WO2000000819A1 - Procede et dispositif d'analyse - Google Patents

Procede et dispositif d'analyse Download PDF

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Publication number
WO2000000819A1
WO2000000819A1 PCT/US1999/014709 US9914709W WO0000819A1 WO 2000000819 A1 WO2000000819 A1 WO 2000000819A1 US 9914709 W US9914709 W US 9914709W WO 0000819 A1 WO0000819 A1 WO 0000819A1
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WO
WIPO (PCT)
Prior art keywords
donor
acceptor
modulation
analyte
sample
Prior art date
Application number
PCT/US1999/014709
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English (en)
Inventor
Henry Szmacinski
Qing Chang
Original Assignee
Fluorrx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fluorrx, Inc. filed Critical Fluorrx, Inc.
Priority to AU49628/99A priority Critical patent/AU4962899A/en
Publication of WO2000000819A1 publication Critical patent/WO2000000819A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—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
    • 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
    • G01N21/78—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 producing a change of colour
    • G01N21/80—Indicating pH value

Definitions

  • This invention relates generally to the Held of assays and more particularly to fluorescence intensity assays.
  • Optical measurements of pH are of wide interest in analytical and clinical chemistry. At present, optical sensing of pH is based on measurements of steady-state fluorescence intensity, as determined by the absorptive and emission properties of a sample. Time-resolved fluorescence has also been demonstrated for pll sensing using a number of lifetime pll sensitive probes. However, most lifetime-pH sensitive probes display relatively short lifetime in the range of several nanoseconds which makes their use difficult in practical inexpensive pH sensing. Though there are several reports about long lifetime luminescent inorganic complexes that show pH-sensitive luminescence characteristics, these Ru(II) based pH- sensitive complexes show pH-dependent emissions, mostly in the low pH range from 0 to 6.
  • MCL metal ligand complex
  • FRET fluorescence energy transfer
  • the sensors contain a fluorescence donor and a pH-sensitive acceptor.
  • the fluorescence donor can be selected for its absorption, emission, quantum yield, and decay time characteristics without concern for its sensitivity to pll.
  • the acceptor can be fluorescent or non fluorescent, but must display absorption spectral change in response to pll in the wavelength range of the donor emission, and be photostable.
  • the donor and acceptor molecules can be covalent ly linked or simply mixed together.
  • the rate of the FRET depends on the integral overlap of the donor emission with the acceptor pH-dependent absorption, the distance between donor and acceptor and the relative orientation of the donor and acceptor transition dipoles.
  • Metal-ligand probes display attractive spectral properties: are excitable with LED and diode lasers, exhibit large Stokes shift ( ⁇ 200 nm), and have acceptable quantum yields.
  • the most attractive property of MLC probes is long luminescence lifetime in the order of several hundreds nanosecond to microseconds. The long lifetime allows of using low modulation frequency in phase-modulation technique. The low modulation frequency in the range of several hundred kHz is desirable in terms of the cost of practical device for sensing.
  • the long lifetime of donor allows for significant molecular diffusion of donor and acceptor molecules which enhances the FRET. This may results in using lower acceptor concentrations than calculated based on F ⁇ rster theory. Therefore, the FRET based pH sensors with MLC as donors may require lower acceptor concentrations compared to that with a nanosecond lifetime donors. It is known that for sufficient energy transfer (more than 0.70). the acceptor concentrations need to be in the range of 3 -50 mM for donor-acceptor system with a F ⁇ rster distance Ro from about 50 to 20 I, respectively. Such acceptor concentrations results in high optical densities at the excitation and emission wavelengths making intensity measurements difficult to use in a quantitative manner.
  • the present invention provides a FRET arrangement for sensing the concentration of protons supported by the acceptor.
  • Energy is generated by subjecting a donor molecule comprising a fluorescent MLC to exciting radiation.
  • the resulting energy will result in detectable fluorescence that may be diminished if acceptor molecules in the vicinity of the energy-producing MLC have a modified hydrogen ion status sufficient to cause them to cause them to accept more energy.
  • Changes in the fluorescence produced are detected and correlated with the presence or amount of the analyte responsible for the change in the pH or hydrogen ion environment of the acceptor.
  • MLC's such as Ru(II) complexes are useful as a donors and pH-sensitive indicators such as phthaleins are useful as acceptors.
  • the donors and acceptors may he held in polymeric supports.
  • Induced changes in H + within sensing element can be transduced via energy transfer to measure an analyte concentration in the sample. This can be usually achieved by using a proper polymers which are designed for specific analytes.
  • FIG. 1 shows absorption spectra of pH indicator Cholophenol Red in pH 4.16 ( ) and pH 8.35
  • FIG. 2 shows Intensity decays of [Ru((dpp)(SO3Na 2 ) 3 ] + in the presence of pH indicator. CP at its several concentrations in EC films at pH 8.35. Intensity decays are represented as a frequency- dependent phase angles and modulations.
  • FIG. 3 shows pH-dependent intensity decays of [Ru((dpp)(SO 3 Na 2 ) 3 ] 2+ in the presence of 8.8 mM of CPR in EC films. Intensity decays are represented as a frequency-dependent phase angles and modulations.
  • FIG 4 shows pH calibration curves for FRET-based sensor [Ru((dpp)(SO 3 Na 2 ) 3 ] 2+ -CPR using time- resoh/ed data, phase angles and modulations at frequency of 363 kHz.
  • FIG 5 shows pH calibration curve for FRET-based sensor with two pH indicators as acceptors [Ru((dpp)(SO 3 Na 2 )3] 2+ -(CPR+TBPSP). Arrows indicate the ranges where CPR and TBPSP are sensitive to the pH.
  • FIG 7 shows Effect of polymeric support on performance of [Ru((dpp)(SO 3 Na 2 ) 3 ] 2+ -TBPSP FRET pH sensor. PH-dependent phase angles were measured at frequency of 300 kHz. Dashed line shows the pH dependent absorbance of TBPSP in buffer.
  • (Ru[dpp)(S ⁇ 3Na2)]3 Q was used as a donor.
  • the synthesis and spectral characteristics of (Ru[dpp)(S ⁇ 3Na)2J3 Ck has been recently described.
  • the FRET acceptors were chosen from the pH-sensitive phthaleins like m-cresol purple (mCP), bromothymol blue (BTB), chlorophenol red (CPR), 3,4,5,6-tetrabromophenolsulfophthalein CTBPSP), and phenolphthalein (PP) to cover wide range of pH values from 1 to 10 were purchased from Aldrich (Milwaukee, WI). The pH indicators were used without further purification.
  • ethyl cellulose EC
  • TEOS tetraethyl orthosilicate
  • PPMA poly(2-hydroxypropyl methacrylate
  • TBP tributyl phosphate TBP
  • TOABr tetraoctylammonium bromide
  • final coating solution contained 70 mM of TOABr.
  • Sensor films were made by applying 4-5 ⁇ l of the given coating solution onto the surface of glass support, and let it be dried at ambient atmosphere for about 10 minutes.
  • the precursor solution was then sonicated at room temperature for 2 h, and kept at about 4° C for 12 h.
  • the sensor film was then made from the solution by dip-coating.
  • Multi-frequency phase and modulation data were collected on the frequency-domain instrumentation ISS K2 (ISS Inc., Urbana Champagne, IL).
  • ISS K2 ISS Inc., Urbana Champagne, IL
  • blue LED Nathode
  • This light source was chosen because it is practical and inexpensive for analytical or clinical sensing.
  • Emission light was collected through a long pass filter above 595 nm. The measurements were carried out at room temperature of 22° C.
  • phase modulation fluorometry the sample is excited with an intensity-modulated light and the fluorescence is emitted with the same modulation frequency.
  • the experimental observables are the
  • excitation light source [28]. These quantities may change from 0 to 90 degree and from 1 to 0 and are
  • Phase and modulation of the donor emission reflects the changes in an intensity decay due to FRET from donor to the pH-sensitive acceptor.
  • a functional sensor for pH does not require measurement of a complete frequency response. Measurement at single-modulation frequency is adequate for quantitative pH sensing [20].
  • Fig.l shows the representative absorption and emission spectra of Ru[(dpp)(SO 3 Na 2 )]3 Cl (Donor) and pH indicator, CPR (Acceptor) in EC films.
  • D-A system One of the most important variables of D-A system is the degree of spectral overlap between emission spectrum of the donor and absorption spectrum of acceptor. The importance of integrated spectral overlap of pH sensor can be understood by examining Fig.l. Protonation of CPR shifts its absorption maximum from 592 nm (large spectral overlap, indicated by grey area) to 405 nm (very small spectral overlap, not shown). Therefore, as the pH value decreases, the energy transfer efficiency from donor to acceptor will also decrease. In consequence the intensity and the lifetime of the donor should be pH dependent.
  • D-A The distance between D and A which allows for FRET to occurs. FRET occurs if the D and A distance is comparable to the critical F ⁇ rster distance, Ro, and not be larger than about 2Ro [24].
  • Ro critical F ⁇ rster distance
  • In this report we have mixed donor and acceptor molecules in liquid solution and then formed the solid polymers. We anticipated that acceptor molecules are uniformly and randomly distributed around the excited donor molecules. In such case the steady-state intensity and the intensity decay of the donor emission can be described as a functions of acceptor concentration [24]. Valuable information about the acceptor concentration needed is the value of critical acceptor concentration, Co, which can be calculated from the value of Ro.
  • acceptor concentration For polymeric matrices it is somewhat difficult to estimate the acceptor concentration because of several factors such as reduced volume during drying of the polymer, expected micro heterogeneity of polymers, and possible molecular diffusion. For optimal pH sensor performance the acceptor concentration need to be determined experimentally. Using time-resolved data the experimental values for Ro can also be determined and compared with those calculated from spectral characteristics of donor and acceptor molecules.
  • Figure 2 illustrates intensity decays (phase angles) of [Ru(dpp(S0 3 Na 2 ) 3 ] in the presence of various acceptor concentrations (CPR) in EC films at pH 8.35 (base form of CPR).
  • the acceptor concentrations in the EC films were estimated to be approximately 8-fold higher than that in the coating solution due to the reduced volume of EC after drying. It have been found that the experimental values of Ro for [Ru(dpp(SO 3 Na 2 )3]-CPR in EC were comparable to that expected from spectral overlap (Table 1 ). This agreement indicates that the donor and acceptor molecules are distributed randomly and there is no translational motion of molecules.
  • Figure 3 shows pH dependent intensity decay of [Ru(dpp(SO 3 Na 2 ) 3 ] - CPR in the pH range from 4.16 to 8.35.
  • the estimated concentration of acceptor in EC film was 8.8 mM. It is evident that with increased pH value from 4.16 to 8.35, the FRET also increased, resulting in shorter lifetimes. The increase of FRET is due to increased concentration of the base form of CPR at higher pH values. Phase angles and modulation dependencies on pH, which are shown in Figure 3, allow choosing a modulation frequency at which there are substantial pH sensitivity on phase and modulation. For
  • sensing pH range usually will be limited to the 2 pH units and in some cases to the 3 pH units i.e.
  • TBPSP TBPSP were used (Figure 5).
  • the optical pH sensor allow pH measurement in the extended range from pH 5 to almost 10.
  • a multiple pH indicators to cover even wider pH range to be measured.
  • a number of pH sensitive donor-acceptor systems have been characterized.
  • the FRET between the Ru(II) donor and the pH sensitive acceptors as a transduction mechanism has been used to demonstrate the pH sensing using phase and modulation of donor luminescence.
  • Several pH indicators with different pKa values were chosen to design the pH optical sensors using a Ru(LT) complex with a high quantum yield and long lifetime.
  • the wide pH range, from 1 to 11 can be measured with the same optical and electronic rearrangement of the instrumentation.
  • pH sensor from 3.5 ⁇ s (EC) to 5.2 ⁇ s (PVC) in the absence of oxygen.
  • Observed apparent pKa of pH sensor may be shifted of about 1 pH unit by changing the polymeric supports.
  • FRET-based pH sensors can be utilized for design an optical sensors for a variety other analytes that induce the pH changes within a sensing element.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne une analyse par transfert d'énergie en fluorescence, pour laquelle on utilise des complexes de ligands métalliques fluorescents comme molécules donneuses, afin d'analyser le pH ou de déceler des analysats dont la présence peut modifier le pH dans l'environnement d'une molécule receveuse.
PCT/US1999/014709 1998-06-29 1999-06-29 Procede et dispositif d'analyse WO2000000819A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49628/99A AU4962899A (en) 1998-06-29 1999-06-29 Assay method and device

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US9106798P 1998-06-29 1998-06-29
US60/091,067 1998-06-29

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WO2000000819A1 true WO2000000819A1 (fr) 2000-01-06

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1271133A1 (fr) * 2000-02-28 2003-01-02 Daiichi Pure Chemicals Co., Ltd. Procede de mesure dans lequel on utilise la fluorescence de longue duree du type d'excitation
US7524974B2 (en) 2002-07-08 2009-04-28 Tetsuo Nagano Fluorescent probe
US7541467B2 (en) 2004-01-09 2009-06-02 Shigenobu Yano Fluorescent zinc ion sensor
US7696245B2 (en) 2003-03-28 2010-04-13 Sekisui Medical Co., Ltd. Fluorescent probe for zinc

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5628310A (en) * 1995-05-19 1997-05-13 Joseph R. Lakowicz Method and apparatus to perform trans-cutaneous analyte monitoring
US5648269A (en) * 1991-05-03 1997-07-15 Joseph R. Lakowicz pH and pCO2 sensing by luminescent lifetimes and energy transfer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648269A (en) * 1991-05-03 1997-07-15 Joseph R. Lakowicz pH and pCO2 sensing by luminescent lifetimes and energy transfer
US5628310A (en) * 1995-05-19 1997-05-13 Joseph R. Lakowicz Method and apparatus to perform trans-cutaneous analyte monitoring

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SIPIOR J, ET AL.: "A LIFETIME-BASED OPTICAL CO2 GAS SENSOR WITH BLUE OR RED EXCITATIONAND STOKES OR ANTI-STOKES DETECTION", ANALYTICAL BIOCHEMISTRY., ACADEMIC PRESS INC., NEW YORK., vol. 227, 1 January 1995 (1995-01-01), NEW YORK., pages 309 - 318, XP002919346, ISSN: 0003-2697, DOI: 10.1006/abio.1995.1286 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1271133A1 (fr) * 2000-02-28 2003-01-02 Daiichi Pure Chemicals Co., Ltd. Procede de mesure dans lequel on utilise la fluorescence de longue duree du type d'excitation
EP1271133A4 (fr) * 2000-02-28 2006-02-08 Daiichi Pure Chemicals Co Ltd Procede de mesure dans lequel on utilise la fluorescence de longue duree du type d'excitation
JP4589588B2 (ja) * 2000-02-28 2010-12-01 哲雄 長野 長寿命励起型蛍光を用いる測定方法
US7524974B2 (en) 2002-07-08 2009-04-28 Tetsuo Nagano Fluorescent probe
US7696245B2 (en) 2003-03-28 2010-04-13 Sekisui Medical Co., Ltd. Fluorescent probe for zinc
US7541467B2 (en) 2004-01-09 2009-06-02 Shigenobu Yano Fluorescent zinc ion sensor

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