Development of an Electrochemical Immunosensor for Direct Detection of Interferon-γ at the Attomolar Level

摘要

An electrochemical immunosensor for direct detection of the 15.5-kDa protein interferon-γ (IFN-γ) at attomolar level has been developed. Self-assembled monolayers (SAMs) of cysteine or acetylcysteine are formed on electropolished polycrystalline Au electrodes. IFN-γ adsorbs physically to each of these SAMs. With injections of 100 mM KCl, IFN-γ can be removed in the flow without damaging the acetylcysteine SAM. However, the cysteine SAM is affected by these KCl injections. In an on-line procedure in the flow, a specific antibody (MD-2) against IFN-γ is covalently attached following carbodiimide/succinimide activation of the SAM. The activation of the carboxylic groups, attachment of MD-2, and deactivation of the remaining succinimide groups with ethanolamine are monitored impedimetrically at a frequency of 113 Hz, a potential of +0.2 V versus SCE, and an ac modulation amplitude of 10 mV. Plots of the real (Z′) and imaginary (Z″) component of the impedance versus time provide the information to control these processes. In the thermostated setup (23.0℃), samples of unlabeled IFN-γ (in phosphate buffer pH 7.4) are injected and the binding with immobilized MD-2 is monitored with ac impedance or potential-step methods. While the chronoamperometric results are rather poor, the ac impedance approach provides unsurpassed detection limits, as low as 0.02 fg mL~(-1) (～1 aM) IFN-γ. From a calibration curve (i.e. Z″ versus the amount injected), recorded by multiple 50-μL injections of 2 pg mL~(-1) of IFN-γ, a dynamic range of 0-12 pg mL~(-1) could be derived. However, when nonspecific adsorption is taken into account, which has been found to be largely reduced through injections of 100 mM KCl, a much smaller dynamic range of 0-0.14 fg mL~(-1) remains. The immunosensor can be regenerated by using a sequence of potential pulses in the flow by which the SAM with attached MD-2 and bound IFN-γ is completely removed. When the developed procedures described above are repeated, the response of the immunosensor is reproducible within 10%.