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Abaxial leaf surface-mounted multimodal wearable sensor for continuous

plant physiology monitoring

Article in Science Advances · April 2023

DOI: 10.1126/sciadv.ade2232

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ENGINEERING Copyright © 2023 The

Authors, some
Abaxial leaf surface-mounted multimodal wearable rights reserved;
exclusive licensee
sensor for continuous plant physiology monitoring American Association
for the Advancement
of Science. No claim to
Giwon Lee1,2,3, Oindrila Hossain1, Sina Jamalzadegan1, Yuxuan Liu2, Hongyu Wang2, original U.S. Government
Amanda C. Saville4, Tatsiana Shymanovich4, Rajesh Paul1, Dorith Rotenberg4,5, Works. Distributed
Anna E. Whitfield4,5, Jean B. Ristaino4,5, Yong Zhu2*, Qingshan Wei1,5* under a Creative
Commons Attribution
Wearable plant sensors hold tremendous potential for smart agriculture. We report a lower leaf surface-attached NonCommercial
multimodal wearable sensor for continuous monitoring of plant physiology by tracking both biochemical and License 4.0 (CC BY-NC).
biophysical signals of the plant and its microenvironment. Sensors for detecting volatile organic compounds
(VOCs), temperature, and humidity are integrated into a single platform. The abaxial leaf attachment position is
selected on the basis of the stomata density to improve the sensor signal strength. This versatile platform
enables various stress monitoring applications, ranging from tracking plant water loss to early detection of
plant pathogens. A machine learning model was also developed to analyze multichannel sensor data for quan-
titative detection of tomato spotted wilt virus as early as 4 days after inoculation. The model also evaluates
different sensor combinations for early disease detection and predicts that minimally three sensors are required
including the VOC sensors.

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INTRODUCTION Recently, several different sensor technologies have been devel-
The United Nations announced that 2020 is the International Year oped for plant health monitoring, such as imaging and spectro-
of Plant Health (IYPH), emphasizing the importance of plant health scopic methods (8, 9), bionanosensors (10), and smartphone-
to end hunger, reduce poverty, and protect the environment. Ac- based devices (11, 12). Imaging or spectroscopic sensors are
cording to the Food and Agriculture Organization (FAO), it is esti- among the few possible solutions that are capable of real-time and
mated that by 2050, the productivity of food needs to be increased noninvasive monitoring. However, the imaging techniques are in-
by about 60% to feed approximately 10 billion people all over the direct by measuring the optical signature of the plant. Such mea-
world (1). Plant diseases cause around 20 to 40% of global crop surement has some obvious limitations, such as poor sensitivity/
loss annually (2, 3). Plant diseases not only cause notable loss in selectivity and complicated processing of raw images or spectral
food production but also reduce species diversity, affect mitigation data. Other approaches, including remote sensing (13) and electro-
of people, increase control costs, and pose negative influence on physiological sensing (14), have also been proposed for continuous
human health and global food security (1). In this regard, sensor monitoring. However, remote sensing, in general, lacks spatial sol-
technologies for early disease diagnosis are essential to shorten ution and is not specific for particular diseases (13), while electro-
stakeholder response time, identify threat before pathogen physiological sensors have only been demonstrated for tracking
spreads, and reduce pesticide usage by optimizing application water stress or the nycthemeral rhythm of the plant (14). Therefore,
timing and choice of pesticides. advanced sensor technologies that can track the status of plant
Tomatoes are one of the most widely consumed agriculture health in real-time and dissect various biotic/abiotic stresses are
products (4). Tomato plants are susceptible to many different needed to detect pathogens early, prevent disease outbreak, and
types of pathogens, including fungi, viruses, and bacteria, which improve plant growth and yield.
substantially reduce the yield and quality of fruit (5, 6). In addition In recent years, wearable electronics have been researched
to biotic stress, abiotic stresses such as high nighttime temperature widely. Applications of wearable technologies range from health
due to climate change, mechanical damage, or inappropriate use of monitoring (15, 16), human-machine interface (17), to soft robotics
pesticides could also result in crucial losses of tomato yield (7). In (18). Wearable electronics for plant health monitoring are also
this study, we choose tomatoes as a model system to design and test emerging. These flexible sensor devices can be attached to various
new sensor technologies that can monitor and predict the status of parts of host plants such as roots, stems, and leaves (19–21) to
the tomato plant health and can be applied to other plants monitor the plant’s microenvironment or physiological host re-
and crops. sponses. The physiological status of a plant is related to multiple
factors. Each plant grows via a set of biological processes such as
photosynthesis, transpiration, respiration, and gas exchange
through the regulation of leaf epidermal pores called stomata (22,
1
Department of Chemical and Biomolecular Engineering, North Carolina State Uni- 23). To accurately monitor plant health status, many of those bio-
versity, Raleigh, NC 27695, USA. 2Department of Mechanical and Aerospace Engi- logical processes and associated environmental conditions need to
neering, North Carolina State University, Raleigh, NC 27695, USA. 3Department of be investigated simultaneously.
Chemical Engineering, Kwangwoon University, Seoul 01897, Republic of Korea.
4
Department of Entomology and Plant Pathology, North Carolina State University,
Several multimodal plant wearables have been demonstrated re-
Raleigh, NC 27695, USA. 5Emerging Plant Disease and Global Food Security Cluster, cently for continuous plant health monitoring. Nassar et al. (24)
North Carolina State University, Raleigh, NC 27695, USA. presented a multifunctional wearable plant sensor by integrating
*Corresponding author. Email: qwei3@[Link] (Q.W.); yzhu7@[Link] (Y.Z.)

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temperature- and humidity-sensing functions, which was used to diagnose plant diseases noninvasively for the first time (26).
understand the optimal growth conditions by sensing surrounding However, the previously developed sensor patch only detects
environments. Zhao et al. (25) developed a stretchable multifunc- plant volatile organic compound (VOC) signals and lacks multi-
tional sensor device for detecting both microclimates (hydration, functionality. To date, a truly multifunctional and real-time
temperature, and light intensity) and growth rate of the plant. sensor device that can track both biochemical (e.g., plant VOCs)
Most recently, Lu et al. (19) demonstrated a multimodal flexible and biophysical (e.g., temperature, humidity, etc.) signals of the
sensor system for measuring microclimates (relative humidity, plant and/or surrounding environments with high sensitivity and
light intensity, and temperature) and the perception of leaf specificity has not been demonstrated yet.
surface humidity. This device was able to detect changes in leaf In this study, we demonstrate an abaxial leaf surface-attachable
surface humidity by a sensor array. However, no chemical or biolog- multimodal wearable plant sensor patch that can continuously and
ical information related to the status of plant health was monitored. simultaneously measure leaf VOCs, leaf surface temperature/hu-
On the other hand, we have recently demonstrated a plant wearable midity, and environmental humidity, with high sensitivity and se-
sensor that can measure leaf volatile emissions and be used to lectivity. This goal was achieved with several material innovations,

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Fig. 1. A multimodal wearable plant sensor. (A) Schematic illustration of the sensor attached to a plant leaf. Our multimodal sensor is attached to the abaxial leaf
surface to simultaneously monitor various physiology data from the leaf. Blue and orange arrows represent emissions of water and VOCs through stomata, respectively.
Different colors of the leaf represent the variation of leaf surface temperature. (B) Overview of the wearable sensor design, which consists of four VOC sensors, one leaf
surface relative humidity sensor, one leaf temperature sensor, and one environmental humidity sensor. All seven individual sensors were integrated with AgNW inter-
connects on a PDMS substrate. (C) Photograph of the actual sensor. VOC sensors with different sensing materials are labeled. (D) Side view of the wearable sensor patch.
(E) Photographs of an actual sensor patch attached to the lower epidermis of the tomato leaf. The environmental humidity sensor (red arrow) is the only sensor mounted
outside the leaf surface area in the air near the plant.

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including the newly designed VOC sensing materials by using a those materials. Moreover, these materials are highly conductive,
network of three dimensional (3D) structured nanowire and nano- stable, and easy to process because of their well-established chem-
tube hybrid to enable sensitive plant VOC detection in real time and istry properties. Synthesized Au@AgNWs were characterized with a
gold-coated silver nanowires (Au@AgNWs) for high stability transmission electron microscope (TEM) and an energy-dispersive
against humidity and solvent exposure. In addition, this study spectrometry (EDS) detector, as shown in Fig. 2B. Compared to
differs from our previous work (26) in several ways: (i) This new- bare AgNWs, Au@AgNWs have a thin gold layer (~30 nm) on
generation wearable sensor is multifunctional, incorporating VOC, the surface, enabling reaction with thiolated surface ligands. More-
temperature, and humidity sensors for measuring both biochemical over, the gold coating on AgNWs can improve stability under
and biophysical signals of plants simultaneously. (ii) The sensor lo- various chemical environments (27). The Au@AgNWs were
cation was optimized to the lower surface of the leaf to maximize the coated with various halothiophenol ligands [e.g., fluorothiophenol
sensor performance. (iii) This wearable device was tested on live (FTP), chlorothiophenol (CTP), bromothiophenol (BTP), and io-
tomato plants (cv. Mountain Fresh Plus) in both laboratory and en- dothiophenol (ITP)] to selectively detect leafy aldehydes through
vironmentally controlled plant growth chamber environments to reversible halogen bonding (Fig. 2A) (26). The surface modifica-
detect various types of stressors such as abiotic stresses (e.g., me- tions were characterized with ultraviolet-visible (UV-vis) spectro-
chanical injury, drought, overwatering, salinity, and absence of scopy to optimize the surface ligand density (fig. S3). The
light) and pathogen infections [e.g., tomato spotted wilt virus Au@AgNWs created a rough 3D surface layer that greatly increased
(TSWV)]. (iv) An unsurprised machine learning framework was the effective sensor surface area for VOC capturing (Fig. 2C and fig.
developed to process multichannel real-time sensor data for quan- S6). As shown in Fig. 2C, ligand-functionalized Au@AgNWs were
titative disease assessment and prediction of the best sensor combi- further wrapped and interconnected with MWCNTs because of the
nation. To the best of our knowledge, this represents the first report size and modulus differences between the two materials, which to-
of a multifunctional wearable plant sensor coupled with machine gether formed a robust heterogeneous sensing network. On top of

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learning data analysis. these active sensing materials, a hydrophobic and nanoporous sol-
gel layer was uniformly coated by drop casting (fig. S4). The water
contact angle of the sensing film was 82° and 111° before and after
RESULTS the sol-gel coating, respectively (fig. S5). We also measured scan-
Design and characterization of multifunctional plant ning electron microscopy (SEM) images with top and cross-section-
wearable sensor al views to investigate the morphology of the VOC sensing
Stomata are the primary gate of leaf tissues to exchange various composite materials coated with the sol-gel layer (fig. S6). The
types of molecules, including water, oxygen, carbon dioxide, and sol-gel coating substantially increases the mechanical stability of
VOCs. For this reason, the plant leaf is the primary location for the VOC sensor, reduces cross-talk to humidity interference
sensor attachment to monitor the plant biophysical properties. (Fig. 2F), and also minimizes sensor baseline drift (fig. S7), which
While many previous wearable sensors are attached to the upper will be discussed in more detail later.
surface of plant leaves (adaxial surface), our sensor patch is attached We then tested the performance of the VOC sensors (namely,
to the lower epidermis of the leaf surface (abaxial surface) to max- FTP, CTP, BTP, and ITP sensors) by measuring the electrical resis-
imize the sensing capability for capturing biologically relevant tance under the exposure of acetone or hexanal vapors, which are
targets that pass the stomata (e.g., VOCs and water molecules), used to mimic ketone and aldehyde-based VOCs emitted from the
which have a higher density on the lower epidermis (Fig. 1A). In plant (Fig. 2, D and E, and fig. S8) (28). The VOC sensors are op-
addition to leaf VOCs, our wearable patch can also monitor erated as chemiresistive sensors, where the electrical resistance of
several biophysical parameters such as leaf surface humidity, leaf MWCNTs varies upon the attachment of VOC molecules on
temperature, and environmental humidity in parallel. To achieve their surface, which creates a doping effect on the carbon nanoma-
this goal, the sensor patch consists of seven sensors in total, includ- terials (29). When solvent vapor concentration was reduced from
ing four resistive VOC sensors, one capacitive leaf surface humidity 500 to 100 parts per million (ppm), the sensor response (ΔR/R0) de-
sensor, one resistive leaf temperature sensor, and one capacitive en- creased accordingly, as a result of the reduced interaction between
vironmental humidity sensor (Fig. 1, B and C). Among them, the VOC gas and sensing materials (Fig. 2, D and E). Furthermore, for
leaf temperature sensor is in direct contact with the leaf surface; both acetone and hexanal vapors, the sensitivity of FTP and CTP
VOC and leaf humidity sensors face the leaf with a small gap, sensors is the highest among the four, followed by BTP and ITP
while the environmental humidity sensor extends from the leaf, sensors. This is due to the decreasing electronegativity of the
open to the environment (Fig. 1, D and E). Active sensing materials halogen atoms, following the order of F > C > B > I. For the final
(e.g., carbon nanotubes, Au@AgNWs, Nafion, etc.) are deposited wearable sensor patch, only FTP and CTP VOC sensors (termed
on the as-patterned interdigitated electrodes made of AgNWs that VOC_F and VOC_C, respectively) were integrated because of
are embedded below the surface of a soft polydimethylsiloxane their higher detection sensitivity.
(PDMS) substrate (fig. S1). The detailed sensor fabrication To characterize the potential cross-talk from other stimuli such
process is described in Materials and Methods and fig. S2. as humidity and temperature, the VOC sensors were tested under
The sensing materials for VOC detection are composed of a different humidity and temperature conditions. As shown in
hybrid network of Au@AgNWs and multiwalled carbon nanotubes Fig. 2F, the presence of a hydrophobic and gas-permeable sol-gel
(MWCNTs) embedded in a hydrophobic sol-gel layer made of layer on the top of the VOC sensors can prevent the interference
methyltrimethoxysilane (MTMS) and tetramethyl orthosilicate of water molecules up to 90% relative humidity (Fig. 2F, black
(TMOS; Fig. 2A). Both AgNWs and MWCNTs have a high aspect curve), whereas the control sensor without the sol-gel coating
ratio that can maintain the percolation state under deformation of showed a clear response to the environmental humidity (Fig. 2F,

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Fig. 2. Characterization of VOC sensors. (A) Optical photograph of VOC sensor and schematic of the sensing materials. Au@AgNWs with surface ligands and MWCNTs
were covered with a hydrophobic sol-gel layer. Surface ligands are halothiophenols immobilized on the gold surface, which selectively interact with leaf aldehydes. (B)
TEM and EDS images to compare the morphology of AgNWs and Au@AgNWs. (C) SEM image of the Au@AgNW and MWCNT composite showing MWCNTs were wrapping
on the Au@AgNW surface. (D and E) Electrical resistance changes of different surface ligand–functionalized VOC sensors (e.g., FTP, CTP, BTP, and ITP) under exposure of (D)
acetone and (E) hexanal, respectively. Vapor concentrations are theoretical concentrations at the nozzle tip of the gas-mixing system. (F) Humidity interference test of the
VOC sensor with or without the sol-gel layer. The hydrophobic sol-gel layer prevents interaction of water molecules with the VOC sensor. (G) Temperature interference test
of the VOC sensor. HAADF, high-angle annular dark-field.

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red curve). The sol-gel layer also improved the repeatability of the the few that can detect biochemical and physical parameters in par-
VOC sensor response by enhancing the stability of the sensing ma- allel (table S1).
terials on PDMS substrate and reducing baseline signal drifting (fig.
S7). For temperature interference, the VOC sensor showed minimal Abaxial leaf surface attachment
dependence on the external temperature (Fig. 2G), owing to the op- Next, the influence of the sensor location, namely, the adaxial
posite temperature coefficient of resistance values of MWCNTs (upper surface of leaf ) attachment versus the abaxial (lower
(−0.33%/K) (30) and AgNWs (0.26%/K; fig. S9) (31). Although surface of leaf ) attachment, was evaluated on a live tomato plant.
the resistance of VOC sensors slightly fluctuated with the temper- In Fig. 3E, the photographs show that the sensor can be directly at-
ature variation (from 40° to 60°C), this change was less than 0.7% tached to the backside of the leaf. The difference in stomata density
for 40°C and lower temperatures (Fig. 2G), exhibiting acceptable between the adaxial and abaxial surfaces of the leaf was observed via
temperature stability for real plant applications. bright-field optical microscopy (Fig. 3F and fig. S10). As shown in
Figure 3A shows the sensing performance of the leaf temperature Fig. 3G, the density of stomata on the abaxial surface is approxi-
sensor, composed of as-synthesized Au@AgNWs (without func- mately 73% higher than that of the adaxial leaf. This result agrees
tionalization) as the sensing agent. Because of the temperature sen- with the previous study, where a higher density of stomata on the
sitivity of Au@AgNWs and the thermal expansion of PDMS lower surface of tomato leaf was reported (36). Moreover, we com-
substrate, the nanowire-based temperature sensor can detect tem- pared various sensor responses from adaxial and abaxial leaf surfac-
perature changes from room temperature to 60°C. To investigate es (fig. S11). For leaf surface humidity and VOC sensors, the sensor
the effect of PDMS thermal expansion on temperature sensing, responses with abaxial surface position were, on average, 10 to 20%
we varied the elastic modulus of PDMS by tuning the mixing higher than those of adaxial surface (Fig. 3H). Here, the leaf surface
ratio of prepolymer and curing agent from 5:1 to 20:1 (Fig. 3A). humidity sensor measured the leaf-emitted water under normal
As the mixing ratio increased to 20:1 (resulting in softer PDMS), growth conditions, while the VOC sensor signal was induced by

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the sensitivity of the temperature sensor slightly increased the leafy VOC profile change upon mechanical leaf cutting. The
because of larger thermal expansion of PDMS, which decreased signal difference was attributed to the difference in stomata
the percolation between Au@AgNWs (32). Moreover, the interfer- density between the upper and lower surfaces of the leaf
ence of different humidity levels or solvent exposure to the temper- (Fig. 3G). For better sensor performance, we therefore chose the
ature sensor was also examined (Fig. 3B). The slope of each response abaxial epidermis of the leaf as our sensor attachment location for
curve did not change obviously when various humidity levels (rel- the rest of the experiments in this work. In addition, we tested the
ative humidity, 25, 50, and 75%) or 500 ppm of acetone were wearability and biocompatibility of sensor for nearly 3 weeks on a
applied, indicating the excellent stability of the temperature live tomato plant. The data showed no apparent indication of plant
sensor. These results can be explained by the protective effect of stress after sensor attachment (fig. S12).
the gold layer on the AgNW core, which does not interact with Before testing the plant stress conditions in the next section, we
water or common VOC vapors because of its chemical inertness. first monitored the leaf surface temperature and relative humidity of
The performance of the humidity sensors was also characterized. a healthy tomato plant during sunny and rainy days continuously
In this case, Nafion film was used as the active humidity sensing using the wearable sensor patch (Fig. 3I). As the light intensity in-
material by measuring its capacitance changes between two elec- creased during the daytime, the leaf surface humidity signals
trodes. The thickness of the Nafion film was controlled to optimize reached the maximum in the middle of the day because of the in-
the detection sensitivity (Fig. 3C). Nafion is known to absorb water creased number of open stomata to release the water molecules to
molecules, resulting in an increase in the capacitance value by re- the environment (Fig 3I). This indicates that the intensity of the
placing air with water molecules within the film (33). As the thick- sunlight can modulate the opening of stomata on leaf surface
ness of the film was increased, the sensitivity was enhanced because (37). The leaf temperature increased under the sunlight condition
of the higher capacity of absorbing water molecules (Fig. 3C). during the daytime, followed by a decrease in temperature that is
However, when the thickness was above 5 μm, the film was delam- attributed to the enhanced leaf transpiration (Fig 3I and fig. S13).
inated from the substrate because of a larger stiffness mismatch However, such a circadian rhythm was much less obvious during
between the film and the substrate (34). Therefore, a 2-μm-thick rainy days when the plant was less influenced by the sunlight with
Nafion layer was selected as the optimized thickness. To test the high relative humidity of the air (Fig 3I). The reading error of the
cross interference, we applied various temperatures (10°C, room wearable humidity sensor was within 10% when compared to a
temperature, and 40°C) or 500 ppm of acetone vapor to the humid- commercial environmental sensor (table S2). These results show
ity sensor (Fig. 3D). With the increase of temperature, larger that this multimodal sensor patch can be potentially used for the
thermal movement of water molecules would induce higher polar- study of plant chronobiology in addition to disease and stress detec-
ization, resulting in a larger capacitance (35). This means that the tion as described below.
absolute capacitance value (C ) would change as a function of the
temperature. However, the relative capacitance response (ΔC/C0) Monitoring abiotic stresses on tomato plants
is quite consistent for temperatures between 10° and 40°C. In the To demonstrate the usability of the multimodal sensor, the patch
case of acetone exposure, the sensitivity of the humidity sensor was mounted on a live tomato plant for monitoring various
was reduced slightly because of water molecule absorption in the abiotic stresses, including drought, overwatering, salinity, and dark-
presence of acetone. We also compared the sensor performance ness (Fig. 4). Figure 4A shows the schematic illustration of the ex-
against previously published multifunctional wearable sensor perimental setup. Four stressors were introduced sequentially to the
patches, which demonstrated that our sensor platform is one of same tomato plant wearing the sensor patch to minimize host dif-
ference. For this experiment, we used a multimodal wearable sensor

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Fig. 3. Characterization of temperature and humidity sensors. (A) Resistance changes under increasing temperature with various mixing ratio of PDMS (error bar
represents n = 3 measurements). The inset is an optical image of the actual temperature sensor. (B) Humidity and vapor interference tests for the temperature sensor. The
temperature response was measured under relative humidity (RH) of 25, 50, and 75% or in the presence of 500 ppm of acetone vapor (error bar represents n = 3 mea-
surements). (C) Capacitance changes under increasing humidity with various thickness of Nafion film. The inset is a photo of the actual humidity sensor. (D) Temperature
and vapor interference tests for the humidity sensor. The humidity was measured at diverse temperatures [10°C, room temperature (RT), and 40°C] or in the presence of
500 ppm of acetone vapor (error bar represents n = 3 measurements). (E) Photographs of the sensor attached underneath the leaf. Front view (top row) and side views
(bottom row). Blue arrows indicate the sensor. (F) Optical microscopy image of the stomata on the abaxial tomato leaf surface. Red arrows indicate the presence of
stomata on the abaxial leaf surface. (G) Comparison of stomata density between the upper and lower surface of the leaf. Error bars are SDs from five samples. (H)
Output signal differences of leaf surface humidity and VOC sensor with different sensor attachment positions. Error bars are SDs from five samples. (I) Real-time mon-
itoring of leaf surface relative humidity and leaf surface temperature of a healthy tomato plant. D and N represent the daytime and nighttime, while gray and carnation
colors indicate rainy day and sunny day, respectively.

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results in Fig. 3I. After 7 days, the plant was watered normally to
reduce the water stress, and the plant recovered for another day
before the next experiments. On day 9, excessive water was
applied to create an overwatering condition. Immediately, the
surface humidity of the leaf increased about 1.5 times as water
content inside the host decreased as increased water evaporation oc-
curred through the leaf surface (Fig. 4B, second panel). On day 10,
salinity stress was introduced by using 150 mM salted water. Typi-
cally, under high-salt conditions, the transpiration of the plant
would be reduced because of the reduction of water absorption
from roots as a result of the osmotic pressure difference between
soil and root (38). This was confirmed from our experimental
data, where the suppression of leaf surface humidity change and
rapid increase of leaf temperature due to reduced transpiration
was observed (Fig. 4B, second and fourth panel). For the last exper-
iment (day 12 and after), the plant was placed in complete darkness
by covering the plant with a box. In this case, notably increased
VOC emission, leaf surface humidity, and leaf temperature were ob-
served, which probably resulted from the induced stress due to the
lack of photosynthesis. The plant eventually died at the end of the
experiment while showing high levels of VOC emissions and in-

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creased leaf temperature. The elevated leaf surface humidity after
light blocking appears contradictory to the conventional plant
physiology theory. However, we attribute this phenomenon to the
plausible evapotranspiration effect of a dying plant. To validate our
results, we also repeated the abiotic stress experiments with single
stressors (one stress at a time), and the plant responses were record-
ed by both wearable sensors (figs. S14 to 17) and a commercial leaf
porometer (figs. S18 and S19). Both devices showed good agree-
ment with each other. Briefly, the shortage and excessive watering
moderately decreased or increased the stomatal conductance and
leaf surface humidity, respectively (fig. S19 and table S3). Watering
with high salt concentration inhibited the transpiration because of
the imbalance of the osmotic pressure in roots (fig. S19 and table
S3). Last, blocking the light hindered the photosynthesis process
and therefore resulted in the reduction of leaf surface humidity
and temperature (fig. S19 and table S3). Each stress was measured
Fig. 4. Plant wearable for monitoring abiotic stresses. (A) Schematic illustration three times, and their sensor response trend was summarized in
of the experimental setup for sequential abiotic stress monitoring, including table S3.
drought, overwatering, salinity, and no light. (B) Real-time sensor data from the In addition, we also monitored the plant response to mechanical
same tomato plant exposed to various abiotic stresses. VOC_C1 and C2 correspond damage on leaves by the wearable sensor patch (fig. S20). When
to CTP sensors, while VOC_F1 and F2 are FTP sensors. Black arrows represent cutting or detaching the leaf, the VOC sensors detected more
specific times for applying different conditions. Carnation color indicates the obvious and immediate response signals than other sensors (e.g.,
daytime of measurement. Gray color represents the rainy day. temperature and humidity sensors; fig. S20), indicating that the bio-
chemical sensor was more sensitive than biophysical sensors in the
composed of four VOC sensors (CTP sensors: VOC_C1 and C2; detection of acute plant tissue damages.
FTP sensors: VOC_F1 and F2), one leaf surface humidity sensor,
one environmental humidity sensor, and one leaf surface tempera- Monitoring biotic stress and early pathogen detection
ture sensor. By applying the sequential stimuli, the responses of the Next, we tested the wearable sensor patch for monitoring different
sensor with multiple electrical signals were simultaneously moni- pathogens on live tomato plants. TSWV (a viral pathogen) and early
tored by a multichannel data recorder for up to 14 days (Fig. 4B). blight (Alternaria linariae, a fungal pathogen) were selected because
First, watering was prevented for 7 days to mimic a drought envi- of their prevalence in tomato production (fig. S21) (1, 39, 40). As
ronment. In this stage, the leaf surface humidity gradually reduced shown in fig. S22, the experiments were conducted in a growth
as water was conserved inside the plant over time, while the VOC chamber at the North Carolina State University (NCSU) Phytotron,
signals increased slightly (Fig. 4B, first and second panel). Although maintained at a constant 23°C with a 16-hour photoperiod and
the plant was grown indoors, we were able to capture a rainy day constant carbon dioxide concentration (400 ppm). TSWV inocula-
(day 3) by observing the depression of leaf transpiration that was tion was first performed, and the plant response was monitored by
modulated by the different lighting conditions, similar to the the wearable sensor. By measuring the electrical signals with the
wearable sensor, differentiable VOC signals can be seen after

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around 5 days post inoculation (dpi; inoculated on day 4 and detect- and Methods). According to the real-time RT-LAMP assay results,
able VOC signals on day 9; n = 3 replicated measurements; Fig. 5A successful inoculation of the plant generated consistent positive
and fig. S23). To compare our electrical sensing method with the nucleic acid test results 7 dpi (Fig. 5B), which is later than the wear-
conventional molecular diagnostic method, a TSWV-specific real able sensor patch. More quantitative early detection data were de-
time reverse-transcription loop-mediated isothermal amplification termined by the machine learning analysis described below. In
(RT-LAMP) assay was performed in parallel (Fig. 5B and Materials addition, a rapid change of VOC signals and a decrease in leaf

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Fig. 5. In-situ measurement of tomato plant health under biotic stresses (pathogen infections) in phytotron. (A) Real-time wearable sensor data of a live tomato
plant after TSWV inoculation. VOC_C1 and C2 correspond to CTP sensors, while VOC_F1 and F2 are FTP sensors. Black arrows represent specific time for conducting the
inoculation. Carnation color indicates the daytime of measurement. (B) Real-time LAMP assay results, verifying the presence of TSWV pathogens after different days of
inoculation. After 7 dpi, a positive result was detected by the RT-LAMP assay for three of four plants. (C) Wearable VOC sensor data and the conventional Horsfall-Barratt
scale (black line) for the inoculation experiment with A. linariae. Heatmap of sensor data after 5 hours of different abiotic stresses (D) and heatmap of sensor data on days 3
and 6 of the tomato plant with various pathogens (E). ∆R/R0, ∆LSH, and ∆LT represent the resistance change due to leaf VOC emission, leaf surface humidity variation
(LSH), and leaf temperature (LT) change, respectively. a.u., arbitrary units.

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surface relative humidity was captured right after the inoculation confirming that this instant VOC response is indeed induced by
(days 4 and 5). These signal perturbations were attributed to the me- mechanical damage to the leaf. Furthermore, during the entire
chanical damage to the leaf surface during the TSWV rub-inocula- period, both leaf surface relative humidity and temperature re-
tion process. The VOC signals returned to the baseline on day 7, mained mostly constant, which also indicated that the plant was
indicating the recovery of the plant from mechanical damage. In ad- not infected by TSWV. The unsuccessful inoculation was lastly con-
dition to VOCs, leaf surface relative humidity rapidly decreased and firmed by the RT-LAMP assay (fig. S25).
the leaf temperature slightly increased after inoculation [Fig. 5A To demonstrate the feasibility of detecting fungal pathogen in-
(second and fourth panel) and fig. S23], which can be related to fection using the same sensor patch, we also performed inoculation
the mechanical damage of the leaf surface and also the closure of experiments with A. linariae (n = 3 replicated measurements;
the stomata after pathogen infection (41). When a plant is infected Fig. 5C figs. S26 and S27). Because the inoculation of this fungal
by TSWV, stomata generally close as a result of invasion by the pathogen was performed by spraying the pathogen solution on
pathogen, resulting in a lower transpiration rate (42). Although the leaf surface without mechanical damage, no instant VOC
less specific, these biophysical signals (e.g., leaf surface relative hu- signal changes were observed after inoculation in this case
midity and temperature) are easy to monitor compared to VOC (Fig. 5C and fig. S27). For A. linariae, our wearable sensor demon-
signals and hence could be useful plant health indicators on strated the capability of detecting pathogen infection before the
their own. visual assessment method. Conventionally, the visual symptoms
Monitoring host plant response using the patch sensors also were quantified using the Horsfall-Barratt scale for assessing
allowed us to determine whether inoculations were successful. disease severity (table S4) (43). On the basis of the scale, the infec-
Failed inoculations resulted in response patterns that varied tion could be confirmed visually 4 dpi for A. linariae (Fig. 5C, black
widely from successful inoculations that developed into visible squares). However, the wearable VOC sensor was able to capture
symptoms (fig. S24). In the case of mock inoculation, the VOC elevated VOC emissions after 2 days of infection, approximately 2

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signals did not increase after 5 dpi (fig. S24). The rapid increase days before the visible assessment method. Leaf surface humidity
of VOC signals immediately after the inoculation was still observed, and temperature signals also showed different patterns before and

Fig. 6. Machine learning analysis of the real-time TSWV sensor data. (A) Schematic diagrams of the process of machine learning using PCA method. (B) A repre-
sentative graph showing 3D PCA analysis from days 0 to 15 for the six-sensor combination (VOC_C1, C2, F1, F2, H, and T). VOC_C1, C2, F1, F2, H, and T denote four VOC
sensors, leaf surface relative humidity sensor, and leaf temperature sensor, respectively. (C) Average discriminability values with different numbers of sensors as a function
of infection days. Error bars represent the SDs of each number of sensors. (D) Discriminability with the best sensor composition for each number of sensors. VOC_C1, C2,
F1, F2, H, and T denote four different types of VOC sensors, leaf surface relative humidity sensor, and leaf temperature sensor, respectively.

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after infection (figs. S26 and S27). Together, these results (Fig. 5, A As shown in Fig. 6B, a three-component PCA for a total of six
and C) suggested that the wearable sensor technology was able to sensors [namely, VOC_C1, VOC_C2, VOC_F1, VOC_F2, leaf
detect different kinds of plant pathogens (viral and fungal) 2 to 3 surface relative humidity sensor (H), and temperature sensor (T)]
days earlier than conventional detection methods, such as the from days 0 to 15 shows the gradual separation of sensor signals
nucleic acid testing by LAMP assays (Fig. 5B) and the visible inspec- from day 0 (green dots) throughout the TSWV infection process.
tion in Fig. 5C. Our previous work also demonstrated that the wear- In the early days, the most obvious cluster separation occurred on
able sensor could detect infection by the oomycete pathogen day 5 (cyan dots) because of the mechanical damage induced by the
Phytophthora infestans (26). As a comparison, healthy tomatoes inoculating method (Fig. 6B). All PCA data with other sensor com-
sprayed with water instead of the spore solution showed no binations (e.g., five sensors, four sensors, three sensors, etc.) are also
sensor response at all (fig. S28). shown in fig. S29.
Moreover, by combining all the sensor signals, the wearable Using discriminability (capturing different signal changes), we
sensor patch was capable of distinguishing biotic stress from are able to quantitatively differentiate diseased plants from
abiotic factors such as mechanical cutting, drought, overwatering, healthy controls and determine the accurate early detection day.
salinity, and light deficiency. To demonstrate that, heatmaps were Simply, if the discriminability value is positive, the two sensor clus-
generated using the sensor data at specific time points for differen- ters are considered distinguishable, resulting in a positive diagnosis
tiating all abiotic and biotic stresses that have been screened. As result. On the other side, if the value is negative, that means the two
shown in Fig. 5D, the VOC, leaf surface relative humidity, and tem- clusters under comparison are overlapped, resulting in a negative
perature data were combined and depicted with different color detection result. We applied the PCA to all possible sensor combi-
scales. Distinct electrical responses of the plant wearable sensor nations using six individual sensors (total 63 combinations) and
under different abiotic stresses can be seen after a constant exposure calculated discriminability for each sensor combination (Fig. 6C
time period of 5 hours. Moreover, similar to the case of abiotic and fig. S30). Figure 6C shows the averaged discriminability value

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stress, all three tested pathogens could be discriminated from each for each sensor combination, and the SD represents the different
other and also from the healthy plants by using the same sensor performance when the number of sensors is fixed but the sensor
patch through the combination of the sensor signals (VOC, leaf composition is different. On the basis of the data, it clearly suggests
temperature, and humidity) at specific time points (e.g., days 3 that the more sensor channels that were used, the higher the dis-
and 6; Fig. 5E). Such a distinct sensor pattern could be recognized criminability value, which also means higher confidence in a posi-
and classified by machine learning methods in the future for rapid tive detection. Excluding the day 5 data (positive but mainly because
differentiation. of mechanical perturbation), the six-sensor channel combination
can clearly detect TSWV infection as early as day 8, which is 4
Machine learning for quantitative early detection and dpi, much earlier than the RT-LAMP results (7 dpi; Fig. 5B).
prediction of best sensor combination Figure 6C displayed a large error bar for each fixed number of
To quantitatively assess our multimodal sensors for the early detec- sensors, suggesting that the actual sensor channel combination is
tion of pathogens, an unsupervised machine learning approach equally if not more important to the total number of sensors. The
based on principal components analysis (PCA) was used to best combination for each number of sensors is presented in
analyze the real-time sensor data (Fig. 6) (44, 45). PCA is one of Fig. 6D. According to the discriminability values, a minimum of
the most well-known statistical algorithms in data analysis and three sensors (VOC_C2, VOC_F1, and H) is needed for the early
image processing projects for multivariate variable dimensionality detection of TSWV after 4 dpi (Fig. 6D, blue triangle). In addition,
reduction with impactful benefits such as feature selection and the results suggest that for effective disease detection, the biochem-
event classification. In comparison with other conventional ical VOC sensor is probably the most important sensor that is
methods such as t-distributed stochastic neighbor embedding (t- needed in each sensor combination; in addition, the leaf surface hu-
SNE), the PCA approach is more applicable in multivariable midity sensor works slightly more effectively than the leaf temper-
sensor systems (46). Figure 6A depicts the schematic illustrations ature sensor in disease detection (Fig. 6D). Such a machine learning
of the PCA-based data analysis pipeline for processing real-time analysis can help find the most impactful sensor (and sensor com-
sensor data. For the demonstration, we used the TSWV inoculation bination) for a particular application and potentially reduce the
data (Fig. 5A) as an example. The multichannel wearable sensor total number of redundant sensors, which would be particularly
data from the same plant was first divided into different days useful to reduce sensor cost while maintaining sensor performance.
(e.g., days 0, 1, 2, 3, etc.). Day 0 data were used as the healthy
control and compared to other days. Data from different days
were clustered by PCA with reduced data dimensions. Then, the DISCUSSION
centroid and Euclidean distance between two centroids of clusters Multifunctional and miniaturized sensor technology for continu-
(two different days) were calculated (see details in Materials and ous plant physiology monitoring is of great interest for early
Methods). The separation of the clusters was quantitatively assessed disease detection, stress sensing, and growth prediction. However,
by a parameter called “discriminability” (D), as defined by the fol- many existing wearable sensor technologies can only detect physical
lowing equation growth of the plant or environmental parameters of the atmosphere
(table S1). None of these sensor platforms has been developed to
D¼E ðRSTD;1 þ RSTD;2 Þ monitor multiple signals that can inform plant health. Here, a mul-
where D, E, and R denote discriminability, Euclidean distance, and timodal plant wearable sensor patch capable of detecting both bio-
radius (or SD) of the cluster, respectively. chemical and biophysical parameters of individual plants, namely,
leafy VOCs, leaf surface humidity, leaf surface temperature, and

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S C I E N C E A D VA N C E S | R E S E A R C H A R T I C L E

environmental relative humidity, was demonstrated for continuous, Sigma-Aldrich. PDMS (SYLGARD 184) was purchased from
on-plant physiology monitoring. This versatile device used a newly Dow Corning.
designed 3D nanohybrid sensing network to capture leaf VOC
signals and greatly minimized the cross-talk between VOC, temper- Synthesis of nanowires
ature, and humidity signals. The performance of each sensor com- The Au@AgNWs were synthesized by a modified chemical solvent
ponent integrated on the patch (e.g., detection sensitivity and range) method based on the previously reported procedure (27). The
matches well with what has been demonstrated in the existing mul- AgNWs are prepared by a modified polyol method and dispersed
tiplexed sensor platforms and has room to approach those mono- in deionized (DI) water for the following steps (53).
functional sensors (table S1). Moreover, our sensor was mounted For the preparation of solution A, 10 ml of AgNW aqueous sol-
underneath the leaf surface (abaxial surface) to maximize the ution (10 mg/ml), 70 ml of 5 weight % polyvinylpyrrolidone
output signals from the plant, which is different from many previ- aqueous solution (Mw, 40,000; Sigma-Aldrich), 14 ml of 0.5 M
ous sensor applications. With this multimodal sensor patch, we L-ascorbic acid aqueous solution (Sigma-Aldrich), 14 ml of 0.5 M
demonstrated the monitoring of various types of plant stresses sodium hydroxide (Sigma-Aldrich), 3.5 ml of 0.1 M Na2SO3
from drought, overwatering, salinity, light deficiency, mechanical (Sigma-Aldrich) aqueous solution, and 80 ml of DI water were uni-
damage, and pathogenic infection (virus and fungus) in both labo- formly mixed with a glass rod. The prepared solution was denoted
ratory and greenhouse conditions. In particular, the wearable sensor as solution A.
patch demonstrated capability for the early detection of plant path- For the preparation of solution B, 10 ml of 0.1 M sodium sulfite
ogens (2 to 3 days earlier) when compared to conventional nucleic aqueous solution, 3.5 ml of 0.5 M sodium hydroxide aqueous sol-
acid LAMP-based reactions or visual assessment techniques. ution, and 100 ml of DI water were mixed first. Then, 1.5 ml of 0.25
In addition, a machine learning analysis framework based on the M hydrogen tetrachloroaurate(iii) hydrate aqueous solution
PCA approach was developed to quantitatively determine the early (HAuCl4·xH2O; Sigma-Aldrich) was added to the mixed solution.

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detection capability and screen the best combination among multi- The solution was then stirred gently with a glass rod. The prepared
ple sensors. In recent years, applying machine learning approaches solution is denoted as solution B.
such as supervised and unsupervised learning have been greatly in- Solution B was then immediately but slowly poured into solution
creased in the biosensor area because of their outstanding benefits A to produce a mixture that appeared light purple with a metallic
in data analysis and noise reduction. Specifically, integrating gloss. Then, the beaker of the mixed solution was sealed and left for
machine learning with biosensors for plant disease detection, 12 hours. After the reaction, the Au@AgNWs turned light brown
stress phenotyping, and predictive analysis showed meaningful and precipitated out of the solution. The remaining solution
results (47). In this research, we conducted PCA as one of the turned clear black. These nanowires were then collected and
most common unsupervised machine learning algorithms to washed with 95% ethanol three times using centrifugation (1000
reduce the dimensions of multichannel sensor data and also classify rpm for 1 min) and dispersed in ethanol for further use.
the roles of each sensor in combination to find out the best combi-
nation candidates to predict plant disease sooner. This data analyt- Preparation of VOC sensors
ics–coupled sensor platform could be used for various applications The VOC sensor’s performance depends heavily on the amount of
related to plant health monitoring and crop loss prevention in ag- ligands attached to the surface of the nanomaterials. To optimize
ricultural settings. the concentration and the amount of ligands decorated to the
For practical field applications, the size of sensor patches should surface of Au@AgNWs, four different concentrations (0.01, 0.1, 1,
be further miniaturized using higher-resolution fabrication tech- and 10 μM) of FTP ligands were tested. We found a higher tendency
niques such as photolithography, micromolding in capillaries of agglomeration of the nanowire solutions at the higher ligand con-
(even on curved surfaces) (48), or direct laser writing (49). The ge- centration. However, if the amount of surface ligand is too low, then
ometry of sensor patch also needs to be flexible to fit diverse shapes the reactivity toward VOC analytes will also be reduced. Therefore,
of attaching leaves. In multimodal sensors, cross-sensitivity inhibits 0.1 μM ligand solutions were eventually chosen to functionalize
the precise measurement of a specific target when multiple stimuli Au@AgNWs without notable aggregation. After that, we optimized
are present at the same time. Therefore, it is critical to implement the amount of ligand solutions by changing the volume of ligand
decoupled sensing mechanisms such as different sensing materials, solutions (e.g., 300, 500, and 1000 μl). The 500-μl solution was se-
sensor layouts, and signaling principles in future sensor design and lected to balance the ligand density and nanomaterial stability. Sim-
development (50). Moreover, a fully standalone sensor device will ilarly, 500 μl of 100 nM ITP, BTP, and CTP ligand solutions were
be expected in the future, which requires the integration of other used to prepare other VOC sensors. Every functionalization reac-
essential components such as thin-film batteries, self-powered tion was continued for 8 hours with slight shaking at room temper-
units (51), and functional circuits (52) for wireless signal transmis- ature. The supernatant was then collected to measure their UV-vis
sion with the sensing elements on the same patch. The robustness of spectrum. After that, ligand-attached Au@AgNW solutions were
the sensor patch will also need to be thoroughly tested in the green- added to MWCNTs at a 5:1 mixing weight ratio.
house and field trails. The sol-gel film was prepared by mixing MTMS, TMOS, meth-
anol, and Nanopure water in the molar ratio of [Link]. The solu-
tion was stirred at room temperature for 2 hours. The final
MATERIALS AND METHODS formulation was diluted 10 times by adding methanol before
Materials and reagents drop-casting.
All materials and reagents were used without further purification.
MWCNTs, Nafion, hexanal, and acetone were purchased from

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Fabrication of a multimodal wearable plant sensor A. linariae inoculation

As shown in fig. S2, AgNWs were spray-coated on the polyimide An isolate of A. linariae (JD1B) was maintained on potato dextrose
(PI) substrate using a stencil mask for patterning interdigitated elec- agar throughout the experiment. To generate conidia, pieces of agar
trodes and interconnect. After patterning, PDMS solution was with active A. linariae culture were broken up in potato dextrose
poured to transfer AgNWs from PI to PDMS. The PI substrate broth and spread onto sporulation agar (0.2 g of CaCO3, 100 ml
was removed when the PDMS was fully cured. With a patterned of V8 juice, 20 g of Difco Bacto agar, and 1 liter of dH2O). Plates
AgNW substrate, sensing materials for each sensor (e.g., were incubated at 20°C under constant light for 2 weeks. Conidia
Au@AgNWs for temperature sensor, Nafion for leaf surface humid- production was then stimulated by brushing the plates with a dry
ity sensor and environmental humidity sensor, and functionalized sterile cell spreader and incubating at room temperature for 1 to 2
Au@AgNW+MWCNTs for VOC sensors) were selectively depos- days with the lids ajar in an inoculation box. To harvest conidia, 2
ited onto the interdigitated electrodes. ml of sterile distilled water was added to the plate and brushed with
a cell spreader. The liquid was then removed and quantified using a
Characterization of the developed sensor hemocytometer. The conidia solution was diluted to 2000 conidia/
The electrical resistance changes were measured by a digital mul- ml using distilled water. Four plants were sprayed with 2 ml of the
timeter (DAQ970A, Keysight) and recorded by the software Bench- conidia solution over the entire surface of the plant, while four
Vue 2018. The capacitance variation was captured by the Capacitive control plants were sprayed with 2 ml of distilled water. Plants
to Digital Converter Evaluation Module (FDC1004EVM, Texas In- were covered with clear plastic bags to maintain humidity. In addi-
struments). The temperature was controlled by a hot plate. Humid- tion to monitoring by the sensor, visual symptoms were observed
ity was produced with a wet nitrogen gas stream. VOC gases were daily for 1 week by measuring the percent leaf area diseased
generated by bubbling nitrogen gas through the corresponding (%LAD) using a modified Horsfall-Barratt scale (table S4).
organic solvents. The concentration of VOC vapors was modulated

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by MKS mass flow controllers. For VOC sensor measurement, the TSWV inoculation
sensor was exposed to VOC vapors at a fixed concentration for 2 A week before the experiment, a 2-week-old tomato Mountain
min, followed by pure nitrogen gas purging for another 2 min for Fresh seedling, susceptible to TSWV, was placed in a growth
baseline recovery. Morphologies of sensing materials were mea- chamber at 23°C with a 16-hour light/8-hour dark schedule. The
sured by SEM (Thermo Fisher Scientific Quanta 3D FEG), TEM plant in a pot was also enclosed in a plastic container with paper
(Thermo Fisher Scientific Talos F200X), and EDS (Thermo Fisher towels soaked in a nutrient solution. As the experiment started,
Scientific Super-X EDS with the four silicon drift detectors). the seedling was inoculated with a wild-type TSWV strain that orig-
Sensors were attached onto the leaf surface using double-sided inated from California using a mechanical leaf-rub method. First, all
tape (2477p, 3M) and connected to the data acquisition system leaves were sprinkled with carborundum (39). Second, several
with thin copper wires (FIXFANS) and silver adhesive epoxy young leaves from TSWV-infected tomatoes were ground in an
(MG Chemicals) for interconnection. A commercial sensor device ice-cold mortar with approximately 5 to 10 ml of sodium sulfite sol-
(Amprobe, THWD-5) was used for measuring relative humidity ution (63 mg per 50 ml of tap water) as a buffer. Next, two cotton
and temperature of the environment. A leaf porometer (Decagon applicators were repeatedly dipped into the ground tissue mix and
Devices Inc., SC-1 leaf porometer) was used to measure the stomatal rubbed on each leaf using a gloved hand to support the leaf and
conductance, leaf surface humidity, and leaf surface temperature of ensure small wounds were made on the leaf surface. The leaf area
the tomato plants under various abiotic stress conditions to validate with the sensor attached was avoided. Ten minutes after this proce-
the sensor measurement. For the reproducibility of porometer mea- dure, the tomato plant was sprayed with DI water to remove the re-
surement, two healthy tomato plants (5 to 6 weeks old) for each maining carborundum. The plant was kept enclosed in a controlled
stress condition were used, and for each plant, three different leaflets chamber for 14 days. Plants were fertilized with nutrient solution
were measured to minimize signal variation. In total, six measure- three times per week. For the negative control, mock inoculation
ments (from six different leaflets) were performed for each stressor, was performed with healthy leaf tissue, and the experiment schedule
and 12 plants were used for the experiments. For each measure- was kept the same.
ment, the porometer data were collected every 30, 60, or 120 min,
and the measurements were continued for 2 to 4 days for different TSWV LAMP assay
stressors. At the experiment termination, one young leaf was used for DNA/
RNA extraction. Polymeric microneedle patch was pressed on a leaf
Preparation of host tomato plants and rinsed with 60 μl of DI water (12). To detect TSWV, 25-μl
Susceptible tomato plants (cv. Mountain Fresh Plus) were grown LAMP reactions with EvaGreen fluorescent and hydroxy naphthol
from seed at the NCSU Phytotron with a combination of natural blue colorimetric dye were performed on a Bio-Rad CFX96 real-
light and artificial light (14-hour photoperiod) and a 26°C day time machine (12). For each reaction, 2 μl of microneedle extraction
and 22°C night temperature cycle. Approximately 1 week before was used for analysis. For positive controls, we used 2 μl of RNA
the experiment, 5- to 6-week-old plants were transferred to a extracted with Total RNA (Plant) Kit (IBI Scientific) from sympto-
growth chamber with a 16-hour photoperiod kept at 23°C (both matic tomato plants maintained in the laboratory. For no template
day and night) for the entire experimental period. controls, 2 μl of molecular grade DI water was added to the reac-
One plant was selected for each inoculation. The plant was then tions. Positive reactions were detected by green fluorescence with
placed in an inverted inoculation box lined with moist paper towels Ct values recorded, as well as by a color change from violet to
to provide water and humidity throughout the experiment. light blue.

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Principal components analysis 12. R. Paul, E. Ostermann, Y. Chen, A. C. Saville, Y. Yang, Z. Gu, A. E. Whitfield, J. B. Ristaino,
Q. Wei, Integrated microneedle-smartphone nucleic acid amplification platform for in-field
We performed PCA, data analysis, and plots in the Project Jupyter
diagnosis of plant diseases. Biosens. Bioelectron. 187, 113312 (2021).
platform using Python programming language. Then, we found the 13. T. Liu, L. Wang, S. Zuo, C. Yang, Remote sensing dynamic monitoring system for agricultural
centroid of each cluster by using centroid function in the k-means disaster in Henan province based on multi-source satellite data. Agric. Sci. Technol. 14,
clustering approach to calculate the Euclidean distance. In a three- 155–161 (2013).
component PCA space, Euclidean distance D was defined as 14. D. Tran, F. Dutoit, E. Najdenovska, N. Wallbridge, C. Plummer, M. Mazza, L. E. Raileanu,
C. Camps, Electrophysiological assessment of plant status outside a Faraday cage using
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
supervised machine learning. Sci. Rep. 9, 17073 (2019).
Dðp; qÞ ¼ ðp1 q1 Þ2 þ ðp2 q2 Þ2 þ ðp3 q3 Þ2 15. S. Yao, P. Swetha, Y. Zhu, Nanomaterial-enabled wearable sensors for healthcare. Adv.
Healthc. Mater. 7, 1700889 (2018).
where p and q are known as the centroids of cluster 1 and cluster 2, 16. G. Lee, G. Y. Bae, J. H. Son, S. Lee, S. W. Kim, D. Kim, S. G. Lee, K. Cho, User-interactive
respectively. The discriminability value (D) is calculated by sub- thermotherapeutic electronic skin based on stretchable thermochromic strain sensor. Adv.
tracting the summation of the SDs (RSTD,1 + RSTD,2) of a pair of clus- Sci. 7, 2001184 (2020).
ters from the corresponding Euclidean distance (E) 17. W. Zhou, S. Yao, H. Wang, Q. Du, Y. Ma, Y. Zhu, Gas-permeable, ultrathin, stretchable epi-
dermal electronics with porous electrodes. ACS Nano 14, 5798–5805 (2020).
D¼E ðRSTD;1 þ RSTD;2 Þ 18. N. Lu, D. H. Kim, Flexible and stretchable electronics paving the way for soft robotics. Soft
Robot. 1, 53–62 (2014).
Positive discriminability values indicate the cluster separation 19. Y. Lu, K. Xu, L. Zhang, M. Deguchi, H. Shishido, T. Arie, R. Pan, A. Hayashi, L. Shen, S. Akita,
without overlaps. For four to six sensors, we applied three-compo- K. Takei, Multimodal plant healthcare flexible sensor system. ACS Nano 14,
nent PCA. For three and two sensor combinations, we applied two- 10966–10975 (2020).
component and one-component PCA approaches, respectively. In 20. H. Yin, Y. Cao, B. Marelli, X. Zeng, A. J. Mason, C. Cao, Soil sensors and plant wearables for
smart and precision agriculture. Adv. Mater. 33, 2007764 (2021).
addition, we calculated the capture variance of each component
21. G. Lee, Q. Wei, Y. Zhu, Emerging wearable sensors for plant health monitoring. Adv. Funct.
of PCA and showed them on each axis.

Downloaded from [Link] on May 08, 2023

Mater. 31, 2106475 (2021).
22. F. Brilli, F. Loreto, I. Baccelli, Exploiting plant volatile organic compounds (VOCs) in agri-
culture to improve sustainable defense strategies and productivity of crops. Front. Plant Sci.
Supplementary Materials 10, 264 (2019).
This PDF file includes: 23. M. Haworth, C. Elliott-Kingston, J. C. McElwain, Stomatal control as a driver of plant evo-
Figs. S1 to S30 lution. J. Exp. Bot. 62, 2419–2423 (2011).
Tables S1 to S4 24. J. M. Nassar, S. M. Khan, D. R. Villava, M. M. Nour, A. S. Almuslem, M. M. Hussain, Compliant
References plant wearables for localized microclimate and plant growth monitoring. npj Flex. Electron.
2, 24 (2018).
View/request a protocol for this paper from Bio-protocol. 25. Y. Zhao, S. Gao, J. Zhu, J. Li, H. Xu, K. Xu, H. Cheng, X. Huang, Multifunctional stretchable
sensors for continuous monitoring of long-term leaf physiology and microclimate. ACS
Omega 4, 9522–9530 (2019).
26. Z. Li, Y. Liu, O. Hoosain, R. Paul, S. Yao, S. Wu, J. B. Ristaino, Y. Zhu, Q. Wei, Real-time
REFERENCES AND NOTES monitoring of plant stresses via chemiresistive profiling of leaf volatiles by a wearable
1. J. B. Ristaino, P. K. Anderson, D. P. Bebber, K. A. Brauman, N. J. Cunniffe, N. V. Fedoroff,
sensor. Matter 4, 2553–2570 (2021).
C. Finegold, K. A. Garrett, C. A. Gilligan, C. M. Jones, M. D. Martin, G. K. MacDonald,
27. M. Yang, Z. D. Hood, X. Yang, M. Chi, Y. Xia, Facile synthesis of Ag@Au core–sheath
P. Neenan, A. Records, D. G. Schmale, L. Tateosian, Q. Wei, The persistent threat of
nanowires with greatly improved stability against oxidation. Chem. Commun. 53,
emerging plant disease pandemics to global food security. Proc. Natl. Acad. Sci. U.S.A. 118,
1965–1968 (2017).
e2022239118 (2021).
28. R. M. C. Jansen, J. Wildt, I. F. Kappers, H. J. Bouwmeester, J. W. Hofstee, E. J. van Henten,
2. E. C. Oerke, Crop losses to pests. J. Agric. Sci. 144, 31–43 (2006).
Detection of diseased plants by analysis of volatile organic compound emission. Annu. Rev.
3. S. Savary, L. Willocquet, S. J. Pethybridge, P. Esker, N. McRoberts, A. Nelson, The global
Phytopathol. 49, 157–174 (2011).
burden of pathogens and pests on major food crops. Nat. Ecol. Evol. 3, 430–439 (2019).
29. C. Tasaltin, F. Basarir, Preparation of flexible VOC sensor based on carbon nanotubes and
4. B. Cara, J. J. Giovannoni, Molecular biology of ethylene during tomato fruit development
gold nanoparticles. Sens. Actuators B Chem. 194, 173–179 (2014).
and maturation. Plant Sci. 175, 106–113 (2008).
30. L. Liu, X. Ye, K. Wu, R. Han, Z. Zhou, T. Cui, Humidity sensitivity of multi-walled carbon
5. M. D. Campos, M. R. Felix, M. Patanita, P. Materatski, C. Caranda, High throughput se-
nanotube networks deposited by dielectrophoresis. Sensors 9, 1714–1721 (2009).
quencing unravels tomato-pathogen interactions towards a sustainable plant breeding.
31. Z. Cui, F. R. Poblete, Y. Zhu, Tailoring the temperature coefficient of resistance of silver
Hortic. Res. 8, 171 (2021).
nanowire nanocomposites and their application as stretchable temperature sensors. ACS
6. M. C. Picanco, L. Bacci, L. B. Crespo, M. M. M. Miranda, J. C. Martins, Effect of integrated pest
Appl. Mater. Interfaces 11, 17836–17842 (2019).
management practices on tomato production and conservation of natural enemies. Agric.
32. J. Bang, W. S. Lee, B. Park, H. Joh, H. K. Woo, S. Jeon, J. Ahn, C. Jeong, T. I. Kim, S. J. Oh, Highly
For. Entomol. 9, 327–335 (2007).
sensitive temperature sensor: Ligand-treated Ag nanocrystal thin films on PDMS with
7. H. N. Fones, D. P. Bebber, T. M. Chaloner, W. T. Kay, G. Steinberg, S. J. Gurr, Threats to global
thermal expansion strategy. Adv. Funct. Mater. 29, 1903047 (2019).
food security from emerging fungal and oomycete crop pathogens. Nat. Food 1,
33. C. Sapsanis, U. Buttner, H. Omran, Y. Belmabkhout, O. Shekhah, M. Eddaoudi, K. N. Salama,
332–342 (2020).
A nafion coated capacitive humidity sensor on a flexible PET substrate, in 2016 IEEE 59th
8. F. M. Padilla, M. Gallardo, M. T. Pena-Fleitas, R. Souza, R. B. Thompson, Proximal optical
International Midwest Symposium on Circuits and Systems (MWSCAS) (2016), pp. 1–4.
sensors for nitrogen management of vegetable crops: A review. Sensors 18, 2083 (2018).
34. A. Karimi, Y. Wang, T. Cselle, M. Morstein, Fracture mechanisms in nanoscale layered hard
9. V. Singh, A. K. Misra, Detection of plant leaf diseases using image segmentation and soft
thin films. Thin Solid Films 420-421, 275–280 (2002).
computing techniques. Inf. Process. Agric. 4, 41–49 (2017).
35. J. Wang, X.-h. Wang, X.-d. Wang, Study on dielectric properties of humidity sensing
10. T. T. S. Lew, V. B. Koman, K. S. Silmore, J. S. Seo, P. Gordiichuk, S. Y. Kwak, M. Park, M. C. Y. Ang,
nanometer materials. Sensors Actuators B Chem. 108, 445–449 (2005).
D. T. Khong, M. A. Lee, M. B. Chan-Park, N. H. Chua, M. S. Strano, Real-time detection of
36. E. Latkowska, Z. Lechowski, J. Bialczyk, J. Pilarski, Photosynthesis and water relations in
wound-induced H2O2 signalling waves in plants with optical nanosensors. Nat. Plants 6,
tomato plants cultivated long-term in media containing (+)-usnic acid. J. Chem. Ecol. 32,
404–415 (2020).
2053–2066 (2006).
11. Z. Li, R. Paul, T. B. Tis, A. C. Saville, J. C. Hansel, T. Yu, J. B. Ristaino, Q. Wei, Non-invasive plant
37. O. Halperin, A. Gebremedhin, R. Wallach, M. Moshelion, High-throughput physiological
disease diagnostics enabled by smartphone-based fingerprinting of leaf volatiles. Nat.
phenotyping and screening system for the characterization of plant–environment inter-
Plants 5, 856–866 (2019).
actions. Plant J. 89, 839–850 (2017).

Lee et al., Sci. Adv. 9, eade2232 (2023) 12 April 2023 13 of 14

S C I E N C E A D VA N C E S | R E S E A R C H A R T I C L E

38. R. Munns, D. P. Schachtman, A. G. Condon, The significance of a two-phase growth re- 53. K. E. Korte, S. E. Skrabalak, Y. Xia, Rapid synthesis of silver nanowires through a CuCl- or
sponse to salinity in wheat and barley. Aust. J. Plant Physiol. 22, 561–569 (1995). CuCl2-mediated polyol process. J. Mater. Chem. 18, 437–441 (2008).
39. P. Nachappa, J. Challacombe, D. C. Margolies, J. R. Nechols, A. E. Whitfield, D. Rotenberg, 54. S. M. Khan, S. F. Shaikh, N. Qaiser, M. M. Hussain, Flexible lightweight CMOS-enabled
Tomato spotted wilt virus benefits its thrips vector by modulating metabolic and plant multisensory platform for plant microclimate monitoring. IEEE Trans. Electron Devices 65,
defense pathways in tomato. Front. Plant Sci. 11, 575564 (2020). 5038–5044 (2018).
40. N. Bessadat, R. Berruyer, B. Hamon, N. Bataille-Simoneau, S. Benichou, M. Kihal, D. E. Henni, 55. J. J. Kim, L. K. Allison, T. L. Andrew, Vapor-printed polymer electrodes for long-term, on-
P. Simoneau, Alternaria species associated with early blight epidemics on tomato and demand health monitoring. Sci. Adv. 5, 463 (2019).
other Solanaceae crops in northwestern Algeria. Eur. J. Plant Pathol. 148, 181–197 (2017). 56. D. Lo Presti, S. Cimini, C. Massaroni, R. D’Amto, M. A. Caponero, L. De Gara, E. Schena, Plant
41. M. Wang, N. Ling, X. Dong, Y. Zhu, Q. Shen, S. Guo, Thermographic visualization of leaf wearable sensors based on FBG technology for growth and microclimate monitoring.
response in cucumber plants infected with the soil-borne pathogen Fusarium oxysporum Sensors 21, 6327 (2021).
f. sp. cucumerinum. Plant Physiol. Biochem. 61, 153–161 (2012). 57. W. Ahmad, B. Jabbar, I. Ahmad, B. M. Jan, M. M. Stylianakis, G. Kenanakis, R. Ikram, Highly
42. V. Ramegowda, M. Senthil-Kumar, The interactive effects of simultaneous biotic and sensitive humidity sensors based on polyethylene oxide/CuO/multi walled carbon nano-
abiotic stresses on plants: Mechanistic understanding from drought and pathogen com- tubes composite nanofibers. Materials 14, 1037 (2021).
bination. J. Plant Physiol. 176, 47–54 (2015). 58. W. P. Shih, L. C. Tsao, C. W. Lee, M. Y. Cheng, C. Chang, Y. J. Yang, K. C. Fan, Flexible tem-
43. K. M. Gold, P. A. Townsend, E. R. Larson, I. Herrmann, A. J. Gevens, Contact reflectance perature sensor array based on a graphite-polydimethylsiloxane composite. Sensors 10,
spectroscopy for rapid, accurate, and nondestructive Phytophthora infestans clonal lineage 3597–3610 (2010).
discrimination. Phytopathology 110, 851–862 (2020). 59. A. Cusano, M. Consales, A. Crescitelli, M. Penza, P. Aversa, P. Delli Veneri, M. Giordano,
44. A. Leal-Junior, L. Avellar, A. Frizera, C. Marques, Smart textiles for multimodal wearable Charge transfer effects on the sensing properties of fiber optic chemical nano-sensors
sensing using highly stretchable multiplexed optical fiber system. Sci. Rep. 10, based on single-walled carbon nanotubes. Carbon 47, 782–788 (2009).
13867 (2020).
45. I. T. Jolliffe, J. Cadima, Principal component analysis: A review and recent developments. Acknowledgments: We also thank A. Locke for providing the leaf porometer device. Funding:
Phil. Trans. R. Soc. A 374, 20150202 (2016). We gratefully acknowledge the funding support from the NCSU Game-Changing Research
46. P. Aghdaie, B. Chaudhary, S. Soleymani, J. Dawson, N. M. Nasrabadi, Detection of morphed Incentive Program for the Plant Science Initiative (GRIP4PSI), USDA (no. 2019-67030-29311),
face images using discriminative wavelet sub-bands. arXiv:2106.08565 [Preprint] [[Link]]. USDA APHIS Farm Bill grant (no. 3.0096), and NSF (nos. 1728370 and 2134664). Author

Downloaded from [Link] on May 08, 2023

(16 June 2020). contributions: G.L., Y.Z., and Q.W. designed and initiated the project. Y.L and O.H. synthesized
47. S. Raza, H. K. Smith, G. J. J. Clarkson, G. Taylor, A. J. Thompson, J. Clarkson, N. M. Rajpoot, and characterized AgNW, Au@AgNW, and functionalized Au@AgNW. G.L. fabricated the
Automatic detection of regions in spinach canopies responding to soil moisture deficit multimodal wearable sensor and performed on-plant measurements. H.W. characterized the
using combined visible and thermal imagery. PLOS ONE 9, e97612 (2014). morphology of sensing materials. A.C.S., T.S., J.B.R., and A.E.W. grew the tomato plants and
48. Y. Liu, M. Zheng, B. O’Connor, J. Dong, Y. Zhu, Curvilinear soft electronics by micromolding performed the inoculation of different pathogens and LAMP tests. S.J. conducted PCA of the
of metal nanowires in capillaries. Sci. Adv. 8, 6996 (2022). real-time sensor data. R.P. helped with the plant pathogen test in the laboratory. D.R. provided
valuable discussion on the experimental design and results analysis. G.L., Y.Z., and Q.W. wrote
49. A. C. Castonguay, N. Yi, B. Li, J. Zhao, H. Li, Y. Gao, N. N. Nova, N. Tiwari, L. D. Zarzar, H. Cheng,
the manuscript. All the authors read and revised the manuscript. Competing interests: The
Direct laser writing of microscale metal oxide gas sensors from liquid precursors. ACS Appl.
authors declare that they have no competing interests. Data and materials availability: All
Mater. Interfaces 14, 28163–28173 (2022).
data needed to evaluate the conclusions in the paper are present in the paper and/or the
50. R. Yang, W. Zhang, N. Tiwari, H. Yan, T. Li, H. Cheng, Multimodal sensors with decoupled
Supplementary Materials.
sensing mechanisms. Adv. Sci. 9, 2202470 (2022).
51. S. Zhang, J. Zhu, Y. Zhang, Z. Chen, C. Song, J. Li, N. Yi, D. Qiu, K. Guo, C. Zhang, T. Pan, Y. Lin, Submitted 1 August 2022
H. Zhou, H. Long, H. Yang, H. Cheng, Standalone stretchable RF systems based on asym- Accepted 16 March 2023
metric 3D microstrip antennas with on-body wireless communication and energy har- Published 12 April 2023
vesting. Nano Energy 96, 107069 (2022). 10.1126/sciadv.ade2232
52. N. Yi, Y. Gao, A. L. Verso Jr., J. Zhu, D. Erdely, C. Xue, R. Lavelle, H. Cheng, Fabricating
functional circuits on 3D freeform surfaces via intense pulsed light-induced zinc mass
transfer. Mater. Today 50, 24–34 (2021).

Lee et al., Sci. Adv. 9, eade2232 (2023) 12 April 2023 14 of 14

 Abaxial leaf surface-mounted multimodal wearable sensor for continuous plant
physiology monitoring
Giwon Lee, Oindrila Hossain, Sina Jamalzadegan, Yuxuan Liu, Hongyu Wang, Amanda C. Saville, Tatsiana Shymanovich,
Rajesh Paul, Dorith Rotenberg, Anna E. Whitfield, Jean B. Ristaino, Yong Zhu, and Qingshan Wei

Sci. Adv., 9 (15), eade2232.

DOI: 10.1126/sciadv.ade2232

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