Patent Application: US-46339400-A

Abstract:
a method and device for fluorimetric determination of a biological , chemical or physical parameter of a sample utilize at least two different luminescent materials , the first of which is sensitive to the parameter , at least with respect to luminescence intensity , and the second of which is insensitive to the parameter , at least with respect to luminescence intensity and decay time . the luminescent materials have different decay times . the time - or phase behaviour of the resulting luminescence response is used to form a reference value for determination of a parameter .

Description:
preferably , both luminescent materials will absorb light in the same range of wavelengths , which will enable them to be excited into luminescence by means of a single light source . emission spectra will preferably be in the same spectral region . it will be possible , for example , to excite both luminescent materials with blue light at a wavelength of 450 nm , one luminescent material emitting green light at 520 nm and the other one red light at 600 nm , as both signals can still be measured with one and the same detector . it will also be possible , however , to simultaneously measure the luminescence of two luminescent materials whose emission spectra differ from each other significantly . the measuring method described has the advantage that the long - life luminescent material need not exhibit analyte - specific response but solely acts as carrier of a constant background signal with long decay time , which is modulated by the short - life luminescent material . for this reason a large number of phosphorescent compounds described in the literature are suitable for this purpose . the long - life luminescent material need not interact with sample , analyte , or fluorescence indicator , and can thus be immobilized such that it is inert to all sample components , which will exclude a priori any potential interference by chemical parameters . following is a more detailed description of the invention by means of examples . fig1 shows the dependence of the measured phase angle φ m on the relationship between intensity of the fluorescence indicator and the reference luminescent material ; a strong fluorescence signal , b weak fluorescence signal . labels used are flu = variable fluorescence signal , ref = constant reference signal , ges = measured total signal ; fig2 shows a computed relationship between the measured phase angle φ m or its cot ( φ m ) and the amplitude ratio r of the two luminescent materials ; fig3 shows spectral properties of a suitable pair of fluorescence indicator and reference luminescent material . the hatched areas indicate optimum spectral windows for excitation of the luminescence signal and measurement of the emitted light ; fig4 shows a time - resolved measurement of the ratio of signal intensities during the excitation pulse ( i 1 ) and during luminescence decay ( i 2 ), the ratio r being independent of the total signal amplitude , as it is only a function of the chemical parameter being monitored ; fig5 shows ph calibration curves of a ph sensor according to example 1 with different amounts of hpts ( a : low hpts ; b : high hpts ), measured as phase shift at a modulation frequency of 80 khz . a blue led is used as a light source , and a photodiode as a detector ; and fig6 shows four possible combinations of the short - lived , chemically sensitive luminescent material ( a ) and the inert , long - lived luminescent material ( b ) in an optical sensor . suitable luminescent materials with long decay times , which are inert to the analyte include : transition metal complexes with ruthenium ( ii ), rhenium ( i ), or osmium and iridium as central atom and diimine ligands ; phosphorescent porphyrins with platinum , palladium , lutetium or tin as central atom ; phosphorescent complexes of rare earths , such as europium , dysprosium or terbium ; phosphorescent crystals such as ruby , cr - yag , alexandrite , or phosphorescent mixed oxides such as magnesium fluoro - germanate . suitable short - life fluorescent materials include all dyes whose excitation and emission spectra overlap with the spectra of the long - life luminescent materials referred to above , and whose fluorescence intensity depends on the parameter being monitored . the long - life luminescent material may be integrated into the sensor in different ways ( fig6 ): by directly dissolving the luminescent material in the analyte - sensitive layer ( fig6 example 3 ) by incorporation in a polymer which serves as a primer for the sensor layer ( fig6 example 1 ) by incorporation in a polymer which is dispersed in the sensitive layer in micro - or nanoparticles ( fig6 example 2 ) by incorporating luminescent dyes in sol - gel glass , followed by sintering , pulverizing and dispersing the glass in the sensor layer ( fig6 example 2 ) by employing pulverized phosphors which are dispersed in the sensitive layer ( fig6 example 2 ) by coating the outside of a sensor foil , avoiding contact with the sample ( fig6 example 4 ) by covalent or electrostatic - bonding of the fluorescence indicator to the surface of luminescent particles which are either dispersed in a polymer layer or directly distributed in the sample by dispersing particles in the sample in which the fluorescence indicator is included as a solute . if phase modulation techniques are employed at frequencies in the khz range , it is important to note that it will not be possible to obtain more than a mean phase shift with this type of sensors . although splitting into both decay time components would be feasible in principle , this would involve considerable instrumentation expense due to the high frequencies required . due to the strong differences in decay times of the two co - immobilized indicators , time - resolved measurement , which , upon providing an excitation light pulse , is exclusively concerned with the decay characteristic of the luminescence signal after the excitation pulse has been switched off , will often fail to produce a useful parameter , as the short - lived component will decay too quickly and can only be detected at considerable expense . it is further possible to perform separate time - resolved measurements of signal intensity - during the excitation pulse and in the decay period , and to compute the ratio r of the two signals . as is seen in fig4 this ratio depends solely on the intensity ratio r of the two luminescent components and is completely independent of the total intensity of the signal . phase modulation techniques can also be employed to determine the mean phase shift of the luminescence signal . the measuring frequency is adapted to the decay time of the luminescent material and lies between 0 . 5 and 100 khz . as is shown below , equation ( 1 ) indicates that the measured phase angle φ m depends only on the ratio of the two signal intensities but not on the absolute signal level and will hence permit the referencing of the intensity of the short - lived fluorescence component . additive superposition of signals ( index ref = reference signal , index flu = fluorescence signal , index m = measured value )   a m · cos   φ m = a ref · cos   φ ref + a flu · cos   φ flu a m · sin   φ m = a ref · sin   φ ref + a flu · sin   φ flu the longer decay time ist significantly greater than the shorter decay time : τ ref & gt ;& gt ; τ flu if the modulation frequency has been chosen such that it is optimal for t ref i . e ., one finds for φ flu : tan   φ flu = 2   π · f mod · τ flu = 2   π · τ flu 2   π · τ ref = τ flu τ ref under the above assumption there results for the angle φ flu : tan   φ flu = τ flu τ ref  → τ flu  & lt ;& lt ;  τ ref  0 ⇒ φ flu → 0 the decay time of the dye exhibiting longer decay time is constant for the measurement of interest : τ ref = constant ⇒ tan   φ ref = constant ⇒ φ ref = constant dividing the first equation by the second results in : a m · cos   φ m a m · sin   φ m = cot   φ m = a ref · cos   φ ref + a flu a ref · sin   φ ref = a ref a ref · ( cos   φ ref + a flu / a ref sin   φ ref )   cot   φ m = cos   φ ref + a flu / a ref sin   φ ref = cot   φ ref + 1 sin   φ ref · a flu / a ref ( 1 ) plotting φ m against the amplitude ratio thus shows a linear dependence between the cotangent of the measured phase angle φ m and the amplitude ratio r ( and thus intensity ratio ) of the two luminescent materials ( cf fig2 ). cot φ m represents an intensity ratio without the necessity of separately measuring two signal intensities . easy conversion of previously optimized fluorescence sensors to decay time measurement by simple admixture of the long - life luminescent material . for a set of sensors for a variety of analytes one and the same long - life luminescent material may be employed . different sensors may thus be evaluated with the same optoelectronicsystem . since the shape of the calibration curve will only depend on the ratio of the two intensities , the sensitivity range of an individual sensor may be optimized simply by varying the amount of luminescent material added . the same purpose may be achieved by optimum selection of the spectral windows for both excitation and emission . the cross - sensitivity of long - life luminescence to oxygen may be eliminated by incorporating the indicators into materials that are impermeable to gases . if phosphorescent solids or glass - embedded luminescent materials are employed , any influence on the signal due to chemical luminescence parameters in the sample will be completely excluded . since oxygen cannot quench luminescence no reactive singlet oxygen will be produced in the membrane . as a consequence , photo - decomposition will be reduced and sensor stability improved . incorporation of long - life luminescent materials in a glass matrix or as solids will completely prevent leaching . moreover , their photostability is exceptionally strong . whereas in measuring principles based on analyte deactivation of the excited state of the long - life luminescent material ( such as pet effect , dynamic quenching , or energy transfer ) a decrease in mean decay time will always be accompanied by a decrease in signal intensity , which will impair the signal - to - noise ratio , signal intensity will rise with a decrease in decay time with the type of sensors discussed here . as a consequence , the signal - to - noise ratio will improve significantly over the entire measuring range . since the characteristic of these sensors exclusively depends on the ratio of signal components of the two indicators , the following conditions should be met : 1 ) none of the two indicators should undergo leaching during measurement . 2 ) the two indicators should not exhibit different rates of photo - decomposition during measurement . 3 ) the concentration ratio of the two indicators should be maintained constant during membrane fabrication . 4 ) the decay time of the long - life luminescent material should always be constant . hpts adsorbed on cellulose with quaternary ammonium groups and incorporated in poly - hyroxyethylmethacrylate ( phema ) hydrogel ru ( phen ) 3 cl 2 in sol - gel ( sintered , ground , and dispersed in hydrogel ). ( fig5 shows the calibration curve of this sensor , measured as phase shift at a frequency of 80 khz .) aminofluorescein covalently attached to sol - gel particles with incorporated ru ( phen ) 3 cl 2 ( sintered , ground , and dispersed in hydrogel ). hpts - cta 3 ionic pair dissolved in ethyl cellulose with tetraoctyl - ammoniumhydroxide as buffer ( cf 6 ) the invention thus permits optical monitoring of a chemical , biological or physical parameter of a sample with the use of an optical sensor . the sensor includes two luminescence indicators in co - immobilized form , one of which acts as carrier of a background luminescence signal , which is characterized by long luminescence lifetime ( preferably in the range of at least 100 nanoseconds to several milliseconds ) and whose intensity and decay time will remain unaffected by the parameter being monitored . the second indicator exhibits shorter - lived fluorescence , preferably in the range of several nanoseconds , which will superpose on the long - lived luminescence signal and whose intensity is a function of the parameter being monitored . with the use of phase modulation techniques or time - resolved measuring methods a reference value is determined , which will represent the ratio of the two individual luminescence intensity components ; this reference value is independent of the total intensity of the luminescence signal and will thus permit referencing of the short - lived , analyte - dependent fluorescence component . 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