Anal. Chem. 2001, 73, 6083-6087
Technical Notes
Monodisperse Plasticized Poly(vinyl chloride) Fluorescent Microspheres for Selective Ionophore-Based Sensing and Extraction Ioannis Tsagkatakis,† Shane Peper,† Robert Retter,‡ Michael Bell,‡ and Eric Bakker*,†
Department of Chemistry, Auburn University, Auburn, Alabama 36849, and Beckman Coulter, Inc., 200 South Kraemer Boulevard, Brea, California 92822
A convenient method for the preparation of monodisperse, plasticized poly(vinyl chloride) particles based on an automated particle casting technique is described. The particles are made highly selective for a number of ions by doping them with ionophores and other active components, in complete analogy to thin-film or fiber-optic chemical sensors. The approach used here produces spheres of high monodispersity at a rate of ∼20 000 particles/s. The casting process is based on a reproducible polymer drop formation and precipitation process, and the particles are formed under very mild, nonreactive conditions. This allows one to conveniently incorporate known amounts of different active components into the polymers. As an initial example, the particles are doped with three optical sensing components, the sodium ionophore tert-butylcalix[4]arene tetraethyl ester, the H+chromoionophore ETH 5294, and the anionic additive sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate. The particles are found to be of spherical shape with a diameter of ∼10 µm. They respond individually and selectively to sodium according to classical optode theory, as determined by fluorescence microscopy. With a RSD of 1.6%, sensing reproducibility from particle to particle is excellent. This technique may allow the development of mass-produced chemically selective microspheres on the basis of bulk extraction processes. Microsphere-based analytical assays and recognition principles are an important research direction in analytical chemistry.1,2 They offer localized recognition chemistry, extremely small amounts of required reagents, and a number of different readout formats. The microspheres can be interrogated in a massively parallel fashion with imaging techniques3 or manipulated with fluidics, for example, on chip4 or with flow cytometry.5 They, therefore, †
Auburn University. Beckman Coulter, Inc. (1) Walt, D. R. Acc. Chem. Res. 1998, 31, 267. (2) Lu, J.; Rosenweig, Z. Fresenius J. Anal. Chem. 2000, 366, 569. (3) Dickinson, T. A.; Michael, K. L.; Kauer, J. S.; Walt, D. R. Anal. Chem. 1999, 71, 2192. ‡
10.1021/ac010694+ CCC: $20.00 Published on Web 11/10/2001
© 2001 American Chemical Society
represent an extremely versatile, adaptable road to perform chemical assays. So far, however, microsphere-based chemistry has been limited to surface-attached reaction schemes3,6 or simple polymer swelling mechanisms.7 This research has the goal of significantly expanding the chemical palette of available recognition chemistries for microsphere-based chemical analysis by developing polymeric microspheres with selective extraction properties. We have recently introduced a simple casting technique to produce spherical plasticized poly(vinyl chloride) particles, ∼6 µm in size.8 The particles show a selective fluorescence response as a function of the chemical composition of the surrounding solution, because they are doped with extremely selective hydrophobic complexing agents (ionophores). The basic recognition principle is adapted from thin-film or optical fiber sensor principles.9,10 Indeed, a large variety of anions, cations, and neutral molecules can be measured by selective extraction into a polymeric environment doped with an ionophore, if coupled to an optical readout mechanism.11 One popular approach, for example, is to relate the extraction of a cation into a polymer to the release of a hydrogen ion from the polymer into the sample by an ion-exchange mechanism.12 This coupling mechanism is based on the charge balance requirement of the hydrophobic polymer containing a lipophilic ion exchanger. If this polymer contains two ionophores, one selective for the cation and the other for the hydrogen ion, and the H+-ionophore changes its absorbance or fluorescence properties upon deprotonation, the sensor is selective and an optical readout of the extraction process is obtained. In past years, such principles have been adapted to the development of a number of optical sensing (4) Oleschuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y.; Harrison, D. J. Anal. Chem. 2000, 72, 585. (5) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R. Clin. Chem. 1997, 43, 1749. (6) Michael, K. L.; Taylor, L. C.; Schultz, S. L.; Walt, D. R. Anal. Chem. 1998, 70, 1242. (7) Seitz, W. R.; Rooney, M. T. V.; Miele, E. W.; Wang, N.; Kaval, N.; Zhang, L.; Doherty, S.; Milde, S.; Lenda, J. Anal. Chim. Acta 1999, 400, 55. (8) Tsagkatakis, I.; Peper, S.; Bakker, E. Anal. Chem. 2001, 73, 315. (9) Seiler, K.; Simon, W. Anal. Chim. Acta 1992, 266, 73. (10) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805. (11) Bu ¨ hlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593. (12) Morf, W. E.; Seiler, K.; Rusterholz, B.; Simon, W. Anal. Chem. 1990, 62, 738.
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formats, including highly miniaturized systems.13,14 While the basic chemistry is available to develop chemically selective microspheres on the basis of extraction processes, numerous challenges still exist to make this technique attractive for routine use. They include eliminating the leaching of microsphere components (active species as well as plasticizer). For this purpose, we continue to study the feasibility of producing microspheres based on plasticizer-free polymeric materials.15 Of the utmost importance, however, is that adequate techniques become available to mass-produce monodisperse microspheres under mild, nonreactive conditions. Traditionally, microspheres have been synthesized by a number of polymerization techniques, including dispersion16 and emulsion polymerization.17 These techniques involve thermally or photochemically initiated polymerization and provide a reactive environment in which many active sensing components may disintegrate. This paper introduces a different approach for mass-producing monodisperse hydrophobic sensing particles under nonreactive conditions, which is based on a polymer droplet forming instrument originally described for a different purpose.18 EXPERIMENTAL SECTION Reagents. Poly(vinyl chloride) (PVC), bis(2-ethylhexyl) sebacate (DOS), tert-butyl calix[4]arene tetraethyl ester (sodium ionophore X), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl borate (NaTFPB), and 9-(diethylamino)-5-octadecanoylimino-5Hbenzo[a]phenoxazine (chromoionophore I, ETH 5294) were Selectophore quality from Fluka (Milwaukee, WI). Cyclohexanone (99.8%, Aldrich), dichloromethane (ACS grade, Fisher), 1-chloronaphthalene (90%, Aldrich), poly(ethylene glycol) (PEG; MW 600; Polysciences), and xylenes (ACS grade; Fisher) were obtained from the indicated suppliers. Silanizing reagents and all salts and buffers were of puriss quality from Fluka. Nanopure water (18 MΩ cm) was used for the preparation of all solutions. Particle Preparation. A particle caster was built according to ref 19 (see Figure 1 and Results and Discussion). The polymer solution (dissolved in an organic solvent) and an aqueous sheath flow are directed by hydrostatic pressure from storage bottles, through pressure valves, to a ceramic tip with an orifice diameter of 46 µm. A piezoelectric crystal (24 mm × 19 mm × 1 mm) is mounted ∼85 mm above the ceramic tip orifice and controls the rapid formation of polymer droplets. The crystal is affixed to the top of the mixing chamber, perpendicular to the direction of polymer flow, and is controlled by a frequency generator. The solvent from the droplets gradually dissolves into the surrounding aqueous solution, leaving precipitated polymer particles. Specifically, a PVC membrane cocktail containing 52.98 mg of PVC, 108.1 mg of DOS, 1.75 mg of ETH 5294, 15.33 mg of Na+ Ionophore X, and 4.10 mg of NaTFPB was prepared and dissolved in 5 mL of (13) Shortreed, M.; Bakker, E.; Kopelman, R. Anal. Chem. 1996, 68, 2656. (14) Brasuel, M.; Kopelman, R.; Miller, T. J.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 2001, 73, 2221. (15) Peper, S.; Tsagkatakis, I.; Bakker, E. Anal. Chim. Acta 2001, 442, 25. (16) Tseng, C. M.; Lu, Y. Y.; El-Aasser, M. S.; Vanderhoff, J. W. J. Polym. Sci., Part A: Polym. Chem. 1986, 24, 2995. (17) Clark, H. A.; Hoyer, M.; Philbert, M.; Kopelman, R. Anal. Chem. 1999, 71, 4831. (18) Fulwyler, M. J.; Perrings, J. D.; Cram, L. S. Rev. Sci. Instrum. 1973, 44, 204. (19) Fulwyer, M. J.; Hatcher, C. W.; Coulter Electronics, Inc., 1981.
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Figure 1. Schematic representation of the particle caster used in this study. A polymer solution (active components, polymer, plasticizer, and organic solvent) are guided by hydrostatic pressure to an orifice, where the constant-frequency oscillation of a piezoelectric crystal leads to the formation of polymer solution droplets. These droplets enter into a mixing chamber, where a surrounding water stream leads to the formation of a jet. The uniform polymer droplets are collected in a water reservoir, where the organic solvent gradually dissipates to leave monodisperse, active polymer spheres.
cyclohexanone under mild heating and diluted with 100 mL of dichloromethane and 1 mL of xylenes. The cocktail was filtered with a 0.45-µm syringe filter and transferred to the polymer solution bottle. Deionized water was used as the sheath liquid, and a solution of 3% PEG was added dropwise every ∼5 s to the particle collection reservoir. The polymer cocktail and the sheath liquid flow rates were adjusted with a pressure regulator to 0.20 and 51 mL/min, respectively. The frequency was set at 17.5 kHz with a frequency generator (BK Precision model 4011, Placentia, CA). The particles were collected in a larger reservoir (5-gal container) and were allowed to settle to the bottom overnight. The catch liquid was decanted to ∼500 mL and filtered with a 10-µm nylon mesh filter (Small Parts Inc, Miami Lakes, FL). The particles were subsequently recovered by rinsing the nylon filter with an aqueous solution of 0.1% PEG. Instrumentation. The Pariss imaging spectrometer (LightForm, Inc., Belle Mead, NJ) in combination with a Nikon Eclipse E400 microscope with an epifluorescence attachment (Southern Micro Instruments, Marietta, GA) was used to optically characterize the particles (with 510-560 nm excitation filter, 565-nm dichroic mirror, 590-nm long-pass emission filter).8 The system was equipped with a motorized stage (Prior Optiscan ES9, Fulbourn, Cambs, U.K.), controlled by the data acquisition software, to record individual fluorescence spectra of a number of particles in the field of view. Scanning electron micrographs (SEMs) were obtained using a Zeiss DSM 940 scanning electron microscope at 5 kV. A drop of the particle stock solution was deposited onto an aluminum
Figure 3. Fluorescence images of plasticized PVC particles.
same particle was imaged as a function of the sample composition in order to record a complete calibration curve for individual particles. The same procedure was followed for the multiparticle measurements with the automated scanning mode of the microscope. Spectral acquisitions were obtained every 1.25 µm, and the slide with the same particles was scanned reproducibly with six different solutions.
Figure 2. Scanning electron micrographs of plasticized PVC particles.
stub. After solvent evaporation, the sample was sputter-coated with Au/Pd using a Pelco SC-7 autosputter coater (Ted Pella, Inc., Redding, CA). Au/Pd deposition of 20-40 nm occurred in two 60-s cycles. Measurements. Microscope glass slides were silanized as described8 by dipping them in 15% hexamethyldisilazane and 8% trimethylchlorosilane in dry 1-chloronaphthalene for ∼2 min, followed by oven drying at 230 °C for 45 min. An aliquot of 50 µL of the particle stock solution was then deposited onto the silanized microscope slide and placed in the dark until all solvent had evaporated. After solvent evaporation, the particles adhered to the slides and could be used for measurements. Sodium, potassium, and calcium chloride standard solutions (1.0-10-5 M) were prepared and mixed 1:1 with a 4 mM pH 7.4 Tris buffer. A 50-µL drop of the standard was placed onto a slide with adhered particles. The slide was covered with a cover glass and characterized with the fluorescence microscope as described.8 Subsequent sample solutions were measured by removing the cover glass with a stream of deionized water and repeating the previous step with a different sample. With this procedure, the
RESULTS AND DISCUSSION A particle casting apparatus18 was used for the high-throughput production of plasticized PVC sensing particles under nonreactive chemical conditions. A schematic of the instrument and the principle of the method are shown in Figure 1. The particle caster consists of two pressurized solution bottles, a flow chamber, a pressure regulation unit, a frequency generator, and a reservoir for the collection of the particles.18 One bottle contains the polymer solution, here a typical PVC membrane cocktail for optical sensors dissolved in dichloromethane, and the other bottle contains deionized water as the sheath liquid. Both solutions are passed under pressure through the flow chamber. The polymer solution is forced through a microtip that is placed close to the lower edge of the chamber. This surrounds the polymer solution by a rapid sheath flow and forms a liquid jet. The flow rates of the two liquids are controlled, by adjusting the pressure in the bottles from the pressure regulation unit. The liquid jet is periodically disturbed by an oscillating piezoelectric crystal, which forms polymer droplets. The frequency is adjusted by a frequency generator and leads to the formation of ∼20 000 drops/s. The droplets are collected into the reservoir by an aqueous poly(ethylene glycol) solution catch liquid. The polymer particles are formed during the curing process, where the polymer solvent and sheath liquid diffuse into the receiving solution. The particles precipitate, settle to the bottom of the container, and are separated by decanting. Figure 2 shows scanning electron micrographs of plasticized PVC particles that were obtained using the particle casting apparatus. The particles are uniform in size, they exhibit a spherical shape, and their surface appears to be perfectly smooth. In Figure 3 the corresponding fluorescence images are shown. Analysis with a commercial sizing instrument found a size distribution of 10.0 ( 1.0 µm, which is consistent with the diameters found in Figure 2. The size can be adjusted from about Analytical Chemistry, Vol. 73, No. 24, December 15, 2001
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Figure 4. Recorded fluorescence intensity ratios (at 650 and 687 nm) of five single particles taken simultaneously using the scanning mode of the imaging spectrometer and fluorescence microscope. Error bars represent the standard deviations of each data set (n ) 5).
2 to 30 µm by the instrument controls (flow rates, frequency change, and nozzle diameter), as well as the polymer solution concentration.18 In pure water solution, the particles form strong aggregates due to the hydrophobic nature of the polymer. This is eliminated by adding poly(ethylene glycol) to the solution. Other tested surfactants, such as dodecyl sulfate or Triton-X 100, interfered with the sensing chemistry. A neutral carrier-based sodium-selective system was chosen to demonstrate the functionality of microsphere-based sensors prepared via the particle casting apparatus. A batch of PVC-DOS plasticized particles was prepared that contained the H+-fluoroionophore ETH 5294, a sodium-selective calix[4]arene ionophore, and the ionic sites NaTFPB. For the spatially resolved spectral measurements, a previously described imaging spectrometer and fluorescence microscope was used.8 In this configuration, particles are immobilized on microscope slides and an epifluorescence microscope is used to excite the fluorophore in the particles and to collect the emitted light. With the same objective, the collimated light is directed toward the entrance slit of an imaging spectrometer, which is coupled to a CCD detector for spectral acquisitions. Single particles that are immobilized on silanized slides can be used to generate a complete calibration curve. As reported recently,8 the spectral acquisition yields a collection of 240 spectra from a selected slice of the field of view. For the present setup using a 40× objective, the spatial distance between adjacent spectra is ∼0.41 µm. Therefore, for a particle with a diameter of 10 µm, ∼25 individual spectra are used to spatially resolve its fluorescence emission characteristics. The spectra collected from each particle were found to be identical, with the fluorescence intensity increasing from the edges toward the center of the particle. This suggested a uniform distribution of the chromoionophore throughout the particle. The average of these single spectra, which corresponds to the particle fluorescence response, was used for calculation. Two peaks were observed at 650 and 687 nm, with a shoulder at 735 nm. The first peak corresponds to the protonated 6086
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Figure 5. Response curves of three single sodium-selective particles to sodium (open circles), potassium (filled circles), and calcium (rectangles) at pH 7.4. The lines are theoretical responses (eq 3).
form of the fluoroionophore, while the other two peaks were assigned to the deprotonated form. The peaks at 650 and 687 nm were used for ratiometric measurements in order to minimize any effects of photobleaching and variations in positioning, size, and lamp intensity. Particle-to-particle reproducibility was assessed by using the fluorescence microscope and imaging spectrometer in a scanning mode. The microscope slide was incrementally moved by a motorized stage, and spectral acquisitions were obtained at every position via software control. After each complete scan, the stage was returned to the initial position and the same particles were scanned with a different sample solution. A separate calibration curve was recorded for each of a total of five particles, and the results are shown in Figure 4. The intensity ratios at 650/687 nm obtained from the five particles produced a RSD of 1.6%, demonstrating good particle-to-particle reproducibility. The degree of protonation of the fluoroionophore (1 - R) is given from the following equation:20
1-R)
(
)
Rmax - R [IndH+] )1- 1+ IndT R - Rmin
-1
(1)
where R, Rmax, and Rmin are the fluorescence intensity ratios at a given equilibrium and at maximum and minimum protonation of the fluoroionophore, respectively. The response mechanism is based on the following ion-exchange equilibrium process:
Ind(org) + LNa+(org) + H+(aq) + R-(org) h IndH+(org) + L(org) + Na+(aq) + R-(org) (2)
where Ind is the neutral chromoionophore, L is the sodium (20) Kurihara, K.; Ohtsu, M.; Yoshida, T.; Abe, T.; Hisamoto, H.; Suzuki, K. Anal. Chem. 1999, 71, 3558.
ionophore, R- is the anionic site, and H+ and Na+ are the hydrogen and sodium ions, respectively. The organic and aqueous phases are indicated as (org) and (aq), respectively. The response function of this system has been described previously:1
aNa+/aH+ ) Kexch-1
RT - (1 - R)IndT R 1 - R LT - (RT - (1 - R)IndT)
(3)
Three response curves of three single particles to Na+, K+, and Ca2+ are shown in Figure 5. Measurements were done in 2 mM Tris-buffered solutions at pH 7.4 with varying concentrations of the cation chloride salt. The degree of protonation 1 - R is plotted as a function of the logarithm of the cation activity. The points correspond to experimental data, and the lines denote the theoretical curves according to eq 3. The response curve for sodium ion corresponds very well to the theoretical response, as with other optical sensing systems.8,9 The sensing particles show excellent selectivity for sodium over potassium and calcium, which is in accordance with the corresponding ion-selective electrode membrane responses.21 (21) Cadogan, A. M.; Diamond, D.; Smyth, M. R.; Deasy, M.; McKervey, M. A.; Harris, S. J. Analyst 1989, 114, 1551.
CONCLUSIONS Monodisperse plasticized PVC particles were prepared with a particle casting apparatus. The particles can be mass-fabricated and easily doped with known amounts of extraction and fluorescence reagents. Particles fabricated using this technique were tested for the selective determination of sodium ions by using an imaging spectrometer coupled with a fluorescent microscope. The sensing particles were shown to be fully functional and to exhibit a selective fluorescence response with very high reproducibility. This mild, high-throughput particle preparation method is a promising tool for mass-producing highly selective polymer-based microspheres that function via bulk extraction principles. ACKNOWLEDGMENT The authors thank the National Institutes of Health (GM59716) and Beckman Coulter, Inc. for financial support. We thank Eric Sinclair and Jorge Quintana (Beckman Coulter, Miami) for technical help with the particle caster and Martin Telting-Diaz (Auburn University) for preparing the particles shown in Figure 3. Received for review June 20, 2001. Accepted October 4, 2001. AC010694+
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