Method for the production of poly(methyl methacrylate) (PMMA) membranes and uses thereof

Abstract

A poly(methyl methacrylate) (PMMA) membrane having a highly porous, reticulated, 3-D structure suitable for lateral flow diagnostic applications is described. Also described is a method for producing a poly(methyl methacrylate) (PMMA) membrane that comprises the steps of mixing a suitable amount of PMMA, a solvent and a optionally one of either a co-solvent or a non-solvent to produce a solution, casting a thin film of the solution onto a support, and removal of the solvent from the solution to produce the PMMA membrane. A lateral flow diagnostic device comprising a highly porous PMMA membrane as a reaction membrane is also described.

Claims

1. A method for producing a homogenous and symmetric poly(methyl methacrylate) (PMMA) membrane on a solid support material, the method comprising the steps of: (i) mixing a suitable amount of PMMA, a solvent, and a C.sub.2-C.sub.6 co-solvent to produce a PMMA solution with increased sensitivity to temperature fluctuations, wherein the C.sub.2-C.sub.6 co-solvent functions as a solvent above a certain temperature and as a non-solvent below said certain temperature; (ii) casting a thin film of the PMMA solution onto a solid support material; and (iii) affecting the temperature of the cast PMMA solution on the solid support material so that the cast PMMA solution becomes unstable and separates out into two phases, wherein the C.sub.2-C.sub.6 co-solvent transitions from a co-solvent in the solution to a non-solvent, thereby producing the homogenous and symmetric PMMA membrane on the solid support material.

2. The method according to claim 1, in which the PMMA solution is heated to a temperature above an upper critical solution temperature of the solution in step (i), the PMMA membrane is then TIPS-cast in step (ii), wherein the cast solution is cooled by casting onto a surface below its upper critical solution temperature followed by removal of the solvent/co-solvent.

3. The method according to claim 1, in which the PMMA membrane is VIPS-cast in step (ii), wherein solvent and co solvents is removed from the PMMA solution in a sequential manner by evaporation that is controlled by air flow over the PMMA solution.

4. The method according to claim 1, in which the PMMA membrane is LIPS-cast in step (ii) and the thin film of the PMMA solution is immersed into a coagulation bath containing a non-solvent and optionally a solvent, whereby exchange of solvent and non-solvent results in the formation of a symmetric porous PMMA membrane on a solid support material and avoids skin layer formation; wherein the non-solvent is selected from the group consisting of ethyl ether, water, glycerol, ethylene glycol, methanol and ethanol, or a combination thereof.

5. The method according to claim 1, in which the PMMA membrane is hybrid-cast in step (ii), the solvent and co-solvent are removed from the PMMA solution by evaporation that is controlled by air flow over the PMMA membrane, and the PMMA membrane is then immersed into a coagulation bath containing a non-solvent whereby the final PMMA membrane structure is fixed.

6. The method according to claim 1, in which the PMMA membrane is temperature- and evaporation-cast, wherein the PMMA solution is heated to a temperature above an upper critical solution temperature of the solution in step (i); the PMMA membrane is then TVIPS-cast in step (ii), wherein the PMMA solution is cooled by casting onto a surface below its upper critical solution temperature, and wherein solvent and co-solvent are removed from the cast solution in a sequential manner by evaporation that is controlled by air flow over the film.

7. The method according to claim 1, in which the support is cooled such that a temperature difference of 10 C. exists between the support temperature and the upper critical solution temperature of the PMMA solution.

8. The method according to claim 1, in which the solution comprises 2-14 wt % PMMA and 55-96 wt % solvent/co-solvent.

9. The method according to claim 1, wherein the solvent is selected from the group consisting of dichloroethane, acetic acid, acetone, iso-propanol, n-propanol, n-butanol, chloroform, toluene, 1,4 dioxane, tetrahydrofuran, ethyl acetate, methyl ethyl ketone or a combination thereof.

10. The method according to claim 1, wherein the C.sub.2-C.sub.6 co-solvent is an alcohol selected from the group consisting of n-propanol, iso-propanol, 1-butanol, 2-butanol, tert-butanol, 2-methylbutan-2-ol, 3-methylbutan-2-ol, 2,2-dimethylpropan-1-ol, pentan-3-ol, pentan-2-ol, pentan-1-ol, 1-hexanol, 2-hexanol, 3-hexanol, 2-methyl-2-pentanol, 2,3-dimethyl-2-butanol, 3-methyl-3-pentanol and cyclo-hexanol.

11. The method according to claim 10, in which the PMMA membrane on the solid support material is rendered hydrophilic by treatment with any one of the following: hydrolysis, aminolysis, silanisation, aqueous solutions of surfactants, ultraviolet radiation, plasma treatment, electron beam radiation and ozonation, or combinations thereof while maintaining the ability to bind proteins via hydrophobic interactions.

12. A porous homogeneous and symmetric poly(methyl methacrylate) (PMMA) membrane on a solid support material produced by a method comprising the steps of: (i) mixing a suitable amount of PMMA, a solvent, and a C.sub.2-C.sub.6 co-solvent to produce a PMMA solution with increased sensitivity to temperature fluctuations, wherein the C.sub.2-C.sub.6 co-solvent functions as a solvent above a certain temperature and as a non-solvent below said certain temperature; (ii) casting a thin film of the PMMA solution onto a solid support material; and (iii) affecting the temperature of the cast PMMA solution on the solid support material so that the cast PMMA solution becomes unstable and separates out into two phases, and wherein the C2-C6 co-solvent transitions from a co-solvent in the solution to a non-solvent, thereby producing the homogenous and symmetric PMMA membrane on the solid support material.

13. The porous symmetric poly(methyl methacrylate) (PMMA) membrane on a solid support material according to claim 12 having a symmetric reticulated 3-D matrix structure and a porosity of at least 85%, as determined by weight volume calculations.

14. The porous symmetric poly(methyl methacrylate) (PMMA) membrane on a solid support material according to claim 12, wherein the membrane has an average pore size of 0.5-30 m.

15. A lateral flow diagnostic device comprising a reaction membrane, characterized in that the reaction membrane comprises a symmetric porous poly(methyl methacrylate) (PMMA) membrane and solid support composite according to claim 12.

16. A device for performing an immunoassay that comprises a homogenous and symmetric poly(methyl methacrylate) membrane on a solid support produced by a method comprising the steps of: (i) mixing a suitable amount of PMMA, a solvent, and a C.sub.2-C.sub.6 co-solvent to produce a PMMA solution with increased sensitivity to temperature fluctuations, wherein the C.sub.2-C.sub.6 co-solvent functions as a solvent above a certain temperature and as a non-solvent below said certain temperature; (ii) casting a thin film of the PMMA solution onto a solid support material; and (iii) affecting the temperature of the cast PMMA solution on the solid support material so that the cast PMMA solution becomes unstable and separates out into two phases, and wherein the C2-C6 co-solvent transitions from a co-solvent in the solution to a non-solvent, thereby producing the homogenous and symmetric PMMA membrane on the solid support material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:

(2) FIG. 1A is a scanning electron microscope (SEM) image of a typical PMMA membrane made by the methods described herein, illustrating the formation of highly porous networks through the membrane (Scale bar 50 m), with FIG. 1B showing the air-side of the membrane, and FIG. 1C showing the composite-side of the membrane.

(3) FIGS. 2A and 2B are top down SEM images of a typical PMMA membrane made by the methods described herein, illustrating the formation of highly porous networks through the membrane.

(4) FIG. 3 is a cross-sectional SEM showing the formation of a bicontinuous PMMA membrane with a skin layer at the air interface and the underlying porous structure.

(5) FIG. 4 is photograph of a typical PMMA membrane produced by the methods described herein, illustrating bead mobility. Bead mobility was assessed using 40 nm gold particles. The gold nanoparticles displayed mobility through the membrane.

(6) FIG. 5 is a photograph of a typical PMMA membrane produced by the methods described herein, illustrating the functionality of the membrane in a bioassay. Two common assays were tested, namely, the Human Chorionic Gonadotropin (hCG) and Hepatitis B Surface Antigen (HBsAg) assays. This photograph illustrates an hCG assay of various analyte concentration for both commercial nitrocellulose membrane and PMMA membrane described herein.

(7) FIG. 6 is a photograph of a typical PMMA membrane produced by the methods described herein, illustrating the functionality of the membrane in a bioassay. Two common assays were evaluated, namely, the Human Chorionic Gonadotropin (hCG) and Hepatitis B Surface Antigen (HBsAg) assays. This photograph illustrates an HBsAg assay of various analyte concentrations for both commercial nitrocellulose membrane and PMMA membrane.

DETAILED DESCRIPTION OF THE INVENTION

(8) The most optimum membranes were formed from PMMA grades with a high molecular weight due to the increased viscosity of solutions with low polymer concentration. These low polymer concentration/high viscosity solutions resulted in more open structures formed upon phase inversion.

(9) Solution productionSolutions for all experiments were produced as follows. The components were weighed into a glass jar and a magnetic stirring bar was placed into the jar. The jar was sealed and the solution was stirred on a magnetic stirring plate at speeds ranging from 50-400 rpm for three days at room temperature. Before casting, solutions were left to stand for 24 hours to degas.

(10) VIPS-castSolutions were cast onto a polyester support using a k-control paint coater at a casting thickness of 250 m. The cast solutions were left to form a membrane in ambient atmosphere. Once dried, the membranes were stored in a sealed dry environment.

(11) Hybrid-castMembranes were VIPS-cast as described above. After a certain period of time of between 1 second and 15 minutes, the forming membrane was transferred into a coagulation bath consisting of either water, ethanol, methanol, acetone, toluene, 1,4,dioxane, tetrahydrofuran or a mixture thereof to complete the formation process. The coagulation bath was kept at a temperature of between 0 C. and 35 C. The formed membrane was then dried in ambient air and stored as before.

(12) TIPS-castSolutions were formed into desired thin film shapes as described above. The films were then placed in contact with a cooling substrate with a temperature difference of between 0 C. and 10 C. below that of the upper critical solution temperature of the solution. The liquid components of the solutions were then removed through evaporation.

(13) In some embodiments, PMMA membrane described herein is produced according to a 3 step process. Step one entails the production of a specialised solution (a mixture of polymer, solvent and non-solvent). Step 2 involves casting a thin film of this specialised solution onto a polyester support and passing this through a controlled atmosphere to induce formation and evaporate the solvent creating the polymeric membrane. Finally, step 3 involves the surface modification of the formed membrane towards lateral flow functionality.

(14) The optimum range for the composition was found to have a PMMA concentration of 4-14 wt % and between 31-75 wt % solvent (typically acetone) (or 55-96 wt % solvent/co-solvent when used in combination); 20-65 wt % non-solvent/co-solvent combination (i.e. ethanol and 1-butanol) or 0-30 wt % non-solvent (i.e. water). In these systems, the acetone acts as a solvent for the PMMA while the aliphatic alcohols act initially as a co-solvent and then as a non-solvent. The membrane forms through a phase inversion process induced by a sudden drop in temperature via contact with a cooling substrate that is at a temperature below the UCST of the solution. Once phase inversion has occurred, the remaining solvent, co-solvents and non-solvents are removed by evaporation. This formation process results in the most open membranes being formed when the temperature difference between the cooling substrate and the UCST is large.

(15) The addition of co-solvent(s) alone to the composition (from 20-65 wt %), while optional, result in a better control of the formation process. The co-solvent(s) in question are generally alcohols, typically C2-C6 alcohols, ideally selected from the group consisting of ethanol, n-propanol, iso-propanol, 1-butanol, 2-butanol, tert-butanol. 2-Methylbutan-2-ol, 3-Methylbutan-2-ol, 2,2-Dimethylpropan-1-ol, Pentan-3-ol, Pentan-2-ol, Pentan-1-ol, 1-Hexanol, 2-Hexanol, 3-Hexanol, 2-Methyl-2-pentanol, 2,3-Dimethyl-2-butanol, 3-Methyl-3-pentanol and cyclo-hexanol. Optimum results in this case, however, were found for C3 to C4 alcohols namelyn-propanol, 2-propanol, 1-butanol, 2-butanol and tert-butanol. The addition of these co-solvents boosts the solubility of PMMA in the resulting solutions and increases the solutions sensitivity to temperature fluctuations.

(16) These solutions are cast onto a polyester support and passed under a casting knife set to a preferred height 250 m, however, a height range of 50-400 m was found to be effective. Upon passing under the casting knife the formed shape is cooled via contact with a cooling substrate. The solvent and non-solvents are then removed via evaporation or extraction in a non-solvent coagulation bath.

(17) Highly porous reticulated networks are formed in a typical PMMA membrane produced by the method described herein, as illustrated in FIGS. 1 and 2. The % porosity of the membrane is greater than 60% as determined by weight and volume calculations, described previously.

(18) Membrane surface modification to optimise functionality toward lateral flow diagnostics was carried out by a number of different methods, including treatment with aqueous solutions of surfactants, whereby the membrane is immersed in a surfactant solution for 1 minute, removed and dried in air.

(19) In another approach, the membrane is exposed to ultraviolet radiation at wavelengths within the range of 100-400 nm for periods ranging from 30 minutes to 3 hours.

(20) In yet another approach, the membrane is exposed to an oxygenated environment which is subsequently converted to ozone upon UV irradiation. Over a period of 30 minutes to 3 hours, the ozone produced oxidises the membrane surface rendering it more functional.

(21) In still another approach, the membrane is treated with an acidic or a basic aqueous solution or a combination thereof to hydrolyse the surface. The membrane is treated in the acidic or basic solutions, at concentrations within the range of 0.1 M to 10 M, for periods of 1 hour to 24 hours with continuous agitation rendering the surface more hydrophilic.

(22) Surface modification of the membrane was carried out on dried membrane by immersion in a surfactant bath followed by drying in air. The resulting membrane exhibited good functionality in promoting lateral flow of aqueous solutions through the membrane without deleteriously affecting the protein binding of the membrane.

(23) The membrane produced by the method of the present invention can be used in lateral flow diagnostic assays. An example of such assays are medical diagnostics (HIV, hepatitis B, hepatitis C, flue etc), women's health (pregnancy & ovulation), blood banking (blood typing), animal health (heartworm, FIV, rabies, tuberculosis) and food safety (Salmonella, E. coli, Listeria etc.).

(24) Bicontinuous Structural Formation

(25) An example of a bicontinuous PMMA membrane produced by this method is given in FIG. 3. It was based, in part, on the discovery that a bicontinuous structure consisting of a dense skin layer at the air interface with an underlying porous structure could easily be produced by a simple alteration of the period of time between when the solution is formed into the desired shape and when it undergoes spinodal decomposition. Skin layers can be produced with a thickness in the range of 1 m to 30 m, through the thermodynamically induced precipitation of polymer at the air interface during evaporation of volatile solution components. The method follows the same process as described above with the inclusion of this slight delay in spinodal decomposition. The delay required was found to be greater than 10 seconds. The best method for achieving this delay was found to be altering the temperature difference between the solution UCST and the cooling platform temperature, so that the solution's UCST is in the order of 2 to 15 C. below the temperature of cooling platform. This allows for appropriate evaporation of solution components prior to spinodal decomposition. The structures formed, as illustrated in FIG. 3, are free of macrovoid defects that are commonly observed in PMMA bicontinuous membranes produced prior art methods.

Example 1

(26) Fabrication of Porous PMMA Membrane

(27) All solvents and non-solvents used were purchased from Sigma-Aldrich Ireland Ltd, Wicklow, Ireland. PMMA was purchased from Arkema Italy, Milan, Italy.

(28) A typical PMMA membrane, as described herein, was formed as follows: A solution was formed of the following components: 5 wt % PMMA; 40 wt % acetone; 27.5 wt % ethanol; and 27.5 wt % 1-butanol. This solution is temperature sensitive whereby below a certain temperature, i.e., the upper critical solution temperature (UCST), the solution becomes unstable and separates out into two phases. Above the UCST, the solution is homogeneous and stable. In the process described herein, the UCST is used to induce the structural formation of the porous membrane. It is believed that the UCST is the point at which the aliphatic alcohol components, especially the 1-butanol, transition from being a co-solvent in the system to becoming a non-solvent (when transitioning through the UCST from higher to lower temperatures). In this example, the solution is formed into a desired shape and then cooled through contact with a substrate that is 0-10 C. below the UCST of the system. The remaining solvent and non-solvent is then removed via evaporation in a low humidity environment <30% RH at 25 C. This formation process results in the largest pore size membrane when using a large UCSTcooling substrate temperature difference, i.e. 10 C. Large temperature differences can, however, cause delamination from the support material and visual defects in the membrane. Using low temperature differences 0-3 C. result in more homogeneous structures with lower pore sizes.

(29) Highly porous reticulated networks are formed in a typical PMMA membrane produced by the method described herein, as illustrated in FIGS. 1 and 2. The % porosity of the membrane is greater than 60% as determined by weight and volume calculations, described previously.

(30) Membrane surface modification to optimise functionality toward lateral flow diagnostics was carried out by a number of different means including, treatment with aqueous solutions of surfactants, whereby the membrane is immersed in a surfactant solution for 1 minute, subsequently removed and dried in air.

(31) Surface modification of the membrane was carried out on dried membrane by immersion in a surfactant bath followed by drying in air. The resulting membrane exhibited good functionality in promoting lateral flow of aqueous solutions through the membrane without deleteriously affecting the protein binding of the membrane.

(32) There is provided, as described herein, a fast method for producing highly porous (>85%) structures with open air interfaces, as illustrated in FIG. 1. All previous reported methods for the production of PMMA membranes result in much lower porosity with macrovoid formation and/or a bicontinuous structure.

(33) The PMMA structures described herein have shown excellent application results in lateral flow diagnostics (see FIGS. 5 and 6). The production of structures for use in lateral flow diagnostics is problematic as it requires spatially homogeneous structures with large pore sizes in the range of between 0.5 to 30 micrometers and a porosity of greater than 85%. Large pore sizes are required to enhance capillary flow, reducing the time for the test to complete, and to allow for the passage of large detector particles through the structure. High porosity is required to enhance the sensitivity of the assay.

(34) The methods disclosed herein for the production of porous PMMA structures address some of problems in the industry, as outlined below; The fast formation process decreases the reliance of the process on the polymer raw material properties. This will allow for greater production speeds and a reduction in lost product from quality control. The polymer, PMMA, is a non-hazardous polymer which simplifies handling, shipping and storage considerations. PMMA does not show the same propensity towards decomposition as nitrocellulose, thus allowing for longer product shelf life.

Example 2

(35) Use of Porous PMMA Membrane as Reaction Membrane in Lateral Flow Diagnostic Assay

(36) Membrane produced by VIPS-casting was assessed for application performance. Application performance was assessed under two categoriesbead mobility and assay functionality. Bead mobility was assessed using 40 nm gold nano particles diluted in a solution of Phosphate Buffer Solution, Tween 20 and Bovine Serum Albumin.

(37) Membrane samples 4.5 mm25 mm were placed in 25 L of bead solution and visually assessed for bead mobility. The gold nanoparticles showed good mobility through the membrane with no bead/liquid front separation (FIG. 4).

(38) Functionality was assessed using a hepatitis B and pregnancy lateral flow test using membranes produced by TIPS-casting. PMMA membrane samples were spotted with a capture (against a target analyte) and control antibody and then fully dried to fix the antibodies in place. Membrane samples were then assembled with a conjugate pad (gold or latex detector particle conjugated with a detection antibody), sample pad (treated with buffer, surfactant and blocking solution) and absorbent pad. Membrane cards were cut into 5 mm wide test strips and run using 150 L of positive (two signals) and negative (one signal) analyte (antigen in urine/serum/blood) solution. As the positive analyte solution passed through the conjugate pad, it re-mobilised the dried conjugate, and the antigen interacted with the conjugate (detector antibody/gold particle), both migrated through the porous network until they reached the capture and control zone. At the capture zone the antigen and conjugate were captured by the fixed antibodies and a red signal caused by the gold or latex detector particle was observed. With a negative solution the detector particle was not captured by the fixed antibody as there was no antigen present and the sandwich complex did not form (FIG. 5 and FIG. 6).

(39) As used herein, the terms comprise, comprises, comprised and comprising or any variation thereof and the terms include, includes, included and including or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.

(40) The invention is not limited to the embodiments described herein, but may be varied in both construction and detail.