Patent Application
20130005047
Solid-state polymeric materials based on target-analyte-sensitive photoluminescent dyes are widely used as optical target-analyte sensors and probes. See, for example United States Published Patent Applications 2009/0029402, 2008/8242870, 2008/215254, 2008/199360, 2008/190172, 2008/148817, 2008/146460, 2008/117418, 2008/0051646, and 2006/0002822, and U.S. Pat. Nos. 7,569,395, 7,534,615, 7,368,153, 7,138,270, 6,689,438, 5,718,842, 4,810,655, and 4,476,870. Such optical sensors are available from a number of suppliers, including Presens Precision Sensing, GmbH of Regensburg, Germany, Oxysense of Dallas, Tex., United States, and Luxcel Biosciences, Ltd of Cork, Ireland.
Target-analyte partial pressure of a fluid system can be ascertained by placing a target-analyte quenchable luminescent probe into fluid communication with the system of interest (e.g., the enclosed retention chamber of a Petri dish, the interior of modified atmosphere packaged foodstuffs, or the headspace of a bottled beverage) and interrogating luminescence characteristics of that probe with a sensing instrument. See, for example United States Published Patent Application 2009/0028756.
Typical sensing instruments expose the probe to excitation radiation over time, measure radiation emitted by the excited probe over time and convert at least some of the measured emissions to a target-analyte concentration based upon a known conversion algorithm.
Radiation emitted by the excited probe can be measured in terms of intensity and/or lifetime (rate of decay, phase shift or anisotropy), with measurement of lifetime generally preferred as a more accurate and reliable measurement technique when seeking to establish a concentration of target-analyte by measuring the extent to which a luminescent dye has been quenched by the target-analyte.
Sensing instruments that measure radiation emitted by an excited probe in terms of luminescence lifetime must be calibrated, which is typically achieved by empirically generating a Stern-Volmer plot from a plurality of luminescence lifetime data points obtained by interrogating a target-analyte quenchable probe exposed to a different known concentration of target-analyte with the instrument being calibrated, and employing the slope of the generated Stern-Volmer plot to calibrate the instrument.
Atoms and molecules can be excited by the absorption of a photon. Such excited particles can return to a ground state by a number of routes. One route is the radiative emission of a photon of light, producing luminescence. Alternatively, such particles return to ground by non-radiative means such as collisions with other atoms or molecules (known as dynamic quenching) or traveling along a down-hill energy path that involves multiple coupled vibrational and electronic energy states.
In a system containing strongly luminescent molecules A, a temporary concentration of excited state molecules [A*] can be generated by exposing the system to radiant energy of the proper wavelength. If there are no quenching agents present in the system (i.e., there are no species present in the system that can quench luminescence through bimolecular collisions), then A* can return to the ground state by luminescence
A*
A+hv (1)
and by non-radiative decay
A*
A (2)
where k1 and k2 are the rate constants for these two processes.
With only these two paths to ground state available, the rate equation for [A*] can be written as
d[A*]/dt=−k
1
[A*]−k
2
[A*]=−(k1+k2)[A*] (3)
Rearrangement and integration of equation (3) with respect to initial conditions: t=0 and [A*]=[A*]0 gives
[A*]=[A*]
0
e
-(k1+k2)t (4)
According to this result, the concentration of excited species [A*] (and therefore luminescence) is expected to decay in an exponential fashion, with the rate constants k1 and k2 quantifying the rate of such decay.
For convenience, we will define a ‘fluorescence lifetime in the absence of quencher’ (τ0) as:
τ0=1/(k1+k2) (5)
where τ0 is the amount of time that it takes for the luminescence intensity to decay to 1/e or 36.8% its initial value.
If a quenching agent (q) is present in solution, then a third path becomes available for returning A* molecules to the ground state;
A*+Q
A (6)
and the rate equation for [A*] becomes
d[A*]/dt=−(k1+k2+kq[Q])[A*] (7)
Where kq is the quenching constant.
Assuming [Q] is much greater than [A*], [Q] can be treated as a constant, allowing equation (7) to be integrated to give
[A*]=[A*]
0
e
-(k1+k2+kq[Q])t (8)
with ‘luminescence lifetime in the presence of quencher’ (τ) defined as:
τ=1/(k1+k2+kq[Q]) (9)
To isolate the effects of quenching, luminescence lifetime measurements are carried out over a range of known quenching agent concentrations (including [Q]=0). A luminescence decay curve is recorded for each trial and each decay curve is fit to an exponential function, yielding a lifetime for each trial.
Dividing equation (9) into equation (5) gives
τ0/τ=(k1+k2+kq[Q])/(k1+k2)
or, upon simplification
τ0/τ=1+kqτ0[Q] (10)
According to equation (10), a plot of τ0/τ versus [Q] should be linear with an intercept equal to one, and a slope equal to kqτ0, thereby permitting the quenching rate constant kq to be ascertained. Such a plot is known as a Stern-Volmer plot with kq comprising the calibration constant for each instrument used to measure luminescence lifetime of an excited probe.
Current systems and techniques for generating Stern-Volmer plots used to calibrate optical instruments are subject to various vagaries that produce nonlinear Stern-Volmer plots, significantly complicating calibration efforts and typically producing calibration error.
Accordingly, a substantial need exists for a system and technique of generating accurate linear or substantially linear Stern-Volmer plots for use in calibrating instruments that measure radiation emitted by an excited probe in terms of luminescence lifetime.
A first aspect of the invention is a method of calibrating an instrument effective for optically interrogating a luminescence target-analyte probe capable of emitting radiation at a first wavelength when exposed to excitation radiation, and determining target-analyte partial pressure from a luminescence lifetime measurement obtained from the probe.
A first embodiment of the first aspect of the invention includes the steps of (i) empirically generating a Stern-Volmer plot from a plurality of luminescence lifetime data points obtained by interrogating a target-analyte quenchable probe exposed at different known concentrations of target-analyte with excitation energy generated by an excitation energy source onboard the instrument is filtered to remove radiation at the first wavelength from the excitation energy prior to transmission of the excitation energy onto the probe, and (ii) calibrating the instrument from the generated Stern-Volmer plot.
A second embodiment of the first aspect of the invention includes the steps of (i) empirically generating a Stern-Volmer plot from a plurality of luminescence lifetime data points obtained by interrogating a target-analyte quenchable probe exposed at different known concentrations of target-analyte, with each luminescence lifetime comprising a time period measured from a starting time comprising a time at which an excitation energy source onboard the instrument is shut-off—delayed by a predetermined decay delay time, until an ending time comprising a time at which the luminescence intensity at the starting time has decayed a predetermined percentage, and (ii) calibrating the instrument from the generated Stern-Volmer plot.
A third embodiment of the first aspect of the invention includes the steps of (i) empirically generating a Stern-Volmer plot from a plurality of luminescence lifetime data points obtained by interrogating a target-analyte quenchable probe exposed at different known concentrations of target-analyte, with each luminescence lifetime comprising a time period measured from a starting time to an ending time, wherein the ending time comprises a time at which a luminescence intensity at the starting time has decayed a predetermined percentage of between 30% and 60%, and calibrating the instrument from the generated Stern-Volmer plot.
A second aspect of the invention is a method of optically interrogating a target-analyte probe effective for emitting luminescent radiation at a first wavelength when exposed to excitation radiation at a second wavelength.
A first embodiment of the second aspect of the invention includes the steps of (i) exposing the probe to excitation radiation from which radiation at the first wavelength has been filtered, to generate an excited probe, (ii) measuring the intensity of radiation emitted by the excited probe after such exposure, and (iii) measuring and reporting luminescence lifetime of the probe comprising that time period measured from a starting time comprising a time at which the luminescence intensity of emitted radiation is proximate a maximum value until an ending time comprising a time at which the luminescence intensity of emitted radiation has decayed a predetermined percentage from the luminescence intensity at the starting time. Such measured and reported luminescence lifetime is indicative of target-analyte partial pressure in fluid communication with the probe.
A second embodiment of the second aspect of the invention includes the steps of (i) exposing the probe to excitation radiation from an excitation energy source, to generate an excited probe, (ii) measuring the intensity of radiation emitted by the excited probe after the exposure, and (iii) measuring and reporting luminescence lifetime of the probe comprising that time period measured from a starting time comprising a time at which the excitation energy source is shut-off—delayed by a predetermined decay delay time, until an ending time comprising a time at which the luminescence intensity of emitted radiation has decayed a predetermined percentage from the luminescence intensity at the starting time. Such measured and reported luminescence lifetime is indicative of target-analyte partial pressure in fluid communication with the probe.
A third embodiment of the second aspect of the invention includes the steps of (i) exposing the probe to excitation radiation from an excitation energy source, to generate an excited probe, (ii) measuring the intensity of radiation emitted by the excited probe after the exposure, and (iii) measuring and reporting luminescence lifetime of the probe comprising that time period measured from a starting time to an ending time, wherein the starting time comprises a time at or after maximum luminescence intensity, and the ending time comprises a time at which the luminescence intensity at the starting time has decayed a predetermined percentage of between 30% and 60%. Such measured and reported luminescence lifetime is indicative of target-analyte partial pressure in fluid communication with the probe.
A fourth embodiment of the second aspect of the invention includes the steps of (i) exposing the probe to excitation radiation from an excitation energy source, to generate an excited probe, (ii) measuring the intensity of radiation emitted by the excited probe after the exposure, and (iii) measuring and reporting luminescence lifetime of the probe comprising a time period measured from a starting time comprising that time at which the excitation energy source is turned-on—delayed by a predetermined rise delay time, until an ending time comprising a time at which the luminescence intensity of emitted radiation has risen a predetermined percentage from the luminescence intensity at the starting time. Such measured and reported luminescence lifetime is indicative of target-analyte partial pressure in fluid communication with the probe.
A fifth embodiment of the second aspect of the invention includes the steps of (i) exposing the probe to excitation radiation from an excitation energy source, to generate an excited probe capable of emitting a peak luminescence intensity, (ii) measuring the intensity of radiation emitted by the excited probe after the exposure, and (iii) measuring and reporting luminescence lifetime of the probe comprising that time period measured from a starting time to an ending time, wherein the starting time comprises a time at or after minimum luminescence intensity, and the ending time comprises a time at which luminescence intensity has risen to a predetermined percentage of between 30% and 60% of peak luminescence intensity. Such measured and reported luminescence lifetime is indicative of target-analyte partial pressure in fluid communication with the probe.
As used herein, including the claims, the phrase “decay delay” means the period of time it takes for the intensity of luminescence emitted by a probe to commence natural logarithmic rate of decay after the excitation energy source has been shut off.
As used herein, including the claims, the phrase “rise delay” means the period of time it takes for the intensity of luminescence emitted by a probe to commence exponential rise after the excitation energy source has been turned on.
As used herein, including the claims, the phrase “target analyte” means a molecule whose presence-absence is detected and measured. Typical target-analytes are oxygen O2 and carbon dioxide CO2.
As used herein, including the claims, the phrase “essentially 100%” means containing only trace amounts of contaminants.
Construction
The invention involves calibration and use of an optical target-analyte sensing system 10. An embodiment of such an optical target-analyte sensing system 10 is depicted in
For purposes of simplicity only, and without intending to be limited thereto, the balance of the description may default to oxygen O2 as the target-analyte since O2-sensitive probes 120 are the most commonly used types of optically active probes 120.
The detection instrument 20 is configured and arranged to optically interrogate a target-analyte-sensitive probe 120 by generating and directing excitation energy E1 having a first wavelength onto the probe 120, followed by detection and measurement of the intensity of radiant energy E2 having a second wavelength different form the first wavelength emitted by the excited probe 120 over time (t). For purposes of discussion, the detection instrument 20 is separated as between the optical components 30 shown in
Referring to
A beam splitter 33 reflects the excitation energy E1 generated by the source of excitation energy 31 down a primary channel 39 and out through a distal end (unnumbered) of the instrument 20.
An optical filter 32 is provided between the source of excitation energy 31 and the primary channel 39 for blocking or attenuating radiant energy generated by the source of excitation energy 31 having a wavelength that matches the wavelength of the radiant energy E2 emitted by a probe 120 to be interrogated by the instrument 20.
A probe 120 contacted by a focused beam of excitation energy E1 emanating from the instrument 20 will luminesce and emit radiant energy E2 having a wavelength that is different from the wavelength of the excitation energy E1. For example, an oxygen sensitive platinum(II)-octaethylporphine-ketone (PtOEPK) probe 120 is excited by radiant energy E1 at a wavelength of 590 nm and emits radiant energy E2 at a wavelength of 760 nm, and an oxygen sensitive platinum(II)-tetrakis(pentafluorophenyl)porphine (PtPFPP) probe 120 is excited by radiant energy E1 at wavelengths of both 525 and 400 nm and emits radiant energy E2 at a wavelength of 650 nm. Emitted energy E2 generated by the excited probe 120 will travel up the primary channel 39, unabated through the beam splitter 33, and into contact with a photodiode 36 capable of sensing the intensity of the emitted energy E2 over time and generating an electrical signal representative of the intensity of the emitted energy E2 reaching the photodiode 36.
A lens 34 is preferably provided in the primary channel 39 for focusing the emitted radiant energy E2 traveling up the primary channel 39 onto a small sensing area on the photodiode 36. This allows use of a photodiode 36 with a small sensing area (not shown) without loss of signal level. A smaller sensing area requires less capacitance, thereby making a larger bandwidth available—resulting in more accurate lifetime luminescence decay curves.
The photodiode 36 may be selected from any of the wide variety of photodiodes 36 including, but not limited to UV enhanced, high speed epitaxail, low dark current, low capacitance, quadrant and black photodiodes, as well as avalanche types such as high speed, IR enhanced, blue enhanced and Geiger. The photodiode 36 of choice is a low capacitance, high speed photodiode with the largest possible area within the limits of practicality.
To prevent stray radiant energy from reaching the photodiode 36 and contaminating the electrical signal, an optical filter 35 is provided between the beam splitter 33 and the photodiode 36 for blocking or attenuating radiant energy with wavelengths other than the wavelength of the radiant energy E2 emitted by a probe 120 interrogated by the instrument 20.
Referring to
The preamplifier 42 is preferably a high speed (e.g., at least 100 MHz) operational amplifier with a gain of 100K-150K. The preamplifier 42 can feed directly into another preferably high speed operational amplifier 45 with a gain of about 100 for purposes of maintaining a bandwidth of about 10 MHz.
The signal from the preamplifier 42 can be split to allow both intensity and lifetime measurements to be made. The intensity measurement can be of interest in some applications, and can also be used to make small corrections or adjustments to the lifetime measurement. One of the split signals from the preamplifier 42 is communicated to an automatic gain control (AGC) 43 to normalize the amplitude of the signal and provide downstream components with a fixed range or gain. The signal is AC coupled 44 to reduce bias, inverted 46 to produce an inverted curve E2′ of emitted radiant energy to center the signal around zero and analyzed in a comparator 47 for ascertaining the time tx at which the primary signal curve and the inverted signal curve cross. This allows LED shut off tOff to be used as the starting time tstart for measuring decay luminescence lifetime τDecay and allows a 50% loss of luminescence to be used as the ending time tEnd for measuring decay luminescence lifetime τDecay as the circuitry can detect a 50% loss of luminescence as this is the point in time tx at which the primary signal curve and the inverted signal curve cross. Employing these points as the starting time tStart and ending time tEnd for measuring decay luminescence lifetime τDecay produces a more accurate measurement of decay luminescence lifetime τDecay as it provides a rapid, reliable and consistent starting and stopping point that avoids the need to detect luminescence and calculate % luminescence loss after a loss of greater than 60% luminescence—which is a time period fraught with excessive fluctuations in the luminescence signal.
Since the rate of luminescence rise is a mirror image of the rate at which luminescence decays—as least for the initial 50% of rise and decay—the electronic signal processing circuitry 40 also allows LED turn on tOn to be used as the starting time tStart for measuring growth luminescence lifetime τRise and allows a 50% gain of luminescence to be used as the ending time tEnd for measuring growth luminescence lifetime τRise as the circuitry can detect a 50% rise of luminescence as this is the point in time tx at which the primary signal curve and the inverted signal curve cross. Employing these points as the starting time tStart and ending time tEnd for measuring growth luminescence lifetime τRise produces a more accurate measurement of growth luminescence lifetime τRise as it provides a rapid, reliable and consistent starting and stopping point along the growth portion of the luminescence lifetime curve that truthfully mimics the corresponding decay portion of the luminescence lifetime curve.
Electronic signals indicative of the values of measured decay luminescence lifetimes τDecay and/or growth luminescence lifetimes τRise are counted and accumulated 48 before being sent to an A/D converter 49 and a microprocessor 50 for processing.
The electrical signal processing system 40 allows construction of a portable low cost detection instrument 20 as it permits rapid and accurate measurement of decay luminescence lifetime τDecay with a low speed A/D converter 49 and microprocessor 50 and requires limited power. It also allows the instrument 20 to communicate via a USB port (not shown).
Referring to
Referring to
Referring to
The probe 120 is sensitive to the partial pressure of a target analyte (most commonly the partial pressure of oxygen) and therefore useful for optically ascertaining the partial pressure of oxygen (PO2) within an enclosed space, such as the retention chamber of a hermetically sealed package (not shown). Such probes 120 include a thin film of a solid state photoluminescent composition (not independently shown) coated onto a support layer (not independently shown). The solid state photoluminescent composition includes an oxygen partial pressure sensitive (PO2 sensitive) photoluminescent dye (not independently shown) embedded within an oxygen permeable polymer matrix (not independently shown).
The oxygen-sensitive photoluminescent dye used in the solid state photoluminescent composition may be selected from any of the well-known PO2 sensitive photoluminescent dyes. One of routine skill in the art is capable of selecting a suitable dye based upon the intended use of the probe. A nonexhaustive list of suitable oxygen sensitive photoluminescent dyes includes specifically, but not exclusively, ruthenium(II)-bipyridyl and ruthenium(II)-diphenylphenanothroline complexes, porphyrin-ketones such as platinum(II)-octaethylporphine-ketone, platinum(II)-porphyrin such as platinum(II)-tetrakis(pentafluorophenyl)porphine, palladium(II)-porphyrin such as palladium(II)-tetrakis(pentafluorophenyl)porphine, phosphorescent metallocomplexes of tetrabenzoporphyrins, chlorins, azaporphyrins, and long-decay luminescent complexes of iridium(III) or osmium(II).
Typically, the oxygen-sensitive photoluminescent dye is compounded with a suitable oxygen-permeable hydrophobic carrier matrix. Again, one of routine skill in the art is capable of selecting a suitable oxygen-permeable hydrophobic carrier matrix based upon the intended use of the probe 120 and the selected dye. A nonexhaustive list of suitable polymers for use as an oxygen-permeable hydrophobic carrier matrix includes specifically, but not exclusively, polystyrene, polycarbonate, polysulfone, polyvinyl chloride and some co-polymers. The photoluminescent composition may be provided as a dispersed material, for example as aqueous suspension or powder of polymeric microparticles or nanoparticles impregnated with an oxygen-sensitive photoluminescent dye.
The support layer may be selected from any of the materials commonly employed as a support layer for a PO2 sensitive photoluminescent solid state composition. One of routine skill in the art is capable of selecting the material based upon the specific analyte to be detected and the intended use of the probe 120. A nonexhaustive list of substrates includes specifically, but not exclusively, cardboard, paperboard, polyester Mylar® film, non-woven spinlaid fibrous polyolefin fabrics, such as a spunbond polypropylene fabric.
The support layer is preferably between about 30 μm and 500 μm thick.
Luminescence lifetimes τ of a PtOEPK probe 120 exposed to known concentrations of O2 as set forth in Table One, were ascertained by measuring and accumulating approximately 300 τRise and τDecay employing the ΔtRise Delay, ΔtDecay Delay and the % Luminescence at tEnd as set forth in Table One. Three sets of accumulated values were averaged to obtain a raw measured τ time count set forth in Table One. The ΔtRise Delay and ΔtDecay Delay set forth in Table One are added together and subtracted from each raw measured τ time count to obtain a corrected τ time count as set forth in Table One. A Stern-Volmer Ratio was calculated at each O2 concentration by dividing the corrected τ time count obtained at an O2 concentration of 0 (τ0) by the corrected τ time count obtained at the given O2 concentration (τ) and subtracting 1 from the obtained quotient. A Stern-Volmer plot of O2 concentration v. Stern-Volmer Ratio is set forth in
This application claims the benefit of U.S. Provisional Application No. 61/317,509, filed Mar. 25, 2010
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/27878 | 3/10/2011 | WO | 00 | 8/30/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61317509 | Mar 2010 | US |
