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In recent years, low-temperature plasma (LTP) has been widely applied in biological and medical fields. This paper is aimed to investigate the deactivation effect of LTP on hepatitis B virus (HBV). Dielectric barrier discharge (DBD) is employed to generate the atmospheric-pressure LTP for treatment of HBV. HBV serum collected from hepatitis B patients with HBsAg, HBeAg, and anti-HBc positive is used as the treatment model. LTP treatment time intervals are set as 10, 20, 30, and 40 s, respectively. Concentrations of HBsAg and S/N value for each different treatment are calculated. The liver function and HBV deoxyribonucleic acid (DNA) copy number are also detected. It is found that, compared with the control group, the concentrations of HBsAg and HBV DNA copy number of different treatment groups have statistically significant differences. With time going of LTP treatment, both the concentration of HBsAg and the copy number of HBV DNA gradually decrease, and all the S/N values are less than 2.1, indicating that HBsAg becomes negative after LTP treatment. Comparing the liver functions (i.e., enzymatic, proteinic, and bilirubin indicators) before and after LTP treatment, all the data are in the range of normal reference values. It is concluded that LTP induced by DBD is effective for HBV deactivation, and the liver function is kept normal during the process of plasma killing HBV.