Development of a Nanostructured Electrochemical Genosensor for the Detection of the K-ras Gene.

In the scientific literature, it has been documented that electrochemical genosensors are novel analytical tools with proven clinical diagnostic potential for the identification of carcinogenic processes due to genetic and epigenetic alterations, as well as infectious diseases due to viruses or bacteria. In the present work, we describe the construction of an electrochemical genosensor for the identification of the k12p.1 mutation; it was based on use of Screen-Printed Gold Electrode (SPGE), Cyclic Voltammetry (CV), and Atomic Force Microscopy (AFM), for the monitoring the electron transfer trough the functionalized nanostructured surface and corresponding morphological changes. The sensitivity of the genosensor showed a linear response for the identification of the k12p.1 mutation of the K-ras gene in the concentration range of 10 fM to 1 µM with a detection limit of 7.96 fM in the presence of doxorubicin (Dox) as DNA intercalating agent and indicator of the hybridization reaction. Thus, the electrochemical genosensor developed could be useful for the identification of diseases related with the K-ras oncogene.

Discovery of a new non-substrate inhibitor of the 26.5 kDa glutathione transferase from Taenia solium by virtual screening.

The inappropriate use of anthelmintics, such as praziquantel and albendazole, has generated resistance and the need to develop new drugs. Glutathione transferases, GSTs, are bisubstrate dimeric enzymes that constitute the main detoxification mechanism against electrophiles, drugs and oxidative damage in Taenia solium. Therefore, GSTs are important targets for the development of new anthelmintics. In this work, we reported a successful virtual screen aimed at the identification of novel inhibitors of a 26.5 kDa GST from T. solium (TsGST26). We found that a compound, i7, able to inhibit selectively TsGST26 concerning human GSTs, showing a non-competitive inhibition mechanism towards substrate glutathione with a Ki (GSH) of 55.7 µM and mixed inhibition towards the electrophilic substrate 1-chloro-2,4-dinitrobenzene with a Ki (CDNB) of 8.64 µM. These results are in agreement with those of docking simulations, which showed i7 binds a site adjacent to the electrophilic site and furthest from the glutathione site.

Development of a Nanostructured Platform for Identifying HER2-Heterogeneity of Breast Cancer Cells by Surface-Enhanced Raman Scattering.

Biosensor technology has great potential for the detection of cancer through tumor-associated molecular biomarkers. In this work, we describe the immobilization of the recombinant humanized anti-HER2 monoclonal antibody (trastuzumab) on a silver nanostructured plate made by pulsed laser deposition (PLD), over a thin film of Au(111). Immobilization was performed via 4-mercapto benzoic acid self-assembled monolayers (4-MBA SAMs) that were activated with coupling reagents. A combination of immunofluorescence images and z-stack analysis by confocal laser scanning microscopy (CLSM) allowed us to detect HER2 presence and distribution in the cell membranes. Four different HER2-expressing breast cancer cell lines (SKBR3 +++, MCF-7 +/-, T47D +/-, MDA-MB-231 -) were incubated during 24 h on functionalized silver nanostructured plates (FSNP) and also on Au(111) thin films. The cells were fixed by means of an ethanol dehydration train, then characterized by atomic force microscopy (AFM) and surface-enhanced Raman scattering (SERS). SERS results showed the same tendency as CLSM findings (SKBR3 > MCF-7 > T47D > MDA-MB-231), especially when the Raman peak associated with phenylalanine amino acid (1002 cm-1) was monitored. Given the high selectivity and high sensitivity of SERS with a functionalized silver nanostructured plate (FSNP), we propose this method for identifying the presence of HER2 and consequently, of breast cancer cells.