Metallic nanoparticles became subject of intensive research because of their potential antibiotic properties. Nanoparticles such as silver, gold, or zinc oxide particles are easily and cost-effectively synthesized by blending metal salts with plant extracts that reduce the metal. However, the resulting nanoparticles differ in size and antimicrobial capacity depending on the plant extract used. Hence, different extracts, varying in the plant or the part of the plant used for the extract, are currently investigated in regard to their capacity to form nanoparticles and their antimicrobial efﬁcacy. The formation of nanoparticles can be veriﬁed by UV-Vis spectroscopy due to surface plasmon resonance of the particles that lead to a characteristic spectrum deﬁned by the underlying metal and particle size. Subsequent analysis of nanoparticles on microbial growth is typically tested by methods based on absorbance changes. Here, we present how the spectrometer-based BMG LABTECH instruments are used to quickly conﬁrm Ag and ZnO nanoparticle formation and their inhibitory effect on the diarrhea-causing bacteria Vibrio cholerae and enterotoxic Escherichia coli (ETEC).
Materials & Methods
- Plant extract (from fruits and leaves of Calotropis procera)
- Zn(NO3)2 and AgNO3 (Sigma-Aldrich)
- LB (lysogeny broth) growth medium
- Disposable UV-Vis cuvettes (Sarstedt Article 742)
Ag and ZnO nanoparticles were synthesized by adding AgNO3 or Zn(NO3)2 to aqueous extracts of plants or leaves of the bush C. procera and subsequent heating and stirring of the mixtures. Generation of particles was veriﬁed upon washing by UV-Vis spectrum analysis.
- Microplates (96 wells, clear, U-bottom, SterilinTM)
- Microbial strains (V. cholerae and ETEC)
- 0.1 % crystal violet (Carl Roth T123.1)
A potential inhibitory effect of the nanoparticles on bioﬁlm generation was determined by growing the bacteria in microplates, addition of nanoparticles, thorough washing and crystal violet-staining of the bioﬁlm. Upon solubilization with 96 % EtOH, absorbance was measured at 595 nm.
|Nanoparticle detection||Biofilm assay|
|Absorbance spectrum 220-800 nm||Absorbance at 595 nm|
|Scan resolution 1 nm|
Results & Discussion
The formation of Ag and ZnO nanoparticles that were synthesized with the help of fruit or leave plant extracts was veriﬁed by UV-Vis spectrometry. Silver nanoparticles showed a characteristic absorbance spectrum with a peak around 340 nm, which was expected based on material and size (diameter of 100-150 nm) of the particle. Generation of zinc oxide nanoparticles was veriﬁed by a characteristic absorption spectrum with a peak around 370 nm. The phytosynthesis approach yielded different nanoparticle concentrations with fruit and leave extracts. Broadly speaking, the leave extract resulted in a higher nanoparticle concentration than the fruit extract, and the difference was displayed in a higher absorption for leave-extract-synthesized nanoparticles.
Having proven the formation of nanoparticles, they were tested regarding their ability to impair bioﬁlm formation of the diarrhea-causing bacteria Vibrae cholerae and enterotoxic Escherichia coli.
Staining the bioﬁlm with crystal violet and solubilizing it with ethanol results in a homogenous absorbance of the chromophore which is proportional to the bioﬁlm. Reading the assay on a SPECTROstar Nano revealed that silver nanoparticles synthesized with leave extract inhibit V. cholera and ETEC-driven bioﬁlm formation (Fig 2.). Furthermore, zinc oxide nanoparticles inhibit ETEC driven bioﬁlm formation irrespective of the extract (fruit or leave) used for synthesis (Fig 2).
Antibacterial nanoparticles can cost-effectively be synthesized by reducing metal salts with the help of plant extracts making it attractive for treating drinking water. Together with other approaches, such as bacteriophages, they are promising options to circumvent bacterial antibiotic resistances. Veriﬁcation of nanoparticle formation as well as its effect on microbial growth, in particular on bioﬁlm formation, is reliably measured by the SPECTROstar Nano absorbance reader. The spectrometer-based instrument acquires absorbance spectra in less than a second, making it the ideal instrument for quickly detecting nanoparticles by UV-Vis spectra.
- Salem, W., Leitner, D.R., Zingl, F.G., Schratter, G., Prassl, R., Goessler, W., Reidl, J., Schild, S. (2015) Antibacterial activity of silver and zinc nanoparticles against Vibrio cholerae and enterotoxic Escherichia coli. Int. J. Med. Microbiol. 305:85–95