Flow cell for efficient staining and washing of biological samples

Abstract

The invention is directed to a flow cell wherein the hydrostatic pressure generated between the level of input fluid in the input reservoir (12) relative to the level of the output fluid in the output reservoir (13) generates a flow of input fluid from the input reservoir (12) through the examination chamber (11) into output reservoir (13) and wherein the output reservoir (13) is provided at least in part with a hydrophilized surface having a contact angle of less than 70 °.

Description

FLOW CELL FOR EFFICIENT STAINING AND WASHING OF BIOLOGICALSAMPLESBACKGROUND

[0001] It is long known practice in biology and medicine that tissue sections and suspension cells can selectively be labeled using fluorescent markers (e.g. antibody conjugates) and then analyzed using a fluorescence microscope. The markers are usually applied as aqueous solution for example in a salt-containing buffer to the biological sample, which interacts with it during the incubation period. The biological samples are usually applied to microscope slides, which as common practice are attached to a carrier (usually irreversibly) such that separate areas (wells) containing individual samples are formed, that allow pipetting of the fluorescent markers into the liquid proof wells.

[0002] Many different flow cells are commercial available for spatial biology or fluorescence microscopy, which differ mainly in the size and number of sample chambers and the way in which the sample is attached to a carrier. Some flow cells use pre-applied adhesive films, others already contain the slide in the carrier itself, while others use mechanical connections. The trend is currently moving towards microfluidics in order to achieve high efficiency with low volume. For example, flow cells such as "p-Slide Luer" from Ibidi or the "CellScape Chip" from Canopy Bioscience form a small microfluidic channel over the sample. Fluid is actively pumped through the microfluidic channel over the sample via a Luer connection until it is removed through a second Luer connection downstream of the sample. This process requires the application of an external pressure (pipette or pneumatics, for example), as otherwise the fluid will not flow continuously through the sample channel. Further, the sample cannot then be removed from the carrier again.

[0003] EP 3711859A1 discloses another disposable for a fluorescence microscopy providing wells for reagents and an examination chamber in which the tissue to be examined is brought into contact with the reagents by pipetting.

[0004] Depending on the size of this area and the hydrophilicity of the materials used, a certain level of liquid is required to wet the entire area including the entire sample. The larger the volume to wet the entire sample, the more reagents have to be used, which are largely responsible for the costs of the experiment.

[0005] After pipetting, the fluorescent markers stay on top of the biological sample for a defined incubation time. This staining time depends on the liquid level and theconcentration of the fluorescent marker. After staining of the sample, the unbound fluorescent markers need to be washed away in order to avoid fluorescent background. This washing step is usually done by multiple pipetting and aspiration of buffer into the well.

[0006] The known flow cells have several disadvantages: Either they require an external pumping system to control the volume of fluids provided to the sample or they require manual interaction by pipetting fluids to and from the sample. Accordingly, the aim of this invention is to minimize the experimental costs by a build-in control of the flow of reagents to / from the sample without the use of a dosing equipment, thereby reducing the amount of reagents required.OBJECT OF THE INVENTION

[0007] It was found that a flow cell having a rather small hydrostatic pressure between an input reservoir and the output reservoir is sufficient to create a steady flow of liquid over a sample if the liquid is drawn away from the output opening preventing blocking or hindering the flow i.e. the opening in the output reservoir. Due to the high surface tension of water and the very low pressure difference in flow cells, droplets are hard to remove once they have been build up.

[0008] To this end, the surface of the output reservoir should be provided hydrophilic enough to reduce the contact angle of the fluid with the surface. This effect can further be enhanced by providing the output opening with channels like structures which lead the fluid away from the opening.

[0009] Object of the invention is a flow cell comprising a top member (10) and a closure member (30) providing a volume as examination chamber (11) wherein the top member (10) is provided with at least one input reservoir (12) for input fluids and one output reservoir (13) for output fluids and the examination chamber (11) is provided with at least one input opening (14) in fluidic communication with at least one input reservoir (12) and at least one output opening (15) in fluidic communication with the output reservoir (13) wherein the hydrostatic pressure generated between the level of input fluid in the input reservoir (12) relative to the level of the output fluid in the output reservoir (13) generates a flow of input fluid from the input reservoir (12) through the examination chamber (11) into output reservoir (13) characterized in that the output reservoir (13) is provided at least in part with a hydrophilized surface having a contact angle of less than 70 ° and the inlet opening (18) of output reservoir (13) is provided with at least one channel (22) designed to lead output fluid into the output reservoir (13).

[0010] Due to the hydrophilic surface and the channel at the input opening of the output reservoir, building of drops preventing the flow of fluids can be avoided and / or only a quite low hydrostatic pressure is necessary to achieve a constant flow of fluids through the examination chamber.

[0011] In another variant, capillary forces are used to draw the fluid into the output reservoir (13). To this end, the inlet opening (18) of output reservoir (13) may be provided with at least one capillary notch (22) designed to lead output fluid into the output reservoir (13) by capillary forces.

[0012] The term “capillary notch (22)” refers to any groove- or channel-like structure which is small enough to create capillary forces easing the flow of liquid away from the inlet opening (18) i.e. avoiding formation of drops on the opening.

[0013] In a variant thereof, the inlet opening (18) of output reservoir (13) may be provided with two capillary notches (22) having an orientation of 45°-135°, preferable perpendicular (90°) to each other. This variant is shown in Fig. 7.

[0014] Preferable, the at least one capillary notch (22) has an edge radius R of 0.01 - 5 pm, more preferable of 0.01 - 2 pm. The meaning of the term “edge radius R” and its position is shown in Fig. 8. The edge radius R of the capillary notch (22) is measured adjacent to the inlet opening (18) of output reservoir (13).

[0015] The flow cell of the invention is a device having preferable a shape as sketched in the drawings. The device is intended as disposable microfluidic structure and can be made of plastic material like PE, PS, PMMA or COP. COP is preferred because of its inertness and low autofluorescence together with silicone seals. The flow cell of the ivention can be easily manufactured by 3D printing or injection moulding.

[0016] Preferable, the flow cell comprises a fluidic seal connecting the top member (10) and the closure member (30) with each other thereby providing the volume for the examination chamber (11).

[0017] The hydrophilized surface having a contact angle of less than 70° may be obtained by standard procedures like plasma activation followed by treatment with PVP (polyvinylpyrrolidone) dissolved in 2-propanol.

[0018] The examination chamber (11) should be as small as possible to reduce diffusion length but large enough to minimize sample impact (ideally 10 pm<d<500 pm).

[0019] Further, the flow cell of the invention can be assembled by the end user to take up the sample and, after use, disassembled again to retain the sample for further use. Preferable, the biological samples are applied to a standard microscope slide, which can bemechanically connected and disconnected with the other parts of the flow cell as often as required.

[0020] Preferable, the hydrostatic pressure generated between the level of input fluid in the input reservoir (12) relative to the level of the output fluid in the output reservoir (13) can be less than 50 mm water gauge, more preferable between 5 and 25 mm water gauge (measured at room temperature).BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 - 3 show schematic three embodiments of the invention

[0022] Fig. 4 shows a side view of the flow cell, omitting the closure member (30)

[0023] Fig. 5 shows a exploded view of the flow cell

[0024] Fig. 6 shows examination chamber (11) with rampard (20)

[0025] Fig. 7 shows the channel (22) as capillary notch on the input opening (18)

[0026] Fig. 7 shows the edge radius of capillary notch (22) at the input opening (18)

[0027] Glossar to the darwings:(10) top member(11) examination chamber(12) input reservoir for input fluids(13) output reservoir for output fluids(14) input opening of examination chamber (11)(15) output opening of examination chamber (11)(16) channel between input reservoir (12) and examination chamber (11)(17) channel between examination chamber (11) and output reservoir (13)(18) inlet opening of output reservoir (13)(19) outlet opening of input reservoir (12)(20) rampart in the examination chamber (11)(21) capillary notches on / at inlet opening (18) of output reservoir (13)(30) closure member(40) base member(41) interconnecting means(42) openings (42) for interconnecting means (41)DETAILED DESCRIPTION

[0028] As disclosed, the hydrostatic pressure is generated by the different levels of input fluid vs output fluids. In a flow cell, this is somewhat influenced on the relative position of input and output reservoir.

[0029] In a first embodiment, the input reservoir (12) is positioned at a level relative to the output reservoir (13) such that the bottom level of the input reservoir (12) is positioned at the same level (20) as the bottom level of output reservoir (13).

[0030] In a variant thereof, the output reservoir (13) is provided with an inlet opening (18) which is in fluid communication with the at least one output opening (15) of examination chamber (11) wherein the inlet opening (18) is positioned at the bottom level of the output reservoir (13).

[0031] In a second embodiment, the output reservoir (13) is provided with an inlet opening (18) which is in fluid communication with the at least one output opening (15) of examination chamber (11) wherein the inlet opening (18) is positioned above the bottom level of the output reservoir (13). For example, the inlet opening (18) may be positioned above the bottom level at a height of between 5 and 20% of the total height of the output reservoir (13).

[0032] In a third embodiment, the output reservoir (13) is provided with an inlet opening (18) which is in fluid communication with the at least one output opening (15) of examination chamber (11) wherein the inlet opening (18) is positioned at the top of an overflow structure (21) extending above the bottom level of the output reservoir (13). For example, the overflow structure (21) may extend into the output reservoir (13) between 5 and 20% of the total height of the output reservoir (13).

[0033] In any of the embodiments, the output reservoir (13) may be provided at least at the inlet opening (18) with a hydrophilized surface having a contact angle of less than 70 °.

[0034] As shown in the drawings, the input reservoir (12), examination chamber (11) and output reservoir (13) are fluidic connected via microfluidic channels (16) and (17).

[0035] Once the input fluids are completely transferred through the channels to the output reservoir (13), the flow of fluid stops, preferable with the outlet opening (19) of input reservoir (12) staying filled with fluids. This can be achieved providing the input reservoir (12) with a decreasing surface area towards the outlet opening (19), for example with by a conical shape.

[0036] In this variant, it is possible to refill the input reservoir (12) without the generation of bubbles in the outlet opening (19) of input reservoir (12). This is necessary for switching from staining to washing or for multiple different staining fluids.

[0037] In order to achieve a homogeneous staining and washing of the complete sample, a constant laminar flow has to be created in examination chamber (11). In yet another embodiment shown in Fig. 6, the input opening (14) and / or the output opening (15) of the examination chamber (11) is provided with a rampart (20) extending over the width of the examination chamber (11).

[0038] The rampart (20) ensures bubble-free filling of the complete width of the examination chamber (11) by forming a stop for the fluids, thereby dominating the impedance and defining the flow rate. The rampart might include vertical capillary notches for an even better defined filling of the sample area. Downstream of the rampart, the height of the examination chamber is increased to 10-500 pm over the sample area. Optional, an additional rampart downstream of the sample further ensures bubble-free filling of the sample area. Then the fluid is guided to the output opening (18) into the output reservoir (13).

[0039] The capacity of the output reservoir (13) should be is larger than the volumes intended for the staining or washing steps, so the total process time is reduced by eliminating the need for pipetting steps.

[0040] Top member (10) and closure member (30) may be combed directly to each other, for example by an adhesive.

[0041] However, more preferred is that the flow cell of the invention comprises a top member (10) and a base member (40) sandwiching the closure member (30) wherein top member (10) and base member (40) are provided with mechanically interconnecting means, thereby providing a detachable mechanical connection with each other.

[0042] In alternative, the top member (10) and the base member (40) may be provided with an adhesive, thereby providing a non-detachable mechanical connection with each other.

[0043] Preferable, the top member (10) is provided with openings (42) and base member (40) with interconnecting means (41), wherein the openings (42) and interconnecting means (41) are configured to mechanically interlock with each other by lateral movement of the top member (10) against the base member (40).

[0044] The interconnecting means (41) of the base member (40) and the openings (42) of the top member (10) can be configured as bay onetted joint or snap-on connector.

[0045] The closure member (30) may be transparent to allow optical detection and / or imaging of the sample in the examination chamber (11). Preferable, closure member (30) is a microscopic slide glass.

Claims

CLAIMS1. A flow cell comprising a top member (10) and a closure member (30) providing a volume as examination chamber (11) wherein the top member (10) is provided with at least one input reservoir (12) for input fluids and one output reservoir (13) for output fluids and the examination chamber (11) is provided with at least one input opening (14) in fluidic communication with at least one input reservoir (12) and at least one output opening (15) in fluidic communication with the output reservoir (13) wherein the hydrostatic pressure generated between the level of input fluid in the input reservoir (12) relative to the level of the output fluid in the output reservoir (13) generates a flow of input fluid from the input reservoir (12) through the examination chamber (11) into output reservoir (13) characterized in that the output reservoir (13) is provided at least in part with a hydrophilized surface having a contact angle of less than 70 °2. Flow cell according to claim 1 characterized in that the input reservoir (12) is positioned at a level relative to the output reservoir (13) such that the bottom level of the input reservoir (12) is positioned at the same level (20) as the bottom level of output reservoir (13).

3. Flow cell according to claim 1 or 2 characterized in that the output reservoir (13) is provided with an inlet opening (18) which is in fluid communication with the at least one output opening (15) of examination chamber (11) wherein the inlet opening (18) is positioned at the bottom level of the output reservoir (13).

4. Flow cell according to claim 1 characterized in that the output reservoir (13) is provided with an inlet opening (18) which is in fluid communication with the at least one output opening (15) of examination chamber (11) wherein the inlet opening (18) is positioned above the bottom level of the output reservoir (13).

5. Flow cell according to claim 1 characterized in that the output reservoir (13) is provided with an inlet opening (18) which is in fluid communication with the at least oneoutput opening (15) of examination chamber (11) wherein the inlet opening (18) is positioned at the top of an overflow structure (21) extending above the bottom level of the output reservoir (13).

6. Flow cell according to any of claims 1 to 5 characterized in that the output reservoir (13) is provided with an inlet opening (18) wherein the output reservoir (13) is provided at least at the inlet opening (18) with a hydrophilized surface having a contact angle of less than 70 °.

7. Flow cell according to any of claims 1 to 6 characterized in that the hydrostatic pressure generated between the level of input fluid in the input reservoir (12) relative to the level of the output fluid in the output reservoir (13) is less than 50 mm water gauge.

8. Flow cell according to any of claims 1 to 7 characterized in that the inlet opening (18) of output reservoir (13) is provided with at least one capillary notch (22) designed to lead output fluid into the output reservoir (13) by capillary forces.

9. Flow cell according to claim 8 characterized in that the inlet opening (18) of output reservoir (13) is provided with two capillary notches (22) having an orientation of 45°- 135° to each other.

10. Flow cell according to claim 8 or 9 characterized in that the at least one capillary notch (22) has an edge radius adjacent to the inlet opening (18) of output reservoir (13) of 0.01 - 2pm.

11. Flow cell according to any of claims 1 to 10 characterized in that the input opening (14) and / or the output opening (15) of the examination chamber (11) is provided with a rampart (20) extending over the width of the examination chamber (11).

12. Flow cell according to any of claims 1 to 11 characterized in that the input reservoir (12) is provided with decreasing surface area towards the outlet opening (19).

13. Flow cell according to any of claims 1 to 12 characterized in that the top member (10) and the closure member (30) are connected with each other by a fluidic seal providing the volume for the examination chamber (11).

14. Flow cell according to any of claims 1 to 13 characterized in that the top member (10) and the base member (40) are provided with mechanically interconnecting means, thereby providing a detachable mechanical connection with each other.

15. Flow cell according to claim 14 characterized in that the the top member (10) is provided with openings (42) and base member (40) with interconnecting means (41), wherein the openings (12) and interconnecting means (41) are configured to mechanically interlock with each other by lateral movement of the top member (10) against the base member (40).

16. Flow cell according to any of claims 1 to 15 characterized in that the top member (10) and the base member (40) are provided with an adhesive, thereby providing a non- detachable mechanical connection with each other.

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