WO1999047918A1 - Detection, basee sur la duree de vie, du sodium et du potassium - Google Patents

Detection, basee sur la duree de vie, du sodium et du potassium Download PDF

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Publication number
WO1999047918A1
WO1999047918A1 PCT/US1999/005980 US9905980W WO9947918A1 WO 1999047918 A1 WO1999047918 A1 WO 1999047918A1 US 9905980 W US9905980 W US 9905980W WO 9947918 A1 WO9947918 A1 WO 9947918A1
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WIPO (PCT)
Prior art keywords
sample
probe
concentration
lifetime
sodium
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Application number
PCT/US1999/005980
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English (en)
Inventor
Joseph R. Lakowicz
Henryk Szmacinski
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University Of Maryland, Baltimore
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Publication date
Application filed by University Of Maryland, Baltimore filed Critical University Of Maryland, Baltimore
Priority to EP99912686A priority Critical patent/EP1073897A4/fr
Priority to CA002325140A priority patent/CA2325140A1/fr
Publication of WO1999047918A1 publication Critical patent/WO1999047918A1/fr
Priority to US09/666,721 priority patent/US6472221B1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • 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
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—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 the field of optically measuring sodium or potassium concentrations in solutions.
  • Measurement of electrolytes in the blood is an important aspect of clinical chemistry.
  • the most common techniques used for measuring electrolytes in aqueous environments are flame photometry or ion selective electrodes (ISE) .
  • ISE ion selective electrodes
  • Both flame photometry and ISE are highly evolved technologies which provide good precision and accuracy over a wide range of concentrations. These methods require good operator skills and meticulous maintenance of equipment for optimal performance. Additionally, these methods require the handling of blood, which is expensive and associated with significant health risks to the operator.
  • Fluorescence assays have been shown to be approximately three orders of magnitude more sensitive than absorption methods and the fluorescence assays also permit analysis using smaller amounts of probe in the assay solution. Moreover, in contrast to absorption methods, fluorescence probes do not require additional chemical reagents and complex sample manipulation. 2
  • a method in which a luminescent ligand is added to a sample to be analyzed in the form of a photoluminescent probe having intrinsic analyte-induced lifetime changes is known in the art. Lifetime measurements are advantageous over intensity measurements because they can be performed in optically dense samples or turbid media and are independent of and/or insensitive to photo bleaching, probe wash out or optical loss. The lifetime changes are measured using known time-resolved or phase-modulation fluorometry methods. A description of the phase modulation fluorometry methods are found in U.S. Patent No. 5,624,847 ('847 patent) which is incorporated by reference herein in its entirety.
  • the step of adding a luminescent ligand (i.e., probe) to the sample to be analyzed requires matching a particular probe to a particular analyte, so that at least a portion of the sample will be non- covalently bound to the probe resulting in both bound and unbound species of the probe.
  • a luminescent ligand i.e., probe
  • the present invention provides a method of optically measuring Na + or K + in a sample such as blood, containing concentrations of up to 6.5 mM of K + and of up to 160 mM of Na + .
  • a photoluminescent ligand probe having intrinsic sodium- or potassium-induced lifetime changes is added to the sample to be analyzed.
  • the probe is non-covalently bound to the 4 ionic solute of either sodium or potassium to form a Na + -bound or K + -bound probe species wherein bound and unbound species of the probe exist in the sample and the probe has intrinsic Na + - induced or K + -induced lifetime changes.
  • the sample is excited with radiation and the resulting emission beams from the bound and unbound species are detected.
  • the apparent luminescence lifetime of the emission is calculated to determine concentration of either Na + or K + in the sample. Because of the similar chemical properties of Na + and K + , probes are utilized such that the presence of high levels of Na + allows the measurement of K + and the presence of K + allows the measurements of Na + .
  • Figure 1 chemical structures of SBFO and CD 222.
  • Figure 2 absorption and emission spectra of SBFO.
  • Figure 3 sodium-dependent fluorescence intensity of SBFO at various excitation wavelengths (top) and excitation intensity ratios (for excitation wavelengths see Fig. 2) . Dashed vertical lines illustrate critical concentrations of sodium in the blood.
  • Figure 4 frequency-domain intensity decays of SBFO.
  • Figure 5 sodium-dependant phase angles (top) and modulations (bottom) of SBFO at several excitation wavelengths.
  • FIG 8 frequency-domain intensity decays of CD222 for various K + concentrations. Dashed lines represent intensity decay at the presence of 100 mM of sodium. Excitation wavelength was 380 nM and the emission above 440 nM. Figure 9, cation-dependent phase angles (top) and modulations (bottom) of CD222 for various solution compositions .
  • Cation-dependent intensities, phase angles and modulations for SBFO and CD 222 were measured at several excitation wavelengths because both probes display shifts in their absorption spectra upon cation binding.
  • the samples were freshly prepared before measurements in the buffers, 5 mM Hepes (pH 7.2) for SBFO, and 30 mM Tris (pH 7.25) for CD 222.
  • the ionic strength of the samples started from 100 mM.
  • the buffers contained 100 mM of the tetramethylammonium chloride, TMA(Cl). The measurements were carried out at room temperature of 22°C.
  • the frequency-domain data were used to determine the intensity decay law using the multi-exponential model (J.R. Lakowicz, E. Gratton, G. Laczko, H. Cherek, and M. Limkeman, Analysis of Fluorescence Decay Kinetics from Variable- Frequency Phase Shift and Modulation Data, Biophys . J. 46, 463-477, 1984; E. Gratton, J.R. Lakowicz, B. Maliwal, H. Cherek, G. Laczko and M. Limkeman, Resolution of Mixtures of Fluorophores Using Variable-Frequency Phase and Modulation Data, Biophys . J. 46, 479-486, 1984)
  • ⁇ 2 are the preexponential factors (amplitudes)
  • ⁇ 2 are the decay times
  • n the number of exponential components.
  • the intensity decays of CD 222 were also fitted to a global model in which the decay times were assumed to be independent 7 of the Na + or K + concentration, but the amplitudes of decay times variable to reflect changes in the fractional amounts of each species for various concentrations of Na + or K + .
  • Such analyses have been described previously for calcium (J.R. Lakowicz, H. Szmacinski, and M.L. Johnson, Calcium Imaging Using Fluorescence Lifetimes and Long-Wavelength Probes. J. Fluoresc. 2, 47-62, 1992; H. Szmacinski and J.R. Lakowicz, Possibility of simultaneous measuring low and high calcium concentration using Fura-2 and lifetime-based method. Cell Calcium 18, 64-75, 1995) and magnesium probes (H.
  • [M + ] is the ion concentration and X indicates the measured (or calculated) ion-dependent parameter.
  • the apparent dissociation constant is the parameter linking the fluorescence observable to the ion concentrations. It should be noted that only the ion-dependent intensities yield a true dissociation constant reflecting the equilibrium between free and ion-bound forms of probe.
  • the dissociation constants calculated from the ion-dependent time-resolved data phase angle, modulation or mean decay time
  • the apparent dissociation . constant is the more important parameter because this value defines the useful range of ion concentrations which can be measured using a particular spectral observable.
  • the concentration range over which a probe produces an observable response with a chosen parameter is approximately from 0.1K D X to 10K D X .
  • SBFO sodium binding benzofuran oxazole
  • SBFO was used for lifetime-based sensing of sodium in blood.
  • the lifetime of SBFO was determined from the frequency-domain data Figure 4.
  • the mean lifetime increased from 1.50 ns for the sodium-free form to 2.16 ns for the sodium bound form.
  • the mean lifetime of SBFO was 1.85 ns in the presence of 500 mM of potassium.
  • Table 1 shows the intensity decays of sodium and potassium probes .
  • Table I Intensity Decays of Sodium and Potassium Probes
  • CD 222 free 0.04 0.15 0.82 0.424 0.481 0.1 0. .17
  • Sodium concentrations can be determined from the phase and modulation values measured at a single light modulation 10 frequency ( Figure 5) .
  • the Na + -sensitive range can be adjusted by the choice of the excitation wavelength.
  • the apparent dissociation constants calculated from the phase angle using equation (4), where the X is replaced by the phase angle are 18.5 mM (345 nm) , 81 mM (380 nm) , and 154 mM (400 nm) .
  • the Na + -sensitive range using modulation is shifted toward lower Na + concentrations ( Figure 5, bottom) , which is usually observed with phase-modulation sensing (H. Szmacinski and J.R.
  • phase angle and modulation require measurements of phase angle and modulation with an accuracy of 0.06 degrees and 0.09% (at 380 nm excitation). Such accuracy may be achievable with a dedicated single modulation frequency instrument using present optoelectronic technology. For instance, commercially available phase-modulation instruments with a wide range of modulation frequencies provide measurements with an accuracy of 0.1-0.2 degrees and 0.3 -0.5% for phase angle and modulation, respectively.
  • CD 222 Absorption and emission spectra of CD 222 are shown in Figure 6. This probe can be excited at longer wavelengths than PBFI and displays a much larger shift in its absorption spectrum than PBFI.
  • the absorption spectrum displays a 30 nm blue shift upon binding either of K + or Na + (dashed line) with a decreasing extinction coefficient above 350 nm.
  • the emission spectra show a minor blue shift on cation binding.
  • the quantum yield of CD 222 fluorescence increases 3.7-fold 11 for the K + -bound form and only 1.4-fold for the Na + -bound form.
  • the K D of CD 222 for K + determined from the intensity is 0.8 mM in the absence of Na + . This value is in close agreement with reported values of 1.0 (R. Crossley, Z.
  • Figure 7 shows the K + -dependent intensities of CD 222 at various excitation wavelengths in the presence of 135 mM Na + .
  • This concentration of Na + was used to mimic that found in whole blood.
  • phase and modulation sensing for measuring K + is shown in Figure 9, where potassium dependent changes in phase and lifetime are observed even in the presence of 135 mM of sodium.
  • Data at the modulation frequency of 286 MHZ was used to determine the K + -dependent phase angles and modulations at presence of 135 mM of Na + .
  • the choice of modulation frequency of 286 MHZ is of course arbitrary. A frequency higher than 500 MHZ could be chosen, but this may require a faster detector than standard photomultiplier tube (PMT) , such as a microchannel plate PMT which is an expensive device, or a photodiode which is typically less sensitive.
  • PMT standard photomultiplier tube
  • the apparent dissociation constants from the phase angles are 0.54 mM for K + and 1.74 mM for Na + , and 0.35 mM for K + and 1.3 mM for Na + from the modulation data. More important are changes in the phase angle and modulation in the presence of both the cations. These changes are 32.4 degrees in phase and 29.8 % in modulation for K + binding, and only of 3.4 degrees and 4.9 % for Na + binding to the CD 222 at 286 MHz.
  • the K + induced changes in the phase angle and modulation are excellent for measurements of K + , particularly at frequencies higher than 200 MHz.
  • the dynamic range for K + is somewhat decreased by the presence of sodium, but the range of phase and modulation values is still adequate for lifetime-based sensing of K + .
  • phase and modulation data indicate that Na + binding does not cause a significant change in the lifetime of CD 222. This is an important observation because almost the entire dynamic range in the phase and modulation remains available for K + sensing. However, the presence of sodium in the solution has a large impact on binding of K + . The K + -sensitive range is dramatically shifted toward higher K + concentration in the presence of 135 mM Na + , resulting in apparent dissociation constants for K + of 34.2 mM and 15.5 mM from phase and modulation, respectively. This means that the apparent binding affinity for K + decreased 63-fold from phase angle and 44-fold from modulation. In spite of decrease of K + affinity, the phase angle and modulation data for CD 222 are promising parameters for K + sensing in the blood.
  • Measurements with an accuracy of 0.12 degree in phase and 0.2 % in modulation are sufficient to fulfill the required tolerance of 0.2 mM in the range from 2.5 to 6.5 mM of K + concentration.
  • accuracy for 14 phase and modulation measurements can be obtained with commercial frequency-domain instruments. The accuracy can be improved if excitation wavelengths shorter than 380 nm are used.

Abstract

L'invention porte sur un système et un procédé de mesure optique du Na+ et de K+ d'un échantillon, par exemple de sang, en contenant de fortes concentrations jusqu'à 160 mM de Na+ et jusqu'à 6,5 mM de K+, en recourant à une sonde photoluminescente présentant des variations intrinsèques de durée de vie sous l'effet de l'analyte. L'invention porte spécifiquement sur l'utilisation de la détection, basée sur la durée de vie, du Na+ et du K+ présents à des concentrations extracellulaire dans du sang entier ou dans du sérum sanguin. L'exécution préférée recourt à la fluorométrie à modulation de phase.
PCT/US1999/005980 1998-03-20 1999-03-19 Detection, basee sur la duree de vie, du sodium et du potassium WO1999047918A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99912686A EP1073897A4 (fr) 1998-03-20 1999-03-19 Detection, basee sur la duree de vie, du sodium et du potassium
CA002325140A CA2325140A1 (fr) 1998-03-20 1999-03-19 Detection, basee sur la duree de vie, du sodium et du potassium
US09/666,721 US6472221B1 (en) 1998-12-09 2000-09-20 Lifetime-based sensing of sodium and potassium

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7885698P 1998-03-20 1998-03-20
US60/078,856 1998-03-20
US11155498P 1998-12-09 1998-12-09
US60/111,554 1998-12-09

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US09/666,721 Continuation US6472221B1 (en) 1998-12-09 2000-09-20 Lifetime-based sensing of sodium and potassium

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020725A1 (fr) * 2001-08-31 2003-03-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Composes coumariniques

Citations (3)

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Publication number Priority date Publication date Assignee Title
US5134232A (en) * 1988-11-14 1992-07-28 The Regents Of The University Of California Fluorescent indicator dyes for alkali metal cations
US5624847A (en) * 1991-05-03 1997-04-29 Joseph R. Lakowicz Method for optically measuring chemical analytes
WO1998016656A1 (fr) * 1996-10-11 1998-04-23 Lakowicz Joseph R Mesure a deux photons ou multiphotonique d'analytes contenus dans des tissus et des fluides animaux et humains

Patent Citations (4)

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US5134232A (en) * 1988-11-14 1992-07-28 The Regents Of The University Of California Fluorescent indicator dyes for alkali metal cations
US5624847A (en) * 1991-05-03 1997-04-29 Joseph R. Lakowicz Method for optically measuring chemical analytes
WO1998016656A1 (fr) * 1996-10-11 1998-04-23 Lakowicz Joseph R Mesure a deux photons ou multiphotonique d'analytes contenus dans des tissus et des fluides animaux et humains
US5759767A (en) * 1996-10-11 1998-06-02 Joseph R. Lakowicz Two-photon and multi-photon measurement of analytes in animal and human tissues and fluids

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Title
AMORINO G P, FOX M H: "INTRACELLULAR NA+ MEASUREMENTS USING SODIUM GREEN TETRAACETATE WITHFLOW CYTOMETRY", CYTOMETRY, ALAN LISS, NEW YORK., US, vol. 21, 1 January 1995 (1995-01-01), US, pages 248 - 256, XP002918871, ISSN: 0196-4763, DOI: 10.1002/cyto.990210305 *
CROSSLEY R, GOOLAMALI Z, SAMMES P G: "SYNTHESIS AND PROPERTIES OF A POTENTIAL EXTRACELLULAR FLUORESCENT PROBE FOR POTASSIUM", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 2., CHEMICAL SOCIETY. LETCHWORTH., GB, 1 January 1994 (1994-01-01), GB, pages 1615 - 1623, XP002918868, ISSN: 1472-779X, DOI: 10.1039/p29940001615 *
HAROOTUNIAN A T, ET AL.: "FLUORESCENCE RATIO IMAGING OF CYTOSOLIC FREE NA+ IN INDIVIDUAL FIBROBLASTS AND LYMPHOCYTES", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 264, no. 32, 15 November 1989 (1989-11-15), US, pages 19458 - 19467, XP002918870, ISSN: 0021-9258 *
LAKOWICZ J R, SZMACINSKI H, BERNDT K W: "FLUORESCENCE LIFETIME-BASED SENSING OF BLOOD GASES AND CATIONS", OPTOMECHATRONIC MICRO/NANO DEVICES AND COMPONENTS III : 8 - 10 OCTOBER 2007, LAUSANNE, SWITZERLAND, SPIE, BELLINGHAM, WASH., vol. 1648, 1 January 1992 (1992-01-01), Bellingham, Wash., pages 50 - 63, XP002918867, ISBN: 978-1-62841-730-2, DOI: 10.1117/12.58296 *
MINTA A, TSIEN R Y: "FLUORESCENT INDICATORS FOR CYTOSOLIC SODIUM", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 264, no. 32, 15 November 1989 (1989-11-15), US, pages 19449 - 19457, XP002918869, ISSN: 0021-9258 *
SATOH H, ET AL.: "QUANTIFICATION OF INTRACELLULAR FREE SODIUM IONS BY USING A NEW FLUORESCENT INDICATOR, SODIUM-BINDING BENZOFURAN ISOPHTHALATE IN GUINEA PIG MYOCYTES", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 175, no. 02, 15 March 1991 (1991-03-15), US, pages 611 - 616, XP002918872, ISSN: 0006-291X, DOI: 10.1016/0006-291X(91)91609-G *
See also references of EP1073897A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020725A1 (fr) * 2001-08-31 2003-03-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Composes coumariniques

Also Published As

Publication number Publication date
CA2325140A1 (fr) 1999-09-23
EP1073897A1 (fr) 2001-02-07
EP1073897A4 (fr) 2006-10-25

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