Ají Amarillo Chilli-Assisted Phytosynthesis of Silver Nanoparticles and Their H2O2 Sensing Ability


 Scope of green chemistry in nanobiotechnology is generating research interest in the development of an ecofriendly synthesis of nanoparticles and their wide range of applications in engineering and biomedical field. In the present study, we report a simple and ecofriendly phytosynthesis of silver nanoparticles (AgNPs) using Ají amarillo chilli (Capsicum baccatum L.) fruit, where phytoconstituents of C. baccatum behaves as a reducing as well as a stabilizing agent. The phyotsynthesized AgNPs were characterized through various spectroscopic and microscopic techniques. The produced AgNPs showed surface plasmon resonance (SPR) at λmax = 458 nm and stable for 10 days, confirmed by UV-vis spectroscopy. Transmission electron microscopy (TEM) explained that produced AgNPs were almost spherical in shape with average size of 10-30 nm along with small aggregation. Aggregation of AgNPs was indicated in the dynamic light scattering (DLS) analysis. The results also demonstrate that as-synthesized AgNPs are low-cost and rapid alternative as optical chemical sensors and showed >50% H2O2 quenching/ sensing activity for 15 mins at room temperature.


Introduction
Chilli peppers are one of the most popular vegetable and present in almost every diet throughout the world. There is archaeological evidence at sites located from the Bahamas to southern Peru that chilli peppers were domesticated more than 6000 years ago, [1] and were one of the rst self-pollinating crops cultivated in Central and South America [2]. Capsicum baccatum L. (Aji amarillo chilli) is a traditional vegetable crop in Ecuador, Argentina, Bolivia, Brazil, Chile, Peru, Costa Rica, and Hawaii. C. baccatum fruit is distinguished from other species by the yellow, red, orange, brown, or dark green colour and valued for their sensory attributes of colour, avor and high pungency [3]. C. baccatum fruit are are economically important and good sources of many bioactive compounds, such as capsaicinoids, polyphenolic compounds, carotenoids, protein/enzymes, polysaccharides, amino acids and vitamins help the human body to function and are responsible for the growth of plants [4][5][6] (Fig. 1). Hence, these phytoactive contents of the C. baccatum fruit may be used as reducing and stabilizing agent for the synthesis of nanoparticles and other applicibality in nanoscience eld.
During last two decades, synthesis of noble metal nanoparticles (MNPs) via green methods has attracted much attention due to their ecofriendly nature and wide range of properties including a high surface-areato-volume ratio, biocompatibility [7], and low toxicity [8]. Nanoparticles (NPs) are particles that range 1-100 nm in size. Green synthesized silver nanoparticles (AgNPs) are widely studied metal among other MNPs and e ciently utilized in a wide range of scienti c and industrial applications including catalysis [9], sensing, molecular imaging [10], electronics [11], antimicrobial [12], therapeutics [13], tissue engineering [14], solar cells [15], drug and gene delivery [16], wastewater remediation [17], etc. Notably, absorption and scattering plasmonic spectra (colors) of colloidal gold nanoparticles (AuNPs) and AgNPs have been used as colorimetric assays for sensing of DNA, proteins and metal ions using the naked eye, showing the importance of design of colors (absorption and scattering optical properties) of colloidal To address the environmental concern in nanoscience, scientists have been working throughout the year for improving the nano synthesis method by using biomolecules/ phytochemicals, and enhance the surface properties of AgNPs by functionalization of its surface with bioactive compounds. Surface functionalization of AgNPs having different morphology alters the physico-chemical properties [19,20], which may play a crucial role in various bioengineering applications [9,21]. Recently, different MNPs were synthesized by using Capsicum species of different geographical origin including C. baccatum [3] and C. annuum fruit [22] for AuNPs [3], C. annuum [23][24] and C. frutescence fruit for AgNPs [25], C. annuum leaves for AuNPs [26], C. annuum var annuum (Jalapeño Chili) for AgNPs [27], Capsicum annuum for ZnO nanoparticles [28,29], etc has been already reported.
To the best of our knowledge, there are no reports on the synthesis of AgNPs using aqueous extract of C. baccatum L. (Aji Amarillo) cultivated in Ecuador and investigation of its H 2 O 2 sensing activity. The current study illustrates an ecofriendly attempt made to phytosynthesize and characterize the synthesized AgNPs with UV-vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and fourier transform infrared spectroscopy (FTIR) to generate evidence of morphology, surface and optical property. The results could provide a new scope about the potential utility of C.
baccatum fruit as a raw material source for the direct reduction of Ag + ion, formation of AgNPs and their utilization as low-cost optical chemical sensors for detection H 2 O 2 .

Materials
All chemicals were of analytical grade and used without any puri cation. Silver nitrate (AgNO 3 99.8 %) and H 2 O 2 (35 %) were purchased from Spectrum, USA. Milli-Q water was used in all experiments. Fresh Ají amarillo chilli /C. baccatum fruit was purchased from the popular market (January 2014) near Universidad de las Fuerzas Armadas ESPE, Sangolqui, Ecuador.

Ají amarillo chilli/ C. baccatum fruit extract and AgNPs synthesis
After being washed thoroughly, C. baccatum /Aji fruit (5g) was cut nely and stirred (23-25°C) in 50 mL of deionized water for 120 mins. After cooling, the light yellow color extract was ltered using Whatman No.1 paper and stored at 4°C for further use. For the phytosynthesis of AgNPs, 3 mL of C. baccatum fruit extract was mixed with 10 mL of AgNO 3 (1 mM) solution at room temperature (22-25°C). Phytoreduction of Ag + to Ag 0 indicated by the appearance of orange red colour after 3 hrs and studied the formation of the AgNPs at different time interval. Ao is the initial absorbance of the AgNPs solution and At is the absorbance of the AgNPs at time t.

Visual and UV-visible analysis
The optical properties and progression of the reaction was monitored visually by the appearance of orange or wine red color in a reaction mixture from colorless solution, after addition of C. baccatum fruit extract to AgNO 3 ..This change of colour is the primary visual indicator of phytosynthesis of AgNPs ( Fig. 3, Inset) and their formation further con rmed by UV − vis spectroscopy (Fig. 3a). This colour change of solution mixture may be attributed to the surface plasmon resonance (SPR) of the AgNPs appeared at 458 nm on completion of the reaction [20]. A broad single absorption band was observed between 300-600 nm corresponds to presence of larger size, spherical and polydisperse AgNPs [19]. Figure 3b showed the absorption spectrum of C. baccatum fruit extract and peak observed at 300-600 nm can be assigned to its bioactive compounds such as capsaicinoids, phenolic compounds, carotenoids, protein/enzymes, polysaccharides, amino acids and vitamins [19,22,27]. In Fig. 3b, the increase in absorption peak in the range of 400-600 nm con rming the involvement of bioactive compounds of C. baccatum fruit works as reducing and stabilizing agent for Ag + to Ag 0 conversion. No change in absorption peak of AgNPs at 458 nm was recorded for 10 days, indicating their stability. The reaction and phytosynthesis of AgNPs is depicted as follows in using Eq.

TEM analysis
TEM is a primary means to determine the sizes and shapes of nanoparticles. Figure 4 show almost spherical shaped and polydispersed AgNPs with average size of 10-30 nm along with small aggregation.

DLS analysis
DLS analysis showed a hydrodynamic diameter of dispersed nanoparticles. In Fig. 5, the average particle size of as-synthesized AgNPs was found to be 132.3 ± 75.5 nm. The PDI of the AgNPs was 0.326, ie-PDI > 0.1, clearly indicating a broad size distribution and polydispersed nature of AgNPs. DLS displayed a size greater than TEM results because of the inter-particle interactions and causes high aggregation or screening of smaller molecules by a bigger one [3,20,30]. The obtained TEM images of AgNPs justifying the observed DLS results.

FTIR analysis
FTIR analysis was performed to recognize the possible phytochemicals involved in the reduction of the metal nanoparticles as well as the capping of the formed nanoparticles. In Fig. 6, the strong broad spectrum at 3350 cm − 1 correspond to O-H stretching vibration of avonoids/ polyphenolic/ capsaicin [26]. The peaks at 2131 and 1638 cm − 1 represent the presence of CN/ -CO-Ag linkages and C = C/C = O groups, whereas 719 cm − 1 attributed to the bending vibration of alkane (C-H) of corresponding saturated hydrocarbons [33]. A variety of IR vibration are present in as-synthesized AgNPs, indicated that -OH and -CHO groups may bind with the Ag + and responsible for the reduction and also con rm the presence of organic coatings/ C. baccatum fruit phytochemical on the surface of AgNPs [22].

H 2 O 2 Sensing of AgNPs
The SPR based optical sensor for the determination of H 2 O 2 represents an important topic in textile, paper, cosmetics, pharmaceutical and food industries [34]. H 2 O 2 is an oxygen metabolite and exposure of small amount in process streams result oxidative stress and associated with aging and cancer and also environmental hazards due to its toxicity [35].

Conclusion
The present study exhibited a simple, cost-effective and green method for the synthesis of AgNPs using C. baccatum fruit extract in aqueous media through the in-situ reduction pathway. As-synthesized AgNPs showed a SPR band at 458 nm in UV-visible spectroscopy and almost spherical, aggregated with size distribution from 10 to 30 nm is con rmed by TEM and DLS analyses. From the FTIR spectrum, the avonoids/ polyphenolic/ capsaicin in the plant extract are more responsible in the reduction as well as capping/stabilization of AgNPs. The phytofunctionalized AgNPs showed a moderate colorimetric sensing activity for the decomposition of H 2 O 2 (> 50%, 15 mins) at concentration of 100 mM. In future, C.
baccatum fruit based AgNPs may be used as optical sensor for the determination of ROS• in different environmental, biomedical and food processing applications.

Con ict of Interests
The authors con rm they have no con ict of interests.

Figure 1
Major bioactive molecules present in Capsicum fruit.

Figure 2
Scheme for the phytosynthesis of AgNPs using C. baccatum fruit extract and their H2O2 sensing ability. TEM images of as-synthesized AgNPs.

Figure 5
DLS pattern of as-synthesized AgNPs.   Optical sensing spectra of AgNPs with time due to the introduction of 100 mM H2O2.

Supplementary Files
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