Screen-Printed Red Luminescent Copolymer Film Containing

Article pubs.acs.org/IECR

Screen-Printed Red Luminescent Copolymer Film Containing Cyclometalated Iridium(III) Complex as a High-Permeability Dissolved-Oxygen Sensor for Fermentation Bioprocess Pengwei Jin, Zhiqian Guo, Ju Chu, Jun Tan, Siliang Zhang, and Weihong Zhu* State Key Laboratory of Bioreactor Engineering, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, P. R. China S Supporting Information *

ABSTRACT: The novel hydrophobic luminescent copolymer P(Ir-TFEMA) was developed as an online dissolved-oxygen (DO) sensor. The phosphorescent moiety of cyclometalated iridium(III) complex exhibits red emission near 650 nm with a large Stokes shift of about 245 nm and minimal optical interference from the fermentation system. The covalent incorporation of the chromophore into the polymeric matrix rather than physical doping was used to avoid phase-separation and leaching problems. The low molar ratio between the introduced chromophore and polymeric matrix within the range of 1:135−1:250 was confirmed to have little influence on the luminescence response ability. To assess its potential utility, this copolymer was applied to the online monitoring of DO during the cephalosporin C fermentation process. The screen-printing technique was utilized as a rapid and reliable automatic approach to preparing sensor films with good photostability and fatigue resistance, showing promise in bioprocess monitoring as a low-cost DO indicator for high-throughput microbioreactors.

1. INTRODUCTION Microbioreactors with integrated sensors combining the features of small volumes and high degrees of parallelization have become promising tools for rapid, high-throughput, and cost-effective bioprocessing.1−4 They are of significant interest in the development of novel microbial cell cultivation technologies for high-throughput screening as well as for bioprocess development. Oxygen monitoring is of high importance in the fermentation bioprocess because it affects metabolism in a multitude of ways.5−10 The traditional determination of trace oxygen with electrochemical techniques (such as Clark electrodes) depends on the current at the electrode surface where oxygen is electrochemically reduced. Although Clark electrodes are sensitive to a wide temperature range, the limitation of miniaturization and online monitoring restricts their application,11 which is quite incapable of monitoring high-throughput bioprocessing in miniature microbioreactor arrays, especially in fluid dynamics. Therefore, the combination of automated robotic systems equipped with miniaturized dissolved-oxygen (DO) detectors has become critical for low-cost high-throughput microbioreactors.12,13 Obviously, optical DO sensors have become attractive because of their many advantages over traditional Clark electrodes, including nonconsumption of oxygen;11 convenient miniaturization; remote sensing; real-time monitoring; and low sensitivity or susceptibility to flowing, signal drift, and electromagnetic fields, even in stirring fermentation systems.14 As an optical sensor film for aqueous fermentation systems, the performance of a sensor is largely dependent on its oxygensensitive luminophores and supporting matrixes. It is known that high hydrophobicity is beneficial to gas permeability and low fouling in reactors. The common method is to dope the luminophores physically into matrixes such as polymers or sol− gel glasses.15 Introducing chemosensors into polymeric back© 2013 American Chemical Society

bones through covalent links can avoid the phase separation and concentration quenching of chromophores, and such structures can be easily fabricated into devices fixed onto microbioreactors.16−19 Controlling the molar ratio in a polymerization process is a difficult but important task because the characterized result is not always equal to the feed molar ratio, which can greatly affect the material performance.20 In particular, screen-printing, as a technique for creating a twodimensional pattern, has been touted as the most versatile techniques for film fabrication with simple, fast, and reproducible characteristics.21 Cyclometalated iridium(III) complexes are attractive because of their highly efficient oxygen-concentration-dependent phosphorescence and long lifetime up to microseconds.22 Herein, we report a red luminescent copolymer DO sensor film, P(Ir-TFEMA) (where TFEMA = trifluoroethyl methacrylate), especially for high-throughput microbioreactors (Scheme 1). It was specifically synthesized from the copolymerization of an iridium(III) complex with trifluoroethyl methacrylate (TFEMA) given its high permeability for oxygen. It can be successfully applied in the online monitoring of DO in fermentation systems. The low molar ratio between the chromophore and the polymeric matrix within the range from 1:135 to 1:250 was confirmed to have minimal influence on the luminescence response ability. The long-wavelength red luminescence with a large Stokes shift of about 245 nm can ensure little interference from biological fermentation samples.23−25 Received: Revised: Accepted: Published: 3980

January 22, 2013 February 28, 2013 March 5, 2013 March 5, 2013 dx.doi.org/10.1021/ie4002525 | Ind. Eng. Chem. Res. 2013, 52, 3980−3987

Industrial & Engineering Chemistry Research

Article

1H), 8.44 (dd, J1 = 8.4 Hz, J2 = 0.8 Hz, 1H), 8.37 (s, 1H), 8.20−8.17 (m, 1H), 8.00−7.95 (m, 2H), 7.89−7.80 (m, 5H), 7.71−7.66 (m, 6H), 7.50−7.45 (m, 2H), 7.39 (d, J = 8.4 Hz, 1H), 7.25−7.20 (m, 2H), 7.15−7.07 (m, 2H), 6.96 (dd, J1 = 8.0 Hz, J2 = 1.2 Hz, 1H), 6.84−6.79 (m, 2H), 6.77−6.71 (m, 2H), 6.60−6.54 (m, 2H), 6.11 (s, 1H), 5.64 (s, 1H), 2.08 (s, 3H). 13 C NMR (CDCl3, 100 MHz, ppm): δ 168.34, 163.43, 163.26, 154.03, 153.81, 152.74, 152.17, 148.32, 147.47, 146.95, 144.79, 144.29, 144.23, 140.81, 140.47, 140.31, 140.17, 139.39, 139.22, 138.86, 136.00, 135.42, 135.25, 134.04, 131.97, 131.61, 131.44, 131.08, 130.99, 130.81, 130.76, 130.57, 130.35, 130.11, 129.57, 128.93, 127.70, 126.56, 126.38, 123.41, 122.93, 122.77, 121.11, 18.52. HRMS (ESI): calcd for C56H39IrN7O ([M − PF6−]+) 1018.2845, found 1018.2850. 2.3. Synthesis of P(phen-TFEMA). A mixture of phen-MA (46.9 mg, 0.178 mmol), TFEMA (3 g, 17.8 mmol), Nmethylpyrrolidone (NMP) (3 mL), and AIBN (6.5 mg, 0.040 mmol) was added into a Schlenk tube (25 mL) under anhydrous oxygen-free conditions at −78 °C. The solution was degassed by three freeze−pump−thaw cycles and stirred at 75− 80 °C for 24 h. The resulting mixture was dissolved in CH2Cl2 (5 mL), and pink floccules precipitated after addition of methanol (400 mL). The dissolution and precipitation cycle was repeated three times to obtain a pale pink solid (2.2 g, yield 72.2%). Several P(phen-TFEMA) materials with various molar ratios of monomers were synthesized by a similar method except using different amounts of phen-MA. 2.4. Synthesis of P(Ir-TFEMA). A mixture of [(dpq)2IrCl]2 (42 mg, 0.0266 mmol), P(phen-TFEMA) (1.0 g), and CH2Cl2 (20 mL) was refluxed for 4 h under argon protection. After the mixture had been cooled to room temperature, NH4PF6 (50 mg, 0.31 mmol) was added, and the mixture was stirred for 2 h at room temperature. The resulting mixture was dissolved in CH2Cl2 (5 mL), and pink floccules precipitated after addition of methanol (400 mL). The dissolution and precipitation cycle was repeated three times to obtain a pale pink solid (0.85 g, yield 81.6%). 2.5. Microorganisms, Medium, and Culture. Acremonium chrysogenum (purchased from China General Microbiological Culture Collection Center with the accession number of CGMCC 3.4008) was grown on PMM agar medium (per liter, 10.0 g of peptone, 12.0 g of malt extract, 40.0 g of maltose, 18.0 g of agar; pH 7.0). The seed medium contained (per liter) 5.0 g of glucose, 35.0 g of sucrose, 0.5 g of methionine, 8.0 g of (NH4)2SO4, 50.0 g of corn steep liquor, 5.0 g of CaCO3, and 5.0 mL of soybean oil (pH 6.5). The fermentation medium contained (per liter) 20.0 g of glucose, 30.0 g of starch, 6.0 g of methionine, 50.0 g of corn steep liquor, 13.0 g of (NH4)2SO4, 3.0 g of urea, 3.0 g of MgSO4·7H2O, 10.0 g of CaCO3, 9.0 g of KH2PO4, 40.0 mL of soybean oil, 0.02 g of CuSO4·5H2O, 0.02 g of ZnSO4·7H2O, 0.02 g of MgSO4·7H2O, and 0.08 g of FeSO4·7H2O (pH 6.2). Acremonium chrysogenum was precultured in a 500 mL shake flask containing 50 mL of seed medium at 28 °C and 220 rpm for 3 days. Precultures (10%, v/ v) were inoculated in the microbioreactor at 28 °C. 2.6. Preparation of DO Sensor Films. The DO sensor film array was prepared by screen-printing a 50 wt % CH2Cl2/ MeCN (1:1, v/v) solution on a thoroughly washed quartz substrate. In the beginning, pure CH2Cl2 was used as the ink because of its excellent solubility; however, its extremely high volatility restricted its use in screen printing. The film border was fixed by strips of adhesive tape. AFM was employed to visualize the surface and roughly measure the film thickness.

Scheme 1. Chemical Structure of Copolymer P(Ir-TFEMA)

2. EXPERIMENTAL SECTION 2.1. Chemicals and Instruments. 1H and 13C NMR spectra were obtained on a Bruker AVANCE III 400 MHz spectrometer with tetramethylsilane (TMS) as the internal standard. High-resolution mass spectroscopy (HRMS) was performed on a Waters LCT Premier XE spectrometer [electrospray ionization (ESI)]. Absorption and fluorescence spectra were measured on a Varian Cary 100 spectrometer and a Horiba Scientific FluoroMax-4 spectrometer, respectively. The weight-average molecular weight (Mw) and molecular weight distribution (Mw/Mn, where Mn is the number-average molecular weight) were obtained with a Waters 1515 gel permeation chromatography (GPC) system with tetrahydrofuran (THF) as the mobile phase. Microsecond time-resolved decay-time data were acquired using an Edinburgh Instruments FLS900 fluorometer equipped with a microsecond flashlamp (μF920H), and a 470-nm cutoff filter was used before the receiver to avoid the scattering of excitation light. The film morphology and thickness were characterized by atomic force microscopy (AFM) with a MicroNano D 3000 scanning probe microscope using tapping mode. A Maya2000 Pro spectrometer from Ocean Optics Inc. was used as the CCD spectrometer in the online setup. Trifluoroethyl methacrylate and IrCl3 were purchased from Aladdin Reagent Co., Ltd. Methacryloyl chloride was freshly prepared before use, and 2,2′-azobisisobutyronitrile (AIBN) was recrystallized from anhydrous ethanol. All other chemicals used in this study were of analytical reagent grade. Benzil, diphenylquinoline (dpq), [(dpq)2IrCl]2, phen-NO2 (phen = 1,10-phenanthroline), phen-NH2, phen-MA (MA = methacrylamide), and methacryloyl chloride were prepared according to literature methods (Scheme S1 in the Supporting Information).26−29 2.2. Synthesis of [Ir(dpq)2phen]PF6. A mixture of [(dpq)2IrCl]2 (70 mg, 0.044 mmol), phen-MA (26 mg, 0.099 mmol), CH2Cl2 (12 mL), and methanol (6 mL) was refluxed for 4 h under argon protection. After the mixture had been cooled to room temperature, NH4PF6 (50 mg, 0.310 mmol) was added, and the mixture was stirred for 2 h at room temperature. The solvent was removed by vacuum, and the residua were purified by column chromatography on silica gel (CH2Cl2) to obtain a red powder (60 mg, yield 67%). 1H NMR (CDCl3, 400 MHz, ppm): δ 9.02 (d, J = 4.0 Hz, 1H), 8.95 (dd, J1 = 4.8 Hz, J2 = 0.8 Hz, 1H), 8.84 (d, J = 8.8 Hz, 1H), 8.78 (s, 3981

dx.doi.org/10.1021/ie4002525 | Ind. Eng. Chem. Res. 2013, 52, 3980−3987


Article

The sensor film was estimated to be about 1 μm thick, and the area of each film was 0.5 × 0.5 cm2. 2.7. Spectral Determination of Copolymer Film on DO Response. The film-coated quartz as a sensor patch was firmly attached to the bottom of the custom-made microbioreactor with the film side facing toward the inside. A QF600-8-VIS/ NIR bifurcated optic fluorescence fiber bundle from Ocean Optics Inc. was used to realize portable online and noninvasive monitoring (Figure 1). The optic probe contained one flat fiber

Moreover, the large Stokes shift of about 245 nm can effectively eliminate the interference of the excitation light during the detection of luminescence emission. The signal acquisition time was set as 0.1 s with fixed slit and integration numbers. 2.8. DO Sensing Principles. The quenching of the intensity and lifetime of DO luminescence is due to collisional quenching by oxygen and can be described by the classic Stern−Volmer equation Iini τ = ini = 1 + KD[Q] I τ

(1)

where Iini and τini are the initial luminescent intensity and lifetime, respectively, of dye in an inert atmosphere and I and τ are the corresponding quantities in the presence of O2. KD is the Stern−Volmer quenching constant, and [Q] is the concentration of O2. In a heterogeneous environment, the quenching curves are analyzed in terms of two components, that is, the two-site model Stern−Volmer equation with two discrete sets of quenching parameters I Iini

Figure 1. Schematic diagram of online DO sensing system based on oxygen-dependent phosphorescence for fermentation bioprocess.

=

f1 f2 τ = + 1 τini 1 + K sv[O2 ] 1 + K sv2[O2 ]

(2)

where f1 and f 2 are the fractions of the total emission for each component (with f1 + f 2 = 1) and K1sv and K2sv are the Stern− Volmer constants for the two components. The polymeric quenching response (QDO) to DO is defined as

for detection and seven angled fibers for directing excitation energy to the region in front of the detection fiber. Therefore, the optic probe combined excitation and detection in one optical fiber, which can eliminate the interfering effect of total reflected light in the traditional 90° optical design in fluorescence spectrometers for the light source and detector.

Q DO = I0/I100

(3)

Scheme 2. Syntheses of Monomer [Ir(dpq)2phen]PF6 and Copolymer P(Ir-TFEMA)

3982



Article

where I0 and I100 correspond to the luminescent intensities of the N2- and O2-saturated states, respectively.

parallel experiments were on the same level and fully comparable. As a key characteristic of oxygen sensors, the values of QDO (eq 3) were determined for the four copolymers and were found to be 16.8, 17.5, 17.8, and 17.0 (Table 1). These results suggest that, during the parallel copolymerization, the feed molar ratio (m/n > 50) between TFEMA and cyclometalated iridium(III) complex had minimal influence on QDO, so that we no longer needed to pay much attention to the feed molar ratio (m/n) of the monomers during the copolymerization process. It is difficult to precisely control the monomer molar ratio in the resulting copolymers because of the difficult batch-to-batch reproducibility during polymerization. As discussed in a later section, molar ratios of about 135−250 between the matrix and the luminophore monomer can exhibit ideal oxygen-dependent luminescence, which can be expected to apply for the online monitoring of DO in a fermentation system. Sensor 3 with a feed molar ratio of 100 (entry 3 in Table 1) was taken as representative of P(Ir-TFEMA) for systematic study in that context. 3.3. Optical Response of Sensing Films to DO. The common method of film preparation is spin-coating, which can produce very thin and uniform films. However, to fabricate large amounts of film samples with good repeatability, the screen-printing technique is a better choice, making industrialscale production possible.21 Hence, a P(Ir-TFEMA) film array was screen-printed, and its luminescence under excitation at 405 nm is presented in Figure 2. Because it is a phosphorescent

3. RESULTS AND DISCUSSION 3.1. Design and Synthesis of P(Ir-TFEMA). The luminescent properties of cyclometalated iridium(III) complexes with high luminescence quantum yields are of particular interest because of their extremely promising applications in organic light-emitting diodes (OLEDs),30 upconversion,31−33 long lifetimes up to microseconds, and convenient emission modulation with ligands.34 The majority of studies on DO sensors so far have focused on doping the luminophores into a polymer or sol−gel matrix physically, which can lead to serious phase-separation and leaching problems.35,36 Accordingly, introducing the chromophore chemically through a covalent bond to the matrix is desirable.37,38 Because of the high hydrophobicity of fluorinated polymers,14 we selected fluorinesubstituted TFEMA as the matrix monomer to copolymerize with a long-wavelength-emission cyclometalated iridium(III) complex. For the synthetic design, a pendent vinyl group was provided for the sake of polymerization (Scheme 2). The purpose of synthesizing monomer [Ir(dpq)2phen]PF6 was for characterization of the structure and determination of the molar ratio in the polymer. The rational synthesis of P(Ir-TFEMA) through coordinating P(phen-TFEMA) with iridium complex [(dpq)2IrCl]2 rather than directly copolymerizing monomer [Ir(dpq)2phen]PF6 with TFEMA can significantly reduce the cost. Here, the fluorine-substituted TFEMA is expected to enhance the polymeric hydrophobicity for preferable permeability to oxygen. 3.2. Molar Ratio (m/n) Optimization of Monomers. To establish the relationship of the molar ratio (m/n) between cyclometalated iridium(III) complex and TFEMA, four parallel experiments were conducted in the polymerization process (Table 1). The molar ratio (m/n) of the two monomers, which Table 1. Parameters of P(Ir-TFEMA) with Various Feed Molar Ratios between TFEMA and Cyclometalated Iridium(III) Complex

entry sensor sensor sensor sensor

1 2 3 4

feed molar ratio (m/n)

absorbancedetermined molar ratio (m/n)

solution QDO

50 80 100 150

135.1 182.7 203.4 247.9

16.8 17.5 17.8 17.0

weightaverage molecular weight

polydispersity

× × × ×

1.36 1.43 1.39 1.23

1.32 1.33 1.53 1.76

105 105 105 105

Figure 2. (a) Screen-printed P(Ir-TFEMA) film array. (b) Luminescence of film array under excitation at 405 nm. Note: Quartz glass is placed on a blue substrate for high image contrast.

chromophore, its emission intensity is dependent on the concentration of oxygen. In an aqueous system, the phosphorescence is due to collisional quenching by oxygen and can be described by the Stern−Volmer equation (see eq 1). In the study of the film optical response to DO, it was found that the emission intensity of the P(Ir-TFEMA) film was quenched by 2.5-fold from the N2-saturated state to the O2saturated state (QDO = 2.5, Figure 3a). In addition, the O2 sensitivity of the copolymer in acetonitrile solution was examined (Q DO = 17.8, Figure 3b). The different O 2 sensitivities of P(Ir-TFEMA) in the film and solution states can be attributed to the great difference in the diffusion of oxygen in these media. The DO response time of P(IrTFEMA) film was further studied in aqueous systems. Water was saturated with N2 and O2 in turn for five cycles, and the luminescence at 650 nm was monitored each second automatically (Figure 3c). It was found that the P(IrTFEMA) film can be toggled repeatedly between the N2- and O2-saturated states, exhibiting good fatigue resistance, which is

could be determined from the standard working curve in the absorption spectra (Figure S1a in the Supporting Information), was easily tuned by varying the initial feed. Because the absorption at 475 nm arose solely from the cyclometalated iridium(III) complex, the molar ratio (m/n) of the two monomers could easily be determined from variations in the copolymer absorbance (Figure S1b in the Supporting Information). As reported in Table 1, the obtained molar ratios (m/n) between TFEMA and cyclometalated iridium(III) complex for the four copolymers were 135.1, 182.7, 203.4 and 247.9. Obviously, these values are higher than the initial feed molar ratios, which might be due to the different reactivities of the two monomers. The weight-average molecular weights and polydispersities determined by GPC indicated that the four 3983



Article

Figure 3. Luminescence intensities of P(Ir-TFEMA) in N2-, air-, and O2-saturated solutions at λex = 405 nm: (a) film in water medium, QDO = 2.5; (b) polymer in acetonitrile solution, QDO = 17.8. (c) Luminescence intensity response of P(Ir-TFEMA) film in water to O2/N2 switching cycles, λem = 650 nm. (d) Partial enlarged drawing of the plot in panel c. Note: The response time was calculated when the 95% emission intensity changed.

vital for long-time monitoring. The response times of P(IrTFEMA) film in 95% luminescence quenching and 95% luminescence enhancement were 1.0 and 2.0 min (Figure 3d), respectively. Moreover, the value of QDO and the luminescence of P(Ir-TFEMA) remained stable after the film was irradiated every 10 s at 405 nm for 2 h (Figure 4a). No distinct degradation or leaching problem occurred over a one-month period under ambient air or in water, thus making P(IrTFEMA) films satisfactory for long-time processes in fermentation microbioreactors. Figure 4b shows the line slope of the oxygen-dependent luminescence. In Figures 3a and 4b, the experimental conditions are comparable, and the quenching responses to DO (QDO) are around 2.5. In Figure 3a, QDO can be calculated as the ratio of the emission intensities in the N2- and O2saturated states. In Figure 4b, QDO can be determined from the highest point, which is indicative of the N2-saturated state versus the O2-saturated state. Here, the 30 ppm oxygen concentration (the highest point) can be treated as the same condition as for I100 (O 2 -saturated state). When the concentration of O2 is increased, the emission intensity (I) of the sensor film decreases, thus resulting in an increase in I0/I with an upward curve (Figure 4b). Obviously, the high slope of the curve is an indication of a high DO sensitivity. The curve at low oxygen concentration is nonlinear, whereas it is linear at high oxygen concentrations greater than 5 ppm. This finding can be well explained by the fact that, in contrast to homogeneous solutions, the microenvironment of the polymer matrix is usually inhomogeneous for all dye molecules. At low oxygen concentrations, only the outer-layer luminophore molecules can interact with oxygen, leading to a high sensitivity, whereas at high oxygen concentrations, the outer-layer luminophore molecules are largely quenched, and the quenching sensitivity is dominated by the luminophore molecules in the less accessible areas, which is determined by the diffusion speed of oxygen in the copolymer matrix.39

Figure 4. (a) Photostability test of P(Ir-TFEMA) film. The film was irradiated at 405 nm every 10 s in N2-saturated water, and for the rest of the time, the shuttle before the excitation light was closed automatically. λem = 650 nm. (b) Two-site model fit of the oxygendependent luminescence for the P(Ir-TFEMA) film. Note: The highest point (30 ppm) represents the O2-saturated state.

In consideration of the unique properties of cyclometalated iridium(III) complexes, the luminescence lifetimes of the DO sensing film were investigated in N2-, air-, and O2-saturated states. It was found that the luminescence lifetime curves were best fitted with a double-exponential decay equation (Figure S2 in the Supporting Information) 3984



Article

R(t ) = A1e−t / τ1 + A 2 e−t / τ2

consume oxygen at a very high rate. Consequently, their dissolved-oxygen level will drop to near zero once the microbioreactor stops bubbling, making timely DO measurement difficult. Moreover, in the traditional right-angle configuration of fluorescence spectrometers, a certain angle of the sensing film (generally in the range of 30−60°, except for 45°) is tuned to receive the excitation light and to reflect the emission light. However, most fermentation broths are highly viscous fluids that are difficult for light to penetrate, and the fermentation will strongly scatter both excitation and emission light. Fortunately, these problems could be avoided in our setup, in which the sensing film patch was attached to the bottom of the microbioreactor with the film side facing toward the inside. The optical fiber could easily detect the luminescence without interference, as no fluid was present between the optical fiber and the sensor patch (Figure 1). Cephalosporin C fermentation broth cultivated in a shake flask was transferred to our system and saturated with N2 for 10 min. Then, O2 was bubbled into the microbioreactor at a constant rate until the fermentation broth was saturated with O2, during which time the luminescence was monitored at short intervals (1 s, Figure 5a). As expected, the luminescence became weak as the O2 content increased. Here, the downward trend of the luminescence indicates that the DO sensor film based on P(IrTFEMA) can provide a practical response during a fermentation process. Furthermore, to assess its potential utility, the proposed method was applied as an online DO indicator during the cephalosporin C fermentation process (Figure 1). The fermentation was cultivated as usual, and a parallel experiment was conducted in a conventional fermentation bioreactor equipped with a DO electrode. Except for the reactor volume, the two fermentation systems remained in the same conditions as much as possible. The variation trends in the two types of measurements exhibited a good consistency (Figure 5b),

(4)

The molar contribution of the lifetime can be calculated by the equation Aτ αi = n i i ∑i = 1 Ai τi (5) where Ai represents the amplitude of the exponential decay equation and τi represents the lifetime. In view of the fact that α1 and α2 are far more than 3% (Table 2), this result cannot be ignored as an error to being a Table 2. Parameters of Luminescence Lifetime of DO Sensing for P(Ir-TFEMA) Films conditiona

τ1 (μs)

τ2 (μs)

α1b (%)

α2b (%)

τwc (μs)

O2 air N2

0.18 0.28 0.30

0.78 1.08 1.25

12.3 13.0 10.9

87.7 87.0 89.1

0.71 0.98 1.15

Water was saturated with the corresponding gas over 30 min. bα1 and α2 are the fractions of the total emission for each component (with α1 + α2 = 1). cPre-exponential weighted lifetime. a

single-exponential decay. On the contrary, the doubleexponential decay indicates precisely that there are two processes during the course of luminescence decay, which is consistent with the work plot. Here, the two processes can be designated as quenching at sites that are easy and difficult for oxygen to access. 3.4. Potential Application. Finally, the feasibility of online measurements of DO in the cephalosporin C (CPC) fermentation system was checked. In microbioreactors, no DO electrode is available, and the fermentation broth has to be removed for measurements to be conducted. However, many fermentation broths, such as that of Cephalosporins acremonium,

Figure 5. (a) Luminescence intensities of P(Ir-TFEMA) film in cephalosporin C fermentation with increasing O2. λex = 405 nm, QDO = 2.5. (b) Online monitoring of DO in cephalosporin C fermentation broth monitored by sensing film and electrode equipped in a conventional fermentation bioreactor. Note: The luminescence intensity is reversed vertically for convenience of comparison. (c,d) Luminescence intensity responses of P(IrTFEMA) film upon excitation at 405 nm in N2-saturated aqueous system as functions of (c) temperature (20−80 °C) and (d) pH (λem = 650 nm). 3985



■

further confirming the potential application of our DO sensor in microbioreactors. In the culture process of cephalosporin C fermentation, the DO content of the fermentation first declined gradually to its minimum value, during which the cell made use of starch as the carbon source. Afterward, the DO rose again because the starch ran out before the soybean oil was introduced to be used as the second carbon source. Finally, when the soybean oil started to be used, the DO dropped again as Acremonium chrysogenum consumed large amounts of oxygen while making use of the soybean oil. In addition, the luminescence can be converted to DO content quantitatively utilizing a standard working curve (Figure 4b). Importantly, a practicable sensor should have a high specificity in the detection process. To qualify the sensor film as a microbioreactor DO indicator, it must be demonstrated that the polymer film sensor based on cyclometalated iridium(III) complex is responsive only to DO and not to other species (or temperature or pH). The effect of temperature on the luminescence of P(Ir-TFEMA) film was studied preferentially because all luminescent probes display more or less temperature-dependent signals (thermal quenching). Predictably, the luminescence intensity decreased with increasing temperature (Figure 5c). However, the fermentation temperature was always kept between 25 and 28 °C during the whole measurement. Moreover, the effect of temperature drift can be neglected because luminescence changes slightly from 20 to 40 °C (Figure 5c). Of course, a correction factor might be needed when the film is used in a wide temperature window; a modified Stern−Volmer model might be needed to compensate for the effects of temperature drift;40 or furthermore, multiple or colorimetric chemical sensors might need to be used.41 At the same time, pH was demonstrated to have no effect on the luminescence of the film (Figure 5d). Moreover, during the beginning of cephalosporin C cultivation, the pH of the fermentation broth rose from 6.5 to 7.0 and then stabilized at 5.5 for the most of time, indicating minute interference in the film luminescence. Obviously, the probable effects caused by temperature and pH can be eliminated during the continuous online measurement of DO in the cephalosporin C fermentation system. Accordingly, with optical stability and response specificity, the copolymer film of P(Ir-TFEMA) could potentially be applied as a low-cost microbioreactor DO indicator in high-throughput bioprocessing.

Article

ASSOCIATED CONTENT

S Supporting Information *

Synthetic route; 1H NMR, 13C NMR, and HRM spectra of monomer [Ir(dpq)2phen]PF6; and standard working curves and luminescent lifetime of copolymer P(Ir-TFEMA). This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Fax: (+86) 21-6425-2758. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank NSFC/China, National 973 Program (2013CB733700), Oriental Scholarship, National Major Scientific Technological Special Project (2012YQ15008709), Fundamental Research Funds for the Central Universities (WK1013002, WJ1114013), SRFDP 20120074110002, and Open Funding Project of the State Key Laboratory of Bioreactor Engineering for providing financial support to this project.

■

REFERENCES

(1) Zhang, Z. Y.; Szita, N.; Boccazzi, P.; Sinskey, A. J.; Jensen, K. F. A well-mixed, polymer-based microbioreactor with integrated optical measurements. Biotechnol. Bioeng. 2006, 93, 286−296. (2) Pohar, A.; Ž nidaršič-Plazl, P.; Plazl, I. Integrated system of a microbioreactor and a miniaturized continuous separator for enzyme catalyzed reactions. Chem. Eng. J. 2012, 189, 376−382. (3) Titmarsh, D.; Cooper-White, J. Microbioreactor array for fullfactorial analysis of provision of multiple soluble factors in cellular microenvironments. Biotechnol. Bioeng. 2009, 104, 1240−1244. (4) Sharma, S.; Rangaiah, G. P. Modeling and optimization of a fermentation process integrated with cell recycling and pervaporation for multiple objectives. Ind. Eng. Chem. Res. 2012, 51, 5542−5551. (5) Wang, X. D.; Stolwijk, J. A.; Lang, T.; Sperber, M.; Meier, R. J.; Wegener, J.; Wolfbeis, O. S. Ultra-small, highly stable, and sensitive dual nanosensors for imaging intracellular oxygen and pH in cytosol. J. Am. Chem. Soc. 2012, 134, 17011−17014. (6) Zou, X.; Hang, H. F.; Chu, J.; Zhuang, Y. P.; Zhang, S. L. Oxygen uptake rate optimization with nitrogen regulation for erythromycin production and scale-up from 50 L to 372 m3 scale. Bioresour. Technol. 2009, 100, 1406−1412. (7) Nikakhtari, H.; Hill, G. A. Enanced oxygen mass transfer in an external loop airlift bioreactor using a packed bed. Ind. Eng. Chem. Res. 2005, 44, 1067−1072. (8) Lee, H. L. T.; Boccazzi, P.; Ram, R. J.; Sinskey, A. J. Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control. Lab-on-a-Chip 2006, 6, 1229−1235. (9) Liu, H. J.; Liu, D. H.; Zhong, J. J. Oxygen limitation improves glycerol production by Candida krusei in a bioreactor. Process Biochem. 2004, 39, 1899−1902. (10) Chen, X. Q.; Tian, X. Z.; Shin, I.; Yoon, J. Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species. Chem. Soc. Rev. 2011, 40, 4783−4804. (11) Wang, X. D.; Chen, H. X.; Zhao, Y.; Chen, X.; Wang, X. R. Optical oxygen sensors move towards colorimetric determination. Trends Anal. Chem. 2010, 29, 319−338. (12) Wang, Z. J.; Wang, H. Y.; Li, Y. L.; Chu, J.; Huang, M. Z.; Zhuang, Y. P.; Zhang, S. L. Improved Vitamin B12 production by stepwise reduction of oxygen uptake rate under dissolved oxygen limiting level during fermentation process. Bioresour. Technol. 2010, 101, 2845−2852.

4. CONCLUSIONS In summary, a screen-printed red luminescent hydrophobic copolymer P(Ir-TFEMA) based on oxygen-sensitive phosphorescence has been developed as a DO indicator for bioprocess monitoring, in which a covalently immobilizable cyclometalated iridium(III) complex is used as a DO-sensitive unit and fluorinated monomer TFEMA is chosen as the polymeric matrix because of its high hydrophobicity. The low molar ratio between the introduced chromophore and polymeric matrix within the range of 1:135−1:250 has little influence on the luminescence response ability, making polymerization synthesis much easier. Moreover, the screen-printing technique for film preparation enables industrial production. The long-wavelength emission reaching the near-infrared range could ensure minimal interference from the biological fermentation system. Given the convenience of film preparation, good photostability, and fatigue resistance, the sensor film of P(Ir-TFEMA) can be successfully applied as an online DO indicator for highthroughput bioprocesses. 3986



Article

(13) Wu, D.; Hao, Y. Y.; Chu, J.; Zhuang, Y. P.; Zhang, S. L. Inhibition of degradation and aggregation of recombinant human consensus interferon-α mutant expressed in Pichia pastoris with complex medium in bioreactor. Appl. Microbiol. Biotechnol. 2008, 80, 1063−1071. (14) Wang, X. D.; Wolfbeis, O. S. Fiber-optic chemical sensors and biosensors (2008−2012). Anal. Chem. 2013, 85, 487−508. (15) Xiang, H. F.; Zhou, L.; Feng, Y.; Cheng, J. H.; Wu, D.; Zhou, X. G. Tunable fluorescent/phosphorescent platinum(II) porphyrinfluorene copolymers for ratiometric dual emissive oxygen sensing. Inorg. Chem. 2012, 51, 5208−5212. (16) Shen, L. J.; Lu, X. Y.; Tian, H.; Zhu, W. H. A long wavelength fluorescent hydrophilic copolymer based on naphthalenediimide as pH sensor with broad linear response range. Macromolecules 2011, 44, 5612−5618. (17) Kim, H. N.; Guo, Z. Q.; Zhu, W. H.; Yoon, J.; Tian, H. Recent progress on polymer-based fluorescent and colorimetric chemosensors. Chem. Soc. Rev. 2011, 40, 79−93. (18) Chen, X. Q.; Zhou, G. D.; Peng, X. J.; Yoon, J. Biosensors and chemosensors based on the optical responses of polydiacetylenes. Chem. Soc. Rev. 2012, 41, 4610−4630. (19) Cross, A. J.; Davidson, M. G.; Garcia-Vivo, D.; James, T. D. Well-controlled synthesis of boronic-acid functionalised poly(lactide)s: A versatile platform for biocompatible polymer conjugates and sensors. RSC Adv. 2012, 2, 5954−5956. (20) Peracchia, M. T.; Desmaële, D.; Couvreur, P.; D’Angelo, J. Synthesis of a novel poly(MePEG cyanoacrylate-co-alkyl cyanoacrylate) amphiphilic copolymer for nanoparticle technology. Macromolecules 1997, 30, 846−851. (21) Krebs, F. C.; Jørgensen, M.; Norrman, K.; Hagemann, O.; Alstrup, J.; Nielsen, T. D.; Fyenbo, J.; Larsen, K.; Kristensen, J. A complete process for production of flexible large area polymer solar cells entirely using screen printing−first public demonstration. Sol. Energy Mater. Sol. Cells 2009, 93, 422−441. (22) Marín-Suár ez, M.; Curchod, B. F. E.; Tavernelli, I.; Rothlisberger, U.; Scopelliti, R.; Jung, I.; Di Censo, D.; Grätzel, M.; Fernández-Sánchez, J. F.; Fernández-Gutiérrez, A.; Nazeeruddin, M. K.; Baranoff, E. Nanocomposites containing neutral blue emitting cyclometalated iridium(III) emitters for oxygen sensing. Chem. Mater. 2012, 24, 2330−2338. (23) Guo, Z. Q.; Nam, S.; Park, S.; Yoon, J. A highly selective ratiometric near-infrared fluorescent cyanine sensor for cysteine with remarkable shift and its application in bioimaging. Chem. Sci. 2012, 3, 2760−2765. (24) Yuan, L.; Lin, W. Y.; Zheng, K. B.; He, L. W.; Huang, W. M. Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging. Chem. Soc. Rev. 2013, 42, 622−661. (25) Guo, Z. Q.; Zhu, W. H.; Tian, H. Dicyanomethylene-4H-pyran chromophores for OLED emitters, logic gates and optical chemosensors. Chem. Commun. 2012, 48, 6073−6084. (26) Lin, W. H.; Sun, W. L.; Yang, J.; Sun, Q. H.; Shen, Z. Q. Fabrication of magnetic thin films by a layer-by-layer self-assembly approach. J. Phys. Chem. C 2009, 113, 16884−16895. (27) Hwang, F. M.; Chen, H. Y.; Chen, P. S.; Liu, C. S.; Chi, Y.; Shu, C. F.; Wu, F. I.; Chou, P. T.; Peng, S. M.; Lee, G. H. Iridium(III) complexes with orthometalated quinoxaline ligands: Subtle tuning of emission to the saturated red color. Inorg. Chem. 2005, 44, 1344− 1353. (28) Beheshtiha, Y. S.; Heravi, M. M.; Saeedi, M.; Karimi, N.; Zakeri, M.; Tavakoli-Hossieni, N. Efficient and green synthesis of 1,2disubstituted benzimidazoles and quinoxalines using brønsted acid ionic liquid, [(CH2)4SO3HMIN][HSO4], in water at room temperature. Synth. Commun. 2010, 40, 1216−1223. (29) Xu, C. J.; Li, B. G.; Wan, J. T.; Bu, Z. Y. Synthesis and characterization of a Eu-containing polymer precursor featuring thenoyltrifluoroacetone and 5-acrylamido-1,10-phenanthroline. Spectrochim. Acta A 2011, 82, 159−163.

(30) Chen, Y. M.; Zhang, A. G.; Wang, K. Z. Cyclometalated iridium(III) complex of 6-hydroxydipyrido[3,2-a:2′,3′-c]phenazine: Synthesis, and acid−base and avid DNA binding properties. Appl. Organomet. Chem. 2011, 25, 521−529. (31) Sun, J. F.; Wu, W. H.; Zhao, J. Z. Long-lived room-temperature deep-red-emissive intraligand triplet excited state of naphthalimide in cyclometalated Ir(III) complexes and its application in triplet−triplet annihilation-based upconversion. Chem.Eur. J. 2012, 18, 8100− 8112. (32) Yao, L. M.; Zhou, J.; Liu, J. L.; Feng, W.; Li, F. Y. Iridiumcomplex-modified upconversion nanophosphors for effective LRET detection of cyanide anions in pure water. Adv. Funct. Mater. 2012, 22, 2667−2672. (33) Ma, L. H.; Guo, H. M.; Li, Q. T.; Guo, S.; Zhao, J. Z. Visible light-harvesting cyclometalated Ir(III) complexes as triplet photosensitizers for triplet−triplet annihilation based upconversion. Dalton Trans. 2012, 41, 10680−10689. (34) Adachi, C.; Baldo, M. A.; Forrest, S. R.; Lamansky, S.; Thompson, M. E.; Kwong, R. C. High-efficiency red electrophosphorescence devices. Appl. Phys. Lett. 2001, 78, 1622−1624. (35) Xiong, Y.; Zhu, D. Q.; Chen, S. H.; Peng, H.; Guan, Y. F. A fiber-optic evanescent wave O2 sensor based on Ru(II)-doped fluorinated ORMOSILs. J. Fluoresc. 2010, 20, 269−274. (36) Lu, H. G.; Jin, Y. G.; Tian, Y. Q.; Zhang, W. W.; Holl, M. R.; Meldrum, D. R. New ratiometric optical oxygen and pH dual sensors with three emission colors for measuring photosynthetic activity in cyanobacteria. J. Mater. Chem. 2011, 21, 19293−19301. (37) Payne, S. J.; Fiore, G. L.; Fraser, C. L.; Demas, J. N. Luminescence oxygen sensor based on a ruthenium(II) star polymer complex. Anal. Chem. 2010, 82, 917−921. (38) Wang, D. M.; Li, X. H.; Wang, W. J.; Gong, X.; Li, B. G.; Zhu, S. P. Kinetics and modeling of semi-batch RAFT copolymerization with hyperbranching. Macromolecules 2011, 45, 28−38. (39) Zhao, Y.; Ye, T. X.; Chen, H. X.; Huang, D. P.; Zhou, T. Y.; He, C. Y.; Chen, X. A dissolved oxygen sensor based on composite fluorinated xerogel doped with platinum porphyrin dye. Luminescence 2011, 26, 29−34. (40) Lo, Y. L.; Chu, C. S.; Yur, J.; Chang, Y. C. Temperature compensation of fluorescence intensity-based fiber-optic oxygen sensors using modified Stern−Volmer model. Sens. Actuators B: Chem. 2008, 131, 479−488. (41) Stich, M. I. J.; Fischer, L. H.; Wolfbeis, O. S. Multiple fluorescent chemical sensing and imaging. Chem. Soc. Rev. 2010, 39, 3102−3114.

3987


Screen-Printed Red Luminescent Copolymer Film Containing

Recommend Documents