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IEEE SENSORS JOURNAL, VOL. 14, NO. 1, JANUARY 2014

Fabrication of Arrayed Flexible Screen-Printed

Glucose Biosensor Based on
Microfluidic Framework
Jung-Chuan Chou, Member, IEEE, Ya-Li Tsai, Tsung-Yi Cheng, Yi-Hung Liao, Member, IEEE,
Guan-Chen Ye, and Shu-Ying Yang

Abstract This paper uses a simple, fast, and low-cost method

to fabricate an arrayed flexible glucose biosensor device. The
ruthenium dioxide (RuO2 ) sensing membranes were deposited
on the flexible polyethylene terephthalate substrate by radio
frequency sputtering technology. The silver paste was fabricated
as conducting wire and the epoxy was fabricated as insulation
layer by screen-printed technique. The glucose oxidase and
nafion were prepared as glucose sensing membrane. The glucose
sensing membranes were dropped on RuO2 sensing membranes
by entrapment method. The arrayed flexible glucose biosensor
was measured in different concentrations of glucose solution.
Finally, the arrayed flexible glucose biosensor was integrated in
microfluidic to measure characteristics at the dynamic condition.
All experimental results demonstrated that the arrayed flexible
glucose biosensor based on microfluidic had a fast potentiometric
response (<10 s) to glucose, high sensitivity, and a good linear
voltage-time relation over a wide range of glucose concentrations
from 100 to 400 mg/dL.
Index Terms Radio frequency sputtering technology,
polyethylene terephthalate, screen-printed technique, ruthenium
dioxide, microfluidic.

I. I NTRODUCTION

HE determination of glucose concentration is very important in biology, chemistry, food processing and fermentation, as well as in clinic area for diagnosing diabetics [1].
In recent years the lifestyles and diet habits changed, more
and more people suffered from diabetes in the world. The
diabetes have become the modern disease gradually. According
to the world health organization (WHO) expresses that there

Manuscript received May 28, 2013; revised July 27, 2013; accepted
August 19, 2013. Date of publication September 5, 2013; date of current
version November 1, 2013. This work was supported by the National Science
Council of China under Contracts NSC 100-2221-E-224-017, NSC 101-2815C-224-033-E, NSC 101-2221-E-224-046, and NSC 102-2221-E-224-075.
The associate editor coordinating the review of this paper and approving it
for publication was Prof. Manuel Quevedo-Lopez.
J.-C. Chou, T.-Y. Cheng, and G.-C. Ye are with the Graduate School of Electronic and Optoelectronic Engineering, National Yunlin University of Science
and Technology, Douliou 64002, Taiwan (e-mail: choujc@yuntech.edu.tw;
m10013312@yuntech.edu.tw; g9918722@yuntech.edu.tw).
Y.-L. Tsai is with the Department of Electronic Engineering, National
Yunlin University of Science and Technology, Douliou 64002, Taiwan (e-mail:
u9813043@yuntech.edu.tw).
Y.-H. Liao is with the Department of Information Management, Transworld
University, Douliou 64002, Taiwan (e-mail: liaoih@twu.edu.tw).
S.-Y. Yang is with the Department of Information Management, Fortune Institute of Technology, Kaohsiung 83160, Taiwan (e-mail: allison@
center.fotech.edu.tw).
Color versions of one or more of the figures in this paper are available
online at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JSEN.2013.2280455

are around 347 million people suffering from diabetes in

2012 [2]. Diagnosis and treatment of diabetes require a tight
daily monitoring of the blood glucose level. Fast and accurate
determination of glucose concentration are very important.
But it is wasting time and money consuming for diabetics
to go to hospital for routine examination. Moreover, it needs
lots of blood volumes and operates with professionals. These
are inconvenient to allow some patients to achieve real-time
measurement. If the glucose biosensor can be miniaturized and
reduced cost, that are beneficial for development. Thus we
use screen-printed technique to fabricate the arrayed flexible
glucose biosensor. This can reduce cost due to its mass
production technology [3], [4]. Also we devoted to develop a
glucose biosensor which can keep high measurement region,
improve rate of detection, portability, simple to use and
improve reproducibility that are beneficial for diabetics selfmonitoring.
In the production of cost-effective biosensors, thick film
screen-printed technique is considered essential. Generally, a
paste or ink is screened onto a flat substrate by a squeegee
through the openings on a polymeric screen transferring a
designed pattern onto the substrate. This mass production technology has been used to produce planar patterns of electrodes
cost-effectively and with high degree of reproducibility [5].
The screen-printed technique originated from Nagata and
Karube et al. [6] that was used to fabricate the amperometric
biosensor for detecting glucose in 1995. The measuring range
of glucose biosensor was from 0.5 mg/ml to 2.0 mg/ml.
In addition, Laschi and Miscoria et al. [7] used screen-printed
technology to fabricate the rhodium (Rh) metal for working
electrode to detect the glucose concentration in 2006. The
measuring range was from 0.0045 mg/ml to 2.70 mg/ml.
Ruthenium dioxide (RuO2 ) is one of the most promising
materials and has been more widely used in pH sensors,
biosensors and super capacitors due to its chemical stability
and high conductivity which inhibits the space charge accumulation [8]. The literatures [9], [10] proposed the advantages
of RuO2 , such as high conductivity, high surface area, and
excellent enzyme attachment. Therefore, the RuO2 is suitable
for fabricating the membrane of biosensor.
In the recent years, people have great attention to microfluidic. Because it has outstanding advantages for many related
productions [11] such as reducing sample volume [12][14],
reducing weight and portability [15], [16]. Microfluidic is
a new and emerging science and technology field dealing

1530-437X 2013 IEEE

CHOU et al.: FABRICATION OF ARRAYED FLEXIBLE SCREEN-PRINTED GLUCOSE BIOSENSOR

with miniaturised systems that process, or manipulate small

volumes offluids [16]. Microfluidic technology can be used
to supply and transfer media, buffers, and even air while the
waste products by cellular activities are drained in a way
resembling the human circulatory system [17]. Nowadays,
polymeric materials such as polydimethylsiloxane (PDMS),
polymethylmethacrylate (PMMA), and polycarbonate (PC) are
often used for microfluidic system fabrication since they
enable the low-cost and mass production of these systems
through commercially available processes such as casting, hotembossing, imprinting etc. Polymeric microfluidic systems are
easy to fabricate but they lack thermal stability and resistance
to organic solvents [18]. Therefore, in this study, the PDMS
was used in microfluidics due to its hydrophobic, biocompatibility, small contact angle, flexible and other properties
like a very small dependence of viscosity to temperature
variations [19].
To sum up these advantages, in this study, the polyethylene
terephthalate (PET) which has advantages of anti-acid, high
biocompatibility, flexible and lightweight [20] was used as
a substrate. The flexible material of PDMS was used in
microfludics in order to prevent liquid leakage and weight
reduction. And RuO2 which has the advantages of high
conductivity and excellent enzyme attachment was used as the
sensing membranes. The screen-printed technique was used
to fabricate the arrayed flexible glucose biosensor. Finally we
integrated arrayed flexible glucose biosensor with microfluidic
to measure the dynamic characteristic.

179

Fig. 1.

Structure of microfluidic.
TABLE I

D EPOSITION PARAMETERS OF THE RuO 2 S ENSING M EMBRANES

Insulation layer

30 mm

II. E XPERIMENTAL
A. Materials

Silver leading wires

40 mm

Polyethylene terephthalate (PET) as the substrate of

the arrayed flexible pH biosensor was purchased from
Zencatec Corporation (Taiwan). Silver paste was purchased
from Advanced Electronic Materials Inc. (Taiwan). Ruthenium
target was purchased from Ultimate Materials Technology
Co., Ltd. (Taiwan). Potassium phosphate monobasic and potassium phosphate dibasic were purchased from Nihon Shiyaku
Industries (Japan). Nafion was purchased from Sigma-Aldrich
(America). Glucose powders were purchased from J. T. Baker
(America). Polydimethylsiloxane (PDMS) and curing agents
were purchased from Dow Coming (America).

RuO2 thin
membranes
PET substrate
(a)

(b)

Fig. 2. (a) Schematic diagram of arrayed flexible pH biosensor. (b) Finished

product of arrayed flexible pH biosensor.

B. Fabrication of Microfluidic
First, the pattern of microfluidic was designed by AutoCAD
software and the microfluidic mold was fabricated by computer numerical control (CNC). Then, the pre-polymer was
prepared by mixing a curing agent and PDMS pre-polymer at
1:10 weight ratio, and the liquid state PDMS was poured
subsequently in the microfluidic mold and PDMS was placed
in the vacuum chamber with 20 minutes to remove all bubbles.
The microfluidic mold placed in oven to solidify the PDMS.
The PDMS was peeled off from the microfluidic mold. The
inlet and outlet of PDMS layer were drilled with 3 mm
diameter cavern. The structure of the microfluidic is shown
in Fig. 1, the width, the length and the depth of microfluidic
are 1 mm, 50 mm and 10 mm, respectively.

C. Fabrication of Arrayed Flexible pH Biosensor

First, the PET substrate was cut into dimension 30 mm
40 mm. Then, the PET substrate was cleaned by ethanol and
distilled (D. I.) water in ultrasonic vibrator for 10 minutes.
And the nitrogen was used to remove water. The RuO2 sensing
membranes were deposited by radio frequency sputtering technology. The deposition parameters were shown in Table I [21].
Then, the PET substrate was put in the oven set at 120 C
for 30 minutes. Furthermore, the PET substrate was packaged
by screen-printed technique using insulation layer with epoxy.
Finally, the PET substrate was placed in the oven set at 120 C
for 90 minutes. Fig. 2(a) and (b) shows the fabricated pH
biosensor.

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TABLE II
C OMPARISON OF THE pH S ENSORS [22][24]

D. Immobilization of Glucose Oxidase Enzyme

The 0.1 M potassium phosphate dibasic (K2 HPO4 ) and
0.1 M potassium phosphate monobasic (KH2 PO4 ) were mixed
in the D.I. water to obtain 0.1 M phosphate buffer saline
(pH 7). Then, the 3 mg glucose oxidase enzyme was dissolved
in 5 ml of 0.1 M phosphate buffer solution by magnetic stirring
for 10 minutes. Then, the sensing membranes of arrayed
flexible pH biosensor were cleaned by D.I. water. The glucose
sensing membranes were prepared by mixing glucose oxidase
enzyme and nafion. Then, the mixture was dropped on the
sensing membranes. The sensing membranes were dried and
stored at 4 C until use.
E. Measurement System
In this study, the characteristics of the arrayed flexible
biosensor was measured by V-T measurement system which
consists of front part of sensing device and back part of signal
process. The front part of sensing device is the arrayed flexible
biosensor which was immersed in a test solution. Then, the
back part of signal process including eight instrumentation
amplifiers (AD620) and a data acquisition card (DAQ card)
(Model: NI USB-6201, National Instrument Corp., U.S.A.)
were connected with PC of analysis software (Model: LabVIEW 2009, National Instrument Corp., U.S.A.) to estimate
the sensitivity and linearity.
III. R ESULTS AND D ISCUSSION
A. Measurement of Arrayed Flexible pH Biosensor
First, to ensure the stability of sensing membranes, which
were measured by immersing in the different pH solutions
from pH 1 to pH 13. The linearity and the sensitivity were
described by the software of origin 7.0. The sensitivity and the
linearity of arrayed flexible pH biosensor were 53.36 mV/pH
and 0.96, respectively. The result was shown in Fig. 3.
Comparisons of previous literatures [22][24] were shown in
Table II.

Fig. 3. Arrayed flexible pH biosensor was measured by different pH solutions

from pH 1 to pH 13. The sensitivity and the linearity were 53.36 mV/pH and
0.96, respectively.

glucose oxidase enzyme and nafion at 4 mL:3 mL that were

dropped on RuO2 sensing membranes. The arrayed flexible
glucose biosensor was immersed in the different concentrations (100 mg/dL400 mg/dL) of glucose solutions to measure
the characteristics of the arrayed flexible glucose biosensor.
The sensing mechanism of glucose sensors were based on
an enzymatic reaction catalyzed by GOD according to the
following [25]:
GOD

H2 O + O2 + -D-glucose -gluconolactone + H2 O2 (1)

As a result of this reaction, -gluconolactone and hydrogen
peroxide were produced. These two products and the oxygen
consumption can be used for the glucose determination. With
H2 O availability in the reaction, gluconolactone is spontaneously converted to gluconic acid, which at neutral pH, form
the charged products of gluconate and proton (H+ ), according
to the equation below [25]:
spontaneous

B. Static Measurement of Arrayed Flexible Glucose

Biosensor
After ensuring the stability of RuO2 sensing membranes,
the glucose sensing membranes were prepared by mixing

-Gluconolactone gluconate + H+

(2)

Concentration changes of ions will change the electrode

potential. Therefore we used Eq. (2) for the determination of
the glucose concentration. The sensitivity and the linearity of

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181

TABLE III
S ENSITIVITY AND THE L INEARITY OF A RRAYED F LEXIBLE G LUCOSE
B IOSENSOR W ERE M EASURED AT D IFFERENT F LOW R ATES

Fig. 4.
Arrayed flexible glucose biosensor was measured by different
concentrations of glucose solutions from 100 mg/dL to 400 mg/dL. The
sensitivity and the linearity were 0.267 mV(mg/dL)1 and 0.973, respectively.

Fig. 6. Arrayed flexible glucose biosensor was measured by different concentrations of glucose solutions from 100 mg/dL to 400 mg/dL at 30 mL/min
flow rate.

Fig. 5.
Arrayed flexible glucose biosensor was measured by different
concentrations of glucose solutions from 100 mg/dL to 400 mg/dL and with
different flow rates from 5 mL/min to 30 mL/min.

arrayed flexible glucose biosensor were 0.267 mV(mg/dL)1

and 0.973, respectively. The result was shown in Fig. 4.
The inset in Fig. 4 showed a response time of the static
measurement. The arrayed flexible glucose biosensor achieved
95% of steady-state voltage in less than 50 s.
C. Dynamic Measurement of Arrayed Flexible
Glucose Biosensor
In order to obtain the characteristics of the arrayed flexible
glucose biosensor at the dynamic measurement, the arrayed
flexible glucose biosensor was integrated in microfluidic. And
then the arrayed flexible glucose biosensor was measured in
the different concentrations of glucose with different flow
rates (5 mL/min30 mL/min). The result was shown in
Fig. 5 and Table III. The Table III was shown the flow
rate at condition of 30 mL/min had the best sensitivity
and linearity. In comparison with the literatures [23], [26],

and [27], the arrayed flexible glucose biosensor integrated

with microfluidic had a wide range of glucose concentration
(100 400 mg/dL), higher correlation coefficient (0.999),
higher sensitivity (0.272 mV/(mg/dL)) (Table IV). The results
were demonstrated that the sensitivity and linearity at the
dynamic conditions were better than at the static conditions.
Also the Fig. 6 showed that the response time was shorter than
at static measurement. The arrayed flexible glucose biosensor
achieved 95% of steady-state voltage in less than 10 s. The factor can be explained with membrane diffusion resistance [28].
The membrane diffusion resistance exists in the outer of
sensing membranes and the reactant transmits to the internal
of sensing membranes by diffusion method. However the
well-known warburg impedance can be represented as the
membrane diffusion resistance in many fields [29]. Therefore,
according to the warburg impedance equation below
Z w = /1/2 j /1/2

(3)

and the warburg coefficient, , is given by

RT
=
(4)

2
2
n F ACb 2D
where is the angular frequency, R is the ideal gas constant,
T is the absolute temperature, F is the Faraday constant,

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TABLE IV
C OMPARISON OF THE G LUCOSE B IOSENSORS [23], [26], [27]

n is the valence, D is the diffusion coefficient of the species,

Cb is the bulk concentration, and A denotes the surface area.
According to the Stokes-Einstein relation, D is proportional
to the squared velocity of the diffusing particles, which
depends on the temperature, viscosity of the fluid and the size
of the particles. Therefore the membrane diffusion resistance
will be decreased by increasing the flow rate.
IV. C ONCLUSION
In this study, the screen-printed technique which has advantages of mass production, low cost and simple was used to
fabricate the biosensor. Besides the arrayed flexible glucose
biosensor was integrated in microfluidic to measure characteristics at the dynamic conditions. The results show that
the linearity, sensitivity and response time at the dynamic
conditions were better and shorter than at the static conditions.
Furthermore the arrayed flexible glucose biosensor integrates
with microfluidic can improve the sensing characteristics and
reduce sample requirement.
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Yi-Hung Liao (M12) was born in Yunlin, Taiwan,

on January 10, 1963. He received the bachelors
degree in electronic engineering from the National
Taiwan Institute of Technology, Taipei, Taiwan, in
1990, the M.S. degree in electronic engineering
from the National Yunlin Institute of Technology,
Yunlin, in 1997, and the Ph.D. degree from the
Graduate School of Engineering Science and Technology, National Yunlin University of Science and
Technology, Yunlin, in 2010.
His current research interests include the chemical
sensors and its applications, the array sensors and multisensors for biosensing,
the characterization of biosensors, and implementing home care systems with
PIC microprocessors.

Jung-Chuan Chou (M01) was born in Tainan,

Taiwan, on July 13, 1954. He received the B.S.
degree in physics from Kaohisung Normal College,
Kaohsiung, Taiwan, in 1976, the M.S. degree in
applied physics from Chung Yuan Christian University, Chung-Li, Taiwan, in 1979, and the Ph.D.
degree in electronics from National Chiao-Tung University, Hsinchu, Taiwan, in 1988.
He was with Chung Yuan Christian University
from 1979 to 1991. Since 1991, he has been an Associate Professor with the Department of Electronic
Engineering, National Yunlin University of Science and Technology, Yunlin,
Taiwan. Since 2010, he has been a Professor with the Department of Electronic
Engineering, National Yunlin University of Science and Technology. From
1997 to 2002, he was Dean of the Office of Technology Cooperation with the
National Yunlin University of Science and Technology. From 2002 to 2009,
he was a Chief Secretary with the National Yunlin University of Science
and Technology; from 2009 to 2010, he was the Director of the library
with the National Yunlin University of Science and Technology. From 2010
to 2011, he was the Director of the Office of Research and Development
at the National Yunlin University of Science and Technology. Since 2011,
he has been a Distinguished Professor with the Department of Electronic
Engineering, National Yunlin University of Science and Technology. His
current research interests include sensor material and device, biosensor and
system, microelectronic engineering, optoelectronic engineering, solar cell,
and solid state electronics.

Guan-Chen Ye was born in Hsinchu, Taiwan,

on April 29, 1988. He received the bachelors
degree from the Department of Electronic Engineering, National United University, Miaoli, Taiwan, in 2010, and the masters degree in microelectronic and optoelectronic engineering from the
Graduate School of Electronic and Optoelectronic
Engineering, National Yunlin University of Science
and Technology, Douliou, Taiwan, in 2012. His
current research interests include MEMS technology,
biosensors, and their applications.

Ya-Li Tsai was born in Tainan, Taiwan, on November 1, 1990. He received the bachelors degree
from the Department of Electronic Engineering,
National Yunlin University of Science and Technology, Douliou, Taiwan, in 2013.
His current research interests include MEMS technology, biosensor and their applications.

Tsung-Yi Cheng was born in Taichung, Taiwan

on September 20, 1988. He received the bachelors
degree from the Department of Electronic Engineering, National United University, Miaoli, Taiwan, in
2011, and the masters degree in microelectronic
and optoelectronic engineering from the Graduate
School of Electronic and Optoelectronic Engineering
from the National Yunlin University of Science and
Technology, Douliou, Taiwan.
His current research interests include MEMS technology, biosensor and their applications.

Shu-Ying Yang was born in Kaohsiung, Taiwan, on

September 19, 1973. She received the B.S. degree
from the Department of Electronics Engineering,
National Yunlin Institute of Technology, Douliou,
Taiwan, in 1997, the M.S. degree from the Institute of Electronics and Information Engineering,
National Yunlin University of Science and Technology, Douliou, in 1999, and the Ph.D. degree from the
Department of Electrical Engineering, National Sun
Yat-Sen University, Kaohsiung, Taiwan, in 2009.
She was a Lecturer with the Department of Information Management, Fortune Institute of Technology, from 2010 to 2009.
Since 2009, she has been an Assistant Professor. Her current research interests
include semiconductor processing, sensor material and device, microelectronic
engineering, optoelectronic material and device, solar cell, solid state electronics, electrochemical analysis, electrodeposited technology, programming,
database management, network database system, website design, integrated
website system, and e-commerce.