Fast colourimetric detection of H₂O₂ by biogenic silver nanoparticles synthesised using Benincasa hispida fruit extract

2.1 Preparation of silver nanoparticles

Benincasa hispida (common name: winter melon) fruit (shown in Figure 1) was collected from local market for preparing extract. A piece of 50 g of this fruit was taken, cleaned and chopped before crushing inside a grinder. After grinding, it was filtered and centrifuged at 2000 rpm for 5 min to get clear soup of B. hispida fruit extract. Pure silver nitrate required for this research was purchased from Merck India Limited, Mumbai, India. Now for preparing 20 mm stock solution of AgNO₃, 0.34 g pure silver nitrate was dissolved in 100 ml de-ionised (DI) water. To prepare silver nanoparticles, the silver ions present in the AgNO₃ solution were reduced by adding 100 ml fruit extract of B. hispida dropwise to it at room temperature. The reacting solution was then incubated in darkness, and a gradual change in the colour of the solution was observed after a few hours of incubation (shown in Figure 2). The solution was scanned using a UV-Vis spectroscope at regular intervals to get an insight of the formation of silver nanoparticles. Once the reaction was completed (after 12 h of incubation), the reacting colloidal mixture was centrifuged at 20,000 rpm for 30 min to separate the colloidal particles formed during interaction in the medium. The supernatant obtained after centrifugation was decanted, and the pellet formed at the bottom of the centrifuge tube was carefully re-dispersed in small amount of DI water before centrifuging it at 10,000 rpm for 15 min. This process was repeated to remove maximum biomass residue attached to the surface of silver nanoparticles. At the end, the precipitation of colloidal particles was collected carefully from the bottom of the tube and dried inside a vacuum drier to get dry powder of biogenic silver nanoparticles.

Figure 1:

Fresh Benincasa hispida fruit.

Figure 2:

Change in colour of the reacting solution.

2.2 Characterisation of Ag nanoparticles

Formation of nanosilver in the reacting solution was monitored by studying the absorption spectra of the solution at regular intervals using a Perkin Elmer ultraviolet-visible spectrometer (USA). The X-ray diffraction of the dried Ag nanoparticles was carried out by a Rigaku Ultima-III X-ray diffractometer (operating voltage 40 kV; λ=0.154 nm). FTIR spectroscopy of the dry nanoparticles was performed with the help of an IR-Prestige FTIR spectroscope (Shimadzu, Japan). To prepare the grid for the HRTEM study, the dry nanoparticles were suspended in DI water maintaining a concentration of nearly 50 μg/ml. Then a few drops of this suspension were placed on the copper grid and dried in a desiccator before scanning under high-resolution TEM (JEOL-2010; operating voltage 200 kV). EDX spectroscopy was carried out by placing two to three drops of this suspension on the EDX sample holder and drying it in the air before scanning under an EDX machine (Inca X-stream; Oxford Instruments).

2.3 Procedure for colourimetric sensing of H₂O₂

To study the hydrogen peroxide sensing ability of these biogenic silver nanoparticles, 10 mm aqueous stock solution of H₂O₂ (Merck India Ltd.) was taken and 1 ml of this solution was added to the light brown suspension of silver nanoparticles. After addition, the colour of the nano-suspension changed from light brown to colourless instantly. To assess the minimum detectable concentration of H₂O₂ by this method, the stock solution of hydrogen peroxide (10 mm) was diluted in the required amount of de-ionised water to prepare five different solutions of H₂O₂ with various concentrations, i.e. 1 mm, 2 mm, 4 mm, 6 mm and 10 mm. Then these different concentrations of H₂O₂ were added to the light brown suspension of silver nanoparticles and the UV-visible spectra of each sample were obtained by a UV-Vis spectrometer (Perkin Elmer, USA).

3.1 Green synthesis of silver nanoparticles

Fruit of Benincasa hispida is a proven source of functional bioactive molecules that can reduce the silver cations present in the reacting solution [32]. After an hour of the addition of fruit extract to the silver nitrate solution, a gradual change of the colour of the reacting solution was noticed. The colour of the reacting solution changed from colourless to light brown after an incubation period of 3–4 h (shown in Figure 2). The colour intensified with time and eventually became dark brown after 12 h of observation. This colour change may arise due to the surface plasmon resonance of the silver nanoparticles formed in the solution [33]. The formation of silver nanoparticles in the reacting solution was monitored by recording the absorption spectra of the solution at regular intervals (every 2 h) using a UV-Vis spectrometer (shown in Figure 3A). Maximum absorbance was noticed near wavelength 450 nm, which further confirmed the production of Ag nanoparticles. The peak absorbance was observed to be increasing with reaction time, and its variation is shown in Figure 3B. It can be inferred from the inset figure that the absorbance of the reacting solution continued to rise linearly up to 8 h. This may be due to the generation of more number of colloidal particles in the solution [34]. After 12 h, the rate of reaction was saturated, denoting the completion of reaction.

Figure 3:

(A) UV-Vis absorption spectra of the reacting solution. (B) Variation of peak absorbance with reaction time.

3.2 Characterisation of biogenic Ag nanoparticles

The X-ray diffraction curve of the dry Ag nanoparticles (shown in Figure 4) exhibits five distinct peaks at 2θ=27.85°, 32.06°, 46.27°, 54.85° and 76.92°, which may correspond to the (220), (122), (231), (331) and (311) planes of face centred cubic (fcc) structures of Ag, respectively (ref. to JCPDS: File card no. 04-0783). This result further confirms the crystallinity and purity of the green synthesised silver nanoparticles.

Figure 4:

XRD curve of biogenic Ag nanoparticles.

Figure 5 shows the high-resolution TEM images of the biosynthesised silver nanoparticles. It may be observed from the images that the particles are nearly spherical with an average diameter of 10 nm. The lattice fringes observed here indicate high crystallinity with an interplanar spacing of almost 0.282 nm that may be assigned to the (122) planes of the fcc phase of Ag nanoparticles.

Figure 5:

TEM images of green synthesised silver nanoparticles.

The FTIR spectra of the biogenic silver nanoparticles and the fruit extract are shown in Figure 6. The spectrum of fruit extract (recorded in absorbance mode) reveals four distinct peaks between 500 cm^-1 and 4000 cm^-1. Bands observed at 1614 and 1390 cm^-1 may be attributed to the C=O stretching vibration of amides and C–H bending vibration of alkanes, respectively [35]. Other two bands found near 1053 and 3282 cm^-1 may correspond to the stretching of C–N bonds and O–H bonds present in amines and aromatic compounds (such as phenol), respectively [36]. The IR spectrum of biosynthesised nanoparticles shows no specific peaks throughout the entire region, indicating high level of purity of the silver nanoparticles. From FTIR analysis, it may be inferred that the functional biomolecules such as amines, alkanes and phenols present in the fruit extract of Benincasa hispida probably reduced the silver ions and stabilised the colloidal particles during interaction in the reacting solution [29, 37].

Figure 6:

FTIR spectra of (a) fruit extract of Benincasa hispida and (b) biogenic silver nanoparticles.

EDX spectroscopy was performed in order to manipulate the atomic and weight percentage of metallic nanosilver obtained by this method. The EDX data (shown in Figure 7) reveal a couple of silver peaks that further confirmed the production of silver, and its amount was found to be nearly 21.5% as shown in the table embedded inside the curve. Small amounts of carbon and oxygen were also found due to the presence of capping agents on the surface of nanoparticles.

Figure 7:

EDX analysis biosynthesised silver nanoparticles.

3.3 Study of H₂O₂ detection

The light brown suspension of silver nanoparticles was studied as a colourimetric sensor for detecting H₂O₂ by the addition of various concentrations of hydrogen peroxide to it. The suspension of nanosilver decolourised after the addition of H₂O₂ (shown in Figure 8), and thus colourimetrically detected the presence of hydrogen peroxide instantly. The minimum detectable concentration of H₂O₂ for colourimetric sensing was evaluated by adding different concentrations of H₂O₂ to the suspension of silver nanoparticles and recording the variation of absorbance in the UV-Vis spectra of the solutions. Figure 9A shows that the minimum H₂O₂ concentration (i.e. 1 mm) added to the suspension of Ag nanoparticles reduced the intensity of absorbance slightly. The intensity of the absorbance peak gradually decreased with higher concentration of H₂O₂ and eventually disappeared at a concentration of more than 10 mm, as revealed in Figure 9A. The brown suspension of Ag nanoparticles was also exposed to a real natural sample of H₂O₂ (with concentration approx. 10 mm) for practical verification, and their interaction showed a similar result as stated above.

Figure 8:

Colourimetric sensing of H₂O₂ by suspension of Ag nanoparticles.

Figure 9:

(A) UV-Vis spectra of Ag nanoparticle suspension with different H₂O₂ concentrations. (B) Variation of peak absorbance with the concentration of H₂O₂.

The mechanism for sensing hydrogen peroxide by suspension of nanosilver may be explained in terms of high oxidising ability of H₂O₂. Hydrogen peroxide is a strong oxidiser that can oxidise Ag nanoparticles to Ag⁺¹ while reacts and forms silver oxide [38, 39]. As a result, the suspension of nanosilver eventually decolourises from light brown to colourless (see Figure 8) [40]. The chemical reaction is provided below:

Ag+H2O2=Ag2O+H2O

The colourimetric detection was performed using various concentration of H₂O₂ and the results indicate that the minimum detectable concentration of H₂O₂ that may be visually detected by this process is 1 mm (see Figure 9B). Thus, the biosynthesised stable Ag nanoparticles can be used to detect hazardous hydrogen peroxide successfully in real water samples as well [41].