Biosensors and Bioelectronics 24 (2009) 14171423

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Biosensors and Bioelectronics
j our nal homepage: www. el sevi er . com/ l ocat e/ bi os
Wireless enzyme sensor system for real-time monitoring of blood glucose
levels in sh
Hideaki Endo
a,
, Yuki Yonemori
a
, Kyoko Hibi
a
, Huifeng Ren
a
, Tetsuhito Hayashi
a
,
Wakako Tsugawa
b
, Koji Sode
b
a
Department of Ocean Sciences, Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
b
Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan
a r t i c l e i n f o
Article history:
Received 13 April 2008
Received in revised form 9 August 2008
Accepted 11 August 2008
Available online 2 September 2008
Keywords:
Biosensor
Enzyme sensor
Glucose
Blood
Wireless
Telemetory
Real-time
Monitoring
Fish
a b s t r a c t
Periodic checks of sh health and the rapid detection of abnormalities are thus necessary at sh farms.
Several studies indicate that blood glucose levels closely correlate to stress levels in sh and represent
the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to
determine blood glucose levels in sh. It can be rapidly and conveniently monitored using the newly
developed needle-type enzyme sensor, consisting of a PtIr wire, Ag/AgCl paste, and glucose oxidase.
To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor
response, the PtIr electrode was coated with Naon, and then glucose oxidase was immobilized on the
coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl,
and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor sh glucose levels. The
sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The uid
of the inner sclera of the sh eyeball (EISF) was a suitable site for sensor implantation to obtain glucose
sample. There was a close correlation between glucose concentrations in the EISF and those in the blood.
Glucose concentrations in sh blood could be monitored in free-swimming sh in an aquariumfor 3 days.
2008 Elsevier B.V. All rights reserved.
1. Introduction
In recent years, mass outbreaks of pathogenic bacterial infec-
tions have killed large quantities of sh, causing great nancial
damage toshfarms. Major disease outbreaks typically occur when
the balance among the sh, the etiologic agent, and the environ-
ment is disturbed. To prevent or reduce the severity of epizootics,
impaired disease resistance in sh must be detected at the earli-
est opportunity. Periodic checks of sh health for early detection
of abnormalities are thus necessary at sh farms. Further, it is
important to establish farming techniques with minimal depen-
dence on antibiotics and antimicrobial agents for farming safety
and high-quality cultured sh. To address this problem, heme-
analysis methods for sh blood have been a recent focus in sh
physiology. Several studies have demonstrated that blood glucose
levels closely correlate to the stress levels in sh and are rep-
resentative of the respiratory and nutritional state (Carballo et
al., 2005; Chowdhury et al., 2004; Jentoft et al., 2005; Trenzado
et al., 2003; Van Anholt et al., 2004). In addition, blood choles-

Corresponding author. Tel.: +81 3 5463 0616; fax: +81 3 5463 0616.
E-mail address: endo@kaiyodai.ac.jp (H. Endo).
terol levels are a useful indicator of reduced resistance to bacterial
infection (Maita et al., 1998a,b). Therefore, monitoring of blood
glucose and cholesterol levels is important for ascertaining sh
health. These tests are currently conducted using clinical labora-
tory test kits designed for humans. Furthermore, as each sample
needs to be analyzed separately, testing is time- and labor-
intensive. For sh health testing at sh farms, it is necessary
to test as many sh as possible in a fast and convenient man-
ner.
Recently, researchers have beenactively investigating the devel-
opment of biosensors for measuring blood components such as
glucose (Hiratsuka et al., 2001; Ishikawa et al., 1998; Jeong et al.,
2003; Quinto et al., 2000; Wang and Zhang, 2001), cholesterol
(Bertrand et al., 1979; Bongiovanni et al., 2001; Karube et al., 1982;
Motonaka and Faulkner, 1993; Satoh et al., 1997) and lactic acid
(Wu et al., 2000). Wang and Zhang (2001) developed a miniature
needle-type enzyme sensor suitable for simultaneous amperomet-
ric monitoring of glucose and insulin. The sensor was constructed
from dual modied carbon-paste working electrodes inserted into
a 14-G needle. Wu et al. (2000) reported an enzyme sensor for
whole blood lactate monitoring. The sensor consisted of a stainless
steel needle with the surface modied for enzyme immobilization
using a polymer material. Studies of non-invasive glucose sen-
0956-5663/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2008.08.038
1418 H. Endo et al. / Biosensors and Bioelectronics 24 (2009) 14171423
sors have also been undertaken for application in patients with
diabetes (Caduff et al., 2003; Gourzi et al., 2003, 2005). Caduff
et al. (2003) developed a glucose monitoring system based on
impedance spectroscopy. Changes inglucose concentrations canbe
monitored by varying the frequency in the radio band over a range
optimized to measure the impact of glucose on the impedance pat-
tern. Most sensor systems, however, are designed for humans or
livestock.
We recently developed a biosensor to measure cholesterol lev-
els in sh plasma in an attempt to establish rapid and convenient
methods for ascertainingshhealth(Endoet al., 2003). Becausethis
method is based on ow injection analysis (FIA), continuous mea-
surement is possible and only 5min is required for analysis of each
sample, markedly reducing testing time. Even this method, how-
ever, requires a certain amount of skill to draw blood, and because
the method is based on FIA, the measurement apparatus itself is
unwieldy and complicated. Tests can be performed in a laboratory,
but testing at sh farms is difcult and inconvenient. To establish
a simple and rapid method to determine blood glucose levels in
sh, we developed a needle-type enzyme sensor system (Endo et
al., 2006). The sensor consists of a needle-type hollow container,
immobilized enzyme membrane, and ber optic probe contain-
ing a ruthenium complex. The enzyme membrane was prepared
from glucose oxidase, AWP (azide-unit dependent water-soluble
photopolymer), and ultra-thin dialysis membrane. The ber optic
probe was inserted into the rolled enzyme membrane placed in
the needle-type hollow container. The calibration curve was linear
in the range of 10180mg/dl glucose in sh blood (Nile tilapia).
The sensor was inserted into the caudal vein of the sh to mea-
sure blood glucose levels. A very strong correlation was observed
between values determined by the sensor and conventional meth-
ods, in the range of 48157mgdl
1
(correlation coefcient: 0.9474,
y =0.8452x 3.4018). As the sensor, spectrometer, and personal
computer are small; this test system is easily transportable for use
at sh farms. It is still necessary, however, to capture sh using a
spoon-net from the sh tank to obtain the blood sample for each
measurement. Further, it is difcult to measure blood components
inreal-timebecausesensor output decreases over timeduetoblood
coagulation and protein (e.g., albumin, -globulin) coalescing on
the sensor.
Glucose biosensor systems have developed remarkably recently
in the medical eld. Minimed Inc. developed a continuous glucose
monitoring system that allows blood glucose to be continuously
monitored for 72h (Mastrototaro, 2000). The system does not
measure blood glucose levels, but rather, the glucose levels in
the interstitial uid (ISF) of subcutaneous tissue surrounding the
sensor. Briey, the method is based on the principle that ISF
glucose levels in subcutaneous tissue reect blood glucose lev-
els. Glucose kinetics can be described using a two-compartment
model of the separated capillary blood glucose and tissue bed
(Rebrin and Steil, 2000). Even in sh, the two-compartment model
might be applicable for continuous glucose monitoring in subcu-
taneous tissue. We focused on the uid-lled inner eyeball sclera
(eyeball sclera ISF; EISF) and hypothesized that if the glucose dif-
fusion rate is constant in the EISF as in the two-compartment
model, scleral ISF can be used for monitoring glucose levels in
sh.
The present paper describes the steps toward developing a
rapid and convenient method for real-time monitoring blood glu-
cose levels in sh, as follows: (1) investigation of the relationship
between EISF and blood glucose levels in sh, (2) preparation
of needle-type enzyme sensor using exible wire electrodes for
implanting into the sh body, (3) wireless monitoring of blood
glucose levels under free-swimming conditions in an aquar-
ium.
2. Materials and methods
2.1. Reagents
Glucose oxidase (from Aspergillus niger; E.C. 1.1.3.4, type VII-
S; 197000unit/g) was purchased from SigmaAldrich (St. Louis,
MO). Bovine serum albumin (BSA) and 2-phenoxy ethanol were
purchased from Wako Pure Chemical Industries (Tokyo, Japan) d-
glucose was dissolved in 0.1Mphosphate buffered saline (PBS) and
allowedto mix by the rotationfor 24hbefore use. All other reagents
used for the experiments were commercial- or laboratory-grade.
2.2. Relationship between blood and EISF glucose levels
Nile tilapia (Oreochromis niloticus) cultured at Tokyo University
of Marine Science & Technology was used as the model sh. A test
was performed to conrm whether the temporal change in blood
glucose levels is reected by a change EISF glucose levels. A 50-
l sh tank (60cm30cm36cm) was used. During the test, the
water temperature was 30

Cwithcontinuous aerationandrecircu-
lation through a biologic lter. The photoperiod was set with lights
on from 10:00 to 20:00h provided by a uorescent light. The sh
(body weight, ca. 300g; body length, ca. 25cm) were netted from
the preserve andanesthetizedwith0.1%2-phenoxy ethanol by bath
exposure for 5min. Blood was then sampled from the Cuvierian
duct (Ikeda et al., 1985) using a heparinized syringe tted with
a 27-G needle. The blood samples (100500l) were centrifuged
(650g, 10min) to separate the plasma. EISF sampling was per-
formed as follows; while slightly pushing on one eye, a blister-like
membrane rose up to the surface. A heparinized syringe tted with
a 27-G needle was then inserted into the blister-like membrane.
EISF samples (50100l) were obtained from the ISF of the eye-
ball sclera. To reduce measurement error due to the stress induced
by handling the sh, the sampling procedure was restricted to less
than 5min per sh. EISF samples were transferred to test tubes and
stored at 80

C before use. Blood and EISF samples were collected

at the same hour each day using the procedure described above.
The test was performed over 27 days and the sh were fed at days
5 and 16, 4h before glucose measurement, to evaluate whether
the change in the blood glucose concentration was continuously
reected by the EISF glucose concentration.
Glucose levels were determined using an enzymatic colorimet-
ric method (C-II glucose test; Wako Pure Chemical Industries).
When each sample (20l) was mixed with 3ml PBS (pH 7.1) con-
taining enzymes (glucose oxidase, horseradish peroxidase) and the
color-producing reagent, the glucose was oxidized, producing glu-
conolactone and hydrogen peroxide. The hydrogen peroxide was
reacted with 4-aminoantipyrine and phenol to produce a red color.
Glucose concentrations were measured using a UV/vis spectropho-
tometer (JASCO Co., Tokyo, Japan)
2.3. Enzyme sensor preparation
A needle-type enzyme sensor was prepared for subcutaneous
glucose monitoring in sh. Fig. 1(a) shows the schematic of the
needle-type enzyme sensor. A working electrode was made using
an18mmlengthof Teon-coatedplatinumiridiumwire. The Teon
was stripped at one end to expose 1mm of the PtIr wire as a
sensing cavity. Copper wire was wrapped around the Teon coated
surface. An Ag/AgCl paste (BAS, Tokyo, Japan) wire was used as a
reference electrode/counter electrode. The tip of the wire (2mm)
was sealed with epoxy resin. The sensing cavity was dipped in 5%
Naon solution and dried for 10min. The Naon-coated electrode
was dipped in 6% acetyl-cellulose solution and dried for 10min.
An enzyme solution containing 2mg (352unit/ml) glucose oxi-
H. Endo et al. / Biosensors and Bioelectronics 24 (2009) 14171423 1419
Fig. 1. Schematic diagramof the wireless monitoring systemfor sh. 1, needle-type
enzyme sensor; 2, waterproof sheet; 3, wireless potentiostat; 4, nylon threads; 5,
receiver; 6, personal computer; 7, sample sh (Nile tilapia).
dase and 20mg BSA dissolved in 1ml 0.1M phosphate buffer (pH
7.8) was freshly prepared. The Naon and acetyl-cellulose-coated
electrode was dipped in the enzyme solution and air-dried for
10min; this procedure was repeated twice. The electrode was held
in a vertical position in a vial, and 100-l glutaraldehyde (50%)
was added to induce cross-linking between the glucose oxidase
and BSA. The electrodes were stored at 30

C for 6h. The sensors

were soaked and stored in PBS (pH 6.5) overnight at 4

C before
use.
The prepared sensor was connected to a wireless potentiostat
(3102BP, Pinnacle Technology Inc., Lawrence, KS), and the measure-
ment was performedusingareceiver (3100RX, PinnacleTechnology
Inc.) and a personal computer (Dimension 4700C, DELL, Round
Rock, TX). A 650-mV potential (vs. Ag/AgCl) was applied by the
potentiostat to the PtIr working electrode for the amperometric
glucose measurement. Hydrogenperoxide, producedby anenzyme
reaction inducing glucose oxidation, was detected by an electrocat-
alytic oxidation reaction at the sensing cavity of the PtIr working
electrode, producing an electric output current.
2.4. Measurement of glucose standard solution
For the amperometric glucose measurements, the sensor was
immersedintoacell consistingof 10ml of 0.1MPBS(pH6.5) at 25

C
and dissolved oxygen was saturated with stirring. The background
output current was allowed to stabilize before measurement. A
glucose standard solution was then added to the cell and the
response time to reach 90% steady state level (T90) was mea-
sured. When the sensor output had stabilized, the procedure was
repeated until the sensor output reached the rate-limiting enzyme
phase.
2.5. Sensor implantation and device immobilization
A schematic diagram of the wireless monitoring system is
shown in Fig. 1(b). The systemwas constructed using a needle-type
enzyme sensor, a wireless potentiostat, a receiver, and a personal
computer. Sensor implantation and xation was performed using
the following procedure. Nile tilapia was starved for 24h before
continuous glucose monitoring. First, the sh were anesthetized in
a water bath containing 0.1% 2-phenoxy ethanol for 5min. A 22-
G catheter consisting of an outer Teon layer and inner puncture
needle (23mm, SerowTM, Terumo Co, Tokyo, Japan) was inserted
into the ISF of the eyeball sclera for sensor implantation. The inner
puncture needle was removed and the excess exposed catheter
on the skin was removed. The sensor was then inserted into the
outer Teon layer and xed in place using biomedical adhesive
(Aronalpha-A Sankyo, Toagosei, Tokyo, Japan), which contained
an ethylcyanoacrylate monomer as the major ingredient. Water-
proong the device was essential because the wireless potentiostat
was not designed to work under water. The potentiostat was cov-
ered using a waterproof polypropylene sheet (Honda Motor Co.
Ltd., Tokyo, Japan) and sealed with a thermo-compression bond-
ing device (NL-101J, Ishizaki Electric Mfg Co. Ltd., Tokyo, Japan).
The waterproofed wireless potentiostat was attached to the dorsal
andpectoral ns of the shusing nylonthreads andthenconnected
to the sensor.
2.6. Monitoring of blood glucose levels in sh
Nile tilapia equipped with the sensor systemwas transferred to
a 50-l sh water tank (60cm30cm36cm) and allowed to swim
freely. Dissolved oxygen in the water tank was saturated using an
air pump. At rst, to compare between directly measured blood
glucose levels and sensor-estimated glucose levels, blood was col-
lected and blood glucose levels were measured using a Glucocard
(Arkray KDK Co. GT 1640, Kyoto, Japan). After a few minutes, the
sh were placed back in the tank, and the sensor output current
was recorded via computer-controlled data acquisition software
(Pinnacle Acquisition Laboratory, Pinnacle Technology).
The estimation of blood glucose levels was performed using a
one-point calibration method or a two-point calibration method
in vivo. For the one-point calibration method, the sensor sensitiv-
ity S was determined from a single blood glucose measurement
as the ratio between the sensor output current I and the blood
glucose level G. Glucose levels were then estimated fromcurrent
I using the equation G(t) =I(t)/S. The sensor output current was
rst allowed to stabilize after the sensor was implanted into the
sclera. Bloodglucosewas thensampledfromtheCuvierianduct and
the blood glucose level G was measured. These parameters (I, G)
wereusedas thecalibrationreference. For thetwo-point calibration
method, the sensor sensitivity S, expressed in (nA)/(mg/dl), and
the intercept I
0
with the sensor output (current, expressed in nA)
axis, i.e., the theoretical sensor output that would be observed in
the absence of glucose in ISF as I
0
=I
1
G
1
S. These parameters S
and I
0
were subsequently used to transformthe sensor output I(t)
to estimate glucose levels G(t) using the equation G(t) =(I(t) I
0
)/S.
A method for determining S and I
0
takes into account, during a
change in blood glucose levels G from G
1
to G
2
, the change
in the sensor current I from I
1
to I
2
: S =(I
2
I
1
)/(G
2
G
1
), and
I
0
=I
1
(S G
1
).
1420 H. Endo et al. / Biosensors and Bioelectronics 24 (2009) 14171423
Fig. 2. Relationship between blood and eyeball ISF (EISF) glucose levels. Nile tilapia
was bred in a 50-l sh tank. The water temperature was 30

C with continuous
aeration and recirculation through a biologic lter. The photoperiod was set with
lights on from 10:00 to 20:00h provided by a uorescent light.
3. Results and discussion
3.1. Relationship between blood and EISF glucose levels
As described above, continuous monitoring of the blood com-
ponents is difcult using a biosensor because the sensor output
decreases over time due to blood coagulation and coalescing pro-
tein on the sensor. Additionally, implantation of enzyme sensors
into sh blood vessels needs expertise due to their ne vascular
anatomy. Therefore, we focused on EISF and hypothesized that if
the glucose diffusion rate is constant in the EISF as in the two-
compartment model, the scleral ISF can be used as the sensor
implantation site in sh. The relationship between blood and EISF
glucose levels was therefore investigated. Continuous changes in
glucose levels following feeding were investigated in the blood and
EISF. Fig. 2 shows the temporal changes in glucose levels in Nile
tilapia. Blood glucose levels were almost equal to EISF glucose lev-
els. Additionally, after feeding, glucose levels in the EISF increased
along with an increase in the blood glucose levels. These glucose
levels were4050mg/dl higher thannormal glucoselevels. Theglu-
cose levels returnedto the normal 30hafter feeding. These ndings
suggest that blood glucose levels are rapidly reected by EISF glu-
cose levels. The glucose levels ranged from20 to 60mg/dl. Because
the average glucose level in Nile tilapia is generally in the range of
50mg/dl (Nolanet al., 1999), these measuredvalues are reasonable.
These ndings indicated that blood glucose levels can be estimated
from the EISF in sh.
3.2. Response curve and calibration curve of the sensor
The most common glucose amperometric enzyme sensors are
based on the electrochemical detection of hydrogen peroxide pro-
duced by the enzymatic oxidation of glucose. Within the living
body, there is also concomitant electro-oxidation of many inter-
fering species, such as uric acid and ascorbic acid. Naon lm is
highly selective due to its negatively charged sulfonic functional
groups (Harrison et al., 1988; Hu et al., 1994). In this study, to pre-
vent the effects of these interfering species on the sensor response,
thePtIr electrodewas coatedwithNaonandthenglucoseoxidase
was immobilized on the coated electrode. Fig. 3(a) shows a typical
response curve of the needle-type enzyme sensor equipped with
a Naon-coated membrane for various substrates, such as glucose,
ascorbic acid, and uric acid. The sensor was immersed into a cell
containing 10ml of 0.1M PBS (pH 6.5) at 25

C and dissolved oxy-

gen was saturated with stirring and then each substrate solution
was added to the cell. For the glucose solution (0.1mM; 18mg/dl),
the sensor output increased within 30s after adding the sample,
and a maximum current was obtained within 60s. When ascorbic
acid (0.1mM) or uric acid solution (0.1mM) was added to the cell,
the sensor output current did not change. Without Naon, the sen-
sor produced a clear response when ascorbic acid or uric acid was
addedtothecell (Fig. 3(b)). Therefore, theNaoncoatingis required
for in vivo glucose monitoring.
We investigated the relationship between sensor output and
the glucose standard solution concentration (Fig. 4). The cali-
bration curve of the glucose concentration was linear from 0.18
to 144mg/dl (y =0.3315x +1.013, R=0.9983), and the detection
limit was 0.18mg/dl. The reproducibility of the sensor output was
demonstrated by using a single electrode to make 20 measure-
ments of an 18-mg/dl glucose solution (Table 1). Five needle-type
enzyme sensors were examined. The sensors were evaluated based
Fig. 3. Typical response curves of a needle-type enzyme sensor with and with-
out Naon membrane. (a) Enzyme sensor with Naon; (b) enzyme sensor without
Naon. Sample solution was added in pH 6.5 PBS under a +650-mV potential (vs.
Ag/AgCl) for 25

C.
H. Endo et al. / Biosensors and Bioelectronics 24 (2009) 14171423 1421
Fig. 4. Relationship between sensor output and the glucose standard solution con-
centration. The assay conditions were the same as those described for Fig. 5.
on the relative standard deviation because sensor output current
varies fromlot to lot. The sensors had good reproducibility for stan-
dard glucose measurement. (R.S.D.%=1.93). The low R.S.D. values
indicated that these glucose biosensors can be used to determine
glucose withsatisfactory precisionandcantherefore provide stable
and accurate continuous in vivo glucose measurements.
3.3. Correlation of actual blood glucose levels and glucose levels
estimated using the wireless monitoring system
The needle-type enzyme sensor was used for wireless mon-
itoring of blood glucose levels in sh. To compare actual blood
glucose levels and sensor-estimated glucose levels for calibra-
tion, Nile tilapia was anesthetized with 0.1% 2-phenoxy ethanol
by bath exposure for 5min, and then the wireless sensor system
was attached. The sh was transferred to a 50-l sh water tank
containing 1ppmof 2-phenoxy ethanol as a light-anesthetic agent.
After 30min, the output current of the sensor was monitored when
the shbeganswimming. After continuous monitoring for 180min,
blood samples were collected from sh several times and the sh
were placed back into the tank. Fig. 5 shows the response curves
for in vivo continuous glucose monitoring over 180min with sh
underlight-anesthesia. As shown in Fig. 5(a), initial glucose lev-
els were 85mg/dl (point (o)). The initial sensor output current
increased with an increase in blood glucose levels.
Sensor-calibratedglucose levels were estimatedusing either the
one-point or two-point calibration method. Fig. 5(b) and (c) rep-
Table 1
Reproducibility of glucose measurements using needle-type enzyme sensor
Electrode no. Standard
deviation (S.D.)
Relative standard
deviation (R.S.D.%)
1 1.31 1.38
2 0.70 2.29
3 0.61 2.77
4 2.97 1.66
5 2.70 1.55
Average 1.66 1.93
resents blood glucose levels and sensor-calibrated glucose levels
calculated using both calibration methods. The estimated glucose
levels using both calibration methods paralleled the blood glucose
levels. In Fig. 5(b), one-point calibration (1) was used as calibra-
tion value (G
1
, I
1
) in which G
1
was the initial blood glucose level
of 88mg/dl and I
1
was the subsequently obtained sensor output
current (1.59nA) at 30min (0min on the monitoring time) after
sensor implantation (point (o)). The one-point calibration (2) was
calculated using as calibration value (G
2
, I
2
) in which the blood glu-
cose level and sensor output current obtained 45min (15min on
the monitoring time) after sensor implantation (G
2
, 261mg/dl; I
2
,
7.81nA; point (p)). As shown in the gures, the one-point calibra-
tion (2) yielded more accurate glucose monitoring than one-point
calibration (1), except at 30min of the monitoring time, possibly
due to the saturation of the sensor output current when the actual
blood glucose level was 430mg/dl because the calibration curve
Fig. 5. Wireless monitoring of glucose levels for short-term underlight anesthe-
sia. (a) Comparison between the change in blood glucose levels and sensor output
current; (b) Comparisonbetweenbloodglucose levels andsensor-calibratedglucose
levels calculatedusingone-point calibration; (c) Comparisonbetweenbloodglucose
levels and sensor-calibrated glucose levels calculated using two-point calibration.
Sensor implantation and xation was performed at 30min.
1422 H. Endo et al. / Biosensors and Bioelectronics 24 (2009) 14171423
of the sensor was linear for glucose levels ranging from 0.18 to
144mg/dl (Fig. 4). Moreover, in the one-point calibration method,
not enough time was allowed for the background output current
(I
0
) to stabilize, resulting in an inaccurate calibration reference (I,
G). In animals, approximately 2h is necessary for the background
current to stabilize (Rebrin et al., 1999). Therefore, the one-point
calibration method should be performed at least 45min after sen-
sor implantation.
On the other hand, the two-point calibration utilized the rst
parameter (I
1
; 1.59nA, G
1
; 88mg/dl) (point (o)) at 30min and the
second parameter (I
2
; 7.81nA, G
2
; 261mg/dl) (point (p)) at 45min
after sensor implantation (Fig. 5(c)). The sensor-estimated glucose
levels and actual blood glucose levels were well-correlated. How-
ever, despite its theoretical superiority, the two-point calibration
method is a time-consuming procedure and is susceptible to mea-
surement errors (Choleau et al.). Moreover, although the two-point
calibration procedure can be used to determine sensor sensitiv-
ity (S) and the background current (I
0
) by obtaining the current
responses (I
1
, I
2
) to two different glucose levels (G
1
, G
2
), it is very
difcult to obtain blood samples from unanesthetized sh under
free-swimming conditions and without an anesthetic. Therefore, in
this study, the one-point calibration method (the calibration time
point was better than 45min after sensor implantation) was used
to monitor the glucose levels in sh.
3.4. Wireless monitoring of glucose levels in sh
We used the needle-type enzyme sensor to wirelessly monitor
glucose levels in free-swimming sh in an aquarium. Nile tilapia
was anesthetized with 0.1% 2-phenoxy ethanol, and then the wire-
less sensor systemwas attached. The sh were transferred to a 50-l
sh water tank containing no anesthetic. The sh became active
within 30min. The time courses of the output current of the sen-
sor and sensor-calibrated glucose levels are shown in Fig. 6. The
calibrationtime point was 2hafter sensor implantationfor the one-
point calibration method. Initially, the glucose levels were high,
which may have been caused by stress induced by xation of the
sensor or by the sensor itself. The levels decreased gradually and
stabilized after 24h. After feeding, glucose levels again increased
and the maximum value was 70mg/dl at 48h. Subsequently, the
glucose levels decreased gradually and stabilized over 60h. In this
experiment, the glucose levels infree-swimmingshcouldbe mea-
sured for 3 days inthe aquarium. Inmore than10 replications of the
experiment, the sensor response was similar. Although the sensor
was not sterilized prior to implantation into the sh, there was no
apparent effect on the active conditions of the sh. However, mon-
itoring with the wireless potentiostat could not continue for more
Fig. 6. Wireless monitoring of blood glucose levels in sh. Nile tilapia equipped
with the sensor systemwas placed in a 50-l sh water tank without anesthesia and
allowed to swim freely.
than3days because of insufcient battery power. This newly devel-
oped sensor provided rapid and convenient real-time monitoring
of glucose levels in sh.
4. Conclusion
The present ndings conrm that blood glucose levels in sh
can be rapidly and conveniently monitored using the newly devel-
opedneedle-typeenzymesensor, consistingof aPtIr wire, Ag/AgCl
paste, and glucose oxidase. To prevent the effects of interfering
anionic species, such as uric acid and ascorbic acid, on the sen-
sor response, the PtIr electrode was coated with Naon, and then
glucose oxidase was immobilized on the coated electrode. The cal-
ibration curve of the glucose concentration was linear, from 0.18
to 144mg/dl, and the detection limit was 0.18mg/dl. Naon coat-
ing was useful for monitoring the glucose concentration in vivo.
The sensor was used to wirelessly monitor sh glucose levels. The
sensor-calibrated glucose levels and actual blood glucose levels
were in excellent agreement. The uid of the inner sclera of the
sh eyeball (Nile tilapia) was a suitable sampling site to obtain ISF.
Therewas aclosecorrelationbetweenglucoseconcentrations inthe
EISF and those in the blood. Glucose concentrations in sh blood
could be monitored in free-swimming sh in an aquarium for 3
days. In future studies, we aimto solve the rapid battery consump-
tion problem and apply the sensor system to various types of sh
such as carp, rainbow trout, and eel.
Acknowledgments
This research was supported in part by a Grant-in-Aid for Scien-
tic Research (B) from The Ministry of Education, Culture, Sports,
Science and Technology, and a Grant-in-Aid from the Faculty of
Marine Science, Tokyo University of Marine Science and Technol-
ogy.
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