A Novel Fiber Optic Biosensor

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A Novel Fiber Optic Biosensor

for the Determination
of Adrenaline Based on
Immobilized Laccase Catalysis
a a a a
Jun Huang , Hua Fang , Cheng Liu , Erdan Gu
a
& Desheng Jiang
a
Key Laboratory of Fiber Optic Sensing Technology
and Information Processing, Wuhan University of
Technology, Ministry of Education , Wuhan, China
Published online: 10 Jul 2008.
To cite this article: Jun Huang , Hua Fang , Cheng Liu , Erdan Gu & Desheng
Jiang (2008) A Novel Fiber Optic Biosensor for the Determination of Adrenaline
Based on Immobilized Laccase Catalysis, Analytical Letters, 41:8, 1430-1442, DOI:
10.1080/00032710802119525
To link to this article: http://dx.doi.org/10.1080/00032710802119525
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DOI: 10.1080/00032710802119525
SENSORS
A Novel Fiber Optic Biosensor for the

Determination of Adrenaline Based on

Immobilized Laccase Catalysis
Jun Huang, Hua Fang, Cheng Liu, Erdan Gu, and Desheng Jiang
Key Laboratory of Fiber Optic Sensing Technology and Information Processing,
Wuhan University of Technology, Ministry of Education, Wuhan, China
Abstract: A novel fiber optic biosensor for the determination of adrenaline

based on immobilized laccase catalysis and fluorescence quenching was designed
and fabricated. The immobilized laccase formed by the immobilization of laccase
on the CuTAPc-Fe3O4 nanoparticles composite were used to catalyze the oxi-
dation of adrenaline and the fluorescent oxygen-sensing membrane was used to
detect the consumption of oxygen. The effects of pH and temperature on laccase
activity using adrenaline as the substrate were studied. The optimal pH and tem-
perature for the activity of immobilized laccase are 5.0 and 55C, respectively. The
immobilized laccase has good thermal, storage and operation stability. The lock-
in technology was used to detect the change of the life time of the oxygen-sensing
membrane. By using ABTS as the electron mediator, the biosensor showed a
response time of 30 sec. The biosensor has good performance in the adrenaline
concentration ranges of 2.0 10 7 to 9.0 10 7 mol=l and 1.0 10 8 to
9.0 10 8 mol=l, and it also shows good stability.
Keywords: Adrenaline; Fiber optic biosensor; Immobilized laccase
Received 16 February 2008; accepted 26 March 2008.

The authors wish to acknowledge the following financial supports: The
National Natural Science Foundation of China (Project No. 60377032) and
Key Project (No. 60537050); the Program for Changjiang Scholars and Innova-
tive Research Team in University (PCSIRT, No. IRT0547); and the Ministry
of Education, China.
Address correspondence to Jun Huang, Key Laboratory of Fiber Optic Sensing
Technology and Information Processing, Wuhan University of Technology,
Ministry of Education, Wuhan 430070, China. E-mail: hjun@whut.edu.cn
1430
Novel Fiber Optic Biosensor 1431
INTRODUCTION
Adrenaline is one of the best-known catecholamines biosynthesized in the

adrenal medulla and sympathetic nerve terminals and plays an important
role in the central nervous system as neurotransmitters. Pharmaceutically,
it is widely used for the treatment of neutral disorders (Deftereos et al.
1993), acute allergy, asthma, bradycardia, cardiac arrest, glaucoma, and
hemorrhage (Goodman and Gilman 1996). An early diagnosis based on

adrenaline determination in plasma and urine could prevent an unnecess-
ary drug treatment (Hollenbach et al. 1998). Nowadays, numerous
methods have been applied to determine the adrenaline concentration,
such as spectrophotometry (Sorouraddin et al. 1998), high-performance
liquid chromatography (HPLC) (Chen et al. 2007; Kartsova et al. 2004),
flow-injection chemiluminescence (Wolyniec et al. 2007; Yao et al.
2006; Yu et al. 2006), chemiluminescence (CL) (Lin et al. 2007), and
electrochemical detection with various modified electrodes (Nikolelis
et al. 2005; Nikolelis et al. 2003; Felix et al. 2006, Salimi et al. 2005;
Marazuela et al. 1999). Although sensitive, these methods are time-
consuming and are not satisfactory for on-line and real-time monitoring.
Fiber optic biosensors have many advantages such as high sensitivity,
fast response, low cost, immunity from electrical and magnetic distur-
bances, small size and lightweight, and the ability to provide an alterna-
tive to laboratory-based measurement methods and be used for online
and real-time monitoring of many parameters. Fiber optic biosensors
are composed of a biological recognition element (biocomponent), a
transducer to convert the biocomponent response to an optical signal
and associated electronics. Laccases (E.C. 1.10.3.2) are blue multicopper
oxidases and can catalyze the oxidation of some compounds, causing the
reduction of molecular dioxygen to water (Yaropolov et al. 1994;
Solomon et al. 1996). For the fiber optic biosensors based on laccase
catalysis, which have great application in clinical medicine detection,
the immobilized laccase formed by the immobilization of enzyme on a
carrier could act as the sensing biocomponent and will have tremendous
influence to the sensor properties.
In our previous study, we used copper tetra-aminophthalocyanine
(CuTAPc)-Fe3O4 nanoparticle composite to immobilize laccase. The
immobilized laccases have good activity and stability by using ABTS as
the substrate, and could be easily separated from solution for reuse
because of the magnetic properties of the carrier (Huang et al. 2006).
In this paper, we present a novel fiber optic biosensor for the determi-
nation of adrenaline based on the immobilized laccase catalysis and
fluorescence quenching. With the catalysis of immobilized laccase, the
adrenaline in the solution was oxidized by dissolved oxygen and
1432 J. Huang et al.
fluorescent oxygen-sensing membrane was used to detect the consump-

tion of oxygen and therefore to determine the adrenaline concentration.
The lock-in technology was used to detect the lifetime of the oxygen-
sensing membrane, which can improve the sensor stability. The effect
of pH and temperature to immobilized laccase and their stability have
been studied by using adrenaline as the substrate. ABTS was used as
the mediator in this system to accelerate the catalysis rate. This biosensor
is sensitive, fast response, and stable, suggesting that it should be

promising in the determination of adrenaline.
EXPERIMENTAL
Materials
Pycnoporus sanguineus laccase (E.C.1.10.3.2), a white-rot fungus laccase

with a molecular weight of 64 kDa, was kindly presented by the Institute
of Microbiology of the Chinese Academy of Sciences, and its purity was
tested by electrophoresis with a single protein band. ABTS were obtained
from Sigma Chem. Co. (Deisenhofen, Germany). Adrenaline hydrochlor-
ide solutions (1 mg=ml) were purchased from Wuhan Grand Pharmaceuti-
cal Group Co., China. All other chemicals were of analytical grade and
were used without further purification. Tridistilled water was used through-
out our experiments. Adrenaline standard solutions were freshly prepared
every 24 h from a stock solution (100 mg=l adrenaline in phosphate buffer,
pH 7.0). The stock solution was used for 7 days and stored at 4C.
Apparatus
UV-2450 spectrophotometer (Shimadzu, Japan) was used for the assay

of the laccase activity. Lock-in amplifier (SR830, Stanford Research Sys-
tems, USA) was used for measuring the phase delay of the sensor head.
Principle of Biosensors
Adrenaline is an o-diphenol containing a hydroxyl group (He et al. 2000)

and can be easily oxidized by molecular oxygen (Palop et al. 2002) with
the catalysis of laccase. If the sensor head with an oxygen-sensitive mem-
brane is put in the adrenaline solution containing laccase, an oxygen
gradient will be created on the oxygen-sensitive membrane due to the
consumption of the dissolved oxygen caused by the adrenaline oxidation,
which leads to a fluorescence change because molecular oxygen acts as a
dynamic quencher of fluorescence. This can be mathematically described

by using Stern-Volmer equation:
I 0 s0
1 Ksv Q 1
I s
where I0 and I are the fluorescence intensities of the sensor in the absence
and presence of the oxygen quencher, respectively, and Ksv is the Stern-
Volmer constant. s0 and s are the lifetime of the fluorescence in the

absence and presence, respectively, of the oxygen quencher in the sample
solution. [Q] is oxygen concentration.
Since a novel lock-in amplifier is used, light signal from LED is
supplied as sine modulated signal, and the fluorescence signal thus also
appears a sine signal but a phase delay. Light signal containing the fluores-
cence lifetime change can be converted into phase change in the lock-in
amplifier according to the relationship between s and u as shown below:
tan / 2pf s 2
where f is the exciting frequency.
From Eqs. (1) and (2) we can get Eq. (3):
tan /0
1 Ksv Q 3
tan /
By collecting the data of phase delay u, the quantification of adrenaline is
achieved.
Preparation of Biosensors
The CuTAPc-Fe3O4 nanoparticle composite was prepared by our pre-

vious method (Huang et al. 2006). Using glutaraldehyde, laccase was
covalently immobilized on the surface of the composite. The oxygen-
sensitive membrane was prepared by using tris(2,20 -bipyridyl)dichloro-
ruthenium (II) hexahydrate as the fluorescence indicator and cellulose
acetate as the matrix according to our previously work (Zhang et al. 2002).
The detecting system consists of a lock-in amplifier, a LED with the
excitation wavelength of 416 nm as the light source, a sensor head with an
oxygen-sensing membrane and a computer for data processing (see Fig. 1).
Measurements
The activities of the free and immobilized laccase at different pH and

temperature were assayed by our previous method (Huang et al. 2006),
using adrenaline as the substrate. Free and immobilized laccase were
incubated at 55C and pH 7.0 (phosphate buffer) for variable periods.
Figure 1. Schematic diagram of the fiber optic sensor.
The samples were withdrawn every 30 min and the residual activity was
immediately assayed to determine their thermal stability.
For the detection of adrenaline concentration, measurements were
performed with the setup shown schematically in Fig. 1. Oxygen consum-
ption occurred in the enzymatic reaction catalyzed by immobilized laccase
was detected by the sensor head with oxygen-sensing membrane. The sen-
sor head was placed into a tiny reaction cell which is available for a small
quantity of sample. In order to eliminate the interference of oxygen from
the open air, an entire airtight reaction cell was introduced. All the mea-
surements were carried out at 25C under continuous and constant stirring.
The enzymatic reaction was completed in 1 min, and the fluorescence signal
was collected by PIN and guided to the lock-in amplifier through the out-
put bundle, and then transferred to phase-delay u collected by the com-
puter. The following measurement could be performed after a simple
washing of the sensor head and immobilized laccase with buffer solution.
RESULTS AND DISCUSSION
Effect of pH and Temperature on Laccase Activity Using Adrenaline

as the Substrate
According to our previous study (Huang et al. 2006), CuTAPc disper-

sed randomly onto the surface of Fe3O4 nanoparticles to form the
CuTAPc-Fe3O4 nanoparticles composite and there are chemical combi-

nations between CuTAPc and Fe3O4 nanoparticles. The particles with
the average diameter of 50 nm are roughly spherical and almost super-
paramagnetic, which is beneficial to the laccase immobilization.
We studied the effects of pH and temperature on the activity of the
free laccase and the laccase immobilized on CuTAPc-Fe3O4 nanoparticles
composite using ABTS as the substrate. The optimal pH values of the
free and immobilized laccase were the same at pH 3.0, and the optimal
temperatures for the free and immobilized laccase were 30C and 45C,
respectively (Huang et al. 2006). In this work, by using the similar pro-
cedure, we determined the pH and temperature effects on the free and
immobilized laccase using adrenaline as the substrate. Figure 2 shows
that in the weakly acidic environment, the optimal pH for the activities
of free and immobilized laccase are both at 5.0. That is, for the oxidation
of both adrenaline and ABTS catalyzed by laccase, the optimal pH would
not change when free and immobilized laccase were used, indicating that
the microenvironments influencing the pH of the free and immobilized
laccases are the same. It can also be seen that with the increase of buffer
pH, the activity of the immobilized laccase decreases more slowly than
Figure 2. pH effect on the activity of free and immobilized laccase. A: free

laccase; B: immobilized laccase. (t 25 C)
Figure 3. Temperature effect on the activity of free and immobilized laccase.

A: free laccase; B: immobilized laccase (pH 5.0).
that of free laccase and it still has 50% of its initial activity at pH 7.0,
while that of free laccase is only 15% of its initial activity. Since the
pH of the human serum is around 7.0, the immobilized laccase is more
advantageous to the determination of the adrenaline in serum.
Figure 3 shows the relationship between the activities of free and
immobilized laccase and temperature. It can be seen that, at pH 5.0,
the optimal temperatures for the catalysis of adrenaline by free laccase
is 50C, while that for the catalysis of adrenaline by immobilized laccase
is 55C, 5C higher than the former, which is different from those when
ABTS were used as the substrate because the enzyme reaction is different.
Figure 3 also shows that after the immobilization on the carrier, the lac-
case has better activity than free laccase in the range of 2040C, showing
its advantages in biological detection.
Stability of the Immobilized Laccase
The stability of the immobilized laccase was studied using adrenaline as the
substrate. As shown in Fig. 4, when the incubation temperature was 55C,
with the increase of incubation time the activity of free enzyme decreased
Figure 4. Thermal stabilities of free and immobilized laccase. A: free lactose;

B: immobilized laccase. (t 55C).
significantly and finally dropped to 28.2% after 240 min incubation.

However, while the activity of immobilized enzyme quickly increased from
to 22.7% to 100% after 150 min incubation and then decreased to 94.3% in
the following 90 min, showing a high thermal stability. The results were in
agreement with those of Al-Adhami et al. who reported on temperature
stabilities of laccase immobilized on modified cellulose and acrylic carriers
(Al-Adhami et al. 2002). This could be explained in terms of activation-
period inactivation. Immobilization limits drastic conformational changes
of the enzyme, thus resulting in increased stability towards denaturation
(Klibanov 1979; Leonowicz et al. 1988; Weetall 1976). This outstanding
thermal stability of the immobilized laccase will make it suitable for the
application at elevated temperatures.
After stored at 4C for 1 month, the activity of the immobilized lac-
case still had 85% of its initial activity. However, under the same storage
conditions, the free laccase could only maintain 30% of its initial activity.
The immobilized laccase has better storage stability than free laccase.
Another important property in assessing the value of immobilized
laccase is its ability to be recycled for successive applications. Since the
main contents of the carriers are magnetic Fe3O4 nanoparticles, the
immobilized laccase can be separated easily from solution. After 10 con-

secutive operations, the immobilized laccase could still retain 84% of its
initial activity, showing that the immobilized laccase has good reusability.
Properties of Biosensor
Due to the fact that the pH of the human serum is neutral and that the
oxygen-sensitive membrane could only be used at the temperature below
40C according to our study, the biosensor was performed at pH 7.0
(phosphate buffer) and t 25C. The immobilized laccase could catalyze
the oxidation of adrenaline effectively at these conditions since it had
50% of its maximal activity at pH 7.0 and 63% of its maximal activity
at 25C as described above.
The oxidation process of adrenaline catalyzed by laccase takes more
than 20 minutes. However, this process could be much faster when a
small number of ABTS (e.g., 10 ml ABTS in 3 ml phosphate buffer con-
taining 80 ml adrenaline) was used as the electron mediator. The electron
transfer from the mediator to the enzyme is followed by electron dona-
tions from the target molecule to oxidized mediator (Potthast et al.
2001), causing the regeneration of the mediator and the accelerating of
the oxidation process of adrenaline. Therefore, the response time of the
biosensor could be reduced to 30 seconds. The experimental results also
showed that after the addition of ABTS, the immobilized laccase still had
the same activity at the working conditions (pH 7.0 and t 25C) as that
in the system without ABTS, indicating that this biosensor can perform
well for the adrenaline detection when ABTS was added.
A linear relationship between the phase delay u and adrenaline con-
centration was observed in the concentration range of 2.0 107 to 9.0
107 mol=l (see Fig. 5), and the linear graph was defined by the equation
of y 0.016533x 1.30882 and R2 0.988. In the concentration range
of 1.0 108 to 9.0 108 mol=l the calibration curves showed deviation
from linearity, and the graph was defined by the equation of y
0.01366x2 0.01736x 0.06384 and R2 0.993. It could be expected that
by improving the catalyzing characteristics of the immobilized laccase, the
determination of lower adrenaline concentration can be achieved.
Repeatability of the sensor was tested for the detection of adrenaline
using ABTS as the electron mediator. In the concentration range of
2.0 10 7 to 9.0 10 7 M and 1.0 10 8 to 9.0 10 8 M, the stand
deviation (SD) value was 4.9 10 9 (n 5) and 3.8 10 9 (n 5) M.
The stability of the biosensor depended on the stability of the
oxygen-sensing membrane and the immobilized laccase. Before the
Figure 5. Relationship between adrenaline concentration and phase delay of the

sensing membrane ((a) 6 ml pH 7.0 phosphate buffer: 0.25 mg/ml ABTS: 10 U
immobilized laccase: t 25C; (b) 6 ml pH 7.0 phosphate buffer: 0.1 mg/ml
ABTS: 5 U immobilized laccase: t 25C).
determination, we immersed oxygen-sensitive membrane in the buffer for

1 h to make the indicators connected weakly with the matrix leak out.
Then the oxygen-sensing membrane was immersed in the reaction cell
containing working buffer solution for 72 h at optimized working con-
ditions and no decrease of its oxygen sensitivity was observed. On the
other hand, stored at 4C for 1 month, the immobilized laccase still
retains 85% of its initial activity. As mentioned above, the immobilized
laccase also has good reusability and can be used for 10 consecutive
operations without notable decrease of its initial activity. Therefore, in
can be concluded that this biosensor has good stability.
CONCLUSIONS
A novel fiber optic biosensor consisting of an immobilized laccase and an

oxygen-sensing membrane was investigated for adrenaline determination
based on fluorescence quenching. The immobilized laccase was formed
by the immobilization of laccase on the CuTAPc-Fe3O4 nanoparticles
composite. ABTS was used as the electron mediator to accelerate the
oxidation of adrenaline. The detection of adrenaline with this biosensor
is simple, fast, and stable, and its detecting precision is outstanding,
showing the delightful application prospect of this kind of sensor.
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Biographies
Jun Huang graduated from Wuhan University, China, in 1982 .Dr Huang
completed his PhD from Wuhan University of Technology, China, in
1999. He is now a full professor of Wuhan University of Technology.
His research interests focus primarily on fiber optical sensing materials,
fiber optic chemical and biological sensors, and nanocomposite materials.
Hua Fang received her M.S. degree from Hubei University, China, in
1999. She is currently pursuing the PhD degrees in Wuhan University
of Technology, China, in the field of biomaterials and biosensors.
Cheng Liu is a postgraduate student of Wuhan University of Technology.

He is engaged in the research of fiber optical biological sensors.
Erdan Gu obtained his PhD degree form University of Aberdeen, UK, in

1992. His research interests include thin film physics, thin film growth,
device fabrication and characterization, photonics and sensor device
applications.
Desheng Jiang graduated from Wuhan University of Technology, China,

in 1975. He is now the chief professor of Wuhan University of
Technology. His research interests focus primarily on optical sensing
materials and devices, fiber optic sensors and systems, and materials
processing.

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A Novel Fiber Optic Biosensor

To link to this article: http://dx.doi.org/10.1080/00032710802119525

PLEASE SCROLL DOWN FOR ARTICLE

A Novel Fiber Optic Biosensor for the

Determination of Adrenaline Based on

Abstract: A novel fiber optic biosensor for the determination of adrenaline

Keywords: Adrenaline; Fiber optic biosensor; Immobilized laccase

Received 16 February 2008; accepted 26 March 2008.

Adrenaline is one of the best-known catecholamines biosynthesized in the

hemorrhage (Goodman and Gilman 1996). An early diagnosis based on

fluorescent oxygen-sensing membrane was used to detect the consump-

is sensitive, fast response, and stable, suggesting that it should be

Pycnoporus sanguineus laccase (E.C.1.10.3.2), a white-rot fungus laccase

UV-2450 spectrophotometer (Shimadzu, Japan) was used for the assay

Adrenaline is an o-diphenol containing a hydroxyl group (He et al. 2000)

dynamic quencher of fluorescence. This can be mathematically described

Volmer constant. s0 and s are the lifetime of the fluorescence in the

The CuTAPc-Fe3O4 nanoparticle composite was prepared by our pre-

The activities of the free and immobilized laccase at different pH and

Figure 1. Schematic diagram of the fiber optic sensor.

RESULTS AND DISCUSSION

Effect of pH and Temperature on Laccase Activity Using Adrenaline

According to our previous study (Huang et al. 2006), CuTAPc disper-

CuTAPc-Fe3O4 nanoparticles composite and there are chemical combi-

Figure 2. pH effect on the activity of free and immobilized laccase. A: free

Figure 3. Temperature effect on the activity of free and immobilized laccase.

Stability of the Immobilized Laccase

Figure 4. Thermal stabilities of free and immobilized laccase. A: free lactose;

significantly and finally dropped to 28.2% after 240 min incubation.

immobilized laccase can be separated easily from solution. After 10 con-

Figure 5. Relationship between adrenaline concentration and phase delay of the

determination, we immersed oxygen-sensitive membrane in the buffer for

A novel fiber optic biosensor consisting of an immobilized laccase and an

A.Y. 2004. Determination of catecholamines by capillary electrophoresis

Wolyniec, E., Niedzwiedzka, U., and Kojlo, A. 2007. Flow-injection chemi-

1994. Laccase properties, catalytic mechanism, and applicability. Appl.

Cheng Liu is a postgraduate student of Wuhan University of Technology.

Erdan Gu obtained his PhD degree form University of Aberdeen, UK, in

Desheng Jiang graduated from Wuhan University of Technology, China,

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