Anal. Chem. 1994,66, 3895-3899
Organic Phase Enzyme Electrode Operated in Water-Free Solvents Shaojun Dong' and Yithu Guo
Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China
A new material, polyhydroxyl cellulose, and a refrigerating immobilization method were used to construct HRP-mediator electrode for determination of hydrogen peroxide in waterfree organic solvents. Rapid and sensitive response was obtained. The enzyme electrode had a prolonged lifetime of at least 2 months. A stable and high catalytic activity could be retained for the enzyme immobilized in this way. The material and immobilization described offer a new and effective way for enzyme stabilization in biosensor fabrication, especially for organic phase enzyme electrodes, which suffer a more hostile and harsh environment. Since the introduction of the first enzyme electrode over 30 years ago, growing interest in biosensor development has resulted in increasingly widespread applications. However, the applications for biosensors are currently limited to the measurement of analytes soluble in aqueous solutions to prevent denaturation of the protein.' Enzymatic assays in organic phases are recently gaining an increasing interest.*-" Organic phase biocatalytic sensing possesses distinct advantages, including monitoring of hydrophobic substrates, elimination of microbial contamination, reduction of side reactions, enhanced thermostability, and relative ease of enzyme immobilization based on their insolubility in organic solvents. Organic phase enzyme electrodes (OPEEs) are expected to facilitate biomonitoring in many industrial and environmental applications and in biotechnological processes. Thus, a variety of enzymes5-" have been used to construct OPEEs for measurement of analytes in nonaqueous phases including chloroform, chlorobenzene, dioxane, and alcohols. However, these "nonaqueous phases" were not actually water-free organic solvents, but had deliberately and previously been saturated with aqueous buffer or mixed with certain amounts of water prior to use. ( I ) Saini, S.;Hall, G. F.; Downs, M. E. A,; Turner, A. P. F. Anal. Chim.Acta 1991, 246, 1. (2) Danielsson, B.; Flygare, L.;Velev, T. Anal. Lett. 1989, 22, 1417. (3) Wang, J.; Wu, L.; Angnes, L. Anal. Chem. 1991, 63, 2993. (4) Wang, J.; Lin, Y . Anal. Chim.Acta 1993, 271, 53. (5) Schubert, F.; Saini, S.;Turner, A. P. F. Anal. Chim.Acta 1991, 245, 133. ( 6 ) Miyabayashi, A.; Reslow, M.; Adlereutz, P.; Mattiasson, B. Anal. Chim. Acta 1989, 219, 27. (7) Wang, J.; Naser, N.; K won, H. S.;Cho, M. Y .Anal. Chim. Acta 1992,264, 7. ( 8 ) Hall, G. F.; Best, D. J.; Turner, A. P. F.; Scheller, F. Enzyme Microb. Technol. 1988, IO, 543. (9) Schubert, F.; Saini, S.;Turner, A. P. F. Sens. Actuators B 1992, 7 , 408. (IO) Hall, G. F.; Tuner, A. P. F. Anal. Lett. 1991, 24, 1375. (11) Wang, J.; Lin, Y . Anal. Lett. 1993, 26, 197.
0003-2700~94~0366-3895$04.50/0 0 1994 American Chemical Society
Strictly, it is impossible for enzymes to retain catalytic activity in a water-free environment. Enzymes on the OPEEs must retain a thin aqueous film, essential for their catalytically active conformation in organic media. However, in organic solvents, especially in water-miscible solvents, the essential water layer is easily distorted or dehydrated, and the enzyme is readily deactivated. In order to provide and retain the essential hydration layer for enzymes immobilized (generally adsorbed) on OPEEs, water has been added to the organic solvents prior to organic phase analysis. Therefore, the OPEEs previously reported5-" were not operated in absolute nonaqueous media, which causes inconvenience and problems in many actual operations, especially for determination and monitoring in situ or on line. The development of OPEEs is at its transition stage, during which they are operated in mixtures of water and organic solvents. Now we are coming to the turning point. The utility of a new material and an immobilization method enables the OPEEs to be operated in water-free organic solvents, while the immobilization material itself provides a suitable aqueous microenvironment for the enzyme. We have prepared a polyhydroxyl cellulose (PHC) whose aqueous solution can be refrigerated to form a hydrogel. This cryohydrogel can retain its water molecules in organic solvents. A reagentless enzymemediator ~ y s t e mwhere , ~ the mediated amperometric enzyme electrode incorporated horseradish peroxidase (HRP) for the determination of hydrogen peroxide in organic solvents, was chosen to demonstrate the feasibility and workability of the cryohydrogel for constructing OPEEs.
EXPERIMENTAL SECTION Materials. Horseradish peroxidase (HRP; EC 1.1 1.1.7, 90 units/mg) was purchased from Sigma. The following organic solvents were used: chlorobenzene, chloroform, ethylene dichloride, acetonitrile, dioxane, methanol, cyclohexane, ethanol, tetrahydrofuran, N,N-dimethylformamide (all Analytical Reagent Grade, and made in China). They were desiccated before use with a maximum water content of 0.001%. Hydrogen peroxide was a 30% solution (A.R., made in China). Polyhydroxy1 cellulose (PHC) was prepard by mixing 6 6 9 0 % (by weight) poly(viny1 alcohol) (PVA) with 1 6 4 0 % carboxymethyl hydroxyethyl cellulose (CMHEC). Poly(viny1 alcohol) was PVA-217 (A.R., made in China). CMHEC was prepared in our laboratory. Analytical Chemistry, Vol. 66,No. 22, November 15, 1994 3895
Electrode Preparation. The graphite electrode was prepared by inserting a spectrographic graphite rod (5-mm diameter) into a Teflon shrinking tube. The graphite electrode was polished on wet, fine emergy paper, ultrasonicated in deionzed water and acetone, successively, and then allowed to dry at room temperature. For preparation of the enzymemediator electrode, 3 mg of H R P were dissolved in 200 p L of 10% PHC solution (containing 0.01 M potassium hexacyanoferrate(I1)). An aliquot (20 pL) of the mixture was spread over the graphite electrode surface. The electrode was then refrigerated at -4 to -10 OC for 12-24 h and stored at 4 OC when not in use. The enzyme electrode was rehydrated with 10 pL of water and dried in ambient atmosphere for 0.5 h prior to use. Electrochemical Measurements. Amperometric measurements were performed at room temperature with a conventional three-electrode system with continuous stirring. The three-electrode system was similar to that described previ0us1y.~ The glass cell well covered with a PTFE lid was installed in a ventilating closet for safety consideration, since chloroform and DMF are suspect carcinogens. A Pt plate and Ag/AgCl (salurated aqueous KC1) were used as the counter electrode and reference electrode, respectively. Unless otherwise indicated, the electrolyte solutions contained 0.1 M tetrabutylammonium perchlorate (TBAP). No water or buffer was deliberately added into the electrolyte solutions. Hydrogen peroxide standard solution was prepared using acetonitrile. The electrode potential was controlled with a potentiostat, and current was recorded with an X-Y(-t) recorder as a function of time. RESULTS AND DISCUSSION Properties of the PHC Cryohydrogel. The PHC aqueous solution can be refrigerated to produce a hydrogel; however, this cryohydrogel has properties different from the commonly referred hydrogel (gel swollen by water). It has a semiinterpenetrating network, and its structure can be controlled by the concentration of PHC and the refrigerating history. The hydrogel has a relatively high mechanical strength and adheres tightly to the graphite surface. There are threedifferent states of water in the hydrogel,I2 Le., “nonfreezing” water, “bound” water, and “free” water. The stability of the hydrogel in organic solvents varies with the hydrophilicity of the solvents. It is more stable in water-immiscible solvents than in watermiscible solvents. The cryohydrogel can retain its water molecules to some extent in organic media and can be easily recovered by soaking it in water. Effect of Solvents. The enzyme-mediator electrode was tested with respect to its performance in different organic solvents in which the electrolyte TBAP can readily be dissolved. Table 1 shows that the HRP-mediator electrode operated in water-immiscible solvents has greater responses than in watermiscible solvents. This result is consistent with the stability of the PHC hydrogel in different solvents. The behaviors of the hydrogel in different solvents have been investigated by soaking the hydrogel in organic solvents and weighing it (12) Feng, Y . D.Sichuan Daxue Xuebao. Ziran Kexueban ( J . Sichuan Uniu. Nat. Sci. E d . ) 1989, 26, 470.
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Table 1. Response of the HRP-Mediator Electrode to H202(0.25 mM) in Different Organic Solvents solvent response, nA solvent response, nA
chlorobenzene chloroform ethylene dichloride acetonitrile acetone dioxane
925 740 705 100 100 50
methanol cyclohexane ethanol tetrahydrofuran DMF
34 30 6 5 0
Table 2. Response of the HRP-Medlator Electrode to H202(0.25 mM) in DMF-Chloroform and DMF-AcetonHrlle solvent response, nA solvent response, nA
DMF-chloroform 1:3 1:l 3: 1
160 100 40
DMF-acetonitrile 1:3 1:l 3:l
66 27 5
intermittently. The experimental results demonstrated that the weight of the hydrogel decreased gradually and the hydrogel was more difficult dehydrated in water-immiscible solvents than in water-miscible solvents. An exception was observed for N,N-dimethylformamide (DMF), in which the weight of hydrogel increased first and then decreased, although the DMF molecules should penetrate into the hydrogel readily, while other organic solvents tested would not. This might suggest why the enzyme electrode showed no response in DMF, because DMF molecules readily disturb the essential water layer of the catalytically acth e conformation of the enzyme entrapped in the hydrogel. Though no enzyme activity was observed in pure DMF, responses of the enzyme electrode to hydrogen peroxide were found in DMF-chloroform and DMFacetonitrile mixtures. D M F is an excellent solvent for many analytes, and results in Table 2 show that OPEEs can be operated in mixtures of D M F and other solvents; thus the OPEEs can find a wider range of analytes and applied situations. The enzyme-mediator electrode also has a relatively high response in water-contained organic phases and in aqueous solution. Thisdemonstrates the flexibility of thecryohydrogel immobilization method that will favorably facilitate the application of OPEEs in practical operations, especially monitoring in situ or on line. Response Properties of the HRP-Mediator Electrode. Figure 1 shows the variation of the response of the enzyme electrode with the applied potential. Sensitive response can be obtained in the vicinity of 0.0 V. Figure 2 shows the currenttime curves of hydrogen peroxide responses obtained at the enzyme electrode in chloroform. A stable base current was obtained after an equilibration time of 5-15 min, which is much more rapid than that previously describeds (1-1.5 h) with a shorter response time of 0.5-2 min. The useful measuring range is up to 2.5 mM with a detection limit of 5 x 10-7 M. Figure 3 shows the dependence of the amperometric response of the enzyme-mediator electrode on the concentration of H202 in acetonitrile. Figure 4 shows the calibration plots for H202 in acetonitrile. As can be seen, the enzyme electrode has an even shorter response time (within 10 s) and a wider measuring range (up to 7.5 mM) operated in
2.4.
16-
O , * ! .
-200
,
,
-100
0
L 100
200
J
300
Potential / mV Flgure 1. Dependenceof the response to H202(0.5 mM) of the HRPmediator electrode upon the applied potential. Conditions: solution, chloroform containing 0.1 M TBAP; stirring rate, 250 rpm.
Time Flgure3. Amperometric responseof the graphite electrodeImmobilized with PHC only (a) and HRP-mediator-PHC (b) to successive additions of 5.0 X lo-' M H1O,. Conditions: solution, acetonitrile containing 0.1 M TBAP (b,) or 0.7 M NaCIO, (b2);applied potential, 30 mV, stiring rate, 250 rpm.
/
1.2.
.
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,
u 0.4
0.0
2.0
4.0
6.0
8.0
Concentration / mM
a
1.0 2.0 3.0 Concentration I mM
Time Flgure2. Amperometric response of the graphite electrodeimmobilized with PHC only (a) and HRP-mediator-PHC (b) to successive additions of 3.33 X loJ M H202. Also shown (inset) is the calibration plot. Conditions: solution, chloroform containing 0.1 M TBAP; applied potential, 4-30 mV; stirring rate, 250 rpm.
acetonitrile than in chloroform, while the sensitivity decreased from 2.96 mA/mM H202 in chloroform to 0.4 mA/mM in acetonitrile. This might be due to the solubility of H202 in different solvents. The H202 standard solution is much more miscible with acetonitrile than with chloroform; therefore, in acetonitrile, equilibration is more readily reached after injection of H202 standard solution, and the response time is shorter. The improved measuring range and decreased sensitivity could be interpreted in the same way. In fact, there exists a partition coefficient of H202 in the two-phase (organic solvent-water in hydrogel) system. The solubility of H202 in water is much higher than in chloroform, so there is an extraction and concentration effect in the waterchloroform system. The concentration of H202 is much higher in water contained in the hydrogel than in the chloroform background solution. Thus, very low detection limits can be
Flgure 4. Dependence of the amperometric response upon the concentration of H202in acetonitrile. Conditions as in Flgure 3.
obtained in chloroform. The solubility of H202 in water and acetonitrile, however, is similar, so the concentrations of Hz02 in hydrogel and in background solution, acetonitrile, are almost the same when the equilibration is reached. In fact, the sensitivity of the enzyme electrodeoperated in aqueous solution is 0.54 mA/mM H202. Therefore, in chloroform, the enzyme electrode has a much higher sensitivity and is more readily saturated with a narrow measuring range. In previous work5-I1 no activity was found for OPEEs in water-free solvents unless the solvents were mixed with certain amounts of water. However, the enzyme electrodes prepared in this way have excellent responses in water-free organic media. This is an innovation for the OPEEs. Effect of Electrolyte. The electrolytes generally used for OPEEs are expensive synthesized organic compounds, such as tetraethylammonium p-toluene sulfonate (TEATS) or TBAP. Two methods have been tried to reduce the costs of OPEEs in practical operations. The first involves reducing the amounts of organic salts, and the second involves the use of less expensive inorganic salts. The effect of the concentration of the organic salt TBAP on the response shows that the sensitivity decreased from 2.96 to 2.16 and to 0.04 mA/mM H202 when the concentration of TBAP was reduced from 0.1 Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
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6.0,
f i
25
35
45
55
65
75
Temperature / “C Figure 5. Temperature effect on the amperometric response to H202 (0.5 mM): (a) The enzyme-mediator modified electrode operated In chloroform. Conditionsas In Figure 2. The dots (e)denote operations after triangles(A). (b) A bare graphite electrode operatedin phosphate buffer(0.1M, pH6.98)withdlssolvedHRPandmediator. OthercondAions as in (a).
to 0.01 and to 0.001 M. At the same time the measuring range became more narrrow. Therefore, reducing the amounts of organic electrolyte is not acceptable. On the other hand, some inorganic electrolytes, such as LiC104 and NaC104, can dissolve in acetonitrile and DMF. Figure 3 shows that there is small difference in the responses of the enzyme electrode to H202 in acetonitrile containing 0.1 M TBAP or NaC104; however, the sensitivity, Le., the response slope in the linear range, with NaC104 (0.22 pA/mM H202) is about 50% lower than with TBAP (0.40 pA/mM H202). The dection limit is 4.0 X 10“ M with NaC104 and 7.0 X 10“ M with TBAP. This is the first time inorganic electrolytes have been used for OPEEs. Inorganic electrolytes are an economical and convenient alternative because they are generally much cheaper and easier available.
backbone of the hydrogel. The “elastically locked” catalytically active conformation could thus not easily be disturbed, and the immobilized enzyme would not readily be deactivated. Lifetime of the Enzyme Electrode. Compared with adsorption immobilization,5 the cryohydrogel immobilization has a distinct improvement on the enzyme electrode stability or the lifetime. The sensitivity of Schubert’s electrode decreased to 40% of its initial value in 2 weeks; however, no obvious deterioration in the sensing properties was observed for our enzyme electrode in the same period. The cryohydrogel enzyme electrode was used daily for 2 months, and the electrode retained a sensitivity of 60% of its initial value. This may be attributed to two aspects: On one hand, because H R P and mediator are physically and chemically entrapped in the threedimensional interpenetrating network and are insoluble in water-free organic solvents, they would not readily leach out of the hydrogel that adheres tightly to the graphite electrode surface. On the other hand, the hydrogel itself provides an appropriate aqueous microenvironment for the enzyme to maintain its activity. It was reported that14 chymotrypsin lyophilized from aqueous solution containing certain ligands had an enhanced activity in organic solvents because of the ligand “locking” the enzyme into a more active conformation. Also polyhydroxyl compounds‘ when dried with enzymes tend to stabilize activity because of hydroxyl groups substituted for the “bound” water essential for the retention of the tertiary structure of the protein and the subsequent activity of the molecule. In this paper, during electrode preparation, the enzyme experienced processing somewhat similar to lyophilization. During the refrigeration process, the interpenetrating network formed gradually, and the active conformation of the enzyme in aqueous solution was maintained in the hydrogel due to its interaction with the polyhydroxyl compound. Therefore, the enzyme immobilized on the electrode has and can retain a relatively high and stable catalytic activity.
Enhanced Thermostability. Curve a in Figure 5 shows the temperature effect on current responses of the enzyme electrode to H202 in chloroform. It can be noted that the OPEEs demonstrated an increasing response from 10 to 45 OC,and the response was greatly enhanced at temperature greater than 45 “C. Furthermore, following operations at high temperatures, the enzyme electrode still maintained a similar response without obvious deactivation. It was shown previously that the OPEEs behaved similarly in aqueous solution.13 Curve b shows the temperature effect on current response of the bare graphite electrode. The responses were obtained with free H R P and mediator in aqueous buffer. As can be seen, the current increased gradually with temperature up to 60 OC and then the current decreased. The results imply that enhanced thermal stability could be obtained for an enzyme electrode prepared in this way. The enzyme molecules immobilized in the cryohydrogel may by “locked” by hydrogen bondings, formed between the carboxyl groups, amino groups, and phenol groups of the enzyme molecules and the carboxyl groups and hydroxyl groups on the polymer
One of the most important problems in development of a commercial biosensor is the stabilization of the enzymes. Although many different biosensors have been constructed, few have been developed in commercially successful instruments except the glucose biosensor, because glucose oxidase is a very stable enzyme. One of the major problems in expanding the use of enzyme sensors beyond that for glucose has been protein stabilization, especially when it has been necessary to dry and then rehydrate on the transducer’s surface before use.15 The use of the P H C cryohydrogel to construct enzyme electrodes solves the problem simply and effectively. The enzyme electrodes prepared in this way can be conveniently stored in a dry state and simply rehydrated before use. Results reported above imply that the PHC is an effective stabilizer for preserving the enzyme’s catalytic activity and that refrigerating immobilization is a useful method for keeping the catalytically active conformation of the enzyme. In conclusion, the above results demonstrate that the application of polyhydroxyl cellulose and refrigerating immobilization for constructing organic phase enzyme electrodes is very promising. Rapid and sensitive responses can thus be
(13) Dong, S.J.; Guo, Y . Z.,to be submitted.
(14) Zaks, A,; Klibanov, A. M. J. Bo/. Chem. 1988, 263, 3194
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obtained in organic solvents containing no (deliberately added) water. The enzyme electrodes have a prolonged storage and lifetime. Applicability to other enzymes, substrates, and solvent systems can be easily envisioned. Potential application to commercial biosensors may be reasonably expected. (15) Gibson, T. D.; Woodward, J. R. Biosensors and Chemical Sensors; Edelman, P. G., Wang, J., Eds.; ACS Symposium Series 487; American Chemical Society: Washington, DC, 1992; Chapter 5.
ACKNOWLEDGMENT The financial support of the National Nature Science Foundation of China and the discussion with Ms. Q. Deng are gratefully appreciated. Received for review March 11, 1994. 994."
Accepted August 10,
*Abstract published in Aduance ACS Absrracrs, September 15, 1994.
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