The high sensitivity and specificity of terahertz (THz) biosensing are both promising and challenging in DNA sample detection. This study produced and refined a flexible THz MM biosensor for ultrasensitive detection of HBV in clinical serum samples based on a gold magnetic nanoparticle-mediated rolling circle amplification (GMNPs@RCA) sandwich assay under isothermal conditions. Typically, solid-phase RCA reactions mediated by circular padlock probes (PLPs) are triggered under isothermal conditions in the presence of HBV DNA, resulting in long single-stranded DNA (ssDNA) with high fidelity and specificity. Then, the resultant ssDNA was conjugated with detection probes (DPs) immobilized on gold nanoparticles (DP@AuNPs) to form GMNPs-RCA-AuNPs sandwich complexes. The HBV DNA concentrations were quantified by introducing GMNPs-RCA-AuNPs complexes into the metasurface of a flexible THz metamaterial-based biosensor chip and resulting in a red shift of the resonance peak of the THz metamaterials. This biosensor can lead to highly specific and sensitive detection with one-base mismatch discrimination and a limit of detection (LOD) down to 1.27E + 02 IU/ml of HBV DNA from clinical serum samples. The HBV DNA concentration was linearly correlated with the frequency shift of the THz metamaterials within the range of 1.27E + 02∼1.27E + 07 IU/ml, illustrating the applicability and accuracy of our assay in real clinical samples. This strategy constitutes a promising THz sensing method to identify virus DNA. In the future, it is hoped it can assist with pathogen identification and clinical diagnosis.

Li et al. presented a new terahertz (THz) biosensing strategy for serum Hepatitis B virus DNA (HBV DNA) detection in clinical samples based on a flexible MM chip with an ultrathin polyethylene terephthalate (PET) substrate and gold magnetic nanoparticle-mediated rolling circle amplification (GMNPs@RCA) sandwich assay under isothermal conditions. This THz biosensing strategy demonstrates excellent analytical performance towards serum HBV DNA with high sensitivity, ultralow detection limit, excellent specificity and accuracy, as well as good stability, suggesting a promising THz sensing platform for virus DNA in clinical diagnosis, pathogen detection, and environmental monitoring in the future.

The rapid detection of viruses is becoming increasingly important to prevent widespread infections. However, virus detection via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is time-consuming, as it involves independent nucleic acid extraction and complementary DNA synthesis. This process limits the potential for rapid diagnosis and mass analysis, which are necessary to curtail viral spread. In this study, a simple and rapid thermolysis method was developed to circumvent the need for extraction and purification of viral RNA. The developed protocol was applied to one-chip digital PCR (OCdPCR), which allowed thermolysis, RT, and digital PCR in a single unit comprising 20,000 chambers of sub-nanoliter volume. Two viruses such as tobacco mosaic virus and cucumber mosaic virus were tested as model viral particles. First, the temperature, exposure time, and template concentration were optimized against tobacco mosaic viral particles, and the most efficient conditions were identified as 85°C, 5 min, and 0.01 μg/nL with a cycle threshold of approximately 33. Finally, the OCdPCR analysis yielded 1,130.2 copies/µL using 10⁻² μg/nL of viral particles in a 30 min thermolysis-RT reaction at 70°C. This novel protocol shows promise as a quick, accurate, and precise method for large-scale viral analysis in the future.