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Publication Number
US-10760111-B2
Publication Date
2020-09-01
Expiration Date
Abstract
A dry reagent composition that includes an active redox enzyme that oxidizes an analyte as a specific substrate to produce an inactive reduced form of the enzyme; and a salt of ferricyanide provides improved performance in electrochemical test strips such as those used for detection of glucose. The salt of ferricyanide consists of ferricyanide and positively-charged counter ions, and the positively charged counter ions are selected such that the salt of ferricyanide is soluble in water, and such that the salt of ferricyanide or the crystalline phase of the salt of ferricyanide has a solubility in water and/or a lower E0eff at a concentration of 100 mM than potassium ferricyanide. For example, the salt of ferricyanide may be tetramethylammonium ferricyanide.
Core Innovation
The invention provides a dry reagent composition for analyte detection that includes an active redox enzyme that oxidizes an analyte as a specific substrate to produce an inactive reduced form of the enzyme. The dry reagent composition further includes tris-tetramethylammonium ferricyanide, forming an electrochemical redox system based on the ferricyanide/ferrocyanide redox couple. The dry reagent is configured to dissolve when a liquid sample is applied in test strip architectures, supporting electrochemical detection.
A key aspect is the use of tris-tetramethylammonium ferricyanide rather than potassium ferricyanide, with described attention to water solubility and to reducing or controlling the effective ferri/ferro electrode potential (E0eff) relative to potassium ferricyanide. The described behavior includes ferricyanide-to-ferrocyanide conversion that can cause baseline drift, and the invention addresses this by improving baseline signal stability while maintaining enzyme activity retention during storage.
The dry reagent compositions also optionally include a non-ferricyanide electron transfer mediator in an oxidized form having an electrochemical potential in aqueous medium sufficient to oxidize the inactive reduced form of the enzyme to regenerate active redox enzyme. The mediator is exemplified by osmium coordination complexes, including Os(dmbpy)2PicCl. Zwitterion buffering components with positively charged buffer counter ions and buffer conjugate bases are described, with essentially all positively-charged buffer counter ions being tetramethylammonium, and the overall dry formulation is presented as shelf-stable.
A method is described for forming a dry shelf stable electrochemical test reagent by selecting an oxidoreductase enzyme that oxidizes/reduces the analyte as a specific substrate and combining the selected enzyme with tris-tetramethylammonium ferricyanide, with the selected enzyme identified as glucose oxidase. The background problem being solved is baseline signal drift arising from ferricyanide-to-ferrocyanide conversion during storage, which undermines electrochemical test performance and comparability over time.
Claims Coverage
The independent claims cover dry reagent compositions and a method of forming a dry shelf-stable electrochemical test reagent. Across the independent claims, the inventive features center on combining an active redox enzyme with tris-tetramethylammonium ferricyanide, optionally adding an oxidized non-ferricyanide electron transfer mediator such as Os(dmbpy)2PicCl, and using zwitterion buffering with essentially all tetramethylammonium as the positively-charged buffer counter ion.
Active redox enzyme oxidizing analyte to inactive reduced enzyme form with tris-tetramethylammonium ferricyanide
A dry reagent composition comprising an active redox enzyme that oxidizes an analyte as a specific substrate to produce an inactive reduced form of the enzyme, and tris-tetramethylammonium ferricyanide.
Non-ferricyanide oxidized electron transfer mediator for regenerating active redox enzyme
The dry reagent further includes an electron transfer mediator in oxidized form that is not a salt of ferricyanide, the oxidized form having an electrochemical potential in aqueous medium sufficient to oxidize the inactive reduced form of the enzyme to regenerate active redox enzyme.
Os(dmbpy)2PicCl oxidized electron transfer mediator
The oxidized electron transfer mediator is Os(dmbpy)2PicCl.
Zwitterion buffering agent with essentially all tetramethylammonium positively-charged counter ions
A zwitterion buffering agent comprising a positively-charged buffer counter ion and a buffer conjugate base, wherein essentially all of the positively-charged buffer counter ion is tetramethylammonium.
Active redox enzyme with tris-tetramethylammonium ferricyanide and zwitterion buffering with essentially all tetramethylammonium
A dry reagent composition comprising an active redox enzyme that oxidizes an analyte as a specific substrate to produce an inactive reduced form of the enzyme; tris-tetramethylammonium ferricyanide; and a zwitterion buffering agent with a positively-charged buffer counter ion and a buffer conjugate base, wherein essentially all of the positively-charged buffer counter ion is tetramethylammonium.
Method of forming a dry shelf stable electrochemical test reagent using glucose oxidase and tris-tetramethylammonium ferricyanide
A method for forming a dry shelf stable electrochemical test reagent for use in detection of an analyte comprising selecting an oxidoreductase enzyme that oxidizes/reduces the analyte as a specific substrate, and combining the selected enzyme and tris-tetramethylammonium ferricyanide to form a dry shelf stable electrochemical test reagent for use in detection of the analyte, wherein the selected enzyme is glucose oxidase.
Overall, the independent claims define dry shelf-stable electrochemical reagent compositions that couple an active redox enzyme with tris-tetramethylammonium ferricyanide, optionally include an oxidized non-ferricyanide mediator with an electrochemical potential sufficient to regenerate the enzyme, including Os(dmbpy)2PicCl, and incorporate zwitterion buffering where essentially all positively charged counter ions are tetramethylammonium. One independent claim further covers forming the dry test reagent by selecting the oxidoreductase enzyme, glucose oxidase, and combining it with tris-tetramethylammonium ferricyanide.
Stated Advantages
Improved baseline signal stability of an electrochemical test strip over storage by reducing baseline drift associated with ferricyanide-to-ferrocyanide conversion.
Improved shelf stability of the dry electrochemical test reagent formulation when using tris-tetramethylammonium ferricyanide.
Maintains enzyme activity retention during storage.
Documented Applications
Dry shelf-stable electrochemical test reagent for use in detection of an analyte, including analyte detection using a dry electrochemical test strip architecture where the dry reagent dissolves upon application of a liquid sample.
Detection of an analyte using glucose oxidase as the selected oxidoreductase enzyme in a dry shelf stable electrochemical test reagent.
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