2-(Dibutylamino)ethyl acrylate as a highly efficient co-reactant of Ru(bpy)3 2+ electrochemiluminescence for selective detection of cysteine

Article abstract of DOI:10.1016/j.electacta.2019.135117

A highly efficient co-reactant of Ru(bpy)₃ ²⁺ electrochemiluminescence (ECL), 2-(dibutylamino)ethyl acrylate(DBAEA), was successfully synthesized by a straightforward one-step reaction between dibutylaminoethanol and acrylyl chloride. On account of the interaction between the olefin group of DBAEA and Pt electrode, the ECL intensity utilizing DBAEA as the co-reactant was increased dramatically, nearly as 10 times and 17 times as conventional co-reactants dibutyaminoethanol and tripropylamine. Since cysteine can remove the acrylate group of DBAEA specifically through conjugated addition/cyclization reaction, the ECL intensity of Ru(bpy)₃ ²⁺/DBAEA was declined selectively. Therefore, cysteine was detected through this novel ECL system with high selectivity towards other amino acids. The linear range was between 10 μM and 200 μM with a limit of detection of 1.15 μM. The cysteine in Dulbecco's Modified Eagle Medium (DMEM) was detected and satisfying recovery was obtained.

Full text of DOI:10.1016/j.electacta.2019.135117

Journal Pre-proof
2+
2-(Dibutylamino)ethyl acrylate as a highly efficient co-reactant of Ru(bpy)
3
electrochemiluminescence/.;'e for selective detection of cysteine
Fan Yuan, Kai Hao, Shu Sheng, Tadesse Haile Fereja, Xiangui Ma, Feng Liu,
Muhammad Nadeem Zafar, Baohua Lou, Huayu Tian, Guobao Xu
PII:
S0013-4686(19)31988-7
DOI:
https://doi.org/10.1016/j.electacta.2019.135117
EA 135117
Reference:
To appear in: Electrochimica Acta
Received Date: 19 September 2019
Revised Date: 18 October 2019
Accepted Date: 19 October 2019
Please cite this article as: F. Yuan, K. Hao, S. Sheng, T.H. Fereja, X. Ma, F. Liu, M.N. Zafar, B.
2+
Lou, H. Tian, G. Xu, 2-(Dibutylamino)ethyl acrylate as a highly efficient co-reactant of Ru(bpy)
3
electrochemiluminescence/.;'e for selective detection of cysteine, Electrochimica Acta (2019), doi:
https://doi.org/10.1016/j.electacta.2019.135117.
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2019 Published by Elsevier Ltd.

2
-(Dibutylamino)ethyl acrylate as a highly efficient co-reactant of
2
+
Ru(bpy)3
cysteine
electrochemiluminescence for selective detection of
a,c
b,c
b,d
a,d
Fan Yuan, Kai Hao, Shu Sheng, Tadesse Haile Fereja, Xiangui
a,c
b,d
a,e
*a,c
Ma, Feng Liu, Muhammad Nadeem Zafar, Baohua Lou, Huayu
*
b,c
*a,c
Tian, Guobao Xu
a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR
China
b
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences, Changchun, Jilin 130022, PR China
c
University of Science and Technology of China, Hefei, Anhui 230026, PR China
d
University of the Chinese Academy of Sciences, Chinese Academy of Sciences,
Beijing 100049, PR China
e
Department of Chemistry, University of Gujrat, Gujrat, 50700 Pakistan
E-mail: guobaoxu@ciac.ac.cn (GB Xu); thy@ciac.ac.cn (HY Tian); loubh@ciac.ac.cn
BH Lou)
Phone (+86) 431-85262747; Fax: (+86) 431-85262747
(
*
Abstract
2
+
A highly efficient co-reactant of Ru(bpy)3 electrochemiluminescence (ECL),
-(dibutylamino)ethyl acrylate(DBAEA), was successfully synthesized by a
2
straightforward one-step reaction between dibutylaminoethanol and acrylyl chloride.
On account of the interaction between the olefin group of DBAEA and Pt electrode,

the ECL intensity utilizing DBAEA as the co-reactant was increased dramatically,
nearly as 10 times and 17 times as conventional co-reactants dibutyaminoethanol and
tripropylamine. Since cysteine can remove the acrylate group of DBAEA specifically
through the conjugated addition/cyclization reaction, the ECL intensity of
2
+
Ru(bpy) /DBAEA was declined selectively. Therefore, cysteine was detected
3
through this novel ECL system with high selectivity towards other amino acids. The
linear range was between 10 ꢀM to 200 ꢀM with a limit of detection of 1.15 ꢀM. The
cysteine in Dulbecco's Modified Eagle Medium (DMEM) was detected and satisfying
recovery was obtained.
Key words: Electrochemiluminescence; 2-(Dibutylamino)ethyl acrylate; Cysteine;
Co-reactant; Olefin group
1
. Introduction
Electrochemiluminescence (ECL) is an efficient analytical method with the
advantages of low background, high sensitivity, simplicity, rapidity, easy
2
+
miniaturization and integration [1]. Ru(bpy)3 has good stability, solubility and ECL
2
+
efficiency in aqueous solutions. In addition, Ru(bpy)3 ECL allows the detection of
various co-reactants such as proteins, amino acids, DNA and pharmaceuticals, making
it one of the mostly used luminophores [2-5].
Cysteine (Cys) plays an important role in metabolism, detoxification and protein
synthesis [6-8]. The amount of Cys in the body is related to various diseases, such as
cardiovascular disease, Alzheimer’s disease and liver damage [9, 10]. Hence, it is
necessary to develop efficient methods to detect Cys quantitatively. Currently, several
ECL-based methods for the detection of Cys have been reported [11-13]. However,

those methods depend on the interactions, such as hydrogen bond, Van der Waal’s
force and electrostatic attraction between Cys and luminophores or co-reactants,
which limits the selectivity.
Herein, a new ECL co-reactant, 2-(dibutylamino)ethyl acrylate(DBAEA), was
synthesized through the reaction between dibutylaminoethanol (DBAE) and acrylyl
2
+
chloride. DBAEA can dramatically increase the ECL intensity of Ru(bpy)3 on Pt
electrode compared with tripropylamine (TPA) and DBAE. This increase was
attributed to the strong interaction of olefinic group with Pt electrode, which made the
electro-oxidation of DBAEA easier. Cys can remove the acrylate group of DBAEA
through a Michael addition/cyclization reaction between thiol and acrylate group
[
14-16], leading to the decrease of ECL intensity. As a consequence, Cys can be
quantitatively detected by the decrease of ECL intensity (Scheme 1). It is worth
mentioning that this simple detection system exhibited outstanding selectivity towards
amino acids and possessed high sensitivity.
2
. Experimental
2
.1 Chemicals and Apparatus
Both dibutylaminoethanol and tris(2,2'-bipyridyl) ruthenium(II) chloride were
purchased from Sigma-Aldrich. Tripropylamine and acryloyl chloride were obtained
from Aladdin (Shanghai, China). Cysteine was provided by Shanghai Yuanju
biological company (China). Other amino acids, such as alanine, glutamic, aspartic,
were bought from Beijing Dingsheng biological company (China). Na HPO ꢀ12H O
2
4
2
and NaH PO were obtained from Beijing Chemical Reagent company(China).
2
4
Dichloromethane and dimethyl sulfoxide (DMSO) were provided by Tianjin Fuyu
Fine Chemical Company (China). Dulbecco's Modified Eagle Medium (DMEM) was

obtained from Beijing Wobisen Technology Company. All the reagents and chemicals
were of analytical grade without any further purification. The deionized water and 0.1
M phosphate buffer solution (PBS) were utilized throughout the whole experiment.
The structure of DBAEA was confirmed by high-resolution mass spectrometry (MS)
1
1
and proton nuclear magnetic resonance ( HNMR). The HNMR spectra were recorded
on a Bruker Avance 400 MHz NMR spectrometer. High-resolution mass spectrum
was determined on an Bruker Autoflex ꢁ MALDI-TOF mass spectrometer.
2
.2 Synthesis of DBAEA
DBAEA was synthesized according to the reported method (Scheme 2) [17]. Briefly,
0
1
.1 mol DBAE, 0.1 mol triethylamine and 0.001 mol hydroquinone were dissolved in
00 mL tetrahydrofunan (THF) into a dry flask. Then 0.1 mol acryloyl chloride was
added dropwise under room temperature. After the reflux for 2 hours, triethylamine
salt was removed by filtration and the solvent was evaporated. The obtained reaction
product was distilled in vacuum between 83 ꢀ to 87 ꢀ. The final product DBAEA
was presented as yellow liquid and dissolved in DMSO for further adoption.
2
.3 Procedure of ECL detection
2
+
In a typical ECL experiment, 50 ꢀL Ru(bpy) , 50 ꢀL DBAEA of desired
3
concentration and 0.1 M PBS were pipetted into the ECL cell to make the final
solution 500 ꢀL. The cyclic voltammetry (CV) was performed on a CHI 800B
potentiostat with the potential ranging from 0 V to 1.5 V and a scan rate of 100 mV/s
unless special illustration. The ECL intensity was detected on an ultra-weak
luminescence analyzer. The PMT was biased at 1000 V.

2
.4 The detection of Cys
In each experiment of Cys detection, 50 ꢀL DBAEA (50 mM) and 50 ꢀL Cys were
mixed with 100 ꢀL 0.1 M pH 7.0 PBS and the mixture was kept for a period to react
2
+
completely. Subsequently, the solution was mixed with 50 ꢀL Ru(bpy)3 (10 ꢀM) and
2
50 ꢀL 0.1 M pH 7.0 PBS into the ECL cell.
3
. Results and discussion
DBAEA was prepared by Michael addition reaction between DBAE and acrylyl
1
chloride (Scheme 2). The structure of DBAEA was confirmed by H NMR (Figure S1)
and MS (Figure S2).
3
.1 The ECL and CV performances of TPA, DBAE and DBAEA as co-reactants on
different electrodes
2
+
The ECL intensities of Ru(bpy)3 with different co-reactants were compared on Pt
electrode, glassy carbon electrode (GCE) and Au electrode (Figure 1). The
2
+
2+
Ru(bpy) /DBAEA ECL intensity is nearly 17 times and 10 times of Ru(bpy) /TPA
3
3
2
+
and Ru(bpy) /DBAE on Pt electrode and 7 times and 4 times on Au electrode.
3
However, the difference between co-reactants is not obvious on GCE. The CV curves
of three ECL systems on different electrodes were also studied (Figure 2). The
oxidation current intensity of DBAEA is the highest, and next come DBAE and TPA
separately on three electrodes. Olefin is commonly used to modify metal electrodes
especially Pt electrode through chemical adsorption [18-20]. The interaction between
the olefinic group of DBAEA and metal electrodes may affect the amount of electric

charge passed through the solution. Moreover, the olefinic group of DBAEA may
2
+
enable more efficient generation of the excited Ru(bpy) * during electron transfer
3
3
+
•
annihilation between electrogenerated Ru(bpy)3 and DBAEA produced by proton
•
+
abstraction from DBAEA (Scheme 3). Therefore, DBAEA shows much stronger
ECL than DBAE and TPA at Pt and Au electrodes.
2
+
3
.2 The ECL intensity of Ru(bpy)3 with different concentrations of TPA, DBAE
and DBAEA as co-reactants
2
+
The Ru(bpy)3 ECL intensities with TPA, DBAE and DBAEA as co-reactants of
different concentrations on Pt electrode were compared in Figure 3. TPA has the
weakest increasing effect and the slowest increasing speed. The ECL intensity of
2
+
Ru(bpy)3 increases with DBAE concentrations and becomes the strongest when the
concentrations exceed 20 mM, which is consistent with the previous work of our
2
+
group [21]. However, the ECL intensity of Ru(bpy) /DBAEA is much more higher
3
2
+
2+
and reaches 17 times and 10 times as that of Ru(bpy) /TPA and Ru(bpy) /DBAE at
3
3
a co-reactant concentration of 5 mM. The slight decrease of ECL intensity of
2
+
Ru(bpy) /DBAEA at DBAEA concentrations higher than 5 mM may be ascribed to
3
side reactions, such as radical annihilation by olefinic group of DBAEA. Therefore, 5
mM of DBAEA was adopted as optimized concentration in the following
experiments.
2
+
3
.3 The ECL mechanism of Ru(bpy) /DBAEA
3
2
+
The ECL mechanism of Ru(bpy) /DBAEA was inferred as demonstrated in
3
Scheme 3 according to some common tertiaryamine co-reactants [22]. Firstly,

2
+
3+
•
Ru(bpy)3 and DBAEA are electro-oxidized as Ru(bpy)3 and DBAEA , respectively.
•
3+
2+*
Then DBAEA reacts with Ru(bpy) , generating the excited state of Ru(bpy)
.
3
3
2
+*
Finally Ru(bpy)3 turns back to the ground state accompanied by light emitting. It
has been proved that the direct oxidation of co-reactants is dominant at low
2
+
2+
concentration of Ru(bpy)3 and the electro-oxidation of co-reactant by Ru(bpy)3 is
2
+
dominant at high concentration of Ru(bpy)3 [23-26]. Since the concentration of
2
+
Ru(bpy)3 (1 ꢀM) in this work is much lower than that of DBAEA (5 mM), the direct
electrooxidation of DBAEA is dominant. As shown in Figure S3, the anodic current
1
/2
varies approximately linearly with the square root of the scan rate (v ),
demonstrating that diffusion process plays important role in ECL in the presence of
the relatively high concentration of DBAEA.
2
+
3
.4 The ECL property of Ru(bpy) /DBAEA system with and without Cys
3
2
+
The influence of Cys on the ECL intensity of Ru(bpy) /DBAEA system was
3
2
+
investigated. As demonstrated in Figure 4A, both individual Ru(bpy)3 and DBAEA
2
+
has weak ECL intensity and the ECL intensity of Ru(bpy) /DBAEA is much higher.
3
After the reaction between Cys and DBAEA, the ECL intensity is declined by nearly
7
times. The current intensity of different components (Figure 4B) is consistent with
2
+
the results of ECL system. That is, the current intensity of Ru(bpy) /DBAEA system
3
2
+
is higher than that of Ru(bpy)3 and DBAEA, and the addition of Cys can decrease
the current. It is obvious that the characteristic oxidation peak of Cys at around 0.6 V
cannot be seen in the reacting mixture. This phenomenon suggests that the decrease of
ECL is caused by the reaction between Cys and DBAEA.
3
.5 The mechanism of the reaction between Cys and DBAEA

It has been reported that Cys can remove the acrylate group of compound specifically
through Michael addition/cyclization reaction [14-16]. The mechanism of the reaction
between DBAEA and Cys is displayed as two steps according to the previous report
(Scheme 4). The first step is Michael addition. The nucleophilic reagent thiol attacks
and links to the unsaturated β-carbon atom of carbonyl. The electron moves along
carbon chain and generates unstable oxygen anion. Next it turns into carbon anion and
combines with a proton to become saturated carbonyl carbon. The second step is
cyclization. Briefly, amine serves as a nucleophilic reagent to attack the carbonyl
carbon to generate a heptatomic ring. Finally, the ring leaves and the acrylate is
removed successfully.
To prove the mechanism, we mixed DBAEA and Cys for over 10 minutes and
1
checked the H NMR spectrum (Figure S4). As we can see, the characteristic olefin
1
peaks disappeared compared with the H NMR spectrum of DBAEA, indicating that
the olefin group was removed by Cys successfully.
3
.6 The optimization of the conditions for the detection of Cys
To obtain higher sensitivity for Cys detection, the influence of several experimental
conditions were studied to find out the optimized ones (Figure S6). For the balance of
wide linear detection range and low LOD, each detection was performed under the
concentration of 5 mM DBAEA.
2
+
The optimized concentration of Ru(bpy)3 was studied by measuring the ECL
intensity before and after the reaction of DBAEA and Cys under different
2
+
concentrations of Ru(bpy)3 (0.5 µM, 1 µM, 2 µM, 3 µM, 5 µM) (Figure S5A). The
2
+
ECL intensity ratio of Ru(bpy) /DBAEA in the presence and absence of Cys is the
3
2
+
.
biggest when the concentration of Ru(bpy)3 is 1 µM (Figure S5B). Therefore, the

2+
concentration of Ru(bpy)3 was set as 1 ꢀM in subsequent experiments.
The influence of pH on the detection of Cys was also investigated in the pH range
from 6.5 to 10.0 (Figure S5C). It can be observed that the ECL intensity of
2
+
Ru(bpy) /DBAEA is the strongest when pH is 7.5 with or without Cys. However,
3
2
+
the ECL intensity ratio of Ru(bpy) /DBAEA in the presence and absence of 5 mM
3
Cys is the biggest when pH is 7.0 (Figure S5D). It is because the thiol of Cys can be
easily oxidized in alkaline condition, restricting the reaction of Cys and DBAEA, and
Michael addition is difficult to proceed in acid condition. To get higher sensitivity, a
pH of 7.0 was utilized in the following experiments.
The reaction time is another crucial condition for Cys detection. As illustrated in
Figure S5E, the ECL intensity increases with time and levels off after 10 minutes,
indicating the reaction proceeds completely within 10 minutes. Hence, each ECL
detection was performed after mixing Cys and DBAEA for 10 minutes.
3
.7 The detection of Cys
Cys was detected under optimized conditions by studying the ECL intensity decrease
caused by Cys. As demonstrated in Figure 5A, the decreasing value (I -I ) increases
1
2
with the concentrations of Cys. There is a linear relationship between the logarithm of
I -I ) and the logarithm of Cys concentrations from 10 ꢀM to 200 ꢀM (Figure 5B).
(
1
2
2
The linear equation is log (I -I ) = 3.871 + 0.816 log C (R = 0.994), with a limit of
1
2
detection (LOD) of 1.15 ꢀM. The comparison of our method with other methods for
Cys detection (Table 1) suggests that this method is sensitive.
2
+
3
.8 The selectivity of Ru(bpy) /DBAEA ECL system
3
In order to prove the excellent selectivity of this method towards Cys, we studied

2
+
the influence of other amino acids on the ECL intensity of Ru(bpy) /DBAEA
3
following the same procedure as Cys. As shown in Figure 6, only Cys can lead to
obvious decrease in the ECL intensity and the influence of other amino acids is
negligible. This result confirmed that thiol plays a determinate role in the reaction
between Cys and DBAEA. Therefore, this method shows good selectivity for Cys
among other native amino acids.
3
.9 The detection of Cys in real sample
As is well known, Cys is an essential nutrition that needs to be supplemented during
the cell metabolism. To test the feasibility of this method, we detected Cys in
Dulbecco's Modified Eagle Medium (DMEM) (without pretreatment except diluting
for 10 times) using for the cultivation of Mouse embryonic fibroblasts (L929) under
the optimized conditions. As shown in table 2, the concentration of Cys in DMEA was
found as 0.210 mM. With standard addition method, the recoveries range from
9
3.5%-105.6, indicating the present method is feasible for real sample detection.
4
. Conclusion
2
+
In this work, we synthesized DBAEA as a new co-reactant of Ru(bpy)3 through a
one-step method. It can increase the ECL intensity dramatically on Pt electrode
compared with conventional co-reactants. This method can be used to detect Cys
quantitatively based on the conjugate addition/cyclization reaction between Cys and
DBAEA, and it shows high selectivity and good sensitivity among other native amino
acids. This work will broaden the ECL application by synthesizing new co-reactant
from a design perspective and promote the sensitivity of analysis. It also shows great
potential applications in biological detection, clinical analysis and environmental

monitoring.
Conflict of interest
The authors declare no competing financial interests.
Acknowledgements
We thank the support from National Natural Science Foundation of China (Nos.
2
1750110447, 21804127, 21675148, 21874126), the National Key Research and
Development Program of China (No. 2016YFA0201300), the Chinese Academy of
Sciences (CAS)-the Academy of Sciences for the Developing World (TWAS)
President’s Fellowship Program.
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30] A. Abbaspour, A. Ghaffarinejad, Electrocatalytic oxidation of l-cysteine with a stable copper–
cobalt hexacyanoferrate electrochemically modified carbon paste electrode, Electrochimica Acta, 53
[
(
[
2008) 6643-6650.
31] J. Elistratova, B. Faizullin, N. Shamsutdinova, A. Gubaidullin, I. Strelnik, V. Babaev, K. Kholin, I.
Nizameev, E. Musina, R. Khairullin, A. Karasik, A. Mustafina, Synthesis of Au(I) complex-based
aqueous colloids for sensing of biothiols, Inorganica Chimica Acta, 485 (2019) 26-32.

Legends
2
+
Scheme 1. ECL detection of Cys based on the ECL of Ru(bpy) /DBAEA on Pt
3
electrode
Scheme 2. The synthesis method of DBAEA
2
+
2+
Fig. 1. ECL intensity of 1 ꢀM Ru(bpy) /5 mM TPA (a)ꢂ1 ꢀM Ru(bpy) /5 mM
3
3
2
+
DBAE (b), 1 ꢀM Ru(bpy) /5 mM DBAEA (c) on Pt electrode (A), GCE (B) and Au
3
electrode (C), pH = 7.5.
2
+
2+
Fig. 2. CV curve of 1 ꢀM Ru(bpy) /5 mM TPA (a)ꢂ1 ꢀM Ru(bpy) /5 mM DBAE
3
3
2
+
(
b), 1 ꢀM Ru(bpy) /5 mM DBAEA (c) on Pt electrode (A), GCE (B) and Au
3
electrode (C), pH = 7.5.
Fig. 3. The dependence of ECL intensities on the concentrations of TPA, DBAE,
2
+
DBAEA, C (Ru(bpy) ) = 1 ꢀM, pH = 7.5, Pt electrode.
3
2
+
Scheme 3. ECL mechanism of Ru(bpy) /DBAEA
3
2
+
Fig. 4. The ECL intensity (A) and CV curve (B) of 1 ꢀM Ru(bpy)3 (a), 5 mM
2
+
2+
DBAEA (b), 1 ꢀM Ru(bpy) /5 mM DBAEA + 5 mM Cys (c) 1 ꢀM Ru(bpy) /5
3
3
mM DBAEA (d), 5 mM Cys (e), pH = 7.0, Pt electrode.
Scheme 4. The reaction mechanism of DBAEA and Cys.
2
+
Fig. 5. (A) The ECL decrease of Ru(bpy) /DBAEA in the presence of different
3
concentrations of Cys (10 ꢀM, 30 ꢀM, 50 ꢀM, 70 ꢀM, 0.1 mM, 0.2 mM, 0.3 mM, 0.5
mM, 1 mM, 3 mM, 5 mM, 10 mM, 30 mM, 50 mM and 70 mM). (B) The linear
relationship of the logarithm of ECL decrease against the logarithm of C (Cys) from
1
0 ꢀM to 200 ꢀM. I and I represent the ECL intensity with and without Cys,
2 1
separately, pH = 7.0, Pt electrode.
Fig. 6. The ECL intensity decrease of Ru(bpy) /DBAEA in the presence of different
2
+
3

amino acids on Pt electrode. All the experiments were operated under the same
condition: reaction solution: 50 ꢀL amino acids (50 mM), 50 ꢀL DBAEA (50 mM)
2
+
and 150 ꢀL pH 7.0 PBS; ECL solution: 200 ꢀL pH 7.0 PBS + 50 ꢀL Ru(bpy)3 (10
ꢀ
M). I and I represent the ECL intensity before and after the reaction of DBAEA and
2
1
amino acids.
Table 1. Comparison of different methods used for Cys detection.
Table 2. The detection of Cys in DMEM

Table 1. Comparison of different methods used for Cys detection.
Method
Probe
Linear range
LOD
Ref.
[27]
[28]
[29]
[30]
Colorimetry
Fluorescence
Au nanoparticles
Fluorogenic dye
1 µM
100 µM-1.0 mM 39 µM
Electrochemistry Pt/Fe O nanoparticles 100 µM-1.0 mM 10 µM
3
4
Electrochemistry Copper–cobalt
hexacyanoferrate
6 µM-1.0 mM
5 µM
Fluorescence
Au(I) complex-based 0-20 µM
colloids
1 µM
[31]
2
+
ECL
Ru(bpy) /DBAEA
10 µM-2 mM
1.149 µM This
work
3

Table 2. The detection of Cys in DMEM
Cys added
mM)
Cys founded
(mM)
RSD (%)
Recovery (%)
(
0
.210
4.54
6.35
5.10
4.30
0
.05
0.275
0.290
0.373
105.6
93.5
0
.1
0.15
103.7