Anal. Chem. 1997, 69, 4092-4098
Photo-Cross-Linked Decyl Methacrylate Films for Electrochemical and Optical Polyion Probes Theresa M. Ambrose and Mark E. Meyerhoff*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Potentiometric and optical polyion probes based on photocross-linked thin films of decyl methacrylate (DMA) are described, and the effects of film composition on the response toward heparin are examined in detail. In accordance with existing theory governing potentiometric polyion response, lowering the amounts of plasticizer and tridodecylmethylammonium chloride ion exchanger within the film enhances its sensitivity toward heparin. Varying the cross-linker content of a DMA-based film, however, provides an additional mechanism to regulate its physical structure and, hence, the observed potentiometric polyion response. Films with low hexanedioldimethacrylate crosslinker content yield optimal potentiometric heparin detection limits (0.04 µM), suggesting a lower diffusion coefficient within such films, apparently due to interactions between adjacent pendant decyl groups. Increasing crosslinker content interrupts these interactions and facilitates diffusion. This knowledge is applied to optimize optical heparin sensing via DMA films covalently attached to glass substrates. When used in a limited volume/fixed exposure time measurement mode, such optically sensitive films can detect clinically relevant levels of heparin (0.55.0 units/mL) in undiluted human plasma.
While polymer membrane-based potentiometric sensors for small ions (e.g., K+, Na+, Ca2+, etc.) have been in use for two decades, analogous electrochemical devices capable of detecting polyionic species (such as heparin, protamine, and carrageenan) at submicromolar levels have been reported only recently.1-7 Polyion-sensitive electrodes, now commercially available, are poised to become important tools for fundamental biomedical studies including novel enzyme activity assays and determinations of binding affinities between certain polyions and biological macromolecules.2,5,7 The potentiometric response of polyionsensitive electrodes is known to be highly dependent on membrane composition.3 Limited diffusion of a polyion/ion-exchanger complex within the membrane phase causes a steady-state (1) Ma, S. C.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1992, 64, 694-697. (2) Ma, S. C.; Yang, V. C.; Fu, B.; Meyerhoff, M. E. Anal. Chem. 1993, 65, 2078-2084. (3) Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 2250-2259. (4) Ma, S. C.; Yang, V. C.; Meyerhoff, M. E.; Yun, J. H.; Fu, B. Electroanalysis 1993, 5, 719-724. (5) Meyerhoff, M. E.; Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C. Anal. Chem. 1996, 68, 168A-175A. (6) Fu, B.; Bakker, E.; Yang, V. C.; Meyerhoff, M. E. Macromolecules 1995, 28, 5834-5840. (7) Yun, J. H.; Meyerhoff, M. E.; Yang, V. C. Anal. Biochem. 1995, 224, 212220.
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accumulation of polyion at the membrane-sample interface, resulting in a change in the phase boundary potential. To achieve potentiometric response to polyions in analytically useful concentration ranges, membrane viscosity must be increased in order to control membrane-phase polyion diffusion. In all previously described polyion probes, based primarily on solvent-cast polymers such as poly(vinyl chloride) (PVC), this is accomplished by lowering the plasticizer content of the membrane. In contrast, optical sensing of polyions (via the same extraction chemistry) is achieved using conventional high-plasticizer levels.8 Recently, a novel photopolymerizable membrane matrix for preparing ion-selective electrodes has been described: decyl methacrylate (DMA) cross-linked with hexanedioldimethacrylate (HDDMA).9 This matrix is an attractive candidate for polyion detection since membrane-phase diffusion can be controlled by changing the cross-linker content. Indeed, the physical structure of DMA membranes can be readily modified by varying the relative amounts of monomer, cross-linker, and plasticizer. Another key advantage of this material over solvent-cast membranes is its potential for use in fabricating solid-state potentiometric and optical sensor designs, since DMA can be patterned photolithographically and covalently attached to appropriately pretreated glass or silica substrates. In this work, cross-linked decyl methacrylate is investigated in detail as a matrix for preparing polyanion sensing films. Specifically, the effects of membrane composition (ion-exchanger level, plasticizer content, and cross-linker/monomer ratio) on polyion response properties are examined. In addition, crosslinked films of this material will be shown to exhibit unusual behavior with respect to diffusion rates as a function of crosslinker content. The unique physical characteristics of DMA are further exploited to fabricate optical chemically sensitive probes based on covalently attached thin films of DMA on glass substrates. Such sensing films are robust and inexpensive and can be used to detect clinically relevant levels of the polyanionic anticoagulant drug heparin in undiluted human plasma. EXPERIMENTAL SECTION Materials. Decyl methacrylate was obtained from Pfaltz & Bauer (Waterbury, CT). Hexanedioldimethacrylate, benzophenone, benzoyl peroxide, and [2-(methacryloyloxy)propyl]trimethoxysilane were purchased from Aldrich (Milwaukee, WI). Tridodecylmethylammonium chloride (TDMAC), dioctyl sebacate (DOS), and Chromoionophore IV (ETH 2412, 3-hydroxy-4-(4nitrophenylazo)phenyloctadecanoate) were obtained from Fluka (Ronkonkoma, NY). Beef lung heparin and tris(hydroxymethyl)(8) Wang, E.; Meyerhoff, M. E.; Yang, V. C. Anal. Chem. 1995, 34, 522-527. (9) Ambrose, T. M.; Meyerhoff, M. E. Electroanalysis 1996, 8, 1095-1100. S0003-2700(97)00345-4 CCC: $14.00
© 1997 American Chemical Society
Table 1. Compositions of Membranes Used To Prepare Heparin-Sensitive Electrodes
c
membrane labela
TDMAC
5/30/40 3/40/40 3/40/30 3/30/50 3/30/40 3/30/30 3/20/40 3/20/30 1/30/40 0.5/30/40
5 3 3 3 3 3 3 3 1 0.5
weight percentage of component HDDMA DOS DMA BPb 30 40 48 25 31 36 20 25 31 31
40 41 31 50 40 30 42 30 42 43
22 13 15 19 22 28 32 39 23 23
2 2 2 2 2 2 2 2 2 2
BΦc 1 1 1 1 1 1 1 1 1 1
a Weight percent TDMAC/HDDMA/DOS. b Benzoyl peroxide. Benzophenone.
aminomethane (Tris buffer) were obtained from Sigma (St. Louis, MO). Fragmin, a low molecular weight heparin preparation, was generously donated by Dr. Wahr (Department of Anesthesiology, University of Michigan). All other chemicals were of analytical reagent grade. DMA and HDDMA were purified by extraction with aqueous NaOH using a previously described procedure;9 all other chemicals were used as received. Solutions were prepared with doubly distilled deionized water. Evaluation of Potentiometric Response Characteristics. The procedure for preparation of DMA-based potentiometric sensors has been described previously.9 Briefly, small aliquots of each membrane cocktail listed in Table 1 were outgassed and photopolymerized for 30 min at 365 nm using a 100-W UV lamp. Small disks of the resulting 75-100-µm-thick films were mounted in glass electrode bodies, with an internal Ag/AgCl wire reference electrode and 0.12 M NaCl as the inner filling solution. Membrane resistance was evaluated after 5 h of soaking in 0.12 M NaCl using a Model 6310 Princeton Applied Research electrochemical impedance analyzer as described previously.9 For potentiometric measurements, an attempt was made to ensure that all electrodes experienced the same analyte mass transfer to the films via a controlled stir rate and a specially designed beaker with a rounded bottom. After presoaking at least 5 h in 0.12 M NaCl, potentiometric response was measured by adding aliquots of polyion stock solutions to 50.0 mL of stirred 0.12 M NaCl. The transient response of each polyion electrode was recorded (vs an external double-junction Ag/AgCl reference electrode) using the previously described multichannel LabView setup9 at the rate of either 30 or 60 data points/min. The transient response profiles of DMA-based polyion electrodes were similar to those obtained with PVC-based electrodes; that is, for all but the lowest heparin concentrations, the EMF signal reaches a stable, steady-state value after a short (3-5 min) measuring time.1-3 Thus, in accordance with previously established experimental procedure, the EMF values used to construct calibration curves were recorded 5 min after each polyion aliquot addition by averaging the recorded signal over a 6-s interval. At least three disks of each membrane composition were evaluated, and polyion response was measured using only fresh membranes. Differential Scanning Calorimetry (DSC) of DMA Membranes. DMA membrane samples ranging from 1.5 to 8 mg were sealed into aluminum sample holders. For some samples, removal of plasticizer and ion exchanger was accomplished by presoaking in tetrahydrofuran followed by air-drying. DSC traces were
recorded using a Perkin-Elmer DSC 7 controlled by a PerkinElmer TGA 7/DX at a scan rate of 40°/min over various temperature ranges encompassing 0-350 °C. Characterization of Polyion Diffusion Using Optically Sensitive Films. Membrane cocktails for polyion-sensitive optical films were prepared using 40 wt % DOS, 2 wt % TDMAC, 1.6 wt % chromoionophore, 3 wt % photoinitiator, and the same three HDDMA/DMA ratios used to prepare films for potentiometric devices (see Table 1). When necessary, the cocktail was gently heated to facilitate dissolution of the chromoionophore. Glass strips (9 mm × 4 cm) were silanized by immersion in a 10% (v/v) solution of [2-(methacryloyloxy)propyl]trimethoxysilane in toluene at 70 °C for 1 h. Small aliquots (2.5 µL) of the membrane cocktail were placed on a glass strip, covered with an untreated glass slide, and simultaneously photopolymerized and covalently attached to the glass by exposure to 366-nm UV light for 6-8 min. The films were stored in a dry, dark drawer until use. Each film was immersed in stirred Tris buffer (0.05 M, pH 7.4) for 20 min prior to optical measurements. Absorbance spectra were recorded at a scan rate of 1200 nm/min using a Beckman DU-70 spectrophotometer. The dynamic response to heparin (indicative of polyion diffusion into the film) was measured in 10.0 mL of Tris buffer by recording the absorbance spectra at various time intervals. Each film was transferred to a cuvette containing an identical solution while spectra were recorded and then immediately returned to the stirred sample solution. The absorbance values at 536 nm were normalized to yield the fraction, R, of deprotonated chromoionophore according to the following expression:
R ) (A - A0)/(A1 - A0)
(1)
where A is the measured absorbance at 536 nm, A1 was measured in buffer, and A0 was measured in air prior to immersion in buffer. At least two fresh sensing films were tested for each membrane composition and heparin concentration evaluated. Due to a 25% variation in film thickness, A1 and A0 were measured individually for each sensing film tested. Diffusion of Small Ions through Cross-Linked Membranes. Two blank DMA membrane cocktails (without ionic additives) were prepared, with 40 wt % DOS and either 20 or 40 wt % HDDMA. A disk was cut from each resulting photopolymerized film and mounted in a diffusion cell containing 15 mL of 0.1 M citrate buffer (pH 4.5) in both chambers. After 5 h of exposure to the buffer with continual stirring in both chambers, sodium salicylate was added to chamber A of the diffusion cell, yielding an initial concentration of 0.1 M sodium salicylate. At various time intervals, 100-µL aliquots of solution were removed from chamber B of the diffusion cell and assayed for salicylate by measuring the absorbance at 298 nm (using an experimentally determined calibration curve). The apparent diffusion coefficient of salicylate in the membrane phase is related to the rate of concentration increase of chamber B. Following the salicylate assay, the film thickness (corresponding to the diffusion path length) was measured using an Olympus inverted optical microscope. Response of Optically Sensitive Films to Heparin and Small Ions. Optical polyanion probes were prepared with the same compositions as the optically sensitive films described above, containing 40 wt % HDDMA. Films used for construction of heparin calibration curves and for small-ion-selectivity measureAnalytical Chemistry, Vol. 69, No. 20, October 15, 1997
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Table 2. Potentiometric Response Characteristics toward Heparin of DMA-Based Electrodesa membrane label
detection range midpoint µMb %c
0.5/30/40 1/30/40 3/30/40 5/30/40 3/20/30 3/20/40 3/30/30 3/30/50 3/40/30 3/40/40
0.24 0.63 1.29 2.25 0.31 1.26 0.60 2.17 0.67 1.45
detection limitd units/mL µM
10.7 28.0 57.3 100.0 13.8 56.0 26.7 96.4 29.8 64.4
