Method for analyzing the ability of a substance to cross a bilayer membrane
The present invention relates to a method to analyse the ability of a substance ( 3 ) to cross a bilayer membrane comprising the following steps: (i) providing in a hydrophobic medium ( 1 ) a first droplet D 1 of an aqueous solution AS 1 , wherein the droplet D 1 is surrounded by a monolayer of amphiphilic molecules ( 2 ), (ii) providing in the said hydrophobic medium ( 1 ) a second droplet D 2 of an aqueous solution AS 2 containing the substance ( 3 ) to be analysed, wherein the droplet D 2 is surrounded by a monolayer of amphiphilic molecules ( 2 ), (iii) putting the first droplet D 1 and the second droplet D 2 into contact so as to form a bilayer of amphiphilic molecules ( 2 ) in the contact area and to have a contact angle 2θ between the two droplets D 1 and D 2 comprised between 10° and 180°, (iv) adding to the hydrophobic medium ( 1 ) a solvent capable of separating the droplets D 1 and D 2 put into contact, (v) collecting the droplet D 1 , and (vi) analysing the content of the droplet D 1 in order to determine the presence or not of the substance ( 3 ).
1. A method to analyse the ability of a substance to cross a bilayer membrane comprising the following steps:
(i) providing in a hydrophobic medium a first droplet D 1 of an aqueous solution AS 1 , wherein the droplet D 1 is surrounded by a monolayer of amphiphilic molecules,
(ii) providing in said hydrophobic medium a second droplet D 2 of an aqueous solution AS 2 containing the substance to be analysed, wherein the droplet D 2 is surrounded by a monolayer of amphiphilic molecules,
(iii) putting the first droplet D 1 and the second droplet D 2 into contact so as to form a bilayer of amphiphilic molecules in the contact area and to have a contact angle 2θ between the two droplets D 1 and D 2 comprised between 90° and 180°,
(iv) adding to the hydrophobic medium a solvent capable of separating the droplets D 1 and D 2 put into contact, wherein the solvent capable of separating the droplets D 1 and D 2 put into contact is selected from the group consisting of a halogenated hydrocarbon, an alcohol, an ether, a fatty acid, a diglyceride, an ester, a low molecular weight hydrocarbon, and a mixture thereof,
(v) collecting the droplet D 1 , and
(vi) analysing the content of the droplet D 1 in order to determine the presence or not of the substance,
wherein the halogenated hydrocarbon is a linear or branched, saturated or unsaturated hydrocarbon comprising 1 to 6 carbon atoms, in which one or more hydrogen atoms have been replaced by a halogen atom,
wherein the alcohol is a linear or branched, saturated or unsaturated hydrocarbon comprising 1 to 12 carbon atoms, in which a hydrogen atom has been replaced with an OH group,
wherein the ether is a molecule of formula A 4 —O—A 5 , wherein A 4 and A 5 each represent independently of one another a monovalent linear or branched, saturated or unsaturated hydrocarbon chain comprising 1 to 12 carbon atoms, or A 4 and A 5 form together a divalent linear or branched, saturated or unsaturated hydrocarbon chain comprising 1 to 12 carbon atoms and optionally containing another oxygen atom,
wherein the fatty acid is a linear, saturated or unsaturated carboxylic acid comprising from 4 to 18 carbon atoms,
wherein the diglyceride is an ester derived from glycerol and two fatty acids which are identical or different,
wherein the ester is a molecule of formula A 7 —OC(O)—A 8 , wherein A 7 and A 8 each represent independently of one another a monovalent linear or branched, saturated or unsaturated hydrocarbon chain comprising 1 to 12 carbon atoms, and
wherein the low molecular weight hydrocarbon is a linear, branched or cyclic, saturated or unsaturated hydrocarbon containing no more than 9 carbon atoms.
2. The method according to claim 1 , wherein the hydrophobic medium is an oil, triglycerides, a silicone oil, a high molecular weight hydrocarbon or a mixture thereof;
optionally in admixture with a solvent selected from a halogenated hydrocarbon, an alcohol, an ether, a fatty acid, a diglyceride, an ester, a low molecular weight hydrocarbon and a mixture thereof.
3. The method according to claim 1 , wherein the amphiphilic molecules are phospholipids, glycolipids, monoglycerides, cholesterol, or a mixture thereof.
4. The method according to claim 3 , wherein the amphiphilic molecules are phospholipids.
5. The method according to claim 4 , wherein the amphiphilic molecules are selected from dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and mixtures thereof.
6. The method according to claim 1 , wherein the aqueous solutions AS 1 and AS 2 contain a buffering agent.
7. The method according to claim 1 , wherein the first and second droplets D 1 and D 2 have a diameter comprised between 0.5 μm and 1000 μm.
8. The method according to claim 1 , wherein step (i) is performed by:
providing the aqueous solution AS 1 ,
providing the hydrophobic medium further containing the amphiphilic molecules, and
forming a droplet of the aqueous solution AS 1 in the hydrophobic medium which contains the amphiphilic molecules.
9. The method according to claim 1 , wherein step (ii) is performed by:
providing the aqueous solution AS 2 containing the substance,
providing the hydrophobic medium further containing the amphiphilic molecules, and
forming a droplet of the aqueous solution AS 2 containing the substance in the hydrophobic medium which contains the amphiphilic molecules.
10. The method according to claim 1 , wherein the contact angle 2θ is comprised between 120° and 180° .
11. The method according to claim 1 , wherein the droplets D 1 and D 2 are maintained into contact in step (iii) during at least 1 min.
12. The method according to claim 1 , wherein step (vi) is performed by means of fluorescence imaging, HPLC or mass spectrometry.
13. The method according to claim 1 , used in high-throughput.
14. The method according to claim 1 , performed by means of a microfluidic analysis system comprising:
a microfluidic device comprising:
a first microfluidic channel comprising one inlet and one outlet, through which alternate droplets D 1 and D 2 can flow in the hydrophobic medium from the inlet to the outlet of the first microfluidic channel and can be put into contact,
a second microfluidic channel comprising one inlet and one outlet, through which the droplets D 1 and D 2 can flow in the hydrophobic medium from the inlet to the outlet of the second microfluidic channel and the droplets D 1 and D 2 put into contact can be separated, the inlet of the second microfluidic channel being connected to the outlet of the first microfluidic channel,
at least two outlets connected to the outlet of the second microfluidic channel, wherein:
the first outlet of the microfluidic device is further connected to a first receiver container intended to receive the droplets D 1 to be analysed, and
the second outlet of the microfluidic device is further connected to a second receiver container intended to receive the droplets D 2 ,
at least five inlets:
wherein four inlets of the microfluidic device are connected to the inlet of the first microfluidic channel,
the first two inlets being further connected respectively to a reservoir intended to contain the aqueous solution AS 1 and to a reservoir intended to contain the hydrophobic medium and the amphiphilic molecules, and
the last two inlets being further connected respectively to a reservoir intended to contain the aqueous solution AS 2 and the substance and to a reservoir intended to contain the hydrophobic medium and the amphiphilic molecules, and
wherein one inlet of the microfluidic device is connected to the inlet of the second microfluidic channel and is further connected to a reservoir intended to contain the solvent capable of separating the droplets D 1 and D 2 put into contact,
at least four reservoirs adapted for containing respectively (i) the aqueous solution AS 1 , (ii) the aqueous solution AS 2 and the substance, (iii) the hydrophobic medium and the amphiphilic molecules, and (iv) the solvent capable of separating the droplets D 1 and D 2 put into contact,
at least two receiver containers adapted for receiving respectively (i) the droplets D 1 to be analyzed, and (ii) the droplets D 2 , and
an analyzing device to analyze the content of droplets D 1 .
15. The method according to claim 4 , wherein the amphiphilic molecules are phosphatidylcholines (PC), phosphatidylethanolamines (PE), dioleoylphosphatidylglycerol (PG), dioleoylphosphatidylserine (DOPS), or a mixture thereof.
16. The method according to claim 7 , wherein the first and second droplets D 1 and D 2 have a diameter comprised between 50 μm and 200 μm.
17. The method according to claim 11 , wherein the droplets D 1 and D 2 are maintained into contact in step (iii) between 10 and 30 min.
18. The method according to claim 1 , wherein the solvent capable of separating the droplets D 1 and D 2 put into contact is selected from chloroform, dichloromethane, butanol, diethyl ether, 1,4-dioxane, oleic acid, glyceryl dioctanoate, glyceryl dioleate, ethyl acetate, isopropyl acetate, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, and a mixture thereof.
19. The method according to claim 18 , wherein the solvent capable of separating the droplets D 1 and D 2 put into contact is chloroform.