An electrochemical aptamer-based (E-AB) biosensor has the ability to generate an electrochemical signal in response to specific target binding in vivo The signal is measured by a change in Faradaic current passed through an electrode. E-AB sensors are advantageous over previously reported aptamer-based sensors, such as fluorescence generating aptamers, due to their ability to detect target binding in vivo with real-time measurements. An E-AB sensor is composed of a three-electrode cell: an interrogating electrode, a reference electrode, and a counter electrode. A signal is generated within the electrochemical cell then measured and analyzed by a potentiostat. There are several biochemical and electrochemical parameters to optimize signal gain for E-AB biosensors. The density packing of DNA or RNA aptamers, the ACV frequency administered by the potentiostat, and the chemistry of the SAM are all factors that determine signal gain as well as the signal to noise ratio of target binding. E-AB biosensors provide a promising mechanism for in-situ sensing and feedback-controlled drug administration.
Signal generation
The DNA or RNA aptamers are fixed on the interrogating electrode, where a redox reaction is reported by a redox tag. Gold is often used as the probe surface for interrogating electrodes. The surface of the gold electrode is packed with redox-tagged DNA or RNA aptamers. The redox reporter is often methylene blue. Upon target binding, the aptamer changes structure by folding, bringing the redox reporter closer to the gold electrode. This increase in proximity from the redox-reporter to the electrode enables faster electron transfer from the redox tag to the gold electrode. The increase in speed of electron transfer contributes to a change in Faradaic current that is detected by the potentiostat.
The reference electrode is the site of a known chemical reaction that has a known redox potential. For example, a reference electrode that harbors the reaction of silver-silver chloride (Ag/AgCl) has a fixed redox potential and is the measuring point for the redox potential of the interrogating electrode. The counter electrode (or auxiliary electrode) acts as a cathode or anode to the interrogating electrode. The applied voltage is not passed through the reference electrode due to an impedance supplied by the potentiostat. Therefore, the potential generated within the electrochemical cell is attributed to the interrogating electrode. Current is measured as potential of the interrogating electrode vs. the fixed potential of the reference electrode. The difference in potential is what produces the current in the external circuit and generates a signal. The signal quantifies target binding depending on electron transfer that is stoichimetrically proportional to target binding.
Four electrode method has also been demonstrated in an electrochemical nanoporous alumina membrane sensor, where the aptamer was grafted onto the membrane and not on the electrode. The binding of the aptamer with the target protein produces a change in impedance of the membrane which is picked up by the electrochemical sensor using an impedance spectroscopy analyzer. This approach could be beneficial in cases where the electric field of the electrode can change the aptamer structure or the biointerface which may decrease the sensing ability.
Signal optimization
There are several parameters to consider for optimization of binding-induced electrochemical signal gain. The aptamer probe packing density, the nature of the self-assembling monolayer, and the ACV frequency are factors that affect detecting and measuring of signal. Two main factors are considered when fabricating the packing density on the probe surface. The concentration of aptamer and the surface chemistry of the self-assembling monolayer (SAM) enable variations of desired probe packing density.