The document describes the design and fabrication of an amperometric biosensor for glucose monitoring. 1) Microfabrication techniques were used to fabricate the sensor on a glass substrate, which consisted of a silver-silver chloride reference electrode, working electrode, and counter electrode. 2) Glucose oxidase was immobilized on the working electrode to catalyze the oxidation of glucose. When a potential was applied, the current produced was directly proportional to the glucose concentration. 3) Characterization of the reference electrode showed potential variations of less than 1mV between two identical electrodes fabricated for testing. Microfabrication allows mass production of low-cost, disposable glucose sensors.

1. Microfabrication of Amperometric Biosensor for
Glucose Monitoring
Aminuddin Debataraja, Nur Fauzi Soelaiman, Latif mawardi.
Electrical Engineering State Polytechnic of Jakarta University of Indonesia,
Kampus Baru UI DEPOK, 16424, Phone: (021)7863531; Fax: (021)7863531
E-mail: adebataraja@yahoo.com

Abstract— The objectives of this research are to sensor research is a relative mature and well worked
design and fabricate glucose sensor using enzyme research field. The majorities of sensors are based on
based amperometric sensor on glass substrate. electrochemical principles and employ enzymes as
Microfabrication technology was used to fabricate the biological components for molecular recognition.
electrode. The biosensor developed initially to Several new techniques for glucose sensing have been
determine glucose in aqueous solutions, with later developed in clinical practice as well as in
application to others analytes. The glucose electrode biotechnology and the food industry. Glucose sensors
itself consists of a silver silver chloride reference can be broadly classified in three main categories
electrode, a working electrode and a counter electrode. depending on the number of applications under
Characterization of the reference electrode has been investigation: (1). The first and by far the largest
evaluated. The output potential and noises are category consist of the enzyme-based needle-type
observed at random times and the potential variation electrochemical glucose sensors. The detection
of the two reference electrodes is less then 1 mV. It is principle of these sensors is based on the monitoring
due to copper wire silver paste coated was used but of the enzyme-catalyzed oxidation of glucose. The
with unknown purity. category includes glucose sensors using amperometric
or potentiometric operating principles (hydrogen-
I. INTRODUCTION peroxide electrode based, oxygen-electrode based,
mediator-based and potentiometric-electrode based).
A glucose test is one of many tests performed in a
(2). The second category consists of glucose sensors
clinical laboratory. The concept of a glucose sensor
based on the direct electro-oxidation of glucose on
was first introduced by Clark & Lyons in 1962 [1]. In
noble metal electrodes (electrocatalytic glucose
their article dealing with continuous monitoring of
sensors). (3). The third category consists of glucose
blood chemistry, they suggested that a thin layer of
sensors based on a number of different detection or
soluble enzyme might be retained at the surface of an
glucose extraction techniques. This category includes
oxygen electrode using a dialysis membrane. Glucose
affinity-based glucose sensors, coated wire glucose
and oxygen would diffuse into the enzyme layer from
electrodes, reverse ionophoresis based glucose
the sample site and the consequent depletion of
sensors, suction effusion fluid based glucose sensors
oxygen would provide a measurement of the glucose
and microdialysis based glucose sensors.
concentration. Since this pioneer work in the 1960s,
reasonable research effort has been devoted to the The objective of this present research is to design
development of glucose sensors by a number of and fabricate glucose sensor using enzyme based
process for fabrication of the electrode is also amperometric sensor on glass substrate. The aim of
explained. this paper is to describe the design and fabrication of
Starting from publication of Clark and Lyons in amperometric glucose sensor. The fabrication of the
1962, the amperometric biosensors became one of the silver-silver chloride reference electrode is presented
popular and perspective trends in biosensor. The and the microfabrication configuration as shown in
amperometric biosensors measure the changes of the figure 1. To the working electrode (cathode, negative)
current of indicator electrode by direct is applied a potential which is constant relative to the
electrochemically oxidations or reduction of the reference electrode, which itself serves as a reference
products of the biochemical reaction [2]. In potential. As this potential is applied, the current
amperometric biosensors the potential at the electrode between the working electrode and counter electrode
is held constant while the current is measured. A is monitored. If there is no electroactive substance in
simple circuit involves a two-electrode configuration the electrolyte solution then no current occur
although more precise control of the applied potential (essentially). Cottrell equation indicates in the
can be achieved by using a potentiostat and a three- presence of an electroactive substance a current
electrode research groups worldwide. Today, glucose ensues. The magnitude of the current is directly

2. proportional to the concentration of that electroactive stoichiometric relationship with its substrate or target
substance if all else, such area, diffusion coefficient analytes. Amperometric biosensors are typically
and the underlying assumptions in deriving the inexpensive and less critically dependent of an
equation, remain constant. The amperometric accurate reference electrode [6]. The amperometric
biosensors are known to be reliable, less critically biosensor allows the electrochemical reaction
dependent of an accurate reference electrode, typically (oxidation or reduction) to proceed at the electrode
inexpensive and highly sensitive for environment, surface, giving rise to a current. Amperometric
clinical and industrial purposes [3]. biosensors operate at a fixed potential with respect to a
reference electrode and the current generated by the
oxidation or reduction of the substrate at the surface of
the working electrode is measured. This current is
directly related to the bulk substrate concentration. It
is important that electron transfer between the
biological molecules and electrode material be
facilitated. The design of amperometric biosensors
involves several strategies. These can be divided into
two main groups: those, which require a soluble
enzyme and those where the enzyme is immobilized at
the electrode surface [7]. The enzyme layer can be
sandwiched between two permeable membranes. A
layer-by-layer deposition technique may be used to
optimize enzyme loading in bi-enzyme systems [8]. An
Figure 1. Electrode configuration (a) two electrode (b) three
outer cellophane, polycarbonate, or cellulose acetate
electrode.
membrane served to exclude larger molecules or
molecules like ascorbate. The most common enzymes
used in monoenzymatic systems are oxidases, which
catalyze the model oxidation eraction
Oxidases are usually flavoproteins that use O2, the
natural electron acceptor, to regenerate the reduced
Where, i = current (A), D = Diffusion Coefficient,
A = Area, C = Analyte concentration, n = number of
electrons involved in the electrode reaction, F =
Faraday’s constant (96485 C/mol). enzyme during the reaction. New systems have
A reference electrode is used in measuring the recently been developed in which a chemical mediator
working electrode potential of an electrochemical cell replaces oxygen. Among them, ferrocene-ferricinium
[4]. The precision and accuracy of the measurement ion couples or derivatives are the most frequently
depend strongly on the effective performance of the employed [8, 9]. Glucose oxidase is widely used for
reference electrode, which affects the results through the determination of glucose in body fluids and in
fluctuations in its own potential and through the removing residual glucose and oxygen from beverages
liquid-junction potential at the interface with the test and foodstuffs. Furthermore, Glucose oxidase-
solution. Therefore, a reference electrode should have producing moulds such as aspergillus and penicillum
a stable electrochemical potential as long as no current species are used for the biological production of
flows through it. The purpose of the reference gluconic acid.
electrode is to complete the measuring circuit and
provide a stable and reproducible potential against II. EXPERIMENTAL
which the indicator electrode is compared [5]. The
Design and construction technology and materials
contact is made through a liquid junction that allows science are intimately linked in biosensor
the reference electrolyte to contact the sample. The development. Sensor design, including materials, size
silver-silver chloride electrode is the most common shape and methods of construction, are largely
due to its ease of manufacture or simple, inexpensive, dependent upon the principle of operation of the
non-toxic, very stable and superior temperature range, transducer, the parameters to be detected and the
actually usable even above 130oC. The electrode is a working environment [2, 8, 10]. Materials used in
silver wire that is coated with a thin layer of silver electrochemical biosensors are classified as (1).
chloride either by electroplating or by dipping the wire Materials for the electrode and supporting substrate,
in molten silver chloride. In an amperometric enzyme (2) materials for the immobilization of biological
electrode the function of the enzyme is to generate (or recognition elements, (3) materials for the fabrication
to consume) an electroactive species in a of the outer membrane and (4) biological elements,

3. such as enzymes, antibodies, antigens, mediators, and
cofactors. In amperometric electrochemical processes
are generally complex and may be considered a
succession of electron transfers and chemical events.
Amperometric biosensors function by the production
of a current when a potential is applied between two
electrodes. For example, the overall oxygen reduction
involves different steps (such as oxygen reduction to
hydrogen peroxide, hydrogen peroxide reduction to
H2O, and dismutation of hydrogen peroxide), and Figure 2. Glucose sensor layout
numerous parameters influence the rates of these
reactions (the potential, the nature of electrode metal,
and the operating conditions). The overall sensor
current is thus dependent on many factors; including
charge transfer, adsorption, chemical kinetics,
diffusion, convection, and substrate mass transport
[11-13]. The understanding of the kinetic peculiarities Figure 3. Glucose sensor structure
of the biosensors is of crucial importance for their
design. One of the most critical characteristics of using IC technology, has been extremely challenging,
biosensors is their stability. The operational stability mainly due to process incompatibility issues,
of a biosensor response may vary considerably packaging problems, failure to incorporate a true
depending on geometry and method of sensor reference electrode and the difficulties involving
preparation, a transducer use and some other some patterning relatively thick organic layers such as ion
other parameters [2]. Furthermore it is strongly depend selective membrane and hydrogel [11]. In addition,
on the response rate-limiting factor, i.e. substrate microfabrication techniques can also be used to either
diffusion and enzymatic reaction rate [13]. significantly improve sensor characteristics (with
In this research, the layout of the glucose sensor respect to conventionally fabricated devices) or to
to be constructed is shown in figure 2. Three different develop devices with new functionality, which cannot
patterns and sizes of glucose electrode are used to be realised in conventional fabrication technology.
evaluate the sensor response rate (A1= 9.63 mm2, A2= Chemical sensors or biosensors usually consist of a
4.60 mm2, A3= 3.97 mm2). The base transducer sensitive layer or coating and a transducer or
consists of H2O2 sensor, which is essentially the same electrode. Various enzyme materials serve as
as the oxygen sensor. The enzyme glucose oxidase is biologically sensitive layers that can be coated onto
immobilized in front of the H2O2 sensor between two the different transducers. The set of microfabrication
membranes. The inner membrane is a permeselective processes used for coating a sensitive layer for
membrane that allows passage of H2O2 where as the
biosensors is completed by various deposition
outer membrane separates the biosensor from
techniques for biologically sensitive layers such as
measurement medium (figure 3).
dispensing or spray coating, polymerisation, a sol-gel
In order to coat a thin sensing membrane onto a
gold electrode, appropriate membrane materials have process, printing and by sputtering. For example
to choose. The requirements of the membrane are electrochemical polymerisation is an attractive
good adhesion to the electrode surface, good approach for enzyme immobilisation. This is a simple
permeability of the membrane are good adhesion to procedure where a suitable monomer is oxidised in the
the electrode surface, good permeability of glucose presence of an enzyme. The enzyme is confined to a
and adequate mechanical strength. Albumin layer adjacent to the electrode. The techniques used
crosslinked by glutaraldehyde and cover with a for the production of electrode can be roughly
cellulose acetate adhesion layer may be use in this classified as: (1) printing, (2) deposition, (3)
research. Microfabrication processes are used to polymerisation, (4) plasma induced polymerisation,
produce devices with dimensions in the micrometer to (5) photolithography and (6) nano technology. In this
millimetre range [10]. Over the past few years, research project, deposition and photolithography
microfabrication technology has emerged as a techniques were used to fabricate and pattern the
promising technology for miniaturising and integrating glucose electrode. Flow diagram and fabrication steps
chemical or biosensor systems. Microfabrication of for fabrication of biosensor glucose sensor are shown
electrochemical sensors, in figures 4(a) and (b). The next steps are
manufacturing the reference electrode (Ag|AgCl) and
enzyme coating using spray coating. The silver/silver
chloride reference electrode is produced by
chloridising the gold wire coated with silver paste in

4. 0.1 M Potassium chloride (KCl) solution. The the cathode for electrolysis, with the copper wire silver
following are the fabrication steps involved for paste coated electrode as the anode. A current of ± 50
amperometric glucose electrode on glass substrate: µA was passed through the electrode for
 Deposition of chrome/gold electrode using approximately 120 mins in 0.1 M KCl, where a
sputtering system magnetic stirrer was employed to keep homogeneity of
 Patterning of gold electrode using the solution. The electrode was then immediately
photolithography rinsed in deionised water and subsequently stored in
 Etching (wet or dry) the plastic bottle. Before potential measurements, the
 Enzyme coating using spy coating or sol-gel reference electrode was immersed in the test solution
process for about 1 hour to establish a stable
electrode/electrolyte interface. The reversible
electrode reaction consists of silver ions going into
solution and then combining with the chloride ions to
form silver chloride. Thus its potential is determined
by the following reactions:
Ag  Ag + e- (3.5)
Ag+ + Cl-  AgCl(s) (3.6)
Ag + Cl  AgCl(s) + e
- -
(3.7)
Figure 4. (a) Flow diagram of the fabrication of glucose
sensor used. (b) Glucose sensors fabrication steps.
Figure 5. (a) Schematic Diagram reference electrode
Different approaches of fabrication of silver- experimental set-up (b) Photograph of reference electrode
silver chloride reference electrode have been cited in experimental set-up
literatures [14-18]. In this research, the reference
electrode used was a Ag|AgCl electrode. The The key feature of a reference electrode is its
silver/silver chloride reference electrode was produced reproducible and stable potential that is not influenced
by chloridising the copper wire coated with silver by the measuring solution. The fabricated reference
paste but to minimise the contamination hazard and to electrode developed during this research was of a
achieve good surface contact, the copper wire was first Ag|AgCl type. The simplest way to test an unknown
rinsed with 10% HNO3 solution followed by acetone reference electrode is to compare its potential with a
(CH3COCH3) solution. The copper wire coated with known good reference electrode in a beaker containing
silver paste (drying at ± 150 0C for 120 mins) was an electrolyte and connect the two electrodes to the
chemically chloridized in 0.1 M potassium chloride inputs of a good quality voltmeter. Ideally the
(KCl) solution. The experimental set-up is illustrated voltmeter’s input impedance should be 100 MW or
in figure 5(a) & (b). A gold coated wire was used as greater. Since we don’t have a good reference

5. electrode, in order to make a valid assessment of an electrodes is less than 1 mV. Temperature of the setup
integrated reference electrode, two identical reference during the experiment was not taken into account.
electrodes (Ref. A and Ref. B) were fabricated and
then immersed in electrolyte. Read the open circuit
voltage of the system. This is a measure of the
potential difference between the two reference
electrodes. This enables a comparison to be made
between electrodes of the same type as a consistency
check.
III. RESULTS AND DISCUSSIONS
The quality of the AgCl layer depends on the Figure 8. The response potential of two identical reference
current density and length of time during its growth. electrodes
The length of time the electrode is submerged was
observed to be directly proportional to the thickness of
the AgCl layer. Figure 6 shows the fabricated of IV. CONCLUSIONS
reference electrode. In this present research glucose sensor using
enzyme-based amperometric sensor was designed and
fabricated. Microfabrication technology was used to
fabricate the electrode. The biosensor developed
initially to determine glucose in aqueous solutions,
with later application to others analytes. The glucose
electrode itself consists of a silver silver chloride
reference electrode, a working electrode and a counter
electrode. The intention during this research was to
Figure 6. Images of two reference electrodes made of develop a miniature reference electrode that was fully
copper wire coated with silver paste (I = 50 A, T=120 integrated with the working and counter electrode.
mins) However, since silver coating cannot be made in PIDC
Figure 7 shows a schematic diagram of the – Hsinchu Taiwan and silver wire (with purity
experimental set-up for stability testing of the 99.99%) is not available also, copper wire silver paste
fabricated reference electrode. The measurement coated was used but with unknown purity. This raises
apparatus consisted of a multimeter (BRYMEN question on the integrity of the results. From
BM859CF) with a resolution of 0.01 mV was selected preliminary test the output potential and noises are
to measure the voltage generated by the electrode and observed at random times. The potential variation of
was interfaced with a PC. The test solution was 0.1 M the two reference electrodes is less then 1 mV.
KCl with magnetic stirrer at the bottom. Both Temperature of the setup during the experiment was
fabricated reference electrodes Ref. A and Ref. B were not taken into account. Due to limitation of time the
immersed in test solution at room temperature for glucose sensor developed cannot be completed. The
more than 2.5 hours and the output signal was enzyme coating and the performance or response rate
recorded. of the glucose sensor cannot be done during this time.
AKNOWLEDMENT
Multimeter
The authors would like to thank Dr. Hiskia for
…. mV
theoretical calculations and technical support. Part of
Ref. A Ref. B
the research was supported by DP2M-Dikti (Hibah
Bersaing).
0.1 M KCl
Magnetic stirrer
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