Carbon nanotube transistors show tremendous potential for electronic detection of biomolecules in solution. However, the nature and magnitude of the sensing signal upon molecular adsorption have so far remained controversial. Here, the authors show that the choice of the reference electrode is critical and resolves much of the previous controversy. The authors eliminate artifacts related to the reference electrode by using a well-defined reference electrode to accurately control the solution potential. Upon addition of bovine serum albumin proteins, the authors measure a transistor threshold shift of −15 mV which can be unambiguously attributed to the adsorption of biomolecules in the vicinity of the nanotube. © 2007 American Institute of Physics. ͓DOI: 10.1063/1.2775090͔Biosensors based on nanoscale field-effect transistors have the potential to significantly impact drug discovery, disease screening, biohazard screening, and fundamental science.1 The adsorption of biomolecules on the sidewall of a semiconducting carbon nanotube ͑CNT͒ or nanowire causes changes in local electrostatic environment, thereby changing the conductance of the nanomaterial. Pioneering work has indicated that this modulation of conductivity can be utilized to build CNT-based 2-11 and nanowire-based 1,12 sensors for real-time electrical detection of proteins or DNA. These sensors must be carefully designed to give reliable measurements of biomolecule binding. Of critical importance is the electrostatic potential of the solution which strongly affects the conductivity of the nanomaterial. The solution potential was not well controlled in many CNT biomolecule-binding experiments.3-10 Reported conductance changes can have little or no relationship to interactions between biomolecules and the CNT transistor, leading to unreliable sensors and hindering efforts to determine sensing mechanisms. We show that a major biosensing artifact can be removed by using a well-defined reference electrode to accurately control the solution potential.Carbon nanotube biosensors are generally constructed as shown in Fig. 1͑a͒. [3][4][5][6][7][8][9][10] The device is exposed to solution, allowing protein adsorption on the semiconducting CNT. A metal wire is used to control the electrostatic potential of the solution. A gate voltage V g , applied to the metal wire, can tune the conductance of the CNT, while a small bias eV bias Ͻ k B T is used to monitor the CNT conductance ͓for example, see Fig. 1͑b͒, curve 1͔. The electrostatic potential difference between the solution and the CNT is determined by the applied gate voltage V g and the interface potential at the metal-liquid interface V interface . This interface potential depends sensitively on electrochemical reactions occurring at the metal-liquid interface. Larrimore et al. have recently tested CNT sensors in solutions where this electrochemistry was controlled using a high concentration of a potentialdetermining redox couple.13 Sensors for biomolecule binding are generally operated in buffer solutions where ...