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Title:
SPECTROSCOPY
Document Type and Number:
WIPO Patent Application WO/1990/008949
Kind Code:
A1
Abstract:
Detection and/or quantification of analytes in test samples is effected by optothermally observing changes in electromagnetic radiation absorption by the samples caused by changes in particle density during physically or chemically induced sedimentations.

Inventors:
ELWING HANS BJOERNE OLAUS (SE)
HELANDER PER OLOF FOLKESSEN (SE)
LUNDSTROEM KURT INGEMAR (SE)
MASSON PARVESH (SE)
Application Number:
PCT/EP1990/000212
Publication Date:
August 09, 1990
Filing Date:
February 01, 1990
Export Citation:
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Assignee:
HOLMES MICHAEL JOHN (GB)
VARILAB AB (SE)
International Classes:
G01N15/04; G01N33/543; G01N15/05; G01N33/48; G01N33/53; (IPC1-7): G01N15/05; G01N25/20
Domestic Patent References:
WO1986005275A11986-09-12
Foreign References:
EP0049918A11982-04-21
Download PDF:
Claims:
CLAIMS :
1. 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.
2. A method as claimed in claim 1 in which the transparent heat conducting solid element has a first surface for contacting a sample, a radiation input surface and a radiation path between said surfaces, detector means being ffrovided close to the said first surface without obstructing the radiation path and responding to heat produced by the sample due to absorption of the incident radiation.
3. A method as claimed in claim 2 in which the detector means comprise piezoelectric means attached to the transparent heat conducting element and which provide an electric signal on expansion and/or contraction of said element.
4. A method as claimed in claim 2 in which the detector means comprises thermal detector means which provide a signal responsive to heat from the sample conducted through the heat conducting element.
5. A method as claimed in any of the preceding claims in which the incident radiation is modulated and a signal produced by heat from the sample is sampled at intervals synchronised with said modulation.
6. A method as claimed in any of the preceding claims in which the electromagnetic radiation is light.
7. A method as claimed in any of the preceding claims in which the sample is blood treated with a reagent causing coagulation and the rate of sedimentatio of erythrocytes is measured.
8. A method as claimed in any of claims 1 to 6 in which the particles are produced by or are involved in an agglutination reaction.
9. A method as claimed in claim 8 in which the sample comprises antibodies coupled to coloured particles, colloidal metals or colloidal metal compounds or cells, said antibodies combining with the analyte to be determined whereby the sedimentation rate of the particles is changed.
10. A method as claimed in claim 8 in which the binding partners in said agglutination reactions are antibodies and antigens, antibodies and haptens, protein A and IgG, corresponding nucleic acids, sugars and lectins, metals and chelating agents, biotin and avidin or streptavidin and such binding partners when linked to other compounds.
Description:
"Spectroscopy"

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.

Alterations in suspensions of particles such as cells or aggregates are commonly measured using instruments detecting alterations in dispersion of light, or are observed visually using the same principle.

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.

The formation of 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.

Using optothermal spectroscopy 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. ("Principles of optothermal spectroscopy" by P. Helander, Uppsala J Med Sci 9: 155-158, 1986).

It has also been observed that erythrocytes sedimenting towards the surface of a photoacoustic sensor cause an increased signal ("Whole blood -a sedimenting sample studied by photoacoustic spectroscopy" by P.Helander and I. Lundstrδm, J.

Photoacoustics 1:203-215, 1982). However, alteration in sedimentation properties in general due to chemical or physical interactions involving cells or particles has not been recognized as a diagnostic tool and particularly not in combination with optothermal spectroscopy.

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 problem with all mechanical methods is that the strength of the clot formed varies between the samples. Low clot strength will give rise to longer coagulation times in a mechanical system than is observed visually or by turbidimetry. Turbidimetric measurement is not affected by clot strength in the same way , but cannot be applied to whole blood.

We have now found that a great deal of information concerning sedimentation of particles in a sample can be obtained more simply and accurately by using an optothermal sensor to detect changes in radiation absorption due to changes in particle density during sedimentation.

According to 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.

- A -

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. When 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

Kingdom Patent Application No. 8902415.2, the disclosure of which is incorporated herein by reference.

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. In one case, 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. This reduces background noise arising from temperature fluctuations between the modulations. Furthermore, since heat from within the sample takes a finite time to reach the detector, 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

3 is analysed, typically only a few mm . This definition of the volume facilitates quantification of the light absorbing substance if this is required. When a particle is present in a solution it will sediment at a certain rate dependent on its size, shape and relative weight. When such a solution is applied above the window of an optothermal sensor and exposed to light pulses, the layer of particles in close proximity to the window will lead to certain signals as the particles then sediment towards the surface of the window; thus the absorption of light near the window will increase as a function of time and sedimentation properties of the particles. The optothermal sensor can also be placed with the sample below the window and with the light coming from above. In this case the signal will decrease due to the sedimentation. 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 thrombθplastin 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. According to the present invention, 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.

If the amount of analyte is zero, 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. Using optothermal spectroscopy 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. As with coagulation, 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.

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

The following Examples are given by way of illustration only:

Example 1

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. For the analysis, 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) . To test linearity, 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. For method comparisons, patient samples were analysed with LODE LC-61 and optothermal spectrometry and the results compared by the least square regression analyses.

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. When 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.

The coefficients of variation for within- series analyses varied between 4.6 and 13.9%. The linearity extended over the range 48 to 220 seconds. This represents coagulation activity of prothrombin complex (PT) ranging from 45 % to less than 5%. The comσarison between the routine

method and optothermal spectrometry is shown in Fig. 2. The coagulation times were not affected by lipaemia using either method.

Example 2

ABO antigen detection on erythrocytes using haemagqlutination and optothermal spectroscopy.

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. 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.

When a stock solution was diluted to 10% with 0.15 mol/L NaCl and mixed with /3 volume of monoclonal anti-A type antibody (Biological Corp. of America, Div. of Cooper diagnistics Inc., West Chster, USA) the sedimentation of the erythrocytes increased. This is shown by plot 2 in Fig. 3. as an increased signal rate compared to the control.

Complete ABO-typing of blood may be performed by combining anti-A and anti-B monoclonal antibodies. Heparinized blood is diluted to 10 % using 0.15 mol/L NaCl and one of the antibodies is added. After one hour the blood was resuspended and added to the optothermic spectrometer. Positive reaction with either anti-A, or anti-B, or both, was indicated by a significantly more rapid increase in the signal compared to the control, relative to blood groups A, B, or AB respectively. No reaction was related to group 0. This test may also be done at one measuring point by adding samples to the instrument, waiting for 30 seconds, and measuring. A signal elevated about two times the signal from a control indicates a positive test towads the antibody used.

Example 3

Quantitive aspects of agglutination measured by optothermic spectroscopy.

Blood samples and antibodies were obtained and prepared as shown below to demonstrate ABO antigen detection in erythrocytes.

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.