Disposable Stainless-Steel Wire-Based Electrochemical Microsensor for In Vivo Continuous Monitoring of Hydrogen Peroxide in Vein of Tomato Leaf

As one of the pivotal signal molecules, hydrogen peroxide (H2O2) has been demonstrated to play important roles in many physiological processes of plants. Continuous monitoring of H2O2 in vivo could help understand its regulation mechanism more clearly. In this study, a disposable electrochemical microsensor for H2O2 was developed. This microsensor consists of three parts: low-cost stainless-steel wire with a diameter of 0.1 mm modified by gold nanoparticles (disposable working electrode), an untreated platinum wire with a diameter of 0.1 mm (counter electrode), and an Ag/AgCl wire with a diameter of 0.1 mm (reference electrode), respectively. The microsensor could detect H2O2 in levels from 10 to 1000 µM and exhibited excellent selectivity. On this basis, the dynamic change in H2O2 in the vein of tomato leaf under high salinity was continuously monitored in vivo. The results showed that the production of H2O2 could be induced by high salinity within two hours. This study suggests that the disposable electrochemical microsensor not only suits continuously detecting H2O2 in microscopic plant tissue in vivo but also reduces the damage to plants. Overall, our strategy will help to pave the foundation for further investigation of the generation, transportation, and elimination mechanism of H2O2 in plants.


Introduction
Hydrogen peroxide (H 2 O 2 ), a simple molecular compound discovered by Louis Jacques Denard a hundred years ago, is one of the crucial members of the reactive oxygen species (ROS) [1,2]. Compared to the other ROS members such as 1 O 2 , O 2 , and OH, H 2 O 2 is more stable. H 2 O 2 is generally produced in various cellular components such as chloroplast, peroxisomes, and mitochondria, where many metabolic pathways occur in plants [2][3][4][5][6]. The levels of H 2 O 2 in the fresh leaf extraction were significantly different in different plants under normal growth conditions [7]. For example, the concentrations of H 2 O 2 ranged from 0.15 µmol·(g·FW) −1 in tobacco leaves to 6 µmol·(g·FW) −1 in marigold leaves under normal conditions [8,9]. Tewari et al. reported that the localization of H 2 O 2 in different regions of maize leaf exhibited an increasing pattern from the base to the leaf tip [10]. In general, there is a balance between the production and elimination of H 2 O 2 . However, this balance can be broken by stresses or mechanical wounding [11]. H 2 O 2 is also

Material Preparation
HAuCl 4 ·3H 2 O (10 g/L) was distilled by the double distilled water to prepare 0.5 mmol/L HAuCl 4 ·3H 2 O for electroplating. Thirty percent of H 2 O 2 was diluted to different concentrations by 0.2 M phosphate-buffered solution (PBS) with pH 7.0 for detection. Tomato plants were grown in a mixture of perlite: plant ash: vermiculite (1:2:6) in a growth room at 22 • C. The lighting regime for the tomato plants was 16 h of light/8 h dark at a total light intensity of around 540 µmol/m 2 /S. For analysis of the production and transportation of H 2 O 2 in the vein of tomato leaf response to high salinity stress, six-week-old tomato was treated with 0.4 M NaCl solution. As shown in Figure S1, the lateral stem of tomato was cut and the end of the stem was wrapped with cotton soaked in water (control) or 0.4 M NaCl solution (high salinity stress).

The Preparation of SS Wire-Based Electrochemical Microsensor
The SS wire, Pt wire, and silver wire were cut into 50 mm segments and washed with ethanol, water under ultrasonic for 10 min, and dried at room temperature, respectively. The capillaries (d = 0.3 mm) were cut into 35 mm segments and an SS wire was placed into the capillary (Scheme 1A). One end of the SS wire was exposed to a length of 2 mm for sample testing, and the other end of the SS wire was exposed to a length of 13 mm for linking to the electrochemical workstation. The SS wire was fixed in the capillary by the 3M super glue. Finally, copper conductive tape was applied to the end of the 13 mm SS wire (Scheme 1B). Then, electrodeposition of nano-Au on the bare SS was carried out in a 0.5 mmol/L HAuCl 4 solution under the potential from −0.8 to 0 V by cyclic voltammetry (CV) (Scheme 1C). The modified SS wire was used as a working electrode (Au/SS electrode). The unmodified Pt wire (counter electrode) was fixed in the capillary in the same way as the SS wire. For the reference electrode, the 0.1 mm silver wire was fixed in the capillary in the same way as the Pt electrode. Then, the fixed-silver wire was soaked in 84 disinfectant (the main composition is NaClO) for 30 min to generate a silver/silver chloride electrode (Ag/AgCl).

Electrochemical Measurements
All electrochemical experiments were carried out on a CHI 1240C electrochemical station. The electrocatalytic behaviors of the electrochemical detection system toward H2O2 were investigated by CV with the potential from −1.0 to 0.6 V. H2O2 was detected by an amperometric i-t curve with an initial potential of −0.6 V, sample interval of 0.1 s, running time of 1500 s, quiet time of 0 s, scales during a run of 1 on the SS wire-based electrochemical microsensor.

qRT-PCR Analysis of Gene Expression
In order to evaluate the level of H2O2 in the vein of tomato leaves, the expression of SLRbohA and SLRbohB was analyzed. Total RNA was extracted from the vein of tomato leaves (0.20 g fresh weight) with TRIzol (Invitrogen, Shanghai, China) and then treated with RNase-free DNase (TaKaRa, Dalian, China). First-strand cDNAs were synthesized by AMV reserve transcriptase (TaKaRa, Dalian, China) as instructed. The obtained cDNA was used for gene expression analysis with qRT-PCR. Each qPCR reaction contained 0.1 µg cDNA, 7.5 pmol of each gene-specific primer (Table S1), and 12.5 µL × 2 SYBR premix Ex TaqTM (TaKaRa, Dalian, China) in a final volume of 25 µL. The qRT-PCR was performed in a Roche LC480 real-time quantitative polymerase chain reaction detection system. The relative gene expression levels were calculated using the 2 −ΔΔCT method as described [37]. Three independent biological replicates were analyzed.

Characterization of theModified Electrodes
The surface characterization of the electrode was observed using a scanning electron microscope (ZEISSSEM300, Oberkochen, Germany). As shown in Figure 1, the surface of the bare SS wire electrode with a diameter of 0.1 mm is smooth ( Figure 1A).The electrodeposition methods were used to deposit gold nanoparticles on the bare SS wire electrode.

Electrochemical Measurements
All electrochemical experiments were carried out on a CHI 1240C electrochemical station. The electrocatalytic behaviors of the electrochemical detection system toward H 2 O 2 were investigated by CV with the potential from −1.0 to 0.6 V. H 2 O 2 was detected by an amperometric i-t curve with an initial potential of −0.6 V, sample interval of 0.1 s, running time of 1500 s, quiet time of 0 s, scales during a run of 1 on the SS wire-based electrochemical microsensor.

qRT-PCR Analysis of Gene Expression
In order to evaluate the level of H 2 O 2 in the vein of tomato leaves, the expression of SLRbohA and SLRbohB was analyzed. Total RNA was extracted from the vein of tomato leaves (0.20 g fresh weight) with TRIzol (Invitrogen, Shanghai, China) and then treated with RNase-free DNase (TaKaRa, Dalian, China). First-strand cDNAs were synthesized by AMV reserve transcriptase (TaKaRa, Dalian, China) as instructed. The obtained cDNA was used for gene expression analysis with qRT-PCR. Each qPCR reaction contained 0.1 µg cDNA, 7.5 pmol of each gene-specific primer (Table S1), and 12.5 µL × 2 SYBR premix Ex TaqTM (TaKaRa, Dalian, China) in a final volume of 25 µL. The qRT-PCR was performed in a Roche LC480 real-time quantitative polymerase chain reaction detection system. The relative gene expression levels were calculated using the 2 −∆∆CT method as described [37]. Three independent biological replicates were analyzed.

Characterization of theModified Electrodes
The surface characterization of the electrode was observed using a scanning electron microscope (ZEISSSEM300, Oberkochen, Germany). As shown in Figure 1, the surface of the bare SS wire electrode with a diameter of 0.1 mm is smooth ( Figure 1A).The electrodeposition methods were used to deposit gold nanoparticles on the bare SS wire electrode. Compared to the bare SS electrode, the 5~15 nm gold nanoparticles homogeneously dis-tributed throughout the surface of the bare SS wire electrode ( Figure 1C). The element composition of the modified electrodes was also characterized by an energy dispersive spectrometer (Oxford Instruments, UK). As shown in Figure 1B, the main element of bare SS wire electrode was Fe, above 60.9%. After electrodepositing of Au, the elemental composition of Fe decreased to 52.8%, while the Au increased from 0% to 7.2% ( Figure 1D). All of these results suggested the gold nanoparticles were electroplated on the bare SS wire electrode.
Biosensors 2022, 12, x FOR PEER REVIEW 5 of 12 Compared to the bare SS electrode, the 5~15 nm gold nanoparticles homogeneously distributed throughout the surface of the bare SS wire electrode ( Figure 1C). The element composition of the modified electrodes was also characterized by an energy dispersive spectrometer (Oxford Instruments, UK). As shown in Figure 1B, the main element of bare SS wire electrode was Fe, above 60.9%. After electrodepositing of Au, the elemental composition of Fe decreased to 52.8%, while the Au increased from 0% to 7.2% (Figure1D). All of these results suggested the gold nanoparticles were electroplated on the bare SS wire electrode.

The Electrochemical Performance of the Au/SSWire-Based Electrochemical Microsensor
The electrocatalytic behavior of the Au/SS wire-based electrochemical microsensortoward H2O2 reduction was investigated using CV. There was only one reduction peak of CV curves at around −0.7 V on the bare SS wire electrode in the presence of 100 µM H2O2 ( Figure 2A).Compared to the bare SS wire electrode, the CV curves changed with an obvious reduction peak at around−0.7 V and −0.2 V on the Au/SS electrode ( Figure 2A). Such results showed that Au nanoparticles possess exceptional electrical conductivity and electrocatalytic activity, which could improve the electrochemical response of H2O2. Moreover, with the addition of H2O2 from concentrations of 0 to 1500 µM, the reduction current of H2O2 at around−0.7 V was enhanced gradually, while there was no obvious increase at −0.2 V, which suggested that the electrocatalytic reaction of H2O2 mainly occurs at −0.7 V ( Figure 2B). Figure 2C shows the curves of the SS wire-based electrochemical microsensor system with the increase in scanning rates from 10 to 300 mV/s. The results showed that the reduction peak current of 100 µM H2O2 decreased linearly with the increase in the scan rates, which suggested that the redox process of the modified electrode surface is an adsorption-controlled process with an irreversible electroreduction of H2O2. In addition, the reduction peak potential of H2O2 at around −0.7 V gradually moves toward the negative

The Electrochemical Performance of the Au/SSWire-Based Electrochemical Microsensor
The electrocatalytic behavior of the Au/SS wire-based electrochemical microsensortoward H 2 O 2 reduction was investigated using CV. There was only one reduction peak of CV curves at around −0.7 V on the bare SS wire electrode in the presence of 100 µM H 2 O 2 ( Figure 2A).Compared to the bare SS wire electrode, the CV curves changed with an obvious reduction peak at around −0.7 V and −0.2 V on the Au/SS electrode ( Figure 2A). Such results showed that Au nanoparticles possess exceptional electrical conductivity and electrocatalytic activity, which could improve the electrochemical response of H 2 O 2 . Moreover, with the addition of H 2 O 2 from concentrations of 0 to 1500 µM, the reduction current of H 2 O 2 at around −0.7 V was enhanced gradually, while there was no obvious increase at −0.2 V, which suggested that the electrocatalytic reaction of H 2 O 2 mainly occurs at −0.7 V ( Figure 2B). Figure 2C shows the curves of the SS wire-based electrochemical microsensor system with the increase in scanning rates from 10 to 300 mV/s. The results showed that the reduction peak current of 100 µM H 2 O 2 decreased linearly with the increase in the scan rates, which suggested that the redox process of the modified electrode surface is an adsorption-controlled process with an irreversible electroreduction of H 2 O 2 . In addition, the reduction peak potential of H 2 O 2 at around −0.7 V gradually moves toward the negative direction with the increase in scanning rates, which is possibly caused by the torpid electron transfer kinetics. Biosensors 2022, 12, x FOR PEER REVIEW 6 of 12

Amperometric Detection of H2O2 with the Au/SS Wire-Based Electrochemical Microsensor
Considering the great influence of potential on the sensitivity, the amperometric i-t method was used to estimate the influence of the applied potential on the Au/SS wirebased electrochemical microsensor. As shown in Figure 3A, the influence of different applied potentials (−0.9, −0.8, −0.7, −0.6, −0.5, and −0.4 V) on the amperometric response of the Au/SS wire-based electrochemical microsensor system was investigated by continuously adding 100 µM H2O2 into 0.2 M PBS solution. The signal-to-noise ratio (a ratio of the current response signal to background noise, S/N ratio) can reflect the sensitivity of the detection system. It was observed that the S/N ratio increased and reached a maximum value at −0.6 V, then gradually decreased ( Figure 3B). Therefore, an applied potential of −0.6 V for the amperometric i-t method was selected in the following experiments. Whether the untreated stainless-steel wires could be used as reference electrodes and counter electrodes was studied. Compared to the present electrode system, the untreated stainless-steel wire could not be used as reference electrode and counter electrodes due to the big background noise ( Figure S2). The influence of working electrodes fabrication with the different steel wire diameters (0.1, 0.05, and 0.01 mm) on the electrochemical detection of H2O2 was also studied. The results showed that the highest current response was the steel wire with a diameter of 0.1 mm ( Figure S3).

Amperometric Detection of H 2 O 2 with the Au/SS Wire-Based Electrochemical Microsensor
Considering the great influence of potential on the sensitivity, the amperometric i-t method was used to estimate the influence of the applied potential on the Au/SS wire-based electrochemical microsensor. As shown in Figure 3A, the influence of different applied potentials (−0.9, −0.8, −0.7, −0.6, −0.5, and −0.4 V) on the amperometric response of the Au/SS wire-based electrochemical microsensor system was investigated by continuously adding 100 µM H 2 O 2 into 0.2 M PBS solution. The signal-to-noise ratio (a ratio of the current response signal to background noise, S/N ratio) can reflect the sensitivity of the detection system. It was observed that the S/N ratio increased and reached a maximum value at −0.6 V, then gradually decreased ( Figure 3B). Therefore, an applied potential of −0.6 V for the amperometric i-t method was selected in the following experiments. Whether the untreated stainless-steel wires could be used as reference electrodes and counter electrodes was studied. Compared to the present electrode system, the untreated stainless-steel wire could not be used as reference electrode and counter electrodes due to the big background noise ( Figure S2). The influence of working electrodes fabrication with the different steel wire diameters (0.1, 0.05, and 0.01 mm) on the electrochemical detection of H 2 O 2 was also studied. The results showed that the highest current response was the steel wire with a diameter of 0.1 mm ( Figure S3).
The typical amperometric (i-t) curves of the Au/SS wire-based electrochemical microsensor were recorded by the successive addition of H 2 O 2 with different concentrations into 0.2 M PBS solution at an applied potential of −0.6 V (Figure 4). The response of the H 2 O 2 on the bare SS and Au/SS wire electrodes was also compared. As shown in Figure 4A, the response of H 2 O 2 on the Au/SS wire electrode was higher than the SS wire, which showed that Au nanoparticles modified SS wire could improve electrochemical detection of H 2 O 2 .The current response value increased with the increase in H 2 O 2 concentration ( Figure 4B). There was a good linear relationship between the current variation of H 2 O 2 and the concentration of H 2 O 2 in the range of 10 µM to 1000 µM ( Figure 4C). After adding 500 µM H 2 O 2 in stirring PBS solution, the reduction current of the Au/SS wire-based electrochemical microsensor system showed a significant increase and obtained the maximum steady-state current within 1.6 s ( Figure 5A). The possible interference from the plant molecules in plant fluids for the determination of H 2 O 2 in plants was also investigated. Figure 5B shows that no significant interferences were observed for the determination of H 2 O 2 in the presence of salicylic acid (SA), indole-3-acetic acid (IAA), methyl jasmonate (MeJA), abscisic acid (ABA), ascorbic acid (AA), malic acid (H 2 MA), succinic acid, and citric acid (CA) with 100 µM, respectively. These results suggested that the Au/SS wire-based electrochemical microsensor system can be used for the determination of H 2 O 2 in real samples. The reproducibility of the Au/SS wire-based electrochemical microsensor was evaluated from the response on 100 µM H 2 O 2 using six working electrodes. The relative standard deviation was about 5.8% and indicated a good reproducibility of the Au/SS wirebased electrochemical microsensor. The limit of detection (LOD) was 3.97 µM. The stability of the Au/SS wire-based electrochemical microsensor was also tested. The results showed that the Au/SS wire-based electrochemical microsensor could be used at least seven times and maintained stability for at least seven days ( Figure S4). These results suggested that the Au/SS wire-based electrochemical microsensor system can be used for the determination of H 2 O 2 in real samples. Compared with previous reports [28][29][30][31][38][39][40], the disposable SS wire-based electrochemical microsensor was superior regarding the low cost (about USD 0.02, suitable for mass production), simple preparation (less than 20 min to prepare this system), good analytical performance, and in vivo and continuous electrochemical monitoring of H 2 O 2 in plants (Table S2).

Amperometric Detection of H2O2 with the Au/SS Wire-Based Electrochemical Microsensor
Considering the great influence of potential on the sensitivity, the amperometric i-t method was used to estimate the influence of the applied potential on the Au/SS wirebased electrochemical microsensor. As shown in Figure 3A, the influence of different applied potentials (−0.9, −0.8, −0.7, −0.6, −0.5, and −0.4 V) on the amperometric response of the Au/SS wire-based electrochemical microsensor system was investigated by continuously adding 100 µM H2O2 into 0.2 M PBS solution. The signal-to-noise ratio (a ratio of the current response signal to background noise, S/N ratio) can reflect the sensitivity of the detection system. It was observed that the S/N ratio increased and reached a maximum value at −0.6 V, then gradually decreased ( Figure 3B). Therefore, an applied potential of −0.6 V for the amperometric i-t method was selected in the following experiments. Whether the untreated stainless-steel wires could be used as reference electrodes and counter electrodes was studied. Compared to the present electrode system, the untreated stainless-steel wire could not be used as reference electrode and counter electrodes due to the big background noise ( Figure S2). The influence of working electrodes fabrication with the different steel wire diameters (0.1, 0.05, and 0.01 mm) on the electrochemical detection of H2O2 was also studied. The results showed that the highest current response was the steel wire with a diameter of 0.1 mm ( Figure S3).  The typical amperometric (i-t) curves of the Au/SS wire-based electrochemical microsensor were recorded by the successive addition of H2O2 with different concentrations into 0.2 M PBS solution at an applied potential of −0.6 V (Figure 4). The response of the H2O2 on the bare SS and Au/SS wire electrodes was also compared. As shown in Figure  4A, the response of H2O2 on the Au/SS wire electrode was higher than the SS wire, which showed that Au nanoparticles modified SS wire could improve electrochemical detection of H2O2.The current response value increased with the increase in H2O2 concentration (Figure 4B). There was a good linear relationship between the current variation of H2O2 and the concentration of H2O2 in the range of 10 µM to 1000 µM ( Figure 4C). After adding 500 µM H2O2 in stirring PBS solution, the reduction current of the Au/SS wire-based electrochemical microsensor system showed a significant increase and obtained the maximum steady-state current within 1.6 s ( Figure 5A). The possible interference from the plant molecules in plant fluids for the determination of H2O2 in plants was also investigated. Figure  5B shows that no significant interferences were observed for the determination of H2O2 in the presence of salicylic acid (SA), indole-3-acetic acid (IAA), methyl jasmonate (MeJA), abscisic acid (ABA), ascorbic acid (AA), malic acid (H2MA), succinic acid, and citric acid (CA) with 100 µM, respectively. These results suggested that the Au/SS wire-based electrochemical microsensor system can be used for the determination of H2O2 in real samples. The reproducibility of the Au/SS wire-based electrochemical microsensor was evaluated from the response on 100 µM H2O2 using six working electrodes. The relative standard deviation was about 5.8% and indicated a good reproducibility of the Au/SS wire-based electrochemical microsensor. The limit of detection (LOD) was 3.97 µM. The stability of the Au/SS wire-based electrochemical microsensor was also tested. The results showed that the Au/SS wire-based electrochemical microsensor could be used at least seven times and maintained stability for at least seven days ( Figure S4). These results suggested that the Au/SS wire-based electrochemical microsensor system can be used for the determination of H2O2 in real samples. Compared with previous reports [28][29][30][31][38][39][40], the disposable SS wire-based electrochemical microsensor was superior regarding the low cost (about USD 0.02, suitable for mass production), simple preparation (less than 20 min to prepare this system), good analytical performance, and in vivo and continuous electrochemical monitoring of H2O2 in plants (Table S2).

In Vivo and Continuous Electrochemical Monitoring of H2O2 in Vein of Tomato Leaf
To study the production of H2O2 in the veins of the tomato leaf under high salinity stress, H2O2 was continuously monitored in vivo and based on the Au/SS wire-based electrochemical microsensor. As shown in Figure 6A, the working electrode, counter electrode, and reference electrode of the Au/SS wire-based electrochemical microsensor were placed into the vein of the tomato leaf, followed by transferring 5 µL 0.2 M PBS (pH 7) on the surface of electrodes for electrochemical detection. The typical amperometric i-t detection curves of H2O2 in the vein of tomato with or without high salinity stress for 2 h are shown in Figure 6B. The results showed that the records of control remained flat during the continuous measurement which suggested a stable H2O2 concentration in the vein of tomato leaf. With the high salinity stress, H2O2 was observed to increase about 40 min and resulted in a large peak at 60 min ( Figure 6B). Figure 6C illustrates the dynamic change in H2O2 in the vein of tomato leaf from 6.7 to 678.6 µM. In vivo H2O2 monitoring with high salinity stress was repeated three times and each experiment yielded similar results of excess H2O2 accumulation ( Figure S5). Respiratory burst oxidase homologs (Rbohs) also known as NADPH oxidases, are key enzymes that can catalyze the generation of ROS in plants [41]. Rbohs-generated ROS has been shown to regulate responses to many abiotic stresses such as drought, heat, cold, and salinity [42,43]. Stresses are shown to alter the levels of different plant hormones that in turn regulate RBOHs activity and ROS production via many different mechanisms [43]. Eight SlRboh genes (SlRbohA~SlRbohH) were identified in tomato [44]. Among them, SLRbohB has been proven to positively regulate resistance to B. cinerea and drought stress tolerance in tomato [44]. Therefore, the expression analysis of SlRbohA and SlRbohB in our studies was performed to confirm our detection results. Comparing the control treatments, the expression levels of SlRbohA and SlR-bohB in the veins of tomato leaves under high salinity treatment showed a > 8-fold increase at 1 h post treatment, which related to the increase in H2O2 in the vein of tomato under high salinity stress ( Figure 6D). These results suggested that our approach might provide an effective way for in vivo and continuous detection of H2O2 in plants.

In Vivo and Continuous Electrochemical Monitoring of H 2 O 2 in Vein of Tomato Leaf
To study the production of H 2 O 2 in the veins of the tomato leaf under high salinity stress, H 2 O 2 was continuously monitored in vivo and based on the Au/SS wire-based electrochemical microsensor. As shown in Figure 6A, the working electrode, counter electrode, and reference electrode of the Au/SS wire-based electrochemical microsensor were placed into the vein of the tomato leaf, followed by transferring 5 µL 0.2 M PBS (pH 7) on the surface of electrodes for electrochemical detection. The typical amperometric i-t detection curves of H 2 O 2 in the vein of tomato with or without high salinity stress for 2 h are shown in Figure 6B. The results showed that the records of control remained flat during the continuous measurement which suggested a stable H 2 O 2 concentration in the vein of tomato leaf. With the high salinity stress, H 2 O 2 was observed to increase about 40 min and resulted in a large peak at 60 min ( Figure 6B). Figure 6C illustrates the dynamic change in H 2 O 2 in the vein of tomato leaf from 6.7 to 678.6 µM. In vivo H 2 O 2 monitoring with high salinity stress was repeated three times and each experiment yielded similar results of excess H 2 O 2 accumulation ( Figure S5). Respiratory burst oxidase homologs (Rbohs) also known as NADPH oxidases, are key enzymes that can catalyze the generation of ROS in plants [41]. Rbohs-generated ROS has been shown to regulate responses to many abiotic stresses such as drought, heat, cold, and salinity [42,43]. Stresses are shown to alter the levels of different plant hormones that in turn regulate RBOHs activity and ROS production via many different mechanisms [43]. Eight SlRboh genes (SlRbohA~SlRbohH) were identified in tomato [44]. Among them, SLRbohB has been proven to positively regulate resistance to B. cinerea and drought stress tolerance in tomato [44]. Therefore, the expression analysis of SlRbohA and SlRbohB in our studies was performed to confirm our detection results. Comparing the control treatments, the expression levels of SlRbohA and SlRbohB in the veins of tomato leaves under high salinity treatment showed a >8-fold increase at 1 h post treatment, which related to the increase in H 2 O 2 in the vein of tomato under high salinity stress ( Figure 6D). These results suggested that our approach might provide an effective way for in vivo and continuous detection of H 2 O 2 in plants.

Conclusions
In this study, we developed a disposable Au/SS wire-based electrochemical microsensor to detect H2O2 in vivo and continuously. Compared to the traditional methods, the complicated and time-consuming treatment processing for quantification of H2O2 in the plant samples was avoided, which could ensure continuously obtaining the H2O2 concentration in vivo. With our approach, the dynamic change in H2O2 in the vein of tomato leaf under high salinity was continuously monitored in vivo. Our strategy will help to pave the foundation for further investigation of the generation, transportation, and elimination mechanisms of H2O2 in plants.

Conclusions
In this study, we developed a disposable Au/SS wire-based electrochemical microsensor to detect H 2 O 2 in vivo and continuously. Compared to the traditional methods, the complicated and time-consuming treatment processing for quantification of H 2 O 2 in the plant samples was avoided, which could ensure continuously obtaining the H 2 O 2 concentration in vivo. With our approach, the dynamic change in H 2 O 2 in the vein of tomato leaf under high salinity was continuously monitored in vivo. Our strategy will help to pave the foundation for further investigation of the generation, transportation, and elimination mechanisms of H 2 O 2 in plants.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/bios12010035/s1, Table S1: The qRT-PCR primers; Table S2: List of analytical performance of electrochemical methods for detection of H 2 O 2 in the plants; Figure S1: The method of high salinity stress for the tomato; Figure S2