Biosynthesis of AgNPs. Conditions of bio- reduction
Influence of the initial plant material
In the synthesis technologies of AgNPs based on biological processes, where the source of reducing compounds is of botanical origin, plants or parts of plants (e.g. leaves, flowers, seeds, bulbs, fruits) constitute the fundamental raw material. Therefore, in addition to the abundance of the compounds responsible for the reaction it is essential to know all the factors that influence the concentration and quality of these compounds. Drying processes are unitary operations that are part of the processing of plant material in order to guarantee the reproducibility of an industrially scalable process and the protection of this biological material from microbial contamination (bacteria and fungi). Although it is known that the composition of secondary metabolites in plants varies with the edaphoclimatic conditions (Pino et al. 2013), the influence of drying on the synthesis of AgNPs was evaluated in the present study.
The influence of the drying of the plant material on the synthesis of AgNPs was demonstrated, not only by the color change observed with respect to the organoleptic properties of the extracts obtained (Figure 1 C and G), but also by the differences in speed and yields of the bio-reduction reaction. In this sense, the reaction with the extract of fresh leaves started instantly (before 5 minutes), and the color drastically changed to dark brown (Figure 1 D), while the reaction with the dry leaves was after one hour (Figure 1H). Regarding the yields of the crude suspension, they were statistically superior in the synthesis with the fresh plant material p ˂ 0.05 (Figure 2).
Kinetics of the bio-reduction
The results of the kinetic study of the synthesis reaction of AgNPs at different times (t0, 5, 30, 60, 120, 180, 240, 360, 720, 1080, 1320, 1440, 2160, and 4320 minutes) showed different behavior depending on the starting plant material (fresh and dry) (Figure 3).
Monitoring of the UV-Vis spectra confirmed that the reaction with the fresh material started instantaneously (Figure 3 and 4 A), given by the occurrence of a sudden color change from light green to dark brown, and by the presence of a well-defined peak at λmax 440 nm. Likewise, the increase in absorbance over time was evident until 1320 minutes (end of the reaction), which indicated a concentration increase of AgNPs over time what points to compliance with the Beer-Lambert law. From this time on the absorbance remained constant.
However, monitoring of the UV-Vis spectra with the dry material showed the beginning of the reaction after 180 minutes (Figures 3 and 4 B), made evident by the presence of the characteristic peak with λmax of 440 nm. In this case, the increase in absorbance over time was lower with respect to fresh plant material, until 1320 minutes (end of the reaction in both cases), suggesting the degradation of the reducing compounds and the loss of their properties. These results point to the need to carry out stability studies of the plant raw material in order to establish the useful life time and the optimal storage conditions.
The results were confirmed by monitoring the variation of pH over time (Figure 5) and observing the decrease of this indicator from the beginning of the reaction in the case of fresh plant material. As the AgNPs are formed, the pH decreases due to the release of H+ by the formation of nitric acid, which slightly acidifies the medium. In the case of the dry material, no changes in pH were practically observed until 180 minutes, which corroborated that the reaction started at this time.
Numerous studies have shown the influence of pH on the morphology and stability of AgNPs. It is suggested that with increases in pH to basic values the concentration of AgNPs increases. Likewise, it was shown that the production of larger NPs occurs at acid pH, while the formation of smaller and slightly stable NPs is favored by basic pH (Singh et al. 2020; Oluwasogo et al. 2019; Nasar et al. 2019; Handayani et al. 2020), so that the basic pH is ideal for the formation of AgNPs (Nasar et al. 2019). Other authors showed the influence of pH on nanostructure shapes, and they reported shapes between spherical and rods at basic pH (pH = 11.1), while triangular plate nanostructures or polygonal particles were obtained at acidic pH (pH = 5) (Handayani et al. 2020).
The results of the study of the kinetics of the bio-reduction reaction when the differential method was applied revealed a complex phenomenology where different kinetic mechanisms were expected to occur due to the presence of dissimilar families of secondary metabolites. These metabolites could have reducing properties and the possible occurrence of concurrent or non-concurrent parallel reactions, self-catalysis, etc., were likely to occur (Figure 6 A and B). Further studies should clarify the possible reactions and end products.
The results obtained point to the technical feasibility of using fresh plant material for the synthesis of AgNPs with L. coccinea because of the higher yields and a faster bioreduction reaction. For this reason, subsequent studies were performed on AgNPs biosynthesized with fresh leaves.
Characterization of the AgNPs
The morphology of the AgNPs synthesized at different times was examined by SEM, and abundant NPs were observed to have spherical shapes and diameters less than 100 nm at all times (6, 12, and 24 hours) of the synthesis (Figure 7A-D). The magnification of the images showed average diameters of 70-90 nm. Many authors agree on the strong agglomeration due to the electrostatic interactions between metal ions (Chen et al. 2016).
Determination of particle size
The particle size determinations by the DLS method for AgNPs synthesized with fresh plant material showed diameters less than 100 nm (Figure 8), and the high agglomeration that the nanostructures showed in the aqueous medium (-21.30 mV ) (Figure 8 B) ), which corroborated the results obtained by SEM.
Analysis by EDX
The results of the qualitative analysis using EDX (Oxford Instrument) at different times (6, 12 and 24 hours) showed a high percentage of Ag from the first six hours of the reaction (n=3), made evident by the strong signals in the range of 2 -4 keV and the presence of carbon and oxygen indicative of hybrid NPs, with an organic component (e.g., flavonoid) covalently bound to the metallic element (Figure 9). Likewise, the high compaction demonstrated its crystalline nature (Singh et al. 2019; Kasithevar et al. 2017; Oluwasogo et al 2019; Moodley et al. 2018) due to the reduction of Ag cations by the reducing compounds contained in the aqueous extract of L. coccinea leaves.
Influence of surfactant-stabilizing agents
MNPs are characterized by the easy conversion of monomeric and dimeric forms, to more stable aggregate forms in aqueous medium. However, numerous studies have shown that this natural tendency of AgNPs to form aggregates and agglomerates affects the biological activity (Bélteky et al. 2019). In this sense, the use of surfactants and stabilizers are studied with the aim of achieving the greatest stability of the nanostructures in the environment. The results of evaluating different surface active agents in the stabilization of the suspension of AgNPs, by determining the particle size (Figure 10) showed that all the evaluated surface active agents kept this indicator less than 100 nm, except Mt with which an increasing trend from 89.62 to 122.4 nm was observed. Similarly, although there were no significant differences, an decreasing trend from 102.10 to 81.61 nm was observed over time with SDS.
On the other hand, the results by determining the Z potential (Malvern zetasizer) (Figure 11) showed that, excepting Tw 80, all the substances improved the stabilization of the nanostructures with respect to the aqueous medium, being SDS the best. A colloidal suspension is considered stable when the Z potential is outside the range ± 30 mV because the surface charge prevents the particles from aggregating (Duval et al.2019; Salvioni et. 2017). These results agree with those obtained by other studies on AgNPs synthesis with plants reporting similar Z potentials (Roy and Anantharaman 2017; Mohanta et al. 2017).
The Z potential provides the electrokinetic potential in colloidal systems because its value can be related to the stability of colloidal dispersions and indicates the degree of repulsion between adjacent charged particles in a dispersion (Salvioni et. 2014). Because of the agglomeration tendency of AgNPs, due to the electromagnetic forces between them, destabilization and sedimentation are frequent, depending on the size of the aggregates, and affect the biological activity (Bélteky et al. 2019). For this reason, numerous studies are carried out to design stabilized systems based on AgNPs; however, their results not always are in agreement (Bae et al. 2011; Chen et al. 2016).