Subject(s)
Biosensing Techniques/instrumentation , DNA, Viral/analysis , DNA/genetics , Gold/chemistry , Oligonucleotide Array Sequence Analysis/instrumentation , Viruses/genetics , Viruses/isolation & purification , Biosensing Techniques/methods , DNA/chemistry , DNA, Viral/genetics , Equipment Design , Equipment Failure Analysis , Nanotechnology/instrumentation , Nanotechnology/methods , Oligonucleotide Array Sequence Analysis/methodsABSTRACT
The ultrasensitive detection of DNA is achieved by PCR-induced evolution of a DNAzyme.
Subject(s)
DNA Primers/genetics , DNA Primers/metabolism , DNA, Catalytic/metabolism , Base Sequence , Molecular Sequence Data , Oxidation-Reduction , Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Nucleic acids include substantial information in their base sequence and their hybridization-complexation motifs. Recent research efforts attempt to utilize this biomolecular information to develop DNA nanostructures exhibiting machine-like functions. DNA nano-assemblies revealing tweezers, motor, and walker activities exemplify a few such machines. The DNA-based machines provide new components that act as sensitive sensors, transporters, or drug delivery systems.
Subject(s)
DNA/chemistry , Nanotechnology/methods , Base Sequence , Biosensing Techniques/methods , Catalysis , DNA/genetics , DNA, Catalytic/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid HybridizationSubject(s)
Bacteriophage M13/genetics , Bacteriophage M13/isolation & purification , DNA, Single-Stranded/chemistry , DNA, Viral/chemistry , Nucleic Acid Amplification Techniques/methods , Base Sequence , Catalysis , DNA, Catalytic/metabolism , DNA, Single-Stranded/analysis , DNA, Single-Stranded/genetics , DNA, Viral/analysis , DNA, Viral/genetics , Nucleic Acid ConformationABSTRACT
Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasi-reversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by approximately 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold.
Subject(s)
Biosensing Techniques/methods , Protein Engineering , Superoxide Dismutase/analysis , Biosensing Techniques/instrumentation , Electrochemistry , Humans , Sensitivity and SpecificityABSTRACT
A novel multilayer cytochrome c electrode for the quantification of superoxide radical concentrations is introduced. The electrode consists of alternating layers of cytochrome c and poly(aniline(sulfonic acid)) on a gold wire electrode. The formation of multilayer structures was proven by SPR experiments. Assemblies with 2-15 protein layers showed electrochemical communication with the gold electrode. For every additional layer, a substantial increase in electrochemically active cytochrome c (cyt. c) was found. For electrodes of more than 10 layers, the increase was more than 1 order of magnitude as compared to monolayer electrode systems. Thermodynamic and kinetic parameters of the electrodes were characterized. The mechanism of electron transfer within the multilayer assembly was studied, with results suggesting a protein-protein electron-transfer model. Electrodes of 2-15 layers were applied to the in vitro quantification of enzymatically generated superoxide, showing superior sensitivity as compared to a monolayer-based sensor. An electrode with 6 cyt. c/PASA layers showed the highest sensitivity of the systems studied, giving an increase in sensitivity of half an order of magnitude versus the that of the monolayer electrode. The stability of the system was optimized using thermal treatment, resulting in no loss in sensor signal or protein loading after 10 successive measurements or 2 days of storage.
Subject(s)
Cytochromes c , Superoxides/analysis , Animals , Biosensing Techniques , Cattle , Electrochemistry/methods , Electrodes , Erythrocytes/enzymology , Horses , Superoxide Dismutase/bloodABSTRACT
The in vitro superoxide scavenging activity (as determined by electrochemical measurement) and the in vivo antioxidant potential (as determined by a mouse model of carbon tetrachloride (CCl(4)) hepatotoxicity) of methanolic extracts prepared from 10 Chinese tonifying herbs were compared. Electrochemical measurement using a cytochrome c (Cyt. c) sensor showed that all of the tested herbal extracts exhibited a medium superoxide scavenging activity of different potency, as indicated by their IC(50) values. The in vivo measurement demonstrated that 80% of the herbal extracts displayed in vivo antioxidant potential, as assessed by the percentage of protection of the activity of plasma alanine aminotransferases and the hepatic glutathione regeneration capacity under CCl(4)-intoxicated condition. Although the in vitro antioxidant activity did not correlate quantitatively with the in vivo antioxidant potential, for 8 out of 10 samples a similar tendency was found. The rapid amperometric assessment of antioxidant potential by Cyt. c sensor may offer a convenient and direct method for screening as well as the quality control of herbal products.
