# US 2019/0170744 A1 · Micropatterning assembly, methods for micropatterning, and micropatterned devices

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- Number: US 2019/0170744 A1
- Document ID: US20190170744A1
- Status: Published application
- Kind: A1
- Application number: 16/324,728
- Filed: 2017-08-10
- Published: 2019-06-06
- Earliest claimed: 2016-08-12
- Assignees: Ist Austria (Institute of Science and Technology Austria)
- Applicants: Ist Austria - Institute of Science and Technology Austria
- Inventors: MICHAEL SIXT; Robert HAUSCHILD; Alexander Jan SCHWARZ; Maria NEMETHOVA
- Web page: https://patentgrep.com/patent/US20190170744A1

## CPC

- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding}
- G01N 33/569: for microorganisms, e.g. protozoa, bacteria, viruses
- C12N 2535/10: Patterned coating
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to the field of micropatterning. In particular, the present invention provides micropatterning assemblies and methods for micropatterning. Moreover, the present invention provides micropatterned devices obtained by using the micropatterning assemblies and/or methods of the invention. Furthermore, the present invention provides methods for using said devices.

## Claims

### Claim 1

An assembly for micropatterning a transparent solid carrier, the assembly comprising:
i) a stage adapted for mounting a transparent solid carrier thereto;
ii) a spatially light modulating optics comprising
a) a light source for providing light to said carrier along an optical path, wherein the light source comprises one or more LEDs;
b) a spatial light modulator (SLM) for generating a binary or greyscale pattern image of light, wherein the SLM is positioned in said optical path between said light source and said carrier;
c) first optical means for directing light from said light source along the optical path to said SLM; and
d) second optical means comprising an objective for directing the pattern image generated by said SLM along the optical path to said carrier such that the pattern image is projected onto said carrier; and
iii) control means having a digital input and connected to said SLM for providing control signals to said SLM to cause said SLM to generate a pattern image of light.

### Claim 2 (depends on 1)

The assembly of claim 1, wherein the stage is a motorized stage configured for positioning the transparent solid carrier in X-, Y-, and Z-direction.

### Claim 3 (depends on 1, 2)

The assembly of claim 1 or 2, further comprising a computer comprising software, wherein the software comprises a pattern generation system configured for generating pattern image data, and wherein the computer is configured for providing drive signals corresponding to said pattern image data to said control means.

### Claim 4 (depends on 3)

The assembly of claim 3, wherein the software is adapted to control the motorized stage.

### Claim 5 (depends on 3, 4)

The assembly claim 3 or 4, wherein the software is further adapted to control the light intensities of the one or more LEDs.

### Claim 6 (depends on 4, 5)

The assembly of any of claims 4 and 5 further comprising an auto-focus system.

### Claim 7 (depends on 6)

The assembly of claim 6, wherein the auto-focus-system is confocal.

### Claim 8 (depends on 7)

The assembly of claim 7, wherein the confocal auto-focus system comprises
a) an infrared light source, preferably an infrared LED, for providing infrared light to the carrier/air or carrier/liquid interface along an optical path;
b) third optical means for directing light from said infrared light source along the optical path to said carrier/air or carrier/liquid interface;
c) light detection means configured to convert the light into electric signals; and
d) fourth optical means for directing light reflected by the carrier/air and/or carrier/liquid interface to said light detection means, wherein the fourth optical means comprise a pinhole arranged in front of said light detection means and configured to suppress out-of-focus light such that only light from the focal plane passes to said light detection means;
wherein the computer is adapted to receive and process said electric signals from the light detection means and the software is configured to correlate said electric signal with the z-position of the stage and to instruct the computer to generate and transmit an output signal to said motorized stage to position said stage such that the carrier is in the desired focal plane.

### Claim 9 (depends on 6, 7, 8)

The assembly of any of the claims 6-8, wherein the assembly is capable of compensating for a tilted position of the carrier by a tilt correction function.

### Claim 10

The assembly of any of the preceding claims, wherein the SLM is selected from DMD and LCD.

### Claim 11

The assembly of any of the preceding claims, wherein the one or more LEDs have a wavelength of 302 nm, 365 nm, 470 nm, or 560 nm.

### Claim 12

The assembly of any of the preceding claims, comprising at least three LEDs with distinct wavelengths.

### Claim 13

The assembly of any of the preceding claims, wherein at least one LED has a wavelength in the UV range, wherein preferably at least one LED has a wavelength of 302 nm or 365 nm.

### Claim 14 (depends on 1)

The assembly of any of the preceding claims, wherein the optical path between the light source of claim 1 ii) a) and the SLM does not comprise a pinhole.

### Claim 15

The assembly of any of the preceding claims, wherein the optical path does not comprise any observation optics.

### Claim 16

The assembly of any of the preceding claims, wherein the assembly does not comprise any optical filters.

### Claim 17

The assembly of any of the preceding claims, wherein all components are arranged integrally within the assembly.

### Claim 18

A method for micro-patterning a solid carrier, the method comprising:
i) providing a transparent solid carrier, wherein one surface side of said carrier is covered with a liquid phase comprising adaptor molecules;
ii) projecting a desired light pattern onto the carrier/liquid interface using an assembly comprising the following elements:
a) a stage adapted for mounting a specimen thereto;
b) a spatially light modulating optics comprising:
a light source for providing light to the carrier along an optical path;
a spatial light modulator (SLM) for generating a binary or greyscale pattern image of light, wherein the SLM is positioned in said optical path between said light source and said carrier;
first optical means for directing light from said light source along the optical path to said SLM; and
second optical means comprising an objective for directing the pattern image generated by said SLM along the optical path to said carrier such that the pattern image is projected onto said carrier;
c) control means having a digital input and connected to said SLM for providing control signals to said SLM to cause said SLM to generate a pattern image of light, whereby the adaptor molecules are covalently attached to the solid carrier surface by photo-immobilization according to the projected light pattern; and
iii) attaching a coupling molecule to the adaptor molecule.

### Claim 19 (depends on 18)

The method of claim 18, wherein the light source is comprises one or more lasers, one or more LEDs and/or one or more mercury (Hg) lamps.

### Claim 20 (depends on 18, 19)

The method of claim 18 or 19, wherein the assembly of step ii) comprises a motorized stage configured for positioning the transparent solid carrier in X-, Y-, and Z-direction.

### Claim 21 (depends on 18, 19, 20)

The method of any one of claims 18 to 20, wherein the assembly of step ii) further comprises a computer comprising a software, wherein the software comprises a pattern generation system configured for generating pattern image data, and wherein the computer is configured for providing drive signals corresponding to said pattern image data to said control means.

### Claim 22 (depends on 21)

The method of claim 21, wherein the assembly of step ii) comprises a motorized stage configured for positioning the transparent solid carrier in X-, Y-, and Z-direction, and wherein the software is adapted to control the motorized stage.

### Claim 23 (depends on 21, 22)

The method of claim 21 or 22, wherein the software is further adapted to control the light intensities of the one or more light sources.

### Claim 24 (depends on 21, 22, 23)

The method of any one of claims 21 to 23, wherein the assembly of step ii) further comprises an auto-focus system.

### Claim 25 (depends on 24)

The method of claim 24, wherein the auto-focus-system is confocal.

### Claim 26 (depends on 25)

The method of claim 25, wherein the confocal auto-focus system comprises
a) an infrared light source, preferably an infrared LED, for providing infrared light to the carrier/air or carrier/liquid interface along an optical path;
b) third optical means for directing light from said infrared light source along the optical path to said carrier/air or carrier/liquid interface;
c) light detection means configured to convert the light into electric signals; and
d) fourth optical means for directing light reflected by the carrier/air and/or carrier/liquid interface to said light detection means, wherein the fourth optical means comprise a pinhole arranged in front of said light detection means and configured to suppress out-of-focus light such that only light from the focal plane passes to said light detection means;
wherein the computer is adapted to receive and process said electric signals from the light detection means and the software is configured to correlate said electric signal with the z-position of the stage and to instruct the computer to generate and transmit an output signal to said motorized stage to position said stage such that the carrier is in the desired focal plane.

### Claim 27 (depends on 24, 25, 26)

The method of any one of claims 24 to 26, wherein the assembly of step ii) is capable of compensating for a tilted position of the carrier by a tilt correction function.

### Claim 28 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

The method of any one of claims 18 to 27, wherein the assembly of step ii) comprises a SLM selected from DMD and LCD.

### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of claim 18, wherein the assembly of step ii) is the assembly of any one of claims 1 to 17.

### Claim 30 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)

The method of any one of claims 18-29, wherein in step iii) the coupling molecule is covalently or non-covalently attached.

### Claim 31 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The method of any one of claims 18-30, wherein in step i) the solid carrier surface covered with said liquid phase comprises a passivating polymeric coating.

### Claim 32 (depends on 31)

The method of claim 31, wherein the passivated polymeric coating comprises or consists of a hydrophilic polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene glycol (PEG), and polyhydroxyethylmethacrylate (polyHEMA), bovine serum albumin (BSA) and derivates of any of the foregoing.

### Claim 33 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The method of any one of claims 18-32, wherein the transparent solid carrier is selected from glass, plastics, hydrogel, elastomers, glass slide, polymeric slide, cover slip, microtiter plate, cuvette, micro array slides, microfluidic chips, test tubes, and polymeric chambers.

### Claim 34 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)

The method of any one of claims 18-33, wherein the adaptor molecule comprises a photoreactive moiety capable of surface immobilization by photobleaching with any organic and inorganic light accessible surfaces thereby immobilizing the adaptor molecule.

### Claim 35 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)

The method of any one of claims 18-34, wherein the adaptor molecule comprises a moiety for a cycloaddition reaction with a counterpart reactive group present on the coupling molecule, preferably wherein the corresponding pair of moiety/counterpart reactive group is any of the reacting pairs selected from the group consisting of azides reacting with terminal alkynes, cyclic alkynes, transcyclooctenes, norbornenes, cyclopropenes; and tetrazines reacting with terminal alkynes, cyclic alkynes, trans-cyclooctenes, norbornenes, and cyclopropenes.

### Claim 36 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The method of any of claims 18-35, wherein the coupling molecule is a biologically active molecule.

### Claim 37 (depends on 36)

The method of claim 36, wherein the biologically active molecule is a cell binding molecule.

### Claim 38 (depends on 37)

The method of claim 37, wherein the cell binding molecule comprises a cell binding moiety capable of being recognized by a cellular surface structure or cell surface receptor selected from the group consisting of adhesion receptors such as integrins, cadherins and selectins; and cell signaling receptors such as immunoglobulins, G-protein coupled receptors, receptor tyrosine kinases, receptor serine/threonine kinases; receptor guanylyl cyclases and histidine kinase associated receptors.

### Claim 39 (depends on 38)

The method of claim 38, wherein said cell binding molecule is an extracellular signaling molecule, preferably a peptide comprising any of the RGD motif derivatives, formyl-methionyl-leucyl-phenylalanine (fMLP), chemokines, G-protein coupled receptor ligands, receptor tyrosine kinase ligands, receptor serine/threonine kinase ligands; receptor guanylyl cyclase ligands and histidine kinase associated receptor ligands.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

Use of the assembly of any of claims 1-17 for micro-patterning, opto-genetics, 3D printing, and/or fluorescence recovery measurements (FRAP).

### Claim 41

A cell binding device offering defined patterns comprising
a) a passivating polymeric coating that is covalently attached to the surface of a solid carrier;
b) an adaptor molecule covalently bound by directed photo-immobilization to a predetermined area of the surface coating, and
c) a cell binding molecule covalently bound to said adaptor molecule.

### Claim 42 (depends on 41)

The device of claim 41, wherein the passivating coating comprises or consists of a hydrophilic synthetic polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene glycol (PEG) and polyhydroxyethylmethacrylate (polyHEMA), bovine serum albumin (BSA) and derivatives of any of the foregoing.

### Claim 43 (depends on 41, 42)

The device of claim 41 or 42, wherein the adapter molecule comprises a dye moiety capable of surface immobilization by photobleaching with any organic and inorganic light accessible surfaces thereby immobilizing the adaptor molecule.

### Claim 44 (depends on 41, 42, 43)

The device of any of claims 41 to 43, wherein the adaptor molecule comprises a moiety for a cycloaddition reaction with a counterpart reactive group present on the cell binding molecule, preferably wherein the corresponding pair of moiety/counterpart reactive group is any of the reacting pairs selected from the group consisting of azides reacting with terminal alkynes, cyclic alkynes, trans-cyclooctenes, norbornenes, cyclopropenes and tetrazines reacting with terminal alkynes, cyclic alkynes, trans-cyclooctenes, norbornenes, cyclopropenes.

### Claim 45 (depends on 41, 42, 43, 44)

The device of any of claims 41 to 44, wherein the cell binding molecule comprises a cell binding moiety capable of being recognized by a cellular surface structure or cell surface receptor selected from the group consisting of adhesion receptors such as integrins, cadherins, selectins or immunoglobulins and cell signaling receptors such as G-protein coupled receptors, receptor tyrosine kinases, receptor serine/threonine kinases; receptor guanylyl cyclases and histidine kinase associated receptors.

### Claim 46 (depends on 45)

The device of claim 45, wherein said cell binding moiety is an extracellular signaling molecule, preferably a peptide comprising any of the RGD motif derivatives, formyl-methionyl-leucyl-phenylalanine (fMLP), chemokines, G-protein coupled receptor ligands, receptor tyrosine kinase ligands, receptor serine/threonine kinase ligands; receptor guanylyl cyclase ligands and histidine kinase associated receptor ligands.

### Claim 47 (depends on 41, 42, 43, 44, 45, 46)

The device of any of claims 41 to 46, wherein the cell binding molecule is comprised within a predetermined area of the device, preferably at a certain density of the receptor molecule or pattern.

### Claim 48 (depends on 41, 42, 43, 44, 45, 46, 47)

The device of any of claims 41 to 47, which is any of an analytical, diagnostic, medical, or industrial device, preferably selected from the group consisting of a microscopy slide, affinity matrix, cell culture support, diagnostic array, medical implant, cell migration applications such as chemotaxis and haptotaxis applications, and microfluidic applications.

### Claim 49 (depends on 41, 42, 43, 44, 45, 46, 47, 48)

A method of producing the device of any of claims 41 to 48, comprising the steps:
a) passivating the surface of a solid carrier by covalently attaching a polymeric coating;
b) covalently binding an adaptor molecule by directed photo-immobilization to a predetermined area of the coating, and
c) covalently binding a cell binding molecule to the adaptor molecule.

### Claim 50 (depends on 49)

The method of claim 49, wherein the photo-immobilization is directed to a predetermined area thereby obtaining a cell behavior influencing area on the surface of the device suitable for activating cell surface receptors.

### Claim 51 (depends on 41, 42, 43, 44, 45, 46, 47, 48)

Use of the device of any of claims 41 to 48 for immobilizing and processing viable single cells within a predetermined area, preferably single cell analysis and cell population analysis.

### Claim 52 (depends on 41, 42, 43, 44, 45, 46, 47, 48)

A preparation of bioactive target cells specifically binding onto the device of any of claims 41 to 48, preferably wherein the target cells are specifically binding as a monolayer and/or cell clusters.

### Claim 53 (depends on 52)

The preparation of claim 52, wherein the target cells are movable or migrating on the surface of the predetermined area without consuming the cell binding molecule.

### Claim 54 (depends on 52, 53)

The preparation of claim 52 or 53, wherein the target cells are selected from the group consisting of epithelial cells, tumor cells, leukocytes, mesenchymal cells, stem cells.

### Claim 55 (depends on 41, 42, 43, 44, 45, 46, 47, 48, 52, 53, 54)

A kit for preparing a preparation of any of claims 52 to 54, comprising
a) the device of any of claims 41 to 48, and
b) means for preparing a suspension of cells from a cellular sample,
c) preferably wherein the cellular sample is obtained from a biological sample of a subject, or from a cell culture.