JP2013072687A - Suspension container for binding particles for isolating biological material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a suspension container for binding particles for the isolation of biological material.
SOLUTION: Binding particle suspension for isolation of biological material Inner wall (2) forming an elongated groove (3) in the bottom of the suspension container (1)
A cover (4) having an opening (5) and / or through which a linear arrangement of a number of pipettes or pipette tips can be penetrated, said openings being arranged above and in parallel with said elongated groove,
A suspension container (1).
[Selection figure] None

Description

The present invention belongs to the field of sample preparation for analytical purposes, in particular for the separation and / or isolation of biological materials such as nucleic acids or proteins in or from complex mixtures. Within the field, the present invention relates to the provision of binding particles for the separation and / or isolation of biological materials.

BACKGROUND OF THE INVENTION Isolation of biological material such as nucleic acids or proteins from complex biological mixtures such as clinical samples has become of considerable importance especially for diagnostic purposes.

  A number of different methods have been developed in the art, such as denaturation, precipitation and removal of unwanted components in a sample, followed by precipitation and isolation of the analyte of interest (e.g., alcohol-based precipitation of nucleic acids). ing.

  Another approach is to bind the biological material to be isolated to a solid support material that can be provided, for example, in the form of a chromatography column.

  Bound particles are often used for diagnostic purposes, particularly for the automated isolation of biological material that is later subjected to medium- or high-throughput analysis. Such particles may have a functionalized surface, i.e., often coated with antibodies, nucleic acid capture probes, etc. to bind the desired analyte. Alternatively, it may have an unmodified surface, such as a glass surface for nucleic acid isolation in particular.

  Such bound particles, in conjunction with automated analyzers, are often provided as suspensions in containers in which the particles are collected and distributed with the aid of a pipetting tool. US Pat. No. 6,099,077 suggests an approach for providing particles from a container for isolation of biological material, the use of a container in the form of a plastic bottle that is agitated by a shaker, and particle distribution. The use of pipettes for is disclosed.

  In the present invention, an improved approach is used that exhibits several advantages.

EP 1 614 475

  The object of the present invention is to provide a suspension container for binding particles for the isolation of biological material.

That is, the gist of the present invention is as follows:
[1] suspension of bound particles for the isolation of biological material inner wall (2) forming an elongate groove (3) in the bottom of the suspension vessel (1)
A cover (4) having an opening (5) and / or through which a plurality of pipettes or linear arrangements of pipette tips can be penetrated, said opening being located parallel to the top of the elongated groove;
Suspension container containing (1),
[2] The suspension container according to [1], wherein the inner walls are inclined at an angle to each other, thereby forming the elongated groove,
[3] The suspension container according to [1] or [2], wherein the binding particles include nucleic acid binding particles,
[4] The suspension container according to any one of [1] to [3], wherein the binding particles include magnetic glass particles,
[5] In addition,
A filling opening (6) for filling the suspension of binding particles into the container
A suspension container according to any of the above [1] to [4], comprising one or more elements selected from the group of removable foils that seal the cover,
[6] The suspension container according to any one of [1] to [5], wherein the cover has an opening, and the opening is covered with a septum that can pass therethrough.
[7] The suspension container according to any one of the above [1] to [6], which is adapted to hold a volume of at least 100 ml,
[8] The suspension container according to any one of [1] to [7], including one or more fasteners for reversibly fixing the container to a shaker,
[9] The suspension container according to any one of the above [1] to [8], wherein the installation surface (footprint) of the container is an elliptical shape,
[10] A method for pipetting a suspension of binding particles for the isolation of biological material comprising the steps of:
agitating the container (1) containing the suspension of bound particles, the container comprising an inner wall (2) forming an elongated groove (3) in the bottom of the container, the container further comprising an opening (5) and / or a cover (4) through which a linear arrangement of multiple pipettes or pipette tips can be penetrated,
b. introducing a linear arrangement of the multiple pipettes or pipette tips into the suspension from the openings and / or through-penetrating covers
c. including an automated process of aspirating at least a portion of the suspension with a linear arrangement of multiple pipettes or pipette tips and releasing the aspirated suspension or portions thereof into multiple reservoirs. ,Method,
[11] The method according to [10], wherein in step c., The linear arrangement of the multiple pipettes or pipette tips simultaneously releases the suspension into the multiple tanks.
[12] In step c., The linear arrangement of the multiple pipettes or pipette tips is individually suspended by manipulating one or more of the multiple pipettes or pipette tips in the linear arrangement. The method according to the above [11], wherein a liquid is discharged into the multiple tanks.
[13] An analytical system for providing bound particles for the isolation of biological materials,
A linear arrangement of a number of pipettes or pipette tips; a container containing a suspension of binding particles for binding of the biological material, the container including an elongated cavity at the bottom thereof, the container being Further comprising a cover, wherein the cover has an opening and / or a linear arrangement of the multiple pipettes or pipette tips can be penetrated, and includes a shaker that stirs the container and suspends the bound particles , Analysis system,
[14] The analysis system according to [13], further including a plurality of tanks for receiving a suspension of the binding particles from a linear arrangement of the multiple pipettes or pipette tips.
[15] The analysis system according to [13] or [14], further including a separation module for isolation of the biological material with the binding particles,
[16] In addition,
Reaction modules containing components of chemical and / or biochemical reactions Detection modules for detecting signals caused by analytes Storage modules for reagents and / or disposables Controls for controlling system components The analysis system according to any one of [13] to [15], comprising one or more elements selected from the group consisting of units.

  According to the present invention, a suspension container for bound particles for the isolation of biological material can be provided.

FIG. 1 is a perspective view from above of a suspension container with a cover. FIG. 2 is a perspective view from above of a suspension container without a cover. FIG. 3 is a cross-sectional view of a suspension container without a cover. FIG. 4 is a perspective view from below of the suspension container. FIG. 5 is a perspective view of a suspension container placed on a shaking device. FIG. 6a is a multipipetter interacting with the suspension vessel. FIG. 6b is a multipipetter interacting with the suspension vessel. FIG. 6c is a multipipetter interacting with the suspension vessel.

DESCRIPTION OF THE INVENTION The present invention relates to providing bound particles for separation and / or isolation of biological materials.

  In particular, suspension containers, methods and analysis systems for the above purposes are provided.

Among the advantages provided by certain embodiments of the invention are, for example, the following:
-Uniform distribution of bound particles from the container to the vessel for downstream processing, in particular for the isolation of biological materials-Parallelization of the provision of bound particles, thereby particularly for automated systems • Avoiding contamination • Increases suspension stability • Avoids loss of suspension material due to vessel agitation • From suspension in that vessel • Quantitative recovery of • Throughput flexibility • Individual distribution of bound particles.

  Which aspects provide which particular advantages are shown below.

In the first aspect, the present invention provides:
A suspension of binding particles for the isolation of biological material an inner wall forming an elongated groove in the bottom of the suspension vessel an opening and / or a linear arrangement of a number of pipettes or pipette tips A cover that is possible, the opening is located parallel to the top of the elongated groove,
Providing a suspension container comprising: provides the specific advantages described above.

  The suspension container according to the invention ensures a reduced risk of contamination due to the presence of a cover that protects the suspension of bound particles from potential contaminants.

  In the context of isolating biological material for subsequent analysis, contaminants in the sample often have serious consequences, particularly with respect to clinical samples. In particular, in the case of substances (eg nucleic acids) that are amplified during analysis, already very small amounts of contaminants can lead to false positive results in qualitative assays or overestimation of titers in quantitative assays. Both problems can have a significant impact on the diagnosis of the disease or the respective treatment. Also, for example, the introduction of an inhibitory substance such as a specific protein may hinder analytical experiments so that no results are obtained. In addition, substances that reduce the stability of the suspension, or substances that affect physicochemical properties such as concentration, ionic strength, etc. can be introduced.

  Furthermore, the use of a cover is quite advantageous when the suspension container is agitated. Agitation of the suspension of bound particles is often performed to resuspend the particles. Whenever the container is not moved, particle settling occurs due to the effects of gravity. However, in multiple suspension draws, a suspension of bound particles is provided in a uniform manner so that when an equal volume of suspension is withdrawn, an essentially equal number of bound particles are recovered. It is important to do. As a result, the concentration of the entire suspension must be essentially constant during the various draw events.

  In the prior art, a container with a suspension of bound particles for the isolation of biological material is stirred, for example, on a shaker or mixed using a stirrer. Stirrers are another potential source of contaminants and introduce the problem of losing suspension due to spillage due to stirring. On the other hand, in an automated solution, the placement of the cover on the container is associated with the problem that suspension removal should be possible in a manner that is as easy as possible. For example, removing the cover before each extraction may require additional steps in the method for providing a suspension of binding particles, and opening the container increases the risk of contamination.

  Thus, one advantage of the suspension container according to the present invention is that it includes a cover that has an opening and / or through which a linear arrangement of multiple pipettes or pipette tips can be penetrated. In this manner, the risk of both spilling and contamination is significantly reduced without having to remove the cover to extract the suspension of bound particles. The suspension container does not need to be opened and can be used to provide the suspension until it is empty.

  The cover can be advantageously used for parallelization of the provision of a suspension of binding particles, since it has an opening and / or a linear arrangement of multiple pipettes or pipette tips can be penetrated. The use of multiple pipettes allows one skilled in the art to dispense a suspension of binding particles simultaneously into multiple baths. In particular, in the context of automated solutions, media for high sample throughput is often desirable. As an example, clinical sample diagnostic tests, such as blood screening in the context of in vitro diagnostics for blood banks, require analysis of multiple samples within a limited period of time. In addition, laboratories that serve as a central facility for the analysis of clinical samples from several hospitals need to provide analysis results quickly. The latter is important for treatment after analysis of the sample. Parallelization can result in a decrease in the number of single trials performed to make a diagnosis, potentially reducing the length of hospital stay (e.g., if the diagnosis can be provided earlier, antimicrobial therapy is needed). Patients who receive the therapy receive earlier and therefore recover faster). Moreover, antibiotic over-prescription can be avoided if a negative result is provided earlier.

  In embodiments where the cover has an opening, the opening is disposed in parallel on an elongated groove formed in the bottom of the container by its inner wall.

  In the sense of the present invention, an “opening” used in connection with a cover introduces a pipette tip into the container and thus allows the suspension to be withdrawn without removing the cover. ). In a preferred embodiment of the present invention, the opening is a hole. Preferably, the holes are round but can be any other geometric shape that allows passage of a pipette or a linear arrangement of pipette tips.

  “Penetrable” means closed, but still capable of penetrating. More specifically, with respect to the suspension container cover, “pierceable” means that the cover is essentially closed, but still allows passage of a linear arrangement of pipettes or pipette tips. means. As an example, the cover may be a partition made of rubber or rubber-like material through which the pipetting needle can penetrate. The septum used in the context of the present invention is made of an essentially flexible material. Preferably, the cover through which the linear arrangement of the pipette or pipette tip can penetrate is a partition wall. Preferably, the cover is airtight. The hermetic cover has the advantage of effectively preventing the introduction of contaminants into the container.

  The above groove facilitates complete removal of the suspension from the container because the residual volume collects in the groove. According to the invention, the elongated form of the groove is adapted to a linear arrangement of pipettes or pipette tips. Therefore, the latter can be put into the container through the opening and the residual volume can be sucked from the groove. This ensures that there is little residual volume remaining in the container and discarded without being used. In particular, in automated systems that include a cover where the pipette or its corresponding tip typically needs to be introduced into the container in a vertical and straight line, this can be more difficult without such an elongated groove. For example, a residual volume of suspension can be discarded without being used because it can remain in a portion of the bottom of the container that the pipette cannot reach. In addition, when the container is agitated to achieve or maintain a uniform distribution of bound particles, a “dead corner” can result in a design that is free of elongated grooves as used in the present invention. . This means that, for example, in a container with a flat bottom and, for example, a corner at the boundary between the bottom and the side wall orthogonal to each other, the bound particles can be kept at said corner and therefore the flow caused by the stirring of the container does not reach. means. Such a phenomenon can lead to a non-uniform distribution of bound particles, thus changing the concentration of particles extracted by pipetting. The larger the container, the more likely the above problems will occur and the more serious it will be.

  The elongate groove can be provided, for example, by a parabolic or another concave arrangement on the inner wall of the container. Said groove is preferably essentially in the middle of the bottom. In this manner, no edges or corners are created in the container.

  Also, an elongated groove can be formed as a result of the two inner walls forming a boundary at a particular angle with respect to each other. This embodiment can be advantageous for making containers, where the relatively sharp-edged groove is a residual suspension volume into which a pipette or pipette tip can be introduced for efficient withdrawal of the residual suspension volume. Can serve as a well-defined cavity for the collection of In addition, the relatively sharp groove created by two inner walls that are inclined at an angle to each other has a baffle-like structure that can be advantageous in providing a uniform distribution of binding particles in the container. be introduced. It is known in the art that baffle plates contribute to a uniform distribution in a suspension in a stirred vessel such as an Erlenmeyer flask.

  Accordingly, a preferred aspect of the present invention is the suspension container described above, wherein the inner walls are inclined at an angle with each other, thereby forming the elongated groove.

  The “angle” (represented by “α” in FIG. 3) is larger than 0 ° and smaller than 180 °. If the walls together form a completely flat bottom, the angle can be 180 ° and there are no grooves. If the angle is 0 °, no bottom can be formed. Preferably, the angle is between 90 ° and 180 °.

  In connection with the present invention, when the pipette forms a linear arrangement of pipettes or pipette tips, the pipette is preferably a pipetting needle. Such needles are often used in conjunction with automated diagnostic systems, for example because of their advantageously small diameter and accuracy that can be handled, for example, by a robotic arm. Also, if the pipette or pipette tip linear arrangement is formed by a pipette tip, the pipette tip needs to be fixed to the pipette in order to serve the purpose of pipetting the suspension contained in the container . In this embodiment, the pipette tip is preferably a disposable pipette tip from which the suspension is aspirated and redistributed. Such pipette tips can be discarded or replaced after several uses. Often, but not necessarily, such pipette tips are made of plastic. Disposable pipette tips are generally known in the art.

  “Linear arrangement” means that pipettes or pipette tips are arranged in a line, preferably in a straight line. The arrangement includes a bearing to which a corresponding pipette is fixed. Preferably, the bearing is also adapted to manipulate and move the linear arrangement of pipettes or pipette tips in all dimensions, i.e. the x, y and z axes. Preferably, the bearing is a robotic arm that is preferably adapted to facilitate aspiration and dispensing of the suspension from the container.

  As mentioned above, the present invention provides the opportunity for high analytical sample throughput and rapid results by parallelization. Accordingly, it is preferred that the linear arrangement of pipettes or pipette tips comprises at least a certain number of pipettes or pipette tips, preferably at least 4, more preferably at least 8, most preferably 8 pipettes or pipette tips.

  A “binding particle for the isolation of biological material” is a solid phase on which biological material can be immobilized. The particles can include different materials and shapes. For example, particles such as beads or fleece having a diameter in the nano-, micro or millimeter range may be suitable for binding biological materials. “Biological material” in the sense of the present invention includes all types of biological molecules, for example proteins or nucleic acids, but also other molecules that are naturally occurring or are derivatives or synthetic analogues or variants thereof. Including. Furthermore, the term “biological material” includes viruses and eukaryotic cells and prokaryotic cells. Often, such cells or viruses are bound to the binding particles by biomolecules, particularly proteins, present in their respective cell membranes or capsids. For the purpose of binding biological material, often the surface of the binding particle is modified by coating with a biomolecule having specific or non-specific affinity for the biological material to be bound. Is advantageous. As an example, such a coating can include streptavidin that specifically binds to biotin. The latter is often chemically bonded to biomolecules to bind the biomolecule to a streptavidin-containing surface, such as the surface of each modified binding particle. For the same purpose, the interaction between the histidine tag and nickel or between the antibody and its antigen or epitope can be utilized. In situations where the nucleic acid is the biological material to which it is bound, so-called capture probes can be used advantageously. These capture probes are nucleic acids themselves, usually oligonucleotides, that bind by Watson-Crick base pairing to essentially complementary nucleic acid sequences, often about 15-25 nucleotides in length. Methods involving binding particles coated with specific conjugates have the advantage that specific biological materials such as specific proteins or nucleic acids can be isolated but other proteins or nucleic acids are not bound. This can contribute to the isolation of such specific biological material for analytical purposes. Those skilled in the art will appreciate that there are various interactions between the specific coatings of biological particles and binding particles that can be used within the spirit of the present invention. Such binding particle coating methods are described in the prior art.

  Of particular importance for nucleic acid extraction purposes is the adsorption of nucleic acids to the glass surface, although other surfaces are possible. In recent years, many procedures have been proposed for isolating nucleic acids from their natural environment through the use of properties that bind to the glass surface. When an unmodified nucleic acid is the target, direct binding of the nucleic acid to a substance having a silica surface is preferred because, for a number of reasons, it is not necessary to modify the nucleic acid and even natural nucleic acids can be bound. These methods are described in detail in various publications such as Vogelstein B. et al., Proc. Natl. Acad. USA 76 (1979).

  In the sense of the present invention, the term “isolation”, “purification” or “extraction” of biological material refers to the following: before biological material such as nucleic acids can be analyzed in a diagnostic assay, eg by amplification. In particular, it relates to having to be purified, isolated or extracted from a biological sample containing a complex mixture of different components. Often, in the first step, a method is used that allows for enrichment of the material. In order to release the contents of the cells or virus particles, they are treated with enzymes or chemicals to lyse, degrade or denature the cell walls or virus particles. This process is commonly referred to as dissolution. The resulting solution containing such dissolved material is referred to as a lysate. A problem often seen during lysis is that other enzymes that degrade the component of interest, such as deoxyribonucleases or ribonucleases that degrade nucleic acids, come into contact with the component of interest during the lysis procedure. These degrading enzymes can also be present extracellularly or can be spatially separated in different cell compartments prior to lysis. As lysis occurs, the components of interest become exposed to the degrading enzyme. Other components released during this process can be, for example, endotoxins that belong to the lipopolysaccharide family, are toxic to cells and can cause problems for products intended for use in human or animal therapy.

  There are various means to address the above problems. When it is intended to liberate nucleic acids, it is common to use guanidinium thiocyanate or chaotropic agents such as anionic, cationic, zwitterionic or nonionic surfactants. It is also advantageous to use proteases that rapidly degrade the aforementioned enzymes or unwanted proteins. However, this may lead to another problem that the substance or enzyme may interfere with the reagent or component in the next step.

  Enzymes that can be used advantageously in such lysis or sample preparation processes described above are those that cleave the amide bond of the protein substrate and are classified as proteases or (interchangeably) peptidases. In particular, the enzyme esperase, a powerful protease that maintains its activity both at high alkalinity and high temperatures, is advantageous for use in the lysis or sample preparation process described above (EP 1 201 753).

  In the sample preparation step after the dissolution step, the target component is preferably further enriched by using binding particles contained in the suspension container according to the present invention.

  For example, procedures for binding nucleic acids involve the selective binding of nucleic acids to glass surfaces of binding particles in chaotropic salt solutions and the separation of nucleic acids from contaminants such as agarose, proteins or cell debris. In order to separate the glass particles from the contaminants, either the particles can be centrifuged or the liquid is drawn through a glass fiber filter. The use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is more advantageous, eg, Alderton RP et al., S., Anal. Biochem. 201 (1992) 166-169 and It is described in PCT GB 91/00212. Also, a magnetic porous glass is commercially available that is porous, particularly containing magnetic particles in a glass matrix and covered with a layer containing streptavidin. This product can be used to isolate biological materials, such as proteins or nucleic acids, if they have been modified in a complex preparation process to covalently bind to biotin. Certain magnetizable adsorbents have proven to be very efficient and suitable for automated sample preparation. Ferrimagnetic and ferromagnetic and superparamagnetic pigments are used for this purpose.

  A preferred aspect of the present invention is the above-described suspension container, and the binding particles include nucleic acid binding particles. More preferably, it contains magnetic glass particles. Even more preferably, it comprises magnetic glass particles having an unmodified glass surface. In addition, preferably, magnetic glass particles produced by a sol-gel method are included. Most preferably, it comprises magnetic glass particles made by a sol-gel method and having an unmodified glass surface. In a preferred embodiment, the binding particles are nucleic acid binding particles, preferably magnetic particles and / or glass particles. In a more preferred embodiment, the binding particles are preferably magnetic glass particles made by a sol-gel method and / or having an unmodified glass surface.

  The most preferred magnetic particles and their use are those described in WO 01/37291. Briefly, these magnetic glass particles are a solid dispersion of small magnetic cores in glass. The particles are also relatively small and substantially spherical. The non-magnetic fine particle content of the composition of magnetic glass particles is very low due to its preparation method. This has the effect that these magnetic glass suspensions settle slowly and can therefore be used advantageously in processes in molecular biology that can be automated.

“Glass” according to the present invention is to be understood as an essentially amorphous material containing silicon. Glass, for _{example, B 2 O 3 (0~30%} ), Al 2 O 3 (0~20%), CaO (0~20%), BaO (0~10%), K 2 O (0~20 _{%), Na 2 O (0~20} %), MgO (0~18%), Pb 2 O 3 (0~15%) may include other substances, such as. The glass may also contain a small percentage (0-5%) of several other oxides, such as Mn ₂ O ₃ , TiO ₂ , As ₂ O ₃ , Fe ₂ O ₃ , CuO, CoO, and the like.

  Particularly preferred are glasses that are formed using the gel sol process described in WO 96/41811 and then dried and compressed.

  The method according to R. Boom et al. (J Clin Microbiol. 28 (1990), 495-503) is particularly useful for the isolation of nucleic acids in the context of the present invention.

  After purification or isolation of a nucleic acid potentially containing a specific target nucleic acid from its natural environment, the nucleic acid or specific target nucleic acid can be detected.

  Optionally, prior to detection, the nucleic acid can be amplified by techniques such as polymerase chain reaction (PCR, eg, as described in US Pat. No. 4,683,202). PCR typically uses two or more oligonucleotide primers that bind to a selected nucleic acid template (eg, DNA or RNA).

  Nucleic acid amplification reactions other than PCR include ligase chain reaction (LCR; Wu DY and Wallace RB, Genomics 4 (1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991) 189- 193); polymerase ligase chain reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO 90/01069); repair chain reaction (EP 0439182 A2), 3SR (Kwoh DY et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli JC, et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808), And NASBA (US 5,130,238). In addition, strand displacement amplification (SDA), transcription-mediated amplification (TMA), and Qβ-amplification (for review see, for example, Whelen AC and Persing DH, Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson RD and Myers TW, Curr Opin Biotechnol 4 (1993) 41-47).

  Appropriate nucleic acid detection methods are known to those skilled in the art and include Sambrook J. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 and Ausubel F. et al .: Current Protocols in It is described in standard textbooks such as Molecular Biology 1987, J. Wiley and Sons, NY. There may be further purification steps such as a precipitation step before performing the nucleic acid detection step. Examples of the detection method include, but are not limited to, binding of a specific dye such as ethidium bromide or intercalation of the dye, which intercalates between double-stranded DNA and subsequently changes its fluorescence. Purified nucleic acids can also be separated by electrophoresis, optionally after restriction digestion, and then visualized. Some probe-based assays take advantage of oligonucleotide hybridization to specific sequences and subsequent detection of the hybrid. It is also possible to sequence the nucleic acid after further steps known to those skilled in the art. Useful template-dependent nucleic acid polymerases are ZO5 DNA polymerase and variants thereof. Other template-dependent nucleic acid polymerases include, for example, Taq polymerase and Tth polymerase.

In a preferred embodiment of the present invention, the above-mentioned suspension container further comprises
A filling opening for filling the container with a suspension of binding particles, comprising one or more elements selected from the group of removable foils that seal the cover.

  While it is possible to refill the container without departing from the scope of the present invention, the suspension container of the present invention is preferably disposable, i.e. suspended until there is no more suspension therein. Using a turbid container, the container is then discarded. This has the advantage that no impurities can be introduced by refilling the container.

  In order to initially fill the container with a suspension of binding particles for the isolation of biological material, it is preferred that the cover has a filling opening. The filling opening may have any geometric shape. Preferably, the filling opening is wider than the opening for the linear arrangement of multiple pipettes or pipette tips. In a preferred embodiment, the filling opening is used only once for filling the container and then sealed. Preferably, the opening remains sealed until the container is discarded. Also preferably, the opening is sealed in a sterile manner. For sealing the filling opening, various materials such as plastic and metal can be used. Preferably, the filling opening is sealed with a sterile foil.

  Furthermore, regarding the avoidance of contamination, a preferred aspect of the present invention is the above-described suspension container, wherein the cover has an opening, and the opening is covered with a penetrable partition wall. In this manner, the advantages of the opening and the penetrable partition are combined. The opening alone, for example, in a particularly preferred embodiment, if the diameter of the opening is not significantly larger than that required for the pipette or pipette tip to pass, the cover that is closed when the pipette does not pass is The advantage is that the pipette or pipette tip can pass through while most of the source of contamination is eliminated. Furthermore, the opening is easily introduced into the cover, which provides a stable barrier between the inside and the outside of the container. On the other hand, the septum can essentially seal any opening, such as an aperture in the container of the present invention. Since the septum used in the present invention is made of an essentially flexible material such as rubber or other suitable material, the opening in the septum has a linear arrangement of pipettes or pipette tips. It is formed only when penetrating the partition, but after such penetration, the formed opening is essentially closed again. When combined, the cover containing the opening provides a stable barrier at the top of the container of the present invention while introducing a linear arrangement of pipettes or pipette tips into the container, while an additional septum opens the opening of the cover. Sealing essentially contributes to a reduced risk of contamination, but the pipette or pipette tip can penetrate. Preferably, the septum is fixed above or below the opening and seals the opening before and preferably after the introduction of a pipette or pipette tip linear arrangement. In a further preferred embodiment, the cover comprising the opening preferably consists of two layers of hard material, such as plastic, and a septum sandwiched between the layers of hard material, thereby sealing the opening. The septum is preferably made of rubber or a rubber-like material so that it essentially closes the hole after the pipette or pipette tip is withdrawn from the opening and septum. In this context, “essentially closed” means that the pierced septum can preferably still prevent the introduction of contaminants. Preferably, the septum is still airtight at normal pressure.

  Since it is important that the suspension container of the present invention be provided so that contamination is avoided as much as possible, it is preferred that the container includes a removable foil that seals the cover. Preferably, the foil is removed prior to the first use of the container and thus the suspension so that a linear arrangement of pipettes or pipette tips can be introduced into the container. Preferably, the foil is then discarded. Alternatively, the foil may remain above or below the cover if a linear arrangement of pipettes or pipette tips can be penetrated.

  With regard to packaging of the container, for example to transport or provide a suspension container to a user, the container is provided in a hermetic plastic packaging that is sterile and free of biological material, but other packaging. Substances may also be appropriate. Preferably, such packaging allows one skilled in the art to insert such a container into an automated system, such as a device, without physical contact with the suspension of binding particles in the container.

  Another preferred aspect of the invention is a suspension container as described above, said container being adapted to hold a volume of at least 100 ml, preferably 100-500 ml, more preferably 150-300 ml.

  In particular, when aiming at an automated high-throughput solution in which sample preparation and analysis reaction are made parallel, it is preferable to use a suspension container having a relatively large amount of suspension of binding particles. Such a container can be used to provide a suspension of bound particles for multiple experiments, after which the container needs to be discarded and actually replaced. For example, the opening and closing of the device is preferably less frequent because the risk of contamination is most likely to be introduced.

  As described above, using a container having a relatively large volume involves the difficulty that even though the inner surface is large, only a small residual volume remains in the container without being used. This problem is solved by the container of the present invention with an elongated groove with an opening and / or penetrable cover as described above.

  Moreover, the problem that the suspension is spilled and lost when the container is stirred is also solved by the present invention by the cover included in the container of the present invention. This is also a more frequent problem when using containers with large volumes.

  For stirring, the suspension container is preferably placed on a shaker. Laboratory shakers are known in the art and achieve a variety of movements suitable for resuspension of potentially precipitated binding particles. A preferred aspect of the present invention for providing a stable connection between a container and a shaker during shaking is the above-described suspension container, which is for reversibly fixing the container to the shaker. Includes one or more fasteners. The fastener may be attached to the suspension container during or after manufacture of the suspension container.

  The container of the present invention can be made of various materials. For example, if the container is made of plastic, the manufacturing process preferably includes injection molding, so that the fastener can be introduced during the manufacturing process. Preferably, the container is made of polypropylene. In a manufacturing method useful in connection with the present invention, the container is made in a two-step procedure. Initially, the bottom of the container, i.e. the tray containing the suspension later, is made by a one-component injection molding process using polypropylene and a suitable molding tool. In another process, the cover is manufactured from two components, first the basic form is made of polypropylene by injection molding, and then the material forming the partition (eg rubber) is added and combined with the cover.

  With respect to filling the container with a suspension of bound particles, the suspension first weighs the solid material and adds a predetermined amount to a suitable container, preferably the aforementioned container of the present invention, and then It is preferable to prepare by adding each liquid matrix of fixed quantity. In this way, substantially uniform amounts of liquids and particles and suspensions of substantially uniform morphology can be provided in various containers. Furthermore, adding the particles first before the liquid has the advantage that the formation of lumps that are likely to occur when the particles are added to the liquid is avoided. Instead of pre-forming the suspension and then transferring it to the container, preparing the solution in the container of the present invention has yet another advantage: When the suspension is added in this way, various In order to provide a substantially uniform suspension in the container, it is necessary to stir the suspension prior to and / or during the transfer of the suspension to the container. This can be avoided by first performing the filling by adding the above-mentioned binding particles.

  Preferably, the shaker includes elements such as indentations or recesses corresponding to fasteners on the container to allow stable fixation. Preferably, the fixation can be reopened. Preferably, the container of the present invention is disposable so that it can be removed from the shaker after the container is empty and can be replaced with a new suspension container filled with a suspension of binding particles.

  In a further preferred aspect of the present invention, the above-mentioned suspension container footprint is elliptical. This shape facilitates a homogeneous distribution of particles when the container is agitated. In addition, the elliptical mounting surface facilitates the recovery of the remaining volume of suspension and also potentially deposited particles in the elongated groove at the bottom of the container. Other geometric shapes that achieve these objectives may be used, such as an essentially circular mounting surface. The combination of the mounting surface described above, the elongated groove and the cover is particularly advantageous in order to provide a homogeneous distribution of binding particles.

Another preferred aspect of the invention is a method of pipetting a suspension of binding particles for the isolation of biological material, said method comprising:
agitating the container (1) containing the suspension of binding particles, wherein the container comprises an inner wall (2) forming an elongated groove (3) in the bottom of the container, the container further comprising Including a cover (4) having an opening (5) and / or through which a linear arrangement of multiple pipettes or pipette tips can be penetrated
b. introducing a linear arrangement of the multiple pipettes or pipette tips into the suspension through the openings and / or penetrable covers
c. including an automated step of sucking at least a portion of the suspension with a linear arrangement of a number of pipettes or pipette tips and releasing the sucked suspension or portions thereof into a number of vessels.

  With respect to the method, it is preferred to stop stirring before step b. In this way, the linear arrangement of multiple pipettes or pipette tips has good access to the suspension and may not damage the suspension by moving parts of the container. Alternatively, the opening of the container cover is sufficiently wide to allow pipetting through the cover during agitation so that the linear arrangement of multiple pipettes or pipette tips does not contact the edge of the opening.

  In sucking up the suspension, it is preferred that enough of the suspension is drawn up at once, allowing a large number of pipetting into several reaction vessels. Preferably, each pipette or pipette tip is pipetted by discharging the sucked suspension into multiple portions. This aspect contributes to the parallelization of the method.

  A preferred aspect of the present invention is the method described above, wherein in step c. The linear arrangement of the multiple pipettes or pipette tips releases the suspension simultaneously into the plurality of vessels. This means that all pipettes or pipette tips in the linear arrangement release the sucked suspension or part of it simultaneously. Thus, for example, multiple wells of a multi-well plate or deep well plate can be filled simultaneously with the suspension.

  In another preferred embodiment, the present invention relates to the aforementioned method, wherein in step c. Said multiple pipettes are operated by independently operating one or more of said multiple pipettes or pipette tips in said linear arrangement. Alternatively, the linear arrangement of pipette tips individually releases the suspension into the multiple tanks.

  In this way, only selected wells of the multi-well plate or deep well plate can be filled simultaneously with the suspension. Such pipetting in a coordinated manner can be controlled by the control unit. The control unit allows the various components of the analysis system to operate and interact accurately with the correct timing, for example to move components such as pipettes or pipette tips along the linear arrangement in a coordinated manner May include software to ensure The control unit may also include a processor that operates a real-time operating system (RTOS), which is a multitasking operating system for real-time applications. In other words, the system processor can manage operational deadlines from real-time forcing, ie events, to system responses not related to system load. The control unit controls in real time that the various units in the system operate and respond accurately according to predetermined instructions.

The present invention also provides a corresponding analysis system. Accordingly, another preferred aspect is an analytical system for providing bound particles for the isolation of biological material, the analytical system comprising:
A linear arrangement of a number of pipettes or pipette tipsa container containing a suspension of binding particles for binding to the biological material, the container comprising an elongated cavity at the bottom thereof, further comprising a cover, The cover has an opening and / or a linear arrangement of the multiple pipettes or pipette tips can be penetrated. It includes a shaker for stirring the container and suspending the bound particles.

  In the analytical system, the components interact as described above with respect to the containers and methods of the present invention.

  Another preferred aspect of the present invention is an analytical system as described above, wherein the system further comprises a plurality of vessels for receiving the suspension of binding particles from a linear arrangement of the multiple pipettes or pipette tips. Including.

  The multiple pipettes or pipette tips preferably discharge the blotted suspension into multiple reservoirs rather than one reservoir to facilitate parallelization of the supply of binding particle suspension. Many of these vessels can contain, for example, reagents and / or different samples from different sources, but all vessels are suspensions of the same bound particles for the isolation of the biological material to be analyzed. Use liquid.

  In this regard, the linear arrangement of the pipettes or pipette tips comprises at least a certain number of pipettes or pipette tips, preferably at least 4, more preferably at least 8, most preferably 8 pipettes or pipette tips. It is preferable.

  Another preferred aspect of the present invention is the analytical system described above, which further comprises a separation station for isolating the biological material with the binding particles.

  A “separation station” is a device or component of an analytical system that allows the isolation of bound particles from other materials present in a liquid sample. Such separation stations may include, for example, without limitation, centrifuges, racks with filter tubes, magnets, or other suitable components. In a preferred embodiment of the present invention, the separation station includes one or more magnets. Preferably, one or more magnets are used for the separation of magnetic particles, preferably magnetic glass particles. For example, when a liquid sample and binding particles are combined together in a well in a multi-well plate, one or more magnets included in the separation station to attract magnetic particles and subsequently separate them from the surrounding liquid For example, by introducing a magnet into the well, the liquid sample itself can be contacted, or the one or more magnets can approach the outer wall of the well.

  In a preferred embodiment, the separation station comprises a multiwell plate comprising a bath with an opening on the top surface of the multiwell plate and a closed bottom. The vessel includes an upper portion, a central portion and a bottom portion, the upper portion being connected to the top surface of the multiwell plate and preferably including two long sides and two short sides. The central part preferably has a substantially vertical cut surface with two long sides, and the vessels are preferably arranged in a row. Preferably, a continuous space is arranged between two adjacent rows to selectively contact at least one magnet mounted on the fixture with the sides in at least two Z positions. The separation station preferably further comprises a magnetic separation station comprising at least one fixture. Preferably, the fixture includes at least one magnet that generates a magnetic field. Preferably, there is a moving mechanism for moving the at least one fixture including the at least one magnet vertically between at least a first and a second position with respect to the reservoir of the multiwell plate. Preferably, the at least two Z positions of the tub include a side wall and a bottom of the tub. The magnetic field of the at least one magnet preferably attracts magnetic particles to the inner surface of the container adjacent to the at least one magnet when the at least one magnet is in the first position. The effect of the magnetic field is less when the at least one magnet is in the second position than when the at least one magnet is in the first position. Preferably, the fixture including the at least one magnet includes a frame. Preferably, the separation station includes a frame for receiving the multiwell plate and a latch-clip for mounting the multiwell plate. Preferably, the separation station includes two types of magnets. A second preferred embodiment includes a spring that applies pressure to the frame containing the magnet, and the magnet is pressurized against the multi-well plate reservoir. Preferably, the first magnet is constructed and arranged to interact with a multi-well plate reservoir to apply a magnetic field to a large volume of liquid containing retained magnetic particles in the vessel. Preferably, the second magnet is constructed and arranged to interact with a multi-well plate reservoir to apply a magnetic field to a small volume of liquid containing magnetic particles maintained in the reservoir. Preferably, the first and second magnets can be moved to different Z positions.

An even more preferred aspect of the invention is the analysis system described above, wherein the system further comprises:
A reaction module containing components of a chemical reaction and / or a biochemical reaction a detection module for detecting a signal caused by an analyte one selected from the group consisting of a storage module for reagents and / or disposables Including the above elements.

  A “reaction module” is a module in which reactions such as polymerase chain reaction (PCR) or antibody binding for analysis of a sample or specific analyte occur. For example, the reaction module can include various vessels such as tubes or plates. The outer boundary or wall of such a container is chemically inert and therefore does not interfere with the analytical reaction taking place therein. If the material to be analyzed is a nucleic acid, there are various methods applicable in this regard, one very significant method being the polymerase chain reaction described above.

  A “detection module” is a module in which detection of a signal, preferably a signal caused by an analyte or control, is performed. The detection module can be, for example, an optical detection unit for detecting the result or effect of an analytical procedure. The optical detection unit may include a light source, for example an optical device such as a xenon lamp, a mirror, a lens, an optical filter, an optical fiber for guiding and filtering light, one or more reference channels, or a CCD camera.

  The “storage module” stores reagents necessary to cause a chemical or biological reaction important for the analysis of the sample of interest. The storage module may also include additional components useful in the methods of the invention, such as disposable items such as pipette tips or tanks used as reaction vessels in the reaction module.

  Preferably, the analysis system of the present invention further includes a control unit for controlling the system components.

  Such a control unit is described above with respect to the method of the present invention.

  It will be appreciated that the preferred embodiment described for the suspension container also applies to the method and analysis system of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS FIG. 1 shows an example of a suspension container (1) of the present invention viewed from above an angle. In this embodiment, the container comprises a frame (7) fitted with a reservoir (8) containing a suspension (not shown) of binding particles. The frame further includes a recess (9) for placing a container in a device such as a shaker, the device including an indentation that fits into the recess. Further, the frame includes an indentation (11) for securing the container. The reservoir includes a knob (12) that can also function on its outer wall for securing or handling the suspension container.

  Also shown is an inclined wall (2) that forms an elongated groove (3) in the bottom of the container at the inner surface of the reservoir. The groove indicated by the dotted line is arranged linearly under and in parallel with the opening (5) in the cover (4) of the container, and the opening includes a penetrable partition wall (10) and passes through the partition wall. The pipette or pipette tip can then be introduced into the suspension in the reservoir.

  The filling opening (6) functions to initially fill the container with a suspension of binding particles, but is preferably subsequently sealed and in this embodiment is not used for recovery of the suspension.

  The flat corner (18) provides a horizontal pole in the cassette so that, for example, the grasper can determine in which direction the container should be inserted in the device, or in which direction, for example, on the shaker. Can understand.

  FIG. 2 is a perspective view from above of a suspension container without a cover. Although the cover is part of the suspension container of the present invention, this figure shows the container without the cover for clarity. The frame (7) includes an outer edge (13) and an inner edge (14), and the inner edge functions as a platform for supporting a cover (not shown).

  The cross-sectional view of FIG. 3 shows the inside of a container holding a suspension of binding particles (15). The binding particles (16) are homogeneously dispersed in the liquid matrix in this figure. When a portion of the suspension is withdrawn from the container, the rest of the suspension collects in an elongated groove (3) formed by the slanted inner wall (2) so that the remaining suspension can be removed from the pipette or pipette tip. It can be quantitatively extracted from the container by a linear arrangement.

  The perspective view of FIG. 4 shows the container having the frame (7), the reservoir (8), the recess (9), the notch (11) and the knob (12) as described above. Furthermore, from this perspective view, a cross-shaped notch (17) can be seen, and one of the bars is arranged below and parallel to the inner groove (3). Cross-shaped notches are useful for mounting containers on devices such as shakers.

  FIG. 5 is a perspective view of the container installed on the shaker (19). It can be seen that the side knob (12) functions to secure the container on the shaker by interaction with the gripping structure (20) on the shaker. It may be important that the container is well secured in response to each movement of the shaker. Each of the indentations (9) and (17) seen in FIG. 1 or 4 may also be useful for securing the container on a shaker.

  FIG. 6 shows the interaction of a multipipette (21) comprising a suspension container and a pipetting needle (22). In 6a, the needle is placed outside the container in an empty state. In 6b, the needle is introduced into the container through a cover opening containing a septum, and in 6c the pipettor containing the needle is lowered considerably so that the needle reaches the elongated groove (3) in the bottom of the container. In this embodiment, the groove is shown to be adapted for interaction with the indicated multipettor so that the suspension can be drawn in an effective manner.

Claims (16)

Suspension of binding particles for isolation of biological materialInner wall (2) forming an elongated groove (3) at the bottom of the suspension container (1)
A cover (4) having an opening (5) and / or through which a linear arrangement of a number of pipettes or pipette tips can be penetrated, said openings being arranged above and in parallel with said elongated groove,
Suspension container containing.   The suspension container of claim 1, wherein the inner walls are inclined at an angle to each other, thereby forming the elongated groove.   The suspension container according to claim 1 or 2, wherein the binding particles include nucleic acid binding particles.   The suspension container according to claim 1, wherein the binding particles include magnetic glass particles. A filling opening for filling the container with a suspension of binding particles (6)
The suspension container according to any of claims 1 to 4, further comprising one or more elements selected from the group of removable foils that seal the cover.   The suspension container according to claim 1, wherein the cover has an opening, and the opening is covered with a penetrable partition wall.   7. Suspension container according to any of claims 1 to 6, adapted to hold a volume of at least 100 ml.   8. A suspension container according to any one of the preceding claims, comprising one or more fasteners for reversibly fixing the container to a shaker.   The suspension container according to any one of claims 1 to 8, wherein an installation surface of the container is elliptical. A method of pipetting a suspension of binding particles for the isolation of biological material comprising:
a. stirring the container (1) containing the suspension of the binding particles, the container includes an inner wall (2) forming an elongated groove (3) in the bottom of the container, and has an opening (5) And / or a cover (4) through which a linear arrangement of multiple pipettes or pipette tips can penetrate
b. introducing a linear arrangement of the multiple pipettes or pipette tips into the suspension through the opening and / or penetrable cover.
c. A method comprising the automated step of sucking at least a portion of the suspension with a linear arrangement of a number of pipettes or pipette tips and releasing the sucked suspension or portion thereof into a number of vessels.   11. The method of claim 10, wherein in step c., A linear arrangement of the multiple pipettes or pipette tips simultaneously discharges the suspension into the multiple reservoirs.   In step c., By linearly operating one or more of the multiple pipettes or pipette tips in the linear arrangement, the linear arrangement of the multiple pipettes or pipette tips may cause the suspension to flow into the multiple tanks. The method according to claim 11, wherein each is released individually. An analytical system for providing bound particles for the isolation of biological material comprising:
A linear arrangement of a number of pipettes or pipette tipscontainers containing a suspension of binding particles for binding to the biological material, the container comprising an elongated cavity at the bottom thereof, further comprising a cover, The analysis system comprising an opening and / or through which a linear arrangement of the multiple pipettes or pipette tips can be penetrated, comprising a shaker for stirring the container and suspending the bound particles.   The analysis system of claim 13, further comprising a plurality of vessels for receiving a suspension of the binding particles from a linear arrangement of the multiple pipettes or pipette tips.   15. The analytical system according to claim 13 or 14, further comprising a separation module for isolation of the biological material with the binding particles. Reaction modules containing components of chemical and / or biochemical reactionsDetection modules for detecting signals caused by analytesStorage modules for reagents and / or disposablesControlling system components The analysis system according to claim 13, further comprising one or more elements selected from the group consisting of:

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