A membrane is an essential step in the evolution of the first living organisms (Rasmussen et al. 2004). Prebiotic membranes are often hypothesized to be composed of single-chain amphiphiles that can self-assemble into membrane structures called vesicles (Fig. 1, for review see Maurer and Monnard 2011). These amphiphiles have a variety of headgroups and tail lengths, the identity of which determines specific properties of a vesicle.

Fig. 1
figure 1

Vesicles formed from single-chain amphiphiles. a Chemical structure of decanoic acid (1) and glycerol monodecanoate (2). b Fluorescent micrograph of decanoic acid vesicles. The membranes are stained with Nile red. Scale bar =10 μm

Perhaps the most popular single-chain amphiphiles, as well as the first considered for the origins of life, are fatty acids (Fig. 1a; Gebicki and Hicks 1973). Fatty acids, like decanoic acid, form membranes when the pH is adjusted to the apparent pK a, which increases with increasing hydrophobic tail length. It has also been shown that fatty acids membranes are less stable than phospholipid membranes in many ways, including precipitating under high salt conditions (Monnard et al. 2002). The addition of other amphiphiles, for example glycerol monodecanoate, has been shown to improve stability of prebiotic vesicles under salt stress.

Salts, used here to mean ionic solutes, are known to affect the solubility of organic molecules, as well as nanoparticulates. Organic nonpolar molecule solubility is primarily controlled by the hydrophobic effect. When salt is added to water, entropic forces become more favorable to exclude hydrophobic structures resulting in lower solubility. For example, the solubility of benzene in water decreases 20 % when 0.5 M NaCl is added (for review see Xie et al. 1997). Charged particles are affected by the addition of salt as well, where electric shielding (or electric double layer) allows the charged surfaces to become more stable in an aqueous environment (Shih et al. 2012). In fatty acid vesicles, where half of the headgroups are negatively charged carboxylates, salts have the potential to affect both the solubility of the hydrocarbon tail and the interaction of carboxylate headgroups.

There are many methods of describing stability of membranes, from permeability to encapsulation ability. One common method of assessing the likelihood of membrane formation is using the critical vesicle concentration (CVC). This has been measured for fatty acid vesicles many times and has been shown to rely on the pH, buffer, and determination method (Table 1). All of the methods used rely on an increase in the intensity of the signal above a “no vesicle” baseline value upon vesicle formation. While turbidity and light scattering both detect aggregates in solution by the scattering of light, Merocyanine 540 (MC540) specifically detects the formation of a hydrophobic environment. When in an aqueous phase, MC540 has a peak maximum at 530 nm; in a hydrophobic phase, like a membrane, the peak is at 570 nm (Dixit and Mackay 1983). MC540 cannot distinguish between oil-in-water emulsions, micelles, and vesicle phases, so this detection method must also be coupled with microscopy.

Table 1 Literature values for the critical vesicle concentration (CVC) of decanoic acid

As ideal environments for abiogenesis, prebiotic chemistry often uses “warm ponds”, which would undergo cycles of dehydration to concentrate molecules also concentrating salts. Yet the impact of salts in prebiotic aggregation behavior is only partially understood, and salts are often excluded from buffer systems when designing experiments (as all of the experiments in Table 1). When culturing cells, salt is often essential for growth, suggesting the same may be true for protocells. We report here the importance of salt addition to prebiotic membrane experiments, in terms of the CVC determined by MC540. Previous reports have suggested detriment to vesicle systems upon salt addition; we suggest the opposite: monovalent salts could have improved the stability of the first cell membranes.



Decanoic acid and oleic acid were obtained from Acros Organics (>99 % purity; New Jersey, USA). Glycerol monodecanoate (GMD or monocaprin) was purchased from Tokyo Chemical International (>97 % purity; Oregon, USA). The salts, NaCl, KCl, and NaBr, were obtained from JT Baker (USA). HEPES free acid was used as buffer in decanoic and GMD samples at 10 mM and pH 7.2 (Research Products International Corp., Illinois, USA). Bicene (99 %, Alfa Aesar, USA) was used as buffer for the oleic acid samples at pH 9.2 and 10 mM. All solutions were prepared with ultrapure water (MilliQ AcademicA10, Millipore, USA). Merocyanine 540 (MC540) was obtained from AnaSpec Inc. (California, USA).

Vesicle Preparation

Stock amphiphile suspensions were prepared as follows, in volumetric glassware to a final concentration of 75 mM for decanoic acid and 3 mM for GMD in 10 mM of HEPES buffer, or 4 mM oleic acid in 10 mM bicine buffer. Solid amphiphile was disbursed in buffer using heating, vortexing and sonication. Decanoic acid needed to be pH adjusted to pH 7.2 with 5 M NaOH before it would mix into buffer. Care was taken to not go over pH 7.2 because acid adjustment would result in NaCl concentrations in solution to increase. Stock salt solutions were also prepared at 500 mM NaCl and 10 mM of HEPES buffer for dilution to determine CVC.

Aliquots of stock amphiphile and stock salt solutions were put into microcentrifuge tubes and the volume was brought to 1.2 mL with buffer. The concentration of amphiphile in the dilution series was adjusted to give several points below the CVC and several points just after the CVC before the absorbance at 570 nm plateaued. MC540 was dissolved in 1:1 ethanol/water for an approximate concentration of 1 mg/mL. To each 1.2 mL of vesicles, 4 μL of MC540 solution was added. The vesicle suspension was just slightly orange/pink to the eye, as too much MC540 may have resulted in lower sensitivity.

Sample Analysis

Samples were analyzed using a Hewlett Packard 8453 spectrophotometer (USA) in quartz cuvettes. Each CVC was determined in duplicate and values differed by no more than 6 %. The entire spectrum was obtained for each sample (190–1100 nm) however the region of interest is from 400 to 700 nm. The baseline was then subtracted out at 650 nm, and the 530 nm value was normalized to 100 AU (Fig. 2a). The normalized intensity at 570 nm was then used to determine the CVC. This is proportional to the ratio of intensity at 570 nm to intensity at 530 nm. To calculate the precise value for the CVC, a straight line is fitted to the values between the baseline below the CVC and the plateau far above the CVC (Fig. 2b). The CVC is determined as the value for which the line intersects the baseline value.

Fig. 2
figure 2

Determination of CVC by Merocyanine 540. a Normalized absorbance spectra of GMD from dark at low concentration to light lines at high concentration. Peak at 570 nm showing the formation of a hydrophobic membrane. b CVC determination from a. Two lines were drawn: one for the baseline “no vesicle” condition and one for the increasing absorbance at 570 nm indicating vesicles were forming. The intersection of this line was mathematically determined as the CVC


Determination of critical vesicle concentration (CVC) was performed for decanoic acid, oleic acid, and glycerol monodecanoate (GMD) while varying the concentration of salt. In the example shown the CVC was 0.85 mM of glycerol monodecanoate (Fig. 2). The full results are shown in Table 2.

Table 2 CVC of single-chain amphiphiles with increasing concentrations of salt

The CVC for fatty acid vesicles was dependent on the amount of NaCl added to the solution. The lowest concentration of salt examined is 10 mM of NaCl, as vesicles are not stable in the absence of salt and produce widely variable results, possibly due to a greater pH sensitivity. All samples also contain 10 mM of buffer, which was used in low concentrations to prevent a significant impact on the CVC. Decanoic acid vesicles showed a reduction in the CVC as the concentration of NaCl increased. A similar trend is observed with oleic acid. The CVC of decanoic acid levels out at 100 mM and further increases in salt concentration (200 mM) begin to impact the stability of the structures, therefore the CVCs at higher salt concentrations are not possible. In fact, in 500 mM NaCl complete aggregation of the vesicles occurs. This is likely a “salting out” effect of the NaCl, and has been reported elsewhere (Monnard et al. 2002).

The CVC of GMD and oleic acid are much lower than the CVC of decanoic acid because they are less water-soluble. To compare the relative impact of salt on GMD to fatty acid, the ratio of the CVCs was taken (Fig. 3). If the salt impacted the two amphiphiles in the same way the ratio would have been the same for all of the samples. However GMD vesicle formation is less dependent on the salt concentration, specifically at lower concentrations of salt. Interestingly, at 50 and 100 mM of NaCl the CVC ratios were the same. Also, GMD will form stable structures at higher salt concentrations (200 mM).

Fig. 3
figure 3

Impact of concentration of NaCl on ratio of CVC between fatty acid and glycerol monodecanoate. a Decanoic acid ratio and b oleic acid ratio with GMD indicates the sensitivity differences at each salt concentration

The CVC was also compared between ionic compounds to determine the role of specific anions or cations in stabilization. NaBr and KCl were therefore also tested and compared to the CVC of NaCl for both decanoic acid and GMD (Fig. 4). There was no difference found in the CVC of decanoic acid or GMD between ionic species.

Fig. 4
figure 4

Ionic species impact on vesicle formation. 570 nm peak indicates vesicles occurring in almost the same concentration for each monovalent salt: NaCl (✕), KCl (☐), and NaBr ()


The results obtained clearly show salt dependent vesicle formation. In general, salts are known to affect colloidal solubility, in both nanoparticulates and micelles. Two driving forces are attributed to this stabilization: electric shielding of the charged or polar surface and increasing the entropic contributions to aggregation.

The first effect is seen more strongly with decanoic acid, which only forms vesicles when some of the acid is in the carboxylate form. These negatively charged bilayers are likely stabilized by the addition of cations to form an electric double layer, in the same manner as cations stabilize negatively charged micelles and nanoparticulates (Ruso et al. 2000; Shih et al. 2012; Zhai et al. 2006). The ratio of the CVC of decanoic acid to the CVC of GMD decreases as the concentration of salt increases, but plateaus at 50 mM. We expect this is due to the incomplete formation of the electric double layer on the surface of the decanoic acid vesicles 10 and 30 mM of NaCl. The uncharged headgroup is expected to have a weaker interaction with ions, and so this effect is not as pronounced in the GMD vesicles. Once the layer is formed, the CVCs of GMD and decanoic acid respond the same way to further increases in salt, with equal ratios at 50 and 100 mM.

The hydrophobic effect also stabilizes vesicles upon salt addition. It is well known that salts effect the phase partitioning of non-polar organic substances (Xie et al. 1997), which contributes to bilayer formation in amphiphiles. This is due to the salt increasing the entropy of water interactions making hydrophobic interactions with water less favorable. Interestingly, at higher concentrations of salt, both amphiphile CVCs continue to decrease even though the ratio between the two CVCs remains the same, perhaps indicating that the major contribution to self-assembly is the hydrophobic effect which would impact the decyl groups similarly.

Often when solubility changes with ionic strength, the identity of the ions is important. This is known as the Hofmeister or lyotrophic effect. However we did not observe the phenomenon with amphiphiles, although the effect may not be measureable in this experimental setup. These amphiphiles are relatively small compared to other biomolecules, and therefore the variation may be outside of our limit of detection.

The CVC of vesicles composed of di-acyl phospholipids, which are more common model systems for cell membranes, are not affected by salts because of very already low CVC values (Tanford 1973). Di-lauroylphosphatidylcholine for example has a CVC of 0.025 μM (Buboltz and Feigenson 2005). This resulted in the assumption that the impact of salts on the CVC of single-chain amphiphiles was also insignificant. We show here that there is a significant impact on vesicle formation when monovalent salts are added, contrary to phospholipid CVCs. Our results indicate that while high concentrations of salt need to be avoided when using single-chain amphiphiles to model origins of life, some monovalent ions in a “warm pond” would have been advantageous to self-assembly.

Additionally, the method generally cited for producing stable fatty acid vesicles is pH vesiculation. This method increases the pH to well above the apparent pK a with NaOH, the lowers the pH to the pK a with HCl (Monnard and Deamer 2003). It was believed that the generation of micelles at high pH was essential as the precursor to bilayer formation. pH vesiculation also would add at about half as much salt to the solution as fatty acid concentration (e.g. 50 mM NaCl for 100 mM decanoic acid). In fact, the experiments performed here included known amounts of salts in the buffer and the formation was identical as the pH vesiculation method, which we have previously used (Maurer et al. 2009; Maurer et al. 2011). This result indicates that future experimentation on single-chain amphiphile structures should include at least 50 mM NaCl in the buffer solution to assist in self-assembly, especially if diluting preformed vesicles to lower concentrations.


A consequence of salt dependent vesicle formation is two-fold. Experimentally, buffer for single-chain amphiphile experiments should include at least a small amount of NaCl, but not more than 100 mM, to prevent fatal aggregation. Perhaps more importantly, astrobiologists have considered wet/dry cycles a problem for vesicle formation due to salt build up over time due to the salt sensitivity of single-chain amphiphiles. The results shown here indicate that instead of a hurdle, salts may have been a requirement for the self-assembly of the first cell membranes.