A novel electrochemical biosensing method with double-layered polymer brush modified electrode.

We developed a novel electrochemical biosensor electrode that has a potential to reduce background noise for which we constructed an original conductive substrate modified with a double-layered polymer brush structure that is water impermeable and can control biomolecules adsorption/desorption. In this study, a hydrophobic poly(tert-butyl methacrylate) brush layer was prepared on a gold electrode, and then, the tert-butyl group near the outermost surface was dissociated by the acid treatment to obtain a hydrophilic carboxy group, thereby fabricating a conductive substrate with the double-layered polymer brush structure. Formation of the double-layered polymer brush structure was indicated by surface wettability and optical analyses.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands.

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies.

Electrical Monitoring of Methylated DNA Based on Its Conformational Change to G-Quadruplex Using a Solution-Gated Field-Effect Transistor.

Methylated DNA is not only a diagnostic but also a prognostic biomarker for early-stage cancer. However, sodium bisulfite sequencing as a "gold standard" method for detection of methylation markers has some drawbacks such as its time-consuming and labor-intensive procedures. Therefore, simple and reliable methods are required to analyze DNA sequences with or without methylated residues.

Methods for Improving Aptamer Binding Affinity.

Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities.

Simultaneous improvement of specificity and affinity of aptamers against Streptococcus mutans by in silico maturation for biosensor development.

In silico evolution with an in vitro system can facilitate the development of functional aptamers with high specificity and affinity. Although a general technique known as systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method for aptamer selection, it sometimes fails to identify aptamers with sufficient binding properties. We have previously developed in silico maturation (ISM) to improve functions of aptamers based on genetic algorithms.

Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing.

We selected DNA aptamers against insulin and developed an aptameric enzyme subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. In vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round.

Improvement of Aptamer Affinity by Dimerization.

To increase the affinities of aptamers for their targets, we designed an aptamerdimer for thrombin and VEGF. This design is based on the avidity of the antibody, whichenables the aptamer to connect easily since it is a single-strand nucleic acid. In this study,we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer.

Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method.

Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF(165)) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting.

Selection and characterization of DNA aptamers against VEGF165 with aptamer blotting method and its application.

We have isolated two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165). To select aptamers that have high specificity for VEGF165, we used another protein as a competitor in the SELEX process. In addition, we evaluated the screened library with aptamer blotting.

Screening of DNA aptamer against mouse prion protein by competitive selection.

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody.