The Molecular Characterization and Immunity Identification of Rhoptry Protein 22 of Toxoplasma gondii as a DNA Vaccine Candidate Against Toxoplasmosis.

Toxoplasma gondii (T. gondii) rhoptry proteins (TgROPs) have been considered main targets and indicator molecules for immune diagnosis and prophylaxis since they initially present during the process of invasion. In this study, the effect of intramuscularly injecting the genetic vaccine pVAX-ROP22 was evaluated, made by inserting the TgROP22 sequence into the eukaryotic expression vector of pVAX I, into BALB/c mice. The levels of IgG, IgG1, and IgG2a in pVAX-ROP22 vaccinated animals were integrally increased. It was uncovered by cytokine profile analyses that the levels of IFN-γ and IL-2 were significantly increased, while no significant changes were detected in IL-4 and IL-10 levels. In addition, we found that immunization with pVAX-ROP22 significantly prolonged the survival time (13.80 ± 1.75 d) of mice after challenge infection with the virulent T. gondii RH strain, in comparison with those of control animals (died within 10 d). Moreover, the number of brain cysts (1,406 ± 277) in the animals subjected to pVAX-TgROP22 vaccination decreased remarkably (P < 0.05) compared with the blank control mice (2,333 ± 473), and the size of brain cysts in pVAX-TgROP22 group was significantly smaller than the groups of blank, PBS and pVAXI. These results suggested that TgROP22 as DNA vaccine could trigger strong humoral and cellular responses and induce partial protection against toxoplasmosis.

A Sensitive Signal-on Supersandwich DNA Biosensor Based on the Enhancement of Poly(aniline-luminol) Nanowires Electrochemiluminescence by Ferrocene.

A signal-on supersandwich type of electrochemiluminescence (ECL) DNA biosensor was developed based on the poly(aniline-luminol) nanowires (PALNWs) modified electrode and enhancement of ferrocene (Fc) on ECL of luminol. Aminated capture DNA was covalently linked to the PALNWs on the electrode surface by the crosslinking of glutaraldehyde. In presence of target DNA, its 3' terminus hybridizes with the capture probe and the 5' terminus hybridizes with ferrocene labeled DNA (Fc-DNA) to form a long DNA concatamer supersandwich structure. The ECL intensity of the prepared biosensor was clearly improved by increasing the concentration of target DNA due to the enhancement of ferrocene on luminol ECL. The difference of the ECL intensity in the absence and presence of target DNA was used to monitor the hybridization event. The difference of ECL linearly increased with the logarithm of target DNA concentration in the range from 1.0  × â€¯10-16 - 1.0  × â€¯10-8 mol L-1 with a detection limit of 5.8  × â€¯10-17 mol L-1. The sensor had high sensitivity and wide linear relationship for the detection of target DNA.

A label-free electrochemiluminescence immunosensor for carbohydrate antigen 153 based on polypyrrole-luminol-AuNPs nanocomposites with bi-catalysis.

Polypyrrole-luminol-AuNPs nanocomposites were prepared and used to develop a sensitive label-free electrochemiluminescence (ECL) immunosensor for carbohydrate antigen 153 (CA153) detection. Firstly, polypyrrole (PPY) nanoparticles were synthesized by a chemical oxidation method using FeCl3 as an oxidizing agent, then luminol and gold nanoparticles (AuNPs) were combined with PPY nanoparticles through electrostatic interaction to form PPY-luminol-AuNPs nanocomposites. The nanocomposites were characterized by transmission electron microscopy (TEM), UV-Vis absorption spectra, atomic emission spectrometry (AES), X-ray diffraction (XRD) and electrochemical impedance spectroscopy (EIS). Especially, iron element was also detected in the nanocomposites. The PPY-luminol-AuNPs nanocomposites showed excellent ECL activity due to the bi-catalysis of iron ion and gold nanoparticles on the ECL of luminol. Furthermore, the nanocomposites showed good film-forming property, and it can be fixed on electrode surface without the assistance of other film-forming materials. On this basis, an ECL immunosensor for CA153 was constructed by covalently immobilizing anti-CA153 on PPY-luminol-AuNPs modified indium-doped tin oxide (ITO) electrode. In the presence of CA153, a remarkable decrease in ECL signals was observed due to the formation of anti-CA153/CA153 complex. The immunosensor showed a good linear relationship in the concentration range of 0.001 to 700 U/mL for CA153, and the detection limit was 5.8 × 10-4 U/mL (S/N = 3). Furthermore, the ECL immunosensor was applied to the determination of CA153 in practical human serum sample.