Patent Application
20130284609
1. Field of the Invention
The present invention relates, in general, to medical devices and, in particular, to enzymatic electrochemical-based sensors.
2. Description of 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, ketone bodies, lactate, cholesterol, lipoproteins, triglycerides, acetaminophen and/or HbA1c concentrations in a sample of a bodily fluid such as urine, blood, plasma or interstitial fluid. Such determinations can be achieved using sensors, based on, for example, visual, photometric or electrochemical techniques. Conventional electrochemical-based analytical test strips are described in, for example, U.S. Pat. Nos. 5,708,247, and 6,284,125, each of which is hereby incorporated in full by reference.
The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate presently preferred embodiments of the invention, and, together with the general description given above and the detailed description given below, serve to explain features of the invention, in which:
The following detailed description should be read with reference to the drawings, in which like elements in different drawings are identically numbered. The drawings, which are not necessarily to scale, depict exemplary embodiments for the purpose of explanation only and are not intended to limit the scope of the invention. The detailed description illustrates by way of example, not by way of limitation, the principles of the invention. This description will clearly enable one skilled in the art to make and use the invention, and describes several embodiments, adaptations, variations, alternatives and uses of the invention, including what is presently believed to be the best mode of carrying out the invention.
As used herein, the terms “about” or “approximately” for any numerical values or ranges indicate a suitable dimensional tolerance that allows the part or collection of components to function for its intended purpose as described herein.
In general, nicotinamide adenine dinucleotide (NAD) polymeric coenzymes that are employed in enzymatic electrochemical-based sensors according to embodiments of the present invention include NAD moieties covalently bound as pendent groups to a polymer backbone (e.g. polyacrylamide). Such NAD polymeric coenzymes are beneficial in that they can be readily incorporated as a redox coenzyme into enzymatic electrochemical-based biosensors (such as analytical test strips) according to embodiments of the present invention by employing, for example, techniques to immobilize the polymeric NAD coenzyme and a polymeric electron transfer agent within an analyte detection matrix. Such enzymatic electrochemical-based biosensors include continuous biosensors and beneficially combine NAD polymeric coenzymes with polymeric electron transfer agents (see, for example, U.S. Patent Publication 2006/0069211, which is hereby incorporated in full be reference).
Once appraised of the present disclosure, one skilled in the art will recognize that although
The synthesis depicted in those figures are described below with reference to the figures. The synthesis of N6-carboxymethyl-NAD+ was accomplished using the following 3 step process:
To a 5 ml Biotage microwave reaction tube, added NAD+ (1.0 g, 1.51 mmol) pre-dissolved in 0.1M pH 7.0 sodium phosphate buffer (3.5 ml) and iodoacetic acid (1.5 g, 8.06 mmol, 5.34 eq). The pH was adjusted to 7.0 by using 5.0M NaOH aqueous solution. The reaction vessel was sealed and the mixture was heated to 50° C. for 10 minutes using microwave irradiation.
The resultant pink solution (c.a. 5 ml) was acidified to pH3.0 using 5M HCl aqueous solution before being poured into a pre-cooled (−5° C.) mixture of acetone/IMS (1:1) (25 ml). The resulting precipitate was filtered, washed first with IMS (5 ml), then dry diethyl ether (15 ml) before air drying under dry nitrogen for 10 minutes. Further drying overnight in a desiccator over fused CaCl2 afforded N1-carboxymethyl-NAD+ as a pink amorphous solid (1.62 g) (crude).
The above prepared crude N1-carboxymethyl-NAD+ (9.1 g, c.a. 10.57 mmol) was dissolved in 1.3% w/v NaHCO3 in aqueous solution (450 ml) and the solution deoxygenated by sparging with nitrogen for 10 minutes. Sodium dithionite (3.5 g, 20.1 mmol) added in one portion and the mixture stirred at ambient temperature to effect reduction of the nicotinamide moiety (i.e. conversion of oxidized state NAD+ to reduced state NADH). After 1.0 hour, the solution color had changed from pink to yellow. The solution was then sparged with air for 10 minutes to destroy any excess dithionite and the pH brought to 11.0 by using 5M NaOH aqueous solution. The mixture was heated at 70° C. for 90 minutes, to promote Dimroth rearrangement to N6-carboxymethyl-NADH, before cooling to 25° C. Thin-layer chromatography (silica gel, isobutryic acid/water/32% NH4OH (aq), 66/33/1.5 by volume) showed no evidence for the presence of N1-carboxymethyl-NADH at this stage.
The reaction mixture containing N6-carboxymethyl-NADH was treated with 3M TRIS buffer (pH7.0) (17.5 ml) and the pH adjusted to 7.5 using 5M HCl aqueous solution (c.a. 4.9 ml). Acetaldehyde (3.5 ml, 62.6 mmol) was added, immediately followed by yeast alcohol dehydrogenase (from saccharomyces cerevisiae) (˜300 U/mg) (10.5 mg, c.a. 3150 U of enzyme) before allowing to stir at ambient temperature to deoxidize the nicotinamide moiety (i.e. conversion of NADH to NAD+). After 18 hours, the reaction mixture (c.a. 485 ml) was concentrated in vacuo (30° C./10-15 bar) to approximately ⅓ volume and poured into a pre-cooled (−5° C.) mixture of acetone/IMS (1:1) (1800 ml). The fine slurry was left to age for 18 hours at 3° C. The resulting precipitate was collected by centrifugation and washed on a glass sinter with IMS (40 ml) then dry diethyl ether (120 ml) before air-drying under dry nitrogen for 10 minutes. Further drying overnight in a desiccator over fused CaCl2 afforded N6-carboxymethyl-NAD+ as a tan colored hygroscopic solid (3.99 g) (crude).
The crude N6-carboxymethyl-NAD+ (1.0 g) was taken up in water (20 ml) and passed through a Sephadex G10 gel filtration column (2×10 cm, 20 ml). All eluted fractions containing UV active material were combined (60 ml total volume) and added to a column of Dowex 1-X2 ion exchange resin (Cl−; 4×50 cm, 200 ml) which had been pre-equilibrated with water. A linear gradient of 0-50 mM LiCl (buffered to pH 3.0), at 10 ml/min over 65 min, was applied using “Presearch Combiflash Companion” chromatography equipment. The fractions eluted between 25-35 mM were combined (c.a. 100 ml), neutralized to pH 7.0 with 5M LiOH and evaporated to approximately ⅓ volume and poured into a pre-cooled (−5° C.) mixture of acetone/IMS (1:1) (300 ml). The fine slurry was left to age for 18 hours at 3° C. The resulting precipitate was collected by centrifugation and washed on a glass sinter with IMS (30 ml) then dry diethyl ether (50 ml) before air-drying under dry nitrogen for 10 minutes. Further drying overnight in a desiccator over fused CaCl2 afforded purified N6-carboxymethyl-NAD+ as a cream colored hygroscopic solid (0.307 g). Extrapolated total yield=1.225 g, 14%.
NAD monomer was prepared as follows and as depicted in
The pH was monitored and maintained at between pH7.0 and pH7.5. The reaction mixture was then diluted with distilled water (30 ml) and passed through a pre-prepared bed of Sephadex gel (15 g equilibrated with 150 ml distilled water) and eluted with distilled water (80 ml). The eluate (approximately 110 ml) was loaded onto a pre-prepared column (Dowex 1-X2 (Cl− form)) which was set up to run through an automated chromatography system (Presearch Combiflash Companion) and was equilibrated with 5 column volumes of water at 40 ml/minute. The flow rate was reduced to 20 ml/minute prior to loading the crude compound (via the pump).
A linear gradient of 0-50 mM of aqueous lithium chloride (buffered at pH3) over 60 minutes was applied (i.e. binary pump configuration; A: water, B: 50 mM LiCl (pH 3), 100:0 A:B to 0:100 A:B over 60 min). The eluted fractions were analysed by TLC and the desired fractions (eluted between 40-50 mM, 50 ml) concentrated to half volume (25 ml) and added dropwise to a stirred solution of ethanol (375 ml, 15 volumes) at 2° C. The resulting precipitate was aggregated by centrifugation (Genevac EZ2+, 3600 rpm, 3 minutes) collected by filtration and washed with cold ethanol (50 ml). The precipitate was dried at ambient temperature under vacuum, in a desiccator over calcium chloride and subsequently stored in the freezer.
The synthesis of copolymer of the NAD monomer and acrylamide (i.e., an NAD polymeric coenzyme according to an embodiment of the present invention) was conducted as follows and depicted in
The measurement of
5B, and 5C are simplified cross-sectional end and, perspective and exploded perspective views of an enzymatic electrochemical-based sensor 100 (i.e., an electrochemical based analytical test strip) according to an embodiment of the present invention.
Referring to
Electrically-insulating substrate layer 102 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.
Patterned insulation layer 104 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 conductor layer 106 can be formed of any suitable electrically conductive material including, but not limited to, electrically conductive carbon ink materials.
Enzymatic reagent layer 108 can include, in addition to the aforementioned NAD polymeric coenzyme and polymeric electron transfer agent, 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 108 can include NAD-dependent 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.
The polymeric electron transfer agent can be any suitable polymeric electron transfer agent including, for example, a high molecular weight redox polymer comprising a hydrophilic polymer with ionic portions and a plurality of attached redox mediators (for example, ferrocene). The molecular weight of such an ionic hydrophilic high molecular weight polymer can beneficially be, for example, greater than 16 Kg/mol. Such ionic hydrophilic high molecular weight polymers are described in U.S. Patent Publication 2006/0069211, which is hereby incorporated in full be reference.
Electrochemical-based biosensors according to embodiments of the present invention are particularly beneficial in that, for example, the inclusion of both a nicotinamide adenine dinucleotide (NAD) polymeric coenzyme and a polymeric electron transfer agent enables the novel use of dehydrogenase enzymes in a continuous biosensor by, for example, employing them both in an immobilized configuration.
Electrochemical-based analytical test strip 100 can be manufactured, for example, by the sequential aligned formation of patterned insulation conductor layer 106, patterned insulation layer 104 and enzymatic reagent layer 108. 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.
Method 200 further includes determining the analyte in the bodily fluid sample based on an electronic signal produced by the enzymatic electrochemical-based biosensor (see step 220 of
Once apprised of the present disclosure, one skilled in the art will recognize that method 600 can be readily modified to incorporate any of the techniques, benefits and characteristics of enzymatic electrochemical-based biosensors according to embodiments of the present invention and described herein.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. 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 compositions, devices and methods within the scope of these claims and their equivalents be covered thereby.
