WO2012013316A1

WO2012013316A1 - Method and device for passively separating and sorting drops, in particular in a microfluidic system, by using non-optical markers for reactions within the drops

Info

Publication number: WO2012013316A1
Application number: PCT/EP2011/003640
Authority: WO
Inventors: Christian Böhm
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2010-07-26
Filing date: 2011-07-20
Publication date: 2012-02-02
Also published as: DE102010032203A1

Abstract

The invention relates in particular to a method for passively separating and sorting drops by using non-optical markers for reactions within the drops. Such a method preferably comprises a) providing a first phase of primary drops, which comprise a reaction medium and in which certain reactions occur, in an incubation chamber, which contains a second liquid carrier phase, which is not miscible with the first phase; b) allowing the reactions to occur in the primary drops, whereby at least some of the primary drops are converted to secondary drops, which differ from the primary drops by a change in at least one non-optical physical parameter of the drops; and c) passively separating the primary and secondary drops according to differences of the at least one non-optical physical parameter of the drops by contacting the drops with a separating apparatus, which has a specified geometric form and/or surface shape, in such a way that drops differing in regard to the at least one non-optical physical parameter experience a different interaction with the geometric form and/or surface of the separating device and are thereby separated.

Description

Method and apparatus for the passive separation and sorting of drops, in particular in a microfluidic system, by using non-optical markers for reactions within the drops

The present invention relates to methods for passively separating and sorting drops by using non-optical markers for reactions within the drops.

The methods are particularly suitable for microfluidic systems with high throughput. The methods of the invention are particularly suitable for use in screening methods for the analysis, selection and identification of a broad spectrum of analytes, including cells, catalysts, enzymes and other substances that can influence or control physical, chemical or biological reactions.

Microfluidic systems have been used increasingly successfully for such examination and screening methods recently.

Microfluidic systems offer the unique ability to handle the smallest fluid volumes in micron-sized channel structures. Due to the small dimensions, a large number of channels, reaction spaces and control elements can be integrated in the smallest space, which enables the possibility of an efficient parallelization of reactions. An important prerequisite for high-throughput processes is thus fulfilled. In addition to size reduction, micro fluidic systems are characterized by further physical properties, which give access to new applications: The flow in such systems is usually laminar. As a result, parameters such as substance concentration and temperature can be manipulated selectively and locally. The large surface-to-volume ratio, another characteristic of microfluidics, enables efficient temperature exchange and thus controlled reaction conditions. Icon drops are easily generated and can be used as microreactors, in which, for example, individual cells or all reactants of interest can be included.

By using several liquid phases in microfluidic systems, the range of applications can be expanded further: The production of monodisperse drops with production rates of up to 10,000 drops per second is made possible by hydrodynamic focusing of two immiscible liquids.

Such drops can be used as isolated Picoliter reaction vessels: Different drops of different contents can be fused together or split into daughter drops of different sizes. The flexible design options and unique physical properties, combined with the ability to control system parameters at the cellular level, make microfluidic technology a promising tool, especially for cell biology applications, because of the ability to examine individual cells in separated reaction volumes.

The ability to create and analyze smallest volumes offers significant benefits in a variety of screening applications, especially in biological assays: ^• The required sample volume is drastically reduced (a microdip with a diameter of 25 pm has a volume of ~ 8 pl, so that more than 120,000 drops can be generated from 1 μΐ sample solution).

5 · A variety of reactions can be parallelized and carried out in the smallest space.

• The small sample volume offers the possibility to include single cells in the drops. This allows whole cell populations to be analyzed at the individual cell level.

L0

The use of drop microfluidics for high throughput analysis has been described, for example, by Agresti et al. (PNAS 107, 4004-9 (2010)).

L5 While the reaction steps in current microfluidic approaches are fully miniaturized, analysis and control continue to be performed using classical techniques that require active and external control. So be locked at ^¬ play as classic fluorescent markers for assay analysis

.0 (as described, for example, in US 2009/0068170 A1). The fluorescence signal of each individual drop is detected and evaluated {externally). The drops with corresponding signal are finally sorted out by means of electric fields (active).

25

The lab-on-chip of the reaction step thus becomes again a macroscopic system in which the necessary control and analysis unit exceeds the size and cost of the reaction module many times over.

30

Against this background, the object of the present invention is to provide new improved methods for analysis, separation and sorting of drops in which chemical, biological or physical reactions take place or have taken place, and analytes contained therein, in particular in a microfluidic system, which are suitable for performing high-throughput screening and analysis methods and no longer have the disadvantages of the prior art and in particular the advantages of a much simpler handling and high cost efficiency Offer .

This object is achieved according to the invention with the method according to claim 1 and 21 and the device according to claim 25. More specific embodiments and further aspects of the invention are the subject of the further claims.

The inventive method according to claim 1 comprises the following steps:

a) providing a first phase of primary droplets comprising a reaction medium and in which chemical, biological or physical reactions take place in an incubation room containing a second liquid carrier phase which is immiscible with the first phase;

b) draining the chemical, biological or physical reactions into the primary droplet, whereby at least a portion of the primary droplets are transferred to secondary droplets that differ from the primary droplets by a change in at least one non-optical physical parameter of the droplets,

c) passive separation of the primary and secondary drops as a function of differences in the at least one non-optical physical parameter of the drops by contacting the drops with a separator having a predetermined geometric shape and / or surface design such that at least one non-optical one physical parameter different droplets have a different interaction with the geometric see shape and / or surface of the separator experienced and thereby separated.

A significant advantage of the method according to the invention over most of the separation methods of the prior art is that it is possible to dispense with the customary optical markers for detecting a reaction which has taken place in the drops and the associated external detection systems. According to the invention, a modified non-optical physical parameter of the drops, preferably a geometric or mechanical parameter, is used as the marker.

More specifically, this physical parameter is selected from the group consisting of volume, specific gravity, adhesion to or affinity for particular surfaces, surface tension, viscoelasticity, or shape of the drops.

Most preferably, the physical parameter is a volume change, i. a reduction or increase in volume of the drops. In this case, the separation of droplets of different size is preferably carried out by a separator comprising a surface with one or more openings (s) of predetermined diameter. This surface may for example be part of a screening or filtering device and / or consist of the surface of a perforated plate or perforated membrane.

In a special and particularly advantageous embodiment of the method according to the invention, the separating device is formed by at least one part of at least one boundary surface, in particular cover and / or base, of the incubation space. If the droplets have a lower specific gravity than the FLÜS ^¬ SiGe carrier phase, they accumulate on the apertured ceiling of the incubation room, and there will be automatically separated according to size. Conversely, drops of greater specific gravity than the liquid carrier phase collect on the bottom where they can be separated by size.

Even in a process design in which the altered physical parameter of the droplets is a change in the specific density, a separation of different droplets by separation devices on the floor and / or the ceiling of the incubation chamber can take place in a simple and efficient manner. In ^¬ play, the drops can with the higher density on the ground and the droplets are collected and with the lower density on the ceiling removed.

In a process design in which the altered physical parameter of the droplets is the droplet shape, the separator may e.g. comprise a surface with openings of predetermined size and shape which allows only drops of a particular shape to pass through.

In a process design in which the altered physical parameter of the droplets is the adhesion to or affinity to particular surfaces, the separator may comprise a surface which exclusively or preferably only the adhesion of particular droplets, for example only primary droplets or only secondary droplets or only a kind of secondary drops, allowed.

When changing the viscoelastic drop properties:

Drops of predominantly viscous character are easily deformed and can also be filled through openings smaller than the drop diameter, with corresponding pressure differences. squeeze through. This does not succeed or only at a significantly higher pressure difference with elastic drops. Elastic drops can be formed, for example, by polymerization / gellation of the droplet medium, triggered by a chemical or biological reaction within the droplets. The different deformation behavior of the drops finally allows easy separation.

When the surface tension of the drops changes:

A change in the surface tension of microdroplets can be caused by a reaction-induced change of the surfactants used in the droplets for stabilization or by the formation of further surface-active substances. As a result, droplets can be stabilized or destabilized: destabilized droplets can fuse together and form larger droplets. The stabilized drops retain their original size and do not fuse with other drops. These drops, in turn, can be separated from the fused large drops by a filtering process.

In a preferred embodiment of the method according to the invention, the incubation space and / or the separation device is part of a microfluidic system. In this case, the separating device can also be advantageously formed by at least one inner surface of a microfluidic channel. Such a microfluidic system can use a plurality of channels and thus also of incubation chambers and / or separation devices in parallel.

In a process design in which the altered physical parameter of the droplets is the volume, this will have at least one inner surface of the microfluidic channel at least one opening with a predetermined diameter. Due to the possible parallel use of many channels Also, parallel separators, each with only one orifice (or a few orifices), can result in very efficient and rapid separation and sorting of droplets.

In a specific embodiment of the invention, the altered physical parameter of the drops is the result of a mass transfer between the drops and their surroundings.

Typically, this mass transfer is caused by an osmotic pressure differential between the droplets and their environment built up by the flow of chemical, biological or physical reactions in the droplets.

The osmotic pressure difference between the drops and their surroundings leads to an outflow or inflow of water or another solvent of the reaction medium from the drops and thus to a reduction in volume or increase in volume of the drops. The type of volume change is determined by the reactions taking place in the drops: increasing the total number of dissolved substances in the drops, for example by enzymatic polymer degradation, leads to a solvent influence and thus to an increase in volume. Conversely, a reduction in the total number of solutes in the drops, for example, by glucose consumption by cells, leads to a reduction in volume.

The mass transfer between the drops and their environment may take place with a reaction medium-containing carrier phase and / or with at least one reaction medium-containing surface of the incubation space. The latter alternative is usually preferred, as it is also the use allows such carrier phases, which can absorb little or no reaction medium.

The reaction medium-containing surface preferably has the same reaction medium as that of the droplet phase adsorbed thereto and / or absorbed therein. The material of this surface is not particularly limited and may be selected depending on the kind and composition of the reaction medium. A specific, non-limiting example of such a material having hydrophilic properties is polydimethylsiloxane which works well with aqueous media, e.g. Cell media, can be saturated.

The reaction medium-containing surface may also comprise or consist of a membrane permeable to the solvent of the reaction medium which separates the droplets from a reaction medium reservoir, so that the mass transfer can occur through this membrane.

In one embodiment of the invention, another droplet phase may also serve as the reaction medium-containing environment. These drops, hereinafter referred to as inactive drops, contain only the reaction medium or reaction medium together with an inactive or weakly active catalyst or an inactive or weakly active cell. There is therefore no reaction or only a weak reaction in the inactive drops. Thus, an osmotic pressure difference is built up between the inactive drops and the active drops, in which desired chemical, biological or physical reactions take place, which finally causes a mass transfer between these drops. The change in volume of the inactive droplets behaves in the same way as that of the active droplets: A solvent flow takes place from the active droplets into the inactive droplets or vice versa Direction instead. The relative size difference between active and inactive drops is thereby enhanced, thus facilitating the selection of active drops.

After chemical, biological or physical reactions take place in the active droplets, these droplets contain all the necessary reactants and auxiliaries for the respective reaction. In addition to the educts of the chemical, biological or physical reactions, they preferably contain at least one further component which may have an influence on the respective chemical, biological or physical reactions. This component often simultaneously constitutes an analyte which is to be investigated or identified by means of the method according to the invention.

More specifically, this component is selected from the group of catalysts including enzymes, cocatalysts and inhibitors. The method according to the invention can be carried out such that the chemical, biological or physical reactions in step b) occur for a predetermined time. This period of time will be at least as long that the sequence of the respective reaction leads to a sufficient change in the intended physical parameter of the droplets. This period of time may vary widely depending on the particular reaction and reaction conditions, but can be easily determined by one skilled in the art in routine experiments.

In a particular embodiment, the drops contain entities in or on which chemical, biological or physical reactions occur. These structures can, for example, be natural or artificial cells, particles, beads, vesicles or capsules, or other molecular aggregates.

In a preferred embodiment, these are biological cells whose cell activity, in particular metabolic activity and / or division activity, is to be investigated.

The cells to be examined are enclosed in drops by hydrodynamic flux-focusing of a cell suspension (LI) (FIG. 1). As the inert oil (L2), for example, perfluorinated hydrocarbons having a higher density than water (e.g., FC-40 of 3, about 1.9 g / ml) are used. However, in principle all types of solvents can be used which do not mix with the drop phase and thus allow a two-phase system (microemulsion): hydrocarbons, mineral oils etc.

Due to metabolic activities, the composition of the drop fluid changes: For example, the concentration of small nutrient molecules drops drastically outside the cells. These are taken up by the active cells, metabolized and finally used to build up intracellular structures. This depletion of the droplet medium leads to the formation of an osmotic pressure difference between the droplet medium and the medium-saturated microfluidic environment. The environment consists for example of polydimethylsiloxane (PDMS), an elastic polymer which can be easily saturated with aqueous solutions, a solvent-permeable membrane which separates the droplets from a medium reservoir, or other droplets containing only droplet medium, without an active component that causes a reaction within the drops. The osmotic pressure difference leads to a water outflow from the drops into the environment (PDMS walls). The loss of water compensates for the concentration difference again and leads to a reduction of the drops. Finally, the size change serves as a new microfluidic marker for the biological activity of the cells enclosed in the drops.

Selection and sorting into active and non-active cells can now be carried out in a purely passive manner by suitable microstructuring: the drops have a lower density than the surrounding carrier oil. The drops rise. Smaller drops can be separated and removed from the rest by a filter cover (Fig. 2).

For selection, no external and active analysis ^¬ methods are necessary, for example, measure a fluorescence signal of each drop, evaluate and sort out drops based on it. All it takes is a simple integrated microfluidic system.

In a typical embodiment of the method according to the invention, several types of primary droplets of different composition are provided in step a) and in step b) different secondary droplets are generated, which differ from one another by at least one physical parameter of the droplets, and then in step c) a passive separation and sorting of various secondary drops.

In the various primary drops, for example, different chemical, biological or physical reactions may take place or the same chemical, biological or physical reactions in different ways, eg different speeds, expire. For example, these differences may be due to the presence of a particular component, analyte, and may be used to identify a component having certain desired properties.

In a specific embodiment, the different primary drops contain different types of cells which differ in at least one cell activity, and L0 the separation of the different secondary drops formed due to the different cell activity enables discrimination and sorting of active and non-active cells.

L5 The size of the drops used in the process according to the invention is not particularly critical and can be adapted to the systems to be investigated and reaction conditions.

In a preferred embodiment of the method, this is carried out in a microfluidic system and the droplet diameters are in the size range produced by conventional microfluidic techniques. This range is from about 1 micron to 500 microns. Typically, the droplet size ranges from 5 microns to 100 microns, more preferably from 10 microns to 50 microns. For droplets containing entities such as biological cells, the minimum size becomes that size And droplet size typically ranges from 10 microns to 50 microns.

30

For drops without structure, the lower limit depends essentially only on the drop generation system used. Basically, the use of the smallest possible drop, as far as still manageable, preferred as material-saving and therefore cheaper. How to As stated above, a major advantage of all micro-fluidic methods, and in particular of the method according to the invention, is the possibility of carrying out a large number of reactions and subsequent analysis and separation steps in a small space in parallel.

An essential aspect of the present invention is the use of the described separation method for the selection and identification of cells which have or lack a particular cell activity, or for the selection and identification of substances which have a certain qualitative or quantitative influence on certain have chemical, biological or physical reactions or not. These selection and analysis methods are preferably high throughput methods.

More specifically, the substances studied are substances from the group of catalysts, including enzymes, cocatalysts and inhibitors.

A more specific method of selecting and identifying cells that have or do not have a particular cell activity, or for selecting and identifying substances that have or do not have a particular qualitative or quantitative influence on particular chemical, biological or physical reactions,

comprises the steps a) -c) of the method according to any one of claims 1-18, wherein in step a) primary drops are provided, of which at least a part contains the cells or substances to be examined, and further

d) isolating drops with a predetermined physical target parameter and identifying the cells or substances present in these drops. In a preferred embodiment of this selection or screening method, the method is carried out several times in succession, the cells or substances selected after the first passage of the method being subjected to further selection passes. In these further runs, they may, for example, be subjected to other, in particular more stringent, reaction conditions, to thereby obtain a higher degree of selection. In addition, the serial procedure is also suitable for optimizing substances by "directed evolution": After each reaction step, the selected cells and substances are subjected to a change, in particular by mutagenesis. Subsequently, a further selection and modification takes place it is possible to achieve a gradual optimization of the desired properties.

Advantageous application possibilities are, for example, enzyme screening and enzyme optimization by "directed evolution" for various purposes, for example cellulose degradation, ethanol production, detergent enzymes, etc.

Another aspect of the invention relates to a device, in particular for carrying out the method according to one of claims 1-24, comprising

a) an incubation room used to hold a first

Phase in the form of droplets comprising a reaction medium and a second liquid carrier phase which is immiscible with the first phase;

b) a separating device, which has a predetermined geometric shape and / or surface design, such that different droplets have a different interaction with the geometric shape and / or surface learn chengestaltung the separator and thereby can be separated.

In a preferred embodiment, in particular for the separation of drops with different diameters, the device is characterized in that the separating device comprises a surface with one or more openings (s) with a predetermined diameter. This surface may for example be part of a sieve or filter device or the surface of a perforated plate or membrane.

In a special and particularly advantageous embodiment of the device according to the invention, the separating device is formed by at least one part of at least one boundary surface, in particular cover and / or base, of the incubation space. If the drops have a lower specific gravity than the liquid carrier phase, they accumulate at the ceiling of the incubation room provided with openings or other selecting surface and are automatically stored there, e.g. separated according to the size. Conversely, drops of greater specific gravity than the liquid carrier phase collect on the bottom and can be separated there.

Even in the event that the altered physical parameter of the droplets is a change in the specific gravity, at least one separating device can be provided on the bottom and / or the ceiling of the incubation chamber for the separation of different droplets.

In the event that the altered physical parameter of the droplets is the droplet shape, for example, the separator may comprise a surface having openings of predetermined size and shape which will allow only droplets of a particular shape to pass through. In the event that the altered physical parameters of the drops of the adhesion to or affinity for certain surfaces, the separating means may comprise a surface which exclusively or preferably only the adhesion of be ^¬ voted drops, for example, only primary drops or only secondary drops or only one kind of secondary drops allowed.

In a preferred embodiment of the device according to the invention, the incubation space and / or the separation device is part of a microfluidic system. In this case, the separating device can also be advantageously formed by at least one inner surface of a microfluidic channel. Such a microfluidic system can use a plurality of channels and thus also of incubation chambers and / or separation devices in parallel.

In a process design in which the altered physical parameter of the droplets is the volume, this will have at least one inner surface of the microfluidic channel at least one opening with a predetermined diameter. Due to the possible parallel use of many channels, parallel separators, each having only one exit opening (or a few openings), can lead to a very efficient and rapid separation and sorting of drops.

In a further preferred embodiment, the device according to the invention is characterized in that the incubation space comprises at least one surface which has the same reaction medium as that of the droplet phase adsorbed thereto and / or absorbed therein. The incubation space may also have a surface containing a permeable to the solvent of the reaction medium membrane, which the drops in the Separation incubation room from a reaction medium reservoir separates, includes or consists of. Such a surface can also be considered in a broader sense as a reaction medium-containing surface.

FIG. 3 shows a compact novel micro fluidic system according to the invention, which allows the generation of droplets, incubation with concomitant change in size of the droplets and selection through a perforated membrane.

The realization of the present invention in a microfluidic system is based on four core steps in order to miniaturize the reactions to be investigated and the subsequent detection and selection: The target parameter (I) to be detected is transformed into a microfluidic variable, a change of specific drop characteristics ( II). This is done by the interaction of microdrops with their environment, which is characterized by a suitable geometry and material properties. The microfluidic marker (III) finally allows a purely passive selection (IV), which requires no active and external control. The system works autonomously.

Fig. 4 shows in panel A the representation of a cell screen with reference to the four core steps discussed above. After incubation, the volume of the drops with division-active yeast cells (Nos. 1, 2, 3 and 6) has significantly decreased (Panel B). Small droplets are selected by passive buoyancy through a microfilter (box C). Panel D shows a prototype for drop production, incubation and subsequent filtering (corresponding to Fig. 3).

The inventive methods presented here are based on a fundamentally new approach: The use of passive sorting using special markers, which is a non-optical physical parameter of the droplets, which is modified by the course of the reaction so that it can be used to distinguish and separate from different droplets, eliminates the Need for active and external observation and control of the drops.

The preferred application in a microfluidic system has further advantages:

a) not only the reactions to be investigated are miniaturized, but also the subsequent steps of analysis and control / sorting;

b) the miniaturization of analysis and sorting is carried out by the use of special microfluidic markers in combination with a suitable microfluidic environment;

c) this combination leads to a passive sorting, which requires no active and external control and thus the number of required micro-macro interfaces is reduced to a minimum.

The present invention provides access to a new universal base and platform technology that can be used for a variety of microfluidic applications (and other applications) and is not limited to a product niche.

The reduction of the micro-macro interfaces and the greatest possible avoidance of external control elements increase the robustness considerably. A property that is decisive for a successful application, especially in microfluidics.

In addition, the absence of active controls and the simple basic concept reduces the costs of the system drastically: Da the inventive concept can reduce the necessary analysis elements on microstructured geometries and material properties, production costs for an OF INVENTION ^¬ to the invention chip are essentially used by the Materi- determined al: in the case of PDMS currently used about 1 € per unit.

Novel microfluidic markers allow the selection of global system properties without the need to develop specific, often costly assays: the overall efficiency of enzymatic polymer degradation can be measured as a target, regardless of the molecular details (cut position, fragment length, substrate modifications or substrate heterogeneity). The system works "assay-free".

The simple design also allows the realization of compact and mobile products for flexible use in research laboratories in the form of simple consumables ("Ki t concept"): Already a 20 x 20 cm ² system allows the incubation and the parallel selection of approx 2.5 x 10 ⁷ drop containers Furthermore, the simple design makes it easy to parallelize and cascade

1 shows a diagram of the generation of microdroplets by flux focusing of two immiscible liquids

Fig. 2: Schematic representation of a passive sorting according to the invention based on the drop size by simply tilting the device with the drops

3 shows a plan view of an apparatus for carrying out the method according to the invention Fig. 4: schematic representation of an embodiment of the method according to the invention

Fig. 5: Generation of microdroplets by flow focusing of water and oil

FIG. 6: Volume change of model drops due to osmotic pressure differences between drops and PDMS environment; FIG.

6a: salt gradient; 6b: sugar gradient

Figures 7 and 8: volume reduction of drops with enclosed yeast cells; Fig. 7: micrograph of the drops in micro-fluidic channels, Fig. 8: graphical representation of the volume reduction

Fig. 9: Volume reduction of drops with entrapped E. coli bacteria

Fig. 10: Growth curve of E. coli bacteria in the drops compared to conventional aerated cultures

Fig. 11: Volume enlargement of drops with enclosed algae

Fig. 12: Drop formation for cell-free enzyme systems

13: Volume enlargement of drops with a cell-free enzyme system (DNAsel)

Fig. 14: DNAsel control

Fig. 15: view of a separating device according to the invention

FIG. 16: Detailed view of the separator of FIG. 15 with two drops passing through it. FIG

The following non-limiting examples are intended to illustrate the invention in more detail.

In these examples, the mechanism of size change was tested on various cell systems and enzyme systems. It has been shown that the inventive method is not only on cell systems, but also on non-cell systems for the detection of chemical reactions within the drops, eg enzymatic synthesis and degradation of polymers, can be successfully applied.

In the following experiments, drops with a diameter of 20 to 50 μm were used. These were made by the microfluidic flow focusing method (Figure 5). An inert perfluorinated carrier oil was used (FC-40, 3M). The aqueous phase contained the surfactant Zonyl FSO-100 for stabilization. Further, the ammonium salt of fluorinated surfactant Krytox 157 FSL was added to the oil phase (2% by weight) to increase the drop stability. FC-40 has a density of 1.855 g / ml. Aqueous drops experience a buoyancy that allows filtering through a holey ceiling.

In order to create a defined system for the exchange of water between droplets and the environment, a microfluidic channel system was used whose walls and ceiling consist of PDMS and are saturated with the appropriate aqueous solution (eg cell medium). For saturation, the PDMS system was incubated for at least one day in the appropriate solution. The PDMS absorbs the solution during this time, thus providing a reservoir of defined osmolarity that allows the exchange of water with drops and the PDMS system through the oil phase and into direct contact with the drops. EXAMPLE 1

Volume change due to different substance concentrations inside and outside the drops

The possibility of changing the droplet size by osmotic pressure differences in a suitable microfluidic system was characterized by defined model systems:

To further investigate the water exchange between the droplets and the solution-saturated PDMS environment, various static systems, each with a fixed solution composition, were used in terms of the number of solutes within and outside the droplets. Fig. 6 shows the change in size of drops containing a 1% solution of zonyl in a microenvironment of PDMS treated with the same solution or a 100 mM NaCl; 1% zonyl solution was saturated.

After about 10 h of incubation, the cross-sectional area has reduced to about 80% of the initial size of the drops, which corresponds to a volume change of about 30% (FIG. 6a).

Using a 1 M sugar solution in the droplets in the same saturated PDMS environment increased the droplet cross-sectional area by more than 20% after 48 hours (Figure 6b).

EXAMPLE 2

Volume reduction by yeast cells

The inclusion and subsequent incubation of yeast cells results in a noticeable change in drop size within a few hours (FIG. 7). The extent of volume decrease per Drops are determined by the metabolic activity, the ability of the cells to divide and the number of cells enclosed in the drops at the start of drop formation (FIG. 8).

EXAMPLE 3

Volume reduction by bacteria

Also E. coli bacteria show the effect of volume reduction by incubating the bacteria in the drops (Figures 9 and 10B).

Interestingly, the bacteria in the drops show a higher growth rate and reach a higher cell density compared to classical cell culture conditions (cell incubation in the incubator with shaking in milliliter volumes) (FIG. 10).

EXAMPLE 4

Volume increase by algae

The mechanism of volume change of the drops by changing the osmotic pressure difference allows for sugar-building organisms to increase the volume. In fact, this was actually observed in experiments with algae that are capable of photosynthesis and thus of the production of glucose: Drops of algae (Figure 11, bottom right) increased in size, while drops containing yeast became smaller.

EXAMPLE 5

Volume increase by enzyme systems

To demonstrate the volume effect in non-living reaction systems, a DNA / DNase system for the drops was prepared. wraps. For this, DNase was attached to polystyrene beads and enclosed in drops together with a DNA solution using a double-flow microfluidic system. The double-feed system ensures that the enzyme and substrate are in contact only immediately prior to drop formation (FIG.

12) come. This ensures that the polymer degradation of the DNA only begins with the formation of droplets.

The binding of the DNase to beads allows the simple production of a "binary" drop system, with drops containing either beads and thus DNase or containing no enzyme, and the beads also allow easy visualization (FIG.

13).

The activity of the DNase bead systems was checked by gel electrophoresis (FIG. 14): After incubation with the DNAase beads, the previously added lambda DNA was broken up into small fragments.

Description of FIG. 14:

Control 1: DNasel in solution; Reaction 1-3: DNasel beads in various concentrations (0.67 mg, 3.33 mg, 0.33 mg); control 2: supernatant of beads with DNA; control 3: heat inactivation; control 4: only DNA; ladder (rightmost track): DNA ladder (bands from bottom to top: 1-10 kb in increments of 1 kb and 15 kb).

The expected increase in volume of the drops due to polymer degradation and the associated increase in osmolarity was only slight due to the very small amount of DNA used (90 μΜ) (FIG. 13). Nevertheless, this clearly demonstrated the applicability of the method to enzyme systems. Applications therefore exist for the enzyme Screening and Enzyme Optimization by Directed Evolution for a Wide Range of Purposes.

EXAMPLE 6

Passive filtering based on drip buoyancy

through a grid

The passive filtering of droplets of different size produced by one of the methods described above could be demonstrated by means of a simple metal grid (TEM-Grid) placed as a separation filter between two microfluidic channels (Figure 15).

This system made it easy to separate by the drift of the drips: Deformation of the droplets does not take place under static conditions or at low flow velocities. Only drops with a diameter smaller than the hole size of the grid pass from the lower chamber into the overlying channel.

The size separation of the droplets is effected by the permeation of sufficiently small droplets through a lattice structure, the separation diameter being determined by the lattice geometry and the flow velocities in the channels leading back and forth. The grid and an overlying collecting geometry replace the duct ceiling in a certain area of the flow cell. If the diameter is sufficiently small, droplets flowing down the channel rise driven by the buoyancy force through the grid and can be sucked out of the collecting channel.

FIG. 15 shows a possible structure of the separation geometry in a flow cell: A lower PDMS layer contains a channel structure in which drops flow along. An upper PDMS Layer with a collecting channel closes the flow cell tight. In the area where the channels of upper and lower PDMS layers overlap, there is a thin hexagonal TE grid. In Fig. 16, the rise of two small drops is shown coming from below flow under the grid and then ascend through the grid, recognizable by the running out of focus.

Claims

A method for separation and passive sorting of droplets in a multi-phase system, comprising

c) passive separation of the primary and secondary drops as a function of differences in the at least one non-optical physical parameter of the drops by contacting the drops with a separator having a predetermined geometric shape and / or surface design such that at least one non-optical one physical parameter different droplets experience a different interaction with the geometric shape and / or surface of the separator and thereby separated.

2. The method according to claim 1, characterized in that the non-optical physical parameter is a geometric or mechanical parameter of the droplets.

3. The method according to claim 1 or 2, characterized in that the physical parameter from the group is selected, which summarizes the volume, specific gravity, adhesion to or affinity for ^¬ to certain surfaces, surface tension, viscoelasticity or shape of the drops.

A method according to claim 3, characterized in that the altered physical parameter of the drops is a volume change of the drops and the passive separation of drops of different sizes by a separator comprising a surface with one or more openings (s) of predetermined diameter, he follows.

5. The method according to any one of claims 1 to 4, characterized in that the separating device is formed by at least a part of at least one boundary surface, in particular ceiling or floor of the incubation room.

6. The method according to any one of claims 1 to 5, characterized in that the drops contain structures in which run chemical, biological or physical reactions.

7. The method according to claim 6, characterized in that the structures are natural or artificial cells, particles, ves sikel or capsules, or other molecular aggregates.

8. The method according to any one of claims 1-7, characterized in that the primary drops in addition to the educts of the chemical, biological or physical reactions contain at least one further component which may have an influence on the respective chemical, biological or physical reactions.

9. The method according to claim 8, characterized in that the further component from the group of catalysts, including enzymes, cocatalysts and inhibitors is selected.

10. The method according to any one of claims 1-9, characterized in that the altered physical parameter of the drops is the result of a mass transfer between the drops and their environment.

11. The method according to claim 10, characterized in that the mass transfer is caused by an osmotic pressure difference between the drops and their environment, which was built up by the course of the chemical, biological or physical reactions in the drops.

12. The method according to claim 11, characterized in that the osmotic pressure difference between the droplets and their surroundings leads to an outflow or inflow of water or other solvent of the reaction medium from the droplets and thus to a reduction in volume or Volumenerhö ^{¬ increase of} the drops.

13. The method according to any one of claims 10 to 12, characterized in that the mass transfer between the droplets and a reaction medium-containing carrier phase and / or a reaction medium-containing further droplet phase and / or between the droplets and at least one reaction medium-containing surface of the incubation space takes place.

14. The method according to any one of claims 1-13, characterized in that the chemical, biological or physical see reactions in step b) for a predetermined time.

15. The method according to any one of claims 1-14, characterized in that in step a) several types of primary droplets are provided with different composition and in step b) different secondary droplets are generated, which are characterized by at least one physical parameter of the droplets from each other distinguish, and in step c) a passive separation and sorting of the various secondary drops takes place.

16. The method according to any one of claims 1-15, characterized in that the primary drops contain different types of cells that differ in terms of at least one cell activity, and in that the separation of the different secondary drops formed due to the different cell activity a distinction and Sorting of active and non-active cells allows.

17. The method according to any one of claims 1-16, characterized in that the incubation space and / or the separating device is or are part of a microfluidic system.

18. The method according to claim 17, characterized in that the separating device is formed by at least one inner surface of a microfluidic channel, which has at least one opening with a predetermined diameter.

19. Use of the method according to any one of claims 1-18 for the selection and identification of cells that have or do not have a certain cell activity, or for the selection and identification of substances that have a have or do not have a specific qualitative or quantitative influence on certain chemical, biological or physical reactions.

20. Use according to claim 19, characterized in that the substances are selected from the group consisting of catalysts, including enzymes, cocatalysts and inhibitors.

21. Methods for the selection and identification of cells which have or do not have a certain cell activity, or for the selection and identification of substances which have or do not have a specific qualitative or quantitative influence on specific chemical, biological or physical reactions,

which selection method comprises steps a) -c) of the method according to any one of claims 1-18, wherein in step a) primary drops are provided, at least a portion of which contains the cells or substances to be examined, and which selection method further comprises

d) isolating drops with a predetermined physical target parameter and identifying the cells or substances present in these drops.

22. The method according to claim 21, characterized in that the substances to be investigated are selected from the group consisting of catalysts, including enzymes, cocatalysts and inhibitors.

23. The method according to claim 21 or 22, characterized in that the method is carried out several times in succession, wherein the cells or substances selected after the first passage of the method are subjected to further selection passages.

24. The method of claim 23, wherein the further rounds of selection are performed, among others, especially more stringent, Re ^¬ action conditions, to thereby obtain a higher level of selection, and / or wherein the selected after one pass or multiple passes of the process cells, or Substances are subjected to random changes between the selection rounds, eg by mutagenesis, in order thereby to achieve a directed evolution and / or optimization of the substances or cells.

25. Device, in particular for carrying out the method according to one of claims 1-24, comprising

a) an incubation room adapted to receive a first phase in the form of droplets comprising a reaction medium and a second liquid carrier phase immiscible with the first phase;

b) a separating device, which has a predetermined geometric shape and / or surface design, such that different drops experience a different interaction with the geometric shape and / or surface design of the separator and thereby can be separated.

26. The device according to claim 25, characterized in that the separating device comprises a surface with one or more openings (s) with a predetermined diameter.

27. The device according to claim 26, characterized in that the surface of at least one part of at least one boundary surface, in particular ceiling or floor, of the incubation space is formed.

28. Device according to one of claims 25 to 27, characterized in that the incubation space and / or the separating device is or are part of a microfluidic system.

29. The device according to claim 28, characterized in that the separating device is formed by at least one inner surface of a microfluidic channel, which has at least one opening with a predetermined diameter.

30. Device according to one of claims 25 to 29, characterized in that the incubation chamber comprises at least one surface which has the same reaction medium as that of the droplet phase adsorbed thereto and / or absorbed therein.