The authors gratefully acknowledge the ANR for financial support (ANR Sysa).

1. Fabricating micropipettes
NOTE: Here, micropipettes with an inner diameter ranging from 6 to 12 &#181;m and a taper length around 3-4 mm are necessary. A detailed method of manufacturing micropipette is described in the following.
<ol>
	<li>Place the borosilicate glass capillary in the drawbar of the puller and fix one of the ends by tightening the knob.</li>
	<li>Carefully slide the glass through the holes at the side of the heater chamber.</li>
	<li>Tighten down the clamping knob at the other end.</li>
	<li>Control the size of the tip and the taper length to achieve the desired specifications. For that, optimize technical parameters such as heating temperature, pull, velocity, delay and pressure. Here is an example of a program used: 
	Heat: 550 &#176;C 
	Pull: 10 (Range of the machine: 0-255 in arbitrary units) 
	Velocity: 30 (Range: 0-255 in arbitrary units) 
	Delay: 1 (Range: 0-255 in arbitrary units) 
	Pressure: 500 (Range: 0-999 in arbitrary units)</li>
	<li>Click on PULL to execute the events defined by the program. The capillary is then separated into two micropipettes, whose dimensions have to be adjusted using a microforge.</li>
	<li>Insert the micropipette into the metal pipette holder of the microforge (see Figure 1). By using 10x objective, adjust the microscope stage and the pipette manipulator (vertical and horizontal movement) until the pipette tip is close to the glass bead surface.</li>
	<li>Press the foot switch to melt the glass bead. Lower the tip and put it in contact with the molten glass bead. Molten glass will flow into the pipette by capillary action. Wait a few seconds until the level of the molten glass reaches a certain height as shown in the insert of the Figure 1.</li>
	<li>Stop the heating by removing the pressure on the foot switch, and quickly pull the tip away using the vertical pipette manipulator to cause a sharp break.</li>
	<li>Repeat steps 1.7 and 1.8 until the desired diameter is obtained (6 to 12 &#181;m). 
	NOTE: To improve the accuracy of the diameter measurement, during the last step, use a 32x objective equipped with a reticle.</li>
</ol>
2. Coating pipette tips with BSA (bovine serum albumin)
<ol>
	<li>To prepare a 0.1 M solution of glucose containing 1% wt. BSA in pure water.
	<ol>
		<li>Weigh 180 mg of glucose powder, place in a 15 mL polypropylene conical tube and complete with 10 mL of pure water.</li>
		<li>Add 0.1 g of BSA powder and shake gently using a test tube rotatory mixer until complete dissolution (approximately 4 h).</li>
	</ol>
	</li>
	<li>Take the solution with a 10 mL disposable Luer syringe fitted with a needle. Once filled, remove the needle and install a 0.22 &#956;m acetate cellulose filter. Fill several polypropylene micro-centrifuge tubes (1.5 mL) that will be used to immerse the tip.</li>
	<li>Place the capillaries vertically into holders. Lower the holder and immerse the tip into the glucose/BSA solution overnight. The solution should rise about 1 cm high by capillary action.</li>
	<li>Remove the pipette tip from the glucose/BSA solution. Prepare 5 mL of 0.1 M glucose solution (dilute 90 mg of glucose powder in 5 mL of pure water) and filter through a 0.22 &#956;m acetate cellulose filter.</li>
	<li>Fill the pipette with the glucose solution by using a 500 &#181;L glass syringe equipped with a flexible fused silica capillary. Then, remove all the glucose solution by sucking it back and discard it (Figure 2). Repeat this step several times to remove the unbounded BSA.</li>
</ol>
3. Formation of GUVs and GHUVs by electroformation
NOTE: Electroformation is a commonly used technic developed by Angelova9. The procedures to obtain an electroformation chamber, deposit a lipid or polymer film (or both for GHUVs (Giant Hybrid Unilamellar Vesicles)) and hydrate the film under an alternative electric field are described in the following. The procedure to collect the GUV obtained is also described.
<ol>
	<li>Amphiphile, sucrose and glucose solutions preparation
	<ol>
		<li>Prepare an amphiphile solution at a concentration of 1 mg/mL. Weigh 10 mg of amphiphile and dissolve in 10 mL of chloroform. Keep the solution in sealed vials to avoid solvent evaporation.</li>
		<li>Prepare a stock solution of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rhod) at 1 mg/mL in chloroform.</li>
		<li>Add 10 &#181;L of fluorescent lipid solution to the amphiphile solution. Keep the solutions in sealed vials to avoid solvent evaporation.</li>
		<li>Prepare sucrose and glucose solutions at a concentration of 0.1 M. Weigh 342 mg and 180 mg of sucrose and glucose, respectively, and dissolve them in 10 mL of pure water.</li>
	</ol>
	</li>
	<li>Preparation of the electroformation chamber 
	NOTE: Different conductive materials can be used to make an electroformation device (e.g., platinum wires10, stainless needles11). The electroformation chamber is composed of two ITO slides separated by an O-ring rubber spacer that has been cut on one side to create an aperture. The slides are connected to a voltage generator via two electric wires (Figure 3 and Figure 4A).
	<ol>
		<li>Clean the ITO slides with organic solvent (e.g., chloroform). Identify the conductive surface using an ohmmeter.</li>
		<li>Attach the electrical wires on the conductive side using adhesive tape.</li>
		<li>Amphiphile solution deposition
		<ol>
			<li>Dip a capillary in the solution until the level increases by capillary action and collect about 5 &#956;L of the solution.</li>
			<li>Put the capillary in contact with the center of the ITO glass plate and gently spread the solution. Wait 10 seconds to ensure complete solvent evaporation (Figure 4A).</li>
			<li>Repeat this procedure 3 times for each side.</li>
		</ol>
		</li>
		<li>Add a layer of silicon-free grease on both sides of the opened O-ring spacer. Put it around the area of deposition. Place the conductive face of the second ITO glass plate on the top of the spacer.</li>
		<li>Place the electroformation chamber under vacuum for 3 hours to remove any traces of organic solvent.</li>
	</ol>
	</li>
	<li>Electroformation procedure
	<ol>
		<li>Plug the electric wires to the generator.</li>
		<li>Use the following settings for the generator: 
		Alternative sinusoidal tension 
		Frequency: 10 Hz 
		Amplitude: 2 Vpeak-to-peak 
		NOTE: Optimum voltage frequency and duration must be found for each system.</li>
		<li>Ensure that the voltage is applied before injection of the solution in the chamber.</li>
		<li>Inject 1 mL of solution using a syringe with 0.8 mm inner diameter needle to fill the chamber. Remove eventual bubbles.</li>
		<li>Let the chamber under the applied voltage/frequency for 75 min (Figure 4B).</li>
	</ol>
	</li>
	<li>GUVs harvest
	<ol>
		<li>Switch off the generator.</li>
		<li>Using 1 mL syringe with 0.8 mm inner diameter needle, suck a small part of the solution in order to produce an air bubble inside the chamber. Slightly tilt the chamber in order to make this bubble move inside the chamber. This can help the GUVs to detach from the ITO surface (Figure 4C).</li>
		<li>Suck all the solution and transfer it into a 1 mL plastic tube.</li>
		<li>Remove the wires and clean the ITO slides with organic solvents (toluene then chloroform).</li>
	</ol>
	</li>
</ol>
4. Micromanipulation set up
NOTE: The principle of micropipette aspiration is to suck a single vesicle through a glass micropipette by applying a depression. The length of the tongue inside the pipette is measured as a function of the suction pressure. The pipette coating with BSA, described previously, is essential to prevent or minimize any adhesion between vesicles membranes and the pipette.
The protocol is illustrated below.
<ol>
	<li>Pipette and water filled reservoir connection 
	NOTE: The water filled tank and the micrometer are fixed to a sliding plate. A digital counter with a micrometer head allows a vertical displacement of the device in a range of 0 to 2.5 cm and an accuracy of 1 &#181;m. Displacement along an aluminum optical rail is possible up to 1 meter in length. A silicon tubing connects the reservoir and the capillary holder (Figure 5A).
	<ol>
		<li>Fill the tank with pure water. Connect a disposable 5 mL syringe to the capillary metal holder via silicone tubing and aspirate to create a water flow from the tank to the holder.</li>
		<li>Touch the tube slightly to eliminate air bubbles. Simultaneously, raise the water tank to create a positive pressure. The 5 mL syringe is still attached to the holder.</li>
		<li>After coating and cleaning steps described previously (see step 2), fill a capillary with glucose solution until a drop forms at the end. Remove the syringe tubing from the metal holder to create a slight water flow at its end.</li>
		<li>Turn the capillary upside down and connect the glucose drop with the water flow from the holder. Fix the capillary and the holder by screwing them together.</li>
	</ol>
	</li>
	<li>Position a pipette 
	NOTE: During this operation, the water tank is still positioned on the aluminum rail to maintain a positive pressure.
	<ol>
		<li>Take the homemade aluminum stage equipped with two glass slides (previously cleaned with ethanol) and glue them with vacuum grease on each side. Install it on the microscope stage and form a meniscus between the two slides by using a 1 mL syringe containing 0.1 M of glucose as shown in Figure 5B,C.</li>
		<li>Place the pipette and its holder on the motor unit of the micromanipulator and tighten down clamping knob.</li>
		<li>Use the control panel joystick in coarse mode to lower the micropipette near the glucose meniscus. Adjust the position of the tip to the center of the meniscus using the fine mode.</li>
		<li>Hold the tip immersed in glucose for a few minutes to clean its outer and inner surface (as a positive pressure is maintained, the water flow will rinse the inner surface of the pipette to remove uncoated BSA).</li>
		<li>Store the position of the tip on the micromanipulator keyboard and withdraw it from the meniscus.</li>
		<li>Remove the glucose meniscus and replace it by a fresh one. Suck 2 &#956;L of GUVs in 0.1 M sucrose by using a 20 &#956;L micropipette and put it in the fresh glucose meniscus. Observe in DIC mode (differential interference contrast) the GUVs located at the bottom due to the difference of density between sucrose (mainly inside the GUVs) and glucose (mainly outside the GUVs).</li>
		<li>When the vesicles are slightly deflated, reinsert the suction pipette and focus on the tip. Set the height H0 of the water tank for which pressure is almost 0. For that, take advantage of small vesicles or particles which are naturally present in solution and adjust the water tank height until the motion of these particles is stopped.</li>
		<li>At this point, surround the meniscus with mineral oil to prevent water evaporation, see Figure 5D. 
		NOTE: The room temperature should be controlled between 20-22 &#176;C by using air conditioning.</li>
	</ol>
	</li>
	<li>Micropipette aspiration experiment
	<ol>
		<li>Lower the pipette tip (6-12 &#181;m in diameter) and create a small suction (-1 cm) to aspirate a vesicle (15-30 &#181;m in diameter). The membrane of the selected vesicle should slightly fluctuate, and must not present any visible defects (no bud or filament) (Figure 6).</li>
		<li>Raise the pipette to a higher level to isolate the aspirated vesicle from the other vesicles, by using the micromanipulator and keep this position during the whole experiment.</li>
		<li>Pre-stress the vesicle by lowering the water filled tank to approximately -10 cm, then decrease the pressure to return to its initial value (-1 cm). Repeat this step several times to remove membrane excess and small defects from the membrane.</li>
		<li>From a height of -0.5 cm defined by the position of the water tank, slowly decrease the suction pressure with the micrometer to reach a regime in which the membrane fluctuates. Then increase the pressure to clearly visualize a tongue in the tip with a significant projection length (a few microns). 
		NOTE: The lowest applied pressure (P0) that allows sucking up the smallest membrane projection length (L0) will define the reference point &#945;0 (Figure 7A). All points of the curve will be measured according to this reference (&#916;L = L-L0 and &#916;P = P-P0).</li>
		<li>Increase the suction pressure with the micrometer in a stepwise manner until reaching 0.5 -0.8 mN/m. At each step, wait 5 s and take a snapshot of the tongue. This procedure at low tension enables determination of the bending modulus.</li>
		<li>Keep on increasing the suction pressure from 0.5 mN/m to the rupture tension by adjusting the height of the water filled sliding on the rail (ranging from -2 to -50 cm) (Figure 7B-D). From this experiment at high tension regime, the area compressibility modulus, lysis tension and lysis strain will be measured.</li>
		<li>Stretch about 15-20 vesicles to acquire significant statistics. Each micropipette aspiration experiment takes between 7 and 10 minutes. Perform image analysis using LASAF software for measuring the projection length of the tongue, the diameter of vesicles and the radius of the capillary.</li>
	</ol>
	</li>
	<li>Measuring bending modulus, area compressibility modulus, lysis tension and lysis strain
	<ol>
		<li>To access these parameters, use the formalism established by E. Evans12. Calculate the suction pressure applied over the membrane from equation 1: 
		&#916; P = &#961;wg (h&#8722;h0) (1) 
		where g is the gravitational acceleration (9.8 m&#8729;s&#8722;2), &#961;w is the density of the water (&#961; = 1 g&#8729;cm&#8722;3), h is the position of the water tank and h0 is the initial position where pressure is equal to zero.</li>
		<li>Calculate the membrane tension from the Laplace equation: 
		<img alt="Equation 1" src="/files/ftp_upload/60199/60199eq1.jpg" /> (2) 
		where &#916;P is the suction pressure on the micropipette, Rp and Rv are the micropipette and vesicle radii (outside the micropipette) respectively. The surface area strain (&#945;) of the membrane is defined as: 
		<img alt="Equation 2" src="/files/ftp_upload/60199/60199eq2.jpg" /> (3) 
		<img alt="Equation 3" src="/files/ftp_upload/60199/60199eq3.jpg" /> being the membrane area of the vesicle at the lower suction pressure.</li>
		<li>Calculate &#945; from the increase in projection length &#916;L of vesicle inside the capillary tip according to Equation 412: 
		<img alt="Equation 4" src="/files/ftp_upload/60199/60199eq4.jpg" /> (4)</li>
		<li>Under a very low tension regime, smoothing of thermal bending undulations dominates the apparent expansion. Plot ln(&#963;) vs &#945;. At low-&#963; values (typically 0.001&#8211;0.5 mN.m&#8722;1 13), this gives a straight line whose slope is linked to the bending modulus, Kb (first term of the equation 5)14: 
		<img alt="Equation 5" src="/files/ftp_upload/60199/60199eq5.jpg" /> (5) 
		NOTE: Under high tensions (&#62; 0.5 mN.m&#8722;1), membrane undulations are completely suppressed and membrane area increases as the result of increased spacing between molecules. In this regime, the second term of equation 5 dominates and give access to area compressibility modulus Ka (Figure 8 and Figure 9).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The coating of the micropipette is one of the key points to obtain reliable measurements. Adhesion of the vesicle to the micropipette must be prevented, and a coating is commonly used in literature17,18,19,20,21, with BSA, &#946;-casein or surfasil. Details of the coating procedure are rarely mentioned.
Dissolution of the BSA should...

Giant vesicles obtained from phospholipids (liposomes) have been widely used since the 1970s as the basic cell membrane model1. In the late 1990s, vesicular morphologies obtained from the self-assembly of copolymers, named polymersomes in reference to their lipid analogs2,3, rapidly appeared as an interesting alternative to liposomes that possess weak mechanical stability and poor modular chemical functionality. However, their cell biomimetic character is rather limited compared to liposomes since the latter are composed of phospholipids, the main component of the cell membrane. Furthermore, their low membrane permeability can be an issue in some applications like drug delivery where controlled diffusion of species through the membrane is required. Recently, the association of phospholipids with block copolymers to design hybrid polymer-lipid vesicles and membranes has been the subject of an increasing number of studies4,5. The main idea is to design entities that synergistically combine the benefits of each component (bio-functionality and permeability of lipid bilayers with the mechanical stability and chemical versatility of polymer membranes), which can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, development of bio-inspired micro-/nano-reactors.
Nowadays, different scientific communities (biochemists, chemists, biophysicists, physico-chemists, biologists) have increasing interest in development of a more advanced cell membrane model. Here, our goal is to present, as detailed as possible, existing methodologies (electroformation, micropipette aspiration) to obtain and characterize the mechanical properties of giant vesicles and the recent &#34;advanced&#34; cell membrane models that are hybrid polymer lipid giant vesicles4,5.
The purpose of these methods is to obtain reliable measurement of the area compressibility and bending moduli of the membrane as well as their lysis stress and strain. One of the most common techniques existing to measure bending rigidity of a giant vesicle is fluctuation analysis6,7, based on direct video microscope observation; but this requires large visible membrane fluctuation, and is not systematically obtained on thick membranes (e.g. polymersomes). Area compressibility modulus can be experimentally determined using the Langmuir Blodgett technique but most often on a monolayer8. The micropipette aspiration technique allows the measurement of both moduli on a bilayer forming giant unilamellar vesicle (GUV) in one experiment.
The following method is appropriate for all amphiphilic molecules or macromolecules able to form bilayers and, consequently, vesicles by electroformation. This requires a fluid character of the bilayer at the temperature of electroformation.

<table>
	<tbody>
		<tr>
			<td>Required equipment and materials for micropipette design</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td>external and internal diameter of 1mm and 0.58 mm respectively.</td>
		</tr>
		<tr>
			<td>Filament installed</td>
			<td>Sutter Instrument Co.</td>
			<td>FB255B</td>
			<td>2.5mm*2.5mm Box Filament</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>NARISHGE Co.</td>
			<td>MF-900</td>
			<td>fitted with two objectives (10x and 32x)</td>
		</tr>
		<tr>
			<td>Materials for coating pipette tips with BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>10735078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>62.1612</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 10 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>626616</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 5 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>62.5607</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable acetate cellulose filter</td>
			<td>Cluzeau Info Labo</td>
			<td>L5003SPA</td>
			<td>Pore size: 0.22&#181;m, diameter: 25mm</td>
		</tr>
		<tr>
			<td>Flexible Fused Silica Capillary Tubing</td>
			<td>Polymicro Technologies.</td>
			<td>TSP530660</td>
			<td>Inner Diameter 536&#181;m, Outer Diameter 660&#181;m,</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 500 &#181;L luer Lock GASTIGHT</td>
			<td>Hamilton Syringe Company</td>
			<td>1750</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube rotatory mixer</td>
			<td>Labinco</td>
			<td>28210109</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulation Set up</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Optical Rail, 1000 mm Length, M4 threads, X48 Series</td>
			<td>Newport</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Damped Optical Table</td>
			<td>Newport</td>
			<td></td>
			<td>used as support of microscope to prevent external vibrations.</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Eppendorf</td>
			<td>Patchman NP 2</td>
			<td>The module unit (motor unit for X, Y and Z movement) is mounted on the inverted microscope by the way of an adapter.</td>
		</tr>
		<tr>
			<td>Micrometer</td>
			<td>Mitutoyo Corporation</td>
			<td>350-354-10</td>
			<td>Digimatic LCD Micrometer Head 25,4 mm Range 0,001 mm</td>
		</tr>
		<tr>
			<td>Plexiglass water reservoir (100 ml)</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP5 inverted confocal microscope (DMI6000) equipped with a resonant scanner and a water immersion objective (HCX APO L 40x/0.80 WU-V-I).</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X48 Rail Carrier 80 mm Length,with 1/4-20, 8-32 and 4-40 thread</td>
			<td>Newport</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for sucrose and amphiphile solution preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.sigmaaldrich.com/catalog/substance/2oleoyl1palmitoylsnglycero3phosphocholine760082685331611" target="_blank">2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine</a></td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>22711.244</td>
			<td></td>
		</tr>
		<tr>
			<td>L-&#945;-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl)</td>
			<td>Sigma-Aldrich</td>
			<td>810146C</td>
			<td>Rhodamine tagged lipid</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroformation set up</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L glass capillary ringcaps</td>
			<td>Hirschmann</td>
			<td>9600110</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml syringe Luer Tip</td>
			<td>Codan</td>
			<td>62.1612</td>
			<td></td>
		</tr>
		<tr>
			<td>H Grease</td>
			<td>Apiezon</td>
			<td>Apiezon H Grease</td>
			<td>Silicon-free grease</td>
		</tr>
		<tr>
			<td>Indium tin oxide coated glass slides</td>
			<td>Sigma-Aldrich</td>
			<td>703184</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>AN2138R1</td>
			<td>0.8 x 38 mm</td>
		</tr>
		<tr>
			<td>Ohmmeter (Multimeter)</td>
			<td>Voltcraft</td>
			<td>VC140</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>VWR</td>
			<td>28676.297</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltage generator</td>
			<td>Keysight</td>
			<td>33210A</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtention of giant unilamellar hybrid vesicles by electroformation and measurement of their mechanical properties by micropipette aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

With the protocol aforementioned, we have studied different synthetic giant unilamellar vesicle (GUV), obtained from a phospholipid: 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), a triblock copolymer: Poly(ethyleneoxide)-b-Poly(dimethylsiloxane)-b-Poly(ethyleneoxide) (PEO12-b-PDMS43-b-PEO12) synthesized in a previous study13, and a diblock copolymer Poly(dimethylsiloxane)-b...

Watch this Scientific Journal Video about Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration at JoVE.com

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

obtention-giant-unilamellar-hybrid-vesicles-electroformation

Research

JoVE Journal

Chemistry

8.3K Views.  CNRS, Bordeaux INP, LCPO, UMR 5629. The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Video: Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Fabricating Nanogaps by Nanoskiving

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two electrodes-and generating them in practical quantities-is still challenging. The preparation of nanogap electrodes using nanoskiving, which is a form of edge lithography, is a fast, simple and powerful technique. This method is an entirely mechanical process which does not include any photo- or electron-beam lithographic steps and does not require any special equipment or infrastructure such as clean rooms. Nanoskiving is used to fabricate electrically addressable nanogaps with control over all three dimensions; the smallest dimension of these structures is defined by the thickness of the sacrificial layer (Al or Ag) or self-assembled monolayers. These wires can be manually positioned by transporting them on drops of water and are directly electrically-addressable; no further lithography is required to connect them to an electrometer.

The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties

We demonstrate a novel synthesis strategy for the preparation of Pr-doped SrTiO3 ceramics via a combination of solid state reaction and spark plasma sintering techniques. Polycrystalline ceramics possessing a unique morphology can be achieved by optimizing the process parameters, particularly spark plasma sintering heating rate. The phase and morphology of the synthesized ceramics were investigated in detail using X-ray diffraction, scanning electron microcopy and energy-dispersive X-ray spectroscopy. It was observed that the grains of these bulk Pr-doped SrTiO3 ceramics were enhanced with Pr-rich grain boundaries. Electronic and thermal transport properties were also investigated as a function of temperature and doping concentration. Such a microstructure was found to give rise to improved thermoelectric properties. Specifically, it resulted in a significant improvement in carrier mobility and the thermoelectric power factor. Simultaneously, it also led to a marked reduction in the thermal conductivity. As a result, a significant improvement (> 30%) in the thermoelectric figure of merit was achieved for the whole temperature range over all previously reported maximum values for SrTiO3-based ceramics. This synthesis demonstrates the steps for the preparation of bulk polycrystalline ceramics of non-uniformly Pr-doped SrTiO3.

Synthesis of Non-uniformly Pr-doped SrTiO3 Ceramics and Their Thermoelectric Properties

A protocol for the synthesis and processing of polycrystalline SrTiO3 ceramics doped non-uniformly with Pr is presented along with the investigation of their thermoelectric properties.

We demonstrate a novel synthesis strategy for the preparation of Pr-doped SrTiO3 ceramics via a combination of solid state reaction and spark plasma ...

Synthesis and Characterization of Fe-doped Aluminosilicate Nanotubes with Enhanced Electron Conductive Properties

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe aims at lowering the band gap of imogolite, an insulator with the chemical formula (OH)3Al2O3SiOH, and at modifying its adsorption properties towards azo-dyes, an important class of organic pollutants of both wastewater and groundwater.
Fe-doped nanotubes are obtained in two ways: by direct synthesis, where FeCl3 is added to an aqueous mixture of the Si and Al precursors, and by post-synthesis loading, where preformed nanotubes are put in contact with a FeCl3&#8226;6H2O aqueous solution. In both synthesis methods, isomorphic substitution of Al3+ by Fe3+ occurs, preserving the nanotube structure. Isomorphic substitution is indeed limited to a mass fraction of ~1.0% Fe, since at a higher Fe content (i.e., a mass fraction of 1.4% Fe), Fe2O3 clusters form, especially when the loading procedure is adopted. The physicochemical properties of the materials are studied by means of X-ray powder diffraction (XRD), N2 sorption isotherms at -196 &#176;C, high resolution transmission electron microscopy (HRTEM), diffuse reflectance (DR) UV-Vis spectroscopy, and &#950;-potential measurements. The most relevant result is the possibility to replace Al3+ ions (located on the outer surface of the nanotubes) by post-synthesis loading on preformed imogolite without perturbing the delicate hydrolysis equilibria occurring during nanotube formation. During the loading procedure, an anionic exchange occurs, where Al3+ ions on the outer surface of the nanotubes are replaced by Fe3+ ions. In Fe-doped aluminosilicate nanotubes, isomorphic substitution of Al3+ by Fe3+ is found to affect the band gap of doped imogolite. Nonetheless, Fe3+ sites on the outer surface of nanotubes are able to coordinate organic moieties, like the azo-dye Acid Orange 7, through a ligand-displacement mechanism occurring in an aqueous solution.

Here, we present a protocol to synthesize and characterize Fe-doped aluminosilicate nanotubes. The materials are obtained by either sol-gel synthesis upon addition of FeCl3&#8226;6H2O to the mixture containing the Si and Al precursors or by post-synthesis ionic exchange of preformed aluminosilicate nanotubes.

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

TiO2-coated Hollow Glass Microspheres with Superhydrophobic and High IR-reflective Properties Synthesized by a Soft-chemistry Method

This manuscript proposes a soft-chemistry method to develop superhydrophobic and highly IR-reflective hollow glass microspheres (HGM). The anatase TiO2 and a superhydrophobic agent were coated on the HGM surface in one step. TBT and PFOTES were selected as the Ti source and the superhydrophobic agent, respectively. They were both coated on the HGM, and after the hydrothermal process, the TBT turned to anatase TiO2. In this way, a PFOTES/TiO2-coated HGM (MCHGM) was prepared. For comparison, PFOTES single-coated HGM (F-SCHGM) and TiO2 single-coated HGM (Ti-SCHGM) were synthesized as well. The PFOTES and TiO2 coatings on the HGM surface were demonstrated through X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive detector (EDS) characterizations. The MCHGM showed a higher contact angle (153&#176;) but a lower sliding angle (16&#176;) than F-SCHGM, with a contact angle of 141.2&#176; and a sliding angle of 67&#176;. In addition, both Ti-SCHGM and MCHGM displayed similar IR reflectivity values, which were about 5.8% higher than the original HGM and F-SCHGM. Also, the PFOTES coating barely changed the thermal conductivity. Therefore, F-SCHGM, with a thermal conductivity of 0.0479 W/(m&#183;K), was quite like the original HGM, which was 0.0475 W/(m&#183;K). MCHGM and Ti-SCHGM were also similar. Their thermal conductivity values were 0.0543 W/(m&#183;K) and 0.0543 W/(m&#183;K), respectively. The TiO2 coating slightly increased the thermal conductivity, but with the increase in reflectivity, the overall heat-insulation property was enhanced. Finally, since the IR-reflecting property is provided by the HGM coating, if the coating is fouled, the reflectivity decreases. Therefore, with the superhydrophobic coating, the surface is protected from fouling, and its lifetime is also prolonged.

TiO2-coated Hollow Glass Microspheres with Superhydrophobic and High IR-reflective Properties Synthesized by a Soft-chemistry Method

This manuscript proposes a soft-chemistry method to synthesize superhydrophobic, TiO2-coated hollow glass microspheres (HGM) with high IR-reflective properties.

This manuscript proposes a soft-chemistry method to develop superhydrophobic and highly IR-reflective hollow glass microspheres (HGM). The anatase TiO2 ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...