Enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes.
Voltammetric enzyme genosensors on streptavidin-modified screen-printed carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin and autolysin genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate were used as the enzymatic label and substrate, respectively. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 degrees C on the surface of the SPCEs. After the hybridization reaction with FITC-labeled complementary targets, the enzyme is captured using an anti-FITC antibody conjugated to AP. In nonstringent experimental conditions, these genosensors can detect 0.49 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In the presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected.
Hernández-Santos
David
D
Departamento de Química Física y Analítica, Facultad de Química, Universidad de Oviedo. 33006 Oviedo, Asturias, Spain.
Díaz-González
María
M
González-García
María Begoña
MB
Costa-García
Agustín
A
D000327:Adsorption; D000469:Alkaline Phosphatase / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D002244:Carbon / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis; D004563:Electrochemistry; D004566:Electrodes; D004867:Equipment Design; D007211:Indoles / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*; D013499:Surface Properties
D004279:DNA, Viral; D007211:Indoles; D015345:Oligonucleotide Probes; C034083:indoxyl phosphate; D001710:Biotin; D002244:Carbon; D019809:Streptavidin; D000469:Alkaline Phosphatase