Biosensors and Chemical Sensors - American Chemical Society

Chapter 24

Swelling of a Polymer Membrane for Use in a Glucose Biosensor 1

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Marian F. McCurley and W. Rudolf Seitz 1

National Institute of Standards and Technology, Gaithersburg, MD 20879 Department of Chemistry, University of New Hampshire, Durham, NH 03824

Downloaded by SUNY STONY BROOK on October 31, 2014 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch024

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A membrane for afiberoptic biosensor based on polymer swelling and optical displacement has been investigated. A copolymer of polyacrylamide and 2-(dimethylamino) ethylmethacrylate (DMAEM), lightly crosslinked with Ν,Ν-methylenebisacrylamide (MBA) served as the membrane for immobilizing glucose oxidase. The swelling was found to be a function of the glucose concentration in the physiological range. The final degree of swelling varies inversely with the extent of crosslinking and directly with the amine content. Analyte induced swelling of polymers can serve as a transduction mechanism in biosensors. The changes in polymer size may be measured either electrically or optically and related to analyte concentration. For example, we recently described afiberoptic sensor for salt concentration based on polymer swelling (i). Changes in polymer size were detected as changes in the intensity of light reflected into an optical fiber. Other investigators have described a sensor for organic solvents in which small changes in polymer size are detected interferometrically (2). Fiber optic sensors based on polymer swelling offer several potential advantages. They can be designed so that the optical measurement is separated from the polymer by a diaphragm so that the measurement can not be affected by the optical properties of the sample. Unlikefiberoptic sensors based on indicator absorbance or luminescence, photodegradation is not a potential source of sensor instability. Measurements can be made in the near infrared region of the spectrum and take advantage of inexpensive components available for fiber optic communications. Membranes that swell in the presence of glucose have been prepared by immobilizing glucose oxidase in an amine-containing 0097-6156/92/0487-0301$06.00/0 © 1992 American Chemical Society

In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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acrylate polymer hydrogel (3,4). The enzyme catalyzes the oxidation of glucose to gluconic acid, which protonates the amine. Electrostatic repulsion between charged amine groups causes the gel to expand. The degree of swelling increases with glucose concentration. Studies on glucose-induced polymer swelling have focussed on developing membranes that could serve in systems for controlled delivery of insulin to diabetics (3,4). It has been shown that hydrophobic methacrylate copolymers undergo a sharp swelling transition as the pH is decreasedfrom7 to 6 (3-7). However, the kinetics of the transition are too slow for the proposed application to glucose delivery. We are interested in further investigating the potential of biosensors based on polymer swelling. We report here experiments with glucose oxidase impregnated membranes prepared from polyacrylamide copolymerized with (dimethylamino) ethylmethyacrylate. Because this polymer is more hydrophilic than the hydrophobic methacrylate gels investigated previously (3-7), it may provide faster response. The pH dependence of the membrane with no enzyme is reported. The response of the membrane with immobilized glucose oxidase to glucose is then described. In addition the effects of percent crosslinking and percent of amine moieties in the membrane on swelling are also investigated. Experimental Apparatus. The diameters of gel disks were measured with a Cambridge Instruments Stereoscope with a micrometer reticle. An Ohaus GA200D balance was used to weigh swollen gel disks. The volume was calculated from the weight assuming a density of 1.00. An Orion Research pH meter (Model EA920) and electrode were used to monitor pH. A BioRad electrophoresis chamber was used to form the gel slab. Reagents. Anhydrous d-glucose and glucose oxidase Type VII Aspergillus Niger were purchased from Sigma. Acrylamide 97%, potassium persulfate 99%, 2-((hmethylamino)ethylmethacrylate 98% (DMAEM), N,N-methylenebisacrylamide 99% and NaOH were from Aldrich. NaCl was from Mallinckrodt. HC1 was purchased from Fisher. Procedures. Polymer Preparation. The polymer gel was prepared by combining specified volumes of a filtered 40% acrylamide solution, a 2% Ν,Ν-methylenebisacrylamide solution and 98% DMAEM. Total volumes ranged from 3 to 5 mL. The mixture was deoxygenated by bubbling N2 through it for 15 minutes. Ten to twenty microliters of a saturated potassium persulfate solution were added to initiate polymerization. At this point 0.0010 g of glucose oxidase, dissolved in 0.010 mL saline, was added to those gel solutions that were used to measure glucose concentration. The gel solution was then transferred


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Swelling of a Polymer Membrane

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to the mold which consisted of two glass plates separated by 0.50 mm spacers. The solidified gel slab was removed from the glass plates and allowed to swell in distilled water. A cork borer was used to cut gel disks from the slab. Polymer Swelling. The swelling of the gel as a function of solution pH, percent crosslinking and percent DMAEM was measured as follows. Gel disks were placed in citrate/phosphate buffer solutions ranging in pH from 2.6 to 7.0. These buffers were prepared according to published methodology (8). In addition, they each contained 0.010 M NaCl. At specific time intervals the gel was taken from the solution, excess water was removed with a tissue, and the change in diameter was measured using a stereoscope. This was repeated until the gel diameter reached a constant value. The gel disks used to measure glucose contained 40% (w/w) DMAEM and 3.3% (w/w) N,N-methylenebisacrylamide. The gel was placed in aqueous glucose standard solutions, and the change in diameter was measured as above. Results and Discussion Water in contact with freshly prepared polymer has a pH close to 7. This is lower than expected for aqueous solutions of aliphatic amines which typically have pKb values close to 9. Since the polymer preparation procedure does not involve acids which could protonate the amine groups, we conclude that the amine moieties must not be in full contact with the water. Figure 1 plots the percent increase in disk diameter vs. time at various pH values for gels with 5% crosslinking agent and 40% amine. The data show that swelling is slow; the disks take an hour or more to reach a constant volume. Also, decreasing the pH from 4 to 2.6 causes a large increase in the constant volume observed after two hours of contact with buffer. Since a dissolved aliphatic amine would be fully protonated over the range from 2.6 to 7, the data in Figure 1 also suggest that the amine is not in full contact with the solvent, confirm that the gel is sensitive to changes in pH. Figure 2 shows volume of the gel vs. pH for a polymer gel with 2% crosslinking agent and 40% amine. In Figure 2a the gel was initially exposed to base, then titrated with 0.10 M HC1. In Figure 2b the gel was initially exposed to acid, then titrated with 0.10 M NaOH. The pH effect has a high degree of hysteresis. If polymer pH is lowered by adding acid, then swelling occurs between pH 4 and pH 2.6. However, if the direction of the pH change is reversed by adding base, then shrinking occurs between pH 6 and 10, a range much closer to that expected based on amine pKb values. Figure 3 plots percent increase in disk diameter vs. time after exposure to a pH 2.6 phosphate/citrate buffer for polymers containing 1% crosslinking agent and 10, 20 and 40% amine. It illustrates that the final degree of swelling is highly dependent on the percent of amine, in the form of DMAEM, in the membrane. As the amine content



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120 Time (min)

Figure 1. Effect of pH on the swelling of a 5% crosslinked polyacrylamide gel with 40 % DMAEM. The percent increase in disk diameter vs time at various pH values.




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6

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Figure 2a. Swelling ratio of a 2% crosslinked polymer gel with 40% DMAEM as a function of pH. The gel was initially exposed to base, then titrated with 0.10 M HC1.

Figure 2b. Swelling ratio of a 2% crosslinked polymer gel with 40% DMAEM as a function of pH. The gel was initially exposed to acid, then titrated with 0.10 M NaOH.



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increased the disk diameter increased. The polymer containing 40% amine undergoes substantial swelling, while formulations with 10 and 20% amine swell only slightly. According to theory, in a swollen polymer gel the electrostatic component of swelling should vary with the number of charges to the 1.2 power(9). The data in Figure 3 do not show this relationship. Instead, there appears to be something unique about the polymer with 40% amine that causes it to swell as the pH is reducedfrompH 7 to 2.6. Figure 4 plots the swelling ratio vs. time after exposure to a pH 2.6 phosphate/citrate buffer for polymers containing 1, 2 and 5% crosslinking agent and 40% amine. As would be expected the final swelling ratio is greatest for the polymer with the lowest amount of crosslinker and smallest for the polymer with the highest crosslinker percentage. However, the differences in the final swelling ratio are relatively small. Furthermore, the rate of swelling is significantly faster for the more highly crosslinked polymer. Normally, one would expect mass transfer processes to be faster in the more loosely crosslinked material. Figure 3 and 4 show that when exposed to a buffered solution, polymer volume reaches a constant value after approximately 20 minutes with little or no further change observed. Figure 5 shows percent increase in diameter of a 3.3% crosslinked polyacrylamide gel with 32% DMAEM as a function of glucose concentration vs. time. After exposure to glucose, gel placed in pH 7.4 phosphate buffer took about 60 minutes to shrink to its original size. The data in Figure 5 confirm that the amine-modified polyacrylamide membranes swell in response to glucose and thus are potentially useful for biosensors. The enzyme catalyzes the oxidation of glucose to gluconic acid, which protonates the amine. Electrostatic repulsion between charged amine groups causes the gel to expand. The degree of swelling increases with glucose concentration. Our data is similar to that reported for membranes prepared using 2-hydroxyethyl methacrylate (HEMA) rather than acrylamide (3). Response is reversible but the rate of deswelling is quite slow. Proposed Model. We proposed the following model for gel behavior. The hydrophobic aminoesters associate with each other, forming a separate phase within the polymer. However, the absence of any visible cloudiness in the membrane implies that the aminoester domains must be significantly smaller than the wavelengths of visible light. According to this model, the reason that the solution in contact with freshly prepared polymer doesn't become basic is because the amine groups are tied up in a separate phase. Furthermore, it is known that the crosslinking reagent, Ν,Ν'-methylenebisacrylamide, also forms a separate phase in polyacrylamides (10). It may be miscible with the amine phase. The pH has to be adjusted below 4 to protonate the amine to the extent that the amine phase is disrupted and the amines enter the aqueous phase. This explains the swelling observed as the pH is reduced below 4.




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Figure 3. Effect of amine concentration on swelling. Percent increase in diameter as a function of time after exposure to a pH 2.6 phosphate/citrate buffer for 1% crosslinked polyacrylamide gels with 10, 20 and 40% DMAEM.

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Figure 4. Effect of percent crosslinking on swelling. Swelling ratio vs. time after exposure to a pH 2.6 phosphate buffer for 1, 2 and 5% crosslinked polyacrylamide gels with 40% DMAEM.


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Figure 5. Effect of glucose on the swelling of a 3.3% crosslinked polyacrylamide gel with 32% DMAEM. Percent increase in diameter as a function of glucose concentration vs. time. Once formed, the protonated amine groups remain in the aqueous phase until the pH is increased to the point that the proton is removed. At this point the microdomains of amine reform. This accounts for the hysteresis in the titration curves that is shown in Figure 5. This model is consistent with the observation that only the polymer with 40% amine undergoes significant swelling at low pH. In the polymers with lower amine percentages, the amine level may be too low to allow formation of a separate phase. Only at the highest level is there enough amine present for the formation of separate amine domains. The phase structure of the polymer may also be affected by the degree of crosslinking. The observation that the highly crosslinked polymer swells more rapidly may indicate that the amine domains are smaller in this material. Implications for Biosensor Design. This study indicates that aliphatic amine containing polymers are not appropriate for biosensing. The proposed model suggests that formation of a separate amine phase is necessary to see swelling as the pH decreasesfrom7 to lower values. However, the formation of a separate amine phase is also linked to slow rates of response and hysteresis in the variation of volume vs. pH when measured after equilibration for 40 minutes. The original motivation for using amines in glucose sensitive polymers was because of the intended application to drug delivery. This requires that polymer permeability increase with glucose concentration. This requires a functional group that becomes charged as the pH decreases due to gluconic acid formation. In biosensors, however, this is not required. Therefore, it may prove advantageous to use other pH sensitive functional groups to prepare glucose sensitive polymeric membranes. One possibility is to hydrolyze the amide group In Biosensors and Chemical Sensors; Edelman, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.



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into a carboxylic acid moiety. The swelling characteristics of this system as a function of pH have been well established by Tanaka et al.(ii) At high pH the carboxylate group is uncharged and the gel is swollen. The decrease in volume observed as the solvent pH is lowered is due to protonation of the carboxylate group which leaves the polymer uncharged. Similar behavior was observed in this laboratory. The amine polymers used to prepare glucose sensitive polymers are heterogeneous. The rate of response is slow because of the slow rate of amine transfer from a hydrophobic phase to the aqueous phase. Faster rates of response should be possible with systems that do not require phase transformations. Certain commercial equipment, instruments or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the equipment, instruments or materials are necessarily the best available for the purpose.

Literature Cited 1. 2. 3. 4. 5.

McCurley, M.F.; Seitz, W.R. Anal. Chim. Acta, 1991, 249, 373-380 Butler, M.A.; Ricco, A.J.; Buss, R. J. Electrochem. Soc. 1990, 137, 1325-1326 Albin, G; Horbett, T.A.; Ratner, B.D. J. Controlled Release 1985, 2, 153-164 Siegel, R.A.; Firestone, B.A. J. Controlled Release 1990, 11, 181-192 Siegel, R.A.; Falamazian, M.; Firestone, B.S.; Moxley, B.C. J . Controlled Release 1988, 8, 179-182

6. 7. 8. 9. 10. 11.

Siegel, R.A.; Firestone, B.A.; Johannes I.; Cornejo, J. Polymer Preprints 1990, 31, 231-232 Siegel, R.A.; Firestone, B.A. Macromolecules 1988, 21, 3254-3259 Methods In Enzymology; Colowick, S.P.; Kaplan, N.O., Eds.; 1955, Vol. 1 Flory, P.J. Principles of Polymer Chemistry; Cornell University Press, Ithaca, NY, 1953 Parreno, J.; Pierola, I.F. Polymer 1990, 31, 1768-71 Tanaka, T.; Fillmore, D.; Sun, Shao-Tang; Nishio, I.; Swislow, G. and Shah, A. Phys. Rev. Lett 1980, 45, 1636-1639

R E C E I V E D November 26, 1991


Biosensors and Chemical Sensors - American Chemical Society

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