Development and characterization of fluorescent probes derived from BODIPY for application in bioconjugation with proteins

Name: Vanessa Gobbi Amorim
Type: MSc dissertation
Publication date: 09/12/2020
Advisor:

Namesort descending Role
Juliana Barbosa Coitinho Goncalves Co-advisor *
Lucas Cunha Dias de Rezende Advisor *

Examining board:

Namesort descending Role
André Romero da Silva Internal Examiner *
Bianca Prandi Campagnaro External Examiner *
Juliana Barbosa Coitinho Goncalves Co advisor *
Lucas Cunha Dias de Rezende Advisor *

Summary: Fluorescence is part of a phenomenon of light emission by cold bodies, known as luminescence. Fluorescence is widely used in the analyses and detection of substances, diseases and other processes. Its application requires the use of compounds capable of absorbing light and emitting it in the form of fluorescence at different wavelengths. However, only a few compounds have natural fluorescence and, in many fluorescence analyzes it is necessary to add fluorescent probes. Depending on the specific application of the fluorescent probe, functional groups can be inserted into its structure. In this work, aiming at the development of fluorescent probes for bioconjugation with proteins, two compounds of the borondipyrrometene class (BODIPY) were used, functionalized with the isothiocyanate group. These compounds were tested by spectrophotometry and spectrofluorimetric analyses to evaluate the photophysical properties of the developed fluorescent probes. The applicability of fluorescent probes in bioconjugation was tested using a serum bovine albumin as model protein. The bioconjugation product was characterized by spectrophotometry, spectrofluorimetric and SDS-PAGE. In addition, an initial test was performed to evaluate a possible fluorophore application with conjugation to a primary antibody in an immunofluorescent assay by Western Blotting. The results of bioconjugation are promising, WHERE it was found that the two compounds used in the study showed the ability to conjugate with the protein and emit fluorescence. It was also verified the interference of the thiourea bond formed after conjugation of each compound with the serum bovine albumin in the fluorescence intensity, observing its reduction. In the Western Blotting assay, it was observed that there was conjugation with the primary antibody and recognition of the chosen protein. However new analyses need to be carried out to standardize their applicability. The results of the present study represent an advance in the functional applications of BODIPY, generating perspectives for new prospects regarding its molecular uses.

Keywords: BODIPY, fluorimetry, isothiocyanate, thiourea, albumin.

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