FR3127810A1 - Drop sorting process - Google Patents

Abstract

The invention relates to a method for sorting drops implemented in a system which comprises: A chamber (1) formed by two substrates (100, 101) facing each other, Means for generating several independent electrostatic fields and juxtaposed between the two substrates,A control and power supply unit (2) configured to individually generate each electrostatic field,A fluidic inlet (11) and a fluidic outlet (12) opening into the chamber (1),Said method comprising:Generating at least one electrostatic field to create a non-zero electrical potential difference with a view to fixing a drop of interest (40) on said first substrate in its determined position,Injecting a fluid flow (F1) into the chamber through said inlet channel to evacuate the other drops of the emulsion, outside of said chamber through the outlet channel. Figure to be published with abstract: Figure 1

Description

Drop sorting process

Technical field of the invention

The present invention relates to a method for sorting drops, using in particular an electrowetting principle.

State of the art

It is known to want to analyze cells or molecules by isolating them in drops. Certain drops then constitute micro-reactors and it is thus possible to quickly scan a set of drops to detect and isolate those in which a reaction takes place. Thus, one can easily isolate the cells which may be of interest, for example, for the bio-production of therapeutic molecules.

However, it is necessary to be able to sort the drops of interest from among a set of several drops. And it turns out to be interesting to be able to sort drops and to separate the drops of interest with a high throughput. The challenge is to obtain very high sorting rates, for example several hundred or even several thousand drops per second.

To manipulate drops and move them, it is known to use the principle of electrowetting (EWOD in English for "Electowetting-on-dielectric"). It makes it possible to move drops by activating electrodes. The forces used are electrostatic forces. Patent application FR2841063A1 describes in particular this principle using a catenary placed opposite several juxtaposed electrodes. The electrodes are individually activated to move the drop along the catenary. Patent application WO2006/070162A1 also describes a device for dispensing drops using this principle of electrowetting to move the drops.

Drop sorting systems are also known, for example systems using a method of the DLD type (“Deterministic lateral displacement” – patent applications EP3318328A1 and FR3078637A1 ). Other methods are also known, such as those which use a channel comprising a bifurcation forming two distinct branches, an external action making it possible to direct the drops towards one or the other of the two branches. However, this last solution remains difficult to operate, complex to automate and requires qualified operators.

In general, the prior solutions do not necessarily make it possible to perform a sorting of the drops of interest with a high sorting rate.

The object of the invention is therefore to propose a solution making it possible to carry out a selection of drops of interest from among several drops, at a high rate, this solution being reliable and easy to implement, without involving qualified operators. .

This goal is achieved by a process for sorting drops implemented in a system which comprises:

• A chamber formed by two facing substrates, a first substrate and a second substrate,
• Means for generating several independent and juxtaposed electrostatic fields between the two substrates,
• A control and power supply unit configured to individually generate each electrostatic field,
• A fluidic inlet and a fluidic outlet opening into the chamber,

Said method comprising:

• A step of introducing an emulsion into said chamber, said emulsion comprising several drops juxtaposed over two dimensions, among which at least one so-called drop of interest having a determined position on said matrix of electrodes,
• Determination of the position of said drop of interest between the two substrates,
• Generation of at least one electrostatic field to create a non-zero electrical potential difference with a view to fixing said drop of interest on said first substrate in its determined position,
• Injection of a fluid flow into the chamber through said inlet channel to evacuate the other drops of the emulsion, outside of said chamber through the outlet channel.

According to one feature, the method comprises a step of generating at least one electrostatic field associated with at least one drop adjacent to the drop of interest, to coalesce said drop of interest with said adjacent drop, in order to form a larger drop.

According to a particular embodiment, the method includes a step of injecting a fluid flow to evacuate said at least one drop of interest from the chamber.

According to another particular embodiment, the method comprises a step of generating several electrostatic fields to move said at least one drop of interest towards said fluid outlet.

According to another feature, the step of determining the position of the drop of interest is implemented by fluorescence detection.

The invention also relates to a fluidic system used for implementing the method as defined above, the system comprising:

• A chamber formed by two facing substrates,
• Means for generating several independent and juxtaposed electrostatic fields between the two substrates,
• A control and power supply unit configured to individually generate each electrostatic field,
• A fluidic inlet and a fluidic outlet opening into the chamber.

According to a particular embodiment, the means for generating several electrostatic fields comprise a matrix of several juxtaposed electrodes placed in the first substrate, and at least one counter-electrode common to all the electrodes of said matrix placed in the second substrate.

According to another particular embodiment, the first substrate is produced by a stack of layers in which a photoconductive layer is excitable by a light beam.

According to one feature, the first substrate comprises an outer layer made of a hydrophobic material.

According to another feature, the light beam is generated by an OLED type display.

According to another feature, the system comprises a valve and pump control system controlled by the control and electrical power supply unit and configured to control each fluidic flow injected into the fluidic inlet and the fluidic outlet.

Brief description of figures

Other characteristics and advantages will appear in the detailed description which follows given with regard to the appended drawings in which:

• There represents the system used for the implementation of the method according to the invention;
• There illustrates the principle of electrowetting employed in the method of the invention;
• There schematically represents the structure of an emulsion, formed by the juxtaposition of drops in two dimensions;
• Figures 4A to 4F illustrate the different steps of the method according to the invention implemented in the system of the ;
• There shows an alternative embodiment of the method illustrated by FIGS. 4A to 4F;
• THE to 6D illustrate a variant of use of the system of the to implement a method for sorting drops;
• There shows an embodiment of the fluidic component used in the sorting system;

Detailed description of at least one embodiment

In the rest of the description, the terms “lower”, “upper”, “above”, “below” are to be understood by taking into account an axis traced vertically in the plane of the sheet.

The method of the invention is implemented in a system allowing the sorting of the drops. In a non-limiting way, the size of the drops is for example between 50 μm and 100 μm.

The system is of the fluidic type. It mainly includes:

• A fluidic component comprising a chamber 1,
• A control and power supply unit 2,
• Advantageously, a fluidic system comprising one or more reservoirs 50, 51, 52 connected to the chamber,
• A control system 3 of pumps and valves, controllable by the control and electrical power supply unit 2 to inject or evacuate one or more fluid flows respectively into the chamber 1 or out of the chamber 1 from the fluid system.

The fluidic component may comprise a fluidic inlet 11 opening into the chamber 1 and a fluidic outlet 12 exiting from the chamber. The fluidic outlet can lead to one or more reservoirs 51, 52 of the fluidic system.

The fluidic component can be formed by superimposing two parallel substrates 100, 101 facing each other.

The system operates by electrostatic effect and comprises means for generating several independent and juxtaposed electrostatic fields between the two substrates 100, 101. The electrostatic fields are generated perpendicular to the two substrates.

Depending on the embodiment envisaged, these means may comprise a matrix of so-called real electrodes or a matrix of electrodes created virtually.

Figures 1 to 5 illustrate the operating principle of the invention implemented using a matrix of real electrodes.

In these figures, a first substrate, called lower 101, comprises a matrix of electrodes 10 optionally surmounted by a hydrophobic layer, and a second substrate, called upper 100, comprises a counter-electrode CE.

The two faces of chamber 1 facing each other are flat and parallel to each other. The lower face is designated the bottom of chamber 1 and incorporates the matrix of electrodes 10 and the upper face incorporates the counter-electrode CE.

In the matrix of electrodes 10, the electrodes E_i are juxtaposed so as to occupy the entire surface of the bottom of the chamber 1.

We will see below different embodiments of the structure of each substrate 100, 101.

The electrode array 10 is connected to the control and power supply unit 2.

The control and power supply unit 2 is configured to control each electrode E_i of the matrix 10 individually. The control and power supply unit 2 comprises hardware and software means for controlling and supplying each electrode E_i individually. In a non-limiting manner, it may comprise an electrical power source and a solution of the programmable automaton type comprising input/output modules and switching means connected to said input/output modules, the switching means being configured to activating or deactivating each electrode E_i, respectively by connecting or disconnecting it from a power supply circuit connected to said power supply source. The control and power supply unit 2 is also configured to control the control system 3 with a view to activating/deactivating the valves and pumps during the implementation of the method of the invention.

The activation of an electrode E_i of the matrix by the control and electrical supply unit 2 makes it possible to place said electrode E_i at an electrical potential distinct from the electrical potential of the counter-electrode CE (this latter electrical potential being by null example) and thus to create a potential difference between the activated electrode and the counter-electrode CE. This principle can be applied to each electrode E_i of the matrix 10. The control and power supply unit 2 can thus activate one or more electrodes E_i of the matrix 10. The illustrates this principle. On this , you can see :

At T0: The selected electrode E_i is not yet activated by the control and power supply unit 2. The drop 4 located opposite is therefore not subjected to any electrostatic field and retains its shape initial.

At T1: Electrode E_i is activated and a potential difference (V1-V0) appears between activated electrode E_i and counter electrode CE. The electrostatic forces generated make it possible to fix the drop 4, which tends to spread out on the bottom of the chamber 1. This principle of fixing the drop is used within the framework of the method of the invention.

Starting from this structure and this principle, the method of the invention is described in connection with FIGS. 4A to 4F.

E1- : Chamber 1 of the component is seen from above.

An emulsion is introduced into the chamber of the system through the fluid inlet 11 of the component.

By emulsion is meant a set of several juxtaposed drops 4 which touch each other, but which do not coalesce. These advantageously form only a single two-dimensional layer of juxtaposed drops 4, this layer being placed in the chamber between the upper face of the lower substrate and the lower face of the upper substrate. There schematically shows the structure of such an emulsion used for implementing the method of the invention, respectively in top view V_1 and in side view V_2.

In general, the surfaces of the substrates which are in contact with the emulsion are hydrophobic in order to avoid wetting of the drops. Note that there is generally a film of oil between the drops and the surface which means that the drops do not wet the surface naturally. This helps in the formation of a well-compacted drop network which can be transported in the chamber without some drop clinging to the surface (when the electrical system is at rest).

Furthermore, to prevent the drops which touch each other coalescing naturally, it may be important to add suitable surfactants well known in the state of the art.

Initially, no matrix electrodes are active.

Among the 4 drops of the emulsion, one or more are said to be of interest. In a non-limiting manner, a so-called drop of interest is a drop in which a reaction occurs. The selection of the drops of interest present in the emulsion can be done by any technique, for example by fluorescence detection. The two substrates 100, 101 of the component can in particular be chosen transparent so as to be able to visualize the interior of the chamber 1.

E2 – : Chamber 1 of the component is seen from above.

By way of example, a single drop of interest 40 present in the emulsion is identified. Of course, there could be many more and the process would remain the same.

The position of this drop of interest is determined by the control and power supply unit 2. The determination of the position is for example carried out during the scanning of the chamber by an optical beam and by fluorescence detection on a sensor . An optical detection method with a camera placed above the chamber with suitable lighting to observe the fluorescence or the color of the drops can also be considered. Other detection solutions could be considered. These solutions are not the subject of the invention.

E3- : Chamber 1 of the component is viewed from the side.

The control and power supply unit 2 selects the electrode E_i or the electrodes of the matrix 10 to be activated in order to fix the drop of interest 40 on the bottom of the chamber 1. If the drop of interest 40 is at straddling several electrodes of the matrix, the control and power supply unit 2 may be required to select all of the electrodes E_1 of the matrix 10 covered by the drop of interest 40 in order to fix it. However, it is preferable for the size of the drops to be greater than the size of the electrodes. Several electrodes can thus be covered by a single drop.

E4 – : Chamber 1 of the component is viewed from the side.

The control and power supply unit 2 activates the electrode E_i or the electrodes selected during step E3. The other electrodes of matrix 10 remain inactive.

Each activated electrode E_i passes to an electrical potential distinct from that of the counter-electrode CE, thus making it possible to create a non-zero potential difference between the activated electrode and the counter-electrode, creating an electrostatic force, and therefore to generate a force electrostatic wetting known as EWOD, as previously described.

Subjected to this electrostatic force, the drop of interest 40 is fixed in its position, against the bottom of the chamber. As illustrated by the , the drop of interest 40 is thus flattened or in other words spread out against the bottom of the chamber.

E5- : Chamber 1 of the component is seen from above.

The drop of interest 40 being fixed, the control and power supply unit 2 controls the injection of a fluid flow F1 into the chamber 1, for example through the fluid inlet 11. This fluid flow F1, by example composed of an oil flow, makes it possible to evacuate all the drops towards the outlet channel, with the exception of the drop of interest 40 which is kept fixed by the electrostatic forces.

E6 – : Chamber 1 of the component is seen from above.

The control and power supply unit 2 deactivates each electrode selected during step E3.

The control and power supply unit 2 is configured to evacuate the drop of interest 40 present in the chamber 1. Two solutions are for example possible:

• The control and power supply unit 2 can control the injection of a fluid flow F2 into the chamber 1 to push the drop of interest 40 towards the fluid outlet 12.
• The control and power supply unit 2 selects and activates the electrodes of the matrix of electrodes 10 to advance the drop of interest 40 towards the fluid outlet. It thus uses the principle of electrowetting already described in the state of the art to move a drop.

According to a variant embodiment illustrated by the , after step E3 and before step E4, it is possible to enlarge the targeted drop of interest, in order to fix it better during step E4. For this, the control and power supply unit 2 actuates one or more electrodes E_i associated with drops located around the drop of interest 40 in order to make the drop of interest coalesce with one or more adjacent drops and create thus a bigger drop. The control and power supply unit 2 will then be led to select the electrodes of the matrix 10 which make it possible to fix this new large drop of interest.

It should be noted that the drops 4 are manufactured upstream by known techniques, preferably a flow method which makes it possible to achieve very high rates in a component that is simple to implement. The flow method consists in creating the drops using two phases, a so-called continuous phase (for example oil) and a dispersed phase (another solution immiscible with the oil). The two phases are injected into two separate channels which meet, forming a crossroads between them. Drops 4 are formed at the junction of the two phases at the crossroads. Other known techniques for forming the emulsion can be envisaged, the various techniques not forming part of the invention.

It can also be noted that the drops are generally made in the form of an aqueous solution. The drops float in a continuous phase, usually oil. The sample is therefore an emulsion. When this emulsion is injected into a two-dimensional chamber, the drops thus confined tend to clump together, forming a more or less organized crystal ( ).

Advantageously, the component may comprise two distinct fluidic outlets, a first outlet to evacuate the drops of interest from the emulsion and a second outlet to evacuate the other drops from the emulsion. It is also possible to provide a single outlet channel from the chamber splitting into two separate reservoirs (as in the appended figures). The pump and valve control system 3 makes it possible to select the fluidic inlets and outlets which open into the chamber 1.

The method described above may also have a variant embodiment.

In this variant embodiment, steps E1 and E2 described above remain identical, that is to say that the control and power supply unit 2 has identified each drop of interest 40 present in the emulsion and their positions. In the example, the emulsion only has for example a single drop of interest.

With reference to FIGS. 6A to 6D, the new steps are then as follows:

E30 – : Chamber 1 of the component is viewed from the side.

The control and power supply unit 2 is configured to create 4 larger drops in the emulsion, with the exception of the drop of interest 40. For this, the control and power supply unit 2 controls the electrodes of the matrix 10 which are located opposite all the drops, apart from those located opposite the drop of interest. The control and power supply unit 2 must actuate the electrodes in groups and according to a given sequence in order to make the drops coalesce and thus form larger drops.

E40 – : Chamber 1 of the component is viewed from the side.

The drops 4 located opposite the activated electrodes coalesce, forming larger drops than the drop of interest 40.

E50 – : Chamber 1 of the component is seen from above.

All the electrodes E_i of matrix 10 are deactivated. The control and power supply unit 2 controls the injection of a fluidic flow F3 into the chamber 1 to evacuate all of the drops 4, including the drop of interest 40, towards the fluidic outlet 12.

E60 – : Chamber 1 of the component is seen from above.

The drops 4, 40 are evacuated via the fluid outlet to a sorting device 5 making it possible to sort the drops by size. The smallest drops are then those which have not coalesced, i.e. the drops of interest 40.

It is therefore a question of using the system of the invention to prepare a sorting of drops to be carried out downstream.

This latter process could also be implemented by causing the drop of interest to coalesce, without acting on the other drops of the emulsion. The sorting carried out downstream would then make it possible to distinguish the drop of interest 40 which is larger than the other drops 4 of the emulsion.

As indicated above, the component, in particular the matrix of electrodes, can be manufactured according to different variant embodiments.

A first embodiment variant consists in using an active matrix of the TFT type (for “Thin Film Transistor”). Patent applications WO200345556A and US2014202863A1 describe this type of solution.

Another variant is to use a matrix of virtual electrodes called "Opto-EWOD" as described in the referenced publication P. Y. Chiou , H. Moon, H. Toshiyoshi , CJ Kim, and MC Wu, “Light actuation of liquid by optoelectrowetting ,” Sensors Actuators , A Phys., vol. 104, no. 3, p. 222–228, 2003 .

In this variant, the component thus comprises a stack of layers, including a photoconductive layer. Light radiation makes it possible to make the photoconductive layer locally conductive, temporally and locally defining a virtual electrode. The light beam therefore locally induces the electrowetting effect on the scale of a drop without having to manufacture a matrix of electrodes in a concrete way. There is thus a matrix of virtual electrodes. The light source can be a laser source or an overhead projector which projects an image (in order to define light zones (activated zone) and dark zones (deactivated zone)). It is also possible to use an OLED type display placed near under the component, capable of generating light beams F.

There gives an example of a stack of layers forming the component, according to this "Opto-EWOD" principle.

The upper substrate is composed of a layer of glass C1, on which is deposited a thin layer of ITO (for Indium Tin Oxide) C2, of about 100 nm, or another transparent conductive material, then forming the counter- EC electrode. A very thin layer C3 of a material is then deposited, for example chosen to be very hydrophobic (for example PTFE for Polytetrafluoroethylene (Teflon – Registered trademark)). Substrate 100 is flipped over to place layer C3 down.

The lower substrate 101 comprises a layer of C10 glass, on which a layer of ITO C20 is deposited. The photoconductive layer C30 is then deposited on the ITO layer. A C40 insulating layer is then deposited on the photoconductive layer and then a chosen hydrophobic C50 layer (for example made of PTFE). The two substrates 100, 101 are placed face to face and joined from the outside to form the chamber 1 intended to receive the drops. The distance separating the two hydrophobic layers C3, C50 in the direction of the height is chosen sufficient to be able to place only one drop and not a stack, thus making it possible to be able to detect the drops of interest and to associate each drop with a single matrix electrode or a group of several matrix electrodes.

The OLED type display A can be used to generate each localized light beam through the lower substrate and allow the activation of each virtual electrode of the matrix. The size of the light beam which defines a virtual electrode must preferably be less than or equal to the size of the drops.

As the principle of the invention does not work by moving the drops on a surface (as in the documents of the state of the art) but by fixing the drops of interest, it is not necessarily necessary to use outer layers C3, C50 hydrophobic. We can indeed be satisfied with a layer of PTFE (Teflon-registered trademark) or a chemical treatment, for example with a silane such as FDTS (for Perfluorodecyltrichlorosilane).

It is understood from the foregoing that the method of the invention has many advantages, including:

• A solution allowing rapid sorting at high speeds;
• A solution that can be fully automated, not requiring the intervention of qualified operators;
• A solution that uses principles that are already known and already proven;
• A versatile solution that can use different sorting protocols with or without coalescence in the same device;

Claims (11)

Method for sorting drops implemented in a system which comprises:

• A chamber (1) formed by two substrates (100, 101) facing each other, a first substrate and a second substrate,
• Means for generating several independent and juxtaposed electrostatic fields between the two substrates,
• A control and power supply unit (2) configured to individually generate each electrostatic field,
• A fluid inlet (11) and a fluid outlet (12) opening into the chamber (1),

Characterized in that said method comprises:

• A step of introducing an emulsion into said chamber, said emulsion comprising several drops (4) juxtaposed over two dimensions, among which at least one so-called drop of interest (40) having a determined position,
• Determination of the position of said drop of interest (40) between the two substrates,
• Generation of at least one electrostatic field to create a non-zero electric potential difference in order to fix said drop of interest (40) on said first substrate in its determined position,
• Injection of a fluidic flow (F1) into the chamber via said fluidic inlet (11) to evacuate the other drops of the emulsion, outside of said chamber via the fluidic outlet (12).

Method according to claim 1, characterized in that it includes a step of generating at least one electrostatic field associated with at least one drop adjacent to the drop of interest (40), to coalesce said drop of interest with said adjacent drop, to form a larger drop. Method according to Claim 1 or 2, characterized in that it comprises a step of injecting a fluid flow (F2) to evacuate the said at least one drop of interest (40) from the chamber. Method according to Claim 1 or 2, characterized in that it comprises a step of generating several electrostatic fields to move the said at least one drop of interest towards the said fluid outlet (12). Method according to one of Claims 1 to 4, characterized in that the step of determining the position of the drop of interest is implemented by fluorescence detection. Fluidic system used for implementing the method as defined in one of Claims 1 to 4, characterized in that it comprises:

• A chamber (1) formed by two substrates (100, 101) facing each other,
• Means for generating several independent and juxtaposed electrostatic fields between the two substrates,
• A control and power supply unit (2) configured to individually generate each electrostatic field,
• A fluid inlet (11) and a fluid outlet (12) opening into the chamber (1).

System according to Claim 6, characterized in that the means for generating several electrostatic fields comprise a matrix (10) of several juxtaposed electrodes (E_i) placed in the first substrate, and at least one counter-electrode (CE) common to all the electrodes of said matrix placed in the second substrate. System according to Claim 6 or 7, characterized in that the first substrate is produced by a stack of layers in which a photoconductive layer (C30) is excitable by a light beam (F). System according to Claim 8, characterized in that the first substrate comprises an outer layer made of a hydrophobic material. System according to claim 8 or 9, characterized in that the light beam is generated by an OLED type display (A). System according to one of Claims 6 to 10, characterized in that it comprises a valve and pump control system (3) controlled by the control and electrical supply unit (2) and configured to control each fluid flow injected into the fluid inlet (11) and the fluid outlet (12).