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25 August 2020 Silver nanoparticles synthesis using biomolecules of habanero pepper (Capsicum chinense Jacq.) as a reducing agent
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Abstract

A simple, low-cost, and ecofriendly method of obtaining silver nanoparticles was obtained from AgNO3 in dilution for the first time with a habanero pepper infusion. Two sets of experiments were carried out. The samples obtained in the first experiment were analyzed by SEM and TEM. Results showed that the samples prepared using 0.001 M and 0.005 M of AgNO3 and particles with an average size of 19.3 and 26.4 nm were obtained. Furthermore, the nanoparticles prepared with 0.005 M showed an absorption peak shifted toward a larger wavelength, and according to its distribution, it also exhibited larger particle sizes. In the second experiment, different concentrations of habanero pepper (10, 50, and 100 mL) were explored and the pH and oxidation reduction potential were monitored. The sample with 100 mL concentration showed the most intense absorption peak and the highest decrease in pH, which is explained by biomolecules deprotonation.

1.

Introduction

The number of papers published on the synthesis of silver nanoparticles has been increasing due to the optical and electronic properties these materials present on a nanometric scale. This has been a very important application as bactericidal, catalytic, and biological sensors.13 One of the main characteristics of silver nanoparticles is its absorption peak in the visible-ultraviolet around 400 nm, which is not observed in bulk silver.4

The method commonly used in the synthesis of this kind of nanoparticles is through the chemical reduction of the silver nitrate salt AgNO3. This method consists essentially of preparing a molar concentration of this salt as a precursor of silver ions in dissolution with chemical reagents that work as reducing and stabilizing agents.5,6 The first agent changes the silver ions to the neutral state, favoring the formation of nanoparticles, while the second stabilizing agent prevents the agglomeration of nanoparticles.

However, it has been reported that the reagents usually used in this method have toxic properties. Because of this downside, in the first decade of the 2000s, synthesis of biological reduction was used as a method to obtain these nanoparticles. Although the basis of this method is the chemical reduction of salt as it uses AgNO3, the difference is that this method, instead of using chemical reagents, uses organic products.711 Likewise, this green method has been used to prepare other metallic nanoparticles, such as gold, platinum, and palladium. The reasons for continuing to explore this green method are that it is ecofriendly and low cost.1214 This could be improved in the application of silver nanoparticles for bactericidal use if it were obtained as a nontoxic medium given that the reported toxic activity of these nanomaterials published in biological systems has been contrary to what was expected.15

Biological processes play a key role in the size and stability of silver nanoparticles. For instance, in the synthesis from a molar solution of AgNO3 in dilution with an extract of aloe vera, nanoparticles showed a mean size of 15.2.16 Meanwhile, in an extract based on garlic cloves, nanoparticles had a mean size of 7.3 nm, and an average size of 23 nm was obtained in coconut extract.17,18 On the other hand, in a green tea infusion the size of nanoparticles increases depending on the concentration on the tea extract in the samples, whereas using coffee grains they had an average size of 30 nm.19,20 In Table 1, there are more reported results in the scientific literature about spherical silver nanoparticles. This indicates that a wide variety of natural ingredients was used for a biological process in the synthesis of silver nanoparticles.

Table 1

Biosynthesis of nanoparticles using different plant extracts.

ExtractAverage sizea (nm)Reference
Aloe vera leaf15.28
Garlic7.317
Capsicum annumm L.4531
Olive leaf20 to 2532
Marigold flower10 to 9033
Rosa rugosa1234
Mango peel7 to 2735
Guava leaf10 to 9036
Cycas leaf2 to 637
Prusno persica40 to 9838
Apple30.2539
Onion33.640
Lemon25 to 5041
Mandarin peel5 to 2042
Watermelon1443
Orange peel7.3644
Habanero pepper19.3 to 26.4(This study)

aThe sizes was taken directly from the publications.

Notably, in biosynthesis, biomolecules play an important role as reducing and stabilizing agents.2123 We have as an example the Fourier-transform infrared spectroscopy method (FTIR), where, as reported, the detection of certain biomolecules belonging to amides, carboxyl, amino, and polyphenols groups on the surface of the nanoparticles synthesized in the tea.24 In Ref. 25, it was shown that the reduction of silver ions was due to terpenoids, polysaccharides, and flavonoids present in the leaf extract of Cinnamomum camphora. In addition, some studies highlight the antioxidant capacity of the plants or the fruit used in the synthesis. This is because it promotes the reduction of silver ions, and therefore, increases the formation of silver nanoparticles. For example, it has been suggested that green tea, pineapple, and Elephantopus scaber leaf are good antioxidants. As a result, these natural products were used to obtain silver nanoparticles.19,2628

The aim of this study was to explore the effect of habanero pepper in the synthesis of the silver nanoparticles. We chose this fruit because it is not only a great antioxidant of Mexican origin, where it is grown and consumed, but also because of its economic importance and its nutritional value.29

In addition, habanero pepper is a traditional crop in southeastern Mexico. In 2012, 955.6 hectares were planted with habanero pepper, resulting in the production of 5138.2 tons. The state of Yucatan is the leading producer of this kind of pepper with a planted area of 708.4 hectares and a production volume of 3295. 2 tons, followed by the states of Tabasco, Campeche, and Quintana Roo.30

In our experiments, we used the infusion of habanero pepper in dilutions with AgNO3 and, to explain the kinetic performance of the nanoparticles, we measured the pH and the oxidation reduction potential (ORP) in real time. Furthermore, we propose an explanation on effect of the biomolecules in the silver nanoparticles’ formation. The samples were analyzed by UV–Vis spectroscopy and electronic microscopy (SEM and TEM).

2.

Materials and Methods

2.1.

Extraction of Habanero Pepper Infusion

In order to prepare the infusion, 4 g of habanero pepper were rinsed and cut into small pieces, then boiled with 200 mL of bidistilled water in a volumetric flask. This infusion was filtered to remove any debris, and the supernatant was stored at 4°C for further analysis.

2.2.

First Experimental Setup

Figure 1(a) shows the scheme of the first experiment designed to explore the effect of the molar concentration of silver nitrate (AgNO3) in the synthesis. For this, we prepared two samples with molar concentrations of 0.001 M and 0.005 M of AgNO3 in dilution with 150 mL of bidistilled water. In each one, 50 mL of the infusion of habanero pepper was added in a final working solution of 200 mL. According to Fig. 1(a), the dilution was stirred at 40°C and 300 rpm for 10 min.

Fig. 1

Experimental setup for the synthesis of nanoparticles. (a) First experimental setup employed to explore the effect of silver nitrate in the synthesis. (b) Second experimental setup employed to explore the effect of habanero pepper in the synthesis, as well as to measure the pH and ORP in real time.

JNP_14_3_036012_f001.png

These samples were analyzed with a Shimadzu spectrometer, and the nanoparticles obtained were observed by the TEM using a JEOL JEM1010 at 100 kV. The size distributions were obtained through analyses of the electron micrographs. For the analysis, we measured the areas of the nanoparticles and determined that their diameter was that of a circle. Likewise, X-ray analysis by energy dispersion was performed using a SEM (JSM-6390V JEOL).

2.3.

Second Experimental Setup

Figure 1(b) shows a scheme of the second experiment where three samples were prepared in batch reactions using a molar concentration of 0.005 M of AgNO3 in dilution with habanero pepper. Different concentrations of the infusion were used (10, 50, and 100 mL), with a final working volume of 200 mL. Another difference from the first experiment was that the synthesis was carried out at room temperature. In each one, the pH and the ORP were measured for 10 min. In addition, the UV–vis absorption spectra of three stable samples are shown. In the same case, they were obtained with a Shimadzu spectrometer.

3.

Results and Discussion

During the synthesis in both experiments, we observed a color change at the beginning. It transformed from transparent to a deep yellow color. According to scientific articles, if the samples turn this color, this suggests that it is due to the presence of silver nanoparticles.

3.1.

Biosynthesis of Nanoparticles Using Different Concentrations of AgNO3

Figure 2(a) shows two absorption spectra of the samples prepared according to the first experiment using different molar concentrations of AgNO3 in dilution with the infusion of habanero pepper. It should be mentioned that in both samples, an absorption peak in the region Vis–UV was clearly observable, where the absorption maximum is around 400 nm. This is a characteristic of silver nanoparticles in suspension.44 The absorption peak of the sample prepared with 0.005 M was more intense. Because this sample had a greater amount of AgNO3 used, an increase in silver nanoparticles’ production was expected. This can be explained using the linear relation from Lambert–Beer; A=αlC, where A is the absorbance, α is the absorption coefficient, l is the path length that the radiation travels in the sample, and C is the concentration of the absorbent, which corresponds to the suspended nanoparticles. This linear relation indicates that a greater concentration of nanoparticles in suspension corresponds with a larger absorbance.45 Furthermore, another difference in the spectra was that the absorption peak of silver nanoparticles obtained at 0.005 M shifted toward 432 nm. This behavior could be due to larger nanoparticles suspended in the dilution with habanero pepper. This can be explained by the Mie theory for metallic nanoparticles with a spherical shape, where theoretically, the extinction efficiency of a single nanoparticle was calculated since this efficiency is related to the absorbance. For instance, Haiss et al. showed that the largest gold nanoparticles show a position peak toward larger wavelengths.46 In addition, a similar behavior was reported for silver nanoparticles, where the simulations were performed by the modification of FORTRAN 77 code by Haiss et al.47

Fig. 2

UV–Vis absorbance of nanoparticles obtained with concentrations of 0.001 M and 0.005 M in dilution with an infusion of habanero pepper. (a) Day of the synthesis and (b) day of the synthesis and after 1 week.

JNP_14_3_036012_f002.png

Figure 2(b) shows the comparison of spectra of the samples of the first day of the reaction versus 1 week after the synthesis. Clearly, we can observe that the absorption peak is more intense after the synthesis, suggesting that during this time period, the nanoparticles remained stable in the suspension, and that there was an increase of nanoparticles’ production. The resulting stability can be explained by factors such as the dipole moment of the solvent in combination with the biological molecules, which generate a stronger electric double layer in the nanoparticles’ surfaces, increasing the repulsive force between nanoparticles.44 This stability is favored by the infusion of habanero pepper and its protein, as explained by Li et al. An extract of Capsicum annum (this is a kind of pepper) was employed in the synthesis of silver nanoparticles, and its resulting stability is attributed to the protein. This knowledge was obtained from the characterization of these samples by the FTIR, detecting biomolecules on the surfaces of nanoparticles.31

Figures 3(a) and 3(b) show the distribution size of nanoparticles synthesized from molar concentrations of 0.001 M and 0.005 M in dilution with the infusion of habanero pepper, showing the transmission electron micrographs as insets. The number of particles used on these distributions was 100 [Fig. 3(a)] and 132 [Fig. 3(b)], respectively. According to the results in the size distributions, the nanoparticles obtained by a higher concentration of AgNO3 (0.005 M) presented diameters larger than 40 nm. This reinforces the predicted results made by the analysis of the absorption peaks of these two samples [see Fig. 2(a)], where it is mentioned that the absorption peak tends to be shifted toward the larger wavelength, corresponding to nanoparticle suspensions obtained with 0.005 M of AgNO3, and this sample presented the largest nanoparticles.

Fig. 3

Size distributions of nanoparticles obtained with concentrations of 0.001 M and 0.005 M in dilution with an infusion of habanero pepper.

JNP_14_3_036012_f003.png

In addition, Fig. 4 shows an elemental analysis by energy dispersive X-ray spectroscopy (EDS) of nanoparticles obtained using a molar concentration of 0.005 M of AgNO3 in dilution with habanero pepper, showing the scanning electron micrograph as an inset. We can observe a strong characteristic energy peak for silver (Ag0) at 2.98 keV, indicating that a representative concentration of nanoparticles was structured from this element. As a result of silver ions, reduction was generated by biological molecules where Ag+1 passed to their neutral state Ag0.

Fig. 4

EDS spectra of nanoparticles obtained with a concentration of 0.005 M in dilution with an infusion of habanero pepper.

JNP_14_3_036012_f004.png

3.2.

Biosynthesis of Nanoparticles Using Different Concentrations of Habanero Pepper Infusion

For this set of experiments, three syntheses were carried out using 0.005 M of silver nitrate concentration in dilution with different amounts of habanero pepper of 10, 50, and 100 mL, in a working volume of 200 mL, respectively. See Fig. 1(b) for more details of this experiment, which was done at room temperature. During each synthesis, the pH and the ORP were measured.

Figure 5 shows the absorption spectra of the three samples obtained using different amounts of the infusion of habanero pepper. As can be seen, the absorption peak increased with the habanero pepper, although in each synthesis the same molar concentration of AgNO3 was employed. This result suggests that during the aqueous dilution, having more biological molecules of habanero pepper increases the number of silver ions reduced (Ag+) to their neutral state (Ag0), generating a greater production of silver nanoparticles.

Fig. 5

UV–Vis absorbance of nanoparticles obtained with a concentration of 0.005 M in dilution with different amounts of infusions of habanero pepper.

JNP_14_3_036012_f005.png

3.2.1.

Effect of pH and ORP during the biosynthesis of AgNPs

When pH and ORP were monitored in real time, the initial value of the three samples started as slightly acidic (6.33±0.2). In the first minute, all the samples’ behaviors were very similar. They tended to decrease and remain in a steady state during the last 9 min, until reaching pHs of 5.73 and 5.79 for 10 and 50 mL samples, respectively [Figs. 6(a) and 6(b)]. For the 100-mL sample, the drop was more noticeable. In fact, it was the concentration that reached the highest range, decreasing to a pH of 5.83 [Fig. 6(c)]. This experiment suggests that the biomolecules are working as nucleophiles and the silver nanoparticle are working as electrophiles, i.e., biomolecules are deprotonating, giving H+ and electrons to the medium for the reduction of the silver ions (Ag+) to the neutral state (Ag0), thus aiding the production of nanoparticles.

Fig. 6

pH (○) and ORP (□) kinetics using different concentration of habanero extracts with 0.005 M of AgNO3. (a) 10 mL of habanero extract, (b) 50 mL of habanero extract, and (c) 100 mL of habanero extract.

JNP_14_3_036012_f006.png

The REDOX potential is a simple way of measuring the oxidation–reduction chemical energy using an electrode, transducing the signal in electric energy. This potential is positive when the oxidation occurs in the microenvironment and negative when the reduction occurs. Although both reactions go together, molecule A is oxidized while molecule B is reduced. Figures 6(a)6(c) show the kinetics of the REDOX potential of these three samples prepared with different amounts of infusion of habanero pepper. We observe that the lower the concentration of habanero, the higher the initial value of oxidation. This is only for the 10 and 50 mL concentrations with values of 37.2 and 41.5, respectively. With respect to the higher concentration (100 mL), the initial value was very low (2.8). This could be explained due to the fact that this concentration has a greater number of biomolecules, which could be precipitated to the bottom of the beaker. When the agitation started, the biomolecules interacted with silver nitrate and this interaction began to be detected by the electrode. As can be seen during the first minute, the kinetic reaction reaches values similar to those of the other two concentrations (50 and 10 mL). Through the remaining 9 min, the behavior of the kinetic was comparable in the three dilutions (53.03±8.13). All three dilutions tended to increase during the process. The oxidation reactions acidified the medium due to the release of H+ from the biomolecules, which corroborates and reaffirms the pH experiments.

In addition, Fig. 7 shows a representation of the silver nanoparticles’ formation from the dilution of silver nitrate with the infusion of habanero pepper. There are silver ions (Ag+) by molecular dissociation of AgNO3 and hydrogen ions (H+) due to biomolecules deprotonation, generating many free electrons (e). Afterward, silver ions are reduced by the acceptance of these electrons. In other words, at this stage, the bioreduction is beginning. In consequence, the nucleation of silver atoms (Ag0) occurs, favoring the silver nanoparticles’ production.

Fig. 7

Schematic of the silver nanoparticles’ formation by the biosynthesis method.

JNP_14_3_036012_f007.png

All the biological systems continuously produce reactive oxygen species (ROS), where the chloroplasts are the main sites of production under normal conditions in plants. For this reason, the plants have evolved with marvelous antioxidant machinery, mediated by enzymes (SOD, POD, CAT, APX, MDHAR, and GR) and nonenzymatic antioxidants (ascorbate, glutation, tioredoxine, carotenoids, and tocopherols) and as the scavenger of electrons produced by ROS.48,49 The role of this antioxidant machinery in plants reduces the impact of oxidative stress, where the antioxidants play a key role in stabilizing molecules by means of reducing and scavenging electrons. Green synthesis is due to multiple molecules as described above. Antonious et al.50 analyzed the phytochemical content of Capsicum chinense Jacq., where the Mexican variety reached the highest content of phenolic compounds (up to 349  μg/g). Adyani and Soleimani52 evaluated the AgNPs production using Punica granatum extracts with its phenolic compounds. They evaluated the antioxidant and scavenger activity of these compounds and established the reducing role of the phenolic compounds and their activity during nanoparticle production.

4.

Conclusions

In conclusion, in this work, silver nanoparticles were successfully obtained using a simple and ecofriendly method. Notably, the concentration of silver nitrate in antioxidants such as the habanero pepper has an influence over the production of nanoparticles, as indicated by the absorption peaks of the nanoparticles in suspension. Measuring the pH and ORP in real time during the synthesis can be used to gain knowledge about the role that biomolecules play in the silver ion reduction because it is a fundamental stage in the nanoparticles’ formation process.

Acknowledgments

This study was partially supported by PRODEP program UDG-PTC-1463. The authors want to thank CONACYT, CuValles-UDG, and UCOL for facilities to realize this research. The authors want to thank Martha Beatriz Guzman-Aburto and Israel Ceja-Andrade for technical assistance.

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Biography

David Omar Oseguera-Galindo received his PhD from the University of Guadalajara Guadalajara, Jalisco, Mexico, in 2013. He obtained a CONACYT postdoctoral fellowship at the Ensenada Center for Scientific Research and Higher Education (CICESE) in cooperation with at Center of Nanoscience and Nanotechnology (CNyN) of the National Autonomous University of Mexico (UNAM), Ensenada, Baja California, Mexico. Currently, he is an associate professor at the University of Guadalajara in Ameca, Jalisco, Mexico. His research interest is nanoscience. He is currently working on the biosynthesis and characterization of silver nanoparticles.

Edén Oceguera-Contreras received his PhD from the University of Guadalajara in Guadalajara, Jalisco, Mexico, in 2012. Then he received a postdoctoral fellowship in bioprocessing of biofuels at IPICyT A.C. in San Luis Potosí, SLP, México. He was also a postdoc in bioprocessing at McGill University in Montreal, QC, Canada. Currently, he is an associate professor at the University of Guadalajara in Ameca, Jalisco, Mexico. He is working on the optimization of operational conditions of biological systems mainly focused on the biotechnological upstream process of biotransformation and the dynamics of metabolic flux.

Dario Pozas-Zepeda received his MSC degree from Baja California University in 2018. He is currently working as a research assistant at the University of Colima in the Laboratory of Electronic Microscopy. His research interests include the synthesis and characterization of inorganic compounds.

© 2020 Society of Photo-Optical Instrumentation Engineers (SPIE) 1934-2608/2020/$28.00 © 2020 SPIE
David O. Oseguera-Galindo, Eden Oceguera-Contreras, and Dario Pozas-Zepeda "Silver nanoparticles synthesis using biomolecules of habanero pepper (Capsicum chinense Jacq.) as a reducing agent," Journal of Nanophotonics 14(3), 036012 (25 August 2020). https://doi.org/10.1117/1.JNP.14.036012
Received: 29 May 2020; Accepted: 6 August 2020; Published: 25 August 2020
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