Enzyme and Microbial Technology 36 (2005) 91–99

A biosensing method with enzyme-immobilized eggshell membranes

for determination of total glucosinolates in vegetables
Martin M.F. Choia,∗ , May M.K. Lianga , Albert W.M. Leea,b,1
a Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China
bCentral Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis,
Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China

Received 3 September 2003; received in revised form 20 June 2004; accepted 30 June 2004

Abstract

An optical biosensing method using enzyme-immobilized eggshell membranes for glucosinolate determination has been developed.
Myrosinase-immobilized eggshell membranes were fabricated and applied to hydrolyze glucosinolates at 37 ◦ C for 16 h. The glucose produced
was subsequently determined by an optical glucose biosensor which was constructed with a glucose oxidase-immobilized eggshell membrane
and an oxygen-sensitive optode membrane. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to
glucose solution. The rate change in fluorescence intensity of the oxygen-sensitive membrane was monitored and related to the glucose con-
centration. The effect of myrosinase loading, egg white concentration, incubation time, temperature, pH, and phosphate buffer concentration
on the hydrolysis of sinigrin has been studied in detail. The myrosinase-immobilized eggshell membrane demonstrated a long shelf-life of at
least 6 months and the results showed good repeatability. It has been successfully applied to determine glucosinolates contents in cruciferous
and non-cruciferous vegetable samples. The results show that only cruciferous vegetables contain various amounts of glucosinolates whereas
non-cruciferous vegetables do not contain any.
© 2004 Elsevier Inc. All rights reserved.

Keywords: Biosensor; Eggshell membrane; Glucosinolates

1. Introduction cells are injured, and catalyze the hydrolysis of glucosinolates

to form glucose, isothiocyanates (ITCs), and sulfate [3]:
Organic isothiocyanates (ITCs), also known as mustard
oils, are commonly found in plants and their seeds and are
responsible for the pungent taste of many condiments such as
mustard and horseradish. Many of these compounds occur in
high concentrations in vegetables in the form of their glucosi-
nolate precursors [1]. Glucosinolates are an important class
of naturally occurring thioglucosides [2] and usually occur
together with the plant myrosinase (thioglucoside glucohy-
drolase, EC 3.2.3.1) which is widespread in seeds and tissues Glucosinolates are widely distributed in cruciferous veg-
of the Cruciferae family. Myrosinase is released when plant etables that are consumed by humans, such as broccoli, cab-
bage, cauliflower, radish, turnip, and watercress [2,4]. It has
been reported that ITCs, the hydrolytic products of glucosino-
∗ Corresponding author. Tel.: +852 3411 7839; fax: +852 3411 7348. lates, have positive effects on cancer prevention. This cancer
E-mail address: mfchoi@hkbu.edu.hk (M.M.F. Choi). protective effect is attributed to the ability of ITCs to inhibit
1 An area of Excellence of the University Grants Committee, Hong Kong
phase I enzymes that are responsible for the bioactivation of
SAR, China.

0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2004.06.009
92 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99
carcinogens [5] and to induce phase II detoxification enzymes
[6]. A recent report on the study of lung-cancer risk from a co-
hort of 18,224 men in Shanghai, followed from 1986 to 1997,
also supported the chemopreventive effects of dietary ITCs
[7]. In addition, epidemiological studies indicated the con-
sumption of high quantities of cruciferous vegetable in hu-
man diets could reduce the risk of developing certain types of
cancer [8–11]. Moreover, glucosinolates play an extremely
important role in the food industry since their breakdown
products produce flavor and aroma that are also important
for a nutritional diet. Hence, the quantification of the total
glucosinolates in vegetables is of particular importance.
To date the determination of total glucosinolates in cru-
ciferous vegetables includes mainly chromatographic and en-
zymatic methods. Chromatographic techniques such as gas
chromatography, supercritical fluid chromatography, high-
performance liquid chromatography and electrokinetic capil-
lary chromatography have been published. While these chro-
matographic methods provide good information about the
types of glucosinolates, they are labor-intensive, and suffer
from low throughout and high overall cost. A sensor approach
was developed to achieve a higher degree of miniaturization,
repeatability, faster determination and lower cost.
Sensors are devices that provide information about the
concentrations and chemical states of the species present
within a sample. Research in the development of chemosen-
sors and biosensors is growing rapidly. In clinical diagnosis
and the food industries, a glucose sensor is very desirable for
analysis and process control purposes. Clark reported the first
successful electrochemical biosensor using immobilized glu-
cose oxidase in conjunction with an oxygen electrode nearly
50 years ago [12]. Since then, reports on the development
of electrochemical biosensors have been accumulating at a
very high speed. Electrochemical biosensing methods for the
determination of glucosinolates have also been reported in
the literature [13–16]. The serious drawback of electrochem-
ical sensors is that the weak electrical signal will easily suf-
fer from electric and magnetic interferences especially when
they perform in a hash environment. Optical sensors provide
a promising alternative as they possess certain advantages
over conventional electrochemical sensors or devices. Some
of the attractive features of optical sensors are often related
to simple sensor design, easy operation, free from electric
and magnetic interference, and are suitable for ‘in situ’ or
‘online’ remote monitoring.
Enzyme-based biosensors are currently a major area for
research and development since essentially all chemical re-
actions in living systems are catalyzed by enzymes. For a
re-usable enzyme-based biosensor, an enzyme has to be im-
mobilized on, or in close proximity to, the surface of a trans-
ducer. Very often, the problem of long-term operational life
and storage stability present crucial hurdles for development.
The shelf-life of an enzyme-based biosensor usually depends
on how long the biological activity of the enzyme can be re-
tained and this may vary from days to months. It has been
reported that some biomaterials consisting of silk [17–19],
collagen [20,21] and eggshell membrane [22–27] were em-
ployed as platforms for the immobilization of enzymes and
the life-spans of the immobilized enzymes were much ex-
tended. Eggshell membranes possessing excellent gas- and
water-permeability can be an ideal bio-platform for enzyme
immobilization. In this study, we explore the possibility of
employing glucose oxidase- and myrosinase-immobilized
eggshell membranes to indirectly determine the total glucosi-
nolates in cruciferous vegetables. To our knowledge, this is
the first report on the application of eggshell membranes for
optically biosensing glucosinolates. Myrosinase was initially
immobilized onto the surface of a fresh eggshell membrane,
which was subsequently employed to hydrolyze glucosino-
lates with the production of glucose. The generated glucose
was then measured by an optical glucose biosensor consist-
ing of a glucose oxidase-immobilized eggshell membrane
and an oxygen-sensitive optode membrane mounted into a
flow cell. The effects of myrosinase concentration, incuba-
tion time, pH, temperature, and buffer concentration on the
hydrolysis of glucosinolate by enzyme-immobilized eggshell
membranes were investigated. The properties of storage, sta-
bility and repeatability of the biosensing method were also
studied in detail. In this report, the whole biosensing method
showed good stability and sensitivity to glucosinolate. The
proposed method was successfully applied to determine glu-
cosinolate content in some vegetable samples.
2. Materials and methods
Glucose oxidase (EC 1.1.3.4 from Aspergillus niger) with
a specific activity of 18,500 U per gram of solid, thioglu-
cosidase (myrosinase, EC 3.2.3.1 from Sinapis alba) with
a specific activity of 324 U per gram of solid and sinigrin
monohydrate from horseradish were obtained from Sigma
(St. Louis, MO). Fifty percent (w/w) glutaraldehyde solu-
tion in water was purchased from Aldrich (Milwaukee, WI).
␤-d-Glucose was from Acros Organics (Geel, Belgium). All
other reagents were of analytical-reagent grade and were used
without further purification. Eggs were purchased from a lo-
cal market. The buffer solution for preparing glucose stan-
dards was 50 mM sodium phosphate solution at pH 7.0. All
solutions were prepared with deionized (DI) water.
2.1. Preparation of myrosinase-immobilized eggshell
membrane
An eggshell membrane was carefully peeled off from a
broken fresh eggshell after the albumen and yolk had been
removed. It was cleaned with a copious amount of DI water.
The membrane was placed in a clean watch glass and it was
cut into a circle of a diameter of 25 mm. A 200 ␮l aliquot of
5% (w/v) myrosinase in phosphate buffer solution (pH 6.6,
0.1 M) was added; after 10 min, a teaspoon (0.3 g) of egg
white was added and mixed with the enzyme solution. After
a further 10 min, 50 ␮l of 30% (w/w) glutaraldehyde solution
 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99
was dropped onto the surface of the membrane and left for
5 min. A spatula was gently used to spread the glutaraldehyde
solution thoroughly onto the membrane. The membrane was
washed with a 0.1 M phosphate buffer (pH 7.0) for 5 min.
Then, the myrosinase-immobilized eggshell membrane was
kept and stored in a 0.1 M phosphate buffer (pH 7.0) at 4 ◦ C
until further use.
2.2. Immobilization of glucose oxidase on eggshell
membrane
An eggshell membrane was peeled off from a broken fresh
eggshell after the albumen and yolk had been removed. It
was cleaned with a copious amount of DI water. The mem-
brane was placed in a clean watch glass and it was cut into
a circle of a diameter of 15 mm. A 200 ␮l aliquot of 0.8%
(w/v) glucose oxidase solution at pH 7.0 phosphate buffer
(25 mM) was added; after 20 min, 10 ␮l of 25% (w/w) glu-
taraldehyde solution as cross-linking agent was dropped onto
the surface of the membrane and stood for 5 min. A spatula
was gently used to spread the glutaraldehyde solution thor-
oughly onto the membrane. The membrane was then rinsed
with a 25 mM pH 7.0 phosphate buffer for 5 min. Finally, the
glucose oxidase-immobilized eggshell membrane was kept
and stored in a 25 mM pH 7.0 phosphate buffer at 4 ◦ C until
further use.
2.3. Assembly of optical glucose biosensor
The construction of the optical glucose biosensor was fully
described in our previous article [25]. In brief, the biosensor
was fabricated from a laboratory-made flow cell consisting
of an oxygen-sensitive membrane and a glucose oxidase-
immobilized eggshell membrane. The flow cell was posi-
tioned in a spectrofluorometer (Photon Technology Interna-
tional, London, Ontario, Canada) for fluorescence measure-
ments. A 1.0 ml aliquot of a freshly prepared standard or
sample glucose solution was injected into the flow cell with
the use of a 5.0 ml syringe. The fluorescence emission in-
tensity at 602 nm was monitored at an excitation wavelength
of 468 nm for 5 min. Afterwards, the fluorescence signal was
brought back to the baseline by passing a stream of phosphate
buffer through the biosensor before the next measurement
was undertaken. When the optical glucose biosensor was not
in use, the glucose oxidase-immobilized eggshell membrane
was taken from the flow cell and stored in a 25 mM phosphate
solution (pH 7.0) at 4 ◦ C. All the pH measurements were
taken on a combined pH glass electrode (Orion, Chicago,
IL).
2.4. Glucosinolate determination in vegetable samples
Glucosinolates are commonly found in cruciferous veg-
etables such as watercress, choi sum and kai choi and were
chosen for this study. For non-cruciferous vegetable, spinach
was also taken for analysis and comparison. All the vegetable
93
samples were bought from local markets. To prepare the veg-
etable sample, 100 g of vegetable sample was blanched in
the boiling water (100 ◦ C) for 5 min to inactivate the endoge-
nous myrosinase. Then it was washed under tap water and
cut into small pieces. The cooked vegetable was blended
with 200 ml of 0.1 M phosphate buffer (pH 6.5) for 1 min.
The mixture was wrapped with a filter cloth and squeezed
to release the solution into a 500 ml beaker. The sample so-
lution was centrifuged at 4000 rpm for 15 min and 100 ml
of the green supernatant was collected to mix successively
with the following cleaning reagents: (1) 2 ml of 10% (v/v)
NH4 OH, (2) 1 ml of 10% (v/v) acetic acid, (3) 3 ml of 22%
(w/v) zinc acetate, and (4) 3 ml of 10.6% (w/v) potassium
ferrocyanide 3-hydrate. After mixing, the mixture left for
15 min and then was centrifuged at 4000 rpm for 15 min.
The clear pale yellow supernatant was collected. A 20 ml
aliquot of this solution was adjusted to pH 6.5 by addition
of 0.1 M phosphate buffer (pH 6.5) and transferred into a
50 ml beaker containing a myrosinase-immobilized eggshell
membrane. The solution was incubated with the myrosinase-
immobilized membrane at 37 ◦ C for 16 h unless otherwise
stated. After the hydrolysis of glucosinates in the sample
solution, the amount of produced glucose from the sample
solution was subsequently determined by the optical glucose
biosensor. Another portion of sample solution not incubated
with the myrosinase-immobilized membrane, was similarly
analyzed for its background level of glucose.
3. Results and discussion
3.1. Response behavior of optical glucose biosensor
In this work, an optical glucose biosensor was employed to
determine the hydrolytic product, glucose, of glucosinolates
as displayed in Eq. (1). The detection scheme of the opti-
cal glucose biosensor is based on the depletion of dissolved
oxygen level when glucose is enzymatically oxidized, with
a concomitant increase in the fluorescence intensity of the
oxygen-sensitive membrane. An oxygen-sensitive membrane
was employed to measure the rate of oxygen consumption in
the enzymatic oxidation of glucose [25].
D-glucose + O2 + H2 O
glucose-oxidase
−→ D-glucose acid + H2 O2 (2)
The magnitude of the analytical signal of the glucose biosen-
sor is determined by the glucose concentration inside the oxy-
gen membrane, the amount or activity of glucose oxidase on
the eggshell membrane and the temperature of the biosensor.
The typical response curve of the glucose biosensor is dis-
played in Fig. 1. The relative signal change of the glucose
biosensor is much greater when the glucose solution is at the
stop flow mode than at the flowing mode. In the stop flow
mode, a depletion zone of oxygen forms in the enzyme layer
as glucose and oxygen react with a subsequently faster rate of
94 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99

Fig. 1. Response curves of the glucose biosensor fabricated from enzyme-immobilized eggshell membrane on exposure to various concentrations of glucose
solution: (1) 0.1, (2) 0.2, (3) 0.5, (4) 1.0, (5) 1.5, (6) 2.0, (7) 3.0, and (8) 4.0 mM glucose.

increase in fluorescence signal. In the flowing mode, oxygen
and glucose are constantly replenished and so only a smaller
depletion layer of oxygen forms. As a result, the stop flow
mode was applied to detect glucose in the sample solution. It
was observed that the rate of change in the fluorescence inten-
sity was related to the glucose concentration. The response
rate of the biosensor, R, is defined as:
(Iglucose − Ib )/Ib
R=
t after the incubation with a myrosinase-immobilized eggshell
where Ib and Iglucose represent the detected fluorescence sig-
nals from the glucose biosensor exposed to buffer solution
and glucose solution, respectively, and t is the time duration.
For our biosensor, t was taken as 5 min since a constant sig-
nal could only be obtained after at least 30 min. A calibration
curve using R (min−1 ) against G, concentration of glucose
(mM) was plotted and shown in Fig. 2. The curve has a sig-
moid shape, R = 0.354−[0.349/(1 + e(G−1.37)/0.282 )], ranging
from 0.0 to 4.0 mM. For glucose concentration at and above
2.0 mM, the biosensor showed lower sensitivity and the re-
sponse rate started to saturate. The response of the biosensor
to glucose is reversible by exposing the sensor to a phosphate
buffer for 1–2 min. The glucose biosensor has been demon-
strated to possess a long shelf-life and can be successfully
applied to the determination of glucose content in real sample
[25]. As such, it was used to accurately determine the glu-
cose concentrations of the vegetable sample both before and
membrane.
3.2. Effect of myrosinase loading on hydrolysis of
glucosinolate
Since the hydrolysis of glucosinolate depends on the en-
zymatic activity of the immobilized-enzyme on the eggshell
membrane, any change in the amount of myrosinase on im-

Fig. 2. Calibration curve of the glucose biosensor.

 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99
mobilization would affect the amount of glucose produced
from the sample solution. Myrosinase-immobilized eggshell
membranes were prepared by adding various amounts of
myrosinase on the eggshell membranes. Sinigrin was cho-
sen as the testing substrate in this work because it is a
kind of glucosinolates which can be purchased easily from
commercial market with high purity (>99.9%). In addi-
tion, it has very good water solubility and is convenient
for preparation of calibration standards and testing samples.
The myrosinase-immobilized eggshell membranes were in-
cubated with 2.0 mM sinigrin as substrate in 0.1 M phosphate
buffer (pH 6.5) at 37 ◦ C for 2 h. After the hydrolytic reaction,
the glucose generated was determined by the glucose biosen-
sor. The response rate of each solution was calculated. In
general, the amount of glucose produced from the hydroly-
sis reaction increased with the increase in enzyme loading
on the eggshell membrane. However, only a small increase
in the glucose produced was observed when the amount of
myrosinase reached 10 mg. Further increase in the amount
of enzyme used for immobilization did not produce better
result. Hence, 10 mg myrosinase was chosen as the optimum
enzyme loading for the eggshell membrane.
3.3. Effect of concentration of egg white on hydrolysis of
glucosinolate
As the hydrolytic efficiency of glucosinolate strongly de-
pends on the amount of immobilized enzyme on the eggshell
membrane, any change in the immobilized enzyme concen-
tration would affect the amount of glucose produced. How-
ever, the glucose production from sinigrin was only about
11% when the myrosinase and glutaraldehyde solutions were
used for enzyme immobilization process. Hence, some pro-
tein in natural material would be added to improve the ef-
ficiency of enzyme immobilization since it can form cross-
linking network with the enzyme and egg protein fiber such
that a larger amount of enzyme can be kept on the eggshell
membrane. Egg white is protein in nature and is cheap so it
was chosen for further improvement on the enzyme immobi-
lization method. Various concentrations of egg white ranging
25–100% (w/v) were added to each eggshell membrane in the
enzyme immobilization procedure. These membranes were
incubated with 2.0 mM sinigrin in 0.1 M phosphate buffer
(pH 6.5) at 37 ◦ C for 2 h. After the hydrolytic reaction, the
glucose generated was determined with the glucose biosen-
sor. The response rate of each solution was determined. The
amount of glucose produced from the hydrolysis of sinigrin
increased with the increase in egg white on the eggshell mem-
brane. The results illustrate that egg white can serve as a sup-
port material for binding or cross-linking of eggshell mem-
brane and myrosinase after the addition of glutaraldehyde
and myrosinase. It is also possible that egg white can reduce
the inactivation of the enzyme by glutarladehyde. After the
addition of glutaraldehyde and egg white, a thin layer of gel
was formed on the surface of the eggshell membrane, which
thus can retain a certain amount of myrosinase on the eggshell
95
membrane. The generation of glucose from the hydrolysis of
sinigrin increased 9-fold compared with that without the egg
white in the enzyme immobilization. As a result, egg white
was applied for myrosinase immobilization on the eggshell
membrane.
3.4. Effect of incubation temperature on hydrolysis of
glucosinolate
The enzymatic reaction rate of myrosinase on sinigrin at
different temperatures was investigated. The enzyme activ-
ity was strongly affected by the change in working temper-
ature due to its protein in nature. In general, the reaction
rate is faster with increase in working temperature. However,
the rate will decrease if the temperature is too high because
most enzymes can easily be denatured at high temperatures.
In this study, myrosinase-immobilized eggshell membranes
were subject to 1.6 mM of sinigrin at temperatures 32–60 ◦ C
for 2 h incubation. After the hydrolytic reaction, the glucose
generated was determined by the glucose biosensor. The re-
sponse rate of each solution at various temperatures was de-
termined and is shown in Fig. 3. After the completion of the
first set of experiments, all the membranes would be reused
for another set of experiments. The purpose of this study was
to investigate the possibility of reusing the membranes af-
ter low and high incubation temperature usages. For the first
set of experiments, the response rate increased from 32 to
55 ◦ C and then rapidly decreased at above 55 ◦ C. It is possi-
ble that the enzyme can acquire higher enzyme activities at
higher working temperatures. Thus, sinigrin is hydrolyzed at
a faster rate and produce more glucose in the enzymatic reac-
tion. On the other hand, the enzyme also starts to be denatured
at high temperatures with subsequent effect on decreasing the
enzyme activity. For the second set of experiments, the re-
sponse rate was fairly constant from 37 to 55 ◦ C and then
decreased at above 55 ◦ C. But the response rates were lower
than that of the first set of experiments especially when the
temperatures were high (50–60 ◦ C). This observation illus-
trates that high working temperatures can increase enzyme
activity but at the same time decreases enzyme working life-
time. At a lower temperature range such as 32–37 ◦ C, the re-
sponse rate was more or less constant. As such, the optimum
working temperature for myrosinase-immobilized eggshell
membrane was set at 37 ◦ C to maintain adequate enzymatic
reaction rate and thermal stability.
3.5. Effect of incubation time on hydrolysis of
glucosinolate
In general when the concentration of enzyme and sub-
strate is kept constant, the longer the incubation time, the
higher the product yield obtained. The effect of incubation
times, ranging between 8 and 24 h, on the hydrolysis of glu-
cosinolate was investigated by subjecting the myrosinase-
immobilized eggshell membranes to various concentrations
of sinigrin (0.2–1.6 mM) solutions containing 0.1 M phos-
96 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99

Fig. 3. Effect of incubation temperature on myrosinase-immobilized eggshell membrane: The membrane was subject to (a) first incubation test, and then it was
used for (b) second incubation test again.

phate buffer at pH 6.5. According to reaction (1), 1 mol of
glucose can be produced from 1 mol of sinigrin. Fig. 4 depicts
the recovery of glucose against the concentration of sinigrin
used at 8, 16 and 24 h incubation periods, respectively. For
an 8 h incubation period, the recovery of glucose was be-
low 80% for all amounts of sinigrin. For a 16 h incubation
period, the recovery of glucose was above 90% for all con-
centrations of sinigrin and reached almost 100% when the
amount of sinigrin was above 12 ␮mol. For a 24 h incuba-
tion period, the recovery of glucose reached 100% for all
the amounts of sinigrin. These results demonstrate that the
longer the incubation time, the higher amount of glucose is
formed. The hydrolytic reaction was incomplete especially
when the amount of sinigrin was high and a short incuba-
tion period of 8 h was employed. In this work, a 16 h incu-
bation time was sufficient to convert most of the sinigrin to
glucose.
3.6. Effect of buffer concentration on hydrolysis of
glucosinolate
The effect of phosphate buffer concentration on the hy-
drolysis of glucosinolate at 37 ◦ C for 16 h was investigated by
subjecting the myrosinase-immobilized eggshell membrane
to 1.2 mM sinigrin standards containing 10–200 mM phos-
phate at pH 6.5. The amount of glucose produced was then
determined by the glucose biosensor. The amount of glu-
cose generated was relatively constant throughout the range

Fig. 4. Effect of incubation time on the efficiency of myrosinase-immobilized eggshell membrane: (a) 8, (b) 16, and (c) 24 h incubation.
 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99
of 20–200 mM phosphate buffer. But when the concentra-
tion of phosphate buffer was 10 mM, the amount of glucose
produced was about 50% of that obtained from the other
phosphate concentrations. Thus, a sufficient concentration of
phosphate is required to maintain good enzymatic reaction of
immobilized myrosinase on sinigrin. In our previous article
[25], it was observed that the optimal phosphate concentra-
tion for the glucose biosensor was 1.0–100 mM. When the
concentration of phosphate buffer was increased to above
100 mM, the response rate of the glucose biosensor started to
decline upon the increase in phosphate concentration. As a
result, 20–100 mM phosphate buffer concentration was most
suitable for this proposed work in order to make the best
use of both glucose oxidase- and myrosinase-immobilized
eggshell membranes.
3.7. Effect of pH on hydrolysis of glucosinolate
It is well known that most enzyme activities are affected
by the pH value of the substrate solutions. The pH effect on
the hydrolysis of 1.2 mM sinigrin in 0.1 M phosphate buffer at
37 ◦ C for 16 h was investigated over the pH range of 5.0–8.0.
The amount of glucose produced was then determined by the
glucose biosensor. Fig. 5 shows the normalized response rate
of the biosensor against pH when the biosensor was subject to
glucose solutions, which were obtained from the hydrolysis
of sinigrin at various pH phosphate buffers. When the pH was
low or high, the amount of glucose produced was low. The re-
sponse rate could be maintained above 90% for the pH range
6.0–7.0. The results showed that the optimal pH value was
6.5 and this finding is in agreement with data previously de-
termined with other enzyme-immobilized membranes [14].
The myrosinase-immobilized eggshell membrane can func-
tion satisfactorily in the pH range from 6.0 to 7.0 and has a
relatively narrow pH working range.
Fig. 5. Effect of pH on myrosinase-immobilized eggshell membrane.
97
3.8. Shelf-life of myrosinase-immobilized eggshell
membrane
The shelf-life of the myrosinase-immobilized eggshell
membrane was tested over a 6-month period at 4 ◦ C. The
activity of the myrosinase-immobilized eggshell membrane
was evaluated each week by exposing it to 2.0 mM sinigrin
standards (pH 6.5) at 37 ◦ C for 2 h. The amounts of glucose
produced were then determined by the glucose biosensor. The
amount of glucose generated was relatively constant through-
out the investigation. The activity of the immobilized enzyme
was found to be above 95% of its initial value over the 6-
month period. There was no significant decrease in the ac-
tivity of the immobilized enzyme. The excellent shelf-life of
the immobilized myrosinase on eggshell membrane is pos-
sibly related to the biological compatibility of the eggshell
membrane with the enzyme. Eggshell membrane is mainly
composed of biological molecules and protein fibers, which
may supply polycations to stabilize the enzyme [28]. The net-
veined structure and the gas-permeable hydrophilic property
of eggshell membrane can provide an excellent biological
micro-environment for the enzyme to survive and maintain
its enzymatic activity.
3.9. Reproducibility test of the proposed method
In this work, the response time of the glucose biosensor
was taken as 5 min to record the change of the fluorescence
signal when the biosensor was in contact with a buffer solu-
tion and then switched to a known concentration of glucose
standard solution. The glucose biosensor response was com-
pletely reversible after a running buffer had flown through
for 1–2 min as displayed in Fig. 1. The results demonstrated
that it had a highly reproducible and reversible response
to glucose. The reproducibility of the method was assessed
98 M.M.F. Choi et al. / Enzyme and Microbial Technology 36 (2005) 91–99

Table 1
Reproducibility test for sinigrin using the biosensor and spectrophotometric methods
Concentration of sinigrin used (mM) Concentration of sinigrin (mM) determined by
Biosensora R.S.D. (%) Spectrophotometrya R.S.D. (%)
1.20 1.15 0.87 1.22 1.6
a Average value from 10 determinations.

in 10 different days. The proposed method was tested ten
times by subjecting the myrosinase-immobilized membrane
to 1.2 mM sinigrin solutions at 37 ◦ C for 16 h. The concen-
trations of glucose produced were measured by the glucose
biosensor and they were directly related to the initial sinigrin
concentrations (reaction 1). The results of the sinigrin analy-
sis obtained from the proposed method were also compared
with that of a spectrophotometric method [29] and are sum-
marized in Table 1. The above results show that this method
agrees well with the spectrophotometric method. It confirmed
that the proposed biosensor method exhibited very desirable
analytical features of excellent reproducibility and accuracy.
3.10. Sample analysis
Epidemiology studies suggest that brassica vegetables
have protective effects on cancers of lungs and alimentary
tract. Cruciferous vegetables are the dietary source of glu-
cosinolates. These compounds remain intact unless brought
into contact with the enzyme myrosinase by grinding, food
processing, or chewing. Myrosinase releases glucose and
isothiocyanates which inhibit mitosis and stimulate apoptosis
in human tumor cells [30]. Therefore, some cruciferous and
non-cruciferous vegetable samples imported from mainland
China were bought from local markets in different days to de-
termine their glucosinolate contents by the proposed biosen-
sor method. The results are depicted in Table 2. There were
some variations of the glucosinolate contents in the vegetable
samples possibly due to different sources and batches of the
samples. However, watercress was found to contain the high-
est content of glucosinolates among the vegetables examined.
Table 2
Determination of glucosinolate contents in various vegetables using the
biosensing method
Sample class Vegetable
Cruciferous Watercress Sample 1
Sample 2
Sample 3
Kai choi Sample 1
Sample 2
Sample 3
Choi sum Sample 1
Non-cruciferous Spinach Sample 1
Sample 2
a Not detected.
No detectable glucosinolates was found in the spinach sam-
ple. All the results were consistent with the findings in the
literature [31].
4. Conclusion
The biosensor method described was successfully applied
to determine of glucosinolate content in some vegetables.
The myrosinase-immobilized eggshell membrane shows ex-
cellent stability with a long shell-life of at least 6 months
and its enzyme activity is still being monitored. The enzyme
membrane can be used repeatedly for many months with no
sign of degradation in enzymatic activity. In essence, the pro-
posed work demonstrates successfully the use of a bioma-
terial, such as eggshell membrane, as a promising enzyme
immobilization platform.
Acknowledgments
The authors gratefully acknowledge the financial sup-
port provided by the HKBU Faculty Research Grant through
project FRG/01-02/II-08.
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