Towards the development of a sensitive and selective electrochemical aptamer-based ampicillin sensor

[Display omitted] •A systematic approach was used to design a “signal-on” E-AB ampicillin sensor.•Effects of displacement probe length and Tm on sensor performance were analyzed.•The sensor fabricated with a 12-base displacement probe is the optimal design.•The sensor responds to the target rapidly; it is also sensitive and specific.•The sensor functions well in 50% urine, saliva, milk, and aquifer water samples. We report the design and fabrication of a “signal-on” electrochemical aptamer-based (E-AB) sensor for detection of ampicillin (AMP). The conventional E-AB sensor is fabricated on a gold disk electrode using a thiolated and methylene blue (MB)-modified DNA aptamer probe. Binding of AMP alters the probe conformation and flexibility, resulting in an increase in the MB signal. However, this target-induced change in conformation and flexibility is insufficient to generate a large signal gain. To circumvent the issue with limited signal gain, we employed a displacement-based approach to the design of this sensor. For the displacement-based E-AB sensor, the aptamer probe is first hybridized to a displacement probe (DP) of a specific length and/or Tm; binding of AMP to the aptamer probe displaces the DP from the DNA duplex. This change in probe conformation and flexibility should produce a substantial increase in the MB signal. The extent of the signal gain, however, is dependent on the length and/or Tm of the DP. Among the nine DPs used in this study, the 12-base S7 probe is ideal for the displacement-based sensor design. The S7 sensor has a lower limit of detection and a broader linear dynamic range when compared to the conventional E-AB sensor. Despite being a displacement-based sensor, the response time of the S7 sensor is short, and signal saturation can be achieved in