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Sensors and Materials, Vol. 29, No. 12 (2017) 1703–1711
MYU Tokyo
S & M 1463

Characterization of Electron Mediator Preference of
Aerococcus viridans-Derived Lactate Oxidase for Use in
Disposable Enzyme Sensor Strips
Noya Loew,† Maya Fitriana,† Kentaro Hiraka, Koji Sode,1 and Wakako Tsugawa*
Department of Biotechnology and Life Science, Graduate School of Engineering,Tokyo University of Agriculture and Technology,
2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
1
Institute of Global Innovation Research, Tokyo University of Agriculture and Technology,
3-8-1 Harumi-cho, Fuchu, Tokyo 184-8538, Japan(Received August 30, 2017; accepted December 7, 2017)

Keywords: lactate oxidase, screen-printed carbon electrode, electron mediator, 1-methoxy-5-methyl
phenazinium methyl sulfate, potassium ferricyanide, hexaammineruthenium(III) chloride, biomedical
engineering

Monitoring blood lactate concentration is important in clinical diagnosis and evaluationof fitness care. Electrochemical enzyme sensors are commonly used for the measurement oflactate owing to their simplicity and sensitivity. Lactate oxidases (LOxs) are widely used for theconstruction of lactate enzyme sensors, especially the LOx derived from Aerococcus viridans(AvLOx). To minimize interference from redox substances, most lactate sensors utilize artificial
mediators. However, reports on the comparison of different mediators in lactate measurements
are limited. In this study, the preferences of AvLOx for electron mediators were investigatedelectrochemically. We concluded that the charge of the mediator plays a significant role indetermining its suitability as a mediator for AvLOx. The relatively uncharged 1-methoxy-5-methyl
phenazinium methylsulfate (mPMS) was the preferred mediator for AvLOx, the strongly negative
ferricyanide was a less preferential mediator, and the strongly positive hexaammineruthenium(III)was not utilized as a mediator by AvLOx. This suggests that the protein scaffold of AvLOx canalmost completely block the access to the co-factor for very small electron acceptors, depending on
their charge.

1.

Introduction

Determination of the blood lactate concentration level is widely used in clinical diagnosis andevaluation of exercise in sports medicine.(1,2) The normal blood lactate level in people at rest is
0.5–1.8 mM.(1) An increased lactate level is indicative of hypoxia due to medical problems orexercise.(1,2) With extreme exercise, the lactate level can increase to up to 25 mM.(2) Commonly,
handheld blood lactate analyzers are used for the measurement of capillary blood lactate.(1)These analyzers play a major role in lactate monitoring in the biomedical engineering field. The
analyzers are based on amperometry and measure electric current generated by lactate oxidation
using the enzyme lactate oxidase (LOx). LOx is a flavoenzyme that harbors flavin mononucleotide*

Corresponding author: e-mail: tsugawa@cc.tuat.ac.jp
These authors contributed equally to this work.
http://dx.doi.org/10.18494/SAM.2017.1731†

ISSN 0914-4935 © MYU K.K.

1704

Sensors and Materials, Vol. 29, No. 12 (2017)(FMN) as the cofactor, and catalyzes the oxidation of lactate to pyruvate while flavin is reduced(in the reductive half-reaction).(3,4) In the subsequent oxidative half-reaction, the reduced flavinis re-oxidized by oxygen, resulting in oxidized cofactor and hydrogen peroxide.(3,4) Alternatively,
electrons can be transferred from the reduced flavin in LOx to artificial electron mediators.
Therefore, second-generation lactate sensors utilizing LOx can be developed, which employ
artificial electron mediators to avoid the interference of redox substances in the blood by allowing
the application of a lower potential than that necessary to oxidize hydrogen peroxide.
To date, many artificial mediators for LOx sensors have been reported, such as ferrocene
derivatives,(5–11) indophenol derivatives,(5,7) indoaniline derivatives,(10,11) phenoxazine derivatives(i.e., Meldola blue),(6) tetrathiafulvalene,(12) hydroquinone,(5) and metal complexes including
ferricyanide,(5) Ni(II)-cyclam,(5) and Os complexes.(7,11,13) Some reports compared different
mediators regarding their suitability for LOx-based lactate sensors.(5,6,8,11,13) However, only few
papers discussed influential factors other than the local mediator concentration at the electrode/solution interface.(5,11,13)
One of the most common artificial electron mediators used in commercially available enzyme
sensors is potassium ferricyanide. Ferricyanide is a small, water-soluble, inorganic metal complex
with reversible electron transfer kinetics. However, ferricyanide is easily reduced by lightirradiation, which leads to measurements that are too high. Recently, hexaammineruthenium(III)chlor ide has been at t racting at tention as a mediator because it allows for a lower
application potential and because it is more stable under light irradiation than ferricyanide.
Hexaammineruthenium(III) is also a small, water-soluble, inorganic metal complex with reversible
electron transfer kinetics. Although these mediators are commonly used, there is no report thatcompares these mediators scientifically.
In this paper, we compare the com monly used elect ron mediators fer r icyanide,
hexaammineruthenium(III), and 1-methoxy-5-methyl phenazinium methylsulfate (mPMS;
the electroactive form of phenazinium methylsulfate, an electron acceptor often used inspectrophotometric assays) in combination with Aerococcus viridans-derived LOx (AvLOx). We
use end-point assays with disposable film electrodes mimicking enzyme sensor strips and discuss
the preference of AvLOx for these mediators.

2.

Materials and Methods

2.1

Materials and apparatus

2,6-dichlorophenolindophenol (DCIP) was purchased from Merck (Darmstadt, Germany).
4-Aminoantipyrine (4AA), 5-methyl-phenazinium methylsulfate (PMS), and potassium
ferricyanide {K3[Fe(CN)6]} were purchased from Kanto Chemical (Tokyo, Japan). 3-(N-ethyl3-methylanilino)-2-hydroxy-propanesulfonate (TOOS) and mPMS were purchased from Dojindo
Laboratories (Kumamoto, Japan). Peroxidase (POD) was obtained from Amano Enzyme (Gifu,
Japan). Sodium l-lactate and hexaammineruthenium(III) chloride {[Ru(NH3)6]Cl3} werepurchased from Sigma-Aldrich (St. Louis, MO, USA).
Screen-printed carbon electrodes (SPCEs; working electrode: carbon 2.4 mm2; referenceelectrode: Ag/AgCl; counter electrode: carbon) were kindly supplied by i-SENS (Seoul, Republic ofKorea). All electrochemical measurements were carried out with a VersaSTAT4 potentiostat from
Princeton Applied Research (AMETEC Inc., Berwyn, PA, USA).

Sensors and Materials, Vol. 29, No. 12 (2017)

2.2

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Enzyme preparation

AvLOx was prepared by recombinant expression in Escherichia coli.(3) The Km of AvLOx was
0.61 mM and the Vmax was 101 U/mg based on the oxidase activity (Table 1), which are comparable
to those in the literature.(3,4) The dehydrogenase activity was 135 U/mg (Table 1).
2.3

Enzyme activity

The oxidase activity was measured using 1.5 mM 4AA, 1.5 mM TOOS, 2 U/ml POD, and
0–25 mM lactate in 10 mM potassium phosphate buffer (PPB; pH 7.0). The enzymatic reaction
was initiated by the addition of lactate. One unit (U) of enzyme activity was defined as the amountof enzyme that catalyzes the production of 1 µmol of H2O2 per minute using 20 mM lactate at
25 ℃ (the absorbance change at 555 nm due to the formation of quinone imine dye was monitored).
Dehydrogenase activity was measured using 4 mM PMS, 0.06 mM DCIP, and 0–25 mM lactate in
10 mM PPB (pH 7.0). One unit (U) of enzyme activity was defined as the amount of enzyme thatcatalyzes the reduction of 1 µmol DCIP per minute using 20 mM lactate at 25 ℃ (the absorbancechange at 600 nm was monitored).
2.4

Electrochemical measurements

Chronoamperometry measurements were carried out. SPCEs were prepared by attaching aspacer and cover to form a µL-volume capillary space above the electrodes. A premix (4 µL)
was prepared containing a mediator [ferricyanide, mPMS, or hexaammineruthenium(III)] andAvLOx in 100 mM PPB (pH 7.0). The enzyme reaction was started by adding lactate (1 µL) to thepremix and 1.8 µL of the mixture was loaded onto an SPCE. The final concentration of lactate
was 0, 0.5, 1, 2, 5, 10, 20, 30, or 50 mM. The amount of enzyme was adjusted so that 0.1 U was
loaded onto the SPCE. A potential [+400 mV for ferricyanide, +200 mV for mPMS, 0 mV for
hexaammineruthenium(III)] was applied 60 s after the addition of lactate (the wait time) and thecurrent was monitored for another 60 s. For the standard curves, the current at 10 s (samplingtime) after application of the potential was plotted against the lactate concentration.

3.

Results and Discussion

3.1

Electrochemical measurements with ferricyanide as mediator

Electrochemical measurements were carried out, which mimicked blood lactate sensors. For
these end-point assays, the enzyme, mediator and lactate were mixed before loading onto theTable 1
Properties of purified AvLOx. Parameters determined from Hanes–Woolfs plots.
Oxidase activity Dehydrogenase activity
0.61
0.58
Km (mM)
101
135
Vmax (U/mg)
167
232
Vmax/Km (U/mg∙mM)

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Sensors and Materials, Vol. 29, No. 12 (2017)

electrode strip. A potential was applied after a sufficient time to allow the complete conversion of
the substrate (the wait time). Thus, the electrochemical measurement is that of a specific amount of
reduced mediator, which reflects the initial amount of substrate. In these types of measurements,
the current is described by the Cottrell equation, that is, the current is initially high but decreases
with time.
Figure 1 shows the time courses of measurements with 100 mM ferricyanide as a mediatorand various concentrations of lactate. The amount of enzyme was chosen so that the complete
conversion of the substrate was achieved within 60 s for all lactate concentrations. With increasing
lactate concentration, the response current increased, indicating that a lactate sensor could beconstructed with ferricyanide as the mediator. This was expected, as ferricyanide has been shown
to be an electron acceptor for LOx.(5)Corresponding measurements were carried out with other concentrations of ferricyanide, andthe current at 10 s was plotted against the lactate concentration (Fig. 2). Without ferricyanide(0 mM), no response current was observed, confirming that ferricyanide transfers electrons
from the enzyme to the electrode. Similar sensitivities (slope of line; 0.64 μA/mM for 100 mM
ferricyanide) within the linear range were achieved with the different concentrations offerricyanide, and the linear range extended to higher lactate concentrations for higher ferricyanide
concentrations (Fig. 2). For each oxidation of one molecule of lactate, the LOx reduces twomolecules of ferricyanide to two molecules of ferrocyanide. In this end-point assay, the enzyme
reaction is finished before the potential is applied, which means that there are either no more lactate
molecules or no more ferricyanide molecules. In the case of low initial lactate concentrations(<20 mM), all lactate molecules are oxidized. The resulting amount of ferrocyanide molecules
only depends on the initial lactate concentration, which is evidenced by the fact that the response
currents do not depend on the ferricyanide concentration. When the initial lactate concentration
exceeds half of the initial ferricyanide concentration, the amount of ferricyanide molecules
becomes the limiting factor. This results in response currents depending on the ferricyanide
concentration and not on the lactate concentration, and thus a loss of linearity. For high lactate

60

Current (μA)

50
40
30
20
10
0

d

c
a b
0
10

e

f

g

20

30
Time (s)

40

50

60

Fig. 1. (Color online) Time courses of response currents for various lactate concentrations.
Mediator: 100 mM ferricyanide; applied potential: +400 mV; enzyme: 0.1 U per sensor strip; wait time: 60 s; n = 3.
Lactate concentration: (a) 0, (b) 2, (c) 5, (d) 10, (e) 20, (f) 30, and (g) 50 mM.

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Sensors and Materials, Vol. 29, No. 12 (2017)25

Current (μA)

20
15
10
5
0

0

10
20
30
40
Lactate concentration (mM)

50

Fig. 2. (Color online) Standard curves of response currents vs lactate concentration for various ferricyanide
concentrations. Wait time: 60 s; enzyme: 0.1 U per sensor strip; applied potential: +400 mV; sampling time: 60 s; n = 3.
Inset: blow-up of low lactate concentrations.

concentrations (50 mM), the obtained response currents were somewhat lower than expected for
100 and 200 mM ferricyanide, indicating an incomplete turnover of lactate. A possible reason
for this incomplete turnover is an insufficient amount of time. The time needed for the complete
turnover of a given lactate amount (concentration) depends on the amount of enzyme. Therefore,
further optimization (increasing) of the wait time or of the amount of AvLOx might lead to a wider
linear range.
3.2

Electrochemical measurements with different mediators

Corresponding measurements to those described above for ferricyanide were carried out with
mPMS and hexaammineruthenium(III) as mediators, and the currents at 10 s were plotted against
the lactate concentration (Figs. 3 and 4). The properties of the mediators used in this study aresummarized in Table 2.
Ferricyanide is a small inorganic compound and has a strong negative charge and a redox
potential of +0.23 V vs Ag/AgCl.(14) Hexaammineruthenium(III) is also a small inorganic
compound, but with a strong positive charge and a redox potential of −0.11 V vs Ag/AgCl.(15)The third mediator used in this study, mPMS, is an organic compound with either a weak
positive charge (oxidized form) or no charge (reduced form), and a similar redox potential tohexaammineruthenium(III) (−0.14 V vs Ag/AgCl).(16)With mPMS as a mediator, which is used as one mediator in a commercial sensor based on
LOx,(17) higher currents were achieved than with ferricyanide (Fig. 3). In contrast to ferricyanide,
the LOx reduces only one molecule of mPMS for each oxidation of one molecule of lactate.
Therefore, less mPMS is needed and a wide linear range can be achieved with lower mPMS
concentrations. Furthermore, the wait time was long enough for the complete turnover of up to
50 mM lactate, indicating that the electron transfer from the FMN of AvLOx to mPMS was fasterthan that to ferricyanide. However, the background current was high and increased with increasing
mPMS concentration. This indicates that the mPMS used contained some interfering substance1708

Sensors and Materials, Vol. 29, No. 12 (2017)

80
70
Current (μA)

60
50
40
30
20
10
0

0

10
20
30
40
Lactate concentration (mM)

50

Fig. 3. (Color online) Standard curves of response currents vs lactate concentration for various mPMS
concentrations. Wait time: 60 s; enzyme: 0.1 U per sensor strip; applied potential: +200 mV; sampling time: 10 s; n = 3.

25

0 mM
25 mM
50 mM
100 mM
200 mM

Current (μA)

20
15
10
5
0

0

10
20
30
40
Lactate concentration (mM)

50

Fig. 4. (Color online) Standard curves of response currents vs lactate concentration for various
hexaammineruthenium(III) concentrations. Wait time: 60 s; enzyme: 0.1 U per sensor strip;
applied potential: 0 mV; sampling time: 10 s; n = 3.

Table 2
Properties of mediators used in this study. All mediators are water soluble.
Structure/Formula
Electron mediatorType(oxidized form)
Small inorganic
Potassium ferricyanide
K3[Fe3(CN)6]
metal complex
Hexaammineruthenium(III)
Small inorganic[Ru(NH3)6]Cl3
chloride
metal complex

mPMS

*

oxidized form/reduced form

Small organic
compound(phenazine derivative)

Redox potential(vs Ag/AgCl)

Charge(Ox/Red)*+0.23 V(14)−3/−4−0.11 V(15)+3/+2−0.14 V(16)+/0Sensors and Materials, Vol. 29, No. 12 (2017)

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that could be oxidized at the electrode. Although mPMS is more stable than PMS,(16) mPMS is
known to degrade in non-acidic solutions.(18) Therefore, the interfering substance might be a
degradation product. Another possibility is that a portion of the mPMS was in its reduced state.
With hexaammineruthenium(III), no response currents were obtained (Fig. 4). This indicatesthat AvLOx does not utilize hexaammineruthenium(III) as an electron acceptor.
Whether a mediator can accept electrons from an enzyme should depend on the redox potential;
mediators with a high redox potential should accept electrons more readily. Indeed, the mediatorwith the highest redox potential in this study, ferricyanide, does accept electrons readily from
AvLOx. However, although the redox potentials of mPMS and hexaammineruthenium(III)are similar, mPMS accepts electrons from AvLOx, while hexaammineruthenium(III) does not.
Nevertheless, the redox potential of hexaammineruthenium(III) should be high enough to accept
electrons from the active center of AvLOx, FMN (redox potential: −0.42 vs Ag/AgCl).(19) Thus, theredox potential does not explain the phenomena observed in this study.
The few previous reports on the comparison of mediators hint that factors other than theredox potential influence the suitability of a mediator. In 1988, Taniguchi et al. mentioned “somespecific interaction”, but did not go into more detail.(5) Later, in 2001, Hirano et al. stated thatthe chemical structure of the mediator is crucial, but they, too, did not go into more detail.(11)Finally, in 2013, Nieh et al. compared metal complexes bound to polymers and concluded that theelectrostatic repulsion of negative ligands and the local surface charge of the enzyme, as well as
steric hindrance, lead to less efficient mediators.(13)The mediators used in this study are all small and of similar size, so steric hindrance does not
explain any differences. Therefore, the most likely explanation is the electrostatic interactionof the mediators and the local surface charge of AvLOx. It seems that, especially for small
mediators with strong charges, the charge of the mediator and the sign of the charge have a great
influence on its suitability as a mediator. The relatively uncharged mPMS is the most effective
mediator for AvLOx in this study. The negatively charged ferricyanide does function as a mediator
for AvLOx; however, it is not as effective as mPMS. Therefore, AvLOx can tolerate negative
charges fairly well, although the negative charge of the ferricyanide seems to hinder the electrontransfer slightly, which is in accordance with the results of Nieh et al.(13) The positively charged
hexaammineruthenium(III) is not utilized as an electron acceptor by AvLOx. This suggests thatthe access of the hexaammineruthenium(III) to the active center of AvLOx is blocked, despite thesmall size of the mediator.

4.

Conclusions

In this study, the preferences of AvLOx for mediators that have a strong positive charge[hexaammineruthenium(III)], a strong negative charge (ferricyanide), or are relatively uncharged(mPMS) were investigated electrochemically. For this, recombinantly expressed AvLOx wasused. The relatively uncharged mediator mPMS was most effective. With 100 mM mPMS, near
perfect linearity was achieved up to 50 mM lactate. However, mPMS is known to be unstable,
so that such high mPMS concentrations are impractical for commercial use.(18) The negatively
charged ferricyanide was less effective than mPMS but did successfully transfer electrons from
AvLOx to the electrode. This suggests that AvLOx tolerates negative charges fairly well. The
linear range (up to 30 mM) and sensitivity (0.64 μA/mM) achieved with 100 mM ferricyanide

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Sensors and Materials, Vol. 29, No. 12 (2017)

should be sufficient for blood lactate sensors (normal range 0.5–1.8 mM, maximal with exercise
25 mM).(1,2) Ferricyanide is already the most common mediator in commercial enzyme sensors.
Finally, AvLOx did not utilize the hexaammineruthenium(III) as an electron acceptor, suggestingthat the access of the positively charged hexaammineruthenium(III) to the FMN in the active
center of AvLOx is blocked by the protein scaffold. Therefore, hexaammineruthenium(III) cannot
be used as the primary electron acceptor in enzyme sensor strips utilizing AvLOx. However,hexaammineruthenium(III) is the most desirable mediator for commercial use. Detailed structuralinvestigations of AvLOx might reveal how the access of the hexaammineruthenium(III) to
the active center is blocked, which might lead to strategies to engineer the enzyme to utilize
hexaammineruthenium(III) as a mediator.

Acknowledgments
The authors thank i-SENS Inc. (Seoul, Republic of Korea) for their kindness in providing the
SPCEs.

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About the Authors
Noya Loew received her M.S. and Ph.D. degrees from the University of
Potsdam, Germany, in 2003 and 2008, respectively. From 2008 to 2014, she wasa postdoctoral researcher at Tokyo Institute of Technology, Japan. Since 2015,
she has been a specially appointed assistant professor/postdoctoral researcher at
Tokyo University of Agriculture and Technology. Her research interests are inbiosensors, bioelectronics, and bioelectrochemistry.

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. Her research interests arein bioengineering, biomolecular engineering, and biosensors.

Kentaro Hiraka received his B.S. degree from Tokyo University of Agriculture
and Technology, Japan, in 2016. Since 2016, he has been a master course student
at Tokyo University of Agriculture and Technology. His research interests are inbioengineering, biomolecular engineering, and biosensors.

Koji Sode received his B.S., M.S., and Ph.D. degrees from the Tokyo Institute ofTechnology, Japan, in 1983, 1986, and 1988, respectively. Since 2000, he has
been a professor at Tokyo University of Agriculture and Technology. Since
2003, he has been a science and technology advisor at Ultizyme International
Ltd. His research interests are in bioengineering, biomolecular engineering, andbiosensors.

Wakako Tsugawa received her B.S. degree from the 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.