Dense Layer of Bacteriophages Ordered in Alternating Electric Field and Immobilized by Surface Chemical Modification as Sensing Element for Bacteria Detection

Abstract

Faster and more sensitive environmental monitoring should be developed to face the worldwide problem of bacterial infections. To remedy this issue, we demonstrate a bacteria-sensing element that utilizes dense and ordered layers of bacteriophages specific to the given bacteria strain. We combine (1) the chemical modification of a surface to increase the surface coverage of bacteriophages (2) with an alternating electric field to greatly increase the number of properly oriented bacteriophages at the surface. Usually, in sensing elements, a random orientation of bacteriophages results in steric hindrance, which results in no more than a few percent of all receptors being available. An increased number of properly ordered phages results in the optimal performance of phage receptors, manifesting in up to a 64-fold increase in sensitivity and a limit of detection as low as 100 CFU mL. Our sensing elements can be applied for selective, sensitive, and fast (15 min) bacterial detection. A well-studied pair T4 bacteriophage-bacteria Escherichia coli, was used as a model; however, the method could be adapted to prepare bacteriophage-based sensors for detection of a variety of bacterial strains.

Phage-based Electrochemical Sensors: A Review

“…What is more, the zeta potential of T4 bacteriophages suspended in SM buffer had been detected as −9.75 ± 0.96 mV [71]. Therefore, the electric dipole properties and the negative surface charge of the bacteriophages provide the possibility of anchoring phages onto the sensor surface in a proper orientation either due to the electrostatic interaction [70,72,73] or in the presence of an external electric field [65,74].…”

“…Another possible reason was that both phages studied here were asymmetric, therefore, the orientation of the deposited phage would affect the accessibility of the receptors on their tail fibers, hence, control the ability to capture the target bacteria cells [54]. The random orientation would also result in the steric hindrances between deposited neighboring virions, suppressing the availability of the receptors [65].…”

“…When L D is smaller than the size of the phages, the orientation occurs almost due to electrostatic interactions while phages will be manipulated to align along the electric field lines when L D is larger than the virion size [74]. Therefore, this group later utilized an alternating electric field with the trapezoidal pulses (10 V, 16.67 kHz) in combination with chemical surface functionalization (cysteamine/glutaraldehyde and DTSP) to immobilize T4 phages [65]. Compared to the constant voltage application, the AC electric field will expand during the inter-pulse periods, increasing the exposure time of bacteriophages to the actual electric field and improving the manipulation efficiency [71].…”

“…Compared to the constant voltage application, the AC electric field will expand during the inter-pulse periods, increasing the exposure time of bacteriophages to the actual electric field and improving the manipulation efficiency [71]. The application of external electric field resulted in a 33-fold increase in the phage surface density compared to just chemical surface modification with DTSP and a 64-fold increase in the sensor sensitivity in comparison to physical adsorption immobilization method [65]. The combination of chemical functionalization with EDC/NHS chemistry and AC electric orientation was also performed by Xu et al to improve the phage deposition protocol with the study of effect of Debye length as well [71].…”

Electrochemical Methodologies for the Detection of Pathogens

“…Bacteriophages (called simply phages) are natural host-specific nanostructured particles. Some reports concern oriented phage immobilization using charge-directed immobilization 120,130 . Most phages show an overall negative charge, with a negatively charged head and positively charged tail.…”