EP2928605B1 - Récipient de réaction à fermeture magnétique - Google Patents

Récipient de réaction à fermeture magnétique Download PDF

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Publication number
EP2928605B1
EP2928605B1 EP13805819.3A EP13805819A EP2928605B1 EP 2928605 B1 EP2928605 B1 EP 2928605B1 EP 13805819 A EP13805819 A EP 13805819A EP 2928605 B1 EP2928605 B1 EP 2928605B1
Authority
EP
European Patent Office
Prior art keywords
reaction vessel
subdivision
state
magnetic field
movable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Not-in-force
Application number
EP13805819.3A
Other languages
German (de)
English (en)
Other versions
EP2928605A1 (fr
Inventor
Federico Buersgens
Joachim Stehr
Lars Ullerich
Tobias Andres
Matthias Graessel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GNA Biosolutions GmbH
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GNA Biosolutions GmbH
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Filing date
Publication date
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Publication of EP2928605A1 publication Critical patent/EP2928605A1/fr
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Publication of EP2928605B1 publication Critical patent/EP2928605B1/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/049—Valves integrated in closure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts

Definitions

  • the invention relates to a reaction vessel having a first and a second subdivision, wherein in a first state of the reaction vessel, the first is separated from the second subdivision and in a second state, the first is connected to the second subdivision. Furthermore, the use of the reaction vessel in a method for the detection of proteins and / or nucleic acids and in a method for the amplification of nucleic acids is part of the invention. The invention further includes a method for converting the one into the other of the two states of the reaction vessel.
  • the patent application WO 00/12675 describes an apparatus having a first hollow elongate cylinder with a closed end and therein a plurality of chambers, a second hollow elongated cylinder internally positioned contiguously adjacent to the first cylinder and capable of relative rotation.
  • a closed position A a sample is introduced into the second cylinder.
  • the second cylinder contains reagents for extraction of nucleic acids.
  • the second cylinder is rotated to an open position B.
  • the extracted nucleic acid remains in a solid state in an upper chamber as the fluid flows into a reservoir.
  • the second cylinder is rotated to the closed position A so that the second cylinder is sealed, water is added, and a cover is closed.
  • a reaction bead from one chamber is added to the second cylinder.
  • the reaction bead contains those for amplification necessary enzymes that are resuspended in the water.
  • the amplification takes place on the solid phase, which contains the extracted nucleic acids.
  • the second cylinder is rotated relative to the first cylinder to a washout position C.
  • the reaction mixture of the amplification may enter the detection chamber and is absorbed by a pad.
  • the reaction mixture is absorbed by a strip.
  • colored microparticles bind to the haptens from the amplification reaction and move to the capture zone on a membrane where they form a visible detection line if the target sequence is present.
  • the patent application WO 2006/050636 A1 describes a PCR reaction tube having a tubular body and a cap, wherein the inner surface of the cap is provided with an inner tube and the inner tube is provided with at least one hole.
  • the reaction vessel is suitable for performing nested PCR, whereby reagents for the nested inner part of the PCR in the inner tube can be provided. After the first part of the PCR is complete, the reaction vessel can be centrifuged or exposed to vibration so that the reagents are released from the inner tube. Thereafter, the second part of the PCR can be performed.
  • the U.S. Patent 5,576,197 discloses a reaction vessel for PCR in which wax is applied to the inner surface.
  • the wax is at a position that is approximately at the height of the meniscus of a filled PCR mix.
  • the wax does not completely close the volume of the vessel.
  • the wax is solid at temperatures below 40 ° C and liquid at PCR temperatures. When the wax liquefies, it spontaneously covers the surface of the PCR mix.
  • the wax is present in an amount sufficient so that when the wax is liquefied, it completely covers the surface of the PCR mix.
  • the molten fat or wax creates a vapor barrier to minimize evaporation of the solution during the thermal PCR cycles and, upon cooling, after the amplification is complete, again forms a solid barrier that, inter alia, reduces the likelihood of contamination of the following reactions.
  • the patent application WO 2008/047272 A2 discloses an apparatus suitable for use in the amplification and detection of nucleic acids.
  • the device comprises a simple sample tube having a chamber which is preferably open at the top so that its contents flow out when the sample tube is turned upside down.
  • the sample tube further comprises a lid containing a microarray for nucleic acids to which amplified samples can hybridize DNA.
  • a bio-valve thin-film device which comprises a plurality of chambers, for example a preparation chamber, a buffer chamber, a reaction chamber and a waste chamber. Between the chambers arranged magnetic valves can be opened and closed by the force of a movable permanent magnet.
  • the patent US 3,424,820 discloses a method in which two substances are mixed together by the action of a magnetic field. In a first state, these substances are separated by a liquid having a certain density. A perforator can be moved by means of magnetic fields in such a way that the separating liquid perforated and as a result, the two substances are mixed together.
  • the patent application US 2006 0116270 A1 discloses a system for maintaining a liquid separation comprising a liquid container, a solenoid-operated valve within the liquid container, and a centrifuge that positions the valve so that liquid can flow past the valve, while the centrifuge is running.
  • the valve contains an iron material that is placed in a plug.
  • the plug interacts with a solenoid of a valve actuator. This interaction causes the valve to be selectively opened and closed. When the valve is open, the flow of a sample around the valve is possible. When the valve is closed, the flow of the sample around the valve is blocked.
  • the invention has for its object to provide an improved reaction vessel and a use for such an improved reaction vessel.
  • an object of the invention is to provide a method using the improved reaction vessel available.
  • the object is achieved by a reaction vessel with the features of claims 1, 2 and 3.
  • the task is also solved by use of the reaction vessel in a process for purification, modification and / or detection of proteins and / or nucleic acids.
  • a further solution of the object according to the invention consists in a method for transferring one into the other of the two states of the reaction vessel.
  • the reaction vessel is a vessel capable of reacting in it.
  • the reaction vessel is closable, z. B. with a lid.
  • the preferred closed reaction vessel is liquid-tight with respect to its environment, particularly preferably it is gas-tight.
  • the reaction vessel can in principle assume any shape and size.
  • the reaction vessel is substantially cylindrical, circular cylindrical or conical, more preferably, the reaction vessel has both a substantially circular cylindrical portion and a substantially conical portion.
  • the reaction vessel z.T. plane walls, in particular e.g. a rectangular base, as in commercial cuvettes.
  • the volume of the reaction vessel is less than 500 ml, more preferably less than 100 ml, more preferably less than 50 ml, more preferably less than 25 ml, more preferably less than 10 ml, most preferably less than 5 ml, most preferably less than 2 , 5 ml, particularly preferably less than 1 ml, more preferably less than 0.5 ml and most preferably less than 0.25 ml.
  • the preferred volume of the reaction vessel is greater than 0.05 ml, more preferably greater than 0 , 1 ml.
  • the reaction vessel is at least partially, particularly preferably in its entirety made of one or more inert materials such as glass or plastic, preferably polyethylene, polypropylene or silicone.
  • the reaction vessel is preferably transparent, so that an optical inspection of the opening mechanism or the opening success is possible.
  • the reaction vessel has at least two subdivisions.
  • the first In the first state, the first is separated from the second subdivision, so that no liquid - preferably also no gases - can flow from one of the two subdivisions into the other.
  • the first In the second state, the first is connected to the second subdivision and liquids - preferably also gases - can flow from one of the two subdivisions into the other.
  • One subdivision may adjoin the other, one may comprise the other.
  • the partitions may also be spaced apart so that they do not abut each other.
  • the sealed reaction vessel encloses the subdivisions.
  • the subdivisions are fixed in the reaction vessel.
  • the subdivisions are connected. This can be done reversibly, so that the reaction vessel from the second can be converted back into the first state.
  • the second state is irreversibly produced, for. When a wall separating the first from the second partition is destroyed in the state change.
  • the reaction vessel may preferably have further subdivisions.
  • the further subdivisions may be in the first and / or second state with the first and / or the second subdivision and / or interconnected or separated from each other.
  • the reaction vessel may preferably assume further states.
  • one or more of the further subdivisions may preferably be connected to or separate from one or more of the further subdivisions and / or with the first and / or the second subdivision.
  • the first and / or second state can be converted into the other of the two states by the action of one magnetic field.
  • the first can be separated from the second subdivision or the first can be connected to the second subdivision.
  • the reaction vessel can be used in a process for the purification, modification and / or detection of proteins and / or nucleic acids.
  • a first reaction can take place in the first subdivision, which is initially separated from the second subdivision.
  • the first reaction may, for. Example, the proliferation of nucleic acids by PCR or the purification of nucleic acids or proteins from a sample.
  • the second reaction may, for. B. be a detection reaction to a specific nucleic acid or a specific protein. In this way, nucleic acids and proteins can be analyzed in a particularly simple and low-contamination manner.
  • the modification may be, for example, in a digestion of a sample or label of a protein or a nucleic acid.
  • the reaction vessel can be used according to the invention in a method for the amplification of nucleic acids.
  • a part of the reagents required for the duplication are present in the first subdivision.
  • the remaining part is preferably contained exclusively in the second subdivision.
  • the first partition may be connected to the second partition by the action of a magnetic field. Due to the separate storage of the two parts a higher shelf life of the reagents can be achieved.
  • the particular desired reaction conditions can be optimally adjusted by the targeted presence or absence and the concentration of the respective reagents in each of the two parts.
  • the invention further includes a method for changing the state of the reaction vessel.
  • the reaction vessel is exposed to a magnetic field.
  • a magnetic field In this case, either no magnetic field is present before exposure or a previously existing magnetic field is changed when exposed to alignment and / or field strength. This can be accomplished by moving the reaction vessel relative to an existing magnetic field.
  • a non-constant magnetic field may be used, e.g.
  • a magnetic field can be generated or changed by means of an electromagnet.
  • the invention it is advantageously possible to store two different liquids in two subdivisions of a reaction vessel without the liquids mixing.
  • one partition can be connected to the other so that the two liquids can mix with one another.
  • the abandonment of the opening of the reaction vessel for mixing the liquids can be the automation of Simplify reactions.
  • waxes are used to separate two liquids in a reaction vessel, which are heated to mix the liquids; as soon as the wax cools down and hardens, mixing is no longer possible or greatly impeded.
  • an inert material can be used to separate the liquids in the subdivisions, no mixing is necessary for mixing the liquids, whereby an impairment of reactions can be avoided.
  • the invention makes it possible to produce a structurally simple, inexpensive reaction vessel.
  • the first and second subdivisions are separated by a movable shutter.
  • the movable closure may in principle take any shape, but preferably the movable closure is spherical or cylindrical in shape.
  • the connection of the first with the second subdivision can be made reversible.
  • a separation of initially associated subdivisions can be brought about by a movement of the movable closure.
  • Movement of the movable shutter which leads to the change of state of the reaction vessel, may consist of a translatory or rotational movement or a combination of the two types of movement.
  • the movable shutter may consist of a single or multiple elements.
  • the movable shutter is a ball that closes off the first partition from the second partition.
  • a one-piece movable closure can advantageously be produced particularly easily.
  • the movable closure consists of a flap and a latch.
  • the flap can separate the two divisions against each other, the latch can lock the flap.
  • the latch In order to bring about the state change, in this embodiment, first of all, the latch must be moved, so that the flap is released. Only then can the flap be moved. With such a Two-piece movable closure can be prevented that the change of state, for example, during an optional centrifugation or shocks generated accidentally, for example, by an un-padded case on the ground.
  • Such an unintentional, premature change of state can preferably be prevented by the bar according to the invention also in cases in which a high pressure builds up in the closed subdivision (be it by a high temperature, by the use of very volatile chemicals, by reaction-induced gas formation or by low external pressure eg during transport).
  • the movable shutter includes a magnetic component.
  • the movable closure consists of the magnetic component.
  • the preferred magnetic component is cylindrical or spherical.
  • the magnetic component of the movable shutter can be moved. In an inhomogeneous magnetic field, the magnetic component may move from a weaker location to a stronger magnetic field location.
  • the magnetic component of the movable shutter can be aligned by the action of the magnetic field along the field line of the magnetic field. This can be done both in an inhomogeneous as well as in a homogeneous magnetic field. Due to the magnetic component of the movable shutter, the state change can be achieved particularly easily by the action of the magnetic field.
  • the movable shutter may e.g. consist of a magnetic component (e.g., a ferromagnetic material) provided with a plastic coating for better sealing and / or corrosion protection.
  • the movable shutter may be e.g. Also, consist of a non-magnetic body, which is provided with a ferromagnetic layer, which represents the magnetic component.
  • the movable shutter with the magnetic component may be incorporated in a partition wall between the first and second divisions.
  • the magnetic component By acting on the magnetic field, the magnetic component can move and / or align so as to form a connection between the first and second subdivisions.
  • parts or the entire partition wall between the first and second Subdivision of the magnetic component Preferably, parts or the entire partition wall between the first and second Subdivision of the magnetic component. In this way, the reaction vessel can be produced with a particularly low cost of materials.
  • the magnetic component may include a ferromagnetic material or be made of a ferromagnetic material.
  • the magnetic component contains iron. By using iron in the magnetic component, the manufacturing cost of the reaction vessel can be kept particularly low.
  • the magnetic component is at least partially permanent magnetic.
  • the magnetic component consists entirely of a permanent magnet.
  • an applied magnetic field it is possible for an applied magnetic field to exert a greater force on the magnetic component than if the magnetic component consists solely of a ferromagnetic, non-magnetized material.
  • a permanent magnet that aligns the magnetic component in a magnetic field.
  • the magnetic component may align in a magnetic field along the field lines.
  • the alignment along the field lines produces a rotational movement of the magnetic component, by means of which the first and the second subdivision can be connected to one another or separated from one another.
  • the movable closure in the first or second state is non-positively connected to the reaction vessel.
  • an area of the movable shutter on a surface of the reaction vessel is such that a force along the surface normal generates sufficient friction between the surfaces to hold the movable shutter in the reaction vessel.
  • exposure to the magnetic field may exert sufficient force on the magnetic component of the movable shutter to overcome the friction of the two surfaces, such that the movable shutter moves in the reaction vessel and the first and second subdivisions are connected or disconnected can.
  • the movable closure and the adjacent parts of the reaction vessel can take any shape.
  • the movable closure of a magnetic rotationally symmetrical body preferably of a permanent magnetic circular cylinder or a permanent magnetic ball.
  • the diameter of the body is preferably between 0.1 and 10 mm, more preferably between 1 and 6 mm and most preferably between 2 and 4 mm.
  • the body can be inserted into a flexible tube which is closed at one end.
  • the tube is made of silicone.
  • the ratio of the diameter of the body to the inner diameter of the tube is preferably 1: 1, particularly preferably 1.01: 1, particularly preferably 1.03: 1, particularly preferably 1.05: 1, more preferably 1.1: 1, more preferably 1.2: 1, most preferably 1.3: 1, and most preferably 1.5: 1.
  • the hose is stretched during insertion of the body so that a tight connection between the hose and the body is formed.
  • the tube preferably has a wall thickness between 0.1 and 5 mm, more preferably between 0.5 and 3 mm, and most preferably between 0.7 and 1.5 mm.
  • the first subdivision in which a liquid can be located.
  • the stiction holding the body in the tube can be overcome so that the body is removed from the tube and the first partition is connected to a second partition comprising the tube.
  • the movable closure in the first or second state is positively connected to the reaction vessel.
  • a first portion of the movable shutter is arranged to obstruct a second portion of the movable shutter the path that must be traveled to connect the first subdivision with the second subdivision.
  • the first portion is a bar which blocks the second portion so that no connection between the first and the second partition can occur.
  • the second portion is a hinged lid and the first portion is a bar containing the magnetic component.
  • the opening of the lid can by a suitable elastic element, such as a steel spring, or be relieved at a corresponding material of the lid by biasing the lid.
  • a suitable elastic element such as a steel spring
  • the positive connection of the movable closure allows a particularly secure separation of the first from the second subdivision in the first state. An accidental release of the movable closure, for example in an optional centrifugation step, can be avoided.
  • the movable closure in the first or second state is firmly bonded to the reaction vessel.
  • the movable closure is irreversibly joined to the rest of the reaction vessel.
  • a state change in which the first and second subdivisions are connected together can only be achieved by destroying parts of the movable shutter.
  • the movable closure z. B. consist of a film with which the first subdivision is closed.
  • a magnetic component present in the film can, by the action of the magnetic field, transmit a force to the film sufficient to destroy the film and thereby connect the first to the second subdivision.
  • the movable closure - can be made in one piece with the rest of the reaction vessel except for the magnetic component, preferably predetermined breaking points are attached to the movable closure, so that by the action of the magnetic field, the magnetic component on the movable closure exerts a force to destroy the predetermined breaking points and thus the state change of the reaction vessel leads.
  • At least one of the subdivisions is at least partially bounded by a cover of the reaction vessel. With the lid open, the reaction liquid can be filled into at least one of the subdivisions. After closing the lid both subdivisions are preferably closed on all sides. In this embodiment, the filling of liquids is particularly simple.
  • the reaction vessel is manufactured in one piece with at least one of the two subdivisions.
  • both subdivisions are made in one piece with the reaction vessel.
  • the moving one Closure may preferably be made in one piece with the rest of the reaction vessel. In this type of production, the reaction vessel can be produced particularly cheap.
  • At least one of the two subdivisions is located in a hollow body which is movable in the reaction vessel.
  • this hollow body is not attached in the reaction vessel, but is located loosely in the reaction vessel.
  • Such a subdivision may, for. B. are designed as a hose which is closed on one side by the movable closure and on the other side by a solid wall and is not attached to the rest of the reaction vessel.
  • an already closed subdivision is introduced into the reaction vessel, which may be advantageous when using particularly volatile reagents.
  • the magnetic component may thereby move in the inhomogeneous magnetic field of zones of low magnetic field strength to higher magnetic field strength, wherein preferably the first subdivision is connected to the second subdivision or these two are separated from each other.
  • these can be aligned in the direction of the field lines in the inhomogeneous magnetic field and the state change can thus be achieved.
  • the state change of the reaction vessel is brought about by a homogeneous magnetic field.
  • the magnetic component is preferably permanently magnetic, so that it rotates in the magnetic field and aligns along the field lines.
  • the magnetic component can be mounted so that the movable shutter is opened by its rotation.
  • the movable shutter may consist of a magnetized ball having a bore.
  • the ball may be rotatably mounted in a bearing, so that in an orientation of the ball, the bore is sealed by the bearing.
  • the orientation of the permanent magnetic sphere may change so that the bore is no longer obscured and the first is connected to the second subdivision via the bore of the sphere.
  • connection of the subdivisions can be made reversible in this way.
  • the state change of the reaction vessel is done using a permanent magnet or an electromagnet.
  • a permanent magnet By using a permanent magnet, the state change of the reaction vessel can be brought about without an electric device. This is particularly advantageous for mobile use of the reaction vessels.
  • An electromagnet can generate the magnetic field necessary for the change of state in a relatively small size. Since the magnetic field can be switched on and off as desired by the electromagnet, no relative movement of the reaction vessel to the electromagnet is necessary to trigger the change of state. Rather, the reaction vessel can be placed firmly in place and the magnetic field switched on at the appropriate time.
  • the state change of the reaction vessel using two permanent and / or electromagnets which are arranged such that between the permanent and / or electromagnets, a gap is formed.
  • the inventors have found through simulations that such an array of magnets can create an inhomogeneous field whose spatial variation in magnetic flux density is sufficient to provide the necessary force for the change in state of the reaction vessel to the magnetic component.
  • the magnitude of the magnetic field gradient is preferably between 0.001 and 10 T / mm, more preferably between 0.01 and 1 T / mm and most preferably between 0.03 and 0.3 T / mm.
  • the substances presented in the subdivisions may preferably mix or react with one another.
  • the substances preferably come into contact after the connection has been established by gravity.
  • it may be necessary to shake the reaction vessel, vibrate or suspend in a suitable form of centrifugation so that the liquid flows out.
  • FIG. 1 shows a reaction vessel 1, which is made in this embodiment of polypropylene, polyethylene, silicone or other plastic.
  • the reaction vessel 1 consists of an upwardly open circular cylindrical and a lower conical part which is rounded towards the lower end.
  • the outer opening 2 on the circular cylindrical part of the reaction vessel 1 can be closed by a cover 3.
  • a hose 4 is fixed for example by clamping, gluing or welding.
  • the tube 4 is made of silicone and clamped in the lid 3.
  • the tube 4 can also be made of other materials, preferably plastics.
  • the lid 3 is produced in one piece together with the tube 4 made of polypropylene.
  • a liquid or a solid, such as a powder can be filled.
  • the tube 4 can be closed by a movable cylindrical closure 5 in this case.
  • the tube 4 thus encloses, together with the lid 3 and the movable closure 5, the first partition 6, which is surrounded by the remaining reaction vessel 1 as the second partition 7.
  • the circular cylindrical movable shutter 5 includes a magnetic component 8 made of a ferromagnetic or ferrimagnetic material such as iron or ferrite.
  • the entire movable closure 5 consists of the magnetic component 8.
  • the movable closure 5 can also be provided with a plastic coating or consist of a plastic in which magnetic particles are introduced.
  • FIG. 1 In the partial illustration on the top right, a schematic external permanent magnet 9 is brought into the vicinity of the reaction vessel 1 and thus generates an inhomogeneous magnetic field in the reaction vessel.
  • the movable shutter 5 moves in the inhomogeneous field in the direction of higher magnetic field strength to the permanent magnet 9 and is removed from the tube 4.
  • the liquid previously contained in the tube 4 flows downwards out of the tube 4 out and falls on the liquid which is in the second subdivision 7.
  • a centrifugal acceleration of preferably less than 10,000 g (g 9.81 m / s 2 ), particularly preferably less than 1000 g, particularly preferably less than 100 g and very particularly less than 10 g, is usually required for this. It may be sufficient to pick up the reaction vessel and make a suitable circular rapid movement of the hand.
  • the reaction vessel 1 can now be moved.
  • the movable shutter 5 can also be set in motion by an alternating magnetic field ( FIG. 1 , bottom left) so as to mix the liquids, such as is usually done with a magnetic fish and a magnetic stirrer. Subsequently, by a lateral placement of the external permanent magnet 9, the movable shutter 5 can be removed from the liquid mixture in order to perform an absorption measurement of the liquid with a laser 10 or another light source, eg an LED ( FIG. 1 , below, in the middle and on the right).
  • a laser 10 or another light source eg an LED
  • reaction vessel 1 is particularly suitable for the detection of nucleic acids using nanoparticles.
  • a sample to be tested can be filled into the lower part of the reaction vessel 1 together with PCR mix including primers and polymerase.
  • the nucleic acids required for the detection reaction, conjugated with nucleic acids nanoparticles can be filled as a suspension in the tube 4 on the lid 3.
  • the lid 3 is now closed with the movable closure 5 and placed on the remaining reaction vessel 1.
  • the subdivisions 6, 7 are initially separated from each other.
  • the reaction vessel 1 passes through the temperature cycles necessary for carrying out a PCR in a thermocycler. Thereafter, the amplified nucleic acid is present in the liquid at the bottom of the reaction vessel 1.
  • the movable shutter 5 is removed from the tube 4 by the action of an inhomogeneous magnetic field, so that the nanoparticle suspension is released and flows to the bottom of the reaction vessel (optionally by exposing the reaction vessel to suitable accelerations, such as by shaking).
  • a low-frequency alternating magnetic field is applied, whereby the movable shutter 5 mixes the liquids at the bottom of the reaction vessel 1.
  • the low-frequency alternating field can preferably also be generated by movement of the reaction vessel in the vicinity of a (permanent) magnet.
  • the nanoparticles can specifically bind to the nucleic acids amplified by PCR via nucleic acids conjugated to them, whereby a change in the absorption spectrum of the liquid can be produced.
  • the movable shutter 5 is removed from the liquid by the application of an inhomogeneous magnetic field and held in the upper region of the reaction vessel 1. Subsequently, the concentration of the desired nucleic acids by absorption measurement by means of a laser 10 is determined.
  • FIG. 2 schematically illustrates a method with which in.
  • Reaction vessel 1 shown a change in state can be brought about.
  • a gap 12 is located between two external permanent magnets 9, which in this case each consist of three individual, joined individual magnets 11.
  • a reaction vessel 1 with a separate first 6 and second subdivision 7 is moved from left to right through the gap 12 between the two external permanent magnet 9 moves.
  • the field strength of the magnetic field in the center of the gap 12 is much stronger than at its edge, so that the movable shutter 5 is pulled out of the tube 4 due to the magnetic component 8 contained therein and the partitions 6, 7 are connected.
  • the liquid contained in the tube 4 can thus mix with the liquid present at the bottom of the reaction vessel 1.
  • the state change can be brought about here without the use of electricity.
  • FIG. 3 shows a reaction vessel 1 in a further embodiment.
  • the first subdivision is provided by a tube 4 made of silicone, which is bounded by the lid 3 of the reaction vessel 1 on one side and by the movable shutter 5 on the other side.
  • the movable shutter 5 is not designed here as a circular cylinder 13 but as a ball 14.
  • the ball 14 may for example consist of a ferromagnetic material or contain a ferromagnetic material. For better sealing and / or for better corrosion protection, the ball 14 may be coated with a plastic material (eg PTFE).
  • the diameter of the ball 14 is slightly larger than the inner diameter of the tube 4, so that it is expanded by the ball 14 and thus the sealing effect between the tube 4 and movable closure 5 is improved. Due to the symmetry properties of the ball 14, the movable shutter 5 can be particularly easily inserted into the tube 4 in this embodiment, since it does not have to pay attention to whether the axis of rotation of the movable shutter 5 coincides with the axis of the tube 4. To change the state, an inhomogeneous magnetic field is also applied here, which has a higher field strength, for example, at the bottom of the reaction vessel 1, so that the ball 14 is pulled out of the tube 4 and the subdivisions 6, 7 are connected.
  • the magnetic field is selected such that the strongest gradient of the magnetic field strength is at the location of the sphere.
  • the liquid flows from the tube 4 to the bottom of the reaction vessel 1.
  • an alternating magnetic field eg, by the relative movement of the reaction vessel to a permanent magnet
  • the ball 14 mixes the two liquids.
  • the ball 14 need not be removed from the liquid, since it is small enough that the light of the laser 10 above the ball 14 can radiate through the liquid.
  • FIG. 4 shows an alternative embodiment of the reaction vessel 1 with a fixed to the cover 3 first subdivision 6.
  • a recess 15 in which the movable closure is introduced in the form of a ball 14.
  • the ball 14 By the action of a magnetic field, the ball 14 can be pulled down from the first partition 6 and then stored laterally on the reaction vessel 1. The liquid contained in the first subdivision 6 can thus flow into the remaining reaction vessel 1 and mix with the liquid at the bottom of the reaction vessel 1.
  • the magnetic field can also be designed so that the ball 14 is drawn into the first subdivision 6 and the state change of the reaction vessel 1 is thereby brought about.
  • the first partition 6 is not attached to the lid 3, but at an upper wall portion 18 of the reaction vessel 1.
  • the reaction vessel 1 is designed so that, when the lid 3 is open, advantageously liquids can be filled both directly into the first subdivision 6 and the second subdivision 7.
  • the first partition 6 is closed at its lower end with a spherical movable shutter 5. Again, the inhomogeneous magnetic field can be generated so that in the state change of the movable shutter 5 is pulled into the first 6 or in the second division 7.
  • a reaction vessel 1 is divided into an upper, first subdivision 6 and a lower, second subdivision 7 by a partition wall 19 which runs parallel to the plane of the cover 3.
  • the movable shutter 5 is in the form of a ball 14. Liquid can be filled only in the second partition 7 when the ball 14 is removed.
  • the first subdivision 6 should be empty, in order to avoid that liquid from the first subdivision 6 already gets into the second subdivision 7 during the filling of the liquids.
  • FIG. 7 shows a further embodiment of a reaction vessel 1 with a vertical partition wall 19 between the first 6 and second partition 7.
  • the partition 19 is located in a plane of symmetry of the reaction vessel 1.
  • a spherical, movable shutter 5 can be pulled by the action of a magnetic field in the first 6 or second partition 7, so that the two partitions 6, 7 are connected and at sufficient filling level, the liquids in the partitions 6, 7 are mixed together.
  • FIG. 8 the reaction vessel 1 according to the invention is shown, which has a first subdivision 6, which is surrounded by the outer walls of the reaction vessel 1 including the lid 3.
  • a second, freely movable subdivision 7 can be added in the first subdivision 6, a second, freely movable subdivision 7 can be added.
  • the second partition 7 is formed by a movable hollow body 20, which is permanently closed at one end and in which a movable closure 5 can be sealingly introduced at the other end. If the subdivisions 6, 7 are to be connected to one another, an inhomogeneous magnetic field can be applied, so that the movable shutter 5 with the second subdivision 7 rests against the wall of the reaction vessel 1.
  • spacers 21 are mounted, which can keep the opening of the second partition 7 at least as far away from the inner wall of the first partition 6 when acting on the inhomogeneous magnetic field, that there is sufficient space to pull out the movable shutter 5.
  • FIG. 9 shows a further embodiment of the reaction vessel 1 according to the invention with an outer first subdivision 6 and a second freely movable therein Division 7 in a movable hollow body 20.
  • the second partition 7 is cylindrical and with a spherical, movable closure 5 closed.
  • the pulling out of the movable shutter 5 upon the action of an inhomogeneous alternating magnetic field is based here on the lower inertia of the movable shutter 5 in comparison to the remaining second subdivision 7
  • the movable shutter 5 has a substantially lower mass than the second subdivision including the liquid 7 contained therein.
  • reaction vessel 1 has a lower conical portion 22, in which after filling a liquid, a movable closure 5 - can be pressed - in the form of a ball 14 - here.
  • a movable closure 5 - can be pressed - in the form of a ball 14 - here.
  • the ball 14 is pulled upwards, so that the subdivisions 6, 7 connect.
  • an elastic ring 23 is introduced, which preferably consists of silicone.
  • the outer diameter of the ring 23 corresponds to the inner diameter of the reaction vessel 1 and the inner diameter of the ring 23 corresponds to the outer diameter of a circular cylinder 13 (or is slightly smaller), which constitutes the movable shutter 5.
  • the movable shutter 5 is pressed into the ring 23.
  • the ring 23 is pressed with the movable shutter 5 in the reaction vessel 1, whereby two subdivisions 6, 7 arise.
  • the movable shutter can be removed from the ring 23, so that the partitions 6, 7 are connected to each other.
  • FIG. 12 is a first subdivision 6 on the lid 7 of the reaction vessel 1 by a film 24 created.
  • a magnetic component 8 which in this embodiment is realized as a ball 14.
  • a liquid is additionally included in the first subdivision 6.
  • the movable shutter 5 in this case consists of two components: the magnetic component 8 (the ball 14) and the film 24.
  • the reaction vessel 1 is in an inhomogeneous field, the ball 14 can be pulled down so that the film 24 breaks and a connection between the two divisions 6, 7 arises.
  • the connection of the two subdivisions 6, 7 is irreversible, the movable shutter 5 is partially destroyed by the action of the magnetic field.
  • the inhomogeneous magnetic field is generated here by a suitable magnet, for example an electromagnet 26. Therefore, if the state change is to be brought about, the reaction vessel 1 does not have to be moved, but rather the electromagnet 26 can simply be switched on.
  • Reaction vessel 1 shown is a 0.2 ml standard PCR tube (Molecular Bioproducts, Inc., San Diego, Calif., USA) and corresponds to that described in US Pat FIG. 3 shown embodiment.
  • the lid 3 of the reaction vessel 1 continues on the inside in a cylindrical projection 25, which is introduced when closing the lid 3 in the opening 2 of the reaction vessel 1 and contributes to a good sealing effect of the lid 3.
  • a tube 4 is pressed, which has a length of 8 mm and is made of silicone.
  • the inner diameter of the tube 4 is 2.45 mm, the outer diameter is 5 mm (quality SL601 and hardness 60 ° Shore, J. Lindemann GmbH, Helmstedt).
  • the pressed-in the lid 4 end of the tube 4 is sealed by the lid 3.
  • the still open end of the tube 4 is closed by the movable shutter 5 in the form of a Teflon coated ball 14 made of magnetic stainless steel (Nirosta material 1.4034) with a diameter of 2.54 mm.
  • the movable shutter 5 can be removed again in an inhomogeneous magnetic field, which preferably has a gradient of at least 0.05 T / mm, particularly preferably> 0.1 T / mm.
  • FIG. 14 An arrangement is shown with which such an inhomogeneous magnetic field can be generated.
  • the ball can preferably be brought into a region of the reaction vessel through a small permanent magnet 9 (eg 5 mm diameter and 10 mm length), where it does not impair further investigation.
  • a small permanent magnet 9 of NdFeB is shown, which is guided directly to the wall of the reaction vessel 1, so that the ball 14 approaches the permanent magnet 9.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Claims (15)

  1. Récipient de réaction (1) comportant une première subdivision (6) et une seconde subdivision (7), dans lequel, dans un premier état du récipient de réaction (1), la première subdivision (6) est séparée de la seconde subdivision (7) et, dans un second état, la première subdivision (6) est reliée à la seconde subdivision (7), dans laquelle l'un des deux états peut être transféré dans l'autre par l'action d'un champ magnétique, et, dans le premier état, la première subdivision (6) et la seconde subdivision (7) sont séparées l'une de l'autre par une fermeture mobile (5) ayant un composant magnétique (8), caractérisé en ce que, dans le premier état, la fermeture mobile (5) est reliée au récipient de réaction (1) par liaison de matière.
  2. Récipient de réaction (1) comportant une première subdivision (6) et une seconde subdivision (7), dans lequel, dans un premier état du récipient de réaction (1), la première subdivision (6) est séparée de la seconde subdivision (7) et, dans un second état, la première subdivision (6) est reliée à la seconde subdivision (7), dans laquelle l'un des deux états peut être transféré dans l'autre par l'action d'un champ magnétique, et, dans le premier état, la première subdivision (6) et la seconde subdivision (7) sont séparées l'une de l'autre par une fermeture mobile (5) ayant un composant magnétique (8), caractérisé en ce qu'au moins une des deux subdivisions (6, 7) est située dans un corps creux (20) mobile dans le réacteur (1).
  3. Récipient de réaction (1) comportant une première subdivision (6) et une seconde subdivision (7), dans lequel, dans un premier état du récipient de réaction (1), la première subdivision (6) est séparée de la seconde subdivision (7) et, dans un second état, la première subdivision (6) est reliée à la seconde subdivision (7), dans laquelle l'un des deux états peut être transféré dans l'autre par l'action d'un champ magnétique, caractérisé en ce que la première subdivision (6) est créé sur un couvercle du récipient de réaction par un film (24), et qu'un composant magnétique (8) est situé à l'intérieur de la première subdivision (6).
  4. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce que le composant magnétique (8) est au moins partiellement un aimant permanent.
  5. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce que, dans le premier état, la fermeture mobile (5) est reliée au récipient de réaction (1) par adhérence.
  6. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce que, dans le premier état, la fermeture mobile (5) est reliée au récipient de réaction (1) par engagement positif.
  7. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce que, dans le premier état, la fermeture mobile (5) est reliée au récipient de réaction (1) par liaison de matière.
  8. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce qu'au moins une des subdivisions (6, 7) est au moins partiellement limitée par un couvercle (3) du récipient de réaction (1).
  9. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce que le récipient de réaction (1) est fabriqué d'une seule pièce avec au moins une des deux subdivisions (6, 7).
  10. Récipient de réaction (1) selon l'une des revendications précédentes, caractérisé en ce qu'au moins une des deux subdivisions (6, 7) est située dans un corps creux (20) mobile dans le récipient de réaction (1).
  11. Utilisation du récipient de réaction (1) selon l'une des revendications précédentes dans un procédé pour la purification, la modification et/ou la détection de protéines et/ou d'acides nucléiques.
  12. Utilisation du récipient de réaction (1) selon l'une des revendications précédentes dans un procédé d'amplification d'acides nucléiques.
  13. Procédé pour changer l'état du récipient de réaction (1) selon l'une des revendications 1 à 10.
  14. Procédé selon la revendication 13, caractérisé en ce que le changement d'état du récipient de réaction (1) est effectué à l'aide d'un aimant permanent (9) ou d'un électroaimant (26).
  15. Procédé selon l'une des revendications 13 ou 14, caractérisé en ce que le changement d'état du récipient de réaction (1) est effectué à l'aide de deux aimants permanents (9) et/ou électroaimants (26) qui sont disposés de telle sorte qu'il existe un espace (12) entre les aimants permanents (9) et/ou électroaimants (26).
EP13805819.3A 2012-12-05 2013-12-05 Récipient de réaction à fermeture magnétique Not-in-force EP2928605B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102012222351.7A DE102012222351A1 (de) 2012-12-05 2012-12-05 Reaktionsgefäß mit magnetischem Verschluss
PCT/EP2013/075637 WO2014086912A1 (fr) 2012-12-05 2013-12-05 Récipient de réaction à fermeture magnétique

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EP2928605A1 EP2928605A1 (fr) 2015-10-14
EP2928605B1 true EP2928605B1 (fr) 2019-09-04

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DE (1) DE102012222351A1 (fr)
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US20220314214A1 (en) * 2019-06-13 2022-10-06 Chin Hung Wang Centrifugal reaction microtube, centrifugal reaction device and its centrifugal examination method

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WO2006020182A2 (fr) * 2004-07-16 2006-02-23 Smart Medical Technologies, Llc Système de centrifugation
WO2006050636A1 (fr) * 2004-11-10 2006-05-18 Capitalbio Corporation Tube de reaction pcr nette

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EP2928605A1 (fr) 2015-10-14
WO2014086912A1 (fr) 2014-06-12

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