EP0467978A4 - The use of fluid insoluble oxidizing agents to eliminate interfering substances in oxidation-reduction measuring systems - Google Patents
The use of fluid insoluble oxidizing agents to eliminate interfering substances in oxidation-reduction measuring systemsInfo
- Publication number
- EP0467978A4 EP0467978A4 EP19900906723 EP90906723A EP0467978A4 EP 0467978 A4 EP0467978 A4 EP 0467978A4 EP 19900906723 EP19900906723 EP 19900906723 EP 90906723 A EP90906723 A EP 90906723A EP 0467978 A4 EP0467978 A4 EP 0467978A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- fluid
- redox
- insoluble
- oxidizing compound
- interfering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
Definitions
- the invention generally relates to more accurate redox measuring devices. Analytical devices based on redox reactions can provide inaccurate measurements due to the presence of redox-active contaminants.
- This invention relates generally to a system for removing, eliminating or rendering non-interfering redox- active contaminants contained in fluids by the use of redox active insoluble compounds (hereinafter, RAIC), methods for performing the removal and the resulting fluid free of the interfering redox-contaminants.
- RAIC redox active insoluble compounds
- the invention also relates to diagnostic kits whi contain the redox-active insoluble compound used in accordance with the invention. Improved assays are obtaine as a result of the invention.
- the invention relates to disposable, machine- independent devices and to machine or apparatus-dependent devices, not disposable after each use.
- the fluid free of the interfering redox-active contaminants is then suitable for a variety of uses includi a redox reaction based analysis of analytes in the fluid using redox measuring systems.
- the analysis is more accura than if performed without the use of the redox-active insoluble compound.
- oxidation-reduction chemical reactions for the removal of the unwanted or contaminating redox-active components.
- redox-active insoluble compounds and the interfering redox- active contaminants which are sought to be rendered non- interfering (or inert) in the analytical reaction for the analyte (as well as various analysis techniques) can be understood by reference to the general principles of oxidation-reduction reactions.
- Insoluble - a material is insoluble if a fluid that passes over (or contacts it) picks up an amount of said material that is wholly insignificant compared to the concentration of the analyte(s) of interest in said fluid. As a general matter, insoluble material will not dissolve to an extent in excess of 10 pp , regardless of analyte concentration.
- Redox reaction - a reaction in which electrons are liberated by one substance while simultaneously being consumed by another substance.
- the substance that loses the electrons is the reducing agent, or reductant, and is itself oxidized.
- the substance that gains electrons is the oxidizing agent, or oxidant, emd is itself reduced.
- neither oxidation nor reduction can occur alone. It can also be defined in terms of change in oxidation number - the oxidizing agent undergoes an algebraic decrease in oxidation number, and the reducing agent undergoes an algebraic increase.
- redox reactions are typically catalyzed. Reactions catalyzed by proteins include enzymatic reactions; reactions can also be - -
- Redox measurement system a redox reaction, or a set of redox reactions that act on some redox-active analyt to give a product of reaction; the product then being converted to some signal that can be interpreted and used t detect the absence or presence and determine the concentration of the analyte.
- this measurement include colori etric measurement, as in the case where the product of the reaction is a color change in a dye molecule and electronic measurement, as in the case where the redox reaction is linked to a semiconductor on a biosensor.
- Redox-active - compounds which can undergo oxidation or reduction in redox reactions under the conditions present in the fluid, i.e., reducing and oxidizi agents.
- Analyte - a redox-active substance whose absence presence or concentration is measured by a redox measuremen system.
- Fluid - any liquid substance either aqueous, non aqueous or any mixture of the two, that can be contacted wi an insoluble redox-active material.
- the fluid may be subsequently analyzed in a redox measurement system, but do not have to be so analyzed.
- a redox based analysis of a fluid with an interfering substance would be less accurate than if the analysis were performed without an interfering substance.
- This term in the invention is interchangeable with "contaminating”.
- "interfering" means causing error or non-ideal performance in the use of the product fluid.
- the "interfering" substance will generally cause a lower reading of the concentration of the analyte, unless rendered “non-interfering" in accordance with the invention.
- Render non-interfering - refers to the effect the insoluble redox-active compound has on the redox based analysis of an analyte containing fluid, which is to make such analysis more accurate. This is achieved by the interaction of the insoluble redox-active agent with redox- active contaminants in the fluid. As a result the contaminant is oxidized if the RAIC is an oxidizing agent or the contaminant reduced if the RAIC is a reducing agent. This interaction causes the contaminant or contaminants to b inert, inactive or to not interact with the compounds responsible for the redox based analytical determination. The presence of the RAIC in the system causes the analytical reaction to be as accurate as if there were no contaminant present.
- the RAIC reacts with respect to the interfering contaminant by way of a redox reaction, whic precedes the principal redox reaction which involves the analytical determination of the analyte. It would appear that there is a net positive electromotive potential between the RAIC emd redox-active contaminant and a pathway exists for electron tremsfer between them while no effective pathwa exists for electron tremsfer between the RAIC and analyte of interest. Thus the RAIC reacts selectively with the contaminants while not affecting the analyte. When oxidized the reducing interfering compound is not interfering in the following redox reaction.
- Oxidation - reduction (redox) reactions are very common emd importemt chemical reactions. Me y different types of compounds can undergo redox reactions. As is well known, a molecule is oxidized when it undergoes an algebraic increase in oxidation number, and a molecule is reduced when it undergoes an algebraic decrease in oxidation number. Oxidation or reduction cannot occur without the other also occurring. Thus a substance cannot be reduced without another substance being oxidized. When a substance is reduced, it is responsible for the oxidation of another substance, so the substance that is reduced is called the oxidizing agent. Conversely, the substance that is oxidized is called the reducing agent, because it is responsible for the reduction of another substance.
- the second condition is important. It is well known that most organic compounds of intermediate or high reduction state are essentially stable in the presence of atmospheric oxygen, yet they could give up energy if a redo reaction occurred between them emd oxygen.
- An aqueous solution containing ethyl alcohol and glucose, for instemce could oxidize to carbon dioxide and water by reaction with oxygen, as is well known by those skilled in the art.
- energy barrier exists for the tremsfer of electrons from these molecules to oxygen such that these potential redox reactions do not occur or occur at insignificant rates unde conditions of neutral pH and temperatures of 0-40"C. Redox reaction between these materials emd oxygen can be facilitated by a rise in ambient temperature that overcomes the energy barrier, or by the addition of catalysts to provide a pathway for electrons between interacting redox compounds.
- Enzymes are especially import a catalysts of organic redox reactions under conditions restricted to the neutral pH range and temperatures from 0 about 40*C. Enzymes catalyzing redox reactions fall into classes including, but not limited to, the dehydrogenases, the oxidases, the oxygenases and other oxidoreductases, as well known in the art emd described in Biochemistry. 2nd e A.L. Lehninger, Worth Publishers, Inc. N.Y. (1975) pp. 477- 508.
- catalysts that facilitate redox reactions of organic molecules with each other, or with inorganic or organometallic redox partners.
- These catalysts can generall be termed electron tremsfer agent(s) (etas) since they reversibly transfer electrons among the redox-active compounds being alternatively oxidized and reduced in a redo reaction. In these reactions, the etas are not consumed but recycle between the oxidized emd reduced forms, facilitating the reaction.
- reduced chemical compounds can be selectively oxidized in the presence of each other by addition of redox catalysts that selectively interact with one or the other reduced compound, for whatever reason.
- redox catalysts that selectively interact with one or the other reduced compound, for whatever reason.
- the addition of iron (III) ion to the aqueous solution of alcohol and glucose mentioned above will facilitate oxidation of the glucose, with concomitemt reduction of oxygen, but will not similarly facilitate oxidation of the ethemol.
- addition of enzyme catalysts such as glucose oxidase will cause oxidation of glucose without oxidation of ethemol, emd addition of alcoho oxidase will cause oxidation of the alcohol without oxidizin the glucose.
- Selective oxidation of reduced analytes in mixtures of compounds by the addition of a redox catalyst that selectively interacts with em emalyte of interest is a commo and powerfil meems to determine the presence and to determin the concentration of analytes, as is well known to those skilled in the art.
- the electrons provided by change in oxidation state of the analyte is converted to a measuretble signal by a number of means, such as coupling the oxidation to a colori etric system as was done in the test used to measure glucose, disclosed in U.S. Patent 4,391,906.
- a signal cem be generated by coupling the oxidation to an electronic system, as is done with enzyme electrodes.
- the signals obtained in colorimetric and electronic systems typically result from the total of electrons available from redox- active subste ces in the system.
- Non analyte redox-active materials may be present in the sample that will give up their electrons to the colorimetric or electronic signal producing system, emd thereby provide a falsely high signal compared to the concentration of emalyte.
- Another common means to correct for the presence o the interfering contaminants is to destroy them prior to initiation of the analysis by addition of an excess of an oxidizing agent. Rendering interfering reducing substances non- interfering by addition of oxidants is fraught with difficulty.
- the oxidemt added must not oxidize the analyte of interest. Thus, it must be of an energy level able to spontaneously react with the interfering reducing substances and it must have a pathway for electrons from interfering materials to itself, but not from the analyte of interest. More importantly, addition of an oxidant typicall leaves a residue of oxidant in the sample.
- This residue itself constitutes an interfering contaminant because it is capable of accepting electrons from the oxidation of the analyte in the emalysis step, resulting in em incorrect, low signal relative to the emalyte.
- the oxidemt added to remove interfering reducing substances must itself be removed, without any significant residue, by a back titration with a known reducing agent.
- the fluid treated by the oxidizing agent may be left with the residual agent in place, but the fluid is not subsequently used in a manner involving redox reactions. This way of correcting for the interfering materials is not satisfactory.
- NADH plus iodophenyl nitrophen phenyltetrazoliu (INT) in the presence of a diaphorase wil yield NAD emd INT-formazan, which is dark red.
- the amount red color produced by this redox reaction is proportional t the concentration of NAD(P)H; however, if em interfering reducing agent were present in this seunple, then a more intense signal will be generated, thereby, causing an overestimation of the concentration of NADH.
- This technology is utilized by U.S. Patent Numbers 3,867,259; 4,024,021; 4,247,833; emd 4,556,834, all of which are subject to interference by reducing substances as described.
- Another redox measurement technology that determines metabolite or chemical levels in a fluid utilizes enzyme oxidases.
- enzyme oxidases Numerous examples of this technology exist in the patent literature, for example see U.S. Patent 4,391,906; U.S. Patent 3,164,534; and U.S. Patent 4,544,249 which deal with methods for eliminating bilirubin interference, and U.S. Patent 4,186,251. All of these patents involve the very well known assay system in which the compound to be measured is contacted with em oxidase in the presence of oxygen, resulting in hydrogen peroxide production. The resulting hydrogen peroxide is then utilized to oxidize a chromogen to produce em indicative dye.
- Still emother technology that makes use of redox reactions is the enzyme electrode.
- the oxidation of glucose will produce hydrogen peroxide, the rat of whose production can be measured by a current at the surface of the electrode.
- One such apparatus is disclosed i U.S. Patent No. 4,340,448, which discusses the potentiometri determination of hydrogen peroxide.
- U.S. Patent No. 4,547,280 which describes a maltose sensor
- U.S. Patent No. 4,356,074 which describes substrate-specific galactose oxidase electrodes. Since these electrodes are dependent on redox reactions, interfering substances that are redox-active may generate incorrect readings.
- Newer measurement systems that use redox chemistry can come in the form of biosensors.
- This is a generic term that is not specific to any measurement type, but one embodiment of biosensors involves attaching a redox-active biological material to a semiconductor.
- the behavior of the semiconductor and, therefore, the signal generated by the semiconductor is dependent upon the redox state of the biological material.
- it is important that the redox state of the particular measurement system, in this case, the redox-active biological material be affected only by the analyte. Therefore, if an interfering redox-active substance is present in the fluid t be measured, an erroneous signal will be generated.
- Pending patent application serial number 075,817 discloses a method for decreasing the color produced by colorimetric redox-measuring systems.
- the disclosure discusses the oxidation of NAD(P)H emd the simultemeous reduction of a chromogen, the reaction being catalyzed by diaphorase.
- the concentration of NAD(P)H is such that too much color would be produced by the chromogen, so that the concentration of NAD(P)H cannot be determined. Therefore, em alternate electron acceptor is introduced tha competes for electrons with the chromogen.
- the alternate electron acceptor is by definition an oxidizing agent. Therefore, problems may arise when a contaminating reducing agent is present; the conteuninating agent can reduce the alternate electron acceptor, thereby giving a false reading.
- U.S. Patent No. 4,342,740 describes the use of a soluble oxidizing agent which is added to facilitate the labeling of red blood cells with Technetium - 99 M.
- the resulting fluid contains residual oxidizing agent and the fluid is not subsequently analyzed by redox analysis.
- U.S. Patent No. 4,180,592 describes a process for decolorizing blood by the addition of the soluble oxidizing agent hydrogen peroxide emd the removal of excess peroxide.
- the end use of the decolorized blood product is not analyzed by redox methods.
- U.S. Patent No. 4,298,688 describes a test strip t measure glucose in body fluids.
- the strip contains an oxidizing agent to destroy interferents.
- the residual oxidizing agent and glucose are separated by chromotography.
- the patent does not teach nor suggest the use of insoluble oxidizing agents and their removal based on insolubility.
- U.S. Patent No. 4,255,385 describes a method, reagent emd test kit for the determination of glycosylated hemoglobin with use of the soluble oxidizing agent, ferricyemide.
- the treated fluid contains residual oxidizing agent and is not analyzed by redox reactions.
- U.S. Patent No. 3,894,844 describes a method for determining the amount of triglycerides, cholesterol emd phospholipids present in blood by using oxidation reactions. The three analytes of interest are separated in a procedure which removes interfering reducing subst ⁇ mces as well.
- U.S. Patent No. 4,645,660 describes the addition o a soluble reducing agent to a radioactive diagnostic agent wherein iron ions compete with the desired radioactive elements for formation of desired chelate compounds, emd a reducing agent is added during formation of these chelates t reduce the iron to a form that does not effectively chelate.
- a reducing agent is added during formation of these chelates t reduce the iron to a form that does not effectively chelate.
- the resulting fluid contains the residual reducing agents and the fluid is not subsequently analyzed b redox methods.
- U.S. Patent No. 4,720,385 describes the sterilization of therapeutically or immunologically active protein solutions by contacting these solutions with a metal chelate complex such as copper phenemthroline and a reducing agent, such as ascorbic acid or a thiol.
- a metal chelate complex such as copper phenemthroline and a reducing agent, such as ascorbic acid or a thiol.
- the resulting solution contains the soluble residual reducing agent and is not subsequently analyzed by redox methods.
- Patents dealing with the addition of oxidizing agents to solutions containing various contaminants, for the purpose of removing the contaminants include U.S. Patent No. 4,572,797, describing the addition of soluble oxidizing agents to aqueous solutions containing trace metals.
- the oxidized product of the trace contaminant is insoluble in the liquid is then separable from the fluid as precipitate. The residual oxidizing agent remains in solution and the resulting fluid is not emalyzed by redox methods.
- U.S. Patent No. 4,431,847 describes th addition of an oxidizing agent to fluids containing halogenated phenolic compounds, to cause the polymerization and insolubilization of these compounds.
- the resulting polymeric form of the conteuninants are easily removed as precipitates.
- the resulting fluid contains the residual oxidizing agent and is not subsequently emalyzed by redox methods.
- U.S. Patent No. 4,249,939 describes the removal of copper from spent solutions of complexing agents containing cuprous aluminum tetrahalides. In this patent, the copper is oxidized to the water soluble cupric form by contacting the fluid with em oxidizing agent.
- Organic materials in the solution are removed by extraction with an organic solvent, and the copper is subsequently recovered by contacting the aqueous phase with a metal of higher electromotive potential them copper, plating the copper out of the solution.
- the reducing agent, the metal of higher potential is an insoluble agent serving as a specific means of recovering the copper from solution.
- This invention relates to a system including kits, for the inactivation or the rendering non-interfering of interfering redox-active substemces in a fluid by the use of redox-active compounds, a method for using the redox-active insoluble compounds to render inert the interfering substances in the fluid emd the purified fluid free of the interfering compounds.
- the system of the invention provides for highly improved emalytical results such as would be obtained in the absence of the contaminant under comparable conditions.
- the invention relates to disposable, non-machine or instrumentation-independent disposable devices, and to machine or instrumentation dependent devices, which are not customarily discarded after each use.
- the invention disclosed overcomes the difficulties presented by reducing agents contaminating a fluid.
- the invention involves a process for providing an amount of an oxidizing agent in excess of that required to render the conteuninants non-interfering, of energy level enabling the spontaneous reaction with the contaminant reducing agent.
- the fluid also provides optionally an electron transfer agen to catalyze passage of electrons from the reducing agent to the added oxidizing agent.
- the oxidizing agent has the distinctive property to be insoluble in the fluid, so that i can be simply removed from the treated fluid by rapid, uncomplicated means such as filtration, decantation, sedimentation or centrifugation or other methods known in th art.
- the fluid treated in this manner is then prepared or ready by subsequent emalysis or other use free from interference by either the conteuninating reducing substances or residual oxidizing agent.
- This invention is particularly useful in the pretreat ent of certain biological fluids including, but not limited to, blood serum or plasma, cerebrospinal fluid, semen, saliva, tears, sinovial fluid, lymph or urine since these contain not only many important analytes, but also contain unknown emd variable amounts of contaminating reducing agents.
- the invention is also useful for veterinar applications in the emalysis of the biological fluids of animals.
- the invention may also be applied to complex fluid containing contaminating oxidizing agents, wherein these contaminants would be destroyed by the addition of electron transfer agent and an insoluble reducing agent, that would b removed on the basis of its insolubility prior to use or analysis of the fluid.
- the invention is applicable to system and method of rendering non-interfering either oxidizing contaminants or reducing contaminants by an insoluble oxidizing agent or reducing agent, respectively.
- the fluid to be treated can be aqueous partially aqueous or a non-aqueous (or organic) fluid which contains a analyte to be assayed as well as redox-active contaminants.
- the invention is broadly applicable to fluids which are single or multi-phase; multi-phase fluids may be considered emulsions or suspensions.
- the system of the invention provides for the interfering-substance containing fluid to be contacted with the redox-active agent which is insoluble in the fluid.
- the interfering substance can be a reducing or an oxidizing compound.
- the insoluble redox-active compound undergoes a redox reaction with the interfering substance.
- Insoluble redox agent remaining in the fluid is then selectively removed from the fluid.
- the resulting fluid free of the interfering substemce cem then be emalyzed for a particular analyte by oxidation-reduction analysis methods without the interfering redox contaminant that would cause inaccurate results.
- the emalysis performed on the purified fluid is more reliable and accurate.
- any other use made of the fluid cem be without the distorting or interfering effect that the redox-active contaminants would cause.
- the invention is applicable for use in machine-dependent diagnostic systems and methods whereby more accurate analysis of a fluid's emalyte or analytes is possible.
- machine-independent devices are discarded after each use, the machine-dependent are not.
- kits include a system for bringing the emalyte containing fluid into contact with the RAIC so that the RAIC renders non-interfering substemtially all the redox-active contaminemts in the fluid.
- the kit provides a means for the selective passage of the treated fluid into a separate compartment or vessel without any residual RAIC.
- the kit provides compounds for em oxidation-reduction based emalysis to take place in the compartment. This analysis is performed on the fluid which has had any redox-active contaminemts as well as residual RAIC removed. Thus an accurate redox-based analysis for a particular analyte in th fluid cem then be performed.
- the invention also provides a method by oxidation for the removal, inactivation or the rendering non- interfering of interfering, redox-active contaminants which are contained in fluids.
- the invention also encompasses fluids such as biological fluids free of contaminants that would interfere in a redox reaction and render a redox-based analysis inaccurate.
- the invention encompasses biological fluids which are suitable for redox analysis of the fluid's redox-active analytes.
- the system (including kits) and method of the invention also optionally contemplates the use of active electron transfer agents to facilitate the oxidation- reduction reaction between the insoluble redox-active agent and the redox-active interfering contaminant.
- active electron transfer agents to facilitate the oxidation- reduction reaction between the insoluble redox-active agent and the redox-active interfering contaminant.
- th electron transfer agent or agents in conjunction with the insoluble redox-active purifying compounds more effectively subject the interfering compound to a redox reaction.
- the fluid to b purified is pretreated with the insoluble redox-active agent
- the redox-active agent reacts with the redox-active contaminemts contained in the fluid.
- the fluid is assayed for the particular analyte sought to be determined in a redox measuring system.
- redox based measuring systems for analyzing a fluid include but are not limited to those described earlier in this application.
- the fluid to be purified emd then emalyzed passes into two reaction chambers.
- the first chamber (or area) contains the insoluble redox agent and may or may not contain an electron transfer agent.
- the fluid containing the contaminants passes through the first cheunber where the redox conteuninants are rendered non-interfering and then into a second chamber without carry over of the insoluble redox agent, where the fluid is assayed for particular analytes.
- These analytes are detected and concentration determined by a redox measuring system for analyzing a fluid including but not limited to those described eaxlier in this application.
- This embodiment permits the fluid or emalyte to be treated and measured in one closed system, in one procedure emd without having to recover the purified fluid from one system and transferring it to another system.
- the insoluble redox-active agents selectively react with the redox-active contaminant when the emalyte and the insoluble redox-active agent are present in the same fluid; they do not affect the fluid's emalyte which is subsequently to be analyzed.
- the insoluble redox agent can be considered substantially inert with respect to the fluid's analyte to be emalyzed.
- the eunount of insoluble redox-active agent used is that amount sufficient to eliminate the advers effect of any interfering redox-active contaminant in the fluid to be emalyzed.
- the eunount of insoluble redox-active agen used to treat one ml of bodily fluid ranges from about 1 to about 100 g, prefer ⁇ ibly from about 5 to about 20mg.
- the insoluble redox-active agent is placed in a physical state that is most effective to render the fluid's contaminants non-interfering. It is desirable to increase a much as possible its reactive surface areas. For instance. such a physical state includes that of a finely ground material.
- insoluble redox-active agents are: lead (IV) dioxide (PbO,), cuprous sulfide (Cu-S), eerie dioxide (CeO_), silver sulfide (Ag,S), silver ferrocy ⁇ mide (Ag 4 Fe(CN) 6 ), silver oxide (Ag 2 0) , manganese dioxide (Mn ⁇ 2 ), barium permemg ⁇ mate (BaMn0 4 ), cuprous carbonate, potassium dichrornate (K_Cr 2 0 7 ), potassium permanganate (KMn0 4 ), cuprous ferrocyanide, lead permanganate and the peroxyacetic acid derivative of carboxymethyl cellulose.
- the particular insoluble redox-active agent selected will depend on the fluid emd emalyte to be analyzed.
- redox-active agents in the invention belong to classes of compounds that can be identified as follows:
- electron transfer agents are generally used in an amount in the range of about 0.1-lOmM.
- the preferred range of amount of electron transfer agent is about 0.5mM- 2mM.
- this invention has special application where there is a need for an accurate analysis of a biological fluid.
- blood, plasma or serum is often analyzed for particular analytes of interest such as cholesterol, blood urea nitrogen, ketones, glucose, lactate or triglycerides.
- analytes of interest such as cholesterol, blood urea nitrogen, ketones, glucose, lactate or triglycerides.
- Interfering contaminants with which this invention deals include, without limitation the following classes of compounds: redox active acids, thiols emd bilirubin.
- these may include organic acids like ascorbic acid, as well as other substemces such as uric acid, gentisic acid, bilirubin, glutathione, cysteine, and thiol- containing peptides and proteins such as albumins.
- organic acids like ascorbic acid, as well as other substemces
- uric acid gentisic acid
- bilirubin glutathione
- cysteine and thiol- containing peptides and proteins
- albumins proteins
- the specific nature of many contaminants that interfere in the redox-based analysis of a fluid are generally unknown.
- proteins themselves may be associated with contaminants which may be substituents thereon. In those circumstances, these are oxidized.
- the assaying or analyzing of other bodily fluid for glucose, or monosaccharides is also of interest for patients with diabetes. Interfering contaminants in such bodily fluids include uric acid, ascorbic acid and thiol-containing proteins and peptides.
- the invention provides a system of treatment or method performed on a fluid which can be aqueous, non- aqueous, or a mixture.
- the fluid generally contains a redox active analyte or analytes which fluid contains a redox- active interfering contaminant.
- These contaminants are interfering in the sense defined earlier in that they interfere in the accurate analysis of a redox-active analyte in the fluid which is sought to be analyzed by a redox measurement system.
- the contaminemts are also interfering i the sense that they cause error or non-ideal performance in the use of the fluid.
- the invention provides a system and method whereby the fluid is treated with em insoluble redox-active agent. It is important to note that regardless of whether the fluid is aqueous, non-aqueous or partially aqueous, the redox- active agent used in this invention should be insoluble in the fluid.
- the eunount of insoluble redox-active agent introduced will depend on the fluid itself and the redox- active component to be inactivated by the redox reaction.
- the amount of insoluble redox-active agent used is that amount which is sufficient to render interfering redox-active contaminants non-interfering. In general an amount of insoluble redox-active agent is added which is in excess of that needed to insure the complete oxidation-reduction reaction of the insoluble redox-active agent with the redox- active contaminemts.
- the temperature at which the reaction is carried out is generally ambient temperature such as in the range of about 10*C to 50'C.
- reaction between the insoluble redox-active agent or agents or the redox-active contaminant or contaminants can be facilitated by the introduction of one or more electron tremsfer agents to the fluid.
- any remaining insoluble redox-active agent is removed. This removal is accomplished by means based on the insolubility of the redox-active agent.
- the resulting fluid is free of redox-active contaminemts as well as residual redox-active agent.
- This fluid is ideal for accurate analysis for redox-active analyte by redox-reaction measurement systems.
- the invention provides a fluid which is free in general of any residual non-specific reducing or oxidizing agent which would constitute an interference for the subsequent use of the fluid.
- the kit, system emd method disclosed herein generally involves a set of two sequential redox reactions.
- the first reaction involves the RAIC emd interfering redox- active contamin ⁇ mt.
- the second reaction is generally a NAD- NADH dependent redox reaction or a non-NAD-NADH oxido- reductase (enzymatic) redox reaction coupled to color changing reactions which involves the determination of the concentration or presence of an analyte.
- a large number of color changing reactions such as the peroxidase-chromogen reactions for hydrogen peroxide can be coupled to these redox reactions.
- the kit includes both those compounds necessary for rendering the contaminants non-interfering, and those compounds for a redox-based analysis.
- the kit typically includes a physical support or housing for two layers of filters between which is contained the RAIC.
- the fluid to be treated is generally passed through the first filter which may contain an electron transfer agent.
- the fluid dissolves the electron tremsfer agent, contacts the RAIC emd passes through the second filter with the RAIC retained.
- This invention is useful for removing interfering reducing substances from a liquid medium.
- This invention is particularly useful if the fluid to be treated is a sample that is to be analyzed by redox chemistry, or a sample where residual non-specific reducing or oxidizing agent constitute an interference for the subsequent use of the fluid.
- the invention is especially useful if the fluid is a bodily flui such as blood serum or plasma, amniotic fluid, cerebrospinal fluid, sinovial fluid, urine, saliva, tears, semen and the like, emd the subsequent use is the emalysis of the fluid fo the concentration of a component of the fluid by enzymatic and other redox reactions.
- analyte When an analyte is redox-active, it can be used in redox reactions to detect its presence and determine its concentration. Determination of em emalyte by redox reactions, however, opens the possibility of interference by emy non-emalyte redox-active contaminant in the sample. Therefore, it is useful to have a means by which interfering redox-active substances can be eliminated without affecting the emalytes that will subsequently be assayed by a redox reaction, and without leaving a new redox-active contaminant in the fluid, such as residual oxidizing agent.
- insoluble means the inability to dissolve to any appreciable extent in the fluid of interest.
- appreciable solubility refers to the residual concentration, if any, of the insoluble agent relative to the analyte of interest, and in typical situations, the residual will be less than 1/1000 of the emalyte concentration, and in any case, this residual concentration will typically be less them about 10 ppm in the product fluid.
- the oxidizing agent used in this invention is insoluble in the fluid to be treated or analyzed.
- em oxidizing agent as for any chemical composition, will depend upon the nature of the fluid.
- Manganese dioxide for example, is insoluble in water but soluble in UN HC1.
- Potassium dichromate is soluble in water but insoluble in alcohols.
- Solubilities of organic and inorge ic oxidizing (emd reducing) agents in a wide variety of solvents are well known to those skilled in the art, and are extensively compiled, for instance, in the Tables of Physical Constant of Inorganic and of Organic Compounds, found in the Hemdbook of Chemistry and Physics, CRC Publications, Cleveland, OH.
- oxidizing agents will mean any chemical composition having an electromotive potential such that it would react with emother chemical composition, called the reducing agent, having different electromotive potential, where electrons are tr ⁇ sferred spontaneously from the reducing agent to the oxidizing agent if a means or pathway for the electron tremsfer was available.
- em oxidizing compound may be used with an electromotive potential such that it can react with an interfering reducing conteuninemt.
- insoluble oxidizing (emd reducing) agents can be so made it is not obvious that insoluble oxidizing (or reducing) agents would actually effectively react with other redox partners. This may be because the insignificemt solution concentration of insolub agents would abolish the inherent redox capability or that the reaction would be prevented by the necessity for elect transfer across a physical phase barrier, or for other specific reasons.
- TCA trichloracetic acid
- lead (IV) dioxide insoluble oxidation agent is capable of destruction of all the contaminating reducing activity in plasma.
- An electron transferring substance is not needed for the action of PbO,' an ⁇ b 0 ⁇ * 1 the TCA soluble and insoluble contaminants are destroyed.
- Pb0 2 should be em acceptable agent.
- the standard oxidation potential of lead is 1.455 volts, considerably greater than the 1.229 volt reduction potential of oxygen.
- lead (IV) dioxide has the energetic capacity to split water, suggesting that it might not be stable in an aqueous solution. Stability is observed, however, probeibly due to the extreme insolubility of the lead oxidizing agent.
- the removal of contaminating reducing (or oxidizing) agent was accomplished by the pretreatment of a fluid with em insolubl oxidizing (or reducing) agent, especially when the fluid was also treated with a soluble electron transfer agent to provide more effective pathways for electrons to the insoluble agent.
- the resultant fluid is free of both oxidizing and reducing activity and will be especially suitable for uses where such residual activity constitutes a interference (but where the presence of the electron transfe agent does not constitute such interference).
- the invention is not intended to be limited to analytical uses of fluids pretreated in accordance with the invention, emd indeed, the general spirit and teaching of this invention me ⁇ ces obvious multitude of other uses to those of average skill in the ar
- the system comprises the treatment of some fluid, that fluid either hydrophilic (aqueous solutions), hydrophobic (lipid-like), e y mixture of the two.
- An insoluble oxidizing agent is introduced into the fluid so that the agent will contact th interfering contaminants.
- Some of the more preferred insoluble redox-active agents are: cuprous sulfide (Cu.S), eerie dioxide (Ce0 2 ), silver sulfide (Ag 2 S), silver ferrocyanide (Ag 4 Fe(CN) g ), silver oxide (Ag 2 0) , manganese dioxide (Mh0 2 ), barium permanganate (BaMn0 4 ), potassium dichromate (K 2 Cr 2 0 7 ), potassivun permanganate (KMnO.), cuprous ferrocy ⁇ mide, lead permanganate and the peroxyacetic acid derivative of carboxymethyl cellulose.
- the use of a particular insoluble redox-active agent will depend on the fluid emd emalyte to be analyzed.
- the amount of insoluble oxidizing agent to be used is that amount sufficient to render non- interfering any interfering reducing agents in the fluid to be emalyzed, but is usually an amount in excess of that eunount needed to insure the complete oxidation of all contaminants.
- This eunount is a function of the surface area the insoluble redox-active agent provides for effective contact with the interfering contaminant in the fluid. Since the redox-active agent is insoluble it cem only react with the interfering conteuninemt at its surface area in contact with the interfering conteuninemt.
- the liquid medium itself emd the contaminant to be rendered non-interfering also affect the amount of insoluble redox-active agent to be used.
- the reaction of the insoluble oxidizing (or reducing) agent may be further facilitated by inclusion of small amounts, usually in the remge of about 0.1-2 mM of emy of a variety of electron tremsfer agents, not limited to but including the group consisting of phenazine methosulfate, 2,6- dichlorophenolindophenol, substituted 1,4-benzoquinones including 1,4-benzoquinone, mono-, di-, tri- and tetra- substituted 1,4-benzoquinones where the substituents may include, but are not limited to, simple alkyl groups containing 1 to 5 carbons, halogens, alkoxyl radicals with l to 4 carbons, among other possible substitutions.
- Substituted 1,4-benzoquinones of particular utilit in this regard are 2,5- and 2,6-dimethyl-l,4-benzoquinone, 2,5- emd 2,6-dichloro-l,4-benzoquinone, 2-isopropyl-5-methyl 3,6-dibromo-l,4-benzoquinone, 2-methoxy-5-methyl-l,4- benzoquinone, emd 2,3-dimethoxy-5-methyl-1,4-benzoquinone.
- Substituted and unsubstituted 1,4-naphthoquinones, emd 1,2- benzoquinones are also generally useful as electron transfer agents in this invention.
- a large collection of redox-activ dyes generally members of the phenazine, phenoxazine, phenothiazine and indamin families of dyes also function wel as electron tremsfer agents, although they are usually not preferred because they impart a color to the final treated fluid.
- emalyte is passed over a be of the insoluble oxidizing (or reducing) agent.
- the bed may contain an amount of oxidizing agent so that all contaminating reducing agents are oxidized as the emalyte passes over the bed.
- the surface area of the bed is prefere ⁇ ly sufficiently large so that all analyte comes in contact with oxidizing agent as the emalyte flows through th bed.
- the bed of oxidizing agent may be supported on a porou filter matrix that retains the oxidizing agent while allowin the fluid to pass through rapidly.
- the insoluble oxidizing agent is sandwiched between two membranes.
- the analyte is introduced through the top of one membrane, flows through the layer of oxidizing agent, then passes through the second membrane.
- the membrane allows the rapid passage of fluid, but retain the oxidizing agent so that it is separated from the fluid.
- the kit represents a specific physical disposable embodiments of the system.
- the oxidizing agent is bound to a substrate.
- the substrate is insoluble, and can consist of, but is not limited to membranes, polymers, and columns of some supporting material. Binding the oxidizing agent to an insoluble support has the effect of rendering the oxidizing agent insoluble. Analyte is then passed through the oxidizing agent which is attached to the support substrate. In this embodiment, filtration is not required since the emalyte will not carry any of the bound oxidizing agent with it.
- insoluble oxidizing agent and analyte can be mixed together in some container, then mixed for an amount of time sufficient to allow all analyte to be contacted by the oxidizing agent.
- the oxidizing agent can then be removed from the analyte by filtration, decantation, sedimentation or centrifugation.
- the electron tremsfer agent may be introduced to the fluid dried on a support layer that constitutes the first layer of the sandwich, such that the fluid contacts this layer just prior to contacting the oxidizing agent. In such an embodiment, some of the electron transfer agent leaches into the fluid in passage to the oxidizing agent.
- the embodiment involving a support layer with electron transfer agent dried on, followed by a sandwich of oxidizing agent may, in certain configurations, permit the fluid to be actively driven through the structure holding the sandwich layers. This embodiment is particularly efficient and rapid for pretreating the fluid.
- the amount of insoluble oxidizing agent used is set by the dimensions of the support system.
- the support system is designed in such a way to offer enough surface area of insoluble oxidizing agent so that all of the analyte flowing through the system will be contacted by the oxidizing agent.
- the length of contacting time has to be sufficiently long to allow the oxidation of all contaminating reducing agents.
- reaction chambers into which the emalyte is made to pass.
- a chamber in this case consists of any enclosed are designed to contain the fluid being emalyzed, and can be open at one or both ends and be of any desired shape and size.
- the first chamber contains the insoluble oxidizing agent, and is designed for the emalyte to be introduced into one end of the chamber, then pass through to the other end readily, where it enters the second chamber.
- This second chamber contains the redox measuring system, and can include but is not limited to one of the redox measuring systems described in the Background and Prior Art Section of this patent application.
- the first chamber or pretreatment chamber contains enough insoluble oxidizing agent so that all contaminating reducing substances are rapidly reduced as the fluid passes through. If the oxidizing agent used consists of loose, insoluble particles, there is provided a filter between the two chamber. If the oxidizing agent is bound to an insoluble support, such filter is unnecessary. It is an advantage of this invention that the analyte does not have t first be treated separately by one procedure wholly apart from the redox measuring system, then introduced into the measuring system is a separate step - the emalyte is treated then passes directly to the measuring system. It is also an advantage that treatment is rapid, and does not interfere with obtaining accurate results from the redox measuring system.
- filters, filter matrices, and membranes which have been discussed in connection with embodiments of the invention are insoluble, hydrophilic, synthetic or natural polymers.
- a solution of 100 mg/dL glucose was prepared, and dosed with 1 mM dithiothreitol (DTT). This solution was then measured with a glucose enzyme electrode. An erroneous result was obtained based on the known amount of glucose put in the solution.
- DTT dithiothreitol
- insoluble silver (I) compounds such as silver ferrocyanide (Ag 4 Fe(CN) 6 ) emd silver oxide (Ag 2 0), were also found to perform similarly.
- a particular reducing sugar assay monosaccharides are determined by oxidation in an alkaline solution by potassium ferricyanide, and the reduction of ferricyemide is measured by the decrease in absorbance at 420 nm.
- This is a redox measurement system that does not employ a protein catalyst.
- An insoluble oxidizing filter was formed by making a slurry of Mn0 2 emd cellulose fibers, then drying down to form the filter.
- the mannose/uric acid solution was passed through this filter at neutral pH.
- the solution was made basic by the addition of sodium hydroxide, since mannose is not reducing unless the pH is high, then run in the reducing sugar assay again, this time with the expected results. Therefore, uric acid was oxidized but mannose was not.
- Example 7 Some oxidizing agents are soluble in aqueous solutions, but they are not soluble in an organic solvent, that is, non-aqueous fluid. This is true for potassium dichromate (K-Cr.O-), which is soluble in water, but not in ethemol.
- K-Cr.O- potassium dichromate
- Example 8 Human plasma was analyzed for its lactate concentration by incubating an aliquot of freshly prepared plasma in a reaction mixture containing:
- the plasma contained an interfering reducing activity equivalent to 0.5 mM lactate in the native plasma.
- Example 9 Measurement of plasma cholesterol in a machine- independent, disposable device. 50 ul of human plasma were placed in a plastic tube containing a sandwich of filter papers. Suitable filter papers were prepared from cellulose, nitrocellulose, nylon, fiberglass or polycarbonate polymers.
- the first filter paper contained 1 umol of 2-isopropyl-5- methyl-3,6-dibromo-l,4-benzoquinone. Beneath this paper a bed 1 mm thick of finely ground Mh0 2 was contained, held in place a bed 1 mm thick of finely ground MnO_ was contained, held in place by a second filter paper.
- This tube was constricted, mechanically holding the sandwich in place, with an opening going to a 20 ul capillary containing a dried film that, when rehydrated by entrance of aqueous fluid, would give a solution of 0.1 M potassium phosphate, pH 7.4, 2% sodium cholate, 200 U/ml porcine pancreatic cholesterol esterase, 20 U/ml cholesterol oxidase, 1 mM thiazolylblue tetrazolium (MTT), 3% trans-l,2-cyclooctanediol, and 12.4 mM potassivun ferricyanide.
- a firmly fitting plunger is used to drive the plasma through the sandwich and into the capillary.
- the plasma hydrates the reagents, causing hydrolysis of cholesterol esters by the cholesterol ester hydrolase, and oxidation of the cholestero by the oxidase.
- the electrons made available by the oxidation reduce the ferricyemide.
- electrons then emd only then reduce the tetrazolium dye giving a sharp color change by formation of the highly colored fomazan dye.
- concentration of cholesterol in a particular plasma was 230 mg/dl, equal to 5.94 mM.
- a solution of 0.05 M Tris chloride, pH 7.7 contained 2.4 mM hydrogen peroxide and 0.4 mM ascorbic acid.
- the hydrogen peroxide content of the solution was measured by dilution of 1 volume into 19 volumes of a solution containing 20 U/ml horseradish peroxidase, 0.1 M potassium phosphate pH 7.3, 0.8 mM 4- aminoantipyrine and 20 mM 4-hydroxybenzoic acid.
- the solution was allowed to react for 10 min, emd the etbsorbance was read at 500 nm in a spectrophotometer.
- 1,4-benzoquinone emd a 1 mm deep bed of Mn0 2 the resultant plasma measured 5.23 mM glucose in the glucose oxidase, peroxidase/benzidine color reaction described above.
- the interfering reducing substances present in the blood were removed from the plasma by the insoluble oxidizing agent and electron tremsfer agent prior to analysis, permitting an essentially correct measurement of the glucose content.
- the glucose content measure without pretreatment by insoluble oxidizing agent was 1.82 m glucose.
- the glucose content was found t be 2.45 mM and in the oxygen electrode, the content was determined to be 2.45 mM.
- cerebrospinal fluid is subject to greater relative error in glucose determination them is blood due to the typically lower glucose concentrations and the typically higher content of reducing substances like ascorbic acid.
- a sample of human plasma was diluted 20-fold into a measurement reaction containing 0.1 M Tris chloride pH 7.5, 20 U/ml pancreatic cholesterol esterase, 20 mM sodium cholate, 0.2% Triton X- 100, 5 U/ml Nocardia cholesterol oxidase, 0.4 mM 4- aminoemtipyrine, 10 M phenol and 25 U/ml horseradish peroxidase.
- the mixture was incubated for 20 min at 37*C emd the color determined at 500 nm in the ususal manner.
- the color of the test mixture was related to cholesterol concentration.
- the cholesterol content was found to be 4.87 mM (equivalent to 188 mg/dl).
- oxygen consumption corresponded to a cholesterol content of 5.23 M (202 mg/dl). If the plasma was pretreated by amendment with 0.5 mM of 2,5-dichloro-
- the measured cholesterol content was found to be 5.20 mM, in excellent agreement with the value determined by the electrode.
- the pretreatment with insoluble oxidizing agent and electron transfer agent removed unidentified reducing substemces in the plasma that were responsible for the falsely low measurement.
- Interfering reducing substemces were rendered non-interfering by an organic oxidizing agent attached to an insoluble matrix.
- the peroxyacetic acid derivative of carboxymethyl cellulose (CMC) was prepared by incubating 100 g CMC with 100 ml 90% hydrogen peroxide for 6 h at room temperature, and the derivatized product washed extensively with water to remove the hydrogen peroxide.
- the wet peroxyacetic derivative was mixed with an equal weight of cellulose carrier and the mixture dried to 50% relative humidity at 20*C. Twenty mg of this matrix was placed in the interior of a glass tube emd used for the pretreatment of 100 ul of the plasma described in example 12, by forcing the plasma through the tube with a plunger.
- the cholesterol content of the plasma following this treatment was measured by the esterase/oxidase/peroxidase color system described in example 12 to be 5.24 mM, in excellent agreement with the correct value.
- this insoluble orgemic oxidizing agent also destroyed the contaminating reducing substances in the plasma, without affecting the emalyte concentration.
- Pretreatment of Plasma by Lead (IV) Dioxide Prior to Cholesterol Measurement A small circle of glass fiber filter material precoated with lead (IV) dioxide, as discussed above, was inserted into the bottom of a 1 ml disposable plastic syringe. A seunple of plasma, of approximately 0.5 ml was added to the syringe, emd the plasma pushed through the filter at such a rate so that at least 1 sec of filter time occurred. The cholesterol content of the treated plasma was subsequently measured in a Ciba Corning Impact 400E clinical analyzer, using the manufacturer's cholesterol reagent and run accoring to the manufacturer's specifications.
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Abstract
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Application Number | Priority Date | Filing Date | Title |
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US33762489A | 1989-04-13 | 1989-04-13 | |
US337624 | 1989-04-13 | ||
US50055490A | 1990-03-28 | 1990-03-28 | |
US500554 | 1990-03-28 |
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EP0467978A1 EP0467978A1 (en) | 1992-01-29 |
EP0467978A4 true EP0467978A4 (en) | 1992-03-11 |
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Application Number | Title | Priority Date | Filing Date |
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EP19900906723 Withdrawn EP0467978A4 (en) | 1989-04-13 | 1990-04-12 | The use of fluid insoluble oxidizing agents to eliminate interfering substances in oxidation-reduction measuring systems |
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Country | Link |
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EP (1) | EP0467978A4 (en) |
JP (1) | JPH04504504A (en) |
AU (1) | AU5527090A (en) |
CA (1) | CA2051119A1 (en) |
WO (1) | WO1990012113A1 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
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US4954451A (en) * | 1989-06-26 | 1990-09-04 | Miles Inc. | Agent for diminishing ascorbate interference in reagent systems and method relating thereto |
US6162647A (en) * | 1997-10-14 | 2000-12-19 | Stephens; James Matthew | Method for removing interfering substances from a urine sample using a chemical oxidant |
US6352835B1 (en) | 1998-11-17 | 2002-03-05 | Kyoto Daiichi Kagaku Co. Ltd. | Method of measuring substance in sample using a redox reaction |
US7045098B2 (en) | 2001-02-02 | 2006-05-16 | James Matthew Stephens | Apparatus and method for removing interfering substances from a urine sample using a chemical oxidant |
US7153666B2 (en) | 2003-07-17 | 2006-12-26 | General Atomics | Methods and compositions for determination of glycated proteins |
DE602006010084D1 (en) | 2005-08-05 | 2009-12-10 | Bayer Healthcare Llc | METHOD FOR THE DISTINCTION OF ELECTROCHEMICAL SENSORS |
US7855079B2 (en) | 2006-07-25 | 2010-12-21 | General Atomics | Methods for assaying percentage of glycated hemoglobin |
US7943385B2 (en) | 2006-07-25 | 2011-05-17 | General Atomics | Methods for assaying percentage of glycated hemoglobin |
US8673646B2 (en) | 2008-05-13 | 2014-03-18 | General Atomics | Electrochemical biosensor for direct determination of percentage of glycated hemoglobin |
CN112226483A (en) * | 2020-11-05 | 2021-01-15 | 洛阳恒恩生物科技有限公司 | High-stability reduced nicotinamide coenzyme determination reagent and preparation method thereof |
CN114371165B (en) * | 2022-01-04 | 2023-07-25 | 杭州英普环境技术股份有限公司 | Method for detecting silicon dioxide content in high-chroma and reductive wastewater |
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DD143379A3 (en) * | 1978-07-25 | 1980-08-20 | Kallies Karl Heinz | INDICATOR TUBES FOR GLUCOSE DETERMINATION |
US4310626A (en) * | 1980-06-02 | 1982-01-12 | Miles Laboratories, Inc. | Interference-resistant composition, device and method for determining a peroxidatively active substance in a test sample |
US4897346A (en) * | 1986-07-15 | 1990-01-30 | Beckman Instruments, Inc. | Stabilized liquid enzyme composition for glucose determination |
-
1990
- 1990-04-12 CA CA 2051119 patent/CA2051119A1/en not_active Abandoned
- 1990-04-12 EP EP19900906723 patent/EP0467978A4/en not_active Withdrawn
- 1990-04-12 AU AU55270/90A patent/AU5527090A/en not_active Abandoned
- 1990-04-12 JP JP50644390A patent/JPH04504504A/en active Pending
- 1990-04-12 WO PCT/US1990/001887 patent/WO1990012113A1/en not_active Application Discontinuation
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WO1990012113A1 (en) | 1990-10-18 |
CA2051119A1 (en) | 1990-10-14 |
JPH04504504A (en) | 1992-08-13 |
EP0467978A1 (en) | 1992-01-29 |
AU5527090A (en) | 1990-11-05 |
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