Electrochemical Determination of Salivary N-Acetylneuraminic Acid by Miniaturized Capillary Electrophoresis Coupled with Sample Stacking

Article abstract of DOI:10.1002/cjoc.201600189

Due to their important biological role as markers for different pathologies, sialic acid (SA) analyses are important for clinical research. In this work, a miniaturized capillary electrophoresis with amperometric detection (mini-CE-AD) was developed for the determination of N-acetylneuraminic acid (NANA), which is the most widespread form of SAs. NANA was first oxidized by periodic acid in an acidic solution, and then the oxidation product β-formyl pyruvic acid was derivatized with electroactive 2-thiobarbituric acid (TBA) to form an electroactive NANA-TBA adduct, which could be readily determined by mini-CE-AD. The limit of detection (LOD) of NANA-TBA could achieve 0.50 μg/mL (1.6 μmol·L^?1, S/N=3) based on an online enrichment approach of moving chemical reaction boundary. The proposed method was successfully applied to the analysis of NANA in human saliva, and the recoveries were in the range of 91.8% –109% with RSDs of 1.8% –3.9%. Due to its simple design and construction, low cost and portability, the mini-CE-AD device will possess more practicability in more field work as an alternative to conventional and microchip CE approaches.

Full text of DOI:10.1002/cjoc.201600189

FULL PAPER
DOI: 10.1002/cjoc.201600189
Electrochemical Determination of Salivary N-Acetylneuraminic
Acid by Miniaturized Capillary Electrophoresis Coupled with
Sample Stacking
Yiliang Zheng, Tingting Wang, Jiaxing Kong, Yaolu Ma, Yi Heng,
Yujuan Ren, Jiannong Ye, and Qingcui Chu*
School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China
Due to their important biological role as markers for different pathologies, sialic acid (SA) analyses are im-
portant for clinical research. In this work, a miniaturized capillary electrophoresis with amperometric detection
(mini-CE-AD) was developed for the determination of N-acetylneuraminic acid (NANA), which is the most wide-
spread form of SAs. NANA was first oxidized by periodic acid in an acidic solution, and then the oxidation product
β-formyl pyruvic acid was derivatized with electroactive 2-thiobarbituric acid (TBA) to form an electroactive
NANA-TBA adduct, which could be readily determined by mini-CE-AD. The limit of detection (LOD) of
NANA-TBA could achieve 0.50 μg/mL (1.6 μmol•L1, S/N＝3) based on an online enrichment approach of moving
chemical reaction boundary. The proposed method was successfully applied to the analysis of NANA in human sa-
liva, and the recoveries were in the range of 91.8%－109% with RSDs of 1.8%－3.9%. Due to its simple design
and construction, low cost and portability, the mini-CE-AD device will possess more practicability in more field
work as an alternative to conventional and microchip CE approaches.
Keywords miniaturized capillary electrophoresis with amperometric detection, human saliva, sample stacking,
N-acetylneuraminic acid, 2-thiobarbituric acid derivatization
Introduction
due to the lesser cost involved, the greater reproducibil-
ity and the convenience of use. Then, the resulting mix-
tures were often determined by liquid or gas chroma-
tography and capillary electrophoresis (CE) based on
derivatization with a chromophore or fluorophore for
more sensitivity and selectivity compared with colori-
metric methods. Several reviews have summarized the
characteristics of the relevant methods applied for bio-
logical tissues and fluids.[2,3] And CE, as a promising
alternative to HPLC, has a broad prospect on the sepa-
ration of complex system due to its high efficiency and
speed, and small consumptions of sample and solvent.
CE combined with ultraviolet (UV),[6-8] LIF[9] and
MS[10,11] has been applied to the analyses of SAs in
glycoprotein, sialoglycan and human serum samples.
Saliva is one of accessible, informative and
non-invasive biofluids, and the contents of some species
in saliva could be used as the indicators for the diagno-
ses of physiological and biological conditions,[12] mak-
ing it ideal for the primary disease diagnoses involved
cardiovascular, renal, and autoimmune diseases, partic-
ularly for cancers. For example, Trivedi et al.[13] found
that the salivary levels of free and protein bound SAs
were significantly increased in oral cancer patients as
Sialic acid (SA) is the generic name given to a fami-
ly of the nine-carbon sugar neuraminic acid derivatives,
which generally exist as the components of glycopro-
teins, polysaccharides, and glycolipids in nature, and
very little parts present as free form.[1] Among the over
40 SA derivatives, N-acetylneuraminic acid (NANA)
and its hydroxylated form, glycolylneuraminic acid, are
two major SAs found in nature.[2,3] NANA is the most
widespread form and almost the only one found in hu-
man bodies, while glycolylneuraminic acid is not ex-
pressed in humans because of the evolutionary loss of
the gene encoding CMP-NANA hydroxylase converting
NANA into its hydroxylated form.[4] Some research has
shown that the total amount of SAs in human serum
could be a potential index of clinical diagnosis, espe-
cially as a tumor marker.[5,6] Due to their important bio-
logical role as markers for different pathologies, SA
analyses for clinical research are of high importance.
The detection methods of SAs in biological samples
usually involve two main steps. First of all, releasing the
non-free SAs could be accomplished by enzymatic and
acid hydrolysis, and the latter is the most common way
*
E-mail: qcchu@chem.ecnu.edu.cn; Tel.: 0086-021-54340048; Fax: 0086-021-54340048
Received March 30, 2016; accepted June 8, 2016; published online August 10, 2016.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201600189 or from the author.
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compared with normal healthy volunteers, and that ra-
diotherapy could contribute to significant decrease of
the same parameters as compared to the values observed
before radiotherapy. Furthermore, the salivary SA level
in smokers was obviously higher than that in nonsmok-
ers,[14] and that passive smoking was associated with the
increase in amylase activity and SA level of stimulated
whole saliva in young children.[15] Therefore, the deter-
mination of salivary SAs is of great importance in bio-
chemistry and clinical chemistry. Now, LC-UV,[16]
LC-LIF[17] and MEKC-UV[18] have been reported for
the analyses of salivary SAs. In addition, amperometric
detection (AD) can offer high sensitivity and selectivity
for electrochemical substances,[19-21] good compatibility
with advanced micromachining technologies, and low
cost and power requirements,[22,23] while to our best
knowledge so far CE combined with AD has not been
employed for this analytical purpose.
In this work, a new method has been developed for
the analysis of salivary NANA based on miniaturized
CE-AD coupled with 2-thiobarbituric acid (TBA) deri-
vatization. Due to its good electroactivity, TBA has been
selected as an ideal derivative reagent for electrochemi-
cal detection of low-molecular-weight carbonyl com-
pounds in our previous studies.[24-26] Various parameters
influencing hydrolysis and derivative efficiency of
NANA, as well as electrophoretic separation and am-
perometric detection, were optimized, respectively. And
a stacking technique of moving chemical reaction
boundary was used for further improving the detection
sensitivity. This proposed method has been applied to
the saliva analyses for both nonsmokers and smokers.
prepared as the running buffer. The separation voltage
was 2.8 kV, the inject time was 4 s (at 2.8 kV), and the
potential applied to the carbon disc electrode was set at
＋950 mV (vs. SCE). All electrophoretic experiments
were carried out at room temperature in an air-condi-
tioned room.
Sample preparation
Saliva samples were collected from volunteers in the
age range of 18－25 years in our campus, including 10
female nonsmokers, 10 male nonsmokers and 10 male
smokers. And all saliva samples were collected at 1 h
after eating or drinking. The hydrolysis[2] and derivative
methods[27,28] were further optimized and the detailed
procedures were as follows.
Firstly, 300 μL of fresh saliva sample after double
dilution with 200 mmol•L1 H2SO4 was acid hydrolyzed
in a thermostatic water bath at 90 ℃, and the hydrolysis
time was 45 min with the assistance of magnetic stirring
kept at 800 r/min (the optimization of the hydrolysis
parameters was provided in Figure S1 of Supporting
Information). Secondly, 400 μL of hydrolysate, 260 μL
of HIO4, 30 μL of H2SO4 (1 mol•L1) and 10 μL of de-
ionized water were successively added into a 1.5 mL of
centrifuge tube at 37 ℃ of water bath, and the oxida-
tion reaction was carried out for 15 min. And then, 150
μL of Na2S2O3 used as a reductant for superfluous HIO4
was added and shaken for a moment. Subsequently, 125
μL of TBA and some deionized water were added into a
total volume of 1.2 mL, and then the reaction proceeded
for 30 min in a thermostatic water bath at 90 ℃. Finally,
the reaction product was centrifuged for 5 min at 10000
r/min, 100 μL of the supernatant was diluted five-fold
with 200 mmol•L1 NaOH, and then injected directly to
the mini-CE-AD system for analysis. And the online
enrichment mechanism of NaOH was as described in
our previous work.[25] Before use, all sample solutions
were stored at 4 ℃.
Experimental
Reagents and chemicals
The standard compound of NANA (98%) and other
chemical reagents including periodic acid (HIO4), so-
dium hyposulfite (Na2S2O3) and sodium hydroxide
(NaOH) were purchased from Shanghai Jingchun Sci-
entifical Co., Ltd. (Shanghai, China), TBA was pur-
chased from J&K Scientific Ltd. (Beijing, China), and
sulfuric acid (H2SO4, 98%) was from Shanghai Chemi-
cal Reagent Co. Ltd. (Shanghai, China). All chemicals
were at least of analytical grade.
The stock solutions of NANA (2.0 mg/mL), HIO4
(1.9 mg/mL) and Na2S2O3 (35.0 mg/mL) were prepared
with deionized water, and that of TBA (7.25 mg/mL)
was prepared in ethanol solution (Vethanol∶Vwater＝1∶1).
The standard stock solutions and working solutions
were stored at 4 ℃ before using.
Results and Discussion
The derivative mechanism of NANA is shown in
Figure 1. NANA released by hydrolytic process was
first oxidized by HIO4 in an acidic solution, the excess
of HIO4 was then reduced with Na2S2O3. Subsequently,
the oxidation product β-formyl pyruvic acid with car-
bonyl groups was reacted with electroactive TBA to
form a stable adduct expressed as NANA-TBA, finally
Apparatus and electrophoretic procedure
The principles and details of the high-integrated
mini-CE-AD system were similar to those reported pre-
viously.[21,25,26] The analyses were carried out in a 19.5
cm fused-silica capillary (25 μm id×360 μm od), and
80 mmol•L1 H3BO3-Na2B4O7 buffer (pH＝9.0) was
Figure 1 The derivative mechanism of NANA.
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Electrochemical Determination of Salivary N-Acetylneuraminic Acid
this electroactive adduct could be readily detected by
mini-CE-AD. The parameters affecting the oxidation
and derivative conditions for NANA were investigated,
respectively.
range of 20－60 ℃ (as shown in Figure S2A of Sup-
porting Information). The results showed that the peak
area of NANA-TBA has a slight decrease with the in-
creasing temperature. So, 37 ℃ of a water bath was
selected as the oxidative temperature, which is proximal
to the normal temperature of human body.
Optimization of oxidation conditions
A strongly acidic environment is necessary to obtain
an optimal oxidative efficiency, and a most possible
explanation is that strong acid is required to remove the
acetyl from the amino group of NANA before oxida-
tion.[27] Moreover, subsequent derivatization also needs
to be carried out in a strong acid system.[24-26] Therefore,
100 mmol•L1 H2SO4 was used as the acidic solution for
further investigation, which was consistent with the hy-
drolysis system.
Since arsenite used as a reductant in TBA colorimet-
ric method[27,28] belongs to the carcinogens, a safety
substitute Na2S2O3 was selected as the reductant to re-
duce the excess oxidant (HIO4). In order to obtain the
maximum derivative yield, the mole ratios of the oxi-
dant and reductant versus NANA were studied, respec-
tively. As shown in Figures 2A and 2B, when the mole
ratio of HIO4, Na2S2O3 versus NANA was 2∶25∶1,
the peak area of NANA-TBA could achieve the maxi-
mum value. Thus, this mole ratio n(HIO4)∶n(Na2S2O3)∶
n(NANA)＝2∶25∶1 was selected for the subsequent
experiments.
The oxidative time was also studied in the range of
0－60 min (as shown in Figure S2B of Supporting In-
formation). The results showed that when the reaction
time was shorter than 15 min, the adduct yield increased
fast, while the reaction time exceeded 15 min, the ad-
duct yield levelled off. Therefore, 15 min was chosen as
the oxidative time. And the excess of HIO4 could be
quickly reduced by Na2S2O3 (the effect of reduction
time on the peak area of NANA-TBA adduct was shown
in Figure S2C of Supporting Information). Besides, the
oxidation in the dark had no effect on the final deriva-
tive efficiency.
Optimization of derivative conditions
Temperature is an important parameter affecting the
derivative efficiency. In order to investigate the effect of
temperature on the reaction yield of NANA-TBA, a se-
ries of derivative experiments were conducted at 80, 85,
90, 95 and 100 ℃, respectively (as shown in Figure
S2D of Supporting Information), and 90 ℃ was se-
lected for the further studies considering the stability
and reaction yield of the adduct.
The reaction temperature was also investigated in the
Figure 2 Effects of (A) the oxidation ratio of HIO4 versus NANA, (B) the reduction ratio of Na2S2O3 versus NANA, (C) the derivation
ratio of TBA versus NANA, and (D) derivation time on the peak area of NANA-TBA adduct.
Chin. J. Chem. 2016, 34, 925—930
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The effect of TBA amount on the peak area of
NANA-TBA was studied to obtain optimum derivative
efficiency. Different volume of TBA was added into the
reaction solution keeping the amount of NANA un-
changed. As shown in Figure 2C, when the mole ratio of
TBA versus NANA was 5∶1, the adduct yield could
reach the peak. Therefore, the optimum mole ratio of
TBA versus NANA maintained at 5∶1. Once formed,
the adduct was stable at 4 ℃ in a dark within 12 h.
Reaction time is another important parameter signif-
icantly affecting the adduct yield. The effect of the reac-
tion time was also investigated in the range of 0－60
min in a water bath at 90 ℃ as shown in Figure 2D.
When the reaction time was lower than 30 min, the ad-
duct yield raised quickly with the increasing reaction
time, and then levelled off as the time continued to ex-
tend. Therefore, 30 min was chosen as the optimum
derivation time
The reproducibility of this proposed method was
evaluated by intraday precision (n＝7) and interday
precision (n＝5) at three different concentrations (5.0,
20.0, 100.0 μg/mL), respectively (the assay data were
summarized in Table S1 of Supporting Information).
The results showed that the average RSDs of peak area
were within 2.0% and 3.3% for intraday and interday
precision, respectively; and those of migration time
were within 1.6% and 2.4% for intraday and interday
precision, respectively.
To further evaluate the reliability of the proposed
method, recovery experiments were also performed by
standard addition method at three different concentra-
tion levels (5.0, 20.0, and 100.0 μg/mL) with the real
saliva samples of healthy volunteers and smokers (the
assay data were summarized in Table S2 of Supporting
Information). The recoveries were in the range of
91.8%－109% with corresponding RSDs of 1.8%－
3.9%, which indicated that the mini-CE-AD method
was sufficiently accurate for the determination of
NANA-TBA adduct.
Method validation
Under the optimum conditions, a series of the stand-
ard solutions were tested in the range of 2.0－200.0
μg/mL to determine the linearity of NANA-TBA. The
regression analysis showed that the peak area and con-
centration of the analyte presented a linear response at
two orders of magnitude. The regression equation for
NANA-TBA was y＝0.12＋0.10x with a correlation
coefficient of 0.9990, in which y was the peak area (V•s)
and x was the concentration (μg/mL). Furthermore, the
sensitivity was further improved by a stacking technique
of moving chemical reaction boundary[25] (the effect of
NaOH concentration on the peak area of NANA-TBA
was shown in Figure S3 of Supporting Information),
and the LOD of NANA-TBA could achieve 0.50 μg/mL
(1.6 μmol•L1, S/N＝3). As showed in Table 1, the pro-
posed mini-CE-AD method could provide much lower
or nearly equivalent LOD compared with most reported
electrophoretic methods.[6-8,10,11,18] Although its LOD
was higher than that of several reported methods,[14,16,17]
this proposed method could fully meet the requirements
of real saliva sample analysis with no need of additional
sample purification.
In practice, fucose[11] and 2-deoxy-D-ribose[27] are
two main interferences in spectrophotometry because
their TBA adducts possess similar colors and maximum
absorption wavelength to NANA-TBA. So, the effects
of potential coexisting interferences, including fucose,
2-deoxy-D-ribose, the main aliphatic aldehydes (for-
maldehyde, acetaldehyde, propaldehyde) and glucose
existing in saliva samples, on NANA-TBA analysis
were also investigated in this work (as shown in Figure
S4 of Supporting Information). The assay results
showed that fucose and 2-deoxy-D-ribose could be
identified by standard addition method, while it is diffi-
cult to assign the corresponding peaks for the above
aliphatic aldehydes and glucose, which might be due to
their side reactions in oxidation-reduction procedure
before TBA derivatization, however, no interference
peaks were found in the peak position of NANA. As a
result, NANA-TBA could be well separated from the
TBA adducts of fucose, 2-deoxy-D-ribose and unkown
interferences as well as excess derivative reagent TBA,
although the contents of the interferences were much
higher (above 2－3 orders of magnitude) than those in
actual saliva samples. Therefore, the interference ex-
periments showed that mini-CE-AD method was accu-
rate enough to detect NANA-TBA adduct.
Table 1 Comparison of this method with the reported methods
for the determination of NANA in biological samples
Method
CE-UV
LOD/(μmol•L1) Recovery/% Reference
9.6
2
95.6±8.4
－
[6]
[7]
Sample analyses
CZE-UV
Under the optimum conditions, this proposed meth-
od was applied to determining NANA in saliva samples
collected from nonsmokers and smokers. The typical
sample electrophoretograms were shown in Figures 3A
and 3B. By a standard addition method and comparing
the migration time of target analyte with that of the
mixed standard electropherogram (Figure S4), NANA
could be determined in all tested saliva samples. As seen
from the sample electrophoretograms, NANA-TBA
could be well separated from the co-existing substances
CE-UV
81
－
[8]
CE-MS/HPLC-MS
CZE-MS
1
－
[10]
[11]
[18]
[14]
[16]
[17]
6.5
－
MEKC-UV
SPE/HPLC-FD
HPLC-UV
9.7
100－102
－
0.0086
0.060
0.00032
1.6
90－102
91.20
SPE/μ-LC-LIF
mini-CE-AD
91.8－111 This work
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Electrochemical Determination of Salivary N-Acetylneuraminic Acid
Table 2 Contents of NANA in the tested saliva samples (n＝3)
Content/(μg•mL1) Average/(μg•mL1)
Group
P-value
－
Male nonsmokers (n＝10)
Female nonsmokers (n＝10)
Male smokers (n＝10)
14.2－137.6
33.8－123.4
180.1－388.8
69.9±29.1
88.3±36.3
301.9±77.2
0.226
＜0.001
in the saliva samples under the optimum experimental
conditions.
vide much better or nearly equivalent sensitivity com-
pared with most reported electrophoretic methods, and
relatively good recoveries (91.8%－111%) were ob-
tained for saliva samples. Thirdly, due to its simple de-
sign and construction, low cost, and portability, the
miniaturized device will possess more practicability in
more field work as an alternative to conventional and
microchip CE approaches. Further characterization and
application of this mini-CE-AD system are continuing
in our laboratory.
Acknowledgement
This work was supported by the National Natural
Science Foundation of China (No. 21205042), the Daxia
Foundation of East China Normal University (No.
2015DX-284) and the Students Innovative Experimental
Project of Shanghai Municipality (No. 201610269070).
The authors have declared no conflicts of interest.
Figure 3 Typical sample electrophoretograms of (A) male
nonsmoker No. 4 and (B) male smoker No. 3. a: blank saliva
without TBA derivation, b: blank saliva with TBA derivation, c:
spiked saliva with 5.0 μg/mL NANA, d: spiked saliva with 20.0
μg/mL NANA, e: spiked saliva with 100.0 μg/mL NANA. Peak
identifications: (1) NANA-TBA, and (2) TBA.
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Chin. J. Chem. 2016, 34, 925—930