Voltammetric DNA biosensor for cystic fibrosis based on a modified carbon paste electrode.
Carbon paste electrodes modified by the inclusion of either octadecylamine or stearic acid were used as solid phases to which DNA was covalently bound. Immobilized DNA was detected by voltammetry of solutions containing submillimolar quantities of Co(bpy)3(ClO4)3, Co(phen)3(ClO4)3, and Os(bpy)3-Cl2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline), all of which associate reversibly with immobilized DNA and yield increased peak currents at DNA-modified electrodes. Immobilization onto octadecylamine-modified electrodes was performed using a water-soluble carbodiimide, and at high DNA concentrations in the reaction mixture, it resulted in visible polymerization of DNA on the surface. Optimization of the deoxyguanosine- (dG-) selective immobilization reaction for stearic acid-modified electrodes, using water-soluble carbodiimide and N-hydroxysulfosuccinimide reagents to activate carboxylate groups on the surface, yielded conditions of 4.5% (w/w) stearic acid and 10 micrograms/mL DNA. Polythymidylic acid of 4000-base average length (poly(dT)4000) was immobilized at stearic acid-modified electrodes following enzymatic elongation with dG residues at the 3'-end. These DNA-modified electrodes were used to study hybridization with analyte poly(dA)4000 by in situ voltammetry of 60 microM Co(bpy)3(ClO4)3 at low ionic strength (20 mM NaCl), and by voltammetry of the same complex, following exposure of the electrode to poly(dA)4000 in a separate hybridization step conducted at high ionic strength (0.5 M NaCl). Results indicate slow (> or = 1 h) hybridization at low ionic strength and fast (< or = 10 min) hybridization at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry and Biochemistry, Concordia University, Montreal, Canada.
K M
KM
Millan
A
A
Saraullo
S R
SR
Mikkelsen
D001483:Base Sequence; D015374:Biosensing Techniques; D002244:Carbon / Q000737:chemistry*; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D008839:Microelectrodes; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry
D009838:Oligodeoxyribonucleotides; D002244:Carbon; D004247:DNA