Electrochemical phagemid assay for the specific detection of bacteria using Escherichia coli TG-1 and the M13KO7 phagemid in a model system

Tova Neufeld, Adrian Schwartz Mittelman, Virginia Buchner, Judith Rishpon

Research output: Contribution to journalArticlepeer-review

Abstract

We describe a reporter phagemid system for the specific amperometric detection of bacteria. We constructed a phagemid a bacteriophage containing a bacterial plasmid using the M13K07 helper phage and a commercial plasmid, pFLAG-ATS-BAP, which contains a gene encoding for a reporter enzyme, alkaline phosphatase. In the bacteria, the enzyme reacts with the substrate, p-aminophenyl phosphate, in the periplamic space that separates the outer plasma membrane from the cell wall. Thus, the activity of the reporter enzyme can be measured directly in an electrochemical cell without further treatment. The product of the enzymatic activity, p-aminophenol, diffuses out and is oxidized at the working electrode with an applied potential of 220 mV vs the reference electrode Ag/AgCl. The lower detection limit was 1 cfu/mL E. coli TG1 in less than 3 h in a very specific manner. The use of plasmid alkaline phosphatase as the reporter increased the sensitivity by 10-fold over our earlier electrochemical lyric phage method. Such a system can be used for the rapid detection of any strain of bacteria using the appropriate bacteriophage and reporter gene.

Original languageEnglish
Pages (from-to)652-657
Number of pages6
JournalAnalytical Chemistry
Volume77
Issue number2
DOIs
StatePublished - 15 Jan 2005

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