Patent Application
20100068820
The present invention is directed to molecularly imprinted polymers, and the use thereof in diagnostic devices for the analysis of target molecules.
Molecular recognition is critical for the functioning of biological systems. Biological systems depend on molecular recognition to perform specific functions. Molecular recognition systems such as enzyme-substrate interactions, antibody-antigen interactions, DNA replication and cellular replication are examples of biological systems dependent on specific molecular interactions. Biomolecules such as enzymes and antibodies are used in in-vitro diagnostic devices to detect and quantify a specific target molecule which is indicative of a specific disease or biological function. For example, the enzyme glucose oxidase is used in diagnostic test strips to quantify the concentration of glucose in biological fluid such as blood, serum and interstitial fluid. Glucose oxidase reacts specifically with glucose. Diabetics routinely use glucose test strips to monitor the glucose level in their blood.
Molecularly imprinted polymers (MIPs) are synthetic compounds created with receptor sites that exhibit selectivity to a target compound. The synthesis of a polymer with the functionality to recognize a specific target molecule enables the polymer to capture and to concentrate the target molecule. Alternatively, a molecularly imprinted polymer containing target molecules can be made to controllably release these molecules from the polymer network.
Applications for molecularly imprinted polymers include: chromatographic adsorbents, membranes, sensors and drug delivery systems. (references: Molecular Imprinting at the edge of the Third Millennium, Sergey A. Piletsky, et al., Trends in Biotechnology, vol 19(1), pages 9-12, January 2001. and “Polymers and Gels as Molecular Recognition Agents”, Nicholas A. Peppas and Yanbin Huang, Pharmaceutical Research, vol. 19(5), pages 578-587, May 2002; “Molecular Imprinting Science and Technology: A Survey of the Literature for the Years up to and including 2003”, Alexander et al, Journal of Molecular Recognition, 2006, Vol. 19, pp 106-180.
The synthesis of molecularly imprinted polymers involves the polymerization and cross-linking of functional monomers in the presence of a template molecule to capture the imprint of the template. The template molecules may be extracted from the polymer to create 3-dimensional sites within the polymer matrix with functional groups that are complementary to those of the template molecule. (Reference, “Molecular Imprinting: State of the Art and Perspectives”, Jean Daniel Marty and Monique Mauzac, Advances in Polymer Science, vol. 172 pages 1-35, 2005.)
Highly cross-linked polymer networks are rigid structures that can exhibit high specificity to the target molecule. This high specificity make these rigid polymers ideal for analytical methods and separation techniques that require an exact complementary match of molecular functional groups and the position and orientation of the groups on a target molecule. Consequently, molecularly imprinted polymers may be used as chromatographic column packing used to separate enantiomers from racemic mixtures.
Rigid MIPs have the advantage of being highly specific to a single target molecule. They have the disadvantage that it is difficult to extract the template molecule from the highly cross-linked polymer network due to strong complementary interactions between the polymer and the target molecules. In addition, the complementary functionality and orientation requirements reduce the rate of reaction or the time for the template molecule to be adsorbed at the imprinted site. Reducing the cross-link density of a MIP reduces the specificity of the capture sites. However, the rate of template capture is increased.
Many patents and articles describe the use of specific polymer-biomolecule molecule interactions in biosensors. Per Bjork et al in Biosensors & Bioelecelectronics 20 (2005) pages 1764-1771 describes a biosensor based on the electrostatic and hydrogen bonding interaction between polythiophene and oligonucleotides. US patent publication No. 2004/0053425 describes an on-chip assay based on molecular recognition between a peptide or protein and a monoclonal antibody. These applications are based on molecular recognition rather than a molecularly imprinted polymer.
U.S. Pat. No. 6,638,498 by Green et al describes MIPs to bind and remove toxins from the gastrointestinal tract.
Chin-Shiou Huang in U.S. Pat. No. 6,680,210 teaches techniques for making polymers that imprint for macromolecules. The MIPs may be used for detecting and quantifying the amount of each macromolecule in a complex biological source.
U.S. Pat. No. 6,762,025, assigned to Molecular Machines, Inc. teaches the use of oligonucleotides for molecular recognition.
U.S. Pat. No. 6,807,842 describes a molecular recognition sensor for detecting an analyte using a semiconductive polymer film. The polymer film is imprinted with the analyte and the resistance of the film changes when exposed to the analyte and interferents.
U.S. Pat. No. 6,582,971 by Singh et al describes a method for molecular imprinting polymers with large biomolecules such as proteins. Using biphasic polymerization, a target biomolecule is molecularly imprinted in the polymer at the interface between an aqueous and an organic phase.
U.S. Pat. No. 6,461,873 by Catonia et al describes a caffeine detector that uses at least two molecularly imprinted polymers in first and second zones on a paper strip. The MIP in the first zone removes substances that may interfere with the analysis of caffeine. The second zone is coated with a MIP that selectively adsorbs caffeine and chromogenic reagents that provide colorimetric quantification of caffeine.
A polymer is provided containing receptor sites that have complementary structural and chemical moieties which enable molecular recognition of a target molecule. Such polymers can be used with advantage in diagnostic devices.
A polymer with molecularly imprinted sites can be formulated into an adhesive. The molecularly imprinted adhesive may be used in in-vitro diagnostic devices to concentrate a target analyte in a sensing area. In addition, a molecularly imprinted adhesive may be used to extract or bind interfering compounds from fluids where they contact the adhesive and remove these compounds in a biosensor. By concentrating an analyte in the sensing area or by removing interfering compounds the accuracy, sensitivity and detection level of the device can be improved.
Alternatively, an adhesive that is made using a polymer that has been synthesized to imprint for a specific molecule may controllably release the target molecule as required. For example, an adhesive imprinted with a surfactant may retain the surfactant in the adhesive network and then release the surfactant in a diagnostic device to reduce the surface tension of fluids. Surfactant imprinted adhesives may be used in lateral flow devices to reduce the surface tension of biological fluids such as blood and sputum to increase flow rates and reduce sensor response time.
Further, the present invention comprises a method of conducting an analysis of a liquid sample containing a component to be analyzed as to identity and/or amount comprising contacting said liquid sample with a cross-linked polymer having been imprinted with said component, and conducting said analysis based on the amount of said component absorbed by said polymer having previously been imprinted with said component.
Alternatively, the method of conducting an analysis of a liquid sample containing a component to be analyzed as to identity and/or amount comprising contacting said liquid sample with an adhesive comprised of a cross-linked polymer having been imprinted with said component, and conducting said analysis based on the amount of said component absorbed by said polymer having previously been imprinted with said component.
b is a schematic diagram of the lateral flow diagnostic device of
The present invention is directed to polymers (such as adhesives) containing at least one molecularly imprinted polymer (MIP). A molecularly imprinted polymer is a cross-linked polymer created with a specific molecular recognition site. The molecular recognition site is complementary to the shape and functionality of a target or receptor molecule.
The present invention is based on the combined effect of the use of a target molecule, functional monomers in the polymer which are complementary to the target molecule, and the use of a cross-linking agent.
The target molecule may be an analyte, an interferent compound, or a compound to be collected. In addition, the molecularly imprinted polymer of the present invention may be used to enable chemical release of an imprinted component (such as an antimicrobial compound), or drug delivery by means of rate programming (diffusion), activated release, or regulated release. The MIP may be used to collect a target molecule, or release a target molecule.
During formation of the MIP, functional monomers are polymerized and cross-linked in the presence of a template molecule to produce a molecularly imprinted resin.
Advantageously, the polymer resin may be comprised of one or more of the following functional monomers: acrylic acid, methacrylic acid, trifluoro-methacrylic acid, 4-vinylbenzoic acid, itaconic acid, 1-vinylimidazole, 2-vinylpyridine, 4-vinylpyridine, 4(5)-vinylimidazole, 4-vinylbenzyl-iminodiaceditc acid, and 2-acrylamido-2-methyl-1-propane sulphonic acid. This listing of functional monomers is not intended to be exhaustive, and other functional monomers can be employed as deemed appropriate. The selection of suitable functional monomers is well within the skill of the routineer in the art.
Other exemplary monomers that may be employed include but are not limited to hydroxyl ethyl methacrylate, 1-vinylimidazole, vinyl acetic acid, acrylamide, and diacetone acrylamide.
A cross-linking agent is also employed in order to control the morphology of the polymer matrix, stabilize the binding site, provide mechanical stability, and is generally present in an amount of from 25-90% by weight based on the total weight of the reactants used to form the polymer.
Exemplary cross-linking agents include but are not limited to 4-divinylbenzene, N,N′-methylene-bisacrylamide, N,N′-phenylene-bisacrylamide, 2,6-bisacrylamidopyridine, ethylene glycol dimethacrylate, poly(ethylene glycol) dimethacrylate, and trimethylolpropane trimethacrylate.
Such monomers can be employed to yield polymers such as poly(hydroxyethylmethacrylate); poly(acrylic acid); poly(methacrylic acid); polypyrrole; copolymers of vinyl acetic acid, acrylamide, and allyl benzene; poly(4-vinyl pyridine); polystyrene-co-acrylamide; copolymer of acrylamide and 4-vinylpyridine.
The polymer reactants and cross-linking agent may be combined together in the presence of a solvent or porogen. The solvent or porogen brings all reactants together during polymerization, is responsible for creating pores in the polymer (either gel-type polymers which may be amorphous or glassy), macroporous or microgel particles. Typical solvents include but are not limited to acetonitrile or water.
Solvent-based or water-based initiators may also be employed to enhance the polymerization of the reactants. A suitable solvent-based initiator is azobis-isobutyronitrile, and a water-based initiator is 2,2′-azobis-cyanovaleric acid.
The resin is used to formulate an adhesive that may be coated onto various carrier films. The template molecule may be retained in the resin, allowing the template molecule to be controllably released from the adhesive. Alternatively, the template molecule may be extracted from the resin prior to formulation into an adhesive.
Adhesives may be formulated to contain multiple molecularly imprinted polymers. An adhesive containing multiple MIPs can be made to extract different template molecules (e.g., interferents) from a fluid as the interferents in the fluid contact the adhesive surface.
The MIP compositions of the present invention may be used in a variety of different ways. The MIP composition, once formed, can be ground to the size of a powder. The MIP powder can then be admixed into an adhesive composition to form an adhesive having uniformly dispersed therein the MIP component. The MIP powder may also be applied to the surface of an adhesive layer, blended into an adhesive solution either as solids or in the form of a solvated mixture of solvent/solids, suspended in a solution (either dissolved or not), and cast or spray coated onto a surface such as an adhesive surface.
Different MIP compositions may be blended in the presence of a suitable solvent, and then cast or otherwise formed into a solid. The solid can then be ground into a suitable particle size. The polymer can also be polymerized in the presence of two or more molecules.
Alternatively, assuming that the resulting imprinted polymer is sufficiently soft to serve as a pressure sensitive adhesive, the molecularly imprinted polymer may itself constitute the adhesive layer.
The identity of the adhesive component with which the MIP may be blended is not critical. A variety of adhesives such as heat sealable adhesives and pressure sensitive adhesives may be employed, the identity of which is known to those of ordinary skill in the art.
For instance, a variety of adhesives including but not limited to polyvinyl ethers, acrylic adhesives, poly-alpha-olefins, and silicone adhesives, as well as blends thereof may be used. By way of example, polyvinyl ether pressure sensitive adhesives generally comprise blends of vinyl methyl ether, vinyl ethyl ether or vinyl iso-butyl ether, or homopolymers of vinyl ethers and acrylates. Acrylic pressure sensitive adhesives may comprise, for example, a C3-12 alkyl ester component and a polar component such as (meth)acrylic acid, N-vinyl pyrrolidone, etc. Such adhesives may be tackified. Poly-alpha-olefin adhesives may comprise an optionally cross-linked C3-18poly(alkene) polymer, which is either self-tacky or may include a tackifier. Silicone pressure sensitive adhesives comprise a polymer or gum constituent and a tackifying resin.
More specifically, the acrylic pressure sensitive adhesive is preferably comprised of a polymer formed from the reaction product of at least one acrylate A and a B monomer different from the A monomer.
The at least one A monomer preferably comprises a monomeric (meth)acrylic acid ester of a non-tertiary alcohol where the alcohol portion has from 1 to 30 carbon atoms. Exemplary A monomers include but are not limited to esters of acrylic acid or methacrylic acid with non-tertiary alcohols such as 1-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 1-methyl-1-pentanol, 2-methyl-1-pentanol, 3-methyl-1-pentanol, 2-ethyl-1-butanol, 3,5,5-trimethyl-1-hexanol, 3-heptanol, 2-octanol, 1-decanol, 1-dodecanol, etc. Such monomers are well-known to those skilled in the art. The least one A monomer component (if more than one A monomer is present) will preferably exhibit an average number of carbon atoms in the alcohol portion of the total acrylic or (meth)acrylic acid esters of from 3 to 16.
One or more polymerizable B monomers different from the A monomer may be incorporated in the polymer which B monomer(s) is copolymerizable with the A monomer. Such additional B monomer(s) may be either hydrophilic or hydrophobic.
Exemplary optional B monomers include vinyl monomers having at least one nitrogen atom. Such monomers (each of which exhibit a Tg of >20° C.) include but are not limited to N-mono-substituted acrylamides such as acrylamide, methacrylamide, N-methylacrylamide, N-ethylacrylamide, N-methylolacrylamide, N-hydroxyethylacrylamide, and diacetone acrylamide; N,N-disubstituted acrylamides such as N,N-dimethylacrylamide, N,N-diethylacrylamide, N-ethyl-N-aminoethyl acrylamide, N-ethyl-N-hydroxyethylacrylamide, N,N-dimethylolacrylamide, and N,N-dihydroxyethylacrylamide, etc.
Other optional B monomers may include, for example, various vinyl monomers such as (meth)acrylic acid, itaconic acid, crotonic acid, methoxyethyl (meth)acrylate, ethyoxyethyl (meth)acrylate, glycerol (meth)acrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, beta-carboxyethyl acrylate, vinyl pyrrolidone, vinyl caprolactam and caprolactam acrylate. One or more B monomers may be employed.
Such pressure sensitive adhesives are well known to one of ordinary skill in the art and may be easily selected by such persons for use in the present invention.
Advantageously, such MIPs may be used in diagnostic devices such as lateral flow devices or other types of diagnostic devices as depicted in the Figures as discussed below. It has been found that indigenous interferents present in a fluid which contacts the diagnostic device may interfere with the obtaining of an accurate diagnostic result. For example, as discussed below, the presence of the interferent uric acid and/or ascorbic acid in blood interferes with the obtaining of an accurate determination of the amount of glucose present in the blood. By use of a MIP which has been imprinted with uric acid and/or ascorbic acid, uric acid and/or ascorbic acid can be removed from the fluid during diagnosis, thus enhancing the accuracy of the determination of glucose in the fluid. The same advantage exists with respect to other interferents that may be present.
The target molecule must be non-reactive under the conditions of polymerization employed to form the imprinted polymer. A typical weight ratio of monomer/target molecule is 4:1, although the amount of target molecular which is employed is not critical to practice of the claimed invention.
A method for preparing a molecularly imprinted polymer with glucose recognition sites is described in Hasoo Seong et al. in the Journal Biomaterial Science Polymer Education, Vol. 13(6), pages 637-649 (2002), herein incorporated by reference in its entirety.
A glucose imprinted polymer was synthesized using functional monomers currently used to make pressure sensitive adhesives. Examples 1-7 describe various formulations and conditions used to make glucose imprinted MIPs as well as control systems. The cross-linker and cross-linker concentration were selected to control the rigidity of the glucose receptor site.
The present invention is further described in connection with the following Examples, which are to be viewed as being merely illustrative of the invention and not limiting by nature.
Example 1 is a conventional polymer formulation which is made without the presence of a glucose agent, and is made as a control composition.
The above reactants are mixed with 1% by weight of the monomers with the initiator 2,2′-azobis(2,4-dimethylpentanenitrile), and polymerized for 4 hours at 60° C. under nitrogen atmosphere.
Example 2 contains the same polymer components as the formulation of Example 1, but also includes a D-glucose component.
The above reactants are mixed with 1% by weight of the monomers with the initiator 2,2′-azobis(2,4-dimethylpentanenitrile), and polymerized for 4 hours at 60° C. under nitrogen atmosphere.
Upon completion of the polymerization the polymer of Examples 1 and 2 was dried to remove the solvent and then ground into a fine powder.
The polymer powder of Example 2 was washed with water to remove any glucose. After the water wash, the polymers were re-dried. To determine the ability of the imprinted sites to extract and concentrate D-glucose the polymer was exposed to an aqueous solution containing 10% glucose. Thermal gravimetric analysis using a Universal V2.6D from TA instruments was used to measure the shift in polymer thermal stability caused by adsorption of glucose by the polymer. By measuring the shift in the decomposition temperature of the polymer with or without glucose it was found that 70% of the theoretical sites were formed during the polymerization, and 50% of these sites remained viable for entrapment of glucose after glucose removal.
Example 3 is another conventional polymer formulation which is made without the presence of a glucose agent, and is made as a control composition.
The above reactants are mixed with 1% by weight of the monomers with the initiator 2,2′-azobis(2,4-dimethylpentanenitrile), and polymerized for 4 hours at 60° C. under nitrogen atmosphere.
Example 4 contains the same polymer components as the formulation of Example 3, but also includes a D-glucose component.
The above reactants are mixed with 1% by weight of the monomers with the initiator 2,2′-azobis(2,4-dimethylpentanenitrile), and polymerized for 4 hours at 60° C. under nitrogen atmosphere.
As noted above, the polymer synthesized in Example 3 contains no glucose imprinted sites and is used as a control. Example 4 is a similar polymer. However, the polymerization and cross-linking occurs in the presence of glucose as the template molecule.
Thermal gravimetric analysis could not be used for Examples 3 and 4 since the shift in the polymer decomposition was similar to glucose. To measure glucose imprinting in Example 4, a Glucose G2 Autokit Glucose kit was obtained from WAKO Chemicals USA, Inc. This kit employs an enzymatic method using mutarotase and glucose oxidase to generate hydrogen peroxide during the oxidation of glucose. The hydrogen peroxide then induces oxidative condensation between phenol and 4-aminoantipyrine in the presence of peroxidase to create a red color. By measuring the absorbance of the red color the concentration of glucose can be determined.
A washing and drying procedure similar to Examples 1 and 2 was followed. The polymers from Examples 3 and 4 were exposed to a 10% aqueous glucose solution then washed and a sample of the water extracts from the control and imprinted polymers were collected. The water samples were prepared following the glucose kit directions and the absorbance was tested on a Lambda 9000 UV/VIS/NIR spectrophotometer from Perkin Elmer. Using a known concentration standard include in the kit, the glucose concentration in the water samples was determined. This value was related to the theoretical imprinted glucose sites in the polymers. It was calculated that 80% of the theoretical sites were formed during the polymerization for Example 4 and 70% of these sites remained viable for entrapment after glucose removal.
Examples 2 and 4 form rigid to soft polymers that were formulated into an adhesive and coated onto a polyester carrier. The adhesive film was used in a lateral flow device to support a nitrocellulose membrane under a sensing area. The sensing area on the nitrocellulose membrane had been treated with colorimetric reagents which respond to glucose. One drop of a 1% aqueous glucose solution was passed through the membrane and the color in the sensing area responded within 2 seconds. A similar lateral flow device was constructed without the MIP adhesive using the controls of Examples 1 and 3, and the colorimetric reagents in the sensing area did not respond until 10 seconds.
Example 5 also contains the same polymer components as the formulation of Example 2, but also includes a uric acid component instead of a D-glucose component. Uric acid is a known interferent in glucose determinations as discussed above.
The above reactants are mixed with 1% by weight of the monomers with the initiator 2,2′-azobis(2,4-dimethylpentanenitrile), and polymerized for 4 hours at 60° C. under nitrogen atmosphere.
Example 5 shows a MIP polymer imprinted for uric acid by removal of uric acid from the resulting polymer by exhaustive water washing. Uric acid has been reported to interfere with the response of blood glucose test strips. The uric acid MIP was formulated into an adhesive and the adhesive tape was used as a cover over the blood channel of an Accu-Chek Comfort Curve blood glucose test strip manufactured by Roche Diagnostics Corporation, Indianapolis, Ind. A test solution containing 130 mg/decaliter glucose and 8 mg/decaliter uric acid was allowed to wick through the blood channel.
More accurate glucose measurements were obtained when the channel was enclosed by the uric acid imprinted adhesive in comparison to a control test strip which did not contain the MIP adhesive.
Other compounds such as acetaminophen and ascorbic acid are also known to interfere with blood glucose analysis. Other molecules which can be imprinted include but are not limited to caffeine, melatonin, morphine or other drugs of abuse, etc. MIPs similar to Example 5 can be prepared using these interferent compounds as template molecules. For instance, individual polymers for each interferent can be prepared in the same manner noted above. Alternatively, one polymer with imprinted sites for multiple interferent compounds can synthesized by incorporating different interferents in a single reaction batch.
Example 6 contains another conventional solvent-free photocurable pressure sensitive adhesive polymer formulation for use as a control.
The above reactants are mixed with 1% by weight of the monomers with the initiator 2,2′-azobis(2,4-dimethylpentanenitrile), and polymerized for 4 hours at 60° C. under nitrogen atmosphere.
Example 7 contains the same polymer components as the formulation of Example 6, but also includes a surfactant component—i.e., Aerosol OT, an anionic surfactant, obtained from Cytec Industries.
After free radical photocuring Example 7 produces a hydrophilic pressure sensitive adhesive. The surfactant molecule is used as a template to create imprinted sites within the adhesive matrix. The surfactant molecules may be controllably released from the adhesive during use to reduce the surface tension of fluids that contact the adhesive. Since the surfactant molecules are entrapped within the polymer matrix they are less labile and the adhesive retains its hydrophilic properties even after rinsing the adhesive under flowing tap water.
A pressure sensitive adhesive tape was made by coating the imprinted polymer from Example 7 on a 3 mil polyester film. The surfactant-imprinted tape was used as a cover to enclose fluidic channels molded into a polyethylene substrate. Water flowed through the channels while a similar construction prepared using a pressure sensitive adhesive tape made using the control polymer in Example 6 did not. Water flow through the channels could be repeated multiple times illustrating the retention of the surfactant in the imprinted adhesive polymer.
The molecularly-imprinted polymers of the present invention may be used with advantage with diagnostic devices such as those disclosed in PCT publication WO 02/085185. Such devices include lateral flow devices, micro-fluidic in-vitro diagnostic devices, and in-vitro diagnostic devices comprised of a microplate having a base plate having disposed therein a multitude of microholes or cavities, and at least one cover placed in sealing relationship to said microholes or cavities.
PCT publication WO02/085185 teaches the combination of a surfactant with a polymer composition in order to yield a hydrophilic polymer. However, the noted publication merely teaches the admixing of the surfactant with a solvated polymer. This is in contrast to the present invention where the surfactant is admixed with the mixture of monomers which are then copolymerized and crosslinked in the presence of the surfactant to form an imprinted polymer.
Lateral flow devices as shown in
Adhesive backings are typically used in the construction of lateral flow devices to support the various components of the device including the conjugate pad, a microporous membrane with specific reagents and an absorbent pad as shown in
The present invention may be employed with advantage in a variety of in-vitro diagnostic devices, both of the lateral flow and of the capillary flow type, with devices of the lateral flow rate type of
Advantageously, in accordance with the present invention, the backing strip between the first and second ends may be a molecularly imprinted polymer film which may be adhesive by nature. The backing strip 7 may be, e.g., heat-sealable or exhibit pressure sensitive adhesive properties. If the backing strip 7 exhibits pressure sensitive adhesive properties, and is molecularly imprinted with a surfactant, the hydrophilic character of the material serves to avoid reducing the effectiveness of any membrane 9 attached to the backing strip in the event that migration of the adhesive into the membrane occurs.
By way of further advantage, if the backing strip is imprinted with a surfactant, it may be possible to avoid use of the membrane 9, instead relying solely on the hydrophilic character of the backing strip itself to wick the sample from the sample introduction point to the sample collection point. In such an embodiment, the reagent with which the sample must contact or react with will either be applied directly to the backing strip for contact with the sample, or be introduced to the surface of the backing strip from a reservoir attached to the backing strip in a conventional manner.
Port 11 may be employed to provide access for another material such as a buffer to be applied to absorbent pad 13. The sample once added to port 3 contacts absorbent pad 15. The assembly of the backing strip and associated attached components may be positioned within a bottom portion 17 of the housing. The housing cover 1 includes view port 20 for viewing the visual result of the reaction between the sample and the reagent present in the device.
Another embodiment of the device of the present invention is depicted in
Various modifications can be undertaken with advantage in such an embodiment. As discussed above, selective areas of hydrophilic/hydrophobic surface character can be provided on the surface of the backing material by molecular imprinting to modify the flow characteristics of the fluid sample, either by directing the sample longitudinally along the backing strip toward the fluid collection point, or by causing the fluid sample to contact adjacent hydrophilic/hydrophobic areas to slow the flow rate of the fluid sample along the backing strip. In such an instance, for example, the reagent may be placed on the hydrophobic portion where the wicking of the fluid sample would be slower to permit a longer contact time with between the fluid sample and the reagent. In terms of this discussion, the term hydrophobic is not intended to mean that the portion of the backing would be entirely hydrophobic, but could also mean that that the area is more hydrophobic than the adjacent hydrophilic portion of the backing strip (i.e., both portions would have varying degrees of hydrophilicity so that the wicking of the fluid sample would still be encouraged to travel from the sample inlet to the sample collection area).
Accordingly, in the context of
As discussed above, in an embodiment where the backing strip comprises a hydrophilic pressure sensitive adhesive layer, the membrane can still be used with advantage due to the hydrophilic character of the adhesive without fear of diminishment of the ability of the membrane to function due to migration of the adhesive. However, it is still possible to avoid the use of the membrane, with the hydrophilic adhesive layer serving as the transport medium for the sample from the sample pad to the absorbent pad. Any reagents desired to be contacted with the sample may be applied directly to the surface of the hydrophilic adhesive layer. The adhesive character of the backing strip can also be employed with advantage to bond the respective sample/conjugate/absorbent pads to the backing strip. This facilitates the manufacture of the device. Such a device would typically be contained in a suitable housing that generally includes a viewing window to determine the extent of the reaction of the sample and the reagent (e.g., to determine extent of reaction due to color formation or the intensity of the color formed).
In the context of a microfluidic diagnostic device which employs capillary transport of the fluid sample during the analysis procedure, such devices typically include microfluidic channels molded in a suitable polymeric substrate (see
The microfluidic diagnostic device is generally comprised of a substantially planar base portion having one or more microfluidic channels, passages, chambers or conduits therein. A variety of materials may comprise the base portion, including polymeric materials such as polymethylmethacrylate, polycarbonate, polytetrafluoroethylene, polyvinylchloride, polydimethylsiloxane, polysulfone, and silica-based substrates such as glass, quartz, silicon and polysilicon, as well as other conventionally-employed substrate materials.
Such substrates are manufactured by conventional means, such as by injection molding, embossing or stamping, etc. The microfluidic passages or channels may be fabricated into the base portion by conventional microfabrication techniques known to those skilled in the art, including but not limited to photolithography, wet chemical etching, laser ablation, air abrasion techniques, injection molding, embossing, and other techniques. The base material is selected on the basis of compatibility with the desired method of manufacture as well as for compatibility with the anticipated exposure to materials and conditions, including extremes of pH, temperature, salt concentration, and the application of electric fields. The base material may also be selected for optional properties including clarity and spectral characteristics.
An enclosure surface or cover is placed over the top portion of the base substrate to enclose and otherwise seal the microfluidic passages or channels. In the context of the present invention, the channels or passages are covered with a substrate according to the present invention the surface of which is molecularly imprinted which covers the passages or channels in the base substrate. A surfactant imprinted cover can be used to enhance the flow of the liquid through the microfluidic passages and channels.
Such devices typically include optical detector means positioned adjacent to a detector window whereby the detector senses the presence or absence of an optical characteristic from within the microfluidic passage or channel resulting from flow of the liquid sample through the passage or sample. The optical detector may comprise any of a variety of detector means such as fluorescent, colorimetric or video detection systems, which include an excitation light source (laser or LED), etc. A variety of optically detectable labels can be employed to provide an optically detectable characteristic such as colored labels, colloid labels, fluorescent labels, spectral characteristics and chemiluminescent labels.
As discussed above, an alternative to otherwise having to ensure that the channels possess sufficient hydrophilicity to cause the fluid sample to travel along the capillary tube, the top portion of the channel is covered with a hydrophilic material which has been molecularly imprinted with a surfactant in accordance with the present invention. That is, a heat-sealable polymeric film having hydrophilic surface characteristics may be applied over the open cavity of the channel to both enclose the channel and provide the necessary hydrophilic character so that the fluid sample will be caused to wet the channel. As an alternative, the polymeric film may include a pressure sensitive adhesive coating which is also hydrophilic in character to provide the necessary hydrophilicity to cause the fluid sample to wet the channel by being molecularly imprinted with a surfactant. The use of such materials in the construction of the microfluidic diagnostic device also serves to simplify the manufacturing of the device. In the context of the present invention, the entire facing surface of the covering layer need not be hydrophilic; instead, only that portion of the covering layer that serves to enclose the microfluidic channels or passages is required to be hydrophilic. Of course, as is the case with lateral flow devices, certain portions of the covering layer that enclose the microfluidic channels or passages may be rendered less hydrophilic than other portions to modify the flow rate of the fluid sample.
A typical microfluidic device which has been prepared in accordance with the present invention is depicted at
By way of an alternative embodiment depicted in
The spacer layer 71 preferably is an adhesive layer which is bonded to the opposing base layers, either as a result of pressure sensitive adhesive properties of the spacer layer or as a result of being heat-sealed to each of the base layers. If pressure sensitive, the spacer layer may be used in the form of a transfer film or as a double face construction. As discussed above, if the base layers are not hydrophilic in character, the spacer layer may possess the requisite hydrophilic character to assist wetting of the fluid channel by the fluid sample. The fluid channels 73 in the spacer layer may be die-cut into the spacer layer or provided by any other means effective to provide a spacer layer with the requisite fluid channels. One advantage of such a construction is that the micro-fluidic device may be constructed easily without the need to mold the fluid channels into the base layers as in the embodiment of
Microplates of the present invention include various embodiments such as microwell-containing microplates as shown in
An alternative microplate embodiment is shown in
The present invention may employ a multitude of polymeric films which can be molecularly imprinted to provide desired properties. Polymers which can be modified in this manner are well known in the art. Exemplary of such polymers are the following polymers: polyolefins, including but not limited to polyethylene, polystyrene, polyvinyl chloride, polyvinyl acetate, polyvinylidene chloride, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyethyl acrylate, polyacrylamide, polyacrylonitrile, polypropylene, poly(1-butene), poly(2-butene), poly(1-pentene), poly(2-pentene), poly(3-methyl-1-pentene), poly(4-methyl-1-pentene), 1,2-poly-1,3-butadiene, 1,4-poly-1,3-butadiene, polyisoprene, polychloroprene, ethylene-vinyl acetate copolymer, polycarbonate, ethylene-isobutyl acrylate copolymer, as well as random or block copolymers of two or more polyolefins or a polyolefin and a non-olefin. Similarly, blends of two or more polymers may also be employed.
The polymer may also comprise a polyester such as polyethylene terephthalate, polyethylene isophthalate-terephthalate, copolymers of poly-(1,4-cyclohexane dimethylene)terephthalate, poly(1,4-cyclohexane dimethylene) isophthalate, and isophthalate-terephthalate copolymers; poly(1,4 phenylene) terephthalate and isophthalate and copolymers; poly(1,4-phenylene)-4,4′ diphenyl dicarboxylate; polyesters derived from aliphatic dibasic acids, such as maleic, adipic and sebacic acids and polyhydroxy compounds such as polyethylene glycol, neopentyl glycol, butylene glycol, glycerol, pentaerythritol, and cellulose. Preferably, the film-forming polymers used in the present invention exhibit a Tg or Tc sufficient to permit the polymer to be film-forming as well as to enable the resulting polymer film to be heat sealable at a sufficiently low temperature (e.g., in the range of from 70 to 100° C.).
A variety of surfactants may be used to molecularly imprint the polymer. Surfactants which are suitable for use in the present invention include any surfactant which effectively imparts hydrophilic surface properties to the hydrophobic polymer film. While the identity of such surfactants is not critical to the practice of the present invention, anionic surfactants are preferred. However, exemplary of such surfactants (without limitation) are ammonium salts or sodium salts of alkyl phenoxy (polyethylene oxy) ethanol, ammonium perfluoroalkyl sulfonates, etc. Exemplary surfactants preferably include one or more hydroxyl, carboxylic acid, sulfonic acid, and amine functionalities. A detailed discussion of surfactants resides in Kirk-Othmer, Encyclopedia of Chemical Technologies, 2nd Edition, Vol. 19, pages 512-564, herein incorporated by reference.
The surfactant may be admixed in an amount of, for example, up to about 15% by weight, based on the total weight of the polymer and surfactant, such as in an amount of from 0.05 to 15% by weight. Preferably, the surfactant is admixed with the polymer in an amount in the range of from about 3 to 6% by weight.
It is thus apparent that a molecularly imprinted adhesive may be used with advantage in the above-described devices by providing a surface which includes, for example, a molecularly imprinted surfactant to provide enhanced hydrophilic surface properties, a molecularly imprinted adhesive surface which is imprinted to collect a specific interferent(s) from a liquid to be analyzed, or a molecularly imprinted adhesive surface to collect a specific component(s) for which an analysis is to be made. Advantageously, the molecular imprinted properties of the adhesive layer can easily be tailored to meet the desired end result consistent with the objects of the present invention.
With respect to the “bulk” embodiment, the concentration of MIP is 5% in the bulk of the adhesive. 5% by weight of the MIP to adhesive solids is mixed into the adhesive solution prior to coating the adhesive onto a carrier film.
With respect to the “surface” embodiment, the concentration of MIP is 5% on the surface of the adhesive by suspending 5% by weight of MIP to adhesive solids in a solvent and coating the suspension onto a release film. The adhesive solution is coated onto a carrier film, and after drying and curing, the adhesive film is laminated onto the surface of the MIP coated liner. Alternatively, the adhesive may be directed coated on top of the MIP coated liner. Alternatively, the MIP suspension may be directly coated or sprayed onto the top of the adhesive coating.
The results of
For instance,
It is accordingly an advantage of practice of the present invention to provide a method for the analysis of a liquid sample whereby significantly increased efficiency of analysis can be achieved by use of a molecularly imprinted polymer having been imprinted with an interferent present in the liquid sample is used to accomplish the analysis.
By way of further explanation of
Referring to
Referring to
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/24219 | 6/22/2006 | WO | 00 | 12/19/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60692559 | Jun 2005 | US |
