Electrochemical detection of fucosidase activity in human serum

Article abstract of DOI:10.1002/jccs.202000376

Liver cancer is one of the most common malignant tumors globally and is the second leading cause of cancer deaths in Taiwan. The activity of α-fucosidase (AFU) in the human body has been confirmed by many clinical studies and experiments to be applicable in the early diagnosis of primary liver cancer. Herein, an electrochemical method for determining AFU activity was developed based on measuring p-nitrophenol (pNP), which is a product of the reaction between AFU and p-nitrophenyl-α-L-fucopyranoside. The electrochemical detection of pNP in serum samples exhibited a wide linear range (2.5–250 μM), which corresponds to approximately 2.5–250 U/L AFU. The recovery of AFU measured upon enzyme addition to standard serum was 97–110%. Moreover, no potential interferants had a significant effect on pNP quantification. In contrast, an optical absorption method was subject to interference by the serum color. Further, the linear range for pNP was only 2.5–50 μM and the recovery of AFU was 165–219%. The performance of this absorption method could lead to a misjudgment of the disease course. Therefore, the electrochemical method for AFU activity determination has greater potential for use in clinical applications.

Full text of DOI:10.1002/jccs.202000376

Received: 31 August 2020
Accepted: 29 October 2020
DOI: 10.1002/jccs.202000376
A R T I C L E
Electrochemical detection of fucosidase activity in
human serum
1
,2
1
1
3
Hweiyan Tsai
| Chen Yu Yu | Chien-Hong Chen
| Chwan Bor Fuh
1
Department of Medical Applied
Chemistry, Chung Shan Medical
University, Taichung, Taiwan
Abstract
Liver cancer is one of the most common malignant tumors globally and is the
2
Department of Medical Education,
second leading cause of cancer deaths in Taiwan. The activity of α-fucosidase
Chung Shan Medical University Hospital,
Taichung, Taiwan
(AFU) in the human body has been confirmed by many clinical studies and
3
experiments to be applicable in the early diagnosis of primary liver cancer.
Herein, an electrochemical method for determining AFU activity was devel-
oped based on measuring p-nitrophenol (pNP), which is a product of the reac-
tion between AFU and p-nitrophenyl-α-L-fucopyranoside. The electrochemical
detection of pNP in serum samples exhibited a wide linear range (2.5–250 μM),
which corresponds to approximately 2.5–250 U/L AFU. The recovery of AFU
measured upon enzyme addition to standard serum was 97–110%. Moreover,
no potential interferants had a significant effect on pNP quantification. In con-
trast, an optical absorption method was subject to interference by the serum
color. Further, the linear range for pNP was only 2.5–50 μM and the recovery
of AFU was 165–219%. The performance of this absorption method could lead
to a misjudgment of the disease course. Therefore, the electrochemical method
for AFU activity determination has greater potential for use in clinical
applications.
Department of Applied Chemistry,
National Chi Nan University, Puli,
Nantou, Taiwan
Correspondence
Hweiyan Tsai, Department of Medical
Applied Chemistry, Chung Shan Medical
University, Taichung, Taiwan.
Email: annetsai@csmu.edu.tw
Chwan Bor Fuh, Department of Applied
Chemistry, National Chi Nan University,
Puli, Nantou, Taiwan.
Email: cbfuh@ncnu.edu.tw
Funding information
Ministry of Science and Technology,
Taiwan, Grant/Award Number: MOST
1
07-2113-M-040-002
K E Y W O R D S
electrochemical technique, liver cancer, p-nitrophenol, α-Fucosidase activity
1
| INTRODUCTION
operator's experience. In addition to AFP, several other
biomarkers (such as squamous cell carcinoma antigen–
Hepatocellular carcinoma (HCC) is one of the most com-
mon malignant tumors in some areas of the world and
has been the second leading cause of cancer-related
immunoglobulin M complexes, α-fucosidase (AFU),
chromogranin A, human hepatocyte growth factor, and
insulin-like growth factor) have been proposed as
[1,2]
[4]
deaths worldwide over the past 10 years.
Indeed,
markers for the early detection of HCC. Since the
more than 8,000 patients die as a result of liver cancer
1980s, hepatocarcinoma cells have been found to cause
an abnormal increase in the activity of AFU in vivo, as
[3]
each year in Taiwan.
Currently, the recommended
[
4–13]
screening strategy for patients suffering from cirrhosis
includes the determination of serum α-fetoprotein (AFP)
levels and an abdominal ultrasound every 6 months to
detect HCC at an early stage. However, as a marker, AFP
has poor sensitivity and specificity, and the results of
abdominal ultrasounds are highly dependent on the
confirmed by numerous clinical studies.
It has been
suggested that the activity of AFU is highly sensitive and
selective for the diagnosis of primary HCC and
fucosidosis. Therefore, several techniques have been
developed to determine the activity of AFU, such as spec-
trophotometric methods using p-nitrophenyl-α-L-
J Chin Chem Soc. 2020;1–8.
http://www.jccs.wiley-vch.de
© 2020 The Chemical Society Located in Taipei & Wiley-VCH GmbH
1

2
TSAI ET AL.
[
14,15]
fucopyranoside (pNPF) as the enzyme substrate,
fluorescence spectrometric methods using 4-methyl-
umbelliferyl-α-L-fucoside as the enzyme substrate
one unit (1 U) of AFU activity is defined as the produc-
tion of 1 μmol of pNP per minute in the reaction with
pNPF, the quantitative determination of pNP is used to
calculate the AFU activity. In addition, the electrochemi-
cal detection method is compared with a traditional opti-
cal absorption technique for measuring AFU activity in
serum. A number of common electroactive species pre-
sent in human serum, such as tyrosine, glucose, ascorbic
acid, urea, and uric acid are also examined to determine
any interference effects.
[
16–18]
to
release a fluorescent methylumbelliferyl derivative, CdTe
[19]
[11,20]
quantum dots,
immunoassays,
and potentiometric
methods using 2-chloro-4-nitrophenol–rhodamine B ion
pair chemical recognition in PVC membrane sensors.
[
21]
One of the most commonly employed methods for
detecting AFU activity is based on a colorimetric tech-
nique; however, the yellow color of serum and urine can
easily interfere with this method. Although other alterna-
tive methods have been employed for AFU detection, they
involve the use of expensive reagents, complex processes,
or the application of noble metals, some of which are less
sensitive than expected. Therefore, the development of an
efficient and low-cost method for the detection of AFU
activity in blood is necessary for the diagnosis of liver
cancer.
2 | RESULTS AND DISCUSSION
2.1 | Optimization of reaction and
detection conditions
2.1.1 | Optimization of alkali solution
Because p-nitrophenol (pNP) exhibits electroactivity
in addition to its optical properties, several studies have
investigated its detection using electrochemical
Generally, the AFU activity was measured based on the
amount of pNP produced by the AFU-catalyzed hydroly-
sis of pNPF at pH 4.0. pNP is colorless under acidic con-
ditions but dissociates to 4-nitrophenolate (Scheme 1b),
which is yellow and exhibits a maximum absorption at
405 nm, under basic conditions. Therefore, an alkali solu-
tion was added to stop the enzymatic reaction at the end
of the desired reaction time and to form 4-nitrophenolate
for optical measurements.
[22–25]
methods.
Indeed, electrochemical methods are pop-
ular analytical techniques owing to their sensitivity, con-
venience, and low cost. Herein, an electrochemical
sensing strategy for AFU activity determination is
designed based on the use of pNPF as a substrate and
subsequent detection based on the electrochemical prop-
erties of pNP. More specifically, cyclic voltammetry, a dis-
posable screen-printed carbon electrode, and a simple
electrochemical pretreatment method are employed. As
The changes in the absorbance of the reaction solution
following the addition of various concentrations of sodium
SCHEME 1 (a) AFU-
catalyzed hydrolysis of pNPF to
produce terminal nonreducing
α-L-fucose. (b) Dissociation of
pNP to 4-nitrophenolate

TSAI ET AL.
3
carbonate are shown in Figure 1. The results show the rela-
tionship between the equilibration time and the alkali con-
centration used to stop the enzymatic reaction and to
achieve pNP dissociation. Following sodium carbonate addi-
tion, the absorbance reached a plateau after 13 min, and no
significant changes were observed after this point upon vary-
ing the sodium carbonate concentration. As 1.2 M sodium
carbonate was able to immediately quench the enzymatic
reaction and transform pNP to 4-nitrophenolate, this con-
centration was selected for future experiments.
2.1.3 | Optimization of solution pH for
the enzymatic reaction
As reported in the literature, the optimized reaction condi-
tions for the enzymatic reaction of AFU are the use of a
0.1 M acetate buffer containing 0.1% BSA at pH 4.0. How-
ever, as the pH of human serum is 7.2–7.4, the AFU activ-
ity at pH 4.0 was compared with that at pH 7.4. As shown
in Figure 4, a greater AFU activity was observed at pH 4.0.
Thus, to achieve the more sensitive detection of AFU in
human serum and reduce the amount of serum required
for detection, the serum sample was diluted 10 times using
pH 4.0 acetate buffer prior to the reaction with pNPF.
Figure 2a shows the cyclic voltammograms of pNPF and
pNP. In the initial step, pNP undergoes an irreversible
reduction reaction (R1) to produce 4-hydroxylaminophenol,
and the subsequent oxidation of 4-hydroxylaminophenol
(
O1, E ~ 0.0 V) produces 4-nitrosophenol. Finally, a revers-
p
ible reduction reaction occurs (R2, E ~ −0.1 V) (Figure 2b).
2.1.4 | Optimization of enzymatic
p
pNPF has a nitro group that can undergo similar reactions;
however, the redox potentials are somewhat shifted owing
to the different substituent at the para position of the ben-
zene ring. As the R2 peak of pNP was found to be
completely separated from that of pNPF, the R2 peak was
used for the quantitative determination of pNP formed dur-
ing the enzymatic reaction.
reaction time
Experiments revealed (data not shown) that the forma-
tion of pNP increased upon increasing the reaction time
during the initial 30 min of the reaction, after which, the
depletion of pNPF led to the curve leveling off. The
enzyme activity was then calculated based on the forma-
tion of pNP (see Equation ( )). The optimal reaction time
1
for the enzymatic reaction was considered to be 10 min,
2.1.2 | Optimization of starting potential
The starting potential of cyclic voltammetry will affect
the amount of 4-hydroxylaminophenol formed, and so
will subsequently affect the currents of the O1 and R2
peaks. As indicated in Figure 3, a more negative starting
potential resulted in greater O1 and R2 peak currents.
FIGURE 2 (a) Cyclic voltammograms for pNP ( – ) and pNPF
(red dots) (potential range (1.4 V to 0.5 V; scan rate: 100 mV/s). The
experiment was carried out in a mixture of 0.1 M acetate buffer and
1.2 M sodium carbonate (84:16, v/v). Before the mini-cell was
assembled, the screen-printed electrode was cleaned by cyclic
voltammetry in the potential range of 1.8 to −0.6 V at a scan rate of
0.3 V/s for 20 cycles in phosphate buffer solution (pH 7.0).
FIGURE 1 Changes in the absorbance of the reaction solution
following the addition of various concentrations of sodium
carbonate. The reaction between pNPF and AFU in 0.1 M acetate
buffer (pH 4.0) containing 0.1% BSA was examined. All reactions
ꢀ
were carried out at 40 ± 1 C. After allowing the enzymatic reaction
to proceed for 10 min, a solution of sodium carbonate was added
[26]
(
acetate buffer/sodium carbonate = 84:16, v/v)
(b) Electrochemical reactions of pNP

4
TSAI ET AL.
at which point the formation of pNP was proportional to
the AFU activity and a sufficiently strong electrochemical
signal was produced.
this system upon diluting 10 times with pH 4.0 buffer
and reacting with pNPF at 40 C over 10 min (calculated
ꢀ
based on Equation ( )). The linear detection range of
1
AFU activity was 2.5–250 U/L.
2.2 | pNP calibration curves
2.3 | Measurement of serum AFU
Calibration curves were constructed using pNP standards
in a mixture of buffer at pH 4.0 containing 10% human
serum (HS) and 1.2 M sodium carbonate (Figure 5). As
outlined in Table 1, the electrochemical quantification of
activity
[13]
According to a report by Wang et al. in 2004, the average
serum AFU levels were 59.7 ± 39 U/L for 148 liver cancer
patients and 22.8 ± 7.1 U/L for 518 healthy adults. Various
amounts of AFU were therefore spiked into commercial
normal serum to mimic clinical samples with AFU concen-
trations ranging from 10 to 70 U/L. Figure 6 shows the
cyclic voltammograms of the serum samples. The enlarged
part of figure was inserted to show the peaks used to quan-
tify the activity of AFU. The recovery results (Table 2)
clearly show that electrochemical detection resulted in high
recoveries (97–110%), while the optical measurements gave
exaggerated results (165–219%), which could lead to mis-
judgments regarding the course of the disease.
2
pNP exhibited good linearity (R > 0.995) and a wide lin-
ear range (2.5–250 μM). The limit of detection (LOD) for
pNP was 2.1 μM (S/N = 3), thereby indicating that a
serum AFU activity of 2.1 U/L can be measured using
2.4 | Effect of storage conditions on the
activity of serum AFU
Figure 7 shows the changes in serum enzyme activity
with time under different storage conditions (i.e., 4 and
ꢀ
FIGURE 3 Effects of starting potential on the redox peak
currents (i.e., O1 and R2) of 4-hydroxylaminophenol
−20 C). The serum AFU activity remained high over
ꢀ
2 days when stored at −20 C but decreased rapidly with
time upon storage at 4 C. These results therefore indicate
ꢀ
that the serum sample must either be measured
FIGURE 4 Comparison of the AFU activities at pH 4.0 and
7
.4. The AFU concentration was calculated directly as indicated on
the commercial product without considering activity decay. The
pNPF and AFU solutions were prepared using pH 4.0 acetate buffer
and pH 7.4 phosphate buffered saline, respectively. After 10 min, a
FIGURE 5 Plots of peak current (blue circles) and absorbance
(red squares) versus pNP concentration in pH 4.0 buffer with 10% HS
and 1.2 M sodium carbonate (buffer/sodium carbonate = 84:16, v/v)
1
.2 M solution of sodium carbonate was added to the mixture, and
the produced pNP was determined using cyclic voltammetry

TSAI ET AL.
5
TABLE 1 Parameters for the pNP
calibration curves
2
Method
LOD (μM)
R
Linear range (μM)
2.5–250
m
Slope (±S )
Cyclic voltammetry
Optical absorption
2.1
2.9
0.9986
0.9953
21.98 ± 0.36
19.34 ± 0.77
2.5–50
FIGURE 6 The cyclic voltammograms of the serum samples. Various amounts of AFU were spiked into commercial normal serum to
mimic clinical samples with AFU. The samples were diluted 10 times with a 0.1 M acetic buffer solution (pH 4.0) and mixed with pNPF
ꢀ
(
1 mM), then incubated at 40 C for 10 min. Subsequently, the enzymatic reaction was stopped by the addition of 1.2 M sodium carbonate.
The CV measured at room temperature, potential range: −1.4 to 0.5 V; scan rate: 100 mV/s. The enlarged part of figure was inserted to show
the peaks used to quantify the activity of AFU
TABLE 2 Recovery values for serum AFU using different
techniques
Recovery (%)
Serum
AFU
Electrochemical
measurement (R at
E = −0.1 V)
p
2
Optical absorption
(at 405 nm)
(
U/L)
1
4
6
3.9
1.7
9.6
105 ± 8.4
110 ± 3.1
97 ± 5.8
165 ± 2.0
213 ± 0.1
219 ± 0.1
ꢀ
immediately or maintained at −20 C after collection from
the patients.
FIGURE 7 Variation in the relative activity of serum AFU
with time under different storage conditions
2.5 | Interference studies
Figure 8a shows the relative signals obtained in the pres-
ence of interfering substances, such as urea, vitamin C,
uric acid, tyrosine, and glucose, in the pNP standard solu-
tion. These substances generally exist in human serum
and are known to be electroactive. The concentrations of
the interferants were chosen based on the maximum
values in clinical reports. Interestingly, none of the
potential interferants had a significant effect on pNP
quantification. In addition, tyrosine and L-dopa contain a
similar ring structure to pNP, while glucose has a struc-
ture similar to fucose. However, upon the addition of
these substances to a mixture of AFU and pNPF, no effect

6
TSAI ET AL.
purchased from Taoyuan Major Science (Taoyuan, Tai-
wan). A SH-U830 Vis-spectrophotometer (Shishin Tech-
nology, Taipei, Taiwan) was used for absorption
measurements.
3.2 | Chemicals
Disodium hydrogen phosphate, urea, and D(+)-glucose
were purchased from Showa Kako Corp. (Osaka, Japan).
α-(1–2,3,4,6)-L-Fucosidase from Homo sapiens (AFU) and
pNPF were purchased from Megazyme (Chicago, IL).
BSA, uric acid, and pNP were purchased from Acros
Organics (Illkirch Cedex, France). Sodium carbonate, L
−
3,4-dihydroxyphenylalanine (L -dopa), L-ascorbic acid,
and sodium phosphate monobasic monohydrate
(
NaH PO ꢁH O, ACS reagent, ≥98%) were purchased
2
4
2
from Sigma-Aldrich (St. Louis, MO). L-Tyrosine (≥98.5%)
was obtained from Alfa Aesar (Heysham, UK). Sodium
acetate 3-hydrate (≥99%) and acetic acid (99%) were pur-
chased from Scharlab (Barcelona, Spain).
FIGURE 8 Relative signal intensities in the presence of
possible interfering substances in (a) 0.05 mM pNP standard
solution and (b) a mixture of AFU (10 U/L) and pNPF (4 mM). The
concentrations of interferants were 7.8 mM urea, 220 μM
vitamin C, 430 μM uric acid, 62 mM tyrosine, 20 μM L-dopa, and
3.3 | Serum sample preparation
7
.8 mM glucose
Normal HS from Jackson ImmunoResearch (West Grove,
PA) was employed in this study. Varying amounts of
AFU were spiked into this serum to mimic clinical sam-
ples. The sample was diluted 10 times with a 0.1 M acetic
buffer solution (pH 4.0) and then mixed with pNPF. All
on the AFU activity was observed (Figure 8b). These
results indicate that no additional pretreatment steps are
required for serum samples and that pNP quantification
can be carried out rapidly.
ꢀ
samples and reagents were preheated to 40 C and
maintained in an MD-Mini heating dry bath incubator
ꢀ
3
3
| EXPERIMENTAL
.1 | Apparatus
(40 C) for the desired amount of time. Subsequently, the
enzymatic reaction was stopped by the addition of 1.2 M
sodium carbonate. Finally, the enzyme activity was deter-
mined based on the formation of pNP, as measured by
cyclic voltammetry at room temperature (25 ± 1 C). All
experiments were repeated in triplicate.
ꢀ
Each electrochemical experiment was carried out using a
computer controlled by a CH Instruments Electrochemi-
cal Workstation 660C System (Austin, TX). A homemade
mini-cell (see supporting information) was equipped with
a screen-printed carbon working electrode, a platinum
wire as the auxiliary electrode, and a Ag/AgCl electrode
with 3 M NaCl solution as the reference electrode. For
measurements with this homemade mini-cell, only
3.4 | Theory and calculation
AFU, which is named α-L-fucoside oligosaccharide
hydrolase (EC 3.2.1.51) by the EC (International Union
of Biochemistry and Molecular Biology's Enzyme Com-
mission), is distributed in various tissue cells and fluids
within the human body. It is a lysosomal acid hydro-
lase with multiple catalytic pockets
the metabolism of fucosyl-based molecules in the body.
As described above, 1 U of AFU activity is defined as the
amount of enzyme required to release 1 μmol of pNP per
minute from pNPF (1 mM) in sodium acetate buffer
~0.5 ml solution was required. The screen-printed carbon
electrode (SE100, diameter: 5 mm; electrode surface area:
2
[7]
0
.196 cm ) was purchased from Zensor R&D (Taichung,
[
27]
Taiwan), while the Ag/AgCl electrode (RE-1B) and the
platinum wire (CHI115) were purchased from CH Instru-
ments (Austin, TX). An MP220 Mettler Toledo pH meter
was used to measure the pH values of the buffer solu-
tions. A MD-Mini heating dry bath incubator was
that is involved in

TSAI ET AL.
7
(100 mM) at pH 4.0 at the temperature indicated. This
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ORCID
Hweiyan Tsai https://orcid.org/0000-0002-4070-0058
Chien-Hong Chen https://orcid.org/0000-0001-6459-
[
4
467
[

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How to cite this article: Tsai H, Yu CY,
Chen C-H, Fuh CB. Electrochemical detection of
fucosidase activity in human serum. J Chin Chem
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