Method for analyzing the activity of an ion channel

Patent metadata

Field	Value
Publication number	US-10690680-B2
Application number	US-201615776685-A
Country	US
Kind code	B2
Filing date	Nov 17, 2016
Priority date	Nov 17, 2015
Publication date	Jun 23, 2020
Grant date	Jun 23, 2020

Abstract

A method to analyze the activity of an ion channel comprising: providing in a hydrophobic medium a first droplet of an aqueous solution AS 1 comprising a concentration q of an ion I with c 1≥ 0, wherein the droplet is surrounded by a monolayer of amphiphilic molecules; providing in the hydrophobic medium a second droplet of an aqueous solution AS 2 comprising a concentration c 2 of the ion I with c 2≥ 0, and c 2 ≠c 1 , wherein the second droplet is surrounded by a monolayer of amphiphilic molecules further comprising the ion channels to an analyzed; bringing the first droplet and the second droplet into contact so as to form a bilayer of amphiphilic molecules, wherein the bilayer further comprises ion channels to be analyzed, and measuring the radius of the two droplets when they are brought into contact referred to as initial state; and maintaining the first droplet and the second droplet in contact until equilibrium is reached and measuring the radius of the two contacted droplets or determining the number of resulting droplet(s) at the equilibrium state, wherein the ion channel is inactive when the difference of the radius of at least one droplet between its initial state and its equilibrium state is at least 10% or when only one droplet is obtained at the equilibrium state.

First claim

The invention claimed is:

1. A method to analyze the activity of an ion channel comprising:
(i) providing in a hydrophobic medium a first droplet D 1 of an aqueous solution AS 1 comprising a concentration c 1 of an ion I with c 1 ≥0, wherein the droplet D 1 is surrounded by a monolayer of amphiphilic molecules,
(ii) (ii) providing in the said hydrophobic medium a second droplet D 2 of an aqueous solution AS 2 comprising a concentration c 2 of the ion I with c 2 ≥0 and c 2 ≠c 1 , wherein the droplet D 2 is surrounded by a monolayer of amphiphilic molecules further comprising the ion channels to be analyzed,
(iii) (iii) bringing 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, wherein the bilayer of amphiphilic molecules further comprises ion channels to be analyzed, and measuring the radius of the two droplets when they are brought into contact referred to as initial state, and
(iv) (iv) maintaining the first droplet D 1 and the second droplet D 2 into contact until equilibrium is reached, wherein at equilibrium the size of the two droplets does not evolve anymore and the osmolarity between the two droplets is in equilibrium;
and measuring the radius of the two contacted droplets or determining the number of resulting droplet(s) at the equilibrium referred to as equilibrium state,

wherein the ion channel is inactive when the difference of the radius of at least one droplet between its initial state and its equilibrium state is at least 10% or when only one droplet is obtained at the equilibrium state.

2. The method according to claim 1 , wherein the ion channel is a sodium channel, a potassium channel, a calcium channel, a proton channel or a chloride channel and the ion I is Na + , K + , Ca 2+ , H + or Cl − respectively.

3. The method according to claim 1 , wherein the hydrophobic medium is an oil; triglycerides; silicone oil; a hydrocarbon; or a mixture thereof.

4. The method according to claim 1 , wherein the amphiphilic molecules are phospholipids, glycolipids, cholesterol, or a mixture thereof.

5. The method according to claim 4 , wherein the amphiphilic molecules are phospholipids.

6. The method according to claim 5 , wherein the amphiphilic molecules are selected from dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylglycerol (DOPG), and mixtures thereof.

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 the concentration c 1 or c 2 is 0 or the concentrations c 1 and c 2 satisfy the following equation:
Ic 1 −c 2 I /( c 1 +c 2 )>0.1.

9. The method according to claim 1 , wherein the higher concentration of c 1 and c 2 is comprised between 1 mM and 1 M.

10. 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 in which the amphiphilic molecules are present.

11. The method according to claim 1 , wherein the ion channels are soluble in the aqueous solution AS 2 and step (ii) is performed by:
providing the aqueous solution AS 2 further containing the ion channels,
providing the hydrophobic medium further containing the amphiphilic molecules, and
forming a droplet of the aqueous solution AS 2 which contains the ion channels in the hydrophobic medium which contains the amphiphilic molecules.

12. The method according to claim 1 , wherein step (ii) is performed by:
providing the aqueous solution AS 2 further containing the ion channels present in liposomes,
forming a droplet of the aqueous solution AS 2 which contains the ion channels ( 3 ) present in liposomes in the hydrophobic medium containing no amphiphilic molecule,
shaking the droplet thus formed, and
adding the amphiphilic molecules to the hydrophobic medium.

13. The method according to claim 1 , wherein the radius of the droplets is measured by means of an optical detection device.

14. The method according to claim 1 , used in high-throughput.

15. The method according to claim 1 , performed by means of a microfluidic analysis system comprising:
a microfluidic device comprising:
a main 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 microfluidic channel,
at least two outlets connected to the outlet of the main microfluidic channel and wherein:
the first outlet of the microfluidic device is further connected to a first receiver container intended to receive the droplets analyzed as containing active ion channels, and
the second outlet of the microfluidic device is further connected to a second receiver container intended to receive the droplets analyzed as containing inactive ion channels,
at least four inlets connected to the inlet of the main microfluidic channel and wherein:
the first two inlets of the microfluidic device are 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 are further connected respectively to a reservoir intended to contain the aqueous solution AS 2 and the ion channels optionally present in liposomes and to a reservoir intended to contain the hydrophobic medium and optionally the amphiphilic molecules,
wherein, when the ion channels are present in the aqueous solution AS 2 in liposomes, the microfluidic device comprises a fifth inlet connected to the inlet of the main microfluidic channel and to a reservoir intended to contain the amphiphilic molecules and optionally the hydrophobic medium,
at least three reservoirs adapted for containing respectively (i) the aqueous solution AS 1 , (ii) the aqueous solution AS 2 and the ion channels optionally present in liposomes, and (iii) the hydrophobic medium and/or the amphiphilic molecules,
at least two receiver containers adapted for receiving respectively (i) the droplets analyzed as containing active ion channels, and (ii) the droplets analyzed as containing inactive ion channels, and
a detection device placed at the end of the main microfluidic channel.

16. The method according to claim 3 , wherein the hydrophobic medium is a mixture of triglycerides and hydrocarbon.

17. The method according to claim 5 , wherein the phospholipids are phosphatidylcholines (PC), phosphatidylethanolamines (PE) or a mixture thereof.

18. The method according to claim 6 , wherein the amphiphilic molecules are selected from DOPC, DOPE and mixtures thereof.

19. 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.

20. The method according to claim 14 , used in a high-throughput screening.

Assignees

• Paris Sciences Lettres Quartier Latin
• Univ Paris Diderot Paris 7
• Commissariat Energie Atomique

Inventors

• Thiam Abdou Rachid
• Schaack Béatrice

Classifications

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title

• B01L2200/0652

  Sorting or classification of particles or molecules · CPC title

• G01N33/6872 Primary

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• G01N33/92

  involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} · CPC title