Integrated system for chemical, biochemical or biological reactions in microplates subjected to a temperature gradient

Abstract

a disposable including a well; one or more gradient tempering units, each comprising a tempering block including a plurality of temperable Peltier elements contacting a thermally conductive block opposite a Peltier element, the gradient tempering units having a flat surface area for positioning the disposable on the thermally conductive block, the tempering blocks generating a set linear temperature profile for the well upon contacting the thermally conductive block; and a control unit for controlling the gradient tempering units; wherein the gradient tempering block includes a Peltier element capable of tempering at a temperature T1 and a Peltier element capable of tempering at a temperature T2, T1 being higher than T2, and both elements contacting the thermally conductive block outside of the positioning area for the disposable such that a linear temperature profile can be generated in the thermally conductive block between the two elements and conducted to the disposable through contact with the thermally conductive block.

Description

[Technical Field]

[0001] The present invention relates to an integrated system for chemical, biochemical or biological reactions requiring one or more set-point reaction temperatures / gradients in microplates following a defined protocol, such as, but not limited to, Cellular Thermal Shift Assay (CETSA®). [Background technology]

[0002] In the Cellular Thermal Shift Assay (CETSA®), cells are divided into different reaction vessels, and each vessel is heated to a different temperature. The samples are then cooled, the cells are lysed, and the amount of soluble protein is quantified by different means. In vessels with a higher initial temperature, the amount of soluble protein is smaller. This is caused by heat-induced protein aggregation, and protein stabilization by compound interactions increases the amount of soluble protein by shifting protein aggregation to higher temperatures.

[0003] The CETSA® method is described, for example, in EP 2 699 910 A1, in which a differential heating step is carried out in an apparatus adapted for the polymerase chain reaction (also called a PCR apparatus or PCR cycler).

[0004] Assays are typically performed in microplates comprising multiple wells, preferably high-density microtiter plates having a large number of wells. Reliable and accurate method execution on samples located in a microplate requires precise and uniform application of process parameters across the microplate.

[0005] The uniform process parameter in this application refers to a homogeneous linear temperature profile across the microplate along the heating line during heating. The temperature profile of the microplate can be obtained by measuring the surface temperature of the microplate in the tempering unit using an infrared camera to determine the temperature line scan across the microplate (e.g., Figure 4A / B). Such a profile is represented by a temperature gradient in the microplate, which indicates the direction and rate of temperature change expressed in temperature units per unit length.

[0006] The solutions of the state of the art present the following limitations or major drawbacks: Commercially available PCR cyclers are compatible with conical-bottom microtiter plates with 96 or 384 reaction vessels, which are not suitable for culturing adherent cells and have a rather low throughput compared to 1536 plates. After the heating step, the samples are transferred to another (micro)plate compatible with the luminescence reader used to quantify the amount of soluble protein by luminescence reading. These additional pipetting steps increase the standard deviation of the assay. The temperature range for the gradient in the PCR cycler is rather small (less than 21°C). Proteins that aggregate over a temperature range greater than 21°C cannot be analyzed in a single experiment. PCR cyclers are suitable for generating the required linear temperature profile across a microplate on a heating block. The temperature profile is generated by at least two heating and two cooling Peltier elements, typically six Peltier elements, distributed under a metal block opposite the positioning area that contacts the microtiter plate from below (see Figure 7). This configuration compromises the PCR cycler's primary function of providing a homogeneous temperature distribution. However, this compromise leads to an S-shaped temperature gradient that leads to an uneven distribution of temperature data points within the columns of the microplate. To detect low-affinity complex protein interactions, very short heating and cooling times are required (e.g., Seashore-Ludlow et al., SLAS Discovery 2020, Vol. 25(2) 118-126; Seashore-Ludlow et al., Biochemistry 2018, 57, 6715-6725). When commercial PCR cyclers perform a sample heating gradient and subsequent cooling on the same heating block, the heating and cooling times make this method rather slow. The CETSA protocol performed on a PCR cycler cannot be performed with a 3-minute heating and cooling time; the reaction becomes heterogeneous at shorter times. Figure 7 shows a diagram of a CETSA® experiment performed on a commercial PCR cycler. The experiment shows that a total of five microtiter plates and three pipetting steps are required for one CETSA® experiment.

[0007] German Utility Model No. 8814398U1 describes an apparatus for generating a temperature gradient, the temperature gradient being generated in a thermally conductive block extending between two Peltier elements at different temperatures. The apparatus is configured to rapidly modify the temperature in a disposable comprising a plurality of wells arranged in a row perpendicular to the temperature gradient by moving the disposable along the thermally conductive block at a position on the gradient having the desired temperature and pressing the disposable against the thermally conductive block at said position using a spring fixed on the thermally conductive block. The apparatus is unable to implement a linear temperature profile in the disposable.

[0008] US Patent Application Publication No. 2021 / 041379A1 shows an example of an apparatus comprising two temperature elements having hot and cold surfaces for creating a substantially linear temperature gradient in one or more sample holders, each sample holder positioned with a first end in contact with a first temperature element and a second, opposing end in contact with a second temperature element, and heat flow occurring through the sample holder between the contact area between the body of the sample holder and the temperature element. The solution can be used with sample holders comprising multiple wells arranged horizontally or vertically relative to the temperature gradient.

[0009] Applicants hypothesize that some of the aforementioned limitations are caused by non-uniform temperature profiles during heating in PCR cyclers.

[0010] Thus, there remains a need for a solution that allows for the required accurate and uniform application of process parameters across microplates, even using high-density microtiter plates that address the cost, thermal response, and uniformity, particularly for CETSA® experiments. Preferably, the solution would allow for processing of the method in one microplate without transferring samples between plates with different characteristics. The solution would be compatible with flat-bottom microtiter plates and be able to provide the temperature range for CETSA® experiments in one microplate. Summary of the Invention [Problem to be solved by the invention]

[0011] Said problem has been solved by a system and method as claimed. Exemplary embodiments of the solution can be gathered from the respective dependent claims.

[0012] Solutions will be described below without distinction as to claimed subject matter (method or system). To the contrary, the following description is intended to apply analogously to all claimed subject matter, regardless of the context in which they occur.

[0013] A system is claimed for conducting one or more chemical, biochemical or biological reactions (collectively referred to as reactions) in a disposable article comprising one or more wells at one or more set-point reaction temperatures. The disposable article is preferably a microtiter plate or microplate.

[0014] As used herein, "biological reaction" refers to a molecular biological cellular reaction, and a reaction using cellular tissue refers to the assembly of cells, viruses or phages.

[0015] As used herein, "cell" refers to a living cell such as an animal cell, a plant cell, a yeast, a fungus, or a bacterium.

[0016] The system is comprised of independent modules that are operatively linked to one another by transport means for the transfer and placement of disposables from and into the modules as needed for the automated execution of predefined reaction protocols.

[0017] The system of the present invention comprises: at least one disposable comprising one or more wells in a body, the wells being capable of acting as containers for one or more reactions requiring one or more defined reaction temperatures according to defined protocols, the body comprising a flat bottom side (also referred to as body floor) establishing a first heated surface capable of conducting heat homogeneously to the wells, and a flat top side comprising well openings establishing a second heated surface capable of conducting heat homogeneously in the wells; one or more gradient tempering units GTUx, each comprising at least one tempering block comprising at least two temperable Peltier elements, a thermally conductive block in contact with the temperable Peltier elements, a flat surface area for positioning at least one disposable on the thermally conductive block, the tempering block being configured to generate a linear temperature profile of the well of the disposable(s) in contact with the thermally conductive block; a control unit comprising one or more processors configured to activate the gradient tempering unit(s) for the implementation of a defined protocol for one or more chemical, biochemical or biological reactions; Here, the tempering block with a linear temperature profile comprises at least one Peltier element capable of tempering at a first temperature T1 and at least one Peltier element capable of tempering at a second temperature T2, T1 being higher than T2, and both Peltier elements contacting the thermally conductive block outside the positioning area for the disposable item such that a linear temperature profile can be generated in the thermally conductive block between the two elements and conducted to the disposable item contacting the thermally conductive block.

[0018] That is, the Peltier element contacts one side of the thermally conductive block at a first end and a second end of the block, while the positioning region is located on the opposite side of the block between the first and second ends.

[0019] In embodiments, at least one tempering block(s) is / are positioned to temper the first heated surface and / or the second heated surface of a disposable placed in the positioning area. In a preferred embodiment, the disposable may be subjected to a linear temperature profile on both heated surfaces by a tempering block positioned with a linear temperature profile.

[0020] The terms "temper" or "tempering" as used herein refer to maintaining a temperature at a set point(s). A "tempering block" is an arrangement capable of reaching (heating and cooling) a temperature and maintaining said temperature. A "temperable" element is a means capable of tempering, reaching a temperature and maintaining said temperature.

[0021] The term "positioning area" as used herein refers to a functional surface of the thermally conductive block that contacts the positioning area and is capable of tempering the disposable. The positioning area is also referred to as an active area. The positioning area can be adapted for better positioning of the disposable, for example in the form of a positioning stage.

[0022] In an embodiment, the first Peltor element at the first temperature T1 is a heating Peltier element, which means that the first temperature T1 is set higher than room temperature, and the second Peltor element at the second temperature T2 is a cooling Peltier element, which means that the first temperature T2 is set lower than room temperature.

[0023] In an embodiment, the at least one tempering block is positioned to temper the first heated surface and / or the second heated surface of the disposable when placed in the positioning area.

[0024] In an embodiment, the disposable may be subjected to a linear temperature profile on both heating surfaces by positioned gradient tempering blocks set with the (same) linear temperature profile.

[0025] In an embodiment, the system further comprises at least one further tempering unit TUx capable of tempering at a temperature Tx comprising at least one tempering block (also referred to as temperature control block) comprising at least one Peltier element in contact with a thermally conductive block opposite the Peltier element(s), the flat surface area being for positioning at least one disposable item on the thermally conductive block, the tempering block being configured to uniformly temper the disposable item when in contact with the thermally conductive block.

[0026] In an embodiment, the disposable when placed in the tempering unit (TUx) can be tempered on both heating surfaces by tempering blocks positioned thereon.

[0027] In an embodiment, the system comprises at least one tempering unit TUx according to a set point reaction temperature of a defined reaction protocol.

[0028] In an embodiment, the system comprises one tempering unit TUx for fast temperature removal, also called cooling unit.

[0029] The control unit is configured to activate the cooling unit(s) for the execution of a defined protocol for one or more chemical, biochemical or biological reactions, in particular the disposable is preferably in contact with the thermally conductive block of the tempering unit.

[0030] In an embodiment, the system further comprises a transport means comprising a carrier, preferably a carrier frame, on which the disposables are placed.

[0031] In an embodiment, the transport means further comprises moving means for moving the disposable to and / or from the gradient tempering unit GTUx and / or tempering unit TUx and aligning it with the at least one positioning area(s) respectively within the interior space.

[0032] In an embodiment, the gradient tempering unit(s) GTUx and / or tempering unit TUx comprises a second tempering block arranged such that the disposable can be positioned in the interior space between the first thermally conductive block and the second thermally conductive block and can be uniformly fixed between said blocks using a moving means.

[0033] In embodiments, the system further comprises a moving means for contacting one or more positioning areas of one or more thermally conductive blocks with one or both heating surfaces of a disposable positioned within the interior space.

[0034] In an embodiment, the system further comprises one or more securing mechanisms capable of securing the disposable between the first tempering block and the second tempering block.

[0035] Preferably, the control unit is configured to activate the transport means, movement means and / or fixation means for the execution of a defined protocol for one or more chemical, biochemical or biological reactions.

[0036] In an embodiment, the gradient tempering units GTUx are configured to create a linear temperature gradient range from the shorter left side of the disposable to the shorter right side of the disposable, i.e., the disposable is placed in the gradient tempering units GTUx such that the direction of the temperature gradient is along the larger side of the disposable, creating a larger temperature gradient.

[0037] In an embodiment, the system further comprises a readout / imaging unit I, the imaging unit I comprising an imaging device capable of capturing an image of a scene comprising at least one well of the disposable from a well opening side. In an embodiment, the imaging unit I is configured to capture an image of a scene covering all wells of the disposable.

[0038] In an embodiment, the imaging unit I also comprises one or more lighting elements for proper illumination of the disposable item during image acquisition.

[0039] In an embodiment, the surface of at least the positioning area of ​​the thermally conductive block is complementary to the respective heating surface (flat or structural) of the disposable. Most preferably, the bottom of the disposable is flat.

[0040] Most preferably, the disposable is selected to be suitable for cell culture / attachment and / or suitable for one or more readout methods of the readout unit / imaging unit I, most preferably both, so that fluid movement is limited or avoided. In a preferred embodiment, the disposable is suitable for luminescence / fluorescence readout.

[0041] In embodiments, the readout of the disposable may be performed in a mass spectrometer, for example for proteome analysis by measuring the aggregation of all proteins in a cell sample.

[0042] In embodiments, the well opening of the disposable may be sealed by a thin transparent sealing foil, which constitutes the second heating surface.

[0043] A further subject of the present application is a method for the use of a system according to one of the preceding claims, comprising the following steps: loading one or more reaction mixtures into the wells of the disposable and optionally sealing the top side of the disposable with a sealing foil; Introducing a disposable into the system and transporting and positioning it in one of the one or more gradient tempering units GTUx according to a defined protocol.

[0044] As used herein, the term "reaction mixture" refers to a fluid comprising several biological or chemical components or elements, including but not limited to tissues, cells, compounds and / or substances, which together are capable of causing a reaction and transformation of the original components.

[0045] In an embodiment, one gradient tempering unit GTUx is used according to a reaction temperature gradient defined by a reaction protocol, and said gradient tempering unit GTUx is set to the defined temperature gradient by the control unit.

[0046] In an embodiment, the disposable is subjected to a linear temperature profile on both heating surfaces by a positioned tempering block set with a linear temperature profile.

[0047] In an embodiment, the linear temperature profile of the tempering block in contact with the second heated surface of the disposable is set by at least one Peltier element capable of tempering at a first temperature T'1 and at least one Peltier element capable of tempering at a second temperature T'2, T'1 being higher than T'2 and T'1-T'2 being equal to T1-T2.

[0048] In an embodiment, one tempering unit TUx is used according to a defined reaction temperature of the reaction protocol, and said tempering unit TUx is set to a defined temperature by the control unit, which is advantageous for achieving the most rapid cooling of the entire microplate coming out of the gradient tempering unit GTUx.

[0049] Those skilled in the art will recognize that the gradient tempering unit GTUx can be used as a tempering unit for uniform tempering of disposable items by setting the temperatures T1=T2 and / or T'1=T'2.

[0050] In an embodiment, the method further comprises transferring / positioning the disposable to / into an imaging unit I and capturing an image of at least one well of the disposable.

[0051] The described solution is particularly suitable for, but not limited to, the Cellular Thermal Shift Assay (CETSA®).

[0052] The described solution is particularly suitable for high-throughput reactions or assays in disposables where a gradient is required. [Means for solving the problem]

[0053] The solutions will now be described in more detail. The use of any examples or exemplary language (e.g., "such as") provided herein is intended merely to better elucidate the invention and does not limit the scope of the solutions unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0054] Preferred embodiments of this invention are described above, including the best mode known to the inventors for carrying out the invention.

[0055] Variations of those preferred embodiments may become apparent to those skilled in the art to use such variations where appropriate, and the inventors intend the solutions to be implemented other than as specifically described herein.

[0056] 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 herein unless otherwise indicated herein or clearly contradicted by context.

[0057] In an embodiment, a disposable comprising 1536 wells can be used, each well having a volume of at most 10 μL, particularly 0.3-6 μL, particularly 0.5-4 μL, particularly 5-4 μL, particularly 1 μL. Any well cross-section can be used, typically rectangular or circular. It will be apparent to those skilled in the art that other disposable formats can be used.

[0058] In embodiments, the disposable is a microtiter plate (also called an MTP or microplate) or a picotiter plate.

[0059] In the context of the present invention, disposables exhibiting a format and pattern in accordance with the recommendations of the Society for Biomolecular Laboratory Automation and Screening are most preferred (ANSI / SBLAS1-2004, ANSI / SBLAS2-2004, ANSI / SBLAS3-2004, and ANSI / SBLAS4-2004). The disposables can be of any format and pattern including at least 96, 384, 1536, or 3456 wells, preferably 1536 wells, in the 96-well format (12 x 8), 384-well format (24 x 16), 1536-well format (48 x 32), or 3456-well format (48 x 72) established by the ANSI standards of the Society for Laboratory Automation and Screening.

[0060] In this solution, the wells of the disposable are positioned perpendicular to the flat bottom side and therefore perpendicular to the linear temperature profile when the disposable is within the positioning area of ​​the thermally conductive block, with each row of wells at a different position along the gradient and therefore at a slightly different temperature than the other wells.

[0061] In embodiments, the body is made of a thermoplastic polymer that can be sealed with a sealing foil, with or without an added heat-conducting medium, and is preferably made of polystyrene, polypropylene, or COC (cycloolefin copolymer). The disposable may comprise a frame support made of polycarbonate or polystyrene. In embodiments, the flat bottom of the disposable may be defined by a transparent membrane fixed to the body comprising the well, said transparent membrane allowing image acquisition from the bottom side.

[0062] In embodiments, after the reactants are loaded into the wells, the disposable product can be sealed with a transparent thin sealing foil that is established in the art. The transparent thin sealing foil can be made of polycarbonate, polypropylene, cyclic olefin, or other plastic materials known to those skilled in the art, or a multi-layer film made of two or more transparent materials with the desired barrier properties, as is well established in the art. Sealing can be achieved by welding the sealing foil to the body of the disposable product.

[0063] In both the gradient tempering unit GTUx and the tempering unit TUx, heat exchange between the heating surface(s) of the disposable and the respective thermally conductive block(s) is achieved to thermally treat the liquid sample contained therein.

[0064] In an embodiment, the first heating surface of the disposable is selected to fit the surface of a corresponding positioning area of ​​the tempering unit.

[0065] In embodiments, flat-bottom disposables are used. Flat bottoms are particularly advantageous for automating the transport and / or fixation of the disposables. The bottom thickness can be any thickness, but conventional bottom thicknesses are less than 1000 μm.

[0066] Most preferably, the disposables are selected to be suitable for culturing cells and / or suitable for one or more readout methods, in particular luminescence / fluorescence readout, most preferably both, so that fluid movement is limited or avoided.

[0067] Commercially available microplates such as, but not limited to, 1536-well Microplate, PS, μClear®, LoBase (Greiner Bio-One, Cat. No. 783092), 384-well Microplate, PS, μClear® (Greiner Bio-One, Cat. No. 781092) can be used.

[0068] In an embodiment, the gradient tempering unit(s) GTUx and / or the cooling / tempering unit(s) TUx comprise a second tempering block arranged such that a disposable can be positioned in the interior space between the first and second thermally conductive blocks and uniformly fixed between said blocks using a moving means. In this embodiment, the disposable can be tempered following contact with both tempering surfaces.

[0069] In an embodiment, the positioning area of ​​the second thermally conductive block that contacts the second heating surface of the disposable is planar and preferably uniform, regardless of whether the wells are sealed by a sealing foil or not.

[0070] In an embodiment, the temperature gradient of the thermally conductive block contacting the first heated surface (bottom side) of the disposable in the gradient tempering unit GTUx is a linear temperature gradient (also called temperature profile) TGx.

[0071] In an embodiment, the temperature T1 is typically set at 37° C., and T2 is set at T1+TGx. Those skilled in the art will recognize that the exact parameters for the temperature gradient TGx, T1 and T2, will depend on the defined protocol for the reaction of interest.

[0072] In an embodiment, a temperature gradient is established along the length of the disposable.

[0073] It has been found that the solution of the present application is capable of providing a homogenous linear gradient along the length of a standard size disposable from about 20 to 45°C, preferably 25 to 35°C.

[0074] In an embodiment, the tempering blocks of the gradient tempering units 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 a thermally conductive block outside the positioning area for the disposable from opposite sides of said positioning area (Figure 1 or Figure 6), so that a linear temperature profile is generated between the two arrays and conducted to the disposable when contacting the positioning area of ​​the thermally conductive block. Those skilled in the art will recognize that the appropriate number of Peltier elements per array can be optimized in terms of performance in relation to the shape of the disposable.

[0075] In an embodiment, the tempering block(s) of the tempering unit TUx comprises a thermally conductive plate on which the disposables are placed, and an array of Peltier elements (i.e., individual physical units that convert electrical current into heat / cold) in contact with the thermally conductive plate is used. Considering that a standard MTP has an aspect ratio of 3:2, an array of six Peltier elements is most preferred.

[0076] In an embodiment, the temperature of the Peltier elements is monitored using a temperature sensor, e.g., a thermal resistor. For the most homogeneous tempering, it is preferred that each Peltier element is provided with a thermal sensor and each Peltier element is controlled separately.

[0077] In an embodiment, the tempering block of the gradient tempering unit GTUx and / or the tempering block of the tempering unit GTUx also comprises a heat sink that dissipates the total power converted to heat or cold and carries the Peltier element. A heat sink / fan combination can also be used.

[0078] In an embodiment, the thermally conductive block in the gradient tempering unit GTUx contacting the second heated surface of the disposable is set with the same linear temperature profile TGx as the thermally conductive block contacting the first heated surface (obtained by the difference between T1 and T2).

[0079] In an embodiment, the thermally conductive block of the gradient tempering unit GTUx that contacts the second heated surface of the disposable can be set to exhibit the same linear temperature profile TGx with temperatures T'1 and T'2 slightly higher (e.g., +1°C) than the temperatures T1 and T2 set for the thermally conductive block that contacts the bottom of the disposable. When a thin transparent sealing foil is used as the second heated surface, the higher temperatures have been shown to prevent condensation on the sealing foil inside the wells at the higher temperatures of the profile.

[0080] In an embodiment, alignment of the linear profile may be enhanced by a positioning / alignment rim on the thermally conductive block for accurate positioning of a corresponding structural element of the disposable in the positioning area of ​​the tempering unit.

[0081] The gradient tempering unit(s) GTUx described above have been shown to be capable of quickly creating large linear temperature gradients across flat-bottom microtiter plates up to 1536-well format, ranging from the shorter left side of the microtiter plate to the shorter right side of the microtiter plate (see Figure 4B).

[0082] In an embodiment, the temperature of the thermally conductive block contacting the thin transparent sealing foil on the second heated surface of the disposable in the tempering / cooling unit TUx is kept constant at a temperature slightly higher than the temperature of the thermally conductive block contacting the bottom side of the disposable, which has been shown to prevent condensation on the sealing foil inside the well.

[0083] In one embodiment, a thermally conductive block(s) made of metal, for example aluminum or glass, preferably metal, can be used.

[0084] In an embodiment, the thermally conductive block in contact with the second heated surface is made of glass. The imaging / reading means can be used to acquire images through the glass plate / block, for example to monitor fluorescence changes. It is also possible to monitor temperature changes in disposable items.

[0085] In an embodiment, the system comprises an imaging unit I, and the imaging unit I comprises an imaging device.

[0086] The imaging device may be a mechanical, digital, or electronic viewing device such as a still camera, camcorder, movie camera, scanner, or any other instrument, device, or format capable of recording, storing, or transmitting a visual image of an object. In examples, a CCD camera, e.g., an sCMOS camera, or an image intensifier camera may be used.

[0087] In embodiments, a camera with an objective lens can be used. In embodiments, a 35 mm F1.6 C-mount objective lens can image the entire disposable. In embodiments, a microscope objective lens can be used. In embodiments, the imaging unit I can be configured to acquire images of a single well. In embodiments, the system can be realized for multiplexed immunofluorescence reading, as known, for example, from the PhenoCycler System from Akoya Biosciences. This allows the detection of aggregation profiles of up to 100 different proteins in a single CETSA® experiment.

[0088] In an embodiment, the imaging unit I also comprises one or more lighting elements for proper illumination of the disposable during image acquisition. In an embodiment, a ring light is used positioned for homogenous illumination of the disposable across all cavities during image capture.

[0089] In an embodiment, a filter may be used.

[0090] Those skilled in the art will recognize that the objective lenses and / or filters depend on the use of the device, especially excitation and emission filters for image acquisition using photosensitizers or genetically encoded luminescent or fluorescent reporters. For example, if a compact device is required, the imaging unit I may comprise optical lens(es) and / or mirror(s).

[0091] In an embodiment, the imaging unit I comprises support and calibration means for positioning the imaging device, lighting elements and / or filters in relation to the disposable items for optimal image acquisition.

[0092] In an embodiment, the imaging device may be positioned to capture images from the second heated surface of the disposable.

[0093] In an embodiment, the imaging device may be positioned to acquire images through one or more glass plates of the gradient tempering units GTUx and / or tempering units TUx.

[0094] In an embodiment, the readout unit / imaging unit I may comprise a tempering block for tempering the disposable from the side opposite the well opening or sealing foil.

[0095] In an embodiment, the transport means is capable of transporting and positioning the disposable in the readout unit or imaging unit I for image capture of at least a portion of the disposable, most preferably the entire well opening side of the disposable.

[0096] In an embodiment, the imaging unit I comprises a fixing frame or a transparent fixing plate, and the disposable item can be fixed between the tempering block and the fixing plate or frame. For this purpose, a fixing mechanism can be used.

[0097] 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 the fixation mechanism as needed for optimal image acquisition.

[0098] In an embodiment, the transport means comprises a moving mechanism or means for moving disposables to and / or from one or more internal spaces of the gradient tempering unit GTUx, the tempering unit TUx and / or the imaging unit I, said internal spaces being defined as the alignment space between the first and second thermally conductive blocks (which position the area) in the gradient tempering unit GTUx or the tempering unit TUx, or the alignment space between the tempering block and the fixed plate or frame in the imaging unit I (Figure 4).

[0099] In an embodiment, the transport means comprises at least one horizontal drive and the disposables are positioned on a moving carrier for transport and positioning in the gradient tempering unit(s) GTUx, tempering unit TUx and / or imaging unit I as required by the reaction protocol.

[0100] In some embodiments, the carrier may be movable between the interior space and an external disposable loading position outside the instrument frame / casing for loading and / or unloading of microplates to / from the carrier. Specifically, in some embodiments, the carrier is movably mounted to a base for repeatable, bidirectional movement between the interior disposable position and the exterior disposable position. In some embodiments, the movement mechanism is configured as a carrier drive mechanism for driving the carrier in either of two directions, i.e., as a means for driving the carrier to the interior and exterior space(s) or microplate position, respectively.

[0101] In an embodiment, the carrier is slidably mounted to a horizontal drive(s) that allows repeatable, bidirectional movement between a processing position inside the system for thermally processing the reaction product and a loading position outside the system for loading or unloading disposables to / from the carrier.

[0102] In an embodiment, the transport means comprises one or more stop plates arranged in a horizontal plane for accurate positioning of the carrier and placement within the interior space of the unit in alignment with its positioning region. In an embodiment, one stop plate per unit is used.

[0103] Such sliding mechanisms are well known to those skilled in the art and need not be further elucidated herein. In some embodiments, the system comprises an automated carrier drive mechanism, such as a motor-based belt drive or wheel drive, for automatically moving the carrier between the processing position and the loading position. Such drive mechanisms are well known to those skilled in the art and need not be further elucidated herein. In embodiments, horizontal and vertical motors, for example, servo motors or stepper motors, are used to transport the microplate assembly (meaning the microplate and carrier) into contact with the top surface of the tempering block.

[0104] In an embodiment, the disposable, in the case of a tempering block, is positioned within a carrier on a metal heating fixture shaped to closely fit the disposable, particularly its frame support, and positioning stage / area.

[0105] In an embodiment, the system comprises one or more fixing mechanisms for fixing disposables within the interior space of the gradient tempering unit(s) GTUx, of the tempering unit(s) TUx, and / or in the imaging unit I.

[0106] In the tempering unit (GUTx or TUx), a fixing mechanism is capable of moving one or both tempering blocks relative to each other so as to make it possible to fix the disposable between the heating blocks after they have been appropriately positioned in the interior space between them by the transport system. Thus, full contact on both sides of the disposable can advantageously be obtained by the fixing.

[0107] In an embodiment, the imaging unit I can comprise a tempering block. In an embodiment, the fixing mechanism can move the tempering block relative to the fixing plate / frame for positioning the disposable in the imaging unit I.

[0108] At least one of the tempering blocks in each tempering unit (GTUx or TUx) or the tempering block or fixing plate / frame in imaging unit I is preferably spring mounted for smooth fixation of the disposable. In an embodiment, force measurement can be realized using the spring constant and the stroke of the spring-mounted element.

[0109] In embodiments, securing the disposable may be achieved by a vertical motor or lever that moves the lower tempering block upward along a vertical drive and presses the disposable against a spring-loaded upper tempering block. Force measurement may be achieved using the spring constant and stroke of the upper tempering block.

[0110] In an embodiment, the same fixing mechanism is used to fix the disposables in the tempering unit (GTUx and / or TUx) and / or imaging unit I. In particular, one vertical motor or lever may be used to fix the lower tempering block(s) in a different unit (tempering GTUx and / or TUx and / or imaging unit I) upward. In this embodiment, spacing / alignment blocks may be used to achieve aligned positioning of the disposables in the units, in particular by aligning the positioning areas of the tempering unit (GTUx and / or TUx) and / or imaging unit I. In an embodiment, the tempering blocks may be attached to spacing / alignment blocks for better positioning.

[0111] Those skilled in the art will recognize that additional embodiments for securing the disposable within the interior space of the unit may be used, for example, the securing means may force the disposable down onto the tempering block and / or lower temperature platen.

[0112] In an embodiment, the control unit may be configured to activate and / or control the tempering unit, the transport means and / or the fixation mechanism according to a predefined reaction protocol.

[0113] Compared to static embodiments (one temperature controlled heating / cooling unit), this embodiment has the advantage that the tempering block does not have to wait for the transition to the next set point temperature, which can significantly speed up the test time.

[0114] In an embodiment, the system of the present invention may comprise a module for providing all reagents necessary for a reaction in the correct concentrations in a disposable item. A pipetting robot or a pipetting unit can be used as a unit for providing all reagents in the present invention.

[0115] In some embodiments, the systems of the invention are instruments for incubating, thermally treating, or otherwise processing liquid samples, such as an automated thermocycler that allows a liquid reaction mixture to be subjected to a series of temperature excursions to perform, for example, a Cellular Thermal Shift Assay (CETSA®), a Purified Protein Thermal Shift Assay (TSA), or any other protein denaturation or aggregation assay.

[0116] In some embodiments, the device is used to chemically process a liquid sample, for example, by performing a test or assay related to an immunochemical or clinical chemistry analysis.

[0117] To perform all these steps, the system typically requires a user programmable computer system configured to control the system via a control unit.

[0118] In embodiments, a user inputs a reaction protocol into the system or selects an appropriate reaction protocol from a database comprising a collection of reaction protocols via a user interface.

[0119] The solution of the present invention is particularly useful for automated performance of chemical, biochemical or biological reactions, especially high-throughput reactions and assays.

[0120] A further object of the present invention is a method for the use of the system of the present invention as described above, comprising the following steps: loading the reaction mixture into the wells of the disposable and optionally sealing the top side of the disposable with a sealing foil; Introducing a disposable (optionally sealed) in the system and transporting and positioning it in one of the gradient tempering units GTUx and / or tempering units TUx according to the defined protocol.

[0121] In a further embodiment, the method further comprises transferring and positioning the disposable in an imaging unit I, and capturing an image of at least one well of the disposable.

[0122] It is preferred to use one gradient tempering unit GTUx per defined reaction temperature gradient of the reaction protocol.

[0123] It is preferred that the TUx temperature is below room temperature to achieve rapid cooling of the sample after the ramp step. It is preferred to use one tempering unit TUx per reaction temperature defined in the reaction protocol.

[0124] It is also preferred that the temperature of the second tempering block (in contact with the sealing foil) in the tempering unit TUx is set to a temperature slightly higher than the highest reaction temperature specified in the reaction protocol.

[0125] The preferred settings for the gradient tempering units GTUx are as described above.

[0126] Therefore, the objectives of this application are as follows: 1. A system for conducting one or more chemical, biochemical or biological reactions in one or more wells of a disposable item at one or more set point reaction temperatures, comprising: at least one disposable comprising one or more wells in a body, the wells being capable of acting as containers for one or more chemical, biochemical or biological reactions requiring one or more defined reaction temperatures according to a defined protocol, said body comprising a flat bottom side constituting a first heated surface capable of conducting heat homogeneously in the wells, and a flat top side comprising a well opening, optionally sealed by a thin sealing foil, constituting a second heated surface capable of conducting heat homogeneously in the wells; one or more gradient tempering units GTUx, each comprising at least one tempering block comprising at least two temperable Peltier elements in contact with a thermally conductive block, with a flat-surface positioning area opposite the Peltier elements configured for positioning at least one disposable item on the thermally conductive block, the tempering block configured to generate a set linear temperature profile in the well of the disposable item(s) when in contact with the thermally conductive block; a control unit comprising one or more processors configured to control the gradient tempering unit(s) GTUx for the implementation of a defined protocol for one or more chemical, biochemical or molecular biological reactions, Here, the heating block with a set linear temperature profile comprises at least one Peltier element capable of being tempered at a first temperature T1 and at least one Peltier element capable of being tempered at a second temperature T2, T1 being higher than T2, and both Peltier elements contacting the thermally conductive block outside of the positioning area for the disposable item such that a linear temperature profile can be generated in the thermally conductive block between the two elements and conducted to the disposable item when in contact with the thermally conductive block.

[0127] In an embodiment, the at least one tempering block is positioned to temper the first heated surface and / or the second heated surface of the disposable when placed on the positioning area.

[0128] In an embodiment, the disposable may be subjected to a linear temperature profile on both heating surfaces by a positioned heating block set with a linear temperature profile.

[0129] In an embodiment, the system further comprises at least one tempering unit TUx capable of tempering at a temperature Tx comprising at least one heating block comprising at least two Peltier elements in contact with a thermally conductive block opposite a Peltier element, the flat surface area being for positioning at least one disposable item on the thermally conductive block, the heating block being configured to uniformly temper the disposable item when in contact with the thermally conductive block.

[0130] In an embodiment, the disposables when placed in the tempering unit TUx may be tempered on both heating surfaces by the heating blocks positioned thereon.

[0131] In an embodiment, the system further comprises transport means capable of moving the disposable to and / or from the gradient tempering unit GTUx and / or tempering unit TUx and aligning it with at least one positioning area(s) within the interior space.

[0132] In embodiments, the system further comprises a moving means configured to contact one or more positioning areas of the one or more thermally conductive blocks with one or both heating surfaces of the disposable when positioned within the interior space.

[0133] In embodiments, the system further comprises one or more securing mechanisms capable of securing the disposable between the first heating block and the second heating block.

[0134] In an embodiment, the control unit is configured to control the transport means, the movement means and / or the fixation mechanism for the execution of a defined protocol for one or more chemical, biochemical or biological reactions.

[0135] In an embodiment, the system further comprises an imaging unit I, wherein the imaging unit I comprises an imaging device capable of capturing an image of at least one well of the disposable from the sealed side.

[0136] A further object of the present application is a method for the use of the above system, comprising: loading one or more reaction mixtures into the wells of the disposable and optionally sealing the top side of the disposable with a sealing foil; · Introducing sealed disposables in the system; ·Transporting and positioning disposables in one of one or more gradient tempering units GTUx according to the defined reaction protocol.

[0137] In an embodiment, one gradient tempering unit GTUx is used according to a reaction temperature gradient defined by a reaction protocol, and said gradient tempering unit GTUx is set to the defined temperature gradient by the control unit.

[0138] In an embodiment, the disposable is subjected to a linear temperature profile on both heating surfaces by positioned heating blocks set at a defined temperature gradient.

[0139] In an embodiment, the system further comprises one tempering unit TUx according to a defined reaction temperature of a reaction protocol, said tempering unit TUx being set to a defined temperature by the control unit, and disposables being transported and positioned in the tempering unit TUx for tempering, preferably cooling, according to the reaction protocol.

[0140] In an embodiment, the method further comprises transferring / positioning the disposable to an imaging unit I and capturing an image of at least one well of the disposable.

[0141] In particular, the solution can be used for Cellular Thermal Shift Assay (CETSA®), Purified Protein Thermal Shift Assay (TSA), or any other protein denaturation or aggregation assay. The solution allows for high-throughput reactions or assays.

[0142] The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (particularly in the context of the claims which follow) shall be construed as covering the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "including," "having," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise specified.

[0143] The use of any examples or exemplary language (e.g., "etc.") provided herein is intended merely to better elucidate the invention and does not limit the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0144] Preferred embodiments of this invention are described above, including the best mode known to the inventors for carrying out the invention.

[0145] Variations of those preferred embodiments may become apparent to those skilled in the art to employ such variations where appropriate, and the inventors intend the invention to be practiced other than as specifically described herein. Accordingly, this invention 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 above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context. [Brief explanation of the drawings]

[0146] [Figure 1] A three-dimensional view of one of the gradient tempering blocks of the gradient tempering unit GTUx showing the positions of the Peltier elements that can be tempered at T1 and T2, respectively, where the disposable items are positioned outside the positioning area that contacts the aluminum block (heat conduction block). [Figure 2] A perspective view of the mounting diagram of the system according to the present invention. Explanation of symbols: MTP Microplate / Disposable item / Microtiter plate GTUx Gradient tempering unit 10, 10a, 10b Gradient tempering blocks 11, 11a Heat conduction blocks 12, 12a, 12b Peltier elements that can be tempered at the first temperature T1 13, 13a, 13b Peltier elements that can be tempered at the second temperature T2 14, 14a, 14b (not shown) Positioning area or stage / Active area 15 Supply line for the temperature detector (Pt100) for the Peltier element 16, 16a Screws 17a, 17b Heat sinks 19 Tempering block housing 20 Internal space 21 Positioning / alignment trim 30 Horizontal drive device 31 Horizontal lever 32 Vertical drive device 33 Vertical lever TUx Tempering unit 40, 40a, 40b Temperature control blocks 41a, 41b Heat conduction blocks 42a, 42b Peltier elements 44a, 44b Positioning area / stage 46a (not shown), 46b Heat sinks 50 Carrier 51 Loading position 52 Support frame 53 Vertical drive device 54 Sliding part I Imaging unit 60 Imaging device 70 Frame 71 Partition wall 80 Heat conduction stage 81 Conductive block 82a, 82c Outer Peltier elements tempered at discrete temperatures T1, T2 or T3 where T1 < T2 < T3 82b Intermediate Peltier element tempered at the midpoint [Figure 3]Shown are experimental curves obtained with the Androgen Receptor CETSA® in a flat-bottom 1536-plate using a qPCR cycler by Screening Technology (2A) and with the published Androgen Receptor CETSA® in a V-bottom 384-plate using a commercially available PCR cycler (2B, Shaw et al, Scientific Reports, (2018) 8:163 | DOI:10.1038 / s41598-017-18650-x). [Figure 4A] 1 shows the temperature gradient of a 1536-well microplate obtained using a Sensoquest® gradient cycler. [Figure 4B] 1 shows the temperature gradient in a 1536-well microtiter plate obtained using the system of the present invention. [Figure 5] 5A and 5B show thermal aggregation curves for androgen receptor CETSA® with suspension (5A) or adherent (5B) cells seeded on flat-bottom 1536-well microplates treated according to the present invention. [Figure 6] FIG. 1 shows a schematic diagram of a CETSA® experiment using the solution of the present invention, where all necessary steps can be performed on / in the same microtiter plate. [Figure 7] 1 shows a schematic diagram of a CETSA® experiment using a commercially available PCR cycler. DETAILED DESCRIPTION OF THE INVENTION

[0147] FIG. 1 shows a three-dimensional open-view schematic diagram of the tempering block 10 of the gradient tempering unit GTUx. When the tempering block 10 is operational, Peltier elements 12 and 13, capable of tempering at T1 and T2, respectively, are positioned outside the positioning stage 14. The Peltier elements 12 and 13 are housed by a tempering block housing 19 positioned above a heat sink 17 (not shown), from which heat is removed. In this embodiment, two Peltier elements are used for each temperature, and each Peltier element is controllable by a temperature sensor supplied by a supply line 15. A Pt100 detector can be used. For operation, the thermally conductive block 11 is in contact with the heating element by positioning screws 16 for precise alignment. When positioned, the disposable MTP contacts the aluminum block 11 (thermally conductive block 11) on the positioning stage 14. In this embodiment, the thermally conductive block 11 comprises a positioning rim 21 for accurate positioning of the disposable MTP on the positioning stage 14 .

[0148] 2 shows a perspective view of a montage diagram of a system comprising one gradient tempering unit GTUx and one tempering unit TUx. The gradient tempering unit GTUx and tempering unit TUx each comprise a first / lower and second / upper tempering block 10a, 10b and 40a, 40b, respectively. During operation of the system, disposable MTPs are transported in either tempering unit GTUx / TUx in an internal space 20 (not shown) between the first and second tempering blocks and fixed between the tempering blocks for a period defined by the experimental protocol. For automated transport, disposable MTPs are transported between a loading position 51 for introduction of the disposable MTP into the apparatus and the tempering units using horizontal transport means for transport along a transport lane connecting the internal spaces 18 of the tempering units GTUx and GTUx. In this embodiment, the disposable MTP is placed on a carrier 50 movable on a slide 53 by a vertical drive 53, which can be moved into and out of the interior space 18 (not shown) of each tempering unit on the horizontal drive 30 using a horizontal lever 31. During an experiment driven by an automated software program in a control unit (not shown), the disposable MTP coupled to the carrier 50 is transported by the horizontal lever 31 along the horizontal drive 30 into the interior space 18 (not shown) of the gradient tempering unit GTUx, aligned by positioning areas / stages 14a, 14b (a stepper motor can be used), which are pressed and held against said positioning areas by moving the gradient tempering blocks 10a, 10b relative to the disposable MTP (also referred to as a fixation mechanism). Reactions can be performed according to a predefined protocol.The disposable MTP in the carrier 50 is released from the gradient tempering unit GTUx by opening the locking mechanism and moves into the interior space of the tempering unit TUx between the tempering blocks 40a, 40b in alignment with the positioning areas / stages 44a, 44b for homogeneous cooling according to a predefined protocol. The disposable MTP can be transferred to the imaging unit I (not shown) for image acquisition by mass spectrometry or protein quantification.

[0149] Gradient tempering blocks 10a, 10b are positioned in gradient tempering unit GTUx and comprise thermally conductive blocks 11a, 11b tempered by Peltier elements temperable at a first temperature T1 and a second temperature T2 to establish the desired temperature gradient in the positioning area / stage and the disposable MTP, with heat being dissipated from each gradient tempering block by heat sinks 17a, 17b, respectively. In this embodiment, tempering blocks 40a, 40b of tempering unit TUx comprise thermally conductive blocks tempered by Peltier elements operable at a setpoint temperature, with heat being dissipated by heat sinks 46a (not shown), 46b. In the embodiment of Figure 2, securing of the disposable MTP is achieved by moving one or both tempering blocks along a vertical drive 32 using horizontal lever 33 (a vertical motor can be used) to press the tempering block(s) against the disposable MTP. The tempering blocks can be spring mounted and force measurement can be realized 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 units GTUx and the tempering units TUx are mounted on a frame 70 and the tempering units can be separated from each other except for the transport lane by segments of a dividing wall 71.

[0150] experiment In the example, the solution is used for CETSA®, which example is explained in somewhat more detail below, but which does not in any way limit the invention to this embodiment.

[0151] CETSA® experiments were performed according to the protocol of Shaw et al., using a cell line containing a luminescence detector for the stability of the protein of interest (androgen receptor). In contrast, Shaw et al. used detection of soluble protein by AlphaScreen® technology, rather than a luminescence detector.

[0152] Several flat-bottom 1536-well microplates heated at different homogenous temperatures using a Screening Technology qPCR cycler were used to construct a complete thermal melting curve for a protein of interest (3A) by connecting the results from microplates at different temperatures. The small molecule DHT led to thermal stabilization of the androgen receptor.

[0153] For this purpose, each 1536-well flat-bottom microplate was filled according to the protocol of Shaw et al. (2018) and sealed with an optically clear, permanently adhesive membrane (Applied Biosystems, 4311971). Commercially available 1536-well flat-bottom microplates were used. Each microplate was centrifuged, and the assay was run in a Screening Technology qPCR cycler.

[0154] Figure 3 shows experimental curves obtained with the androgen receptor CETSA® in flat-bottom 1536-well plates tempered at several discrete temperatures over a predefined range using a qPCR cycler as described in International Application No. PCT / EP2022 / 077322, compared to the published androgen receptor CETSA® in V-bottom 384-well plates using a commercially available PCR cycler (3B, Shaw et al., 2018). The resulting curves are side-by-side. Heating the flat-bottom 1536-well plates leads to thermal aggregation of the protein of interest, and binding of small molecules reduces aggregation, thus confirming that flat-bottom 1536-well plates are suitable for CETSA®.

[0155] Using the homogenous heating of microplates in Screening Technology's qPCR cycle, 14 microplates heated at discrete temperatures were required to generate a complete melting curve for the protein of interest.

[0156] Comparison of the Sensoquest® Gradient Cycler with the system of the present invention using flat-bottom microplates: To the inventors' knowledge, the Sensoquest® Gradient cycler is the only commercially available device designed to generate the required gradient across flat-bottom microplates, and was selected for further comparative experiments.

[0157] A microtiter plate (1536-well Microplate, PS, μClear®, LoBase (Greiner Bio-One, Cat. No. 783092) was placed on a Sensoquest® gradient cycler for 3 minutes. Immediately after the run, the surface temperature across the microplate was acquired using an infrared camera. A temperature line scan across the microplate (Figure 4A) shows the temperature gradient of a flat-bottom 1536-well microtiter plate generated by the Sensoquest® gradient cycler. Brackets indicate the position on the plate where a linear temperature gradient occurs. We found that the Sensoquest® cycler only generated a fairly linear temperature gradient (very small) with poor homogeneity, of approximately 13°C after 5 minutes.

[0158] It was assumed that the region with the appropriate linear gradient was limited by the heating and cooling Peltier elements located directly below the thermally conductive block in contact with the microplate floor. Furthermore, the heated lid pressing the plate onto the tempering block may only produce a homogenous temperature, which may interfere with the gradient created by the tempering block in contact with the microplate floor.

[0159] To generate a melting curve using only one microtiter plate, a device for applying a temperature gradient across the microtiter plate was required. For comparative experiments, a system according to Figure 2 was used.

[0160] Figure 4B shows the temperature gradient of a 1536-well microtiter plate obtained in the system of the present 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. Immediately after heating, the surface temperature of the microplate was acquired using an infrared camera. Figure 4B shows the temperature gradient / line scan across the plate. The system used generates a linear temperature gradient of approximately 26°C on the 1536-well plate and heats the plate within 60 seconds. This is a major improvement compared to the line scan across the plate in the Sensoquest® cycler (4A).

[0161] Using the solution of the present invention and a luminescence detector for protein stability, the androgen receptor CETSA® experiment of Shaw et al. was repeated.

[0162] Androgen receptor cell lines were seeded on flat-bottom 1536-well microplates and measured either in suspension or after adhering to the microplate.In either case, cells were heated for 1 minute in the system of the present invention, and then incubated with DHT or DMSO for 1 hour before cooling for 1 minute.After adding CETSA reagent, both plates were measured in a luminescence reader.

[0163] Figure 5 shows the thermal aggregation curves of androgen receptor CETSA® with suspension cells (5A) or adherent cells (5B) seeded on a flat-bottom 1536-well microplate treated according to the present invention. In both cases, a clear shift in the thermal aggregation curve was observed for cells treated with DHT. This experiment demonstrates that the present invention makes it possible to perform CETSA® on a single flat-bottom 1536-well microtiter plate. Only one microtiter plate was required per experiment.

[0164] FIG. 6 shows a schematic diagram of a CETSA® experiment using the solution of the present invention, with the main system components represented in cross section. The system comprises a gradient tempering unit GTUx, a tempering unit TUx, and an imaging unit I. The horizontal transport lanes are indicated by horizontal arrows; the fixation mechanisms are not shown. The simplified imaging unit I comprises an imaging device 60. The tempering units GTUx and TUx comprise first / lower and second / upper tempering blocks 10a, 10b, and 40a, 40b, respectively. During operation of the system of the present invention, a disposable MTP is positioned within the interior space 20 of either tempering unit, fixed between positioning areas 14a, 14b or 44a, 44b, respectively, for a period as defined by the experimental protocol, and 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 figure also shows in cross section the arrays of Peltier elements 12a, 13a and 12b, 13b associated with thermally conductive blocks 11a, 11b and positioning areas 14a, 14b in the gradient tempering unit GTUx compared to the arrays of Peltier elements 42a and 42b associated with thermally conductive blocks 41a, 41b and positioning areas 44a, 44b in the tempering unit TUx.

[0165] Regarding one CETSA® experiment schematically shown in FIG. 7 as described in Shaw et al 2018 using a commercially available PCR cycler, the arrows indicate the movement to the next plate for further processing. Cells are cultured (MTP1), the cells are heated with the reaction mixture (MTP2, MTP3), and different plates are required for luminescence / fluorescence readings (MTP4 and MTP5), so it is necessary to transfer samples between different plates. The temperature gradient that the Sensoquest® cycler can generate in microplates is not large enough, so it is necessary to divide the sample in two plates (MTP2, MTP3) for the heating step. Two V-bottom PCR plates MTP2 and MTP3 for heating in the high temperature range (from T2 to T3) and heating in the low temperature range (from T1 to T2) are used respectively. The gradient is achieved by Peltier elements 82 distributed to temper the thermally conductive block 81 and the thermally conductive stage 80. The low range gradient is achieved by the outer Peltier elements 82a, 82c set to temperatures T1 and T2 respectively, T1 < T2, assisted by the intermediate Peltier element(s) 82b set at the midpoint. Similarly, the high range gradient is achieved by setting the outer Peltier elements to T2 and T3 respectively, T2 < T3, each assisted by the intermediate Peltier element at the midpoint. The contents of each microplate MTP2 and MTP3 are transferred to flat bottom plates MTP4 and MTP5 respectively for reading. A total of five microtiter plates (MTP) are required to obtain the data necessary for a complete melting curve.

[0166] As far as the inventors are aware, the system of the present invention is the first system that enables CETSA® of adherent cells on one single microplate.

[0167] 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 those skilled in the art in view of the teachings of this invention that certain changes and modifications may be made therein without departing from the scope of the appended claims.

Claims

1. 1. A system for conducting one or more chemical, biochemical, or biological reactions in one or more wells of a multi-use plate (MTP) at one or more set-point reaction temperatures, comprising: at least one disposable product (MTP) comprising said one or more flat-bottom wells in a body, said wells being capable of acting as containers for said one or more chemical, biochemical or biological reactions requiring one or more defined reaction temperatures according to defined protocols, said body comprising a flat bottom side constituting a first heating surface capable of homogeneously conducting heat to said wells, and a flat top side comprising well openings constituting a second heating surface capable of homogeneously conducting heat in said wells, said second surface optionally being sealed by a thin sealing foil; one or more gradient tempering units (GTUx), each comprising at least one gradient tempering block (10) comprising at least two temperable Peltier elements (12, 13), a thermally conductive block (11) in contact with the at least two temperable Peltier elements (12, 13), a flat-surface positioning area (14) configured for positioning the at least one disposable item (MTP) on the thermally conductive block (11), the tempering block (10) being arranged to contact the flat-surface positioning area (14) of the thermally conductive block (11) and configured to generate a set linear temperature profile in a well of the disposable item (MTP); a control unit comprising one or more processors configured to control said gradient tempering unit(s) (GTUx) for the implementation of said defined protocol for said one or more chemical, biochemical or biological reactions; Including, The tempering block (10) configured to generate the set linear temperature profile comprises at least one Peltier element (12) capable of tempering at a first temperature T1 and at least one Peltier element (13) capable of tempering at a second temperature T2, where T1 can be set higher than T2, and both Peltier elements (12, 13) are arranged to contact the thermally conductive block (11) outside the positioning area (14) for the disposable item (MTP) so that the linear temperature profile can be generated in the thermally conductive block (11) between the two Peltier elements and conducted to the disposable item (MTP) in contact with the thermally conductive block (11).

2. 2. The system of claim 1, wherein the at least one tempering block (10) is positioned to temper the first heated surface and / or the second heated surface of the disposable item (MTP) positioned to contact the positioning area (14a, 14b).

3. 3. The system according to claim 1 or 2, wherein the disposable item (MTP) can be subjected to the linear temperature profile on both heating surfaces by positioned tempering blocks (10a, 10b) set by the linear temperature profile.

4. 4. The system according to claim 1, further comprising at least one tempering unit (TUx) capable of tempering at a temperature Tx, said at least one tempering unit (TUx) comprising at least one tempering block (40) comprising at least two Peltier elements (42), a thermally conductive block (41) in contact with said at least two Peltier elements (42), a flat surface area (44a) for positioning said at least one disposable item (MTP) on said thermally conductive block (41), said tempering block being configured to contact said thermally conductive block (41) and to uniformly temper said disposable item (MTP).

5. 5. The system according to claim 4, comprising tempering blocks (41a, 41b) capable of tempering the disposable items (MTP) placed in the tempering unit (TUx) on both heating surfaces between the tempering blocks (41a, 41b).

6. 6. The system according to claim 1, further comprising transport means capable of moving the disposable article (MTP) to and / or from the gradient tempering unit (GTUx) and / or the tempering unit (TUx) and aligning it with the at least one positioning area(s) (14, 14a, 14b, 44a, 44b) within the interior space (20).

7. 7. The system according to claim 1, further comprising a moving means for moving the disposable item (MTP) in the interior space (20) between the thermally conductive blocks (11, 11a, 11b, 41a, 41b) to bring the one or more positioning areas (14, 14a, 14b, 44a, 44b) of the one or more thermally conductive blocks (11, 11a, 11b, 41a, 41b) into contact with the one or both heating surfaces of the disposable item (MTP).

8. 8. The system according to any one of claims 1 to 7, further comprising one or more securing mechanisms capable of securing the disposable item (MTP) between the first tempering block and the second tempering block (10a, 10b, 40a, 40b, respectively).

9. 9. The system of claim 6, wherein the control unit is configured to control the transport means, the movement means and / or the fixation mechanism for the execution of the defined protocol for the one or more chemical, biochemical or biological reactions.

10. The system according to any one of claims 1 to 9, further comprising an imaging unit I, wherein said imaging unit (I) comprises an imaging device or a readout unit for mass spectrometry capable of capturing an image of at least one well of said disposable item.

11. A method for the use of the system according to any one of claims 1 to 10, comprising the following steps: - loading one or more reaction mixtures into the wells of a disposable product (MTP) and optionally sealing the top side of the disposable product (MTP) with a sealing foil; - introducing the disposable item (MTP) in the system; Transporting and positioning said disposables (MTPs) in one of said one or more gradient tempering units (GTUx) according to a defined reaction protocol; subjecting said disposable article (MTP) to a linear temperature profile on at least one heated surface by a positioned gradient tempering block (10) set to a defined temperature gradient according to said defined reaction protocol; comprising the 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, T1 being higher than T2, and both Peltier elements (12, 13) are arranged to contact the thermally conductive block (11) outside the positioning area (14) for the disposable item (MTP) such that the linear temperature profile is generated in the thermally conductive block (11) between the two Peltier elements (12, 13) and is conducted to the disposable item (MTP) when in contact with the thermally conductive block (11).

12. 12. The method according to claim 11, wherein one gradient tempering unit (GTUx) is used according to a reaction temperature gradient defined by the reaction protocol, and the gradient tempering unit (GTUx) is set to the defined temperature gradient by the control unit.

13. 13. The method according to any one of claims 11 to 12, wherein the disposable is subjected to the linear temperature profile on both heating surfaces by positioned gradient tempering blocks (10a, 10b) set to the defined temperature gradient.

14. 14. The method according to any one of claims 11 to 13, wherein the system further comprises one tempering unit (TUx) according to a defined reaction temperature of the reaction protocol, the tempering unit (TUx) being set to the defined temperature by the control unit, and the disposable item (MTP) being transported and positioned to the tempering unit (TUx) for tempering, preferably cooling, according to the reaction protocol.

15. 15. The method according to any one of claims 11 to 14, further comprising transferring / positioning the disposable item (MTP) in an imaging unit (I) and capturing an image of at least one well of the disposable item (MTP) in a readout unit for mass spectrometry.

16. 16. The method of any one of claims 11 to 15, wherein the chemical, biochemical or biological reaction is for performing a Cellular Thermal Shift Assay (CETSA®), a Purified Protein Thermal Shift Assay (TSA) or any other protein denaturation or aggregation assay.

17. The method according to any one of claims 11 to 16, wherein the method is used for high-throughput reactions or assays.

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