Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin Al c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Auichitosan/Fe3O4, which was synthesized with easy handling feature of Fe3O4 by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe3O4 magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immuno-reactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 mu g mL(-1), with the detection limit of 0.025 mu g mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c. (C) 2011 Elsevier B.V. All rights reserved.