Decyl Methacrylate-Based Microspot Optodes - ACS Publications

Nov 12, 2005 - Thus, the microspot size can be controlled based on the hydrophobicity of the surface and the size of the tip used for spotting. Calibr...
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Anal. Chem. 2006, 78, 524-529

Decyl Methacrylate-Based Microspot Optodes Amanda S. Watts, Aaron A. Urbas, Timothy Finley, Vasilis G. Gavalas, and Leonidas G. Bachas*

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055

Optode sensing membranes employing decyl methacrylate cross-linked with 1,6-hexanediol dimethacrylate as the polymer support were fabricated by a direct microspotting method on several surfaces. Photopolymerization was used to attach the microspots to the substrate. Using this method, diameters in the micrometer domain were obtained. Silanized glass, poly(methyl methacrylate) (PMMA), polycarbonate, and poly(dimethylsiloxane) were tested as possible substrates. Both polypropylene tips and the steel tips of drafting pens were used for spotting. It was determined that both silanized glass and PMMA gave working optodes, but the ones on PMMA did not fit the theoretical model. Diameters of 994 ( 80 and 1279 ( 85 µm were obtained on silanized glass and PMMA, respectively, using the polypropylene tips for spotting. Different size optodes were fabricated using 0.35- and 0.50-mm steel drafting pen tips. The 0.35-mm tips produced diameters of 895 ( 26 and 688 ( 54 µm on silanized glass and PMMA, respectively, and the 0.50mm tips produced diameters of 1274 ( 94 µm on silanized glass and 839 ( 28 µm on PMMA. Thus, the microspot size can be controlled based on the hydrophobicity of the surface and the size of the tip used for spotting. Calibration plots of potassium optode microspots indicated that miniaturization does not alter response characteristics, such as selectivity, response time, and dynamic range, of the optodes. There has been an increasing interest in the development of miniaturized systems for multianalyte detection. This has led to the development of both micrometer-1,2 and nanometer-sized3-6 optical sensors. Sensor miniaturization has become more prevalent, largely because it enables analysis in small volumes, improves detection limits, and allows for localized sensing and single-cell analysis. Additionally, miniaturized sensors can be integrated with microfluidics to design micro total analysis systems (µTAS). The appeal of these systems is based on advantages such as low power * Corresponding author: (e-mail) [email protected]; (phone) (859)257-6350; (fax) (859)323-1069. (1) Shortreed, M.; Bakker, E.; Kopelman, R. Anal. Chem. 1996, 68, 26562662. (2) Lindner, E.; Buck, R. P. Anal. Chem. 2000, 72, 336A-345A. (3) Brasuel, M.; Kopelman, R.; Miller, T. J.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 2001, 73, 2221-2228. (4) Park, E. J.; Brasuel, M.; Behrend, C.; Philbert, M. A.; Kopelman, R. Anal. Chem. 2003, 75, 3784-3791. (5) Tsagkatakis, I.; Peper, S.; Retter, R.; Bell, M.; Bakker, E. Anal. Chem. 2001, 73, 6083-6087. (6) Barker, S. L. R.; Thorsrud, B. A.; Kopelman, R. Anal. Chem. 1998, 70, 100104.

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and space requirements, the small amount of reagent and sample needed, faster analysis, and disposability due to their low cost.7,8 Both centrifugal7,9 and on-chip,8,10 microfluidic platforms have been paired with ion-selective electrodes and optodes for use in ion analysis. However, the difficulty of integrating ion-sensing systems in such platforms11 prompted us to explore new methods to interface ion optode chemistry with microfluidics devices. By miniaturizing optode sensors, optode microarrays could be developed using different sensing chemistries for the simultaneous analysis of various ions in complex samples. Ion-selective optodes have traditionally been fabricated either as thin films or as spherical particles. Thin films have been formed by photopolymerizing aliquots of a cocktail containing all necessary components between one silanized and one untreated glass slide.12,13 Another approach involves spin coating a cocktail containing all necessary components onto a silanized glass slide.14 In both cases, the silanized glass slide with the membrane can be incorporated into a flow cell using a fluorometer as the detection device. New opportunities for optodes have emerged with the development of plasticized poly(vinyl chloride) (PVC) microspheres,5,15 which were incorporated into flow cytometry by Bakker,16 fluorescent spherical nanosensors, or PEBBLEs (probe encapsulated by biologically localized embedding), by Kopelman4,6 and ion-selective luminescent quantum dots by Rosenzweig.17 This move toward spherical optodes has risen from the realization that it would be advantageous to detach the instrument from the sensing element, allowing for a noninvasive method to monitor processes inside living cells after injecting the sensing particles into them.15 PVC is currently the most commonly used polymer for membrane-based ion-selective electrodes because of its strength, compatibility with ionophores, and chemical inertness.12 However, it has some limitations in applications such as optical sensors because of its poor adhesion to solid supports. Upon long exposure (7) Johnson, R. D.; Badr, I. H. A.; Barrett, G.; Lai, S.; Lu, Y.; Madou, M. J.; Bachas, L. G. Anal. Chem. 2001, 73, 3940-3946. (8) Tantra, R.; Manz, A. Anal. Chem. 2000, 72, 2875-2878. (9) Badr, I. H. A.; Johnson, R. D.; Madou, M. J.; Bachas, L. G. Anal. Chem. 2002, 74, 5569-5575. (10) Hisamoto, H.; Horiuchi, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 1382-1386. (11) Harris, C. M. Anal. Chem. 2001, 73, 475A. (12) Ambrose, T. M.; Meyerhoff, M. E. Electroanalysis 1996, 8, 1095-1100. (13) Ambrose, T. M.; Meyerhoff, M. E. Anal. Chim. Acta 1999, 378, 119-126. (14) Badr, I. H. A.; Johnson, R. D.; Diaz, M.; Hawthorne, M. F.; Bachas, L. G. Anal. Chem. 2000, 72, 4249-4254. (15) Tsagkatakis, I.; Peper, S.; Bakker, E. Anal. Chem. 2001, 73, 315-320. (16) Retter, R.; Peper, S.; Bell, M.; Tsagkatakis, I.; Bakker, E. Anal. Chem. 2002, 74, 5420-5425. (17) Chen, Y.; Rosenzweig, Z. Anal. Chem. 2002, 74, 5132-5138. 10.1021/ac051652e CCC: $33.50

© 2006 American Chemical Society Published on Web 11/12/2005

to aqueous environments, PVC membranes can peel off the substrate. Further, the preparation of PVC membranes often involves the use of solvents, such as tetrahydrofuran, that are incompatible with some of the plastics used in microfluidic devices. Methacrylic-acrylic materials generated by chemical or optical methods of polymerization have been used in ion sensing in the form of either films12,13,18,19 or microspheres20,21 and are better suited for integration into microfluidic devices. In this paper, we have employed decyl methacrylate (DMA) cross-linked with 1,6-hexanediol dimethacrylate (HDDMA) as the polymer support to fabricate photopolymerizable optode microspots on various substrates. The long alkyl chains in DMA enhance the mechanical stability of the membranes compared to shorter chain methacrylates.12 The methacrylate group also allows for covalent attachment to a pretreated substrate during photopolymerization. Additionally, use of a structurally similar solid substrate, such as poly(methyl methacrylate) (PMMA) enhances adherence of the photopolymerized material through noncovalent interactions. Optode sensing membranes of diameters in the micrometer domain were fabricated by a direct microspotting method on silanized glass and PMMA. Microspotting has been an effective method of microfabricating protein and DNA arrays,22-27 and this method is extended, herein, to the microfabrication of sensing, microdome-shaped, optodes. The membrane size can be controlled based on the hydrophobicity of the surface and the size of the tip used for spotting. Factors to be taken into consideration when miniaturizing the sensing components include the reproducibility of the diameter and mechanical strength of the microspot, the most suitable substrate on which to apply the microspot, and whether the response characteristics are altered by miniaturization. The optodes could be miniaturized using this microspotting approach while maintaining their selectivity and low detection limits. EXPERIMENTAL SECTION Materials. Bis(2-ethylhexyl) sebacate (DOS), 9-(diethylamino)5-(2-octadecylimino)benzo[a]phenoxazine (Chromoionophore III, ETH 5350), and potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB) were obtained from Fluka (Milwaukee, WI). Benzoyl peroxide, benzophenone, 3-(trimethoxysilyl)propyl methacrylate (MPTS), and HDDMA were purchased from SigmaAldrich (St. Louis, MO). Valinomycin was obtained from Acros Organics (Cincinnati, OH). DMA was purchased from Pfaltz and Bauer (Waterbury, CT). Tris(hydroxymethyl)aminomethane (Tris), ultrapure, molecular biology grade, was obtained from Research Organics (Cleveland, OH). Potassium phosphate monobasic and toluene were purchased from Mallinckrodt (Paris, KY). All (18) Heng, L. Y.; Hall, E. H. A. Anal. Chim. Acta 1996, 324, 47-56. (19) Heng, L. Y.; Hall, E. H. A. Anal. Chim. Acta 2000, 403, 77-89. (20) Peper, S.; Tsagkatakis, I.; Bakker, E. Anal. Chim. Acta 2001, 442, 25-33. (21) Peper, S.; Ceresa, A.; Qin, Y.; Bakker, E. Anal. Chim. Acta 2003, 500, 127136. (22) Cho, E. J.; Tao, Z.; Tehan, E. C.; Bright, F. V. Anal. Chem. 2002, 74, 61776184. (23) Shumaker-Parry, J. S.; Zareie, M. H.; Aebersold, R.; Campbell, C. T. Anal. Chem. 2004, 76, 918-929. (24) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (25) Lee, K.; Park, S.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (26) Ito, Y.; Nogawa, M. Biomaterials 2003, 24, 3021-3026. (27) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681-5687.

aqueous solutions were prepared with 18-MΩ deionized reverseosmosis water obtained by a Milli-Q water purification system (Millipore, Bedford, MA). Glass slides used in the experiment were obtained from Gold Seal Products (Portsmouth, NH). Polycarbonate was purchased from United States Plastic Corp. (Lima, OH). PMMA was obtained from Plastifab (Poway, CA). A poly(dimethylsiloxane) (PDMS) Sylgard 184 elastomer kit (Dow Corning Corp., Midland, MI) was used to fabricate the PDMS used in the experiments. Polypropylene tips were purchased from VWR International, Inc. (Finntip 10, Item 53515-954, Batavia, IL), and the Rapidograph drafting pens were manufactured by KohI-Noor (Bloomsbury, NJ). Silanization of Glass Slides. An 80% (v/v) solution of MPTS/ toluene was made and placed in an evaporating dish. The glass slides and the evaporating dish were placed inside a vacuum desiccator. After 1.5 h, the vacuum was turned off, and the slides were left inside the desiccator for another 2 h. Preparation of PDMS. The Sylgard 184 elastomer kit contains a curing agent and silicone elastomer. A 1:10 ratio of curing agent to silicone elastomer was prepared and degassed under vacuum for 40 min. The gel was poured into a large Petri dish and placed in an oven at 65 °C for 3 h. The slides were then cut to size. Preparation of Optode Membranes. The membrane cocktail was prepared with the following composition: 100 mg of DOS, 98 mg of HDDMA, 32 mg of DMA, 5.0 mg of benzoyl peroxide, and 2.5 mg of benzophenone. The solution was vortexed to dissolve the components. To 100 mg of this cocktail, 1.9 mg of valinomycin and 1.5 mg of KTFPB were added and the resultant mixture sonicated to dissolve. Finally, 1.0 mg of chromoionophore III was added and vortexed to dissolve. Optode membranes were spotted onto the substrates using either a polypropylene tip (inner diameter of 0.4 mm) or Rapidograph drafting pen tips, measuring 0.35 mm and 0.50 mm in diameter. It should be mentioned that a micropipet was not used to deliver a fixed volume of cocktail. The microspots were simultaneously photopolymerized and covalently attached using a 100-W ultraviolet lamp at 366 nm (model B100AP Black-Ray, Upland, CA) in an oxygen-free environment for 8 min. Profilometry. An Alpha-Step 500 Surface Profiler (Tencor Instruments, Mountain View, CA) was used to obtain the profiles of the optode microspots on each of the substrates. This instrument characterizes the surface by scanning it with a diamond stylus, producing a trace, which represents a cross-sectional view with high vertical and spatial resolution. Contact Angle Measurements. The contact angles of the membrane cocktail on the different substrates were measured. A 4-µL drop was used for each measurement, and three samples were analyzed on each substrate. Calibration Measurements. Fluorescence measurements were taken using a Zeiss Axioskop (Carl Zeiss) with an attached Axiocam MRm. An allophycocyanin filter set (Chroma Technology Corp., Rockingham, VT) with excitation at 595 nm, emission at 660 nm, and a bandwidth of 40 nm was used. According to the manufacturer, the transmittance of the filters at 595 and 660 nm is approximately 80 and 90%, respectively. A 50-W mercury arc lamp was used as the excitation source. Solutions ranging from 1 × 10-7 to 1 M KCl were prepared in 0.05 M Tris/H2SO4 buffer, pH 7.4. Additionally, a 0.01 M NaOH solution and a 0.01 M KH2Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

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PO4/HCl buffer, pH 2.9, were prepared. The microspots were first conditioned in 0.05 M Tris/H2SO4 buffer, pH 7.4, for 30 min. Two methods were used for the calibration measurements. In the first, five microspots on a single silanized glass slide were chosen, and measurements were taken of the same five microspots each time. The second method involved using sets of five microspots on different silanized glass slides for each individual concentration. In both methods, the microspots were equilibrated in a vial containing the appropriate KCl solution for 5 min, with a measurement taken after each equilibration. To obtain the fluorescence values for the fully protonated and deprotonated forms of the chromoionophore, measurements were taken after the microspots were conditioned in vials of the KH2PO4/HCl buffer and the NaOH solution, respectively, for 30 min. In all instances, after the incubation time, the slide containing the microspots was placed on the microscope stage and focusing was done on a single microspot. The CCD camera was used to take a snapshot of the microspot and the Axiocam software was used to determine the amount of fluorescence exhibited in the image. This procedure was repeated for each microspot. Response Time Measurements. A laser scanning confocal microscope (Leica TCS NT SP) was used in conjunction with a flow cell to perform response time measurements of the microspots on silanized glass. The body of the flow cell used in this initial characterization was made of Lexan and contained a 3.8 × 0.4 × 0.2 cm open channel that had an inlet and an outlet to which tubing was attached to facilitate the flow of solution. A microscope coverslip was used for the top of the flow cell so that light could pass through. The flow cell was mounted onto the silanized glass slide containing the microspots, and aquarium glue was used to seal the coverslip and the glass slide to the flow cell. Initially, buffer was pumped through the flow cell to condition the microspot optodes. Measurements were taken with buffer alone to establish a baseline. Then, a potassium chloride solution was pumped through the flow cell, with the first measurement taken as soon as the solution came in contact with the microspots. Selectivity Measurements. To determine selectivity, the optodes were conditioned in 1 × 10-5, 1 × 10-3, and 0.1 M solutions of LiCl, NaCl, CsCl, NH4Cl, and CaCl2. All solutions were made in 0.05 M Tris/H2SO4 buffer, pH 7.4. The experiment was conducted in the same manner as described for the calibration. RESULTS AND DISCUSSION Theory. The sensing mechanism for the microspotted optodes is based on ion-exchange where potassium ions from the aqueous phase exchange with hydrogen ions in the microspot. The equilibrium reaction is as follows: + + + K+ (aq) + L(org) + CH(org) + R(org) h LK(org) + C(org) + H(aq) +

R(org) (1)

where L represents the potassium-selective ionophore (valinomycin), C represents the chromoionophore (ETH 5350), and Rrepresents the anionic lipophilic additive tetrakis[3,5-bis-(trifluoromethyl)phenyl]borate. The chromoionophore’s fluorescence emission maximum and intensity shift upon protonation. The optode response mechanisms have been described in detail 526

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previously.28,29 In the system described in this paper, involving equimolar concentrations of ionophore, lipophilic additive, and chromoionophore, the response function can be expressed in terms of activity (a), the extraction coefficient (Kex), and the relative portion of unprotonated chromoionophore (R) using the following equation:

aI )

aH+ R2 Kex 1 - R2

(2)

The following equation was used to relate the measured fluorescence, F, at given activity to R:1

R ) (FP - F)/(FP - FD)

(3)

where FP and FD are the fluorescence emission signals of the fully protonated and fully deprotonated forms of the chromoionophore, respectively. To calculate the selectivity coefficient, Kopt IJ , where I is the primary ion and J is the interfering, or secondary, ion at a chosen pH value, the following equation can be used:28,29

Kopt IJ ) aI/aJ

(4)

where aI and aJ are the activities of I and J, respectively, at a given degree of protonation of the chromoionophore. This equation is valid when the stoichiometries of cation to ionophore are 1:1, which is the case for the valinomycin based system. Characterization of Microspots. Microspots were applied to silanized glass, PMMA, polycarbonate, and PDMS. Diameters and heights were measured only for the microspots on silanized glass and PMMA, because the microspots did not fully polymerize onto the polycarbonate, and the membrane components diffused into the PDMS matrix upon application. Profilometry measurements of the microspots on silanized glass using the polypropylene tip gave a diameter of 994 ( 80 µm (average ( standard deviation, n ) 8) and a height of 38.5 ( 8.3 µm. Similarly, the resulting microspots on PMMA had a diameter and height of 1279 ( 85 and 7.4 ( 2.0 µm, respectively. This difference in the diameter and height of the microspots could be due to the differences in the hydrophobicity of the substrate. This is further substantiated by the contact angle of the membrane cocktail on the substrates, which was measured to be 30.8 ( 2.0° on silanized glass and 9.2 ( 2.6° on PMMA. The hydrophobicity of the substrate is not the only parameter that controls the amount dispensed or the size of the microspot. As will be demonstrated below with the use of the drafting pen tips, parameters such as the diameter and material of the tip used for spotting significantly affect the size of the microspot. Using the polypropylene tip, a relative standard deviation of 8% or less was observed in the diameters of the microspots on both substrates. This illustrates that the fabrication of the microspots can be performed reproducibly. Additionally, the microspots remained attached to the silanized glass when stored in buffer for more than 1 year without peeling off the substrate. To detach the microspots from the silanized glass, it (28) Bakker, E.; Buhlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132. (29) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805-1812.

Figure 1. Surface profile of a representative microspot on silanized glass generated with MATLAB and profilometry data. The y-axis represents the height of the microspot. The broken lines represent the scans taken by the profilometer.

was necessary to expose them to 0.01 M KH2PO4/HCl buffer, pH 2.9, for a prolonged period of time. The detachment is most likely the result of hydrolysis of the siloxane bonds between MPTS and the silanized glass, which is more effective at low pH, and indicates the presence of covalent bonds between the microspots and the silanized glass surface. Rapidograph drafting pens with steel tips were also used for spotting the optode membranes onto silanized glass and PMMA. The 0.35-mm pen tip produced an average diameter (n ) 10) of 895 ( 26 µm and a height of 27 ( 2 µm on silanized glass, compared to a diameter (n ) 5) of 688 ( 54 µm and a height of 12 ( 4 µm when the spotting was done on PMMA. Employing a 0.50-mm pen tip resulted in microspots with an average diameter (n ) 10) of 1274 ( 94 µm and a height of 36 ( 5 µm on silanized glass and microspots of diameter (n ) 5) of 839 ( 28 µm and height of 11 ( 1 µm on PMMA. Both microspotted surfaces show a relative standard deviation of less than 8% in microspot diameter. The diameters of the microspots depend on the interaction (surface tension) of the membrane cocktail with both the tip used for spotting and the substrate.30 Relative wetting (tip vs substrate) favored the release of more liquid on certain substrates. Several vertical and horizontal scans were taken of the microspot on silanized glass using the Alpha-Step 500 surface profiler. A program, written in MATLAB, was used to align these scans, which was composed of a collection of three-dimensional points across the surface. Edge correcting was done to fit an ellipse to the surface to create a base where the microspot meets the substrate. Then, GRIDDATA was used to fit a surface to these points. In intersections where the profiles did not always match up perfectly, a small discontinuity could be seen as a spike since the program saw this as an identical point with two z-axes. Smoothing was performed using a cubic smoothing spline, which fits polynomials to the surface and does not change the membrane shape. Figure 1 shows the profile of a microspot having a diameter (30) Weibel, C. JALA 2002, 7, 91-96.

Figure 2. Optode response for K+ calibration on PMMA (2) and on silanized glass using (0) the same five microspots for the entire experiment and (b) a different set of five microspots for each calibration solution. The solid line denotes the theoretical response of the optode toward potassium using the same five microspots, and the dashed line represents the theoretical response using a difference set of microspots. The dotted line represents the best fit of eq 2 using the PMMA data and indicates that PMMA-supported optodes do not follow conventional optode theory. Error bars represent 1 standard deviation (n ) 5).

of 980.7 µm and a height of 41.4 µm generated by the program using the original data. The microspots had an elliptical shape. Calibration. Calibrations were performed using microspots applied on silanized glass and PMMA (Figure 2). Groups of five microspots on PMMA and silanized glass were exposed sequentially to solutions ranging from 1 × 10-7 to 1 M KCl in 0.05 M Tris/H2SO4, pH 7.4, to obtain quintuplicate calibration data. A second method of calibration was explored utilizing a different set of microspots on silanized glass for each K+ concentration. For the latter studies, these optodes were exposed to solutions ranging from 1 × 10-5 to 0.1 M KCl. Figure 2 shows the results of the calibration with K+ using both methods for microspots on silanized glass along with the corresponding theoretical response Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

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curves for the optode membrane toward potassium obtained by fitting each set of data into eq 2. The two data sets are statistically indistinguishable, and the experimental results fit well with the theoretical simulations (solid and dashed lines in Figure 2), supporting the notion that the ion-exchange mechanism of bulk optodes (see Theory section) also governs the response of the microspots. The data also demonstrate that the microspots can be fabricated reproducibly since there is no significant difference in the responses of different microspots. For these measurements, microscope optics were used to collect light, and the intensity of the collected light was sufficient to allow the accurate analysis of different concentration samples. For example, the absolute signal was 368 ( 52 (arbitrary units, n ) 5) for a solution of 1 × 10-5 M KCl and was 164 ( 18 (n ) 5) for a 1 × 10-2 M KCl solution. In comparison, the signals corresponding to the high-pH and lowpH solutions, which determine the response range of the optode were 149 ( 11 and 410 ( 50 (n ) 5), respectively. It is expected that, with the miniaturization of the optodes, the absolute signal of the sensing element will be reduced. The dynamic range of the optode membrane extends over ∼2 decades of concentration (from 6 × 10-6 to 3 × 10-4 M). By changing the pH or the relative amounts of the ionophore components in the membrane, the dynamic range could be adjusted if needed for a particular application.28 The observed extraction coefficients calculated from the experimental points and eq 2 are 3.1 × 10-4 for the first calibration method and 2.4 × 10-4 for the second method. Both of the values of the extraction coefficient are on the same order of magnitude as bulk optodes based on PVC and containing the same ionophore but a different chromoionophore.31 As can be seen from Figure 2, the microspots on PMMA did not give good response, most probably because diffusion of the active components of the microspot into the substrate occurred. The shape of the calibration plot is not characteristic of optode membranes. Indeed, fitting eq 2 to the data using an algorithm that minimizes deviation of each data point from the calculated plot gave unacceptably large deviation values. One of the limiting factors for optode sensors is the loss of signal from the decomposition of the chromoionophore as a result of photochemical processes.32 Consequently, the amount of time the microspots are exposed to light should be kept to a minimum. To study the stability of the signal, a set of microspots was used to perform two calibration measurements in series using the first calibration method. It was determined that a decrease in the fluorescence signal of ∼20% had occurred between calibrations. This decrease indicates that, although there is photobleaching or leaching of the membrane components, the microspots’ performance is not significantly affected, especially since these microspots are designed for one-time use. However, this loss in signal could be avoided by using different sets of microspots to perform each analysis as in the second calibration method. It should be mentioned that the small difference in the two extraction coefficient values given above could be a result of such photobleaching of the chromoionophore, which is more probable with the first calibration method, where the microspots are exposed to radiation multiple times. In contrast, the microspots are only (31) Suzuki, K.; Ohzora, H.; Tohda, K.; Miyazaki, K.; Watanabe, K.; Inoue, H.; Shirai, T. Anal. Chim. Acta 1990, 237, 155-164. (32) Puyol, M.; Miltsov, S.; Salinas, I.; Alonso, J. Anal. Chem. 2002, 74, 570576.

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Figure 3. Response time of the optode microspot on silanized glass to 1 × 10-2 ([) and 1 × 10-4 M (9) KCl. An arrow is used to depict the point of injection of KCl.

exposed once to radiation when using the second calibration method. Since the fabrication and the fluorescence signal of the microspots are reproducible, the second method provides analytically useful data even when using different microspots for calibration and sample analysis. Response Time. The response time of the optodes was measured using solutions of 1 × 10-2 and 1 × 10-4 M KCl. The confocal microscope has the ability to collect fluorescence from only a specific volume of the sample that has a diameter and height preselected by the operator (i.e., optical slice). This microscope was used to take fluorescence measurements of 10 slices of the membrane of ∼6.6-µm thickness each. The optode was conditioned in buffer, and a baseline was established by taking measurements of each slice. Then, the KCl solution was introduced into the flow cell, and measurements of each slice were taken every 30 s for the first 2 min, and then every minute after for 7 min more. First, the number of active pixels exhibiting fluorescence was determined and each pixel in the image acquired was assigned a value based on its fluorescence intensity. Only the nonzero values were retained. The total fluorescence intensity for each image was obtained by summing up the values for all of the pixels in the image. For each specific time scan, the total sum of the pixel intensities for each slice was multiplied by the number of pixels determined to be exhibiting fluorescence. These are added together to get a total intensity for the entire membrane. Figure 3 depicts the response of a typical microspot toward potassium. As seen in the figure, immediately after potassium is introduced, there is a sharp response toward it. Also, 90% of the response takes place within ∼1 min at high concentrations and within 4 min for the lower concentrations. Selectivity. To study the selectivity of the optode membrane for potassium, the optode was tested in solutions of LiCl, NaCl, NH4Cl, and CsCl. For each cation studied, a different set of five optodes on a silanized glass slide was used for fluorescence measurements. Figure 4 depicts the calibration plots for K+, Cs+, NH4+, Na+, and Li+. From eq 4, at constant pH, Kopt KJ can be obtained graphically as the horizontal distance between separate calibration curves for a given R on the R versus log C plot.29,33 In this case, R ) 0.5 was used to calculate the log Kopt KJ values (33) Wang, E.; Zhu, L.; Ma, L.; Patel, H. Anal. Chim. Acta 1997, 357, 85-90.

Table 1: Selectivity Coefficients (Kopt KJ ) of Microspotted Optodes in 0.05 M Tris/H2SO4, pH 7.4, in Comparison to Prior Work with Bulk Optodes and DMA, Polyurethane/ PVC (PU/PVC), and PVC Matrixesa Kopt KJ K+ Cs+ NH4+ Na+ Li+

present study

DMAb

PU/PVCc

0 0 0 (5.1 ( 3.2) × 10-4 (1.8 ( 1.2) × 10-4 1.1 × 10-2 2.5 × 10-3 (8.8 ( 3.8) × 10-5