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WO1990008949A1 - Spectroscopie - Google Patents

Spectroscopie Download PDF

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Publication number
WO1990008949A1
WO1990008949A1 PCT/EP1990/000212 EP9000212W WO9008949A1 WO 1990008949 A1 WO1990008949 A1 WO 1990008949A1 EP 9000212 W EP9000212 W EP 9000212W WO 9008949 A1 WO9008949 A1 WO 9008949A1
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WO
WIPO (PCT)
Prior art keywords
sample
radiation
particles
sedimentation
heat
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1990/000212
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English (en)
Inventor
Hans Björne Olaus ELWING
Per Olof Folkessen Helander
Kurt Ingemar LUNDSTRÖM
Parvesh Masson
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VARILAB AB
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VARILAB 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 VARILAB AB filed Critical VARILAB AB
Publication of WO1990008949A1 publication Critical patent/WO1990008949A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/04—Investigating sedimentation of particle suspensions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/04—Investigating sedimentation of particle suspensions
    • G01N15/05—Investigating sedimentation of particle suspensions in blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for the detection or quantification of analytes in test samples based on alterations which take place in suspensions of particles due to chemical or physical interactions.
  • Light dispersion techniques are based on exposure of the sample to a light beam of a certain wavelength.
  • the dispersion of light by particles such as cells or aggregates is read either as reduction in the amount of light which passes through the sample (turbidimetry) or as the amount of light reflected at 90° to the light beam (nephelometry) .
  • the dispersion of light is altered when for example the particles aggregate or when aggregates are formed in a clear solution.
  • particle ⁇ aggregates may be observed by the naked eye.
  • One such reaction is the aggregation of latex beads utilized in many immunochemical assays.
  • Another is the formation of cellular aggregates, e.g. aggregation of erythrocytes with blood-type specific antibodies. In these methods a negative result is observed as maintenance of a homogeneous suspension, whereas a positive result is associated with formation of large aggregates.
  • One particular technique is to assay an antigen by mixing it with antibodies whereby aggregates may be isolated by filtration or centrifugation; alternatively they may be visualized by light dispersion or visual examination of the solution.
  • the amount of a haemoglobin may be measured directly in a blood sample without lysing the erythrocytes. This is possible because only that part of the sample which is in close proximity to the sensor is measured. The method is not dependent on absorption of light in a diluted sample, and may hence be utilized in very dense samples.
  • Coagulation of blood may be measured by various techniques.
  • a common method is to perform a visual observation of the clotting time of blood (or plasma) mixed with an activator (e.g. thromboplastin or elagic acid) in a thermostated water-bath.
  • the clotting time may also be measured using mechanical devices like metal spheres rotating by means of a magnetic field, and hooks which go up and down in the reaction mixture. In these cases the formation of a clot will lead to alteration of the mechanical properties of the reaction mixture which may be registered; the metal spheres will stop rotating and disturb the magnetic field, which disturbance may be measured, or the formation of the gel will increase the viscosity, which in turn may be monitored.
  • Coagulation may also be measured optically.
  • One method is that a rotating glass tube is exposed to a light beam. When the reaction mixture forms a clot, this sticks to the wall of the glass tube and follows the rotation of the glass, thereby breaking the beam.
  • Another method is the use of turbidimetric measurements which may be performed using spectrophoto- meters. This is based on increased transparency when the clot is formed.
  • the present invention therefore we provide a method of detection or quantification of analytes in test samples based on alterations in the sedimentation of particles in the sample due to chemical or physical interactions, wherein said sedimentation is detected or quantified using an optothermal sensor wherein electromagnetic radiation is passed through a transparent heat conducting solid element to irradiate a sample in contact therewith, absorption of said radiation producing heat which is detected or quantified, said sample being positioned vertically above or below that surface of said solid element with which it is in contact, sedimentation causing an increase or decrease in the absorption of said radiation and consequently of the heat detected or quantified.
  • an optothermal sensor wherein electromagnetic radiation is passed through a transparent heat conducting solid element to irradiate a sample in contact therewith, absorption of said radiation producing heat which is detected or quantified, said sample being positioned vertically above or below that surface of said solid element with which it is in contact, sedimentation causing an increase or decrease in the absorption of said radiation and consequently of the heat detected or quantified.
  • the electromagnetic radiation may be ultraviolet, visible or infrared light.
  • the optothermal sensor may be a thermoacoustic system of the kind described in EP49918.
  • This method is based on a sample cell which consists of a transparent heat conducting material with a high temperature coefficient of expansion, connected to piezo-electric crystals and equipped with a light source.
  • a sample is put on one side of the transparent material and exposed to light pulses from the opposite side through the support, the light may be absorbed by the sample.
  • This leads to a pulsed temperature increase which in turn expands and contracts the transparent support and affects the piezo-electric crystals, resulting in a signal which is related to the light absorption.
  • the principle of optothermal spectroscopy may also utilize other types of sample cells. If the piezo-electric crystals are replaced by temperature-sensitive detectors positioned adjacent to the sample, the absorbance of light is registered as an increase in heat conducted to such detectors rather than expansion of the material. Such a system is described in our copending application of even date herewith corresponding to United
  • both such systems have in common the use of a transparent heat conducting solid element which has a first surface for contacting the sample, a radiation input surface and a radiation path between said surfaces, detector means being provided close to said first surface without obstructing the radiation path but responding to heat produced from the sample due to absorption of the incident radiation.
  • the detector means comprises piezoelectric crystals in combination with the heat conducting solid element, while in the other case the thermal detector is a thermooptic or thermo ⁇ electric device such as a thermistor, a thermocouple or a temperature responsive laser.
  • the above* optothermal devices are advantageously used in such a way that the incident electromagnetic radiation is modulated and the signal produced by the heat from the sample is sampled at intervals which are synchronised with these modulations.
  • the time interval between incidence of the radiation and sampling of the signal limits the signal to that produced by heat from zones of the sample very close to the sensor.
  • the maximum depth within the sample from which heat contributes to the signal is called the 'thermal diffusion length' and defines the volume of the sample which
  • Alterations in sedimenting properties may thus be monitored and constitute a basis for monitoring chemical reactions. If a blood sample is mixed with a reagent which triggers the coagulation mechanism, the clotting time may be indicated as the time to reach the point when the rate of increase in the signal declines. The reason for this change in the signal is that further sedimentation of erythrocytes is prevented by formation of fibrin gel. If the mixture of blood and reagent is under proper temperature control, the clotting time may be used for monitoring the coagulant properties of blood.
  • Appropriate reagents which may be mixed with citrated blood include thromboplastin reagent, activated coagulation factors and elagic acid reagents.
  • the thromb ⁇ plastin induced reaction makes possible the examination of the extrinsic coagulation system and the monitoring of oral anticoagulant therapy.
  • Addition of activated coagulation factors may be used for measuring the thrombin time and the fibrinogen concentration.
  • Addition of activated Factor X may be used for the indirect measurement of low molecular weight heparin.
  • Thrombin may be used for indirect measurement -of unfractionated heparin.
  • Elagic acid induces a reaction which may be used for the examination of the intrinsic coagulation system and for the monitoring of heparin therapy.
  • a further common type of analysis is the so-called agglutination reaction in which members of receptor-ligand pairs coupled to spheres, cells or macroscopic particles cause a visible formation of aggregates. Agglutination may also take place in direct reactions between receptor-ligand pairs, among which the antigen-antibody reactions are the most widespread.
  • Coloured particles which may be coloured latex beads or any other beads of polymeric material, colloid metals or metal compounds such as colloidal gold and silver, or cells such as erythrocytes, are coupled to antibodies directed against an analyte present in a sample.
  • the suspension of particles and the sample may be mixed in a certain ratio and applied to the window of an optothermal spectrometer.
  • the light wavelength is selected to be well absorbed by the particles.
  • the particles will sediment at a certain rate causing the signal to vary as a function of time.
  • the optothermal method has many advantages compared to other methods. It may be used directly on whole blood, and it avoids mechanical disturbance of the reaction mixture. Furthermore, since the reaction measured takes place in close proximity to the window surface due to the short thermal diffusion length, the system is nearly independent of the sample size for all practical purposes. As indicated above, agglutination of particles may easily be followed on an optothermic spectrometer. Although light dispersion may be used to follow such reactions instrumentally, this technique is dependent on mechanical stirring of the reaction mixture. Hence, variations in the samples (viscosity, or interfering substances) cause variations in the result which makes this method less precise. Furthermore, it may only be used in a narrow range of particle concentrations, and the technique is of limited sensitivity.
  • agglutination may be used for a wide range of particle concentration; it is not dependent on mechanical stirring, and it provides an easy method of instrumental verification of agglutinaton which also permits quantification.
  • the volume of the reaction mixture is of little importance.
  • the sensitivity range may also be varied by alteration of particle concentration, particle size and particle density.
  • Combination of colours on the particles may also be utilized, e.g. small coloured particles which are normally not sedimenting may be coupled to larger, non-coloured particles by specific receptor- ligand reactions, thereby causing the coloured substance to sediment and gradually modify the optothermal signal.
  • analyte If the analyte is present in a certain concentration it will induce an agglutination of the particles which in turn will cause a more rapid increase in sedimentation and hence a more rapid signal modification, which may be monitored and will be related to the concentration of the analyte in the sample. In the case of antibody-antigen reactions in the absence of particles, there will be no sediment ⁇ ation when the amount of analyte is zero. When the analyte is present, aggregates will form and precipitate to produce a signal. All types of agglutination or aggregation may be monitored using optothermal spectroscopy. The most frequently used reactants inducing such reaction are, as mentioned above, antibodies and antigens. However, all receptor-ligand pairs may be used.
  • Such pairs may be Staphylococcus protein A and IgG, corresponding nucleic acids, sugars and lectins, metals and chelating agents, enzymes and correspnding inhibitors, biotin and avidin, biotin and streptavidin, and all combinations of these pairs of compounds linked to other compounds
  • Blood samples collected in sodium citrate were analysed within four hours of sample collection.
  • the contents of one vial of lyophilised Thrombotest reagent (batch no 313, Nycomed AS, Oslo, Norway) were dissolved in 11 ml of a solution containing 3.2 mmol/1 CaCl 2 and frozen at -20°C as 100 1 aliquots.
  • an aliquot was warmed to room temperature and 20 1 of blood added.
  • a 40 1 portion of the mixture was transferred immediately to the sapphire window of the optothermal spectrometer as described in Fig. 2 of EP49918 referred to above (2 Hz frequency, 37 °C wavelength 540 + 40 n ) and the light source coupled to the spectrometer was started at the same time as the chart recorder coupled to the spectrophotometer (adjustment 2 volts, chart speed 1 cm/min) .
  • a sample with a coagulation time of ca. 50 seconds was diluted with 150 mmol/1 NaCl and analysed in triplicate with a conventional spectrophotometer (LODE LC-61, Holland) and by the above optothermal spectrometry.
  • patient samples were analysed with LODE LC-61 and optothermal spectrometry and the results compared by the least square regression analyses.
  • erythrocytes analysis as haemoglobin
  • Fig. 1 The sedimentation of erythrocytes (analysis as haemoglobin) in a sample containing 20 1 blood and 100 1 of isotonic sodium chloride (0.15 mol/litre) is shown in Fig. 1.
  • the sodium chloride is replaced by thrombotest reagent, the sedimentation rate is altered as soon as clot formation starts (Fig. 1 b) .
  • the point at which this rate of sedimentation alters is measured and represents the coagulation time in seconds.
  • Blood anticoagulated with heparin was used.
  • the erythrocytes were washed twice in 0.15 mol/L NaCl by centrifugation. A concentrated solution of A-typed erythrocytes was used as stock solution.
  • An optothermal spectrophotometer as described in Fig. 2 of EP49918, equipped with piezoelectric crystals for determination of heat-induced expansion of a sapphire window was used.
  • the instrument was used with light pulses at a frequency of 2 Hz.
  • a 20W halogen lamp was used as light source and band pass filter (Schott, West Germany, filter BG18, 540 + 40 nm) was used.
  • band pass filter Schott, West Germany, filter BG18, 540 + 40 nm
  • the instrument was adjusted so that the solvent for erythrocytes gave zero signal and the stock solution of erythrocytes gave a signal of 100 arbitrary units.
  • Various dilutions of erythrocytes in 0.15 mol/L NaCl resulted in an increasing signal as a function of time - plot 1 in Fig. 3.
  • the gradual increase in the signal reflected sedimentation of erythrocytes towards the surface of the sapphire window.
  • the aim of this example is to demonstrate that optothermic detection may be used for the quantification of an agglutinating agent; in this case the use of varying concentrations of antibody leads to a variable degree of sedimentation towards the sensor.
  • the instrument used was the same as in the before-mentioned example.
  • a suspension of A-erythrocytes was diluted to 5 % and the anti-blood group A antibodies were added in dilutions ranging from 1/1 to 1/16. After incubation for one hour the suspensions were applied to the optothermic spectrometer and the sedimentation rates were recorded for 30 seconds. The rates in arbitrary units are illustrated in Fig. 4, which shows that there is a clear relationship between the concentration of antibodies and the sedimentation rate. The entire effect appears to be between 1/1 and 1/4 dilution of antibodies in which range the relationship fits to a half-logarithmic plot.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
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  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Dispersion Chemistry (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

On procède à la détection et/ou à la quantification d'analytes dans des échantillons de test par observation optothermique de changements se produisant dans l'absorption de rayonnement électromagnétique par lesdits échantillons, provoquée par des changements de densité des particules pendant des sédimentations induites physiquement ou chimiquement.
PCT/EP1990/000212 1989-02-03 1990-02-01 Spectroscopie Ceased WO1990008949A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8902417.8 1989-02-03
GB898902417A GB8902417D0 (en) 1989-02-03 1989-02-03 Spectroscopy

Publications (1)

Publication Number Publication Date
WO1990008949A1 true WO1990008949A1 (fr) 1990-08-09

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PCT/EP1990/000212 Ceased WO1990008949A1 (fr) 1989-02-03 1990-02-01 Spectroscopie

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AU (1) AU5165390A (fr)
GB (1) GB8902417D0 (fr)
WO (1) WO1990008949A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006897A1 (fr) * 2006-07-14 2008-01-17 Alifax Holding Spa Appareil intégré détectant les états inflammatoires présents dans un échantillon de sang entier
WO2013088131A1 (fr) * 2011-12-12 2013-06-20 Vivacta Ltd Procédé et dispositif permettant de mesurer la coagulation d'un échantillon de produit sanguin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0049918A1 (fr) * 1980-10-10 1982-04-21 Ab Varilab Procédé de mesure photothermique pour l'étude de l'absorption de lumière par un échantillon d'une substance
WO1986005275A1 (fr) * 1985-03-04 1986-09-12 Labsystems Oy Procede de mesure de la sedimentation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0049918A1 (fr) * 1980-10-10 1982-04-21 Ab Varilab Procédé de mesure photothermique pour l'étude de l'absorption de lumière par un échantillon d'une substance
WO1986005275A1 (fr) * 1985-03-04 1986-09-12 Labsystems Oy Procede de mesure de la sedimentation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008006897A1 (fr) * 2006-07-14 2008-01-17 Alifax Holding Spa Appareil intégré détectant les états inflammatoires présents dans un échantillon de sang entier
US8425843B2 (en) 2006-07-14 2013-04-23 Alifax Holding Spa Integrated apparatus and method to detect inflammatory states present in a sample of whole blood
WO2013088131A1 (fr) * 2011-12-12 2013-06-20 Vivacta Ltd Procédé et dispositif permettant de mesurer la coagulation d'un échantillon de produit sanguin

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Publication number Publication date
GB8902417D0 (en) 1989-03-22
AU5165390A (en) 1990-08-24

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