The Fabrication And Applications Of SERS Active Nanoprobes For Single Cells Detection And Imaging

The integration of nanotechnology with biology has brought in new prospects and generated exciting developments.Nanotechnology has provided new tools for studying the biological process,especially at the cellular and molecular levels.The combination of nanotechnology with surface-enhanced Raman scattering(SERS),which is pursuing the characteristic advantages of specificity,selectivity and sensitivity has become one of the most incisive analytical approaches for chemical and biochemical detection and analysis at cellular levels.The current SERS-active nanoprobes designs share some limitations of size,fabrication complications,or high background signal derived from functionalization.In current work SERS-active glass nanopipette and silver nanoparticles are developed to meet the intracellular metal ion detection and imaging.Additionally,the capability of SERS-active nanopipettes for highly localized study is demonstrated in the cancer cells by using aptamer based detection method.Primarily,a ligand affinity based approach is used to detect heavy metal ions in cell.The Nanoprobe is composed of gold coated thin film on a 100 nm inner diameter pulled glass capillary that was functionalized with metal chelating ligand.Cyanide is well-known metal capturing ligand is selected in this study due to its stable characteristic peak in cell silent region of Raman spectra.Exceptionally,chemically modified nanoprobe with organic cyanide derivative was subjected for free trivalent iron ion as well as for metalloprotein detection with high selectivity(against di valent iron ion)and good reusability.Based on simple fabrication design the quantitation of free iron was attained by using peak intensity ratio within the cell silent region of Raman spectra.Also,the organic cyanide was highly stable in environmental condition,which overcomes the cyanide toxicity too.The nanoprobe has substantial binding affinity and superb tolerance against interferants.Aside these,the nanoprobe are sufficiently adroit to differentiate the iron in divalent and trivalent,as well in free and bound state.All these characteristics make the nanoprobe applicable to use in cellular microenvironment.Secondly,the gold film decorated glass nanopipette was used for the highly localized study of important cancer biomarker within in single living cells.The designed nanoprobe was working on the principal of SERS signal quenching(signal off response)after capturing the target.The glass nanoprobe was immobilized with specific thiolated DNA aptamer AS1411 then hybridized with silver nanoparticles pre-modified with complementary thiolated cDNA and Raman Reporter.The hybridized gold nanoprobe was precisely inserted in various region of the cell including nucleus and cytoplasm of cancer and normal cells,after extraction the signal on nanoprobe is measured by Raman spectroscopy.The nanoprobe showed spatial distribution of cancer biomarker(Nucleolin)in single cell and also exhibit significant signal variation with high selectivity among the cancer and normal cells.Finally,a novel SERS nanoprobe was developed for tracking the natural metal ion recycling process in single cell.The SERS nanoprobe was constructed with silver nanoparticles(55 nm)functionalized with optimal concentration of SH-PEG and metal ion ligand reporter molecule.The Fe-CN chemistry was applied with SERS intensity ratio for SERS imaging.Initially,the red blood cells(RBCs)were labeled with SERS nanoprobes for a required passage of time then these labeled RBCs incubated with macrophages for phagocytosis assay.The SERS nanoprobe showed good binding affinity for iron ion in free of protein bound state,thus different recycling step of hemoglobin to iron are accessible for it to provide understanding of the natural cellular processes.