Anal. Chem. 2006, 78, 7511-7516
Slab Optical Waveguide High-Acidity Sensor Based on an Absorbance Change of Protoporphyrin IX Tomonari Umemura,*,† Hiroki Hotta, Takahiko Abe, Yoshihito Takahashi, Hiromi Takiguchi, Masayuki Uehara, Tamao Odake, and Kin-ichi Tsunoda
Department of Chemistry, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
A sensitive and fast-responsive evanescent wave absorption sensor has been constructed for pH measurements in highly acidic ranges. This sensor is based on a pHdependent color change of protoporphyrin IX (PPIX). For the sensitive detection, a visible attenuated total reflection spectrometer with a slab optical waveguide (SOWG) was laboratory-made, and the guiding layer surface was modified with a PPIX-immobilized acrylamide-based thin membrane. The sensing membrane with a thickness of ∼1 µm was directly fabricated on the SOWG glass surface by copolymerization of acrylamide, N,N′-methylene bisacrylamide, and PPIX in the narrow space confined by a cover plate. PPIX possesses two double bonds in its structure, and so it can be covalently incorporated into the membrane. The response characteristics of the PPIX-immobilized optode membrane were explored using aqueous solutions with different concentrations of HNO3 or HCl. The optode membrane provided characteristic Soret band absorption spectra depending on the hydrogen ion concentration; the absorbance at 410 nm increased with increasing the concentrations in the range of 0.15-2 M, corresponding to the range of pH -0.3 to 0.8. The absorption signal reached 90% of its final value within 10 s, while the absorption signal was quite readily returned to background level simply by passing 2 mL of distilled water through a flow cell with a volume of 16.5 µL placed on the SOWG. Due to the rapid response and reversibility, this sensor could be operated in a flow-through mode as well as in a conventional static mode, where deionized water was conveniently used as a carrier and conditioning solution. In terms of the stability and precision, this sensor showed no significant change in response even after 100 assays and after being stored in a dry condition for over 6 months. Relative standard deviations for 10 replicate measurements were less than 1.8% in the linear range, and the detection limit calculated from 3 times of the standard deviation was 0.02 pH unit. Development and applications of pH sensors have still attracted much attention because of the importance of pH measurements and the wide applicability in various scientific research and in * To whom correspondence should be addressed. E-mail: umemura@ apchem.nagoya-u.ac.jp. Tel.: +81-52-789-5288. Fax: +81-52-789-4665. † Present address: Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. 10.1021/ac0606150 CCC: $33.50 Published on Web 09/29/2006
© 2006 American Chemical Society
industry.1,2 The applications include the sensing of an enzyme reaction by measuring protons generated or consumed by the reaction. With the expanding applications, there arises an increasing demand for special pH sensors that allow real-time continuous monitoring and measurements in severe environments and in confined narrow spaces.3 In order to satisfy such analytical demands, optical pH sensors have eagerly been explored,4 because they offer many advantages over traditional potentiometric sensors, including immunity from electromagnetic interference, intrinsic safety in the use of harsh environments, ease of miniaturization, and the resultant possibility of remote sensing and in situ monitoring. The advantage of optical sensor also includes the applicability to pH measurements in highly acidic or basic ranges, where glass electrodes suffer from significant errors and chemical deterioration.5-8 To date, a variety of optical sensing approaches have been proposed, and several promising sensing devices such as fiberoptic and optical waveguide devices have been exploited.9-12 As one of such devices, we have so far constructed a visible attenuated total reflection (ATR) spectrometer with a slab optical waveguide (SOWG) and applied it to rapid and sensitive colorimetric detection of iron(II), silicic acid, or anionic surfactant.13-15 This SOWG measurement system based on evanescent wave absorption has potential to concurrently achieve high selectivity and sensitivity by concentrating analytes at the waveguide surface modified with host substances (or analyte selective dye) and by increasing the number of internal reflection.16,17 Furthermore, in (1) Wolfbeis, O. S. Anal. Chem. 2002, 74, 2663-2678. (2) Lin, J. Trends Anal. Chem. 2000, 19, 541-552. (3) Grant, S. A.; Glass, R. S. Sens. Actuators, B 1997, 45, 35-42. (4) Wolfbeis, O. S. Trends Anal. Chem. 1996, 15, 225-232. (5) Safavi, A.; Bagheri, M. Sens. Actuators, B 2003, 90, 143-150. (6) Safavi, A.; Abdollahi, H. Anal. Chim. Acta 1998, 367, 167-173. (7) Allain, L. R.; Sorasaenee, K.; Xue, Z. Anal. Chem. 1997, 69, 3076-3080. (8) Allain, L. R.; Xue, Z. Anal. Chem. 2000, 72, 1078-1083. (9) Toth, K.; Nagy, G.; Lan, B. T. T.; Jeney, J.; Choquette, S. J. Anal. Chim. Acta 1997, 353, 1-10. (10) Kosch, U.; Klimant, I.; Werner, T. Wolfbeis, O. S. Anal. Chem. 1998, 70, 3892-3897. (11) Bradshaw, J. T.; Mendes, S. B.; Saavedra, S. S. Anal. Chem. 2002, 74, 17511759. (12) Puyol, M.; Salinas, I.; Garces, I.; Villuendas, F.; Llobera, A.; Dominguez, C.; Alonso J. Anal. Chem. 2002, 74, 3354-3361. (13) Tsunoda, K.; Itabashi, H.; Akaiwa, H. Anal. Chim. Acta 1995, 299, 327332. (14) Tsunoda, K.; Yamamoto, E.; Akaiwa, H. Chem. Lett. 1996, 919-920. (15) Umemura, T.; Kasuya, Y.; Odake, T.; Tsunoda, K. Analyst 2002, 127, 149152. (16) Matsuda, N.; Takatsu, A.; Kato, K. Chem. Lett. 1996, 105-106.
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this ATR mode measurement, the absorption sensitivity is independent of the sensing membrane thickness down to the order of several hundreds nanometers, and then high-throughput analysis is feasible by preparing thinner sensing membranes that usually lead to rapid sensor response. For these reasons, this SOWG measurement system can be the most promising and convenient sensing tool. In addition to the importance of sensing devices, it is well known that the development of novel indicator dyes and stable immobilization are key to the success of optochemical sensing. In order to extend the pH response range, multiple pH indicators were coimmobilized,22 while an indicator with multiple steps of acid dissociation was employed.23,24 Some conductive polymers such as polyaniline and polypyrrole that exhibited color changes in a wide pH range were also exploited.25,26 The control of membrane thickness and permeability (porosity) is also an important subject.27 In the case of pH sensing, hydrophilic membranes may be desirable because the hydrophilicity promotes proton diffusion in the membrane, resulting in rapid response. So far, some promising membrane materials as well as traditional sol-gel glass films or plasticized poly(vinyl chloride) membranes have eagerly been exploited. Cellulose-based membranes, which have a high permeability for water and ion and also have resistance to acid and alkali, were extensively explored,28,29 while plasticizer-free methacrylate-based polymer membranes with thin thickness and high porosity were successfully employed.30-32 In the present work, a polyacrylamide-based hydrogel, which is popularly used in polyacrylamide gel electrophoresis, is chosen as a hydrophilic polymer matrix because of its ease in handling and its nonadsorptive properties that lead to low irreversible fouling. As for an indicator dye, protoporphyrin IX (PPIX) is employed,33,34 which, from our preliminary experiments, has been found to exhibit novel pH-dependent absorbance changes around 410 nm in the highly acidic range between pH 0.1 and 1.0. PPIX possesses two double bonds in its structure, and so it may be covalently immobilized into the polymer matrix by copolymeri(17) Edmiston, P. L.; Lee, J. E.; Wood, L. L.; Saavedra, S. S. J. Phys. Chem. 1996, 100, 775-784. (18) Puyol, M.; Miltsov, S.; Salinas, I.; Alonso, J. Anal. Chem. 2002, 74, 570576. (19) Hazneci, C.; Ertekin, K.; Yenigul, B.; Cetinkaya, E. Dyes Pigm. 2004, 62, 35-41. (20) Hisamoto, H.; Tsubuku, M.; Enomoto, T.; Watanabe, K.; Kawaguchi, H.; Koike, Y.; Suzuki, K. Anal. Chem. 1996, 68, 3871-3878. (21) Hisamoto H.; Manabe Y.; Yanai H.; Tohma H.; Yamada T.; Suzuki K. Anal. Chem. 1998, 70, 1255-1261. (22) Schulman, S. G.; Chen, S.; Bai, F.; Leiner, M. J. P.; Weis, L.; Wolfbeis, O. S. Anal. Chim. Acta 1995, 304, 165-170. (23) Lin, J.; Liu, D. Anal. Chim. Acta 2000, 408, 49-55. (24) Gupta, B. D.; Sharma, S. Opt. Commun. 1998, 154, 282-284. (25) Pringsheim, E.; Terpetschnig, E.; Wolfbeis, O. S. Anal. Chim. Acta 1997, 357, 247-252. (26) Sotomayor, M. D. T.; DePaoli, M. A.; deOliveira, W. A. Anal. Chim. Acta 1997, 353, 275-280. (27) Adhikari, B.; Majumdar, S. Prog. Polym. Sci. 2004, 29, 699-766. (28) Werner, T.; Wolfbeis O. S. Fresenius’ J. Anal. Chem. 1993, 346, 564-568. (29) Safavi, A.; Pakniat, M. Anal. Chim. Acta 1996, 335, 227-233. (30) Citterio, D.; Minamihashi, K.; Kuniyoshi, Y.; Hisamoto, H.; Sasaki, S.; Suzuki, K. Anal. Chem. 2001, 73, 5339-5345. (31) Qin, Y.; Peper, S.; Radu, A.; Ceresa, A.; Bakker, E. Anal. Chem. 2003, 75, 3038-3045. (32) Heng, L. Y.; Alva, S.; Ahmad, M. Sens. Actuators, B 2004, 98, 160-165. (33) Igarashi, S.; Kuwae, K.; Yotsuyanagi, T. Anal. Sci. 1994, 10, 821-822. (34) Gupta, V. K.; Kumar, A.; Mangla, R. Sens. Actuators, B 2001, 76, 617-623.
7512 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006
Figure 1. Schematic diagram of visible ATR spectrum measurement system with SOWG. 1, Xe arc lamp; 2, convex lens; 3, optical fiber; 4, slide glass; 5, flow cell; 6, coupling prism; 7, guiding layer (fused silica sheet, 50 µm thick, nD ) 1.459); 8, poly(tetrafluoroethyleneco-hexafluoropropylene film (25 µm thick, nD ) 1.338); 9, sample inlet; 10, sample outlet; 11, sample injector; 12, HPLC pump; 13, multichannel CCD detector; 14, personal computer; θi, incident angle of source light.
zation with acrylamide (AA) and N,N′-methylene bisacrylamide (BIS), resulting in being free from dye leaching. In order to fabricate a thin and flat polymer membrane, the copolymerization was carried out in a narrow space between two flat plates, one of which was a SOWG glass. The pH response of the sensing membrane was evaluated through the measurement of different concentrations of HNO3 and HCl by a laboratory-made visible ATR spectrometer, which can be operated in a flow-through mode as well as in a conventional static mode. The present SOWG absorption sensor fulfills most of the requirements for ideal optical sensing, such as rapid response, high sensitivity and selectivity, easy handling, good repeatability, and long lifetime, and thus, a simple and practical maintenance-free flow analytical system is realized. EXPERIMENTAL SECTION Reagents. Acrylamide (AA) and BIS forming a hydrophilic polymer matrix, ammonium persulfate (APS), and N,N,N′,N′tetramethylethylenediamine (TEMED) as a polymerization initiator and accelerator were purchased from Amersham Biosciences (Piscataway, NJ). PPIX used as a pH indicator dye was obtained from Sigma-Aldrich (St. Louis, MO). (3-Methacryloxypropyl)trimethoxysilane used for chemically anchoring the sensing membrane fabricated onto a waveguide glass was purchased from Shin-etsu Chemicals (Tokyo, Japan). These reagents were of commercially available highest purity and used as received. Nitric acid, hydrochloric acid, and inorganic salts were of reagent grade from Wako Pure Chemicals (Osaka, Japan). Deionized water was prepared with a Millipore Milli-Q system (Milford, MA). Apparatus. A schematic illustration of a visible ATR spectrum measurement system is shown in Figure 1. The system used was basically similar to that in our previous paper.15 An economical
microscopic cover glass (120-160 µm thick, from Musashino Fine Glass) was employed as a guiding layer, and it was fixed on a thick glass substrate to reinforce the mechanical strength. The surface of the waveguide glass was modified with a PPIXimmobilized polymer membrane by a method described below. On the modified waveguide glass, a laboratory-made flow cell was placed. The flow cell was made of a poly(tetrafluoroethylene) (PTFE) block with a central hollow for sample reservoir and two lateral holes for sample inlet and outlet (1/16 in.). The cell volume was 16.5 µL (10 mm length × 3 mm width × 0.55 mm height). This SOWG device was mounted upon a 360° rotational stage with X-Y-Z translation to regulate the incident angle (θi). In this experiment, the angle of 40° near the critical angle for total reflection was used. A xenon arc lamp (150 W, from Hamamatsu Photonics) was employed as a light source, and the light was coupled into the waveguide glass with a coupler prism (La-SF08, nD ) 1.8785, from Kogakugiken, Japan) after collimation with an optical fiber collimator. The guided light was out-coupled through another coupler prism and then introduced to a charged-coupled device (CCD) detector (PMA-11, from Hamamatsu Photonics) through an optical fiber. The signal was processed by a personal computer to obtain visible ATR spectra. This measurement system was operated in both a standard static mode and a flow-through mode. For the flow-through mode measurement, an HPLC pump (model LC-10ADVP, Shimadzu, Kyoto, Japan) and an injector (model 7520, Rheodyne, Cotati, CA) with a sample loop (20, 100, 200, 400, or 1000 µL) were connected to the SOWG sensing device through a short length of PTFE tubing. Preparation of PPIX-Immobilized Polymer Membrane. A thin and flat PPIX-immobilized polymer membrane was directly fabricated on the surface of the SOWG glass (microscopic cover glass) by radical polymerization of AA, BIS, and PPIX. The total monomer concentration (%T) and the concentration of cross-linker (i.e., BIS) were fixed as 3.3 %T and 2.7 %C, respectively. The concrete conditions were as follows. 0.291 g of AA, 0.009 g of BIS, and 0.03 g of PPIX were dissolved in ∼10 mL of 0.1 M Tris-HCl buffer (pH 7). After purging with N2 for 3 min, 60 µL of 10% APS and 15 µL of TEMED were added to the monomer solution. The 5-µL aliquot of the monomer solution was then dropped on the center of the waveguide glass pretreated with (3-methacryloxypropyl)trimethoxysilane (30% v/v in acetone) and was immediately covered crosswise with a flat plate (a slide glass pretreated with Repel-silane). After allowing to stand for 12 h at 25 °C, the slide glass was detached and the membrane-coated SOWG glass was washed with a plenty of water to fully remove any unreacted chemical species. And the produced sensing plates were preserved in dry form at room temperature. Sample Preparation. Concentrated nitric acid (∼13.5 M) and hydrochloric acid (∼12 M) were appropriately diluted with distilled water to obtain various pH solutions with the pH values of -0.5 to 2. The pHs of the most diluted HNO3 and HCl solutions (10 mM) were measured by a Horiba pH meter to validate the pH of the prepared solutions. Measurement of ATR Absorption Spectrum of PPIX Immobilized in Polymer Matrix. Absorption spectrum of PPIX exposed to distilled water was conveniently used as a reference, indicating the zero level, because PPIX exhibited relatively low
Soret band absorption spectra around the pH (5) of distilled water (strictly speaking, at a pH value higher than 1.0, as mentioned in detail later). In static mode operation, sample solutions were manually introduced into the 16.5-µL-volume flow cell with a syringe. After each measurement, the flow cell was rinsed with distilled water as quickly as possible to prevent damage to the sensing membrane and experimental setup by strong acids. In flow-through mode operation, distilled water was used as a carrier solution and pumped with an HPLC pump, and sample solutions were injected into the carrier stream through an HPLC injector. RESULTS AND DISCUSSION Preparation of a Thin and Highly Permeable Membrane. The permeability of the gel matrix to the diffusion species should have significant influence on the sensor response, which may be affected by its thickness and pore size. It is considered that the membrane with a thickness comparable to the penetration depth of the evanescent wave may be most suitable for sensitive and rapid measurement. Under the present experimental conditions of the incident angle of 40°, the penetration depth of the evanescent wave was estimated to be ∼800 nm from our previous work.35 To obtain such a thin and flat polymer membrane, we employed the pinching method, in which component monomers were copolymerized in a narrow space pinched between two flat glass plates. This preparation method was almost the same as that of slab gels in polyacrylamide gel electrophoresis,36 except that a spacer for setting membrane thickness was not used. In this study, the PPIX-immobilized polyacrylamide membrane with a thickness of ∼1 µm, which was estimated from the micrograph of scanning electron microscopy (SEM; see Figure S1 Supporting Information), was produced. The pore size of the gel matrix was also investigated. It is known that the pore size is strongly dependent on the total monomer concentration (%T) and the cross-linking degree (%C). According to previous papers,37,38 the polyacrylamide gels prepared under the conditions of more diluted %T or of lower %C have larger pore sizes, which promote mass transfer in gels. For example, the pore radii were reported to be 123 nm for the gel of 3.5%T and 3%C, 60 nm for that of 3.5%T and 4%C, and 70 nm for that of 10.5%T and 3%C, respectively.37 As expected from these reports, the response time should certainly be reduced with decreasing the %T, %C, or both. In the preliminary experiments using simple polyacrylamide gel without PPIX (i.e., only AA and BIS), the gelation was not completed and peeled from the waveguide glass under the conditions of
