S-Click Reaction for Isotropic Orientation of Oxidases on Electrodes to Promote Electron Transfer at Low Potentials

Article abstract of DOI:10.1002/anie.201909203

Electrochemical sensors are essential for point-of-care testing (POCT) and wearable sensing devices. Establishing an efficient electron transfer route between redox enzymes and electrodes is key for converting enzyme-catalyzed reactions into electrochemical signals, and for the development of robust, sensitive, and selective biosensors. We demonstrate that the site-specific incorporation of a novel synthetic amino acid (2-amino-3-(4-mercaptophenyl)propanoic acid) into redox enzymes, followed by an S-click reaction to wire the enzyme to the electrode, facilitates electron transfer. The fabricated biosensor demonstrated real-time and selective monitoring of tryptophan (Trp) in blood and sweat samples, with a linear range of 0.02–0.8 mm. Further developments along this route may result in dramatic expansion of portable electrochemical sensors for diverse health-determination molecules.

Full text of DOI:10.1002/anie.201909203

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201909203
German Edition: DOI: 10.1002/ange.201909203
Enzyme–Electrode Interfaces
Hot Paper
S-Click Reaction for Isotropic Orientation of Oxidases on Electrodes to
Promote Electron Transfer at Low Potentials
Lin Xia, Ming-Jie Han, Lu Zhou, Aiping Huang, Zhaoya Yang, Tianyuan Wang, Fahui Li,
Lu Yu, Changlin Tian, Zhongsheng Zang, Qing-Zheng Yang, Chenli Liu, Wenxu Hong, Yi Lu,
Lital Alfonta, and Jiangyun Wang*
Abstract: Electrochemical sensors are essential for point-of-
care testing (POCT) and wearable sensing devices. Establish-
ing an efficient electron transfer route between redox enzymes
and electrodes is key for converting enzyme-catalyzed reac-
tions into electrochemical signals, and for the development of
robust, sensitive, and selective biosensors. We demonstrate that
the site-specific incorporation of a novel synthetic amino acid
(2-amino-3-(4-mercaptophenyl)propanoic acid) into redox
enzymes, followed by an S-click reaction to wire the enzyme
to the electrode, facilitates electron transfer. The fabricated
biosensor demonstrated real-time and selective monitoring of
tryptophan (Trp) in blood and sweat samples, with a linear
range of 0.02–0.8 mm. Further developments along this route
may result in dramatic expansion of portable electrochemical
sensors for diverse health-determination molecules.
of wearable sensors for monitoring of sweat components was
recently reported.[4] However, important metabolites present
in blood and sweat, such as amino acids, are rarely studied in
the POCT field because real-time sensitive analysis tech-
niques are lacking.
Enzymatic electrochemical biosensors provide a conven-
ient, low cost, and real-time approach for measuring analyte
concentration, and they are essential for diabetes manage-
ment.[5] An efficient enzymatic electrochemical biosensor has
several key requirements: high surface density, long-term
enzymatic stability, and perhaps most challenging, an efficient
electron transfer pathway between the enzyme active site and
the electrode. Although great effort has been devoted to
improving the robustness, selectivity, and activity of the
enzyme to meet the primary requirements for practical
sensing applications, highly selective and sensitive electro-
chemical sensors for most health-determining biomolecules
are still underdeveloped.[6,7] To achieve this goal, it is
important to improve the electron transfer between the
enzyme and the electrodes. Efficient electron transfer
between the enzymeꢀs redox active cofactor and the electrode
requires a short distance (typically less than 14 ꢁ),[8] redox
mediators,[9] and electrode-modifying redox polymers.[10]
Using mediators in enzymatic electrochemical biosensors is
a robust approach for improving electron transfer, and yet,
the use of mediators usually leads to an increased over-
potential relative to the original redox potential of the
enzyme. Moreover, the redox mediators are generally non-
selective, as they facilitate electron transfer between elec-
trode and protein, as well as various interfering molecules.[11]
Amino acids are essential metabolic intermediates and
cellular signaling molecules.[1] Aberrant amino acid metabo-
lism leads to many severe diseases, therefore real-time amino
acid analysis is of great importance for diagnosis and
medicine.[2] For example, tumor cells over-express indole-
amine 2,3-dioxygenase (IDO), which mediates tryptophan
(Trp, required for T cell activation) depletion to suppress
immune response and the effectiveness of cancer immune
therapy.[2] Conventional analytical procedures for measuring
amino acid concentration are based on non-portable spectro-
scopic or chromatographic[3] instruments, which are not
suitable for POCT and healthcare-monitoring biosensors
that are wearable.[4] Interest is growing in personalized
medicine through continuous health monitoring, and the use
[*] Dr. L. Xia, L. Zhou, Z. Zang, Prof. C. Liu
Institute of Synthetic Biology, Shenzhen Institutes of Advanced
Technology, Chinese Academy of Sciences, 1068 Xueyuan Ave
Shenzhen (China)
Prof. Q.-Z. Yang
College of Chemistry, Beijing Normal University, Beijing (China)
Dr. W. Hong
Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center
Shenzhen (China)
Dr. M.-J. Han, Dr. A. Huang
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences, Tianjin (China)
Prof. Y. Lu
Department of Chemistry, University of Illinois
Urbana-Champaign, IL 61801 (USA)
Z. Yang, T. Wang, Dr. F. Li, Z. Zang, Prof. J. Wang
Institute of Biophysics, Chinese Academy of Science
Chaoyang District, Beijing (China)
Prof. L. Alfonta
Department of Life Sciences and Ilse Katz Institute for Nanoscale
Science and Technology, Ben-Gurion University of the Negev
Beer-Sheva (Israel)
E-mail: jwang@ibp.ac.cn
Dr. L. Yu, Prof. C. L. Tian
High Magnetic Field Laboratory
Chinese Academy of Sciences, Hefei (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201909203.
Prof. C. L. Tian
Hefei National Laboratory of Physical Sciences at Microscale and
School of Life Sciences, University of Science and Technology of
China, Hefei (China)
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Besides, for in vivo applications, mediator immobilization is
required to ensure biocompatibility. Enhancing the direct
electrical communication across the enzyme–electrode inter-
face with nanomaterials has made great contributions to the
realization of third generation biosensors;[12] however, the
random orientation of the enzyme with respect to the
electrode surface leads to large variations in the electron
transfer efficiency. Attachment of enzyme cofactors to con-
ductive nanoparticles can achieve higher catalytic turnover
rate than the unmodified system,[13] yet this approach cannot
be applied to an enzyme that has its cofactor completely
buried within a protein. Site-specific wiring of an enzyme to
the electrode through cysteine-based surface ligation requires
one unique cysteine to be present in its surface.[14] Genetic
code expansion[15] has been used to introduce synthetic amino
acids into enzymes to facilitate enzyme wiring[16] to electro-
des; typically using carcinogenic linkers or expensive gold
electrodes. Therefore, a fully biocompatible and cost-effective
approach is highly desirable for making broadly applicable
POCT biosensors.
We developed an efficient and broadly applicable wiring
strategy to transform redox enzymes into electrochemical
sensors. Our strategy involved site-specific incorporation of
the synthetic amino acid 2-amino-3-(4-mercaptophenyl)
propanoic acid (or p-thiolphenylalanine, TF), as a unique
enzyme-anchoring point, to two different amino acid oxi-
dases, glycine oxidase (GlyOx, PDB code 1NG4) and
l-tryptophan oxidase (TrpOx, PDB code 5G3T). Importantly,
TF differs from tyrosine (Tyr) by only one atom, which
introduces minimal perturbation to the target enzyme. We
then used boron-dipyrromethene (Bodipy373)[17] as an
enzyme/electrode wiring linker, which reacts with TF specif-
ically through a thiol–chlorine nucleophilic substitution
reaction (S-click reaction, Figure 1). Distinct from other
biorthogonal reactions,[16] Bodipy373 undergoes a dramatic
bathochromic shift (Supporting Information, Figure S3) after
reaction with TF, which facilitates convenient characteriza-
tion of Bodipy373-labeled redox enzymes. The modified
enzyme can be attached onto the carbon electrode surface
where it generates an efficient electron transfer biocatalytic
current sufficient to transform specific substrates at a potential
close to the original redox potential of the enzyme, thus
yielding improved selectivity.
Figure 1. A) The structure of TrpOx and the positions for TF incorporation.
The distances between TF and FAD are indicated. B) A schematic depiction
of the wiring of TrpOx from the TF395 site through a Bodipy373 linker to
the CNT surface. C) A schematic depicting the S-click reaction; sulfur atom
in TF (S), chlorine atom in Bodipy373 (Cl).
(SDS-PAGE) showed that full-length GlyOx and TrpOx
were expressed only in the presence of TF (Figure 2A),
indicating that TF was recognized specifically by TFRS. The
purified protein was then subjected to trypsin digestion, and
mass spectrometry (MS–MS) analysis of the product mixture
revealed the site-specific incorporation of TF into the target
protein (Figure 2C). Enzymatic assays show that the wild-
type and mutant TrpOx have similar activity, indicating that
the site-specific incorporation of TF cause minimal perturba-
tion to enzyme function (Supporting Information, Figure S2).
To demonstrate that Bodipy373 selectively reacts with the
thiophenol group of TF, wt GlyOx, GlyOx-266TF, wt TrpOx,
and TrpOx-395TF were incubated with 50 mm Bodipy373 in
pH 8.0 Tris buffer, at 48C for 60 minutes. SDS-PAGE analysis
revealed fluorescent bands associated with GlyOx-266TF and
TrpOx-395TF, but none were observed for wt GlyOx and wt
TrpOx (Figure 2B). Notably, wt GlyOx and wt TrpOx both
contain four surface cysteine residues, which did not react
with Bodipy373 under the condition tested.[17] A titration
experiment shows that TF reacts with Bodipy373 at a rate of
5.6mÀ1 sÀ1. By contrast, Bodipy373 reacts with N-acetyl-
cysteine about 1000-fold slower, at a rate of 0.0057mÀ1 sÀ1
(Supporting Information, Figure S3). The much lower pKa of
TF (6.4) relative to cysteine (8.4) accounts in part for this
remarkable reactivity, which allows for the selective modifi-
cation of TF in the presence of surface exposed cysteines.
Fluorescence measurements demonstrate that Bodipy373
fluorescence is quenched 60-fold (Supporting Information,
Figure S4) when incubated with carbon nanotubes (CNTs),
which is likely a consequence of strong pi–pi interactions
between Bodipy373 and CNT, and photoinduced electron
transfer (PET) quenching.[19a] The site-specific attachment of
TrpOx to the carbon electrode was further characterized by
atomic force microscopy (AFM). In the absence of Bodipy373
modification, the coverage of physically adsorbed TrpOx-
395TF enzyme on the highly oriented pyrolytic graphite
The synthesis of TF consists of four steps starting from
4-nitrobenzyl bromide (Supporting Information, Figure S1).
To obtain an orthogonal transfer ribonucleic acid (tRNA)/
aminoacyl-tRNA synthetase pair that selectively charges TF
in response to amber suppressor, three rounds of positive and
two rounds of negative selections with a Methanocaldococcus
jannaschii (Mj) tyrosyl-tRNA synthetase (MjTyrRS) library
were performed, as described previously.[18] A TF-specific
TyrRS mutant, termed TFRS, was identified. To further verify
that TF was incorporated into the target protein with high
efficiency and fidelity, an amber stop codon was substituted
into GlyOx and TrpOx. Mutant GlyOx and TrpOx expression
Tyr
were carried out with TFRS and Mj tRNACUA in the
presence of 1 mm TF, or in the absence of TF as a negative
control. Analysis of the purified GlyOx-266TF and TrpOx-
395TF by sodium dodecyl sulphate gel electrophoresis
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of 300 mV, it is not practical to perform
selective Trp measurement at this potential
in biological systems because of strong
interference. We demonstrated the detec-
tion of Trp at a much lower potential
by TrpOx-395TF-Bodipy373-wired CNT-
modified glassy carbon electrode (TrpOx-
395TF-Bodipy373/CNT/GCE). As shown
in Figure 3D, a significant increase in the
oxidative current was observed with a half-
wave potential of À94 mV upon the addi-
tion of 2 mm Trp. To exclude the possibility
that Bodipy373 served as a redox mediator,
the electrochemical signal of wt TrpOx in
the presence of Bodipy373- or Bodipy373-
modified electrode without enzyme modi-
fication was measured; no catalytic current
was observed (Supporting Information,
Figure S7). Moreover, the same TrpOx-
395TF mutant without Bodipy373 wiring to
the electrode (Figure 3C) showed negligi-
ble oxidative current upon Trp addition,
indicating that Bodipy373 wiring is
required for efficient electron transfer.
To investigate the origin of the oxida-
tion current, the absorption spectra of
TrpOx-395TF was measured before and
after addition of Trp substrate. A new
absorption peak at about 320 nm was
observed during the oxidation of Trp
(Supporting Information, Figure S8A), cor-
responding to formation of one-electron-
Figure 2. Genetic incorporation of TF into protein and site-specific modification using the
S-click reaction. A) A coomassie-blue-stained SDS-PAGE gel of GlyOx and TrpOx mutants
expressed in the absence or presence of TF (1 mm). B) A coomassie-blue-stained (top) and
fluorescence imagine (bottom) of SDS-PAGE gel of TrpOx-395TF and GlyOx-266TF mutants
incubated in the presence or absence of Bodipy373. C) MS spectra of TrpOx-395TF and
GlyOx-266TF.
(HOPG) surface (Figure 3A) was much lower and less
uniform than that of TrpOx-395TF-Bodipy373 (Figure 3B).
Quantitative analysis of the protein coverage was further
performed by using the Nanoscope IIIa software function;[19b]
the calculated result for physically adsorbed TrpOx-395TF
was 12.3%, which is much lower than the calculated value of
51.5% for TrpOx-395TF-Bodipy373. An absorption peak at
570 nm associated with the purified TrpOx-395TF-Bodipy373
complex was used to quantify the protein before and after
immobilization. The calculated amount of enzyme immobi-
lized on the HOPG electrode was 2.1 pmolcmÀ2 for TrpOx-
395TF-Bodipy373 complex and 0.52 pmolcmÀ2 for physically
adsorbed TrpOx-395TF. These results suggest, with the aid of
Bodipy373 linker as a “sticker” to the carbon surface, that the
modified enzyme has significantly increased surface coverage
on the HOPG surface. Molecular dynamics simulations were
performed to elucidate the binding mechanism between
Bodipy373 and CNT. The binding energy between
Bodipy373 and the CNT surface was calculated to be
À152 kJmolÀ1, which is stronger than that of pyrene
(À110 kJmolÀ1). The more precise PM6-DH + semiempirical
quantum mechanical method gave similar results (Supporting
Information, Figures S5 and S6).
reduced radical semiquinones (FAD·) in flavoproteins, as
Figure 3. AFM images of A) TrpOx-395TF absorbed on a HOPG sur-
face. B) TrpOx-395TF-Bodipy373 absorbed on a HOPG surface. Cyclic
voltammetry (CV) of C) TrpOx-395TF absorbed on CNT/GCE and
D) TrpOx-395TF-Bodipy373 absorbed on CNT/GCE before (red) and
after (black) the addition of Trp (2 mm). CV conditions: phosphate-
Having established site-specific wiring of TrpOx to the
electrode, we then performed real-time electrochemical
measurement of Trp. While Trp is an electrochemically
active amino acid that can be oxidized at an overpotential
buffered saline (PBS), pH 7.4, scan rate 5 mVsÀ1
.
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previously reported.[20] Low-temperature electron paramag-
netic resonance (EPR) measurements were also performed to
verify the presence of FAD·. The EPR spectra of TrpOx
during reaction with its substrate are presented in Figure S8B
(Supporting Information). The signal was centered at g =
2.0046 with a linewidth (peak-to-peak) of 11.2 Gauss, in
agreement with those reported for FAD·.[21] The reported
oxidation potential of FAD· was around À200 mv (vs.
standard calomel electrode (SCE)), which is close to the
observed oxidation potential in this study.[20] Therefore,
considering the inevitable protein barrier, and also the
voltage drop caused by the molecular bridge between the
enzyme and the electrode, the half-wave potential of À94 mV
indicates an efficient communication between the electrode
and the FAD center, which arises from the re-oxidation of
FAD·. Further electron transfer rate analysis and kinetic
study of the wired TrpOx mutants indicates the importance of
proper enzyme alignment on the electrode to favor electron
transfer (Supporting Information, Table S3). To illustrate the
generality of the approach, we show that Bodipy373 wiring is
required for efficient electron transfer between GlyOx-
266TF-Bodipy373 and the electrode (Supporting Informa-
tion, Figure S9D).
We then measured the amperometric response of the
TrpOx-395TF-Bodipy373/CNT/GCE for Trp, at an applied
potential of À0.1 V (Figure 4A). The dynamic range was
0.02–2.5 mm, and the linear range was 0.02–0.8 mm (Fig-
ure 4B). Compared with the current response to 30 mm of Trp,
signals corresponding to physiological levels of dopamine,
ascorbic acid, and uric acid (0.1 mm, respectively) were
negligible (Supporting Information, Figure S10). This anti-
interference ability was attributed to the low operating
potential that was applied for the selective detection of Trp.
Subsequently, we tested whether the TrpOx-395TF-
Bodipy373/CNT/GCE is capable of selective real-time mon-
itoring of Trp concentrations in blood. As shown in Figure 4C,
after adding 0.03 mm Trp to mice blood, a rapid current
increase was observed within 5 seconds. After injection of
IDO (a Trp degradation enzyme, 0.1 mm in PBS buffer) a fast
drop of steady-state current was observed, indicating that the
Trp concentration decreased. We then used TrpOx-395TF-
Bodipy373/CNT/GCE electrode to monitor the Trp concen-
tration in the HeLa cell culture. As shown in Figure 4D, in the
absence of IFN-g, the Trp concentration dropped slowly
during the growth stage from 48–54 h. However, in the
presence of 50 ngmLÀ1 IFN-g, more IDO was expressed in
HeLa cells and a significant decrease in Trp concentration was
observed.
Figure 4. A) Amperometric response of TrpOx-395TF-Bodipy373/CNT/
GCE upon addition of Trp; applied potential À0.05 V in pH 7.4 PBS
buffer. B) The steady-state currents versus Trp concentration. C) The
Trp concentration measurement in vitro, and after the injection of Trp,
Tyr, and IDO. D) Real-time and on-line Trp concentration measurement
during HeLa cell growth, from 48–54 h; blank culture media (blue
dot), HeLa cells in the absence of IFN-g stimulation (black dot), HeLa
cells after 50 ngmLÀ1 IFN-g stimulation for 24 h (red dot). Prior to
measurement, the culture media were renewed by a RPMI 1640 growth
medium with a Trp concentration adjusted to 60 mm.
In conclusion, we have demonstrated an efficient and
biocompatible approach for wiring redox enzymes site-
specifically to carbon-based electrodes using an S-click
reaction. This approach can be applied to a wide range of
redox enzymes, thereby leading to a much improved enzyme–
electrode communication. This method will bring bio-
electrochemical systems closer to real-world applications
and facilitate a deeper understanding of redox enzyme/
electrode interactions.
Acknowledgements
We are grateful for the financial support from the National
Key Research and Development Program of China
(2016YFA0501502), the National Natural Science Foundation
of China (21750003 to J.W.; U1532150, 91640106, and
31971200 to F.L.; 21503268 to M.-J.H.), and the Sanming
Project of Medicine in Shenzhen (No. SZSM201811092).
GlyOx-266TF-Bodipy373/CNT/GCE and TrpOx-395TF-
Bodipy373/CNT/GCE sensors were then used to measure
glycine and Trp concentrations in human sweat and blood
samples. As shown in Tables S4 and S5 (Supporting Informa-
tion), all the measured values from amperometry have a good
linear correlation to the reference value obtained by LC-MS
(Supporting Information, Figure S12). Considering the sim-
plicity and rapidity of amperometry measurement, our
approach demonstrates great potential in human sweat and
blood sample analysis and real-time health status monitoring.
Conflict of interest
The authors declare no conflict of interest.
Keywords: amino acids · biosensors · electrochemistry ·
electron transfer · genetic code expansion
4
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Manuscript received: July 23, 2019
Revised manuscript received: August 26, 2019
Version of record online: && &&, &&&&
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Communications
Enzyme–Electrode Interfaces
L. Xia, M.-J. Han, L. Zhou, A. Huang,
Z. Yang, T. Wang, F. Li, L. Yu, C. L. Tian,
Z. Zang, Q.-Z. Yang, C. Liu, W. Hong,
Y. Lu, L. Alfonta, J. Wang*
&&&& — &&&&
S-Click Reaction for Isotropic Orientation
of Oxidases on Electrodes to Promote
Electron Transfer at Low Potentials
Blood, sweat & Trp : Electrochemical
sensors are essential for point-of-care
testing and wearable sensing devices.
Efficient electron transfer from a redox
enzyme to an electrode is established by
wiring the two together with S-click
chemistry. These biosensors are sensitive
and selective for tryptophan and glycine
in blood and sweat. Key: l-tryptophan
oxidase (TrpOx), boron-dipyrromethene
(Bodipy), reduced semiquinone in flavo-
protein (FAD·).
6
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Angew. Chem. Int. Ed. 2019, 58, 1 – 6
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