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Study of biomolecules by Surface-enhanced Raman microspectroscopy
Šimáková, Petra ; Procházka, Marek (advisor) ; Šmejkal, Petr (referee)
This work deals with study of biomolecules using surface-enhanced Raman scattering (SERS) microspectroscopy. For that purpose surfaces based on gold and silver colloidal nanoparticles immobilized either by silane or by drying on glass plate were tested. As a model molecule cationic free-base 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin was chosen. The aim was primarily to improve sensitivity and spectral reproducibility of the mentioned SERS-active systems using an integrated Raman microspectrometer which has several advantages (better signal-to-noise ratio, shorter collection time, mapping, confocality) over a classical Raman spectrometer. The obtained results show that gold and silver nanoparticles immobilized by silane give the limits of detection of porphyrin ~ 1×10-8 M, moreover in the intact free-base form. While gold surfaces also show excellent spectral reproducibility, in the case of silver surfaces there is a great problem of frequent presence of spurious bands originating apparently from contamination by carbonaceous compounds and decomposition of silane or porphyrin itself. In the case of silver nanoparticles, drops that form a ring of aggregates with extremely high SERS enhancement after drying on glass proved more suitable.
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Surface-enhanced Raman microspectroscopy of biomolecules and biological systems
Šimáková, Petra ; Procházka, Marek (advisor) ; Michl, Martin (referee) ; Šloufová, Ivana (referee)
The aim of this thesis was using surface-enhanced Raman scattering (SERS) microspectroscopy for the study of biomolecules and biological systems. The main probe molecule was cationic porphyrin H2TMPyP, however, other porphyrins, tryptophan and two lipids were also used. The sensitivity and reproducibility of several solid SERS substrates: (i) Au and Ag nanoparticles (NPs) immobilized via a bifunctional linker, (ii) AgNPs immobilized by drying, (iii) highly ordered Au and Ag film-over-nanosphere (FON) and (iv) Ag-coated insect wings were compared. On most of the solid substrates, the lowest detected H2TMPyP concentration was ~10-8 M. The highest sensitivity was provided by the dried drops of AgNPs/analyte mixture, where concentrations 1×10-10 M TMPyP, 1×10-5 M tryptophan, 2×10-7 M DSPC and 3×10-7 M DMTAP were detected. Nevertheless, the spectral reproducibility was decreased due to porphyrin metallation and perturbation of the lipid spectra in comparison to their Raman spectra from solution. The highest reproducibility was achieved by AuFON and Ag-coated insect wings. Finally, the AgNPs modified by PEG polymers were tested for intracellular application using HeLa cancer cells. Metallation of H2TMPyP served to probe the accessibility of PEG- AgNPs surface. The results proved that the accessibility...
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Surface-enhanced Raman microspectroscopy of biomolecules and biological systems
Šimáková, Petra ; Procházka, Marek (advisor) ; Michl, Martin (referee) ; Šloufová, Ivana (referee)
The aim of this thesis was using surface-enhanced Raman scattering (SERS) microspectroscopy for the study of biomolecules and biological systems. The main probe molecule was cationic porphyrin H2TMPyP, however, other porphyrins, tryptophan and two lipids were also used. The sensitivity and reproducibility of several solid SERS substrates: (i) Au and Ag nanoparticles (NPs) immobilized via a bifunctional linker, (ii) AgNPs immobilized by drying, (iii) highly ordered Au and Ag film-over-nanosphere (FON) and (iv) Ag-coated insect wings were compared. On most of the solid substrates, the lowest detected H2TMPyP concentration was ~10-8 M. The highest sensitivity was provided by the dried drops of AgNPs/analyte mixture, where concentrations 1×10-10 M TMPyP, 1×10-5 M tryptophan, 2×10-7 M DSPC and 3×10-7 M DMTAP were detected. Nevertheless, the spectral reproducibility was decreased due to porphyrin metallation and perturbation of the lipid spectra in comparison to their Raman spectra from solution. The highest reproducibility was achieved by AuFON and Ag-coated insect wings. Finally, the AgNPs modified by PEG polymers were tested for intracellular application using HeLa cancer cells. Metallation of H2TMPyP served to probe the accessibility of PEG- AgNPs surface. The results proved that the accessibility...
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Study of biomolecules by Surface-enhanced Raman microspectroscopy
Šimáková, Petra ; Procházka, Marek (advisor) ; Šmejkal, Petr (referee)
This work deals with study of biomolecules using surface-enhanced Raman scattering (SERS) microspectroscopy. For that purpose surfaces based on gold and silver colloidal nanoparticles immobilized either by silane or by drying on glass plate were tested. As a model molecule cationic free-base 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin was chosen. The aim was primarily to improve sensitivity and spectral reproducibility of the mentioned SERS-active systems using an integrated Raman microspectrometer which has several advantages (better signal-to-noise ratio, shorter collection time, mapping, confocality) over a classical Raman spectrometer. The obtained results show that gold and silver nanoparticles immobilized by silane give the limits of detection of porphyrin ~ 1×10-8 M, moreover in the intact free-base form. While gold surfaces also show excellent spectral reproducibility, in the case of silver surfaces there is a great problem of frequent presence of spurious bands originating apparently from contamination by carbonaceous compounds and decomposition of silane or porphyrin itself. In the case of silver nanoparticles, drops that form a ring of aggregates with extremely high SERS enhancement after drying on glass proved more suitable.
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