Page 79 - ITPS-8-3

INNOSC Theranostics and
Pharmacological Sciences Activity of green-synthesized nanoparticles

mutations in the target bacteria – an effect that raises 2.3. Antibacterial analysis
important concerns regarding the possible development of E. coli 1946 (ATCC 25922) and K. pneumoniae NCTC 9633
resistance to nanoparticle-based antimicrobial agents. (ATCC 13883) were cultured in nutrient broth medium

2. Materials and methods at 37°C for 24 h with shaking at 150 rpm in a shaking
incubator. The antibacterial activity of biosynthesized silver
2.1. Materials nanoparticles from CBW at concentrations ranging from 0

In this study, carpenter bees were obtained from Winston- to 100 µM was evaluated against E. coli and K. pneumoniae,
Salem State University. The two Gram-negative bacterial following protocols adapted from previous studies. 24,25
strains – E. coli 1946 and K. pneumonia – were obtained After 24 h of incubation, bacterial growth was assessed by
from the American Type Culture Collection (ATCC; measuring the optical density at 600 nm using a 98-well
United States). The following analytical-grade chemicals plate format with a GloMax Multiplate Reader (Promega,
were used: deionized water, 70% ethanol (Fisher Scientific, United States). In addition, SEM was used to analyze the
USA), 1 mM silver nitrate (Fisher Scientific, USA), 0.1 M morphological changes in the treated bacterial strains . 24,25
sodium hydroxide (Fisher Scientific, USA), nutrient broth 2.4. Genomic analysis
(Fisher Scientific, USA), phosphate-buffered saline (Fisher
Scientific, USA), and glutaraldehyde solution (Fisher Whole genome sequencing (WGS) was performed
Scientific, USA). Additional materials included a 98-well to investigate the genetic alterations in E. coli and
plate (Fisher Scientific, USA), centrifuge tubes (Fisher K. pneumoniae following exposure to biosynthesized
Scientific, USA), Ziploc bags, a field emission scanning silver nanoparticles. 25,26 Briefly, after 24 h of treatment,
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electron microscope (JEOL Ltd., Japan), a GENESYS 180 genomic DNA was extracted from both the control group
ultraviolet-visible (UV-vis) spectrophotometer (Fisher (untreated bacteria) and the nanoparticle-treated cells
Scientific, United States), a DNeasy 96 PowerSoil Pro using the DNeasy 96 PowerSoil Pro QIAcube HT Kit,
QIAcube HT Kit (QIAGEN, USA), and an Isotemp 2300 following the instructions provided by the manufacturer.
Digital Water Bath (Fisher Scientific, USA). Genomic libraries were prepared and sequenced using
the NextSeq2000 system (manufacturer, country) with
2.2. Extract preparation and synthesis of silver a 300-cycle flow cell kit to generate 2 × 150 base pair
nanoparticles paired-end reads. Read demultiplexing, trimming, and
run analytics were performed using DRAGEN v4.2.7, the
Dead carpenter bees (X. virginica) were collected in June onboard analysis software integrated with the NextSeq2000
2022 from Winston-Salem State University, Winston- system.
Salem, North Carolina, United States of America, and
transported to the laboratory in Ziploc bags. The wings 2.5. Statistical analysis
were carefully removed using sterilized forceps, then All statistical analyses were conducted using GraphPad
sterilized in 70% ethanol, washed with deionized water, Prism version 8.01 (manufacturer, country). Data are
and air-dried at room temperature.
presented as the mean ± standard error of the mean.
Silver nanoparticles were synthesized using the Statistical comparisons between groups were conducted
following protocols adapted from previous studies. 24,25 using the Student’s t-test, and differences were considered
Briefly, 0.1 g of bee wings was weighed and hydrolyzed in statistically significant at p<0.05.
0.1 M sodium hydroxide at 90°C using an Isotemp 2300
Digital Water Bath for 60 min. After hydrolysis, the mixture 3. Results
was cooled and centrifuged at 8,000 rpm for 10 min. The 3.1. Characterizations of biosynthesized silver
supernatant pH was adjusted to neutral, and 1 mL of nanoparticles from CBWs
this extract was added to 49 mL of 1 mM silver nitrate
solution in a 100 mL beaker. The mixture was incubated The biosynthesis of silver nanoparticles is visually
at 28 ± 1°C for 60 min, during which the color change confirmed by a color change in the treated extract, turning
from light yellow to dark brown indicated the formation from light yellow to dark brown. The UV-vis spectral
of silver nanoparticles. The UV-vis absorbance spectrum analysis reveals a strong surface plasmon resonance (SPR)
of the synthesized nanoparticles was measured in the peak at approximately 420 nm, indicating the successful
25
200 – 1,000 nm range using a GENESYS 180 UV-vis formation of silver nanoparticles (Figure 1).
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spectrophotometer. Scanning electron microscopy (SEM) The shape, size, morphology, and composition of the
was performed using a field emission SEM to characterize synthesized nanoparticles directly influence the SPR bands.
the morphology and size distribution of the nanoparticles. SEM images reveal agglomeration of the biosynthesized

Volume 8 Issue 3 (2025) 73 doi: 10.36922/ITPS025080007