Engineering Biorthogonal Phage-Based Nanobots for Ultrasensitive, In Situ Bacteria Detection

Abstract

Advances in synthetic biology, nanotechnology, and genetic engineering are allowing parallel advances in areas such as drug delivery and rapid diagnostics. Although our current visions of nanobots may be far off, a generation of nanobots synthesized by engineering viruses is approaching. Such tools can be used to solve complex problems where current methods do not meet current demands. Assuring safe drinking water is crucial for minimizing the spread of waterborne illnesses. Although extremely low levels of fecal contamination in drinking water are sufficient to cause a public health risk, it remains challenging to rapidly detect Escherichia coli, the standard fecal indicator organism. Current methods sensitive enough to meet regulatory standards suffer from either prohibitively long incubation times or requirement of expensive, impractical equipment. Bacteriophages, tuned by billions of years of evolution to bind viable bacteria and readily engineered to produce custom proteins, are uniquely suited to bacterial detection. We have developed a biosensor platform based on magnetized phages encoding luminescent reporter enzymes. This system utilizes bio-orthogonally functionalized phages to enable site-specific conjugation to magnetic nanoparticles. The resulting phage-based nanobots, when combined with standard, portable field equipment, allow for detection of <10 cfu/100 mL of viable E. coli within 7 h, faster than any methods published to date.

Strategies for Surface Immobilization of Whole Bacteriophages: A Review

“…Since filamentous phages in the Ff class (e.g. fd and M13) typically have several thousand identical, helically-tessellated copies of the pVIII coat protein 213 , they are the most frequent examples of genetic modification for the purpose of immobilization 38,79,80,[84][85][86][87]214 , although examples of such modification of T4 81,83,88,89 and T7…”

“…While the 50-residue pVIII protein of filamentous phages are typically modified [212][213][214] (but also pIII 79 ), in the case of phages T4 and T7 it is rather Soc and Hoc proteins of the phage head that are targeted for modification 81,216,217 .…”

Biosensors and Point‐of‐Care Devices for Bacterial Detection: Rapid Diagnostics Informing Antibiotic Therapy

“…[133] A case in point is the publication by Zurier and colleagues where a phage-based magnetic nanobots were designed to detect E. Coli bacteria. [134] A luminescent reporter bacteriophage (T4) was ) sample is filtered on cellulose 7) luminescence arising from T4 phage is measured. [134] (Reproduced with permission.…”

“…[134] A luminescent reporter bacteriophage (T4) was ) sample is filtered on cellulose 7) luminescence arising from T4 phage is measured. [134] (Reproduced with permission. [134] Copyright 2020, American Chemical Society); B) impedance sensing platform by Brosel-Oliu and coworkers.…”

“…[134] (Reproduced with permission. [134] Copyright 2020, American Chemical Society); B) impedance sensing platform by Brosel-Oliu and coworkers. [138] (Reproduced with permission.…”