The low therapeutic index of digoxin necessitates careful monitoring of its 
serum levels. Most of digoxin immunoassays suffer from interferences with 
digoxin-like immunoreactive substances. Since aptamers have been shown to be 
highly specific for their targets, the aim of this study was to develop DNA 
aptamers for this widely used cardiac glycoside. Digoxin was coated onto the 
surface of streptavidin magnetic beads. DNA aptamers against digoxin were 
designed using Systematic Evolution of Ligands by Exponential enrichment method 
(SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin 
magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR 
amplification. The PCR product was cloned and sequenced. Binding affinity was 
determined using digoxin-BSA conjugate, coated onto ELISA plate. Inhibitory 
effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium. 
Three aptamers (D1, D2 and D3) were identified. Binding studies of 
fluorescein-labeled truncated (without primer binding region) D1 and D2 and full 
length D1 anti-digoxin aptamers were performed and their corresponding 
dissociation constants values were 8.2×10(-9), 44.0×10(-9) and 17.8×10(-9) M, 
respectively. This is comparable to what other workers have obtained for 
interaction of monoclonal antibodies raised against digoxin. There was little 
difference in binding affinity between full length and truncated anti-digoxin D1 
aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the 
isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between 
digoxin and ouabain in both tissue study and binding experiments. Our finding 
indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further 
studies might show its suitability for use in digoxin assays and as a 
therapeutic agent in life-threatening digoxin toxicity.