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Phosphorescent polymer films for optical oxygen sensors
Biosensors& Bioelechmics 7 (1991) W-206
Phosphorescent polymer films for optical oxygen sensors Dmitry B. Papkovsky”,*, Janos Olahb, lgor V. Troyanovsky”, Nikita A. Sadovsky? Valentina D. Rumyantsevab, Andrey F. Mironovb, Alexander I. Yaropolov” and Alexander P. Savitsw “Bakh Institute of Biochemistry, USSR Acad. Sci., Moscow, Russia ‘Lomonosov Institute of Fine Chemical Technology, Moscow, Russia %omonosov Moscow State University, Moscow, Russia (Received 4 February
1991; revised version received 13 June 1991; accepted 17 June 1991)
Abstract: Hydrophobic phosphorescent Pt-porphyrins have been used for the development of luminescent polymer films designed for tibre-optic oxygen sensors. Luminescent and quenching characteristics of several Pt-porphyrins incorporated into polymer matrices have been studied to optimize the preparation of sensitive coatings for tibre-optical sensors. The films thus obtained have been used for libre-optical oxygen monitoring in solutions. They proved to be effective and have some practical advantages in comparison with the oxygen-sensitive probes and coatings used at present. biosensor, Keywords: oxygen, libre-optical Pt-porphyrin, quenched phosphorescence.
polymer
film,
in solutions but they were not effective for tibreoptical constructions (Papkovsky et al., 1990a). At high concentrations of these dyes in aqueous solutions (above 1 FM) essential self-quenching occurred (Vanderkooi et al., 1987) whereas lower concentrations could not provide stable and high-luminescent signals (Papkovsky et al., 199Oa). These disadvantages could be overcome with a thin luminescent polymer film approach (Sharma & Wolfheis, 1988; Shah et al., 1988; Trettnak et al., 1988) where highly concentrated dye solutions in solid polymers giving high and stable signals could be used. Polynuclear aromatic dyes (Sharma & Woltbeis, 1988; Shah et al., 1988; Trettnak er al., 1988) possessing longdecay fluorescence are commonly used. But these
INTRODUCTION Luminescent dyes with efficient and longwavelength excitation and emission bands and decay times within the microsecond range are optimal for the development of oxygen optical biosensors working at physiological oxygen concentrations (Papkovsky ef al., 1990a). Pt(II)and Pd(IT)-porphyrins possessing intense phosphorescence at room temperature were found to be suitable for these purposes (Vanderkooi et al.. 1987; Khalil et al.. 1989; Papkovsky et al., 199Oa, 1990b). Water-soluble metalloporphyrins were used for oxygen sensing *Present address: Research Centre of Molecular Diagnostics, Simpheropolskii blvd. 8,113 149 Moscow, Russia 0965-5663/92/$05.00 0 1992 Elsevier Science Publishers
luminescent
Ltd.
199
D. B. Papkovsky,et al.
compounds do not display ideal spectral and decay characteristics for use in fibre-optical sensors (Papkovsky et al., 1991). Fluorescent organic complexes of Ru(I1) have better spectral characteristics (Woltbeis et al.. 1986; Lippitsch er al., 1988) but their decay times and ‘absolute sensitivity’ are not large enough. Pt- and Pdporphyrin plastic coatings were suggested for use in tibre-optical oxygen sensors (Khalil eral.. 1989). The aim of this work was a comparative study on the spectral-luminescent and quenching properties of several hydrophobic Pt-porphyrins in thin polymer films with the view of optimization of working characteristics of such films for practical use in tibre-optical oxygen biosensors.
EXPERIMENTAL Materials Pt-coproporphyrin-III tetraethyl ester (Pt-CPPt-octaethylporphin (Pt-OEP), PtTEE), tetraphenylporphin (Pt-TPP) and Pt-ethyoporphyrin (Pt-EP) were synthesized at the Lomonosov Institute of Fine Chemical Technology (Moscow) according to the modified method (Eastwood & Gouterman, 1970). Fractionated polystyrene (mol. wt 5 MDa, from Waters) and vulcanized polydimethylsiloxane were used as polymer matrixes. All salts and solvents used were of reagent grade. Preparation of the luminescent polymer films Three per cent (w/w) stock solution of the polymer in toluene was prepared and a small amount of concentrated solution of Pt-porphyrin in toluene or chloroform added to obtain the desired final concentration of the dye (20.001 II’IM, controlled spectrophotometrically). Fixed volumes of Pt-porphyrin polymer solution were applied on the horizontal glass slides (10 X 20 X 2 mm) and left for 24 h at room temperature to evaporate the solvent. The average thickness of the film and Pt-porphyrin concentration were calculated using known concentrations of the components in start solutions, volumes and the slide area. Coating of the quartz monotibres (50 mm length, 10OOpm diameter) was performed as follows. A 20-mm portion of the monofibre core 200
Biosensors& Bioelectronics
was exposed and silicone cladding removed by 3 h incubation in concentrated sulphuric acid. Then the exposed core was dipped into a solution of Pt-porphyrin and polystyrene in toluene put out and left to evaporate the solvent. Optical measurements For measurement of optical properties of the luminescent polymer films we used coated glass slides positioned into standard 10 X 10 mm quartz cuvettes containing aqueous solutions. Absorption spectra monitored on a Hitachi-557 spectrophotometer were in the range 350-600 nm. Molar extinction of Pt-porphyrins was determined in toluene solutions. An LS-50 luminescent spectrometer (Perkin Elmer) was used for the excitation spectra (350-600 nm). emission spectra (600-750 nm) and decay time measurements performed with the coated slides put in air-saturated or deoxygenated aqueous solutions. Dissolved oxygen was removed from aqueous solutions by addition of 5 mg/ml sodium bisulphite (Garcia & Sanz-Medel, 1986). Fihre-optical measurements The frbre-optical sensing part was made from a 500-mm bifurcated glass bundle having a black of 25pm polymer core and consisting monotibres. Its excitation and emission arms (200 mm length, 1.5 mm diameter) were positioned in the cuvette compartment of the tibre-optical well-plate reader accessory of the LS-50 spectrometer. The end of the bundle (300 mm length, 2 mm diameter) was terminated with a connector to which the disposable quartz monofibres (50 mm length, 1OOOpm diameter) coated with luminescent polymer films were linked. The tibre-optical sensing part, coated with the luminescent film, was dipped into aqueous sample solutions where oxygen concentration was monitored. All luminescent measurements were performed in the dark.
RESULTS AND DISCUSSION Selection of polymer matrixes Dealing with the preparation of the luminescent oxygen-sensitive films some factors should be taken into account. The main factors are:
Biosensors & Bioelectronics
(1) the diffusion rate for oxygen in the polymer
Phosphorescent polymerjlms
for optical oxygen sensors
Intensity
material and partition of oxygen between the polymer and aqueous phases; (2) solubility, aggregation state and phosphorescence quenching of the dye in the polymer matrix; effects (oxygen solubility, (3) temperature diffusion, viscosity, mechanics, etc.) in the polymer matrix; optical, adhesive and mechanical properties (4) of the polymer material, biocompatibility, etc. Polystyrene was chosen as a rigid polymer with good optical properties and moderate diffusion and partition coefficients for oxygen. It seems to be suitable for the further immobilization of various proteins (Pesce et al., 1977) and the development of tibre-optical biosensors. At the same time mechanical and adhesive properties of polystyrene are not quite so good. Silicone rubber is known to have good mechanical and adhesive properties, biocompatibility, and a high diffusibility for oxygen. Siloxanes were used in tibre-optical oxygen sensors with fluorescent dyes (Sharma & Wolfbeis, 1988; Trettnak et al.. 1988). The high solubility of oxygen in siloxanes, however, should result in undesirably strong quenching of longdecay phosphorescent dyes in the polymer phase at physiological oxygen concentrations.
Wavelength.
rim
(a) Intensity
Optical properties of Pt-porphyrins
Characteristic excitation and emission spectra for normal and meso-substituted Pt-porphyrins are shown in Fig. 1. Luminescent properties of Ptporphyrins in toluene solutions and in polymer matrixes are summarized in Table 1. The values of molar extinction coefficients, absorption maxima and lifetimes (t,) correspond to the monomeric forms of Pt-porphyrins. All Pt-porphyrins incorporated in polystyrene or silicone showed intensive red phosphorescence at room temperature which was sensitive to oxygen concentration in the polymer matrix. Luminescence spectral and decay parameters were similar to those of Pt-porphyrins in organic solvents. Phosphorescence quantum yields for Pt-porphyrins are known to be as high as 0.6-O-9 in the absence of quenchers (Eastwood & Gouterman, 1970). The efficiency of oxygen quenching for Ptporphyrins incorporated in thin polystyrene
i i
-AL 0
Wavelength.
nm
@I Fig. 1. Characten’stic luminescent spectra of Pt-porphyrin (Pt-OEP. curve I) and meso-substituted R-porphine (R-TPP. curve 2) in polystyrene matrix: (a) excitation spectra: (b) emission spectra. 201
D. B. Papkovsky,et al. TABLE 1 Luminescent Porphyrin
Pt-CP-TEE
Biosensors& Bioelectronics properties of Pt-porphyrins
Medium
Toluene
Polystyrene Silicone Pt-TPP
Toluene Polystyrene
Pt-OEP
Toluene Polystyrene
Pt-EP
Toluene
True and quenched nm., not measured.
Excitation maximum, (molar extinction,
(20°C)
nm
Optimization of working characteristics of oxygen-sensitive polystyrene films thickness
determines
the response
time to
oxygen concentration changes. For 2pm and 6pm films 100% response was achieved in less than 10 s (Fig. 2 (a) and (b)). The time resolution interval of about 5-10 s was limited by the procedure of transition of the film from the cuvette with air-saturated solution to a deoxygenated one. For the 2pm thick film, the 202
Quenched decay time, tg>ps
(amplitude) 647
n.m.
n.m.
647
87.5
23.7 (3.69)
84.7
6.5 (13.0)
647 662
n.m.
n.m.
662
68.8
15.1 (4.56)
647
n.m.
n.m.
647
94.7
26.3 (360)
647
n.m.
n.m.
in deoxygenated
films can be seen from t, data in Table 1. At 20°C in air-saturated conditions phosphorescence of Pt-porphyrins is smaller by a factor of 3.6-4.6 compared to deoxygenated solvents. As a result, polystyrene matrix is quite optimal to provide stable signals and maximal system response (Papkovsky et al., 199Oa). In silicone matrix the amplitude of oxygen quenching is several times greater (13 times). Pt-porphyrin-silicone films are expected to be more useful for measurement of decreased oxygen concentrations (e.g. in anaerobic process control).
Film
True decay time, t, (w)
mM)
381 (280) 501 (12.8) 535 (50) 382 535 376 535 401(380) 508 (43.9) 404 508 380 (290) 534 (62) 383 535 380 (276) 535 (60)
decay times are measured
Emission maximum (nm)
and in air-saturated
media respectively.
100% response was observed within seconds. For the 30 pm thick films response time increased to 2 min (see Fig. 2 (c) and (d)). Since very thin films are mechanically unstable, 2-6pm thick films were chosen as optimal for fibre-optical biosensors. Equilibrium values of c, and t, for films of different thickness were the same (Fig. 3). To provide stable signals and effective excitation the concentration of the dye in the film should be as high as possible so as to provide absorbance of l-2 AU at the excitation wavelength. In such conditions efficiency of light absorption was 90-99%. On the other hand the maximal concentration of the dye is limited by its solubility both in polymer and in solvent and by quenching and aggregation processes. For different concentrations of Pt-OEP in polystyrene within the range 0.03-67 mM phosphorescence decay times t, and tq were found to be similar (Fig. 4). Dependence between Pt-porphyrin concentration in the film and its absorbance was close to linear. Therefore one can conclude that in all cases true solutions of
for optical oxygen
Phosphorescent polymer films
Biosensors & Bioelectronics
40
80
120
sensors
160
1
rir.. ‘CC
(a)
Cc)
rntmsity
xntansity
,’ 150
100
50
-A 0.
J 120
SO
40
160
:
Tlrr?. JCC
@I
Cd)
Fig 2. Signal response to oxygen concentration changes for polystyrene-Pt-OEPflms of dt@erent thickness. The@ns were transitionedfrom air-saturated solution to a deqvgenated one. Kinetics of luminescence intensityfor 2 pm (a), 6 pm (b), 18 pm (c) and 30 urn thick (d) films.
100
decay time, WJ
Absorbenee 10
1
80 I
80 0.1
40 -
20 -A c
0.01
A
”
01.....,1
0
0.001
5
10
15
20
film thickness, -A- +-saturated
25
30
35
/ 0.01
-+- deoxygenated
Fig 3. E$ect of Pt-OEP-polystyrenef thickness on the phosphorescence decay characteristics.
I
0.1
Urn
10
Pt-OEP, +
535 nm
100
mM -
380 nm
Fig. 4. E&t of Pt-OEP concentration in polystyrenejlm (6 pm thick) on the luminescence decay characteristics 203
D. B. Papkovsky, et al. decay 1001
Biosensors & Bioelectronics
time. us
oxygen
conch uM
(400
- 350 - 300 250
leads mainly to the increase of the quenching rate constant. The absolute values of bimolecular quenching rate constant, k,, for different temperatures could be calculated according to the following equation (derived from the Stern-Volmer equation): k,
to - t9
=
t, x tq x - 200 - 150 - 100
\___
_ - 50
u-
”
0
10
20
30
40
50
60
70
temp. C Fig. 5. Effect of temperature on luminescence decay characteristics of R-OEP in polystyrene Jilm(6 pm thick). Curve I: in deoxygenated aqueous solution; curve 2: in airsaturated aqueous solution. Temperature changes of the concentration of dissolved oxygen in air-saturated aqueous solution are shown on curve 3.
Pt-porphyrins in polystyrene monomeric obtained and no self-quenching occurred. Threemicrometer thick films at a concentration of 67 mM Pt-porphyrin would have an absorption of about 8 AU at the Soret band or 2 AU at the 535~nm band. The effect of temperature was studied for PtOEP-polystyrene film within the range 9-65°C. In deoxygenated media (see Fig. 5. curve 1) C, varied slightly within the range and diminished by about 7% at increased t,. This corresponded to a temperature coefficient of less than 0.14% per “C for t,. The effect of temperature on cq is stronger than that for t,. Curve 2 shows temperature dependence of tq in air-saturated solution. It reflects both changes in oxygen concentration in solution (see curve 3) in bimolecular quenching rate constant and in t,. It is also seen that the trend in the change oft, is inverse to that expected only from the oxygen solubilty curve. Decrease in oxygen concentration in solution alone should result in an increase of t, and luminescence intensity. Looking at the curves shown in Fig. 5 one can conclude that raising of temperature 204
(1)
IQ1
where t,, tq and [Q] are the true and quenched decay times and concentration of the quencher, respectively. The true k, value at 25°C for polystyrene was found to be 3-87 X 10’ M-I s-’ (calculated using tabulated values of oxygen solubility in polystyrene). Since the concentration of oxygen in the thin polystyrene film obtained could not be determined precisely for different temperature values, we used ‘effective’ k, values. They were calculated using tabulated values of oxygen solubility in air-saturated aqueous solution at different temperatures (see Fig. 5, curve 3). The effect of temperature on k, is shown in Fig. 6. Within this range, k, varied from 5 X lo7 to 30 X lo7 M-’ s-‘, which corresponds to the temperature coefficient of 5 X lo6 M-i s-i per “C. At ambient temperature (about 25°C) this corresponds to a value of about 5% per “C. This value means that, for example, if temperature is
k, l/(uM’s) 300.
250 -
200 -
,’
A+
100 -
50 -
/ .1”” +C q
True
E:
30
40
0 0
10
20
temp. +
effective
50
60
70
C k
Fig. 6. Effect of temperature on k, for Pt-OEP-poly.styrene film (5 pm thick).
Phosphorescent polymer films for optical oxygen sensors
Biosensors & Bioelectronics
varied by 5°C at 25“C, the deviation in oxygen concentration which is determined by quenched luminescence measurements would be about 7% (or 1.2% per “C). The last value of temperature coefficient is several times less than that for electrochemical oxygen detection. Time stability of the signal for Pt-OEPpolystyrene film is shown in Fig. 7 both for airsaturated and deoxygenated solutions. Under illumination with blue light (385 nm) produced by the 8.3 W lamp of the LS-50 spectrometer a decrease of about 10% in luminescence intensity was observed during an interval of 100 min (or about 0.1% per min). A similar course of photobleaching in air-saturated and in deoxygenated media enable one to suppose that the process of photodestruction did not essentially involve singlet oxygen. Fibre-optical oxygen sensing Monitoring of the dissolved oxygen concentration in aqueous solution using fibreoptical equipment and polystyrene Ptoctaethylporphin coating (see Experimental) is shown in Fig. 8. Changes in phosphorescence
z
0,”
,,,,,,,v
1
2
,,,,
3
4
Tire. min Fig. 8. Fibre-optical oxygen monitoring with Pt-OEPpolystyrene luminescent~lm. Excitation at 535 nm, registration at 648 nm. Bars indicate the time of oxygen concentration change (transitionfrom air-saturated medium to deoxygenated one and back).
intensity (at a time of about 1 min and 4 min) correspond to the transfer of the sensor from a deoxygenated solution to an air-saturated one and back. The system possessed a 100% response time of about 20 s and good signal stability under illumination. CONCLUSION
-----_._ I
-- ._ ___
-I
I
I
0
EO-
Intmcity
100
90
Intensity
’
I
I
33
I
I
I
66 Time,
I 99
min
Fig. 7. Time stability of the luminescent signal of Pt-OEPpolystyrene JIlm (6 urn thick. 10 rnM of Pt-OEP) in airsaturated media (curve 1) and in deoxygenated media (curve 2). Luminescence excitation at 380 nm and registration at 647 nm.
The polystyrene luminescent films described above seemed to be very useful for application in tibre-optical oxygen sensors and for developing of enzyme optrodes. Contrary to most aromatic dyes possessing long-decay phosphorescence at room temperature, no fluorescence in the visiblenear-infrared spectral region was observed for pure Pt-porphyrin samples. This fact simplifies phosphorescent signal monitoring, which could be made by steady-state fluorimetry. A large shift between excitation and emission bands (more than 100 nm) enables one to discriminate easily between excitation and emission light using broad-band and/or cut-off filters. Pt-porphyrins (Pt-CP-TEE. Pt-OEP, Pt-EP) and meso-substituted porphin derivatives (PtTPP) differ in their luminescent spectral properties (See Fig. 2) whereas decay times and effectiveness of oxygen quenching are similar. Spectral differences of normal and mesosubstituted Pt-porphyrins could be used for optimization of the optoelectronic registration
D. B. Papkovsky, et al.
system for biosensors. Despite the longwavelen~h excitation band (electron transfer S&t, with a maximum at 535 nm) for normal Ptporphyrins being four to five times less intense than the Soret band (electron transfer Se-&, maximum at 380 nm) (Gurinovich etal.. 1968) it is more useful for fibre-optical devices. This enables use of cheap and available plastic or glass optical fibres and miniature green light-emitting diodes. The parameters of photochemical stability of Pt-porphyrin-polystyrene tilms seems to be quite satisfactory for fibre-optical biosensors, where the energy of exciting light sources is usually as small as 1 mW. They can be improved by further increase of the dye concentration in polystyrene to provide conditions of total absorption of the exciting light. Thus luminescent polymer coatings with high concentration of Pt-porphyrin could work for prolonged times with periodical recalibration. For optical sensors based on lifetime measurement the factor of photobleaching is not essential. At the same time a microsecond range of decay time changes for Ptpo~hy~ns is technically more suitable for timeresolved oxygen biosensors than a submicrosecond range of fluorescent oxygen probes. Polystyrene luminescent films possessed moderate adhesion on glass. Thick films had low adhesion and washed off with water. We failed to improve adhesion by silanization of glass before the film application. The problem of adhesion of ~lys~rene films to the solid support could be using polymethylmethac~late overcome waveguides. ACKNOWLEDGEMENT D. P. wishes to thank Professor Otto S. Wolfbeis for helpful discussion of the results. REFERENCES Eastwood, D. % Gouterman, M. (1970). Porphyrins. XVII. Luminescence of Co, Ni, Pd, Pt complexes. J Molec. Spectrosc.. 35, 359-375.
206
Biosensom & Bioelectronics
Garcia, M. E. D. & Sanz-Medel, A. (1986). Facile chemical deoxygenation of micellar solutions for Mom-tem~m~re phosphorescence. Anul. C/rem., 58, 1436-1440. Gurinovich. G. P.. Sevchenko, A N. & Solov’ev K. N. (1968). Spectroscopy of chlorophyll and related compounds. (In Russian.) Nauka i tekhnika, Minsk, 148-224.
Khalil, G. E., Gouterman, M. P. & Green. E. (1989). Method for measurement oxygen concentration. US Patent no. 4.810655. Lippitsch, M. E., Pusterhofer, J., Leiner, M. J. P. & Wolfbeis. 0. S. (1988). Fibre-optic oxygen sensor with the fluorescence decay time as the information carrier. Anal. Chim. Acta, 205, l-6. Papkovsky. D. B., Savitsky, A P. & Yaropolov, A. I. (1990a). Oxygen and glucose optical biosensors based on phosphorescence quenching of me~llopo~h~ns. Zhum~~An~lyt. Khimii~~SSR), 45, 1441-1445.
Papkovsky, D. B.. Savitsky, A. P. & Yaropolov. A. I. (199Ob). Optical biosensors (review). Prikl. Biokhim. Mikrobiol. (USSR), 26,435~444.
Papkovsky, D. B.. Savitsky,A. P. &Yaropolov,k Let al. (1991). Flow-injection glucose determination using long-wavelength luminescent oxygen probes. Biomed Sei., 2, 63-66. Pesce, A. J., Ford, D. J., Gaizutis, M. % Rolak V. E. (1977). Binding of protein to polystyrene in solidstate immunoassays. Biochim. Biophys. Acta, 492, 399-407.
Shah, R., Margerum, S. C. & Gold, M. (1988). Grafted hydrophylic polymers as optical sensor substrates. Proc. SPIE Int. Sot. Opt. Eng. (Opt. Fibers Med. 3), 906,65-73.
Sharma, A. & Wohbeis. 0. S. (1988). Fibre-optic oxygen sensor based on fluorescence quenching and energy transfer. Appl. Spectrosc.., 42, 1009-1011. Trettnak, W., Leiner, M. J. P. & Wolfbeis, 0. S. (1988). Optical sensors. Part 34. Fibre-optical glucose sensor with an oxygen optode as the transducer. Analyst, 113,1519-1523.
Vanderkooi, J., Maniara, G., Green, T. J. &Wilson, D. F. (1987). An optical method for measu~ment of dioxygen concentration based upon quenching of phosphorescence. J. Biol. Chem., 262,5476-5482. Wolfbeis, 0. S., Leiner, M.J. P. & Posch. H. E. (1986). A material for optical oxygen new sensing measurement with the indicator embedded in an aqueous phase. Mikrochim. Acta. III, 359-366.
Phosphorescent polymer films for optical oxygen sensors Dmitry B. Papkovsky”,*, Janos Olahb, lgor V. Troyanovsky”, Nikita A. Sadovsky? Valentina D. Rumyantsevab, Andrey F. Mironovb, Alexander I. Yaropolov” and Alexander P. Savitsw “Bakh Institute of Biochemistry, USSR Acad. Sci., Moscow, Russia ‘Lomonosov Institute of Fine Chemical Technology, Moscow, Russia %omonosov Moscow State University, Moscow, Russia (Received 4 February
1991; revised version received 13 June 1991; accepted 17 June 1991)
Abstract: Hydrophobic phosphorescent Pt-porphyrins have been used for the development of luminescent polymer films designed for tibre-optic oxygen sensors. Luminescent and quenching characteristics of several Pt-porphyrins incorporated into polymer matrices have been studied to optimize the preparation of sensitive coatings for tibre-optical sensors. The films thus obtained have been used for libre-optical oxygen monitoring in solutions. They proved to be effective and have some practical advantages in comparison with the oxygen-sensitive probes and coatings used at present. biosensor, Keywords: oxygen, libre-optical Pt-porphyrin, quenched phosphorescence.
polymer
film,
in solutions but they were not effective for tibreoptical constructions (Papkovsky et al., 1990a). At high concentrations of these dyes in aqueous solutions (above 1 FM) essential self-quenching occurred (Vanderkooi et al., 1987) whereas lower concentrations could not provide stable and high-luminescent signals (Papkovsky et al., 199Oa). These disadvantages could be overcome with a thin luminescent polymer film approach (Sharma & Wolfheis, 1988; Shah et al., 1988; Trettnak et al., 1988) where highly concentrated dye solutions in solid polymers giving high and stable signals could be used. Polynuclear aromatic dyes (Sharma & Woltbeis, 1988; Shah et al., 1988; Trettnak er al., 1988) possessing longdecay fluorescence are commonly used. But these
INTRODUCTION Luminescent dyes with efficient and longwavelength excitation and emission bands and decay times within the microsecond range are optimal for the development of oxygen optical biosensors working at physiological oxygen concentrations (Papkovsky ef al., 1990a). Pt(II)and Pd(IT)-porphyrins possessing intense phosphorescence at room temperature were found to be suitable for these purposes (Vanderkooi et al.. 1987; Khalil et al.. 1989; Papkovsky et al., 199Oa, 1990b). Water-soluble metalloporphyrins were used for oxygen sensing *Present address: Research Centre of Molecular Diagnostics, Simpheropolskii blvd. 8,113 149 Moscow, Russia 0965-5663/92/$05.00 0 1992 Elsevier Science Publishers
luminescent
Ltd.
199
D. B. Papkovsky,et al.
compounds do not display ideal spectral and decay characteristics for use in fibre-optical sensors (Papkovsky et al., 1991). Fluorescent organic complexes of Ru(I1) have better spectral characteristics (Woltbeis et al.. 1986; Lippitsch er al., 1988) but their decay times and ‘absolute sensitivity’ are not large enough. Pt- and Pdporphyrin plastic coatings were suggested for use in tibre-optical oxygen sensors (Khalil eral.. 1989). The aim of this work was a comparative study on the spectral-luminescent and quenching properties of several hydrophobic Pt-porphyrins in thin polymer films with the view of optimization of working characteristics of such films for practical use in tibre-optical oxygen biosensors.
EXPERIMENTAL Materials Pt-coproporphyrin-III tetraethyl ester (Pt-CPPt-octaethylporphin (Pt-OEP), PtTEE), tetraphenylporphin (Pt-TPP) and Pt-ethyoporphyrin (Pt-EP) were synthesized at the Lomonosov Institute of Fine Chemical Technology (Moscow) according to the modified method (Eastwood & Gouterman, 1970). Fractionated polystyrene (mol. wt 5 MDa, from Waters) and vulcanized polydimethylsiloxane were used as polymer matrixes. All salts and solvents used were of reagent grade. Preparation of the luminescent polymer films Three per cent (w/w) stock solution of the polymer in toluene was prepared and a small amount of concentrated solution of Pt-porphyrin in toluene or chloroform added to obtain the desired final concentration of the dye (20.001 II’IM, controlled spectrophotometrically). Fixed volumes of Pt-porphyrin polymer solution were applied on the horizontal glass slides (10 X 20 X 2 mm) and left for 24 h at room temperature to evaporate the solvent. The average thickness of the film and Pt-porphyrin concentration were calculated using known concentrations of the components in start solutions, volumes and the slide area. Coating of the quartz monotibres (50 mm length, 10OOpm diameter) was performed as follows. A 20-mm portion of the monofibre core 200
Biosensors& Bioelectronics
was exposed and silicone cladding removed by 3 h incubation in concentrated sulphuric acid. Then the exposed core was dipped into a solution of Pt-porphyrin and polystyrene in toluene put out and left to evaporate the solvent. Optical measurements For measurement of optical properties of the luminescent polymer films we used coated glass slides positioned into standard 10 X 10 mm quartz cuvettes containing aqueous solutions. Absorption spectra monitored on a Hitachi-557 spectrophotometer were in the range 350-600 nm. Molar extinction of Pt-porphyrins was determined in toluene solutions. An LS-50 luminescent spectrometer (Perkin Elmer) was used for the excitation spectra (350-600 nm). emission spectra (600-750 nm) and decay time measurements performed with the coated slides put in air-saturated or deoxygenated aqueous solutions. Dissolved oxygen was removed from aqueous solutions by addition of 5 mg/ml sodium bisulphite (Garcia & Sanz-Medel, 1986). Fihre-optical measurements The frbre-optical sensing part was made from a 500-mm bifurcated glass bundle having a black of 25pm polymer core and consisting monotibres. Its excitation and emission arms (200 mm length, 1.5 mm diameter) were positioned in the cuvette compartment of the tibre-optical well-plate reader accessory of the LS-50 spectrometer. The end of the bundle (300 mm length, 2 mm diameter) was terminated with a connector to which the disposable quartz monofibres (50 mm length, 1OOOpm diameter) coated with luminescent polymer films were linked. The tibre-optical sensing part, coated with the luminescent film, was dipped into aqueous sample solutions where oxygen concentration was monitored. All luminescent measurements were performed in the dark.
RESULTS AND DISCUSSION Selection of polymer matrixes Dealing with the preparation of the luminescent oxygen-sensitive films some factors should be taken into account. The main factors are:
Biosensors & Bioelectronics
(1) the diffusion rate for oxygen in the polymer
Phosphorescent polymerjlms
for optical oxygen sensors
Intensity
material and partition of oxygen between the polymer and aqueous phases; (2) solubility, aggregation state and phosphorescence quenching of the dye in the polymer matrix; effects (oxygen solubility, (3) temperature diffusion, viscosity, mechanics, etc.) in the polymer matrix; optical, adhesive and mechanical properties (4) of the polymer material, biocompatibility, etc. Polystyrene was chosen as a rigid polymer with good optical properties and moderate diffusion and partition coefficients for oxygen. It seems to be suitable for the further immobilization of various proteins (Pesce et al., 1977) and the development of tibre-optical biosensors. At the same time mechanical and adhesive properties of polystyrene are not quite so good. Silicone rubber is known to have good mechanical and adhesive properties, biocompatibility, and a high diffusibility for oxygen. Siloxanes were used in tibre-optical oxygen sensors with fluorescent dyes (Sharma & Wolfbeis, 1988; Trettnak et al.. 1988). The high solubility of oxygen in siloxanes, however, should result in undesirably strong quenching of longdecay phosphorescent dyes in the polymer phase at physiological oxygen concentrations.
Wavelength.
rim
(a) Intensity
Optical properties of Pt-porphyrins
Characteristic excitation and emission spectra for normal and meso-substituted Pt-porphyrins are shown in Fig. 1. Luminescent properties of Ptporphyrins in toluene solutions and in polymer matrixes are summarized in Table 1. The values of molar extinction coefficients, absorption maxima and lifetimes (t,) correspond to the monomeric forms of Pt-porphyrins. All Pt-porphyrins incorporated in polystyrene or silicone showed intensive red phosphorescence at room temperature which was sensitive to oxygen concentration in the polymer matrix. Luminescence spectral and decay parameters were similar to those of Pt-porphyrins in organic solvents. Phosphorescence quantum yields for Pt-porphyrins are known to be as high as 0.6-O-9 in the absence of quenchers (Eastwood & Gouterman, 1970). The efficiency of oxygen quenching for Ptporphyrins incorporated in thin polystyrene
i i
-AL 0
Wavelength.
nm
@I Fig. 1. Characten’stic luminescent spectra of Pt-porphyrin (Pt-OEP. curve I) and meso-substituted R-porphine (R-TPP. curve 2) in polystyrene matrix: (a) excitation spectra: (b) emission spectra. 201
D. B. Papkovsky,et al. TABLE 1 Luminescent Porphyrin
Pt-CP-TEE
Biosensors& Bioelectronics properties of Pt-porphyrins
Medium
Toluene
Polystyrene Silicone Pt-TPP
Toluene Polystyrene
Pt-OEP
Toluene Polystyrene
Pt-EP
Toluene
True and quenched nm., not measured.
Excitation maximum, (molar extinction,
(20°C)
nm
Optimization of working characteristics of oxygen-sensitive polystyrene films thickness
determines
the response
time to
oxygen concentration changes. For 2pm and 6pm films 100% response was achieved in less than 10 s (Fig. 2 (a) and (b)). The time resolution interval of about 5-10 s was limited by the procedure of transition of the film from the cuvette with air-saturated solution to a deoxygenated one. For the 2pm thick film, the 202
Quenched decay time, tg>ps
(amplitude) 647
n.m.
n.m.
647
87.5
23.7 (3.69)
84.7
6.5 (13.0)
647 662
n.m.
n.m.
662
68.8
15.1 (4.56)
647
n.m.
n.m.
647
94.7
26.3 (360)
647
n.m.
n.m.
in deoxygenated
films can be seen from t, data in Table 1. At 20°C in air-saturated conditions phosphorescence of Pt-porphyrins is smaller by a factor of 3.6-4.6 compared to deoxygenated solvents. As a result, polystyrene matrix is quite optimal to provide stable signals and maximal system response (Papkovsky et al., 199Oa). In silicone matrix the amplitude of oxygen quenching is several times greater (13 times). Pt-porphyrin-silicone films are expected to be more useful for measurement of decreased oxygen concentrations (e.g. in anaerobic process control).
Film
True decay time, t, (w)
mM)
381 (280) 501 (12.8) 535 (50) 382 535 376 535 401(380) 508 (43.9) 404 508 380 (290) 534 (62) 383 535 380 (276) 535 (60)
decay times are measured
Emission maximum (nm)
and in air-saturated
media respectively.
100% response was observed within seconds. For the 30 pm thick films response time increased to 2 min (see Fig. 2 (c) and (d)). Since very thin films are mechanically unstable, 2-6pm thick films were chosen as optimal for fibre-optical biosensors. Equilibrium values of c, and t, for films of different thickness were the same (Fig. 3). To provide stable signals and effective excitation the concentration of the dye in the film should be as high as possible so as to provide absorbance of l-2 AU at the excitation wavelength. In such conditions efficiency of light absorption was 90-99%. On the other hand the maximal concentration of the dye is limited by its solubility both in polymer and in solvent and by quenching and aggregation processes. For different concentrations of Pt-OEP in polystyrene within the range 0.03-67 mM phosphorescence decay times t, and tq were found to be similar (Fig. 4). Dependence between Pt-porphyrin concentration in the film and its absorbance was close to linear. Therefore one can conclude that in all cases true solutions of
for optical oxygen
Phosphorescent polymer films
Biosensors & Bioelectronics
40
80
120
sensors
160
1
rir.. ‘CC
(a)
Cc)
rntmsity
xntansity
,’ 150
100
50
-A 0.
J 120
SO
40
160
:
Tlrr?. JCC
@I
Cd)
Fig 2. Signal response to oxygen concentration changes for polystyrene-Pt-OEPflms of dt@erent thickness. The@ns were transitionedfrom air-saturated solution to a deqvgenated one. Kinetics of luminescence intensityfor 2 pm (a), 6 pm (b), 18 pm (c) and 30 urn thick (d) films.
100
decay time, WJ
Absorbenee 10
1
80 I
80 0.1
40 -
20 -A c
0.01
A
”
01.....,1
0
0.001
5
10
15
20
film thickness, -A- +-saturated
25
30
35
/ 0.01
-+- deoxygenated
Fig 3. E$ect of Pt-OEP-polystyrenef thickness on the phosphorescence decay characteristics.
I
0.1
Urn
10
Pt-OEP, +
535 nm
100
mM -
380 nm
Fig. 4. E&t of Pt-OEP concentration in polystyrenejlm (6 pm thick) on the luminescence decay characteristics 203
D. B. Papkovsky, et al. decay 1001
Biosensors & Bioelectronics
time. us
oxygen
conch uM
(400
- 350 - 300 250
leads mainly to the increase of the quenching rate constant. The absolute values of bimolecular quenching rate constant, k,, for different temperatures could be calculated according to the following equation (derived from the Stern-Volmer equation): k,
to - t9
=
t, x tq x - 200 - 150 - 100
\___
_ - 50
u-
”
0
10
20
30
40
50
60
70
temp. C Fig. 5. Effect of temperature on luminescence decay characteristics of R-OEP in polystyrene Jilm(6 pm thick). Curve I: in deoxygenated aqueous solution; curve 2: in airsaturated aqueous solution. Temperature changes of the concentration of dissolved oxygen in air-saturated aqueous solution are shown on curve 3.
Pt-porphyrins in polystyrene monomeric obtained and no self-quenching occurred. Threemicrometer thick films at a concentration of 67 mM Pt-porphyrin would have an absorption of about 8 AU at the Soret band or 2 AU at the 535~nm band. The effect of temperature was studied for PtOEP-polystyrene film within the range 9-65°C. In deoxygenated media (see Fig. 5. curve 1) C, varied slightly within the range and diminished by about 7% at increased t,. This corresponded to a temperature coefficient of less than 0.14% per “C for t,. The effect of temperature on cq is stronger than that for t,. Curve 2 shows temperature dependence of tq in air-saturated solution. It reflects both changes in oxygen concentration in solution (see curve 3) in bimolecular quenching rate constant and in t,. It is also seen that the trend in the change oft, is inverse to that expected only from the oxygen solubilty curve. Decrease in oxygen concentration in solution alone should result in an increase of t, and luminescence intensity. Looking at the curves shown in Fig. 5 one can conclude that raising of temperature 204
(1)
IQ1
where t,, tq and [Q] are the true and quenched decay times and concentration of the quencher, respectively. The true k, value at 25°C for polystyrene was found to be 3-87 X 10’ M-I s-’ (calculated using tabulated values of oxygen solubility in polystyrene). Since the concentration of oxygen in the thin polystyrene film obtained could not be determined precisely for different temperature values, we used ‘effective’ k, values. They were calculated using tabulated values of oxygen solubility in air-saturated aqueous solution at different temperatures (see Fig. 5, curve 3). The effect of temperature on k, is shown in Fig. 6. Within this range, k, varied from 5 X lo7 to 30 X lo7 M-’ s-‘, which corresponds to the temperature coefficient of 5 X lo6 M-i s-i per “C. At ambient temperature (about 25°C) this corresponds to a value of about 5% per “C. This value means that, for example, if temperature is
k, l/(uM’s) 300.
250 -
200 -
,’
A+
100 -
50 -
/ .1”” +C q
True
E:
30
40
0 0
10
20
temp. +
effective
50
60
70
C k
Fig. 6. Effect of temperature on k, for Pt-OEP-poly.styrene film (5 pm thick).
Phosphorescent polymer films for optical oxygen sensors
Biosensors & Bioelectronics
varied by 5°C at 25“C, the deviation in oxygen concentration which is determined by quenched luminescence measurements would be about 7% (or 1.2% per “C). The last value of temperature coefficient is several times less than that for electrochemical oxygen detection. Time stability of the signal for Pt-OEPpolystyrene film is shown in Fig. 7 both for airsaturated and deoxygenated solutions. Under illumination with blue light (385 nm) produced by the 8.3 W lamp of the LS-50 spectrometer a decrease of about 10% in luminescence intensity was observed during an interval of 100 min (or about 0.1% per min). A similar course of photobleaching in air-saturated and in deoxygenated media enable one to suppose that the process of photodestruction did not essentially involve singlet oxygen. Fibre-optical oxygen sensing Monitoring of the dissolved oxygen concentration in aqueous solution using fibreoptical equipment and polystyrene Ptoctaethylporphin coating (see Experimental) is shown in Fig. 8. Changes in phosphorescence
z
0,”
,,,,,,,v
1
2
,,,,
3
4
Tire. min Fig. 8. Fibre-optical oxygen monitoring with Pt-OEPpolystyrene luminescent~lm. Excitation at 535 nm, registration at 648 nm. Bars indicate the time of oxygen concentration change (transitionfrom air-saturated medium to deoxygenated one and back).
intensity (at a time of about 1 min and 4 min) correspond to the transfer of the sensor from a deoxygenated solution to an air-saturated one and back. The system possessed a 100% response time of about 20 s and good signal stability under illumination. CONCLUSION
-----_._ I
-- ._ ___
-I
I
I
0
EO-
Intmcity
100
90
Intensity
’
I
I
33
I
I
I
66 Time,
I 99
min
Fig. 7. Time stability of the luminescent signal of Pt-OEPpolystyrene JIlm (6 urn thick. 10 rnM of Pt-OEP) in airsaturated media (curve 1) and in deoxygenated media (curve 2). Luminescence excitation at 380 nm and registration at 647 nm.
The polystyrene luminescent films described above seemed to be very useful for application in tibre-optical oxygen sensors and for developing of enzyme optrodes. Contrary to most aromatic dyes possessing long-decay phosphorescence at room temperature, no fluorescence in the visiblenear-infrared spectral region was observed for pure Pt-porphyrin samples. This fact simplifies phosphorescent signal monitoring, which could be made by steady-state fluorimetry. A large shift between excitation and emission bands (more than 100 nm) enables one to discriminate easily between excitation and emission light using broad-band and/or cut-off filters. Pt-porphyrins (Pt-CP-TEE. Pt-OEP, Pt-EP) and meso-substituted porphin derivatives (PtTPP) differ in their luminescent spectral properties (See Fig. 2) whereas decay times and effectiveness of oxygen quenching are similar. Spectral differences of normal and mesosubstituted Pt-porphyrins could be used for optimization of the optoelectronic registration
D. B. Papkovsky, et al.
system for biosensors. Despite the longwavelen~h excitation band (electron transfer S&t, with a maximum at 535 nm) for normal Ptporphyrins being four to five times less intense than the Soret band (electron transfer Se-&, maximum at 380 nm) (Gurinovich etal.. 1968) it is more useful for fibre-optical devices. This enables use of cheap and available plastic or glass optical fibres and miniature green light-emitting diodes. The parameters of photochemical stability of Pt-porphyrin-polystyrene tilms seems to be quite satisfactory for fibre-optical biosensors, where the energy of exciting light sources is usually as small as 1 mW. They can be improved by further increase of the dye concentration in polystyrene to provide conditions of total absorption of the exciting light. Thus luminescent polymer coatings with high concentration of Pt-porphyrin could work for prolonged times with periodical recalibration. For optical sensors based on lifetime measurement the factor of photobleaching is not essential. At the same time a microsecond range of decay time changes for Ptpo~hy~ns is technically more suitable for timeresolved oxygen biosensors than a submicrosecond range of fluorescent oxygen probes. Polystyrene luminescent films possessed moderate adhesion on glass. Thick films had low adhesion and washed off with water. We failed to improve adhesion by silanization of glass before the film application. The problem of adhesion of ~lys~rene films to the solid support could be using polymethylmethac~late overcome waveguides. ACKNOWLEDGEMENT D. P. wishes to thank Professor Otto S. Wolfbeis for helpful discussion of the results. REFERENCES Eastwood, D. % Gouterman, M. (1970). Porphyrins. XVII. Luminescence of Co, Ni, Pd, Pt complexes. J Molec. Spectrosc.. 35, 359-375.
206
Biosensom & Bioelectronics
Garcia, M. E. D. & Sanz-Medel, A. (1986). Facile chemical deoxygenation of micellar solutions for Mom-tem~m~re phosphorescence. Anul. C/rem., 58, 1436-1440. Gurinovich. G. P.. Sevchenko, A N. & Solov’ev K. N. (1968). Spectroscopy of chlorophyll and related compounds. (In Russian.) Nauka i tekhnika, Minsk, 148-224.
Khalil, G. E., Gouterman, M. P. & Green. E. (1989). Method for measurement oxygen concentration. US Patent no. 4.810655. Lippitsch, M. E., Pusterhofer, J., Leiner, M. J. P. & Wolfbeis. 0. S. (1988). Fibre-optic oxygen sensor with the fluorescence decay time as the information carrier. Anal. Chim. Acta, 205, l-6. Papkovsky. D. B., Savitsky, A P. & Yaropolov, A. I. (1990a). Oxygen and glucose optical biosensors based on phosphorescence quenching of me~llopo~h~ns. Zhum~~An~lyt. Khimii~~SSR), 45, 1441-1445.
Papkovsky, D. B.. Savitsky, A. P. & Yaropolov. A. I. (199Ob). Optical biosensors (review). Prikl. Biokhim. Mikrobiol. (USSR), 26,435~444.
Papkovsky, D. B.. Savitsky,A. P. &Yaropolov,k Let al. (1991). Flow-injection glucose determination using long-wavelength luminescent oxygen probes. Biomed Sei., 2, 63-66. Pesce, A. J., Ford, D. J., Gaizutis, M. % Rolak V. E. (1977). Binding of protein to polystyrene in solidstate immunoassays. Biochim. Biophys. Acta, 492, 399-407.
Shah, R., Margerum, S. C. & Gold, M. (1988). Grafted hydrophylic polymers as optical sensor substrates. Proc. SPIE Int. Sot. Opt. Eng. (Opt. Fibers Med. 3), 906,65-73.
Sharma, A. & Wohbeis. 0. S. (1988). Fibre-optic oxygen sensor based on fluorescence quenching and energy transfer. Appl. Spectrosc.., 42, 1009-1011. Trettnak, W., Leiner, M. J. P. & Wolfbeis, 0. S. (1988). Optical sensors. Part 34. Fibre-optical glucose sensor with an oxygen optode as the transducer. Analyst, 113,1519-1523.
Vanderkooi, J., Maniara, G., Green, T. J. &Wilson, D. F. (1987). An optical method for measu~ment of dioxygen concentration based upon quenching of phosphorescence. J. Biol. Chem., 262,5476-5482. Wolfbeis, 0. S., Leiner, M.J. P. & Posch. H. E. (1986). A material for optical oxygen new sensing measurement with the indicator embedded in an aqueous phase. Mikrochim. Acta. III, 359-366.