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JMD 2007, Vol. 9, No. 2
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Technical Advance

Development of an Advanced Electrochemical DNA Biosensor for Bacterial Pathogen Detection

Joseph C. Liao^*, Mitra Mastali, Yang Li^*, Vincent Gau, Marc A. Suchard^¶||, Jane Babbitt, Jeffrey Gornbein^||, Elliot M. Landaw^||, Edward R.B. McCabe^¶**, Bernard M. Churchill^* and David A. Haake

From the Departments of Urology, * Medicine, Human Genetics, ^¶ Biomathematics, ^|| and Pediatrics, ** David Geffen School of Medicine, University of California, Los Angeles; Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles; and GeneFluidics, Inc., Monterey Park, California

Abstract

Electrochemical sensors have the capacity for rapid and accurate detection of a wide variety of target molecules in biological fluids. We have developed an electrochemical sensor assay involving hybridization of bacterial 16S rRNA to fluorescein-modified detector probes and to biotin-modified capture probes anchored to the sensor surface. Signal is generated by an oxidation-reduction current produced by the action of horseradish peroxidase conjugated to an anti-fluorescein monoclonal Fab. A previous study found that this electrochemical sensor strategy could identify uropathogens in clinical urine specimens. To improve assay sensitivity, we examined the key steps that affect the current amplitude of the electrochemical signal. Efficient lysis and release of 16S rRNA from both gram-negative and -positive bacteria was achieved with an initial treatment with Triton X-100 and lysozyme followed by alkaline lysis, resulting in a 12-fold increase in electrochemical signal compared with alkaline lysis alone. The distance in nucleotides between the target hybridization sites of the detector and capture probes and the location of fluorescein modification on the detector probe contributed to a 23-fold change in signal intensity. These results demonstrate the importance of target-probe and probe-probe interactions in the detection of bacterial 16S rRNA using an electrochemical DNA sensor approach.