Analyticn Chimk Actc, 131 (1981) 123-132 Ehevicr Scientific publishing Company, Amsterdam - Printed in The Netherlands

CHEMICALLY MODIFIED GRAPHITE ELECTRODE WITH IMMOBILIZED ENZYME AS A POTENTIOMETRIC SENSOR FOR SOME L-AMINO ACIDS

ROBERT M. IANNIELLO and ALEXANDER M. YACYNYCH*

Department of Chemistry. Rutgers, The State University of New Jersey, New Brunswick, NJ 08903 (U.S.A .)

(Received 9th April 1981)

SUMMARY

A chemically modified electrode with immobilized enzyme was constructed by covalent attachment of L-amino acid oxidase (E-C!. 1.4.3.2) to a graphite rod via chemical modification of the electrode surface. Logarithmic response with concentration of selected L-amino acids was observed in the lo-‘-10“ M range. The electrodes displayed slopes of 24-29 mV/decade over the tested concentration range for L-phenylalarine, L-mcthionine, and I.-leucine. The electrode slope degraded by 33% after 78 days under the defined storage conditions. Intemction of hydrogen peroxide with surface groups generated during cyanuric chloride modification appears to be the major contributor to the poten- tiometric response. Cations change the electrode potential but have essentially no effect on the electrode slope. A plausible model describing the mechanism of response is pre- sented.

The immobilization of various enzymes onto electrochemical sensors to yield substrate-selective sensors is well documented [ 11. In general, these hybrid devices function by selectively sensing a product or reactant of the enzymatic reaction which can be subsequently related to the initial substrate concentration. These sensors can be classified into two main types: reaction systems coupled to electrodes that respond potentiometrically to the change in concentration of a substance produced by the enzymatic reaction, and reaction systems coupled to electrodes that act amperometrically, with oxidation or reduction of a product or a reactant of the enzymatic reaction. Amperometric detectors have been used almost exclusively to monitor enzymecatalyzed oxidation/reduction reactions. This paper reports the use of a chemically modified electrode with covalently-bound enzyme as a potentiometric sensor to monitor enzyme-catalyzed oxidation/reduction reactions.

In recent years, there has been a large amount of research activity in the field of chemically modified electrodes. Such chemical modification involves the strong binding (via irreversible adsorption, polymer coating, or covalent bonding) of a chemical reagent to the surface of an electrical conductor or semiconductor in order to give the electrode surface some particular charac-

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teristic. Usually a series of chemical reactions is used in order to obtain a

sufficient number of binding sites. It has been shown by RsZi&a and co-workers [2, 31 and Ansaldi and

Epstein [4] that graphite, in physical contact with an electroactive species, can serve as an ion-sensitive electrode_ Cyanuric chloride has been successfully used as a linking agent for bonding varicus species to graphite [ 5-81, and it has been used for bonding enzymes to water-insoluble supports for affinity chromatography [g-12].

With the above facts in mind, a chemically modified electrode with immobilized enzyme was constructed by covalently bonding ~amino acid oxidase (E.C. 1.4.3.2) to a chemically modified graphite electrode via a cyanuric chloride linkage. The electrode potential varied logarithmically with variations in concentration of Lphcnylalanine (LPhe), Lmethioninc (L-Met), and Lleucine (~Leu). Hydrogen peroxide appears to be the major con- tributor to the change in electrode potential, but cations also significantly influence the signal. A plausible explanation, based on previous work, is presented.

EXPERIMENTAL

Apparatus and materials All potential measurements were made with either a Fisher Model 525

Digital pIl/ion meter or a Coming Model 135 pH/ion meter. The meter output was recorded on a Fisher Recordall Series 5000 recorder so that variations of potential versus time could be monitored. A saturated calomel electrode (Coleman Instruments inc.) was used as the reference electrode for all trials. All solutions were kept at 25 i O.l”C in a thermostated water bath.

L-Amino acid oxidase (Type VI, from Crotalus atrox venom) and amino acids (Sigma grade) were used as obtained from Sigma Chemical Co. The enzyme, reported to have an activity of 0.33 1-U. mg-‘, was stored desiccated in a refrigerator at -20°C. A stock solution of sodium-potassium phosphate buffer (0.1 M, pH 7.30) was prepared in a routine manner. Cyanuric chloride (2,4,6-trichloros-triazine) and butylamine were of analytical grade (Aldrich). The carbon rods (0.5 cm diameter X 15 cm; spectroscopic electrodes; National Carbon Co.) were cut into 5cm segments, extracted with anhydrous methanol for 24 h, dried at 15O”C, and stored in vacua at 2°C before use. Triple-distilled water was used for all solutions and in all washing procedures. All other chemicals were of reagent grade.

Procedures Electrode preparation. Cyanuric chloride was covalently attached to the

carbon surface as described by Kuwana et al. [ 51. When storage was necessary between any of the synthetic steps of the sequence or after completion of the synthetic scheme, the electrodes were vacuum-desiccated at 2°C. L-Amino

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acid oxidase (LAAO) was bound to the cyanuric chloride-modified electrodes by a modified procedure of Wilson et al. [ 13]_ The electrode was first reacted with a 0.2 M N-(3aminopropyl)diethanolamine in phosphate buffer solution at 25°C for 7.5 min to remove excess of unreacted cyanuric chloride. Reaction with 100 mg of LAAO in 7 ml of pH 7.30 phosphate buffer at 25°C for 30 min followed, after which the electrode was washed three times each with cold water, 1 M NaCI, and phosphate buffer. In order to insure proper electrical contact to the meter, one end of the electrode was polished with emery cloth. In addition, a small portion of the exposed electrode was coated with parafilm so that the total surface of all electrode exposed to any given substrate solution would be reproducible. The LAAO electrode thus produced is shown in Fig. 1. This electrode was preconditioned by soaking in 10m4 M L-Phe (in pH 5.80 phosphate buffer solution containing 0.01 M chloride) overnight at 2°C before use. This preconditioning procedure provided a more reliable electrode response. When not in use, the enzyme was stored in pH 5.80 buffer containing 0.01 M chloride. It has been reported [ 143 that this solution composition is effective in the prevention of LAAO destabilization.

Control electrodes were constructed so that all steps and conditions were identical to those used for the construction of the enzyme electrode. Virgin spectroscopic-grade graphite was extracted with anhydrous methanol, dried at 12O”C, and.soaked in pH 7.30 phosphate buffer for 30 min. Chemically reduced graphite was prepared by refluxing virgin graphite, previously treated with oxygen plasma, with lithium aluminlum hydride in ether for 3 h. The electrodes were then washed three times each with ether, cold 1 M HNOa, and cold water, and soaked in pH 7.30 phosphate buffer. Cyanuric chloride- bound graphite electrodes were prepared as described previously, followed by immersion in pH 7.30 phosphate buffer. Butylaminebound electrodes were prepared by reaction of the cyanuric chloride electrodes in a 1 :l (v/v) solution of n-butylamine in pH 7.30 phosphate buffer. All control electrodes

-.-- Gmtoct to ptl/mV meter

-- P0llstwl md

Fig. 1. Construction and dimensions of the LkAO electrode system.

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were washed three times each with cold water, 1 M NaCl, and pH 7.30 phosphate buffer, and then stored in pH 5.80 phosphate buffer (with 0.01 M chloride) at 2°C overnight before use.

Standard solutions. Stock solutions of 0.1 M L-Phe, &Met, L-Leu, L-Cys- HCI, and n-Phe in pH 7.30 phosphate buffer were prepared. All standard solutions were then prepared by serial dilution immediately before use because dilute amino acid solutions are known to be unstable. Stock solutions of 0.1 M NaCl, KCl, and NH&l were prepared in 0.1 M pH 7.30 Tris buffer for use in the cation control experiments. A stock solution of 0.994 M hydrogen peroxide in 6.1 M pH 7.30 phosphate buffer was prepared by dilution of 30% hydrogen peroxide. The concentration of this stock solution was determined by titration with standardized potassium permanganate.

Standard solutions were allowed to come +a thermal equilibrium by immersion in a water bath for 10 min. During this time, the solutions were saturated with osygen because it is required for the enzymatic reaction, and it is also necessary to maintain constant oxygen concentration_ After immer- sion of the electrodes into these stirred solutions, millivolt readings were recorded when steady state was obtained (2-3 min). The electrode was rinsed and stored in buffer between trials.

RESULTS AND DISCUSSION

L-Amino acid oxidase is known as a specific catalyst for the decomposition of L-amino acids in the overall reaction

LAAO R-C,H<OO- + Hz0 + O2 -R-$-COO- i- NH,’ + H102 (I)

NH; 0

It has been reported [ 151 that LAAO exhibits maximum activity in the pH range 7.0-7.5. In addition, L_4AO is known to demonstrate the highest degree of catalytic activity with r.-Phe as substrate [ 161.

Potentiometric response of the electrode to some selected amino acids is illustrated in Fig. 2. I,-Phenylalanine, L-methionine, and L-leucine are shown to give useful logarithmic response with variation in concentration from lo-’ M to lo-’ M. The lack of selectivity of the electrode towards L-Phe, I.-Leu, and L-Met agrees with results from other potentiometric sensor methods [ 171 for the LAAO system. The potentiometric response of the electrode generally agrees with the catalytic selectivity of LAAO towards L-Phe, L-Leu, and I.-Met. However, it should be noted that the electrode was preconditioned with r_-Phe before use with other substrates. A more detailed description of the response characteristics for these systems is summarized in Table 1. The non-linear response of the electrode for these systems at 10-l M could be due to enzyme inhibition caused by excessive substrate concentration [18] and/or ionic strength variation. In addition, the lower sensitivity at the 10W6 M substrate level may be attributed to the

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.‘.___ L-

-6 -5 -4 -3 -2 -I

L9.g Iomlytel

Fig. 2. Response of the LAAO electrode to (A) L-Leu; (0) ~Cys; (0) D-Phe.

TABLE 1

Response characteristics of the proposed electrode

various amino acids: (= ) r.-Phe; (0) L-Met;

Analytc Response time at lo-’ Ma (min)

Drift (mV)

Imprecision at lo-’ M (mV)

Slopeb (mV/decade)

s b.c YX

L-Phe 2 0.1 1 29.3 1. 0.13 0.29 L-Met 2 0.1 2 24.9 ? 1.95 4.36 L-Leu 2.5 0.2 3 29.4 1 2.17 6.70

‘96% of steadystate potential. bRegression values, concentration range lo-'-lo-' M. CStandard error of the estimate.

unstable nature of dilute amino acid solutions [ 18]_ As expected, the elcc-

trade did not respond to n-Phe. The lack of response of the electrode towards L-Cys is consistent with literature claims that certain LAAO pre- parations lack L-Cys activity [ 181. Finally, the spurious response at 10-l M L-Cys is most likely due to changes in pH and ionic strength of solution caused by L-Cys- HCl dissociation.

In preliminary experiments, diminished response (17 mV/decade) was observed for LAAO electrodes having a large contact time (24 h) between

128

enzyme and the cyanuric chloride-modified electrode. This seems to be in agreement with the work of Kay and Crook [lo] relating decreased enzyme activity with increased contact time. It is interesting to note that the control, consisting of an electrode containing a covalently-bound butylamine moiety on its surface, exhibited a basically flat response. As the alkyl group is inert towards L-phenylalanine, this result indicates that the graphite surface is not substrate-sensitive except in the presence of enzyme_ Thus, interaction of the substrate with the graphite surface {via adsorption and/or chemical reaction) can be considered to be negligible ir. contributing to the potential response.

The stability of the LAAO electrode was examined in order to ascertain the useful lifetime of the device. Table 2 summarizes the response of the electrode to L-phenylalanine after storage. It appears that the electrode may be useful for about 2.5 months, provided t’nat a calibration curve for recent trials is available.

In order to ascertain the response mechanism of the LAAO electrode, it was necessary to examine its potentiometric response in the presence of added hydrogen peroxide_ As the 11202/02 redox couple is irreversible, it would not be expected to display Nemstian response when a conventional (i.e. metallic) redox indicator electrode is employed. This is demonstrated in Table 3. With platinum as the indicator electrode, an almost flat response was observed for hydrogen peroxide concentrations in the range 1O-5-lO-’ M. This result would indicate that the lIZ02/02 redox couple is not the primary redox pair responsible for the potcntiometric response (at least not in the

normal Nernstian fashion) of the IAAO electrode. However, the hydrogen

peroxide may interact with the enzyme electrode surface in such a way as to produce a potentiometric response. With this in mind, the response of a number of control electrodes to added hydrogen peroxide was examined and the results are presented in Table 3.

It has been shown [ 7, 15, 191 that various preparations of graphite can contain a multitude of different surface-functional groups. Interaction of these groups with hydrogen peroxide could be a possible contributor to the potentiometric response of the LAAO electrode_ The response of virgin graphite to hydrogen peroxide is so different, in both range of potential and direction of slope, from the LAAO-substrate system as to eliminate

TABLE 2

Stability of the LAAO electrode

Slope (mV/decado)

Electrode 1 Electrode 2 Electrode 3

Range for

L-Phe

(M)

Initial 29.3 : 0.13 29.0 ! 0.21 28.0 ? 0.19 10-“-lo-’

After 25 days 28.2 c 0.27 - - 10”-10” After 78 days - 20.8 l 0.74 - 10-:_10-

After 106 days - - 10.3 ? 0.41 5 x lo’-10-s

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TABLE 3

Response of the control electrodes to hydrogen peroxide

Electrode H,O, range Potential ranse No. of slwe” s (M) (mV) poinrt (mV/decade)

Pt 2.4 X 10a’6.3 X IO-’ 217-200.0 8 4.4 f 0.54 GmphitJJ 2.4 X lo-‘-6.3 X IO-’ 184.6-382.4 8 44.3 f. 4.57 1 LiAIHjreduced mphite 2.5 X 10-‘-l X IO-’ -1524 to -126.7 10 9.4 +- 0.93 Cyanuric chloride (CC) graphite 2.5 X 10sL1.7 X lo-’ 5.8-218.5 7 79.0 f 4.91 1 Butyhmhe~C wphite 2.4 X lo-‘-1.6 X 10-t 24.6-230.4 7 71.5 f 2.10 LAAOdC 1~8phite 2.4 X lo-‘-E.3 X 10-l 93.7-216.2 9 40.6 f 3.84 I

“Regression values. bSpectroscopic grade, untreated.

these “virgin ” surface species as potentiometric response contributors. Catalytic decomposition of hydrogen peroxide at the graphite surface is a possible cause of the observed response of the virgin graphite electrode [ 201. The next step in the chemical modification scheme (chemical reduction of virgin graphite with lithium aluminum hydride) led to radical alterations in the potentiometric response (Table 3), presumably through vast changes in the identity of the surface species. This result, however, essentially elimi- nates’ the reduction step as the primary cause of the potentiometric response of the LAAO electrode. The next step in the immobilization, bonding of cyanuric chloride to the electrode surface, gave an electrode displaying a 79.0 mV/decade change in potential_ Bonding of an inert alkyl chain (butyl- amine) to this electrode only slightly changed the potentiometric response. The range of potential in both cases was essentially identical_ It would appear that interaction of hydrogen peroxide with surface species generated during cyanuric chloride bonding (possibly including cyanuric chloride itself) is primarily responsible for the observed potentiometric response of the cyanuric chloride control electrodes_ The final electrode, LAAO-cyanuric chloride graphite, displayed a reduced-potential variation to hydrogen peroxide compared to the above cyanuric chloride controls. The ranges of potential observed, however, were quite similar in all three cases. It may be concluded that, in the case of the LAAO electrode, while HiOz-cyanuric chloride interaction (possibly including other species that may have been generated after the reduction step) is a major contributor to the potentiometric response, other enzyme-solution interactions must be present which either alter or compete with the above interaction. Some novel probe methods are currently being employed in an attempt to identify the various surface moieties gener- ated during the immobilization reactions. The results of these experiments wiI.l be presented in a future report. The competing enzyme-solution interactions were suspected to be primarily due to cation-enzyme inter- actions and prompted investigation of the influence of cations on the observed potential of the LAAO electrode.

Majer et al. [21] have examined the potentiometric response of various virgin and oxidized, carbons and graphite. In particular, oxidized graphite

130

(which contains a large variety of surface groups, e.g., COO-) exhibited sizable variations in potential at pI4 >5 to many alkali metal a-d alkaline e&h cations. This observation has been utilized in the development of a useful electrode for potassium ion titrations [ 221. Because of this, it was suspected that the presence or absence of monovalent cations would contri- bute to the potentiometric response. Figure 3A shows the influence of added Na’, K*, and NH; on the LAAO electrode potential in the absence of substrate. It is apparent, because each cation generates different electrode potentials at all tested concentrations, that interaction of cations with the enzyme (presumably via ion exchange with the enzyme moieties) is a partial contributor to the observed potential. The response of a butylamine control electrode (Fig. 3B) supports the cation response model. In this case, because the alkyl group is unable to undergo ion exchange with the tested cations, one would not expect to observe any significant variation in potential. The response of the LAAO electrode to L-phenylalanine in the presence of 0.01 M Na*, K’, and NH: is presented in Fig. 4. Because the cation con- centration is identical for any tested L-Phe concentration, the influence of the cation on the electrode potential would be expected to be constant. However, because the different cations most probably have different affinities for the available ion exchange sites, an absolute potential shift would be expected, as is observed. Interestingly, the electrode still maintains a 29 mV/ decade response to L-phenylalanine. The influence of the keto acid product on the potentiometric response was the final suspected interferent which was investigated. Figure 4 shows the response of the LAAO electrode to L-phenylalanine in the presence of 0.01 M P-phenylpyruvate. This is the keto acid product of the LAAO--L-Phe system. As indicated, the electrode maintains its response to L-phenylalanine, with no apparent interference from the keto acid. An attempt was made to “swamp out” the cation interference by adding escess of cation (1 M) so that the ion-exchange sites

. .- - +.-t .* *. .Q*.--p,~- ‘*-t - 0 --err

---A.4 . . - . A 4

Lop [cotloo Log kotlull

Fig. 3. Effect of Na*, K’, and NH; on (A) the LAAO electrode potential and (B) the butylnmine electrode potential in the absence of substrate. ‘D-is buffer (0.1 M. pH 7.30) used for a!1 trials. (0) Na’; (A) K’;(I) NH,‘.

131

-I%- = 1 _

_i; -

-150;

L --i. __I

-4 -.3 _.>

Log [L-PHEI

Fig. 4. Response of the LAnO electrode to L-phenylalanine in the presence of added ions at the 0.01 M level: (0) Na’; (A) K’; (0) NH.*; (A) p-phenylpyruvatc. Concentration was maintained at 0.01 M. Tris buffer (0.1 M. pH 7.30) used for all trials.

Fig. 5. Response of the LMO electrode to L-phenylalanine in the presence of added ions at the 1 M level: (0) Na’; (A) K’; (a) NH,‘. Slopes for Na’, K’, and NH,’ are 34.2 2 3.4,

38.9 * 3.7 and 7.9 2 0.7, respectively. The corresponding error estimates are 10.0. 10.8,

and 2.1.

would become saturated. While the displacements in potential were greatly reduced in all cases, the response range was considerably narrowed. These results are illustrated in Fig. 5. The magnitude of response of the LAAO electrode in the presence of 1 M Na’ and K’ is surprisingly similar to that obtained for the LAAO-H202 experiment described above. This may indicate that the ion-exchange sites are indeed saturated for these cases. However, the reduction of the response range (when [ Na’, K’] = 1 M) may

indicate the occurrence of enzyme inhibition via a salt effect of the first or second kind [ 231. For the case of 1 M NH,*, a drastic loss of response is observed. The results in this case indeed indicate that 1 M NH,’ is an effective inhibitor of the LAAO-L-phenylalanine system.

The influence of Na’, K*, and NH,’ on the electrode potential is quite sizable, as indicated by the above results. On the stlrface, this would seem to be quite a drawback when attempting to use the LAAO electrode as a quantitative substrate probe. Iiowever, if employed in a sample addition mode, where the background buffer volume is large compared to that of the sample, satisfactory results are obtained. Standard additions of L-phenyl- alanine to 0.1 M Na’-K’ phosphate buffer solutions gave satisfactory correlations obeying the equation Ck;az = 0.895 f 0.018 C”L”p”h’,” + 4.8 X 10m5 + 1.7 X IO-‘. Standard additions to 0.1 M Tris buffer solutions obeyed the equation CrbPht Ound = 0.868 f 0.012 CFPyed + 6.3 X 10m5 f 1.1 X lo-‘. The corresponding standard errors of estimate were 2.7 X lo-’ and 1.7 X lo-‘, respectively. These results show that, when employed in a standard addition mode, satisfactory quantitative assays may be obtained.

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This technique offers a viable potentiometric alternative to amperometric enzyme electrodes for the analysis of enzyme catalyzed redox reactions. In addition, unlike conventional potentiometric enzyme electrodes, which utilize

an internal sensor, this electrode may be used at the optimum pH of the enzyme-substrate system, without regard to the pH-response dependence of an internal sensor.

A.M.Y. thanks Rutgers Research Council, Biomedical Research Support Grants, the National Institutes of Health (grant number GM 28125-Ol), and the National Science Foundation (grant number CHE-8022237) for research support. R.M.I. acknowledges the support of the American Chemical Society, Division of Analytical Chemistry, and the Olin Corporation Charitable Trust for the award of a Summer Fellowship during the course of this work.

REFERENCES

1 See, e.g. P. W. Carr and L. D. Bowers, Immobilized Enzymes in Analytical and Clinical Chemistry, in Chemical Analysis, P. J. Elving and 2. D. Winefordner (Eds.), Wiley- Interscience, New York, 1980, Vol. 56. pp. 197-310.

2 J. Rt%Eka and C. Lamm, Anal. Chim. Acta, 54 (1971) 1; 53 (1971) 206. 3 J. RtX%Eka and K. Raid, Anal. Chim. Acta, 53 (1971) 1. 4 A. Ansaldi and S. Epstein, Anal. Chem., 45 (1973) 595. 5 D. Tse, T. Kuwnna and G. Royer, J. Electra-I. Chem., 98 (1979) 345. 6 M. Dautartas, J. Evans and T. Kuwana, Anal. Chem., 50 (1978) 104. 7 A. M. Yacynych and T. Kuwana, Anal. Chem., 50 (1978) 640. 8 A. Lin, P. Yeh, A. M. Yacynych and T. Kuwana, J. Electroanal. Chem., 84 (1977)411. 9 G. Kay, M. Lilly, A. Sharp and R. J. II. Wilson, Nature, 217 (1968) 641.

10 G. Kay and E. M. Crook, Nature, 216 (1967) 514. 11 K. Wheeler, B. Edwards and R. Wbittam, Biochim. Biophys. Acta, 191 (1969) 187. 12 P. Monsan and G. Durand. FEBS Lett., 16 (1971) 39. 13 R. J. H. Wilson, G. Kay and M. Lilly, Biochem. J., 109 (1968) 137. 14 E,. Kearney and T. Singer, Arch. B&hem., 33 (1951) 971. 15 G. G. Guilbault and E. Hrabankova, Anal. Lett., 3 (1970) 53. 16 G. G. Guilbault and J. Hieserman, Anal. Biochem., 26 (1968) 1. 17 G. G. Guilbault and E. Hnbankova, Anal. Chem., 42 (1970) 1779. 18 H. V. Mahnstadt and T. Hadjiioannou, _4nal. Chcm., 35 (1963) 14. 19 R. A. Kamin and G. 8. Wilson, Anal. Chem., 52 (1980) 1198. f0 Y. K. Cho and J. E. Bailey, Biotechnol. Bioeng., 19 (1977) 769. 21 C. Majer, J. Vesely and R. Stulik, J. Electroanal. Chem., 45 (1973) 113. 22 R. Geyer and P. Stein, Wiss. 7.. Tech. Hochsch., Carl Schorlemmer, Lenna-Merseburg,

17 (1975) 364. 23 G. G. Guilbault, Enzymatic Methods of Analysis, Pergamon, New York, 1970, p. 17.