WO2013117880A1 - Formulation and method for the printing of biological materials - Google Patents
Formulation and method for the printing of biological materials Download PDFInfo
- Publication number
- WO2013117880A1 WO2013117880A1 PCT/GB2013/000042 GB2013000042W WO2013117880A1 WO 2013117880 A1 WO2013117880 A1 WO 2013117880A1 GB 2013000042 W GB2013000042 W GB 2013000042W WO 2013117880 A1 WO2013117880 A1 WO 2013117880A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- printing
- formulation
- nitrocellulose
- printed
- buffer
- Prior art date
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B41—PRINTING; LINING MACHINES; TYPEWRITERS; STAMPS
- B41M—PRINTING, DUPLICATING, MARKING, OR COPYING PROCESSES; COLOUR PRINTING
- B41M1/00—Inking and printing with a printer's forme
- B41M1/26—Printing on other surfaces than ordinary paper
- B41M1/30—Printing on other surfaces than ordinary paper on organic plastics, horn or similar materials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B41—PRINTING; LINING MACHINES; TYPEWRITERS; STAMPS
- B41M—PRINTING, DUPLICATING, MARKING, OR COPYING PROCESSES; COLOUR PRINTING
- B41M1/00—Inking and printing with a printer's forme
- B41M1/02—Letterpress printing, e.g. book printing
- B41M1/04—Flexographic printing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B41—PRINTING; LINING MACHINES; TYPEWRITERS; STAMPS
- B41M—PRINTING, DUPLICATING, MARKING, OR COPYING PROCESSES; COLOUR PRINTING
- B41M1/00—Inking and printing with a printer's forme
- B41M1/10—Intaglio printing ; Gravure printing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/03—Printing inks characterised by features other than the chemical nature of the binder
- C09D11/033—Printing inks characterised by features other than the chemical nature of the binder characterised by the solvent
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D11/00—Inks
- C09D11/02—Printing inks
- C09D11/10—Printing inks based on artificial resins
- C09D11/106—Printing inks based on artificial resins containing macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
Definitions
- the present invention relates to a formulation and method for the printing of biological materials and in particular but not exclusively to the printing of antibodies.
- Patterning proteins with piezoelectric inkjet requires a very narrow operational window in terms of rheological properties and suitable additives, and is unpredictable and has a very slow print speed.
- Flexographic printing is a high speed reel-to-reel process traditionally used for graphics printing in applications such as packaging. Flexographic printing has been focused on electronics applications as flexographic printing to date would not be suitable for the printing of biological materials due to the high shear rates with such rapid processes. For biological inks such as antibody inks these are generally water based and are thus difficult materials to use with plastic films onto which antibodies are generally printed as they struggle to adequately wet the surface. There is also a need to protect the biological materials during the printing process which induces high shear and interfacial stress.
- the present invention seeks to overcome the problems of high speed printing of biological materials using flexographic techniques by providing ink compositions that can be used with biological materials whilst still allowing for high printing throughput. Furthermore, the invention provides coatings that can be used with the printing process to improve the quality and accuracy of printing whilst still allowing for a high speed process.
- a printing formulation for the printing of biological materials by way of a rotary printing press process comprising a biological material a high molecular weight poly (vinyl alcohol) and a buffer.
- PVA poly(vinyl alcohol)
- the buffer is a carbonate bicarbonate buffer, and preferably it is a 0.05 M carbonate-bicarbonate buffer.
- the buffer has a pH 9.6 at 25 °C
- the rotary printing press process is flexographic printing. However as an alternative gravure printing may be used.
- the biological material is an antibody.
- the buffer an alkaline buffer such as a carbonate bicarbonate buffer with alkaline pH but others such as phosphate buffered saline, pH 7.4 can be used.
- alkaline buffer such as a carbonate bicarbonate buffer with alkaline pH but others such as phosphate buffered saline, pH 7.4 can be used.
- the PVA is present in an amount between 1% and 5% of the formulation. More preferably between 2 and 3% of the formulation and more particularly 2.5% of the formulation.
- a method of printing a biological material on a surface said method involving providing a nitrocellulose layer, transferring a printing formulation including a biological material, PVA and a buffer onto said layer using a rotary printing press.
- the rotary printing press is a flexographic printing or gravure printing.
- the nitrocellulose film is A or E grade nitrocellulose. It is envisaged that the nitrocellulose is in an alcohol based solvent with some acetate to aid solubility.
- the majority of the solvent is propan-2-ol.
- a printed film having a nitrocellulose layer and a printed antibody layer thereon wherein the printed antibody layer is printed using a printing formulation according to a first aspect of the invention.
- Figure 1 shows a schematic view of flexographic printing apparatus
- Figure 2 shows a comparison of depositions of Anti-Sheep IgG (whole molecule)- Peroxidase, visualised by chemiluminescence, on acrylic (left) and nitrocellulose surfaces (right) to indicate greater binding on nitrocellulose.
- Figure 3 shows printing of dye in PVA and buffer on nitrocellulose (left) and acrylic (right) using 8 mL/m 2 anilox and fast print speed (top) and 24 mL/m 2 anilox and slow print speed (bottom); to illustrate enhanced image quality on nitrocellulose
- Figure 4 shows Printed anti-FITC serum visualised by binding anti-sheep-peroxidise then chemiluminescence - 10 seconds exposure
- flexography uses a photopolymer relief plate as an image carrier with a raised printing surface onto which ink is transferred using an engraved cylinder known as an anilox.
- a substrate 1 has and image 2 printed thereon.
- An anilox roller 5 takes up ink from a reservoir 5 and surplus ink is removed from the anilox top surface using doctor blade 7.
- the ink is transferred to a plate cylinder which counter rotates with an impression cylinder to deliver the pattern 2 to the substrate 1 as it passes through rollers 3 and 4.
- the substrate may be a paper or plastic film.
- the frequency and size of cells engraved in the anilox determines the volume of ink that can be held in the cells and is the main controlling factor in the amount of ink transferred.
- a formulation for printing biological materials such as an antibody "ink” has two purposes, firstly to act as a vehicle to apply the antibody to a substrate and secondly to allow the antibody to survive the stresses imposed by printing (due to shearing, film splitting etc.) and drying while permitting binding to a surface.
- the ink needs to have, i.e. to provide the appropriate rheological properties for the printing process while not compromising the activity of the material.
- Flexography typically uses a viscosity range of approximately 0.05 to 0.5 Pa s which necessitates the addition of polymeric binders and the presence of excessive polymer binders is not desirable as they may impair diffusion of the antibodies to the surface and subsequently analytes to the antibody.
- Low molecular weight forms of polymers such as PVA poly(vinyl alcohol) and poly(ethylene glycol) were found in preliminary experiments to hinder adhesion of antibodies to a surface because they do not allow for the securing of biological materials to a surface.
- Low molecular weight PVA (Mw 9000 to 10,000 Sigma Aldrich 360627), also exhibited a noticeable surfactant effect, and due to their limited effect on viscosity, were needed in greater concentrations in order to facilitate printing.
- High molecular weight PVA was found to have good properties.
- the ink used typically 2.5% by mass high molecular weight PVA (average Mw 130,000 Sigma Aldrich 563900) as a rheology modifier/binder in 0.05 M carbonate-bicarbonate buffer, pH 9.6 at 25 °C (Sigma Aldrich C3041) which is a buffer typically used to coat microwell plates in ELISA testing.
- This gave a viscosity of 0.01 Pa s which did not vary with shear rate (cone and plate, average viscosity between 1 and 100 s "1 ).
- High molecular weight PVA did not exhibit a strong surfactant effect unlike low molecular weight polymers.
- Surfactants are useful in reducing the surface tension of water-based systems to facilitate the transfer of ink through the various stages of the printing process as well as the subsequent wetting of the liquid onto a surface. However, because surfactants migrate to interfaces they will interfere with adsorption of antibodies and will also increase drop spreading. Surfactants are also reported to denature proteins at high concentrations, especially with ionic surfactants.
- Nitrocellulose has been known to be used in the coating of materials used in processes needing the adhesion of protein such as membranes for lateral flow and Western blots. The invention found that certain nitrocellulose coatings can be made which when used with a printing process allow for improved adhesion.
- Nitrocellulose is available in a range of grades depending on the nitrogen content. However, the degree of nitration determines which solvents can be used to dissolve and dilute the resin. The higher the nitrogen, content the more aggressive the solvent required.
- highly nitrated E-grade nitrocellulose (RS 120, Nobel NC Co Ltd. Bangkok, Thailand) was dissolved in n-butyl acetate then further diluted using propan-2-ol (at lower concentrations than the acetate) to make a coating with suitable viscosity. This coating was applied to plastic film using an automated bar coater (K Control Coater, RK PrintCoat Instruments Ltd., Herts, UK) to give a thin layer of dry nitrocellulose.
- a labeled antibody 0.25 % vol Anti-Sheep IgG (whole molecule)-Peroxidase antibody produced in donkey, (Sigma Aldrich A3415), was added to 2.5% high molecular weight PVA in carbonate-bicarbonate buffer, and applied in 3 drops using a pipette and allowed to dry at 37°C. This was washed and then visualised by spraying on a chemiluminescent substrate (Immobilon Western Chemiluminescent HRP Substrate, Millipore, WBKLS0100) which was activated by the peroxidise enzyme.
- the chemiluminescence imager was a ChemiDoc XRS+ System, Bio-Rad.
- the presence of the peroxidase tagged antibody is denoted by the colour intensity of the droplet, with a darker colour signifying greater presence of peroxidase, and hence antibody, on the nitrocellulose coated sample.
- the alkaline carbonate buffer performed better on the nitrocellulose surface than a neutral buffer (phosphate buffered saline, pH 7.4, at 25 °C (Sigma Aldrich P4417)).
- Figure 3 shows printing tests were carried out using the IGT Fl .
- the figure compares printed images on nitrocellulose and acrylic surfaces as the optimum settings; 8 mL/m 2 anilox and fast print speed and the worst settings; 24 mL/m 2 anilox and slow print speed.
- Preliminary tests were carried out to establish the optimum print settings using dye in place of antibodies in order to make the printed features visible (other ink ingredients were 2.5% by mass high molecular weight PVA (average Mw 130,000 Sigma Aldrich 563900) in 0.05 M carbonate- bicarbonate buffer, pH 9.6 at 25 °C (Sigma Aldrich C3041)).
- PVA average Mw 130,000 Sigma Aldrich 563900
- the print quality was substantially reduced on the uncoated substrate due to dewetting of the ink formulation. This was mitigated by using an increased print speed which reduced the time available for dewetting of the antibody ink.
- the nitrocellulose coated film reduced the sensitivity of print quality to the printing parameters and gave substantially better printed images than uncoated films, especially at high anilox volumes and low printing speeds. Again as the coating was more receptive to the ink, it removed the need to add surfactant as a means of improving print quality and preventing dewetting. Both E and A grade nitrocellulose coatings permitted good print quality for the antibody ink formulation.
- nitrocellulose has the benefit of reducing the amount of acetate that is needed in the ink which could potentially damage the photopolymer plate used in the printing process.
- a labelled secondary antibody was attached and visualised using the following steps: the printed antibodies were washed three times with a blocking buffer (Sigma Aldrich B6429) to remove any unattached protein (3 x 5 minute washes at 75 rpm on a shaker). The printed samples were then incubated in 0.1 % vol enzyme labelled secondary antibody (Anti-Sheep IgG (whole molecule)-Peroxidase antibody produced in donkey, Sigma Aldrich A3415) in blocking buffer to bind to the printed anti-FITC serum under gentle agitation for a period of one hour. This was washed and visualised by spraying on the chemiluminescent substrate. Images of the visualised printed features are shown in Figure 4; from an exposure time of 10 seconds. The images confirm that the printing methods and formulation were appropriate to obtain sufficient detail and antibody transfer. Features such as dots, squares, text and fine lines were reproduced effectively.
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1412879.7A GB2512787A (en) | 2012-02-09 | 2013-02-04 | Formulation and method for the printing of biological materials |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1202261.2 | 2012-02-09 | ||
GB201202261A GB201202261D0 (en) | 2012-02-09 | 2012-02-09 | Formulation and method for the printing of biological materials |
Publications (1)
Publication Number | Publication Date |
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WO2013117880A1 true WO2013117880A1 (en) | 2013-08-15 |
Family
ID=45929881
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2013/000042 WO2013117880A1 (en) | 2012-02-09 | 2013-02-04 | Formulation and method for the printing of biological materials |
Country Status (2)
Country | Link |
---|---|
GB (2) | GB201202261D0 (en) |
WO (1) | WO2013117880A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997043644A1 (en) * | 1996-05-09 | 1997-11-20 | Applied Research Systems Ars Holding N.V. | Method of assay |
US20050009116A1 (en) * | 2003-07-07 | 2005-01-13 | Syngenta Participation Ag | Reagents, methods and kit for detecting feed enzymes |
US20080064032A1 (en) * | 2006-09-13 | 2008-03-13 | Syngenta Participations Ag | Polynucleotides and uses thereof |
-
2012
- 2012-02-09 GB GB201202261A patent/GB201202261D0/en not_active Ceased
-
2013
- 2013-02-04 WO PCT/GB2013/000042 patent/WO2013117880A1/en active Application Filing
- 2013-02-04 GB GB1412879.7A patent/GB2512787A/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997043644A1 (en) * | 1996-05-09 | 1997-11-20 | Applied Research Systems Ars Holding N.V. | Method of assay |
US20050009116A1 (en) * | 2003-07-07 | 2005-01-13 | Syngenta Participation Ag | Reagents, methods and kit for detecting feed enzymes |
US20080064032A1 (en) * | 2006-09-13 | 2008-03-13 | Syngenta Participations Ag | Polynucleotides and uses thereof |
Non-Patent Citations (1)
Title |
---|
LAURA GONZALEZ-MACIA ET AL: "Advanced printing and deposition methodologies for the fabrication of biosensors and biodevices", THE ANALYST, vol. 135, no. 5, 8 January 2010 (2010-01-08), pages 845 - 867, XP055060853, ISSN: 0003-2654, DOI: 10.1039/b916888e * |
Also Published As
Publication number | Publication date |
---|---|
GB201412879D0 (en) | 2014-09-03 |
GB201202261D0 (en) | 2012-03-28 |
GB2512787A (en) | 2014-10-08 |
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