Oxidative coupling dye for spectrophotometric quantitive analysis of analytes

A method of determining analyte concentration in a liquid which comprises:

• (a) quantitatively measuring baseline reflectance from a first surface of a reagent element comprising an inert, porous, hydrophilic, substantially reflective, single-layer matrix having pores of a size sufficient to exclude red blood cells and a reagent system which interact with said analyte to produce a light-absorbing reaction product, said reagent system being impregnated in the pores of said matrix, prior to application of said liquid to said reagent element;
• (b) applying said liquid to a second surface of said reagent element and allowing said liquid to migrate from said second surface to said first surface;
• (c) quantitatively measuring reaction reflectance from said first surface of said reagent element without removing excess sample or non-migrating components of said sample from said second surface;
• (d) quantitatively measuring reflectance of interfering substances from said first surface of said reagent element using a wavelength of light reflected by interfering substances and different from the wavelength of light used to measure said reaction product reflectance in order to correct for background reflectance at the reaction product wavelength caused by interfering substances;
• and
• (e) calculating a value expressing said analyte concentration from said reflectance measurements, wherein said reaction system includes a dye couple consisting essentially of 3-methyl-2-benzothiazolinone hydrazone and 8-anilino-l-naphthalene sulfonate and at least one reagent capable of reacting with said analyte to produce a strong oxidizing agent which reacts with said dye couple to form a blue dye.

A method of determining analyte concentration in a liquid which comprises:

• (a) quantitatively measuring baseline reflectance from a first surface of a reagent element comprising an inert, porous, hydrophilic, substantially reflective, single-layer matrix having pores of a size sufficient to exclude red blood cells and a reagent system which interact with said analyte to produce a light-absorbing reaction product, said reagent system being impregnated in the pores of said matrix, prior to application of said liquid to said reagent element;
• (b) applying said liquid to a second surface of said reagent element and allowing said liquid to migrate from said second surface to said first surface;
• (c) quantitatively measuring reaction reflectance from said first surface of said reagent element without removing excess sample or non-migrating components of said sample from said second surface;
• (d) quantitatively measuring reflectance of interfering substances from said first surface of said reagent element using a wavelength of light reflected by interfering substances and different from the wavelength of light used to measure said reaction product reflectance in order to correct for background reflectance at the reaction product wavelength caused by interfering substances;
• and
• (e) calculating a value expressing said analyte concentration from said reflectance measurements, wherein said analyte comprises a substance that reacts with an enzyme to produce hydrogen peroxide and said reagent system comprises said enzyme, peroxidase and 3-methyl-2-benzothiazolinone hydrazone and 8-anilino-1-naphthalene sulfonate.

In a method for determining glucose in a blood sample employing a membrane and a signal-producing system which reacts with glucose to produce a light-absorptive dye product, said system being bound to the membrane, and in which the amount of said dye product is determined by means of a reflectance measurement from a surface of said membrane, said method comprising:

• (a) applying an unmeasured whole blood sample to a first surface of a single-layer, substantially reflective, porous, hydrophilic membrane having pores of a size sufficient to exclude red blood cells and which contains said signal-producing system;
• (b) making said reflectance measurement on a second surface of said membrane other than the surface to which said sample is applied without removing excess sample or red blood cells from said first surface;
• and
• (c) determining the concentration of glucose in said sample from said reflectance measurement, wherein the improvement comprises employing as said signal-producing system glucose oxidase, peroxidase and 3-methyl- 2-benzothiazolinone hydrazone and 8-anilino-1-naphthalene sul-fonate.

A method for determining glucose comprising the steps of:

• (a) applying a whole blood sample to an application site on a reagent element wherein said reagent element comprises a single-layer, substantially reflective, porous, hydrophilic matrix which filters out red blood cells and to which is bound a signal-producing system comprising glucose oxidase, peroxidase, and a dye indicator, which signal-producing system reacts with glucose to form a reaction dye product;
• (b) allowing the sample to migrate to a reading site on said membrane different from said application site;
• (c) monitoring reflectance at said reading site for a decrease in reflectance indicative of sample presence in said reading site in order to initiate timing of an incubation period;
• and
• (d) determining the change in reflectance at said reading site during the incubation period as a measure of dye product formed to determine the amount of glucose in said sample, in which all reflectance measurements at said reading site are performed without removing excess sample or red blood cells from said application site and at least one measurement is taken at a wavelength at which light is absorbed by said dye product, wherein said dye indicator comprises 3-methyl-2-benzothiazolinone hydrazone and 8-anilino-1-naphthalene sulfonate.