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., fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation, but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

Phage-based biosensors: in vivo 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). An efficient platform to evaluate the promoter activity of reporter T4 phages was developed. The ability to upregulate Nluc within T4 phages was evaluated using 15 native T4 promoters. Data indicates a six-fold increase in reporter enzyme signal from integration of the selected promoters. Collectively, this work demonstrates that fine tuning the expression of reporter enzymes such as Nluc through optimization of transcription can significantly reduce the limits of detection.

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 Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli 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. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

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. This strategy is based on the use of engineered bacteriophages (phages) to separate and concentrate target E. coli when conjugated with magnetic beads, and to facilitate the detection by expressing gold binding peptides fused alkaline phosphatase (GBPs-ALP). The fusion protein GBPs-ALP has both the enzymatic activity and the ability to directly bind onto a gold surface. This binding-peptide mediated immobilization method provided a novel and simple approach to immobilize proteins on a solid surface, requiring no post-translational modifications. The concentration of E. coli was determined by measuring the activity of the ALP on gold electrodes electrochemically using linear sweep voltammetry (LSV). This approach was successfully applied in the detection of E. coli in drinking water. We were able to detect 105 CFU mL-1 of E. coli within 4 hours. After 9 hours of preincubation, 1 CFU of E. coli in 100 mL of drinking water was detected with a total assay time of 12 hours. This approach compares favorably to the current EPA method and has the potential to be applied to detect different bacteria in other food matrices.

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. Based on the fluorescent intensity, the detection limit of TEV protease is 51 ng/µL. Additionally, we have utilized the sensing system to detect bacteria cells. Bacteriophages, which were engineered to carry TEV protease genes, were used to infect target bacteria (Escherichia coli) resulting in the translation and release of the protease. This allowed the estimation of bacteria at the concentration of 104 CFU/mL. This strategy has the potential to be developed as a multiplex detection platform of multiple bacterial species. Graphical abstract.

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. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 × 105 CFU, equating to 1.2 × 103 CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 101 CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays. Graphical abstract.

Integrating recognition elements with nanomaterials for bacteria sensing.

Pathogenic bacterial contamination is a major threat to human health and safety. In this review, we summarize recent strategies for the integration of recognition elements with nanomaterials for the detection and sensing of pathogenic bacteria. Nanoprobes can provide sensitive and specific detection of bacterial cells, which can be applied across multiple applications and industries.

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 (ß-gal). The T7lacZ phages can infect E. coli, and have the ability to trigger the overexpression of ß-gal during the infection of E. coli. The use of the engineered phages resulted in a more sensitive detection of E. coli by (1) overexpression of ß-gal in E. coli during the specific infection and (2) release of the endogenous intracellular ß-gal from E. coli following infection. The endogenous and phage-induced ß-gal was detected using the electrochemical method with 4-aminophenyl-ß-galactopyranoside (PAPG) as a substrate. The ß-gal catalyzed PAPG to an electroactive species p-aminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 105 CFU/mL in these aqueous samples in 3 h and 102 CFU/mL after 7 h. This strategy has the potential to be extended to detect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.

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. Gold particles (∼500 nm) were synthesized by an oxidation-reduction reaction between gold precursors and residue EDOT monomers on the surface of the PEDOT nanofibers. In order to investigate the electrochemical performance of these electrodes, ascorbic acid was chosen as an analyte model. Our results indicated that PEDOT nanofiber electrodes showed an enhanced response with respect to bare gold electrodes. Furthermore, the OSVP PEDOT nanofibers with gold particles demonstrated the highest sensitivity at low ascorbic acid concentrations. These hierarchically assembled, gold particle-decorated, conductive polymer nanofibers were further fabricated into flexible electrodes, demonstrating a potential in advanced applications such as wearable electronics.

Colorimetric Detection of Escherichia coli Based on the Enzyme-Induced Metallization of Gold Nanorods.

A novel enzyme-induced metallization colorimetric assay is developed to monitor and measure beta-galactosidase (ß-gal) activity, and is further employed for colorimetric bacteriophage (phage)-enabled detection of Escherichia coli (E. coli). This assay relies on enzymatic reaction-induced silver deposition on the surface of gold nanorods (AuNRs). In the presence of ß-gal, the substrate p-aminophenyl ß-d-galactopyranoside is hydrolyzed to produce p-aminophenol (PAP). Reduction of silver ions by PAP generates a silver shell on the surface of AuNRs, resulting in the blue shift of the longitudinal localized surface plasmon resonance peak and multicolor changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for ß-gal is 128 pM, which is lower than the conventional colorimetric assay. Additionally, the assay has a broader dynamic range for ß-gal detection. The specificity of this assay for the detection of ß-gal is demonstrated against several protein competitors. Additionally, this technique is successfully applied to detect E. coli bacteria cells in combination with bacteriophage infection. Due to the simplicity and short incubation time of this enzyme-induced metallization colorimetric method, the assay is well suited for the detection of bacteria in low-resource settings.

Development of a novel bacteriophage based biomagnetic separation method as an aid for sensitive detection of viable Escherichia coli.

The application of bacteriophage combined with the use of magnetic separation techniques has emerged as a valuable tool for the sensitive identification and detection of bacteria. In this study, bacteriophage T7 labelled magnetic beads were developed for the detection of viable bacterial cells. Fusion of the biotin acceptor peptide (BAP) with the phage capsid protein gene and the insertion of the biotin ligase (BirA) gene enabled the display of the BAP ligand and the expression protein BirA during the replication cycle of phage infection. The replicated Escherichia coli specific bacteriophage was biotinylated in vivo and coated on magnetic beads via streptavidin-biotin interaction. Immobilization efficiency of the recombinant phage was investigated on magnetic beads and the phage-bead complex was evaluated by detecting E. coli from inoculated broth. When compared to the wild type phage, the recombinant phage T7birA-bap had a high immobilization density on streptavidin-coated magnetic beads and could capture 86.2% of E. coli cells from broth within 20 min. As this phage-based biomagnetic detection approach provided a low detection limit of 10(2) CFU mL(-1) without pre-enrichment, we believe this assay could be further developed to detect other bacteria of interest by applying host-specific phages. This would be of particular use in detecting bacteria which are difficult to grow or replicate slowly in culture.

Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli.

A large fraction of foodborne illnesses are linked to (∼46%) leafy green vegetables contaminated by pathogens harbored in agricultural water. To prevent this, accurate point-of-production detection tools are required to identify and quantify bacterial contaminants in produce before consumers are impacted. In this study, a proof-of-concept model was engineered for a phage-based Escherichia coli detection system. We engineered the coliphage T7 to express alkaline phosphatase (ALP) to serve as the signal for E. coli detection. Wild type phoA (T7ALP) and a dominant-active allele, phoA D153G D330N (T7ALP*) was inserted into the T7 genome, with engineered constructs selected by CRISPR-mediated cleavage of unaltered chromosomes and confirmed by PCR. Engineered phages and E. coli target cells were co-incubated for 16 hours to produce lysates with liberated ALP correlated with input cell concentrations. A colorimetric assay used p-nitrophenyl phosphate (pNPP) to demonstrate significant ALP production by T7ALP and T7ALP* compared to the vector control (T7EV) (p≤ 0.05). Furthermore, T7ALP* produced 2.5-fold more signal than T7ALP (p≤ 0.05) at pH 10. Due to the increase in signal for the modified ALP* allele, we assessed T7ALP* sensitivity in a dose-responsive manner. We observed 3-fold higher signal for target cell populations as low as ∼2 × 10(5) CFU mL(-1) (p≤ 0.05 vs. no-phage control).

Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy.

Here we presented a simple, rapid and label-free surface-enhanced Raman spectroscopy (SERS) based mapping method for the detection and discrimination of Salmonella enterica and Escherichia coli on silver dendrites. The sample preparation was first optimized to maximize sensitivity. The mapping method was then used to scan through the bacterial cells adsorbed on the surface of silver dendrites. The intrinsic and distinct SERS signals of bacterial cells were used as the basis for label-free detection and discrimination. The results show the developed method is able to detect single bacterial cells adsorbed on the silver dendrites with a limit of detection as low as 10(4) CFU mL(-1), which is two orders of magnitude lower than the traditional SERS method under the same experimental condition. The time needed for collecting a 225 points map was approximately 24 minutes. Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica subsp. enterica BAA1045 and Escherichia coli BL21 from a mixture sample using principle component analysis. Our results demonstrate the great potential of the label-free SERS mapping method to detect, identify and quantify bacteria and bacterial mixtures simultaneously.

Dehydration of bacteriophages in electrospun nanofibers: effect of excipients in polymeric solutions.

Bacteriophages are viruses capable of infecting and lysing target bacterial cells; as such they have potential applications in agriculture for decontamination of foods, food contact surfaces and food rinse water. Although bacteriophages can retain infectivity long-term using lyophilized storage, the process of freeze-drying can be time consuming and expensive. In this study, electrospinning was used for dehydrating bacteriophages in polyvinylpyrrolidone polymer solutions with addition of excipients (sodium chloride, magnesium sulfate, Tris-HCl, sucrose) in deionized water. The high voltage dehydration reduced the infectivity of bacteriophages following electrospinning, with the damaging effect abated with addition of storage media (SM) buffer and sucrose. SM buffer and sucrose also provided the most protection over extended storage (8 weeks; 20 °C; 1% relative humidity) by mitigating environmental effects on the dried bacteriophages. Magnesium sulfate however provided the least protection due to coagulation effects of the ion, which can disrupt the native conformation of the bacteriophage protein coat. Storage temperatures (20 °C, 4 °C and -20 °C; 1% relative humidity) had a minimal effect while relative humidity had substantial effect on the infectivity of bacteriophages. Nanofibers stored in higher relative humidity (33% and 75%) underwent considerable damage due to extensive water absorption and disruption of the fibers. Overall, following storage of nanofiber mats for eight weeks at ambient temperatures, high infective phage concentrations (106-107 PFU ml-1) were retained. Therefore, this study provided valuable insights on preservation and dehydration of bacteriophages by electrospinning in comparison to freeze drying and liquid storage, and the influence of excipients on the viability of bacteriophages.

Rapid screening of waterborne pathogens using phage-mediated separation coupled with real-time PCR detection.

Escherichia coli O157:H7 is a ubiquitous pathogen which can be linked to foodborne outbreaks worldwide. In addition to the significant illnesses, hospitalizations, and deaths resulting from the outbreaks, there can be severe economic consequences to farmers, food manufacturers, and municipalities. A rapid detection assay which can validate sanitation and water quality would prove beneficial to these situations. Here, we report a novel bacteriophage-mediated detection of E. coli O157:H7 which utilizes the specific recognition between phages and their host cell as well as the natural lysis component of the infection cycle for DNA release. Carboxylic acid-functionalized magnetic beads were conjugated with bacteriophage and used to separate and concentrate E. coli O157:H7. The effects of bead incubation time, salinity, pH, and temperature on the bio-magnetic separation were investigated and compared to an antibody-based counterpart. The conditions of 0.01 M PBS, pH 7.0, and 20 min of reaction at 37 °C were found to be optimal. The capture efficiency of the coupled assay was approximately 20 % higher than that of antibody-based separation under extreme conditions. The resulting bead-phage-bacteria complexes were quantitatively detected by real-time PCR (qPCR). Our results demonstrated that the use of phage-based magnetic separation coupled with qPCR improved the sensitivity of detection by 2 orders of magnitude compared that without phage-based pre-concentration. Specificity and selectivity of the assay system was evaluated, and no cross-reactivity occurred when Salmonella typhimurium, Staphylococcus aureus, and Pseudomonas aeruginosa were tested. The total assay time was less than 2 h.

Detection of Escherichia coli in drinking water using T7 bacteriophage-conjugated magnetic probe.

In this study, we demonstrate a bacteriophage (phage)-based magnetic separation scheme for the rapid detection of Escherichia coli (E. coli) in drinking water. T7 phage is a lytic phage with a broad host range specificity for E. coli. Our scheme was as follows: (1) T7 bacteriophage-conjugated magnetic beads were used to capture and separate E. coli BL21 from drinking water; (2) subsequent phage-mediated lysis was used to release endemic ß-galactosidase (ß-gal) from the bound bacterial cells; (3) the release of ß-gal was detected using chlorophenol red-ß-d-galactopyranoside (CRPG), a colorimetric substrate which changes from yellow to red in the presence of ß-gal. Using this strategy, we were able to detect E. coli at a concentration of 1 × 10(4) CFU·mL(-1) within 2.5 h. The specificity of the proposed magnetic probes toward E. coli was demonstrated against a background of competing bacteria. By incorporating a pre-enrichment step in Luria-Bertani (LB) broth supplemented with isopropyl ß-d-thiogalactopyranoside (IPTG), we were able to detect 10 CFU·mL(-1) in drinking water after 6 h of pre-enrichment. The colorimetric change can be determined either by visual observation or with a reader, allowing for a simple, rapid quantification of E. coli in resource-limited settings.

Electrochemical nanoparticle-enzyme sensors for screening bacterial contamination in drinking water.

Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacterial detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles were released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1 h) with high sensitivity (10(2) CFU mL(-1)).

A hybrid paper and microfluidic chip with electrowetting valves and colorimetric detection.

Sequential fluid delivery with minimized external equipment is vital towards a point-of-care diagnostic device. In this work, we have further developed the On-chip Electrowetting Valves concept for the sequential delivery of the reagents to the reaction site in a miniaturized capillary-driven microfluidic chip. Specifically, a disposable polymeric microfluidic device was developed containing capillary force driven microchannels. The device was fabricated using laser ablation and inkjet printing and required no external pumping equipment. The assay was conducted on the microchip containing microfluidic channels with embedded electrowetting valves and a porous membrane patterned with capture molecules and colloidal gold labels. To conduct the assay, the microchip was connected with a low voltage supply which was capable of sequentially opening the valves, delivering the sample and the rinsing reagent to generate visual results. Using T7 bacteriophage as a model, we have demonstrated the development of the device, operation of the valves and execution of the automated assay.

Electrospun water-soluble polymer nanofibers for the dehydration and storage of sensitive reagents.

The ability to preserve and deliver reagents remains an obstacle for the successful deployment of self-contained diagnostic microdevices. In this study we investigated the ability of bacteriophage T7 to be encapsulated and preserved in water soluble nanofibers. The bacteriophage T7 was added to mixtures of polyvinylpyrrolidone and water and electrospun onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning and during storage. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage.

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. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.