DNA biosensor based on optical detection for environmental control

Resumo

The main goal of the project is to develop a miniaturized optical device for the detection of DNA for multiple applications: early detection of Zebra Mussels (ZM) in river basins and detection of contaminated food by pathogens. The optical device based on DNA detection should be easy to operate, low-cost and allow portability. This project rely on the colorimetry features of gold combined with the sensing ability of recognition elements. The specific objectives of the project include: 1) Synthesis and tuning of nanomaterials The intensity and peak location of the absorption and scattering spectra are highly dependent on the metal nanoparticles (NP) geometry, material, size, inter-particle distance and surrounding media. In this project, two strategies for DNA detection might be used; based on localized surface plasmon resonance coupling for naked-eye detection and/or based on refractive index change provided by DNA or other biomolecules. Gold nanoparticles will be synthesized accordingly to the desired maximum plasmon peak position. 2) Characterization of the nanomaterials Characterization of metal NP should be performed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS) and UV-vis Spectroscopy. 3) Identification of specific targets and probes Identification of target/probe DNA sequences compatible with conventional (PCR) and alternative (enzymatic) amplification methods. The specificity of the selected probes and targets will then be experimentally tested in optimal working conditions and if necessary new sequences will be re-design in an iterative process until reaching the best analytical response. Databases classically used in molecular biology (GeneBank, European Nucleotide Archive (ENA)-EMBL, etc.) will be used to identify a specific sequence for the target and complementary probes. Target and complementary DNA sequences will also be analysed using Geneious software. 4) Chemical functionalization of NP with DNA recognition element DNA sequences will be modified with suitable chemical groups and oligonucleotides to allow their chemical immobilization on the nanoparticles surface. The optimization of DNA surface coverage, spacer effect and salt conditions to prevent aggregation are crucial to achieve the best hybridization efficiency. 5) Evaluation of NP-based detection approach The evaluation of the NP-based detection approach will involves the assessment of the optical response, the evaluation of the specificity of the selected DNA sequences, analysis and comparison of both strategies for target recognition and testing of the selected detection strategies with real and spiked samples. Spiked and real samples which undergo amplification will be used to study matrix effects on the analytical response of the biosensor. Interference tests will also be performed, using chemical compounds or DNA from other microorganisms by competitive assays. 6) Integration of the optical detection approach on a miniaturized device The best approach for the recognition of the target DNA is selected for the development of a miniaturized devices for in situ detection and the device is then used to evaluate the following analytical responses: 6.1. Integration of the optical sensor with a microfluidic device The best optical sensor to record quantification of specific DNA targets is then integrated on a microfluidic device that allows the use of small volumes and rapid analysis of the target solutions. 6.2. Evaluation of naked eye detection on multichannel microfluidic devices The development of a multi task device could be achieved by integrating the device with other analytical devices previously developed as modules (DNA purification or amplification) towards a Micrototal Analysis System (µTAS). The technical studies such as SEM, TEM, DLS, Atomic Force Microscopy (AFM), Spectroscopy Fluorescence and Micro spotter will be a part of the PhD project.