Optical detection and structural analysis of dna via direct surface-enhanced raman scattering

Resumo

Recognition of alterations in DNA sequences is of outmost importance for diagnosis and prognosis of genetic diseases. However, DNA is usually found at low concentrations and requires the use of extremly sensitive and accurate analytical techniques capable of detecting known and unknown modifications. Such sensitivity and specificity is not always fulfilled by traditional detection methods, which often involve pre-amplification steps, complicated procedures or the use of fluorescent reporters. In recent years, surface-enhanced Raman scattering (SERS) spectroscopy has developed into a mature and reliable spectroscopic technique for the analysis and identification of nucleic acids. SERS combines the specificity and experimental versatility of Raman spectroscopy with an increased sensitivity, as a result of the unique optical signal amplification properties of plasmonic nanomaterials. Despite the potential of SERS for ultrasensitive detection of analytes, even down to single molecule detection, its implementation to DNA analysis has been mainly restricted to indirect SERS strategies. These strategies mainly involve DNA hybridization events and the use of extrinsic Raman labels, to report the presence of the target DNA sequence. However, indirect approaches dismiss the structural information intrinsically contained in the Raman spectra of DNA. On the other hand, while direct SERS approaches offer higher potential in terms of specificity as they can provide such vibrational information, they have traditionally experienced key issues associated with low sensitivity and/or limited reproducibility. In recent years, great efforts have been devoted to circumvent these limitations. On this basis, the general objective of this dissertation is the application of label-free direct surface-enhanced Raman scattering of DNA sequences for genetic screening applications and structural analysis of DNA/nanoparticles conglomerates. To this end, a novel method based on the use positively charged spermine-coated silver nanoparticles (AgSp NPs) as plasmonic materials is exploited. Cationic AgSp NPs are used as efficient SERS substrates to electrostatically entrap negatively-charged DNAs at the interparticle junctions, yielding highly SERS-active and long-term stable clusters in suspension. In this way, intense and highly reproducible SERS signal from DNA are obtained down to the nanogram regime. The dissertation is organized according to the following specific objectives: I) synthesis, characterization and implementation of AgSp NPs to the direct SERS analysis of DNA; II) detection and conformational classification of point mutations in a relatively long sequence of the K-Ras gene; and III) direct structural analysis of native DNA/RNAs upon electrostatic interactions with AgSp NPs. Accordingly, the dissertation will comprise four chapters: Chapter 1 provides the basic theoretical background to understand diseases triggered by punctual alterations (mutations) in specific genes, such as the case of cancer. In addition, the advantages and limitations of conventional methods for detection of point mutations in DNA sequences is reviewed. In the second part of this chapter, the integration of SERS as a sensing tool for the exploration of nucleic acids is presented, as well as the theoretical basis to understand the technique. In Chapter 2, the implementation of AgSp NPs as the sensing platform for SERS analysis of nucleic acids is discussed. Firstly, the plasmonic substrate is fully characterized by several techniques, such as UV-visible absorption and transmission electron microscopy, and dynamic light scattering. Subsequently, representative single and double stranded DNA sequences are selected as model analytes to to assess the performance of AgSp NPs in the direct SERS study of nucleic acids and determine the corresponding optimal working conditions. This chapter also includes the vibrational assignment of the SERS spectral features of DNAs, based from previous literature reports. Chapter 3 highlights the clinical potential of the direct SERS method for discrimination of DNA point mutations. In particular, point mutations in Ras oncogenes are routinely screened for diagnostics and treatment of tumors (especially in colorectal cancer). Therefore, the determination of this punctual mutations in a sensitive, fast and inexpensive manner is essential for diagnosis and prognosis. Herein, direct SERS coupled with chemometrics is used as a tool for the study of the specific conformations that single-point mutations impose on a relatively large fragment of the K-Ras gene (141 nucleobases). Finally, in Chapter 4 the underlying mechanisms of interaction between DNA (as well as for RNA) with AgSp NPs in this direct SERS method are investigated in depth. Here, plasmon-based spectroscopies and theoretical simulations are combined to directly investigate the role of the cooperative binding of cationic nanoparticles with different surface charges on the structural integrity of a large variety of nucleic acids.