Figure 3: DNA-based molecular circuits allow complex signal processing in antibody detection.

(a) Multiplexed antibody detection. Two orthogonal translator modules process the anti-HA and anti-HIV antibodies into two unique ssDNA outputs. Each output is coupled to a downstream TMSD reaction to displace a different colour reporter duplex, allowing the identification of each antibody in a dedicated fluorescent channel (anti-HA=red, anti-HIV=green). (b,c) OR and NOR gates. Both input antibodies generate the same output sequence, which react with a single reporter duplex, resulting in increased (OR) or decreased (NOR) fluorescence when either one or both input antibodies are present. (d) AND gate. Sequential toehold exchange and toehold-mediated strand displacement reactions of the HIV and HA specific oligonucleotides displace a fluorescently labelled strand from the reporter complex. (e) NAND gate. Sequential toehold exchange and strand displacement reaction result in the quenching of an internally fluorescently modified reporter complex. Fluorescence levels were measured after 3 h incubation and corrected for background fluorescence measured at t=0 h for the multiplex, OR and AND gates, or the fluorescence measured upon addition of an excess of output oligonucleotides for the NOR and NAND gates. Experiments were performed using [BO]=5.5 nM, [I]=5 nM, [Ab]=10 nM and [Gate/Rep]=1 nM at 28 °C in TE/Mg²⁺ buffer supplemented with 1 mg ml⁻¹ BSA. Error bars represent the s.d. calculated from triplicate measurements.