Biotechnology Letters Vol 7 No 6 419-422 (1985)

"BANANATRODE" - AN ELECTROCHEMICAL BIOSENSOR FOR DOPAMINE

J. S. Sidwell and G. A. Rechnitz

Department of Chemistry
University of Delaware
Newark, Delaware 19716

SUMMARY

A new type of biocatalytic membrane electrode utilizing banana

pulp tissue slices for sensing catecholamine neurotransmitters is
described. This amperometric-based probe for the measurement of
dopamine exhibits high biocatalytic activity, good time stability,
and favorable selectivity.

INTRODUCTION

Various types of biocatalysts have been shown to be useful

in the construction of biocatalytic membrane electrodes ( Rechnitz,
1981; Arnold, 1983 ). Such biocatalysts include isolated enzymes,
subcellular fractions, intact bacterial cells, and whole sections of
mammalian and plant tissues. Both potentiometric and amperometric
electrodes have been employed in the fabrication of plant: tissue
based biocatalytic membrane sensors ( Arnold, 1984; Kuriyama and
Rechnitz, 1981; Kuriyama et. al., 1983; Schubert et. al., 1983;
Schubert et. al., 1984; Smit and Rechnitz, 1984 ). The majority of
these biosensors are designed for the determination of amino acids.
We Now extend this work to an important new class of compounds, e.g.
catecholamine neurotransmitters, through the development of a novel
biosensor having selective response for dopamine.
The new sensor uses a Clark-type oxygen electrode in conjunction
with banana pulp tissue slices which serve as a sensitive and selec-
tive biocatalyst for the quantification of dopamine. The principle
of the sensor involves the so-called browning reaction of banana
tissue ( Palmer, 1963 ) in which the enzyme polyphenol oxidase
catalyzes the conversion of dopamine to its corresponding quinone
and subsequent reactions occur spontaneously. The consumption of
oxygen in this reaction gives rise to an electrical signal, via the

419
oxygen electrode, in proportion to the concentration of dopamine pres-
ent
The physical construction of this biocatalytic membrane electrode
is shown schematically in Figure i. A 0.25 mm to 1.0 mm thick slice
of banana pulp tissue is placed on the gas permeable membrane of the
oxygen electrode and held in place with a dialysis membrane. The
substrate dopamine can readily diffuse through the dialysis membrane
into the banana tissue to undergo catalytic conversion by membrane
bound polyphenol oxidase. The depletion of ambient oxygen in this
enzymatic conversion yields a steady-state response as in conven-
tional enzyme electrodes.

ELECTRODE

PERMEABLE
BANANA j • / GMEMBRANE
AS
R"~'DIALYSIS MEMBRANE

FIGURE i. Schematic diagram of Electrode Assembly

MATERIALS AND METHODS

All chemicals used were of reagent grade. Dopamine Hydro-

chloride (Sigma) was dissolved in milllmolar Sulfuric acid to
prevent autoxidation ( Varozuaux, 1979 ). Distilled, doubly
deionized water was used in the preparation of all solutions.
Bananas were obtained in the local market and allowed to ripen
for four to five days. This ripening period allows indigenous
dopamine to be scavenged while not compromising polyphenol oxidase
activity ( Weaver and Charley, 1974 ). Typical banana pulp slices
were obtained by cutting sections of approximately 0.75 mm thick-
ness with a razor blade. The biocatalytic membrane electrode was
prepared by placing the banana tissue slice on the gas permeable
membrane of an Orion ( Model 97-08 ) oxygen electrode and securing
it with a Technicon type C dialysis membrane as shown in Figure I.
Measurements were carried out in a cell thermostated at 25~ in
pH 6.50, 0.10M phosphate buffer media. The solutions were air
equilibrated~y stirring. Signals were measured with a C o m i n g
model 12 meter and a Heath strip chart recorder.

420
 RESULTS AND DISCUSSION

The poiyphenol oxidase ( EC 1.14.18.1 ) catalyzed oxidation

of dopamine to dopamine quinone occurs quite rapidly with the
subsequent reactions proceeding nonenzymaticaliy. Since dopamine is
prone to autoxidation, stock solutions ( 0.05~ ) were freshly prepared
daily in I x 10-32 H2SO 4. Steady-state measurements obtained in
O. IOM, pH 6.50 phosphate buffer are shown in Figure 2. When stored
in buffer, this sensor is stable for at least two weeks with no
detectable deterioration in response characteristics. If the
biocatalytic activity in the banana Slice toward dopamine were in the
saturation region one would expect a substrate slope independent of
slice thickness. Such constant slopes ( • 3% ) were indeed observed
for slices ranging from 0.25mm to 1.0mm in thickness. Comparison of
biocatalytic activity for dopamine with other polyphenol oxidase
containing plants, e.g. mushrooms, confirms the exceptionally high
enzyme levels present in banana pulp tissue, but little difference
was found between different sources or brands of bananas.

10

~ g
8
O
E
_=6
._t
Z
~4

! ! f ! 1 I
~2 ~4 0.6 0.8 1.0 1.2
[DOPAMINE] (mm0[ -~1)

FIGURE 2. Typical dopamine response of the Banana based

electrode
Steady-state response =imes for the fully assembled banana
slice electrode are on the order of one to three minutes, while
homogeneous experiments carried out on homogenized banana pulp
tissue, using the oxygen electrode as a monitor, give response
times in the 30 - 40 second range. The slewer response of the
immobilized electrode reflects the additional diffusional steps

421
required at or in the biocatalyst barrier. The response of the
electrode is practically independent of pH in the 6.0 to 8.0 range,
but experiments were carried out at pH 6.50 to minimize autoxidation
of the substrate.
Polyphenol oxidases have been reported as copper containing
enzymes of rather broad specificity ( Yasunobu, 1959 ), but each
enzyme from a particular source, e.g. bananas, tends to oxidize
one substrate more readily then others and banana polyphenol oxidase
enzyme has been shown to utilize dopamine as its primary substrate
( Palmer, 1984 ).
The present sensor might be useful for measurements in urine,
where dopamine is found in micromolar levels, after preconcentration
on alumina or gels ( Krstulovic, 1979 ) which also serves to remove
interferences. The drug DOPA, structurally similar to dopamine,
yielded a much smaller response in our experiments as expected on the
basis of K values ( Palmer, 1963 ). Further selectivity studies
m
and analytical evaluation of this type of biosensor are currently in
progress with particular emphasis on rapid neurotransmitter measure-
ments.

ACKNOWLEDGMENT

We gratefully acknowledge the support of National Science

Foundation Grant CHE-8318192.

REFERENCES

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