Determination of low-abundance human immunodeficiency virus (HIV) enzymes is essential for early detection of the viral infection. Designed for the rapid detection of the virus in human serum, a rapid and ultrasensitive electrochemical assay for HIV-1 reverse transcriptase (HIV-1 RI) is presented in this article. The assay format is based on the formation of a self-assembled monolayer (SAM) of a synthesized ferrocene (Fc)-labeled lipoic acid onto a gold electrode. X-ray photoelectron spectroscopy (XPS) and Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) were employed to confirm the binding of the Fc-labeled lipoic acid to the gold via the Au-S bond. A short RT-specific peptide, VEAIIRILQQLLFIH, was covalently attached to the Fc-labeled lipoic acid. Square wave voltammetry (SWV) offered a two-dimensional measurement of RI based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RI in the range of 100-500 pg mL(-1), equivalent to 85.5-427.4 fM. with a detection limit of 50 pg mL(-1) (42.7 fM). The developed biosensor is inexpensive, easy to prepare and operate, and allows a highly selective detection of RI in 20s. (C) 2011 Elsevier Ltd. All rights reserved.