Cyclic voltammetry: A powerful tool to follow redox processes in biologically relevant systems (original) (raw)
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Optical and electrochemical detection of DNA
Biomaterials, 1988
There is a growing demand for the production of a DNA biosensor with applications in medicine, the food industry, agriculture, veterinary science and environmental science. In this paper we describe methods for the optical and electrochemical detection of DNA using the enzyme horseradish peroxidase (EC 1.11.1.7) and glucose oxidase . We have used bis-methylacridinium nitrate and luminol for the optical detection of DNA using a purpose built, inexpensive luminometer. Using this system detection limits of 1 O-" g of plasmid DNA have been obse~ed. Electrochemical detection of DNA was carried out by the use of a fluoride ion selective electrode and stripping voltamet~. DNA was detected down to 1 OVs-l O-lo g of DNA by the enzymatic release of halogen ions from organohalogen compounds.
Bio- and chemiluminescence in bioanalysis
Fresenius' Journal of Analytical Chemistry, 2000
Analytical chemiluminescence and bioluminescence represent a versatile, ultrasensitive tool with a wide range of applications in diverse fields such as biotechnology, pharmacology, molecular biology, clinical and environmental chemistry. Enzyme activities and enzyme substrates and inhibitors can be efficiently determined when directly involved in luminescent reactions, and also when they take part in a reaction suitable for coupling to a final light-emitting reaction. Chemiluminescence detection has been exploited in the fields of flow-injection analysis and column-liquid chromatographic and capillary-electrophoretic separative systems, due to its high sensitivity when compared with colorimetric detection. It has widely been used as an indicator of reactive oxygen species formation in cells and whole organs, thus allowing the study of a number of pathophysiological conditions related to oxidative stress. Chemiluminescence represents a sensitive and rapid alternative to radioactivity as a detection principle in immunoassays for the determination of a wide range of molecules (hormones, food additives, environmental pollutants) and in filter membrane biospecific reactions (Southern, Northern, Western, dot blot) for the determination of nucleic acids and proteins. Chemiluminescence has also been used for the sensitive and specific localization and quantitation of target analytes in tissue sections and single cells by immunohistochemistry and in situ hybridization techniques. A relatively recent application regards the use of luminescent reporter genes for the development of bioassays based on genetically engineered microorganisms or mammalian cells able to emit visible light in response to specific inorganic and organic compounds. Finally, the high detectability and rapidity of bio-and chemiluminescent detection make it suitable for the development of microarraybased high throughput screening assays, in which simultaneous, multianalyte detection is performed on multiple samples.
“Multicolor” Electrochemical Labeling of DNA Hybridization Probes with Osmium Tetroxide Complexes
Analytical Chemistry, 2007
Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2′ -bipyridine, 1,10-phenanthroline derivatives or N,N,N′,N′tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.
Chemiluminescent detection of DNA: application for DNA sequencing and hybridization
Nucleic Acids Research, 1989
A non-radioactive DNA detection chemistry is described and its application is shown for DNA hybridization and standard dideoxy DNA sequencing. The method employes a biotin-streptavidin system which binds an enzyme specifically to a target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is capable of detecting DNA in the hundred attomol range. INlTRODUCTION The growing interest in DNA mapping and sequencing has initiated the development of numerous new cloning and sequencing techniques (1). These have improved and partially automated the two basic methods of DNA sequencing, the enzymatic dideoxy method (2) and the chemical degradation method (3). One area of active research in this field is the development of non-radioactive, fluorescent (4, 5, 6, 28) or colorimetric (7, 8) detection chemistries. Here we report a third non-radioactive detection chemistry for DNA sequencing based on chemiluminescence. Luminescence has been used successfully for detection in DNA hybridization and other applications (20, 21, 22) but to date has not been applied to DNA sequencing. Similar to colorimetric staining, our chemiluminescent detection relies on a biotin/streptavidin interaction (9). A 5'-biotinylated oligonucleotide (19, 23) is used in a primer extension reaction for DNA sequencing or as a probe for hybridization to a target DNA. Biotinylated alkaline phosphatase is bound to the biotinylated 5'-end of the oligonucleotide via a streptavidin bridge and catalyzes the light reaction by cleaving off a phosphate group from an added chemiluminescent substrate (10). The emitted photons are detected by a Polaroid or X-ray film. The application of the chemiluminescent detection chemistry is shown for hybridization and standard dideoxy DNA sequencing and further applications are discussed. MATERIALS AND METHODS Materials: Single stranded M 13mp8 DNA was obtained from Bethesda Research Laboratories (BRL), Gaithersburg, MD and American BioTechnologies (ABT), Inc., Cambridge, MA. Deoxyand dideoxytriphosphates were from BRL and Pharmacia, Piscataway, NJ. Biol l-dCTP was from Enzo Biochem, Inc NY. DNA polymerase I large fragment (Klenow fragment) was from Boehringer Mannheim, Indianapolis, IN. Direct blotting electrophoresis was performed with a home made DBE apparatus (8). Streptavidin and biotinylated alkaline phosphatase were from Bethesda Research Laboratories (BRL kit # 8239SA). The purity of streptavidin and alkaline phosphatase seems to be essential for a good signal to noise Volume 17 Number 13 1989
Analytica Chimica Acta, 2002
An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide propyl-N -ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described. .tr (M. Ozsoz). low-cost point-of-care detection of specific nucleic acid sequences .
DNA hybridization at microbeads with cathodic stripping voltammetric detection
Talanta, 2002
In electrochemical DNA hybridization sensors generally a single-stranded probe DNA was immobilized at the electrode followed by hybridization with the target DNA and electrochemical detection of the hybridization event at the same electrode. In this type of experiments nonspecific adsorption of DNA at the electrode caused serious difficulties especially in the case of the analysis of long target DNAs. We propose a new technology in which DNA is hybridized at a surface H and the hybridization is detected at the detection electrode (DE). This technology significantly extends the choice of hybridization surfaces and DEs. Here we use paramagnetic Dynabeads Oligo(dT) 25 (DBT) as a transportable reactive surface H and a hanging mercury drop electrode as DE. We describe a label-free detection of DNA and RNA (selectively captured at DBT) based on the determination of adenines (at ppb levels, by cathodic stripping voltammetry) released from the nucleic acids by acid treatment. The DNA and RNA nonspecific adsorption at DBT is negligible, making thus possible to detect the hybridization event with a great specificity and sensitivity. Specific detection of the hybridization of polyribonucleotides, mRNA, oligodeoxynucleotides, and a DNA PCR product (226 base pairs) is demonstrated. New possibilities in the development of the DNA hybridization sensors opened by the proposed technology, including utilization of catalytic signals in nucleic acid determination at mercury (e.g. signals of osmium complexes covalently bound to DNA) and solid DEs (e.g. using enzyme-labeled antibodies against chemically modified DNAs) are discussed.