Particle sorting system with multiway valve based on a cantilever beam with multiple actuation states

Abstract

The invention is directed to a micromechanical device for sorting at least two distinct fractions of target particles and at least one non-target target particle from each other, all suspended in a fluid, comprising: - a source of magnetic flux (1) - a movable member (2) comprising permeable magnetic material (3) wherein the movable member (2) is moved from a first position (4) to a second position (5) and / or one or more third positions (6) by applying a magnetic flux to the movable member (2) - a sample inlet channel (7) through which the fluid flows into the micromechanical device - a first output channel (8) which the movable member (2) diverts the non-target particles while being in the first position (4) - a second output channel (9) into which the movable member (2) diverts one distinct fraction of the target particles while being in the second position (5) - one or more third output channels (10) into which the movable member (2) diverts further distinct fractions of the target particles while being in one of the one or more third position (6) - a spring (11) attached to the movable member (2) providing mechanical resistance to the movable member (2) when moving from the first position (4) to the second position (5) and / or to one or more of the third positions (6) characterized in that the spring (11) has non-linear elastomechanical properties when the movable member (2) is at the second position (5) and / or at one or more of the third positions (6).

Description

PARTICLE SORTING SYSTEM WITH MULTIWAY VALVE BASED ON ACANTILEVER BEAM WITH MULTIPLE ACTUATION STATESBACKGROUND

[0001] The invention is directed to a cell-sorting valve having a spring (11) with nonlinear elastomechanical properties thereby enabling multi channel sorting.

[0002] Currently, only droplet-sorters enable a fast and reliable separation of cell suspensions into more than one dedicated sorting fraction plus waste.

[0003] Multisort valves are known for example from US 9,404,838 and US20200179931A1. The multipath sorting as disclosed here is achieved by variation of the hydraulic resistance of the microchannels for each sorted fraction (sort channels). To select the corresponding sort channels, it is required to hold the sorting mechanism at a certain position for sufficiently long hold-times so that sorted particles are guided to the dedicated sort channel. This reduces the sorting speed. In addition, cross talk between the sorting channels is a severe risk that causes reduced purity of the sorted fractions. The cell sorter chips disclosed in this prior art still use the simple cantilever beams as shown in US 20180065120A1.

[0004] US2015093817 discloses a different device for multi channel cell sorting using a valve provided with two magnetic drives. These magnetic drives can change the actuation direction by biasing one of the magnets. Actuating two magnets requires additional controlling mechanisms and is difficult to control at high switching speeds. A pure mechanical solution would be preferable.

[0005] Additionally, there is prior art for a different chip-based microfluidic particle- / cell-sorter technology that relies on a vortex generator to redirect the fluid flow (see for example Microfluidic Particle Sorter (GB2585584A) and Appartus for sorting microfluidic particles (US20220234042A1). The technology described has the potential to be used for multiway sorting by application of a manifold and proper arrangement of the vortex generators with respect to corresponding microfluidic junctions.

[0006] The arrangement and interconnection of multiple cell-sorting mechanisms require more space on the chip and therefore lead to higher costs. This present approach, however, is targeted at multiway cell-sorting chips with a minimum footprint and subsequently low costs.

[0007] In general, a cell-sorter-chip utilizes a cantilever beam with a largely linear elastomechanic characteristic curve as a cell-sorting valve. Due to the largely linear elastomechanic curve, the cantilever cannot robustly be actuated and held at intermediate actuation states for multiway-sorting actuations. Even with an adjusted microfluidic channel network, this mechanism cannot be used for robust multiway-sorting.

[0008] Surprisingly, it was found that by providing sorter chips with cantilever beams having a non-linear elastomechanic characteristic curve more than one stable actuation state can be achieved, resulting in robust and fast sorting valves.OBJECT OF THE INVENTION

[0009] The object of this invention is to provide a microfluidic multiway cell-sorting chip that is compatible with the known cell sorting devices like the MACSQuant Tyto system available from Miltenyi Biotec B.V. & Co KG. This mainly relies on a movable member (2) that acts as a cell-sorting valve with multiple stable actuation states. In combination with an adjusted microfluidic channel layout, the stable actuation states of the valve can be used to redirect cells to dedicated sort chambers.

[0010] This is realized by a non-linear elastomechanic characteristic curve of the spring (11) that is part of the movable member (2). The non-linearity can be a discontinuity within the characteristic curve, that is caused by a change in the stiffness of the spring (11) after the cell-sorting mechanism has travelled a defined distance.

[0011] Accordingly, object of the invention is a micromechanical device for sorting at least two distinct fractions of target particles and at least one non-target target particle from each other, all suspended in a fluid, comprising: a source of magnetic flux (1) a movable member (2) comprising permeable magnetic material (3) wherein the movable member (2) is moved from a first position (4) to a second position (5) and / or one or more third positions (6) by applying a magnetic flux to the movable member (2) a sample inlet channel (7) through which the fluid flows into the micromechanical device a first output channel (8) which the movable member (2) diverts the non-target particles while being in the first position (4) a second output channel (9) into which the movable member (2) diverts one distinct fraction of the target particles while being in the second position (5)one or more third output channels (10) into which the movable member (2) diverts further distinct fractions of the target particles while being in one of the one or more third position (6) a spring (11) attached to the movable member (2) providing mechanical resistance to the movable member (2) when moving from the first position (4) to the second position (5) and / or to one or more of the third positions (6) characterized in that the spring (11) has non-linear elastomechanical properties when the movable member (2) is actuated to the second position (5) and / or to one or more of the third positions (6).

[0012] In general, the number of distinct fractions of target particles, output channels, positions of the movable member and occurrences of non-linear elastomechanical properties of the spring (11) are not limited. Due to size restrictions and manufacturing limitations of micromechanical devices, 3-5 distinct fractions of target particles, output channels, positions of the movable member and occurrences of non-linear elastomechanical properties of the spring (11) are preferred.

[0013] In a preferred embodiment of the invention, the micromechanical device is characterized in that fractions of first target particles, second target particles and non-target target particles are sorted from each other, all suspended in a fluid and wherein the comprises micromechanical device:- a source of magnetic flux (1)- a movable member (2) comprising permeable magnetic material (3) wherein the movable member (2) is moved from a first position (4) to a second position (5) and / or a third position (6) by applying a magnetic flux to the movable member (2)- a sample inlet channel (7) through which the fluid flows into the micromechanical device- a first output channel (8) which the movable member (2) diverts the non-target particles while being in the first position (4)- a second output channel (9) into which the movable member (2) diverts a first distinct fraction of the target particles while being in the second position (5)- a third output channel (10) into which the movable member (2) diverts a second distinct fraction of the target particles while being in the third position (6)- a spring (11) attached to the movable member (2) providing mechanical resistance to the movable member (2) when moving from the first position (4) to the second position (5) and / or third position (6)wherein the spring (11) has non-linear elastomechanical properties when the movable member (2) is at the second position (5) and / or third position (6).

[0014] In the following, the terms “cantilever beam” refers to the “movable member (2)” and vice versa.

[0015] The term “distinct fractions of target particles” refers to fractions of particles that can be distinguished i.e. sorted from each other. Distinguishing properties are for example fluorescence emission as well known in the technical field of cell sorting. With the device of the invention, a suspension of for example two sorts of target particles (like cells) can be sorted into two different channels while simultaneously non-target particles (cells) are sorted into a third (waste) channel.

[0016] The term “cantilever tip” refers to the end of the cantilever beam or movable member (2) which connects the input channel with one of the output channels. Depending on the architecture of the device i.e. the position of the input and output channels, the cantilever tip may have an symmetric or asymmetric design.

[0017] In combination with driving time-modulated magnetic forces, the cantilever beam is held at stable intermediate actuation states.

[0018] To utilize the elastomechanical characteristics of the cantilever beam for multiway sorting applications, a corresponding routing of the fluidic channels is mandatory. By proper design of the cantilever tip, the input channel is only in fluidic contact with one selected output channel. The cross-talk from the input to each output channel is reduced by the design of the cantilever tip as well.

[0019] The device of the invention can be used for sorting fractions of any kind of target particles form and non-target particles as long as the particles have at least one property which can trigger the sorting process (like color, emission upon irradiation, size). Preferable, the device is used to sort cells suspended in media, especially mammalian cells with are marked by one or more fluorescent dyes. The fluorescent dyes (usually antibody-conjugated dyes) and media are well known from immunofluorescence staining and the various sorting processes in this art. The presence or absence of fluorescent marked cells triggers the magnetic source which in turn controls the movement of the movable member.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig.l (A) shows a schematic curve of the elastomechanical properies of a two- way sorting system with a corresponding mechanism shown in Fig. 1(B).

[0021] Fig. 2 shows a schematic illustration of an asymmetric 3-way-sorting approach according to the invention with a first variant of the path a flow between the input and output channels as indicated by the arrows.

[0022] Fig. 3 shows another schematic illustration of an asymmetric 3-way-sorting approach according to the invention with a second variant of the path a flow between the input and output channels as indicated by the arrows.

[0023] Fig. 4 shows the simulated displacement of the movable member (2) over time that is caused by the time modulated magnetic forces that result from an electromagnet that is powered by the plotted time modulated input current (dashed line).

[0024] Fig. 5 shows another embodiment of the invention with a symmetric 3-way- sorting approach with direction inverter.

[0025] It should be noted that the one output channel in the devices shown in Fig. 2, 3 and 5 is positioned perpendicular to the input channel and the tow other output channels (i.e. perpendicular to the paper plane.DETAILED DESCRIPTION

[0026] By exploiting the discontinuity within the elastomechanical characteristic curve of the spring (11), it is possible to hold the mechanism in an additional intermediate position. In this context, time modulated magnetic forces can be used to hold the mechanism at the desired position. After applying higher forces, the valve travels to its final stable position at an appropriate hard stop.

[0027] In a first embodiment of the invention, the non-linearity within the elastomechanical characteristic curve of the spring (11) is introduced by an additional boundary stop-condition to the mechanism that leads to a discontinuity. In Fig.l (A), a characteristic curve for a two-way sorting system is shown with the corresponding mechanism in Fig. 1(B).

[0028] By application of a magnetic force to the movable member (2) (see Fig. 1(B)), the movable member (2) propagates towards the source of magnetic flux (1). Initially, it reaches the first hard stop (12). After being in contact with the first hardstop (12), the stiffness of the spring (11) is increased for further movement of the movable member (compare Fig. 1(A)). This is beneficial, since it is well known that the driving magnetic forces increase with decreasing gap difference between fixed source of magnetic flux (1) and the comprising permeable magnetic material (3) of the movable member (2).

[0029] The actuation position (05) serves as an intermediate actuation state and enables multiway-sorting. By applying higher magnetic forces to the comprising permeable magnetic material (3), the movable member (2) reaches the second hard stop (13) where its actuation range ends (06).

[0030] In a variant, the spring (11) is provided with non-linear elastomechanical properties such that the movement of the movable member (2) from the first position (4) to the second position (5) is in the same direction as the movement of the movable member (2) from the second position (5) to the third position (6).

[0031] This variant is shown in Fig. 1 as schematic illustration of an asymmetric 3- way-sorting device. (A) The moveable member (2) is positioned at rest with no load applied(4). Due to the design of the cantilever-tip, the inlet (7) fluid flow (indicated by arrows) is guided towards the first output channel (8) which is intended for non-target particles. (B) By application of magnetic forces, the cell-sorter mechanism is held at an intermediate position(5). The inlet (7) fluid flow (indicated by arrows) is guided towards the second output channel (9) for first order target particles. (C) By application of high magnetic forces, the movable member (2) reaches its final position (6). The inlet (7) fluid flow (indicated by arrows) is subsequently guided to the third output channel (10) for second order target particles.

[0032] In another variant of the invention, the cantilever-tip is adjusted so that the fluid flow directions with respect to the actuation positions (4,5 and 6) are redistributed enabling a symmetric switching from non-sort to sort positions.

[0033] This variant is shown in Fig. 3 as schematic illustration of an symmetric 3- way-sorting approach. (A) The movable member positioned at rest (4) with no load applied. The inlet (7) fluid flow (indicated by arrows) is guided towards the third output channel (10) for second order target particles. (B)The movable member (2) is held at an intermediate state(5) by application of a suitable driving magnetic force. The inlet (7) fluid flow (indicated by arrows) is subsequently guided towards the first output channel (8) for non-target particles. (C) By application of high magnetic force, the movable member (2) reaches its final position(6). The inlet (7) fluid flow (indicated by arrows) is subsequently guided to the channel (9) for first order target particles.

[0034] For these variants, spring (11) is preferable provided with non-linear elastomechanical properties by providing the device and / or the spring (11) with a first hard stop, which is positioned to be in contact with the spring (11) when the movable member (2) is at the second position (5). Further, the spring (11) may be provided with non-linear elastomechanical properties by providing the device and / or the spring (11) with a second hardstop which is positioned to be in contact with the spring (11) when the movable member (2) is at the third position (6).

[0035] In another embodiment of the invention, the spring (11) is provided with nonlinear elastomechanical properties such that the movement of the movable member (2) from the first position (4) to the second position (5) is in the diametral direction of the movement of the movable member (2) from the second position (5) to the third position (6).

[0036] Preferable, the source of magnetic flux (1) and the movable member (2) form a wedge shaped gap when the movable member (2) is at the second position (5).

[0037] This embodiment can be achieved by providing the spring (11) with non-linear elastomechanical properties by utilizing the source of magnetic flux (1) as a second hard stop (13) which is positioned to be in contact with the movable member (2) when the movable member (2) is at the third position (6).

[0038] Preferable, the spring (11) is provided with non-linear elastomechanical properties by utilizing the source of magnetic flux (1) as first hard stop and / or second hard stop which are positioned to be in contact with the permeable magnetic material (3) when the movable member (2) is at the first (5) and / or second position (6).

[0039] This embodiment is shown in Fig. 5 as schematic illustration of an symmetric 3-way-sorting approach with direction inverter. (A) The movable member (2) is positioned at rest (4) with no load applied. The inlet (7) fluid flow (indicated by arrows) is guided towards third output channel (10) for second order target particles. (B) The movable member (2) is held at an intermediate state (5) by application of a suitable magnetic force. The inlet (7) fluid flow (indicated by arrows) is subsequently guided towards the first output channel (8) for non-target particles . (C) By application of high magnetic forces, the movable member (2) moves in the opposite direction to reach position (6). This direction inversion is enabled by a hinge (14) that is attached to the spring(l 1). The inlet (7) fluid flow (indicated by arrows) is subsequently guided to the second output channel (9) for first order target particles.

[0040] In this embodiment, a hinge or joint (14) is added to the spring (11) that enables the movement of the movable member (2) from the second position (5) to the third position (6) in the diametral direction of the movement of the movable member (2) from the first position (4) to the second position (5).

[0041] The device of the invention makes use of a “holding” step or “holding position” of the movable member for the position of the movable member between the position with “no magnetic forces” and “maximum magnetic force” being applied.

[0042] The graph in Fig. 4 shows the simulated displacement of the movable member (compare Fig. 1(B)) that is actuated by the magnetic force caused by an electromagnet that is driven by the time-modulated input current (two rectangular pulses) that is visualized in Fig. 4 as well.

[0043] The numerical simulation results plotted in Fig. 4 show that it is possible to hold the movable member in an intermediate position due to the non-linear elastomechanical curve of the spring in combination with suitable time modulated driving magnetic forces.

[0044] In Fig. 5, holding the mechanism in its intermediate position (5) causes a flow to the first output channel (8) for non-target particles (see Fig.5(B)). Returning to the unbiased (at rest) position (4) redirects fluid from the inlet (7) to the third output channel (10) for second order target particles (compare Fig.5 (A)). Switching from the intermediate (5) position to the position (6) requires an inversion of the travelling direction (see Fig.5 (C)) enabled by the hinge (14) attached to the spring (11). With this approach, the size of the cantilever-tip can be drastically reduced. Together with the wedge-shaped gap formed by the comprising permeable magnetic material (3) of the movable member (2) and the fixed source of magnetic flux (1), high magnetic forces occur that enable a fast switching from position (5) to (6).

[0045] By application of magnetic forces, the movable member (2) moves until it hits the fixed source of magnetic flux (1) that acts in this context as a first hard stop (12) (see Fig. 5 (B)). By increasing the magnetic forces, the hinge (14) that is attached to the spring (11) flexes and the movable member (2) closes the wedge shaped gap by moving form position (5) to (6) and hits the second hard stop (13). This causes an inversion of the actuation direction (compare Fig. 5 (C)).Glossar a source of magnetic flux (1) a movable member (2) comprising permeable magnetic material (3) first position (4) , second position (5) third position (6) a sample inlet channel (7) first output channel (8) , second output channel (9) third output channel (10) spring (11) first hard stop (12)second hard stop 13 additional hinge or joint 14

Claims

Claims1. A micromechanical device for sorting at least two distinct fractions of target particles and at least one non-target target particle from each other, all suspended in a fluid, comprising: a source of magnetic flux (1) a movable member (2) comprising permeable magnetic material (3) wherein the movable member (2) is moved from a first position (4) to a second position (5) and / or one or more third positions (6) by applying a magnetic flux to the movable member (2) a sample inlet channel (7) through which the fluid flows into the micromechanical device a first output channel (8) which the movable member (2) diverts the non-target particles while being in the first position (4) a second output channel (9) into which the movable member (2) diverts one distinct fraction of the target particles while being in the second position (5) one or more third output channels (10) into which the movable member (2) diverts further distinct fractions of the target particles while being in one of the one or more third position (6) a spring (11) attached to the movable member (2) providing mechanical resistance to the movable member (2) when moving from the first position (4) to the second position (5) and / or to one or more of the third positions (6) characterized in that the spring (11) has non-linear elastomechanical properties when the movable member (2) is at the second position (5) and / or at one or more of the third positions (6).

2. The micromechanical device of claim 1, characterized in that the hydrodynamic resistance of first output channel (8) is lower than the hydrodynamic resistance of second output channel (9).

3. The micromechanical device of claim 1 characterized in that fractions of first target particles, second target particles and non-target target particles are sorted from each other, all suspended in a fluid and wherein the comprises micromechanical device: a source of magnetic flux (1) a movable member (2) comprising permeable magnetic material (3) wherein the movable member (2) is moved from a first position (4) to a second position (5) and / or a third position (6) by applying a magnetic flux to the movable member (2) a sample inlet channel (7) through which the fluid flows into the micromechanical device a first output channel (8) which the movable member (2) diverts the non-target particles while being in the first position (4) a second output channel (9) into which the movable member (2) diverts a first distinct fraction of the target particles while being in the second position (5) a third output channel (10) into which the movable member (2) diverts a second distinct fraction of the target particles while being in the third position (6) a spring (11) attached to the movable member (2) providing mechanical resistance to the movable member (2) when moving from the first position (4) to the second position (5) and / or third position (6) wherein the spring (11) has non-linear elastomechanical properties when the movable member (2) is at the second position (5) and / or third position (6).

4. The micromechanical device of claim 1 or 2, characterized in that the non-linear elastomechanical properties of the spring (11) are provided by non-linearly increasing the elastomechanical stiffness of the spring (11) when the movable member (2) is at the second and / or third position (6).

5. The micromechanical device of any of claims 1 to 3, characterized in that the spring (11) is provided with non-linear elastomechanical properties by providing the device and / or the spring (11) with a first hard stop, which is positioned to be in contact with the spring (11) when the movable member (2) is at the second position (5).

6. The micromechanical device of any of claims 1 to 4, characterized in that the spring (11) is provided with non-linear elastomechanical properties by providing the device and / or the spring (11) with a second hard stop which is positioned to be in contact with the spring (11) when the movable member (2) is at the third position (6).

7. The micromechanical device of any of claims 1 to 3, characterized in that the spring (11) is provided with non-linear elastomechanical properties by providing the source of magnetic flux (1) as first hard stop which is positioned to be in contact with the movable member (2) when the movable member (2) is at the second position (5).

8. The micromechanical device of claim 6, characterized in that the spring (11) is provided with non-linear elastomechanical properties by providing the source of magnetic flux (1) as second hard stop which is positioned to be in contact with the movable member (2) when the movable member (2) is at the third position (6).

9. The micromechanical device of claim 6 or 7, characterized in that the spring (11) is provided with non-linear elastomechanical properties by providing the source of magnetic flux (1) as first hard stop and / or second hard stop which are positioned to be in contact with the permeable magnetic material (3) when the movable member (2) is at the first and / or second position (5).

10. The micromechanical device of any of claims 6 to 8, characterized in that the source of magnetic flux (1) and the movable member (2) form a wedge shaped gap when the movable member (2) is at the second position (5).

11. The micromechanical device of any of claims 1 to 9, characterized in that the movable member (2) moves between the first, second and third position (6) in an actuation plane and wherein at least one of the first, second and third output channel(10) is orientated perpendicular to the actuation plane.

12. The micromechanical device of any of claims 1 to 10 characterized in that the movable member (2) is in the first position (4) when no magnetic flux (1) is applied and diverts non-target particles into the first output channel (8).

13. The micromechanical device of any of claims 1 to 11 characterized in that the movable member (2), the spring (11), the first, second and third output channel (10) are formed on a surface of a substrate.

14. The micromechanical device of any of claims 1 to 12 characterized in that the spring(11) is provided with non-linear elastomechanical properties such that the movement of the movable member (2) from the first position (4) to the second position (5) is inthe same direction as the movement of the movable member (2) from the second position (5) to the third position (6).

15. The micromechanical device of any of claims 1 to 13 characterized in that the spring (11) is provided with non-linear elastomechanical properties such that the movement of the movable member (2) from the first position (4) to the second position (5) is in the diametral direction of the movement of the movable member (2) from the second position (5) to the third position (6).

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