High-resolution gravure printing of graphene for biomedical applications
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1 High-resolution gravure printing of graphene for biomedical applications Thorsten Knoll Fraunhofer-Institut für Biomedizinische Technik IBMT Main Department of Biomedical Engineering Düsseldorf, 3 rd March 2016
2 Basic idea Roll-to-roll gravure printing of biosensors Electrically conductive structures Adherence proteins 1. Printing of graphene electrodes on polymer foils for impedimetric or electrochemical measurements 2. Drying of printed graphene 3. Printing of protein patterns on conductive electrodes for improved cell adherence and proliferation (expensive resource efficiency) 4. Drying of printed protein 1/18
3 Biosensor Combination of surface functionalized foils with cell culture substrates, e.g. with bottom-less well plates Graphene/protein micro pattern combine with 128 mm 2/18
4 Objectives Biocompatible and electrically conductive graphene ink suited for gravure printing Fabrication of gravure printing cylinders with structure size < 10 µm Roll-to-roll process for graphene/protein printing Two-colour roll-to-roll production line for surface functionalisation of large area polymer foils Evidence of suitability of printed biosensors for Vaccine and antiviral drug validation Cell based toxicitiy tests 3/18
5 Project consortium Name Fraunhofer IBMT SAUERESSIG GmbH + Co. KG Haydale Ltd. Tasks R2R gravure printing process (graphene and proteins), biocompatibility tests R2R two-colour printing machine, laser-based method for micro patterning of gravure printing cylinders Biocompatible graphene ink for gravure printing AiCuris GmbH & Co. KG cellasys GmbH Application: antiviral drug validation Application: repeated dose toxicity 4/18
6 Ink development Basis: screen printing graphene ink (HDPlas IGSC02002) Viscosity: ~ 5.5 Pa*s Thickness before / after drying: ~ 13 µm / 7 µm Sheet resistivity: ~ 12 /sq. (normalised to 25 µm) Target values for gravure printing on polymer foil Viscosity: ~ 100 mpa*s (gravure printing ink ~ 40 mpa*s) Surface tension: < 58 mn/m (gravure printing ink ~ 32 mn/m) Modifications Solvent type Solvent content Resin content Milling procedure 5/18
7 Fabrication of gravure printing cylinders Different common fabrication methods Electromechanical engraving and direct engraving in zinc not suitable for printing of graphene or proteins Masking and chemical etching strongly defined structures, high ink transfer (low electrical resistance) Direct engraving in copper with ultrashort pulse laser microstructures < 10 µm Directly structured dots with diameter 8 µm 6/18
8 Fabrication of gravure printing cylinders First prototypes of graphene cylinders 1 mm 1 mm Etched IDE structure, line width 40µm 7/18
9 R2R gravure printing with graphene ink PET foil (50 µm, 300 mm) Corona activation (600 W) Ink volume: ~ 10 ml Speed: 25 m/min One-colour printing machine for flexo and gravure printing (FREE-1) 8/18
10 Thickness (µm) R2R gravure printing with graphene ink Printed graphene patterns with first test cylinder 200 mm 7,00 6,50 6,00 5,50 5,00 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 20 µm 40 µm 60 µm 80 µm Cell depth 9/18
11 R2R gravure printing with graphene ink Printed lines (width 0.05 mm and 1.0 mm), various cell depths 20 µm 40 µm 60 µm 80 µm 10/18
12 R2R gravure printing with graphene ink Orientation dependency (0.05 mm lines, different cell depths) Cell depth 20 µm Cell depth 80 µm 11/18
13 R2R gravure printing with graphene ink Printing with first biosensor printing cylinder 1 mm IDE structures (nominal width 50 µm) 1 mm IDE structures (nominal width 100 µm) 12/18
14 Sheet resistance ( /sq.) Electrical characterization Resistivity measurements Line width: ~1.05 mm, length: 10 mm Angles: 45 and 135, cell depth: 60 µm Graphene ink thickness: ~ 4 µm Normalized sheet resistance (25 µm) 10 0 R sq. = /sq. 13/18
15 SEM images of graphene patterns Cell depth 20 µm Cell depth 80 µm 14/18
16 Cytotoxicity testing of graphene ink Samples printed on PET foil and sterilized by ethylene oxide (EO) 1. PET foil reference (PET) 2. Graphene (pure) 3. Graphene + solvent 1 4. Graphene + solvent 2 5. Graphene + solvent 3 10 mm Determination of cellular behaviour after exposure to testing substance Cells: human fibroblasts (MRC5) Following ISO 10993: 1. Quantitative determination of the metabolic activity (WST-1) 2. Quantitative determination of the rate of cell division (BrdU) 3. Qualitative direct contact test (live/dead staining) 15/18
17 Relative growth [%] Indirect contact test WST-1 and BrdU WST-1 BrdU sample PET sample GA sample GA sample GD sample GDA WST-1 = metabolic activity BrdU = cell proliferation 16/18
18 Cell morphology and direct contact test Cell morphology after 24 h incubation Control PET Graphene Direct contact test after 48 h incubation Graphene (pure) Graphene + collagen type IV Graphen + matrigel 17/18
19 Summary Successful initial gravure printing with graphene Developed graphene ink is not cytotoxic Enhancement of cell adhesion by additional protein coating on graphene structures Next steps Set-up of first biosensors for validation Two-colour printing machine 18/18
20 Acknowledgements
21 Thank you for your attention.
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