EP0618980A1 - Determination colorimetrique de la peroxydase et du peroxyde - Google Patents

Determination colorimetrique de la peroxydase et du peroxyde

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Publication number
EP0618980A1
EP0618980A1 EP92906329A EP92906329A EP0618980A1 EP 0618980 A1 EP0618980 A1 EP 0618980A1 EP 92906329 A EP92906329 A EP 92906329A EP 92906329 A EP92906329 A EP 92906329A EP 0618980 A1 EP0618980 A1 EP 0618980A1
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EP
European Patent Office
Prior art keywords
dye
peroxidase
hydrogen peroxide
reaction system
test sample
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.)
Withdrawn
Application number
EP92906329A
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German (de)
English (en)
Inventor
Steven Dale Salt
Neal Arthur Siegel
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Abbott Laboratories
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Abbott Laboratories
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Publication date
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Publication of EP0618980A1 publication Critical patent/EP0618980A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives

Definitions

  • the present invention relates to the colorimetric determination of peroxidases and peroxide.
  • the present invention relates to the colorimetric determination of peroxidases, peroxide, compounds which can be directly or indirectly oxidized to yield a stoichiometric quantity of hydrogen peroxide, and catalysts which directly or indirectly oxidize compounds to yield a stoichiometric quantity of hydrogen peroxide.
  • the present invention provides a method to determine the presence or amount of peroxidase, hydrogen peroxide (HO ), and substances which are capable of generating hydrogen peroxide, or catalyzing the generation of hydrogen peroxide.
  • a sulfonephthalein dye or a phthalein dye having a major absorbance peak in the visible region, preferably at a wavelength from between about 500 nanometers to about 700 nanometers is subjected to oxidative extinguishment without strong alkali treatmen .
  • the method is performed by forming a reaction system comprising such sulfonephthalein dye or phthalein dye and: (a) a test sample containing an unknown amount of peroxidase; and an amount of H 2 0 2 sufficient to cause extinguishment of the absorbance peak of such dye whereby the amount of peroxidase can be determined by monitoring such extinguishment; or
  • test sample containing an unknown amount of a compound which is capable of undergoing a reaction with oxygen to form H 2 0 2 ; peroxidase; and, if necessary, ' an amount of an enzyme or other catalyst which causes the compound to undergo the reaction which forms HO , wherein the HO which is generated causes extinguishment of the absorbance peak of such dye whereby the amount of the compound can be determined by monitoring such extinguishment; or
  • extinguishment of the absorbance peak of such dye has been monitored and the value thereof determined, such extinguishment value is compared with previously prepared standards to determine the unknown amount.
  • the extinguishment of the absorbance peak can be monitored by an instrument or may be visually monitored by observing the change of hue.
  • the method of the present invention is particularly useful for determining the presence or amount of peroxidase which may be bound to, for example, an antibody or " analyte, such as in an immunoassay method or system, or for determining an analyte or an enzyme or other catalyst which participates in the generation of hydrogen peroxide by, for example, enzymatic oxidation, wherein the amount of hydrogen peroxide so produced, or rate of production thereof, is related to the amount of analyte or enzyme present in a test sample.
  • the dye employed according to the present invention may be present in solution as a free form thereof, or it may be bound, absorbed or otherwise affixed to a wettable, solid matrix according to methods known in the art.
  • Figs. 1-3 are graphs which illustrate changes in absorbance of a dye in a liquid phase spectrophotometric assay for a peroxide generating analyte according to the present invention.
  • Figs. 4-5 are graphs which illustrate the general principal of the change in color hue of dyes employed according to the present invention.
  • Fig. 6 illustrates the general structure of nonionized sulfonephthalein dyes.as contemplated by the present invention.
  • Fig. 7 illustrates the general structure of ionized sulfonephthalein dyes as contemplated by the present invention.
  • Fig. 8 illustrates the general structure of nonionized phthalein dyes as contemplated according to the present invention.
  • Figs. 9-13 are graphs which illustrate the absorbance of reaction mixtures employed in liquid phase kinetic assays for peroxidase according to the present invention.
  • Figs. 14-17 are graphs which illustrate the effect of pH when performing the method according to the present invention.
  • H 0 2 means hydrogen peroxide; "percent" and “parts” refer to percent and parts by weight, unless otherwise indicated; g means gram or grams; mg means milligram or milligrams; ⁇ g means microgram or micrograms; ng means nanogram or nanograms; cm means centimeter or centimeters; mm means millimeter or millimeters; nm means nanometer or nanometers; A means absorbance; 1 means liter or liters; ⁇ l means microliter or microliters; ml means milliliter or milliliters; means mole percent, and equals 100 times the number of moles of the constituent designated in a composition divided by the total number of moles in the composition; V means percent by volume; M means molar and equals the number of gram moles of a solute in 1 liter of a solution; mM means illimolar and equals the number of millimoles of a solute in 1 liter
  • certain sulfonephthalein dyes and phthalein dyes are progressively oxidized by HO in the presence of a suitable catalyst, such as a peroxidase enzyme, and as a result of such oxidation, undergo a succession of changes in color hue.
  • a suitable catalyst such as a peroxidase enzyme
  • the change of color hue is caused by the oxidative extinguishment of the absorbance peak of sulfonephthalein dyes and phthalein dyes at a wavelength in the visible region, preferably from between about 500 nanometers and about 700 nanometers.
  • sulfonephthalein dyes include, but are not intended to be limited to, bromcresol green, bromcresol purple, bromcresol blue, bromthymol blue, thymol blue, phenol red, and the like
  • phthalein dyes include, but are not intended to be limited to, phenophthalein, and the like.
  • bromcresol green and bromcresol purple are yellow when the amount of H 2 0 2 is sufficient to cause complete oxidation and when there is an excess of HO .
  • the hue is that which results from a blending of the yellow oxidized dye with the unoxidized dye, which is blue in the case of bromcresol green and purple in the case of bromcresol purple. Accordingly, by carrying out a plurality of such oxidations of differing, known amounts of bromcresol green or of bromcresol purple and observing the hue subsequent ' to oxidation, it is possible to select a known amount of the dye which is sufficient to react with the amount of HO formed by enzymatic oxidation of the unknown sample or, alternatively, to estimate the amount of unknown analyte in a sample by visually determining the extent of color change in a known quantity of dye.
  • the rate at which hue changes as a result of the oxidation is a direct function of the concentration of the peroxidase or of any substance to which peroxidase is stoichiometrically bound.
  • a peroxide or an an Jyte such as glucose or cholesterol which can undergo an enzymatic reaction which produces an amount of H 2 0 2 or other peroxide stoichiometrically related to the amount of the analyte, can be combined in a reaction system with peroxidase, a dye as heretofore described, and, in the case of, for example, glucose, cholesterol or the like, an enzyme which causes the glucose, cholesterol or the like to react, to produce H0.
  • the extent of the enzymatic oxidation of such dye by the HO and the extent of the change in hue are a measure of the amount of the analyte present in the aqueous system.
  • the amount of free or bound peroxidase, or the unknown amount of a catalyst is determined by monitoring the rate of change of hue, preferably by monitoring the rate of change of absorbance at a given wavelength at which oxidation of the dye extinguishes absorbance. It is to be understood that the amount of H_,0 2 or the amount of an H 2 0 2 -generating analyte can be determined by monitoring such rate of change either visually or with an instrument.
  • a reaction system is formed, which may or may not include solid state chemically inert components to determine sample volume, deliver reagents, separate components or perform other functions, comprising peroxidase, a dye according to the present invention, and either a sample containing the peroxide to be determined or a sample of the analyte compound to be determined, and an enzyme, if required, which causes the compound to undergo a reaction which forms H 2 0 2 .
  • the system is then observed to determine the hue.
  • SUBSTI determination Although it is preferred to employ an instrument to measure absorbance when peroxidase is being kinetically determined, a semiquantitative visual determination of peroxide, glucose, cholesterol or the like substances capable of generating peroxide, may be accomplished as well.
  • Fig. 6 illustrates non-ionized sulfonephthalein dyes
  • Fig. 7 illustrates ionized sulfonephthalein dyes
  • Fig. 8 illustrates non-ionized phthalein dyes
  • Fig. 6 illustrates non-ionized sulfonephthalein dyes
  • Fig. 7 illustrates ionized sulfonephthalein dyes
  • Fig. 8 illustrates non-ionized phthalein dyes
  • such dyes possess the following properties: (a) -an oxidatively extinguishable light absorbance peak in the range of 500 to 700 nm (green to violet) of sufficient magnitude to mask a non-oxidatively bleachable peak of much lower magnitude at ca.
  • Phenol red as shown in Fig. 5, possesses an oxidatively extinguishable absorbance peak at ca. 560 nm while a peak at ca. 430 nm is resistant to oxidative bleaching.
  • the 430 nm (yellow-orange) peak is of substantially greater magnitude than the 560 nm peak so that peroxidase-catalyzed oxidation results in a hue change (scarlet-orange to orange) which can be followed instrumentally at 560 nm or visually with some degree of difficulty;
  • SUBSTITUTESHEET (b) a p a of ionization of the phenolic hydroxyls (and, consequently, a shift from generally yellow acid pH color to green to violet alkaline pH color) of less than 8, preferably less than 7, so that the dye will be in the ionized form and susceptible to oxidation at a pH within the range where peroxidase is active (pH 5 to 8).
  • bromcresol green When used as pH indicators, bromcresol green ionizes and changes color at pH 3.8 to 5.4; bromcresol purple at pH 5.2 to 6.8; bromthymol blue at pH 6.0 to 7.6; phenol red at pH 6.8 to 8.2; thymol blue at pH 8.0 to 9.2; and phenolphthalein at pH 8.5 to 10. Furthermore, it has been found that while phthalein and sulfonephthalein dyes may be oxidatively bleached at any pH above the lower limit of their color shift ranges (e.g. pH 5.2 for bromcresol purple), that the kinetics (speed) of oxidation are greatest at a pH close to the bottom of the range of their color shift.
  • pH 5.2 for bromcresol purple
  • the magnitude of the oxidatively bleachable absorbance peak at 500-700 nm is quite low at a pH near the lower limit of that at which the color change occurs and increases rapidly with increase in pH to a maximum at and above the upper pH limit of the color shift range.
  • sensitivity of assays is a compromise between maximum speed (kinetics) at a pH near the lower limit at which the color shift begins (phenolic ionization) and maximum discrimination between pre-oxidation and post- oxidation absorbance peak intensity (500-700 nm) at a pH near or above the upper pH limit at which the color shift occurs.
  • the pH of the buffer in which the assay is run can be manipulated to maximize (1) the speed of reaction (a pH toward the lower end of the color shift range; desirable for many instrumental applications or for assays for analytes expected to be present in low concentrations) , or (2) the magnitude of the difference between initial and final absorbance values and, thus, the apparent extent of hue changes (a pH toward the higher end of the color shift range, desirable for many visual se iquantitative assays, or for assays for analytes expected to be present in relatively high concentrations and thus requiring a large dynamic range for quantitation) .
  • the optimal pH compromise between speed and magnitude of color change appears to be in the upper third of the pH-induced pre-oxidation ionization range, e.g., pH 7.5 to 7.8 for phenol red, pH 5.2 to 5.6 for bromcresol green, pH 6.5 for bromcresol purple, and pH 7.4 for bromthymol blue.
  • pH 7.4 in a 50 mM sodium phosphate buffer solution
  • pH 6.0 in a 50 mM potassium 2-(N-morpholino) ethanesulfonate ["MES"] buffer solution.
  • the present invention can be modified to measure different ranges of analyte concentrations simply by altering dye concentration so as to make the range between 0% and 100% oxidized dye the same order of magnitude as the range in anticipated analyte concentrations.
  • the present invention will now be illustrated, but is not intended to be limited, by the following examples:
  • a spectrophotometric assay for the kinetic determination of glucose was performed employing molecular oxygen, glucose oxidase and bromcresol purple.
  • the glucose oxidase served as a catalyst to oxidize glucose in solution to gluconic acid and hydrogen peroxide wherein the hydrogen peroxide in the presence of the peroxidase oxidized the bromcresol purple.
  • Reactions were conducted by preparing mixtures at ambient temperature (ca. 22°) in 2 ml spectrophotometric cuvettes by mixing 1.5 ml of the bromcresol purple solution, 20 ⁇ l of the glucose oxidase solution, 15 ⁇ l of the horseradish 0 peroxidase solution and 0.5 ml of water or 0.5 ml of water containing varying amounts of D-glucose, mixing rapidly, and monitoring light absorbance at 589 nm as a function of time.
  • the amounts of D-glucose ranged from 0.0 to 3.6 mg (0-20 /-mole) .
  • 1, 2, and 3 are graphs which 5 illustrate absorbance at 589 nm as a function of time for reaction mixtures which contained, respectively, 0, 0.25 and 0.5 imole/ml glucose.
  • Fig. 1 shows that, with no peroxide added, the absorbance at 589 nm remained the same, slightly under 3.0, for 5 minutes.
  • Fig. 2 shows a decrease 0 in absorbance, when the reaction mixture contained 0.5
  • Fig. 3 shows a faster decrease in absorbance, when the reaction mixture contained 1.0 /anole glucose, the slope of the curve being estimated to be 2.8. 5
  • the three traces shown in Figs. 1, 2 and 3, as well as others described herein, were taken with a Beckman DU- 70 spectropho-tometer (Beckman Instruments), 1.0 cm pathlength, with an incandescent light source, and scanning was at a rate of 15 nm per second.
  • Example 1 the procedures described above in Example 1 have not been, but could be, optimized, and that they illustrate reactions that could be used to provide a reliable analysis for H 2 0 2 , for glucose, and for other analytes which react with molecular oxygen in the presence of oxidative enzymes to produce H 2 0 2 .
  • the reactions could also be used to provide a reliable analysis for other analytes which undergo an enzymatic or non- enzymatic oxidation that produces H 2 0 2 , so long as the reaction does not involve the use of a catalyst or the like which interferes with the oxidation of bromcresol purple or the like by the peroxide.
  • the procedures described above can be repeated using amounts of glucose in the reaction mixture ranging from 0.4 /"mole to 1.0 /-mole to provide data for a curve showing the slope of a curve plotting absorbance at 589 nm as a function of glucose content.
  • a differentiating spectrophotometer which plots rate of change of absorbance at 589 nm can be used.
  • lower concentrations of bromcresol purple can be used to develop data where glucose in the reaction mixture is less than 0.4 /oriole or higher concentrations can be used to develop data where glucose is higher than 1.0 /jmole, and like techniques can be used to develop data to be used to analyze for peroxides or for other analytes.
  • the following solutions were prepared; (a) 0.04 mg/ml bromcresol green in 50 mM sodium phosphate buffer (pH 6.8); and (b) 10 mg/ml horseradish peroxidase in 50 mM sodium phosphate buffer (pH 6.8).
  • a reaction mixture was prepared by mixing 1.5 ml of the bromcresol green solution, 10 ⁇ l 0.1 M H 2 0 2 and, after a spectrophotometer trace was taken, 5 ⁇ l of the horseradish peroxidase solution was added. Two more spectrophotometer traces of the reaction mixture were taken, a first one taken two minutes after the peroxidase addition, and a second one taken four minutes thereafter.
  • Fig. 4 is a graph which illustrates the three traces taken as described in the preceding paragraph, 325 nm to 700 nm, wherein curve A is the pre-oxidation trace taken before the peroxidase addition, curve B is the trace taken two minutes after the peroxidase addition, and curve C is the trace taken four minutes after the peroxidase addition. It is readily apparent from Fig. 4 that oxidation of bromcresol green progressively extinguishes absorption at 612 nm (blue) while affecting neither the magnitude nor the position of the absorption peak at ca. 400 nm (yellow) .
  • the reaction may be followed at a single wavelength, the progressively diminishing peak being at 612 nm.
  • the major absorbance peak for bromcresol purple is at 589 nm
  • the minor absorbance peak is at 400 nm. It will be appreciated that traces similar to those of Fig. 4 of the system described in Example 1 which contained 1.0 /zmole glucose would have the general appearance of Fig. 4 and from the data of Fig.
  • a reaction mixture was then prepared by mixing 1.5 ml of the phenol red solution, 50 ⁇ l of 0.1 M H 2 0 2 and, after a spectrophotometer trace was taken, 15 ⁇ l of the horseradish peroxidase solution. Another spectrophotometer trace of the reaction mixture was taken fifteen minutes after the peroxidase addition.
  • Fig. 5 is a graph which illustrates the two traces taken as described in the preceding paragraph (400 to 700 nm) wherein curve A is the pre-oxidation trace taken before the peroxidase addition, and curve B is the trace taken fifteen minutes after the peroxidase addition. It will be seen from Fig. 5 that phenol red follows a path similar to that followed by bromcresol green, but differs in that before oxidation, the "yellow" (actually orange) peak near 430 nm is quantitatively much larger than the oxidatively reduced "red” peak near 560 nm.
  • bromcresol green has two major light absorbance bands, one at about 625 nm and one at about 405 nm.
  • bromcresol purple has a major light absorbance band at about 589 nm and another at about 400 nm.
  • the absorbances at 625 nm and at 589 nm are markedly greater than those at the wavelengths of the other major absorbance bands.
  • SUBSTITUTESHEET 625 nm and 589 nm are extinguished and, as a consequence, the changes in hue and in absorbance are large. Bromthymol blue functions similarly with absorption maxima at 618 nm (extinguished by oxidation) and 405 nm (not extinguished) . 5
  • Filter paper discs (Whatman No. 1), 6.5 mm in 10 diameter, were impregnated with a 5 ⁇ l portion of a solution of 7.5 mg/ml bromcresol green in ethanol, and dried. Each of the dried discs were then impregnated with a 5 ⁇ l portion of an aqueous solution which contained 0.08 percent of horseradish peroxidase in 60 mM sodium phosphate 15 buffer (pH 7.4), and dried. Finally, each of the discs was impregnated with a 5 ⁇ l portion of an aqueous 0.1 N sodium nonanoate solution and dried.
  • the dried discs were placed in wells of standard 96-well matrix microtiter plates, and a 5 ⁇ l portion of water or of up to 20 mM hydrogen peroxide 20 in water was titrated onto each disc. After the color development reached equilibrium (ca. 20 minutes at room temperature, ca. 22°), the discs were dried at room temperature and their colors were observed. There was a visually observable, successive progression in color on the 25.discs from deep royal blue (0 mM peroxide) through shades of blue-green, green and yellow-green (1-5 mM peroxide) to yellow (8 mM peroxide) to orange-yellow (10-20 mM peroxide) .
  • a qualitatively similar, nonoptimized, assay for cholesterol/cholesterol esters in plasma was performed in which cholesterol/cholesterol ester-containing blood plasma was applied to a porous polystyrene cylinder impregnated with cholesteryl ester esterase and cholesterol oxidase (oxidizing cholesterol in the presence of atmospheric oxygen to cholestanone plus hydrogen peroxide) and placed upon similarly treated paper discs in place of the water or hydrogen peroxide solution. The degree of "yellowness" of the discs appeared to be directly proportional to the amount of cholesterol applied.
  • a liquid phase kinetic assay for peroxidase was performed by first preparing the following solutions: (a) 0.067 mg/ml bromcresol purple in 60 mM sodium phosphate buffer (pH 7.4);
  • Figs. 9, 10, 11, 12 and 13 are graphs which illustrate the data, showing absorbance at 589 nm as a function of time for reaction mixtures which contained 1.0, 0.5, 0.2, 0.1 and 0.05 mg/ml horseradish peroxidase, respectively, and include tangential lines which represent the slopes of the straight portions of the curves.
  • the slopes calculated for each the curves are set forth in Table I below:
  • assays for peroxides and chemical analytes susceptible to oxidative production of peroxides can be performed either kinetically or as end-point assays
  • the embodiment of the invention as an assay for substances catalyzing the oxidation of said substances with the consumption of peroxides can only be performed in a kinetic format, with the rate of the color change reaction (extinguishment of one of he absorbance peaks) proportional to the peroxidase or the like activity present, provided the concentration of dye is sufficient to saturate the enzyme reaction.
  • peroxidase enzyme that obtained from horseradish roots
  • numerous other natural sources in plant and animal tissues and microbes are known, e.g., potatoes, white blood cells, and various bacteria, and the enzyme is likely widespread in many other biological materials.
  • many other protein and non- protein substances, e.g., heme exhibit peroxidase activity though with generally lower speed and substrate specificity than classical peroxidase enzymes.
  • Figures 14, 15, 16 and 17 illustrate graphs of the data, showing absorbance at 612 nm as a function of time for the four reaction mixtures.
  • Figs. 14 through 17 demonstrate the dramatic effect of pH upon both initial absorbance values and rates of oxidative extinguishment of the higher wavelength light absorbance peak of sulfonephthalein dyes.
  • the initial absorbance at 612 nm is 1.2 at pH 5.6, 2.6 at pH 6.0 and 2.8 at pH 6.5 and pH 7.4.
  • the pH-dependent color change range of bromcresol green as a pH indicator dye is "3.8 (yellow) to 5.4 (blue)", although it still appears green-blue at pH 5.6 and royal blue only above pH 6.
  • maximum kinetics of oxidation were fastest at pH 5.6 (6-7 OD/min).
  • the rate was 2.1

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Abstract

Procédé permettant de déterminer la présence ou la quantité de peroxydase ou de peroxyde d'hydrogène dans lequel on utilise un colorant à la sulfonephtaléine ou un colorant à la phtaléine présentant une pointe d'absorbance dans le champ visible, de préférence à une longueur d'onde comprise entre environ 500 nanomètres et environ 700 nanomètres, cette pointe d'absorbance subissant un degré d'extinction de l'oxydation sans recevoir un fort traitement à l'alcali. Dans ce procédé on forme un système réactionnel comprenant ledit colorant à la sulfonephtaléine ou le colorant à la phtaléine, et un échantillon de test contenant une quantité indéterminée de (i) peroxyde, (ii) une peroxydase, (iii) un composé qui est à même d'être oxydé pour produire du peroxyde d'hydrogène, ou (iv) un catalyseur participant à l'oxydation; ledit peroxyde d'hydrogène provoquant l'extinction catalysée par le peroxydase de la pointe d'absorbance dudit colorant, on peut par conséquent déterminer la quantité indéterminée qu'on désire mesurer en surveillant cette extinction.
EP92906329A 1991-12-09 1991-12-09 Determination colorimetrique de la peroxydase et du peroxyde Withdrawn EP0618980A1 (fr)

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JP3155685B2 (ja) * 1995-07-20 2001-04-16 財団法人山形県企業振興公社 ペルオキシダーゼの測定方法
DE19533072C1 (de) * 1995-09-07 1997-04-24 Fraunhofer Ges Forschung Nachweissystem und Verfahren für die qualitative und quantitative Bestimmung von Wasserstoffperoxid, Substraten, aus denen unter der Einwirkung von Oxidasen Wasserstoffperoxid gebildet wird, und Halogeniden
EP1218748A2 (fr) * 1999-08-06 2002-07-03 IMI International Medical Innovations Inc. L'analyse d'espaces de couleurs dans des dosages biochimiques et immunologiques
CN104596954B (zh) * 2014-12-10 2017-11-17 中国科学院上海微系统与信息技术研究所 一种利用邻对位醛基取代的芳基酚检测过氧化物的方法
CN105158219B (zh) * 2015-07-28 2018-03-13 中国科学院上海微系统与信息技术研究所 一种利用含醛基的硼酯化合物检测过氧化物的方法

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DE2826965A1 (de) * 1978-06-20 1980-01-17 Boehringer Mannheim Gmbh Diagnostisches mittel zum nachweis von leukozyten in koerperfluessigkeiten und dafuer geeignete chromogene
US4274832A (en) * 1979-02-12 1981-06-23 Eastman Kodak Company Analytical element and method for analysis of multiple analytes

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