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J. Electroanal. Chem., 222 (1987) 343-346 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

BIOSENSORS USING FLOWER PETAL STRU- *

SHUNICHI UCHIYAMA l * and G.A. RECHNITZ

Department of Chemistry Vniversiq of Delaware, Newark, DE 19716 (U.S.A.)

(Received 30th September 1986)

INTRODUCTION

A major theme in the development of electrochemical biosensors has been the use of natural materials as biocatalytic layers coupled to potentiometric or arnperomet- ric elements. Both animal and plant substances [l] have been used for this purpose. Only recently has it been recognized, however, that the specialized structures of plants, e.g. leaves [2], fruit [3], etc., offer particularly attractive properties as biocatalysts because structures related to growth, reproduction, and nutrient storage concentrate and stabilize highly selective biocatalytic activity.

Very recently it was shown [4] that the component structures of blossoms from chrysanthemum and carnations can be effective biocatalysts when coupled to potentiometric gas-sensing membrane electrodes. Selective deamination responses for certain amino acids were found, but the fragility of the flower structures involved required the use of minced tissue as the biocatalytic layer. We now report the first example of a potentiometric biosensor constructed using intact portions of flower petals, where longitudinal slices of magnolia petals are held directly at the surface of a potentiometric ammonia-gas-sensing electrode. It will be seen that this arrangement eliminates the need for support membranes and, thus, results in a sensor having rapid dynamic response characteristics. The magnolia petal also displays a unique pattern of biocatalytic activity not previously reported for biosensors.

EXPERIMENTAL

All potentiometric measurements were made with a Corning Model 12 meter in conjunction with a Heath-Schhrmberger SR-240 strip chart recorder. A Haake Model FS water bath circulator was employed to thermostat the experimental cell;

* Dedicated to the memory of Don Smith. ** On leave from Department of Environmental Engineering, Saitama Institute of Technology, .Japan.

0022-0728/87/$03.50 0 1987 Elsevier Sequoia S.A.

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10 ml sample volumes were used for all experiments. Orion Model 95-10 ammonia gas electrodes were used in the construction of the biosensors. Analytical grade reagents were employed for all experiments. Ammo acids were purchased from Sigma and test solutions prepared freshly each day in phosphate buffer.

Flower-petal biosensors were constructed as follows. Magnolia flower petals were taken directly from blooming local trees (M. grundifloru). The white blossoms, up to 20 cm in diameter, are represented schematically in Fig. 1, which also shows the component structures of the flower. Owing to the rather large size of the blossom, individual petals can be detached readily and subdivided without perforation. For purposes of biosensor construction, petals were sliced longitudinally (Fig. 1) to remove the outer hydrophobic skin and prepare sections 0.1-0.4 mm in thickness. These sections are cut further into 12 mm diameter discs; each disc fits exactly on the sensing tip of the Orion 95-10 ammonia-gas-sensing electrode (Fig. 1) in contact with the gas-permeable membrane of the electrode. No dialysis or other support membranes were employed in the construction of the biosensors, which are ready for operation after approximately 3 h conditioning in the pH 7.4 phosphate buffer.

RESULTS AND DISCUSSION

Preliminary screening tests on the new biosensor, consisting of a magnolia petal slice held at the tip of the potentiometric NH3 electrode, were carried out for the essential amino acids and some other compounds (creatine, creatinine) which can be deaminated by biocatalytic action. Primary responses were found for L-glutamine and L-asparagine, with a lesser response to the D-isomers of these amino acids. A slight interference from L-aspartic acid was observed, while L-isoleucine, L- threonine, L-histidine, L-arginine, L-omithine, L-citrulline, L-phenylalanine, L-

f

Fig. 1. Schematic diagram of magnolia (M. grrurdifloru) flower and construction of biosensor. (a) petal, (b) stamen, (c) carpel, (d) leaf, (e) petal tissue slice, (f) tissue disc, (g) hydrophobic gas-permeable membrane, (h) electrode body.

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0 7.0 7.5 8.0 8.5

P”

Fig. 2. pH profile of magnolia flower petal tissue biosensor response to L-glutamine (0) and L-aspara- gine (O), 33O C.

methionine, creatine, creatinine, L-serine, L-glycine, L-valine, L-leucine, and urea gave negligible responses.

Such a response pattern is very unusual and, indeed, has not been reported for any previous biosensor. It suggests that the primary biocatalytic activity involves the enzyme glutamine-(asparagine-)ase, E.C. 3.5.1.38, known from bacterial systems [5]. A principal feature of this enzyme is that it catalyzes the hydrolysis of the D-isomers of glutami.ne and asparagine at one-third the rate of the L-isomers, which is in accord with our observations. Further confirmation is provided by the pH profile (Fig. 2) of the magnolia petal biosensor showing maximum activity in the pH range 7.4-7.8 for both substrates, as predicted for the glutamine-(asparagine)-ase enzyme [5].

The analytical response to the primary substrates is very good, with response slopes of 48-50 mV per concentration decade, a tenfold linear range at the lOa M

level, and a detection limit of 8 x lob6 M and 1 X 10e5 M for L-glutamine and L-asparagine, respectively. The optimum response is obtained in phosphate buffer of pH 7.4, after some hours of conditioning in this buffer. This observation suggests that the biocatalytic processes involved may be activated by phosphate.

The response slopes to the primary substrates were unchanged (relative standard deviation < 2%) for at least ten days. After two weeks of operation there is some decrease in response slope (to approx. 43 mV per decade) and some softening of the petal tissue layer can be observed. Such operating lifetimes are typical of plant-tis- sue-based biosensors [1,2,3].

The most important advantage derived from using an intact tissue slice from flower petals is a significant shortening of response times owing to the elimination of additional support membranes in the construction of the biosensor. Previous plant-tissue-based biosensors had response times in the lo-20 min range [l-4] with

TABLE 1

Dynamic response behavior of magnolia petal biosensor

Tissue thickness/mm lo4 Concentration/M Steady state response time/mm

L-ghrtamine NH,Cl

0.1 0.5 6.5 1 5 2 4.5 2.8 5 4

10 3

0.2 2 8 7

0.4 2 13 13

the dynamic response being determined by diffusional steps through the sandwich structure of the biocatalytic layer and its support membranes. It can be seen from Table 1 that the flower-petal-slice biosensor described in this communication has substrate response times as short as 3 min at the millimolar level at 0.1 mm tissue thickness. As expected, response times increase with decreasing substrate concentra- tions and increasing tissue thickness. The diffusional limit of overall sensor response times is reflected in the values obtained by exposure to NH&l samples (Table 1) where no biocatalytic processes are involved; these response times indicate the lower limits, which could be obtained with the present sensor geometry if the biocatalytic conversion of substrate to ammonia were infinitely fast. It can be seen that the substrate response times for L-glutamine are only slightly longer than those for NH,Cl at 0.1 mm tissue thickness and become indistinguishable for the thicker (0.2-0.4 mm) tissue slices. These results indicate that the level of specific biocata- lytic activity in the magnolia tissue is very high and that the biosensor constructed with 0.1 mm tissue thickness is very nearly optimized in terms of dynamic response.

ACKNOWLEDGEMENT

We greatly appreciate the financial support of NSF grant CHE-8318192.

REFERENCES

1 G.A. Rechnitz, Science, 214 (1981) 287. 2 N. Smit and G.A. Rechnitz, Biotechnol. Lett., 6 (1984) 209. 3 J.S. Sidwell and G.A. Rechnita, Biotechnol. Lett., 7 (1985) 419. 4 S. Uchiyama and GA. Rechnitz, unpublished results. 5 J. Roberts, J.S. Holdenberg and W.C. Dolowy, J. Biol. Chem., 247 (1972) 84.