Flow cell for sequencing

Abstract

The invention is directed to a flow cell (100) for examination of a tissue sample wherein the flow cell (100) is provided with at least 2 fixing points (56) capable of fixing the flow cell in a consistent position in a support structure.

Description

FLOW CELL FOR SEQUENCINGBACKGROUND

[0001] This invention relates to a flow cell, especially for sequencing or analyzing biological specimens by providing an examination chamber with fluidic access for reagents and transparent covering to allow optical detection techniques.

[0002] A lot of techniques for analyzing biological specimens depend on optical detection and therefore require transparent examination hardware. Such techniques further require that different reagents like antibodies, fluorescent dyes or media can be at best automatically applied to the biological specimens.

[0003] To this end, many devices comprising wells or reservoirs for fluid reagents, in part attached to a standard microscope inspection slide, have been developed. So called “chambered slides” are for example disclosed in EP0014007A1, EP0210071B1, EP1222462B1, GB2127577A, US3883398 and DE2157150.

[0004] EP3711859A1 discloses chambered slides which can be easily assembled / disassembled and which provides one or more wells for reagents that can be applied to the sample to be examined.

[0005] Such chambered slides are also known as “flow cells” because of their design to provide a flow of reagents over a sample. Flow cells are further disclosed in US2004037739A1 and US2017014824.

[0006] However, all the chambered slides or flow cells of the prior art are designed for pipetting and not for automated processing.SUMMARY

[0007] The flow cell is preferable used for optical analysing of tissue samples. To this end, the flow cell is placed in a device for optical analysing and fluids are pumped through the flow cell which might result in vibrations causing optical disturbances. To allow precise optical analysing, any kind of movement of the flow cell within the device for optical analysing should be avoided. Accordingly, the flow cell was provided with fixing points complementary with the device for optical analysing i.e. a “support structure”.

[0008] Object of the invention is a flow cell (100), comprising a top glass (70), fluidic seal (10) and base glass (20) positioned in top frame (80) and base frame (50), wherein- the fluidic seal (10) is provided with an opening as examination chamber (11)- the base glass (20) is provided with at least two openings (21, 22) positioned to provide fluidic access to the examination chamber (11) and wherein stack (30) comprising the top glass (70), the fluidic seal (10) and the base glass (20) is provided in the base frame (50) to form a composite (60) and wherein the composite (60) is provided with the top frame (80) and wherein the base frame (50) and the top frame (80) are provided with complementary interconnecting means (51, 81) configured to mechanically interlock with each other thereby pressing the base frame (50) against the base member and the top frame (80) thereby closing the examination chamber (11) characterized in that the flow cell is provided in a support structure and wherein the base frame (50) is provided with at least 2 fixing points (56) capable of fixing the flow cell in a consistent position in the support structure.

[0009] Further object of the invention is the use of the flow cell for optical analysing tissue samples for example of a human being. To this end, first composite 60 is assembled by either by production or the user. Then, the flow cell of the invention is prepared for optical analysis by the user as follows: combining the fluidic seal (10) and the base glass (20) to form a stack (30) combining the stack(30) in the base frame (50) to form a composite (60) placing a tissue sample on the top glass (70) and positioning on the composite (60) such that tissue sample is located in the examination chamber (11) closing the flow cell by providing the composite (60) with the top frame (80) by mechanically interlocking the complementary interconnecting means (51, 81) of the base frame (50) and the top frame (80) with each other preferable by lateral movement of the top frame (80) against composite (60).

[0010] The fluidic seal (10) may be covalently bonded to base glass (20) using no added chemicals or adhesive, wherein the surface of the glass and the fluidic seal (10) is modified using oxygen plasma and when the two surfaces touch they chemically bond with one another:BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Fig. 1 shows the assembly of stack (30) comprising the fluidic seal (10) with opening or recess as examination chamber (11) and base glass (20) is provided with at least two openings (21, 22).

[0012] Fig. 2 shows base frame (50) with interconnecting means (51) for the top frame (80) (not shown), adjustment points (52) and fixing points (56).

[0013] Fig. 3 shows an exploded view of a variant of composite (60) comprising and stack (30), seal (40) and base frame (50)

[0014] Fig. 4 shows the full flow cell assembly with composite (60), top glass (70) and top frame (80) via with interconnecting means (81) of the top frame (80) engaging opening is the base frame of the composite (60)

[0015] It should be understood that the drawings are not necessarily to scale, and that like numbers may refer to like features.DETAILED DESCRIPTION

[0016] The flow cell as disclosed herein is especially useful for sequencing “on tissue” i.e. on the surface of at least one biological specimen. So called spatial sequencing is for example disclosed in EP3936623A1 or EP4039822A1.

[0017] Further the flow cell of the invention can be used in a multi-staining and de-staining process, for example as disclosed in US 7741045 B2, EP 0810 428 Bl, DE10143757, EP3037821A1, EP2725359B1 or WO2017144338A1. It should be noted that the terms “sample”, “biological tissue” or “biological sample” are used synonymous hereinafter.

[0018] The flow cell of the invention is especially designed for spatial sequencing of tissue. Such experiments require first that the tissue is placed in the examination chamber of the flow cell , i.e. the flow cell should be easily opened and closed to insert the tissue. Then, reagents need to be provided and removed to the tissue without leaking. After the experiment, the tissue should be recoverable from the flow cell. If the experiment involves obtaining of spatial information, it is essential that the tissue stays in a consistent position to the optics. This can be achieved by fixing the glass sheets in a consistent position in the frames and taking care that the whole flow cell stays in a consistent position during examination. The consistent positions are achieved by providing the flow cell or parts thereof with appropriate mechanical fixing means as disclosed later. The term “consistent position” refers to fixed and not changeable position of the respective components in a way that the spatial position of the tissue does not change during examination.

[0019] In a first embodiment of the flow cell, the complementary interconnecting means (51,81) are provided as openings (51) in the base frame (50) and complementarybay onetted joints (81) in the top frame (80) wherein both are designed to interconnect by lateral movement of the base frame (50) and the top frame (80) against each other.

[0020] In a second embodiment, the base frame (50) is provided with at least 3 (for example 3 to 12) adjustment points (52) capable of fixing the stack in a consistent position in the frame (50).

[0021] In a third embodiment, the flow cell is provided with at least 2 flat spring as fixing points (56) capable of fixing the flow cell in a consistent position in the support structure.

[0022] First, second and third embodiment are combinable in any possible combination.

[0023] After the optical analysis of the tissue is performed, the flow cell of the invention may be disassembled, so that the sample located on the top glass (70) can be retrieved and optionally stored for further examination.

[0024] Accordingly, the interconnecting means (51, 81) are configured to be mechanically disengaged from each other, for example by lateral movement of the base frame (50) against the top frame (80).

[0025] Essential for optical analysis of the tissue sample covered or at least wetted by a fluid is the water-tight assembly of the parts (members) of the flow cell. This requires a sufficient pressure on the fluidic seal (10) combining the base frame (50) and the top frame (80).

[0026] To this end, the interconnecting means (81) of the top frame (80) and the openings (51) of the base frame (50) are preferable configured as bay onetted joint. As shown in Fig. 4, the interconnecting means (81) of the top frame (80) and the openings of the base frame (50) are provided with means configured to mechanically interconnect with each other. Such means can be for example one or more notches and a corresponding noses and or a groove-and-spring design.

[0027] Preferable, the interconnecting means (81) of the top frame and the openings (51) are configured as to create an increasing pressure (force) between the top frame (80) and base frame (50) during the closing process, i.e. during lateral movement of the of the top frame (80) against the base frame (50).

[0028] In a first variant, increasing pressure (force) between the top frame (80) and base frame (50) is achieved by providing the interconnecting means (81) of the top frame (80) and the openings (51) with a tilted shape. In another variant, eitherthe interconnecting means (81) of the top frame (80) or the openings (51) are provided with a tilted shape

[0029] In a further embodiment of the invention, the increasing pressure (force) between the base member (40) base frame (50) is provided by the fluidic seal (10), which is slightly compressed during the closing process, i.e. during lateral movement of the of the base member (40) against the base frame (50). In this embodiment, it is not necessary to provide the interconnecting means (81) of the base member (40) or the openings (12) with a tilted shape.

[0030] In a further embodiment of the invention, composite (60) comprises a further adhesive layer (40) positioned between the stack (30) and base frame (50). In another variant, base frame (50) may be provided with a recess for the adhesive layer (40). The recess for the adhesive layer (40) may be provided only around or at the outer circumference of the examination chambers. In a variant thereof, if a plurality of examination chambers is used, the recess and the adhesive layer (40) may be provided around or at the position of each examination chamber.

[0031] Of course, the shape and / or geometry of the recess of the fluidic seal (10) determines the shape of the examination chamber. One geometry is shown by way of example in Fig.l. It should be clear that the shape of recess / the examination chamber can be adjusted according to the required volume and fluidics. The fluidic seal may have 2 to 5 recesses, leading to 2 to 5 examination chambers. The recesses are all in parallel or sequential (or combination thereof) fluidic communication with each other.

[0032] The fluidic seal (10) may be manufactured from any rubber material known in the art, like silicone rubber.

[0033] To aid assembly and disassembly of the slide chamber, the top frame (80) may be provided with at least one grip for example as shown with (85) in Fig. 4.

[0034] The top frame (80) and the base frame (50) can be manufactured from any thermoplastic material like PET, PS, PE, PP or COP, for example by injection moulding.USE OF THE INVENTION

[0035] The slide chamber according to the invention may be used in all application of biology or medicine where cells or biological tissue is subjected to optical examination under wet conditions. This includes microscopy, live cell microscopy or ultrasonic microscopy, preferable involving a process comprisingsequencing, staining, incubation, washing and removal of stain on the biological sample.

[0036] The flow cell according to the invention is especially useful for space-resolved optical detection methods of a tissue sample. Preferable, the at least two openings (21, 22) in the base glass (20) are connected with vacuum chucks to the appropriate optical detection device.

Claims

WHAT IS CLAIMED IS:

1. Flow cell (100), comprising a top glass (70), fluidic seal (10) and base glass (20) positioned in top frame (80) and base frame (50), wherein- the fluidic seal (10) is provided with an opening as examination chamber (11)- the base glass (20) is provided with at least two openings (21, 22) positioned to provide fluidic access to the examination chamber (11) and wherein stack (30) comprising the top glass (70), the fluidic seal (10) and the base glass (20) is provided in the base frame (50) to form a composite (60) and wherein the composite (60) is provided with the top frame (80) and wherein the base frame (50) and the top frame (80) are provided with complementary interconnecting means (51, 81) configured to mechanically interlock with each other thereby pressing the base frame (50) against the base member and the top frame (80) thereby closing the examination chamber (11) characterized in that the flow cell is provided in a support structure and wherein the base frame (50) is provided with at least 2 fixing points (56) capable of fixing the flow cell in a consistent position in the support structure.

2. Flow cell according to claim 1 characterized in that the complementary interconnecting means (51,81) are provided as openings (51) in the base frame (50) and complementary bay onetted joints (81) in the top frame (80) wherein both are designed to interconnect by lateral movement of the base frame (50) and the top frame (80) against each other.

3. Flow cell according to claim 1 or 2 characterized in that composite (60) comprises an adhesive layer (40) positioned between the stack (30) and base frame (50).

4. Flow cell according to any of claims 1 to 3 characterized in that the base frame (50) is provided with at least 3 adjustment points (52) capable of fixing the stack (30) in a consistent position in the frame (50).

5. Flow cell according to any of claims 1 to 4 characterized in that the fixing points (56) are provided as flat spring.

6. Flow cell according to any of claims 1 to 5 characterized in that the base frame (50) and the top frame (80) are connected by at least one hinge.

7. Flow cell according to claim 6 characterized in that the base frame (50) and the top frame (80) are connected by at least one hinge and wherein the at least hinge allows lateral movement of the base member and the top member against each other.

8. Flow cell according to any of claims 1 to 7 characterized in that the top glass (70) is provided on the inner surface with a coating reflecting infrared radiation.

9. Flow cell according to any of claims 1 to 8 characterized in that the top glass (70) is provided on the inner surface with a coating promoting cell adhesion.

10. Flow cell according to claim any of claims 1 to 9 characterized in that the fluidic seal (10) is provided from plasma-activated silicone material.

11. Flow cell according to any of claims 1 to 10 characterized in that the fluidic seal (10) is provided with at least one channel providing fluidic access to the examination chamber (11) and wherein at least two openings (21,22) of the base glass (20) are positioned to provide fluidic access to the at least one channel.

12. Use of the flow cell according to any of claims 1 to 11 for space-resolved optical detection methods of a tissue sample.

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