The drastically increasing consumption of petroleum-derived plastics hasserious environmental impacts and raises public concerns. Poly(ethylene terephthalate) (PET) is amongst the most extensively produced synthetic polymers. Enzymatic hydrolysis of PET recently emerged as an enticing path for plastic degradation and recycling. In-lab directed evolution has revealed the great potential of PET hydrolases (PETases). However, the time-consuming and laborious PETase assays hinder the identification of effective variants in large mutant libraries. Herein, we devise and validate a dual fluorescence-based high-throughput screening (HTS) assay for a representative IsPETase. The two-round HTS of a pilot library consisting of 2850 IsPETase variants yields six mutant IsPETases with 1.3-4.9 folds improved activities. Compared to the currently used structure- or computational redesign-based PETase engineering, this HTS approach provides a new strategy for discovery of new beneficial mutation patterns of PETases.
Phage Display technology provides a mechanism for us to make bio-recognition elements on biosensors for detection of Salmonella enterica serovars. In the procedure, the filamentous M13 bacteriophage is used for acquiring peptides that have a high affinity for the target recognition. Our approach in this study was to develop peptide structures in the pIII region of this thread-shaped virus. A phage pIII library was used to perform biopanning for the phage clones to bind the target Salmonella serovars. The clones were bound, washed, eluted and amplified four times. Then, the phage peptides were sequenced tested for specificity using ELISA procedures. In this project to make a biosensor for all relevant Salmonella enterica serovars, we used common LPS salmonellae antigens as targets in the biopanning procedure. This enabled us to have a phage probe specific for all serovars of Salmonella enterica excluding the typhoid organisms. The final phage was then immobilized onto an electromagnetic platform to complete the biosensor, which gives us the real-time ability to measure resonance changes that indicate mass loading. The mass loading is an indication of binding to the target cells. Our current data with an ELISA procedure show the phage probe's high affinity for salmonellae, very low cross-reactivity with Escherichia coli, Shigella, and no cross-reactivity to Staphylococcus aureus and Listeria monocytogenes. The biosensor with the phage showed that the capture ability for Salmonella serovars is thirty times higher than the control sensor. This biosensor is a candidate for detection of Salmonella in food and other settings.
This paper investigates the use of wireless, magnetoelastic (ME) biosensors for the surface-scanning detection of Salmonella Typhimurium on fresh produce (eggs, spinach leaves, tomatoes, etc.). The ME biosensor consists of a ME resonator as the sensor platform and E2 phage as the bio-recognition element. The E2 phage is genetically engineered to specifically bind with Salmonella Typhimurium. The ME biosensor is actuated into resonance by an externally applied magnetic field. A microfabricated planar coil was used to measure the resonant frequencies of multiple ME biosensors. The resonant frequency of the biosensors before and after the exposure to the spiked fresh produce was measured. When the Salmonella cells bind with the ME biosensors, the mass of the resonator increases, resulting in a decrease in the sensor's resonant frequency. Real time, in-situ bacteria detection on fresh produce surfaces was demonstrated and compared with previous detection results.
This paper presents an investigation into magnetoelastic (ME) biosentinels that capture and detect low-concentration pathogenic bacteria in stagnant liquids. The ME biosentinels are designed to mimic a variety of white blood cell types, known as the main defensive mechanism in the human body against different pathogenic invaders. The ME biosentinels are composed of a freestanding ME resonator coated with an engineered phage that specifically binds with the pathogens of interest. These biosentinels are ferromagnetic and thus can be moved through a liquid by externally applied magnetic fields. In addition, when a time-varying magnetic field is applied, the ME biosentinels can be placed into mechanical resonance by magnetostriction. As soon as the biosentinels bind with the target pathogen through the phage-based biomolecular recognition, a change in the biosentinel's resonant frequency occurs, and thereby the presence of the target pathogen can be detected. Detection of Bacillus anthracis spores under stagnant flow conditions was demonstrated.
Abstract This paper investigates phage-coated magnetoelastic (ME) biosentinels that capture and detect low-concentration pathogenic bacteria in stagnant liquid. These biosentinels are composed of a freestanding ME resonator platform coated with a landscape phage that specifically binds with the pathogens of interest. These biosentinels can be moved through a liquid by externally applied magnetic fields. When a time-varying magnetic field is applied, the ME biosentinels can be placed into mechanical resonance by magnetostriction. As soon as the biosentinels bind with the target pathogen through the phagebased biomolecular recognition, a change in the biosentinel’s resonant frequency occurs, and thereby the presence of the target pathogen can be detected. Detection of Bacillus anthracis spores under stagnant flow conditions was demonstrated.
The magnetoelastic (ME) biosensors are used to detect pathogen in fresh juice or milk by solenoid coil, and also developed for real-time, direct pathogen detection on food surfaces by surface-scanning coil. This paper presents blocking effect of different reagents on non-specific binding for detecting Salmonella typhimurium in apple juice using phage-based magnetoelastic biosensors. Three different blocking reagents of Bovine serum albumin, Superblock blocking buffer and blocker BLOTTO were used and evaluated. The results shows that blocker BLOTTO has the best blocking effect on non-specific binding.
This paper investigates the use of wireless magnetoelastic (ME) biosensors for the direct detection of Salmonella Typhimurium on fresh produce (tomatoes, spinach leaves and shell eggs). The ME biosensor consists of an ME resonator as the sensor platform and E2 phage as the bio-recognition element. The E2 phage is genetically engineered to specifically bind with S. Typhimurium. The ME biosensor, a wireless sensor, is actuated into resonance by an externally applied magnetic field. When the biosensor binds with Salmonella cells, the mass of the resonator increases, resulting in a decrease in the sensor's resonant frequency. Multiple sensors can be wirelessly and remotely monitored. Multiple measurement and control sensors were placed on fresh produce that was spiked with S. Typhimurium solutions of different known concentrations (5 × 10 1 to 5 × 10 8 CFU/ml (colony-forming unit/ml)). The resonant frequency of the sensors before and after the exposure to the spiked fresh produce was measured. The resonant frequency change of the measurement sensors was significantly different from the control sensors, indicating Salmonella contamination. Despite the different surface topographies of the fresh produce, similar ME biosensor measurement results were obtained for the three fresh produces. Scanning electron microscopy was used to confirm binding of the Salmonella to the biosensor surface and the resulting resonant frequency changes.
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