WO1984000609A1 - Assemblage de fluorometre ameliore et procede - Google Patents

Assemblage de fluorometre ameliore et procede Download PDF

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Publication number
WO1984000609A1
WO1984000609A1 PCT/US1983/001012 US8301012W WO8400609A1 WO 1984000609 A1 WO1984000609 A1 WO 1984000609A1 US 8301012 W US8301012 W US 8301012W WO 8400609 A1 WO8400609 A1 WO 8400609A1
Authority
WO
WIPO (PCT)
Prior art keywords
excitation energy
fluorometer
fluorescence
assembly
source
Prior art date
Application number
PCT/US1983/001012
Other languages
English (en)
Inventor
Zindel Herbert Heller
Original Assignee
American Hospital Supply Corp
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 American Hospital Supply Corp filed Critical American Hospital Supply Corp
Publication of WO1984000609A1 publication Critical patent/WO1984000609A1/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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • 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

  • This invention is directed to an improved apparatus and to a method. More specifically, this invention concerns itself with an improved fluorometer assembly and a method for continuously monitoring the performance of the source of excitation energy within said assembly during the operation thereof.
  • ratioing a technique similar to that employed in split-beam spectrophotometry.
  • the source of excitation energy is split into two beams: one beam being directed, to a standard (i.e., a stable fluorophore), while the l ⁇ second beam .from the same source of excitation energy being directed ' onto the specimen.
  • a standard i.e., a stable fluorophore
  • Comparison of the secondary emissions from successive excitation of the standard provides a ratio upon which to compensate for drift, thus, enabling meaningful comparison of emissions from the specimen with a standard curve.
  • additional objects of this invention include a method for continuously monitoring the performance of a fluorometer during the opera- tion thereof; and, for the compensation for such variation in fluorometer performance at the time of interpretation of the level of fluorescence from the irradiated specimen.
  • a fluorometer assembly comprising a source of excitation energy and means for monitoring the output of the source of excitation energy.
  • Such monitoring means are to be disposed along the optical pathway of the source of excitation energy.
  • a reflecting means typically a dichroic mirror - preferably oriented at -.5 degrees to the direction of propagation - which is capable of deflecting at least a portion of the excitation energy to a specimen containing the analyte of interest while at the same time permit- ting the uninterrupted transmission of at least a portion of said excitation energy to the monitoring means.
  • the fluorescence emission from the fluorophore contained in the specimen will also be transmitted through the dichroic mirror to a means for detection of the level of such emission (typically a photomultiplier tube).
  • a means for detection of the level of such emission typically a photomultiplier tube.
  • such assembly can also include one or more lenses to further refine the focusing of the various beams of excitation energy and fluorescence emissions; and filters (i.e., band pass, infrared absorption, etc.) to screen out unwanted wavelengths of light.
  • Fig. 1 is a perspective view of an analytical instrument designed for performing batch processing of patient samples by fluorometric analysis.
  • Fig. 2 is a diagramatic representation of the fluorometer assembly of the 10 type incorporated within the instrument illustrated in Fig. 1.
  • the automated batch analyzer illustrated in Fig. 1 utilizes a unique sel -stacking reagent tab which is stored within the analyzer at tab load station 2.
  • a single reagent tab (not shown) is peeled from the stack stored within the load station by a pick-off finger (not shown) of the carousel assembly.
  • Th carousel assembly contains two (2 ) r,r, separate and independently rotatable sections which move in registration with one another through the various processing stations of the analyzer. Initially, a reagent tab is transferred from the load station onto a rotating platform of the carousel which precisely orients it relative to each of the three (3) fluid dispensing stations 8, 12 and 16 and fluorometer assembly 60
  • this analyzer can be tailored to the particular type of test that is to be performed and thus may vary from one analytical protocol to another; and, the particular demands of the chemistries which are unique to such protocol.
  • EIA enzyme immunoassay
  • a series of cams position the syringe mechanism of pump assembly 8 over sample cup 6 containing a patient specimen.
  • a microprocessor within the instrument activates the syringe
  • OMPI of the dispensing tip and rate of application of patient sample are precisely controlled by the analyzer logic.
  • the carousel transfers the reagent tab to the next station within the instrument.
  • the rate of transfer of the tab from one station to the other is carefully programmed to allow adequate time for incubation of the sample with the reagents of the reagent tab.
  • the incubation period between dispensing of sample onto the reagent tab and the. next fluid dispensing station is critical in order to allow for sufficient binding of the analyte of interest from the patient sample to an antibody which is immobilized within the reagent tab.
  • the ambient environment within the analyzer is also carefully controlled in order to optimize this interaction of patient sample and reagent in the tab.
  • the dispensing tip of the sample pump arm is repositioned and flushed of residual patient sample with water from a reservoir located at wash station 10.
  • the reagent tab containing the patient sample is precisely positioned under the next aperture in the carousel housing (partially obscured by boom of sample dispensing pump).
  • the dispensing tip of the pump aspirates a precise quantity of antigen-enzyme conjugate from reservoir 1 and delivers it to the reagent tab through the aperture in the carousel housing.
  • the constraints built into the tab transport mechanism of the carousel assembly precisely position the reagent tab relative to this station so as to enable application of the conjugate to the center of the reagent tab.
  • the manner of application of conjugate by the conjugate dispensing station is controlled in a similar fashion as was the case in the sample dispensing station.
  • the quantity of conjugate, and its rate of application and distance of the dispensing tip of the syringe from the reagent tab are also precisely controlled in order to insure essentially uniform flow of the conjugate from the dispensing tip into the reagent tab.
  • ⁇ he reagent tab is then positioned under the aperture in the carousel housing at the substrate dispensing station 16 while a series of cams positions the syringe mechanism of the pump 18 over reservoir 20 containing a substrate fluid and a microprocessor activates the syringe through a series of stepping motors to aspirate and then dispense the required aliquot of this 5 fluid onto the reagent tab in a manner similar to that performed previously at the sample and conjugate dispensing stations.
  • the amount of substrate dispensed onto the reagent tab is sufficient to effect radial elution of unbound materials out of the anticipated field of view of the fluorometer at the center of the reagent tab.
  • the substrate 10 interacts with the enzyme portion of the conjugate molecule bound at the center of the tab releasing a fluorophore.
  • the reagent tab containing the various solutions which have been applied to it at the three (3) immediately preceding dispensing stations, is 5 " now transferred, in relatively rapid succession, to a fluorometer read station of the type diagmatically illustrated in Fig. 2.
  • the field of view of the fluorometer is controlled by aperature 0 and thusly confined to but a small portion of the center of the reagent tab which coincides with the point of fluid application.
  • reagent tab 6 is illuminated with
  • excitation energy from light source 42 is directed to dichroic mirror 4 which reflects a substantial portion of said energy onto that portion of the reagent tab which has not been masked from such energy by aperture 40.
  • the excitation energy striking the tab causes the fluorophore In the tab to emit fluorescence radiation, a fraction of which is directed
  • fluorescence sensing means 48 such as a photomultiplier tube.
  • the level of fluorescence is monitored over a finite period in order to construct a curve, the slope of which being indicative of the concentration of analyte in the patient sample.
  • the assembly illustrated in Fig. 2 continuously monitors the output of said source by placement of a monitoring means 50, such as a reference photo diode, along the optical pathway of the excitation energy.
  • a monitoring means 50 such as a reference photo diode
  • dichroic mirror is inten ⁇ tionally designed so as to permit uninterrupted transmission of a pre- determined proportion of excitation energy to the monitoring means.
  • One of the principle functions of this dichroic mirror is to split the excitation energy into any usable ratio.
  • transmission of from 2 to 5% and reflection of 35 to 98% of the excitation energy provides an acceptable and workable ratio; however, such ratio could in fact be the inverse and still provide an operative system (depending of course upon the intensity of the fluorescence emission from the patient sample and the sensitivity of the means for monitoring such emission.) Any deviation from design specifica ⁇ tions in the intensity of energy transmitted through the dichroic mirror to the monitoring means is relayed to a microprocessor 52 in the instrument which notes such variation. The microprocessor can automatically compensate for such difference in its comparison of the fluorescence emitted from the irradiated portion of the reagent tab to a standard curve which is stored within the microprocessor.
  • a series of filters, 54, 56 and 58 are also positioned between the source of excitation energy and •the various detectors (reference photo diodes and photomultiplier tubes).
  • lense assemblies may also be positioned in a similar fashion to further refine and focus the various beams of electromagnetic radiation before they impinge upon the element at which each is targeted.
  • the fluorometer assembly can be contained within a monolithic block which would not only serve to maintain precise positioning of components, but also render the components of such assembly less sensitive to temperature changes.

Abstract

Assemblage de fluoromètre (60) pouvant contrôler en continu les caractéristiques de sa source d'énergie d'excitation pendant son fonctionnement. Une photodiode de référence (50), ou l'équivalent d'un tel dispositif, est placée le long du chemin optique de l'énergie d'excitation et détecte en continu la sortie de la source de cette énergie en même temps que la déviation d'une partie de cette énergie sur un échantillon contenant la substance à analyser. Dans l'éventualité d'une détérioration de la source d'énergie d'excitation provoquée par le vieillissement ou des variations de température, la diode de référence (50) détecte une telle modification et fournit des informations adéquates au travers d'un circuit de contrôle pour compenser ces variations au moment de l'interprétation du niveau de fluorescence de l'échantillon irradié. Cet assemblage de fluoromètre est très sensible et permet d'obtenir une commande par réaction en temps réel des caractéristiques de fonctionnement du fluoromètre, permettant ainsi d'utiliser celui-ci avec une instrumentation analytique de précision.
PCT/US1983/001012 1982-07-26 1983-06-16 Assemblage de fluorometre ameliore et procede WO1984000609A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US40167082A 1982-07-26 1982-07-26

Publications (1)

Publication Number Publication Date
WO1984000609A1 true WO1984000609A1 (fr) 1984-02-16

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1983/001012 WO1984000609A1 (fr) 1982-07-26 1983-06-16 Assemblage de fluorometre ameliore et procede

Country Status (2)

Country Link
EP (1) EP0114866A1 (fr)
WO (1) WO1984000609A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986004988A1 (fr) * 1985-02-20 1986-08-28 Commonwealth Scientific And Industrial Research Or Agencement fluorimetrique
US4841156A (en) * 1987-05-15 1989-06-20 Electronic Instrumentation And Technology, Inc. Measurement of the thickness of thin films
WO1989007757A2 (fr) * 1988-02-12 1989-08-24 Martin Snook Fluorimetres
US5798085A (en) * 1996-02-21 1998-08-25 Biomerieux Vitek, Inc. Optical reader and sample card transport stations for biological sample testing machine
US5853666A (en) * 1997-02-12 1998-12-29 Biomerieux Vitek, Inc. Optical reader and sample card transport stations for biological sample testing machine
WO2009147232A1 (fr) * 2008-06-05 2009-12-10 Bohle Ag Dispositif de détermination par fluorescence de la présence d'éléments sur une surface
EP1856509B1 (fr) * 2005-03-03 2011-12-28 QIAGEN Lake Constance GmbH Appareil de mesure de la fluorescence
WO2013043780A1 (fr) * 2011-09-23 2013-03-28 Nalco Company Procédé fluorométrique pour contrôler des additifs de surface dans un processus de fabrication de papier

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3854050A (en) * 1973-09-11 1974-12-10 Department Of Health Education High precision fluorometer for measuring enzymatic substrates in tissue
US3975098A (en) * 1975-08-05 1976-08-17 Applied Photophysics Limited Spectrofluorimeter
US4076420A (en) * 1974-02-22 1978-02-28 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Apparatus for investigating fast chemical reactions by optical detection
US4087685A (en) * 1977-01-11 1978-05-02 International Business Machines Corporation Fluorescent microanalytical system and method for detecting and identifying organic materials
US4125828A (en) * 1972-08-04 1978-11-14 Med-El Inc. Method and apparatus for automated classification and analysis of cells
US4171956A (en) * 1977-06-13 1979-10-23 General Electric Company Laser immunoassay
US4176963A (en) * 1975-11-28 1979-12-04 Hartman & Braun AG. Device for determining the nitrogen oxide concentration in a gaseous mixture
US4200801A (en) * 1979-03-28 1980-04-29 The United States Of America As Represented By The United States Department Of Energy Portable spotter for fluorescent contaminants on surfaces

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4125828A (en) * 1972-08-04 1978-11-14 Med-El Inc. Method and apparatus for automated classification and analysis of cells
US3854050A (en) * 1973-09-11 1974-12-10 Department Of Health Education High precision fluorometer for measuring enzymatic substrates in tissue
US4076420A (en) * 1974-02-22 1978-02-28 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Apparatus for investigating fast chemical reactions by optical detection
US3975098A (en) * 1975-08-05 1976-08-17 Applied Photophysics Limited Spectrofluorimeter
US4176963A (en) * 1975-11-28 1979-12-04 Hartman & Braun AG. Device for determining the nitrogen oxide concentration in a gaseous mixture
US4087685A (en) * 1977-01-11 1978-05-02 International Business Machines Corporation Fluorescent microanalytical system and method for detecting and identifying organic materials
US4171956A (en) * 1977-06-13 1979-10-23 General Electric Company Laser immunoassay
US4200801A (en) * 1979-03-28 1980-04-29 The United States Of America As Represented By The United States Department Of Energy Portable spotter for fluorescent contaminants on surfaces

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986004988A1 (fr) * 1985-02-20 1986-08-28 Commonwealth Scientific And Industrial Research Or Agencement fluorimetrique
US4841156A (en) * 1987-05-15 1989-06-20 Electronic Instrumentation And Technology, Inc. Measurement of the thickness of thin films
WO1989007757A2 (fr) * 1988-02-12 1989-08-24 Martin Snook Fluorimetres
WO1989007757A3 (fr) * 1988-02-12 1989-12-14 Martin Snook Fluorimetres
US5888455A (en) * 1996-02-21 1999-03-30 Biomerieux, Inc. Optical reader and sample card transport stations for biological sample testing machine
US5798085A (en) * 1996-02-21 1998-08-25 Biomerieux Vitek, Inc. Optical reader and sample card transport stations for biological sample testing machine
US5925884A (en) * 1996-02-21 1999-07-20 Biomerieux, Inc. Fluorescence station for biological sample testing machine
US5955736A (en) * 1996-02-21 1999-09-21 Biomerieux, Inc. Reflector assembly for fluorescence detection system
US5853666A (en) * 1997-02-12 1998-12-29 Biomerieux Vitek, Inc. Optical reader and sample card transport stations for biological sample testing machine
EP1856509B1 (fr) * 2005-03-03 2011-12-28 QIAGEN Lake Constance GmbH Appareil de mesure de la fluorescence
US8269966B2 (en) 2005-03-03 2012-09-18 Qiagen Lake Constance Gmbh Fluorescence meter
EP2522986A1 (fr) * 2005-03-03 2012-11-14 QIAGEN Lake Constance GmbH Appareil de mesure à fluorescence
WO2009147232A1 (fr) * 2008-06-05 2009-12-10 Bohle Ag Dispositif de détermination par fluorescence de la présence d'éléments sur une surface
WO2013043780A1 (fr) * 2011-09-23 2013-03-28 Nalco Company Procédé fluorométrique pour contrôler des additifs de surface dans un processus de fabrication de papier

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