Label-Free Real-Time Detection of HBsAg Using a QCM Immunosensor.

Abstract

Hepatitis B virus surface antigen (HBsAg) is an important protein in both diagnosis and prevention of hepatitis B infection. In the current study, a piezoelectric immunosensor based on antibody-antigen interaction was designed to detect HBsAg. A quartz crystal microbalance system was employed to detect antibody-antigen interaction. At first, an oscillator was designed to measure the resonant frequency affected by the reactants using IC 74LVC1GX04. Antibody against HBsAg was immobilized on 10 MHz AT-cut quartz crystal. The surface modifications were monitored by atomic force microscopy (AFM) and contact angle measurements. Different concentrations of antibody were used for surface immobilization and the frequency shifts were assessed. The system stability was studied by evaluating the stability of the crystal and the immobilized antibody. The adsorption of antibody onto the crystal was analyzed using AFM and changes in the resonance frequency. Further, a direct immunoassay was performed with this immobilized antibody to identify HBsAg solutions at different concentrations. Finally, specific and non-specific responses were investigated using hepatitis B (HBsAg) and hepatitis C (HCV Ag) antigens, respectively. Antibodies against HBsAg were successfully immobilized on 10 MHz AT-cut quartz crystal. The stability tests of crystal immobilized with antibody and unimmobilized crystal revealed that both forms of crystals were stable. Theoretical and experimental frequency assays were compared. A decrease in the contact angle indicated the hydrophilicity of surface after modifications. AFM images illustrated a more uniform surface after antibody adsorption and the surface roughness (RMS) reduced from 1.13 to 0.99 nm. Changes in the frequency were detected after the physical adsorption of HBsAb on the designed chip. The standard curve of antigen revealed the frequency changes depend on concentration of antigen. Finally, the specificity test confirmed the specificity of the designed biosensor for the detection of HBsAg from HCV Ag. The quantization of immobilized antibody was characterized by the frequency shift of the QCM. The obtained results were compared with ELISA assay. The correlation coefficients of HBsAg dilution between QCM and ELISA was 0.9821. This study is a new step to meet the challenges regarding HBsAg detection. Physical adsorption used in this study was effective as the simplest immobilization method to design a QCM-based immunosensor for HBsAg detection. Facilitated, fast, and simple detection of HBsAg by an antibody-based QCM biosensor is our main objective.