WO2023117864A1

WO2023117864A1 - Micro-droplet generating apparatus

Info

Publication number: WO2023117864A1
Application number: PCT/EP2022/086593
Authority: WO
Inventors: Jean Roman; Sylvain URSUEGUI; Rémi DANGLA; Etienne FRADET
Original assignee: Stilla Technologies
Priority date: 2021-12-20
Filing date: 2022-12-19
Publication date: 2023-06-29

Abstract

The invention relates, among others, to a micro-droplet generating apparatus for analyzing biological material and to a continuous oily phase for use in microfluidic devices. The applicant indeed identified a new category of surfactants and an oil/surfactant system which are compatible with molecular biology, improve digital PCR performances and limit sample wasting and dead volume.

Description

Micro-droplet generating apparatus

Field of the Invention

The invention pertains to the technical field of micro-droplet generating apparatus, and more particularly to micro-droplet generating apparatus for dispensing droplets of an aqueous phase into a continuous oily phase.

Background of the Invention

In the mid-1990s, advances in microfabrication techniques allowed the production of devices with feature sizes as small as few microns. Micromachining, photolithography, etching of silicon and glass substrates, all produced networks of flow channels that could sort reagents and molecules, partition chemical reactions, and act as droplet generators for mixture of aqueous and oil-based fluids. These microfluidic devices are well suited for performing biochemical assays, such as single-cell assays, studies of macromolecules e.g. proteins and nucleic acids, and polymerase chain reactions (PCR).

By the early 2000s, water-in-oil emulsions were being studied for potential applications designed to partition biological materials into nanoliter/pico liter-scale droplets. A significant advance was the use of surfactants in the oil-based continuous phase to maintain the integrity of droplets formed after the introduction of the discontinuous aqueous phase. Thanks to the use of surfactants in the oil-filled microfluidic devices, droplets can be pooled in widened channels or reservoirs, touching each other without breaking or fusing. Extensive fluidic networks of oil-and-surfactant filled channels can be designed to coordinate the intentional mixing of plugs or droplets to mediate biochemical reactions.

Fluorinated oils combined with fluorinated surfactants are particularly useful for handling biomolecules within aqueous droplets (Holtze et al., Biocompatible surfactants for water-in- fluorocarbon emulsions, 2008, 8(10), 1632-1639).

Emulsions containing fluorinated oil, however, can suffer from various disadvantages identified in the art. For example, aqueous droplets are typically buoyant in fluorinated oil (the density of fluorinated oil being higher than that of water), which may increase the complexity of droplet formation and manipulation: in some cases, the PCR mix deposition needs to be visually inspected; emulsions may require removal of excess oil below the droplets to position the droplets closer to a heat source, and/or the PCR mix position in the inlet well may be sensitive to inclination of the microfluidic chip.

Furthermore, the buoyant droplets may be more likely to be damaged by exposure to air above the emulsion, particularly when heated, the PCR mix is sensitive to evaporation leading to time constraints between sample deposition and sample analysis, and in some instances to the use of a sealing oil to prevent evaporation is necessary. Hence, alternative systems have been proposed such as in WO2014/145582 which describes emulsions comprising aqueous droplets disposed in a continuous phase that includes a silicone oil and a silicone surfactant, wherein the silicone surfactant is described by the following general formula [SILICONE BACKBONE] [ALKYL]x[POLYETHER]y[POLYSILOXANE]z, where x is 0-5, y is 1- 35, and z is 2-50.

WO2017070363 also discloses fdler fluids for microfluidic devices comprising a silicone oil and a siloxane block co-polymer solubilized in the silicone oil, wherein the siloxane block co-polymer is substantially immiscible with an aqueous liquid.

However, the identification of a suitable surfactant/oil system, compatible with molecular biology, is a major aim for improving digital PCR performances.

The compatibility with molecular biology means that the system should not impair with the main reaction protocols used in molecular biology, such as PCR.

Digital PCR performances encompass, notably, droplets stability (meaning that the droplets are substantially prevented from mixing and merging when in contact with each other), increase of the number of droplets for a given volume of PCR mix, with the consequential increase of the analyzed volume, and finally, avoidance of buoyant droplets.

Further, the accurate dispensing of micro-droplets is widely required in these applications. There are several options to generate micro-droplets but they mostly make use of a surfactant/oil system to dispense aqueous micro-droplets into a continuous oily phase.

Some of them make use of a container comprising a continuous oily phase and a micro-droplet dispenser with a dispensing tip and an actuator. The action of the actuator onto the dispensing tip filled with an aqueous phase causes the formation of micro-droplets which discharge out of a nozzle of the dispensing tip into the continuous oily phase.

It is also a significant goal to simplify, when loading microfluidic chips, PCR mix deposition and positioning, to prevent PCR mix evaporation to avoid sample wasting, and finally to minimize dead volume, i.e. the volume of sample that cannot be analyzed.

Summary of the Invention

The applicant identified a new category of surfactants and an oil/surfactant system, which are compatible with molecular biology, which improve digital PCR performances and limit sample wasting and dead volume and which are usable with micro-droplet generating apparatuses.

The applicant indeed demonstrated that surfactants according to the invention can be used in the micro-droplet generating apparatuses of the invention to prevent droplets from mixing and merging when in contact with each other and increase the droplets number and the analyzed volume compared to other surfactants. Surprisingly exceptional results have been obtained: sample partitioning has been demonstrated to achieve performances similar to those obtained using microfluidic technology, such as droplet size, droplet monodispersity, and droplet count.

This increase of droplets number and analyzed volume ensures the precision of digital PCR quantification and the separability of fluorescence signals between different populations, which are key parameters to determine robustness and precision of digital PCR.

Furthermore, the density ratio of the oil/surfactant system according to the invention is reversed compared to a fluorinated system, thus facilitating chip loading, preventing sample wasting and minimizing dead volume. These are major improvements for assay accuracy and reproducibility using the micro-droplet generating apparatuses according to the invention.

Finally, due to the density ratio inversion of a silicone oil compared to a fluorinated oil, the PCR mix is also protected against evaporation, thus allowing an extended operating time between chip loading and processing.

Drawings

Further details and advantages are described in the following detailed description with reference to the attached drawings, among which:

FIG. 1 schematically illustrates an exemplary micro-droplet generating apparatus according to the invention;

FIG. 2 is a magnified view around the dispensing tip of the micro-droplet generating apparatus of FIG. 1;

FIG. 3 schematically illustrates an exemplary embodiment of the dispensing tip with both outer and inner surfaces being frustoconical in shape;

FIG. 4 schematically illustrates an exemplary nozzle of the dispensing tip of FIG. 3, with a restriction;

FIG. 5 is a magnified view of an example of the nozzle of the dispensing tip of FIG. 4, with an angled nozzle free end;

FIG. 6 is a magnified view of an example of the nozzle of the dispensing tip of FIG. 4, with a straight portion leading to the nozzle free end;

FIG. 7 schematically illustrates an exemplary nozzle of the dispensing tip of FIG. 3, with a restriction and smooth transition between the core portion of the dispensing tip and the restriction; FIG. 8 is a magnified view of an exemplary nozzle of the dispensing tip of FIG. 7, with a cylindrical passage leading to the nozzle free end and smooth transition of the inner surface from the core portion up to the nozzle free end;

FIG. 9 schematically illustrates an exemplary nozzle of the dispensing tip of FIG. 3, with a frustoconical core portion and frustoconical nozzle portion of the inner surface, with smooth transition between both portions;

FIG. 10 schematically illustrates an exemplary dispensing tip in the shape of a tube;

FIG. 11 schematically illustrates an exemplary dispensing tip in the shape of a bended tube;

FIG. 12 schematically illustrates an exemplary dispensing tip in the shape of a needle;

FIG. 13 schematically illustrates an exemplary combination of reservoir and dispensing tip formed in one piece of material, the reservoir having a flat bottom;

FIG. 14 schematically illustrates an exemplary combination of reservoir and dispensing tip formed in one piece of material and in the shape of a funnel;

FIG. 15 schematically illustrates an exemplary combination of reservoir and dispensing tip, wherein the reservoir and the dispensing tip are connected through tubing;

FIG. 16 schematically illustrates an exemplary micro-droplet generating apparatus with a sealing bell;

FIG. 17 schematically illustrates a dock with a ring shape in one piece;

FIG. 18 schematically illustrates a dock with a ring shape in more than one piece;

FIG. 19 schematically illustrates an exemplary embodiment with an interface to contact the dispensing tip and transfer acoustic energy from the piezo element to the dispensing tip, and an inertial mass;

FIG. 20 schematically illustrates an exemplary embodiment with a piezoelectric actuator inside the dispensing tip;

FIG. 21 schematically illustrates an exemplary micro-droplet generating apparatus with an electromagnetic vibrator to vibrate the nozzle free end at the oil/air interface;

FIG. 22 schematically illustrates an exemplary micro-droplet generating apparatus with a motion actuator to set the dispensing tip in movement in the direction of its longitudinal axis;

FIG. 23 is a graph showing the speed v of the nozzle free end as a function of time when it has the shape of a square wave;

FIG. 24 schematically illustrates an exemplary micro-droplet generating apparatus with a motion actuator to set the dispensing tip in movement in a direction perpendicular to its longitudinal axis; FIG. 25 schematically illustrates an exemplary micro-droplet generating apparatus with a motion actuator to set the dispensing tip in an oscillating movement;

FIG. 26 schematically illustrates an exemplary micro-droplet generating apparatus with a single particle dispensing module;

FIG. 27 is a magnified schematical view of the nozzle showing a first portion and a second portion used for single particle testing in the micro-droplet generating apparatus of FIG. 26; and

FIG. 28 schematically illustrates an exemplary micro-droplet generating apparatus with a driver.

Although some embodiments are illustrated in the drawings as distinct, these are merely nonlimiting illustrations of the invention. From the following detailed description, the skilled person would understand that combinations thereof are possible and are part of the present disclosure.

Detailed Description

Micro-droplet generating apparatus

The present invention relates to a micro-droplet generating apparatus, which will be descripted in the following with reference to FIG. 1 to 28. The micro-droplet generating apparatus 1 comprises: a container 3 comprising a continuous oily phase 31, the continuous oily phase 31 comprising a silicon oil; a micro-droplet dispenser 2 comprising a reservoir 21 filled with an aqueous phase 211, a dispensing tip 22, and an actuator 23, and optionally a controller 24 for controlling the operation of the micro-droplet dispenser 2 and in particular the actuator 23.

The dispensing tip 22 comprises a nozzle 221. The actuator 23 is coupled to the dispensing tip 22 to generate micro-droplets 9 of aqueous phase at the nozzle 221 and dispense the micro-droplets 9 into the continuous oily phase 31. At least one of the continuous oily phase 31 and the aqueous phase 211 comprises a surfactant which is a block copolymer comprising blocks [A], [B] and [C] : block [A] of the surfactant corresponds to a block of formula (I)

wherein Ri is a Ci-Cis alkyl, and m is comprised between 10 and 100; block [B] of the surfactant corresponds to a block of formula (II)

wherein q is comprised between 2 and 18, n is comprised between 1 and 50, and R2 is selected from the group consisting of compounds of • formula (III)

• formula (V)

wherein r, s, t, u, v and w are comprised between 1 and 32 and R3 is selected from the group consisting of -OH, -O-CH3 and -O-CH2CH3; block [C] of the surfactant corresponds to a block of formula (VII)

wherein p is comprised between 5 and 300; wherein terminal groups are selected from the group consisting of -H, -CH3 and -Si(CH3)3; and wherein the general formula of the surfactant is selected from [A]-[B]-[C], [B]-[C]-[A], [C]- [A]-[B], [B]-[A]-[C], [A]-[C]-[B] and [C]-[B]-[A],

In an embodiment, the container 3 may have any shape suitable to the applications. For example, the container 3 may be a well plate of any size such as 30 to 500,000 wells for example: 96 wells, 384 wells, 200,000 wells, etc. The container may be made in a transparent or opaque material. The container preferably has a transparent bottom.

Although the drawings show only one dispensing tip 22, the micro-droplet dispenser 2 may be provided with a plurality of dispensing tips 22.

In an embodiment, the dispensing tip 22 may be described as having an outer surface 222 and inner surface 223 (FIG. 3).

The outer surface 222 may have a tubular or a frustoconical shape, for example corresponding to a cone with an apex angle between 0 to 10°, with the apex towards the ground.

The inner surface 223 may also have a core portion 2231 with a tubular or frustoconical shape, for example corresponding to a cone with an apex angle between 0 to 10°, with the apex towards the grounds. In some embodiments (FIG. 4 to 8), the inner surface 223 has a core portion 2231 and a nozzle portion, the nozzle portion comprising a restriction 224 (a short section with a reduced cross-sectional area) forming the nozzle 221 of the dispensing tip 22. This is a well-known technique to improve the dispenser’s drop generation performance. The restriction 224 is progressively diminishing the diameter of the inner surface 223 from the core portion 2231 to the nozzle free end 2211.

For example, the restriction 224 can create an acutely angled nozzle free end (FIG. 4 and 5). In other words, the inner surface 223 at the restriction 224 has a frustoconical shape corresponding to a cone with an apex angle greater than that of the core portion 2231, for example from 20 to 60°. This has the advantage of having a low fluid impedance, allowing the jet to operate with a lower amplitude of voltage applied to the actuator 23. It is also less prone to clogging (with wanted or un-wanted particles) than other designs because of the low aspect ratio of the final section. However, the nozzle free end 2211 of this jet is very delicate because of the very thin sharp edge used to form the nozzle free end 2211 itself. With this arrangement the directionality of the micro-droplets 9 produced may be poor in some applications.

In another example (FIG. 4 and 6), the restriction 224 has a straight portion 2241 leading to the nozzle free end 2211. In other words, the inner surface 223 at the restriction 224 can be divided into two portions, an intermediate portion 2242 with a frustoconical shape corresponding to a cone with an apex angle greater than that of the core portion, for example from 20 to 60°, and a straight portion 2241 leading to the nozzle free end with a cylindrical shape. The straight portion 2241 is at least 10 times shorter than the intermediate portion 2242, preferably at least 15, 20, 30, 40 or 50 times shorter. This arrangement has a higher fluid impedance and thus requires more applied energy to eject a droplet 9. This arrangement is more prone to clogging, and so it may not be suitable for some applications in which the dispensed liquid includes particles. However, directionality of the droplets 9 ejected is generally increased and is generally more consistent.

In still another example (FIG. 7 and 8), the transition between the core portion 2231 and the restriction 224 up to the nozzle free end 2211 is smooth. In other words, when seen in a longitudinal section, the inner surface from the core portion 2231 up to the nozzle free end forms a curve, the first derivate of which is continuous. The restriction 224 then has a central portion 2243 with a shape close to a conical frustum and corresponding to a cone with an apex angle greater than that of the core portion, for example from 20 to 60°. The restriction 224 has an end portion 2241 leading to the nozzle free end 2211, the inner surface at the aperture has a longitudinal section forming an angle of 0 to 20° with the longitudinal axis. This example has performance which is a compromise between the two examples described above. It has a relatively low fluid impedance, because of its very short end section and the smooth transition of its inner diameter from the core portion 2231 to the nozzle free end 2211. There is good directionality because of a short end section 2241. There is reasonable resistance to clogging because of high aspect ratio of narrow section. The aperture design is less fragile, which should help the manufacturing process.

In still another example (FIG. 9), the inner surface 223 has an end portion 2233 leading to the nozzle free end 2211 with a frustoconical shape corresponding to a cone with an apex angle smaller than that of the core portion 2231. An intermediate portion 2232 smoothly connects the core portion 2231 to the end portion 2233.

In an example (FIG. 10 and 11), the dispensing tip may be a simple tube. In such case, the tube may be straight (FIG. 10). Alternatively, the tube may be curved (FIG. 11): the tube has a first straight portion 226, a second curved portion 227 and a third straight portion 228 bearing the nozzle free end 2211. The third straight portion 228 is much shorter than the first straight portion 226. The angle a formed between the first and third straight portions 226, 228 may be 30° and 60°, for example 45°. In an example (FIG. 12), the dispensing tip 22 may be described as having a needle shape, in other words, it has a tubular shape terminated with a bevel 225.

In an embodiment, the reservoir 21 is located just above the dispensing tip 22 with the lower surface 212 of the reservoir connecting to the inner surface 223 of the dispensing tip 22 (FIG. 1, 13 and 14). The lower surface 212 of the reservoir 21 may be flat (FIG. 13). The combination of the reservoir 21 and the dispensing tip 22 may have a funnel shape. For example, the lower surface 212 may form with the longitudinal axis AA of the dispensing tip 22 an angle of 30 to 90° (FIG. 14). In some examples, the reservoir 21 is formed in the continuity of the dispensing tip 22. In other words, the upper part of the dispensing tip 22 acts as a reservoir 21.

In other embodiments (FIG. 15), the reservoir 21 may also be disposed otherwise, with a tubing 25 feeding the dispensing tip 22 from the reservoir 21. The aqueous phase 211 may be filled into the dispensing tip 22 through the tubing 25 by a pump 26. The controller 24 may be configured to control the operation of the pump 26 to fill the dispensing tip 22. In some cases, the aqueous phase 211 may be filled into the dispensing tip 22 by capillary forces (not illustrated).

In some embodiments, the dispensing tip 22, optionally together with the reservoir 21, may be removable from the micro-droplet dispenser 2. In such case, the micro-droplet dispenser 2 may have a dock 27 with an orifice to receive the dispensing tip 22 in a sealed manner. In such case, a preferred embodiment provides the combination of dispensing tip 22 and reservoir 21 in a funnel shape as described above. The dispensing tip 22 and the reservoir 21 are filled with the aqueous phase 211 and the top of the reservoir may be closed with a seal. In such case, the micro-droplet dispenser 2 may comprise a needle configured to pierce the seal. Alternatively, the reservoir 21 is closed with a lid provided with a vent 213 and the vent 213 may be closed with a seal. The micro-droplet dispenser 2 may comprise a sealing bell 214 configured to form a sealed chamber around the combination of dispensing tip 22 and reservoir 21 above the dock 27, thus enabling control of the pressure inside the reservoir (FIG. 16).

In an embodiment, the actuator 23 is a piezoelectric actuator to generate acoustic waves, wherein the piezoelectric actuator 23 and the dispensing tip 22 are coupled so that the generated acoustic waves propagate through the dispensing tip 22 up to the nozzle 221, thereby generating micro-droplets 9 at the nozzle free end 2211. In other words, the actuator 23 is configured to couple acoustic energy to the aqueous phase 211 in the dispensing tip 22 to dispense said aqueous phase through the nozzle free end 2211 as a droplet 9. The generated acoustic waves may be transmitted to the aqueous phase through a water column in direct contact with the aqueous phase. Preferably, the column is a degassed water column. Such water column may have a volume of 1 to 5 pL, such as 2 pL. 3 pL and 4 pL. Alternatively, the generated acoustic waves may be transmitted to the aqueous phase through a wall of the dispensing tip 22. The piezoelectric actuator 23 may be controlled by the controller 24 to set or change at least one of piezo frequency, pulse duration and intensity. This makes it possible to control at least one of the size and frequency of the dispensed micro-droplets 9.

The piezoelectric actuator 23 may be configured to engage with and disengage from the dispensing tip 22. The piezoelectric actuator 23 may be formed by one or more piezo elements, which may be in the form of piezo stacks.

The micro-droplet dispenser 2 may further comprise an actuator assembly comprising the piezoelectric actuator 23 and the dock 27 to receive the dispensing tip 22. The dock 27 may have a ring shape to receive the dispensing tip 22. The ring shape may be in one piece (FIG. 17) or a plurality of pieces such as ring elements 271 (FIG. 18). In this latter case, the ring shape may not be continuous around the dispensing tip 22 but the ring elements may be separated from each other by a small gap. The ring elements 271 may form at least one movable jaw and the other being static jaws, the at least one movable jaw being displaceable from an open position to a closed position and vice versa, for example through a jaw drive mechanism preferably controlled by the controller 24. In the closed position, the jaws are engaged with the dispensing tip 22 for dispensing. In the open position, the at least one movable jaw enables loading of the dispensing tip 22 onto the dock 27. The actuator assembly may be configured to enable top or side loading of the dispensing tip 22 onto the dock 27. In such case, the micro-droplet dispenser 2 may comprise a docking mechanism to load the dispensing tip 22 onto the dock 27 either from the top or the side.

The number of j aws may be 2, 3 , 4, 5 or more . Preferably, at least some of the j aws may be movable . Preferably, the ring shape dock 27 is equally divided into the number of jaws, i.e. all jaws are equally dimensioned. At least one jaw is provided with a piezo element 231 forming the piezoelectric actuator, preferably at least two. In the preferred embodiment, all jaws are provided with a piezo element.

The piezo element 231 may be in direct contact with the dispensing tip 22, in the case of movable jaws when in the closed position. Alternatively, an interface 232 may be provided to contact the dispensing tip 22 and for transferring acoustic energy from the piezo element 231 to the dispensing tip 22 (FIG. 19). In such case, the piezo element 231 contacts the interface 232.

The actuator assembly may be configured to apply a static mechanical pressure against the dispensing tip 22. The static mechanical pressure may be a bias force upon which the piezoelectric actuator 23 applies a pressure wave. The actuator assembly may comprise an inertial mass 272 for the piezo element 23 to act against in order to couple pressure waves into the dispensing tip 22 (FIG. 19).

The dispensing tip 22 may be in a friction fit into the dock 27 when received therein. The actuator assembly may comprise a contact surface inclined in cross-section to an axis of the dispensing tip 22 at an angle in the range of 0° to 5° and preferably 1° to 1.5°.

The actuator assembly may comprise a movement amplifying mechanism (not shown) for amplifying piezo element movement, preferably controlled by the controller 24. The movement amplifying mechanism may comprise a base and a pivoting link arm, one end of which is acted upon by the piezo element 231 and the other end of which has a face for engagement with the dispensing tip 22. Alternatively, the piezo-electric actuator 23 may be provided inside the dispensing tip 22, in the space formed by the inner surface 223 thereof (FIG. 20).

Preferably, the piezo element 231 is positioned at the dispensing tip 22 level above the nozzle 221 thereof.

The actuator assembly may be configured to provide controlled heating in a limited region around the nozzle free end 2211, preferably controlled by the controller 24.

The actuator 23 may be an electromagnetic vibrator to vibrate the nozzle 221 of the dispensing tip 22 when this latter is dipped into the continuous oily phase 31 and while the aqueous phase 211 is fed to the dispensing tip 22, thereby dispensing micro-droplets 9 into the continuous oily phase 31 (FIG.

21). In such case, the dispensing tip 22 may be configured to translate along a vertical axis. The electromagnetic vibrator 23 may be positioned so that although the dispensing tip 22 is partially submerged in the continuous oily phase 31, the vibrator 23 remains out of the continuous oily phase 31. The electromagnetic vibrator 23 may be configured to vibrate at a constant frequency and peak amplitude, preferably between 1 and 5 mm, for example 2, 3 or 4 mm. The electromagnetic vibrator 23 may be controlled by a waveform generator. The electromagnetic vibrator 23 may be configured to vibrate the nozzle free end 2211 of the dispensing tip 22 at the oil/air interface.

The actuator 23 may be a motion actuator to set the dispensing tip 22 into motion according to at least two stages, a volume-increasing stage during which a micro-droplet 9 is formed at the nozzle 221 of the dispensing tip 22, and a micro-droplet detaching stage during which the formed micro-droplet 9 breaks free from the nozzle 221 of the dispensing tip 22. The motion actuator 23 may set the dispensing tip into a periodic motion.

The motion actuator 23 may be configured to set the dispensing tip 22 into a translational motion or a rotational motion. For example, the motion actuator 23 may be configured to set the dispensing tip 22 into motion so that its nozzle 221 periodically moves below the surface 311 of the continuous oily phase 31 in the container 3 and in the direction of the longitudinal axis AA of the dispensing tip 22 (FIG.

22). The speed of the nozzle free end 2211 may vary in the shape of a square wave (FIG. 23); wherein a speed of the nozzle free end 2211 in a first half motion period p'/₌,i and that in a second half motion period p>/^ are identical but in opposite directions. The nozzle free end 2211 remains in the continuous oily phase 31 during the periodic motion. In another example, the direction of the periodic motion is not in the direction of the longitudinal axis AA of the dispensing tip 22 but perpendicular thereto (FIG. 24).

In another example, the motion actuator 23 may be configured to set the dispensing tip 22 into motion so that the nozzle free end 3311 of the dispensing tip 22 periodically oscillates between two top positions by moving the dispensing tip 22 around an oscillation axis while maintaining the nozzle free end 2211 in the continuous oily phase 31 (FIG. 25).

The conditions for detachment are explained in EP 3738671 Al. In all cases, there exists a maximum adhesion force f? between the micro-droplet in formation and the nozzle free end 2211. The value of f? is related to the surface free energy of nozzle, the surface tension of the droplet and the geometric dimension of the nozzle free end 2211.

The motion actuator 23 may be configured to move the dispensing tip 22 with an instantaneous acceleration during the micro-droplet detaching stage. The instantaneous acceleration is typically an acceleration of the dispensing tip 22 when this latter changes direction. The instantaneous acceleration causes the micro-droplet 9 to detach. Although this may be the case, this does not necessarily mean that at each instantaneous acceleration of the dispensing tip 22, a micro-droplet 9 will detach. In such case, the speed of the dispensing tip 22 at the time the direction is changed, is zero (the speed changes from being negative to positive or from being positive to negative). The condition for detachment is when the instantaneous acceleration ai becomes greater than fi/m. m being the weight of the micro-droplet 9: |f₃| < |ai| . Thus, during the volume-increasing stage, the volume of the micro-droplet 9 increases as well as its weight m. At each direction change |f₃|/m decreases until it becomes lower than the instantaneous acceleration and the micro-droplet 9 breaks free. Control of the instantaneous acceleration may also be used to control micro-droplet size.

One period of the periodic motion may include a plurality of instantaneous acceleration with the same value, the instantaneous accelerations are then preferably equally spaced apart in time. Thus, a plurality of micro-droplets may be dispensed during one periodic motion when the value of the instantaneous acceleration is correctly chosen, for example so that a droplet detaches at each instantaneous acceleration. In one example, the periodic motion includes two instantaneous accelerations and the moving trajectory of the nozzle free end 2211 is a straight line or an arc. In one example, the periodic motion includes more than two instantaneous accelerations and the moving trajectory of the nozzle free end 2211 is a regular polygon such as a regular triangle, a square, a regular pentagon, a regular hexagon, and so on. It is also possible to determine an instantaneous acceleration for detachment of the micro-droplet 9 every 'A p, I p, 1,5 p, 2 p, etc.: p being the duration of one period of the periodic motion. Preferably, between two subsequent instantaneous accelerations, the speed of the dispensing tip 22 is constant.

The motion actuator 23 may be configured to move the dispensing tip 22, below the surface 311 of the continuous oily phase 31, with changing velocity. More particularly, the motion actuator 23 may be configured to move the dispensing tip 22 so that the speed of the nozzle free end 2211 changes monotonously in both a first half period and a second half period of the periodic motion. The monotonously changing means that the speed of the nozzle free end 2211 at a subsequent moment is always greater than or equal to (or, less than or equal to) the speed at a previous moment. In such case, the condition for detachment of the micro-droplet is |f₃|<|f2|, where fz is the viscous resistance force applied on the droplet moving in the continuous oily phase 31 and satisfies fz = 67rr|rv, wherein q is the viscous coefficient of the continuous oily phase 31, r the radius of the droplet 9, and v its speed. Thus, during the volume increasing stage, the volume of the micro-droplet 9 increases as well as its radius r. At some point, the value of fz will supersede the value of f₃. Control of the speed of the motion of the dispensing tip 22 provides control of the time when the micro-droplet 9 breaks free. Control of the speed may also be used to control micro-droplet size.

The motion actuator 23 may be coupled with a discharger configured to cause the aqueous phase to flow out of the nozzle 221 at a constant flow rate.

The micro-droplet generating apparatus 1 may comprise a loading station. The loading station may particularly be used with a reservoir 21 formed from the upper part of the dispensing tip 22. The loading station may be in the form of a well plate. The well plate may be for example a 96-well plate. In one example, at least two wells comprise different aqueous phases. In such case, the micro-droplet generating apparatus 1 may also comprise a cleaning station for cleaning the dispensing tip 22, for example by flushing.

The actuator and the dispensing tip may form a piezo dispense capillary (PDC). In such case, at least the nozzle of the dispensing tip may be optically transparent. The PDC may have an inner diameter of 50 to 120 pm. Lower and upper values may be one of 60 pm, 80 pm, 90 pm and 100 pm. The PDC may be surrounded by a metallic sheath bringing rigidity to the PDC. The actuator is preferably a piezo element surrounding the PDC.

The micro-droplet generating apparatus 1 may further comprise a single particle dispensing module 4, in particular when the aqueous phase 211 comprises particles. Such particles may be micro-sized or nano-sized beads. For example, the beads are made of transparent polymethylmethacrylate (PMMA). PMMA provides an excellent optical brilliance and a low autofluorescence compared to other materials. The beads may be functionalized, for example carboxylated beads or aldehyde modified beads. The beads may be fluorescence encoded beads. They may be color encoded with up to six fluorescent dyes (fluorophores). The fluorophores may be directly incorporated into the core of the beads during the microparticle formation. This ensures a much more homogeneous distribution of the dyes within the beads when compared to conventional diffusion controlled dyeing processes. Additionally, the fluorophores may be caged within the PMMA matrix and thus less likely to leak out. The size of the beads may be 100 nm to 20 pm. In some applications, the bead size may be 100 nm to 500 nm. In other applications, the bead size may be 2 pm to 20 pm.

The micro-droplet dispenser 2 may comprise a driver 5 to drive the dispensing tip over the container 3, and preferably controlled by the controller 24 (FIG. 28). The driver 5 may be configured to move the dispensing tip 22 in translation along one, two or three axes. In case the displacement is along more than one axis, the driver 5 enables combined displacement along the axes. When movement along three axes are enabled, there are two horizontal axes x, y and one vertical axis z. Therefore, the driver 5 may be configured to displace the dispensing tip 22 all over the container 3 to dispense droplets 9 in multiple locations of the container 3. The driver 5 may also be configured to bring the dispensing tip 22 above the container 3 from a location away from the container 3, and from a location away from the container to the container. The driver 5 may be coupled to the controller 24 to displace the dispensing tip 22 after dispensing of each droplet 9. Thus, stacking of droplets 9 can be avoided, although in some applications stacking of droplets 9 is desired.

The driver 5 may be coupled to the controller 24 to displace the dispensing tip 22 over the loading station and the cleaning station.

The single particle dispensing module 4 is configured to:

- detect whether a single particle is comprised within a volume of aqueous phase closest to the nozzle 221;

- control the micro-droplet dispenser 2 and the drive 5 to dispense the volume of aqueous phase closest to the nozzle 221 into the continuous oily phase if a single particle is detected therein; and

- control micro-droplet dispenser 2 and the drive 5 to dispense the volume of aqueous phase closest to the nozzle into a recovery reservoir 6, otherwise (FIG. 26).

For example, the single particle dispensing module 4 may comprise a nozzle camera enabling imaging of the aqueous phase 211 in the nozzle 221 of the dispensing tip 22, and preferably controlled by the controller 24.

The nozzle camera, like e.g. the camera IDS UI2220 SE, may be provided with a CCD-based device and a camera optic, and it is configured for imaging the nozzle 221 and further for a visualization and monitoring of the drop formation prior to dispensing. As an example, the nozzle camera may be adapted for detecting particles in the nozzle 221 of the dispensing tip 22 over an axial length of e.g. 700 pm to 800 pm. When the micro-droplet dispenser 2 comprises a plurality of dispensing tips 22, each may be provided with a nozzle camera.

The single particle dispensing module 4 may comprise an irradiation device, e. g. an LED device is provided, which is adapted for illuminating the nozzle 221, and preferably controlled by the controller 24, so that the image intake by the nozzle camera can be improved. Advantageously, the irradiation device provides a stable and consistent light source adapted to the nozzle camera.

The single particle dispensing module 4 may comprise a testing unit, preferably controlled by the controller 24, coupled with the nozzle camera and configured to test a single particle condition in a first portion 2212 of the nozzle 221 closest to the nozzle free end 2211, the first portion 2212 typically having a volume equal to about the volume of dispensed micro-droplets (FIG. 27). The testing unit may be configured to communicate with the controller 24 to indicate whether the single particle condition is fulfilled. In such case, the controller 24 may be configured to control the micro-droplet dispenser 2 to dispense a micro-droplet 9 into the container 3 if the single particle condition is fulfilled and to dispense a micro-droplet 9 into a collection reservoir 6 otherwise.

The single particle condition may be the presence of one single particle inside the first portion 2212 of the nozzle 221. The single particle condition may be the combination of the presence of one single particle inside the first portion 2212 and the absence of any particle in a second portion 2213 of the nozzle adjacent to the first portion 2212. Thus, not only the first portion 2212 closest to the nozzle free end 2211 is tested but also its surrounding as testing is extended to a portion adjacent the first portion 2212 of the nozzle 221. When the second portion 2213 comprises a particle, this particle can travel to the first portion 2212 after testing and before dispensing. By controlling the second portion 2213, it is possible to avoid dispensing the micro-droplet 9 into the continuous oily phase 31 in such situation.

Alternatively, the single particle dispensing module 4 may be a module to dispense droplets with a set number of particles of 2 or more, such as 3, 4, 5, 10, 20, etc. In such case, the single particle condition is replaced by a P particles condition, P being a set number of particles, which may be the presence of P particles in the first portion 2212 or the presence of P particles in the combined volume formed by the first and second portions 2212, 2213.

The testing unit may be configured to provide optical feedback and modeling of individual particle’s behaviors within the nozzle 221. In particular through a prior training of the particle’s behaviors within the dispensing tip 22, the testing unit can accurately identify single particles, for example in at least one of the first and second portions 2212, 2213 or even in the dispensing tip 22, which possess a high probability of encapsulation in a proceeding droplet. Once a particle has been identified, the testing unit provides feedback to the micro-droplet dispenser 2 to selectively deposit the single particle encapsulating micro-droplet 9 while discarding all other remaining droplets.

Image data of the first and second portions 2212, 2213 of the nozzle 221 may be pre-stored in the testing unit. Advantageously, this facilitates conducting the testing by comparing a current image of the nozzle 221 during the operation of the micro-droplet dispenser 2 with the pre-stored image data. The image data may comprise fixed sets of first portion image data and second portion image data if the dimensions of the first and second portions 2212, 2213 do not change during operation. Alternatively, the image data may include multiple sets of at least one of first and second portion image data if the dimensions of these portions 2212, 2213 dynamically change during operation as a result of changing dispensing conditions.

The micro-droplet generating apparatus 1 may further comprise a camera to capture images of the micro-droplets 9. The camera may be placed above the container 3 to capture images of the microdroplets 9 from the top of the container 3. The camera may be placed below the container 3 with a transparent bottom to capture images of the micro-droplets 9 from the bottom of the container 3. The micro-droplet generating apparatus 1 may further comprise a display for displaying images captured by the camera.

The micro-droplet generating apparatus 1 may further comprise a light source to illuminate the micro-droplets 9 inside the continuous oily phase 31 in the container 3. When fluorophores are used, the light source may be adapted to the absorption spectrum of each fluorophore. Therefore, it is possible to capture separate images for each fluorophore.

Although the present description refers to only one controller 24, a plurality of controllers 24 may be provided to control each element of the micro-droplet generating apparatus 1. The micro-droplet generating apparatus 1 may further comprise a digital PCR module to carry out PCR inside each micro-droplet 9 in the continuous oily phase 31.

In the above general formula [A], [B] and [C], unless specified otherwise: alkyl denotes a straight-chain or branched group containing 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18 carbon atoms. Examples of suitable alkyl radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, etc. Hence, a Ci-Cis alkyl corresponds to an alkyl group containing 1 to 18 carbon atoms.

In an embodiment, m is comprised between 10 and 35, preferably between 10 and 17 or between 17 and 25.

In an embodiment, m is comprised between 25 and 75.

In an embodiment, Ri is a C12 alkyl.

In an embodiment, Ri is a Ci6 alkyl.

In an embodiment, Ri is a C12 alkyl and m is comprised between 10 and 35, preferably between 10 and 17 or between 17 and 25.

In an embodiment, Ri is a Ci6 alkyl and m is comprised between 25 and 75.

In an embodiment, block [B] of the surfactant corresponds to a block of formula (II)

wherein q is comprised between 2 and 18, n is comprised between 1 and 50, and R2 is selected from the group consisting of compounds of

• formula (III)

formula (V)

wherein r, s, t, u, v and w are comprised between 1 and 32 and R3 is selected from the group consisting of -OH, -O-CH3 and -O-CH2CH3.

In an embodiment, q is 3.

In an embodiment, n is comprised between 1 and 15, preferably 1 and 5.

In an embodiment, n is comprised between 5 and 15.

In an embodiment, r, s, t, u, v and w are comprised between 1 and 18, preferably between 1 and

12 and more preferably between 6 and 9.

In an embodiment, r, s, t, u, v and w are independently equal to 1 or 10.

In an embodiment, R3 is -OH.

In an embodiment, R2 is a compound of formula (III)

In an embodiment, R2 is a compound of formula (V)

wherein r is comprised between 6 and 9 and R3 is -OH.

In an embodiment, R2 is a compound of formula (V)

wherein t is 10, u is 1 and R3 is -OH.

In an embodiment, block [C] of the surfactant corresponds to a block of formula (VII)

wherein p is comprised between 5 and 300.

In an embodiment, p is comprised between 5 and 200, preferably between 75 and 175 and more preferably between 95 and 140.

In an embodiment, p is comprised between 100 and 300.

In an embodiment, the block copolymer comprising blocks [A], [B] and [C] comprises terminal groups selected from the group consisting of -H, -CH3 and -Si(CH3)3.

In an embodiment, the terminal groups are -CH3 or -Si(CH3)3.

In an embodiment, the surfactant is a block polymer comprising blocks [A], [B] and [C], wherein block [A] of the surfactant corresponds to a block of formula (I)

wherein Ri is a C12 alkyl and m is comprised between 10 and 35; block [B] of the surfactant corresponds to a block of formula (II)

wherein r is comprised between 6 and 9 and R3 is -OH; block [C] of the surfactant corresponds to a block of formula (VII)

(VII), wherein p is comprised between 75 and 175; wherein terminal groups are selected from the group consisting of H, CH3 and -Si(CH3)3.

wherein Ri is a Ci6 alkyl and m is comprised between 25 and 75; block [B] of the surfactant corresponds to a block of formula (II)

wherein q is 3, n is comprised between 1 and 5 or between 5 and 15, and R2 is a compound of formula (V)

wherein u is 1, t is 10 and R3 is -OH; block [C] of the surfactant corresponds to a block of formula (VII)

(VII), wherein p is comprised between 100 and 300; and wherein terminal groups are selected from the group consisting of H, CH3 and -Si(CH3)3. In an embodiment, the general formula of the surfactant is [A]-[B]-[C].

In an embodiment, the general formula of the surfactant is [B]-[C]-[A] .

In an embodiment, the general formula of the surfactant is [C]-[A]-[B] .

In an embodiment, the general formula of the surfactant is [B]-[A]-[C] .

In an embodiment, the general formula of the surfactant is [A]-[C]-[B] . In an embodiment, the general formula of the surfactant is [C] - [B] -[ A] .

According to an embodiment, surfactants that can be used in the system according to the invention can be selected from the group consisting of the surfactant commercialized by Gelest® under the commercial name ABE 3642 (CAS No 212335-52-9), the surfactant commercialized by Siltech Corporation, under the commercial name Silube®J208-812 (CAS No 212335-52-9) and the surfactant also commercialized by Siltech Corporation, under the commercial name Silube® T310-A-16 (CAS No 145686-34-6).

In a preferred embodiment, the surfactant commercialized by Gelest® under the commercial name ABE 3642 (CAS No 212335-52-9) can be used as a surfactant according to the present invention.

In a preferred embodiment, the surfactant comprises 15 to 50% by weight of the surfactant of block

[A], preferably 18 to 50%, more preferably 20 to 40%.

In a preferred embodiment, the surfactant comprises 3 to 10% by weight of the surfactant of block

[B].

In a preferred embodiment, the surfactant comprises 20 to 40% by weight of the surfactant of block [A], 3 to 10% by weight of the surfactant of block [B], and the remaining weight of the surfactant of block [C].

In an embodiment, the surfactant comprises 30 to 35% by weight of the surfactant of block [A], 7 to 10% by weight of the surfactant of block [B], and the remaining weight of the surfactant of block [C] .

Typically, the percentages by weight are estimated by RMN 1H analysis.

According to the invention, the continuous oily phase comprises a silicone oil.

A silicone oil as used herein encompasses molecules having a skeleton structure of alternating silicon and oxygen atoms bonded one to another with hydrocarbon attached to silicon atoms. The silicon atoms may be substituted by various moieties, such as hydrocarbon moieties.

The silicon oil may be chosen according to its viscosity and optionally according to the design of the microfluidic device in which the continuous oily phase may be used.

Viscosity can be measured by any method known by the one skilled in the art. Typically, the viscosity can be measured using usual viscometer such as a rotational viscometer. Typically, the viscosimeter can be a viscosimeter adapted to low viscosity material. For example, Brookfield viscosimeters (AMETEK Brookfield, 11 Commerce Blvd., Middleboro, MA 02346 USA) such as LVT, LVDV-E, DV1MLV, DV2TLV, DV3LTV can be used, together with Brookfield low viscosity accessories.

In an embodiment, the continuous oily phase comprises a silicone oil with a viscosity between 0.5 and 500 centistokes at 25°C.

In an embodiment, the continuous oily phase comprises a silicone oil with a viscosity between 0.5 and 100 centistokes at 25°C.

In an embodiment, the continuous oily phase comprises a silicone oil with a viscosity between 0.5 and 50 centistokes at 25°C. In an embodiment, the continuous oily phase comprises a silicone oil with a viscosity between 0.5 and 10 centistokes at 25°C.

In an embodiment, the continuous oily phase comprises a silicone oil with a viscosity between 0.7 and 5 centistokes at 25°C.

In an embodiment, the viscosity of the continuous oily phase is comprised between 0.8 and 4.0 centistokes at 25°C, preferably between 0.9 and 3.0 centistokes at 25°C, and more preferably between 1.0 and 2.0 centistokes at 25°C.

Typically, the viscosity of the continuous oily phase is between 1. 1 and 1.5 centistokes at 25°C.

Silicone oils with a viscosity between 0.7 and 5 centistokes at 25°C are particularly advantageous when used in Opal™ microfluidic devices, described, for example, in patent applications W02020/109379 and W02020/109388.

In an embodiment, the silicone oil comprises decamethyltetrasiloxane.

In another embodiment, the silicone oil comprises at least 60% by weight relative to the total weight of the silicone oil of decamethyltetrasiloxane, preferably 70% by weight, relative to the total weight of the silicone oil of decamethyltetrasiloxane, more preferably 80% by weight relative to the total weight of the silicone oil of decamethyltetrasiloxane and even preferably 90% by weight relative to the total weight of the silicone oil of decamethyltetrasiloxane.

In another embodiment, the silicone oil further comprises octamethyltrisiloxane and/or dodecamethylpentasiloxane .

In an embodiment, the continuous oily phase comprises 1 to 10% by weight of octamethyltrisiloxane and/or 1 to 10% by weight of dodecamethylpentasiloxane.

In an embodiment, the product DMS-T01.5 (CAS No: 63148-62-9) commercialized by Gelest® can be used as the silicone oil.

In an embodiment, the continuous oily phase comprises the surfactant according to the invention.

In an embodiment, the continuous oily phase comprises 0,5 to 5% by weight, relative to the total weight of the continuous oily phase, of the surfactant as previously defined, preferably, 0,5 to 2.5% by weight, relative to the total weight of the continuous oily phase, of the surfactant as previously defined, and more preferably, 0,5 to 1.5% by weight, relative to the total weight of the continuous oily phase, of the surfactant as previously defined.

In an embodiment, the continuous oily phase comprises about 1.0% by weight, relative to the total weight of the composition, of the surfactant as previously defined. In a preferred embodiment, the continuous oily phase comprises a silicone oil comprising at least 60% of decamethyltetrasiloxane and a surfactant which is a block copolymer surfactant comprising blocks [A], [B] and [C], wherein block [A] of the surfactant corresponds to a block of formula (I) 2 alkyl and m is comprised between 10 and 35; ] of the surfactant corresponds to a block of formula (II)

wherein q is 3, n is comprised between 1 and 15, and R2 is a compound of formula (III)

(VII), wherein p is comprised between 75 and 175; and wherein terminal groups are selected from the group consisting of H, CH3 and -Si(CH3)3. In another preferred embodiment, the continuous oily phase comprises silicone oil comprising at least 60% of decamethyltetrasiloxane and a surfactant which is a block copolymer surfactant comprising blocks [A], [B] and [C], wherein block [A] of the surfactant corresponds to a block of formula (I) 6 alkyl and m is comprised between 25 and 75; ] of the surfactant corresponds to a block of formula (II)

(VII), wherein p is comprised between 100 and 300; and wherein terminal groups are selected from the group consisting of H, CH3 and -Si(CH3)3. In an embodiment, the dispersed phase comprises water and any water miscible co-solvent, such as for example ethers glycol and polyether glycols, dimethyl sulfoxide (DMSO), short organic alcohols, acetone, short fatty acids, glycerol short organic amines, hydrogen peroxide, or organic and inorganic acids.

In an embodiment, the dispersed phase comprises reagents for performing a biological reaction and biological material.

Biological material refers, without limitation, to organisms, organs, tissues, cells (including eukaryotic and prokaryotic cells), viruses or virus particles, nucleic acids (including double stranded and single stranded DNA or RNA), plasmids, proteins, peptides, antibodies, enzymes, hormones, growth factors, carbohydrates and lipids, and derivatives, combinations, or polymers thereof. A "biological material" according to the present invention may be a material of natural or of synthetic origin. A "biological material" may be extracted, recovered, or obtained directly from a biological sample, such as, without limitation, blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool, tissues or cells.

In a preferred embodiment, biological material refers to nucleic acids.

"Emulsion" refers to a composition comprising at least two liquids, each of them being substantially immiscible in the other, wherein one of the liquids (which is referred to as the "dispersed phase") is partitioned into the other liquid (which is referred to as the "continuous phase"). The dispersed phase of an emulsion is typically suspended in the form of colloids, micelles, capsules and/or droplets. The dispersed phase and the continuous phase are typically fully immiscible. Emulsions are typically stabilized by inclusion in the emulsion of one or more surfactants and/or emulsifying agents.

In an embodiment, the present invention also relates to a population of droplets comprising an aqueous phase, dispersed in a continuous oily phase comprising a silicone oil and a surfactant.

All features detailed in the previous paragraphs entitled “dispersed aqueous phase”, “continuous oily phase” and “surfactant” also apply to the emulsion and the population of droplets.

In an embodiment, the step of processing the biological material comprises amplifying the biological material, preferably by digital PCR, more preferably digital PCR in droplets.

Digital PCR may be seen to encompass three technologies, as follows:

Digital PCR in Microarrays:

With microfluidic-based digital PCR becoming well-known and more widely practiced, in 2006 Fluidigm Corporation commercialized the technology in an integrated microfluidic circuit. The BioMark system was based on the 1275 Digital Array, a chip of 12 panels, wherein each panel partitioned the reaction fluid into 765 6-nL chambers. After loading the PCR reaction mixture through 12 carrier inputs, the chip was thermocycled, fluorescence was detected, and the signal was processed and analysed by the Digital PCR Analysis software.

Digital PCR in Micro-droplets:

An alternative approach that further enhanced throughput and sensitivity while addressing the cost per reaction limitation was to generate picoliter-sized microdroplet reactors by flow-focusing offering many more partitions than the chamber-based systems. Droplets are generated in a microfluidic system, thermocycled to perform single -molecule digital PCR within the droplets, and end-point amplification is detected and quantified via real-time fluorescence curves. These droplet-based lab-on-chip systems were also adapted to perform reverse transcription PCR (RT-PCR) to detect single copies of RNA genomes and to perform multiplex reactions directed to multiple targets within a single droplet.

This technology was for example commercialized by QuantaLife, Inc. as the QX100 ddPCR™ System in 2011 . The microfluidic consumables used on the ddPCR™ platform could accommodate up to eight samples per chip, generating 14,000-16,000 droplets per sample.

Crystal Digital PCR^TM:

An advanced digital PCR equipment, the Naica® System was launched in 2016 by Stilla Technologies. This system performs digital PCR by partitioning the sample, using a confinement gradient, into a large 2D array of droplets, also called a droplet crystal. A PCR reaction occurs in each of the partitioned 25-30,000 droplets that make up the droplet crystal, and a fluorescence read-out is performed at end point by taking high resolution image of the crystal.

Hence, and as used herein, the term “digital PCR” encompasses but is not limited to Digital PCR in Microarrays, Digital PCR in Micro-droplets and Crystal Digital PCR™, and the term “digital PCR in droplets” encompasses Digital PCR in Micro-droplets and Crystal Digital PCR™.

In an embodiment, the step of processing the biological material comprises amplifying the biological material, preferably by digital PCR and more preferably by digital PCR in droplets.

Examples

Material and Methods

Protocol used in examples 1 and 2:

Thaw the reagents - PCR Mix (Buffer A and B), Oligo Master Mix, H2O and DNA stock.

Thoroughly vortex and centrifuge each tube at least 10 sec.

Prepare the following mix:

Use the following set up for 48 reactions of the 3-color run, using three Opal chips. Set up the PCR mix for all 48 samples in a batch mix to ensure equal conditions in each reaction.

Vortex and centrifuge the reaction mix.

Use in Opal chips with 7 pU in each inlet port.

Use in the naica® Geode with the “Opal Template_PCR_45 cycles” program.

Read chips on the Prism3 reader. - Open data with Crystal Miner, check cluster separation in all three channels, as well as all the other criteria for assessing suitability of the tested surfactant/oil mixture for use in dPCR.

Protocol used in example 3 - 3-plex assay

• Thaw the reagents - PCR Mix (Buffer A and B), Oligo Master Mix, H2O and DNA stock.

• Thoroughly vortex and centrifuge each tube at least 10 sec. • Prepare the following mix :

• Vortex and centrifuge the reaction mix.

• Use in Opal chips with 5 pL in each inlet port.

• Use in the naica® Geode with a PCR 45 cycles program. • Read chips on the Prism3 reader.

• Open data with Crystal Miner, check cluster separation in all three channels, as well as all the other criteria for assessing suitability of the tested surfactant/oil mixture for use in dPCR.

• Export quantification and separability data in a excel file. Protocol used in example 3 - 6-plex assay

• Thoroughly vortex and centrifuge each tube at least 10 sec.

• Prepare the following mix :

Use the following set up for 48 reactions of the 6-color run, using three Opal chips. Set up the PCR mix for all 48 samples in a batch mix to ensure equal conditions in each reaction.

• Vortex and centrifuge the reaction mix.

• Use in Opal chips with 5 pL in each inlet port.

• Use in the naica® Geode with a PCR 45 cycles program. • Read chips on the Prism6 reader.

• Open data with Crystal Miner, check cluster separation in all three channels, as well as all the other criteria for assessing suitability of the tested surfactant for use in dPCR.

• Export quantification and separability data in an excel file.

Results: Example 1 : Droplet analysis

In a first set of PCR experiments, 8 different surfactants were used at a concentration of 5% w/w in decamethyltetrasiloxane .

Results obtained for 4 chambers (four replicates) per surfactant are described in the table below:

ABE 3642 advantageously increases the droplets number and the analyzed volume, compared to the other surfactants tested. In a second set of experiments, three new surfactants were screened, together with ABE 3642 as reference, at 1% w/w (as detailed below) in decamethyltetrasiloxane, following the same procedure previously used.

Results obtained for 16 chambers (sixteen replicates) per surfactant are described in the following table:

The best results were obtained with ABE 3642 which enables to obtain the highest number of droplets and thus the highest analyzed volume. It is noteworthy that Silube J208-812 and Silube T310- A16 are also suitable to generate stable water-in-oil droplets while no stable droplets were observed with CMS-222 at 1% w/w or at 0. 1% w/w.

CMS-222 is a siloxane block co-polymer represented by the following formula. This surfactant does not allow to obtain stable droplets.

Without wishing to be bound by theory, it is hypothesized that polyethylene glycol chain of the [B] block within the surfactants according to the invention is a key parameter for preventing droplets from mixing and merging when in contact with each other and therefore a key parameter for increasing the droplets number and the analyzed volume compared to other surfactants. The alkyl chain of the [A] block within the surfactants according to the invention is potentially also a key parameter for preventing droplets from mixing and merging when in contact with each other and therefore a key parameter for increasing the droplets number and the analyzed volume compared to other surfactants.

Best results were obtained when the percentage by weight of surfactant of block [A] is in the range going from 20% to 40% by weight of surfactant and the percentage by weight of surfactant of block [B] is in the range going from 3% to 10% by weight of surfactant.

Example 2: Surfactant concentration

The ABE 3642 surfactant was studied at a range of concentrations from 0.05% to 10% in decamethyltetrasiloxane in Opal chips.

Stability of the 2D monolayer can be observed for surfactant concentrations of 0.5% w/w and above (as described in the table above), while concentrations above 5% w/w may be detrimental to biocompatibility (PCR could be inhibited by the too high concentration of surfactant).

Example 3 : Molecular biology compatibility experiments

In addition to the number of droplets, the precision of dPCR quantification, and the separability of fluorescence signals between different populations, were then assessed. All these experiments were performed with ABE 3642 at 1% (w/w) in decamethyltetrasiloxane.

The quantification accuracy was directly evaluated by comparison with results obtained with Opal chips filled with fluorinated chemistry.

For each channel, a satisfactory separability between negative and positive droplets was observed (separability index calculated by the Crystal Miner software of the naica® Digital PCR suite > 4). No deviation of the quantification was observed compared to the reference concentration obtained with the chips filled with the fluorinated chemicals.

In addition to the 3-plex assay described above, a 6-plex assay was also performed to confirm suitability of the oil/surfactant mixture with other biological objects. This 6-plex assay uses Phage Lambda (blue channel), PhiX174 (teal channel), pUC18 MCS LI (green channel), pBR 322 (yellow channel), Entero Cloaca (custom plasmid based on pUC57, red channel) and ALB (infra-red channel). The quantification accuracy was evaluated by comparison with target concentrations.

For all six channels, a satisfactory separability between negative and positive droplets was observed (separability > 4). No deviation of the quantification was observed compared to the target concentrations.

Example 4: Evaluation of time of use between chip loading and chip processing

A comparative study was performed between the fluorinated system (Opal chips) and the silicon system (ABE 3642 1% in DMS-T01.5) to evaluate the effect of incubation time between chip loading and processing. For this, the chambers of each chip were loaded with the PCR mixture at 2-minute intervals and the chips were then processed in PCR such that the first chamber loaded had 26 minutes of incubation between chip loading and PCR while the last chamber loaded was processed immediately. Two chips per chemical system were processed with the 3-plex assay validation and the related protocol described for Example 3.

A clear increase in calculated concentration with the incubation time was observed in the fluorinated system, most likely due to evaporation of the PCR before injection, while calculated concentrations remained stable with the silicon system (i. e. an increase of 25% was observed in the green channel after 26 minutes for the fluorinated system while no deviation was observed with the silicon system).

In addition to the comparative studies with the fluorinated system, extended incubation times between chip loading and PCR have been evaluated with the silicon system (ABE 3642 1% in DMS- T01.5). The chips were loaded at the same time and then processed in dPCR after 6 hours or 10 hours of incubation at room temperature. The changes in calculated concentrations were evaluated by comparison to the results obtained when the chips are processed immediately after loading. The 3-plex validation assay and the related protocol described for Example 3 were also used for these tests.

Results obtained for 24 chambers. Chambers with less than 10K droplets were excluded before the analysis.

A small deviation in quantification of less than 10% for the three channels was observed after 6h and lOh of incubation at room temperature between chip loading and dPCR processing.

These results demonstrate that the silicon chemistry system according to the invention allows for an extended operating time between chip loading and processing, contrary to the fluorinated system of the state of the art.

Claims

1. A micro-droplet generating apparatus (1), comprising: a container (31) comprising a continuous oily phase (3), the continuous oily phase comprising a silicon oil; a micro-droplet dispenser (2) comprising a reservoir (21) filled with an aqueous phase (211), a dispensing tip (22) fluidly connected to the reservoir and an actuator (23), wherein the dispensing tip comprises a nozzle (221), wherein the actuator is coupled to the dispensing tip to generate micro-droplets of aqueous phase at the nozzle and dispense the micro-droplets into the continuous oily phase; wherein at least one of the continuous oily phase and the aqueous phase comprises a surfactant which is a block copolymer comprising blocks [A], [B] and [C] : block [A] of the surfactant corresponding to a block of formula (I)

wherein Ri is a Ci-Cis alkyl, and m is comprised between 10 and 100; block [B] of the surfactant corresponding to a block of formula (II)

• formula (III)

formula (IV)

• formula (V)

• formula (VI)

wherein r, s, t, u, v and w are comprised between 1 and 32 and R3 is selected from the group consisting of -OH, -O-CH3 and -O-CH₂CH₃; block [C] of the surfactant corresponding to a block of formula (VII)

wherein p is comprised between 5 and 300; wherein terminal groups are selected from the group consisting of -H, -CH3 and -Si(CH3)3; and wherein the general formula of the surfactant is selected from [A]-[B]-[C], [B]-[C]-[A], [C]-[A]-

[B], [B]-[A]-[C], [A]-[C]-[B] and [C]-[B]-[A],

2. The micro-droplet generating apparatus of claim 1, wherein block [A] of the surfactant corresponds to a block of formula (I)

wherein p is comprised between 75 and 175; wherein terminal groups are selected from the group consisting of H, CH3 and -Si(CH3)3.

3. The micro-droplet generating apparatus of claim 1, wherein block [A] of the surfactant corresponds to a block of formula (I)

wherein Ri is a Ci6 alkyl and m is comprised between 25 and 75; - block [B] of the surfactant corresponds to a block of formula (II)

wherein p is comprised between 100 and 300; wherein terminal groups are selected from the group consisting of H, CH3 and -Si(CH3)3.

4. The micro-droplet generating apparatus of anyone of claims 1 to 3, wherein the viscosity of the silicone oil is comprised between 0.7 and 5.0 mm²/s at 25°C.

5. The micro-droplet generating apparatus of anyone of claims 1 to 4, wherein the silicone oil comprises decamethyltetrasiloxane.

6. The micro-droplet generating apparatus of anyone of claims 1 to 5, wherein the silicone oil comprises at least 60% by weight of decamethyltetrasiloxane, relative to the total weight of the silicone oil.

7. The micro-droplet generating apparatus of anyone of claims 1 to 6, wherein the silicone oil further comprises octamethyltrisiloxane and/or dodecamethylpentasiloxane.

8. The micro-droplet generating apparatus of anyone of claims 1 to 7, wherein the continuous oily phase comprises 1 to 10% by weight of octamethyltrisiloxane and/or 1 to 10% by weight of dodecamethylpentasiloxane relative to the total weight of the silicone oil.

9. The micro-droplet generating apparatus of anyone of claims 1 to 8, wherein the surfactant is ABE 3642.

10. The micro-droplet generating apparatus of anyone of claims 1 to 9, wherein the actuator is a piezoelectric actuator to generate acoustic waves, wherein the piezoelectric actuator and the dispensing tip are coupled so that the generated acoustic waves propagate through the dispensing tip up to the nozzle, thereby generating micro-droplets at the nozzle.

11 . The micro-droplet generating apparatus of anyone of claims 1 to 9, wherein the actuator is an electromagnetic vibrator to vibrate the nozzle of the dispensing tip when this latter is dipped into the continuous oily phase and while the aqueous phase is fed to the dispensing tip, thereby dispensing microdroplets into the continuous oily phase.

12. The micro-droplet generating apparatus of anyone of claims 1 to 9, wherein the actuator is a motion actuator to set the dispensing tip into motion according to at least two stages, a volume -increasing stage during which a micro-droplet is formed at the nozzle of the dispensing tip, and a micro-droplet-detaching stage during which the formed micro-droplet breaks free from the nozzle of the dispensing tip; preferably, the motion actuator is configured to set the dispensing tip into a translational motion or a rotational motion.

13. The micro-droplet generating apparatus of anyone of claims 1 to 12, further comprising a driver (5) to drive the dispensing tip over the container.

14. The micro-droplet generating apparatus of claim 13, further comprising a single particle dispensing module (4) configured to:

- detect whether a single particle is comprised within a volume of aqueous phase closest to the nozzle;

- control the driver and the actuator to dispense the volume of aqueous phase closest to the nozzle into the continuous oily phase if a single particle is detected therein; and

- control the driver and the actuator to dispense the volume of aqueous phase closest to the nozzle into a recovery reservoir, otherwise.

15. The micro-droplet generating apparatus of anyone of claims 1 to 14, further comprising a digital PCR module to carry out PCR inside each micro-droplet in the continuous oily phase.