Improvements in polymer membranes and improvements relating to polymer membranes

Abstract

Disclosed is a method for producing a polymeric porous membrane suitable for liquid filtration or analyte capture, comprising the steps of providing a flowable composition (100) on a substrate (220), the composition comprising at least photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules; providing one or more laser light pulses (L) at at least one focal point in the composition with sufficient energy to locally polymerize the composition; moving the or each focal point to a plurality of additional locations relative to the already polymerized composition in a sequential or stepwise predetermined manner; and repeating the pulses at the additional locations to polymerize a three-dimensional matrix of the composition while leaving unpolymerized regions the same size as the pores of conventional polymeric membranes.

Description

[Technical field]

[0001] The present invention relates to polymeric porous membranes and methods for making same, particularly but not exclusively for use in flow-through filtration of fluids or capture of analytes. [Background technology]

[0002] Porous membranes have many biological applications, including applications in bioprocessing, biopharmaceutical processing, and cell and gene therapy. The ever-increasing understanding of biological mechanisms and cellular activity requires more controlled membrane filtration to better distinguish between retentate and filtrate resulting from filtration. Typically, these retentate or filtrate are target molecules or substances, such as cells, proteins, peptides, DNA, RNA, antibodies, viruses, bacteria, or phages, or other microorganisms or macromolecules. Commercially available polymeric membranes consist of a generally random array of pores formed by casting a liquid web of polymer dissolved in a solvent that spreads over a supporting substrate, drying the web until the solvent evaporates, and leaving behind a residual solid polymer structure with a large number of pores. One method of manufacture is described in more detail in U.S. Patent Application Publication No. 20180104655(A1), which is incorporated herein or relied upon in conjunction with the publications cited therein as an enabling disclosure of the general configuration of known polymeric membranes.

[0003] The pore size, pore size uniformity, pore shape, pore size deviation from one side of the membrane to the other, and number of pores per unit area are highly dependent on the composition of the initial casting solution, the thickness of the web, and the drying conditions of the web. Making membranes of new thicknesses or with new pore sizes, or with new materials, requires some amount of trial and error, even for experienced membrane manufacturers. Typically, membranes with thicknesses ranging from 4 μm to 1 mm are used, with pores averaging 0.1 to 10 μm in size.

[0004] Another technique for producing membranes with very small pore sizes, e.g., pore sizes of 0.1 μm and 10 μm, is to irradiate polymers such as polycarbonate with pulses of electron beams, similar to shotgun blasting. This technique is known as "track etching" and an example is described in U.S. Patent No. 5,449,917. This technique results in a highly random pattern of fine pores with inconsistent properties, even when consistent fabrication parameters are used.

[0005] A three-dimensional (3D) microfabrication method is known that uses ultrashort laser pulses to create solid objects based on the so-called two-photon polymerization of a volume of photosensitive material. Focusing on a volume of photosensitive material, the pulses initiate polymerization via two-photon absorption and subsequent polymerization. In a dimensionally controlled environment, the photosensitive material is irradiated with a laser in a pattern similar to the desired 3D shape, and after subsequent washing away of the non-irradiated external areas, a polymerized material of the desired 3D shape remains. This allows the processing of any computer-generated 3D structure by direct "recording" with the laser in the volume of photosensitive material. Under these conditions, fabrications with elements with dimensions of about 100 nm or more are possible.

[0006] 3D fabrication of membranes is reviewed but largely rejected in "Perspective on 3D printing of separation membranes and comparison to related unconventional fabrication techniques", Journal of Membrane Science, 2017, 523, pp. 596-613. However, the time required to fabricate such pores by the aforementioned conventional techniques is deemed impractical, and thus 3D fabrication of micropores in membranes is largely rejected. For example, the above paper does not mention pore density, but lists the time required to fabricate a 100 mm x 100 mm membrane sample with pores of 100 nm size, which is estimated to be a practically useful pore density, as several months. Also, no practical techniques for fabricating such membranes are disclosed.

[0007] Conventional membranes rely on a variety of filtration mechanisms, including both surface filtration, based on sieving action, and depth filtration, where filtration relies at least in part on physical retention by blocking pore structure, specific or non-specific surface interactions, or surface modification with affinity ligands. Known depth filtration structures may include sandwiches of multiple layers of membranes, where the pore size, pore structure, porosity, tortuosity, and other physical pore properties in each layer may be varied to create asymmetries along the paths of liquids and particles passing through the membrane.

[0008] Traditional filtration mechanisms include retention of waste products (clarification or flow-through chromatography), retention and subsequent release of targets such as proteins, cells, etc. in depth filtration (also called purification), or retention of products in surface filtration upstream of size exclusion filters (commonly called ultrafiltration or diafiltration, including cell washing). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 20180104655(A1) [Patent Document 2] U.S. Patent No. 5,449,917 [Non-patent literature]

[0010] [Non-Patent Document 1] "Perspective on 3D printing of separation membranes and comparison to related unconventional fabrication techniques", Journal of Membrane Science, 2017, 523, pp. 596-613 Summary of the Invention [Problem to be solved by the invention]

[0011] Because the conventional filtration mechanisms described in the above two paragraphs are part of conventional processes, any improved membrane must be compatible with such mechanisms and therefore must have roughly equivalent physical properties. [Means for solving the problem]

[0012] The inventors have addressed this problem in a novel way by employing an entirely new approach to fabricating polymeric membranes. The inventors have realized that in one approach, low energy laser photopolymerization of monomeric or oligomeric compositions can be used to create polymeric structures or portions thereof having solid photopolymerized regions similar to the polymeric structures of conventional polymeric membranes, and can leave behind non-polymerized regions similar to the pores left by solvent evaporation in conventional polymeric membranes, which can be removed from the remaining polymerized regions by washing with a solvent. In a modification of this approach, the photopolymerized regions can each surround a non-photopolymerized region, such that once the non-polymerized regions (pores) have been washed away, the surrounded non-polymerized regions can be subjected to extensive photopolymerization to reduce processing time. In another approach, low energy laser photopolymerization of compositions can be used to create male polymeric structures having solid polymerized regions similar to the pores of conventional polymeric membranes, leaving behind non-polymerized regions of the composition similar to the polymeric structures of conventional polymeric membranes. This is followed by washing of the non-polymerized composition, leaving only the male regions, followed by casting of additional solid material around at least the male polymer structures, which are then removed, leaving only the additional solid material, thereby providing pores left by the removal of the male polymer structures that resemble the pores of conventional polymer membranes. In fact, the term "similar to" in this paragraph means "has the same effect as", since the pores and surrounding material of conventional polymer membranes are obtained as a result of solvent evaporation or electron beam irradiation, and therefore have a generally random morphology, while photopolymerization is carried out in a dimensionally controlled manner, and therefore does not need to have randomness unless desired. And this means that the pore size, pore shape, pore geometry (e.g., helical / curved pores), pore density, and pore size uniformity can be achieved virtually arbitrarily within the manufacturing tolerances. Moreover, this means that the manufacturing method described herein allows for complete control of the important geometry of the pores to be created without the randomness of conventional membranes, and therefore does not have to accept the shortcomings of conventional polymer membranes.For example, the tortuous paths formed by solvent evaporation during the manufacture of nitrocellulose membranes can be optionally eliminated or emulated.

[0013] According to a first aspect of the invention, the invention provides a method for producing a polymeric porous membrane suitable for filtering liquids or trapping an analyte, comprising the steps of: The method includes providing a flowable composition on a substrate, the composition comprising at least photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules; providing one or more pulses of laser light at at least one focal point in the composition of sufficient energy to locally polymerize the composition; and moving the or each focal point to a plurality of additional locations in a continuous or stepwise predetermined manner relative to the already polymerized composition and repeating the pulse at the additional locations such that a three-dimensional matrix of the composition is polymerized while leaving unpolymerized regions the same size as the pores of a conventional polymer film.

[0014] In this manner, polymeric membranes with predictable pore sizes and pore shapes can be produced and the unpredictable nature of conventional evaporation processes can be largely eliminated, the only limitations being the manufacturing tolerances of the methods just described.

[0015] In one embodiment, the composition is a solution containing photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules, and the substrate is immersed in the solution, for example in a bath of the solution or a replenishment amount of the solution.

[0016] In one embodiment, the laser light pulses are delivered by a laser through an objective lens that focuses light at a focal point or multiple focal points along a Z axis generally parallel to the direction of propagation of the laser light through the lens, and further through optical elements that can move the focal point or each focal point along the Z axis and / or transversely to said Z axis, e.g. along an X axis perpendicular to said Z axis and / or in a Y axis perpendicular to both the Z and X axes, and at least some of said multiple positions are provided by control of the laser and optical elements.

[0017] In one embodiment, the substrate and polymerized composition are moved in the X, Y, and / or Z directions to provide a plurality of locations, or a further portion of the plurality of locations.

[0018] Thus, the polymerization focal position of the laser light can be moved relative to the composition by optical means, and / or the already polymerized polymer composition can be moved relative to the position of the laser light by mechanical means. Since optical operations are relatively fast and accurate, but relatively limited in size, one small first area of ​​the film, or one layer of the film, can be produced by optically repositioning the light before mechanically moving the area where the new part of the film was formed a relatively long distance to a second area adjacent to the first area, so that the optical repositioning operation can start again in the second area. Such a process can be repeated many times in subsequent areas until multiple areas of the formed film, or the entire film formed from the parts formed by optically repositioning the polymerization position, are produced. However, for small areas, only optical repositioning of the laser light can be performed with good results. Once the film has been moved to the second or subsequent region, a microscope and image recognition software can be used to recognize the pore (or partial pore) pattern in order to realign the polymerized film to the laser focus, and then the film can be precisely aligned with respect to the laser focus by further moving the film or adjusting the focus position so that the pore pattern created in the second or subsequent region matches the pore pattern already created. Similar techniques can be used after a layer has been created to provide a datum for subsequent layers, such that asymmetric pores can be created, for example, by intentionally moving one layer away from the layer below, forming zigzag pores, for example.

[0019] In one embodiment, the focused laser light pulses have a low degree of photon count consistency, eg, dominated by two-photon absorption.

[0020] In one embodiment, the at least one focal point is a split laser beam and / or multiple focal points resulting from multiple laser sources. Conveniently, the focal points are a linear or two-dimensional array, spaced apart at a pitch of the desired pore spacing or a multiple of said pitch. Conveniently, the linear or two-dimensional array of focal points are optically movable together to maintain their spacing.

[0021] In one embodiment, polymerization is only performed at locations intended to become pore walls. The gaps between the polymerized pore walls remain unpolymerized. In that case, the method then further comprises removing the non-polymerized composition from the areas of the membrane corresponding to the pores, and then extensively photopolymerizing the non-polymerized gaps by applying a polymerizing light over the entire membrane or to a contiguous area thereof. Conveniently, the top and bottom of the membrane are also formed prior to said further extensive photopolymerization.

[0022] In this way, only the pore walls and outer surfaces of the membrane need to be photopolymerized under dimensionally controlled conditions, essentially forming a shell, while the interior regions of the final membrane structure remain liquid. Large-area photopolymerization of the liquid composition of the remaining interior space can be achieved by using a broad-area light source, such as a high-energy laser or multiple high-energy lasers, once the non-polymerized composition of the pores has been flushed out. Polymerization by heat or UV light is also possible. This technique significantly reduces manufacturing time, since much of the polymerization can be done without dimensional control.

[0023] According to a second aspect, the present invention provides a method for producing a polymeric porous membrane suitable for filtering liquids or trapping an analyte, comprising the steps of: providing a flowable composition on a substrate, the composition comprising at least photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules; providing one or more pulses of laser light at at least one focal point in the composition with sufficient energy to locally polymerize the monomer; and moving the focal point or focal points of the composition to a number of locations in a continuous or stepwise predetermined manner and repeating the pulsing at those locations so that three-dimensional pillars are formed that are the same size as the pores in the membrane; Including, removing the non-polymerized composition; filling the area around the pillars with membrane material; and then removing the pillars from the membrane material to leave openings in the membrane material. The method further comprises:

[0024] The present invention covers any and all combinations of features disclosed herein, whether or not such combination is expressly referred to in the specification. Furthermore, where two or more features are referred to in combination, it is intended that such features may be claimed separately without broadening the scope of the invention.

[0025] The invention can be implemented in many ways, and example embodiments thereof are described below with reference to the drawings. [Brief description of the drawings]

[0026] [Figure 1] FIG. 2 shows a micrograph of a membrane according to the present invention. [Diagram 2] FIG. 13 shows micrographs of further membranes. [Diagram 3] FIG. 13 shows micrographs of further membranes. [Figure 4a] FIG. 4 illustrates a technique for producing the membrane shown in FIG. 1, FIG. 2 or FIG. 3. [Figure 4b] FIG. 4 illustrates a technique for producing the membrane shown in FIG. 1, FIG. 2 or FIG. 3. [Figure 4c] FIG. 4 illustrates a technique for producing the membrane shown in FIG. 1, FIG. 2 or FIG. 3. [Diagram 5] FIG. 1 illustrates a manufacturing facility for producing the membranes described. [Figure 6] FIG. 1 illustrates a manufacturing facility for producing the membranes described. [Figure 7] FIG. 4 shows a variation of the membrane manufacturing technique shown in FIGS. 4a to 4c. [Figure 8] FIG. 13 shows a variation of the completed membrane. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention, together with its objects and advantages, may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numbers or reference numbers having the same last two digits indicate similar elements, and in which:

[0028] Referring to Figure 1, a micrograph of a printed polymer film 10 having pores 2, produced according to the procedure described below, is shown in plan view. In this example, roughly circular pores 12 are shown as black regions about 2.5 μm in diameter, with a white "halo". The "halo" is due to the effect of charge concentration at sharp edges, which saturates the microscope's pixels as the dynamic range of the optical system is not wide enough to show the edges.

[0029] Referring to FIG. 2, a photomicrograph of a membrane 20 formed from regions A, B, and C with different sized printed test holes, each printed with intentionally drawn lines, is shown, all at a smaller magnification than membrane 10 shown in FIG. 1. The pore sizes are approximately 4.5, 4, and 1.6, 2.5 μm, respectively. The multiple pores shown are suitable for use as membranes of the type mentioned above. Using the procedure described below, the pores were produced by solidifying a liquid polymer layer by layer, leaving the pore size regions unsolidified, and then washing the unsolidified polymer away from the pore size holes to reveal the pores. Each region, such as A, B, and C, was printed sequentially using optical manipulation before moving to the next region by mechanical movement of a portion of the membrane against the optical element used to form each pore.

[0030] FIG. 3 shows a further membrane 30 made in the same way as above, but with different regions D and E also having a plurality of pores 32 with a much smaller diameter, in this case pores of 2 μm and 1 μm, respectively. It will be clear that the spacing between the pores 12, 22, 32 shown may be increased or decreased to some extent, and the repeated lattice pattern may be an irregular or different pattern, for example a tessellation pattern of regularly shaped pores. The pore shapes may also be other than circular, for example hexagonal pores may be used, where the hexagonal pores are tessellated like a honeycomb to reduce the amount of material polymerized. In this way, membranes with the desired properties similar to those of membranes produced by conventional methods can be achieved. The pore density may be adjusted depending on the application of the membrane, but can be up to 70% of the pores relative to the solid material density, for example with regular rows of pores spaced apart by about 0.018 D, where D is the average pore diameter and the rows are staggered.

[0031] Referring to FIG. 4a, an example of a manufacturing technique is shown in schematic form. A cross-section of a portion of a bath of liquid composition 100 containing a silicone rubber substrate bed 220 is shown. The composition includes a mixture consisting of at least photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules, as described below. In this figure, linear continuous pieces 114 of polymerized composition 100 are formed using a laser beam L having femtosecond laser pulses activated by a controller (FIG. 5, 250) focused to a fixed piece height in the Z axis when the focal point is aligned with the desired XY coordinate axis. Here, Z is up and down the page of the figure, X is left and right of the figure, and Y is into and out of the figure. Such pulses are controlled to form overlapping polymerized pieces 114, but leave unpolymerized "pores" 112, and the laser is then optically repositioned to an adjacent location to repeatedly effect solidification of the next continuous piece 114. In the figure, a laser focus P is scanned in the X direction (from left to right) and is energized periodically to create successive strips 114, but not at the locations where holes 112 are intended to be placed.

[0032] In Figure 4b, a second layer of polymerized particles is created that slightly overlaps the first layer shown in Figure 4a by rescanning the focus P in the same pattern as Figure 4a, but raised in Z by a height less than one particle size so as to overlap the first successive particle, but again without current passing over the pore region 112. This layer-by-layer polymerization can be repeated many times to create a film of any desired thickness with pores of almost any size and with any pore pattern.

[0033] FIG. 4c shows the final membrane 140 formed with more flake layers formed on top of the previous layer with non-polymerized pore regions 112. In reality, by washing the membrane 140 with a solvent, the non-polymerized composition is removed and these pore regions 112 are left empty. For clarity, the flakes are shown much larger than they will actually be formed. The interstitial regions 118 formed between the edges 120 of the pore regions 112 are shown polymerized. However, it is within the scope of the invention to leave at least a portion of those interstitial regions 118 unpolymerized, and once the pore regions 112 have been washed away, a final extensive polymerization can be performed that also polymerizes the regions 118.

[0034] For convenience, pore region 112 is shown as a simple vertical pore path, but other shapes of pore paths are just as easily fabricated, e.g., helical pores, zigzag pores, pseudorandom paths, etc., that may actually be better shapes for retaining or capturing analytes of interest, e.g., large molecules such as proteins or cells, but allowing other materials to pass through the membrane. Such tortuous paths are provided for the purposes of depth filtration as discussed above.

[0035] It should be understood that Figures 4a, 4b and 4c are schematic representations of cross-sections of the membrane with the pores 112 exaggerated relative to the solidified regions 118 for ease of understanding.

[0036] The pore size may be less than 5 μm or greater than 5 μm, with typical pore path lengths (not necessarily the membrane thickness) being 5, 10, 20, 50, or 100 times the pore size. For long pores with relatively small diameters, if only the pore edges 120 are polymerized and the regions 118′ are left liquid, the long hollow structure will be fragile once the liquid in the pore regions 112 is washed away. It is possible to strengthen these solidified edges 120, for example by polymerizing braces 121 that join adjacent edges 120 of adjacent pore regions 112. To increase strength and stiffness, multiple braces 121 may be used between the pore edges 120, for example cross braces 121 as shown in FIG. 4b and FIG. 4c.

[0037] FIG. 5 shows an example of an apparatus 200 used to produce the aforementioned films. In this example, a laser 202 is directed to transmit a coherent light beam L to two galvanic reflecting mirrors 204 and 206, each under the influence of a controller 250. An adjustable objective lens or multiple objective lenses 208, also under the influence of a controller 250, are used to precisely focus the beam L at a polymerization point P in the chamber 100 for the aforementioned polymerization. The focal point P is rapidly repositioned by controlled movement of the mirrors 204 and 206, and rapid firing of the laser with femtosecond laser pulses achieves the aforementioned polymerized continuous particles 114 within the illustrated area A. Layers of particles can be created by adjusting the height Z of the lens 208 using a linear actuator 212 under the control of the controller 250. The chamber 100 is repositioned in the X-axis direction using a further linear actuator 210, and in the Y-axis direction by a similar arrangement not shown. In either case, the actuator is under the influence of a controller 250 so that the next regions B and C etc. can be polymerized in the same way before the next region is selected by rapid movement of the aforementioned mirror. For better accuracy, the space between the objective lens 208 and the glass layer 125 on the vessel 100 is filled with optically transparent oil 105 so that most of the optical path of the laser light pulse L passes through a material with approximately the same refractive index.

[0038] Although the production by the above apparatus is sufficiently fast, it is desirable to produce faster. Therefore, it is preferable to use multiple laser beams L, as shown in FIG. 6. The multiple laser beams L come from multiple lasers 211 in the apparatus 200' in this example, but can also be generated by splitting a more powerful laser beam into multiple beams using multiple beam splitters. The latter approach is cheaper, but requires more optical alignment, so multiple lasers are preferred. The apparatus 200' shown in FIG. 6 operates in a similar manner to that described above in connection with FIG. 5, except that multiple lasers 211 are collected and their output beams are formed into a focused pattern. In this example, nine lasers are arranged in a two-dimensional pattern of 3×3. The beams propagate towards a bath of polymer composition 100 as described above, and can be moved in stages to form polymerized pieces as detailed above.

[0039] To reduce heat generation, the laser beam L (FIG. 5) or multiple laser beams L' (FIG. 6) may move a distance greater than the width between adjacent pores (i.e., pore pitch) and / or the beams L' may be spaced apart by more than one pore pitch. Thus, for example, if the beam L' moves X-1 pitches in steps spaced apart by X pitches, the remaining pores, except for the end pores, may be formed with less heat generation than if they were moved only one pitch at a time. Although FIG. 6 shows multiple lasers, it would be possible to use LED laser arrays instead of these lasers.

[0040] Regardless of which polymerization method is used, successive layers of polymerized material can result in reduced transparency and diffraction issues. To mitigate these issues, laser light systems can utilize accurate modeling of the underlying 3D structure by evaluating and optimizing the local light fluence, angle, etc. and input energy that are the main causes of non-uniformity in the structure, diffraction pattern, etc.

[0041] Another membrane fabrication technique is shown in Figure 7, where a male mold with features 312 is formed by photopolymerization using the same technique as described above. The unpolymerized remainder of the polymer 318 is washed away leaving the male mold. The voids 318 can now be filled with a membrane-forming material, such as a filler thermosetting polymer. Once the filler polymer has cured, the mold may be removed mechanically, by washing with a suitable solvent, or using heat, leaving behind pores corresponding to the pores 12, 22, 32, 112, etc. previously described.

[0042] An example of composition 100 and manufacturing procedure is provided below. In one example, the flowable composition 100 includes a transparent photoactivatable acrylate monomer resin doped with up to 3% of a photoactivatable initiator, such as an acylphosphine oxide, such as a 2,4,6-trimethylbenzoyldiphenylphosphine oxide molecule, or a benzophenone, xanthone, or quinone, or combinations of these molecules, and a photoactivatable quencher, such as a tertiary amine molecule. The laser may be an exciplex laser (also known as an excimer laser) with an output wavelength of about 800 nm and a pulse length of 10-100 femtoseconds (fs), although a pulse length of about 40-60 fs, e.g., 50 fs, is preferred, with a possible repetition rate of about 5 MHz.

[0043] When the pores are dimensionally most critical, the gaps between the pores can be filled with lower resolution, for example by using a higher energy laser light, which allows a larger area to be photopolymerized faster, thereby speeding up the manufacturing process, or by the introduction of material by jetting, i.e. the introduction of a liquid thermoplastic (which does not necessarily have to be the same material as the material surrounding the pores).

[0044] The energy required to cause localized polymerization is provided by a laser pulse focused at at least one focal point in the composition 100, which causes two-photon absorption polymerization, i.e. two or more photons are simultaneously absorbed by the light-activated initiator (photoinitiator) described above, generating active species that initiate the polymerization of the monomer resin. Under these conditions, multiphoton absorption occurs only in the areas where the light intensity is at a maximum. This limits the polymerization to the volume (known as a voxel) of the focused laser beam. In this way, small pieces of polymerized material are created that overlap slightly, for example 25%. Quenching molecules quench the fluorescence and inhibit or stop the dendritic spreading of the polymer branches, resulting in a more integrated and well-defined polymerized mass. The unsolidified parts of the resin are washed away using an organic solvent to reveal the film.

[0045] The use of the above techniques and materials results in a suitable equivalent of a conventional layered nitrocellulose membrane or a Track-Etched membrane, including its membrane thickness and pore density. However, it is believed possible to provide modifications that improve the properties of the membranes made according to the present invention. For example, as shown in FIG. 8, a membrane 10' is shown having fibers 411, e.g., nanofibers, introduced into the composition bath 100 during manufacture that do not unduly impede the formation of the pores described above. The finished membrane 10' has improved mechanical strength. Additionally or alternatively, a mesh or web of material (not shown) having a relatively loose weave compared to the size and arrangement of the membrane's pores may be immersed in the bath prior to polymerization to achieve the same effect. Once the pores are formed, the mesh or the like is incorporated into the membrane, which likewise significantly strengthens the membrane, thereby increasing the transmembrane pressure.

[0046] The aforementioned substrate support 220 is intended in the above example to have a removable surface for forming the membrane. The surface of the substrate 220 may also be used to place one or more microsensors 400 (FIG. 8) thereon, and if necessary, electrical conductive paths 410 for communicating with and / or powering those sensors are formed on the surface of the support 220, for example by 3D printing, before the polymer bath is placed. By peeling the support 220 from the finished membrane, or vice versa, the sensor and any conductor tracks are left in place and ready to be used on or in the finished membrane. In this case, the microsensor 400 is a capacitive gap sensor that measures the transmembrane pressure difference, which can give an indication of the membrane's filter performance. Other sensors, such as other pressure sensors, flow sensors, conductivity sensors, pH sensors, osmolality sensors, chemical composition or concentration sensors, etc., may also be used, which can give data in real time as the filtration is performed, such as the membrane's performance. In another example (not shown), the membrane may be formed on a substrate that includes an absorbance sensor, where the membrane includes an inlet and an outlet for the sensor, so that the light absorption performance of the fluid passing through the membrane can be monitored remotely. This allows monitoring of protein concentration, etc. In yet another variation, the membrane may be formed on a microfluidic valve and on a pressure sensor that can generate a signal to open the valve, for example, when the transverse pressure difference across the membrane exceeds a predetermined amount. Other sensors may be used, such as electrical conductivity sensors or temperature sensors that send a signal indicating a membrane failure when an electrical path is interrupted, such as when the membrane breaks. More conventional additive manufacturing of low-resolution materials to manufacture additional sensors or other membrane accessories, such as frames or other physical supports, can be used with good results in conjunction with the membrane manufacturing methods described herein.

[0047] Although the illustrated embodiment results in a planar membrane, other configurations of membranes may also be useful, such as tubular membranes that function as hollow fibers in hollow fiber filtration, and thus the term membrane as used herein is intended to include sheet-like and other thin materials that are not necessarily planar.

[0048] While a number of embodiments have been described and illustrated, it will be apparent to those skilled in the art that additions, omissions, and modifications can be made to these embodiments without departing from the scope of the invention as claimed.

[0049] The techniques described above can be used to achieve asymmetry in single layer membranes, which may also have multiple asymmetries in physical properties (e.g., wider then narrower then wider pores) and / or undulating surface characteristics to facilitate surface or depth filtration, such as funnel-shaped pore openings or constricted pore openings.

[0050] Chemical ligands or anchors for binding ligands can then be printed, allowing for controlled placement and subsequent modification of anisotropic and asymmetric properties to improve functionality and / or make more efficient use of (expensive) ligands.

[0051] The above techniques require a relatively small optical head scanning distance to create a small polymerization area and then move the optical head to another area to be polymerized or move the material to be polymerized relative to the head. It is then desirable to have a matching pore pattern in each area, but this is not necessary. It is also possible to have pore breaks, folds, or pleats at the boundaries between areas. The folds of the membrane can be attached to a relatively rigid part to form a filter cassette or filter cartridge.

[0052] With respect to Fig. 8, above, techniques have been described for printing on known sensors or other hardware, thereby incorporating such hardware into the so-printed membrane. In other embodiments, it is possible to form the sensor or part of the sensor from a polymerized material. For example, with respect to the pressure sensor described above, it is possible to form a flexible, compressible cavity with walls formed from a polymerized material, where the sensing surface is precisely formed as a flexible pressure-sensitive membrane, and the remainder of the cavity acts as a fluid conduit to the pressure-sensitive sensor element, thereby allowing measurement of fluid pressure in a portion of the interior of the membrane.

[0053] Furthermore, in some embodiments, it is possible to provide a light conduit or light guide. Such a light guide can provide a second polymerization step, for example inside a less transparent structure. If the light has a terminal light diffuser or lens, the light guided to the partially polymerized film forming the structure can be used to fully polymerize the film.

[0054] Although the manufacture of individual sheet membranes has been described and illustrated, it will be apparent that other techniques may be used, for example continuous manufacturing techniques may be used, for example the completed membrane may be peeled from its support 220, rinsed to produce pores, and then wound into a roll.

[0055] Although the above assumes polymerization with single or multiple laser beams, multiple light paths can be created using holographic systems or beam splitters and multiple lenses. The polymerization materials can be selected to be sterilizable, for example with gamma radiation. Additionally or alternatively, sterilization can be achieved by manufacturing the membrane under aseptic closed conditions and using pre-sterilized materials. The sterile finished membranes are suitable for single-use applications, and if biocompatible materials are used, they can be part of the Good Manufacturing Process (GMP) and are suitable for medical applications such as cell and gene therapy. [Explanation of symbols]

[0056] 10, 10', 20, 30, 140 membranes 12, 22, 32, 112 pores 100 compositions 105 Oil 114 Continuous Pieces 118 Interstitial area 120 Edge 121 Brace 125 Glass Layer 200, 200' equipment 202, 211 Laser 204, 206 Mirror 208 Lens 210, 212 Linear actuator 220 Substrate 250 Controller 312 Processing 318 Residue, vacancy 400 Microsensor 411 Fiber L Pulse P focus

Claims

1. 1. A method for producing a polymeric porous membrane suitable for filtering liquids or trapping an analyte, comprising the steps of: providing a flowable composition (100) on a substrate (220), the composition comprising at least photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules; providing one or more pulses of laser light (L) at at least one focal point in the composition of sufficient energy to locally polymerize the composition; moving the or each focal point to a plurality of additional locations relative to the already polymerized composition in a continuous or stepwise manner; repeating the pulse at these additional locations so that the three-dimensional matrix of the composition is polymerized leaving unpolymerized regions of 0.1 μm to 10 μm in size; and removing said non-polymerized regions by solvent washing. Including, the photoactivatable monomer molecules comprise a transparent photoactivatable acrylate monomer resin; the photoactivatable initiator molecule comprises an acylphosphine oxide, a benzophenone, a xanthone, or a quinone, or a combination of these molecules; The method, wherein the photoactivated quencher molecules comprise tertiary amine molecules.

2. The method of claim 1, wherein the composition is a solution containing the photoactivatable monomer molecules, the photoactivatable initiator molecules, and the photoactivatable quencher molecules, and the substrate is immersed in the solution.

3. 3. The method according to claim 1 or 2, wherein the laser light pulses are provided by a laser light source, via an objective lens which focuses the light in one or more focal points along a Z-axis generally parallel to the propagation direction of the laser light through the lens, and via further optical elements which are capable of moving the or each focal point along an X-axis perpendicular to the Z-axis and / or in a Y-axis perpendicular to the Z-axis and the X-axis, and wherein at least some of the multiple further positions in the first region are provided by controlling the laser light source and the optical elements, and wherein the further optical elements are galvanic reflecting mirrors.

4. The method of claim 3 , further comprising repositioning the substrate and polymerized composition relative to the lens in at least the X and Y directions to irradiate a second area with the pulse of laser light.

5. The method according to any one of claims 1 to 4, wherein a laser pulse having a pulse length of 10 femtoseconds to 100 femtoseconds is used.

6. The method according to claim 1 , wherein the at least one focal point is a multiple focal point of a split laser beam and / or multiple laser sources.

7. 7. The method of claim 6, wherein the multiple focal points are arranged in a linear or two-dimensional array, the multiple focal points being spaced apart by a pitch of a desired pore spacing or a multiple of the pitch.

8. The method of claim 7 , wherein the linear or two-dimensional array of focal points are optically movable together while maintaining a spacing between the focal points.

9. 9. The method of any one of claims 1 to 8, wherein polymerization occurs only in locations intended to become the pore walls (120), with all or most of the gaps (118) between the polymerized pore walls remaining unpolymerized, the method further comprising the steps of removing non-polymerized composition from areas of the membrane corresponding to the pores, and then photopolymerizing the unpolymerized gaps (118) throughout the membrane by applying a polymerization light throughout the membrane.

10. 10. The method of claim 9, comprising polymerizing the top and bottom of the film prior to photopolymerizing throughout the film.

11. 10. The method of claim 9, wherein the gap includes overlapping braces (121) that act to support one pore wall (120) with another pore wall (120).

12. 1. A method for producing a polymeric porous membrane suitable for filtering liquids or trapping an analyte, comprising the steps of: providing a flowable composition on a substrate, the composition comprising at least photoactivatable monomer molecules, photoactivatable initiator molecules, and photoactivatable quencher molecules; providing one or more pulses of laser light at at least one focal point in the composition of sufficient energy to locally polymerize the monomer; and moving the or each focal point to a plurality of locations on the composition in a continuous or stepwise manner and repeating the pulsing at those locations to form three-dimensional columnar polymerized regions having dimensions between 0.1 μm and 10 μm; Including, removing the non-polymerized composition; filling the areas around the three-dimensional columnar polymerized regions with membrane material; and then removing the three-dimensional columnar polymerized regions from the membrane material to leave open pores in the membrane material. Further comprising: the photoactivatable monomer molecules comprise a transparent photoactivatable acrylate monomer resin; the photoactivatable initiator molecule comprises an acylphosphine oxide, a benzophenone, a xanthone, or a quinone, or a combination of these molecules; The method, wherein the photoactivated quencher molecules comprise tertiary amine molecules.

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