Phytotoxicity, Uptake, and Translocation of Fluorescent Carbon Dots

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Phytotoxicity, uptake and translocation of fluorescent carbon dots in mung bean plants Wei Li, Yinjian Zheng, Haoran Zhang, Zulang Liu, Wei Su, Shi Chen, YingLiang Liu, Jianle Zhuang, and Bingfu Lei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07268 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

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ACS Applied Materials & Interfaces

Phytotoxicity, uptake and translocation of fluorescent carbon dots in mung bean plants Wei Li1, Yinjian Zheng1,2, Haoran Zhang1, Zulang Liu1, Wei Su2, Shi Chen1, Yingliang Liu1*, Jianle Zhuang1, and Bingfu Lei1,2*

1. Guangdong Provincial Engineering Technology Research Center for Optical Agriculture, College of Materials and Energy, South China Agricultural University Guangzhou 510642, China 2. College of Horticulture, South China Agricultural University Guangzhou 510642, China E-mail: [email protected] Keywords: Carbon dots, fluorescent, phytotoxicity, mung bean, bioimaging

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Abstract Fluorescent carbon dots (CDs) have been widely studied in bioscience and bioimaging, but the effect of CDs on plants has been rarely studied. Herein, mung bean was adopted as a model plant to study the phytotoxicity, uptake, and translocation of red emissive CDs in plants. The incubation with CDs at a concentration range of 0.1 to 1.0 mg/mL induced physiological response of mung bean plant and imposed no phytotoxicity on mung bean growth. The lengths of the root and stem presented an increasing trend up to the treatment of 0.4 mg/mL. Confocal imaging showed that CDs were transferred from the roots to the stems and leaves by the vascular system through the apoplastic pathway. The uptake kinetics study was performed and demonstrated that the CDs were abundantly incubated by mung beans during both germination and growth periods. Furthermore, In vivo visualization of CDs provides potential for their successful application as delivery vehicles in plants based on its unique optical properties.


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1. INTRODUCTION Nanomaterials, possess at least one dimension ranges from 1 to 100 nm, are becoming intensively studied due to the unique physicochemical properties emerging at the nanoscale. Specific characteristics can be obtained by varying their size, morphology, structure and surface composition, which enable the development of nanomaterials in the areas of energy, electronics and biomedicine. These nanomaterials differ from bulk materials because they have larger surface area, higher surface energy and are under quantum confinement.1 Generally, nanomaterials exhibit increased reactivity and toxicity compared to their bulk counterparts.2 Rapid development and widespread applications of nanomaterials has increased likelihood of their deliberate or accident release to the surroundings, highlighting the urgency to understand their fate and potential impact on the environment.3 Now, the short and medium term investigations of the environmental impact of nanomaterials are an emerging area for further exploration. Fluorescent carbon dots (CDs), receive wide attention as a fascinating class of fluorescence-based nanoparticles (NPs), which have been spotlighted to be a promising substitute to conventional semiconductor quantum dots (QDs) and organic dyes. The great issue associated with QDs and organic dyes is the toxic elements of components, which gives rise to significant health and environmental concerns. QDs have been reported to degrade with oxygen and ultraviolet light with unstable structure, freeing toxic Cd2+, Se2− or SeO32−.4 The CDs are notably advantageous in their facile synthesis, robust chemical inertness, easy modification, high light stability



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and low cytotoxicity.5 CDs have broad application prospects in bioimaging,6 sensing,7 and other optoelectronic device applications.8-9 As a new technology applied for biosystems, CDs have gained increased attention in live imaging of tissues. Plants are cornerstones of most ecosystems and play an essential role in the fate and transport of nanomaterials in the environment via plant uptake and bioaccumulation.10 Researches have been reported on the interactions of nanomaterials with plant, including carbonaceous nanomaterials (fullerenes11 and nanotubes12), metal oxides13, zero-valent metals14, nanopolymers15, QDs16 and other NPs (Ni(OH)217 and NaYF418). Once NPs are taken by plants through an aerial or root pathway, they could result in beneficial or adverse effect on plants growth. Most NPs had negative effects on plants growth at high concentrations while enhanced plants growth at low concentrations. The positive effects of nanomaterials on plants were mainly reported for Au or Ag NPs, Cu NPs, Al related NPs, TiO2 NPs, CeO2 NPs, SiO2 NPs and carbon nanotubes.19-24 However, once changed the composition, concentration, size and coating of NPs, the results were different. An increasing tendency appears to apply nanomaterials as bio-markers in plant science. Even though much works have been studied on imaging of mammalian cells, only very limited researches have been reported about the successful uptake and application of fluorescent NPs in live plant imaging. In vivo nanoparticles imaging is of great importance for their successful application as delivery vehicles in plants. To date, luminescent materials have been used as imaging agents in live plant tissues, including organic dyes,25 rare-earth doped nanoparticles26 and QDs.16 These materials


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often suffer from a variety of drawbacks, such as potential long-term toxicity, poor water solubility and low chemical stability. Most of them are not dispersible in water and thus not appropriate for bio-applications. In addition, there are no reactive functional groups available on the surface of these NPs to link any biomolecule. Surface modification is necessary to render them hydrophilic with functional groups. In addition, heavy metal ions dissolving from the QDs and the leakage of rare-earth ions could prove to cause concerns for biocompatibility.18, 27 CDs are novel fluorescent nanomaterial with greater frequency applied in various areas, they could potentially enter the total environment via intended (e.g., biological applications) or incidental routes (e.g., release into atmosphere, rivers, soil, etc). Thus, it is necessary to explore their synthesis, properties, toxicity, applications, and delivery systems in biology. Owing to the unique properties of CDs, they are helpful tools for live imaging in plant system, such as visualizing the plant structure, investigating the dynamic plant system processes and tracking the particles movement in the plant tissues. The application of CDs as markers for plant bioimaging would be beneficial because of their high aqueous solubility and flexibility in surface modification. CDs exhibit unique luminescence characteristics such as wide and continuous absorption spectra, symmetrical and narrow fluorescence emission, and excellent photostability. Strong blue and green luminescent CDs with high quantum yields are commonly achieved, however, efficient long-wavelength (e.g. orange and red-light region) emissive CDs are very scarce. Efficient red emissive CDs would be advantageous as biolabels in plants, which are effective for separating the CDs



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fluorescence from blue and green endogenous fluorescence of most plant tissues, and thus can be used to investigate CDs distribution inside the plants. Few research has been detailed report the assessment of the phytotoxicity and the potential uptake of CDs in plants. In this study, mung bean was adopted as a model plant to investigate the dose-response on the phytotoxicity of CDs, uptake, translocation of CDs in plants. Mung bean is a fast-growing vegetable, which enables us to observe the physiological effects of CDs within a relative short exposure time. The efficient red emissive CDs were prepared from p-phenylenediamine through solvothermal method via column chromatography according to a reported method,28 for tracking particles uptake and movement in mung bean sprouts. This study represents a simple system to understand the uptake and translocation of CDs by optical analysis in vivo, indicating the possibility of applying them for live imaging in plant system and offering great support of their use as bio-markers for further research in live plant.

2. EXPERIMENTAL SECTION 2.1 Synthesis of CDs. The CDs were prepared on the basis of a reported method.28 Briefly, 0.6 g p-phenylenediamine was dissolved in 60.0 mL water, and the obtained solution was transferred into a 100 mL Teflon-lined stainless steel autoclave. After heating at 180 °C in oven for 12 h, the autoclave was naturally cooled down. The obtained dark-red suspension was purified through silica column chromatography employing a mixture of ethyl acetate and petroleum ether as the eluent. Afterward, the obtained CDs were redispersed in deionized water with different concentration.


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2.2 Plant growth. Mung bean seeds were selected with nearly sizes and plumpness and then sterilized by treating them with a 10% NaClO solution for 10 minutes. Subsequently, the seeds were washed with deionized water for three times to remove the residual and then were arranged in 100 mL beakers (20 seeds for each group). In the control experiments, pure water and CDs solution with different concentration (0.1, 0.4, 0.7, 1.0 mg/mL) were added into the above beakers, respectively, and then covered and transferred into a growth chamber at 25 °C without the light. After 5 days, the bean sprouts were harvested. The experiment was repeated three times. The germination was calculated and the root length and stem length were measured. Standard deviations are showed as error bars for data sets. Experimental data were calculated by the Student’s t-test with two-tailed distribution to investigate the statistical differences. P values below 0.05 or 0.01 are denoted as significantly statistical different. 2.3 Exposure experiment. Mung bean seeds were surface sterilized and washed with deionized water to remove the residual. And then the seeds were transferred to pure water for germination under the same condition mentioned above. After 3 days, the seedlings were then transferred into 100 mL beaker, in which contains CDs solution with concentration of 1.0 mg/mL. The beaker was covered and placed in a growth chamber at 25 °C without the light. At 20-minute intervals, a mung bean sprout was took out and cleaned with deionized water thoroughly, and then cut the stem into transverse section, the cut site was three centimeter from the bottom of the stem. The section was analyzed by a laser scanning microscope. The luminescence



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spectra of CDs solution remained in the beaker were collected every other 20 minute within 160 minutes. 2.4 Characterizations. The Fourier transform infrared (FT-IR) spectra were taken on a Nicolet Avatar 360 FT-IR spectrophotometer. Transmission electron microscopy (TEM) images were taken with a FEI Tecnai 12 transmission electron microscope. High-resolution transmission electron microscopy (HRTEM) images were collected in a JEOL-2010 electron microscope. Photoluminescence spectra were recorded with a F-7000 Hitachi fluorescence spectrofluorometer. Laser scanning microscopy (LSM) was performed with LSM 710, Zeiss, equipped with 514 nm laser for excitation. X-ray photoelectron spectroscopy (XPS) spectrum was carried out by employing a X-ray photoelectron Spectroscope (AXIS ULTRA DLD, Kratos).

3. RESULTS AND DISCUSSION Most engineered nanoparticles (NPs) are attached with functional groups to improve their water solubility, biocompatibility and stability. These surface coatings reduce the tendency of NPs aggregation and precipitation, relieve the release of potentially toxic components, and regulate NPs mobility when released into ecosystem.29 Given the superior performance of CDs regarding the above aspects, the CDs used herein were synthesized without any surface coatings based on a modified previously reported method,28 including solvothermal reaction and purification. The morphology of CDs that dispersed in water was studied by high-resolution transmission electron microscopy (HRTEM) and atomic force microscopy (AFM). It can be clearly found that the CDs display as uniform, microspherical particles without


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significant aggregation with an average diameter of 4 nm as shown in Figure S1(a) and (b) (Supporting Information). AFM image (Figure S1(c), Supporting Information) shows that the CDs exhibit similar particle heights of approximately 3-4 nm. The zeta potential (ζ) is a vital parameter of a stabilized colloid system, which helps the prediction of the stability of a nanoparticle system in solutions.30 The CDs in aqueous solution had a positive ζ-potential centered at +33.8 mV owing to the amino groups exist on the CDs surface. A large magnitude (usually ±25 mV) of the zeta potential represents a stable colloid system.

Figure 1. (a) FTIR spectra of CDs. (b)XPS of CDs. (c) Emission spectra of CDs excited at 283 nm, 483 nm, and 365 nm, respectively, and excitation spectrum of CDs monitored at 633 nm. The inset shows photographs of CDs taken under daylight and UV (365 nm) irradiation, respectively. The FTIR spectra of the CDs is shown in Figure 1(a), broad absorption bands at 3050-3650 cm-1 are assigned to ν(O-H) and ν(N-H). Peaks emerge at about 1226 cm-1, 1311 cm-1, 1645 cm-1 and 2850 cm-1 can be attributed to C-O, C-N=, N-H, and



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aliphatic C-H stretching vibrations, respectively. As shown in Figure 1(b), the X-ray photoelectron spectroscopy (XPS) spectra of CDs reveal the presence of C, N and O. The photoluminescence (PL) excitation and emission spectra of CDs were investigated as shown in Figure 1(c). The PL excitation spectrum of CDs displays continuously wide excitation band peaked at 283 nm, 365 nm, and 483 nm, respectively, monitored at 633 nm. The CDs exhibit reddish-orange luminescence with excitation-wavelength-independent PL in water. More intense emission centered at 633 nm for CDs can be obtained if optimal excitation wavelength is employed. The images of the CDs dispersion under daylight and UV light (365 nm) show clear solution and reddish-orange fluorescence, respectively.


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Figure 2. Effects of CDs on the growth and development of mung beans after incubation for 5 days. Mung beans were grown in water, CDs solution with concentration of 0.1, 0.4, 0.7, 1.0 mg/mL, respectively (from left to right). (a) Digital photo of mung bean sprouts under daylight. (b) Digital photo of mung bean sprouts under 365 nm UV light. (c) Length of root, (d) length of stem, (e) fresh weight and (f) moisture level of mung bean sprouts grown on different concentrations of CDs. Error bars correspond to standard deviation. Marked with “*” (P < 0.05) and “**” (P < 0.01) indicate significant differences from control, respectively. CDs closely contact with their surrounding environment system and plants are a fundamental base component of ecosystems, thus plants are potentially subject to CDs exposure and these effects such as uptake, translocation and accumulation in plants will highly affect their growth. On the other hand, CDs have constant and unique



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optical properties that provide CDs with the potential for high resolution and long timescale imaging of plants. Mung bean was adopted as a model plant and uptake was studied hydroponically to guarantee that the CDs are readily available for uptake and the delivery of CDs to the root surface less limitedly. Mung bean seeds of similar size were selected and incubated with different concentrations of CDs solutions (0.1, 0.4, 0.7, 1.0 mg/mL), the seeds grown in pure water were set as a control group. After cultivation at 25 °C for 5 days, the mung bean sprouts were harvested. On the basis of the U.S. EPA guideline for nanotoxicity assay of plant, seed germination and seedling elongation are often used to evaluate exposure effects.31 On germinating mung bean seeds with different concentrations of CDs, it turned out that the seeds in CDs solution and pure water all germinated with radicles. As shown in Figure 2(a), there is an obvious difference of development of mung bean sprouts between the control group and CDs-treated groups. A promotion on mung bean growth was found in the concentration range of CDs from 0 to 1.0 mg/mL. It turned out that a concentration threshold (0.4 mg/mL) is presented, below which a little promotion is found. The digital image of mung bean sprouts under daylight visually shows the concentration-dependent promotion effect. Mung bean sprouts grown in CDs solution emitted clear concentration-dependent reddish-orange fluorescence over the entire cotyledons under 365 nm UV light (Figure 2(b)), thus providing evidence for increased absorption of CDs by mung bean.


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514 nm excitation

Bright field

Overlay

Root

Stem

Cotyledon

Leaf

Figure 3. Laser scanning microscopy (LSM) images of transverse sections from root, stem, cotyledon, and leaf in mung beans, cultured with CDs (1.0 mg/mL) for 5 days. All images were collected under same exposure conditions. To further investigate the dose-response on the phytotoxicity of CDs during mung bean growth period, the root and stem elongation were also investigated (Figure 2(c) and (d)). Compared with the control group, the root and the stem also elongated to



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some varying degrees grown in different concentration of CDs. The lengths of the root and stem presented an increasing trend up to the treatment of 0.4 mg/mL which significantly increased (p

Phytotoxicity, Uptake, and Translocation of Fluorescent Carbon Dots

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