Dioxin-Binding Pentapeptide for Use in a High-Sensitivity On-Bead Detection Assay
journal contributionposted on 01.12.2005 by Chikashi Nakamura, Yasuhiro Inuyama, Hiroki Goto, Ikuo Obataya, Nao Kaneko, Noriyuki Nakamura, Noriaki Santo, Jun Miyake
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The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3‘,4‘-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.