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Engineering, 2012, 5, 129-134
doi:10.4236/eng.2012.410B034 Published Online October 2012 (http://www.SciRP.org/journal/eng)
Copyright © 2012 SciRes. ENG
Fast Determination of Vitamin B2 Based on Molecularly
Imprinted Electrochemical Sensor
Peidong X u, C hangsheng Qia o, Shufeng Y ang, Lijuan Liu, Mingwei Wang and Jua nkun Zhang*
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Bioengineering,
Tianjin Key Laborat ory of Indust ry Microbi ology, C ollege of Bioengi neerin g,
Tianjin University of Sci ence & Techn ology, TUST, Tianjin, P.R.China
Under the condition of weak acidity of pH 5.2, a sensitive vitamin B2 electrochemical sensor based on molecularly imprinted non-
conducting polymer of o-aminophenol by potentiostatic polymerization in the presence of template(vitamin B2) on a glassy carbon
electrod e was p rep ared , an d i ts p erfor mance was studied. The sensor exhibited good sensitivity and selectivity to VB2. The detection
limit went down to 2.3851nM, and a linear relationship between the current incremental and the concentration was found in the range
of 10～120nM. And the sensor could use in detection of VB2 real sample for a long time and show good reproducibility. The aver-
age recovery rate to VB2 was 98.41%.
Keywords: Vitamin B2; Molecularly Imprinted Technique; Potentiostatic Polymerization; O-aminophenol; Drug Determination
Vitamin B2(VB2), also named riboflavin(RF), is B-group wa-
ter-soluble vitamin. Its structure is exhibited in Figure 1.
VB2 is stable in acidic aqueous solution and its isoelectric
point(PI) is 6. Human beings as well as all animals requires a
constant supply of VB2 for the optimal maintenance of large
number of metabolic processes requiring the flavin coenzymes,
such as flavin mononucleotide(FMN) and flavin adenine di-
nucletide(FAD). FMN and FAD are important cofactor of
dehydrogenase in vivo. But VB2 can not be synthesized in vivo,
only be obtained from vegetables, fruit, health care food and
drug etc. What is worse, man and all animals have little ability
to store VB2 and excess VB2 will be urinated out of body.
Hence, VB2 should be intake and supplemented in time to
avoid shortage. Recently, many counterfeit vitamin drugs (in-
cluding veterinary medicine) that mainly consist of not enough
quantity frequently appear on market. Thus, determination of
VB2 is very significant in monitoring the amount of VB2 in
Figure 1. Chemical structure of VB2.
Currently, different methods and techniques have been used
to determine concentration of VB2, including chemilumines-
cence[ 3-4], chromatography[5-7], voltammetry, fluor escence
, flame atomic absorption spectrometry, and combina-
tion of these methods[11-13]. But these methods are not only
sophisticated but nonspecific, worse still, some expensive and
broad-scale equipments are needed. Molecularly imprinted
electrochemical sensor, however, based on “molecular memo-
ry”, combining the advantages of molecularly imprinted me-
thod(MIM) with electrochemical measurement technique, but
has not been reported to used in determination of VB2 yet.
The synthesis of molecularly imprinted polymers(MIPs) and
artificial recognition sites are key and core content in MIM.
They are establ ished by chemical and electro chemical meth ods.
These sites tailor-formed in situ by copolymerization of func-
tional monomers and cross-linkers around the template. The
print molecules are subsequently removed from the polymer,
living accessi ble complementary binding cavity. According
to the interaction between template and polymer, MIM can be
divided into covalent imprinting and noncovalent imprinting.
Noncovalent interaction consists of hydrogen bond, electrostat-
ic force, metal ion coordination interaction, Van der Waals
Selection of functional monomer plays an essential role in
preparing sensitive film in MIM. O-aminophenol(oAP), with
important functional groups of –OH and –NH2, has been used
to prepare kinds of sensitive films of biosensor. In contrast
to conducting polymers(polyaniline, polypyrrole, poly(3,
4-ethylenedioxythiophene) etc.), the electrosynthesis of non-
conducting polymers, take PoAP as an example, is self-limited
and inevitably ceased when the polymer film on the electrode
grows thick enough to resist electron transfer between the elec-
trode and monomer molecules. Moreover, electrochemical
polymerization is very easy to achieve i n electrolyte mixed with
P. D. XU ET AL.
Copyright © 2012 SciRes. ENG
functional monomer and template. The thickness of films could
be controlled precisely by adusting current density and goes
down to nanometers scale. PoAP film was selected in this
study because of its relative cheapness, easy availability and
Therefore, in this paper, VB2 molecularly imprinted electro-
chemical sensor was prepared through electropolymerizaton of
oAP with VB2 under pH 5.2. Meanwhile, the effects of percen-
tage of oAP on polymerization were investigated by UV-vis
spectrophotometry, and elution time of template, performance
of this sensor, etc. were also investigated in detail.
Materials and Methods
1) Chemicals and reagents
O-aminophenol(oAP) was from Tianjin recovers the fine
chemical research institute after appropriate recrystallization
wherever necessary. Perchloric acid was purchased from Alad-
din(Shanghai, China). Riboflavin(RF or VB2), ascorbic ac-
id(VC), vitamin B6(VB6) came from Tianjin Ruijinte Chemical
Co., Ltd. Flavin mononucleotide(FMN) was from Beijing
dingguo changsheng biotech Co. Ltd. Acetic acid, sodium hy-
droxide, potassium hexacyanoferrate, sodium phosphate dib asic
dodecahydrate, sodium dihydrogen phosphate, sulfuric acid and
other reagents were of analytical reagent grade. All solutions
were prepared with deionized water(conductivity≤0.06μS/cm).
Phosphate buffer solution(PBS) with various pH values and
concentration were prepared by mixing standard stock solutions
of NaH2PO4 and Na2HPO4 and adjusting the pH with NaOH
or HCl. Acetate buffer solution(HAc-NaHAc, pH 5.0) was
made by mixing HAc and NaOH.
All electrochemical measurements were performed on
LK2005A Electrochemistry Workstation (LANLIKE, Tianjin,
China). A standard tree-electrode system, a glassy carbon(GC)
electrode(3 mm in diameter) or its modified electrode, a
Ag/AgCl with saturated KCl solution and a Pt001 disk elec-
trode(2 mm in diameter) were used as working electrode, ref-
erence electrode and counter electrode, respectively. TU-1810
UV-Vis Spectrofluorimeter(PERSEE, Beijing, China),
FA2004A Electronic Analytical Balance(JIN GTI AN, Sh angh ai,
China), SB3200DT Ultrasonic Cleaning Machine(SCIENTZ,
Ningbo, China) and PHS3BW pH meter(LIDA, Shanghai,
China) were utilized in this study.
3) Pretreatment of glassy carbon electrode
Prior to modification, GC electrode was polished with 0.1μm
and 0.05μm alumina slurry successively. After rinsed with
deionized water, the electrode was treated in Piranha solu-
tion(30% H2O2 mixed with H2SO4 at the volume rate of 3 to
7 ) for 15min, followed by rinsing and sonication in 1:1(V/V)
HNO3 aqueous solution, 10%(W/V) NaOH solution, ethanol
and acetone for 10min, respectively. Repeated above process
triplicately. And then the three-electrode system was put into a
single-compartment cell with 0.2M H2SO4 solution, cyclic
scaning from potential of -0. 5 to 1.5V several cycles at th e rate
of 0.1V/s to achieve steady state for activation.
4) Preparation of VB2 molecularly imprinted polymers
VB2 molecularly imprinted polymers modified elec-
trode(VB2-/PoAP/GCE) was prepared by traditional
tree-electrode system. A oAP stock solution(0.1M) was pre-
pared by dissolving the oAP in 0.5M HClO4 solution. A VB2
stock solution(1mM) was prepared by dissolving the VB2 in a
1%(V/V) acetic acid aqueous solution at refrigerator(4℃). A
10mL volume of polymerization solution was prepared by
mixing 8 mL of oAP and 2 mL of VB2 and adjusting the pH
to 5.2 with 1M NaOH solution, and then standing in dark place
for 20min to prereaction. After that, three electrodes was in-
serted into this mixed solution. Polymerization was performed
by potentiostatic polymerization technology. The parameters
were set as follows: potential 0.65V, time 1200s, sampling time
interval 0.1125s. After polymerization, the modified electrod
was rinsed by deio nized water and immersed in 0.8M H2SO4
for 8h to remove template molecules. Nonmolecularly im-
printed modified electrode(PoAP/GCE) was prepared by the
same proced u re in th e ab sence o f V B2 , 2mL of 1%(V / V ) aceti c
acid in stead , for co mpariso n. When not in use th e elect rode was
stored in 0.01M pH 7.0 PBS at room temperature(RT).
5) Electrochem ical measurements
All electrochemical measurements were performed in 10mL
single-compartment cell with 4mM K3[Fe(CN)6] as electro-
chemical probe at RT and potential range from -0.1V to 0.6V.
All parameters of cyclic voltammetry(CV) were scan rates of
0.05V/s. All parameters of di fferenti al pulse vo ltammetry(DP V)
were potential incremental of 0.004V, pulse amplitude of 0.05V,
pulse width of 0.06V. All parameters of square wave voltam-
metry(SWV) were potential incremental of 0.004V,
square-wave frequency of 15Hz, square-wave amplitude of
0.025V. All potentials refered to Ag/AgCl with saturated KCl
solution. The tested solutions were deoxygenated by high pure
nitrogen bubbling for 20min just before experiments and the
nitrogen atmosphere was kept over the cell during all mea-
surement s .
3. Results and Discussion
3.1. Current-time curve of forming VB2 imprinted
Figure 2 i s Current-time(I -t) curve of copolymerization of oAP
and VB 2 on GC electrode. As shown in Figure 2, at the begin-
nin g of electrochemical polymeri zation (0s) the value o f current
decreases sharply from 80μA which indicates the fast formation
of nonconducting polymer. As increasing of electrolysis time
the current decreases gradually slow, and then down to a plat-
form. Finally the current decreases close to zero. This is the
result of electrochemical oxidation of oAP to form PoAP on
GC electrode, which is demonstrated that a layer of dense po-
lymer membrane blocking electrons transferring from bulk
solu tion to sur face of electrod e is formed and VB2 molecu lar is
freezed in PoAP at the same time. Thi s thanks to “self-limited”
electrodeposition process of nonconductive PoAP film
3.2. Selection of Polymerization Time
Here three modified electrode were prepared in 600s, 1200s and
1800s, respectively. CV was performed in PBS with 4mM
P. D. XU ET AL.
Copyright © 2012 SciRes. E NG
Figure 2. I-t curve of molecularly imprinted copolymerization
proc es s o f oA P an d VB2 on GC electr od e.
K3[Fe(CN)6] to characterize their properties. It was indicated
that the formed polymer modified electrode of polymerization
of 600s and 1200s show no redox peak in K3[Fe(CN)6] solu-
tion, but the response current of the former is larger than the
latter. When polymerization of 1800s, however, there are a
couple of weak redox peaks which indicates the conductivity of
this polymer. This result is corresponding to Ref. , i.e.,
PoAP can transfer between insulator and conductor. Therefore
1200s(equivalent to 15 cycles in CV) is selected in this study
which is closed to the result of Chi et al(14 cycles in CV
pol ymerizati on on carbon paste electrode) .
3.3. Electrochemical Characterization of VB2
Imprinted Se nsor
CV was performed in PBS with 4mM K3[Fe(CN)6] to charac-
terize property of this sensor. [Fe(CN)6]3- can successfully
diffuse from the bulk solution to the surface of electrode and
quasi-reversible redox reaction take place on naked GC elec-
trode(Fig ure 3a). After elution of the template molecu-
lar(VB2-/PoAP/GCE), though there is redox peak the peak is
very weak, the redox current is very small too(Fig ure 3b).
Moreover, the CV curve shape is poor, which indicates the
template has been eluted by H2SO4 living imprinted sites that
pave the way for a portion of [Fe(CN)6]3- transferring. But
uneluted electrode(VB2+/PoAP/GCE) and nonimprinted elec-
trode(PoAP/GCE) have no response to [Fe(CN)6]3-(Figure 3c,
3d, respectively), which also demonstrated that PoAP thin film
is very dense and nonconducting, blocking diffusion of
[Fe(CN)6]3- compl etely.
3.4. Mechanism of Interaction between o A P and VB2
Reference reported that PoAP thin film formed by electro-
chemical oxidation polymerization was electric inert when pH
﹥3 and in form of oxidized state in the absence of Cl- in pH 5.
In this case, PoAP was insulated completely and showed no
redox activity itself. Hence, PoAP, in weak acid environment,
can not transfer electrons through redox process to be conduct-
Reference reported that the first and the second proton
binding constant of VB2 were 1010 and 106, respectively.
Therefore, when VB2 in solution of pH﹤6, it positively
charged(RFH22+), b alance state equation defined as :
Hence, it is speculated that hydrogen bond must be formed
between oAP and VB2.
In this text, UV-vis spectrometry was performed to analyze
mechanism of interaction between oAP and VB2 due to its low
cost, ease of operation, cheapness of instrument and so on.
Volume percentage of oAP changed from 0 to 100%(0, 10%,
20%, 40%, 80% and 100%, respectively) and scanning curve
from 300nm to 700nm was recorded as shown in Figure 4.
From these scanning curve, adding of oAP did not alter the
absorbance peak shape of VB2 and it was found the value of
maximum absorption wavelength shifted to long-wave direc-
tion(redshift, from 374nm to 384nm) with absorbance value
increasing(from 2.245 to 2.816) as increasing percentage of
oAP from 0 to 80%. This was due to hydrogen bond has impact
on the first π-π* absorption band of chromophore as a proton
donor molecule. It was conformation of hydrogen bond forma-
tion between oAP and VB2.
Thus imprinted process of VB2 was sketched in Figure 5.
When mixing oAP with VB2 and standing for 20min, there
were numerous hydrogen bonds producing(process of prepoly-
merization). And then electrochemical oxidation polymeriza-
tion reaction was performed to form PoAP and VB2 was im-
printed in PoAP thin film simultaneously. Followed by elution
process, i.e. under the diluted sulfuric acid(0.8M) condition the
hydrogen bond was destroyed, which lead to separation of VB2
from the film, living cavity memorized for VB2.
Figure 3. Cyclic voltammograms of different modified electrode in
a→f: 80%, 40%, 20%, 10%, 0(1mM VB2) and 100%(0.1M oAP)
Figure.4. UV-vis scanning curve of different percentage of oAP in
P. D. XU ET AL.
Copyright © 2012 SciRes. ENG
3.5. Selection of Elution Time
Diluted strong acid solution is a powerful tool to destroy the
hydrogen bond. 0.8M H2SO4 was used in this study and its
elution kinetics was also studied. It was indicated that the re-
duced peak current incremental value(ΔI) increased with dilu-
tion time at the range from 0 to 80min. Up to 140min ΔI
achieved a platform. After elution of 480min(8h) ΔI was almost
unch anged which in dicated elution came to equilib rium. Hence
8h of elution was selected in this study.
3.6. Eval uat ion of the Imprinting Effe ct
PoAP/GCE and VB2+-PoAP/GCE had no response to target
molecule(VB2) in 0.2M pH 5.0 HAc-NaHAc with 1mM VB2
and 4mM K3[Fe(CN)6](Figure 5d, 5e). VB2--PoAP/GCE had
good response to VB2 and the cathode peak current to target
molecule decreased from 14.183μA to 12.786μA (Figure 6b,
6c). Compared to nGCE(Figure 6a) respons e current decreased
significantly which demonstrated VB2 molecule binded to im-
printed cavity by hydrogen bond and closed the empty site,
blocking [Fe(CN)6]3- diffused from bulk solution to the surface
of electrode. Thus concentration of [Fe(CN)6]3- on the surface
of electrode decreased significantly, leading to reduced current
decreased signific antly.
PP: Prepolymerization; ECP: Electrochemical Polymerization; E: Elution
Figure 5. Schematic illustra tion of imprinted process of VB2 o n GC
a: nGCE; b: before rebinding of VB2; c: after rebinding of
VB2 d: PoAP/GCE；e: VB 2+-PoAP/GCE
Figure 6. DPV curve of different modified electrode in potassium
ferricyanide with VB2.
3.7. Drawing of Calibration Curve of VB2 Rebinding
SWV with high sensitivity was performed to draw calibration
curve of VB2 rebinding as shown in Figure 7. The curve
showed nonlinear overall from 20nM to 80μM, and the curve
tended to steady at 80μM with increasing concentration of VB2,
which indicated there was saturation effect of the sensor res-
ponding to VB2. To obtain good linear relationship between the
concentration and ΔI, low VB2 concentration was selected to do
rebianding test again. The result was shown in Figure 8. The
linear response range of this imprinted sensor to VB2 concen-
tration was from 10nM to 120nM with a correlation coefficient
of 0.9989(n=7). The linear regression equation was
ΔI=-0.03381[VB2]+15.76 94. The detect ion limit was calcu lated
according to equation as follows:
where SD and B were standard deviation and slope of linear
regression equ ation, r es pectively. LOD of VB2 was 2.3851nM.
VB2 concentration from 20nM to 80μM(20nM, 40nM, 60nM, 80nM,
20μM, 40μM, 60μM and 80μM, respectively )
Figure 7 Relationship between VB2 concentration and current of
VB2 concentration from 10nM to 120nM(10nM, 20nM, 40nM, 60nM,
100nM, 110nM, and 120nM, respectively ). Inset: SWV testing curve of
dif ferent concentration of VB2
Figure 8. Calibration curve of VB2 rebi nding to imprinted sensor.
P. D. XU ET AL.
Copyright © 2012 SciRes. E NG
Table 1. The determination and recovery result in vitamin B2 tablet.
Real amount/mg Adding a mount o f st an dard/mg Detected valu e/mg Re cover rat e /% Average recover ra te/ % RSD/%
4.98 5.18 4 .89 94.42
4.97 10.32 9.30 90.12
5.01 15.12 14.93 98.75
4.95 20.34 21.04 103.45
4.95 25.20 26.60 105.00 98.41 4.75
5.02 30.15 29.33 97.28
4.96 35.40 35.30 99.72
4.99 40.44 41.05 101.52
5.04 45.70 43.61 95.43
3.8. Selectivity of Imprinted Se nsor
Water-soluble vitamin, such as VB6 and VC, were selected to
be disruptors to rebind to imprinted cavity site competitively
due to their consist of a large number of hydroxy group. And
FMN was also selected because of its structure similarity with
VB2. The absorption isotherm was made in absorption equili-
brium time of 100min. It was found that VB6, VC and FMN
could rebind to imprinted cavity site by hydrogen bond , but
compared to VB2, the quantity was very low showed in terms
of ΔI. Hence the sensor has good selectivity.
3.9. Real Sample Analysis
VB2 tablets were grinded to powder in a mortar and then dis-
solved by 1%(V/V) acetic acid solution. A quarter of above
solution was tested by SWV according to the methods as men-
tioned before. As shown in Table 1, the average recovery rate
to VB2 was 98.41%.
3.10. Reproducibility and St ability of the V B2
Imprinted Se nsor
Reproductive testing of 8 times in 40μM successively(absorbed,
tested and eluted , repeated 8 times), showed an acceptable
reproducibility with a RSD of 4.1%. The sensor could retain
94.8% of acivity to VB2 after a storage of 5 weeks i n 0. 01M pH
7.0 PBS at RT, which indicated good stability.
On the surface of naked GC electrode , oAP could electro-
chemical oxidation polymerization with VB2 by potentiostatic
method. Trough controlling polymerization parameters of po-
tential, time, pH of solution and percentage of monomer, oAP
as monomer, VB2 as template molecule, electrons as initiator,
VB2 electrochemical molecular imprinting sensor was estab-
lished. The sensor showed good recognition to VB2 , LOD went
down to 2.3851nM and exhibited good selectivity, reproduci-
bility and stability. Hydrogen bond plays important role betreen
the host(PoAP) and the guest(VB2). This sensor was used to
test VB2 tablet with good recover y rate.
The authors would like to thank the Ministry of Science and
Technology of the People’s Republic of China (2009GJA-
10047), Tianjin Municipal Science and Technology Commis-
sion (09ZCZDSFO4200), Tianjin Municipal Education Com-
mission (SB20080035) and the Tianjin University of Science
and Technology to support the work.
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