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WO1993010437A1 - Methode de quantification d'une substance par fluorescence - Google Patents

Methode de quantification d'une substance par fluorescence Download PDF

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Publication number
WO1993010437A1
WO1993010437A1 PCT/SE1992/000792 SE9200792W WO9310437A1 WO 1993010437 A1 WO1993010437 A1 WO 1993010437A1 SE 9200792 W SE9200792 W SE 9200792W WO 9310437 A1 WO9310437 A1 WO 9310437A1
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WO
WIPO (PCT)
Prior art keywords
lambda
emission
fluorescence
measured
wavelength
Prior art date
Application number
PCT/SE1992/000792
Other languages
English (en)
Inventor
Håkan DREVIN
Aija Mattila-Fjelner
Erik Ringberg
Original Assignee
Kabi Pharmacia Ab
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 Kabi Pharmacia Ab filed Critical Kabi Pharmacia Ab
Publication of WO1993010437A1 publication Critical patent/WO1993010437A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction

Definitions

  • the invention pertains to a method of expanding the range within which the concentration of a substance (analyte) contained in a sample can be quantified by means of a fluorescence measuring process.
  • Fluorescence measuring methods are now widely used for the quantitative determination of low-concentration analytes.
  • the methods involve relating measured fluorescence signals to the amount of analyte present (concentration, content, etc.), with the aid of a standard curve or with the aid of standard values, where fluorescence (emission, signal
  • analyte concentration (the amount of analyte present).
  • the analyte concerned may be the
  • test system may be designed so that the strength of measured fluorescence will increase or decrease with increasing amounts of analyte.
  • a standard curve which is obtained by measuring the emission values for different part-intervals I 1 , ....I a , .I n of the curve, either for different excitation wavelengths or different emission wavelengths.
  • the emission values of a part-interval (I a ) corresponding to lower emission in a conventional standard curve are obtained by measuring emission subsequent to excitation at a wavelength at which the fluorophore has higher extinction, compared with a part-interval (I a+1 ) corresponding to higher emission in the conventional standard curve.
  • the emission for the part-interval (I a ) is measured at a wavelength at which the emission of the fluorophore is higher than the emission at the
  • the different intervals are measured, or determined, preferably at a common emission wavelength.
  • excitation for the different intervals preferably takes place at a common excitation wavelength.
  • Intervals having a lower index a correspond to fluorescence (emission) that has lower measured values in a conventional standard curve.
  • Normalization is conveniently effected towards the part-interval which corresponds to the lowest measured fluorescence. See the experimental part.
  • the measured fluorescence (emission) intervals of the standard curve are corresponded by a concentration range which can be divided into part-intervals C 1 . . . ... . .
  • the left and the right limits in I a+1 are higher than the corresponding limits in I a .
  • the invention thus relates to a method of quantifying with the aid of fluorescence emitted from a fluorophore the presence of a substance (analyte) in a sample incorporated into an assay medium, said method comprising the steps of comparing measured fluorescence values with a standard curve where the signal strength (emission) is a function of the amount of analyte.
  • excitation for a part-interval (C a+1 ) of the concentration range has occurred at different excitation wavelengths (lambda ex(a) and
  • lambda ex(a+1) respectively); said lambda ex(a) and lambda ex(a+1) being selected so that the extinction of the fluorophore at lambda ex(a) is greater than at lambda ex(a+1) , or b. are measured at the same excitation wavelength
  • say medium is meant the sample plus all constituents present in the medium on which the fluorescence measurement is performed.
  • the indexes a and a+1 for wavelength (lambda) refer to the part-interval I a and C a and I a+1 and C a+1 , respectively.
  • wavelengths shall be chosen so as to obtain with the emission an acceptable signal/noise ratio.
  • the wavelength for excitation maximum (lambda exmax ) is
  • lambda ex(a+1) lambda ex(a)
  • lambda ex(a+1 ) ⁇ lambda ex(a+1 ) .
  • Lambda ex(a) and lambda ex(a+1) are preferably chosen in the same excitation peak, such that the quotient between the extinction at lambda ex(a) and lambda ex(a+1) will be > 2, preferably 10 or more.
  • (lambda emmax ) is preferably smaller than or equal to
  • lambda em(a) which is smaller than lambda em(a+1) , i.e.
  • lambda emmax lambda em(a) ⁇ lambda em(a+1) .
  • Lambda em(a) and lambda em(a+1) are preferably chosen in the same emission peak, so that the quotient between emission at lambda em(a) and lambda em(a+1) will be > 2, preferably 10 or more.
  • FIGS 1 and 2 illustrate superimposed excitation
  • Figure 1 illustrate a preferred embodiment according to alternative i:a.
  • Figure 2 illustrates a preferred embodiment according to alternative i:b. The significance of the various lambda will be evident from the Figures.
  • the fluorescent substance measuxed in the assay shall preferably exhibit an emission maximum at 300 nm or at a lower wavelength. Stokes shift should be greater than 10 nm, and preferably above 30 nm.
  • the substance may be organic or inorganic. Those compounds of most interest include compounds that have an umbelliferone structure, a rhodamine structure, a fluorescein structure, etc., and fluorescent lanthanide chelates (primarily Eu 3+ . Tb 3+ , Sm 3+ and Dy 3+ ). These latter substances in particular have a large Stokes shift with emission maxima which are well-separated from their respective excitation maxima and from emission wavelengths of proteins and other substances present in biological samples. The fluorescence of lanthanide chelate is often long-lived, which renders the chelates more suited for assaying with time-resolved fluorescence spectroscopy.
  • the invention can be applied with several different types of fluorescence techniques.
  • the fluorescence signal is directly related to the amount of analyte present (concentration) in the sample in those
  • an elevated fluorescence signal may, in this particular case, indicate either a higher or a lower analyte concentration.
  • reagent cofactors, coenzyme, enzyme activity, substrate, cosubstrate, etc.
  • bio-affinitive methods include ligand-receptor methods which utilize a bio-affinitive reactant which is labelled with a fluorophore group or with a group which can give rise to fluorescence, so as to form a receptor-ligand complex in an amount which is related to measured fluorescence and the amount of analyte present.
  • methods which use bio-affinitive ligand-receptor-pairs are antigen/hapten and antibodies (immuno assays).
  • the fluorescence signal is normally a measure of the concentration of fluorophore in the assay medium. This applies to methods where the fluorophore is an analyte, enzymedetermining methods which utilize a fluorogenic substrate, heterogeneous receptor-ligand methods which utilize
  • determining fluorescence are independent of the analyte concentration in the assay medium, but with which the signal is modulated (amplified or reduced) because of direct or indirect interactions with the analyte (homogeneous
  • receptor-ligand assays for instance homogeneous fluoroimmuno assays.
  • the ratio of the extinction for lambda em(a) to lambda em(a+1) and the ratio of the emission at lambda em(a) and the emission at lambda em(a+1) is directly proportional to the "breadth" of the concentration of the composite
  • fluorescing substance takes part, are liable to set limits which render the range more narrow.
  • concentration range in the type of sample concerned.
  • the invention is therewith particularly beneficial in respect of samples that have been taken from living material, such as whole blood, serum, plasma, urine, cerebro spinal fluid, etc.
  • the method can also be applied to assaying environmental contaminants in air, water, soil and living material.
  • receptor-ligand methods the primary use of the invention is found in so-called heterogeneous variants, e.g. heterogeneous immunoassays of, e.g., "sandwich” or competitive (inhibition) types.
  • Example 1 Determining 4-methyl-umbelliferone in aqueous solution. Excitation at different wavelengths. Fluorescence measurement at a common wavelength.
  • the measured values for ex 412 were multiplied by 32, in order to provide a continuous standard curve from the emission values (lambda 365 and lambda 412 ) obtained with excitation at 365 nm and 412 nm respectively.
  • the normal measuring range of 4-methylumbelliferone is 0.008-0.98 Nm (120 times measuring range) with the cuvette concerned and without dilution. Combination measuring according to the invention extended the measuring range to 0.008-125 Nm (about 15,000 times).
  • Example 2 Determining 4-methyl-umbelliferone in aqueous solution. Excitation at the same wavelength. Fluorescence measurement at different wavelengths.
  • Example 3 Determining enzymatic activity when the enzyme is a label group in an enzyme immuno assay .
  • the test used was a commercially available test designated Pharmacia CAP IgE FEIA (Kabi Pharmacia Diagnostics AB, Uppsala, Sweden).
  • the test protocol involved incubating a solid-phase-bound anti-IgE antibody with a serum sample, wherein IgE present in the sample binds to the solid phase (the matrix). The matrix was then washed and incubated with galactosidase-labelled anti-IgE antibody so as to form the complex
  • 4-methyl-umbelliferone was eluted from the matrix and assayed fluorometrically, in our case by excitation at 365 nm and 412 nm and measuring emission at 450 nm for respective excitation wavelengths.
  • the test is designed so that the fluorescence measured (liberated 4-methylumbelliferone) becomes a function of the amount of IgE present in the sample.
  • the matrix used had a sponge-like character and the matrix pores were able to accommodate the whole of the liquid volume in which the immune reaction was carried out.
  • the fluorescence-assaying process was carried out in an FC 96 Fluorocounter (Pharmacia Diagnostics AB, Uppsala, Sweden).
  • the upper measurement signal that can be used is then 60,000 FU, which is about three times more than what the instrument used can measure.
  • the lower part of the assaying range is limited by the non-specific signal. When the non-specific signal contains 0.2% of the total activity, good precision is required in order to be able to distinguish between the lowest standard point (10 KU/l) and the blank value.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

On décrit une méthode pour mesurer la fluorescence émise par un fluorogène dans le dosage d'une substance dans un échantillon, dans laquelle un signal de fluorescence mesuré est comparé avec une courbe représentant la variation de l'intensité du signal (emission) en fonction de la concentration en substance dosée. La méthode est caractérisée essentiellement en ce que (i) les valeurs de fluorescence qui correspondent à l'intervalle de concentration (c) de la substance dosée sur la courbe étalon sont mesurées à la même longueur d'onde d'émission (lambdaem) ou à la même longueur d'onde d'excitation (lambdaex), l'intervalle (c) étant divisé en un nombre fini d'intervalles partiels C1, ... Ca, ... Cn, où a^- est un nombre entier 1 « a « n et n^- est un nombre entier » 2, et que (ii) le signal (fluorescence) émis par l'échantillon est mesuré respectivement pour la longeur d'onde d'excitation et à la longueur d'onde d'émission correspondant respectivement aux longueurs d'onde utilisées lorsqu'on mesure des valeurs correspondantes sur la courbe étalon.
PCT/SE1992/000792 1991-11-20 1992-11-18 Methode de quantification d'une substance par fluorescence WO1993010437A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9103427A SE9103427D0 (sv) 1991-11-20 1991-11-20 Foerfarande vid kvantifiering av en substans med hjaelp av fluorescens
SE9103427-2 1991-11-20

Publications (1)

Publication Number Publication Date
WO1993010437A1 true WO1993010437A1 (fr) 1993-05-27

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Application Number Title Priority Date Filing Date
PCT/SE1992/000792 WO1993010437A1 (fr) 1991-11-20 1992-11-18 Methode de quantification d'une substance par fluorescence

Country Status (3)

Country Link
AU (1) AU3053592A (fr)
SE (1) SE9103427D0 (fr)
WO (1) WO1993010437A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0794433A1 (fr) * 1996-03-05 1997-09-10 Texaco Development Corporation Estimation de l'index API par mesure de fluorescence multiple
WO1998015814A1 (fr) * 1996-10-10 1998-04-16 Cambridge Imaging Limited Methode et dispositif pour analyser des tests biologiques
CN116924596A (zh) * 2023-05-30 2023-10-24 深圳市新西林园林景观有限公司 一种海绵城市污水处理用电化学装置及其处理方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0154743A2 (fr) * 1983-11-15 1985-09-18 Gail Ann Rock Méthode et appareil pour effectuer une analyse automatique, double fluorochromique de la toxicité anti-lymphocytes
EP0257559A2 (fr) * 1986-08-21 1988-03-02 Becton, Dickinson and Company Analyse à fluorescence de plusieurs couleurs avec excitation de longueur unique d'onde
EP0362435A1 (fr) * 1987-02-26 1990-04-11 Nalco Chemical Company Dispositif de surveillance de traitement chimique utilisant des traceurs fluorescents
EP0454886A1 (fr) * 1989-04-26 1991-11-06 Foxs Labs Méthode et appareil améliorés pour mesurer la concentration en oxygène

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0154743A2 (fr) * 1983-11-15 1985-09-18 Gail Ann Rock Méthode et appareil pour effectuer une analyse automatique, double fluorochromique de la toxicité anti-lymphocytes
EP0257559A2 (fr) * 1986-08-21 1988-03-02 Becton, Dickinson and Company Analyse à fluorescence de plusieurs couleurs avec excitation de longueur unique d'onde
EP0362435A1 (fr) * 1987-02-26 1990-04-11 Nalco Chemical Company Dispositif de surveillance de traitement chimique utilisant des traceurs fluorescents
EP0454886A1 (fr) * 1989-04-26 1991-11-06 Foxs Labs Méthode et appareil améliorés pour mesurer la concentration en oxygène

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0794433A1 (fr) * 1996-03-05 1997-09-10 Texaco Development Corporation Estimation de l'index API par mesure de fluorescence multiple
WO1998015814A1 (fr) * 1996-10-10 1998-04-16 Cambridge Imaging Limited Methode et dispositif pour analyser des tests biologiques
CN116924596A (zh) * 2023-05-30 2023-10-24 深圳市新西林园林景观有限公司 一种海绵城市污水处理用电化学装置及其处理方法
CN116924596B (zh) * 2023-05-30 2024-06-07 深圳市新西林园林景观有限公司 一种海绵城市污水处理用电化学装置及其处理方法

Also Published As

Publication number Publication date
SE9103427D0 (sv) 1991-11-20
AU3053592A (en) 1993-06-15

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