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The development of fluorescent silica nanoparticles (SNP-RB) from natural amorphous silica and its performance as an <i>Escherichia coli</i> (<i>E. coli</i>) biosensor is described in this paper. SNP-RB was derived from silica recovered from geothermal installation precipitation and modified with the dye, Rhodamine B. The Fourier Infrared (FTIR) confirms the incorporation of Rhodamine B in the silica matrix. Transmission Electron Microscopy (TEM) micrographs show that the SNP-RB had an irregular structure with a particle diameter of about 20–30 nm. The maximum fluorescence spectrum of SNP-RB was recorded at 580 nm, which was further applied to observe the detection performance of the fluorescent nanoparticles towards <i>E. coli</i>. The sensing principle was based on the fluorescence-quenching mechanism of SNP-RB and this provided a wide linear <i>E. coli</i> concentration range of 10–10⁵ CFU/mL with a limit detection of 8 CFU/mL. A rapid response time was observed after only 15 min of incubation of SNP-RB with <i>E. coli</i>. The selectivity of the biosensor was demonstrated and showed that the SNP-RB only gave quenching response only to live <i>E. coli</i> bacteria. The use of SNP-RB as a sensing platform reduced the response time significantly compared to conventional 3-day bacterial assays, as well having excellent analytical performance in terms of sensitivity and selectivity.