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_____________________________________________________________ Results and Discussion immobilization time employed in the experiment (30 s) is not optimal and already leads to a too high ssDNA coverage for the employed detection scheme resulting in a decreased hybridization efficiency. By doing this the rest of the electrodes were longer exposed to immobilization by incubation allowing to investigate, how a possible contamination influences the quality of the chip preparation. After potential-assisted immobilization of FRIZ ssDNA and passivation with MCU, region a was also exposed to E. coli ssDNA. There was no signal upon hybridization with E. coli tDNA showing the high integrity of the formed FRIZ/MCU films. Region b was exposed to FRIZ ssDNA solution, and subsequently to the MCU passivating solution and finally to the E. coli ssDNA solution, with immobilization occurring by incubation in all cases. Prior to the final potential-assisted passivation with MCU, the electrodes were cleaned using the potential-assisted desorption method. Thus, this region should be modified only with MCU. Figure 3.50, b and c confirm this by showing the absence of any signal from FRIZ and E. coli tDNA (grey curve). In order to prove that the absence of signals is due to a successful desorption and not due to an undetectable amount of immobilized DNA, region d was exposed to same conditions, without performing any desorption step. That region should be modified with both FRIZ and E. coli ssDNA sequences and passivated completely with MCU. In both cases (Figure 3.50, b and c, beige curve) a very small amount of tDNA is detected upon hybridization with both FRIZ and E. coli tDNA, however, this signal is negligible as compared to the signal obtained in the regions with potential-assisted immobilization. Nevertheless, the amount of contaminating DNA is measurable, proving that desorption in region b was done successfully. Finally, region c was initially exposed to FRIZ ssDNA solution at OCP, after which the electrodes were cleaned by potential-assisted desorption and subsequently immobilized with E. coli ssDNA and passivated with MCU by the potential-assisted method. Therefore, it is the only region that should show a signal upon hybridization with the E. coli tDNA sequence (Figure 3.50, c, green curve). The absence of any peak upon hybridization with FRIZ tDNA (Figure 3.50, b, green curve) also indicates that desorption was performed successfully in this region as well. Potential-assisted methods for immobilization and desorption allow for a fully electrochemically controlled preparation of DNA chips within a very short time. The big advantage of the potential-assisted immobilization method is that it provides the desired DNA coverage and blocking of the surface tremendously faster than the process at OCP and by this 3.4 Potential-assisted preparation of DNA sensors 94
_____________________________________________________________ Results and Discussion it does not allow undesired contamination, which guarantees a high quality of the prepared DNA chips. Additionally, the construction of DNA chips in this way allows for the use of even smaller electrodes. The size of the electrode does not need to be limited by the size of the droplet used for modification or its evaporation rate since there is no need for covering individual electrodes for their modification with the solution of interest. 3.4 Potential-assisted preparation of DNA sensors 95
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Jambrec Daliborka - 2016 DISSERTATI
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5.1 Materials and consumables …..
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2. Aims of the Work
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