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Red Light-Excitable Oxygen Sensing Materials Based on Platinum(II) and Palladium(II) Benzoporphyrins S. M. Borisov,* G. Nuss, and I. Klimant Institute of Analytical Chemistry and Radiochemistry, Graz University of Technology, Stremayrgasse 16, 8010 Graz, Austria New optical oxygen-sensing materials make use of highly luminescent NIR platinum(II) and palladium(II) complexes with benzoporphyrins. Bulk optodes based on polystyrene and sensing nanobeads based on poly(styreneblock-vinylpyrrolidone) and polysulfone are prepared and characterized. The versatility of the new materials is demonstrated. The features include excellent compatibility with most common excitation sources, high brightness, and suitability for subcutaneous oxygen monitoring. Oxygen is one of the most important analytes on earth and its determination is of extreme importance in numerous fields of science and technology. Nowadays, it is routinely monitored with the help of optical sensors that are noninvasive or minimally invasive, disposable, can be easily miniaturized, and allow for imaging of oxygen tension over a surface or in volume.1 UV-vis oxygen indicators are fairly established and are almost exclusively represented by luminescent metal complexes.2 These include ruthenium(II) polypyridyl complexes,3-8 platinum(II) and palladium(II) porphyrins,9-12 and cyclometalated complexes of platinum(II) and iridium(III).13,14 The recently reported cyclometalated complexes of iridium(III) with coumarins15 are distinguishable for their excellent brightness (defined as the product of molar
absorption coefficient ε and luminescence quantum yield Φ), while other oxygen indicators possess moderate to low brightness (BS < 10 000) upon excitation in visible. Although the oxygen sensors based on the above indicators are fully adequate for a variety of applications, they, of course, have certain limitations. These sensors are poorly suited for measurements in highly scattering media and in those containing certain fluorescent substances such as chlorophyll. Moreover, the UV-vis indicators cannot be used for measurements in subcutaneous tissue because of high scattering and the presence of blood, which efficiently absorbs light in visible region. Implantable glucose sensors or “smart tattoos”16 hold great promise in diabetes care17 and can rely on oxygen indicators as transducers.18 So far, optical sensors based on platinum(II) and palladium(II) complexes with porphyrin lactones19-21 and porphyrin ketones22-25 were reported. These indicators efficiently absorb at 570-600 nm (which is still not fully adequate for subcutaneous measurements); however, they possess only moderate emission.22 Vinogradov and co-workers reported water-soluble oxygen probes based on substituted platinum(II) and palladium(II) tetrabenzoporphyrins and tetranaphthoporphyrins.26-29 Such dyes are very interesting candidates for designing optical sensors. That is particularly true for the platinum(II) complexes. In fact, the general trend in
* To whom correspondence should be addressed. E-mail: sergey.borisov@ tugraz.at. Telephone: +43 316 873 4326. Fax: +43 316 873 4329. (1) Wolfbeis, O. S. J. Mater. Chem. 2005, 15, 2657–2669. (2) Amao, Y. Microchim. Acta 2003, 143, 1–12. (3) Garcia-Fresnadillo, D.; Marazuela, M. D.; Moreno-Bondi, M. C.; Orellana, G. Langmuir 1999, 15, 6451–6459. (4) Amao, Y.; Okura, I. Sens. Actuators, B 2003, 88, 162–167. (5) Ji, J.; Rosenzweig, N.; Jones, I.; Rosenzweig, Z. Anal. Chem. 2001, 73, 3521–3527. (6) Xu, H.; Aylott, J. W.; Kopelman, R.; Miller, T. J.; Philbert, M. A. Anal. Chem. 2001, 73, 4124–4133. (7) McEvoy, A.; McDonagh, C.; MacGraith, B. J. Sol-Gel Sci. Technol. 1997, 8, 1121–1125. (8) Roche, P.; Al-Jowder, R.; Narayanaswamy, R.; Young, J.; Scully, P. Anal. Bioanal. Chem. 2006, 386, 1245–1257. (9) Lee, S.; Okura, I. Anal. Commun. 1997, 34, 185–188. (10) Borisov, S. M.; Vasylevska, A. S.; Krause, Ch.; Wolfbeis, O. S. Adv. Funct. Mater 2006, 16, 1536–1542. (11) Kimura, F.; Khalil, G.; Zettsu, N.; Xia, Y.; Callis, J.; Gouterman, M.; Dalton, L.; Dabiri, D.; Rodriguez, M. Meas. Sci. Technol. 2006, 17, 1254–1260. (12) Koese, M. E.; Carrol, B. F.; Schanze, K. S. Langmuir 2005, 21, 9121– 9129. (13) Vander Donckt, E.; Camerman, B.; Herne, R.; Vandeloise, R. Sens. Actuators, B 1996, 32, 121–127. (14) DeRosa, M.; Mosher, P.; Yap, G.; Foscaneanu, K.; Crutchley, R.; Evans, C. Inorg. Chem. 2003, 42, 4864–4872. (15) Borisov, S. M.; Klimant, I. Anal. Chem. 2007, 79, 7501–7509.
(16) Stein, E. W.; Grant, P. S.; Zhu, H.; McShane, M. J. Anal. Chem. 2007, 79, 1339–1348. (17) Pickup, J. C.; Hussain, F.; Evans, N. D.; Sachedina, N. Biosens. Bioelectron. 2005, 20, 1897–1902. (18) Borisov, S. M.; Wolfbeis, O. S. Chem. Rev. 2008, 108, 423–461. (19) Zelelow, B.; Khalil, G. E.; Phelan, G.; Carlson, B.; Gouterman, M.; Callis, J. B.; Dalton, L. R. Sens. Actuators, B 2003, 96, 304–314. (20) Khalil, G.; Gouterman, M.; Ching, S.; Costin, C.; Coyle, L.; Gouin, S.; Green, E.; Sadilek, M.; Wan, R.; Yearyean, J.; Zelelow, B. J. Porphyrins Phthalocyanines 2002, 6, 135–145. (21) Gouterman, M.; Callis, J.; Dalton, L.; Khalil, G.; Mebarki, Y.; Cooper, K. R.; Grenier, M. Meas. Sci. Technol. 2004, 15, 1986–1994. (22) Papkovsky, D. V.; Ponomarev, G. V.; Trettnak, W.; O′Leary, P. Anal. Chem. 1995, 67, 4112–4117. (23) Kolle, C.; Gruber, W.; Trettnak, W.; Biebernik, K.; Dolezal, C.; Reininger, F.; O’Leary, P. Sens. Actuators, B 1997, 38, 141–149. (24) Cao, Y.; Koo, Y.-E. L.; Kopelman, R. Analyst 2004, 129, 745–750. (25) Park, E. J.; Reid, K. R.; Tang, W.; Kennedy, R. T.; Kopelman, R. J. Mater. Chem. 2005, 15, 2913–2919. (26) Vinogradov, S. A.; Wilson, D. F. J. Chem. Soc., Perkin Trans. 2 1995, 103– 111. (27) Dunphy, I.; Vinogradov, S. A.; Wilson, D. F. Anal. Biochem. 2002, 310, 191–198. (28) Finikova, O. S.; Cheprakov, A. V.; Beletskaya, I. P.; Carroll, P. J.; Vinogradov, S. A. J. Org. Chem. 2004, 69, 522–535. (29) Finikova, O. S.; Cheprakov, A. V.; Vinogradov, S. A. J. Org. Chem. 2005, 70, 9562–9572.
10.1021/ac801521v CCC: $40.75 2008 American Chemical Society Published on Web 11/12/2008
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photophysical properties of metalloporphyrins indicates that platinum(II) complexes possess ∼2-3 times higher emission quantum yields than the respective palladium(II) derivatives.22,29 Moreover, the shorter luminescence decay time of the former is better suited for designing sensors operating in the dynamic range from 0 to 100% air saturation. They can also be applied in OLEDs, which was demonstrated recently.30 To the best of our knowledge, so far no platinum(II) complexes with benzoporphyrins have been used in solid-state optical oxygen sensors. Recently, we reported the synthesis and photophysical properties of the new platinum(II) and palladium(II) complexes with fluorinated benzoporphyrin derivatives.31 They were found to benefit from intense absorption in the red region, very high luminescence brightness, but also high photostability. Here we present various materials (bulk optodes and nanobeads) that are based on the new indicators and demonstrate their broad applicability for optical sensing. EXPERIMENTAL SECTION Materials. Polysterene (PS; M ) 250 000) and polysulfone (PSulf, M ) 26 000) were obtained from Fischer Scientific (www.fishersci.com) and Aldrich (www.sigmaaldrich.com), respectively. Platinum(II) and palladium(II) complexes with 5,10,15,20tetrakis-(2,3,4,5,6-pentafluorophenyl)porphyrin (PtTFPP and PdTFPP, respectively) and platinum(II) complex with octaethylporphyrin (PtOEP) were purchased from Frontier Scientific (www.frontiersci. com). Platinum(II) complex with octaethylporphyrin ketone (PtOEPK) was a friendly gift from Prof. Dmitri Papkovsky (University of Cork). Poly(ethylene glycol terephthalate) support (Mylar) was from Goodfellow (www.goodfellow.com). All solvents were obtained from Roth (www.carl-roth.de). Nitrogen and synthetic air (all of 99.999% purity) were obtained from Air Liquide (www.airliquide.at). Human blood samples were kindly provided by the Medical University of Graz. The synthesis of the iridium(III) complex with 3-(benzothiazol2-yl)-7-(diethylamino)coumarin (Ir(CS)2(acac)) is reported elsewhere.15 The synthesis of the platinum(II) and palladium(II) complexes with meso-tetraphenyltetrabenzoporphyrin (TPTBP), meso-tetra(4-fluorophenyl)tetrabenzoporphyrin (TPTBPF), and mesotetra(3,5-difluorophenyl)tetrabenzoporphyrin (TPTBPF2) was reported earlier.31 Preparation of the Bulk Optodes. A 1.5-mg sample of a benzoporphyrin complex and 200 mg of polystyrene were dissolved in 1.8 g of chloroform. The “cocktail” was knife-coated onto a dust-free polyester support, and the solvent was let to evaporate in ambient air to result in ∼2-µm-thick sensing films. In the case of Ir(CS)2(acac) and PtTFPP, the concentration of the indicators in PS was 1.5% w/w; for PtOEPK, 1.5% w/w. Preparation of the Sensing Nanobeads. The oxygen indicators were incorporated into the core of the poly(styrene-blockvinylpyrrolidone) nanobeads according to the published method.32 The polysulfone nanobeads were prepared according to the following procedure. A 1.5-mg sample of a porphyrin complex and (30) Borek, C.; Hanson, K.; Djurovich, P. I.; Thompson, M. E.; Aznavour, K.; Bau, R.; Sun, Y.; Forrest, S. R.; Brooks, J.; Michalski, L.; Brown, J. Angew. Chem., Int. Ed. 2007, 46, 1109–1112. (31) Borisov, S. M.; Nuss, G.; Haas, W.; Saf, R.; Schmuck, M.; Klimant, I. J. Photochem. Photobiol., A, in press; DOI: 10.1016/j.jphotochem.2008.10.003. (32) Borisov, S. M., Klimant, I. Michrochim. Acta, in press; DOI 10.1007/s00604008-0047-9.
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200 mg of PSulf were dissolved in 150 mL of tetrahydrofuran. Then, 200 mL of water was added dropwise under vigorous stirring. Finally, tetrahydrofuran and part of the water were removed under reduced pressure. Sterilization of the nanobeads was conducted in an autoclave from Systec (www.systec-lab.de). Each cycle was performed at 121 °C for 20 min in 5-mL glass vials (3 mL of bead suspension). Measurements. Emission spectra were acquired on a Hitachi F-7000 fluorescence spectrometer (www.inula.at) equipped with a red-sensitive photomultiplier R 928 from Hamamazu (www. hamamatsu.com). The emission spectra were corrected for the sensitivity of the PMT, which was calibrated using a halogen lamp. Absorption spectra were measured on a Cary 50 UV-vis spectrophotometer (www. lzs-concept.com). Luminescence decay times were determined in frequency domain. The luminescence phase shifts for the sensor films and the bead dispersions were measured with a two-phase lock-in amplifier (SR830, Standford Research Inc., www.thinksrs.com). Excitation was performed with the light of a 435-nm LED (Roithner, www.roithner-laser.com) filtered through a BG 12 glass filter (Schott, www.schott.com), which was sinusoidally modulated at a frequency of 5 kHz (in the case of the platinum(II) complexes) or at a frequency of 800 Hz (in the case of the palladium(II) complexes). A bifurcated fiber bundle was used to guide the excitation light to the vial and to guide back the luminescence after passing an RG 9 glass filter (Schott). The luminescence was detected with a photomultiplier tube (H5701-02, Hamamatsu, www.sales.hamamatsu.com). Temperature was controlled by a cryostat ThermoHaake DC50. Gas calibration mixtures were obtained using a gas mixing device (MKS, www.mksinst.com). Relative luminescence quantum yields in polystyrene (1-3µm-thick layers, 0.5% of dye w/w) were determined using a lockin Amplifier (PreSens, www.presens.de) equipped with a silicon photodiode. Excitation was performed with a 5-mm violet (405nm) LED. A BG 12 filter (Schott) was used for excitation and an OG 540 filter (Schott) for emission. The measurements were performed at a modulation frequency of 916 Hz, and the values were corrected for the amount of the absorbed light and demodulation. Photostability was determined using a lock-in Amplifier from PreSens. The sensor foils were positioned at an angle of 45° to the photodiode and the light sources. Continuous irradiation was performed by unmodulated light from 1-W 405-nm LED (300 mA) from Roithner. For the interrogation we used a second violet LED (405 nm) modulated at 916 Hz. BG 12 and a RG 630 filters were used respectively for the excitation and the emission. The subcutaneous conditions for the interference tests were simulated by using biological “filters”: ∼1-mm chicken skin, 5-mmthick chicken tissue, and a 1-mm cuvette filled with 25% blood in water. Sensor films based on PtTPTBPF, PtTFPP, PtOEPK, and Ir(CS)2(acac) in PS were used. Excitation of Ir(CS)2(acac) was performed with a 470-nm LED (Roithner); a BG-12 and a OG 590 glass filter (Schott) were used for the excitation and the emission, respectively. Excitation of PtTFPP was performed with the light of a 505-nm LED filtered through a plastic Lagoon Blue filter (www.leefilters.com). An RG 630 filter was used for the emission. A 590-nm LED and a 617-nm LED was used for the excitation of PtOEPK and PtTPTBPF, respectively; a NIR-blocking Calflex X
Figure 1. Chemical structures of the oxygen indicators.
filter (Linos Photonics, www.linos.com) was used for the excitation, and a RG-9 (Schott) filter for the emission. The luminescence was detected using a lock-in Amplifier (PreSens, www.presens.de) equipped with a silicon photodiode. Blood samples were deoxygenated by adding glucose (50 mg/mL of blood) and glucose oxidase (0.1 mg/mL of blood). RESULTS AND DISCUSSION Indicators. Brightness of indicators is of extreme importance in optical sensing since it is directly related to the S/N ratio. Indicators having high luminescence brightness can be used for manufacturing of very thin fast-responding optodes, magnetic beads, or nanobeads, which still provide reliable data in interfering media. Both molar absorption coefficients ε and luminescence quantum yields Φ contribute to overall brightness and should be considered. It is useful to compare the spectral properties and the brightnesses of platinum(II) and palladium(II) benzoporphyrins (structures shown in Figure 1) with those of the wellestablished oxygen indicators. Since luminescence quantum yields in solutions and in rigid matrix can vary significantly, we determined relative quantum yields of the indicators in polystyrene, which is one of the most common matrixes for designing optical sensors. On the other side, we used the ε values reported for solutions. Table 1 gives an overview of ε, relative Φ, and calculated brightnesses (in polystyrene) for some most common indicators used in optical sensing. As can be seen, the classical indicators such as PtTFPP, PtOEP, and PdTFPP possess a very intense Soret band in the UV region (ε > 200 000 M-1 cm-1) and much less intense Q-bands in the visible. Since visible excitation is strongly preferable, the brightness of these indicators is low to moderate. Relative quantum yields for PtOEP are very high; however, the intense narrowband peaking at 536 nm is not compatible with LEDs available for 505 and 525 nm. Molar absorption coefficients for the blue light-excitable ruthenium(II) polypyridyl complexes are significantly lower than for spectrally similar iridium(III) complexes with coumarins15 and typically do not exceed 30 000 M-1 cm-1.33 As can be seen from Table 1, the iridium(III) complexes show excellent brightnesses in PS. The platinum(II) and palladium(II) complexes with porphyrin lactones and porphyrin ketones have intense absorption bands in the orange part of the spectrum (λmax 570-605 nm, ε 50 000-70 000 M-1 cm-1).20,22 However, brightness in PS for a platinum(II) complex with octaethylporphyrin ketone (PtOEPK) is rather low. Compared to other oxygen indicators, the complexes of platinum(II) and palladium(II) with meso-substituted benzoporphyrins are much more versatile since they can be excitable both (33) Alford, P. C.; Cook, M. J.; Lewis, A. P.; McAuliffe, S. G.; Skarda, V.; Thomson, A. J. J. Chem. Soc. Perkin Trans. II 1985, 5, 705–709. (34) Lai, S.-W.; Hou, Y.-J.; Che, C.-M.; Pang, H.-L.; Wong, K.-Y.; Chang, C. K.; Zhu, N. Inorg. Chem. 2004, 43, 3724–3732.
in the blue and in the red parts of the spectrum (Figure 2). Upon excitation in the blue region, the respective platinum complexes show even higher brightness than the iridium(III) coumarins. Considering excitation in the red region, the benzoporphyrin complexes absorb at longer wavelengths than the respective complexes of porphyrin ketones and porphyrin lactones. The significantly higher molar absorption coefficients and emission quantum yields in the case of the benzoporphyrin complexes result in ∼3-10-fold improvement in brightness compared to PtOEPK. Figure 2 illustrates the versatility of the new indicators and shows the excitation spectra of the platinum(II) and palladium(II) complexes with TPTBPF in polymer nanobeads together with some of the most common excitation sources used in optical sensing and microscopy. As can be seen, the indicators are nicely compatible with bright blue LEDs (425 nm, 435 nm, and also with a 450-nm LED in the case of PdTPTBPF) and a 625-nm red LED. This makes the indicators particularly promising for application in (fiber optic) sensors. The indicators are also extremely efficiently excitable with the 436-nm line of a mercury lamp, which is the most common light source in microscopy. In scattering media with a high level of autofluorescence, the microscopic imaging can also be performed using the 578-nm line of mercury. Considering that molar absorption coefficients for the second Q-band are as high as 16 000-20 000 M-1 cm-1,31 the indicators are still able to produce higher intensities than the classical metalloporphyrins. Lasers can also be used for efficient excitation of the complexes (such as a 405-nm diode laser and a 632.8-nm He-Ne laser). This makes the indicators suitable for confocal microscopy. Finally, the palladium(II) benzoporphyrin complex can be efficiently excited with red laser diodes (such as a 635-nm or even 645-nm diodes). As can be seen, the excitation of PtTPTBPF by a 635-nm laser diode is much less efficient; however, sufficient brightnesses can still be obtained. Bulk Optodes. Bulk optodes make use of the platinum(II) and palladium(II) benzoporphyrins embedded in polystyrene. These optodes provide valuable information on the behavior of the indicators in polymers and can be easily compared to many oxygen sensors reported in the literature. The trends established for polystyrene bulk optodes usually are applicable to many other polymers in which the indicators can be embedded. As an example, Figure 3 shows the Stern-Volmer plots for a palladium(II) complex (PdTPTBP) and a platinum(II) complex (PtTPTBPF). As expected, the palladium(II) complexes show significantly higher sensitivity to oxygen than the respective platinum(II) dyes. When contained in polystyrene, the latter are ideally suitable for measurements from 0 to 100% air saturation. Evidently, the palladium(II) complexes are more adequate for trace oxygen sensing, since their luminescence is quenched almost completely at air saturation. If to be used to measure oxygen up to air saturation, the palladium(II) complexes may be incorporated in polymers with lower oxygen permeability, such as poly(styrene-co-acrylonitrile). As can be observed from Table 2, the palladium(II) benzoporphyrins are much more prone to thermal quenching than the platinum(II) complexes. In the case of the palladium(II) complexes the temperature dependence of the triplet-state decay time (estimated in the absence of oxygen) is as high as 0.33-0.38%/K. On the other hand, the respective platinum(II) complexes show almost negligible thermal quenching Analytical Chemistry, Vol. 80, No. 24, December 15, 2008
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Table 1. Spectral Properties of the Oxygen Indicators in Solutions and Relative Brightnesses in Polystyrene Films indicator
λmaxabs, nm
ε, M-1 cm-1
λmaxem, nm
Ir(CS)2(acac)
444 472 394 509 540 382 501 536 398 592 430 615 410 522 554 443 629
86800 92800 220000 13400 18100 286000 12600 62400 86200 55100 212000 146000 218000 23100 19200 268000 115000
562
CHCl3
1
649
benzene
0.42
647
toluene
1.12
758
CHCl3
0.22
773
toluene
0.70
673
toluene
0.20
800
toluene
0.23
PtTFPP PtOEP PtOEPK PtTPTBPF PdTFPP PdTPTBPF a
solvent
relative Φa
relative BS in PS 87000 93000 92000 5600 7600 320000 14000 70000 19000 12000 149000 102000 43600 4620 3800 62000 27000
ref 15 34 this work 22 31 this work 31
Φ for Ir(CS)2(acac) in PS was arbitrarily set as 1.
The Stern-Volmer constants KSV are summarized in Table 2. The “two-site model”35,36 assumes localization of an indicator in two different environments and thus explains the nonlinear character of the quenching curves. The equation from this model was adapted in which the Stern-Volmer constant for the second site was set as 0: I τ f +1-f ) ) I0 τ0 1 + KSV[O2]
Figure 2. Excitation spectra of the platinum(II) and palladium(II) benzoporphyrin complexes (λem ) 770 and 800 nm, respectively) in PS-PVP and PSulf nanobeads, respectively, and common light sources.
Figure 3. Stern-Volmer plots for the oxygen polystyrene optodes. Squares, 1 °C; circles, 25 °C; triangles, 50 °C. Lines indicate a fit according to eq 1.
(0.06-0.11%/K). For comparison, the temperature dependence in the case of PtTFPP in polystyrene was determined to be 0.28%/ K,15 while for Ru-dpp in the same polymer, it is as high as 0.46%/ K. Of course, it is evident that the new optodes still have to be compensated for the temperature effects, since quenching by oxygen is always temperature-dependent. 9438
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(1)
where f and KSV are respectively the fraction of the total emission and the Stern-Volmer constant for the first site. The equation fits the experimental plots very well and the correlation coefficient exceeds 0.999. As can be seen from Table 2, the oxygen-sensing properties of different porphyrin derivatives are rather similar. However, both platinum(II) and palladium(II) complexes with TPTBPF are more efficiently quenchable than the respective complexes with TPTBP and TPTBPF2. Since the fluorination results in a bathochromic shift of the Q-bands (Table 2), the optodes based on PtTPTBPF2 become better suitable for excitation with a 635-nm laser diode. Solubility of indicators in polymers is an important parameter for their application in optical sensors. Porphyrins often are prone to self-quenching due to aggregation. Planar π-extended porphyrins aggregate readily, but substituents in the meso-position of the porphyrin ring may significantly improve solubility.37 We have found that the complexes of meso-substituted benzoporphyrins show very low tendency to aggregation in nonpolar polymers such as polystyrene. For example, in the case of PtTPTBP, the decay time decreases by ∼0.5% when the concentration of the dye in PS increases from 0.5 to 1.5% w/w. An increase in the concentration to 2, 3, and 4% w/w results, respectively, in a 1.5, 2.2, and 4.2% shortening of the lifetime. The data indicate that the indicators can be embedded in rather high concentrations so that (35) Carraway, E. R.; Demas, J. N.; DeGraff, B. A.; Bacon, J. R. Anal. Chem. 1991, 63, 337–342. (36) Sacksteder, L.; Demas, J. N.; DeGraff, B. A.; Bacon, J. R. Anal. Chem. 1993, 65, 3480–3483. (37) Finikova, O. S.; Aleshchenkov, S. E.; Brinas, R. P.; Cherpakov, A. V.; Carroll, P. J.; Vinogradov, S. A. J. Org. Chem. 2005, 70, 4617–4628.
Table 2. Properties of the Polystyrene Optodes Based on the Benzoporphyrin Complexes (0.75% Dye w/w) KSV (τ0), kPa-1 (µs) a
indicator
λmax (abs), nm
λmax (em), nm
f
PtTPTBP PtTPTBPF PtTPTBPF2 PdTPTBP PdTPTBPF PdTPTBPF2
432, 566, 615 431, 566, 617 425, 570, 619 445, 581, 630 444, 583, 631 439, 583, 630
772 777 785 800 802 812
0.87 ± 0.02 0.85 ± 0.01 0.83 ± 0.03 0.87 ± 0.01 0.85 ± 0.01 0.89 ± 0.02
a
1 °C 0.123 (55.9) 0.167 (53.4) 0.136 (53.6) 0.78 (390) 0.89 (384) 0.71 (392)
25 °C 0.165 (55.3) 0.218 (52.6) 0.188 (52) 0.92 (356) 1.04 (356) 0.91 (363)
50 °C 0.231 (54.2) 0.274 (51.6) 0.254 (50.8) 1.05 (327) 1.16 (323) 1.10 (318)
∆τ/K, % 0.06 0.07 0.11 0.33 0.32 0.38
Corrected spectra.
Figure 4. Photodegradation of the polystyrene optodes. Continuous irradiation is performed with a 405-nm 1-W LED.
very thin sensing films can be manufactured. In fact, in the case of 3% w/w composition, a sensing layer of only 0.7 µm thick would result in a 50% light absorption upon excitation in the Q-band. A layer of this thickness would respond virtually in real time to changes in oxygen concentration, but would still produce a very high signal because of the high emission quantum yields of the indicators. This property makes the benzoporphyrin complexes particularly suitable for ultrafast monitoring of oxygen tension. Photostability. Previously we investigated photobleaching of the new benzoporphyrin complexes in poly(vinyl chloride).31 The photostability of the platinum(II) benzoporphyrins was found to be comparable to that of PtTFPP, which is known for its exceptional photostability.9 We also found that the respective palladium(II) complexes bleached faster than PtTFPP but still significantly slower than PtOEP. Here we compared photobleaching of the PS optodes based on PtTPTBPF, PtOEP, and PtTFPP (Figure 4). As can be seen, the trends established previously are also valid for polystyrene. PtTPTBPF shows excellent photostability which is only slightly lower than that of PtTFPP. Commonly used PtOEP shows ∼10-fold faster photodegradation. Oxygen-Sensing Nanobeads. Nanosensors are smart materials that combine flexibility of dissolved indicators with reliability and robustness of bulk optodes.38 They are ideally suitable for imaging purposes. As was shown previously,32,39 poly(styreneblock-vinylpyrrolidone) is an excellent matrix for simple preparation of optical nanosensors. These uncharged nanobeads (average i.d. ) 245 nm) are robust in complex media (such as those used in biotechnology) and do not aggregate at high ionic strength. The calibration plot for PtTPTBPF embedded in PS-PVP beads (38) Borisov, S. M.; Klimant, I. Analyst. 2008, 133, 1302–1307. (39) Borisov, S. M.; Mayr, T.; Klimant, I. Anal. Chem. 2008, 80, 573–582.
Figure 5. Stern-Volmer plots for the oxygen nanosensors based on benzoporphyrin complexes (25 °C). Lines indicate a fit according to eq 1.
is shown in Figure 5. The indicator shows optimal sensing dynamics in the beads, which are suitable for monitoring oxygen tension from 0 to 100% air saturation. Similar to the bulk optodes, the dye can be incorporated into the beads in a rather high concentration. In fact, the calibration plots for the beads containing 0.75 and 2.6% w/w of the dye (1.2 and 4% w/w, respectively, in the polystyrene core) are virtually identical. The exceptional brightness of the resulting nanosensors means that only a small amount of the beads is required to achieve sufficient signal. This further minimizes the potential interferences to biological systems. The beads retain their properties if sterilized by 20-min autoclaving at 121 °C. The Stern-Volmer constants KSV (eq 1) for the beads before and after autoclaving were determined to be 0.173 and 0.163 kPa-1, respectively (f ) 0.95). The decay time τ0 in beads is higher than in polystyrene (59.6 vs 52.6 µs) and does not change after sterilization. As was mentioned above, the palladium(II) benzoporphyrins with τ0 ∼ 350 µs at room temperature are too sensitive to be suitable for sensing oxygen from 0 to 100% air saturation if embedded in polystyrene. Analogous to the bulk polystyrene optodes, the PS-PVP beads based on the palladium(II) complexes are mostly suitable for trace oxygen sensing. We have found that benzoporphyrins can be incorporated into PSulf nanobeads using a precipitation procedure. The resulting uncharged beads (average i.d. ) 380 nm) are stable in water and do not aggregate. They can also be freeze-dried and dispersed in such matrixes as silicon or polyurethane hydrogels. However, the freeze-dried beads are not fully dispersible in water. The calibration plot for the PdTPTBPF embedded in PSulf particles is shown in Figure 5. We have found that autoclaving significantly reduces the sensitivity of the beads to oxygen (τ0/τair sat was 5.3 and 3.9 before and after Analytical Chemistry, Vol. 80, No. 24, December 15, 2008
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sterilization, respectively). Interestingly, subsequent 20-min sterilization cycles lead to increase in sensitivity (τ0/τair sat was measured to be 4.3 and 4.7 after the second and the third cycles, respectively). That is rather unexpected since polysulfone is known to have a glass transition point much above the autoclaving temperature. Notably, sterilization does not alter luminescence decay time (τ0 ) 354 µs), luminescence intensity, and the size of the beads so that the reasons for the phenomenon are currently unclear. However, it is evident that the beads can be used after sterilization although one-point recalibration (e.g., at air saturation) is necessary. It should also be mentioned that no alteration of the decay time and sensitivity was observed after 3-months storage at 4 °C. Because of the excellent compatibility of PdTPTBPF with the 632.8-nm line of the He-Ne laser, the beads are potentially promising for application in confocal microscopy. The PSulf beads with embedded PtTPTBPF behave similarly to those based on the palladium(II) complex. Again, a drop in the sensitivity is observed after the first autoclaving, but the sensitivity increases after subsequent cycles. The PtTPTBPF/ PSulf beads clearly are less suitable for measurements at low oxygen tensions but may be promising in some biological systems where oxygen concentration exceeds 100% air saturation (e.g., in photosynthesis). Oxygen Optodes as “Smart Tattoos”. As was already mentioned, the NIR indicators are promising as a basis for designing smart tattoos, i.e., sensors that are implanted into subcutaneous tissue to be used for continuous noninvasive monitoring of vital analytes, like oxygen or glucose (Figure 6). Such “tattoos” are excited through the skin and interrogated with the help of a photodiode (in a single point) or a CCD camera (for imaging over a certain area). The main interferences originate from absorption by the skin pigments and capillary blood, and from reflection and scattering in skin and tissue. Capillary blood effectively filters both excitation and emission light in visible due to presence of oxyhemoglobin (HbO2) and hemoglobin (Hb). Figure 7a shows the absorption spectra of these blood components. As can be observed, Hb has a broad absorption spectrum and, thus, interferes in the whole visible region extending into the NIR. However, it should be considered that even in the case of venous blood oxygen partial pressure is ∼5.5 kPa, which equals ∼80% of oxyhemoglobin and ∼20% of hemoglobin.40 Since the capillary blood can be roughly described as the 1:1 mixture of the venous and arterial blood, the amount of hemoglobin is ∼10% while the rest is in the form of oxyhemoglobin. Evidently, HbO2 is a very efficient “filter” for both excitation light and the emission of the UV-vis indicators such as Ir(CS)2(acac) and PtTFPP (Figure 7b). In the case of the NIR PtOEPK, the interference on the excitation side is still very pronounced. On the other side, in the case of the benzoporphyrin complexes such as PtTPTBP, HbO2 filters off the excitation light to a much less extent. In this work, we attempt to estimate the degree of interferences by simulating the subcutaneous conditions and using a platinum(II) benzoporphyrin complex along with three other oxygen indicators. The experimental setup is shown in Figure 6. The “filters” included ∼1-mm-thick chicken skin, ∼5-mm-thick chicken tissue, and a 1-mm cuvette filled with diluted blood (25% blood in
water, since the fractional volume of blood per volume of tissue usually ranges from 0.05 to 0.15).41 These filters were placed in front of and behind a sensor foil (indicator in polystyrene) to establish the influence on the excitation and the emission, respectively. The luminescence intensities for the sensor foils are collected in Table 3. As can be observed, substantial loss of the excitation light and the emission occurs because of absorption
(40) Aberman, A.; Cavanilles, J. M.; Trotter, J.; Erbeck, D.; Weil, M. H.; Shubin, H. J. App. Physiol. 1973, 35, 570–571.
(41) Cui, W.; Ostrander, L. E.; Lee, B. Y. Trans. Biomed. Eng. 1990, 37, 632– 639.
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Figure 6. Schematic representation of sensing with help of smart tattoos (upper part) and optical setup used for the interference tests (lower part).
Figure 7. Spectral properties of the blood components (a) and oxygen indicators (b): 1, Ir(CS)2(acac); 2, PtTFPP; 3, PtOEPK; 4, PtTPTBP.
Table 3. Luminescence Intensities (%) for the Oxygen Sensors in Simulated Subcutaneous Conditionsa complex PtTPTBPF PtOEPK Ir(CS)2(acac) PtTFPP
type of interference
tissue scattering
HbO2
Hb
tissue + 90% HbO2 + 10% Hb
excitation emission excitation emission excitation emission excitation emission
39 67 20 62 1 50 13 58
59 72 28 68 1.5 75 7 80
20 53 8 50 2.5 46 7 48
21 47 5 41
