Modified Electrodes with Grafted DNA and Oligonucleotides for Detection and Quantification of Peroxynitrate
Date of Award
Master of Science in Chemistry
Peroxynitrite (ONOO-) is a strong oxidizing and nitrating agent, and its formation has been correlated with many pathological conditions. It is generated in-vivo through the diffusion-controlled reaction between nitric oxide and superoxide. Peroxynitrite has been linked to nitration of protein and DNA as well as to DNA strand breaks. Accumulation of mutations and/or other kinds of DNA damage represent a carcinogenic risk. The accurate measurement of peroxynitrite concentration has been a challenge since this analyte is very unstable and reacts with many cellular targets. Development of analytical techniques capable of rapid and sensitive detection of this fast-reacting and damaging agent is an important research target to determine the chemical damage by this oxidant both at the tissue and the cellular levels.
In this work, we develop DNA films as sensitive sensing platforms to detect and quantify ONOO- DNA damage. We have used two methods for DNA immobilization on the electrodes surfaces: (1) electrochemical grafting and (2) layer-by-layer (LBL) deposition methods. In the first method, we generate carboxylic acid groups on the electrode surface via electrochemical reduction of trans-4cinnamic acid diazonium tetrafluoroborate, followed by coupling of pre-activated carboxylic groups with amino terminated oligonucleotide. In the LBL deposition method, we construct films of alternate layers of posittively charged poly(diallyl dimethyl ammonium) and the target DNA as a negatively charged counterpart on the surface of the graphite electrode. On both platforms (grafted oligos and DNA films), we assess the effect of defined exogenous levels peroxynitrite metabolite on the electrochemical response of the DNA interface. Particularly for the grafted DNA oligonucleotides, we focused on detecting the differential response of complementary strands versus DNA helices with a single base mismatch. We show in the current work that electrodes modified with DNA oligonucleotides show sensitive responses towards micromolar range concentrations of PON. Additionally, we found that the mismatch oligonucleotides immobilized on the electrode surfaces are more sensitive biosensors for detection of peroxynitrite. To this end, we have used chronocoulometry, cyclic voltammetry, and square wave voltammetry to monitor the effect of the mismatch on the sensitivity of the modified electrodes towards peroxynitrite through defined electrocatalytic processes mediated by the grafted oligonucleotides.
Salim, Heba Azmy, "Modified Electrodes with Grafted DNA and Oligonucleotides for Detection and Quantification of Peroxynitrate" (2016). ETD Archive. 894.