WO2011157895A1

WO2011157895A1 - Method for manufacturing a sheet-like reaction plate, reaction plate and its use

Info

Publication number: WO2011157895A1
Application number: PCT/FI2011/050567
Authority: WO
Inventors: Petri Ihalainen; Anni MÄÄTTÄNEN; Jouko Peltonen
Original assignee: Åbo Akademi University
Priority date: 2010-06-15
Filing date: 2011-06-15
Publication date: 2011-12-22
Also published as: FI20105692A0

Abstract

The invention relates to a method for manufacturing a sheet-like reaction plate such as paper for chemical and/or biochemical analysis. Method comprises selecting a sheet-like substrate comprising fibres of natural origin such as paper and having a hydrophilic surface, which has a contact angle of water < 90°, selecting a ink material, which has a hydrophobic surface when in layer form, whereby a contact angle of water is > 9 0°, and directly forming uncoated reaction regions ( 3', 3'') on the surface of the sheet-like substrate by applying a layer of the ink material (2) on the surface of the sheet-like substrate in a pre-determined pattern. The invention relates also to a sheet-like reaction plate and its use.

Description

METHOD FOR MANUFACTURING A SHEET-LIKE REACTION PLATE, REACTION PLATE AND ITS USE

The present invention relates to a method for manufacturing a sheet-like reaction plate, a reaction plate and its use according to preambles of the enclosed claims.

TECHNICAL AREA

In chemical and biochemical analysis a vast variety of different reaction plates and vessels are employed. One example of a commonly used reaction plate type is a microplate or multiwell plate, which is a three-dimensional panel with multiple sample wells which are used as small test tubes. Each sample well may hold between tens of nanolitres to several milli litres of liquid. The wells can be either circular or square, and normally the number of wells in a microplate is 6, 24, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix. Some microplates have been manufactured even with 3456 or 9600 wells.

Microplates are standard tools in analytical research and in diagnostic testing. They can be manufactured from a variety of materials, such as polystyrene, polypropylene, polycarbonate or cyclo-olefins. Typically, the microplates are manufactured by injection moulding, which manufacturing process is used for polystyrene, polypropylene and cyclo-olefins, or by vacuum forming, which manufacturing process is used for softer plastics, such as polycarbonate. Commonly used reaction plates and vessels have, however, some drawbacks. They can be cumbersome to manufacture, especially if the inner volume of the vessel or a microplate well should be functionalised, e.g. by incorporation of reagents. Also, the final deposition or destruction of the used plates and vessels may be problematic, if dangerous or toxic samples are analysed or dangerous or toxic reagents are used in analysis.

It would also be advantageous if the size of the reaction plates could be minimised or freely adjusted. For example, when samples are analysed in field, it would be a great advantage if the three-dimensional size, i.e. volume, of the reaction plate was as small as possible.

OBJECTS AND SHORT SUMMARY OF THE INVENTION

Therefore an object of this invention is to minimise or even eliminate the disadvantages existing in the prior art.

One object of the invention is to provide a method, with which it is simple and easy to manufacture various reaction plates suitable to be used in chemical and/or biochemical analysis.

A further object of this invention is to provide a reaction plate that is simple to manufacture for various analytical or diagnostic purposes.

These objects are attained with a method and an arrangement having the characteristics presented below in the characterising parts of the independent claims. Typical method according to the present invention for manufacturing a sheet-like reaction plate for chemical and/or biochemical analysis, comprises

- selecting a sheet-like substrate comprising fibres of natural origin and having a hydrophilic large surface, which has a contact angle of water < 90°,

- selecting a ink material, which has a hydrophobic surface when in layer form, whereby a contact angle of water is > 90°, and

- applying layer of the ink material on the surface of the sheet-like substrate in a pre-determined pattern, whereby the applied layer defines uncoated regions on the surface of the sheet-like substrate.

Typical reaction plate according to the present invention for chemical and/or biochemical analysis comprises

- a sheet-like substrate comprising fibres of natural origin and having a hydrophilic large surface, having a contact angle of water < 90 °, and - a layer of ink material having a hydrophobic surface, having a contact angle of water > 90°, the layer of ink material being applied on the large surface of the sheet-like substrate so that the ink material defines directly uncoated reaction regions in a predetermined pattern on the large surface of the sheet-like substrate.

Typically a reaction plate according to the present invention is used for chemical and/or biochemical analysis or for diagnostics.

DETAILED DESCRIPTION OF THE INVENTION

Now it has been surprisingly found out that a sheet-like reaction plate can be obtained by applying a hydrophobic ink material to a hydrophilic substrate surface, simultaneously creating on the substrate surface uncovered and uncoated reaction regions, defined and limited by the hydrophobic ink layer. When a liquid sample then comes into contact with the reaction plate according to the invention, the hydrophobic layer of the ink material restricts the liquid sample to the uncoated reaction regions, i.e. areas which are free from hydrophobic ink layer. The sheetlike reaction plate may be considered to be in practice nearly two-dimensional, which makes it easy to store and transport e.g. by regular mail. According to the invention it is also easy to produce reaction plates where the uncoated reaction regions have different size and/or geometry and the number of uncoated regions in one reaction plate may be freely chosen.

In this context uncoated reaction regions means areas on the substrate surface, which are uncovered by the hydrophobic ink material layer. The term "uncoated reaction region" is interchangeable with the terms "uncovered reaction area" or "uncoated well area".

The ink layer, when applied on the surface of the substrate, forms a hydrophobic surface. The layer of ink material has a contact angle (0.1 - 0.5 s) of water > 95°, or > 100°, typically > 105°, more typically > 1 10°, preferably > 1 15°, occasionally even > 120° or > 130°. The contact angles are measured by using the standard method TAPPI 565 pm-96. An increase in the contact angle indicates an increased water repellence of the measured surface.

The hydrophobic ink layer covers the surface of the substrate only partially, i.e. the ink layer on the substrate surface is non-continuous in the sense that there exist uncoated reaction regions. Thus uncoated reaction regions are formed directly on the substrate surface as a result of a one-step printing process and no removal or modification of the ink layer is required after it is formed on the substrate surface. The uncoated reaction regions are formed solely by applying the ink material on the substrate surface. The uncoated reaction regions are preferably irreversible, i.e. the size, design, pattern and form of the uncoated regions cannot be altered after the ink layer is applied on the substrate surface. The sheet-like reaction plate thus comprises a number of uncoated reaction regions, which are unconnected to each other and separate from each other. Adjacent uncoated reaction regions are separated by an area of hydrophobic ink layer and the ink layer defines the shape and size of the individual and isolated uncoated regions. The substrate is preferably selected so that no transport of sample or reactants is occurring between the uncoated reaction regions inside the substrate underneath the ink layer.

One preferred embodiment comprises directly forming uncoated reaction regions on the large surface of the sheet-like substrate by applying a layer of the ink material on the surface of the sheet-like substrate in a pre-determined pattern. After application on the substrate surface the ink layer may be cured, preferably thermally. According to one preferred embodiment of the invention, the manufacturing method consists of only two steps, namely application of the ink to the substrate surface according to the desired pattern and curing of the ink layer. Typical curing time needed is 7 - 60 s, preferably 10 - 30 s.

According to one preferred embodiment of the invention the ink layer and the substrate surface are not chemically reacting with each other. This means that it is not necessary to create chemical bonds between the substrate surface and the ink layer material, i.e. the resulting sheet-like reaction plate is essentially free from chemical bonds between the ink layer and substrate. Preferably the hydrophobic ink layer does not penetrate or impregnate into the substrate material, but the ink layer is essentially confined on its surface.

According to one embodiment of the invention the ink material is prepared in a manner free from organic solvents by mixing a crosslinker to a solution of polymer, and thereafter adding suitable catalyst to the mixture. Preferably the preparation of the ink material is completely free from organic solvents. The crosslinker may be a solvent-free hydrogen polysiloxane comprising a high percentage of reactive Si-H groups and comprising, for example, 1 .3 - 1 .9 weight-% of reactive hydrogen groups. The catalyst, which is preferably used in preparing the hydrophobic ink material is a highly-active platinum complex used for the thermal curing of addition-crosslinking, solvent-based and solventless silicones. The platinum content of the catalyst is 0.1 - 1 .3 weight-%, typically around 1 weight-%, sometimes as low as 0.1 13 - 0.1 15 weight-%. The highly active platinum catalyst shortens the curing time of the hydrophobic ink, and improves the production efficiency and economics. According to one preferred embodiment the hydrophobic ink material comprises a polymer, which may be selected from the group comprising silicone, silicon rubber, polystyrene and crosslinked polyvinyl alcohol. In one embodiment of the invention the polymer is an essentially solvent-free or solventless, addition-curing polydimethylsiloxane with an oily consistency intended for coating paper and other substrates, especially suitable for solvent-free or solventless coating of paper. Polymer preferably possesses optimum release properties once it has undergone complete crosslinking, which means that even if the uncoated reaction regions would be covered by a protective tape prior or after the use of the reaction plate, the tape may be peeled off without damaging the ink material layer. Subsequent postcuring of the polymer is not necessary. The mixing ratio polymer:crosslinker:catalyst may be 100 : (2 - 4) : (1 - 3), given in weight-%. In one embodiment the mixing ratio polymer:crosslinker:catalyst is 100:2.5:1 , given in weight-%. The reaction plate substrate according to the invention is hydrophilic, i.e. wettable by water. On to the substrate is applied a hydrophobic ink material in order to form a patterned layer, which is hydrophobic, i.e. water repellent, on the substrate surface. Thus the reaction plate substrate comprises at least one, preferably a plurality of uncoated reaction regions, where the substrate surface is visible and exposed and available for example for further functionalisation or for direct contact with liquid sample. The uncoated reaction regions are encircled by the layer of ink material, which defines the size and geometry of the uncoated regions. Liquid, preferably aqueous, sample spreads on the hydrophilic uncoated regions and is contained therein by the patterned hydrophobic ink material layer. According to one embodiment of the invention the substrate has a hydrophilic surface, where the contact angle of water (0.1 - 0.5 s) is < 75°, typically < 65°, more typically < 45°, preferably < 30°. The contact angles are measured by using the standard method TAPPI 565 pm-96.

According to one embodiment of the invention the sheet-like substrate having a hydrophilic surface, is selected so that it has a contact angle of water (0.1 - 0.5 s) < 75°, and the ink material, which has a hydrophobic surface when in layer form, is selected so that it has a contact angle of water (0.1 - 0.5 s) is > 100°. The contact angles are measured by using the standard method TAPPI 565 pm-96.

The substrate may be any substrate with suitable water retention properties. For example, the sheet-like substrate comprising fibres of natural origin may be selected from natural fibre-based substrates including unsized paper, internally sized paper, surface sized paper, uncoated paper, coated paper, glassine paper (transparent air and water resistant paper), filter paper, cellulose sheet and starch sheet. The substrate may be any substrate, on which the hydrophobic ink material is applicable, the substrate comprising fibres originating from the nature, such as cellulose and/or wood fibres. The substrate may include super calendered (SC) paper, ultralight weight coated (ULWC) paper, light weight coated (LWC) paper, medium weight coated (MWC) paper, heavy weight coated (HWC) paper, machine finished coated (MFC) paper, film coated offset (FCO) paper, woodfree coated (WFC) paper, light weight coated (LWCO) printing paper, SC offset (SCO) printing paper, machine finished specialties (MFS), multilayer coated paper, inkjet paper, copy paper or newsprint paper, but not limited to these. Typical coated paper, such as LWC, which may be used as a substrate, comprises mechanical pulp around 40 - 60 weight-%, bleached softwood pulp around 25 - 40 weight-% and fillers and/or coating agents around 20 - 35 weight-%. SC paper comprises mechanical pulp around 70 - 90 weight-% and long fibered cellulose pulp around 10 - 30 %. Typical grammage values for different paper grades may be: 40 - 80 g/m² for SC, 40 - 70 g/m² for LWC, 70-130 g/m² for MWC, 50 - 70 g/m² for MFC, 40-70 g/m² for FCO, 70-90 g/m² for MWC, 100-135 g/m² for HWC, 80 - 140 g/m² for WFC. The substrate may comprise also recycled fibres. For example, MFS paper grades are uncoated papers made of mechanical fibres and also at least partly of recycled fibres. Different paper grades are generally good substrates for producing sheet-like reaction plates according to the present invention. Paper is easy to print or coat with hydrophobic ink material, and the uncoated well areas are easily functionalised e.g. by printing electric circuits or by bonding bioactive agents to the paper surface. According to one embodiment of the invention the use of a hydrophilic paper as the substrate offers also a high recyclability and lower unit cost due to the possibility for roll-to-roll mass-manufacturing compared to the currently used manufacturing methods of the reaction plates.

According to one embodiment of the invention the sheet-like substrate is paper, which has low water permeability and/or low porosity. This restricts or eliminates the transport of sample or reactants between the uncoated reaction regions inside the substrate underneath the ink layer. One possible method for preparing a paper that is suitable for the sheet-like substrate is described in WO2010/08651 1 , which is hereby incorporated as reference.

The sheet-like substrate may also be selected from cellulose sheet or starch sheet, such as filter paper. The sheet-like substrate may also be selected from synthetic polymeric membranes, such as cellulose acetate membrane, nitrocellulose membrane, polyvinyl difluoride (PVDF) membrane, polyvinyl alcohol (PVOH) membrane or nylon membrane.

The substrate material may be selected depending on the intended use of the sheet-like reaction plate. For example, if biomaterials and/or electronics are to be printed on the uncoated well areas of the reaction plate, substrate is preferably selected from different paper substrates, such as uncoated or coated paper, or glassine paper. Paper substrate provides good printing properties, such as resolution and/or patterning, for these purposes. Membrane-type substrates are suitable for applications, where the sheet-like reaction plate is employed in microfluidics. Cellulose sheets and starch sheets are especially suitable to be used as a substrate in different bioapplications.

In one embodiment the substrate may also be a paperboard like folding boxboard (FBB), white lined chipboard (WLC), solid bleached sulphate (SBS) board, solid unbleached sulphate (SUS) board or liquid packaging board (LPB), but not limited to these. Boards may have grammage from 120 to 500 g/m² and they may be based 100 % on primary fibres, 100 % on recycled fibres, or to any possible blend between primary and recycled fibres.

The hydrophobic ink material may be applied on the surface of the substrate by printing or coating. It may be applied on the surface of the substrate by inkjet printing, by flexographic printing, by screen printing or by reverse gravure coating or by gravure printing. Preferably the hydrophobic ink material is applied on the surface of the substrate by printing. In this context printing is understood as a process, where an image or pattern is transferred or reproduced on a substrate surface, preferably paper, from an original. Examples of printing processes are relief printing, including letterpress and flexography; planographic printing, including offset lithography; screenless lithography, collotype, and waterless printing; intaglio, including gravure, steel-die, and copper-plate engraving; stencil and screen printing; and electronic printing, including electrostatic, magnetographic, ion or electron deposition, and ink-jet printing. Preferable printing processes comprise flexographic printing and inkjet printing. Different printing methods may be applied for different purposes. For example, inkjet printing makes it possible to produce patterns with very high resolution, whereas flexographic printing provides a fast method for creating printed patterns on to the substrate. Printing provides fast and economically advantageous method of forming pre- determined patterns on substrate surface.

The ink material may be applied on one side of the reaction plate substrate or it may be applied on both sides of the substrate. Same or different ink material may be applied on the first and second large surface of the reaction plate substrate. The ink material may be applied in amount 1 - 15 g/m², typically 3 - 1 1 g/m², more typically 5 - 8 g/m².

The number, dimensions and shape of the uncoated reaction regions on the surface of the substrate may be freely chosen according to the needs of the end application. For example, the effective volume and/or depth of the uncoated reaction region may be adjusted either by changing the thickness of the hydrophobic layer or by changing the porosity of the substrate. The thickness of the pure hydrophobic ink layer may be 30 - 3000 nm, preferably 100 - 1000 nm. One embodiment of the sheet-like reaction plate according to the present invention may comprise uncoated reaction regions, which are arranged in the similar pattern than the wells of the conventional three-dimensional microtiter plate. The number of the uncoated regions may be 6, 24, 96, 384 or 1536 arranged in a 2:3 rectangular matrix. Thus the reaction plate according to the present invention may be made compatible with existing analytical equipment. The minimum distance between two uncoated regions may be 2 mm. For example, in a reaction plate having 96 uncoated regions which are arranged in a 2:3 rectangular matrix, the distance between adjacent uncoated regions may be 5 mm. The diameter of the uncoated reaction region on the substrate surface may be 0.3 - 5 mm, typically 0.5 - 3 mm, more typically 0.75 - 1 .5 mm. By using the present invention it is possible to choose the size and/or the shape of the uncoated region freely to suit the specific application. As the "wells" of the sheet-like reaction plate are created by coating or printing the hydrophobic ink material on to the hydrophilic substrate, the size and/or shape of the "wells" is easily adjusted according to the needs of the end application or analysis method. The uncoated reaction regions may also be in form of channels, which may be used to connect two adjacent uncoated regions in form of circular or rectangular "sample wells". The uncoated well regions may be functionalised with different reagents and the uncoated channel region may be left untreated or it may be treated with a substance which enhances the transfer of the sample from one uncoated well region to the adjacent second well region.

After applying the layer of the ink material to the substrate it may be cured, for example by using thermal treatment. Curing time is typically 5 - 90 s. The applied layer may be heated to a temperature in the range 100 - 180 °C. The hydrophobic ink material may alternatively be cured by radiation, for example by using IR or UV radiation. In this application the term IR-radiation comprises radiation wavelengths in the range from 0.7 to 300 μιτι, and the UV radiation comprises radiation wavelengths from 10 to 400 nm. The curing may occur also as self-curing under prolonged period of time, without any application of heat or light. According to one embodiment of the invention the uncoated reaction region of the substrate is functionalised, whereby the uncoated regions on the substrate surface comprise functional groups. The uncoated region of the substrate surface may also be functionalised with e.g. bioactive material to enhance anchoring of the analyte. The bioactive material is preferably covalently bound to the substrate surface. For example, it is possible to print an electronic circuit or to arrange an organic thin-film on the uncoated region of the substrate.

On the uncoated region may be bonded or applied a layer of conductive metal, such as silver, carbon, a layer of insulating material, a layer of sensor material, such as porous silicon, a layer of light sensitive material, such as fluorescence material or luminescence material, a layer of conductive and/or semiconductive material, such as poly(3-hexylthiophene) (P3HT), polyaniline (PANI), Poly(3,4- ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS), or a layer of biomaterial, such as different antibodies or streptavidin. The bonded or applied layer may be continuous, i.e. it may cover the whole area of the uncoated region, or it may be discontinuous, whereby a functional pattern may be formed on the uncoated region.

According to one embodiment of the invention it is possible to form uncoated reaction regions on both large surfaces of the sheet-like substrate by applying a layer of the ink material on both large surfaces of the sheet-like substrate in a predetermined pattern. In other words, the sheet-like reaction plate may comprise uncoated reaction regions both on the first surface and on the second, opposite surface, the uncoated reaction regions being defined by the hydrophobic ink layer.

FIGURES The invention is more closely explained in the following non-limiting schematical figures:

Figure 1 and 2 show schematically a sheet-like reaction plate according to one embodiment of the invention.

Figure 1 shows a sheet-like reaction plate 1 comprising a layer 2 of ink material 2 having a hydrophobic surface. The uncoated reaction regions 3, 3' are defined and encircled by the layer 2 of ink material, which defines the size and geometry of the uncoated reaction regions 3, 3'. Liquid, preferably aqueous, sample spreads on the hydrophilic uncoated reaction regions 3, 3' and is contained therein by the patterned hydrophobic ink material layer 2.

Figure 2 shows a similar sheet-like reaction plate 1 as in Fig. 1 . The numbering in Figure 2 corresponds to the numbering of Fig. 1 . A sheet-like substrate is indicated with letter A. EXAMPLE

Preparation of ink material Ink material is prepared as follows:

Polymer: Dehesive® 920; Crosslinker: V24; Catalyst: OL; all chemicals from Wacker silicones. Mixing ratio of polymer:crosslinker:catalyst is 100:2.5:1 weight- %. Solution of 20 weight-% polymer is poured into the mixing vessel and crosslinker is added. The mixture is stirred thoroughly for 5 min. Catalyst is added slowly under stirring and the resulting mixture is stirred for 10 min.

Preparation of reaction plate

A desired pattern geometry is printed on a substrate by using ink material prepared above. The ink material is crosslinked in the oven in 150 °C, for 10 s. After this the reaction plate is ready for use. Even if the invention was described with reference to what at present seems to be the most practical and preferred embodiments, it is appreciated that the invention shall not be limited to the embodiments described above, but the invention is intended to cover also different modifications and equivalent technical solutions within the scope of the enclosed claims.

Claims

1 . Method for manufacturing a sheet-like reaction plate for chemical and/or biochemical analysis, comprising

- selecting a sheet-like substrate comprising fibres of natural origin and having a hydrophilic surface, which has a contact angle of water < 90°,

- selecting a ink material, which has a hydrophobic surface when in layer form, whereby a contact angle of water is > 90°,

2. Method according to claim 1 , characterised in curing, preferably thermally, the layer of the ink material after applying it to the substrate, the curing time being 5 -

90 s.

3. Method according to claim 1 or 2, characterised in functionalising the uncoated reaction region of the substrate.

4. Method according to claim 1 , 2 or 3, characterised in preparing the ink material in a manner free from organic solvents by mixing a crosslinker to a solution of polymer, and thereafter adding a catalyst to the mixture.

5. Method according to any of the preceding claims, characterised in selecting the sheet-like substrate comprising fibres of natural origin from natural fibre-based substrates including unsized paper, internally sized paper, surface sized paper, uncoated paper, coated paper, glassine paper, filter paper, cellulose sheet and starch sheet.

6. Method according to any of the preceding claims, characterised in applying the ink material on the surface of the substrate by printing, for example by inkjet printing, by flexographic printing or by screen printing.

7. A reaction plate for chemical and/or biochemical analysis, comprising

- a sheet-like substrate comprising fibres of natural origin and having a hydrophilic surface, having a contact angle of water < 90°, and

- a layer of ink material having a hydrophobic surface, having a contact angle of water is > 90°, the layer of ink material being applied on the surface of the sheet like substrate so that the ink material directly defines uncoated reaction regions in a predetermined pattern on the surface of the sheet-like substrate.

8. Reaction plate according to claim 7, characterised in that the layer of ink material has a contact angle of water > 95°, or > 100°, typically > 105°, more typically > 1 10°, preferably > 1 15°.

9. Reaction plate according to claim 7 or 8, characterised in that the substrate has a hydrophilic surface, where the contact angle of water is < 75°, typically <

65°, more typically < 45°, preferably < 30°.

10. Reaction plate according to one of claims 7 - 9, characterised in that the sheet-like substrate comprising fibres of natural origin is selected from natural fibre-based substrates including unsized paper, internally sized paper, surface sized paper, uncoated paper, coated paper, glassine paper, filter paper, cellulose sheet and starch sheet.

1 1 . Reaction plate according to any of the preceding claims, characterised in that the ink material is free from organic solvents and prepared by mixing a crosslinker, such as a solvent free hydrogen polysiloxane comprising a high percentage of reactive Si-H groups, to a solution of polymer, such as addition-curing silicone, and thereafter adding a catalyst to the mixture.

12. Reaction plate according to any of the preceding claims, characterised in that the mixing ratio polymer:crosslinker:catalyst is 100 : (2 - 4) : (1 - 3), given in weight-%

13. Reaction plate according to any of the preceding claims characterised in that the thickness of the ink layer is 30 - 3000 nm, preferably 100 - 1000 nm.

14. Reaction plate according to any of the preceding claims characterised in that the uncoated reaction region on the substrate surface comprises functional groups.

15. Use of a reaction plate according to any of the claims 7

and/or biochemical analysis or for diagnostics.