Abstract: A new group of Os(II) and Os(III) compounds useful as redox mediators in electrochemical biosensors. These compounds have 1) low oxidation potential, 2) fast reaction kinetics between the electroactive center of an enzyme and the compound, 3) slow oxidation of osmium by oxygen, and 4) excellent solubility in aqueous medium. These mediators are particularly useful as a component of a reagent used in an electrochemical biosensor, wherein the biosensor is useful for measuring analytes from a biological fluid, such as blood.

Abstract: An enzyme composition for dissociating tissues is disclosed. The composition comprises purified collagenase I and collagenase II from Clostridium histolyticum in a mass ratio of about 0.3 to 0.6 and a neutral protease such that the ratio of total FITC casein units of activity to Wunsch units of activity in the composition is about 250:1 to about 600:1. The preferred neutral protease is thermolysin.

Abstract: An electrochemical biosensor test strip that has a minimum volume blood sample requirement of about 9 microliters. The test strip has working and counter electrodes that are substantially the same size and made of the same electrically conducting material placed on a first insulating substrate. Overlaying the electrodes is a second insulating substrate, which includes a cutout portion that forms a reagent well. The cutout portion exposes a smaller area of the counter electrode than the working electrode. A reagent for analysis of an analyte substantially covers the exposed areas of working and counter electrodes in the reagent well. Overlaying the reagent well and affixed to the second insulating substrate is a spreading mesh that is impregnated with a surfactant. The small cutout portion of 4 millimeters by 4.2 millimeters, small mesh of 6 millimeters by 5.8 millimeters, and small amount of reagent, 4 microliters before drying, allow the test strip to analyze a whole blood sample of about 9 microliters.

Abstract: A purified enzyme mixture useful for isolating cells or cell clusters from tissue is disclosed. The mixture includes at least two collagenase enzymes, at least two other proteases, and additional non-protease components. The mixture is purified by removing at least some of the non-protease components. The purified mixture may then be used to isolate cells or cell clusters from tissue. Also disclosed are the essential components of the purified enzyme mixture as well as preferred ranges and ratios of these essential components for isolating cells or cell clusters from tissue. Finally, a dissociation system is disclosed that can be used with the purified enzyme mixture to dissociate tissue and recover cells or cell clusters.

Abstract: A new reagent and methods for measuring the concentration of (or detecting the presence of) an analyte in a sample. The reagent includes a phenazine-containing compound and an enzyme. The phenazine-containing compound must be of sufficient type to form a semiquinoid (the color indicator) by reaction involving the enzyme, analyte, and phenazine-containing compound. Importantly, the phenazine-containing compound must be in sufficient amount to correlate the concentration of semiquinoid to the concentration of analyte in the sample or to detect the presence of the analyte in the sample. The reagent may further include a buffer and a surfactant. The reagent may be incorporated into a film and may be provided in kit form.

Abstract: The invention is a stable reagent used in assay systems that form a colored ferric ion complex, such as Prussian Blue. Such reagents are useful for the detection or measurement of an analyte from a fluid sample. Surprisingly, it has been found that the inclusion of certain ferric ion chelating agents, such as 3-sulfobenzoic acid, will inhibit formation of the blank reaction in the reagent.

Abstract: A new group of Os(II) and Os(III) compounds useful as redox mediators in electrochemical biosensors. These compounds have 1) low oxidation potential, 2) fast reaction kinetics between the electroactive center of an enzyme and the compound, 3) slow oxidation of osmium by oxygen, and 4) excellent solubility in aqueous medium. These mediators are particularly useful as a component of a reagent used in an electrochemical biosensor, wherein the biosensor is useful for measuring analytes from a biological fluid, such as blood.

Abstract: A stable reagent for assaying an analyte from a fluid sample which includes an enzyme, a mediator, a tetrazolium salt and an oxidizing agent selected from the group consisting of sodium chlorate, 2,5-dimethylhexane-2,5-dihydroperoxide, benzoyl peroxide, t-butyl peroxide, sodium iodate, N-ethylmaleimide, t-butylperoxyacetate, nickel acetylacetonate, stannic chloride, rhodium (III) trichloride hydrate, and t-butylperbenzoate is described. Inclusion of an oxidizing agent from the group specified above stabilizes the reagent, thereby preventing a high blank reaction because a high blank introduces error into the assay of an analyte. The reagent may be provided in solution form by adding water, or may be included in a film by adding a film-forming agent. The reagent may be utilized to perform the assay of an analyte from a fluid sample.

Abstract: Novel derivatives of procainamide and N-acetylprocainamide (NAPA) are disclosed having the following formula: ##STR1## wherein: X=hydrogen or acetyl;n=1 to p where p=MW of Z/1000;Z=a poly(amino acid) or polysaccharide; andR3=a bond or ##STR2## wherein R2=an alkyl, cycloalkyl or aryl group having 2 to 10 carbon atoms. The derivatives include maleimide conjugates of proteins or poly(amino acids), enzymes, enzyme donor polypeptides and labeling substances. Novel activated hapten intermediates useful in the preparation of the conjugates and methods for synthesis of the hapten intermediates and derivatives are also disclosed.

Abstract: A new electrochemical biosensor and method of its use. The electrochemical biosensor has a pair of electrodes consisting of a working electrode and a counter electrode made of the same electrically conducting materials. The counter electrode may be the same size or smaller than the working electrode. The biosensor includes a sample receiving portion wherein a portion of the electrode surfaces is covered by a reagent that includes a redox mediator and an enzyme. A fluid that contains an analyte is added to the sample receiving portion. A reaction involving the analyte, enzyme and redox mediator occurs. After this reaction is complete, an electrical potential difference is applied between the electrodes. Diffusion limited current is measured and correlated to the concentration of analyte in the fluid.

Abstract: Muteins of enzyme acceptor polypeptide fragments of .beta.-galactosidase are provided which exhibit substantially increased kinetic complementation activity with no significant loss in stability. A preferred enzyme acceptor fragment has an amino acid other than cysteine located at position 500 of the natural sequence. An especially preferred substitution is serine or valine. Other preferred muteins have an amino acid other than methionine located at position 443, with leucine being especially preferred, or an amino acid other than cysteine at position 76, with leucine being an especially preferred substitution. Also provided are methods for producing the novel muteins, reagent compositions comprising the novel muteins, and immunoassay methods for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

Abstract: A mutein of an enzyme acceptor polypeptide fragment of .beta.-galactosidase which is resistant to oxidation is provided. The enzyme acceptor fragment has an amino acid other than cysteine located at position 602 of the natural sequence. An especially preferred substitution is serine. Also provided are a method for producing the novel mutein, a reagent composition comprising the novel mutein, and an immunoassay method for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

Abstract: Novel derivatives of procainamide and N-acetylprocainamide (NAPA) are disclosed having the following formula: ##STR1## wherein: X=hydrogen or acetyl;R.sub.1 =an alkyl group having 1 to 3 carbon atoms;m=an integer from 2 to 10;R.sub.2 =an alkyl, cycloalkyl or aryl group having 2 to 10 carbon atoms;Z=a poly(amino acid); andn=1 to p where p=MW of Z/1000.The derivatives include maleimide conjugates of proteins or poly(amino acids), enzymes, enzyme donor polypeptides and labeling substances. Novel activated hapten intermediates useful in the preparation of the conjugates and methods for synthesis of the hapten intermediates and derivatives are also disclosed.

Abstract: A method for fabricating high-resolution, biocompatible electrodes is disclosed, allowing production of an electrochemical sensor which is capable of precise analyte concentration determination on a very small sample size. Electrically conducting material is affixed to a first insulating substrate. A second insulating substrate is then affixed to the electrically conducting material and patterned using photolithography to define an electrode area. Alternatively, the electrically conducting material may be screen printed directly onto a standard printed circuit board substrate in the case of a counter or reference electrode. In either case, the substrate may be rigid or flexible. When the electrodes produced in accordance with the present invention are then used in an electrochemical sensor which includes a reagent, the small and highly-defined electrode areas permit highly-accurate electrochemical analyte measurements to be performed on very small sample sizes.

Abstract: An immunoassay diagnostic kit, method, and apparatus for electrochemically determining the concentration of an analyte in a sample. A mixture is formed which includes the sample, an enzyme-acceptor polypeptide, an enzyme-donor polypeptide linked to an analyte analog (enzyme-donor polypeptide conjugate), a labeled substrate, and an antibody specific for the analyte to be measured. The analyte and the enzyme-donor polypeptide conjugate competitively bind to the antibody. When the enzyme-donor polypeptide conjugate is not bound to antibody, it will spontaneously combine with the enzyme acceptor polypeptide to form an active enzyme complex. The active enzyme hydrolyzes the labeled substrate, resulting in the generation of an electroactive label, which can then be oxidized at the surface of an electrode. A current resulting from the oxidation of the electroactive compound can be measured and correlated to the concentration of the analyte in the sample.

Abstract: A biosensor and method for determining the hematocrit level of a whole blood sample using electrochemistry. The biosensor includes working and counter electrodes. A porous membrane is impregnated with an electroactive compound and is spatially displaced from the surface of the electrodes. When a whole blood sample is applied to the porous substrate, a mixture of the electroactive compound and the blood is formed. The mixture settles on the electrodes and a potential difference is applied sufficient to oxidize or reduce the electroactive compound and generate a current. This current can be measured and correlated to hematocrit level.

Abstract: A biosensor that includes working and counter electrodes of substantially the same size and made of the same electrically conducting material. A reagent is contained in a reagent well in the biosensor. The reagent includes a redox mediator, an enzyme, and a buffer, and covers substantially equal surface areas of portions of the working and counter electrodes. When a sample, containing the analyte to be measured, is added to the reagent well, a reaction occurs that oxidizes the analyte and reduces the redox mediator. After this reaction is complete, an electrical potential difference is applied between the electrodes. The amount of oxidized form of the redox mediator at the counter electrode and the applied potential difference must be sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode.

Abstract: The operative site of an assay device is dosed with a controlled volume of a liquid specimen by a holding device which holds the controlled volume of specimen in contact with the operative site, although the holding device remains out of contact with the operative site. A sink device is in contact with the margin of the holding device and moves excess specimen away from the holding device. In one embodiment the device makes possible a non-wipe teststrip architecture. The device can be used in the detection of analytes including glucose or cholesterol in blood or urine and can be interrogated visually, optically or electrically, depending on the reagent used at the operative site.

Abstract: Non-aqueous, polymeric reagent film compositions for use with analytical test devices of the dry chemistry type are described. The film compositions are prepared from an organic solution of a copolymer formed by interaction of at least two monomers wherein(a) the first monomer is a hydroxylated acrylate of the general formula ##STR1## where R1 is hydrogen or, more preferably, methyl, and R2 is a hydroxyalkyl group having from 1 to 5 carbon atoms, and(b) the second monomer is a neutral acrylate of the general formula ##STR2## where R1 is hydrogen or, more preferably, methyl, and R3 is a substituted or unsubstituted alkyl group having from 1 to 8 carbon atoms.Preferably, a third monomer is also included which is an amine-containing acrylate of the general formula ##STR3## where R1 is hydrogen or, more preferably, methyl, and R4 is a substituted or unsubstituted aminoalkyl or glycidyl group having from 1 to 5 carbon atoms or a surfactant polyethylene glycol group having from 10 to 30 carbon atoms.

Abstract: The present invention relates to a novel method and apparatus for the amperometric determination of an analyte, and in particular, to an apparatus for amperometric analysis utilizing a novel disposable electroanalytical cell for the quantitative determination of biologically important compounds from body fluids.