Real-Time Monitoring of Pesticide Translocation in Tomato Plants by

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Real-time monitoring of pesticide translocation in tomato plants by surface-enhanced Raman spectroscopy Tianxi Yang, Jeffery J Doherty, Huiyuan Guo, Bin Zhao, John M. Clark, Baoshan Xing, Ruyan Hou, and Lili He Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04522 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Analytical Chemistry

Real-time monitoring of pesticide translocation in tomato plants by surface-enhanced Raman spectroscopy Tianxi Yanga, Jeffery Dohertyb,c, Huiyuan Guod, Bin Zhaoa, John M Clarkb,c, Baoshan Xingd, Ruyan Houe and Lili He*a a Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States b Department of Veterinary & Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, United States c Massachusetts Pesticide Analysis Laboratory, Amherst, Massachusetts 01003, United States d Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States e State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui 230036, China ABSTRACT: Understanding the behavior of pesticide translocation is significant for effectively applying pesticides and reducing pesticide exposures from treated plants. Herein, we applied surface enhanced Raman spectroscopy (SERS) for real-time monitoring of pesticide translocation in tomato plant tissues, including leaves and flowers, following root exposure in hydroponic and soil systems. Various concentrations of the systemic pesticide, thiabendazole, was introduced into hydroponic systems used for growing tomato plants. At selected time internals, tomato leaves and flowers were picked and thiabendazole measured directly under a Raman microscope after pipetting gold nanoparticle-containing solution onto the plant tissue. We found that the pesticide signals first appeared along the midrib in the lowest leaves and moved distally to the edge of the leaves. As the concentration of pesticide applied to the root was increased, the time necessary to detect the signal was decreased. The SERS surface mapping method was also able to detect thiabendazole in the trichomes of the leaves. In addition, we found a unique SERS peak at 737 cm-1 on both leaves and flowers at 4 and 6 days following the application of 200 mg/L of thiabendazole to the hydroponic system. This peak appears to be coming from adenine-containing materials and may be related to the plant’s response to pesticide toxicity, which could be used as a potential marker for monitoring plant responses to stresses. These results demonstrate a successful application of SERS as a rapid and effective way to study the real time translocation behavior of pesticides in a plant system.

Pesticides continue to be widely used in modern agriculture.1–3 As chemicals, they are comprise a wide variety of structures, and therefore, have differences in their mode of action, uptake, translocation and degradation.4 Translocation is one of the important behaviors of pesticides and understanding pesticide translocation is significant for effective and safe applications of pesticides on plants.5–8 Vascular plants have two different types of transport tissue. Xylem transports water and solutes from the roots to the leaves and phloem transports soluble organic compounds made during photosynthesis from the leaves to the rest of the plant.9 Transpiration is the process by which water evaporates from the leaves, which results in more water being drawn up from the roots.10 Systematic pesticides are capable of entering plants and being transported into the vascular system.11 Their distribution in plants can be achieved following foliar application as well as by uptake via roots.12 Root-to-shoot translocation is more common but less studied due to the technical difficulties associated with root experiments.13 During the root-to-shoot translocation process, systemic pesticides can be transported to stems and leaves through xylem after root uptake with transpiration being the main driving force.14,15 Once pesticides get to the leaves, leaf veins are the main transport system16,17 and systemic pesticide can also reach flowers. The accumulation of pesticides in flowers may result in

contaminated pollen and nectar, putting bees and other needed pollinators at risk.18 Although pesticide translocation is a wellstudied phenomenon, the techniques used to date are invasive to the plant and are not capable of ‘real time’ estimates, leading sometimes to unrealistic conclusions. Thus, a method to study the dynamic translocation and real time distribution of pesticides in plant tissues is a critical need. Understanding this process is crucial for both effectively applying pesticides and minimizing pesticide residues in plants. Chromatographic techniques (e.g., gas or liquid chromatography) are traditionally carried out to analyze pesticide translocation in plants.10,13,19 For example, Ronnie Juraskea. et al. reported estimated insecticide imidacloprid uptake and translocation in tomato plants using highperformance liquid chromatography (HPLC).12 Fernando Sicbaldi. et al. described the application of HPLC to study the root-to-shoot translocation of non-radiolabelled pesticides in soybean.13 Unfortunately, the complex procedures of sample treatment are time-consuming and involve laborious and costly manipulations. Additionally, these techniques cannot determine where pesticide translocate in real time and the dynamics of pesticide distribution in plant tissues. Understanding the dynamic pesticide translocation allows us to predict time dependent pesticide concentrations in different plant tissues. Herein, we aimed to develop a novel method that can realize

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real-time monitoring of pesticide translocation in plants without pre-labelling using surface-enhanced Raman scattering (SERS) method. SERS has become a powerful analytical tool that enables direct identification of analytes in contact or in close proximity with plasmonic nanostructures such as gold (Au) or silver nanoparticles (NPs).20–23 SERS has a broad range of applications in both agriculture and food science because of its attractive features such as rapid data collection under real time conditions, ease of use, non-invasive determinations and spectroscopic fingerprint identification of food additives, pesticides, etc.24–26 We previously reported the use of an innovative SERS method for monitoring penetration behavior of pesticides.27–32 The real time penetration behaviors of multiple classes of pesticide were investigated in a variety of fresh produce (i.e. apple, grape, spinach and basil) with penetrable AuNPs as a probe to enhance pesticide signals. In this study, we will expand the use of SERS as an effective approach to monitor pesticide translocation behavior. The aim of the present study is to apply innovative SERS techniques for real-time monitoring of pesticide translocation behaviors in plant and examining the dynamics pesticide distribution in plant tissues. Tomato plant and thiabendazole were chosen for study because the tomato plant is widely grown worldwide and thiabendazole is a typical systemic pesticide belong to benzimidazole family, which provides good control of tomato diseases.33 Liquid chromatography tandem mass spectrometry (LC−MS/MS) methods was utilized to determine the amounts of pesticides on plants after translocation. To the best of our knowledge, it is the first study that applied SERS method to study pesticide translocation in plants. Understanding this information is important to provide insights into the development of better strategies to effectively applying pesticides and reducing pesticide exposures from plants. EXPERIMENTAL SECTION Materials. Thiabendazole (systemic fungicide: 2-(4thiazolyl)-1H-benzimidazole, ≥99%, analytical grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Citratecapped AuNPs colloids (50 nm) were purchased from NANO PARTZ™ Inc. (Loveland, CO, USA). Hoagland modified basal salt mixture was purchased from PhytoTechnology Laboratories (Lenexa, KS, USA). 50 % Hoagland solution was prepared with 0.815 g of Hoagland modified basal salt mixture per liter of distilled water. Tomato seeds were purchased from W. Atlee Burpee & Co. (Warminster, PA, USA). Vermiculite potting media were provided by the Greenhouses Center at University of Massachusetts (Amherst, MA, USA). Ultrapure water (18.2 MΩ.cm) was produced using a Thermo Scientific Barnstead Smart2Pure Water Purification System (Waltham, MA, USA) and used for the preparation of all solutions. Plant culture. Pesticide translocation in tomato plants was performed in both hydroponic and soil systems. Prior to growth in the hydroponic system, tomato seedings were first germinated and cultivated in vermiculite potting medium. Uniform tomato seeds were sowed in small plastic pots filled with moisturized vermiculite in a greenhouse (25°C/light/16 hours) and 18 °C/dark/8 hours, light intensity 200 μmol photons m−2 s−1, and relative humidity 50 %-60 %). After 30 days, every 12 plants were transferred to a container with 4 L of 50 % Hoagland solution. The container was covered by aluminium foil paper to protect plant roots from light exposure. After 7 days, each plant was transferred to an individual aluminum foilcovered vial with 100 mL of 50 % Hoagland solution containing

increasing concentrations of thiabendazole (10, 50, 100 and 200 ppm) for translocation studies. 50 % Hoagland solutions were replenished in each vial every 8 h to maintain the volume at 100 mL. The soil system was established using same protocol as above for growing plants for 30 days. Then each plant was transferred to an individual pot. Each pot was watered with 50 mL of 50 % Hoagland solutions every 24 h. After 7 days, plants were exposed to 100 mL of 50 % Hoagland solution containing 200 ppm of thiabendazole for translocation studies. Twenty mL of 50 % Hoagland solution were added directly to the surface of the soil in each pot every 8 h. There were four replicate plants per treatment. Characterization of the SERS Signals of Thiabendazole in Tomato Leaves. For characterization of SERS signals of thiabendazole in tomato leaves, harvested leaves from pesticide-free tomato plants were first carefully rinsed with about 200 mL of ultrapure water and dried on a clean glass slide at room temperature before the experiment. A 1000 mg/L (ppm) thiabendazole stock solution was prepared with ultrapure water and methanol (v/v, 1:1) and diluted to the final desired concentrations with ultrapure water before use. A 50 μL aliquot of the 100 ppm thiabendazole solution was mixed with 50 μL of a 250 mg/L solution of 50 nm AuNPs for 1 h at room temperature to ensure effective pesticide complexation with AuNPs through Au-thiol or Au-amino bond.34 A 5 μL aliquot of the pre-prepared thiabendazole/AuNPs solution was pipetted onto tomato leaves and air-dried in a fume hood for 10 min. Solutions of AuNPs without thiabendazole and thiabendazole alone were also pipetted onto leaves as control treatments for comparison. Raman spectra were then collected individually from each treatment spot. To evaluate of the feasibility of SERS detection of thiabendazole on tomato leaves, 5 μL aliquots of the preprepared thiabendazole solutions of varying concentrations were first pipetted onto the tomato leaf surface. The leaves were then quickly dried with air for 5 min minimizing the loss of pesticide from the leaf surface due to penetration.30,31 Then, a 5 μL aliquot of the AuNPs solution was pipetted onto the same position where pesticides were applied. After quickly drying for 5 min, SERS spectra were collected from the treated leaves. Pesticide Translocation Study. A schematic illustration is shown in Scheme 1 used for the measurements of the translocation behaviors of thiabendazole in tomato plants grown using a hydroponic culture system. For each experiment, 100 mL of a 50 % Hoagland solution, each containing increasing concentrations of thiabendazole (10 ppm, 50 ppm, 100 ppm and 200 ppm), were prepared fresh and added to the roots of individual tomato plants growing in the hydroponic system. Thiabendazole was absorbed by roots and then translocated to other plant tissues (i.e. leaves and flowers) through vascular tissue (step 1). A target leaf was selected from the first branch of the plant and was the closest to the stem. The target leaf was selected for SERS analysis of pesticide translocation and distribution in leaves because it provided us the earliest time point for detecting pesticide-dependent SERS signals. Thiabendazole signals on target leaves were monitored following different exposure time periods from 2 hours to 6 days. After each time interval, a target leaf was cut from the plant using a sharp knife and placed flat onto a glass slide with the adaxial (top) side facing upwards. Five μL aliquots of a 250 ppm AuNPs solution were pipetted onto different positions of leaf surface (step 2). After drying with air for 10 min, SERS


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Analytical Chemistry spectra for the detection of thiabendazole were collected from each position on the leaf surface using a Raman spectrometer (step 3). Each treatment was replicated four times with four different leaves coming from two different plants. The two

leaves, which were nearest to the main stem and on the first branch of the plant, were chosen as the target leaves of a single plant. SERS mapping method was also applied to show thiabendazole distribution in trich-

Scheme 1. Schematic illustration of SERS method for monitoring of pesticide translocation on tomato plants in a hydroponic system.

omes. Thiabendazole translocation to flower was likewise monitored following the insertion of the roots into 100 mL of 50% Hoagland solution containing 200 ppm of thiabendazole. SERS spectra were collected over time after adding AuNPs on the surface of flowers. To study translocation of thiabendazole in a soil system, 100 mL of 50 % Hoagland solution containing 200 ppm of thiabendazole were prepared and applied to the soil surface of a pot with a single tomato plant for root uptake. Thiabendazole signals on leaves were monitored over time as in the hydroponic system described above. Raman Instrumentation and Data Analysis. A DXR Raman microscope (Thermo Fisher Scientific, Madison, WI, USA) with a 780-nm laser and a 20 × long distance microscope objective, 50 μm slit aperture and 3 mW laser was used in this study. Each sample was scanned from 400 to 2000 cm-1 for a 2-s exposure time. For detecting thiabendazole within the area where a dried droplet of AuNPs solution was placed on either a tomato leaf or flower, 10 discrete locations within each area were chosen and scanned. Each discrete location gave one independent SERS spectrum. TQ Analyst (version 8.0) was used to analyze the spectra. The final SERS spectrum was an average spectrum of the 10 spectra collected from 10 locations within the area where the AuNPs solution was placed. This averaging was done using the average function in the software package in order to reduce noise and produce a more representative spectrum for each individual area scanned. For detecting thiabendazole on tomato trichomes, SERS surface mapping method was applied to an area (140 μm × 190 μm). The step size of the mapping was 10 µm and one image contained 300 scanning spots. Raman images were integrated based on the characteristic peaks of thiabendazole at 1010 cm-1

in the SERS spectra using the Atlµs Function in the OMINCS software (Thermo Fisher Scientific). LC-MS/MS. The amounts of thiabendazole on tomato leaves were determined using LC-MS/MS. Thiabendazole residues on leaf tissues were first extracted by QuEChERS standard operating procedure based on a published study and Agilent Application Notebook.35 Thiabendazole in each extraction was measured on Waters Alliance LC equipped with Waters Acquity TQD MS/MS system at Massachusetts Pesticide Analysis Laboratory. The analytical column was Atlantis T3, 2.1 x 100 mm, maintained at 30 °C. Mobile phases consisted of 0.1 % formic acid/water (Phase A) and 0.1 % formic acid acetonitrile (Phase B). Chromatography started with 95:5 A : B, held for 0.5 min, ramped to 95 % B at 7 min, held until 12 min, ramped to 95% A at 13 min, and held until 18 min to equilibrate. The flow rate was 0.2 mL/min and the injection volume were 10 μL. Capillary voltage was kept at 3000 V. High purity argon (99.999 %) was used as collision gas. Ion source temperature was 250 °C, with nitrogen for desolvation. Chromatograms were obtained in the positive ion and multiple reactions monitoring mode (MRM). MRM conditions: positive ionization ES+, collision gas 0.2 mL/min. Retention time: 7.85 min, Parent ion 201.96, Quantifying ion 65, Qualifying ion 131. RESULTS AND DISCUSSION Measurement of SERS Spectra from Thiabendazole on Tomato Leaf Surfaces. SERS spectra in the combined presence of 100 ppm of thiabendazole and 50 nm AuNPs were obtained on tomato leaf surfaces (Figure 1A). The characteristic SERS peaks of thiabendazole were clearly determined at 1275, 1010 and 780 cm-1 (Figure 1A-a). AuNPs alone resulted in some peaks around 1600 cm-1, likely coming from AuNPs aggregation (Figure 1A-b). Little or no signals were observed from thiabendazole without mixing with AuNPs, or from



tomato leaf alone (Figure 1A-c and d, respectively). The peak at 1010 cm-1 of thiabendazole was selected as the characteristic peak for monitoring and image integration in the following studies. The corresponding SERS spectra of thiabendazole on tomato leaves were obtained with increasing concentration from 20 ppb to 100 ppm (Figure 1B). As shown in Figure 1B, Raman signals of thiabendazole can still be observed even as low as ppb levels, indicating the ultra-high sensitivity of the developed SERS approach for the determination of thiabendazole on tomato leaves.

positions (1-6) on the leaf were chosen for SERS measurements (Figure 2A-a). Position 1, 2 and 3 were selected on the midrib and position 1 was located where is the lowest on the leaf and close to the petiole. Position 4 was chosen on the side vein and close to position 1. Position 5 and 6 were on the lamina. The corresponding SERS spectra of each position in Figure 2A-b show that the characteristic peaks of thiabendazole are clearly seen in positions 1-4 whereas little or no peaks of thiabendazole are presented in either position 5 or 6, indicating that thiabendazole was transported to positions 1-4 through veins but little or no transport occurred to position 5 or 6 in the lamina after 20 hours exposures. After 4 days of exposure, eight positions (1-8) were chosen on the leaf as shown in Figure 2Ba. Positions 1-4 were the same locations on the leaf as in Figure 2A-a, while positions 5, 6, 7 were on the lamina, close to the margin, and position 8 was on the apex. The corresponding SERS spectra obtained at positions 1-8 are presented in Figure 2B-b, which clearly show the fingerprint information of thiabendazole at positions 5-8 with no obvious thiabendazole signatures at positions 1-4. These results demonstrate that pesticide transport from veins towards the leaf margins (edges) and tend to accumulate there after a 4-day exposure. Control experiments without thiabendazole exposure were also performed and the SERS spectra of leaves at position 1 after 20 hours (Figure S-1a) and position 8 after 4 days (Figure S-1b) did not show characteristic peaks of thiabendazole.

Figure 1. (A) Raman spectra on tomato leaves. (B) Concentrationdependent SERS spectra of thiabendazole on tomato leaves.

Real-Time Monitoring of the Translocation of Thiabendazole in Tomato Plants in the Hydroponic System. Pesticides can be absorbed by root and then translocated to plant leaves and flowers through vascular tissue. Upon reaching the leaves, leaf veins transport the pesticide. The amount of pesticide in leaf veins should be higher than other parts of leaves because pesticides first accumulate in veins, which consist of xylem and phloem, and are then translocated to other locations, such as spongy mesophyll, palisade mesophyll or epidermis.13 For monitoring pesticide translocation to the leaves following root exposure, we focused on collecting pesticide signals on different positions on the surface of leaves because surface detection is simple and rapid. Based on our previous study31 where we applied AuNPs on the leaf surface, some AuNPs penetrated inside (Scheme 1) but most remained on the surface and enhanced pesticide signals, which are then easily acquired by Raman instrument. SERS results of thiabendazole distribution on the target leaf following different exposure times (20 hours and 4 days) with 200 ppm of thiabendazole applied in a hydroponic system are displayed in Figure 2. After 20 hours exposure, different


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Figure 2. (A) (a) Schematic illustration of the leaf structure showing different positions (1-6) on leaf surface and (b) the corresponding SERS spectra of selected positions. (B) (a) Schematic illustration of different positions (1-8) on the leaf surface and (b) the corresponding SERS spectra of selected positions. (C) (a) Bright light scattering image of a trichome on tomato leaf surface. (b) SERS surface mapping image of trichome (using the intensity of SERS peak of thiabendazole at 1010 cm-1). Step size is 10 µm and one image contains 300 scanning points. (c) SERS spectra of selected positions on the mapping image.

Figure 3. SERS spectrum from flowers (a), SERS spectra from flowers following different exposure times (b-e) and Raman spectra of flowers with (f) and without AuNPs (g), following a 100 ppm thiabendazole exposure of roots using the hydroponic system.

In addition, there were many trichomes on tomato leaf surfaces and SERS mapping method can be utilized to image the pesticide distribution in trichomes (Figure 2C). Figure 2Ca shows the bright light image of a trichome with an area of 140 μm × 190 μm on a leaf following a 20-hour exposure of roots to 200 ppm thiabendazole using the hydroponic system. The corresponding SERS surface mapping image is given in Figure 2C-b. Figure 2C-c presents SERS spectra at positions 1-3 chosen from the SERS mapping image. The diagnostic fingerprint of thiabendazole was clearly seen from the SERS spectra on positions 1 and 2, while there was no signal seen from position 3, indicating that the thiabendazole concentration was very low or the amount of AuNPs was very small there. Pesticide translocations to both upper and lower surfaces of leaves were also investigated with SERS method. After a 4-day exposure, thiabendazole signals were detected on both surfaces (Figure S-2). The intensity of SERS signal at 1010 cm-1 on the upper surface was higher than the lower surface, indicating that more thiabendazole was present near the upper surface. This finding was expected because xylem tissues, which were located closer to the upper surface, was the transport system for pesticides and therefore, more pesticide moved to the upper surface due to the shorter distance. SERS techniques were also used to monitor pesticide translocation into flowers. SERS spectra of thiabendazole on flowers following the exposure of roots to 100 ppm of thiabendazole using the hydroponic system was obtained and clearly shows the characteristic peaks at 1275 and 1010 cm-1 (Figure 3a). In the flower control without thiabendazole

exposure, there was a small peak around 1010 cm-1 with AuNPs (Figure 3f) but no peak at 1275 cm-1 was seen in flowers with or without AuNPs (Figure 3f and 3g). Therefore, the characteristic peak at 1275 cm-1 was used to indicate the presence of thiabendazole on flowers. No thiabendazole signals were detected following the first day of exposure (Figure 3b). After 2 days, the SERS signals of thiabendazole appeared (Figure 3c) and became more apparent on day 3 and 4 (Figure 3d and 3e), indicating that thiabendazole translocated to flowers after 2 days and increased in flowers after 3-4 days. Concentration Effects on Pesticide Translocation using the Hydroponic System. In order to monitor the real time translocation of pesticides on tomato leaves, increasing concentrations (10, 50, 100, and 200 ppm) of thiabendazole were added to the roots of tomato plants growing in the hydroponic system and the SERS method used to detect the target leaf signals over time. Position 1 on the target leaf (Figure 2A-a) was determined to be the earliest time that thiabendazole was detected in leaves. The first time thiabendazole was detected following translocation was 48, 38, 24 and 12 hours for 10, 50, 100, and 200 ppm of thiabendazole, respectively. The corresponding SERS spectra clearly show the Raman signatures of thiabendazole (Figure S-3A). The time course when thiabendazole distributed into the whole target leaf was monitored using eight positions (1-8) for detection as shown in Figure 2B-a. When SERS spectra from all the positions showed the Raman signatures of thiabendazole, it meant that the pesticide had translocated into the whole leaf. The results showed that thiabendazole translocated to whole leaves after 5, 3, 2 and 1 days following a 10, 50, 100, or 200 ppm thiabendazole exposure, respectively (Figure S-3B). In order to further confirm the effects of pesticide concentration on translocation, the first time that pesticide was detected in both the lowest midrib and in the whole leaf following exposure to increasing concentrations of thiabendazole were determined in Table 1. The results showed that higher concentrations of thiabendazole when applied in the hydroponic systems took less time less time to translocate to tomato leaves. LC-MS/MS method was then performed to determine the amount of thiabendazole transported to the target leaf when first detected by SERS on the lowest midrib of leaves following exposure of the roots to either 10 or 200 ppm of thiabendazole. The analysis showed that after 48 hours of a 10 ppm or 12 hours of a 200 ppm thiabendazole exposure, the amount of thiabendazole transported to the target leaves were the same, which was ~2 ppm (μg/g). This result was expected and shows again the relatively high sensitivity of the SERS method to detect thiabendazole. Whereas the LC-MS/MS method measured all the pesticide in the entire target leaf, the SERS method only measured the pesticide at certain limited positions/areas on the leaf. Thus, the amount of thiabendazole at these positions was much less than that in the whole leaf but still was detected by SERS. Plant Response to Pesticide Exposure in the Hydroponic System. When pesticide translocation was monitored following a 200-ppm exposure to thiabendazole over time (2 hours to Table 1. The time for pesticide translocation to tomato leaves following exposure of the roots to increasing concentrations of thiabendazole using the hydroponic system.



Figure 4. (A) SERS spectra of tomato leaves following different exposure time periods (a-d) and SERS spectra of NAD (e) and adenine (f) on tomato leaves. (B) SERS spectra of tomato flowers following different exposure time periods.

6 days) in the hydroponic system, we found that leaves gradually turned yellow around their margins (edges). SERS spectra were collected at the margins of leaves after different exposure time periods (Figure 4A (a-d)). After 24 hours, the characteristic SERS peaks of thiabendazole were present, indicating that the pesticide had translocated to the leaves (Figure 4A-b). After 4 days, besides the SERS spectrum displayed the SERS peak of thiabendazole at 1010 cm-1, another peak at 737 cm-1 appeared (Figure 4A-c). This peak increased in size at 6 days post-exposure and the characteristic peaks of thiabendazole disappeared (Figure 4A-d). Based on published studies, the peak at 737 cm-1 may be attributed to pyridine nucleotides, such as nicotinamide adenine dinucleotide NAD(H) and nicotinamide adenine dinucleotide phosphate NADP(H), or other adenine containing materials. NAD(H) and

NADP(H) are essential metabolites involved in numerous redox reactions and regulate various cellular processes in all living cells.36 In plants, they function as the central metabolites involved in directing plant cellular redox homeostasis. These nucleotides play vital roles in systems controlling adaptation to environmental stresses such as UV irradiation, salinity, heat shock and drought.37–39 When pesticides, such as thiabendazole, are applied to the roots of tomato plants and translocate as shown above, they likely result in physiological stress. As the metabolic balance of the plants is disrupted, the leaves may have responded to the stress over time by producing more adenine-containing materials. To better understand this phenomenon, we collected SERS signals from tomato leaves exposed to 100 ppm solutions of NAD or adenine in the presence of AuNPs (Figure 4e and f). Exposure to either NAD or adenine resulted in the appearance of the characteristic peak of adenine-containing materials at 737 cm-1 and the SERS patterns of adenine were very similar with that of leaves after 6 days exposure to thiabendazole. The same phenomena were also observed on tomato flowers (Figure 4B). After 5 days, the SERS spectra showed the characteristic peak for thiabendazole at 1010 cm-1. However, this peak disappeared following 6- and 7-days post-exposure along with the specific peak at 737 cm-1 increasing in size. Thus, tomato plants appear to respond to thiabendazole-induced stress and adenine-containing materials could be used as potential biomarkers for monitoring plant stress response using the SERS method. Monitoring Pesticide Translocation in Soil Systems. Pesticide translocation to tomato leaves was also investigated in a soil system following a 200 ppm thiabendazole exposure to the roots. Figure S-4 shows the SERS spectra of the lowest midrib of the target leaf. SERS spectra following a 10-ppm exposure to thiabendazole with AuNPs and SERS spectra with only AuNPs on leaves were presented as controls. After 4 days, no thiabendazole signals were observed from the spectra showing that the pesticide has not translocated into the leaves and reached the detectable concentration (2 μg/g). The SERS signal of thiabendazole at 1010 cm-1 appeared after 7 days, indicating that pesticide had accumulated to above 2 μg/g in the lowest leaves at this time. Control experiments without thiabendazole exposure were also performed. After 7 days, the SERS spectra of the lowest midrib of the target leaf did not show characteristic peaks of thiabendazole (Figure S-5). CONCLUSION The present study demonstrates the application of SERS for real time monitoring of pesticide translocation in tomato plant tissues, including leaves and flowers, in hydroponic and soil systems. The SERS signals of the systemic pesticide, thiabendazole, in leaves were initially detected at the lowest midrib of the target leaf and subsequently translocated to the margins of the leaves over time. When thiabendazole was applied at higher concentrations, less time was required to translocate to tomato leaves. LC-MS/MS analysis determined that there was ~2 μg/g of thiabendazole in leaves at a time when the SERS method first detected thiabendazole translocation onto the leaves after root exposure. SERS surface mapping technique was likewise used to detect the distribution of thiabendazole in tomato plant leaf trichomes following translocation. In addition, a unique SERS peak at 737 cm-1 was detected on leaves and flowers, which appears to be coming from adenine-containing materials and may reflect a plant stress response to pesticide exposure. The detection of these types of


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Analytical Chemistry materials by SERS could be useful as potential biomarkers to indicate plant response to environmental stresses. The information obtained from this study could provide insights and tools necessary to more effectively and safely apply pesticides to plants. Future studies will focus on the use of this method to study pesticide translocation behaviors into additional plant tissues, such as edible plant fruits. ASSOCIATED CONCENT Supporting information The supporting information is available free of charge on the ACS publication website. Figure S-1. SERS spectra of leaves after 20 hours (a) and 4 days (b) in hydroponic system. Figure S-2. SERS spectra of pesticides on upper and lower surfaces after translocation. Figure S-3. (A) SERS spectra at lowest midrib of tomato leaves after various concentrations of thiabendazole translocation following different exposure time periods. (B) SERS spectra of tomato leaves after various concentrations of thiabendazole translocation following different exposure time periods. Figure S-4. SERS spectra of 10 ppm thiabendazole and pure AuNPs on leaves as well as SERS spectra at lowest midrib of leaves after 4 days and 7 days translocation. Figure S-5. SERS spectra of leaves after 7 days in soil system. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Tel: +1 413 545 5847. Notes The authors declare no competing financial interest.

This work was supported by the National Institute of Food and Agriculture of the U.S. Department of Agriculture (USDA-NIFA, grant no.: 2016-67017-24458) and National Natural Scientific Foundation of China (No. 31772076)

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