Bacteriophage-Modified Microarrays for the Direct Impedimetric Detection of Bacteria

Abstract

A novel method is presented for the specific and direct detection of bacteria using bacteriophages as recognition receptors immobilized covalently onto functionalized screen-printed carbon electrode (SPE) microarrays. The SPE networks were functionalized through electrochemical oxidation in acidic media of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) by applying a potential of +2.2 V to the working electrode. Immobilization of T4 bacteriophage onto the SPEs was achieved via EDC by formation of amide bonds between the protein coating of the phage and the electrochemically generated carboxylic groups at the carbon surface. The surface functionalization with EDC, and the binding of phages, was verified by time-of-flight secondary ion mass spectrometry. The immobilized T4 phages were then used to specifically detect E. coli bacteria. The presence of surface-bound bacteria was verified by scanning electron and fluorescence microscopies. Impedance measurements (Nyquist plots) show shifts of the order of 10(4) Omega due to the binding of E. coli bacteria to the T4 phages. No significant change in impedance was observed for control experiments using immobilized T4 phage in the presence of Salmonella. Impedance variations as a function of incubation time show a maximum shift after 20 min, indicating onset of lysis, as also confirmed by fluorescence microscopy. Concentration-response curves yield a detection limit of 10(4) cfu/mL for 50-microL samples.

Electrical impedance spectroscopy (EIS) for biological analysis and food characterization: a review

“…Thus, R ct variation due to bacteriophages binding to the host bacteria is somewhat different from other bioreceptors in that at the beginning R ct increases due to bacterial binding hindering the redox reaction at the WE surface, while later it decreases due to cell lyses allowing the release of cell ionic material. Bacteriophages have been used as immobilizing bioreceptors in biosensors for different bacterial species (Shabani et al, 2008;Gervais et al, 2007;Tolba et al, 2012), as well as for an unconventional approach to IM where bacteriophages are integrated into the culture broth, and the monitored variation of electrolyte resistance is mainly due to conductivity increase induced by cell lyses (and not only due to bacterial metabolism) (Mortari et al, 2015). According to the authors, this approach results in much better performances with a detection limit of 1 CFU mL −1 or lower and response time less than 1 h.…”

Bacteriophage based self-assembled monolayer (SAM) on gold surface used for detection of Escherichia coli by electrochemical analysis

“…The images were obtained with the SEM instrument (Hitachi S-3400N, Japan) (Shabani et al, 2008). FTIR spectrum taking using model Perkin Elmer (spectrum 400) under 400 to 4000 wavelength of Gold surface (Rawson et al, 1989).…”

“…Figure 4B indicates that modified gold surface was successfully linked to the Au-SAM surface. Figure 4C are aggregates of the bacteriophage over the Au-SAM surface (Shabani et al, 2008;Garcıa-Gonzalez et al, 2008;Chai et al, 2013).…”

“…When gold surface are considered, preferred strategies via self-assembled monolayer (SAM) and covalent binding provide options for strong and stable biomolecules immobilization in the very near vicinity of the sensor surface (Yang and Bashir, 2008;Shabani et al, 2008;Mejri et al, 2010, Yang andLi, 2005;Yang et al, 2004;Ruan et al, 2002;Radke and Alocilja, 2005). As gold surface is relatively homogeneous and provides suitable reactive groups for the directed conjugation of capture biocomponents it is used as a substrate (Baldrich et al, 2008).…”

Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow

“…Bacteriophages are specific to their target host, self-replicate, have extensive shelf lives, are inexpensive (38), and infect only metabolically active cells (39). Bacteriophage amplification technology has emerged as a means of rapid bacterial detection, using modern protein analytical techniques to monitor changes in sample bacteriophage concentration (40 -43).…”