A multi-functional microfluidic device and methods of providing and using such device

Abstract

The invention provides a microfluidic probe (100) comprising a cantilever (150) and a cantilever support (130), wherein the cantilever (150) comprises a cantilever channel (165) and wherein the cantilever support (130) comprises a support channel (135) configured in fluidic contact with the cantilever channel (165); wherein the cantilever (150) comprises a support end (151) and a cantilever end (152) remote from the cantilever support (130); wherein the cantilever (150) comprises a protrusion (154) and a cantilever aperture (156) both configured on a first side (51) of the cantilever (150), wherein the protrusion (154) and the cantilever aperture (156) are configured closer to the cantilever end (152) than to the support end (151), and wherein the protrusion (154) and the cantilever aperture (156) are spatially separated; wherein the cantilever aperture (156) is configured in fluidic contact with the cantilever channel (165); wherein the protrusion (154) comprises a protrusion tip (155).

Description

[0001] A multi-functional microfluidic device and methods of providing and using such device

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a microfluidic probe. The invention further relates to a multi-functional microfluidic device comprising the microfluidic probe. The invention further relates to a method for 3D printing a device. The invention further relates to a method for operating the multi-functional microfluidic device and a use of the multi-functional microfluidic device.

[0004] BACKGROUND OF THE INVENTION

[0005] Microfluidic AFM probes are known in the art. For instance, WO2022146549A1, describes a multipurpose scanning microscopy probe, comprising: a probe holder; a cantilever connected to the probe holder; and a probe tip connected to the cantilever, wherein the probe tip is a three-dimensional geometry, and wherein the probe tip is a 3D printed part.

[0006] Kramer, et al. (2020): “Multiscale 3D-printing of microfluidic AFM cantilevers”, Lab Chip, 20, p. 311-319, describes Microfluidic atomic force microscopy (AFM) cantilever probes having all the functionalities of a standard AFM cantilever along with fluid pipetting. They have a channel inside the cantilever and an aperture at the tip and are useful for precise fluid manipulation at a desired location, for example near or inside cells. The described probes are made using two different 3D additive manufacturing techniques, stereolithography and two-photon polymerization, to directly print ready-to-use microfluidic AFM cantilever probes. Using these 3D printed probes, AFM imaging of a surface, puncturing of the cell membrane, and aspiration at the single cell level have been demonstrated.

[0007] Deladi etal. (2004): “Micromachined fountain pen for atomic force microscopebased nanopatteming”, Applied Physics Letters, 85, no. 22, p. 5361-5363, describes a tool for AFM consisting of a reservoir micromachined into the probe support that is connected to the fluidic channels embedded in a V-shaped cantilever. Via the fluidic channels the liquid reached the tip.

[0008] Furthermore, US20130105034A1 describes a cantilever having a tip with an opening and a microchannel extending through the cantilever in its longitudinal direction, said microchannel being fluidly connected to the opening at the tip of the cantilever. D3 further describes providing suspension means for holding the cantilever and spatially moving the cantilever along a predetermined spatial path. SUMMARY OF THE INVENTION

[0009] As indicated by Robert C.L.N. Kramer et al. in Lab Chip, 2020, 20, 311, DOI: 10.1039 / c91c00668k, which is herein incorporated by reference, and also referring to references cited therein, “No two cells are identical. Moreover, cells are constantly responding to internal and external signals which result in highly dynamic cellular characteristics, for example in cell type and cellular state. This means that even purified cell populations are often composed of a heterogeneous mix of cells and bulk analyses often result in an averaged representation of a cell population's cellular characteristics. Fully unravelling cellular complexity in both healthy and diseased states will therefore require characterization and manipulation at single-cell resolution. Microsystem technologies are suitable for this task due to their similar size scale to the cells under study. To sort cells, passive methods such as micropillar arrays or active methods like microfluidic acoustic resonance have been used. To accurately quantify single cell characteristics, micro / nano-mechanical resonators have been used to determine cell density, growth rate and response to drugs. To manipulate single cells, atomic force microscopy (AFM) based methods are suitable. This manipulation of single cells can provide information on cellular characteristics such as elastic modulus, adhesion strength, and response to mechanical stimuli. With the advent of the FluidFM technique, simultaneous precise force control and (sub)picolitre volume fluid manipulation inside a cell has become possible. The technique uses an AFM cantilever with an internal channel and aperture at the tip."

[0010] AFM cantilevers are generally exchanged for different measurements and each measurement takes a relatively long processing time. Therefore, by the time the cantilevers are exchanged, the object of interest (e.g. a targeted cell) may be lost or the properties of the object may have changed.

[0011] There appears to be a desire to (further) improve production of microfluidic devices and / or to provide microfluidic devices with multi-functional application, i.e., that can be used for multiple different types of measurements, which allow a higher resolution and / or which are better controllable or with which processes can be executed in a better controlled way.

[0012] Hence, it is an aspect of the invention to provide an alternative multi-functional microfluidic device, which preferably further at least partly obviates one or more of abovedescribed drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. In a first aspect, the invention provides a microfluidic probe comprising a cantilever and a cantilever support. In embodiments, the cantilever may comprise a cantilever channel. Especially, in embodiments, the cantilever may be hollow. Similarly, in embodiments, the cantilever support may comprise a support channel. Especially, in embodiments, the cantilever support may be hollow. The cantilever channel and the support channel may especially be configured in fluidic contact with each other. In embodiments, the cantilever may comprise a support end and a cantilever end. The cantilever end may especially be remote from the cantilever support. Further, in embodiments, the cantilever may comprise a protrusion and a cantilever aperture. The protrusion and the cantilever aperture may, in embodiments, both be configured on a first side of the cantilever. Especially, the protrusion and the cantilever aperture may be configured closer to the cantilever end than to the support end. Furthermore, in embodiments, the protrusion and the cantilever aperture may be spatially separated. The cantilever aperture may, in embodiments, be configured in fluidic contact with the cantilever channel. Further, the protrusion may comprise a protrusion tip. Hence, in embodiments, the invention provides a microfluidic probe comprising a cantilever and a cantilever support, wherein the cantilever comprises a cantilever channel and wherein the cantilever support comprises a support channel configured in fluidic contact with the cantilever channel; wherein the cantilever comprises a support end and a cantilever end remote from the cantilever support; wherein the cantilever comprises a protrusion and a cantilever aperture both configured on a first side of the cantilever, wherein the protrusion and the cantilever aperture are configured closer to the cantilever end than to the support end, and wherein the protrusion and the cantilever aperture are spatially separated; wherein the cantilever aperture is configured in fluidic contact with the cantilever channel; wherein the protrusion comprises a protrusion tip.

[0013] With such a microfluidic probe microfluidic experiments may be performed. For instance, the microfluidic probe may be used for AFM imaging applications or other applications where a microscale cantilever may be needed. A microfluidic probe as described here, may especially be multi-functional as it comprises means for microfluidic modulation, imaging, and measuring mechanical properties of substrates.

[0014] Microfluidic probes are known in the art. Amongst others, the invention provides a microfluidic probe comprising a suspending cantilever, e.g. for AFM or microfluidic applications. Especially, the invention provides a microfluidic AFM cantilever. Hence, in embodiments, the microfluidic probe comprises a microfluidic AFM cantilever. However, the invention is not limited to microfluidic AFM cantilevers.

[0015] Here below embodiments in relation to the microfluidic probe are described. As indicated above, the microfluidic probe comprises a cantilever and a cantilever support. The cantilever may especially comprise a suspending cantilever, i.e., the cantilever may have an extended shape configured suspended in the air. Especially, in embodiments, the cantilever support may function as an anchor for the cantilever. The cantilever support may be attached to a surface or element, and may be configured to keep the cantilever in place, especially, to keep the cantilever suspended. The cantilever support may, in embodiments, have essentially any shape. In particular embodiments, the cantilever support may have a dome shape. However, other shapes may also be possible. In embodiments, the cantilever support may approximate a dome shape.

[0016] In embodiments, the cantilever may be 3D printed. Further, in embodiments, the cantilever support may also be 3D printed, though this is not necessarily the case. The cantilever support and the cantilever may be of different materials, but may also comprise essentially the same materials. Using essentially the same materials may facilitate a good connection between the cantilever support and the cantilever.

[0017] Furthermore, in embodiments, the cantilever support may comprise a hollow structure. Especially, in such embodiments, the cantilever support may comprise a support channel configured to provide passage for a fluid through the cantilever support. Similarly, in embodiments, the cantilever may also comprise a hollow structure. Especially, in such embodiments, the cantilever may comprise a cantilever channel configured to provide passage for a fluid through the cantilever. The cantilever channel and the cantilever support channel may, in embodiments, be configured in fluidic contact with each other. The term “fluidic contact” may especially indicate that a fluid may flow between the elements which are indicated to be in fluidic contact. Especially, herein, a gas (mixture) or a liquid, like water, may flow between the two elements. As such, in embodiments, a fluid, such as a gas (mixture, e.g. air) or a liquid (e.g. water) may pass from the cantilever channel to the support channel and vice versa.

[0018] The cantilever may comprise a support end and a cantilever end. Especially, in embodiments, the support end may be functionally coupled with, such as physically attached to, the cantilever support. In embodiments, the cantilever end may be configured remote from the cantilever support. The cantilever may, in embodiments, have an elongated shape. Especially, in embodiments, the cantilever may have a cylindrical shape. However, other elongated shapes may be possible too. For example, in embodiments, the cantilever may have a shape approximating the shape from one of the group comprising a rectangular shape, an parallelepiped shape, an polygonal shape, a half-cylindrical shape, a needle-like shape, a homed shape, etc. etc.

[0019] The term “approximate” and its conjugations herein, such as in “to approximate a shape”, may refer to being nearly identical to, especially identical to, the following term, for example nearly identical to a circular sector or a semi-cylindrical shape. For example, a cantilever may define a cylindrical sector but for a defect. In particular, an object approximating a first shape may herein refer to: a first shape realization encompassing the object, wherein the first shape realization is defined as the smallest encompassing shape of the (2D or 3D, respectively) object wherein the first shape realization has the shape of the first shape, wherein a ratio of the area (volume) of the first shape realization to the area (volume) of the object may be < 1.2, especially < 1.1, such as <1.05, especially <1.02. For instance, a cantilever may approximate a semi-cylindrical shape, wherein the first shape realization may be defined as the smallest encompassing semi -cylindrical shape of the cantilever, wherein a ratio of the volume of the first shape realization to the volume of the welding section is < 1.2, especially, especially < 1.1, such as <1.05, especially <1.02, including 1. Further, if the dimensions of the first shape are defined, the term approximate may refer to the object and the first shape being superimposable (in 2D or 3D, respectively) such that an intersection between the object and the first shape covers at least n% of the object and at least n% of the shape, wherein n is at least 90%, such as at least 95%, especially at least 98%, such as at least 99%, including 100%.

[0020] In embodiments, the cantilever may especially have a shape comprising one or more sides. Especially, the cantilever may comprise a first side. In embodiments, the cantilever aperture and the protrusion may both be configured on the same side, i.e., may both be configured on the first side of the cantilever. In particular, in embodiments, one or more of, especially both of, the cantilever aperture and the protrusion may be configured closer to the cantilever end than to the support end. Hence, in such embodiments, the cantilever aperture and the protrusion may be configured remote from the cantilever support.

[0021] Further, in embodiments, the cantilever aperture may comprise an opening in the cantilever. Especially, the cantilever aperture may provide an inlet and / or outlet for fluids in the cantilever. As such, in embodiments, the cantilever aperture may be configured in fluidic contact with the cantilever channel. Furthermore, in embodiments, the cantilever aperture may be configured in fluidic contact with the support channel, via the cantilever channel. Further yet, in embodiments, the cantilever aperture may be configured in fluidic contact with an external of the cantilever. Further, in embodiments, the cantilever aperture and the protrusion may be spatially separated. Hence, in embodiments, the cantilever aperture may not be configured in the protrusion. As such, in embodiments, the protrusion may not be configured in fluidic contact with the cantilever channel. However, fluidic contact of the protrusion with the cantilever channel is herein not excluded.

[0022] In embodiments, the protrusion and the cantilever aperture may be configured side by side in a direction perpendicular to a direction of elongation of the cantilever (especially perpendicular to a cantilever arm length axis (LCA), see also further below). However, in other embodiments, the protrusion and the cantilever aperture may also be configured side by side in a direction parallel to the direction of elongation of the cantilever. Especially, in such embodiments, the protrusion may be configured closer to the cantilever end than the cantilever aperture, and thus the cantilever aperture may be configured closer to the support end than the protrusion. Such embodiments may be beneficial for the use of the probe in experiments where a substrate (such as a biological cell) may first need to undergo imaging / force-spectroscopy followed by extraction of the substrate (i.e. the biological cell). However, in other embodiments, the cantilever aperture may be configured closer to the cantilever end than the protrusion, and thus the protrusion may be configured closer to the support end than the cantilever aperture. Such embodiments may be beneficial for the use of the probe in experiments where a substrate (such as a biological cell) may first be extracted, for example in case of a substrate in a 3D format such as spheroids or organoids, followed by imaging / forcespectroscopy on the substrate (i.e. the biological cell). Other configurations of the cantilever aperture and the protrusion relative to each other and relative to the direction of elongation of the cantilever may be possible as well. The configuration may especially be selected in favor of the following experimental steps. Especially, in embodiments, the cantilever aperture and the protrusion are spatially separated.

[0023] Furthermore, in embodiments, the protrusion may comprise a protrusion tip. Especially, the protrusion tip may herein refer to a position in the protrusion placed furthest away from the first side of the cantilever.

[0024] In embodiments, the protrusion may define a protrusion axis (Ap). Especially, in embodiments, the protrusion axis (Ap) may be defined perpendicular to the first side (of the cantilever) and intersecting the protrusion tip. Furthermore, in embodiments, the protrusion tip may extend from the first side (of the cantilever) by a second distance (d2). The second distance may in embodiments also be indicated as “height”. In embodiments, the second distance (d2) may especially be selected from the range of 0.5 pm < d2 < 20 pm. Hence, in embodiments, the protrusion may define a protrusion axis (Ap), wherein the protrusion axis (Ap) may be defined perpendicular to the first side and intersecting the protrusion tip; and wherein the protrusion tip may extend from the first side by a second distance (d2), wherein 0.5 pm < d2 < 20 pm.

[0025] Such embodiments may be beneficial as the extension of the protrusion from the first side of the cantilever may enable the protrusion tip to approach and / or touch (or e.g. puncture) a substrate prior to contact of said substrate with the cantilever aperture. The combination of the spatial separation of the cantilever aperture and the protrusion with the extension of the protrusion may allow for careful separation of their functions. In other words, the microfluidic probe as described here may be applied in a variety of experiments, such as microfluidic modulation or mechanical measurement, without the need to switch, renew or wash the probe in between measurements. The ability to reuse the same microfluidic probe for a variety of different measurement and experiments may be beneficial as there may be less precious time wasted in between experiments, which may provide improved measurement accuracy.

[0026] In embodiments, the protrusion may thus define a protrusion axis (Ap). The protrusion axis (Ap) may especially be defined as an imaginary line leading from the first side through the protrusion and intersecting with the protrusion tip. Similarly, in embodiments, the cantilever aperture may define an aperture axis (AA). The aperture axis (AA) may especially be defined as an imaginary line perpendicular to the first side and leading through (the geometrical center of) the cantilever aperture. In embodiments, the protrusion axis (Ap) and the aperture axis (AA) may be essentially parallel. However, in other embodiments, the protrusion axis (Ap) and the aperture axis (AA) may have a (slight) angle relative to each other. Hence, in such embodiments, the protrusion axis (Ap) and the aperture axis (AA) may have a nonzero angle relative to each other. Especially, in embodiments, the protrusion axis (Ap) and the aperture axis (AA) may have an axis mutual angle (OLA) selected from the range of 2-45°, such as from the range of 3-40°, like from the range of 5-30°. For example, in embodiments, the protrusion axis (Ap) and the aperture axis (AA) may have an axis mutual angle (O.A) selected from the range of 10-25°. Note that herein the ranges for the axis mutual angle (O A) may refer to the acute (or small) angle of the respective axes at their intersection point. Conversely, the axis mutual angle (OLA) may be described as the (or large) angle of the respective axes at their intersection point. In such a case, the axis mutual angle (OLA) may be selected from the range of 135-178°, such as from the range of 140-177°, like from the range of 150-175°. The above described embodiments may be beneficial as the protrusion may function as an anchor during e.g. experiments where a substrate is to be injected using the microfluidic probe, e.g. by keeping the substrate and the aperture in close contact with each other. Hence, in embodiments, the protrusion may help in adhering (or even trapping) a substrate to the aperture (during operation of the microfluidic probe).

[0027] As described above, the protrusion tip may extend from the first side of the cantilever by a second distance (d2). Especially, in embodiments, the second distance (d2) may be at least 0.1 pm, such as at least 0.25 pm, especially at least 0.5 pm, like at least 1 pm, including at least 5 pm. Further, in embodiments, the second distance (d2) may be at most 50 pm, such as at most 30 pm, like at most 25 pm, especially at most 20 pm, including at most 15 pm. In embodiments, the second distance (d2) may be selected from the range of 0.5 pm < d2 < 20 pm, for example approximately 10 pm.

[0028] Further, in embodiments, the cantilever aperture may comprise an aperture edge. Hence, in embodiments, the aperture may be defined as an opening surrounded by an aperture edge. In embodiments, a first (shortest) distance (dl) between the protrusion tip and the aperture edge may be selected from the range of at least 1 pm and / or selected from the range of at maximum about 50 pm, such as selected from the range of 1 pm <dl<50 pm. Hence, in embodiments, the cantilever aperture may comprise an aperture edge, wherein a first (shortest) distance (dl) between the protrusion tip and the aperture edge may be selected from the range of 1 pm <dl< 50 pm.

[0029] Such embodiments may be beneficial as the distance between the protrusion tip and the cantilever aperture may have an influence on measurements when using the microfluidic probe. For instance, a too short distance between the aperture and the protrusion tip may cause (unwanted) interaction between the cantilever aperture and the substrate, when aiming to protrude the substrate with the protrusion tip. On the other hand, a too large distance between the aperture and the protrusion tip may also be undesired, as for example in viscoelasticity experiments, the aperture and the protrusion tip may be configured to cooperatively manipulate the substrate.

[0030] The cantilever aperture may thus, in embodiments, be defined by an aperture edge. Especially, in embodiments, a first distance (dl) may be defined between the protrusion tip and the aperture edge. The first distance (dl) may especially, in embodiments, be a first shortest distance (dl), i.e., the first distance may be measured as the distance of the most direct and shortest path from the protrusion tip to the aperture edge. Hence, the first distance (dl) may herein especially be defined as the first shortest distance (dl) between the protrusion tip and the aperture edge. In embodiments, the first (shortest) distance (dl) may especially be at least 100 nm, such as at least 500 nm especially at least 1 pm, like at least 5 pm, including at least 8 pm. Further, in embodiments, the first (shortest) distance (dl) may be at most 50 pm, such as at most 40 pm, like at most 25 pm, especially at most 20 pm, like at most 15 pm. In embodiments, the first (shortest) distance (dl) may be selected from the range of 1 pm < dl < 30 pm, for example approximately 25 pm.

[0031] In embodiments, as mentioned above, the cantilever aperture and the protrusion may be spatially separated. Therefore, in some embodiments, the first (shortest) distance (dl) may especially be larger than the second distance (d2). However, this may not necessarily be the case. For example, in embodiments, the protrusion may have a cone-shape and the protrusion tip may extend from the first side with d2=2 pm. In such embodiments, especially, the first distance (dl) may, e.g., be 8 pm, i.e., dl>d2. Hence, the first distance may provide a quantification of the spatial separation of the cantilever aperture and the protrusion.

[0032] In embodiments, the cantilever aperture may thus comprise an aperture edge. The aperture may, in embodiments, have an aperture shape approximating a circle. Hence, in such embodiments, the aperture edge may be an essentially circular edge. However, in embodiments, the aperture may also comprise a different shape, such as e.g. a shape approximating one of the group comprising a triangle, a rectangle, a polygon, an oval, or an amorphous shape. The cantilever aperture may comprise an aperture diameter (DAP). In particular, in embodiments, the aperture diameter (D P) may be defined as an equivalent circular diameter of the cantilever aperture. The equivalent circular diameter (or ECD) (or “circular equivalent diameter”) of an (irregularly shaped) two-dimensional shape is the diameter of a circle of equivalent area. For instance, the equivalent circular diameter of a square with side a is 2*a*SQRT(l / 7t). For a circle, the diameter is the same as the equivalent circular diameter. Would a circle in an xy-plane with a diameter D be distorted to any other shape (in the xy-plane), without changing the area size, then the equivalent circular diameter of that shape would be D. Here, the circular equivalent diameter may be refer to the diameter of a circle with an area equal to the area of the cantilever aperture. In embodiments, the aperture diameter (DAP) may especially be selected from the range of 0.5- 60 pm, such as from the range of 1-50 pm, like from the range of 2-25 pm, such as from the range of 5-15 pm. Especially, in embodiments, the aperture diameter (DAP) may be at most 60 pm, such as at most 50 pm, especially at most 40 pm, like at most 30 pm, especially at most 20 pm. Hence, in embodiments, the cantilever aperture may comprise an aperture diameter (DAP) selected from the range of 2-60 pm.

[0033] The microfluidic probe may further, in embodiments, comprise a cantilever head and a cantilever arm. Especially, in embodiments, the cantilever head and the cantilever arm may be associated to each other. Further, in embodiments, the support end may comprise at least part of the cantilever arm. Yet further, in embodiments, the cantilever head may comprise one or more of the protrusion, the cantilever aperture, and the cantilever end, such as especially all of the protrusion, the cantilever aperture, and the cantilever end.

[0034] In embodiments, the cantilever head may be configured to taper from the cantilever arm along the direction of elongation of the cantilever to the cantilever end. In such embodiments, in a cross-sectional plane of the cantilever head (defined perpendicular to the direction of elongation of the cantilever), the cantilever head may thus have a slanted side. In embodiments, the slanted side may comprise the cantilever aperture. In other words, in embodiments, the cantilever aperture may be configured in the slanted side. Optionally, in embodiments, the slanted side may further comprise the protrusion. The slanted side may, in embodiments, have a slanted side length (Ls) selected such that the slanted side may accommodate at least the cantilever aperture. Therefore, in embodiments, the slanted side length (Ls) may be at least two times the aperture diameter (DAP), such as at least three times the aperture diameter (D P), like at least four times the aperture diameter (DAP). Further, the slanted side length (Ls) may be at most twenty times the aperture diameter (DAP), such as at most ten times the aperture diameter (DAP).

[0035] Furthermore, in embodiments, the aperture axis (AA) (see also further above) and the cantilever arm length axis (LCA) may have a first mutual angle (P). In some embodiments, the first mutual angle (P) may be about 90°, i.e., the aperture axis (AA) and the cantilever arm length axis (LCA) may be orthogonal relative to each other. Alternatively, in embodiments where the cantilever head may have a slanted side comprising the cantilever aperture, the first mutual angle (P) may be selected from the range of 20-90°, such as from the range of 30-80°, like from the range of 40-70°.

[0036] Conversely, in embodiments, the protrusion axis (Ap) (see also further above) and the cantilever arm length axis (LCA) may have a second mutual angle (y). In some embodiments, the second mutual angle (y) may be about 90°, i.e., the protrusion axis (Ap) and the cantilever arm length axis (LCA) may be orthogonal relative to each other. Alternatively, in embodiments, the second mutual angle (y) may be selected from the range of 20-90°, such as from the range of 30-80°, like from the range of 40-70°, especially from the range of 75-90°. Note that herein the ranges for the first mutual angle (P) and the second mutual angle (y) may refer to the acute (or small) angle of the respective axes at their intersection point. Conversely, the first mutual angle (P) and the second mutual angle (y) may be described as the (or large) angle of the respective axes at their intersection point. In such a case, the first mutual angle (P) and the second mutual angle (y) may be individually selected from the range of 90-160°, such as from the range of 100-150°, like from the range of 110-140°. Especially, in such embodiments, in embodiments, the second mutual angle (y) may be selected from the range of 90-105°.

[0037] In specific embodiments, the cantilever head may taper from the cantilever arm along the direction of elongation of the cantilever to the cantilever end, wherein the cantilever head may have a slanted side, wherein the cantilever aperture may be configured in the slanted side; wherein the cantilever aperture may define an aperture axis (AA), wherein the aperture axis (AA) and the cantilever arm length axis (LCA) may have a first mutual angle (P), wherein the first mutual angle (P) may be selected from the range of 30-80°; and wherein the protrusion axis (Ap) the cantilever arm length axis (LCA) have a second mutual angle (y), wherein the second mutual angle (y) may be selected from the range of 75-90°. Such embodiments may be beneficial as during use of such a probe the configuration as described here may allow both (i) the cantilever aperture to be provided substantially parallel (or level) to substrate, and (ii) the cantilever protrusion to be provided at an angle (relative to the substrate) optimized for puncturing and / or trapping a substrate. Especially, in such embodiments, the cantilever protrusion may function as a trap or an anchor, while the cantilever aperture may efficiently (provide fluids to or) aspirate the (trapped) substrate.

[0038] The cantilever arm may further, in embodiments, comprise a cross-sectional equivalent circular diameter (DCA). Furthermore, in embodiments, the cantilever arm may comprise a cantilever arm length axis (LCA). Especially, the cantilever arm length axis (LCA) may be defined as an imaginary line through the cantilever arm in the direction of elongation of the cantilever. In embodiments, the cantilever arm length axis (LCA) and the cross-sectional equivalent circular diameter (DCA) may be configured essentially perpendicular relative to each other. Further, in embodiments, the cantilever head may comprise a cantilever face part comprising a cantilever head dimension (DCH) configured perpendicular to the cantilever arm length axis (LCA). More especially, in embodiments, the cantilever head dimension (DCH) may be larger than the cross-sectional equivalent circular diameter (DCA), especially, DCH>2*DCA. Hence, in embodiments, the microfluidic probe may comprise a cantilever head and a cantilever arm associated to each other; wherein the support end may comprise at least part of the cantilever arm, wherein the cantilever head may comprise the protrusion, the cantilever aperture and the cantilever end, wherein the cantilever arm may comprise a cross-sectional equivalent circular diameter (DCA) and a cantilever arm length axis (LCA), wherein the cantilever head may comprise a cantilever face part comprising a cantilever head dimension (DCH) configured perpendicular to the cantilever arm length axis (LCA), and wherein DCH>2*DCA. Such embodiments may be beneficial as introducing a cantilever head being wider than the cantilever cross-sectional diameter may provide a larger surface for measurement. For example, Atomic Force Microscopy measurements rely on reflection of laser light on the cantilever to perform measurements. By providing a wider cantilever head, reflection of laser light on the cantilever head may be improved, which may lead to a higher intensity signal in the AFM measurement.

[0039] The microfluidic probe may thus comprise a cantilever head and a cantilever arm associated to each other. Especially, in embodiments, the cantilever head and the cantilever arm may be functionally coupled to form the cantilever comprising the cantilever channel. In embodiments, the cantilever head and the cantilever arm may be a monolithic structure. Hence, in such embodiments, the cantilever may comprise a support end (optionally) comprising at least part of the cantilever arm. However, in other embodiments, the cantilever head and the cantilever arm may be separately produced (e.g. 3D printed) and functionally coupled (e.g. glued together) to provide the cantilever. Furthermore, in embodiments, the cantilever head may comprise one or more of the protrusion, the cantilever aperture and the cantilever end. Especially, in embodiments, the protrusion and the cantilever aperture may be configured on the cantilever head.

[0040] In embodiments, the cantilever arm may comprise a cross-sectional equivalent circular diameter (DCA). For example, in embodiments, the cantilever arm may have a circular cross-sectional shape having a diameter (D). In other embodiments, the cantilever arm may have a cross-sectional shape different from circular. In such embodiments, the cantilever arm may have a cross-sectional equivalent circular diameter (DCA). In embodiments, the cross- sectional equivalent circular diameter (DCA) may be selected from the range of 0.2-60 pm, like at least about 1 pm, such as selected from the range of 2-50 pm, like selected from the range of 5-25 pm.

[0041] The cantilever head may, in embodiments, comprise a cantilever face part comprising a cantilever head dimension (DCH). The cantilever face part may comprise a face of the cantilever head, which may have essentially any shape. In particular, in embodiments the cantilever face part may have a shape approximating one of the group comprising a semicircular shape, an oval shape, a parabola shape, a rectangular shape, a trapezoid shape, a horseshoe shape, and an amorphous shape. In embodiments, during operation of the microfluidic probe, the cantilever face part may be configured facing the substrate, i.e., the cantilever face part may essentially comprise the first side, comprising the cantilever aperture and the protrusion. In other embodiments, during operation of the microfluidic probe, the cantilever face part may (also) be configured facing away from the substrate, e.g. facing towards a laser in AFM.

[0042] The cantilever face part may, in embodiments, comprise a cantilever head dimension (DCH). Especially, in embodiments, the cantilever head dimension (DCH) may be defined perpendicular to the cantilever arm length axis (LCA). In embodiments, the cantilever head dimension (DCH) may comprise one of a width, a length, and a diameter. In embodiments, the cantilever head dimension (DCH) may be larger than the cross-sectional equivalent circular diameter (DCA). Especially, in embodiments, DCH>DCA, such as DCH>2*DCA, like DCH>3 *DCA. For example, in embodiments, the cantilever head dimension (DCH) may be selected from the range of 5-150 pm, such as from the range of 10-100 pm, like from the range of 20-80 pm. Further, in embodiments, the second distance (d2) may be selected smaller than the cantilever head dimension (DCH), i.e., d2<Dcn, such as d2 / 2<Dcn. However, in other embodiments, the second distance (d2) may also be selected larger than the cantilever head dimension (DCH), i.e., d2>DcH, such as d2>2*Dcn.

[0043] The cantilever head dimension (DCH) may, in embodiments, especially be at least larger than the cross-sectional equivalent circular diameter (DCA). In embodiments, the cantilever arm may comprise a cantilever arm part having a length parallel to the cantilever arm length axis (LCA). Note that, in embodiments, a length of the cantilever, or even the cantilever arm may thus be larger than the length of the cantilever arm part. Especially, in embodiments, the length of the cantilever arm may comprise the sum of the length of the cantilever arm part and a length of the cantilever head (defined in a direction parallel to the cantilever arm length axis (LCA)). In embodiments, cross-sectional dimensions of the cantilever arm part over its length may be constant. Further, in embodiments, one or more cross-sectional dimensions of the cantilever head may be larger than one or more cross-sectional dimensions of the cantilever arm part. Hence, in embodiments, at least part of the cantilever head may have a cross-sectional dimension larger than a cross-sectional dimension of the cantilever arm part (measured in the same respective direction as for the cross-sectional dimension of the cantilever head). Hence, in embodiments, the cantilever arm may comprise a cantilever arm part having a length parallel to the cantilever arm length axis (LCA), wherein cross-sectional dimensions of the cantilever arm part over its length may be constant; and wherein one or more cross-sectional dimensions of the cantilever head may be larger than one or more cross-sectional dimensions of the cantilever arm part.

[0044] In embodiments, the cantilever arm part may comprise at least 20% of a full length of the cantilever, such as at least 30%, like at least 40%, especially at least 50%, more especially at least 60%. Further, in embodiments, the cantilever arm part may have cross- sectional dimensions, i.e., when taking a cross-section of the cantilever arm part perpendicular to its length (i.e. perpendicular to the cantilever arm length axis (LCA) , the cross-section may have one or more dimensions such as e.g. a length, a width, or a diameter. In embodiments, the cross-sectional dimensions of the cantilever arm part may be constant over the length of the cantilever arm part. In other words, in embodiments, the cantilever arm part may have a constant cross-sectional shape, such as e.g. a (semi-)circular cross-sectional shape or a rectangular cross-sectional shape. Further, in embodiments, when taking a cross-section perpendicular to the cantilever length axis (LCA) of the cantilever head, the cantilever head may also have cross-sectional dimensions such as e.g. a length, a width, or a diameter. In embodiments, the cross-sectional dimensions of the cantilever head may be constant over a length (parallel to the cantilever length axis (LCA)) of the cantilever head. However, in other embodiments, the cross-sectional dimensions of the cantilever head may not be constant over the length (parallel to the cantilever length axis (LCA)) of the cantilever head. For example, in embodiments, the cantilever head may have a tapering shape in a direction along the cantilever length axis (LCA). Especially, in embodiments, the cantilever head may broaden in a direction from the support end to the cantilever end. More especially, in embodiments, the cantilever may have a cantilever arm part with a (substantially) constant cross-sectional shape and a cantilever head with a cross-sectional shape increasing in size relative to the cantilever arm part. Hence, in embodiments, the cantilever head may be broader than the cantilever arm part.

[0045] Further, in embodiments, the cantilever face part may comprise a radiation reflective material. Especially, in embodiments, the radiation reflective material may be reflective for radiation having a wavelength selected from the range of visible and infrared radiation. More especially, in embodiments, the radiation reflective material may be reflective for radiation having a wavelength selected from the range of 300-2000 nm. Hence, in embodiments, the cantilever face part may comprise a radiation reflective material, wherein the radiation reflective material may be reflective for radiation having a wavelength selected from the range of visible and infrared radiation, especially a wavelength selected from the range of 300-2000 nm.

[0046] Such embodiments may be beneficial as, for example in AFM measurements, laser radiation may be provided to the cantilever face part, and hence, a cantilever face part comprising a radiation reflective material may provide reflection of the laser radiation to provide a signal for the AFM measurement. By providing a larger total surface area of the cantilever face part (comprising radiation reflective material) the reflection of the laser light, and thus the sensitivity of the AFM measurement may be improved. However, by making the total surface area of the cantilever face part too large, the cantilever may become too bulky and thus less agile in application.

[0047] In embodiments, the cantilever face part may comprise a radiation reflective material. For example, in embodiments, the cantilever face part (i.e., at least part of the cantilever, see also further below) may be 3D printed using a radiation reflective 3D printing material. In other embodiments, the cantilever face part (i.e. at least part of the cantilever) may be 3D printed using a 3D printing material comprising radiation reflective particles. Radiation reflective particles may for example, in embodiments, comprise particles comprising one or more metals. In yet other embodiments, a radiation reflective coating may be applied to the cantilever face part (after the cantilever has been provided, e.g. 3D printed). In embodiments, a radiation reflective coating may, e.g., be applied via physical vapor deposition of metal to provide a radiation reflective metallic layer. The radiation reflective material may especially, in embodiments, be reflective for radiation having a wavelength selected from the range of visible and infrared radiation. Especially, in embodiments, the radiation reflective material may be reflective for radiation having a wavelength selected from the range of 300-2000 nm, such as from the range of 300-1000 nm, like from the range of 380-780 nm.

[0048] As mentioned above, in embodiments, the cantilever face part may comprise 3D printed material. In embodiments, one or more of the cantilever support and the cantilever may be (at least partly) 3D (laser) printed. Especially, in embodiments, one or more of the cantilever support and the cantilever may comprise a laser printed polymeric material.

[0049] In embodiments, at least part of the microfluidic probe may be provided using a 3D printing technique, such as one or more of the group comprising: two-photon polymerization (2PP), digital light processing (DLP), stereolithography (SLA), and fused deposition modelling (FDM). In embodiments, the cantilever may be provided using a 3D printing technique, such as one or more of the group comprising: two-photon polymerization (2PP), digital light processing (DLP), and stereolithography (SLA), such as especially two- photon polymerization. For example, in embodiments, one or more of the cantilever and the cantilever support may be provided using a 3D printing technique. Therefore, in embodiments, the microfluidic probe may comprise a plurality of second 3D printed layers. Especially, the plurality of second 3D printed layers may comprise, in embodiments, a second material selected from the group comprising cured photosensitive materials. The second 3D printed layers may, in embodiments, have thicknesses independently selected from the range of 0.05-15 pm, such as from the range of 0.1-10 pm. Suitable photosensitive materials may also comprise thiol- ene / yne photopolymerizable material. In embodiments, the photosensitive material may comprise material that is curable into acryl polymers or methacryl polymers. Further, the photosensitive material may comprise initiators. Yet further, in embodiments, the photosensitive material for the microfluidic probe may comprise additives, such as TiCh, or other (pigment) additives (such as UV absorbing (nano)particles or reflecting (nano)particles).

[0050] Herein, the term “cured photosensitive” material may refer to 3D printed material. The 3D printed material may in embodiments comprise cured acryl polymers and / or cured methacryl polymers. Hence, the second (3D printed) material may comprise cured acryl polymers and / or cured methacryl polymers. Hence, herein the term “3D printable material”, and similar terms, may refer to uncured material and the term “3D printed material”, and similar terms, may refer to the cured (3D printable) material. In embodiments, the former may e.g. comprise one or more of acrylatemethacrylate monomers and acrylate-methacrylate oligomers, and the latter may comprise acrylate-methacrylate polymers. In embodiments, the former may e.g. comprise thiol-ene / yne photopolymerizable material, and the latter may comprise acryl polymers and / or methacryl polymers.

[0051] The phrase “thicknesses independently selected from”, and similar phrases, may indicate that in embodiments different layers may have different thicknesses but may also indicate that in (other) embodiments two or more layers may have the same thicknesses. Instead of the terms “layer thickness” or “thickness”, also the term height may be applied.

[0052] The layer widths may in embodiments be in the order of the layer thicknesses or larger. Hence, the layer widths may be selected from the range of 75-500% of the respective layer thicknesses, such as selected from the range of 100-400% of the layer thickness (i.e. 1-4 times the thickness).

[0053] Experiments using the microfluidic probe as described herein may thus include AFM experiments making use of reflection of laser light from the probe. Further, the microfluidic probe may also be used in microfluidics and measuring mechanical characteristics such as cell adhesion, toughness, and viscoelasticity. Therefore, in embodiments, the microfluidic probe may comprise the protrusion as described above. Especially, in embodiments, the protrusion may have a shape approximating a cone shape. More especially, in embodiments, the protrusion may have a cone shape. Therefore, in embodiments, the protrusion may have a half angle (ap) relative to the protrusion axis (Ap). Especially, in embodiments, the half angle (ap) may be an acute angle. More especially, in embodiments, the half angle (ap) may be selected from the range of 20-60°. Hence, in embodiments, the protrusion may be (relatively) sharp with a relatively small contact surface with a substrate (during operation of the probe). Such embodiments may especially be beneficial for example in experiments where soft matter (such as e.g. a biological cell) may need to be punctured (e.g. to inject matter into the cell). However, in embodiments, the half angle (ap) may also be an obtuse angle. Hence, in embodiments, the protrusion may have a shape approximating a cone shape, wherein the protrusion may have a half angle (ap) relative to the protrusion axis (Ap), wherein the half angle (ap) may be an acute angle; wherein the half angle (ap) may be selected from the range of 20-60° . Hence, in embodiments, the protrusion may be (relatively) sharp with a larger (than the previously described protrusion) contact surface with a substrate (during operation of the probe). Such embodiments may especially be beneficial for example in experiments where soft matter (such as e.g. a biological cell) may need to be approached in a less disruptive yet highly accurate manner.

[0054] Hence, such embodiments may be beneficial as having a sharp tip on the microfluidic probe may enable use of the microfluidic probe to protrude or puncture a substrate, e.g. to measure toughness and / or surface tension or to subsequently inject the substrate with a fluid. Further, having a sharp tip on the microfluidic probe may reduce the contact area between the probe and the substrate, facilitating a higher resolution measurement. Hence, such embodiments may provide additional functionalities to the microfluidic probe.

[0055] In specific embodiments, the protrusion may have a shape approximating a (polygonal) pyramid shape. For example the microfluidic probe may be applied in experiments on a relatively soft substrate having a low surface tension. In such experiments, a pyramid shape of the protrusion may provide the benefit that undesirable slipping of the protrusion over the surface of the soft substrate may be reduced, due to increased friction imposed by the ribs of the pyramid shape (as compared to a cone shape). Furthermore, such embodiments may be beneficial as a pyramid shape may be rather easily fabricated using e.g. a (silicon) lithography process. Hence, in embodiments, where the protrusion has a shape approximating a pyramid shape, the pyramid may comprise a polygonal pyramid, i.e., the pyramid may have a crosssection (or base) comprising three of more sides, such as three sides, like four sides, especially five sides, more especially six sides, or even up to twelve sides. Especially, in specific embodiments, the protrusion may have a (polygonal) pyramid shape. Therefore, in embodiments, the half angle (ap) relative to the protrusion axis (Ap) may also be referred to as an apex angle (ap). Especially, in embodiments, the apex angle (ap) may be an acute angle. More especially, in embodiments, the apex angle (ap) may be selected from the range of 20- 60°. However, in embodiments, the apex angle (ap) may also be an obtuse angle. Hence, in embodiments, the protrusion may have a shape approximating a pyramid shape, wherein the protrusion may have an apex angle (ap) relative to the protrusion axis (Ap), wherein the apex angle (ap) may be an acute angle.

[0056] The protrusion tip may thus, in embodiments, comprise a sharp tip. Further, in embodiments, the protrusion may have a shape approximating one of: a cone shape, a pyramid shape, a polygonal shape, and a needle shape. Especially, the protrusion may have a half angle (ap) of at least 5°, such as at least 10°, especially at least 15°, like at least 20°, especially at least 25°, including at least 30°. In specific embodiments, the protrusion may have a half angle (ap) of (about) 45°. Further, in embodiments, the protrusion may have a half angle (ap) of at most 80°, such as at most 70°, like at most 65°, especially at most 60°, more especially at most 55°. In embodiments, the protrusion may have an asymmetric shape, such as e.g. a slanted cone-shape. In other embodiments, the protrusion may have more than one half angle (ap), such as e.g. when the protrusion has a pyramid-like shape. Hence, in embodiments, the protrusion may have one or more half angles (ap) selected from the range of 10-80°, such as from the range of 20-70°, like from the range of 35-55°, especially from the range of 40-50°.

[0057] Further, in embodiments, it may be beneficial, especially for mechanical measurements, to have a rounded or blunt protrusion tip. Therefore, in embodiments, the protrusion may also have a rounded tip. For example, in embodiments, the protrusion may have a shape approximating one of: a dome, a rounded cone, a rounded pyramid, a rounded (or curved) polygon, a rounded (or curved) parallelepiped, and a rounded (or curved) cylinder. Yet in other embodiments, the protrusion may have a blunt protrusion tip. Therefore, in embodiments, the protrusion may have a shape approximating one of: a flat-topped cone, a flat- topped pyramid, a flat-topped polygon, a flat-topped parallelepiped, and a flat-topped cylinder. Such embodiments may be beneficial as the rounded or blunt protrusion tip (rather than a sharp protrusion tip) may be less disruptive to soft-tissues during mechanical measurement experiments. For example, when measuring adhesion strength of e.g. a biological cell to a substrate, the use of a probe comprising a rounded or blunt protrusion may prevent damage to the biological cell (compared to a sharp tip). On the other hand, a probe comprising a sharp protrusion may be desired for example in experiments where the biological cell may need to be punctured (e.g. to inject matter into the cell).

[0058] As indicated above, in embodiments, the cantilever may comprise a cantilever arm. The cantilever (especially the cantilever head), may further comprise a visual indicator. The visual indicator may, in embodiments, be configured to indicate a location of one or more of the protrusion and of the aperture relative to the cantilever arm length axis (LCA). The visual indicator may especially, in embodiments, be configured on a second side of the cantilever (especially of the cantilever head) different from the first side. Hence, in embodiments, the cantilever may comprise (a) the cantilever arm and (b) a visual indicator; wherein the cantilever arm may comprise the cantilever arm length axis (LCA); wherein the visual indicator may be configured to indicate a location of one or more of the protrusion and the aperture relative to the cantilever arm length axis (LCA), wherein the visual indicator may be configured on a second side f the cantilever (especially of the cantilever head) different from the first side.

[0059] Such embodiments may be beneficial as during operation of the microfluidic probe it may be difficult to pinpoint whether the cantilever aperture and the protrusion are positioned correctly, as these may be concealed from vision by the bulk of the cantilever head. Implementing visual indicators on another side of the cantilever (head) may help the user to more quickly bring the cantilever in the desired position, therewith helping improve measurement efficiency.

[0060] The cantilever, especially the cantilever head, may thus, in embodiments, comprise a visual indicator. Especially, in embodiments, the cantilever head may comprise a visual indicator configured to indicate a location of the protrusion. Similarly, in embodiments, the cantilever head may also comprise a visual indicator configured to indicate a location of the cantilever aperture. Hence, in embodiments, the cantilever head may comprise one or more visual indicators. In embodiments, the cantilever head may comprise one or more indicators configured to indicate a location of one or more of the protrusion and the aperture relative to the cantilever length axis (LCA). The visual indicator may especially, in embodiments, be configured on a second side of the cantilever (head) different from the first side. For example, in embodiments, the visual indicator may comprise a (relatively small) shape, such as e.g. a triangular shape or a semicircular shape, extending from a side of the cantilever head (or extending from the cantilever face part) in a direction perpendicular to the cantilever length axis (LCA). In other embodiments, the visual indicator may comprise a (relatively small) shape, such as e.g. a triangular shape or a semicircular shape, indented into a side of the cantilever head (or indented into the cantilever face part) in a direction perpendicular to the cantilever length axis (LCA). In yet other embodiments, the visual indicator may comprise a marking provided on a side of the cantilever head (different from the first side, i.e., different from the cantilever face part). For example, in such embodiments, the marking may comprise a different color and / or material relative to the rest of the cantilever head. The skilled person will understand that different visual indicators than the examples described here may be possible as well. In specific embodiments, the cantilever head may comprise two triangular shapes extending from opposite sides of the cantilever head in a direction perpendicular to the cantilever length axis (LCA) and positioned such that an imaginary line from one of the triangles to the other one of the triangles may intersect with the protrusion (and / or the protrusion axis (Ap)). Similarly, in specific embodiments, the cantilever head may comprise two semicircular shapes sticking out from opposite sides of the cantilever head in a direction perpendicular to the cantilever length axis (LCA) and positioned such that an imaginary line from one of the semicircles to the other one of the semicircles may intersect with the aperture (and / or the aperture axis (AA)).

[0061] In a further specific aspect, the invention provides a method for 3D printing at least part of a device. In embodiments, the device may comprise (a) a fluidic interface element and (b) the microfluidic probe as described above. The fluidic interface element may, in embodiments, comprise an interface element channel. Further, in embodiments, the fluidic interface element may comprise a hole configured in fluidic contact with the interface element channel. The method may, in embodiments, especially comprise a resin providing stage, a microfluidic probe 3D printing stage, a resin removal stage, and a sealing stage. The resin providing stage may, in embodiments, comprise providing a photosensitive resin configured to cure when exposed to first radiation. The microfluidic probe 3D printing stage may, in embodiments, comprise 3D printing a 3D printable polymeric material, using the photosensitive resin, to provide the microfluidic probe. The resin removal stage may, in embodiments, comprise removing uncured photosensitive resin from the fluidic interface element and the microfluidic probe by flushing the interface element channel of the fluidic interface element with a washing fluid. Especially, in embodiments, the interface element channel may be configured in fluidic contact with the (flushed) cantilever channel. Further, in embodiments, the sealing stage may comprise sealing the hole in the washed fluidic interface element. Hence, in specific embodiments, the invention may provide a method for 3D printing at least part of a device, wherein the device may comprise (a) a fluidic interface element and (b) the microfluidic probe; wherein the fluidic interface element may comprise an interface element channel, wherein the fluidic interface element may comprise a hole configured in fluidic contact with the interface element channel; wherein the method may comprise: (A) a resin providing stage comprising providing a photosensitive resin configured to cure when exposed to first radiation; (B) a microfluidic probe 3D printing stage comprising 3D printing a 3D printable polymeric material, using the photosensitive resin, to provide the microfluidic probe; (C) a resin removal stage comprising removing uncured photosensitive resin from the fluidic interface element and the microfluidic probe by flushing the interface element channel of the fluidic interface element with a washing fluid; wherein the interface element channel may be configured in fluidic contact with the cantilever channel; and (D) a sealing stage comprising sealing the hole in the washed fluidic interface element. In this way, also the support channel and the cantilever channel can be made essentially free from uncured photosensitive resin.

[0062] In a yet further aspect, the invention provides a method for 3D printing a device comprising a fluidic interface element and the microfluidic probe as described above. The method may especially, in embodiments, comprise a resin providing stage comprising providing a photosensitive resin. The photosensitive resin may especially be configured to cure when exposed to first radiation. Further, in embodiments, the method may comprise a fluidic interface element 3D printing stage comprising exposing part of the photosensitive resin to first radiation to provide the fluidic interface element. In embodiments, the fluidic interface element may especially comprise an interface element channel, i.e., the fluidic interface element may be hollow. Further, in embodiments, the fluidic interface element may comprise a first face and a hole. In embodiments, the hole may be configured in the first face of the fluidic interface element. Especially, the hole may, in embodiments, be configured in fluidic contact with the interface element channel. The method may further, in embodiments, comprise a resin removal stage comprising removing uncured photosensitive resin from the fluidic interface element. Especially, in embodiments, the uncured photosensitive resin may be removed from the fluidic interface element by flushing the (hollow) fluidic interface element with a washing fluid. The method may further, in embodiments, comprise a sealing stage comprising sealing the hole in the (hollow) washed fluidic interface element. Further, in embodiments, the method may comprise a microfluidic probe 3D printing stage comprising 3D printing a 3D printable polymeric material to provide the microfluidic probe. In specific embodiments, the microfluidic probe may be 3D printed onto the first face of the fluidic interface element, however, this may not necessarily be the case. Hence, in embodiments, the invention may provide a method for 3D printing a device comprising a fluidic interface element and the microfluidic probe as described herein; wherein the method may comprise: (A) a resin providing stage comprising providing a photosensitive resin configured to cure when exposed to first radiation; (B) a fluidic interface element 3D printing stage comprising exposing part of the photosensitive resin to first radiation to provide the fluidic interface element, wherein the fluidic interface element may comprise an interface element channel, and wherein the fluidic interface element may comprise a first face and a hole (configured in fluidic contact with the interface element channel); (C) a resin removal stage comprising removing uncured photosensitive resin from the fluidic interface element by flushing the (hollow) fluidic interface element with a washing fluid; (D) a sealing stage comprising sealing the hole in the (hollow) washed fluidic interface element; and (E) a microfluidic probe 3D printing stage comprising 3D printing a 3D printable polymeric material to provide the microfluidic probe.

[0063] Such embodiments may be beneficial as providing a vent hole in the fluidic interface element during fabrication and sealing the hole after 3D printing is completed may reduce clogging in the microfluidic interface element, especially in the cantilever. Especially, the vent hole may allow for development liquid, i.e., in embodiments 3D printable material (of the printing procedure) to pass through the interface element channel to the cantilever without clogging (or building up) in the interface element channel and / or the cantilever. By sealing the hole at a later stage, a device may be provided wherein the only flow path for fluid from outside of the device will be through the aperture in the cantilever and a fluid inlet / outlet (see also further below) configured in the fluidic interface element and fluidically connected with the cantilever aperture.

[0064] As described above, the invention may provide a method for 3D printing a device. Especially, in embodiments, the invention may comprise a method for 3D printing a device comprising a fluidic interface element and the microfluidic prove as described above.

[0065] The method may comprise multiple stages, especially at least a resin providing stage, a microfluidic probe 3D printing stage, a resin removal stage, and a sealing stage. In embodiments, the resin providing stage may comprise providing a photosensitive resin. Especially, the photosensitive resin may be configured to cure (especially as a result of crosslinking) when exposed to first radiation. The photosensitive resin may especially, in embodiments, comprise a photosensitive polymeric material. For example, in embodiments, the photosensitive resin may comprise one or more of the group comprising: epoxides, urethanes, polyethers, and polyesters. Especially, in embodiments, the photosensitive resin may comprise one or more of multi-functional acrylates and multi-functional methacrylates. Note that, during the resin providing stage, the photosensitive resin that is provided is thus not yet cured. Curing of the photosensitive resin may occur during the microfluidic probe 3D printing stage, see also further below. Therefore, in embodiments, the photosensitive resin may herein also be referred to as photosensitive resin prior to curing, or photosensitive resin precursor. Hence, in such embodiments, the invention may provide a method for 3D printing at least part of a device, wherein the device may comprise (a) a fluidic interface element and (b) the microfluidic probe; wherein the fluidic interface element may comprise an interface element channel, wherein the fluidic interface element may comprise a hole configured in fluidic contact with the interface element channel; wherein the method may comprise: (A) a resin providing stage comprising providing a photosensitive resin (precursor) configured to cure when exposed to first radiation; (B) a microfluidic probe 3D printing stage comprising 3D printing a 3D printable polymeric material, using the photosensitive resin (precursor), to provide the microfluidic probe; (C) a resin removal stage comprising removing uncured photosensitive resin (precursor) from the fluidic interface element and the microfluidic probe by flushing the interface element channel of the fluidic interface element and the cantilever channel (with a washing fluid; wherein the interface element channel may be configured in fluidic contact with the cantilever channel; and (D) a sealing stage comprising sealing the hole in the washed fluidic interface element.

[0066] In embodiments, the microfluidic probe 3D printing stage may subsequent the resin providing stage. The microfluidic probe 3D printing stage may especially, in embodiments, comprise exposing part of the photosensitive resin to first radiation. In embodiments, the first radiation may especially comprise radiation having a wavelength selected from one of the visible and the UV wavelength range. Especially, in embodiments, the first radiation may comprise radiation having a wavelength selected from the range of 100-800 nm, such as from the range of 200-700 nm, like from the range of 300-450 nm, especially from the range of 350-400 nm. The first radiation may especially, in embodiments, be selected in dependence of the used photosensitive resin, such that the first radiation may induce crosslinking of the photosensitive resin. In embodiments, the first radiation may especially be provided using a targeted relatively narrowly focused beam of radiation, such that the photosensitive resin may be cured to provide the (shape of the) microfluidic probe. Hence, in embodiments, the microfluidic probe may be provided by curing the photosensitive resin through cross-linking induced by the first radiation.

[0067] In embodiments, the resin removal stage may subsequent the microfluidic probe3D printing stage. The resin removal stage may especially, in embodiments, comprise removing uncured photosensitive resin from the fluidic interface element and the microfluidic probe. Such removal may, in embodiments, be achieved through flushing of the (hollow) fluidic interface element and the (hollow) microfluidic probe with a washing fluid. Hence, in embodiments, a washing fluid may be forced through the fluidic interface element and the microfluidic probe, such that the washing fluid along with the uncured photosensitive resin may exit the fluidic interface element and the microfluidic probe. In embodiments, the washing fluid may comprise a gas or a liquid. Especially, in embodiments, the washing fluid may comprise a gas mixture. In other embodiments, the washing fluid may comprise a liquid mixture. In yet other embodiments, the resin removal stage may comprise flushing the fluidic interface element with multiple washing fluids, wherein the multiple washing fluids may comprise one or more of: gases, gaseous mixtures, liquids, and liquid mixtures. For example, in embodiments, the washing fluid may comprise one or more of the group comprising isopropyl alcohol, tripropylene glycol monomethyl ether, dipropylene glycol monomethyl ether, acetone, and water. For example, in embodiments, the resin removal stage may comprise flushing the fluidic interface element with compressed air and isopropyl alcohol.

[0068] In specific embodiments, the sealing stage may be performed (last, i.e., may be performed) subsequent to all of the resin providing stage, the fluidic interface 3D printing stage (see also further below), the resin removal stage, and the microfluidic probe 3D printing stage.

[0069] In embodiments, the sealing stage may comprise sealing the (vent) hole in the (hollow) washed fluidic interface element. Especially, in embodiments, the sealing stage may comprise providing a seal over the hole, such that a fluid may no longer pass from the fluid inlet through the interface element channel to the (vent) hole and vice versa. Hence, in embodiments, the sealing stage may comprise providing a seal over the hole, wherein the seal may be configured to block fluids from passing through the hole. In embodiments, the seal may be provided through 3D printing of a 3D printable material (e.g. photocurable material) inside or over the hole (and curing the photocurable material). In other embodiments, the hole may be sealed by providing (e.g. gluing) a layer or element over the hole. Hence, the seal may prevent that liquid escapes via the (vent) hole.

[0070] The term “stage” and similar terms used herein may refer to a (time) period (also “phase”) of a method and / or an operational mode. The different stages may (partially) overlap (in time). However, generally, the stages may be executed sequentially. For instance, in embodiments, the resin providing stage may be finished prior to starting the fluidic interface 3D printing stage, which may be finished prior to starting the resin removal stage.

[0071] In embodiments, the fluidic interface element 3D printing stage may precede one or more of the resin providing stage, and the microfluidic probe 3D printing stage. In other embodiments, the fluidic interface element 3D printing stage may be performed simultaneously with one or more of the resin providing stage, and the microfluidic probe 3D printing stage. In yet other embodiments, the fluidic interface element 3D printing stage may subsequent one or more of the resin providing stage, and the microfluidic probe 3D printing stage.

[0072] In embodiments, the fluidic interface element may especially comprise an interface element channel. The fluidic interface element may thus, in embodiments, be a hollow element. In embodiments, dimensions of the fluidic interface element may especially be selected such that the fluidic interface element may fit in an AFM holder, i.e., the fluidic interface element may function as a connector for functionally coupling the microfluidic probe as described above to a device, like an AFM apparatus. Furthermore, in embodiments, the fluidic interface element may comprise a first face and a (vent) hole. In specific embodiments, the hole may be configured in the first face of the fluidic interface element, however, this may not necessarily be the case. The hole may, however, in embodiments, be configured in fluidic contact with the interface element channel. Yet further, in embodiments, the fluidic interface element may comprise a fluid inlet (especially a first opening, see also further below). The fluid inlet may be configured in fluidic contact with the interface element channel and optionally the hole. Hence, in such embodiments, a fluid may pass from the fluid inlet through the interface element channel to the (vent) hole and vice versa.

[0073] The microfluidic probe 3D printing stage may especially, in embodiments, comprise 3D printing a 3D printable polymeric material to provide the microfluidic probe. Especially, in embodiments, the microfluidic probe 3D printing stage may comprise 3D printing a 3D printable polymeric material onto the first face of the fluidic interface element to provide the microfluidic probe (onto the fluidic interface element). Hence, in such embodiments, the microfluidic probe 3D printing stage may at least subsequent the fluidic interface element 3D printing stage. Furthermore, in such embodiments, the microfluidic probe may thus be 3D printed directly onto the fluidic interface element. In other embodiments, the microfluidic probe may be 3D printed separately from the fluidic interface element and at a later stage may be functionally coupled (such as e.g. glued) to the fluidic interface element.

[0074] For the microfluidic probe printing stage, especially two-photon polymerization may be used, which allows a relatively high precision. Two-photon polymerization as a direct laser writing technique allows for creating complex three-dimensional structures down to feature sizes in the order of about 100 nm, or even lower. Key elements of two-photon polymerization are lasers providing femtosecond pulses, suitable photosensitive materials (photoresists), a precise positioning stage and a computer to control the procedure. Two-photon polymerization is a non-linear optical process based on the simultaneous absorption of two photons in a photosensitive material (photoresist). This process changes the photosensitive material, i.e., it leads to a polymerization, especially by activating so-called photo-initiators in the resist. These may turn into radicals that polymerize the resist locally. In a subsequent step, the non-polymerized photoresist may be washed out to uncover the structure. Two-photon absorption may require relatively high intensities. Hence, to this end lasers may be applied, with relatively focused beams, to obtain the high resolution. As two-photon absorption is proportional to the square of the intensity, it only takes place in the focus of the laser beam, providing high spatial resolution. Hence, in specific embodiments, the microfluidic probe printing stage may comprise two-photon-polymerization. However, in embodiments, other 3D printing techniques may also be applied, such as e.g. one or more of stereolithography (SLA), digital light processing (DLP), or fused deposition modelling (FDM).

[0075] Further, as indicated above, the interface element may also be 3D printed. The 3D printed layers thereof may be thicker than that of the cantilever, as a high resolution may be less necessary for the interface element, than for the cantilever. Layer thicknesses (or heights) may e.g. be in the order of 15-500 pm, such as selected from the range of 20-250 pm, like in embodiments selected from the range of about 20-200 pm, such as selected from the range of about 25-100 pm. In embodiments, the interface element may comprise a plurality of first 3D printed layers, wherein the first 3D printed layers comprise a first material selected from the group comprising cured photosensitive materials, and wherein the first 3D-printed layers have thicknesses independently selected from the range of 15-500 pm. Suitable photosensitive materials may comprise thiol-ene / yne photopolymerizable material. In embodiments, the photosensitive material may comprise material that is curable into acryl polymers or methacryl polymers. Further, the photosensitive material may comprise initiators, and optionally also additives, like TiCh. TiCh may increase the strength, and may e.g. increase the Young’s modulus of the interface element. Hence, the first material may comprise a first additive selected from the group comprising TiCh and other UV absorbing (nano)particles or reflecting (nano)particles. In embodiments, the interface element may (also) be provided by one or more of stereolithography (SLA), digital light processing (DLP), or fused deposition modelling (FDM). Hence, in embodiments, the fluidic interface element 3D printing stage may comprise one or more of stereolithography and digital light processing. Note that the fluidic interface element could also be made out of glass-based materials or silicon-based materials fabricated by printing or cleanroom fabrication methods.

[0076] As described above, in embodiments, the method may comprise providing a device comprising a fluidic interface element comprising an interface element channel. Furthermore, the device may comprise the microfluidic probe as described above, i.e., comprising the cantilever channel and the support channel. Therefore, in embodiments, the method may comprise 3D printing the device comprising a microfluidic channel structure. Especially, in embodiments, the microfluidic channel structure may comprise one or more of the cantilever channel, the support channel, and the interface element channel configured in fluidic contact with each other. Hence, in embodiments, the device may comprise a microfluidic channel structure, wherein the microfluidic channel structure may comprise the cantilever channel, the support channel, and the interface element channel configured in fluidic contact with each other.

[0077] Further, in embodiments, the microfluidic channel structure (of the device) may comprise a first opening and a second opening. The first opening may be configured in the fluidic interface element. Especially, in embodiments, the first opening may comprise a fluid inlet and / or fluid outlet. More especially, in embodiments, the first opening may be configured to fluidically couple the microfluidic channel structure to a fluid control system. Further, in embodiments, the cantilever aperture may comprise the second opening. Hence, in embodiments, the microfluidic channel structure (of the device) may comprise a first opening and a second opening, wherein the first opening may be configured in the fluidic interface element, and wherein the first opening may be configured to fluidically couple the microfluidic channel structure to a fluid control system, and wherein the cantilever aperture (of the microfluidic probe) may comprise the second opening.

[0078] Such embodiments may be beneficial as the microfluidic channel structure may provide a fluid path from a fluid control system to the cantilever aperture, which may allow for microfluidic modulation of a substrate in a controlled manner. Especially, the fluid control system may allow a user to easily switch between providing (e.g. pumping) a fluid from the fluid control system to the cantilever aperture (e.g. to inject the fluid into a substrate), and providing suction at the cantilever aperture via the microfluidic channel structure. In embodiments, the fluid control system may be essentially comprised by the device. However, in other embodiments, the fluid control system may be external of the device.

[0079] In embodiments, the microfluidic channel structure may thus be fluidically coupled to a fluid control system. The fluid control system may especially, in embodiments, be configured to control an operational mode of the device, i.e., the fluid control system may be configured to provide a fluid from the fluid control system to a substrate through the microfluidic channel structure or to provide suction at the cantilever aperture via the microfluidic channel structure. Furthermore, in embodiments, the fluid control system may be configured to control one or more features of the fluid, such as e.g. flow rate, temperature, pressure, and fluid composition.

[0080] In embodiments, the device may be applied for microfluidic applications. In such embodiments, e.g. the fluidic interface element may comprise a fluid inlet, and the cantilever may comprise, especially at the cantilever head, an outlet (i.e. the cantilever aperture). The cantilever aperture (or outlet) may be in fluid communication with the fluid inlet at the interface element via the microfluidic channel structure. In further embodiments, the device, especially the microfluidic probe, may be configured for application in experiments (e.g. atomic force microscopy) to determine substrate characteristics such as e.g. the elastic modulus, adhesion strength, and viscoelasticity of said substrate (e.g. a cell). Hence, in specific embodiments, the invention may (essentially) comprise a method for 3D printing a device, wherein the device may comprise a multi-functional microfluidic device.

[0081] In a further aspect, the invention provides a multi-functional microfluidic device comprising a fluidic interface element, the microfluidic probe as described herein, a support, and an actuator. In embodiments, the fluidic interface element may comprise an interface element channel. Further, in embodiments, the fluidic interface element may be configured to support the microfluidic probe. Especially, in embodiments, the interface element channel may be configured in fluidic contact with the cantilever channel. Further, in embodiments, the actuator may be configured to control a relative position of the microfluidic probe and the support. Hence, in embodiments, the invention may provide a multi-functional microfluidic device comprising (a) a fluidic interface element, (b) the microfluidic probe, (c) a support, and (d) an actuator; wherein the fluidic interface element may comprise an interface element channel and may be configured to support the microfluidic probe, wherein the interface element channel may be configured in fluidic contact with the cantilever channel; wherein the actuator may be configured to control a relative position of the micro fluidic probe and the support.

[0082] Such embodiments may be beneficial as the device may be applied in a plurality of different experimental measurement, without the need to switch the microfluidic probe between the different experiments. Not having to switch the microfluidic probe between experiment reduces the time in between experiments, i.e., increases experimental throughput. Additionally, reducing the loss of time in between experiments may lead to improved experimental results with improved sensitivity in time-sensitive measurements. The present device may e.g. be used for one or more of dip pen nanolithography, nanoscale dispensing, as nano fountain probe, scanning ion pipette, dispensing probe, scanning ion conductance microscope, as bio plume, or as aspirating micro / nano pipette. Furthermore, by providing the first side of the cantilever level relative to the substrate to be measured, modulation of the substrate with improved control and sensitivity may be provided.

[0083] In embodiments, the multi-functional microfluidic device may comprise the fluidic interface element. The fluidic interface element may be used to functionally couple the microfluidic probe as described above to a device, like an AFM apparatus. The fluidic interface element may also be a microfluidic chip. In embodiments, the interface element may be used to mount on an atomic force microscopy (AFM) holder or may be used for connection between the 3D printed microfluidic probe (especially comprising an AFM cantilever) and another device.

[0084] Further, in embodiments, the fluidic interface element may especially comprise an interface element channel. The fluidic interface element may thus, in embodiments, be a hollow element. Furthermore, in embodiments, the fluidic interface element may be configured to support the microfluidic probe. Especially, in embodiments, the microfluidic probe may be configured onto the fluidic interface element. More especially, the microfluidic probe may be configured onto the fluidic interface element such that microfluidic probe and the fluidic interface element may be fluidically coupled. Especially, in embodiments, the interface element channel may be configured in fluidic contact with the cantilever channel. Hence, in such embodiments, the first opening (i.e. the fluid inlet), the interface element channel, the support channel (of the cantilever support), the cantilever channel, and the cantilever aperture may be configured in fluidic contact with each other.

[0085] Yet further, in embodiments, the support may be configured to support a sample (or substrate). Hence, in embodiments, the sample to be measured (e.g. in AFM experiments) may be configured on the support. During operation of the multi-functional microfluidic device the support and the microfluidic probe may, in embodiments, be configured to move relative to each other. Especially, in embodiments, the actuator may be configured to move the support relative to the (stationary) microfluidic probe. In other embodiments, the actuator may be configured to move the microfluidic probe relative to the (stationary) support. In yet other embodiments, the actuator may be configured to move both the cantil microfluidic probe ever and the support relative to each other.

[0086] Further, the fluidic interface element may especially, in embodiments, be configured to, during an operational mode of the multi-functional microfluidic device, position the microfluidic probe such that the first side of the cantilever is level relative to the support. In other words, the fluidic interface element may especially, in embodiments, be configured to, during an operational mode of the multi-functional microfluidic device, position the microfluidic probe such that the first side of the cantilever is parallel to the support. Hence, in embodiments, the microfluidic probe of the multi-functional microfluidic device may approach the support such that the first side of the probe may be parallel to the substrate.

[0087] Further, in embodiments, the multi-functional microfluidic device may comprise the microfluidic channel structure such as described in more detail above. Hence, in embodiments, the multi-functional microfluidic device may comprise a microfluidic channel structure, wherein the microfluidic channel structure may comprise the cantilever channel, the support channel, and the interface element channel configured in fluidic contact with each other.

[0088] Yet further, in embodiments, the microfluidic channel structure (of the multifunctional microfluidic device) may comprise a first opening and a second opening. The first opening may be configured in the fluidic interface element. Especially, in embodiments, the first opening may comprise a fluid inlet and / or fluid outlet. More especially, in embodiments, the first opening may be configured to fluidically couple the microfluidic channel structure to a fluid control system. Further, in embodiments, the cantilever aperture may comprise the second opening. Hence, in embodiments, the microfluidic channel structure (of the multifunctional microfluidic device) may comprise a first opening and a second opening, wherein the first opening may be configured in the fluidic interface element, and wherein the first opening may be configured to fluidically couple the microfluidic channel structure to a fluid control system, and wherein the cantilever aperture (of the microfluidic probe) may comprise the second opening.

[0089] One of the possible functions of the multi-functional microfluidic device may be for atomic force microscopy imaging applications. Therefore, in embodiments, the multifunctional microfluidic device may comprise a laser configured to provide second radiation to the cantilever (especially to the cantilever head). Further, in such embodiments, the multifunctional microfluidic device may comprise a detector configured to (i) collect second radiation reflected by the cantilever (especially the cantilever head) and (ii) provide an (electrical) signal with an intensity proportional to a displacement of the cantilever. Hence, in embodiments, the multi-functional microfluidic device may comprise a laser configured to provide second radiation to the cantilever, and a detector configured to (i) collect second radiation reflected by the cantilever and (ii) provide a signal with an intensity proportional to a displacement of the cantilever.

[0090] Such embodiments may be beneficial as inclusion of a laser in the multifunctional microfluidic device may allow for its use in AFM cantilever-beam deflection measurements. AFM cantilever-beam deflection measurements using the herein described multi-functional microfluidic device may provide high sensitivity with a relatively low noise floor.

[0091] In embodiments, the multi-functional microfluidic device may comprise a laser diode, i.e., a solid-state diode, configured to provide second radiation to the cantilever (especially the cantilever head). The cantilever (especially the cantilever head) may be configured to reflect the second radiation back off the cantilever. The reflected second radiation may subsequently, in embodiments, be collected by a (position-sensitive) detector. Especially, in embodiments, the (position-sensitive) detector may comprise two closely spaced photodiodes, whose output signal may be collected by a differential amplifier. The differential amplifier may, in embodiments, provide an output signal which may be proportional to a deflection of the cantilever. Hence, in embodiments, the multi-functional microfluidic device may comprise a laser diode, and a position-sensitive detector.

[0092] Yet further, in embodiments, the multi-functional microfluidic device may comprise one or more piezoelectric devices. Especially, in embodiments, the actuator may comprise the one or more piezoelectric devices. In embodiments, the one or more piezoelectric devices may be configured to control a position of the microfluidic probe relative to the support. Especially, in embodiments, the one or more piezoelectric devices may be configured to control a distance between the protrusion tip and the support by a lateral movement of the microfluidic probe relative to the support. Additionally or alternatively, in embodiments, the one or more piezoelectric devices may be configured to control a position of the microfluidic probe relative to the support. Especially, in embodiments, the one or more piezoelectric devices may be configured to control a distance between the protrusion tip and the support by a rotational movement of the microfluidic probe relative to the support. Hence, in embodiments, the multifunctional microfluidic device may comprise one or more piezoelectric devices configured to control a position of the microfluidic probe relative to the support, wherein one of more of the following may apply: (a) the one or more piezoelectric devices may be configured to control a distance between the protrusion tip and the support by a lateral movement of the microfluidic probe relative to the substrate, and (b) the one or more piezoelectric devices may be configured to control a distance between the protrusion tip and the support by a rotational movement of the microfluidic probe relative to the substrate.

[0093] Such embodiments may be beneficial as piezoelectric elements may facilitate small but accurate and precise movement on command, which may enable precise scanning when using the multi-functional microfluidic device.

[0094] In embodiments, the piezoelectric element may especially comprise an element comprising a piezoelectric material, such as a material selected from the group comprising a ceramic material, a crystal (e.g. lead zirconate titanate), and a biological material (e.g. bone, DNA or a protein). The one or more piezoelectric elements may especially, in embodiments, be configured to oscillate such that, during an operational mode of the multi-functional microfluidic device, a lateral movement of the microfluidic probe (especially the cantilever) relative to the support is achieved. In embodiments, the one or more piezoelectric elements may be configured to, during an operational mode of the multi-functional microfluidic device, move the microfluidic probe in a lateral fashion along the x, y, or z-direction relative to the support. Additionally or alternatively, in embodiments, the one or more piezoelectric elements may be configured to oscillate such that, during an operational mode of the multi-functional microfluidic device, a lateral movement of the support relative to the microfluidic probe (especially the cantilever) is achieved. In such embodiments, the one or more piezoelectric elements may be configured to, during an operational mode of the multi-functional microfluidic device, move the support in a lateral fashion along the x, y, or z-direction relative to the microfluidic probe.

[0095] Further, in embodiments, the one or more piezoelectric elements may be configured to, during an operational mode of the multi-functional microfluidic device, move the microfluidic probe in a rotational fashion relative to the support. Additionally or alternatively, in embodiments, the one or more piezoelectric elements may be configured to, during an operational mode of the multi-functional microfluidic device, move the support in a rotational fashion relative to the microfluidic probe.

[0096] In a yet further aspect, the invention relates to a method for operating the multifunctional microfluidic device as described herein. Especially, the method may, in embodiments, comprise providing a fluid (via one or more of the cantilever channel and the interface channel) through the second opening in the microfluidic probe. Further, in embodiments, the method may comprise providing suction at the second opening of the microfluidic probe. Yet further, in embodiments, the method may comprise applying pressure with the protrusion tip on an element supported by the support. Hence, in embodiments, the invention may provide a method for operating the multi-functional microfluidic device as described herein, wherein the method may comprise one or more of (i) providing a fluid through the second opening in the microfluidic probe; (ii) providing suction at the second opening of the microfluidic probe, (iii) applying pressure with the protrusion tip on an element supported by the support.

[0097] Such embodiments may be beneficial as a single microfluidic probe may be applied for a wide range of experimental applications without the need to switch between different probes, thereby saving time and increasing accuracy in time-sensitive measurements.

[0098] In embodiments, the method may especially comprise applying a modulation to the element supported by the support using the multi-functional microfluidic device. In some embodiments, the element may especially comprise a biological cell, though non-biological samples may also be possible. Hence, in embodiments, the method may comprise a method for operating the multi-functional microfluidic device to provide modulations to a biological cell, e.g. in single-cell force spectroscopy analysis. In embodiments, the modulation may comprise one or more of: picking up the cell; adhering the microfluidic probe to the cell (e.g. to measure adhesion strength); puncturing the cell with the protrusion tip; and injecting fluid into the cell. Hence, in embodiments, (he method may comprise applying a modulation to the element supported by the support using the multi-functional microfluidic device, wherein the element may comprise a cell, wherein the modulation may comprise one or more of: picking up the cell; adhering the microfluidic probe to the cell; puncturing the cell with the protrusion tip; and injecting fluid into the cell.

[0099] In a yet further aspect, the invention relates to a use of the multi-functional microfluidic device as described herein in one or more of Atomic Force Microscopy and Atomic Force Spectroscopy. Especially, in embodiments, the invention may relate to use of the multi-functional microfluidic device as described herein for ex vivo measuring physical and / or mechanical properties of a cell. Especially, in embodiments, the multi-functional microfluidic device may be used in one or more of force measurement, topographic imaging, and substrate manipulation.

[0100] BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

[0102] Fig. l schematically depicts an embodiment of the microfluidic probe 100. Fig.

[0103] 2 schematically depicts some features of the microfluidic probe 100. Fig. 3 schematically depicts a method of the invention. Fig. 4 schematically depicts some features of the multifunctional microfluidic device 1500. Fig. 5 schematically depicts a method of the invention. Fig. 6 schematically depicts some further specific features of the microfluidic probe 100. The schematic drawings are not necessarily on scale.

[0104] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0105] Fig. 1 schematically depicts a microfluidic probe 100. Especially, the microfluidic probe 100 may comprise a cantilever 150, and a cantilever support 130. The cantilever 150 may comprise a cantilever channel 165 and the cantilever support 130 may comprise a support channel 135. The cantilever channel 165 and the support channel 135 may, in embodiments, be configured in fluidic contact with each other. Further, the cantilever 150 may comprise a support end 151 and a cantilever end 152 remote from the cantilever support 130. Yet further, the cantilever 150 may comprise a protrusion 154 and a cantilever aperture 156. In embodiments, the protrusion 154 and the cantilever aperture 156 may both be configured on a first side 51 of the cantilever 150. Further, in embodiments, the protrusion 154 and the cantilever aperture 156 may be configured closer to the cantilever end 152 than to the support end 151. The protrusion 154 and the cantilever aperture 156 may especially, in embodiments, be spatially separated. Furthermore, in embodiments, the cantilever aperture 156 may be configured in fluidic contact with the cantilever channel 165. Yet further, the protrusion 154 may comprise a protrusion tip 155. Yet further, the protrusion 154 may define a protrusion axis Ap. The protrusion axis Ap may especially be defined as an imaginary line leading from the first side 51 through the protrusion 154 and intersecting with the protrusion tip 155. Similarly, in embodiments, the cantilever aperture may define an aperture axis AA, see also Fig. 3. The aperture axis AA may especially be defined as an imaginary line perpendicular to the first side 51 and leading through (the geometrical center of) the cantilever aperture 156. In embodiments, the protrusion axis Ap and the aperture axis AA may be essentially parallel, as depicted in Figs. 1, 3 and 5. However, in other embodiments, the protrusion axis Ap and the aperture axis AA may have a (slight) angle relative to each other, as depicted in Fig. 6.

[0106] Reference 170 may herein refer to a microfluidic channel structure, see also further below. Further, reference 172 may herein refer to a second opening, see also further below.

[0107] As depicted in Fig. 1, in embodiments, the protrusion 154 may be configured closer to the cantilever end 152 than the cantilever aperture 156 and thus the cantilever aperture 156 may be configured closer to the support end 151 than the protrusion 154. In other embodiments, not depicted, the cantilever aperture 156 may be configured closer to the cantilever end 152 than the protrusion 154 and thus the protrusion 154 may be configured closer to the support end 151 than the cantilever aperture 156.

[0108] Further, in embodiments, the cantilever aperture 156 may comprise an aperture edge 157. In embodiments, a first (shortest) distance dl between the protrusion tip 155 and the aperture edge 157 may be selected from the range of 1 pm < d2 <20 pm (see also Fig. 6).

[0109] Yet further, in embodiments, the microfluidic probe 100 may comprise a plurality of second 3D-printed layers 232. Especially, in embodiments, the second 3D-printed layers 232 may comprise a second material selected from the group comprising cured photosensitive materials. Further, in embodiments, the second 3D-printed layers 232 may have thicknesses independently selected from the range of 0.05-15 pm. As depicted in Fig. 1, in embodiments, the microfluidic probe 100 may comprise a cantilever head 158 and a cantilever arm 160 associated to each other. In embodiments, the support end 151 may comprise at least part of the cantilever arm 160. Further, in embodiments, the cantilever head 158 may comprise (one or more of) the protrusion 154, the cantilever aperture 156 and the cantilever end 152.

[0110] Fig. 2 schematically depicts some further embodiments of the microfluidic probe 100. Especially, in embodiments, the cantilever arm 160 may comprise a cross-sectional equivalent circular diameter DCA and a cantilever arm length axis LCA. The cantilever head 158 may, in embodiments, comprise a cantilever face part 159 comprising a cantilever head dimension DCH. The cantilever head dimension DCH may especially be configured perpendicular to the cantilever arm length axis LCA. Especially, in embodiments, the cantilever head dimension DCH may be equal to or larger than twice the cross-sectional equivalent circular diameter DCA, i.e., DCH>2*DCA.

[0111] The cantilever arm 160 may further, in embodiments, comprise a cantilever arm part 161 having a length parallel to the cantilever arm length axis LCA. Note that, in embodiments, a length of the cantilever 150, or even the cantilever arm 160 may thus be larger than the length of the cantilever arm part 161. Especially, in embodiments, the length of the cantilever arm 160 may comprise the sum of the length of the cantilever arm part 161 and a length of the cantilever head 158 (defined in a direction parallel to the cantilever arm length axis LCA). In embodiments, cross-sectional dimensions of the cantilever arm part 161 over its length may be constant. Further, inn embodiments, one or more cross-sectional dimensions of the cantilever head 158 may be larger than one or more cross-sectional dimensions of the cantilever arm part 161.

[0112] Reference 40 may herein refer to a radiation reflective material 40. Especially, in embodiments, the cantilever face part 159 may comprise a radiation reflective material 40. The radiation reflective material may, in embodiments, be reflective for radiation having a wavelength selected from the range of visible and infrared radiation, especially a wavelength selected from the range of 300-2000 nm.

[0113] Furthermore, in embodiments, the cantilever face part 159 may have a shape approximating a shape selected from the group comprising: a circle, a semi-circle (such as depicted in subfigure I), an oval, a trapezoid, a parabola, and a horse-shoe shape (such as depicted in subfigures II and III).

[0114] Yet further, in embodiments, the cantilever 150 (especially the cantilever head 158) may comprise (a) the cantilever arm 160 comprising the cantilever arm length axis LCA and (b) a visual indicator 15. In embodiments, the visual indicator 15 may be configured to indicate a location of one or more of the protrusion 154 and the aperture 156 relative to the cantilever arm length axis LCA. AS depicted in Fig. 2 subfigures II and III, the visual indicator 15 may comprise one or more visual indicators 15 (e.g. 4 visual indicators 15). Here especially, in embodiments, the visual indicator 15 may be configured on a second side 52 of the cantilever 150 (especially of the cantilever head 158) different from the first side 51. Subfigure II especially depicts visual indicators 15 extending from the cantilever head 158, whereas subfigure III especially depicts visual indicators 15 indented into the cantilever head 158.

[0115] In embodiments, one or more of the cantilever support 130 and the cantilever 150 may comprise a laser printed polymeric material.

[0116] Further, in embodiments, the cantilever aperture 156 may comprise an aperture diameter DAP selected from the range of 2-60 pm. In embodiments, the cantilever head 158 may be configured to taper from the cantilever arm 160 along the direction of elongation of the cantilever 150 to the cantilever end 152. In such embodiments, in a cross-sectional plane of the cantilever head 158 (defined perpendicular to the direction of elongation of the cantilever 150 as depicted in Figs. 1 and 6), the cantilever head 158 may thus have a slanted side 58. In embodiments, the slanted side 58 may comprise the cantilever aperture 156. The slanted side 58 may thus, in embodiments, have a slanted side length Ls selected such that the slanted side 58 may accommodate at least the cantilever aperture 156. Therefore, in embodiments, the slanted side length Ls may be at least two times the aperture diameter DAP.

[0117] Fig. 3 schematically depicts some embodiments of the microfluidic probe 100 as well as some applications of the microfluidic probe 100.

[0118] Especially, in embodiments, the protrusion 154 may define a protrusion axis Ap. More especially, the protrusion axis Ap may be defined perpendicular to the first side 51 and intersecting the protrusion tip 155. The protrusion tip 155 may, in embodiments, extend from the first side 51 by a second distance d2. Especially, in embodiments, 0.5 pm < d2 < 20 pm.

[0119] As depicted in Fig. 3, in embodiments, the protrusion axis Ap and the aperture axis AA may be essentially parallel. Furthermore, in embodiments, the aperture axis AA and the cantilever arm length axis LCA may have a first mutual angle p. In some embodiments (not depicted), the first mutual angle P may be about 90°, i.e., the aperture axis AA and the cantilever arm length axis LCA may be orthogonal relative to each other. Alternatively, in embodiments where the cantilever head 158 may have a slanted side 58 comprising the cantilever aperture 156 (such as e.g. depicted in Figs. 1,3 and 6), the first mutual angle P may be selected from the range of 30-80°. Conversely, in embodiments, the protrusion axis Ap and the cantilever arm length axis LCA may have a second mutual angle y. In some embodiments (such as e.g. depicted in Fig. 6), the second mutual angle y may be about 90°, i.e., the protrusion axis Ap and the cantilever arm length axis LCA may be orthogonal relative to each other. Alternatively, in embodiments (such as e.g. depicted in Figs. 1 and 3), the second mutual angle y may be selected from the range of 75-90°.

[0120] Reference ap may herein refer to a half angle, see also further below.

[0121] Fig. 4 schematically depicts some embodiments of a device 1000.

[0122] Especially, in a further aspect, the invention provides a method for 3D printing at least part of a device 1000. In embodiments, the device 1000 may comprise (a) a fluidic interface element 200 and (b) the microfluidic probe 100 as described above. In embodiments, the fluidic interface element 200 may comprise an interface element channel 205. Further, in embodiments, the fluidic interface element 200 may comprise a first face 210, and a (vent) hole 220 configured in fluidic contact with the interface element channel 205. In some embodiments, the (vent) hole 220 may especially be configured on the first face 210 of the fluidic interface element 200, such as depicted in Fig. 4 subfigure II.

[0123] In embodiments, the method may especially, comprise a resin providing stage comprising providing a photosensitive resin configured to cure when exposed to first radiation. Further, in embodiments, the method may comprise a microfluidic probe 3D printing stage comprising exposing part of the photosensitive resin to first radiation to provide the microfluidic probe 100. Yet further, in embodiments, the method may comprise a resin removal stage comprising removing uncured photosensitive resin from the fluidic interface element 200 and the microfluidic probe 100 by flushing the interface element channel 205 of the (hollow) fluidic interface element 200 (and the cantilever channel 165) with a washing fluid. In embodiments, the interface element channel 205 may especially be configured in fluidic contact with the (flushed) cantilever channel 165. Yet further, in embodiments, the method may comprise a sealing stage comprising sealing the hole 220 in the (hollow) washed fluidic interface element 200.

[0124] Yet further, in embodiments, the method may comprise a fluidic interface element 3D printing stage comprising exposing part of the photosensitive resin to first radiation to provide the fluidic interface element 200. In embodiments, the fluidic interface element 3D printing stage may comprise one or more of stereolithography and digital light processing. In specific embodiments, the microfluidic probe 3D printing stage may comprise two-photon- polymerization. In specific embodiments, the device 1000 may comprise a multi-functional microfluidic device 1500.

[0125] Hence, in a further aspect, as depicted in Fig. 4, the invention provides a multifunctional microfluidic device 1500 comprising (a) the fluidic interface element 200, (b) the microfluidic probe 100 as described herein, (c) a support 50, and (d) an actuator 360. Fig. 4 subfigure I schematically depicts a cross-sectional view of the device 1000, especially the multi-functional microfluidic device 1500. In embodiments, the fluidic interface element 200 may comprise an interface element channel 205. Further, in embodiments, the fluidic interface element 200 may be configured to support the microfluidic probe 100. Especially, in embodiments, the interface element channel 205 may be configured in fluidic contact with the cantilever channel 165. Further, in embodiments, the actuator 360 may be configured to control a relative position of the microfluidic probe 100 and the support 200. The fluidic interface element 200 may especially, in embodiments, be configured to, during an operational mode of the multi-functional microfluidic device 1500, position the microfluidic probe 100 such that the first side 51 of the cantilever 150 is level (i.e. parallel) relative to the support 50.

[0126] Further, in embodiments, the fluidic interface element 200 may comprise a plurality of first 3D-printed layers 132. Especially, in embodiments, the first 3D- printed layers 132 may comprise a first material selected from the group comprising cured photosensitive materials. Further, in embodiments, the first 3D-printed layers 132 may have thicknesses independently selected from the range of 15-500 pm.

[0127] Fig. 4 subfigure II schematically depicts a top view of the fluidic interface element 200. In an aspect, the invention relates to a use of the multi-functional microfluidic device 1500 as described herein (and especially depicted in Fig. 4 subfigure I) in one or more of Atomic Force Microscopy and Atomic Force Spectroscopy. Especially, in embodiments, the invention may relate to use of the multi-functional microfluidic device 1500 as described herein for ex vivo measuring physical and / or mechanical properties of a cell.

[0128] As depicted in Fig. 4, the device 1000, especially the multi-functional microfluidic device 1500 may further comprise a microfluidic channel structure 170. In embodiments, the microfluidic channel structure 170 may comprise (one or more of) the cantilever channel 165 (see e.g. Fig. 1), the support channel 135 (see e.g. Fig. 1), and the interface element channel 205 configured in fluidic contact with each other. Especially, in embodiments, the microfluidic channel structure 170 may comprise a first opening 171 and a second opening 172. In embodiments, the first opening 171 may be configured in the fluidic interface element 200. Especially, in embodiments, the first opening 171 may be configured to fluidically couple the microfluidic channel structure 170 to a fluid control system 350. Further, in embodiments, the cantilever aperture 156 may comprises the second opening 172.

[0129] Yet further, in embodiments, the multi-functional microfluidic device 1500 may comprise one or more piezoelectric devices (not depicted) configured to control a position of the microfluidic probe 100 relative to the support 50. Especially, in embodiments, the one or more piezoelectric devices may be configured to control a distance between the protrusion tip 155 and the support 50 by a lateral movement of the microfluidic probe 100 relative to the support 50. Additionally or alternatively, in embodiments, the one or more piezoelectric devices may be configured to control a distance between the protrusion tip 155 and the support 50 by a rotational movement of the microfluidic probe 100 relative to the support 50.

[0130] Fig. 3 and Fig. 5 schematically depict some applications of the device 1000, especially of the microfluidic probe 100.

[0131] Especially, in a yet further aspect, the invention relates to a method for operating the multi-functional microfluidic device 1500 as described herein. Fig. 3 especially schematically depicts some embodiments of such operations. Especially, the method may comprise one or more of: (i) providing a fluid through the second opening 172 in the microfluidic probe 100 (via one or more of the cantilever channel 165 and the interface channel 205); (ii) providing suction at the second opening 172 of the microfluidic probe 100, (iii) applying pressure with the protrusion tip 155 on an element 30 supported by the support 50 (as depicted especially in Fig. 3 subfigure III).

[0132] In embodiments, the method may especially comprise applying a modulation to the element 30 using the multi-functional microfluidic device 1500. In embodiments, the element 30 may comprise a cell. In embodiments, Fig. 5 subfigure I schematically depict the microfluidic 100 approaching the element 30 in a lateral movement. In embodiments, the modulation of the element 30 may comprise one or more of: picking up the cell (as depicted in Fig. 5 subfigure IV and V); adhering the microfluidic probe 100 to the cell (e.g. to measure adhesion strength) (as depicted in Fig. 5 subfigure III and IV); puncturing the cell with the protrusion tip 155 (as depicted in Fig. 5 subfigure II); and injecting fluid into the cell (as depicted in Fig. 5 subfigure III). Fig. 5 subfigure VI schematically depicts placement of an element 30 after the element 30 had been picked up using the microfluidic probe 100.

[0133] Further, in embodiments, the multi-functional microfluidic device 1500 may comprise a laser 20configured to provide second radiation 21 to the cantilever 150 (especially to the cantilever head 158). Yet further, in embodiments, the multi-functional microfluidic device 1500 may comprise a detector 300 configured to (i) collect second radiation 21 reflected by the cantilever 150 and (ii) provide an (electrical) signal with an intensity proportional to a displacement of the cantilever 150.

[0134] Fig. 6 schematically depicts some further embodiments of the cantilever 150. Especially, in embodiments, the protrusion 154 may have a shape approximating a cone shape, such as depicted in Fig. 6. Especially, in embodiments such as depicted in subfigures I and IV, the protrusion 154 may have a (normal) cone shape. In other embodiments such as depicted in subfigures II and V, the protrusion 154 may have a rounded cone shape. In yet other embodiments such as depicted in subfigure III, the protrusion 154 may have a flat-topped cone shape. In other embodiments, the protrusion 154 may have a shape approximating a (polygonal) pyramid. Especially, in embodiments, the protrusion 154 have a polygonal pyramid shape (not depicted), e.g. having a rectangular base.

[0135] Further, the protrusion 154 may have a half angle ap relative to the protrusion axis Ap. In embodiments, the half angle ap may be an acute angle, such as depicted in subfigures I, II, and III. Especially, in such embodiments, the half angle ap may be selected from the range of 20-60°. However, in other embodiments, such as depicted in subfigures IV and V, the half angle ap may be an obtuse angle.

[0136] As depicted in Fig. 6, in embodiments, the protrusion axis Ap and the aperture axis AA may have a nonzero angle relative to each other. Especially, in embodiments, the protrusion axis Ap and the aperture axis AA may have an axis mutual angle aA selected from the range of 2-45°. As depicted in the schematic examples in Fig. 6, in embodiments, the protrusion axis Ap and the aperture axis AA may have an axis mutual angle aA of about 20°. Additionally, in embodiments, as depicted in Fig. 6, the first mutual angle P may be about 70°, whereas the second mutual angle y may be about 90°. Hence, in such embodiments, the cantilever 150 may taper, such that the cantilever aperture 156 configured in the slanted side 58 may have a non-orthogonal angle with the cantilever arm length axis LCA.

[0137] Experiments

[0138] Unless specified otherwise, the experiments described herein are performed using the materials and methods described hereinafter.

[0139] Fluidic interface element printing - The fluid interface element was manufactured using a DLP printer (Prusa SL1) with a layer height of 25 pm, employing a standard recipe alongside an orange-colored methacrylate / acrylate-based photoresist (Prusa orange). Following printing, the fluidic interface element underwent cleaning with IPA (99.8% Honeywell, Riedel-de-Ha' en) and was post-cured with UV light (Photopol A5406, Dentalfarm). Subsequently, apertures (or holes) were fashioned through laser ablation (WS Starter micromachining laser system, Optec S.A.), employing a laser power of 15W at 50kHz, with a writing speed of 15mm s-1 and 15 repetitions. The final preparatory step before 2PP printing involved briefly sputtering the fluidic interface element with a 3nm gold layer (JEOL JFC1300 sputter coating system) using a sputtering current of 10-15 mA for 5 s, maintaining a target-substrate distance of 20 mm. This process augmented the surface’s reflectivity, facilitating automatic interface identification within the 2PP printer.

[0140] Following DLP printing, a thorough inspection of the fluidic interface element was conducted to identify any evident defects before proceeding with the process. Subsequently, upon laser drilling of the apertures (for fluidic contact with the cantilever), each aperture was meticulously inspected, and its diameter was measured using an optical microscope (Motic). The fluidic interface element was checked for fluidics by applying DI water, and a small meniscus was observed at the laser drilled aperture location. The majority of the water flowed out from the development hole (i.e. a hole that is present as an artefact of the development process), but a well-prepared sample always had some liquid coming out from the laser drilled aperture. Only interfaces that passed this test were moved on to the 2PP printing step.

[0141] 2PP printing - The cantilever was fabricated using two-photon-polymerization (2PP) atop the fluid interface element with 200 nm resolution, employing the Photonic Professional GT Laser Lithography System by Nanoscribe GmbH. Employing the dip-in-laser- lithography (DiLL) configuration, a 63x magnifying objective was utilized along with the IP- Dip resin from Nanoscribe GmbH. To ensure robust attachment and prevent leakage, the dome was printed 5 pm deeper than the fluid interface element surface. Subsequently, the cantilever was printed (layer by layer) on a slanted channel opening of the dome. Utilizing a printing strategy involving subsequent overlapping slabs at a 45° angle prevented the sagging of the bottom layers and facilitated the production of the cantilever of the invention.

[0142] After completing the cantilever print, the samples were removed from the 2PP printer. To develop the samples, they were initially immersed in propylene-glycol-methyl- ether-acetate (PGMEA, Sigma-Aldrich) for 30 minutes, followed by immersion in isopropanol (IP A, Sigma-Aldrich) for 5 minutes, and finally in methyl perfluoropropyl ether, 1-m ethoxy - heptafluoropropane (Novec, Sigma-Aldrich) for 10 minutes, with gentle compressed air blow- drying between each step. Subsequently, the samples underwent inspection using an optical microscope to ensure clearance of all microfluidic channels. For precise measurements of the smallest features such as the individual tip radius and aperture size, a scanning electron microscope (SEM, Jeol JSM6010LA) was utilized on models with an exposed cross-section, printed using the same parameters as the complete models. In some batches, an additional development cycle was necessary to ensure thorough channel development.

[0143] To expedite the assessment of the microfluidic probe functionality postdevelopment and sealing (e.g. plugging) of the hole (in the fluidic interface element, i.e. a purge channel), individual devices were connected to a lOmL syringe via Tygon tubing and submerged in IPA. Manual pressure was then applied with the syringe to observe the occurrence of droplets at the cantilever aperture.

[0144] Atomic Force Microscopy - The JPK Nanowizard 4 was used for indentation and cell adhesion experiments. The indentation experiments were conducted in contact mode using the force spectroscopy module, and the data was analyzed using the proprietary JPK data processing software. The Elveflow pressure controller MK31+, which can handle pressure ranges between -1000 to 4000 mbar, was used for fluidic operations. Fluidic connections were made through silicone tubing. The cell adhesion module from JPK AFM was utilized for this purpose. The experiments were observed using a Zeiss Axio observer microscope with the PIFOC arrangement, equipped with a 40X objective lens with a numerical aperture (NA) of 0.55.

[0145] Indentation and imaging - The tip radius (of the cantilever protrusion) on the cantilever had been constantly around 600nm radius, making it ideal for measurements in the micron scale measurement, especially cell and indentation experiments. Experiments were performed with different cantilever configurations, especially (i) wherein the protrusion may be configured closer to the cantilever end than the cantilever aperture, and (ii) wherein the cantilever aperture may be configured closer to the cantilever end than the protrusion. Both configurations appeared to show identical results, although visibility during the indentation was improved in the case of the protrusion being configured closer to the cantilever end than the cantilever aperture. The imaging experiments confirmed imaging by the cantilever capturing features in the microns range, therewith making it ideal for measuring the topography as well as the mechanical property characterization at 1-100 microns range. The indentation capacity of the cantilever was tested in multiple ways on different substrates indicating that the protrusion is capable of capturing the viscoelastic behavior of the cell where the hysteresis in the approach, and the retract during indentation is captured. In other substrates like hydrogel and PDMS, which are stiffer compared to cells, the protrusion accurately measures the values of the young modulus within the reported range in the literature, see also below table. As evidenced by the table, the measured Young’s modulus is well within the value reported in literature.

[0146] For the hydrogel experiments, a structure was designed and printed using hydrogel via the two-photon polymerization method. This structure was tailored to the scale of cells and was tailored to possess specific properties to provide the above comparisons.

[0147] Conclusions - a multifunctional probe was developed, which was proven capable of performing both indentation and cell adhesion measurements on a unified interface. The experiments showcase the cantilever protrusion’s capacity to assess both topography and changes in Young’s modulus within a single structure. Essentially, this validates the protrusion’s capability to indent and measure cytoskeletal properties at various locations within a single cell. The spatial differences in Young’s modulus, spanning a few hundred nanometers, along with topographical variations, underscore the unique potential of the protrusion to characterize mechanical properties across distinct regions within individual cells.

[0148] The probe design features versatile configurations, with the aperture and protrusion components interchanged as needed for different experiments. To validate the efficacy of the probe, various tests demonstrating fluidics functionality and cell culture experiments were conducted, showcasing the successful operation of both the fluidic channel and the aperture. Indentation experiments were carried out across a range of substrates with Young’s modulus spanning from 2 kPa to 3 MPa, affirming the robustness of the protrusion design. Furthermore, the spatial resolution was assessed by simulating cell-like hydrogel samples, yielding promising results in detecting changes in Young’s modulus and accurately measuring surface topography. Hence, fluidics, cell adhesion, and elastic modulus measurements with the microfluidic probe have been confirmed.

[0149] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

[0150] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0151] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0152] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

[0153] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0154] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0155] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0156] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0157] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0158] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0159] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0160] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0161] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0162] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A microfluidic probe (100) comprising a cantilever (150) and a cantilever support (130), wherein the cantilever (150) comprises a cantilever channel (165) and wherein the cantilever support (130) comprises a support channel (135) configured in fluidic contact with the cantilever channel (165); wherein the cantilever (150) comprises a support end (151) and a cantilever end (152) remote from the cantilever support (130); wherein the cantilever (150) comprises a protrusion (154) and a cantilever aperture (156) both configured on a first side (51) of the cantilever (150), wherein the protrusion (154) and the cantilever aperture (156) are configured closer to the cantilever end (152) than to the support end (151), and wherein the protrusion (154) and the cantilever aperture (156) are spatially separated; wherein the cantilever aperture (156) is configured in fluidic contact with the cantilever channel (165); wherein the protrusion (154) comprises a protrusion tip (155); wherein the cantilever (150) comprises a cantilever head (158) and a cantilever arm (160) associated to each other; wherein the support end (151) comprises at least part of the cantilever arm (160), wherein the cantilever head (158) comprises the protrusion (154), the cantilever aperture (156), and the cantilever end (152), wherein the cantilever arm (160) comprises a cross-sectional equivalent circular diameter (DCA) and a cantilever arm length axis (LCA), wherein the cantilever head (158) comprises a cantilever face part (159) comprising a cantilever head dimension (DCH) configured perpendicular to the cantilever arm length axis (LCA), and wherein DCH>2*DCA; and wherein the cantilever face part (159) comprises a radiation reflective material (40), wherein the radiation reflective material (40) is reflective for radiation having a wavelength selected from the range of visible and infrared radiation.

2. The microfluidic probe (100) according to claim 1, wherein the protrusion (154) defines a protrusion axis (Ap), wherein the protrusion axis (Ap) is defined perpendicular to the first side (51) and intersecting the protrusion tip (155); and wherein the protrusion tip (155) extends from the first side (51) by a second distance (d2), wherein 0.5 pm < d2 < 20 pm.

3. The microfluidic probe (100) according to any one of the preceding claims, wherein the cantilever aperture (156) comprises an aperture edge (157), wherein a first distance (dl) between the protrusion tip (155) and the aperture edge (157) is selected from the range of 1 pm < dl <50 pm.

4. The microfluidic probe (100) according to any one of the preceding claims, wherein the cantilever arm (160) comprises a cantilever arm part (161) having a length parallel to the cantilever arm length axis (LCA), wherein cross-sectional dimensions of the cantilever arm part (161) over its length are constant; and wherein one or more cross-sectional dimensions of the cantilever head (158) are larger than one or more cross-sectional dimensions of the cantilever arm part (161).

5. The microfluidic probe (100) according to claim 4, wherein the radiation reflective material (40) is reflective for radiation having a wavelength selected from the range of 300-2000 nm.

6. The microfluidic probe (100) according to any one of the preceding claims 1-5, wherein the protrusion (154) has a shape approximating a cone shape, wherein the protrusion (154) has a half angle (ap) relative to the protrusion axis (Ap), wherein the half angle (ap) is an acute angle; wherein the half angle (ap) is selected from the range of 20-60°.

7. The microfluidic probe (100) according to any one of the preceding claims 1-5, wherein the protrusion (154) has a shape approximating a pyramid.

8. The microfluidic probe (100) according to any one of the preceding claims, wherein the cantilever aperture (156) is configured closer to the cantilever end (152) than the protrusion (154) and wherein the protrusion (154) is configured closer to the support end (151) than the cantilever aperture (156).

9. The microfluidic probe (100) according to any one of the preceding claims, wherein the cantilever head (158) tapers from the cantilever arm (160) along the direction of elongation of the cantilever (150) to the cantilever end (152), wherein the cantilever head (158) has a slanted side (58), wherein the cantilever aperture (156) is configured in the slanted side (58); wherein the cantilever aperture (156) defines an aperture axis (AA), wherein the aperture axis (AA) and the cantilever arm length axis (LCA) have a first mutual angle (P), wherein the first mutual angle (P) is selected from the range of 30-80°; and wherein the protrusion axis (Ap) as defined in claim 2 and the cantilever arm length axis (LCA) have a second mutual angle (y), wherein the second mutual angle (y) is selected from the range of 75-90°.

10. The microfluidic probe (100) according to any one of the preceding claims, wherein the cantilever (150) comprises (a) the cantilever arm (160) comprising the cantilever arm length axis (LCA), and (b) a visual indicator (15); wherein the visual indicator (15) is configured to indicate a location of one or more of the protrusion (154) and the aperture (156) relative to the cantilever arm length axis (LCA), wherein the visual indicator (15) is configured on a second side (52) of the cantilever (150) different from the first side (51).

11. The microfluidic probe (100) according to any one of the preceding claims, wherein one or more of the cantilever support (130) and the cantilever (150) comprise a laser printed polymeric material.

12. The microfluidic probe (100) according to any one of the preceding claims, wherein the cantilever aperture (156) comprises an aperture diameter (D P) selected from the range of 2-60 pm.

13. A method for 3D printing at least part of a device (1000), wherein the device (1000) comprises (a) a fluidic interface element (200) and (b) the microfluidic probe (100) according to any one of the preceding claims 1-12; wherein the fluidic interface element (200) comprises an interface element channel (205), wherein the fluidic interface element (200) comprises a hole (220) configured in fluidic contact with the interface element channel (205); wherein the method comprises: a resin providing stage comprising providing a photosensitive resin configured to cure when exposed to first radiation; a microfluidic probe 3D printing stage comprising 3D printing a 3D printable polymeric material, using the photosensitive resin, to provide the microfluidic probe (100); a resin removal stage comprising removing uncured photosensitive resin from the fluidic interface element (200) and the microfluidic probe (100) by flushing the interface element channel (205) of the fluidic interface element (200) and the cantilever channel (165) with a washing fluid; wherein the interface element channel (205) is configured in fluidic contact with the cantilever channel (165); and a sealing stage comprising sealing the hole (220) in the washed fluidic interface element (200).

14. The method according to claim 13, further comprising a fluidic interface element3D printing stage comprising exposing part of the photosensitive resin to first radiation to provide the fluidic interface element (200).

15. The method according to claim 14, wherein the fluidic interface element 3D printing stage comprises one or more of stereolithography and digital light processing.

16. The method according to any one of the preceding claims 13-15, wherein the microfluidic probe printing stage comprises two-photon-polymerization.

17. The method according to any one of the preceding claims 13-16, wherein the device (100) comprises a microfluidic channel structure (170), wherein the microfluidic channel structure (170) comprises the cantilever channel (165), the support channel (135), and the interface element channel (205) configured in fluidic contact with each other.

18. The method according to claim 17, wherein the microfluidic channel structure(170) comprises a first opening (171) and a second opening (172), wherein the first opening(171) is configured in the fluidic interface element (200), wherein the first opening (171) is configured to fluidically couple the microfluidic channel structure (170) to a fluid control system (350), and wherein the cantilever aperture (156) comprises the second opening (172).

19. The method according to any one of the preceding claims 13-18, wherein the device (1000) comprises a multi-functional microfluidic device.

20. A multi-functional microfluidic device (1500) comprising (a) a fluidic interface element (200), (b) the microfluidic probe (100) according to any one of the preceding claims 1-12, (c) a support (50), and (d) an actuator (360); wherein the fluidic interface element (200) comprises an interface element channel (205) and is configured to support the microfluidic probe (100), wherein the interface element channel (205) is configured in fluidic contact with the cantilever channel (165); wherein the actuator (360) is configured to control a relative position of the microfluidic probe (100) and the support (50).

21. The multi-functional microfluidic device (1500) according to claim 20, wherein the fluidic interface element (200) is configured to during an operational mode of the multi-functional microfluidic device (1500) position the microfluidic probe (100) such that the first side (151) of the cantilever (150) is level relative to the support (50).

22. The multi-functional microfluidic device (1500) according to any one of the preceding claims 20-21, wherein the multi-functional microfluidic device (1500) comprises a microfluidic channel structure (170), wherein the microfluidic channel structure (170) comprises the cantilever channel (165), the support channel (135), and the interface element channel (205) configured in fluidic contact with each other; and wherein the microfluidic channel structure (170) comprises a first opening (171) and a second opening (172), wherein the first opening (171) is configured in the fluidic interface element (200), and wherein the first opening (171) is configured to fluidically couple the microfluidic channel structure (170) to a fluid control system (350), and wherein the cantilever aperture (156) comprises the second opening (172).

23. The multi-functional microfluidic device (1500) according to any one of the preceding claims 20-22, wherein the multi-functional microfluidic device (1500) comprises a laser configured to provide second radiation to the cantilever (150), and a detector configured to (i) collect second radiation reflected by the cantilever (150) and (ii) provide a signal with an intensity proportional to a displacement of the cantilever (150).

24. The multi-functional microfluidic device (1500) according to any one of the preceding claims 20-23, wherein the multi-functional microfluidic device (1500) comprises one or more piezoelectric devices configured to control a position of the microfluidic probe (100) relative to the support (50), wherein one or more of the following applies: (a) the one or more piezoelectric devices are configured to control a distance between the protrusion tip (155) and the support by a lateral movement of the microfluidic probe (100) relative to the support (50), and (b) the one or more piezoelectric devices are configured to control a distance between the protrusion tip (155) and the support (50) by a rotational movement of the microfluidic probe (100) relative to the support (50).

25. A method for operating the multi-functional microfluidic device (1500) according to any one of claims 20-24, wherein the method comprises one or more of (i) providing a fluid through the second opening (172) in the microfluidic probe (100), (ii)providing suction at the second opening (172) of the microfluidic probe (100), (iii) applying pressure with the protrusion tip (155) on an element (30) supported by the support (50).

26. The method according to claim 25, wherein the method comprises applying a modulation to the element (30) supported by the support (50) using the multi-functional microfluidic device (1500), wherein the element (30) comprises a biological cell, wherein the modulation comprises one or more of: picking up the biological cell; adhering the microfluidic probe (100) to the biological cell; puncturing the cell with the protrusion tip (155); and injecting fluid into the biological cell.

27. Use of the multi-functional microfluidic device (1500) according to any one of the preceding claims 20-24 in one or more of Atomic Force Microscopy and Atomic Force Spectroscopy.

28. The use according to claim 27 for ex vivo measuring physical and / or mechanical properties of a biological cell.

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