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WO1999060383A1 - Amelioration de l'angle de phase et de la modulation en analyse fluorimetrique - Google Patents

Amelioration de l'angle de phase et de la modulation en analyse fluorimetrique Download PDF

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Publication number
WO1999060383A1
WO1999060383A1 PCT/US1999/010874 US9910874W WO9960383A1 WO 1999060383 A1 WO1999060383 A1 WO 1999060383A1 US 9910874 W US9910874 W US 9910874W WO 9960383 A1 WO9960383 A1 WO 9960383A1
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WIPO (PCT)
Prior art keywords
fluorescent
analyte
lifetime
concentration
radiation
Prior art date
Application number
PCT/US1999/010874
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English (en)
Inventor
Henryk Szmacinski
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.
Publication of WO1999060383A1 publication Critical patent/WO1999060383A1/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
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence

Definitions

  • Fluorescence-based sensing is one of the promising techniques because of fluorescence sensitivity, providing number of sensitive and specific fluorescent probes for a variety of analytes and their fabrication with fiber optics .
  • intensity-based sensing devices are based on the standard intensity- based methods, in which the intensity of the fluorescence produced by the probe molecule changes in response to the analyte of interest. These intensity changes can be induced by an analyte due to changes in extinction coefficient, changes in quantum yield, absorption and emission spectral shifts, or simply due to the inner filter effects. While intensity measurements are simple and accurate in the laboratory, they are often inadequate in real-world situat'ions . This is because the sample may be turbid, the optical surfaces may be imprecise and become dirty and optical alignment may vary from sample to sample. A significant disadvantage of intensity based sensing is the problem of referencing the intensity measurements.
  • the fluorescence intensity measurement depends on the intensity of exciting light, the optical density at the excitation and emission wavelengths, the light loses in the optical path length, detector sensitivity and the concentration of the fluorophore .
  • a number of lifetime-sensitive probes have been characterized for several analytes such as pH, Ca 2+ , Mg 2+ , K ⁇ , Na * .
  • Practically all of the known analyte lifetime-sensitive probes excluding the probes for O 2 sensing display short lifetimes, most often in the range of 1- 5 ns .
  • Using probes with short lifetimes requires high modulation frequencies in the range of 50 - 300 MHz in order to obtain sufficient changes in phase and modulation for analyte sensing.
  • inexpensive light sources such as LED' s can be modulated in that range of frequencies, the cost of phase modulation device is still sufficiently expensive to inhibit broad commercial use .
  • sensing can be at a relatively lower modulation frequency, for example, in the range of 10-1000 kHz.
  • a phase and/or modulation instrument based on the use of longer lifetime fluorescent materials, such as the metal-ligand complex based materials can be designed tc use inexpensive components due mainly to the lower frequencies required.
  • the present invention makes use of the discovery that relatively low frequency phase and modulation techniques can be used to assay for analytes by employing, in combination, a fluorescent probe that shows a fluorescent intensity response in the presence of the analyte but has a fluorescent lifetime that is substantially unaffected by the presence of the analyte and a fluorescent material having a fluorescent lifetime that is different from the fluorescent lifetime of the probe, the fluorescent material being substantially unaffected in both fluorescence lifetime and fluorescence intensity by the presence of the analyte.
  • phase angle and modulation of the sample depend on values of lifetime and fractional intensities of components.
  • the changes in phase angle and modulation can be as result of changes in fractional intensities without changes in the lifetime of both component.
  • an analyte sensitive fluorophore short lifetime or long lifetime
  • an analyte sensitive probe can be created.
  • the expected analyte induced changes in phase angle and modulation can be as large as 90 degree and 1.0, respectively.
  • the modulation frequency can be at a lower range, determined by the long lifetime component .
  • the controlled mixing of two fluorophores allows using any intensity-based fluorophore regardless of its lifetime as a lifetime-based probe using a phase and modulation technique.
  • the analyte-induced changes in fractional intensities of two components allow the determination of the analyte concentration from the phase and/or modulation at a single modulation frequency.
  • Figs, la and Fig. lb show the expected frequency responses of phase angle and modulation for long lifetime and short lifetime fluorophores.
  • Fig. 2a and Fig 2b show the expected frequency responses of phase angle and modulation of fluorescence that consist of fraction of long lifetime and fraction the short lifetime fluorescence.
  • Fig. 3a and Fig. 3b show the expected frequency responses of phase angle and modulation where the value of short lifetime is changed from 0.5 to 10 ns in several steps.
  • Fig. 4a and Fig 4b show the expected frequency responses of phase angle and modulation where the value of long lifetime is 100, 500, and 5000 ns and short lifetime fluorescence of 10 ns .
  • Figs. 5a, 5b, 6 and 7 illustrate Example 1.
  • Figs. 8, 9 and 10 illustrate Example 2.
  • Figs. 11, 12, 13, 14 and 15 illustrate Example 3.
  • Figs. 2a and Fig 2b show the expected frequency responses of phase angle and modulation of fluorescence that consist of a fraction of long lifetime and a fraction the short lifetime fluorescence.
  • the values from 0 to 1 represent the fractional intensity of short lifetime fluorescence in the measured signal.
  • the steeples part of the modulation value is equal the fractional intensity of short lifetime fluorescence.
  • fractional-dependent phase angle and/or modulation can be used to measure the intensity of a desired fluorophore in the sample using in most cases only one modulation frequency.
  • the changes in fractional intensity can be induced by the analyte; (1) by affecting the absorption spectra (extinction coefficient and/or spectra shift), (2) by affecting the emission spectra (quantum yields and/or spectra shifts) .
  • absorption spectra absorption coefficient and/or spectra shift
  • emission spectra quantum yields and/or spectra shifts
  • the fractional intensity of the short fluorescence lifetime component in each case is the same for 0.15 in Fig. 3a and 0.5 in Fig.3b.
  • the important observations from these figures are that the phase angle and modulation below certain frequency are not sensitive to the value of short lifetime fluorescence in the sample. This is similar to the gating technique in the pulse method where, by applying a certain delay after pulse excitation, only the signal from long lifetime fluorescence is detected. In the phase-modulation technique it is impossible to measure only long the lifetime component . Analytical methods have been developed for background correction in phase-modulation fluorometry based on the measurements of the background sample or based on known intensity decay of background and it contribution in the sample signal.
  • the desired intensity of the long lifetime component can be obtained by measuring the phase and/or modulation at single modulation frequency regardless of intensity decay of the background or autofluorescence or scattered light until the mean lifetime is short enough compared to a long lifetime fluorophore.
  • This feature can be used also to determine the anisotropy of the long lifetime component in turbid media with scattered light or with background fluorescence having a known value of anisotropy. This may find immediate application in detecting binding of high molecular weight macromolecules labeled with a metal-ligand complexes or in i munoassays . It is also important for" chemical sensing that changes in the lifetime due to presence of an analyte for short lifetime indicators have no effect of fractional intensity and thus on sensing of analyte concentration.
  • Example 1 demonstrates the phase and modulation sensitivity when the fractional intensity of sample is varied by various relative concentrations of two dyes in the sample.
  • Example 2 demonstrates the possibility to determine the intensity of flurophore of interest in presence of various amoun of background or autofluorescence from the solvent .
  • Example 3 demonstrate how the sensing probe can be created when pH induced changes in fractional intensities of a probe contained pH intensity sensitive indicator and long lifetime fluorophore con be measured by phase angle and modulation.
  • Two fluorophores have been chosen, one with a long lifetime fluorescence from metal-ligand complexes like [Ru (bpy) 2 dcbpy] 2 with a lifetime in glycerol of 1060 ns and the second with short lifetime like many organic fluorophores Texas Red Hydrazide with a lifetime of 3.4 ns in glycerol.
  • the two dyes were mixed at various relative concentrations to induce the various fractional intensities in the sample
  • Fig. 5a show the absorption spectra of long lifetime fluorophore [Ru (bpy) 2 dcbpy] 2+ and short lifetime fluorophore Texas Red Hydrazide (TRH) (solid lines and their mixture at concentrations specified in Figure. It is shown that any excitation wavelength shorter than about 640 nm will excite both fluorophores. The resulting fractional intensities from both fluorophores will be strongly dependent on the choice of excitation wavelength. One can imagine that value of extinction coefficient or shift in absorption spectrum will result in changes of fractional intensities that can be monitored with phase and/or modulation measurements. One excitation wavelength has been chosen as 488 nm (Argon- ion laser) . The total concentration of dyes were low to avoid the inner filter effects. The changes in absorption was induced by using various concentration combination of both fluorophores.
  • TRH Texas Red Hydrazide
  • Fig. 5b shows the emission spectra of [Ru (bpy) 2 dcbpy] 2+ and TRH at one selected concentration combination.
  • the emission spectra overlap well and for phase and modulation measurements we used the long pass filter above 550 nm.
  • Fig. 6 show the frequency responses of phase angle for long lifetime fluorophore [Ru (bpy) 2 dcbpy] 2* with a lifetime of 1060 ns and the short lifetime TRH of 3.4 ns when mixed together at a specified relative concentrations from 0 to 12.8.
  • the obtained values for fractional intensities are in good agreement with those expected from steady-state measurements of full emission spectra.
  • Fig.7 show the frequency responses of modulation for long lifetime fluorophore [Ru (bpy) 2 dcbpy] " + with a lifetime of 1060 ns and the short lifetime TRH of 3.4 ns when mixed together at a specified relative concentrations from 0 to 12.8.
  • the purpose of this example was to demonstrate the calculation of intensity of long lifetime fluorophore in presence of background fluorescence from the solvent.
  • Long lifetime fluorophore was the same as in Examplel [Ru (bpy) 2 dcbpy] 2 with a lifetime in glycerol of 1060 ns .
  • the glycerol (from Fluka) displayed a background fluorescence that overlaps with the emission of ruthenium. In many applications the requirements are for very low dye concentration which posses the difficulties for increased background corrections.
  • the increased contribution of background fluorescence from solvent was obtained by the dilutions of the ruthenium sample with glycerol . Fig.
  • Fig. 9 show frequency responses of phase angle of the samples with increased contributions of background fluorescence. The obtained values are in good agreement with those from steady-state measurements. The small difference are because of different excitation sources (xenon lamp and monochromator in steady-state, and Ar- ion laser in phase-modulation measurements) . It should be noted that phase angle is related only to fractional intensity at modulation frequencies lower than 1 MHz . The glycerol displayed a complex intensity decay with a mean lifetime shorter than 3.5 ns . These experimental data confirm that presented in Fig 3a where the short lifetime component do not contribute to changes in phase angle for certain low modulation frequencies.
  • Fig 10 show frequency responses of modulation of the samples with increased contributions of background fluorescence. The steeples part of modulation indicate good separation between the fluorescence of ruthenium and that of glycerol and can be easy used to determine the absolute intensity of the ruthenium. These results confirm that discussed in Fig. 3b.
  • the goal of this example is to demonstrate the great opportunity of designing the fluorescence probe for measuring a large variety of chemical species where the change in fluorescence intensity can be " obtained.
  • Fig. 11 shows the emission spectra of a mixture of ruthenium and Naphtofluorescein at various values of pH.
  • the increased pH values affect the fractional intensities from both of dyes which is displayed as decreased fluorescence from the ruthenium and increased contribution from the Naphtofluorescein.
  • the fractional intensities in the sample can be selected by the cutt off filter or by band pass filter. We have chosen use long pass filter above the 595 nm.
  • the excitation source was a blue LED with a maximum intensity at 475 nm.
  • Fig. 12 shows the frequency responses of phase angle of such pH sensor. There are observed remarkably large changes in phase angle at modulation induced by the pH of a sample.
  • the pH phase-based sensing can be performed at low modulation frequencies in spite of very short lifetime of Naphtofluorescein of about 0.45 ns frequencies below 10 MHz.
  • Fig. 13 shows large changes in modulation induced by pH of the sample. There is a wide range of modulation frequencies where modulation value is related only to the pH value even not to modulation frequency. This is because the difference in lifetimes of ruthenium and Naphtofluorescein is very large about 1000-fo ' ld. It is again important to note that long lifetime value determines the useful low modulation frequency for sensing.
  • Fig. 14 shows pH-dependent phase angle for several modulation frequencies. It should be noted the magnitude of phase angle changes up to 69 deg (see values in the brackets) .
  • Fig. 15 shows pH-dependent modulations for several modulation frequencies .
  • the pH induced changes in modulation ( values in the brackets) are large and significantly depends on the choice of modulation frequency.
  • the apparent pKa is slightly dependent on modulation frequency.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Il est possible d'analyser la présence ou la concentration d'analysats dans un échantillon en observant, avec des techniques liées à la durée de vie de la fluorescence (par exemple, modification de l'angle de phase et changement de modulation), la réponse combinée à l'excitation d'une sonde fluorescente en présence de l'échantillon et d'un matériau fluorescent, sachant que la sonde présente une certaine réponse d'intensité de fluorescence devant une présence ou une concentration d'anlysat, et que, par ailleurs, la sonde et le matériau ne manifestent pas de changement substantiel quant à la durée de vie de la fluorescence en réponse à une présence ou une concentration d'analysat, et que le matériau possède en réponse à l'excitation une durée de vie de fluorescence qui diffère de celle de la sonde.
PCT/US1999/010874 1998-05-15 1999-05-17 Amelioration de l'angle de phase et de la modulation en analyse fluorimetrique WO1999060383A1 (fr)

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US8554598P 1998-05-15 1998-05-15
US60/085,545 1998-05-15

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6982431B2 (en) 1998-08-31 2006-01-03 Molecular Devices Corporation Sample analysis systems
US7070921B2 (en) 2000-04-28 2006-07-04 Molecular Devices Corporation Molecular modification assays
US7452457B2 (en) 2003-06-20 2008-11-18 Roche Diagnostics Operations, Inc. System and method for analyte measurement using dose sufficiency electrodes
US7556723B2 (en) 2004-06-18 2009-07-07 Roche Diagnostics Operations, Inc. Electrode design for biosensor
US7569126B2 (en) 2004-06-18 2009-08-04 Roche Diagnostics Operations, Inc. System and method for quality assurance of a biosensor test strip
US7597793B2 (en) 2003-06-20 2009-10-06 Roche Operations Ltd. System and method for analyte measurement employing maximum dosing time delay
US7604721B2 (en) 2003-06-20 2009-10-20 Roche Diagnostics Operations, Inc. System and method for coding information on a biosensor test strip
US7632651B2 (en) 1997-09-15 2009-12-15 Mds Analytical Technologies (Us) Inc. Molecular modification assays
DE10101576B4 (de) * 2001-01-15 2016-02-18 Presens Precision Sensing Gmbh Optischer Sensor und Sensorfeld

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007245A1 (fr) * 1990-10-10 1992-04-30 The University Of Maryland Procede et appareil pour mesurer en phase la duree de vie d'une emission fluorescente, utilises dans la cytometrie en milieu fluide
WO1992013265A1 (fr) * 1991-01-24 1992-08-06 The University Of Maryland Procede et appareil d'imagerie multidimensionnelle a duree de vie de fluorescence a modulation de phase
US5270548A (en) * 1992-07-31 1993-12-14 The United States Of America As Represented By The United States Department Of Energy Phase-sensitive flow cytometer
US5315122A (en) * 1992-08-25 1994-05-24 Becton, Dickinson And Company Apparatus and method for fluorescent lifetime measurement

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007245A1 (fr) * 1990-10-10 1992-04-30 The University Of Maryland Procede et appareil pour mesurer en phase la duree de vie d'une emission fluorescente, utilises dans la cytometrie en milieu fluide
WO1992013265A1 (fr) * 1991-01-24 1992-08-06 The University Of Maryland Procede et appareil d'imagerie multidimensionnelle a duree de vie de fluorescence a modulation de phase
US5270548A (en) * 1992-07-31 1993-12-14 The United States Of America As Represented By The United States Department Of Energy Phase-sensitive flow cytometer
US5315122A (en) * 1992-08-25 1994-05-24 Becton, Dickinson And Company Apparatus and method for fluorescent lifetime measurement

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7632651B2 (en) 1997-09-15 2009-12-15 Mds Analytical Technologies (Us) Inc. Molecular modification assays
US6982431B2 (en) 1998-08-31 2006-01-03 Molecular Devices Corporation Sample analysis systems
US7070921B2 (en) 2000-04-28 2006-07-04 Molecular Devices Corporation Molecular modification assays
DE10101576B4 (de) * 2001-01-15 2016-02-18 Presens Precision Sensing Gmbh Optischer Sensor und Sensorfeld
US7452457B2 (en) 2003-06-20 2008-11-18 Roche Diagnostics Operations, Inc. System and method for analyte measurement using dose sufficiency electrodes
US7597793B2 (en) 2003-06-20 2009-10-06 Roche Operations Ltd. System and method for analyte measurement employing maximum dosing time delay
US7604721B2 (en) 2003-06-20 2009-10-20 Roche Diagnostics Operations, Inc. System and method for coding information on a biosensor test strip
US7556723B2 (en) 2004-06-18 2009-07-07 Roche Diagnostics Operations, Inc. Electrode design for biosensor
US7569126B2 (en) 2004-06-18 2009-08-04 Roche Diagnostics Operations, Inc. System and method for quality assurance of a biosensor test strip
US9410915B2 (en) 2004-06-18 2016-08-09 Roche Operations Ltd. System and method for quality assurance of a biosensor test strip

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