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MYU Tokyo
S & M 3429

Enzyme-immobilized Screen-printed Sensor
for One Drop of Sample Using Porous Silica Spheres
Shunsuke Fujii,1,2 Akiko Yoshida,3,4 Tracy T. Chuong,2 Yuka Minegishi,2
Yuta Nishina,5 Hirotomo Nishihara,4 Kouji Masumoto,1* and Tetsuji Itoh2**
1Department of Pediatric Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
2National Institute of Advanced Industrial Science and Technology (AIST), Sendai, Miyagi 983-8551, Japan
3R & D Department, Techno Medica Co., Ltd., Yokohama, Kanagawa 224-0041, Japan

4Institute of Multidisciplinary Research for Advanced Materials (IMRAM)/Advanced Institutefor Materials Research (WPI-AIMR), Tohoku University, Sendai, Miyagi 980-8577, Japan

5Graduate School of Natural Science and Technology, Okayama University, Kita-ku, Okayama 700-8530, Japan(Received April 27, 2023; accepted August 24, 2023)

Keywords: biosensor, enzyme electrode, mediator, screen-printed device, porous silica

A sensor chip that can detect enzyme electrode reactions with a sample volume as small as 20
μL has been developed using screen-printing technology with porous silica spheres (PSSs). PSSs
are spherical particles of silica with mesoporous pores, a material with a large specific surface
area. Other material properties of PSSs include good dispersibility, high mechanical strength,
and the ability to be synthesized with precisely controlled mesopore sizes. These advantages
support the increased adsorption of enzymes to create highly sensitive sensors. The electrodewas designed as follows: carbon paste (CP) with PSSs was used as the working electrode,glucose dehydrogenase (GDH) was used as the enzyme, and quinoline-5,8-dione (QD) was usedas the mediator of electron transfer to induce a glucose dehydrogenation reaction. The sensitivityof the sensor was demonstrated through the detection of glucose. The glucose sensor showedreliable sensitivity between 1 and 60 mM glucose, which is comparable to the detection range ofa traditional working electrode such as platinum. Furthermore, the GDH had a minimal
reduction in activity over repeated usage. This device demonstrates the ability to create anequally sensitive but less expensive and reusable sensor that has the potential to better suit
general applications.

1.

Introduction

Recent efforts have been directed toward the development of biosensors that combine
biological detection with physical transducers.(1–6) In particular, amperometric biosensors using
immobilized enzyme electrodes can provide high sensitivity, high selectivity, and short responsetime without the need for large or complex equipment. These sensors have been shown to detect
glucose or lactate(7–12) at low concentrations reliably and reproducibly. Parallel to biosensor
development, active development is also taking place to simplify sensor production, specifically*Corresponding author: e-mail: kmasu@md.tsukuba.ac.jp**Corresponding author: e-mail: tetsu-itou@aist.go.jp

https://doi.org/10.18494/SAM4473

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

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on the stability and longevity of the sensors as analytical instruments. Stability is mainly
dependent on the rate of denaturation or inactivation of the enzyme employed.(13–16) Novel
immobilization methods(4,16–18) used to enhance enzyme stability and optimize the surrounding
microenvironment through material choice(19–22) have been investigated. One of the best
immobilization methods to stabilize enzymes to retain good catalytic activity is to encapsulate
the enzyme in mesoporous silica (MPS) materials. MPS materials inherently have high surface
areas and pore volumes, tunable pore sizes to accommodate enzymes of different dimensions,and excellent mechanical stability.(23,24) Our previous studies using an enzyme-immobilized
electrode in a stirred-batch cell showed that high-performance biosensors can be constructedusing immobilized enzymes within MPS materials.(25–31)
However, when this technique is combined with screen-printing, a range of complications
occur. At minimum, the particles can be non-uniformly distributed, resulting in the poor
reproducibility of the detection quality; at worst, the pores can be clogged, which inhibits the
diffusion of the substrates and the electron mediator, resulting in device failure. We exploredporous silica spheres (PSSs) with a uniform particle size as candidate materials to resolve thisissue. The pore and particle sizes of PSSs can be controlled over a wide range while maintaininga small variance, which allows them to be arranged uniformly on the electrode. The enzyme canthen be immobilized on the PSS layer, and stability can be obtained as demonstrated in ourprevious papers,(25,29) where carbon-coated PSS electrodes were arranged in a very small
area.(32)
In this paper, we report on the development of a highly stable, sensitive, and reusable sensor
using PSSs as an enzyme immobilization host, fabricated by screen-printing.

2.

Materials and Methods

2.1

Materials

Pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) (1040 U/mg) frommicroorganisms was purchased from Toyobo Co., Ltd. in Japan. PQQ-GDH was dissolved inwater at a concentration of 2.8 U/μL. Sodium dihydrogen phosphate (NaH2PO4) and disodiumhydrogen phosphate (Na2HPO4) were obtained from Kanto Chemical Co., Ltd., Japan. Phosphatebuffer (50 mM, pH 7) was prepared by mixing NaH2PO4 and Na2HPO4. Potassium ferricyanide(PF) and quinoline-5,8-dione (QD), a water-soluble quinone mediator, were used as mediators ofelectron transfer.(32,33) PF was purchased from Sigma-Aldrich, and the synthesis of QD is
described below (see Sect. 2.2). PF or QD was dissolved in phosphate buffer to 5 mM, pH 7. D(+)-glucose was obtained from Fujifilm Wako Pure Chemicals Co., Ltd. and dissolved inphosphate buffer to 100 mM, pH 7.
2.2. Synthesis of QD(32,33)
A solution of 5-hydroxyquinoline (312 mg, 2.14 mmol) in acetonitrile (30 mL) was cooled to 0
℃ under argon atmosphere. Then, [bis(trifluoroacetoxy)iodo] benzene (PIFA) (2 g, 4.7 mmol)

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was dissolved in 2:1 acetonitrile–water solution (60 mL). The PIFA solution was added dropwise
to the precooled solution of 5-hydroxyquinoline and the mixture was stirred for 1 h. The reaction
mixture was concentrated under reduced pressure, resulting in a dark brown aqueous solution.
The aqueous solution was extracted with ethyl acetate (20 mL) three times. The organic extract
was washed with brine (50 mL), dried over magnesium sulfate, filtered, and concentrated under
reduced pressure to give a dark yellow residue. The residue was purified by silica gel column
chromatography eluting with 1:4 ethyl acetate–hexanes to give crystals.
2.3. Fabrication of three-electrode sensor chips
A sensor chip consisting of a three-electrode system was fabricated by screen-printing(Fig. 1). On an aluminum oxide substrate, a working electrode and a counter electrode wereplaced with sintering platinum (Pt) paste (Tanaka Kikinzoku Kogyo K.K., Japan) and wired withsintering silver paste (Mitsuboshi Belting Ltd., Japan). A Ag/AgCl reference electrode wascreated by overlaying silver chloride on silver. Thermosetting silver paste was used for thecontacts between the silver wiring part and the Pt electrode part. Thermosetting-type epoxy
resin paste and polyester resin paste were laminated thereon to define the area of the electrodeFig. 1. (Color online) Fabrication of electrode sensor chip. (a) Actual photo of sensor chip (sensor chip size:
5 × 1 cm 2, working electrode: ϕ 1 mm). (b) Wiring diagram of sensor chip. (c) Structure of sensor chip laminated byscreen-printing. (d) A CP electrode was fabricated by the screen-printing of CP on a platinum working electrode.
(e) Electrochemical analysis with electrode sensor chip. Electrochemical analysis was performed by connecting theconnecting terminal of the sensor chip to a potentiostat and dropping 20 µL of sample (including enzymes, mediator,and electrolyte) onto the electrode.

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section and to protect the wiring section. Carbon paste (CP) working electrodes were preparedby screen-printing CH-N carbon ink (Jujo Chemical Co., Ltd., Japan) onto the Pt working
electrode.
2.4. Electrochemical analysis
For electrochemical analysis, the sensor chip was used in combination with a potentiostat
HX-5000 (Hokuto Denko Corporation, Japan) [Fig. 1(e)]. First, a mixture of 5 μL of PQQ-GDH
solution and 5 μL of mediator solution was placed on the working electrode. Then, a mixture of
5 μL of D-glucose and 5 μL of mediator solution was added to induce the following enzymatic
reaction:
GDH

D-glucose + acceptor → D-glucono-1,5-lactone + reduced acceptor.

(1)

The final concentrations of PQQ-GDH and the mediator in the mixed solution were 0.7 U/μL
and 2.5 mM, respectively. Note that glucose was diluted fourfold from its original concentrationin the mixed solution. The sensing performance for the above enzymatic reaction was examinedby cyclic voltammetry and amperometry. Cyclic voltammetry was performed for six cycles in a
potential window of −0.5 to 0.8 V, and the redox currents at the third cycle were compared. For
amperometry, a constant potential (0.3 V) was applied, and after the background current
stabilized for 120 s, the D-glucose solution was dropped onto the working electrode. Fifty
seconds after the D-glucose solution was dropped (t = 170 s), the output currents were compared.
A calibration curve was created by plotting the change in output current across a range of
glucose concentrations tested on a single sensor chip. To demonstrate the longevity and stabilityof the fabricated device, the same sensor chip was used across the experiments, where theelectrode was washed with distilled water after each measurement.
2.5. Adhesion of PSSs on CP electrodes
PSSs with a particle size of 10 μm and a pore size of 20 nm were purchased from Fuji Silysia
Chemical Ltd., Japan. The specific surface area and pore size of the PSSs were obtained bydeaeration at 150 ℃ for 3 h at 10−3 mmHg or less and the adsorption/desorption of nitrogen at−196 ℃ on a Belsorp-MAX II (Microtrac BEL Co. Ltd., Japan). The specific surface area wascalculated by the Brunauer–Emmett–Teller (BET) method. The pore size was estimated by the
Barrett–Joyner–Halenda (BJH) method applied to adsorption isotherms. The experiment was
conducted in triplicates (N = 3), and the mean and standard deviation (SD) were determined.
PSSs were sprinkled on the CP working electrode of the sensor chip immediately after the CH-N
carbon ink was printed and when the ink has not yet dried and fully dried at 120 ℃ for 30 min
for adhesion. Scanning electron microscopy (SEM) was used to observe the working electrodesurface using an S-4800 (Hitachi, Ltd., Japan) system without a metal coating at an acceleration
voltage of 1.5 kV.

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3.

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Results and Discussion

3.1. Sensing of enzyme reactions using sensor chip
We first developed a sensor chip that can detect the enzymatic electrode reaction with a small
amount of sample by screen-printing (Sect. 2.3, Fig. 1). The electrode part consists of a threeelectrode system comprising a Pt or CP working electrode, a platinum counter electrode, and a
Ag/AgCl reference electrode. The working electrode was 1 mm in diameter. Electrochemical
analysis was performed by dropping 20 μL of sample (including enzyme, mediator, andelectrolyte) onto the electrode [Fig. 1(e)].
The sensor chip was used to evaluate the electrochemical sensing performance of GDH in the
glucose dehydrogenation reaction [see Eq. (1)]. Figure 2(a) shows potassium ferricyanide (PF)Fig. 2. (Color online) Evaluation of sensing GDH enzyme response with sensor chip. The blue bars, lines, andplots (A) indicate measurements with a Pt working electrode and the orange bars, lines, and plots (B) with a CP
working electrode. (a) Comparison of output currents for different types of electron transfer mediator. (b)Comparison of cyclic voltammograms. (c) Comparison of amperometric responses. The dashed lines (blue C andorange D) show drops of solution without glucose, and the solid lines show drops of solution with glucose (blue A
and orange B). (d) Calibration curve of output current at varying glucose concentration.

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and QD as mediators of electron transfer since PF was considered the conventional mediator forelectrochemical sensors.(5,6) No obvious output current could be detected when PF was used as amediator. In contrast, significant output currents could be detected when QD was used as amediator. QD was better than PF in this sensor system. Therefore, QD was used as a mediator in
the following experiments.
In cyclic voltammograms [Fig. 2(b)], stable redox currents were detected at both the Pt andCP working electrodes. Here, especially in the CP working electrode, the QD reduction peak issignificantly shifted to the minus side, and the oxidation peak is shifted to the plus side.
However, its mechanism is unclear, and there is probably some interaction between carbon and
QD. Similar output currents were obtained at both working electrodes in the amperometric
reaction [Fig. 2(c)]. Figure 2(d) shows the calibration curve obtained by plotting the output
current at varying glucose concentration. For the Pt working electrode, the plot values were
nearly linear over the glucose concentration range of 1–80 mM; for the CP working electrode,they were nearly linear from 1 to 60 mM. The sensor’s sensitivity is superior to the previously
reported results of a glucose sensor using FAD-GDH and QD on a gold working electrode,which had an upper detection limit of 55.5 mM glucose.(33)
3.2. Enzyme immobilization on working electrode of sensor chip
Although we have used enzyme solutions in our evaluations with sensor chips so far, it wasnecessary to immobilize enzymes on the working electrode for practical applications to improvestorage and repeated stabilities. We focused on PSSs, whose particle and pore sizes can becontrolled, which was advantageous for enzyme immobilization because their well-defined pore
structure and large surface area allowed for the uniform modification and encapsulation of
enzymes. Figure 3(a) shows the N2 adsorption/desorption isotherm of PSSs. Hysteresis wasobserved in the pressure range of 0.8–1.0, confirming the mesoporous structure of PSSs.
Figure 3(b) shows the pore size distribution calculated by the BJH method. The PSS pore size
averaged 22.5 nm with no significant variation (standard deviation of 0.64). Table 1 shows thepore diameter, pore volume, and specific surface area of PSSs.
However, the PSS itself was not suitable as an electrode material because it has no
conductivity. If PSSs can be immobilized on an electrode and an optimal mediator can be used,
the problem of electrical conductivity can be solved, and an enzyme-immobilized electrode canbe produced. In fabricating the sensor, PSSs were sprinkled directly on the CP electrodeimmediately after the CP was printed. The layer was allowed to fully dry for the particles toadhere to the CP electrode. Figure 4 shows SEM images of the CP electrode without PSSs andthe CP electrode with PSSs adhered (PSS-CP electrode). For the CP electrode without PSSs, theSEM image visually confirmed that the CP was uniformly printed [Fig. 4(a)]. When PSSs
adhered to the CP electrode, the particles were self-arranged in a single layer on top of the CP,with the CP layer observable through the gaps between the particles [Fig. 4(b)].
Fifty microliters of GDH solution (28 U/μL) prepared at 10 times the concentration was
dropped onto the PSS-CP electrode and left for 24 h at 4 ℃ to allow the enzymes to adsorb intoand onto the PSSs. After the GDH solution was removed, the GDH-free mediator solution wasSensors and Materials, Vol. 35, No. 10 (2023)

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Fig. 3. (a) N2 adsorption-desorption isotherms of PSSs measured at −196 ℃. The black and white circle plots show
the adsorption and desorption sides, respectively. (b) Pore size distribution plot of PSSs estimated by BJH methode.
Table 1
Porous properties of PSSs. The means and standard deviations (SD) of pore size, pore volume, and specific surface
area obtained by three nitrogen gas adsorption methods are shown.
Pore size (nm)Pore volume (cm3 g−1)
Specific surface area (m 2 g−1)
Mean
22.5
0.665
150
SD
0.64
0.003
11

Fig. 4. (a) SEM image of CP working electrode without PSSs. (b) SEM image of CP electrode with PSSs adhered(PSS-CP electrode).

placed on the PSS-CP electrode. The target glucose solution was added onto the electrode, andthe output current was examined by amperometric reaction [Fig. 5(a), orange B line]. The results
were compared with the same setup on a CP electrode without PSSs (blue A line). Figure 5(a)shows that there was little change in reaction rate immediately after introducing glucose(comparable slope between Lines A and B). Figure 5(b) shows a calibration curve plotting the4776

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B

A

A
B

Fig. 5. (Color online) Evaluation of sensing of GDH enzyme reaction on GDH-immobilized PSS-CP working
electrode. The blue lines (A) and plots (A) show measurements with GDH solution on the CP working electrode, andthe orange lines (B), plots (B), and bars show measurements with GDH-free solution on the GDH-immobilized CPPSS working electrode. (a) Comparison of amperometric responses. The glucose concentration was 30 mM, and the
glucose solution was added at the arrow. (b) Calibration curve of output current at varying glucose concentration.
(c) Stability of GDH-immobilized PSS-CP electrode for repeated measurements.

output current at varying glucose concentration at a GDH-immobilized CP-PSS electrode[orange (B) plot]. The plotted values for the CP-PSS electrode maintained a linear relationshipfrom 1 to 60 mM, although the output current was moderately lower than that obtained with the
GDH solution on a CP electrode [blue (A) plot]. This means that GDH was successfully
immobilized on a CP electrode without loss of GDH activity. To evaluate the stability of PSSs on
the CP electrode, we examined to what extent the output current was maintained when samples
were repeatedly removed and dropped at the electrode [Fig. 5(c)]. A single sensor chip was used,
and only the dropping solution was changed between measurements, with no washing process in
between. No significant change in output current was observed after repeated measurements up
to about 10 times. Thereafter, a gradual decrease in output current was observed, and after about
20 times, the output current decreased by half. This was likely due to the gradual detachment of
PSSs from the CP electrode rather than enzyme inactivation, leaching or denaturation (Fig. S1).
This is the first step to demonstrating a reusable device using PSSs on the CP electrode. Moreinvestigations are ongoing to improve the technique of retaining the PSSs on CP.

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Fig. 6. (Color online) Schematic of GDH-immobilized PSS-CP electrode. A large amount of GDH is immobilizedon the PPS owing to the pore structure of the PSS. The PPSs are arranged in a single layer on the CP electrode so thatthe electrons generated via the mediator in the GDH enzyme reaction are efficiently transferred to the CP electrode.

4.

Conclusions

In this study, we presented an amperometric biosensing platform based on the immobilizationof enzymes in PSSs that were screen-printed on a CP electrode. We demonstrated the sensor
functionality using the GDH enzyme for glucose detection, but the sensor can be repurposedwith other enzyme/substrate combinations. In conclusion, the methods described in this study
demonstrate that PSS materials can be aligned by screen-printing on electrodes to provide asuitable support for the immobilization of enzymes (Fig. 6). This technique produces practicaland easy-to-fabricate electrochemical biosensors that improve the tools’ performance in terms ofsensitivity, selectivity, and reusability. In addition, we are currently using a drop system, but inthe future, we plan to form a flow path and incorporate it into repeat use.

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Electronic supplementary information

Fig. S1. SEM image of PSS-CP electrode surface after repeating the experiment 20 times. Figures 4(b) and S1 weretaken at the same magnification.