CNT Binding Peptides Selected by the Phage Display Method.

Using the M13 phage display method, 236 amino acid sequences (peptide aptamers) that could specifically adsorb to CNTs were selected. These peptide aptamers had abundant hydrophobic amino acids and evenly dispersed charged amino acids. The hydrophobic amino acids were postulated to contribute to CNT adsorption, while the charged amino acids contribute to their aqueous solubility. The frequency of proline amino acids, which causes the amino acid main chain bending, was slightly higher than in nature, suggesting that some conformational constraint might be required. Four peptide aptamers with a high frequency of occurrence in the selected sequences were further studied. Hydrophobicity scores were periodic along the amino acid sequence. 3D structure predictions by PEP-FOLD3 indicated that these aptamers would take a helical structure with hydrophobic amino acid residues on one side, suggesting that the aptamers bind hydrophobically to the CNT. The adsorption of these four aptamers to the carbon electrode was confirmed by electrochemical impedance spectroscopy, which demonstrated the effectiveness of the phage display method. At the same time, it was shown that even for selected peptides, the adsorption performance varied, and verification was needed.

Electrochemical Impedimetric Real-Time Polymerase Chain Reactions Using Anomalous Charge Transfer Enhancement.

We developed a new electrochemical impedimetric method for the real-time detection of polymerase chain reactions (PCR) based on our recent discovery that the DNA intercalator, [Ru(bpy)2DPPZ]2+, anomalously enhances charge transfer between redox mediators, K4[Fe(CN)6]/K3[Fe(CN)6], and a carbon electrode. Three mM [Fe(CN)6]3-/4- and 5 µM [Ru(bpy)2DPPZ]2+ were added to the PCR solution, and electrochemical impedance spectroscopy (EIS) measurements were performed at each elongation heat cycle. The charge transfer resistance (Rct) was initially low due to the presence of [Ru(bpy)2DPPZ]2+ in the solution. As PCR progressed, amplicon dsDNA was produced exponentially, and intercalated [Ru(bpy)2DPPZ]2+ ions, which could be detected as a steep Rct, increased at specific heat cycles depending on the amount of template DNA. The Rct increase per heat cycle, ΔRct, showed a peak at the same heat cycle as optical detection, proving that PCR can be accurately monitored in real time by impedance measurement. This simple method will enable a cost-effective and portable PCR device.