Resumen de Dna biosensors based on integrated isothermal amplification-detection strategies
Justification and Needs for the Research The enormous amount of genetic information brought by extensive genome sequencing has raised the need for simple, rapid, cost-effective and high-throughput miniaturised and mass-producible analytical devices to address the growing market of molecular diagnostics, thus accomplishing the basic criteria for decentralised DNA testing. Biosensors are expected to play an important role in the trend of decentralisation of molecular diagnostics by bridging the gap existing between traditional nucleic acid tests and the definitive integration of point of care devices through lab on a chip solutions. All in all, biosensors represent a new approach to molecular diagnostics as they provide an intrinsic miniaturisation over the traditional nucleic acid tests surpassing their drawbacks, and allowing easier, faster and cheaper results whilst keeping high sensitivity and specificity of detection.
the Methodology Used The main goal of this Doctoral Thesis is to present alternative approaches in the field of DNA biosensors, designing and building new detection platforms that combine the amplification and quantification of targeted DNA while overcoming some of the current limitations. In order to achieve this objective, a variety of different strategies of effective solid-phase immobilisation strategies and isothermal enzymatic amplification have been explored to achieve lower detection limits with rapid and easy to execute assays.
This work presents a convenient, rapid, simple, easy to integrate yet robust biosensing detection platform that can bring new ideas for the integration of nucleic acid tests in point of care devices through the use of lab on a chip solutions. Ultimately, the vision underlining this work is to humbly contribute to the concept of decentralisation, allowing molecular diagnostics to move away from laboratories, providing bioanalytical information in situ. We report the work performed to achieve the specific objectives of this Doctoral Thesis: the use of solid-phase recombinase polymerase amplification strategy, as a hybrid concept that combines DNA amplification and detection, the optimisation of this idea, the detection of real samples, the exploiting of the surface chemistry and DNA to overcome the limitations found, and the use of ring resonators for the label-free and real-time monitoring of the recombinase polymerase amplification mechanism.
Conclusions First of all we have demonstrated the success of the isothermal solid-phase RPA concept as a DNA amplification method combined with detection to enhance DNA biosensing sensitivity. The general objective of this doctoral thesis was the development of a detection platform by exploiting a combined strategy of isothermal solid-phase amplification technique with genosensor detection for a simple, sensitive and rapid analysis of genetic material. Typically LOD of 1.3•10-15 M within a dynamic range of 5 decades were obtained in less than 1 h. Further optimisation allowed us to improve the analytical parameters of the platform in terms of LOD, signal-to-noise ratio and steps required. Moreover, our solid-phase RPA approach overcomes not only the limitations present in regular PCR amplification techniques, but also many of the regular RPA-based published methods, due to the simplicity and genericity of the system.
The concept has been applied for the electrochemical detection of genomic DNA from pathogens in the analysis of real samples, and the device has proven its robustness when successfully amplified and detected genomic DNA of Francisella tularensis and Piscirickettsia salmonis from extracted from real samples.
The DNA amplification in solid-phase bridge structures have been achieved by exploiting the knowledge of surface primer immobilisation and pushing the bending limits of dsDNA in a way that opens possibilities for exploring the selectivity of solid-phase bridge recombinase polymerase amplification to perform multiplex analysis of different nucleic acid targets simultaneously due to the fact that the system avoids the formation of primer-dimer formations.
Detection of the amplified material has been carried out by hybridisation with labelled primers, incorporation of ferrocene labelled dNTPs during the amplification and also in real time using ring resonators. The main added value of this thesis is that it presents a flexible solution for detecting DNA with biosensors, exploiting a general concept of solid-phase amplification and detection, thus integrating two nucleic acid tests, PCR and microarrays, in one single device.
