Electrochemical One-Step Immunoassay Based on Switching Peptides and Pyrolyzed Carbon Electrodes

Abstract

Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance (C dl ), high electron transfer rate (k app ), and wide electrochemical window. In addition, the factors influencing the amperometry of switching peptides labeled with ferrocene were analyzed according to the hydrodynamic radius, the number of intrahydrogen bonds, dipole moments, and diffusion coefficients. Finally, the applicability of the electrochemical one-step immunoassay for the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was assessed.

Carbon electrode obtained via pyrolysis of plasma-deposited parylene-C for electrochemical immunoassays

“…The current signals for anti-human hepatitis-C virus (hHCV) IgG ELISA kit analysis were calculated at the peak potentials of TMB oxidation (0.55 V vs. Ag/AgCl) and reduction (0.49 V vs. Ag/AgCl). 17 The square wave voltammetry (SWV) was performed with the initial potential and final potential of 0.7 and 0.3 V, respectively. The step potential ( E step ) was set as 4 mV, the pulse amplitude (Δ E ) was 25 mV, and the frequency was 15 Hz ( τ = 70 ms).…”

“…The pyrolyzed carbon electrodes were prepared via both deposition methods. 16,17 In the first step, thermally and plasma-deposited parylene-C was deposited on a Si wafer (10 × 10 mm 2 , 1 µm SiO 2 on top) with a thickness of 3 µm and 600 nm, respectively. The two types of parylene-C were then pyrolyzed in a tube furnace at 900 °C for 1 h under N 2 gas and a vacuum of 2.7 Pa (20 mTorr).…”

“…The E step was set as 10 mV, the pulse amplitude was 10 mV, the pulse time of 20 ms, and the scan rate was 50 mV s −1 . 17 The limit of detection (LOD) was calculated by adding three times of standard deviation to the mean value for a negative (blank) sample. 24,25 The mean value and the standard deviation were calculated by more than three times repetition (n = 3).…”

“…In this study, parylene-C obtained via plasma deposition was applied in preparing carbon electrodes via pyrolysis of parylene-C samples obtained via both deposition methods at 900 °C under N 2 gas. 16,17 The carbon film preparation from the pyrolysis of parylene-C film has advantages for the preparation of thin films far thinner than 1 μm on complicated shaped substrates. 15 Usually, screen-printed carbon films are usually made for a carbon layer with the thickness of micrometers 18 and the laser scribing method has difficulty in making carbon films on complicated shaped substrates.…”

One-step immunoassay for the detection of food-poisoning related bacteria using a switching peptide

“…Recently, a one-step immunoassay based on switching-peptides has been reported for the detection of target analytes. 19,20 As shown in Fig. 1(a), the switching-peptides are known to bind to the binding pocket of IgG and are quantitatively released from the immobilized antibodies when binding to the antigens.…”