CA1309876C - Method and composition for measuring oxygen concentration - Google Patents

Abstract

METHOD AND COMPOSITION FOR MEASURING OXYGEN CONCENTRATION
Abstract Methods and compositions are described for measuring oxygen concentration, particularly for monitoring oxygen in the blood with a fiber optic catheter. Oxygen concentration is determined by observing quenching of the emission from a luminescent (phosphorescent or fluorescent) molecule embedded in oxygen-premeable plastic. A test fluid of unknown oxygen concentration is contacted with a plastic film containing at least one luminescent substance. Thefilm is subjected to irradiation over som eperiod of time by light of a wavelength that is strongly absorbed by the luminescent substance, and a measure of the time dependence of luminescent emission intensity I(t) is obtaned. Three modes modes of determining oxygen concentration from I(t) are described.
(i) Subsequent to a brief (approximately 5 µs) flash of light I(ti) is determined by use of a transient recorder and fit to Eq. (6). An average decay rate ? = (A1k1 + A2K2)/(A1 + A2) is determined, and ? used for the Stern-Volmer plot of Eq. (1). (ii) The period of linear decay of luminescent emission is determined from the I(t) data, and that intensity versus time profile is referenced against similarly obtained profiles for reference fluids of known oxygen concentration, using shapes, intensity or time setpoints. (iii) The sample is irradiated for some time interval (generally under 50 µs) using a flash lamp or a light emitting diode. Intensity segments of emission, I1 and I2, are determined during two time intervals defined with respect to the time of irradiation. These two intensity segments are compared to form a ratio R, and a calibration plot of R versus oxygen pressure is obtained using solutions of known oxygen concentration. Three different methods for determining I1, I2, and R are presented. These methods (i-iii) of measuring quenching are insensitive to variation in plastic thickness and emitter concentration of the probe and to decomposition during operation. Methods (i-iii) also take into account the non-exponential decay of the emission, and thus extend the pressure range over which the probe is sensitive. Method (iii) requires at most one point calibration against atmospheric oxygen.
Also disclosed are photostable luminescent molecules for use with the subject method. In a preferred embodiment platinum tetra(pentafluoro-phenyl)porphyrin, Pt(TFPP), serves as the luminescent oxygen quenching-sensitive molecule. Pt(TFPP) has a strong absorbance in the visible region, a strong phosphorescence with lifetime of roughly 100 µs, and is photostable.
Photostability is provided by the substitution of fluorine atoms in the periphery of the synthetic prophyrin ring. Other suitable fluorinated luminescent molecules include metallo derivatives, particularly platinum and palladium derivatives, of partially or fully fluorinated octaethylporphyrin, tetraphenyl-porphyrin, tetrabenzoporphyrin, or the chlorins, bacteriochlorins, or isobacteriochlorins thereof. The latter reduced porphyrins have the advantage that their absorption is red-shifted to a region for which light emitting diodescan be used for excitation.

Description

~3~ 16 ~ 2839-925 METHOD AN~ COMPOSITION FOR MEASURING OXYGEN CONC~NTRATIO'N
Field of the Invention This invent,ion relates generally to the measurement oE
oxygen concentration using the quenching of emission of a luminescent aromatic molecule embedded in a plastic medium.
Background of the Invention It is known that a luminescent aromatic molecule embedded in plastic is subject to quenching by oxygen present in the gas or liquid in contact with the plastic. This phenomenon was reported by Bergman (Nature 218:396, 1966), and a study of oxygen diffusion in plastic was reported by Shaw ~Trans. Faraday Soc. 63:2181~2189, 1967). Stevens, in U.S. Patent No. 3,612,866, ratios the luminescence intensities from luminescent materials dispersed in oxygen-permeable and oxygen-impermeable plastic films to determine oxygen concentration. Lubbers et al. in U.S. Patent No. 4,003,707 proposed the possibility of positioning -the emitting substance at the end o an optical fiber. Peterson et al. in U.S. Patent No. 4,476,870 also employs the quenching of an emitting molecule in plastic at t'he end of an optical fiber. Both Lubbers and Peterson reference emission against scattered exciting lightO
The quenching of the luminescence of an emitter at the end of an optical fiber has been used in temperature sensors. For temperature probes the emitters are generally solid phosphors rather -than an aromatic molecule embedded in plastic, since access by molecules from the environment is not desirable. ~arious methods have been used to measure the amount of quenching~

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- 2 - 62839-g25 Quick et al. in U.S. Patent No. 4,223,226 ratios the intensity at one wavelength of the emission against another; (ii) Quick et al~
also proposes determining the length of time it takes for the signal to fall from one level to another, (iii) Samulski in U.S.
Patent No. 4,245,507 (reissued as Patent No. Re. 31,832) proposes to measure quenching by determining the phase of the emitted life.
In a very recent patent for temperature sensing at the end of an optical fiber, Hirschfeld in U.S. Patent No. 4,~42,987 proposes, in addition to method (i), that (iv) emission lifetime be used to measure quenching and that (v) Raman scattered light can be used as a reference~
Eastwood and Gouterman (1970) noted generally with respect to Pd and Pt porphyrin complexes that their "relatively high [emission] yields and short triplet lifetimes... may make these systems useful as... biological probes for the presence of oxygen." More recently, Bacon and Demas in UK Patent Application No. 2,13234~A propose the use of, inter alia, porphyrin complexes of vo2+, Cu2+, Pt2+, Zn2+ and Pd2~ or dimeric Rh, Pt, or Ir comple~es for monitoring oxygen concentra-tion by emission quenching of intensity or life-time. Suitable ligands would reportedly by etioporphyrin, octaethylporphin, and porphin~
Summary of the Invention The present invention provides a method of measuring oxygen concentration in a fluid, comprising the steps of:

.
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- 2a 62839-925 (a) contacting a test fluid with a sensor composition comprising at least one luminescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light containing wavelengths strongly absorbed by the luminescent substance, (c) terminating the irradiation of step (b), (d) measuring the flux of luminescent light emitted by the sensor composition during at least two time intervals comprising at least one time interval subsequent to step (c), (e) comparing thee flux values measured in step (d) to obtain a rationing R value, (f) determining the oxygen concentration in the test fluid by comparing the R value obtained in step (e) with a similarly obtained R value for a-t least one reference fluid of known oxygen concentration.
The invention further provides a photostable, fiber-optic oxygen sensor comprising a partially or wholly fluorine-substituted luminescent substance, whose luminsecent emission is sensitive to quenching by oxygen, disposed in a light path of at least one optical fiber.
Methods and compositions are described for measuring oxygen concentration, particularly -for monitoring oxygen in the blood with a -fiber optic catheter. Oxygen concentration is determined by observing quenching of the emission from a luminescent (phosphorescent or fluorescent) molecule embedded in ..~.~

7 ~
- 2b - 62839-925 oxygen-permeable plastic. A test fluid of unknown oxygen concentration is contacted with a plastic film containing at least one luminescent substance. The film is subjected to irradia-tion over some period of time by light of a wavelength tha-t is strongly absorbed by the luminescent substance, and a measure of the time dependence of luminescent emission intensity I(t) is obtained.
Three modes of determining oxygen concentration from I(t) are described. (i) Subsequent to a brief (approximately 5 ~s) flash of light I(ti) is determined hy use of a transient recorder and fit to Eq. (6). An average decay rate k=(Alkl+A2k2)/(Al~A2) (7) is determined, and k used for the Stern-Volmer plot of Eq. (1).
(ii) 'rhe period of linear decay of luminescent emission is determined from the I(t) data, and that intensity versus time profile is referenced against similarly obtained profiles for reference fluids of known oxygen concentration, using slopes, in-tensity or time setpoints. (iii) The sample is irradiated for some tlme interval (generally under 5 ~s) using a flash lamp or light emitting diode. Intensity segments of emission, Il and I2, are determined during two time intervals defined with respect to the time of irradiation. These two intensity segments are compared to form a ratio R, and a calibration plot of R versus oxygen pressure is obtained ~O~, . . . ~ .

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~~' iL3~38~7 using solutions of known oxygen concentration. Three different methods for determining Il, I2, an~ R are presented. These methods (i-iii) of meQ~uring quenching are insensitive to variation in plastic thickness and emitter concentration of the probe and to decom7position during operation. Methods (i-iii) also take into account the non-exponenti~l decay of the emission, Qnd thus extend the pressure range over which the probe is sensitive. Method (iii) requires at most one point c~libration against atmospheric oxygen.
Also disclosed are photostable luminescent molecules for use with the subject method. In a preferred embodiment platinum tetra(pentafluoro-phenyl)porphyrin~ Pt(TFPP), serves as the luminescent oxygen quenching-sensitive molecule. Pt(TFPP) has a strong absorbance in the visible region, a strong phosphorescence with lifetime of roughly 100 ,u s, and is photostable.
Photostability is provided by the substitution of fluorine atoms in the periphery of the synthetic porphyrin ring. Other suitaMe fluorinated luminescent molecules include metallo derivatives, particularly platinum 7and palladium derivatives, of partially or fully fluorinated octaethy}porphyrin, tetra-phenylporphyrin, tetrabenzoporphyrin, or the chlorins, bacteriochlorins, or isobacteriochlorins thereof. The latter reduced porphyrins have the advantage that their absorption is red-shifted to a region for which light emitting diodescan be used for excitation.
Brief Description of the Drawings FIGURE 1 is a block diagram of a representative system 10 for measuring oxygen concentration by the emission lifetime method of this invention;
FIGURE a is a graph showing phosphorescence emission of Pt(TFPP) decay curves as a function of m7olecular oxygen pressures, as describedin Example 1;
FIGURE 3 is a graph plotting k/ko and To/T versus P02 for the decay curves shown in FIGURE 2 and the datQ presented in Table t;
FIGURE 4 is a representative plot showing the two intensity segments Il and Ia QS used in Equation t9) in conjunction with system 10;
FIGURE 5 is a plot of 1~ versus PO2 for the em7~odiment shown in FIGURE 4;
FIGURE 6 is a block di7~gram oî representative systems 10' and 10"
suitable for monitoring oxygen concentration in the bloodstrenm;
FIGURE 7 is a representative plot showing the two intensity segments I1' and I2' as used in EquRtion (10) in conjunction with syste7n 107~

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- ~ ~ 62839-925 FIGURE 8 is a plo-t of R' versus P02 for the embodimen-t shown in FIGU~E 7;
FIGURE 9 is a .represen-tative plot showing -the two intensity segments Il" and I2l' as used in Equation (ll) in conjunction with system lO";
FIGURE 10 is a plot of R" versus P02 for the embodiment shown in FIGURE 9;
FIGURE 11 presen~s Stern-Volmer plots for P-t(TFPP) in various film matrices containing various amounts of plasticizer, as described in Example 3, and, FIGURE 12 presents Stern-Volmer plots of k/ko for films containing various mixtures o Pt(TFPP) and Pd(TFPP), as described in Example 4.
Detailed Description of the Preferred Embodiment The present invention addresses several previously unrecogni~ed problems inherent in the prior art. In particular, we have found that the molecules studied by Eastwood and Gou-terman are subjec-t to photodecomposition when exposed to light and oxygen for extended periods of time. For best pho-tostability the previously unreported fluorinated derivative platinum tetra(penta-fluorophenyl)porphyrin Pt(TFPP), must be used. ~As used herein, the term "tetra(pentafluorophenyl)porphyrin" refers -to the identical compound as the term "tetraperfluorophenylporphyrin".
Furthermore, we have found that the phosphorescent emission decay in plastic is not a simple exponential, and so conventional methods of analysis fail to provide a measure of : .

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- 4a - ~2839-925 quenching that is useful over a wide range of oxygen pressures.
In the next paragraphs we review the luminescence and quenching of aromatic molecules and then present our method for using the observed nonexponen-tial decay to accurately and conveniently determine oxygen concentration.
Photoexcitation of many molecular systems leads to metastable excited states that relax to the ground state by emitting light. Metasta'ole photoexcited states are of two types:
(i) those wi-th the same spin as the ground state and (ii) those o~
different spin from the ground state. Fmission from metastable excited states of type (i~ is called fluorescence and is generally accomplished in under 0.2 microseconds. Emission from metastable excited states of type (ii) is called phosphorescence and is generally accomplished in times ranging from 1 microsecond to 20 seconds.

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.
Both types of emission nre quenched by oxygen. The first order dec~y rQte of emission ~k) of an excited state in the presence of oxygen is given by:
knat + kd ~ kq [ 2] = ko ~ kq [ 2] = 1/T (1) wherein knat is the natural radiative lifetime of the metastable excited state, kd is the rate of any intrinsic rQdiationless decay processes, kq is the quenching rate of oxygen, [021 is the oxygen concentration, and ko is the decay rate in the absence of oxygen. The inverse of k is called the emission lifetime, T.
The amount of light emitted by a sample is called the guantum yield, ~, ~nd is defined as:
~ = (photons emitted/photon Qbsorbed) = knat/k. (2) It follows from Equations (1) and (2) that the quantum yield in the absence of oxygen,~O, divided by the quantum yield in the presence of oxygen,~, is given by:
~ 0/~ kq~O2]. (3) Equations (1) nnd (3) are two forms of the Stern-Volmer equation for the e~fect of oxygen on quantum yield. This relationship is the basis for oxygen monitors that study the intensity of emission (1), which is directly proportionQl to quQntum yield as follows:
Io/l = 1 ~ kq [ 2] (41 wherein Io and I are respectively the emission intensities in the absence and - presence of oxygen.
It can be seen that oxygen quenching can be determined by - monitoring either decQy rate QS in Equation (1) or decay intensity QS in Equation (4). For both types of measurements it is necessary to calibrate the sensor against reference solutions of known oxygen concentrations in order to determine the quenching p~rameter kq. If decay rate is to be used, it is necessary to measure the emission intensity at a series of times, t1, t2, t3 ....
after the exciting light has terminated. ~or a simple exponential decay two suchtimed measurements will suffice, and decQy rate is determined from the -~` equation:
- k = (t2-tl~ 1 ln[I(t1)/I(t2)] . (5) If intensity is used as in Equation (4), then it is necessary to reference intensity I to the intensity Io in the absence of oxygen. ~s mentionedQbove, Stevens proposed obtaining Io from a second probe from which oxygen is excluded by an oxygen impermeable varnish. The Stevens configuration is impractical for fiber optic probes. Lubbers and Peterson determine Io from scattered light. Both methods of measuring lo have the intrinsic limit~tion thatthey do not t~ke ~ccount of photodegrad~tion of the sensing molecule th~t m~y ~'~" ... . .

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~3~76 occur during operation and that would reduce light output. Also, depending on the ability to m~nufacture exactly ;dentical sensors, each pQrticular probe mQy need calibr~tion. Accordingly, Peterson cclibrates each probe llt three points:
no oxygen, oxygen ~t the pressure of air, and at an intermediate oxygen pressure.
In contrast to the intensity measurement of Equation (~), the decay rate measurement of Equ~tion (5) ~voids difficulties cQused by variability in sensor manufacture and by photodegr~dation. Ilowever, the oxygen sensors of Stevens, Lubbers et al., and Peterson et al. are all based on the quenching of fluorescence, which decays gener~Uy in under 0.1 microsecond (~IS) and so requires fast electronics and fast light flashes to measure decay rate. This muyaccount for their preferred use of the intensity ratio of Equution (4). The molecule used in the preferred embodiment of our oxygen sensor has Q decay time in the lO0 s time range, SQ that slower el~ctronics and exciting light flashes can be used, makillg the study of quenching through dec~y rate very practical. Furthermore, the use of decQy rate as in Equation ~1) rather than intensity ratio as in Equation (4) in principle makes our sensor insensitive to both variation in the probe manufacture teg., small variations in plastic thickness and emitter concentration) and to the formation of nonluminescent photoproducts during operation. In principle no calibration is necessary, but in practice we have found that one point calibration in air increases the accuracy of measurement by different probes.
While Equations (1) and (4) ~re generally considered to be equivalent measures oî oxygen quenching, a serious problem arises in determining decay rate of aromatic molecules in plastic, since we find that suchdecay is generally nonexponentiQI. That is, the decay time of luminescent aromatic molecules in plastic cannot be fit by a simple exponential but must rather be considered as n sum of two exponentials:
I(t) = A1e k1t ~ A2e k2t (6) wherein e is the exponentiEIl function. The four fitting parameters Al, k~, A2, k2 can be fit if I(t) is measured at many times t following an interval of photoexcitation. This type of determination requires considerable instrumentation and software analysis that may not be practical for routine operation. Furthermore, even given the knowledge of these four p~rameters, it is not clear how best to employ them to determine oxygen concentration because in the absence of an exponential decny the Stern-Volmer Equations (1) ~nd ~3-) no longer apply.
To overcome these problems, we provide moIecules h~ving R decay time sufficiently long so that oxygen concentration can be conveniently . ..
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~7--determined through a measurement of the time dependence of emission intensity, i.e., I~t). We thereby avoid problems due to variatiors in probe construction and photodegradation that arise using methods th~t me~lsure total intensity and its ratio to a reîerence, i.e., ns in Equation (4). We also provide convenient methods of determining oxygen concentration from the function I(t), even thou~h I(t) is non-exponential.
FIGURE 1 shows a representative system 10 for determining the oxygen concentration of gaseous samples by meQsuring the quenching of various emitting sensor compositions in pl~stic. A flashing light source 12 (e.g., Strobota-~ Model No. 1538A) provides time dependent light exclt~tion (indicated by dashed arrow 14) of an oxygen quenchin~sensitive composition, which is sequestered in film 16 in this embodiment. Film 16 is mounted inside the fluid, depicted QS vapor 18 here, that is to be sampled. Phosphorescent light (dashed arrow 20) emitted from film 16 impinges on a photodetector 22 (e.g., RCA 7265).
A housing 24 containing windows 26, 28 can be used to isolate film 16 inside thefluid 18 being monitored, which is typically not identical with the environment of the rest of system 10. Windows 26, 28 can be made of quartz or glass.
Key components of system 10 are filters 30, 32. Filter 30 is a band-p~ss filter (e.g., EDling 35-3649) that ~llows only shorter wavelength light 14, e.g., wavelengths in the range 480 to 600 nanometers (nm), to impinge on film 16. The range of band-pass filter 30 is chosen to match the region of strong absorption by the phosphorescing compound thQt is sequestered in film 16.Filter 32 is a cutoff filter (e.g., Corning 261) that allows only long wavelength light 209 e.g., wavelengths longer than 620 nm, to impinge on photodetector 2a and is chosen to allow the phosphorescent Iight 20 to reach the photodetector 22.
In a preferred embodiment, the ban~pass of filter 30 and the cutoff of filter 32are complementary such that no light 14 from flashing light source 12 reaches photodetector 22.
The electric output (arrow 38) of photodetector 22 passes into preamp~ifier 40 of standard design. In the embodiment shown here, the output (arrow 42) of preamplifier 40 passes into a transient recorder 44 (e.g., Biomation Model No. 805) that is capable of sampling intensity, I(ti), Qt time intervals below a micro~econd. The timing of system 10 is under control of microcomputer 36. In Q preferred mode of operation that provides a zero baseline, microcomputer 36 puts out a trigger (arrow 46) to activate transient recorder 44 and after ~ slight delay on the order of 20 microseconds puts out anothet trigger (arrow 48) to start flash light source 12. TrQnsient re¢order 4 thus collects data relating to l~ti), the intensity I o~ ph~phore~sccnce at variQus .~

3~87~ --times ti before and after the flash, which data are read (as indicated by arrow 50) into the computer 36.
FlRsh rates using system 10 are typically on the order of 100 per second. The time interval between successive flQshes 14 is sufficiently long that 5 a decay time and hence oxygen concentration can be cQlculated after each f~ash 1~. It should be noted that the l;miting time of response is set by the diffusion rate of oxygen into the monitor film 16, which is typically under one second. This diffusion rate becomes faster, permitting response times on the order of milliseconds, with thinner films 16 and by adding plasticizer 10 to the carrier matrix~ as described below. Thus, real-time meQsurements of oxygen concentrations can be made by this method.
Pursuant to this disclosure, measurem ent is made of the intensity, I~ti), of phosphorescence 20 emitted by the film 16 at a series of times, t1, ta~ t3 . . ., after the flash 14. The light intensities I(ti) at any 15 particular time ti following several flashes can be averaged. A decay rate, k, of the phosphorescence is calculated from these data by the computer 36 using various ~Igorithms. In particular, with a full set of values I(ti) we can fit the decay to Eq. (6) above. Decay curves for Pt(TFPP) in polyvinyl chloride with plasticizer are shown in FIGURE 2, wherein t' is the period of linear decay, i.e., 20 where ~I/at has a constant slope at Qny particular oxygen pressure of interest, and wherein t" is the remaining period of the detectable emission, during which al/at is not constant. l~epresentative values found for the fitting parameters A1~ k1, A2, k2 ure listed in Table 1.

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g DRta analysis of PtTFPP in PLS/PVCa'b'C.

P2 A1 kl A2 k2 k T k/ko To/T
.
1. 13.1 13.1 ,76.3 1. 1.
0.~8 13.260.12 2~.2 14.6 71.3 1.11 1.07 0.78 15.030.22 26.5 17.6 60.3 1.3~ 1.27 100 0.6~ 16.230.36 31.4 21.7 50.9 1.66 1.50 20û 0.63 18.250.37 41.S 26.8 ~3.4 2.05 1.76 300 0.58 l9.a30.~2 ~5.4 30.2 39.4 2.30 1.94 400 0.33 17.00.67 39.~ 32.0 36.5 2.44 2.09 500 0.3~5 17.20.695 ~1.1 33.~ 3~.6 2.58 2.20 a. PLS/PVC = plasticized polyvinyl chloride; see ExQmple 1 for details.
b. Data taken by system 10.
c. The k's are in (ms) 1, and ~ is in ~ s.
... .... .......... ... .. _ .. ... .... . . _ Two measures for the time decay can be defined: AverRge decay rQte, k, is given by:
k = (A1k1 ~ A2k2)/(AI ~ A2)-Average decay time, T, corresponds to the normalized integral of the emission intensity, I(t3, as follows:
~ ~ (A kl-1 + A2k2 )/(A1 + A2). (8) T represents the total amount of light emitted oUowing termination of excita-tion, which hQs been the principal measure of oxygen quenching in the prior art;however, the prior art did not consider the disclosed nonexponentiQl decay phenomenon. The value 1~ corresponds to the normalized decay rate at t=0. In FIGURE 3 we plot representative To/T and k/ko curves using the data listed in Table 1. These curves would be identical for an exponential decay. FIGURE 3 shows that k/ko gives a more lineQr Stern-Yolmer plot; hence k provides a more accurate`determination of oxygen concentration at higher oxygen pressures.
The double exponentiQl decay of Eq. (6) can be understood as resulting from two types of emitting molecules: Those with larger kl are more subject Qnd those with smaller k2 are less subject to quenehing by oxygen. The uverage decOEy time, ~, gives heavier weight to the unquenched molecules, .~

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.
.

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whereas the average decay rate, k, gives heavier weight to the quenched population and provides a better me~sure of oxygen quenching.
In a related method, the slope of the emission profile during the period of lineQr decay is compQred with similarly obtained slopes for fluids of 5 known oxygen concentrations. Due to the double exponential nature of the luminescent decay curve, the referenced slope values must not encompass any of the tail region (t" in Fig. 2) of the emission profile. Once this relationship is established, the time it takes for the intensity of emission to fall to Qny particular level within time t' can provide a convenient readout of PO2.
10 AlternQtively, the intensity measured with the test fluid at any particular time t less than t' can be compared with standard curves of intensity versus time (again, less than t') for a series of fluids of known oxygen concentrations.
We have also discovered other convenient methods of measuring PO2, taking into consideration the nonexponential decay of 15 luminescent molecules in plastic. The following methods reference one time segment of emission against ~nother and thus provide measurements that are independent of sensor configurRtion or light path variation. With system 10, thebest measure for oxygen quenching is the intensity ratio, R, given by:
R = (11 - I2)/(I1 12)-20 wherein 11 and I2 Qre the sums of the intensities, or total flux of luminescent light! measured by the transient recorder during two time periods that together substanti~lly encompass the period of line~r decay of the luminescent emission once the light source 12 is turned off. With system 10, the time intervals for I1 ~nd I2 can bc re~dily optimized for particulQr combinations of luminescent 25 substance, m~trix, and oxygen pressure range of interest by samplin~ time intervQls Qnd selecting those that provide the best resolution within the lineardecay period. For example, with n Pt(TFPP) film 16, 11 CQn be the flux of phosphorescence detected over times from 0 to 10 lls, and 12 can be the sum of the intensities over times from 10 to 20 IJS, with time zero for both 30 measurements being the end of the period of illumination. FIGURE 4 illustrates the two intensity segments Il and I2, and a corresponding plot o' R versus P02 ~oxygen pressure) is shown in FIGURE 5. It is not necessary for the selected time intervals to encompass the entire period of lineQr decay. However, Il and 12 preferaMy subdivide the period of linear decay (t' in Fig. 2) into two equQl 35 time periods.
The subject method can be used to measure oxy~en concentrQtion in a fluid, meaning gas and/or liquid. For exQmple, the oxygen concentration in closed and semiclosed atmospheres 18 such as aircraft Qnd mines can be . - '. ~

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mensured using a detection system similar to 10, shown in FIGURE 1. Oxygen concentrations in liquids such as blood, seawater, and sewage can also be monitored by the method of this invention.
FIGURE 6 shows representntive systems 10', lû" for monitoring oxygen in the bloodstreQm 18'. The following components are identical to those described above for system 10: sensing composition 16, phototube 22, filters 30,32~ microcomputer 36, and preamplifier 40. In systems 10' and 10" the film 16containing the oxygen quenching-sensitive composition is positioned at the end of ~n optical pipe 56, which carries the exciting light to and the emitted light from the film 16. In system 10' the exciting light source 12' is a flash lamp, while in system 10" the exciting light source 12" is a light emitting diode (LEV). The exciting light from light source 12' or 12" passes into light pipe 55. The emitted light exits through light pipe 57. The light pipes 55, 56, 57 are connected by astandard three-way optical coupler 58 (e.g., GTE ATA No. OCL~0102-X). In system 10' the output ~2 from the preamplifier 40 passes into a fast AlD
converter 44' (e.g., the I.AB-40 manufactured by Computer Continuum, D~ly City, CA 94015). In system 10" the output from the preamplifier 40 passes into aspeci~lly designed electronic circuit 4~", termed herein an integral ratio determinator, which calculates R" using Eq. (11) below.
l~sing system 10', oxygen quenching is mensured as the intensity - ratio, R', given by:
R~ /I2~ (10) wherein Il' a~id I2' represent the fluxes of luminescence over two time periods thnt substantially encompnss the period of detectnble luminescent emission once 25 the light sollrce 12' is turned off. With system 10', time intervals are selected from the lamp-off regions of linear t' and nonlinear t" decay to provide ratioing R values of Il' and I2' that give the best resolution within the oxygen pressurernnge of interest. I1' preferably encompasses the period of linear decay of the luminescent emission. For example, with a Pt(TFPP) film 16, Il' can be the sum 30 of the intensities I(ti) digitized by the fast A/D converter over times from 0 to about 20 l~s, and I2' can be the sum of the intensities over times from 20 to 300 ~s, with time zero for both mensurements being the end of the period of illumination. FIGURE 7 illustr~tes the two intensity segments Il' and I2', and acorresponding a plot of R' versus P02 is shown in FIGURE 8.
With system 10" the LED is turned on, for exnmple, from time 0 to approximately 50 us. Here the measure of oxygen quenehing is the intensity ratio, R", given by:
E~ t'=I "/I " ( 11) . ~
. ~ ", "~,,. j ~

.

wherein 11" is the nux of luminescence during a first time interval taken before- the light 12" i9 turned off, and I2" is the flux of emitted light during Q second time interval taken subsequent to extinguishing the light source 12". I1"
preferably encompQsses the entire time period during which light source 12" is 5 turned on, and I2" preferQbly encompasses substantially the entire period of detectable luminescent emission subsequent to turning of ~ the light source 12".The light source 12" is most preferably turned on only until the detected luminescent intensity plRteaus, meaning nttains its maximum intensity, QS shown in FIGURE 9. For the representative Pt(T~PP) embodiment, I1" cnn be the 10 intensity of emission gQthered over the time period from ~ to 50 ~s, i.e., while the LED is on, and I2" can be the intensity of emission gathered from 50 ~s to 300 ~ s, sfter which there is no more emitted light. In the integr~l ratio determinator 44" the signal 42 from the preamp 40 is split by two balQnced synchronous demodulators into the pulse Qnd decay (light 12" on und off) 15 components. Circuit ~4" then integrntes the pulse and decay components separQtely, passes the signals through V/F converter channels, and respectively counts each component for the digitized equivalent. FIGURE 9 illustrates the two intensity segments I1" and I2", and a corresponding plot of R" versus P02 isshown in FIGURE lû. We found with system 10" that QppQrently identical 20 sensors give calibrQtion plots Of P02 versus R" that are parQilel but shifted from one another. Thus, to obtain Qccurate values of P02 each sensor should be c~librated Qt one pressure, preferably the oxygen pressure of air.
The disclosed method uses QS emitters met~llo-organic molecules, in particular Pt ~nd Pd derivatives of porphyrins. The organic region of these 25 molecules is where light absorption and emission occurs, and this provides Q
strong ~bsorption coefficient. Moreover, their natural phosphorescence decay rate, lcnat, has been greRtly increased by the metal atom so they have a high quantum yield of phosphorescence. Furthermore, their phosphorescence decQy time is sufficiently short so that it is in a convenient range to monitor for 30 oxygen quenching.
A suitable oxygen quenching-sensitive monitor 16, or sensor com position, f or practicing this invention contQins at least one species of phosphorescent molecule having the following properties (a) through (i3:
(a) The phosphorescent molecule should hQve R high absorption 35 coefficient for light obtained from a convenient flashing light source~
~ b) The quantum yield of phosphorescence should be large so that there is sufficient light to worlc with, roughly ~ > 0.2.

. . .
j, , ~ .

~1 ~3~g76 (c~ The phosphorescence lifetime in the absence of oxygen should be in the range 50 microseconds to 5 milliseconds. With too short a lifetime, app~ratus becomes more expensive. With too long a lifetime, the emission will tend to be entirely quenched in higher oxygen ranges of interest.
(d) The wavelengths of strong Qbsorption Rnd strong emission should be well sep~rated so that complementary filters can be obtained to isolate these spectral regions.
(e) The emission should be in the red, with wavelengths longer than 600 nanometers, as this avoids competition from extraneous emission that might occur from the sample or from windows, filters, and opticQl pipes.
(f) The phosphorescent molecule should be stable to photooxidation, since they will be used under illuminQtion and with oxygen present.
(g) The phosphorescent molecule should be insoluble in the fluid being monitored so that it does not leach out of the monitor. ~or example, for monitoring blood the molecule should not be water soluble. On the other hnnd, the molecule must have solubility suitable for monitor fabrication.
(h) The nQtursl and radiationless decay times, knQt and kd in Eq. (1), should be insensitive to sm~ll temperature changes.
(i) The emission properties should not be sensitive to common molecules, e.g., H2O, CO2, Na~ and to other molecules likely to be present in the fluid to be monitored, e.g., halothane or N2O in blood.
The phosphorescent molecule(s) having the foregoing properties cQn be sequestered in a carrier matrix to form an oxygen monitor lG. The following characteristics (j) through (l) apply to both the phosphorescent molecule and the carrier matrix:
(j) I`he phosphorescent molecule(s) and the carrier matrix should be chemically stable, so that they do not deteriorate.
(k) The phosphorescent molecule(s) and the carrier matrix should not be toxic.
(l) The phosphorescent molecule(s) ~nd the carrier matrix should be conveniently synthesized or purchased.
The following prop2rties (m) through (s) apply to the carrier matrix, which is preferably an oxygen-permeable plastic:
(m) The c`arrier matrix should dissolve or bond with the phosphorescent molec~e and be easily cast onto the substrate which holds it.
(n) The carrier matrix must be porous to oxygen and should equilibrate over convenient times, since this equilibration rate determines the "~
, ~

:

response time of the oxygen monitor. We have found that the response time can be shortened by the amount of plQsticizer in a PVC carrier matrix.
(o) For oxygen concentrations, [2] fl~ of interest in the fluid being monitored, the equilibrium oxygen concentration in the c~rrier matrix, 5 ~2] mx~ and the quenching constant, kq, should be of size:
kq[2] mx knat + kd wherein these quantities are those of Eq. (1). If the left side of Eq. (9) is much larger th~n the right side, then little emission is observed; if the left side is much sm~ller than the right side, then little quenching is observed. Thus the 10 ` product kq[2] ~T~x must be adjusted to match the parameters knE~t ~ kd of the phosphorescing molecule and the oxygen concentration [ 2] n in the fluid to be monitored.
(p) The carrier matrix should stick to the substrate on which it is mounted, e.g., the end of a light pipe 56 as in FIG. 6.
(q) The carrier matrix should be nonvolatile and insoluble in the fluid being monitored.
(r) The carrier matrix should be chemically stnble with respect to the fluid being monitored, the phosphorescent molecule in its ground state, and the phosphorescent molecule in light in the presence of oxygen (s) Finally, the carrier matrix should be transparent to the exciting light ~nd to the phosphorescent light.
Suitable oxygen quenching-sensitive phosphorescent molecules for practicing this invention include porphyrins, meaning those compounds that contain the porphyrin ring structure (Monograph No. 7468, Tenth Edition of The 25 Merck Index, Merck ~ Company, Inc., Rahway, NJ, 1983), chlorins, bacteriochlorins, and isobacteriochlorins. The porphyrin ring structure gives rise to intense optical absorp~ion and emission in the wavelength r~nge of interest.
The wavelengths for absorption and emission can be shifted by various chemic~l modificQtions to the porphyrin ring structure. In addition, the emission lifetimes 30 and quantum yields are strongly dependent on any metal incorporated into the center of the ring. The preferred synthetic rings for employment in the practiceof this invention are tetra(pentafluorophenyl)porphyrin (TFPP), octaethylpor-phyrin (OEP), tetraphenylporphyrin (TPP), ~nd tetrabenzporphyrin (TBP~
compounds. Preferred metals are second and third transition row metals with 35 electron configurQtions d6 or d8, including Ru(II), Rh(IIIj, Pd(II), Os(II), Ir(III), Pt(lI), and Au(III). All of the aforementioned metalloporphyrins show phosphorescence with moderate quantum yields and suitable lifetimes. However, the Pd(II) and Pt(IIl derivatives ure the most preferred since only they are free of ....
~. . ~ .

`J ~ 3 ~

nxiQl ligands ~nd/or counter ions, which can complicate the synthesis and can introduce instabilities either during film prep~ration or during operation underlight; moreover, they have the highest quRntum yields of emission. Some other metalloporphyrins may also be suitable; for example, we have had some success using Hf(IV) octaethylporphyrin.
Pd(II) and Pt(II) complexes of tetra(pentafluorophenyl)porphyrin (TFPP), octaethylporphyrin (OEP), tetraphenylporphyrin (TPP)~ and tetrabenz-porphyrin (TBP) have proven in our hands to be the best phosphorescent molecules for practicing this invention as they tend to satisfy most of the abov~
stated c~iteria (a) to (i). Moreover, the palladium, Pd(lI), porphyrins have lifetimes in the absence of oxygen of 1-2 milliseconds and the platinum, Pt(II),porphyrins of Qround 100 microseconds. As Q result, these Pd (II) and Pt(II) porphyrins are most sensitive in different regions of oxygen concentration: In particular, the pQlladium porphyrins are best used under 10 torr pressure, whilethe platinum porphyrins cnn best be used above 50 torr pressure. By employing in the oxygen quenchin~sensitive composition ~ combination of different phosphorescent molecules having different phosphorescent lifetimes, e.g., any ofthe above-stated Pd porphyrins along with any of the above-stated Pt porphyrins,n wider range of oxygen pressures can be monitored than by use of either phosphorescent molecule alone.
We have ~lso discovered that the range of sensitivity for any particulRr phosphorescent molecule used in the oxygen quenching-sensitive composition can be adjusted by choice of the carrier matrix ~nd also the amount of plasticizer dissolved in the matrix. Polyvinyl chloride with variable amounts- 25 Of plasticizer provide suitable carrler matrices, as does polymethyl rnethacrylate without plasticizer. Other suitable oxygen-permeable matrices can be made of cellulo6e acetate or silicone-polybicarbonate copolymer (Petrarch MB).
We hQve also discovered that Pd and Pt tetraphenylporphyrin Qnd tetru(pentafluorophenyl)porphyrin show a specific ~bsorption band proportional to the deterioration of the compound. This bnnd lies in the wavelength range 550-620 nm, depending on the compound. This new band may be due to Q photo-oxidation product. The appearance of the specific band upon prolonged irradiation can be used to provide a quality control check of photodeteriorationto determine when the monitor film should be changed.
As mentioned above, the Pd(lI) and Pt~II) porphyrins with the rings OEP, TBP, TFPP, and TPP ~re the preferred phosphorescent molecules for use in the disclosed measurement methods. All of these preferred molecules are in addition reasonably stable when freshly synthesized Qnd over the time course of 7 ~

typical laboratory analyses of absorption and emission properties. However, we found that some of these eight species proved less stable than others when subjected to extended illumination and ambient oxygen. Not all of the Pd and Pt porphyrins in polyvinyl chloride containing plasticizer are sufficiently stable 5 under extended illumination to be suitable for oxygen sensors. The OEP and TBPrings deteriorated so readily that they were entirely gone, as evidenced by lackof absorption spectrum, after fifteen hours of illumination. The TPP ring provedmore hardy and showed a survival of emission intensity and lifetime slightly poorer than perylene dibutyrate (PDB), the molecule of choice for a fluorescent 10 oxygen sensor described in U.S. Patent No. 4,476,870 and in Anal.Chem. 56:62-67, 1984. The TFPP ring proved more hardy than PDB under -the same test conditions. The survival of the emission intensity of Pd(TFPP) wascomparable to that of PDB, while that of Pt(TFPP) was bett~r. The emission intensity of the Pt(TFPP) after 15 hours o~ illumination was 80% of its initi~l 15 value while the lifetime was 95% of its initial value.
Since luminescent compounds nre quite generally photooxidized in the presence of oxygen, it is critical to select a relatively photostable phos-phorescent or fluorescent molecule for use in luminescent oxygen sensors. The most preferred phosphorescent molecules for monitoring oxygen concentration 20 by any of the previously reported or subject methods therefore include Pd(TFPY) and Pt(T~PP).
Photooxidation of aromatic molecules is one of the most important processes by which compounds are degraded and undergo permanent chemical transformations. In general, photooxidation reactions of aromatic compounds 25 nre enhanced by more extended conjugation, higher electron density, and loweroxidation potentials. Porphyrin reactivity with molecular oxygen in the presenceof light is influenced by the inductive effects of the functionnl group attachedeither at the periphery of the porphyrin ring or in extraplanar ligands. In the case of TFPP systems we observed that the electron withdrawing effect of the 30 pentafluorophenyl substituents raises the oxidation potential and reduces theelectron density. These factors make the TFPP molecule less reactive toward photooxidation process and increase its photostability. The measured oxidation potentials of a series of free base porphyrins are shown in T~ble 2.

~,, ,~

~ 3~7~

Porphyrin oxidation potential.

Compound EX[Volts] Reference -H2OEP 0.81 J.Amer.Chem.Soc. 95:140,1973.
H2ETIO 0.77 J.Ph~s.Chem. 229:259, 1964.
H2TPP 0.97 J.Phys.Chem. 229:259, 1964.
H2TFPP 1.6 Our unpublished data.

__ Recently we have optically studied the pi-cation Eormed when thin film samples of various porphyrins, inlcuding Zn(TFPP), are exposed to various partial pressures of molecular oxygen and ligh-t. This work clearly showed that the concentration of cation formed depends upon the porphyrin ring oxidation potential. The Zn(TFPP) showed no evidence of cation formation when exposed to 760 -torr oxygen and white light -for 24 hours.
Pursuant to this aspect of the invention, other aromatic molecules can be made more photostable by subs-tituting fluorine atoms on the periphery of the synthetic, ring. Such complete or partial fluorine substitutions make the luminescent molecule less susceptible to photodeterioration. Photodeterioration is indicated by a diminution following exposure to illumination of the molecule's absorption spectrum and/or its emission peak ratios and li~etime ratios. The first step in photodeterioration probably involves electron loss from the luminescent molecule to ambient oxygen molecules, and we believe that the fluorinated sidegroups inhibit such transfers. We ~ave also observed that ~ .

:~ 3 ~ 6 ~ 2839-925 such fluorination serves to protect -the integri-ty of the phosphorescent molecule's emission lifetime profile more -than its emission intensity. Thus, these fluorinated porphyrins are particularly well-suited for monitoring oxygen concentration using the disclosed emission lifetime method.
Closely related molecules to Pd(TFPP) and Pk(TFPP) that may retain the advantage of photostability are the reduced ring chlorin [dihydroporp~yrin], bacteriochlorin [opposite tetrahydroporphyrin], and isobacteriochlorin [adjacent tetrahydroporphyrin], described in M~ Gouterman, Chapter 1, pp.
1-165, in the Porphyrins, Vol. III, D. Dolphin, Ed., Academic Press, N.Y. 1978. We have effected ring reduction of Pt(TFPP) using a two-fold excess o-f para-toluenesulfonylhydrazine, K2C03 (anhydrous in pyridine) at 100C for a few hours, using the method of Whitlock et al., J.Amer.Chem.Soc. 91(26 :7485-74~9, 1969. The reduced porphyrins o-f these and the other mentioned prophyrins have the advantage that their absorption is red-shifted to a region for which light emitting diodes can be used for excitation.
Another preferred group of photostable, phosphorescent molecules for incorporation in oxygen quenching-sensitive compositions must include Pd(II) and Pt(II) derivatives of fluorine substituted tetrabenzporphyrin (TBP), shown below and abbreviated TFBP.

.,~
.,~,,~; ,, , - 18a - 62~39-925 X X
x -~X
X ~ X X - H- TBP
X ~ I ~ X All of X = F: TFBP II
X 1 ~ ~ Any of X = F III

X~-X

X X

Because of the greater electron withdrawing power oE fluorine with respect to hydrogen, TFBP molecules are expected to prove more stable than TBP to photooxidation when exposed to light and oxygen. Par-tial fluorination to give compounds of formula III
should also enhance photostability.
Although the Pd and Pt tetrabenzporphyrins proved unstable under extended illumination, they exhibit certain other advantages as compared with the TFPP derivatives, In particular, since the TBP absorption maxima are further to the red, they are more suitable for excitation by available light emit-tiny diodes.
Also, since their emission is further to the red than Pt(TFPP) it is less absorbed by optical piping and is less subject to interference by extraneous emission. Thus it is contemplated that Pd(lI) and Pt(II) derivatives of the above-stated molecules II or III will provide photostability as well as a more convenient spectral range.
The following illustrative but nonlimiting examples further illustrate the invention.

- . _. , .

~ 3 ~

- 18b - 62~39 925 E~AMPLE 1 Pt(TFPP) in PVC.
Free base tetra(pen-tafluorophenyl)porphyrin, H2(TFPP), was made following Longo et al., J.Heteroc~cl.Chem. 6:927, 1969.
H2(TFPP) was purified , ~ .~ . . , 7 ~

by the procedure of Spellane et al., Inorg.Chem. 19:386, 1980, Q paper that describes preparation of Pd(TFPP). Pt(TFPP) WQS made from H2(TFPP) and a 10 times molar excess of PtC12 (Aldrich Chemical Co.) refluxed for 2~ hours in benzonitrile. The product was chromatographed on neutral alumina column with CH2Cl2 as eluant. 10 mg of the Pt(TFPP) were then dissolved in Q 25 ml aliquot of Q PVC stock solution made by dissolving 3 grQms of polyvinyl chloride (PVC;
E~.F. Goodrich) in 70 ml tetrahydrofuran and 200 ul of 2-nitrophenyl-octylether (Fluka AG) as plas~icizer. Samples are prep~red by cQsting the resulting solution on a glass slide Qnd allowing the tetrahydrofuran to evaporate. Films prepared in this manner were smooth and transp~rent.
One of the films WRS mounted inside of ~n aluminum sample chamber thRt WRS then evacuated. The chamber consisted of 3 optic~l flats to Qllow Qbsorption and emission datQ acquisition, ~nd~had Q valve assembly for controlling the pressure of Qmbient gases. This chamber allowed the lifetime Qnd intensity of the emission to be monitored QS functions of 2 pressure.
Measurements were made using the system 10 shown in FIGURE 1.
The decay of phosphorescence intensity QS a function of time at several oxygen pressures for this Pt(TFPP) in PVC film is shown in FIGURE 2, wherein curves 1 to 10 are respectively for 2 pressures of 1, 10, 50, 100, 200,300, ~oo, 50b, 600, and 730 torr. Plots of k/ko and To/T for this Pt(TFPP) in PVC film as ~ function of 2 pressure are shown in FIGURE 3.
EXA~MPLE 2 Pd(TFPP) in PVC.
Pd(TFPP) was synthesized following the procedure described in Inorg.Chem. 19:386, 1980. 10 n~g of Pd(TFPP) w~s then dissolved in a 25 ml aliquot of the PVC stoclc solution described in Example 1. Films of Pd~TFPP) in PVC were made as in Example 1. Phosphorescence emission intensity decay curves, I(t), of the Pd(TFPP) in plasticized PVC film were determined at variousmolecular oxygen pressures ranging from 1 to 600 torr. Stern-Volmer plots of k~ko and To/~ were also made for this Pd(TFPP) in PVC film as a function of oxygen pressure. The datR ~re shown in Table 3.

.

.
.. . ~ ;.

~3~7 -ao-Data an~lysis of PdTFPP in PLS/P~Ca'b'C.
P02 A1 k1 A2 k2 k T k/ko To/ T
. ~

0.91 1.050.09 0.79 1 ~ 02984 .1 0.80 1.340.20 2.92 1.656691.61 1. ~7 0.37 2.020.63 5.56 4.25295.5 4.16 3.33 100 0.32 2.92Q.68 9.43 7.351827.18 5.~1 200 0.26 3.730.7~ 14.08 11.3~ 123 11.1 8.0 300 0.30 4.890.70 18.52 14.~g8~4.1 10.0 500 0.15 5.240.85 19.61 17. ~ 72 17.2 13.7 Q. PLS/PVC = plasticized polyvinyl chloride as described above.
b. Data taken by system 10.
c. The k's are in (ms) 1, and T iS in us.

Alternative matrices nnd the effect o~plnsticizer.
Other polymer matrices such as polymethyl methacrylnte (PMM),cellulose acetQte (CA), and silicone polymer can be used as matrices for the Pt(TPPP,~, Pd(TFPP), or other luminescent aromatic molecule. In fact, these polymers are more permeable to gases than PVG and so cnn be used without plasticizer. For example, films of these matrices were made as described above from the following stock solutions that each contained 10 mg of Pt(TFPP):
PMM - 1 g of polymethyl methacrylRte ~Aldrich) and ao ml of tetrahydrofuran;
CA - I g of cellulose acet~te (Aldrich) and 20 ml of acetone; and, PMB - I g of dimethylsiloxane-bisphenol A-polycarbonate block copolymer (Petrarch Systems, Inc.) and 20 ml of tetrahydrofuran.
Quenching plots of the Pt(TFPP) in PMM, CA, and silicone PME~
films as a function of 2 pressure are shown in FIGURE 11.

.. ~ .

;. ~

~ 3 ~ 6 FIGUl?E 11 also shows data from two PVC films with different amounts of pl~sticizer, thus indicating the influence of plastici~er concentration on oxygen quenching. These films were made as described in Example 1 PVC-1 was made using 200 microliters of the plasticizer 2-nitrophenyloctaethylether, and PVC-2 WQS made using 500 microliters of the plasticizer.

Pt(TFPP) and Pd(TFPP) mixture in PVC.
Films of Pt(TFPP) and Pd(TFPP) mixture with different ratios of the phosphorescent species were made from the PVC stock solution described in Example 1. Quenching plots as functions of 2 pressure are shown in FlGURE 12 for two plasticized PVC films with 10:1 and 1:1 ratios of the Pd:Pt mixture.
Oxygen pressure sensitivity range can be adjusted by changing the concentration ratios of Pt(TFPP) and Pd(TFPP,l.

Light sensitivity st(!dies.
The stability of VRrious oxygen quenching-sensitive compositions under extended illumination was determined. Various metalloporphyrins were cast in polymer films and then exposed to extended illumination as follows: The light from a 2DO watt reflection bulb was shined on the films after passing through a water container, the glass bottom of the w~ter container, Rnd the plQ~tic of Q transpQrent pl~stic stand. Air was blown over the films as well. The purpose of the water and the airstream was to make sure that the films were not subject to heat stress. Also, by using the tungsten lamp nnd passing the light through water and plastic any high energy ultraviolet radiation was attenuated.
Short wave UV would not be present under the conditions of this setup.
Pd and Pt derivations of OEP and TPP were synthesized as described in J.Mol.Spectroscopy 35(3):359-375, 1970. Pd(TBP) and Pt(TBP) were synthesized as described in J.Amer.Chem.50c. 104.6278-6283, 1982. Pt(TFPP) and Pd(TFPP) were synthesized as described in Examples 1 and 2 above. The various metalloporphyrins were individunlly cast into polymer films as describedin Example 1.
The metalloporphyrins and films were prepared fresh for this test and were subjected to 15 hours o~ illumination in the above-described setup.
Absorptions, emission intensities, and emission lifetimes were measured before and after the 15 hour illumination period. ThP results are summarized in Table 4.

~, -'~ ~ 3 ~

SummQry of survival ~fter 15 hours of illuminQtion.
Compound Absorption Emissiona~ b LifetimesC
__ . . . . . . _ Pd(OEP) gone 5 Pt(OEP) gone Pd(TBP) gone Pt(TBP) gone Pd(TFPP) survives 65% 90-94~6 PtlTFPP) survives 8096 92-100%
10 Pd(TPP) survives 65~6 62-83%
Pt(TPP) survives 20% 58-63%
a) Average emission peulc intensity rutios nfter/before exposure to illumination.
b) Pd~OEP), Pt(OEP), Pd(TBP), Pt(TBP) showed no phosphore~scence emission after exposure to illumination.
c) k or T with system 10.

The absorption spectrum is a minimal test for survival of the compound. If the spectrum is gone, the compound is gona. The Qbsorption spectra of neither the OEP nor the TE~P rings survived 15 hours of illumination.The Qbsorption spectra for Pd(TFPP), Pt(TFPP), Pd(TPP), and Pt(TPP) showed growth of an impurity with an absorption ~t approximQtely 600 nm. For both the TFPP and the TPP rings the impurity appeared slightly blue shifted from Pd to Pt (with the shift more marked for the TPP rings). This Nould suggest that the impurity somehow contains or is associQted with the metal.
~or the TPP films the amount of emission was much more e~ensively quenched thQn for the TFPP fiIms. Since the test for 2 pressure depends on emission intensities Qnd lifetimes, it appeQrs that the TFPP
molecules Qre the only suitable rings with respect to survival of emission properties after extended illumination. Furthermore, the lifetimes of the TFPP
molecules appear to be the most h~rdy test for the amount of 2~ in the sense ofremaining constant aIter extended illumination.

... ~ ' . ' '.

. .

3~87~

Extended illumination of perylene dibutyr~te.
Peterson in U.S. Patent 4,~76,870 and in Anal.Chem. 56:62-67, 1984, describes an oxygen sensor based on the fluorescence quenching of 5 perylene dibutyrate (PDB). The latter paper reports: "A 5-day test showed an average loss of sensitivity of 6.5% per day due to continuous exposure to the blue excitation light when connected to a fiber optic sensor." We prepared Q sample of this same dye in PVC with plasticizer and subjected it to 15 hours of iUumination in the setup of Example 5. We observed that ~he emission decreased lO about 30% over this time period. Given the very different geometries of illumination for a fiber optic sensor and that of our setup, it is difficult to Icnow the relative light fluxes. However, it is not unreasonable to assume that we cantake our observations of PDB deterioration to mean that our extended 15 hours of illumination is roughly equivalent to 5 days on a light pipe. Thus we 15 contemplate that the emission lifetimes of the 'rFPP molecules provide a much more stable measure of oxygen pressure than the emission intensities of Peterson's fluorescent dye of cholce. We further contemplate that PDB and other oxygen-quenchable fluorescent substances known in the art can be used with our disclosed methods, which provide rneasurements oî oxygen quenching 20 that are insensitive to photodeterioration of the luminescent substance during use.
The term "plastic" QS used herein refers to ~ polymeric product of large molecular weight that can be shaped by flow, including p~incipally Qt least one polymeric starting materiQl and permissible amounts of plastic~zer as 2 5 described above.
While the present invention has been described in conjunction with a preferred embodiment~and illustrative examples, one of ordinary skill Qfter reading the foregoing specification will be able to effect various changes, substitutions of equivalents, and other ~lterations to the methods and 30 compositions set forth herein. It is therefore intended that the protection ~ranted by Letters Patent hereon be limited only by the definition contQined in the appended claims and equivalents thereo.
.

Claims (41)

1. A method of measuring oxygen concentration in n fluid, comprising the steps of:
(a) contacting a test fluid with a sensor composition comprising at least one luminescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light containing wavelengths strongly absorbed by the luminescent substance, (c) terminating the irradiation of step (b), (d) measuring the flux of luminescent light emitted by the sensor composition during at least two time intervals comprising at least one time interval subsequent to step (c), (e) comparing the flux values measured in step (d) to obtain a ratioing R value, (f) determining the oxygen concentration in the test fluid by comparing the R value obtained in step (e) with a similarly obtained R value for at least one reference fluid of known oxygen concentration.

2. The method of Claim 1 wherein the time intervals for flux measurement in step (d) substantially encompass the period of linear decay of the luminescent emission subsequent to step (c).

3. The method of Claim 2 wherein the time intervals subdivide the period of linear decay into substantially equal time intervals.

4. The method of Claim 2 wherein the time intervals are two in number.

5. The method of Claim 4 wherein the ratioing R value is given by:
R = (I1 - I2)/(I1 + I2) wherein 11 is the flux of luminescent emission measured during a first time interval and I2 is the flux of luminescent emission measured during a second time interval.

6. The method of Claim 1 wherein the time intervals for flux measurement in step (d) substantially encompass the period of detectable luminescent emission subsequent to step (c).

7. The method of Claim 6 wherein at least one of the time intervals substantially encompasses the period of linear decay of the luminescent emission subsequent to step (c).

8. The method of Claim 6 wherein the ratioing R value is given by:
R = I1/I2 wherein I1 is the flux of luminescent emission measured during the period of linear decay of the luminescent emission subsequent to step (c) and I2 is the flux of luminescent emission measured during the remaining period of detectable luminescent emission.

9. The method of Claim 1 wherein step (b) comprises at least one of the time intervals for flux measurement.

10. The method of Claim 9 wherein at least one first time interval substantially encompasses the period leading up to the emission intensity plateau during step (b) and at least one second time interval substantially encompasses the period of detectable luminescent emission subsequent to step (c).

11. The method of Claim 10 wherein the ratioing R value is given by:
R = I1/I2 wherein I1 is the flux of luminescent emission measured during the first time interval and I2 is the flux of luminescent emission measured during the second time interval,

12. A method of measuring oxygen concentration in a fluid, comprising the steps of:

(a) contacting a test fluid with a sensor composition comprising at least one phosphorescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light containing wavelengths strongly absorbed by the phosphorescent substance, (c) terminating the irradiation of step (b), (d) measuring the intensity of phosphorescent light emitted by the sensor composition at a plurality of times subsequent to step (c), (e) fitting the measured intensity values as follows:

I(t) = A1ek1t+A2e-k2t wherein I(t) is the measured intensity at time t, e is the exponential function,and A1, k1, A2, and k2 are fitting parameters, (f) determining the average decay rate, ?, from the fitting parameters determined in step (e) as follows:

? = (A1k1 + A2k2)/(A1 A2), (g) determining the oxygen concentration in the test fluid by comparing the average decay rate determined in step (f) with a similarly obtained ? value for at least one reference fluid of known oxygen concentration.

13. A method of measuring oxygen concentration in a fluid, comprising the steps of:
(a) contacting a test fluid with a sensor composition comprising at least one phosphorescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light containing wavelengths strongly absorbed by the phosphorescent substance, (c) terminating the irradiation of step (b), (d) measuring the intensity of phosphorescent light emitted by the sensor composition at a plurality of times subsequent to step (c), (e) fitting the measured intensity values as follows:

I(t) = A1e-k1t+A2e-k2t wherein I(t) is the measured intensity at time t, e is the exponential function,and A1, k1, A2, and k2 are fitting parameters, (f) determining the average decay time, ?, from the fitting parameters determined in step (e) as follows:

? = (A1k1-1 + A2k2-1)/(A1 + A2), (g) determining the oxygen concentration in the test fluid by comparing the average decay time determined in step (f) with a similarly obtained ? value for at least one reference fluid of known oxygen concentration.

14. A method of measuring oxygen concentration in a fluid, comprising the steps of:
(a) contacting a test fluid with a sensor composition comprising at least one luminescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light containing wavelengths strongly absorbed by the luminescent substance, (c) terminating the irradiation of step (b), (d) measuring the intensity of luminescent light emitted by the sensor composition at a plurality of times subsequent to step (c), (e) determining the slope of emission decay during the period of linear decay measured in step (d).
(f) determining the oxygen concentration in the test fluid by comparing the slope value obtained in step (e) with a similarly obtained slope value for at least one reference fluid of known oxygen concentration.

15. A method of measuring oxygen concentration in a fluid, comprising the steps of:
(a) contacting a plurality of reference fluids of known oxygen concentration with a sensor composition comprising at least one luminescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light containing wavelengths strongly absorbed by the luminescent substance, (c) terminating the irradiation of step (b), (d) measuring the intensity of luminescent light emitted by the sensor composition during a plurality of times subsequent to step (c), (e) determining for each reference fluid the intensity versus time profile of emission decay during the period of linear decay measuredin step (d), (f) contacting a test fluid of unknown oxygen concentra-tion with the sensor composition and repeating steps (b) and (c) with the test fluid, (g) measuring the time it takes for the intensity of luminescent light emitted by the sensor composition subsequent to step (f) to fall to a predetermined intensity value which can be referenced against the intensityversus time profiles determined in step (e), and (h) determining the oxygen concentration in the test fluid by comparing the time value measured in step (g) with the intensity versustime profiles determined in step (e) for the reference fluids of known oxygen concentrations.

16. A method of measuring oxygen concentration in a fluid, comprising the steps of:
(a) contacting a plurality of reference fluids of known oxygen concentration with a sensor composition comprising at least one luminescent substance admixed in an oxygen-permeable matrix, (b) irradiating the sensor composition with light con-taining wavelengths strongly absorbed by the luminescent substance, (c) terminating the irradiation of step (b), (d) measuring the intensity of luminescent light emitted by the sensor composition during a plurality of times subsequent to step (c), (e) determining for each reference fluid the intensity versus time profile of emission decay during the period of linear decay measuredin step (d), (f) cantacting a test fluid of unknown oxygen concentra-tion with the sensor composition and repeating steps (b) and (c) with the test fluid, (g) measuring the intensity of luminescent light emitted by the sensor composition at a given time interval subsequent to step (f), whichtime interval falls within the period of linear decay for the sensor composition, and (h) determining the oxygen concentration in the test fluid by comparing the intensity value measured in step (g) with the intensity versus time profiles determined in step (e) for the reference fluids of known oxygen concentrations.

17. The method or Claim 1, 12, 13, 14, 15 or 16 wherein the test fluid is blood.

18. The method of Claim 1, 12, or 13 wherein the test fluid is blood and the reference fluid is air.

19. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the irradiating light and the emitted light are transmitted by fiber optic means.

20. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the irradiated light and the emitted light are transmit-ted by fiberoptic means and the sensor composition is positioned in the light path of a single optical fiber.

21. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the luminescent substance is a phosphorescent of fluorescent substance.

22. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the luminescent substance is a metallo derivative of a porphyrin, chlorin, bacteriochlorin, or isobacteriochlorin.

23. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the luminescent substance is octaethylporphyrin, tetraphenylporphyrin, tetra(pentafluorophenyl)porphyrin, tetrabenzporphyrin, or the chlorins, bacteriochlorins, or isobacteriochlorins of said porphyrins.

24. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the luminescent substance is a platinum or palladium derivative of a porphyrin chlorin, bacteriochlorin or isobacteriochlorin.

25. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the luminescent substance is partially or wholly fluorine substituted.

26. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the sensor composition comprises one or both of platinum derivative and palladium derivative of tetra(pentafluorophenyl)porphyrin.

27. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the oxygen-permeable matrix is a plastic.

28. The method of Claim 1, 12, 13, 14, 15 or 16 wherein the oxgen-permeable matrix is a plastic which comprises one or more of polyvinyl chloride, polymethyl methacrylate, cellulose acetate, and silicon-polybicarbonate copolymer, with or without a plasticizer.

29. A method of measuring oxygen concentration in a fluid, comprising the steps of:
(a) contacting a test fluid with an oxygen quenching-sensitive composition comprising at least one fluorinated luminescent substance, (b) irradiating the composition with light containing wavelengths strongly adsorbed by the fluorinated luminescent substance, (c) terminating the irradiation of step (b), (d) measuring the decay intensity or decay rate of luminescent light emitted by the composition, and (e) determining the oxygen concentration in the test fluid by comparing the decay intensity or decay rate value measured in step (d) with a similarly obtained decay intensity or decay rate value for at least one reference fluid of known oxygen concentration.

30. The method of Claim 29 wherein the fluorinated luminescent substance is selected from the group consisting of metallo derivatives of partially or fully fluorinated octaethylporphyrin, tetraphenylporphyrin, tetrabenzporphyrin, or the chlorins, bacteriochlorins, or isobacteriochlorins of said porphyrins.

31. The method of Claim 30 wherein the metallo derivative is platinum or palladium derivative.

32. The method of Claim 29 wherein the fluorinated luminescent substance comprises one or both of platinum derivative and palladium derivative of tetra(pentafluorophenyl)porphyrin.

33. A photostable, fiber-optic oxygen sensor comprising a partially or wholly fluorine-substituted luminescent substance, whose luminescent emission is sensitive to quenching by oxygen, disposed in a light path of at least one optical fiber.

34. The sensor of Claim 33 wherein the partially or wholly fluorine-substituted luminescent substance is selected from the group consisting of metallo derivatives of partially or fully fluorinated octaethylporphyrin, tetraphenylporphyrin, tetrabenzporphyrin, or the chlorins, bacteriochlorins, or isobacteriochlorins of said porphyrins.

35. The sensor of Claim 34 wherein the metallo derivatives are platinum or palladium derivatives.

36. The sensor of Claim 33 wherein the partially or wholly fluorine-substituted luminescent substance comprises one or both of a platinum derivative and a palladium derivative of a tetra(pentafluorophenyl)porphyrin.

37. The sensor of Claim 33 wherein the partially or wholly fluorine-substituted luminescent substance is admixed in an oxygen-permeable matrix.

38. The sensor of Claim 37 wherein the oxygen-permeable matrix comprises one or more of polyvinyl chloride, polymethyl metacrylate, cellulose acetate and silicone-polybicarbonate copolymer, with or without a plasticizer.

39. The sensor of Claim 37 wherein the oxygen-permeable matrix is an oxygen-permeable plastic.

40. The method of Claim 29 wherein the sensor composition further comprises the photostable fluorinated luminescent substance admixed in an oxygen-permeable matrix.

41. The method of Claim 40 wherein the oxygen-permeable matrix is an oxygen-permeable plastic.