The formed complex is capable of triggering a strand displacement reaction that releases the GOx-DNA hybrid strand from a preformed duplex. To optimize this step, it is crucial to find the optimal conditions under which hybridization between the two antigen-conjugated DNA strands occurs only upon bivalent binding of the target antibody. To this end, we first used the small molecule digoxigenin (Dig) and the antibody anti-Dig as antigen and target antibody, respectively (Figure ). We then synthesized a series of Dig-conjugated DNA strands with complementary domains of different lengths. We tested lengths from 0 to 14 bases corresponding to estimated values of hybridization free energy from 0 to -23 kcal mol -1 , respectively. For each set of Digconjugated strands, we performed the reaction in the absence and presence of a fixed concentration of anti-Dig antibody (10 -8 M) and measured the electrochemical signal generated by the GOx-DNA output strand on a disposable electrode as described previously (Figure ). As expected, Dig-conjugated strands with short complementary portions (<4 nucleotides) yield low signals in both the absence and presence of anti-Dig antibody. With longer complementary portions (>8 nucleotides) we observe higher background signals as the two antigen-conjugated strands tend to hybridize spontaneously even in the absence of the target antibody. A complementary domain of 6 nucleotides provides the highest signal change in the presence of the antibody and was therefore selected for future experiments. Using these Dig-conjugated DNA strands and previously described amplification step, we tested increasing concentrations (from 10 -15 to 10 -6 M) of anti-Dig antibody. In this way, we obtained a dose-response curve with a dynamic range (defined as the concentration range in which we obtain signals between 10% and 90% of the maximum signal) between 3 x 10 -13 and 3 x 10 -11 M of anti-Dig antibody with a detection limit (LOD) (defined as the concentration that reaches a signal three standard deviations above a blank) of 1 x 10 -12 M and an average RSD% of 11 % (Figure ). The observed current saturation signal in these conditions is smaller compared to that obtained from direct hybridization of the GOx-DNA strand (see Figure , Figure ). This difference is likely due to the fact that the antibody-responsive reaction is not 100% efficient as the same target antibody can bind to two antigen-conjugated strands thus not providing any signal. ELIDIS works well also in complex media. To demonstrate this, we performed the reaction step (step 1) in 80% bovine serum and we obtained similar results in terms of dynamic range (between 4 x 10 - 13 and 2 x 10 -11 M), K1/2 (3 ± 1 x 10 -12 M), LOD (1 x 10 -12 M). We note here that a recently reported antibodyresponsive strand displacement reaction coupled with electrochemical detection without an enzymatic amplification (the output strand binds to a redox-reporter modified complementary strand immobilized on a disposable electrode) provides poorer sensitivity (LOD in the nM range) and requires longer assay time thus demonstrating the crucial role of the enzymatic amplification step to achieve the high sensitivity displayed by ELIDIS.