VOLTAMMETRIC DNA BIOSENSOR FOR CYSTIC-FIBROSIS BASED ON A MODIFIED CARBON-PASTE ELECTRODE

Carbon paste electrodes modified by the inclusion of either octadecyla mine or stearic acid were used as solid phases to which DNA was covale ntly bound. Immobilized DNA was detected by voltammetry of solutions c ontaining submillimolar quantities of Co(bpy)(3)(ClO4)(3), Co(phen)(3) (ClO4)(3), and Os(bpy)(3)-Cl-2 (bpy = 2,2'-bipyridine; phen = 1,10-phe nanthroline), all of which associate reversibly with immobilized DNA a nd yield increased peak currents at DNA-modified electrodes. Immobiliz ation onto octadecylamine-modified electrodes was performed using a wa ter-soluble carbodiimide, and at high DNA concentrations in the reacti on mixture, it resulted in visible polymerization of DNA on the surfac e. Optimization of the deoxyguanosine- (dG-) selective immobilization reaction for stearic acid-modified electrodes, using water-soluble car bodiimide and N-hydroxysulfosuccinimide reagents to activate carboxyla te groups on the surface, yielded conditions of 4.5% (w/w) stearic aci d and 10 mu g/mL DNA. Polythymidylic acid of 4000-base average length (poly(dT)(4000)) was immobilized at stearic acid-modified electrodes f ollowing enzymatic elongation with de residues at the 3'-end. These DN A-modified electrodes were used to study hybridization with analyte po ly(dA)(4000) by in situ voltammetry of 60 mu M Co(bpy)(3)(ClO4)(3) at low ionic strength (20 mM NaCl), and by voltammetry of the same comple x, following exposure of the electrode to poly(dA)(4000) in a separate hybridization step conducted at high ionic strength (0.5 M NaCl). Res ults indicate slow (greater than or equal to 1 h) hybridization at low ionic strength and fast (greater than or equal to 10 min) hybridizati on at high ionic strength. At high ionic strength, a detection limit o f 2.5 ng of Poly(dA)(4000) was obtained, which corresponds to 250 pg o f a typical 400-base polymerase chain reaction product. The results ar e applied to the selective detection of the cystic fibrosis Delta F508 sequence in an 18-base oligodeoxynucleotide sample.