Biosynthesis of silver nanoparticles using lavender leaf and their applications for catalytic, sensing, and antioxidant activities

2.1 Materials

Lavender leaves were collected from the local garden of Universidad de las Fuerzas Armadas ESPE, Sangolqui, Ecuador. Silver nitrate (AgNO₃, 99.8%), 4-nitrophenol (>99.5%), and H₂O₂ (35%) were purchased from Spectrum, USA. Sodium borohydride (NaBH₄, >99.5%) and DPPH (>99.5%) were purchased from Sigma Aldrich, St. Louis, MO, USA. All solutions were prepared in ultrapure Milli Q water. All chemicals were reagent grade and used without further purification.

2.2 Preparation of the extract

Thoroughly washed lavender leaves (2 g) were chopped and ultrasonicated in 25 ml of water for 3 min. After ultrasonication, the yellowish lavender leaf extract (LLE) was filtered using Whatman paper no. 1 and stored at 4°C for further use. Ultrasonication was performed with ultrasonic processors (DAIGGER GE 505, 500 W, 20 kHz) immersed directly into the reaction solution. The operating condition was at 30-s pulse on/30-s pulse off time with amplitude of 72% at 25°C for 3 min.

2.3 Biosynthesis of AgNPs

For the biosynthesis of AgNPs, 2.0 ml of LLE was completely dissolved in 20 ml of 1.0 mm AgNO₃ solution by vigorous stirring, and heated at 60–65°C for 30 min in the presence of visible light (55–60 cd/m²). Reduction of Ag⁺ to AgNPs occurs by the appearance of a wine red color of the reaction mixture after 3 h of incubation period, and as-synthesized AgNPs were characterized further using different analytical instruments.

2.4 Catalytic activity of AgNPs for the reduction of 4-NP to 4-AP

To test the catalytic efficacy of AgNPs, reduction of 4-NP was carried out as a model reaction and monitored as follows: about 0.2 ml of 4-NP (2 mm), 0.2 ml of as-synthesized AgNPs, and 3.4 ml of H₂O were mixed in a 4-ml standard quartz cuvette. To this reaction mixture, 0.2 ml of NaBH₄ (0.05 mm) solution was added and UV-vis absorption spectra were recorded at 1 min intervals, leading to a color change from pale yellow to colorless. The observed percentage of the reduction process was determined by analyzing the change in the intensity of the peaks at 400 nm as a function of time.

2.5 Optical sensing activity of AgNPs for H₂O₂

The optical sensing activity of AgNPs to detect H₂O₂ was assessed by the method adapted from Raja et al. [27], with slight modifications. In this assay, 0.5 ml of 100 mm H₂O₂ was added to 2.5 ml of diluted AgNP (5%) solution, and thoroughly mixed. The spectral absorbance was measured at regular intervals after the addition of H₂O₂.

2.6 Antioxidant activity of LLE and AgNPs

The antioxidant activity of the LLE and AgNPs was measured by using the DPPH assay adapted from Kumar et al. [9], [10], with slight modifications. The LLE/AgNPs (1000–200 μl) or control and 1000–1800 μl of H₂O were mixed with 2.0 ml of 0.2 mm DPPH in 95% ethanol. The mixture was vortexed vigorously and allowed to reach a steady state in dark incubation at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. The lower absorbance of the reaction mixture indicated a higher percentage of scavenging activity, and the percent inhibition was calculated by using the following equation:

where A₀ is the absorbance of the control (blank, without extract or AgNPs) and A₁ is the absorbance in the presence of the LLE or AgNPs. The final result was expressed as percentage of DPPH free radical scavenging activity (ml).

2.7 Characterization of AgNPs

The synthesized AgNPs were primarily characterized with the help of a UV-vis single beam spectrophotometer (GENESYS 8; Thermo Spectronic, England). TEM and selected area electron diffraction (SAED) measurements were recorded digitally (Tecnai G2 Spirit TWIN, FEI, Holland). The samples for TEM were prepared by depositing a drop of colloidal solution on a carbon-coated copper grid and drying at room temperature. The hydrodynamic size distributions and polydispersity index (PDI) of nanoparticles were analyzed by using DLS instrumentation (LB-550; Horiba, Japan). XRD studies on thin films of the nanoparticle was carried out to identify the phase and purity, and to determine the crystallite size using a diffractometer (EMPYREAN, PANalytical) and a θ-2θ configuration (generator-detector), wherein a copper X-ray tube emitted a wavelength of λ=1.54059 Å. The intensity data for the nanoparticle solution deposited on a glass slide were collected over a 2θ range of 5–100°. The catalytic, sensing, and antioxidant activities of AgNPs were evaluated with the help of a UV-vis spectrophotometer (SPECROD S600; Analytika Jena, Germany).

3.1 Visual and UV-vis study

The biosynthesis of AgNPs in the colloidal solution with LLE was monitored periodically at 3, 24, 48, and 96 h by absorbance measurement using UV-vis spectroscopy and visual inspection at 96 h (Figure 1). It was observed that, in comparison with the colorless AgNO₃ and the pale yellow LLE, the color of the colloidal solution changed to light yellow and then wine red. It is a direct proof for the reduction of Ag⁺ to Ag⁰ and the formation of AgNPs. The UV-vis spectra indicated the appearance of a single surface plasmon resonance (SPR) band for the AgNPs at λ_max=440 nm, which is due to the collective oscillation of the electrons in the conduction band of AgNPs [28], [29]. Also, the single SPR band for AgNPs increased with time and shifted from 440 to 460 nm. This confirms that the AgNPs have a spherical form and that the size increases with time (shift of monodisperse to polydisperse particles) [9]. Therefore, UV-vis spectra were applied to characterize the biosynthesis of AgNPs. The difference in the standard reduction potential between Ag⁺ (E^o_red=+0.80 V) and flavonoids/polyphenolic compounds present in the leaf extract (E^o_red=~+0.3–0.8) [30] was believed to be responsible for the reduction of Ag⁺ to Ag⁰ and the subsequent formation of AgNPs [Eq. (2)]. During the formation of Ag⁰ in an aqueous reaction mixture, biosynthesis of Ag₂O was also suggested. It may be due to the presence of molecular oxygen (O₂) in the reaction medium, which oxidizes Ag⁰ to Ag⁺ and leads to the formation of Ag₂O NPs [Eq. (3)].

Figure 1:

UV-visible absorption spectra of as-synthesized AgNPs using LLE at different incubation times at room temperature. (Inset) Visual picture of (A) 1 mm AgNO₃, (B) LLE, and (C) as-synthesized AgNPs after 96-h incubation at room temperature.

3.2 TEM and SAED studies

The morphology, size, and crystallinity of as-synthesized AgNPs were investigated by TEM and SAED measurements. In Figure 2A,B, the TEM images indicate the formation of spherical AgNPs of diameter in the range of 10–80 nm, and that AgNPs are surrounded by LLE. The AgNPs are well separated from each other and show the absence of aggregation. This may be due to the association of LLE biomolecules on the surface of the AgNPs, and makes the surface repulsive. The observed circular white dot-like fringe patterns in SAED (Figure 2C) suggest the formation of spherical and crystalline AgNPs [31].

Figure 2:

(A, B) TEM pictures and (C) SAED pattern of AgNPs.

3.3 DLS study

In Figure 3, the AgNPs synthesized with LLE are in the range of 20–120 nm, and the average particle size was found to be 56.9±23.8 nm. The PDI of the AgNPs was 0.174, indicating a broad size distribution. The size of AgNPs observed in DLS was slightly bigger than the result obtained from TEM analysis. This may be due to the screening of smaller molecules by a bigger one and functionalization of the AgNP surface either by nitrate ions or by LLE biomolecules [14].

Figure 3:

DLS size distribution histogram of as-synthesized AgNPs using LLE.

3.4 XRD study

It is vital to understand the exact crystal structure of the AgNPs formed, and this can be achieved by evaluating the XRD spectra of the samples. The crystalline property of AgNPs was confirmed by XRD analysis at 2θ values ranging from 5° to 100° (Figure 4). The Bragg reflection peak at 38.038° corresponds to the (111) lattice planes of face-centered cubic metallic Ag⁰, which is in agreement with ICSD no. 98-018-0878. Besides the metallic Ag, another reflection peak at 32.22° corresponds to the (111) lattice planes of Ag₂O, which matched well with ICSD no. 98-003-5662. Both peaks corresponding to (111) were broad and had higher intensity than other lattice planes, suggesting the nanocrystalline nature and predominant growth of AgNPs along the (111) direction. The XRD pattern reveals that the prepared AgNPs are a mixture of metallic Ag⁰ and Ag₂O.

Figure 4:

XRD pattern of AgNPs.

3.5 Catalytic activity of AgNPs

To test the catalytic activity of the biosynthesized AgNPs, we studied the reduction of 4-NP to 4-AP using aqueous NaBH₄ by UV-vis spectroscopy. As shown in Figure 5, 4-NP shows an absorbance peak at 317 nm and 4-nitrophenolate ion in the alkaline medium (after addition of NaBH₄) appeared at 400 nm. The addition of NaBH₄ and AgNPs to the aqueous 4-NP solution produced a rapid decrease in their absorption spectra within 10 min at 400 nm with the concomitant appearance of a new peak at 298 nm indicating the formation of 4-AP. Though the reduction of 4-NP to 4-AP using aqueous NaBH₄ is thermodynamically favorable [E₀ for 4-NP/4-AP=−0.76 V and H₃BO₃/BH₄⁻=−1.33 V vs. normal hydrogen electrode (NHE)], the presence of the kinetic barrier due to the large potential difference between donor and acceptor molecules decreased the feasibility of this reaction. It is well known that the metal nanoparticles catalyze this reaction by facilitating electron relay from the donor BH₄⁻ to an acceptor 4-NP to overcome the kinetic barrier. The conversion from 4-NP to 4-AP occurs via an intermediate 4-nitrophenolate ion formation, and NaBH₄ and AgNPs act as reducing agent and catalyst, respectively [32], [33].

Figure 5:

Catalytic activity of AgNPs for the reduction of 4-NP to 4-AP.

3.6 Optical sensing of H₂O₂

H₂O₂ is an oxygen metabolite that induces oxidative stress and associated with aging and cancer. Moreover, H₂O₂ is widely used as a strong oxidizing agent in the food, pharmaceutical, cosmetics, wood, and pulp industries. However, the exposure to and the presence of even a small amount of H₂O₂ in process streams result in various health and environmental hazards due to its toxicity [26], [34]. The quantitative sensing of H₂O₂ has received enormous interest in the development of biosensors. Figure 6 displays the optical sensing spectra of AgNPs in the presence of H₂O₂. It shows a decreasing trend in absorbance as the time increased, and eventually the characteristic SPR peak of AgNPs at 434 nm disappeared. The maximum quenching of H₂O₂ was found to be 95% for 30 min. It is due to the spontaneous redox reaction of AgNPs with H₂O₂, and H₂O₂ can efficiently oxidize AgNPs in both acidic and basic media. The redox potential of the H₂O₂/H₂O couple (1.763 V in acidic medium) or H₂O₂/OH⁻ couple (0.867 V in basic medium) is higher than that of the Ag⁺/Ag⁰ couple (0.8 V) [35]. Thus, as-synthesized AgNP-based optical sensors for H₂O₂ could be potentially applied in the determination of reactive oxygen species and toxic chemicals.

Figure 6:

H₂O₂ optical sensing activity of as-synthesized AgNPs.

3.7 Antioxidant activity

Polyphenols and their modified nanoparticles have powerful antioxidant activity and are capable of scavenging a wide range of free radicals [7], [25]. The evaluation of the antioxidant activity of LLE and AgNPs by the DPPH method is shown in Figure 7. It could be noted that the DPPH scavenging activity for LLE and AgNPs increases in a dose-dependent manner. At lower concentrations, the scavenging activity of AgNPs (5.33%, 0.1 ml; 12.91%, 0.2 ml; 29.5%, 0.4 ml; 35.18%, 0.6 ml; and 34.95%, 0.8 ml) was higher than that of LLE (1.83%, 0.1 ml; 4.73%, 0.2 ml; 7.70%, 0.4 ml; 14.09%, 0.6 ml; and 22.27%, 0.8 ml), whereas, at 1 ml concentration, it is 27.48% and 34.28% for AgNPs and LLE, respectively. It is due to the synergistic result of an active physicochemical interaction of Ag atoms with the functional groups of the LLE and, at higher concentration, less solubility of AgNPs [14], [19].

Figure 7:

Antioxidant activity of (A) LLE and (B) AgNPs after 96 h.