Patent Application
20240329059
The present invention relates to a device that is a reaction vessel suitable for exerting defined shear force onto a liquid sample, a method for producing the reaction vessel, and to a process for exerting a defined shear force onto a sample, preferably including optical analysis of the sample through a portion of the reaction vessel.
The reaction vessel contains a rotor run on a bearing, preferably on exactly one bearing, the rotor having an axle which at one end has a driving section, suitable for receiving rotational torque from a drive motor that can be arranged adjacent to or at a distance from the driving section. Preferentially the bearing is arranged at a distance from a level of the liquid sample to be arranged in the vessel such that the bearing is not in contact with the liquid.
The reaction vessel contains a stator arranged co-axially with the rotor yielding a defined gap between rotor and stator, allowing the application of a defined shear stress to the liquid. The shear stress is proportional to the rotation frequency of the rotor, the radius of the rotor, and the viscosity of the liquid, and the shear stress is inversely proportional to the gap width between rotor and stator.
The reaction vessel allows for the circulation of the liquid sample when driven by the rotation of the rotor. The geometry of the vessel is designed in a way that the maximum shear stress is applied to the liquid in the section with the minimal gap width between rotor and stator.
The reaction vessel comprises a detection section, preferably arranged at its bottom end, which is at least in part optically transmissible, e.g. for light irradiating the inner volume of the reaction vessel and for detecting radiation passing through and/or radiation emitted from the inner volume of the reaction vessel.
WO 2012/110570 A1 schematically shows a reaction vessel in which a rotor is run on an axle that is run on a bearing located at the lid covering the vessel and a second bearing located at the bottom of the lid, with the rotor having a conical form that is parallel to the conically tapering bottom section of the vessel. In the alternative, the vessel is run on one sleeve arranged in the upper half of a cylindrical vessel, with the rotor extending into a tube section, open at both ends, which tube section is spaced from the rotor and is arranged in the lower half of the vessel with a spacing from the vessel bottom.
WO 2016/001334 A1 shows a reaction vessel within which a stator is inserted with a spacing from the vessel wall and spacing from the central rotor. The rotor is cylindrical with chamfered circumferential edges. The stator has extensions below its portion enclosing the rotor, and a light source and a detector are directed at 90° to one another and at 45° to the common central axis, and are directed into the space formed by the extensions.
An object of the invention is to provide an alternative reaction vessel that is suitable for exerting shear stress onto a liquid, and an alternative process for exerting shear force onto a liquid by using the reaction vessel. A preferred object is to provide a reaction vessel that is set up for effectively exerting shear force on the entire volume of liquid contained in the vessel, and to provide a vessel that allows optical detection of a representative portion of the liquid.
The invention achieves the object by the features of the claims, and specifically by providing a reaction vessel that is suitable for subjecting the entire volume of a liquid contained in the vessel to shear force, while the vessel is set up for circulating the liquid through the vessel, the vessel comprising
The ring-shaped gap of constant radius between the cylindrical rotor section and the cylindrical stator section forms an annular gap with constant cross-section.
Generally, the rotor, the circumferential collar of the rotor, the stator, the optional extension pipe, and the first bearing and the optional second bearing as well as the magnetic shaft drive are coaxial to the shaft, preferably all of the constituents of the vessel are coaxial to a common longitudinal axis, which is e.g. the longitudinal axis of the shaft.
In a detection section having two window sections, the window sections preferably are arranged at 90°, more preferably arranged parallel to one another in opposite sides of the housing wall.
Generally, the stator is arranged with a spacing from the housing, the spacing of the stator from the housing forming a channel for liquid to flow in the spacing from one end section of the stator, e.g. from its first end cross-section, to the opposite end section of the stator, e.g. to its second end cross-section, or to the opening of the extension pipe opposite to the stator. Accordingly, also the extension pipe is arranged with a spacing from the housing. As there is no relative movement between the stator and the housing in the spacing that forms the channel for liquid, there is significantly lower shear force in this channel, e.g. essentially no shear force in comparison to the shear force generated between the rotor and the stator, so that in the channel between the stator and the housing liquid can return to the gap between rotor and stator without being exposed to any relevant shear force. Preferably, the spacing of the stator from the housing is at least as large as the radius of the ring-shaped gap between the rotor and the stator in order to generate the maximum shear force between rotor and stator and to avoid significant shear forces when liquid flows through the spacing between stator and housing. The extension pipe is arranged coaxially to a nozzle holding section of the housing, wherein preferably the extension pipe is arranged with a constant spacing from the nozzle holding section of the housing.
Preferably, the cross-sectional area of the extension pipe is at least as large as the cross-sectional area of the ring-shaped gap between the rotor and the stator. Preferably, the extension pipe at each of its cross-sections or levels is circular.
The webs connecting the stator to the housing with a spacing can be formed with one of the stator and the housing with clamping connection between the webs and the respective other one of stator and housing, or the webs can be formed with both of the stator and the housing in one piece, e.g. by additive manufacturing, e.g. using a 3D-printer, for example using the fused deposition molding (FDM) technique. The embodiment, in which the stator, optionally the extension pipe connected to the second end of the stator, and the housing including the connecting webs are formed in one piece has the advantage of less strain and warp on the stator, e.g. compared to a stator clamped by webs into the housing.
Preferably, the rotor is spaced from the first bearing so that for the spacing the shaft is not covered. The spacing of the first bearing from the rotor provides for less contamination of the first bearing by liquid contacting the rotor.
Preferably, the housing in its section between the level of the lid or of the first bearing, e.g. the level of first bearing arranged in the lid, and the level of the rotor, has a diameter which is smaller than the outer or the inner diameter of the stator, and/or smaller than the diameter of the rotor, especially smaller than the diameter of the circumferential collar of the rotor. Such a section of smaller diameter is also interchangeably referred to as a collar section of the housing. Preferably, in the collar section, especially covering the section of the housing between the lid or the first bearing, e.g. the first bearing arranged in the lid, and the rotor, the shaft is not covered by the first bearing nor by the rotor. In a process using the vessel, preferably liquid is introduced into the housing up to at maximum the smallest cross-section of the collar section, and preferably up to a level completely filling the annular gap between the rotor and the stator.
The housing at its first end section preferably has a recess, preferably of cylindrical cross-section, for receiving a lid, e.g. for receiving cylindrical side walls of the lid, preferably with frictional fit, optionally including meshing of grooves and ridges of the outside of cylindrical side walls of the lid with grooves and ridges of the inside surface of the recess of the housing first end section.
Optionally, the housing in its section at the level of the circumferential collar of the rotor has an enlarged inner cross-section, e.g. broadens, compared to its section that is at the level of the second end of the stator and/or compared to the cross-section of housing section between the level of the lid or the level of first bearing, e.g. the first bearing arranged in the lid, and the level of the rotor. An enlarged inner cross-section of the housing at the level of the circumferential collar of the rotor has the advantage of guiding the liquid flow that is moved radially outwards by the rotating rotor, especially by its rotating circumferential collar, into the spacing between the stator and the housing, e.g. for generating a return flow of liquid to the second end cross-section of the stator, or to the cross-sectional opening of an extension pipe arranged opposite to the rotor and/or opposite to the stator.
The shaft preferably is a cylindrical stainless steel rod. Alternatively, the shaft can be a cylindrical rod of high-performance plastic.
Preferably, the first bearing is fixed, e.g. clamped, to the lid, e.g. the first bearing is clamped inside a recess formed by cylindrical side walls of the lid. The first bearing can be a bore in a plate, the bore providing a friction bearing for the shaft, optionally with radial guidance only and/or without axial guidance. The magnetic shaft drive preferably comprises or consists of a holder containing magnets, which holder is fixed, e.g. by clamping, to the shaft. The holder preferably has recesses for holding magnets, preferably recesses for holding one or two pairs of magnets. Along the shaft, the magnetic shaft drive is preferably arranged between the lid, the lid forming a second bearing for the first end of the shaft, and the first bearing which is fixed to the lid, e.g. by clamping in a recess formed by side walls of the lid, so that the magnetic shaft drive holds the shaft between the first bearing and the second bearing also when the first bearing is a bore that allows the shaft to axially slide. For restraining the axial movement of the shaft in the first bearing, the magnetic shaft drive can be fixed to the shaft in a position, in which the magnetic shaft drive is arranged adjacent to the first bearing, e.g. that the magnetic shaft drive contacts the first bearing, and in which position the first end of the shaft runs in the second bearing with the front face of the shaft contacting the recess of the lid with sufficient gap for allowing the shaft to rotate. Preferably, the magnets are neodymium magnets, e.g. cylindrical magnets arranged in borings of the holder. Alternatively other high performance magnets can be used, including hig performance composite magnets fabricated by injection molding
Preferably, the shaft is a cylindrical metal rod, the magnets of the magnetic shaft drive are neodymium magnets, and all of the other constituents of the vessel are of synthetic resin, preferably optically transparent, e.g. polystyrene, polyethylene, polylactic acid, polyethylenterephthalate, polycarbonate, or acrylnitril-butadien-styrol, or nylon.
Preferably, the constituents of synthetic resin that are fixed to one another by clamping on their surfaces that are sliding over one another e.g. when mounting constituents, have grooves that are in perpendicular to the common longitudinal axis, e.g. in perpendicular to the longitudinal axis of the shaft. The grooves are e.g. produced by additive manufacturing, preferably by 3D-printing of a liquified resin 3d-printing, for example using high precision fused deposition molding (FDM) technique.
The magnetic shaft drive is drivable by a magnetic drive that has correspondingly arranged magnets, preferably the same number of pairs of magnets as the shaft drive.
Preferably, the first bearing is connected to the lid, preferably inside a recess formed by cylindrical side walls of the lid, e.g. by clamping, and the lid is connected to the first end section of the housing by clamping. This allows the vessel to be provided as separate elements which are simple to connect by clamping without additional fixation means necessary, e.g. without additional glue, sealing or mechanical fastening devices.
The vessel can be provided as a combination of
The reaction vessel has the advantage that it is set up to exert a shear force between the rotor and the stator and, by rotating the rotor, to provide a pumping action onto the liquid that results in a homogenous processing of the entire liquid within the vessel, resulting in presence of liquid that is representative for the entire liquid in the detection section of the housing. The circumferential collar extending over the radius of the cylindrical rotor section upon rotation can generate sufficient centrifugal force to the liquid for exerting a pumping action onto the liquid such that the liquid circulates through the gap between rotor and stator and returns through the channel formed by the spacing between the housing and the stator.
The pumping action is provided only by rotation of the rotor, and accordingly the driven, e.g. rotating, elements of the vessel preferably consist of the rotor arranged on the second end of the shaft and the shaft drive arranged on the first end of the shaft. Further, the vessel has the advantage of exerting significant shear force onto the liquid essentially only between the rotor and the stator, so that the shear force can be controlled by controlling the rotation speed of the rotor only, while the vessel is set up to avoid generating relevant shear forces outside of the defined ring-shaped gap between rotor and stator. Accordingly, the vessel is set up for subjecting liquid to maximal shear force only in the ring-shaped gap between rotor and stator. The vessel is set up for circulating the entire volume of the liquid through the ring-shaped gap between rotor and stator when rotating the rotor, exerting the maximal shear force for converting a native conformation prion protein to its aggregated state only in the ring-shaped gap between rotor and stator, while the flow of liquid through the vessel other than this ring-shaped gap does not provide shear force sufficient to significantly affect the conversion from native conformation prion protein to its aggregated state.
The ring-shaped gap between the rotor and the stator preferably is in the range of 0.2 to 0.5 mm, and/or between 5% and 30% of the radius of the rotor. The shear stress is proportional to the rotation frequency of the rotor, the radius of the rotor, and the viscosity of the liquid, and the shear stress is inversely proportional to the gap width between rotor and stator.
Preferably, at least two vessels are arranged in parallel and are connected to one another, wherein their housings and the connections between them are one piece, e.g. produced as one piece, and further preferably the material of the housings and of the connections between them is continuous. Therein, the at least two vessels that are parallel and connected to one another are arranged such that their optically transparent windows are arranged in common planes, e.g. their windows are arranged in a plane that is parallel to the line of the row along which the vessels are arranged in the arrangement.
Further, the invention provides for an analytical process in which a liquid sample is introduced into the vessel, preferably by introducing a sample into the housing and/or stator and subsequently arranging the rotor inside the stator, preferably by arranging a rotor mounted on a shaft that is run on a first bearing fixed in a lid, while arranging the lid at the first end section of the housing, rotating the magnetic shaft drive for rotating the rotor, optically detecting the sample at the detection section of the housing, and preferably transmitting the detection result or a medical indication derived from the detection result to the originator of the sample. The originator of the sample can be a medical laboratory, a physician or the patient from whom the sample was taken. Further, the analytical process can be used to determine the effect of a compound for its efficacy in retarding or reversing the formation of aggregated conformation prion protein by adding a compound to the sample or to an aliquot of the sample, and comparing the rate of formation of aggregated conformation prion protein during the process. Accordingly, the process can be used for analysing a sample originating from a patient by adding a compound suspected to have activity on the formation of aggregated conformation prion protein to the sample that originates from a specific patient for detecting the efficacy of the compound to at least delay aggregated prion protein formation in the sample of the originator. The efficacy of a compound to delay the formation of aggregated prion protein includes prevention of, retarding, suppressing and/or reversing the formation of aggregated prion protein. Therein, the process can be used to select a compound for its efficacy in delaying, e.g. retarding, supressing, preventing or reversing the formation of aggregated conformation prion protein for a specific patient sample. Generally, a sample can be a liquid or solid biopsy obtained from a patient, e.g. liquor, serum, tissue. The patient can be a human patient or, especially for research, an animal or a tissue culture. The process is an in vitro process or assay, and the process can be used as a translational assay system e.g. during drug discovery.
The process using the device of the invention can also be employed for screening and selecting compounds for their activity and efficacy in retarding, supressing or reversing the formation of aggregated conformation prion protein, the process comprising the step of introducing a liquid sample comprising native prion protein and/or aggregated conformation prion protein, and adding at least one compound to be screened into the housing and arranging the rotor inside the stator, rotating the magnetic shaft drive for rotating the rotor, optically detecting the sample at the detection section of the housing, for detecting a compound having activity for delaying the formation of aggregated prion protein.
The invention is now described in greater detail by way of an example and with reference to the figures, which show in
Generally, each feature described with reference to the figures is a separate feature of the vessel of the invention, independent from other features.
Inside the housing 1, a stator 10 is arranged, which from its open first end cross-section 11 extends to an opposite open second end cross-section 12, having a cylindrical inner surface 15. At the second end cross-section 12, an extension pipe 13 is connected, which tapers towards the second end section 3 of the housing 1. The extension pipe 13 serves to guide liquid that flows along the extension pipe 13 and the stator 10 through the detection section 5. Preferably, as shown for the extension pipe 13, the extension pipe 13 tapers towards the second end section 3 of the housing 1, forming a nozzle.
The stator 10 is arranged to the housing with a spacing by webs 14. The stator 10 extends along a stator holding section 8 of the housing 1, and preferably the webs 14 extend between the housing 1 and the stator 10 and along the stator holding section 8.
The rotor 20 is arranged co-axially inside the stator 10, the spacing between rotor 20 and cylindrical inner surface of the stator 11 forming a ring-shaped gap, in which upon rotation of the rotor 20 shear force is applied onto liquid. The rotor as a cylindrical section 21 which is arranged within the stator 10, and the cylindrical section 21 at its first end 22 has a terminal circumferential collar 23 extending over the radius of the cylindrical rotor section 21. The rotor collar 23 in accordance with a preferred embodiment also extends over the ring-shaped gap between the rotor 20 and the stator 10.
The opposite second end 24 of the rotor has a flat front surface 25 with a circumferential bevel 26.
The rotor 20 is arranged on the second end 32 of a shaft 30. Opposite the second end 32, the first end 31 of shaft 30 carries a magnetic shaft drive 33, which includes at least two magnets 34. The magnetic shaft drive 33 is fixed to the shaft 30, which runs in a first bearing 35, wherein the shaft 30 optionally is axially displaceable. In the embodiment shown, the magnetic shaft drive 33 has a flat front surface 36 which can run with friction over an adjacent flat front surface 37 of the first bearing 35, which limits the axial movement of the shaft 30. The first bearing 35 is held by clamping inside a cylindrical section 41 of a lid 40, which cylindrical section 41 is clamped to the first end section 2 of housing 1. The lid 40 also closes the cross section of the first end 2 of the housing 1.
In the embodiment shown, all the elements are arranged co-axially to the longitudinal axis 39 of shaft 30.
As preferred,
The section of the housing 1 in which the extension pipe 13 is arranged, is also referred to as nozzle holding section 83 of the housing. Preferably, the extension pipe 13 is only connected to the stator 10, i.e. without webs extending between the extension pipe 13 and the nozzle holding section 83. Preferably, a first section 83a of the nozzle holding section 83 extends for the axial extension of the extension pipe 13 and is spaced from the extension pipe 13 by a constant distance, and the housing 1 adjacent to the first section 83a in a second section 83b of the nozzle holding section 83 can taper towards the second end section 3 of the housing 1. Therein, the second section 83b of the nozzle holding section 83 connects the first section 83a to the second end section 3 of the housing 1 across the portion, into which the extension pipe 13 does not extend.
Generally, the Figures show an embodiment of the vessel according to the invention, in which the vessel is provided as two separate elements, each of which is pre-assembled to be connected to one another to form the hermetically closed reaction vessel by clamping the lid 40 onto the second end section 2 of housing 1, whereby the lid 40 closes the cross section spanned open by the second end section 2 of housing 1, while arranging the rotor 20 co-axially within stator 10.
For the analytical process of the invention, it is preferred to introduce a liquid sample into the housing 1 prior to attaching the lid 40 to the housing.
As an exemplary sample, a recombinant aggregated prion protein derived from a shear-force induced reaction of recombinant native prion protein with a post-mortem brain homogenate sample originating from a synucleopathy disease patient was mixed at a dilution of 1/100000 with 1.5 mg/ml native conformation human α-synuclein in PBS containing 1% Triton X-100 (positive control). The buffer composition and the procedural details were analogous to the previous disclosures WO 2012/110570 A1 and WO 2016/001334 A1. The Fluorophore used for aggregated prion protein detection was Thioflavin T. As a negative control, the same reaction composition was used without the addition of recombinant aggregated prion protein. The vessel generally corresponded to
In the positive controls, aggregated conformation prion protein was building up after about 4 hours (±0.5 hours). In the negative control, aggregated conformation prion protein was beginning to build up after about 14 hours in some replicas, while most did not show signs of aggregated conformation prion buildup during the time of observation. The measurement results are shown in
Generally, detection can be measured through the detection section of the vessel by the change of fluorescence of a fluorescent dye added to the mixture, the dye being specific for aggregated conformation prion protein. Exemplary dyes are e.g. Thioflavin T, Thioflavin S, Congo Red, thiophene-based amyloid ligands like luminescent conjugated polythiophenes (LCP), polythiophene acetic acid (PTAA) and luminescent conjugated oligothiophenes (LCO), Pittsburgh compound B, Aminonaphthalene 2-Cyanoacrylate (ANCA) probes, pegylated phenylbenzoxazole derivatives, Pinacyanol, Chrysamine G, and dyes containing at least one of the following scaffolds: Chalcone, Flavone, Aurone, Stilbene, Diphenyl-1,2,4-oxadiazole, Diphenyl-1,3,4-oxadiazole, Benzothiazole, Benzooxazole, Benzofuran, Imidazopyridine, Benzimidazole, Quinoline, Naphthalene.
In the alternative, the native conformation prion protein can be labelled with a fluorescent dye, e.g. by binding of a fluorescent dye to the native conformation prion protein directly or by an intermediate spacer, e.g. fluorophore derivatives that contain reactive chemical groups such as isothiocyanates (reactive to primary amines, e.g. Lysines), succinylimide esters (reactive towards amino groups to form amido bonds, e.g. N-terminal amino acids), maleimide (reactive to free sulfhydryl groups, e.g. cysteine). Such fluorophore derivatives include cyanines, flurescein, rhodamine, Alexa Fluors, Dylight fluors, ATTO-Tec Dyes, BODIPY Dyes, SETA Dyes, SeTau Dyes, DYOMICS Dyes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21183830.5 | Jul 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/068360 | 7/3/2022 | WO |
