Sample reaction vessel

Abstract

A reaction vessel comprising, comprising a housing and a base a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample and the base further includes a substrate support between a heated surface and the substrate. The substrate support is thermally conductive and is operable to allow thermal communication between the heated surface and the substrate to heat the sample to perform an assay reaction.

Description

SAMPLE REACTION VESSELBACKGROUND

[0001] Aspects of the present disclosure relate generally to devices, systems, and methods providing biological or chemical analysis. Various protocols in biological or chemical research involve performing a number of controlled reactions on local support surfaces or within predefined reaction chambers. The designated reactions may then be observed or detected, and subsequent analysis may help identify or reveal properties of chemicals involved in the reaction.

[0002] The ability to determine the identity and location of target analytes in a biological sample is highly desirable. Obtaining sequence information from the 5’ end and central regions of a target nucleic acid (e.g., an RNA molecule), however, remains challenging.

[0003] The emerging field of spatial transcriptomics is being driven by the development of new technologies that allow the mapping of single cell transcriptomes to their spatial locations in a tissue slice. Spatial transcriptomics is used to study gene expression patterns within a tissue sample, allowing researchers to understand how different cell types are spatially arranged and interact with each other within their native tissue environment, providing insights into complex biological processes that cannot be understood from analyzing cells in isolation, like cell-cell communication and tissue development dynamics across different disease states or developmental stages.Beyond measuring mRNA transcripts, spatial analysis can also (or alternatively) be used to measure other molecules of interest such as proteins, DNA, or metabolites.

[0004] While a variety of devices, systems, and methods have been made and used to perform biological or chemical analysis, it is believed that no one prior to the inventor(s) has made or used the devices and techniques described herein.SUMMARY

[0005] Some aspects of the present invention relate to a reaction vessel comprising a housing, a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample. The base further includes a substrate support between a heated surface and the substrate wherein the substrate support is thermally conductive and is operable to allow thermal communication between the heated surface and the substrate to heat the sample to perform an assay reaction. In another example, the substate support is a thermally conductive thermoplastic. In another example, the substrate support is Makrolon. In another example, the base is a thermally conductive thermoplastic. In some examples, the base is a metal or metal alloy. In some examples the metal or metal alloy is aluminum or an aluminum alloy.

[0006] Other aspects of the present invention relate to a reaction vessel comprising a housing and a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample. The reaction vessel further comprises a hinge coupled to the housing and the base to allow the housing and the base to move relative to each other from a first position where the housing and base are open to allow access to the substrate to a second position where the housing and base are closed. The reaction vessel further comprises a releasable connector coupled to the housing and base that releasably couples the housing and the base to each other when in the second position. In one example, releasable connector is a snap fit coupling. In another example, the hinge is operable to releasably couple the housing and base so that the base can be separated from the housing.

[0007] Other aspects of the present invention relate to a reaction vessel comprising a housing having an opening in a first surface, a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample and a stopple releasably attached to the housing to form a closure to the opening. In one example, the stopple is a flexible film. In one example, the stopple is a rigid cover. In one example, the stopple is a flexible cover.

[0008] Other aspects of the present invention relate to a reaction vessel comprising a housing, a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample and wherein the substrate defines an active area for sample attachment and assay processing of about 7.5 cm2. In one example, a reaction volume of the reaction vessel is larger in the X / Y dimension than the active area to mitigate edge effect.

[0009] Other aspects of the present invention relate to a reaction vessel comprising a housing including a shoulder, a shoulder stop surface and a sealing member, and a base including a base wall and wall stop surface, the base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample, wherein the shoulder stop surface overlaps in the Z-direction with the wall stop surface and the housing and base are operable to provide a hard stop to relative movement between the base and the housing during compression by of the reaction vessel by a thermocycler. In one example, the hard stop is operable to restrict further compression of the sealing member.

[0010] Other aspects of the present invention relate to a reaction vessel comprising a housing and a base releasably attached coupled to the housing wherein the attached coupled housing and base define a cavity for receiving a substrate having at least one sample, wherein the substrate includes a patterned region. In one example, the substrate includes a flowcell. In one example, the flowcell includes a region that is sequenced within the flowcell.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depicts an isometric view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0012] FIG. 2a depicts an isometric view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0013] FIG. 2b depicts an isometric view of another example of a reaction vessel that may beused to provide biological or chemical analysis.

[0014] FIG. 3 a depicts a top plan view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0015] FIG. 3b depicts a side view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0016] FIG. 3c depicts a bottom view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0017] FIG. 4a depicts a top plan view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0018] FIG. 4b depicts a top plan view of another example of a reaction vessel that may be used to provide biological or chemical analysis.

[0019] FIG. 4c depicts a top plan view of another example of a reaction vessel that may be used to provide biological or chemical analysis.

[0020] FIG. 5 depicts an isometric view of an example of a reaction vessel that may be used to provide biological or chemical analysis.

[0021] FIG. 6a depicts a cross-sectional view of an example of a reaction vessel that may be used to provide biological or chemical analysis located on a thermocycler.

[0022] FIG. 6a depicts a cross-sectional view of an example of the reaction vessel of FIG. 6a that may be used to provide biological or chemical analysis located on a thermocycler.

[0023] FIG. 7 depicts an isometric view of an example of a reaction vessel that may be used to provide biological or chemical analysis placed inside of a thermocycler located on a thermal block of a thermocycler.

[0024] FIG. 8 depicts a top plan view of an example of a substrate that may be used with the reaction vessels depicted in FIGS. 1-7 to provide biological or chemical analysis.DETAILED DESCRIPTION

[0025] The following detailed description of certain examples will be better understood when read in conjunction with the appended drawings. To the extent that the figures illustrate diagrams of the functional blocks of various examples, the functional blocks are not necessarily indicative of the division between hardware components. Thus, for example, one or more of the functional blocks (e.g., processors or memories) may be implemented in a single piece of hardware (e.g., a general purpose signal processor or random access memory, hard disk, or the like). Similarly, the programs may be standalone programs, may be incorporated as subroutines in an operating system, may be functions in an installed software package, and the like. It should be understood that the various examples are not limited to the arrangements and instrumentality shown in the drawings.

[0026] It is to be understood that the subject matter described herein is not limited in its application to the details of construction and the arrangement of components set forth in the description herein or illustrated in the drawings hereof. The subject matter described herein is capable of other implementations and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. As used herein, an element or step recited in the singular and proceeded with the word “a” or “an” should be understood as not excluding plural of said elements or steps, unless such exclusion is explicitly stated. Furthermore, references to “one example” are not intended to be interpreted as excluding the existence of additional examples that also incorporate the recited features. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0027] When used in the claims, the term “set” should be understood as one or more things which are grouped together. Similarly, when used in the claims “based on” should be understood as indicating that one thing is determined at least in part by what it is specified as being “based on.” Where one thing is required to be exclusively determined by another thing, then that thing will be referred to as being “exclusivelybased on” that which it is determined by.

[0028] Unless specified or limited otherwise, the terms “attached,” “mounted,” “connected,”“supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings. Also, it is to be understood that phraseology and terminology used herein with reference to device or element orientation (such as, for example, terms like “above,” “below,” “front,” “rear,” “distal,” “proximal,” and the like) are only used to simplify description of one or more examples described herein, and do not alone indicate or imply that the device or element referred to must have a particular orientation. In addition, terms such as “outer,” “inner,” “exterior” and “interior” are used herein for purposes of description and are not intended to indicate or imply relative importance or significance.

[0029] It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-described examples (and / or aspects thereof) may be used in combination with each other. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the presently described subject matter without departing from its scope. While the dimensions, types of materials and coatings described herein are intended to define the parameters of the disclosed subject matter, they are by no means limiting and instead illustrations. Many further examples will be apparent to those of skill in the art upon reviewing the above description. The scope of the disclosed subject matter should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. In the appended claims, the terms “including” and “in which” are used as the plain-English equivalents of the respective terms “comprising” and “wherein.” Moreover, in the following claims, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects. Further, the limitations of the following claims are not written in means — plus-function format and are not intended to be interpreted based on 35 U.S.C. §112(f) paragraph, unless and until such claim limitations expressly use the phrase“means for” followed by a statement of function void of further structure.

[0030] FIG. 1 illustrates a reaction vessel 10 for use, in one example, in ex-situ spatial transcription assays on fresh-frozen tissue or formalin-fixed paraffin embedded (FFPE) sample slices. The reaction vessel 10 includes a base 100 and a housing 150. Details of the various components are provided below. In general terms, however, the base 100 and the housing 150 are coupled with each other to retain a substrate 200 having at least one sample 210 mounted thereto. Further, the base 100 and the housing 150 are coupled with each other to define a cavity 180 that further defines at least one reaction volume 182 for performing assay reactions on the at least one sample 210 mounted to the substrate 200.

[0031] The base 100 includes a recess 110 that is sized appropriately for receiving a substrate 200. In one example, the substrate 200 is a conventional glass slide to which fresh- frozen or FFPE tissue samples slices may be mounted. In another example, the substate 200 is a custom glass or silicon slide patterned with chemistries such as hydrogel and DNA molecules to which fresh-frozen or FFPE tissue samples slices may be mounted. The base 100 further includes retention tabs 130 that extend from the exterior 101 of the base 100 in a direction away from the cavity 180. The retention tabs 130, as described in more detail below, provide a mating surface 135 for the housing 150 to be releasably coupled to the base 100. The base includes a substrate support 115 proximal to the substrate 110 and is arranged between the substrate 110 and a heated thermal block 320 of a thermocycler 300 to support the substrate 110. In one example, the substrate support 115 partially defines the recess 110. In one example, the base 100 is heat conductive and provides thermal communication between heat provided by a thermocycler 300 to the substrate 200 and the at least one reaction volume 182. In some examples the base 100 is thermally conductive to transfer heat from a heated thermal block 320 of the thermocycler 300 to allow reactions to occur on the at least one sample 210 on the substrate 200. In some examples, the base is partially thermally conductive. In some examples the substate support 115 of the base is all or partially thermally conductive to transfer heat from a heated thermal block 320 of the thermocycler 300 to allow reactions tooccur on the at least one sample 210 on the substrate 200. In some examples, the base, or portions thereof, or the substrate support 115, or portions thereof, are made of a thermally conductive polymer. In some examples, the thermally conductive polymer is capable of being injection molded. In some examples the thermally conductive polymer is Makrolon from Covestro. In some examples, the base is a metal or metal alloy. In some examples the metal or metal alloy is aluminum or an aluminum alloy.

[0032] The base 100 may optionally include a first hinge coupling 143, such as a hinge bar, to interface with a second hinge coupling 162 of the housing 150, such as one or more hinge tabs. The first hinge coupling 143 and the second hinge coupling 163 are coupled with each other to form a hinge 165 so as to allow the housing 150 and base 100 to rotate relative to each other between a first position 20 that is open to allow access to the substrate 200 and a second position 30 that is closed (see FIGS. 2a and 2b) to enclose the substrate. As described further below, the hinge 165 in one example is optional to the reaction vessel 10 as the base 100 and the housing 150 can be releasably secured to each other through other structures. The base may also optionally include a grip 145 that extends from the base 100 to allow a lab technician to conveniently handle the reaction vessel 10 with one hand. In one example, the first hinge coupling 143 and the second hinge coupling 163 are releasably secured to each other such that the housing 150 can be separated from the base 100. In one example, the second hinge coupling 163 includes open ended hinge tabs to releasably couple to the hinge bar of the first hinge coupling 162. In this example, the base 100 can be used independently of the housing 150 to hold a substrate 200 with or without sample 210 mounted thereon. In one example use, after performing assay reaction steps outside of the reaction vessel 10, such as fixation and H&E staining, users can place a wet substrate 200 onto the base 100 and then place this base 100 (without a housing 150 coupled to it) into a thermocycler 300 and use the thermocycler 300 to heat the substrate 200 in order to dry it. The user could then use this dry substrate 200 for imaging (e.g. on a microscope). After imaging, the substrate 200 and base 100 can be coupled to the housing 150 as described herein for further assay reactions as described below.

[0033] With continued reference to FIG. 1, the housing 150 includes a top surface 153 defining at least one opening 158 to access the cavity 180. The housing 150 further includes at least one interior wall 155 that extends from the opening 158 toward the base 100 when coupled to the base 100. The housing 150 further includes a bottom surface 154 proximal to the interior wall 155. A sealing member 160 is coupled to the bottom surface 154 such that when the reaction vessel 10 is in the second position 30, the sealing member 160 contacts the substrate 200 (see also FIGS. 6a-6b). In one example, the combination of the interior wall 155, the sealing member 160 and recess 110 defines the cavity 180. In one example, the combination of the sealing member 160, interior wall 155 and substrate 200 defines at least one reaction volume 182 for performing assay reactions on the at least one sample 210 mounted to the substrate 200. The sealing member 160 seals against the substrate 200 so that liquid remains inside the reaction volume 182 during an assay reaction.

[0034] In one example, as shown in FIG. 1, the reaction vessel may have one opening wherein the interior wall 155 and sealing member 160 at least partially form one cavity 180 and one reaction volume 182. In one example with one reaction volume 182, the reaction volume 182 can be about 5.3 cm x 1.8 cm in the X / Y dimension. These dimensions allow for an active area 215 for optimal placement and analysis of sample 210 on the substrate 200 smaller than the X / Y dimension of the reaction volume 182. It has been observed that reagent evaporation at the assay reactions edges of the reaction volume 182 proximal to the substrate 200 surface during assay reactions can reduce the sensitivity of detecting (cDNA) reads from tissue in that region. This may be referred to as “edge effect.” As a result, the X / Y dimensions of the reaction volume 182 on the substrate 200 may be 3mm longer and wider than the dimensions of the active area 215 of the substrate 200. This is useful for mitigating “edge effect” from reagent evaporation at the edges of the reaction volume 182 proximal to the substrate 200 surface during reagent exchanges by moving the evaporation to areas outside of the active area 215. In this regard, in one example, a reaction volume that is 5.3 cm in length by 1.8 cm in width may have an active area 215 of 5 cm in length x 1.5 cm in width. In the example above, the active area 215 for placement of sample 210 on the substrate 200 can be about 5 cm x 1.5 cm in theX / Y dimension which provides an active area 215 of about 7.5 cm2. This amount of area conveniently allows the lab technician to place varied numbers and sizes of sample on the substrate.

[0035] With continued reference to FIG. 1, the base 100 and the housing 150 can be releasably coupled to each other via a releasable attachment 168. In this way, an operator can conveniently place the substate 200 in the base 100, releasably attach the housing 150 to the base to perform assay operations, and then, when complete, conveniently uncouple the housing 150 from the base 100 to retrieve the substrate 180. In one example, the releasable attachment 168 includes snap-fit connectors where the base 100 and housing 150 have mating snap-fit connections that can be released. In one example, the releasable attachment 168 is a butterfly snap connection 169. In this example the housing 150 includes closure members 170 that are attached to the housing 150, but are rotatable relative to the housing 150. The closure members can be rotated toward the base 100 such that a first mating surface 135 on retention tabs 130 of the base can be engaged by a second mating surface 175 on the closure member 170 to form a snap-fit connection to releasably couple the housing 150 to the base 100. To release the housing 150 from the base 100 the closure member 170 may be pulled away from the base 100 to release the first mating surface 135 and second mating surface 175 snap-fit connection. In one example, the first mating surface 135 and the second mating surface 175 are in the form of complementary hooks that can be snapped together to secure the housing 150 to the base 100 and can be pulled apart to release the housing 150 from the base 100. It should be understood that a releasable coupling in the present context means that the base 100 and the housing 150 can be separated from each other to allow access to the substrate 200; however, the base 100 and housing 150 can maintain attachment otherwise such as through a hinge 165.

[0036] With reference to FIG. 2a, a reaction vessel 10 in the second position 30 is shown.The reaction vessel further includes a stopple 190. The stopple 190 overlays the opening 158 to form a closure to the opening 158 to substantially seal the opening 158. In this way, the reaction volume 182 can be sealed to prevent evaporation ofliquid or contamination of the sample during assay reactions. In one example, the stopple 190 is a flexible film that is coupled to the top surface 153 of the housing 150. In one example, the stopple 190 is coupled through friction. In another example, the stopple 190 is coupled by an adhesive. In one example, the stopple 190 is a rigid or flexible cover that can be releasably couple to or removably inserted into the opening 158 to form a closure to the opening 158 to substantially seal the opening 158. With also reference to FIG. 5, the stopple 190 in this example can be applied for assay reactions, but can be peeled off to allow for additional reagents necessary for assay reactions to be added. Another stopple 190 may then be reapplied to seal the opening 158 again for additional assay reactions. In another example, the same stopple can be reapplied to seal the opening 158 again for additional assay reactions. With reference to FIG. 5, a reaction vessel 10 is shown according to one example with a flexible film stopple 190 partially peeled from the top surface 153.

[0037] With reference to FIG 2b, another example of the reaction vessel 10 is shown. In FIG.2b, the stopple 190 is not shown as coupled to the reaction vessel 10, but can be coupled during assay reactions as shown in FIG. 2a. In this example, the top surface 153 of the housing 150 defines a plurality of openings 158 to access a plurality of reaction volumes 182. A plurality of interior walls 155 extends from the openings 158 toward the base 100 and partially define a plurality of cavities. The housing 150 further includes a bottom surface 154 proximal to the interior walls 155. A sealing member 160 (see FIGS. 1 and 6a-b) is attached to the bottom surface 154 such that when the reaction vessel 10 is in the second position 30, the sealing member 160 contacts the substrate 200. In one example, the combination of the interior walls 155, sealing member 160 and recess 110 defines the plurality of cavities 180. In one example, the combination of the sealing member 160, interior wall 155 and substrate 200 defines a plurality of reaction volumes 182 for performing assay reactions on a plurality of samples 210 mounted to the substrate 200 when the substrate is within the recess 110. The sealing member 160 seals each reaction volume 182 against the substrate 200 so that liquid remains inside the respective reaction volume 182 during an assay reaction. In one example, the plurality of cavities 180 and reaction volumes 182 is six, but a greater or fewer number may be used. In one example, the pluralityof cavities 180 and reaction volumes 182 have a pitch of about 9 mm apart so that conventional multi-channel pipettors having a 9 mm pitch can be used to dispense reagent. In one example, the pitch of the plurality of cavities 180 and reaction volumes 182 can be larger or smaller than 9 mm but still accommodate 9 mm pitch multi-channel pipettors. In one example, each of the plurality of cavities 180 and reaction volumes 182 are 6.7 mm in length by 15 mm in width and are separated from each other by 2 mm. In this example, the pitch of the plurality of cavities 180 and reaction volumes 182 is 8.7 mm. Despite not having a 9 mm pitch, 9 mm pitch multichannel pipettors can still be used to dispense reagents into the plurality of reaction volumes 182 at the same time.

[0038] With reference to FIG 3 a, an example of the reaction vessel 10 with a plurality of openings 158 is shown. With reference to FIGS. 3b and 3c, the base 100 includes thermal block interfaces 120. These thermal block interfaces 120 are sized and positioned so that the reaction vessel 10 can be placed on a variety of conventional thermal blocks 320 of a thermocycler 300 so that the reaction vessel base 100 can sit substantially flush on the thermal block 320 while the thermal block interfaces 120 reside with the wells of the thermal block 320. By residing within the wells of the thermal block 320, the thermal block 320 interfaces 120 prevent the reaction vessel 10 from sliding across the thermal block 320 during loading and thermocycling. By sitting substantially flush with the thermal block 320, heat transfer from the heated thermal block 320 to the sample 210 on the substrate 200 can be achieved to allow for the assay reactions to occur. In one example, the thermal block interfaces 120 are cylindrical and have a diameter of about 3.8mm and a length of about 3.5 mm. In one example the base 100 has four thermal block interfaces 120 whose centers and separated by about 72 mm and about 18 mm. In this way, the reaction vessel of this example is able to be received within thermal blocks having well diameters of greater than 4 mm, depths greater than 3.5 mm and thermal blocks having wells with pitches of about 9 mm. In another example, the thermal block interfaces 120 can be spaced by another multiple of 9 mm, including, without limitation, 9 mm x 36 mm, 9 mm x 54 mm or 18 mm x 36 mm. As would be understood by one of skill in the art, other spacing for the thermal block interfaces may be used as well including spacing thethermal block interfaces 120 at multiples of thermal block 320 wells having pitches other than 9 mm.

[0039] FIGS. 4a-c depict sample 210 arrangements on the substrate 200 according to various examples of the present invention. With reference to FIG. 4a, a substrate is shown with a plurality of samples 210 on the substrate. In this example, all samples 210 are in one reaction volume 182. In this example, the same assay reaction can be performed on all samples. With reference to FIG. 4b, a substrate is shown with a plurality of samples 210 on the substrate. In this example, the samples 210 are completely or partially within one or more of a plurality of reaction volumes 182. In this example, different assay reactions can be performed on each sample 210 or portions of each sample 210. With reference to FIG. 4c, a substrate is shown with a large single sample 210 on the substrate in one reaction volume 182. In this example, an assay reaction can be performed on a large sample 210 in a single assay reaction. In one example, a large sample is one that may be greater than about 10mm in length, greater than about 20 mm in length or greater than about 40 mm in length.

[0040] With reference to FIGS. 6a-b, FIG. 6a depicts a reaction vessel 10 prior to compression by a thermocycler 300. In operation, a reaction vessel 10 is placed within a thermocycler 300 during an assay process. Due to the weight of the lid 310 and the size of the gap between the lid 310 and the thermal block 320, the reaction vessel 10 may be compressed when the lid 310 is closed. Over-compression of the reaction vessel 10 in the Z-direction can result in over-compression of the sealing member 160 against the substrate 200 resulting in damage to the sealing member 160 or the substrate 200. In one example of the present invention, as shown in FIG. 6a, the housing 150 includes a shoulder 192 and a shoulder stop surface 193 that overlaps in the Z-direction, as shown by the dotted line in the figure, with a base wall 146 and wall stop surface 147 of the base 100. As shown in FIG. 6b, during compression of the reaction vessel 10 in the Z-direction by the lid 310 of the thermocycler 300, the shoulder stop surface 193 contacts the wall stop surface 147 to provide a hard stop to relative movement between the base 100 and the housing 150 due to rigidity of the base 100 and housing 150. As a result, the hard stop is operable to restrict furthercompression of the sealing member 160, mitigating over-compression and resulting damage to the substrate 200 or sealing member 160.

[0041] FIG. 7 shows an example of two reaction vessels 10 placed on the thermal block 320 of a thermocycler 300. The reaction vesselslO are substantially flush with the thermal block 320 and the thermal block interfaces 120 are within wells of the thermal block.

[0042] FIG. 8 shows an example substrate 200 that is a custom glass or silicon slide including a patterned region patterned with chemistries such as hydrogel and DNA molecules. The substrate 200 can include a flowcell 220, that has been debonded after sequencing. In one example, the flowcell 220 is large enough to correspond to the active area 215. In this regard, in one example, the substrate 200 may be sized similarly to a conventional microscope slide, having dimensions such as 75 mm in length by 25 mm in width. In one example, the substrate is 1 mm in thickness. In another example, the substrate 200 is 1.1 mm in thickness. In another example, the substrate is greater than 1.1 mm in thickness.

[0043] The flowcell 220 may be sized to correspond partially or entirely to the area of the substrate 200. In one example, the flowcell 220 of a substrate that is 75 mm in length by 25 mm in width may be at least 50 mm in length by 15 mm in width with an area of at least 750 mm2. In another example, a partial region that is sequenced within the flowcell 220 of a substrate that is 25 mm in width by 75 mm in length may be at least 15 mm in width by 50 mm in length with an area of at least 750 mm2._0ne of skill in the art will understand that the size of the substrate 200 and the size of the flowcell 220, however, may be smaller or larger depending on the application. In other examples, the substrate 200 including a flowcell 220 may be used with a reaction vessel 10 having one or having a plurality of reaction volumes 182 wherein the flowcell 220 may be sized to correspond to one reaction volume 182 or to a plurality of reaction volumes 182, including having a plurality of reaction volumes 182 encompassing one or more flowcells 200 or having a plurality of reaction volumes 182 each having a flowcell 220 from a plurality of flowcells 220. In this regard, in additional examples, one flowcell 220 may be used with one reaction volume 182,one flowcell 220 may be used with a plurality of reaction volumes 182, such as six shown in FIG. 2b, or a plurality of flowcells 220 may be used with a plurality of reaction volumes 182 on one substrate 200. In a further example, after assay reactions, the flowcell 220 can be re-bonded and used for sequencing.

[0044] In operation, in one example, the reaction vessel 10 is a consumable cartridge for holding a substrate 200 with tissue sample(s) mounted on it, and for enabling users to fluidically address the surface of the substrate to perform biochemical reactions. The reaction vessel 10 is compatible with workflows for processing fresh-frozen tissues, as well as formalin-fixed paraffin embedded (FFPE) tissues. For fresh-frozen tissues, tissue such as kidney, brain, or potentially a whole organism is embedded in a compound and frozen into a block. This block can be attached to a cryostat and thin (10-20 pm) slices are cut from the block and mounted onto the substrate slide. With the tissue slices on the slide, users then go through a process of fixation and H&E staining of the tissue. Following imaging the stained tissue with a microscope, the substrate 200 with tissue mounted on it is then loaded into base of the reaction vessel 10. The reaction vessel 10, in one example, is then closed by rotating the housing 150 of the reaction vessel 10 such that sealing member 160 contacts the substrate 200. The user then, in one example, further secures the reaction vessel 10 and substrate by pressing down on the closure members 170 of the butterfly snap connection 169 on both sides of the housing 150 until they bend and the second mating surface 175, in the form of complementary hooks, couple to the first mating surface 135, in the form of complementary hooks, on the base 100 and snap into place. This assembly provides the force and compression of the sealing member 160 needed to create a fluidic seal on top of the substrate 200. The reaction vessel 10 top features a large opening 158, or multiple smaller openings 158, and, when sealed against a substrate 200, create at least one reaction volume 182 that has the substrate 200 with sample 210 at the bottom and is open at the top when the stopple 190 is not attached to the housing 150. Users can then use pipettes or other liquid handling techniques to add and remove liquid reagents into the at least one reaction volume 182 containing sample 210. While in the reaction vessel 10, users can add reagents to permeabilize the sample 210 and release mRNA. This mRNA will diffuse to the surface of thesubstrate 200 where it is captured. Users then go through a series of steps where they add and remove reagents to convert the mRNA to cDNA that contains additional sequences that encode the spatial location of the mRNA on the substate 200 (which corresponds to the spatial location the mRNA was within the tissue), as well as sequences that enable the copying and amplification of the cDNA strands to prepare a library for DNA sequencing. For each reaction, users will pipette a liquid reagent into the at least one reaction volume 182. They will then use the stopple 190 to seal the at least one opening 158 of the at least one reaction volume 182 to prevent evaporation. The reaction vessel 10 is placed onto a thermocycler 300 to control the temperature of the reaction. Following the reaction, the reaction vessel 10 is removed from the thermocycler 300, the stopple 190 is peeled off the housing 150, and a pipette is used to remove the reagent, perform washes, and add the next reagent. At the end of the reaction vessel 10 workflow, the spatially encoded cDNA is released from the surface of the substrate 200 and is transferred by pipette to a microtube for further processing towards creating a library for sequencing. Sequencing and data analysis is used to create a virtual image showing the location of all mRNA transcripts and this can be overlay ed with the microscopy image of tissues to see which transcripts were in which part of the tissue. The steps can be different depending on if the user is measuring mRNA, proteins, or using fresh-frozen or FFPE tissues.

[0045] The following claims recite aspects of certain examples of the disclosed subject matter and are considered to be part of the above disclosure. These aspects may be combined with one another.

Claims

What is claimed is:

1. A reaction vessel comprising:a housing,a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample, the base further including a substrate support between a heated surface and the substrate wherein the substrate support is thermally conductive and is operable to allow thermal communication between the heated surface and the substrate to heat the sample to perform an assay reaction.

2. The reaction vessel of claim 1, wherein the substate support is a thermally conductive thermoplastic.

3. The apparatus of claim 2, wherein the substrate support is Makrolon.

4. The apparatus of claim 2, wherein the base is a thermally conductive thermoplastic.

5. A reaction vessel comprising:a housing,a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample, a hinge coupled to the housing and the base to allow the housing and the base to move relative to each other from a first position where the housing and base are open to allow access to the substrate to a second position where the housing and base are closed; anda releasable connector coupled to the housing and base that releasably couples the housing and the base to each other when in the second position.

6. The reaction vessel of claim 5 wherein the releasable connector is a snap fit coupling.

7. The reaction vessel of claim 5 wherein the hinge is operable to releasably couple the housing and base so that the base can be separated from the housing.

8. A reaction vessel comprising:a housing having an opening in a first surface,a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample, a stopple releasably attached to the housing to form a closure to the opening.

9. The reaction vessel of claim 8 wherein the stopple is a flexible film.

10. The reaction vessel of claim 8 wherein the stopple is a rigid cover.

11. The reaction vessel of claim 8 wherein the stopple is a flexible cover.

12. A reaction vessel comprising:a housing,a base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample, wherein the substrate defines an active area for sample attachment and assay processing of about 7.5 cm2.

13. The reaction vessel of claim 12 further comprising a reaction volume that is larger in the X / Y dimension than the active area.

14. The reaction vessel of claim 13 wherein the reaction volume that is larger in the X / Y dimension than the active area is operable to mitigate edge effect.

15. A reaction vessel comprising:a housing including a shoulder, a shoulder stop surface and a sealing member, and a base including a base wall and wall stop surface, the base releasably coupled to the housing wherein the coupled housing and base define a cavity for receiving a substrate having at least one sample,wherein the shoulder stop surface overlaps in the Z-direction with the wall stop surface and the housing and base are operable to provide a hard stop to relative movement between the base and the housing during compression by of the reaction vessel by a thermocycler.

16. The reaction vessel of claim 15 wherein the hard stop is operable to restrict further compression of the sealing member.

17. A reaction vessel comprising:a housing,a base releasably attached coupled to the housing wherein the attached coupled housing and base define a cavity for receiving a substrate having at least one sample, wherein the substrate includes a patterned region.

18. The reaction vessel of claim 17 wherein the substrate includes a flowcell.

19. The reaction vessel of claim 17 wherein the flowcell includes a region that is sequenced within the flowcell.

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