EP1027593A1 - Methode et dispositif de dosage immunologique fluorometrique - Google Patents

Methode et dispositif de dosage immunologique fluorometrique

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Publication number
EP1027593A1
EP1027593A1 EP98959766A EP98959766A EP1027593A1 EP 1027593 A1 EP1027593 A1 EP 1027593A1 EP 98959766 A EP98959766 A EP 98959766A EP 98959766 A EP98959766 A EP 98959766A EP 1027593 A1 EP1027593 A1 EP 1027593A1
Authority
EP
European Patent Office
Prior art keywords
light
optical waveguide
optical
piston
cylinder
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.)
Withdrawn
Application number
EP98959766A
Other languages
German (de)
English (en)
Inventor
Andreas Katerkamp
Ulrich Kunz
Frank Grawe
Göran KEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut fuer Chemo und Biosensorik Muenster eV ICB
Original Assignee
Institut fuer Chemo und Biosensorik Muenster eV ICB
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 Institut fuer Chemo und Biosensorik Muenster eV ICB filed Critical Institut fuer Chemo und Biosensorik Muenster eV ICB
Publication of EP1027593A1 publication Critical patent/EP1027593A1/fr
Withdrawn legal-status Critical Current

Links

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
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • 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"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G01N2021/7706—Reagent provision
    • G01N2021/7709—Distributed reagent, e.g. over length of guide
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/808—Optical sensing apparatus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property

Definitions

  • the invention relates to a device and a method for carrying out fluorescence immunoassays, light from at least one light source being directed onto a surface at one end of an optical waveguide and fluorescence from at least one with the light coupled into the optical waveguide by evanescent field excitation on the surface of the optical waveguide marker substance bound to a chemical or biochemical partner of a general receptor-ligand system is excited and the fluorescent light is partially coupled into the optical waveguide and is coupled out of the optical waveguide from the surface into which the exciting light has been coupled and via optics an optical detector is directed, with which the intensity of the fluorescent light is measured.
  • biochemical tests on general receptor-ligand systems such as e.g. Antibody antigen can be performed.
  • the tests are used to quantitatively determine chemical or biochemical substances in liquid samples.
  • Antibodies can thus be labeled with a specific marker (fluorophore), the marker in question being able to be optically excited at a specific excitation wavelength of the light and the fluorescent light obtained by the excitation, which in turn with a different wavelength occurs, detected with a suitable optical detector and the intensity of the fluorescent light is used to determine the respective proportion of the chemical or biochemical substance from the sample liquid.
  • a specific marker fluorophore
  • the evanescent field that forms at an interface is used for the fluorescence excitation.
  • the known physical relationships of the evanescent field and in particular the required total reflection of the excitation light must be taken into account.
  • WO 97/10 506 discloses an optical device for carrying out fluorescence immunoassays, in which an optical fiber is used, into which light from a light source is coupled and fluorescence of a sample is generated by evanescent field excitation.
  • the surface of the optical waveguide is accordingly coated with a chemical or biochemical component before the respective test, ie the introduction of the sample liquid into a container in which the optical fiber is accommodated, is carried out.
  • a very complex and complex optic is required, which consists of a plurality of individual optical components.
  • the light from the light source used is directed via a lens system onto a semitransparent mirror and part of the light is coupled as a reference signal to an optical detector and the other part of the light is coupled into the fiber via a further lens.
  • the end of the optical fiber opposite the coupling and decoupling surface is mirrored, so that most of the excitation and fluorescent light again is coupled out of the optical fiber. This leads to the fact that the ratio of excitation light to fluorescence light for the evaluation with the photodetector deteriorates and consequently the measuring accuracy is undesirably impaired.
  • a further disadvantage of this known device is that the light is to pass through the lens arranged in front of the coupling surface of the optical fiber, so that light enters the optical fiber at a defined main angle within a narrow angular range, which is advantageously required for evanescent field excitation. if at all very difficult to reach.
  • the fluorescence immunoassays are then carried out according to this known solution in such a way that the prepared, coated optical fiber accommodated in the container is brought into contact with the sample liquid by perforation in the lid of the container and the sample liquid enters and the connection to that on the surface of the optical fiber immobilized complementary partner of the receptor-ligand system can take place. After the connection, fluorescence is then excited by irradiation of the light from the light source used and its intensity is measured with the optical detector.
  • connection Since the respective mass transfer to the optical waveguide surface is important for the connection process and convection and diffusion must be taken into account, measurement errors occur in the solution known from WO 97/10 506, since the sample volume accommodated in the container is constant and the penetration the sample liquid via the perfo- rations happens very quickly and the connection is made in a still liquid. In this case, the connection is mainly influenced by diffusion, which, in addition to other disadvantages, also leads to the measurement time being extended.
  • the device according to the invention builds on the known state of the art already described and likewise uses at least one light source, with which light, for fluorescence excitation via the evanescent cente field, is coupled into an optical waveguide and the intensity of the fluorescent light of a marking substance, which is bound to a partner of a general receptor-ligand system, is determined with an optical detector.
  • the fluorescent light and part of the excitation light are coupled out from the same area into which the excitation light has been coupled.
  • the optical waveguide is received in a measuring chamber which is formed in a piston of a piston-cylinder unit.
  • the measuring chamber of the piston has an inflow into the interior of the cylinder which leads through the piston.
  • Such a piston-cylinder unit can be designed at least approximately like a conventional syringe, only the piston having to be modified accordingly with the measuring chamber.
  • a further improvement can be achieved by forming an additional sample collection chamber in the piston, which is connected to the measurement chamber. This creates a unit in which the incubation and the measurement can be carried out and after the respective test has been carried out the sample is safely recorded and the corresponding one
  • Piston-cylinder unit transported and can be disposed of without major problems and hazard formation.
  • the optical waveguide can have a surface which closes the bulb in this direction. It also forms a conclusion for the measuring and sample collection chamber. It also serves to fix the optical fiber on this side of the Piston.
  • the optical waveguide and the surface can advantageously be made in one piece from the same material, such as, for example, a polymer, such as polyethacrylate (PMMA).
  • a light absorber is advantageously arranged, which preferably consists of a black-colored plastic.
  • the light absorber can be dimensioned and shaped in such a way that it aligns and fixes the optical waveguide in the lower region of the measuring chamber.
  • Another favorable effect can be achieved with the light absorber, which improves the ratio of excitation light and fluorescent light to a value that is favorable for the measurement.
  • the light absorber With the help of the light absorber, almost all of the excitation light is absorbed, while the fluorescent light is only reduced by half, so that the ratio of the two light components is shifted in favor of the fluorescent light. This means that very sensitive light detectors can be used to measure the weak fluorescent light.
  • optical waveguide it is also possible to subdivide the optical waveguide into a number of optical waveguiding segments which run parallel to the light propagation in the optical waveguide.
  • the individual segments are spatially separated from each other by a thin layer.
  • the refractive index n 2 of this layer is smaller than that of the light-wave-guiding segments in order to achieve light guidance through total reflection.
  • a multi-analyte measurement is possible with such a segmented optical waveguide, since a fluorescence immunoassay is carried out with each segment carrying light waves can.
  • the closure e.g. can be a thin film or a corresponding stopper, which can be removed if necessary, which will be explained later, and the opening can be opened.
  • the respective sample liquid can advantageously be supplied via an inflow opening in the cylinder, as is also the case with conventional syringes.
  • the inflow opening should advantageously be closable with a valve, in order to avoid an undesired escape of sample liquid from the cylinder, after it has been filled.
  • Another favorable embodiment of the device according to the invention uses an absorbent material which is accommodated at least in the sample collecting chamber, wherein a part of the absorbent material can also protrude into the measuring chamber.
  • Filters and / or an immune column and / or a membrane which can be permeable or semi-permeable, can be present in front of or in the already mentioned inflow through the piston into the measuring chamber, in order to keep certain components contained in the sample liquid away from the measurement or otherwise targeted influence on the measurement with the use of an immune column and / or membrane and / or the filter. to be able to take.
  • the various fluorescence immunoassays can now be carried out in such a way that the surface of the optical waveguide contains various chemical or biochemical components, such as e.g. Antibody is coated, the coating being able to take place, for example, in a two-stage batch process.
  • Adsorptive immobilization of the respective components including prior cleaning of the fiber optic surface.
  • This can be carried out in a simple immersion process, with which it is easily and effectively possible to process a relatively large number of such optical fibers at the same time. Since the optical waveguides can also be produced by conventional injection molding processes and, consequently, a large number of optical waveguides are available connected to one another after the injection molding, this process can of course be carried out extremely effectively.
  • optical waveguides coated in this way are then inserted into the measuring chamber of a piston designed as already described, the opening in the surface at one end of the optical waveguide, which closes the piston, also being closed.
  • This surface can then be connected to the piston by gluing, an airtight seal being intended to be ensured.
  • the end face of the piston used for example with a biocomponent, before it is inserted into the cylinder.
  • the surface of the inside of the cylinder can be coated with a lyophilized biocomponent. It is also suitable to cover the surface of the inflow of the cylinder.
  • the device according to the invention can be made available for the respective need, and longer storage times are also readily possible under appropriate conditions.
  • the piston-cylinder unit prepared in this way can then be drawn up like a syringe and filled with sample liquid if necessary, a defined amount of sample liquid being able to be drawn into the cylinder.
  • the sample liquid remains exclusively in the cylinder and cannot get into the measuring chamber through the inflow in the piston and consequently not into the sample collection chamber, since the air contained therein cannot be displaced because the opening that allows air to escape is still closed.
  • the appropriately pretreated sample can be guided into the measuring chamber and past the optical waveguide if the air outlet opening on the end face is opened. In the case of a film used for this, this can be done by removing or piercing it. The air can escape from the measuring chamber and sample collection chamber and the chambers are filled by capillary forces, the sample liquid flowing continuously past the surface of the optical waveguide.
  • the flow of the sample liquid through the measuring chamber with a defined flow rate can also be achieved by a correspondingly targeted movement of the cylinder with the syringe plunger fixed. It is also not absolutely necessary for the air outlet opening on the end face to be opened, since the air in the measuring chamber and in particular in the sample collection chamber can be compressed and thus the sample can also flow past the surface of the optical waveguide. In this operating mode, an air outlet opening on the end surface is not necessary.
  • the syringe plunger When the pre-incubated sample liquid flows through the measuring chamber, the syringe plunger must be held in a defined manner so that the light from the light source for the excitation light hits the surface at the correct angle and can be coupled into the optical waveguide. If an optical fiber with a diameter of approx. 1 mm is used, relatively large compromises can be made in terms of position accuracy without the respective measurement results being adversely affected.
  • the valve at the inflow into the cylinder already mentioned prevents the sample liquid accommodated in the cylinder from undesirably escaping again and the entire amount being able to be used for the measurement; this is particularly important when the cylinder is moved to flow through the measuring chamber.
  • the fluorescent light excited by means of the light source already mentioned, which is coupled out from the end face of the optical waveguide, is released is detected with the optical detector already mentioned and its intensity is measured.
  • the optical structure to be used for this purpose will be specified further later.
  • Another improved possibility for guiding the sample liquid preincubated, as described, past the optical waveguide through the measuring chamber can also be done in such a way that in the sample collecting chamber a material which is highly absorbent for liquid, such as e.g. Vlies is included.
  • This material protrudes into the measuring chamber and the pre-incubated sample liquid can get into the measuring chamber from the cylinder after opening the closure, the opening in the surface, by capillary force and will continue to be caused by capillary forces in the liquid-absorbing material. chamber guided until the sample collection chamber is filled. This leads to a more stable liquid transport.
  • the fluorescence intensity is measured in the period in which the sample liquid flows along the optical waveguide through the measuring chamber. If a device designed in this way is used, the valve already mentioned at the inflow into the cylinder can be dispensed with, so that, for example, a conventional syringe body is used as the cylinder can be used, which is available inexpensively.
  • device with certainty. This is firstly a hydrodynamic layer at a certain distance from the wall and secondly a diffusive layer, the respective distances between the layers depending on the wall, the viscosity and the flow rate of the fluid.
  • the diffusive layer i.e. at a greater distance from the wall, the chemical and biochemical components are brought up to the surface by convection, and within the diffusive layer, ie in the direction of the wall, the movement then takes place via diffusion processes.
  • Mass transport by convection is much larger than that which can be achieved by diffusion. This for the relatively fast antigen-antibody reaction means that the reaction process is greatly slowed down by the relatively slow diffusion process and this can lead to a time of about 1 hour being required for an immunoassay, for example, in order to obtain a sufficiently accurate measurement result determine.
  • the thickness of the diffusive layer can be reduced, thus increasing the influence of the convective mass transfer to the wall to which the chemical or biochemical components are to be attached. This greatly improves the response behavior of the immunoassay and consequently reduces the measurement time required.
  • the thickness of the channel-like flow can also be reduced if the flow rate is not increased, in order to reduce the extent of the diffusive layer, as can be advantageously designed in the arrangement according to the invention comprising the measuring chamber and the optical waveguide.
  • the distance between the optical fiber surface and the inner surface of the measuring chamber is kept as small as possible. A distance of less than 2 mm is advantageous, preferably between 0.1 - 0.05 mm.
  • a device according to the invention designed in this way can be inexpensively manufactured as a mass product and prepared in advance coated with corresponding biocomponents. Another advantage is that contact of the respective sample liquid with a person is avoided and the person is kept securely closed even after the test has been carried out, which is particularly advantageous in medical use.
  • the lens and an optical filter which is transparent to the coupled fluorescent light in front of the optical detector in the beam path of the light coupled out, the excitation light being blocked by the optical filter.
  • the outcoupled light should be directed in parallel through the respective filter in the direction of the optical detector.
  • the optical part of the device according to the invention can be further improved by arranging an additional second lens in the beam path of the outcoupled light following the optical filter already mentioned, with which the fluorescent light guided through the filter is now focused on the detector.
  • the optical parameters and the distance between the lens and the detector must be selected or adjusted accordingly so that the image of the fluorescent light coupled out of the optical waveguide is imaged on the optical detector.
  • Filters for the respective fluorescent light should advantageously be arranged and manipulated in such a way that they can be moved alternately into the beam path of the outcoupled light, so that there is always only one of the two filters for the respective fluorescent light.
  • the corresponding filter When the corresponding filter is introduced into the beam path of the fluorescent light, it is advantageous to interrupt the beam path of the other excitation source in parallel, so that no light from this source enters the optical waveguide.
  • a further advantageous development of the invention is to arrange a diaphragm in front of the detector, the opening of which can be positioned at variable times in the beam path in front of the detector by translation and / or rotation of the diaphragm.
  • each individual segment of the optical waveguide can be measured with this arrangement.
  • a linear or areal arrangement of detectors e.g. to use a CCD camera to measure the individual segments of the optical fiber.
  • FIG. 1 shows an optical waveguide in several views for a device according to the invention
  • Figure 2 shows an inventive device in a
  • FIG. 3 shows an example of a piston for a device according to the invention
  • Figure 4 shows another example of a piston for a device according to the invention
  • Figure 5 shows an example of a device according to the invention with an additional valve in different operating positions
  • Figure 6 shows an example of the optical part of a device according to the invention.
  • FIG. 1 shows an optical waveguide 1 to be used in a device according to the invention in different views.
  • the optical waveguide 1 has on one of its two end faces a light absorber 5 made of a dark, preferably black plastic material, which absorbs a very large part of the incident light.
  • the light absorber 5 has approaches which allow guidance and fixing when the optical waveguide 1 is introduced into a measuring chamber 9, which will be described in more detail below. It is also possible to attach one or more such approaches to the optical waveguide 1.
  • FIG. 1 shows a possible way of dividing the optical waveguide 1 into a number of segments 27 that conduct light waves.
  • the single ones Segments 27 are separated from one another by a thin layer 28, the refractive index n 2 of which is smaller than that of the light wave-guiding segments n j .
  • the side view in FIG. 1 again illustrates the possible type of subdivision of the optical waveguide 1 shown.
  • a surface 2 is formed on the other end face of the optical waveguide 1, the diameter of which is substantially larger than that of the optical waveguide 1 and closes off one side, towards the surroundings, of a piston 6, which will also be described in more detail below.
  • a closure 4 can be, for example, a peelable or pierceable film.
  • the side view shown in FIG. 1 also makes clear the area of surface 2 to which the light is to be directed in order to excite the fluorescence.
  • the optical waveguide 1 thus completed can be introduced into a measuring chamber 9 of a piston 6 and glued to the piston on the surface 2, as can be seen in FIG. 2.
  • a chemical or biochemical component is already immobilized on the surface of the optical waveguide 1.
  • At least one sample collection chamber 10 is also formed, which is connected to the measuring chamber 9, the sample collection chamber 10 being ring-shaped. can be formed around the measuring chamber 9.
  • the sample collection chamber 10 should be dimensioned such that it can accommodate all or a large part of the sample liquid volume.
  • an inflow 8 which represents a connection between the measuring chamber 9 and the interior of the cylinder 7, in which the piston 6 with the optical waveguide 1 has been inserted.
  • the cylinder 7 has a further inflow 11 through which sample liquid can get into the interior of the cylinder 7.
  • Cylinders 7 arrive, as is the case with conventional syringes or other piston-cylinder arrangements. Since the opening 3 in the surface 2 is closed with the closure 4, no sample liquid can get into the measuring chamber 9 through the inflow 8. Only at the moment when the opening 3 is at least partially opened can the air contained in the sample collection chamber 10 and the measurement chamber 9 escape and be displaced by the sample liquid, which now flows through the inflow 8, the measurement chamber 9 into the sample collection chamber 10 can reach.
  • the cylinder 7 is preferably moved with the piston 6 fixed, so that the free volume inside the cylinder 7 is reduced and, at a defined speed of this movement, the corresponding flow rate of the sample liquid through the measuring chamber 9 can also be set in a defined manner.
  • the outflow of the sample liquid through the inflow 11 should be prevented with the help of a valve 15, which will be explained below.
  • the opening 3 in the surface can be dispensed with.
  • the sample liquid displaces the air in the measuring chamber 9 and compresses it in the closed sample collecting chamber 10, so that the sample liquid, depending on the direction of movement of the cylinder 7, flows through the measuring chamber 9.
  • a sample flow can take place solely by capillary forces into the measuring chamber 9 and from there into the sample collecting chamber 10 if the closure 4 has been removed or pierced.
  • FIG. 3 shows another example of a piston 6 for a device according to the invention.
  • the piston seal 13 can also be clearly seen in this illustration.
  • the sample collection chamber 10 is filled with a material 12 that is particularly absorbent for the sample liquid, it not being clearly evident in this illustration that a part of the absorbent material 12 can at least partially protrude into the measuring chamber 9.
  • the liquid transport of the sample liquid through the measuring chamber can be supported by the capillary action of the absorbent material, the sample liquid also flowing first through the measuring chamber 9 when the opening 3 in the surface 2 is at least partially cleared, and air can thereby escape is and the inflow 11 is open.
  • FIG. 4 shows that a filter or an immune column 14 can be arranged in the inflow 8 of the piston 6, through which the sample liquid must be passed before the actual entry into the measuring chamber 9. It is also possible for a filter or a membrane to cover the inflow 8 on the piston surface 23.
  • FIG. 5 shows the arrangement and function of a valve 15 which blocks or releases the inflow 11 into the interior of the cylinder 7.
  • a simple flap valve with a non-return effect is used, which is arranged in the interior of the cylinder 7 in the region of the inflow 11.
  • sample liquid can get into the interior of the cylinder 7 through the inflow 11 and the opened valve 15.
  • valve 15 closes and thus prevents sample liquid from being able to escape from the interior of the cylinder 7 via the inflow 11 in an undesirable manner.
  • two laser diodes 16 and 17 are used as light sources in order to either determine two different analytes in parallel or to additionally carry out a reference measurement.
  • the laser diode 16 directs light onto the coupling surface of the optical waveguide 1 at a fixed angle, so that the light for fluorescence excitation is coupled into the optical waveguide 1 and guided at a defined angle.
  • a laser diode 16 was used, which emits light with a wavelength with which fluorescence can be excited in the fluorophore Cy5.
  • the detector 22 can be a photodiode, a photo-avalanche diode or a photomultiplier.
  • a filter 19 is located in the beam path in front of the detector and is used for the fluorescent light of the fluorescent light. rophors is permeable and does not allow the proportions of excitation and stray light to pass through.
  • the lens 18 is provided with two slit-shaped cutouts through which the excitation light of the laser diodes 16 and 17 can be directed directly onto the coupling-in surface of the optical waveguide 1 without the light being refracted or deflected through the lens 18.
  • the laser diode 17 emits light with a wavelength with which the fluorophore Cy7 can be excited.
  • the detectable proportion of fluorescent light that can ultimately reach detector 22 is relatively small, it is favorable to alternate the already mentioned filter 19 and a second filter 20, which is permeable to fluorescent light from the fluorophore Cy7, into the beam path to move in front of the detector, consequently of course the respective measurement must also take place alternately and the measurement signals of the detector 22 must be synchronized with the movement of the two filters 19 and 20.
  • FIG. 6 also shows that an additional lens 21, for bundling the fluorescent light onto the detector 22 into the beam path of the light coupled out of the optical waveguide 1, is arranged after the filters 19 or 20.
  • the two laser diodes 16 and 17 are arranged diametrically opposite one another, but they can also be at another almost any angle to be arranged to each other.
  • the angle of incidence of the respective light beam of the laser diodes 16 and 17 must be maintained, which can be different in order to ensure an almost optimal coupling of the respective excitation light into the optical waveguide 1.
  • the beam path of the light source that is not required is also advantageously interrupted.
  • the laser diode 16 and the filter 19 are operated together, while the light from the laser diode 17 cannot enter the optical waveguide 1, since the beam path through the filter 20 has been interrupted.
  • the filters 19 and 20 should therefore both be chosen so that they are opaque to light from the laser diodes 16 and 17.
  • the diameter of the lens 18 can be reduced so that the light beams of the laser diodes 16 and 17 are not influenced by the lens 18 while maintaining all set angles.
  • optical filters 24 and 25 can be seen in FIG. 6, which are arranged directly behind the laser diodes 16 and 17 in their beam path and through which the respective excitation light is filtered in accordance with its design.
  • the laser diodes 16 and 17 used as a unit with appropriate optics that align the light beam direction in parallel.
  • the individual segments 27 can be measured in that there is a movable diaphragm 26 in the beam path in front of the detector 22.
  • the opening of the aperture 26 is positioned in the beam path by translation and / or rotation of the aperture 26 such that only fluorescent light from a segment 27 of the optical waveguide 1 reaches the detector 22.
  • the diaphragm 26 is movable, the fluorescence luminosity from the individual segments 27 can be measured with the detector 22 one after the other by translating and / or rotating the diaphragm 26.
  • a detector 22 which consists of a linear or areal arrangement of light-sensitive detectors. The fluorescent light from all segments 27 of the optical waveguide 1 can thus be measured virtually simultaneously.

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

Abstract

La présente invention concerne une méthode et un dispositif de dosage immunologique par fluorescence. La lumière est dirigée depuis au moins une source de lumière sur une surface à une extrémité d'une fibre optique, et la lumière couplée dans la fibre optique est utilisée de telle manière qu'une substance de marquage liée à un partenaire chimique ou biochimique d'un système général de ligands récepteurs devient fluorescente grâce à une excitation de champ évanescente à la surface de la fibre optique. Cette solution devrait permettre d'effectuer rapidement et à faible coût des dosages immunologiques fluorométriques de grande précision. Pour ce faire, on découple la lumière fluorescente de la fibre optique et on la dirige par des moyens optiques sur le détecteur optique, afin d'en mesurer l'intensité. La fibre optique est contenue dans une chambre de mesure formée dans le piston d'un ensemble piston-cylindre et relié à l'intérieur du cylindre par une orifice d'admission aménagé dans ledit piston.
EP98959766A 1997-10-28 1998-10-27 Methode et dispositif de dosage immunologique fluorometrique Withdrawn EP1027593A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19747572A DE19747572C1 (de) 1997-10-28 1997-10-28 Vorrichtung und Verfahren zur Durchführung von Fluoreszenzimmuntests
DE19747572 1997-10-28
PCT/DE1998/003154 WO1999022222A1 (fr) 1997-10-28 1998-10-27 Methode et dispositif de dosage immunologique fluorometrique

Publications (1)

Publication Number Publication Date
EP1027593A1 true EP1027593A1 (fr) 2000-08-16

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EP98959766A Withdrawn EP1027593A1 (fr) 1997-10-28 1998-10-27 Methode et dispositif de dosage immunologique fluorometrique

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US (1) US6440748B1 (fr)
EP (1) EP1027593A1 (fr)
JP (1) JP2001521167A (fr)
CA (1) CA2308248A1 (fr)
DE (1) DE19747572C1 (fr)
RU (1) RU2213341C2 (fr)
WO (1) WO1999022222A1 (fr)

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Also Published As

Publication number Publication date
DE19747572C1 (de) 1999-04-08
US6440748B1 (en) 2002-08-27
CA2308248A1 (fr) 1999-05-06
WO1999022222A1 (fr) 1999-05-06
RU2213341C2 (ru) 2003-09-27
JP2001521167A (ja) 2001-11-06

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