Carbon Fiber Microelectrodes As DNA Sensors Using Fast Scan Cyclic Voltammetry

Over the past five decades, since the discovery of the structure of nucleic acids (DNA and RNA), different technological advancements have been achieved to understand the overall biochemical properties of nucleic acids. However, as nucleic acids are the profound and vital biomolecules of all living things, they are still the center of intensive research. The building blocks of DNA are purine bases, adenine and guanine and pyrimidine bases, thymine and cytosine, where each base is attached to ribose sugar with phosphate group. The electrochemical reactivity of these nucleobases defines the overall reactivity of DNA. So far, the technological progress on electrochemical DNA biosensors has been applied on different biomedical and environmental analyses which includes, analysis of infectious diseases, diagnosis and treatment of cancer and extensive studies on neurodegenerative and genetic diseases.Fast scan cyclic voltammetry (FSCV) is electroanalytical technique which applies a voltage in a fast and cyclic manner to a working carbon fiber-microelectrode (CFME) and records the resultant current produced by oxidation of analyte on the electrode surface. Previous studies by Venton and colleagues has shown that the electroactive nucleosides can be oxidized at a voltage of specific waveform using CFME[1]. The applied voltage can only oxidize specific ionizable nucleobases, such as purines, and the peak oxidative current is analyzed to quantify the concentration of the analyte.When co-detecting the purine nucleobases using traditional triangle waveform, which ramps up from -0.4 V to 1.45 V and back at 400 V/s, the two primary oxidative peaks overlap. As a result, a scalene waveform, initially developed by Ross et. al [2], has been used to co-detect purine bases adenine and guanine in DNA. The used waveform sweeps up at 150 V/s from -0.4 to 1.45 V and sweeps back at 400V/s vs Ag/AgCl reference electrode. Three oxidative peaks for adenine (for 3 sequences of oxidation) at 0.2 V and 0.95 V for the tertiary and secondary peaks respectively and the primary peak is at 1.4 V on the backward scan, and two oxidative peaks for guanine at 0.65 V for the secondary and 1.15 V for the primary peak (both on forward scan) were recorded. Forward scan rates lower than 150 V/s were less sensitive to detect the nucleosides, especially at lower concentration. Moreover, upon detecting annealed DNA and native bacterial DNA it was possible to detect cytosine at a potential 0.6 V. Cytosine peak, not only overlaps with guanine secondary peak, it shows low sensitivity using scalene waveform. The structure of DNA sample, single stranded DNA (ssDNA), double stranded DNA (dsDNA), synthetic DNA, bacterial DNA, short base pairs and longer base pairs, determines the shape and measurement of the peak oxidative current (POC) of adenine and guanine. Based on the analyzed data from this experiment, it was possible to detect DNA using FSCV for the first time. It was also obtained that, CFMEs are more sensitive towards adenine in DNA than adenosine as a nucleoside, but for guanosine it was the nucleoside that has higher peak than guanine in DNA. In addition, this study shows that shorter base pairs and single stranded DNA samples have higher POC than longer base pairs and double stranded or annealed DNA samples. Hence, this analytical method and measurement can be used in detecting different physical and structural DNA damages and paves a way for studies on a potentially new diagnostic and treatment mechanisms and new class of DNA biosensors.