To prepare tubeimoside Ⅲ nanoemulsion (TBM Ⅲ-NE) and evaluate its adjuvant effect in vaccines.

TBMⅢ-NE was prepared using low-energy emulsification. Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE, and transmission electron microscopy (TEM) was employed to observe the morphology. CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells (BMDCs). The in vitro safety of TBMⅢ-NE was evaluated using a hemolysis assay. The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy. After co-incubation of TBMⅢ-NE with BMDCs, the expression levels of CD40, CD86, MHC-Ⅰ, and CCR7 on the surface of BMDCs were detected using flow cytometry, and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay (ELISA). After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBM Ⅲ-NE, ELISA was conducted to determine the serum levels of specific IgG, IgG2a, and IgG1 antibodies. The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot (ELISpot) assay.

The prepared blank nanoemulsion (BNE) and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm, and a polydispersity index of 0.214 and 0.125, respectively. TEM displayed that TBM Ⅲ-NE was in uniform sphere and well dispersed. When the TBMⅢ-NE adjuvant was diluted by 400-fold, the survival rate of BMDCs was approximately 86%. Compared with free TBM Ⅲ, the hemolytic toxicity of TBM Ⅲ-NE was significantly reduced (P < 0.01). TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significantly increased the expression of CCR7 on the surface of BMDCs (P < 0.05), indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes. TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs (P < 0.05). When combined with RBD, TBMⅢ-NE significantly increased the levels of specific IgG, IgG2a, and IgG1 antibodies in mouse serum (P < 0.01) and promoted the secretion of specific IFN-γ in splenocytes (P < 0.01), indicating that TBM Ⅲ-NE could enhance specific cellular immune responses.

A stable and highly effective TBM Ⅲ-NE that can induce humoral and cellular immune responses is successfully prepared.

To prepare a high performance electrochemical immunosensor for detecting Brucella abortus (B. abortus).

Prussian blue (PB), multi walled carbon nanotubes (MWCNTs) and gold nanoparticles (GNPs) (PB-MWCNTs-GNPs) nanocomposites were prepared, and appropriate antibody was used to construct the immunosensor for detecting B. abortus samples. The optimal conditions were clarified by examining the key factors in sensor construction, and then the performance of the sensor was evaluated.

The optimal construction conditions were determined as follows: the ratio of MWCNTs-PB was 1 ∶5, the drying temperature was 37 ℃, the pH value of buffer system was 7.5, and the incubation time of antibody and sample was 1 h and 30 min, respectively. B. abortus exhibited a good linear relationship, when ranging from 10 to 1×10⁵ CFU/mL. The sensor had good anti-interference ability, repeatability, stability and high accuracy.

Our prepared PB-MWCNTs GNPs nanomaterials modified electrochemical immunosensor for detecting B. abortus is easy to prepare, has good performance, and can provide reference for the early clinical diagnosis of brucellosis.