Characterization of DNA hybridization procedures using an optical biosensor

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In order to reduce sample volume and increase sample number output in routine analysis, there is a need to develop other instruments besides the ones used in conventional laboratory, with simpler methods, enabling real time monitoring in the field. The biosensors development has increased recently mainly due to their interesting features, such as, robustness and short detection time, meeting the needs requested for day-to-day analysis. Optical fiber sensors based on Long Period Gratings (LPG) are very sensitive devices considering the fiber surrounding refractive index with potential to be applied for the development of biosensors using label-free DNA. The aim of the present study was to characterize the DNA hybridization process using a LPG fiber optic biosensor. In order to achieve this goal several parameters were investigated: a) probe specificity and; b) target sequence size effect. The probe was immobilized with Poly-L-Lysine witch, in turn, was deposited over the fiber surface. In the end the fiber surface was washed with nitric acid, being an efficient and safe method for removing all the layers laid on the fiber surface without damaging in the short-term the fiber. Five oligonucleotide sequences were used considering their complementarity to the probe, Target 1 (complementary sequence), Target 2 (non-complementary sequence), Target 3 (with a single nucleotide polymorphism), Target 4 (3 base polymorphism) and Target 5 (complementary sequence with a tail). The ability to detect a real matrix was also tested using DNA extracted from leaf samples of Vitis vinifera L. Target 1 was positively detected and the absence of hybridization with Targets 2, 3 and 4 revealed the high specificity of the present biosensor. Hybridization of Target 5 revealed that the sensor is able to detect sequences larger than the probe despite the loss of signal compared to Target 1. The detection of the real matrix was positive but the signal assumed a lower value when compared to Targets 1 and 5. Summarizing, the optimization of the biosensor used here may lead to the development of an excellent method for DNA detection comprising the required advantages for the biosensor development, such as, low cost, reusable and short time detection.
Dissertação de Mestrado em Biotecnologia e Qualidade Alimentar
Biossensores , DNA , Hibridação