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Sensitive CE-ECL method with AuNPs-enhanced
signal for the detection of b-blockers and the study
of drug–protein interactions
Hong-Bing Duan,
ab
Jun-Tao Cao,*
ab
Hui Wang
ab
and Yan-Ming Liu*
ab
A sensitive capillary electrophoresis (CE) system coupled with electrochemiluminescence (ECL) of tris(2,20-
bipyridyl) ruthenium(II) is described for the detection of propranolol (Pro) and acebutolol (Ace). AuNPs were
added to the end column luminescent reagents solution to enhance the sensitivity of the CE-ECL system.
Experimental conditions of separation and detection as well as the amount of AuNPs were optimized. Under
the optimum conditions, the linear range of Pro is 0.01 to 100 mmol L
1
with the LOD of 3.6 10
9
mol L
1
(S/N ¼3) and with the LOQ of 1.1 10
7
mol L
1
in human urine samples (S/N ¼10). For Ace, the linear
range is 0.02 to 100 mmol L
1
with the LOD of 5.0 10
9
mol L
1
(S/N ¼3) and with the LOQ of 9.5
10
8
mol L
1
in human urine samples (S/N ¼10). RSDs (n¼3) of the migration time for Pro and Ace are
from 1.9 to 2.4% intraday and from 2.9 to 3.8% interday. RSDs of the peak area for Pro and Ace are from
3.2 to 3.7% intraday and from 4.3 to 4.6% interday, respectively. The developed method was successfully
used to determine two analytes in human urine samples. In addition, the interaction between Pro as
a model analyte and human serum albumin (HSA) was investigated; the number of binding site and the
binding constant of Pro with HSA are 1.0 and 2.3 10
4
L mol
1
, respectively.
Introduction
Propranolol (Pro) and acebutolol (Ace) are nonselective b-
adrenergic blocking drugs (b-blockers) which are commonly
used to treat several diseases, such as cardiacarrhythmia,
hypertension, angina pectoris, sinus tachycardia, anxiety
disorders, thyrotoxicosis, hypertrophic subaortic stenosis,
myocardial infarction, and migraine in the clinic.
1,2
These drugs
are sometimes used with other drugs in poly-pharmacy. They
are also misused in some sports to reduce cardiac frequency,
contraction force and coronary ow.
3
The International Olympic
Committee has identied Pro as a doping substance and
included it in the list of forbidden substances.
4
In addition, an
overdose of b-blockers may lead to bradycardia, hypotension,
aggravation of cardiac failure, bronchospasm, hypoglycemia,
and fatigue.
5
The increasing demand for b-blocker detection
have driven tremendous efforts in the development of sensitive
and selective methods for the determination of b-blockers in
pharmaceutical formulations.
6,7
Therefore, sensitive and accu-
rate analytical methods for the quantication of these
compounds in biological uids are required. Several methods,
such as solid-phase extraction coupled to gas chromatography-
mass spectrometry (SPE-GC-MS),
8
GC-MS,
9
ow injection-
chemiluminescence (FI-CL),
10
high performance liquid
chromatography-photodiode-array UV (HPLC-UV)
11
and solid-
phase extraction-differential pulse voltammetry (SPE-DPV)
12
have been reported for the determination of b-blockers.
However, these methods require the use of expensive instru-
mentation
9
and lack sufficient sensitivity;
8,10–12
also, some of
them involve long analysis times
8,9,11
and tedious preliminary
procedures such as pre-concentration in an organic solvent,
ltration, extraction and derivatization.
12
Therefore, the devel-
opment of sensitive and selective analytical methods to detect b-
blockers in biological uids is necessary.
Human serum albumin (HSA), the most abundant protein in
blood plasma, is a main carrier protein with high affinity to
bind a wide range of endogenous and exogenous compounds,
such as fatty acids, metals, toxic metabolites, hormones, amino
acids, bile acids, and drugs.
13,14
HSA has long been a focus of the
pharmaceutical industry due to its ability to bind various drug
molecules. The interaction of HSA with drugs plays an essential
role in biological activity in the human body. Among its phar-
macokinetic functions, drug distribution is controlled by HSA
because most drugs transported in blood bind to serum
albumin and thereby reach the target tissues.
15
Studying the
interaction of drugs with HSA is of considerable importance to
understand the processes of drug transportation and predict
the concentration of free drug.
Capillary electrophoresis (CE) has attracted many
researchers' attention because of its advantageous properties of
a
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang
464000, China. E-mail: liuym9518@sina.com; jtcao11@163.com; Fax: +86-376-
6392889; Tel: +86-376-6392889
b
Key Laboratory of Simulation and Control for Dabie Mountains Population Ecology,
Xinyang Normal University, Xinyang 464000, China
Cite this: RSC Adv.,2016,6, 45533
Received 16th March 2016
Accepted 25th April 2016
DOI: 10.1039/c6ra07003e
www.rsc.org/advances
This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6, 45533–45539 | 45533
RSC Advances
PAPER
high separation efficiency, short analysis time, low reagent
consumption, and ease of installation, operation and mainte-
nance.
16–18
Electrochemiluminescence (ECL) occurs when elec-
trochemically generated reactants undergo a high-energy
electron transfer reaction to generate an excited state. ECL is
well known as a powerful analytical technique with the advan-
tages of high sensitivity, wide linear range, and simple instru-
mentation. ECL has been widely used in the areas of
immunoassay, food and water testing and biowarfare agent
detection.
19
Ru(bpy)
32+
-based ECL has received considerable
attention among a number of ECL systems, owing to its low
background signal, excellent chemical stability and high ECL
efficiency in aqueous and non-aqueous solvents.
20
Therefore,
CE coupled with Ru(bpy)
32+
-ECL detection has been applied to
separate and detect compounds such as alkaloids,
21
hormones,
22
biogenic amines,
23
protein,
24
drugs
25
and small
organic molecules.
26
Over the past decade, gold nanoparticles (AuNPs) have
attracted much attention in biomolecular detection and clinical
diagnostic applications because of their facile synthesis, good
biocompatibility, relatively large surface and strong catalytic
properties. It has been found that the larger surface area of
AuNPs and the catalytic ability of AuNPs will improve the
number of transmission states, which is an important param-
eter for the intensity of ECL emission.
27,28
Inspired by the useful applications of AuNPs, a sensitive CE-
Ru(bpy)
32+
-ECL method based on the enhancement of AuNPs
was developed for the separation and detection of Pro and Ace.
The factors inuencing CE and ECL, including the pH and
concentration of the running buffer, separation voltage and
detection potential as well as the amount of AuNPs were
investigated systematically. The proposed method was
successfully applied to the analysis of two analytes in human
urine samples and the study of the interaction between the
drugs and the protein. The number of binding site and the
binding constant of the drug and protein were obtained.
Experimental
Apparatus
The MPI-A CE-ECL system was produced by Remax Electronic
Science-Tech Co. Ltd. (Xi'an, China). The detection system
consists of four main parts: a high-voltage power, a potentiostat,
a multifunction chemiluminescence detector and a data
collection analyzer. All data collection was performed with MPI-
A analysis soware and recorded on a computer. CE separation
was performed in a 55 cm length 50 mm i.d. and 375 mm o.d.
uncoated fused-silica capillary obtained from Yongnian Optical
Conductive Fiber Plant (Hebei, China). ECL detection was
employed using a three-electrode system: a 500 mm diameter
platinum disk as the working electrode, a Pt wire as the auxiliary
electrode, and a Ag/AgCl electrode (in saturated KCl solution) as
the reference electrode. The transmission electron microscopy
(TEM) images were recorded using a Tecnai G
2
F20 TEM system
(FEI Co., America). UV-visible detection was carried out using an
UVmini-1240 UV-vis spectrophotometer (Shimadzu Corp.,
Kyoto, Japan).
Chemicals
All the reagents used were commercially available and of
analytical grade. Standard Ace was achieved from J&K Chemical
Ltd. (Shanghai, China). Standard Pro was purchased from the
National Institute for the Control of Pharmaceutical and Bio-
logical Products (Beijing, China). Hydrogen tetra-
chloroaurate(III) trihydrate (AuCl
3
$HCl$4H
2
O, Au > 47.8%) was
obtained from Alfa Aesar (A Johnson Matthey Company, Ward
Hill, MA). Tris(2,20-bipyridyl)-ruthenium(II) chloride hexahy-
drate (Ru(bpy)
3
Cl
2
) and HSA (>96%, M
w
¼66 kDa) were
purchased from Sigma-Aldrich (St Louis, MO, USA). Tween 20
was obtained from Seebio Biotech (Shanghai, China). Ultrapure
fresh water (18.2 MUcm) used was processed with an ultrapure
water system (Kangning Water Treatment Solution Provider,
China). All solutions were ltered through 0.22 mm cellulose
acetate membrane lters (Shanghai Xingya Purication Mate-
rial Factory, China) and stored under refrigeration at 4 C before
use.
Procedure
Before rst use, the new capillary was lled with 2.0 mol L
1
CH
3
OH–NaOH, 1.0 mol L
1
NaOH, 1.0 mol L
1
HCl, H
2
O, and
nally with phosphate buffer solution (PBS) for 30 min,
respectively. At the beginning use of each day, the capillary was
rinsed with 0.1 mol L
1
NaOH, water, and the corresponding
separation buffer for 10 min successively until the baseline of
chemiluminescence was at to maintain an active and repro-
ducible capillary inner surface prior to a series of analyses. The
photomultiplier tube (PMT CR105, Beijing Binsong Photonics,
China) positioned under the detection cell for collecting the
ECL signal was set at 850 V in the process of detection. The
detection potential applied to the working electrode was xed at
1.25 V. Electrokinetic injections were performed at 10 kV for 10
s. The inlet end of the capillary was held at a positive potential
and the outlet end was maintained at ground. 5 mmol L
1
Ru(bpy)
32+
with 50 mmol L
1
PBS was added into the detection
cell as the ECL reagent. Prior to the experiments, the Pt disc
working electrode was polished with 0.3 and 0.05 mmAl
2
O
3
in
sequence, then cleaned with ultrapure water in an ultrasonic
cleaner. Elimination of the oxide layer from the Pt electrode was
performed in 50 mmol L
1
pH 7.0 PBS by cycling the potentials
of the working electrode from 0.5 to 0.0 V. At a scan rate of 100
mV s
1
, more than 10 cycles were required.
Preparation of AuNPs
All the glassware in the following experiments were cleaned in
freshly prepared HNO
3
: HCl (1 : 3) solution, rinsed with water
and dried in air. AuNPs were prepared according to a literature
report.
29
Briey, 5 mL of 40 mmol L
1
sodium citrate was rapidly
added to 50 mL of boiling 0.05% AuCl
3
$HCl$4H
2
O solution
under vigorous stirring; the colour of the solution changed from
pale-yellow to wine-red. Boiling was continued for an additional
15 min, and then the heating was stopped. The solution was
stirred for another 15 min until it cooled to room temperature.
Aer cooling, the synthesized AuNPs were ltered through
45534 |RSC Adv.,2016,6, 45533–45539 This journal is © The Royal Society of Chemistry 2016
RSC Advances Paper
a 0.22 mm cellulose membrane and stored in a dark glass bottle
at 4 C before use.
Preparation of human urine sample
The urine samples were provided by a healthy male volunteer in
Xinyang Normal University. The male volunteer gave written
informed consent, and complete ethical approval was obtained.
The study was performed in compliance with the relevant laws
and institutional guidelines and was also approved by the
Institutional Review Board of Xinyang Normal University. Prior
to analysis, the fresh urine samples were immediately centri-
fuged at 2000 rpm for 15 min to remove as much precipitation
as possible. Then, the urine sample was diluted 100-fold with
water and then ltered through 0.22 mm cellulose acetate lters.
Results and discussion
AuNPs enhanced ECL of Ru(bpy)
32+
in the presence of Pro and
Ace
To investigate the effect of AuNPs (Fig. 1) on the ECL intensity in
the detection of Pro and Ace, cyclic voltammetry (CV) and ECL
measurements were conducted. Fig. 2 shows the CV and cor-
responding ECL curves before and aer adding analytes and
analytes with AuNPs, respectively. As is clearly shown in Fig. 2,
the ECL intensities of the two analytes increased markedly in
the presence of AuNPs. We can conclude that Pro and Ace can
react with the ruthenium species in the ECL process, and the
AuNPs can enhance the emitted light intensity. The enhance-
ment of the ECL emission in Ru(bpy)
32+
system can be inter-
preted as follows. In the ECL detection, the interaction of
negatively charged AuNPs with the positively charged
Ru(bpy)
32+
may decrease the ligand p–p*energy gap, which
determines the rate and efficiency of the charge transfer tran-
sitions between a d-orbital on the ruthenium and a p*anti-
bonding orbital on the ligand.
30
This interaction makes the
transition of the excited species Ru(bpy)
32+
*into the ground
state easier and more efficient, resulting in an enhancement of
the ECL emission. In addition, the large surface area and the
good catalytic ability of the AuNPs will also improve the number
of transmission states of Ru(bpy)
32+
, which is another impor-
tant parameter affecting the ECL intensity.
To achieve highly sensitive detection of Pro and Ace, the
amount of AuNPs in the detection cell was optimized. First, 300
mL of 5 mmol L
1
Ru(bpy)
32+
with 50 mmol L
1
PBS was injected
into the detection cell, and the ECL curve was recorded. Then,
30 mL solutions were extracted from the detection cell, followed
by the addition of 30 mL of AuNPs solution. The solutions were
immediately mixed with an injector and the corresponding ECL
spectrum was recorded. These actions were repeated eight
times. Fig. 3 shows the relationship of ECL intensity with the
volume ratio of AuNPs and Ru(bpy)
32+
solution for (a) Pro and
(b) Ace. As can be seen, with increasing ratio, the ECL intensity
for those two analytes exhibits approximately the same
tendency, an initial increase followed by a decrease, and the
maximum ECL intensity was obtained at a volume ratio of 0.7.
This can be explained as follows: at the rst stage, an increase in
AuNPs can greatly enhance the quantity of transmission states
of Ru(bpy)
32+
*; thus, the ECL intensity increases. However,
Fig. 1 TEM image of the as-prepared citric-acid stabilized AuNPs.
Fig. 2 Cyclic voltammograms (inset figure) and their corresponding
ECL curves. (a) 5 mmol L
1
Ru(bpy)
32+
+ 50 mmol L
1
PBS (pH 8.0); (b)
a + 0.1 mmol L
1
Pro; (c) a + 0.1 mmol L
1
Ace; (d) b + AuNPs; (e) c +
AuNPs; scan rate, 100 mV s
1
.
Fig. 3 Variation of the intensity of 5 mmol L
1
Ru(bpy)
32+
based ECL
emission in the presence of 0.1 mmol L
1
Pro (a) and Ace (b) with the
volume ratio of the suspension of AuNPs to Ru(bpy)
32+
solution.
This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6, 45533–45539 | 45535
Paper RSC Advances
continuously increasing the amounts of AuNPs dilutes the
concentration of Ru(bpy)
32+
, causing the ECL intensity to
decrease; thus, a volume ratio of 0.7 was selected.
Effects of CE separation conditions
The concentration and pH of PBS play important roles in CE
separation. In order to acquire good CE separation of the two
analytes, the effects of the CE running buffer on separation were
investigated. In this work, PBS was chosen as the running
buffer. The buffer concentration was tested from 10 to 30 mmol
L
1
, and 20 mmol L
1
was selected. It was found that baseline
separation results were not obtained when PBS alone was used
as the running buffer. In order to further improve the separa-
tion and detection sensitivity, we choose Tween 20, Tween 80,
CTAB, PEG-20000, Brij-35 and PVP-K30 as additives respective
to the running buffer. It was found that there were no obvious
improvements in resolution for any of these additives except for
Tween 20. Tween 20 concentrations ranging from 0 to 0.07% (v/
v) were tested. The results indicated that 0.03% Tween 20 in the
buffer was good to increase the resolution of the two analytes.
The effect of the pH value of the running buffer was also
investigated. The results showed that the ECL signal increased
with the running buffer pH up to 8.0 and then decreased when
the pH was further increased from 8.0 to 8.5. Therefore, 8.0 was
chosen as the pH of the running buffer for further experiments.
The separation voltage has a great effect on the efficiency of
the separation and the migration time of analytes. The voltage
over the range of 10 to 16 kV was investigated. For two analytes,
the ECL intensity and the resolution reached a maximum value
up to a separation voltage at 13 kV. However, when the sepa-
ration voltage was greater than 13 kV, the baseline noise
increased. Furthermore, the increasing joule heat in the capil-
lary caused peak broadening and adversely affected the sepa-
ration effectiveness. Therefore, 13 kV was chosen as the
separation voltage.
Effect of detection potential
In ECL, the intensity depends on the oxidation rate of
Ru(bpy)
32+
and the potential applied to the working electrode.
Therefore, the relationship of ECL intensity with the applied
potential over the range of 1.10 to 1.35 V was studied. The
results indicated that when the applied potential increased
from 1.0 to 1.25 V, the ECL intensity increased; it reached
a maximum value at 1.25 V and then decreased slightly.
Therefore, the applied potential was set at 1.25 V.
Performance characteristics of the method
The optimal experimental conditions were used for the detec-
tion of Pro and Ace. The parameters were conrmed as follows:
20 mmol L
1
PBS containing 0.03% Tween 20 at pH 8.0 as
running buffer; a volume ratio of AuNPs to 5 mmol L
1
Ru(bpy)
32+
with 50 mmol L
1
PBS of 0.7 at pH 8.0 as ECL reagent
solution in the detection cell; electrokinetic injection for 10 s at
10 kV; separation voltage at 13 kV; and detection potential at
1.25 V. A typical electropherogram of Pro and Ace is shown in
Fig. 4b; baseline separation of the two analytes could be ach-
ieved in 7 min.
The separation efficiencies of the two analytes were evalu-
ated by the theoretical plate numbers, which were calculated
according to the following equation: N¼5.54 (t
R
/W
1/2
)
2
,
where Nis the theoretical plate number, t
R
is the migration time
and W
1/2
is the width at half the maximum peak height of the
analyte. The numbers of theoretical plates obtained for Pro and
Ace are 7.2 10
4
and 4.6 10
4
plates per m. The linear cali-
bration ranges, regression equations, limits of detection (LODs)
and limits of quantitation (LOQs) of the two analytes were also
examined, and the results are listed in Table 1. As can be seen
from Table 1, the linear range is 0.01 to 100 mmol L
1
for Pro
with the LOD of 3.6 10
9
mol L
1
(S/N ¼3) and with the LOQ
of 1.1 10
7
mol L
1
in urine samples (S/N ¼10). For Ace, the
linear range is 0.02 to 100 mmol L
1
with the LOD of 5.0 10
9
mol L
1
(S/N ¼3) and with the LOQ of 9.5 10
8
mol L
1
in
Fig. 4 Electropherograms of 1 10
7
mol L
1
propranolol (peak 1)
and 1 10
7
mol L
1
acebutolol (peak 2) without (a) and with (b)
AuNPs in the detection cell. Conditions: running buffer, 20 mmol L
1
PBS containing 0.03% Tween 20 at pH 8.0; injection, 10 kV 10 s;
separation voltage, 13 kV; detection potential, 1.25 V. In the ECL
detection solution, the volume ratio of AuNPs to 5 mmol L
1
Ru(bpy)
32+
with 50 mmol L
1
PBS is 0.7 at pH 8.0.
Table 1 The performance characteristics of the proposed method
Analytes
Linear range
(mmol L
1
)
Calibration curves
LOD
(mol L
1
)
LOQ in urine
(mol L
1
)Slope Intercept R
Pro 0.01–100 2958 16 714 0.998 3.6 10
9
1.1 10
7
Ace 0.02–100 2607 32 363 0.995 5.0 10
9
9.5 10
8
45536 |RSC Adv.,2016,6, 45533–45539 This journal is © The Royal Society of Chemistry 2016
RSC Advances Paper
urine samples (S/N ¼10). The relative standard derivations
(RSDs, n¼3) of the migration time for Pro and Ace (concen-
tration: 1 10
7
mol L
1
) are from 1.9 to 2.4% intraday and
from 2.9 to 3.8% interday. The RSDs of the peak area for Pro and
Ace are from 3.2 to 3.7% intraday and from 4.3 to 4.6% interday,
respectively.
Analytical applications
The proposed CE-ECL method was applied to the determination
of Pro and Ace in human urine samples. The analytes are not
found in urine samples of healthy person because the analytes
are exogenous substances. To evaluate the precision of the
proposed method, spiked urine samples were investigated. All
of the urine samples were spiked at three concentration levels to
determine the recoveries of the targeted analytes. For each
concentration level, ve replicates were conducted. The elec-
tropherograms of the blank urine sample from a healthy person
and a urine sample spiked with 5 10
7
mol L
1
Pro and Ace
are illustrated in Fig. 5a and b; no interferences from the
sample matrix overlapped with the two target analytes. The
recovery results are listed in Table 2. It can be seen that the
recoveries of Pro and Ace in urine samples are in the range of
98.0 to 106.7% and the RSD of the corresponding ECL peak
areas were less than 4.2%.
Comparison with other methods
A comparison of the proposed CE-ECL method with previous
reports
8–12
is listed in Table 3. Compared with other methods,
the proposed CE-ECL method exhibits high sensitivity and
a wide linear range, although the LOQ is lower in ref. 9. The
results demonstrated that the proposed CE-ECL method has
excellent application prospects in the eld of drug analysis.
Study of the binding of HSA with Pro
It is oen of great interest to investigate the interaction of HSA
with drugs. HSA-drug binding has become an important
research eld in chemistry, the life sciences and clinical medi-
cine; it is of great interest to evaluate this binding during the
drug development process. Therefore, the binding behaviour
between HSA and Pro as a model analyte was researched in this
work.
In order to obtain the equilibrium time, a mixture solution of
100 mL20mmol L
1
Pro and 100 mL 200 mmol L
1
HSA in
a dialysis bag was incubated at 37 C. The solution outside of
the dialysis bag was 4 mL PBS and the ECL intensity of Pro
outside of the bag was detected every half an hour until no
remarkable change in the ECL intensity was observed (a total of
about 8 h), which indicates that the binding has already reached
its equilibrium. The results show that the equilibrium time is
about 5 h. In order to explore whether Pro can react with HSA,
100 mL 20 mmol L
1
Pro, 100 mL 200 mmol L
1
HSA, and
a mixture of 100 mL 20 mmol L
1
Pro with 100 mL 200 mmol L
1
HSA balanced from the start to the end solution were subse-
quently examined by UV-vis spectrophotometry for comparison,
respectively. As shown in Fig. 6, there was a marked difference
in absorbance between the balanced solution (curve d) and the
starting mixture of HSA and Pro (curve c). Both facts conrmed
the binding of HSA with Pro under the experimental conditions.
Fig. 5 Electropherograms of a blank urine sample (a) and a urine
sample spiked with Pro and Ace (b). Peak 1: Pro, peak 2: Ace, peak X:
unknown compounds. Other conditions are the same, as shown in
Fig. 4.
Table 2 Recoveries in human urine at different spiked levels
Analytes
Added
(mmol L
1
)
Found
(mmol L
1
)
Recovery
(%)
RSD (%)
(n¼5)
Pro 0.5 0.51 102.0 4.2
25 24.8 99.2 2.8
75 75.8 106.7 3.3
Ace 0.5 0.49 98.0 1.9
25 25.6 102.4 3.6
75 74.3 99.1 2.4
Table 3 Comparison of other methods for Pro and Ace determination with the proposed CE-ECL method
Methods
Linear range (mmol L
1
) LOD (mol L
1
) LOQ (mol L
1
)
Ref.Pro Ace Pro Ace Pro Ace
SPE-GC-MS 20–3400 20–3400 6.8 10
3
6.8 10
3
2.0 10
2
2.0 10
2
8
GC-MS 0.068–17 —2.1 10
8
—6.3 10
8
—9
FI-CL 0–6.8 10
4
—1.3 10
7
———10
HPLC-UV 85–3400 85–3400 2.0 10
5
2.0 10
5
8.5 10
5
8.5 10
5
11
SPE-DPV 5–100 20–150 5.0 10
6
2.1 10
5
1.2 10
5
4.2 10
5
12
CE-ECL 0.01–100 0.02–100 3.6 10
9
5.0 10
9
1.1 10
7
9.5 10
8
This work
This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6, 45533–45539 | 45537
Paper RSC Advances
Pro and HSA bind to form a complex structure; an improved
equation
31
was used to calculate the number of binding sites (n)
and the binding constant (K).
r¼½Cbound
½Ptotal¼X
m
i¼1
ni
KiCfree
1þKiCfree(1)
where [C
bound
], [P
total
], and [C
free
] represent the concentrations
of bound drug, total HSA and free drug, respectively; ris the
fraction of bound drug molecules per protein molecule, n
i
represents the number of sites of class iand K
i
is the binding
constant.
32
Drug protein data analysis oen assumes one type of
binding site on the protein; thus, eqn (1) can be simplied
to (2):
r¼n
KCfree
1þKCfree(2)
A series of different volumes (ranging from 40 to 160 mL, with
increments of 20 mL) of 20 mmol L
1
Pro was mixed with 100 mL
200 mmol L
1
HSA in the dialysis bag and incubated in 4 mL PBS
at 37 C. Aer equilibrium was reached, the ECL intensity
outside the dialysis bag was measured and the concentration of
the free Pro could be estimated. According to eqn (2), the
number of binding sites and binding constant can be obtained;
the binding curves of Pro with HSA were established, as shown
in Fig. 7. According to the calculated results, the number of
binding site and the binding constant of Pro with HSA are 1.0
and 2.3 10
4
L mol
1
, respectively.
Conclusions
In this study, a new and sensitive CE-ECL method for the
detection of Pro and Ace was described. The experimental
conditions were discussed in detail. The introduction of AuNPs
enhanced the detection sensitivity. Compared with other
methods, the proposed CE-ECL method has high sensitivity and
a wide linear range for Pro and Ace analysis. The applicability of
the proposed method was demonstrated in the determination
of Pro and Ace in human urine samples and investigation of the
interaction between drug and protein. The proposed method is
promising for biochemical analysis.
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (Grant 21375114, 21405129) and Nan Hu
Young Scholar Supporting Program of XYNU.
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