Multiple enzyme layers on carbon nanotubes for electrochemical detection down to 80 DNA copies

Signal amplification using enzyme multilayers on carbon nanotube (CNT) templates is shown to yield a remarkably sensitive electrochemical detection of proteins and nucleic acids. The electrostatic layer-by-layer (LBL) self-assembly onto CNT carriers maximizes the ratio of enzyme tags per binding event to offer the greatest amplification factor reported to date. Absorption spectroscopy, TEM, and electrochemical characterization confirm the formation of LBL enzyme nanostructures on individual CNT carriers. The enzymatic activity is found to increase with the number of enzyme layers. The new protocol is illustrated for monitoring sandwich hybridization and antibody-antigen interactions in connection with alkaline phosphatase tracers. Factors affecting the enzyme loading and the analytical performance have been optimized. Such amplified bioelectronic assays allow detection of DNA and proteins down to 80 copies (5.4 aM) and 2000 protein molecules (67 aM), respectively. Given the enormous amplification afforded by the new CNT-LBL biolabel, such route offers great promise for ultrasensitive detection of infectious agents and disease markers.