Protocol for building synthetic protocell membranes that sense redox using synthetic phospholipids and natural lipids

Summary

Sensing is a critical function of artificial cells; however, this is challenging to realize using bottom-up approaches. Here, we present a protocol for building protocell membranes that sense cues important for redox biochemistry and signaling by combining synthetic phospholipids and natural lipids. We detail procedures for building giant unilamellar vesicles as protocell models that fluoresce in response to the biologically significant redox agents peroxynitrite, hydrogen peroxide, and hydrogen sulfide.

For complete details on the use and execution of this protocol, please refer to (i) Gutierrez and Aggarwal et al.¹ as well as (ii) Erguven and Wang et al.²

Graphical abstract

Highlights

• •Preparation of diverse synthetic lipids with modular designs from a common precursor
• •Generation of giant unilamellar vesicles (GUVs) with chemically functional lipids
• •Demonstration by confocal imaging of how GUVs appear before and after redox treatment

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Sensing is a critical function of artificial cells; however, this is challenging to realize using bottom-up approaches. Here, we present a protocol for building protocell membranes that sense cues important for redox biochemistry and signaling by combining synthetic phospholipids and natural lipids. We detail procedures for building giant unilamellar vesicles as protocell models that fluoresce in response to the biologically significant redox agents peroxynitrite, hydrogen peroxide, and hydrogen sulfide.

Before you begin

We recommend researchers to handle the described chemicals with caution to maintain their integrity and function. Specifically, it is essential to keep the lipids (Figure 1) dry (without solvent) at ultra-low temperatures (e.g., −80°C) to minimize degradation (e.g., hydrolysis or side reactions from trace impurities) prior to use. Store the fluorogenic lipids in amber vials to protect them from light and minimize photobleaching. Aliquot the lipids into multiple batches to avoid reusage and potential warming-cooling cycles. Redox agents should be handled with care. We recommend the use of either NaSH or Na₂S as the source of H₂S, which is highly toxic when used in bulk.

Figure 1.

Structures of the phospholipids used in the described methods

(A) Synthetic routes to the probes DPPC-TC-ONOO^–, DPPC-TF-H₂O₂, and DPPC-TC-H₂S from the easily accessible 1,2-dipalmitoyl-rac-glycerol.

(B) The key amphiphilic module used for lipidation of all the redox-sensing modules.

(C) Activity-based sensing of redox cues using phospholipid-based probes and their fluorescently activated redox products. DPPC-TC-ONOO^–: [1,2-Dipalmitoyl-rac-glycero-3-phosphocholine]–[triazole-coumarin]–[peroxynitrite-reactive module]; DPPC-TF-H₂O₂: [1,2-Dipalmitoyl-rac-glycero-3-phosphocholine]–[triazole-fluorescein]–[hydrogen peroxide-reactive module]; DPPC-TC-H₂S: [1,2-Dipalmitoyl-rac-glycero-3-phosphocholine]–[triazole-coumarin]–[hydrogen sulfide-reactive module]; DPPC-TC: [1,2-Dipalmitoyl-rac-glycero-3-phosphocholine]–[triazole-coumarin]. DPPC-TF: [1,2-Dipalmitoyl-rac-glycero-3-phosphocholine]–[triazole-fluorescein]. Each redox-sensing module is connected with alkynyl rac-DPPC through a copper-catalyzed azide−alkyne cycloaddition (“click”) reaction.⁶

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
1,2-Dipalmitoyl-rac-glycerol	MilliporeSigma	CAS # 40290-32-2
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)	Avanti Polar Lipids	CAS # 384833-00-5
3-Dimethylamino-1-propyne	MilliporeSigma	CAS # 7223-38-3
Triethyl amine		CAS # 121-44-8
Magnesium sulfate (anhydrous)	VWR	CAS # 7487-88-9
Chloroethylene phosphate	Alfa Aesar	CAS # 6609-64-9
Ammonium hydroxide	VWR	CAS # 1336-21-6
3-Azido-7-hydroxy coumarin	MilliporeSigma	CAS # 817638-68-9
Pyridine	Thermo Scientific Chemicals	CAS # 110-86-1
Tris(3-hydroxypropyltriazolylmethyl)amine (THTPA)	MilliporeSigma	CAS # 760952-88-3
Copper sulfate	Thermo Scientific Chemicals	CAS # 7758-98-7
Potassium iodide	VWR	CAS # 7681-11-0
Potassium bicarbonate	VWR	CAS # 298-14-6
Sodium bicarbonate	MilliporeSigma	CAS # 144-55-8
5-aminofluorescein	MilliporeSigma	CAS # 3326-34-9
Sodium nitrite	Thermo Scientific Chemicals	CAS # 7632-00-0
Sodium azide	Thermo Scientific Chemicals	CAS # 26628-22-8
4-Hydroxymethylphenylboronic acid pinacol ester	Bachem	CAS # 302348-51-2
Triphosgene	MilliporeSigma	CAS # 32315-10-9
4-Azidobenzyl bromide	Combi-Blocks	CAS # 74489-49-9
Hydrogen peroxide (30% w/w in water)	MilliporeSigma	CAS # 7722-84-1
Sodium sulfide nonahydrate (Na2S·9H2O)	Strem Chemicals	CAS # 1313-84-4
Other
Norm-Ject syringe 1 mL Luer-lock, non-sterile	VWR	Cat # 89174-491
ITO-coated float glass unpolished slides, 30–60 Ω 25 × 25 mm	Structure Probe, Inc.	Cat # 06409-AB
Microscope cover glasses 18 × 18 mm	VWR	Cat # 16004-308
Thin layer chromatography (TLC) plate, aluminum back, 0.2 mm coating, binder polyacrylate-Na	ChemScene	Cat # CS-E0008
FlashPure EcoFlex Diol 12 g	Büchi	Cat # 140000068
Glass microscope slide, frosted one end, 25 × 75 mm – 1.2 mm thick	Electron Microscopy Sciences	Cat # 71870-01
Eppendorf tube, DNA LoBind, 2 mL	Labviva	Cat # 4043-1048
DDS signal generator/counter, frequency meter 200 MSa/s (15 MHz)	Koolertron	SKU # GH-CJDS66-A
Orion Star A211 pH benchtop meter	Thermo Scientific	Cat # 13-645-611
Leica TCS SP8 tauSTED confocal microscope	Leica Microsystems
Software and algorithms
ChemDraw 22.2.0	PerkinElmer	v22.2.0
GraphPad Prism	GraphPad	v8
ImageJ	National Institutes of Health and the Laboratory for Optical and Computational Instrumentation	https://imagej.nih.gov/ij/
Adobe Illustrator	Adobe	v2021
BioRender	BioRender	https://www.biorender.com/
MestReNova	Mestrelab Research	https://mestrelab.com/download/mnova/

Step-by-step method details

Synthesis of alkynyl rac-DPPC

Timing: 1 week

• 1.
  Phosphorylation of 1,2-dipalmitoyl-rac-glycerol (Figure 2).

  • a.
    The day prior, dry a 25 mL round bottom flask and stir bar overnight in a 115°C oven.
  • b.
    To the cooled flask add 500 mg (0.75 mmol, 1 equiv) of 1,2-dipalmitoyl-rac-glycerol and the stir bar, and cap the flask with a rubber septum.
  • c.
    Using a needle connected to a vacuum gas manifold line (Schlenk line), exchange the flask’s atmosphere by alternating vacuum and dry N₂ gas three times.
  • d.
    Add 5 mL of anhydrous benzene via syringe and cool the reaction vessel in an ice bath with stirring for 10 min.
  • e.
    Via syringe add 0.5 mL (3.3 mmol, 4.4 equiv) of triethyl amine to the reaction vessel dropwise and let stir for another 10 min.
  • f.
    Via syringe add 0.75 mL (7.1 mmol, 9.5 equiv) of ethylene chlorophosphate to the reaction vessel dropwise and let stir for another 10 min.
  • g.
    Remove the reaction vessel from the ice bath and let stir for 24 h.
  • h.
    The following day quench the reaction via the addition of 10 mL of saturated sodium bicarbonate solution.
  • i.
    Transfer the reaction mixture to a 50 mL separatory funnel and add 15 mL of ethyl acetate.
  • j.
    Extract the product with ethyl acetate 3 × 15 mL collecting organic layers together.
  • k.
    Add anhydrous magnesium sulfate to the organic solution until it no longer clumps and let sit for 5 min before decanting to a round bottom flask.
  • l.
    Concentrate the organic solution with a vacuum to give a white product.
  • m.
    Confirm product formation and purity via proton and phosphorus NMR.

Note: Further purification of this reaction was deemed unnecessary, as the excess reagents were removed during extraction. Pre-drying benzene with activated molecular sieves the week prior can increase product yield.

Note: Toluene can be used as a less toxic alternative to benzene.

• 2.
  Alkyne tagging of 1,2-dipalmitoyl-rac-glycerol.

  • a.
    The day prior, dry a 10 mL pressure flask and stir bar overnight in a 115°C oven.
  • b.
    Add 540 mg (6.4 mmol, 43 equiv) of 3-dimethylamino-1-propyne to dry 10 mL round bottom flask along with a small scoop of sodium sulfate and 3 mL acetonitrile. Cap and bubble nitrogen through this solution for 30 min.
  • c.
    Add 100 mg (0.15 mmol, 1 equiv) of 3-((2-oxido-1,3,2-dioxaphospholan-2-yl)oxy)propane-1,2-diyl dipalmitate from the prior step to the pressure tube along with 2 mL of acetonitrile and bubble nitrogen through the solution for 30 min.
  • d.
    Add the 3-dimethylamino-1-propyne solution to the pressure tube and cap the reaction.
  • e.
    Heat the reaction to 80°C behind a blast shield for 60 h.
  • f.
    Cool the reaction mixture to room temperature and dilute with methylene chloride.
  • g.
    Concentrate reaction using a vacuum pump overnight to give golden yellow oil.
  • h.
    Purify the reaction using a diol column using dichloromethane/(methanol:7 M NH₄OH 6:1 v/v) gradient from 0 to 25%.
  • i.
    Perform TLC (70:29:1 chloroform/methanol/water) using molybdenum blue stain to visualize pure fractions to collect white solid product.
  • j.
    Confirm purity via proton and phosphorus NMR spectroscopy.

Note: These reactions are extremely moisture sensitive; preparation of this reaction in a glove box is encouraged if available. Additionally, the addition of molecular sieves to the reaction can be used when moisture is suspected.

Note: Drying the mixture of the amine can be using activated molecular sieves the week prior.

Figure 2.

Synthesis of alkynyl rac-DPPC

Synthesis of TC-ONOO^– (clickable peroxynitrite-reactive module)

Timing: 2 days

• 3.
  Peroxynitrite recognition moiety (Figure 3)

  • a.
    The day prior, dry a 10 mL round bottom flask and stir bar overnight in a 115°C oven.
  • b.
    To the cooled flask add 81 mg (0.28 mmol, 0.60 equiv) of triphosgene and a stir bar and cap with rubber septum.
  • c.
    Using a needle connected to a vacuum gas manifold (Schlenk line) exchange the atmosphere of the flask by alternating vacuum and dry N₂ gas three times.
  • d.
    Add 2 mL of anhydrous toluene via syringe and cool the reaction vessel in an ice bath and stir for 10 min.
  • e.
    Via syringe add 56 μL (0.70 mmol, 1.5 equiv) of pyridine to the reaction vessel dropwise and let stir for another 10 min.
  • f.
    Make a solution of 100 mg (0.47 mmol, 9.5 equiv) of 1,1,1-trifluoro-4-(4-hydroxyphenyl)butan-2-one (the CF₃-ketone, Figure 3) in 1 mL of toluene.
  • g.
    Via syringe add the solution of 1,1,1-trifluoro-4-(4-hydroxyphenyl)butan-2-one to the reaction vessel dropwise and let stir for another 10 min.
  • h.
    Remove the reaction vessel from the ice bath and let stir. TLC reaction to track progress (2–3 h).
  • i.
    Once the reaction is complete, remove solvent using a vacuum from the reaction to give an oily chloroformate intermediate.
  • j.
    Redissolve the intermediate in dry dichloromethane and purge it with nitrogen for 10 min and cool in an ice bath.
  • k.
    Add 64 mg (1 equiv) of 3-azido-7-hydroxy coumarin to 2 mL of anhydrous acetonitrile and add 56 μL (0.70 mmol, 1.5 equiv) of pyridine.
  • l.
    Add this solution dropwise to the reaction under a nitrogen environment. Stir for 4 h at room temperature.
  • m.
    Concentrate the reaction with a vacuum to give a white crude product.
  • n.
    Purify crude reaction using normal phase column chromatography using dichloromethane/methanol 0–2%.
  • o.
    Perform TLC (70:29:1 chloroform/methanol/water) using molybdenum blue stain to visualize pure fractions to collect TC-ONOO^– as a white solid product.
  • p.
    Confirm purity via proton and phosphorus NMR spectroscopy.

Note: The reaction is extremely moisture sensitive, preparation of this reaction in a glove box is encouraged if available. Further, the addition of molecular sieves to the reaction can be used when moisture is suspected.

Figure 3.

Synthesis of TC-ONOO^–

Synthesis of TF-H₂O₂ (clickable hydrogen peroxide-reactive module)

Timing: 5 days

• 4.
  Synthesis of the hydrogen peroxide-reactive site (Figure 4).

  • a.
    The day prior, dry a 10 mL round bottom flask and stir bar overnight in a 115°C oven.
  • b.
    To the cooled flask, add 81 mg (0.28 mmol, 0.60 equiv) of triphosgene and a stir bar and cap the flask with a rubber septum.
  • c.
    Using a needle connected to a vacuum gas manifold (Schlenk line) exchange the atmosphere of the flask by alternating vacuum and dry N₂ gas three times.
  • d.
    Add 2 mL of anhydrous dioxane/toluene (1:1 v/v) via syringe and cool the reaction vessel in an ice bath for 10 min.
  • e.
    Via syringe add 56 μL (0.70 mmol, 1.5 equiv) of pyridine to the reaction vessel dropwise and let stir for another 10 min.
  • f.
    Make a solution of 100 mg (0.54 mmol, 1.2 equiv) of 4-hydroxymethylphenylboronic acid pinacol ester in 1 mL of toluene.
  • g.
    Via syringe add the solution of 4-hydroxymethylphenylboronic acid pinacol ester to the reaction vessel dropwise and let stir for another 10 min.
  • h.
    Remove the reaction vessel from the ice bath and let stir. TLC (70:29:1 chloroform/methanol/water) reaction to track progress (2–3 h).
  • i.
    Once the reaction is complete, remove solvent using a vacuum from the reaction to give an oily chloroformate intermediate.

Note: Reaction is extremely moisture sensitive; preparation of this reaction in a glove box is encouraged if available. Additionally, the addition of molecular sieves to the reaction can be used when moisture is suspected.

• 5.
  Synthesis of the clickable fluorophore.

  • a.
    Add 100 mg (0.29 mmol, 1 equiv) of 5-aminofluorescein and a stir bar to a 100 mL beaker and 25 mL of DI water and cooled in an ice bath.
  • b.
    Add 30 mg (0.43 mmol, 1.5 equiv) sodium nitrite to the reaction, once dissolved add 10 mL of 6 M HCl dropwise while keeping the reaction cool. Let stir at 0°C for 20 min.
  • c.
    Add 38 mg (0.58 mmol, 2 equiv) of sodium azide. Let stir at 0°C for 90 min.
  • d.
    Quench reaction with 1 M NaOH to pH ∼6 and collect product 5-azidofluorescein via filtration. The high purity was confirmed via TLC and ¹H-NMR.
• 6.
  Conjugation of clickable hydrogen peroxide-reactive module.

  • a.
    The day prior, dry a 10 mL round bottom flask and stir bar overnight in a 115°C oven.
  • b.
    To the cooled flask add 105 mg (0.28 mmol, 1 equiv) of 5-azidofluorescein and 2.5 mL of tetrahydrofuran and cool in an ice bath.
  • c.
    To the round bottom flask, add 250 mg (0.84 mmol, 3 equiv) of the chloroformate from procedure-4 dissolved in 1 mL of tetrahydrofuran dropwise.
  • d.
    Seal reaction and let stir at room temperature for 24 h.
  • e.
    Dry reaction crude under reduced pressure.
  • f.
    Purify the reaction using a column using an isocratic gradient of 100% dichloromethane.
  • g.
    TLC to visualize pure fractions to collect white solid product.
  • h.
    Confirm purity via proton NMR spectroscopy.

Figure 4.

Synthesis of TF-H₂O₂

Clicking redox-sensitive modules to alkynyl rac-DPPC

Timing: 2 days

• 7.
  General “click” procedures toward functionalized DPPC (Figure 5).

  • a.
    In a 25 mL flask with a stir bar add 0.05 mmol (1 equiv) of alkynyl rac-DPPC, 0.07 mmol (1.3 equiv) of azido-labeled recognition moiety, and 5 mL tetrahydrofuran.
  • b.
    Bubble N₂ gas through tetrahydrofuran solution for 15 min to exclude dissolved oxygen.
  • c.
    Add 0.03 mmol (0.5 equiv) of copper sulfate and 0.06 mmol (1 equiv) of THTPA as a solution in 0.4 mL of water and continue to bubble N₂ gas.
  • d.
    Let the reaction stir for 12 h.
  • e.
    Concentrate the reaction under vacuum.
  • f.
    Purify the reaction using a diol column using dichloromethane/(methanol:water 30:1 v/v) gradient from 0 to 35%.
  • g.
    Perform TLC (70:29:1 chloroform/methanol/water) to visualize pure fractions, which will provide a white solid product after concentration.
  • h.
    Confirm purity via proton and phosphorus NMR spectroscopy.

Figure 5.

Synthesis of DPPC-TC-ONOO^– and DPPC-TF-H₂O₂

Synthesis of DPPC-TC-H₂S (H₂S/HS^– probe)

Timing: 5 days

• 8.
  Synthesis of DPPC-TC (Figure 6).

  • a.
    Synthesize DPPC-TC, the click product of alkynyl rac-DPPC and 3-azido-7-hydroxy coumarin using procedure-7.
• 9.
  Synthesis of DPPC-TC-H₂S (Figure 6).

  • a.
    In a 25 mL flask with a stir bar add 75 mg (0.08 mmol, 1 equiv) of DPPC-TC, 33.5 mg (0.16 mmol, 2 equiv) of 4-azidobenzyl bromide, and 2 mL acetonitrile/chloroform (3:1, v/v).
  • b.
    Stir reaction and add 32.7 mg (0.24 mmol, 3 equiv) of potassium bicarbonate and 1.3 mg (0.008 mmol, 0.1 equiv) potassium iodide (used for enhancing nucleophilic substitution reaction).
  • c.
    Let the reaction stir for 4 days at 55°C for optimal results.
  • d.
    Concentrate the reaction under vacuum.
  • e.
    Purify the reaction using a diol column using dichloromethane/(methanol:water 30:1 v/v) gradient from 0 to 35%.
  • f.
    Perform TLC to visualize pure fractions, which will provide a white solid product after concentration.
  • g.
    Confirm purity via proton and phosphorus NMR spectroscopy.

Figure 6.

Synthesis of DPPC-TC-H₂S

Formation of GUV-based sensors

Timing: 4–5 h

• 10.
  Preparing ITO coated glass slides (Figure 7):

  • a.
    Clean two ITO coated slides (each electroformation setup requires two slides) with 2% neutral soap solution and then rinse with water.
  • b.
    Clean them with acetone, then dry using Kimwipes.
  • c.
    Identify the conductive side of ITO slides using an ohmmeter. The conductive side will show an electric resistance around 100 Ω (numbers will vary according to the slides), and the non-conductive side will not give a reading and the units will be in the MΩ range. Mark the conductive side for your convenience.
  • d.
    Cut the silicon gasket in the desired shape (empty in the centered region and contains an opening for liquid transfer) and wash them with 2% neutral soap solution and then with water. The gasket is finally washed with acetone, dried by squeezing the liquid out and then left in a fume hood for 30 min.

Note: Ensure that both the ITO slides and silicon gasket are in undamaged condition, as if the gasket is unclean or ripped, and if the slides are unclean, the final readings will be inaccurate.

• 11.
  Coat lipids onto the ITO coated slides:

  • a.
    Prepare lipid stock solutions. POPC, POPG, and cholesterol stock solutions were prepared in chloroform, at concentrations of 5 mM, 2 mM, and 2 mM, respectively. Lipid probes (DPPC-TC-ONOO^–, DPPC-TF-H₂O₂, and DPPC-TC-H₂S) were prepared in chloroform:methanol (3/1 v/v) at 250 μM.
  • b.
    Mix the lipids at a final total lipid amount of 50 nmol.

    Note: For each GUV system the lipid composition is as follows: A) GUVs that sense ONOO^–: 98.5% POPC, 1% DPPC-TC-ONOO^–, 0.5% Liss-Rhod PE. B) GUVs that sense H₂O₂: 48.5% POPC, 21% POPG, 30% cholesterol, and 0.5% of DPPC-TF-H₂O₂. C) GUVs that sense H₂S/HS^–: 48% POPC, 21% POPG, 30% cholesterol, and 1% DPPC-TC-H₂S. These latter two GUVs contain only ∼0.1 mol% of Liss-Rhod PE, which can be considered as negligible for convenience.

  • c.
    Use a Hamilton syringe to spread the lipid mixture on the conductive side of the ITO slide (not on the whole slide, but only in the center).

    Note: total solution volume may vary for different GUV systems, and the total lipid amount should be 50 nmol.

  • d.
    After spreading the lipid, keep the ITO in a high vacuum for an hour.

    Note: Keep the lipid stock solutions at −80°C for long-term storage. Keep the solutions on ice when preparing the lipid-coated slides to prevent organic solvent evaporation. There is no particular reason for not using POPG or cholesterol in the formation of peroxynitrite-sensing GUVs. Researchers can choose which natural lipid is suitable.

• 12.
  Assembling the Electroformation Chamber:

  • a.
    Attach one copper tape or aluminum foil tape to each side of the silicon gasket as electrodes
  • b.
    Place the silicon gasket with electrodes on the conductive side of the uncoated ITO.
  • c.
    Remove the lipid coated ITO from the high vacuum and place it on the top of the silicon gasket to form a chamber, with the conductive side attaching the gasket.
  • d.
    Clamp the chamber with 3 binder clips from the three sides to avoid any leakage. The opening is positioned towards up to provide an input for liquid.
  • e.
    Slowly fill the chamber with 200 μL of hydrating buffer (5 mM Tris, 300 mM Sucrose, pH 7.5) avoiding any air bubbles.
  • f.
    Seal the chamber using a small ball of clay to prevent leakage.

Note: For solutions at high ionic strength (e.g., Tris-buffered saline), increase the frequency of the function generator to 500 Hz will be more optimal for vesicle electroformation.

• 13.
  Electroformation Process:

  • a.
    Connect the chamber using a function generator. Attach the alligator clips of the generator to the electrodes to complete the circuit.
  • b.
    Use the frequency of 10 Hz and voltage of 2 V (V_p, peak voltage) for 3 h.
  • c.
    After the electroformation program has been run, pipet out the solution slowly and transfer into a 2 mL Eppendorf tube.

Figure 7.

Preparation workflow of GUV-based sensors

(A) Cleaning of the slides.

(B) Testing for conductive and nonconductive sides using an ohmmeter.

(C) Attaching aluminum metal strips to either opposing side of the foam piece.

(D) Combining the two ITO (Indium-Tin-Oxide) slides (conductive sides facing each other) with the foam piece between them.

(E) Attaching all of the corresponding clips starting with the largest one on the bottom.

(F) Fill the chamber with 200 μL lipid hydration solution.

(G) Seal the chamber with fingernail sized amount of clay.

(H) Attach each alligator clip to one of the protruding metal strips and place in container.

(I) Cover the container thoroughly to seal off any light.

Visualizing the response of GUVs for redox cues

• 14.
  Preparing the stock solutions of redox species.

  • a.
    The stock solution of ONOO^–: In a glass vial, vigorously stir 50 mM (0.5 mL) of NaNO₂ and 50 mM (0.5 mL) of H₂O₂. Rapidly add 1 M HCl solution (1 mL), followed by 1.5 M NaOH solution (1 mL).
  • b.
    The stock solution of H₂O₂ (10 mM): Dilute H₂O₂ solution (30% w/w in water, 9.8 M) with water.
  • c.
    The stock solution of H₂S (10 mM): Dissolve Na₂S·9H₂O in water and then mix with Tris buffer (pH 7.4 or 7.5).

Note: Details on the preparation and handling of ONOO^– solution can be found in Gutierrez and Aggarwal et al.¹

CRITICAL: We recommend researchers to prepare these stock solutions fresh. For ONOO^–, it is critical to have a strongly basic stock solution (pH ≥ 10) after the addition of NaOH.³ The concentration of ONOO^– depends on how fast the final NaOH addition is. Therefore the concentrations may vary and should be determined by UV absorbance prior to usage.¹ The highly basic stock solution of ONOO^– can be stored at ultra-low (e.g., −80°C) temperatures.

• 15.
  Incubation with Redox Species and Microscopy (Figure 8):

  • a.
    Incubate the vesicles (19 μL of solution) with the redox species (1 μL) of interest in a PCR tube at room temperature.
  • b.
    Take a rectangular microscopy slide, then apply 5 μL of the GUV solution in the center.
  • c.
    Cover it with a cover slip. Seal the four edges with nail polish.
  • d.
    Put the slides upside down on the stage of the confocal microscope and start imaging.

Note: The final concentrations of redox species at time of mixing with vesicles are 100 μM for ONOO^–, 100 μM for H₂O₂, and 500 μM for Na₂S. Incubation time is 5 min for ONOO^–, 90 min for H₂O₂, and 60 min for Na₂S.

CRITICAL: Image the vesicles using a controlled laser power. Strong laser power and long-term exposure may cause significant photobleaching.

Figure 8.

Confocal images of functional GUVs that sense redox cues

(A) Peroxynitrite-sensing of GUVs built from 98.5% POPC, 1% DPPC-TC-ONOO^–, and 0.5% Liss-Rhod PE. Coumarin channel: 405 nm excitation.

(B) Hydrogen peroxide-sensing of GUVs built from 48.5% POPC, 21% POPG, 30% cholesterol, and 0.5% of DPPC-TF-H₂O₂. Fluorescein channel: 490 nm excitation.

(C) Hydrogen sulfide-sensing of GUVs built from 48% POPC, 21% POPG, 30% cholesterol, and 1% DPPC-TC-H₂S. Coumarin channel: 405 nm excitation. Liss-Rhod PE dye (rhodamine channel, 560 nm excitation) was used to label vesicle membranes, at lipid ratios of 0.5% in (A) and 0.1% in both (B) and (C). Scale bars = 5 μm.

Expected outcomes

Using electroformation to create GUVs, then imaging with a confocal microscope should result in clear images of vesicles at around 10 μm in diameter. Electroformation is specifically used, as it is one of the best methods to generate larger vesicles (i.e., vesicles greater than 10 μm, and no less than 2 μm). Under confocal microscope, the vesicles will present a bright circle in the “rhodamine” channel due to Liss-Rhod PE membrane dye. The vesicle circle in the probe channel (“coumarin” for both DPPC-TC-ONOO^– and DPPC-TC-H₂S and “fluorescein” for DPPC-TF-H₂O₂) will be relatively dim before incubation with the target redox cue, yet will become brighter after incubation.

Limitations

Synthesis of lipids remains challenging due in part to their insolubility in common solvents, and difficulty to separate. The protocols described above work well with the described lipid, however variations of either the synthetic lipid probes or other lipids used in GUV electroformation may greatly impact the effectiveness of described techniques. These protocols will require slight adjustments of solvents and procedures for alternative lipid designs. Additionally, many steps are highly water sensitive, and the lack of water free solvents and procedures will impact reaction yields.

Photostability of the lipid-based probes is of important note. Fluorescein is relatively cost effective and easy to chemically modify. However, it is considered to be less photostable compared to other commonly used fluorophores.⁴ Confocal images were collected under exposure times of seconds. For longer exposure times, lipid-based probes with a fluorophore that is more photostable than fluorescein can be utilized.

The irreversible probe activation mechanism described in these methods poses a limitation for the application of GUVs in dynamic redox conditions. GUV designs that allow for reversible redox sensing would be of particular interest for future protocell models.

Troubleshooting

Problem 1

Lipid probes have poor solubility in organic solvents or aqueous buffers.

Potential solution

First, use a mixture of chloroform and methanol (e.g., in 3:1 volumetric ratio) to dissolve the lipid at ∼100 μM. The resulting mixture should go into a fully homogenous state with gentle shaking. The deuterated form of this solvent system (CDCl₃/d4-methanol) can be used for NMR spectroscopic authentication of lipid probes.

Problem 2

Carbonate-containing probes have limited stability in aqueous, physiologically relevant solutions.

Potential solution

To mitigate hydrolysis of the carbonate, keep all solvents for synthetic steps dry, avoid the use of basic medium for work up or purification, and minimize time with compounds in aqueous work up conditions.

Problem 3

Un-clicked lipid purification difficult.

Potential solution

The unclicked lipids do not absorb standard wavelengths and are thus difficult to monitor during chromatography. The use of auto columns with evaporative light scattering devices (ELSD) is extremely useful to identify fractions containing product.

Additionally, as mentioned above molybdenum blue stain is excellent at staining phosphate-containing compounds however it has a short shelf life due to instability with light and a long activation time (∼10–15 min to develop the stain). Keep solutions of the stain covered and consider making fresh stain from molybdenum(IV) oxide and sulfuric acid regularly.

Problem 4

Poor electroformation efficiency observed (not many vesicles formed).

Potential solution

Decrease the molar percentage of the probes to less than 1% of the total lipid composition. Increase the electroformation time. Decrease the voltage of electroformation.

Problem 5

Mobility of GUVs makes them challenging to image.

Potential solution

Larger GUVs are more suitable to image, as their mobility is less than those with smaller size. To this end, increase the vesicle size by adjusting the parameters in electroformation mentioned above. Furthermore, there are reported methods for GUV immobilization that can help mitigate this problem, such as the avidin-biotin method.⁵

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Enver Cagri Izgu (ec.izgu@rutgers.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Liming Wang (lw641scarletmail.rutgers.edu) and Mark J. Dresel (mjd458@scarletmail.rutgers.edu).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact upon reasonable request with a completed Materials Transfer Agreement.

Data and code availability

• •Any primary data in this study can be requested from the lead contact.
• •This study did not generate any new code.
• •Any additional information to reanalyze the data can be requested from the lead contact.