Development of electrochemical dna sensors based on the incorporation ferrocene labelled datp
- Autores: Ivan Magriñá Lobato
- Directores de la Tesis: Ciara K. O'Sullivan (dir. tes.), Mayreli Ortiz Rodríguez (codir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2019
- Idioma: español
- Tribunal Calificador de la Tesis: Anne Varene (presid.), Mònica Campàs i Homs (secret.), Luis Antonio Tortajada-Genaro (voc.)
- Programa de doctorado: Programa de Doctorado en Nanociencia, Materiales e Ingeniería Química por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
The detection of specific DNA sequences has a plethora of diverse applications in many fields including clinical diagnostics, disease monitoring, food analysis, forensics, bioterrorism and environmental control. However, traditional DNA analysis methods are multiple-step, time-consuming, expensive, require bulky equipment and require properly trained personnel, thus restricting their use. As a consequence, there is a considerable time-lag between sample acquisition and response, limiting the decision-making capacity, which in some cases might be life-threatening. To overcome this problem, miniaturized, accurate, simpler and affordable analytical devices (biosensors) have been developed during the last decades. DNA electrochemical sensors are excellent candidates for point-of-need DNA analysis that combine the high sensitivity and robustness of electrochemistry based sensors with the selectivity provided by the specific DNA hybridization that occurs between complementary DNA sequences. Nevertheless, electrochemical DNA sensors use as point-of–need devices is still limited because they require several steps including, the amplification of the target sequence, the generation of single stranded DNA following amplification, target hybridization with a complementary capture probe and finally other reporting steps to detect the hybridized target DNA sequence. In order to reduce the number of steps and bring electrochemical DNA sensors closer to the point-of-need we firstly proposed to eliminate the reporting step by using ferrocene labelled dATP (dAEFcTP) to obtain already labelled target amplicons. To eliminate the need to generate single stranded DNA we pursued two different strategies, a) The use of tailed primers during the liquid phase DNA amplification step to obtain amplicons with a ssDNA tail ready to be hybridized, and b) Solid-phase amplification The methodology employed in this thesis was systematic and included an optimization of the capture probe/primer vs backfiller for the development of hybridization/solid-phase-amplification electrochemical DNA sensors, respectively.
The first and simplest approach using dAEFcTP consisted of primer extension, hybridization and electrochemical detection of a primer extension product using a synthetic DNA sequence. Once it had been demonstrated that dAEFcTP can be incorporated in primer extension we moved to a more challenging approach in which we performed amplification, hybridization and electrochemical detection of two different targets: K. armiger, a toxic microalgae, and B. anthracis, the causative agent of anthrax. First, we optimized the methods using synthetic DNA, and then we moved forward to the detection of genomic DNA extracted from real samples. Results were validated with the gold standard qPCR method. The detection of B. anthracis was more challenging because it required the amplification of two different targets in a single-pot reaction as well as the hybridization of both targets in a single electrode array.
Additionally, the effects of various parameters were studied: a) dAEFcTP:dATP ratio on DNA amplification yield, ferrocene peak intensity and limit of detection.
b) Electrolyte solution on ferrocene peak intensity c) Electrode stability with time d) Confinement/diffusion free ferrocene on cyclic voltammetry results Finally, we performed isothermal solid-phase amplification of a target DNA synthetic sequence using recombinase polymerase amplification.
We concluded that dAEFcTP can be incorporated with KODXL polymerase at different dAEFcTP:dATP ratios in both primer extension and amplification experiments with success. Tailed primers in combination of dAEFcTP have been employed to develop two electrochemical DNA sensors, one for K. armiger and another one for B. anthracis.
We determined that the increase in dAEFcTP:dATP ratio increase the ferrocene peak intensity obtained and the limit of detection, but it decreases the DNA amplification yield.
We also discovered that the ferrocene electron transfer and ferrocene oxidation peak obtained are strongly dependent on the electrolyte composition and concentration, requiring the presence of divalent or trivalent cations to be effective.
