Nanochemistry and biotechnology
Plant-mediated synthesis of Ag2S nanoparticles, their surface modification and genotoxic properties
Borova M1., Kapush O2., Horiunova I1., Plokhovska S1., Pushkareva N1., Yemets A1. 
1Institute of Food Biotechnology and Genomics, NAS of Ukraine,
Osypovskogo str. 2a., Kyiv 04123, Ukraine, marie0589@gmail.com

2V.E. Lashkaryov Institute of Semiconductor Physics NAS of Ukraine, Pr. Nauki 45, Kyiv 02000

Ag2S quantum dots attract research interest in recent years due to their appealing near-infrared (NIR) photoluminescence in the second (NIR-II) window, which promises in vivo fluorescence imaging with higher penetration depth. It is very important that the nanoparticles be in a water-dispersible form for their use in any biological applications. Water-soluble nanoparticles are produced through surface modification of nanoparticles with biocompatible materials. The surface modification of  nanoparticles increases their luminescent quantum yields, expand their stability, prevent their aggregation and facilitates usage in biological research. Living organisms show a unique potential in environmentally friendly production and accumulation of nanoparticles with different shapes and sizes. The main benefit of using plant extract for nanoparticles synthesis is that they are easily available, nontoxic, economically, and have a wide variety of metabolites that can assist in the reduction of metal ions [1]. The aim of this study was to synthesize Ag2S nanoparticles by hairy root extract of Linaria maroccana, characterize their structural, morphological and optical features, functionalize the surface of obtained nanoparticles using BSA and L-cysteine, and investigate also genotoxic properties of silver sulfide quantum dots on a model using Allium-test.
It should be noted that «green» synthesis methodology was previously developed by us for cadmium-based quantum dots. All experimental details are presented in [2]. Luminescence spectra were measured at room temperature using the serial spectrophotometer Cary Eclipse (Varian Inc., Agilent Tech. USA). Characterization of Ag2S quantum dots was performed using electron microscope JEOL JEM-2100 F (Japan) with accelerating voltage 200 kV. Each sample was dispersed ultrasonically to separate individual particles, and some drops of the suspension were deposited onto carbon coated copper grids. For surface modification experiments we have used BSA and L-cysteine solutions at concentration 10 mg/mL. Freshly prepared solutions were added to the nanoparticles samples in the ratio 2:1. Surface modification methodology was described in [3]. To determine the genotoxicity of Ag2S nanoparticles, the roots of 4-day onion roots were treated under 4˚C for 2-4 h for mitosis synchronization in meristematic root apex cells. Then roots were fixed with Clark's fixer (a mixture of 96% ethyl alcohol and glacial acetic acid as 1:4 ratio) for 1 h at room temperature and washed out in 70% ethyl alcohol). Fixed roots were treated with silver sulfide nanoparticles (concentration 3mg/mL) for 24h. Further squashed samples were prepared and analyzed using a light fluorescence microscope Axioskop 40 (Carl Zeiss, Germany). Influence of Ag2S nanoparticles in concentrations from 0.3 to 3 mg/mL on mitosis progression in cells of A. cepa apical meristem was evaluated. 
The morphology and size distribution of Ag2S nanoparticles were investigated by transmission electron microscopy (TEM). It has been found that synthesized Ag2S nanoparticles have a spherical morphology, and a specific shell around the nucleius of nanoparticles, which is probably formed as a result of “biological” synthesis. Produced silver-based nanoparticles have no structural defects. Their diameter was in the range of 10-20 nm. Optical studies have shown that synthesized Ag2S quantum dots have intense luminescence in NIR region, namely from 1100 to 1600 nm, that is a characteristic feature of such nanoparticles. Surface modification of these nanoparticles was made in order to develop further applications for binding to proteins or antibodies in immunoassays. In this case, Raman spectra were analyzed. Raman band centered at about 220 cm-1. However that peak was observed at blue (488 nm) or green (514-532 nm) excitation, while at red excitation (633 or 647 nm) a mode at 200 cm-1 was observed. The study of genotoxicity of Ag2S nanoparticles is important also for understanding the prospects for their employment in cell biology and biomedical applications. For this purpose, the standard Allium-test was used. It was found that Ag2S nanoparticles do not cause significant genotoxic abnormalities in the meristematic cells  of A. cepa roots in tested concentrations. It was revealed found that the mitotic index value in the experimental samples does not differ from the control, and was in the range of 91-99%. Thus, the mitotic activity in cells after treatment with different concentrations of silver sulfide nanoparticles did not change significantly compared to the control, indicating that they did not have a negative effect on the duration of the mitosis progression in A. cepa cells. Therefore, our study demonstrated the possibility of synthesis obtaining the stable homogeneous Ag2S nanoparticles by hairy root culture as the matrix. Produced nanoparticles had spherical morphology, intense luminescence and were not genotoxic which allows them to be used as luminescent probe to create bioconjugates with proteins or antibodies in immunohistochemical different cell biology studies. 

1.	Sibiya P.N., Moloto M.J.  Green synthesis of Ag2S nanoparticles: effect of pH and capping agent on size and shape of NPs and their antibacterial activity // Dig. J. Nanomater. Biostruct. – 2018. – 13. – P. 411-418.
2.	Borovaya M.N., Naumenko A.P., Matvieieva N.A., Blume Ya.B., Yemets A.I. Biosynthesis of luminescent CdS quantum dots using plant hairy root culture // Nanoscale Res. Lett. – 2014. – 9. – P.1-7.
3.	Beatriz Pelaz, Pablo del Pino, Pauline Maffre, Raimo Hartmann et al. Surface functionalization of nanoparticles with  polyethylene glycol (PEG): effects on protein adsorption and cellular uptake // ACS Nano. – 2015. – 9. – P. 6996-08.