Patent Application
20160017399
1. Field of the Invention
The present invention relates, in general, to medical devices and, in particular, to electrochemical-based analytical test strips and enzymatic reagents for use therein.
2. Description of Related Art
The determination (e.g., detection and/or concentration measurement) of an analyte in, or a characteristic of, a fluid sample is of particular interest in the medical field. For example, it can be desirable to determine glucose, ketone bodies, cholesterol, lipoproteins, triglycerides, acetaminophen, hematocrit 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 analytical test strips, 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 tolerance that allows a component or collection of components to function for its intended purpose as described herein.
An electrochemical-based analytical test strip for the determination of an analyte (such as glucose) in a bodily fluid sample according to embodiments of the present invention include an electrically insulating base layer, an electrically conductive layer disposed on the electrically insulating base layer and including at least one electrode, an enzymatic reagent layer disposed on the at least one electrode, a patterned spacer layer, and a top layer. Moreover, the enzymatic reagent layer includes at least one naphthoquinone-based mediator. Such a naphthoquinone-based mediator can be, for example, 1,2-naphthalenedione-4-(3-mercapto-1-propane sulfonic acid) and 1,2-naphthalenedione-4-(3-mercaptopropionic acid), and FAD-GDH enzyme.
Electrochemical-based analytical test strips according to embodiments of the present invention are beneficial in that the combination of at least one naphthoquinone-based mediator (e.g., at least one of 1,2-naphthalenedione-4-(3-mercapto-1-propane sulfonic acid) and 1,2-naphthalenedione-4-(3-mercaptopropionic acid)), and FAD-GDH enzyme creates an enzymatic reagent layer that is biochemically efficient (for example, the mediators are 2 electron acceptors, results in an enzymatic reaction that does not proceed via a free-radical route, and has a first order rate constant is, for some naphthoquinone-based mediators, greater than that of the conventional FAD-GDH mediator potassium ferricyanide) and that is not susceptible to interference from uric acid, acetaminophen, glutathione and ascorbic acid.
Enzymatic reagents for use in electrochemical-based analytical test strips according to the present invention comprise at least one naphthoquinone-based mediator such as, for example, at least one of (i) 1,2-naphthalenedione-4-(3-mercapto-1-propoane sulfonic acid) and (ii) 1,2-naphthalenedione-4-(3-mercaptopropionic acid); and flavin adenine dinucleotide dependant glucose dehydrogenase enzyme, herein abbreviated to FAD-GDH enzyme. Such enzymatic reagents are beneficial in that they creates an enzymatic reagent layer that is biochemically efficient (i.e., the mediators are 2 electron acceptors and the enzymatic reaction does not proceed via a free-radical route) and not susceptible to interference from uric acid, acetaminophen, glutathione and ascorbic acid. Moreover, the enzymatic reagents can be applied during manufacturing of electrochemical-based analytical test strip using conventional techniques such as ink jet printing and screen printing.
Referring to
In the embodiment of
Electrically-insulating base layer 102 can be any suitable electrically-insulating base layer known to one skilled in the art including, for example, a nylon base layer, a polycarbonate base layer, a polyimide base layer, a polyvinyl chloride base layer, a polyethylene base layer, a polypropylene base layer, a glycolated polyester (PETG) base layer, or a polyester base layer. The electrically-insulating base layer 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.
Electrically-insulating base layer 102 provides structure to electrochemical-based analytical test strip 100 for ease of handling and also serves as a base for the application (e.g., printing or deposition) of subsequent layers (e.g., a patterned electrically conductor layer and an enzymatic reagent formed by ink jet printing or screen printing of an enzymatic reagent according to the present invention and described herein).
Patterned electrically conductive layer 104 is disposed on the electrically-insulating base layer 102 and includes a first electrode 104a, a second electrode 104b and a third electrode 104c. First electrode 104a, second electrode 104b and third electrode 104c can be, for example, configured as a counter/reference electrode, a first working electrode and a second working electrode, respectively. Therefore, the second and third electrodes are also referred to herein as working electrodes 104b and 104c and the first electrode as counter electrode 104a. Although, for the purpose of explanation only, electrochemical-based analytical test strip 100 is depicted as including a total of three electrodes, embodiments of electrochemical-based analytical test strips, including embodiments of the present invention, can include any suitable number of electrodes.
Patterned electrically conductive layer 104, including first electrode 104a, second electrode 104b and third electrode 104c, of electrochemical-based analytical test strip 100 can be formed of any suitable conductive material including, for example, electrically conducting carbon-based materials including carbon inks. It should be noted that patterned electrically conductive layers employed in electrochemical-based analytical test strips according to embodiments of the present invention can take any suitable shape and be formed of any suitable materials including, for example, metal materials and conductive carbon materials.
Referring in particular to
Enzymatic reagent layer 108 is disposed on at least a portion of patterned electrically conductor layer 104 (see
where R is any suitable organic substituent.
Particularly beneficial examples of naphthoquinone-based mediators suitable for use in embodiments of the present invention are 1,2-naphthalenedione-4-(3-mercapto-1-propane sulfonic acid) [also referred to herein as Compound A] and 1,2-naphthalenedione-4-(3-mercaptopropionic acid) [also referred to herein as Compound B]. However, once apprised of the present invention, one of skill in the art could employ routine experimentation to select suitable organic substituents (i.e., suitable “R” groups) to create other naphthoquinone-based mediators that have beneficial reaction kinetics, suitable aqueous solubility and that are not susceptible to interference effects. In this respect, a suitable R group may, for example, be sufficiently hydrophilic to impart a suitable aqueous solubility to the mediator.
Compound A has the molecular formula C13H12S2O5, a molecular weight of 312.36 and the following structure:
Compound A is an orange amorphous solid, soluble in base and neutral phosphate buffer at room temperature. Compound A has the following beneficial electrochemical characteristics:
Compound A was synthesized as follows. 1,2-Naphthoquinone-4-sulfonic acid sodium salt (10 mmol) was added to distilled water (100 ml) and stirred until a clear solution developed. 3-Mercapto-1-propanesulfonic acid monosodium salt (10 mmol) was added at once creating a dark brown solution. The solution stood for 12 hours after which the clear brown solution was evaporated to dryness to yield a dark yellow solid. The dark yellow solid was washed with chloroform and dried under vacuum to yield 1,2-naphthalenedione-4-(3-mercapto-1-propane sulfonic acid) (1.48 g, 47%) as an orange solid. Mass spectra analysis confirmed the composition. However, once apprised of the present disclosure, one skilled in the art may develop other methods for synthesizing Compound A.
Compound B has the molecular formula C13H10S2O4, a molecular weight of 262.28 and the following structure:
Compound B is an orange amorphous solid, soluble in base and neutral phosphate buffer at room temperature. Compound A has the following beneficial electrochemical characteristics:
Compound B was synthesized as follows. 1,2-Naphthoquinone (10 mmol) was added to methanol (50 ml) and stirred for 5 minutes in ice. 3-Mercaptopropionic acid (10 mmol) was added and within 5 minutes the solution went clear and was evaporated to dryness. The dark brown solid was purified in silica gel using ethyl acetate as eluent to yield 1,2-naphthalenedione-4-(3-mercaptopropionic acid) (1.19 g, 51%). Mass spectra analysis confirmed the composition. However, once apprised of the present disclosure, one skilled in the art may develop other methods for synthesizing Compound B.
An exemplary, but non-limiting, example of an enzymatic reagent according to the present invention that includes Compound B includes the following components:
Another exemplary, but non-limiting example of an enzymatic reagent according to the present invention includes Compound A as follows:
Referring in particular to
The data of
Referring in particular to
Once apprised of the present disclosure, one skilled in the art will recognize that a variety of suitable enzymatic reagents containing Compound A and/or Compound B and FAD-GDH can be formulated. Such suitable enzymatic reagents can include, for example, tri-sodium citrate, citric acid, polyvinyl alcohol, hydroxyl ethyl cellulose, antifoam, fumed silica (either with or without a hydrophobic surface modification), PVPVA, and water. Further details regarding reagent layers in general, and electrochemical-based analytical test strips in general, are in U.S. Pat. Nos. 6,241,862 and 6,733,655, the contents of which are hereby fully incorporated by reference.
Referring to
Patterned spacer layer 110 can be formed, for example, from a screen-printable pressure sensitive adhesive commercially available from Apollo Adhesives, Tamworth, Staffordshire, UK. In the embodiment of FIG. and 2, patterned spacer layer 110 defines outer walls of the sample-receiving chamber 118. Patterned spacer layer 110 can have a thickness of, for example, approximately 110 microns, be electrically nonconductive, and be formed of a polyester material with top and bottom side acrylic-based pressure sensitive adhesive.
Top layer 112 can be, for example, a clear film with hydrophilic properties that promote wetting and filling of electrochemical-based analytical test strip 100 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. and Coveme (San Lazzaro di Savena, Italy). Top layer 112 can be, for example, a polyester film coated with a surfactant that provides a hydrophilic contact angle <10 degrees. Top layer 112 can also be a polypropylene film coated with a surfactant or other surface treatment. In such a circumstance, the surfactant coating serves as hydrophilic sub-layer 114. Top layer 112 can have a thickness, for example, of approximately 100 μm.
Electrochemical-based analytical test strip 100 can be manufactured, for example, by the sequential aligned formation of patterned electrically conductive layer 104, patterned insulating layer 106, enzymatic reagent layer 108, patterned spacer layer 110, and top layer 112. Any suitable techniques known to one skilled in the art can be used to accomplish such sequential aligned formation, including, for example, screen printing, ink-jet printing, photolithography, photogravure, chemical vapour deposition and tape lamination techniques. However, enzymatic reagents according to embodiments of the present invention are particularly beneficial in that they can be formulated as aqueous compositions suitable for relatively low-cost and otherwise conventional ink jet and screen printing techniques. Such enzymatic reagents can, for example, be employed to create enzymatic reagent layers with a linear response between electrochemically-generated current and glucose concentration in a whole blood sample up to at least a glucose concentration of 700 mg/dL.
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 devices and compositions of matter within the scope of these claims and their equivalents be covered thereby.
