Electrochemical Detection Methodologies

Krull IS, Ding XD, Selavka C, et al. 1984. Explosives and other nitro compounds determined by liquid chromatography with photolysis-electrochemical detection. Methodological Surveys in Biochemistry and Analysis 14 365-366. 

The system developed in this work establish a general electrochemical detection methodology that can be applied to a variety of immunosystems and DNA detection systems, including lab-on-a-chip technology, with special interest for further applications in clinical analysis, food quality and safety as well as other industrial applications. 

Adsorption is an easy way to attach nucleic acids to solid surfaces, since no reagents or modified-DNA are required. These features have promoted extensive use of adsorption as immobilization methodology in genetic analysis. The mainly claimed disadvantages of adsorption with respect to covalent immobilization are that (i) nucleic acids may be readily desorbed from the substrate and (ii) base moieties may be unavailable for hybridization if they are bonded to the substrate in multiple sites [55]. However, the electrochemical detection strategy based on the intrinsic oxidation of DNA requires the DNA to be adsorbed in close contact with the electrochemical substrate by multisite attachment. This multi-site attachment of DNA can be thus detrimental for its hybridization but is crucial for the detection based on its oxidation signals. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

There are many substances which would appear to be good candidates for LC-EC from a thermodynamic point of view but which do not behave well due to kinetic limitations. Johnson and co-workers at Iowa State University used some fundamental ideas about electrocatalysis to revolutionize the determination of carbohydrates, nearly intractable substances which do not readily lend themselves to ultraviolet absorption (LC-UV), fluorescence (LC-F), or traditional DC amperometry (LC-EC) [2], At the time that this work began, the EC of carbohydrates was more or less relegated to refractive index detection (LC-RI) of microgram amounts. The importance of polysaccharides and glycoproteins, as well as traditional sugars, has focused a lot of attention on pulsed electrochemical detection (FED) methodology. The detection limits are not competitive with DC amperometry of more easily oxidized substances such as phenols and aromatic amines however, they are far superior to optical detection approaches. 

Anton AH, Berk AI. Evaluation and improvement of homovaniUic acid methodology, and a critique of liquid chromatography with electrochemical detection. Chn Chem 1987 33 600-3. 

The first successful application of this methodology involved glassy carbon surfaces derivatized with single-stranded DNA. " Electrostatically-bound octahedral complexes of Co(lll) were used to quantitiate immobilized DNA and to monitor the binding of a complementary oligonucleotide. This work verified that electrochemical detection of target sequences was feasible. 

Electrochemical detection with ion-pairing adaptations of reversed-phase chromatography are the most common methodologies, and many techniques for measuring urinary catecholamines and metanephrines have been... 

Electrochemical detection is possible for analytes amenable to oxidation or reduction at moderate electrode potentials. The selectivity can to some extent be adjusted through electrode potential. However, this methodology has scarcely been used for the detection of carbamate pesticides. Thus, Anderson et al. determined various carbamates in water samples by LC using a cyclic voltammetry detector or a micro-array electrochemical flow detector. ... 

In spite of the overall importance of biological amines, the methodological difficulties have confined the determinations of such compounds to specialized biomedical laboratories. While the derivatization methods still appear to be far from being optimized, the high-sensitivity determinations done with expensive equipment (e.g., a mass spectrometer) are outside the reach of many laboratories. More recently, HPLC has been under development as a very attractive alternative for this class of compounds fluorometric and, in particular, the electrochemical detection in combination with HPLC have enjoyed much attention [417]. Although the GC methodological approaches to the analysis of biological amines may yield to HPLC in time, a fair number of GC studies are still reported in the literature. 

Campbell, J.M. E.A. Flickinger G.C. Fahey, Jr. A comparative study of dietary fiber methodologies using pulsed electrochemical detection of monosaccharide constituents. Sem. Food Anal. 1997,2, 43-53. 

Very few biological materials have been analyzed for the presence of MBOCA or its metabolites. MBOCA and its metabolites have been measured in urine of exposed humans and experimental animals. Hemoglobin adducts have also been measured in the blood of exposed animals. The most frequently used techniques are gas chromatography (GC) with electron capture detection (ECD) and high-performance liquid chromatography (HPLC) with electrochemical detection (ED). Detailed methodologies from selected studies are presented in Table 6-1. 

DNA probes, the first one linked with paramagnetic beads and the second one modified with Au-NPs via biotin-streptavidin affinity reactions. Zheng et al. [67] employed this detection methodology for the development of a specific electrochemical aptasensor for the detection of thrombin. The sensor was based on a sandwich format of magnetic nanoparticle/thrombin/Au-NP and signal amplification by forming network-like thiocyanuric acid/Au-NPs. A detection limit of 7.82 aM was achieved. 

Potentiometry is the most common method of electrochemical detection used in flow injection analysis. This methodology creates more favorable conditions for potentiometric measurements when compared to batch procedures. In FIA measurements, it is easier to avoid incidental contamination or that resulting from leakage of the solution from the reference electrode. Moreover, deactivation of the sensing surface of the indicator electrode due to adsorption, precipitation, or corrosion is minimized greatly as a consequence of the short contact time with the sample... 

Multidimensional chromatography is one of the best options to prevent unwanted coelutions of analytes with other matrix components present in a variety of complex samples, and also to incorporate automated online sample preparation in the analytical process. Nowadays, well-developed methodologies already exist using multidimensional modes as LC-LC or SPE-LC in combination with spectroscopic or electrochemical detection for the selective and sensitive determination of many analytes, mainly in the biomedical, pharmaceutical, environmental, and food fields. 

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