We report a new label-free, homogenous detection of viral protein, HCV helicase and replicase using rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of three parts. The first part, the malachite green (MG) binding aptamer as signaling domain was fused to the second part, each HCV helicase or replicase binding aptamer as recognition domain and the third part, inducing arm was added to make a conformational change of MAAS. Inducing arm was designed to transmit the recognition signal of target binding aptamer to make the signaling domain active enough to capture MG, resulting in higher fluorescence emission of MG due to reduced vibrational de-excitation. Only three energy states, corresponding to each free aptamer sensor, target bound sensor, and both target and dye bound sensor were starting point for our rational design, and the energy difference between each state was the main driving force for sensor activation. Constructed MAAS could selectively detect HCV helicase and replicase, and activation of sensor was proportional to the amount of target protein present. It also showed good performance in bead based, non-homogenous detection of HCV replicase using similar dye, TMR.

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