Biology Reference
In-Depth Information
9.4.1
Enzymatic Genosensors on Streptavidin-Modified
Screen-Printed Carbon Electrode
This section outlines the development of genosensors on screen-
printed carbon electrodes (SPCEs) for the identification of nucleic
acid determinants exclusively present in the genome of the
pathogen
Streptococcus pneumoniae
. Orientation of the strands in
the sensing phase is achieved by modifying the surface of the
electrode with streptavidin by physical adsorption followed by the
immobilizationofbiotinylatedoligoprobes.Thephysicaladsorption
of streptavidin must be performed at a constant temperature above
the room temperature. Moreover, the electrode surface must be
previously electrochemically pretreated at an anodic potential in
acidic medium to improve its adsorptive properties. In this way,
reproducible,sensitive,andstablesensingphasesareobtained[66].
The biotinylated oligo nucleic acid probes used in this work target
the pneumolysin (ply) gene. This target is randomly labeled with
the Universal Linkage System (ULS). This labeling system consists
of the use of a platinum (II) complex that acts as a coupling agent
betweenDNAstrandsandalabelmolecule,usuallyfluorescent.This
platinumcomplexisamonofunctionalderivateofcisplatin(apotent
anticancer agent used in the treatment of a variety of tumors) that
binds to DNA at the N7 position of guanine with release of one Cl
ion per molecule of the complex. The label molecule used in this
study was fluorescein (FITC). Electrochemical detection is achieved
using two strategies. One of them is carried out using an anti-FITC
alkaline phosphatase-labeled antibody and 3-indoxyl phosphate (3-
IP) as enzymatic substrate of AP. The resulting enzymatic product is
indigo blue, an aromatic heterocycle insoluble in aqueous solutions.
Its sulfonation in acidic medium gives rise to indigo carmine IC, an
aqueous soluble compound that shows an electrochemical behavior
similartoindigoblue.Both3-IPandIChavealreadybeenstudiedon
SPCEs[67,68].However,althoughthesegenosensorsarestableand
sensitivedevicesforthedetectionofspecificnucleicacidfragments,
the need of two additional steps to obtain the analytical signal
resultedinalargetime-consuminganalysis.Thisfactcanbeavoided
using the second strategy for detection. In this case the analytical
signal is directly obtained from platinum (II) complex, which is
