Patent Application
20070266871
Reference will now be made in detail to various embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.
Embodiments of the present invention relate to methods for manufacturing diagnostic test media using microcontact printing. Microcontact printing is a technique that has been used in the biotechnology industry for various purposes. To briefly summarize, the technique entails creating a stamp with a pattern of interest. In certain embodiments, the stamp is formed using a master with the inverse pattern of interest as a template. The stamp is then coated with an “ink” and stamped onto a substrate, depositing the “ink” onto the substrate in the pattern of interest.
It has been found that microcontact printing could be used to transfer a monolayer of alkanethiolates onto a gold or silver film to study, for example, wetting, adhesion, protein adsorption, and cell adhesion (Whitesides, et al., Ann. Rev. Biomed. Eng., 3:335 (2001)). It has also been found that microcontact printing could be used to transfer an ethanolic solution of catalytic ink to facilitate carbon nanotube growth on a silicon substrate (Nilsson and Schlapbach, Langmuir, 16:6877 (2000)). More recently, scientists have found microcontact printing can transfer proteins, dendrimers, and other biomolecules for producing, for example, protein and DNA microarrays (Inerowicz et al., Langmuir 28:5263 (2002); Hong et al, Bull. Korean Chem. Soc. 24:1197 (2003)).
Prior techniques of contact printing are primarily concerned with the application of Self Assembled Monolayers (SAMs) on a substrate surface that is usually comprised of gold or silver (see Zhao et al. J. Mater Chem., 1997, (7), 1069-1074). The application of SAMs to the target substrate layer occurs through a process of coating a stamp with a hexadecanethiol ink, after which, the inked stamp is brought into contact with the target gold or silver substrate layer. Through this contact, the sulfur end of the hydrocarbon chain is chemisorbed onto the surface through the formation of a stable thioether bond between alkanethiol molecule and underlying gold or silver film. The monolayer of hexadecanethiolate (CH3(CH2)15S−) is further stabilized by Van der Waals forces between adjacent alkyl chains. Micrometer scale patterns (and sometimes even smaller) are formed by these processes, whereby, the SAM patterns provides a protective barrier over the metal layer it covers. Therefore, after a chemical etching process, the metal patterns protected by the SAM material will remain in the underlying stamped pattern of interest with the surrounding metal layers being removed.
The present disclosure uses a novel microcontact printing technique for the manufacture of diagnostic test media. The test media of the present disclosure may be used with a suitable test meter to detect or measure the concentration of one or more analytes. An exemplary electrochemical biosensor is described in U.S. Pat. No. 6,743,635 (the '635 patent) which is incorporated by reference herein in its entirety. The '635 patent describes an electrochemical biosensor used to measure glucose level in a blood sample. The electrochemical biosensor system is comprised of a test strip and a meter. The test strip includes a sample chamber, a working electrode, a counter electrode, and fill-detect electrodes. A reagent layer is disposed in the sample chamber. The reagent layer contains an enzyme specific for glucose, such as, glucose oxidase or glucose dehydrogenase, and a mediator, such as, potassium ferricyanide or ruthenium hexaamine.
In one exemplary measurement technique, when a user applies a blood sample to the sample chamber on the test strip, the reagents react with the glucose in the blood sample and the meter applies a voltage to the electrodes to cause redox reactions. The meter measures the resulting current that flows between the working and counter electrodes and calculates the glucose level based on the current measurements. As noted above, the ease of test media production as well as additional factors such as cost, a desire for size reduction, and a reproducible uniform electrode pattern and area, are all considerations addressed by the test media systems and methods of the current application.
Examples of suitable meters are illustrated in
The strip 10 includes a conductive pattern with electrodes formed at a proximal end 12, which then extend to corresponding conductive contacts close to the distal end 14. For example, in one embodiment, the conductive pattern forms a cathode electrode region 16, an anode electrode region 18, and first and second fill detect electrode regions 20 and 22 respectively, all of which are in contact with some portion of a sample cavity reception location 24. The four electrode regions 16, 18, 20, and 22, each lead to a corresponding conductive contact, 26, 28, 30, 32, for interfacing with a meter system. As will be described in more detail below, in one embodiment, a distal region 34 of strip 10 includes an additional contact pattern providing additional contacts for reception by a corresponding meter interface.
As shown in
According to an illustrative embodiment, a conductive layer 40 is disposed on base layer 36. As will be described in more detail below, the conductive layer 40 can be applied according to a novel process of contact printing and/or transfer molding. Conductive layer 40 defines the electrodes 16-22 described above, the plurality of electrical contacts 26-32 described above, and a plurality of conductive regions electrically connecting the electrodes to the electrical contacts.
The next layer in the illustrative test strip 10 is a spacer layer 64 disposed on conductive layer 40. The spacer layer 64 is composed of an electrically insulating material, such as polyester. The spacer layer 64 can be about 0.10 mm thick and cover portions of the electrodes 16-22, but in the illustrative embodiment does not cover a distal portion of electrical contacts 26-32. For example, spacer layer 64 can cover substantially all of conductive layer 40 thereon, from a line just proximal of contacts 26-32 all the way to proximal end 12, except for a slot 52 extending from proximal end 12. In this way, slot 52 can define an exposed portion of the cathode electrode region 16, an exposed portion of anode region 18, and an exposed portion of electrodes 20-22.
A cover 72, having a proximal end 74 and a distal end 76, can be attached to spacer layer 64 via an adhesive layer 78. Cover 72 can be composed of an electrically insulating material, such as polyester, and can have a thickness of about 0.075 mm. Additionally, the cover 72 can be transparent.
Adhesive layer 78 can include a polyacrylic or other adhesive and have a thickness of about 0.02 mm. Adhesive layer 78 can consist of sections disposed on spacer 64 on opposite sides of slot 52. A break 84 in adhesive layer 78 extends from the distal end 70 of slot 52 to an opening 86. Cover 72 can be disposed on adhesive layer 78 such that its proximal end 74 is aligned with proximal end 12 and its distal end 76 is aligned with opening 86. In this way, cover 72 covers slot 52 and break 84. In another arrangement, opening 86 can be replaced by a hole that is formed in cover 72 itself. Such a hole in the actual cover 72 provides a vent pathway to allow air in the chamber to be displaced by the fluid sample.
Slot 52, together with base layer 36 and cover 72, defines a sample chamber 88 in test strip 10 for receiving a blood sample for measurement in the illustrative embodiment. Proximal end 12 of slot 52 defines a first opening in sample chamber 88, through which the blood sample is introduced into sample chamber 88. Slot 52 is dimensioned such that a blood sample applied to its proximal end 68 is drawn into and held in sample chamber 88 by capillary action, with break 84 venting sample chamber 88 through opening 86, as the blood sample enters. Moreover, slot 52 can advantageously be dimensioned so that the blood sample that enters sample chamber 88 by capillary action is about 1 micro-liter or less. For example, slot 52 can have a length (i.e., from proximal end 12 to distal end 70) of about 0.140 inches, a width of about 0.060 inches, and a height (which can be substantially defined by the thickness of spacer layer 64) of about 0.005 inches. Other dimensions could be used, however. As noted above, in another arrangement the opening 86 can be replaced by a hole that is formed in cover 72 itself. In such an arrangement, the hole in cover 72 allows for a fluid sample to be drawn into the sample chamber 88 via capillary action in the same manner as that resulting from break 84.
A reagent layer 90 is disposed in sample chamber 88. In the illustrative embodiment, reagent layer 90 covers at least exposed portion of the cathode electrode region 16. Further according to the illustrative embodiment, reagent layer 90 also at least contacts an exposed portion of the anode electrode region 28 and preferably fully covers the anode. Reagent layer 90 includes chemical constituents to enable the level of glucose or other analyte in the test fluid, such as a blood sample, to be determined electrochemically. Thus, reagent layer 90 can include an enzyme specific for glucose, such as glucose oxidase or dehydrogenase, and a mediator, such as potassium ferricyanide or ruthenium hexamine. Reagent layer 90 can also include other components, such as buffering materials (e.g., potassium phosphate), polymeric binders (e.g., hydroxypropyl-methyl-cellulose, sodium alginate, microcrystalline cellulose, polyethylene oxide, hydroxyethylcellulose, and/or polyvinyl alcohol), and surfactants (e.g., Triton X-100 or Surfynol 485).
With these chemical constituents, reagent layer 90 reacts with glucose in the blood sample in the following way. The glucose oxidase initiates a reaction that oxidizes the glucose to gluconic acid and reduces the ferricyanide to ferrocyanide. When an appropriate voltage is applied to the cathode electrode region 16, relative to anode electrode region 18, the ferrocyanide is oxidized to ferricyanide, thereby generating a current that is related to the glucose concentration in the blood sample.
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Although
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To manufacture the test media using microcontact printing, in certain embodiments, a master may be created and patterned by standard lithography procedures known to one of ordinary skill in the art. In short, photoresist (either negative or positive) is applied to a silicon wafer, although any suitable material may be used. Then a mask with the pattern of interest is placed on top of the wafer. The photoresist is then exposed, which, depending on whether it is negative or positive photoresist, will either polymerize or degrade the exposed regions of photoresist. (Alternatively, instead of using a mask, a laser may be used to selectively expose a desired pattern directly onto the photoresist.) The mask (if applicable) is then removed, and the unreacted photoresist is washed or etched away.
In certain embodiments, the stamp is then fabricated using the master as a template. To prevent the stamp from adhering to the master, the master may be treated be gas phase silanization, plasma flourination, or other suitable techniques. The stamp may be made from (poly)dimethylsiloxane (“PDMS”), but any suitable material may be used. When using PDMS, PDMS precursors, including a curing agent, are mixed and put into a vacuum chamber to remove any oxygen bubbles, which may distort the stamp and affect the deposition of the ink. Afterwards, the mixed precursors are poured over the master. As an example, the PDMS can then be cured (e.g., at 60 degrees Celsius for one or more hours). After curing, the PDMS with the pattern of interest is peeled away from the master, thereby creating the stamp. The surface pattern features of the PDMS are the inverse of those present on the master.
As illustrated in
Other polymer materials suitable for curing over a master may be used for the stamp. Once formed into a patterned stamp, the polymer material should be reusable and should not react with a subsequently described “ink,” which may contain biomolecules. Likewise, the polymer stamp material should not interfere with the electroactive or chemical properties of the “ink.” Moreover, the material should not be too stiff to hinder removal from the master or ink transfer to the substrate.
After the stamp is created, a substance, also known as an “ink,” is applied to the stamp. The ink may be applied using any number of methods known to one of ordinary skill in the art. In certain embodiments, the ink may be applied by spraying or misting the ink onto the substrate. The ink may also be applied by dipping the stamp either completely or partially into the ink. Any excess ink may be removed using a blade, such as a razor, or other appliance for scraping the excess ink away. In other embodiments, the ink is applied directly by, for example, painting or spreading the ink onto the stamp using a brush, roller, or other suitable ink-applying utensil. As noted above, in embodiments where the final product test media requires a chemistry reagent, a reagent “ink” substance will be applied and formed after the application of an electroactive “ink” substance such that at least a portion of the applied reagent covers at least one of the electrodes formed by an electroactive “ink.”
Generally, the PDMS stamp surface will exhibit hydrophobic properties. This may hinder the transfer of the ink to the underlying substrate, depending on the type of ink used. Therefore, before use, the PDMS stamp can be treated with an oxygen plasma to create a hydrophilic surface. This will increase the propensity of the ink material to transfer it from the stamp to a surface to be printed, as well as the ink to more uniformly coat the stamp. Any plasma treatment device that is commercially available may be used to treat the stamp (e.g., Harrick Plasma bench top plasma cleaner, PVA TePla Plasma Pen, and ScanArc Plasma Technologies treaters). For purposes of this application, after this plasma process, the stamp is considered to be “plasma treated.”
The “ink” is the material that will be applied to a substrate material through microcontact printing, which will form the underlying conductive layer 40, described above. As described above, prior art procedures used inks containing SAM precursors to print SAM structures containing, for example, hexadecanethiol. In the following systems and methods, the microcontact printing is different in that the applied ink is an electrically conductive material and not a SAM. In addition, a feature or features printed from the ink may form a multi-layer structure as opposed to a monolayer structure. Moreover, the substrate of interest may be a polymer (e.g., a polyethylene terephthalate (PET) material) and not a gold or silver layer as used in earlier techniques. Since the ink materials, and the preferred surface materials, differ from those described with regard to earlier microcontact printing techniques, the mechanism of attachment between the ink and printed substrate, and the mechanism of layer formation within as-printed features, are also necessarily different from those related to earlier techniques.
The ink for the electrode pattern may comprise a suitably transferable form of any electroactive substance, including palladium, gold, silver, carbon, platinum, copper, doped silicon, conductive polymers, and/or any other suitable electrode material. The ink may comprise a single electroactive substance, or may comprise a mixture of electroactive substances. The electroactive ink may also be a custom organometallic ink (e.g., available from Gwent Electronic Materials, Ltd.) created for a particular purpose or characteristic, such as, for example, preventing conglomeration, or for its heat-treating properties. The ink may be in any form that allows for transfer onto a substrate, including liquid, paste, or powder form. The use of the word “ink,” on its own, is not intended to impart or imply any particular method of application or formation of the “ink” material.
For example, the mechanism of attachment between the ink and the polymer substrate is based on a mechanism of physical adsorption of the ink upon the polymer substrate. In some embodiments, the substance within the ink that provides the conductive properties will need to be mixed with a polymeric agent. When used, the polymeric agent provides a mechanism of cross-linking that results in a curing of the ink substance that provides one aspect of the attachment mechanism.
In one embodiment, the ink materials need only consist of an electrically conductive material, such as conductive metal particles or carbon powder, provided in a liquid-paste consistency state. The conductive material can be provided in a liquid-paste consistency, with the desired viscosity level of the ink controlled as desired with the addition of known chemical substances, as would be apparent to one having ordinary skill in the art. The substance in which the conductive material is dispersed can be comprised of an organic medium. For example, organic binders based on cellulose material such as ethyl cellulose and hydroxyethyl cellulose, acrylic resins such as polybutylmethacrylate, polymethylmethacrylate, and polyethylmethacrylate, epoxy resin, phenol resin, alkyd resin, polyvinyl alcohol, polyvinyl butyral or the like; and organic solvents, for example, ester solvents such as butyl cellosolve acetate, butyl carbitol acetate, ether solvents such as butyl carbitol, ethyleneglycol and diethyleneglycol derivatives, toluene, xylene, mineral spirit, terpineol, and methanol, can be used.
In another aspect of this application, the chemical reagent layer described above, can be applied in the form of a stamped ink material. The ink for the reagent layer may be any chemical substance that, once printed, may be used to facilitate the detection of one or more analytes. The ink may include one or more enzymes (e.g., glucose oxidase, cholesterol oxidase). Furthermore, the ink may include other chemical substances such as electrochemical mediators (e.g., potassium ferricyanide, ruthenium hexaamine), buffers (e.g., potassium phosphate), polymeric binders (e.g., hydroxypropyl-methyl-cellulose, sodium alginate, microcrystalline cellulose, polyethylene oxide, hydroxyethylcellulose, and/or polyvinyl alcohol), surfactants (e.g., Triton X-100 and/or Surfynol 485), enzyme stabilizers, color indicators, and/or any other chemical substance needed to facilitate production of a suitable test reaction. In some embodiments, due to the properties of the chemistry solution, applying the chemistry solution via the printing processes described in this application does not require any plasma treatment of the stamp prior to printing.
As shown in
As illustrated in
In certain embodiments, printing the electroactive ink creates one or more electrode patterns. The one or more electrode patterns may include one or more electrodes (e.g. a cathode electrode, an anode electrode, a fill-detect cathode, and/or a fill-detect anode), one or more electrical contacts (e.g., extending from each of the electrodes), and/or one or more conductive traces connecting the one or more electrodes to the corresponding electrical contact. Other electrode patterns that may be deposited include a conductor that detects the contact with the meter and automatically turns the meter on.
Once the electroactive ink is deposited on the substrate, the ink on the substrate may be cured by baking, sintering, UV treatment, or by any number of suitable techniques. The curing conditions will vary depending on the properties of the ink applied. For example, in certain embodiments, a custom organometallic ink from Gwent Electronic Materials, Ltd. (GEM), is sintered at 500 degrees Fahrenheit. In the case of commercially available carbon and gold inks (GEM, Dupont), the material is cured at 60 degrees Celsius for 1-5 minutes.
As noted above, in certain embodiments, chemistry reagent layers may be deposited using this microcontact printing technique. Dilute solutions of the chemistry components may be used for the ink. The chemistry components may be added separately or simultaneously, and any appropriate drying technique may be used. In certain embodiments, the stamp deposits the chemistry layer in the sample well area of the test media.
For example,
The stamp 600 is then placed in contact with a substrate 400 (which can constitute any of the materials described above, such as, for example, PET). The ink material 500 is then acted upon through a process that leaves the ink material in a solid form. For example, the ink material 500 can be subjected to a process of curing through illumination with ultra-violet light (UV illumination is not used when applying the chemistry reagent, however) or by the application of heat through either baking or sintering. In one embodiment, the application of a chemistry reagent is effectuated by employing a low temperature baking process to prevent denaturing of enzymes therein. As shown in
The conductive pattern formed on base layer 36, through one of the methods described above, extends along test strip to include the distal strip contact region 700. As illustrated in
The first plurality of electrical strip contacts 26-32 are divided, for example, through breaks 754 formed through the underlying conductive pattern in the test strip 10. These breaks could be formed in the conductive pattern during the contact printing or transfer molding procedures, described above. In addition, other processes of forming conductive breaks by removing a conductor in the test strip 10 may be used as would be apparent to one having ordinary skill in the art. One break 754 divides conductive region 710 from conductive region 34 within distal strip contact region 700, and a further break 754 separates the upper right-hand portion of distal strip contact region 700 to form a notch region 756, as will be described more fully in detail below.
In
As described in commonly owned co-pending U.S. patent application Ser. No. 11/181,778 filed Jul. 15, 2005 (the entire contents of which are hereby incorporated by reference), the contacting pads 758, 760, 762, 764, and 766 are configured to be operatively connected to the second plurality of connector contacts 740 within a meter connector 750 (see
Further to the invention, the disclosed method may be normalized through various means to allow for mass production of test strips. As illustrated in
The following portion of the application provides a few examples of conductive patterns and chemistry layers provided with the system and methods described above. Microcontact printed patterns according to embodiments of the invention may have features with spatial resolutions on the order of 1 micron or larger. As a non-limiting example, contact printed electrodes and chemistry layers for biosensors would, in some embodiments, have minimum spatial resolutions on the order of 25-1500 microns, and more preferably, on the range of between about 50-1000 microns.
In the systems and methods described throughout this application, the minimal spatial resolution of the underlying pattern formed is dependent on a number of factors. Optimization and modification of any of these factors can ultimately improve the dimensions of the printed features, as well as their resolution. For example, resolution and uniformity of the printed pattern features is dependent on the underlying quality and resolution of the features of the stamp, and ultimately the master from which the stamp is cast. Irregular features or edges on the surface features of the master (produced from ragged edges on the exposed photoresist) can limit the feature resolution that will be resolvable on the stamp and on the final print.
In addition, the rigidity of the stamp structure can affect the resulting pattern formed. For example, if the features of a polymer material forming the stamp are too soft, the stamp can compress too greatly upon contacting the substrate, which leads to deformation and undesired spreading of the applied ink material.
The solvent compatibility of the stamp is another factor that can affect spatial resolution. For example, organic solvents present on in ink may tend to expand the stamp, thereby also undesirably expanding the resulting stamped features.
As another example, the underlying particle size of the conductive substances in the ink limit the minimum spatial resolution achievable for a pattern. That is, the printed features can be no smaller than the individual particles present in the ink.
Contact Printing of Carbon and Gold Electrodes
As a non-limiting exemplary procedure, the gold and carbon electrodes were formed in one experiment as follows. A PDMS stamp was prepared using a silicone elastomer curing agent and base (Sylgard 184 silicone elastomer kit, available from Dow Corning Corporation) which were mixed together in a 1:10 ratio and poured evenly over patterned and surface treated silicon wafer masters (Premitec). The resulting PDMS material was then baked at 65 degrees F. for two hours. The cured PDMS material was removed from the masters and cut into individual stamps. A PDMS stamp was prepared and cut as described above. The stamp was treated with oxygen plasma (for about 30 seconds) prior to stamping. The stamps were then coated with a thin layer of either gold or carbon polymer paste (C2041206D2, C2000802D2, Gwent Electronic Materials Ltd.). A drop of hexane was used to reduce the viscosity of the paste materials to a desired level. Stamps were inked and then placed into contact with a polyester film substrate (Hostaphan W54B, available from Mitsubishi) for approximately 15 seconds. The PDMS stamp was then carefully removed to reveal the electrode features printed with ink. The printed electrode features were then baked at 65 degrees F. for approximately 30 minutes to form the final electrodes.
Contact Printing of Chemistry Layer
As a non-limiting exemplary procedure, the chemistry layer was formed in one experiment as follows. A PDMS stamp was prepared and cut as described above. The stamp was treated with oxygen plasma (for about 30 seconds) prior to stamping. An ink comprising the chemistry solution was applied to the stamp with a cotton swab and allowed to dry. An exemplary chemistry solution comprised: 0.05% Silwet, 0.05% Triton-x, 0.25% methocel F4M, 100 mM potassium phosphate buffer, 5% sucrose, 190 mM ruthenium hexaamine chloride, and 5000 u/ml glucose dehydrogenase, pH 7.25. The inked stamp was then applied to a 30 nm Au layer on a polyethylene terephthalate (PET) substrate for approximately 20 seconds and the stamp removed. The chemistry solution was then allowed to dry, thereby forming the final printed features.
One having ordinary skill in the art will appreciate that the present invention is adaptable for testing any analyte. Such possible analytes include, but are not limited to, glucose, cholesterol, lactate, blood urea nitrogen, TSH, T4, pharmaceuticals, and nontherapeutic drugs. It should be noted that the microcontact printing and microtransfer molding procedures described in this application can be used solely for the preparation of either the conductive electrode layer or the chemistry layer. Alternatively, the microcontact printing and microtransfer molding procedures described above can also be used in combination in order to provide test media with both a conductive layer and a chemistry layer thereon.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
