Signal amplification of a quartz crystal microbalance immunosensor by gold nanoparticles-polyethyleneimine for hepatitis B biomarker detection.

The procedures currently used for hepatitis B (HB) detection are not suitable for screening, clinical diagnosis, and point-of-care testing (POCT). Therefore, we developed and tested a QCM-based immunosensor by surface modification with AuNP-PEIs to amplify the signal and provide an oriented-immobilization surface. The AuNP-PEIs were characterized by ICP-Mass, UV/Vis, DLS, FE-SEM, and ATR-FTIR. After coating AuNP-PEIs on the gold electrode surface, anti-HBsAg antibodies were immobilized using NHS/EDC chemistry based on response surface methodology (RSM) optimization. The efficiency of the immunosensor was assessed by human sera and data were compared to gold-standard ELISA using receiver-operating-characteristic (ROC) analysis. FE-SEM, AFM, EDS, and EDS mapping confirmed AuNP-PEIs are homogeneously distributed on the surface with a high density and purity. After antibody immobilization, the immunosensor exhibited good recognition of HBsAg with a calibration curve of ∆F = - 6.910e-7x + 10(R2 = 0.9905), a LOD of 1.49 ng/mL, and a LOQ of 4.52 ng/mL. The immunosensor yielded reliable and accurate results with a specificity of 100% (95% CI 47.8-100.0) and sensitivity of 100% (95% CI 96.2-100.0). In conclusion, the fabricated immunosensor has the potential as an analytic tool with high sensitivity and specificity. However, further investigations are needed to convert it to a tiny lab-on-chip for HB diagnosis in clinical samples.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker.

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1-1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.