无标记实时检测乙肝表面抗原的 QCM 免疫传感器

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

出版信息

Clin Lab. 2022 Apr 1;68(4). doi: 10.7754/Clin.Lab.2021.210537.

DOI:10.7754/Clin.Lab.2021.210537

Abstract

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.

摘要

背景

乙肝病毒表面抗原（HBsAg）是诊断和预防乙肝感染的重要蛋白。本研究设计了一种基于抗体-抗原相互作用的压电免疫传感器来检测 HBsAg。采用石英晶体微天平系统检测抗体-抗原相互作用。

方法

首先，设计了一个振荡器，通过 IC 74LVC1GX04 测量受反应物影响的谐振频率。将抗 HBsAg 抗体固定在 10MHz AT 切石英晶体上。通过原子力显微镜（AFM）和接触角测量监测表面修饰。使用不同浓度的抗体进行表面固定，并评估频率变化。通过评估晶体和固定抗体的稳定性来研究系统的稳定性。使用 AFM 分析抗体在晶体上的吸附以及谐振频率的变化。此外，使用这种固定的抗体进行直接免疫测定，以识别不同浓度的 HBsAg 溶液。最后，分别使用乙型肝炎（HBsAg）和丙型肝炎（HCV Ag）抗原研究特异性和非特异性反应。

结果

成功地将抗 HBsAg 抗体固定在 10MHz AT 切石英晶体上。对固定抗体的晶体和未固定晶体的稳定性测试表明，两种形式的晶体都很稳定。比较了理论和实验频率检测。接触角的降低表明修饰后表面的亲水性。AFM 图像表明，抗体吸附后表面更加均匀，表面粗糙度（RMS）从 1.13nm 降低到 0.99nm。HBsAb 物理吸附在设计芯片上后检测到频率变化。抗原的标准曲线表明，抗原浓度决定频率变化。最后，特异性试验证实了设计的生物传感器对 HCV Ag 中 HBsAg 的检测具有特异性。通过 QCM 的频率变化来表征固定抗体的定量。将获得的结果与 ELISA 检测进行比较。QCM 和 ELISA 之间 HBsAg 稀释的相关系数为 0.9821。