ABSTRACT
Nucleic acid amplification testing is a very powerful method to perform efficient and early diagnostics. However, the integration of a DNA amplification reaction with its associated detection in a low-cost, portable, and autonomous device remains challenging. Addressing this challenge, the use of screen-printed electrochemical sensor is reported. To achieve the detection of the DNA amplification reaction, a real-time monitoring of the hydronium ions concentration, a byproduct of this reaction, is performed. Such measurements are done by potentiometry using polyaniline (PAni)-based working electrodes and silver/silver chloride reference electrodes. The developed potentiometric sensor is shown to enable the real-time monitoring of a loop-mediated isothermal amplification (LAMP) reaction with an initial number of DNA strands as low as 10 copies. In addition, the performance of this PAni-based sensor is compared to fluorescence measurements, and it is shown that similar results are obtained for both methods.
Subject(s)
Aniline Compounds/chemistry , DNA/analysis , Nucleic Acid Amplification Techniques/methods , Electrochemical Techniques , Electrodes , Hydrogen-Ion Concentration , MicrofluidicsABSTRACT
This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly OH radicals, one-electron transfer process and singlet oxygen (1O2). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA N-glycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed.
