ABSTRACT
Using a mechanically grinded pyrolytic graphite electrode in edge orientation, a sensitive electrochemical method was developed for simultaneous determination of uric acid (UA), xanthine (XAN), hypoxanthine (HYP) (products of purine catabolism in human), allopurinol (ALO), and oxypurinol (OXY) (a drug used in treatment of purine catabolism disorders and its metabolite, respectively). It is demonstrated that differential pulse voltammetry in connection with this electrode can serve as a simple and efficient tool for monitoring transformation of purine catabolites (HYP --> XAN --> UA) catalyzed by xanthine oxidase (XO) as well as inhibition of this pathway by ALO being enzymatically converted to OXY. Our protocol is based on direct electrochemical measurement of oxidation peaks for each of the substances during in vitro reactions in a single detection step by the same electrode system. In addition, we show that the proposed electrochemical technique can be applied to parallel detection of metabolites involved in the XO pathway excreted in urine without any pretreatment of the clinical samples.
Subject(s)
Allopurinol/analysis , Electrochemical Techniques/methods , Oxypurinol/analysis , Purinones/analysis , Purinones/metabolism , Xanthine Oxidase/metabolism , Biosensing Techniques/economics , Biosensing Techniques/methods , Carbon/chemistry , Electrochemical Techniques/economics , Electrodes , Enzyme Inhibitors/analysis , Humans , Hypoxanthine/analysis , Hypoxanthine/metabolism , Hypoxanthine/urine , Purinones/urine , Sensitivity and Specificity , Uric Acid/analysis , Uric Acid/metabolism , Uric Acid/urine , Xanthine/analysis , Xanthine/metabolism , Xanthine/urine , Xanthine Oxidase/antagonists & inhibitorsABSTRACT
Polyhedral boron clusters are proposed as new, chemically and biologically stable, versatile redox labels for electrochemical DNA hybridization sensors. Selective and sensitive detection of the redox labeled DNA-probe was achieved by means of covalently attached electroactive marker 7,8-dicarba-nido-undekaborate group. A nanomolar concentration of boron cluster-labeled DNA was recognized. High specificity of the analysis with the boron cluster-labeled DNA probe, including detection of single-base mismatch, was demonstrated. The above findings, together with proposed earlier use of metallacarboranes as an electrochemical label for biomolecules opens the door for a "multicolor" electrochemical coding of DNA with boron clusters and simultaneous detection of several DNA targets.
Subject(s)
Borates/chemistry , Boron/chemistry , DNA/chemistry , Nucleic Acid Hybridization/methods , Uridine/chemistry , Base Pair Mismatch , Base Sequence , Biosensing Techniques , Borates/chemical synthesis , DNA/analysis , Electrochemistry , Electrodes , Oxidation-Reduction , Uridine/chemical synthesisABSTRACT
A cathodic stripping transfer voltammetric procedure for trace determination of DNA and its components is described. The method is based on the DNA acid hydrolysis with subsequent electrochemical determination of released purine bases. In the first step, DNA is hydrolyzed for 30 min in 0.5 M perchloric acid at 75 degrees C. The electrochemical step involves generation of Cu(I)-purine base complex on a mercury electrode surface, transfer of electrode with accumulated complex into supporting electrolyte where voltammetric measurement is performed. Analysis is carried out in 14-microl drop volume (two-electrode connection) or in 30-microl drop (three-electrode connection) on a platinum plate, which is used as a counter electrode. Blank electrolyte contains 0.05 M borate buffer, pH 9.2 with 6.3 microM Cu(II). We could observe voltammetric signal at hydrolyzed nucleosides, nucleotides, ODN, and DNA containing purine bases. We are able to accumulate under the controlled potential and determine subnanomolar concentration of DNA corresponding to the amount of 200 pg of DNA.
Subject(s)
Biosensing Techniques/methods , Copper/chemistry , DNA/analysis , DNA/chemistry , Electrochemistry/methods , Microchemistry/methods , Purines/chemistry , Electrodes , Oligodeoxyribonucleotides/analysis , Oligodeoxyribonucleotides/chemistry , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of the DNA sensors. We show that subnanomolar concentrations (related to monomer content) of unlabeled DNA can be determined using copper solid amalgam electrodes or hanging mercury drop electrodes in the presence of copper. DNA is first treated with acid (e.g., 0.5 M perchloric acid), and the acid-released purine bases are directly determined by the cathodic stripping voltammetry. Volumes of 5-3 microL of acid-treated DNA can easily be analyzed, thus making possible the determination of picogram and subpicogram amounts of DNA corresponding to attomole and subattomole quantities of 1000-base pair DNA. Application of this determination in DNA hybridization detection is demonstrated using surface H for the hybridization (superparamagnetic beads with covalently attached DNA probe) and the mercury electrodes only for the determination of DNA selectively captured at surface H.
