An electrochemical immunosensor based on reduced graphene oxide/multiwalled carbon nanotubes/thionine/gold nanoparticle nanocomposites for the sensitive testing of follicle-stimulating hormone.

Follicle-Stimulating Hormone (FSH) is a kind of gonadotropin which can promote human reproduction and development. Abnormal FSH levels may lead to endocrine disorders and infertility. Sensitive determination of FSH is very significant for the clinical diagnosis of these diseases. Here, an electrochemical immunosensor based on a screen-printed electrode (SPE) was developed for the detection of FSH. Nanocomposites, compounded with reduced graphene oxide (rGO), multiwalled carbon nanotubes (MWCNTs), thionine (Thi) and gold nanoparticles (AuNPs), were used for increasing the specific surface area to adsorb molecules and amplify signals. The rGO/MWCNTs/Thi/AuNP nanocomposites, anti-FSH and BSA were successively assembled onto a SPE to fabricate the immunosensor. Electrochemical performance of the modified immunosensor was studied by differential pulse voltammetry (DPV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). FSH testing was based on the principle that the insulating FSH antigen-antibody immunocomplex could retard the electron transfer of Thi which led to the decrease of the DPV current response. Under optimum conditions, the rGO/MWCNTs/Thi/AuNP modified immunosensor exhibited high sensitivity and accuracy for the determination of FSH in a linear range from 1 mIU mL-1 to 250 mIU mL-1, and the detection limit was 0.05 mIU mL-1 at a signal-to-noise ratio of 3. The immunosensor was successfully applied for the determination of quality serum samples with a recovery of 94.0-109.8%. The electrochemical immunosensor could be utilized for testing other gonadotropins.

A paper-based electrochemical immunosensor with reduced graphene oxide/thionine/gold nanoparticles nanocomposites modification for the detection of cancer antigen 125.

A paper-based electrochemical immunosensor was developed for the detection of cancer antigen 125 (CA125) by screen-printing technique. The reduced graphene oxide/thionine/gold nanoparticles (rGO/Thi/AuNPs) nanocomposites were compounded and coated onto the working electrode of immunosensor for CA125 antibody (anti-CA125) immobilization and detection signal amplification. The detection principle was based on the fact that the immunocomplex formed by specify binding of CA125 antibody and antigen could reduce the current response of thionine, which was proportional to the corresponding concentration of CA125 antigen. The immunoassay results showed that the linear range of CA125 was from 0.1 U mL-1 to 200 U mL-1 with the limit of detection (LOD) of 0.01 U mL-1 at signal to noise of 3. Quality control serum samples measured by our proposed immunosensor showed acceptable agreement with traditional ELISA method with the relative error less than 8.05%. The immunosensor exhibited good electrochemical performance with high reproducibility, reliability, stability and accuracy. The proposed immunosensor could be used for the determination of CA125 and had the potential for point-of-care testing (POCT) of other tumor marker.

[Cloning of the gene encoding a thermostable alpha-amylase from bacillus licheniformis CICIM B0204 and functional identification of its promoter].

Thermostable alpha-amylase, catalyzing the hydrolyzation of starch to dextrin, maltose and glucose at higher temperature, is one of the most industrial important enzymes. Several species of Bacillus have been found and genetic improved to produce the thermostable alpha-amylases. In present study, a gene, amyL, coding for a thermostable alpha-amylase with its flanking sequences was cloned from an industrial Bacillus licheniformis CICIM B0204 by using a combination of routine polymerase chain reactions (PCR) and inverse PCR with a pair of initial primers derived from the highly conserved region of bacterial alpha-amylase genes and the functional identifications of the cloned amyL and the activities of its promoter and signal peptide in Escherichia coli were investigated. The amyL was composed of 1539 bp with 180 bp at upstream for its promoter and 160 bp at downstream for its terminator. The deduced mature peptide of the a-amylase was composed of 512 amino acid residues and its signal peptide 29 amino acid residues at N-terminal. The nucleotide and deduced amino acid sequences of amyL were extremely similarity to those from Bacillus species with three amino acid residues difference (Arg163-->Leu, Ser339-->Gly, Ala349-->Ser) comparison to that from a laboratory strain B. licheniformis 584. Under the control of T7 promoter, the structural region of amyL was functionally expressed in Escherichia coli. Additionally, the structural region of the gene coding for a beta-mannosidase from B. licheniformis CICIM B2004 was inframely inserted into the downstream of the promoter and signal sequence of amyL and expressed in E. coli. The amyL promoter and signal sequence was functionally directed the expression and secretion of the beta-mannosidase in E. coli cells with the expression level of 295 U/mL.