Evaluation of Silver Nanoparticles Based on Fresh Cocoa Pods ( Theobroma Cacao ) Extracts as New Potential Electrode Material

Tis work focuses on the synthesis of silver nanoparticles using fresh cocoa pods from the “ Teobroma cacao ” extract plant through the reduction of silver ions (Ag + ) into Ag (0) by a green chemistry process, subsequently used as an electrode material. Reaction factors such as pH, incubation time, and silver ion concentration were optimized during the formation of nanoparticles. Te synthesized nanoparticles were characterized by ultraviolet spectrophotometry (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), powder X-ray difraction (PXRD), scanning electron microscopy (SEM)


Introduction
Nanotechnology is an area of extensive research which plays an important role in the manufacturing of new materials for application in nano-health delivery systems [1].Nanomaterials, specifcally metallic nanoparticles made of single or multiple metals (alloy nanoparticles), have signifcant interest in various felds ranging from materials science to biotechnology [2][3][4].Tey are widely used in medicine for antimicrobial medical products and personal care, in industry for the manufacture of building materials and wastewater fltration, in food processing, cosmetics, and in clothing manufacturing [5,6].Among those metallic nanoparticles, silver nanoparticles (AgNPs) are mostly used because nanometric silver is produced in good quantities.Silver nanoparticles are synthesized under various approaches, including chemical and biological methods [7].Te chemical approach requires styling agents (chemicals) for the stabilization of the nanoparticles which are toxic to organisms and the environment.Tus, the biocompatibility of the resulting AgNPs is too low for application in biological systems [8].Te biological method uses biological agents such as actinomycetes, bacteria, algae, fungi, plants, viruses, and yeasts [9].Moreover, not all biological entities can be used for the synthesis of nanoparticles because of their enzymatic activities and intrinsic metabolic processes.Terefore, an appropriate selection of the biological entity is needed to produce nanoparticles with interesting properties such as specifc size and morphology.For both chemical and biological methods, the use of costly chemicals or reagents is required, and the lifetime of some resulting nanoparticles is low [10].Terefore, the green chemistry process is proposed today by researchers as an alternative for the synthesis of silver nanoparticles, which is eco-friendly and sustainable [8].Te literature survey shows some work published recently for the synthesis of AgNPs by a green chemistry process through the use of plant extracts of Megaphrynium macrostachyum [11], Eucalyptus hybrid Myrtaceae [12], Helianthus annuus, Asteraceae [13], Oryza sativa, Poaceae [14], Mentha piperita, and Lamiaceae [15].However, the above AgNPs synthesized have never been used in the electroanalysis domain.Terefore, in the present work, apart from having as novelty the synthesis of silver nanoparticles from fresh cocoa pods (Teobroma Cacao), this work would show for the frst time that it is possible to use the resulting AgNPs as electrode material for the analysis of pathological compounds such as ascorbic acid (AA) and uric acid (UA), which are important and urgent analytes to be daily tested clinically.
Ascorbic acid and uric acid are coexisting constituents of pathological samples, which always appear at the same potential, making it difcult to separate them for their identifcation.In physiological samples, the normal range of ascorbic acid is 11.5-115 μmol/L and that of uric acid is 160-500 μmol/L [16,17].Te amount of these species is a marker of the health condition of human beings, as both the low level as well as the high level can cause diferent adverse health efects.Even if ascorbic acid is generally well tolerated by the humans in the normal range of 0.6-2 mg/dL, large doses may cause gastrointestinal discomfort, headache, trouble in sleeping, and fushing of the skin [16].Unusual concentrations of uric acid (high-levelinduced hyperuricemia) result in diseases such as arthritis, diabetes, gout, high blood pressure, hypertension, Lesch-Nyhan syndrome, neurological diseases, and obesity.Low levels of uric acid (low-level induced hypouricemia) in serum can result in cardiovascular disease, multiple sclerosis, Parkinson's, schizophrenia, and scurvy [18,19].
Given the above healthy efects and the industrial and biomedical importance of ascorbic acid and uric acid, it is important to develop a method for the daily monitoring of products consumed by humans.Tis passes through the elaboration of point-of-care testing (POCT) device for clinical applications.Several works have been published on this aim [16][17][18][19][20], but due to the lack of good stability, sensitivity, and electrocatalytic ability for the separation of pathological compounds, the research in this aspect is still ongoing.Te present work has following objectives: 1.To develop a green method for the synthesis of silver nanoparticles from Teobroma cacao extracts.2. To use the resulting AgNPs as electrode material to test their electrocatalytic ability for the separation of ascorbic acid and uric acid.Te societal implication of this work would then be the valorization of the plant, since, due to their large availability, they are usually considered waste for farms or as frewood.

Collection of Material and Preparation of Extracts.
Teobroma cocoa (Figure 1) was collected in the Santchou district (West region, Cameroon) and authenticated at the National Herbarium of Cameroon, compared to the reference sample of the herbarium according to the reference Teobroma cocoa Linn No. 60111/HNC.Fresh cocoa pods (Teobroma cacao) were frst washed with water, then cut into very small sizes, cleaned, and dried at room temperature.15 g of dried cocoa pods (CP) were introduced into a fask of water (W) (ratio 10 : 1 W/CP) previously heated to 80 °C for 10 min.Te mixture was stirred for 5 min.After being cooled to room temperature, the mixture was sieved under cotton, and the resulting extract was fltered using Whatman flter paper Nr1 and stored in the refrigerator at 4 °C.

Synthesis of Silver Nanoparticles.
Te procedure used is adapted to that proposed by Eya'ane et al. [21] with slight modifcations [21].In practice, diferent solutions of silver nitrate (1 mM, 2 mM, and 3 mM in 100 mL) were prepared and stored in tightly closed bottles to prevent the oxidation of silver.Subsequently, the aqueous extracts of Teobroma cocoa (TCC) were introduced into several vials numbered A to C, followed by the addition of silver nitrate solution in the volume ratio 5 : 1 (AgNO 3 /TCC); the mixture was left at room temperature for the bioreduction of silver ion.Te dark incubation time was set for 30 min, 1 h, 24 h, and 48 h to minimize the photoactivation of silver nitrate; the pH of the solution was adjusted to 6, 8, and 10 using 0.1 M NaOH or H 2 SO 4 .After each incubation time, the nanoparticle solutions were centrifuged at 6000 rpm for 1 h and washed twice with distilled water and once with ethanol (99.99%).Te fnal product obtained was dried in an oven at 60 °C for 24 h and stored in an eppendorf for future uses.

Physicochemical Characterization Techniques and
Apparatus.Te formation of silver nanoparticles was monitored after each incubation time, recording the UVvisible spectrum of the reaction mixture using a UV line 9100 UV-vis spectrophotometer.Te presence of various functional groups was identifed using FT-IR spectroscopy at ambient temperatures in the range of 4000 to 600 cm −1 with a Bruker Vertex 70.Scanning electron microscope (SEM) images were taken using the Zeiss SIGMA FE-SEM device to study the texture of the formed nanoparticles, and EDX 2 Journal of Chemistry spectra were carried out using a Zeiss SIGMA FE-SEM device to fnd the chemical elements and morphology of the nanoparticles based on extracts of fresh cocoa pods.Te elemental analysis was carried out using a thermo Scientifcfash 2000; the analysis by XRD was carried out at room temperature using a Bruker-AXS D8 X-ray difractometer.

Electrochemical Procedure and Equipment.
Prior to its modifcation, the glassy carbon electrode (GCE) was polished with diferent sizes of alumina powder (1; 0.3, and 0.05 µm sizes) and then sonicated in a 1 : 1 ethanol-water solution for 5 min.5 mg of the synthesized nanoparticles were dispersed with ultrasound for 10 min in 1 mL of distilled water.Ten 5 μL of AgNP suspension were then dropcoated on the surface of the clean GCE and dried at 50 °C.Te obtained electrode, namely, GCE/AgNPs, was used as a working electrode for the simultaneous determination of ascorbic acid and uric acid.For comparison, the bare GCE was also used for that purpose.Platinum wire and Ag/Ag + were used as counter and pseudo-reference electrodes, respectively.Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed using a PalmSens potentiostat equipped with the PS Trace software.Voltammograms were recorded with potential ranges of −0.4 V to 0.6 V, −0.5 V to 0.2 V, and 0.4 , respectively, at a concentration of 5 × 10 −3 M, with a scan rate of 20 mV/s.EIS was performed in a solution of 0.1 M KCl, 10 −3 M ions [Fe (CN)6] 3/4− with a frequency range of 0.1 Hz to 1000 Hz at a potential of 0.2 V.For the simultaneous determination of ascorbic acid and uric acid, 10 −4 M of their mixture was prepared in Briton Robinson bufer at pH 7, and the voltammograms were recorded in the potential range from 0.0 V to 1.80 V.

Determination of the Concentration of Nanoparticles in Solution.
Te nanoparticles concentration was determined by centrifugation, according to Eya'ane et al. [21]; inside three tubes, 30 mL solutions of the nanoparticles obtained at pHs 6, 8, and 10 were centrifuged for 30 min.A large amount of nanoparticles was produced at pH 10 compared to the quantities obtained at pH 6 and 8. Table 1 summarizes the concentrations of nanoparticles in each solution.Te study confrmed that basic pH is favorable for the synthesis of nanoparticle-mediated plant extracts.Tis can be explained by the fact that several metabolites of the extract reacted at this pH 10 for the reduction of silver ions.

Physico-Chemical Characterization of Silver
Nanoparticles Based on Teobroma cocoa

Visual Observation of Synthesized Silver Nanoparticles.
Te extract of fresh cocoa pods was subjected to incubation in contact with silver ions of concentration 1 mM in proportions 5 :1.A visual observation followed by the UV-Vis measurement was made.Initially, the extract was colorless, but after 30 min of stirring the silver solution with Teobroma cocoa extract, the solution changed from colorless to pale yellow (Figure 2(b)), then to brown yellow (Figure 2(c) and (d)), which suggests the formation of nanoparticles [21].Cocoa pods are made up of several secondary metabolites such as saponins, cellulose, favonoids, tannins, triterpenes, and steroids [22].Tese secondary metabolites are the origin of the bioreduction of the Ag + ion into Ag 0 to form intermediate quinones.

UV-Visible Spectral Analysis.
To optimize the synthesis of nanoparticles, the UV-Vis spectra of fresh cocoa pod extracts, silver nitrate solution, and its mixture were recorded (Figure 3).Diferent incubation times (30 min, 1 h, 24 h, and 48 h), pH 6, 8, and 10, and silver nitrate concentrations of 1 mM, 2 mM, and 3 mM were used.Te solutions exhibit a 400 nm plasmon resonance signal that indicates the presence of nanoparticles.Tis value is consistent with the plasmon resonance obtained using plants and silver ion [21].At pH 6 (Figure 4(a)), the plasmonic resonance absorbance increases with the incubation time, which is due to the increase of nanoparticle density in the solution [21].Tey are located between 423 and 436 nm, which is consistent with the formation of silver nanoparticles.At 48 h of incubation time, the curve shows signs of agglomeration with the presence of peaks (Figures 4(a)-4(c)).Tis suggests that the secondary metabolites have reacted totally and the nanoparticles started to grow with diferent kinetics due to the Brownian movement.At higher silver nitrate concentrations of 3 mM (pH 6), all curves show signs of agglomeration.
At 2 mM (Figure 4(b)), the formation of silver nanoparticles is slow compared to 1 mM and 3 mM.In general, it was observed that surface plasmon resonance bands increase with Ag + ion concentration [23,24].Tis could be explained by the fact that there is strong competition between the reducing molecules, which play an additional role as nanoparticle stabilizers.Chandran and collaborators using aloevera leaves in [25] pointed out that the concentration of the reaction medium could considerably infuence the shape of the nanoparticles [19].As postulated by Mie's theory, spherical nanoparticles appear as a single plasmon resonance surface band in the absorption spectra.Furthermore, anisotropic particles exhibit two or more bands, depending on the shape of the particles [26].Tese bands correspond to the  4 Journal of Chemistry appearance of a polydispersion due to the fact that the particles react with each other, but also to the simultaneous formation of particles of diferent nature such as Ag and AgCl [21].A similar observation was made with the pH adjusted to 8 and 10 (Figure 5).Te results obtained confrm that more nanoparticles are formed when the pH, the concentration of silver nitrate, and the incubation time increase.

Characterization by the FT-IR Spectroscopy.
To study the diferent functional groups of metabolites at the surface of nanosilver, we characterized the nanoparticles by the FT-IR spectroscopy, and the results are shown in Figure 6.Te FT-IR spectrum of raw cocoa pod powder (Figure 6(a)) shows a broad band that appears in the region of 3600 cm −1 to 3000 cm −1 with a maximum of around 3398 cm −1 .Tis maximum is attributable to the stretching of hydroxyl group (−OH) of carboxyl and phenol in lignin or cellulose.Te two bands observed at 1765 cm −1 and 1612 cm −1 were attributed, respectively, to the elongation vibrations of C=O and C=C, characteristic of the carboxylic acid metabolites.A wide band also appears between 1750 cm −1 and 1000 cm −1 with a maximum of 1031 cm −1 attributed to the C-O stretching vibration in the phenol, acid, and lactone groups [27].In the spectra of the nanoparticles (Figure 6(b)), the peaks situated at 1612 cm −1 and 1031 cm −1 of fresh Teobroma cocoa pods are displaced to lower frequencies 1525 cm −1 and 1010 cm −1 , respectively.Furthermore, the -OH peak observed at 3398 cm −1 in the raw pods is absent in the spectra of the nanomaterial, indicating that Ag + ions have been reduced by the hydroxyl group of the molecule in the extract.Tis shifting to lower frequencies indicates the adsorption of C-O, C=O, and C=C groups of alcohol and carboxylic acid metabolites of Teobroma cocoa extract to the surface of the silver nanoparticles [27].

Characterization by SEM.
Scanning electron microscopy was performed to determine the morphology of both raw cocoa pods (A) and AgNPs (B) and the size   7(c)).Tere is a variation in particle size with nearly 5% of particles in the 1 nm range, 10% of particles in the 1.5 nm range, 25% of particles in the 2 nm range, 45% in the 2.3 nm range, and 12% in the 3 nm range.A very small percentage of nanoparticles exist in the long 3.5 nm range.

EDX Analysis of Silver Nanoparticles.
Figure 8 shows the EDX spectrum of silver nanoparticles based on the extracts of fresh cocoa pods.EDX analysis was performed to confrm the formation of silver nanoparticles with the elemental mapping.Observation of the characteristic peaks of silver, carbon, oxygen, and chlorine is made.Te latter peaks, such as silica, aluminum, carbon, and oxygen, come from the phytochemicals present at the surface of the nanosilver, while chlorine is contained in AgCl nanoparticles.Table 2 summarizes all the chemical elements found in AgNPs as well as their mass and atomic concentration.A mixture of silver and silver chloride has been obtained using Selaginela myosorus plant extract.[29].Two peaks located at the binding energy of 1.5 KeV belonging to aluminum were observed as well as that of Silicon at 1.7 KeV.Furthermore, the sample contained a high concentration of silver nanoparticles and a high atomic percentage.

X-Ray Difraction of Nanoparticles from Aqueous
Extract of Fresh Cocoa Pods and Silver Nanoparticle-Mediated Fresh Cocoa Pods. Figure 9 shows the difractogram obtained from the fresh cocoa pods (a) and silver nanoparticles (b).Te difractogram of fresh raw cocoa pods shows wide difraction lines of an amorphous structure.Te PXRD pattern of the synthesized silver nanoparticles presents a crystalline peak at a 2θ value of 37.8 °that can be indexed to the plane (111) of the face-centred cubic structure (JCPDS fle: 65-2871).Te PXRD pattern also showed the presence of the cubic phase of silver chloride at 2θ values of 27.8 °, 32.3 °, 46.2 °, 54.8 °, 57.4 °, and 76.7 °corresponding to the (111,200,220,311,222) and (420) planes, respectively (JCPDS fle: 31-1238) (Table 3).Te calculated average crystalline particle size was found to be 17.60 and 30.40 nm for silver and silver chloride, respectively, using the Debye-Scherrer equation (1) [16] (equation ( 1)): where Dv is the average crystalline size; K is a dimensionless shape factor, with a value close to unity (0.99); λ is the wavelength of CuKα; β is the full width at half-maximum of the difraction peaks; and θ is Bragg's angle.Te average crystallinity size is 30.40 nm for AgCl and 17.60 nm for Ag.Te difraction pattern also shows unidentifed crystalline phases of silver at 23.10, 34.50, and 42.80 positions of 2θ.shown in Figures 10(a) and 10(b) prove the efciency of the nanoparticles synthesized in this work, which are performant in acid, neutral, and base media when drop-coated on GCE in comparison to the peak current obtained with the bare GCE.Te system is fast and enhanced in neutral medium compared to the acidic or basic medium.Moreover, the silver nanoparticle flm deposited on the surface of the GCE presents a parasitic oxidation signal, which appears around 0.03 V. Te presence of this parasitic signal is due to the oxidation of silver metal present in the nanoparticles.

Electrochemical Characterization of Silver Nanoparticles
Analysis of the [Ru (NH 3 ) 6 ] 3+ ion has also been achieved, and the results are shown in Figures 10(c) and 10(d).As in the case of ferricyanide, the system for ruthenium hexamine is also reversible with bare GCE, and when modifed with AgNPs, the background currents for oxidation and reduction are similar to those of GCE, even if the peaks are not well defned.
When using a neutral redox probe ([Fe (MeOH) 2 ]), the modifed GCE remained higher in intensity and faster than bare GCE at diferent pH, as shown in the Figures 10(e) and 10(f ).Te results obtained from this characterization confrm that silver nanoparticles based on fresh cocoa pods are very porous conductor and can fx mostly negative and    Journal of Chemistry neutral analytes in solution by electrostatic attraction and afnity phenomena, while repelling positive analytes.Tus, this result promotes the use of GCE/AgNPs as an electrochemical sensor for negative analytes in solution.12 shows the electrochemical behavior of ascorbic acid towards uric acid in the bufer solution of Briton Robinson at pH 7 using unmodifed GCE in the absence of ascorbic and uric acid (green).Analysis of this fgure reveals the presence of an oxidation peak centred at 0.30 V with GCE refecting the oxidation of ascorbic acid or uric acid or both.On the other hand, for GCE/AgNPs, two peaks of oxidation potential are observed around 0.37 V and 1.13 V corresponding, respectively, to ascorbic acid and uric acid [30].Tis result confrms the efectiveness of AgNPs against negatively charged analytes in solution, and the good electrocatalytic behavior of the fabricated electrode, as shown in Table 4 where the comparison in terms of the potential diference between ascorbic acid and uric acid is given with the diferent sensors.Te mechanism of their oxidation is given by Scheme 1.

Conclusion
Te synthesis and characterization of silver nanoparticles based on extracts of fresh cocoa pods have been reported.Te pH, incubation time, and concentration of silver nitrate greatly infuence the formation of nanoparticles.Characterization methods such as UV-vis, FT-IR spectroscopy including structural (XRD) and morphological (SEM) studies suggest that fresh cocoa pod extract plays an essential role during the bioreduction of silver ions and in the stabilization of silver nanoparticles.Electrochemical characterizations have shown that silver nanoparticles based on extracts of Teobroma cacao can be used as an electrode material for the electroanalysis of negatively or neutrally charged analytes in solution, since the electrostatic interaction will be favorable between the ion metallic Ag + and the negatively charged analyte.Electrochemical impedance spectroscopy showed a low charge-transfer resistance to GCE/AgNPs, which involves a rapid electron transfer.Te fabricated electrode shows a good electrocatalytic behavior for the separation of ascorbic acid and uric acid (around 0.60 V).Tis work allows us to propose a new axis of synthesis of electrode materials for various applications, in particular the simultaneous detection of certain organic compounds in drugs, which is currently being studied by our research team.[34].Journal of Chemistry

Figure 1 :
Figure 1: Fresh cocoa pods harvested in a feld located in the district of Santchou (West region Cameroon).

Figure 2 :Figure 3 :
Figure 2: Aqueous extract of fresh cocoa pod (a) and of silver nanoparticles after 30 min incubation time (b); after 24 h (c); and after 48 h (d).

Figure 7 :
Figure 7: Scanning electron microscopy of fresh cocoa pods (a) and silver nanoparticles synthesized from fresh cocoa pod extract (b) and size distribution (nm) of AgNPs (c).

Figure 8 :
Figure 8: EDX spectrum of silver nanoparticles based on extracts of fresh cocoa pods.

Scheme 1 :
Scheme 1: Proposed mechanism of the interaction between GCE/AgNPs and the two analytes.

Table 1 :
Determination of the nanoparticle concentration of solutions.

Table 2 :
Elemental analysis of silver nanoparticles based on Teobroma cacao.

Table 3 :
Main characteristics of the difractogram of the nanoparticles obtained with the aqueous extract of Teobroma cacao.
Spectroscopy.Te results obtained from the Nyquist graph for GCE and GCE/AgNPs are presented in Figure11.Te electrolysis (Re) and charge transfer (Rt) resistance for bare GCE and GCE/AgNPs were Re � 0 kΩ, R ct � 972.22 Ω, Re � 0 kΩ, and R ct � 333.33 Ω, respectively.Te low value of the R ct obtained with GCE/AgNPs refects a rapid electron transfer on GCE-AgNPs compared to GCE, which can be explained by the shielding of AgNPs by negatively charged [Fe (CN) 6 ] 3−/4− in solution as previously demonstrated in CV.
3.3.3.Application of GCE/AgNPs to the Simultaneous Detection of AscorbicAcid and Uric Acid.GCE/AgNPs have been applied for the simultaneous detection of ascorbic acid (AA) and uric acid (UA).Figure

Table 4 :
Comparison of the potential diference of AA-UA using diferent electrodes.