Bimetallic nanoparticle production using Cannabis sativa and Vitis vinifera waste extracts

Preparation of plant extract

The materials utilized for biosynthesis consisted of dried Vitis vinifera canes which were collected during dormancy period and waste material after Cannabis sativa cannabinoid extraction, both located from sources in the Czech Republic. Additional materials included silver nitrate and tetrachlorauric acid obtained from Sigma Aldrich, ethanol (40%), and distilled water, ultrapure water.

Nanoparticle characterization

Using extracts from waste of Vitis vinifera and Cannabis sativa, bimetallic nanoparticles were successfully prepared, with their formation monitored initially by the change in visible light absorbance, followed by analytical confirmation by UV-vis spectrophotometry. In the spectra, absorbances at wavelengths of 550 nm (Vitis vinifera) and 520 nm (C. sativa) are evident, which are caused by the plasmon resonance of the formed nanoparticles (see Fig. 2). In the study by Abbasi et al. (2018), the synthesis of AgAuNPs using an extract from Cannabis sativa leaves was investigated, with the absorption maximum observed at a wavelength of 538 nm.¹⁸ Another study synthesized silver, gold, and bimetallic nanoparticles using an extract from Dovyalis caffra fruits. Absorption peaks were found at wavelengths of 430 nm (silver), 532 nm (gold), and 540 nm (bimetallic).¹⁹ The synthesis of AgAuNPs was also achieved using an extract from Stigmaphyllon ovatum leaves, and a time-dependent experiment showed a shift in the absorption maximum from 534 nm after 30 minutes to 542 nm after 45 minutes.²⁰ Regarding the structure of bimetallic nanoparticles, literature suggests that the presence of a single absorption peak indicates an alloy nature.²¹ The absorbance of 550 nm (V. vinifera) and 520 nm (C. sativa) observed in our work therefore corresponds to the range in which gold–silver alloy nanoparticles are found. The difference between nanoparticles synthesized by respective plant extract can be attributed to the difference in their size and shape profile (as demonstrated by TEM analysis, see below) which is a direct result of differences in composition of extracts from various plants.

Fig. 2

UV-vis absorption spectra of AgAuNPs prepared using Vitis vinifera (A) or Cannabis sativa (B) waste extract; ■ – AgAuNPs,  – plant extract. The data show a distinctive peak at wavelengths characteristic for bimetallic alloy nanoparticles between 500 and 550 nm.

The metal concentrations in the synthesized dispersion of AgAuNPs prepared using Vitis vinifera extract were determined by AAS to be 215.6 mg L⁻¹ of silver and 393.3 mg L⁻¹ of gold, while the concentrations of the synthesized dispersion of AgAuNPs prepared using Cannabis sativa extract were determined by AAS to be 106.4 mg L⁻¹ of silver and 179.0 mg L⁻¹ of gold. Specific descriptions of methods for determining the concentration of metal nanoparticles are relatively scarce in the literature. A large portion of experimental studies does not address the determination of nanoparticle concentration at all, and the input concentration of the precursor metal is often used as the concentration for subsequent experiments (e.g., antimicrobial activity tests). This assumes 100% conversion of metal ions without knowledge of the synthesis process mechanism or the actual yield of nanoparticle production.²² However, there are some studies that focus on determining the actual concentration of nanoparticle dispersions. In a study investigating the production of gold and silver nanoparticles using an extract from Cannabis sativa, the concentration was determined using inductively coupled plasma mass spectrometry (ICP-MS). Through analysis of individual particles, it was found that the number of nanoparticles per milliliter ranged from 26 to 83 billion. When converted to mass concentration, the concentrations ranged from 0.7 to 4.5 mg mL⁻¹.²³ In a study on the synthesis of copper nanoparticles using an extract from Magnolia kobus leaves, a linear relationship was found between the nanoparticle concentration measured by ICP-MS and the size of the absorption peak caused by surface plasmon resonance.²⁴ A special subtype of ICP-MS (single particle ICP-MS or spICP-MS) is an emerging methodological trend, where concentration, composition and size of nanoparticles can be analysed on single particle-level and then statistically evaluated.^25–27

The basic morphology of AgAuNPs created using Vitis vinifera extract was explored using transmission electron microscopy, revealing high heterogeneity with nanoparticles exhibiting spherical, rod-like, triangular, polygonal, and ellipsoidal shapes. Similarly, the basic morphology of AgAuNPs, created using Cannabis sativa extract was investigated, showing predominantly spherical nanoparticles with occasional polygonal or triangular shapes (see Fig. 3). TEM photographs were utilized for image analysis, resulting in size histograms of the produced nanoparticles. For nanoparticles created using Vitis vinifera extract, the particle size ranged from 6 to 94 nanometers, with the highest frequency (42%) observed between 14 and 23 nm. Similarly, for nanoparticles created using Cannabis sativa extract, the majority (63%) of the produced nanoparticles fell within the size range of 1–7 nm, while the largest synthesized nanoparticles had an average diameter of 26–29 nm (1%) (see Fig. 4). In terms of size, a study utilizing an extract from Triticum aestivum observed nanoparticles with diameters ranging from 5 to 40 nm. In other studies using fungal xylanase or an extract from Lawsonia inermis seeds, nanoparticles with sizes ranging from 10 to 52 nm were observed.^28–30 Therefore, the AgAuNPs (V. vinifera) in our work showed the highest variability in size when compared with literature, while AgAuNPs (C. sativa) synthesized in our work displayed unusually small size which, according to literature, may lead to higher antimicrobial properties.

X-ray photoelectron spectroscopy (XPS) of bimetallic nanoparticles produced using Vitis vinifera extract revealed the presence of carbon, oxygen, silver, and gold on their surface layers, with relative compositions of 35.7%, 46.9%, 4.9%, and 12.5%, respectively, and 33.3%, 29.1%, 23.5%, and 14.1%, respectively for bimetallic nanoparticles produced using Cannabis sativa extract. In the case of bimetallic nanoparticles, a study utilizing an extract from Diopyros kaki leaves confirmed the presence of gold and silver through peaks at 83.9 and 87.6; 367.7 and 373.7 eV, respectively, in the X-ray photoelectron spectroscopy spectra.^31,32 In a study where an extract from Antigonon leptopus leaves was used for the synthesis of bimetallic nanoparticles, in addition to gold and silver, carbon, oxygen, and nitrogen were also identified through peaks at 284.8; 532.7; and 399.9 eV, respectively.³³ Consistent with the literature, this work identified the presence of gold, silver, carbon, and oxygen through peaks at 83.8; 373.8; 284.6; and 532.6 eV for the Vitis vinifera extract, and 84.4; 368.0; 285.2; and 532.4 eV for the Cannabis sativa extract.

X-ray diffractograms of bimetallic nanoparticles synthesized using Cannabis sativa extract exhibited diffraction peaks at 2θ = 38.3°, 44.5°, 64.7°, and 77.7°, corresponding to the indexing of (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of metallic gold. Additionally, diffraction peaks at 2θ = 38.2°, 44.3°, 64.6°, and 77.4° can be indexed to (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes of metallic silver, while peaks at 2θ = 27.9°, 32.3°, 46.3°, 54.9°, 57.5°, 67.6°, and 76.8° can be attributed to (1 1 1), (2 0 0), (2 2 0), (3 3 1), (2 2 2), (4 0 0), and (4 2 0) planes of silver chloride (see Fig. 5). The X-ray diffraction data of bimetallic nanoparticles synthesized using Vitis vinifera extract display diffraction peaks at 2θ = 38.3°, 44.5°, 64.7°, and 77.7°, corresponding to the indexing of (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of metallic gold. Additionally, diffraction peaks at 2θ = 38.2°, 44.3°, 64.6°, and 77.4° can be indexed to (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes of metallic silver, while peaks at 2θ = 27.9°, 32.3°, 46.3°, 54.9°, 57.5°, 67.6°, and 76.8° can be attributed to (1 1 1), (2 0 0), (2 2 0), (3 3 1), (2 2 2), (4 0 0), and (4 2 0) planes of silver chloride (see Fig. 5). The crystallographic analysis of bimetallic nanoparticles synthesized using an extract from Dovyalis caffra identified peaks at 2θ values of 38.1, 44.3, 64.5, and 77.5° corresponding to the planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1) of the face-centered cubic lattice of gold. Additionally, peaks at 2θ values of 38.1, 64.4, and 76.5° were observed, corresponding to the planes (1 1 1), (2 2 0), and (3 1 1) of the face-centered cubic lattice of silver. Furthermore, peaks at 2θ values of 32.0 and 46.0° were observed, corresponding to the planes (2 0 0) and (2 2 0) of silver chloride. Similar results, except for the presence of silver chloride, were obtained in studies investigating the preparation of bimetallic nanoparticles using extracts from Moringa oleifera and Piper betle.^19,34,35 The X-ray diffractograms of bimetallic nanoparticles synthesized using Cannabis sativa, Vitis vinifera, and Dovyalis caffra extracts show similar diffraction peaks corresponding to the planes of metallic gold and silver. However, the nanoparticles synthesized using Dovyalis caffra extract exhibited a unique pattern with the absence of certain peaks corresponding to silver chloride, a characteristic also observed in nanoparticles synthesized using Moringa oleifera and Piper betle extracts. This suggests a potential influence of the extract type on the formation and composition of the bimetallic nanoparticles. Both AgAuNPs in this study exhibit characteristics indicative of a solid-solute type structure, as opposed to being intermetallic. This structural formation can be hypothesized to be influenced by the role of strong d-orbital interactions and anisotropic ordering in bimetallic nanoparticle synthesis. Additionally, the thermal and kinetic conditions during synthesis, such as temperature and reduction rates, significantly influence the alloying process and final structure. Our experimental conditions, particularly the unique properties of the plant extracts used, may have favored the formation of a solid solute structure by altering these synthesis dynamics.^36–38

Fig. 5

XRD spectrum of bimetallic nanoparticles synthesized using Vitis vinifera (A) or Cannabis sativa (B) waste extract. The x-axis represents the diffraction angles (2θ), while the y-axis denotes the intensity of the diffracted X-rays. The diffractograms confirmed the presence of metallic  silver,  gold and  chlorargyrite in both tested samples.

Fourier-transform infrared spectroscopy (FTIR) was used to identify functional groups present on the surface of bimetallic nanoparticles synthesized using Vitis vinifera extract (see Fig. 6). The broad absorption band at 3373 cm⁻¹ indicates the presence of O–H groups, aromatic compounds, or CC double bonds. The combined valence vibrations at 1609 and 1107 cm⁻¹ confirm the presence of these CC groups. Additional absorption bands at 1609 and 1512 cm⁻¹ confirm the presence of proteins. The width of the right shoulder of the absorption band at 3373 cm⁻¹, along with valence vibrations at 1609 and 1512 cm⁻¹, suggests the presence of aromatic compounds. Deformation vibrations at 820 cm⁻¹ indicate substitution of these aromatic compounds in the para position. Deformation vibrations at 1445 and 1107 cm⁻¹ are associated with the presence of C–H and C–O groups, specifically indicating the presence of alcohols, ethers, carboxylic groups, or esters. For AgAuNPs synthesized using Cannabis sativa extract (see Fig. 6), the absorption band at 3306 cm⁻¹ suggests the presence of O–H bonds. The combination with valence vibrations at 1640 cm⁻¹ confirms the presence of CC bonds, and the absorption at 1550 and 1050 cm⁻¹ indicates the valence vibration of C–O bonds. Valence vibrations of C–H bonds were observed at 2925 and 2855 cm⁻¹, along with deformation vibrations at 1450 and 1374 cm⁻¹. Absorption bands at 3306 and 2855 cm⁻¹ confirm valence vibrations of primary and secondary amines, while bands at 3306 and 2925 cm⁻¹ confirm valence vibrations of hydroxyl groups. Valence vibrations at 1640 cm⁻¹ indicate the presence of carbonyl groups. The broad absorption band at 1050 cm⁻¹ may signify valence vibrations of C–N bonds in aliphatic amines.

In studies investigating the dispersion of silver or gold nanoparticles synthesized using Cannabis sativa extract, absorption bands were detected at 3332 cm⁻¹ indicating the presence of O–H groups, valence vibration bands at 1741 cm⁻¹ or 1604 cm⁻¹ indicating the presence of CC bond, and an increased absorption band at 1019 cm⁻¹ indicating the valence vibration of the C–O bond. In addition, clear absorption bands of valence vibrations of carbonyl groups, which may belong to proteins, were observed at 1635 cm⁻¹. Valence vibrations of C–N groups of amines were also observed at 1045 cm⁻¹. In this study, the presence of O–H groups was confirmed with an absorption band at 3305 cm⁻¹, and the presence of CC bond was indicated by valence vibration band at 1641 cm⁻¹. Other identified groups included C–O bonds with absorption bands at 1536 cm⁻¹ and 1050 cm⁻¹, and a broader absorption band of aliphatic amines at 1050 cm⁻¹. For silver nanoparticles and bimetallic nanoparticles synthesized in this study using Cannabis sativa extract, the presence of valence vibration of the C–Cl bond with absorption at 700 cm⁻¹ was detected. Similar results were obtained in a study examining the synthesis of silver nanoparticles using Cannabis sativa, where peaks at 769 cm⁻¹, 687 cm⁻¹, and 526 cm⁻¹ were attributed to these vibrations. The reducing and stabilizing properties were attributed to reducing sugars or proteins, while the purely reducing activity likely resulted from terpenoids and flavonoids.^23,39

In the case of nanoparticles synthesized from Vitis vinifera extract, wide absorption bands were identified in the literature at 3421–3469 cm⁻¹, indicating the presence of O–H groups. In combination with the valence vibration at 1650 cm⁻¹ characteristic of CC bonds in the aromatic ring, the participation of phenolic compounds in nanoparticle synthesis can be considered. Additionally, a deformation vibration at 820 cm⁻¹, suggesting para-substitution, was identified in this study. Absorption bands in the range of 1315 cm⁻¹ and 1730 cm⁻¹ are characteristic of valence vibrations of CO and C–O groups, indicating the presence of carboxylic acids (e.g., gallic or apple acids). The involvement of proteins was confirmed in the literature through the identification of absorption peaks indicating the presence of amides at 1655 cm⁻¹, combined with the aforementioned CO groups.^40–42 In this study, the presence of O–H groups was confirmed by a wide absorption band at 3326 cm⁻¹, which also provides an indication of the occurrence of aromatic compounds. These findings were further supported by the identification of valence vibrations of CC groups at 1605 cm⁻¹ and 1108 cm⁻¹. Our results also confirmed the participation of proteins in nanoparticle synthesis, as additional peaks were observed at 1605 cm⁻¹ and 1514 cm⁻¹. Deformation vibrations at 1446 cm⁻¹ and 1108 cm⁻¹, associated with the presence of C–H and C–O groups, were also observed, indicating the involvement of primary and secondary alcohols.