EP4378586A1 - Système intégré pour réactions chimiques, biochimiques ou biologiques dans une microplaque soumise à un gradient de température - Google Patents

Système intégré pour réactions chimiques, biochimiques ou biologiques dans une microplaque soumise à un gradient de température Download PDF

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Publication number
EP4378586A1
EP4378586A1 EP22210890.4A EP22210890A EP4378586A1 EP 4378586 A1 EP4378586 A1 EP 4378586A1 EP 22210890 A EP22210890 A EP 22210890A EP 4378586 A1 EP4378586 A1 EP 4378586A1
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EP
European Patent Office
Prior art keywords
disposable
tempering
block
mtp
gradient
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.)
Pending
Application number
EP22210890.4A
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German (de)
English (en)
Inventor
Pascal Lambertz
Torsten Feller
Mike Küster
Philipp PFLÜGER
Loretta Hamacher
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.)
Bayer AG
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Bayer AG
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Filing date
Publication date
Application filed by Bayer AG filed Critical Bayer AG
Priority to EP22210890.4A priority Critical patent/EP4378586A1/fr
Priority to PCT/EP2023/082841 priority patent/WO2024115272A1/fr
Publication of EP4378586A1 publication Critical patent/EP4378586A1/fr
Pending legal-status Critical Current

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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
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/54—Heating or cooling apparatus; Heat insulating devices using spatial temperature gradients

Definitions

  • the present invention is directed to an integrated system for chemical, biochemical, or biological reactions requiring one or more/ a gradient of set point reaction temperatures in a microplate according to a prescribed protocol, for example but not limited to cellular thermal shift assay (CETSA ® ).
  • a prescribed protocol for example but not limited to cellular thermal shift assay (CETSA ® ).
  • CETSA ® a cellular thermal shift assay
  • the CETSA ® method is described for example in EP 2 699 910 A1 , wherein the differential heating step is conducted in an apparatus adapted for Polymerase Chain Reaction (also referred to as PCR-apparatus or PCR-cycler).
  • an apparatus adapted for Polymerase Chain Reaction also referred to as PCR-apparatus or PCR-cycler.
  • Assays are typically conducted in microplates comprising multiple wells, preferably high-density-microtiter plateshaving a large number of wells.
  • the implementation of a reliable and precise method on samples located in a microplate requires a precise and over the entire microplate uniform application of the process parameters.
  • Uniform process parameters within the present application means a homogenous linear temperature profile across the microplate along a heating line during heat up.
  • Temperature profile of a microplate can be obtained by measuring surface temperature of the microplate in the tempering unit using an infrared camera and computing a temperature linescan across the microplate (e. g. Fig. 4A/B ). Such a profile is described by a temperature gradient of the microplate, describing the direction and rate at which the temperature changes expressed in temperature units per unit length.
  • Applicant assumed that some limitations mentioned above are caused by an inhomogeneous temperature profile during heat up in a PCR cycler.
  • the solution shall allow processing the method in one microplate, without transferring samples between plates with different characteristics.
  • reaction A system for conducting one or more chemical, biochemical, or biological reactions (together referred to as reaction) in a disposable comprising one or more wells at one or more set point reaction temperatures is claimed.
  • the disposable is preferably a microtiter plate or microplate.
  • Bio reactions refers to molecular biological, cellular reactions, reaction using cellular tissue, means an ensemble of cells, viruses or phages.
  • Cells refers to biological cells, such as animal cells, plant cells, yeast, fungi, or bacteria.
  • the system is composed of independent modules which are operatively linked to each other by transport means for transport and placement of a disposable from and into the modules as required for automated implementation of a predefined reaction protocol.
  • the system of the invention comprises:
  • the tempering block with the linear temperature profile comprises at least one Peltier element temperable at a first temperature T1 and at least one Peltier element temperable at a second temperature T2, wherein T1 is higher than T2, and both Peltier elements are contacting the thermoconductive block outside of the positioning area for the disposable so that the linear temperature profile can be generated in the thermoconductive block between the two elements and conducted to the disposable when contacting the thermoconductive block.
  • the at least one tempering block(s) are positioned to temper the first and / or the second heating surface of the disposable placed in the positioning area.
  • the disposable can be submitted to the linear temperature profile on both heating surfaces by way of positioned tempering blocks with the linear temperature profile.
  • emper or "tempering” as used herein refers to maintaining temperature at set point(s).
  • a “tempering block” is an arrangement capable of reaching (heating, cooling) and maintaining temperature.
  • a “temperable” element is capable of tempering, means capable of reaching and maintaining a temperature.
  • positioning area refers to the functional surface of a thermoconductive block capable of tempering the disposable while contacting the positioning area.
  • the positioning area is also referred to active area.
  • the positioning area can be adapted for better positioning of the disposable e. g. in form of a positioning stage.
  • the first pelter element at the first temperature T1 is a heating Peltier element, means first temperature T1 is set higher than room temperature and the second the second pelter element at the second temperature T2 is a cooling Peltier element, means first temperature T2 is set below room temperature.
  • the at least one tempering block are positioned to temper the first and / or the second heating surface of the disposable when placed in the positioning area.
  • the disposable can be submitted to the linear temperature profile on both heating surfaces by way of positioned gradient tempering blocks set with the (same) linear temperature profile.
  • the system further comprises at least one further tempering unit TUx temperable at a temperature Tx comprising at least one tempering block (also referred to as temperature controlled blocks) comprising at least one Peltier element contacting a thermoconductive block, opposite to the Peltier element(s) a flat-surfaced area for positioning the at least one disposable on the thermoconductive block, said tempering block being configured to uniformly temper the disposable when contacting the thermoconductive block.
  • at least one tempering unit TUx temperable at a temperature Tx comprising at least one tempering block (also referred to as temperature controlled blocks) comprising at least one Peltier element contacting a thermoconductive block, opposite to the Peltier element(s) a flat-surfaced area for positioning the at least one disposable on the thermoconductive block, said tempering block being configured to uniformly temper the disposable when contacting the thermoconductive block.
  • the disposable when placed in the tempering unit (TUx) can be tempered on both heating surfaces by way of positioned tempering blocks
  • system comprises at least one tempering unit TUx per set point reaction temperatures of the prescribed reaction protocol.
  • the system comprises one tempering unit TUx for rapid temperature removal also referred to as cooling unit.
  • control unit is configured to activate the cooling unit(s) for the implementation of the prescribed protocol for the one or more chemical, biochemical, or biological reactions, in particular while the disposable is contacting the thermoconductive block of the tempering unit.
  • system further comprises transport means comprising a carrier, preferred a carrier frame, in which the disposable is placed.
  • the transport means further comprises moving means for moving the disposable into and/or out of the gradient tempering unit GTUx and /or the tempering unit TUx, aligning it with the respective at least one positioning area(s) in an internal space.
  • the gradient tempering unit(s) GTUx and / or the tempering units TUx comprise a second tempering block arranged so that the disposable can be positioned in an internal space between the first and the second thermoconductive blocks and evenly clamped between said blocks using the moving means.
  • system further comprises moving means for contacting the one or more the positioning areas of the one or more thermoconductive blocks with the one or both heating surfaces of the disposable positioned in the internal space.
  • system further comprises one or more clamping mechanism capable of clamping the disposable between the first and the second tempering blocks.
  • control unit is configured to activate the transport means, the moving means and / or the clamping means for the implementation of the prescribed protocol for the one or more chemical, biochemical, or biological reactions.
  • the gradient tempering unit GTUx is configured to create a linear temperature gradient ranging from the left shorter side of the disposable to the right shorter side of the disposable.
  • the disposable is placed in the gradient tempering unit GTUx so that the direction of the temperature gradient is along the large side of the disposable, so a large temperature gradient is created.
  • the system further comprises a read-out/imaging unit I, wherein said imaging unit I comprises an imaging device capable of capturing an image of a scene comprising at least one well of the disposable from the well openings side.
  • said imaging unit I is configured to capture an image of a scene covering all the wells of the disposable.
  • the imaging unit I also comprises one or more lighting elements for adequate illumination of the disposable during image acquisition.
  • the surface of at least the positioning area of the thermoconductive block is complementary to the respective heating surface of the disposable (flat or structured). It is most preferred that the bottom of the disposable is flat.
  • the disposable is chosen to be suitable for culturing/adhering of cells and / or appropriate to one or more read-out methods of the read-out unit/imaging unit I, most preferably both, so that fluid transfer is limited or avoided.
  • the disposable is suitable for luminescent / fluorescent readout.
  • read-out of the disposable can be conducted in a mass spectrometer, for example for proteome analysis by way of measurements of aggregation of all proteins within a cell sample.
  • the well openings of the disposable can be sealed by means of a thin transparent sealing foil, the sealing foil building the second heating surface.
  • reaction mixture means a fluid comprising several biological or chemical items or elements, including but not limited to tissues, cells, compounds and / or substances, together able to cause reaction and transformation of original items.
  • one gradient tempering unit GTUx is used per reaction temperature gradient prescribed by the reaction protocol, said gradient tempering unit GTUx being set to the prescribed temperature gradient by the control unit.
  • the disposable is submitted to the linear temperature profile on both heating surfaces by way of positioned tempering blocks set with the linear temperature profile.
  • the linear temperature profile of the tempering block contacting the second heating surface of the disposable is set with at least one Peltier element temperable at a first temperature T'1 and at least one Peltier element temperable at a second temperature T'2, wherein T'1 is higher than T'2 and wherein T'1 minus T'2 equals T1 minus T2.
  • one tempering unit TUx is used per prescribed reaction temperature of the reaction protocol, said tempering unit TUx being set to the prescribed temperature by the control unit.
  • the method further comprises transporting the disposable to / positioning the disposable within the imaging unit I and capturing an image of at least one well of the disposable.
  • the solution as described is particularly suitable for cellular thermal shift assays (CETSA ® ) but not limited thereto.
  • the solution as described is particularly suitable for high throughput reactions or assay in a disposable, wherein a gradient is needed.
  • a disposable comprising 1536 wells can be used, wherein the wells of the disposable each have a volume of at most 10 ⁇ l, in particular 0.3 to 6 ⁇ l, in particular 0.5 to 4 ⁇ l, in particular 5 4 ⁇ l, in particular 1 ⁇ l.
  • Any well section can be used; typically, rectangular or circular is used. It is clear to the skilled person that other disposable formats can be used.
  • the disposable is a microtiter plate (also called MTP or microplate) or a picotiter plate.
  • disposables showing format and patterns according to the recommendation of the Society for Biomolecular Laboratory Automation and Screening are most preferred (ANSI / SLABS 1-2004, ANSI / SBLAS 2-2004, ANSI / SBLAS 3-2004 and ANSI / SBLAS 4-2004).
  • the disposable can be of any formats and patterns, including least 96, 384, 1536 or 3456 wells, preferred 1536 wells, in the established 96 well format (12 ⁇ 8), 384 well format (24 ⁇ 16), 1536 wells format (48 ⁇ 32) or 3456 well format (48x72) of the ANSI standard of the Society for Laboratory Automation and Screening.
  • the body is made of thermoplastic polymer capable of sealing with the sealing foil, preferred made of polystyrene, polypropylene or COC (Cycloolefin-Copolymer) with or without added thermally conductive medium.
  • the disposable may comprise a frame support made of polycarbonate or polystyrol.
  • the flat bottom of the disposable may be defined by a transparent film fixed to the body comprising the wells, said transparent film allowing image acquisition from the bottom side.
  • the disposable can be sealed with a transparent thin sealing foil as established in the art.
  • the transparent thin sealing foil may be made from polycarbonate, polypropylene, cyclic olefins or other plastic materials known to those skilled in the relevant art or from multi-layer films made from two or more clear materials with desired barrier properties as well established in the art. Sealing can be achieved by welding of the sealing foil to the body of the disposable.
  • thermoconductive block(s) Both in the gradient tempering unit GTUx and in the tempering unit TUx, a thermal communication between the heating surface(s) of the disposable and the respective thermoconductive block(s) is achieved to thermally process the liquid samples contained therein.
  • the first heating surface of the disposable is chosen to be adapted to the surface of the corresponding positioning area of the tempering units. In an embodiment.
  • flat-bottom disposable are used.
  • Flat bottom is inter alia advantageous for automation of disposable transport and/or clamping.
  • Bottom thickness can be any, conventional bottom thickness being below 1000 ⁇ m.
  • the disposable is chosen to be suitable for culturing of cells and / or appropriate to one or more read-out methods, in particular luminescent / fluorescent readout, most preferably both, so that fluid transfer is limited or avoided.
  • microplates can be used, such as 1536 Well Microplate, PS, ⁇ Clear ® , LoBase (Greiner Bio-One, Cat. No. 783092), 384 Well Microplate, PS, ⁇ Clear ® (Greiner Bio-One, Cat. No. 781092) without being limited thereto.
  • the gradient tempering unit(s) GTUx and/or the cooling/tempering unit(s) (TUx) comprise a second tempering block arranged so that the disposable can be positioned in an internal space between the first and the second thermoconductive blocks and evenly clamped between said blocks using the moving means.
  • the disposable can be tempered per contact on both tempering surfaces.
  • the positioning area of the second thermoconductive block contacting the second heating surface of the disposable is planar, preferably even, regardless of the wells being sealed by a sealing foil or not.
  • the temperature gradient of the thermoconductive block contacting the first heating surface (bottom side) of the disposable in the gradient tempering unit TUx is a linear temperature gradient (also called temperature profile) TGx.
  • temperature T1 is typically set at 37 °C and T2 is set at T1+TGx.
  • T1 is typically set at 37 °C and T2 is set at T1+TGx.
  • the skilled person will appreciate that the exact parameters for the temperature gradient TGx, T1 and T2 depends on the defined protocol for the reaction of interest.
  • the temperature gradient is set along the length side of the disposable.
  • the solution of the application was found to be capable of providing a homogeneous linear gradient along the length of a standard-sized disposable of about 20 to 45 °C, preferred 25 to 35 °C.
  • the tempering blocks of the gradient tempering unit GTUx each comprise an array of two or three Peltier elements operable at a first temperature T1 and an array of two or three Peltier elements operable at a second temperature T2, said arrays contacting the thermoconductive block outside of the positioning area for the disposable from the opposite side of said positioning area ( Fig. 1 or 6 ), so that the linear temperature profile is generated between the two arrays and conducted to the disposable when contacting the positioning area of the thermoconductive block.
  • the adequate number of Peltier elements per array can be optimized in view of the performance thereof in relation to the geometry of the disposable.
  • the tempering block(s) of the tempering unit TUx comprise a thermoconductive plate, on which the disposable rests, and an array of Peltier elements (i. e. the individual physical unit that converts electrical current into heat/cold) contacting the thermoconductive plate is used Most preferred is an array of six Peltier elements, considering standard MTPs have an aspect ratio of 3:2.
  • the temperatures of the Peltier elements are monitored using temperature sensors, e. g. thermal resistors.
  • temperature sensors e. g. thermal resistors.
  • each Peltier element is provided with a thermal sensor and each Peltier element is controlled separately.
  • the tempering blocks of the gradient tempering unit GTUx and/or the tempering blocks of tempering unit GTUx also comprises a heat sink that dissipates the entire electrical power converted into heat or cold and carries the Peltier elements. Also, a heatsink/fan combination can be used.
  • thermoconductive block in the gradient tempering unit GTUx contacting the second heating surface of the disposable is set with the same linear temperature profile TGx as the thermoconductive block contacting the first heating surface (obtained by the difference between T1 and T2).
  • thermoconductive block in the gradient tempering unit GTUx contacting the second heating surface of the disposable can be set to show the same linear temperature profile TGx at a slightly higher temperature (e. g. +1°C), T'1 and T'2 than the temperatures T1 and T2 set for thermoconductive block contacting the bottom of the disposable.
  • a slightly higher temperature e. g. +1°C
  • T'1 and T'2 the temperatures T1 and T2 set for thermoconductive block contacting the bottom of the disposable.
  • the higher temperature was shown to prevent condensation on the sealing foil inside of the well at the higher temperatures of the profile.
  • alignment of the linear profiles can be improved by way of positioning/alignment rims on the thermoconductive blocks for accurate positioning of corresponding structural elements of the disposable in the positioning areas of the tempering units.
  • the gradient tempering unit(s) GTUx described above was shown to be capable of swiftly creating a large linear temperature gradient ranging from the left shorter side of the microtiter plate to the right shorter side of the microtiter plate across the flat bottom microtiter plates up to the 1536 well format (cf. Fig. 4B ).
  • the temperature of the thermoconductive block for contacting the thin transparent sealing foil on the second heating surface of the disposable in the tempering/cooling unit TUx is constant at a slightly higher temperature than the temperature of the thermoconductive block contacting the bottom side of the disposable. The higher temperature was shown to prevent condensation on the sealing foil inside of the well.
  • thermoconductive block(s) made of metal, e.g. aluminium, or glass, preferred metal, can be used.
  • thermoconductive block contacting the second heating surface is made of glass.
  • An imaging/read-out mean can be used for acquiring images through the glass plate/block, for example for monitoring fluorescence changes. Also, temperature changes in the disposable can be monitored.
  • the system comprises an imaging unit I, wherein said imaging unit I comprises an imaging device.
  • Imaging devices may be mechanical, digital, or electronic viewing device, such as still camera, camcorder, motion picture camera, scanner or any other instrument, equipment, or format capable of recording, storing, or transmitting visual images of an object.
  • a CCD camera e. g. a sCMOS camera, or an image amplifier camera can be used.
  • a camera with an objective can be used.
  • a 35mm F1.6 C-mount objective so full disposable can be imaged.
  • a microscopic objective can be used.
  • imaging unit I can be configured so that single well images are acquired.
  • the system can be implemented for multiplexed immunofluorescence readout, as known for example from PhenoCycler System of Akoya Biosciences. Thereby, the aggregation profile of up to 100 different proteins in a single CETSA ® experiment could be detected.
  • the imaging unit I also comprises one or more lighting elements for adequate illumination of the disposable during image acquisition.
  • a ring light positioned for homogeneous lighting of the disposable over all cavities during image capture is used.
  • filters can be used.
  • the person skilled in the art will appreciate that objective and/or filters depend on use of the device. Among others, excitation and emission filters for image acquisition using photosensitive substances or genetically encoded luminescent- or fluorescent reporters.
  • the imaging unit I may also comprise optical lens(es) and / or mirror(s) for example in case compact device is required.
  • the imaging unit I comprises a support and calibration means for positioning the imaging device, the lighting elements and / or the filters in relation to the disposable for optimal image acquisition.
  • the imaging device can be positioned to acquire images from the second heating surface of the disposable.
  • the imaging device can be positioned to acquire images though a glass plate of one or more of the gradient tempering unit GTUx and / or tempering unit TUx described above.
  • the read-out unit / imaging unit I can comprise a tempering block for tempering the disposable from the side opposite the well openings or sealing foil.
  • the transport means are capable of transporting and positioning the disposable in the read-out unit or imaging unit I for image capture of at least part of the disposable, most preferred of the whole well openings side of the disposable.
  • the imaging unit I comprises a clamping frame or a transparent clamping plate, so the disposable can be clamped between the tempering block and the clamping plate or frame.
  • a clamping mechanism can be used.
  • control unit is configured to control the imaging unit I.
  • Controlling the imaging unit I comprises activating, deactivating or positioning the imaging device, the lighting device, the filters and / or a clamping mechanism as needed for optimal image acquisition.
  • the transport means comprises a moving mechanism or moving means for moving the disposable into and/or out of the internal space of one or more of the gradient tempering units GTUx, of the tempering unit TUx and / or of the imaging unit I, said internal space being defined as the alignment space between the first and the second thermoconductive blocks (positioning areas thereof) in the gradient tempering units GTUx or in the tempering unit TUx or the alignment space between a tempering block and a clamping plate or frame in the imaging unit I ( Fig 4 ).
  • the transport means comprises at least one horizontal drive and the disposable is positioned on a moving carrier for transport and positioning in the gradient tempering unit(s) GTUx, in the tempering unit(s) TUx and/or in the imaging unit I as required by the reaction protocol.
  • the carrier can be movable between the internal spaces and an external disposable loading position outside an instrument frame/casing for loading and/or unloading the microplate to/from the carrier.
  • the carrier is movably mounted to a base for performing a repetitive, bidirectional movement between the internal and external disposable positions.
  • the moving mechanism is configured as carrier driving mechanism for driving the carrier in either of the two directions, means for driving the carrier into the internal and external space(s) or microplate positions, respectively.
  • the carrier is slidably mounted to the horizontal drive(s) enabling a repetitive, bidirectional movement between the processing positions inside the system for thermally processing the reaction products and a loading position outside the system for loading or unloading the disposable on/from the carrier.
  • the transport means comprise one or more stopping plates arranged on the horizontal for precise positioning of the carrier and disposal within the internal space of a unit in alignment with its positioning areas. In an embodiment one stopping plate per unit is used.
  • the system comprises an automated carrier driving mechanism such as a motor-based belt-or wheel-drive for automatically moving the carrier between the processing and loading positions. Since such driving mechanism is well-known to those of skill in the art, it need not be further elucidated herein.
  • an automated carrier driving mechanism such as a motor-based belt-or wheel-drive for automatically moving the carrier between the processing and loading positions. Since such driving mechanism is well-known to those of skill in the art, it need not be further elucidated herein.
  • horizontal and vertical motors e. g. servo or stepper motors, are used to transport the microplate assembly (means microplate and carrier) to contact with the top surfaces of the tempering blocks.
  • the disposable is positioned in the carrier on a metal heating fixture shaped to closely conform to the disposable, in particular to its frame support, and to the positioning stage/areas if the tempering blocks.
  • the system comprises one or more clamping mechanisms for clamping the disposable in the internal space of the gradient tempering unit(s) GTUx, of the tempering unit(s) TUx and/or in the imaging unit I.
  • the clamping mechanism is capable of moving one or both tempering blocks in relation to each other to allow clamping the disposable between the heating blocks after the disposable is properly positioned in the internal space between these by the transport system. Accordingly, a full contact on both sides of the disposable can advantageously be obtained by clamping.
  • the imaging unit I can comprise a tempering block.
  • a clamping mechanism can move the tempering block in relation to a clamping plate/frame for positioning of the disposable in the imaging unit I.
  • At least one of the tempering blocks in each tempering unit (GTUx or TUx) or one of the tempering blocks or the clamping plate/frame in the imaging unit I is spring mounted for smooth clamping of the disposable.
  • a force measurement can be implemented using the spring constant and the stroke of the spring-mounted element.
  • clamping of the disposable can be achieved by a vertical motor or a lever moving the lower tempering block upwards along a vertical drive and pressing the disposable against a spring-mounted upper tempering block.
  • a force measurement may be implemented using the spring constant and the stroke of the upper tempering block.
  • the same clamping mechanism is used for clamping the disposable in the tempering units (GTUx and/or TUx) and/or in the imaging unit I.
  • one vertical motor or lever can be used to the lower tempering block(s) in the different units (tempering GTUx and/or TUx and/or imaging unit I) upwards.
  • spacing/alignment blocks can be used to achieve aligned positioning of the disposable in the units, in particular by way of aligning the positioning areas of the tempering units (GTUx and/or TUx) and / or of the imaging unit I.
  • a tempering block may be mounted on a spacing/alignment block for better positioning.
  • means for clamping can cause the disposable to be pressed down onto the tempering block and / or lower temperature-controlled platen.
  • control unit can be configured to activate and / or control the tempering unit, the transport means and / or the clamping mechanism according to the predefined reaction protocol.
  • this embodiment Compared to a static embodiment (one temperature-controlled heating/cooling unit), this embodiment has the advantage of not having to wait for a tempering block to transition to next set point temperature, which can speed up test time significantly.
  • the system of the invention may comprise a module for providing all reagents necessary for the reaction at in the right concentration into the disposable.
  • a pipetting robot or a pipetting unit can be used as unit for providing all reagents within the present invention.
  • the system of the invention is an instrument for incubating, thermally treating or otherwise processing liquid samples such as an automated thermocycler enabling liquid reaction mixtures to be put through a series of temperature excursions, e.g., for performing cellular thermal shift assays (CETSA ® ), purified protein thermal shift assays (TSA) or any other protein denaturation or aggregation assays.
  • CETSA ® cellular thermal shift assays
  • TSA purified protein thermal shift assays
  • any other protein denaturation or aggregation assays any other protein denaturation or aggregation assays.
  • the instrument is being used for chemically processing liquid samples, e.g., by performing tests or assays related to immunochemical or clinical-chemical analysis items.
  • the system typically requires a user programmable computer system which is configured for controlling the system through a control unit.
  • the user enters the reaction protocol into the system or selects adequate reaction protocol from a database comprising a collection of reaction protocols by way of a user interface.
  • the solution of the invention is particularly useful for automatically conducting chemical, biochemical or biological reactions in particular high throughput reactions and assays.
  • a further object of the invention is a method for use of the system of the invention described above, comprising the following steps:
  • the method further comprises transporting to and positioning the disposable in imaging unit I and capturing an image of at least one well of the disposable.
  • one gradient tempering unit GTUx is used per prescribed reaction temperature gradient of the reaction protocol.
  • the temperature of the TUx is below room temperature to achieve fast cooling of the samples after the gradient step. It is preferred that one tempering unit TUx is used per prescribed reaction temperature of the reaction protocol.
  • the temperature of the second tempering block (contacting sealing foil) in a tempering unit TUx is set at a slightly higher temperature than a highest prescribed reaction temperature of the reaction protocol.
  • a system for conducting one or more chemical, biochemical or biological reactions in one or more wells of a disposable at one or more set point reaction temperatures comprising:
  • the at least one tempering block are positioned to temper the first and / or the second heating surface of the disposable when placed on the positioning area.
  • the disposable can be submitted to the linear temperature profile on both heating surfaces by way of positioned heating blocks set with the linear temperature profile.
  • the system further comprises at least one tempering unit TUx temperable at a temperature Tx comprising at least one heating block comprising at least two Peltier elements contacting a thermoconductive block, opposite to the Peltier elements a flat-surfaced area for positioning the at least one disposable on the thermoconductive block, said heating block being configured to uniformly temper the disposable when contacting the thermoconductive block.
  • at least one tempering unit TUx temperable at a temperature Tx comprising at least one heating block comprising at least two Peltier elements contacting a thermoconductive block, opposite to the Peltier elements a flat-surfaced area for positioning the at least one disposable on the thermoconductive block, said heating block being configured to uniformly temper the disposable when contacting the thermoconductive block.
  • the disposable when placed in the tempering unit TUx can be tempered on both heating surfaces by way of positioned heating blocks.
  • system further comprises transport means capable of moving the disposable into and/or out of the gradient tempering unit GTUx and /or the tempering unit TUx, aligning it with the at least one positioning area(s) in an internal space.
  • system further comprises a moving means configured for contacting the one or more the positioning areas of the one or more thermoconductive blocks with the one or both heating surfaces of the disposable when positioned in the internal space.
  • system further comprises a one or more clamping mechanism capable of clamping the disposable between the first and the second heating blocks.
  • control unit is configured to control the transport means, the moving means and / or the clamping mechanism for the implementation of the prescribed protocol for the one or more chemical, biochemical, or biological reactions.
  • the system further comprises an imaging unit I, wherein said imaging unit I comprises, an imaging device capable of capturing an image of at least one well of the disposable from the sealed side.
  • a further object of the present application is a method for use of the system described above, comprising:
  • one gradient tempering unit GTUx is used per reaction temperature gradient prescribed by the reaction protocol, said gradient tempering unit GTUx being set to the prescribed temperature gradient by the control unit.
  • the disposable is submitted to the linear temperature profile on both heating surfaces by way of positioned heating blocks set to the prescribed temperature gradient.
  • the system further comprises one tempering unit TUx per prescribed reaction temperature of the reaction protocol, said tempering unit TUx being set to the prescribed temperature by the control unit and wherein the disposable is transported to and positioned in the tempering unit TUx for tempering, preferably cooling, according to the reaction protocol.
  • the method further comprises transporting the disposable to / positioning the disposable in an imaging unit I and capturing an image of at least one well of the disposable.
  • the solution can be used for cellular thermal shift assays (CETSA ® ), purified protein thermal shift assays (TSA) or any other protein denaturation or aggregation assays.
  • CETSA ® cellular thermal shift assays
  • TSA purified protein thermal shift assays
  • the solution allows high throughput reactions or assays.
  • FIG. 1 shows a schematic representation of 3-dimensional open view of a tempering block 10 of the gradient tempering unit GTUx.
  • Peltier elements 12, 13 respectively temperable at T1 and T2, are positioned outside of the positioning stage 14, when tempering block 10 is operable.
  • the Peltier elements 12, 13 are housed by tempering block housing 19 positioned above an heatsink 17 (not shown); heat is evacuated from heatsink 17.
  • two Peltier elements are used for each temperature; each Peltier element is controllable by way of a temperature sensor fed by feedline 15. Pt100 sensors can be used.
  • thermoconductive block 11 is contacted with the heating elements by way of positioning screws 16 for accurate alignment.
  • the disposable MTP contacts aluminium block 11 (thermoconductive block 11) on positioning stage 14.
  • thermoconductive block 11 comprises positioning rims 21 for accurate positioning of disposable MTP on positioning stage 14.
  • FIG. 2 shows an oblique view of a montage diagram of a system comprising one gradient tempering unit GTUx and one tempering unit TUx.
  • Gradient tempering unit GTUx and tempering unit TUx each comprise a first/lower and a second/upper tempering block 10a, 10b and 40a, 40b respectively.
  • the disposable MTP is transported in one or the other tempering units GTUx / TUx in the internal space 20 (not shown) between the first and the second tempering blocks and clamped between said tempering blocks for a period prescribed by an experiment protocol.
  • disposable MTP is transported between a load position 51 for the introduction of disposable MTP in the device and the tempering units using a horizontal transport means for transport along a transport lane connecting the internal spaces 18 of the tempering units GTUx and GTUx.
  • disposable MTP is placed on a carrier 50 movable on a sleigh 53 by way of a vertical drive 53, said sleigh 53 being movable into / out of the internal space 18 (not shown) of each tempering unit on a horizontal drive 30 using a horizontal lever 31.
  • disposable MTP engaged in carrier 50 is transported by horizontal lever 31 (a stepper motor can be used) along the horizontal drive 30 into the internal space 18 (not shown) of the gradient tempering unit GTUx, aligned with the positioning areas/stages 14a, 14b, where it is pressed and held against the said positioning areas by moving the gradient tempering blocks 10a, 10b against the disposable MTP (also referred to as clamping mechanism). Reaction is allowed to take place according to the predefined protocol.
  • the disposable MTP in carrier 50 is released from the gradient tempering unit GTUx by opening the clamping mechanism and moved into the internal space of the tempering unit TUx, between the tempering blocks 40a,40b in alignment with positioning areas/stages 44a, 44b for homogeneous cooling according to the predefined protocol.
  • Disposable MTP can be transported to an imaging unit I (not shown) for image acquisition or protein quantification via mass spectrometry.
  • Gradient tempering blocks 10a, 10b comprise thermoconductive block 11a, 11b tempered by Peltier elements temperable at a first temperature T1 and Peltier elements temperable at a second temperature T2 to build desired temperature gradient in the positioning area/stage and in the disposable MTP while positioned in the gradient tempering unit GTUx; heat is released from each gradient tempering block by way of heatsinks 17a, 17b respectively.
  • tempering blocks 40a, 40b of the tempering unit TUx comprise thermoconductive blocks tempered by Peltier elements operable at a set point temperature; heat is released by way of heatsinks 46a (hidden), 46b.
  • clamping of disposable MTP is achieved by moving one or both tempering blocks along vertical drive 32 using horizontal lever 33 (a vertical motor can be used) and pressing the tempering block(s) against the disposable MTP.
  • the tempering blocks can be spring mounted; a force measurement can be implemented using the spring constant and the respective stroke of the tempering block(s) (not shown).
  • the fixed elements of the gradient tempering unit GTUx and tempering unit TUx are mounted on frame 70, the tempering units can be isolated from each other except for the transport lane by way of segments of separating wall 71.
  • each 1536 flat-bottom microplate was filled according to the to the protocol of Shaw et al 2018 and sealed with an optically clear, permanently adhesive film (Applied Biosystems, 4311971). Commercially available 1536 flat-bottom microplates were used. Each microplate was centrifuged, and the assay was conducted in a qPCR cycler of Screening Technology.
  • Figure 3 shows experimental curves obtained with Androgen receptor CETSA ® on flat bottom 1536 plates tempered at several discrete temperatures over a predefined range using a qPCR cycler as described in PCT/EP2022/077322 compared to published androgen receptor CETSA ® on V-bottom 384 plates using a commercially available PCR cycler (3B, Shaw et al 2018). Obtained curves are in line. Heating of flat bottom 1536 well plates leads to thermal aggregation of the protein of interest and binding of a small molecule reduces aggregation, thus confirming that flat bottom 1536 well plates are amenable for CETSA ® .
  • Sensoquest ® Gradient cycler is the only commercially available device designed to perform needed gradient across a flat-bottom microplate. Said cycler was chosen for further comparison experiments.
  • the microtiter plate (1536 Well Microplate, PS, ⁇ Clear ® , LoBase (Greiner Bio-One, Cat. No. 783092) was left on the Sensoquest ® gradient cycler for three minutes. Directly after the run, the surface temperature across the microplate was acquired using an infrared camera. Temperature linescan across the microplate ( Fig. 4A ) shows the temperature gradient of a flat-bottomed 1536 microtiter plate produced by the Sensoquest ® gradient cycler. The bracket indicates the positions on the plate, where a linear temperature gradient occurs. Sensoquest ® cycler was found to only generate a poorly homogene adequately linear thermal gradient of about 13 °C (very small) after 5 minutes.
  • the region with adequate linear gradient is limited by the heating and cooling Peltier elements being located directly under the thermoconductive block contacting the floor of the microplate.
  • the heating lid that presses the plate onto the tempering block can only perform a homogenous temperature which; this may interfere with the gradient produced by the tempering block contacting the floor of the microplate.
  • Figure 4B shows the temperature gradient of a 1536 well microtiter plate obtained in the system of the invention.
  • the microplate was pressed between gradient tempering block 10a and gradient tempering block 10b for 5 seconds, 30 seconds, 60 seconds or 180 seconds respectively.
  • the surface temperature of the microplate was acquired using an infrared camera
  • Fig. 4B show a temperature gradient/linescan across the plate. Used system generates a linear temperature gradient of about 26 °C on 1536 well plates and heats up the plate within 60 seconds. This is a maj or improvement compared to the linescan across the plate in a Sensoquest ® cycler (4A).
  • the androgen receptor cell line was seeded on flat-bottom 1536 well microplates and either measured in suspension or after adhering to the microplate. In both cases, cells were incubated with DHT or DMSO for one hour prior to heating the cells for one minute and cooling the cells for one minute in the system of the invention. After addition of CETSA reagents, both plates were measured in a luminescence reader.
  • Fig 5 shows thermal aggregation curves in Androgen receptor CETSA ® with suspension cells (5A) or adherent (5B) cells seeded on flat-bottom 1536 well microplates processed according to the invention. In both cases, a clear shift of the thermal aggregation curve was observed for cells treated with DHT.
  • This experiment shows that the current invention enables conducting CETSA ® on one flat bottom 1536 well microtiter plate. Only one microtiter plate was required per experiment.
  • FIG. 6 shows a schematic representation of a CETSA ® experiment using the solution of the invention, wherein main system components are represented in cross section.
  • Said system comprises a gradient tempering unit GTUx, a tempering unit TUx and an imaging unit I.
  • the horizontal transport lane is represented by the horizontal arrows; the clamping mechanism is not represented
  • the simplified imaging unit I comprises imaging device 60.
  • Tempering units GTUx and TUx comprise a first/lower and a second/upper tempering blocks 10a, 10 and 40a, 40b respectively.
  • the disposable MTP is positioned in the internal space 20 of one or the other tempering units and clamped between the positioning areas 14a, 14b or 44a, 44b respectively for a period as prescribed by the experiment protocol, then transported to the next tempering unit for further reaction or to the imaging unit I for image acquisition. All necessary steps can be performed on/in the same disposable MTP.
  • the representation also shows in cross section the array of Peltier elements 12a, 13a and 12b, 13b in relation to thermoconductive block 11a, 11b and positioning areas 14a, 14b in the gradient tempering unit GTUx compared to the array of Peltier elements 42a and 42b in relation to thermoconductive block 41a, 41b and positioning area 44a, 44b of the tempering unit TUx.
  • Two V-bottom PCR plates MTP2 and MTP3 for high temperature range heating (T2 to T3) and low temperature range heating (T1 to T2) respectively are used.
  • Gradient is achieved by way of Peltier elements 82 distributed to temper the thermoconductive block 81 and thermoconductive stage 80.
  • Low range gradient is achieved by outer Peltier elements 82a, 82c set at temperature T1 and T2 respectively, with Tl ⁇ T2, assisted by middle Peltier element(s) 82b set at midpoint.
  • high range gradient is achieved by setting the outer Peltier elements at T2 and T3 respectively, with T2 ⁇ T3 respectively assisted by middle Peltier elements at midpoint.
  • Content of each microplate MTP2 and MTP3 is transferred to flat bottom plates MTP4 and MTP5 respectively for read out.
  • a total of 5 microtiter plates (MTP) is needed for obtaining data required for a full melting curve.
  • the system of the invention is the first system that allows CETSA ® of adherent cells on one single microplate.

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  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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EP22210890.4A 2022-12-01 2022-12-01 Système intégré pour réactions chimiques, biochimiques ou biologiques dans une microplaque soumise à un gradient de température Pending EP4378586A1 (fr)

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EP22210890.4A EP4378586A1 (fr) 2022-12-01 2022-12-01 Système intégré pour réactions chimiques, biochimiques ou biologiques dans une microplaque soumise à un gradient de température
PCT/EP2023/082841 WO2024115272A1 (fr) 2022-12-01 2023-11-23 Système intégré pour réactions chimiques, biochimiques ou biologiques dans une plaque de microtitration soumise à un gradient de température

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EP22210890.4A EP4378586A1 (fr) 2022-12-01 2022-12-01 Système intégré pour réactions chimiques, biochimiques ou biologiques dans une microplaque soumise à un gradient de température

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Citations (6)

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Publication number Priority date Publication date Assignee Title
US4679615A (en) * 1984-03-02 1987-07-14 Advanced Products Ltd. Method and apparatus for heating and/or cooling objects simultaneously at different preselected temperatures
DE8814398U1 (de) * 1988-11-17 1989-02-16 Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV, 3400 Göttingen Thermostatiergerät
WO1994001217A1 (fr) * 1992-07-10 1994-01-20 Vertex Pharmaceuticals Incorporated Calorimetre a gradient de temperature
US6767512B1 (en) * 1996-11-08 2004-07-27 Eppendorf Ag Temperature-regulating block with temperature-regulating devices
EP2699910A1 (fr) 2011-04-18 2014-02-26 Evitraproteoma AB Procédés de détermination de la liaison d'un ligand à une protéine cible à l'aide d'un essai de variation thermique
US20210041379A1 (en) * 2018-03-12 2021-02-11 The Penn State Research Foundation Method and apparatus for temperature gradient microfluidics

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4679615A (en) * 1984-03-02 1987-07-14 Advanced Products Ltd. Method and apparatus for heating and/or cooling objects simultaneously at different preselected temperatures
DE8814398U1 (de) * 1988-11-17 1989-02-16 Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV, 3400 Göttingen Thermostatiergerät
WO1994001217A1 (fr) * 1992-07-10 1994-01-20 Vertex Pharmaceuticals Incorporated Calorimetre a gradient de temperature
US6767512B1 (en) * 1996-11-08 2004-07-27 Eppendorf Ag Temperature-regulating block with temperature-regulating devices
EP2699910A1 (fr) 2011-04-18 2014-02-26 Evitraproteoma AB Procédés de détermination de la liaison d'un ligand à une protéine cible à l'aide d'un essai de variation thermique
US20210041379A1 (en) * 2018-03-12 2021-02-11 The Penn State Research Foundation Method and apparatus for temperature gradient microfluidics

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Title
SEASHORE-LUDLOW ET AL., BIOCHEMISTRY, vol. 57, 2018, pages 6715 - 6725
SEASHORE-LUDLOW ET AL., SLAS DISCOVERY, vol. 25, no. 2, 2020, pages 118 - 126
SHAW ET AL., SCIENTIFIC REPORTS, vol. 8, 2018, pages 163

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