Cation exchange paper

Abstract

Disclosed herein are methods of phosphorylating a cellulose filter paper in an aqueous solution comprising phosphate and urea for preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays with comparable results to Whatman® P81. Also disclosed herein is a phosphocellulose cation exchange filter paper obtained by the method of this disclosure. Further disclosed herein are methods of preparing unsupported phosphorylated cellulose paper for radiolabelled ATP assays and methods of conducting radiolabelled ATP assays on unsupported phosphorylated cellulose paper.

Description

[0001] CATION EXCHANGE PAPER

[0002] Field

[0003] This disclosure relates to methods of preparing cation exchange paper, in particular cation exchange paper for radiolabelled ATP kinase assays, and to cation exchange papers obtained by the methods. The disclosure also relates to phosphorylated cation exchange papers and methods of wax printing on cation exchange papers, and methods of performing radiolabelled ATP kinase assays on wax-printed phosphorylated cation exchange paper.

[0004] Background

[0005] The human kinome encodes approximately 520 protein kinases and approximately 20 lipid kinases, which play a central role in health and diseases such as cancer. Protein kinases constitute the largest proportion of targets in drug development programs in the biotechnology and pharmaceutical industry. To date, 121 protein kinase inhibitors are currently approved for clinical use. For example, Imatinib / Gleevec was a game changer for patients with chronic myelogenous leukemia and heralded the age of targeted therapies and precision oncology.

[0006] There is an ongoing requirement for reliable protein kinase assays for the identification and profiling of protein kinase inhibitors.

[0007] Radiometric assays are considered the “gold standard” for protein kinase assays because of their direct readout, high sensitivity, reproducibility, reliability, and very low background signals. There are numerous companies around the world that deliver kinase assays as a commercial service.

[0008] Non-radiometric high throughput screening assays include luciferase-based and fluorescence-based assays. These assays are indirect and typically measure a physical or chemical phenomenon that is a downstream effect of phosphorylation. Therefore, nonradiometric assays are affected by factors that interfere with these physical or chemical effects, such as background fluorescence and interference with coupling enzymes, and are consequently inferior to radiometric assays discussed above.

[0009] Radiometric ATP kinase assays rely on Whatman® P81 grade phosphocellulose cation exchange filter paper (P81 paper) for capturing peptide and protein analytes on the phosphorylated surface of the paper, after which unreacted material is washed off prior to analysis via scintillation counting. The global production of Whatman® P81 phosphocellulose cation exchange filter paper has been discontinued, and stockpiled supplies are running out. Although alternatives have been reported, such as Macherey-Nagel® LSA-50 paper, they do not match the performance of P81 paper. LSA-50 filter paper features a strong acidic cation exchange resin. The matrix is polystyrene cross-linked with 8.5% divinyl benzene and the active groups are SO3H. A common issue experienced in practice with alternatives to Whatman® P81 paper is that they do not produce accurate results because the observed binding is worse than that of Whatman® P81 paper for both peptide and protein substrates under varied concentrations of radiolabelled ATP.

[0010] The gold standard performance of Whatman® P81 paper has resulted in several small-scale producers of phosphocellulose paper, but the supply does not currently meet the demand and prices of these alternative phosphocellulose papers are high. Therefore, there is an urgent need to provide alternative phosphocellulose cation exchange paper for continuing performing fundamental research.

[0011] Methods to phosphorylate cellulose were initially developed to flameproof cloth. The phosphate ester favours dehydration of cellulose and therefore the formation of char, which is responsible for flame retardance (Scheme 1).

[0012] Scheme 1 : Proposed mechanism for thermally stable char formation of phosphorylated cellulose.

[0013] These initial reports used phosphorous oxychloride and pyridine or phosphoric acid and urea as reagents, however the use of phosphorylating agents including POCh, H3PO4, P2O5, (NH )2HPO4, NH4H2PO4, and organophosphates, and amines including N,N- dimethylformamide, pyridine, and urea have been reported to date. However, the use of these protocols is potentially risky as they involve dangerous reagents and therefore can only be performed by trained personnel under strict safety conditions.

[0014] Recent advances in the functionalisation of nanocellulose have renewed interest in the aqueous preparation of phosphocellulose using ammonium phosphates and urea since this approach is safer and more environmentally friendly. However, these current approaches are suitable for the phosphorylation of bulk cellulose. There remains a need for simple, safe and economical methods for producing phosphocellulose cation exchange filter paper.

[0015] The present disclosure provides methods that address at least some of these problems. Summary

[0016] Described herein is a safe, cost effective, aqueous method of preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays. The paper produced by this method is suitable for use in radiometric protein kinase assays with comparable results to those achieved with the Whatman® P81 paper. Advantageously, the method employs user-friendly, safe reagents and can easily be performed with minimal skill and basic equipment.

[0017] In a first aspect there is provided a method of phosphorylating a cellulose filter paper in an aqueous solution comprising phosphate and urea for preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays.

[0018] Phosphorylation of cellulose takes place by reacting the anhydroglucose units (AGU) of cellulose with a phosphate source in the presence of a base (Scheme 2).

[0019] Scheme 2: Phosphorylation of cellulose.

[0020] The method may comprise the steps of: a) Determining the number of anyhydroglucose units in the cellulose (AGU) of a filter paper; b) Incubating the filter paper in an aqueous solution comprising a phosphate source and a base; c) Swelling the cellulose of the filter paper; d) Optionally drying the filter paper; e) Curing the filter paper; f) Washing the filter paper; and g) Drying the filter paper.

[0021] The drying step d is optional, however it is desirable to obtain consistent results. Omitting the drying step reduces control of the reaction since curing occurs in the absence of water. When wet papers are inserted in the oven to cure, the incubation period would be equal to the drying time prior to phosphorylation. If the differences in the amount of water may vary between runs, thus introducing an error into the timing of the curing step. Therefore, it is advisable to dry the filter paper after steps b) and c) (e.g. with a hot gun) prior to the curing step. Steps b) and c) combined may be subsequently described as heat soak pretreatment. In other words, the method may comprise the following steps: a) Determining the number of anyhydroglucose units in the cellulose (AGU) of a filter paper; b) Subjecting the filter paper to a heat soak pretreatment step by submerging the filter paper in an aqueous solution comprising a phosphate source and a base at an elevated temperature; c) Optionally drying the filter paper; d) Curing the filter paper; e) Washing the filter paper; and f) Drying the filter paper.

[0022] The cellulose filter paper may be any filter paper or cellulose based material, such as wood composite. The cellulose filter paper may be any suitable grade, such as grade 1, grade 2, grade 3, grade 4, grade 5, grade 50 Hardened Ashless, grade 541 Hardened Ashless, 1 Chr, or 3 mm Chr, for example. Most preferably, the cellulose filter paper may be grade 1 filter paper such as Whatman® 1 Chr filter paper, Appleton® grade 1 filter paper, Supertek® grade 1 filter paper, smith filters® grade 1 filter paper, and the like. Whatman® grade 1 Chr Cellulose Chromatography Paper may be preferable in some instances because it is an analytical grade of filter paper.

[0023] The cellulose filter paper may have any shape and dimensions. For example, the paper may be compatible with 96-well plates. The cellulose filter paper may be round. The cellulose filter paper may have a diameter from about 5 mm to about 7 mm. The cellulose filter paper may be square or rectangular. For example, the cellulose filter paper may be about 210 x 297 mm (A4).

[0024] The base may be any suitable base such as an amine, a hydroxide salt, a carbonate salt, etc. Preferably, the base may not be pyridine. Preferably, the base may be urea. Without wishing to be bound by theory, using urea may be advantageous because, due to its melting point, urea is liquid at the reaction temperatures and may act as a solvent for the phosphate source. Phosphorylation with urea may render phosphorylated filter papers with higher charge densities compared to using different bases.

[0025] The phosphate may be provided by any suitable water soluble phosphate source, such as a phosphate salt, phytic acid or phosphoric acid. The phosphate salt may be mono basic, dibasic or tribasic. The phosphate may be H3PO4, [H2PC>4]', [HPC ]2', [PO4]3; or an organophosphate (e.g. fertiliser grade organophosphate). Preferably, the phosphate may be selected from [H2PO4]' or [HPC ]2'. The phosphate source may be selected from calcium a phosphate salt, a potassium phosphate salt, a sodium phosphate salt, an aluminium phosphate salt, an ammonium phosphate salt. The phosphate may be provided by an ammonium phosphate salt, such as ammonium phosphate monobasic (NH4H2PO4), or ammonium phosphate dibasic ((NH4)2HPO4). Without wishing to be bound by theory, the inventor has discovered that employing ammonium phosphate salts leads to faster phosphorylation than other phosphate sources.

[0026] The aqueous solution may further comprise a buffer. The buffer may be an acidic buffer or a basic buffer. In some cases, the buffer may be selected from phosphate buffered saline (PBS) based buffer or tris-buffered saline (TBS).

[0027] The amount of reagents required for the method will depend on the number of anyhydroglucose units (AGU) of the cellulose and the degree of phosphorylation of cellulose required. Therefore, the method may comprise an initial step (i.e. prior to the phosphorylation step) of determining the number of anhydroglucose units (AGU) in the cellulose.

[0028] Method for determining the AGU of cellulose

[0029] The concentration of anyhydroglucose units in cellulose is based on 162 g mol-1for AGU, or 6.2 mmol AGU per gram of cellulose.

[0030] For determining the AGU of the cellulose (e.g. cellulose filter paper), the cellulose filter paper may be weighed and the AGU may be calculated using the formula:

[0031] AGU in cellulose (mol) = [weight of cellulose (g)] ■ 6.2 ■ 10-3(mol ■ g~ )

[0032] In other words, if the degree of polymerisation of the cellulose is relatively high (in the case of the filter paper it’s about 8-1 OK), the calculation tends to be performed by essentially ignoring the molecular weight of the end groups and calculating the ratio instead based solely on the molecular weight of the AGU repeat unit (CeHwOs; 162.14 g / mol).

[0033] The AGU : phosphate source : base molar ratio and the wt.% cellulose may be varied to alter the degree of phosphorylation. The ratio of AGU : phosphate: urea may be 1 :0.6:3.2. The ratio of AGU : phosphate: urea may be 1 :2.5:10. The weight % of cellulose may be calculated by taking into account the weight of the filter paper and the weight of water in the solution. For example, the wt.% of a filter paper weighing 1 g would be 1 % when the filter paper is placed in a solution containing 100 mL (100 g) of water.

[0034] The molar ratio of AGU in the cellulose filter paper to phosphate source (AGU : phosphate source molar ratio) may be selected from about 1 :0.3 to about 1 :3, or from about 1 :0.3 to about 1 :5, or from about 1 :0.3 to about 1 :4, from about 1 :0.3 to about 1 :3, from about 1 :0.3 to about 1 :2.5, from about 1 :0.3 to about 1 :2, or from about 1 :0.3 to about 1 :1 , or from about 1 :0.5 to about 1 :3, from about 1:0.6 to about 1 :2.5, or from about 1:0.5 to about 1:1.5, or from about 1 :0.3 to about 1 to 1.35. The molar ratio of AGU in the cellulose filter paper to phosphate source (AGU : phosphate source molar ratio) may be about 1:0.3, or about 1:1.35, or about 1 :3.

[0035] The molar ratio of AGU in the cellulose filter paper to base (AGU : urea molar ratio) may be selected from about 1 :1.6 to about 1:15, or from about 1 :1.6 to about 1 :6.9, or from about 1:6.9 to about 1 :15, or from about 1:2.5 to about 1 :12, or from about 1 :2. to about 1 :11 , from about 1 :2.5 to about 1:10, from about 1:3 to about 1 :12, from about 1:4 to about 1:12, or from about 1 :5 to about 1 :12, or from about 1 :5 to about 1 :10, from about 1 :3.2 to about 1:10, or from about 1 :3 to about 1 :10.5.

[0036] The phosphate source may be ammonium phosphate monobasic, the base may be urea, and the ratio of AGU : ammonium phosphate monobasic: urea may be 1:0.6:3.2. The unadjusted pH in those embodiments may be at least 2.5, or at least 3, for example 4. Without wishing to be bound by theory, the reaction should be able to proceed at any pH but certain pH will result in greater degree of cellulose phosphorylation.

[0037] The phosphate source may be ammonium phosphate dibasic, the base may be urea, and the ratio of AGU : ammonium phosphate dibasic: urea may be 1:2.5:10. The unadjusted pH in those embodiments may be at least 7, or at least 8, for example 8.6.

[0038] The method may comprise heat soaking (incubating + swelling) the cellulose filter paper in the aqueous solution. Incubating cellulose in an aqueous solution may comprise maintaining the cellulose inside the aqueous solution for a period of time sufficient to allow the cellulose to be impregnated with the reagents. The cellulose filter paper may be maintained in the aqueous solution from about from about 10 minutes to about 90 minutes, or from about 20 minutes to about 90 minutes, or from about 20 minutes to about 80 minutes, or from about 20 minutes to about 70 minutes, or from about 20 minutes to about 60 minutes, or from about 10 minutes to about 60 minutes, or from about 10 minutes to about 45 minutes, or from about 10 minutes to about 30 minutes, or from about 15 minutes to about 30 minutes, or about 20 minutes to about

[0039] 35 minutes, or about 20 minutes to about 30 minutes, or about 25 minutes to about 35 minutes. The cellulose may be maintained in the aqueous solution for about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes. Preferably, the cellulose may be maintained in the aqueous solution for about 25 to about 30 minutes.

[0040] Incubating the cellulose filter paper in an aqueous solution may comprise immersing filter paper in the aqueous solution, covering the system, and maintaining the closed system for a period of time as discussed above. The incubation step may be performed while the system is stationary or while it is agitated (e.g. on a rocker, shaker, or via stirring (e.g. with a glass rod or magnetic stirrer) or the like). Incubating cellulose in an aqueous solution may be performed at any desired temperature, e.g. from about 15 °C to about 105 °C, or from about 15 °C to about 90 °C, or from about 15 °C to about 60 °C, or from about 15 °C to about 40 °C, or from about 15 °C to about 30 °C, or from about 15 °C to about 25 °C, or from about 18 °C to about 25 °C, or from about 18 °C to about 30 °C, or from about 30 °C to about 60 °C, or from about 60 °C to about 90 °C, or from about 70 °C to about 105 °C, or from about 80°C to about 90 °C. Incubating cellulose in an aqueous solution may be performed at room temperature (i.e. from about 18 °C to about 25 °C). Incubating cellulose in an aqueous solution may be performed at about 80 °C or at about 90 °C). Incubation at about 90 °C may render slightly higher charge densities than incubating at other temperatures. In some embodiments, the incubation step may be carried out at about 80 °C or at about 90 °C for about 25-30 minutes.

[0041] The method may further comprise the step of swelling the cellulose of the filter paper. Swelling the cellulose of the filter paper in the aqueous solution may be performed at any suitable temperature. For example, it may be performed at room temperature (i.e. from about 18 °C to about 25 °C), or it may be performed at a raised temperature, for example from about 30 °C to about 200 °C, or from about 70 °C to about 180 °C, or from about 80 °C to about 160 °C, or from about 70 °C to about 100 °C, or from about 100 °C to about 160 °C, or from about 120 °C to about 150 °C, or from about 30 °C to about 90 °C, or from about from about 30 °C to about 90 °C. Swelling cellulose in an aqueous solution may be performed at about 70 °C , or at about 80 °C, or at about 100 °C, or at about 120 °C, or at about 150 °C. In some embodiments, swelling cellulose in an aqueous solution may be performed from about 80 °C to about 150 °C, for example at 80 °C, 90 °C, or at 150 °C.

[0042] The incubating and / or the swelling steps separately or combined (e.g. heat soak step) may last from about 10 min to about 90 minutes, or from about 20 minutes to about 80 minutes, or from about 20 minutes to about 60 minutes, or from about 20 minutes to about 50 minutes, or from about 20 minutes to about 40 minutes, or from about 20 minutes to about 30 minutes, or from about 25 minutes to about 30 minutes, or from about 60 minutes to about 90 minutes, or from about 80 minutes to about 90 minutes, or from about 60 minutes to about 80 minutes, or from about 25 minutes to about 45 minutes. Preferably, the incubating and / or the swelling steps may last for about 25 minutes or for about 30 minutes.

[0043] The heat soak step may take place at a temperature of from about 50 °C to about 120 °C, or from about 70 °C to about 120 °C, or from about 80 °C to about 100 °C, or from about 70 °C to about 90 °C, or from about 75 °C to about 95 °C, or at about 80 °C, or at about 90 °C. Urea is liquid between 140-160 °C (melting point at 133 °C). Therefore in the range of 140- 160 °C urea will be fully liquefied and mostly available for performing the reaction. For this reason, in this temperature urea can swell cellulose and act as a solvent for phosphoric acid all whilst buffering cellulose from phosphoric acid to reduce degradation If ammonium phosphates are used as the phosphorylating agent instead, the sample only needs to be cured at high temperatures for less than an hour, which makes this approach practical for functionalising sheets of filter paper whilst maintaining the characteristics of the filter paper.

[0044] The swelling and incubation steps may be combined into a single step. This may be called the heat soak step. The swelling and incubation steps (or heat soak step) may last from about 10 minutes to about 90 minutes (preferably about 30 minutes) and take place at from about 80 °C to about 90 °C (preferably 90 °C).

[0045] The water content of the aqueous solution in the heat soak step may be from about 0.1 wt% to about 22 wt. %, or from about 0.5 wt. % to about 22 wt. %, or from about 0.1 wt. % to about 10 wt. %, or from about 1.5 wt. % to about 5 wt. %, or from about 1 wt.% to about 22 wt.%, or from about 1 wt.% to about 20 wt.%, or from about 1 wt.% to about 18 wt.%, or from about 1 wt.% to about 15 wt.%, or from about 1 wt.% to about 12 wt.%, or from about 1 wt.% to about 10 wt.%, or from about 1 wt.% to about 8 wt.%, or from about 1 wt.% to about 5 wt.%, or from about 5 wt.% to about 22 wt.%, or from about 10 wt.% to about 22 wt.%, or from about 15 wt.% to about 22 wt.%, or from about 17 wt.% to about 20 wt.%, or from about 5 wt.% to about 15 wt.%, or from about 12 wt.% to about 18 wt.%, or from about 5 wt.% to about 10 wt.%.

[0046] The weight percentage of cellulose in the heat soak pretreatment may be from about 0.1 wt.% to about 10 wt.%, or from about 0.1 wt.% to about 9 wt.%, or from about 0.1 wt.% to about 8 wt.%, or from about 0.1 wt.% to about 7 wt.%, or from about 0.1 wt.%, to about 6 wt.%, or from about 0.1 wt.% to about 5 wt.%, or from about 0.1 wt.% to about 4 wt.% or from about 0.1 wt.% to about 3 wt.%, or from about 0.1 wt.% to about 2 wt.%, or from about 0.1 wt.% to about 1 wt.%, or from about 0.5 wt.% to about 1.5 wt.% , or about 0.5 wt.%, or from about 1 wt.% to about 5 wt.%, or from about 1 wt.% to about 3 wt.%, or from about 5 wt.% to about 10 wt.%, or from about 7wt.% to about 10 wt.%, or from about 1 wt.% to about 2 wt.%, or about 1 wt.%, or about 1.5 wt.%, or about 2 wt.%, or about 3 wt.% , or about 4 wt.%, or about 5 wt.%. Preferably, the weight percentage of cellulose in the reaction may be about 1-2 wt.%, for example about 1.5 et. %.

[0047] The temperature of the heat soak step may be from about 50 °C to about 100 °C, or from about 70 °C to about 100 °C, or from about 80 °C to about 100 °C, or from about 80 °C to about 90 °C, or about 80 °C, or about 90 °C. The length of the heat soak step may be from about 10 minutes to about 45 minutes, or from about 20 minutes to about 40 minutes, or from about 25 minutes to about 35 minutes, or from about 20 minutes to about 35 minutes, or from about 25 minutes to about 35 minutes, or from about 25 minutes to about 30 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes. Preferably, the heat soak step may be 25 minutes or 30 minutes, most preferably 30 minutes.

[0048] The method may further comprise the step of drying the filter paper (after the incubation step - i. e. drying the incubated cellulose paper). Prior to drying, the filter paper may be removed from the aqueous solution. Advantageously, removing the filter paper from the aqueous solution may not require filtering the filter paper. For example, the filter paper may be lifted out from the aqueous solution by any suitable means such as with tweezers, spatula, by hand using gloves, or the like. The aqueous solution may be poured out of the container. The aqueous solution may be allowed to evaporate (e.g. at room temperature or at a raised temperature). Will evaporation of solvent at room temperature results in higher degrees of phosphorylation because more reagent remains with the paper into the curing step. However, evaporating off a large volume of water is not practical, and therefore removing the filter paper from the solution is the most practical approach.

[0049] The aqueous solution may be decanted out of the system comprising the aqueous solution and filter paper.

[0050] In some embodiments, the drying step is not required. For example, in embodiments in which the wt.% of cellulose is high (e.g.: about 10-20 wt.%), the filter paper may undergo the curing step directly after incubation step. However, in embodiments in which the wt.% of cellulose is low (e.g. from about 0.1 wt.% to about 1.5 wt.%), the filter paper may undergo a drying step between the swelling and curing steps. Without wishing to be bound by theory, drying the filter paper between the swelling and curing steps may increase the degree of phosphorylation achieved by the method. This is the case for a direct comparison on the degree of phosphorylation of a wet and a dry paper over the same curing times. The wet paper will undergo an induction period until most of the water has evaporated. However, a wet paper from a high wt.% heat soak could contain more phosphate and urea reagents, and consequently yield a higher charge density. Suitability of the paper to perform in kinase assays may be determined by reviewing the charge density of the phosphorylated filter paper and comparing it to a control (e.g. Whatman® P81 paper).

[0051] Drying may be performed at room temperature (i.e. from about 18 °C to about 25 °C) or at a raised temperature (e.g. from about 50 °C to about 120 °C, for example at about 100 °C). The drying step may be performed in an oven. The drying step may be performed under vacuum. The drying step may be performed in ambient conditions (e.g. on a plate, airer, dish or the like). Drying may be assisted (e.g. to speed up the drying process) by an air current such as compressed air, or by placing the filter paper on a funnel and applying vacuum. Drying may be assessed by weighing the filter paper at time intervals until the weight of the paper has stabilised (i.e. until the weight of the paper does not change over time).

[0052] The drying step may be performed for as long as it is required until the filter paper is dry (i.e. until the weight of the filter paper is stable).

[0053] The method may further comprise the step of curing the filter paper. Without wishing to be bound by theory, phosphorylation may take place during the curing step. Curing the paper may be performed at a raised temperature (e.g. in an oven). Curing the paper may be performed from about 130 °C to about 200 °C, or from about 130 °C to about 160 °C, or from about 130 °C to about 160 °C, or from about 130 °C to about 150 °C, or from about 150 °C to about 170°C, or at about or at about 130 °C, or at about 140 °C, or at about 150 °C, or at about 160 °C.

[0054] The curing step may be performed for any length of time required for phosphorylation to take place.

[0055] The curing step may be performed for about 30 seconds, about 1 minute, about 2 minutes, or about 5 minutes, or about 7 minutes, or about 10 minutes to about 45 minutes, or for about 15 minutes to about 40 minutes, or for about 20 minutes to about 35 minutes, or for about 20 minutes to about 30 minutes. The curing step may be performed for about 10 minutes to about 180 minutes, or from about 20 minutes to about 60 minutes, or about 10 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes, or from about 15 minutes to about 45 minutes, or for about 30 minutes to about 60 minutes, or from about 45 minutes to about 180 minutes, or for about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes. The curing step may last from about 1 minute to about 90 minutes, or about 5 minutes to about 70 minutes, or from about 10 minutes to about 80 minutes, or from about 10 minutes to about 60 minutes, or from about 20 minutes to about 50 minutes, or from about 20 minutes to about 40 minutes, or from about 20 minutes to about 30 minutes, or from about 25 minutes to about 30 minutes, or from about 60 minutes to about 90 minutes, or from about 80 minutes to about 90 minutes, or from about 60 minutes to about 80 minutes, or from about 25 minutes to about 45 minutes. The curing step may be performed for 5 minutes, 10 minutes, 30 minutes, or 60 minutes. Preferably, the curing step may last for about 25 minutes or for about 30 minutes.

[0056] The curing step may be performed at about 150 °C for about 20-30 minutes. The curing step may be performed from about 150 °C to about 170 °C for about 20-90 minutes. The curing step may be performed at about 165 °C for up to 10 minutes. However, experimental design (discussed later) revealed that reaction time is not as impactful as other variables.

[0057] The reaction pH may be from about 2 to about 9, or from about 5 to about 9, or from about 7 to about 9, or from about 8 to about 9, or from about 2 to about 5, or from about 2 to about 4, or from about 3 to about 4, or from about 2.5 to about 4.5, or from about 2.5 to about 3.5. Preferably the reaction pH may be about 4. In some embodiments, the phosphate source may be ammonium phosphate monobasic and the ratio of AGU : ammonium phosphate monobasic: urea may be 1 :0.6:3.2. The pH in those embodiments may be at least 2.5, or at least 3, for example 4. The pH may be about 8 or about 9, for example about 8.6. The curing time required for these embodiments may vary, but in some examples the curing step may be maintained for about 20 minutes.

[0058] In some embodiments, the phosphate source may be ammonium phosphate dibasic and the ratio of AGU : ammonium phosphate dibasic: urea may be 1:2.5:10. The unadjusted pH in those embodiments may be at least 7, or at least 8, for example 8.6. The curing time required for these embodiments may vary, but in some examples the curing step may be maintained for about 30 minutes.

[0059] Without wishing to be bound by theory, increasing the temperature at which any of the steps is carried out may decrease the time required for completing said step(s).

[0060] The method may comprise the step of washing the (functionalised) filter paper with any suitable solvent. Deionised water may be preferred. Washing the filter paper may remove unreacted reagents from the filter paper, for example to prevent contamination or crossreaction with excess reagents during assays. The washing step may be performed at any required temperature, but room temperature may be preferred (e.g. about 18 °C to about 22 °C). The washing step may be performed by rinsing the filter paper with the solvent or by immersing the filter paper in the solvent and draining the solvent.

[0061] The method may comprise drying the (functionalised) filter paper. The drying step may be performed at room temperature or at a raised temperature. For example the drying step may be performed (e.g. in an oven) at a temperature between about 30 °C and about 105 °C, for example at about 100 °C. Additionally or alternatively, the drying step may be assisted, for example by washing with an organic solvent (e.g. acetone). The drying step may be performed under vacuum. The drying step may be performed in ambient conditions (e.g. on a plate, airer, dish or the like). Drying may be assisted (e.g. to speed up the drying process) by an air current such as compressed air, or by placing the filter paper on a funnel and applying vacuum. Advantageously, the method may not require filtration of the cellulose filter paper as opposed to methods of phosphorylating cellulose pulp. The present method allows simpler and easier handling of the starting material (filter paper) compared to loose cellulose pulp or fibres. The reagents and reaction conditions employed in the present method are safer than other methods of cellulose phosphorylation in the art. Furthermore, the method does not require a complex and cumbersome step of forming paper sheets as it employs off the shelf filter paper, therefore simplifying the procedure, for example enabling any lab to perform it in house. Therefore, the present method provides a fast and convenient method for phosphorylating cellulose filter paper which can be subsequently used in radiometric protein kinase assays.

[0062] The degree of substitution and grafting mode (i.e.: mono, disubstituted, trisubstituted, or crosslinked phosphoester) depends on variables including the: 1) molar ratio between the phosphorylation agent and the cellulose monomer, 2) reaction time, and 3) reaction temperature. The design of experiments also revealed that the amount of urea is crucial. Also, the wt.% cellulose during the heat soak and reaction pH are very influential. The degree of phosphorylation not only influences the charge density but also the tensile strength and appearance of the filter paper product. The inventor has found that the proposed reaction conditions provide suitable phosphorylation levels with minimal reaction times and temperatures, therefore providing a simple, safe, cost-effective method for preparing functionalised phosphocellulose cation exchange filter paper which can be used for radiolabelled ATP kinase assays.

[0063] In a second aspect there is provided a phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays.

[0064] The phosphocellulose cation exchange filter paper may be obtained directly by the method of phosphorylating a cellulose filter paper described above.

[0065] The phosphocellulose cation exchange filter paper may have comparable performance to Whatman® P81 filter paper in radiometric protein kinase assays.

[0066] In a third aspect there is provided a phosphocellulose cation exchange paper suitable for use in radiometric protein kinase assays.

[0067] Without wishing to be bound by theory, the phosphocellulose cation exchange filter paper may have more than >50% strong acid, even just slightly from some crosslinking. This would be advantageous for reasons discussed in terms of reactivity.

[0068] The phosphocellulose cation exchange filter paper may have a charge density of from about 90 pmol g-1to about 500 pmol g-1, or about 100 pmol g-1to about 450 pmol g-1, or about 100 pmol g-1to about 400 pmol g-1, or about 100 pmol g-1to about 350 pmol g-1, or about 100 pmol g-1to about 300 pmol g1, or about 100 pmol g-1to about 250 pmol g-1, or about 100 pmol g1to about 200 pmol g1, or about 100 pmol g-1to about 150 pmol g1, or about 120 pmol g-1to about 250 pmol g-1, or about 200 pmol g-' to about 300 pmol g-1, or about 98 pmol g-1, or about 117 pmol g-1, or about 140 pmol g-1, or about 150 pmol g-1, or about 160 pmol g-1, or about 200 pmol g-1, or about 240 pmol g-1, or about 350 pmol g-' , or about 420 pmol g-1. The charge density of the phosphocellulose paper may be measured according to the conductometric titration method described herein.

[0069] The phosphocellulose cation exchange paper may be from about 0.1 mm to about 0.6 mm thick, or from about 0.1 mm to about 0.4 mm thick, or from about 0.1 to about 0.3 mm thick, or from about 0.1 to about 0.2 mm thick, or from about 0.2 mm to about 0.4 mm thick, or about 0.2 mm thick, or about 0.3 mm thick, or about 0.4 mm thick.

[0070] The tensile strength of the phosphocellulose cation exchange paper may decrease from dry to wet. The strength of the phosphocellulose cation exchange paper when dry may be from about 6.5 MPa to about 13.0 MPa, or from about 8.0 MPa to about 10.0 MPa, or from about 8.0 MPa to about 10.0 MPa, or from about 8.5 MPa to about 10.0 MPa, or about 8.5 MPa, or about 9.0 MPa, or about 9.5 MPa, or about 10 MPa.

[0071] The tensile strength of the phosphocellulose cation exchange paper when wet may be from about 100 kPa to about 300 kPa, or from about 100 kPa to about 250 kPa, or from about 100 kPa to about 200 kPa, or from about 150 kPa to about 250 kPa, or about 150 kPa, or about 200 kPa, or from about 200 kPa to about 250 kPa, or about 130 kPa, or about 150 kPa, or about 170 kPa, or about 200 kPa, or about 210 kPa, or about 220 kPa, or about 230 kPa, or about 240 kPa.

[0072] The strain of the phosphocellulose cation exchange paper when dry may be from about 2% to about 5%, or from about 2% to about 4%, or from about 2% to about 3%, or from about 3% to about 4%, or from about 4% to about 5%, or from about 3.0% to about 4.0%, or from about 2.5% to about 4.5%, or about 2%, or about 3%, or about 4%.

[0073] The strain of the phosphocellulose cation exchange paper when wet may be from about 1% to about 4%, or from about 1 % to about 3%, or from about 1 % to about 2%, or from about 2% to about 4%, or from about 1.2% to about 3.0%, or from about 1.5% to about 3.5%, or from about 1.4% to about 2.8%, or about 1.2%, or about 1.3%, or about 1.4%, or about 1.5%, or about 1.6%, or about 1.7%, or about 2%, or about 2.5%, or about 2.7%, or about 2.8%, or about 3.0%.

[0074] In a fourth aspect there is provided a method of manufacturing unsupported phosphocellulose cation exchange paper for radiolabelled ATP assays. The method comprises providing a phosphocellulose cation exchange paper and depositing a boundary of hydrophobic material on the phosphocellulose cation exchange paper to receive sample for performing a radiolabelled ATP assay. The boundary is configured to contain a sample and prevent the sample from spreading or otherwise spilling outside the area enclosed by the boundary.

[0075] The boundary may be deposited in any suitable shape of form. For example, the boundary may be deposited as a circle, a square, triangle, rhomboid, rectangle or any suitable shape. The boundary may be closed (to prevent sample from exiting the area enclosed by the boundary).

[0076] The method may comprise depositing a single boundary or multiple boundaries. For example, the method may comprise depositing a row of boundaries (e.g. a row of circles), or multiple rows of boundaries. The method may comprise depositing a 96 well pattern of boundaries on the phosphocellulose cation exchange paper.

[0077] The hydrophobic material may be any suitable hydrophobic material which may be deposited permanently on phosphocellulose cation exchange paper and be stable (e.g. not dissolve or decompose) in the conditions employed in radiolabelled ATP kinase assays. For example, the hydrophobic material may be a wax based material, such as Xerox ColorQube ink or other solid ink printer ink, a polymeric material, a resin (or natural or synthetic origin), such as rosin, or a combination thereof. Preferably, the hydrophobic material may be a wax-based hydrophobic material.

[0078] Deposition of the hydrophobic material on the phosphocellulose cation exchange paper may be performed by any suitable means. For example, the hydrophobic material may be printed, 3-D printed, screen printed, painted manually (e.g. with a gel pen), or the like. Preferably, deposition of the hydrophobic material may comprise printing the hydrophobic material on the phosphocellulose cation exchange paper in the desired boundary shape and pattern. Printing the hydrophobic material on the phosphocellulose cation exchange paper may require a step of fluidizing the hydrophobic material prior to printing, for example by heating the hydrophobic material above its melting point, dissolving, or dispersing the hydrophobic material in a carrier or solvent.

[0079] The phosphocellulose cation exchange paper may be any phosphocellulose cation exchange paper suitable for performing radiolabelled ATP assays. For example, the phosphocellulose cation exchange paper may be Whatman® P81 or the phosphocellulose cation exchange paper of any of the preceding aspects. Preferably, the phosphocellulose cation exchange paper is one of the preceding aspects. In preferred embodiments, the method comprises wax printing one or more boundaries (e.g. circle(s) on a phosphocellulose cation exchange paper of one of the preceding aspects. In most preferred embodiments, the method comprises wax printing a 96 well pattern of wax on a phosphocellulose cation exchange paper of one of the preceding aspects.

[0080] In a fifth aspect there is provided an unsupported phosphocellulose cation exchange paper for a radiolabelled ATP assay having a boundary of hydrophobic material for receiving a sample for radiolabelled ATP assay. The unsupported phosphocellulose cation exchange paper may be directly obtained by the method of the fourth aspect and / or may comprise any of the characteristics described in the fourth aspect. The unsupported phosphocellulose cation exchange paper may comprise multiple boundaries of hydrophobic material for receiving a sample for a radiolabelled ATP assay. For example, the unsupported phosphocellulose cation exchange paper may comprise a pattern of boundaries of hydrophobic material, such as a 96 well plate pattern.

[0081] In a sixth aspect there is provided a method of performing a radiolabelled ATP assay, the method comprising providing an unsupported phosphocellulose cation exchange paper according to the fifth aspect. The method may comprise depositing a sample inside the boundary or boundaries of the hydrophobic material (this may be done manually (e.g. with a single or multi-channel pipette) or employing an automated sampler), optionally placing the cation exchange paper in a frame or support. The method may comprise washing the unsupported phosphocellulose cation exchange paper with the sample (the washing step can be performed with water orwith orthophosphoric acid); optionally placing the phosphocellulose cation exchange paper in a support or frame; optionally drying the phosphocellulose cation exchange paper; sealing the phosphocellulose cation exchange paper with liquid scintillation fluid cocktail, optionally wherein sealing is performed in a flexible transparent container.

[0082] Brief Description of the Figures

[0083] Figure 1: Image of functionalised filter paper and control filter paper incubated in aqueous CuSC>4 and washed with boiling water (Left: functionalised and washed with NaCI; Middle: functionalised; Right: untreated control).

[0084] Figure 2: Quantitation of radiation on cation exchange papers via scintillation for the kinase assay of EP975 using PKA (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

[0085] Figure 3: Quantitation of radiation on cation exchange papers via scintillation for the kinase assay of: a) EP4562 using Aurora A, b) POLY using BRK, and c) c-kit peptide using c-kit (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

[0086] Figure 4: Quantitation of radiation on cation exchange papers via scintillation for a kinase assay and I C50 curve of Sapk2b with inhibitor SB203580 using 20 pM33P ATP (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

[0087] Figure 5: Quantitation of radiation on cation exchange papers via scintillation for the kinase assay of PKBa using 5 pM33P ATP (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

[0088] Figure 6: Quantitation of radiation on cation exchange papers via scintillation for the kinase assay of Sapk2b using 20 pM33P ATP (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

[0089] Figure 7: Quantitation of radiation on cation exchange papers via scintillation for the kinase assay of SRPK1 using 50 pM33P ATP (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

[0090] Figure 8: Conductometric titration curve of Whatman P81 filter paper (0.1241 g; 4 cm x 4 cm) in acidic media titrated with NaOH. A) Full titration curve, showing conductivity and pH. B) Region of interest between conductometric equivalence points.

[0091] Figure 9: Conductometric titration curve of Inventive Paper 1 (0.1423 g; 4 cm x 4 cm) in acidic media titrated with NaOH. A) Full titration curve, showing conductivity and pH. B) Region of interest between conductometric equivalence points.

[0092] Figure 10: Conductometric titration curve of Inventive Paper 2 (0.1428 g; 4 cm x 4 cm) in acidic media titrated with NaOH. A) Full titration curve, showing conductivity and pH. B) Region of interest between conductometric equivalence points.

[0093] Figure 11 : Thickness of dry and wet filter papers.

[0094] Figure 12: Tensile stress-strain curves of dry filter papers.

[0095] Figure 13: Tensile stress-strain curves of wet filter papers.

[0096] Figure 14: Tensile strength (a) values of a) dry, and b) wet filter papers. Figure 15: Percent tensile strain (E) values for a) dry, and b) wet filter papers.

[0097] Figure 16: Young’s modulus (E) values for a) dry, and b) wet filter papers.

[0098] Figure 17: Spotting of Orange G (24 mM in water) on Whatman® P81 and Inventive Paper 2.

[0099] Figure 18: Image of wax printing on Inventive Paper 2 a) before, and b) after heating in a 150 °C oven for 2 minutes.

[0100] Figure 19: Image of various volumes (5-20 pL) of Orange G (24 mM in water) spotted into wax printed Inventive Paper 2 wells.

[0101] Figure 20: T ransfer of reaction mixtures from a 96 well plate onto wax printed cation exchange filter paper using a multi-channel pipette.

[0102] Figure 21: Images of loading of wax printed cation exchange filter paper in scintillation cocktail fluid into the back of a plate prior to reading on a TopCount NXT microplate scintillation counter.

[0103] Figure 22: Quantitation of radiation on Whatman® Unifilter P81 filter 96 well microplate devices via scintillation for the IC50 curve of CLK2 with selected inhibitors using 20 pM33P ATP (count time = 1 minute; reaction mixture volume analysed = 25 pL).

[0104] Figure 23: Quantitation of radiation on 96 well wax printed Inventive Paper 2 via scintillation for the IC50 curve of CLK2 with selected inhibitors using 20 pM33P ATP (count time = 1 minute; reaction mixture spot volume = 10 pL).

[0105] Figure 24: Properties of wet filter papers. A: Thickness of wet filter papers. B: Tensile strength (a) values of wet filter papers.

[0106] Figure 25: Properties of wet filter papers. A: Percent tensile strain (s) values for wet filter papers. B: Charge density of filter papers.

[0107] Figure 26: Pareto chart of standardised effects on charge density.

[0108] Figure 27: Chart of main effects on charge density.

[0109] Figure 28: Chart of interacting effects on charge density.

[0110] Figure 29: A: Pareto chart of standardised effects on tensile strength (o). B: Chart of main effect on tensile strength (o).

[0111] Figure 30: A: Pareto chart of standardised effects on kinase assay performance; B: Chart of main effect on kinase assay performance.

[0112] Figure 31: Pareto chart of standardised effects on wet filter paper thickness. Figure 32: Chart of main effect on wet filter paper thickness.

[0113] Figure 33: Plot of filter paper tensile strength (a) as a function of charge density.

[0114] Figure 34: Charge density and tensile strength (a) values for Inventive Papers. The red line denotes a strength threshold, below which papers maintain insufficient strength for use in routine kinase assays.

[0115] Figure 35: Relationship between wet filter paper thickness and tensile strength.

[0116] Figure 36: Binding capacity observed in kinase assay as a function of filter paper tensile strength (o) and charge density.

[0117] Figure 37a: Conductometric titration curve of Whatman P81 filter paper (0.1310 g) titrated with NaOH; b: Conductometric titration curve of Inventive Paper 1 (0.5696 g) titrated with NaOH.; c: Conductometric titration curve of Inventive Paper 2 (0.2816 g) titrated with NaOH.

[0118] Detailed Description

[0119] Disclosed herein is a safe, cost effective, aqueous method to functionalise cellulose filter paper to prepare phosphocellulose cation exchange filter paper for radiolabelled ATP kinase assays.

[0120] The method may comprise the steps of: a) Determining the number of anyhydroglucose units in the cellulose (AGU) of a filter paper; b) Incubating the filter paper in an aqueous solution comprising a phosphate source and a base (e.g. urea); c) Swelling the cellulose of the filter paper; d) drying the filter paper; e) Curing the filter paper; f) Washing the filter paper; and g) Drying the filter paper.

[0121] Steps b) and c) may be performed together at elevated temperature as a heat soak step. The advantage of keeping the incubation step separately is that agitation / rocking / stirring can happen at this stage before placing in the oven and may lead to greater phosphorylation rates. However, performing a single heat soak step at elevated temperature may also increase charge densities and simplify the process.

[0122] The conditions and reagents of the method are discussed in the summary section of this application.

[0123] Test Methods Conductometric Titration for determination of charge density

[0124] Conductometric titration experiments were performed in duplicate according to the Canadian Standards Association protocol for titration of cellulose functionalised with weak acids (CSA Z5100-14) using Mettler Toledo Seven Excellence conductometer to determine the charge density of the phosphocellulose papers. The pH was also monitored during the titrations. Prior to analysing the papers, counterions were exchanged into protons by incubating the papers in 200 mM HCI with rocking at room temperature overnight, washing the papers with deionised water, and drying the papers in an oven at 100 °C. Phosphocellulose filter papers in acidic aqueous solution were titrated with NaOH. The initial step to the first equivalent point is associated with deprotonation of the free acid, which is followed by deprotonation of the acidic analyte to the second equivalence point. Thereafter, the increase in conductivity is associated with excess NaOH. The total analyte acid content is calculated from the volume of NaOH titrated between the strong acid and analyte acid equivalence points and is used to calculate the charge density of the phosphocellulose papers.

[0125] Tensile Testing

[0126] Mechanical properties of cation exchange and Whatman® Grade 1 Chr filter papers were assessed using a Instron 34SC-1 universal testing machine equipped with a 500 N load cell. Specimens were cut into 80 mm x 20 mm rectangular strips and conditioned at ambient temperature and humidity for 3 days prior to testing for dry samples or saturated in deionised water for 24 hours at ambient temperature prior to testing for wet samples, after which specimen thickness was evaluated using a micrometer. The gauge length was set to 30 mm and specimens were tested at a crosshead speed of 0.25 mm min-1.

[0127] Typical stress-strain curves were obtained, and the tensile strength (o), strain at break (c), and Young’s modulus (E) were calculated by taking the average across 5 specimens.

[0128] Determination of ratios of strong to weak protons by conductometric titrations

[0129] Conductometric titration experiments were performed using a Mettler Toledo Seven Excellence conductometer to determine the ratio between strong and weak acid content in phosphocellulose papers. The pH was also monitored during the titrations. Prior to analysing the papers, counterions were exchanged into protons by incubating the papers in 200 mM HCI with rocking at room temperature overnight, washing the papers with deionised water, and drying the papers in an oven at 100 °C. Phosphocellulose filter papers in aqueous solution were adjusted to an initial conductivity above 200 pS cm-1using 5% NaCI and were titrated with 10 mM NaOH. The initial step to the first equivalent point is associated with deprotonation of the strong acid, which is followed by deprotonation of the weak acid to the second equivalence point. Thereafter, the increase in conductivity is associated with excess NaOH. The ratio of analyte acid content is calculated from the relative volumes of NaOH titrated up to the first equivalence point, corresponding to strong acid A1 , and between equivalence points, corresponding to weak acid A2. The results are presented in Figure 37a (Whatman ® P81), 37 b (Inventive Paper 1) and 37c (Inventive Paper 2).

[0130] Examples

[0131] The following method protocol was followed to prepare phosphocellulose cation exchange filter papers.

[0132] Phosphorylation Method Protocol

[0133] 1. Weigh Whatman® grade 1 Chr paper (or another grade of cellulose filter paper) and calculate the number of anhydroglucose units (AGU).

[0134] 2. Incubate the filter paper in a glass dish containing an aqueous solution of ammonium phosphate monobasic or dibasic and urea on a rocker at room temperature. The AGU : phosphate source : urea molar ratio and wt.% cellulose (i.e.: the amount of water) can be varied within the ranges discussed above to alter the degree of phosphorylation (incubation step).

[0135] 3. Heat the covered mixture in an oven at 80 °C-90°C for 25-30 minutes to swell the paper (swelling step).

[0136] 4. Remove the solution from the dish or remove the filter paper from the solution and place it in a fresh glass dish. Alternatively, where the wt.% cellulose is high (i.e.: 10-20 wt.% compared to 1 or 0.1 wt.%, for example), the paper can be dried from solution to increase the degree of phosphorylation.

[0137] 5. Dry the paper in an oven at 100 °C until the weight has stabilised (i.e.: the paper is dry) (drying step). This step is performed to increase the degree of phosphorylation as the presence of water may decrease the degree of phosphorylation with urea and it is preferred that the curing step occurs in the absence of water or when the water content is stable

[0138] 6. Cure the paper in an oven at 150 °C for 20-30 minutes. Note: This is the step during which cellulose is functionalised, and varying the cure time and temperature impacts the charge density (curing step).

[0139] 7. Wash the paper extensively in deionised water at room temperature (washing step).

[0140] 8. Dry the paper at ambient temperature. Alternatively, drying can be accelerated by using an oven, vacuum or organic solvent such as acetone (drying step). Example 1

[0141] Reagent ratios: AGU : ammonium phosphate monobasic : urea = 1 :0.6:3.2 (the unadjusted pH is acidic, pH « 4).

[0142] Curing conditions in step 6: cured at 150 °C for 20 minutes.

[0143] Example 2

[0144] Reagent ratios: AGU : ammonium phosphate dibasic : urea = 1 :2.5:10 (the unadjusted pH is basic, pH ~ 8.6).

[0145] Curing conditions in step 6: cured at 150 °C for 30 minutes.

[0146] Characterisation of examples, comparison with Whatman® P81 and optimisation of reaction conditions

[0147] The most important features of phosphocellulose filter paper for radiometric assay applications are a) the charge density of the paper for assay performance, and b) the physical properties of the paper such that it is suitable for the application in that it has sufficient tensile strength and is not soluble in water.

[0148] Although Inventive Papers 1 and 2 both exhibited similar cation exchange performance in radiolabelled ATP kinase assays as Whatman® P81 cation exchange filter paper, differences in their charge densities and mechanical properties were determined by conductometric titrations and tensile strength testing, respectively, such that Inventive Paper 1 exhibited superior mechanical properties whereas Inventive Paper 2 maintained a higher charge density. This suggests that, in the phosphorylation of cellulose filter paper using urea as a catalyst, there exists a trade-off between charge density and tensile strength. Since sufficient charge density is required for performance in cation exchange assays whilst sufficient wet tensile strength is required to handle the wet filter papers when performing an assay, reaction conditions should optimise charge density and wet tensile strength.

[0149] Independent Variables:

[0150] • Paper choice: although there are many grades and thicknesses of filter paper that can be functionalised, Whatman® grade 1 Chr Cellulose Chromatography Paper was chosen by the inventor for the following reasons: o It is an analytical grade of filter paper with uniform wicking capability o It is available in large sheets (58 x 68 cm) that can be cut to any desired size.

[0151] The inventor had a preference for A4 size paper as it can be run through a printer for wax printing and is a convenient size for researchers, storage and shipping, in addition to swelling and curing in an oven. o Its 0.18 mm thickness and smooth surface is convenient for analytical techniques and wax printing o It is composed of pure cellulose produced from cotton linters and is additive free. It therefore does not require any pre-treatment and can be used directly to prepare phosphocellulose. o It is widely available at a reasonable price (£280 for 100 ea. 58 x 68 cm sheets, from which 5 A4 sheets can be cut) o Stronger commercial filter papers were considered, such as Whatman® Quantitative hardened ashless filter paper, however were found to be significantly more expensive, often not available in large sheets (> A4), and sometimes contain additives (for example, Whatman® Grade 1573 Qualitative Filter Papers have high wet strength, however contain a resin which could interfere with either the preparation of phosphocellulose or assays performed using phosphocellulose).

[0152] • Phosphate source: Numerous phosphate sources are available for the phosphorylation of cellulose using urea such as phosphoric acid, phosphate salts, and phytic acid, to name a few. Both ammonium phosphate monobasic and ammonium phosphate dibasic were selected by the inventor for further investigation for the following reasons: o Absence of hazards (safer than phosphoric acid) o Low cost (alternatives such as phytic acid are significantly more expensive) o Wide availability o Reactivity: Different phosphate salts were considered but ammonium salts were selected for the phosphorylation of cellulose. Without wishing to be bound by theory, phosphates salts with ammonium counterion may be more reactive and in turn result in higher degrees of phosphorylation because of the lower affinity of the ammonium counterion to the acid. However, any other phosphate salt or phosphoric acid may be used for the phosphorylation reaction. o Forms an ammonium counterion in the resulting phosphocellulose, which is desirable for phosphocellulose reactivity and mechanical properties o The smaller the counterion, the stronger the binding affinity to the acid. Since ammonium is larger than sodium and lithium, for example, it has less affinity for the acid, and consequently its rate of counterion exchange is quicker, enhancing the capture rate of the cation exchange filter paper. o Cellulose fibers with a quaternary ammonium counterion may exhibit improved mechanical properties relative to lithium or sodium counterions. Fiber strength may increase with ion size since the reduction in binding affinity enhances hydrogen bonding between fibers and sliding between fibers, enabling further deformation without a loss in strength. Therefore, quaternary ammonium counterions ultimately improve inelastic deformation, yielding tougher, more ductile cation exchange filter papers.

[0153] • Base: Although other bases may be used, urea was selected as the base because of its absence of hazards, low cost, and performance in green, aqueous phosphorylation methods. Without wishing to be bound by theory, phosphorylation of cellulose in the presence of urea has may yield higher charge densities than phosphorylation in the absence of urea.

[0154] • Molar ratios of phosphate and base (e.g. urea): The molar ratios of phosphate and base selected may have been tailored to the cellulose source and desired properties of the resulting phosphocellulose. o Although any base could be used in the phosphorylation reaction, the inventor chose urea as preferential base for the experiments because it may 1) enhance cellulose fiber swelling, increasing penetration of the phosphate salt, 2) catalyse the phosphorylation reaction, since reactions performed in the absence of urea result little charge content, 3), increase charge content, and 4) prevent degradation of cellulose during curing. o Amount of phosphate salt selected for further investigation: 0.3, 1.35, and 3. These values were selected to explore the effect on the phosphate concentration on reactions and quality of papers for being above and below those used for Inventive Papers 1 and 2 o Amount of urea selected for further investigation: 1.6, 6.9, and 15. Low medium, and high concentrations of base (urea in this case) were selected for further investigation.

[0155] • Amount of water during heat soaking pretreatment (wt.% filter paper / cellulose): Filter paper cellulose fibers are impregnated with reagents via a heat soak pretreatment in aqueous solution containing phosphate salt and urea. Thereafter, excess reagent is decanted and the filter paper dried in an oven at 100 °C. A direct correlation between the amount of water used and the charge density obtained in the final product was noted such that increasing the amount of cellulose (wt.%) increases the charge density, presumably since the amount of reagents absorbed by the fibers increases as the concentration of reagents in solution is increased (data not shown). Although various amounts of water used in the heat soaking pretreatment have been investigated (0.1-10 wt.% cellulose), the amount of water used in this process was optimised for the preparation of A4 size sheets (« 5.48 g). In this study, A4 size sheets were soaked in a tray with 548 mL of aqueous solution of phosphate salt and urea to completely cover the filter paper sheet (1 wt.% cellulose). Therefore, the amount of cellulose used in the reaction was set to 1 wt.%.

[0156] • Heat soak temperature: A heat soak temperature of 90 °C was selected to maximise the charge density of the phosphorylated filter paper.

[0157] • Heat soak time: A 30 minute heat soak time was selected to maximise charge density while prevent damage to the cellulose fibre crystallinity.

[0158] • Curing temperature: Phosphorylation of cellulose using urea is a reaction in which urea acts as both a catalyst and solvent. As such, the reaction should be performed above the melting point of urea (133 °C). A reaction temperature of 150 °C was selected for the following reasons: o Performing the reaction at a lower temperature (150 °C relative to 165 °C) decreases the rate of the reaction, enabling greater precision over the products formed by performing the reaction within the time frame of minutes instead of seconds, increasing reproducibility. o The inventor note that the reaction may be performed at lower temperatures if the base employed is other than urea.

[0159] • Curing time:. Since favourable charge densities were noted for 30 and 60 minute reaction times, other reaction times were investigated for reactions using 2.5:10 and 1.2:4.9 ammonium phosphate dibasic:urea and ammonium phosphate monobasic:urea, respectively, at 150 °C, reaction times of 10, 30, and 60 minutes at 150 °C were selected for further investigation.

[0160] • Reaction pH: Reactions are typically performed at unadjusted pH values. For the design of experiments, the inventor employed acidic reaction mixtures (with pH adjusted) for monobasic and dibasic ammonium phosphate). The pH of heat soak solutions was set to 4 for maximising the charge content of the resulting phosphocellulose paper.

[0161] Dependent Output Variables:

[0162] • Strength of wet filter paper, as determined by tensile testing

[0163] • Charge density, as determined by conductometric titrations

[0164] • Whether or not the paper passes or fails a radiolabelled ATP assay

[0165] Charge density of phosphorylated filter paper

[0166] Conductometric titrations of the inventive phosphorylated filter papers and Whatman® P81 as controlled were performed following the test method described above.

[0167] Results of the conductometric titration curves of inventive papers 1 and 2 and of Whatman® P81 are presented in Table 1. The profile of the conductometric titration curve for Whatman® P81 paper reveals three inflection points and two plateaus, indicating that two -OH groups were titrated (Figure 8). The profile of the conductometric titration curves for both Inventive Papers 1 and 2 reveal two inflection points and one plateau, indicating that one -OH group was titrated (Figures 9 and 10).

[0168] Conductometric titrations indicate that the structure of Whatman® P81 filter paper clearly exhibits phosphate groups with two acidic protons and the inventive papers appear to exhibit phosphate groups with one acidic proton. Possible phosphorylated cellulose structures are presented in Scheme 3. Whatman® P81 paper exists predominantly as structure 1 whereas the Inventive Papers 1 and 2 contain a mixture of structures, especially structures... (Scheme 3).

[0169] Cellulose-

[0170] 1

[0171] Cellulose-

[0172] Cellulose-

[0173] 3

[0174] Cellulose- Cellulose

[0175] 4

[0176] Cellulose- Cellulose

[0177] Scheme 3: Potential phosphorylated cellulose structures, including phosphorylation of cellulose C2 and C6 carbons (1,2,3), and crosslinked possibilities (4,5). Strong acidic protons are denoted in red and weak acidic protons are denoted in blue.

[0178] Where two acidic protons are present, as in structure 1 (Scheme 3), one -OH group will act as a strong acid and the other will act as a weak acid. This is consistent with the pH values recorded for the conductometric titration of Whatman® P81 filter paper (Figure 8). Where one acidic proton is present, as in structures 4 and 5, all -OH groups act as a strong acid (Scheme 3). This is consistent with the pH values recorded for the conductometric titration of Inventive Papers 1 and 2 (Figures 9 and 10). Therefore, although the charge density of Inventive Papers 1 and 2 is lower than that of Whatman® P81 filter paper, their phosphorylated functionality predominates in its more reactive, strong acid form. An advantage of structures 4 and 5 over structure 1 is that the same performance of phosphocellulose filter paper can be achieved with less charge density, which helps to preserve the physical integrity of the filter paper for manual handling (Scheme 3).

[0179] Conductometric titrations determined that the charge densities of Inventive Papers 1 and 2 are significantly lower than that of Whatman® P81 paper (Table 1).

[0180] From Figure 37a it can be observed that the expected ratio of strong to weak acid in Whatman® P81 filter paper is approximately 50%. The calculated value from the conductometric titration is approximately 43%.

[0181] The calculated ratio of strong to weak acids in Inventive Paper 1 and Paper 2 is approximately 77% and 70%, respectively (Figures 37b and c respectively), indicating that a significant proportion of the phosphocellulose is in its strong acid form.

[0182] The higher strong acid content of Inventive Papers 1 and 2 relative to Whatman® P81 paper may explain their excellent performance as cation exchange filter papers in radiolabelled ATP assays despite having significantly lower charge densities relative to Whatman® P81 paper

[0183] Table 1 : Charge densities of phosphocellulose filter papers as determined by conductometric titrations.

[0184] Tensile Strength of phosphorylated filter paper

[0185] In order to evaluate the mechanical properties of phosphorylated filter papers, the tensile strength of unphosphorylated Whatman® Grade 1 Chr, Whatman® P81 and Inventive Papers 1 and 2 was measured following the tensile strength test method described above. The stress-strain curves obtained are presented in Figures 12 and 13, and the tensile strength (a), strain at break (E), and Young’s modulus (E) were calculated by taking the average across 5 specimens (Figures 14-16). These values are also provided in Table 2.

[0186] Table 2: Mechanical properties of Whatman® Grade 1 Chr and cation exchange filter papers.

[0187] Whatman® P81 paper is thicker and more absorbent than Whatman® grade 1 Chr and cation exchange papers derived thereof.

[0188] All filter papers tested are significantly weaker when wet compared to when dry. Inventive Papers 1 and 2 are not as strong as Whatman® P81 paper when dry. However, phosphorylating Whatman® grade 1 Chr filter paper increases its strength and renders it more ductile, although it decreases its elasticity, as indicated by its Young’s modulus. Inventive Paper 1 is stronger (212 kPa) and more ductile (2.75% strain) than Whatman® P81 paper (171 kPa; 1.80% strain) when wet whilst Inventive Paper 2 is weaker than both (131 kPa; 1.37% strain)). Inventive Papers 1 and 2 are not as elastic as Whatman® P81 paper and therefore are slightly more brittle. In summary, Inventive Paper 1 exhibits preferred mechanical properties to Whatman® P81.

[0189] Evaluation of phosphorylated papers in radiometric kinase assays

[0190] The cation binding properties of the functionalised filter paperwas first confirmed by incubating the paper in aqueous CuSC and washing it with boiling water to yield green, copper-bound paper (Figure 1).

[0191] Thereafter, the functionalised paper was tested in a radiolabelled32P ATP kinase assay against Whatman® P81 cation exchange filter paper in triplicate (Scheme 4). Comparable results were obtained (Figure 2). Samples obtained by the methods of Example 1 (using ammonium phosphate monobasic) and Example 2 (using ammonium phosphate dibasic) (namely Inventive paper 1 and Inventive paper 2, respectively) were tested using a range of different protein and peptide substrates for suitability in radiolabelled ATP kinase assays.

[0192] Scheme 4: Radiolabelled32P ATP kinase assay of EP975 using PKA.

[0193] In one assay, both paper samples were compared against Whatman® P81 paper in a33P ATP kinase assay using three very different substrates, both of which performed similarly to Whatman® P81 paper for all substrates tested in duplicate (Figure 3).

[0194] The paper samples were also compared against Whatman® P81 paper in the following33P ATP kinase assays, and comparable results were obtained in each assay:

[0195] 1. IC50 curve, 7 datapoints, single shot (Figure 4)

[0196] 2. Low [33P ATP], peptide substrate, performed in triplicate (Figure 5)

[0197] 3. Medium [33P ATP], protein substrate, performed in triplicate (Figure 6)

[0198] 4. High [33P ATP], peptide substrate, performed in triplicate (Figure 7)

[0199] These experiments show that the same trends in data are observed for the papers obtained with the novel method and for Whatman® P81 paper.

[0200] Discussion of results

[0201] Although Inventive Papers 1 and 2 both exhibited similar cation exchange performance in radiolabelled ATP kinase assays as Whatman® P81 cation exchange filter paper, differences in their charge densities and mechanical properties were determined by conductometric titrations and tensile strength testing, respectively, such that Inventive Paper 1 exhibited superior mechanical properties whereas Inventive Paper 2 maintained a higher charge density. This suggests that, in the phosphorylation of cellulose filter paper using urea as a catalyst, there exists a trade-off between charge density and tensile strength. Since sufficient charge density is required for performance in cation exchange assays whilst sufficient wet tensile strength is required to handle the wet filter papers when performing an assay, reaction conditions were investigated to optimise charge density and wet tensile strength.

[0202] Conclusions on Experiments 1 and 2 and characterisation of the inventive papers

[0203] • Cellulose starting material: o Cellulose can be sourced from wood, plant, algae, animal, or bacteria, which influences its microstructural organisation and properties, including degree of polymerisation and crystallinity. Furthermore, cellulose can exist in fiber, microfibril / nanofibril, and micro / nanocrystalline morphological forms following its source and processing, including extraction and pretreatment methods. Wood and plant cellulose is comprised of microfibrils that are assembled into cellulose fibers. Tyes of cellulose fibers include 1) strand fibers, composed of single cells (20-100 cm), staple fibers, composed of many cells (« 60 mm), and pulp fibers, treated to remove lignin (1-10 mm). Interestingly, cotton linters are fibers that remain adhered to the cotton seed and are only a few millimetres in length. o Choice of cellulose starting material influences how it is handled. For example, it can have impurities that need to be removed, be wet or dry, or be redispersible in water. Furthermore, cellulose starting material also influences the structure and properties of the final material. o Phosphorylation of cellulose has been performed on many types of cellulose, including wood, pulp sheets, pulp, cellulose sludge, microcrystalline cellulose, and blended Whatman® Grade 1 Chr filter paper, to name a few examples. o Where the intention is to generate a film or sheet of phosphocellulose instead of an aqueous dispersion, for example, the phosphocellulose product is typically mechanically processed, where needed, and formed into a film via solvent casting or a sheet using a sheet former. Although Noguchi et al. (2017) phosphorylated pulp sheets, post-phosphorylation, the sheets were suspended in water, forming a slurry, neutralised via the addition of NaOH, and mechanically disintegrated to form nanofibers. o The present inventive method of directly phosphorylating filter paper instead of bulk cotton linters eliminates steps involved in processing the phosphorylated product, such as homogenization, and paper making (sheet formers are specialist equipment that is not readily available in most labs, and once the sheet is formed, a press and dryer are also required to finish the process). Formation of a sheet via solvent casting would be cumbersome and could results in an imperfect surface. Furthermore, the inventive approach described herein does not require any development to obtain particular, uniform filtration characteristics. Therefore, the inventive method is significantly quicker, cheaper, and does not require specialist expertise or extensive, expensive equipment.

[0204] • Concentration of charge density at the surface: o Although Inventive Papers 1 and 2 exhibit significantly lower charge densities than Whatman® P81 cation exchange filter paper, their performance matches that of Whatman® P81 cation exchange filter paper in radiolabelled ATP kinase assays in part because their charge densities are likely to be concentrated at the surface following the inventive method of phosphorylation (i.e.: more accessible cellulose hydroxyl groups at the surface of the filter paper are preferentially phosphorylated over those embedded in the filter paper). o In addition, when employing urea as the base, it is preferable to have no water or a constant / stable amount of water at the curing step. The speed of evaporation of water molecules generated during the phosphorylation reaction may differ between the inner and outer surfaces of pulp sheets, and as such, can result in non-uniform degrees of phosphorylation throughout the thickness of the sheets, which may further trigger concentration of charge at the surface of the paper.

[0205] • Improved mechanical properties of Inventive Paper 1 over Whatman® P81 cation exchange filter paper

[0206] • Presence of more reactive monobasic protons

[0207] Further experimental design

[0208] Of the independent variables listed below, 1) phosphate source (i.e.: mono or dibasic ammonium phosphate), 2) molar equivalents of phosphate salt (i.e.: 0.3, 1.35, 3.), 3) molar equivalents of urea (i.e.: 1.2, 6.9, and 15 eq.), and 4) curing time (i.e.: 10, 30, and 60 minutes) were investigated via a full factorial design of experiments. The charge density and wet tensile strength dependent outputs were measured.

[0209] The inventor performed a full factorial design of experiments with 4 factors to determine the main effects of reaction variables, namely phosphate (ammonium phosphate monobasic or ammonium phosphate dibasic, phosphate molar equivalents, urea molar equivalents, and reaction time) on reaction outcomes (namely charge density and strength) (Table 3). Tensile strength results are reported as the average of 3 specimens. This design of experiments provided an overview of how various inputs affect outputs. Table 3: List of reactions performed.

[0210] The results obtained are detailed in Table 4.

[0211] Table 4: List reaction outcomes.

[0212] Effects on Charge Density

[0213] • The amount of urea and phosphate used to prepare phosphocellulose were determined to affect the charge density of the resulting phosphocellulose (Figure 26). Specifically, charge density increases as the amount of phosphate and urea increases (Figures 27 and 28).

[0214] Effects of Tensile Strength

[0215] • The amount of urea was determined to affect tensile strength (Figure 29a). Specifically, as the amount of urea is increased, the resulting tensile strength decreases (Figure 29 b).

[0216] Effects on Percent Strain at Break

[0217] • None of the inputs investigated were determined to have a statistically significant effect on strain at break.

[0218] Effects on Kinase Assay Performance

[0219] • Papers generated in reaction numbers 17, 22, 27, 35, 36, 47, 48, 51 , and 53 were determined to have excellent signal to noise ratios in MAPKAP-K2 kinase assays and therefore were qualified as inventive papers for kinase assays.

[0220] • The only input correlated to kinase assay performance is the amount of phosphate such that the kinase assay performance increases as the amount of phosphate is increased (Figures 30 a and b). • Despite some papers having low charge density (even much lower than Whatman® P81) strength when wet, they still performed well in radiolabelled ATP assays.

[0221] • Tensile strength of the papers when wet is an important parameter, especially for high- throughput kinase assays. Any phosphorylated papers with tensile strengths below 50 kPa could rip in high throughput assays and are suboptimal.

[0222] Effects on Wet Filter Paper Thickness

[0223] See Figures 31 and 32.

[0224] Charge Density and Tensile Strength

[0225] • In general, as charge density is increased, tensile strength decreases.

[0226] • As the paper gets thicker, it requires more washes to reduce background noise (wash out unreacted radio labelled ATP). In addition, thicker papers tend to show lower tensile strength (see Figure 35).

[0227] • Many of the papers in Figure 33 were expected to perform well in kinase assays, however, did not. The working hypothesis is that the chemical structure, and therefore function, of the resulting phosphocellulose depends on reaction inputs and requires further investigation.

[0228] • Papers with at least 50 kPa tensile strengths tended to be simpler to work with and more durable for performing the assays.

[0229] Other Notes

[0230] • Choice of phosphate salt between ammonium phosphate monobasic and ammonium phosphate dibasic did not have a statistically significant effect on any of the outcomes investigated.

[0231] • Previously, only white phosphocellulose papers were prepared. In this experiment, several off-white and pink papers were prepared. This is correlated to the amount of phosphate such that as the amount of phosphate is increased, the likelihood of generating a pink paper is increased.

[0232] • Figure 34 presents a summary of the correlation between the charge density and tensile strength of the different phosphorylated papers prepared in the experimental optimisation experiment. Papers 6, 7, 10 and 11 present insufficient tensile strength for use in routine kinase assays, while paper 3 is acceptable and papers 1 , 2, 4, 5, 8 and 9 present sufficient tensile strength for use in routine kinase assays. The best inventive papers had charge densities between 60 and 240 pmol / g. Wax Printed Inventive Phosphocellulose for 96 Well Plate Applications

[0233] • Discontinued Whatman® Unifilter P81 filter 96 well microplate devices (well diameter ~ 6 mm) are routinely used for high throughput radiolabelled ATP kinase assays.

[0234] • A low cost, simpler alternative to fitting Inventive Paper into a 96 well filter plate was sought.

[0235] • The wettability of various grades of cellulose filter paper was investigated to determine if it is possible to spot assay mixtures directly onto a single sheet of Inventive Paper without the mixtures bleeding into each other so that samples can be read in a 96 well format by a TopCount NXT microplate scintillation counter. A typical kinase assay mixture is 25 pL and therefore < 25 pL of water was spotted onto various grades of cellulose filter paper and the diameter of the spot measured (Table 5).

[0236] Table 5: Quantitation of Whatman® filter paper wettability.

[0237] • Analysis of 3 pL aliquots of33P ATP kinase assay mixtures directly onto a sheet of Whatman® P81 filter paper for reading in a 96 well format was determined to be more sensitive than using Whatman® Unifilter P81 filter 96 well microplate devices. Without wishing to be bound by theory, washing a sheet of filter paper on a rocker may be gentler than automated washes run on a Harvester96™ instrument when working in a 96 well format, the former of which increases the sensitivity and accuracy of radiolabelled ATP kinase assays for this type of kinases and therefore requires fewer assay repeats.

[0238] • Interestingly, aliquots spotted onto Inventive Papers 2 exhibited sharper spots, with more defined edges, than those on Whatman® P81 paper (Figure 17).

[0239] • In order to spot more than 3 pL samples onto a single sheet of Inventive Paper 2 in a 96 well format, hydrophobic barriers were introduced into Inventive Paper 2 in a 96 well format.

[0240] • To test whether wax can be patterned on phosphocellulose filter paper and determine if it interferes in kinase assays, ~ 2 cm diameter circles were drawn on Inventive Paper 2 squares using coloured wax ink (i.e. wax with pigment) and the wax melted into the paper by heating on a hot plate set to 150 °C for 2 minutes. A33P ATP kinase assay was performed as per standard protocol with the exception that reaction mixtures were spotted within the wax barrier on phosphocellulose cation exchange filter paper. The results obtained (not shown) match expected values and a solid wax printer was employed for further work.

[0241] • Wax printing or patterning hydrophobic wax barriers into hydrophilic paper, was performed using a Xerox ColorQube 8570N printer. Xerox ColorQube wax was printed in a 96 well design onto a single surface of A4 size Inventive Paper 2. The 96 well pattern was designed using black circular lines (1 pt line size, » 1 mm) on a white background using Microsoft PowerPoint software. The wax was melted into the paper using an oven set at 150 °C for 2 minutes to form a complete barrier. The line width after spreading of the molten wax in paper was ~ 2 mm (Figure 18). It was possible to print four 96 well patterns per A4 sheet.

[0242] • The wax barrier was tested by spotting various volumes of Orange G (24 mM aqueous solution) into wax printed Inventive Paper 2 (Figure 19).

[0243] • The performance of wax printed Inventive Paper 2 was compared with Whatman® Unifilter P81 filter 96 well microplate devices in radiolabelled IC50 assays (Figures 22 and 23). Similar results were obtained, indicating that printed Inventive Paper 2 can be used in place of discontinued Whatman® Unifilter P81 filter 96 well microplate devices for high throughput radiolabelled ATP kinase assays performed in a 96 well format.

[0244] • Similar wax printing results can be achieved using any solid ink printer, design software, and method of heating the ink to form a complete barrier, including heating on a hotplate. Furthermore, wax printing in any design can be easily achieved and is not limited to a 96 well format.

[0245] • To the inventor’s knowledge, this is the first example of wax printing on phosphocellulose.

[0246] Performance of high throughput radiolabelled ATP assays: Method comparison using Whatman® Unifilter P81 filter 96 well microplate devices and wax printed cation exchange filter paper

[0247] The assay method performed is identical to the high throughput radiolabelled ATP assay described above, modified only in the manner on which the sample is captured onto cation exchange filter paper and processed prior to reading in a scintillation counter.

[0248] Sample capture using Whatman® Unifilter P81 filter 96 well microplate devices

[0249] • The assay is performed in a 96 well plate (a plate with a smaller or larger number of wells may be printed). • The samples / reaction mixtures are transferred onto a Whatman® Unifilter P81 filter 96 well microplate device via vacuum and washed with water or orthophosphoric acid using a Harvester96™ instrument.

[0250] • The Whatman® Unifilter P81 filter 96 well microplate device is removed from the Harvester96™ instrument and transferred to a Thermo Scientific® Multidrop Dispenser, where liquid scintillation fluid cocktail (MicroScint-O™) is added to the wells.

[0251] • The wells are sealed by placing specialist films on the top and back of the plate.

[0252] • A barcode is added to the plate, which can be read by the scintillation counter. This matches the data collected to the plate when performing large assays with multiple plates.

[0253] • The plate is placed into the TopCount NXT microplate scintillation counter for analysis. The counts per well are read and the data analysed.

[0254] Capture using wax printed cation exchange filter paper

[0255] • The assay is performed in a 96 well plate.

[0256] • The samples are transferred onto a wax printed cation exchange filter paper using a multichannel pipette (Figure 20).

[0257] • The paper is then placed in a plastic container and washed 3 x 5-10 minutes in water or orthophosphoric acid on a rocker.

[0258] • The paper is then dried.

[0259] • Where33P ATP is used and scintillation fluid is required for scintillation counting, the paper is then placed in a plastic sample bag and liquid scintillation fluid cocktail (MicroScint-O™) is added to the bag. The bag is rolled to remove air bubbles, sealed using Astrapac heat sealing machinery, and trimmed to the edges of the paper.

[0260] • The sample bag is then placed into a plastic frame to be read by the scintillation counter (Figure 21). The TopCount NXT microplate scintillation counter is designed to read plates of this specific dimension. Note that scintillation fluid cocktail, which is essential to count 33P and not32P, contains organic solvents and consequently dissolves the wax used in wax printing - this does not disrupt the sample, which is bound to the paper, or interfere with scintillation counting.

[0261] • A barcode is added to the plate, which can be read by the scintillation counter. This matches the readings to the plate when performing assays using multiple plates. Custom barcodes can be used.

[0262] • The plate is the placed into the TopCount NXT microplate scintillation counter for analysis. The counts per well are read and the data analysed.

[0263] Advantages of the wax printed approach: • Significantly Cheaper

[0264] • Greener: The Whatman® Unifilter P81 filter 96 well microplate device is a disposable product. Each plate weighs « 37.65 g. Therefore, the inventor’s current consumption generates 3.77-11.30 Kg of radioactive waste per month. Where wax printed cation exchange filter papers are used, only the paper is disposed of (the plastic shell to read the paper is re-used). Producing less radioactive waste will also reduce the cost of disposing of this waste.

[0265] • Increased sensitivity and precision: Where capture is performed using the wax printed cation exchange filter papers, more consistent and reliable results are obtained (fewer wells with no reading or statical outliers are obtained), which results in fewer repeat assays being required, ultimately saving time and money. It is hypothesized that washes using the Harvester96™ instrument are more aggressive than washes on a rocker, which may contribute to the discrepancy in sensitivity / precision. Although it takes longer to perform radiolabelled ATP assays using the wax printed cation exchange filter papers, because fewer assay repeats are required using this approach, in practice it is ultimately quicker, cheaper, and more efficient than the approach using the Whatman® Unifilter P81 filter 96 well microplate devices.

[0266] • Requires less storage space

[0267] Other notes:

[0268] • The Whatman® Unifilter P81 filter 96 well microplate device contains individual discs of filter paper sandwiched in place - this is what stops samples from bleeding into neighbouring wells as excess sample washes off through the holes in the backplate before being absorbed onto a neighbouring disc.

[0269] • Prior to the application of wax printing to cation exchange filter paper, experiments were performed in a 96 well format by pipetting onto Whatman® P81 paper with the top and bottom rows marked in pencil. The addition of wax printing not only prevents bleeding of neighbouring wells into each other, it increases pipetting precision and helps align the wells into the plastic shell prior to reading on a TopCount NXT microplate scintillation counter.

[0270] • A solid ink printer was selected as this technology is routinely used to prepare paperbased analytical devices (PADs). The wax printing was performed with a Xerox ColorQube printer and ColorQube wax-based inks.

[0271] The inventor has herein proven that methods according to the present disclosure yield phosphocellulose cation exchange filter paper which can be used in radiometric protein kinase assays with comparable results to Whatman® P81 paper. Therefore, the methods described herein address the urgent need to provide stocks of phosphocellulose cation exchange filter paper that can replace the discontinued Whatman® P81 product in order to enable the diagnostic and research communities to continue using the gold-standard radiolabelled ATP kinase assays employed to date.

Claims

Claims1- A method of phosphorylating cellulose filter paper in an aqueous solution comprising phosphate and urea for preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays, the method comprising the steps of: a. determining the number of anyhydroglucose units in the cellulose (AGU) of the filter paper by weighing the filter paper and calculating the AGU using the formula:AGU in cellulose (mol) = [weight of cellulose (g)] ’ 6.2 ■ 10-3(mol ■ g~r) b. subjecting the filter paper to a heat soak pretreatment step by submerging the filter paper in an aqueous solution comprising a phosphate source and a base at an elevated temperature; c. drying the filter paper; d. curing the filter paper; e. washing the filter paper; and f. drying the filter paper.2- The method of claim 1 wherein the base is selected from an amine, a hydroxide salt, and a carbonate salt, optionally wherein the base is an amine, further optionally wherein the base is urea.3- The method of claim 1 or 2, wherein the phosphate source is a phosphate salt, optionally wherein the phosphate salt is selected from ammonium phosphate monobasic (NH4H2PO4) or ammonium phosphate dibasic ((NF^ HPC ).4- The method of any preceding claim, wherein the molar ratio of AGU in the cellulose filter paper to phosphate source (AGU : phosphate source molar ratio) is selected from about 1 :0.3 to about 1:5, optionally wherein the molar ratio of AGU in the cellulose filter paper to phosphate source (AGU : phosphate source molar ratio) is selected from about 1:0.6 to about 1:2.5.5- The method of any preceding claim wherein the molar ratio of AGU in the cellulose filter paper to urea (AGU : urea molar ratio) is selected from about 1 :2.5 to about1 :12., optionally wherein the molar ratio of AGU in the cellulose filter paper to base (AGU : base molar ratio) is selected from about 1:3.2 to about 1 :10.6- The method of claim 1 or2, wherein the phosphate source is ammonium phosphate monobasic and the ratio of AGU : ammonium phosphate monobasic: urea is 1 :0.6:3.2, optionally wherein the pH in those is at least 2.5, or at least 3, for example 4.7- The method of claims 1 to 3, wherein the phosphate source is ammonium phosphate dibasic and the ratio of AGU : ammonium phosphate dibasic: urea is 1 :2.5:10, optionally wherein the pH in those embodiments may be at least 7, or at least 8, for example 8.6.8- The method of any one of claims 1 to 5 wherein one of: the phosphate source is ammonium phosphate monobasic and the ratio of AGU : ammonium phosphate monobasic: urea is 1:0.6:3.2, optionally wherein at least one of: the pH is at least 3 and / or wherein the curing time is about 20 minutes; or the phosphate source is ammonium phosphate dibasic and the ratio of AGU : ammonium phosphate dibasic: urea is 1 :2.5:10, optionally wherein at least one of: the pH is at least 7 or wherein pH is below7; and / or the curing time is about 30 minutes.9- The method of any preceding claim, wherein at least one of: the step of subjecting the filter paper to a heat soak pretreatment step is performed from about 10 minutes to about 90 minutes; the step of subjecting the filter paper to a heat soak pretreatment step is performed at a temperature from about 80 °C to about 100 °C.10- The method of any preceding claim, wherein the drying steps c and / or f are performed by at least one of: evaporation of solvent at room temperature, evaporation of solvent at a raised temperature (e.g. in an oven at about 100 °C), under vacuum, washing with organic solvent and evaporating the solvent at room temperature.11- The method of any preceding claim, wherein at least one of: curing the paper is performed from about 130 °C to about 200 °C, optionally from about 150 °C to about 170 °C; and / orthe curing step is performed for from about 5 minutes to about 90 minutes, optionally for about 5-40 minutes.12- A phosphocellulose cation exchange filter paper suitable for use in radiolabelled ATP assays obtained directly by the method of any preceding claim.13- A phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays wherein the phosphocellulose cation exchange filter paper has a charge density of from about 0.5 pmol crrr2to about 1.5 pmol cm-2.14- The phosphocellulose cation exchange filter paper of claim 13, wherein the phosphocellulose cation exchange filter paper has a charge density of from about 90 pmol g-1to about 500 pmol g-1.15- The phosphocellulose cation exchange filter paper of claim 13 or 14, wherein the phosphocellulose cation exchange filter paper has a tensile strength when wet of from about from about 100 kPa to about 300 kPa.16- The phosphocellulose cation exchange filter paper of any one of claims 13 to 16, wherein the phosphocellulose cation exchange filter paper has a strain when wet of from about 1% to about 4%.17- A method of manufacturing unsupported phosphocellulose cation exchange paper for radiolabelled ATP assays, the method comprising providing a phosphocellulose cation exchange paper and depositing a boundary of hydrophobic material on the phosphocellulose cation exchange paper to receive sample for performing a radiolabelled ATP assay, wherein the boundary is configured to contain a sample and prevent the sample from spreading or otherwise spilling outside the area enclosed by the boundary.18- The method of claim 17, wherein the hydrophobic material is a wax-based hydrophobic material, and wherein the boundary is a 96 well pattern of wax printed on a phosphocellulose cation exchange paper according to any one of claims 12 to 16.19- A method of performing a radiolabelled ATP assay, the method comprising:Providing an unsupported phosphocellulose cation exchange paper according to claims 17 or 18; depositing a sample for analysis in the radiolabelled ATP assay inside the boundary or boundaries of the hydrophobic material; washing the unsupported phosphocellulose cation exchange paper with the sample, optionally wherein washing is performed with water or with orthophosphoric acid; optionally placing the phosphocellulose cation exchange paper in a support or frame; optionally drying the phosphocellulose cation exchange paper; optionally soaking phosphocellulose cation exchange paper in liquid scintillation fluid cocktail; optionally sealing the phosphocellulose cation exchange paper with liquid scintillation fluid cocktail, optionally wherein sealing is performed in a flexible transparent container; and performing the scintillation counting according to any suitable protocol.

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