1. Field of the Invention
This invention relates, in general, to analytical devices and, in particular, to electrochemical-based analytical test strips and associated methods.
2. Description of the Related Art
The determination (e.g., detection and/or concentration measurement) of an analyte in a fluid sample is of particular interest in the medical field. For example, it can be desirable to determine glucose, cholesterol, acetaminophen and/or HbA1c concentrations in a sample of a bodily fluid such as urine, blood or interstitial fluid. Such determinations can be achieved using analytical test strips, based on, for example, photometric or electrochemical techniques, along with an associated meter. For example, the OneTouch® Ultra® whole blood testing kit, available from LifeScan, Inc., Milpitas, USA, employs an electrochemical-based analytical test strip for the determination of blood glucose concentration in a whole blood sample.
Typical electrochemical-based analytical test strips employ a plurality of electrodes (e.g., a working electrode and a reference electrode) and an enzymatic reagent to facilitate an electrochemical reaction with an analyte of interest and, thereby, determine the concentration of the analyte. For example, an electrochemical-based analytical test strip for the determination of glucose concentration in a blood sample can employ an enzymatic reagent that includes the enzyme glucose oxidase and the mediator ferricyanide. Further details of conventional electrochemical-based analytical test strips are included in U.S. Pat. No. 5,708,247, which is hereby incorporated in full by reference.
A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
An embodiment of an electrochemical-based analytical test strip according to the present invention includes an electrically-insulating substrate and at least one metal electrode (e.g., a gold metal electrode) disposed on a surface of the electrically-insulating substrate. In addition, the metal electrode has an upper surface with hydrophilicity-enhancing chemical moieties thereon and an enzymatic reagent layer disposed on the upper surface. Details, characteristics and benefits of such an electrochemical-based analytical test strip are described with respect to the further embodiments discussed below.
Electrically-insulating substrate 12 can be any suitable electrically-insulating substrate known to one skilled in the art including, for example, a nylon substrate, polycarbonate substrate, a polyimide substrate, a polyvinyl chloride substrate, a polyethylene substrate, a polypropylene substrate, a glycolated polyester (PETG) substrate, or a polyester substrate. The electrically-insulating substrate can have any suitable dimensions including, for example, a width dimension of about 5 mm, a length dimension of about 27 mm and a thickness dimension of about 0.5 mm.
Insulation layer 16 can be formed, for example, from a screen printable insulating ink. Such a screen printable insulating ink is commercially available from Ercon of Wareham, Mass. U.S.A. under the name “Insulayer.” Patterned adhesive layer 20 can be formed, for example, from a screen-printable pressure sensitive adhesive commercially available from Apollo Adhesives, Tamworth, Staffordshire, UK.
Hydrophilic layer 22 can be, for example, a clear film with hydrophilic properties that promote wetting and filling of electrochemical-based analytical test strip 10 by a fluid sample (e.g., a whole blood sample). Such clear films are commercially available from, for example, 3M of Minneapolis, Minn. U.S.A. Top film 24 can be, for example, a clear film overprinted by black decorative ink. A suitable clear film is commercially available from Tape Specialities, Tring, Hertfordshire, UK.
Enzymatic reagent layer 18 can include any suitable enzymatic reagents, with the selection of enzymatic reagents being dependent on the analyte to be determined. For example, if glucose is to be determined in a blood sample, enzymatic reagent layer 18 can include oxidase or glucose dehydrogenase along with other components necessary for functional operation. Further details regarding enzymatic reagent layers, and electrochemical-based analytical test strips in general, are in U.S. Pat. No. 6,241,862, the contents of which are hereby fully incorporated by reference.
Electrochemical-based analytical test strip 10 can be manufactured, for example, by the sequential aligned formation of patterned conductor layer 14, insulation layer 16 (with electrode exposure window 17 extending therethrough), enzymatic reagent layer 18, patterned adhesive layer 20, hydrophilic layer 22 and top film 24 onto electrically-insulating substrate 12. Any suitable techniques known to one skilled in the art can be used to accomplish such sequential aligned formation, including, for example, screen printing, photolithography, photogravure, chemical vapour deposition and tape lamination techniques.
Counter electrode 26, first working electrode 28 and second working electrode 30 can be formed of any suitable electrode metal including, for example, gold, palladium, platinum, indium and titanium-palladium alloys. The formation of such metal electrodes typically results in a metal electrode with a smooth, albeit hydrophobic, surface.
Counter electrode exposed portion 26′, first working electrode exposed portion 28′ and second working electrode exposed portion 30′ can have any suitable dimensions. For example, counter electrode exposed portion 26′ can have a width dimension of about 0.72 mm and a length dimension of about 1.6 mm, while first working electrode exposed portion 28′ and second working electrode exposed portion 30′ can each have a width dimension of about 0.72 mm and a length dimension of about 0.8 mm.
Following formation of insulation layer 16, patterned conductive layer 14, and the disposition of hydrophilicity-enhancing moieties on the counter electrode exposed portion 26′, the first working electrode exposed portion 28′ and the second working electrode exposed portion 30′, enzymatic reagent layer 18 is applied over counter electrode exposed portion 26′, first working electrode exposed portion 28′ and second working exposed portion 30′. Details regarding the use of such electrodes, electrode exposed portions and enzymatic reagent layers for the determination of the concentrations of analytes in a fluid sample, albeit without the hydrophilicity-enhancing moieties described in this disclosure, are in U.S. Pat. No. 6,733,655, which is hereby fully incorporated by reference.
During use of electrochemical-based analytical test strip 10 to determine an analyte concentration in a fluid sample (e.g., blood glucose concentration in a whole blood sample), counter electrode 26, first working electrode 28 and second working electrode 30 are employed to monitor an electrochemical reaction induced current of interest. The magnitude of such a current can then be correlated with the amount of analyte present in the fluid sample under investigation.
The current measured by a working electrode is governed by the following simplified equation:
i=nFAJ Eq. 1
where:
and
Based on equation 1 above, a reliable and accurate determination (e.g., quantification) of an analyte concentration in a fluid sample requires knowledge of the area of the working electrode at which the reaction occurs. It has been determined that the sensing area of an electrode in an electrochemical-based analytical test strip is dependent on the uniformity and adherence of an enzymatic reagent layer to the electrode throughout manufacturing and during use. In addition, it has been determined that employing a metal electrode with hydrophilicity-enhancing moieties thereon improves the uniformity and adherence of enzymatic reagent layers and, thus, the reproducibility and accuracy of results obtained with electrochemical-based analytical test strips that employ such metal electrodes.
The reaction that occurs between a gold metal electrode surface and the thiol (—SH) group of hydrophilicity-enhancing composition 44 is described by a general reaction sequence of the form:
X—R—SH+Au→X—R—S−Au++½H2 Seq. 1
where:
and
In sequence 1 above, R can be beneficially limited to the range of C1 to C5 to provide a hydrophilicity-enhancing composition that is soluble, yet avoids the formation of self-assembled monolayers on the gold metal electrode surface. Self-assembled monolayers of hydrophilicity-enhancing moieties need not necessarily be avoided, but their formation is difficult to control, often slow and can require an electrode surface that is “atomically” clean. The manufacturing of such self-assembled monolayers is, therefore, more difficult than the non-self-assembled disposition of hydrophilicity-enhancing moieties that occurs spontaneously by dip coating an electrode surface with a MENSA solution as described elsewhere in this disclosure.
Furthermore, the thiol group (also referred to as a “tail” group) enables a conjugation between the gold metal electrode surface and the hydrophilicity-enhancing composition to occur. In addition, the polar, positively charged or negatively charged side group “X” (also referred to as a “head” group) provides for a hydrophilic interaction with an enzymatic reagent layer, thereby improving the uniformity and adherence of the enzymatic reagent layer to the metal electrode upper surface. Examples of suitable head groups include, but are not limited to, the following groups: NH2 (amine) group, COOH (carboxy) group, and SO2OH (sulphonate) group.
As noted above, the length of the “R” group (also referred to as a “spacer chain”) is a factor in determining whether or not the hydrophilicity-enhancing moieties are disposed on the electrode surface as a self-assembled monolayer.
Although
Enzymatic reagents are formulated such that they readily mix with common fluid samples (such as a whole blood or other bodily fluid sample) and, therefore, typically consist of components that are readily soluble in aqueous solutions. It has been determined that such components have an affinity for hydrophilic or at least amphiphilic surfaces.
A variety of metal electrode surfaces are naturally hydrophobic. In other words, such metal electrode surfaces tend to repel water, aqueous solutions, and solutions with significant hydrophilic component content (such as enzymatic reagents). However, it has been determined that such metal electrode surfaces can be rendered more hydrophilic (i.e., be hydrophilically-enhanced) by treating the metal electrode surfaces with a hydrophilicity-enhancing composition that disposes hydrophilicity-enhancing moieties on the metal electrode surface.
Examples of hydrophilicity-enhancing compositions are compositions that contain 2-mercaptoethanesulphonic acid (MESNA), 3-mercaptopropanesulphonic acid, 2,3-dimercaptopropanesulphonic acid and its homologues, bis-(2-sulphoethyl)disulphide, bis-(3-sulphopropyl)disulphide and homologues; mercaptosuccinic acid, cysteine, cysteamine, and cystine. When such hydrophilicity-enhancing compositions include a compound with a sulphonate moiety (e.g., MESNA) or a compound with an amino moiety (e.g., cysteamine), the adhesion of a enzymatic reagent layer to the upper surface of a metal electrode is particularly enhanced.
As depicted in
Table 1 below lists the water contact angle of gold substrate surfaces that had received various treatments. For treatments 1-15 of Table 1, cleaned gold substrates were exposed to MESNA solutions as indicated in the Table. Treatment 16 consisted of cleaning a gold substrate surface but no exposure to MESNA and treatment 17 involved no cleaning or exposure to MESNA. The data of Table 1 indicate that a significant reduction in water contact angle and, thus, enhancement in hydrophilicity and enzymatic reagent layer adhesion and uniformity, can be achieved with an exposure to MESNA for a time period as short as 1 minute. The data of Table 1, therefore, indicate that the manufacturing of metal electrodes with hydrophilicity-enhancing moieties on their upper surfaces could be accomplished using continuous web-based processes (such as the processes described in WO 01/73109, which is hereby incorporated in full by reference) that have been modified to include a metal electrode upper surface treatment module.
To demonstrate characteristics and benefits of electrochemical-based analytical test strips according to embodiments of the present invention, a comparison between an electrochemical-based analytical test strip with gold electrodes in the absence of hydrophilicity-enhancing moieties (i.e., a comparison electrochemical-based analytical test strip) and an electrochemical-based analytical test strip with gold metal electrodes according to an exemplary embodiment of the present invention was undertaken.
As is evident from
As noted above with respect to
Hydrophilicity-enhancing moieties were disposed on counter electrode exposed portion 206, first working electrode exposed portion 208, second working electrode exposed portion 210 by submerging them in a 4 g/L aqueous solution of MESNA for 2 minutes followed by a water rinse. This exposure occurred prior to the application of enzymatic reagent layer 212.
A comparison of
Subsequently, the upper surface of each of the at least one metal electrodes is treated with a hydrophilicity-enhancing composition to form a treated upper surface of the metal electrode with hydrophilicity-enhancing chemical moieties thereon, as set forth in step 420. The treatment can be accomplished using, for example, any suitable treatment technique including dip coating techniques, spray coating techniques, and inkjet coating techniques. Any suitable hydrophilicity-enhancing composition can be employed including those described above with respect to electrochemical-based analytical test strips according to the present invention.
The following two examples are illustrate, in a non-limiting manner, treatment technique sequences that can be employed in treatment step 420 of process 400:
(a) clean upper surface of the metal electrode(s) by placing them in a 2% v/v aqueous solution of degreasant (e.g. Micro-90®) for 2 minutes at room temperature.
(b) Rinse the metal electrodes with water to remove excess degreasant.
(c) Dip the metal electrodes into a 4 g/L aqueous solution of MESNA) for two minutes.
(d) Rinse the metal electrodes with water to remove excess aqueous solution.
(e) Dry the metal electrodes in a clean environment.
(a) Place the metal electrodes into an ultrasonic bath with an aqueous solution containing 2% v/v degreasant (e.g. Micro-90®) and 4 g/L MESNA).
(b) Sonicate the two minutes in an ultrasonic bath at a temperature of 50° C.
(c) Rinse the metal electrodes with water to remove excess degreasant and MESNA.
(d) Dry the metal electrodes.
Thereafter, at step 430 of process 400, an enzymatic reagent layer is applied to the treated upper surface of the at least one metal electrode.
It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that structures and methods within the scope of these claims and their equivalents be covered thereby.