Label-free chemiresistive immunosensors for viruses

Abstract

We report development, characterization, and testing of chemiresistive immunosensors based on single polypyrrole (Ppy) nanowire for highly sensitive, specific, label free, and direct detection of viruses. Bacteriophages T7 and MS2 were used as safe models for viruses for demonstration. Ppy nanowires were electrochemically polymerized into alumina template, and single nanowire based devices were assembled on a pair of gold electrodes by ac dielectrophoretic alignment and anchored using maskless electrodeposition. Anti-T7 or anti-MS2 antibodies were immobilized on single Ppy nanowire using EDC-NHS chemistry to fabricate nanobiosensor for the detection of corresponding bacteriophage. The biosensors showed excellent sensitivity with a lower detection limit of 10(-3) plaque forming unit (PFU) in 10 mM phosphate buffer, wide dynamic range and excellent selectivity. The immunosensors were successfully applied for the detection of phages in spiked untreated urban runoff water samples. The results show the potential of these sensors in health care, environmental monitoring, food safety and homeland security for sensitive, specific, rapid, and affordable detection of bioagents/pathogens.

Figure 1

a) Photo image of 16 electrodes chip (inset optical image showing gap between two electrodes). Schematic of b) aligned and c) anchored Ppy nanowire on the gold electrodes with 3 µm gap (inset: blown up schematic & SEM image). Schematics with blown ups of d) anti-T7 functionalized Ppy nanowire. e) BSA blocking after antibody-functionalization f) T7 phage interacts with anti-T7 antibody on the nanowire surface.

Figure 2

A: Wet I–V characteristic curves of a a) bare Ppy nanowire b) after anti T7 immobilization and BSA blocking and c) after subsequent incubation with T7 phage (10⁹ PFU). Solid lines represent the linear fit. B: Wet I–V characteristic curves of a a) bare Ppy nanowire b) after anti MS2 immobilization and BSA blocking and c) after subsequent incubation with MS2 phage (10⁹ PFU). Solid lines represent the linear fit.

Figure 2

A: Wet I–V characteristic curves of a a) bare Ppy nanowire b) after anti T7 immobilization and BSA blocking and c) after subsequent incubation with T7 phage (10⁹ PFU). Solid lines represent the linear fit. B: Wet I–V characteristic curves of a a) bare Ppy nanowire b) after anti MS2 immobilization and BSA blocking and c) after subsequent incubation with MS2 phage (10⁹ PFU). Solid lines represent the linear fit.

Figure 3

AFM image and height profile measured along the length of (a) a bare single Ppy nanowire, (b) Ppy nanowire functionalized with anti-T7 antibody and blocked with BSA, and (c) anti-T7 functionalized and BSA blocked Ppy nanowire after T7 bacteriophage binding.

Figure 4

Sensing response of anti-T7 immobilized Ppy nanowire towards various concentrations of bacteriophages in PB and lake water samples.

Figure 5

Sensing response of anti-MS2 immobilized Ppy nanowire towards various concentrations of bacteriophages in PB and lake water samples.