WO1989006284A1 - Reactif de diagnostic sous forme de poudre seche et ses applications - Google Patents

Reactif de diagnostic sous forme de poudre seche et ses applications Download PDF

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Publication number
WO1989006284A1
WO1989006284A1 PCT/US1988/004698 US8804698W WO8906284A1 WO 1989006284 A1 WO1989006284 A1 WO 1989006284A1 US 8804698 W US8804698 W US 8804698W WO 8906284 A1 WO8906284 A1 WO 8906284A1
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WO
WIPO (PCT)
Prior art keywords
reagent
powder
enzyme
indicator
powdered
Prior art date
Application number
PCT/US1988/004698
Other languages
English (en)
Inventor
Arthur M. Usmani
Mark T. Skarstedt
Original Assignee
Boehringer Mannheim Corporation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Boehringer Mannheim Corporation filed Critical Boehringer Mannheim Corporation
Publication of WO1989006284A1 publication Critical patent/WO1989006284A1/fr

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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

Definitions

  • This invention relates to a dry reagent useful in analytical chemistry, apparatus which employ this reagent, processes for preparing both the reagent and the apparatus, and methods for performing analysis using these.
  • the field of analytical and diagnostic chemistry has grown tremendously in recent years.
  • the field relates to analysis of body fluids, such as blood, serum, urine and the like, food substances, such as milk, drinking water, and other fluid substances.
  • Applications of the field include medical diagnosis, purity testing, forensic science, etc.
  • the apparatus employed in analytical chemistry of the type discussed herein is generally a device referred to at times as a test strip, a dip stick, or other terms which will be familiar to those skilled in the art.
  • the device contains various substances incorporated therein, which react with a particular analyte, or substance to be determined, with formation of a determinable or detectable signal. Frequently, but not always, this is a color. Color formation, or change in color, permits one to give a "yes-no" answer to the question of whether a substance or analyte is present in a test sample. Degree of color formed, or the actual color, can be used as an indication of how much analyte is present.
  • the sample may be applied to the test device, or the device may itself be dipped in or otherwise contacted to the test sample.
  • One very common test which is presented as exemplary of the type of assay performable with test devices and dry chemistry is a determination of blood glucose level. This, of course, is important in many situations, such as monitoring of diabetic and hypoglycemic patients.
  • a test strip is prepared which contains the enzymes glucose oxidase and a peroxidase, as well as the indicator 3,3 ' ,5,5'-tetramethylbenzidine, also referred to as "TME". If glucose is present in the sample, it reacts with oxygen in the presence of glucose oxidase to form gluconic acid and hydrogen peroxide.
  • the peroxide reacts with the TMB in the presence of the peroxidase, oxidizing the TMB.
  • TMB in unoxidized form, is colorless. When oxidized, it is blue, red or purple, depending upon the change transfer complex formed. Thus, one can determine if glucose is present, and. how much, by observing formation of color.
  • the system is analyte specific, because hydrogen peroxide is not a normal component of the sample, and only forms when the glucose specific enzyme glucose oxidase acts on its substrate.
  • a test device of the type used in the analysis under consideration contains three “layers".
  • the first which plays no actual part in the assay, is a base or carrier layer.
  • This layer is inert, and is generally constructed from materials such as thermoplastic films.
  • a second layer which will be referred to as the "reflective layer” , is employed to aid in reading the detectable signal when it forms. This is especially helpful in systems which use a reflected light detector, as discussed. supra.
  • This layer contains some material which reflects light not absorbed by the detectable signal generator.
  • this iaver takes the form of a coating, foar , membrane, paper, or metal foil which is reflective. Substance useful for this layer include TiO ? and ZrOfact .
  • the third layer generally referred to as the reagent layer contains the ingredients which cause formation of the detectable signal, and it is this layer which is actually "read” to determine the analyte.
  • Various enzymes, substrates, receptors, and binding partners can be incorporated in this layer, such as labeled antibodies, indicator molecules, fluorescent agents, capping or quenching agents, and so forth.
  • This layer is generally constructed of paper or other fibrous materials, non-fibrous materials such as films of polymeric substances, as well as hybrid layers containing both.
  • the system can also be used via deployment of a visible light detecting system.
  • the test device is placed in a "reader", in which incident light is directed to the reagent layer. Some will be absorbed by the signal generator, and some will be reflected, forming the "signal”. This signal is reflected light, which is read by a detector. Reading the wavelength and intensity of the light at the detector is how the sample is analyzed.
  • the practice of this invention involves preparing a mixture of powdered enzyme and powdered indicator to form a homogeneous reactive powder reagent composition containing both.
  • Powdered indicators and enzymes are both readily available materials, purchasable from any number of suppliers. Additional materials may be added to the uniform powdered composition, as desired. Most desirably, a surfactant may be added.
  • Surfactants in powder form, are useful for several reasons. While they help to bring together the test sample and the indicator/enzyme powder mix, some possess additional useful properties.
  • the surfactant sodium dodecyl-benzene sulfonate for example, helps to stabilize the color forming species following reaction of the indicator and enzyme to form a signal.
  • red blood cells frequently interfere with the assay, because of their strong color. If a suitable surfactant is chosen, red blood cells do not tend to stick thereto and do not interfere with the reaction being studied. Additionally, rupture of these red blood cells can be mi-igated by choosing surfactants which impede hemolysi ⁇ . Among the preferred surfactants . in addition to sodiur.
  • dodecvl-be zene ⁇ ulfcnate are non-ionic surfactants including, but not limited to, ethylene oxide/propylene oxide block copolymers; ethyleneoxy ethanol; ethylene oxide adduct ⁇ ; alkoxylated lauryl alcohol; nonylphenoxypolyethanol; alkylphenoxypoly- oxyethylene ethanol; ethoxylated fatty alcohols; ethoxylated alcohol; and ethoxylated octylphenol.
  • Useful amphoteric surfactants include the sulfobetaines .
  • Other anionic surfactants include sodium dodecylsulfate, triethanol a ine alkylbenzene sulfonate and sodium alkylbenzene sulfonate.
  • Polymers are also useful in the powder reagent. These act as fillers, rather than binders, which is the normal role of polymers in the art.
  • the polymer or polymers chosen, if chosen should be hydrophilic and not contain significant amounts of group ⁇ which interfere with enzyme activity, such as carboxylic acid group ⁇ .
  • suitable polymers include polyvinyl alcohol; polyvinyl acetate; polyvinyl propionate; vinyl acetate/vinyl chloride copolymers; acrylic resin ⁇ (acrylate and methacrylate) ; lightly hydroxylated acrylics; ethylene oxide/propylene oxide block copolymers; polyvinyl chloride; styrene acryonitrile resins; polyvinyl- pyrrolidone; hydroxyethyl cellulose; cellulose acetate; cellulose acetate propionate; alginate ⁇ ; xanthans; cro ⁇ linked polyvinyl alcohol, and polymerized and copolymerized diallyl phthalate.
  • Other useful materials will be evident to the skilled artisan.
  • Additional useful materials include fluorinated substances, as they are useful in repelling red blood cells.
  • Useful fluorinated powders include polyvinylidene fluoride, especially of a particle size of 3-5 /u; vinylidene fluoride/hexafluoro propylene copolymers (3-5 ⁇ ) ; and teflon.
  • Cationic polymers may be used as well as anionic polymers, as may mixes of these. This is unusual, because, in solutions of ingredients, mixtures of anionic and cationic materials lead to undesirable ficcculatior.. Such is not the case in this invention.
  • the anionic substances are useful in repelling negatively charged red blood cells. Exemplary of such useful anionic materials include anionic compounds, surfactants, and polymers.
  • Buffers may optionally be used, for their known effect.
  • a typical dry powder, in accordance with the invention, and the broad ranges over which the various components may extend, is as follows:
  • the ranges given are in weight percent. It must be understood as well that the only essential ingredients in the reagent are the enzyme or enzymes (in this case GO and POD: glucose oxidase and peroxidase, respectively) , and the indicator (in this case TMB, 3,3 ' ,5,5 r -tetramethylbenzidine) . It will be noted by the skilled artisan that a reagent of this composition could not be prepared in aqueous form. TMB is net very soluble in water.
  • the adhesive surface is a film of polycarbonate cr another thermoplastic tc which has been applied a pressure sensitive emulsion.
  • This emulsion gives the film "tack” or, in other terms, makes it sticky.
  • the emulsion should it contain water, can be treated at temperatures of up to 80-90°C to drive the water off. Such treatment, of course, is not possible if the enzyme has already been applied to the carrier, as this temperature would deactivate the enzyme.
  • Pressure sensitive emulsions are well known to the art.
  • Examples include “Flexbond 149”, and “Flexbond 150” of Air Products, “Aroset” produces of Ashland Chemicals, and so forth. These pressure sensitive emulsions are generally polymers and resins. “Flexbond 149”, for example, is a vinyl acetate copolymer, and Flexbond 150 is a saturated vinyl acetate polymer.
  • One common form of pressure sensitive emulsion will be recognized as “scotch tape”, and other forms of sticky tape will be known to the skilled artisan as well. 21 C.F.R. ⁇ 175.105, “adhe ⁇ ive ⁇ ", which i ⁇ incorporated by reference, lists many adhe ⁇ ive ⁇ which are usable in the invention. The choice of adhesive will, of course vary, depending on availability and the particular system being used.
  • the carrier on which the adhesive is applied and to which the dry composition adhered may be a polycarbonate. Typically, this is about 140 uxa in thickne ⁇ s, but the thickness of the carrier and adhesive emulsion may cover a broad range of thickness. Typically, a range of from about 90 m to about 250 urn is preferred. Other materials may also be used.
  • nylon block co-polymers examples include polyester ⁇ , polyvinyl-chloride ⁇ , polypropylene-ethylene-vinylacetate blend. These may include a reflective agent, such as TiO tenure, but thi ⁇ is not required.
  • the pressure sensitive emulsion is applied to the carrier, by any conventional method. Once this is done, any water is removed by, e.g., evaporation under ambient or reduced pressure conditions. Once this is accomplished, the reagent powder is applied.
  • the carrier containing the pressure sensitive emulsion can be pulled via, e.g., a conveyor belt past a hopper which distributes the powdered reagent uniformly thereover. Exces ⁇ powder i ⁇ brushed away, and recycled. Simple sprinkling, dry powder nozzle dispensing, and fluidized bed application are all possibi ⁇ lities, as are electrostatic applications, such as with an electrostatic gun.
  • Particle size of the reagent powder can be controlled by various sizing methods, e.g., a ball mill. Varying the applicator rate will result in varying the concentration of the powder.
  • this element is prepared in which the reagent i ⁇ incorporated into the carrier, it can be u ⁇ ed by itself, or it may also be cut, shaped, or otherwise styled to be part of different test strip embodiments. Once the element or the test strip is prepared, it may then be used to evaluate analytes in various fluid samples.
  • enzymes and indicators may be used.
  • these enzymes which are used in test system ⁇ of thi ⁇ type and which will be recognized by the skilled artisan are amylase, beta galactosidase, alkaline phosphatase, catalase, urica ⁇ e, and so forth.
  • Different substrates include CPRG, ail galactopyranoside derivatives, resorufin, ABT£, _-meth ⁇ -_y naphthci galactopyrancside, MBTS, AAP (4-amino antipyrine) , TOOS (N-ethyl-N- (2- hydroxy-3-sulfopropyl) -m-toluidine, TMB derivatives, such as TMB dihydrochloride, 3, 3 ' ,5,5 ' -tetraethylbenzidine and its hydrochloride, 4AAP/MAOS (3, 5-dimethyl-N-ethyl-N- (2-hydroxy-3-sulfopropyl)-aniline) , 4AAP/ADOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine) , 4AAP/ALPS (N-ethyl-N-sulfopropylaniline
  • Nonionic surfactant 1.500g 1.500c
  • each of these formulations was applied to a previously prepared polycarbonate film.
  • the film had applied thereto the polymeric adhesive Flexbond 149, at thicknes ⁇ of 200 m ⁇ and 40 m ⁇ .
  • the ⁇ e were allowed to dry overnight under a hood. Following removal of additional water, a tacky film re ⁇ ulted to which the formulation ⁇ were applied.
  • the dry film ⁇ were about 100 m ⁇ and 20 m ⁇ . thick when dry.
  • the powdered reagent ⁇ were then ⁇ alted onto the film ⁇ .
  • the films had the powder adhering thereto, they were tested against controls containing 60, 200, and 600 mg/dl glucose, in saline solutions as well as gluco ⁇ e ⁇ piked blood.
  • the solutions were applied to the elements in suitable configurations, and allowed to develop. The results obtained compared to those with strips made from equivalent, waterborne formulations, as is now explained.
  • Formulas 2, 4 and 6 applied by dry form technique were found to maintain their enzymatic activity after strip preparation whereas waterborne formulations partially lost their activity during the drying proces ⁇ .
  • the dry powder formulas gave more intense color (percent reflectance vs. wavelength and glucose concentration plots) with no or minimal adhesion of the red blood cells to the test strip surface. Also, the color reaction was rapid, reached end-point quickly and the sensitivity was excellent. Thu ⁇ , results obtained with dry technique ⁇ trips were better than obtained with other test systems.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Un réactif sous forme de poudre sèche comprend une enzyme ainsi qu'un indicateur en poudre destinés à être utilisés pour évaluer un analyte dans divers échantillons de fluide. On a mis au point un procédé de préparation du réactif sous forme de poudre sèche, ainsi qu'un appareil de test comprenant une surface adhésive sur laquelle on colle ledit réactif sous forme de poudre sèche par exemple, simplement en le saupoudrant sur la surface adhésive.
PCT/US1988/004698 1988-01-11 1988-12-29 Reactif de diagnostic sous forme de poudre seche et ses applications WO1989006284A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14250488A 1988-01-11 1988-01-11
US142,504 1988-01-11

Publications (1)

Publication Number Publication Date
WO1989006284A1 true WO1989006284A1 (fr) 1989-07-13

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PCT/US1988/004698 WO1989006284A1 (fr) 1988-01-11 1988-12-29 Reactif de diagnostic sous forme de poudre seche et ses applications

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EP (1) EP0398941A4 (fr)
JP (1) JPH03501681A (fr)
CA (1) CA1338525C (fr)
WO (1) WO1989006284A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19608129A1 (de) * 1996-03-02 1997-09-04 Karl H A Dr Schmidt Selbstklebender Indikator
WO2004031760A1 (fr) * 2002-09-30 2004-04-15 3M Innovative Properties Company Element capteur colorimetrique reflechissant
US7449146B2 (en) 2002-09-30 2008-11-11 3M Innovative Properties Company Colorimetric sensor
US7767143B2 (en) 2006-06-27 2010-08-03 3M Innovative Properties Company Colorimetric sensors

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB808742A (en) * 1955-06-09 1959-02-11 Miles Lab Glucose test indicator
US3963442A (en) * 1974-12-04 1976-06-15 Bullard Wade A Colorimetric indicator compositions and method of manufacture
US3993451A (en) * 1970-07-28 1976-11-23 Miles Laboratories, Inc. Test for a given constituent in a liquid
US4106991A (en) * 1976-07-07 1978-08-15 Novo Industri A/S Enzyme granulate composition and process for forming enzyme granulates
US4258001A (en) * 1978-12-27 1981-03-24 Eastman Kodak Company Element, structure and method for the analysis or transport of liquids
US4421719A (en) * 1980-06-20 1983-12-20 Minnesota Mining And Manufacturing Company Colorimetric indicators
US4438067A (en) * 1978-05-17 1984-03-20 Battelle Memorial Institute Test strips for analyzing dissolved substances
US4548906A (en) * 1981-11-02 1985-10-22 Fuji Shashin Film Kabushiki Kaisha Integral multilayer analytical element for the analysis of ammonia or an ammonia forming substrate and a method for the detection thereof using the same
US4615972A (en) * 1983-11-04 1986-10-07 Immuno Concepts, Inc. Stabilization of indicators for detecting enzyme activity

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3095277A (en) * 1956-05-21 1963-06-25 Miles Lab Diagnostic composition
USRE29498E (en) * 1972-05-12 1977-12-20 Istituto Sieroterapico e Vaccinogeno Toscano "SCLAVO", S.p.A. Process for the enzymatic determination of glucose with a glucose-oxydazed/peroxidazed enzyme system
US3884764A (en) * 1974-03-25 1975-05-20 Eastman Kodak Co Method and composition for blood serum cholesterol analysis
DE3411997A1 (de) * 1984-03-31 1985-10-17 Boehringer Mannheim Gmbh, 6800 Mannheim Neue redox-indikatoren

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB808742A (en) * 1955-06-09 1959-02-11 Miles Lab Glucose test indicator
US3993451A (en) * 1970-07-28 1976-11-23 Miles Laboratories, Inc. Test for a given constituent in a liquid
US3963442A (en) * 1974-12-04 1976-06-15 Bullard Wade A Colorimetric indicator compositions and method of manufacture
US4106991A (en) * 1976-07-07 1978-08-15 Novo Industri A/S Enzyme granulate composition and process for forming enzyme granulates
US4438067A (en) * 1978-05-17 1984-03-20 Battelle Memorial Institute Test strips for analyzing dissolved substances
US4258001A (en) * 1978-12-27 1981-03-24 Eastman Kodak Company Element, structure and method for the analysis or transport of liquids
US4421719A (en) * 1980-06-20 1983-12-20 Minnesota Mining And Manufacturing Company Colorimetric indicators
US4548906A (en) * 1981-11-02 1985-10-22 Fuji Shashin Film Kabushiki Kaisha Integral multilayer analytical element for the analysis of ammonia or an ammonia forming substrate and a method for the detection thereof using the same
US4615972A (en) * 1983-11-04 1986-10-07 Immuno Concepts, Inc. Stabilization of indicators for detecting enzyme activity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0398941A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19608129A1 (de) * 1996-03-02 1997-09-04 Karl H A Dr Schmidt Selbstklebender Indikator
WO2004031760A1 (fr) * 2002-09-30 2004-04-15 3M Innovative Properties Company Element capteur colorimetrique reflechissant
CN100425987C (zh) * 2002-09-30 2008-10-15 3M创新有限公司 反光比色的传感元件
US7449146B2 (en) 2002-09-30 2008-11-11 3M Innovative Properties Company Colorimetric sensor
US8506902B2 (en) 2002-09-30 2013-08-13 3M Innovative Properties Company Colorimetric sensor
US7767143B2 (en) 2006-06-27 2010-08-03 3M Innovative Properties Company Colorimetric sensors

Also Published As

Publication number Publication date
CA1338525C (fr) 1996-08-20
EP0398941A1 (fr) 1990-11-28
EP0398941A4 (en) 1991-04-10
JPH03501681A (ja) 1991-04-18

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