DE102020102389A1 - METHOD AND SYSTEM FOR THE ISOLATION OF MOLECULE FRACTIONS - Google Patents

Abstract

The present invention relates to a method for isolating one or more molecular fractions from a sample, comprising (i) introducing the sample into a separation unit, (ii) separating molecules contained in the sample into molecular fractions in a separation channel of the separation unit filled with a separation medium, ( iii) Extraction of one or more molecular fractions from an extraction area of the separation channel by activating a dosing unit, the extraction area being fluidically coupled to an opening and, by activating the dosing unit, medium located in the extraction area is ejected from the opening as a medium fraction, the medium fraction being a molecular fraction or parts of which may include.

Description

introduction

The present invention relates to a method for isolating one or more molecular fractions from a sample, comprising (i) introducing the sample into a separation unit, (ii) separating molecules contained in the sample into molecular fractions in a separation channel of the separation unit filled with a separation medium, ( iii) Extraction of one or more molecular fractions from an extraction area of the separation channel by activating a dosing unit, the extraction area being fluidically coupled to an opening and, by activating the dosing unit, medium located in the extraction area is ejected from the opening as a medium fraction, the medium fraction being a molecular fraction or parts of which may include.

State of the art

In the biological-chemical context, the term purification often describes a multi-stage process for the enrichment and purification of certain molecular species. However, especially for more complex proteins, the process is usually both time consuming and costly. For many downstream analyzes (e.g. protein structure analysis, ligand binding, etc.) as well as for use as a therapeutic agent (e.g. insulin, albumin, ...), this step is absolutely necessary in order to avoid undesirable side effects or incorrect results, among other things.

In addition to extraction / precipitation and filtration, chromatography and electrophoresis are the most powerful and efficient technologies for purification, whereby the method is selected according to the respective application. Nevertheless, these approaches have some disadvantages in principle, which are mainly in the preparation and post-processing effort.

Preparative slab gel electrophoresis, for example, offers a relatively cost-effective solution for the extraction of molecular fractions. In practice, however, the method is mostly only used for nucleic acids, as extraction from the hydrogels usually used is carried out by melting these gels, which would affect other, more sensitive samples such as proteins. In addition, preparative gel electrophoresis is complex because it involves a comparatively high amount of preparation and follow-up work. The process itself also takes a comparatively long time with an expenditure of time in the region of at least one to two hours.

Compared to preparative gel electrophoresis, capillary electrophoresis takes less time for the process itself, but it also involves a lot of preparation and post-processing and is associated with high costs both for the devices and for each sample run [ Zhu, Z., Lu, JJ, & Liu, S. Analytica chimica acta 2012, 709, 21-31 ].

In chromatography (e.g. HPLC), the individual fractions can be isolated well using a downstream collector, but the preparation, post-processing and analysis times are comparatively long and the costs for equipment and sample runs are very high.

In addition, all known purification methods lead to the isolated molecular fractions being present in a volume that is at least in the microliter (µL) range, often even in the milliliter (mL) range. In order to save sample (isolated molecular fractions), additional pipetting steps must therefore always be carried out for subsequent analyzes.

In the prior art, methods are also known in which individual cells can be isolated from a sample [ Schoendube, J., Wright, D., Zengerle, R., & Koltay, P, Biomicrofluidics 2015, 9 (1), 014117 ; US2008 / 0286751A1 ]. However, these methods do not allow the separation and isolation of molecular fractions of a sample.

There is thus a need for the development of methods and devices for the purification of molecular fractions from a sample, which are associated with a low expenditure of time, both in terms of implementation and preparation and postprocessing. In particular, it would be desirable if the purification of molecular fractions were also possible in very small target volumes, for example in the pico liter (pL) range, so that post-processing is simplified for certain subsequent uses that require a small volume. In addition, new processes should be able to be carried out cost-effectively and be suitable for automation.

Description of the invention

The present invention is therefore based on the object of providing a preferably automatable method and / or a device for purifying or isolating molecular fractions from a sample, which is time and cost efficient and enables a low target volume in the nL-pL range.

The technical problem on which the invention is based is solved by the method according to the invention described here and the system according to the invention.

• - Einbringen der Probe in eine Separationseinheit,
• - Auftrennung von in der Probe enthaltenen Molekülen in einem mit einem Separationsmedium gefüllten Separationskanals der Separationseinheit in Molekülfraktionen ,
• - Extraktion einer oder mehrerer Molekülfraktionen aus einem Extraktionsbereich des Separationskanals mittels Aktivierung einer Dosiereinheit,

wobei der Extraktionsbereich fluidisch an eine Öffnung gekoppelt ist und durch Aktivierung der Dosiereinheit im Extraktionsbereich befindliches Medium als Mediumfraktion aus der Öffnung ausgestoßen wird, wobei die Mediumfraktion eine Molekülfraktion oder Teile davon umfassen kann.The invention relates to a method for isolating one or more molecular fractions from a sample, comprising

• - Introducing the sample into a separation unit,
• - Separation of molecules contained in the sample in a separation channel of the separation unit filled with a separation medium into molecule fractions,
• - Extraction of one or more molecular fractions from an extraction area of the separation channel by activating a dosing unit,

wherein the extraction area is fluidically coupled to an opening and by activating the dosing unit in the extraction area medium located in the extraction area is expelled from the opening as a medium fraction, wherein the medium fraction can comprise a molecular fraction or parts thereof.

The method according to the invention is preferably a microfluidic method. In embodiments, the expelled medium fraction comprises at least part of the medium located in the extraction area. In embodiments, the expelled medium fraction comprises all of the medium located in the extraction area.

The present invention is based on the combination of several operating units in one, preferably microfluidic, system. The term “microfluidic system” denotes the entirety of an arrangement of different operating units or fluidic elements such as channels, chambers, mixers, etc., which serve the purpose of preparing or analyzing a liquid sample. Examples of this are electrophoresis or chromatography systems as well as complete analysis systems which are also referred to as micro-total analysis systems or lab-on-a-chip systems.

Surprisingly, the previously unknown combination of a separation unit with a dosing unit has the great advantage that individual types of molecules or molecular fractions can be isolated from a sample quickly, concentrated, in small volumes and inexpensively and made available for subsequent applications. This results in many advantageous and, in the light of the known prior art, surprising application possibilities, such as, for example, the fast and efficient production of special microarrays.

In addition, the invention enables a particularly high degree of flexibility in the fractionation, i.e. when dividing a sample volume, given by at least one isolated molecular fraction, into a large number of smaller volumes, the concentration of the large number of smaller volumes differing depending on the spatial distribution of the molecular fraction in the separation channel before the fractionation. A very fine-grained, concentration-dependent isolation of partial volumes of a molecular fraction is therefore conceivable.

The present invention uses, on the one hand, a separation unit in order to split up or separate the molecules contained in a sample. For this purpose, the separation unit contains a separation medium, which can be liquid, gel-like or solid, for example, or can contain combinations thereof, such as solid particles surrounded by liquid. The separation medium is arranged in a separation channel into which a sample is introduced. The molecules contained in the sample can be separated due to different migration speeds within the separation channel, into fractions of molecules which have similar or identical properties with regard to the separation criteria and therefore migrate at the same speed along the separation channel.

The molecules of a sample to be separated can be any organic or inorganic molecules or compounds which, due to different interactions with the separation medium and / or the wall surface of the separation channel, have different migration speeds within the separation channel. In particular, the biomolecules contained in a sample, such as, for example, biopolymers, can be separated in the separation unit, in particular according to physical or chemical or other properties, such as, for example, binding properties. This applies in particular to DNA, RNA, proteins, lipids, lipoproteins, polysaccharides and other biomolecules and biopolymers which are known to the person skilled in the art.

A separation can take place, for example, by means of electrophoresis via the specific charge of the molecules. For this purpose, the system according to the invention can comprise electrodes for generating an electrical field along the separation channel.

In embodiments of the invention, a pressure-driven separation / separation of the molecules can also take place. In embodiments, the separation can be carried out by means of LC (liquid chromatography), in particular HPLC (high performance liquid chromatography).

Further possibilities of separation within the scope of the present invention include separation by means of a magnetic field, magnetic particles can be used; Affinity chromatography or other separation methods in which the migration speed of molecules in a sample within the separation channel is influenced on the basis of specific affinities of the molecules for a medium present in the separation channel, which can for example include certain molecules on its surface.

In the context of the invention, the terms separation channel, separation channel and migration channel are used synonymously and are interchangeable with one another. The same applies to the terms separation medium, separation medium and migration medium.

Following the separation of the molecules contained in the sample into different fractions within the separation channel, individual fractions can be extracted or isolated from the separation channel within the scope of the method or system according to the invention. A dosing unit is used for this.

In embodiments, the components of the sample, such as different biomolecules, are separated during the migration through the separation medium on the basis of specific properties of the respective molecules, which include the electrical properties (eg charge), the isoelectric point, the size, the morphological structure and include the molecular weight. In addition, the separation can be influenced by the pore size, the electrical and fluidic properties (e.g. conductivity, viscosity, surface tension and contact angle) of the, for example, liquid or gel-like separating medium, as well as the surface material of the separating channel or, for example, a solid separating medium such as a matrix. In addition, the separation is influenced by combined effects such as molecular interactions between (bio) molecules and the separation medium and / or the surface material.

In the context of the present invention, extraction or isolation of molecules or molecular fractions is understood to be a process in which molecules contained in the sample, which migrate into fractions with similar separation properties after being separated in the separation channel, are removed or removed in a controlled manner after separation from the separation channel . be removed or detached. The present invention thus makes it possible to isolate certain individual molecules or groups of molecules from a complex mixture of molecules that are present in a sample, which molecules have specific properties and are of particular interest to the user.

In embodiments of the invention, the metering unit comprises a metering channel.

In embodiments of the invention, the method additionally comprises depositing the expelled medium fraction. For this purpose, a storage unit can be used in the system according to the invention.

In addition, within the scope of the method according to the invention, a detection unit can detect a molecule fraction in the separation channel, for example by reading out a fluorescence signal. In embodiments of the invention, the metering unit is activated by the detection of a molecular fraction, preferably the detection of a molecular fraction in the extraction area of the separation channel. In embodiments, this activation takes place by the detection unit, the detection unit being able to send an activation signal to the dosing unit, for example.

• - Einbringen der Probe in eine Separationseinheit,
• - Auftrennung von in der Probe enthaltenen Molekülen mittels eines mit einem Separationsmedium gefüllten Separationskanals der Separationseinheit, wobei die Moleküle in unterschiedlichen Molekülfraktionen entlang des Separationskanals migrieren können,
• - Extraktion von einer oder mehrerer Molekülfraktionen aus dem Separationskanal mittels Aktivierung einer Dosiereinheit umfassend einen Dosierkanal, wobei der Dosierkanal den Separationskanal durchquert und auf einer ersten Seite des Separationskanals eine mit Flüssigkeit gefüllte Dosierkammer umfassen kann, wobei durch Aktivierung der Dosiereinheit eine fluidische Strömung bevorzugt aus der Dosierkammer durch den Dosierkanal erzeugt wird, wodurch im Durchquerungsbereich von Dosierkanal und Separationskanal befindliches Medium auf einer zweiten Seite des Separationskanals aus einer Öffnung des Dosierkanals als Mediumfraktion ausgestoßen wird, wobei die ausgestoßene Mediumfraktion eine Molekülfraktion aus dem Separationskanal enthalten kann,
• - und bevorzugt Ablegen der ausgestoßenen Mediumfraktion.

Embodiments of the invention also include a preferably microfluidic method for isolating one or more molecular fractions from a sample, comprising

• - Introducing the sample into a separation unit,
• - Separation of molecules contained in the sample by means of a separation channel of the separation unit filled with a separation medium, whereby the molecules can migrate in different molecule fractions along the separation channel,
• Extraction of one or more molecular fractions from the separation channel by activating a metering unit comprising a metering channel, the metering channel passing through the separation channel and being able to include a liquid-filled metering chamber on a first side of the separation channel, whereby a fluidic flow is preferably emitted by activating the metering unit Dosing chamber is generated by the dosing channel, whereby medium located in the area where the dosing channel and separation channel pass through is ejected on a second side of the separation channel from an opening of the dosing channel as a medium fraction, wherein the expelled medium fraction can contain a molecule fraction from the separation channel,
• - and preferably depositing the expelled medium fraction.

In embodiments, the dosing unit comprises one with liquid, for example one Medium, filled dosing chamber. In embodiments, the activation of the dosing unit generates a fluid flow from the dosing chamber through the dosing channel.

In the context of embodiments of the invention with a metering channel, the extraction area of the separation channel can correspond to the area where the metering channel and separation channel pass through. The medium located in the extraction area or the traversing area can be a separation medium, but also another medium, such as, for example, the liquid that is present in a dosing chamber of the dosing unit.

In embodiments, the opening for ejecting the medium fraction is arranged at the end of the dosing channel opposite the dosing chamber.

The metering channel can run transversely to the separation channel and crosses the separation channel. Correspondingly, the metering channel runs through the separation channel, so that there is a crossover area for the two channels. In embodiments of the invention, the metering channel runs approximately orthogonally to the separation channel. However, it is also possible for the metering channel to run obliquely or diagonally to the separation channel, for example at an angle between 90 ° and 0 °. It is not necessary here for the separation channel and / or the metering channel to run straight. Both channels can also have a curved or “curvy” course, for example. However, it is preferred that both channels form a cross-over area which can be referred to as the point of intersection of the two channels and, in embodiments, corresponds to the extraction area of the separation channel.

In embodiments, the metering channel comprises a metering chamber on a first side which is filled with a suitable liquid, for example a buffer solution or the liquid separation medium. This dosing chamber serves as a reservoir for liquid, so that when the dosing unit is activated, a defined, suitable volume of liquid can be ejected from the dosing chamber that enters the dosing channel, so that a fluid flow is created within the dosing channel when the dosing unit is activated. As a result, the medium that is located in the area where the separation channel and the metering channel pass, which may be a buffer solution located in the metering chamber, for example, is displaced from this area and flows along the direction of flow of the fluid flow in the metering channel into a second part of the metering channel, the is on the other, second side of the separation channel on which the dosing chamber is not located.

At the end of this second part of the metering channel there is an opening from which the medium located in the metering channel can exit when a fluid flow is generated. The system or method according to the invention is preferably set in such a way that when the dosing unit is activated, so much liquid is expelled from the dosing chamber that the medium located in the extraction area, i.e. in the area where the two channels cross, comes out of the opening located on the Can be located at the end of the second area of the metering channel, exits. This makes it possible to isolate a molecular fraction that is located in the area where the separation channel and metering channel crosses from the separation channel by being ejected from the opening of the metering channel when the metering unit is activated.

In embodiments, the part of the metering channel with the metering chamber is above the separation channel, and the part of the metering channel with the opening is below the separation channel. Other arrangements will be apparent to a person skilled in the art based on the description herein.

In embodiments, the activation of the dosing unit takes place, as described above, by the detection unit as a result of an activation unit that can be viewed and integrated as part of the dosing unit, but also as an external unit that interacts with the dosing unit, exerting a force on the dosing unit as a result of which the liquid is set in motion and as a result liquid emerges from the nozzle at the end of the metering channel. This exit can be forced by the activation unit, for example, by exerting / acting on a mechanical force, pressure, thermal expansion, acceleration or pulse transmission or oscillation. The activation unit that generates this force can be attached above, below, inside or behind the dosing chamber and also at a point along the dosing channel. In the case of attachment below the dosing chamber, i.e. along the dosing channel, the liquid volume would first be ejected from the opening and at almost the same time (due to capillary forces) liquid would flow from the dosing chamber into the dosing channel. Other arrangements will be apparent to a person skilled in the art based on the description herein.

The volume ejected from the opening, i.e. the medium fraction, which in particular comprises the medium of the extraction area or the traversing area and the molecules contained therein or the molecule fraction contained therein, is deposited after ejection or during the ejection from the opening. With larger Dimensioning of the dosing channel, it is likely that several successive activations of the dosing unit are necessary until the desired volume of the crossing area of the dosing and separation channel is expelled, since a smaller volume may be expelled compared to the volume of the dosing channel on the second side.

In the context of the invention, the term “depositing” encompasses non-contact depositing, for example in which the medium is ejected from the opening as one or more free-flying drops and is collected on a collecting device, for example a collecting vessel or a collecting substrate, without a direct contact occurs between the opening and the collecting device or a fluid bridge is formed between these structures. In embodiments of the invention, the medium fraction is ejected from the opening as a free-flying droplet.

In addition, “depositing” also includes a collecting process in which a contact or a fluid bridge is created, for example by means of a contact or half-contact method. In the context of the invention, depositing thus comprises all types of conventional metering methods which can be used in particular in microfluidic systems for depositing or applying liquids and which are known to a person skilled in the art.

In embodiments, the opening is a nozzle for ejecting the medium fraction. The type of opening of the metering channel can be adapted within the scope of the present invention depending on the type of the desired deposit mechanism. For example, it is preferred that the opening is formed by a nozzle which enables or simplifies the formation of free-flying droplets. This is particularly desirable in cases where non-contact filing is desired or necessary. The nozzle opening can, as far as advantageous, assume any geometry. These include, for example, round, rectangular, square, star-shaped, oval and mixed forms of these. In addition, the opening can also be located on an area which is many times larger than the nozzle opening area or can be formed by the end of a capillary-like structure (such as a cannula, for example). Suitable opening shapes for the various deposit variants are known to the person skilled in the art.

Dosing can be implemented, for example, by means of a diaphragm deformation in the dosing chamber (repeated pressing and subsequent relaxation) using a piezo actuator [ Schoendube, J., Wright, D., Zengerle, R., & Koltay, P. Biomicrofluidics 2015, 9 (1), 014117 ]. Furthermore, the dosage can take place according to the bubble jet principle, the inkjet principle due to inertia, pressure-driven, thermally by punctual heating of the liquid in the dosing chamber or via a valve control (e.g. pneumatic, etc.). Furthermore, the dosage can take place by capillary forces, for example by placing the nozzle on a substrate or the bottom of a collecting vessel, so that the liquid and the substrate come into contact and the liquid is drawn out of the nozzle via the wetting of the substrate material. Suitable metering units and metering chambers are known to a person skilled in the art.

The separation unit comprises a separation channel, which is preferably a microchannel that is filled with a preferably liquid or gel-like separation medium. This can also be filled with a liquid separation medium in one sub-area and with a gel-like separation medium in another sub-area. Within the scope of the invention, a separation channel can comprise different cross-sectional shapes, for example round, oval, rectangular, square, polygonal or any other conceivable suitable shape. The cross-sectional shape can also change along the separation channel. In addition, depending on the application, different cross-sectional sizes of the separation channel are suitable for the present invention. For channels with an approximately round cross-section, for example, diameters of 10-2000 μm are suitable, in particular also diameters of 15-1800, 20-1600, 30-1400, 40-1200, 50-1000, 60-900, 70-800, 80 -600, 90-500, 100-480, 120-460, 140-440, 160-420, 180-400, 200-380, 220-360, 240-340, 260-320, 280-300 µm. Corresponding resulting cross-sectional areas are also suitable for other forms of separation channels and are obvious to a person skilled in the art within the scope of the present disclosure. Corresponding cross-sectional variants and dimensions can also apply to the metering channel in embodiments. In addition, the separation channel can contain restriction elements which lead to an increased fluidic resistance. A separation channel is also conceivable, which is laid out in a meander shape, for example.

In embodiments, the separation channel contains a different medium in the extraction area than in other or all other areas of the separation channel. The respective media can be selected in such a way that the separation medium does not flow into the extraction area after activation of the dosing unit. For example, the separation medium can be in gel form, whereas the medium in the extraction area is liquid.

In the context of the invention, the separation channel can be located in a capillary, similar to that in capillary electrophoresis. In embodiments of the invention, the separation unit comprises a capillary that serves as a separation channel. In such embodiments, the molecular fractions of the sample are preferably separated along an electric field within the capillary filled with the separation medium. Correspondingly, within the scope of the invention, the separation can take place by means of capillary electrophoresis. In another embodiment, the driving force for moving the molecules can also be a pressure gradient.

The term “separation medium” or “separation medium” in the context of the present invention relates to any type of suitable medium with the aid of which it is possible to separate molecules in a separation channel according to certain chemical or physical properties of the molecules. Liquid or aqueous solutions and gels are particularly suitable for this purpose. In addition, solid media can also be used as the separation medium, for example those which are known to a person skilled in the art from chromatography columns, and these can also be used in conjunction with a liquid mobile phase or liquid or gel-type separation medium. Correspondingly, all common separation media that are used in chromatography and electrophoresis processes can be used as separation media within the scope of the invention, in particular together with a solid medium, such as the matrix of a chromatography column. Correspondingly, the term separation medium in embodiments of the invention can also be understood, for example, as the combination of a solid medium and a liquid mobile phase.

Liquid and gel-like separation media for the purposes of the invention include any type of aqueous water-based solution or gels that require water or other liquids such as inorganic or organic solvents or additives such as DMSO, DMF or glycerol for their production. Examples of liquid and gel-like separation media according to the present invention include water-based gels, buffers, salt solutions, liquid gels, solid gels, liquid or solid gels based on polyacrylamide, linear or (cross) cross-linked gels based on polyacrylamide, agarose-based gels, hydrogels such as alginate , Block polymers, block copolymers, hydroxyethyl cellulose or derivatives thereof, pluronic, gelatine or chitin hydrogels, gradient gels and buffer solutions and mixtures of the aforementioned media and gel types. In addition, a separation channel can contain more than one separation medium, for example more than one liquid and / or a gel and / or solid materials, for example chromatography materials. For example, the separation channel can be composed of different parts of different types of liquid or gel. For example, a separation channel can comprise a section formed from polyacrylamide which is connected to a section formed from agarose. It is a great advantage of the present invention that in particular all types of liquids and gels with electrical conductivity are suitable for serving as liquid separation media.

The present invention offers significant advantages over other methods for isolating molecules or molecular groups or molecular fractions from a sample with a complex molecular composition.

On the one hand, only a very small amount of starting material or sample volume is required. On the other hand, however, large sample volumes can also be introduced, for example via suitable reservoirs for receiving samples. Regardless of the initial volume, it is possible to focus molecules of a large sample volume in the separation channel or when entering the separation channel and to separate them into sharply separated molecule fractions. The invention thus gives the user the opportunity to analyze both extremely small and relatively large sample volumes. In the case of larger sample volumes, the repeated purification of several smaller sub-volumes sequentially one after the other is advisable. Suitable sample volumes, which can be introduced into the system or the method in embodiments, are in particular on the picoliter to the microliter scale, although other volumes can also be used.

In addition, the method or system according to the invention enables a separation and isolation of molecules using very small amounts of consumables, so that the costs for the analysis and separation of a sample can be kept very low. In addition, the present invention enables the separation and isolation of molecules or molecular fractions in a single, uniform process, which saves a considerable amount of time. In addition, the resulting isolated molecule fractions can be available in very small volumes after isolation, which are already suitable for subsequent use, and concentration-dependent isolation can also take place. Since a molecular fraction usually also represents a spatial concentration gradient, i.e. with a maximum concentration in the middle of the fraction and a decreasing concentration at the flanks (in front of and behind the middle of the fraction), precisely the area can be isolated from a molecular fraction that corresponds to a subsequent application has a suitable concentration or from which this results depending on the isolated volume.

The process can be adapted to the needs of the user and thus requires very little preparation and follow-up times.

Different types of available samples can be used as starting material in the method, and the volume and the buffer in which the resulting isolated molecular fractions are present after the method can be adapted to the needs and subsequent applications, in particular via those in the dosing chamber and in the Liquid present in the dosing channel, such as a buffer solution. It is possible that isolated molecular fractions are present in very small volumes of a few picoliters or nanoliters.

In addition, the isolated molecular fractions can be deposited in different vessels or on different substrates or different substrate positions, depending on the desired subsequent application. For example, different fractions can be collected in different pots (wells) of a microtiter plate or deposited at different locations on a substrate, such as a slide suitable for microarrays. Both non-contact filing and other methods such as semi-contact writing [Gutzweiler et al. J. Micromech. Microeng. 26 (2016) 045018 (10pp)]. The pots of the microtiter plate can also be prefilled with suitable buffers or other liquids in order to reduce the evaporation of the small ejected or deposited volumes.

Due to the possible small resulting volumes, the method according to the invention enables, for example, the simple and fast production of microarrays, for example protein or DNA microarrays, by a simple and integrated method in which molecules of a sample, for example proteins, are separated and applied to a Substrate are possible without an intermediate step within a process.

In embodiments of the invention, a molecular fraction or parts thereof are deposited or metered into the sample feed of a second microfluidic system. This can be a second system according to the invention, which differs from the first microfluidic system from which the molecular fraction was isolated in that other separation or cleaning conditions, for example through the use of a different separation medium or a different ionic strength or a different pH Value are given, so that the molecular fraction is separated into sub-fractions which could not be separated in the first system and these are isolated according to the invention. With the system described according to the invention, a method corresponding to 2-dimensional (gel) electrophoresis or 2-dimensional chromatography could be implemented.

In embodiments of the method according to the invention, the separation takes place on the basis of one or more physical and / or chemical properties of the molecules, for example according to charge, molecular weight, size, function, structure, isoelectric point, chemical or physical interaction with the separation medium and / or the surface of the separation channel .

In embodiments of the invention, the surfaces in the separation channel are modified or functionalized. With suitable functionalization of the surface, for example by coating the channel walls, undesired interactions, such as, for example, unspecific bonds, between the surfaces and the molecules can be prevented. In addition, surface functionalization can also be used to bind unwanted molecule fractions by means of capture molecules bound to the channel wall or to filter them out through specific bonds. An (additional) separation or filtering or purification can thus be achieved by functionalization. In addition, in embodiments, the channel wall can be modified in such a way that the surface charge is influenced in order to consciously generate an electro-osmotic flow for chromatographic separations or to prevent this in the case of electrophoretic separations.

In a preferred embodiment of the invention, the sample volume (ie the so-called “loaded volume) is about 1 nL to 100 µL, preferably about 10 nL to 10 µL, about 100 nL to 2 µL, or about 1 µL.

The range from about 1 nL to 100 µL is particularly advantageous because it relates to small sample volumes which lead to a high separation resolution. The range from approx. 10 nL to 10 µL is also preferred because, in addition to the advantage of high separation resolution, a smaller maximum volume is required, which leads to more efficient use of the sample material. The sample volume range of approx. 100 nL to 10 µL is preferred because, in addition to the advantages mentioned above, it enables precise metering of the sample volume, while the maximum volume is still small enough to enable the use of very thin or fine separation channels can accommodate the sample. In addition, this sample volume enables high sensitivity or detection of the Sample components with very high separation accuracy at the same time. A sample volume of about 1 μL is particularly suitable, since the amounts of all other materials involved in the method of the present invention can be reduced due to the small volume of the sample.

In embodiments of the invention, the separation unit comprises electrodes for generating an electric field along the separation channel, preferably at the ends of the separation channel or within the metering chamber.

The system or method according to the invention can preferably comprise electrodes, for example an anode and a cathode, which are in contact with the separating medium, i.e. are electrically connected to the separating medium, so that an electric field can be applied to the separating medium via the respective contact points. This enables a voltage to be applied between the electrodes within the separation medium, which leads to the formation of an electric field and to migration and ultimately to separation of the components in a sample which has been introduced into the separation unit and the separation medium and is enclosed therein. The components can be separated in particular on the basis of their size, their molecular weight, their charge and / or their isoelectric point (pI, pH (I), IEP).

In preferred embodiments, the present invention includes more than one anode and more than one cathode. For example, the device of the present invention can comprise two anodes and two cathodes which enable parallel analysis of two samples in independent separation channels connected to different electrodes. In such embodiments, the remaining components of the system can also be present in correspondingly multiple designs.

In addition, it is possible to apply different electrical fields across the separation channel and the metering channel, so that, for example, undesired diffusion of molecules within the separation medium, in particular in the area where the separation channel and metering channel pass through, can be prevented. For example, by applying a suitable electric field in the area of the crossing point of separation and dosing channel, undesired molecular fractions can be prevented from diffusing into the dosing channel or the dosing chamber, so that it is ensured that these fractions migrate through the crossing area along the separation channel. Such fractions migrating through can be collected in a suitable reservoir at the end of the separation channel.

Embodiments of the invention include an electric field which is aligned along the metering channel and prevents the diffusion of molecules or molecule fractions contained in the sample into the metering channel. A corresponding repulsive electric field can be applied along the various structures of the invention in order to ensure the desired direction of migration of the molecules contained in the sample. For example, undesired migration into the metering channel or in the direction of the metering chamber can be prevented.

The electrical field applied for the separation can be switched off in the meantime, preferably during the brief activations of the dosing unit to expel the molecule fractions, or vice versa in order to prevent subsequent molecule fractions from migrating from the separation channel into the crossover area between the dosing channel and the separation channel or through the Dosing unit possibly generated and counteracting flow along the separation channel, which has a negative effect on the purification.

A detection unit is preferably used in the method according to the invention. In embodiments, the invention comprises a detection unit which detects a molecule fraction in the separation channel. In embodiments, the detection unit detects a molecular fraction in the extraction area, for example in the area where the metering channel and the separation channel pass through.

The detection can include UV / Vis, fluorescence, conductivity and / or impedance detection. In embodiments of the invention, the system according to the invention or at least parts thereof, such as, for example, the casing of the separation channel, is transparent. This enables the detection of molecular fractions, for example by detecting a fluorescence signal, also by a detection unit arranged outside the separation channel.

A detection unit to be used within the scope of the invention is suitable for detecting molecular fractions within the separation channel and in particular in the area where the metering channel and separation channel pass through by means of conventional detection methods and sensors, with UV / Vis, fluorescence, conductivity and / or impedance detection and corresponding suitable sensors can be used within the detection unit. The person skilled in the art is able to provide appropriate detection units which can be oriented towards the molecules and molecule fractions to be isolated.

The conductivity detection can be used as a universal detector in electromigratory separation techniques that can be used within the scope of the present invention, such as, for example, capillary electrophoresis. If the detection is carried out without contact, very stable and reproducible measurements can be achieved, since the electrodes cannot be contaminated by materials used in the method or system according to the invention. For example, with the help of conductivity detection on microfluidic chips, the separation of molecular fractions can also be monitored in the context of multi-dimensional separations. In this case, a potential check can also only be carried out at the crossover area or shortly before the crossover area of the metering channel and the separating channel.

The visualization and / or quantification of molecules or molecule fractions in the separation channel can take place by means of laser-induced fluorescence. Therefore, in the context of this embodiment, the molecules to be detected should be labeled fluorescent. This can be achieved by coupling a molecule to a fluorophore. Fluorophores (or fluorochromes) which can be used in the context of the present invention are fluorescent chemical compounds which can emit light again when excited by light. Fluorophores for use as labels for the generation of labeled biomolecules of the invention include, without claiming to be exhaustive, rhodamine and derivatives such as Texas Red, fluorescein, and derivatives such as 5-bromomethylfluorescein, Lucifer Yellow, IAEDANS, 7-Me2N-coumarin-4 acetate, 7-OH-4-CH3-coumarin-3-acetate, 7-NH2-4CH3-coumarin-3-acetate (AMCA), monobromobimane, pyrene trisulfonates, such as Cascade Blue, and monobromotrimethyl-ammoniobimane, FAM, TET, CAL Fluor Gold 540, HEX, JOE, VIC, CAL Fluor Orange 560, Cy3, NED, Quasar 570, Oyster 556, TMR, CAL Fluor Red 590, ROX, LC Red 610, CAL Fluor Red 610, Texas Red, LC Red 610 , CAL Fluor Red 610, LC Red 640, CAL Fluor Red 635, Cy5, LC Red 670, Quasar 670, Oyster645, LC Red 705, Cy5.5, BODIPY FL, Oregon Green 488, Rhodamine Green, Oregon Green 514, Cal Gold , BODIPY R6Gj, Yakima Yellow, JOE, HEX, Cal Orange, BODIPY TMR-X, Quasar-570 / Cy3, TAMRA, Rhodamine Red-X, Rhodamine B, Rhodamine 6G, Redmond Red, BODIPY 581/591, Cy3.5, Cal Red / Texa s Red, BODIPY TR-X, BODIPY 630/665-X, Pulsar-650, Quasar-670 / Cy5.

Alternatively, the molecules of the present invention can be intrinsically fluorescent, for example fluorescent protein. In a further preferred embodiment, fluorescent molecules can be linked to the molecule to be detected. Non-limiting examples of fluorescent proteins which can be used in the context of the invention are photoproteins such as aequorin; Obelin; Aequorea fluorescent proteins, such as green fluorescent proteins (GFP, EGFP, AcGFP1), blue fluorescent proteins (CFP, ECFP), blue fluorescent proteins (BFP, EBFP), red fluorescent proteins (RFP), yellow fluorescent proteins (YFP, EYFP ), ultraviolet fluorescent protein (GFPuv), their fluorescence enhancement variants, their peptide destabilization variants, and the like; fluorescent proteins from the coral reef, such as Discosoma red fluorescent proteins (DsRed, DsRed1, DsRed2 and DsRed-Express), Anemonia red fluorescent proteins (AsRed and AsRed2), Heteractis far-red fluorescent proteins (HcRed, HcRed1), Anemonia cyan fluorescent proteins (AmCyan, AmCyan1), Zoanthus green fluorescent proteins (ZsGreen, ZsGreen1), Zoanthus yellow fluorescent proteins (ZsYellow, ZsYellow1), their fluorescence enhancement variants, their peptide destabilization variants, and the like; Renilla reniformis green fluorescent protein (Vitality hrGFP), its fluorescence enhancement variants, its peptide destabilization variants, and the like; and Great Star Coral fluorescent proteins such as g, Montastrea cavernosa fluorescent protein (Monster Green® Fluorescent Protein), its fluorescence enhancement variants, its peptide destabilization variants, and the like. One skilled in the art understands that these and a variety of other fluorescent proteins may be useful as fluorescent protein in aspects of the invention (J. ennifer Lippincott-Schwartz & George H. Patterson, Development and Use of Fluorescent Protein Markers in Living Cells, 300 (5616) Science 87-91 (2003) ; other Jin Zhang et al., 3 (12) Nat. Rev. Mol. Cell. Biol. 906-918 (2002) ). One skilled in the art will also appreciate that these and many other fluorescent proteins, including species orthologues and paralogues of the naturally occurring fluorescent proteins described above, as well as artificial fluorescent proteins, may be useful as the fluorescent protein disclosed in aspects of the present specification.

In a further preferred embodiment of the present invention, intercalating fluorescent dyes can be used for the detection of molecules, for example of nucleic acid molecules. These dyes include in particular ethidium bromide, propidium iodide, Hoechst 33258, Hoechst 33342, acridine orange, ethidium bromide homodimers, EverGreen and all dyes of the SYBR and SYTO families. These dyes also include POPO, BOBO, YOYO, and TOTO from Molecular Probes (Eugene, Oreg.).

Further possible detection means can include specific markings, such as fluorescent markings, luminous markings, specific reactive chemical groups, gold, silver, magnetic, metal oxide particles, beads with marker particles, non-fluorescent dyes or radioactive markings. In another embodiment, the molecules are detected by ultraviolet (UV) absorption detection during migration. In addition, the molecules can be detected electrochemically during the migration via the impedance detection, which requires a specific measuring electrode structure that can be implemented in the system according to the invention and in particular the detection unit, without any markings.

In embodiments of the method according to the invention, the detection of a molecular fraction in the area where the metering channel and separation channel pass through activates the metering unit. Correspondingly, the dosing unit can be activated by the detection unit when the detection unit detects or indicates the presence of a molecule fraction in the area where the dosing channel and separation channel pass through.

In embodiments, the detection unit controls the activation of the dosing unit, with activation of the dosing unit leading to the expulsion of the medium located in the extraction area, comprising the molecular fraction contained therein. In this case, activation of the dosing unit is triggered by the detection unit when the presence of a molecular fraction that is to be isolated is detected by the detection unit within the area where the dosing channel and separation channel pass through.

In this case, the detection unit can be arranged in such a way that a detection of molecular fractions is detected shortly before or in the area where the metering channel and separation channel pass through. In cases where the detection unit is arranged in front of the traversing area and detects a molecule or a molecular fraction before entering the traversing area, the dosing unit can be activated with a suitable delay after the detection, so that the molecular fraction in the period between detection and activation in the traversal area has migrated. In addition, the detection unit can also control the switching off or reversing of the applied electrical field used for separation as well as the application of an electrical field between an electrode located in front of the dosing unit and the electrode in the sample introduction, so that the migration of subsequent fractions is stopped for the period of activation of the dosing unit or at least delayed.

A person skilled in the art is able to design or configure various embodiments of the invention in which the units of the invention are arranged relative to one another in such a way that the detection unit controls the activation of the dosing unit in a suitable manner in order to isolate specific detectable or detected molecule fractions to guarantee.

In embodiments of the invention, the metering unit is activated continuously. In these embodiments, medium fractions are thus constantly ejected from the metering channel, with only the desired drops or volumes of separation medium being deposited on a collecting device, for example in a collecting vessel or on a substrate, whereas the remaining ejected medium is discarded, for example sucked off. It is possible here for the detection unit to indicate when an ejected drop is to be collected or deposited, and when ejected drops are to be sucked off or discarded.

Such embodiments are advantageous because the continuous metering or the continuous ejection can prevent undesired diffusion of the separation medium and migrating molecular fractions into the metering channel.

In embodiments of the method, the opening of the metering channel comprises a nozzle for ejecting the separation medium located in the region where the metering channel and separation channel pass through. The use of a nozzle in the area of the opening of the metering channel is particularly advantageous for embodiments in which contact-free collection of the ejected medium is desired, since nozzles promote the formation of free-flying drops and are particularly suitable for this. In embodiments, the molecular fraction to be isolated is ejected with the medium as one or more flying drops from the metering channel. Accordingly, the laying down can take place without contact by ejecting flying droplets. This form of contact-free depositing is particularly advantageous, since it can minimize the risk of contamination, since there is no contact between the collecting device and the opening of the metering channel, not even in the form of a fluid bridge.

In general, the term “nozzle” describes a technical device for influencing a fluid when a pipe flow passes into the free space. The nozzle often forms the end of the pipeline. Correspondingly, an open end of a hose or a capillary can also be referred to as a (free-standing) nozzle. The nozzle edge does not necessarily have to be thin-walled as with free-standing nozzles such as for example capillaries or cannulas, but can be many times larger in the plane of the nozzle surface compared to the nozzle diameter. A nozzle can have the same cross-sectional area over its entire length, widen, taper or have other complex shapes. A nozzle does not normally do any work, but instead converts between speed and static pressure. Through a nozzle, a fluid can be accelerated along a pressure gradient, a solid or viscous mass can be formed (see extruder) or a liquid substance can be atomized. Various nozzle shapes which can be used at the opening of the metering channel within the scope of the present invention are known to the person skilled in the art.

Alternatively, the molecular fraction to be ejected can be deposited via a capillary bridge between the opening of the metering channel and a surface. It is preferred here that there is a height h> 0 mm between the opening and the depositing surface.

Within the scope of the invention, a hose nozzle (PipeJet) or a drawn chip nozzle can also be used at the opening of the metering channel, which are preferred for embodiments in which the ejected separation medium is deposited using the contact or half-contact method.

In further embodiments, contact can be made between the opening of the metering channel and the surface of the deposit when it is deposited.

In the context of the invention, the metering unit can be arranged at the end of the separation channel. In other embodiments, the metering unit is arranged in the first third, approximately halfway, or after the second third of the separation channel. Further embodiments are readily conceivable and implementable for a person skilled in the art.

In the context of the invention, however, it is preferred that a suitable or adequate separation / separation of the molecules contained in a sample in the separation channel is ensured upstream of the dosing unit. The separation channel can continue after the crossing point of the metering channel and the separation channel. Embodiments are possible which comprise more than one metering unit, it being possible for the metering unit to be arranged in different regions of the separation channel.

It is also possible that the separation unit according to the invention comprises one or more reservoirs, for example for separation medium, liquids, buffer solutions and / or samples. Individual reservoirs can fulfill several functions here, for example serve as a contact point for electrodes and as an introduction point for samples.

In the context of the invention, the term “reservoir” relates to an area of the separation unit in which there is an expansion or expansion of the separation medium. These can be, for example, contact points between the separation medium and the electrodes of the system according to the invention.

It is advantageous to have reservoirs made of separation medium or electrolyte solution (e.g. buffer) at these contact points, as they offer flexibility in the placement of the electrodes and otherwise it can be difficult to use the electrodes with the sometimes very small-sized structures of the separation channel To bring dosing channel in connection. In addition, the large contact area, which can be made possible with the aid of reservoirs between the electrodes and the separating medium, prevents the development of heat and thus the formation of gas bubbles, which can lead to current instabilities.

In addition, reservoirs supply an increased number of charge carriers in order to keep the current stable and thus to keep the migration speed constant.

Furthermore, reservoirs can serve as introduction sites for the sample in the system according to the invention. The introduction of samples into the system can be difficult, especially in microfluidic systems in which all structures are very small. The introduction can be simplified in that reservoirs of separation medium are present at the introduction points, from which the molecules or molecular fractions contained in the sample migrate into the separation channel, for example by applying an electric field. Here, the electric field can be applied in such a way that the sample molecules are focused at the point of entry into the separation channel, so that a sharp separation of the molecules is possible even with comparatively large sample volumes.

In addition, reservoirs can also be present at the end of the separation channel as collection points for molecules and molecular fractions that were not isolated from the separation channel with the aid of the dosing unit, but that have migrated through the crossing point of the separation channel and dosing channel.

In embodiments, one of the electrodes, with the aid of which an electric field can be applied across the separation channel, can be in the Be positioned in the dosing chamber of the dosing unit. In addition, the metering chamber can serve as an ion reservoir in the context of the system according to the invention. In such embodiments it is possible for the molecules or molecular fractions to enter the metering channel, for example the area above the opening.

In addition, it is preferred in embodiments of the invention that the liquid in the dosing chamber, the medium in the dosing channel and the separation medium are identical or similar liquids and / or have the same conductivity or ionic strength.

In the context of the invention, the term “liquid” also includes liquid-based gels.

In certain embodiments of the invention, the temperature of the separation unit and / or the metering unit is actively regulated. Corresponding embodiments can include one or more temperature regulating units.

One or more temperature regulating units can regulate the temperature of the separation channel and / or the metering channel and / or parts of these channels or the media contained therein. In particular, a temperature regulating unit can be used which can regulate the temperature in the area where the separation channel and the metering channel pass through. A temperature regulating unit can be part of the separation unit or the dosing unit or both. For example, within the scope of the method according to the invention, one or more temperature regulating units can be used which regulate the temperature of the separation unit, the metering unit and / or certain areas of the arrangement.

If, for example, the separation medium is a gel that is solid or gel-like at the usual ambient temperature in which the method according to the invention is carried out, but becomes liquid at an elevated temperature, it is advantageous if the temperature in the area where the metering channel passes through when the metering unit is activated and separation channel is temporarily increased to such an extent that liquefaction of the separation medium takes place in this area, so that the liquefied separation medium can be displaced from the traversing area without problems by the induced fluid flow. Furthermore, the temperature regulation unit can be used to fill channel structures with gels that are solid at room temperature and liquid at elevated temperatures. Conversely, cooling the system can allow the channel structure to be filled with separating media that solidify at higher temperatures. Correspondingly, a cooling of the dosing unit in the area where the dosing channel and separation channel crosses would also facilitate the displacement of the separation medium by the induced fluid flow from the area where it crosses. An example of such a separation medium is Pluronic® F127.

In the context of the method according to the invention, the ejected molecule fraction can be stored on or in a collecting device, for example a reaction vessel, in particular a well of a microtiter plate, a tube or reaction vessel (e.g. 200 µL Eppendorf tubes) or on a suitable collecting substrate / substrate, are preferably placed on an array substrate such as a slide made of glass, metal, plastic or similar material. In addition, the collecting device can be a porous membrane, for example a (nitro) cellulose, cellulose acetate, polyethersulfone or the like, a solid hydrogel, the opening of a capillary or chromatographic column or a reservoir described according to the invention for introducing samples.

In particular, it is possible according to the invention for different medium or molecule fractions to be deposited in different reaction vessels or at different positions on a substrate. For this purpose, the use of a storage unit in the context of the method or system according to the invention is preferred. In embodiments, a depositing unit provides a different reaction vessel or a different position on the substrate after depositing a medium fraction.

A depositing unit controls the positioning of the respective collecting device used, such as a multiwell plate or a slide relative to the separation and / or dosing unit used, from which the medium fraction is ejected. The activation and position shifting of the depositing unit and / or the separation and / or metering unit can be synchronized with the activation of the metering unit. If necessary, both the separation and / or metering unit and the depositing unit are controlled or activated by the detection unit.

A depositing unit thus enables the collecting device to be displaced relative to the opening from which the medium fractions are ejected. For example, after activating the dosing unit and depositing the ejected medium in or on a collecting device, a time-delayed relative position shift of the depositing unit and the collecting device held by it relative to the opening can take place, so that when the dosing unit is subsequently activated, the then ejected medium is transferred to another Position of the catching device is placed. Accordingly, it is advantageous if, within the scope of the invention, a depositing unit is used for depositing which, after depositing a molecular fraction, provides a different reaction vessel or a different position on the substrate. In this way, a further or subsequent molecular fraction can be deposited in this different vessel or in this different position. A 2- or 3-axis system, for example, can be used to position the catching device.

In particular, the present invention makes it possible to produce a microarray by depositing a multiplicity of preferably different separated molecular fractions on different positions on a substrate.

The invention therefore also relates to a method for producing a microarray, the method according to the invention being used to isolate one or more molecular fractions from a sample, and several molecular fractions from one or more samples being deposited at different positions on a substrate suitable for a microarray, preferably by non-contact dosing, such as ejecting the medium from the extraction area from a nozzle as free-flying droplets. The use of a depositing unit which controls the relative positioning of the collecting device with respect to the opening is advantageous here.

• - eine Separationseinheit umfassend
• 1. i. einen Separationskanal, der bevorzugt mit einem Separationsmedium gefüllten ist, und optional
  2. ii. ein oder mehrere Reservoire für Flüssigkeiten und/oder Proben, und/oder
  3. iii. Elektroden zur Erzeugung eines elektrischen Feldes entlang des Separationskanals,
• - eine Dosiereinheit, und optional
• - eine Detektionseinheit und/oder
• - eine Ablegeeinheit.

The present invention also relates to a system for isolating one or more molecular fractions from a sample, comprising

• - Comprising a separation unit
• 1. i. a separation channel, which is preferably filled with a separation medium, and optionally
  2. ii. one or more reservoirs for liquids and / or samples, and / or
  3. iii. Electrodes for generating an electric field along the separation channel,
• - a dosing unit, and optional
• - a detection unit and / or
• - a filing unit.

In embodiments of the system, the metering unit comprises a metering channel which crosses the separation channel and comprises a liquid-filled metering chamber on a first side of the separation channel and an opening, preferably a metering nozzle, on a second side of the separation channel.

The system according to the invention is preferably a microfluidic system. In embodiments of the system that use separation by means of pressure, the separation unit comprises suitable connections for applying a suitable pressure to the separation channel from an external pressure source.

The various units of the system are not necessarily connected to one another in a uniform device and can be combined with one another as separate devices. It is possible, for example, for the separation unit to be a microfluidic chip which, in the case of electrophoretic separation, is inserted into a holder with electrodes, for example, or receives electrodes itself. A separation chip can include the dosing unit in a single device, or it can be combined with a dosing unit, the dosing unit forming a physically separate device. The same applies to the detection unit and the deposit unit.

The invention further relates to a kit for microfluidic isolation of one or more molecular fractions from a sample, comprising one or more components of a system according to the invention, such as a separation unit comprising a separation channel, a dosing unit comprising a dosing channel with a dosing chamber, a detection unit, a collecting device and / or a storage unit.

The use of all the features of the invention disclosed here for the method according to the invention is also possible within the framework of the system or kit according to the invention and is hereby disclosed. Conversely, the same also applies to features that have been described in the context of the system according to the invention.

Specific description of the invention

The invention is described in more detail by the following figures and examples. These do not limit the scope of the invention, but rather represent preferred embodiments of aspects of the invention and serve to better illustrate the invention described herein.

Figure list

• 1 : Representation of the basic principle of the separation of different biomolecule species from a mixed solution on the basis of a specific embodiment. In the 1 The illustrated embodiment of the invention shows a microfluidic system for separating biomolecule fractions and consists in the following of two functional operating units:

List of reference symbols

1

= (Bio) mixed molecules = sample with sample components

1a, 1b and 1c; 1a, b & c

= Separated biomolecule fractions (sample components);

2

= Separation channel;

3

= Separation medium;

5

= Dosing chamber;

6th

= Dosing channel;

8th

= Extraction area = crossing area separation channel / dosing channel;

9

= Opening / nozzle at the end of the dosing channel;

10a

= expelled medium fraction without biomolecule fraction;

10b

= expelled medium fraction with biomolecule fraction 1c ;

11

= Depositing unit / collecting unit.

An operating unit 1 (Separation unit) serves to separate the individual molecular fractions ( 1a , 1b , 1c ), especially as shown here biomolecule fractions ( 1a , 1b , 1c ), from a mixed solution ( 1 ) within a microchannel / separation channel ( 2 ), for example using suitable methods for separation according to molecular weight, size, isoelectric point, charge, etc ...

An operating unit 2 (Dosing unit) enables the purification of individual (bio) molecular fractions ( 1a , 1b , 1c ). Here is the operating unit 2 behind operating unit 1 switched and serves to separate or extract the respective separated (bio) molecule fractions ( 1a , 1b , 1c ) from the microchannel ( 2 ) during the migration process, preferably as shown here in the form of free-flying droplets ( 10b ) using non-contact dosing principles (inkjet, bubblejet, etc.). The respective fractions are, for example, placed in separate reaction vessels ( 11 ) or pots of a microtiter plate or in array format dosed onto special substrates.

The invention can include a detection unit which detects a molecular fraction to be purified shortly before or in the area where the operating unit passes through 1 and operation unit 2 is detected and the dosing unit is activated.

2 : An impedance chip from previous work at the University of Freiburg is shown here [ Schoendube, J., Wright, D., Zengerle, R., & Koltay, P. (2015). Single-cell printing based on impedance detection. Biomicrofluidics, 9 (1), 014117 ]. The chip was originally developed to isolate cells from a sample. The picture shows the chip that is also used in 3 is shown in its entirety. A voltage is applied across the separation channel so that the sample to be separated migrates through the channel.

3 : Marked chip cutout 2 under the fluorescence microscope (Rhodamine B channel). The chip is rotated 180 ° and has a channel width of 40 µm. The channels were filled with a suitable separating gel. A fraction of single-stranded DNA fragments (30 nucleotides / dye label: Atto532 / concentration: 100 μM) was placed in the reservoir as a sample. The applied field was 40 V / cm. The different images were taken at different times during the migration process.

Example 1, electrophoresis + _" Inkiet"

The separation structure described in this example contains reservoirs for liquids and samples as well as a microchannel that is filled with a liquid separation medium and at the ends of which an electric field is applied as a driving force for the migration of the biomolecules. If the sample (or part of it) is introduced into the separation channel, the (bio) molecules migrate at different speeds due to electrokinetic effects according to their size or their charge-to-mass ratio and separate into fractions.

At the end of the separation channel, the separated fractions can be recorded with a detector (examples: UV / Vis, fluorescence or impedance-based detection). The dosing unit is located directly behind the detector. The metering channel, at the lower end of which there is a nozzle and at the other end a chamber filled with liquid, runs transversely to the metering channel.

The dosing unit is activated as soon as or shortly after a (bio) molecule fraction is detected and it is in the dosing channel. Individual drops are continuously dosed into a reaction vessel, a small pot of a microtiter plate or onto another substrate (e.g. a microscope slide) until no more signal from the respective fraction is detected.

As soon as the next fraction is detected, the process is repeated, with the next fraction either being dosed into another reaction vessel / pot or at another point on the substrate (prerequisite: installation of the separation / dosing system on an automation system). The two units are ideally integrated on one chip.

Dosing can be implemented, for example, by means of a diaphragm deformation in the dosing chamber (repeated pressing and subsequent relaxation) using a piezo actuator [ Schoendube, J., Wright, D., Zengerle, R., & Koltay, P. (2015). Single-cell printing based on impedance detection. Biomicrofluidics, 9 (1), 014117 ]. Furthermore, the dosage can take place according to the bubble jet principle, due to inertia or via a valve control (e.g. pneumatic, etc.). Whether one or more fractions are dosed per reaction vessel / potty can be selected individually. Likewise, only one target fraction can be extracted by dosing, while the other fractions migrate further along the separation channel into a collecting reservoir behind the dosing channel.

• Die Auftrennung der Probe und die Herstellung eines Microarrays für die weitere Analyse der Probe kann in einem Schritt und mit einer wesentlich kleineren Probenmenge erfolgen, als bei herkömmlicher Durchführung in zwei aufeinanderfolgenden Schritten. Die Prozesszeit zwischen 5-20 Minuten pro Lauf-je nach Probe - ist relativ gering und stellt neben der geringen erforderlichen Probenmenge einen weiteren Vorteil des Verfahrens dar. Somit entspricht die Aufreinigungszeit ungefähr der benötigten Zeit für die Elektrophorese und ist damit maximal effizient.

In addition, it is possible to generate several microarrays in succession through repeated partial injection processes of the sample, subsequent separation and dosing of the fractions, which represents another relevant application in addition to pure purification and is a decisive advantage and unique selling point compared to established methods:

• The separation of the sample and the production of a microarray for the further analysis of the sample can be carried out in one step and with a significantly smaller amount of sample than with conventional implementation in two successive steps. The process time between 5-20 minutes per run - depending on the sample - is relatively short and, in addition to the small amount of sample required, represents a further advantage of the method. The purification time corresponds approximately to the time required for electrophoresis and is therefore maximally efficient.

Since the separation and dosing structure can be integrated on one chip, the costs amount to approx. <5 € in the case of a reusable chip and approx. 20-50 € in the case of a single-use product. In the case of production using injection molding, the estimated costs could also be significantly reduced.

Example 2, separation unit

In this example, which illustrates the principle feasibility of the invention and through the 2 and 3 is described in more detail, a microfluidic chip originally intended for cell isolation is used to demonstrate the migration of a (bio) molecule fragment across the metering channel. The misappropriated chip, a composite of silicon and glass, has at least parts of the structures required for the invention [ Schoendube, J., Wright, D., Zengerle, R., & Koltay, P. (2015). Single-cell printing based on impedance detection. Biomicrofluidics, 9 (1), 014117 ].

For the in 3 In the series of images shown, charged fluorescence-labeled DNA fragments were moved along the separation channel under the influence of the electric field. The fragments migrate as a broad stream, since there is no corresponding injection structure on the chip used to focus the sample on the chip and thus new sample constantly flows in, which also prevents a visible separation.

During the migration process shown here, which only partially corresponds to the claimed invention, the sample diffuses into the metering channel (that is, into the nozzle and the metering chamber). This can be prevented by applying a repelling electric field to the metering chamber and metering the fractions from the chip nozzle with the aid of a metering unit, which is not possible in the comparative example shown here due to the lack of a metering unit according to the invention.

Likewise, in a method according to the invention or a device according to the invention, a continuous dosing process combined with suction for undesired drops could prevent diffusion into the dosing chamber. In the example shown, the chip was not actuated for reasons of better visualization of the migration process, so no drop ejection was induced. However, the principle of drop dosing has already been demonstrated several times and is already used in various commercial products.

QUOTES INCLUDED IN THE DESCRIPTION

This list of the documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent literature cited

• US 2008/0286751 A1 [0008]

Non-patent literature cited

• Zhu, Z., Lu, J. J., & Liu, S. Analytica chimica acta 2012, 709, 21-31 [0005]
• Schoendube, J., Wright, D., Zengerle, R., & Koltay, P, Biomicrofluidics 2015, 9 (1), 014117 [0008]
• Schoendube, J., Wright, D., Zengerle, R., & Koltay, P. Biomicrofluidics 2015, 9 (1), 014117 [0042]
• Jennifer Lippincott-Schwartz & George H. Patterson, Development and Use of Fluorescent Protein Markers in Living Cells, 300 (5616) Science 87-91 (2003) [0071]
• Jin Zhang et al., 3 (12) Nat. Rev. Mol. Cell. Biol. 906-918 (2002) [0071]
• Schoendube, J., Wright, D., Zengerle, R., & Koltay, P. (2015). Single-cell printing based on impedance detection. Biomicrofluidics, 9 (1), 014117 [0115, 0121, 0124]

Claims (10)

A method for isolating one or more molecular fractions from a sample, comprising a. Introducing the sample into a separation unit, b. Separation of molecules contained in the sample in a separation channel of the separation unit filled with a separation medium into molecule fractions, c. Extraction of one or more molecular fractions from an extraction area of the separation channel by activating a dosing unit, the extraction area being fluidically coupled to an opening and, by activating the dosing unit in the extraction area, medium located in the extraction area is ejected from the opening as a medium fraction, the medium fraction comprising a molecular fraction or parts thereof can. Procedure according to Claim 1 , which is a microfluidic process. Method according to one of the preceding claims, wherein the medium fraction is ejected as a free-flying drop. Method according to one of the preceding claims, wherein a detection unit detects a molecule fraction in the separation channel, the detection preferably comprising UV / Vis, conductivity and / or impedance detection. Method according to the preceding claim, wherein the metering unit is activated by the detection of a molecular fraction, preferably in the extraction area of the separation channel. Method according to one of the preceding claims, wherein the temperature of the separation unit and / or the metering unit is actively regulated. Method according to one of the preceding claims, wherein the expelled medium fraction is deposited in a reaction vessel, preferably a cavity (well) of a microtiter plate, or on a suitable substrate, preferably an array substrate such as a slide, and / or different medium fractions in different reaction vessels or at different positions on a substrate. Method according to the preceding claim, wherein a depositing unit is used for depositing which, after depositing a molecular fraction, provides a different reaction vessel or a different position on the substrate. Method according to the preceding claim, wherein a microarray is produced by depositing a plurality of preferably different separated molecular fractions at different positions on a substrate. A system for isolating one or more molecular fractions from a sample, comprising a. comprising a separation unit i. a separation channel, which is preferably filled with a separation medium, and optional ii. one or more reservoirs for liquids and / or samples, iii. Electrodes for generating an electric field along the separation channel, iv. Suitable connections for applying a suitable pressure to the separation channel from an external pressure source b. a dosing unit, and optional c. a detection unit and / or d. a filing unit.

Images