Anal. Chem. 2007, 79, 7501-7509
Ultrabright Oxygen Optodes Based on Cyclometalated Iridium(III) Coumarin Complexes Sergey M. Borisov* and Ingo Klimant
Institute of Analytical Chemistry and Radiochemistry, University of Technology Graz, Stremayrgasse 16, 8010 Graz, Austria
Cyclometalated iridium(III) coumarin complexes represent new types of probes for optical oxygen sensing. In comparison to the most commonly used ruthenium(II) polypyridyl dyes and porphyrin complexes with platinum group metals, they possess much more efficient visible absorption and higher quantum yields, which results in much higher brightnesses. Spectral properties and oxygen sensitivity can be fine-tuned by varying the nature of the coumarin ligand and using respective monomeric or dimeric complexes. When incorporated in a model polystyrene film the probes show optimal dynamics of luminescence decay time for oxygen monitoring in the range from 0% to 100% air saturation. Cross-sensitivity to temperature is significantly lower than for the commonly used ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline oxygen probe. The probes, however, exhibit significantly lower photostability, which restricts their application. If long-term measurements are not required, the probes can be successfully used for reliable monitoring of oxygen concentration. High brightness of the complexes makes them particularly attractive for application in thin films (for monitoring of fast processes) and various types of nano- and microparticles, including magnetic beads. If temperature compensation is not applied, the novel optodes result in the lower errors in determination of oxygen content. Optical sensing has become very popular in the last decades. A variety of oxygen sensors have been developed, and some of them have been commercialized. However, this area still attracts significant attention of the research community. New methods have appeared recently, for example, those allowing for simultaneous optical sensing and imaging of several analytes.1-6 Since oxygen is one of the key analytes, its monitoring is of extreme importance in process monitoring in biotechnology, environmental monitoring, seawater analysis and marine research, food industry, * Corresponding author. E-mail:
[email protected]. Phone: +43 316 873 4326. Fax: +43 316 873 4329. (1) Zelelow, B.; Khalil, G.; Phelan, G.; Carlson, B.; Gouterman, M.; Callis, J. B.; Dalton, L. R. Sens. Actuators, B 2003, 96, 304-314. (2) Borisov, S. M.; Vasylevska, G. S.; Krause, Ch; Wolfbeis, O. S. Adv. Funct. Mater. 2006, 16, 1536-1542. (3) Borisov, S. M.; Wolfbeis, O. S. Anal. Chem. 2006, 78, 5094-5101. (4) Koese, M. E.; Carrol, B. F.; Schanze, K. S. Langmuir 2005, 21, 91219129. (5) Borisov, S. M.; Krause, Ch; Arain, S.; Wolfbeis, O. S. Adv. Mater. 2006, 18, 1511-1516. (6) Schroeder, C. R.; Polerecky, L.; Klimant, I. Anal. Chem. 2007, 79, 60-70. 10.1021/ac0710836 CCC: $37.00 Published on Web 08/25/2007
© 2007 American Chemical Society
medicine, and many other fields of science and technology. Viable oxygen probes can be divided into several groups. Evidently, the probes based on polyaromatic hydrocarbons,7,8 which were used at the earliest stage of optical sensing technology, are no alternative to the metal complexes, because of the severe drawbacks of fluorescence intensity measurements and too low sensitivity. On the other side, all luminescent metal complexes possess relatively long-lived emission (microsecond to millisecond range), which allows for self-referenced measurements of the luminescence decay time. By far, ruthenium(II) polypyridyl complexes and particularly ruthenium(II)-tris-4,7-diphenyl-1,10phenanthroline (Ru-dpp) have been the most popular oxygen probes.9-15 The ruthenium probes, however, exhibit only moderate brightnesses (Bs; defined as the product of molar absorption coefficient and quantum yield Φ), which do not exceed ∼10 500.16 Moreover, the probes suffer from pronounced crosssensitivity to temperature, since their triplet states are subject to severe thermal quenching.17,18 Another inconvenience includes relatively short decay times of several microseconds, which results in low sensitivity unless highly oxygen permeable polymers (such as silicones,9,19 Ormosils,16,20 or silica gels on which the dyes are adsorbed) are used. On the other side, platinum(II) porphyrin complexes have proved to be excellently suitable for oxygen sensing in the range from 0% to 100% air saturation.21-23 Particu(7) Xu, W.; Schmidt, R.; Whaley, M.; Demas, J.; DeGraff, B.; Karikari, E.; Farmer, B. Anal. Chem. 1995, 67, 3172-3180. (8) Fujiwara, Y.; Amao, Y. Sens. Actuators, B 2004, 99, 130-133. (9) Carraway, E.; Demas, J.; DeGraff, B.; Bacon, J. Anal. Chem. 1991, 63, 337342. (10) Amao, Y.; Okura, I. Sens. Actuators, B 2003, 88, 162-167. (11) Apostolidis, A.; Klimant, I.; Andrzejewski, D.; Wolfbeis, O. S. J. Comb. Chem. 2004, 6, 325-331. (12) McEvoy, A.; McDonagh, C.; MacGraith, B. J. Sol.-Gel Sci. Technol. 1997, 8, 1121-1125. (13) Roche, P.; Al-Jowder, R.; Narayanaswamy, R.; Young, J.; Scully, P. Anal. Bioanal. Chem. 2006, 386, 1245-1257. (14) Vasilevska, A. S.; Borisov, S. M.; Krause, Ch; Wolfbeis, O. S. Chem. Mater. 2006, 18, 4609-4616. (15) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1995, 67, 2650-2654. (16) Alford, P. C.; Cook, M. J.; Lewis, A. P.; McAuliffe, S. G.; Skarda, V.; Thomson, A. J. J. Chem. Soc., Perkin Trans. 2 1985, 5, 705-709. (17) Forster, L. S. Coord. Chem. Rev. 2002, 227, 59-92. (18) Liebsch, G.; Klimant, I.; Wolfbeis, O. S. Adv. Mater. 1999, 11, 1296-1299. (19) Draxler, S.; Lippitsch, M.; Klimant, I.; Kraus, H.; Wolfbeis, O. S. J. Phys. Chem. 1995, 99, 3162-3167. (20) Klimant, I.; Ruckruh, F.; Liebsch, G.; Stangelmayer, A.; Wolfbeis, O. S. Microchim. Acta 1999, 131, 35-46. (21) Papkovsky, D. V.; Ponomarev, G. V.; Trettnak, W.; O’Leary, P. Anal. Chem. 1995, 67, 4112-4117. (22) Mills, A.; Lepre, A. Anal. Chem. 1997, 69, 4653-4659. (23) Park, E. J.; Reid, K. R.; Tang, W.; Kennedy, R. T.; Kopelman, R. J. Mater. Chem. 2005, 15, 2913-2919.
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Figure 1. Chemical structures of the ligands and their dimeric and monomeric complexes with iridium(III).
larly, the platinum(II) complex with 5,10,15,20-tetrakis-(2,3,4,5,6pentafluorphenyl)-porphyrin (PtTFPP) shows excellent photostability24 and, thus, has been widely used in oxygen sensing.4,5,25,26 Again, upon visible excitation (which is strongly preferable compared to the excitation in the intense Soret band due to restrictions of plastic optical fibers and lower level of the background fluorescence) Bs of the probe is only moderate and is compared to the brightness of the ruthenium probes.27 Finally, the third group of probes is comprised of cyclometalated complexes of iridium(III)28-30 and platinum(II).31 Despite usually high emission quantum yields, the brightness of the probes is poor, since molar absorption coefficients in the visible region rarely exceed several thousand liters per mole‚centimeter.32 (24) Lee, S.-K.; Okura, I. Anal. Commun. 1997, 34, 185-188. (25) Amao, Y.; Miyashita, T.; Okura, I. J. Fluorine Chem. 2001, 107, 101106. (26) Puklin, E.; Carlson, B.; Gouin, S.; Costin, C.; Green, E.; Ponomarev, S.; Tanji, H.; Gouterman, M. J. Appl. Polym. Sci. 2000, 77, 2795-2804. (27) 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. (28) Amao, Y.; Ishikawa, Y.; Okura, I. Anal. Chim. Acta 2001, 445, 177-182. (29) Di Marco, G.; Lanza, M.; Mamo, A.; Stefio, I.; Di Pietro, C.; Romeo, G.; Campagna, S. Anal. Chem. 1998, 70, 5019-5023. (30) DeRosa, M.; Mosher, P.; Yap, G.; Foscaneanu, K.; Crutchley, R.; Evans, C. Inorg. Chem. 2003, 42, 4864-4872. (31) Vander Donckt, E.; Camerman, B.; Herne, R.; Vandeloise, R. Sens. Actuators, B 1996, 32, 121-127.
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An iridium(III) complex of coumarin 6 was reported to have high brightness of ∼38 000 upon visible excitation33 and was suggested to be applicable in the OLED technology. A derivative of the complex covalently bound to polymer support, however, exhibited lower brightness of ∼19 000.34 In this work we will demonstrate that cyclometalated complexes of iridium(III) with coumarin ligands (Figure 1) can be viable oxygen probes for sensing and imaging purposes. EXPERIMENTAL SECTION Materials. Iridium(III) chloride hydrate was obtained from ABCR (www.abcr.de); 3-(benzothiazol-2-yl)-7-(diethylamino)-coumarin () CS), sodium 3-(trimethylsilyl)-1-propanesulfonate (NaTMS), 2-ethoxyethanol, silver trifluoromethanesulfonate, acetylacetone () acac), and piperidine were bought from Aldrich (www.sigmaaldrich.com); benzothiazol-2-yl-acetonitrile was from Maybridge (www.maybridge.com); ruthenium(III) chloride hydrate and 4,7-diphenyl-1,10-phenanthroline were (32) Lamansky, S.; Djurovich, P.; Murphy, D.; Abdel-Razzaq, F.; Kwong, R.; Tsyba, I.; Bortz, M.; Mui, B.; Bau, R.; Thompson, M. E. Inorg. Chem. 2001, 40, 1704-1711. (33) Lamansky, S.; Djurovich, P.; Murphy, D.; Abdel-Razzaq, F.; Lee, H.; Adachi, C.; Burrows, P. E.; Forrest, S. R.; Thompson, M. E. J. Am. Chem. Soc. 2001, 123, 4304-4312. (34) DeRosa, M. C.; Hodgson, D. J.; Enright, G. D.; Dawson, B.; Evans, C.; Crutchley, R. J. J. Am. Chem. Soc. 2004, 126, 7619-7626.
obtained from Lancaster (www.lancastersynthesis.com); platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin () PtTFPP) was purchased from Frontier Scientific (www.frontiersci.com); 3-(1-methylbenzoimidazol-2-yl)- 7-(diethylamino)-coumarin () CN), 3-dimethylaminophenol, phosphorus oxychloride, and polystyrene () PS; M ) 250 000) were obtained from Fischer Scientific (www.fishersci.com); 1-methyl-2-pyrrolidone and ethyl cellulose with ethoxyl content of 46% were from Fluka (www.sigmaaldrich.com), 3-(5-chlorobenzooxazol-2-yl)-7-(diethylamino)-coumarin (CO) “Macrolex fluorescent yellow” was bought from Simon and Werner GmbH (www.simon-und-werner.de); poly(ethylene glycol terephthalate) support (Mylar) was obtained from Goodfellow (www.goodfellow.com). All the 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.com; www.sigmaaldrich.com). The preparation of ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline dichloride (Ru(dpp)3Cl2) is described elsewhere.35 Ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline dichloride was converted into its much more lipophilic (TMS) salt () Ru-dpp) by addition of an equivalent amount of Na-TMS to an aqueous solution of the dye and subsequent extraction of the product into chloroform. 3-(Benzothiazol-2-yl)-7-(dimethylamino)-coumarin (CS-Me) was prepared from 4-(dimethylamino)-2-hydroxybenzaldehyde and benzothiazol-2-yl-acetonitrile according to the literature procedure.36 Preparation of 4-(dimethylamino)-2-hydroxybenzaldehyde is reported elsewhere.37 Synthesis of the Iridium Coumarin Complexes. The cyclometalated Ir(III) µ-chloro-bridged dimer coumarin complexes of a general formula (CX)2Ir(µ-Cl)2Ir(CX)2 were prepared according to the procedure of Lamansky et al.33 The dimers were converted to the corresponding acetylacetonate complexes of a general formula Ir(CX)2(acac) following the procedure of DeRosa et al.34 The iridium(III) complex with 3-(5-chlorobenzooxazol-2-yl)-7(diethylamino)-coumarin Ir(CO)2(acac) was prepared in the following manner. Three hundred seventy milligrams of CO and 175 mg of iridium(III) chloride hydrate were dissolved in the mixture of 30 mL of 1-methyl-2pyrrolidone and 10 mL of water. Then, 50 µL of triethylamine (TEA) was added and the solution was refluxed under nitrogen at 150 °C for 15 h. The solution was cooled, and 100 mL of water was added. The precipitate was washed twice with water and then methanol/water mixture (1:2 v/v). The precipitate was dissolved in 40 mL of acetone, and 100 µL of acac and 150 µL of TEA were added. The solution was stirred at room temperature for 5 h, the solvent was evaporated under reduced pressure, and the precipitate was dissolved in dichloromethane. CO and other impurities were washed out with dichloromethane on a short silica gel column, then, the product was collected by passing the mixture of tetrahydrofuran and dichloromethane (1:10 v/v). Yield: 60 mg (11%). The complex was not isolated as a pure substance. (CS)2Ir(µ-Cl)2Ir(CS)2. 1H NMR [500 MHz, (CD3Cl), δ]: 7.88 [d, 4H, H(9)], 7.31 [d, 4H, H(12)], 7.05 [t, 4H, H(10)], 7.02 [t, 4H, H(11)], 6.12 [s, 4 H, H(8)], 5.28 [d, 4H, H(5)], 4.97 [d, 4H, H(6)], 3.06 [q, 16H, 4 × 2CH2], 0.92 [t, 24H, 4 × 2CH3]. Analysis: found C 51.46, H 3.84, N 5.67; calcd C 51.85, H 3.70, N 6.05. (35) Klimant, I.; Wolfbeis, O. S. Anal. Chem. 1995, 67, 3160-3166. (36) Christie, R. M.; Lui, C. H. Dyes Pigm. 2000, 47, 79-89. (37) Baird, W. C.; Schriner, R. L. J. Am. Chem. Soc. 1964, 86, 3142-3145.
(CN)2Ir(µ-Cl)2Ir(CN)2. MS: m/z 1840.9 calcd, 1840.9 found. 1H NMR [500 MHz, (CD3Cl), δ]: 7.63 [d, 4H, H(9)], 6.91 [t, 4H, H(10)], 6.80 [t, 4H, H(11)], 6.75 [d, 4H, H(12)], 6.09 [s, 4 H, H(8)], 5.20 [d, 4H, H(5)], 5.00 [d, 4H, H(6)], 4.25 [s, 12H, 4 × N-CH3], 3.04 [q, 16H, 4 × 2CH2], 0.90 [t, 24H, 4 × 2CH3]. Analysis: found C 54.07, H 4.45, N 8.95, Cl 4.05; calcd C 54.8, H 4.38, N 9.13, Cl 3.85. Ir(CS)2(acac). MS: m/z 990.2 calcd, 990.2 found. 1H NMR [500 MHz, (CD3Cl), δ]: 7.88 [d, 2H, H(9)], 7.60 [d, 2H, H(12)], 7.26 [t, 2H, H(10)], 7.23 [t, 2H, H(11)], 6.30 [s, 2 H, H(8)], 6.06 [d, 2H, H(5)], 5.83 [d, 2H, H(6)], 5.30 [s, 1H, (acac)], 3.21 [q, 8H, 2 × 2CH2], 1.71 [s, 6H, 2CH3 (acac)], 1.04 [t, 12H, 2 × 2CH3]. Analysis: found C 52.62, H 4.18, N 5.25; calcd C 54.58, H 4.17, N 5.66. Ir(CS-Me)2(acac). MS: m/z 934.1 calcd, 934.1 found. 1H NMR [500 MHz, (CD3Cl), δ]: 7.89 [d, 2H, H(9)], 7.59 [d, 2H, H(12)], 7.30 [t, 2H, H(10)], 7.22 [t, 2H, H(11)], 6.31 [s, 2 H, H(8)], 6.08 [d, 2H, H(5)], 5.88 [d, 2H, H(6)], 5.30 [s, 1H, (acac)], 2.87 [s, 12H, 2 × 2CH3], 1.71 [s, 6H, 2CH3 (acac)]. Analysis: found C 52.40, H 4.19, N 5.20; calcd C 52.72, H 3.56, N 6.00. Ir(CN)2(acac). MS: m/z 984.3 calcd, 984.3 found. 1H NMR [500 MHz, (CD3Cl), δ]: 7.43 [d, 2H, H(9)], 7.23 [t, 2H, H(10)], 7.05 [t, 2H, H(11)], 6.44 [d, 2H, H(12)], 6.23 [s, 2 H, H(8)], 5.88 [d, 2H, H(5)], 5.30 [d, 2H, H(6)], 4.72 [s, 1H, (acac)], 4.56 [s, 6H, 2 × N-CH3], 3.20 [q, 8H, 2 × 2CH2], 1.62 [s, 6H, 2CH3 (acac)], 1.04 [t, 12H, 2 × 2CH3]. Analysis: found C 57.24, H 4.95, N 8.13; calcd C 57.36, H 4.81, N 8.54. Preparation of the Sensor Films. Three milligrams of the respective indicator and 200 mg of PS were dissolved in 1800 mg of chloroform. The “cocktails” were knife-coated onto a dust-free polyester support, and the solvent was allowed to evaporate under ambient air to result in an ∼6 µm sensor film. Other films were prepared in a similar manner. Spectral Measurements. Emission and excitation spectra were acquired on a Hitachi F-7000 fluorescence spectrometer (www.inula.at) equipped with a red-sensitive photomultiplier tube (PMT) R 928 from Hamamazu (www.hamamatsu.com). According to the spectral response characteristics of the PMT, its sensitivity at 650 nm is ∼15% less than at 500 nm. The emission spectra were not corrected for the sensitivity of the PMT. Absorption spectra were measured at a Cary 50 UV-vis spectrophotometer (www. lzs-concept.com). Relative luminescence quantum yields were determined according to Demas and Crosby38 using fluorescein solution in 0.1 M NaOH (φ ) 0.90) as a standard. The solutions of the dyes were thoroughly deoxygenated by bubbling nitrogen through. Luminescence phase shifts for the dyes in solutions and for the sensor films were measured with a two-phase lock-in amplifier (SR830, Stanford Research Inc., www.thinksrs.com). The sensor films containing the iridium(III) complexes or Ru-dpp in polystyrene were excited with the light of a blue light-emitting diode (LED) (λmax 470 nm, www.roithner-laser.com) which was sinusoidally modulated at a frequency of 20 kHz (in the case of the iridium(III) complexes) or at a frequency of 45 kHz (in the case of Ru-dpp). The sensor film containing PtTFPP in PS was excited with the light of a violet 405 nm LED, and a modulation frequency of 5 kHz was used. A bifurcated fiber bundle was used to guide the excitation light to the vial and to guide back the luminescence after passing the OG 550 (Schott) glass filter. The luminescence (38) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991-1024.
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Figure 2. Spectral properties of the coumarin ligands (a and b) and the respective iridium(III) complexes (c and d) in chloroform solutions at room temperature.
was detected with a PMT (H5701-02, Hamamatsu, www.sales.hamamatsu.com). Temperature was controlled by a ThermoHaake DC50 cryostat. Gas calibration mixtures were obtained using a gas mixing device (MKS, www.mksinst.com). Photostability tests were performed by irradiating the sensor films with the light from a xenon fiber light source LQX 1800 (180 W, Linos Photonics, www.linos.com) and monitoring absorption, excitation, and emission spectra, as well as the luminescence decay time. Drift of the luminescence intensity and phase shift was also estimated for the microsensors, obtained by coating a 140 µm optical glass fiber (GP Fiber Optics; www.gp-fiberoptics.de) with sensor cocktails (complexes and PS in chloroform). A pH-microdevice from Presens (www.presens.de) was used to read the luminescence from the microsensors. Fitting was performed using Origin version 7.5 (www.originlab .com) software. RESULTS AND DISCUSSION General Considerations. The coumarin structure (Figure 1) represents a highly flexible system in respect to fine-tuning of spectral properties. Spectral properties depend on the nature of the substituent in the 3 position of the coumarin, and significant bathochromic shift is observed in the row benzimidazol-benzooxazol-benzothiazol. Spectral properties also are determined by the nature of the donor group in the 7 position of the coumarin. All the trends should also be preserved in metal complexes. The electron transitions in the complexes are actually of the same nature as in the ligands, apart from the very high probability of the intersystem crossing from the singlet exited state into the triplet state promoted by the presence of the “heavy atom”. Thus, high molar absorption coefficients are preserved when Ir(III) and Pt(II) are coordinated, as well as the sharpness of excitation and emission bands, as was demonstrated by Lamansky et al.33 and DeRosa et al.34 In the work of DeRosa et al. the suitability of an iridium(III) coumarin complex for oxygen sensing was demon7504
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strated. It should be noted that coumarin dimers of general structure (CX)2Ir(µ-Cl)2Ir(CX)2 (Figure 1) were considered as intermediate products and their spectral and oxygen-sensitive properties were not investigated. Apart from the complex with CS no other indicators were reported. Synthesis. We have found that the reported synthetic procedures33,34 were adequate for synthesis of most of the complexes. The notable exception includes the iridium(III) complex with 3-(5chlorobenzooxazol-2-yl)-7-(diethylamino)-coumarin Ir(CO)2(acac). If the ligand and the iridium(III) chloride hydrate are refluxed in 2-ethoxyethanol/water (3:1 v/v) mixture, a nonluminescent redcolored precipitate is formed. The luminescent intermediate product can, however, be obtained by refluxing the components in the 1-methyl-2-pyrrolidone/water (3:1 v/v) mixture. A small amount of TEA also is added to avoid protonation of the nitrogen in the benzooxazol ring which occurs in acidic media. The intermediate product is then converted to the monomeric complex Ir(CO)2(acac) by stirring its solution in acetone with acetylacetone and TEA. Although the literature procedure via formation of the dimeric complex is preferable in most cases, since it can be thoroughly controlled at both steps, it cannot be used for the dyes having low solubility in the 2-ethoxyethanol/water mixture, such as CO. The yield and purity of the complex obtained in the second procedure were, unfortunately, not optimal, but they can be possibly improved by varying the nature of the solvent, water content, etc. Photophysical Properties of the Complexes. Coumarin ligands CN, CO, and CS show efficient visible absorption ( ∼ 50 000 M-1‚cm-1) and emission (quantum yield (QY) ∼ 1.0). The position of absorption and fluorescent bands, however, differ significantly (Figure 2, parts a and b, Table 1). The benzimidazol-based coumarin (CN) is the most short-waved, whereas the benzothiazolbased (CS) is the most long-waved. The same trend is preserved for the iridium(III) complexes of the general formula Ir(CX)2(acac) (Figure 2, parts c and b). Generally, Ir(CO)2(acac) and Ir(CS)2-
Table 1. Photophysical Properties of the Iridium Complexes and the Respective Ligands in Chloroform Solutions at 25 °C dye
λmax abs (nm)
(M-1‚cm-1)
CN
410
CO
454 442 (sh)
CS-Me
458 (sh) 440
59 900
CS
464 446
(CN)2Ir(µ-Cl)2Ir(CN)2
λmax em (nm)
QY
τ0 (µs)
478 53 200
481
1.00
486
0.96
50 400 51 000
491
0.94
463 433
87 600 133 600
567
0.30
9.7
(CS)2Ir(µ-Cl)2Ir(CS)2
482 457
136 500 136 900
587
0.21
13.1
Ir(CN)2(acac)
450 421
57 100 86 900
545
0.53
8.5
Ir(CO)2(acac)
467 443
53 700 47 000
552
0.34
10.7
Ir(CS-Me)2(acac)
471 441
75 400 77 800
564
0.44
11.3
Ir(CS)2(acac)
472 444
92 800 86 800
563
0.54
11.3
(acac) show excellent compatibility with the bright 470 nm LED, whereas Ir(CN)2(acac) can be efficiently excited with the light of a 405 nm LED. All the complexes can also be efficiently excited with 425, 435, and 450 nm LEDs. Thus, if necessary, fine-tuning of the spectral properties of a sensor can be achieved by choosing a complex with a particular coumarin ligand. Such flexibility is of much interest for multianalyte sensing, particularly with the modified dual lifetime referencing method6,39 where simultaneous excitation of a phosphorescent oxygen indicator and a fluorescent (pH, ion, etc.) indicator is essential. All the complexes show highly efficient phosphorescence at room temperature in deoxygenated chloroform solutions and no detectable fluorescence. The phosphorescence bands are bathochromically shifted by ∼70 nm compared to the fluorescence bands of the respective ligands (Figure 2, parts b and d, Table 1). Notably, the absorption spectra change only slightly upon complexation and bathochromic shift does not exceed 10 nm. Phosphorescence QY for deoxygenated Ir(CX)2(acac) solutions in chloroform were measured to be 0.34-0.54 (Table 1). The brightness of the complexes (Bs, which is determined as the product of the molar absorbance and luminescence quantum yield) is very high. For Ir(CS)2(acac) it exceeds 50 000, whereas for Ir(CN)2(acac) it is as high as 46 000 upon excitation in the absorption maxima. For comparison, Bs of the most widely used oxygen probe, ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline (Ru-dpp), is 10 500.15 Platinum(II) and palladium(II) complexes with 5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (TFPP) also are viable oxygen indicators, but their Bs upon excitation in visible does not exceed 10 000.27 It should be mentioned here that brightness of indicators is the key parameter for a successful application in optical sensing. Indicators which possess high (39) Borisov, S. M.; Neurauter, G.; Schroeder, C.; Klimant, I.; Wolfbeis, O. S. Appl. Spectrosc. 2006, 60, 1167-1173.
brightness will enable (a) manufacturing of very thin sensor films which have therefore fast response times, (b) preparation of nanobeads which can be used in small amounts and thus will have little influence on the optical properties of the system, (c) preparation of the magnetic beads with high signal-to-noise ratio, and (d) lower consumption of the costly indicators. Relatively sharp excitation and emission bands of the complexes (compared, e.g., with the bands of Ru-dpp) enable simultaneous monitoring of various species (by using indicators absorbing and emitting in different spectral regions). The dyes can also be efficiently used for encoding perposes.40 We have also found that the dimeric complexes of the general formula (CX)2Ir(µ-Cl)2Ir(CX)2 also are strongly phosphorescent. Although emission quantum yields are lower than for the respective monomeric complexes (Table 1), the Bs is still good (∼15 000-20 000 calculated for one iridium atom). The absorption and emission maxima shift bathochromically compared to those of the respective monomeric complexes (Figure 3). Notably, shift in the absorption is less pronounced (∼10 nm), whereas the emission is long-wave shifted by more than 20 nm compared to that of the respective monomeric complexes. The dimeric complexes thus provide one more possibility for the fine-tuning of the spectral properties. Additionally, (CS)2Ir(µ-Cl)2Ir(CS)2 enables excitation with a 488 nm argon laser and is thus suitable for confocal microscopy. The phosphorescence decay times for both dimeric and monomeric complexes of ∼10 µs (Table 1) make them suitable candidates for optical oxygen quenching. The donor ability of the dimethylamino group is known to be lower than that of the diethylamino group. Therefore, we have investigated another possibility of the fine-tuning of the spectral properties, namely, by varying the donor group in the 7 position of the coumarin ring. In fact, both absorption and emission bands (40) Mayr, T.; Moser, C.; Klimant, I. Anal. Chim. Acta, in press.
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Figure 3. Spectral properties of the CS ligand and the respective monomeric and dimeric complex in chloroform solutions at room temperature.
of 3-(benzothiazol-2-yl)-7-(dimethylamino)-coumarin (CS-Me) were found to be hypsochromically shifted by 4 nm compared to 3-(benzothiazol-2-yl)-7-(diethylamino)-coumarin (Table 1). The complexes with both ligands, however, show practically identical spectral properties. On the other side, it is likely that the coumarin complex containing in the 6 position julolidine instead of diethylamino-group will result in significant bathochromic shift in absorption and emission. Oxygen Optodes. Polystyrene has been by far the most commonly used polymer matrix for preparation of oxygen optodes, and therefore it was also used as a standard matrix here. Advantages of PS include good permeability to oxygen, but complete impermeability to ions, cheapness, and easiness in film manufacturing. The 5 µm thick sensor foils contained 1.5 wt % of an indicator in PS. For estimation of brightnesses, 2 µm thick sensor foils were prepared that contained 0.5 wt % of an indicator (which is the typical concentration in optical sensing) so that the absorption of the foils was kept low (A < 0.2). Spectral properties of the
probes are presented in Table 2. In comparison to the absorption and emission spectra obtained for the complexes in chloroform solutions (Table 1), a small bathochromic shift of the bands is observed in PS, which is attributed to the lower polarity of the polymer. The properties of the novel optodes were compared with those of the standard optodes based on Ru-dpp and PtTFPP in PS, which are currently the most investigated and used ones. Although the response of the oxygen optodes was investigated in the frequency domain, the time domain method can be applied as well. Moreover, spectral properties of the indicators allow for ratiometric measurements which are common in such applications as confocal microscopy and measurements in microplate readers. The emission of the novel oxygen indicators can be easily referenced via the emission of the ligands, since both emissions occur in different spectral regions. Notably, absorption spectra of both are very similar so that simultaneous excitation of both is possible. These two properties make the pair ligand/complex ideally suitable for the ratiometric measurements. Brightness of the Oxygen Optodes. Figure 4 shows the comparative study of the brightnesses of Ir(CS)2(acac)-based oxygen optode and the standard optodes based on Ru-dpp and PtTFPP, which are commonly used oxygen probes. It should be mentioned here that we preferred to use the platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin and not the platinum(II) octaethyl porphyrin (PtOEP), which is even a more widespread oxygen indicator, since the latter suffers from poor photostability. The spectroscopic properties of both PtTFPP and PtOEP are very similar in which the indicators exhibit intense absorption in the UV region (the Soret band) and much less intense Q-bands in the visible region. As can be seen from Figure 4, the iridium(III) dye (M ) 990), indeed, absorbs in visible much more efficiently than Ru-dpp (M ) 1489 for the TMS- form) and PtTFPP (M ) 1168). The emission spectra were obtained by exciting the sensor films with the light of a xenon lamp at the
Table 2. Spectral and Oxygen-Sensitive Properties of the Iridium(III) Coumarin and Standard PtTFPP and Ru-dpp Optodes dye parameters λmax abs (nm) λmax em (nm) τ0 at 25 °C (µs) relative Bs at 0 hPa O2 and 25 °C relative Bs at 212 hPa O2 and 25 °C photostability resolution at 25 °C and 25 hPa O2 (deg/hPa) resolution at 25 °C and 175 hPa O2 (deg/hPa) error at 25 hPa (hPa/°C) error at 175 hPa (hPa/°C) f1 K1 at 1 °C (hPa-1) K1 at 25 °C (hPa-1) K1 at 50 °C (hPa-1)
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Ir(CS)2(acac) Ir(CN)2(acac) Ir(CO)2(acac) Ir(CS-Me)2(acac) (CS)2Ir(µ-Cl)2Ir(CS)2 (CN)2Ir(µ-Cl)2Ir(CN)2
PtTFPP
Ru-dpp
448, 477
421, 455
444, 472
446, 475
457, 484
432, 463
510, 542
452, 475
566
544
554
566
588
567
650
607
9.1
7.5
7.7
10.4
12.6
8.8
55
5.1
1
1.90
0.61
1.33
0.76
1.17
0.067
0.15
0.62
1.21
0.36
0.73
0.36
0.58
0.021
0.12
moderate 0.090
very poor 0.078
poor 0.093
poor 0.110
poor 0.143
very poor 0.130
very good 0.270
very good 0.038
0.044
0.043
0.049
0.060
0.070
0.063
0.063
0.035
0.47
0.54
0.92
0.4
0.45
0.46
0.45
4.2
2.6
3.2
2.8
2.0
2.0
2.43
3.65
5.5
0.81 ( 0.08 0.00322
0.80 ( 0.11 0.00297
0.86 ( 0.04 0.00329
0.87 ( 0.03 0.00392
0.89 ( 0.01 0.00542
0.92 ( 0.05 0.00440
0.86 ( 0.002 0.73 ( 0.084 0.0149 0.00176
0.00428
0.00395
0.00431
0.00518
0.00701
0.00573
0.0181
0.00215
0.00558
0.00606
0.00516
0.00641
0.00826
0.00730
0.0207
0.00259
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
Figure 4. Absorption (left) and emission spectra (right) of 2 µm thick sensor films, containing 0.5 wt % of an oxygen indicator in polystyrene. Excitation of Ir(CS)2(acac), Ru-dpp, and PtTFPP was performed at 477, 475, and 542 nm, respectively (5 nm slit). The emission from the films was measured in deoxygenated solutions at the same PMT voltages.
Figure 5. Stern-Volmer plots for Ir(CS)2(acac) in PS (1.5 wt %) at 25 °C.
wavelengths corresponding to the absorption maxima of the complexes. All the measurements were performed in deoxygenated solutions. Evidently, the Ir(CS)2(acac)-based oxygen optode benefits from much better Bs than the respective Ru-dpp and PtTFPP standard optodes, which results from much higher molar absorptions and luminescence quantum yields. Notably, QY for the coumarin complexes can increase even more in the polymer, which is also the case for the ruthenium(II) polypyridyl complexes, such as Ru-dpp.16 Relative brightnesses of the coumarin probes (Bs of the Ir(CS)2(acac)-based probe under deoxygenated condition was set as 1) are summarized in Table 2. Notably, the Bs of the probes remains very high even at air saturation so that precise determination of the decay time is still possible. It should be mentioned, however, that at significantly higher concentrations of the dyes and thicknesses of the sensor films much more light can be absorbed by all the sensors including those based on Rudpp and PtTFPP, and the difference in Bs will not be so pronounced. It should also be considered that if porphyrin complexes such as PtTFPP or PtOEP are excited into the Soret band (peaking at 392 nm for PtTFPP), much higher brightnesses can be achieved. However, excitation in the visible region is strongly preferred for many practical reasons: (a) much lower background fluorescence from the sample; (b) higher stability and brightness of the visible light sources; (c) restrictions of the cheap plastic fibers which have low transmittance in the UV region.
High concentrations of the indicators and thick sensor films often are not desirable, and the iridium(III) complexes are indicators of choice in these cases. Moreover, they can be potentially act as the indicators of choice in various types of nanobeads (used, e.g., for microscopic purposes) since rather low concentrations of the latter can be used in analyzed media to achieve sufficient signal. Another exciting application includes magnetic beads which possess high internal absorbance and therefore require high indicator brightnesses. Sensitivity of the Oxygen Optodes. Figure 5 shows the comparison of the Stern-Volmer plots (I0/I and τ0/τ vs pO2) for Ir(CS)2(acac) at 25 °C. As expected, the intensity plot shows better linearity than the decay time plot, which is common for the optical oxygen sensors. Response of the optodes to oxygen is illustrated by Figure 6. It is evident that the dimeric complexes show longer luminescence decay times and thus higher sensitivity to oxygen compared to the respective monomeric complexes. The changes of the phase shift in deg/hPa are presented in Table 2 (“resolution at 25 °C”). The modulation frequencies used were 45, 5, and 20 kHz for Ru-dpp, PtTFPP, and the iridium complexes, respectively. Since this parameter is determined by the form of the response curve, the resolution is higher at lower oxygen partial pressures. At high pO2 the coumarin optodes show precision comparable to that of the PtTFPP optode, but their resolution is significantly lower at 25 hPa O2. The Ru-dpp optode shows the lowest precision in the whole dynamic range. It should be noted that the resolution of the coumarin optodes will be increased if polymers with higher oxygen permeability (such as poly(4-tertbutylstyrene), ethylcellulose, or sol-gels) are employed. Although the Stern-Volmer plots for the iridium(III) probes exhibit a high degree of linearity (Figure 5), they are still best described using the following equation from the “two-site model”:41,42
f2 f1 I τ + ) ) I0 τ0 1 + K1[O2] 1 + K2[O2]
(1)
where f1 and f2 are the fractions of the total emission for each component, respectively (with f1 + f2 being 1), and K1 and K2 are the Stern-Volmer constants for each component. Since K2 is usually very small, it can be considered to be 0 for the sake of simplicity. A fit with eq 1 will therefore result in two fit parameters, namely, f1 and K1. The fit parameters for the oxygen optodes are collected in Table 2. Temperature Dependence of the Calibration Curves. Since temperature has a pronounced effect on the behavior of any oxygen sensor2 the response curves were measured at 1, 25, and 50 °C. The response of the standard sensors based on PtTFPP and Ru-phen in PS also was obtained in similar conditions. The results are presented in Figure 7. It was found that in case of the coumarin complexes temperature has only a minor effect on the triplet state decay times in the absence of oxygen (Figure 7a) which is comparable to that of PtTFPP (Figure 7c). On the contrary, Ru-dpp exhibits tremendous temperature dependence of its decay time (Figure 7e). On the other side, for the latter a very moderate response to oxygen is observed in PS. Indeed, in (41) Carraway, E. R.; Demas, J. N.; DeGraff, B. A.; Bacon, J. R. Anal. Chem. 1991, 63, 337-342. (42) Sacksteder, L.; Demas, J. N.; DeGraff, B. A.; Bacon, J. R. Anal. Chem. 1993, 65, 3480-3483.
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
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Figure 6. Calibration curves for the oxygen optodes (1.5 wt % of an indicator in polystyrene) at 25 °C. Left, decay time plots (fitting according to the monoexponential decay model); right, Stern-Volmer plots (fitting according to the two-site model, eq 1).
Figure 7. Calibration curves of the oxygen optodes (1.5 wt % of an indicator in polystyrene) at different temperatures. Left, decay time plots (fitting according to the monoexponential decay model); right, Stern-Volmer plots (fitting according to the two-site model, eq 1). (a and b) Ir(CS)2(acac); (c and d) PtTFPP; (e and f) Ru-dpp.
PS the temperature effect is even more pronounced than quenching by oxygen. For precise and reliable measurements all oxygen sensors require compensation for the temperature effects, which can be determined in the second independent measurement with the help of a thermoelement or a temperature optode, or even using more elegant dual optodes.2,3 However, if temperature fluctuations are relatively small, such second independent measurement can be impractical, since it requires additional apparatus and calculation procedures. In this case, error in determination of oxygen content will be determined by two factors: sensitivity of an optode to oxygen and its cross-talk to temperature. The temperature 7508 Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
Figure 8. Photobleaching curves of Ir(CS)2(acac)/PS microoptode (λexc 470 nm) acquired onto a pH-microdevice. Measurement rate was 1 point/s. The modulation frequency was 5 kHz.
dependence for all the optodes was estimated from measurements at 1, 25, and 50 °C (Figure 7, parts a, c, and e). It is, of course, dependent on oxygen partial pressure. As an example, we estimated the errors caused by variation in temperature by 1 °C, when the measurements are performed at around 25 °C at low pO2 (25 hPa) and at high pO2 (175 hPa). The data are presented in Table 2. Errors caused by the Ru-dpp-based optodes are rather high and are relatively uniform in the whole range of oxygen concentrations, since they are mostly caused by very efficient thermal quenching of the triplet state decay time. Ru-dpp has been the most commonly used oxygen probe; however, it is definitely one of the most unreliable ones due to severe temperature effects. It should be mentioned that in the case of Ru-dpp the temperature effect on the Stern-Volmer plots is lower than for the other indicators which is due to significant decrease of τ0 at higher temperatures. As expected, for the PtTFPP and the iridium(III) coumarin optodes, the error decreases significantly at lower pO2, since diffusional and collisional factors predominate here. Since quenching of PtTFPP is rather efficient, the error at 175 hPa is higher than for the coumarin probes. These optodes are thus excellently suitable for monitoring of oxygen content in cases when compensation for temperature effects is not applied. Photostability of the Oxygen Optodes. Photostability of any optode is an important property, and it becomes a critical one if prolonged measurements are performed. We have found that photostability of the novel optodes is unfortunately much lower than that of the respective Ru-dpp- and Pt-TFPP-based optodes. Since intensity of the excitation light differs significantly for various optodes and setups, and so does the photobleaching rate, we tested the optodes as microsensors, where high light densities (and consequently decomposition rates) are common. The photobleaching curves were acquired onto a pH-microdevice, using a 470 nm LED as an excitation source (modulation frequency 5 Hz) and measurement rate of 1 point/s. Figure 8 shows typical photobleaching curves for the optode which makes use of Ir(CS)2(acac). It appears to be the most photostable and shows similar photobleaching rates in deoxygenated and air-saturated solutions. In fact, under nitrogen degradation in luminescence intensity was 1.9% per 1000 measurement points (mp), and 1.2% in the airsaturated solution. Taking into consideration that intensity in the
Figure 9. Absorption spectra of Ir(CN)2(acac) in PS (1.5 wt %, 5 µm thick films) upon irradiation with a xenon lamp.
latter case is lower, the photobleaching appears to be 1.9% if recalculated to the oxygen-free condition. The observed decrease in the luminescence phase shift was 0.122°/1000 mp under nitrogen, and 0.073°/1000 mp under air. The other optodes were found to exhibit much higher photodegradation rates. In fact, photostability decreased in the row Ir(CS)2(acac) > Ir(CO)2(acac) > Ir(CS-Me)2(acac) > Ir(CN)2(acac), and the latter showed the fastest photobleaching from all the optodes investigated. Notably, the optodes based on the monomeric complexes show similar photobleaching in the absence of oxygen and at air saturation. The situation is different, however, for the dimeric complexes. In the absence of oxygen, the (CS)2Ir(µ-Cl)2Ir(CS)2- and (CN)2Ir(µCl)2Ir(CN)2-based optodes show photobleaching rates similar to the respective Ir(CS)2(acac)- and Ir(CN)2(acac)-based microoptodes, but they bleach much faster at air saturation. Here, photooxidation is presumably the predominant way of photobleaching. It should be noted that photobleaching of all iridium coumarin optodes results in a drift of the decay time, which is, however, always less pronounced than degradation of luminescence intensity. Evidently, measurements of the decay time results in much more reliable measurements. Photobleaching was further investigated in a different experiment, in which the air-equilibrated sensor foils were irradiated with the light from a xenon lamp. The light density of the light source (which contains both UV and visible light) was estimated to be higher than 10 mW/cm2. As can be observed from Figure 9, photobleaching results in notable changes in the absorption spectra of the complexes. A new band appears at 411 nm in the case of Ir(CN)2(acac) and at 416 nm in the case of Ir(CS)2(acac). At the same time, absorbance of the film decreases significantly. Notably, a new band also appears in the excitation spectra; however, no change in the emission spectra is observed. This indicates that the mechanism of the photobleaching is not photodissociation but rather a transformation of the coumarin ligand in the complex in which a new phosphorescent product is produced. The luminescence decay time also decreased significantly and for Ir(CS)2(acac) under nitrogen was as low as 4.4 µs. The same decay time was measured for the Ir(CN)2(acac) in the end of the bleaching experiment. This data correlate excellently with those obtained for the microoptodes where decrease in luminescence intensity and luminescence phase shift was ob-
served. Notably, most photodecomposition occurs in the initial period of irradiation (Figure 9), which is ∼10 min for the Ir(CN)2(acac) sensor films and ∼30 min for those based on Ir(CS)2(acac). Photostability of the ligand CS in PS film also was investigated. The ligand was found not to exhibit detectable bleaching after 1 h of irradiation in similar conditions. Photostability of the complex is thus significantly lower than of the ligand, indicating that both coumarin moieties in the complex are likely to be involved in the photodegradation process. For comparison, photobleaching tests also were performed with Ru-dpp and PtTFPP sensor films. In similar conditions virtually no changes in absorption spectra of the dyes are observed after 1 h of irradiation. In the case of Ru-dpp an ∼10% decrease in the luminescence decay time was observed (from 5.1 to 4.6 µs), while for PtTFPP the decay time remained unaltered. Although the performed tests are only a rough estimation, since no monochromatic light was used but rather the whole spectrum of the xenon lamp (including UV light), these data indicate significantly higher photostability of Ru-dpp and PtTFPP compared to that of the iridium(III) coumarin complexes. The lower photostability, thus, limits the applicability of the novel optodes when prolonged measurements are performed or when high light densities are used (e.g., in microsensors or microscopy). It should be mentioned here that polystyrene itself can be prone to photodegradation, which, however, occurs much slower than photodecomposition of the indicators. It is also the case here, since bleaching of PtTFPP and CS sensor foils revealed no alteration in properties, whereas bleaching of the coumarin complexes resulted in distinct spectral changes. In this work we investigated and compared the optodes which made use of PS. Polymers with higher permeability to oxygen are readily available and of course can also be used for immobilization of the indicators so that the sensor materials with significantly higher sensitivity to oxygen are obtained. Except for this parameter (which is governed by the nature of an indicator and by the gas permeability of a polymer) other properties of these optodes will mostly be determined by the nature of an indicator, so the trends obtained for the PS standard sensors will be preserved. In conclusion, we have shown that novel oxygen-sensitive optodes possess such features as outstanding brightness, low cross-sensitivity to temperature, and suitability for both decay timebased and ratiometric measurements. The sensor materials are nicely compatible with bright LEDs. They can be effectively used for sensing and imaging of oxygen in gas phase and aqueous solutions both in the format of planar sensor foils or in microand nanobeads. Spectral properties of the optodes and their sensitivity to oxygen can be fine-tuned by choosing the appropriate (monomeric or dimeric) complex. The drawbacks include moderate to low photostability of the indicators, which is significantly lower than that of the most commonly used oxygen indicators, namely, ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline and platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin. This limits potential application of the optodes to short-time measurements. Received for review May 24, 2007. Accepted July 20, 2007. AC0710836 Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
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