Biosensors and Bioelectronics 287 (2025) 117674

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

A plant-insertable multi-enzyme biosensor for the real-time monitoring of

stomatal sucrose uptake
Shiqi Wu a , Wakutaka Nakagawa a , Yuki Mori b , Saman Azhari a , Gábor Méhes a ,
Yuta Nishina c,d, Tomonori Kawano b, Takeo Miyake a,*
a
Graduate School of Information, Production and Systems, Waseda University, Kitakyushu, 808-0135, Japan
b
Faculty and Graduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu, 808-0135, Japan
c
Research Institute for Interdisciplinary Science, Okayama University, Okayama, 700-8530, Japan
d
Institute for Aqua Regeneration, Shinshu University, 390-8621, Matsumoto, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Monitoring sucrose transport in plants is essential for understanding plant physiology and improving agricultural
Flexible wearable sensor practices, yet effective sensors for continuous and real-time in-vivo monitoring are lacking. In this study, we
Plant monitoring developed a plant-insertable sucrose sensor capable of real-time sucrose concentration monitoring and demon­
Carbon fiber
strated its application as a useful tool for plant research by monitoring the sugar-translocating path from leaves
Multi-enzyme system
to the lower portion of plants through the stem in living plants. The biosensor consists of a bilirubin oxidase-
based biocathode and a needle-type bioanode integrating glucose oxidase, invertase, and mutarotase, with the
two electrodes separated by an agarose gel for ionic connection. The sensor exhibits a sensitivity of 6.22 μA
mM− 1 cm− 2, a limit of detection of 100 μM, a detection range up to 60 mM, and a response time of 90 s at 100 μM
sucrose. Additionally, the sensor retained 86 % of its initial signal after 72 h of continuous measurement. Day-
night monitoring from the biosensor inserted in strawberry guava (Psidium cattleianum) showed higher sucrose
transport activity at night, following well the redistribution of photosynthetically produced sugars. In addition,
by monitoring the forced translocation of sucrose dissolved in the stable isotopically labeled water, we
demonstrated that a young seedling of Japanese cedar known as Sugi (Cryptomeria japonica) can absorb and
transport both water and sucrose through light-dependently opened stomata, which is the recently revealed path
for liquid uptake by higher plants. These findings highlight the potential of our sensor for studying dynamic plant
processes and its applicability in real-time monitoring of sugar transport under diverse environmental conditions.

1. Introduction et al., 2023; Song et al., 2023; Ye et al., 2020), and point-of-care testing
(Bihar et al., 2016; Kulkarni et al., 2022; Lu et al., 2023). Enzymatic
Enzymatic biosensors have been widely used and developed for biosensors for glucose have also been used to monitor blood sugar levels
various applications, such as medical diagnoses (Huang et al., 2017; in animals (Bruen et al., 2017; Makaram et al., 2014; Miyake et al.,
Ispas et al., 2012), environmental monitoring (Justino et al., 2017; 2011a).
Purcarea et al., 2024), food and beverage industry (Gavrilaş et al., 2022; Despite the wide use of enzymatic biosensors in humans, animals,
Shankaran et al., 2007), and biotechnology and pharmaceuticals (Wang and insects, the applications in plants are relatively few, particularly for
et al., 2023; Yan et al., 2011; Zhou et al., 2023). Thanks to the specific monitoring plant metabolites (Perdomo et al., 2023; Taniguchi et al.,
catalytic character of enzymes, these biosensors could detect and 2020). There are some publications on real-time glucose monitoring
quantify various targeted analytes. At the current state, the research of from sap, root exudates, and even chloroplasts (Table S1). However,
enzymatic biosensors is mainly focusing on human health applications, other than glucose, one of the important and common plant metabolites
including disease detection and diagnosis (Hemdan et al., 2024; Tang is sucrose, which plays a crucial role in plant physiology as a primary
and Ren, 2008), therapeutic drug monitoring (Alvau et al., 2018; Kai product of photosynthesis and a major transport sugar (Huber and
et al., 2017; Ogawa et al., 2015), wearable health monitoring devices (Li Huber, 1992; Lastdrager et al., 2014; Sturm, 1999). In most higher

* Corresponding author.
E-mail address: [email protected] (T. Miyake).

https://doi.org/10.1016/j.bios.2025.117674
Received 11 March 2025; Received in revised form 31 May 2025; Accepted 6 June 2025
Available online 8 June 2025
0956-5663/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Wu et al. Biosensors and Bioelectronics 287 (2025) 117674

plants, glucose produced via photosynthesis is rapidly converted into (Chen et al., 2024),introduced a microneedle-based enzymatic sensor
sucrose in the cytosol before being loaded into the phloem. Sucrose then that enabled real-time glucose monitoring in plant stems. With a re­
acts as the main form of long-distance carbon transport, moving from ported sensitivity of 17 nA μM− 1 cm− 2, the sensor was demonstrated on
source tissues such as mature leaves to sink tissues like roots, young soft-tissue plants such as aloe and tomato. However, due to the fixed
leaves, and developing fruits (Lemoine et al., 2013; Ruan, 2014). Once length and depth of the microneedle array, its applicability to woody or
delivered, sucrose is metabolized by enzymes including invertase and structurally rigid plant tissues remains limited, and the inability to
sucrose synthase, which support its further utilization in energy pro­ adjust the insertion depth restricts its use across diverse plant species
duction and biosynthetic pathways such as starch and cellulose syn­ with varying sap flow depths.
thesis. Real-time monitoring of sucrose concentrations within plants To overcome these limitations and enable real-time monitoring of
could provide valuable data on plant health, growth, and response to dynamic sucrose changes within plant tissues—particularly those
environmental conditions. Nevertheless, the development of enzymatic introduced via light-regulated stomatal water uptake—we developed a
sensors for real-time sucrose monitoring in plants faces several chal­ needle-type implantable enzymatic biosensor capable of minimally
lenges, including the complex and variable plant shape, the need for invasive integration into a wide range of plant structures (Fig. 1),
miniaturization and integration of sensors into plant tissues without achieving sensitivity of 6.22 μA mM− 1 cm− 2, 100 μM limit of detection
causing damage, and ensuring the sensors’ sensitivity and selectivity in (LOD), and operational stability beyond 72 h of continuous measure­
the plant’s environment. In response to these challenges, researchers are ment. The anode uses a multienzyme electrode with a mixture of glucose
exploring more refined monitoring technologies to reveal the complex oxidase (GOD) and mutarotase (MUT) covered with invertase (INV),
responses of plants to environmental changes. while the cathode uses bilirubin oxidase (BOD), the latter reported
One such environmental change involves water exchange process previously (Yin et al., 2019, 2020, 2021). Our biosensor can provide
and the uptake of nutrients dissolved in water(Cao et al., 2015; Trom­ continuous measurements of sucrose levels with a low LOD and wide
melen et al., 2017).Water physiology is very important in higher plants, detection range. To achieve this, the insertion device is designed so that
but there are still many unclear points regarding the fates of water in the oxidation current from sucrose at the anode is lower than the
vivo and the mechanism of water exchange between the aerial parts of reduction current from oxygen at the cathode to extend the operation
plants and the surrounding environments. Conventionally, a model has lifetime. Furthermore, the use of carbon fibers as base electrodes ensures
been used in which the root system is solely involved in the uptake of the a compact, disposable, and environmentally friendly sensor with a form
liquid form of water and the discharge of water through transpiration by factor highly integrable with plants. Thus, our sensor has considerable
which the gaseous form of water (vapor) is released in the air through potential applications in agriculture and horticulture. Moreover, we
the stomatal opening on the leaves. Therefore, it is well considered that applied the sensor for real-time monitoring of sucrose uptake through
regulation of stomatal opening plays a central role in plant water cedar leaf stomata during light and dark exposure cycles. Using this
physiology in living plants. However, numerous recent observations implantable sucrose sensor, we can observe the immediate response of
suggest that plants may also absorb water through alternative pathways. plants to light, wherein sucrose uptake through the stomata is signifi­
Many lower plants can readily absorb water through their surfaces cantly enhanced during light exposure compared to dark periods. This
(Berry et al., 2019; Chin et al., 2023; Dawson and Goldsmith, 2018). In real-time monitoring device provides a method for precision agriculture,
arid environments, the desert plant, Syntrichia caninervis, has been allowing for the optimization of growth conditions and a better under­
observed to capture water from fog (Berry et al., 2019; Pan et al., 2016). standing of plant responses to environmental changes.
Even in higher plants, certain phenomena indicate that the cuticle may
play a role in water absorption (Boanares et al., 2018; Guzmán-Delgado 2. Materials and methods
et al., 2021). For example, fruit growers have often noted that cherries
and other fruits crack after maturing, especially when exposed to pro­ 2.1. Materials
longed rain, due to excess water on the fruit surface (Knoche, 2015;
Louise Hovland * and Sekse, 2004). Moreover, studies have demon­ Carbon fibers were obtained from FC-R&D Corp. (Sagamihara,
strated that blue light induces stomata opening, thereby allowing water Japan). TUBALL SWCNT solution (01RW03) was obtained from Kusu­
uptake into the plant’s water cycle. Our preliminary demonstrations moto Chemicals, Ltd. (Tokyo, Japan). Polyvinylimidazole-[Os
with model plants, such as the hydroponically grown seedlings of let­ (bipyridine)2Cl] (PVI-Os) was donated by Research Core for Interdis­
tuce, revealed that stromata opening in response to light can serve as a ciplinary Sciences, Okayama University. Glucose oxidase (GOD, solid,
pathway for the uptake of liquid water, in addition to the outward from Aspergillus sp., 180 U/mg) and Invertase (INV, solid, from Candida
movement of vapor (Noda et al., 2025). These findings promoted us to sp., 100 U/mg) were purchased from TOYOBO Biotechnology operating
verify a new water physiological model in which stomate contributes to department (Osaka, Japan). Mutarotase (MUT, suspension, from porcine
water exchange. In this preliminary study, we used young seedlings of kidney, 10000 U/mL), monopotassium phosphate, and dipotassium
Japanese cedar known as Sugi (Cryptomeria japonica) as a model to phosphate were purchased from FUJIFILM Wako Pure Chemical Cor­
confirm the movement of liquid water and solute (sucrose) through poration (Osaka, Japan). Bilirubin oxidase (BOD, solid, from Myrothe­
light-regulated stomata. cium sp., 10 U/mg) was purchased from Amano Enzyme Inc. (Nagoya,
However, verifying such a physiological model requires tools Japan). Glutaraldehyde (Grade II, 25 % in H2O) was purchased from
capable of monitoring solute transport—in particular, sucro­ Sigma Aldrich. Charge pump IC (S-882Z20, input voltage (0.3–3 V),
se—continuously, quantitatively, and in real time within intact plant output voltage (VIC, 2V), discharge starting voltage: 2.4 V, and shut­
tissues. This demand highlights the need for biosensing technologies down voltage: 2.5 V) was purchased from Mouser Electronics, Inc.
that can operate stably inside living plants, detect low concentrations of (Texas, USA). Deionized water made by Kitakyushu tap water was used
sucrose, and withstand mechanical and environmental challenges. To as a source of fresh water for plants. For water tracing experiments,
date, enzymatic biosensors for plant sucrose monitoring have made Kitakyushu tap water and commercially available bottled Alaskan
substantial progress in form factor and integration. For example, Diacci glacial water (Alaskan Glacier Products Chugiak, Alaska, USA) sampled
et al. (2021) developed an organic electrochemical transistor (OECT)-­ from Eklutna Lake, Alaska state, USA, was used.
based sensor capable of continuous monitoring for up to 48 h. However,
this platform only enabled qualitative tracking of sucrose levels in plants 2.2. Preparation of sucrose bioanode
and was not suitable for quantitative analysis. In addition, its patch-like
structure limits its applicability in mechanically rigid plant tissues and The preparation of bioanode and related materials’ characterizations
may suffer from low resistance to physical disturbances. More recently was based on our previous work (Yin et al., 2019, 2020, 2021). In short,

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S. Wu et al. Biosensors and Bioelectronics 287 (2025) 117674

Fig. 1. Schematic figure of the needle-type implantable enzymatic biosensor specifically designed for real-time monitoring of sucrose concentrations within
plant tissues.

5 cm long carbon fiber (CF) was dip-coated in a 50 μL SWCNT solution AgCl (3 M KCl) electrode served as the reference electrode. To evaluate
(TUBALL), then dried at 90 ◦ C on a hot plate for 10 min, repeated thrice. the responsivity of the sucrose sensor, the experimental setup involved
The coated SWCNT/CF was then washed in distilled water for 30 min to stirring 8 mL of 22 ◦ C PBS solution while incrementally adding high-
remove loosely attached SWCNT. For further functionalization, the concentration sucrose solutions (0.5 M) in volumes of 1.6 μL (equiva­
SWCNT/CF was immersed into a PVI-Os solution while stirring at 4 ◦ C lent to 0.1 mM) and 16 μL (equivalent to 1 mM). The full sensor was
for 2 h, followed by 30 min of washing in distilled water. The GOD-MUT tested in 5 mL PBS while stirring.
solution was made by mixing GOD (1.5 mg/mL), MUT (12.5 μL/mL),
glutaraldehyde (12.5 μL/mL), and phosphate-buffered saline (PBS, 50 2.5. Preparation and testing of needle-type injection device
mM KH2PO4, pH = 7) solutions. The INV solution was made by mixing
INV (2.5 mg/mL), glutaraldehyde (12.5 μL/mL), and PBS (50 mM). The needle-type injection device is composed of an injection needle
Then, the PVI-Os/SWCNT/CF was immersed into a GOD-MUT solution (18G, Terumo), a custom-made 3D-printed (Form 3+, Formlabs, USA)
under stirring at 4 ◦ C for 2 h, followed by immersing the (GOD-­ needle holder, and enzymatic bioelectrodes (Fig. S1). To expose the
MUT)/PVI-Os/SWCNT/CF in INV solution under stirring at 4 ◦ C for 2 h. enzyme electrodes and increase the surface area available for chemical
reactions, the tip of the needle was modified by creating holes (0.8 mm
2.3. Preparation of O2-Diffusion bilirubin biocathode diameter) with a grinder, as mentioned in previous work (Yin et al.,
2020). The sucrose sensing bioanode length exposed at the needle tip
The O2-diffusion biocathode was fabricated and optimized using the was standardized at 15 mm to ensure consistent performance across
method reported in our previous work (Yin et al., 2019, 2020, 2021). various samples and to maintain the structural integrity of the needle.
The CF fabric was cut into a 15 mm × 15 mm square shape. Then, the CF The agarose solution (1 wt %) was filled and solidified inside the needle
fabric was dip-coated with a 225 μL SWCNT solution (TUBALL) and then and the needle holder, allowing the protons to move through the agarose
dried at 90 ◦ C on a hot plate for 10 min. This process was repeated thrice. gel between bioanode and biocathode. Oxygen diffusion through the
The coated SWCNT/CF was then washed in distilled water for 30 min to holes in the needle holder happens via the agarose gel to the biocathode
remove loosely attached SWCNT. This step will also remove the sur­ as well.
factant contained in the product and make CNT hydrophobic. Then, the
SWCNT/CF was cut into a ring shape with an outer diameter of 12 mm 2.6. Evaluation of water uptake by tracing the stable isotope ratio
and an inner diameter of 4 mm. The SWCNT/CF was then immersed into
a solution of bilirubin oxidase (5 mg/mL) in 50 mM PBS solution, under To investigate plant metabolic pathways, traditional methods such as
stirring at 4 ◦ C for 12 h. fluorescence labeling, isotope labeling, and radioactive tracers have
been widely used. However, fluorescence labeling is often limited by the
2.4. Electrochemical measurements and biosensor assembly and testing natural pigmentation of plant leaves, which can interfere with fluores­
(non-needle form) cence signals and reduce the accuracy of the results. Similarly, radio­
active tracers, while highly sensitive, pose safety concerns and are less
The electrochemical properties of the enzymatic sucrose sensor were practical for experiments involving living plants over extended periods.
systematically evaluated to assess its responsiveness, stability, and To overcome these challenges, we employed isotope labeling as an
suitability for real-time monitoring. Chronoamperometry and cyclic alternative. Isotope labeling is non-invasive, safe, and capable of
voltammetry (CV) were employed to quantify current responses and providing precise insights into metabolic pathways. Specifically, we
characterize the sensor’s behavior under varying sucrose concentrations utilized water from two different regions with distinct oxygen-18 (18O)
and environmental conditions. For bioanode and biocathode testing, isotope compositions. It is well-established that water from different
both electrodes were connected as the working electrode, while a plat­ geographical sources contains varying 18O isotope levels due to envi­
inum wire electrode was used as the counter electrode, and a wire Ag/ ronmental and climatic factors. By using these naturally varying 18O

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S. Wu et al. Biosensors and Bioelectronics 287 (2025) 117674

compositions, we could track water uptake through leaf stomata and similar multi-enzyme system, but in a different electrode material. In our
determine whether plants absorb water directly from their leaf surfaces. multi-enzyme system, GOD is the only enzyme responsible for trans­
This approach not only avoids the limitations of other methods but also ferring electrons to the electrode mediated by PVI-Os. To ensure that the
leverages natural isotopic variations to study plant water absorption glucose generated by INV and MUT could be fully utilized by GOD, we
processes effectively. increased the amount of GOD and set its concentration equal to that of
Physicochemical analysis of water intake by typical coniferous leaf INV. The revised ratio was evaluated using cyclic voltammetry in
tissues of intact living Sugi seedlings was performed according to the Fig. S2. The results showed that the oxidation peak was significantly
protocol proposed by Kawano Lab (Noda et al., 2025). Briefly, the higher with the 2 : 1: 2 ratio compared to the 10 : 5: 1 ratio for our
detection and quantification of stable isotopes of oxygen (18O over electrode, confirming that the new combination improved the overall
oxygen-16 (16O)) in the water samples or leaf extracts (1 μL, each) signal output. Therefore, this ratio was used for all subsequent
loaded to Ag capsules were carried out by using a stable isotope ratio experiments.
mass spectrometer (IsoPrime100; IsoPrime, Cheadle, UK) connected For the multi-enzyme system (Fig. 2a(i)), it is important to properly
with an element (CNSOH) analyzer (vario PYRO cube; IsoPrime, Chea­ arrange the immobilization sequence of the three enzymes to align with
dle, UK). the reaction steps. As shown in Fig. 2a(ii), based on spatial structure
simulation of INV, MUT, and GOD, INV appears as an ellipsoid, MUT as a
3. Results and discussion cylinder, and GOD has a flattened disk shape (Alberto et al., 2004;
Thoden and Holden, 2002; Wohlfahrt et al., 1999). Despite the differ­
3.1. Description of the sensing concept ences in shape, the sizes of these enzymes are similar, allowing us to try
various sequences of immobilizations without worrying that larger en­
Biofuel cell (BFC)-type devices are traditionally employed for power zymes would block smaller ones. Therefore, we tested the following
generation due to their ability to efficiently convert chemical energy enzyme immobilization sequences: 1) immersion in a GOD and MUT
into electrical energy. However, as shown in Fig. 1, in this study, we mixed solution followed by an INV solution ((GOD + MUT),INV); 2)
adapted a BFC for sucrose sensing, leveraging this technology to achieve immersion in a mixed solution of GOD, MUT, and INV all together (GOD
real-time and highly sensitive detection. The enzymatic bioanode and + MUT + INV); 3) immersion first in a GOD solution, followed by a
biocathode were designed to maximize the precision of substrate mixture of MUT and INV (GOD,(MUT + INV)); and 4) immersion in each
detection, ensuring that the device output directly corresponds to the of GOD, MUT, and INV solutions in this order (GOD, MUT, INV).
measured sucrose concentration rather than to energy generation. This Chronoamperometry was employed to measure the current outputs
design offers several advantages over conventional sensing devices. of bioanodes with four different enzyme immobilization sequences in
First, the integration of a multi-enzyme bioanode (glucose oxidase, stirred PBS solutions containing sucrose at concentrations from 0.1 to
mutarotase, and invertase) enables efficient and sequential catalysis of 100 mM in increments of two- or five-fold. Fig. 2b shows that after
sucrose, producing electron transfer directly proportional to sucrose having tried sequences 1–4, sequence 1, i.e., immersing the electrode
concentration. Second, the oxygen diffusion biocathode, based on bili­ first in a GOD and MUT mixed solution, followed by an INV solution (red
rubin oxidase, facilitates stable reduction reactions under air-saturated dots), resulted in the highest current density (12.08 μA/cm2 at 50 mM
conditions. Additionally, the use of PVI-Os as an embedded electron sucrose). We hypothesize that this sequence allows GOD and MUT to be
transfer mediator in the bioanode significantly enhances electron immobilized in the inner layer closer to the electrode material, facili­
transfer efficiency compared to traditional systems that rely on freely tating electron transfer and enabling the newly formed β-glucose to be
diffusing mediators. Together, these features should ensure high sensi­ immediately oxidized by the adjacent GOD. Meanwhile, subsequent
tivity, rapid response times, and a wide detection range. immobilization of INV on the electrode surface maximized the contact
Specifically, the multi-enzyme bioanode acted on sucrose by cata­ area with sucrose, thereby enhancing the conversion of sucrose to
lyzing it into gluconic acid in three steps. First, invertase catalyzed the α-glucose. Moreover, the LOD was determined based on the standard
breakdown of sucrose into α-glucose and fructose. Then, mutarotase deviation of the blank signal and the logarithmic calibration curve.
converted α-glucose into β-glucose. Finally, glucose oxidase catalyzed Using the criterion of S/N = 3, the LOD was calculated to be 100 μM,
the oxidation of β-glucose into gluconic acid at the anode. α-glucose and corresponding to a current response of 1.175 μA.
β-glucose are two isomeric forms of glucose, differing in the orientation At a sucrose concentration of 100 mM, the current level of (GOD +
of the hydroxyl group (Chiba, 1997; Hudson, 1910). Both forms exist MUT), INV was nearly identical to that of (GOD + MUT + INV), sug­
naturally, with an approximate ratio of α-glucose to β-glucose of 9:16 gesting the catalytic limit of the electrode was reached. However, at a
(Weng et al., 2023). Since glucose oxidase can only catalyze the oxida­ sucrose concentration of 50 mM, the current level of the (GOD + MUT),
tion of β-glucose (Mano, 2019), the use of mutarotase to convert INV configuration was significantly higher than that of the other groups.
α-glucose is essential. Therefore, we focused further experiments on ((GOD + MUT), INV)
Our device also incorporates an agarose gel interface to prevent anode in different sucrose concentrations. As shown in the CV graph
short-circuiting while enabling efficient proton transfer between the (Fig. 2c), the oxidation peak current increased linearly with sucrose
bioanode and biocathode. These modifications make the sensor highly concentration in the range from 0 mM to 60 mM, but there was only a
adaptable to varying environmental conditions, such as temperature and little difference between the oxidation peak currents at 60 mM and 70
oxygen availability, and improve its reliability for in vivo plant moni­ mM, indicating that the electrode had reached its catalytic limit at these
toring. The choice of carbon material ensures that the electrodes do not concentrations.
pollute the environment and do not negatively impact the commercial or Since the sucrose sensor developed in this study is a plant-insertable
edible value of plants. By adapting a fuel cell-type technology for sensing device, it should reliably operate under varying temperature conditions
applications, this sensor bridges the gap between energy devices and for real-world applications. These conditions may include day-night
biosensing applications, offering a robust platform for studying dynamic temperature fluctuations or controlled environments such as green­
plant processes with high precision and efficiency. houses. To understand the relationship between temperature and the
sensor’s electrical output, we evaluated its performance at different
3.2. Electrochemical performance of bioanode, biocathode, and the temperatures. In this experiment, a 10 mM sucrose PBS solution was
complete enzymatic sensing system initially cooled in a refrigerator and then stirred on a hot plate. CV
measurements were performed when the solution reached specific
The ratio of INV: MUT: GOD is 2:1:2. A previous study by Hamid J.A. temperatures of 10 ◦ C, 22 ◦ C, 30 ◦ C, and 40 ◦ C. The results, as shown in
et al. (Hamid et al., 1988) reported an effective ratio of 10:5:1 for a Fig. 2d, demonstrate that the sensor generates stable electrical signals

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Fig. 2. Electrochemical characterization of bioanode and biocathode. a) Graphical illustrations of (i) the multi-enzyme system used in the bioanode for sucrose
sensing and (ii) the spatial structure of bioanode. b) Concentration dependence of sucrose conversion by bioanode with different enzyme immobilization sequences in
the range of sucrose concentrations from 0.1 to 100 mM, determined by chronoamperometric measurements at +0.6 V. CV scans of (GOD + MUT), INV bioanode at
10 mV⋅s-1 in PBS (each point is the average and the error bars are the standard deviation between three samples (n = 3)) c) at different sucrose concentrations at
22 ◦ C, and d) in 10 mM sucrose at 10 ◦ C, 22 ◦ C, 30 ◦ C, 40 ◦ C. e) Current response of (GOD + MUT), INV bioanode in 0.1 mM and 1 mM sucrose solutions, respectively.
f) Schematic of the gas-diffusion BOD cathode used in the sensor. g) CV scans of biocathode at 10 mV s− 1 in PBS, PBS saturated with N2, PBS saturated with O2 and on
PBS absorbed agarose gel, at 22 ◦ C. h) Power and polarization curves of the full biosensor in 50 mM sucrose.

across this temperature range. Furthermore, the signal intensity sensitivity (0.56 μA mM− 1) divided by the electrode surface area (0.09
increased with rising temperature, indicating that moderately high cm2), yielding a current density sensitivity of 6.22 μA mM− 1 cm− 2.
temperatures enhance the electrochemical response of the sensor. These Next, to assess the efficiency of our gas-diffusion BOD cathode
findings suggest that the sensor’s performance is temperature- (Fig. 2f), we determined the reduction currents produced from atmo­
dependent, and the observed variations in signal intensity can be spheric oxygen. During these measurements, the cathode was positioned
effectively standardized based on the measurement temperature. This on an agarose gel that had been saturated with PBS, and the other face of
temperature normalization allows for more accurate sucrose concen­ the cathode was exposed to air. Because of the low solubility and slow
tration readings in practical applications, ensuring reliable measure­ diffusion rate of dissolved oxygen in aqueous solutions, the BOD cathode
ments across diverse environmental conditions. relies heavily on an effective gas-diffusion electrode for highly catalytic
To achieve real-time monitoring of sucrose concentrations, the im­ performance. Due to the intrinsic structural properties of the SWCNT,
mediate response of the sensor electrode to target sucrose molecules is the material exhibited high hydrophobicity, effectively promoting air
critical. Thus, we measured the current generated by enzymatic re­ oxygen contact with the electrode’s surface.
actions using chronoamperometry at a constant potential of +0.6 V vs As shown in Fig. 2g, the CV scans performed in PBS solution satu­
Ag/AgCl (3 M KCl). As shown in Fig. 2e, the response time of the rated by N2 (dashed line) displayed no reduction current signal in the
fabricated sucrose sensor was ~90 s and ~2 min for the addition of 0.1 absence of oxygen. When the cathode was immersed into non-degassed
mM and 1.0 mM sucrose, respectively. Compared to other sucrose sen­ PBS (black line), a distinct oxygen reduction peak was observed,
sors relying on hydrogen peroxide-mediated electron transfer, which attributable to the dissolved oxygen in the PBS. In contrast, when the
typically requires around 5 min at the concentration of 5.6 mM (Scheller electrode was placed on an agarose gel (red line), the surface of the
and Renneberg, 1983) for response, the highly efficient electron transfer cathode was exposed to air, resulting in a greater current compared to
of PVI-Os enabled faster electrode responses of 1.5 (0.1 mM) and 2 (1.0 when it was fully submerged in PBS. This enhanced current is indicative
mM) minutes, respectively. This rather fast response shows the potential of improved oxygen availability at the electrode surface. Finally, with
of the sensor for practical real-time measurements, owing to the use of PBS saturated with oxygen (blue line), the dissolved oxygen concen­
PVI-Os that significantly improve the efficiency of electron transfer, tration reached saturation, resulting in a more pronounced enzymatic
reducing the time required for the sensor to detect changes in sucrose response by the BOD catalyst. This further confirmed that the reduction
concentration. Moreover, the sensitivity was calculated as total current peak observed was associated with oxygen reduction, with the reduction

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reaction initiating at approximately +0.55 V vs Ag/AgCl (3 M KCl). using a grinder, as shown in Fig. 3a. Since sucrose was transported in the
Finally, the performance of the sucrose BFC-sensor, constructed from phloem, which is located near the surface of stems and branches, precise
the functionalized multi-enzyme bioanode and oxygen diffusion bio­ insertion of the needle tip into this layer allows the biosensor to detect
cathode is shown in Fig. 2h. In a 50 mM sucrose solution, an open circuit sucrose transported in the sap. The concentration of sucrose in this re­
potential of 0.6 V was observed, which matches the value calculated as gion is regulated by the plant itself and is not affected by the insertion
the sum of the anode (0.1 V, Fig. 2c) and cathode (0.5 V, Fig. 2g) re­ process, as long as the correct tissue layer is reached.
action potentials. The maximum power of the sensor was measured to be To test our sensor, we measured the sucrose concentration in
49 μW at 0.21 V. strawberry guava. Before performing this experiment, as a proof of
Moreover, to verify the temperature stability and reversibility of the concept first we wanted to see whether this BFC-type sensor can
device, we tested the sensor response to 50 mM sucrose solution under generate enough power to light up an LED. Therefore, as described in
varying temperatures with the sequence of 10 ◦ C → 22 ◦ C → 30 ◦ C → our previous work (Miyake et al., 2011a, 2011b), we combined the su­
40 ◦ C → 30 ◦ C → 22 ◦ C → 10 ◦ C (Fig. S3). The results demonstrate crose sensor with a blinking device that includes a charge pump inte­
consistent signals at the same temperature, indicating that the sensor grated circuit (Fig. 3b(i)), a red light-emitting diode (LED), and a 0.47 μF
can maintain measurement accuracy despite temperature fluctuations. capacitor. Since the LED has a driving voltage of 1.7 V and the
open-circuit potential of the sucrose sensor in this study is 0.6 V, we used
a booster circuit to deliver enough voltage to the LED (Fig. 3b(i)). Upon
3.3. Performance of the needle-type sensor in the fruit and stem of insertion into the fruit of the strawberry guava, the sucrose sensor de­
strawberry guava (Psidium cattleianum) tects an electrical signal generated from the sucrose in the sap, which is
then stored in the capacitor. When the capacitor accumulates sufficient
The cross-sectional view of the needle-shaped sucrose sensor devel­ charge, the charge pump circuit discharges, producing a 1.7 V voltage
oped in this study is illustrated in Fig. 3a. It consists of two main parts: that momentarily lights the LED, creating a blink. A higher sucrose
the sensor body, which includes the agarose gel and electrodes, and the concentration results in a stronger electric signal that fills the 0.47 μF
needle section, which partially encloses the bioanode. The sensor body is capacitor in a shorter time, leading to a higher blinking frequency,
fabricated using 3D printing, with a mesh structure in the upper portion which in turn reflects the sucrose concentration in the fruit of strawberry
to allow free oxygen diffusion from the air to the biocathode. The bio­ guava.
cathode, in the form of a disc, is positioned in the middle section, To calibrate the blinking frequency to sucrose concentration, we
beneath which lies the agarose gel immersed into 50 mM PBS solution, counted the number of blinks of the LED for PBS solutions with sucrose
serving as a medium for proton transport from the bioanode to the concentrations of 5, 10, 30, and 40 mM, as well as when inserted into
biocathode. A mesh-like tube surrounding the bioanode separates the strawberry guava, with measured frequencies of 0.46 Hz, 0.77 Hz, 3.34
two electrodes, preventing direct contact and a potential short-circuit Hz, 4.98 Hz, and 4.64 Hz, respectively (Fig. 3b(ii)). The last value (4.64
while permitting free diffusion of liquid and protons. Hz) was measured in strawberry guava, corresponding to a sucrose
The needle portion containing a commercially available standard concentration of ~38 mM.
hypodermic needle with a diameter of 1.2 mm allows for a smooth Fig. 3c(i) shows the real-time sucrose concentration monitoring in
passage of the electrode and proper filling of the agarose gel within the plant sap in the stem of a strawberry guava plant grown in soil over 24 h.
needle. Additionally, as discussed in our previous work (Yin et al., The sensor was operated in a chronoamperometric regime with a fixed
2020), the needle was modified to increase the bioanode’s exposure to potential of +0.6 V, and changes in the measured current correspond to
plant sap at the tip, thereby markedly shortening the response time of changes in sucrose concentration, as shown in Fig. 3c(ii). Sucrose con­
chemical measurements and facilitating the inflow of fresh centration decreased progressively to 2.22 mM during the daytime,
sucrose-containing plant sap and the outflow of the used sap. This while at night, it steadily increased to 4.3 mM, which concentration is
modification was realized by creating four openings in the needle tip

Fig. 3. a) Schematic for the cross-sectional view of the needle-shaped sucrose sensor. b) (i) Schematic of sucrose measurement circuit in the fruit of strawberry guava
with blinking device that includes a charge pump integrated circuit; (ii) blink frequency of the LED measured in strawberry guava and in solutions of different sucrose
concentrations (SI video1). c) (i) Schematic for real-time day-night monitoring of sucrose concentration in the stem of the strawberry guava plant using a poten­
tiostat; (ii) 24-h monitoring of sucrose.

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S. Wu et al. Biosensors and Bioelectronics 287 (2025) 117674

within the physiological range(Secchi and Zwieniecki, 2016). This Moreover, to further investigate the movement of water molecules
pattern suggests that sucrose transport is more active at night. During through the stomatal aperture, we devised a new protocol to identify
the day, under sunlight exposure, the plant leaves produce glucose plant-derived water molecules released into the exogenous bathing
through photosynthesis, generating and accumulating sugars as nutri­ medium. This was achieved by detecting proton (1H) signals from H2O
ents. However, when photosynthesis ceases at night, the accumulated molecules released into deuterium-based water (2H2O, or D2O, purity
sugars are transported through the stem’s vascular system to supply the 99.9 %). After immersing intact Sugi leaves into D2O-filled plastic bags
entire plant. Studies have indicated that plants tend to grow more for up to 60 min, the migration of H2O from the leaves was confirmed by
significantly at night, and our experimental findings support this monitoring the 1H signal in the D2O medium using a benchtop 1H-NMR
observation (Zhu et al., 2021; Zweifel et al., 2021). (NMReady60pro, Nanalysis Corp, Calgary, Canada).
The rate of water efflux was enhanced under light. When the kinetics
of water movement was simulated with the Michaelis-Menten-like Hill-
3.4. Monitoring sucrose uptake through stomata in Japanese cedar type equation (V = (Vmax tn)/(Knt + tn), where V is the 1H signal intensity
(arbitrary units), t is time (min), Kt is the apparent Michaelis constant,
After successfully testing our needle-type BFC sensor for sucrose and n is the Hill coefficient) as a function of time, Kt recorded under the
sensing in strawberry guava, we proceeded to test the real-time moni­ light was 1/3 of the one recorded in the dark (Fig. S4), suggesting that
toring of sucrose uptake through stomata –to the best of our knowledge, the time required for attaining the equilibrium was largely shortened
for the first time. We used Japanese cedar as our model species (Fig. 4a). under illumination. Specifically, the fitted curves revealed a lower Kt
In our experiments, we employed two types of water with stable dif­ under backlight (BL; Kt = 14) compared to dark (Kt = 41), indicating
ferences in the isotopic composition of 18O and 16O: Alaskan water and faster water exchange under light-stimulated stomatal opening. Thus,
Kitakyushu local water. This distinction allowed us to assess water our analysis supports the role of water translocation across the light-
transport through Japanese cedar leaves quantitatively, as explained in stimulated aperture of the stomata in Sugi leaves. Since sucrose is
Section 2.6. water-soluble, we also investigated whether sucrose could enter the
Japanese cedar used in the experiment was ordinarily grown in plant body along with water absorbed through the leaves. To explore
Kitakyushu water. Therefore, the amount of Alaskan water absorbed this, we used the sucrose sensor developed in this study to monitor real-
through the leaf surface could be determined by measuring the level of time sucrose concentrations in Japanese cedar and inserted the needle
18
O (Fig. 4b). We varied light exposure by covering some leaves with into a branch (Fig. S5). Chronoamperometry was used at the potential of
aluminum foil (Fig. 4c) by immersing leaves in Alaskan water within +0.6 V, to determine the current density reflecting the sucrose con­
plastic bags. After 8 h, we measured the 18O content in the plant using an centration (Fig. 4e). The experiment began after 4 h in order for the
isotope analyzer. As shown in Fig. 4d, the experimental group exposed current to reach a plateau and to achieve a stable experimental envi­
to light had a higher 18O content than those kept in darkness, indicating ronment. At the 4-h mark, the Japanese cedar leaves were immersed into
that Japanese cedar leaves can absorb water through the leaf surface a 50 mM sucrose solution, and light exposure was alternated every 4 h.
under light exposure, consistent with previous findings (Noda et al., During the 4 to 8-h measurement period, the sucrose concentration
2025).

Fig. 4. Real-time monitoring of changes in sucrose levels in Japanese cedar reflecting the light-dependent uptake of sucrose solution through stomata.
Evidence of the intake of foreign water differed in isotopic property through intact leaf surface of Sugi seedling: a) intact leaf of Sugi grown in local water with
moderate H18 18 18 18
2 O content (δ O = − 5.05); b) leaf tissue soaked in foreign water (Alaskan water with low H2 O content (δ O = − 16.53); c) leaf soaked in Alaskan
water but covered with aluminum foil as dark control; d) comparison of δ18O values in leaf water extracted after 8 h of incubation with Alaskan water under daylight
or in the dark, average and standard deviation were calculated from n = 3. e): Real-time chronoamperometric (V = +0.6V vs BOD biocathode) monitoring of sucrose
concentration in Japanese cedar soaked in 50 mM sucrose in different light conditions (4–8 h: light-off, 8–12 h: light-on, 12–16 h: light-off, 16–20 h: light-on).

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S. Wu et al. Biosensors and Bioelectronics 287 (2025) 117674

detected under dark conditions showed only minor changes, similar to photosynthetic performance of individual plants under changing envi­
those observed in Fig. 3c(ii), presumably originating in the plant’s ronmental conditions (Okamoto et al., 2016). However, successful sys­
intrinsic metabolic activity. This observation suggests that without light, temic allocation through finely controlled translocation of the
the stomata remain closed, preventing water and sucrose entry into the photosynthetic products among the plant organs meeting the changing
plant, and only the plant’s internal sugar transport was active. Between local demands must be the missing piece among the key parameters to
8 and 12 h, with light exposure, the stomata opened, enabling water and assess the orchestrated growth of whole plants. Therefore, the applica­
sucrose absorption through the leaves, resulting in a marked increase in tion of our novel plant-insertable sensors, allowing the real-time moni­
sucrose concentration. Despite some variability, a clear upward trend in toring of the dynamic translocation of the sugars within the living
sucrose concentration was observed. plants, may provide novel tools for agricultural researchers and field
At 12 h, the light was turned off, and as the absorbed sucrose was agricultural engineers. In future work, applying this platform to other
gradually transported to other parts of the plant, the sucrose concen­ plant organs such as leaves, roots, and seeds, may offer valuable insights,
tration in the measured area decreased rapidly. However, the concen­ for example, sink dynamics, root exudation processes, and sugar mobi­
tration remained higher than that during the 4 to 8-h period, presumably lization during seed germination, enabling a more complete under­
due to the initial high sucrose uptake. This may also be due to the plant’s standing of whole-plant physiological coordination.
instability to transport and store large amounts of sucrose in a short It should be noted that plant sap contains various secondary me­
time. At 16 h, the light was reintroduced, initiating the same water tabolites such as phenolic compounds, which may potentially interfere
absorption process and causing a subsequent rise in sucrose concentra­ with enzymatic reactions. In this system, only GOD is directly involved
tion. Interestingly, the peak sucrose concentration during this period in generating the electrochemical signal. Although some phenolic
(4.5 mM) was comparable to that observed between 8 and 12 h (4.5 compounds have been reported to inhibit GOD activity in peroxidase-
mM), suggesting a saturation limit for sucrose absorption through the coupled colorimetric assays (Wong and Huang, 2014), our sensor uses
leaves of the tested Japanese cedar. Taken together, we successfully direct electrochemical detection, which minimizes such interference.
demonstrated real-time monitoring of sucrose levels in Japanese cedar Furthermore, both enzymes are present in excess and do not participate
(‘Sugi’) introduced through stomatal uptake for the first time using a in redox reactions. Therefore, we consider the impact of such interfer­
BFC-type multienzyme biosensor. ence to be negligible in our measurements. Nevertheless, a more sys­
To further evaluate the reproducibility and operational stability of tematic investigation of interference from plant-derived metabolites
the device, we monitored the sensor’s response over a three-day period would be valuable in future studies.
by inserting it into a Sugi branch with young leaves immersed in 50 mM The current needle-type biosensor was applied in a one-time mea­
sucrose solution. The plant underwent light/dark cycles every 12 h, surement fashion under controlled laboratory conditions, and localized
consistent with standard cultivation conditions. As shown in Fig. S6, the insertion into lignified tissues such as stems or branches was used to
sensor exhibited a response pattern over the first two days that closely minimize physiological impact. Although some tissue disruption is
resembled the trend observed in Fig. 4e, confirming the reproducibility inevitable, the effect remains limited to the insertion site and does not
of the device under similar experimental conditions. On the third day, a significantly influence overall plant function during short-term moni­
moderate fluctuation and decline in signals were observed, which we toring. In future practical applications, the electrode housing and
attribute to partial drying of the agarose gel and possible loss of enzy­ insertion interface can be further optimized depending on the structural
matic activity over time. properties of different plant species. For instance, microneedle-based
However, as demonstrated in the device lifetime test performed in electrodes, thinner tips, or flexible substrates may reduce invasiveness
25 mM sucrose solution Fig. S7, the sensor retained 86 % of its initial and potentially allow repeated measurements. These engineering im­
signal after 72 h of continuous measurement, suggesting that the overall provements would expand the sensor’s applicability for long-term or
operational stability remains acceptable for extended use. field-based monitoring while minimizing plant stress.
It is observed that when the lighting condition changes, the signal
changes with a lag of about 45 min, which is greater than the 90 s 4. Conclusions
response time of the sensor. Therefore, the lag time is limited by the
sucrose transport speed in the phloem of the plant. In plant physiology, In this study, we developed a plant-insertable sucrose sensor
phloem transport of sucrose occurs on the timescale of minutes to hours, featuring a multi-enzyme bioanode integrating glucose oxidase, inver­
depending on the plant species, environmental conditions, and sour­ tase, and mutarotase, paired with a bilirubin oxidase-based biocathode.
ce–sink activity. For instance, studies using radiolabeled carbon or This sensor demonstrated rapid and reliable real-time monitoring of
fluorescent tracers have reported sucrose transport speeds ranging from sucrose uptake through stomatal pathways, with a response time of 90 s
1 to 10 mm per minute in higher plants (Giaquinta, 1983; Turgeon, for 0.1 mM sucrose and 2 min for 1.0 mM sucrose. The efficient electron
2010). As sucrose accumulation or depletion due to processes such as transfer mediated by PVI-Os significantly enhanced the sensor’s
stomatal regulation, photosynthetic activity, or circadian rhythms responsiveness and stability.
typically evolves over periods longer than a few minutes, the sensor’s Experimental results indicated that the sensor could detect sucrose
response time is acceptable for real-time physiological monitoring in concentrations as low as 100 μM, which was identified as the LOD, and a
intact plants. linear detection range of 100 μM–60 mM, covering physiologically
These experimental results highlight the sensor’s capability to relevant sucrose concentrations in plants. Electrochemical measure­
monitor dynamic changes in sugar levels under varying environmental ments confirmed that the sensor remains functional across a tempera­
conditions. Such real-time monitoring opens new possibilities for con­ ture range from 10 ◦ C to 40 ◦ C, with enzymatic activity preserved and
necting plant metabolic activity with practical agricultural manage­ reversible changes observed in signal response, enabling its use under
ment. Photosynthesis and translocation of sugars, chiefly in the forms of varying environmental conditions. Day-night monitoring of strawberry
glucose and sucrose, determine the growth rate and eventually yield of guava (Psidium cattleianum) revealed higher sucrose transport activity
starch and biomass in all agricultural crops. Therefore, monitoring the at night, consistent with the redistribution of photosynthetic sugars.
photosynthetic productivity and utilization of the photosynthetic Furthermore, experiments with isotopically labeled water demonstrated
products within the living plants is the key to predicting the agricultural that Japanese cedar can absorb and transport both water and sucrose
productivity both in the field and under horticultural facilities such as through stomatal pathways under light conditions.
greenhouses. In order to simulate and control plant growth based on These findings highlight the potential of this novel sucrose sensor for
mathematical models (Kawano et al., 2020; Okamoto et al., 2016), studying dynamic plant processes and its applicability in real-time
several researchers are engaged in the monitoring and simulation of monitoring of sugar transport across diverse plant species and

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S. Wu et al. Biosensors and Bioelectronics 287 (2025) 117674

environmental conditions. Looking forward, future work should focus Bruen, D., Delaney, C., Florea, L., Diamond, D., 2017. Glucose sensing for diabetes
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