Instant green synthesis: obtaining stable nanoparticles and understanding the extract's behavior in the particle formation mechanism

Numerous plant extracts are abundant in biomolecules that can be employed in the biogenic synthesis of metallic nanoparticles owing to their potent reducing capabilities. The mechanism by which biomolecules act as reducers and expedite the reduction of silver ions remains poorly understood. This study presents an instantaneous and environmentally friendly synthesis of silver nanoparticles (AgNPs) using varying concentrations of commercially available green tea and concentrations of a dextrose-reducing solution. The AgNPs formed instantaneously, likely due to the competitive reaction between the polyphenols present in green tea and the dextrose. The best AgNPs produced using a diluted green tea solution at a concentration of 0.05 g of tea/ml and 100 μl of dextrose solution exhibited high stability over a period of 90 days, as confirmed by UV–vis spectroscopy and dynamic light scattering. The results of antioxidant properties from diluited tea showed 2,2-diphenyl-1-picrylhydrazyl (DPPH) 0.013 ± (0.1) μmol Trolox Equivalent Anyioxidant Capacity (TEAC) TEAC/g, Ferric Reducing Antioxidant Power (FRAP) 10.3 ± (0.1) μmol TEAC/g and Total Polyphenol Content (TPC) 0.12 ± (.001) μgGAE(Galic Acid Equivalent)/g). The resulting nanoparticles are extremely small, measuring approximately 30 to 50 nm in size, and exhibit a spherical morphology as evidenced by SEM imaging. The plasmon bandwidth is better in more diluted tea and higher proportions of dextrose added than the others condition of synthesis. Probably, the results of 2nd extraction of green tea diluted can be evidence that phenolic compounds, mainly, caffeine and gallic acid, are contributing to forming and stabilizing the silver nanoparticles. This fundamental knowledge showed the method employed is ecologically sound and adheres to green principles.


Introduction
The field of nanoscience has witnessed substantial growth over the last two decades.Scientists consistently investigate how subtle structural changes at the nanoscale can impact the chemical, physical, and mechanical properties of materials.Nanometric particles present intriguing possibilities for research across various domains, including pharmaceutical sciences, healthcare, cosmetics, food, environmental science, mechanics, optics, chemical industries, electronics, space industries, drug-gene delivery, and energy science [1][2][3][4][5].For the synthesis of metallic nanoparticles, such as silver and gold, various methods are employed, including physical, chemical, biological, and green routes [6,7].Recently, green synthesis has gained favor due to it is cost-effective and environmentally friendliness.The utilization of Camellia sinensis (green tea) for the production of silver nanoparticles using is not a new concept; Vilchis-Nestor et al [8] used C. sinensis extract as a reducing and stabilizing agent for the biosynthesis of silver and gold metallic nanoparticles.The author synthesized silver nanoparticles using a basic medium of reaction.The particles obtained were after 4 h of reaction and the author claim that 'was the fastest bio-reducing methods to produce silver nanostructures reported so far' [8,9].In another study, Loo et al [10] synthesized silver nanoparticles using tea leaves from C. sinensis that underwent physical processing for 6 months, forming pu-erh tea.The particles were obtained with an average size of 4.05 nm at room temperature, although the preparation of the leaf for extracting the material is laborious.Recently, Salih [11] used C. sinensis leaves for silver nanoparticles in the synthesis; however, the extraction did not adhere to green chemistry principles, as methanol solvent was employed for obtaining the extracts and the medium for nanoparticle growth.Phenolic acid-type biomolecules (e.g., caffeine and gallic acid) found in C. sinensis and black tea extracts appear to be responsible for the formation and stabilization of silver nanoparticles [9].Green tea has potential applications in cancer prevention by disrupting angiogenesis and blood flow to the tumor enhancing the growth of normal cells and promoting programmed cell death.Catechin (a polyphenol), caffeine, and epigallocatechin-3-gallate (a flavonoid) are the most abundant compounds in green tea, playing crucial roles in its anticancer and antioxidant effects, respectively [9].Hermanto et al [12] obtained silver nanoparticle using C sinensis leaf extract in electrochemical approach.The authors show a method eco-friendly to synthesized spherical Ag nanoparticles using plants extract.The synthesis requires an electrochemical apparatus that can make difficult to obtain the particles.The overarching goal of this study was to synthesize silver nanoparticles (AgNPs) through a green and instantaneous methodology, optimizing the synthesis by varying the concentration of green tea and the alkaline dextrose solution.This study represents a significant advancement by demonstrating, for the first time, the instantaneous production of stable silver nanoparticles using commercial tea bag residues, without the need for changes in synthesis conditions such as temperature and pH.This innovative approach offers a promising solution for efficient synthesis of silver nanoparticles, leveraging accessible and environmentally friendly materials.The ability to maintain unchanged synthesis conditions streamlines the process and enhances the commercial viability of this technique, opening up new prospects for a wide range of applications in science and technology.

Green tea infusion
The infusion was prepared by adding a sachet of green tea (1.5 g) commercial (Dr Otker Brazil ® ) with distilled water at 98 °C, maintaining the temperature for 2 min.Two concentrations were tested for the preparation of green tea infusion: 0.1 g of green tea/ml and 0.05 g of green tea/ml.Another factor evaluated was the effect of the number of infusion on the same sachet (first and second infusion).For use in synthesis, the infusion was cooled to 25 °C for immediate use.The plant was purchased at the local market.The commercial sachet of green tea and its use was by all the relevant guidelines.Table 1 summarizes all conditions of tea preparation.
1.3.Phenolic compounds and antioxidant activity of green tea and silver nanoparticles TPC analysis was carried out by the Folin-Ciocalteu method, according to Singleton, Orthofer e Lamuela-Raventos [13], and the absorbance was read at 760 nm (UV-vis Even, MODELO, Pais).Gallic acid solutions (4-24 μg ml −1 ) were used to obtain the analytical curve (R 2 > 0.99) and the results were expressed in μg of gallic acid equivalent (GAE) per 100 ml of sample (μg GAE 100 ml −1 ).The methods used to analyze the antioxidant activity were: DPPH free radical scavenging, performed according to Brand-Williams, Cuvelier, and Berset [14] and adaptations of Ravichandran, Ahmed, Knorr, and Smetanska [15]; (b) ferric reducing antioxidant power (FRAP) as described by Benzie and Strain [16].Analytical curves with Trolox solutions were prepared with concentrations between 0.05 and 0.6 mM and 0.75-75 mM.The results were expressed in μmol equivalent to Trolox (TE) per 100 ml of sample (μmol TE 100 ml −1 ).

Green synthesis of AgNPs
For the synthesis of silver nanoparticles, 10 ml of AgNO 3 1 mM solution was added in a transparent test tube, followed by the addition of 1 ml of green tea and stirring in a vortex-type stirrer, model XH-CU (Global Trade Technology).With the system stirring, the basic dextrose solution (0, 5 mM) was added, varying the volume from 0 to 100 μl.The synthesis was performed under room conditions (temperature and light), and the stiring continued for 30 seconds after the addition of the dextrose solution.

Characterization details
2.1.UV-vis spectroscopy UV-vis spectroscopy analyses were performed on a spectrophotometer (Thermo Fischer Scientific brand and model Nanodrop@ONE C).The range of 300 to 600 nm was used, with a resolution of 1 nm.For the analyses, quartz cuvettes were used, adding 1.5 ml of the sample to 2.5 ml of water.

Scanning electron microscope
The morphological and surface evaluation of the sample was carried out in a scanning electron microscope (SEM) with field emission MYRA 3 LMH (Tescan).1 μl of the nanoparticle suspensions was added to the sample holder and oven-dried at 36 °C for 24 h.The samples were metalized with gold on the SC7620 mini sputter Coater and the micrographs were obtained after visualization of the samples, using acceleration voltages between 10 and 25 kV.The recording of the images occurred through the equipment software.

Dynamic light scattering (DLS)
For the size distribution analysis, a polystyrene cuvette with four polished faces was used, performed in a particle size analyzer (Anton PAAR brand, model Litesizer 100).The analyses were performed at a temperature of 25 °C.

Identification of caffeine and gallic acid by high-performance liquid chromatography (HPLC)
The chromatographic determinations were carried out on a Jasco ® LC-4000 high-performance liquid chromatograph, with a quaternary pump for RHPLC, UV/Vis detector (UV-4070/75 -deuterium lamp), and 20 μl injection loop.A Fortis ® C18 reversed-phase column (250 mm×4.6 nm, 5 μm particle size) was used to separate the analytes.

Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR)
The AgNPs samples were centrifuged at 14 °C for 30 min at 15.000 rpm (novatecnica, model NT 805) under refrigeration.The pellet was collected, and the aqueous extracts were subjected to drying by lyophilization, for subsequent analysis.The samples were analyzed using an Agilent Cary 630 spectrophotometer equipped with Microlab PC software and an Attenuated Total Reflection (ATR) accessory in the range of 4000 cm-1 to 400 cm-1 with a resolution of 4 cm -1 .

AgNP's stability monitoring
For the experiment, all samples used were obtained from the second infusion of diluted green tea.This choice was based on preliminary optical results, which indicated that this infusion presented the best result.The samples were stored in polypropylene Falcon tubes at a temperature of 5 °C.The stability of the optical properties of AgNPs was evaluated using UV-vis spectroscopy and size distribution analysis.The monitoring time was over 92 days.

Statistical analysis
The entire experiment was conducted in duplicate using a completely randomized design.The analyses of each characteristic of the teas and nanoparticles were determined in triplicate.The TPC and Antioxidant Activity (AA) data were submitted to ANOVA and Tukey's test (α = 5%) using the Statistica 7.1 software (StatSoft, 2006).

Phenolic compounds and antioxidant activity of green tea
There was a higher content of TPC and AA by FRAP in green tea in higher concentration and the infusion obtained by the first extraction (CTFI) (table 2) about DTSI tea (lower tea concentration and second extraction).This result is expected since concentrated teas have a higher tea/water ratio.Thus, the most appropriate choice of plant extract/solvent ratio should be considered in the tea preparation process.
It is also noted that most of the teas that present AA by DPPH were possibly leached both in the first and the second extraction of the diluted tea since no statistical difference was noticed between the AA of the 1st and the 2nd extractions.By FRAP, there was higher AA in the 1st extraction of concentrated tea.This result is justified by the fact that the FRAP and DPPH methods do not employ the same determination mechanism and, according to the analyzed matrix (and the profile of compounds that present AA) may not have a correlation.According to Dudonné et al [17], electron transfer mechanisms prevail in the FRAP and ABTS methods (with radical reduction, in which compounds with redox potential lower than that of the radical itself cannot be evaluated), as well as for the TPC method, while in ORAC and DPPH there is the transfer of a hydrogen atom.Considering that TPC and AA compounds present in C. sinensis can act as reducing and stabilizing agents of AgNPs [18] obtained by green synthesis and, therefore, may be more bioavailable, it is essential to evaluate and study this synthesis.

HPLC analysis
The chromatograms of all teas used in this study and the analytical standards are presented in figure 1.
In a preliminary analysis, it is observed that the chromatogram profiles of both the first and second extractions are similar, differing only in peak intensities.This indicates that the compounds obtained form the various extractions are identical, with only the concentration varying.Additionally, the peaks appearing at retention times of 6.1 and 14 min correspond to gallic acid and caffeine, respectively, confirmed by the chromatogram of the standards.In figure 2, a bar graph is presented correlating the peak area values of gallic acid and caffeine in all tea samples, as well as the standards.
The bars show that the amount of extracted compounds decreased as the extraction was repeated using the same tea bag.This behavior was also observed in the antioxidant activities of the teas, as reusing the tea bag led to the extraction of compounds but in smaller quantities.These data support the HPLC results.b Formulations: CTFI concentrated tea: 0.1 g of green tea/ml in first and second infusion and diluted: 0.05 g of green tea/ml in first and second.

Synthesis, optimization, and characterization of AgNp's
The color of the Ag+ solutions in the reaction flasks changed from colorless to dark brown very fast, which indicates the formation of silver nanoparticles.The UV-vis results for the samples are shown in figure 3.In general, for all the samples obtained, it was possible to observe the presence of the plasmon band, around 433 nm, indicating the formation of NPs.Some methods have used different plant extracts and reducing agents for green synthesis of AgNPs, but the estimated time to obtain NPs varies from 1 to 42 h [19][20][21][22][23], in Salih's work [11], silver nanoparticles were obtained using methanolic and aqueous extracts of C. sinensis, and the results obtained showed that the formation time was long, around 48 h.Another characteristic is that the process for  preparing the plant extract is not green, as it uses solvents such as petroleum ether and methanol.Ahmad et al that found that temperature is an important factor to obtained silver nanoparticles, the authors observed the formation of AgNPs in 40 °C and 100 °C.In this work all synthesis are made at room temperature [24].
In this work, as noted, the achievement was instantaneous, thus promoting time-saving that can contribute to sustainability by reducing resource consumption, waste generation, and energy consumption, and optimizing this process.Patra et al [25] synthesized silver nanoparticles using C sinensis.The authors noted that various synthesis condition influence the obtaining the particles.In this work all synthesis were performed at room temperature and no pH variation.It was also possible to observe a change in the intensity and shape of the plasmonic band when synthesizing the silver nanoparticles, varying the way of obtaining the green tea extract.In figures 3 (B) and (D) are the spectra of the most diluted teas, B first boil and D second boil.A plasmon band form has been observed to vary significantly with the concentration of the tea and the amount of additional reducing agent added.There is a more prominent band in more diluted tea and higher proportions of dextrose added [26].From these observations, it is assumed that, in more diluted teas and larger amounts of reducing solution, dextrose preferentially reacts to silver, leading to the formation of nanoparticles.
For the formation of silver nanoparticles, the process involves the reduction reaction of Ag+ ions by dextrose and the major compound (caffeine) in green tea.The results showed that the optimal medium for nanoparticle formation is tea with lower antioxidant content and a higher quantity of reducing solution (100 μl).To confirm this inference, the Total Phenolic Content (TPC) of the AgNPs prepared with both the more concentrated tea (29.3 ± 1.4 μg ml −1 ) and the more diluted tea (18.8 ± 0.6 μg ml −1 ) was evaluated, confirming a higher TPC in the more concentrated tea.

Particle size by Mie theory
From the UV-vis spectra of the samples, it is possible to correlate the shape of the plasmonic band with particle size, using Mie theory [27], which establishes a relationship between the electromagnetic radiation scattered by spherical particles in solution.To estimate the particle size, equation (1) was used.
Where: d is the diameter; VF is Fermi´s velocity; l P 2 is the maximum absorption to the power of two; C is the speed of light and Δλ is the width at half height.Table 3 shows the results of nanoparticle size calculated by Mie Theory equation (1).
Table 3 shows that the lowest size values are found for the 2nd infusion tea, indicating that the use of a more diluted tea favors the formation of nanoparticles in a very reduced size.

Scnning electron microscope (SEM)
The results obtained by scanning electron microscope for the samples AgNP obtained using concentreted green tea first extraction are shown in figure 4. Nanoparticles are presented as spherical and uniform structures in the images.The size of most nanoparticles was approximately 30 to 50 nm It is possible to observe a degree of agglomeration between the nanoparticles.The images from the photomicrographs presented in figure 5 show that the AgNP obtained using concentreted green tea second extraction has an amorphous, almost spherical appearance.The sizes of the individual AgNP measured by the equipment software indicated sizes close to 100 nm.The nanoparticles appear to agglomerate or collapse.
According to figure 6, AgNP obtained using dilueted green tea first extraction presented a spherical format and the presence of aggregates.They presented average sizes close to 35 to 45 nm.The agglomeration formation can be explained by the plant residual matrix which can keep the nanoparticles connected after the biosynthesis [28].
In figure 7 severe degree of agglomeration of AgNP obtained using dilueted green tea second extraction nanoparticles renders the definition of particle morphology and size very difficult.Amorphous to spherical appearance can be observed in the microphotographs.
The FTIR analyses were conducted to study how tea acts as a stabilizing agent for nanoparticles.Figure 8 shows the FTIR spectra for the silver nanoparticle samples and the spectra of lyophilized tea.
Analyzing the spectra of the obtained teas, it is possible to observe the same band profile for all tea samples, and the approximate assignment of the main bands is shown in table 4.
The spectrum of the nanoparticles shows characteristic bands of the tea, indicating that the compounds present in the extract are on the surface of the particles.Bands at 1691 and 1143 cm-1 are absent in spectrum (a),  suggesting that the bonding between phenolic compounds in the tea and the surface of nanoparticles occurs through N and O atoms, which may be present in caffeine and gallic acid.The results of figure 9 demonstrated that the optical properties of AgNPs in diluted green tea solution (0.05 g of tea/ml) with 100 μl of Dextrose solution were highly stable over a period of 90 days.UV-vis spectroscopic analysis revealed that the absorption spectra of AgNPs remained consistent, with no significant changes over time.This indicates the maintenance of the integrity of the optical characteristics of the particles.According to Li et al [28] good stability of the nanoparticles in solution is due to the presence of chemical compounds in the extract of green tea leaves fixed in the AgNPs.It also infers that the instability of the suspensions occurs in high concentrations of tea extract, showing the importance of a more diluted tea.In addition, the analysis of the size distribution by DLS is presented in figure 9(b).Showed two particle sizes, the initial 1-10 nm, when relating to the results of UV-vis is believed to be the actual size of AgNPs, and the second distribution of 100-350 nm and both remained constant during the first two months of the study, evidencing the preservation of the structural     integrity of the particles and the absence of agglomeration or degradation.In the third month, a slight increase in particle size of 1-30 nm in the initial distribution and 120-300 nm in the second distribution was observed, which reinforces the stability of AgNPs over time.

Stability monitoring of AgNPs
The formation of silver nanoparticles in a reaction medium occurs in two stages, nucleation and growth.For a small size and a homogeneous size distribution, is necessary the formation of several nuclei at the same time, drastically decreases the concentration of the solution so that only growth happens and there is no more formation of new nuclei, preventing the Ostwald phenomenon [29].In the reaction medium, the reduction of Ag+ ions, which will give rise to the nuclei, can happen through two routes.The first would be the reaction with only the tea polyphenols [30], and the second way would be the reaction with the polyphenols and dextrose in the basic medium.The two reaction proposals are shown in equations (2) and (3), ( ) It was experimentally observed that the reaction in a medium with only polyphenols happened slowly, taking around two hours, and, as a result, the UV-vis Spectrum showed a wide and not well-defined band.On the other hand, with the use of the additional reducing agent, the basic dextrose solution, the formation of the nanoparticles occurred instantaneously and the UV-vis spectrum presented a very intense plasmon band.Indicating that the method using dextrose generated the formation of a more homogeneous sample.With higher amounts of dextrose, the nuclei are formed quickly and the growth of the particles is more homogeneous.On the other hand, the reaction with less dextrose, which is significantly influenced by the reaction with the polyphenols, with slower formation of the nucleus and non-homogeneous growth, leaving the larger particles, i.e. the additional reducing agent accelerated the process of formation of the nanoparticles, so it can be inferred that the increase of dextrose in the medium makes the phenolics not participate in the reduction of silver preserved for the stabilization of the nanoparticles formed.Then, it is suggested that the tea acts better as a stabilizer of the nanoparticle than as a reducer of silver ions, since the stability study showed that the dispersions remained stable and with no major changes for more than 90 days.

Conclusion
This study evidenced the viability of green synthesis of instataneous-preparation silver nanoparticles using residues of commercial green tea infusion and alkaline dextrose solution at room temperature and no pH variation.Green tea was characterized by CFT and AA tests by FRAP and DPPH, while synthesized AgNPs were analyzed by UV-vis, DLS, and SEM.The particles demonstrated stability over several months of storage, maintaining their characteristic plasmonic band.In addition, they had an average size of less than 100 nm and exhibited antioxidant activity due to the compounds present in the green tea extract used in the synthesis.The compounds phenolics, in special, caffeine and gallic acid, in minor quantities, and dextrose are responsible for the former of silver nanoparticles.The use of sustainable methods, such as green synthesis, promotes sustainability in the production chain of silver nanoparticles, using only natural materials with low environmental impact.These results indicate the potential of synthesized AgNPs for applications in several areas, such as nanomedicine and environmental nanotechnology.

Figure 1 .
Figure 1.Chromatograms of the teas and analytical standards for caffeine and gallic acid.Where CV1AFC: Concentrated first-boil green tea; CV1AFD: Diluted first-boil green tea; CV2aFC: Concentrated second-boil green tea and CV2aFD: Diluted second-boil green tea.

Figure 2 .
Figure 2. Peak areas of caffeine and gallic acid for the teas and analytical standards.

Figure 3 .
Figure 3. Spectroscopic analysis of Synthesis optimization by UV-vis spectrum of Ag nanoparticles.A: Concentrated tea (tea:water 0.1 g ml −1 ) 1st infusion.B: 2nd infusion of the same concentrated tea.C: Diluted tea (tea:water 0.05 g ml −1 ) 1st infusion.D: 2nd infusion of the same diluted tea.In this graph is show the amount of Dextrose solution (volume range to 10-100 μl).

Figure 4 .
Figure 4. Microphotographs obtained by SEM of the AgNP obtained using concentreted green tea first extraction.

Figure 5 .
Figure 5. Microphotographs obtained by SEM of the AgNP obtained using concentreted green tea second extraction.

Figure 9
shows the UV-vis spectra (figure 9(a)) and size distribution by DLS -Dynamic Light Scattering (figure 9(b)) of silver nanoparticle dispersions aimed at studying the stability of the nanoparticles in dispersion.The samples were stored for 90 days.

Figure 6 .
Figure 6.Microphotographs were obtained by SEM of the AgNP obtained using dilueted green tea first extraction.

Figure 7 .
Figure 7. Microphotographs obtained by SEM of the AgNP obtained using dilueted green tea second extraction.

Table 1 .
Details for all the extract infusion.

Table 3 .
Particle size (nn) calculated by Mie's Theory of Concentrated and Diluted Tea and the variation of Dextrose solution, concentration (10-100 microliters) in its first and second infusion.