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Sensors and Materials, Vol. 34, No. 6 (2022) 2105–2124
MYU Tokyo
S & M 2959

A Thiol-reactive Phenazine Ethosulfate – A Novel RedoxMediator for Quasi-direct Electron-transfer-type Sensors
Maya Fitriana,1,2 Kentaro Hiraka,1,3 Kazunori Ikebukuro,1Koji Sode,4 and Wakako Tsugawa1*
1Department of Biotechnology and Life Science, Graduate School of Engineering,Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan

2Department of Biotechnology, Faculty of Life Sciences and Technology, Sumbawa University of Technology,

Jl. Raya Olat Maras, Batu Alang, Moyo Hulu 84371 Sumbawa Besar, Indonesia
3College of Science, Engineering, and Technology, Grand Canyon University,
3300 W Camelback Rd, Phoenix, AZ 85017, USA
4Joint Department of Biomedical Engineering, The University of North Carolina
at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA(Received March 22, 2022; accepted May 17, 2022)

Keywords: thiol-reactive phenazine ethosulfate, quasi-direct electron transfer, redox probe, enzyme
modification, lactate oxidase, electrochemical enzyme sensor, biomedical engineering

A novel redox mediator, thiol-reactive phenazine ethosulfate (trPES), was used to modify an
enzyme for the first time for biosensor development. Aerococcus viridans-lactate oxidase (LOx),
widely used to study lactate biosensors, was modified with a single trPES molecule. A cysteine
mutation was introduced into the vicinity of the LOx cofactor to enable the modification by
trPES. LOx cysteine mutants were then successfully modified using trPES, thus acquiring quasidirect electron transfer ability. An electrode immobilized with the trPES-LOx A96L/S210C
mutant showed the highest amperometric response currents among the modified LOx cysteine
mutants, indicating efficient electron transfer. The position around residues S210 to N212 on the
LOx surface (distance <14 Å from FMN N5) is important for the mediator to access the reduced
flavin. Then, the performances of the lactate sensor were improved by utilizing LOx A96L/
S210C modified with trPES and arPES, another redox mediator used to modify a lysine residue.
The lactate sensor has a detection range of up to 1 mM, a sensitivity of 6.62 µA/mM.cm2, and alimit of detection of 9.9 µM. Furthermore, Aspergillus f lavus-derived FAD glucose
dehydrogenase was successfully modified with trPES and the response currents were obtained,showing the versatility of trPES for modifying other oxidoreductases.

1.

Introduction

Modification of proteins and peptides using chemical probes is a general experimental
technique used to functionalize these molecules. The modification is performed to analyze thefunctions and dynamics of proteins and peptides, and also to develop biosensing systems using a
combination of spectroscopic or image analyses and/or electrochemical analyses. Similarly to*Corresponding author: e-mail: tsugawa@cc.tuat.ac.jp

https://doi.org/10.18494/SAM3898

ISSN 0914-4935 © MYU K.K.
https://myukk.org/

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Sensors and Materials, Vol. 34, No. 6 (2022)

other chemical probes in general, proteins and peptides are covalently modified by redox probes
directly via amine or thiol groups. The established modification processes are simple because
the chemical probes have functional groups such as (1) the succinimidyl group for targeting
primary amine groups at the side chain of lysine residues(1–18) and the N-termini in proteins andpeptides; or (2) the maleimide group for targeting the thiol group in cysteine residues.(19–23)
A variety of redox probes with different redox potentials have been used as redox mediators
to develop biosensing systems. Most studies on protein modification using redox probes targeted
the amine group of lysine residues of oxidoreductases. In those studies, proteins were modified
with derivatives of ferrocene,(1–8) a ruthenium complex,(2) tetrathiafulvalene,(9) phenothiazine,(10,11)
phenoxazine,(12) quinone,(13) and, as recently reported by our group, phenazine.(14–18) In contrast,
studies of redox probes targeting the thiol group of cysteine residues have been limited to themodification of binding proteins with a ruthenium complex,(19–23) and no studies on themodification of oxidoreductases with a redox mediator targeting the thiol group of cysteine havebeen reported.
Protein modification targeting lysine residues is used for the modification of proteins withmultiple redox molecules,(1–18) and protein modification targeting cysteine residues is used forthe modification with single molecules.(19–23) This is because lysine has a high prevalence inproteins (~6%),(24,25) whereas cysteine has a low prevalence (<2%).(26) The modification withmultiple redox molecules is generally used to facilitate the direct communication of enzymes
and an electrode for the construction of reagentless biosensors. However, the modified proteins
have not been analyzed in detail because of the complexity of analyzing a large number ofattached redox molecules in the proteins. For the detailed analysis of protein functions,bioelectrochemical analyses of redox enzymes, protein dynamics studies, biosensing, andmodification with a single redox molecule at the desired position using a redox probe targeting
cysteine can be used.
Oxidoreductases harboring a flavin cofactor require electron mediators to transfer electrons
to the electrode because flavin is buried inside the enzyme. Therefore, many studies on thedevelopment of biosensors using oxidoreductases in mediated electron transfer (MET)-type
biosensors have been reported. However, the ideal electron transfer system for bioelectrochemicalsensing is the direct electron transfer (DET) type, in which electrons released by substrateoxidation are directly transferred from flavin to the electrode, but this is not feasible foroxidoreductases harboring a flavin cofactor without the presence of an electron mediator. The
mediator can enter the cavity of the enzyme and reach the flavin, then be re-oxidized at theelectrode, thereby mediating electron transfer from flavin to the electrode.
Our research group has reported the application of a novel phenazine derivative redox probe,
1-[3-(succinimidyl oxycarbonyl) propoxy]-5-ethyl phenazinium trifluoromethane sulfonate oramine-reactive phenazine ethosulfate (arPES), with proteins.(14–18) Phenazine derivatives are
suitable mediators for biosensing applications because their redox potential is sufficiently low to
avoid the appearance of bias signals originating from redox substance interference. Phenazine
ethosulfate (PES), one of the derivatives of phenazine and the redox component of arPES, hashigh stability, (27–31) and its versatility has been shown for oxidoreductase-based
biosensors.(31) Through its succinimidyl group, arPES can modify the amine group of lysine inSensors and Materials, Vol. 34, No. 6 (2022)

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oxidoreductases and provide quasi-direct electron transfer (quasi-DET) to the enzyme.(32) Enzyme
modification with this commercially available redox mediator allows electron transfer fromflavin to the electrode via the attached arPES. By immobilizing the modified enzyme on theelectrode, the substrate can be monitored by electrochemical analysis such as chronoamperometry
without the addition of a free mediator.(14–17) The application of arPES led to the successful
development of a glucose sensor based on flavin adenine dinucleotide (FAD)-dependent glucose
dehydrogenase derived from Botryotinia fuckeliana (BfGDH);(14) however, no amperometricresponse was observed when using Aerococcus viridans-lactate oxidase (AvLOx or LOx)
modified by arPES.(14,15) Some lysine residues in BfGDH might be located at a suitable position
to mediate electron transfer from flavin to the electrode, thus enabling amperometric response
currents to be observed. The position of the lysine residue in BfGDH, which has a major impact
on electron transfer from the reduced flavin, cannot be determined. Suzuki et al.(16) predicted
the position of this lysine residue. Then, Hiraka et al.(15) introduced a lysine residue near theentrance to the active site on a LOx surface, and a lactate sensor was successfully developed
using an arPES-modified LOx lysine mutant. In this case, multiple arPES molecules wereattached and the response currents observed were based on the electrons mediated by these
multiple arPES molecules. Here, the response currents mediated by a single arPES molecule
attached on the introduced lysine cannot be determined.
Recently, another novel phenazine derivative redox probe, 1-[3-(2-maleimidoethyl carbamoyl)
propoxy]-5-ethyl phenazinium triflate or thiol-reactive phenazine ethosulfate (trPES) (Scheme1), has become commercially available.(33) Because trPES contains a maleimide group, it can beused to modify cysteine residues in proteins, thus enabling the modification of an enzyme with asingle trPES. In 1993, Badia et al.(6) reported that the locations of a few key ferrocene groups
attached to the enzyme in the vicinity of the cofactor and also the enzyme surface are critical forelectrocatalytic activity, rather than the number of ferrocene groups loaded onto the enzyme.
However, the modification of a protein with a single redox mediator molecule targeting cysteinein oxidoreductases, including the application of trPES for this purpose, has not been reported.
In this study, we demonstrated the application of the novel redox mediator trPES for targeting
cysteine in well-known oxidoreductases, LOx, and we characterized the impact of themodification of the redox enzyme with a single redox mediator molecule near the cofactor.
AvLOx is widely used in the development of l-lactate biosensors. AvLOx harbors the flavinmononucleotide (FMN) as the cofactor and catalyzes the oxidation of l-lactate. Since AvLOx
does not have a cysteine residue, a single cysteine mutation enables one-point modification of
the enzyme with trPES. The results obtained in this study were compared with reported studies

Scheme 1.

Structure of trPES.

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Sensors and Materials, Vol. 34, No. 6 (2022)of the modification of AvLOx with multiple redox mediator molecules of arPES. Following thecommon strategy of modifying proteins with a single redox molecule at a specific position bysite-directed mutagenesis, a cysteine residue was introduced near the active site at the enzyme
surface, which allowed quasi-DET via the attached trPES. This strategy is expected to clearlyshow the impact of attaching a single redox mediator at a specific position in an enzyme.
Furthermore, Aspergillus flavus-derived FAD GDH (AfGDH) was modified with trPES toevaluate the versatility of trPES for modifying other oxidoreductases.

2.

Materials and Methods

2.1

Materials and devices

trPES and arPES were provided by Dojindo Laboratories Inc. (Kumamoto, Japan). Sodium
L-lactate and poly(ethylene glycol) diglycidyl ether (PEGDGE Mn 500) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). 2,6-Dichloroindophenol (DCIP) was purchased from
Merck (Darmstadt, Germany). 5-Methyl-phenazinium methylsulfate (PMS) and D(+)-glucose
were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). An Amicon Ultra-0.5 centrifugal
filter unit (30-KDa cutoff) was purchased from Merck Millipore Ltd. (Carrigtwohill Co., Cork,
Ireland). A glassy carbon (GC) working electrode (WE, 3.0 mm diameter), silver/silver chloride(Ag/AgCl) glass reference electrode (RE), and electrode polishing kit (consisting of 1 µm
polishing diamond and 0.05 µm polishing alumina) were purchased from BAS Inc. (Tokyo,
Japan). A platinum (Pt) wire counter electrode (CE) was purchased from Tanaka Kikinzoku
Kogyo K.K. (Tokyo, Japan). All other chemicals were of reagent grade. All solutions wereprepared using pure water from a Milli-Q water purification system (Millipore Corporation,
Burlington, MA, USA).
A 3D structure analysis of AvLOx was performed using PyMOL Molecular Graphics System
software. Colorimetric evaluations were performed using a UV-1289 UV-Vis spectrophotometer(Shimadzu Corp., Kyoto, Japan). Enzymes were incubated with trPES and/or arPES in an
Eppendorf ThermoMixer R (Thermo Fisher Scientific, Tokyo, Japan). All electrochemicalmeasurements were performed using a PG580RM potentiostat-galvanostat from UniScan
Instruments Ltd. (Buxton, UK).
2.2

Preparation of engineered LOx mutants

A cysteine mutation was introduced in LOx A96L, a mutant with low reactivity toward
oxygen,(34) through site-directed mutagenesis (Supplementary Information Table S1).
Escherichia coli (E. coli) BL21 (DE3) was used for LOx engineering following the methods of
Hiraka et al.(15) The recombinant enzymes were prepared according to the methods of Hiraka etal.(34) with minor modifications.(31) Protein concentration was measured using the Bradford
method.
The dye-mediated dehydrogenase activity of LOx mutants was measured using the PMS/DCIP system. Each enzyme sample was mixed with a premix solution containing (final
concentrations) 4 mM PMS, 0.06 mM DCIP, and 20 mM lactate in 20 mM potassium phosphateSensors and Materials, Vol. 34, No. 6 (2022)

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buffer (PPB, pH 7.0). DCIP reduction was determined by monitoring absorbance at 600 nm. One
unit of enzyme was defined as the amount of enzyme that catalyzes the reduction of 1 µmol
DCIP per min using 20 mM lactate at 25 ℃, based on the molar absorption coefficient at pH 7.0(16.3 mM−1cm−1). Kinetic parameters were calculated using the Hanes–Woolf plot. These assays
were performed in triplicate for each enzyme sample.
2.3

Modification of LOx mutants with redox probes

Purified LOx (0.55 µM) was mixed with trPES (80 µM) in 20 mM PPB (pH 7.0) andsubsequently incubated at 25 ℃ in a thermomixer with 1200 rpm shaking for 2 h. The unboundtrPES was removed by ultrafiltration at 14,000 g and 4 ℃ for 5 min. The centrifugation was
repeated 10 times with 20 mM PPB (pH 7.0) added to obtain trPES-modified LOx (trPES-LOx).
LOx mutants were modified with both trPES and arPES. A mixture containing LOx (0.55µM), trPES (80 µM), and arPES (400 µM) was incubated in 20 mM tricine buffer (pH 8.3). The
modification steps and other conditions were the same as for the above methods. The pH of the
mixture changed during the removal of the unbound mediators, and we obtained trPES- andarPES-modified LOx (tr.arPES-LOx) with neutral pH. LOx mutants were also modified with
arPES only(15) for comparison with other modified enzymes.
2.4

Construction and evaluation of enzyme electrodes utilizing modified LOx enzymes

GC electrodes were polished and sonicated in pure water prior to use. The modified enzymes
were immobilized through cross-linking with PEGDGE following the method of Vasylieva etal.(35) A mixture (6 µl) containing 1 mg/mL modified LOx and 10 mg/mL PEGDGE wasdeposited on the GC electrode layer by layer. The enzyme electrode was dried at 25 ℃ for 15
min before the next layer was added. After adding the last layer, the enzyme electrode was driedat room temperature (RT, around 25 ℃) with very low humidity (less than 1% relative humidity)
for 2 h. Three different electrodes were prepared for each modified LOx variant. The enzyme
electrodes were equilibrated in 20 mM PPB (pH 7.0) at RT for at least 15 min before use.
Cyclic voltammetry (CV) measurements were performed in 2 mL of 100 mM PPB (pH 7.0)
with a sweep range from −0.7 to +0.3 V (vs Ag/AgCl) and a scan rate of 0.05 V/s.
Chronoamperometry (CA) measurements were performed to monitor the response currents withthe successive addition of lactate (0–20 mM) under constant stirring and with an operating
potential of 0 V (vs Ag/AgCl) under atmospheric conditions. Finally, CV measurements in thepresence of lactate were performed to record the behavior of the enzyme electrodes. The
performances of the constructed biosensors such as sensitivity, range of linear detection, andlimit of detection (LOD) were determined following previous studies.(15,31)
2.5

Application of trPES for other enzymes

AfGDH (NCBI reference sequence: XP_002372599.1) and its mutant were also produced byalmost the same method as in a previous report.(36) The amino acid sequence of AfGDH withouta signal peptide was expressed using a plasmid (pET30C) to encode the AfGDH gene in E. coli

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BL21 (DE3) cells. A cysteine mutation was introduced into AfGDH K477 through site-directedmutagenesis (Supplementary Information Table S1). AfGDH wild-type (WT) or AfGDH K477C
mutant was modified with trPES by incubating trPES (80 µM) with the enzyme (1.33 µM) in 20
mM PPB buffer (pH 6.5) following the methods described in Sect. 2.3. Enzyme electrodes basedon modified trPES-AfGDH WT or trPES-AfGDH K477C were prepared according to themethods described in Sect. 2.4 and the electrochemical response current toward glucose was
evaluated in 20 mM PPB buffer with pH 6.5.

3.

Results

3.1

Design, construction, and characterization of LOx cysteine mutants

A cysteine residue was introduced in LOx to enable the modification with trPES. The
residues to be substituted with cysteine were selected on the basis of two criteria:(15) (1) they
were located on the surface of LOx to provide accessibility to the electrode surface; (2) there wasa short distance between the residue (alpha carbon, Cα) and the isoalloxazine ring of FMN (N5)(<30 Å),(37) thus enabling optimal electron transfer from flavin to the electrode through theattached trPES. Five residues, V20, S178, V185, S210, and N212, were chosen for substitution(Fig. 1). Six LOx mutants were produced as recombinant proteins and purified: A96L/V20C,
A96L/S178C, A96L/V185C, A96L/S210C, A96L/N212C, and A96L, where A96L was used as acontrol.
The kinetic parameters of the LOx mutants were determined (Table 1). The Vmax values
indicated the dye-mediated dehydrogenase activity of all LOx mutants, suggesting that the LOx
mutants were in an active form. LOx A96L/S210C exhibited the highest activity (162% of thecontrol), whereas LOx A96L/N212C exhibited the lowest activity among the LOx mutants (38%of the control). In a previous study,(15) residues S178 and N212 were substituted with lysine to

(a)

(b)

Fig. 1. (Color online) Structure of Aerococcus viridans-lactate oxidase (AvLOx) (PDB code: 2E77) showing themutation sites of AvLOx A96L/cysteine mutants: (a) location of mutated residues at the active site entrance on the
enzyme surface and (b) native amino acid residues that were substituted with cysteine and their distance from theisoalloxazine ring of FMN (N5). 1: Valine20 (V20); 2: Serine178 (S178); 3: Valine185 (V185); 4: Serine210 (S210); 5:
Asparagine212 (N212). A96 (red letters): residue mutated to decrease the oxygen reactivity. FMN: AvLOx cofactor.
Pyruvate: product of L-lactate oxidation catalyzed by LOx. All residues in the model are in chain D (light greenarea) of the homo-tetrameric AvLOx, except V20, which is located in chain B (pink area).

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Table 1
Kinetic parameters of LOx mutants before and after modification with trPES.
LOx mutants
A96L
A96L/V20C
A96L/S178C
A96L/V185C
A96L/S210C
A96L/N212C

Kinetic parameters
Vmax (U/mg)
Km (mM)
LOx
trPES-LOx
LOx
trPES-LOx
98 ± 650 ± 30.79 ± 0.10
0.44 ± 0.06
74 ± 433 ± 20.63 ± 0.03
6.80 ± 0.60
78 ± 35±10.60 ± 0.03
2.58 ± 0.40
118 ± 985 ± 30.73 ± 0.11
1.19 ± 0.10
159 ± 176 ± 1
1.26 ± 0.12
2.50 ± 0.28
37 ± 322 ± 51.20 ± 0.08
1.34 ± 0.54

design the arPES modification site, resulting in inactivated LOx A96L/SI78K but high enzyme
activity in LOx A96L/N212K. The cysteine mutation in this study appeared to markedly
decrease the enzyme activity in LOx A96L/N212C. In addition, all LOx cysteine mutants
showed very low oxidase activity (Supplementary Information Fig. S1) because they maintained
the characteristic of the A96L mutation, which exhibited low reactivity to the molecularoxygen,(15) making them suitable for MET- and quasi-DET-type sensor applications.
3.2

Catalytic activity of LOx cysteine mutants after modification with trPESThe LOx mutants were modified by incubation with trPES. The modified trPES-LOx mutants
were expected to have one molecule of trPES covalently attached to each monomer of the homotetrameric LOx, except for trPES-LOx A96L, which lacks a cysteine residue. The kinetic
parameters of the trPES-LOx mutants were determined (Table 1). All modified enzymes retained
dye-mediated dehydrogenase activity, as shown by their Vmax values, although all the Vmax
values after modification with trPES decreased to 72% of the values before modification or
lower, and the Km values were also changed by the modification. trPES-LOx A96L/V185C
showed the highest activity and trPES-LOx A96L/S178C showed the lowest activity. All Km
values after modification increased, except for trPES-LOx A96L, for which it decreased almost
to half of that before modification.
3.3

Electrochemical evaluation of enzyme electrodes using LOx modified with single
mediator molecule

3.3.1 Cyclic voltammetry
The enzyme electrodes constructed using the trPES-LOx mutants were evaluated by CVmeasurement (Fig. 2). All cyclic voltammograms showed redox peaks in both the absence andpresence of lactate. These redox peaks are attributed to trPES because they are similar to theredox peaks of the trPES solution (Supplementary Information Fig. S2). This confirms that trPES
attached on LOx and that the modified enzymes were electrochemically active. The midpoint
potentials (E1/2) were approximately −0.10 V (vs Ag/AgCl), as calculated from the oxidation andreduction peaks, which were observed at approximately −0.06 and −0.14 V, respectively.

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(a)

(b)

(c)

(d)

(e)

(f)

Fig. 2. (Color online) Cyclic voltammograms of modified enzymes: (a) trPES-LOx A96L, (b) trPES-LOx A96L/
V20C, (c) trPES-LOx A96L/S178C, (d) trPES-LOx A96L/V185C, (e) trPES-LOx A96L/S210C, and (f) trPES-LOx
A96L/N212C in 2 ml of 100 mM PPB (pH 7.0) in the absence (dashed line) and presence (solid line) of lactate. Scan
rate: 0.05 V/s.

Catalytic currents were observed in the presence of lactate, suggesting that the electrons weretransferred from the attached trPES to the electrode upon substrate oxidation. trPES-LOx A96L/
V185C [Fig. 2(d)], trPES-LOx A96L/S210C [Fig. 2(e)], and trPES-LOx A96L/N212C [Fig. 2(f)]Sensors and Materials, Vol. 34, No. 6 (2022)

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clearly showed high catalytic current. Therefore, CA measurement was carried out and thelactate concentration dependence was evaluated for each modified LOx mutant.
3.3.2 Chronoamperometry
CA measurements were performed to evaluate the electron transfer from flavin to theelectrode through the attached trPES. The response currents were recorded with the successive
addition of 0–20 mM lactate in a buffer solution without a free mediator [Fig. 3(a)]. The steadystate currents obtained with each addition of lactate were plotted against lactate concentration[Fig. 3(b)].
The enzyme electrodes utilizing trPES-LOx A96L/V20C, trPES-LOx A96L/V185C, trPESLOx A96L/S210C, and trPES-LOx A96L/N212C clearly showed response currents during theaddition of lactate. In contrast, trPES-LOx A96L/S178C and the control, trPES-LOx A96L, did
not show response currents. The observed response currents in the trPES-LOx A96L/cysteine
mutants increased with increasing lactate concentration. The highest response current wasobserved with trPES-LOx A96L/S210C. The response currents in trPES-LOx A96L/V20C,
trPES-LOx A96L/V185C, and trPES-LOx A96L/N212C were approximately 16, 24, and 35%,
respectively, of that observed in trPES-LOx A96L/S210C (calculated from Imax). The response
currents suggested that when lactate was oxidized, the electrons were successfully transferred tothe electrode surface through the attached trPES on the LOx cysteine mutants, indicating
successful quasi-DET in the trPES-LOx A96L/cysteine mutants. Although the dye-mediateddehydrogenase activity of trPES-LOx A96L/S210C was 89% of that obtained by trPES-LOx
A96L/V185C, trPES-LOx A96L/S210C exhibited a higher response current than trPES-LOx
A96L/V185C. In this case, the observed response current depends on the electron transfer abilityof the modified enzymes, not on the enzyme activity, and the optimum electron transfer occurs
in trPES-LOx A96L/S210C. Residue S210 in LOx is thus probably the best position for themodification with trPES because it appears that residue S210 plays an important role in accessing
the electrons from flavin to transfer them to the electrode. In the case of trPES-LOx A96L/
N212C, the low enzyme activity of LOx A96L/N212C appears to be the reason for the low
response currents observed, because the enzyme activity of LOx A96L/N212C before and after

(a)

(b)

Fig. 3. (Color online) (a) Representative time course of response currents and (b) steady-state currents of thetrPES-LOx mutants at various lactate concentrations (n = 3). Applied potential: 0 V (vs Ag/AgCl).

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modification with trPES is approximately 23 and 29%, respectively, of that of LOx A96L/S210C(calculated from Vmax). The low enzyme activity after modification with trPES also resulted in
the low response currents of trPES-LOx A96L/V20C, and even trPES-LOx A96L/S178C showedno response. For trPES-LOx A96L, no response current was observed because no trPES was
attached near the isoalloxazine ring of FMN.
3.4

Response currents of LOx modified with multiple mediator molecules

To improve the performance of the lactate sensor, LOx A96L/S210C and the control mutant,
LOx A96L, were modified with both trPES and arPES, obtaining tr.arPES-LOx A96L/S210C
and tr.arPES-LOx A96L, respectively. The amperometric response of tr.arPES-LOx A96L/
S210C markedly increased to fivefold that of trPES-LOx A96L/S210C (Fig. 4). This is because
the number of attached PES in LOx A96L/S210C increased, resulting in an increased response
current. In contrast, the response current of tr.arPES-LOx A96L was 58% of that obtained for
trPES-LOx A96L/S210C, suggesting that although there are many arPES attached to LOx A96L,
a high response current can be achieved if at least one PES is attached in a position accessible forelectron transfer from flavin, and thus the electrons can be transferred to either the electrode oranother attached arPES. For comparison, LOx A96L and LOx A96L/S210C were also modified
with only arPES to obtain arPES-LOx A96L and arPES-LOx A96L/S210C, respectively. Bothshowed similar response currents of approximately 46% of that obtained by trPES-LOx A96L/
S210C. Such a low response current for arPES-LOx A96L was also observed in a previous
study.(15) This result supports the above assumption that PES should be attached to a crucial
position to achieve a high response current. The properties and performances of lactate sensors
were determined from the observed response currents (Table 2). As expected, the lactate sensor
based on tr.arPES-LOx A96L/S210C exhibited the best performances.

Fig. 4. (Color online) Steady-state response currents of LOx modified with multiple PES molecules at various
lactate concentrations (n = 3). Applied potential: 0 V (vs Ag/AgCl).

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Table 2
Properties of lactate sensors.
Sensitivity(µA/mM.cm 2)*
N.A.

Lactate sensor

Km (mM)

Imax (nA)trPES-LOx A96L

N.A.

N.A.

tr.arPES-LOx A96L

0.43

111

0.89

21.5

arPES-LOx A96L

0.23

89

0.82

22.8

trPES-LOx A96L/S210C

0.69

192

1.40

18.3

tr.arPES-LOx A96L/S210C

1.80

1000

6.62

9.9

arPES-LOx A96L/S210C

0.57

90

0.68

26.8

trPES-LOx A96L/N212C

0.86

68

0.42

27.5

*

Sensitivity for detection range up to 1 mM lactate			Km and Imax were determined using a Hanes–Woolf plot		

3.5

LOD (µM)
N.A.

LOD: Limit of detection
N.A.: Not available

Modification and electrochemical investigation of AfGDH modified with trPES

To evaluate the application of trPES for other enzymes, we modified AfGDH WT (control) or
AfGDH K477C mutant with trPES. Residue K477 was predicted as a suitable position to attach aredox mediator that could allow electron transfer from the cofactor to the electrode through theattached mediator.(16) As in the chosen residues in LOx for cysteine substitution, residue K477 isalso located close to the AfGDH cofactor, FAD, on the enzyme surface. AfGDH K477C mutant
has one cysteine residue located in the enzyme surface; thus, the same approach as for LOx is
acceptable. The response currents of modified trPES-AfGDH WT and trPES-AfGDH K477C
were obtained (Fig. 5). trPES-AfGDH K477C clearly showed a response current with the current
depending on the glucose concentration, whereas no response current was observed from trPESAfGDH WT. The results suggest that the attached trPES at residue K477 successfully mediatedthe electron transfer from the flavin to the electrode.

4.

Discussion

In this study, we modified LOx with trPES to achieve a quasi-DET enzyme. LOx does nothave a cysteine residue. Therefore, to enable the modification with trPES, a cysteine residue wasintroduced at the active site entrance on the LOx surface. Thus, the modified trPES-LOx
cysteine mutant should have one trPES molecule attached near the flavin and the electrode torealize electron transfer. Here, we show the clear impact of modification with a single mediatormolecule on a redox enzyme. The best location for the attachment of the redox mediator onoxidoreductases to enable electron transfer is then confirmed.
The oxygen-insensitive mutant, LOx A96L, a suitable enzyme for MET-type sensors,(34) wasused as a template to design LOx cysteine mutants. Five variants of LOx A96L/cysteine mutant
were prepared and all were active, although they exhibited various enzyme activities upon
cysteine substitution (Table 1). After chemical modification with trPES, the enzyme activity was
obtained, although the activity was decreased by the modification. In a previous study, residues
S178 and N212 were substituted with lysine to obtain LOx A96L/S178K and LOx A96L/N212K,

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(a)

(b)

Fig. 5. (Color online) (a) Representative time course of response currents and (b) steady-state currents of AfGDH
modified with trPES at various glucose concentrations (n = 3). Applied potential: 0 V (vs Ag/AgCl).

respectively.(15) LOx A96L/S178K lost its activity as a result of mutation,(15) whereas in thisstudy, LOx A96L/S178C maintained its activity. However, trPES-LOx A96L/S178C showed adrastic decrease in activity. In contrast to LOx A96L/N212K, which has been reported to exhibit
high enzyme activity,(15) the activity of LOx A96L/N212C in this study dramatically decreased
after mutation. A LOx cysteine mutant with high enzyme activity that is maintained after
modification with trPES was expected to show a high response current in an amperometricevaluation. For this, LOx A96L/V185C and LOx A96L/S210C are potentially suitable mutantsbecause they successfully maintained their high activity after modification with trPES (Table 1).
Additionally, the differences in the Km values of all LOx cysteine mutants before and after
modification might have been caused by the modification with trPES, which might alter theaccessibility of the substrate (lactate) to the active center of enzymes.(16)The LOx mutants were successfully modified with trPES because redox peaks attributed to
trPES were observed in the cyclic voltammograms of all trPES-LOx mutants. Because redox
peaks were also observed in trPES-LOx A96L even though this mutant does not possess acysteine residue, trPES might have attached on its surface during the incubation with the
enzyme. The modification with trPES was performed at neutral pH, which is the optimum
condition for the reaction of maleimide with a thiol group. However, the maleimide group is also
known to react slightly with the amino group at this pH.(25,38–42) Therefore, trPES might modify
not only the cysteine residue but also the lysine residue(s) on the LOx surface. This may occur inenzymes that possess multiple lysine residues on their surface such as LOx. Nevertheless, theamperometric results showed no response current for trPES-LOx A96L, indicating thatspontaneous modification of the lysine surface with trPES does not affect electron transfer fromflavin to the electrode. Therefore, trPES can be used to observe the impact of modification witha single trPES molecule on the active site entrance to enhance electron transfer, thus revealing
the best position for attaching the mediator on a LOx surface.
The response currents toward lactate were observed in CA, indicating that PES can accept
electrons from flavin and successfully transfer them to the electrode. Among the trPES-modified
enzymes, only trPES-LOx A96L/cysteine mutants showed response currents toward lactate,suggesting that only the trPES-modified cysteine residue took part in electron transfer. SinceSensors and Materials, Vol. 34, No. 6 (2022)

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flavin, the redox center of the enzyme, is deeply buried in the enzyme structure, the electrons
from the cofactor are rarely directly transferred to the electrode. To realize electron transfer, anelectron mediator is used as a relay. In this case, the electron transfer occurs in two
steps: (1) intramolecular electron transfer from the enzyme redox center to the electron relay and(2) transfer of electrons to the electrode.(1)In redox proteins, a high intramolecular electron transfer rate is achieved within a distance of
14 Å.(43) The distance between the isoalloxazine ring of FMN and trPES, which acts as theelectron relay center(1) in the modified trPES-LOx, can be assumed to be the distance from N5 offlavin to Cα of cysteine (Fig. 1). Since all the cysteine residues are close to flavin, all the trPESLOx A96L/cysteine mutants were expected to show electron transfer, especially trPES-LOx
A96L/N212C, because arPES-LOx A96L/N212K showed high response currents in a previous
study.(15) trPES-LOx A96L/N212C, trPES-LOx A96L/S178C, and trPES-LOx A96L/S210C were
expected to achieve higher response currents in the amperometric evaluation because thedistance between flavin and residues N212, S178, and S210 is shorter (<14 Å) than that between
flavin and residues V185 and V20.
On the other hand, considering that all the mutations were designed at residues located at the
enzyme surface, the electrons can be readily transferred from the relay to the electrode in all thetrPES-LOx mutants. In fact, only trPES-LOx A96L/S210C exhibited a high response current andshowed clear dependence to 0–20 mM lactate, which can be attributed to the highly efficient
intramolecular electron transfer in the modified enzyme. LOx A96L/N212C showed low
enzyme activity because of the impact of cysteine mutation; thus, trPES-LOx A96L/N212C
exhibited a low response current. trPES-LOx A96L/S178C showed no response current, mostly
because of the drastic decrease in the enzyme activity after modification with trPES. trPES-LOx
A96L/V185C exhibited a lower response current than trPES-LOx A96L/S210C, even thoughboth mutants showed equally high activity both before and after modification with trPES. Thismay be due to the longer distance of residue V185 from flavin (>14 Å), which decreases theelectron transfer rate.(2,44) The longer distance of residue V20 from flavin may also be the reason
for the low response currents in trPES-LOx A96L/V20C, in addition to their low activity after
modification with trPES.
Furthermore, LOx A96L/S210C was modified with trPES and arPES to increase the numberof PES molecules attached on LOx. For tr.arPES-LOx A96L/S210C, the response current was
fivefold that for trPES-LOx A96L/S210C. In this case, the response current was obtained fromcomplex electron transfer, which included intramolecular electron transfer as the first step of theprocess. Thus, the response currents in tr.arPES-LOx A96L/S210C obtained from intramolecularand complex electron transfer were in the ratio 1:4. To elucidate details of the complex electrontransfer, further in-depth study is necessary. Other modified enzymes with many attached PES
molecules, i.e., tr.arPES-LOx A96L, arPES-LOx A96L, and arPES-LOx LOx A96L/S210C,
exhibited similar response currents, which were lower than that obtained for trPES-LOx A96L/
S210C with a single attached PES molecule. These results support the previous statement (Sect.
3.4) that a high response current can be achieved if at least one PES molecule is attached in a
crucial position that enables electron transfer from the reduced flavin. Increasing the number ofattached PES molecules can thus increase the response current, as observed in tr.arPES-LOx

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A96L/S210C. Here, a lactate sensor with high response currents and markedly improved
performances was successfully developed using tr.arPES-LOx A96L/S210C (Table 2). For thesame detection range (up to 1 mM lactate) as the reported lactate sensor using arPES-LOx
A96L/N212K,(15) the LOD was improved to less than 9.9 µM, suggesting that our lactate sensor
can be used to detect such low lactate concentrations. Although the sensitivity of the lactate
sensor (6.6 µA/mM.cm2) is only half that of the sensor reported in Ref. 15, because lactate canbe detected at low concentrations owing to the low LOD, this lactate sensor can be used withconfidence.
We found that residue S210 is the best position for modification with trPES because highelectron transfer was achieved using the LOx modified with trPES attached to the residue. In aprevious study, residue N212 was assumed as the best position for attaching arPES.(15) In the 3D
structure of LOx, residue S210 is located next to residue N212 at the active site entrance on the
LOx surface, and both residues are located at a similar distance from flavin (<14 Å) (Fig. 1).
Combining our findings and the result of a previous study showing that residue N212 isimportant for electron transfer accessibility,(15) we conclude that the positions of residues S210 toN212 are important for mediator access. When PES is attached at a position near residues S210
to N212 on LOx, the PES has high accessibility to electrons from the reduced flavin; thus, highly
efficient electron transfer can be achieved.
In addition, AfGDH, a representative FAD-dependent oxidoreductase, was modified with
trPES and its electrochemical performances were evaluated to demonstrate the versatility of
trPES. Response currents were successfully observed with trPES-AfGDH K477C, suggesting
that, with a similar approach, trPES can be applied to other redox enzymes and thus to otherproteins. On the basis of our structural analysis, FAD (N5) and residue K477 (Cα) are separated
by a distance of 21.8 Å. This relatively long distance may be the reason for the response current
observed in trPES-AfGDH K477C being lower than that observed in trPES-LOx A96L/S210C.
However, this distance is still in the range of long-distance electron tunneling in proteins (<30Å); thus, electron transfer can occur from the enzyme cofactor to the electrode.(37) This distanceis similar to the distance between FAD (N5) and residue I489 (Cα) in AnGOx (21.7 Å), a crucial
residue for mediator access in AnGOx.(16) These results indicate that quasi-DET is possible in a
modified enzyme-redox probe provided the mediator is attached within an acceptable distance
range and in an accessible position for electron transfer.
Initial studies on protein modification using redox probes mostly targeted the amine group of
the lysine residue, and they were performed without considering protein structural analysis(1–5,7)
because the protein crystal structures were not known at that time. Hence, the modification of aprotein with a single redox probe molecule was not possible. In 1993, Badia et al. reported theuse of a ferrocene derivative as a redox probe to modify the amine group of lysine residues in
AnGOx by considering the X-ray structure, which had been reported in the previous year.(6) The
authors stated the importance of a few key ferrocene groups being attached to the enzyme close
to the enzyme cofactor and the protein surface for electrocatalytic activity, rather than thenumber of ferrocene groups attached to the enzyme. This was one of the most importantconsiderations in designing our study for the modification of oxidoreductases with a single
redox probe molecule using trPES.

Sensors and Materials, Vol. 34, No. 6 (2022)

2119

The immobilized lactate sensors can be used in a continuous monitoring system such as forflow injection analysis, as indicated in Ref. 17, and the use of quasi-DET enzymes based on
modified enzyme-redox probes could prevent the leakage of mediators. However, someconsequences of applying redox probes for enzyme modification may occur, such as decreased
enzyme activity due to enzyme mutation or chemical modification.(1,2,6,11,16) Therefore, toanticipate any decrease in enzyme activity, the enzyme should have high activity before
modification with redox probes so that the activity is not reduced after enzyme modification,
enabling appropriate response currents to be achieved in amperometric evaluation.(14)The application of trPES can be expanded to a wider range of applications inbioelectrochemical analysis to develop better electrochemical sensor systems and to analyze thefunctions and dynamics of proteins, peptides, and nucleotides. trPES can be applied to otherredox enzymes whose structures are well characterized. For enzymes that already possess native
cysteine residues on their protein surface, it may be necessary to substitute these cysteines withother residues to introduce a new cysteine residue in the selected position to attach the trPES.
trPES can also be conjugated with other proteins and peptides, as shown previously for binding
proteins(19–23) or nucleotides in recent studies on the application of arPES to aptamer-based
sensors.(45,46) trPES is an alternative to the only previously reported redox probe targeting
cysteine, a ruthenium complex derivative.(19–23) This is because both trPES and the ruthenium
complex derivative have low redox potential,(19,33) and some redox enzymes were reported not toreact with hexaammine ruthenium(III) because of its cationic property.(47–49) In addition, the useof the rare metal ruthenium raises problems related to sustainable usage. Therefore, the organic
compound trPES is more suitable for large-scale and continuous applications. Moreover, trPES is
now easy to obtain because it has become commercially available.

5.

Conclusions

In this study, a novel and now commercially available redox probe, trPES, was applied for the
first time for the development of a quasi-DET-type lactate sensor. LOx, a well-characterized
redox enzyme that is widely used to study lactate sensors, was successfully modified with trPES
to produce a quasi-DET enzyme. The maleimide group of trPES was covalently bound to thethiol group of the cysteine residue in LOx after introducing a cysteine residue near the LOx
cofactor FMN at the entrance to the active site on the LOx surface. Because native LOx does nothave cysteine, the LOx cysteine mutant was modified with trPES to attach a single trPES
molecule at the selected position on LOx. One of the modified enzyme variants, trPES-LOx
A96L/S210C, exhibited high response currents in an amperometric evaluation, indicating theefficient transfer of electrons from flavin to the electrode through the attached trPES. This result
suggests that the modified enzyme was successfully immobilized to realize a lactate sensor.
Highly efficient electron transfer was observed in the LOx cysteine mutant with cysteine
introduced at a residue with distance <14 Å from FMN (N5). A high response current can beachieved provided the LOx cysteine mutant maintains its dye-mediated dehydrogenase activity
after mutation and after modification with trPES. Here, we found that the position aroundresidues S210 to N212 on the LOx surface [distance <14 Å from FMN (N5)] is important to2120

Sensors and Materials, Vol. 34, No. 6 (2022)

ensure the accessibility of the mediator to the reduced flavin. The attachment of a single PES
molecule at these positions resulted in highly efficient electron transfer from the reduced flavinto the electrode. Furthermore, we increased the number of attached PES molecules by modifying
LOx A96L/S210C with trPES and arPES, which are redox mediators used to modify the lysine
residue of proteins, to obtain tr.arPES-LOx A96L/S210C. The response current of the enzyme
electrode based on tr.arPES-LOx A96L/S210C was fivefold that of trPES-LOx A96L/S210C,
thus improving the performances of the lactate sensor. The lactate sensor had a detection rangeof up to 1 mM with a sensitivity of 6.62 µA/mM.cm2 and a LOD of 9.9 µM. Finally, theversatility of trPES for another redox enzyme was evaluated. Following the same strategy,
AfGDH and the mutant AfGDH K477C were successfully modified with trPES to obtain response
currents, indicating that trPES can be applied to other oxidoreductases. AfGDH has one cysteine
residue buried in the enzyme structure; thus, the same approach as that for LOx, which does nothave a cysteine residue, is acceptable. For enzymes that already possess native cysteine residues
on their protein surface, it may be necessary to substitute these cysteines with other residues tointroduce a new cysteine residue in the selected position to attach trPES. We reported theapplication of the new material trPES as a redox chemical for biological materials. The study canbe expanded by applying trPES to other proteins, peptides, or nucleotides for its wider applicationin sensing systems and bioelectrochemical analyses.

Acknowledgments
The authors thank Dojindo Laboratories for their kindness in donating trPES and Mr. Yuma
Hatano for the construction of the AfGDH K477C mutant. The first author (M.F.) acknowledges
the Indonesia Endowment Fund for Education, Ministry of Finance of Indonesia (LPDP RI), forproviding the scholarship.

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About the Authors
Maya Fitriana received her B.S. degree from the State University of Malang,Indonesia, in 2010. Since 2013, she has been an instructor at Sumbawa
University of Technology, Indonesia. Since 2016, she has been a Ph.D. student
at Tokyo University of Agriculture and Technology, Japan. Her researchinterests are in bioengineering, biomolecular engineering, and biosensors forbiomedical applications.
Kentaro Hiraka received his B.S., M.S., and Ph.D. degrees from Tokyo
University of Agriculture and Technology, Japan, in 2016, 2018, and 2021,
respectively. His research interests are in bioengineering, biomolecularengineering, and biosensors for biomedical applications. Currently, he is doing
postdoctoral research in the College of Engineering, Science, and Technology,
Grand Canyon University, USA.
Kazunori Ikebukuro received his B.S., M.S., and Ph.D. degrees from
University of Tokyo, Japan, in 1989, 1992, and 1996, respectively. Since 2009,he has been a professor at Tokyo University of Agriculture and Technology,
Japan. His research interests are in bioengineering, biosensors based on
aptamers and nucleic acids, epigenetic modification sensors, and evolutionary
molecular engineering for biomedical applications.
Koji Sode received his B.S., M.S., and Ph.D. degrees from Tokyo Institute ofTechnology, Japan, in 1983, 1986, and 1988, respectively. Since 2000, he has
been a professor at Tokyo University of Agriculture and Technology, Japan.
Since 2003, he has been a science and technology advisor at Ultizyme
International Ltd. Since 2018, he has been a distinguished professor at The
University of North Carolina, USA. His research interests are inbioengineering, biomolecular engineering, biosensors, and synthetic biology
for biomedical applications and bioprocesses.

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Wakako Tsugawa received her B.S. degree from Tokyo Institute ofTechnology, Japan, in 1987 and her Ph.D. degree from Tokyo University ofAgriculture and Technology, Japan, in 1998. Since 2005, she has been an
associate professor at Tokyo University of Agriculture and Technology. Her
research interests are in bioengineering, biomolecular engineering, andbiosensors for biomedical applications.

Appendix
Table S1
Primers used to design the cysteine mutants.
Enzymes
Forward primers*
Reverse primers*
AvLOx cysteine mutants––
A96L(34)
A96L/V20C
5’- A AG TACAT T G AT G T T t g c A ATAC T 5’- TA AGT C GTA AGTAT Tg c a A ACAT CTACGACTTA -3’
AATGTACTT -3’
A96L/S178C
5’ - T G A C T C A A C T G T T t g t G G A A A C 5’ - G G T C A C G G T T T C C a c a A A C A G T
CGTGACC -3’
TGAGTCA -3’
A96L/V185C 5’- A AC C GTGAC C GTGAT t gcA AGA AT 5’- A AC G A AT T TAT T C T Tg c a AT CAC G
AAATTCGTT -3’
GTCACGGTT -3’
A96L/S210C 5’- ACAG CAG A AG GTAT G t g t T TA A AC 5’- G TAG ATAT T G T T TA A a c a CATAC CAATATCTAC -3’
TTCTGCTGT -3’
A96L/N212C 5’- G A AG G TAT G T CAT TA t g c A ATAT C 5’- AG CAC C G TAG ATAT Tg c aTA AT G A
TACGGTGCT -3’
CATACCTTC -3’
AfGDH cysteine mutant
K477C
5’- G AG AC C t g t C CAG G C C T GT G AG AT 5’- GCCTGGacaGGTCTCCTTCGCGATC
CCCAGCTACCGCAGCGGATG -3’
AGTTTGTTCAGTGGTGCGC -3’*
Lowercase codons were the mutation sites.

Fig. S1. (Color online) Oxidase activity of LOx mutants measured in 20 mM potassium phosphate buffer (PPB; pH
7.0) containing 1.5 mM 4AA, 1.5 mM TOOS, 2 U/ml POD, and 20 mM L-lactate. The oxidase activity wasdetermined by monitoring the formation of quinone imine dye at 555 nm based on the molar absorption coefficient of
TOOS at pH 7.0 (39.2 mM−1 cm−1). One unit (U) of enzyme activity is defined as the amount of enzyme thatcatalyzes the production of 1 µmol H2O2 per min using 20 mM lactate at 25 ℃. 4AA: 4-aminoantipyrine. TOOS:
3-(N-ethyl-3-methylanilino)-2-hydroxy-propanesulfonate. POD: peroxidase. n = 3.

All LOx cysteine mutants showed very low oxidase activity compared with the LOx wild
type.(34,47) They maintained the characteristic of the A96L mutation of low reactivity tomolecular oxygen;(34) thus, they are suitable for MET- and quasi-DET-type sensor applications.

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Fig. S2 (Color online) Cyclic voltammograms of 0.1 mM trPES in 100 mM PPB (pH 7.0). Scan rate: 0.1 V/s.
Potential range: −0.6–+0.6 V (RE: Ag/AgCl; WE: GC; CE: Pt wire).

The oxidation and reduction peak potentials (Epc: cathodic peak potential and Epa: anodic
peak potential) were observed at approximately −0.13 and −0.19 V (vs. Ag/AgCl), respectively;thus, the calculated midpoint potential (E1/2) is −0.16 V. These redox behaviors are similar to
those observed in other PES derivatives: arPES, which was used to modify AnGOx,(16) andmPES.(31) A potential higher than E1/2 was applied in the CA evaluation of the modified
electrodes utilizing modified trPES-enzymes.