An activatable NIR‐II fluorescent probe for tracking heavy‐metal ion and high‐level salt‐induced oxidative stress in plant sprouts

Humans and plants have become enfolded and inseparable. Abiotic stresses in particular oxidative stress caused by heavy‐metal ions or high‐level salt contamination deleteriously impact plants’ growth process and have become a major threat to sustaining food security. Sprouting is the first step in plants’ growth process. When plant sprouts endure oxidative stress induced by toxic heavy‐metal ions or high‐level salt, accelerated generation of reactive oxygen species (e.g., H2O2) occurs inside plant sprouts; hence in‐situ H2O2 in plant sprouts could serve as the in‐vivo biomarker for tracking the oxidative stress in plant sprouts. Herein, we design an activatable probe CT‐XA‐H2O2 to track the oxidative stress in plant sprouts via in vivo NIR‐II fluorescent imaging. In CT‐XA‐H2O2, cyano‐thiazole acts as the electron‐accepting moiety and xanthane‐aminodiphenyl as the electron‐donating moiety, and dioxaborolane as the biomarker‐responsive unit and fluorescence quencher. The probe CT‐XA‐H2O2 shows weak fluorescent emission. When H2O2 is present, the dioxaborolane in the probe is cleaved, consequently, the dye CT‐XA‐OH is generated and brings about significant fluorescent signals for detecting and imaging the in‐situ biomarker. Moreover, the aminodiphenyl group endues the chromophore (the activated probe) with aggregation‐induced emission characteristics, which ensures stronger fluorescence in the aggregated state in the aqueous milieu. The probe CT‐XA‐H2O2 has been employed in the Cd2+‐ion or high‐level salt (NaCl) induced oxidative stress models of soybean sprouts and peanut sprouts, and the experimental results evidently reveal the probe's ability for in‐situ biomarker‐activatable in‐vivo detection and imaging in the plants' sprouts.

consequently sprouting is an important phase of a plant's life, and healthy and robust sprouts are crucial to plants' growth. [3][4][5] To ensure high-quality and robust sprouts, the sprouting process requires pollution-free soil/water for the seeds.
Abiotic stress such as the oxidative stress induced by heavy-metal-ion-polluted or high-level-salt-polluted soil/water deleteriously affects plants' growth and has become a major threat to sustaining food security. [1,2,[6][7][8][9] When plant sprouts endure such abiotic stress as oxidative stress induced by toxic heavy-metal ions or high-level salt, their common response is the accelerated generation of reactive oxygen species (ROS, H 2 O 2 is the most abundant). [10,11] It is known that ROS play a dual role as both beneficial S C H E M E 1 Schematic diagram for the probe CT-XA-H 2 O 2 's fluorescent imaging for soybean sprouts and peanut sprout's Cd 2+ -ion or high-level salt (NaCl) induced oxidative stress via responding toward in-vivo biomarker H 2 O 2 and deleterious species depending on the ROS level in plants, [10,11] when the ROS level surpasses the normal (basal) level (H 2 O 2 at about 30 nmol/g), [12] oxidative stress takes place. Hence in-situ H 2 O 2 in plant sprouts could serve as the in-vivo biomarker for tracking the heavy-metal ioninduced or high-level salt-induced oxidative stress in plant sprouts, and the tracking of H 2 O 2 in-vivo in the plant sprouts can also act as the index for the early warning of heavy-metal ion or high-level salt pollution in soil/water so that preventive and remedial measures can be taken as soon as possible to avoid adverse consequences.
In face of toxic heavy metal ions or high-level salt-induced oxidative stress, rapid and amplified production of ROS (e.g., H 2 O 2 ) occurs inside plant sprouts. The establishment of local ROS overproduction is usually transient as ROS has a short half-life; hence the in-situ, real-time and non-invasive detection and imaging of the ROS biomarker is optimal for accurately pinpointing the site with ROS amplification and accumulation. [13][14][15][16] Fluorescence imaging is a non-invasive technique that can detect and monitor in-situ biological processes including the variation of biomarker levels in a real-time manner. [17][18][19][20][21][22][23][24][25][26][27][28][29] In particular, near-infrared second window fluorescence imaging (emission: 900-1700 nm, which is referred to as NIR-II fluorescence imaging) can realize exceptional imaging performance, [30][31][32][33][34][35][36][37][38][39][40][41] since there exists almost no interference from the plant-related pigments in the NIR-II wavelength range. While such plant pigments as chlorophylls, carotenoids, and anthocyanins would cause serious interference for plant imaging in the visible-light wavelength range (400-700 nm) and in the visible-light or NIR-I (700-900 nm) wavelength range as these pigments' absorptions and fluorescence are mostly in these wavelength ranges. [42,43] Among the probes used for fluorescent imaging, the biomarker-activatable probes are more advantageous compared with inert probes which are unresponsive to biomarkers and give out "always-on" (unvaried) signals and thus intensifying background noise. Whereas biomarkeractivatable probes only generate fluorescent signals when they come across the specific biomarker, that's why imaging with biomarker-activatable probes can achieve much higher sensitivity with inappreciable background noise. [44][45][46][47][48][49][50][51][52][53] Therefore, a biomarker-activatable probe with NIR-II fluorescent imaging would provide a utilitarian means for accurate detection of H 2 O 2 in-vivo in the plant sprouts and the ensuing warning of heavy-metal ion or high-level salt pollution in soil/water.
In light of the above, in this study, we designed a biomarker-activatable NIR-II fluorescent probe (CT-XA-H 2 O 2 ) for fluorescent detection and imaging of the in-situ biomarker H 2 O 2 in plant sprouts in vivo (Scheme 1). As to the probe CT-XA-H 2 O 2 , cyano-thiazole serves as the electron-accepting moiety, xanthene-aminodiphenyl as the electron-donating moiety, while dioxaborolane as both the biomarker recognition unit (responsive unit for H 2 O 2 ) and the fluorescence quencher is integrated into the electron-donating moiety. The probe CT-XA-H 2 O 2 itself shows weak fluorescent emission. But, when H 2 O 2 is present, the dioxaborolane in the probe CT-XA-H 2 O 2 is cleaved, consequently, the dye CT-XA-OH is generated and brings on significant fluorescent signals for detecting and imaging the in-situ biomarker. Moreover, the aminodiphenyl group endues the fluorophore CT-XA-OH with aggregation-induced emission (AIE) feature, and this feature would overcome the aggregation-caused quenching in an aqueous milieu and ensures stronger fluorescence for NIR-II imaging in aqueous media. [54][55][56][57][58][59][60] The probe CT-XA-H 2 O 2 specifically responds to H 2 O 2 and thus turns into the fluorophore CT-XA-OH (namely activated probe) that is AIE active with evident fluorescence in the aqueous milieu. Heavy-metal contamination and high-level salt are the two most important inducing factors for abiotic stresses (oxidative stress). [3][4][5] As cadmium is vastly used in industry (e.g., anti-corrosion coating, plating, pigments, and plastic stabilizers), Cd 2+ ion has become the most important toxic heavy-metal ion pollution in water/soil. [61,62] High-level-saltcontaining water/soil would lead to poor plant sprouting and growth, and sodium chloride (NaCl) is the most common salt in groundwater and soil. [6,7] Therefore, in this study, Cd 2+ ions and NaCl were employed to induce oxidative stress in plant sprouts. As soybean plant is important due to the fact that soybean is the most important bean economically for providing vegetable protein for people and ingredients for various chemical products, and peanuts is a vegetarian nuts rich in healthy fats, protein, fiber, and vitamins, [63,64] hence soybean sprouts and peanuts sprouts are adopted as the models of plant sprouts. The probe CT-XA-H 2 O 2 was employed in the Cd 2+ -ion or NaCl-induced oxidative stress models of soybean sprouts and peanuts sprouts, and the experimental data apparently manifest the probe's ability for in-vivo detection and imaging of in-situ biomarker in the plants' sprouts and thus providing warning/monitoring for the Cd 2+ -ion or NaCl induced oxidative stress.

Probe's synthesis and spectral properties
The synthetic processes for the probe and the dye are displayed in Scheme S1. And the probe was characterized with 1 H NMR, 13 C NMR, and high-resolution mass spectrometry (HR-MS) (Figures S1-S9). The probe CT-XA-H 2 O 2 molecules and the dye CT-XA-OH molecules readily formed nanoaggregates in aqueous media (in water containing 15% dimethyl sulfoxide [DMSO]) due to their hydrophobic nature. The size distribution of the nanoaggregates measured by the dynamic light scattering method is presented in Figure S10, and the probe nanoaggregate and the dye nanoaggregate have an average particle size of about 3 nm.
The AIE feature of the dye CT-XA-OH was evaluated by dissolving the dye molecules in H 2 O/glycerol mixtures (containing 15% tetrahydrofuran), and the solutions' fluorescence spectra were measured ( Figure S11). Viscous glycerol provides the high-viscosity environment which would limit the intramolecular movements of the dye CT-XA-OH and leads to the display of AIE behavior of the dye molecules. As the glycerol fraction is increased, the fluorescence intensity of CT-XA-OH solution increases accordingly, indicating that the dye CT-XA-OH is AIE active and the dye CT-XA-OH (theoretically it is the activatable probe) is advantageous for fluorescent imaging in aqueous media.
As for the spectral properties, first, the absorption and fluorescence spectra of the probe CT-XA-H 2 O 2 and the dye CT-XA-OH were compared. Obviously, the absorption peaks of the probe CT-XA-H 2 O 2 and the dye CT-XA-OH measured in water containing 15% DMSO are at 742 and 896 nm respectively ( Figure S12). Compared to the probe CT-XA-H 2 O 2 , the dye CT-XA-OH contains two electron-donating groups (hydroxy groups) in replace of the two electronwithdrawing groups (dioxaborolane groups) in the probe CT-XA-H 2 O 2 , this is probably the reason why the dye shows the absorption peak at a longer wavelength (896 nm) compared to that of the probe (742 nm). From Figure S13, it can be seen that the probe CT-XA-H 2 O 2 emits weakly in water containing 15% DMSO. Contrarily, the fluorescence intensity of the dye CT-XA-OH (fluorescence peak at 1036 nm) is significantly stronger than that of the probe (measured in water containing 15% DMSO). Next, the absorption and fluorescence spectra for the probe CT-XA-H 2 O 2 in the absence or presence of various levels of H 2 O 2 were recorded in water containing 15% DMSO. As presented in Figure 1A, for CT-XA-H 2 O 2 , before its reaction with H 2 O 2 , its absorption peak is at 742 nm; while upon reaction with 100 µM H 2 O 2 , the absorbance increases, and the absorption peak red-shifts to 892 nm. As for CT-XA-H 2 O 2 with 808 nm excitation, the fluorescence intensity peaking at 1036 nm increases as well with increasing H 2 O 2 level ( Figure 1B,C); and the fluorescent intensity at 1036 nm reach the maximum in about 1 h ( Figure 1D and Figure S14). To investigate whether the probe CT-XA-H 2 O 2 can detect H 2 O 2 without interference from the commonly-existing ions in soil (including Zn 2+ , Na + , Ca 2+ , Mg 2+ , Cu 2+ , K + , Ni + , Cd 2+ , and Fe 3+ ) and some ROS substances, the fluorescent intensities of CT-XA-H 2 O 2 with incubation with H 2 O 2 were measured in the presence of these commonly-existing ions in the soil as well as Cd 2+ and some ROS substances ( Figure S15). The results display that the response of the probe CT-XA-H 2 O 2 toward H 2 O 2 won't be affected by these substances.
Furthermore, the fluorescent intensity at 1036 nm remains almost unchanged for 72 h after responding to H 2 O 2 ( Figure S16). The data reveals that the probe CT-XA-H 2 O 2 after incubating with H 2 O 2 has good stability. The above results concerning CT-XA-H 2 O 2 's concentration-and timedependent fluorescent response toward H 2 O 2 signify that the probe CT-XA-H 2 O 2 can serve as a quite good fluorescent probe for H 2 O 2 .

CT-XA-H 2 O 2 's response mechanism to H 2 O 2
In order to verify CT-XA-H 2 O 2 's response toward H 2 O 2 , high-performance liquid chromatography (HPLC) was conducted ( Figure S17 Figure S20). These results manifest that the probe CT-XA-H 2 O 2 is converted into CT-XA-OH after response to H 2 O 2 .

The probe's detection of Cd 2+ -ion-induced oxidative stress in plant sprouts via NIR-II fluorescence imaging
To evaluate probe CT-XA-H 2 O 2 's applicability in tracking plant sprout's oxidative stress, the models based on soybean sprouts and peanut sprouts were adopted, as soybean plant is important due to the fact that soybean is the most impor-tant bean economically for providing vegetable protein for people and ingredients for various chemical products and peanuts is a vegetarian nut rich in healthy fats, protein, fiber, and vitamins. [63,64] Heavy-metal contamination is one of the most important inducing factors for abiotic stresses (oxidative stress), [3][4][5]7,56,57] hence, in this study the cadmium ion (Cd 2+ ) contaminated water was adopted to induce the oxidative stress in the soybean sprouts and peanut sprouts. For comparison, the process of soybean sprouts germinating from the uncontaminated water was photographed at multiple time points as shown in Figure S21, and the photographs of peanut sprouts coming from the sprouting process in the uncontaminated water at different time points are displayed in Figure  S22.
First, the probe CT-XA-H 2 O 2 was employed to monitor soybean sprouts' and peanut sprouts' oxidative stresses induced by Cd 2+ . Cd 2+ is a toxic heavy metal ion that exerts pernicious effects on plants' growth process, and H 2 O 2 overexpressed upon plants' exposure to this metal ion can facilely act as the endogenous biomarker for stress. [6][7][8][9] As for the oxidative stress induced by Cd 2+ in soybean sprouts, the timeline for the experimental course is displayed in Figure 2A. Soybeans underwent sprouting in the water  Figure 2B,C. It is noticeable that, as the Cd 2+ level enhances and the exposure time lengthens, the sprouts' growth is conspicuously inhibited with part of the sprouts becoming brownish. The NIR-II fluorescence imaging results of these sprouts show that the higher the Cd 2+ level is and the longer the exposure time is, the stronger the fluorescent signals become in the sprouts, which signifies that higher level of this toxic heavy metal ion and longer exposure leads to higher H 2 O 2 level and ensuingly the stronger fluorescence intensities ( Figure 2D,E).
For peanuts, the experimental course for the oxidative stress induced by Cd 2+ is exhibited in Figure 3A. Peanuts underwent sprouting in the water containing Cd 2+ for different times (60 or 96 h), then the sprouts were incubated with the probe CT-XA-H 2 O 2 for 1 h and next fluorescent imaging was operated. Photographs and NIR-II fluorescence images of the peanut sprouts after germinating in the water containing different concentrations of Cd 2+ for 60 or 96 h are shown in Figure 3B,C. It is clear that with the increasing Cd 2+ level, the fluorescence signals steadily enhance in the peanut sprouts, indicating higher Cd 2+ level leads to more severe stress in the peanut sprouts ( Figure 3D,E). These experimental data validate that the probe CT-XA-H 2 O 2 can track plant sprouts' oxidative stress induced by Cd 2+ in soybean sprouts and peanut sprouts via in vivo NIR-II fluorescence imaging.

The probe's detection of NaCl-induced oxidative stress in plant sprouts via NIR-II fluorescence imaging
Next, the probe CT-XA-H 2 O 2 was employed to monitor soybean sprouts' and peanut sprouts' oxidative stresses caused by high-level salt (NaCl). High-level-salt-containing water/soil would lead to poor plant sprouting and growth, [4][5][6][7] and in the abiotic stress (oxidative stress) induced by highlevel salt, H 2 O 2 overproduced in vivo in the plant sprouts can serve as the endogenous biomarker. As for the stress induced by high-level NaCl, the soybeans were sprouting in  Figure 4A. As shown in Figure 4B,C, similar to the stress induced by Cd 2+ , the soybean sprouts' photographs in different groups exhibit that the longer time length for exposure to high-level NaCl leads to shorter length of the sprouts and the sprouts partially turn brownish, and also the fluorescence signals become more significant as the time length for stress is increased and the salt level is enhanced ( Figure 4D,E). These data indicate that high-level salt exposure impedes sprouting to a certain extent.
As for the peanut sprouts' oxidative stress induced by highlevel salt (the experimental process is shown in Figure 5A), the peanut sprouts first germinated in the water containing 0, 50, 60, 70, or 80 mM NaCl at different times (60 or 96 h), then the sprouts were incubated with the probe CT-XA-H 2 O 2 for 1 h and next fluorescent imaging was carried out. Photographs and NIR-II fluorescence images of the peanut sprouts which underwent the sprouting process in the water containing different levels of NaCl for 60 or 96 h are presented in Figure 5B,C. It is clearly visible that with the increasing NaCl level, the fluorescence signals augment in the sprouts, and this is because higher NaCl level results in higher stress in the sprouts, which means a higher level of H 2 O 2 overexpression and accordingly stronger fluorescent signals in the sprouts ( Figure 5D,E). These experimental data corroborate that the probe CT-XA-H 2 O 2 can track the abiotic stress (oxidative stress) induced by high-level salt (NaCl) in soybean sprouts and peanut sprouts via in vivo NIR-II fluorescence imaging.

CONCLUSIONS
In summary, an activatable NIR-II fluorescent probe has been developed for tracking plant sprouts' oxidative stress induced by heavy-metal-ion contamination (Cd 2+ ) or high-level salt (NaCl) via detecting the endogenous biomarker H 2 O 2 in vivo. In the presence of oxidative stresses, the probe turns into the activated probe, whose AIE feature ensures strong fluorescence in the aggregated state for NIR-II imaging in an aqueous milieu. The probe CT-XA-H 2 O 2 can function as a utilitarian tool for tracking plant sprouts' oxidative stress via NIR-II fluorescent imaging, and the approach herein could be extended to the design of other activatable probes for monitoring/tracking other important biomarkers in plants.

Experiments of oxidative stress in plant sprouts
As for the stress induced by Cd 2+ during the sprouting process, the soybeans (seeds) were placed on the seedling tray, and the seedling tray was partially immersed in the water containing Cd 2+ (Cd 2+ : 0, 30, 50, 70, or 100 µM) so that the seeds are half submerged in the water containing Cd 2+ (Cd 2+ : 0, 30, 50, 70, or 100 µM) for sprouting (germinating) for 36 or 60 h. The germination process was carried out in the dark.
While as for the stress by Cd 2+ during the germination of peanuts (seeds), the peanuts were put on the seedling tray, and the seedling tray was partially immersed in the water containing Cd 2+ (Cd 2+ : 0, 30, 50, 70, or 100 µM) so that the seeds are half soaked in water containing Cd 2+ (Cd 2+ : 0, 30, 50, 70, or 100 µM) for germinating for 60 or 96 h. The germination process was carried out in the dark.
As for the stress induced by the salt (NaCl) during the sprouting process, similarly, soybeans (seeds) were placed on the seedling tray, and the seedling tray was partially immersed in the water containing NaCl (NaCl: 0, 50, 60, 70, or 80 mM) so that the seeds are half submerged in the water containing NaCl (NaCl: 0, 50, 60, 70, or 80 mM) for sprouting (germinating) for 36 or 60 h. The germination process was carried out in the dark.
While as for the stress by the salt (NaCl) during the germination of peanuts (seeds), the peanuts were put on the seedling tray, and the seedling tray was partially immersed in the water containing NaCl (NaCl: 0, 50, 60, 70, or 80 mM) so that the seeds are half soaked in water containing NaCl (NaCl: 0, 50, 60, 70, or 80 mM) for germinating for 60 or 96 h. The germination process was carried out in the dark.

Fluorescence imaging for soybean sprouts and peanut sprouts
For soybean sprouts enduring stress (Cd 2+ or NaCl), after sprouting for 36 h, five soybean sprouts from each seedling tray were incubated with the probe CT-XA-H 2 O 2 (CT-XA-H 2 O 2 : 15 µM, in water containing 15% DMSO) for 1 h before fluorescence imaging. Similarly, the sprouts upon sprouting for 60 h underwent the same procedures for imaging.
For peanut shoots grown under stress (Cd 2+ or NaCl), after sprouting for 60 h, the incubation of peanut shoots with the probe CT-XA-H 2 O 2 for 1 h before imaging was conducted (CT-XA-H 2 O 2 : 15 µM, in water containing 15% DMSO). The peanut shoots upon sprouting for 96 h underwent the same procedures for imaging. Each group contains five sprouts.

A C K N O W L E D G M E N T S
This work was supported by NSFC (21788102 and 21875069) and the Fund of Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates (2019B030301003).

C O N F L I C T O F I N T E R E S T
The authors declare that they have no conflict of interest.