WO1989011542A1 - Non-instrument methods for detecting an analyte of interest using peroxide intermediates and light scattering crystals - Google Patents

Non-instrument methods for detecting an analyte of interest using peroxide intermediates and light scattering crystals Download PDF

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Publication number
WO1989011542A1
WO1989011542A1 PCT/US1989/001923 US8901923W WO8911542A1 WO 1989011542 A1 WO1989011542 A1 WO 1989011542A1 US 8901923 W US8901923 W US 8901923W WO 8911542 A1 WO8911542 A1 WO 8911542A1
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Prior art keywords
analyte
interest
peroxide
reaction product
crystals
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Application number
PCT/US1989/001923
Other languages
French (fr)
Inventor
Alfred C. Greenquist
Martin Koocher
Nathan O. Siemers
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Crystal Diagnostics, Inc.
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Publication date
Application filed by Crystal Diagnostics, Inc. filed Critical Crystal Diagnostics, Inc.
Priority to JP1505976A priority Critical patent/JPH0714357B2/en
Publication of WO1989011542A1 publication Critical patent/WO1989011542A1/en

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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
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol

Definitions

  • the present invention is generally directed to improvements in qualitative and quantitative methods for detecting an analyte of interest and is particularly concerned with the generation of peroxides as reaction intermediates and their sub ⁇ sequent use in the formation, growth, and optical detection of light scattering crystals.
  • peroxide forming systems conventionally known, perhaps the best known are the enzymatic reaction systems which combine an enzyme of specific activity with the analyte of interest to yield a peroxide, typically hydrogen peroxide, as one of the reaction products.
  • a peroxide typically hydrogen peroxide
  • Representative of these enzymatic systems are the oxidoreductases as these have been defined and characterized [Dixon and Webb, Enzymes , Academic Press, 1979, - the text of which is expressly incorporated by reference herein].
  • the use of such enzymes for the production of a peroxide and the subsequent use of peroxide as a reactant, typically an oxidizing agent, is described in the now classical Trinder methodology [Ann. Glin. Biochem. j ⁇ :24-27 (1969)].
  • the Trinder assay is a determination of glucose in blood using glucose oxidase and peroxidase enzymes which destroy the glucose and release hydrogen peroxide.
  • the formed hydrogen peroxide is then combined with phenol and 4-amino- phenazone to yield a colored reaction product.
  • the enzymatic decomposition of the glucose is thus determined by quantitative measurement of the color intensity that results from the secondary reaction of hydrogen peroxide with phenol and 4-aminophenazone.
  • the present invention provides a method for detecting an analyte of interest in a fluid sample, this method comprising the steps of: reacting the analyte of interest in the fluid sample with at least one reactant to yield a peroxide as a primary reaction product; adding the peroxide to a composition containing at least one ROH moiety wherein R is a carbon containing entity and to chemcial means for initiating a reaction between the peroxide and the ROH containing composition such that a secondary reaction product containing at least one carbonyl group is generated; combining the carbonyl containing secondary reaction product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ, said nucleating sites being immobilized on the surface of a solid substrate; treating the immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of
  • test kit apparatus which maximizes the convenience and utility of the novel methodology as a whole.
  • Fig. 1 is an overhead view of a preferred film badge apparatus
  • Fig. 2 is a side view of the apparatus illustrated by Fig. 1, partly in cross-section;
  • Fig. 3 is a cross-sectional view of the embodi ⁇ ment illustrated within Fig. 1 along the axis of B B' ;
  • Fig. 4 is a cross-sectional view of the embodi ⁇ ment illustrated within Fig. 1 along the axis C C' ;
  • Fig. 5 is a cross-sectional view of the embodi ⁇ ment illustrated within Fig. 1 along the axis D D' ;
  • Fig. 6 is a cross-sectional view of the embodi ⁇ ment illustrated within Fig. 1 along the axis A A' ;
  • Fig. 7 is a photograph illustrating the derivatizing agent on the surface of the solid substrate in the embodiment of Fig. 1;
  • Fig. 8 is a photograph of the optically detectable crystals grown on the surface of the solid substrate within the embodiment of Fig. 1; and
  • Fig. 9 is a graph illustrating the ability to detect varying concentrations of cholesterol via light scattering crystals using the methodology of the present invention.
  • the present invention is a non-instrument method for detecting an analyte of interest in a fluid sample, quantitatively or qualitatively.
  • the methodology is unique in that it employs a variety of different chemical reactions for generating a peroxide, preferably hydrogen peroxide, as a primary reaction product of the analyte of interest in a fluid sample; reacts the generated peroxide inter ⁇ mediate using enzymatic or conventional chemistry with a oxidizable reagent to generate a carbonyl containing secondary reaction product; and then uses nucleating and crystal growing techniques to form light scattering crystals detectable by the unaided eye for both qualitative and quantitative measure ⁇ ments.
  • a peroxide preferably hydrogen peroxide
  • the methodology can be performed using a test kit apparatus which enables the assay to be employed anywhere and at any time, as needed or desired by the user;
  • the analytical methodology can detect a single analyte of interest in a fluid sample and does not require either pretreatment or fractionation of the sample to exclude other compositions prior to performing the steps of the unique method;
  • the present methodology has broad applica ⁇ tion as a biochemical assay system and is intended primarily for use in the clinical/diagnostic test areas which now require intricate reactions and sophisticated and instrumentation for reliable results;
  • the assay method of the present invention may be performed by the layman without prior technical training or experience in chemical reaction systems.
  • the detail description of the unique analytical method will be provided as individual sections followed by a presentation of empirical data demonstrating the benefits of the methodology under specific test conditions .
  • the unique methodology of the present invention and the test kit apparatus most preferred for its use will utilize any fluid sample from any source of origin believed to contain the analyte of interest. Since a primary application of the present invention is as a diagnostic/clinical analytical methodology, it is expected that the fluid sample will come from humans who are suspected of being afflicted with a disease or disorder yet to be identified. Accordingly, the fluid specimen can take the form of blood, plasma, serum, urine, lymph, cerebrospinal fluid, gastric juices, and any other liquid or semi-solid specimen from the subject.
  • the present methodology is also useful for biochemical and environmental analytical assays, of any other fluid including potable water; waste water; industrial discharge water; or specifically prepared solutions; suspensions; dispersions, colloids, and fluid mixtures.
  • the nature, source, prepara ⁇ tion, or composition of the fluid sample is immaterial so long as it does not substantially interfere with the chemical reactions and sequence of the present methodolog .
  • the analyte of interest to be detected by the present invention will also vary with the intended application and the source of the fluid sample.
  • those analytes typically evaluated for medical purposes using conventionally known chemistry methods and techniques are most suitable for detection and analysis by the present invention.
  • the analytes thus include those materials evaluated by the traditional battery of tests for laboratory diagnostic purposes such as blood sugars, lipoproteins, metabolic products, alcohols, enzymes, and enzyme cofactors which are indicative of normal and abnormal conditions in the human and animals generally.
  • a representative, but incomplete, listing of such analytes for clinical/diagnostic purposes is provided by Table I below. It can be readily understood that the assay of analytes which can be chemically linked to those described in Table I can also be detected by the present invention.
  • lactate dehydro- coupled enzyme assay genase with pyruvate oxidase lactate enzymes esterases coupled assay with ethyl butyrate and alcohol oxidase enzymes catalase ethanol or ethanol enzymes glucose oxidase glucose enzymes glycosidase coupled enzyme assay e.g.
  • beta-gal- with o-nitrophenyl actosidase B-D galacto- pyranoside and galactose oxidase enzymes alkaline phosphatase NADP, yeast alcohol dehydrogenase, ethanol, NADH oxidase cofactors NADH or analogue NAHD oxidase NADPH or analogue NADPH oxidase FAD or analogue apoglucose oxidase and glucose
  • the ability of the present invention to detect a specific analyte of interest depends on the ability to combine the analyte of interest in the fluid sample with one or more individual reactants in a single reaction sequence or in a series of multiple reactions to yield a peroxide as a primary reaction product.
  • the first requirement is the reaction system which will selectively react with the analyte of interest in the fluid sample to yield a peroxide, preferably hydrogen peroxide, as one of the reaction products .
  • the preferred reaction system for combination with the chosen analyte of interest employs enzymes for the generation of a peroxide.
  • the preferred enzymes are those of the class oxidoreductases which provide a wide variety of different enzymes of varying specificity which, alone or in combination, will yield hydrogen peroxide or another peroxide as a primary reaction product.
  • An incomplete listing of preferred enzymes for specific analytes of interest is provided by Table I above and clearly illustrates the range of specificity and variety of reactions provided by enzymatic reaction systems within the present invention. These enzymes are generally useful individually or in coupled reactions to yield at least one peroxide as a primary reaction product.
  • non-enzymatic reaction systems for generating a peroxide from the specific analyte of interest in the fluid sample can also be usefully employed.
  • Such non-enzymatic reactions and systems are conventionally known in the chemical arts and are described in detail with the scientific literature. All such reactions and systems are considered to be within the scope of the present invention.
  • the second manipulative step of the methodology is the generation of a carbonyl containing secondary reaction product from a peroxide.
  • the carbonyl containing reaction product generated is most desirably a volatile composition which is released in gaseous form directly upon formation; alterna- tively, the carbonyl containing reaction product may be retained in liquid form if this is desired under specific circumstances.
  • the reaction of peroxide and its conversion into at least one carbonyl containing reaction product is preferably accomplished by either of two reaction schemes: an enzymatic reaction system; and a metal ion catalyzed reaction system.
  • the enzymatic reaction system combined the generated peroxide with a hydroxyl (-0H) containing compoun'd such as an alcohol and an enzyme to produce an aldehyde.
  • a hydroxyl (-0H) containing compoun'd such as an alcohol and an enzyme to produce an aldehyde.
  • Such enzymatic conversions are conven ⁇ tional in the art [Pesee and Bodowrian, Clin . Chem. J22.:2042-2045 (1976); Haeckel and Perlick, j;. Clin. Chem. Clin. Biochem. _L4:411 (1976); Haeckel, R.,
  • reaction Ila demonstrates the enzymatic production of formaldehyde from methanol and hydrogen peroxide.
  • Reaction Scheme lib demonstrates the production of acetaldehyde by the enzymatic reaction of hydrogen peroxide and ethanol.
  • peroxidases the preferred enzyme class for converting peroxides.
  • a representative listing of this class of enzymes is provided by Table II below. It is intended and expected that each hydroxyl containing composition will be combined with the peroxidase most appropriate for the reaction to proceed and for generation of at least one carbonyl containing secondary reaction product.
  • a second general approach is provided for the reaction of a peroxide, preferably hydrogen peroxide.
  • This reaction system relies upon the oxidation of a hydroxyl bearing entity by the peroxide using one or more metallic ions such as that provided by Fenton's reagent, Fe +2.
  • metallic ions such as that provided by Fenton's reagent, Fe +2.
  • the use of such metallic ions in such a reaction is also conventionally known in the literature (Walling and A arnath, JACS _104: 1185-1189 (1982) and Ingles D.,
  • Me is a metal ion
  • any compound containing at least one hydroxyl group may be employed as a co-reactant.
  • a representative, but incomplete, listing of useful hydroxyl containing compounds is provided by Table III below.
  • the metal ion chosen for use in this reaction system may be any of those useful for this purpose as described in the pertinent literature.
  • the remaining manipulative steps of the present invention are directed to the selective formation and growth of light scattering crystals upon reactive contact with the carbonyl group of the secondary reaction product. Accordingly, the individual steps are: combining the carbonyl containing secondary reaction product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ, these nucleating sites being immobilized on the surface of a solid substrate; treating the immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of the formed crystals as a measure of the analyte of interest in the sample.
  • the derivatizing agent is thus a compound capable of providing multiple nucleating particles after reactive contact with the carbonyl containing composition; and is desirably a chemical entity having a reactive amine which has been deposited onto the surface of a solid substrate.
  • the goal and purpose of the derivatizing agent therefore, is to react with the carbonyl containing secondary reaction product; and to provide thereby a series of nucleating sites formed in-situ for the subsequent formation and growth of optically visible crystals.
  • the derivatizing agent appear on the surface of the solid substrate as a substantially monodispersed system of particles having a size in the micron and submicron range.
  • the use of such derivatizing agents as films of monodispersed particles for subsequent combination and reaction with metastable supersaturated solutions for the detection and identification of small crystals has been described [Vonnegut, B., Science 116 :300-301 (1952); United States Patent Number 4,300,587].
  • a representative, but non-exhaustive listing of derivatizing agents useful with the present invention include the following: 2,4-dinitrophenyl-hydrazine; semicarbazide; dimethone or dimedone; p-nitrophenyl- hydrazine; phenylhydrazine; thiosemicarbazide; oxi es, bromobenzohydrazine ; 2, 4-dinitromethylphenylhydrazine; p-carboxyphenylhydrazine; nitrobenzenesulfonhydrazine; nitrobenzohydrazine; diphenylhdrazine; 2-naphthyl- hydrazine; p-chlorobenzohydrazine; m-chlorobenzo- hydrazine; nitroguanylhydrazine; alpha-(alpha,4-nitro- phenyl)-alpha-methylhydrazine; xenylse icarba
  • the derivatizing agent used in the present inven ⁇ tion preferably is a dispersion of particles substan- tially of the same size which is retained on the surface of a solid substrate with relatively uniform spacing between them, the particles typically ranging from about 0.1-1.0 micrometers in size.
  • a single derivatizing agent may be useful for reaction with a variety of different carbonyl containing compositions in either vaporized or liquid physical states - each of which will react with the chosen derivatizing agent to form a plurality of immobilized nucleating sites in-situ on the surface of the solid substrate. It will be understood that the nucleating sites formed in-situ are themselves crystals which are invisible to the unaided eye but which serve as specific initiation sites for the subsequent formation and growth of optically detectable crystals.
  • a film of monodispersed particles comprising the chosen derivatizing agent may be conveniently prepared by directing an aerosol of the solution comprising the derivatizing agent against the surface of a solid substrate in the conventionally known manner.
  • an aerosol of the solution comprising the derivatizing agent against the surface of a solid substrate in the conventionally known manner.
  • By maintaining very accurate control over the concentration in the aerosol over time and by controlling the speed of delivery of the aerosol to the surface of the solid substrate one may deposit an accurately controlled amount of the derivatizing agent onto the surface.
  • Equipment for forming the aerosol in the desired range of particles is commercially available.
  • the solid substrate upon which the chosen derivatizing agent is located may be shaped into a variety of different forms and configurations. The preferred format will be described in the detailed description for the improved apparatus comprising the present invention. Nevertheless, a variety of differently configured apparatus and articles may be prepared as test kits and film badges which employ the derivatizing agent in the described manner for the purpose of forming a plurality of immobilized nucleating sites in-situ.
  • the plurality of immobilized nucleating sites are preferably treated with a selective, metastable supersaturated (developer) solution such that a plurality of optically detectable crystals are formed on the surface of the solid substrate.
  • the metastable supersatured solution is an aqueous or non-aqueous liquid containing a supersaturated concentration of a crystal growing entity which is both chemically and physically stable for a defined period of time.
  • metastable supersaturated (developer) solution when the metastable supersaturated (developer) solution is combined with the nucleating sites formed in-situ on the surface of the solid substrate, crystallization will occur at each of the individual nucleating sites resulting in the formation and growth of optically detectable crystals.
  • Table V A useful but non-exhaustive listing of selective metastable supersaturated (developer) solutions is provided by Table V in which each developer solution is correlated with a preferred derivatizing agent and an illustrative carbonyl containing composition.
  • the quantities of each chemical composition to prepare a metastable supersaturated (developer) solution in aqueous and non-aqueous liquids are conventionally known in the art.
  • Salicylaldehyde semicarbazide salicylaldehyde semicarbazone It is noteworthy that the listing of Table V indicates that the composition of the metastable supersaturated solution is preferably identical to the condensation reaction product formed by the reaction of the carbonyl containing composition and the derivatizing agent. It is specifically noted that under certain circumstances, the chemical composition of the metastable supersaturated solution need not be chemically identical to the condensation product forming the individual nucleating sites, but may be distinctly different in chemical formula and structure while remaining sufficiently similar for crystallization to occur - a phenomenon termed "epitaxy" .
  • the size and nature of the crystals formed and grown on the surface of the solid substrate as a result of the treatment with the selective developer solution are directly affected by the individual number of nucleating sites formed in-situ by the interaction of the carbonyl containing composition and the derivatizing agent.
  • the peroxide conversion reaction produces only a small quantity of a carbonyl containing composition, relatively few nucleating sites are formed on the surface of the solid; alternatively, if the peroxide converting step produces a relatively large quantity of reaction product, a proportionately larger number of individual nucleating sites will be formed on the surface of the solid.
  • the number of nucleating sites formed will determine the ultimate size of the crystals grown since the amount of crystallizable material in the developer solution will be divided equally among all the nucleating sites which are identical in chemical composition regardless of their number. Accordingly, if a large number of nucleating sites are present, the quantity of crystallizing compound in the developer solution will be distributed over a greater number of nucleating sites and the size of each crystal formed and grown will be relatively small. Conversely, if the number of nucleating sites formed is relatively small, the same concentration of crystallizable material in the developer solution will be equally distributed over a smaller number of individual nucleating sites, thereby causing a greater quantity of crystallizable material to be deposited on each site. This causes the formation of larger sized crystals.
  • the concentration of the crystallizable matter comprising the metastable, supersaturated (developer) solution is important in that the concentration be of sufficient magnitude to provide for the growth of optically detectable crystals - even when the number of individual nucleating sites is at a maximum.
  • metastable supersaturated (developer) solutions are preferably prepared at a concentration ranging from 2-10 times saturation concentration.
  • control of the speed of matter deposition also directly affects the ability of the grown crystals to be optically detected.
  • a rapid deposition of crystallizable material from the developer solution onto the individual nucleating sites tends to build up the crystal face toward an irregular point; whereas more leisurely deposition of crystallizable material produces a crystal line lattice whose crystal face is essentially flat.
  • light is scattered more readily from crystals which have irregular and pointed faces and thus become more "visible” as a result of the light scattering effect; in comparison, flat-faced crystal lattices scatter light minimally and are more difficult to visualize with the unaided eye or with the use of specific crystal detection equipment.
  • the concentration of the chemical compositions used in making metastable supersatured (developer) solution be as concentrated as possible in order to promote rapid deposition of the crystalline material when brought into reactive contact with the individual nucleating sites.
  • concentration of the chemical compositions used in making metastable supersatured (developer) solution be as concentrated as possible in order to promote rapid deposition of the crystalline material when brought into reactive contact with the individual nucleating sites.
  • stabilizers such as polyvinyl alcohol, polyvinyl, pyrrolidone, gelatin, agar, sodium carboxymethyl cellulose, methyl and ethyl cellulose and the like are, useful and desirable for prolonging the effective life of the supersaturated developer solution in the present invention.
  • optically detectable and detectable change which is both a qualitative and quantitative measure of the analyte of interest in the test sample.
  • the degree of optically detectable crystals provides both a qualitative and/or quantita ⁇ tive measure which is correlatable with a calculated amount of an analyte of interest in the fluid sample.
  • quantitative results in the micromolar range or less may be reproducibly obtained within 10-15 minutes by the formation and growth of optically detectable crystals in the heretofor described manner.
  • a chamber 10 preferably in the form of a rigid one-piece molded transparent plastic article illustrated in overhead and side views by Figs. 1 and 2, respec- tively.
  • the chamber 10 is preferably made of a resilient and transparent material such as polstyrene of polycarbonate resin having a closed interior volume which is divided into individual sections and spaces of differing configuration and volume.
  • the interior of the chamber 10 is divided into a U-shaped section 12, a reagent housing 30, a developer solution inlet 50, and an enclosed receptable 60.
  • a plastic sheet 14 forms a common back wall throughout the entirety of the chamber 10 and preferably has a metallized layer 15 bonded to the exterior surface to aid in visualization of the crystals to be formed subsequently in the chamber.
  • the interior surface 16 of the sheet 14 within the U-shaped section 12 acts as the solid substrate upon which is a derivatizing agent 18 is deposited. It is most desirable to closely control the precise dimensions and configuration of the U-shaped section 12 in order to obtain accurately reproducible results. The dimensions and configuration may vary within limits depending upon the specific analyte of interest to be detected, the chosen derivatizing agent, the metastable supersaturated solution to be employed, and the range of concentrations and time of exposure to be accommodated. In most instances, the void dimensions above the interior surface 16 of the sheet 14 has a width in the range of 0.2-1.0 inches and has a height of approximately 0.005-0.03 inches.
  • the developer solution inlet 50 is integrally joined to the U-shaped section 12 and provides means for introduction of the metastable, supersaturated solution into the interior of the chamber 10 such that it can be applied to the interior surface 16 of the sheet 14 in a controlled manner on demand.
  • the developer solution inlet 50 is to be sealed with impervious tape which is removed when the developer solution is introduced.
  • the enclosed receptable 60 is composed of a retainer 62 and a frangible ampule 64 filled with an aqueous salt solution of choice.
  • the receptable 60 is sealed by the retainer 62, a strip of flexible impervious adhesive tape adhering at its outer margins to the receptable.
  • Beneath the frangible ampule 64 is a sponge material 66 which is in fluid communication with a pair of capillary openings 68 and 70 at the bottom of the receptable 60.
  • the capillary openings 68, 70 extend through the interior of chamber 10 along the longitudinal margins serve as capillary channels or troughs for the flow of the aqueous salt solution from the receptacle 60 through the U—shaped section 12. This salt solution serves to maintain a constant relative humidity within the chamber 10. If desired, strips of wicking fibrous or porous material may be placed within the capillary openings 68 and 70 to aid in maintaining a uniform flow of liquid.
  • the reagent housing 30 is shown by Fig. 6. As seen in Fig. 6, the reagent housing 30 is configured as a substantially cylindrical space whose internal volume has been divided into an upper spaced region 40 and a lower, narrow passage 34 which communicates with the interior of the chamber 10.
  • the upper spaced region 40 is occupied by a reagent pad 44 preferably composed of a porous matrix material; preferably contains a single enzyme or a mixture of enzymes able to react with the analyte of interest; and is sufficient to generate a peroxide as a primary reaction product.
  • This reagent pad 44 also contains all the other reactants necessary for reacting such peroxides as are generated to yield a carbonyl containing product in the preferred vaporized form.
  • a fluid sample containing the analyte of interest will enter the spaced region 40; react with the enzymes and other reagents present in reagent pad 44; and subsequently produce a carbonyl containing composition which released into the interior of chamber 10 via the passage 34.
  • the film badge is exposed to the ambient environment by removing the device from a sealed package.
  • the frangible ampule is broken and the aqueous salt solution flows through the capillaries 68, 70.
  • a fluid sample believed to contain the analyte of interest is introduced via the housing 30.
  • the secondary reaction product containing at least one carbonyl group is preferably released from the reagent pad 44 in gaseous form which then travels through the narrow passage 34 and enters the interior of the U-shaped section 12.
  • the vaporized carbonyl containing composition will react with the derivatizing agent 18 present in solid microparticle form to yield a condensation reaction product formed as a plurality of nucleating sites in-situ on the interior surface 16 of the sheet 14.
  • a metastable supersaturated solution is introduced via the developer solution inlet 50 and fills the U-shaped section 12 for reaction with the condensation reaction product formed as the plurality of nucleating sites in-situ.
  • the treatment of these nucleating sites with the selective metastable supersaturated solution forms a plurality of optically detectable crystals on the interior surface 16 of the sheet 14. The presence of these optically detectable crystals serves as a qualitative and/or quantitative measure of the analyte of interest in the initial test sample.
  • Figs. 7 and 8 illustrate the utility and operability of this improved apparatus design and configuration.
  • Fig. 7 illustrates a magnified (300X) view of derivatizing agent 18 deposited on the interior surface 16 of the sheet 14 within the U-shaped section 12 before any reactions have occurred.
  • Fig. 8 is a magnified (300X) view demonstrating the formation of optically detectable crystals on the surface 16 after the apparatus has been exposed to a sample for a predetermined period of time and the manipulated steps of the present methodology have been performed within this apparatus.
  • a scale of arbitrary units can be calibrated as to enable the user to quickly determine the concentration of the analyte of sample.
  • a quantitative determination of cholesterol was undertaken in the following manner: a cholesterol stock solution was prepared containing 1 mg/ml cholesterol in 10 mg/ml of bovine serum albumin (fatty acid free), 3% Triton X-100 and 0.2 M acetate buffer (pH 7.0).
  • Cholesterol oxidase was commercially obtained as a preparation derived from Nocardia erythropolis. This enzyme was prepared at 25 U/mg at 25 C and contained 1 mg/ml of protein.
  • a FeSO, solution was prepared as a 1.32 M preparation in 1 N H_S0,.
  • a Saligenin solution was prepared by dissolving one gram of saligenin in 20 milliliters of water (0.4 M solution) .
  • a derivatizing agent comprising a semicarbazide film was accessible to the vaporized secondary reaction product liberated by the reaction mixture.
  • the reaction was then allowed to proceed for 45 minutes exposure, followed by a 10 minute application of developer solution containing an equal mixture of 10% Knox gelatin containing 0.2% salicylaldehyde in water and 0.15% semicarbazide in water (prepared just before use). Subsequently, the presence of light scattering crystals was measured as millimeter length for each test aliquot. The results are illustrated graphically by Fig. 9 which reveals a dose response curve proportional to the concentration of cholesterol for the range from 0-50 mg/dl respectively. For clinical applications, serum samples would be diluted ten fold in this assay format.
  • catalase Sigma C-3155, 48,900 units/mg protein, 16.3 mg protein/ml
  • cholesterol oxidase solution Boehringer Mannheim 396818 solution in NaCl, pH 6.0, 25 units/ g at 25 C, 1 mg protein/ml
  • 10 micro ⁇ liters of the resulting mixture is then spotted onto 1/4 inch Whatman 3 MM disks. The disks are allowed to dry at room temperature in a dessicated atnrosphere for 6 hours before being placed into the housing 30 of Fig. 6 for use as the reagent pad 44.
  • a 20 microliter sample of cholesterol in 0.1M phosphate buffer (pH 3.5) containing 10% ethanol is applied to the prepared disk serving as reagent pad.
  • a film of derivatizing agent comprising a monodisperse, aerosol generated, coating of 5 parts p-nitrophenylhydrazine and 1 part citric acid has been applied as a 0.07 inch wide stripe to the interior surface of the U-shaped section within the badge.
  • the film within the badge is developed by addition of a metastable supersaturated solution of acetaldehyde p-nitrophenyl- hydrazone in a 20% methanol/water solution.
  • a column of optically visible light scattering crystals 21.8 mm in length was present on the face of the film. This length of crystals was quantitatively evaluated as equivalent to a cholesterol concentration of 0.22 mM in the sample.

Abstract

The present invention provides qualitative and quantitative methods for measuring an analyte of interest in a fluid sample using peroxide intermediates for the subsequent formation and growth of light scattering crystals. The methodology is most useful within clinical/diagnostic analytical assays but also may be employed for biochemical and environmental applications generally. Regardless of application, the methodology provides accurate, reproducible results without need for either instrumentation or technically trained personnel.

Description

NON-INSTRUMENT METHODS FOR DETECTING AN ANALYTE OF INTEREST USING PEROXIDE INTERMEDIATES AND LIGHT SCATTERING CRYSTALS
FIELD OF THE INVENTION The present invention is generally directed to improvements in qualitative and quantitative methods for detecting an analyte of interest and is particularly concerned with the generation of peroxides as reaction intermediates and their sub¬ sequent use in the formation, growth, and optical detection of light scattering crystals.
BACKGROUND OF THE INVENTION Peroxides, R00R' wherein R and R' are hydrogen or a carbon containing entity, have been highly favored as detectable compositions in analytical assay methods for many years, particularly for clinical/diagnostic analyses. All such detection methods follow common principles and procedural steps: introducing the sample containing the analyte of interest into a peroxide, typically hydrogen peroxide, forming system; and then detecting the peroxide using one or more specific detection reagents to yield a reaction product which is chromophoric , fluorescent, chemiluminescent , or otherwise detectable by instru¬ ments able to measure light of specific wavelengths.
Of the peroxide forming systems conventionally known, perhaps the best known are the enzymatic reaction systems which combine an enzyme of specific activity with the analyte of interest to yield a peroxide, typically hydrogen peroxide, as one of the reaction products. Representative of these enzymatic systems are the oxidoreductases as these have been defined and characterized [Dixon and Webb, Enzymes , Academic Press, 1979, - the text of which is expressly incorporated by reference herein]. The use of such enzymes for the production of a peroxide and the subsequent use of peroxide as a reactant, typically an oxidizing agent, is described in the now classical Trinder methodology [Ann. Glin. Biochem. j^:24-27 (1969)]. The Trinder assay is a determination of glucose in blood using glucose oxidase and peroxidase enzymes which destroy the glucose and release hydrogen peroxide. The formed hydrogen peroxide is then combined with phenol and 4-amino- phenazone to yield a colored reaction product. The enzymatic decomposition of the glucose is thus determined by quantitative measurement of the color intensity that results from the secondary reaction of hydrogen peroxide with phenol and 4-aminophenazone. Subsequent developments in this art conven¬ tionally follow the teachings and format of the Trinder reactions. These assay methodologies create or specifically add peroxide, typically hydrogen peroxide, as an oxidizing agent to a reaction mixture which contains one or more dyes or dye precursors to form a reaction product which is detectable by light measuring instruments set at specific wave¬ lengths. Representative of recent innovations following the Trinder principles and format are the following: Japanese Publication No. 61274698-A
(86.12.04) Japanese Publication No. 61174267-A
(86.08.05) Japanese Publication No. 61155758-A
(86.07.15) Japanese Publication No. 61146197-A
(86.07.03) German Publication No. 3600563-A
(86.07.17) U.S. Patent No 4,615,982; Australian
Publication No. 8547303-A (86.04.24) ; Japanese
Publication No. 61074599-A (86.04.16); European
Publication No. 175250-A (86.03.26); Japanese
Publication No. 6-21807-A (85.10.31) ; Japanese
Publication No-. 60178358-A (85.09.12) Japanese
Publication No. 60172298-A (85.09.05) Japanese
Publication No. 60091997-A (85.05.23) Japanese Publication No. 60083599-A (85.05.11); United States Patent No. 3,290,228; United States Patent No. 3,266,868; Japanese Publication No. 73002996-B; German Publication No. 2450726-A (76.05.06); U.S. Patent No. 4,101,381; United States Patent No. 4,202,938; United States Patent No. 4,301,244; and German Publication No. 3114935-A (82.11.25).
Separate and distinct from analytical methods employing dyes for the detection of peroxides are the developments in detection methods which correlates the formation and growth of optically detectable crystals with the presence of an analyte of interest in a fluid sample. To date, these methods have taken two primary forms: methods and compositions for detecting carbonyl containing compositions (an aldehyde or ketone) as described within United States Patent No. 4,380,587; and methods and a conjugate reactant comprising a binding partner having at least one amine group available for reaction which is specific for an analyte of interest, a metallic cation, and a releasable marker substance having at least one carbonyl group available for reaction as described in United States Patent No. 4,727,024. Both methodologies are able to detect an analyte of interest in a fluid sample through the elective formation, growth, and optical detection of light scattering crystals without need for any instrumenta¬ tion whatsoever. Each of these methods, however, is clearly limited to the use of carbonyl containing compositions alone.
SUMMARY OF THE INVENTION The present invention provides a method for detecting an analyte of interest in a fluid sample, this method comprising the steps of: reacting the analyte of interest in the fluid sample with at least one reactant to yield a peroxide as a primary reaction product; adding the peroxide to a composition containing at least one ROH moiety wherein R is a carbon containing entity and to chemcial means for initiating a reaction between the peroxide and the ROH containing composition such that a secondary reaction product containing at least one carbonyl group is generated; combining the carbonyl containing secondary reaction product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ, said nucleating sites being immobilized on the surface of a solid substrate; treating the immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of formed crystals as a measure of the analyte of interest in the sample.
The assay methodology is useful on-site for a variety of different clinical/diagnostic, biochemical, and environmental applications. In addition, a test kit apparatus is provided which maximizes the convenience and utility of the novel methodology as a whole.
BRIEF DESCRIPTION OF THE DRAWING The present invention may be more easily and completely understood when taken in conjunction with the accompanying drawing, in whichi
Fig. 1 is an overhead view of a preferred film badge apparatus; Fig. 2 is a side view of the apparatus illustrated by Fig. 1, partly in cross-section;
Fig. 3 is a cross-sectional view of the embodi¬ ment illustrated within Fig. 1 along the axis of B B' ;
Fig. 4 is a cross-sectional view of the embodi¬ ment illustrated within Fig. 1 along the axis C C' ;
Fig. 5 is a cross-sectional view of the embodi¬ ment illustrated within Fig. 1 along the axis D D' ; Fig. 6 is a cross-sectional view of the embodi¬ ment illustrated within Fig. 1 along the axis A A' ;
Fig. 7 is a photograph illustrating the derivatizing agent on the surface of the solid substrate in the embodiment of Fig. 1; Fig. 8 is a photograph of the optically detectable crystals grown on the surface of the solid substrate within the embodiment of Fig. 1; and
Fig. 9 is a graph illustrating the ability to detect varying concentrations of cholesterol via light scattering crystals using the methodology of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is a non-instrument method for detecting an analyte of interest in a fluid sample, quantitatively or qualitatively. The methodology is unique in that it employs a variety of different chemical reactions for generating a peroxide, preferably hydrogen peroxide, as a primary reaction product of the analyte of interest in a fluid sample; reacts the generated peroxide inter¬ mediate using enzymatic or conventional chemistry with a oxidizable reagent to generate a carbonyl containing secondary reaction product; and then uses nucleating and crystal growing techniques to form light scattering crystals detectable by the unaided eye for both qualitative and quantitative measure¬ ments.
The present invention thus provides a number of major advantages to the user:
(1) The methodology provides for both qualitative and quantitative measurements without need for any instrumentation whatsoever;
(2) The unique methodology is rapid, easy to perform, and provides consistently reliable results and data;
(3) The methodology can be performed using a test kit apparatus which enables the assay to be employed anywhere and at any time, as needed or desired by the user;
(4) The analytical methodology can detect a single analyte of interest in a fluid sample and does not require either pretreatment or fractionation of the sample to exclude other compositions prior to performing the steps of the unique method;
(5) The present methodology has broad applica¬ tion as a biochemical assay system and is intended primarily for use in the clinical/diagnostic test areas which now require intricate reactions and sophisticated and instrumentation for reliable results; and
(6) The assay method of the present invention may be performed by the layman without prior technical training or experience in chemical reaction systems. In order to better understand each of the manipulative steps comprising the present invention and the major chemical reactions comprising each component part o the methodology as a whole, the detail description of the unique analytical method will be provided as individual sections followed by a presentation of empirical data demonstrating the benefits of the methodology under specific test conditions .
I. Reacting The Analyte Of Interest To Yield A Peroxide As A Primary Reaction Product
The Fluid Sample Containing The Analyte of Interest
It is intended that the unique methodology of the present invention and the test kit apparatus most preferred for its use will utilize any fluid sample from any source of origin believed to contain the analyte of interest. Since a primary application of the present invention is as a diagnostic/clinical analytical methodology, it is expected that the fluid sample will come from humans who are suspected of being afflicted with a disease or disorder yet to be identified. Accordingly, the fluid specimen can take the form of blood, plasma, serum, urine, lymph, cerebrospinal fluid, gastric juices, and any other liquid or semi-solid specimen from the subject. The present methodology is also useful for biochemical and environmental analytical assays, of any other fluid including potable water; waste water; industrial discharge water; or specifically prepared solutions; suspensions; dispersions, colloids, and fluid mixtures. Accordingly, the nature, source, prepara¬ tion, or composition of the fluid sample is immaterial so long as it does not substantially interfere with the chemical reactions and sequence of the present methodolog . The analyte of interest to be detected by the present invention will also vary with the intended application and the source of the fluid sample. In view of the present methods particular value and advantages for clinical/diagnostic analytical detec¬ tion, those analytes typically evaluated for medical purposes using conventionally known chemistry methods and techniques are most suitable for detection and analysis by the present invention. The analytes thus include those materials evaluated by the traditional battery of tests for laboratory diagnostic purposes such as blood sugars, lipoproteins, metabolic products, alcohols, enzymes, and enzyme cofactors which are indicative of normal and abnormal conditions in the human and animals generally. A representative, but incomplete, listing of such analytes for clinical/diagnostic purposes is provided by Table I below. It can be readily understood that the assay of analytes which can be chemically linked to those described in Table I can also be detected by the present invention.
Table I
PREFERRED ENZYME
TEST CATEGORY ANALYTE OF INTEREST AND/OR SUBSTRATE blood sugar glucose glucose oxidase blood sugar galactose galactose oxidase blood sugar hexose hexose oxidase lipoprotein cholesterol cholesterol oxidase lipoprotein triglycerides coupled enzyme assay using lipase, glycerol kinase and L-alpha- glycerol phosphate oxidase lipoprotein hydroxy fatty L-2-hydroxyacid acids oxidase metabolic uric acid uncase products metabolic bilirubin bilirubin oxidase products metabolic creatinine coupled enzyme products reaction with creatinine amino- hydrolase, and sarcosine oxidase metabolic creatme coupled enzyme assay products with creatine aminohydrolase and sarcosine oxidase metabolic pyruvate pyruvate oxidase products alcohols ethanol alcohol oxidase ester ethylbutyrate coupled assay with esterase and alcohol oxidase enzymes serum coupled enzyme assay glutamic with pyruvate oxaloacetic oxidase transaminase
(aspartate amino transferase) Table I (Cont ' d )
PREFERRED ENZYME
TEST CATEGORY ANALYTE OF INTEREST AND/OR SUBSTRATE enzymes serum coupled enzyme assay glutamic with pyruvate pyruvic oxidase transaminase
(alanine amino transferase) enzymes lactate dehydro- coupled enzyme assay genase with pyruvate oxidase, lactate enzymes esterases coupled assay with ethyl butyrate and alcohol oxidase enzymes catalase ethanol or ethanol enzymes glucose oxidase glucose enzymes glycosidase coupled enzyme assay e.g. beta-gal- with o-nitrophenyl , actosidase B-D galacto- pyranoside and galactose oxidase enzymes alkaline phosphatase NADP, yeast alcohol dehydrogenase, ethanol, NADH oxidase cofactors NADH or analogue NAHD oxidase NADPH or analogue NADPH oxidase FAD or analogue apoglucose oxidase and glucose
The Peroxide Generating Reaction Systems
It will be clearly recognized and understood that the ability of the present invention to detect a specific analyte of interest depends on the ability to combine the analyte of interest in the fluid sample with one or more individual reactants in a single reaction sequence or in a series of multiple reactions to yield a peroxide as a primary reaction product. The first requirement, therefore, is the reaction system which will selectively react with the analyte of interest in the fluid sample to yield a peroxide, preferably hydrogen peroxide, as one of the reaction products .
The preferred reaction system for combination with the chosen analyte of interest employs enzymes for the generation of a peroxide. The preferred enzymes are those of the class oxidoreductases which provide a wide variety of different enzymes of varying specificity which, alone or in combination, will yield hydrogen peroxide or another peroxide as a primary reaction product. An incomplete listing of preferred enzymes for specific analytes of interest is provided by Table I above and clearly illustrates the range of specificity and variety of reactions provided by enzymatic reaction systems within the present invention. These enzymes are generally useful individually or in coupled reactions to yield at least one peroxide as a primary reaction product.
It is expected that for many individual analytes of interest, a series of multiple enzymatic reactions in sequence will be required. Illustrative of this multiple reaction sequence for generating a peroxide is the detection of cholesterol esters in serum which require the use of two different enzymes in sequence in order to generate a peroxide reaction product. Such a reaction system is provided by Reaction Scheme I below.
Reaction Scheme I
Cholesterol
(la) Cholesterol Esters ^Cholesterol + Fatty Acids
Esterase
Cholesterol
(lb) Cholesterol + 0„ > Cholest-4-en-3-one + H-O^
Oxidase
I*1 all these enzymatic reaction systems for generating a peroxide reaction product, it is intended and expected that all necessary reaction conditions and parameters including pH values, cofactors, temperature, concentration, buffers, and any other factor conventionally recognized in this art as being a meaningful influence will be provided appropriately in order that the enzymatic reaction proceed. All such conditions and parameters are considered to be conventional in the art and need not be described -- detail herein.
Alternatively, non-enzymatic reaction systems for generating a peroxide from the specific analyte of interest in the fluid sample can also be usefully employed. Such non-enzymatic reactions and systems are conventionally known in the chemical arts and are described in detail with the scientific literature. All such reactions and systems are considered to be within the scope of the present invention. II. Reaction Means For Converting Peroxide Into A Secondary Reaction Product Containing At Least One
Carbonyl Group
The second manipulative step of the methodology is the generation of a carbonyl containing secondary reaction product from a peroxide. The carbonyl containing reaction product generated is most desirably a volatile composition which is released in gaseous form directly upon formation; alterna- tively, the carbonyl containing reaction product may be retained in liquid form if this is desired under specific circumstances. In general, the reaction of peroxide and its conversion into at least one carbonyl containing reaction product is preferably accomplished by either of two reaction schemes: an enzymatic reaction system; and a metal ion catalyzed reaction system.
The Enzyme Reaction System
The enzymatic reaction system combined the generated peroxide with a hydroxyl (-0H) containing compoun'd such as an alcohol and an enzyme to produce an aldehyde. Such enzymatic conversions are conven¬ tional in the art [Pesee and Bodowrian, Clin . Chem. J22.:2042-2045 (1976); Haeckel and Perlick, j;. Clin. Chem. Clin. Biochem. _L4:411 (1976); Haeckel, R.,
J_. Clin. Chem. Clin. Biochem. _ :101-107 (1976); and Kageyama, N., Clin. Chem. Acta 31:421-426 (1971)].
Representative of such enzymatic reaction systems in general are Reaction Schemes Ila and lib provided below. Reaction Scheme II
Catalase (Ha) H202 + CH30H *HCHO
Catalase (lib) H202 + CH3CH20H CH3CH0
In each instance, the preferred peroxide, hydrogen peroxide, is combined with an alcohol and an enzyme, preferably catalase. Reaction Ila demonstrates the enzymatic production of formaldehyde from methanol and hydrogen peroxide. Alternatively, Reaction Scheme lib demonstrates the production of acetaldehyde by the enzymatic reaction of hydrogen peroxide and ethanol.
In addition, the preferred enzyme class for converting peroxides is peroxidases. A representative listing of this class of enzymes is provided by Table II below. It is intended and expected that each hydroxyl containing composition will be combined with the peroxidase most appropriate for the reaction to proceed and for generation of at least one carbonyl containing secondary reaction product.
Tab le II
Preferred Peroxidases
Catalase
Peroxidase
Cytochrome Peroxidase 5 Iodide Peroxidase
NAD Peroxidase
NADP Peroxidase
Fatty Acid Peroxidase
Glutathione Peroxidase 10 Chloride Peroxidase
Cytochrome P-450
Ligninase
The Metal Ion Catalyzed Reaction System
Alternatively, a second general approach is provided for the reaction of a peroxide, preferably hydrogen peroxide. This reaction system relies upon the oxidation of a hydroxyl bearing entity by the peroxide using one or more metallic ions such as that provided by Fenton's reagent, Fe +2. The use of such metallic ions in such a reaction is also conventionally known in the literature (Walling and A arnath, JACS _104: 1185-1189 (1982) and Ingles D.,
Aust. J_. Chem. 26.-.1021 (1973); Walling, C, Ace.
Chem. 8_:125 (1975)].
Representative of this alternative reaction system for generation of a carbonyl containing composition are the reactions provided by Reaction
Schemes Ilia, Illb , and IIIc respectively provided below.
Reaction Scheme III
Me
(Ilia) H202 + CH30H »HCH0
Me (Illb) H202 + Saligenin Salicylaldehyde
Me (IIIc) H202 + Mandelic Acid >Benzaldehyde
wherein Me is a metal ion.
It will be recognized that a variety of different co-reactants may be employed to react with the peroxide to produce a carbonyl containing composition. For this purpose, any compound containing at least one hydroxyl group may be employed as a co-reactant. A representative, but incomplete, listing of useful hydroxyl containing compounds is provided by Table III below.
Table III
Useful Hydroxy Carboxylic Acids
Glycolic Acid Lactic Acid Glyceric Acid 5 o j Jp or o Hydroxy Butyric Acids
Tartaric Acid Citric Acid Mandelic Acid
Glucuronic acid and uldonic acids in 10 general
Useful Alcohols And Polyols
Methanol
Ethanol and Ethylene Glycol
Propanol, Isopropanol, and Propylene Glycol 15 Butanol, Isobutanol, and Butylene Glycol
Pentanols and Pentylene Polyols
Hexanols and Hexylene Polyols
Benzyl Alcohol
3,4-Dimethoxybenzyl alcohol 20 Saligenin
Similarly, the metal ion chosen for use in this reaction system may be any of those useful for this purpose as described in the pertinent literature.
A representative listing of such metal ions is provided by Table IV below.
Table IV
Preferred Metallic Cations
Iron (Fe and Fe )
Cobalt (Co+2) Manganese (Mn +2)
5 Molybdenum (Mo )
Tungsten (W+7)
III. Means For Forming And Growing Light Scattering Crystals
The remaining manipulative steps of the present invention are directed to the selective formation and growth of light scattering crystals upon reactive contact with the carbonyl group of the secondary reaction product. Accordingly, the individual steps are: combining the carbonyl containing secondary reaction product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ, these nucleating sites being immobilized on the surface of a solid substrate; treating the immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of the formed crystals as a measure of the analyte of interest in the sample. Each of these specific reactants and steps will now be described in detail.
The Derivatizing Agent
Once the secondary reaction product having at least one carbonyl group available for reaction has been generated, this product is combined with an immobilized derivatizing agent to yield a condensation product formed as a plurality of nucleating sites in-situ. The derivatizing agent is thus a compound capable of providing multiple nucleating particles after reactive contact with the carbonyl containing composition; and is desirably a chemical entity having a reactive amine which has been deposited onto the surface of a solid substrate. The goal and purpose of the derivatizing agent, therefore, is to react with the carbonyl containing secondary reaction product; and to provide thereby a series of nucleating sites formed in-situ for the subsequent formation and growth of optically visible crystals. It is most desirable that the derivatizing agent appear on the surface of the solid substrate as a substantially monodispersed system of particles having a size in the micron and submicron range. The use of such derivatizing agents as films of monodispersed particles for subsequent combination and reaction with metastable supersaturated solutions for the detection and identification of small crystals has been described [Vonnegut, B., Science 116 :300-301 (1952); United States Patent Number 4,300,587]. A representative, but non-exhaustive listing of derivatizing agents useful with the present invention include the following: 2,4-dinitrophenyl-hydrazine; semicarbazide; dimethone or dimedone; p-nitrophenyl- hydrazine; phenylhydrazine; thiosemicarbazide; oxi es, bromobenzohydrazine ; 2, 4-dinitromethylphenylhydrazine; p-carboxyphenylhydrazine; nitrobenzenesulfonhydrazine; nitrobenzohydrazine; diphenylhdrazine; 2-naphthyl- hydrazine; p-chlorobenzohydrazine; m-chlorobenzo- hydrazine; nitroguanylhydrazine; alpha-(alpha,4-nitro- phenyl)-alpha-methylhydrazine; xenylse icarbazide; tolylse icarbazide; phenylsemicarbazide; 1-naphylsemi- carbazide; 2-naphylsemicarbazide; 3,5-dinitrophenyl- se icarbazide; dibromomethone; benzothiazole; benzothiazoleine; hydantoins ; aminomorpholines ; hydrazinobenzoic acid; 1,3-cyclohexadione; 1,2-bis(p-methoxybenzylamino)ethane; adipic dihydrazide; benzoylhydrazine; isonicotinic acid hydrazide; nicotinic acid hydrazide; oxalyl dihydrazide; oxamic hydrazide, and salicylhydrazide. The derivatizing agent used in the present inven¬ tion preferably is a dispersion of particles substan- tially of the same size which is retained on the surface of a solid substrate with relatively uniform spacing between them, the particles typically ranging from about 0.1-1.0 micrometers in size. It will be noted also that a single derivatizing agent may be useful for reaction with a variety of different carbonyl containing compositions in either vaporized or liquid physical states - each of which will react with the chosen derivatizing agent to form a plurality of immobilized nucleating sites in-situ on the surface of the solid substrate. It will be understood that the nucleating sites formed in-situ are themselves crystals which are invisible to the unaided eye but which serve as specific initiation sites for the subsequent formation and growth of optically detectable crystals.
A film of monodispersed particles comprising the chosen derivatizing agent may be conveniently prepared by directing an aerosol of the solution comprising the derivatizing agent against the surface of a solid substrate in the conventionally known manner. By maintaining very accurate control over the concentration in the aerosol over time and by controlling the speed of delivery of the aerosol to the surface of the solid substrate, one may deposit an accurately controlled amount of the derivatizing agent onto the surface. To obtain satisfactory adherence of the aerosol particles to the surface of the solid substrate, it is often desirable (but not necessary) to pretreat the surface by application of an electrostatic charge and/or by temperature regulation of the aerosol. Equipment for forming the aerosol in the desired range of particles is commercially available. It should be noted also that the solid substrate upon which the chosen derivatizing agent is located may be shaped into a variety of different forms and configurations. The preferred format will be described in the detailed description for the improved apparatus comprising the present invention. Nevertheless, a variety of differently configured apparatus and articles may be prepared as test kits and film badges which employ the derivatizing agent in the described manner for the purpose of forming a plurality of immobilized nucleating sites in-situ.
The Metastable Supersaturated (Developer) Solution
Once the nucleating sites have been formed in-situ -on the surface of a solid substrate via the reaction of the oxidized product with the derivatizing agent, the plurality of immobilized nucleating sites are preferably treated with a selective, metastable supersaturated (developer) solution such that a plurality of optically detectable crystals are formed on the surface of the solid substrate. The metastable supersatured solution is an aqueous or non-aqueous liquid containing a supersaturated concentration of a crystal growing entity which is both chemically and physically stable for a defined period of time. Accordingly, when the metastable supersaturated (developer) solution is combined with the nucleating sites formed in-situ on the surface of the solid substrate, crystallization will occur at each of the individual nucleating sites resulting in the formation and growth of optically detectable crystals. A useful but non-exhaustive listing of selective metastable supersaturated (developer) solutions is provided by Table V in which each developer solution is correlated with a preferred derivatizing agent and an illustrative carbonyl containing composition. The quantities of each chemical composition to prepare a metastable supersaturated (developer) solution in aqueous and non-aqueous liquids are conventionally known in the art.
Table V
CARBONYL CONTAINING PREFERRED SELECTIVE METASTABLE
COMPOSITION OF CHOICE DERIVATIZING SUPERSATURATED
FORMED BY OXIDATION AGENT DEVELOPER SOLUTION
Formaldehyde p-hydroxybenzoic formyl-p-hydroxy- acid hydrazide benzoic acid hydrazone
Chloroacetaldehyde semicarbazide chloroacetyl semi- (formaldehyde) carbazone
2—bu oxyacetaldehyde semicarbazide 2-butoxyacetyl semi-carbazone
Butyraldehyde p-hydroxybenzoic butyrl-p-hydroxy- acid hydrazide benzoic acid hydrazone
Cyclohexanone 2, -dinitro- cyclohexanone-2,4- phenyl hydrazine dinitro phenyl- hydrazone
Acetone 2 ,4-dinitro- acetone-2,4-dinitro phenyl hydrazine phenyl hydrazone
Methoxy acetaldehyde semlcarbazide methoxyacetyl semi- carbazone
Benzaldehyde 2,4-dinitro- benzaldehyde 2,4- phenyl hydrazine dinitro phenyl hydrazone Acetaldehyde p-nitrophenyl acetyl-p-nitro- hydrazine phenyl hydrazone
Terephthaldehyde 2 ,4-dinitro- terephthaldehyde phenyl hydrazine 2 ,'4-dinitro- phenyl hydrazone m-chlorobenzaldehyde 2, -dinitro- m-chlorobenzal¬ phenyl hydrazine dehyde 2,4-dinitro- phenyl hydrazone
Acrolein semlcarbazide acrolein semi- carbazone Propionaldehyde p-hydroxybenzoic propionyl p-hydroxy¬ acid hydrazide benzoic acid hydra¬ zone
Salicylaldehyde semicarbazide salicylaldehyde semicarbazone It is noteworthy that the listing of Table V indicates that the composition of the metastable supersaturated solution is preferably identical to the condensation reaction product formed by the reaction of the carbonyl containing composition and the derivatizing agent. It is specifically noted that under certain circumstances, the chemical composition of the metastable supersaturated solution need not be chemically identical to the condensation product forming the individual nucleating sites, but may be distinctly different in chemical formula and structure while remaining sufficiently similar for crystallization to occur - a phenomenon termed "epitaxy" . It is also recognized that the size and nature of the crystals formed and grown on the surface of the solid substrate as a result of the treatment with the selective developer solution are directly affected by the individual number of nucleating sites formed in-situ by the interaction of the carbonyl containing composition and the derivatizing agent. Relatively speaking, if the peroxide conversion reaction produces only a small quantity of a carbonyl containing composition, relatively few nucleating sites are formed on the surface of the solid; alternatively, if the peroxide converting step produces a relatively large quantity of reaction product, a proportionately larger number of individual nucleating sites will be formed on the surface of the solid. The number of nucleating sites formed will determine the ultimate size of the crystals grown since the amount of crystallizable material in the developer solution will be divided equally among all the nucleating sites which are identical in chemical composition regardless of their number. Accordingly, if a large number of nucleating sites are present, the quantity of crystallizing compound in the developer solution will be distributed over a greater number of nucleating sites and the size of each crystal formed and grown will be relatively small. Conversely, if the number of nucleating sites formed is relatively small, the same concentration of crystallizable material in the developer solution will be equally distributed over a smaller number of individual nucleating sites, thereby causing a greater quantity of crystallizable material to be deposited on each site. This causes the formation of larger sized crystals. Quantitatively therefore, the concentration of the crystallizable matter comprising the metastable, supersaturated (developer) solution is important in that the concentration be of sufficient magnitude to provide for the growth of optically detectable crystals - even when the number of individual nucleating sites is at a maximum. For this reason, metastable supersaturated (developer) solutions are preferably prepared at a concentration ranging from 2-10 times saturation concentration. By using developer solution of such concentration magnitude, metastable solutions, rapid crystal formation and growth will occur after one or two minutes contact with the individual nucleating sites. In this manner, the speed of the assay methodology is maintained, and maximum crystal growth is obtained within 2-10 minutes; and is visible complete in all respects in not more than 60 minutes time.
It should also be recognized that the control of the speed of matter deposition also directly affects the ability of the grown crystals to be optically detected. For example, a rapid deposition of crystallizable material from the developer solution onto the individual nucleating sites tends to build up the crystal face toward an irregular point; whereas more leisurely deposition of crystallizable material produces a crystal line lattice whose crystal face is essentially flat. Optically, light is scattered more readily from crystals which have irregular and pointed faces and thus become more "visible" as a result of the light scattering effect; in comparison, flat-faced crystal lattices scatter light minimally and are more difficult to visualize with the unaided eye or with the use of specific crystal detection equipment. For this reason also, it is preferred that the concentration of the chemical compositions used in making metastable supersatured (developer) solution be as concentrated as possible in order to promote rapid deposition of the crystalline material when brought into reactive contact with the individual nucleating sites. In view of the inherent instability of a super¬ saturated solution prepared in maximum concentration, it is advisable to prepare the developer solution immediately prior to use. Furthermore, stabilizers such as polyvinyl alcohol, polyvinyl, pyrrolidone, gelatin, agar, sodium carboxymethyl cellulose, methyl and ethyl cellulose and the like are, useful and desirable for prolonging the effective life of the supersaturated developer solution in the present invention. As a practical matter therefore, it is convenient to have two prepared reagents available, one or both of which is in solution, which may be mixed at a predetermined ratio on-site to form the metastable supersaturated (developer) solution. The mixed solution may then be combined with the individual nucleating sites immobilized on the surface of the solid substrate to form the optically detectable crystals.
The effect of forming and growing crystals in this manner gives rise to an optically observable and detectable change which is both a qualitative and quantitative measure of the analyte of interest in the test sample. The degree of optically detectable crystals provides both a qualitative and/or quantita¬ tive measure which is correlatable with a calculated amount of an analyte of interest in the fluid sample. Typically, quantitative results in the micromolar range or less may be reproducibly obtained within 10-15 minutes by the formation and growth of optically detectable crystals in the heretofor described manner.
Test Kit Apparatus For Practicing The Present Invention
The manipulative steps previously described herein are preferably performed using an apparatus such as the monitoring badge device illustrated by Figs. 1-8, respectively. As shown therein, preferred embodiments of the improved apparatus provide a chamber 10 preferably in the form of a rigid one-piece molded transparent plastic article illustrated in overhead and side views by Figs. 1 and 2, respec- tively. The chamber 10 is preferably made of a resilient and transparent material such as polstyrene of polycarbonate resin having a closed interior volume which is divided into individual sections and spaces of differing configuration and volume. The interior of the chamber 10 is divided into a U-shaped section 12, a reagent housing 30, a developer solution inlet 50, and an enclosed receptable 60. A plastic sheet 14 forms a common back wall throughout the entirety of the chamber 10 and preferably has a metallized layer 15 bonded to the exterior surface to aid in visualization of the crystals to be formed subsequently in the chamber. As shown in Figure 3, the interior surface 16 of the sheet 14 within the U-shaped section 12 acts as the solid substrate upon which is a derivatizing agent 18 is deposited. It is most desirable to closely control the precise dimensions and configuration of the U-shaped section 12 in order to obtain accurately reproducible results. The dimensions and configuration may vary within limits depending upon the specific analyte of interest to be detected, the chosen derivatizing agent, the metastable supersaturated solution to be employed, and the range of concentrations and time of exposure to be accommodated. In most instances, the void dimensions above the interior surface 16 of the sheet 14 has a width in the range of 0.2-1.0 inches and has a height of approximately 0.005-0.03 inches.
As seen in Fig. 4, the developer solution inlet 50 is integrally joined to the U-shaped section 12 and provides means for introduction of the metastable, supersaturated solution into the interior of the chamber 10 such that it can be applied to the interior surface 16 of the sheet 14 in a controlled manner on demand. The developer solution inlet 50 is to be sealed with impervious tape which is removed when the developer solution is introduced.
As seen in Fig. 5, the enclosed receptable 60 is composed of a retainer 62 and a frangible ampule 64 filled with an aqueous salt solution of choice. The receptable 60 is sealed by the retainer 62, a strip of flexible impervious adhesive tape adhering at its outer margins to the receptable. Beneath the frangible ampule 64 is a sponge material 66 which is in fluid communication with a pair of capillary openings 68 and 70 at the bottom of the receptable 60. The capillary openings 68, 70 extend through the interior of chamber 10 along the longitudinal margins serve as capillary channels or troughs for the flow of the aqueous salt solution from the receptacle 60 through the U—shaped section 12. This salt solution serves to maintain a constant relative humidity within the chamber 10. If desired, strips of wicking fibrous or porous material may be placed within the capillary openings 68 and 70 to aid in maintaining a uniform flow of liquid.
The reagent housing 30 is shown by Fig. 6. As seen in Fig. 6, the reagent housing 30 is configured as a substantially cylindrical space whose internal volume has been divided into an upper spaced region 40 and a lower, narrow passage 34 which communicates with the interior of the chamber 10. The upper spaced region 40 is occupied by a reagent pad 44 preferably composed of a porous matrix material; preferably contains a single enzyme or a mixture of enzymes able to react with the analyte of interest; and is sufficient to generate a peroxide as a primary reaction product. This reagent pad 44 also contains all the other reactants necessary for reacting such peroxides as are generated to yield a carbonyl containing product in the preferred vaporized form. In this manner, a fluid sample containing the analyte of interest will enter the spaced region 40; react with the enzymes and other reagents present in reagent pad 44; and subsequently produce a carbonyl containing composition which released into the interior of chamber 10 via the passage 34.
To prepare the apparatus for intended use, the film badge is exposed to the ambient environment by removing the device from a sealed package. The frangible ampule is broken and the aqueous salt solution flows through the capillaries 68, 70. Subsequently, a fluid sample believed to contain the analyte of interest is introduced via the housing 30. After a predetermined period of time, it is expected that the analyte of interest will have passed through the reagent housing 30 and reacted with all the reactants residing within the reagent pad 44. The secondary reaction product containing at least one carbonyl group is preferably released from the reagent pad 44 in gaseous form which then travels through the narrow passage 34 and enters the interior of the U-shaped section 12. Once within the U-shaped section 12, the vaporized carbonyl containing composition will react with the derivatizing agent 18 present in solid microparticle form to yield a condensation reaction product formed as a plurality of nucleating sites in-situ on the interior surface 16 of the sheet 14. After a pre- determined time, a metastable supersaturated solution is introduced via the developer solution inlet 50 and fills the U-shaped section 12 for reaction with the condensation reaction product formed as the plurality of nucleating sites in-situ. The treatment of these nucleating sites with the selective metastable supersaturated solution forms a plurality of optically detectable crystals on the interior surface 16 of the sheet 14. The presence of these optically detectable crystals serves as a qualitative and/or quantitative measure of the analyte of interest in the initial test sample.
The utility and operability of this improved apparatus design and configuration is illustrated by Figs. 7 and 8 respectively. Fig. 7 illustrates a magnified (300X) view of derivatizing agent 18 deposited on the interior surface 16 of the sheet 14 within the U-shaped section 12 before any reactions have occurred. Fig. 8 is a magnified (300X) view demonstrating the formation of optically detectable crystals on the surface 16 after the apparatus has been exposed to a sample for a predetermined period of time and the manipulated steps of the present methodology have been performed within this apparatus. A scale of arbitrary units (not shown) can be calibrated as to enable the user to quickly determine the concentration of the analyte of sample.
Illustrative Examples
Example 1: Quantitative Determination of
Cholesterol Using An Enzymatic Reaction System
To empirically demonstrate the methodology of the present invention, a quantitative determination of cholesterol was undertaken in the following manner: a cholesterol stock solution was prepared containing 1 mg/ml cholesterol in 10 mg/ml of bovine serum albumin (fatty acid free), 3% Triton X-100 and 0.2 M acetate buffer (pH 7.0).
Cholesterol oxidase was commercially obtained as a preparation derived from Nocardia erythropolis. This enzyme was prepared at 25 U/mg at 25 C and contained 1 mg/ml of protein.
A FeSO, solution was prepared as a 1.32 M preparation in 1 N H_S0,.
A Saligenin solution was prepared by dissolving one gram of saligenin in 20 milliliters of water (0.4 M solution) .
Procedurally, aliquots of cholesterol varying in concentration from 16-50 mg/dl were prepared by diluting the cholesterol stock solution with the diluent identical in composition to the cholesterol stock solution but without cholesterol. The total aliquot volume in each instance was 0.5 ml. To each aliquot of cholesterol, 5 microliters of cholesterol oxidase was added and this reaction mixture incubated at room temperature for 8 minutes. Subsequently, 0.5 ml of the saligenin solution was added to the primary reaction products followed by addition of 5.0 microliters of the FeSO, solution. 30 microliters of this reaction mixture was then deposited onto a Porex 4900 pad polyethylene (Porex Industries, Inc., Atlanta, GA) placed within the housing of the test apparatus previously described herein. A derivatizing agent comprising a semicarbazide film was accessible to the vaporized secondary reaction product liberated by the reaction mixture. The reaction was then allowed to proceed for 45 minutes exposure, followed by a 10 minute application of developer solution containing an equal mixture of 10% Knox gelatin containing 0.2% salicylaldehyde in water and 0.15% semicarbazide in water (prepared just before use). Subsequently, the presence of light scattering crystals was measured as millimeter length for each test aliquot. The results are illustrated graphically by Fig. 9 which reveals a dose response curve proportional to the concentration of cholesterol for the range from 0-50 mg/dl respectively. For clinical applications, serum samples would be diluted ten fold in this assay format.
Example 2: Preparation Of A Dry Reagent Pad For Cholesterol Oxidation
8 microliters of catalase (Sigma C-3155, 48,900 units/mg protein, 16.3 mg protein/ml) is mixed with 200 microliters of cholesterol oxidase solution (Boehringer Mannheim 396818 solution in NaCl, pH 6.0, 25 units/ g at 25 C, 1 mg protein/ml). 10 micro¬ liters of the resulting mixture is then spotted onto 1/4 inch Whatman 3 MM disks. The disks are allowed to dry at room temperature in a dessicated atnrosphere for 6 hours before being placed into the housing 30 of Fig. 6 for use as the reagent pad 44.
A 20 microliter sample of cholesterol in 0.1M phosphate buffer (pH 3.5) containing 10% ethanol is applied to the prepared disk serving as reagent pad. A film of derivatizing agent comprising a monodisperse, aerosol generated, coating of 5 parts p-nitrophenylhydrazine and 1 part citric acid has been applied as a 0.07 inch wide stripe to the interior surface of the U-shaped section within the badge. After 15 minutes reaction time, the film within the badge is developed by addition of a metastable supersaturated solution of acetaldehyde p-nitrophenyl- hydrazone in a 20% methanol/water solution. After development, a column of optically visible light scattering crystals 21.8 mm in length was present on the face of the film. This length of crystals was quantitatively evaluated as equivalent to a cholesterol concentration of 0.22 mM in the sample.
The present invention is not be restricted in form nor limited in scope except by the claims appended hereto.

Claims

What we claim is:
1. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of: reacting the analyte of interest in the fluid sample with at least one reactant to yield a peroxide as a primary reaction product; adding said peroxide to: a composition containing at least one ROH moiety wherein R is a carbon containing entity, and chemical means for initiating a reaction between said peroxide and said ROH containing composi¬ tion, such that a secondary reaction product containing at least one carbonyl group is generated; combining said carbonyl containing product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ, said nucleating sites being immobilized on the surface of a solid substrate; treating said immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of said formed crystals as a measure of the analyte of interest in the sample.
2. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of: reacting the analyte of interest in the fluid sample with at least one reactant to yield a peroxide as a primary reaction product; adding said peroxide to: a composition containing at least one hydroxyl group selected from the group consisting of alcohols and hydroxyl organic acids, and an enzyme able to initiate a reaction between said peroxide and said hydroxyl-containing composition, such that a secondary reaction product containing at least one carbonyl group is generated; combining said carbonyl containing product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ, said nucleating sites being immobilized on the surface of a solid substrate; treating said immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of said formed crystals as a measure of the analyte of interest in the sample.
3. A method for detecting an analyte of interest in a fluid sample, said method comprising the steps of: reacting the analyte of interest in the fluid sample with at least one reactant to yield a peroxide as a primary reaction product; adding said peroxide to: a composition containing at least one hydroxyl group selected from the group consisting of alcohols and hydroxy organic acids, and a metallic cation able to initiate a reaction between said peroxide and said hydroxyl containing composition, such that a secondary reaction product containing at least one carbonyl group is generated; combining said carbonyl containing product with a derivatizing agent to yield a tertiary reaction product formed as a plurality of nucleating sites in-situ , said nucleating sites being immobilized on the surface of a solid substrate; treating said immobilized nucleating sites with a metastable supersaturated solution such that a plurality of optically detectable crystals are formed; and optically detecting the presence of said formed crystals as a measure of the analyte of interest in the sample.
4. The method as recited in claim 1, 2, or 3 wherein the analyte of interest is combined with at least one enzymatic reactant to yield a peroxide.
5. The method as recited in claim 2 wherein said enzyme is a peroxidase.
6. The method as recited in claim 3 wherein said metallic cation is selected from the group consisting of iron, cobalt, manganese, molybdenum, and tungsten.
7. The method as recited in claim 1, 2, or 3 wherein said carbonyl containing product is detected in gaseous form.
PCT/US1989/001923 1988-05-20 1989-05-05 Non-instrument methods for detecting an analyte of interest using peroxide intermediates and light scattering crystals WO1989011542A1 (en)

Priority Applications (1)

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JP1505976A JPH0714357B2 (en) 1988-05-20 1989-05-05 A method for detecting a problematic test substance without using an instrument by utilizing a peroxide intermediate and a light-dispersible crystal

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US19645088A 1988-05-20 1988-05-20
US196,450 1988-05-20

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US4202938A (en) * 1976-04-05 1980-05-13 Human Gesellschaft mbH fur Biotopanalytic und Biotopschutz Procedure for the quantitative determination of hydrogen peroxide concentration in aqueous solutions
US4380587A (en) * 1981-08-20 1983-04-19 Arthur D. Little, Inc. Film badge for determining carbonyl compounds
US4492754A (en) * 1981-06-23 1985-01-08 Boehringer Mannheim Gmbh Composition and method for the determination of hydrogen peroxide
US4727024A (en) * 1986-05-12 1988-02-23 Martin Koocher Binding assays involving formation and detection of light scattering crystals
US4816414A (en) * 1987-10-02 1989-03-28 Crystal Diagnostics, Inc. Methods and apparatus for detecting an analyte of interest capable of being converted into a carbonyl-containing composition

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4202938A (en) * 1976-04-05 1980-05-13 Human Gesellschaft mbH fur Biotopanalytic und Biotopschutz Procedure for the quantitative determination of hydrogen peroxide concentration in aqueous solutions
US4492754A (en) * 1981-06-23 1985-01-08 Boehringer Mannheim Gmbh Composition and method for the determination of hydrogen peroxide
US4380587A (en) * 1981-08-20 1983-04-19 Arthur D. Little, Inc. Film badge for determining carbonyl compounds
US4727024A (en) * 1986-05-12 1988-02-23 Martin Koocher Binding assays involving formation and detection of light scattering crystals
US4816414A (en) * 1987-10-02 1989-03-28 Crystal Diagnostics, Inc. Methods and apparatus for detecting an analyte of interest capable of being converted into a carbonyl-containing composition

Non-Patent Citations (5)

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Title
Aust. J. Chem., Vol. 26, 1973, D.L. INGLES, "Studies of Oxidations by Fenton's Reagent using Redox Titration", pages 1021-1029. see Abstract. *
Biochemistry, Vol. 22, 1983, MAZER et al, "Quasi-Elastic Light-Scattering Studies of Aqueous Biliary Lipid Systems", pages 426-440. see Abstract. *
Journal of Lipid Research, Vol. 27, 1986, HALPERN et al, "Vesicle Aggregation in Model Systems of Supersaturated Bile: Relation to Crystal Nucleation and Lipid Composition of the Vesicular Phase", pages 295-306. see Abstract. *
Methods of Enzymatic Analysis; Vol. III, 1983, Editors: BERGMEYER et al., "Catalase", pages 243-277, see page 275. *
See also references of EP0371118A4 *

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JPH02501536A (en) 1990-05-31
AU3688389A (en) 1989-12-12
EP0371118A4 (en) 1990-12-27
JPH0714357B2 (en) 1995-02-22
EP0371118A1 (en) 1990-06-06

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