The Application And Exploration Of DNA Biosensor In Clinical Laboratory

DNA biosensor is a new technique developed in 1990 s. A biosensor by definition is an analytical device that combines the specificity of a nucleic acid sensing element with a transducer to produce a signal proportional to target analyte concentration. The signal is converted by a transducer into measurable respose such as electrochemical fluorescence,SPR and field effects transistor. Compared with traditonal method, DNA biosensor is fast, specificity, sensitivity and easy operation and can be adapted to various purposes including genetic diagnosis, environmental monitoring. In this paper, we describe a turn-on electrochemical biosensor for the detection of methyltransferases(MTases) causing DNA adenine methylation, and attempted to clarify the mechanism of nucleic acid self assembly; further we developed a cascade recognition reaction for nucleic acid detection with high selectivity using fluorescence method.1. Proximity-based electrochemical biosensor for highly sensitive determination of methyltransferase activity using gold nanoparticle-based cooperative signal amplification.We describe a turn-on electrochemical biosensor for the detection of methyltransferases(MTases) causing DNA adenine methylation. Thisbiosensor is based on insertion, methylation-resistant cleavage, signal enrichment caused by gold nanoparticles(AuNPs), and a signal probe dragging strategy. A double-strand DNA(dsDNA) containing identical MTase and methylation-resistant endonuclease(Mbo I) sites was immobilized on the surface of a gold electrode via Au-S covalent binding.The surface was subsequently treated with MTase and Mbo I and then washed. Results revealed that the surface of the electrode contains methylated dsDNA and 12-base nucleotides residual. Depending on biotin-streptavidin interactions that enabled signal probes and nucleotide residue hybridization and AuNP enrichment, a large number of signal probes labeled with ferrocene(Fc) are captured by the electrode. Under optimal conditions, the differential pulse voltammetry signals of Fc tags(at a working voltage of 0.24 V vs. Ag/AgCl) are linearly related to MTase logarithmic concentration ranging from 0.1 to 40 U?mL-1. The dynamic range from 0.05 to 50 U?mL-1 and the limit of detection is 0.024 U?mL-1(at an S/N ratio of 3). The sensor is acceptably stable, reproducible and highly selective. In our preception, this strategy provides a promising approach for simple, sensitive and selective detection of Dam MTase and may be extended to the determination of other DNA MTase by exchanging the corresponding DNA.2. Improved hybridization rate by insertion method on gold electrode surface through thermodynamic and kinetic analysis.Analytical performance of a DNA biosensor is ultimately connected to nanoscale arrangement of the DNA capture probe molecules on sensor surfaces. Self-assembly of a mixed monolayer of thiolated probes and diluents onto gold surfaces is nowadays commonly performed by a two-step backfilling immobilization procedure. In this work, we demonstrated a novel method that ‘‘inserting’’ thiolated DNA into a preformed alkanethiol monolayer. Chronocoulometric measurements were performed in the presence of dissolved hexaammineruthenium(RuHex)cations to clarify the probe surface density and hybridization efficiency prepared by these two methods. We found that probes prepared by insertion method have a better performance compared with backfilling method. To further state the mechanism, the kinetic affinity constant K and the thernodynamic constant gibbs energy change(△G)for the hybridization at25 ℃ were calculated.3. Energy driven cascade recognition for selective detection of nucleic acids with high discrimination factor at roomIn this chapter, we demonstrated a cascade recognition strategy for the detection of single strand nucleic acid with high discrimination factor at room temperature. The cascade recognition strategy contains a toehold mediated strand displacement and a double-toehold mediated double strand displacement reaction, thus enable the high ability to discern point mutation of target. The discrimination factor of the model target is between 45 and109, with the medium of 70. This strategy is homogeneous, easy operation,enzyme-free, isothermal, and can be easily adapted to high-throughput devices without the need of designing complicated instruments.