Bacteria screening, viability, and confirmation assays using bacteriophage-impedimetric/loop-mediated isothermal amplification dual-response biosensors

Abstract

Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coliTuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 103-109 cfu/mL, while the linear response of the LAMP approach was determined to be 102-107 cfu/mL. The limit of detection (LOD) of 8 × 102 cfu/mL in less than 15 min and 102 cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h. © 2013 American Chemical Society.