Label-free Detection Of DNA Hybridization Research Articles

Amplified label-free electrical detection of DNA hybridization.

A new protocol is described for amplifying label-free electrochemical measurements of DNA hybridization based on the enhanced accumulation of purine nucleobases in the presence of copper ions . Such electrical DNA assays involve hybridization of the target to inosine-substituted oligonucleotide probes (captured on magnetic beads), acidic dipurinization of the hybrid DNA, and adsorptive chronopotentiometric stripping measurements of the free nucleobases in the presence of copper ions. Both amplified adenine and guanine peaks can be used for detecting the DNA hybridization. The dramatic signal amplification advantage of this type of detection has been combined with efficient magnetic removal of non-complementary DNA, use of microliter sample volumes and disposable transducers. Factors influencing the signal enhancement were assessed and optimized. A detection limit of 40 fmol (250 pg) was obtained with 10 min hybridization and 5 min adsorptive-accumulation times. The advantages of this procedure were demonstrated by its application in the detection of DNA segments related to the BRCA1 breast cancer gene. The copper enhancement holds great promise not only for the detection of DNA hybridization, but also for trace measurement of nucleic acids.

Magnetic bead-based label-free electrochemical detection of DNA hybridization.

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.

Pencil-based renewable biosensor for label-free electrochemical detection of DNA hybridization

The characteristics and advantages of a renewable DNA hybridization biosensor based on a pencil electrode transducer are described. The surface of this biosensor can be renewed rapidly, by a simple mechanical extrusion, hence, obviating the need for an additional regeneration step. The sensor, thus, responds rapidly to the ‘switching’ between target and non-complementary oligonucleotide solutions, with the use of fresh surfaces erasing memory effects. The intrinsic redox activity of the target DNA is employed for detecting the duplex formation. Relevant experimental parameters were examined and optimized. The selectivity of the new device was demonstrated for the detection of a single-point mutation in the BRCA1 breast cancer gene. Such low-cost, renewable graphite transducers provide an attractive alternative to conventional carbon electrodes used for transducing DNA hybridization. By eliminating the needs for a regeneration step and an external indicator, the device offers a greatly simplified operation and holds promise for decentralized genetic testing.