Bacteriophage-Based Bioanalysis.

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors.

Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria.

Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection of their host bacteria. While the potential impact of magnetized phages is high, the current methods of immobilization are either weak, costly, inefficient, or laborious making them less viable for commercialization. In order to bridge this gap, we have developed a highly efficient, site-specific, and low-cost method to immobilize bacteriophages onto solid supports.

Capsid Engineering of Bacteriophages for Oriented Surface Conjugation.

The current state-of-the-art in bacteriophage (phage) immobilization onto magnetic particles is limited to techniques that are less expensive and/or facile but nonspecific or those that are more expensive and/or complicated but ensure capsid-down orientation of the phages, as necessary to preserve infectivity and performance in subsequent applications (e.g., therapeutics, detection).

Phage Ac3's Genome Annotation and Host Range Analysis Against the ECOR Reference Library.

Host range analyses and genome sequencing/annotation of bacteriophage isolates allow more effective development of tools for applications in medicine, agriculture, and the environment and expand our understanding of phage biology. Here we present the complete sequence of phage Ac3's assembled and annotated genome (accession OK040907). Originally referred to simply as "3," Ac3 has previously been described as a T4-like bacteriophage belonging to the family in the order of tailed bacteriophages.

A phage-based magnetic relaxation switching biosensor using bioorthogonal reaction signal amplification for Salmonella detection in foods.

Phages are uniquely suited for bacterial detection due to their low cost and ability to recognize live bacteria. Herein, our work establishes the proof-of-concept detection of Salmonella in orange juice based on a phage-mediated portable magnetic relaxation switching (MRS) biosensor. The limit of quantification (LOQ) could reach 5 CFU/mL (95 % confidence interval [CI]: 4-7, N = 4) with a linear range of 10-10 CFU/mL, which has improved 10-fold than that without bioorthogonal signal amplification.

Rapid, sensitive, and low-cost detection of Escherichia coli bacteria in contaminated water samples using a phage-based assay.

Inadequate drinking water quality is among the major causes of preventable mortality, predominantly in young children. Identifying contaminated water sources remains a significant challenge, especially where resources are limited. The current methods for measuring Escherichia coli (E.

A microfluidic device and instrument prototypes for the detection of in water samples using a phage-based bioluminescence assay.

Current quantification methods of () contamination in water samples involve long incubation, laboratory equipment and facilities, or complex processes that require specialized training for accurate operation and interpretation. To address these limitations, we have developed a microfluidic device and portable instrument prototypes capable of performing a rapid and highly sensitive bacteriophage-based assay to detect cells with detection limit comparable to traditional methods in a fraction of the time. The microfluidic device combines membrane filtration and selective enrichment using T7-NanoLuc-CBM, a genetically engineered bacteriophage, to identify 4.

Phage SV76 Host Range and Genomic Characterization.

Background: Increasing the quantity and detail of bacteriophage genomic data is critical to broadening our understanding of how bacteriophages operate to allow us to harness their unique properties for biotechnology advancements. Here we present the complete sequence of phage SV76's assembled and annotated genome (Accession OM339528). SV76 has previously been classified as a T4-like bacteriophage belonging to the genus within the family of contractile tailed bacteriophages.

Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay.

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage's ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics.

Engineering Biorthogonal Phage-Based Nanobots for Ultrasensitive, Bacteria Detection.

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.

An Engineered Reporter Phage for the Fluorometric Detection of in Ground Beef.

Despite enhanced sanitation implementations, foodborne bacterial pathogens still remain a major threat to public health and generate high costs for the food industry. Reporter bacteriophage (phage) systems have been regarded as a powerful technology for diagnostic assays for their extraordinary specificity to target cells and cost-effectiveness. Our study introduced an enzyme-based fluorescent assay for detecting the presence of using the T7 phage engineered with the operon which encodes beta-galactosidase (β-gal).

Bacteriophage Capsid Modification by Genetic and Chemical Methods.

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g.

FEAST of biosensors: Food, environmental and agricultural sensing technologies (FEAST) in North America.

We review the challenges and opportunities for biosensor research in North America aimed to accelerate translational research. We call for platform approaches based on: i) tools that can support interoperability between food, environment and agriculture, ii) open-source tools for analytics, iii) algorithms used for data and information arbitrage, and iv) use-inspired sensor design. We summarize select mobile devices and phone-based biosensors that couple analytical systems with biosensors for improving decision support.

Optimization of T4 phage engineering via CRISPR/Cas9.

A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03-3%.

Phage-based biosensors: analysis of native T4 phage promoters to enhance reporter enzyme expression.

Phage-based biosensors have shown significant promise in meeting the present needs of the food and agricultural industries due to a combination of sufficient portability, speed, ease of use, sensitivity, and low production cost. Although current phage-based methods do not meet the bacteria detection limit imposed by the EPA, FDA, and USDA, a better understanding of phage genetics can significantly increase their sensitivity as biosensors. In the current study, the signal sensitivity of a T4 phage-based detection system was improved via transcriptional upregulation of the reporter enzyme Nanoluc luciferase (Nluc).

A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages.

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of () in drinking water is an indication of inadequate sanitation and a significant health risk. As is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation.

Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system.

Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E.

Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection.

Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages.

Detection of protease and engineered phage-infected bacteria using peptide-graphene oxide nanosensors.

A peptide-graphene oxide nanosensor has been developed to detect tobacco etch virus (TEV) protease and bacteria infected with an engineered bacteriophage. In the detection strategy, a peptide (sequence: RKRFRENLYFQSCP) is tagged with fluorophores and graphene oxide (GO) is used to adsorb the peptides while quenching their fluorescence. In the presence of TEV protease, fluoropeptides are cleaved between glutamine (Q) and serine (S), resulting in the recovery of fluorescence signal.

Phage based electrochemical detection of Escherichia coli in drinking water using affinity reporter probes.

The monitoring of drinking water for indicators of fecal contamination is crucial for ensuring a safe supply. In this study, a novel electrochemical method was developed for the rapid and sensitive detection of Escherichia coli (E. coli) in drinking water.

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water.

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E.

Lyophilized Engineered Phages for Escherichia coli Detection in Food Matrices.

Ease of use, low cost, and convenient transport are the key requirements for a commercial bacteria detection kit designed for resource-limited settings. Here, we report the colorimetric detection of Escherichia coli (E. coli) in food samples using freeze-dried engineered bacteriophages (phages).

Development of Engineered Bacteriophages for Escherichia coli Detection and High-Throughput Antibiotic Resistance Determination.

T7 bacteriophages (phages) have been genetically engineered to carry the lacZ operon, enabling the overexpression of beta-galactosidase (β-gal) during phage infection and allowing for the enhanced colorimetric detection of Escherichia coli (E. coli). Following the phage infection of E.

Electrochemical Detection of Escherichia coli from Aqueous Samples Using Engineered Phages.

In this study, an enzyme-based electrochemical method was developed for the detection of Escherichia coli (E. coli) using the T7 bacteriophages engineered with lacZ operon encoding for beta-galactosidase (β-gal). The T7 phages can infect E.

Facile hierarchical assembly of gold particle decorated conductive polymer nanofibers for electrochemical sensing.

In this study, we successfully applied vapor-phase polymerization towards the synthesis of PEDOT nanofibers which were subsequently functionalized with gold particles and used as electrodes for electrochemical sensing. Two methods were used to synthesize the PEDOT nanofibers including (1) electrospinning followed by vapor-phase polymerization (EVP), and (2) one-step vapor-phase polymerization (OSVP). The average diameter of EVP fibers was approximately 350 nm, and OSVP was approximately 200 nm.