| Abstract [eng] |
Monitoring of heterogeneous nucleic acid hybridization in real time is a widely researched field due to its applications in disease diagnostics, environmental monitoring, and fundamental science. Electrochemical hybridization readout methods are particularly promising due to their promise of excellent performance at low cost, as well as preparation and measurement protocol simplicity and applicability for high throughput studies. However, there are only a few studies described that perform electrochemical readout of labelled DNA hybridization in real time, and their temporal resolution, as well as detection limits, are disappointing, This master’s thesis aimed to develop an effective electrochemical tool for monitoring the chemically amplified hybridization signal of oligonucleotides in real time. The goals of this study were: to demonstrate an effective chemical signal amplification method for an electrochemical label attached to single-stranded DNA; evaluate the suitability of the electrocatalytic system to measure DNA hybridization in real time and calculate the hybridization parameters from obtained data; to determine whether the demonstrated electrocatalytic system is suitable for the differentiation of two oligonucleotides of identical sequences with an electrochemical label attached at different ends. Experiments were performed in the laboratories at Life Sciences Center (VU). Light absorbance measurements were conducted with BioDrop DUO and EvolutionTM 300 spectrophotometers to assay several prospective reductors of methylene blue. Reference 600+ potentiostat from Gamry instruments was used to execute cyclic voltammetry and chronoamperometry measurements which were needed to assess the functionality of prepared sensing electrodes, as well as prove the effectiveness of MB electrocatalysis for hybridization signal amplification. Obtained data were analyzed and visualized using Wolfram Mathematica 11.2. The results showed that ascorbic acid is an effective reducer of MB and can enable the electrocatalytic cycle needed to amplify the signal of DNA hybridization with electron transfer rates of up to 1,02 s 1. Hybridization monitoring with 1 s temporal resolution was achieved, and the calculation of hybridization parameters from obtained data was demonstrated. MB placement at different target ssDNA ends resulted in enhanced electron transfer when MB is positioned distally from the electrode after hybridization. |
