WO1992012424A1

WO1992012424A1 - An optical probe and method for monitoring an analyte concentration

Info

Publication number: WO1992012424A1
Application number: PCT/US1991/004015
Authority: WO
Inventors: Ashutosh Sharma
Original assignee: Iowa State University Research Foundation Inc ISURF
Current assignee: Iowa State University Research Foundation Inc ISURF
Priority date: 1991-01-04
Filing date: 1991-06-07
Publication date: 1992-07-23
Anticipated expiration: 1993-07-04
Also published as: AU8720891A

Abstract

An optical probe and a method of monitoring analyte concentration, or the partial pressure of a gas in a sample are described. The optical probe contains at least two luminescent (fluorescent or phosphorescent) molecules whose luminescence is quenched by the analyte being measured. The absorption spectrum of each type of luminescent molecule overlap with the absorption spectrum of each of the other molecules, and the emission spectrum of each molecule overlaps with the emission spectrum of each of the other molecules. The partial pressure of a gas or concentration of an analyte is measured using the quenching of the luminescence of said molecules by the analyte.

Description

AN OPTICAL PROBE AND METHOD FOR MONITORING AN ANALYTE CONCENTRATION

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION Accurate measurement of the concentration of an analyte in a sample is important in various industrial, research, military, safety, medical and environmental applications. Oxygen is a good example. From a medical standpoint, oxygen partial pressure (concentration) measurements are necessary to observe the oxygen transfer mechanism in humans. Further, adequate tissue oxygenation is an extremely important concern in many surgical and/or intensive care situations. Thus, quick response sampling or continuous monitoring of oxygen partial pressure is a necessity.

A number of techniques for measuring oxygen partial pressure are known, but none are entirely satisfactory. The 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.

Recently, the use of fluorescence spectroscopy methods to determine oxygen partial pressure has become more prevalent. A number of optical oxygen probes have been developed which are based on fluorescence quenching. These probes measure oxygen partial pressure through quantitative changes in the fluorescence of an oxygen sensitive fluorescent molecule immobilized on a solid support, when oxygen is allowed to contact the molecule. The advantages inherent in the use of luminescence, such as high sensitivity, selectivity and simplicity of instrumentation are obtained. As compared to the Clark electrode, these probes are less sensitive to temperature and do not require controlled mass transfer of oxygen to the electrode surface. Further, fiber optic probes can be used safely in medical applications, because they do not involve the use of electrical currents in the body. Due to their small size, flexible nature, and the material used in making them, fiber optic probes can be used as implants and can monitor oxygen content in very small blood vessels.

Many of the 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.

However, the use of pyrene and its derivatives have some disadvantages. Many of these molecules require excitation by ultraviolet (UV) radiation as opposed to visible radiation, have small Stoke's shifts, and low fluorescence quenching efficiencies.

In an attempt to overcome these problems, use of other molecules were investigated. 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

On Fluorescence Quenching and Energy Transfer," 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. Further, 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. When the two molecules are exposed to oxygen, the fluorescence of the donor molecule is quenched, resulting in decreased energy available for the acceptor molecule, and thus reduced fluorescence from the acceptor molecule. This reduced fluorescence is further quenched by surrounding oxygen molecules. As a result, the fluorescence of the acceptor molecule is quenched more efficiently than it would be were the acceptor molecule used alone. This system has distinct advantages over earlier probes using a single molecule, such as large Stoke's shifts and higher quenching efficiencies. Because of the large Stoke's shifts, there is reduced interference of excitation light when the fluorescence quenching is being monitored.

Notwithstanding the advantages of this sensor, 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.

Despite the aforementioned developments in the use of optical probes for measuring oxygen partial pressure in a sample, there remains a need in the art for an optical probe and method having increased sensitivity to oxygen.

Similar needs also exist for optical probes and methods for measuring the concentration of analytes other than oxygen in a sample and having increased sensitivity to the analyte of interest.

A further need exists for an optical probe containing luminescent molecules which exhibit improved photostability, good quantum yield of luminescence, and a good measurable luminescence using reduced molecule concentrations. As used herein, the term "luminescent molecules" is defined as fluorescent molecules or phosphorescent molecules, and the term "luminescence" is defined as phosphorescence or fluorescence. The term 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. It is an additional object to provide an optical probe for determining the concentration of an analyte in a sample containing at least two luminescent molecules, which together exhibit good photostability and a good measurable luminescence. It is a further object of the present invention to provide a method for determining the concentration of an analyte in a sample using at least two luminescent molecules which requires lower concentration of individual molecules than was needed previously where only one molecule was used.

It is yet another object of the present invention to provide an optical probe for determining the concentration of an analyte in a sample having an increased use lifetime. It is yet another object of the present invention to provide a method for measuring the concentration of an analyte in a sample which can be quickly and accurately performed.

These and other objects and advantages of the present invention will become more readily apparent upon reading the following description.

SUMMARY OF THE INVENTION

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. In accordance with the invention, 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. Preferably, 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. As a result, lower concentrations of molecules can be used, thereby avoiding solubility problems, such as crystallization. In addition, 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. BRIEF DESCRIPTION OF THE DRAWINGS

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.

While the invention will be described and disclosed in connection with certain preferred embodiments and methods, it is not intended to limit the invention to those specific embodiments. Rather, it is intended to cover all such alternative embodiments and modifications as fall within the scope of the claims.

DETAILED DESCRIPTION OF THE INVENTION Referring to the drawings, and more particularly FIG. 1, an optical probe in accordance with one aspect of the present invention is illustrated. The probe contains two molecules, a first luminescent molecule 1 and a second luminescent molecule 2.

For a pair of luminescent molecules to be suitable for use in the present invention, 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. Also, both luminescent molecules 1 and 2 must have their luminescence quenched by the analyte whose concentration is being measured.

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.

Since at least one major band of the emission spectrum of each molecule also overlaps, 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.

Overlap between the emission spectrum of one molecule with the absorption spectrum of the other molecule (more than 40%) leads to the energy transfer mechanism disclosed in the Sharma & Wolfbeis article discussed previously. In that instance, luminescence from the donor molecule is absorbed by the acceptor molecule. Major absorption bands of one molecule do not overlap with any of the major absorption bands of the second molecule. The same is the case for the luminescence spectra. Those conditions are not needed and are undesirable in the present invention because it

EET will result in luminescence emission only from the acceptor molecule. In Sharma and Wolfbeis. the monitored luminescence will be that of the acceptor molecule only. In the present invention, 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. Also 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. As is known in the art, 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

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;

(3) fluoranthene in combination with benzo(b) fluoranthene or benzo(ghi)perylene; and

(4) ruthenium complexes of conjugated organic molecules, such as tris(2,2 '-bipyridine) ruthenium II dichloride and tris(l, 10-phenthroline) ruthenium II.

Of these molecule pairs, 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:

(1) anthracene, chrysene, pyrene;

(2) benzo(b)fluoranthane, benzo(e)pyrene;

ET (3) fluoranthane, benzo(b) fluoranthane; and

(4) triphenyl methane dyes.

Preferred indicators for determining chlorine include combinations chosen from the group of polycyclic aromatic hydrocarbons and their derivatives, and include:

(1) 2-amino anthracene, 9-methyl anthracene, 1- amino anthroquinone and fluoranthane;

(2) antracene, anthranilic acid and acradine;

(3) acradine, anthracene and 2-amino anthracene; (4) benzo(ghi)parylene, and 9,10- diphenylanthracene; and

(5) dichloroantracene and 9, 10-diphenylanthracene. Preferred indicators for determining the physical parameter, pH, include: (1) fluorescein and 3 (and 6)-carboxy fluorescein;

(2) 2 ' ,7 '-dichlorofluorescein and 5 (and 6)carboxy- 2 ' ,7 '-dichlorofluorescein;

(3) 2 ' ,7 '-dichlorofluorescein and 5(and 6)carboxy- 4 ' ,5'-dimethylfluorescein; (4) fluorescein and l-hydroxypyrene-3,6,8- trisulphonic acid (HPTS) ;

(5) l-hydroxypyrene-3,6,8-trisulphonic acid (HPTS) and carboxy-fluorescein; and

(6) other known pH indicators having the requisite coexcitation and co-emission wavelengths.

It will be appreciated that 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.

Referring to FIG. 1, 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 XAD4₇ 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.

Again, it is not necessary that the 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.

Referring again to FIG. 1, 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

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.

Another embodiment of the optical probe of the present invention is shown in FIG. 3. As discussed previously, 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. When a single fiber is used, it may be necessary to use 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. In this system, 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.

More than two luminescent molecules can also be used in the practice of the present invention. As was the case with the use of two molecules, 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. The use of 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:

(a) pyrene, chrysene, and anthracene,

(b) fluoranthene, benzo(b) fluoranthene, and benzo(ghi) fluoranthene, and

(c) perylene, perylene dibutyrate, and decacylene. The 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:

I l=l+K_SV [CA]

which relates luminescence intensity I to the concentration of the analyte [CA], where I₀ is the luminescence intensity in the absence of analyte and K_sv is the quenching constant.

Therefore, by measuring the combined luminescence intensity I of luminescent molecules 1 and 2 for known partial pressures of the analyte, 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. In this instance, it will be necessary to plot combined intensity versus analyte concentration for numerous known analyte concentrations to obtain a curve which can be used to determine the amount of a specific analyte in a sample based on the combined luminescence intensity I of luminescent molecules 1 and 2. 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.

In accordance with the method aspect of the present invention, 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. It is also preferred that 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. In the practice of the method of the present invention, where the luminescent molecules are embodied in a probe, 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. Where the luminescent molecules are not embodied in a probe, then 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 I₀, 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. In addition to the direct quenching of luminescence of the luminescent molecules, by analyte, 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. To achieve the analysis, two pH sensitive luminescent molecules are used as the indicator and when the analyte is brought in contact with them, quantitative changes in analyte concentration are then monitored by observing the changes in the luminescence of the indicator which results due to the change in pH of the aqueous environment surrounding the pH luminescent molecules. In accordance with another aspect of the present invention, 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 σreatanine, 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. In the indirect method, consumption or production of a species (other than the analyte itself) during the enzymatic reaction is monitored, and the change in the concentration of the species is indicative of the amount of analyte produced or consumed. Most commonly occurring enzyme reactions involve consumption or production of

NADH, oxygen, hydrogen peroxide, an acid (thus change in pH) , ammonia and a change in temperature during the reaction. Accordingly, 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, when coupled with an enzyme (coimmobilized or otherwise incorporated in the probe) may be used for constructing a bio-sensor and/or probe for a specific analyte. By way of illustration of an enzyme assisted method for the determination of a biomedical analyte, the present invention may be used for the determination of

TUTE SHEET glucose. The enzyme reaction involving glucose oxidase is as follows:

glucose oxidase glucose + 0₂ > gluconolactone + H₂0₂

By suitable selection of the luminescent molecules in accordance with the present invention, several glucose sensors and methods for the determination of glucose can be used.

For example, 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. Thus 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.

As with the oxygen probe, 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. Again, as in the above examples, 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. For example, 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.

Table I

Biomedical Analytes Enzyme lactose glucose oxidase p-galactosidase sucrose invertase and pyranose oxidase

1-lactic acid lactate oxidase pyruvic acid pyrarate oxidase ascorbic acid ascorbate oxidase alcohol alcohol oxidase glycerol glycerol kinase and l-«(- glycerophosphate oxidase)

1-amino acids 1-amino acid oxidase

1-lysine lysine oxidase 1-glutamic acid 1-glutamate oxidase glutamate glutamate oxidase NADH NADH oxidase

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.

SUBSTITUTE SHEET Similarly, enzyme sensors/probes based on pH, hydrogen peroxide or temperature measurement can be constructed.

The following example is illustrative of a probe constructed in accordance with the present invention.

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.

Table II excitation fluorescence wavelength fnm) wavelength fnm) perylene dibutyrate

^'420 shoulder ^'485 major band '440 shoulder ^'510 major band '445 shoulder ^'560 shoulder ^'470 major band '485 shoulder

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 πiM 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 μm) , 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.

The results are shown in FIG. 4. As can be seen, the relationship between I and K_sv for perylene dibutyrate and decacyclene is linear, with a K_sv of 0.034%^-1 for the two molecules. Thus, use of the combination of molecules is more quenching efficient, and therefore more sensitive to small changes in oxygen concentration, than use of either molecule alone, since the K_sv for decacyclene is 0.006%^-1 and the K_sv for perylene dibutyrate is 0.02%^-1.

Therefore, use of these two molecules in combination provides for more efficient quenching than the sum of the two indicator molecules. The result is a probe that is highly sensitive to minute changes in oxygen concentration, which is thereby capable of extremely accurate measurements.

Claims

I CLAIM!

1. An optical probe for measuring the concentration of an analyte in a sample comprising an indicator matrix, said indicator matrix comprising at least two different luminescent molecules, each of whose luminescence is quenched by said analyte, each of said luminescent molecules having at least one major band in its absorption spectrum that overlaps with at least one major band in the absorption spectrum of each of the other luminescent molecules, and each of said luminescent molecules having at least one major band in its emission spectrum that overlaps with at least one major band of the emission spectrum of each of the other luminescent molecules so that all the luminescent molecules may be coexcited at a common excitation wavelength and so that the emission luminescence can be monitored at a common wavelength.

2. The optical probe of claim 1 wherein said luminescent molecules are phosphorescent molecules.

3. The optical probe of claim 1 wherein said luminescent molecules are fluorescent molecules.

4. The optical probe of claim 1 wherein said analyte is oxygen.

5. An optical probe for measuring the concentration of an analyte in a sample comprising: (a) an indicator matrix, said indicator matrix comprising at least two different luminescent molecules, each of whose luminescence is quenched by said analyte, each of said luminescent molecules having at least one major band in its absorption spectrum that overlaps with at least one major band in the absorption spectrum of each of the other luminescent molecules, and each of said luminescent molecules having at least one major band in

^T its emission spectrum that overlaps with at least one major band of the emission spectrum of each of the other luminescent molecules so that all the luminescent molecules may be coexcited at a common excitation wavelength and so that the emission luminescence can be monitored at a common wavelength; and

(b) a member permeable by said analyte enclosing said luminescent molecules.

6. The optical probe of claim 5 wherein said luminescent molecules are phosphorescent molecules.

7. The optical probe of claim 5 wherein said luminescent molecules are fluorescent molecules.

8. The optical probe of claim 5 wherein said analyte is oxygen.

9. An optical probe for measuring the concentration of an analyte in a sample comprising:

(a) an indicator matrix, said indicator matrix comprising at least two different luminescent molecules, each of whose luminescence is quenched by said analyte, each of said luminescent molecules having at least one major band of its absorption spectrum that overlaps with at least one major band in the absorption spectrum of each of the other luminescent molecules, and each of said luminescent molecules having at least one major band in its emission spectrum that overlaps with at least one major band of the emission spectrum of each of the other luminescent molecules so that all the luminescent molecules may be coexcited at a common excitation wavelength and so that the emission luminescence can be monitored at a common wavelength; and (b) support means for carrying said luminescent molecules, said luminescent molecules being immobilized on said support means.

10. The optical probe of claim 9 wherein said luminescent molecules are phosphorescent molecules.

11. The optical probe of claim 9 wherein said luminescent molecules are fluorescent molecules.

12. The optical probe of claim 9 wherein said analyte is oxygen.

13. The optical probe of claim 9 wherein said support means is a polymeric material.

14. An optical probe for measuring the concentration of an analyte in a sample comprising: (a) an indicator matrix, said indicator matrix comprising at least two different luminescent molecules, each of whose luminescence is quenched by said analyte, each of said luminescent molecules having at least one major band in its absorption spectrum that overlaps with at least one major band in the absorption spectrum of each of the other said luminescent molecules, and each of said luminescent molecules having at least one major band in its emission spectrum that overlaps with at least one major band of the emission spectrum of each of the other luminescent molecules so that all the luminescent molecules may be coexcited at a common excitation wavelength and so that the emission luminescence can be monitored at a common wavelength;

(b) support means for carrying said luminescent molecules, said luminescent molecules being immobilized on said support means, and

(c) a member permeable by said analyte enclosing said luminescent molecules and said support means.

15. The optical probe of claim 14 wherein said luminescent molecules are phosphorescent molecules.

16. The optical probe of claim 14 wherein said luminescent molecules are fluorescent molecules.

17. The optical probe of claim 14 wherein said analyte is oxygen.

18. The optical probe of claim 17 wherein at least one of said molecules is a conjugated organic molecule.

19. The optical probe of claim 17 wherein at least one of said luminescent molecules is a substituted or unsubstituted polycyclic aromatic hydrocarbon.

20. The optical probe of claim 17 wherein at least one of said molecules is a metal complex of porphyrines.

21. The optical probe of claim 14 wherein an optically transparent plate is attached to said support means.

22. The optical probe of claim 14 wherein said support means is a polymeric material.

23. The optical probe of claim 14 wherein said support means includes an amount of one of said molecules sufficient to supply a measurable emission spectrum and said support means carrying said one molecule being enclosed by a member impervious to said analyte.

24. An optical probe for measuring the concentration of an analyte in a sample comprising:

(a) an indicator matrix, said indicator matrix comprising at least two different luminescent molecules, each of whose luminescence is quenched by said analyte, each of said luminescent molecules having at least one major band in its absorption spectrum that overlaps with at least one major band in the absorption spectrum of each of the other said luminescent molecules, and each of said luminescent molecules having at least one major band in its emission spectrum that overlaps with at least one major band of the emission spectrum of each of the other luminescent molecules so that all the luminescent molecules may be coexcited at a common excitation wavelength and so that the emission luminescence can be monitored at a common wavelength;

(b) support means for carrying said luminescent molecules, said luminescent molecules being immobilized on said support means;

(c) a member permeable by said analyte enclosing said luminescent molecules and said support means; and (d) means to pass excitation radiation to said luminescent molecules and collect luminescence emitted from said luminescent molecules.

25. The optical probe of claim 24 wherein said luminescent molecules are phosphorescent molecules.

26. The optical probe of claim 24 wherein said luminescent molecules are fluorescent molecules.

27. The optical probe of claim 24 wherein said means to pass excitation radiation to said luminescent molecules and collect luminescence emitted from said luminescent molecules are fiber optic means.

28. The optical probe of claim 24 wherein said analyte is oxygen.

29. The optical probe of claim 24 wherein said support means is a polymeric material.

30. The optical probe of claim 24 wherein said support means includes an amount of one of said molecules

HEET sufficient to supply a measurable emission spectrum and said support means carrying said one molecule being enclosed by a member impervious to said analyte.

31. A method for the detection of an analyte or a physical parameter in gaseous, liquid or solid media comprising contacting said medium with at least two different luminescent molecules, said luminescent molecules being both capable of absorbing radiation at an overlapping wavelength of excitation and emitting luminescence at an overlapping wavelength so that said molecules are coexcited and their emission is comonitored, said emission luminescence from each of the said molecules being quenchable by the analyte or physical parameter of interest, exciting the luminescent molecules with radiation, measuring the emission luminescence of said luminescent molecules at a common emission luminescence wavelength and relating the emission luminescence to a standard to determine the presence of the analyte or parameter in said medium.

32. The method of claim 31 wherein said luminescent molecules are phosphorescent molecules.

33. The method of claim 31 wherein said luminescent molecules are fluorescent molecules.

34. The method of claim 31 wherein said analyte is oxygen.

35. The method of claim 34 wherein at least one of said luminescent molecules is a conjugated organic molecule.

36. The method of claim 34 wherein at least one of said molecules is a substituted or unsubstituted polycyclic aromatic hydrocarbon.

37. The method of claim 34 wherein at least one of said molecules is a metal complex of porphyrines.

38. The method of claim 31 wherein said analyte is sulfur dioxide.

39. The method of claim 38 wherein said luminescent molecules are selected from the group consisting of polycyclic aromatic hydrocarbons and their alkoxy, hydroxy, alkyl, keto and nitro derivatives.

40. The method of claim 39 wherein said luminescent molecules are selected from the group consisting of anthracene, chrysene, pyrene; benzo(b)fluoranthane, benzo(e)pyrene; fluoranthane, benzo(b)fluoranthane; and triphenyl methane dyes.

41. The method of claim 31 wherein said analyte is chlorine.

42. The method of claim 41 wherein said indicator comprises luminescent molecules selected from the group consisting of substituted and unsubstituted polycyclic aromatic hydrocarbons.

43. The method of claim 42 wherein said luminescent molecules are selected from the group consisting of 2- amino anthracene, 9-methyl anthracene, 1-amino anthroquinone and fluoranthane; antracene, anthranilic acid and acradine; acradine, anthracine and 2-amino anthracene; benzo(ghi)parylene, and 9,10- diphenylanthracene; and dichloroantracene and 9,10- diphenylanthracene.

44. The method of claim 31 wherein said physical parameter is pH.

45. The method of claim 44 wherein said luminescent molecules are selected from the group consisting of fluorescein and 3(and 6)-carboxy fluorescein; 2' ,7'- dichlorofluorescein and 5(and 6)carboxy-2' ,7'- dichlorofluorescein; and 2' ,7'-dichlorofluorescein and 5(and 6)carboxy-4' ,5'-dimethylfluorescein, and 1- hydroxypyrene-trisulphonic acid.

46. The method of claim 31 wherein the determination of the analyte or parameter in said medium is quantitative.

47. The method of claim 31 wherein said analyte is an acidic gas, and said analyte is determined indirectly by measuring the luminescence quenching of the luminescent molecules in response to a change in the pH of the sample.

48. The method of claim 31 wherein said analyte is a basic gas and said analyte is determined indirectly by measuring the luminescence quenching of the luminescent molecules in response to a change in the pH of the sample.

49. The method of claim 31 wherein said analyte is a biomedical analyte.

50. The method of claim 49 wherein said biomedical analyte is subjected to an enzymatic reaction in the presence of an enzyme and said biomedical analyte is determined by measuring the luminescence quenching of the luminescent molecules in response to a change in the oxygen content of said sample.

51. The method of claim 49 wherein said biomedical analyte is subjected to an enzymatic reaction in the presence of an enzyme and said biomedical analyte is determined by measuring the luminescence quenching of the luminescent molecules in response to a change in the pH of the sample.

52. The method of claim 50 wherein said biomedical analyte is a member selected from the group consisting of lactose, sucrose, 1-lactic acid, pyruvic acid, ascorbic acid, alcohol, glycerol, 1-amino acids, 1-lysine, 1- glutamic acid, glutamate and NADH, and corresponding enzyme for the analyte is a member selected from the group consisting of glucose oxidase, p-galactosidase, invertase, pyranose oxidase, lactate oxidase, pyrarate oxidase, ascorbate oxidase, alcohol oxidase, glycerol kinase, l-«(-glycerolphosphate) oxidase, l-amino acid oxidase, lysine oxidase, 1-glutamate oxidase, glutamate oxidase, and NADH oxidase.

53. The method of claim 51 wherein said biomedical analyte is a member selected from the group consisting of lactose, sucrose, 1-lactic acid, pyruvic acid, ascorbic acid, alcohol, glycerol, l-amino acids, 1-lysine, 1- glutamic acid, glutamate and NADH, and the corresponding enzyme for the analyte is a member selected from the group consisting of glucose oxidase, p-galactosidase, invertase, pyranose oxidase, lactate oxidase, pyrarate oxidase, ascorbate oxidase, alcohol oxidase, glycerol kinase, l-«(-glycerolphosphate oxidase, l-amino acid oxidase, lysine oxidase, 1-glutamate oxidase, glutamate oxidase, and NADH oxidase.

54. The method of claim 49 wherein said biomedical analyte is subjected to an enzymatic reaction in the presence of an enzyme and said biomedical analyte is determined by measuring the luminescence quenching of the luminescent molecules in response to a change in the hydrogen peroxide content of said sample.

TE SHEET

55. The method of claim 49 wherein said biomedical analyte is subjected to an enzymatic reaction in the presence of an enzyme and said biomedical analyte is determined by measuring the luminescence quenching of the luminescent molecules in response to a change in the temperature of said sample.

56. The method of claim 49 wherein said luminescent molecules and an enzyme for the biomedical analyte are immobilized on a support.

57. The method of claim 56 wherein said biomedical analyte is glucose, said enzyme is glucose oxidase and said biomedical analyte is determined by measuring the luminescence quenching of the luminescent molecules in response to a change in the oxygen content of the sample.

58. The method of claim 56 wherein said biomedical analyte is glucose, said enzyme is glucose oxidase and said biomedical analyte is determined by measuring the luminescence quenching of the luminescent molecules in response to a change in the pH content of the sample.

59. The method of claim 49 wherein an enzyme for the biomedical analyte contacts said sample, said enzyme being modified to include both luminescent molecules as an intregal part of said enzyme.