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. coli Tuf 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 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) 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.