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WO1992012424A1 - Sonde optique et procede permettant de surveiller la concentration d'un analyte - Google Patents

Sonde optique et procede permettant de surveiller la concentration d'un analyte Download PDF

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Publication number
WO1992012424A1
WO1992012424A1 PCT/US1991/004015 US9104015W WO9212424A1 WO 1992012424 A1 WO1992012424 A1 WO 1992012424A1 US 9104015 W US9104015 W US 9104015W WO 9212424 A1 WO9212424 A1 WO 9212424A1
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WIPO (PCT)
Prior art keywords
molecules
analyte
luminescent molecules
luminescent
optical probe
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PCT/US1991/004015
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English (en)
Inventor
Ashutosh Sharma
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Iowa State University Research Foundation, Inc.
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Application filed by Iowa State University Research Foundation, Inc. filed Critical Iowa State University Research Foundation, Inc.
Publication of WO1992012424A1 publication Critical patent/WO1992012424A1/fr

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    • 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"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • 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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • 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"
    • G01N2021/6432—Quenching
    • 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"
    • G01N2021/6434—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
    • G01N21/62—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
    • G01N21/62—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
    • G01N2021/6484—Optical fibres
    • 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
    • G01N2021/772—Tip coated light 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

Definitions

  • the present invention relates to an optical probe for measuring the concentration of an analyte in a sample, and more particularly to an optical probe utilizing at least two luminescent molecules whose luminescence (phosphorescence or fluorescence) is quenched by an analyte.
  • the present invention also relates to a method of using at least two luminescent molecules to determine the concentration of an analyte in a sample by luminescence quenching.
  • Clark electrode is the most frequently used instrument for the measurement of oxygen. However, it is not compact in size and its diffusion dependence is subject to calibration and drift problems. In addition, the use of the Clark electrode poses the added danger of using electrical currents in the body.
  • optical probes for oxygen which are based on fluorescence quenching typically contain pyrene and its derivatives as the oxygen quenchable molecule. Pyrene was chosen for its relatively long fluorescent decay time, good quantum yields, and fairly good sensitivity to oxygen.
  • U.S. Patent No. 3,612,866 to Stevens discloses an optical probe containing pyrene. Oxygen partial pressure can be determined by comparing the quenched fluorescence of the pyrene with the fluorescence of oxygen shielded pyrene.
  • UV radiation ultraviolet
  • visible radiation have small Stoke's shifts, and low fluorescence quenching efficiencies.
  • U.S. Patent No. 4,476,870 to Peterson et al. discloses an optical probe using perylene dibutyrate as the oxygen-quenchable molecule. This molecule can be excited by exposure to visible radiation. Although perylene dibutyrate's fluorescence quenching efficiency with oxygen is an improvement over pyrene, there is still room for improvement.
  • researchers have also investigated the use of two molecules in an optical probe for determining oxygen partial pressure.
  • U.S. Patent No. 4,810,655 to Khalil et al. discloses the use of two or more phosphorescent molecules which are sensitive in different regions of oxygen partial pressure. This allows increased sensitivity through a broad range of oxygen partial pressures. However, only one molecule is effectively used for a given oxygen partial pressure.
  • U.S. Patent 4,861,727 to Hauenstein et al. discloses an oxygen sensor containing both oxygen quenchable and non-quenchable fluorescent molecules.
  • the non-quenchable molecule acts as a reference signal for detecting changes in the optical system or source means while the sensor is in use.
  • Sharma & olfbeis "Fiberoptic Oxygen Sensor Based
  • Appl. Spectrscop. , 42, 1009 (1988) discloses an oxygen probe based on energy transfer and fluorescence quenching, which employs a pair of molecules.
  • One molecule (the donor molecule) is efficiently quenched by oxygen, while the other molecule (the acceptor molecule) is less affected by oxygen.
  • the molecules are chosen so that there is a large overlap in the emission spectrum of the donor molecule with the absorption spectrum of the acceptor molecule. This overlap results in an energy transfer from the donor molecule to the acceptor molecule, when the donor molecule is excited by radiation at a wavelength where it has strong absorption.
  • the optical probe is not entirely satisfactory because it is difficult to identify pairs of molecules which satisfy the criteria regarding energy transfer and fluorescence quenching in addition to having other properties required for use in an oxygen probe, such as photostability and a good quantum yield of fluorescence.
  • luminescent molecules are defined as fluorescent molecules or phosphorescent molecules
  • luminescence is defined as phosphorescence or fluorescence.
  • concentration in the context of a gas means the partial pressure of the gas. It is, therefore, an object of the present invention to provide an optical probe for determining the concentration of an analyte in a sample having increased sensitivity to the analyte being measured.
  • the present invention provides an improved method and means for the determination of an analyte in a sample or for the determination of the physical parameters of a sample.
  • the determination may be either qualitative or quantitative.
  • at least two luminescent molecules are used to carry out the determination.
  • the luminescent molecules both absorb radiation at an overlapping wavelength of excitation and emit luminescence at an overlapping wavelength where the emission luminescence is detected.
  • the different luminescent molecules that are used are both coexcited and comonitored.
  • each luminescent molecule has at least one major band of its absorption spectrum that overlaps with at least one major band of the absorption spectrum of each of the other luminescent molecules employed, and has at least one major band of its emission spectrum that overlaps with at least one major band of the emission spectrum of each of the other luminescent molecules so that the different luminescent molecules employed may be coexcited at a common excitation wavelength and their emission may be comonitored at a common emission luminescence wavelength.
  • Each molecule must also have its luminescence quenched by the analyte being measured.
  • the luminescent molecules can be coexcited by the same wavelength of ultraviolet, visible, or infrared radiation using an optical fiber or directly by a light source.
  • the resulting luminescence exceeds the sum of the luminescence for each molecule at a given wavelength throughout the overlapping region.
  • lower concentrations of molecules can be used, thereby avoiding solubility problems, such as crystallization.
  • the combination of at least two luminescent molecules results in a probe having higher sensitivity to the analyte being measured than that which would be obtained if only one luminescent molecule was used, because the relative changes in the emission signal of the molecules are overlapped. Therefore, the change in this signal will be greater than it would be for one molecule.
  • the present invention also provides a method for determining the amount of an analyte present in a sample using at least two different luminescent molecules in accordance with the aforementioned description, which involves measuring the luminescence quenching of the molecules.
  • FIG. 1 is a diagrammatic view of an optical probe in accordance with the present invention.
  • Fig. 2 is an exploded diagrammatic view of two luminescent molecules immobilized in support means
  • Fig. 3 is a diagrammatic view of an optical probe in accordance with the present invention, and illustrating another exemplary embodiment of the invention, that is, an optical probe containing a reference molecule; and Fig. 4 is a graph showing the response of an illustrative embodiment of the optical probe of the present invention toward a specific analyte, oxygen, which is dissolved in water.
  • the probe contains two molecules, a first luminescent molecule 1 and a second luminescent molecule 2.
  • At least one major band of the absorption spectrum of luminescent molecule 1 overlaps with at least one major band of the absorption spectrum of luminescent molecule 2, and at least one major band of the emission spectrum of luminescent molecule 1 must also overlap with at least one major band of the emission spectrum of luminescent molecule 2 so that luminescent molecules 1 and 2 can be coexcited at a common excitation wavelength and so that the emission luminescence of both luminescent molecules 1 and 2 can be monitored at a common wavelength.
  • both luminescent molecules 1 and 2 must have their luminescence quenched by the analyte whose concentration is being measured.
  • each luminescent molecule 1 and 2 The overlap between at least one major band of the absorption spectrum of each luminescent molecule 1 and 2 is needed so that both luminescent molecules are excited by radiation of a single wavelength. It is believed that this "co-excitation" results in improved photostability of the molecules, since the excitation energy is shared among the molecules. It is preferred that the overlap between the absorption spectra of the molecules be as large as possible. This allows a common absorption wavelength to be chosen where both molecules show higher absorption.
  • the luminescence emitted by the molecules can be measured at a single wavelength and yields a high intensity signal. This allows reduced concentrations of the- individual molecules to be used without affecting the overall analytical emission signal. It is preferred that the overlap between the emission spectra of the molecules be as large as possible to allow a common emission wavelength to be chosen where both molecules show high intensity emission.
  • EET will result in luminescence emission only from the acceptor molecule.
  • the monitored luminescence will be that of the acceptor molecule only.
  • at least one of the major absorption bands of one luminescent molecule overlaps with at least one of the major absorption bands of the other luminescent molecules.
  • at least one of the major luminescence bands of one molecule overlaps with at least one of the major luminescence bands of the other luminescent molecules. Therefore, in the present invention, each of the luminescent molecules used will be coexcited at the common wavelength of excitation and the monitored luminescence signal will always contain luminescence from each molecule used.
  • a major band means a Gaussian structure with a well defined shape and having a measurable half-width when expressed in energy units.
  • the present invention can be used to quantitatively determine the concentration of an analyte in a sample or to determine physical parameters of a sample, or both.
  • the various analytes which can be determined using the present invention include sulphur dioxide, oxides of nitrogen, methane, ethane, propane, butane, halothane, ammonia, mustard gas, hydrogen chloride, hydrogen sulfide, chlorine, bromine, iodine, carbon monoxide, carbon dioxide, ozone, metal ions including, for example, sodium, potassium, magnesium, lead, copper, uranium, and the like, hydrocarbons, vitamins, pesticides, moisture, urea, and like.
  • Physical parameters which can be determined using the present invention include temperature, pressure, viscosity, pH, ionic strength, current, voltage, or other parameters such as nuclear radiation.
  • the present invention may be used to quantitatively determine analyte concentration and physical parameters of samples in gaseous, liquid or solid media.
  • the sample may be used to quantitatively determine analyte concentration and physical parameters of samples in gaseous, liquid or solid media.
  • TE SHEET is brought into contact with the luminescent molecules.
  • the molecules are excited by suitable radiation and luminescent emission, quenched by the analyte, is measured.
  • the extent of quenching is then related to the analyte concentration or physical parameter of the sample by comparison to a standard curve for the analyte or physical parameter.
  • the luminescent molecules of the present invention useful where oxygen is the analyte of interest include substituted or unsubstituted conjugated organic molecules such as polycyclic aromatic hydrocarbons; ruthenium complexes of conjugated organic molecules; or metal complexes of porphyrines. If substituted polycyclic aromatic hydrocarbons are used, it is preferred that the substituted functional groups be chosen from the following functional groups: methoxy, methyl, ethyl, -keto, -nitro, -hydroxy, -amino or metal.
  • Preferred indicators for determining oxygen include: (1) perylene dibutyrate and decacyclene; (2) pyrene in combination with anthracene or chrysene;
  • ruthenium complexes of conjugated organic molecules such as tris(2,2 '-bipyridine) ruthenium II dichloride and tris(l, 10-phenthroline) ruthenium II.
  • perylene dibutyrate and decacyclene or tris(2,2 '-bipyridine) ruthenium II dichloride and tris(l, 10-phenthroline) ruthenium II are most preferred.
  • Preferred indicators for determining sulfur dioxide include combinations chosen from the group of polycyclic aromatic hydrocarbons and their alkyl, alkoxy, nitro and amino derivatives. Preferred indicator combinations include:
  • Preferred indicators for determining chlorine include combinations chosen from the group of polycyclic aromatic hydrocarbons and their derivatives, and include:
  • Preferred indicators for determining the physical parameter, pH include: (1) fluorescein and 3 (and 6)-carboxy fluorescein;
  • luminescent molecules may also be inorganic compounds or polymers or liquid crystals.
  • the luminescent molecules can be present in various proportions, but it is preferred that the luminescent molecules 1 and 2 be present in an amount such that their relative luminescence intensities are equal. Therefore, the optimal amount of luminescent molecules will vary depending on the specific molecules chosen.
  • the concentration of the luminescent molecules l and 2 should also be such that the Beer's law plot for those molecules is linear. A non-linear plot indicates that the radiation emitted by the luminescent molecules is being reabsorbed by molecules in close proximity to the emitting molecules before all of the luminescent molecules have been excited. Also, use of higher concentrations of luminescent molecules will result in increased background luminescence because not all the molecules are accessible to the quencher analyte.
  • luminescent molecules 1 and luminescent molecules 2 are immobilized on support means 3. Although immobilizing the molecules on a support means is not required to practice the invention, it is preferred, especially in a transducer type application, such as in the human body or in a process control.
  • the molecules may be immobilized as a mixture, or luminescent molecules 1 and luminescent molecules 2 may be immobilized in separate layers.
  • a high permeability support is desirable to increase exposure of the individual luminescent molecules to analyte collision. As the permeability of the support increases, the response time of the probe becomes faster.
  • Solid support means for carrying the luminescent molecules may be determined by routine testing.
  • Preferred polymer materials include silicone rubber, polyisoprene, cellulose, silica gel, polyvinyl chloride, and Amberlite XAD4 7 a nonionic, hydrophobic polymer available from Rohm & Haas.
  • the luminescent molecules can be chemically immobilized on the support means by using standard methods of covalent bonding or by using ion exchange. Immobilization of the luminescent molecules can also be accomplished physically by various methods, including entrapping the molecules in a polymeric support (shown in FIG. 2) , adsorbing the molecules on a polymer/solid support surface, vaporizing the molecules and depositing them on a support surface, absorbing the molecules into a support material such as filter paper, and forming a molecular layer or layers using the Langimur-Blodgett techniques.
  • the method chosen to immobilize the luminescent molecules on the support means depends upon the molecules used and/or the nature of the support means.
  • the use of chemical immobilization is dependent upon the functional groups available on one or more of the luminescent molecules, and on the support means.
  • Physical immobilization techniques are dictated by properties of the support means, such as solubility, temperature and surface tension, and by properties of the luminescence molecules, such as melting point and adsorption properties.
  • luminescence molecules be immobilized on a support means to practice the present invention.
  • Mixtures of solid molecules can be used, or the molecules can be used in solution.
  • the luminescent molecules 1 and 2 and support means 3 are enclosed by a member 4, shown in FIG. 1.
  • Member 4 is permeable to the analyte of interest. Where oxygen is the analyte, porous polymer materials, such as "Celgard,” a porous polypropylene sheet available from Celanese, heat-sealed into a tube is suitable. Where support means are used, the molecules need not be enclosed by a member. However, use of a selectively permeable enclosure member may be desirable depending on the analyte being measured. If no support means are used, the luminescent molecules are enclosed by an enclosure member.
  • a bifurcated optical fiber bundle 5 is attached to support means 3.
  • the optical fiber bundle 5 carries excitation radiation to luminescent molecules 1 and 2 via fiber 6, and collects the luminescence emitted by the molecules upon excitation via fiber 7.
  • the fiber optic bundle 5 can be used in conjunction with an optical system (not shown) having a light source, a light intensity measuring device, such as photodiode or
  • the HEET photomultiplier necessary power supplies, and an electronic computing circuit.
  • the electronic computing circuit is driven by current/voltage generated by the light intensity measuring device and is arranged to provide a direct, analog/digital computation of the concentration of the analyte being measured based on the luminescence quenching detected.
  • FIG. 3 Another embodiment of the optical probe of the present invention is shown in FIG. 3.
  • luminescent molecules 1 and 2 are immobilized on support means 3. Molecules 1 and 2 and support means 3 are enclosed by member 4.
  • a bif rcated optical fiber bundle 5 is attached to support means 3, with fibers 6 and 7 exposed to luminescent molecules 1 and 2.
  • Fiber 8 is attached to a portion 9 of support means 3, portion 9 immobilizing an amount of luminescent molecules 1 or luminescent molecules 2.
  • Portion 9 and the immobilized molecule (luminescent molecules 1 or luminescent molecules 2) are enclosed by casing 10, which is impervious to the analyte being measured.
  • Fiber 8 is exposed to the immobilized molecule enclosed by coating 10 and transmits its luminescence, which serves as a reference.
  • the optical probe of the present invention can also be used with a single optical fiber. In that case, both the optical excitation and emission radiation are carried by the single fiber.
  • a means such as a splitter plate or the like for splitting the beam.
  • the optical probe of the present invention can also be used without fiber optics. If such a system is used, the luminescent molecules can be immobilized on an optically transparent support means attached to the luminescent molecules in place of the distal end of the fiberoptic bundle.
  • the transparent plate may be glass, quartz, plexiglass, acrylic, or any other optically transparent material.
  • the luminescent molecules are excited by focusing a collimated beam of light on the transparent plate which is incorporated in a flow through device as a window.
  • a laser source also can be used for this purpose.
  • the resulting luminescence is then collected at the same angle as that of excitation or at an angle other than that of excitation on either side of the plate using, for example, a collimating device.
  • the detection or measurement of an analyte can also be made by injecting the chosen indicator molecules in a stream of analyte at a constant rate so as to have a generally uniform and constant concentration of the indicator luminescent molecules in the stream (such as in a combination with flow injection systems) .
  • An optical window can be provided to allow luminescence measurements.
  • each luminescent molecule when more than two luminescent molecules are used, each luminescent molecule must have at least one major band in its absorption spectrum which overlaps with at least one major band in the absorption spectrum of each of the other luminescent molecules so that all of the molecules can be coexcited, and each luminescent molecule must have at least one major band in its emission spectrum that overlaps with at least one major band in the emission spectrum of the other luminescent molecules so that the emission of all the molecules can be co-monitored at a common wavelength with the emission of each of the other luminescent molecules. Further, each luminescent molecule must have its luminescence quenched by the analyte being measured. If oxygen partial pressure is being measured, it is preferred that at least one or more of the molecules be unsubstituted or substituted conjugated organic molecules, such as polycyclic aromatic hydrocarbons. Preferred functional groups which may be substituted on
  • ET the polycyclic aromatic hydrocarbons are methoxy, methyl, ethyl, keto, nitro, hydroxy, amine, or metallic.
  • metal complexes of porphyrines, such as lead complexes is also preferred.
  • Specific combinations suitable for use in the present invention where oxygen is the analyte of interest include:
  • optical probe and method of the present invention may be used to determine the presence of an analyte of interest in a sample qualitatively, or it may be used to determine the content of an analyte in a sample quantitatively, by luminescence quenching.
  • Dynamic luminescence quenching is given by the Stern- Volmer equation:
  • the quenching constant, K sv for the molecules can be determined. If the relationship between the concentration of the analyte and the intensity of the combined luminescence of the particular luminescent molecules 1 and 2 used is linear, the concentration of the analyte can be easily determined using the above equation. However, if the relationship is non-linear, K sv will change with varying analyte concentration.
  • Another aspect of the present invention comprises a method of using at least two luminescent molecules to determine the presence of or concentration of an analyte in a sample or for the detection or quantitative determination of a physical parameter in a sample, based on the luminescence quenching of the molecules.
  • the method may be used in gaseous, liquid or solid media.
  • the luminescent molecules both absorb radiation at an overlapping wavelength of excitation and emit luminescence at an overlapping wavelength where the emission luminescence is detected. Stated another way, both the absorption spectra and the luminescence spectra of the luminescent molecules overlap. Thus, the luminescent molecules are both coexcited at the common wavelength of excitation, and the monitored luminescence signal will always contain luminescence from each of the luminescent molecules. It is preferred that the overlap between the absorption spectra of the molecules be as large as possible to allow a common absorption wavelength to be chosen where the luminescent molecules show higher absorption.
  • the overlap between emission spectra of the molecules be as large as possible to allow a common emission wavelength to be chosen where the luminescent molecules show high intensity emission. It is most preferred that at least one major absorption band of one luminescent molecule overlap with at least one of the major absorption band of the other luminescent molecules and that at least one major luminescence band of one molecule overlap with at least one of the major luminescence bands of the other luminescent molecules.
  • the probe is brought into position to monitor the sample to be analyzed, or the sample is brought into position to be monitored by the probe, exposing luminescent molecules 1 and 2 to the analyte.
  • the sample to be analyzed is brought into proximity of the luminescent molecules 1 and 2. While luminescent molecules 1 and 2 are in proximity to the analyte whether in a probe or not, they are exposed to radiation having a wavelength where both molecules 1 and molecules 2 show analytically determinable absorption and, in the most preferred embodiment, where at least one of the major bands of their respective absorption spectra overlap. Preferably, the wavelength used is that where the combined absorption of luminescent molecules 1 and 2 is at a maximum. These two steps may be reversed; however, the first measurement must be made in the complete absence of an analyte.
  • the combined luminescence given off by molecules 1 and 2 is then measured at a wavelength where both molecules show analytically determinable emission and, in the most preferred embodiment, where at least one of the major bands of their respective emission spectra overlap. Either fluorescence or phosphorescence should be measured; both should not be used. Again, it is preferred to use the wavelength where the combined emission is at a maximum. Finally, in quantitative determinations, the combined luminescence intensity obtained is used with the previously calculated sv and I 0 , or a previously generated curve based on known analyte concentrations to obtain the analyte content of the sample.
  • the concentration of an analyte can also be determined by monitoring the combined luminescence decay rate of the luminescent molecules indicators, as disclosed in U.S. Patent No. 4,810,655 to Khalil et al.
  • some analytes and physical parameters can be determined using indirect methods. For example, an acidic gas, such as sulfur dioxide, chlorine, hydrogen chloride, carbon dioxide, or a basic gas, such as ammonia, can be determined by using pH indicators.
  • bio-sensors and probes may be constructed with the use of an enzyme which is specific to the analyte of interest or the method of the present invention may be employed to determine (qualitatively or quantitatively) biomedical analytes of interest. Numerous biomedical analyte species can be measured using a probe or method in accordance with the present invention and the appropriate enzyme.
  • Analytes include inorganic species, organic species and activities of enzymes.
  • Illustrative inorganic species of analytes are copper ion, cyanate, nitrate, phosphate, thiosulphate, hydrogen peroxide, mercuric ion, fluorate, nitrite, sulphate, and carbon monoxide.
  • Illustrative of organic species of analytes are acetate, acetylcholine, adenosine, acetyl-B- methylcholine, AMP, ADP, ATP, alcohols, aldehydes, mono- and di-amines, L-amino acids, D-amino acids, L-organine, L-asparagine, L-glutanate, L-glutamine, L-histidine, L- lysine, L-methionine, L-phenylalanine, L-threonine, L- tyrosine, a ygdalin, ascorbate, aspartane, butyrylthiocholin, catechol, cellobiose, cephalosporines, choline, cholesterol, choresterol esters, creatine,
  • TE SHEET ⁇ reatanine, formate, fructose, glucose, gentamicin, D- gluconate, glucose, glucose-6-phosphate, glutamine, glutathion, glycerol, glycerol esters, guanine, 3- hydroxybutyrate, hypoxanthine, inosine, myo-inositol, IMP, D-lactate, L-lactate, lactose, lectin, lignin, malate, maltose, NADH, NAD + , oxalate, oxalacetate, parathion, penicillin, phenol, proteins, pyruvate, sucrose, starch, thiamine pyrophosphate, tyramine, urea, uric acid, xanthine, xylose, xylulose.
  • Illustrative of enzymes whose activities may be determined are acid phosphatase, alkaline phosphatase, amylase, arginase, cholinesterase, creatine kinase, glutamate, pyruvate transaminase, lactate dehydrogenase, pyruvate kinase.
  • Such measurement may be direct, as, for example, by monitoring the changes in the luminescence of the suitably chosen luminescent molecules, due to the consumption or production of the analyte during enzymatic reaction.
  • An indirect approach may also be used.
  • a suitable optical probe made according to the present invention for the measurement of such analytes/parameters i.e., oxygen, hydrogen peroxide, pH, temperature or NADH
  • an enzyme coimmobilized or otherwise incorporated in the probe
  • the present invention may be used for the determination of
  • a sensor/probe or method to determine glucose may be based on the detection of oxygen.
  • Glucose oxidase can be immobilized onto the oxygen probe/sensor described in the present invention such that when the sample containing glucose is exposed to the sensor probe, glucose oxidase is in direct contact with glucose and the enzymatic reaction takes place.
  • the changes in glucose concentration will affect the enzymatic reaction and will change the oxygen partial pressure.
  • the change in the oxygen partial pressure in turn changes the luminescence from the luminescent molecules used as the indicator which changes the analytical signal from which the oxygen and in turn the glucose can be determined.
  • the enzyme glucose oxidase may also be held near the oxygen probe, in a separate membrane or even in a pouch made out of material which is permeable to glucose and oxygen to allow the enzymatic reaction.
  • a modified enzyme may also be used to make an oxygen probe for the determination of glucose.
  • the enzyme is modified by coupling the oxygen sensitive luminescent molecules used as the indicator directly onto the enzyme at sites which will not effect its enzymatic activity.
  • the substitution of the luminescent molecules on the enzyme is done according to known chemical methods and without effecting the enzyme activity.
  • the modified enzyme is then immobilized (with the luminescent molecules used as the indicator as an integral part thereof) on to a solid support or fiber.
  • the response time of such a probe would be faster than a probe where glucose oxidase is separate from the oxygen sensitive indicators.
  • Another example of a probe/sensor (or method) for monitoring glucose that may be made according to present invention is to monitor the pH change of the sample. Luminescent molecules that are sensitive to pH are used as the indicator.
  • Glucose oxidase is immobilized in a sensing layer along with the pH sensitive luminescent molecules.
  • the enzyme catalyses the oxidation of glucose to give gluconic acid, which in turn lowers the pH in the micro-environment of the luminescent molecules, thereby allowing the monitoring of the enzymatic reaction and the determination of glucose.
  • glucose oxidase may be modified by coupling the pH sensitive luminescent molecules to the glucose oxidase by known chemical methods and without affecting the enzyme activity.
  • the modified enzyme may be immobilized onto a solid support.
  • Another way to measure glucose concentration in a sample according to the present invention is to monitor the hydrogen peroxide changes during the enzymatic catalysis, using two luminescent molecules as an indicator, selected as disclosed in the present invention, because of their sensitivity to hydrogen peroxide.
  • analysis of glucose may be conducted by immobilizing the luminescent molecules and the enzyme together or in separate layers or modifying the enzyme by attaching the luminescent molecules.
  • Optical probes for the determination of numerous other analytes that are of biomedical interest can likewise be made using the method disclosed in the present invention.
  • an oxygen probe based on enzymes can be made in a manner similar to the oxygen probe for glucose, that is, by incorporating an enzyme
  • UBSTITUTE SHEET for the analyte of interest into the oxygen probe.
  • Examples of analytes and their corresponding enzymes that may be used are set forth in Table I.
  • An enzymatic cycling procedure where two enzymes cycle the sample to be analyzed, in combination with the oxygen or pH sensor disclosed in the present invention, may be employed to construct an enzyme probe.
  • An oxidase-dehydrogenase couple is used to cycle an analyte and the oxygen probe can be used to monitor the changing oxygen partial pressure.
  • Example I This Example illustrates the use of the present invention to construct an optical probe for oxygen, and its use to determine oxygen content in a sample.
  • the luminescent molecules used were two fluorescent molecules, perylene dibutyrate and decacyclene.
  • the excitation and fluorescence emission spectra of decacyclene and perylene dibutyrate in methanol at 18°C and immobilized on Whatman filter paper number 1 is described in Table II.
  • decacyclene " 320 shoulder ' 480 shoulder '345 shoulder ' 510 major band '395 major band ' 550 shoulder '420 shoulder ' 445 shoulder ' 475 shoulder
  • the perylene dibutyrate was dissolved in toluene to form a 3 ⁇ iM solution, while the decacyclene was separately dissolved in toluene to form a 6 mM solution.
  • 0.5 ml of the perylene dibutyrate solution and 0.5 ml of the decacyclene solution were mixed in a petri dish, after which a 5.5 cm diameter circle of Whatman filter paper No. 1 was placed in the petri dish. The filter paper was not removed immediately, but was left for solvent evaporation.
  • the filter paper containing the absorbed decacyclene and perylene dibutyrate was then gently washed with solvent, redried, coated with silicon rubber (thickness less than 10 ⁇ m) , and cut into small circles of 3 mm in diameter.
  • a circle was then attached to one end of a glass sleeve having an outer diameter of 3 mm and an inner diameter slightly greater than 2 mm using quick drying epoxy.
  • a bifurcated fiber optic light guide having an inner diameter of 2 mm was inserted into the glass sleeve and attached to the circle of filter paper containing the perylene dibutyrate and decacyclene molecules.
  • a white light excitation source was used in combination with interference filters and the bifurcated light guide.
  • Oxygen mixed with nitrogen in concentrations from 0 to 21% oxygen, at a pressure of 97.3 kPa was guided to a chamber containing the probe by 3 mm in diameter PVC tubing, thereby contacting the decacyclene and perylene dibutyrate immobilized by the filter paper.
  • Excitation light having a wavelength of 410 nm was focused into the fiber, and the resulting fluorescence, which was measured at a wavelength at 510 nm using an interference filter, was guided via the fiber to photodetector.

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Abstract

Sonde optique et procédé permettant de surveiller la concentration d'un analyte, ou la pression partielle d'un gaz se trouvant dans un échantillon. La sonde optique renferme au moins deux molécules luminescentes (fluorescentes ou phosphorescentes) dont la luminescence est étouffée par l'analyte dont on mesure la concentration. Le spectre d'absorption de chaque type de molécule luminescente recouvre le spectre d'absorption de chacune des autres molécules, et le spectre d'émission de chaque molécule recouvre le spectre d'émission de chacune des autres molécules. On mesure la pression partielle d'un gaz ou la concentration d'un analyte à l'aide de l'étouffement de la luminescence desdites molécules par l'analyte.
PCT/US1991/004015 1991-01-04 1991-06-07 Sonde optique et procede permettant de surveiller la concentration d'un analyte WO1992012424A1 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4304728A1 (de) * 1993-02-13 1994-08-18 Igor Dr Popov Verfahren und Testbesteck zur Bestimmung von Ascorbinsäure in biologischen Proben
WO1995010766A1 (fr) * 1993-10-14 1995-04-20 Minnesota Mining And Manufacturing Company Detecteurs d'extinction d'emission
US5462880A (en) * 1993-09-13 1995-10-31 Optical Sensors Incorporated Ratiometric fluorescence method to measure oxygen
WO1999032878A1 (fr) * 1997-12-19 1999-07-01 Paul Nicholas Capteurs a cristaux liquides
US5919710A (en) * 1996-07-18 1999-07-06 The United States Of America As Represented By The Secretary Of The Air Force Optical method for quantitating dissolved oxygen in fuel
US6127140A (en) * 1999-06-18 2000-10-03 Abbott Laboratories Assay for quantitative measurement of analytes in biological samples
WO2004044865A2 (fr) * 2002-11-08 2004-05-27 Leatech, Llc Matiere sensible a la pression
EP1805503A1 (fr) * 2004-10-25 2007-07-11 F. Hoffmann-Roche AG Systeme de reference multifonctionnel a utiliser pour la detection d'un analyte par fluorescence
US8081313B2 (en) 2007-05-24 2011-12-20 Airbus Operations Limited Method and apparatus for monitoring gas concentration in a fluid
EP2601515A1 (fr) * 2010-08-03 2013-06-12 General Electric Company Détermination simultanée de multiples analytes dans un système de distribution d'eau industriel
EP2728343A1 (fr) * 2012-11-06 2014-05-07 Technische Universität Graz Sonde optique pour la détermination quantitative d'un analyte
EP2778078A1 (fr) * 2013-03-13 2014-09-17 Witt GmbH & Co. Holding und Handels-KG Dispositif de mesure pour des machines d'emballage pour sachets tubulaires
US8852512B2 (en) 2008-07-28 2014-10-07 Airbus Operations Ltd Monitor and a method for measuring oxygen concentration
EP2336753B1 (fr) * 2009-12-07 2019-12-25 Luxcel Biosciences Limited Sonde d'oxygène photo-luminescente avec sensibilité transversale réduite à l'humidité
DE102019124795A1 (de) * 2019-09-16 2021-03-18 Abberior GmbH Optischer pH-Sensor
CN118758893A (zh) * 2024-09-05 2024-10-11 陕西圣唐乳业有限公司 基于光谱分析的奶粉微量元素检测方法及系统

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4810655A (en) * 1985-07-03 1989-03-07 Abbott Laboratories Method for measuring oxygen concentration
US4861727A (en) * 1986-09-08 1989-08-29 C. R. Bard, Inc. Luminescent oxygen sensor based on a lanthanide complex
US4916060A (en) * 1985-09-17 1990-04-10 Massachusetts Institute Of Technology Process for chemical measurement in small volume samples by fluorescent indicators

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4810655A (en) * 1985-07-03 1989-03-07 Abbott Laboratories Method for measuring oxygen concentration
US4916060A (en) * 1985-09-17 1990-04-10 Massachusetts Institute Of Technology Process for chemical measurement in small volume samples by fluorescent indicators
US4861727A (en) * 1986-09-08 1989-08-29 C. R. Bard, Inc. Luminescent oxygen sensor based on a lanthanide complex

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4304728A1 (de) * 1993-02-13 1994-08-18 Igor Dr Popov Verfahren und Testbesteck zur Bestimmung von Ascorbinsäure in biologischen Proben
US5462880A (en) * 1993-09-13 1995-10-31 Optical Sensors Incorporated Ratiometric fluorescence method to measure oxygen
US5681532A (en) * 1993-09-13 1997-10-28 Optical Sensors, Incorporated Optical sensors for the measurement of oxygen
US5728422A (en) * 1993-09-13 1998-03-17 Optical Sensors, Incorporated Ratiometric fluorescence method of making for measuring oxygen
WO1995010766A1 (fr) * 1993-10-14 1995-04-20 Minnesota Mining And Manufacturing Company Detecteurs d'extinction d'emission
US5462879A (en) * 1993-10-14 1995-10-31 Minnesota Mining And Manufacturing Company Method of sensing with emission quenching sensors
US5518694A (en) * 1993-10-14 1996-05-21 Minnesota Mining And Manufacturing Company Emission quenching sensors
US5919710A (en) * 1996-07-18 1999-07-06 The United States Of America As Represented By The Secretary Of The Air Force Optical method for quantitating dissolved oxygen in fuel
WO1999032878A1 (fr) * 1997-12-19 1999-07-01 Paul Nicholas Capteurs a cristaux liquides
US6127140A (en) * 1999-06-18 2000-10-03 Abbott Laboratories Assay for quantitative measurement of analytes in biological samples
US7176272B2 (en) 2002-11-08 2007-02-13 Leatech, Llc Pressure sensitive material
WO2004044865A2 (fr) * 2002-11-08 2004-05-27 Leatech, Llc Matiere sensible a la pression
WO2004044865A3 (fr) * 2002-11-08 2005-04-28 Leatech Llc Matiere sensible a la pression
US8759112B2 (en) 2004-10-25 2014-06-24 Roche Diagnostics Operations, Inc. Multifunctional reference system for analyte determinations by fluorescence
EP1805503A1 (fr) * 2004-10-25 2007-07-11 F. Hoffmann-Roche AG Systeme de reference multifonctionnel a utiliser pour la detection d'un analyte par fluorescence
US8081313B2 (en) 2007-05-24 2011-12-20 Airbus Operations Limited Method and apparatus for monitoring gas concentration in a fluid
US8852512B2 (en) 2008-07-28 2014-10-07 Airbus Operations Ltd Monitor and a method for measuring oxygen concentration
EP2336753B1 (fr) * 2009-12-07 2019-12-25 Luxcel Biosciences Limited Sonde d'oxygène photo-luminescente avec sensibilité transversale réduite à l'humidité
EP3705876A1 (fr) * 2009-12-07 2020-09-09 Mocon, Inc. Sonde d'oxygène photoluminescente présentant une sensibilité transversale réduite à l'humidité
EP2601515A4 (fr) * 2010-08-03 2014-04-30 Gen Electric Détermination simultanée de multiples analytes dans un système de distribution d'eau industriel
EP2601515A1 (fr) * 2010-08-03 2013-06-12 General Electric Company Détermination simultanée de multiples analytes dans un système de distribution d'eau industriel
US9228986B2 (en) 2010-08-03 2016-01-05 General Electric Company Simultaneous determination of multiple analytes in industrial water system
EP2728343A1 (fr) * 2012-11-06 2014-05-07 Technische Universität Graz Sonde optique pour la détermination quantitative d'un analyte
EP2778078A1 (fr) * 2013-03-13 2014-09-17 Witt GmbH & Co. Holding und Handels-KG Dispositif de mesure pour des machines d'emballage pour sachets tubulaires
DE102019124795A1 (de) * 2019-09-16 2021-03-18 Abberior GmbH Optischer pH-Sensor
CN118758893A (zh) * 2024-09-05 2024-10-11 陕西圣唐乳业有限公司 基于光谱分析的奶粉微量元素检测方法及系统

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