Integrated system for carrying out chemical, biochemical or biological reactions in microwell plate subjected to temperature gradient

Abstract

A system for conducting one or more chemical, biochemical or biological reactions at one or more setpoint reaction temperatures in a disposable consumable comprising one or more wells, said system comprising:-at least one disposable consumable comprising one or more wells in a body, wherein the pores can act as containers for performing one or more chemical, biochemical or biological reactions requiring one or more defined reaction temperatures according to a defined procedure, the body comprising a flat bottom side that constructs a first heating surface capable of conducting heat uniformly into the pores; and a flat upper side comprising the opening of the hole, optionally sealed by means of a thin sealing foil, building a second heating surface capable of conducting heat uniformly in the hole; -one or more gradient temperature control units GTUx, each gradient temperature control unit comprising at least one temperature control block comprising at least two temperature-controllable Peltier elements in contact with the heat-conducting block, a flat surface area opposite the Peltier elements for positioning the at least one disposable consumable on the heat-conducting block, the temperature adjusting block is configured to generate a set linear temperature distribution in one or more holes of the disposable consumable when the temperature adjusting block is in contact with the heat conduction block; -a control unit comprising one or more processors configured to control one or more gradient thermoregulation units GTUx to implement a prescribed protocol for said one or more chemical, biochemical or biological reactions; wherein the gradient temperature control block having a set linear temperature distribution comprises at least one Peltier element that can be temperature-controlled at a first temperature T1 and at least one Peltier element that can be temperature-controlled at a second temperature T2, T1 being higher than T2, and both Peltier elements being in contact with the heat-conducting block outside the positioning region of the disposable consumable, thus, a linear temperature profile may be generated in the thermally conductive block between the two elements and conducted to the disposable consumable when the disposable consumable is in contact with the thermally conductive block. In particular, this solution can be used in a cellular thermal displacement assay # imgabs0 # purified protein thermal displacement assay (TSA) or any other protein denaturation or aggregation assay. The solution allows for high throughput reactions or assays.

Description

[0001] The present invention relates to an integrated system for performing chemical, biochemical or biological reactions (such as but not limited to cellular thermal shift assays) that require one or more set point reaction temperatures / a gradient of set point reaction temperatures in a microplate according to a prescribed protocol. ) Background Art

[0002] In cellular thermal shift assays cells are aliquoted into different reaction vessels and each vessel is heated at a different temperature. Subsequently, the samples are cooled, the cells are lysed, and the amount of soluble protein is quantified by different devices. In vessels with a higher initial temperature, the amount of soluble protein is lower. This is caused by the aggregation of proteins under heat; by shifting the aggregation of proteins to a higher temperature, the stabilization of proteins under compound interaction will increase the amount of soluble protein.

[0003] The method is described for example in EP 2 699 910 A1, where the differential heating step is performed in a device adapted for polymerase chain reaction (also called a PCR device or a PCR cycler).

[0004] The assays are typically performed in a microplate containing multiple wells, preferably a high-density microtiter plate with a large number of wells. To perform a reliable and precise method on samples located in the microplate, it is necessary to apply process parameters precisely and uniformly across the entire microplate.

[0005] The uniform process parameters in the present application refer to a uniform linear temperature profile across the entire microplate along the heating line during the heating process. The temperature profile of the microplate can be obtained by measuring the surface temperature of the microplate in the temperature control unit using an infrared camera and calculating the temperature line scan across the entire microplate (e.g. Figure 4A / B). Such a profile is described by the temperature gradient of the microplate, which describes the direction and rate of temperature change, expressed in temperature units per unit length.

[0006] The solutions of the prior art show the following limitations or major drawbacks:

[0007] - 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-well plates. After the heating step and in order to quantify the amount of soluble protein via luminescence readings, the samples are transferred to another (micro)plate compatible with the luminescence reader used. These additional pipetting steps increase the standard deviation of the assay;

[0008] - The temperature range of the gradient in a PCR cycler is rather small (<21 °C). Proteins aggregated in the temperature range >21 °C cannot be analyzed in one experiment.

[0009] - Adjust the PCR cycler to generate a linear temperature profile required for the entire microplate on the heating block; the temperature profile is generated by at least two heating and two cooling Peltier elements, typically six Peltier elements distributed under the metal block opposite the positioning area that contacts the microtiter plate from below (see Figure 7 ). This setting is a trade-off with the main function of the PCR cycler to provide a uniform temperature distribution. However, this trade-off results in an S-shaped temperature gradient, leading to a non-uniform distribution of temperature data points within the same row of the microplate.

[0010] - To detect low-affinity compound-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]. Since commercial PCR cyclers perform the heating gradient and subsequent cooling of samples on the same heating block, the heating and cooling times make this method rather slow. The CETSA protocol conducted in a PCR cycler cannot be run within three minutes of heating and cooling; within a shorter time, the reaction becomes non-uniform. Figure 7 A schematic diagram of an experiment conducted in a commercial PCR cycler is shown. The experiment shows that a total of five microtiter plates and three pipetting steps are required for one experiment.

[0011] DE 88 14 398 U1 describes a device for generating a temperature gradient, where the temperature gradient is generated in a heat-conducting block extending between two Peltier elements at different temperatures. The device is configured to rapidly change the temperature in a disposable consumable containing a plurality of holes arranged in rows perpendicular to the temperature gradient by transporting the disposable consumable along the heat-conducting block along the temperature gradient to a position on the gradient having the desired temperature and pressing the disposable consumable against the heat-conducting block at that position using a spring fixed to the heat-conducting block. The device cannot conduct a linear temperature profile into the disposable consumable.

[0012] ​US 2021 / 041379 A1 shows an example of a device that includes two temperature elements having a hot surface and a cold surface to create a substantially linear temperature gradient in one or more sample holders, where each sample holder is positioned to contact a first end on a first temperature element and a second opposite end on a second temperature element such that heat flow through the sample holder occurs between the contact regions between the body of the sample holder and the temperature elements. This solution can be used for sample holders that contain multiple wells arranged horizontally or vertically with respect to the temperature gradient.

[0013] The Applicant believes that some of the above limitations are caused by non-uniform temperature distribution during heating in a PCR cycler.

[0014] Accordingly, there is still a need for a solution that allows for the desired precise and uniform application of process parameters across an entire microplate and also uses a high-density microtiter plate that will address cost, thermal response, and uniformity, particularly for experiments. Preferably, the solution should allow the method to be performed in one microplate without transferring samples between plates with different characteristics. The solution should be compatible with flat-bottom microtiter plates and be able to provide a temperature range for experiments in one microplate. Summary of the Invention

[0015] The problem is solved by the system and method as claimed. Exemplary embodiments of the solution can be gathered from the corresponding dependent claims.

[0016] The solution will be elucidated hereinafter without distinguishing between the claimed subject matter (method or system). Instead, the following elucidation is intended to apply similarly to all claimed subject matter, regardless of the context in which they appear.

[0017] There is claimed a system for performing one or more chemical, biochemical, or biological reactions (collectively referred to as reactions) in a disposable consumable that includes one or more wells at one or more set-point reaction temperatures. The disposable consumable is preferably a microtiter plate or a microplate.

[0018] As used herein, "biological reaction" refers to molecular biology, cellular reactions, reactions using cell tissue, meaning an aggregate of cells, viruses, or phages.

[0019] As used herein, "cell" refers to biological cells such as animal cells, plant cells, yeast, fungi, or bacteria.

[0020] The system consists of independent modules that are operably connected to each other via a conveying device for transporting and placing disposable consumables from one module to another as required for automatically implementing a predetermined reaction protocol.

[0021] The system of the present invention includes:

[0022] - At least one disposable consumable that includes one or more holes in a body, wherein the holes are capable of serving as containers for one or more reactions that require one or more specified reaction temperatures according to a specified protocol. The body includes a flat bottom side (also referred to as the body bottom plate) that is configured to conduct heat uniformly to the first heating surface in the holes; and a flat upper side that includes hole openings and is configured to conduct heat uniformly in the holes to the second heating surface;

[0023] - One or more gradient temperature control units GTUx, each gradient temperature control unit including at least one temperature control block that includes at least two temperature-adjustable Peltier elements, a heat-conducting block in contact with the temperature-adjustable Peltier elements, and a flat surface area for positioning the at least one disposable consumable on the heat-conducting block. The temperature control block is configured to generate a linear temperature distribution in the holes of one or more disposable consumables in contact with the heat-conducting block;

[0024] - A control unit that includes one or more processors and is configured to activate one or more gradient temperature control units to implement a specified protocol for the one or more chemical, biochemical, or biological reactions;

[0025] Wherein, the temperature control block having a linear temperature distribution includes at least one Peltier element that can be temperature-adjusted at a first temperature T1 and at least one Peltier element that can be temperature-adjusted at a second temperature T2, where T1 is higher than T2, and both Peltier elements are in contact with the heat-conducting block outside the positioning area of the disposable consumable, so that a linear temperature distribution can be generated in the heat-conducting block between the two elements and conducted to the disposable consumable in contact with the heat-conducting block.

[0026] In other words, the Peltier elements are in contact with one side of the heat-conducting block at the first end and the second end of the heat-conducting block, while the positioning area is located on the opposite side between the first end and the second end of the heat-conducting block.

[0027] In one embodiment, the at least one temperature control block is positioned to temperature-control the first and / or second heating surfaces of the disposable consumable placed in the positioning area. In a preferred embodiment, the disposable consumable can be subjected to the linear temperature distribution on the two heating surfaces by the positioning temperature control block having a linear temperature distribution.

[0028] As used herein, the term "temper (or tempering)" means maintaining the temperature at one or more set points. A "tempering block" is a device capable of reaching (heating, cooling) and maintaining a temperature. A "temperable" element can be tempered, i.e., can reach and maintain a certain temperature.

[0029] As used herein, the term "positioning area" refers to the functional surface of a thermally conductive block that can temper a disposable consumable while the disposable consumable is in contact with the positioning area. The positioning area is also referred to as the active area. The positioning area can be adjusted to be suitable for better positioning of the disposable consumable, for example, in the form of a positioning table.

[0030] In one embodiment, the first Peltier element at the first temperature T1 is a heating Peltier element, which means that the first temperature T1 is set above room temperature; the second Peltier element at the second temperature T2 is a cooling Peltier element, which means that the first temperature T2 is set below room temperature.

[0031] In one embodiment, the at least one tempering block is positioned to temper the first and / or second heating surfaces when a disposable consumable is placed in the positioning area.

[0032] In one embodiment, with a positioning gradient tempering block that sets a (same) linear temperature distribution, a disposable consumable can experience this linear temperature distribution on two heating surfaces.

[0033] In one embodiment, the system further includes at least one additional tempering unit TUx that can be tempered at a temperature Tx. The tempering unit TUx includes at least one tempering block (also referred to as a temperature-controlled block). The tempering block includes at least one Peltier element in contact with a thermally conductive block and a flat surface area opposite the Peltier element for positioning the at least one disposable consumable on the thermally conductive block. The tempering block is configured to uniformly temper the disposable consumable when the disposable consumable is in contact with the thermally conductive block.

[0034] In one embodiment, when a disposable consumable is placed in the tempering unit (TUx), the disposable consumable can be tempered on two heating surfaces by the positioned tempering block.

[0035] In one embodiment, for each set-point reaction temperature of a specified reaction protocol, the system includes at least one tempering unit TUx.

[0036] In one embodiment, the system includes a tempering unit TUx for rapid temperature removal, also referred to as a cooling unit.

[0037] The preferred control unit is configured to activate one or more cooling units to implement a specified protocol for the one or more chemical, biochemical or biological reactions, especially while the disposable consumable is in contact with the heat conducting block of the temperature control unit.

[0038] In one embodiment, the system further comprises a conveying device, which comprises a carrier, preferably a carrier frame, in which the disposable consumable is placed.

[0039] In one embodiment, the conveying device further comprises a moving device for moving the disposable consumable into and / or out of the gradient temperature control unit GTUx and / or the temperature control unit TUx and aligning it with at least one corresponding positioning area in the internal space.

[0040] In one embodiment, one or more gradient temperature control units GTUx and / or temperature control units TUx comprise a second heat conducting block, which is arranged such that the disposable consumable can be positioned in the internal space between the first and second heat conducting blocks using the moving device and evenly clamped between the blocks.

[0041] In one embodiment, the system further comprises a moving device for bringing one or more positioning areas of the one or more heat conducting blocks into contact with one or two heating surfaces of the disposable consumable positioned in the internal space.

[0042] In one embodiment, the system further comprises one or more clamping mechanisms, which are capable of clamping the disposable consumable between the first and second heat conducting blocks.

[0043] Preferably, the control unit is configured to activate the conveying device, the moving device and / or the clamping device to implement a specified protocol for the one or more chemical, biochemical or biological reactions.

[0044] In one embodiment, the gradient temperature control unit GTUx is configured to create a linear temperature gradient from the shorter left side of the disposable consumable to the shorter right side of the disposable consumable. In other words, the disposable consumable is placed in the gradient temperature control unit GTUx such that the direction of the temperature gradient is along the large side of the disposable consumable, thereby generating a large temperature gradient.

[0045] In one embodiment, the system further comprises a reading / imaging unit I, wherein the imaging unit I comprises an imaging device, which is capable of capturing a scene image of at least one well containing the disposable consumable from the well opening side. In one embodiment, the imaging unit I is configured to capture a scene image covering all the wells of the disposable consumable.

[0046] In one embodiment, the imaging unit I further comprises one or more illumination elements for providing sufficient illumination to the disposable consumable during image acquisition.

[0047] In one embodiment, the surface of at least the positioning area of the heat-conducting block is complementary to the corresponding heating surface (flat or structured) of the disposable consumable. Most preferably, the bottom of the disposable consumable is flat.

[0048] Most preferably, the disposable consumable is selected to be suitable for cell culture / adhesion and / or suitable for one or more readout methods of the readout unit / imaging unit I, most preferably both, thereby limiting or avoiding fluid transfer. In a preferred embodiment, the disposable consumable is suitable for luminescence / fluorescence readout.

[0049] In one embodiment, the readout of the disposable consumable can be performed in a mass spectrometer, for example, by measuring the aggregation of all proteins in a cell sample for proteome analysis.

[0050] In one embodiment, the well openings of the disposable consumable can be sealed by means of a thin transparent sealing foil, which forms the second heating surface.

[0051] Another subject matter of the present application is a method of using a system according to any one of the preceding claims, which comprises the following steps:

[0052] - Loading one or more reaction mixtures into the wells of the disposable consumable and optionally sealing the upper side of the disposable consumable with a sealing foil;

[0053] - Introducing the disposable consumable into the system, transporting and positioning it in one of the one or more gradient temperature control units GTUx according to a specified protocol.

[0054] As used herein, the term "reaction mixture" means a fluid containing several biological or chemical articles 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 articles.

[0055] In one embodiment, one gradient temperature control unit GTUx is used for each reaction temperature gradient specified by the reaction protocol, and the gradient temperature control unit GTUx is set to the specified temperature gradient by the control unit.

[0056] In one embodiment, the disposable consumable is subjected to the linear temperature distribution on two heating surfaces by a positioning temperature control block with a linear temperature distribution set.

[0057] In one embodiment, the linear temperature profile of the temperature regulating block in contact with the second heating surface of the disposable consumable is set using at least one Peltier element that can be temperature regulated at a first temperature T’1 and at least one Peltier element that can be temperature regulated at a second temperature T’2, where T’1 is higher than T’2, and where T’1 minus T’2 is equal to T1 minus T2.

[0058] In one embodiment, each prescribed reaction temperature of the reaction protocol uses a temperature regulating unit TUx that is set by the control unit to the prescribed temperature. The temperature regulating unit TUx facilitates the fastest cooling of the entire microplate emerging from the gradient temperature regulating unit GTUx.

[0059] Those skilled in the art will understand that the gradient temperature regulating unit GTUx can be used as a temperature regulating unit to uniformly temperature regulate the disposable consumable by setting the temperature T1 = T2 and / or T’1 = T’2.

[0060] In one embodiment, the method further includes transporting the disposable consumable to the imaging unit I / positioning it within the imaging unit I and capturing an image of at least one well of the disposable consumable.

[0061] The solution as described is particularly suitable for cell thermal shift assays but not limited thereto.

[0062] The solution as described is particularly suitable for high-throughput reactions or assays in disposable consumables where a gradient is required. Detailed Description

[0063] The solution is now described in more detail. Any and all use of examples or exemplary language (e.g., “such as”) provided herein is for illustrative purposes only to better explain the invention and does not limit the scope of the solution. No language in the specification should be construed as indicating that any non-claimed element is essential for the practice of the invention.

[0064] Preferred embodiments of the invention are described, including the best mode known to the inventors for practicing the invention.

[0065] Variations of these preferred embodiments will be apparent to those of ordinary skill in the art, and such variations may be adopted as appropriate, and the inventors intend to practice the solutions in ways other than those explicitly described herein.

[0066] Accordingly, the solution includes all modifications and equivalents of the subject matter recited in the appended claims, as permitted by applicable law. Additionally, this specification covers any combination of the described elements in all possible variations thereof, unless otherwise stated herein or clearly contradicted by the context.

[0067] In one embodiment, a disposable consumable having 1536 wells can be used, wherein each well of the disposable consumable has a volume of at most 10 μl, particularly 0.3 to 6 μl, particularly 0.5 to 4 μl, particularly 5 4 μl, particularly 1 μl. Any well cross-section can be used; typically a rectangular or circular cross-section is used. It will be apparent to those skilled in the art that other forms of disposable consumables can be used.

[0068] In one embodiment, the disposable consumable is a microtiter plate (also known as an MTP or microplate) or a picotiter plate.

[0069] For the purposes of the present invention, the most preferred forms and patterns conform to the recommendations of the Society for Biomolecular Laboratory Automation and Screening (ANSI / SLABS1-2004, ANSI / SBLAS2-2004, ANSI / SBLAS 3-2004 and ANSI / SBLAS 4-2004) for disposable consumables. The disposable consumable can be of any form and pattern, including at least 96, 384, 1536 or 3456 wells, preferably 1536 wells, in the established 96-well format (12×8), 384-well format (24×16), 1536-well format (48×32) or 3456-well format (48x72) of the ANSI standards of the Society for Laboratory Automation and Screening.

[0070] In the present solution, the wells of the disposable consumable are provided perpendicular to the flat bottom side, and thus, when the disposable consumable is in the positioning area of the thermally conductive block, the wells are perpendicular to the linear temperature distribution. Each row of wells is at a different position along the gradient and thus at a slightly different temperature from the other wells.

[0071] In one embodiment, the body is made of a thermoplastic polymer capable of being sealed with a sealing foil, preferably made of polystyrene, polypropylene or COC (cycloolefin copolymer), with or without an added thermally conductive medium. The disposable consumable can include a frame support made of polycarbonate or polystyrene. In one embodiment, the flat bottom of the disposable consumable can be defined by a transparent film fixed to the body containing the wells, which allows images to be captured from the bottom side.

[0072] In one embodiment, after filling the reactants into the wells, the disposable consumable can be sealed with a transparent thin sealing foil as determined in the prior art. The transparent thin sealing foil can be made of polycarbonate, polypropylene, cycloolefin, or other plastic materials known to those skilled in the art, or made of a multilayer film composed of two or more transparent materials, which has the desired barrier properties recognized in the art. The sealing can be achieved by welding the sealing foil to the body of the disposable consumable.

[0073] In both the gradient temperature control unit GTUx and the temperature control unit TUx, thermal communication is achieved between one or more heating surfaces of the disposable consumable and the corresponding one or more heat conducting blocks to perform heat treatment on the liquid sample contained therein.

[0074] The first heating surface of the disposable consumable is selected to be adapted to the surface of the corresponding positioning area of the temperature control unit. In one embodiment.

[0075] In one embodiment, a disposable consumable with a flat bottom is used. The flat bottom is particularly beneficial for the automation of the transportation and / or clamping of the disposable consumable. The bottom thickness can be arbitrary, and the conventional bottom thickness is less than 1000 μm.

[0076] Most preferably, the disposable consumable is selected to be suitable for cell culture and / or suitable for one or more readout methods, especially luminescence / fluorescence readout, most preferably both, thereby limiting or avoiding fluid transfer.

[0077] Commercially available microplates can be used, such as 1536-well microplates (PS, LoBase) (Greiner Bio-One, catalog number 783092), 384-well microplates (PS, )(Greiner Bio-One, catalog number 781092), but not limited thereto.

[0078] In one embodiment, one or more gradient temperature control units GTUx and / or one or more cooling / temperature control units (TUx) include a second temperature control block, which is arranged such that the disposable consumable can be positioned in the internal space between the first and second heat conducting blocks using a mobile device and evenly clamped between the blocks. In this embodiment, the disposable consumable can be temperature-controlled each time it contacts the two temperature control surfaces.

[0079] In one embodiment, regardless of whether the wells are sealed with a sealing foil, the positioning area of the second heat conducting block in contact with the second heating surface of the disposable consumable is planar, preferably uniform.

[0080] In one embodiment, the temperature gradient of the heat conducting block in the gradient temperature control unit GTUx that is in contact with the first heating surface (bottom side) of the disposable consumable is a linear temperature gradient (also known as temperature profile) TGx.

[0081] In one embodiment, the temperature T1 is typically set to 37 °C and T2 is set to T1 + TGx. Those skilled in the art should understand that the exact parameters of the temperature gradient TGx, T1, and T2 depend on the defined protocol for the reaction of interest.

[0082] In one embodiment, the temperature gradient is provided along the length side of the disposable consumable.

[0083] It has been found that the solution of the present application can provide a uniform linear gradient of about 20 to 45 °C, preferably 25 to 35 °C, along the length direction of a standard-sized disposable consumable.

[0084] In one embodiment, the temperature control blocks of the gradient temperature control unit GTUx each comprise an array of two or three Peltier elements that can operate at the first temperature T1 and an array of two or three Peltier elements that can operate at the second temperature T2, and the arrays contact the heat conducting block from opposite sides of the positioning area of the disposable consumable outside the positioning area ( Figure 1 or 6), so that a linear temperature profile is generated between the two arrays and conducted to the disposable consumable when the disposable consumable is in contact with the positioning area of the heat conducting block. Those skilled in the art should understand that the appropriate number of Peltier elements in each array can be optimized according to the relationship between the performance of the Peltier elements and the geometry of the disposable consumable.

[0085] In one embodiment, one or more temperature control blocks of the temperature control unit TUx comprise a heat conducting plate on which the disposable consumable is placed, and an array of Peltier elements (i.e., independent physical units that convert electric current into heat / cold) that are in contact with the heat conducting plate. Considering that the standard MTP has an aspect ratio of 3:2, an array of six Peltier elements is most preferred.

[0086] In one embodiment, the temperature of the Peltier elements is monitored using a temperature sensor (e.g., a thermistor). To achieve the most uniform temperature control, it is preferred to provide a thermal sensor for each Peltier element and control each Peltier element individually.

[0087] In one embodiment, the temperature control blocks of the gradient temperature control unit GTUx and / or the temperature control blocks of the gradient temperature control unit GTUx further comprise heat sinks that dissipate all the electric power converted into heat or cold and carry the Peltier elements. Additionally, a heat sink / fan combination can be used.

[0088] In one embodiment, the heat-conducting block in the gradient temperature control unit GTUx that contacts the second heating surface of the disposable consumable is set to have the same linear temperature profile TGx (obtained from the difference between T1 and T2) as the heat-conducting block that contacts the first heating surface.

[0089] In one embodiment, the heat-conducting block in the gradient temperature control unit GTUx that contacts the second heating surface of the disposable consumable can be set to exhibit the same linear temperature profile TGx at temperatures T'1 and T'2 that are slightly higher (e.g., +1 °C) than the temperatures T1 and T2 set for the heat-conducting block that contacts the bottom of the disposable consumable. If a thin transparent sealing foil is used as the second heating surface, this higher temperature has been proven to prevent condensation on the sealing foil within the holes at the higher temperatures of the profile.

[0090] In one embodiment, the alignment of the linear profile can be improved by positioning / alignment of the edges on the heat-conducting block so as to precisely position the corresponding structural elements of the disposable consumable within the positioning area of the temperature control unit.

[0091] One or more of the above-described gradient temperature control units GTUx have been proven capable of rapidly creating a large linear temperature gradient that ranges from the shorter left side to the shorter right side of a microtiter plate, covering the entire flat-bottom microtiter plate up to the 1536-well format (see Figure 4B ).

[0092] In one embodiment, the temperature of the heat-conducting block in the temperature control / cooling unit TUx that is used to contact the thin transparent sealing foil on the second heating surface of the disposable consumable is kept at a temperature slightly higher than the temperature of the heat-conducting block that contacts the bottom side of the disposable consumable. This higher temperature has been proven to prevent condensation on the sealing foil within the holes.

[0093] In one embodiment, one or more heat-conducting blocks made of metal (such as aluminum) or glass, preferably metal, can be used.

[0094] In one embodiment, the heat-conducting block that contacts the second heating surface is made of glass. An imaging / readout device can be used to acquire images through the glass plate / block, for example, for monitoring fluorescence changes. In addition, temperature changes within the disposable consumable can also be monitored.

[0095] In one embodiment, the system includes an imaging unit I, wherein the imaging unit I includes an imaging device.

[0096] An imaging device can be a mechanical, digital or electronic viewing device, such as a still camera, a video camera recorder, a movie camera, a scanner or any other instrument, equipment or form capable of recording, storing or transmitting visual images of an object. In one example, a CCD camera (e.g., an sCMOS camera) or an image magnification camera can be used.

[0097] In one embodiment, a camera with an objective lens can be used. In one embodiment, a 35mm F1.6 C-mount objective lens can be used, so that a complete disposable consumable can be imaged. In one embodiment, a microscope objective lens can be used. In one embodiment, the imaging unit I can be configured to acquire single-well images. In one embodiment, the system can implement multiplex immunofluorescence readout, such as, for example, from PhenoCycler System of Akoya Biosciences as is known. Thus, up to 100 different protein aggregation profiles can be detected in a single experiment.

[0098] In one embodiment, the imaging unit I further includes one or more lighting elements for providing sufficient illumination to the disposable consumable during image acquisition. In one embodiment, a ring light is used, which is positioned to provide uniform illumination to the disposable consumable above all chambers during image capture.

[0099] In one embodiment, a filter can be used.

[0100] Those skilled in the art should understand that the objective lens and / or the filter depend on the use of the device. There are also excitation and emission filters for image acquisition using photosensitive substances or genetically encoded luminescent or fluorescent reporters. For example, if a compact device is required, the imaging unit I can also include one or more optical lenses and / or mirrors.

[0101] In one embodiment, the imaging unit I includes support and calibration equipment for positioning the imaging device, the lighting element and / or the filter relative to the disposable consumable to achieve optimal image acquisition.

[0102] In one embodiment, the imaging device can be positioned to acquire an image from the second heating surface of the disposable consumable.

[0103] In one embodiment, the imaging device can be positioned to acquire an image through the glass plate of one or more of the above-mentioned gradient temperature control unit GTUx and / or temperature control unit TUx.

[0104] In one embodiment, the readout unit / imaging unit I can include a temperature control block for temperature control of the disposable consumable from the side opposite to the well opening or the sealing foil.

[0105] In one embodiment, the conveying device is capable of conveying and positioning a disposable consumable in a reading unit or an imaging unit I for image capture of at least a portion of the disposable consumable, most preferably the entire pore opening side of the disposable consumable.

[0106] In one embodiment, the imaging unit I includes a clamping frame or a transparent splint so that the disposable consumable can be clamped between a temperature control block and the splint or the clamping frame. For this purpose, a clamping mechanism can be used.

[0107] In one embodiment, the control unit is configured to control the imaging unit I. Controlling the imaging unit I includes activating, deactivating, or positioning an imaging device, a lighting device, a filter, and / or a clamping mechanism according to the requirements of optimal image acquisition.

[0108] In one embodiment, the conveying device includes a moving mechanism or a moving device for moving the disposable consumable into and / or out of the internal space of one or more of the gradient temperature control unit GTUx, the temperature control unit TUx, and / or the imaging unit I, where the internal space is defined as the alignment space between a first and a second heat conducting block (its positioning area) in the gradient temperature control unit GTUx or the temperature control unit TUx, or the alignment space between the temperature control block and the splint or the clamping frame in the imaging unit I (Figure 4).

[0109] In one embodiment, the conveying device includes at least one horizontal driver, and the disposable consumable is positioned on a moving carrier for conveying and positioning in one or more gradient temperature control units GTUx, one or more temperature control units TUx, and / or the imaging unit I according to the requirements of the reaction protocol.

[0110] In some embodiments, the carrier can move between the internal space and an external disposable consumable loading position outside the instrument frame / housing to load or unload a microplate onto or from the carrier. Specifically, in some embodiments, the carrier is movably mounted on a base for repeated bidirectional movement between the internal and external disposable consumable positions. In some embodiments, the moving mechanism is configured as a carrier drive mechanism for driving the carrier in either of two directions to drive the carrier into one or more internal and external spaces or microplate positions, respectively.

[0111] In one embodiment, the carrier is slidably mounted to one or more horizontal drivers such that repeated bidirectional movement can be performed between a processing position inside the system for heat-treating reaction products and a loading position outside the system for loading or unloading a disposable consumable onto or from the carrier.

[0112] In one embodiment, the conveying device includes one or more horizontally arranged stop plates for precisely positioning the carrier and setting it in the internal space of the unit in alignment with its positioning area. In one embodiment, one stop plate is used for each unit.

[0113] Since such a sliding mechanism is well-known to those skilled in the art, it is not necessary to elaborate further herein. In some embodiments, the system includes an automated carrier drive mechanism such as a motor-based belt or wheel drive for automatically moving the carrier between the processing position and the loading position. Since such a drive mechanism is well-known to those skilled in the art, it is not necessary to elaborate further herein. In one embodiment, horizontal and vertical motors (e.g., servo motors or stepper motors) are used to convey the microplate assembly (i.e., the microplate and the carrier) into contact with the top surface of the temperature control block.

[0114] In one embodiment, the disposable consumable is positioned in the carrier, on a metal heating fixture, the shape of which closely fits the disposable consumable, especially its frame support, and the positioning table / area of the temperature control block.

[0115] In one embodiment, the system includes one or more clamping mechanisms for clamping the disposable consumable in the internal space of one or more gradient temperature control units GTUx, one or more temperature control units TUx, and / or imaging unit I.

[0116] In the temperature control unit (GUTx or TUx), the clamping mechanism is capable of moving one or two temperature control blocks relative to each other to allow the disposable consumable to be clamped between these heating blocks after the disposable consumable is correctly positioned in the internal space between the heating blocks by the conveying system. Thus, full contact on both sides of the disposable consumable can be advantageously obtained by clamping.

[0117] In one embodiment, the imaging unit I may include a temperature control block. In one embodiment, the clamping mechanism can move the temperature control block relative to the clamping plate / clamping frame to position the disposable consumable in the imaging unit I.

[0118] Preferably, at least one temperature control block in each temperature control unit (GTUx or TUx) or one temperature control block in the imaging unit I or the clamping plate / clamping frame is spring-mounted to smoothly clamp the disposable consumable. In one embodiment, the spring constant and stroke of the spring mounting element can be used to implement force measurement.

[0119] In one embodiment, the clamping of the disposable consumable can be achieved by a vertical motor or a lever that moves the lower temperature control block upward along the vertical drive and presses the disposable consumable against the spring-mounted upper temperature control block. The spring constant and stroke of the upper temperature control block can be used to implement force measurement.

[0120] In one embodiment, the same clamping mechanism is used to clamp the disposable consumable in the temperature control unit (GTUx and / or TUx) and / or the imaging unit I. In particular, a vertical motor or lever can be used to move one or more lower temperature control blocks in different units (temperature control unit GTUx and / or TUx and / or imaging unit I) upward. In this embodiment, spacer / alignment blocks can be used to achieve alignment positioning of the disposable consumable in the unit, particularly by aligning the positioning areas of the temperature control unit (GTUx and / or TUx) and / or the imaging unit I. In one embodiment, the temperature control block can be mounted on the spacer / alignment block for better positioning.

[0121] Those skilled in the art will understand that other embodiments can be used to clamp the disposable consumable in the internal space of the unit. For example, the device for clamping can be such that the disposable consumable is pressed down onto the temperature control block and / or the lower temperature-controlled platen.

[0122] In one embodiment, the control unit can be configured to activate and / or control the temperature control unit, the conveying device, and / or the clamping mechanism according to a predetermined reaction protocol.

[0123] Compared with the static embodiment (a temperature-controlled heating / cooling unit), this embodiment has the advantage of not having to wait for the temperature control block to switch to the next set point temperature, which can significantly speed up the test time.

[0124] In one embodiment, the system of the present invention can include a module for providing all the reagents required for the reaction to the disposable consumable in the correct concentration. As the unit for providing all the reagents in the present invention, a pipetting robot or a pipetting unit can be used.

[0125] In some embodiments, the system of the present invention is an instrument for incubating, heat-treating, or otherwise treating liquid samples, such as an automated thermal cycler capable of subjecting a liquid reaction mixture to a series of temperature changes, for example, for performing cell thermal shift assays Purified protein thermal shift assay (TSA) or any other protein denaturation or aggregation assay.

[0126] In some embodiments, the instrument is used for chemically treating liquid samples, for example, by performing tests or assays related to immuno-chemical or clinical-chemical analysis items.

[0127] To perform all these steps, the system generally requires a user-programmable computer system configured to control the system through the control unit.

[0128] In one embodiment, a user inputs a reaction protocol into the system via a user interface or selects a suitable reaction protocol from a database containing a set of reaction protocols.

[0129] The solution of the present invention is particularly suitable for automatically performing chemical, biochemical or biological reactions, especially high-throughput reactions and assays.

[0130] A further object of the present invention is to provide a method of using the system of the present invention described above, which comprises the following steps:

[0131] - Loading a reaction mixture into the wells of a disposable consumable and optionally sealing the upper side of the disposable consumable with a sealing foil;

[0132] - Introducing the (optionally sealed) disposable consumable into the system, transporting and positioning it in one of the gradient temperature control units GTUx and / or temperature control units TUx according to a specified protocol.

[0133] In a further embodiment, the method further comprises transporting the disposable consumable to the imaging unit I and positioning it in the imaging unit I and capturing an image of at least one well of the disposable consumable.

[0134] Each specified reaction temperature gradient of the preferred reaction protocol uses one gradient temperature control unit GTUx.

[0135] To achieve rapid cooling of the sample after the gradient step, the temperature of the TUx is preferably lower than room temperature. Each specified reaction temperature of the preferred reaction protocol uses one temperature control unit TUx.

[0136] It is also preferred to set the temperature of the second temperature control block (in contact with the sealing foil) in the temperature control unit TUx at a temperature slightly higher than the highest specified reaction temperature of the reaction protocol.

[0137] The preferred settings of the gradient temperature control unit GTUx are as described above.

[0138] Therefore, the object of the present application is:

[0139] A system for performing one or more chemical, biochemical or biological reactions at one or more set-point reaction temperatures in one or more wells of a disposable consumable, the system comprising:

[0140] - At least one disposable consumable, which contains one or more holes in the body, wherein the holes can act as containers for one or more chemical, biochemical or biological reactions that require one or more specified reaction temperatures according to a specified protocol, the body contains a flat bottom side, and the bottom side is constructed to conduct heat evenly to a first heating surface in the holes; and a flat upper side containing hole openings, which is optionally sealed by a thin sealing foil, and is constructed to conduct heat evenly in the holes to a second heating surface;

[0141] - One or more gradient temperature control units GTUx, each gradient temperature control unit contains at least one temperature control block, the temperature control block contains at least two adjustable Peltier elements in contact with a heat conducting block, and a flat surface positioning area opposite the Peltier elements and configured to position the at least one disposable consumable on the heat conducting block, the temperature control block is configured to generate a set linear temperature distribution in the holes of the disposable consumable when the disposable consumable is in contact with the heat conducting block;

[0142] - A control unit containing one or more processors, which is configured to control one or more gradient temperature control units GTUx to implement a specified protocol for the one or more chemical, biochemical or molecular biological reactions;

[0143] Wherein, the heating block with a set linear temperature distribution contains at least one Peltier element that can be temperature-controlled at a first temperature T1 and at least one Peltier element that can be temperature-controlled at a second temperature T2, where T1 is higher than T2, and both Peltier elements are in contact with the heat conducting block outside the positioning area of the disposable consumable, so that a linear temperature distribution can be generated in the heat conducting block between the two elements and conducted to the disposable consumable when the disposable consumable is in contact with the heat conducting block.

[0144] In one embodiment, the at least one temperature control block is positioned to temperature-control the first and / or second heating surfaces when the disposable consumable is placed on the positioning area.

[0145] In one embodiment, through the positioning heating block with a set linear temperature distribution, the disposable consumable can experience the linear temperature distribution on the two heating surfaces.

[0146] In one embodiment, the system further contains at least one temperature control unit TUx that can be temperature-controlled at a temperature Tx, the temperature control unit TUx contains at least one heating block, the heating block contains at least two Peltier elements in contact with a heat conducting block; a flat surface area opposite the Peltier elements for positioning the at least one disposable consumable on the heat conducting block, and the heating block is configured to evenly temperature-control the disposable consumable when the disposable consumable is in contact with the heat conducting block.

[0147] In one embodiment, when the disposable consumable is placed in the temperature control unit TUx, the disposable consumable can be temperature-controlled on two heating surfaces by the positioned heating block.

[0148] In one embodiment, the system further includes a conveying device that can move the disposable consumable into and / or out of the gradient temperature control unit GTUx and / or the temperature control unit TUx and align it with at least one positioning area in the internal space.

[0149] In one embodiment, the system further includes a moving device configured to bring one or more positioning areas of the one or more heat-conducting blocks into contact with one or two heating surfaces of the disposable consumable when the disposable consumable is positioned in the internal space.

[0150] In one embodiment, the system further includes one or more clamping mechanisms that can clamp the disposable consumable between the first and second heating blocks.

[0151] In one embodiment, the control unit is configured to control the conveying device, the moving device, and / or the clamping mechanism to implement a prescribed protocol for the one or more chemical, biochemical, or biological reactions.

[0152] In one embodiment, the system further includes an imaging unit I, wherein the imaging unit I includes an imaging device that can capture an image of at least one hole of the disposable consumable from the sealed side.

[0153] Another object of the present application is a method of using the above system, the method comprising:

[0154] - Loading one or more reaction mixtures into the holes of the disposable consumable and optionally sealing the upper side of the disposable consumable with a sealing foil;

[0155] - Introducing the sealed disposable consumable into the system;

[0156] - Conveying and positioning the disposable consumable in one of the one or more gradient temperature control units GTUx according to a prescribed reaction protocol.

[0157] In one embodiment, each reaction temperature gradient specified by the reaction protocol uses one gradient temperature control unit GTUx, and the gradient temperature control unit GTUx is set to the specified temperature gradient by the control unit.

[0158] In one embodiment, the disposable consumable undergoes the linear temperature distribution on two heating surfaces by the positioned heating block set to the specified temperature gradient.

[0159] In one embodiment, the system further comprises a temperature control unit TUx for each specified reaction temperature of the reaction protocol, the temperature control unit TUx being set to the specified temperature by the control unit, and wherein the disposable consumable is transported to and positioned in the temperature control unit TUx for temperature control according to the reaction protocol, preferably for cooling.

[0160] In one embodiment, the method further comprises transporting the disposable consumable to the imaging unit I / positioning it in the imaging unit I and capturing an image of at least one well of the disposable consumable.

[0161] In particular, the solution can be used for cellular thermal shift assays Purified protein thermal shift assay (TSA) or any other protein denaturation or aggregation assay. The solution allows for high-throughput reactions or assays.

[0162] Unless otherwise specified herein or clearly contradicted by context, the terms "a", "an", "the", and similar references used in the context of describing the invention (especially in the context of the appended claims) shall be construed to cover both singular and plural referents. Unless otherwise indicated, the terms "comprising", "including", "having", and "containing" shall be construed as open-ended terms (i.e., meaning "including but not limited to").

[0163] Unless otherwise stated, the use of any and all example or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating that any non-claimed element is essential for the practice of the invention.

[0164] Preferred embodiments of the invention are described, including the best mode known to the inventors for practicing the invention.

[0165] Variations of these preferred embodiments will be apparent to those of ordinary skill in the art, and such variations may be adopted as appropriate, and the inventors intend to practice the invention in ways other than those explicitly described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the appended claims hereto, as permitted by applicable law. Moreover, the invention covers any combination of the above elements in all possible variations thereof, unless otherwise specified herein or clearly contradicted by context. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1 A three-dimensional view of a temperature control block of a gradient temperature control unit GTUx is shown, showing the positions of Peltier elements that can be temperature-controlled at T1 and T2, respectively, which are located outside the positioning area, where the disposable consumable contacts the aluminum block (heat conduction block) in the positioning area.

[0167] Figure 2 Shows an oblique view of a montage of the system according to the present invention.

[0168] List of reference numerals

[0169] MTP Microwell Plate / Disposable Consumable / Microtiter Plate

[0170] GTUx Gradient Temperature Unit

[0171] 10, 10a, 10b Gradient temperature blocks

[0172] 11, 11a Heat conducting blocks

[0173] 12, 12a, 12b Peltier elements that can be temperature-controlled at the first temperature T1

[0174] 13, 13a, 13b Peltier elements that can be temperature-controlled at the second temperature T2

[0175] 14, 14a, 14b (Hidden) positioning area or positioning table / moving area

[0176] 15 Feeder of the temperature sensor (Pt100) of the Peltier element

[0177] 16, 16a Screws

[0178] 17a, 17b Radiators

[0179] 19 Temperature block housing

[0180] 20 Internal space

[0181] 21 Positioning / alignment edge

[0182] 30 Horizontal driver

[0183] 31 Horizontal lever

[0184] 32 Vertical driver

[0185] 33 Vertical lever

[0186] TUx Temperature unit

[0187] 40, 40a, 40b Temperature-controlled blocks

[0188] 41a, 41b Heat conducting blocks

[0189] 42a, 42b Peltier elements

[0190] 44a, 44b Positioning area / positioning table

[0191] 46a (Hidden), 46b Radiators

[0192] 50 Carrier

[0193] 51 Loading position

[0194] 52 Support frame

[0195] 53 Vertical driver

[0196] 54 Carriage

[0197] I Imaging unit

[0198] 60 Imaging device

[0199] 70 Frame

[0200] 71 Partition wall

[0201] 80 Heat conducting table

[0202] 81 Conductive block

[0203] 82a, 82c External Peltier elements for temperature regulation at discrete temperatures T1, T2 or T3, where T1 < T2 < T3

[0204] 82b Intermediate Peltier element for temperature regulation at the midpoint

[0205] Figure 3 shows the experimental curve (2A) obtained using a qPCR cycler of Screening Technology on a flat-bottom 1536-well plate with androgen receptor and the experimental curve (2B, Shaw et al., Scientific Reports, (2018) 8:163 | DOI:10.1038 / s41598-017-18650-x) obtained using a commercially available PCR cycler on a V-bottom 384-well plate with a published androgen receptor obtained.

[0206] Figure 4A Shows the use of The temperature gradient of a 1536-well microplate obtained using a gradient cycler. Figure 4B Shows the temperature gradient of a 1536-well microtiter plate obtained using the system of the present invention.

[0207] Figure 5 shows the thermal aggregation curve in androgen receptor where suspended cells (5A) or adherent cells (5B) are seeded on a flat-bottom 1536-well microplate treated according to the present invention.

[0208] Figure 6 Shows the use of the solution of the present invention for Schematic diagram of the experiment, where all necessary steps can be carried out on / in the same microtiter plate.

[0209] Figure 7 Shows the schematic diagram of the experiment carried out using a commercially available PCR cycler. Detailed implementation

[0211] Figure 1 Schematic diagram showing a three-dimensional open view of the temperature control block 10 of the gradient temperature control unit GTUx. When the temperature control block 10 is operable, the Peltier elements 12, 13 that can be temperature-controlled at T1 and T2 respectively are positioned outside the positioning table 14. The Peltier elements 12, 13 are housed in the temperature control block housing 19 located above the heat sink 17 (not shown in the figure); heat is dissipated from the heat sink 17. In this embodiment, two Peltier elements are used for each temperature; each Peltier element can be controlled by a temperature sensor powered by the feeder line 15. A Pt100 sensor can be used. During operation, the heat conduction block 11 contacts the heating element through the positioning screw 16 to achieve precise alignment. During positioning, the disposable MTP contacts the aluminum block 11 (heat conduction block 11) on the positioning table 14. In this embodiment, the heat conduction block 11 includes a positioning edge 21 to accurately position the disposable MTP on the positioning table 14.

[0212] Figure 2A perspective view of a montage of a system including a gradient temperature control unit GTUx and a temperature control unit TUx is shown. The gradient temperature control unit GTUx and the temperature control unit TUx each include first / lower temperature control blocks 10a, 40a and second / upper temperature control blocks 10b, 40b. During operation of the system, a disposable MTP is conveyed in one or the other of the temperature control units GTUx / TUx in an internal space 20 (not shown) between the first and second temperature control blocks and is clamped between the temperature control blocks for a period of time specified by the experimental protocol. For automated conveyance, the disposable MTP is conveyed between a loading position 51 for introducing the disposable MTP into the device and the temperature control units using a horizontal conveyance device for conveyance along a conveyance channel connecting the internal space 18 of the temperature control unit GTUx and GTUx. In the present embodiment, the disposable MTP is placed on a carrier 50 which can be moved on a carriage 53 by a vertical drive 53, and the carriage 53 can be moved into / out of the internal space 18 (not shown) of each temperature control unit on a horizontal drive 30 using a horizontal lever 31. During the experiment, driven by an automated software program in a control unit (not shown), the disposable MTP engaged in the carrier 50 is conveyed along the horizontal drive 30 by the horizontal lever 31 (which can use a stepper motor) into the internal space 18 (not shown) of the gradient temperature control unit GTUx and aligned with positioning areas / positioning tables 14a, 14b, where the gradient temperature control blocks 10a, 10b are moved by abutting against the disposable MTP (also referred to as a clamping mechanism) and are pressed against and held on the positioning areas. Reaction is allowed to proceed according to a predetermined protocol. By opening the clamping mechanism, the disposable MTP in the carrier 50 is released from the gradient temperature control unit GTUx and moved into the internal space of the temperature control unit TUx and aligned with positioning areas / positioning tables 44a, 44b between the temperature control blocks 40a, 40b for uniform cooling according to a predetermined protocol. The disposable MTP can be conveyed to an imaging unit I (not shown) for image acquisition or protein quantification via mass spectrometry.

[0213] The gradient temperature control blocks 10a, 10b include heat conducting blocks 11a, 11b which are temperature controlled by a Peltier element temperature controllable at a first temperature T1 and a Peltier element temperature controllable at a second temperature T2, thereby building a desired temperature gradient in the positioning areas / positioning tables and in the disposable MTP when positioned in the gradient temperature control unit GTUx; heat is released from each gradient temperature control block through heat sinks 17a, 17b respectively. In the present embodiment, the temperature control blocks 40a, 40b of the temperature control unit TUx include heat conducting blocks which are temperature controlled by Peltier elements operable at a set point temperature; heat is released through heat sinks 46a (hidden), 46b. In Figure 2In an embodiment, the clamping of the disposable MTP is achieved by moving one or two temperature control blocks along the vertical drive 32 using a horizontal lever 33 (a vertical motor can be used) and pressing the temperature control blocks against the disposable MTP. The temperature control blocks can be spring-mounted; the spring constants and corresponding strokes of one or more temperature control blocks can be used to implement force measurement (not shown). In this embodiment, the fixing elements of the gradient temperature control unit GTUx and the temperature control unit TUx are mounted on the frame 70. Except for the conveying channels, the temperature control units can be separated from each other by segments of the partition wall 71.

[0214] Experiment

[0215] In one example, this solution is used for This example will be explained in somewhat more detail below, but it is not intended to limit the present invention to this embodiment.

[0216] Experiments were carried out according to the protocol of Shaw et al. using a cell line containing a luminescence sensor for detecting the stability of the protein of interest (androgen receptor). In contrast, Shaw et al. did not use a luminescence sensor but detected soluble proteins by technology.

[0217] Using a number of flat-bottom 1536-well microplates heated at different uniform temperatures with a qPCR cycler utilizing Screening Technology, by correlating the results from microplates at different temperatures, a complete melting curve (3A) of the protein of interest was constructed. The small molecule DHT causes thermal stabilization of the androgen receptor.

[0218] 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 transparent permanent adhesive film (Applied Biosystems, 4311971). Commercially available 1536-well flat-bottom microplates were used. Each microplate was centrifuged and assayed in a qPCR cycler of Screening Technology.

[0219] Figure 3 shows the experimental curves obtained with androgen receptor on flat-bottom 1536-well plates temperature-controlled at a number of discrete temperatures within a predetermined range using a qPCR cycler as described in PCT / EP2022 / 077322, and compared with the published androgen receptor on V-bottom 384-well plates using a commercially available PCR cycler compared with the experimental curves obtained (3B, Shaw et al. 2018). The curves obtained are consistent. Heating of the flat-bottom 1536-well plate results in thermal aggregation of the protein of interest, while binding of the small molecule reduces aggregation, thus confirming that the flat-bottom 1536-well plate is suitable for

[0220] For the uniform heating of the microplate used in the qPCR cycle of Screening Technology, 14 microplates heated at discrete temperatures are required to generate the complete melting curve of the protein of interest.

[0221] Comparison of gradient cycler and the system of the present invention using flat-bottom microplates :

[0222] To our knowledge, The gradient cycler is the only commercially available device designed to achieve the required gradient on a flat-bottom microplate. The cycler was selected for further comparative experiments.

[0223] The microtiter plate (1536-well microplate (PS, LoBase) (Greiner Bio-One, catalog number 783092) was left on the gradient cycler for three minutes. After running, the surface temperature of the entire microplate was directly obtained using an infrared camera. The temperature line scan of the entire microplate ( Figure 4A ) showed the temperature gradient of the flat-bottom 1536-well microtiter plate generated by the gradient cycler. The brackets indicate the location where the linear temperature gradient occurs on the plate. It was found that the cycler generated only a sufficient linear thermal gradient with a poor uniformity (homogene) of about 13 °C (very small) after 5 minutes.

[0224] Presumably, the region with a sufficient linear gradient is limited by the heating and cooling Peltier elements located directly below the heat conducting block in contact with the bottom plate of the microplate. Additionally, the heating lid that presses the plate onto the temperature control block may only achieve a uniform temperature; this would interfere with the gradient generated by the temperature control block in contact with the bottom plate of the microplate.

[0225] To generate a melting curve using only one microtiter plate, a device for applying a temperature gradient on the microtiter plate is required. Comparative experiments were carried out using the system according to Figure 2 of.

[0226] Figure 4B The temperature gradient of the 1536-well microtiter plate obtained in the system of the present invention is shown. The microplate was pressed between the gradient temperature control block 10a and the gradient temperature control block 10b for 5 seconds, 30 seconds, 60 seconds, or 180 seconds, respectively. After heating, the surface temperature of the microplate was directly obtained using an infrared camera.Figure 4B shows the temperature gradient / line scan across the entire plate. The system used generates a linear temperature gradient of approximately 26 °C on a 1536-well plate and heats the plate within 60 seconds. This is a significant improvement compared to the line scan (4A) across the plate in a cycler.

[0227] Using the solution of the present invention and a luminescence sensor for detecting protein stability, the experiment of Shaw et al. on androgen receptor was repeated.

[0228] The androgen receptor cell line was seeded on a flat-bottom 1536-well microplate and measured either in suspension or after adhesion to the microplate. In both cases, the cells were first incubated with DHT or DMSO for one hour, then heated for one minute and cooled for one minute in the system of the present invention. After adding the CETSA reagent, both plates were measured in a luminescence reader.

[0229] Figure 5 shows the thermal aggregation curves in androgen receptor wherein suspension cells (5A) or adherent cells (5B) were seeded on a flat-bottom 1536-well microplate treated according to the present invention. In both cases, a distinct shift in the thermal aggregation curve was observed for the cells treated with DHT. This experiment demonstrates that the present invention enables performing Each experiment only requires one microtiter plate.

[0230] Figure 6 shows the use of the solution of the present invention Schematic diagram of an experiment, in which the main system components are presented in cross-section. The system comprises a gradient thermostat unit GTUx, a thermostat unit TUx and an imaging unit I. Horizontal transport channels are presented with horizontal arrows; clamping mechanisms are not presented. The simplified imaging unit I comprises an imaging device 60. The thermostat units GTUx and TUx comprise a first / lower thermostat block 10a, 40a and a second / upper thermostat block 10, 40b, respectively. During operation of the system of the present invention, a disposable MTP is positioned in the interior space 20 of one or the other thermostat unit and is clamped between the positioning areas 14a, 14b or 44a, 44b, respectively, for a period of time as specified in the experimental protocol and then transported to the next thermostat unit for further reaction or transported 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 array of Peltier elements 12a, 13a and 12b, 13b in the gradient temperature control unit GTUx relative to the heat-conducting blocks 11a, 11b and the positioning areas 14a, 14b, compared with the array of Peltier elements 42a and 42b in the temperature control unit TUx relative to the heat-conducting blocks 41a, 41b and the positioning areas 44a, 44b.

[0231] It should be noted that for the PCR assay described by Shaw et al. 2018, a commercially available PCR cycler was used and Figure 7 One of the schematics presented in Experiment, where arrows indicate transfer to the next plate for further processing. Samples must be transferred between different plates because different plates are needed for culturing cells (MTP1), heating cells and reaction mixture (MTP2, MTP3), and luminescence / fluorescence readout (MTP4 and MTP5). For the heating step, the sample must be split onto two plates (MTP2, MTP3) because The temperature gradient that the cycler can generate in the microplate is not large enough. Two V-bottom PCR plates MTP2 and MTP3 are used, for high temperature range heating (T2 to T3) and low temperature range heating (T1 to T2), respectively. The gradient is achieved by distributed Peltier elements 82 to adjust the temperature of the heat block 81 and the heat stage 80. The low range gradient is set at temperatures T1 and T2 (where T1 <T2)下的外部珀尔帖元件82a、82c实现,并由设置在中点处的中间珀尔帖元件82b辅助。类似地,高范围梯度通过将外部珀尔帖元件分别设定在T2和T3(其中T2<T3)下并由中点处的中间珀尔帖元件辅助来实现。每个微孔板MTP2和MTP3的内容物分别被转移到平底板MTP4和MTP5以便读数。总共需要5个微孔板(MTP)才能获得完全熔解曲线所需的数据。

[0232] To the best of our knowledge, the system of the present invention is the first to allow adherent cells on a single microplate.

[0233] Although the foregoing invention has been described in considerable detail for purposes of clarity of understanding by way of illustration and example, it will be apparent to those skilled in the art that certain changes and modifications may be made thereto in light of the teachings of the present invention without departing from the scope of the appended claims.

Claims

1. A system for performing one or more chemical, biochemical, or biological reactions in one or more wells of a disposable consumable (MTP) at one or more set-point reaction temperatures, the system comprising: - at least one disposable consumable (MTP) that includes one or more flat-bottomed wells in a body, wherein the wells are capable of serving as containers for performing the one or more chemical, biochemical, or biological reactions that require one or more specified reaction temperatures according to a specified protocol, the body includes a flat bottom side configured to conduct heat uniformly to a first heating surface in the wells; and a flat upper side including well openings, the upper side configured to conduct heat uniformly in the wells to a second heating surface, the second surface optionally sealed by a thin sealing foil; - one or more gradient temperature units (GTUx), each gradient temperature unit including at least one gradient temperature block (10), the gradient temperature block (10) including at least two temperature-adjustable Peltier elements (12, 13); a heat conducting block (11) contacted by the at least two temperature-adjustable Peltier elements (12, 13); a flat surface positioning area (14) configured to position the at least one disposable consumable (MTP) on the heat conducting block (11), the temperature block (10) being configured to generate a set linear temperature distribution in the wells of the disposable consumable (MTP) when placed in contact with the flat surface positioning area (14) of the heat conducting block (11); - a control unit including one or more processors, configured to control the one or more gradient temperature units (GTUx) to implement a specified protocol for the one or more chemical, biochemical, or biological reactions; Among them, The temperature block (10) configured to generate the set linear temperature distribution includes at least one Peltier element (12) adjustable to a first temperature T1 and at least one Peltier element (13) adjustable to a second temperature T2, where T1 can be set higher than T2, and both Peltier elements (12, 13) are arranged to contact the heat conducting block (11) outside the positioning area (14) of the disposable consumable (MTP), such that the linear temperature distribution can be generated in the heat conducting block (11) between the two Peltier elements and conducted to the disposable consumable (MTP) when the disposable consumable (MTP) is in contact with the heat conducting block (11).

2. The system according to claim 1, wherein the at least one temperature block (10) is positioned to thermally regulate the first and / or the second heating surface of the disposable consumable (MTP) placed in contact with the positioning areas (14a, 14b).

3. The system according to any one of the preceding claims, wherein the disposable consumable (MTP) can be subjected to the linear temperature distribution on two heating surfaces by positioning temperature blocks (10a, 10b) set to have the linear temperature distribution.

4. The system according to any one of the preceding claims, wherein the system further comprises at least one temperature control unit (TUx) capable of temperature control at temperature Tx, the temperature control unit (TUx) comprising at least one temperature control block (40), the temperature control block (40) comprising at least two Peltier elements (42), a heat conducting block (41) contacted by the at least two Peltier elements (42), and a flat surface area (44a) for positioning the at least one disposable consumable (MTP) on the heat conducting block (41), the temperature control block being configured to uniformly control the temperature of the disposable consumable (MTP) when the disposable consumable (MTP) is in contact with the heat conducting block (41).

5. The system according to claim 4, wherein the system comprises two temperature control blocks (41a, 41b), and the temperature control blocks (41a, 41b) are capable of controlling the temperature of the disposable consumable (MTP) placed in the temperature control unit (TUx) on two heating surfaces between the temperature control blocks (41a, 41b).

6. The system according to any one of the preceding claims, wherein the system further comprises a conveying device capable of moving the disposable consumable (MTP) into and / or out of the gradient temperature control unit (GTUx) and / or the temperature control unit (TUx) and aligning it with at least one positioning area (14, 14a, 14b, 44a, 44b) in the internal space (20).

7. The system according to any one of the preceding claims, wherein the system further comprises a moving device for moving the disposable consumable (MTP) in the internal space (20) between the heat conducting blocks (11, 11a, 11b, 41a, 41b) and bringing the one or more positioning areas (14, 14a, 14b, 44a, 44b) of the one or more heat conducting blocks (11, 11a, 11b, 41a, 41b) into contact with the one or two heating surfaces of the disposable consumable (MTP).

8. The system according to any one of the preceding claims, wherein the system further comprises one or more clamping mechanisms capable of clamping the disposable consumable (MTP) between the first and the second temperature control blocks (respectively, 10a, 10b, 40a, 40b).

9. The system according to any one of claims 6 to 8, wherein the control unit is configured to control the conveying device, the moving device and / or the clamping mechanism to implement a prescribed procedure for the one or more chemical, biochemical or biological reactions.

10. The system according to any one of the preceding claims, wherein the system further comprises an imaging unit I, and the imaging unit (I) comprises an imaging device or a mass spectrometry readout unit capable of capturing an image of at least one well of the disposable consumable.

11. A method of using the system according to any one of the preceding claims, the method comprising the following steps: - Load one or more reaction mixtures into the wells of a disposable consumable (MTP) and optionally seal the upper side of the disposable consumable (MTP) with a sealing foil; - Introduce the disposable consumable (MTP) into the system, - Convey and position the disposable consumable (MTP) in one of the one or more gradient temperature control units (GTUx) according to a defined reaction protocol, - Subject the disposable consumable (MTP) to a linear temperature profile on at least one heating surface by a positioning gradient temperature control block (10) set to a defined temperature gradient according to a defined reaction protocol, wherein the gradient temperature control block (10) comprises at least one Peltier element (12) adjustable to a first temperature T1 and at least one Peltier element (13) adjustable to a second temperature T2, where T1 is higher than T2, and both Peltier elements (12, 13) are arranged to contact the heat conducting block (11) outside the positioning area (14) of the disposable consumable (MTP), such that the linear temperature profile is generated in the heat conducting block (11) between the two Peltier elements (12, 13) and conducted to the disposable consumable (MTP) when the disposable consumable (MTP) is in contact with the heat conducting block (11).

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

13. The method according to any one of the preceding claims, wherein the disposable consumable is subjected to the linear temperature profile on two heating surfaces by positioning gradient temperature control blocks (10a, 10b) set to a defined temperature gradient.

14. The method according to any one of the preceding claims, wherein the system further comprises a temperature control unit (TUx) for each defined reaction temperature of the reaction protocol, the temperature control unit (TUx) is set by the control unit to the defined temperature, and wherein according to the reaction protocol, the disposable consumable (MTP) is conveyed to and positioned in the temperature control unit (TUx) for temperature control, preferably for cooling.

15. The method according to any one of the preceding claims, the method further comprising conveying / positioning the disposable consumable (MTP) to an imaging unit (I) and capturing an image of at least one well of the disposable consumable (MTP) or conveying it to a mass spectrometry readout unit.

16. The method according to any one of the preceding claims, wherein the chemical, biochemical or biological reaction is used to perform a cellular thermal shift assay purified protein thermal shift assay (TSA) or any other protein denaturation or aggregation assay.

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

Images