WO2017162708A1 - Aiguilles creuses souples et porte-à-faux et leurs procédés de fabrication - Google Patents

Aiguilles creuses souples et porte-à-faux et leurs procédés de fabrication Download PDF

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Publication number
WO2017162708A1
WO2017162708A1 PCT/EP2017/056755 EP2017056755W WO2017162708A1 WO 2017162708 A1 WO2017162708 A1 WO 2017162708A1 EP 2017056755 W EP2017056755 W EP 2017056755W WO 2017162708 A1 WO2017162708 A1 WO 2017162708A1
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Prior art keywords
layer
shank
range
cantilever
thickness
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PCT/EP2017/056755
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English (en)
Inventor
Janos VÖRÖS
Tomaso Zambelli
Vincent Martinez
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Eth Zurich
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Publication of WO2017162708A1 publication Critical patent/WO2017162708A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B01L2300/123—Flexible; Elastomeric

Definitions

  • the present invention relates to flexible, hollow devices, particularly microneedles or hollow cantilevers with complex tip shape and integrated force sensor, and methods of manufacture and uses thereof.
  • Micropipette-based technologies represent still today the simplest approach for cell injections and manipulations in general. Most techniques for the fabrication of micopipettes are low throughput and rely on heating and pulling principles. They are limited to tubular and straight tips and are therefore not compatible with the realization of tips with complex geometries such as comers, high curvature and several cm in length.
  • conventional micropipettes are usually fabricated of brittle materials (e.g. glass).
  • a further technology targeting single-cell applications evolved from the Atomic Force Microscopy (AFM) technology. Integrating a microchannel into an AFM cantilever allowed broadening the spectrum of AFM applications in liquid towards a force-controlled nanopipette enabling novel range of applications such as controlled spatial manipulation, local injection and dispensing, selective and massive force spectroscopy, and serial perturbation experiments.
  • AFM Atomic Force Microscopy
  • AFM cantilevers are usually made of silicon or silicon nitride, which exhibit high Young's moduli (190 and 385 GPa, respectively). For biological applications, cantilevers are mostly operating in liquid environment with contact mode where a low spring constant is desirable to be less invasive on fragile biomaterials. Replacing silicon by soft polymeric material would allow higher sensitivity for the same considered channel and cantilever thicknesses. On the other hand, thicker channel and cantilever dimensions could potentially enable a larger range of microfluidic applications with similar force detection sensitivity to conventional silicon- based AFM cantilevers.
  • SU-8 IBM is known as the negative-tone photoresist with the highest available epoxide functionality for its eight epoxy sites per monomer component.
  • SU-8 Thanks to the strongly cross- linked nature of this photoplastic material, SU-8 exhibits important mechanical stability as well as chemical resistance to a multitude of solutions, which makes it a good candidate for robust microfluidic systems. SU-8 does not only present advantages in terms of surface chemistry with the possibility of multiple surface functionalisations but also, physical surface modification by laser or plasma treatment among others.
  • the SU-8 presents other interesting features: low Young's modulus of 4.5 GPa, transparent material, ease to tailor the thickness down to 500 nm simply by spin-coating, and high aspect-ratio structuring (> 18).
  • SU-8 can be also directly photo patterned allowing complex designs and potential mass fabrication. After optimization of a working fabrication process, it is rather simple to adapt the design of the device directly by changing the photolithography masks. If one takes into account the formula for the spring constant of a cantilever, geometrical dimensions of the cantilever contribute more to the final stiffness in the case of SU-8 because of its lower Young's Modulus compared to silicon. This provides the possibility for the fabrication of devices with a wider spectrum of spring constants on the same wafer.
  • a force sensor within or at the micropipette or the hollow cantilever is advantageous to avoid the application of too excessice forces on the single cell.
  • a force sensor can help to rupture gently the membrane, maximizing therefore the rate of cell recovery.
  • most force sensors are based on laser reflective methods, which are therefore invasive and non-usable in surgical environment.
  • the present invention is particularly based on the on the novel concept that metal may be used as sacrifial filling material in a photolithographic process using photoresists for the manufacturing of devices in the micrometer scale with microfludic channels. Furthermore, the concept of fabricating a thick hollow needle layer-by-layer, from a side to another, is newly introduced. Thereby, it is possible to provide new designs of flexible hollow needles that particularly overcome the limitations of glass micropipettes in terms of tip geometry, fluidic robustness and fabrication throughput.
  • a method for manufacturing a device comprising a flexible shank
  • the shank comprises a microfluidic channel and an aperture, which is in fluid communication with microfluidic channel.
  • the method comprises the steps of: providing a support covered by a sacrificial release layer;
  • a mould layer of a second photoresist onto the metal seed layer, curing the mould layer within a second defined pattern, thereby yielding a cured mould layer, wherein a part of the metal seed layer covering the bottom layer is not covered by the cured mould layer, and the cured mould layer forms the mould that later defines the microfluidic channel and optionally the aperture and/or a reservoir in fluid communication with the microfluidic channel;
  • the cover layer completely curing the first photoresist comprised within the cover layer and the bottom layer within a third defined pattern, thereby forming sidewalls and a topside of the device or a bottomside, a second sidewall opposite to the first sidewall and a topside of the device, whereby the topside or the second sidewall opionally comprises an opening, the cover layer merge with the bottom layer such that at least one part of the first photoresist delimiting the microfluidic channel is integrally formed in one piece, and the bottomside, the sidewalls and the topside embrace the additional metal layer optionally with the exception of the opening or the aperture;
  • the method of the invention enables the access to new designs of flexible hollow needles, whose tip can be made of a complex shape by mass production.
  • a high throughput fabrication of needles with complex tip shape (corners or high curvature) is enabled, on the contrary of individually made glass
  • micropipettes The fabrication relies on a layer-by-layer photolithography process where bottom or side layer, channel and top or second side layer are accordingly patterned in a three-step protocol.
  • the device of the invention may be produced with the method of the invention from bottom to top or it may be fabricated sideways from a side to another.
  • the sideways fabrication offers more possibilities to get a sharp tip with high curvature, e.g. for single-cell injection or for biopsy-like applications.
  • shank in the context of the present specification particularly refers to a structural element that houses a microfluidic channel and an aperture being in fluidic communication with the microfluidic channel.
  • the shank may be designed as a beam of a hollow cantilever or a shank of a micropipette.
  • photoresists in the context of the present specification particularly refers to a viscous composition essentially consisting of, or comprising, a species of photoreactively polymerisable or depolymerisable compounds, and optionally comprising a solvent, wherein particularly the viscous composition is characterized by a viscosity of at least 100 cSt (centi Stokes), for example in range of 100 cSt to 500 cSt or 10,000 cSt to 60,000 cSt.
  • Such photoreactive compounds may polymerise or be cross-linked upon light expore, such as the monomers comprised within a negative photoresists.
  • Such photoreactive compound may also depolymerise upon light exposure, for example by cleavage of cross-link bondings, such as compounds comprised within a positive photoresist.
  • curing is used in the context of the present specification in the meaning known to the skilled person. It particularly refers to the process of solidifying of the above-mentioned photoresists or the above-mentioned visous composition by means of light exposure
  • Curing may also comprise the post-exposure bake, developing by contacting the cured photoresists with a solvent such as propylene glycol monomethyl ether acetate, and rising with an organic solvent such as isopropanol or acetone, thereby removing unpolymerised photoresists.
  • a post exposure bake may be important to complete the photo-activation process.
  • curing within a defined pattern means in the context of the present specification that the photoresist layer is exposed to light (particularly light in the ultraviolet region) within the defined pattern resulting in polymerization of the photoresist within the pattern, in case of a negative photoresist, or in case of a positive photoresist in
  • part curing in the context of the present specification particularly means that the respective photoresist layer is cured in such a manner that only a fraction of the photoresist is cured, or in other words, that part of the photoresist monomers remains reactive for further polymerisation.
  • a partly cured photoresists layer may be connected with another photoresist layer such that both layer are integrally formed in one piece.
  • such partly curing of the bottom layer may be achieved by limited light exposure and/ or reduced baking time of the bottom layer.
  • the microfluidic channel is characterized by complete tightness or perfect sealing while applying pressure to the channel, particularly up to 6 bar. Additionally, the integrally one-piece design of the device avoids problems such as delamination of the device or adhesion issues.
  • the first photoresist comprised within the partly cured bottom layer and the cover layer is completely cured such that at least a part of the cured photoresist delimiting the microfluidic channel exhibits a homogenous structure.
  • the above-mentioned metal seed layer has the advantage that the metal seed layer effectively separates the partly cured bottom layer of the device from the mould layer, thereby avoiding undesired interaction between the aforementioned layers. Additionally, in case of the bottom layer in made of a negative photoresist and the mould layer is made of a positive photoresist, the bottom layer is shielded by the metal seed layer from light exposure during the curing of the mould layer, thereby avoiding complete curing of the bottom layer.
  • An advantage of using the metal layer as sacrifical filing body that later defines the microfluidic channel and optionally the aperture and/or the reservoir is that the deposition of the additionally metal layer can be performed under conditions, under which a complete curing of the bottom layer can be avoided, in contrast to the use of, for example, expoxy or polydimethylglutaridimide as sacrifical filing material, wherein the high temperatures being applied to evaporate the solubilizing solvent can lead to an undesired complete curing of the bottom layer. Additionally, the additional metal layer does not exhibit any undesired interactions with the first photoresist and is furthermore easily removable.
  • the metal doesn't dissolve into the freshly spin-coated cover photoresist, which may comprise a solvent, particularly when the photoresist layer hasn't undergone baking until complete solvent evaporation. Without the additional metal layer, the freshly spin-coated mould layer may directly dissolve the patterned and only partly cured bottom layer.
  • any material for example a viscous ink or a liquid plastic, may be used as sacricial filling body that does not interact with the first photoresist and optionally can be cured under condition that avoids complete curing of the bottom layer and/or shields the bottom layer form light exposure during curing of the mould layer and/or is easily removable.
  • the mould layer is cured such under such conditions that a complete curing of the bottom layer is avoided, e.g. by reducing the baking time of the mould layer.
  • the second defined pattern defines the shape of mould that later defines the shape and space of the microfluidic channel and optionally of the aperture and/or of the reservoir.
  • the first defined pattern defines the shape of the bottomside of the device of the invention and optionally the position and size of the aperture. In certain embodiments, the first defined pattern defines the shape of a first sidewall of the device of the invention.
  • the third defined pattern defines the shape of the topside of the device of the invention and optionally the position and size of an opening in the topside. In certain embodiments, the third defined pattern defines the shape of a second sidewall opposite to the first sidewall of the device of the invention and optionally the position and size of an opening in the second sidewall.
  • the third defined pattern is identical to the first defined pattern, optionally with the exception of the opening comprised within the topside or second sidewall of the device of the invention.
  • the first photoresist is a negative photoresist.
  • negative photoresists include SU-8 and phenol formaldehyde resins (CAS Nr. 9003-35-4) such as Novolac.
  • the second photoresist is a positive photoresist.
  • positive photoresists include AZ 4620 AZ 4533, AZ 4562, and PMMA.
  • a handling block layer or a handling block is attached onto the cover layer, the topside or the second sidewall of the device of the invention, wherein the handling block layer or the handling block comprises a through hole that is in fluid communication with the microchannel.
  • the handling block layer or the handling block is attached to a part of the topside or second sidewall of the device that does not comprise the flexible shank.
  • the through hole in the handling block layer or the handling block has the same diameter as the opening in the topside or the second sidewall.
  • the handling block layer or the handling block is characterized by a thickness in the range of 100 pm to 300 pm. In some embodiments, the handling block layer is characterized by a thickness of 250 pm.
  • the handling block layer consists of or comprises the first photoresist, is deposited onto the top layer, and the handling block layer is cured.
  • the cover layer may be only partly cured before deposition of the handling block layer, and after deposition of the handling block layer, the first photoresit comprised within bottom layer, the cover layer and the handling block layer is completely cured such the device is integrally formed in one piece.
  • the bottom layer is characterized by a thickness in the range of
  • the bottom layer is characterized by a thickness in the range of 4 pm to 10 pm. In certain embodiments, the bottom layer is characterized by a thickness in the range of 5 pm.
  • the cover layer is characterized by a thickness in the range of 0.5 pm to 100 pm. In certain embodiments, the cover layer is characterized by a thickness in the range of 5 pm to 12 pm. In certain embodiments, the cover layer is characterized by a thickness of 7 pm.
  • the aforementioned thickness of the cover layer particularly refers to the thickness of the part of the coverlayer covering the additional metal layer.
  • the additional metal layer is characterized by a thickness in the range of 0.1 pm to 50 pm. In certain embodiments, the additional metal layer is characterized by a thickness in the range of 0.5 pm to 23 pm. In certain embodiments, the additional metal layer is characterized by a thickness of 3 pm.
  • thicker channels may be manufactured using the method of the invention for a different range of applications e.g. 3D printing, dispensing of micro-objects such as single cells, or robust patch clamping.
  • the sacrificial release layer characterized by a thickness in the range of 5 nm to 500 nm. In certain embodiments, the sacrificial release layer characterized by a thickness in the range of 5 nm to 150 nm. In certain embodiments, the metal seed layer is characterized by a thickness of 150 nm. In certain embodiments, the metal seed layer consisits of or comprises a metal selected from the group comprised of copper, aluminium, chromium, gold, silver, platinum, and zinc. In certain embodiments, the additional metal layer consists of or comprises a metal selected from the group comprised of copper, aluminium, chromium, gold, silver, platinum, and zinc. In certain embodiments, the support is formed by a silicon wafer.
  • the sacrificial layer comprises a bottom sacrificial layer, a top sacrificial layer and optionally a sacrificial interlayer, wherein the top sacrificial layer is removed within the first defined pattern, particularly by etching, before the bottom layer is deposited onto the sacrifical layer.
  • areas of the sacrificial layer lying outside of the first defined pattern are coated with a protective layer, particularly a cured photoresist, areas not coated by the protective layer are contacted with an etchant solution, and the protective layer is removed after etching.
  • the top sacrifical layer and/or the bottom consit of or comprise chromium.
  • the sacrifical interlayer consists of or comprises gold.
  • the bottom layer is deposited by spin coating.
  • the mould layer is deposited by spin coating.
  • the cover layer is deposited by spin coating.
  • the metal seed layer is deposited by thermal evaporation, particularly at low current for low UV radiation. In certain embodiments, the metal seed layer is deposited with an emission current of below 5 A, parti culary below 1 A, more particular at 0.5 A.
  • the additional metal layer is electrochemically deposited in a bath comprising a solution with the respective metal ions, such as, for example, copper sulphate in case of the additional metal layer consist or comprises copper, accompanied by the application of a current.
  • the respective metal ions such as, for example, copper sulphate in case of the additional metal layer consist or comprises copper, accompanied by the application of a current.
  • the bottom layer and the cover layer are cured such with the first defined pattern and the third defines pattern, respectively, that the shaft of the device comprises a tip that extends along a first direction.
  • the angle between the first direction a longitudinal axis of the shank or the microfluidic channel is in the range of 180 to 90°.
  • the tip comprises a plurality of segments extending from the shank, wherein each segment is characterized by a tapered shape, and between each segment a portion of the tip is arranged, and wherein at least one of the portions arranged between the segments is designed as predetermined breaking point.
  • the tip curvature, shape and/or length can directly be patterned with the method of the invention, e.g.
  • the device is in this case manufactured sideways, meaning particulary that the bottom layer becomes the first sidewall of the device and the cover layer becomes the bottomside, the topside and the second sidewall of the device.
  • the bottom layer and the coverlayer are cured such with the first defined pattern and the third defined pattern, respectively, that the shaft exhibits a curved shape, a jagged shape, a zigzag shape or a shape comprising a step, or in other words, the shank comprises one or more corners.
  • a shape comprising a step particularly refers to the shape of a shank that comprises two parts each of them extending along a direction, respectively, wherein one of the two directions is arragned with respect to the other direction at an angle in the range of - 30° to 30 C, particularly in the range of -10 C to 10 C, more particular 0°, and the two parts are connected to each other by a intermediate part extending along a direction, which parallel to none of the aforementioned two direction.
  • the bottom layer and the cover layer are cured such with the first defined pattern and the third defines pattern, respectively, that the shaft of the device comprises a tip that extends along a first direction, the angle between the first direction a longitudinal axis of the shank or the microfluidic channel is in the range of 180 to 90, and the shaft exhibits a curved shape, a jagged shape, a zigzag shape or a shape comprising a step, wherein the curved shape, the jagged shape, the zigzag shape or the shape comprising a step extends within a plane being parallel to the first direction.
  • the device of the invention is manufactured sideways as described above.
  • the production of devices, particularly of cantilevers with complex shapes of the shank and the tip may be performed with the method of the invention, which additionally suitable for mass production.
  • a device having a flexible shank comprising a microfluidic channel and an aperture that is in fluid communication with the microfluidic channel, wherein the microfluidic channel extends through the shank along the longitudinal axis of the shank and is delimited by at least one surface being formed by a polymer
  • the polymer forming the at least one surface delimiting the microfluidic channel is integrally formed in one piece.
  • the polymer forming the at least one surface delimiting the microfluidic channel is characterized by an elasticity in the range of 0.5 GPa to 20 Gpa, preferably in the range of 1 GPa to 10 GPa, more preferable in the range of 4 GPa to 5 GPa, even more preferable 4.5 GPa.
  • the microfluidc channel is configured to pass a fluid stream with pressure of up to 6 bar, and and particularly is fluid tight up a pressure of 6 bar(a).
  • the flexible shank is characterized by a spring constant in the range of 0.01 N * m “1 to 1 ,000 N * m “1 , preferably in the range of 0.5 N * m "1 to 80 NPm "1
  • the microfluidic channel is completely fluid tight up to a pressure of 6 bar(a), when applied to the microfluidic channel.
  • the device of the invention is manufactured by a method according to the first aspect of the invention or any embodiment thereof.
  • the polymer forming the at least one surface delimiting the microfluidic channel exhibits a homogenous structure.
  • the device of the invention is integrally formed in piece, and particularly exhibits a homogenous structure.
  • the device further comprises a handling means with a through hole in fluid communication with the reservoir, wherein particularly the handling means is arranged above the reservoir such that the shank is deformable without be obstructed by the handling means.
  • the reservoir extends to the through hole.
  • the polymer is cured photosensitive polymer, in other words a polymer formed by curing, wherein oligomers are cross-linked upon exposure of light.
  • the polymer is SU-8.
  • the polymer is a blend of at least two photosensitive polymers that are cured such that the blend is integrally formed in one piece.
  • the microfluidic channel has a length in the range of 0.05 mm to 50 mm. In certain embodiments, the microfluidic channel has a length in the range of 0.1 mm to 0.5 mm. In certain embodiments, the microfluidic channel has a length in the range of 1 mm to 5 mm. In certain embodiments, the microfluidic channel has a width in the range of 1 ⁇ 3 ⁇ 4 to 50 ⁇ , particulary from 1 pm to 30 m. In certain embodiments, the microfluidic channel has height in the range of 0.1 ⁇ to 30 ⁇ .
  • the shank has a thickness in the range of 10 m to 50 ⁇ . In certain embodiments, the shank has a thickness of 12 ⁇ . In certain embodiments, the shank has a width in the range of 20 m to 80 pm. In certain embodiments, the aperture is characterized by a diameter in the range of 1 prrs to 50 pm, particularly in range of 5 ⁇ to 35 ⁇ , more particular in the range of 20 ⁇ to
  • the handling means is characterized by a height in the range of 100 ⁇ to 300 ⁇ , particularly 250 ⁇ m.
  • the through hole comprised within the handling means is characterized by a diameter in the range of 100 ⁇ to 500 ⁇ .
  • the means for measuring a mechanical force acting on the flexible shank is formed by a metal layer consisting of at least one noble, non-toxic metal, whereby the metal layer is able to changes its resistance in dependence of a mechanical force acting on the metal layer and thereby on the shanks.
  • the noble, non-toxic metal is gold.
  • the means for measuring a mechanical force acting on the flexible shank is formed by a sensing layer comprising a plurality of nanowires made of metal, wherein the nanowires are partially coated with an isolator such that at least a part of the nanowires are in conductive contact to each other when no mechanical force is acting on the shank, and the at least one part of the nanowires in conductive contact to each other decreases with increase of the mechnical forces acting on the shank.
  • Such means is particularly suitable to detect mechanical forces in the range of pN to nN.
  • the nanowires are characterized by a thickness in the range of 40 nm to 60 nm and/or a length in the range of 5 ⁇ to 20 ⁇ .
  • the nanowires consist of or comprise silver.
  • the isolator is aluminium oxide.
  • the sensing layer is characterized by a thickness in the range of 2 ⁇ to 3 ⁇ , wherein the nanowires comprised within the sensing layer are characterized by a length of 5 ⁇ to 10 ⁇ and a diameter of approx. 60 nm.
  • the means for measuring a mechanical force acting on the flexible shank is formed by a sensing layer comprising a plurality of nanowires made of metal, wherein the nanowires are arranged such within the sensing layer that at least a part of the nanowires are in conductive contact to each other when no mechanical force acts on the shank, and the at least one part of the nanowires in conductive contact to each other decreases with increase of an mechnical forces acting on the shank.
  • Such means is particularly suitable to detect mechanical forces in the range of 100 nN and higher.
  • the nanowires are characterized by a thickness in the range of 40 nm to
  • the nanowires consitst of or comprise silver.
  • the means for measuring a mechanical force acting on said flexible shank is formed by a part of the shank being optically transparent, wherein the part of the shank is configured to change its optical properties, particularly its refractive or diffractive properties, in dependence of a mechanical force acting on the part of the shank.
  • the aperture is comprised within a tip of the shank.
  • the tip extends along a first direction from the shank, wherein the angle between the first direction and the longitudinal axis of the shank is in the range of 180 to 90C
  • the tip is characterized by a cylindrical shape or a tapered shape, particularly a conical or pyramidal shape. Such device may advantageously be used as cantilever for single-cell injections or imaging of soft tissues with high sensitivity.
  • the shank is characterized by a curved shape, a jagged shape, a zigzag shape or a shape comprising a step, wherein particularly the curved shape, the jagged shape, the zigzag shape or the shape comprising a step extends within a plane being parallel to the first direction along which the tip extends from the shank.
  • a tip comprising a plurality of segments extends from the shank, each segment is characterized by a tapered shape, and between each segment a portion of the tip is arranged, and wherein at least one of the portions arranged between the segments is designed as predetermined breaking point.
  • clogging possibly occuring within the tip can be overcome by removing the clogged segment, e.g. by breaking off the clogged segment with a tweezer.
  • the device is designed as a cantilever or a micropipette.
  • the micropipette comprises a re-usable tip such as a tip comprising a plurality of segments extends from the shank, each segment is characterized by a tapered shape, and between each segment a portion of the tip is arranged, and wherein at least one of the portions arranged between the segments is designed as predetermined breaking point.
  • the device of the invention is particularly suitable for for fine surgical applications.
  • a method for spatially manipulating a microscopic object is provided. The method comprises the steps of: providing a device having a flexible shank with a microfluidic channel and an aperture in fluidic communication with said microfluidic channel according to any one of the above aspects or embodiments of the invention, and
  • the microscopic object is located at first postion and captured, and the captured microscopic object is released at a second position, wherein particularly a) the device of the of the invention is positioned such that the microscopic object is capturable at the first position,
  • the device of the invention comprising the captured microscopic object is positioned such that the captured microscopic object is releasable at the second position, and d) the captured microscopic object is released at the second position.
  • the microscopic object is captured by attaching the microscopic object at the aperture. In certain embodiments, the microscopic is released by detaching the microscopic object from the aperture.
  • the microscopic object is captured by sucking the microscopic object into the microfluidic channel. In certain embodiments, the microscopic object is released by pumping the microscopic object out of the microfluidic channel.
  • the microscopic object is capture by sucking the microscopic object through the microfluidc channel into a reservoir in fluid communictation with the microfluid channel and released by pumping the microscopic object out of the reservoir through the microscopic channel.
  • Fig. 1 shows the microfabri cation process for producing micro-channeled SU-8
  • cantilevers with A) deposited Cr/ Au/ Cr release layer on a silicon wafer and patterned SU-8 bottom layer with definition of a circular aperture, B) deposited copper seed layer, C) patterned positive AZ sacrificial photoresist, D) electroplated copper growth layer, E) dissolved positive photoresist and copper seed layer, F) patterned SU-8 top layer with definition of an inlet, G) patterned SU-8 chip layer for handling and H) etching of the channel sacrificial layer and release from the wafer.
  • Fig. 2 shows the characterization of the SU-8 devices with A) optical picture of a cantilever after copper plating, B) and C) SEM views of a cantilever of 12 pm thickness for channel thickness of 3 pm (flipped upside down compared to Figure 1 ), D) fluorescent microscopy top view with fluorescent liquid coming out at the aperture, E) apparent liquid meniscus reaching the transparent cantilever and F) flow rate as a function of pressure applied on the chip with linear fit in red dash line. Uncertainty measured in the flow rate was at most 4% for the same applied pressure. It is not represented on the graph for more clarity.
  • Fig. 3 shows the force adhesion measurements of S. cerevisiae.
  • the yeast cells were measured on bare glass and PDA-coated glass substrates with A) an optical picture of a cantilever with a 6 pm diameter aperture before and B) during force spectroscopy of single targeted yeast.
  • B) force spectroscopy of single targeted yeast.
  • C a typical force spectroscopy curve of a yeast cell on glass substrate showed adhesion forces of 15.8 nN (distance of detachment of 240 nm) as a difference between approach curve in blue and retract curve in red.
  • adhesion forces on the 2 substrates were compared exhibiting forces of 15 ⁇ 7.6 nN on glass and 33 nN ⁇ 1 1 .9 nN on PDA, averaged respectively, over 21 and 26 force curves for 10 and 12 different yeasts and performed with the same cantilever.
  • the mean is represented as a square, the median as a line separating two boxes with each box determined by the 25"' and 75 th percentiles and whiskers determined by the 5 th and 95t h percentiles.
  • Fig. 4 shows examples of forces curves obtained with a SU-8 cantilever in A) air and
  • Fig. 5 shows schematics illustrating the main approaches developed for single-cell patterning using a hollow cantilever.
  • A) A micropipette-like configuration for a best cell placement resolution of 250 pm (configuration 1 ).
  • Fig. 6 shows the precise deposition of single C2C12 cells using configuration 2 with
  • FIG. 7 shows the negative patterning of C2C12 mammalian myoblast cells using configurations 3 and 4, corresponding respectively to top and bottom panels.
  • the cantilever was approached A) on the cell of interest.
  • a large underpressure of -800 mbar was applied to the cantilever for 10 sec to detach the cell in B).
  • Fig. 8 shows the subtractive patterning of primary hippocampal neurons on a fully
  • FIG. 9 shows the subtractive patterning of primary hippocampal neurons using
  • a PDL pattern was locally dispensed in the form of a smiley before backfilling the remainder with PLL-g- PEG for a better adhesive to non-adhesive contrast.
  • AAV adeno-associated virus
  • neurons were removed from PLL-g-PEG surface on a second pattern as well as the half-right of the PDL pattern shown by a bright field image of healthy neurons population with no apparent neuron adhering again on the half-right of the PDL-coated smiley in E) and in F) calcium activity expressed after 12 DIV in GFP.
  • Fig. 10 shows the in situ modifications of an existing pattern of neurons.
  • A the pattern realized in Fig. 9C) is illustrated after 14 DIV in fluorescence light microscopy.
  • the two white encircled areas were subject to removal in B) as well as an eye of the smiley in C), by applying with the cantilever high and constant suction for 10 sec onto each spot (pressure inferior to -800mbar).
  • Another existing pattern is shown in bright field microscopy after 14 DIV before patterning in D), and additional deposition of 3 fresh primary neurons forming a second eye to the smiley, as pointed out in E).
  • Fig. 1 1 shows the upside down SEM images of A) a cantilever used in the
  • micropipette-like configuration with the aperture defined in the front plane for a channel thickness of 22 prn (configuration 1 ) and B) a cantilever with the aperture designed at the bottom plane with same channel thickness for an aperture diameter varying from 20 to 35 m (configurations 2 to 4).
  • Fig. 12 shows in A), B) and C) a layer-by-layer photolithography process, wherein bottom layer, channel and top layer are patterned in a three-step protocol; in D) a global view of the sketched device and in E) an optical image of the finalized device.
  • Fig. 13 shows optical pictures of four different designs of cantilevers microfabricated sideways, scalebar being identical for all.
  • A) shows a curved tip
  • Fig. 14 shows optical pictures of five different micropipette designs.
  • A) and B) depict a geometry of probe, which could act as an diffractometer upon mechanical force,
  • C) and D) have a high and long tip to reach inaccessible locations,
  • E) and F) a sliceable tip for several uses.
  • G) and H) display a long tip with an additional corner.
  • Fig. 15 shows corresponding SEM images of figure 14.
  • Fig. 16 shows SEM images of the sliceable tip before in A) and after cutting in B) at the location represented by a dashed green line.
  • C) depicts a close-up view of
  • FIG. 17 shows SEM images of two different thick cantilevers (respectively at bottom and top panels) demonstrating a pillar-like structure at the aperture-level.
  • Fig. 18 shows the process flow for tube-like tips with in A) patterning of SU-8 bottom layer, B) copper seed layer deposition, C) AZ sacrificial patterning for channel definition, D) copper plating for thickness of 10 ⁇ , E) etching of AZ and copper seed layer and patterning of SU-8 top layer and F) release of the probe.
  • Fig. 19 shows a scheme of the mode of action of one possible force sensor.
  • Fig. 20 schematically shows an embodiment of the force sensor comprised within shank (cantilever beam) of the device of the invention (here designed as cantilever), wherein the force sensor is formed by a composite structure in the shape of a cantilever beam.
  • Fig. 21 schematically shows a method for producing the composite structure depicted in fig. 20.
  • Figs. 22 to 23 show optical images of a cantilever equipped with the force sensor depicted in
  • Fig. 24 shows an embodiment of the device of the invention characterized by a
  • Fig. 25 shows an embodiment of the device of the invention characterized by a biopsy tool-like design; (A) global view from different perspectives and (B) a section of the shank with the tip (upper panel) and a cross section thereof.
  • Fig. 26 shows an embodiment of the device of the invention characterized by a biopsy tool-like design with light sensor; (A) global view from different perspectives and (B) a section of the shank with the tip (upper panel) and a cross section thereof.
  • Fig. 27 shows an embodiment of the device of the invention being a sealed and reusable micropipette with sliceable tips; (A) global view from different perspectives and (B) a section of the shank with the tip (upper panel) and a cross section thereof.
  • Fig. 28 shows an embodiment of the device of the invention characterized by a micropipette design with force sensor; (A) global view from different perspectives and (B) a section of the shank with the tip (upper panel) and a cross section thereof.
  • Fig. 29 shows an embodiment of the device of the invention characterized by a blunt
  • tipss cantilever design
  • A global view from different perspectives
  • B a section of the shank with the aperture (upper panel) and a cross section thereof.
  • Fig. 30 shows an embodiment of the device of the invention characterized by a
  • cantilever design with a triangle tip (A) global view from different perspectives and (B) a section of the shank with the triangle tip (upper panel) and a cross section thereof.
  • Fig. 31 shows an embodiment of the device of the invention characterized by a
  • cantilever design with a rectangle tip; (A) global view from different perspectives and (B) a section of the shank with the rectangle tip (upper panel) and a cross section thereof.
  • Fig. 32 shows an embodiment of the device of the invention characterized by a
  • Fig. 33 shows an embodiment of the device of the invention characterized by a
  • micropipette design for patch-clamp (A) global view from different perspectives and (B) a section of the shank with the tip (upper panel) and a cross section thereof.
  • a novel fabrication method was established to produce flexible, transparent and robust tipiess hollow atomic force microscopy (AFM) cantilevers made entirely from SU-8. Channels of 3 pm thickness and several millimeters length were integrated into 12 pm thick and 40 pm wide cantilevers. Connected to a pressure controller, the devices showed robust sealing performance with no leakage up to 6 bars. Changing the cantilevers length from 100 pm to 500 pm among the same wafer allowed targeting various spring constants range from 0.5 to 80 N/m within a single fabrication run.
  • Such hollow polymeric AFM cantilevers were operated using optical beam deflection method (OBD).
  • SCFS single-cell force spectroscopy
  • SU-8 now offers a new alternative to conventional silicon-based hollow cantilevers with more flexibility in terms of complex geometric design and surface chemistry modification.
  • oligomers from bottom SU-8 of the microchannel must always be present in sufficient quantity to polymerize with top SU-8 covering the channel in order to ensure a watertight sealing at the interface.
  • Figure 1 illustrates the process flow that was established for this fabrication. A novel
  • the first step consisted in depositing a sacrificial release layer 21 made of 5 nm chromium/ 50 nm gold/50 nm chromium on a silicon carrier substrate, known as enhanced sacrificial layer, to ensure a fast release of the probes from the wafer.
  • a 5- pm thick SU-8 bottom layer 22 was photolithographically patterned with the formation of a circular outlet aperture 13 having a diameter down to 4 pm (Fig. 1A).
  • a copper seed layer 23 of 150 nm thickness was deposited on the wafer surface using thermally resistive evaporation for low UV radiation (Fig. 1 B). This layer 23 was consequently thick to support uniform electrical conductivity over the surface.
  • a positive photoresist 24 such as AZ4562 was then patterned to define the shape of the future hollow channel 12 as well as to guarantee straight walls during the sacrificial metal growth 25.
  • the use of a positive photoresist 24 was advantageously so that SU-8 areas 22 on the bottom plane supposed to polymerize subsequently with the cover layer 26 were spared from UV exposure while patterning the positive resist. Reduced baking time was also advantageous to prevent complete polymerization.
  • a sacrificial copper layer 25 was electrochemically deposited in a bath of copper sulfate for a thickness of 3 pm (Fig. 1 D).
  • the wafer Prior to electroplating, the wafer was quickly plasma treated and dipped in sulfuric acid to ensure a better wetting.
  • the positive photoresist 24 was dissolved by immersing the wafer for 5 s in acetone followed by a quick dipping of 3-4 s in a hydrogen peroxide based etchant until the thin copper seed layer 23 disappeared, as seen in Fig. 1 E.
  • a 7 pm thick SU-8 top layer 26 of the probe was spin-coated over the existing structures and patterned accordingly as seen in Fig. 1 F.
  • a 250- ⁇ thick block layer 28 of SU-8 was patterned to form the handling chip part 15 of the probe 100 making a cylindrical reservoir 14, 16 directly connected to the channel 12 (Fig. 1 G).
  • the sacrificial layer 25 forming the channel 12 was then dissolved in a copper etching solution for several days depending on agitation (2-mm long channels).
  • This versatile approach allowed fabricating several millimeter long channels 12 for thicknesses ranging from 100 nm to tens of micrometers.
  • a ferric chloric based copper etchant was preferred over hydrogen peroxide based etchant because the latter one formed oxygen bubbles that stayed confined at the inlet's surface because of SU-8's hydrophobicity.
  • the fabricated probe 100 was released from the carrier wafer by using a chromium etching solution to dissolve the release layer 21 in two hours as shown in Fig. 1 H.
  • Cantilevers showed maximal initial bending of 5 because of their consequently high thickness (> 10 pm). Therefore, there was no need to use a more complex dry release technique to further minimize such bending.
  • probes 100 were squeezed on top of hydrophobic para film substrate for easy handling before gold deposition. A 10-nm thin layer was deposited onto the entire device to ensure a satisfactory laser reflection on top of the cantilever and by doing so, enabling optical beam deflection (OBD) method for force detection. Depending on the use, this thickness may be increased or decreased if one wanted to privilege AFM laser force signal or better visibility of surface objects through the transparent microlever.
  • OBD optical beam deflection
  • this thickness may be increased or decreased if one wanted to privilege AFM laser force signal or better visibility of surface objects through the transparent microlever.
  • probes 100 could be gently peeled off with tweezers from the parafilm substrate.
  • a specific holder with many more conductive pins could also be designed to guarantee even better copper growth uniformity.
  • micropipettes with complex shapes can be manufactured, particularly in a high-through-put manner.
  • SU-8 offers some advantages in terms of electrical isolation in the case of piezoresistive force sensing in comparison with force sensors known in the art. This latter electrical-based sensing only comes with 2 electrical wires to contact the sensing film (described below), limiting de facto the intrusion for surgical applications.
  • Prototypes of SU-8 micropipettes with various designs were realized.
  • Figure 14 depicts in A) and B) possible geometries of a pipette tip, which upon applied force will change the periodicity between the triangles.
  • Thick cantilevers Thick cantilevers with integrated microchannels of 22 pm height were fabricated as well for single-cell patterning purposes. It is a very similar process to the one described above with the difference that the AZ4562 sacrificial photoresist is spin-coated and patterned at a higher thickness of 27 pm for control over the copper growth within the channel. Cantilevers show a total thickness of 50 pm. Interesstingly, some cantilevers randomly selected for SEM imaging showed a perfectly cylindrical pillar formed in the center of the circular aperture, as for example, depicted in Fig. 19. This necessarily has to do with the copper growth during the electroplating process. One hypothesis to explain this phenomenon might result from the fact that the channel thickness is below expected.
  • mechanical strain can cause bending, compression or elongation on the surface of the device.
  • This strain can be measured within 2 configurations based on piezoresistive sensing.
  • the sensing layer is always located on the surface, as far as possible from the neutral axis of the cantilever or pipette for higher force sensitivity.
  • the different configurations can be classified as following (from low to higher strain):
  • a large network of silver nanowires 31 (AgNWs) is first deposited on a surface.
  • a thin aluminum oxide coating (from few angstroms to few nm) is conformally deposited on the surface of the nanowires 31.
  • the AgNWs network 31 with Al 2 0 3 is then embedded in the SU8 polymer (Fig. 21 ).
  • the Al 2 0 3 coating does not alter the initial resistance R 0 of the network; at rest, R 0 is low since all wires are in contact (Fig. 19).
  • the AgNW network is displaced and the single AgNWs slide along each other's insulator coating, causing a proportion of the wires not to be in contact with other wires anymore, but with the insulating Al 2 0 3 layer.
  • the latter results in a significant increase in resistance (AR) resulting in a higher GF and thus higher sensitivity.
  • AR resistance
  • the increase in AR during bending is appointed to the tunneling resistance which arises at the contact interface of two metal wires, as well as to the ratio of wires in contact/out of contact.
  • the only place where the Al 2 0 3 layer is not deposited is on the AgNWs pads as they form the electrical contact points for piezoresistive readout.
  • a system less sensitive using a thick network of nanowires is ideal for such an application.
  • conductive elements 31 are arranged in or on a cantilever beam 1 1 in a specific pattern.
  • the cantilever beam 1 1 is bent, some sections of the beam 1 1 are compressed and other sections are stretched.
  • conductive elements 31 will remain in contact, lose contact or come into contact physically and/or electrically.
  • the conductivity function of the trail for the described bending is a step function: at first zero (or almost zero), then constant (or almost constant) at a higher level, and then again zero (or almost zero).
  • the amount of bending can thereby be measured by measuring the (change in) conductivity in one or more tracks of the composite structure.
  • Fig. 21 illustrates a process scheme for manufacturing the above described force sensor. Step a) A sacrificial layer 21 is deposited.
  • Step b) Metallic pads 30 for better subsequent electric contact are created, e.g. by vapour deposition.
  • An example for a suitable metal is gold.
  • a percolation structure 31 e.g. a silver nanowire (AgNW) network, is
  • a deposition of shell material in particular layer deposition, e.g. atomic layer deposition of aluminium oxide, can be performed.
  • the layer thickness can range from 2-3 angstroms to 50 nm.
  • Step d) A polymer 22, e.g. SU-8 polymer photoresist, is used as the constituent
  • the material of the cantilever beam 1 1 can be photolithographically patterned in the shape of a beam attached to a handling block 15, which makes the handling easier.
  • the thickness of the handling block 15 is further increased by
  • the increased thickness of the handling block 15 can improve the handling of the cantilever beam.
  • Step f) Optionally, after etching of the sacrificial layer 21. a passivation layer (such as parylene) can be deposited thermally to isolate the cantilever beam 1 1 .
  • the pads' 30 contact area on the hanling block 15 can be protected from the passivation layer e.g. by some cover (because it can have a rather big area, e.g. several mm 2 ).
  • the figure f) shows the cantilever beam 1 1 from the bottom side (turned over as compared to the figures d) and e)) after etching and after deposition of the passivation layer. Therefore only the pads' 30 contact areas are visible.
  • the percolation structure 31 is hidden under the passivation layer and the pads' 30 contact areas, respectively.
  • the conductive elements 31 e.g. the AgNW, move relative to each other, thereby physically connecting or disconnecting and/or sliding relative to each other.
  • the conductive elements 31 e.g. the AgNW
  • the elements do not physically touch but are physically close. They can therefore exchange electric charges, this phenomenon is known as the tunnel- effect.
  • conductive elements 31 initially physically touch.
  • strain is typically low, e.g. 5% or smaller. Therefore for such an application the shell's thickness can be tailored with a steep function (resistance dependant on tunnelling effect dependant on shell's thickness).
  • FIG 22 An optical image of the resulting device is displayed in figure 22.
  • the process only differs from figure 23 in the sense that there are no metallic pads, instead the NW network 31 is patterned and etched using a sacrificial photoresist. Holes 32 were created to minimize the gap between filled structures in order to accelerate the release.
  • silver nanowire pads 31 are glued together with a conductive silver epoxy 35 to gold flexible wires 34 for electrical contact.
  • a FITC- based fluorescent solution did fill the microchannel and came out only from the micro aperture after plasma treatment (Fig. 2D).
  • SU-8 channel surface was made hydrophilic. It facilitated the filling of the cantilever, as pointed out in Fig. 2E by the low contact angle of the liquid meniscus.
  • the cantilevers presented laminar flow showing linear relation of flow rate at the aperture for pressure range applied onto the device (Fig. 2F).
  • different cantilever designs could be fabricated on the same wafer with a thickness ranging from 10 pm to 50 pm thickness, lengths varying from 100 mM to 50 mm, widths from 1 pm to 50 pm, apertures from 4 to 20 pm diameter corresponding to channel volumes from 10 to 50 pL.
  • Those different geometries allow targeting wide spring constants range of 0.01 to 1 ,000 N/m calibrated via Sader's method for resonance frequencies measured from 10 to 5 MHz.
  • These soft cantilevers are considered to have a range of spring constants relevant for biological applications, such as imaging of biological samples or single-molecule force spectroscopy applications.
  • Fig. 1 1 depicts the two types of cantilevers used for the proof of concept experiments described below.
  • the micropipette-like cantilever of Fig. 1 1A was realized by patterning the positive photoresist 24 in a way that the SU-8 became shorter in length than the sacrificial copper layer 25, opening the cantilever on the front plane.
  • the second design of Fig. 1 1 B used for the physical confinement of single-cell patterning applications was simply defined by patterning the 6 pm thick bottom SU-8 layer 22 with a circular aperture ranging from 20 to 35 ⁇ in diameter.
  • the typical spring constant of such cantilevers was calculated to range from 150 to 500 N/m according to the different lengths and widths of the cantilevers, with an average of 300 N/m. Based on calculations, the difference in the spring constants of plain and hollow cantilevers is less the 10%, due to the small channel size as compared to the full cross section of the cantilevers.
  • microfabricated devices can potentially be used as micropipettes with or without force- feedback.
  • the SU-8 cantilevers were mounted on an AFM controller and connected to a fluidic system.
  • Cell adhesion forces can be measured with hollow AFM cantilevers, aspiring a single cell by applying negative pressure at the aperture while overcoming and measuring the
  • S. cerevisiae is a yeast species, which is extensively exploited in baking and brewery fermentation processes and one of the most investigated eukaryotic model organism.
  • the adhesion of microbial cells to abiotic or biotic surfaces is mediated by a complex and dynamic process of interactions affecting cell development and survival. It also plays an active role in the infection process and is furthermore of relevance in biotechnological processes, making necessary a deeper understanding of the ongoing cell to substrate interactions.
  • PDA polydopamine
  • the cantilever was positioned optically with the aperture over a single yeast cell and approached the cantilever on it using systematically the same set point, as shown in Figure 3A.
  • the approach was followed by a contact pause where the single yeast was aspired at the aperture before complete retraction of the cantilever (Fig. 3B).
  • surface modification with an antifouling layer of SU-8 for enhanced biofunctionality could be considered.
  • mechanical overpressure was shown sufficient for the release of yeast cells from the aperture.
  • Fig. 4 shows the spring constant calibration of a SU-8 cantilever (with 500 pm length 40 pm width and 10 pm thickness) via Sader method in air environment using JPK SPM software. The latter was chosen to measure the low adhesion force range.
  • the theoretical spring constant is 0.8 N/m. From calculations, the influence of the hollow channel on the total spring constant is limited to 0.1 % because of its relative ly low thickness.
  • 4C shows an example of a typical force-distance curve obtained for single yeast on glass substrate with adhesion forces of 15.8 nN. Averaged adhesion forces were 15 ⁇ 7.6 nN on glass and 33 nN ⁇ 1 1.9 nN on PDA showing twofold higher adhesion of S.
  • FIG. 5 illustrates the different approaches used and developed in order to evaluate the newly developed hollow SU-8 polymeric cantilevers for the application of single-cell patterning.
  • cell deposition from the cantilever is limited by factors like: the lowest reliable external pressure possibly applied to the system, the drag force acting on the cells, as well as dynamical properties such as the response time.
  • a first approach was taking advantage of the highly flexible polymeric cantilever. This technique has not been investigated so far because most fluidic components in printing technologies are mostly made of rigid and brittle materials.
  • the first approach has been demonstrated using fluorescently labeled C2C12 cells, as depicted in Fig. 6.
  • the cantilever was approached onto the surface with cells carried in the reservoir.
  • the approach of the cantilever was performed using fluorescent light microscopy.
  • overpressure of 20 mbar was constantly applied to the cantilever, ejecting individual fluorescent cells while the cantilever was brought closer to the substrate by one- micrometer vertical steps.
  • the z-position was defined at 0 with the stepper for subsequent patterning events (Fig. 6A). Pressure was then stopped before lifting up the cantilever and moving it to the next deposition spot.
  • the main limitation of the present setup is related to cell adhesion and sedimentation. Initially contained into the reservoir of the cantilever, the cells started adhering to the surface after 30 min. The pressure required to push them out needed slowly to be higher. Cell settling within the supply compartment is a common issue observed in bioprinting techniques, which affects directly the long-term stability of the system. To prevent this, other antifouling coatings than Pll-g-Peg may be used.
  • a fluidic system may be as well set up on the back of the device to decouple the reservoir from the cantilever in order to keep a constant flow running for gentle agitation. Embedding cells in bio-inks may be an additional alternative.
  • Fig. 8 Cell patterns of primary hippocampal neurons were entirely realized on adhesive-coated surfaces.
  • Fig. 8 One pattern example is shown in Fig. 8 in the form of a pumpkin-like smiley. Although it was not measured, adhesion of neurons on PDL surface was relatively lower compared to myoblasts: -500 mbar of underpressure was high enough to remove neurons in the 20 pm vicinity of the aperture contour.
  • the cantilever was set at a sufficient height from the surface- level (-10 pm) to avoid moving away the cells on the surface by sweeping the cantilever on the surface. Since neurons easily gather and migrate together, those patterns realized on PDL-coated surface were difficult to track after more than a week in vitro.
  • the removal process has been repeated on pre-patterned surfaces.
  • smileys of 530 pm in diameter were drawn on bare glass surfaces using fluorescently labeled PDL ink and a linewidth of 15 ⁇ .
  • the dishes were then backfilled with PLL-g-PEG, and the neurons were seeded and incubated for 20 min.
  • the cantilever could enable a selective removal of the unwanted cells that were located on the Pll-g-Peg surface, for a better defined pattern of cells (Fig. 9B).
  • More complex patterns, with gap interval between PDL lines down to 30 pm, could be realized such as a brain-like pattern.
  • the pattern from B) remained intact without any cell migrating to the repulsive surface although several neurites started to grow and bridge over the PEG-coated surface.
  • the pattern depicted in bright field D) and GFP D) constituted a loop, which was 2 mm-long with mostly one or small group up to 3 neurons connecting to each other along the circle.
  • the cantilever proved to be a successful tool to modify in situ an existing pattern of adhering and mature cell cultures.
  • the thick cantilevers were robust enough to undergo high underpressure (nearly -900 mbar). Up to 10 sec were necessary to remove portions of the pattern where neurons started to develop a large carpet of neurites over the PEG-coated surface, as depicted in Fig. 10A), B) and C).
  • the cantilever was used to dispense fresh neurons on an existing pattern. As illustrated in Fig. 10D) and E), a smiley pattern was completed by the controlled deposition of additional cells at the right-eye location. This approach could largely profit to extensive mechanical studies of neural networks. Besides, it presents a high potential to study in situ mechanisms of cell growth, migration and interaction over long-term studies.
  • AFM capability of these tipless cantilevers was demonstrated in a biologically relevant application, by performing single-cell force spectroscopy of yeast cells.
  • One main advantage of this invention is the provision of micropipette that overcome the limitation of glass micropipettes limited in terms of throughput and geometry.
  • the present work introduces a novel technology based on highly flexible hollow cantilevers for cell patterning via controlled deposition and selective removal of single cells.
  • the flexibility of the method has been demonstrated using yeast and C2C12 cells, as well as primary hippocampal neurons.
  • Several patterning strategies have been tested in liquid environment with the use of fully cell adhesive and patterned adhesive/repulsive flat surfaces.
  • the key principle behind the method resulting in single-cell patterning with an accuracy of 5 ⁇ relies on the physical cell confinement enabled by the high flexibility of the polymeric cantilevers.
  • the developed precise cell-patterning techniques can become essential tools for studies related to signal processing and computation in neuroscience.
  • Providing single neuron-based networks with arbitrary topology can lead to a better understanding of the relationships between connectivity and function.
  • the versatility of the cantilever was proved by modifying in situ patterns previously realized. Additional cells could be indeed removed or deposited thanks to the robust hollow cantilever. This latter technique allows deeper studies for mechanical and cell interactions of cell networks.
  • the developed highly flexible hollow cantilevers represent a promising new tool for single-cell manipulations and patterning. Compared to other techniques requiring complex setups and feedback, the presented method works with simple components and does not require laser feedback. This makes it possible to parallelize the process, and to operate in complex, highly scattering samples in liquid environment. Photolithography process
  • the microfabrication process was entirely carried out in the BRNC cleanroom facilities (IBM Zurich Laboratories and ETH Zurich, CH).
  • a sacrificial release layer 21 of 5 nm chromium/ 50 nm gold/ 50 nm chromium was deposited onto a silicon wafer used as a carrier substrate (100 mm outside diameter 500 ⁇ thickness, SILTRONIX Silicon Technologies, France) by e-beam deposition (BAK501 , Evatec, CH). Wafers were cleaned in 5 min acetone and 5 min isopropanol in ultrasonic bath (Wet bench, Ramgraber, Germany) before dehydration bake at 200 °C for 5 min (M-HP 200 Hotplate, Ramgraber).
  • Alignment marks for proper alignment of the different SU-8 layers, were patterned by etching away the chromium top layer only at the mark spots using MaN 1405 negative photoresist.
  • MaN photoresist was spun at 3000 rpm for 30 s (OPTIspin SB20,
  • FIG. 1 A The bottom SU-8 layer 22 was patterned with spin-coating at 3000 rpm (SU-8
  • Fig. 1 B The copper seed layer 23 was deposited by thermal evaporation for a thickness of 150 nm in two steps with 75 nm each using a tilted plate at 35 degrees, an emission current of 3.6 A, a pressure of 5x10 "7 bar and a deposition rate of 0.45 nm/s (PLS 500, Pfeiffer, CH).
  • Fig. 1 C Positive AZ4562 photoresist 24 was used to confine the growth according to the following parameters: spin-coating at 4000 rpm for 40 s, soft bake for 1 min at 65 C and 1
  • Fig. 1 D Copper plating 25 was performed using copper sulfate solution (InterVia Cu 8540, Rohm and Haas, USA) and commercial setup (Ludertechnik AG, CH) for current and voltage applied of respectively 160 mA and 10.2 V during 3 min for copper thickness growth of 3 pm, after electrically contacting the wafer edges at eight equidistant locations. Thickness was controlled with a stylus profiler (Dektak 6M, Veeco, USA).
  • AZ4562 24 was stripped within 10 s in an acetone bath before etching of the seed copper layer 23 for another 5 s (Copper etchant, Sigma Aldrich) and rinsing for 2 min in Dl- water.
  • Fig. 1 F The top SU-8 layer 26 was patterned with spin-coating at 4000 rpm for 30 s (SU-8
  • Fig. 1 G The thick block SU-8 layer 28 (SU-8 100, Microchem) was patterned with spin- coating at 1250 rpm for 40 s, break of few hours to flatten the thick layer, soft bake for 30 min at 65 °C and 75 min at 95 C, cooling down on hot plate back to room temperature, exposure
  • SU-8 cantilevers were coated with a thin reflective layer of 2 nm Cr/ 10 nm Au (E306A Coating System, Edwards, England). Parafilm M substrate (Bemis, USA) was used to carry the devices (excluding cantilevers bending). Hollow SU-8 polymeric cantilevers for controlled single-cell deposition and patterning decribed above were fabricated following the protocol described above. The process based on electroplated copper as sacrificial layer was adapted for the cell-related experiments by producing cantilevers with integrated microchannels of 22 ⁇ in height.
  • SU-8 cantilevers were glued on a PMMA clip containing a 25 ⁇ _ reservoir connected to a pressure controller (Cytosurge, CH) through a tubing system allowing to apply a large range of pressure from -800 mbar to + 1000 mbar.
  • a pressure controller Cytosurge, CH
  • the SU-8 device was completely flushed with Dl-water for 15 min after plasma activation for 5 min (PDC-32G, Harrick
  • the flow rate was calibrated using a digital liquid flow sensor placed close to the reservoir (LG16- 0025-D, Sensirion, CH).
  • the AFM experiments were carried at 23 °C with a NanoWizard® I BioAFM from JPK Instruments (Germany) mounted on an optical microscope (Ax i overt 40 MAT, Zeiss).
  • the AFM was operated with a 15 pm range piezo-stage using the force spectroscopy mode.
  • all cantilevers were kept at rest in liquid from 30 min to 1 h until voltage- deflection signal became stable. Drift is inevitable with the use of polymeric SU-8 cantilevers.
  • high overexposure and baking time probably limited the voids rate within the SU-8 matrix, capable of absorbing liquid.
  • the SU-8 cantilever spring constant was calibrated using the thermal noise via the conventional Sader's method in air environment on glass substrate with the SPM Control Software V4 (JPK).
  • JPK SPM Control Software V4
  • the sensitivity deflection measured with regard to the optical lever sensitivity (OLS) followed the configuration where only the end of the cantilever was touching the substrate.
  • Force spectroscopy curves were obtained by selecting optically a single yeast and subsequently approaching the cantilever on top by systematically using a set point fixed at 50 nN. Approach and retraction curves were both recorded over a length of 10 pm for 5 s each (i.e. speed of 2 pm/s). Approach was followed by a contact pause of 5 s during which force was kept constant and an underpressure of -500 mbar was applied to immobilize the yeast cell at the aperture of the cantilever before subsequent retraction. Finally, every yeast was released by applying an overpressure of 1000 mbar before targeting a new yeast.
  • Hollow cantilevers were connected to a pressure controller (Cytosurge, Switzerland) and mounted on an atomic force microscope (NanoWizard 1 Bioscience AFM; JPK Instruments AG, Germany) for controlling their z-position with sub-pm precision (no laser force feedback has been used for the experiments).
  • Free-hanging cantilevers had an inclination angle of 8- 10 with respect to the substrate.
  • the AFM stage was placed on an inverted microscope (Axio Observer.ZI ) equipped with a Colibri LED light source system (both from Carl Zeiss AG, Switzerland), an incubation chamber, and a CCD camera (C9100-13; Hamamatsu, Japan).
  • the temperature control of the incubation chamber has been used only for the neuron related experiments keeping the setup at 37 °C, the other experiments were performed at room temperature.
  • CLSM images were taken using a LSM 510 mounted on an Ax i overt 200 M motorized microscope (Zeiss), while scanning electron microscope (SEM) images were taken with the help of an NVision 40 (Zeiss) using accelerating voltages in the range of 5-10 kV.
  • Fluorescent cell-adhesive solution was prepared by mixing 100 pg/ml Poly-D-lysine (PDL, P6407 70-150kDa; Sigma-Aldrich) with 40 g/ml fluorescently tagged secondary antibody (anti-mouse IgG (H+L), CF 555; Sigma-Aldrich) diluted in phosphate-buffered saline (PBS, pH 7.4; Thermo Fisher Scientific AG, Switzerland).
  • PLL-g-PEG solution was prepared from powder (PLL(20)-g[3.5]-PEG(2); SuSoS AG, Switzerland) and diluted in PBS to a final concentration of 100 g/ml if not indicated otherwise.
  • Glass substrates (GWSB-5040, WillcoWells, NL) were plasma treated for 30 s.
  • PDA-coated substrates were initially plasma treated for 30 s, immersed in a 10 mM TRIS HCL solution pH 8 containing 4 mg/mL PDA (Sigma Aldrich) for 1 h, then intensively washed with filtered PBS and dried with N2-
  • Wildtype Saccharomyces cerevisiae were grown in yeast extractpeptone-dextrose medium (10 g yeast extract (Oxoid), 20 g bactopeptone (BD) and 20 g glucose (Sigma- Aldrich) in 1 L ddH20, with 15 g Agar (Sigma-Aldrich) for solid media).
  • yeast extractpeptone-dextrose medium 10 g yeast extract (Oxoid), 20 g bactopeptone (BD) and 20 g glucose (Sigma- Aldrich) in 1 L ddH20, with 15 g Agar (Sigma-Aldrich) for solid media.
  • the yeasts were plated onto YPD plates and incubated for 2-3 days at 28 °C. Until the force spectroscopies were performed, colony containing plates were kept for up to three month at 4 °C.
  • cantilevers were coated with PLL-g-PEG (0.5 mg/ml in PBS) for 45 min then flushed with fresh PBS to make the surface anti-adhesive.
  • PLL-g-PEG 0.5 mg/ml in PBS
  • individual colonies were resuspended in PBS and injected into the reservoir of the cantilevers at a concentration of 100 ⁇ 00 cells/ml.
  • C2C12 cells were resuspended at a concentration of 100 ⁇ 00 cells/mi in HBSS (Hank's Balanced Salt Solution H8264; Thermo Scientific).
  • Substrate preparation Glass bottom dishes (GWSB-5040; WilICo Wells B.V., The Netherlands) were used for all the experiments.
  • the dishes were oxygen plasma activated for 2 min at 18 W, then were either filled up with PBS, or incubated with a non-fluorescent PDL solution for 1 h, rinsed 3 times with Milli-Q water, and blow dried with nitrogen gas to create a fully adhesive surface.
  • dishes were filled with medium corresponding to the cell type used.
  • cells were seeded at a density of 150 ⁇ 00 cells/cm 2 and incubated for 20 min.
  • Patterned cell-adhesive surfaces for neuronal cultures have been prepared with the help of a hollow cantilever as a probe.
  • a tipless hollow cantilever was filled with the fluorescent cell-adhesive solution, connected to the pressure controller, and then mounted on a standard AFM head (FlexAFM; Nanosurf, Switzerland).
  • 10 mbar overpressure was applied from the pressure controller to continuously push the patterning solution out of the probe.
  • dishes were rinsed with Milli-Q and blow dried with nitrogen gas, and then PLL-g-PEG solution was added in order to increase the contrast between patterned and non-patterned surface regions by backfilling. After 1 h of incubation the dishes were rinsed three times with PBS, then were filled up with cell culture medium and stored in the incubator until cell seeding.

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Abstract

La présente invention concerne un dispositif comprenant une tige souple, et la tige comprend un canal microfluidique ayant une ouverture, en particulier une aiguille souple creuse, plus particulièrement une micropipette souple ou un porte-à-faux creux souple, et son procédé de fabrication, le procédé comprenant un dépôt et un durcissement couche par couche, le canal microfluidique étant formé au moyen d'une couche de corps de remplissage sacrificielle constituée de métal. Ainsi, l'invention se rapporte à la production de formes de pointe complexes en parallèle et, en outre, l'intégration de détection de force sur les dispositifs.
PCT/EP2017/056755 2016-03-22 2017-03-22 Aiguilles creuses souples et porte-à-faux et leurs procédés de fabrication WO2017162708A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014139031A1 (fr) * 2013-03-15 2014-09-18 Concordia University Procédés de fabrication de nanostructures morphologiquement transformées (mtns) et matériaux polymères nanocomposites réglables et dispositifs utilisant lesdits matériaux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014139031A1 (fr) * 2013-03-15 2014-09-18 Concordia University Procédés de fabrication de nanostructures morphologiquement transformées (mtns) et matériaux polymères nanocomposites réglables et dispositifs utilisant lesdits matériaux

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANGELO GAITAS ET AL: "SU-8 microcantilever with an aperture, fluidic channel, and sensing mechanisms for biological and other applications", JOURNAL OF MICRO/NANOLITHOGRAPHY, MEMS, AND MOEMS, vol. 13, no. 3, 15 September 2014 (2014-09-15), US, pages 030501, XP055300803, ISSN: 1932-5150, DOI: 10.1117/1.JMM.13.3.030501 *
VINCENT MARTINEZ ET AL: "Controlled single-cell deposition and patterning by highly flexible hollow cantilevers", LAB ON A CHIP: MINIATURISATION FOR CHEMISTRY, PHYSICS, BIOLOGY, MATERIALS SCIENCE AND BIOENGINEERING, vol. 16, no. 9, 9 February 2016 (2016-02-09), GB, pages 1663 - 1674, XP055300678, ISSN: 1473-0197, DOI: 10.1039/C5LC01466B *
VINCENT MARTINEZ ET AL: "SU-8 hollow cantilevers for AFM cell adhesion studies", JOURNAL OF MICROMECHANICS & MICROENGINEERING, INSTITUTE OF PHYSICS PUBLISHING, BRISTOL, GB, vol. 26, no. 5, 5 April 2016 (2016-04-05), pages 55006, XP020302064, ISSN: 0960-1317, [retrieved on 20160405], DOI: 10.1088/0960-1317/26/5/055006 *

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