Development of quantum dot-based tools for in vitro and biosensing applications

• Autores: Helena Montón Domingo
• Directores de la Tesis: C. Nogués i (dir. tes.), Arben Merkoçi (dir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2015
• Idioma: inglés
• Tribunal Calificador de la Tesis: Salvador Alegret Sanromà (presid.), Merce Marti Gaudes (secret.), Wolfgang Parak (voc.)
• Materias:
  • Ciencias de la vida
    • Biología celular
      • Cultivo celular
• Enlaces
  • Tesis en acceso abierto en:  TESEO  DDD 
• Resumen

  • This PhD thesis describes the use of quantum dots (QDs) in the development of new tools for biological applications. Commercial CdSe/ZnS core/shell QDs with unique optical and electrochemical properties have been used to develop a variety of optical and electrochemical sensors for the detection of proteins, cells and DNA. An optimized protocol to use QDs in immunocytochemistries is described to visualize intracellular proteins such as ß-tubulin (microtubules protein), GM130 (golgi apparatus protein) and EEA1 (endosomes protein). The use of QDs provided a considerable stability and robustness to the technique, proving that they can be routinely used as optical labels in immunocytochemistry. In addition, QDs have been successfully used as dual optical/electrochemical labels to detect apoptotic cells. QDs were conjugated with Annexin-V (AnnV), a protein specific to phosphatidilserine, which is translocated to the outer surface of the plasma membrane in apopototic cells. The resulting label (QD-AnnV) provided excellent fluorescence images using confocal microscopy, high resolution images using scanning electron microscopy and a quantitative measurement of apoptotic cells using flow cytometry. Furthermore square wave voltammetry was applied to develop a novel electrochemical biosensor for a fast, semiquantitative and cheap detection of apoptotic cells. This work has proved the versatility of the QDs, making them a unique tool to be used for a complete study of a biological state of cells, such as apoptosis. Later on efforts were put towards the development of a device based on the use of QDs and microfluidics for drug screening using the same labeling strategy (QD-AnnV) and detecting apoptosis as well. Interconnected microchannels were designed with different geometries to perform specific tasks: the first one to prepare different concentrations of camptothecin (the pro-apoptotic drug used as model for drug screening), the second to carry out the conjugation of QDs with AnnV, and the last to culture the cells and detect the effect of the drug on them. The use of microfluidics did not only made the experiments more robust, since all the steps were mostly automated, but also more economic as less amount of reagents were required.. The successful fluorescence detection of apoptotic cells in the chip demonstrated that the combination of novel tools, such as QDs and microfluidics, allows for a new generation of point of care platforms for drug screening. Finally, QDs were also used for the detection of nucleic acids. QDs were conjugated with specific hairpin structures of DNA so called molecular beacons (MBs). MBs were modified with a quencher so, when QDs were conjugated to them, their fluorescence was turned off. This strategy was used to detect specific DNA targets which, while hybridized with the QDs-MBs hybrids, opened the hairpin structure making the fluorescence of QDs recovery from their quenching state. Furthermore, we integrate all this process in a transparent microfluidic channel, which let us monitor in real time all the steps, from the immobilization of QDs on the channel surface, followed by the conjugation with MBs and up to the hybridization of the target analyte. Thus, QDs are not only able to replace organic dyes as fluorescent labels, but they can also be combined with electrochemical methods and microfluidics, generating whole new alternatives in biosensing and drug screening.