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.
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 β-galactosidase (β-gal) from the bound bacterial cells; (3) the release of β-gal was detected using chlorophenol red-β-d-galactopyranoside (CRPG), a colorimetric substrate which changes from yellow to red in the presence of β-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 β-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.
A novel enzyme-induced metallization colorimetric assay was developed to monitor and measure beta-galactosidase (β-gal) activity, and was 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 β-gal, the substrate p-aminophenyl β-D-galactopyranoside (PAPG) 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 (LSPR) peak and multi-color changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for β-gal was 128 pM, which was lower than the conventional colorimetric assay. Additionally, the assay had a broader dynamic range for β-gal detection. The specificity of this assay for the detection of β-gal was demonstrated against several protein competitors. Additionally, this technique was 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.
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%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.
Research in microfluidic biosensors has led to dramatic improvements in sensitivities. Very few examples of these devices have been commercially successful, keeping this methodology out of the hands of potential users. In this study, we developed a method to fabricate a flexible microfluidic device containing electrowetting valves and electrochemical transduction. The device was designed to be amenable to a roll-to-roll manufacturing system, allowing a low manufacturing cost. Microchannels with high fidelity were structured on a PET film using UV-NanoImprint Lithography (UV-NIL). The electrodes were inkjet-printed and photonically sintered on second flexible PET film. The film containing electrodes was bonded directly to the channel-containing layer to form sealed fluidic device. Actuation of the multivalve system with food dye in PBS buffer was performed to demonstrate automated fluid delivery. The device was then used to detect Salmonella in a liquid sample.
Paper-fluidic devices have become an emerging trend for micro total analysis systems (microTAS) in the bioengineering field due to their ability to maintain the rapid, sensitive and specific attributes of microfluidic devices. Subsequently, paper-fluidic devices are also more portable, have a lower production cost and are easier to use. However, one of the obstacles in developing paper fluidic devices is the limited ability to control the rate of fluid flow during an assay. In our project, we use electrowetting on dielectrics where a dielectric, which is normally hydrophobic, is polarized and becomes hydrophilic. We have fabricated paper-fluidic devices by inkjet printing and spraying conductive hydrophobic electrodes/valves in conjunction with conductive hydrophilic electrodes which are able to stop the fluid front of phosphate buffered saline (PBS). The hydrophobic valves were then actuated by an applied potential which altered the fluorinated monolayer on the electrode. As the applied potential between the electrodes was increased, the amount of time for the fluid front to pass the valve decreased because the monolayer was altered faster. However, we did not observe significant differences in time as we increased the distance between the electrodes. The valves were also incorporated in a lateral flow assay where the device was used to detect Saccharomyces cerevisiae rRNA sequences. With the ability to control the fluid flow in a paper-fluidic device, more complex and intricate assays can be developed, which not only can be applied in the biomedical, food and environmental fields, but also can be used in low resource settings for the detection of diseases.
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