Development Of A Silver Nanocluster Probe Against Foxp3 For Live Tregs Sorting : Using Mcf-7 As A Model
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Date
2018-02
Authors
Lee, Shin Yong
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
DNA-templated silver nanocluster (AgNC), a new promising fluorescence probe has been gaining importance in recent years. Owing to their special properties such as small in size, photostable and water-soluble, these fluorescence probes are favourable for many diagnostic applications. Due to these advantages, these AgNCs were adapted to develop a method for the identification of regulatory T cells (Tregs) through an intracellular transcription factor called forkhead box P3 (Foxp3). Tregs are crucial mediators of immune homeostasis, exerting suppression on effector cells in autoimmune diseases and promoting the cancer growth. Isolation of live Tregs is required for better characterisation. Current isolation of Tregs relies on a multitude of surface markers and a crucial intracellular marker, Foxp3. Isolation of a pure population is currently impossible as intracellular detection of Foxp3 relies on fixation and permeabilisation of cells, which often leads to cell death. Here, an optimised method for the detection of messenger ribonucleic acid (mRNA) of Foxp3 was introduced by hybridising AgNC and G-rich to its target. The hybridised samples were examined under UV illuminator and fluorescence intensity was determined by fluorescence spectroscopy. The successful hybridisation of a three-way junction with AgNC, G-rich and DNA/RNA Foxp3 target, produced an improved fluorescence intensity with spectral shift. The AgNC and GR-rich was successfully delivered into MCF-7 cells (a Foxp3 model), producing red fluorescence images which was corroborated by flow cytometry results. In summary, the silver nanocluster approach has the potential to detect intracellular Foxp3 nucleic acid to establish a non-lethal intracellular detection system for bioimaging.
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Keywords
Development of a silver nanocluster probe , against foxp3 for live tregs sorting