Purines electrochemical methods

Several techniques have been developed for the determination of purine and pyrimidine derivatives in food sample and in particular for hypoxanthine quantification biosensors (220-223) and electrochemical methods making use of immobilized enzyme electrode (224 -227), electrochemical enzymatic-based HA methods (228,229), enzyme reaction with fluorimetric detection (230), radioimmunoassay (231), colorimetric methods (232), capillary electrophoresis (233), and TLC (234). Many HPLC methods have also been developed and are reported in Table 4 (235-247) the most recent ones are described next. 

Faraggi M, Klapper MH (1993) Reduction potentials determination of some biochemically important free radicals. Pulse radiolysis and electrochemical methods. J Chim Phys 90 711-744 Faraggi M, Klapper MH (1994) One electron oxidation of guanine and 2 -deoxyguanosine by the azide radical in alkaline solutions. J Chim Phys 91 1062-1069 Faraggi M, Broitman F, Trent JB, Klapper MH (1996) One-electron oxidation reactions of some purine and pyrimidine bases in aqueous solutions. Electrochemical and pulse radiolysis studies. J Phys Chem 100 14751-14761... 

Head of the Department, Adrien Albert (1907-89), was an international authority on biologically-active acridines, which included the antimalarial substances I had studied earlier, and now he was concerned with the purines and pyrimidines of the nucleic acids and other biochemically-important substances. My role was to extend the physico-chemical methods for studying organic structures and reactions from the electrochemical methods I had used in Oxford to the spectroscopic, using the infrared (IR) and ultraviolet (UV) spectrophotometers newly available commercially. 

Voltammetric measurements confirm that Hg-modified carbon electrodes are suitable for sensitive electrochemical detection of ODN compared to mercury electrodes. In the presence of the copper ions, these electrodes modified by a mercury layer were used for the detection of a picomolar quantity of ODN. The electrochemical step includes a potential-controlled reduction of the copper ions Cu(II) and accumulation of the Cu(I)-purine base residue complex on the Hg-modified carbon surface. The proposed electrochemical method can be used for the determination of different ODN lengths because the stripping current peak of the electrochemically accumulated Cu(I]-purine complex increased linearly with the length of ODN. The optical microscope images were used for the visualization of the surface morphology of the bare and Hg-modified carbon electrodes [64]. 

Adsorption and electrooxidation of ss NAs on a silver electrode were studied by electrochemical methods and surface-enhanced Raman spectroscopy [189, 228]. Using the latter electrode, Ean and coworkers [229] observed an anodic signal in solutions of DNA. This signal was attributed to redox reactions of purine bases, and provided a convenient way to determine DNA. Oxidation of purine bases... 

Compared with other methods, electrochemical ones have a wider range of application, which makes it possible to study the details of the reaction s mechanism. They are suitable for unique syntheses and for the solution of analytical problems. The use of electrochemical methods made it possible to obtain detailed information about the thermodynamics (redox potentials), kinetics (number of electrons, etc.) and mechanism of reactions with the participation of heterocyclic nitrogen compounds (purines, pyrimidines, porphyrines, etc.). [For more details see 2]. Capacity measurements provided important information [see, for example 3] on the adsorption properties of low-molecular and high-molecular bio-logically-active compounds (proteins, DNA, RNA). 

Electrochemical methods are uniquely suited to provide insight into redox and related chemical reactivity of compounds of biological interest. In electrochemical studies of enzymatic redox reactions, an electrode poised at a suitable potential is used to simulate the redox enzyme. Although the similarity is superficial, the value of such studies has been verified by the unique insights into redox reactivity of compounds of biological interest. These include purines and, recently, the drugs mitomycin and acetaminophen . 

HPLC, when compared to other instrumental methods, presents significant advantages for the simultaneous analysis of creatinine and purine derivatives. The variety of instrumental and experimental conditions (columns, buffers, organic modifiers, detectors, etc.) of these methods reported in the literature offers versatility and flexibility. Chromatographic conditions for these analytes are not complicated when reversed-phase columns are employed. New stationary phases with high separation power provide short analysis times. The mobile phases used are also very simple ones (organic-water mixmres with controlled pH) both isocratic elution and gradient elution are recommended. Different sensitivity detectors (UV, electrochemical, fluorescence, and combined techniques such as HPLC-MS) are very valuable for the... 

Electrochemical (anodic) fluorinations can be carried out, but may be difficult to control and over-fluorination and/or fluorination of substituents often results. The mechanism involves conversion of the substrates into radical cations, which are then trapped by fluoride, rather than electrophilic fluorination. Again, the method is more suited to robust systems such as pyrimidinones and purines. 

Several detection systems are utilized in CE for the analysis of nucleoside and nucleotide mixtures. The performances of UV-visible absorption, conductance, electrochemical, a- P radiochemical and fluorescence detectors and mass spectral interfacing have been compared recently. Although UV-visible absorption is generally considered as not very sensitive, low limits of detection (LODs) of 8x10 mol 1 have been reported for purine metabolites using this method. The conductivity technique suffers from poor sensitivity. Electrochemical detection has a higher sensitivity, but its usefulness is limited by the fact that only electroactive species can be detected. Detection by mass spectrometry (MS) leads to poor sensitivity and implies expensive instrumentation. Radiochemical detection has been applied to a- P-labeled thymidine, cytidine, and adenosine... 

Following these pioneering studies on electrochemical and optical chemical sensors based on functionalized poly thiophenes, many researchers decided to address the detection of small molecules of biomedical interest. For instance, in 1998, Bauerle and Emge described a method to detect the binding of purine or pyrimidine bases by covalently attaching a pyrimidine or triazine unit to polymers 6 or 7 (Scheme 22.1) [29]. Here, the addition of small concentrations of a complementary purine or pyrimidine resulted in an increase in the oxidation potential, and also a decrease in the electroactivity. 

At carbon electrodes, purine bases produce well-defined oxidation peaks within a wide pH range (0-12.5) [142,143]. Purine nucleosides and nucleotides are oxidized at potentials more positive than the parent bases [144]. Signals corresponding to the oxidation of purine bases, nucleotides, and nucleotides have also been obtained using chemically modified carbon electrodes [145,146] (for more details see Sect. 12.4.3). Recently, Cai and coworkers [147] proposed a method for trace A determination using an electrochemically/chemically modified (in alkahne sodium nitrate solution) carbon paste electrode (CPE). Pyrimidines are considered to be electroinactive on carbon electrodes however, Oliveira-Brett and Matysik recently reported [148] specific anodic peaks observed in solutions of T and C bases (but not their nucleosides). Sugar components of nucleotides can be oxidized at copper electrodes [149]. 

In this chapter, we have reported some examples related to DNA sensors, especially used in food applications, by employing electrochemical detection techniques. A variety of sensing systems based on label-free techniques utilizing electrochemical and/or surface activity as well as direct methods that rely on the intrinsic electrochemical properties of DNA (the oxidation of purine bases, particularly guanine) have been presented in the different sections of this chapter. 

The information power of electrochemistry can be expanded by coupling it with methods which can determine the chemical identity of intermediates and products of electrode reactions. In-situ information can be obtained by on-line spectroscopic methods such as ultraviolet/visible (UV/vis) thin-layer spectroelectrochemistrys. Recently, effective on-line coupling of an electrochemical cell with a mass spectrometer has been demonstrated and its application to the study of biological redox reactions has been described . Electrochemistry/mass spectrometry as well as off-line methods were used in determining redox and related chemical reactivity of purine drugs. 

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