Sugar-Mediated Green Synthesis of Silver Selenide Semiconductor Nanocrystals under Ultrasound Irradiation

Silver selenide (Ag2Se) is a promising nanomaterial due to its outstanding optoelectronic properties and countless bio-applications. To the best of our knowledge, we report, for the first time, a simple and easy method for the ultrasound-assisted synthesis of Ag2Se nanoparticles (NPs) by mixing aqueous solutions of silver nitrate (AgNO3) and selenous acid (H2SeO3) that act as Ag and Se sources, respectively, in the presence of dissolved fructose and starch that act as reducing and stabilizing agents, respectively. The concentrations of mono- and polysaccharides were screened to determine their effect on the size, shape and colloidal stability of the as-synthesized Ag2Se NPs which, in turn, impact the optical properties of these NPs. The morphology of the as-synthesized Ag2Se NPs was characterized by transmission electron microscopy (TEM) and both α- and β-phases of Ag2Se were determined by X-ray diffraction (XRD). The optical properties of Ag2Se were studied using UV–Vis spectroscopy and its elemental composition was determined non-destructively using scanning electron microscopy–energy-dispersive spectroscopy (SEM–EDS). The biological activity of the Ag2Se NPs was assessed using cytotoxic and bactericidal approaches. Our findings pave the way to the cost-effective, fast and scalable production of valuable Ag2Se NPs that may be utilized in numerous fields.


Introduction
Semiconductor materials are technologically important due to their size-dependent optical, structural and electrical properties [1,2]. Phase-change chalcogenides have the ability to alternate between amorphous and crystalline phases with exposure to controlled energy inputs, making them an important material for a wide range of optical and electronic applications [3][4][5][6]. Silver selenide (Ag 2 Se), also known as naumannite, is a semiconductor material as a bulk and rarely found in nature as a mineral. It belongs to I-VI compounds with an optical band gap of 1.2-1.8 eV [7]. Among semiconductor

Visual Aspect of Ag 2 Se NPs
The initial observation of the formation of Ag 2 Se NPs consists of a visual color change of the samples after ultrasound (US) irradiation. The results clearly show that the reaction mixture containing silver and selenium precursors, in addition to fructose and starch as the reducing and stabilizing agents, respectively, turns from white to brown after US irradiation ( Figure 1A). The US irradiated control containing only 3.4 mM [Ag + ] f and 1.7 mM [Se 4+ ] f aqueous solutions showed no significant change in color ( Figure 1B), confirming the effectiveness of fructose and starch as green agents when combined with US energy to promote the production of Ag 2 Se NPs.

Visual Aspect of Ag2Se NPs
The initial observation of the formation of Ag2Se NPs consists of a visual color change of the samples after ultrasound (US) irradiation. The results clearly show that the reaction mixture containing silver and selenium precursors, in addition to fructose and starch as the reducing and stabilizing agents, respectively, turns from white to brown after US irradiation ( Figure 1A). The US irradiated control containing only 3.4 mM [Ag + ]f and 1.7 mM [Se 4+ ]f aqueous solutions showed no significant change in color ( Figure 1B), confirming the effectiveness of fructose and starch as green agents when combined with US energy to promote the production of Ag2Se NPs.

UV-Vis Spectroscopy
The formation of Ag2Se NPs was confirmed by the appearance of a unique and well-defined absorption band between 385 and 446 nm with a maximum at ~413 nm, similar to that reported in the literature [46,47] (Figure 2A). In this range, the single absorption peak gives information about the shape, size and stability/aggregation of the NPs [48,49].

UV-Vis Spectroscopy
The formation of Ag 2 Se NPs was confirmed by the appearance of a unique and well-defined absorption band between 385 and 446 nm with a maximum at~413 nm, similar to that reported in the literature [45,46] (Figure 2A). In this range, the single absorption peak gives information about the shape, size and stability/aggregation of the NPs [47,48].
It is notable that the intensity of the absorption band of the as-obtained Ag 2 Se NPs increases as the fructose concentration increases from 5 to 40 mg mL −1 , while the other experimental parameters are kept constant (Figure 2A). This confirms the crucial role played by fructose in Ag 2 Se NP formation. Hypothetically, this process may consist of two steps: first, cationic selenium is reduced to Se 2− that, in turn, coprecipitates with cationic silver (Ag + ) to subsequently give rise to Ag 2 Se NPs. The variations in the absorption intensities indicate significant differences in the particle number between the samples [49]. On the other hand, the corresponding UV-Vis absorption spectra of the control experiments C 1 , C 2 and C 3 exhibit no absorption band ( Figure 2B). This denotes either the absence of any Ag 2 Se NP formation or the instability of any formed Ag 2 Se NPs. It is notable that the intensity of the absorption band of the as-obtained Ag2Se NPs increases as the fructose concentration increases from 5 to 40 mg mL −1 , while the other experimental parameters are kept constant (Figure 2A). This confirms the crucial role played by fructose in Ag2Se NP formation. Hypothetically, this process may consist of two steps: first, cationic selenium is reduced to Se 2-that, in turn, coprecipitates with cationic silver (Ag + ) to subsequently give rise to Ag2Se NPs. The variations in the absorption intensities indicate significant differences in the particle number between the samples [50]. On the other hand, the corresponding UV-Vis absorption spectra of the control experiments C1, C2 and C3 exhibit no absorption band ( Figure 2B). This denotes either the absence of any Ag2Se NP formation or the instability of any formed Ag2Se NPs. After confirming their production and the key role of fructose in this reaction, the colloidal stability of the Ag 2 Se NPs obtained from sample E 4 , made of 3.4 mM [Ag + ] f , 1.7 mM [Se 4+ ] f , 40 mg mL −1 fructose and 10 mg mL −1 starch and stored at room temperature (RT) in the dark, was monitored by recording its UV-Vis spectra at 15, 30 and 60 days ( Figure 3). As a result, the absorption bands display similar features although a slight increase in the intensity after 30 and 60 days is noticed. This slight increase in the absorbance intensity might be attributed to either experimental errors or reorganization of the polysaccharidic matrix that surrounds and stabilizes the NPs. Furthermore, no signs of sedimentation were observed [50]. These findings confirm the stability of the as-produced Ag 2 Se NPs over a long period of time.
After confirming their production and the key role of fructose in this reaction, the colloidal stability of the Ag2Se NPs obtained from sample E4, made of 3.4 mM [Ag + ]f, 1.7 mM [Se 4+ ]f, 40 mg mL −1 fructose and 10 mg mL −1 starch and stored at room temperature (RT) in the dark, was monitored by recording its UV-Vis spectra at 15, 30 and 60 days ( Figure 3). As a result, the absorption bands display similar features although a slight increase in the intensity after 30 and 60 days is noticed. This slight increase in the absorbance intensity might be attributed to either experimental errors or reorganization of the polysaccharidic matrix that surrounds and stabilizes the NPs. Furthermore, no signs of sedimentation were observed [51]. These findings confirm the stability of the as-produced Ag2Se NPs over a long period of time.  The plots reveal that the Ag2Se NPs undergo both direct and indirect transitions. In this particular case, the direct transition ( Figure 4A) seems to have two linear segments with a possible second band gap that could come from the two phases determined in XRD (vide infra) and/or may be due to the presence of a bimodal NP population as observed with TEM (vide infra). The occurrence of both direct and indirect transitions was also previously reported for Ag2S NPs and Ag2Se NPs [52]. The indirect band gaps of samples E3 and E4, displayed in Figure 4B, were found to be 1.95 and 1.92 eV, respectively, which suggests optical absorption at wavelengths below 635 and 645 nm, respectively. The direct band gaps of the same samples were found to be 2.41 and 2.45 eV, respectively ( Figure 4A). In other words, there is no significant difference between these two band gap values, therefore the amount of fructose does not seem to have any impact on the band gap of the as-produced Ag2Se NPs. However, the band gap values of these Ag2Se NPs are higher than the ones experimentally estimated for thin films of the same material (1.37 eV) [53] or for their bulk counterpart (0.16 eV) [11], suggesting that it is possible to modify the band gap by tailoring the NP size. The difference between the reported values and the obtained Eg can be attributed to the appearance of a series of discrete states in the conduction and valence bands yielding an Eg increase in quantum size systems [54,55].  The plots reveal that the Ag 2 Se NPs undergo both direct and indirect transitions. In this particular case, the direct transition ( Figure 4A) seems to have two linear segments with a possible second band gap that could come from the two phases determined in XRD (vide infra) and/or may be due to the presence of a bimodal NP population as observed with TEM (vide infra). The occurrence of both direct and indirect transitions was also previously reported for Ag 2 S NPs and Ag 2 Se NPs [51]. The indirect band gaps of samples E 3 and E 4 , displayed in Figure 4B, were found to be 1.95 and 1.92 eV, respectively, which suggests optical absorption at wavelengths below 635 and 645 nm, respectively. The direct band gaps of the same samples were found to be 2.41 and 2.45 eV, respectively ( Figure 4A). In other words, there is no significant difference between these two band gap values, therefore the amount of fructose does not seem to have any impact on the band gap of the as-produced Ag 2 Se NPs. However, the band gap values of these Ag 2 Se NPs are higher than the ones experimentally estimated for thin films of the same material (1.37 eV) [52] or for their bulk counterpart (0.16 eV) [11], suggesting that it is possible to modify the band gap by tailoring the NP size. The difference between the reported values and the obtained E g can be attributed to the appearance of a series of discrete states in the conduction and valence bands yielding an E g increase in quantum size systems [53,54].

X-ray Diffraction
Ag2Se NPs can exist as either an orthorhombic β-phase or a cubic α-phase. According to Kumashiro et al. [56], Ag2Se is a non-stoichiometric compound in both phases that can co-exist with Ag2Se as a single phase. In addition, silver solubility is higher in the α-phase than in the β-phase.

X-ray Diffraction
Ag 2 Se NPs can exist as either an orthorhombic β-phase or a cubic α-phase. According to Kumashiro et al. [55], Ag 2 Se is a non-stoichiometric compound in both phases that can co-exist with Ag 2 Se as a single phase. In addition, silver solubility is higher in the α-phase than in the β-phase.  221) and (222) planes, respectively, which can be assigned to the orthorhombic β-Ag 2 Se phase (JCPDS card no. 98-001-5213) corresponding to the naumannite structure. Additionally, the prominent peak at 2θ = 37.6 • suggests the presence of elemental silver and might correspond to the (111) plane of the face-centered cubic (fcc) structure of metallic silver (JCPDS file no. 03-0921). Furthermore, weak peaks can be attributed to selenium particles. These last two observations suggest that the precursor cations were reduced to their elemental forms in the final sample.  221) and (222) planes, respectively, which can be assigned to the orthorhombic β-Ag2Se phase (JCPDS card no. 98-001-5213) corresponding to the naumannite structure. Additionally, the prominent peak at 2θ = 37.6° suggests the presence of elemental silver and might correspond to the (111) plane of the face-centered cubic (fcc) structure of metallic silver (JCPDS file no. 03-0921). Furthermore, weak peaks can be attributed to selenium particles. These last two observations suggest that the precursor cations were reduced to their elemental forms in the final sample. The presence of both orthorhombic and cubic phases of silver selenide is due to the acoustic cavitation phenomenon which causes the formation of vapor bubbles that, after collapsing, produce localized hot spots with temperatures up to 5000 K, high pressures up to 20 MPa and very high cooling rates [57]. In other words, this method offers optimal conditions for the simultaneous formation of both phases. Furthermore, widening appears in some of the diffraction peaks, mainly due to the small particle size as confirmed by TEM micrographs (vide infra) which yields peak overlapping of the two phases.
The average crystallite size of the as-produced Ag2Se NPs, determined from the half-width of the diffraction peak at 2θ = 31.8° corresponding to the (102) crystallographic plane of the β-phase using the Debye-Scherrer formula (Equation (2)), was found to be 26.5 nm, in very good agreement with the size range of the nanoparticles (5-40 nm) deduced from the TEM micrographs (vide infra). This result is consistent with the size of the crystallite and similar phases previously reported in the literature [53,55,57].

Transmission Electron Microscopy
TEM images revealed that the as-synthesized Ag2Se NPs from sample E3 (3.4 mM [Ag + ]f, 1.7 mM [Se 4+ ]f, 20 mg mL −1 fructose, 10 mg mL −1 starch) are round in shape, well-dispersed and well-defined with a broad size distribution ranging between 5 and 40 nm ( Figure 6A,B). Figure 6C depicts the corresponding mean particle size distribution of the same sample. It shows the presence of a heterogeneous population of Ag2Se NPs with a bimodal size distribution. The average particle sizes estimated from TEM images with a population of 60 NPs were found to be 15 and 28 nm, which could be due to a non-homogeneous reaction medium in terms of temperature, matter composition and processing time. In fact, the longer the ultrasonic radiation treatment, the higher the energy contribution. This triggers a greater cavitation effect that generates higher temperatures resulting in smaller mean NP diameters [58]. The presence of both orthorhombic and cubic phases of silver selenide is due to the acoustic cavitation phenomenon which causes the formation of vapor bubbles that, after collapsing, produce localized hot spots with temperatures up to 5000 K, high pressures up to 20 MPa and very high cooling rates [56]. In other words, this method offers optimal conditions for the simultaneous formation of both phases. Furthermore, widening appears in some of the diffraction peaks, mainly due to the small particle size as confirmed by TEM micrographs (vide infra) which yields peak overlapping of the two phases.
The average crystallite size of the as-produced Ag 2 Se NPs, determined from the half-width of the diffraction peak at 2θ = 31.8 • corresponding to the (102) crystallographic plane of the β-phase using the Debye-Scherrer formula (Equation (2)), was found to be 26.5 nm, in very good agreement with the size range of the nanoparticles (5-40 nm) deduced from the TEM micrographs (vide infra). This result is consistent with the size of the crystallite and similar phases previously reported in the literature [52,54,56].

Transmission Electron Microscopy
TEM images revealed that the as-synthesized Ag 2 Se NPs from sample E 3 (3.4 mM [Ag + ] f , 1.7 mM [Se 4+ ] f , 20 mg mL −1 fructose, 10 mg mL −1 starch) are round in shape, well-dispersed and well-defined with a broad size distribution ranging between 5 and 40 nm ( Figure 6A,B). Figure 6C depicts the corresponding mean particle size distribution of the same sample. It shows the presence of a heterogeneous population of Ag 2 Se NPs with a bimodal size distribution. The average particle sizes estimated from TEM images with a population of 60 NPs were found to be 15 and 28 nm, which could be due to a non-homogeneous reaction medium in terms of temperature, matter composition and processing time. In fact, the longer the ultrasonic radiation treatment, the higher the energy contribution. This triggers a greater cavitation effect that generates higher temperatures resulting in smaller mean NP diameters [57]. Although no UV-Vis absorption band was recorded for the fructose-free control sample C2 (3.4 mM [Ag + ]f, 1.7 mM [Se 4+ ]f, 10 mg mL −1 starch) ( Figure 2B), the TEM micrograph of the same sample displays particles whose morphology varies from spherical to cubic and rod-shaped nanocrystals with a large particle size (Figure 7), as compared to Figure 6. Moreover, large aggregate nanostructures were formed; however, their chemical nature was not determined. This might be attributed to the fact that starch, a natural polysaccharide, can be decomposed into amylose and amylopectin, natural polymers formed by long chains of α-D-glucose molecules. α-D-glucose, like Dfructose, is a natural sugar that acts as a reducing agent in the production of inorganic NPs [59]. Therefore, starch, after its possible degradation, might have partially acted as the reducing agent instead of the stabilizing agent in the presence of fructose. Consequently, aggregated cubic and rod NPs of different sizes were formed since there is little to no stabilizing matrix to control the NP features (size, shape, stability), corroborating the absence of any UV-Vis absorption band. These results confirm the key roles of fructose as a strong reducing sugar and starch as an efficient stabilizing matrix in the ultrasound-assisted production of nanoparticles with interesting optical properties and controlled morphological features. Although no UV-Vis absorption band was recorded for the fructose-free control sample C 2 (3.4 mM [Ag + ] f , 1.7 mM [Se 4+ ] f , 10 mg mL −1 starch) ( Figure 2B), the TEM micrograph of the same sample displays particles whose morphology varies from spherical to cubic and rod-shaped nanocrystals with a large particle size (Figure 7), as compared to Figure 6. Moreover, large aggregate nanostructures were formed; however, their chemical nature was not determined. This might be attributed to the fact that starch, a natural polysaccharide, can be decomposed into amylose and amylopectin, natural polymers formed by long chains of α-D-glucose molecules. α-D-glucose, like D-fructose, is a natural sugar that acts as a reducing agent in the production of inorganic NPs [58]. Therefore, starch, after its possible degradation, might have partially acted as the reducing agent instead of the stabilizing agent in the presence of fructose. Consequently, aggregated cubic and rod NPs of different sizes were formed since there is little to no stabilizing matrix to control the NP features (size, shape, stability), corroborating the absence of any UV-Vis absorption band. These results confirm the key roles of fructose as a strong reducing sugar and starch as an efficient stabilizing matrix in the ultrasound-assisted production of nanoparticles with interesting optical properties and controlled morphological features.

Scanning Electron Microscopy-Energy-Dispersive Spectroscopy
SEM-EDS was used to study the chemical composition and purity of the as-obtained Ag2Se nanomaterial. The EDS spectrum, taken from selected areas of SEM images, exhibits prominent silver, selenium, carbon and oxygen peaks in the as-synthesized Ag2Se NPs (Figure 8). The detected C and O mainly arise from the stabilizing agent (starch) and the carbon grid used to mount the sample. The identification line for the major emission energy of selenium was observed in the range of 1.3-1.4 keV, while silver was observed in the range of 2.8-3.0 keV, corresponding in both cases to the L-α peak of these elements. These selenium and silver peaks, due to their presence in Ag2Se NPs, are in good agreement with elemental peaks reported in the literature [46,60]. Therefore, the EDS spectrum confirms the presence of silver and selenium atoms in the as-synthesized Ag2Se NPs. Moreover, by dividing the peak intensity of Ag by the one of Se, this results in a 2:1 ratio, suggesting the chemical formula Ag2Se.

Scanning Electron Microscopy-Energy-Dispersive Spectroscopy
SEM-EDS was used to study the chemical composition and purity of the as-obtained Ag 2 Se nanomaterial. The EDS spectrum, taken from selected areas of SEM images, exhibits prominent silver, selenium, carbon and oxygen peaks in the as-synthesized Ag 2 Se NPs (Figure 8). The detected C and O mainly arise from the stabilizing agent (starch) and the carbon grid used to mount the sample. The identification line for the major emission energy of selenium was observed in the range of 1.3-1.4 keV, while silver was observed in the range of 2.8-3.0 keV, corresponding in both cases to the L-α peak of these elements. These selenium and silver peaks, due to their presence in Ag 2 Se NPs, are in good agreement with elemental peaks reported in the literature [45,59]. Therefore, the EDS spectrum confirms the presence of silver and selenium atoms in the as-synthesized Ag 2 Se NPs. Moreover, by dividing the peak intensity of Ag by the one of Se, this results in a 2:1 ratio, suggesting the chemical formula Ag 2 Se.

Cytotoxicity Assessment by MTT Assay
The cytotoxicity of the as-synthesized Ag 2 Se NPs was evaluated using the MTT assay considering that viability assays are key to explaining the cellular response to a toxic agent and provide information about the cell survival and metabolic activity. Human fibroblasts from the ATCC cell line were treated with different concentrations of Ag 2 Se NPs for 24, 48 and 72 h. The obtained results indicated a range of cytotoxicity responses when compared to the control groups.

Cytotoxicity Assessment by MTT Assay
The cytotoxicity of the as-synthesized Ag2Se NPs was evaluated using the MTT assay considering that viability assays are key to explaining the cellular response to a toxic agent and provide information about the cell survival and metabolic activity. Human fibroblasts from the ATCC cell line were treated with different concentrations of Ag2Se NPs for 24, 48 and 72 h. The obtained results indicated a range of cytotoxicity responses when compared to the control groups.
The as-synthesized Ag2Se NPs caused a significant reduction in the mitochondrial activity of ATCC cells with respect to the untreated control group (Figure 9). For instance, cultures at the studied NP concentrations of the E3 sample (3.4 mM [Ag + ]f, 1.7 mM [Se 4+ ]f, 10 mg mL −1 starch, 20 mg mL −1 fructose) showed a decrease in the cytotoxic effect with respect to the controls, reaching values close to ~30% of viability after incubation for 24 and 48 h, following a concentration-dependent effect ( Figure 9C). After 72 h post-NP exposure, the cytotoxicity slightly increased as the viability decreased to ~20%, regardless of the lyophilized Ag2Se NP concentration. On the other hand, samples E1 and E2 exhibited a higher cytotoxicity (~10% cell viability) that was independent of the Ag2Se NP concentration and exposure time ( Figure 9A,B). According to these results, the as-synthesized Ag2Se NPs display a very strong biocidal activity against ATCC human fibroblast cell cultures. Moreover, the initial amount of fructose may impact this activity. The as-synthesized Ag 2 Se NPs caused a significant reduction in the mitochondrial activity of ATCC cells with respect to the untreated control group (Figure 9). For instance, cultures at the studied NP concentrations of the E 3 sample (3.4 mM [Ag + ] f , 1.7 mM [Se 4+ ] f , 10 mg mL −1 starch, 20 mg mL −1 fructose) showed a decrease in the cytotoxic effect with respect to the controls, reaching values close to~30% of viability after incubation for 24 and 48 h, following a concentration-dependent effect ( Figure 9C). After 72 h post-NP exposure, the cytotoxicity slightly increased as the viability decreased to~20%, regardless of the lyophilized Ag 2 Se NP concentration. On the other hand, samples E 1 and E 2 exhibited a higher cytotoxicity (~10% cell viability) that was independent of the Ag 2 Se NP concentration and exposure time ( Figure 9A,B). According to these results, the as-synthesized Ag 2 Se NPs display a very strong biocidal activity against ATCC human fibroblast cell cultures. Moreover, the initial amount of fructose may impact this activity.

Bactericidal Tests
The antibacterial activity of Ag2Se NPs was investigated against the following pathogenic microorganisms by the disc diffusion method: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Pseudomonas aeruginosa. The maximum zone of inhibition (MZI), summarized in Table 1, does not exhibit any trend related to the bacterial cell wall type. On the other hand, there

Bactericidal Tests
The antibacterial activity of Ag 2 Se NPs was investigated against the following pathogenic microorganisms by the disc diffusion method: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Pseudomonas aeruginosa. The maximum zone of inhibition (MZI), summarized in Table 1, does not exhibit any trend related to the bacterial cell wall type. On the other hand, there seems to be a trend among the Gram-species. Indeed, the Ag 2 Se NPs are very toxic towards E. coli, moderately toxic towards S. typhimurium and have the lowest toxicity to P. aeruginosa. Moreover, Ag 2 Se NPs exhibit a very high toxicity against the Gram + S. aureus. Additionally, the initial amount of fructose, the only difference in the composition of E 1 , E 2 and E 3 samples, does not seem to impact the bactericidal activity of the as-obtained Ag 2 Se NPs. Furthermore, the increase in lyophilized Ag 2 Se NP concentration expands the MZI for all the pathogenic bacteria (results not shown). Table 1. Maximum zone of inhibition induced by Ag 2 Se NP samples calculated (in mm) for Gram − and Gram + bacteria.

Strain
Maximum Zone of Inhibition (mm) The mechanism of the Ag 2 Se NP bactericidal mode of action might differ according to the bacterial species and the composition of the samples. Gram − and Gram + bacteria show a similar antibiotic effect but at different sample compositions, as seen in the case of P. aeruginosa. In fact, only two Ag 2 Se NP samples, E 1 and E 3 , at their maximum concentration showed a slight bactericidal effect, whereas all samples promoted a strong antibiotic effect at all NP concentrations against E. coli.
Although the antimicrobial mechanism of semiconductor NPs is not entirely understood, studies suggest this might be related to the formation of oxidative stress in cells, causing damage to their biomolecules that yields cell death [2]. In the present study, it is possible that Ag 2 Se NPs penetrate the cell membrane and irreversibly damage its antioxidative systems in a concentration-dependent manner.

Green Synthesis of Silver Selenide NPs
The green synthesis of Ag 2 Se NPs was carried out via a facile co-precipitation method. Selenous acid (0.44 g) and AgNO 3 (1.73 g) were separately dissolved in DIW at room temperature (RT). The preparation of Ag 2 Se NPs and the associated control experiments are described in Table 2. For Ag 2 Se NP preparation, 5 mL of 34 mM H 2 SeO 3 and 10 mL of 34 mM AgNO 3 were added into a 300 mL glass beaker. Immediately after, varying masses of starch and fructose were added to the reaction mixture and the total volume was brought to 100 mL by adding DIW. Finally, ultrasound irradiation was carried out using a Cole-Parmer ultrasonic processor (DAIGGER GE 505, 500 W, 20 kHz, Daigger Scientific, Buffalo Grove, IL, USA) whose probe was directly immersed into the reaction medium. The operating condition was 59-s pulse ON and 5-s pulse OFF with an amplitude of 70% for 30 min. The formation of Ag 2 Se NPs was confirmed by a color change in the reaction mixture.

Ultraviolet-Visible (UV-Vis) Spectroscopy
The absorption characteristics of the as-obtained Ag 2 Se NPs and their colloidal stability over time were determined by recording the UV-Vis spectra from 300 to 600 nm using a LAMBDA 1050 UV-Vis Spectrophotometer (Perkin Elmer, Inc., Shelton, CT, USA). All measurements were performed in a quartz cuvette, using DIW as the blank. Subsequently, all the samples were stored at RT in the dark.
Starting from the UV-Vis spectra of samples E 3 and E 4 , samples with fructose concentrations of 20 and 40 mg L −1 , respectively, the band gap of the as-produced Ag 2 Se NPs was determined using a Tauc plot relating the absorption coefficient (α) and the photon energy (hν), as depicted in Equation (1): where E g is the band gap in eV, h is Planck's constant (6.626 10-34 J s −1 ), ν is the frequency in Hz, A and α are constants and "n" is 1/2, 3/2, 2 or 3 for direct allowed, direct forbidden, indirect allowed and indirect forbidden transitions, respectively. The extrapolation of the linear portion of the curve with the x-axis gives the value of the E g .

X-ray Diffraction (XRD)
XRD (PANanalytical, EMPYREAN, Almelo, The Netherlands) analysis on lyophilized Ag 2 Se NPs was carried out to identify the phase structure and determine the crystallite size using a θ-2θ configuration (generator-detector), with the Cu-Kα excitation source at λ = 1.54059 Å.
Additionally, the broadening of the diffraction peak in the XRD is related to the particle size by the Debye-Scherrer formula (Equation (2)) to obtain the average crystallite size: where λ is the X-ray wavelength (0.154059 nm), B is the line broadening at full width half maximum (FWHM), θ is the diffraction angle, K is the constant related to crystalline shape (0.93 for spherical NPs) and L is the linear dimension of the particle.

Transmission Electron Microscopy (TEM)
TEM (FEI, TECNAI, G2spirit twin, Holland) was employed to determine the size, shape and morphologies of the as-produced Ag 2 Se NPs. The samples were prepared by casting one drop of Ag 2 Se NP solution onto a carbon-coated copper grid and dried in ambient air at RT. TEM micrographs at 200 and 500 nm scales were subjected to particle size distribution analyses using ImageJ software (developed by the National Institutes of Health (NIH), Bethesda, MD, USA) to generate. CSV files with information about the cross-sectional area of the as-synthesized nanoparticles. As Ag 2 Se NPs are spherical, the diameter was calculated from the area (A = πD 2 /4). Then, using OriginPro software, the size distribution histogram of 2 nm resolution was generated and fitted with a Gaussian function to determine the average size of the NPs.

Scanning Electron Microscopy-Energy-Dispersive Spectroscopy (SEM-EDS)
The elemental analysis and mapping of Ag 2 Se NPs were performed non-destructively using SEM (TESCAN, MIRA 3, Brno, Czech Republic) equipped with EDS (Bruker Nano GmbH, Quantax, Berlin, Germany). The samples were prepared by casting one drop of the Ag 2 Se NP reaction media onto a stub previously covered with two layers of double-coated carbon conductive tape and dried in ambient air at RT.

MTT Assay Using Human Fibroblasts
The ATCC human fibroblast (HFF) cell line was grown in DMEM medium supplemented with 10% wt/v fetal bovine serum (FBS) and 1% wt/v penicillin/streptomycin. Cells were incubated at 37 • C in a humidified 5% CO 2 atmosphere. Subsequently, the cells were harvested using 0.25% wt/v trypsin-EDTA (1X) and the cell count was performed using trypan blue and Neubauer chamber. An amount of 100 µL of the cell suspension was dispersed to achieve 10 4 cells/well in 96-well culture plates and incubated under the same conditions for another 24 h. Then, the medium was replaced with a medium containing the lyophilized Ag 2 Se NPs at different concentrations: 0 (control), 25, 50 and 100 µg mL −1 . After 24, 48 and 72 h incubation, the MTT solution at 0.5 mg mL −1 was added to each well and incubated for another 4 h at 37 • C. Then, formazan crystals were solubilized with 100 µL of 10% SDS per well. The absorbance of each well was measured at 570 nm with a Multiskan microplate reader (USA). Each assay, made in duplicate, included a control-containing culture medium. The cell viability percentage was obtained using Equation (3): Cell viability % = (Absorbance of treated cells/Absorbance of untreated cells) * 100

Antibacterial Activity
The antibacterial activity of Ag 2 Se NPs against several species of bacteria was investigated using a disc diffusion assay. First, 100 µL of bacteria were plated on an LB plate to obtain a bacterial lawn. Then, filter paper discs were dipped into colloidal Ag 2 Se NPs at different concentrations of lyophilized Ag 2 Se NPs: 0 (control), 25, 50 and 100 µg mL −1 , before being placed on the Petri dishes that contained the bacterial lawns and incubated overnight at 37 • C. The pathogenic bacteria Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Pseudomonas aeruginosa were used as model strains. On the other hand, 1% wt/v penicillin/streptomycin solution was used as a reference drug. Finally, the maximum zone of inhibition, expressed as diameter in mm, was measured.

Conclusions
The ultrasound-assisted aqueous green synthesis of silver selenide nanoparticles was successful and demonstrated the ability of fructose and starch as biological reducing and stabilizing agents, respectively. The formation of Ag 2 Se NPs was confirmed by a visual color change in the samples after ultrasound irradiation in the presence of both green agents, as well as the UV-Vis spectra with an absorption band in the range of 385-446 nm. The absorbance intensity was observed to be a function of fructose concentration. In addition to obtaining very stable samples, the band gap of these Ag 2 Se NPs is impacted by their size and crystallographic phase. Nonetheless, the fructose amount does not seem to influence their band gap. The XRD patterns showed the presence of both orthorhombic and cubic phases of silver selenide, as well as some prominent peaks that suggest the presence of both elemental silver and selenium. The crystallite size was comparable to the NP size that was determined from TEM micrographs displaying NPs ranging from 5 to 40 nm. Moreover, TEM images confirmed the production of uniform, well-dispersed and spherical Ag 2 Se NPs only in the presence of both natural agents, fructose and starch. The SEM-EDS elemental analysis corroborated the previous results. In addition to C and O peaks due mainly to starch that acted as the capping agent, the EDS spectrum showed the two prominent elemental L-α peaks related to silver and selenium in a 2:1 ratio, confirming Ag 2 Se NP formation. Green-synthesized silver selenide nanoparticles exhibited a high cytotoxic response to the ATCC human fibroblast cell line and a concentration-dependent bactericidal activity against E. coli, S. aureus, P. aeruginosa and P. typhimurium. Overall, our findings' novelty resides in the efficacy of widespread, renewable and cheap biomolecules to promote the ultrasound-assisted aqueous synthesis of functional Ag 2 Se NPs via a one-step, easy and time-efficient methodology whose versatility may benefit the fabrication of a wide range of nanomaterials of unique features and promising bio-applications.