2010 6th International Colloquium on Signal Processing & Its Applications (CSPA)

A Study on Electrode for Amperometric

Measurement of Human Stress with Flow Injection
Analysis Biosensing System
Lee Yoot Khuan#1,Muhamad Hurairah Adha b Kamaruddin#2, Mohd Firdaus Abdullah#3, Nina Korlina
Madzhi#4, Anuar Ahmad*5
#
Faculty of Electrical Engineering, Universiti Teknologi MARA
Shah Alam, Selangor.
1
leeyootkhuan@salam.uitm.edu.my
3
badli_84@yahoo.co.uk
*
Faculty of Electrical Engineering, Universiti Industri Selangor
Batang Berjuntai, Selangor

Abstract—This paper presents a study on electrode for the
amperometric detection of human stress based on salivary alpha
amylase, emulated with different concentration of glucose, with the
flow injection analysis biosensing system. Amperometric detection
is an electrochemical voltammetric measurement approach, where
the current intensity in a detection cell is regarded as a function of
the concentration of the analyte. Flow injection analysis is a
chemical analysis method that injects a test sample into a flowing
carrier stream. The screen printed electrode is used to detect the
amount of glucose concentration, which is proportional to the
amount of output current. The objective of the study is to conduct a
comparative study between the gold and carbon electrode, as a
choice for amperometric detection of human stress with flow
injection analysis biosensing system. It has been found that, the
[initial, steady state current output at first injection of glucose,
current output at second injection of glucose] for carbon and gold
printed electrode are [0-80, 0-20, 0-20] μA and [0-35, 0-10, 0-5] μA
respectively. This shows measurement resolution of the carbon
screen printed electrode is better, which results in higher
measurement sensitivity. However, it is found that the gold printed
electrode exhibits higher rate of reaction with the steeper gradients
and the current reaches its steady state values faster. Yet, in view of
the cost, the gold printed electrode is about three times more than
the carbon printed electrode and also the ease in handling the zero
baseline, the latter is thus made as the choice electrode.
Keywords— Amperometric, Flow Injection Analysis, Human
Stress, Salivary Alpha Amylase, Biosensor.
the advantage that it is non-invasive, making multiple
sampling easy and stress free [1, 3].
Stress detection methods focuses on two major
physiological stress responses exhibit in the autonomic
nervous system, i.e. the sympathetic adrenal medullary system
(SAM) and hypothalamus pituitary adrenocorticol system
(HPA) [4]. Even though amylase is an enzyme that breaks
down starch into glucose to supply the body with energy [5-
7], studies have shown that the amount of alpha amylase, an
enzyme secreted by the salivary gland, is correlated to the
"fight or flight" response in SAM system to stress. It has been
proven that the increase in salivary amylase activity is more
significant and the reaction is more rapid than that of cortisol
when experimented with psychological stressor, suggesting
that it is a better index of stress [6-7].
The following equations illustrate the chain of chemical
reactions, where maltopentose is used as substrate while α-
glucosidase and glucose oxidase as the immobilized enzymes,
to make possible the measurement of salivary alpha amylase.
The alpha-amylase hydrolyses the glucosidic link in the
maltopentose. Decomposition of the hydrogen peroxide thus
produced releases electrons to deliver current (4). This forms
the basis of detection by the amperometric biosensor
employed here [5, 8].
Amylase
Maltopentose ⎯⎯ ⎯→ Maltose + Maltotriose (1)

Maltose + Maltotriose ⎯α⎯

− glu cos idase ( GOD )
⎯⎯ ⎯⎯ ⎯→ 5D − glu cos e (2)
I. INTRODUCTION
Human stress is defined as the response of human to an glu cos e−oxidase(GOD ) (3)
D − glu cos e + O ⎯⎯ ⎯ ⎯ ⎯ ⎯⎯→ D − glu cos eacid + H 2O2
external stimulus. Stress in human results from interaction
with people and their environment, perceived as exceeding
their adaptive capacity to an extent that threatens their well- ⎯→ 2 H + + 2e − + O2
H 2O2 ⎯ (4)
being. Stress can be distinguished into body and cell stress,
psychological and physical stress, as well as eustress and Biosensor is a device integrating two main elements, a
distress [1]. Several biomarkers that are currently in use for molecular recognition element and a transduction element. It
the measurement of physiological stress response are such as, can transform a biological signal into a quantified electronic
skin conductance and resistance, muscle tension, leukocytes signal, for data analysis and to output devices [9-10]. Figure 1
and saliva amylase [2]. Of these, uniquely saliva sampling has shows the generic structure of a biosensor. The biological
recognition element contains enzyme immobilized over the

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 2010 6th International Colloquium on Signal Processing & Its Applications (CSPA)

surface of a device, as a primary detector. The transduction

element translates the velocity of the biochemical reaction
during the biological recognition process into a measurable
response, usually in the form of an electrical signal [11].

Figure 1: Generic Structure of a Biosensor

In order to measure the electrical signal from the

biosensor, amperometric detection is adopted. Amperometric
detection is one of the modes of electrochemical voltametric
measurement, in which the current intensity in a detection cell
is described as a function of concentration of the analyte. For
the class of amperometric biosensors, the quantity of analyte
in the sample corresponds to current generated from the
induced electrochemical reaction, while keeping a constant
potential between the electrodes [11-12]. The value of this
voltage depends on the substance to detect [11].
The flow injection analysis system is a well-established
flow based technology that injects a test sample into a flowing
carrier stream. It has found wide applications, such as
pharmaceutical, process control and drug. It is an assembly of
flow injection device, flow channels of chemical, chemical
reaction method and data acquisition method. It allows
automated handling of sample and reagent solution with a
strict control of reaction conditions. Here, it is used to regulate
the delivery of the substrate, maltopentose, for mixing with
the analyte, salivary amylase, for chemical reactions as
described by equation (1) to (4) to enable the amperometric
detection [7-8].
In this paper, experimental work to study the electrode for
amperometric detection of human stress based on salivary
alpha amylase, emulated with different concentration of
glucose, is described. First, an automated data acquisition
system for flow injection analysis is developed. Then current
output for different concentration of glucose is acquired with
uncoated and coated gold and carbon electrodes. The initial
and steady state current output at first injection of glucose, as
well as current output at second injection of glucose from both
electrodes are discussed and compared, in terms of resolution,
sensitivity, rate of reaction, cost and handling of baseline.
II. METHODOLOGY
A flow injection analysis device is used as an analytical
system to detect glucose activity. It is used to investigate the
amperometric detection of glucose at different concentration,
to emulate the different stress levels of human [1]. The flow of
experimental work is summarized in Figure 2. The current
was first detected using the uncoated gold and carbon screen
printed electrode for the baseline value. The glucose was
injected to be directly absorbed by the bare carbon and gold at
the centre of the screen printed electrode. Then the gold and
carbon screen printed electrode were coated for detection of
current at different concentration of glucose.
Figure 2: Flow chart for experimental work on amperometric
measurement of glucose.
A. Coating of Electrode
Coating of the screen printed electrode is started with 10
microlitres (μl) of a mixture constituting of 5 weight-in-
percent (wt.%) bovine serum albumin of 400μl, 25 wt.%
glutaraldehyde of 20μl, 0.5 wt.% chitosan solution of 20μl
and distilled water of 560μl, being applied onto the surface. A
selective layer was then made by drying the surface at 35 °C
for about 20 minutes. Next, 10μl solution made of
100milligram (mg) of glucose oxidase, 100μg of bovine
serum albumin, 8μl of 25 wt.% glutaraldehyde, and 992μl of
phosphoric acid buffer solution at pH of 7.3 was applied onto
surface of the selective layer. An immobilized enzyme
membrane was produced by drying the surface at 35 °C for
about 20 minutes. This resulted in the weight of glucose
oxidase for the immobilized enzyme membrane to be 0.1mg,
which corresponded to 17Unit. This weight of glucose oxidase
was chosen to obtain 100nano-ampere of current output, even
the immobilization efficiency may be several percent or less
[5].
B. Preparation of Glucose Mixture
Glucose in powder form and water were mixed together to
yield glucose solution at different concentrations. Altogether
six different degrees of concentration were prepared. The
content in parts of glucose and water mixture is shown in
Table I. A mixture of 1 part glucose and 9 part water contains
0.5milli-litre (ml) of glucose and 4.5ml of water. It follows
that a mixture of 5 part glucose and 5 part water contains
2.5ml of glucose and 2.5ml of water.

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A. Uncoated Screen Printed Electrode

TABLE I
MIXTURE CONTENT OF GLUCOSE AND WATER Current output from the carbon and gold screen printed
Glucose: Water electrodes before coating were measured. Table II tabulates
1:9 the current at different concentration of glucose.
2:8 TABLE II
3:7 CURRENT AT DIFFERENT CONCENTRATION OF GLUCOSE FOR UNCOATED
CARBON AND GOLD ELECTRODE
4:6
5:5
6:4

In our experiments, the glucose was injected twice. This

is to ensure the reaction between glucose and the screen
printed electrode is complete. The first injection of glucose is
at the start of experiment while the second injection is applied
600 seconds later. The flow rate when the system is running
was measured by the reverse osmosis water that flow through
the flow injection system against the time, which is 0.0317ml
per second.
C. Principle of Amperometric Detection
Figure 3 shows a screen printed carbon electrode. It is the
most commonly used amperometric detector with three-
electrodes. The potential of the working electrode (WE) is set
relative to the reference electrode (RE). The current drop
between the working and counter electrode (CE) is
compensated by the potentiostat. The current flowing through
the working electrode is the measured signal. The current,
within the pico-ampere to micro-ampere range, is amplified
and recorded as a function of the time flow of the mobile
phase.
As observed from Figure 4 and Figure 5, it has been found
that for the carbon printed electrodes, the current is drifting at
almost zero μA, while for the gold screen printed electrodes, it
displays a stable output between 1.5 and 3 μA. These are the
current before coating. Since coating has not been applied,
chemical reaction is absent, hence giving current output near
to zero for the carbon printed electrode while a small baseline
value for the gold printed electrode. It can also be observed
that the reaction curves of the gold printed electrode display a
steeper gradient. This shows that their rate of reaction is
speedier. The reason being that gold is found to react easier
with glucose [9].

Figure 4: Current for uncoated carbon screen printed electrode at

different glucose concentration
Figure 3: A screen printed carbon electrode

III. RESULTS AND DISCUSSION

The electrode of a flow injection analysis system for the

detection of glucose at different concentration, to emulate the
different stress levels of human [1] was studied. The
characteristic response of two types of screen printed
electrode, gold and carbon, in their uncoated and coated
conditions, is measured and compared. The findings will
contribute to the basis for a choice of electrode for the
amperometric detection of human stress with the flow Figure 5: Current for uncoated gold screen printed electrode at different
injection analysis biosensing system. glucose concentration

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B. Coated Screen Printed Electrode

After coating, experiments show current output from both
types of screen printed electrodes. Table III shows the current
output at different concentration of glucose for the coated
carbon printed electrode while Table IV for the coated gold
printed electrode, at the first and second injection of glucose.
TABLE III
CURRENT AT DIFFERENT CONCENTRATIONS OF GLUCOSE FOR COATED
CARBON ELECTRODE

Figure 6: Time response of current output from coated carbon screen

printed electrode at first injection of glucose for different glucose
concentration.

TABLE IV
CURRENT AT DIFFERENT CONCENTRATIONS OF GLUCOSE FOR COATED
GOLD ELECTRODE

Figure 7: Current at the first and second injection of glucose by coated

carbon screen printed electrode for different glucose concentration.

From Figure 6 and Figure 7, for the carbon screen printed

electrode, the initial current output ranges from 0 to 80μA,
which then decreases exponentially to the steady state current
output at first injection of glucose, that ranges from 0 to
20μA. The current output at second injection of glucose stays
within 0 to 20μA.
From Figure 8 and Figure 9, for the gold screen printed
electrode, the initial current output ranges from 0 to 35μA,
while the steady state current output at first injection of
glucose ranges from 0 to 10μA. The decrement follows an
exponential graph. The current output at second injection of
glucose reads between 0 and 5μA.
In comparison, the measurement resolution of the carbon Figure 8: Time response of current output from coated gold screen printed
electrode for different glucose concentration.
screen printed electrode is observed to be better, resulting in
higher measurement sensitivity.

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 2010 6th International Colloquium on Signal Processing & Its Applications (CSPA)
Figure 9: Current at the first and second injection of glucose by coated
gold screen printed electrode for different glucose concentration.
It can also be observed that the gradients of the
exponential current response graphs of the gold printed
electrode in Figure 8 are consistently steeper than those of the
carbon printed electrode in Figure 6. This illustrates that gold
with better conductivity, exhibiting higher rate of reaction.
Hence, the current reaches its steady state values faster.
In light of the economic aspect, the cost of gold printed
electrode is about 3 times more than the carbon printed
electrode. Since it can only be used once, the number of
electrodes required for general testing and long-term
monitoring of human stress are substantial.
IV. CONCLUSION
An amperometric measurement with flow injection
analysis biosensing system for measuring human stress based
on salivary alpha amylase, emulated with glucose at different
concentration was successfully developed. A comparative
study on the coated and uncoated carbon and gold screen
printed electrode was performed. For the different
concentration of glucose, it has been found that for the carbon
printed electrodes, the current before coating is drifting at
almost zero μA, while for the gold screen printed electrodes, it
displays a baseline of 1.5 to 3 μA. After coating, the [initial,
steady state current output at first injection of glucose, current
output at second injection of glucose] for carbon and gold
printed electrode are [0-80, 0-20, 0-20] μA and [0-35, 0-10,
0-5] μA respectively. This shows the measurement resolution
of the carbon screen printed electrode is better, resulting in
higher measurement sensitivity. However, it is found that the
gold printed electrode exhibits higher rate of reaction as
shown with the steeper gradients and the current reaches its
steady state values faster. Yet, in view of cost, the gold printed
electrode is about three times more than the carbon printed
electrode and also the ease in handling zero baseline, the latter
is thus made as the choice electrode.
978-1-4244-7120-1/10/$26.00 ©2010 IEEE
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The final proceedings copy is as listed in IEEExplore.
ACKNOWLEDGMENT
The authors would like to acknowledge the Minister of
Science, Technology and Innovation (MOSTI), Malaysia and
Dana Cemerlang 600-RMI/ST/DANA 5/3/Dst (33/2009),
Universiti Teknologi MARA, Malaysia for their financial and
equipment assistant for the project research.
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