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Direct Label-free Surface-Enhanced Raman Scattering Analysis of Nucleic Acids

  • Autores: Judit Morlà Folch
  • Directores de la Tesis: Ramón Álvarez Puebla (dir. tes.), Luca Guerrini (codir. tes.), Nicolás Carlos Pazos Pérez (codir. tes.)
  • Lectura: En la Universitat Rovira i Virgili ( España ) en 2017
  • Idioma: español
  • Materias:
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  • Resumen
    • The research has been carried out in the context of Surface-Enhanced Raman Scattering (SERS) spectroscopy for nucleic acids analysis.

      SERS is a powerful analytical technique that combines the structural specificity and high experimental flexibility of Raman spectroscopy with the extremely high sensitivity provided by the metal nanostructure amplification of the optical signal. Nowadays, SERS is a mature technology which finds a continuously larger number of applications in diverse fields as analytical chemistry, biomedicine, environmental or materials chemistry.

      Characterization of nucleic acids (NAs) has become a major goal in fields such as genetics, medicine or drug discovery. NAs are usually found in low amounts which require extremely sensitive techniques to be detected. Sensitivity represents a key requirement for their implementation into easy-to-use, cost-effective, rapid and high accuracy NA sensors. In this regard, the extraordinary sensitivity, in combination with the rich structural information provided by the Raman spectra, turned SERS into a powerful tool for the analysis of NAs.

      SERS analysis of NAs has been largely restricted to indirect approaches, which normally exploit the selective recognition between two complementary strands to identify a specific sequence, and the use of extrinsic Raman labels for SERS readout. However, these strategies completely dismiss the exquisite structural information provided by the direct acquisition of the biomolecular vibrational fingerprint. Contrarily, direct label-free SERS analysis displays an outstanding potential in terms of chemical-specific information. However, reproducibility issues and/or limited sensitivity have been traditionally limited the successful implementation of direct SERS analysis to NAs. Thus, development of new methods capable of overcoming such limitations would represent an important leap forward for the full exploration of the genetic information by SERS.

      In this regard, positively charged silver nanoparticles (AgNP@Sp) were specifically designed for the nucleic acids analysis by direct SERS. Specifically, by using AgNP@Sp the electrostatic interaction between NAs and the colloids is ensured due to the spermine molecules anchored at the surface. This results in the entrapment of the biomolecules at the interparticle gaps of stable nanoparticle clusters in suspension, yielding intense and highly reproducible SERS spectra at the ultrasensitive level without the need of an external aggregating agent. This, combined with the simple colloidal preparation, makes AgNP@Sp an optimal SERS platform for label-free NAs analysis.

      This highly sensitive label-free approach was successfully implemented in various applications of biological interest:

      - Identification and ultrasensitive quantification of chemically-modified nucleobases in single and double stranded DNA. The investigated nucleobase variants included the most common epigenetic modifications in mammalian DNA. Moreover, classification of the SERS spectra was achieved via PLS-DA.

      - Quantification of nucleobase composition in short and genomic DNA. This simple, inexpensive and flexible method for nucleobase quantification was successfully applied to DNA samples independently of their size (from short to long genomic DNA), conformation (single-stranded vs duplex), and base sequence.

      - Vibrational classification of structurally similar small RNA. It was demonstrated the potential of label-free direct SERS analysis to fully disclose the unique vibrational information of a variety of different small RNAs with high sensitivity.

      - Further, it was proven the efficient integration of the direct SERS method into microfluidic chips for precise manipulation of DNA samples at small length scales, allowing drastic reduction of the amount of DNA required per measurement down to the picogram level. Moreover, the combination of direct SERS analysis with statistical techniques such as PLS-DA provided extra discriminatory capacity.

      In summary, this thesis demonstrates the tremendous analytical potential of this novel direct SERS method for nucleic acids analysis, opening new ways for the future development of a fast, low-cost, and high-throughput analytical technique for nucleic acids analysis.

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