Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae Arunrat Khamhaengpol, Sineenat Siri PII: DOI: Reference:
S1011-1344(16)30483-3 doi: 10.1016/j.jphotobiol.2016.09.003 JPB 10555
To appear in: Received date: Accepted date:
23 June 2016 2 September 2016
Please cite this article as: Arunrat Khamhaengpol, Sineenat Siri, Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae, (2016), doi: 10.1016/j.jphotobiol.2016.09.003
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Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae
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Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
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Arunrat Khamhaengpola, Sineenat Sirib,*
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School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima
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30000, Thailand.
* Corresponding Author. Tel.: +66 89 7119112; fax: +66 44 22 4650.
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E-mail address: [email protected] (S. Siri).
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ABSTRACT Alternative to crude plant extracts, a crude protein extract derived from animal cells is one of the potential sources of biomolecules for mediating a reduction of silver ions and a formation of silver
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nanoparticles (AgNPs) under a mild condition, which very few works have been reported. This work demonstrated a use of the protein extract of weaver ant larvae as a bio-facilitator for a simple, green
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synthesis of AgNPs under fluorescent light at room temperature. The protein extract of weaver ant larvae exhibited the reducing and antioxidant activities, which assisted a formation of AgNPs in the
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reaction containing only silver nitrate under light exposure. Transmission electron microscopy images revealed the dispersed, spherical AgNPs with an average size of 7.87 2.54 nm. The maximum
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surface plasmon resonance (SPR) band of the synthesized AgNPs was at 435 nm. The energydispersive X-ray analysis revealed that silver was a major element of the particles. The identity of
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AgNPs was confirmed by X-ray diffraction pattern, selected area electron diffraction and high
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resolution transmission electron microscopy analyses, which demonstrated the planes of face centered cubic silver. The synthesized AgNPs showed antibacterial activity against both Escherichia coli and
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Staphylococcus aureus with the minimum bactericidal concentration (MBC) values equally at 250 µg/ml, suggesting their potential application as an effective antibacterial agent.
activity
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Keywords: Green synthesis, Silver nanoparticles, Weaver ant larvae, Fluorescent light, Antibacterial
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1. Introduction Due to unique catalytic, optical, thermal and electrical properties of nanoparticles (NPs), their syntheses and applications have attracted the interests of many researchers [1]. NPs have been used
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for numerous applications, for instance drug delivery [2], medical devices [3], biosensing [4], catalysis [5] and water treatment [6]. To supply a high demand of NPs in the past few decades, several methods
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have been developed, including physical methods (microwave radiation, ultrasonic irradiation, radiolysis and photochemical synthesis), chemical methods (chemical reduction and electrochemical
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synthesis), and biological methods (microorganisms, plant extracts and biomolecules) [7,8]. Among these methods, chemical synthesis is the most common approach because of its simple process and
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efficient production of highly dispersed, small and uniform nanoparticles. However, this method cannot avoid the use of hazardous chemicals, making the NPs often not suitable for biological
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purposes [9]. Therefore, green synthesis of NPs has been proposed as an alternative approach, which
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is required no sophisticated instrumentation, technical expertise and excessive use of hazardous chemicals. It is also proved to be more economical than other methods [10]. A green production of
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NPs have been reported by using living organisms including bacteria [11], fungi [12], algae [13] and plants [14,15]. Alternatively, biomolecules and extractions of microorganisms and plants have been used as reducing and stabilizing agents to mediate a formation of NPs [16,17].
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Silver nanoparticles (AgNPs) are well-known for their antibacterial activity to a broad spectrum of bacteria [18]. They have been comprised in many commercial products, such as plastics [19], food packaging [20], antiseptic sprays [21], catheters [22], bandages [23] and textiles [24]. Syntheses of AgNPs are often hobbled by the easy oxidization weakness, causing the loss of their antibacterial activity. To overcome this problem, different organic and inorganic templates have been employed to stabilize AgNPs through the formation of nanocomposites. However, those methods traditionally need complex and tedious procedures, and face problems of high cost and poor biocompatibility. Consequently, many plant extracts derived from vegetables and herbal plants were reported as the alternative chemicals for a green synthesis of AgNPs [25]. In addition, few proteins and certain amino acid were reported to aid as stabilizing and reducing agents for a production of AgNPs; casein [26], silk sericin [27], silk fibroin [28], egg white [29] and tryptophan [30]. Through a use of edible
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proteins, a green production of AgNPs should produce less or no toxic residue and be cost-effective, therefore the abundant and edible proteins of weaver ant larvae was purposed in this study. Weaver ants (Oecophylla smaragdina) are abundant in Southern India, Australia and Southeast
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Asia. The larvae of weaver ants mainly contain proteins and essential amino acids for larvae development and nest construction, especially high contents of tryptophan, leucine, threonine,
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methionine and lysine. Moreover, they also contain retinol, tocopherol, thiamine, niacin, riboflavin and ascorbic acid, which are several times higher than those in domestic fowl eggs [31]. Another
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important protein produced by weaver ant larvae is fibroin, the small fibrous protein used for a nest construction, which comprises of high amount of acidic amino acid and few glycine residue [32]. The
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plenty produced proteins in the larvae may be potentially served as reducing and/or stabilizing agents for green synthesis of AgNPs.
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In this article, we demonstrated a simple green synthesis approach of AgNPs by using a protein
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extract of weaver ant larvae as a biotemplate at room temperature under a fluorescent light exposure to activate the protein activity. The antioxidant and reducing properties of the protein extract were also
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investigated. The characterization and antibacterial activity of the synthesized AgNPs were examined.
2. Materials and methods
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2.1 Materials
Fresh weaver ant larvae (O. smaragdina) were collected from trees in the area of Suranaree University of Technology, Nakhon Ratchasima, Thailand. All chemicals used were of analytical grade. D-glucose and Muller Hinton (MH) medium were purchased from VWR (Belgium) and Merck (Germany), respectively. Silver nitrate (AgNO3) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (USA). 2.2 Preparation of the protein extract of weaver ant larvae Fresh weaver ant larvae (5 g) were frozen in liquid nitrogen and finely ground to obtain a homogeneous powder. Deionized water (10 ml) was added and incubated at 4 C for 5 min. The supernatant containing water-soluble proteins was harvested by centrifugation at 12,000 g for 5 min
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at 4 C. The protein concentration was determined by Bio-Rad protein assay (Bio-Rad, USA). The obtained proteins were separated on a 12.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with Coomassie brilliant blue R-250 dye.
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2.3 Determination of reducing power
The reducing power of the protein extract was determined by a modified method of Ferreira and
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colleagues [33]. Different concentrations of the protein extract (2.5 ml) were mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was
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incubated at 50 °C for 20 min prior to an addition of 2.5 ml of 10% trichloroacetic acid (w/v). The mixture was centrifuged at 3,000 g for 10 min. The upper layer of the solution (1.25 ml) was mixed
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with distilled water (1.25 ml) and a freshly prepared 0.1% of ferric chloride (0.25 ml). Color changes were monitored at the absorbance of 700 nm. All determinations were performed in four replications.
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2.4 Determination of DPPH radical scavenging activity
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The antioxidant activity of the protein extract of weaver ant larvae was determined using DPPH as described by Brand-Williams and colleagues [34] with slight modifications. Briefly, 150 µl of 1 10-4
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M DPPH solution was added to 10 µl of the protein extract. After the mixture was well mixed for 2 min, the absorbance at 515 nm was measured, which an ascorbic acid was used as the standard. The DPPH radical scavenging activity of the sample was calculated by the following equation, where Ac
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and As are the absorbance values of the control and sample, respectively. DPPH radical scavenging activity (%) = 100 × (AcAs)/Ac
The antioxidant activity of the protein extract was expressed as IC50, the concentration of the sample required to scavenge half of DPPH free radical. The IC50 is calculated from the graph plotting scavenging activity against sample concentrations, which all determinations were performed in four replications. 2.5 Synthesis of AgNPs by using the protein extract of weaver ant larvae To synthesize AgNPs, the reaction contained the protein extract of weaver ant larvae (0.36 mg/ml), 1 M AgNO3 and distilled water using a volume ratio of 19:1:0.5. The reaction contained an addition of a reducing sugar, 1 ml of 2 M aqueous solution of glucose, was also performed. The reaction mixtures
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were exposed to light using a fluorescent lamp (400 Lux, Philips, Thailand) at room temperature for 6, 12, 24, 48, 60 and 72 h. A formation of AgNPs was monitored by the absorbance scanning from 300900 nm using a spectrophotometer (Analytikjena Specord® 250 Plus, Germany). In addition, the
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reactions containing 1 M AgNO3 and different concentrations of the protein extract of weaver ant larvae (0.09, 0.18, 0.36, 0.72 and 1.44 mg/ml) were also performed for 72 h. A formation of AgNPs
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was also evaluated in a condition without light, which the reaction contained 1 M AgNO3 and the protein extract of weaver ant larvae (0.36 mg/ml) and incubated for 72 h.
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2.6 Characterization of the synthesized AgNPs
The morphology and size of the synthesized AgNPs were determined by a transmission electron
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microscope (TEM) using a Tecnai G2 20 S-Twin (FEI, USA) with operating at accelerating voltage 200 kV. The sample was prepared by placing a drop of colloidal solution on a carbon-coated copper
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grid and dried at room temperature before transferring it to the microscope.
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The crystalline nature of the synthesized AgNPs was analyzed by selected area electron diffraction (SAED) pattern and high resolution transmission electron microscope (HR-TEM) using a Tecnai G2
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S-Twin TEM operating at 200 kV with LaB6 filament. All images were recorded with a Gatan Orius 200 CCD Camera (Gatan, USA).
Elemental composition of the synthesized AgNPs was characterized by energy-dispersive X-ray
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(EDX) spectroscopy carried out on a Tecnai G2 20 S-Twin (FEI, USA). EDX analysis was equipped with an EDAX r-TEM SUTW detector (FEI, USA) operated at accelerating voltage 10 kV. Crystalline nature of the synthesized AgNPs was characterized by using the X-ray diffraction (XRD) pattern (D8 Advance, Bruker, UK) of Cu kα radiation (λ = 1.5418 Å) with a step size 0.02° within the 2θ range of the 3080 radians. Operating X-ray tube of voltage and current were 40 kV and 40 mA, respectively. The sample was coated on a glass coverslip and dried at room temperature before analyzing. 2.7 Evaluation of antibacterial activity The antibacterial activity of AgNPs was analyzed by a disc diffusion method [35] against Gramnegative (Escherichia coli, ATCC 25922) and Gram-positive (Staphylococcus aureus, ATCC 25923) bacteria. A single colony of each bacteria was initially cultured in MH broth at 37 °C and shaken at
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200 rpm for 6 h. The turbidity of the bacterial culture was adjusted to equal 0.5 McFarland standard as 1 × 108 colony-forming units/ml (CFU/ml). The bacteria culture (0.1 ml) mixed with MH agar (20 ml) at 45 °C was poured into a petri dish plate. The filter papers (Whatman No. 1) were punched into
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circular discs (6 mm in diameter) and sterilized by autoclaving at 121 C. Five µl of suspended AgNPs (5 µg/µl) were added onto each circular disc and dried in a laminar flow cabinet. The filter
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papers were placed on the MH agar plate mixed with microorganisms and incubated at 37 °C for 24 h. The zone of growth inhibition on the plate was measured with a caliper. The antibacterial activities
negative and the positive controls, respectively.
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were performed in triplicate that distilled water and antibiotic ampicillin (25 µg/disc) were used as the
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The minimum inhibitory concentration (MIC) of AgNPs was analyzed by using the standard broth dilution method against E. coli and S. aureus. The MIC was the lowest concentration of antimicrobial
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agents that completely visually inhibited 99% growth of microorganisms. A series of two-fold serial
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dilutions of AgNPs (from 250 µg/ml) was prepared and subsequently inoculated with the tested bacteria at a concentration of 5×105 CFU/ml. The cultures were incubated at 37 °C and shaken at 80
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rpm. Bacterial growth was examined by measuring the optical density (OD) at 600 nm. The aliquot of 100 µl from each culture with no visible observation of bacterial growth was spread on MH agar plates and incubated at 37 C for 24 h. After the incubation, the number of colonies grown on the agar
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was counted. The minimum bactericidal concentration (MBC) value was determined, which was defined as the lowest concentration of antimicrobial agent that kill 100% of the initial bacterial population [36]. 2.8 Statistical analysis All of the quantitative data were expressed as means standard deviations. Statistical comparisons were performed using one-way ANOVA with SPSS 11.5 for Windows software (SPSS, USA). Pvalues of less than 0.05 were considered statistically significant.
3. Results and discussion 3.1 The protein extract of weaver ant larvae
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Proteins of weaver ant larvae were extracted in distilled water, which an average of 0.013 g extracted proteins was obtained from 1 g wet weight of the larvae. The protein profile of the weaver ant larvae extract on a 12.5% gradient SDS-PAGE gel is shown in Fig. 1. The extracted proteins of
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molecular weights ranging from 10 to 200 kDa were obtained. Among these, three distinctive protein bands were observed at approximately 27, 76 and 165 kDa, which contained about 12.8% of total
were previously reported in Australian weaver ant [37].
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Fig. 1
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proteins. Based on the sizes, proteins in a range of 4050 kDa might be weaver ant fibroins, which
3.2 Reducing activity and DPPH radical scavenging activity of the protein extract
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Reducing activity of the protein extract was evaluated by the colorimetric reducing assay, which the degree of reducing activity of each compound was determined from a change of yellow color to various shades of green and blue. The presence of reducers (i.e. antioxidants) causes the reduction of
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the Fe3+/ferricyanide complex to the ferrous form, thus a measurement of the formation of Perl’s Prussian blue at 700 nm can monitor the Fe2+concentration [33]. Fig. 2A shows the reducing activity
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of the protein extract of weaver ant larvae, which exhibited a concentration-dependent response. As the concentration of the sample increased, the reducing activity also increased. The result suggested that the protein extract could serve as a good electron donor, thus reacting with free radicals to convert
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them into more stable nonreactive species and to terminate a radical chain reaction [38]. The antioxidant capacity of the protein extract of weaver ant larvae was evaluated using the DPPH radical scavenging assay. DPPH radical scavenging activity assesses the capacity of an extract to donate hydrogen or scavenge free radicals, such as superoxide and hydroxyl radicals. When the DPPH radical is scavenged, the reaction mixture changes from purple to yellow with decreasing of absorbance at 515 nm [39]. Fig. 2B illustrates the percentage DPPH scavenging effect for the protein extract of weaver ant larvae at various concentrations ranging from 0.125-8.0 mg/ml. The DPPH radical scavenging activities of the protein extract of weaver ant larvae increased gradually in a dosedependent manner. At a concentration of 8.0 mg/ml, the protein extract showed 84.1% DPPH radical scavenging activity and IC50 value of 5.3 mg/ml. Therefore, some water-soluble proteins and antioxidants in the extract of weaver ant larvae, such as thiamine, niacin, riboflavin and ascorbic acid
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[31] that possessed antioxidant activity could be used as the reducing and stabilizing agents to facilitate a synthesis of AgNPs. Fig. 2
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3.3 Green synthesis of AgNPs
Based on its reducing and antioxidant activities, the protein extract of weaver ant larvae was
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applied for a green synthesis of AgNPs. The reactions containing the protein extract and AgNO3 solution with or without glucose as the additional reducing agent were carried out at room temperature
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under the fluorescent light. The formation of AgNPs could be observed from the color changes from transparent to brownish. Absorption spectra in a range of 300900 nm were determined to monitor a
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formation of AgNPs. The surface plasmon resonance (SPR) characteristic of AgNPs was determined from the max value between 400500 nm, which was responsive to their size and shape [10].
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Fig. 3 shows the absorption spectra of the synthesized AgNPs at different time intervals of 0, 6, 12,
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24, 48, 60 and 72 h. The formation of AgNPs was observed in both reactions with a presence and absence of glucose, suggesting that the protein extract could serve as good reducing and stabilizing
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agents in these reactions. The characteristic peak of AgNPs was clearly observed in the reactions incubated for 2472 h, both with and without glucose, suggesting a formation of AgNPs. The production of AgNPs was gradually increased until 72 h, as determined by the increasing intensity of
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the SPR peak. The maximum SPR peaks of the reactions with the presence and absence of glucose were similarly at 432 and 435 nm, respectively. Based on the SPR intensity at 72 h, the quantity of synthesized AgNPs in the glucose-containing reaction was slightly higher than the reaction without glucose, suggesting that an addition of glucose as a reducing agent could facilitate a higher formation of AgNPs. In the reaction containing only AgNO3 and glucose, the SPR characteristic peak of AgNPs was not observed, which was likely due to a lack of capping agent to facilitate a formation of stabilized AgNPs [40]. Fig. 3 Different concentrations of the protein extracts of weaver ant larvae were also used in the 72-h reactions to produce AgNPs without the presence of glucose under the fluorescent light as seen in Fig.
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4A. With increasing concentrations of the protein extracts, the increasing intensities of the SPR peak were observed, suggesting that the higher protein content promoted the higher production of AgNPs. To study the effect of fluorescent light, the reactions containing only AgNO3 and the protein extract
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were incubated for 72 h under the conditions with and without fluorescent light. Under a condition without fluorescent light, no characteristic SPR peak was detected (Fig. 4B) and no color change was
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observed (data not shown). This clearly indicated the important role of photocatalytic activity of fluorescent light in a reduction of silver. Therefore, it is very likely that the extracted proteins mediate
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a reduction of metal ions via a photo-induced electron transfer reaction [5]. In the extracted proteins, tryptophan, tyrosine and phenylalanine residues of protein chains are the major amino acids that can
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serve as reducing agents. Under light exposure, the indolic group of tryptophan, phenolic group of tyrosine and benzyl group of phenylalanine can be induced to form indolyl (Trp ), phenoxyl (Try) and benzyl radicals, respectively. This photoactivation process also causes a production of the
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hydrated electron (eaq), which can lead to a reduction Ag to form metallic AgNPs (Ag0) [41-44]. In addition, among many proteins in insect larvae, antioxidant enzymes, such as superoxide dismutase
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(SOD) and catalase (CAT), which exhibit high reducing activity, may be ones of the proteins facilitating a reduction of silver ions and a formation of AgNPs [45-47]. Besides, some water-soluble
Fig. 4
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antioxidants in weaver ant larvae [31], may assist a production of AgNPs.
3.5 Characterization of the synthesized AgNPs The representative TEM image of the synthesized AgNPs is shown in Fig. 5, which clearly revealed spherical, mono-dispersive AgNPs. Their diameters were distributed from approximately 2 to 16 nm, and the average diameter was 7.87 2.54 nm as determined from 50 random individuals. Due to their small sizes, the synthesized AgNPs might exhibit a good antibacterial activity. AgNPs in a range of 110 nm were reported to exhibit a higher antibacterial activity than the larger sizes because of a greater surface area for interacting bacterial surface and a feasible penetration inside the bacterial cells [48]. Fig. 5
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The identity of the synthesized AgNPs was further investigated by using EDX, HR-TEM, TEMSAED, and XRD analyses. The elemental composition of the synthesized AgNPs was detected by a TEM equipped EDX. The EDX spectrum result (Fig. 6A) showed a strong spectral signal of silver at a
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2.9-keV region, indicating that silver was a dominant element of the synthesized particles. In addition, signals of carbon and copper were also detected, which were likely from a carbon-coated copper grid
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that the samples were deposited on. In Fig. 6B, HR-TEM image of individual synthesized AgNPs indicated the d-spacing of the crystallographic plane of 0.23 nm, which agreed well with the
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interplanar separation of the (111) lattice plane of the face-centered cubic silver. The crystalline nature of AgNPs was demonstrated as the result of TEM-SAED in Fig. 6C. The TEM-SAED exhibited
centered cubic structure of AgNPs [49].
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Fig. 6
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patterns corresponding to the (111), (200), (220), (311) and (222) Bragg’s reflection of the face-
X-ray diffraction is normally used to confirm the crystalline nature of particles. The XRD pattern
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of the synthesized AgNPs is illustrated in Fig. 7. The results indicated two diffraction peaks at 2θ
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values of 38.24° and 44.47°, which corresponded to the planes (111) and (200) of the face-centered cubic (fcc) lattice of silver (JCPDS file no. 01-087-0718), respectively [50]. Fig. 7
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3.6 Antibacterial activity of the synthesized AgNPs Antibacterial activity of AgNPs was initially assessed by using a disk diffusion assay. Fig. 8 shows the growth inhibition zones of AgNPs (25 g) against E. coli and S. aureus, which were seen as the clear circles surrounded the filter papers impregnated with AgNPs. The average diameters of clear zones of AgNPs against E. coli and S. aureus were equally at 9.3 ± 0.6 mm. The growth inhibition zone of ampicillin (25 g), the positive control, was 16.3 ± 0.6 mm, while no growth inhibition zone of distilled water, the negative control, was observed. Fig. 8 The bacteriostatic and bactericidal effects of the synthesized AgNPs were further investigated via a micro-broth dilution assay. The synthesized AgNPs of different concentrations (a serial two-fold
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dilution from 16250 μg/ml) were employed to further examine the antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. The effects of AgNPs on growth inhibition of both bacteria in a time course of 24 h are shown in Fig. 9. As the concentration of AgNPs
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was increased, the more inhibition of the bacterial growth was observed in both bacterial strains, suggesting the dose response effect of AgNPs on bacterial growth inhibition. At 24 h of the
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incubation, the minimum inhibitory concentration (MIC) against E. coli and S. aureus were equally at 125 μg/ml and the minimum bactericidal concentration (MBC) against both bacterial strains were
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equally at 250 μg/ml. The antimicrobial activity of the produced AgNPs against both Gram negative and Gram positive bacteria could be beneficial for their application as an antibacterial agent. With
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their small size, they would be easily attached and penetrated inside the bacterial cells. To inhibit bacterial growth, AgNPs could disturb membrane permeability and respiration of the cells. In
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addition, they could block the functions of some proteins and nucleic acids as well as cause a
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formation of free radicals that induce membrane damages, eventually resulting in cell death [48,51]. Fig. 9
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4. Conclusions
This investigation demonstrated a simple, cost-effective, green synthetic approach of AgNPs using only the protein extract of weaver ant larvae and silver nitrate solution under an exposure of
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fluorescent light at room temperature. Reducing and antioxidant activities of the protein extract suggested its roles as a reducing agent to mediate a formation of AgNPs. The protein extract also helped to stabilize the mono-dispersive, spherical AgNPs of approximately 8 nm. The activity of the protein extract to assist a formation of AgNPs required light activation. Without light exposure, a formation of AgNPs was not detected within 72 h of the reaction. The identity of the produced AgNPs was confirmed by TEM-SAED, HR-TEM, EDX and XRD analyses. Antibacterial activity of the produced AgNPs against both Gram-negative E. coli and Gram-positive S. aureus also suggested their potential application as a good antibacterial agent.
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Acknowledgements Funding of this work is supported by Suranaree University of Technology-National Research Fund under Grant No. SUT-1-104-57-24-20. The student academic scholarship is provided by the Science
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Achievement Scholarship of Thailand (SAST) of the Commission on Higher Education, Thailand.
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Figure Captions Fig. 1. Images of waver ant larvae at the 3rd instar stage (A), a comparison of the larvae with the adult ants (B), and the protein extract of weaver ant larvae visualized on a 12.5% SDS-PAGE gel (C).
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Lane M is the standard protein markers.
Fig. 2. Reducing power (A) and scavenging activity (%) on DPPH radicals (B) of the protein extract
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of weaver ant larvae at different concentrations. The data are presented as the mean standard deviation of four replicate samples.
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Fig. 3. UV-Vis spectra of synthesized AgNPs using the protein extract of weaver ant larvae in the presence (A) and absence (B) of glucose. The reactions containing 1 M AgNO3 and 0.36 mg/ml
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protein extract (prot) in the presence or absence of glucose (glu) were exposed to fluorescent light at room temperature.
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Fig. 4. Effects of protein concentrations and fluorescent light on a formation of AgNPs. A) UV-Vis
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spectra of synthesized AgNPs in the reactions containing 1 M AgNO3 and various concentrations of the protein extract of weaver ant larvae under an exposure of fluorescent light
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for 72 h. B) UV-Vis spectra of synthesized AgNPs in the reactions containing 1 M AgNO3 and 0.36 mg/ml protein extract under the conditions with and without fluorescent light exposure for 72 h.
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Fig. 5. TEM image (A) and the histogram of size distribution (B) of the synthesized AgNPs. Fig. 6. TEM-EDX (A), HR-TEM (B) and TEM-SAED (C) analyses of the synthesized AgNPs. Fig. 7. The XRD pattern of the synthesized AgNPs was interpreted with JCPDS (No. 01-087-0718). The diffraction planes at (111) and (200) depicts that the synthesized AgNPs are cubic crystalline in nature. Fig. 8. Growth inhibition zone of E. coli (A) and S. aureus (B) in response to the synthesized AgNPs. 1: the negative control (distilled water), 2: the positive control (ampicillin), and 3: the synthesized AgNPs (25 µg).
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Fig. 9. Growth inhibition curves of E. coli (A) and S. aureus (B) exposed to different concentrations of the synthesized AgNPs in a time course of 24 h. The data are presented as mean standard
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Graphical abstract
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1. AgNPs was synthesized using the protein extract of ant larvae under light exposure. 2. Small AgNPs of approximately 8 nm was produced.
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3. The AgNPs exhibited antibacterial activity against both E. coli and S. aureus.
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S1011-1344(16)30483-3 doi: 10.1016/j.jphotobiol.2016.09.003 JPB 10555
To appear in: Received date: Accepted date:
23 June 2016 2 September 2016
Please cite this article as: Arunrat Khamhaengpol, Sineenat Siri, Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae, (2016), doi: 10.1016/j.jphotobiol.2016.09.003
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Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae
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Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
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Arunrat Khamhaengpola, Sineenat Sirib,*
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School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima
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30000, Thailand.
* Corresponding Author. Tel.: +66 89 7119112; fax: +66 44 22 4650.
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E-mail address: [email protected] (S. Siri).
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ABSTRACT Alternative to crude plant extracts, a crude protein extract derived from animal cells is one of the potential sources of biomolecules for mediating a reduction of silver ions and a formation of silver
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nanoparticles (AgNPs) under a mild condition, which very few works have been reported. This work demonstrated a use of the protein extract of weaver ant larvae as a bio-facilitator for a simple, green
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synthesis of AgNPs under fluorescent light at room temperature. The protein extract of weaver ant larvae exhibited the reducing and antioxidant activities, which assisted a formation of AgNPs in the
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reaction containing only silver nitrate under light exposure. Transmission electron microscopy images revealed the dispersed, spherical AgNPs with an average size of 7.87 2.54 nm. The maximum
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surface plasmon resonance (SPR) band of the synthesized AgNPs was at 435 nm. The energydispersive X-ray analysis revealed that silver was a major element of the particles. The identity of
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AgNPs was confirmed by X-ray diffraction pattern, selected area electron diffraction and high
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resolution transmission electron microscopy analyses, which demonstrated the planes of face centered cubic silver. The synthesized AgNPs showed antibacterial activity against both Escherichia coli and
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Staphylococcus aureus with the minimum bactericidal concentration (MBC) values equally at 250 µg/ml, suggesting their potential application as an effective antibacterial agent.
activity
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Keywords: Green synthesis, Silver nanoparticles, Weaver ant larvae, Fluorescent light, Antibacterial
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1. Introduction Due to unique catalytic, optical, thermal and electrical properties of nanoparticles (NPs), their syntheses and applications have attracted the interests of many researchers [1]. NPs have been used
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for numerous applications, for instance drug delivery [2], medical devices [3], biosensing [4], catalysis [5] and water treatment [6]. To supply a high demand of NPs in the past few decades, several methods
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have been developed, including physical methods (microwave radiation, ultrasonic irradiation, radiolysis and photochemical synthesis), chemical methods (chemical reduction and electrochemical
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synthesis), and biological methods (microorganisms, plant extracts and biomolecules) [7,8]. Among these methods, chemical synthesis is the most common approach because of its simple process and
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efficient production of highly dispersed, small and uniform nanoparticles. However, this method cannot avoid the use of hazardous chemicals, making the NPs often not suitable for biological
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purposes [9]. Therefore, green synthesis of NPs has been proposed as an alternative approach, which
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is required no sophisticated instrumentation, technical expertise and excessive use of hazardous chemicals. It is also proved to be more economical than other methods [10]. A green production of
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NPs have been reported by using living organisms including bacteria [11], fungi [12], algae [13] and plants [14,15]. Alternatively, biomolecules and extractions of microorganisms and plants have been used as reducing and stabilizing agents to mediate a formation of NPs [16,17].
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Silver nanoparticles (AgNPs) are well-known for their antibacterial activity to a broad spectrum of bacteria [18]. They have been comprised in many commercial products, such as plastics [19], food packaging [20], antiseptic sprays [21], catheters [22], bandages [23] and textiles [24]. Syntheses of AgNPs are often hobbled by the easy oxidization weakness, causing the loss of their antibacterial activity. To overcome this problem, different organic and inorganic templates have been employed to stabilize AgNPs through the formation of nanocomposites. However, those methods traditionally need complex and tedious procedures, and face problems of high cost and poor biocompatibility. Consequently, many plant extracts derived from vegetables and herbal plants were reported as the alternative chemicals for a green synthesis of AgNPs [25]. In addition, few proteins and certain amino acid were reported to aid as stabilizing and reducing agents for a production of AgNPs; casein [26], silk sericin [27], silk fibroin [28], egg white [29] and tryptophan [30]. Through a use of edible
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proteins, a green production of AgNPs should produce less or no toxic residue and be cost-effective, therefore the abundant and edible proteins of weaver ant larvae was purposed in this study. Weaver ants (Oecophylla smaragdina) are abundant in Southern India, Australia and Southeast
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Asia. The larvae of weaver ants mainly contain proteins and essential amino acids for larvae development and nest construction, especially high contents of tryptophan, leucine, threonine,
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methionine and lysine. Moreover, they also contain retinol, tocopherol, thiamine, niacin, riboflavin and ascorbic acid, which are several times higher than those in domestic fowl eggs [31]. Another
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important protein produced by weaver ant larvae is fibroin, the small fibrous protein used for a nest construction, which comprises of high amount of acidic amino acid and few glycine residue [32]. The
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plenty produced proteins in the larvae may be potentially served as reducing and/or stabilizing agents for green synthesis of AgNPs.
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In this article, we demonstrated a simple green synthesis approach of AgNPs by using a protein
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extract of weaver ant larvae as a biotemplate at room temperature under a fluorescent light exposure to activate the protein activity. The antioxidant and reducing properties of the protein extract were also
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investigated. The characterization and antibacterial activity of the synthesized AgNPs were examined.
2. Materials and methods
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2.1 Materials
Fresh weaver ant larvae (O. smaragdina) were collected from trees in the area of Suranaree University of Technology, Nakhon Ratchasima, Thailand. All chemicals used were of analytical grade. D-glucose and Muller Hinton (MH) medium were purchased from VWR (Belgium) and Merck (Germany), respectively. Silver nitrate (AgNO3) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (USA). 2.2 Preparation of the protein extract of weaver ant larvae Fresh weaver ant larvae (5 g) were frozen in liquid nitrogen and finely ground to obtain a homogeneous powder. Deionized water (10 ml) was added and incubated at 4 C for 5 min. The supernatant containing water-soluble proteins was harvested by centrifugation at 12,000 g for 5 min
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at 4 C. The protein concentration was determined by Bio-Rad protein assay (Bio-Rad, USA). The obtained proteins were separated on a 12.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with Coomassie brilliant blue R-250 dye.
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2.3 Determination of reducing power
The reducing power of the protein extract was determined by a modified method of Ferreira and
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colleagues [33]. Different concentrations of the protein extract (2.5 ml) were mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was
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incubated at 50 °C for 20 min prior to an addition of 2.5 ml of 10% trichloroacetic acid (w/v). The mixture was centrifuged at 3,000 g for 10 min. The upper layer of the solution (1.25 ml) was mixed
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with distilled water (1.25 ml) and a freshly prepared 0.1% of ferric chloride (0.25 ml). Color changes were monitored at the absorbance of 700 nm. All determinations were performed in four replications.
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2.4 Determination of DPPH radical scavenging activity
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The antioxidant activity of the protein extract of weaver ant larvae was determined using DPPH as described by Brand-Williams and colleagues [34] with slight modifications. Briefly, 150 µl of 1 10-4
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M DPPH solution was added to 10 µl of the protein extract. After the mixture was well mixed for 2 min, the absorbance at 515 nm was measured, which an ascorbic acid was used as the standard. The DPPH radical scavenging activity of the sample was calculated by the following equation, where Ac
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and As are the absorbance values of the control and sample, respectively. DPPH radical scavenging activity (%) = 100 × (AcAs)/Ac
The antioxidant activity of the protein extract was expressed as IC50, the concentration of the sample required to scavenge half of DPPH free radical. The IC50 is calculated from the graph plotting scavenging activity against sample concentrations, which all determinations were performed in four replications. 2.5 Synthesis of AgNPs by using the protein extract of weaver ant larvae To synthesize AgNPs, the reaction contained the protein extract of weaver ant larvae (0.36 mg/ml), 1 M AgNO3 and distilled water using a volume ratio of 19:1:0.5. The reaction contained an addition of a reducing sugar, 1 ml of 2 M aqueous solution of glucose, was also performed. The reaction mixtures
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were exposed to light using a fluorescent lamp (400 Lux, Philips, Thailand) at room temperature for 6, 12, 24, 48, 60 and 72 h. A formation of AgNPs was monitored by the absorbance scanning from 300900 nm using a spectrophotometer (Analytikjena Specord® 250 Plus, Germany). In addition, the
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reactions containing 1 M AgNO3 and different concentrations of the protein extract of weaver ant larvae (0.09, 0.18, 0.36, 0.72 and 1.44 mg/ml) were also performed for 72 h. A formation of AgNPs
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was also evaluated in a condition without light, which the reaction contained 1 M AgNO3 and the protein extract of weaver ant larvae (0.36 mg/ml) and incubated for 72 h.
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2.6 Characterization of the synthesized AgNPs
The morphology and size of the synthesized AgNPs were determined by a transmission electron
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microscope (TEM) using a Tecnai G2 20 S-Twin (FEI, USA) with operating at accelerating voltage 200 kV. The sample was prepared by placing a drop of colloidal solution on a carbon-coated copper
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grid and dried at room temperature before transferring it to the microscope.
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The crystalline nature of the synthesized AgNPs was analyzed by selected area electron diffraction (SAED) pattern and high resolution transmission electron microscope (HR-TEM) using a Tecnai G2
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S-Twin TEM operating at 200 kV with LaB6 filament. All images were recorded with a Gatan Orius 200 CCD Camera (Gatan, USA).
Elemental composition of the synthesized AgNPs was characterized by energy-dispersive X-ray
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(EDX) spectroscopy carried out on a Tecnai G2 20 S-Twin (FEI, USA). EDX analysis was equipped with an EDAX r-TEM SUTW detector (FEI, USA) operated at accelerating voltage 10 kV. Crystalline nature of the synthesized AgNPs was characterized by using the X-ray diffraction (XRD) pattern (D8 Advance, Bruker, UK) of Cu kα radiation (λ = 1.5418 Å) with a step size 0.02° within the 2θ range of the 3080 radians. Operating X-ray tube of voltage and current were 40 kV and 40 mA, respectively. The sample was coated on a glass coverslip and dried at room temperature before analyzing. 2.7 Evaluation of antibacterial activity The antibacterial activity of AgNPs was analyzed by a disc diffusion method [35] against Gramnegative (Escherichia coli, ATCC 25922) and Gram-positive (Staphylococcus aureus, ATCC 25923) bacteria. A single colony of each bacteria was initially cultured in MH broth at 37 °C and shaken at
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200 rpm for 6 h. The turbidity of the bacterial culture was adjusted to equal 0.5 McFarland standard as 1 × 108 colony-forming units/ml (CFU/ml). The bacteria culture (0.1 ml) mixed with MH agar (20 ml) at 45 °C was poured into a petri dish plate. The filter papers (Whatman No. 1) were punched into
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circular discs (6 mm in diameter) and sterilized by autoclaving at 121 C. Five µl of suspended AgNPs (5 µg/µl) were added onto each circular disc and dried in a laminar flow cabinet. The filter
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papers were placed on the MH agar plate mixed with microorganisms and incubated at 37 °C for 24 h. The zone of growth inhibition on the plate was measured with a caliper. The antibacterial activities
negative and the positive controls, respectively.
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were performed in triplicate that distilled water and antibiotic ampicillin (25 µg/disc) were used as the
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The minimum inhibitory concentration (MIC) of AgNPs was analyzed by using the standard broth dilution method against E. coli and S. aureus. The MIC was the lowest concentration of antimicrobial
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agents that completely visually inhibited 99% growth of microorganisms. A series of two-fold serial
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dilutions of AgNPs (from 250 µg/ml) was prepared and subsequently inoculated with the tested bacteria at a concentration of 5×105 CFU/ml. The cultures were incubated at 37 °C and shaken at 80
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rpm. Bacterial growth was examined by measuring the optical density (OD) at 600 nm. The aliquot of 100 µl from each culture with no visible observation of bacterial growth was spread on MH agar plates and incubated at 37 C for 24 h. After the incubation, the number of colonies grown on the agar
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was counted. The minimum bactericidal concentration (MBC) value was determined, which was defined as the lowest concentration of antimicrobial agent that kill 100% of the initial bacterial population [36]. 2.8 Statistical analysis All of the quantitative data were expressed as means standard deviations. Statistical comparisons were performed using one-way ANOVA with SPSS 11.5 for Windows software (SPSS, USA). Pvalues of less than 0.05 were considered statistically significant.
3. Results and discussion 3.1 The protein extract of weaver ant larvae
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Proteins of weaver ant larvae were extracted in distilled water, which an average of 0.013 g extracted proteins was obtained from 1 g wet weight of the larvae. The protein profile of the weaver ant larvae extract on a 12.5% gradient SDS-PAGE gel is shown in Fig. 1. The extracted proteins of
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molecular weights ranging from 10 to 200 kDa were obtained. Among these, three distinctive protein bands were observed at approximately 27, 76 and 165 kDa, which contained about 12.8% of total
were previously reported in Australian weaver ant [37].
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Fig. 1
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proteins. Based on the sizes, proteins in a range of 4050 kDa might be weaver ant fibroins, which
3.2 Reducing activity and DPPH radical scavenging activity of the protein extract
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Reducing activity of the protein extract was evaluated by the colorimetric reducing assay, which the degree of reducing activity of each compound was determined from a change of yellow color to various shades of green and blue. The presence of reducers (i.e. antioxidants) causes the reduction of
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the Fe3+/ferricyanide complex to the ferrous form, thus a measurement of the formation of Perl’s Prussian blue at 700 nm can monitor the Fe2+concentration [33]. Fig. 2A shows the reducing activity
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of the protein extract of weaver ant larvae, which exhibited a concentration-dependent response. As the concentration of the sample increased, the reducing activity also increased. The result suggested that the protein extract could serve as a good electron donor, thus reacting with free radicals to convert
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them into more stable nonreactive species and to terminate a radical chain reaction [38]. The antioxidant capacity of the protein extract of weaver ant larvae was evaluated using the DPPH radical scavenging assay. DPPH radical scavenging activity assesses the capacity of an extract to donate hydrogen or scavenge free radicals, such as superoxide and hydroxyl radicals. When the DPPH radical is scavenged, the reaction mixture changes from purple to yellow with decreasing of absorbance at 515 nm [39]. Fig. 2B illustrates the percentage DPPH scavenging effect for the protein extract of weaver ant larvae at various concentrations ranging from 0.125-8.0 mg/ml. The DPPH radical scavenging activities of the protein extract of weaver ant larvae increased gradually in a dosedependent manner. At a concentration of 8.0 mg/ml, the protein extract showed 84.1% DPPH radical scavenging activity and IC50 value of 5.3 mg/ml. Therefore, some water-soluble proteins and antioxidants in the extract of weaver ant larvae, such as thiamine, niacin, riboflavin and ascorbic acid
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[31] that possessed antioxidant activity could be used as the reducing and stabilizing agents to facilitate a synthesis of AgNPs. Fig. 2
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3.3 Green synthesis of AgNPs
Based on its reducing and antioxidant activities, the protein extract of weaver ant larvae was
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applied for a green synthesis of AgNPs. The reactions containing the protein extract and AgNO3 solution with or without glucose as the additional reducing agent were carried out at room temperature
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under the fluorescent light. The formation of AgNPs could be observed from the color changes from transparent to brownish. Absorption spectra in a range of 300900 nm were determined to monitor a
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formation of AgNPs. The surface plasmon resonance (SPR) characteristic of AgNPs was determined from the max value between 400500 nm, which was responsive to their size and shape [10].
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Fig. 3 shows the absorption spectra of the synthesized AgNPs at different time intervals of 0, 6, 12,
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24, 48, 60 and 72 h. The formation of AgNPs was observed in both reactions with a presence and absence of glucose, suggesting that the protein extract could serve as good reducing and stabilizing
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agents in these reactions. The characteristic peak of AgNPs was clearly observed in the reactions incubated for 2472 h, both with and without glucose, suggesting a formation of AgNPs. The production of AgNPs was gradually increased until 72 h, as determined by the increasing intensity of
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the SPR peak. The maximum SPR peaks of the reactions with the presence and absence of glucose were similarly at 432 and 435 nm, respectively. Based on the SPR intensity at 72 h, the quantity of synthesized AgNPs in the glucose-containing reaction was slightly higher than the reaction without glucose, suggesting that an addition of glucose as a reducing agent could facilitate a higher formation of AgNPs. In the reaction containing only AgNO3 and glucose, the SPR characteristic peak of AgNPs was not observed, which was likely due to a lack of capping agent to facilitate a formation of stabilized AgNPs [40]. Fig. 3 Different concentrations of the protein extracts of weaver ant larvae were also used in the 72-h reactions to produce AgNPs without the presence of glucose under the fluorescent light as seen in Fig.
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4A. With increasing concentrations of the protein extracts, the increasing intensities of the SPR peak were observed, suggesting that the higher protein content promoted the higher production of AgNPs. To study the effect of fluorescent light, the reactions containing only AgNO3 and the protein extract
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were incubated for 72 h under the conditions with and without fluorescent light. Under a condition without fluorescent light, no characteristic SPR peak was detected (Fig. 4B) and no color change was
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observed (data not shown). This clearly indicated the important role of photocatalytic activity of fluorescent light in a reduction of silver. Therefore, it is very likely that the extracted proteins mediate
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a reduction of metal ions via a photo-induced electron transfer reaction [5]. In the extracted proteins, tryptophan, tyrosine and phenylalanine residues of protein chains are the major amino acids that can
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serve as reducing agents. Under light exposure, the indolic group of tryptophan, phenolic group of tyrosine and benzyl group of phenylalanine can be induced to form indolyl (Trp ), phenoxyl (Try) and benzyl radicals, respectively. This photoactivation process also causes a production of the
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hydrated electron (eaq), which can lead to a reduction Ag to form metallic AgNPs (Ag0) [41-44]. In addition, among many proteins in insect larvae, antioxidant enzymes, such as superoxide dismutase
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(SOD) and catalase (CAT), which exhibit high reducing activity, may be ones of the proteins facilitating a reduction of silver ions and a formation of AgNPs [45-47]. Besides, some water-soluble
Fig. 4
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antioxidants in weaver ant larvae [31], may assist a production of AgNPs.
3.5 Characterization of the synthesized AgNPs The representative TEM image of the synthesized AgNPs is shown in Fig. 5, which clearly revealed spherical, mono-dispersive AgNPs. Their diameters were distributed from approximately 2 to 16 nm, and the average diameter was 7.87 2.54 nm as determined from 50 random individuals. Due to their small sizes, the synthesized AgNPs might exhibit a good antibacterial activity. AgNPs in a range of 110 nm were reported to exhibit a higher antibacterial activity than the larger sizes because of a greater surface area for interacting bacterial surface and a feasible penetration inside the bacterial cells [48]. Fig. 5
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The identity of the synthesized AgNPs was further investigated by using EDX, HR-TEM, TEMSAED, and XRD analyses. The elemental composition of the synthesized AgNPs was detected by a TEM equipped EDX. The EDX spectrum result (Fig. 6A) showed a strong spectral signal of silver at a
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2.9-keV region, indicating that silver was a dominant element of the synthesized particles. In addition, signals of carbon and copper were also detected, which were likely from a carbon-coated copper grid
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that the samples were deposited on. In Fig. 6B, HR-TEM image of individual synthesized AgNPs indicated the d-spacing of the crystallographic plane of 0.23 nm, which agreed well with the
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interplanar separation of the (111) lattice plane of the face-centered cubic silver. The crystalline nature of AgNPs was demonstrated as the result of TEM-SAED in Fig. 6C. The TEM-SAED exhibited
centered cubic structure of AgNPs [49].
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Fig. 6
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patterns corresponding to the (111), (200), (220), (311) and (222) Bragg’s reflection of the face-
X-ray diffraction is normally used to confirm the crystalline nature of particles. The XRD pattern
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of the synthesized AgNPs is illustrated in Fig. 7. The results indicated two diffraction peaks at 2θ
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values of 38.24° and 44.47°, which corresponded to the planes (111) and (200) of the face-centered cubic (fcc) lattice of silver (JCPDS file no. 01-087-0718), respectively [50]. Fig. 7
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3.6 Antibacterial activity of the synthesized AgNPs Antibacterial activity of AgNPs was initially assessed by using a disk diffusion assay. Fig. 8 shows the growth inhibition zones of AgNPs (25 g) against E. coli and S. aureus, which were seen as the clear circles surrounded the filter papers impregnated with AgNPs. The average diameters of clear zones of AgNPs against E. coli and S. aureus were equally at 9.3 ± 0.6 mm. The growth inhibition zone of ampicillin (25 g), the positive control, was 16.3 ± 0.6 mm, while no growth inhibition zone of distilled water, the negative control, was observed. Fig. 8 The bacteriostatic and bactericidal effects of the synthesized AgNPs were further investigated via a micro-broth dilution assay. The synthesized AgNPs of different concentrations (a serial two-fold
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dilution from 16250 μg/ml) were employed to further examine the antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. The effects of AgNPs on growth inhibition of both bacteria in a time course of 24 h are shown in Fig. 9. As the concentration of AgNPs
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was increased, the more inhibition of the bacterial growth was observed in both bacterial strains, suggesting the dose response effect of AgNPs on bacterial growth inhibition. At 24 h of the
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incubation, the minimum inhibitory concentration (MIC) against E. coli and S. aureus were equally at 125 μg/ml and the minimum bactericidal concentration (MBC) against both bacterial strains were
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equally at 250 μg/ml. The antimicrobial activity of the produced AgNPs against both Gram negative and Gram positive bacteria could be beneficial for their application as an antibacterial agent. With
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their small size, they would be easily attached and penetrated inside the bacterial cells. To inhibit bacterial growth, AgNPs could disturb membrane permeability and respiration of the cells. In
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addition, they could block the functions of some proteins and nucleic acids as well as cause a
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formation of free radicals that induce membrane damages, eventually resulting in cell death [48,51]. Fig. 9
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4. Conclusions
This investigation demonstrated a simple, cost-effective, green synthetic approach of AgNPs using only the protein extract of weaver ant larvae and silver nitrate solution under an exposure of
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fluorescent light at room temperature. Reducing and antioxidant activities of the protein extract suggested its roles as a reducing agent to mediate a formation of AgNPs. The protein extract also helped to stabilize the mono-dispersive, spherical AgNPs of approximately 8 nm. The activity of the protein extract to assist a formation of AgNPs required light activation. Without light exposure, a formation of AgNPs was not detected within 72 h of the reaction. The identity of the produced AgNPs was confirmed by TEM-SAED, HR-TEM, EDX and XRD analyses. Antibacterial activity of the produced AgNPs against both Gram-negative E. coli and Gram-positive S. aureus also suggested their potential application as a good antibacterial agent.
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Acknowledgements Funding of this work is supported by Suranaree University of Technology-National Research Fund under Grant No. SUT-1-104-57-24-20. The student academic scholarship is provided by the Science
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Achievement Scholarship of Thailand (SAST) of the Commission on Higher Education, Thailand.
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Figure Captions Fig. 1. Images of waver ant larvae at the 3rd instar stage (A), a comparison of the larvae with the adult ants (B), and the protein extract of weaver ant larvae visualized on a 12.5% SDS-PAGE gel (C).
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Lane M is the standard protein markers.
Fig. 2. Reducing power (A) and scavenging activity (%) on DPPH radicals (B) of the protein extract
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of weaver ant larvae at different concentrations. The data are presented as the mean standard deviation of four replicate samples.
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Fig. 3. UV-Vis spectra of synthesized AgNPs using the protein extract of weaver ant larvae in the presence (A) and absence (B) of glucose. The reactions containing 1 M AgNO3 and 0.36 mg/ml
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protein extract (prot) in the presence or absence of glucose (glu) were exposed to fluorescent light at room temperature.
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Fig. 4. Effects of protein concentrations and fluorescent light on a formation of AgNPs. A) UV-Vis
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spectra of synthesized AgNPs in the reactions containing 1 M AgNO3 and various concentrations of the protein extract of weaver ant larvae under an exposure of fluorescent light
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for 72 h. B) UV-Vis spectra of synthesized AgNPs in the reactions containing 1 M AgNO3 and 0.36 mg/ml protein extract under the conditions with and without fluorescent light exposure for 72 h.
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Fig. 5. TEM image (A) and the histogram of size distribution (B) of the synthesized AgNPs. Fig. 6. TEM-EDX (A), HR-TEM (B) and TEM-SAED (C) analyses of the synthesized AgNPs. Fig. 7. The XRD pattern of the synthesized AgNPs was interpreted with JCPDS (No. 01-087-0718). The diffraction planes at (111) and (200) depicts that the synthesized AgNPs are cubic crystalline in nature. Fig. 8. Growth inhibition zone of E. coli (A) and S. aureus (B) in response to the synthesized AgNPs. 1: the negative control (distilled water), 2: the positive control (ampicillin), and 3: the synthesized AgNPs (25 µg).
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Fig. 9. Growth inhibition curves of E. coli (A) and S. aureus (B) exposed to different concentrations of the synthesized AgNPs in a time course of 24 h. The data are presented as mean standard
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Graphical abstract
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1. AgNPs was synthesized using the protein extract of ant larvae under light exposure. 2. Small AgNPs of approximately 8 nm was produced.
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3. The AgNPs exhibited antibacterial activity against both E. coli and S. aureus.
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