EP2203246B1 - Apparatus and method for the uniform distribution of microparticles in a liquid - Google Patents

Apparatus and method for the uniform distribution of microparticles in a liquid Download PDF

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Publication number
EP2203246B1
EP2203246B1 EP08804783A EP08804783A EP2203246B1 EP 2203246 B1 EP2203246 B1 EP 2203246B1 EP 08804783 A EP08804783 A EP 08804783A EP 08804783 A EP08804783 A EP 08804783A EP 2203246 B1 EP2203246 B1 EP 2203246B1
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EP
European Patent Office
Prior art keywords
suspension
storage chamber
microparticles
liquid
mixing head
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EP08804783A
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German (de)
French (fr)
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EP2203246A1 (en
Inventor
Jörg FELDHUSEN
Hartmut SCHLÜTER
Joachim Thiemann
Johannes Lemburg
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Charite Universitaetsmedizin Berlin
Rheinisch Westlische Technische Hochschuke RWTH
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Charite Universitaetsmedizin Berlin
Rheinisch Westlische Technische Hochschuke RWTH
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/50—Circulation mixers, e.g. wherein at least part of the mixture is discharged from and reintroduced into a receptacle
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F35/55—Baffles; Flow breakers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F2101/00—Mixing characterised by the nature of the mixed materials or by the application field
    • B01F2101/23—Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials

Definitions

  • the invention relates to a device for producing a suspension with uniformly distributed microparticles in a carrier liquid or for maintaining a uniform distribution of microparticles in the carrier liquid.
  • the device comprises a storage chamber 1 with the suspension, a mixing head 2 and a pump 3, which is connected to the storage chamber 1 and the mixing head 2, wherein the mixing head 2 at least one cavity 4 with a Strömungsverwirbler 5 and an overflow 6, by a Opening 7 is connected to the storage chamber 1, characterized in that the storage chamber 1, a pin 14 and / or a rotary inlet 15 and a rotary return 16, which are connected to the pump 3 comprises.
  • the device can be used to identify, characterize and purify proteins.
  • the invention also provides a process for the uniform distribution of microparticles in a liquid in which a turbulent, quasistatic state is generated.
  • microparticles so-called beads, which are a few micrometers to about 0.1 mm large plastic spheres and whose surfaces have certain reactive properties. These microparticles are stored after their preparation in a protective carrier liquid, with which they form a suspension.
  • a fundamental problem of the handling of microparticles is the equal quantitative dosage of these particles on a large number of sample carriers. A direct weighing or counting of the microparticles precipitates because they can not be removed from the carrier liquid and allowed.
  • the patent US 5,705,610 teaches a device comprising mixing and reaction vessels, between which can be transported reagents or suspensions, which are mixed by the introduction of gas.
  • the gas input associated with the formation of bubbles is critical both from the point of view of process technology (increase in volume, unequal volumes of liquid during sampling, etc.) and in terms of the sensitivity of biological structures.
  • WO 2007/064635 A1 a device which operates without air passage, by moving a suspension through an opening between two pump mixing chambers and the mixed suspension is removed through a valve port.
  • the device requires a complex Interaction of the two pumps and prevents sampling in the turbulence range.
  • a device for the uniform distribution of microparticles in a liquid which comprises a storage chamber 1 with a suspension of microparticles and liquid, a mixing head 2 for receiving the suspension and a pump 3 connected to the storage chamber 1 and the mixing head 2 for sucking the Suspension of the storage chamber 1 is connected to the mixing head 2, wherein the mixing head 2, at least one cavity 4 with a Strömungsverwirbler 5 and an overflow 6, which is connected through an opening 7 to the storage chamber 1, characterized in that the storage chamber 1 a pin 14 and / or a rotary inlet 15 and a rotary return 16, which are connected to the pump 3 comprises.
  • the point of interest is an interface 8 to the adjacent laboratory system 10, through which the microparticles or structures associated therewith can be removed and / or analyzed.
  • the structures preferably include biomolecules, such as. For example, proteins, nucleic acids, peptides, carbohydrates, polymers or molecules having a molecular weight between 50 and 1000 Da, or microorganisms or eukaryotic cells.
  • the storage chamber 1 has a pin 14 and / or a rotary inlet 15 and a rotary return 16, which are connected to the pump 3 on.
  • These components are particularly advantageous for generating a second suspension cycle which prevents sedimentation of the microparticles in the storage chamber 1.
  • the microparticles transported in the first cycle between the storage chamber 1 and the mixing head 2 are distributed uniformly in the carrier liquid in the cavity 4, their concentration could decrease there over time. This would be the case if, for example, sedimented microparticles are sucked in at the beginning in the storage chamber and, subsequently, a suspension thinned out at particles.
  • the pin 14 and / or rotational inlet 15 and outlet 16 so not only the uniform distribution in a cycle of the first cycle is achieved, but the maintenance of a constantly distributed suspension over the entire time course of operation of the device.
  • the time course is determined by the filling volume of the storage chamber 1 with suspension and their successive removal from the cavity 4.
  • the pump 3 promotes the suspension in two parallel circuits in this embodiment.
  • the added second cycle ensures the rotation of the suspension stored in the storage chamber 1 by the suspension sucked by the rotation return 16, transported in a rotary loop and then transferred to the rotary feed 15 back into the storage chamber. It is preferable to arrange the rotary feed 15 and the rotary return 16 at different heights of the storage chamber 1.
  • the rotating flow can be swirled by means of a flow swirler 5, as represented by the pin 14.
  • a suction 17 is arranged, via which the mixing head 2 suspension is supplied.
  • the device according to the invention also consists of two units, a pump unit 3 and a unit with storage chamber 1 and mixing head 2, which are connected by lines for transporting the suspension between them are. It is understood that each pump 3 has a motor and is driven by an external power source 9.
  • Pantry 1 and mixing head 2 may form an integral component or otherwise secured together.
  • the storage chamber 1 represents a cavity of any geometric shape, for example, has a non-rotationally symmetrical cross section, in particular an elliptical cross section, polygonal cross section or rectangular cross section with a semicircular surface or two semi-circular surfaces on opposite rectangular surfaces.
  • a rotationally symmetrical, cylindrical design is preferred.
  • the cavity should be of a size suitable for storing a certain amount of microparticles for a particular application.
  • the storage chamber 1 is therefore interchangeable by the user.
  • the pantry is filled either by hand or by machine, the latter having the advantage of a possible automation.
  • the pump 3 promotes the suspension in the circulation by first sucking the suspension out of the storage chamber 1.
  • pumps of any type can be used as long as a minimum velocity of the flow and thus a particle transport are effected.
  • the minimum speed depends i.a. on the nature of the line connections and the properties of the suspension, including in particular the amount and mass of the particles and the viscosity of the carrier liquid count.
  • the parameters can be experimentally determined experimentally in routine experiments.
  • a peristaltic pump is used in the device according to the invention.
  • the aspirated suspension is transported by means of transport lines, preferably by means of hoses 11, into the mixing head 2.
  • the mixing head comprises at least one cavity, the so-called cavity 4.
  • the mixing head 2 eight cavities 4, whereby the simultaneous processing of several samples is given.
  • such a mixing head 2 is compatible with the 96-well format, as z. B. represent microtiter plates. Pre-switching the device to high-throughput robots thus enables automation of basic research, pharmaceutical, chemical, diagnostic and biotechnology industries.
  • the cavity 4 comprises an inflow region 12, a region with flow swirler 5 and a turbulence region.
  • the inflow area 12 is at the bottom of the cavity 4, followed by the area with flow swirler 5 and the turbulence area above it.
  • the change from the area with flow swirler 5 to the turbulence area can be fluid.
  • the aforementioned regions have the same geometric shape of the cross section.
  • the geometric shape is not limited, as long as the installation of Strömungsverwirblungs instituten remains possible. With regard to possible shapes, reference is made to the comments on the storage chamber 1.
  • the cavity 4 has a rotationally symmetrical cross section.
  • the cavity 4 is designed such that a turbulent flow can be generated in it, for which purpose a Strömungsverwirbler 5 is installed.
  • a "flow swirler 5" is any constructional measure and / or in the cavity 4, which is both an external component connected to the cavity or not, and the shape of the cavity 4 itself may include.
  • Nonlimiting examples of flow swirlers 5 are stirrers or shakers (as external, non-connected elements), pin or grid (as external, connected elements), discontinuous extensions or tapers of the cross section, and bulges of the sidewalls (as internal elements).
  • the Strömungsverwirbler 5 comprises at least one discontinuous enlargement of the cross section in an extension region 13 above the Zustrom Anlagens 12.
  • discontinuous magnification refers in the context of the invention to a sudden expansion of the diameter or cross section, wherein the Extension on edges and thus in the form of a stage completes.
  • the suspension flows through the inflow region 12, which is followed by the extension region 13. That is, in this embodiment, the area with flow swirler 5 is represented by the extension area 13.
  • the extension region 13 is preferably arranged in the lower half of the cavity 4, more preferably in the lower third thereof, most preferably in the lower fourth.
  • a ground-level arrangement ensures a large range of turbulence.
  • the turbulence needed to obtain a uniformly distributed suspension can be advantageously adjusted by the configuration of discontinuous magnification.
  • the degree of turbulence is determined by the interaction of various parameters, including the relative height of the extension region 13 with respect to the total height of the cavity 4, the extent of the discontinuous magnification and the nature of the step.
  • the relative height of the extension region 13 should be as small as possible in order to ensure a large range of turbulence.
  • the height of the extension region 13 is less than 25% of the height of the cavity 4, more preferably less than 15%. This is determined inter alia by the inclination of the inner walls in the extension region 13.
  • the cross section in the extension region 13 increases linearly, preferably with an increase tan ⁇ of less than 70 °, more preferably less than 50 °, most preferably less than 30 °.
  • the cavity 4 is formed as a cylinder, so that it diverges conically in the extension region 13 with the aforementioned increase.
  • the extent of the discontinuous enlargement should be dimensioned so that there is sufficient space for the demolition of the flow on the inner wall and turbulence and this Room can also be filled with turbulence.
  • the discontinuous enlargement is designed so that the cross-section in the extension region 13 at least doubles, preferably at least fivefold, particularly preferably at least tenfold.
  • the cavity 4 contains an overflow 6, which is located in the upper part of the turbulence zone. This ensures that the pumped inflow does not lead to an overflow of the cavity 4, provided that no withdrawal or only a withdrawal of suspension takes place, the withdrawn volume is less than the inflowing volume.
  • the overflow 6 represents a sheathed cavity in the cavity 4, the length of which corresponds at least to the wall thickness of the cavity 4.
  • the overflow 6 has a length which exceeds the wall thickness of the cavity 4.
  • the overflow 6 is inclined so that the suspension can drain.
  • the mixing head 2 is preferably arranged above the storage chamber 1 in order to allow the suspension, which is at or above the level of the overflow 6, to flow out by gravity into the lower storage chamber 1.
  • the overflow 6 is arranged above the preferred extension area 13 according to the invention.
  • the mixing head 2 it is also conceivable to position the mixing head 2 at the same or even lower height than the storage chamber 1 and to initially collect the overflowing suspension in a basin before it is returned to the storage chamber 1 by means of a pump or by utilizing capillary forces.
  • an opening 7, which represents the upper open surface of the storage chamber 1, or serve another opening, the latter may also be below the liquid level in the storage chamber 1 upon application of an external pressure on the line leading to the opening.
  • the prerequisite for suspension removal is that the cavity 4 has an interface 8 to an adjacent laboratory system 10.
  • This laboratory system 10 may be, for example, a liquid handling robot (liquid removal robot), which by means of removal needles Suspension sucks.
  • the interface 8 can be understood, for example, as the suspension surface.
  • the removal needles dip at the interface 8 in the suspension.
  • an interface 8 at a constant height is desired, as provided by the invention. Due to the inventive arrangement and design of the pump 3, pantry 1 and cavity 4 (each with at least two openings), which allow driving the suspension in the circulation, a quasi-static state with respect to the turbulence and the position of the interface 8 in the cavity 4 is formed ,
  • the invention also relates to the use of the device according to the invention for the uniform distribution of microparticles in a liquid.
  • the invention also relates to the use of the device according to the invention for obtaining a suspension of uniformly distributed microparticles in a carrier liquid and / or for maintaining a uniform distribution of microparticles in a carrier liquid.
  • the invention further relates to the use of the device according to the invention for taking a suspension of uniformly distributed microparticles in a liquid, preferably with a liquid extraction robot.
  • the device is used for the identification, characterization and / or purification of proteins.
  • Proteins are often involved in the development of serious diseases such. As cancer or Alzheimer's involved.
  • microparticles with a diameter of a few micrometers to 0.1 mm are becoming increasingly important. Their surface has several reactive properties that allow the identification and characterization of proteins.
  • the laboratory automation in the field of Characterization and identification of proteins is a growing market, so that high-throughput robots are indispensable for the effective design of microparticle-based proteomics research.
  • the device according to the invention is constructed in such a way that a liquid handling robot can remove the constantly distributed suspension from the wells (cavities 4) of a mixing head 2, preferably from a mixing head with eight wells, by means of removal needles. This makes it possible to connect the device to high-throughput robots.
  • the use of the device according to the invention is preferably suitable for providing constantly distributed quantities of microparticles for carrying out automated protein purification processes by means of chromatography in the 96-well batch format.
  • the device of the present invention is used in the activity determination of enzymes by mass spectrometry. It has been repeatedly demonstrated that mass spectrometry is a fast, sensitive and reliable tool for the determination of enzymatic activities ( Hsieh et al. 1995, Anal. Biochem. 229, 20 ; Bothner et al. 2000, J. Biol. Chem. 275, 13455 ; Wu et al. 1997, Chem. Biol. 4, 653 ).
  • MALDI-MS is characterized by its resistance to buffer solutions and its ability to analyze complex mixtures, making it predestined for direct screening of enzyme activities that require minimal sample preparation.
  • Enzymatic activities in complex protein fractions can be determined with a mass spectrometer using mass spectrometry-assisted enzyme screening (MES).
  • MES mass spectrometry-assisted enzyme screening
  • the enzyme activity is determined by incubating the immobilized proteins with a reaction-specific probe, followed by analysis of the reaction mixture by MALDI-MS after defined incubation times. Only the use of the device according to the invention ensures a constantly distributed suspension of microparticles, as a result, a uniform surface loading and constant protein concentration and the removal of an identical amount of microparticles for incubation with the probe is made possible and reproducible data is generated.
  • Another object of the invention is a method for uniform distribution of microparticles in a liquid in which the device according to the invention is used.
  • the sequence of steps illustrates that the process is circulated, for which in each case two openings of the storage chamber 1 or the mixing head 2 are essential, which are each different from each other. That is, in the storage chamber 1, the suction 17 for sucking the suspension in the direction of mixing head 2 and the opening 7 for receiving from the mixing head 2 overflowed suspension and in the cavity 4 in the mixing head 2, the inflow 13 of the suspension from the storage chamber 1 and the overflow 6 to return the suspension in the storage chamber 1 are located. Step (g) is carried out until the storage chamber 1 is empty or no mixing of the particles is desired, because the removal and / or analysis of the particle suspension is completed.
  • the turbulent flow in step (e) can be effected by any measure that results in a Reynolds number above the critical value of 2300. Suitable measures are in particular the increase of the flow rate of the suspension, the surface roughness of the cavity 4 and / or the density of the suspension and / or the reduction of the dynamic viscosity.
  • the turbulent flow is generated by the installation of a Strömungsverwirblers 5, more preferably by the incorporation of at least one discontinuous increase in the cross-section in an extension region 13 above a Zustrom Anlagens 12th
  • the method can also be carried out in such a way that, after step (e), (f) or (g), a further step (h) is followed, in which the suspension is removed from a fluid extraction robot.
  • an apparatus and a method for the uniform distribution of microparticles in a liquid are provided for the first time.
  • the invention uses the design of a flow-through sampling cavity 4 in order to generate turbulence in a simple and reliable way, which in turn entails a constant mixing of microparticles in a suspension.
  • the separation of storage chamber 1 and mixing head 2 advantageously allows a uniform distribution of the microparticles at the location of particle removal or analysis (cavity 4).
  • this system is coupled to a second circuit which ensures that the distribution of the microparticles remains homogeneous over a relatively long period of time or both at the beginning and at a time when the device has already been in operation for some time.
  • the mixing head 2 has a plurality of cavities 4, which together drive an identical mixing of the microparticles show and thus an automated microparticle quantity measurement are accessible. While only a gas flow is described in the prior art, the flow of the suspension itself is exploited here. Due to the surprising combination of this turbulence with an overflow 6, a liquid circuit is formed, which leads to a quasi-static state in the cavities 4. Apparatus and method of the invention are characterized by a simple and inexpensive handling and open up a variety of application perspectives, of which biochemistry in particular may be mentioned.
  • the prototype of the overall system, as in Fig. 5 is shown, as a power source 9, a 12 V DC power supply, which is the overall system supplied with electrical energy.
  • the power source powers pump 3, which is a Watson Marlow 102R peristaltic pump powered by a Faulhaber 3540K024C motor. Pump 3 and motor are put into operation via a switch.
  • Fig. 1 pump 3 and motor are covered to protect against external influences with a cover 18 which is fastened with screws DIN 912 (M4x10) to a flange plate 19.
  • the screw type is also used for all other fasteners.
  • Both the pump 3 and the suspension mixer cylinder chamber are mounted on a base plate 20.
  • the suspension mixer cylinder chamber comprises the pantry base 21, mixing head 2, cylinder pin 14 type DIN 7 (3x15) and four connectors type connector M6 250-6.
  • the suspension mixer cylinder chamber is screwed to the storage chamber base 21 with the base plate 20.
  • the base 21 has a cylindrical storage chamber 1 in the center and above it, an 8-well mixing head 2 is screwed.
  • Fig. 2 It can be seen that the wells or withdrawal cavities 4 are shaped in such a way that the conveyed inflow becomes turbulent due to a discontinuous tube expansion in the entry area (extension area 13). In this way, the suspension in the cavity 4 is vortexed and mixed. The removal of the suspension by the liquid handling robot reduces the total volume in the system. In order to achieve a constant high liquid level for the removal of suspension, the cavities 4 have an overflow 6.
  • the pump delivers the suspension in two parallel circuits ( Fig. 3 ).
  • the first circuit supplies the withdrawal cavities 4 ( Fig. 2a . 3 ).
  • the suspension is sucked on a cylindrical suction 17 and passed through hoses 11.
  • the suspension enters the mixing head 2 and is transported by means of a conduit system in the inflow region 12 of the individual cavities 4.
  • the overflowing liquid flows through the opening 7 directly into the storage chamber 1. So it is a liquid circulation, in which the cavity 4 a quasi-static state is generated, is removed from the constantly distributed suspension.
  • the second circuit ensures rotation of the stored suspension in the storage chamber 1 (FIG. Fig. 3, 4 ).
  • suspension is sucked through a rotary return 16, transported by a rotation loop in hoses 11 and returned to the storage chamber 1 via a rotary feed 15.
  • the storage chamber 1 has a cylindrical pin 14 which swirls the rotating flow in such a way that no beads can deposit in the center of the chamber (similar to a teacup with tea crumbs).
  • the suction 17 In the dead water of the pen is the suction 17, which feeds the removal cavities 4 through the riser 22.

Abstract

The invention relates to an apparatus for the uniform distribution of microparticles in a liquid, said apparatus comprising a storage chamber 1 including a suspension of microparticles and liquid, a mixing head 2 for receiving the suspension, and a pump 3 which is connected to the storage chamber 1 and the mixing head 2 for sucking the suspension from the storage chamber 1 into the mixing head 2, said mixing head 2 having at least one well 4 with a flow turbulator 5 and an overflow 6 which is connected to the storage chamber 1 via an opening 7, characterized in that the storage chamber 1 has a pin 14 and/or a rotary inlet 15 and a rotary runback 16 which are connected to the pump 3. The apparatus can be used for the identification, characterization and purification of proteins. The invention is also directed to a method for the uniform distribution of microparticles in a liquid, which method generates a turbulent, quasi-static state.

Description

Die Erfindung betrifft eine Vorrichtung zum Herstellen einer Suspension mit gleichmäßig verteilten Mikropartikeln in einer Trägerflüssigkeit bzw. zum Aufrechterhalten einer gleichmäßigen Verteilung von Mikropartikeln in der Trägerflüssigkeit. Die Vorrichtung umfasst eine Vorratskammer 1 mit der Suspension, einen Mischkopf 2 und eine Pumpe 3, die mit der Vorratskammer 1 und dem Mischkopf 2 verbunden ist, wobei der Mischkopf 2 mindestens eine Kavität 4 mit einem Strömungsverwirbler 5 und einem Überlauf 6, der durch eine Öffnung 7 mit der Vorratskammer 1 verbunden ist, aufweist, dadurch gekennzeichnet, dass die Vorratskammer 1 einen Stift 14 und/oder einen Rotationszulauf 15 und einen Rotationsrücklauf 16, die mit der Pumpe 3 verbunden sind, aufweist. Die Vorrichtung kann zur Identifizierung, Charakterisierung und Aufreinigung von Proteinen verwendet werden. Gegenstand der Erfindung ist auch ein Verfahren zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit, in dem ein turbulenter, quasistatischer Zustand erzeugt wird.The invention relates to a device for producing a suspension with uniformly distributed microparticles in a carrier liquid or for maintaining a uniform distribution of microparticles in the carrier liquid. The device comprises a storage chamber 1 with the suspension, a mixing head 2 and a pump 3, which is connected to the storage chamber 1 and the mixing head 2, wherein the mixing head 2 at least one cavity 4 with a Strömungsverwirbler 5 and an overflow 6, by a Opening 7 is connected to the storage chamber 1, characterized in that the storage chamber 1, a pin 14 and / or a rotary inlet 15 and a rotary return 16, which are connected to the pump 3 comprises. The device can be used to identify, characterize and purify proteins. The invention also provides a process for the uniform distribution of microparticles in a liquid in which a turbulent, quasistatic state is generated.

Die fortschreitende Automatisierung von Abläufen in der Labortechnik basiert zu großen Teilen auf paralleler Probenverarbeitung. Zunehmende Bedeutung in der Proteomforschung erhalten dabei Mikropartikel, sogenannte Beads, die wenige Mikrometer bis etwa 0,1 mm große Kunststoff-Kügelchen darstellen und deren Oberflächen bestimmte reaktive Eigenschaften besitzen. Diese Mikropartikel werden nach ihrer Herstellung in einer schützenden Trägerflüssigkeit gelagert, mit der sie eine Suspension bilden. Ein grundlegendes Problem der Handhabung von Mikropartikeln ist die mengenmäßig gleiche Dosierung dieser Partikel auf eine große Anzahl von Probenträgern. Ein direktes Abwiegen oder Zählen der Mikropartikel scheidet aus, da sie nicht aus der Trägerflüssigkeit entnommen werden können und dürfen.The progressive automation of processes in laboratory technology is largely based on parallel sample processing. Increasingly important in proteome research are microparticles, so-called beads, which are a few micrometers to about 0.1 mm large plastic spheres and whose surfaces have certain reactive properties. These microparticles are stored after their preparation in a protective carrier liquid, with which they form a suspension. A fundamental problem of the handling of microparticles is the equal quantitative dosage of these particles on a large number of sample carriers. A direct weighing or counting of the microparticles precipitates because they can not be removed from the carrier liquid and allowed.

Es ist im Stand der Technik bekannt, dass ein indirektes Bemessen der Partikelmenge über das Abmessen des Volumens der Trägerflüssigkeit möglich ist. Diese Art der Mengenmessung geht von der Annahme der konstanten Verteilung der Mikropartikel in der Trägerflüssigkeit aus. Diese Annahme ist jedoch nicht ohne weiteres zulässig, da die Mikropartikel im Allgemeinen eine höhere Dichte als die Trägerflüssigkeit aufweisen und deshalb zum Sedimentieren neigen. In Abhängigkeit vom Ort der Probennahme befinden sich folglich überdurchschnittlich viele Mikropartikel in einer bodennahen Probe bzw. unterdurchschnittlich wenige Mikropartikel in einer oberflächennahen Probe.It is known in the art that an indirect measurement of the amount of particles is possible by measuring the volume of the carrier liquid. This type of quantitative measurement is based on the assumption of the constant distribution of the microparticles in the carrier liquid. However, this assumption is not readily permissible since the microparticles generally have a higher density than the carrier liquid and therefore tend to sediment. Depending on the location of sampling, there are therefore an above-average number of microparticles in a near-surface sample or below-average few microparticles in a near-surface sample.

Zur konstanten Verteilung der Mikropartikel in einer Flüssigkeit sind verschiedene Vorrichtungen und Verfahren im Stand der Technik vorgeschlagen worden. Die Patentschrift US 6,255,166 B1 offenbart eine Vorrichtung, die ein Mischen von Mikropartikeln in Suspension dadurch ermöglicht, dass Flüssigkeit durch das vertikale Hin- und Herbewegen einer Schaufel bewegt wird. Nachteilig ist hier der benötigte Energieaufwand für das externe Rührelement sowie der Fakt, dass sich die Schaufel innerhalb des Gefäßes befindet und damit eine Entnahme oder Analyse der Suspension behindert.For constant distribution of the microparticles in a liquid, various devices and methods have been proposed in the prior art. The patent US 6,255,166 B1 discloses an apparatus that allows mixing of microparticles in suspension by moving liquid by vertically reciprocating a blade. The disadvantage here is the energy required for the external stirring element and the fact that the blade is within the vessel and thus hinders removal or analysis of the suspension.

Die Patentschrift US 5,705,610 lehrt eine Vorrichtung, die Misch- und Reaktionsgefäße umfasst, zwischen denen Reagenzien bzw. Suspensionen transportiert werden können, die durch das Einspeisen von Gas durchmischt werden. Der mit der Blasenbildung einhergehende Gaseintrag ist jedoch sowohl unter verfahrenstechnischen Gesichtspunkten (Volumenvergrößerung, ungleiche Flüssigkeitsvolumina bei der Probenentnahme etc.) als auch im Hinblich auf die Sensibilität biologischer Strukturen kritisch.The patent US 5,705,610 teaches a device comprising mixing and reaction vessels, between which can be transported reagents or suspensions, which are mixed by the introduction of gas. However, the gas input associated with the formation of bubbles is critical both from the point of view of process technology (increase in volume, unequal volumes of liquid during sampling, etc.) and in terms of the sensitivity of biological structures.

Es ist des Weiteren aus WO 2007/064635 A1 eine Vorrichtung bekannt, die ohne Luftdurchlass arbeitet, indem eine Suspension durch eine Öffnung zwischen zwei Pumpen-Mischkammern bewegt und die durchmischte Suspension durch einen Ventilanschluss entfernt wird. Die Vorrichtung erfordert ein aufwendiges Zusammenspiel der beiden Pumpen und unterbindet eine Probennahme im Turbulenzbereich.It is further off WO 2007/064635 A1 a device is known which operates without air passage, by moving a suspension through an opening between two pump mixing chambers and the mixed suspension is removed through a valve port. The device requires a complex Interaction of the two pumps and prevents sampling in the turbulence range.

Der Erfindung liegt die Aufgabe zugrunde, die im Stand der Technik aufgezeigten Nachteile der Durchmischung von Mikropartikeln in Suspensionen zu überwinden und eine Vorrichtung zu entwickeln, die eine zuverlässige Mikropartikelmengenbemessung gewährleistet.It is an object of the present invention to overcome the disadvantages of microparticle mixing in suspensions identified in the prior art and to develop a device which ensures reliable microparticle quantity measurement.

Die Aufgabe der Erfindung wird gemäß den unabhängigen Ansprüchen gelöst. Die Unteransprüche beinhalten bevorzugte Ausführungsformen. Erfindungsgemäß wird eine Vorrichtung zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit bereitgestellt, die eine Vorratskammer 1 mit einer Suspension aus Mikropartikeln und Flüssigkeit, einen Mischkopf 2 zur Aufnahme der Suspension und eine Pumpe 3, die mit der Vorratskammer 1 und dem Mischkopf 2 zum Ansaugen der Suspension von der Vorratskammer 1 in den Mischkopf 2 verbunden ist, umfasst, wobei der Mischkopf 2 mindestens eine Kavität 4 mit einem Strömungsverwirbler 5 und einem Überlauf 6, der durch eine Öffnung 7 mit der Vorratskammer 1 verbunden ist, aufweist dadurch gekennzeichnet, dass die Vorratskammer 1 einen Stift 14 und/oder einen Rotationszulauf 15 und einen Rotationsrücklauf 16, die mit der Pumpe 3 verbunden sind, aufweist.The object of the invention is achieved according to the independent claims. The subclaims contain preferred embodiments. According to the invention, a device for the uniform distribution of microparticles in a liquid is provided which comprises a storage chamber 1 with a suspension of microparticles and liquid, a mixing head 2 for receiving the suspension and a pump 3 connected to the storage chamber 1 and the mixing head 2 for sucking the Suspension of the storage chamber 1 is connected to the mixing head 2, wherein the mixing head 2, at least one cavity 4 with a Strömungsverwirbler 5 and an overflow 6, which is connected through an opening 7 to the storage chamber 1, characterized in that the storage chamber 1 a pin 14 and / or a rotary inlet 15 and a rotary return 16, which are connected to the pump 3 comprises.

Es hat sich gezeigt, dass die Trennung von Vorratskammer 1 und Mischkopf 2, der Einbau eines Strömungsverwirblers 5 in die Kavität 4 des Mischkopfs 2 und das Verschalten der vorgenannten Bauelemente in einem Kreislauf nicht nur zu einer konstanten Mikropartikelverteilung am Ort des Interesses führt, sondern auch einen quasistatischen Zustand in der Kavität 4 erzeugt. Der Ort des Interesses stellt dabei eine Schnittstelle 8 zum angrenzenden Laborsystem 10 dar, durch das die in der Suspension enthaltenen Mikropartikel oder damit verbundene Strukturen entnommen und/oder analysiert werden können. Die Strukturen umfassen vorzugsweise Biomoleküle, wie z. B. Proteine, Nukleinsäuren Peptide, Kohlenhydrate, Polymere oder Moleküle mit einem Molekulargewicht zwischen 50 und 1.000 Da, oder Mikroorganismen oder eukaryotische Zellen. Erfindungsgemäß weist die Vorratskammer 1 einen Stift 14 und/oder einen Rotationszulauf 15 und einen Rotationsrücklauf 16, die mit der Pumpe 3 verbunden sind, auf. Diese Komponenten sind besonders vorteilhaft, um einen zweiten Suspensionskreislauf zu generieren, der ein Sedimentieren der Mikropartikel in der Vorratskammer 1 verhindert. Zwar sind die im ersten Kreislauf zwischen Vorratskammer 1 und Mischkopf 2 transportierten Mikropartikel in der Kavität 4 gleichmäßig in der Trägerflüssigkeit verteilt, jedoch könnte dort ihre Konzentration mit der Zeit sinken. Das wäre dann der Fall, wenn beispielsweise zu Beginn in der Vorratskammer 1 sedimentierte Mikropartikel angesaugt werden und im weiteren Verlauf eine an Partikeln ausgedünnte Suspension. Durch den Stift 14 und/oder Rotationszulauf 15 und -ablauf 16 wird also nicht nur die gleichmäßige Verteilung in einem Zyklus des ersten Kreislaufs erzielt, sondern das Aufrechterhalten einer konstant verteilten Suspension über den gesamten zeitlichen Verlauf des Betriebs der Vorrichtung. Der zeitliche Verlauf wird durch das Füllvolumen der Vorratskammer 1 mit Suspension und deren sukzessive Entnahme aus der Kavität 4 determiniert. Die Pumpe 3 fördert in dieser Ausführungsform die Suspension in zwei parallelen Kreisläufen. Der hinzugetretene zweite Kreislauf sorgt für die Rotation der in der Vorratskammer 1 gespeicherten Suspension, indem durch den Rotationsrücklauf 16 die Suspension angesaugt, in einer Rotationsschleife transportiert und anschließend am Rotationszulauf 15 wieder in die Vorratskammer überführt wird. Es ist bevorzugt, den Rotationszulauf 15 und den Rotationsrücklauf 16 in verschiedenen Höhen der Vorratskammer 1 anzuordnen. Sofern durch den Rotationskreislauf trotzdem nur eine laminare Strömung entsteht, die ein Sedimentieren der Mikropartikel am Boden der Kammer 1 nicht hinreichend unterbindet, kann mittels eines Strömungsverwirblers 5, wie ihn der Stift 14 darstellt, die rotierende Strömung verwirbelt werden. Im Totwasser des Stifts 14 ist eine Absaugung 17 angeordnet, über die dem Mischkopf 2 Suspension zugeführt wird.It has been found that the separation of storage chamber 1 and mixing head 2, the incorporation of a Strömungsverwirblers 5 in the cavity 4 of the mixing head 2 and the interconnection of the aforementioned components in a circuit not only leads to a constant microparticle distribution at the point of interest, but also produces a quasistatic state in the cavity 4. The point of interest is an interface 8 to the adjacent laboratory system 10, through which the microparticles or structures associated therewith can be removed and / or analyzed. The structures preferably include biomolecules, such as. For example, proteins, nucleic acids, peptides, carbohydrates, polymers or molecules having a molecular weight between 50 and 1000 Da, or microorganisms or eukaryotic cells. According to the invention, the storage chamber 1 has a pin 14 and / or a rotary inlet 15 and a rotary return 16, which are connected to the pump 3 on. These components are particularly advantageous for generating a second suspension cycle which prevents sedimentation of the microparticles in the storage chamber 1. Although the microparticles transported in the first cycle between the storage chamber 1 and the mixing head 2 are distributed uniformly in the carrier liquid in the cavity 4, their concentration could decrease there over time. This would be the case if, for example, sedimented microparticles are sucked in at the beginning in the storage chamber and, subsequently, a suspension thinned out at particles. By the pin 14 and / or rotational inlet 15 and outlet 16 so not only the uniform distribution in a cycle of the first cycle is achieved, but the maintenance of a constantly distributed suspension over the entire time course of operation of the device. The time course is determined by the filling volume of the storage chamber 1 with suspension and their successive removal from the cavity 4. The pump 3 promotes the suspension in two parallel circuits in this embodiment. The added second cycle ensures the rotation of the suspension stored in the storage chamber 1 by the suspension sucked by the rotation return 16, transported in a rotary loop and then transferred to the rotary feed 15 back into the storage chamber. It is preferable to arrange the rotary feed 15 and the rotary return 16 at different heights of the storage chamber 1. If, however, only a laminar flow is created by the rotation circuit, which does not adequately prevent sedimentation of the microparticles at the bottom of the chamber 1, the rotating flow can be swirled by means of a flow swirler 5, as represented by the pin 14. In the dead water of the pin 14, a suction 17 is arranged, via which the mixing head 2 suspension is supplied.

Die erfindungsgemäße Vorrichtung besteht darüber hinaus aus zwei Einheiten, einer Pumpeneinheit 3 und einer Einheit mit Vorratskammer 1 und Mischkopf 2, die zum Transport der Suspension zwischen ihnen durch Leitungen verbunden sind. Es versteht sich, dass jegliche Pumpe 3 über einen Motor verfügt und von einer externen Energiequelle 9 angetrieben wird.The device according to the invention also consists of two units, a pump unit 3 and a unit with storage chamber 1 and mixing head 2, which are connected by lines for transporting the suspension between them are. It is understood that each pump 3 has a motor and is driven by an external power source 9.

Vorratskammer 1 und Mischkopf 2 können eine integrale Komponente bilden oder anderweitig aneinander befestigt sein. In jedem Fall muss die Anordnung von Vorratskammer 1 und Mischkopf 2 zueinander gewährleisten, dass Suspension durch einen Überlauf 6 an der Kavität 4 und eine obere Öffnung 7 an der Vorratskammer 1 in letztere gelangen kann. Die Vorratskammer 1 stellt einen Hohlraum beliebiger geometrischer Gestalt dar, der beispielsweise einen nicht rotationssymmetrischen Querschnitt besitzt, insbesondere einen elliptischen Querschnitt, vieleckigen Querschnitt oder rechteckigen Querschnitt mit einer Halbkreisfläche oder zwei Halbkreisflächen an gegenüberliegenden Rechtecksflächen. Im Sinne der Erfindung ist jedoch eine rotationssymmetrische, zylinderförmige Gestaltung bevorzugt. Der Hohlraum sollte eine Größe aufweisen, die zur Speicherung einer gewissen Mikropartikelmenge für eine spezielle Anwendung geeignet ist. In einer Ausgestaltung der Erfindung ist die Vorratskammer 1 deshalb durch den Benutzer austauschbar. Die Vorratskammer wird entweder per Hand oder maschinell befüllt, wobei letzteres den Vorteil einer möglichen Automatisierung aufweist.Pantry 1 and mixing head 2 may form an integral component or otherwise secured together. In any case, the arrangement of the storage chamber 1 and the mixing head 2 must ensure to each other that suspension can pass through an overflow 6 on the cavity 4 and an upper opening 7 on the storage chamber 1 in the latter. The storage chamber 1 represents a cavity of any geometric shape, for example, has a non-rotationally symmetrical cross section, in particular an elliptical cross section, polygonal cross section or rectangular cross section with a semicircular surface or two semi-circular surfaces on opposite rectangular surfaces. For the purposes of the invention, however, a rotationally symmetrical, cylindrical design is preferred. The cavity should be of a size suitable for storing a certain amount of microparticles for a particular application. In one embodiment of the invention, the storage chamber 1 is therefore interchangeable by the user. The pantry is filled either by hand or by machine, the latter having the advantage of a possible automation.

Die Pumpe 3 fördert die Suspension im Kreislauf, indem die Suspension zunächst aus der Vorratskammer 1 angesaugt wird. Hierfür können Pumpen beliebiger Bauart eingesetzt werden, solange eine Mindestgeschwindigkeit der Strömung und damit ein Partikeltransport bewirkt werden. Die Mindestgeschwindigkeit hängt u.a. von der Beschaffenheit der Leitungsverbindungen und den Eigenschaften der Suspension ab, wozu insbesondere die Menge und Masse der Partikel sowie die Viskosität der Trägerflüssigkeit zählen. Die Parameter können durch den Fachmann experimentell in Routineversuchen ermittelt werden. Bevorzugt wird in der erfindungsgemäßen Vorrichtung eine Schlauchpumpe verwendet.The pump 3 promotes the suspension in the circulation by first sucking the suspension out of the storage chamber 1. For this purpose, pumps of any type can be used as long as a minimum velocity of the flow and thus a particle transport are effected. The minimum speed depends i.a. on the nature of the line connections and the properties of the suspension, including in particular the amount and mass of the particles and the viscosity of the carrier liquid count. The parameters can be experimentally determined experimentally in routine experiments. Preferably, a peristaltic pump is used in the device according to the invention.

Die angesaugte Suspension wird mittels Transportleitungen, vorzugsweise mittels Schläuchen 11, in den Mischkopf 2 transportiert. Der Mischkopf umfasst wenigstens einen Hohlraum, die sogenannte Kavität 4. In einer bevorzugten Ausgestaltung der Erfindung weist der Mischkopf 2 acht Kavitäten 4 auf, wodurch das simultane Bearbeiten mehrerer Proben gegeben ist. Insbesondere ist ein solcher Mischkopf 2 kompatibel zum 96-Well-Format, wie es z. B. Mikrotiter-Platten repräsentieren. Das Vorschalten der Vorrichtung an Hochdurchsatzrobotern ermöglicht damit die Automatisierung von Prozessen in der Grundlagenforschung sowie in der pharmazeutischen, chemischen, diagnostischen und biotechnologischen Industrie.The aspirated suspension is transported by means of transport lines, preferably by means of hoses 11, into the mixing head 2. The mixing head comprises at least one cavity, the so-called cavity 4. In a preferred embodiment of the invention, the mixing head 2 eight cavities 4, whereby the simultaneous processing of several samples is given. In particular, such a mixing head 2 is compatible with the 96-well format, as z. B. represent microtiter plates. Pre-switching the device to high-throughput robots thus enables automation of basic research, pharmaceutical, chemical, diagnostic and biotechnology industries.

Die Kavität 4 umfasst einen Zustrombereich 12, einen Bereich mit Strömungsverwirbler 5 und einen Turbulenzbereich. Der Zustrombereich 12 befindet sich am Boden der Kavität 4, gefolgt vom Bereich mit Strömungsverwirbler 5 und dem Turbulenzbereich darüber. Der Wechsel vom Bereich mit Strömungsverwirbler 5 zum Turbulenzbereich kann fließend sein. Es ist bevorzugt, dass die vorgenannten Bereiche die selbe geometrische Form des Querschnitts besitzen. Die geometrische Form ist dabei nicht limitiert, solange der Einbau von Strömungsverwirblungselementen möglich bleibt. Betreffs möglicher Formen sei auf die Ausführungen zur Vorratskammer 1 verwiesen. In einer bevorzugten Ausgestaltung der Vorrichtung weist die Kavität 4 einen rotationssymmetrischen Querschnitt auf.The cavity 4 comprises an inflow region 12, a region with flow swirler 5 and a turbulence region. The inflow area 12 is at the bottom of the cavity 4, followed by the area with flow swirler 5 and the turbulence area above it. The change from the area with flow swirler 5 to the turbulence area can be fluid. It is preferable that the aforementioned regions have the same geometric shape of the cross section. The geometric shape is not limited, as long as the installation of Strömungsverwirblungselementen remains possible. With regard to possible shapes, reference is made to the comments on the storage chamber 1. In a preferred embodiment of the device, the cavity 4 has a rotationally symmetrical cross section.

Die Kavität 4 ist derart gestaltet, dass in ihr eine turbulente Strömung erzeugt werden kann, wozu ein Strömungsverwirbler 5 eingebaut wird. Im Sinne der Erfindung handelt es sich bei einem "Strömungsverwirbler 5" um eine beliebige bautechnische Maßnahme an und/oder in der Kavität 4, die sowohl ein externes Bauelement, dass mit der Kavität verbunden ist oder nicht, als auch die Gestalt der Kavität 4 selbst beinhalten kann. Nicht limitierende Beispiele für Strömungsverwirbler 5 sind Rührer oder Schüttler (als externe, nicht verbundene Elemente), Stift oder Gitter (als externe, verbundene Elemente), unstetige Erweiterungen oder Verjüngungen des Querschnitts sowie Auswölbungen der Seitenwände (als interne Elemente).The cavity 4 is designed such that a turbulent flow can be generated in it, for which purpose a Strömungsverwirbler 5 is installed. For the purposes of the invention, a "flow swirler 5" is any constructional measure and / or in the cavity 4, which is both an external component connected to the cavity or not, and the shape of the cavity 4 itself may include. Nonlimiting examples of flow swirlers 5 are stirrers or shakers (as external, non-connected elements), pin or grid (as external, connected elements), discontinuous extensions or tapers of the cross section, and bulges of the sidewalls (as internal elements).

In einer bevorzugten Ausgestaltung der Erfindung umfasst der Strömungsverwirbler 5 mindestens eine unstetige Vergrößerung des Querschnitts in einem Erweiterungsbereich 13 oberhalb des Zustrombereichs 12. Der Begriff "unstetige Vergrößerung" bezieht sich im Sinne der Erfindung auf eine plötzliche Erweiterung des Durchmessers bzw. Querschnitts, wobei sich die Erweiterung an Kanten und damit in Form einer Stufe vollzieht. Die Suspension strömt durch den Zustrombereich 12, dem sich der Erweiterungsbereich 13 anschließt. Das heißt, in dieser Ausführungsform wird der Bereich mit Strömungsverwirbler 5 durch den Erweiterungsbereich 13 repräsentiert. Der Erweiterungsbereich 13 ist vorzugsweise in der unteren Hälfte der Kavität 4 angeordnet, besonders bevorzugt in deren unterem Drittel, ganz besonders bevorzugt im unteren Viertel. Durch eine bodennahe Anordnung wird ein großer Turbulenzbereich gewährleistet.In a preferred embodiment of the invention, the Strömungsverwirbler 5 comprises at least one discontinuous enlargement of the cross section in an extension region 13 above the Zustrombereichs 12. The term "discontinuous magnification" refers in the context of the invention to a sudden expansion of the diameter or cross section, wherein the Extension on edges and thus in the form of a stage completes. The suspension flows through the inflow region 12, which is followed by the extension region 13. That is, in this embodiment, the area with flow swirler 5 is represented by the extension area 13. The extension region 13 is preferably arranged in the lower half of the cavity 4, more preferably in the lower third thereof, most preferably in the lower fourth. A ground-level arrangement ensures a large range of turbulence.

Die Turbulenz, die benötigt wird, um eine konstant verteilte Suspension zu erhalten, kann vorteilhaft durch die Ausgestaltung der unstetigen Vergrößerung eingestellt werden. Der Grad der Turbulenz wird dabei durch das Zusammenspiel verschiedener Parameter bestimmt, zu denen die relative Höhe des Erweiterungsbereichs 13 bezogen auf die Gesamthöhe der Kavität 4, das Ausmaß der unstetigen Vergrößerung und die Art der Stufe zählen. Prinzipiell ist die relative Höhe des Erweiterungsbereichs 13 möglichst klein zu wählen, um wiederum einen großen Turbulenzbereich zu gewährleisten. Vorzugsweise beträgt die Höhe des Erweiterungsbereichs 13 weniger als 25 % der Höhe der Kavität 4, besonders bevorzugt weniger als 15 %. Dies wird u.a. durch die Neigung der Innenwände im Erweiterungsbereich 13 bestimmt. In einer bevorzugten Ausführungsform der vorliegenden Erfindung vergrößert sich der Querschnitt im Erweiterungsbereich 13 linear, vorzugsweise mit einem Anstieg tan ϕ von weniger als 70°, besonders bevorzugt weniger als 50°, ganz besonders bevorzugt weniger als 30°. In einer besonders bevorzugten Ausführungsform ist die Kavität 4 als Zylinder ausgebildet, so dass sie im Erweiterungsbereich 13 mit dem vorgenannten Anstieg konisch divergiert. Als weiterer Parameter ist das Ausmaß der unstetigen Vergrößerung so zu dimensionieren, dass genug Raum zum Abriss der Strömung an der Innenwand und Verwirbelung vorhanden ist sowie dieser Raum auch mit Turbulenz ausgefüllt werden kann. Die unstetige Vergrößerung ist so gestaltet, dass sich der Querschnitt im Erweiterungsbereich 13 mindestens verdoppelt, vorzugsweise mindestens verfünffacht, besonders bevorzugt mindestens verzehnfacht.The turbulence needed to obtain a uniformly distributed suspension can be advantageously adjusted by the configuration of discontinuous magnification. The degree of turbulence is determined by the interaction of various parameters, including the relative height of the extension region 13 with respect to the total height of the cavity 4, the extent of the discontinuous magnification and the nature of the step. In principle, the relative height of the extension region 13 should be as small as possible in order to ensure a large range of turbulence. Preferably, the height of the extension region 13 is less than 25% of the height of the cavity 4, more preferably less than 15%. This is determined inter alia by the inclination of the inner walls in the extension region 13. In a preferred embodiment of the present invention, the cross section in the extension region 13 increases linearly, preferably with an increase tan φ of less than 70 °, more preferably less than 50 °, most preferably less than 30 °. In a particularly preferred embodiment, the cavity 4 is formed as a cylinder, so that it diverges conically in the extension region 13 with the aforementioned increase. As a further parameter, the extent of the discontinuous enlargement should be dimensioned so that there is sufficient space for the demolition of the flow on the inner wall and turbulence and this Room can also be filled with turbulence. The discontinuous enlargement is designed so that the cross-section in the extension region 13 at least doubles, preferably at least fivefold, particularly preferably at least tenfold.

Als weiteres bauliches Element enthält die Kavität 4 einen Überlauf 6, der sich im oberen Teil des Turbulenzbereichs befindet. Dadurch wird sichergestellt, dass der geförderte Zustrom nicht zu einem Überlaufen der Kavität 4 führt, sofern keine Entnahme oder lediglich eine Entnahme von Suspension stattfindet, deren entnommenes Volumen geringer als das zugeströmte Volumen ist. Der Überlauf 6 stellt einen ummantelten Hohlraum in der Kavität 4 dar, dessen Länge zumindest der Wanddicke der Kavität 4 entspricht. Vorzugsweise hat der Überlauf 6 eine Länge, die über die Wandstärke der Kavität 4 hinausgeht. Des Weiteren ist der Überlauf 6 so geneigt, dass die Suspension abfließen kann. Der Mischkopf 2 ist bevorzugt oberhalb der Vorratskammer 1 angeordnet, um ein Abfließen derjenigen Suspension, die sich auf oder über dem Niveau des Überlaufs 6 befindet, durch Gravitation in die tieferliegende Vorratskammer 1 zu ermöglichen. Insbesondere ist der Überlauf 6 oberhalb des erfindungsgemäß bevorzugten Erweiterungsbereichs 13 angeordnet. Natürlich ist es auch denkbar, den Mischkopf 2 in gleicher oder sogar geringerer Höhe als die Vorratskammer 1 zu positionieren und die überlaufende Suspension zunächst in einem Becken zu sammeln, bevor sie mittels einer Pumpe oder unter Ausnutzung von Kapillarkräften in die Vorratskammer 1 zurückgeführt wird. Hierzu kann eine Öffnung 7, welche die obere offene Fläche der Vorratskammer 1 repräsentiert, oder einen andere Öffnung dienen, wobei letztere bei Anlegen eines äußeren Drucks auf die zur Öffnung führende Leitung auch unterhalb des Flüssigkeitsspiegels in der Vorratskammer 1 liegen kann.As a further structural element, the cavity 4 contains an overflow 6, which is located in the upper part of the turbulence zone. This ensures that the pumped inflow does not lead to an overflow of the cavity 4, provided that no withdrawal or only a withdrawal of suspension takes place, the withdrawn volume is less than the inflowing volume. The overflow 6 represents a sheathed cavity in the cavity 4, the length of which corresponds at least to the wall thickness of the cavity 4. Preferably, the overflow 6 has a length which exceeds the wall thickness of the cavity 4. Furthermore, the overflow 6 is inclined so that the suspension can drain. The mixing head 2 is preferably arranged above the storage chamber 1 in order to allow the suspension, which is at or above the level of the overflow 6, to flow out by gravity into the lower storage chamber 1. In particular, the overflow 6 is arranged above the preferred extension area 13 according to the invention. Of course, it is also conceivable to position the mixing head 2 at the same or even lower height than the storage chamber 1 and to initially collect the overflowing suspension in a basin before it is returned to the storage chamber 1 by means of a pump or by utilizing capillary forces. For this purpose, an opening 7, which represents the upper open surface of the storage chamber 1, or serve another opening, the latter may also be below the liquid level in the storage chamber 1 upon application of an external pressure on the line leading to the opening.

Voraussetzung einer Suspensionsentnahme ist, dass die Kavität 4 eine Schnittstelle 8 zu einem angrenzenden Laborsystem 10 besitzt. Bei diesem Laborsystem 10 kann es sich beispielsweise um einen Liquid-Handling-Roboter (Flüssigkeitsentnahmeroboter) handeln, der mittels Entnahmenadeln die Suspension absaugt. Die Schnittstelle 8 kann beispielsweise als die Suspensionsoberfläche aufgefasst werden. Die Entnahmenadeln tauchen an der Schnittstelle 8 in die Suspension ein. Um ein wiederholtes Anpassen der Eintauchtiefe der Entnahmenadeln zu vermeiden, ist eine Schnittstelle 8 in einer konstanten Höhe erwünscht, wie sie durch die Erfindung bereitgestellt wird. Durch die erfindungsgemäße Anordnung und Ausgestaltung von Pumpe 3, Vorratskammer 1 und Kavität 4 (mit jeweils mindestens zwei Öffnungen), die ein Fahren der Suspension im Kreislauf ermöglichen, wird ein quasistatischer Zustand hinsichtlich der Turbulenz und der Lage der Schnittstelle 8 in der Kavität 4 ausgebildet.The prerequisite for suspension removal is that the cavity 4 has an interface 8 to an adjacent laboratory system 10. This laboratory system 10 may be, for example, a liquid handling robot (liquid removal robot), which by means of removal needles Suspension sucks. The interface 8 can be understood, for example, as the suspension surface. The removal needles dip at the interface 8 in the suspension. In order to avoid repeatedly adjusting the depth of insertion of the removal needles, an interface 8 at a constant height is desired, as provided by the invention. Due to the inventive arrangement and design of the pump 3, pantry 1 and cavity 4 (each with at least two openings), which allow driving the suspension in the circulation, a quasi-static state with respect to the turbulence and the position of the interface 8 in the cavity 4 is formed ,

Gegenstand der Erfindung ist auch die Verwendung der erfindungsgemäßen Vorrichtung zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit. Mit anderen Worten betrifft die Erfindung auch die Verwendung der erfindungsgemäßen Vorrichtung zum Erhalt einer Suspension aus gleichmäßig verteilten Mikropartikeln in einer Trägerflüssigkeit und/oder zur Aufrechterhaltung einer gleichmäßigen Verteilung von Mikropartikeln in einer Trägerflüssigkeit. Die Erfindung bezieht sich ferner auf die Verwendung der erfindungsgemäßen Vorrichtung zur Entnahme einer Suspension aus gleichmäßig verteilten Mikropartikeln in einer Flüssigkeit, vorzugsweise mit einem Flüssigkeitsentnahmeroboter. Die vorherige Lehre der Erfindung und deren Ausführungsformen betreffend die Vorrichtung an sich sind gültig und ohne Einschränkungen auf deren Verwendung anwendbar, sofern es sinnvoll erscheint.The invention also relates to the use of the device according to the invention for the uniform distribution of microparticles in a liquid. In other words, the invention also relates to the use of the device according to the invention for obtaining a suspension of uniformly distributed microparticles in a carrier liquid and / or for maintaining a uniform distribution of microparticles in a carrier liquid. The invention further relates to the use of the device according to the invention for taking a suspension of uniformly distributed microparticles in a liquid, preferably with a liquid extraction robot. The prior teaching of the invention and its embodiments relating to the device per se are valid and applicable without restriction to their use, if appropriate.

In einer Ausgestaltung der erfindungsgemäßen Verwendung wird die Vorrichtung zur Identifizierung, Charakterisierung und/oder Aufreinigung von Proteinen eingesetzt. Proteine sind häufig an der Entstehung schwerwiegender Krankheiten, wie z. B. Krebs oder Alzheimer beteiligt. Bei der Analyse von Proteinen haben Mikropartikel mit einem Durchmesser von wenigen Mikrometern bis 0,1 mm eine zunehmende Bedeutung. Deren Oberfläche hat verschiedene reaktive Eigenschaften, welche die Identifizierung und Charakterisierung von Proteinen ermöglichen. Des Weiteren stellt die Laborautomatisierung auf dem Gebiet der Charakterisierung und Identifizierung von Proteinen einen wachsenden Markt dar, so dass zur effektiven Gestaltung der Mikropartikel-gestützten Proteomforschung Hochdurchsatzroboter unentbehrlich sind. Die erfindungsgemäße Vorrichtung ist so konstruiert, dass ein Liquid-Handling-Roboter mittels Entnahmenadeln die konstant verteilte Suspension aus den Wells (Kavitäten 4) eines Mischkopfs 2, vorzugsweise aus einem Mischkopf mit acht Wells, entnehmen kann. Damit ist eine Vorschaltung der Vorrichtung an Hochdurchsatzroboter möglich. Die erfindungsgemäße Verwendung der Vorrichtung ist vorzugsweise zur Bereitstellung von konstant verteilten Mikropartikelmengen zur Durchführung von automatisierten Protein-Aufreinigungsprozessen mittels Chromatographie im 96-Well-Batch-Fomat geeignet.In one embodiment of the use according to the invention, the device is used for the identification, characterization and / or purification of proteins. Proteins are often involved in the development of serious diseases such. As cancer or Alzheimer's involved. In the analysis of proteins, microparticles with a diameter of a few micrometers to 0.1 mm are becoming increasingly important. Their surface has several reactive properties that allow the identification and characterization of proteins. Furthermore, the laboratory automation in the field of Characterization and identification of proteins is a growing market, so that high-throughput robots are indispensable for the effective design of microparticle-based proteomics research. The device according to the invention is constructed in such a way that a liquid handling robot can remove the constantly distributed suspension from the wells (cavities 4) of a mixing head 2, preferably from a mixing head with eight wells, by means of removal needles. This makes it possible to connect the device to high-throughput robots. The use of the device according to the invention is preferably suitable for providing constantly distributed quantities of microparticles for carrying out automated protein purification processes by means of chromatography in the 96-well batch format.

In einer weiteren bevorzugten Ausgestaltung der Verwendung wird die Vorrichtung der vorliegenden Erfindung bei der Aktivitätsbestimmung von Enzymen mittels Massenspektrometrie genutzt. Dass die Massenspektrometrie ein schnelles, sensitives und zuverlässiges Werkzeug für die Bestimmung enzymatischer Aktivitäten ist, konnte wiederholt demonstriert werden ( Hsieh et al. 1995, Anal. Biochem. 229, 20 ; Bothner et al. 2000, J. Biol. Chem. 275, 13455 ; Wu et al. 1997, Chem. Biol. 4, 653 ). MALDI-MS zeichnet sich durch Widerstandsfähigkeit gegenüber Pufferlösungen und eine hohe Eignung zur Analyse komplexer Mischungen aus, weswegen sie für ein direktes Screening von Enzymaktivitäten prädestiniert ist, die nur eine minimale Probenvorbereitung benötigen. Mittels eines massenspektrometrisch-unterstützten Enzymscreenings ("mass spectrometry assisted enzyme screening", MES) können enzymatische Aktivitäten in komplexen Proteinfraktionen mit einem Massenspektrometer ermittelt werden. Das Prinzip wird hier kurz beschrieben, während sich eine eingehende Beschreibung in den Artikeln von Jankowski et al. 2001, Anal. Biochem. 290, 324 , sowie Schlüter et al. 2003, Anal. Bioanal. Chem. 37, 1102 , findet, die beide in ihrer Gesamtheit in die vorliegende Anmeldung als Referenz aufgenommen werden. Dem analytischen Vorgehen liegt die kovalente Immobilisierung von Proteinen an Mikropartikel zugrunde, wodurch der proteolytischer Abbau verhindert und das Entfernen solcher Moleküle aus der Proteinfraktion erreicht wird, die den massenspektrometrischen Nachweis der enzymatischen Reaktionsprodukte stören würden. Die Enzymaktivität wird durch Inkubieren der immobilisierten Proteine mit einer reaktionsspezifischen Sonde bestimmt, worauf die Analyse der Reaktionsmischung mittels MALDI-MS nach definierten Inkubationszeiten erfolgt. Erst die Verwendung der erfindungsgemäßen Vorrichtung stellt eine konstant verteilte Suspension an Mikropartikel sicher, infolgedessen eine gleichmäßige Oberflächenbeladung und konstante Proteinkonzentration sowie die Entnahme einer identischen Mikropartikelmenge zur Inkubation mit der Sonde ermöglicht wird und reproduzierbare Daten generiert werden.In a further preferred embodiment of the use, the device of the present invention is used in the activity determination of enzymes by mass spectrometry. It has been repeatedly demonstrated that mass spectrometry is a fast, sensitive and reliable tool for the determination of enzymatic activities ( Hsieh et al. 1995, Anal. Biochem. 229, 20 ; Bothner et al. 2000, J. Biol. Chem. 275, 13455 ; Wu et al. 1997, Chem. Biol. 4, 653 ). MALDI-MS is characterized by its resistance to buffer solutions and its ability to analyze complex mixtures, making it predestined for direct screening of enzyme activities that require minimal sample preparation. Enzymatic activities in complex protein fractions can be determined with a mass spectrometer using mass spectrometry-assisted enzyme screening (MES). The principle is briefly described here while getting a detailed description in the articles by Jankowski et al. 2001, anal. Biochem. 290, 324 , such as Schlüter et al. 2003, anal. Bioanal. Chem. 37, 1102 , both of which are incorporated by reference in their entirety into the present application. The analytical procedure is based on the covalent immobilization of proteins on microparticles, which prevents proteolytic degradation and the removal of such molecules from the protein fraction is achieved mass spectrometric detection of the enzymatic reaction products would interfere. The enzyme activity is determined by incubating the immobilized proteins with a reaction-specific probe, followed by analysis of the reaction mixture by MALDI-MS after defined incubation times. Only the use of the device according to the invention ensures a constantly distributed suspension of microparticles, as a result, a uniform surface loading and constant protein concentration and the removal of an identical amount of microparticles for incubation with the probe is made possible and reproducible data is generated.

Ein anderer Gegenstand der Erfindung ist ein Verfahren zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit, in dem die erfindungsgemäße Vorrichtung eingesetzt wird.Another object of the invention is a method for uniform distribution of microparticles in a liquid in which the device according to the invention is used.

Die Erfindung lehrt ferner ein Verfahren zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit mit den folgenden Schritten:

  1. (a) Befüllen einer Vorratskammer 1 mit einer Suspension aus Mikropartikeln und Flüssigkeit,
  2. (b) Ansaugen der Suspension mit einer Pumpe 3 über eine Absaugung 17 aus der Vorratskammer 1,
  3. (c) Transportieren der angesaugten Suspension in einen Mischkopf 2, der mindestens eine Kavität 4 aufweist,
  4. (d) Überführen der Suspension in die Kavität 4,
  5. (e) Erzeugen einer turbulenten Strömung in der Kavität 4,
  6. (f) Überführen von Suspension oberhalb eines Überlaufs 6 durch eine Öffnung 7 in die Vorratskammer 1, und optional
  7. (g) Wiederholen der Schritte (b) bis (f)
The invention further teaches a method for uniformly distributing microparticles in a liquid, comprising the following steps:
  1. (a) filling a storage chamber 1 with a suspension of microparticles and liquid,
  2. (b) sucking the suspension with a pump 3 via an exhaust 17 from the storage chamber 1,
  3. (c) transporting the aspirated suspension into a mixing head 2, which has at least one cavity 4,
  4. (d) transferring the suspension into the cavity 4,
  5. (e) generating a turbulent flow in the cavity 4,
  6. (f) transferring suspension above an overflow 6 through an opening 7 into the storage chamber 1, and optionally
  7. (g) repeating steps (b) to (f)

Dadurch gekennzeichnet, dass parallel zu den Schritten (b) bis (g) ein Kreislauf mit den folgenden Schritten gefahren:

  • (b') Ansaugen der Suspension mit der Pumpe 3 über einen Rotationsrücklauf 16 aus der Vorratskammer 1,
  • (c') Transportieren der angesaugten Suspension in einer Rotationsschleife,
  • (d') Überführen der Suspension über einen Rotationszulauf 15 in die Vorratskammer 1,
  • (e') Erzeugen einer turbulenten Strömung in der Vorratskammer 1, und optional Wiederholen der Schritte (b') bis (e').
Characterized in that in parallel to the steps (b) to (g) a cycle with the following steps:
  • (b ') sucking the suspension with the pump 3 via a rotary return 16 from the storage chamber 1,
  • (c ') transporting the sucked suspension in a rotation loop,
  • (d ') transfer of the suspension via a rotary feed 15 into the storage chamber 1,
  • (e ') generating a turbulent flow in the storage chamber 1, and optionally repeating the steps (b') to (e ').

Die vorherige Lehre der Erfindung und deren Ausführungsformen betreffend die Vorrichtung und deren Verwendung sind gültig und ohne Einschränkungen auf die Verfahren zur gleichmäßigen Verteilung von Mikropartikeln anwendbar, sofern es sinnvoll erscheint.The prior teaching of the invention and its embodiments relating to the device and the use thereof are valid and applicable without limitation to the methods for the uniform distribution of microparticles, if it makes sense.

Die Abfolge der Schritte verdeutlicht, dass das Verfahren im Kreislauf gefahren wird, wofür jeweils zwei Öffnungen der Vorratskammer 1 bzw. des Mischkopfs 2 essentiell sind, die jeweils voneinander verschieden sind. Das heißt, dass sich in der Vorratskammer 1 die Absaugung 17 zum Absaugen der Suspension in Richtung Mischkopf 2 und die Öffnung 7 zur Aufnahme aus dem Mischkopf 2 übergelaufener Suspension sowie in der Kavität 4 im Mischkopfs 2 der Zustrom 13 der Suspension aus der Vorratskammer 1 und der Überlauf 6 zur Rückführung der Suspension in die Vorratskammer 1 befinden. Schritt (g) wird solange ausgeführt, bis die Vorratskammer 1 leer oder keine Durchmischung der Partikel mehr erwünscht ist, weil die Entnahme und/oder Analyse der Partikelsuspension beendet ist.The sequence of steps illustrates that the process is circulated, for which in each case two openings of the storage chamber 1 or the mixing head 2 are essential, which are each different from each other. That is, in the storage chamber 1, the suction 17 for sucking the suspension in the direction of mixing head 2 and the opening 7 for receiving from the mixing head 2 overflowed suspension and in the cavity 4 in the mixing head 2, the inflow 13 of the suspension from the storage chamber 1 and the overflow 6 to return the suspension in the storage chamber 1 are located. Step (g) is carried out until the storage chamber 1 is empty or no mixing of the particles is desired, because the removal and / or analysis of the particle suspension is completed.

Die turbulente Strömung in Schritt (e) kann durch jede Maßnahme bewirkt werden, die eine Reynolds-Zahl über dem kritischen Wert von 2300 zur Folge hat. Geeignete Maßnahmen sind insbesondere die Erhöhung der Strömungsgeschwindigkeit der Suspension, der Oberflächenrauhigkeit der Kavität 4 und/oder der Dichte der Suspension und/oder die Verringerung der dynamischen Viskosität. In einer bevorzugten Ausgestaltung des Verfahrens wird die turbulente Strömung durch den Einbau eines Strömungsverwirblers 5 erzeugt, besonders bevorzugt durch den Einbau mindestens einer unstetigen Vergrößerung des Querschnitts in einem Erweiterungsbereich 13 oberhalb eines Zustrombereichs 12.The turbulent flow in step (e) can be effected by any measure that results in a Reynolds number above the critical value of 2300. Suitable measures are in particular the increase of the flow rate of the suspension, the surface roughness of the cavity 4 and / or the density of the suspension and / or the reduction of the dynamic viscosity. In a preferred embodiment of the method, the turbulent flow is generated by the installation of a Strömungsverwirblers 5, more preferably by the incorporation of at least one discontinuous increase in the cross-section in an extension region 13 above a Zustrombereichs 12th

Das Verfahren kann auch so durchgeführt werden, dass sich nach Schritt (e), (f) oder (g) ein weiterer Schritt (h) anschließt, in dem die Suspension von einem Flüssigkeitsentnahmeroboter entnommen wird.The method can also be carried out in such a way that, after step (e), (f) or (g), a further step (h) is followed, in which the suspension is removed from a fluid extraction robot.

Im Rahmen der vorliegenden Erfindung werden also erstmalig eine Vorrichtung und ein Verfahren zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit bereitgestellt. Die Erfindung nutzt die Ausgestaltung einer durchströmten Entnahmekavität 4, um auf einfachem und zuverlässigem Wege Turbulenz zu erzeugen, die wiederum eine konstante Durchmischung von Mikropartikeln in einer Suspension nach sich zieht. Die Trennung von Vorratskammer 1 und Mischkopf 2 ermöglicht dabei vorteilhaft eine gleichmäßige Verteilung der Mikropartikel am Ort der Partikelentnahme bzw. -analyse (Kavität 4). Dieses System wird erfindungsgemäß gekoppelt mit einem zweiten Kreislauf, der dafür sorgt, dass die Verteilung der Mikropartikel über einen längeren Zeitraum homogen bleibt bzw. sowohl zu Beginn, als auch zu einem Zeitpunkt zu dem die Vorrichtung schon einige Zeit in Betrieb gewesen ist. Darüber hinaus wird eine parallele Probenverarbeitung gewährleistet, indem der Mischkopf 2 eine Vielzahl an Kavitäten 4 besitzt, die zusammen angesteuert eine identische Durchmischung der Mikropartikel zeigen und folglich einer automatisierten Mikropartikelmengenbemessung zugänglich sind. Während im Stand der Technik lediglich eine Durchströmung mit Gas beschrieben ist, wird hier die Strömung der Suspension selbst ausgenutzt. Aufgrund der überraschenden Kombination dieser Verwirbelung mit einem Überlauf 6 bildet sich ein Flüssigkeitskreislauf, der in den Kavitäten 4 zu einem quasistatischen Zustand führt. Vorrichtung und Verfahren der Erfindung zeichnen sich durch eine einfache und kostengünstige Handhabung aus und eröffnen eine Vielzahl von Anwendungsperspektiven, von denen insbesondere die Biochemie genannt sei.In the context of the present invention, therefore, an apparatus and a method for the uniform distribution of microparticles in a liquid are provided for the first time. The invention uses the design of a flow-through sampling cavity 4 in order to generate turbulence in a simple and reliable way, which in turn entails a constant mixing of microparticles in a suspension. The separation of storage chamber 1 and mixing head 2 advantageously allows a uniform distribution of the microparticles at the location of particle removal or analysis (cavity 4). According to the invention, this system is coupled to a second circuit which ensures that the distribution of the microparticles remains homogeneous over a relatively long period of time or both at the beginning and at a time when the device has already been in operation for some time. In addition, a parallel sample processing is ensured by the mixing head 2 has a plurality of cavities 4, which together drive an identical mixing of the microparticles show and thus an automated microparticle quantity measurement are accessible. While only a gas flow is described in the prior art, the flow of the suspension itself is exploited here. Due to the surprising combination of this turbulence with an overflow 6, a liquid circuit is formed, which leads to a quasi-static state in the cavities 4. Apparatus and method of the invention are characterized by a simple and inexpensive handling and open up a variety of application perspectives, of which biochemistry in particular may be mentioned.

Es versteht sich, dass diese Erfindung nicht auf die spezifischen Methoden, Zusammensetzungen und Bedingungen beschränkt ist, wie sie hierin beschrieben sind, da solche Dinge variieren können. Es versteht sich des Weiteren, dass die vorliegend verwendete Terminologie ausschließlich dem Zweck der Beschreibung besonderer Ausführungsformen dient und nicht den Schutzumfang der Erfindung einschränken soll. Wie vorliegend in der Spezifikation einschließlich der anhängigen Ansprüche verwendet, schließen Wortformen im Singular, wie z. B. "ein", "eine", "einer", "der" oder "das" die Entsprechung im Plural ein, sofern der Kontext nicht eindeutig etwas anderes vorgibt. Beispielsweise enthält der Bezug auf "einen Strömungsverwirbler 5" einen einzelnen Verwirbler oder mehrere Verwirbler, die wiederum identisch oder verschieden sein können, oder der Bezug auf "ein Verfahren" schließt äquivalente Schritte und Verfahren ein, die dem Fachmann bekannt sind.It should be understood that this invention is not limited to the specific methods, compositions and conditions described herein since such things may vary. It is further understood that the terminology used herein is for the purpose of description only particular embodiments and is not intended to limit the scope of the invention. As used herein in the specification including the appended claims, word forms in the singular, such as words, include For example, "a", "an", "an", "the" or "that" are plural equivalents unless the context clearly dictates otherwise. For example, the reference to "a flow swirler 5" includes a single swirler or multiple swirlers, which in turn may be identical or different, or the reference to "one method" includes equivalent steps and methods known to those skilled in the art.

Im Folgenden wird die Erfindung anhand von nicht limitierenden Beispielen für konkrete Ausführungsformen näher erläutert.

  • Fig. 1 zeigt eine schematische Zeichnung der Komponenten der Vorrichtung zur gleichmäßigen Verteilung von Mikropartikeln in einer Flüssigkeit ohne den Rotationskreislauf.
  • Fig. 2 zeigt eine Kavität 4 zur Entnahme von Suspension: a) schematisch, und b) im Strömungsprofil.
  • Fig. 3 zeigt eine schematische Zeichnung der Suspensionsmischer-Zylinderkammer einschließlich des Rotationskreislaufs mit den Konstruktionselementen, welche die Strömungskreisläufe verdeutlichen.
  • Fig. 4 zeigt ein CFD der Vorratskammer 1.
  • Fig. 5 zeigt den Prototyp des Gesamtsystems.
In the following, the invention will be explained in more detail with reference to non-limiting examples of specific embodiments.
  • Fig. 1 shows a schematic drawing of the components of the device for uniform distribution of microparticles in a liquid without the rotation circuit.
  • Fig. 2 shows a cavity 4 for the removal of suspension: a) schematically, and b) in the flow profile.
  • Fig. 3 shows a schematic drawing of the suspension mixer cylinder chamber including the rotation circuit with the structural elements, which illustrate the flow circuits.
  • Fig. 4 shows a CFD of the storage chamber. 1
  • Fig. 5 shows the prototype of the whole system.

BEISPIELEXAMPLE

Der Prototyp des Gesamtsystems, wie er in Fig. 5 dargestellt ist, enthält als Energiequelle 9 ein 12 V Gleichspannungsnetzteil, welches das Gesamtsystem mit elektrischer Energie versorgt. Durch die Energiequelle wird die Pumpe 3 angetrieben, bei der es sich um eine Schlauchpumpe des Typs Watson Marlow 102R handelt, die mit einem Faulhaber-Motor 3540K024C angetrieben wird. Pumpe 3 und Motor werden über einen Schalter in Betrieb genommen.The prototype of the overall system, as in Fig. 5 is shown, as a power source 9, a 12 V DC power supply, which is the overall system supplied with electrical energy. The power source powers pump 3, which is a Watson Marlow 102R peristaltic pump powered by a Faulhaber 3540K024C motor. Pump 3 and motor are put into operation via a switch.

Wie aus Fig. 1 ersichtlich wird, sind Pumpe 3 und Motor zum Schutz vor äußeren Einflüssen mit einem Deckel 18 verkleidet, der mit Schrauben DIN 912 (M4x10) an einer Flanschplatte 19 befestigt ist. Der Schraubentyp wird auch für alle weiteren Befestigungen genutzt. Sowohl die Pumpe 3 als auch die Suspensionsmischer-Zylinderkammer sind auf eine Grundplatte 20 montiert. Die Suspensionsmischer-Zylinderkammer umfasst den Vorratskammer-Sockel 21, Mischkopf 2, ZylinderStift 14 Typ DIN 7 (3x15) und vier Verbindungselemente Typ Connector M6 250-6. Die Suspensionsmischer-Zylinderkammer ist am Vorratskammer-Sockel 21 mit der Grundplatte 20 verschraubt. Der Sockel 21 hat im Zentrum eine zylinderförmige Vorratskammer 1 und oberhalb davon ist ein 8-Well-Mischkopf 2 verschraubt.How out Fig. 1 it can be seen, pump 3 and motor are covered to protect against external influences with a cover 18 which is fastened with screws DIN 912 (M4x10) to a flange plate 19. The screw type is also used for all other fasteners. Both the pump 3 and the suspension mixer cylinder chamber are mounted on a base plate 20. The suspension mixer cylinder chamber comprises the pantry base 21, mixing head 2, cylinder pin 14 type DIN 7 (3x15) and four connectors type connector M6 250-6. The suspension mixer cylinder chamber is screwed to the storage chamber base 21 with the base plate 20. The base 21 has a cylindrical storage chamber 1 in the center and above it, an 8-well mixing head 2 is screwed.

In Fig. 2 wird deutlich, dass die Wells oder Entnahmekavitäten 4 derart geformt sind, dass der geförderte Zustrom auf Grund einer unstetigen Rohrerweiterung im Eingangsbereich (Erweiterungsbereich 13) turbulent wird. Auf diese Weise wird die Suspension in der Kavität 4 verwirbelt und durchmischt. Durch die Entnahme der Suspension durch den Liquid-Handling-Roboter wird das Gesamtvolumen in dem System verringert. Um für die Entnahme von Suspension einen konstant hohen Flüssigkeitspegel zu erreichen, weisen die Kavitäten 4 einen Überlauf 6 auf.In Fig. 2 It can be seen that the wells or withdrawal cavities 4 are shaped in such a way that the conveyed inflow becomes turbulent due to a discontinuous tube expansion in the entry area (extension area 13). In this way, the suspension in the cavity 4 is vortexed and mixed. The removal of the suspension by the liquid handling robot reduces the total volume in the system. In order to achieve a constant high liquid level for the removal of suspension, the cavities 4 have an overflow 6.

Die Pumpe fördert die Suspension in zwei parallelen Kreisläufen (Fig. 3). Der erste Kreislauf versorgt die Entnahmekavitäten 4 (Fig. 2a, 3). Die Suspension wird an einer zylinderförmigen Absaugung 17 angesaugt und durch Schläuche 11 geleitet. Durch ein Steigrohr 22 gelangt die Suspension in den Mischkopf 2 und wird mittels eines Leitungssystems in den Zustrombereich 12 der einzelnen Kavitäten 4 transportiert. Die überlaufende Flüssigkeit fließt durch die Öffnung 7 direkt in die Vorratskammer 1. Es handelt sich also um einen Flüssigkeitskreislauf, bei dem in der Kavität 4 ein quasistatischer Zustand erzeugt wird, aus dem konstant verteilte Suspension entnommen wird.The pump delivers the suspension in two parallel circuits ( Fig. 3 ). The first circuit supplies the withdrawal cavities 4 ( Fig. 2a . 3 ). The suspension is sucked on a cylindrical suction 17 and passed through hoses 11. Through a riser 22, the suspension enters the mixing head 2 and is transported by means of a conduit system in the inflow region 12 of the individual cavities 4. The overflowing liquid flows through the opening 7 directly into the storage chamber 1. So it is a liquid circulation, in which the cavity 4 a quasi-static state is generated, is removed from the constantly distributed suspension.

Der zweite Kreislauf sorgt für eine Rotation der gespeicherten Suspension in der Vorratskammer 1 (Fig. 3, 4). Hierzu wird Suspension durch einen Rotationsrücklauf 16 angesaugt, durch eine Rotationsschleife in Schläuchen 11 transportiert und über einen Rotationszulauf 15 wieder in die Vorratskammer 1 gegeben. Die Vorratskammer 1 weist einen zylinderförmigen Stift 14 auf, der die rotierende Strömung derart verwirbelt, dass sich im Zentrum der Kammer keine Beads ablagern können (ähnlich einer Teetasse mit Teekrümeln). Im Totwasser des Stifts befindet sich die Absaugung 17, die durch das Steigrohr 22 die Entnahmekavitäten 4 speist.The second circuit ensures rotation of the stored suspension in the storage chamber 1 (FIG. Fig. 3, 4 ). For this purpose, suspension is sucked through a rotary return 16, transported by a rotation loop in hoses 11 and returned to the storage chamber 1 via a rotary feed 15. The storage chamber 1 has a cylindrical pin 14 which swirls the rotating flow in such a way that no beads can deposit in the center of the chamber (similar to a teacup with tea crumbs). In the dead water of the pen is the suction 17, which feeds the removal cavities 4 through the riser 22.

BEZUGSZEICHENLISTELIST OF REFERENCE NUMBERS

11
Vorratskammerstoreroom
22
Mischkopfmixing head
33
Pumpepump
44
Kavitätcavity
55
Strömungsverwirblerflow turbulator
66
Überlaufoverflow
77
Öffnungopening
88th
Schnittstelleinterface
99
Energiequelleenergy
1010
Laborsystemlaboratory system
1111
Schläuchehoses
1212
Zustrombereichinflow region
1313
Erweiterungsbereichextension area
1414
Stiftpen
1515
Rotationszulaufrotary feed
1616
RotationsrücklaufRotary runback
1717
Absaugungsuction
1818
Deckelcover
1919
Flanschplatteflange
2020
Grundplattebaseplate
2121
Sockelbase
2222
Steigrohrriser

Claims (14)

  1. An apparatus for uniformly distributing microparticles in a liquid, said apparatus comprising a storage chamber (1) including a suspension of microparticles and liquid, a mixing head (2) for receiving the suspension, and a pump (3) which is connected to the storage chamber (1) and the mixing head (2) for sucking the suspension from the storage chamber (1) into the mixing head (2), said mixing head (2) having at least one well (4) with a flow turbulator (5) and an overflow (6) which is connected to the storage chamber (1) via an opening (7),
    characterized in that
    the storage chamber (1) has a rotary inlet (15) and a rotary runback (16) connected to the pump (3), so that a second, parallel circulation of suspension is generated.
  2. The apparatus as claimed in claim 1, characterized in that the storage chamber additionally has a pin (14).
  3. The apparatus as claimed in claim 1 or 2, characterized in that the flow turbulator (5) is at least one discontinuous enlargement of the cross-section in a widening zone (13) above an inflow zone (12).
  4. The apparatus as claimed in claim 3, characterized in that the cross-section in the widening zone (13) increases at least twofold, preferably at least fivefold and more preferably at least tenfold.
  5. The apparatus as claimed in claim 3 or 4, characterized in that the cross-section in the widening zone (13) increases linearly, preferably with a tansy slope of less than 70°, more preferably less than 50°, especially preferably less than 30°.
  6. The apparatus as claimed in any of the preceding claims, characterized in that the well (4) has a rotationally symmetric cross-section and diverges conically in the widening zone (13).
  7. The apparatus as claimed in any of the preceding claims, characterized in that the aspirated suspension in well (4) forms a constant-level interface (8) to a liquid withdrawal robot.
  8. The apparatus as claimed in any of the preceding claims, characterized in that the mixing head (2) has eight wells (4).
  9. Use of an apparatus as claimed in any of claims 1 to 8 for the uniform distribution of microparticles in a liquid.
  10. Use of an apparatus as claimed in any of claims 1 to 8 for the withdrawal of a suspension with uniformly distributed microparticles in a liquid.
  11. The use as claimed in claim 10 for the identification, characterization and/or purification of proteins, preferably for the enzyme activity determination by means of mass spectrometry.
  12. A method for the uniform distribution of microparticles in a liquid, said method including the following steps:
    (a) filling a storage chamber (1) with a suspension of microparticles and liquid,
    (b) aspirating the suspension from the storage chamber (1) with a pump (3) via a suction means (17),
    (c) conveying the aspirated suspension into a mixing head (2) which has at least one well (4),
    (d) transferring the suspension into the well (4),
    (e) generating a turbulent flow in the well (4),
    (f) transferring the suspension above an overflow (6) through an opening (7) into the storage chamber (1), and optionally
    (g) repeating the steps (b) through (f),
    characterized in that a circulation parallel to steps (b) through (g) is conducted, including the following steps:
    (b') aspirating the suspension from the storage chamber (1) with a pump (3) via a rotary runback (16),
    (c') conveying the aspirated suspension in a rotary loop,
    (d') transferring the suspension into the storage chamber (1) via a rotary inlet (15),
    (e') generating a turbulent flow in the storage chamber (1), and optionally repeating the steps (b') through (e').
  13. The method as claimed in claim 12, characterized in that the turbulent flow in step (e) is generated by fitting a flow turbulator (5), preferably by fitting at least one discontinuous enlargement of the cross-section in a widening zone (13) above an inflow zone (12).
  14. The method as claimed in claim 12 or 13, characterized in that step (e), (f) or (g) is followed by an additional step:
    (h) withdrawal of suspension by a liquid sampling robot.
EP08804783A 2007-09-27 2008-09-26 Apparatus and method for the uniform distribution of microparticles in a liquid Not-in-force EP2203246B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007047478A DE102007047478A1 (en) 2007-09-27 2007-09-27 Apparatus and method for uniformly distributing microparticles in a liquid
PCT/EP2008/062905 WO2009043813A1 (en) 2007-09-27 2008-09-26 Apparatus and method for the uniform distribution of microparticles in a liquid

Publications (2)

Publication Number Publication Date
EP2203246A1 EP2203246A1 (en) 2010-07-07
EP2203246B1 true EP2203246B1 (en) 2011-05-25

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Application Number Title Priority Date Filing Date
EP08804783A Not-in-force EP2203246B1 (en) 2007-09-27 2008-09-26 Apparatus and method for the uniform distribution of microparticles in a liquid

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US (1) US20110026356A1 (en)
EP (1) EP2203246B1 (en)
AT (1) ATE510612T1 (en)
DE (1) DE102007047478A1 (en)
WO (1) WO2009043813A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012127257A1 (en) 2011-03-19 2012-09-27 Dental Care Innovation Gmbh System for dissolution of a tablet or granulate in a stream of water
USD825741S1 (en) 2016-12-15 2018-08-14 Water Pik, Inc. Oral irrigator handle

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3105778A (en) * 1959-06-12 1963-10-01 Kaiser Aluminium Chem Corp Heating and mixing methods
DE1275512B (en) * 1962-01-08 1968-08-22 Willi Walbersdorf Sondermaschb Mixing device
GB1189162A (en) * 1966-05-03 1970-04-22 Inst Wlokien Sztucznych I Synt A Process and Apparatus for Dissolving a Solid in a Liquid Solvent therefor
GB1183887A (en) * 1968-08-13 1970-03-11 British Motor Corp Ltd Slurry Treatment Plant
US4211733A (en) * 1978-12-26 1980-07-08 Chang Shih Chih Gas-liquid mixing process and apparatus
DE3844174A1 (en) * 1988-12-29 1990-07-05 Fresenius Ag Plant for the production of concentrates by mixing liquid with soluble solids
DE69011679T2 (en) * 1989-04-21 1995-02-16 Techno Bio Kk LIQUID EMULSION WITHOUT EMULSIFIER AND METHOD AND DEVICE FOR PRODUCING THE EMULSION.
US5252296A (en) 1990-05-15 1993-10-12 Chiron Corporation Method and apparatus for biopolymer synthesis
GB2285588B (en) * 1994-01-17 1997-04-30 Ea Tech Ltd Method and apparatus for mixing a metal matrix composite
US6255166B1 (en) 1999-08-05 2001-07-03 Aalo Lsi Design & Device Technology, Inc. Nonvolatile memory cell, method of programming the same and nonvolatile memory array
DE60123844T2 (en) * 2001-04-27 2007-08-30 Kansai Paint Co., Ltd., Amagasaki METHOD AND DEVICE FOR DISPERSING DYES IN A LIQUID MEDIUM
US6881363B2 (en) * 2001-12-07 2005-04-19 Symyx Technologies, Inc. High throughput preparation and analysis of materials
CN100594382C (en) * 2004-03-23 2010-03-17 松下电器产业株式会社 Stirring method, reaction vessel, measuring apparatus using the reaction vessel, and measuring method
US7763453B2 (en) 2005-11-30 2010-07-27 Micronics, Inc. Microfluidic mixing and analytic apparatus

Also Published As

Publication number Publication date
EP2203246A1 (en) 2010-07-07
WO2009043813A1 (en) 2009-04-09
US20110026356A1 (en) 2011-02-03
DE102007047478A1 (en) 2009-04-16
ATE510612T1 (en) 2011-06-15

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