iGUVs: Preparing Giant Unilamellar Vesicles with a Smartphone and

Laboratory Experiment pubs.acs.org/jchemeduc

iGUVs: Preparing Giant Unilamellar Vesicles with a Smartphone and Lipids Easily Extracted from Chicken Eggs Víctor G. Almendro Vedia,†,‡ Paolo Natale,†,‡ Su Chen,§ Francisco Monroy,†,‡ Véronique Rosilio,∥ and Iván López-Montero*,†,‡ †

Departamento de Química Física I, Universidad Complutense de Madrid, Avenida Complutense s/n, 28040 Madrid, Spain Instituto de Investigación Hospital Doce de Octubre (i+12), Avenida de Córdoba s/n, 28041 Madrid, Spain § Institut Curie, UMR 9187 CNRS, INSERM U1196, Univ Paris-Sud - Université Paris-Saclay, 91405 Orsay, France ∥ UMR 8612 CNRS, Univ Paris-Sud - Université Paris-Saclay, 5 Rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France ‡

S Supporting Information *

ABSTRACT: Since the first report of electroformed micrometersized liposomes in the 1980s, giant unilamellar vesicles (GUVs) have generated a lot of interest in the biophysical and biochemical communities. However, their penetration rate in high school or at the undergraduate level is still limited because of the requirement of specialized materials for their fabrication. The main objective of this article is to translate the manufacture of these interesting microsystems from highly specialized research laboratories to general chemistry or biology laboratories with the help of everyday objects. Vesicles are made of lipids, which can easily be extracted from chicken eggs. Once obtained, the lipids can be reassembled to form giant vesicular structures in a sugar/ aqueous medium by using a do-it-yourself electroformation device. For that, the homemade electroformation chamber is plugged into the audio output of a smartphone or a tablet, which generates audio signals with variable amplitude and frequency. These GUVs prepared with a smart device (iGUVs) are then resuspended into a salt solution for their visualization under a simple microscope. iGUVs bring the opportunity to teachers to stimulate scientific discussion from a wide variety of scientific disciplines such as colloidal chemistry, biophysical chemistry, statistics and cell biology. KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Graduate Education/Research, Biochemistry, Physical Chemistry, Hands-On Learning/Manipulatives, Biophysical Chemistry, Colloids, Lipids, Membranes

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Angelova et al.4 developed the electroformation method to obtain vesicles with diameters larger than 10 μm that can easily be observed through an optical microscope. These vesicles were formed by one bilayer sheet (unilamellar) and thus are called giant unilamellar vesicles (GUVs). The method described by Angelova et al. consists of spreading an organic solution of lipids over electrically conductive platinum electrodes. Hydration of the dried lipid film for several hours in the presence of an alternating current (AC) electric field clusters the lipids and detaches them from the surface to form lipid vesicles in suspension. The electroformation method was standardized by spreading the lipid film onto conductive glass plates.5 The electroformed vesicles are perfectly spherical and can be obtained with different types of lipids. However, the AC electric field is powered with a function generator, which is not usually available to most high school laboratories. We aim to overcome this difficulty and show the production of giant vesicles using everyday materials such as eggs, salt, and a smartphone.

elf-assembly of macromolecular entities seems to be one of the keys to the understanding of a central question in biology: how early life was able to be organized.1 Membrane self-assembly as cell-like capsules has been straightforwardly described for lipids, one of the most elemental structural components of life. The amphiphilic nature of lipids allows them to be organized as many different forms, and lipid polymorphism is usually studied as a prototypical case of molecular self-assembly. Through spontaneous processes lipids can self-assemble into vesicles via hydrophobic interactions.2 Vesicles are containers that separate their inner content from the outside by a lipid membrane, leading to compartmentalization, which is considered an important principle for the development of life. Lipid precursors in primitive Earth could have spontaneously assembled to form vesicle structures. Scientists try to understand the minimal principles that could lead to growth or division of protocells with the aim to construct minimal cells and artificial life.3 Mimicry and bottom-up synthetic approaches represent today the main experimental strategies to reproduce minimal life conditions. Most of these methodologies are founded on vesicle-based science. Many alternatives have been proposed to reproducibly produce cell-sized vesicles in mass. In particular, © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: December 10, 2016 Revised: March 7, 2017

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DOI: 10.1021/acs.jchemed.6b00951 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

The use of smartphones has extended far beyond communication and messaging, as they are being used for numerous exciting experiments in Science, Technology, Engineering, and Mathematics (STEM) classes using simple apps and add-ons. Here we introduce smart devices as a versatile electronic setup to produce GUVs (iGUVs) that are well-suited as a wet-lab platform to familiarize students with theoretical subjects such as the origin of life, statistical data treatment, and colloidal chemistry.6

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Figure 2. Step-by-step protocol for lipid extraction from chicken eggs: (1) 0.5 mL of chicken egg yolk; (2) 1 + 1 mL of water; (3) 2 + 2 mL of MeOH; (4) 3 + 4 mL of CHCl3; (5) 4 after stirring; (6) phase separation of 5; (7) lipid-containing organic phase from 6; (8) 7 after drying; (9) chloroform addition to 8 (approximately 6 mL to reach a lipid concentration of 10 mg/mL); (10) chloroform dilution of 9 to a lipid concentration of 0.5 mg/mL.

EQUIPMENT AND MATERIALS The equipment and materials needed for the fabrication of iGUVs are shown in Figure 1. A more detailed list is given in the Supporting Information I.

(Figure 2, panel 3). Next, 4 mL of CHCl3 is added to the yolk/ water/methanol mixture, and the resulting mixture is vigorously stirred (Figure 2, panels 4 and 5). At this point, the proteins should be found in the polar water phase and the lipids in the chloroform phase. Because of the different polarities and densities of the solvents (water, MeOH, and chloroform), centrifugation (5 min at 100g) leads to complete phase separation with the water-rich phase on top, the MeOH-rich phase in the middle, and finally the chloroform phase at the bottom, seen as an orange-yellow-colored solution (Figure 2, panel 6). The lipid nature of the isolated material was verified by thin-layer chromatography (TLC) (Figure S1). Lipids are thus contained in this colored solution that can be extracted with a needled syringe. The lipid solution is transferred to a previously weighted glass vial and then carefully dried under vacuum (Figure 2, panels 7 and 8). The dried film appears as a yellowish sticky oil. The vial is then weighed again to obtain the yield of the lipid extraction by subtracting this value from the weight of the empty vial. Once the weight of the lipid extract is known, a solution at a known lipid concentration is prepared by addition of chloroform (Figure 2, panel 9). In a typical experiment, 60 mg of EYL extract is obtained from 0.5 mL of chicken egg yolk. The optimal lipid concentration for electroformation is 0.5 mg/mL (Figure 2, panel 10). Other protocols have been reported for purifying and characterizing lipids from food samples.8

Figure 1. Equipment and materials that are needed for the fabrication of giant unilamellar vesicles made from egg chicken lipids using a smartphone (iGUVs).

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HAZARDS Institutional chemical safety practices for organic solvents such as chloroform (CHCl3) and methanol (MeOH) and waste disposal should be observed. Recommended personal protective equipment includes chemical-resistant gloves, safety goggles, and a lab coat. Standard electrical safety practices should be observed.

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RESULTS AND DISCUSSION The realization of this experiment involves several sequential steps that will be described in different subsections for a better understanding of the whole activity.

Wiring Diagram: Connection from the Audio Jack Connector of the Smartphone to the Fabrication Chamber

Smart devices such as smartphones or tablets can transmit electrical signals with variable voltage and frequency through their audio input jack connector. Headphones transform the electrical signal into an audio signal with a particular intensity (volume) and frequency (i.e., the musical note la, A in Anglo-Saxon notation, corresponds to a frequency of 440 Hz). To transform the audio signal into an AC electrical signal, a 3.5 mm stereo tip−ring− sleeve (TRS) minijack connector must be connected to a stereo double wire using tin solder as indicated in Figure 3. Four wires are connected to the TRS connections as follows: one to the tip connection, another to the ring connection, and the remaining two to the sleeve connection. The opposite ends of the wires are connected to crocodile clips as shown in Figure 3. This assembly with two AC pairs allows two independent experiments to be run simultaneously, as each electroformation chamber has two electrodes (Figure 3). The electronic signal can be now transferred from the smartphone to the electroformation chamber.

Egg Lipid Extraction

The accessibility of highly purified or synthetic lipids is restricted in high school laboratories. However, natural lipids can be easily extracted and partially purified from chicken eggs.7 Egg yolk contains, together with proteins, a large amount of lipids. We present a simplified method to obtain an egg yolk lipid (EYL) extract for use without further purification (see Figure 2). To separate the lipids from the proteins, we take advantage of their different solubilities in polar/nonpolar solvents, since lipids dissolve much better than proteins in organic solvents. With this in mind, the extraction process can be described as follows. With a 1 mL syringe, 0.5 mL of chicken egg yolk is mixed with 1 mL of water (preferably distilled or demineralized, but this is not required) in a glass test tube (Figure 2, panels 1 and 2). After dilution and mixing, 2 mL of MeOH is added, and the whole suspension is gently stirred B




Figure 4. (A) Output voltage at the crocodile clamps using a function generator for the electroformation protocol (1.121 V). (B) Maximum output voltage at the crocodile clamps using a smartphone for the electroformation protocol (0.975 V).

Figure 3. (A) Wiring connection scheme for the use of a smartphone to output electric signals to the electroformation chamber. (B) Example of wiring connections.

Signal Generation

For most applications, electroformation requires sinusoidal electric signals at 1.1 V and 10 Hz. To generate sinusoidal waves with a particular intensity and frequency, there are several free apps available for iOS and Android (mobile phone operating systems) that generate audio waves. For example, SGenerator from ScorpionZZZ’s Lab, SigGen from AudioArtillery, and Audio Function Generator have given positive results (webpages are given in the Supporting Information I). These apps can generate wave signals with frequencies ranging from hertz to tens of kilohertz. The output frequency can be verified with an oscilloscope (not mandatory). It is recommended to use a multimeter before starting any experiment to check that the output voltage is ∼1 V. The master volume is used to tune the voltage intensity, reaching a maximal value of ∼0.97 V with the volume control in its maximum position and volume restriction for ear protection disabled if necessary (readers are referred to their user manuals to do this in their individual devices). This output voltage can be easily produced with an Apple iPad or different iPhones. All of these devices were able to obtain output voltages similar to those obtained with a function generator (see Figure 4).

Figure 5. (A) Scheme to build up an electroformation cell. (B) Dried lipids (EYL extract at 0.5 mg/mL) over an ITO-coated glass surface. (C) Electroformation chamber.

Electroformation

stacked and glued at the left side of the conductive face of each ITO slide. On the top of the spacer is placed a copper adhesive strip with dimensions of 0.4 cm × 3 cm. The copper strip is longer than the spacer strips, so it extends over the top of the slide and acts as an electrode to which the crocodile clips are connected. Then 10 μL of 0.5 mg/mL lipid solution is spread in two drops on each conductor surface. Because of the volatility of the organic solvent, the drops dry quickly, producing concentric circular traces (see Figure 5B). After the organic solvent evaporates, the fabrication chamber is sealed using adhesive water-resistant putty, which is laid around

Figure 5A shows a typical electroformation chamber composed of two conducting indium tin oxide (ITO)-coated glass slides (7.5 cm × 2.5 cm; 15 × 25 Ω/sq surface resistivity) that can be purchased from Sigma-Aldrich ($300 for a 10 unit pack). Low-cost ITO-coated slides can be also procured via the Internet (cheaper than $6 each on Adafruit Inc., for example). The conductive slides are spaced 1 mm apart by an adhesive flexible polyester (FEP) tape (Bytac), although any 0.25 mm thick adhesive insulating tape from a local hardware store can be used. To provide the 1 mm separation between slides, eight strips of about 0.5 cm × 2.5 cm are needed. Four of them are C




the lipid film so that an opening is formed through which the hydration medium will be transferred. The use of binder clips helps to ensure hermetic sealing of the cell (see Figure 5C). Then the lipid film is rehydrated with a 200 mM sucrose solution (approximately 700 mg of sugar in 10 mL of distilled water). Because of the small spacing between the two slides, the sucrose solution must be carefully added with a needled syringe to fill the chamber, avoiding the formation of air bubbles. Once the chamber is filled, the opening in the sealing paste is closed to close the chamber. Finally, the electrodes are connected to the smartphone AC power supply (10 Hz, ∼1 V) for at least 2 h to produce a large number of spherical iGUVs.

Figure 7. (A) Sucrose (200 mM) containing EYL iGUVs in a 100 mM NaCl solution. (B) Size distribution of EYL iGUVs electroformed with a smartphone.

Observation

The ITO-coated transparent slides allow real-time monitoring of vesicle formation with an optical microscope. Placing the whole setup on a microscope stage allows the vesicle growth course to be followed (Figure 6A). Figure 6B,C shows typical

observation fields of sediment vesicles are taken, focusing the microscope at different levels. For each vesicle, the true diameter is assumed when a bright halo is present around the vesicle.9 The size distribution of iGUVs can be represented in a histogram obtained from a population of 100 vesicles. As shown in Figure 7B, the average size for EYL iGUVs was 15 μm. Figure S3 shows a comparative analysis of the size and unilamellarity of giant vesicles electroformed using a function generator, using a smartphone, and in the absence of an AC field. Implementation and Potential Experiments with iGUVS

This experimental laboratory was run and tested with students from the first-year course on Physics for Biosciences (Biochemistry grade) at Universidad Complutense de Madrid (eight students during Summer 2016 and 24 students during Fall 2016). The experimental laboratory was scheduled in a series of three practice sessions, 4 hours each, on consecutive days. Students worked in pairs. A complete activity draft with the student lab handouts, the instructor notes, and student’s attempts is included in the Supporting Information II. Before each experimental section, students were asked to answer some prelab questions and then attended a 1 h lecture/discussion on electromechanical transduction, polarity, and lipid self-assembly issues. On day 1, students built the electroformation chamber and the connecting wires. On day 2, students prepared solutions, performed the lipid extraction, lipid spreading, lipid rehydration, and chamber connection to their smartphone. On day 3, students observed under optical microscopy their own iGUVs and performed a quantitative analysis of their size distribution. After the experimental part, a brief scientific evaluation was requested. Additionally, this experimental laboratory may be part of an extended biomembranes practical course, as iGUVs can be used as a veritable tool to show membrane-associated phenomena: (1) Membrane fluctuations can easily be observed with conventional optical microscopy and used to introduce membrane mechanics and its implications for deformability of red blood cells.10 (2) Shape changes of vesicles, including prolates and oblates, can be promoted just by enabling the outer medium to evaporate.11 The inner water of vesicles permeates to equilibrate the osmolarity gradient between the inside and outside by osmosis and permeability processes.12 (3) Budding events can be observed by externally adding different lipophilic molecules to prolate vesicles.11 (4) If lipids can be purchased and fluorescence microscopy is available, phase-separated vesicles can be electroformed

Figure 6. Swelling of vesicles made of EYL in 200 mM sugar with an AC electric field provided by a smartphone, as observed by bright-field microscopy. (A) Setup for live observation of electroswelling. (B, C) Lipid films (B) 2 h and (C) 3.5 h after application of a 10 Hz AC electric field. Dashed white lines indicate a giant vesicle produced by electroformation. The scale bars (10 μm) were calibrated by using the dimension of the screen of the photographic device, the number of pixels of the photo, and the total zoom used.

microscopy images of the electroswelling process of vesicles at 2 and 3.5 h of electroformation. It should be noted that these photos were shot with a smartphone that was carefully aligned to the eyepiece lens. Better image contrast can be obtained with an optical microscope in phase-contrast configuration. For better observation under the optical microscope, iGUVs can be transferred after electroformation to an observation chamber, which is composed of two circular microscope cover slides (diameter ϕ = 25 mm) separated by an O-ring spacer (2.5 mm thickness) (see Figure S2). A drop (50 μL) of isosmolar table salt solution (100 mM NaCl, ∼60 mg of salt in 10 mL of water) was placed at the center of the observation chamber. The density difference between the NaCl medium outside and the sucrose medium inside allowed the vesicles to sediment in the observation chamber, also providing a good optical contrast for bright-field microscopy. Then 25 μL of EYL iGUVs suspension was transferred to the observation chamber, which was immediately sealed with a cover glass to avoid water evaporation and consequent osmotic stress. Figure 7A shows EYL iGUVs as visualized with an inverted optical microscope (Nikon Eclipse TE2000, objective oil immersion 100×) equipped with a cooled CCD camera (Nikon DS-1QM, 14 frames per second (fps), 1 Mpixel). The iGUVs are almost spherical with a variable size ranging from 3 to 60 μm. Quantitative characterization of the EYL iGUVs can be performed by determining the size distribution of a population of giant vesicles. For that, several pictures of the different D



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CONCLUSIONS We have presented a do-it-yourself experiment to produce giant unilamellar vesicles using an egg yolk lipid extract and a smartphone. This experiment is inexpensive and easy to set up and therefore is appropriate to be performed in high school STEM courses or first-year undergraduate laboratory. Because of its interdisciplinary character, this activity is suitable to revisit basic scientific concepts and open discussions from different disciplines such as biophysical chemistry (biological colloids), biology (biomembranes), mathematics (statistics), basic chemistry (osmotic pressure), thermodynamics (phase transitions and transport phenomena), and electricity (AC current).

with complex lipid mixtures composed of sphingolipids, phospholipids, and cholesterol.13 Thermodynamics and the biological concept of “membrane rafts” can be presented. Moreover, phase transitions can be observed live by depleting and incorporating cholesterol with cyclodextrins in phase-separated vesicles.14 (5) Finally, light-driven chemical proton transport across membranes can be observed by reconstituting commercially available membrane proteins such as bacteriorhodopsin.15 Transport phenomena and membrane potential can thus be visually introduced with this experiment.

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PEDAGOGICAL GOALS AND ASSESSMENT The main pedagogic goal of this practical experiment is to acquire or revise various concepts issued from a basic undergraduate biological physical chemistry course through an integrative mini-project (see the Supporting Information II). In this way, the students consolidate basic fundamental knowledge from theoretical courses and their transversal skills such as observation, rationale, and creative (manufacturing) abilities. We confront the students with a particular scientific problem, the electroformation of giant unilamellar vesicles made of natural lipids, to improve their skills in data acquisition, data analysis, and data interpretation a part of their scientific training. On the fundamental point of view, we want to train the students on the following: • the chemical principles of lipid extraction from a natural source and the partition of organic compounds in solvents with different polarities; • the conditions and process of lipid self-assembly and its significance for the composition and organization of the lipid matrix in a cell membrane; • the physical basics of electroformation; • the physics of optical microscopy; • the handling of basic procedures in chemistry and physics, including safety lab procedures, manipulation of solvents, optical microscopy observation, and basics of electronics. To assess the grade of the students’ learning and performance, we have optioned to use two different strategies: concept tests and assessing the group work.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00951. Complete list of materials, more details on the chemical nature of lipids, a figure showing the observation chamber, a figure showing the size and unilamellarity distributions for giant vesicles made by different methods, and cautions and alternative options for experimental procedures (PDF, DOCX) Student lab handouts and instructor notes (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Iván López-Montero: 0000-0001-8131-6063 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I.L.-M. acknowledges the European Research Council (ERC) (Grant ERC-StG-2013-338133 titled “mitochon”) and the “Ramón y Cajal” Program (RyC-2013-12609) from the Spanish Ministry of Economy. We thank MINECO-Spain for financial support under grant FIS2015-70339-C2-1-R.

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Concept Tests

A series of tests have been proposed (see the Supporting Information II) as short, informal, and targeted questions consisting of multiple-choice questions. Students were asked to select the best answer and submit it by raising their hands to get a snapshot of the current understanding of the whole class and not of an individual student. At the end of each practical session, individual tests were distributed to each student. We designed questions to ask students to predict the outcome of a new experimental setup, to apply rules or principles to new molecules, or to report their own results.

REFERENCES

(1) Luisi, P. L.; Walde, P.; Oberholzer, T. Lipid vesicles as possible intermediates in the origin of life. Curr. Opin. Colloid Interface Sci. 1999, 4 (1), 33−38. (2) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; John Wiley & Sons: Somerset, NJ, 1980. (3) Kurihara, K.; Okura, Y.; Matsuo, M.; Toyota, T.; Suzuki, K.; Sugawara, T. A recursive vesicle-based model protocell with a primitive model cell cycle. Nat. Commun. 2015, 6, 8352. (4) Angelova, M.; Soleau, S.; Meleard, P.; Faucon, J. F.; Bothorel, P. Preparation of giant vesicles by external a.c. electric fields. Kinetics and applications. Prog. Colloid Polym. Sci. 1992, 89, 127−131. (5) Mathivet, L.; Cribier, S.; Devaux, P. F. Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys. J. 1996, 70 (3), 1112−1121. (6) Del Bianco, C.; Torino, D.; Mansy, S. S. Vesicle Stability and Dynamics: An Undergraduate Biochemistry Laboratory. J. Chem. Educ. 2014, 91 (8), 1228−1231.

Team Work

During practical sessions, we asked different groups to submit some deliverables (wirings, electroformation chamber, lipid extracts, or visual verification of iGUVs). In this way we assessed the group work, giving the same mark and grade to all group members, thus encouraging coordinated team work during the session. E




(7) Folch, J.; Lees, M.; Sloane Stanley, G. H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957, 226 (1), 497−509. (8) Bendinskas, K.; Weber, B.; Nsouli, T.; Nguyen, H. V.; Joyce, C.; Niri, V.; Jaskolla, T. W. A Teaching Laboratory for Comprehensive Lipid Characterization from Food Samples. J. Chem. Educ. 2014, 91 (10), 1697−1701. (9) Pécréaux, J.; Döbereiner, H. G.; Prost, J.; Joanny, J. F.; Bassereau, P. Refined contour analysis of giant unilamellar vesicles. Eur. Phys. J. E: Soft Matter Biol. Phys. 2004, 13 (3), 277−290. (10) Boal, D. H. Mechanics of the Cell; Cambridge University Press: Cambridge, U.K., 2002. (11) López-Montero, I.; Rodriguez, N.; Cribier, S.; Pohl, A.; Vélez, M.; Devaux, P. F. Rapid transbilayer movement of ceramides in phospholipid vesicles and in human erythrocytes. J. Biol. Chem. 2005, 280 (27), 25811−25819. (12) Seu, K. J. Osmotic Stressing, Membrane Leakage, and Fluorescence: An Introductory Biochemistry Demonstration. J. Chem. Educ. 2015, 92 (9), 1522−1525. (13) Veatch, S. L.; Keller, S. L. Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and CholesterolSeparation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol. Biophys. J. 2003, 85 (5), 3074−3083. (14) Bacia, K.; Scherfeld, D.; Kahya, N.; Schwille, P. Fluorescence Correlation Spectroscopy Relates Rafts in Model and Native Membranes. Biophys. J. 2004, 87 (2), 1034−1043. (15) Dezi, M.; Di Cicco, A.; Bassereau, P.; Lévy, D. Detergentmediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (18), 7276−7281.

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iGUVs: Preparing Giant Unilamellar Vesicles with a Smartphone and

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