EP4378586A1

EP4378586A1 - Integrated system for chemical, biochemical, or biological reactions in a microplate submitted to a temperature gradient

Info

Publication number: EP4378586A1
Application number: EP22210890.4A
Authority: EP
Inventors: Pascal Lambertz; Torsten Feller; Mike Küster; Philipp PFLÜGER; Loretta Hamacher
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 2022-12-01
Filing date: 2022-12-01
Publication date: 2024-06-05
Also published as: CN120282838A; WO2024115272A1; EP4626611A1; JP2025539480A

Abstract

A system for conducting one or more chemical, biochemical or biological reactions in a disposable comprising one or more wells at one or more set point reaction temperatures, comprising:- at least one disposable comprising the one or more wells in a body, wherein the wells are capable of acting as vessel for the one or more chemical, biochemical or biological reactions requiring one or more prescribed reaction temperatures according to a prescribed protocol, said body comprising a flat bottom side building a first heating surface capable of homogeneously conducting heat into the wells and a flat upper side comprising well openings, optionally sealed by means of a thin sealing foil, building a second heating surface capable of homogeneously conducting heat in the wells;- one or more gradient tempering unit GTUx, each comprising at least one tempering block comprising at least two temperable 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 tempering block being configured to generate a set linear temperature profile in the wells of the disposable(s) when contacting the thermoconductive block;- a control unit comprising one or more processors configured to control the gradient tempering unit(s) GTUx for the implementation of the prescribed protocol for the one or more chemical, biochemical, or biological reactions;wherein the gradient tempering block with set 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 while contacting the thermoconductive block.In particular, the solution can be used for cellular thermal shift assays (CETSA<sup>®</sup>), purified protein thermal shift assays (TSA) or any other protein denaturation or aggregation assays. The solution allows high throughput reactions or assays.

Description

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^®).

Background

In a cellular thermal shift assay (CETSA^®) cells are split into different reaction vessels and each vessel is heated at a different temperature. Next, samples are cooled, cells are lysed, and soluble protein amount is quantified by different means. The amount of soluble protein is smaller in vessels where the initial temperature was higher. This is caused by aggregation of proteins under heat; stabilization of a protein by compound interactions increases the amount of soluble protein by shifting the aggregation of the protein to higher temperatures.
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).
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.
Solutions of the art show the following limitations or major drawbacks:

Commercially available PCR cyclers are compatible with cone-bottom microtiter plates with 96- or 384 reaction vessels, which are not suited for culturing adherent cells and rather low throughput compared to 1536 plates. After heating steps, and for quantifying the amount of soluble protein via a luminescent readout, the sample is transferred to another (micro)plate, which is compatible with used luminescence reader. These additional pipetting steps increase standard deviation of the assay;
The temperature range for a gradient in PCR cyclers is rather small (< 21 °C). Proteins that aggregate over a temperature range > 21 °C cannot be analyzed in one experiment.
PCR cyclers are adapted to generate the required linear temperature profile across the microplate over a heating block; the temperature profile is generated by at least two heating and two cooling Peltiers, typically six Peltier elements distributed under a metal block opposite the positioning area that contacts the microtiter plate from below (cf. fig. 7). This setup is a compromise with the primary function of aPCR cycler of providing a homogenous temperature distribution. However, this compromise leads to a sigmoidal shape of the temperature gradient leading to an uneven distribution of temperature data points within a row of the microplate.
To detect low affinity compound protein interactions, a very short heating and cooling time is required [e. g. Seashore-Ludlow et al, SLAS Discovery 2020, Vol. 25(2) 118-126; Seashore-Ludlow et al, Biochemistry 2018, 57, 6715-6725]. As commercial PCR cyclers perform the heating gradient and the subsequent cooldown of the sample on the same heating block, the heat up and cool down times make this approach rather slow. CETSA protocols conducted in PCR cyclers cannot run under three minutes heating and cooling time; the reaction becomes inhomogeneous under shorter times. Fig. 7 shows a diagram of a CETSA^® experiment conducted in a commercial PCR-cycler. Experiments show that a total of five microtiter plates and three pipetting steps are required for one CETSA^® experiment.

Applicant assumed that some limitations mentioned above are caused by an inhomogeneous temperature profile during heat up in a PCR cycler.
Therefore, there is still need for a solution allowing the required precise and uniform application of process parameters over an entire microplate, also using a high-density microtiter plate that would address cost, thermal response, and uniformity, in particular for CETSA^® experiments. Preferably the solution shall allow processing the method in one microplate, without transferring samples between plates with different characteristics.

SUMMARY OF THE INVENTION

The problem was solved by a system and method as claimed. Exemplary embodiments of the solution can be gathered from the respective dependent claims.
The solution will be elucidated below without distinguishing between the claimed subject-matters (method or system). On the contrary, the following elucidations are intended to apply analogously to all the claimed subjects, irrespective of in which context they occur.
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.
"Biological reactions" as used herein refers to molecular biological, cellular reactions, reaction using cellular tissue, means an ensemble of cells, viruses or phages.
"Cells" as used herein 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:

at least one disposable comprising the one or more wells in a body, wherein the wells are capable of acting as vessel for the one or more reactions requiring one or more prescribed reaction temperatures according to a prescribed protocol, said body comprising a flat bottom side (also called body floor) building a first heating surface capable of homogeneously conducting heat into the wells and a flat upper side comprising well openings building a second heating surface capable of homogeneously conducting heat in the wells;
one or more gradient tempering unit GTUx, each comprising at least one tempering block comprising at least two temperable 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 tempering block being configured to generate a linear temperature profile in the wells of the disposable(s) when contacting the thermoconductive block;
a control unit comprising one or more processors configured to activate the gradient tempering unit(s) for the implementation of the prescribed protocol for the one or more chemical, biochemical, or biological reactions;

Wherein 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.
In an embodiment, 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. In a preferred embodiment, 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.
The terms "temper" 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.
The term "positioning area" as used herein 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, the disposable when placed in the tempering unit (TUx) can be tempered on both heating surfaces by way of positioned tempering blocks
In an embodiment the system comprises at least one tempering unit TUx per set point reaction temperatures of the prescribed reaction protocol.
In an embodiment the system comprises one tempering unit TUx for rapid temperature removal also referred to as cooling unit.
It is preferred that the 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.
In an embodiment, the system further comprises transport means comprising a carrier, preferred a carrier frame, in which the disposable is placed.
In an embodiment 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.
In an embodiment, 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.
In an embodiment, the 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.
In an embodiment, the system further comprises one or more clamping mechanism capable of clamping the disposable between the first and the second tempering blocks.
Preferably the 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.
In an embodiment, 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. In other words, 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.
In an embodiment, 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. In an embodiment, the imaging unit I is configured to capture an image of a scene covering all the wells of the disposable.
In an embodiment the imaging unit I also comprises one or more lighting elements for adequate illumination of the disposable during image acquisition.
In an embodiment, 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.
Most preferably 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. In a preferred embodiment, the disposable is suitable for luminescent / fluorescent readout.
In an embodiment, 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.
In an embodiment, 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.
A further subject matter of the present application is a method for use of the system according to one of the preceding claims, comprising the following steps:

loading one or more reaction mixtures in the wells of a disposable and optionally sealing the upper side of the disposable with a sealing foil;
introducing the disposable in the system, transporting and positioning it in one of the one or more gradient tempering unit GTUx in accordance with the prescribed protocol.

The term "reaction mixture" as used therein 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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 person skilled in the art will appreciate that a gradient tempering unit GTUx can be used as a tempering unit for uniform tempering of a disposable by way of setting the temperatures T1 = T2 and / or T'1=T'2.
In an embodiment, 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.

DETAILLED DESCRIPTION:

The solution is now described in more detail. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the solution unless otherwise claimed. No language in the specifications should be constructed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described, including the best mode known to the inventors for carrying out the invention.
Variations of those preferred embodiments can become apparent to those of ordinary skilled artisans to employ such variations as appropriate, and the inventors intend for the solutions to be practiced otherwise than specifically described herein.
Accordingly, the solution includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the described elements in all possible variations thereof is encompassed by the description unless otherwise indicated herein or otherwise clearly contradicted by context.
In an embodiment, a disposable comprising 1536 wells can be used, wherein the wells of the disposable each have a volume of at most 10 µl, in particular 0.3 to 6 µl, in particular 0.5 to 4 µl, in particular 5 4 µl, in particular 1 µl. 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.
In an embodiment, the disposable is a microtiter plate (also called MTP or microplate) or a picotiter plate.
In connection with the present invention, 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.
In an embodiment, 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. In an embodiment, 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.
In an embodiment, after reactants are filled into the wells, 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.
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.
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 µm.
Most preferably 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.
Commercially available microplates can be used, such as 1536 Well Microplate, PS, µClear^®, LoBase (Greiner Bio-One, Cat. No. 783092), 384 Well Microplate, PS, µClear^® (Greiner Bio-One, Cat. No. 781092) without being limited thereto.
In an embodiment, 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. In this embodiment, the disposable can be tempered per contact on both tempering surfaces.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment temperature 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.
In an embodiment 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.
In an embodiment, 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 skilled person will appreciate that 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.
In an embodiment, 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.
In an embodiment, the temperatures of the Peltier elements are monitored using temperature sensors, e. g. thermal resistors. For most homogeneous tempering, it is preferred that each Peltier element is provided with a thermal sensor and each Peltier element is controlled separately.
In an embodiment, 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.
In an embodiment, the 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).
In an embodiment, the 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. In case a thin transparent sealing foil is used as second heating surface, the higher temperature was shown to prevent condensation on the sealing foil inside of the well at the higher temperatures of the profile.
In an embodiment, 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).
In an embodiment, 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.
In an embodiment, thermoconductive block(s) made of metal, e.g. aluminium, or glass, preferred metal, can be used.
In an embodiment, the 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.
In an embodiment, 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. In an example, a CCD camera, e. g. a sCMOS camera, or an image amplifier camera can be used.
In an embodiment a camera with an objective can be used. In an embodiment a 35mm F1.6 C-mount objective, so full disposable can be imaged. In an embodiment, a microscopic objective can be used In an embodiment, imaging unit I can be configured so that single well images are acquired. In an embodiment, 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.
In an embodiment the imaging unit I also comprises one or more lighting elements for adequate illumination of the disposable during image acquisition. In an embodiment, a ring light positioned for homogeneous lighting of the disposable over all cavities during image capture is used.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, the imaging device can be positioned to acquire images from the second heating surface of the disposable.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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. For this purpose, a clamping mechanism can be used.
In an embodiment, the 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.
In an embodiment, 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).
In an embodiment, 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.
In some embodiments, 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. Specifically, in some embodiments, the carrier is movably mounted to a base for performing a repetitive, bidirectional movement between the internal and external disposable positions. In some embodiments, 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.
In an embodiment, 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.
In an embodiment, 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.
Since such sliding mechanism is well-known to those skilled in the art, it need not be further elucidated herein. In some embodiments, 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. In an embodiment, 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.
In an embodiment 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.
In an embodiment, 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.
In a tempering unit (GUTx or TUx) 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.
In an embodiment, the imaging unit I can comprise a tempering block. In an embodiment, 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.
It is preferred that 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. In an embodiment, a force measurement can be implemented using the spring constant and the stroke of the spring-mounted element.
In an embodiment, 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.
In an embodiment, 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. In particular, 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. In this embodiment, 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. In an embodiment, a tempering block may be mounted on a spacing/alignment block for better positioning.
The skilled person skilled will appreciate that further embodiments for clamping the disposable in the internal space of a unit may be used. For example, means for clamping can cause the disposable to be pressed down onto the tempering block and / or lower temperature-controlled platen.
In an embodiment, the 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.
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.
In an embodiment, 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. As unit for providing all reagents within the present invention, a pipetting robot or a pipetting unit can be used.
In some embodiments, 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.
In some embodiments, 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.
To perform all these steps, the system typically requires a user programmable computer system which is configured for controlling the system through a control unit.
In an embodiment, 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:

loading a reaction mixture in the wells of a disposable and optionally sealing the upper side of the disposable with a sealing foil;
introducing the (optionally sealed) disposable in the system, transporting and positioning it in one of the gradient tempering unit GTUx and / or tempering units TUx in accordance with the prescribed protocol.

In a further embodiment, 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.
It is preferred that one gradient tempering unit GTUx is used per prescribed reaction temperature gradient of the reaction protocol.
It is preferred that 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.
It is also preferred that 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.
Preferred settings for the gradient tempering unit GTUx are as described above.
Objects of the present application are therefore:
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:

at least one disposable comprising the one or more wells in a body, wherein the wells are capable of acting as vessel for the one or more chemical, biochemical or biological reactions requiring one or more prescribed reaction temperatures according to a prescribed protocol, said body comprising a flat bottom side building a first heating surface capable of homogeneously conducting heat into the wells and a flat upper side comprising well openings, optionally sealed by means of a thin sealing foil, building a second heating surface capable of homogeneously conducting heat in the wells;
one or more gradient tempering unit GTUx, each comprising at least one tempering block comprising at least two temperable Peltier elements contacting a thermoconductive block, a flat-surfaced positioning area opposite to the Peltier elements configured for positioning the at least one disposable on the thermoconductive block, said tempering block being configured to generate a set linear temperature profile in the wells of the disposable(s) when contacting the thermoconductive block;
a control unit comprising one or more processors configured to control the gradient tempering unit(s) GTUx for the implementation of the prescribed protocol for the one or more chemical, biochemical, or molecular biological reactions;

In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, the disposable when placed in the tempering unit TUx can be tempered on both heating surfaces by way of positioned heating blocks.
In an embodiment, the 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.
In an embodiment, the 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.
In an embodiment, the system further comprises a one or more clamping mechanism capable of clamping the disposable between the first and the second heating blocks.
In an embodiment, the 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.
In an embodiment, 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:

loading one or more reaction mixtures in the wells of a disposable and optionally sealing the upper side of the disposable with a sealing foil;
introducing the sealed disposable in the system;
transporting and positioning the disposable in one of the one or more gradient tempering unit GTUx in accordance with a prescribed reaction protocol.

In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In an embodiment, 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.
In particular, 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. The solution allows high throughput reactions or assays.
The use of terms "a" and " an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "including", "having", and "containing" are to be construed as open-ended terms (i.e. meaning "including but not limited to") unless otherwise noted.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specifications should be constructed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described, including the best mode known to the inventors for carrying out the invention.
Variations of those preferred embodiments can become apparent to those of ordinary skilled in the art to employ such variations as appropriate, and the inventors intend for the inventions to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subj ect matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a 3-dimensional view of one tempering block of the gradient tempering unit GTUx, showing the location of the Peltier elements respectively temperable at T1 and T2, positioned outside of the positioning area, wherein the disposable contacts the aluminium block (thermoconductive block).
Figure 2 shows an oblique view of a montage diagram of a system according to the invention.
LIST OF REFERENCE NUMERALS
- MTP microplate / disposable / microtiter plate
- GTUx Gradient tempering unit
- 10, 10a, 10b gradient tempering block
- 11, 11a thermoconductive block
- 12, 12a, 12b Peltier element temperable at a first temperature T1
- 13, 13a, 13b Peltier element temperable at a second temperature T2
- 14, 14a, 14b (hidden) positioning area or stage/ active area
- 15 feed line for temperature sensors (Pt100) for Peltier element
- 16, 16a screw
- 17a, 17b heatsink
- 19 tempering block housing
- 20 internal space
- 21 positioning/ alignment rims
- 30 horizontal drive
- 31 horizontal lever
- 32 vertical drive
- 33 vertical lever
- TUx tempering units
- 40, 40a, 40b temperature-controlled block
- 41a, 41b thermoconductive block
- 42a, 42b Peltier elements
- 44a, 44b positioning area/stage
- 46a (hidden), 46b heatsink
- 50 carrier
- 51 load position
- 52 support frame
- 53 vertical drive
- 54 sleigh
- I Imaging unit
- 60 Imaging device
- 70 frame
- 71 separating wall
- 80 thermoconductive stage
- 81 conductive block
- 82a, 82c outer Peltier elements tempered at discrete temperatures T1, T2 or T3, wherein Tl<T2<T3 82b middle Peltier elements tempered at midpoint
Figure 3 shows experimental curves obtained with Androgen receptor CETSA^® on flat bottom 1536 plates using the qPCR cycler by Screening Technology (2A) and published androgen receptor CETSA^® on V-bottom 384 plates using a commercially available PCR cycler (2B, Shaw et al, Scientific Reports, (2018) 8:163 | DOI:10.1038/s41598-017-18650-x).
Figure 4A shows the temperature gradient of a 1536 well microplate obtained using a Sensoquest^® gradient cycler. Figure 4B shows the temperature gradient of a 1536 well microtiter plate obtained using the system of the invention.
Figure 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.
Figure 6 shows a schematic representation of a CETSA^® experiment using the solution of the invention, wherein all necessary steps can be performed on/in the same microtiter plate.
Figure 7 shows a schematic representation of a CETSA^® experiment using a commercially available PCR cycler.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT

Figure 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. In this embodiment, 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. For operation thermoconductive block 11 is contacted with the heating elements by way of positioning screws 16 for accurate alignment. When positioned, the disposable MTP contacts aluminium block 11 (thermoconductive block 11) on positioning stage 14. In this embodiment, thermoconductive block 11 comprises positioning rims 21 for accurate positioning of disposable MTP on positioning stage 14.
Figure 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. During operation of the system, 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. For automatized transport, 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. In this embodiment, 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. During experiment, driven by an automated software program in a control unit (not shown), 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. In this embodiment, 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. In the embodiment of Figure 2, 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). In this embodiment, 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.

Experiments

In an example the solution is used for CETSA^®. This example is explained in somewhat greater detail below, but without any intention of restricting the invention to this embodiment.
CETSA^® Experiments according to the protocol of Shaw et al. were conducted using a cell line that contains a luminescent sensor for the stability of a protein of interest (androgen receptor). In contrast, Shaw et al. used not a luminescent sensor but detection of soluble protein by AlphaScreen^® technology.
Several flat-bottom 1536 well microplates heated at different homogenous temperatures using the qPCR cycler of Screening Technology were used to build full thermal melting curve of the protein of interest (3A) by way of connecting the results from the microplates at different temperatures. The small molecule DHT led to a thermal stabilization of the androgen receptor.
For this purpose, 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^®.
Using homogenous heating of the microplate in the qPCR cycle of Screening Technology, 14 microplates heated at discrete temperatures were needed to generate the complete melting curve of the protein of interest.

Comparison of a Sensoquest^® Gradient cycler with the system of the invention using flat-bottom microplates:

To the best of our knowledge, the 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, µClear^®, 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.
It was assumed that 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. In addition, 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.
To generate the melting curve using only one microtiter plate, a device was required that applies a temperature gradient across the microtiter plate. The system according to Fig. 2 was used for comparison experiments.
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. Directly after the heating, 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 CETSA^® experiment of Shaw et al. was repeated using the solution of the invention and a luminescent sensor for protein stability.
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.
Figure 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. During operation of the system of the invention, 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.
It is to be noted that for one CETSA^® experiment as described by Shaw et al 2018 using a commercially available PCR cycler and schematically represented in Fig. 7, wherein the arrows represent the transfer to a next plate for further processing. Samples must be transported between the different plates because different plates are needed for culturing cells (MTP1), heating the cells with the reaction mixture (MTP2, MTP3) and the luminescent/ fluorescent readout (MTP4 and MTP5). For the heating step, the sample must be split on two plates (MTP2, MTP3) because the temperature gradient the Sensoquest^® cycler can generate in a microplate is not large enough. 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. Similarly 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.
To our knowledge, the system of the invention is the first system that allows CETSA^® of adherent cells on one single microplate.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to the skilled person in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the scope of the appended claims.

Claims

A system for conducting one or more chemical, biochemical, or biological reactions in one or more wells of a disposable (MTP) at one or more set point reaction temperatures, comprising:
- at least one disposable (MTP) comprising the one or more wells in a body, wherein the wells are capable of acting as vessel for the one or more chemical, biochemical or biological reactions requiring one or more prescribed reaction temperatures according to a prescribed protocol, said body comprising a flat bottom side building a first heating surface capable of homogeneously conducting heat into the wells and a flat upper side comprising well openings, optionally sealed by means of a thin sealing foil, building a second heating surface capable of homogeneously conducting heat in the wells;

- one or more gradient tempering unit (GTUx), each comprising at least one gradient tempering block (10) comprising at least two temperable Peltier elements (12, 13) contacting a thermoconductive block (11), a flat-surfaced positioning area (14) opposite to the Peltier elements (12, 13) configured for positioning the at least one disposable (MTP) on the thermoconductive block (11), said tempering block (10) being configured to generate a set linear temperature profile in the wells of the disposable (MTP) while placed for contacting the flat-surfaced positioning area (14) of the thermoconductive block (11);

- a control unit comprising one or more processors configured to control the gradient tempering unit(s) (GTUx) for the implementation of the prescribed protocol for the one or more chemical, biochemical, or biological reactions;
wherein the tempering block (10) with set linear temperature profile comprises at least one Peltier element (12) temperable at a first temperature T1 and at least one Peltier element (13) temperable at a second temperature T2, wherein T1 is higher than T2 , and both Peltier elements (12, 13) are arranged for contacting the thermoconductive block (11) outside of the positioning area (14) for the disposable (MTP) so that the linear temperature profile can be generated in the thermoconductive block (11) between the two Peltier elements and conducted to the disposable (MTP) when contacting the thermoconductive block (11).
The system according to claim 1, wherein the at least one tempering block (10) are positioned to temper the first and / or the second heating surface of the disposable (MTP) while placed for contacting the positioning area (14a, 14b).
The system according to one of the preceding claims, wherein the disposable (MTP) can be submitted to the linear temperature profile on both heating surfaces by way of positioned tempering blocks (10a, 10b) set with the linear temperature profile.
The system according to one of the preceding claims, further comprising at least one tempering unit (TUx) temperable at a temperature Tx comprising at least one tempering block (40) comprising at least two Peltier elements (42) contacting a thermoconductive block (41), opposite to the Peltier elements (42) a flat-surfaced area (44a) for positioning the at least one disposable (MTP) on the thermoconductive block (41), said temperingblock being configured to uniformly temper the disposable (MTP) while contacting the thermoconductive block (41).
The system according to claim 4, wherein the disposable (MTP) when placed in the tempering unit (TUx) can be tempered on both heating surfaces by way of positioned temperingblocks (41a, 41b).
The system according to one of the preceding claims, further comprising transport means capable of moving the disposable (MTP) 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) (14, 14a, 14b, 44a, 44b) in an internal space (20).
The system according to one of the preceding claims, further comprising moving means for contacting the one or more the positioning areas (14, 14a, 14b, 44a, 44b) of the one or more thermoconductive blocks (11, 11a, 11b, 41a, 41b) with the one or both heating surfaces of the disposable (MTP) when positioned in the internal space (20).
The system according to one of the preceding claims, further comprising one or more clamping mechanism capable of clamping the disposable (MTP) between the first and the second tempering blocks (10a, 10b respectively 40a, 40b).
System according to one of the preceding claims, wherein the 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.
System according to one of the preceding claims, further comprising 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 or a read-out unit for mass spectrometry.
Method for use of the system according to one of the preceding claims, comprising the following steps:
- loading one or more reaction mixtures in the wells of a disposable (MTP) and optionally sealing the upper side of the disposable (MTP) with a sealing foil;

- introducing the disposable (MTP) in the system

- transporting and positioning the disposable (MTP) in one of the one or more gradient tempering unit (GTUx) in accordance with a prescribed reaction protocol,

- in accordance with the prescribed reaction protocol, submitting the disposable (MTP) to a linear temperature profile on at least one heating surface by way of a positioned gradient tempering blocks (10) set to a prescribed temperature gradient,
wherein said gradient tempering block (10) comprises at least one Peltier element (12) tempered at a first temperature T1 and at least one Peltier element (13) tempered at a second temperature T2, wherein T1 is higher than T2 , and both Peltier elements (12, 13) is arranged for contacting the thermoconductive block (11) outside of the positioning area (14) for the disposable (MTP) so that the linear temperature profile is generated in the thermoconductive block (11) between the two Peltier elements (12, 13) and conducted to the disposable (MTP) when contacting the thermoconductive block (11);.
Method according to claim 11, wherein 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.
Method according to one of the preceding claims, wherein the disposable is submitted to the linear temperature profile on both heating surfaces by way of positioned gradient tempering blocks (10a, 10b) set to the prescribed temperature gradient.
Method according to one of the preceding claims, the system further comprising 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 (MTP) is transported to and positioned in the tempering unit (TUx) for tempering, preferably cooling, according to the reaction protocol.
Method according to one of the preceding claims, further comprising transporting the disposable (MTP) to / positioning the disposable (MTP) to an imaging unit (I) and capturing an image of at least one well of the disposable (MTP) or to a read-out unit for mass spectrometry.
Method according to one of the preceding claims, wherein the chemical, biochemical or biological reactions are for performing cellular thermal shift assays (CETSA^®), purified protein thermal shift assays (TSA) or any other protein denaturation or aggregation assays.
Method according to one of the preceding claims, wherein the method is used for high throughput reactions or assays.