Anal. Chern. 1988, 60, 2467-2472
stituted (4)for (3), the following equations were obtained:
D = ~ex[X-l2[LI(o) log D = log
KeX[X-l2 + log [L](,)
(5)
(6)
This therefore confirms the 1:1complex formation ability of ionophore for Cu(I1) ion. T E T D S ligand has a n almost ideal C-shaped nonmacrocyclic cavity to fit the Cu(I1) ion. But DMDOTDS did not form an ideal coordination complex sphere for Cu(I1) ion compared to TETDS, and hence the sensitivity and selectivity are not so good. On the other hand, TBTDS was observed to have a different behavior in regard to the selectivity of foreign ions. These differences in selectivity are probably due to the steric hindrance of the n-butyl chain which affects the cavity Of the compound* It was confirmed that the selectivity of TBTDS tended to become similar to that of the other two(TETDS,DMDOTDS), with the addition of a bulky alkyl group such as isobutyl chain, On the basis of above results, it is considered that an alkyl chain also plays an important role in sustaining the C-shaped cavity of the compound. CONCLUSION The results presented here clearly demonstrate that high selectivities may be induced with nonmacrocyclic ligands, as Dredicted earlier (25,27). The nonmacrocyclic neutral carriers i r e quite versatile as chelating agents, somewhat superior to the macrocyclic ligand. TETDS has shown better Sensitivity and selectivity than TBTDS and DMDOTDS for Cu(I1) ion. These neutral carriers have almostideal C+haped cavities to fit Cu(I1) ion, and form a 1:l cation/ionophore coordination sphere. Also the alkyl chain Plays an important role in Nutaining the C-shaped cavities for the formations of metal complexes. A highly selective electrode opens several new aspects in Cu(I1) ion analysis. ACKNOWLEDGMENT The authors gratefully acknowledge Professor W. Simon of the Swiss Federal Institute of Technology (ETH) for helpful
2407
discussion and encouragement. LITERATURE CITED (1) Oehme, M.; Simon, W. Anal. Chlm. Acta 1978, 86, 21. (2) Meier, P. C.; Morf, W. E.; Laubli, M., Simon, W. Anal. Chim. Acta 1984. 156, 1. (3) Simon, W.; Pretsch, E.; Morf, W. E.; Ammann, D.; Oesch, U.; Dinten, 0. Analyst (London) 1984, 109, 207. (4) Kimura, K.; Ishlkawa. A.; Tamura, H.; Shono, T. J . Chem. Soc., Perkin Trans. 1984, 2 , 447. (5) Nieman, T. A.; Horvai, G. Anal. Chim. Acta 1985, 170, 359. (6) Kitazawa, s.; Kimura, K.; Yano, H.; Shono, T. Analyst (London) 1985, 110, 295. (7) Karnata, S.; Higo, M.; Kamibeppu, T.; Tanaka, I . Chem. Lett. 1982, 287. ( 8 ) Kamata, S . ; Yamasaki, K.; Higo, M.; Bhale, A,; Fukunaga, Y. Analyst (London) 1988, 113, 45. (9) Morf, W. E.; Ammann, D.; Simon, W. Chimia 1974, 28. (10) Morf, W. E.; Kahr, G.; Simon, W. Anal. Lett. 1974, 7 , 9. (12) (11) Frensdorff, Kamata, S.;H,Ogawa, K, J , Am, F.; Fukumoto, Chem, sot. M. Chem. 1971, 93, Lett. 6oo, 1987, 533. (13) Pedersen, C. J.; Frensdorff, H. K. Angew. Chem., Inf. Ed. Engl, 1972, 1 1 , 16. (14) Christensen, J. J.; Hill, J. 0.; Izatt, R. M. Science (Washington, D.C.) 1971, 774, 459. (15) Izatt. R. M.; Eatough D. J.; Christensen, J. J. Struct. Bonding (Berlin) 1973. 16, 161. (16) Christensen, J. J.; Eatough D. J.; Izatt, R. M. Chem. Rev. 1974, 7 4 , 351. (17) Busch, D. H.; Farmery, K.; Goedken, V.; Katovlc, V.; Melnyk, A. C.; Sperate, C. R.; Tokel, N. A&. Chem. Ser. 1971, No. 100, 52. (18) Martin, L. Y.; DeHayes, L. J.; Zornpa, L. J.; Busch, D. H. J . Am. Chem. SOC. 1974, 96, 4046. (19) Watkins, Jr., D. D.; Reley, D. P.; Stone, J. A,; Busch, D. H. Inorg. Chem. 1976, 15, 387. ~ ~, ~ ~s, c, k s,~c,; l it,~ A, ,M,; ~ ~ D. H.~ (20) H ~ y,; ~~ ~ ~r tL,,i y,; J. Am. Chem. SOC. 1977. 99. 4029. (21) Hinz. F. P.; Margerum. D. W. Am. Chem. SOC. 1974, 96, 4993. (22) Hinz, F. P.; Margerum, D. W. Inorg. Chem. 1974, 13, 2941. (23) Smith, G. F.; Margerum, D. W. J . Chem. Soc ., Chem. Commun. 1975, 807. (24) Prestsch, E.; Ammann, D.; Osswald, H. F.; Guggl, M.: Simon, W. Helv. Chim. Acta 1980, 63, 191. (25) Schefer, U.; Ammann, D.; Pretsch, E.; Oesch, U.; Simon, W. Anal. Chem 1988 58 2282 (26) Thorn,'G, D,; Lud;uig,R: A. The Dithiocarbamates and Related Compounds; Elsevler: Amsterdam and New York, 1962; Chapter 6. (27) Simon, W.; Morf, W. E.; Meier, P. C. Struct. Bonding (Berlin) 1973, 16, 113.
i.
RECEIVED for review March 10, 1988. Resubmitted August 9,1988. Accepted August 9,1988. This work was supported by the Japanese Ministry of Education, Science and Culture.
Role of Monomer in y-Irradiated Dimethyldiallylammonium Chloride Modified Electrodes Edward W. Huber and William R. Heineman* Edison Sensor Technology Center, Biomedical Chemistry Research Center, and Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-01 72 Commerclally available poly(dlmethyldlallylammonlum chloride) (pdyDMDAAC) solutlons were found by NMR to contain DMDAAC monomer. Polymerization of this monomer by y lrradiatlon results In the formation of Insoluble networks that can be Lmmobilzed on electrode surfaces. The lmmoblllzatlon process Is aided by the high porosity of the graphite electrodes used. The permeablllty of these polyDMDAAC electrodes to solution species can be controlled by varying the amount of monomer polymerlzed. The feasibility of ImmobC llzlng macromolecules onto electrode surfaces by polymerization of monomers Is studied by lmmobllizlng polyDMDAAC (no DMDAAC monomer present) In a network of poly(Nvlnylpyrrolldone)/N-vlnylpyrrolldone.
* To whom a l l correspondence should b e addressed.
The modification of electrode surfaces with polymer films has been an active area of research in the past decade (1,2). Ionomer membranes, with ionizable groups attached to organic polymer backbones, are the basis of a type of electrode modification based on ion-exchange processes. As a result of the electrostatic interactions a t the ion exchange sites, these membranes can exhibit a large difference in permeability toward oppositely charged ionic species in comparison to similarly charged species. The immobilization of ionomer membranes on electrode surfaces results in changes in both the sensitivity and selectivity of the electrode to the appropriately charged solution species (3). Du Pont's Nafion perfluorosulfonated polymer membrane has been the polymer of choice for many studies because it is commercially available, is water-insoluble, can be dip-coated
0003-2700/88/0360-2467$01.50/00 1988 American Chemical Society
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,
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
onto many electrode surfaces, and has ion-exchange properties that make it permeable to cations and impermeable to anions ( 4 , 5 ) . These characteristics have resulted in the development of sensors based on Nafion for catecholamine neurotransmitters, such as dopamine. Dopamine, a cation a t physiological pH, is selectively preconcentrated into Nafion films while anionic and neutral species are excluded (6, 7). Analogous examples of cationic films are not as readily available since many cationic films are too water soluble to yield long-lived coatings. Several approaches have been taken to overcome this problem. Block copolymers consisting of polylysine and a less soluble polymer with improved stability on electrode surfaces have been reported (8). Carbon-paste electrodes that are prepared with a mixture of carbon paste and liquid ion exchanger have been successfully used for the quantitative determination of ferri-/ferrocyanide in wine (9). Polymers with quaternary ammonium groups in the polymeric backbone, based on N,N-dialkyl-substituted aniline derivatives, have been prepared by electrochemical polymerization of the corresponding monomers ( 1 0 , I I ) . The resulting films, which were insoluble in all solvents studied, exhibited pHindependent anion-exchange properties and strong attachment to the electrode surface. Electrodes modified with quaternized poly(4-vinylpyridine) have also received some attention (12, 13). These electrodes often have constraints on available pH ranges and counterions used. We have recently reported the immobilization on electrode surfaces of the aliphatic, water-soluble polymer dimethyldiallylammonium chloride (DMDAAC), a quaternary ammonium based pyrrolidine polymer, using y irradiation ( 1 4 , 1 5 ) . y irradiation of a polymer film can result in the formation of cross-links, grafting, copolymerization of the polymer with monomer species present, or polymer degradation (16, 17). Any of these processes may result in the formation of a polymer network that is insoluble in a given solvent but still retains the ability to swell when immersed in that solvent. Increasing the irradiation dosage can increase the cross-linking, grafting, copolymerization, or degradation of the polymer and thereby alter the swelling characteristics of the resulting network. Since they are insoluble, swollen networks of this type can be immobilized on electrode surfaces if anchoring of the film to the electrode is adequate. In our earlier studies (15) polyDMDAAC was immobilized on electrodes by using y irradiation. These electrodes exhibited ion exchange of anions into the polyDMDAAC film with ferri-/ferrocyanide being preconcentrated by a factor of 26X relative to a nonmodified electrode. These ion exchange processes were shown to be affected by the ionic strength and anion type of the supporting electrolyte. We were not able, however, to vary the permeability of the film toward neutral or cationic analytes in polyDMDAAC by controlling the extent of cross-linking by irradiation dosage. In this paper we expand upon our initial investigation of the immobilization of polyDMDAAC using y irradiation to explore the role of DMDAAC monomer in the formation and the permeability of the network, the effect of electrode porosity on anchoring the network, and the feasibility of immobilizing macromolecules in a polymer network. EXPERIMENTAL SECTION Reagents. DMDAAC monomer was purchased from Fluka Chemical Corp. (Ronkonkoma, NY) as a 60% aqueous solution. PolyDMDAAC was obtained from Polysciences, Inc. (Warrington, PA), either as a dry powder or as a 15% aqueous solution and from Aldrich Chemical Co. (Milwaukee, WI) as a 20% aqueous solution. Poly(N-vinylpyrrolidone) (PNVP) and N-vinylpyrrolidone (NVP) were also obtained from Polysciences, Inc. Potassium ferricyanide (Allied Chemical, New York, NY), 1,Cbenzoquinone(BQ, Fluka Chemical Corp., Ronkonkoma, NY), vitamin BI2 (B12, cyanocobalamin, BDH Chem. Ltd., Poole,
England), hexaamineruthenium(II1)chloride (Aldrich Chemical Co., Milwaukee, WI), and the salts used to make supporting electrolyte solutions (MCB, Cincinnati, OH) were all used as received. All solutions were prepared with distilled, deionized, photolyzed water from a Barnstead Organicpure system (Fisher, Cincinnati, OH) unless noted otherwise. Electrode and Polymer Film Preparation. Spectroscopic grade graphite rods (type FXI-365T) 0.46 cm in diameter were obtained from Poco Co. (Dallas, TX) and were pretreated as previously described (15). The 15% aqueous polymer/monomer solutions were prepared with the calculated polymer/monomer ratios by mixing the appropriate amount of 60% (w/v) monomer solution with 15 or 20% (w/v) polymer solution. A 1O-pL aliquot of these solutions was delivered to the surface of each electrode and allowed to dry. If placed into solution (without irradiation), the films rapidly dissolved from the electrode surface. Bulk polymer films were prepared by pouring polymer/monomer solutions into petri dishes and evaporating the water at ambient conditions. The films were then removed from the petri dishes and cut into rectangular strips. Modified electrodes and polymer/monomer films were then exposed to y radiation from a 6oCosource at the University of Cincinnati, Department of Nuclear Engineering, as described elsewhere (15, 18). Apparatus and Procedures. Cyclic voltammetry and square wave voltammetry were conducted with a BAS-100 electrochemical analyzer (BioanalyticalSystems, West Lafayette, IN). For square wave voltammograms (frequency = 15 Hz, pulse amplitude = 25 mV, step increment = 4 mV) background corrections were made with the BAS-100 by subtraction of a stored background scan. The electrochemical cell included a Pt auxiliary electrode and a BAS RE-1 Ag/AgCl (3 M NaC1) reference electrode. All potentials reported are referenced to this electrode. Dilute solution viscosity measurements were made at 22 "C (ambient)by use of an Ostwald viscosimeter with a 1-mm capillary bore. The 1%solutions of irradiated polymer films were prepared in 1.0 M NaCl. Relative viscosities were determined versus distilled, deionized water (0.955 CPat 22 "C, ref 19). NMR spectra were obtained at the Merrell Dow Research Institute (Cincinnati, OH) using a Varian VXR-300 NMR Spectrometer. Solutions were prepared by adding D20 (Aldrich) to the appropriate polymer film. External 2,2-dimethyl-2-sila5-pentanesulfonate (DSS, Aldrich) was used as a chemical shift reference. Scanning electron micrographs were obtained at the Merrell Dow Research Institute with a Jeol JSM 840 scanning electron microscope and recorded as photomicrographs. The samples were observed by using an accelerating voltage of 10 kV. The volumes of swollen, irradiated polymer films were obtained with tall class A 5-mL graduated cylinders. A weighed amount of dry polymer was placed in 0.5 M NaCl until swollen (1 h or more). The resulting polymer gel was then moved by use of a spatula or forceps to an empty graduated cylinder. By use of a second graduated cylinder filled exactly to 5.00 mL with 0.5 M NaC1, solution was transferred until the miniscus of the first cylinder was at 5.00 mL. The volume remaining in the second cylinder was recorded as the swollen volume. This was normalized by dividing by the weight of the polymer used. RESULTS AND DISCUSSION Role of Monomer in Irradiated Polymer Films. The polymer DMDAAC has been immobilized on platinum and graphite electrodes by y irradiation of films cast from commercially available polyDMDAAC solutions (15). Initial attempts to control the permeability of the membranes on these modified electrodes to solution species by altering the irradiation dosage were not successful. Nearly identical properties were observed for all electrodes irradiated regardless of the dosage (1-30 Mrad), yet immobilization occurred only after irradiation. These films apparently were sufficiently swollen so that even cationic species could diffuse through the positively charged polyDMDAAC film to the electrode surface. If immobilization were a function of either the extent of cross-linking or degradation of the polymer, increasing the irradiation dosage should have increased these processes,
2469
ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988 40
-
lI
35-
W
m
CI
0
a
H
c
z
W V
/
1
O O
S
1RRAd;ATION 1bSAGE20[MRA0)25
30
Flgure 1. Relative viscosities of irradiated polyDMDAAC films as a function of irradiation dosage: (m) film from 15% solution; (A)film from
solid polyDMDAAC. resulting in less swollen, less permeable membranes, which was found not to be the case. In an attempt to understand this phenomenon, we have made dilute solution viscosity measurements (20) in 1M NaCl of polyDMDAAC from two different sources. The viscosity results observed for solutions made from irradiated films cast from commercially available 15% solutions, which was the polymer source used previously for immobilization on electrodes (15),as well as from solutions prepared by using purchased solid DMDAAC polymer are shown in Figure 1. The viscosity of the films prepared from the 15% solutions increased significantly even a t low dosages. At greater than a few megarads the films were not sufficiently soluble for dilute solution viscosity measurements to be made. Conversely, the viscosity of dilute solutions from the films prepared from the solid DMDAAC did not change significantly between 0 and 30 Mrad. Proton NMR spectra of these same films before irradiation were obtained as D 2 0 solutions (Figure 2). The significant differences between these two spectra are resonances observed at 2.94, 3.82, 5.61, 5.65, and 5.96 ppm. Through decoupling experiments and analysis of chemical shifts, these resonances were found to be due to the presence of 2-4% of DMDAAC monomer in the purchased 15% solutions but not present in the solid polyDMDAAC. Irradiation of the films resulted in the disappearance of these resonances with no additional changes observed. The electrochemical characteristics of the modified electrodes and the viscosity data are both explained by the presence of this monomer in the purchased polyDMDAAC solutions. y irradiation results in the free radical polymerization of the monomer and corresponding physical entanglement or grafting with the DMDAAC polymer present, yielding an insoluble network. This polymerization process has been systematically followed by proton NMR (the disappearance of the vinyl proton signals of the monomer) and observed a t dosages of less than 1 Mrad (21). The original successful immobilization of polyDMDAAC by y irradiation was due to the fortuitous presence of the monomer impurity in the polymer being used. Attempts to immobilize DMDAAC polymer in the absence of this monomer have failed in the dosage range studied (up to 30 Mrad). With the polymer solutions containing 2-4% monomer, all of the monomer present is polymerized at dosages greater than a few megarads. This polymerization is sufficient for network formation as indicated by the great increase in viscosity as well as the ability to immobilize the polymer membrane on electrode surfaces. Since all of the monomer is polymerized at low dosages, no additional changes in the polymer are observed up to 30 Mrad, as noted by the lack of change in viscosity of the monomer-free
rim 1 ’
7
I
I
II ’ I ’
I
I ’ d ’ ‘ I
’I
I I I’
I ’ I I I‘
‘7”’ I
I
I
’
I
II
’
1 PPH
I
111
0
Flgure 2. ‘H NMR spectra of (top) solid polyDMDAAC and (bottom) polyDMDAAC from 15% solution, 25 mg of polyDMDAAC film/l mL Of DZO.
polyDMDAAC. For this reason, no control over permeability can be achieved through varying irradiation dosage. Increasing the amount of monomer present prior to irradiation (see below) does lead to some control over film permeability. It should be noted that the concept of adding vinyl monomers to polymer films for immobilization onto electrode surfaces by y irradiation has been successful for other systems, including poly(N-vinylpyrro1idone)lN-vinylpyrrolidone,and is the topic of other reports (18, 22). Electrode Substrates Used for Immobilization. Immobilization of polymer films using y irradiation has been attempted on spectroscopic grade graphite rods, platinum foil, and platinum wire, both in the presence and absence of monomer. When monomer is present, a near 100% success rate for immobilization is observed with the graphite rods. Conversely, with platinum a success rate of 50% or lower is observed. These observations are believed to be the result of differences in surface porosity between the two substrates. Platinum foil and wires are relatively smooth so that the polymer networks formed by irradiation may not adhere to the surface in all instances. Spectroscopic graphite, on the other hand, is very porous as shown in the scanning electron micrograph (SEM) shown in Figure 3. Pores ranging in size from < I Km to up to 10 Km are visible. SEM’s of platinum foil or wire a t the same magnification were essentially featureless due to the smoothness of those surfaces relative to the graphite used. Figure 4 is an SEM of an unswollen DMDAAC film on a graphite electrode as viewed from the side. The dry polymer is estimated to be up to 100 Km thick and is uniform in appearance. When placed in supporting electrolyte, the polymer swells, fiiing the pores in the graphite. It is possible that this aids in tightly anchoring the DMDAAC to the graphite electrode surface compared to the smoother platinum surfaces. Electrode Response as a Function of Monomer Added. The above results clearly show that monomer added to polymer is incorporated into the network formed by y irradiation. Thus, the degree of “cross-linking” of the polymer chains by monomer units should vary with the monomerpolymer ratio, which should affect film permeability. This
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ANALYTICAL CHEMISTRY. VOL. 60. NO. 22. NOVEMBER IS. 1988
noun 5.
RelaHve response of nwdiiied e W & h 2 mM hemamineIulbnium(Il1)chbrk%. 0.5 M NaCI. Scan rate 20 mV/s. F W e 3. SEM of
Lhe surface of an unmodified specb-osmpiC F a p i e electrode. original magnification SOOOX.
1
.
10
a
b
i o i oX MONOMER b i oADOEO i o m h b
~I~IN 6. Nonnalred swo*m MLmes of hadated POhpMDAAC fitns
in 0.5 M NaCI.
ngue 4. SEM of a &y pdynmr film on a mphiie eIemcd.3 as viewed from Ihe side, original magnification 100X. effect was studied by the cyclic voltammetric response to hexaamineruthenium(II1) chloride. Electrodes were prepared containing between 0 and 80% added DMDAAC monomer (all the polymer films contained the 2-4% monomer present in the purchased DMDAAC polymer in addition to the added monomer) and irradiated at 8.2 Mrad. At this high a dosage it has been shown that all of the monomer present in the film has been polymerized. The electrodes were soaked in supporting electrolyte for 24 h a n d then placed in a solution containing 2 mM hexaamineruthenium(1II)chloride. The electrode response w&s evaluated by obtaining a cyclic voltammogram after 5 min in solution. The cathodic peak currents observed for the reduction of the ruthenium complex as a function of the percent of monomer added are shown in Figure 5. As monomer content is increased. a decreased peak current is initially 0bse"I. At greater than 20% monomer, the trend is reversed and an increased peak current is observed. These data are explained by examining the degree of swelling of the polymer as a function of the initial monomer content. While differences in swelling of the DMDAAC polymer on the electrode (ranging from barely visible to several millimeters thick) were visually observable by the naked eye. these differences could not be easily quantified. Instead, the swelling characteristies of bulk polymer/monomer films were investigated after irradiation. The volumes of the swollen, irradiated polymer filmwere obtained as described above and are shown in Figure 6. The
extent of network swelling goeg through a minimum a t a film composition of 20% monomer with increased swelling above 20%. When the amount of monomer added is small, the monomer polymerizes and may graft onto the DMDAAC polymer or become physically entangled with the polymer resulting in a dense, less swollen fh. As the percent monomer increases (and therefore percent polymer decreases), there is possibly insufficient polymer for grafting or entanglement upon irradiation and a less dense, more swollen polymer film results. The common shape of the cyclic voltammetric response curve and the polymer volume curve indicates a relationship between the mass transport of the ruthenium complex through the polymer network (response) and the polymer film density (swollen volume). This characteristic is essentially a measurement of the response time of the electrodes and is especially important for applications involving non-steady-state measurements such as high-performance liquid chromatography or flow-injection analysis. Size Exclusion as a Function of Monomer Added. Electrode response is largely dictated by the diffusion of analyte through the irradiated polymer network. Large molecules such as vitamin B,, (molecular weight 1355) should therefore exhibit a reduced response relative to smaller molecules. Square wave voltammograms of vitamin BIZand 1,4-benzoquinone(molecular weight 108) are shown in Figure 7 at a nonmodified electrode as well as a t modified electrodes prepared with 80% and 20% monomer, respectively, prior to irradiation. At the nonmodified electrode the vitamin BIZresponse is twice that of 1,4benzoquinone, as determined by peak height measurements. At both modified electrodes all signals are attenuated but the degree of attenuation is different for the
ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
2471
Table I. Electrode Response as a Function of Added DMDAAC Monomer
electrode coating
visual observation
normalized response" % of control BQ/Bl2 BQ B12
controlb 0.5 100 100 80% monomer very swollen 1.4 70 23 20% monomer less swollen 2.5 47 10 a Background subtracted, square-wave voltammetric peak currents. b Bare, unirradiated graphite electrode. two compounds. These data are summarized in Table I. At the 20% monomer electrode, which was visually observed to be less swollen than the 80% monomer electrode, a factor of 10 decrease was observed for the vitamin B12signal while only a factor of 2 difference in the 1,Cbenzoquinone signal was observed. This net change of a factor of 5 in selectivity for these two species compares to a factor of 3 change at the more swollen 80% monomer electrode. This characteristic not only is important as a means to differentiate between redox species of differing sizes but may also be important in the prevention of fouling of electrodes in biological systems. Many electrochemical-based biosensors suffer from diminished response due to electrode fouling caused by blockage of the electrode surface due to proteins and other macromolecules present in biological media. The irradiated polyDMDAAC coating may protect the electrode from these large molecules while smaller analytes of interest can still diffuse through the polymer to the electrode surface. Previously, polyDMDAAC has been shown to prevent the fouling effect of gelatin on cadmium peak stripping currents at mercury film electrodes (23). Work is currently under way to investigate application of this feature of polyDMDAAC electrodes with biological systems. Entrapment of Macromolecules in Irradiated Polymer/Monomer Films. The development of procedures for the immobilization of complex biological macromolecules such as enzymes and antibodies onto electrode surfaces for biosensors is an active area of research (24). A general strategy has been developed that uses monomer-free polyDMDAAC as a first step in investigating the feasibility of using the polymerization of monomers by y irradiation as a means of immobilizing macromolecules onto electrode surfaces. Attempts to immobilize monomer-free polyDMDAAC (no vinyl signals observed in 'H NMR spectrum) by y irradiation have been unsuccessful. Therefore, it was chosen as a representative macromolecule (reported molecular weight 250 000) and attempts were made to immobilize it through irradiation in a film of poly(N-vinylpyrrolidone)/N-vinylpyrrolidone (PNVP/NVP). PNVP/NVP is a polymer system that can be immobilized onto graphite electrodes by use of y irradiation. Further, in contrast to polyDMDAAC, the electrochemical response for ferricyanide is attenuated a t electrodes modified with this polymer. The successful immobilization of polyDMDAAC in this polymer system can therefore be detected by the charge-trapping characteristics of polyDMDAAC. The methodology for immobilization was simply to mix the POlyDMDAAC with the PNVP/NVP prior to irradiation. Upon irradiation the NVP polymerizes forming an immobilized PNVP gel in which polyDMDAAC is entrapped. Figure 8 shows steady-state voltammograms of ferricyanide a t irradiated electrodes modified with PNVP/NVP, monomer-free polyDMDAAC, and monomer-free polyDMDAAC/PNVP/NVP, respectively. The PNVP/NVP electrode shows a slight decrease in response relative to an unmodified electrode while the polyDMDAAC electrode is essentially
€[VOLT) Figure 7. Square wave voltammograms of 0.3 mM 1,4-benzoquinone (EP= 0.0 V) and 1.0 mM vitamin B,2 (EP= 0.8 V) in 0.5 M NaCI: bare electrode (top):80% monomer, 12 Mrad (middle);and 20% monomer, 12 Mrad (bottom).
unchanged. In contrast, a large increase in response is observed a t the polyDMDAAC/PNVP/NVP electrode shown at the bottom of Figure 8. Further, when these electrodes were placed into supporting electrolyte, charge trapping of the ferricyanide was only observed with the polyDMDAAC/ PNVP/NVP electrode indicating the immobilization of the DMDAAC moiety with the PNVP/NVP gel. While the exact nature of the coimmobilization product is not known at this time, it is demonstrated that the product retains the charge-trapping ability of the polyDMDAAC. The peculiar peak shape of voltammograms at this electrode is attributed to differing levels of interactions between the ferri-/ferrocyanide and the mixed polymer system formed. The immobilization may involve chemical grafting of POlyDMDAAC with the PNVP/NVP or entrapment due to physical entanglement with the PNVP/NVP. In either case, these experiments demonstrate the utility of using y irradiation of polymer/monomer films as an effective means of immobilizing macromolecules onto electrode surfaces. Ex-
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
diation, the polymer’s swelling characteristics and thus the resultant monitored electrode response can be controlled. This has led to the fabrication of electrodes with controllable size exclusion characteristics. Finally, the utility of this immobilization procedure for the entrapment of macromolecules has been demonstrated. ACKNOWLEDGMENT The authors thank the Merrell Dow Research Institute, Cincinnati, for use of their NMR and SEM facilities. LITERATURE CITED
P N V P \ N V p \ ( N O
I
+0.6
I
0.4
1
0.2
D M O A A C M O N O M E R )
I
1
0.0
-0.2
Flgure 8. Steady-state cyclic voltammograms in 2.0 m M ferricyanide, 0.5 M NaCI, 50 mV/s.
tension of this concept to the immobilization of radiationresistant enzymes and antibodies is currently being investigated in this laboratory. CONCLUSIONS PolyDMDAAC has been successfully immobilized onto graphite electrode surfaces. The immobilization procedure is a function of the polymerization of monomer using y irradiation in the presence of the polymer to form a water-insoluble network. Anchoring of this network onto the graphite electrode surface is aided by the porosity of the graphite used. By control of the amount of monomer present prior to irra-
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RECEIVED for review March 29, 1988. Accepted August 22, 1988. This work was supported in part by the Army Research Office, Grant No. DAAG29-82-K-0161,and the Edison Sensor Technology Center.
