MATERIALS

Field of the Invention

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.

Background of the Invention

The printing of biological materials has been performed using ink jet printing but this is a slow process and does not allow for the high throughput of large print runs. Patterning proteins with piezoelectric inkjet, for example, 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.

Summary of the Invention According to a first aspect of the present invention there is provided a printing formulation for the printing of biological materials by way of a rotary printing press process, said formulation comprising a biological material a high molecular weight poly (vinyl alcohol) and a buffer.

It is preferred that there is 2.5% by mass high molecular weight poly(vinyl alcohol) (PVA) (average Mw 130,000).

It is envisaged that 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

It is preferred that the rotary printing press process is flexographic printing. However as an alternative gravure printing may be used.

Preferably the biological material is an antibody.

It is envisaged that 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.

It is preferred that 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.

According to a second aspect of the present invention there is provided 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.

It is preferred that the rotary printing press is a flexographic printing or gravure printing. Preferably 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.

It is preferred that the majority of the solvent is propan-2-ol.

According to a third aspect of the invention there is provided 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.

Brief Description of the Drawings

An embodiment of the invention will now be described, by way of example only and not in any limitative sense, with reference to the accompanying drawings in which:

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

Detailed Description of the Invention

As shown in Figure 1, 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. There are competing properties that 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 cellulose based polymers were found to confer good rheological properties, with only small concentrations of polymer required but they were found to foam upon printing and appeared to wash away, taking much of the antibodies with them.

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 ). This was suitable for printing without overloading the antibody formulation with polymer. This allowed for an ink composition that has the appropriate viscosity to be printed by flexographic printing as well as retaining the functionality of the antibody.

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.

Commercial polymer films are fabricated from a range of different polymers and with different surface coatings which employ a range of additives such as plasticisers and waxes which will affect surface interactions and hence antibody binding. 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. To make a formulation suitable for bar coating, 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. Using a bar wound with 0.0015 inch (0.0381 mm) wire, a dry film thickness of approximately 0.6 micron was obtained. Typically 1 part NC, 3 parts n-butyl acetate and 2 parts propan-2-ol is used. As shown in Figure 2, a greater amount of antibody immobilisation was observed from a nitrocellulose coated film than any of a range of polymer films not having this layer applied. In Figure 2 an acrylic surface from a commercial film is compared with the nitrocellulose coating. The surface is generally shown as 1 and the printed pattern as 10. 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. In tests 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)). At the lowest tested anilox volume (8 mL/m ² ), the choice of substrate coating or speed made only a small difference to print quality. However, as the anilox volume was increased (to 12 and 24 mL/m ² ), 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.

Low nitration, or A-grade, 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.

In order to confirm the presence of bound printed antibodies, 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.

This required careful selection of ink formulation ingredients and printing parameters as well as adaptation of the film surface through the development of a nitrocellulose-based ink receptive coating which both increased antibody adhesion to the substrates whilst improving printing performance. The specific formulation of the invention allowed the antibody to bind to the surface and remain accessible for binding with a secondary antibody and nitrocellulose coatings were produced which improved binding of the antibodies and the coating removes the need for surfactant in the ink and reduces the sensitivity of the print to changes in printing parameters. These developments allow arrays of antibodies to be printed at speeds far in excess of those previously attainable for large scale production of immunoassays. The ability to deposit arrays of antibodies using reel-to-reel processes would greatly increase the speed of production when compared with current patterning process. Faster patterning methods could be exploited for applications such as enzyme linked immunoassay (ELISA) and biosensors. The availability of low cost, disposable assay technology will aid the early diagnosis of disease and facilitate cost effective health management.

It should be noted that the above mentioned embodiment illustrates rather than limits the invention and that alterations or modifications are possible without departing from the scope of the invention as described. It is to be noted that the invention covers not only individual embodiments described but also combinations of those embodiments.