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Master's Dissertation
DOI
Document
Author
Full name
Jayr Henrique Marin
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Temperini, Marcia Laudelina Arruda (President)
Gonçalves, Norberto Sanches
Mazali, Italo Odone
Title in Portuguese
Uso da técnica SERS para imageamento de complexos de cobre (II) em células HeLa e para caracterização do complexo de superfície da 5-nitroisatina em prata
Keywords in Portuguese
Células HeLa
Complexos oxindolimínicos
Raman ressonante
SERS
Abstract in Portuguese
A capacidade de complexos oxindolimínicos de cobre (II) de inibir células cancerígenas HeLa foi determinada no Laboratório de Bioinorgânica, Catálise e Farmacologia (IQ-USP), coordenado pela Profa. Dra. Ana Maria da Costa Ferreira. Nesta dissertação um dos estudos foi obter o imageamento SERS desses compostos no interior de células HeLa vivas com o objetivo de identificar o alvo intracelular desses complexos. Devido a intensificação SERS desses complexos ser baixa e causarem a agregação das nanopartículas de ouro (AuNP) foi necessária a utilização de um marcador Raman (MR) nestes estudos, sendo escolhido o composto DTNB (Ácido 5,5'-ditiobis-(2-nitrobenzóico)). Portanto a sonda SERS desse estudo consiste de AuNP esféricas juntamente com o MR e os complexos de cobre (AuNP/MR/Cu(isatp) ou Cu(nisatp)). No imageamento das células HeLa foram utilizadas duas radiações excitantes: 532 nm, onde não ocorre intensificação SERS originando uma imagem Raman dos constituintes celulares em maior quantidade e 785 nm, que possibilita o monitoramento dos sistemas AuNP/MR/Cu(isatp) ou Cu(nisatp) no interior das células. Para a análise dos espectros desse estudo (cerca de 2000 espectros para cada imageamento) a linha base foi corrigida por WLS (Weighted Least Squares) para os espectros SERS e por EMSC (Extended Multiplicative Scatter Correction) para os espectros Raman, além de ser feito alisamento utilizando um filtro Savitzky-Golay. Os espectros Raman foram submetidos à uma análise multivariada MCR-ALS (Multivariate Curve Resolution - Asymmetric Least Squares) para se obter as imagens de diferentes componentes celulares e uma análise univariada dos espectros SERS, baseada na banda do MR, possibilitando obter as imagens dos sistemas AuNP/MR/Cu(isatp) e AuNP/MR/Cu(nisatp). O imageamento SERS e Raman das células HeLa mostraram que os complexos têm a capacidade de penetrar nas células, juntamente com as nanopartículas de ouro, e causar dano ao núcleo celular, sem a entrada de água na célula, característica não observada ao tratar as células com outros sistemas sem a presença dos complexos. Outro estudo realizado nesta dissertação foi a caracterização do complexo de superfície formado entre a 5-nitroisatina em nanopartículas de prata, através da espectroscopia SERS e Raman ressonante. Foi observado que o complexo de superfície apresenta uma grande deslocalização eletrônica frente às espécies moleculares estudadas para fins comparativos. Essa deslocalização gera um cromóforo distinto no complexo de superfície que possui uma transição eletrônica na região do verde do espectro visível, enquanto que as espécies moleculares apresentam transições na região do azul e ultravioleta.
Title in English
Usage of SERS technique for imaging of copper (II) complexes in HeLa cells and to characterize a surface complex of 5- nitroisatin in silver
Keywords in English
HeLa cells
Oxindoliminic complexes
Resonant Raman
SERS
Abstract in English
The inhibition of cancer HeLa cells by oxindoliminic copper (II) complexes was determined in the Bioinorganic, Catalysis and Pharmacology Laboratory (IQ-USP), coordinated by Profa. Dra. Ana Maria da Costa Ferreira. In this work one of the studies was to identify the intracellular targets of these complexes by SERS imaging of live HeLa cells. Due to the low SERS intensification of these complexes and the aggregation that they cause in the gold nanoparticles (AuNP), it was necessary to use a Raman reporter (MR) in these studies. The reporter chosen was the DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)). The SERS probe was constituted by spherical AuNP together with the MR and the copper complexes. In the imaging of live HeLa cells two excitation radiations were used: 532 nm, to acquire the normal Raman information of the cells and 785 nm, to acquire the localization of the modified AuNP inside the cells. To analyze the spectra (about 2000 spectra to each imaging) the baseline, for the SERS spectra, was corrected by WLS (Weighted Least Squares) and the EMSC (Extended Multiplicative Scatter Correction) was used to correct the Raman spectra. A Savitzky-Golay filter was used for smoothing. The Raman images of the cellular components were obteined by multivariate analysis MCR-ALS (Multivariate Curve Resolution - Asymmetric Least Squares), and the SERS images were obtained by univariate analysis based on the band of the MR. The SERS and Raman images showed that the complexes are capable of entering the cells together with the AuNP, and cause damage to the nuclei, without the entrance of water into the cell, a characteristic not observed in other studied systems. Another realized study was the characterization of a surface complex formed between 5-nitroisatin and silver nanoparticles (AgNP), using SERS and resonant Raman. The surface complex showed a massive electron delocalization when compared to the molecular species. This electron delocalization in the surface complex generated a new chromophore, not seen in the molecular complex. This chromophore has an electronic transition in the green region of the visible spectrum, while the molecular species transitions occur in higher energies.
 
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Publishing Date
2019-10-02
 
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