Paper-Based Analytical Devices for the Rapid and Direct Electrochemical Detection of Hydrogen Peroxide in Tomato Leaves Inoculated with Botrytis cinerea

Hydrogen peroxide (H2O2) is an important signaling molecule and plays key roles in multiple plant physiological processes. The rapid and direct monitoring of H2O2 could improve our understanding of its regulatory mechanisms in plants. In this study, we developed a paper-based analytical device consisting of a disposable nano-gold modified indium tin oxide working electrode to provide a platform for the rapid and direct detection of H2O2. The total analytical time was dramatically shortened to be approximate 3 min due to the avoidance of the time-consuming and complex treatment of plant samples. In addition, the amount of plant samples required was less than 3 mg in our approach. We used this system to monitor the concentrations of H2O2 in tomato leaves infected by Botrytis cinerea within 24 h. Our results showed that the concentration of H2O2 in tomato leaves was increased in the initial phase, peaked at 1.5 μmol gFW−1 at 6 h, and then decreased. The production trend of H2O2 in tomato leaves inoculated with Botrytis cinerea detected with our approach is similar to the 3,3-diaminobenzidine staining method. Taken together, our study offers a rapid and direct approach for the detection of H2O2, which will not only pave the way for the further investigation of the regulation mechanisms of H2O2 in plants, but also promote the development of precision agriculture technology.


Introduction
Hydrogen peroxide (H 2 O 2 ), a simple molecule discovered by Louis Jacques Denard 100 years ago, is one of the primary reactive oxygen species (ROS) in plants [1][2][3]. Compared to other ROS, such as superoxide anion radicals (O2•−) and hydroxyl radicals (OH•), H 2 O 2 is relatively stable, less reactive, and electrically neutral. These characteristics of H 2 O 2 enable the molecule to translocate cell membranes and reach cell locations that are distant from the site of its formation [1][2][3]. H 2 O 2 can the other chemical reagents were of analytical grade. The ITO conductive glass (40.64 × 35.56 × 0.11 cm STN, 10 ohm) was obtained from Nanbo Display Technology Co., Ltd. (Shenzhen, Guangdong, China). The Whatman No. 1 qualitative filter paper was obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA). The Harris Uni-CoreTM (Tip ID 4.0 mm) Miltex ® was purchased from Ted Pella Inc. (Redding, CA, USA). The tomato seeds (Shanghai 906, one-generation hybrid) were purchased from Lintong Changfeng Vegetable Breeding Farm in Xi'an City. The H 2 O 2 was obtained from Shanghai Zhanyun Chemical Co. Ltd. (Shanghai, China). Double-distilled water was used in all the experiments. For analysis of the concentration of H 2 O 2 produced in response to pathogen infection, four-week-old tomato leaves were sprayed with spore suspensions or a buffer as mock inoculation (control). Briefly, spores were collected in 1% maltose buffer from 10-day-old Botrytis cinerea cultures grown on 2 × V8 agar (36% V8 juice, 0.2% CaCO 3 , 2% agar) by passing through two layers of cheesecloth with a spore density of 1 × 10 5 spores mL -1 . The inoculated plants were covered with a transparent plastic film and maintained in a growth chamber with conditions similar to plant growth conditions. Leaves from at least four individual plants were used in each experiment.

Paper-Based Electroanalytical Devices and Electrochemical Detection
For the nano-gold-modified ITO electrode preparation, the ITO glass was cut into 20 × 8 mm pieces and washed using acetone and ethanol ultrasonically for 10 min. The washed ITO glasses were cleaned repeatedly with double-distilled water to remove acetone and ethanol. The cleaned ITO glasses were dried in an oven at 50 • C. As showed in the Scheme 1, 10 µL of HAuCl 4 •3H 2 O solution was dropped on the conductive surfaces of the ITO glasses adhered with a layer of perforated (4 mm) transparent tape. An Ag/AgCl electrode and a platinum wire electrode were used as a reference electrode and counter electrode, respectively. Then, the HAuCl 4 •3H 2 O solution was electroplated on the conductive surfaces of the ITO glasses (4-mm holes) by cyclic voltammetry with the parameters −1-0.2 V potential range, 0.1 V/s scanning speed, 10 scanning segments, 0.001-V sampling intervals, and 2 s standing time. The modified electrode was named a nano-gold-modified ITO electrode.

Material Preparation
HAuCl4·3H2O (10 g/L, 34 μL) was added to 1 mmol/L of KCl (200 μL), then 3766 μL of double-distilled water was added to prepare 4 mL of 0.25 mmol/L HAuCl4·3H2O, and stored in a refrigerator at 4 °C for later use in electroplating. The H2O2 (30% wt. in water) was diluted using 0.1 M of phosphate buffered solution (pH 7.0) for detection. Tomato plants were grown in a mixture of perlite: vermiculite: plant ash (1:6:2) in a growth room at 22 °C under a 16 h light and 8 h dark regime. For analysis of the concentration of H2O2 produced in response to pathogen infection, four-week-old tomato leaves were sprayed with spore suspensions or a buffer as mock inoculation (control). Briefly, spores were collected in 1% maltose buffer from 10-day-old Botrytis cinerea cultures grown on 2 × V8 agar (36% V8 juice, 0.2% CaCO3, 2% agar) by passing through two layers of cheesecloth with a spore density of 1 × 10 5 spores mL -1 . The inoculated plants were covered with a transparent plastic film and maintained in a growth chamber with conditions similar to plant growth conditions. Leaves from at least four individual plants were used in each experiment.

Paper-Based Electroanalytical Devices and Electrochemical Detection
For the nano-gold-modified ITO electrode preparation, the ITO glass was cut into 20 × 8 mm pieces and washed using acetone and ethanol ultrasonically for 10 min. The washed ITO glasses were cleaned repeatedly with double-distilled water to remove acetone and ethanol. The cleaned ITO glasses were dried in an oven at 50 °C . As showed in the Scheme 1, 10 μL of HAuCl4·3H2O solution was dropped on the conductive surfaces of the ITO glasses adhered with a layer of perforated (4 mm) transparent tape. An Ag/AgCl electrode and a platinum wire electrode were used as a reference electrode and counter electrode, respectively. Then, the HAuCl4·3H2O solution was electroplated on the conductive surfaces of the ITO glasses (4-mm holes) by cyclic voltammetry with the parameters −1-0.2 V potential range, 0.1 V/s scanning speed, 10 scanning segments, 0.001-V sampling intervals, and 2 s standing time. The modified electrode was named a nano-gold-modified ITO electrode. For electrochemical detection, tomato leaves inoculated with spore suspensions of Botrytis cinerea or buffer solution (Mock) were obtained using a Miltex Biopsy Punch with a diameter of 4 mm (Scheme 2A). The circular forms of the retrieved leaf samples were weighed and placed on the surface of a nano-gold-modified ITO electrode, then 10 µL of phosphate buffer solution with a pH of 7.0 was dropped on the electrode, and the electrode surface was covered with a piece of filter paper (Scheme 2B). H 2 O 2 was detected in tomato leaf using a CHI 1240C electrochemical workstation (CH Instruments Inc., Austin, TX, USA). A three-electrode system consisting of a modified nano-gold electrode (working electrode), an Ag/AgCl electrode (reference electrode), and a platinum wire electrode (counter electrode) was used (Scheme 2C). The H 2 O 2 was detected using differential pulse voltammetry with a −1.2-0 V potential range, a potential increment of 0.005 V, an amplitude of 0.05 V, a pulse width of 0.2 s, a sampling width of 0.067 s, a pulse period of 0.5 s, and a standing time of 2 s. Before each test, the counter electrode and the reference electrode were washed with double-distilled water thoroughly. The H 2 O 2 concentrations in tomato leaves infected with Botrytis cinerea were determined directly and rapidly (Scheme 2D).
Sensors 2020, 20, x FOR PEER REVIEW 4 of 11 mm (Scheme 2a). The circular forms of the retrieved leaf samples were weighed and placed on the surface of a nano-gold-modified ITO electrode, then 10 μL of phosphate buffer solution with a pH of 7.0 was dropped on the electrode, and the electrode surface was covered with a piece of filter paper (Scheme 2b). H2O2 was detected in tomato leaf using a CHI 1240C electrochemical workstation (CH Instruments Inc., Austin, TX, USA). A three-electrode system consisting of a modified nano-gold electrode (working electrode), an Ag/AgCl electrode (reference electrode), and a platinum wire electrode (counter electrode) was used (Scheme 2c). The H2O2 was detected using differential pulse voltammetry with a −1.2-0 V potential range, a potential increment of 0.005 V, an amplitude of 0.05 V, a pulse width of 0.2 s, a sampling width of 0.067 s, a pulse period of 0.5 s, and a standing time of 2 s. Before each test, the counter electrode and the reference electrode were washed with double-distilled water thoroughly. The H2O2 concentrations in tomato leaves infected with Botrytis cinerea were determined directly and rapidly (Scheme 2d).

Diaminobenzidine Staining
To validate our approach for the detection of H2O2 in tomato leaves, 3-3′ diaminobenzidine (DAB) staining was performed according to the methods described previously [30]. Briefly, tomato leaves were collected from inoculated plants 0, 1, and 6 h after inoculation with Botrytis cinerea spores and dipped into DAB solution (0.5 mg/mL, pH 3.8) for 8 h in the dark at room temperature. The DAB-treated leaves were placed into 95% ethanol at 80 °C for 30 min to remove chlorophyll. Subsequently, the leaves were maintained in 60% glycerol and the accumulation of H2O2 was visualized using a digital camera.

Results and Discussion
Since the nano-gold-modified ITO electrodes was fabricated by electroplating HAuCl4·3H2O on the surface of the ITO electrodes, we investigated the influence of different concentrations of HAuCl4·3H2O at the ITO electrodes on the electrochemical responses of 200 μM of H2O2. We observed that the 0.25 mM of HAuCl4·3H2O-electroplated ITO electrodes exhibited higher

Diaminobenzidine Staining
To validate our approach for the detection of H 2 O 2 in tomato leaves, 3-3 diaminobenzidine (DAB) staining was performed according to the methods described previously [30]. Briefly, tomato leaves were collected from inoculated plants 0, 1, and 6 h after inoculation with Botrytis cinerea spores and dipped into DAB solution (0.5 mg/mL, pH 3.8) for 8 h in the dark at room temperature. The DAB-treated leaves were placed into 95% ethanol at 80 • C for 30 min to remove chlorophyll. Subsequently, the leaves were maintained in 60% glycerol and the accumulation of H 2 O 2 was visualized using a digital camera.

Results and Discussion
Since the nano-gold-modified ITO electrodes was fabricated by electroplating HAuCl 4 Figure 1A). Notably, there were no electrochemical responses to H 2 O 2 on the bare ITO electrodes. The potential of H 2 O 2 further increased from −0.925 V to −0.775 V when the concentration of HAuCl 4 •3H 2 O was increased ( Figure 1B). In addition, the potential of H 2 O 2 on the 0.25 mM of HAuCl 4 •3H 2 O-modified ITO electrodes was more stable. The surface characterizations of the bare ITO electrode and the 0.25mM of HAuCl 4 •3H 2 O-electroplated ITO electrodes were observed using a Hitachi S-3400 II scanning electron microscope. Compared to the bare ITO electrode, the 5~10 nm gold nanoparticles homogeneously distributed throughout the surface of the ITO electrode ( Figure 2).
Sensors 2020, 20, x FOR PEER REVIEW 5 of 11 electrochemical responses to the H2O2 (Figure 1a). Notably, there were no electrochemical responses to H2O2 on the bare ITO electrodes. The potential of H2O2 further increased from −0.925 V to −0.775 V when the concentration of HAuCl4·3H2O was increased (Figure 1b). In addition, the potential of H2O2 on the 0.25 mM of HAuCl4·3H2O-modified ITO electrodes was more stable. The surface characterizations of the bare ITO electrode and the 0.25mM of HAuCl4·3H2O-electroplated ITO electrodes were observed using a Hitachi S-3400 II scanning electron microscope. Compared to the bare ITO electrode, the 5~10 nm gold nanoparticles homogeneously distributed throughout the surface of the ITO electrode ( Figure 2).  In the present study, H2O2 was quantified and verified based on the differential pulse voltammetry peak heights and potentials. In order to study the influence of the air on the H2O2 detection, prior to each experiment a stream of highly pure nitrogen was gently blown inside a plastic bag with the detection device to maintain the nitrogen atmosphere for at least 20 min. Figure  3 illustrates differential pulse voltammetry curves of H2O2 with various concentrations at the nano-gold-modified ITO electrodes in the air or nitrogen atmosphere. There were two peaks at approximately −0.4 and −0.9 V potentials. The peaks at −0.9 V further increased when the H2O2 concentration was increased, while the peaks at −0.4 V were irregular (Figure 3a,c). The oxidation peak currents of H2O2 displayed a linear response to the concentrations of H2O2 from 10 to 1000 μM in the air or nitrogen atmosphere (Figure 3b,d). The linear relationship between the peak current and the concentration was Y = 2.692X + 259.943 (R 2 = 0.9687) in the air atmosphere and Y = 2.296X + 26.66 (R 2 = 0.9897) in the nitrogen atmosphere (X: concentration of H2O2; Y: peak current magnitude). There were no obvious differences except for the base currents between the detection of H2O2 in the electrochemical responses to the H2O2 (Figure 1a). Notably, there were no electrochemical responses to H2O2 on the bare ITO electrodes. The potential of H2O2 further increased from −0.925 V to −0.775 V when the concentration of HAuCl4·3H2O was increased (Figure 1b). In addition, the potential of H2O2 on the 0.25 mM of HAuCl4·3H2O-modified ITO electrodes was more stable. The surface characterizations of the bare ITO electrode and the 0.25mM of HAuCl4·3H2O-electroplated ITO electrodes were observed using a Hitachi S-3400 II scanning electron microscope. Compared to the bare ITO electrode, the 5~10 nm gold nanoparticles homogeneously distributed throughout the surface of the ITO electrode ( Figure 2).  In the present study, H2O2 was quantified and verified based on the differential pulse voltammetry peak heights and potentials. In order to study the influence of the air on the H2O2 detection, prior to each experiment a stream of highly pure nitrogen was gently blown inside a plastic bag with the detection device to maintain the nitrogen atmosphere for at least 20 min. Figure  3 illustrates differential pulse voltammetry curves of H2O2 with various concentrations at the nano-gold-modified ITO electrodes in the air or nitrogen atmosphere. There were two peaks at approximately −0.4 and −0.9 V potentials. The peaks at −0.9 V further increased when the H2O2 concentration was increased, while the peaks at −0.4 V were irregular (Figure 3a,c). The oxidation peak currents of H2O2 displayed a linear response to the concentrations of H2O2 from 10 to 1000 μM in the air or nitrogen atmosphere (Figure 3b,d). The linear relationship between the peak current and the concentration was Y = 2.692X + 259.943 (R 2 = 0.9687) in the air atmosphere and Y = 2.296X + 26.66 (R 2 = 0.9897) in the nitrogen atmosphere (X: concentration of H2O2; Y: peak current magnitude). There were no obvious differences except for the base currents between the detection of H2O2 in the In the present study, H 2 O 2 was quantified and verified based on the differential pulse voltammetry peak heights and potentials. In order to study the influence of the air on the H 2 O 2 detection, prior to each experiment a stream of highly pure nitrogen was gently blown inside a plastic bag with the detection device to maintain the nitrogen atmosphere for at least 20 min. Figure 3 illustrates differential pulse voltammetry curves of H 2 O 2 with various concentrations at the nano-gold-modified ITO electrodes in the air or nitrogen atmosphere. There were two peaks at approximately −0.4 and −0.9 V potentials. The peaks at −0.9 V further increased when the H 2 O 2 concentration was increased, while the peaks at −0.4 V were irregular ( Figure 3A,C). The oxidation peak currents of H 2 O 2 displayed a linear response to the concentrations of H 2 O 2 from 10 to 1000 µM in the air or nitrogen atmosphere ( Figure 3B,D). The linear relationship between the peak current and the concentration was Y = 2.692X + 259.943 (R 2 = 0.9687) in the air atmosphere and Y = 2.296X + 26.66 (R 2 = 0.9897) in the nitrogen atmosphere (X: concentration of H 2 O 2 ; Y: peak current magnitude). There were no obvious differences except for the base currents between the detection of H 2 O 2 in the air and the nitrogen atmosphere.
Considering the importance of the rapid detection of H 2 O 2 from the plant sample, a strategy for the direct detection of H 2 O 2 in the air was selected.
Sensors 2020, 20, x FOR PEER REVIEW 6 of 11 air and the nitrogen atmosphere. Considering the importance of the rapid detection of H2O2 from the plant sample, a strategy for the direct detection of H2O2 in the air was selected. Figure 3. The differential pulse voltammetry curves of H2O2 with different concentrations (0~1000 μM) on the nano-gold-modified ITO electrodes (A) and the calibration curve between the differential pulse voltammetry peak currents and the H2O2 concentrations in the air (B), the differential pulse voltammetry curves of H2O2 with different concentrations (0~1000 μM) on the nano-gold-modified ITO electrodes (C), and the calibration curve between the differential pulse voltammetry peak currents and the H2O2 concentrations in the N2 atmosphere (D). The average values and standard deviations were obtained based on 6 replicates.
The detection limit and reproducibility are important parameters for the evaluation of sensor performance. The detection limit of our system was estimated to be 1 μM based on a signal-to-noise ratio of six. The reproducibility of the nano-gold-modified ITO electrode was estimated from the response to 100 μM of H2O2 using six different electrodes. The relative standard deviation was found to be 5.8%, indicating a good reproducibility for the sensor preparation. We investigated the potential interferences with the determination of H2O2 in the plants from plant signaling molecules, and found that there were no significant interferences in the presence of abscisic acid, indole-3-acetic acid, salicylic acid, jasmonic acid, methyl jasmonate, and ascorbic acid under 100 or 500 μM of H2O2 ( Figure 4). The results indicated that nano-gold-modified ITO electrodes could facilitate the determination of H2O2 in plant samples. More importantly, the nano-gold-modified ITO electrodes could be fabricated as one-time-use disposable electrodes, which could avoid the contamination of electrodes. In addition, the fabrication of the nano-gold-modified ITO electrodes was feasible (less than five minutes to prepare one electrode) and suitable for mass production. Compared with previous reports, our strategy was superior regarding the simple work electrode preparation, good analytical performance, volume of buffer solution, and simple sample preparation for the H2O2 detection in plants (Table 1). Figure 3. The differential pulse voltammetry curves of H 2 O 2 with different concentrations (0~1000 µM) on the nano-gold-modified ITO electrodes (A) and the calibration curve between the differential pulse voltammetry peak currents and the H 2 O 2 concentrations in the air (B), the differential pulse voltammetry curves of H 2 O 2 with different concentrations (0~1000 µM) on the nano-gold-modified ITO electrodes (C), and the calibration curve between the differential pulse voltammetry peak currents and the H 2 O 2 concentrations in the N 2 atmosphere (D). The average values and standard deviations were obtained based on 6 replicates.
The detection limit and reproducibility are important parameters for the evaluation of sensor performance. The detection limit of our system was estimated to be 1 µM based on a signal-to-noise ratio of six. The reproducibility of the nano-gold-modified ITO electrode was estimated from the response to 100 µM of H 2 O 2 using six different electrodes. The relative standard deviation was found to be 5.8%, indicating a good reproducibility for the sensor preparation. We investigated the potential interferences with the determination of H 2 O 2 in the plants from plant signaling molecules, and found that there were no significant interferences in the presence of abscisic acid, indole-3-acetic acid, salicylic acid, jasmonic acid, methyl jasmonate, and ascorbic acid under 100 or 500 µM of H 2 O 2 ( Figure 4). The results indicated that nano-gold-modified ITO electrodes could facilitate the determination of H 2 O 2 in plant samples. More importantly, the nano-gold-modified ITO electrodes could be fabricated as one-time-use disposable electrodes, which could avoid the contamination of electrodes. In addition, the fabrication of the nano-gold-modified ITO electrodes was feasible (less than five minutes to prepare one electrode) and suitable for mass production. Compared with previous reports, our strategy was superior regarding the simple work electrode preparation, good analytical performance, volume of buffer solution, and simple sample preparation for the H 2 O 2 detection in plants (Table 1).   As a necrotrophic pathogen, Botrytis cinerea can colonize senescent or dead plant tissues and cause gray mold disease [34,35]. Botrytis cinerea infects over 200 dicot crop hosts such as tomato, As a necrotrophic pathogen, Botrytis cinerea can colonize senescent or dead plant tissues and cause gray mold disease [34,35]. Botrytis cinerea infects over 200 dicot crop hosts such as tomato, grapes, and strawberry. Therefore, it could cause substantial economic losses [34,35]. In our experiments, typical disease symptoms-e.g., necrotic lesions-could be observed in tomato leaves inoculated with Botrytis cinerea spore suspensions at 3 dpi. Figure 5A illustrates the typical differential pulse voltammetry detection curves of H 2 O 2 in the tomato leaf samples inoculated with Botrytis cinerea spores at different time points. H 2 O 2 in the tomato leaf samples could be identified at a DPV peak potential of −0.90 V. It is necessary to emphasize that the total analytical time was dramatically shortened to approximately 3 min because our method avoided the time-consuming and complex treatment of tomato leaf samples. In addition, the amount of tomato leaf samples was less than 3 mg in our approach. The concentrations of H 2 O 2 in the tomato leaf samples inoculated with B. cinerea increased at the initial phase and peaked at 1.5 µmol (gFW) −1 6 h after inoculation, then decreased gradually until the presence of H 2 O 2 could not be detected 24 h after inoculation ( Figure 5B). In the normal tomato leaves, for comparison, almost no H 2 O 2 could be observed at different time points ( Figure 5B). grapes, and strawberry. Therefore, it could cause substantial economic losses [34,35]. In our experiments, typical disease symptoms-e.g., necrotic lesions-could be observed in tomato leaves inoculated with Botrytis cinerea spore suspensions at 3 dpi. Figure 5a illustrates the typical differential pulse voltammetry detection curves of H2O2 in the tomato leaf samples inoculated with Botrytis cinerea spores at different time points. H2O2 in the tomato leaf samples could be identified at a DPV peak potential of −0.90 V. It is necessary to emphasize that the total analytical time was dramatically shortened to approximately 3 min because our method avoided the time-consuming and complex treatment of tomato leaf samples. In addition, the amount of tomato leaf samples was less than 3 mg in our approach. The concentrations of H2O2 in the tomato leaf samples inoculated with B. cinerea increased at the initial phase and peaked at 1.5 μmol (gFW) −1 6 h after inoculation, then decreased gradually until the presence of H2O2 could not be detected 24 h after inoculation (Figure 5b). In the normal tomato leaves, for comparison, almost no H2O2 could be observed at different time points (Figure 5b). In order to compare and evaluate the ability of our approach in detecting H2O2 production after tomato leaves were inoculated with Botrytis cinerea, DAB staining was also employed. As showed in Figure 6a, no significant accumulation of H2O2 was observed in tomato leaves 0 h after inoculation. One hour post-inoculation, a few spots due to the presence of H2O2 could be noticed (Figure 6b). Six hours after inoculation, much H2O2 could be visualized throughout the leaves. The production trend of H2O2 in tomato leaves inoculated with Botrytis cinerea based on DAB staining was similar in our method. Patykowski and Urbanek also reported that the H2O2 concentrations of tomato leaves inoculated with Botrytis cinerea increased as early as 5 h after inoculation, increasing to 0.2 μmol (gFW) −1 [36]. These results implied that our approach could offer a rapid and effective means of In order to compare and evaluate the ability of our approach in detecting H 2 O 2 production after tomato leaves were inoculated with Botrytis cinerea, DAB staining was also employed. As showed in Figure 6A, no significant accumulation of H 2 O 2 was observed in tomato leaves 0 h after inoculation. One hour post-inoculation, a few spots due to the presence of H 2 O 2 could be noticed ( Figure 6B). Six hours after inoculation, much H 2 O 2 could be visualized throughout the leaves. The production trend of H 2 O 2 in tomato leaves inoculated with Botrytis cinerea based on DAB staining was similar in our method. Patykowski and Urbanek also reported that the H 2 O 2 concentrations of tomato leaves inoculated with Botrytis cinerea increased as early as 5 h after inoculation, increasing to 0.2 µmol (gFW) −1 [36]. These results implied that our approach could offer a rapid and effective means of studying H 2 O 2 dynamics in plants.

Conclusions
In the present study, we developed a platform using paper-based analytical devices coupled with nano-gold-modified ITO working electrodes for detecting H2O2. Our approach could detect H2O2 in plant samples at the milligram scale. In addition, the complex and time-consuming pre-treatment procedures required in conventional methods for the quantification of H2O2 in plant samples were avoided, and thus our method could determine the concentrations of H2O2 in plants more rapidly and directly. Using our approach, differentiable H2O2 concentrations were obtained in tomato leaves after infection with Botrytis cinerea. Our study presents a valid method that not only facilitates the investigation of the regulating mechanisms of H2O2 in plants but also promotes the development of precision agriculture technology.
Author Contributions: L.S. for the study design, literature search, data analysis, data interpretation, and writing. Y.P. and J.W for the data collection, data analysis, and figures. D.Z and M.H for data collection and data analysis. S.Z., X.Z. and D.L for data analysis and data interpretation. F.S. and C.Z for writing. All authors have read and agreed to the published version of the manuscript.

Conclusions
In the present study, we developed a platform using paper-based analytical devices coupled with nano-gold-modified ITO working electrodes for detecting H 2 O 2. Our approach could detect H 2 O 2 in plant samples at the milligram scale. In addition, the complex and time-consuming pre-treatment procedures required in conventional methods for the quantification of H 2 O 2 in plant samples were avoided, and thus our method could determine the concentrations of H 2 O 2 in plants more rapidly and directly. Using our approach, differentiable H 2 O 2 concentrations were obtained in tomato leaves after infection with Botrytis cinerea. Our study presents a valid method that not only facilitates the investigation of the regulating mechanisms of H 2 O 2 in plants but also promotes the development of precision agriculture technology.