*2.2. Characterization of Green Synthesized Nd2Se<sup>3</sup> NPs*

The confirmation of synthesis was ascertained utilizing UV-Vis spectrophotometer from Shimadzu (UV-1601 PC). Fluorescence was performed by exciting the samples at 330 nm, and the emission spectra were logged from 400 to 700 nm using a spectrofluorometer, FLS920, Edinburgh Instruments, UK at a scan rate of 300 nm/min. X-ray diffraction (XRD) patterns were logged in the 2θ range of 20◦–80◦ with a step size of 0.02◦ and 5 s per step using a Philips X'PERT PRO armed with X'celerator, a rapid solid-state detector with iron-filtered Cu Kα radiation (λ = 1.5406 Å) as the source. The Nd2Se<sup>3</sup> NP suspension coated onto carbon coated copper grids was subjected to TEM analysis using an FEI Tecnai 30 TEM operated at 300 kV. The FTIR spectroscopy of bioengineered Nd2Se<sup>3</sup> NP was performed in KBr pellets using a Perkin-Elmer Spectrum One instrument. The spectrometer was operated in the diffuse reflectance mode at a resolution of 2 cm−<sup>1</sup> . To obtain good signal to noise ratio, 128 scans of the film were taken in the range of 450–4000 cm−<sup>1</sup> . The dried powder of bioengineered Nd2Se<sup>3</sup> NPs was used for thermogravimetric analysis on a Q5000V 2.4 Build 223 instrument by applying a scan rate of 10 ◦C min−<sup>1</sup> [26].

#### **3. Results**

The schematic representation of biosynthesis and proposed mechanism of Nd2Se<sup>3</sup> NPs is depicted in Figure 1.

The present study examined the modified and advanced processes for the greener production of nanomaterials using enzyme nitrate reductase purified from the extra cellular broth of *Fusarium oxysporum*, as reported by Kumar et al. [18]. After incubation at 25 ◦C for 12 h under anaerobic circumstances, the reaction mixture (3 mL) in 200 mM phosphate buffer (pH 7.2) containing freshly prepared NdCl2, SeCl4, NaNO3, synthetic peptide (Glu-Cys)n-Gly, NADPH, and nitrate reductase resulted in the creation of Nd2Se<sup>3</sup> NPs (Figure 1). The synthesis was monitored by following the attendance of the absorption band centered at 330 nm, which indicated the formation of Nd2Se<sup>3</sup> NPs [27]. The absorption band at 270–280 nm is visible in Figure 2a, which is attributable to the proteins and α-NADPH used in the reaction. The suspension was very stable, with no sign of aggregation of the NPs even after one month. No absorption band at 330 nm was found without or denatured nitrate reductase or NADPH (data not shown). This observation confirmed the involvement of the enzyme in the oxidation of neodymium (from Nd2+ to Nd3+) and reduction of selenium (Se4+ to Se2+) to produce Nd2Se<sup>3</sup> NPs utilizing nitrate as a substrate and NADPH as a cofactor. We used the fungus *Fusarium oxysporum* for the extracellular biosynthesis of Nd2Se<sup>3</sup> NPs; the same had been used in earlier studies to purify the nitrate reductase (18).

**Figure 1.** Schematic representation for synthesis of neodymium nanoparticles and proposed mech-**Figure 1.** Schematic representation for synthesis of neodymium nanoparticles and proposed mechanism.

the involvement of the enzyme in the oxidation of neodymium (from Nd2+ to Nd3+) and reduction of selenium (Se4+ to Se2+) to produce Nd2Se3 NPs utilizing nitrate as a substrate **Figure 2.** (**a**) UV/Visible absorption spectrum and (**b**) Tauc plot of neodymium selenide nanoparti-**Figure 2.** (**a**) UV/Visible absorption spectrum and (**b**) Tauc plot of neodymium selenide nanoparticles.

and NADPH as a cofactor. We used the fungus *Fusarium oxysporum* for the extracellular biosynthesis of Nd2Se3 NPs; the same had been used in earlier studies to purify the nitrate cles. Eventually, the optical energy band gap of Nd2Se3 NPs was estimated via the Tauc Eventually, the optical energy band gap of Nd2Se<sup>3</sup> NPs was estimated via the Tauc Equation (1) shown below

light frequency, respectively. Further, on the basis of electronic transition, the n-parameter

where Ab is the absorbance of nanocrystals and t is the thickness of the cuvette used for measurements. The optical band gap of a nanocrystals was obtained by extrapolation of the linear region of the plot (αhν)2 vs. hν to the point (αhν)2 = 0 [29]. The calculated band

Fluorescence spectrum of Nd2Se3 NPs was obtained at 330 nm excitation, as depicted in Figure 2. Whereas emission spectra revealed a band at 421 nm with red shift (Figure 3). The emission band at 421 nm is considerably red shifted compared to its absorption onset, which is credited to the band-gap or near-band-gap emission. The small shift in the peak suggests that the NPs possess a continuous surface with most surface atoms exhibiting the coordination and oxidation states of their bulk counterparts. The full width at half

The absorption co-efficient (α) was estimated from the UV-Vis absorbance parame-

could be 1/2 (direct allowed), 2 (indirect allowed), or 3 (indirect forbidden) [28].

gap for Nd2Se3 NPs was found to be 3.75 eV, and is shown in Figure 2b.

maximum (FWHM), which is the extent of the spread of peak, is 88.9 nm.

**Figure 3.** Fluorescence measurement of neodymium selenide nanoparticles.

$$
\alpha \mathbf{h} \mathbf{v} = \mathbf{A} \left( \mathbf{h} \mathbf{v} - \mathbf{E}\_{\mathbf{g}} \right)^{1/\mathbf{n}} \tag{1}
$$

αhν = A (hν − Eg)1/n (1)

α = 2.303 (Ab/t) (2)

anism.

reductase (18).

Equation (1) shown below

ters via the following Equation (2),

cles.

Equation (1) shown below

where α, A, h, and ν are absorption co-efficient, arbitrary constant, Plank constant, and light frequency, respectively. Further, on the basis of electronic transition, the n-parameter could be 1/2 (direct allowed), 2 (indirect allowed), or 3 (indirect forbidden) [28]. where α, A, h, and ν are absorption co-efficient, arbitrary constant, Plank constant, and light frequency, respectively. Further, on the basis of electronic transition, the n-parameter could be 1/2 (direct allowed), 2 (indirect allowed), or 3 (indirect forbidden) [28].

**Figure 2.** (**a**) UV/Visible absorption spectrum and (**b**) Tauc plot of neodymium selenide nanoparti-

Eventually, the optical energy band gap of Nd2Se3 NPs was estimated via the Tauc

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 5 of 11

The absorption co-efficient (α) was estimated from the UV-Vis absorbance parameters via the following Equation (2), The absorption co-efficient (α) was estimated from the UV-Vis absorbance parameters via the following Equation (2),

$$
\alpha = 2.303 \text{ (A}\_{\text{b}}/\text{t}) \tag{2}
$$

αhν = A (hν − Eg)1/n (1)

where A<sup>b</sup> is the absorbance of nanocrystals and t is the thickness of the cuvette used for measurements. The optical band gap of a nanocrystals was obtained by extrapolation of the linear region of the plot (αhν) <sup>2</sup> vs. hν to the point (αhν) <sup>2</sup> = 0 [29]. The calculated band gap for Nd2Se<sup>3</sup> NPs was found to be 3.75 eV, and is shown in Figure 2b. where Ab is the absorbance of nanocrystals and t is the thickness of the cuvette used for measurements. The optical band gap of a nanocrystals was obtained by extrapolation of the linear region of the plot (αhν)2 vs. hν to the point (αhν)2 = 0 [29]. The calculated band gap for Nd2Se3 NPs was found to be 3.75 eV, and is shown in Figure 2b.

Fluorescence spectrum of Nd2Se<sup>3</sup> NPs was obtained at 330 nm excitation, as depicted in Figure 2. Whereas emission spectra revealed a band at 421 nm with red shift (Figure 3). The emission band at 421 nm is considerably red shifted compared to its absorption onset, which is credited to the band-gap or near-band-gap emission. The small shift in the peak suggests that the NPs possess a continuous surface with most surface atoms exhibiting the coordination and oxidation states of their bulk counterparts. The full width at half maximum (FWHM), which is the extent of the spread of peak, is 88.9 nm. Fluorescence spectrum of Nd2Se3 NPs was obtained at 330 nm excitation, as depicted in Figure 2. Whereas emission spectra revealed a band at 421 nm with red shift (Figure 3). The emission band at 421 nm is considerably red shifted compared to its absorption onset, which is credited to the band-gap or near-band-gap emission. The small shift in the peak suggests that the NPs possess a continuous surface with most surface atoms exhibiting the coordination and oxidation states of their bulk counterparts. The full width at half maximum (FWHM), which is the extent of the spread of peak, is 88.9 nm.

**Figure 3. Figure 3.**  Fluorescence measurement of neodymium selenide nanoparticles. Fluorescence measurement of neodymium selenide nanoparticles.

The spherical shape and size (18 ± 1 nm) of the Nd2Se<sup>3</sup> NPs were confirmed by TEM (Figure 4a) and the spherical formation of the nanocrystals may have been due to their dynamic nature. To further verify the crystallinity of Nd2Se<sup>3</sup> nanoparticles, the diffraction patterns of X-ray was recorded from drop cast films of biogenic Nd2Se<sup>3</sup> nanoparticles. The as-synthesized Nd2Se<sup>3</sup> nanoparticles were found to reveal the crystalline nature owing to well-defined Bragg's reflections. The peak position and 2θ values agree with those reported for Nd2Se<sup>3</sup> nanoparticles, almost all peaks in the pattern could be indexed to cubic phase cell parameters, a = b = c = 8.85, α = β = γ = 90◦ [JCPDF # 190823]. The given investigation has confirmed the first ever synthesis of Nd2Se<sup>3</sup> nanoparticles under ambient conditions. The XRD pattern of the Nd2Se<sup>3</sup> NPs showed intense peaks at (211), (220), (310), (321), (420), (422), (521) (611), (620), and (444) in the 2θ range of 20◦–80◦ (Figure 4b) and agrees with those reported for the Nd2Se<sup>3</sup> nanocrystals. The size of particles under XRD was found to be ~16 nm (Figure 4b). Further, the confirmation of encapsulation of proteins over the surface of particles was realized through FTIR. Therefore, the peak at 1635.56 cm−<sup>1</sup> corresponds to characteristic of C=O of amide group of the amide I linkage confirmed the presence of protein. Further, the peak at 3320.62 cm−<sup>1</sup> confirms the N-H stretching vibration. The C-N stretching of aliphatic amines associated with peptide bond was affirmed by extra characteristic peak at 1044.54 cm−<sup>1</sup> (Figure 4c).

firmed by extra characteristic peak at 1044.54 cm−1 (Figure 4c).

The spherical shape and size (18 ± 1 nm) of the Nd2Se3 NPs were confirmed by TEM (Figure 4a) and the spherical formation of the nanocrystals may have been due to their dynamic nature. To further verify the crystallinity of Nd2Se3 nanoparticles, the diffraction patterns of X-ray was recorded from drop cast films of biogenic Nd2Se3 nanoparticles. The as-synthesized Nd2Se3 nanoparticles were found to reveal the crystalline nature owing to well-defined Bragg's reflections. The peak position and 2θ values agree with those reported for Nd2Se3 nanoparticles, almost all peaks in the pattern could be indexed to cubic phase cell parameters, a = b = c = 8.85, α = β = γ = 90° [JCPDF # 190823]. The given investigation has confirmed the first ever synthesis of Nd2Se3 nanoparticles under ambient conditions. The XRD pattern of the Nd2Se3 NPs showed intense peaks at (211), (220), (310), (321), (420), (422), (521) (611), (620), and (444) in the 2θ range of 20°–80° (Figure 4b) and agrees with those reported for the Nd2Se3 nanocrystals. The size of particles under XRD was found to be ~16 nm (Figure 4b). Further, the confirmation of encapsulation of proteins over the surface of particles was realized through FTIR. Therefore, the peak at 1635.56 cm−1 corresponds to characteristic of C=O of amide group of the amide I linkage confirmed the presence of protein. Further, the peak at 3320.62 cm−1 confirms the N-H stretching vibration. The C-N stretching of aliphatic amines associated with peptide bond was af-

**Figure 4.** (**a**) TEM, (**b**) XRD, (**c**) FTIR and (**d**) TGA/DTA analysis of biogenic Nd2Se3 NPs. **Figure 4.** (**a**) TEM, (**b**) XRD, (**c**) FTIR and (**d**) TGA/DTA analysis of biogenic Nd2Se<sup>3</sup> NPs.

The gravimetric analyses were performed to check the thermal stability of capping agents. TGA was performed on as-synthesized Nd2Se3 NPs in a nitrogen gas environment at temperatures ranging from 20 °C to 800 °C to calculate the amount of proteins present on the nanoparticles. As pointed out earlier, the as-synthesized nanoparticles are capped with proteins that stabilize them against aggregation. As a result, it degrades in two stages. Weight loss occurs up to 120 °C due to the evaporation of adsorbed water. In the second stage, loss is attributed to the decomposition of proteins bound on the surfaces of nanoparticles. The coating was found to contribute almost up to 18% which can be inferred via weight-loss when the particles were heated up to 600 °C. A further increase in the temperature shows a loss of weight that can be accounted for the decomposition of nanoparticles (Figure 4d). The gravimetric analyses were performed to check the thermal stability of capping agents. TGA was performed on as-synthesized Nd2Se<sup>3</sup> NPs in a nitrogen gas environment at temperatures ranging from 20 ◦C to 800 ◦C to calculate the amount of proteins present on the nanoparticles. As pointed out earlier, the as-synthesized nanoparticles are capped with proteins that stabilize them against aggregation. As a result, it degrades in two stages. Weight loss occurs up to 120 ◦C due to the evaporation of adsorbed water. In the second stage, loss is attributed to the decomposition of proteins bound on the surfaces of nanoparticles. The coating was found to contribute almost up to 18% which can be inferred via weight-loss when the particles were heated up to 600 ◦C. A further increase in the temperature shows a loss of weight that can be accounted for the decomposition of nanoparticles (Figure 4d).

The DTA study was also performed on the same particles. The endothermic weight loss was found to be around 110 ◦C which indicated the elimination of adsorbed water molecules on the surface of nanoparticles. Again, endothermic weight loss was found around 300 to 400 ◦C due to the decomposition of proteins (Figure 4d).

Additionally, DLS and zeta potential were performed to check the hydrodynamic radii along with different populations of particles and stability of nanoemulsion. The hydrodynamic radii of particles under DLS were found to be ~57 nm with PDI value of 0.440 (Figure 5A). Zeta potential was found to be −9.47mV (Figure 5B) which indicated the substantially high stability due to low value of Hamaker constant; TEM revealed only the size of the inorganic core, whereas DLS offered the hydrodynamic radii (inorganic core plus hydration layers). The suspension was found to be stable up to 3 months and there were no significant changes in DLS and Zeta potential.

no significant changes in DLS and Zeta potential.

**Figure 5.** (**A**) DLS and (**B**) zeta potential for Nd2Se3 NPs. **Figure 5.** (**A**) DLS and (**B**) zeta potential for Nd2Se<sup>3</sup> NPs.

#### *Possible Mechanism Possible Mechanism*

There are several methods to produce semiconductor NPs, but biological methods are rarely used. In the present study, a very simple and effective greener synthesis method for semiconductor NPs was developed. Nd2Se3 semiconductor NPs were synthesized by the instantaneous oxidation of a metal (from Nd2+ to Nd3+) and the reduction of nonmetal ions (Se4+ to Se2+) using purified nitrate reductase enzyme in the presence of the synthetic peptide (Glu-Cys)n-Gly (where n = 5–7), as a capping and stabilizing agent containing repetitive glutamate and cysteine amino acids. There are several methods to produce semiconductor NPs, but biological methods are rarely used. In the present study, a very simple and effective greener synthesis method for semiconductor NPs was developed. Nd2Se<sup>3</sup> semiconductor NPs were synthesized by the instantaneous oxidation of a metal (from Nd2+ to Nd3+) and the reduction of nonmetal ions (Se4+ to Se2+) using purified nitrate reductase enzyme in the presence of the synthetic peptide (Glu-Cys)n-Gly (where n = 5–7), as a capping and stabilizing agent containing repetitive glutamate and cysteine amino acids.

The DTA study was also performed on the same particles. The endothermic weight loss was found to be around 110 °C which indicated the elimination of adsorbed water molecules on the surface of nanoparticles. Again, endothermic weight loss was found

Additionally, DLS and zeta potential were performed to check the hydrodynamic radii along with different populations of particles and stability of nanoemulsion. The hydrodynamic radii of particles under DLS were found to be ~57 nm with PDI value of 0.440 (Figure 5A). Zeta potential was found to be −9.47mV (Figure 5B) which indicated the substantially high stability due to low value of Hamaker constant; TEM revealed only the size of the inorganic core, whereas DLS offered the hydrodynamic radii (inorganic core plus hydration layers). The suspension was found to be stable up to 3 months and there were

around 300 to 400 °C due to the decomposition of proteins (Figure 4d).

Nitrate reductase has a strong reducing property [30] due to its moderate reduction potential (+0.44 V), which participated in the redox reaction where oxidation of Nd+2 into Nd+3 took place and simultaneously reduction of selenium from Se+4 into Se+2 occurred with the synthesis of nano sized materials (Figure 1). During the synthesis, the synthetic peptide [(Glu-Cys)n-Gly, where n = 5–7] served as a stabilizing agent, which not only reduced steric hindrance and static-electronic repulsive forces between metal and nonmetal, but also served as a capping agent responsible for attaining the required sizes and shapes. Synthetic peptides comprising repetitive units of glutamate [31] and cysteine [32] amino acids were designed by virtue of their tendency to interact with inorganic NPs. The designed peptides stabilize semiconductor Nd2Se3 NPs and enable them to interact with Nitrate reductase has a strong reducing property [30] due to its moderate reduction potential (+0.44 V), which participated in the redox reaction where oxidation of Nd+2 into Nd+3 took place and simultaneously reduction of selenium from Se+4 into Se+2 occurred with the synthesis of nano sized materials (Figure 1). During the synthesis, the synthetic peptide [(Glu-Cys)n-Gly, where n = 5–7] served as a stabilizing agent, which not only reduced steric hindrance and static-electronic repulsive forces between metal and nonmetal, but also served as a capping agent responsible for attaining the required sizes and shapes. Synthetic peptides comprising repetitive units of glutamate [31] and cysteine [32] amino acids were designed by virtue of their tendency to interact with inorganic NPs. The designed peptides stabilize semiconductor Nd2Se<sup>3</sup> NPs and enable them to interact with various molecules due to the existence of different functional groups. The interaction of peptides and NPs depends upon the chemical properties of NPs, peptides, and reaction parameters and the dynamics of their interaction is responsible for the long-term stability of peptide-capped NPs. The electronic properties of NPs can be perfectly determined by UV-Vis spectroscopy, and their absorption peak and its width is directly correlated to the chemical composition and size of the particles. The energy of far-UV light is sufficient to excite the electrons of Nd2Se<sup>3</sup> NPs. Therefore, the size and concentration of the Nd2Se<sup>3</sup> NPs were determined by UV-Vis spectroscopy and were further confirmed by DLS (hydrodynamic radii) and TEM (inorganic core) [33].

The overall NP and peptide interaction is a multifunctional phenomenal process, and its properties are determined not only by the characteristics of the NPs, but also by the interacting peptides and the reaction parameters. Particularly, the rate of specific association and dissociation of the involved peptide will determine its longevity in the interaction with the NP surface. Moreover, the synthetic conditions determine the morphological nature and miscibility of a metal and a nonmetal in semiconductor NPs. Generally, semiconductor NPs comprising two dissimilar elements (metal and non-metal) has received greater attention than metallic NPs, both scientifically as well as technologically [34,35]. The blending of two different constituting elements can result in morphological changes in the semiconductor NPs, wherein an extra degree of freedom is developed [36]. However, the constituting elements (metal and nonmetal) and the size determine the behavior of the semiconductor NPs. Mostly, composition and size generally provide the tendency to optimize the energy of the plasmon absorption band of the metal and non-metal blend, which delivers a multipurpose tool for biological applications. Lastly, by means of semiconductor formation, the catalytic nature of the resultant NPs can be enhanced to a reasonable extent, which may not be achievable when employing its corresponding monometallic NPs.

The energy of the incident photon at a wavelength of 300 nm is 4.136 eV, which is responsible for the electronic transitions of Nd2Se<sup>3</sup> NPs. The spectrum obtained describes the chemical composition and particle size. It also confirms the synthesis of the NPs, and their difference from bulk counterparts.

The dependency of the optical nature on particle size is mostly an effect of the internal structure of the nanocrystals. However, as the crystals become smaller, the quantity of involved atoms on the surface increases, which greatly influences optical behaviors. If these surface energy states are for nanocrystal band gaps, they can trap charge carriers at the surface and decrease the overlap between the electron and the hole. The number of electrons excited in the conduction band (CB) is a function of the temperature and magnitude of the energy band gap (Eg), defined as the separation between the maximum energy in the valence band and the minimum energy in the CB. As our biogenic material Nd2Se<sup>3</sup> revealed, the optical intensity at 330 nm correlates with 3.75 eV (band gap), it can also be calculated by the following Equations (3) and (4).

$$\mathbf{E\_{g}} = 1240 / (\lambda \text{ (nm)}) \tag{3}$$

$$\mathbf{E\_{g}} = 1240/330 = \mathbf{3.75} \,\mathrm{eV} \tag{4}$$

The roles of nano-chalcogenide have been exploited in a variety of newer and emerging technologies. Their applications in broad categories are derived from their unique tunable chemical and physical properties, which give rise to their potential uses in the fields of biomedical, nonlinear optics, luminescence, electronics, catalysis, solar energy conversion, optoelectronics, among others. With decrease in size of nano-chalcogenides, the percent of surface atoms and the value of band gaps increase leading to the surface properties playing an important role in the properties of the materials (14). The transition metal nano-chalcogenides have specific applications. The group II–VI chalcogenide semiconductors have their role in optoelectronic light-emitting diodes and optical devices owing to their wide-bandgap. Additionally, V–VI main-group nano-chalcogenides due to their semiconductor nature have applications in television cameras with photoconducting targets, thermoelectric devices, and electronic and optoelectronic devices and in IR spectroscopy (16). The inner transition metal nano-chalogenides are very rare and their synthesis ought to be exploited in detail because of their non-toxic nature (9) which can be explored for their fluorescence properties toward biomedical applications.

#### **4. Conclusions**

The present study enabled the development of environmentally friendly biogenic methodology to produce Nd2Se<sup>3</sup> NPs utilizing biologically relevant molecules. Neodymium selenide NPs were synthesized using enzyme nitrate reductase. They were orthorhombic with a size distribution of 18 ± 1 nm (under TEM); the chemical formula was found to be Nd2Se<sup>3</sup> based on the crystal structure of the fabricated NPs. Our ability to fabricate these NPs via a non-toxic, eco-friendly method presents a significant advancement in developing "greener" technique to produce NPs. Lastly, this study offers an alternative approach for

the environmentally friendly, cost-efficient, and commercial fabrication of water dispersible Nd2Se<sup>3</sup> NPs. These Nd2Se<sup>3</sup> NPs have favorable bio-medical application potential without any additional requirements for further functionalization.

**Author Contributions:** R.S.V.: write-up, editing and revision; A.A.A. and A.S.: Conceptualization, investigation, revision; A.A.A. and A.S.: Methodology, Data curation, resources; A.M.E.: Software, validation, review and editing; A.H.B.: Methodology, Writing-original draft preparation; M.S.K.: Conceptualization, methodology, supervision, project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Researchers Supporting Project number (RSP-2021/ 56), King Saud University, Riyadh, Saudi Arabia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge Integral University and Department of Biosciences for providing facilities and support for the research. The authors extend their appreciation to the Researchers Supporting Project number (RSP-2021/ 56), King Saud University, Riyadh, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**

