Anal. Chem. 1997, 69, 1904-1908
Organic Phase Enzyme Electrodes Based on Organohydrogel Yizhu Guo and Shaojun Dong*
Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China
A dimethylformamide-polyhydroxyl cellulose organohydrogel has been prepared, and its applications for enzyme immobilization in construction of organic phase biosensors have been exploited. With horseradish peroxidase, tyrosinase, and bilirubin oxidase immobilized in the organohydrogel, enzyme electrodes can be operated in various situations, including aqueous buffer, oil/water mixtures, and anhydrous organic solvents, and even in dimethylformamide, to determine analytes of different solubilities, e.g., organic peroxides, phenolic compounds and bilirubin. Biosensing has no restrictions in terms of measuring media and solubilities of analytes. Biocatalysis in organic medium has recently attracted considerable interest in enzymology, biosyntheses and biosensing.1-5 Biosensing with electrodes has generally been based on the measurement of analytes soluble in aqueous solutions. Many important substrates that are poorly soluble or insolube in water are inaccessible to biosensing. In 1988, an amperometric tyrosinase electrode was developed for the determination of phenols in chloroform.6 Since then, organic phase enzyme electrodes (OPEEs) have attracted much attention,7-13 due to some distinct advantages, including the ability to monitor hydrophobic substrates, elimination of microbial contamination, reduction of side reactions, and enhanced thermal stability. OPEEs show promising applications in organic synthesis, biocatalytic processes, environmental monitoring, drug analysis, etc. A variety of enzymes have been employed to construct OPEEs for measurement of analytes in nonaqueous media. However, future development is challenging: since enzymes immobilized on OPEEs are confronted with more hostile situations in nonaqueous media, they must retain an “essential hydration layer” for their biocatalytic activity. Handicapped by the enzyme immobilization methods so far (1) Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114-118. (2) Klibanov, A. M. CHEMTECH 1986, 16, 354-359. (3) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 3194-3201. (4) Ryu, K.; Dordick, D. J. Biochemistry 1992, 31, 2588-2598. (5) Saini, S.; Hall, G. F.; Downs, M. E. A.; Turner, A. P. F. Anal. Chim. Acta 1991, 245, 1-15. (6) Hall, G. F.; Best, D. J.; Turner, A. P. F. Enzyme Microb. Technol. 1988, 10, 543-546. (7) Schubert, F.; Saini, S.; Turner, A. P. F.; Scheller, F. Sens. Actuators B 1992, 7, 408-411. (8) Wang, J.; Lin, Y. Anal. Lett. 1993, 26, 197-207. (9) Hall, G. F.; Turner, A. P. F. Anal. Lett. 1991, 24, 1375-1378. (10) Wang, J.; Reviejo, A. J. Anal. Chem. 1993, 65, 845-847. (11) Iwuoha, E. I.; Smyth, M. R. Anal. Proc. 1994, 33, 19-210. (12) Iwuoha, E. I.; Smyth, M. R.; Lyons, M. P. J. Electroanal. Chem. 1995, 390, 35-45. (13) Campanella, L.; Fortuney, A.; Sammartino, M. P.; Tomassetti, M. Talanta 1994, 41, 1397-1440.
1904 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
adoptedsways used for traditional aqueous biosensors, e.g., physical adsorption, cross-linking with bovine serum albumin by glutaraldehyde, and entrapment in Nafion or Eastman AQ matrix; no OPEEs reported previously were actually operated in pure organic media, some amount of water must be added to the organic solvents, which is inconvenient and causes problems in many operations, especially for determination and monitoring in situ and on line. In view of the special requirements for OPEEs, we have developed a novel enzyme immobilization method.14-16 By immobilizing enzymes in cryohydrogel, OPEEs thus prepared can be applied in various situations, including water-free organic solvents. Due to the high hydrophilicity of the cryohydrogel, the cryoimmobilization method has been demonstrated to be feasible only for water-soluble analytes, e.g., hydrogen peroxide and phenols, and unworkable for hydrophobic substances, e.g., organic peroxides, bilirubin and cholesterol. Using dimethylformamide (DMF), the so-called universal organic solvent,17 we have prepared an organohydrogel; enzymes immobilized in the organohydrogel can retain their biocatalytic activity. This enzyme immobilization method possesses extra advantages over the traditional methods: (a) it provides an aqueous microenvironment for the immobilized enzymes; (b) it has high partition coefficients for analytes to obtain high measuring sensitivity; and (c) it is stable in various solvents. This method has been demonstrated to be a universal method for construction of OPEEs. OPEEs thus prepared can be applied in various situations and different analytes. EXPERIMENTAL SECTION Materials. Horseradish peroxidase (HRP, EC 1.11.1.7, 90 units/mg), tyrosinase (EC 1.14.18.1, 4200 units/mg), and bilirubin oxidase (EC 1.3.3.5, 21 units/mg) were purchased from Sigma. Organic peroxides, phenols, and bilirubin were from Aldrich. Ferrocene carboxylic acid was from Fluka. All organic solvents were distilled and desiccated before use (with a maximum water content of 0.001%). Polyhydroxyl cellulose (PHC) was prepared by mixing 60-90% (by weight) poly(vinyl alcohol) with 10-40% carboxymethyl hydroxyethyl cellulose (CMHEC). Poly(vinyl alcohol) was PVA-217 (A.R., made in China). CMHEC was prepared in our laboratory.14 Electrode Preparation. The graphite and glassy carbon electrodes were polished on wet, fine emery paper, ultrasonicated in deionized water and acetone successively, and then allowed to (14) Dong, S. J.; Guo, Y. Z. Anal. Chem. 1994, 66, 3895-3899. (15) Dong, S. J.; Guo, Y. Z. J. Electroanal. Chem. 1994, 375, 405-407. (16) Dong, S. J.; Guo, Y. Z. J. Chem. Soc., Chem. Commun. 1995, 483-484. (17) The Merck Index, 10th ed.; Merck: Rahway, NJ, 1983; p 473, entry no. 3237. S0003-2700(96)00470-2 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Gelatification diagram of the DMF-PHC-water system. (a) Gelated within 24 h; (b) precipitated; (c) kept in solution.
dry at room temperature. For preparation of enzyme electrodes, certain amounts of enzymes were dissolved in 100 µL of 10% PHC aqueous solution and then mixed with 300 µL of DMF. An aliquot of the mixture was spread over the graphite (5-mm diameter for HRP electrode) or glassy carbon (3-mm diameter for tyrosinase and bilirubin oxidase electrodes) electrode surface. The mixture gelled within 20 min at room temperature. The enzyme electrodes were kept in a refrigerator when not in use. Electrochemical Measurements. Amperometric and voltammetric measurements were performed at room temperature with a conventional three-electrode system. The glass cell covered with a PTFE lid was kept in a ventilated closet for safety considerations. A Pt plate and Ag/AgCl (saturated aqueous KCl) were used as the counter and reference electrodes, respectively. The electrolyte solutions contained 0.1 M tetrabutylammonium perchlorate (TBAP). No water or aqueous buffer was deliberately added into the electrolyte solutions. RESULTS AND DISCUSSION Activity of Enzymes Immobilized in DMF Organohydrogel. Figure 1 shows the gelatification diagram of the DMF-PHCwater system at room temperature: a denotes systems gelated within 24 h, b denotes systems kept in solution for 24 h, and c denotes systems in which PHC precipitated. As can be seen from the diagram, systems with higher water content and lower PHC content or systems with lower DMF content would not be gelated within 24 h; systems with lower water content and higher PHC content separate into two phases and PHC precipitates; only systems with high DMF content and suitable PHC-water compositions would be gelated within 24 h. Meanwhile, the composition of the systems had an effect on the gelation rate, some additives would accelerate or delay the gelatification process. The gelatification of the system might be the synergistic result of hydrogen bonding between solvents and polymer molecules and crystallization of polymer. The DMF organohydrogel showed a moderate mechanic strength (>105 Pa) and relatively high stability in various solvents. (By dipping pieces of organohydrogel in various solvents over a 48 h period, compared with cryohydrogel, the organohydrogel was considered to be stable when no visually obvious dissolution nor shrinkage nor swelling occurred.) For biosensing, the enzyme immobilization matrix should have good solubility or high partition coefficients for analytes, both hydrophilic and hydrophobic, to obtain enough sensitivity. For biocatalysis in organic media,4,8 the hydrophobicity of the matrix should not be high, which causes significant ground-state stabilization of substrates and decreases the catalytic efficiency. From
Figure 2. Steady-state response of 2-butanone peroxide (b1) and tert-butyl hydroperoxide (b2) at the HRP-DMF organohydrogelmodified electrode (b1, b2) and the HRP-free organohydrogelmodified electrode (a) in chloroform. Applied potential: -200 mV. Each injection used 0.5 mM organic peroxides.
the analyses above, DMF, the universal organic solvent with log P ) 0.86, seems to be the best choice for the OPEEs matrix. From the gelatification diagram, the conditions for the DMF (60-90%)PHC-water systems have been optimized. However, can enzymes immobilized in the DMF organohydrogel retain their catalytic activity? After HRP, tyrosinase, and bilirubin oxidase were immobilized in the DMF organohydrogel, their catalytic activities were observed by electrochemical monitoring. Polyols were reported to stabilize enzymes due to preferential hydration of enzymes in polyol-water mixtures.19-21 In DMF organohydrogel, enzymes should be protected by PHC. Organic Phase Horseradish Peroxidase Electrode for Organic Peroxides. The HRP cryohydrogel-modified electrodes failed to detect organic peroxides, e.g., tert-butyl hydroperoxide, 2-butanone peroxide, and tert-butyl peroxybenzoate, in organic media due to the poor solubility of these peroxides in aqueous cryohydrogel. However, with immobilization of HRP in DMF organohydrogel, these organic peroxides have been determined mediatorlessly (no artificial mediators have been deliberately added) and regentlessly in pure organic solvents. Figure 2 shows the typical response of an HRP-DMF organohydrogel-modified electrode polarized at -200 mV in chloroform to successive additions of 2-butanone peroxide and tert-butyl hydroperoxide, and Table 1 demonstrates the response parameters. The enzyme electrode also worked in various solvents, and the response parameters are listed in Table 2. Ryu and Dordick4,18 pointed out that the most important effect of organic solvents on biocatalysis is the significant ground-state stabilization of substrates in organic media as opposed to aqueous buffer, which decreases the partition coefficient of substrates between the microenvironment of enzyme and organic phase, with an increased apparent Michaelis-Menten constant, KMapp. As can be seen in Tables 1 and 2, HRP immobilized in the DMF organohydrogel showed this principle generally. (18) Ryu, K.; Dordick, J. S. J. Am. Chem. Soc. 1989, 111, 8026-8027. (19) Gekko, K.; Morikawa, T. J. Biochem. 1981, 90, 39-50. (20) Gekko, K.; Morikawa, T. J. Biochem. 1981, 90, 51-60. (21) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4667-4676.
Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
1905
Table 1. Response Parameters of Organic Peroxides at the HRP-DMF Organohydrogel-Modified Electrode in Chlorofrom substrate
linear range (M)
detection sensitivity KMapp limit (M) (nA/mM) (mM)
butanone peroxide 8 × 10-6-5 × 10-4 5 × 10-6 butyl hydroperoxide 5 × 10-5-1 × 10-4 2 × 10-5
130 40
2.71 3.0
Table 2. Response Parameters of 2-Butanone Peroxide at the HRP-DMF Organohydrogel-Modified Electrode in Different Solvents solvent
log P
linear range (M)
detection sensitivity KMapp limit (M) (nA/mM) (mM)
butanol 0.88 1 × 10-5-8 × 10-4 5 × 10-6 chloroform 2.0 2 × 10-5-1 × 10-3 1 × 10-5 chlorobenzene 2.84 5 × 10-5-2 × 10-3 3 × 10-5
85 130 50
1.96 2.71 3.51
Figure 4. Cyclic voltammograms of phenol in chloroform at tyrosinase-DMF organohydrogel-modified electrode (a-d) and tyrosinase-free organohydrogel electrode (a′). Phenol concentration: (a) 0, (b) 0.2, (c) 0.4, (d) 0.8, and (a′) 1.0 mM. Sweep rate: 10 mV/s.
Figure 3. Current-time recordings at HRP-DMF organohydrogelmodified electrode upon addition of mercaptoethanol in 0.1 mM concentration steps. Applied potential: -200 mV. Solution: butanol containing 1 mM 2-butanone peroxide.
Many organic compounds are good inhibitors to enzymes.22-25 Taking advantage of the inhibition effect would greatly enlarge the application field of biosensing. Figure 3 shows the inhibition process of the HRP-DMF organohydrogel electrode by mercaptoethanol in butanol. The activity of the enzyme electrode could be restored by a short incubation in the presence of the substrate. From the Scatchard curve, an apparent inhibition constant (Ki′) of 1.4 mM was calculated, slightly lower than literature values (1.7-2.2 mM);23-25 the inhibitor extraction into the gel and the change in the enzyme rate constant due to the change in the environment within the gel might be accounted for by it. Organic Phase Tyrosinase Electrode for Phenolic Compounds. An ultimate goal in the field of gel matrix technology is to produce polymers that can selectively accumulate analytes of interest at transducer surfaces.5 The sensitivity and selectivity of biosensors can be greatly enhanced by utilizing the physico(22) Minh, C. T. Ion-Sel. Electrode Rev. 1985, 7, 41-75. (23) Wang, J.; Dempsey, E.; Eremenko, A. Anal. Chim. Acta 1993, 279, 203208. (24) Adeyoju, O.; Iwuoha, E. I.; Smyth, M. R. Talanta 1994, 41, 1603-1609. (25) Adeyoju, O.; Iwuoha, E. I.; Smyth, M. R. Anal. Lett. 1994, 27, 2071-2081.
1906 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
Figure 5. Cyclic voltammograms of catechol (A: a, 0 mM; b, 0.4 mM) and p-cresol (B: a, 0 mM; b, 1.0 mM) in chloroform at tyrosinase-DMF organohydrogel-modified electrode (a,b) and tyrosinase-free organohydrogel electrode (a′, 1.0 mM substrate). Sweep rate: 10 mV/s.
chemical characteristics of analytes. Polyphenol oxidase (or tyrosinase) can catalyze the hydroxylation of various phenols to catechol and their subsequent dehydrogenation to o-quinone. The application of different solubilities and hydrophobicities can facilitate the determination of different phenols by optimization of detection conditions. Figure 4 shows the catalytic process of phenol at the tyrosinase-DMF organohydrogel-modified electrode in chloroform. A sensitive response was obtained at the enzyme electrode, since the DMF organohydrogel has a high partition coefficient for phenol and phenol can be extracted and preconcentrated in the enzyme membrane. However, no such obvious catalytic waves were observed at the tyrosinase cryohydrogel-modified electrode due to the low solubility of phenol in an aqueous phase. Similar observations for p-cresol and catechol are shown in Figure 5. The voltammetric behavior demonstrates that tyrosinase-DMF organohydrogel electrodes have higher sensitivity for phenolic compounds than tyrosinase cryohydrogel electrodes. This is also
Table 3. Comparision of Response Parameters of Substrates at Tyrosinase Organohydrogel Electrode (TOE) and Tyrosinase Cryohydrogel Electrode (TCE) in Chloroform TCE
TOE
substrate
KMapp (mM)
sensitivity (nA/mM)
KMapp (mM)
sensitivity (µA/mM)
catechol p-cresol phenol
0.06 3.5 0.58
23.7 3.9 10.9
0.15 0.506 0.167
55.0 23.1 40.5
Table 4. Comparision of Response Parameters of Substrates at Tyrosinase Organohydrogel Electrode (TOE) and Tyrosinase Cryohydrogel Electrode (TCE) in Phosphate Buffer (pH 7.0) TCE app
TOE app
substrate
KM (mM)
sensitivity (nA/mM)
KM (mM)
sensitivity (µA/mM)
catechol p-cresol phenol
0.7 0.8 1.1
86.9 39.6 23.1
0.216 0.333 0.350
32.6 34.8 27.9
Figure 6. Steady-state responses of various phenolic compounds at the tyrosinase-DMF organohydrogel-modified electrode (b,c,d) and the tyrosinase-free organohydrogel electrode (a) in octanol. Applied potential: -200 mV. Each injection used 0.12 mM substrates.
indicated from the amperometric measurements, as shown in Tables 3 and 4. Figure 6 shows the typical responses of various phenolic compounds at the tyrosinase-DMF organohydrogel electrodes in octanol. The response parameters of different substrates in various solvents are summarized (Tables 5-7) for comparison. From these data, the following conclusions can be drawn: (a) A substrate has different sensitivities and KMapp values in different solvents. (b) Different substrates have different sensitivities and KMapp values in the same solvent. The differences may arise from the ground-state stabilization between substrates and solvents as stated before. Besides, the sources of changes in KMapp may also include a change in the enzyme rate constant due to the change in the microenvironment within the gel, solvent effects on solute
Figure 7. Steady-state response of bilirubin at the bilirubin oxidaseDMF organohydrogel-modified electrode (b) and the enzyme-free organohydrogel electrode (a) in DMF containing 0.25 mM FcOOH. Applied potential: +200 mV.
diffusion coefficient and mass transport, etc. It was also indicated that substrate specificity changed with reaction media. From the comparison of different sensitivities of phenolic compounds in different solvents, as shown in Table 8, we can see that the selectivity and sensitivity of analytes of interest can be controlled and chosen by the selecting appropriate detection media. For example, due to their different hydrophobicities and solubilities, the sensitivity for phenol was almost twice that for p-cresol when the enzyme electrode was operated in chlorobenzene; however, 30 times higher sensitivity for p-cresol than for phenol can be achieved by using ethanol as the detection medium instead of chlorobenzene. Organic Phase Bilirubin Oxidase Electrode for Bilirubin. The distinguishing superiority of an organic phase enzyme electrode is its capability for biosensing of hydrophobic compounds that are poorly solubile or insoluble in aqueous solution. Using cryohydrogel immobilization, we have tried to detect bilirubin in organic solvents but failed, since bilirubin cannot dissolve in the aqueous cryohydrogel. Mediated by ferrocenecarboxylic acid (FcOOH), a sensitive response of bilirubin was obtained at the bilirubin oxidase-DMF organohydrogel-modified electrode in anhydrous dimethylformamide solution. The steady-state response curve is shown in Figure 7. The detection limit was 5 × 10-7 M, with a sensitivity of 20 µA/mM. Since DMF is a very aggressive solvent to the essential hydration layer of enzymes, only subtilisin has been found to be catalytically active in anhydrous dimethylformamide and trypsin in DMF-water mixtures.26,27 However, many enzymes (e.g., HRP, tyrosinase, and bilirubin oxidase) have been demonstrated to be biocatalytically active in DMF organohydrogel in this work, probably due to the PHC protection effect. For practical application, this investigation achieves the ultimate evolution of the detection medium in biosensingsfrom water buffer to water/oil mixtures, to organic solvents saturated with
Table 5. Response Parameters of Catechol at Tyrosinase-DMF Organohydrogel-Modified Electrode in Different Solvents solvent
ethylene dichloride
chlorobenzene
chloroform
octanol
butanol
ethanol
phosphate buffer (pH 7)
KMapp (mM) sensitivity (µA/mM) linear range (mM)
0.10 74.6 0.04
0.139 62.8 0.06
0.15 55.0 0.08
0.361 24.7 0.14
0.41 10.05 0.30
0.733 6.95 0.35
0.216 32.6 0.15
Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
1907
Table 6. Response Parameters of p-Cresol at Tyrosinase-DMF Organohydrogel-Modified Electrode in Different Solvents
Table 8. Sensitivity (µA/mM) of Tyrosinase Organohydrogel Electrodes for Different Substrates in Various Detection Media
solvent chloro- chlorobenzene form octanol ethanol KMapp (mM) sensitivity (µA/mM) linear range (mM)
0.765 17.93 0.48
0.506 23.1 0.17
0.067 14.62 0.05
0.414 17.7 0.20
phosphate buffer (pH 7) 0.333 34.8 0.10
Table 7. Response Parameters of Phenol at Tyrosinase-DMF Organohydrogel-Modified Electrode in Different Solvents solvent chloro- chlorobenzene form octanol ethanol KMapp (mM) sensitivity (µA/mM) linear range (mM)
0.265 31.0 0.5
0.167 40.5 0.09
0.275 7.4 0.18
phosphate buffer (pH 7)
0.6
0.35 27.9 0.13
water, to anhydrous organic solvents, and finally to anhydrous dimethylformamide, the universal organic solvent. Meanwhile, the analytes vary from water-soluble to poorly soluble and to insoluble, hydrophobic compounds. Furthermore, as is demonstrated above, the DMF organohydrogel immobilization makes it possible to construct biosensors of many types: mediatorless (direct electron transfer between enzyme and electrode), with a (26) Riva, S.; Chopineau, J.; Kieboom, A. P. G.; Klibanov, A. M. J. Am. Chem. Soc. 1988, 110, 584-589. (27) Guinn, R. M.; Blanch, H. W.; Clark, D. S. Enzyme Microb. Technol. 1991, 13, 320-326.
1908 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
catechol
p-cresol
phenol
62.8 55.0 24.7 6.95 32.6
17.93 23.1 14.62 17.7 34.8
31.0 40.5 7.4 0.6 27.9
chlorobenzene chloroform octanol ethanol phosphate buffer
mediator, and a type of detection of the electroactive product of an enzymatic reaction. Therefore, biosensing can be applied in various situations (aqueous buffer, water/oil mixtures, and anhydrous organic solvents) and for analytes of different physicochemical properties. To summarize, an effective enzyme immobilization method has been developed for the construction of organic phase enzyme electrodes; the enzyme-DMF organohydrogel electrodes are applicable to analytes of different solubilities in various situations. ACKNOWLEDGMENT Financial support from the National Natural Science Foundation of China and the helpful suggestion from one of the reviewers are greatly appreciated.
Received for review May 9, 1996. Accepted December 19, 1996.X AC9604705 X
Abstract published in Advance ACS Abstracts, February 1, 1997.
