecule, the cmc determination is not straightforward (see Supporting Information). DPH is insoluble in aqueous solution, and the principle of the method is based on the fact that the formation of micelles leads to a solubilization of DPH, as evidenced by recording the fluorescence of DPH. It is likely that DPH induces the formation of decanoate micelles below the actual cmc (“premicelles”). Furthermore, DPH is a rather bulky probe molecule. However, the “DPH method” works well with a number of sufactants,11 and we have confirmed it with our own measurements for SDS (cmc ) 9 mM), sodium decylsulfonate (cmc ) 40 mM), and SDBS (cmc ) 0.2 mM). The corresponding plots are given in Supporting Information (Figures S-1 to S-3), together with the data for sodium decanoate (Figure S-4). The cmc of ∼50 mM that we have determined for sodium decanoate by 13C NMR, with pinacyanol chloride or pyrene,

or by following the hydrolysis of p-nitrophenyloctanoate (see later on Figure 3b) agrees well with the cmc value determined recently for ammonium decanoate (Table 1). The earlier literature values for sodium decanoate tend to be higher (see Table 1).16 Competitive Chemical Reactions in the Presence of Decanoate Micelles. Micelles often influence chemical reactions.9,21,22 In the case of dodecanoate micelles, for example, it has been shown that the rate of hydrolysis of p-nitrophenyloctanoate at alkaline pH is decreased because p-nitrophenyloctanoate is taken up by the micelles, away from the hydroxide ions in the bulk aqueous medium.9 The negatively charged micelles repel the HOions, thereby decreasing the chance that a p-nitrophenyloctanoate molecule meets a hydroxide ion, leading to a slowing of the reaction.9 We have applied and extended this principle to the smaller decanoate micelles by studying two competitive hydrolysis reactions simultaneously: the alkaline hydrolysis of p-nitrophenylacetate and the alkaline hydrolysis of fluorescein diacetate. The idea is illustrated in Figure 2. Both substrates S1 ()p-nitrophenylacetate) and S2 (fluorescein diacetate) are partitioned between the bulk aqueous phase and the micelle. The binding properties of the two substrates to the micelles are different because of the different chemical structures of S1 and S2. S1 reacts with HO- to yield P1 (acetate) and P2 (p-nitrophenol). S2 reacts with two molecules of HOto give two molecules of P3 (acetate) and one molecule fluorescein (P4). P2 and P4 can be monitored easily by recording the absorption spectrum in the visible range (Figure 3); the absorption maximum is at 400 and 490 nm, respectively. If one compares the reaction rates (VS1 (16) It is well-established that the experimentally determined cmc may depend on the method of measurement, in particular if dyes and fluorescent molecules are used. Patist, A. In Handbook of Applied Surface and Colloid Chemistry; Holmberg, K., Shah, D. O., Schwuger, M. J., Eds.; John Wiley & Sons, Ltd.: Chichester, U.K., 2002; Vol. 2, pp 239249. For sodium decanoate, a cmc of about 100 mM is often tabulated. See, for example, the following: Holmberg, K.; Jo¨nsson, B.; Kronberg, B.; Lindman, B. Surfactants and Polymers in Aqueous Solution, 2nd ed.; John Wiley & Sons: Chichester, U.K., 2003; p 43. (17) Huang, H.; Verrall, R. E. J. Solution Chem. 1997, 26, 135-162. (18) Campbell, A. N.; Lakshminar, G. R. Can. J. Chem. 1965, 43, 1729-1737. (19) Burkitt, S. J.; Ottewill, R. H.; Hayter, J. B.; Ingram, B. T. Colloid Polym. Sci. 1987, 265, 619-627. (20) Clapperton, R. M.; Ottewill, R. H.; Rennie, A. R.; Ingram, B. T. Colloid Polym. Sci. 1999, 277, 15-24. (21) Bunton, C. A.; Gillitt, N. D.; Mhala, M. M.; Moffatt, J. R.; Yatsimirsky, A. K. Langmuir 2000, 16, 8595-8603. (22) Rispens, T.; Engberts, J. B. F. N. J. Org. Chem. 2002, 67, 73697377.

Decanoic Acid Micelles and Vesicles

Langmuir, Vol. 21, No. 14, 2005 6213

Table 1. The cmc Value Determined for Decanoate by Using Different Methods under Different Experimental Conditionsa conditions

method

cmc

remarks

Sodium Decanoate ∼50 mM ∼50 mM not clearly DPH also solubilized below detectable the cmc (see text)

H2O, room temp

pinacyanol chloride pyrene DPH

50 mM sodium phosphate, pH 11.5, room temp

pinacyanol chloride 13C NMR p-nitrophenyloctanoate hydrolysis

40-55 mM 50-60 mM 60 mM

50 mM borate, pH 9.0, 25 °C

pinacyanol chloride conductivity

60 mM 80 mM

pH 11.8, adjusted with NaOH, 25 °C

conductivity

102 mM

pH > 9.5, adjusted with NaOH, 25 °C

apparent molar volume

110 mm

25 °C

conductivity and surface tension

94 and 96 mM

NH4Cl/NH4OH, pH 8.8, I ) 0.1, 25 °C

surface tension

NH4Cl/NH4OH, pH 9.2, I ) 0.1, 25 °C

turbidity

this work

Morigaki et al.6

at 0.299 mm: Nagg ) 34 (71 Å2/headgroup)

Huang and Verrall17 Campbell and Lakshminar18

Ammonium Decanoate 41 mM from SANS at 150 mM: spherical micelles, Nagg ) 73, radius ) 17.8 Å 55 mM

reference

from TLS: Nagg ) 54; radius ) 16.6 Å (64 Å2/headgroup)

Burkitt et al.19

Capperton et al.20

a Abbreviations used: SANS, small angle neutron scattering; TLS, time-average light scattering; N agg, mean aggregation number, number of surfactant molecules per micelle.

Table 2. Alkaline Hydrolysis of p-Nitrophenylacetate (40 µM), p-Nitrophenyloctanoate (40 µM), and Fluorescein Diacetate (10 µM) at pH 11.0 (50 mM Sodium Phosphate)a time needed for 50% hydrolysis (s)b

substrate p-nitrophenylacetate p-nitrophenyloctanoate fluorescein diacetate p-nitrophenylacetate + fluorescein diacetate

no decanoate, pH 11.0

120 mM sodium decanoate, pH 11.0

46 ( 1 138 ( 1 408 ( 43 8621 ( 764 111 ( 3 3049 ( 181 42 ( 1 116 ( 2 141 ( 4 3796 ( 385

a The time needed to hydrolyze 50% of the initial substrate is reported, as determined in measurements either in the absence of surfactant or in the presence of 120 mM sodium decanoate (mean value and standard deviations from three measurements are given). b If two values are given, the first one is for the first substrate listed. The second value is for the second substrate listed.

and VS2) in the (micelle-free) aqueous solution with the reaction rates in the micellar system, then one obtains VS1aqueous/VS2aqueous < VS1micellar system/VS2micellar system (Table 2). This means that the specificity of the reaction is controlled in first approximation by the different partitioning of the reactants between the aqueous phase and the micelles, thereby controlling the reaction product distribution during the course of the reaction. Table 2 compares the reaction times for 50% hydrolysis (t50%) for the different systems investigated, including also data for the hydrolysis of p-nitrophenyloctanoate. While under the conditions used, 50% of the p-nitrophenylacetate molecules are hydrolyzed in aqueous solution after 4246 s, and in the presence of 120 mM sodium decanoate, it takes about 140 s. In the case of fluorescein diacetate, 50% yield is reached after 110-120 s in bulk solution, while in the presence of 120 mM sodium decanoate, 50% substrate is hydrolyzed after 3000-4000 s (see Table 2). With the more hydrophobic p-nitrophenyloctanoate, t50%

in the absence of decanoate is about 400 s, and in the presence of 120 mM sodium decanoate, t50% is about 9000 s (see Table 2). In summary, under the conditions used, the order of efficiency in inhibiting the hydrolysis reactions by decanoate micelles is p-nitrophenyloctanoate > fluorescein diacetate > p-nitrophenylacetate. The presence of micelles leads to a change in the relative reaction velocities as discussed above. The chosen two-substrate model system allowed us to follow the effect of decanoate micelles on the ester hydrolysis simultaneously by simple spectrophotometric measurements. The Formation of Decanoic Acid/Decanoate Vesicles. If HCl is added to an alkaline solution of sodium decanoate, some of the decanoate molecules get protonated until mixed decanoate/decanoic acid bilayers (vesicles) form.4-6 At about 0.5 equiv of HCl, the dominating state of aggregation of the surfactant is vesicles. Figure 4 shows corresponding titration curves for different total decanoic acid + decanoate concentrations (50-300 mM). For comparison, the titration curve of acetic acid/acetate (200 mM) is also shown (pKa of acetic acid is ∼4.7). While all acetic acid/acetate solutions are completely transparent over the entire titration range and the pH steadily decreases with added HCl, the decanoic acid/decanoate samples get turbid as soon as the degree of protonation reaches a certain limit, indicative of the formation of large aggregates (mainly multilamellar vesicles) that scatter visible light. The formation of vesicles is shown by freezefracture electron microscopy (see below). This occurs around pH 8.0-8.1 as indicated with the dashed line in Figure 4a. If the pH is decreased below 6.4-6.0, phase separation is observed (formation of precipitates), as indicated in Figure 4 with the patterned horizontal bar. Figure 4b is a close-up of the pH 6.5-8.5 region of the titration curves. The titration curves in Figure 4 indicate that the apparent pKa of decanoic acid is increased by more than

6214

Langmuir, Vol. 21, No. 14, 2005

Figure 3. (a) Competitive alkaline hydrolysis of p-nitrophenylacetate (40 µM S1) and fluorescein diacetate (10 µM S2) at pH 11.0 (50 mM sodium phosphate). Shown is an illustration of the simultaneous formation of p-nitrophenolate (P2; λmax ) 400 nm) and fluorescein (P4; λmax ) 490 nm) as a function of reaction time in the presence of 120 mM decanoate. The interval between the spectra was 90 s. (b) Effect of the decanoate concentration on the rate of hydrolysis of p-nitrophenlyoctanoate (S1, 50 µM), expressed as time needed to hydrolyze 50% of the initially present S1 (t50%), pH 11.0 (50 mM sodium phosphate). The dotted line indicates the cmc as estimated from this set of measurements (see Table 1).

two pH units in comparison with acetic acid.4-6 The reason for this type of pKa shift in aqueous fatty acid/soap systems has been discussed before.5b,23,24 Furthermore, the data presented in Figure 4 show that the apparent pKa of decanoic acid tends to increase with increasing decanoic acid + decanoate concentration. This can be explained qualitatively as follows: A certain amount of decanoate is always present as nonassociated, monomeric surfactant in the bulk solution (see below). The relative amount of this monomeric surfactant decreases with increasing [decanoic acid + decanoate]. At high concentration (e.g., 300 mM) and at 0.5 equiv of HCl, most of the decanoic acid and decanoate molecules are involved in the formation of decanoic acid/decanoate bilayers (vesicles). The negatively charged surface of the bilayers attracts H3O+ ions from the bulk medium, leading to an increase in the H3O+ concentration close to the bilayer surface (low local pH) and to a decrease in the H3O+ concentration in the bulk medium (increased bulk pH). At high [decanoic acid + (23) Gebicki, J. M.; Hicks, M. Chem. Phys. Lipids 1976, 16, 142160. (24) Haines, T. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 160-164.

Namani and Walde

Figure 4. Decanoic acid/sodium decanoate titration curves determined after equilibration at room temperature for [decanoic acid + decanoate] ) 50 mM (O), 100 mM (9), 150 mM (b), 200 mM (4), and 300 mM (0). While in (a), the entire titration curves are shown, the part between pH 6.5 and 8.5 and 0.0-0.65 equiv of HCl is plotted in (b). For comparison, the titration curve of 200 mM acetic acid/acetate is also shown (/). Above the dotted horizontal line at pH 8.1 in (a), all decanoate/ decanoic acid samples were transparent (micelles and monomers). Below pH 6.4-6.0 (indicated in (a) with a horizontal patterned bar), oil droplets and precipitation were observed. In the intermediate-pH region, the samples were homogeneously turbid. In (b), the high-pH plateau region for 300 mM [decanoic acid + decanoate] is localized between the two dashed vertical lines positioned at 0.20 and 0.35 equiv of HCl. The solid vertical line in (b) marks 0.5 equiv of HCl.

decanoate], the bulk pH at 0.5 equiv of HCl is therefore higher than at low [decanoic acid + decanoate]. This means that the apparent pKa of decanoic acid at 300 mM (7.25) is higher than at 50 mM (6.50). If one neglects a direct influence of the surface microenvironment of a decanoic acid/decanoate bilayer, then the intrinsic pKa of decanoic acid is probably always about the same (close to 4.7, like in the case of acetic acid) independent of the total decanoic acid + decanoate concentration. This explanation is based on the arguments discussed by Haines.24

Decanoic Acid Micelles and Vesicles

Figure 5. Differential scanning calorimetry measurements of 200 mM decanoic acid/decanoate samples of different pH (as indicated above the corresponding curves).

By applying the Gibbs phase rule in exactly the same way as done before,5b we have tried to theoretically predict the region of formation of vesicles (bilayers) within the titration curves. A plateau in the titration curves indicates coexistence of vesicles with micelles (high pH plateau) or vesicles with emulsion droplets (low-pH plateau).25 Clear plateaus are, however, not always obvious. This is certainly at least partly due to the experimental difficulties in measuring the pH in turbid suspensions.12 On the other hand, the salt present (Na+ Cl-) is not considered at all in the theoretical considerations.5b,25 In only the case of [decanoic acid + decanoate] ) 300 mM, a clear and not even perfect high-pH plateau is evident between about 0.2 and 0.35 equiv of HCl (indicated by the dashed lines in Figure 4b). Definitively, for [decanoic acid + decanoate] ) 50 mM, no high-pH plateau is present. This is expected if micelle formation does not occur, and this may indeed be the case since the cmc of sodium decanoate is just about 50 mM (see above). The presence of vesicles within the intermediate pH region of the titration curves has been confirmed by electron and light microscopy (see Figure S-5 in Supporting Information). The vesicles are heterogeneous in size and lamellarity, like in the case of oleic acid/oleate vesicles.26-28 Differential scanning calorimetry measurements of 200 mM samples seem to agree qualitatively with the above interpretation of the titration curves (Figure 5). In the intermediate-pH region (7.15-6.52), one single transition is observed with a maximum of transition that varies between 13.5 and 17.2 °C. At pH 7.47 two transitions are detected, at ∼7 °C (most likely nonspherical micelles) and ∼13 °C (vesicles). At pH 7.64, 7.86, and 7.95, only one (25) The assumption made is that micelles, bilayers (vesicles), and the oil droplets are independent phases from the water phase.5b Gibbs phase rule is F ) C - P + 2, where F is the degree of freedom, C is the number of components, and P is the number of phases. C is fixed: water, soap, fatty acid (C ) 3). At constant temperature and pressure, F ) 3 - P. If two phases are present (e.g., micelles and water, P ) 2), then F ) 1 (the pH). If three phases are present (e.g., micelles, vesicles, water, P ) 3), then F ) 0 (no pH change). Therefore, the Gibbs phase rule predicts the existence of a region (pH ) constant) where bilayers (vesicles) exist in the absence of micelles. Whether this is true remains to be proven experimentally. In the case of the long-chain oleic acid/ oleate system, electron spin resonance measurements indicated that bilayers indeed always seem to exist with nonlamellar aggregates, although the concentrations of these aggregates can be considerably low: Fukuda, H.; Goto, A.; Yoshioka, H.; Goto, R.; Morigaki, K.; Walde, P. Langmuir 2001, 17, 4223-4231. (26) Walde, P.; Goto, A.; Monnard, P.-A.; Wessicken, M.; Luisi, P. L. J. Am. Chem. Soc. 1994, 116, 7541-7547. (27) Walde, P.; Wick, R.; Fresta, M.; Mangone, A.; Luisi, P. L. J. Am. Chem. Soc. 1994, 116, 11649-11654. (28) Blo¨chliger, E.; Blocher, M.; Walde, P.; Luisi, P. L. J. Phys. Chem. B 1998, 102, 10383-10390.

Langmuir, Vol. 21, No. 14, 2005 6215

Figure 6. Ultrafiltration of aqueous decanoic acid/decanoate dispersions by using Centricon-10 microconcentrators (see Experimental Section). The concentration of decanoate in the ultrafiltrate is plotted as a function of measured pH and total decanoic acid + decanoate concentration: (1) region with vesicles and monomers; (2) region with vesicles, micelles, and monomers. Between the dashed lines is the pH range within which (on increasing the pH) a transition between vesicles and micelles starts to occur.

transition is detected at around 7 °C (possibly nonspherical micelles) (see Figure 5). Within the high-pH plateau of the titration curve, the micelles coexisting with the vesicles are expected to be either elongated cylinders or extended bilayer sheets.29 Since there is still a considerable amount of decanoic acid molecules present, the formation of spherical micelles in contrast to elongated aggregates is not likely to occur at this pH (pH 7.4-8.0). Two DSC peaks observed at pH 7.47 may indicate that there are two type of crystals with different decanoic acid/ decanoate ratios formed at low temperature.30 When the samples are heated, one of the two crystalline states melts earlier than the other. In both cases, dispersed decanoic acid/decanoate aggregates appear at higher temperature (nonspherical micelles and vesicles, respectively). In the case of nearly spherical micelles (at pH >8.5), no DSC signal was observed. A similar pH dependency of the DSC curves was also observed for decanoic acid/decanoate samples with a total concentration of 150 and 300 mM, respectively (data not shown). By ultrafiltration, the concentration of decanoate that could pass an ultrafiltration membrane with a cutoff molecular weight of 10 000 Da (see Experimental Section) has been determined as a function of [decanoic acid + decanoate] and as a function of pH (see Figure 6). With increasing pH, the concentration of decanoate appearing in the ultrafiltrate increases steadily with a steep increase at about pH 7.6-7.7 (see the dashed lines in Figure 6). Above this pH, it is likely that (in addition to the monomers) micelles present in the system passed the ultrafiltration membrane. Indeed, if a micellar solution was used (165 mM sodium decanoate prepared in 100 mM phosphate buffer, pH 11.5), the concentrations in the ultrafiltrate and the retentate were approximately the (29) This interpretation is based on the pioneering work of Mitchell and Ninham on the packing concepts in surfactant assemblies: Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601629. (30) See the extensive work on long-chain fatty acid-soap crystals, such as palmitic acid/sodium palmitate: (a) Lynch, M. L. Curr. Opin. Colloid Interface Sci. 1997, 2, 495-500. (b) Lynch, M. L.; Wireko, F.; Tarek, M.; Klein, M. J. Phys. Chem. B 2001, 105, 552-561. Crystals form with the following compositions: (i) one sodium palmitate and two palmitic acids; (ii) two sodium palmitates and three palmitic acids; (iii) one sodium palmitate and one palmitic acid. Palmitic acid is hexadecanoic acid, H3C-(CH2)14-COOH.

6216

Langmuir, Vol. 21, No. 14, 2005

Figure 7. Turbidimetric cvc determinations for sodium decanoate/decanoic acid samples. The absorbance measured at 350 nm is plotted as function of [decanoic acid + decanoate] at different pH: 0, pH 6.6; b, pH 6.8; 4, pH 7.0; 1, pH 7.2; O, pH 7.4; 9, pH 7.6. For all measurements, a 100 mM sodium phosphate buffer was used.

same, indicating that the micelles are forced through the ultrafiltration membranes. In summary, not only within the pH region where micelles exist above the cmc but also within the intermediate pH region where vesicles are formed, there is always a considerable amount of monomeric decanoate present in the bulk aqueous phase, e.g., ∼20 mM at pH 7.0 (for [decanoic acid + decanoate] ) 50-200 mM). Determination of the cvc for Decanoic Acid/ Decanoate Vesicles. If the total concentration of decanoic acid + decanoate is lowered within the pH region of bilayer formation, one will reach a concentration (or concentration range) below which no vesicles exist anymore.6 On the other hand, by steadily increasing at a fixed pH the [decanoic acid + decanoate] from a low starting value, one arrives at a critical concentration (or in a concentration range) at which vesicle formation starts to occur.4,6 This critical concentration for vesicle formation (cvc) has been determined for different pH values by measuring the turbidity (at the arbitrarily chosen wavelength of 350 nm) as a function of [decanoic acid + decanoate] (see Figure 7). The corresponding cvc values determined are 8 mM (pH 6.6), 10 mM (pH 6.8), 14 mM (pH 7.0), 18 mM (pH 7.2), 22 mM (pH 7.4), and 26 mM (pH 7.6). The higher is the pH, the more decanoate is present and the higher is the cvc. In another set of experiments, merocyanine 540 was used as membrane probe. From studies with phospholipid vesicles,31 it is known that merocyanine can be applied to probe the membrane interface. In aqueous solution, merocyanine 540 dimerizes. The absorption spectrum in the visible range is characterized by two peaks, one at 570 nm (dimer) and another one at 530 nm (monomer).31b If merocyanine is adsorbed to phospholipid membranes, the absorption maxima are at 530 nm (dimer) and 570 nm (monomer), respectively.31b The ratio OD570/OD530 is indicative of the physical state of phospholipid membranes.31 We have applied merocyanine 540 as indicator for the formation of vesicles. Figure 8 shows how OD570/ OD530 varies with [decanoic acid + decanoate] at pH 7.0. A break in the curve at 10 mM correlates quite well with the cvc-determined turbidimetrically (14 mM) (see Figure 7). Merocyanine 540 as probe molecule may have lowered the actual cvc.16 Below 10 mM, no vesicles exist; the dye (31) (a) Bernik, D. L.; Disalvo, E. A. Chem. Phys. Lipids 1996, 82, 111-123. (b) Disalvo, E. A.; Arroyo, J.; Bernik, D. L. Methods Enzymol. 2003, 367, 213-233.

Namani and Walde

Figure 8. Determination of the cvc of decanoic acid/decanoate vesicles (100 mM phosphate buffer, pH 7.0) by using merocyanine 540. The ratio of the optical density at 570 nm over the optical density at 530 nm is plotted as a function of the total concentration of decanoic acid + decanoate.

has a low absorption at 570 nm. Furthermore, a high ratio of OD570/OD530 is indicative of a fluid state of the vesicle bilayers.31 The decanoic acid/decanoate vesicle bilayers at ∼25 °C are therefore in a fluid state (above the melting temperature), in agreement with the DSC measurements shown in Figure 5. Preparation and Stability of Large Unilamellar Decanoic Acid/Decanoate Vesicles. Although the concentration of monomeric decanoate molecules is always relatively high (e.g., ∼20 mM at pH 7.0; see above), the use of decanoic acid/decanoate vesicle systems as potential reaction compartments has been investigated. The following question has been primarily addressed: Can homogeneous, stable, submicrometer-sized unilamellar vesicle suspensions be prepared by the extrusion technique? Heterogeneous multilamellar phospholipid vesicles can easily be sized down by the extrusion technique (ET) to yield stable large unilamellar vesicles (LUV) with a mean size of about 100 nm (LUVET100) if polycarbonate membranes that contain pores with a mean diameter of 100 nm are used.13 If the same procedure is applied to heterogeneous decanoic acid/decanoate vesicle suspensions (pH 7.0), the overall turbidity of the suspensions is drastically reduced, resulting in a mean vesicle diameter of about 63 nm, as determined by dynamic light scattering measurements (Figure 9a). In comparison with POPC LUVET100, the polydispersity of extruded decanoic acid/ decanoate vesicles is, however, considerably larger. Freeze-fracture electron micrographs confirm the relatively high polydispersity of decanoic acid/decanoate LUVET100 (see Figure 10). Furthermore, a vesicle diameter considerably below 100 nm indicates that a large quantity of small vesicles with diameters below 100 nm form during extrusion or are already present before extrusion through the 100 nm polycarbonate membranes. The extruded vesicle suspensions are stable for at least 4 days, comparable with POPC LUVET100 (see Figure 9b). In both cases, the LUVET100 are “metastable”. They will at the end transform into larger, mainly multilamellar vesicles upon long-time storage. In comparison with extruded vesicles prepared from oleic acid/sodium oleate in 100 mM bicine buffer, pH 8.5 (see Supporting Information Figure S-6), decanoic acid/ decanoate LUVET100 prepared in 100 mM phosphate buffer, pH 7.0, sometimes appears aggregated in freezefracture electron micrographs (see Figure 10b). If, instead, decanoic acid LUVET100 were prepared in 200 mM bicine

Decanoic Acid Micelles and Vesicles

Figure 9. Dynamic light scattering measurements of decanoic acid/decanoate LUVET100 and POPC LUVET100. (a) Measurements immediately after preparation. Data with the empty symbols are for decanoic acid/decanoate LUVET100. The data with the filled symbols are for POPC LUVET100. The scattering angles were 60° (O, b), 90° (0, 9), and 120° (4, 2). The polydispersity index in the case of decanoic acid/decanoate LUVET100 was around 0.24. In the case of POPC LUVET100, it is 0.10. (b) Vesicle diameters as a function of storage time at room temperature for decanoic acid/decanoate (b) and POPC (O): 100 mM sodium phosphate buffer, pH 7.0, [decanoic acid + decanoate] ) 30 mM, [POPC] ) 1 mM.

(pH 7.0), the vesicles appear less aggregated (data not shown). The reason for this buffer effect is not clear at the moment. By use of the highly water-soluble dye arsenazo III and gel permeation chromatography (see Experimental Section), it has been shown that the vesicles formed from decanoic acid and decanoate are indeed compartments that have an aqueous interior. Figure 11 shows the elution profile obtained with a sepharose 4B column, indicating that the water-soluble dye arsenazo III could be entrapped inside the vesicles that eluted first (peak 1 in Figure 11), followed by the free, nonentrapped dye molecules (peak 2 in Figure 11).32 This is clear proof that the decanoic acid/decanoate vesicles prepared contained an aqueous interior. Preparation and Stability of Decanoic Acid/SDBS Vesicles. To extend the pH range of vesicle formation from decanoic acid to the alkaline pH region (above pH 7.8), decanol can be admixed.4 The alcohol now takes the role of decanoic acid in eliminating the electrostatic repulsions between the charged decanoate molecules, similar to the octanoate/octanol/water system.1b,33 We

Langmuir, Vol. 21, No. 14, 2005 6217

Figure 10. Freeze-fracture electron micrographs (a, b) of decanoic acid/decanoate LUVET100 prepared in 100 mM sodium phosphate buffer, pH 7.0: [decanoic acid + decanoate] ) 100 mM; length of the bar, 200 nm. The vesicles in (b) are mainly present in an aggregated state.

extended the pH range of decanoic acid vesicle formation to the acidic pH region (below pH 6.4) by admixing SDBS. (32) Because of the high monomer solubility, the separation of the vesicles from nonentrapped dye molecules is experimentally not easy. The elution buffer has to contain monomers at the corresponding concentration. Furthermore, a passage of the vesicles through a gel permeation column leads automatically to a dilution of the vesicles. This dilution may end up at a concentration below the cvc: the longer the column, the higher the dilution. If the column is too short, however, no efficient separation of vesicles and free dye can be obtained. Therefore, the only way to perform this type of experiment is to use a highly concentrated suspension and a sufficiently long column. If the concentration is too high, however, the suspensions get very viscous, impossible to handle. We found that [decanoic acid + decanoate] ) 500 mM is optimal, although extrusion is not possible. Therefore, instead of extrusion for vesicle size reduction, we applied probe sonication to a decanoic acid/decanoate suspension (pH 7.0) that contained arsenazo III (1 mM). (33) (a) Fontell, K.; Ekwall, P.; Mandell, L.; Danielsson, I. Acta Chem. Scand. 1962, 16, 2294-2298. (b) Fontell, K.; Mandell, L.; Ekwall, P. Acta Chem. Scand. 1968, 22, 3209-3223. (c) Ekwall, P.; Mandell, L.; Fontell, L. J. Colloid Interface Sci. 1969, 31, 508-529. (d) Ekwall, P.; Mandell, L.; Fontell, K. J. Colloid Interface Sci. 1969, 31, 530-539. (e) Jo¨nsson, B.; Wennerstro¨m, H. J. Phys. Chem. 1987, 91, 338-352.

6218

Langmuir, Vol. 21, No. 14, 2005

Figure 11. Gel permeation chromatographic separation of sonicated arsenazo III-containing decanoic acid/decanoate vesicles (peak 1) from free, nonentrapped arsenazo III (peak 2). Turbidity, as recorded by measuring the optical density at 800 nm (O) and light absorption by the dye at 550 nm (b), is recorded as a function of elution volume. A sepharose 4B column with a diameter of 1.1 cm and a length of 25 cm was used. A volume of 0.5 mL of the vesicle suspension was applied ([decanoic acid + decanoate] ) 0.45 M, [arsenazo III] ) 1 mM, 100 mM phosphate buffer, pH 7.0). The elution buffer (100 mM sodium phosphate, pH 7.0) contained 15 mM decanoate.

Figure 12. Cryotransmission electron micrograph of LUVET100 prepared from decanoic acid/SDBS (1:1, molar ratio) at pH 4.3 (100 mM phosphate buffer): 50 mM decanoic acid; 50 mM SDBS. Length of the bar is 100 nm.

Since the pKa of dodecylbenzenesulfonic acid is below -1, SDBS always remains in a deprotonated state above pH 1. In bilayers formed from decanoic acid and SDBS in the acidic pH region, SDBS takes the role decanoate has in the decanoic acid/decanoate or decanol/decanoate vesicle systems. Vesicle suspensions formed from an equimolar mixture of decanoic acid and SDBS (each 50 mM) at pH 4.3 are (meta)stable for several days. These suspensions can be extruded through polycarbonate membranes such as in the case of decanoic acid/decanoate vesicles. With “100 nm membranes”, the mean vesicle diameter after extrusion is about 78 nm. The measured scattering angle dependency of the hydrodynamic diameter is low: 82 nm at 60°, 78 nm at 90°, and 76 nm at 120°. Figure 12 shows a cryotransmission electron micrograph of a LUVET100 decanoic acid/SDBS (1:1, molar ratio) vesicle suspension, prepared at pH 4.3. The electron micrograph confirms the homogeneity of the extruded vesicle suspension, and it shows that most of the vesicles are unilamellar. During storage at room temperature, the mean diameter of the

Namani and Walde

Figure 13. Visible/NIR spectra of the polyaniline synthesized in the absence (a) and presence (b) of decanoic acid/SDBS vesicles: 5 mM aniline, 0.25 mg/mL horseradish peroxidase, 0.5 mM H2O2, pH 4.3 (100 mM phosphate buffer), 5 mM decanoic acid, 5 mM SDBS. The spectra were recorded from the reaction solutions 20 min after the start of the reaction.

vesicles increases from 78 to 93 nm after 2 days. This size remains almost constant for another 2 days. The scattering angle dependency of the hydrodynamic diameter measured after 2 days is 97 nm at 60°, 93 nm at 90°, and 77 nm at 120°. The presence of an aqueous pool inside decanoic acid/SDBS (1:1) vesicles has been confirmed by using arsenazo III as a water-soluble dye, just like in the case of decanoic acid/decanoate vesicles (see Supporting Information Figure S-7). It is also possible to incorporate inside mixed SDBS/decanoic acid vesicles macromolecules such as peroxidase. Preliminary entrapment experiments indicated, however, that some of the peroxidase molecules (pI ≈ 7.2) are also bound to the negatively charged vesicle bilayers (data not shown). If an excess of decanoic acid over SDBS is used, the vesicle suspension is less stable, leading to precipitation at room temperature within hours. Use of Decanoic Acid/SDBS Vesicles as Templates for the Polymerization of Aniline. Electrically conductive polyaniline can be prepared in aqueous solution at acidic pH from aniline by using hydrogen peroxide and horseradish peroxidase as catalyst and a negatively charged template, like SDBS micelles.15 In a series of preliminary experiments, we have tried to use decanoic acid/SDBS vesicles as templates for the same enzymecatalyzed polymerization reaction. To the best of our knowledge, this is the first attempt to use vesicles as templates for this reaction. One of the results is shown in Figure 13. If 5 mM aniline is polymerized at pH 4.3 with 0.25 mg/mL horseradish peroxidase and 0.5 mM H2O2 (in the absence of any vesicles), a brown polyaniline product is formed that is mainly branched15 and that does not show any absorbance in the near-infrared around 1000 nm (see curve a in Figure 13). In contrast, the presence of decanoic acid/SDBS vesicles leads to the formation of polyaniline, which absorbs around 1000 nm, indicative for the formation of mainly linear, electrically conductive polyaniline.15,34 In comparison with the same reaction carried out in the presence of SDBS micelles,15 however, the vesicle system seems to be “less efficient” (lower intensity around 1000 nm) and needs to be improved in future work.35 (34) Cao, Y.; Smith, P.; Heeger, A. J. Synth. Met. 1989, 32, 263-281.

Decanoic Acid Micelles and Vesicles

Conclusions and Perspective Although chemically simple, decanoic acid forms a number of different phases in water at room temperature, depending on the degree of protonation and on the concentration, rather similar to the well-investigated octanoic acid/sodium octanoate/water system.1 This is particularly interesting from a prebiotic, “origin of life” point of view. Decanoic acid is one of the amphiphiles that were isolated from carbonaceous meteorites.36 Furthermore, decanoic acid can be synthesized by simple FischerTropsch chemistry.37 In the dilute region of the decanoic acid/decanoate/water phase diagram, the formation of vesicles was studied by titrating a micellar solution with HCl, and the titration curves obtained were analyzed by applying the Gibbs phase rule. Since the concentration of nonaggregated decanoate in the systems is always relatively high and since pH measurements in turbid vesicle suspensions are not trivial, DSC measurements were performed in order to get an idea within which pH region decanoic acid/ decanoate vesicles form. The identified region was pH 6.4-7.8. Under these conditions about 50% of the decanoic acid molecules are present in anionic form (-COO-). Again, from a prebiotic point of view, the formation of vesicular compartments is considered as a possible step toward the first cellular systems.38 Therefore, the currently accepted model systems for the precursor structures of the first cells, the protocells, are indeed vesicles.38 With decanoic acid/decanoate vesicles, however, there are two major drawbacks: (i) the high concentration of nonassociated decanoate and (ii) the narrow pH range of vesicle formation. With respect to (i), longer alkanoic acids could be used, e.g., hexadecanoic acid. Since the Krafft point of sodium hexadecanoate is, however, rather high (∼55 °C),39 the vesicle formation would have to be studied at high (35) The peak around 1000 nm is due to the so-called “polaron transition”, arising from cation radicals localized on the nitrogen atoms of the polyaniline backbone structure: Wudl, F.; Angus, R. O., Jr.; Lu, F. L.; Allemand, P. M.; Vachon, D. J.; Nowak, M.; Liu, Z. X.; Heeger, A. J. J. Am. Chem. Soc. 1987, 109, 3677-3684. (36) Shimoyama, A.; Naraoka, H.; Komiya, M.; Harada, K. Geochem. J. 1989, 23, 181-193. (37) McCollom, T. M.; Ritter, G.; Simoneit, B. R. T. Origins Life Evol. Biosphere 1999, 29, 153-166. (38) (a) Deamer, D. W. Microbiol. Mol. Biol. Rev. 1997, 61, 239-261. (b) Luisi, P. L.; Walde, P.; Oberholzer, T. Curr. Opin. Colloid Interface Sci. 1999, 4, 33-39. (c) Hanczyc, M. M.; Szostak, J. W. Curr. Opin. Chem. Biol. 2004, 8, 1-5. (39) Skurtveit, R.; Sjo¨blom, J.; Hoiland, H. J. Colloid Interface Sci. 1989, 133, 395-403.

Langmuir, Vol. 21, No. 14, 2005 6219

temperature. Indeed, it was shown that bilayers form from approximately equimolar mixtures of hexadecanoic acid/ hexadecanoate at 70 °C in water.39 With respect to (ii), it was shown before that the pH range of decanoic acid/ decanoate vesicle formation can be extended to the alkaline region by adding decanol.1b,4,33 A high concentration of nonvesiculized decanoate in the aqueous phase, however, remains (>20 mM). We have demonstrated that the pH region of decanoic acid vesicle formation can be expanded to the acidic pH region by adding an equimolar amount of the surfactant SDBS. In this system, the concentration of nonassociated molecules at low pH remains low (∼0.5 mM SDBS). In preliminary studies, we finally showed that these mixed vesicles can be used as templates for the peroxidasecatalyzed polymerization of aniline. A systematic work on this subject is in progress. With respect to the use of the micelle-forming surfactant SDBS for vesicle formation, it is worthwhile to mention that other SDBS-based vesicle systems containing only single-chain amphiphiles have been known for several years, the most intensively investigated being SDBS/CTAT (cetyltrimethylammonium tosylate).40 In this case, the “partner surfactant” of SDBS is positively charged, while in our system, it is not charged (decanoic acid at low pH). Acknowledgment. We thank Doris Sutter for performing the NMR measurements, Dr. Peter Skrabal for scientific NMR advice, Martin Colussi for helping with the DSC measurements, and Dr. Martin Mu¨ller and Dr. Ernst Wehrli for the electron microscopy analysis. Critical discussions with Dr. Kenichi Morigaki (National Institute of Advanced Industrial Science and Technology, Osaka, Japan) and his careful reading of the manuscript are highly appreciated. Supporting Information Available: Data of cmc determinations for different anionic surfactants, additional freeze-fracture electron micrographs, and a gel permeation chromatogram obtained from a dye entrapment experiment. This material is available free of charge via the Internet at http://pubs.acs.org. LA047028Z (40) (a) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245, 1371-1374. (b) Kaler, E. W.; Herrington, K. L.; Murthy, A. K.; Zasadzinski, J. A. N. J. Phys. Chem. B 1992, 96, 6698-6707. (c) Chiruvolu, S.; Israelachvili, J. N.; Naranjo, E.; Xu, Z.; Zasadzinski, J. A.; Kaler, E. W.; Herrington, K. L. Langmuir 1995, 11, 4256-4266.