A biosensor based on electroactive dipyrromethene-Cu(II) layer deposited onto gold electrodes for the detection of antibodies against avian influenza virus type H5N1 in hen sera

Abstract

This paper describes the development of a biosensor for the detection of anti-hemagglutinin antibodies against the influenza virus hemagglutinin. The steps of biosensor fabrications are as follows: (i) creation of a mixed layer containing the thiol derivative of dipyrromethene and 4-mercapto-1-butanol, (ii) complexation of Cu(II) ions, (iii) oriented immobilization of the recombinant histidine-tagged hemagglutinin, and (iv) filling free spaces with bovine serum albumin. The interactions between recombinants hemagglutinin from the highly pathogenic avian influenza virus type H5N1 and anti-hemagglutinin H5 monoclonal antibodies were explored with Osteryoung square-wave voltammetry. The biosensor displayed a good detection limit of 2.4 pg/mL, quantification limit of 7.2 pg/mL, and dynamic range from 4.0 to 100.0 pg/mL in buffer. In addition, this analytical device was applied for the detection of antibodies in hen sera from individuals vaccinated and non-vaccinated against the avian influenza virus type H5N1. The limit of detection for the assay was the dilution of sera 1: 7 × 10(6), which is about 200 times better than the enzyme-linked immunosorbent assay.

Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies

“…The Cu(II) salt is reduced to the Cu(I) catalyst by sodium ascorbate as a reducing reagent. Due to its high instability, Cu(I) is stabilized by THPTA complexing ligand [ 25 ]. The CuAAC coupling reaction was performed for 30 min at room temperature followed by consecutive washing steps with washing buffer (WB) (200 mM dihydrogen sodium phosphate, 200 mM sodium chloride, 150 mM ethylenediaminetetraacetic acid, and 50 mM ethanolamine at pH 7.5); 0.5% ( w / v ) SDS; PBS; 0.7 M β-mercaptoethanol (BME); and PBS (5 min for each washing step).…”

“…Alternatively, linkages between the protein and the surface can be established in a more controllable way by fusing the protein of interest with an affinity tag, e.g., a polyhistidine by which an interaction with Ni 2+ , Cu 2+ or Co 2+ cations can be established [ 18 ]. A number of studies have reported the use of Cu(II)-complexing layers deposited on gold electrodes to obtain a uniformly oriented conjugation of His 6 -tagged proteins, e.g., a dipyrromethene-Cu(II) layer and Receptor domains for advanced glycation end products (RAGE) [ 23 , 24 ], a dipyrromethene-Cu(II) layer and antibodies against H5N1 influenza virus [ 25 ], a pentetic acid-Cu(II) layer and JAK2 kinase [ 26 ], an iminodiacetic acid-like-Cu(II) layer and RIO1 kinase [ 27 ]. The thiol derivatives of these complexing reagents are firstly linked to the gold surfaces to form the layers, followed by Cu(II) complexation and His 6 -tagged protein immobilization.…”

Electrochemical Sensors for Detections of Influenza Viruses: Fundamentals and Applications

“…In order to avoid the necessity of the redox marker present in the sample solution, the immunosensor-incorporated redox-active layer has been developed [39].…”

Redox-active Monolayers Deposited on Gold Electrode Surface–Universal Platforms for Electrochemical Sensing

“…8). (37) This biosensor was able to detect a humoral response in the sera of hens immunized with DNA vaccine on the basis of the sequence of HA from the H5N1 virus. Its sensitivity was about 20 to 200 times higher than that of ELISA.…”