1974
Anal. Chem. 1981, 53, 1974-1979
(24) Pedersen, K. 0. Scand. J . Clin. Lab. Invest. 1972, 29, 75-83. (25) Fogh-Andersen, Niels Clin. Chem. ( Winston-Salem, N.C.) 1977, 23, 2 122-2126. (26) Osswaid, H. F.; Dohner, R. E.; Meier. T.; Meier, P. C.; Simon, W. Chimls 1977, 31, 50-52. (27) Osswald, H. F.; Asper, R.; Dimal, W.; Simon, W. Clin. Chem. (Wln@ton-Salem,N.C.) 1979, 25, 39-43. (28) Senkyr, Jaroslav; Ammann, Daniel; Meier, P. C.; Morf, W. E.; Pretsch, Ern& Simon, Wllheim Anal. Chem. 1979, 57, 786-790. (29) Guiibault, G. G.; Durst, R. A.; Frant, M. S.; Freiser, H.; Hansen, E. H.; Llght, T. S.; Pungor, E.; Rechnitz, G.; Rice, N. M.; Rohm, T. J.; Simon, W.; Thomas, J. D. R. Pure Appl. Chem. 1976, 48, 127-132. (30) Moody, G. J.; Thomas, J. D. R. "Selective Ion Sensitive Electrodes"; Merrow Publishing Co. Ltd.: Watford, Herts., England, 1971. (31) Meier, P. C.; Ammann, D.; Osswald, H. F.; Simon, W. Med. Pf08. Technol. 1977, 5. 1-22. (32) Ammann, Daniel; Bissig, Ren6; Guggi, Marc; Pretsch, Ern0 Simon,
(33) (34) (35) (36)
Wilheim; Borowltz, Irving J.; Welss, Louis Helv. Chlm. Acta 1975, 58, 1535- 1548. Horning, E. C. I n "Organic Synthesis"; Wiley: New York, 1955; Vol. 111, p 140. Cassaretto, Frank P.; McLafferty, John J.; Moore, Carl E. Anal. Chlm. Act8 1965, 32, 376-380. Schindler, J. G.; Dennhardt, R.; Simon, W. Chimla 1977, 37, 404-407. Simon, W.; Ammann, D.; Osswald, H. F.; Meier, P. C.; Dohner, R. E. I n Advances in Automated Analysis. Technicon International Congress 1976"; Medlad Inc.: Tarrytown, NY, 1977; Voi. 1.
RECEIVED for review May 26,1981. Accepted July 13, 1981. This work was partly supported by the Swiss National Science Foundation. P.A. thanks RhBne Poulenc as well as Orion Research for a grant.
Ascorbate Electrode for Determination of L-Ascorbic Acid in Food Klyoshi Matsumoto, * Kimlmasa Yamada, and Yutaka Osajlma Department of Food Science and Technology, Faculty of Agriculture, 46-09, Kyushu University, Fukuoka, 8 12, Japan
An amperometric sensor for L-ascorbic acld has been made by lmmobilizlng ascorbate oxldase In the reconsiltuted collagen membrane and mountlng the enzyme-collagen membrane on a Clark oxygen electrode. The enzyme was purlfied from the peel of cucumber, Cucumls salivus. The response of the electrode was h e a r between 5 X IO-' and 5 X IO-' M L-ascorbic acld and the preclslon was found to be better than 2.3% by 35 successlve assays. The only limltatlon Is that the electrode responds for iAsoascorblc acid (about 93% relatlve response). The sensor has a lifetime of 3 weeks. After preparation, buffer Is the only reagent needed. Its usefulness for assay of L-ascorblc acld In food Is also described.
Enzymes have been used widely in the field of analytical chemistry owing to their substrate specificity. Enzyme electrodes combine the electrochemical sensor with the immobilized enzymatic film in compact form ( I , 2). Ascorbate oxidase (EC 1.10.3.3, L-ascorbate:oxygen oxidoreductase) catalyzes the reaction L-ascorbic acid
-
amorbate + 1/202 oxidase
dehydroascorbic acid
+ H 2 0 (1)
and can be employed for the determination of L-ascorbic acid present in aqueous solution. The monitoring of oxygen used up in an enzymatic reaction with an oxygen sensor has been reported (3-8). However, no ascorbate electrode which combines the electrochemical sensor with the immobilized ascorbate oxidase film has been developed. In the present paper, ascorbate oxidase was co-cross-linked with the reconstituted collagen fibrin using the bifunctional agent glutaraldehyde, and the ascorbate electrode was constructed by attaching the immobilized enzyme film to a Clark oxygen electrode (9). The method described here is based on the amperometric measurement of the decrease in dissolved oxygen during the enzymatic reaction shown in eq 1. 0003-2700/81/0353-1974$01.25/0
EXPERIMENTAL SECTION Reagent. Collagen was obtained from Sigma Chemical Co. (insoluble type I, from bovine achilles tendon, No. (3-9879). Glutaraldehyde (25%) was obtained from Nakarai Chemical Co. (Nakagyo-ku,Kyoto, 604, Japan). L-Ascorbicacid (99.9%) was obtained from Ishizu Chemical Co. (Higashi-ku, Osaka, 541, Japan) and its solution was prepared by dissolving in 0.1% metaphosphoric acid solution. All other chemicals were analytical reagent grade and were used without further purification. Apparatus. A -2-mL cell was used for the assay. The upper opening of the cell was closed with an acrylic resin male capillary, and the side opening contained a Clark oxygen electrode. A Clark oxygen electrode was purchased from Yellow Springs Instrument Co. (Model No. 5331). The current was measured with a laboratory-madeinstrument which could change the imposed potential from -2.0 V to +2.0 V and could measure the current from 10 nA full scale to 100 pA full scale with five ranges. The precision of the instrument was *0.1% full scale f 1 digit. The recorder and the digital voltmeter used were Toa Electronic Ltd. Model FBR-252A (Shinjuku-ku, Tokyo, 160, Japan) and Iwatsu Electric Co. Model VOAC 747 (Minato-ku, Tokyo, 105, Japan), respectively. A magnetic stirrer assured perfect stirring of the solution. Unless otherwise mentioned, the temperature of all solutions was carefully controlled at 25.00 0.02 "C (Colora cryothermostat WK 5). Procedures. Enzyme Preparation. Ascorbate oxidase was extracted and fractionated from the peel of cucumber, Cucumis satiuus, according to Nakamura et al. (10). The peel of cucumber was homogenized in a Waring blender with acetone cooled to -15 "C. Acetone was rapidly sucked off through a Buchner funnel, and the pulp thus obtained was suspended in cold 25% saturated ammonium sulfate solution. After the suspension was filtered through cheesecloth, solid ammonium sulfate was added to the extract obtained to give 80% saturation. The floating precipitate was collected and centrifuged at 10OOOg for 10 min. The precipitate obtained was suspended in water and centrifuged at 10OOOg for 10 min
*
0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13,NOVEMBER 1981
P
F
t
0
IO
US
20
25
50
Fraction number Flgure 1. Elution diagrams of pooled fractions from DEAE cellulose ascorbate oxidase activity, (0) separated on Sephadex GlSO: (0) peroxidase activity, (0) protein content, (A)copper content; fraction volume, 2.5 mL; flow rate, 25 mL/h. to remove insoluble material. The supernatant solution was again brought to 80% saturation by adding solid ammonium sulfate, and then centrifuged at lOOOOg for 10 min. The precipitate was dissolved in a minimum volume of water and then dialyzed against 0.02 M phosphate buffer (pH 6.0). The resulting dialyzed solution was purified by column chromatography. The dialyzed solution was centrifuged at, 10 OOOg for 10 min and placed on a DEAE cellulose column (9.5 x 1.5 cm) preequilibrated with 0.01 M phosphate buffer (pH 6.0) and eluted with 0.02 M phosphate buffer (pH 6.0). The active fractions (green or greenish blue) were combined and chromatographed on B Sephadex G-150 column (30 X 1.8 cm) preequilibrated and eluted with 0.04 M NaC1-0.02 M phosphate buffer (pH 6.0). The volume of the solution to be chromatographed must not exceed 10% of column bed volume, otherwise the fractionation efficiency may be drastically impaired. The most active fractions (Figure 1)were pooled and stored at 5 "C until we. The activity of ascorbate oxidase did not decrease for about 3 months under the conditions. Enzyme Assays. The enzyme activity, protein content, and copper content were monitored during purification. The enzyme activity was determined electrochemically by measuring the rate of oxygen depletion kinetically with the Clark oxygen electrode. Before the measurement, the Clark oxygen electrode was calibrated with two different methods according to Capietti et al. (11) and to Misra and Fridovich (12). The reaction mixture was composed of 2 mL of 0.1 M phosphate buffer (pH 6.0) and a desired amount of enzyme solution (10-50 pL, about 0.2-1.0 unit). The oxygen in the solution was allowed to come to equilibrium with atmospheric oxygen. Then, 30 pL of 0.2 M L-ascorbic acid was injected into the cell to trigger the reaction. The reaction rate was recorded as nA change per minute. One unit of ascorbate oxidase activity waq defined as the amount of' enzyme that causes an initial rate of oxygen depletion of 10 pL/min under the above conditions, essentially as defined by Dawson and Magee (13). The amount of protein was determined by the method of Lowry et al. (14) with crystalline bovine albumin as a standard. The total copper in the enzyme was determined according to the method of Poillon and Dawson (25). Enzyme Membrane Preparation. Collagen was suspended in 0.4 M acetic acid and homogenized for 20 min at 5 "C, and the mixture was allowed to sit for 1h and homogenized again. After standing overnight, the mixture was homogenized again. The collagen fibrin paste obtained was dialyzed in a cellulose tube against water until the pH of the contents reached 6.0. The dialyzed past was centrifuged at lOO0Og for 20 min. The supernatant and the bottom layer were discarded, and the
1975
homogeneous intermediate layer was used. The collagen paste obtained was appropriately diluted with water and deaerated before use. The collagen fibrin content was determined by weighing the sample after drying it at 105 "C for 12 h. A desired ambunt of the collagen paste was spread on the high-sensitivity Teflon membrane (Yellow Springs Instrument,, No. 5776), which was previously cut into a circle and placed in a Petri dish (9.6 cm2 in area), and dried at 5 "C, 20% relative humidity. Once dry, an appropriate amount of 8% corbate oxidase in phosphate buffer (pH 6.0) was poured on the membrane, and the membrane was dried under the conditions described above and treated with glutaraldehyde After the membrane was dried (20% relative humidity), it wai washed several times, peeled off the Petri dish, and storedl at 5 "C until use. The solution of 0.05 M phosphate buffer (pH 6.0) was used for these purposes. Enzyme Electrode Preparation. The Clark oxygen elec trode was inverted and the half-saturated KC1 solution war3 spotted on the electrode. The enzyme electrode was prepared by attaching the high-sensitivity Teflon membrane (inside) with the immobilized enzyme-collagen membrane (outside:) to the Clark oxygen electrode. Other procedures were carried out as recommended by the manufacturer (YSI 5331 Oxygen Probe, Yellow Springs Instrument Co., Yellow Springs, OH). The enzyme electrode was dipped in phosphate buffer (pH 6.5) at 5 "C until use. Enzyme Electrode Measurement. Enzyme electrode responses were measured by allowing 2 mL of buffer to equilibrate with atmospheric oxygen and then injecting 10-40 p L of L-ascorbic acid solution. The electrode response was recorded as the current decrease from the point of injection against time. The calibration curve of L-ascorbic acid wa,s prepared by plotting the current at 1 min or 5 min against the L-ascorbic acid content. Preoxidase Activity Assay. The activity of peroxidase wm assayed according to the method recommended by Sigma Chemical Co. (St. Louis, MO). One "Purpurogallin Unit"' causes the formation of 1mg of purpurogallin in 20 s under the conditions descrilbed by Sigma Chemical Co. Sodium Dodecyl !>'ulfate-Polyacrylamide Gel Electrophoresis. Sodium dodecyl sulfate (SDS) electrophoresis was carried out by using the procedure and solutions recommended by the manufacturer (Boehringer Mannheim GmbH. Biochemica, Mannheim, West Germany). Gels with a final acrylamide concentrationof 5% were used. Ascorbate oxidase was treated with 0.1% SDS in the absence of a reducing agent and was subjected to electrophoresis.
RESULTS AND DISCUSSION Enzyme Characteristics. The elution patterns of partially purified enzyme solution from DEAE cellulose on Sephader G-150 column are illustrated in Figure 1. Figure 1shows that ascorbate oxidase is well separated from peroxidase. The most active fractions from1 the G-150 column were pooled and designated as the find preparation. The optical absorption spectrum of the final preparation showed the absorptioin maxima at 280 and 607 nm, which are characteristic for ascrobate oxidase, and little absorption at about 400 nm, which indicates little or no peroxidase. The final preparation was characterized as shorn in Table I. Ascorbate oxidase has been assumed to be composed of two identical subunits (16,I T / , and it will be dissociated into two subunits by treating with sodium dodecyl sulfate (SDS). The molecular weight of eaclh subunit was estimated by the SDS-polyacrylamide gel electrophoresis. The value in Table I was a little larger than that (68000) of Strothkamp and Dawson (17). The optimum pH[, KmM,and Kmozwere nearly equal to those (pH 5.7, KmM1.4 mM, Km020.5 mM) obitained by Nakamura et al. (10). Where, KmMand Kmo2represent the Michaelis constant for L-ascorbic
1978
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
Table I. Characteristics of Free Ascorbate Oxidase with Present Work mol wt 72000X 2 of subunit on SDS treatment optimum pH 6.0 K,AA
Kmoz Cu content
1.09 mM 0.59 mM ca. 0.3%
specific activity ca. 2500 units/ mg protein ca. 800 units/ Clg c u mechanism Ping Po% (double displacement)
SDSpolyacrylamide gel electrophoresis phosphate buffer. depletion of 02' depletion of 0, cuproine, colorimetric Enzyme amount, units Flgure 2. Effect of the amount of enzyme used in membrane preparation on the response of ascorbate electrodes: membranes used, E-1 to E-4; conditions, L-ascorbic acid concentration = 0.3 mM, McIhraine buffer (pH 6.5), 1 rnin response.
Table 11. Ascorbate Oxidase Membranes membrane code
glutarcollagen enzyme aldehyde concn,a % amt,b units concn,c %
E-1 E-2 E-3 E-4 c-1 c-2 c-3 c-4
0.30 0.30 0.30 0.30 0.14 0.28 0.42
G-1
0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30
G-2 G-3 G-4
G-5 G-6
0.56
7.2 22 72 216 73 13 73 73 108
108 108 108
108 108
0.05 0.05 0.05 0.05
0.05 0.05 0.05 0.05 0.001 0.003 0.01 0.02 0.03 0.05
108 0.1 G-8 108 0.3 G-9 108 1.0 Two milliliters of collagen fibrin paste of each concentration was cast onto a 9.6-cm2 Petri dish. One milliliter of enzyme solution of each unit was added onto a 9.6-cm2 Petri dish, One milliliter of glutaraldehyde solution of each concentration was added onto a 9.6-cmZ Petri dish. G-7
acid and oxygen, respectively. The values of the copper content and the specific activity suggest that the preparation of this work is a little different from that of Nakamura et al. (10) but no significant deficiency in later studies was observed. Therefore, this preparation was used as the final one. Membrane Preparation. The effect of variations of the amounts of enzyme, collagen fibrin, and glutaraldehyde used for the enzyme layer was investigated. The membrane codes used are listed in Table 11. In this table, it should be noted that an appropriate volume of each concentration of the solutions was cast to a 9.6 cm2 Petri dish. Figure 2 is a plot of the percentage of maximum response for 0.3 mM L-ascorbic acid against the amount of ascorbate oxidase. A rapid increase in response was observed in the amount up to 72 units of ascorbate, which corresponds to 10 units ascorbate oxidase/electrode. Above 72 units of ascorbate oxidase, a large increase in enzyme concentration gave little increase in response of the ascorbate electrode. This type behavior was pointed out by Guilbault (18) and easily predicted by the Michaelis-Menten equation. Optimum enzyme concentration, only for saving the enzyme, is about 70 units (10 units/electrode). The enzyme activity remaining after the immobilizing process was about 510% of the initial enzyme activity. When a crude enzyme is used, for example,the enzyme preparation
Collagen concentration, % Flgure 3. Effect of the concentration of collagen used in membrane
preparatlon on the response of ascorbate electrodes: membranes used, C-1 to C-4; conditions, same as in Figure 2, except responses are at 1 min (0)and 5 min (0).
70-
-
so
-
40
-
30
-
2
f
u
. l
IdS
Ib*
16'
1.0
GlutaralChyde concentration, '/e
the concentration of glutaraldehyde used in m e n brane preparation on the response of ascorbate electrodes: membranes used, 0 1 to G9; conditions, same as in Figure 2, except responses are at 1 min (0)and 5 min (0).
Flgue 4. Effect of
after dialyzation, a large increase in enzyme concentration will give little decrease in response, owing to relatively large amounts of other contaminating proteins. Figure 3 shows the relationship between the electrode response and the collagen fibrin concentration. The electrode response decreased almost linearly with an increase in the collagen fibrin concentration over the range investigated. At high concentrations of collagen fibrin, the system diffusion may be limited by the membrane thickness. The membrane prepared with 0.1% collagen fibrin is mechanically too weak to mount on the oxygen electrode; therefore use of about 0.3% collagen fibrin for membrane preparation is recommended,
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981 0
1
2
3
4
1077
5
.,IhJrCtlon 20.
2
40
A
B
E f
0
60'
C
80.
PH F w r e 5. Effect of pH on the response of an ascorbate electrode. Conditions are the same as in Figure 2, except buffers used are McIlvalne buffer (0)and 0.2 M phosphate buffer (0).
D
.
100.
E
Flgure 7. Typical response curves of an ascorbate electrode. LAscorblc acld concentration is as follows: A = 0.1 mM, B = 0.2 mM, C = 0.3 mM, D = 0.4 mM, E = 0.5 mM; McIhralne buffer (pH 6.5).
u
6o
t
Buffer concentrollon, M
Flgve 6. Effect of the concentration of phosphate buffer (pH 6.5) on the response of an ascorbate electrode. Conditions are as follows: L-ascorbic acM concentration = 0.3 mM, 1 mln (0)and 5 rnin ( 0 )
responses.
depending on the strength and activity of the membrane. Figure 4,which shows the effect of glutaraldehyde concentration on the electrode response, has a maximum at 0.03% glutaraldehyde concentration. This is probably entirely due to diffusional consideration, as discussed by White and Guilbault (19). Optimum concentration was 0.05%, considering the balance between the strength of the membrane and the electrode response. pH Optimum and Buffer Concentration. The effect of pH on the electrode response was studied from pH 4.0 to 8.0 for 0.3 mM L-ascorbic acid in McIlvaine buffer and/or posphate buffer. The results are given in Figure 5. In phosphate buffer, the response was nearly 100% between pH 5.0 and 7.5. The pH-enzyme activity profile of free ascorbate oxidase in phosphate buffer has shown the constant activity between pH 5.5 and 7.0 (10). The result of this experiment means that the enzyme is stabilized by immobilization. On the other hand, the profile in McIlvaine buffer showed nearly 100% in the pH range 6.0-7.5 and pronouncedly decreased at the left side of this region. Gerwin et al. (20) reported that citrate inhibits competitivelywith respect to ascorbate at low substrate concentration and that, of the four forms of citric acid, only the monoanion is an important inhibitor of the enzyme. When this behavior is taken into account, the pronounced decrease in response at pH values less than 6 is quite probable. However, either buffer system will be useful at pH 6.5, because each maximum electrode response in both buffer systems was equal. Figure 6 shows the effect of buffer concentration on the response of an ascorbate electrode. The electrode responses were slightly increased with increasing buffer concentrations.
Flgure 8. Relationships between the response of the sensor and the concentration of ~-ascorblcacld. Conditions are as follows: McIhraine buffer (pH 6.5), 1 min (0)and 5 min ( 0 )responses.
No disadvantage at high phosphate buffer concentration was found in the electrode measurement. Use of the solution of high buffer concentration, on the contrary, is desirable because of its pH constancy against injection of acidic samples (e.g., citrus fruits). Electrode Response. Typical response curves of the electrode at different concentration of L-ascorbic acid are shown in Figure 7. The current changed rapidly just after injection of samples and the change of current leveled off after about 1min. The electrode response was not affected by the stirring speed if the stirrer assured the perfect stirring of the solution. The enzymaticreaction of ascorbate oxidase follows the ping pong (double-displacement)mechanism (10). When the oxygen concentration is set to be constant, the rate equation for this mechanism can be approximated by a type of Michaelis-Menten equation with a little modification. A plot of the reaction rate against the L-ascorbic acid concentration showed only a narrow linear range. Because the concentration of the other substrate, 02,is 0.257 mM, it is not expected that the steady-state behavior would extend to 0.5 mM L-ascorbic acid. No steady-state region, however, was given even in the curves of Figure 7, and accordingly the current decreased at intervals of 1 min or 5 min after injection of a sample was represented as the values of the electrode response. Figure 8 shows the relationships between the response of the sensor (current) and the concentration of Lascorbic acid. The electrode showed a linear response in the range of 0.05-0.5 mM in L-ascorbic acid concentration.
As.A As.A
+ Cu2+ + Fe3*
0.3 0.3
+ 0.1 + 0.01
102.0 94.9
As.A As.A As.A
+ citric acid + citric acid
0.3 + 9.5 0.3 + 14.3 0.3 t 19.0
100.0 101.0
+ citric acid
Precision of Ascorbate Electrode. The precision of an ascorbate electrode for the constant concentration of L-ascorbic acid (0.3 mM) by 35 successive assays at 1 min was determined. The relative standard variation was 2.21% with these assays. The electrode could be used over 100 successive.assays. Specificity a n d Interference. The specificity and interference studies were performed for an ascorbate electrode. The results are summarized in Table 111. According to Lovett-Janison and Nelson (21),ascorbate oxidase has no action toward p-cresol and only shows a slight action toward catechol and hydroquinone a t comparatively high concentration. Further, Dodds (22) studied the comparative substrate specificity of ascorbate oxidase which was partially purified from cucumber press juice. He demonstrated that the catalytic action of the enzyme was approximately the same for Lascorbic acid and D-isoascorbicacid and was about one-third of the activity for the seven-carbon analogue, and the other analogues showed no oxidation. In the present work, only D-isoascorbic acid was investigated. The response for D-isoascorbic acid was about 93% relative to that for the same concentration of L-ascorbicacid. A lack of substrate specificity of ascorbate oxidase for D-isoascorbic acid will be overcome by using the enzyme from the other origin. White and Smith (23) have discovered the spore enzyme from Mytothecium verrucaria, which has a very high degree of specificity. D-Glucose gave no serious interference, even a t a concentration of 300 mM. Cupric and ferric ions, which catalyze the autooxidation of L-ascorbic acid, did not interfere in concentrations up to 0.1 mM and 0.5 mM, respectively. The presence of EDTA-2Na in amount up to 1 mM had no effect on the electrode response a t 0.3 mM L-ascorbic acid. This result is closely consistent with that of Gerwin et al. (20). No effect of citric acid on t,he electrode response was seen in concentrations up to about 24 mM, which corresponds to 0.5%. The buffer system used was 0.6 M phosphate buffer (pH 6.5) and the pH decrease of this system was only 0.24 pH unit in the presence of 0.5% citric acid. As mentioned in the section of pH optimum, the electrode response was constant between pH 5.5 and 7.5, and the important inhibitor of ascorbate oxidase has been assumed to be monoanion of citrate. Because the concentration of citrate monoanion is less than 2% of the original concentration of citric acid at pH 6.5, no effect under the experimental conditions will be quite reasonable. Long-Term Stability. The long-term stability of an ascorbate electrode was studied by testing the response of an electrode to 0.3 mM L-ascorbic acid in phosphate buffer (pH
99.0
c-1
phosphate (0.6 M,pH 6.5)
J.
0
IO
20
30
40
SO
Days
Figure 9. Long-term stability of an ascorbate electrode. Conditions are the same as in Figure 2.
Table IV. Recovery of L-Ascorbic Acid Added to Citrus unshiu Juice
sample Citrus unshiu
As.A As,A added recovered (mg/100 (mg/100 re1 mL) mL) error, % 16.7 16.3 -2.4 36.3 35.6 -1.9 53.6 55.3 3.2
6.5). When not in use the electrode was stored in 0.2 M phosphate buffer (pH 6.5) at 5 "C. The result is shown in Figure 9. The response of the electrode was constant (100% relative response) for 3 weeks and slightly (ca.14%) decreased a t 5 weeks. The decrease in response is due to slow denaturation and possibly also to slow irreversible inhibition. Recovery Studies and Determination of L-Ascorbic Acid in Fruits, Table IV shows the efficiency of recovery of this ascorbate electrode method. The L-ascorbic acid standard solution was added to Citrus unshiu juice and the concentration of L-ascorbic acid in the resulting mixture was determined. The recovery was quite good. Table V shows the results for determination of L-ascorbic acid in fruit samples by use of an ascorbate electrode. These results are compared to those obtained by the 2,g-dichlorophenolindophenol and 2,4-dinitrophenylhydrazinemethod. The results obtained by the 2,6-dichlorophenolindophenol method were a little smaller than those by the present method. The 2,6-dichlorophenolindophenolmethod is unsuitable for colored samples. Overall, the determinations using the ascorbate electrode compare well with the results obained by
Anal. Chem. 1981, 53, 1979-1982
Table V. Comparisons of Present Method with Two Other Methods method method sample l a 2b tomato 12.4 15.2 (mg/100 mL) 36.5 36.7 Citrus unshiu (mg/100 mL) 48.0 44.5 lemon (mg/100 mL) 93.2 strawberry (mg/100 mL)
present method 14.1 40.5 47.7 86.8
Method 1: 2,6-dichlorophenolindophenolmethod. Method 2: 2,4-&nitrophenylhydrazine method.
2,4-dinitrophenylhydrazine method except Citrus unshiu.
LITERATURE CITED ( I ) GuYbault, G. G. “Handbook of Enzymatic Methods of Analysis”; Marcel Dekker: New York, 1976; pp 460-510. (2) Guilbault, G. 0.In “ComprehenskeAnalytical chemistry”; Sevehla, S., Ed.; Elsevler: Amsterdam. 1977; Vol. 8, Chapter 1. (3) Updike, S. J.; Hicks, G. P. Nature (Loncbn) 1967, 214, 986-988.
1979
Updike, S.J.; Hicks, G. P. Sclence 1967, 158, 270-272. Nanjo, M.; Gullbautt, G. 0.Anal. Chem. 1974, 46. 1769-1772. Nanjo, M.; Qullbault, 0.G. Anal. Chlm. Acta 1974, 73, 367-373. NanJo, M.; Guilbault, G. G. Anal. Chlm. Acta 1075, 75, 169-180. Aizawa, M.; Karube, I.; Suzukl, S. Anal. Chlm. Acta 1974, 69, 431-437. Clark, L. C., Jr. Trans.-Am. Soc. Artll. Intern. Organs 1956. 2 , 41-46. Nakamura. T.; Makino, N.; Ogura, Y. J . Bbchem. 1968, 64, 189-195. Capletti, G. P.; Majorinb, 0.F.; Zucchetti, M.; Marcheslnl, A. Anal. Bbchem. 1977, 83, 394-400. Mlsra, H. P.; Frklovich, I. Anal. Bbchem. 1976, 70, 632-634. Dawson, C. R.; Magee, R. J. Methods Enzynwl. 1957, 2 , 831-835. Lowry, 0.H.; Rosenbrough, N. J.; Fan, A. L.; Randall, I?. J. J. Bkl. Chem. 1951, 193. 265-275. Polllon, W. N.; Dawson, C. R. Bbchim. Biophys. Acta 1963, 77, 27-36. Clark, E. E.; Pollbn, W. N.; Dawson, C. R. Bbchlm. Bbphys. Acta 1966, 718, 72-81. Strothkamp, K. 0.;Dawson, C. R. Blochemlstry 1974, 13, 434-440. Gullbauk, G. 0.Pure Appl. Chem. 1071, 25, 727-740. White, W. C.; Guilbault, G. G. Anal. Chem. 1976, 50, 1481-1486. 8.; Burstein, S. R.; Westely, J. J . Bbl. Chem. 1974, 249, 2005-2008. Gerwin, LovettJankon. P. L.; Nelson, J. M. J . Am. Chem. SOC. 1940, 62, 1409-141 2. Dodds, M. L. Arch. Bbchem. 1948, 18, 51-56. White, G. A.; Smith, F. G. Nature(London) 1961, 190, 187-189.
RECEIVED for review April 22,1981. Accepted July 10,1981.
Catalytic Oxidation of Reduced Nicotinamide Adenine Dinucleotide by Graphite Electrodes Modified with Adsorbed Aromatics Containing Catechol Functionalities Hans Jaegfeldt,’ Arne
B. C. Torstensson, Lo G. 0. Gorton, and Glllis Johansson
Analytical Chemistry. Universitv of Lund, P.O. Box 740, S-220 07 Lund, Sweden
4-[2-( 2-Naphthyi)vlnyl]catechol (NSCH?) and 4424 9,lOethanoanthracen-9-yl)vinyi]catechoi (ASCH2) were adsorbed on graphite electrodes. The naphthalene and the ethaneanthracene ring systems were used as anchors to the graphlte. The catechol group is free to move out Into the electrolyte solutlon. The electron transfer between the 1,2hydroquinone functionality and the graphite was fast. The surface coverage was at most Q x 1o-O mol ern-?. The coenzyme reduced nicotinamide adenine dinucleotide (NADH) could be catalytically oxidlzed by the ImmoMllzed medlating groups. The overvoltage of the NADH oxidation decreased from 410 mV vs. SCE at the unmodMed graphne electrode to 185 mV at the NSCH2 covered electrode at pH 7.0. The reactlon rate of the ASCH?covered electrode was bwer than that of the NSCH? electrode. After 30 min of contlnuous electrochemical cycling of pH 7.0, 30% of the original coverage remalned for the NSCH? electrode.
The coenzyme reduced nicotinamide adenine dinucleotide (NADH) can only be oxidized electrochemically at high overvoltages ( I ) . The overvoltage at pH 7 is about 1.1 V at carbon (2) and 1.3 V a t platinum electrodes (3). If the electrode has been pretreated and if the NADH concentration is at most 0.1 mM, the product a t platinum electrodes is enzymatically active NADt (4). Electrodes modified so that NADH could be oxidized more easily should open up a new field of applications both in analysis and in biotechnology. 0003-2700/81/0353-1979801.25/0
About 300 hydrogenases are known which are dependent on NADH or NADPH as redox transfer agents. Oxidation of NADH to enzymatically active NAD’ in homogeneous solution can only be made with a few oxidizing agenta, often called mediators. Surface modifications have been tried as a means to reduce the overvoltage of the electrochemical oxidation. The strategy has been to immobilize functional groups with known mediating properties on the surface. Blaedel and Jenkins (5) could reduce the overpotential with 0.2 V to 0.45V vs. SCE by an electrochemical pretreatment of the electrode. They presumed that hydroxyl, carbonyl, and quinone functionalities were produced. Tse and Kuwana (6) immobilized 1,2hydroquinones via covalent bonds to the surface of pyrolytic graphite and showed that the functionalities had catalytic activity. The overpotential decreased further about 0.2 V compared to Blaedel and Jenkins. The work demonstrated the feasibility of making catalytic electrodes by the immobilization of mediating functional groups. Degrand and Miller (7)made a polymer from poly(methacryloyl chloride) and dopamine which adsorbed on the surface of vitreous carbon. The decrease in overvoltage was about 0.25 V. The surface coverage and lifetime were improved compared to the electrode made by Tse and Kuwana. It has been observed that a number of polycyclic aromatic compounds adsorb strongly to graphite surfaces. Anson and co-workers studied the electrochemistry of 9,lOphenanthrenequinone (8, 9) and [ 1-(9-phenanthrene)-2-(4pyridine)ethenel (10) adsorbed on graphite. Laviron et al. 0 1981 American Chemical Society
