https://doi.org/10.20965/jrm.2023.p1131
Letter:Wearable Biosensor Utilizing Chitosan Biopolymer
for Uric Acid Monitoring
Mizuki Sato∗1 , Tatsuya Kamiyama∗2 , Kenta Iitani∗3 ,Kazuyoshi Yano∗4 , Kohji Mitsubayashi∗3 , and Takahiro Arakawa∗1,∗2∗1 Graduate School of Engineering, Tokyo University of Technology

1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan
E-mail: arakawath@stf.teu.ac.jp∗2 Department of Electric and Electronic Engineering, School of Engineering, Tokyo University of Technology
1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan∗3 Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University
2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan∗4 Graduate School of Bionics, Computer and Media Sciences, Tokyo University of Technology
1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan[Received April 23, 2023; accepted July 27, 2023]

Wearable biosensors leveraging the technology of
microelectromechanical systems (MEMS) have been devised as a viable alternative to costly and intricate analytical apparatus in healthcare, offering compact and pervasive sensing solutions tailored for healthcare applications [1–4]. The seamless integration of biosensors into
the Internet of Things ecosystem is pivotal to advance thefrontiers of healthcare and medical monitoring, with theoverarching goal of enhancing the quality of life (QoL) of
individuals.
Numerous biosensors for application on the externalsurface of human skin have been devised. For instance,biosensors capable of detecting chemical compounds insweat and gases in the skin derived from blood gases havebeen designed [5–9]. Among these, wound sensors thatfacilitate the monitoring of wound conditions have garnered significant interest. Chronic wounds significantly
impact on the QoL of patients and impose a substantial
financial burden owing to the recurring need for wounddressing replacement and prolonged hospital stays. Consequently, innovative wound care technologies, that offer simple monitoring, affordability, and wearability, withbody-attachable sensors to fulfill these requirements are inhigh demand [10–12]. Therefore, a discernible call exists
for the advancement of wound site treatment technologies
at more affordable prices. Given the escalating costs associated with healthcare for patients and the ensuing implications on their QoL, sensors developed for this purposemust be not only cost-effective but also wearable for human use.
Research pertaining to the monitoring of wound sites
has focused on techniques for measuring the chemical
constituents generated at the site of injury, as well as physical parameters such as body temperature and extent ofstrain [13]. Among the various chemical components,
uric acid (UA) has emerged as a noteworthy biomarker,
owing to its robust correlation with wound severity as elucidated in previous studies [14]. Furthermore, UA levels
are known to decrease owing to the action of uricase during bacterial infection. Consequently, UA has been employed as a reliable indicator to assess both the woundcondition and infection status. Kassal et al. successfully
fabricated a cutting-edge wound sensor by incorporating a
Ag/AgCl reference electrode, Prussian blue-carbon working electrode, and counter electrode within a bandage
while immobilizing glutaraldehyde and uricase [15]. Remarkably, this sensor can transmit real-time UA status to
a computer or smartphone via near-field communication,
making it a valuable tool for monitoring the dynamic status of wounds.
The aim of this study was to develop a wearable biosen-

Journal of Robotics and Mechatronics Vol.35 No.5, 2023

1131

A wearable biosensor was specifically engineered tomeasure uric acid, a biomarker present at wound sites.
This biosensor, fabricated as a disposable and wearable device, was seamlessly integrated onto a polyethylene terephthalate (PET) substrate by utilizing
carbon and silver conductive paste as the electrodes.
The enzyme uricase was immobilized onto the working electrode by utilizing chitosan, a biocompatible
material, to create this biosensor. Notably, the uricacid biosensor fabricated with chitosan showcased exceptional performance metrics, including remarkable
output current values and impeccable stability. Thesefindings suggest the prospective utilization of chitosanbased uric acid biosensors for the accurate measurement of uric acid on human skin in future applications.

Keywords: wearable biosensor, uric acid, uricase, chitosan

1. Introduction© Fuji Technology Press Ltd. Creative Commons CC BY-ND: This is an Open Access article distributed under the terms of
the Creative Commons Attribution-NoDerivatives 4.0 InternationalLicense (https://creativecommons.org/licenses/by-nd/4.0/)

Sato, M. et al.

(a)Fig. 1. Schematic diagram of a UA sensor worn on humanskin for wound site monitoring.

(b)

Fig. 3. Image of UA biosensor. (a) Conparison of electrode appearance before (inner) and after chloride treatment.
(b) Immobilization of uricase using chitosan on the carbon
electrode.

off the substrate, and the fabricated silver electrode waschlorinated to form a silver chloride electrode.
A UA biosensor utilizing uricase is based on the specific enzymatic oxidation of UA by oxygen, resulting in
the production of hydrogen peroxide, allantoin, and carbon dioxide. The reaction can be expressed as follows:uricaseUA + H2 O + O2 −−−−→ Allantoin + H2 O2 + CO2 .
The amperometric detection method can rely on the determination of the generated hydrogen peroxide.
Fig. 2. Fabrication of UA biosensor and an image of fabricated UA biosensor on the PET substrate.

2.2. Fabrication and Measurement of UA Biosensor
sor for wound site evaluation. Specifically, we fabricateda biosensor capable of measuring UA levels as an indicator of wound conditions. Our goal was to demonstrate awound sensor by fabricating electrodes using conductive
paint on a commercially available adhesive bandage, withuricase immobilized on the working electrode to enablebiosensor functionality (Fig. 1). The catalytic reaction ofhydrogen peroxide products resulting from the oxidationof UA allows the detection of the output current at lower
potentials. In this letter, we present the response and sensitivity of a biosensor fabricated by immobilizing a biocompatible material chitosan for UA measurement.

2. Experimental Setup
2.1. Fabrication of Electrodes on a Polyethylene
Terephthalate (PET) Sheet
Figure 2 shows the biosensor and fabrication procedure. First, a patterned sheet of electrodes fabricated using a cutting machine (GS-24, Roland) was attached to a
PET substrate (1.5 mm × 2.5 mm, 1 mm thick). Second, asilver paste material (XA-3513, Fujikura Kasei Co., Ltd.)was spread by heating at 65°C for 30 min using a hot
plate, and a paste material of carbon (XC-3050, Fujikura
Kasei Co.) was spread as the working electrode using anelectrode mold and fixed by heating at 65°C for 30 min
using a hot plate. Finally, the patterned sheet was peeled
1132

Uricase was immobilized on a working electrode using chitosan, resulting in the fabrication of a UA sensor. Chitosan, known for its excellent biocompatibility, biodegradability, and non-toxicity, is widely utilized
in biochemistry and medicine for applications such as
wound healing, microneedles, hemodialysis membranes,
and as a support for immobilized enzymes [16, 17].
To prepare the UA sensor, a mixture of 10 mg of chitosan solution (3 wt%) and 1 mg of uricase (3.40 U/mg)
was applied to the surface of the working electrode anddried in a refrigerator for 18 h. In the experimental setup,
drops of UA solution (C6 H12 O6 , 5 mM) were added every
3 min to achieve varying concentrations of UA (500 nM
to 100 mM) in a beaker containing 50 mL of phosphatebuffer (PB, 50 mM, pH 7.0). The corresponding output current values were continuously measured by applying voltage of +0.8 V using potentiostat (EmStat4S,
PalmSens). A calibration curve was constructed using
output current values for each UA concentration.

3. Results and Discussions
Figure 3(a) presents a micrograph of a silver electrode
that was prepared and subsequently treated with chloride, whereas Fig. 3(b) shows a magnified image of theworking electrode after the immobilization of uricase using chitosan. Uricase was specifically immobilized onto
the working electrode using a sheet stencil mask. The
thicknesses of the carbon electrode, silver electrode, andJournal of Robotics and Mechatronics Vol.35 No.5, 2023

Wearable Biosensor for Uric Acid Monitoring

findings suggest that the chitosan-based UA biosensor
holds promise for measuring UA levels at wound sites.
Further investigations should be conducted to optimize
the amount and immobilization of uricase for the fabrication of a stable UA biosensor. By enabling the applicationof the fabricated biosensor to continuously measure thecondition of a wound site, this study could reduce medical costs by decreasing the frequency of dressing changes
and alleviating patient stress.

(a)Acknowledgments
We gratefully acknowledge the partial support provided by the
Tokyo University of Technology Grant A 2022. This study was
based on the Cooperative Research Project of the Research Center for Biomedical Engineering.

References:

In this study, we fabricated and evaluated a UA biosensor using chitosan as an enzyme-immobilizing material.
The UA biosensor fabricated with chitosan exhibited superior performance than did conventional biosensors thatutilize UV cross-linking agents for enzyme immobilization, in terms of output current value and stability. These[1] J. R. Windmiller and J. Wang, “Wearable electrochemical sensors
and biosensors: A review,” Electroanalysis, Vol.25, No.1, pp. 2946, 2013. https://doi.org/10.1002/elan.201200349[2] J. Kim, A. S. Campbell, B. E.-F. de Ávila, and J. Wang,“Wearable biosensors for healthcare monitoring,” Nat. Biotechnol.,Vol.37, No.4, pp. 389-406, 2019. https://doi.org/10.1038/s41587019-0045-y[3] T. Arakawa, D. V. Dao, and K. Mitsubayashi, “Biosensors andchemical sensors for healthcare monitoring: A review,” IEEJ Trans.
Electr. Electron. Eng., Vol.17, No.5, pp. 626-636, 2022. https://doi.org/10.1002/tee.23580[4] K. Ooe, Y. Hamamoto, T. Kadokawa, and Y. Hirano, “Developmentof the MOSFET type enzyme biosensor using GOx and ChOx,” J.
Robot. Mechatron., Vol.20, No.1, pp. 38-46, 2008. https://doi.org/
10.20965/jrm.2008.p0038[5] B. K. Ashley, M. S. Brown, Y. Park, S. Kuan, and A. Koh, “Skininspired, open mesh electrochemical sensors for lactate and oxygen
monitoring,” Biosens. Bioelectron., Vol.132, pp. 343-351, 2019.
https://doi.org/10.1016/j.bios.2019.02.041[6] H. U. Chung et al., “Skin-interfaced biosensors for advanced wireless physiological monitoring in neonatal and pediatric intensivecare units,” Nat. Med., Vol.26, No.3, pp. 418-429, 2020. https://doi.org/10.1038/s41591-020-0792-9[7] T. Arakawa et al., “Real-time monitoring of skin ethanol gas
by a high-sensitivity gas phase biosensor (bio-sniffer) for the
non-invasive evaluation of volatile blood compounds,” Biosens.
Bioelectron., Vol.129, pp. 245-253, 2019. https://doi.org/10.1016/
j.bios.2018.09.070[8] J. Kim et al., “Simultaneous monitoring of sweat and interstitial fluid using a single wearable biosensor platform,” Adv. Sci.,
Vol.5, No.10, Article No.1800880, 2018. https://doi.org/10.1002/
advs.201800880[9] J. Yin et al., “Flexible textile-based sweat sensors for wearable applications,” Biosensors, Vol.13, No.1, Article No.127, 2023. https://doi.org/10.3390/bios13010127[10] A. Pal et al., “Early detection and monitoring of chronic wounds
using low-cost, omniphobic paper-based smart bandages,” Biosens.
Bioelectron., Vol.117, pp. 696-705, 2018. https://doi.org/10.1016/j.
bios.2018.06.060[11] T. R. Dargaville et al., “Sensors and imaging for wound healing:
A review,” Biosens. Bioelectron., Vol.41, pp. 30-42, 2013. https://doi.org/10.1016/j.bios.2012.09.029[12] A. Pusta, M. Tertis, , C. Cristea, and S. Mirel, “Wearable sensors for the detection of biomarkers for wound infection,” Biosensors, Vol.12, No.1, Article No.1, 2022. https://doi.org/10.3390/
bios12010001[13] M. Jose et al., “Stretchable printed device for the simultaneous
sensing of temperature and strain validated in a mouse woundhealing model,” Sci. Rep., Vol.12, Article No.10138, 2022. https://doi.org/10.1038/s41598-022-13834-6[14] S. RoyChoudhury et al., “Continuous monitoring of wound healing
using a wearable enzymatic uric acid biosensor,” J. Electrochem.
Soc., Vol.165, No.8, pp. B3168-B3175, 2018. https://doi.org/10.
1149/2.0231808jes[15] P. Kassal et al., “Smart bandage with wireless connectivity foruric acid biosensing as an indicator of wound status,” Electrochem.
Commun., Vol.56, pp. 6-10, 2015. https://doi.org/10.1016/j.elecom.
2015.03.018

Journal of Robotics and Mechatronics Vol.35 No.5, 2023

1133

(b)
Fig. 4. (a) Temporal changes in the output of UA biosensor.
(b) The calibration curve for UA biosensors.

chitosan film containing the enzyme measured 19.4 µm,
20.0 µm, and 6.1 µm, respectively, as evaluated with acontact-type thickness meter. An excellent response time
was obtained for the UA biosensor by reducing the thickness of the chitosan film.
Figure 4(a) shows the response output of the fabricatedUA biosensor, and Fig. 4(b) shows the calibration curveof the UA biosensor. The calibration curve was preparedusing the average output current, allowing for quantification in the range of 500 nM to 100 µM. As the concentration of UA on the wound site is typically embedded within
the range of 220 µM to 750 µM [18], this biosensor is suitable for measuring UA levels. Notably, the output currentof the chitosan-based UA sensor at 100 µM was approximately four times that of our previous UA biosensor thatutilized immobilization of a UV cross-linkable polymer.

4. Conclusion

Sato, M. et al.

[16] R. Lungu et al., “Biocompatible chitosan-based hydrogels forbioabsorbable wound dressings,” Gels, Vol.8, No.2, Article No.107,2022. https://doi.org/10.3390/gels8020107[17] M. Suzuki, T. Takahashi, and S. Aoyagi, “Fabrication and characterization of a biodegradable hollow microneedle from chitosan,”J. Robot. Mechatron., Vol.32, No.2, pp. 401-407, 2020. https://doi.
org/10.20965/jrm.2020.p0401[18] M. L. Fernandez, Z. Upton, H. Edwards, K. Finlayson, and G. K.
Shooter, “Elevated uric acid correlates with wound severity,” Int.
Wound J., Vol.9, No.2, pp. 139-149, 2012. https://doi.org/10.1111/
j.1742-481X.2011.00870.x

Name:
Kazuyoshi Yano

Affiliation:
Graduate School of Bionics, Computer and Media Sciences, Tokyo University of Technology

Address:
1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan

Name:

Brief Biographical History:

Mizuki Sato

1994- Assistant Professor, The University of Tokyo
2001- Lecturer, The University of Tokyo
2002- Associate Professor, Tokyo University of Technology
2010- Professor, Tokyo University of Technology

Affiliation:
Graduate Student, Graduate School of Engineering, Tokyo University of Technology

Main Works:• K. Yano and A. Iwasaki, “Enhancement of fluorescence-based sandwich
immunoassay using multilayered microplates modified withplasma-polymerized films,” Sensors, Vol.17, No.1, Article No.37, 2017.

Address:
1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan

Name:

Brief Biographical History:

Kohji Mitsubayashi

2022 Received B.E. degree from Tokyo University of Technology

ORCID:
0000-0002-0709-4957

Name:

Affiliation:

Tatsuya Kamiyama

Affiliation:
Department of Electric and Electronic Engineering, Graduate School of Engineering, Tokyo
University of Technology

Professor, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University

Address:
2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Brief Biographical History:
1998- Associate Professor, Tokai University
2003- Professor, Tokyo Medical and Dental University

Main Works:

Address:
1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan

Brief Biographical History:• K. Mitsubayashi, K. Toma, K. Iitani, and T. Arakawa, “Gas-phasebiosensors: A review,” Sens. Actuators B: Chem., Vol.367, Article
No.132053, 2022.

2022 Received B.E. degree from Tokyo University of Technology

Name:
Name:

Takahiro Arakawa

Kenta Iitani
ORCID:
0000-0002-1555-1281

ORCID:
0000-0001-5608-8863

Affiliation:
Affiliation:
Assistant Professor, Institute of Biomaterials andBioengineering, Tokyo Medical and Dental University

Associate Professor, Graduate School of Engineering, Tokyo University of Technology

Address:

Address:

1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan

2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Brief Biographical History:

Brief Biographical History:
2021- Project Assistant Professor, Tokyo Medical and Dental University2022- Assistant Professor, Tokyo Medical and Dental University

2009- Assistant Professor, Tokyo Medical and Dental University
2014- Junior Associate Professor, Tokyo Medical and Dental University2021- Associate Professor, Tokyo University of Technology

Main Works:

Main Works:• K. Iitani, K. Toma, T. Arakawa, and K. Mitsubayashi, “Transcutaneous
blood VOC imaging system (skin-gas cam) with real-time bio-fluorometricdevice on rounded skin surface,” ACS Sens., Vol.5, No.2, pp. 338-345,
2020.

• T. Arakawa et al., “Mouthguard biosensor with telemetry system formonitoring of saliva glucose: A novel cavitas sensor,” Biosens.
Bioelectron., Vol.84, pp. 106-111, 2016.

Membership in Academic Societies:• Institute of Electrical and Electronics Engineers (IEEE)• The Institute of Energy Economics, Japan (IEEJ)• The Japan Society of Applied Physics (JSAP)

1134

Journal of Robotics and Mechatronics Vol.35 No.5, 2023