Construction of Protein Probe with a His‐tag and an Electron‐transfer Peptide for a Target Protein Sensing

Abstract

A protein probe with an electron‐transfer peptide and a His‐tag was designed to electrochemically sense a target protein. We selected tyrosine‐rich (Y4C) and tryptophan‐rich (W4C) peptides for use as electron‐transfer agents. The peak for oxidation was based on the oxidations of the phenolic hydroxy groups in Y4C and on the indole rings in W4C. Asialofetuin (ASF) with galactose residues was the protein probe, and a galactose recognition protein, soybean agglutinin (SBA), was the target protein. A protein probe composed of an amino acid and carbohydrate residue was expected to be biocompatible. When voltammetric measurements were performed using a glassy carbon electrode, the oxidation peaks of H6Y4C and ASF‐H6Y4C appeared at the same potential. The peak current of ASF‐H6Y4C was 4‐fold that of H6Y4C because of the stronger adsorption of ASF‐H6Y4C onto the electrode. The electrode response of ASF‐H6Y4C with SBA was half that of ASF‐H6Y4C alone. By contrast, the peak current of ASF‐Y4CH6 was higher than that of ASF‐H6Y4C, which was the result of a greater degree of contact between the Y4C moieties and an electrode. On the other hand, the voltammetric behaviors of ASF with W4C and a His‐tag were similar to those with Y4C and a His‐tag. The sensitivity of SBA using ASF‐Y4CH6 was at the 10−13 M level. To confirm the function of the sensing system, measurements were performed in human serum with SBA and ASF‐Y4CH6. When SBA was added, the serum had a concentration that ranged between 5.0×10−13 and 4.0×10−12 M, and the amount of SBA that could be recovered ranged from 97 to 101%. Consequently, this system could be applied to the detection of SBA in serum.

Electrochemical sensing of target cells based on a peptide/single‐strand DNA probe

“…The mass spectra of the synthesized forms of DNA and Ac‐H 6 Y 4 C/ss‐DNA aptamer probe were recorded using MALDI‐TOF/TOF‐MS. The mass spectrometer was normalized based on the average of the mass peaks caused by the mixture of several proteins [42]. An ionizing agent of silver trifluoroacetate was added to the sample, and 3‐Hydroxypicolinic acid was used as a matrix substance.…”

Voltammetric Sensing of Soybean Agglutinin Using an Electrode Modified with Electron‐transfer, Carbohydrate‐mimetic/Cross‐linker‐peptide‐collagen Film

“…Next, a sensor was designed using an Ac-W 4 CA 6 /collagenfilm-modified electrode instead of the Ac-Y 4 CA 6 (Figure S5). The cysteine residue in oligotryptophan was introduced at the C-terminals to serve as an electrontransfer peptide [43]. The peak current of the W 4 C moieties did not decrease even when 2.0 × 10 À 10 M SBA was added to a solution.…”

Construction of Biosensing System for Glycated Albumin Using an Electron Transfer Peptide-Modified Protein Probe

“…17) In a previous study, we sensed soybean agglutinin using H 6 Y 4 C and Y 4 CH 6 , in which six histidine residues were introduced into Y 4 C, H 6 W 4 C, and W 4 CH 6 , which also are electron-transfer peptides. 18) The method involves modifying asialofetuin with an electron-transfer peptide and binding it to soybean agglutinin (SBA) to enable detection of SBA. This method has high levels of detection sensitivity and selectivity, which makes it a sensing tool appropriate for medical and other applications.…”