Talanta 234 (2021) 122649

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Electrochemical detection of kinase by converting homogeneous analysis

into heterogeneous assay through avidin-biotin interaction
Yong Chang a, c, 1, Xiaohua Ma b, 1, Ting Sun a, Lin Liu a, b, **, Yuanqiang Hao b, *
a
College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000, People’s Republic of China
b
College of Chemistry and Chemical Engineering, Shangqiu Normal University, Shangqiu, Henan, 476000, People’s Republic of China
c
School of Chemistry and Materials Engineering, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: In the classical heterogeneous electrochemical assay, phosphorylation of peptide substrate is usually performed
Electrochemical biosensor on the solid-liquid surface. However, immobilization of probe on the solid surface may limit the interaction
Kinase between the reaction site of probe and the active center of kinase due to the steric hindrance effect. In this work,
Phosphorylation
we proposed a heterogeneous electrochemical method for kinase detection, in which the probe is immobilization-
Immobilization-free
Avidin-biotin interaction
free during the phosphorylation reaction. A biotinylated peptide was used as the kinase substrate. After phos­
phorylation, the biotinylated phosphopeptide was captured by the neutravidin (NA)-modified electrode through
the avidin-biotin interaction. The phosphate groups on the electrode surface were then recognized by the con­
jugates preformed between biotinylated Phos-tag™ (Bio-tag-Phos) and ferrocene (Fc)-capped NA-modified gold
nanoparticle (Fc-AuNP-NA). The method integrates the advantages of homogeneous reaction and heterogeneous
detection with high simplicity, sensitivity and specificity. The strategy can be applied to design other hetero­
geneous biosensors without the immobilization of probe during the enzyme catalyzed reaction.

1. Introduction resonance light scattering [20], surface plasmon resonance [21] and
surface enhanced Raman spectroscopy [22]. Among them, electro­
Phosphorylation plays an important role in the process of cell signal chemical biosensors have received widespread attentions because of
transduction by regulating and controlling protein activity and function their advantages of high sensitivity, fast response, low cost, and portable
[1]. The reaction is usually catalyzed by kinase to transfer a phosphoryl instrument. In the heterogeneous kinase biosensors, the peptide sub­
group from adenosine triphosphate (ATP) to the hydroxyl group in strates are usually immobilized on an electrode surface [23]. After
serine, threonine or tyrosine residue. Changes in the activity and content phosphorylation, the phosphorylated substrates can be specifically
of kinase are associated with many diseases, such as cancers, metabolic recognized by anti-phosphoserine antibodies, phosphopeptide-binding
disorders and inflammation [2]. Therefore, sensitive and selective proteins, metal chelators, metal ions or metal-based nanomaterials
detection of kinase is of great importance for clinical diagnosis and drug [24–34]. For example, Nie’s group reported the electrochemical detec­
discovery. tion of kinase by employing DNA-functionalized gold nanoparticle
Traditional methods for determination of kinase activity include (AuNP) to load the signal reporter of [Ru(NH3)6]3+, in which Zr4+ was
isotope labeling, mass spectrometry, autoradiography and gel electro­ used as the linker [26]. Yin’s group developed an electrochemical kinase
phoresis. These methods suffer from some shortcomings, such as biosensor through the signal amplification of avidin-conjugated horse­
possible harm to humans, expensive instrument, complicated operation, radish peroxidase (HRP) by using biotinylated phosphate-binding re­
large sample volume, and/or poor sensitivity [3,4]. In recent years, agent Bio-tag-Phos as the linker [34]. Through the signal amplification
various novel heterogeneous biosensors have been developed for kinase of enzymes or nanomaterials, the heterogeneous electrochemical bio­
detection, including electrochemistry [5–13], photoelectrochemistry sensors exhibit high sensitivity and specificity for kinase detection.
[14–16], electrochemiluminescence [17,18], fluorescence [19], However, when the probe is immobilized on the electrode surface, the

* Corresponding author.
** Corresponding author. College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000, People’s Republic of China.
E-mail addresses: [email protected] (L. Liu), [email protected] (Y. Hao).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.talanta.2021.122649
Received 13 April 2021; Received in revised form 16 June 2021; Accepted 19 June 2021
Available online 25 June 2021
0039-9140/© 2021 Elsevier B.V. All rights reserved.
Y. Chang et al. Talanta 234 (2021) 122649

Scheme 1. Schematic representation of the electrochemical method for PKA detection by the signal amplification of Fc-AuNP-NA-Bio-tag-Phos conjugate.

steric hindrance effect may limit the interaction between the reaction Co., Ltd. (Shanghai). Biotinylated Phos-tag™ (Bio-tag-Phos) was or­
site and the active center of enzyme for the enzymatic reaction [35–38]. dered by Seebio Biotech Co., Ltd. (Shanghai). Bovine serum albumin,
For this view, we attempt to develop a heterogeneous electrochemical ATP, glucose and glutathione (GSH) were obtained from Sangon
method for kinase detection in which the probe is immobilization-free Biotech. Co., Ltd. (Shanghai). Tyrosine kinase Src was provided by R&D
for phosphorylation reaction. Systems, Inc. (Minneapolis, MN). Ferrocene (Fc)-labeled dipeptide Asp-
Avidin, streptavidin (SA) or neutravidin (NA) protein can bind up to Cys (Fc-DC) was provided by ChinaPeptides Co., Ltd. (Shanghai). N-[2-
four biotin molecules with strong non-covalent interaction (Kd = 10− 15 (p-Bromocinnamylamino)ethyl]-5-Isoquinoline (H-89), gold chloride
M). Such an interaction has been exploited for many applications in hydrate and other reagents with analytical-grade were purchased from
bioassay and purification fields [39]. Because peptides, nucleic acids Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).
and proteins can be readily labeled by biotin, the avidin-biotin system
has been used for the development of robust and highly sensitive bio­ 2.2. Preparation and modification of gold nanoparticles
sensors. For instance, the commercial SA-coated magnetic beads have
been widely applied for the immobilization of biotin-labeled receptors, Gold nanoparticles (AuNPs) with a diameter of ~33 nm were pre­
thus allowing for the design of various optical and electronic biosensors pared by citrate reduction method. NA protein was attached on the
[39–41]. SA-covered gold chips, electrodes and nanoparticles have been surface of AuNPs through the hydrophobic and Au–S interactions [44].
used to immobilize antibodies or aptamers for sensitive detection of Briefly, 0.5 mL of 8.6 nM AuNPs suspension was mixed with 0.25 mL of
proteins and analysis of molecular interaction [42–44]. Recently, Tan’s 20 nM NA in carbonate buffer (pH 10) for 2 h. Then, 0.25 mL of Fc-DC
group developed an affinity-switchable lateral flow assay by using an (10 μM) in carbonate buffer containing 0.5 mM TCEP was added to the
avidin-modified test strip to capture biotin-labeled probe [45]. We mixture. After incubation for 2 h again, the unbound NA and Fc-DC were
found that the peptide labeled with two biotin tags can induce the in-situ removed by centrifugation. The Fc-capped NA-modified AuNP
assembly of tetrameric SA protein, and then developed a (Fc-AuNP-NA) particles were washed and resuspended in 1 mL of
surface-tethered electrochemical protease biosensor [46]. Herein, we phosphate buffer (10 mM, pH 7.4). Before the assay of PKA, the
proposed a heterogeneous electrochemical method for kinase detection Fc-AuNP-NA was mixed with a given concetration of Bio-tag-Phos in the
based on the avidin-biotin interaction, in which the phosphorylation presence of equivalent Zn2+ to produce the Fc-AuNP-NA-Bio-tag-Phos
reaction was carried out in homogeneous solution. After phosphoryla­ conjugate through the avidin-biotin interaction.
tion, the phosphopeptide was captured by the NA-modified electrode
through the avidin-biotin interaction and then recognized by a metal
chelator-labeled signal reporter, thus converting the homogeneous 2.3. Preparation of sensor electrode
analysis into the signal-amplified heterogeneous assay. The method in­
tegrates the advantages of homogeneous reaction and heterogeneous Gold electrode was first polished with 0.05 μm alumina slurry and
detection through the avidin-biotin interaction. The “immobilization-­ washed by sonication in 50% ethanol. The electrode was then treated by
free” strategy should be valuable for the design of novel heterogeneous consecutive electrochemical scaning in 0.5 M H2SO4 for 20 circles. The
biosensors to monitor different post-translational modification enzymes. potential changed from − 0.2 to 1.3 V with a scan rate of 100 mV/s.
Then, the cleaned electrode was incubated with 1 μM NA protein in
2. Experimental carbonate buffer for 2 h. NA was attached on the electrode surface by
hydrophobic and Au–S interactions. The unreacted gold surface was
2.1. Chemicals and reagents blocked by immersing the electrode in 0.5 mM GSH solution for 30 min
[47]. Finally, the NA-modified sensor electrode was thoroughly washed
Protein kinase A (PKA, catalytic subunit), thrombin, okadaic acid, with carbonate buffer and kept at 4 ◦ C. The sensor electrode was char­
tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), tris(2- actered by cyclic voltammetry (CV) and electrochemical impedance
carboxyethyl)phosphine hydrochloride (TCEP), and trisodium citrate spectroscopy (EIS) in 5 mM [Fe(CN)6]3− or [Fe(CN)6]3− /4− (1:1) solu­
were obtained from Sigma-Aldrich. NA protein was provided by Thermo tion (Fig. S1). The influence of incubation time and NA concentration for
Fisher Scientific. Peptides were synthesized and purified by synpeptides the electrode modification was examined by monitoring the change of
electron-transfer resistance (Ret) (Fig. S2).

2
 Y. Chang et al. Talanta 234 (2021) 122649

(A ) 0 a (B ) Fig. 1. (A) DPV curves acquired at the NA-modified

c electrode after treatment by Bio-GLRRASLG (curve
e a), Bio-GLRRASpLG (curve b), or the Bio-GLRRASLG/
-1
ATP/PKA mixture (curve d), followed by incubation
with Fc-AuNP-NA-Bio-tag-Phos. Curve c corresponds
-2
to that treated by Bio-GLRRASpLG and Fc-AuNP-NA.
Current/ µA

Curve e is acquired at the Bio-GLRRASLG/NA-

-3
modified electrode after incubation with the ATP/
d PKA mixture and Fc-AuNP-NA-Bio-tag-Phos. The
-4
concentrations of Bio-GLRRASLG, Bio-GLRRASpLG
and PKA were 2.5 nM, 5 nM and 100 U/mL, respec­
-5
tively. (B) SEM images of the NA-modified electrode
after treatment by Bio-GLRRASLG (top) and Bio-
-6
b GLRRASpLG (bottom), followed by incubation with
Fc-AuNP-NA-Bio-tag-Phos.
-7
0.4 0.3 0.2 0.1 0.0
Potential/V vs. Ag/AgCl

2.4. Procedures for PKA detection Scheme 1. The sensing interface was obtained by immobilizing NA
protein on the electrode through the hydrophobic and Au–S interactions
The substrate peptide (Bio-GLRRASLG) was phosphorylated by PKA [44]. The unreacted Au surface was blocked by GSH, a natural zwit­
according to the manufacturer’s protocol. The final concentration of terionic small molecule exhibiting good antifouling property and
ATP was 1 mM. After reaction for 2 h, 5 μL of peptide/PKA mixed so­ biocompatibility [47]. The biotinylated peptide (Bio-GLRRASLG) was
lution was cast onto the NA-modified electrode surface to incubate for first phosphorylated by PKA in solution and then captured by the
10 min. Upon washing with Tris buffer, the electrode was exposed to 5 NA-modified gold electrode through the avidin-biotin interaction. The
μL of the Fc-AuNP-NA-Bio-tag-Phos conjugate. After incubation for 10 resulting phosphate group allowed for the capture of
min again, the electrode was thoroughly rinsed with buffer and water Fc-AuNP-NA-Bio-tag-Phos conjugate by the formation of stable chelate
and then placed in 50 mM Na2SO4 for voltammetric determination. All with Zn2+. Since each AuNP was capped with numerous electroactive Fc
the electrochemical experiments were performed on a CHI 660 elec­ tags, the electrochemical signal was significantly amplified and the peak
trochemical workstation (CH Instruments, Shanghai, China). current would be proportional to the concentration of phosphopeptide.
Note that the phosphorylation level is related to the concentration and
2.5. Assays of PKA in cell lysates activity of PKA; this method can be used to determination of PKA and
screening of its potential inhibitors.
The HeLa cells were grown in DMEM media containing 10% fetal
bovine serum in a CO2 (5%) incubator at 37 ◦ C. The cultured HeLa cells 3.2. Feasibility
were washed three times by Tris buffer and then lysed with the lysis
buffer. The lysate was centrifuged at 14,000 rpm for 10 min. The su­ The feasibility of the signal amplification method was explored by
pernatant was collected and stored at − 20 ◦ C for use. For the assay of differential pulse voltammetry. Fig. 1A shows the differential pulse
PKA, the supernatant was diluted by the Tris buffer containing a given voltammogram (DPV) of the NA-modified gold electrode after incuba­
concentration of substrate peptide. Finally, the cell lysates were tion with analysis sample and Fc-AuNP-NA-Bio-tag-Phos conjugate.
analyzed with the procedures as aforementioned. There is no oxidation peak when the sensor electrode was successively
incubated with biotinylated peptide (Bio-GLRRASLG) and Fc-AuNP-NA-
3. Results and discussion Bio-tag-Phos (curve a), demonstrating that the signal labels cannot be
captured by the peptide/NA-covered electrode. However, when the
3.1. Detection principle sensor electrode was successively incubated with the biotinylated
phosphopeptide (Bio-GLRRASpLG) and Fc-AuNP-NA-Bio-tag-Phos, a
The detection principle with PKA as the model analyte is shown in well-defined oxidation peak with high signal intensity was observed

Fig. 2. Dependence of ipa on Bio-GLRRASpLG (A) and Bio-tag-Phos (B) concentration.

3
Y. Chang et al. Talanta 234 (2021) 122649

(A ) (B )
0.0 3.0
-0.5 2.5
-1.0 0.005 U/mL
Current/ µA

2.0 2.0

i pa/µ A
-1.5
1.5
1.5
-2.0 [PKA]

i pa/µ A
1.0
1.0
-2.5
0.5

-3.0 0.5
100 U/mL 0.0
0 5 10 15 20
-3.5 0.0 [PKA] (U/mL)

0.4 0.3 0.2 0.1 0.0 0 20 40 60 80 100

Potential/V vs. Ag/AgCl [PKA] (U/mL)
Fig. 3. (A) DPV curves of the biosensor for detection of various concentrations of PKA (0.005, 0.1, 1, 2, 10, 15, 20, 50 and 100 U/mL). (B) Dependence of ipa on PKA
concentration. The inset shows the linear part of the curve.

(curve b). The result suggested that the Fc-AuNP-NA-Bio-tag-Phos con­

Table 1
jugate can be captured by phosphopeptide but not non-phosphorylated
Analytical performances of various signal-amplified electrochemical biosensors
peptide substrate, which is further confirmed by SEM observation. As
for PKA detection.
shown in Fig. 1B, AuNPs were found at the NA-modified electrode
incubated with Bio-GLRRASpLG and Fc-AuNP-NA-Bio-tag-Phos. We also Signal reporters Detection limit Linear range Ref.
(mU/mL) (mU/mL)
found that the signal was intensified with the increase of biotinylated
phosphopeptide conentration (Fig. 2A), indicating that the peak current rGO-ZrO2-Thi 5 5–2 × 102 [24]
rGO-PDA-AgNPs-Ti4+ 10 10–5 × 102 [25]
was proportional to the phosphopeptide level. Moreover, no oxidation
[Ru(NH3)6]3+/DNA-AuNPs/ 150 Not reported [26]
peak was observed when Fc-AuNP-NA was used instead of Fc-AuNP-NA- Zr4+
Bio-tag-Phos for the signal output (curve c). It was found that the signal [Ru(NH3)6]3+/DNA/Zr4+ 500 5 × 103–5 × 105 [27]
was intensified with the increase of the linker (Bio-tag-Phos) concen­ [Ru(NH3)6]3+/DNA-AuNPs 30 5 × 103–5 × 105 [28]
tration and began to level off or even drop down beyond 5 nM (Fig. 2B). network/Zr4+
Zr4+/carboxylate/eRAFT 1.02 0–1.4 × 102 [32]
The signal decrease for higher concentration of Bio-tag-Phos can be Zr4+/carboxylate/RAFT 1.05 0–1.4 × 102 [33]
attributed to the fact that redundant Bio-tag-Phos in solution may HRP -AuNPs 1 1–10 [30]
compete with Fc-AuNP-NA-Bio-tag-Phos to bind the phosphopeptide on TiO2/AgNPs 200 0–1 × 103 [31]
electrode surface, thus preventing the capture of Fc-AuNP-NA-Bio-tag- HRP-SA-Bio-tag-Phos 150 5–2.5 × 104 [34]
Fc-AuNP-NA-Bio-tag-Phos 5 5–2 × 104 This
Phos. These results demonstrated that the attachment of signal labels
work
on the phosphopeptide/NA-covered electrode surface was strictly
dependent upon the interaction between phosphate group and tag-Phos. Abbreviation: rGO, reduced graphene oxide; Thi, thionine; PDA, polydopamine;
To investigate the feasibility of this method for PKA detection, the AgNPs, silver nanoparticle; MWNTs, multi-walled carbon nanotubes; eRAFT,
electrochemically controlled reversible addition-fragmentation chain transfer.
NA-modified gold electrode was incubated with the mixture of bio­
tinylated peptide, ATP and PKA, followed by exposure to Fc-AuNP-NA-
Bio-tag-Phos. Consequently, a clear oxidation peak was observed (curve good reproducibility of the biosensor. The method shows a wide linear
d). However, when the biotinylated peptide was immobilized in advance range and a high sensitivity. The detection limit was found to be 5 mU/
on the NA-modified electrode and then phosphorylated by PKA, a weak mL by determining the target smallest concentration at which the
oxidation peak was observed after incubation of the electrode with Fc- response was clearly distinguishable from that of blank control. The
AuNP-NA-Bio-tag-Phos (curve e). These results demonstrated that value is comparable to or even lower than those achieved by the signal
immobilization of peptide on the electrode reduced the phosphorylation amplification of enzymes and redox active nanolabels (Table 1). The
efficiency, probably due to the steric hindrance effect. Thus, the high sensitivity can be attributed the well-defined electrochemical
“immobilization-free” method for phosphorylation can be used to design signal of Fc tags, the signal amplification of AuNPs, the high sensitivity
heterogeneous biosensor for PKA analysis with improving sensitivity of differential pulse voltammetry and the high phosphorylation effi­
and detection efficiency. ciency of homogeneous reaction. Thus, the advantages of homogeneous
reaction and heterogeneous assay are integrated in this method. More­
over, the NA-modified electrode only lost 7% detection capability after
3.3. Sensitivity and stability
storage for twenty days. The result is understandable since avidin pro­
tein is extremely stable and the avidin-biotin interaction is unaffected by
The analytical performances of the method for PKA detection were
extremes of pH, temperature, organic solvents and other denaturing
investigated by measuring various concentrations of PKA. As shown in
agents. Thus, multiple NA-modified electrodes can be prepared in par­
Fig. 3A, the electrochemical signal increased gradually with the increase
allel for the detection of different kinases by adopting sequence-matched
of PKA concentration. The oxidation peak current (ipa) was linearly
substrates, and the detection throughput may be improved by using
proportional to PKA concentration in the range of 0.005–20 U/mL
screen-printed electrode arrays.
(Fig. 3B). The linear equation can be expressed as ipa (μA) = 0.078 +
0.095 [PKA] (U/mL) (R2 = 0.998). The relative standard deviations
(RSDs) obtained at three in parallel prepared electrodes for the detection
of different concentrations of PKA were all lower than 6%, indicating the

4
Y. Chang et al. Talanta 234 (2021) 122649

(A) (B) [okadaic acid]/ µM

0.0
0 1 2 3 4 5
100 100
Src, thrombin, BSA,
GLRRASpLG
-0.5

Inhibition effect (%)

80 80

Inhibition effect (%)

Current/ µA

H-89
60 60
-1.0
okadaic acid
40 40

-1.5
20 20
PKA

-2.0 0 0
0.4 0.3 0.2 0.1 0.0 0 10 20 30 40 50

Potential/V vs. Ag/AgCl [H-89]/nM

Fig. 4. (A) Selectivity of the method. The concentrations of PKA, Src, thrombin, BSA, and GLRRASpLG were 10 U/mL, 25 ng/mL, 10 nM, 10 nM and 20 nM,
respectively. (B) Inhibition effect of H-89 and okadaic acid on PKA activity.

activity in HeLa cell lysates. As shown in Fig. 5, the ipa increased with the
2.0 increase of cell number or lysate concentration, However, when the cell
without H-89
lysate was treated by H-89, the ipa was much lower than that without
with H-89
inhibitor. The result indicated that the method can be used to detect
1.5 active PKA in cell lysates. Therefore, the biosensor shows great potential
to monitor PKA activity in complicated biological samples and to screen
effective kinase inhibitor drugs.
i pa/µA

1.0
4. Conclusion

0.5 In summary, we have developed a general electrochemical strategy

for kinase detection by integrating the advantanges of homogeneous
reaction and heterogeneous detection. The method overcomes the lim­
itation of the traditional heterogeneous electrochemical biosensors in
0.0
10 100 500 which the peptide substrate was pre-immobilized on the electrode sur­
face for phosphorylation reaction. The phosphate group in phospho­
Cell number (cells/mL) peptide was recognized by a metal chelator and the signal was amplified
by Fc-capped AuNPs. The detection limit of 5 mU/mL is lower than most
Fig. 5. Effect of H-89 on the activity of PKA in three concentrations of
cell lysates. of the reported values obtained by the signal amplification of enzymes
and redox active nanolabels. Although only PKA was determined in this
work, the strategy can be used to detect other kinases and post-
3.4. Selectivity and inhibition assay
translational modification enzymes by adopting a sequence-specific
substrate. We believe that this work would be useful for enzyme activ­
To evaluate the selectivity, the biosensor was challenged with
ity assay and disease diagnosis.
different biomolecules, including tyrosine kinase Src, protease
thrombin, serum protein BSA, and biotin-free phosphopeptide
Declaration of competing interest
GLRRASpLG. As shown in Fig. 4A, no oxidation peaks were observed for
the tested interferences, indicating that the selectivity of the biosensor is
The authors declare that they have no known competing financial
excellent for PKA detection. To demonstrate the amenability of the
interests or personal relationships that could have appeared to influence
biosensor for inhibitor screening, PKA was incubated with two well-
the work reported in this paper.
known inhibitors and then analyzed respectively. It was found that the
peak current decreased with the increase of inhibitor concentration.
Acknowledgments
Fig. 4B depicts the dependence of ipa on the concentration of H-89 and
okadaic acid. The ipa was reduced with the increase of inhibitor con­
This work was supported by the Program for Innovative Research
centration. The half-maximum inhibition value of IC50 was found to be
Team of Science and Technology in the University of Henan Province
10.2 nM for H-89 and 1.27 μM for okadaic acid. The results are
(21IRTSTHN005) and the National Natural Science Foundation of China
consistent with the previous reports [26,34]. Thus, the biosensor ex­
(21804085).
hibits great potential for the discovery of new inhibitor drugs.

Appendix A. Supplementary data

3.5. Assays of cell lysates
Supplementary data to this article can be found online at https://doi.
PKA is activated in many cancer cells or tissues, which plays an org/10.1016/j.talanta.2021.122649.
important role in the abnormal proliferation of cells. The amenability of
the method for real sample assays was investigated by probing of PKA

5
Y. Chang et al. Talanta 234 (2021) 122649

Credit author statement [24] Z. Chen, Y. Liu, L. Hao, Z. Zhu, F. Li, S. Liu, Reduced graphene oxide-zirconium
dioxide− thionine nanocomposite integrating recognition, amplification, and
signaling for an electrochemical assay of protein kinase activity and inhibitor
Yong Chang: Investigation. Xiaohua Ma: Investigation. Ting Sun: screening, ACS Appl. Bio Mater. 1 (2018) 1557–1565.
Investigation. Lin Liu: Methodology, Writing – review & editing. [25] J. Wang, X. Liu, C. Wang, D. Liu, F. Li, L. Wang, S. Liu, An integral recognition and
Yuanqiang Hao: Conceptualization, Writing – original draft. signaling for electrochemical assay of protein kinase activity and inhibitor by
reduced graphene oxide-polydopamine-silver nanoparticle-Ti4+ nanocomposite,
Front. Bioeng. Biotechnol. 8 (2020) 1–10.
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