Performance of wild, tailed, humidity-robust phage on a surface-scanning magnetoelastic biosensor for Salmonella Typhimurium detection.

A wild, tailed phage (TST) was compared with a genetically modified, filamentous phage (FST) for S. Typhimurium (ST) detection. When both phages were introduced into oppositely charged MUA and MUAM sensors, the RU values of TST showed an obvious increase on the MUAM sensor. The sensitivity of TST [54.78 ΔRU/(log PFU/mL)] was greater than that of FST [48.05 ΔRU/(log PFU/mL)]. The binding affinity (KD = 1.75 × 10-13 M) of TST on MUAM sensor was greater than that of FST. Both phages were specific to only ST, and TST exhibited a persistent binding capability at 50 % RH. When each phage-immobilized sensor was employed on chili pepper, the sensitivity [880.80 Hz/(log CFU/mL)] and detection limit (1.31 ± 0.27 log CFU/mL) of TST were significantly greater than those of FST. The orientation of TST on sensor promoted the uniform capture of bacteria and enhanced the reliable performance of a surface-scanning magnetoelastic biosensor.

3D Phage-based biomolecular filter for effective high throughput capture of Salmonella Typhimurium in liquid streams.

Foodborne illnesses caused by pathogens on fresh produce remain one of the most critical food safety problems the world faces. The recalls of pasta salad in 2018 and pre-cut melons in 2019 imply current methods in identifying the source of pathogens and outbreak prevention are inappropriate and time consuming. In this article, a new technology, called the 3D phage-based biomolecular filter, was developed to simultaneously capture and concentrate foodborne pathogens from large volumes of liquid streams (food liquid or wash water streams). The 3D phage-based filter consisted of phage-immobilized magnetoelastic (ME) filter elements, a filter pipe system, and a uniform magnetic field to fix and align the ME filter elements in the 3D filter column. The closely packed ME filter elements display a 3D layered structure which allows for enhanced surface interaction of the immobilized bacteriophage with specific pathogens in the passing liquid streams. As a result, a pathogen capture rate of more than 90% was achieved at a high flow rate of 3 mm/s with 20,000 ME filter elements. The capability of the 3D phage-based filter to capture pathogens in liquid streams at different filter element packing densities was further validated by experiments, finite element analysis and theoretical calculations. The capture rate increases significantly with larger numbers of ME filter elements placed in the testing pipe, and the turbulence flow induced by the 3D stacking of ME filter elements can further improve the capture efficiency. This technology enables rapid capture and analysis of large volume of water in processing fresh fruit and vegetables for the presence of small quantities of pathogens, which will ultimately benefit producers, the food industry, and society with improved food safety and production efficiency.

Efficient affinity-tagging of M13 phage capsid protein IX for immobilization of protein III-displayed oligopeptide probes on abiotic platforms.

We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011 pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.

Simultaneous Surface Plasmon Resonance/Fluorescence Spectroelectrochemical in Situ Monitoring of Dynamic Changes on Functional Interfaces: A Study of the Electrochemical Proximity Assay Model System.

Understanding the chemical composition and morphology of interfaces plays a vital role in the development of sensors, drug delivery systems, coatings for biomedical implants, and so forth. In many cases, the interface characterization can be performed by a combination of electrochemical and one of the optical techniques. In this study, we further enhanced capabilities in probing interfaces by combining electrochemical characterization with multiple optical techniques, that is, surface plasmon resonance (SPR) and fluorescence spectroscopy. This new combination was utilized to study the electrochemical proximity assay (ECPA)-a recently developed protein recognition strategy for the point-of-care test. The SPR/fluorescence spectroelectrochemical technique has achieved not only recognition of binding components involved in the ECPA model system, estimation of their thicknesses and surface coverages, but more importantly, highly reliable in situ monitoring of dynamic changes of components involved in interfacial binding via cross-validation and confirmation from three simultaneously generated signals-SPR, fluorescence, and electrochemistry. In addition, the obtained corresponding proportions among magnitudes of three signals provide crucial information for future studies on simultaneous characterization of multiple components in one step and differentiation of nonspecific binding events. Another advantage using this technique is that the excitation of fluorescence is not only confined by surface plasmons, but by photons, so the fluorescence information can be also gained as the distance of fluorophores from the surface exceeds the decay length of surface plasmons.

Detection of Salmonella Typhimurium on Spinach Using Phage-Based Magnetoelastic Biosensors.

Phage-based magnetoelastic (ME) biosensors have been studied as an in-situ, real-time, wireless, direct detection method of foodborne pathogens in recent years. This paper investigates an ME biosensor method for the detection of Salmonella Typhimurium on fresh spinach leaves. A procedure to obtain a concentrated suspension of Salmonella from contaminated spinach leaves is described that is based on methods outlined in the U.S. FDA Bacteriological Analytical Manual for the detection of Salmonella on leafy green vegetables. The effects of an alternative pre-enrichment broth (LB broth vs. lactose broth), incubation time on the detection performance and negative control were investigated. In addition, different blocking agents (BSA, Casein, and Superblock) were evaluated to minimize the effect of nonspecific binding. None of the blocking agents was found to be superior to the others, or even better than none. Unblocked ME biosensors were placed directly in a concentrated suspension and allowed to bind with Salmonella cells for 30 min before measuring the resonant frequency using a surface-scanning coil detector. It was found that 7 h incubation at 37 °C in LB broth was necessary to detect an initial spike of 100 cfu/25 g S. Typhimurium on spinach leaves with a confidence level of difference greater than 95% (p < 0.05). Thus, the ME biosensor method, on both partly and fully detection, was demonstrated to be a robust and competitive method for foodborne pathogens on fresh products.

Novel Approach of a Phage-Based Magnetoelastic Biosensor for the Detection of Salmonella enterica serovar Typhimurium in Soil.

To date, there has been no employment of a magnetoelastic (ME) biosensor method to detect Salmonella enterica serovar Typhimurium in soil. The ME biosensor method needs to be investigated and modified for its successful performance. The filtration method, cation-exchange resin method, and combinations of both methods were employed for the extraction of S. Typhimurium from soil. The number of S. Typhimurium and the resonant frequency shift of the ME sensor were then compared using a brilliant green sulfa agar plate and an HP 8751A network analyzer. A blocking study was performed using bovine serum albumin (BSA), polyethylene glycol (PEG), and casein powder suspension. Finally, the modified ME biosensor method was performed to detect S. Typhimurium in soil. The number of S. Typhimurium was significantly decreased from 7.10 log CFU/soil to 4.45-4.72 log CFU/soil after introduction of the cation-exchange resin method. The greatest resonant frequency shift of the measurement sensor was found when employing centrifugation and filtration procedures. The resonant frequency shift of the PEG-blocked measurement sensor was 3,219 ± 755 Hz, which was significantly greater than those of the BSA- and casein-blocked ME sensor. The optimum concentration of PEG was determined to be 1.0 mg/ml after considering the resonant shift and economic issue. Finally, the modified ME biosensor method was able to detect S. Typhimurium in soil in a dose-response manner. Although these modifications of the ME biosensor method sacrificed some advantages, such as cost, time effectiveness, and operator friendliness, this study demonstrated a novel approach of the ME biosensor method to detect S. Typhimurium in soil.

Comparative study of thermal stability of magnetostrictive biosensor between two kinds of biorecognition elements.

Magnetostrictive biosensors specific to Salmonella typhimurium were prepared by immobilizing antibody or phage as biorecognition elements onto the magnetostrictive sensor platform. The sensors were stored at temperatures of 25 °C (room temperature), 45 °C and 65 °C, respectively, and the ability to bind S. typhimurium was detected by testing the resonant frequency shift using a HP network analyzer after exposure to 1 mL of 1×10(9) cfu/mL of S. typhimurium at a predetermined schedule. The binding of S. typhimurium to biosensors was confirmed by Scanning Electron Microscopy (SEM). The results showed that there existed an initial sudden drop in the average density of S. typhimurium bound to the biosensor surface versus duration at different temperatures for the two kinds of recognition elements, and the binding ability to S. typhimurium of phage-immobilized biosensors was much better than that of antibody-immobilized biosensors, with longevity longer than 30 days at all tested temperatures, though decreasing gradually over the testing period. While the longevity of antibody-immobilized biosensors was only about 30, 8 and 5 days at room temperature (25 °C), 45 °C and 65 °C, respectively. Meanwhile, the activation energy of the two kinds of biosensors was investigated, and it was found that phage immobilized sensors showed much higher activation energy than antibody immobilized sensors, which resulted in less dependency on temperature and thus having much better thermal stability than antibody immobilized sensors.

A surface-scanning coil detector for real-time, in-situ detection of bacteria on fresh food surfaces.

Proof-in-principle of a new surface-scanning coil detector has been demonstrated. This new coil detector excites and measures the resonant frequency of free-standing magnetoelastic (ME) biosensors that may now be placed outside the coil boundaries. With this coil design, the biosensors are no longer required to be placed inside the coil before frequency measurement. Hence, this new coil enables bacterial pathogens to be detected on fresh food surfaces in real-time and in-situ. The new coil measurement technique was demonstrated using an E2 phage-coated ME biosensor to detect Salmonella typhimurium on tomato surfaces. Real-time, in-situ detection was achieved with a limit of detection (LOD) statistically determined to be lower than 1.5×10(3) CFU/mm(2) with a confidence level of difference higher than 95% (p<0.05).

A nanoscale co-precipitation approach for property enhancement of Fe-base alloys.

Precipitate size and number density are two key factors for tailoring the mechanical behavior of nanoscale precipitate-hardened alloys. However, during thermal aging, the precipitate size and number density change, leading to either poor strength or high strength but significantly reduced ductility. Here we demonstrate, by producing nanoscale co-precipitates in composition-optimized multicomponent precipitation-hardened alloys, a unique approach to improve the stability of the alloy against thermal aging and hence the mechanical properties. Our study provides compelling experimental evidence that these nanoscale co-precipitates consist of a Cu-enriched bcc core partially encased by a B2-ordered Ni(Mn, Al) phase. This co-precipitate provides a more complex obstacle for dislocation movement due to atomic ordering together with interphases, resulting in a high yield strength alloy without sacrificing alloy ductility.

Rapid and sensitive detection of Salmonella Typhimurium on eggshells by using wireless biosensors.

This article presents rapid, sensitive, direct detection of Salmonella Typhimurium on eggshells by using wireless magnetoelastic (ME) biosensors. The biosensor consists of a freestanding, strip-shaped ME resonator as the signal transducer and the E2 phage as the biomolecular recognition element that selectively binds with Salmonella Typhimurium. This ME biosensor is a type of mass-sensitive biosensor that can be wirelessly actuated into mechanical resonance by an externally applied timevarying magnetic field. When the biosensor binds with Salmonella Typhimurium, the mass of the sensor increases, resulting in a decrease in the sensor's resonant frequency. Multiple E2 phage-coated biosensors (measurement sensors) were placed on eggshells spiked with Salmonella Typhimurium of various concentrations (1.6 to 1.6 × 10(7) CFU/cm(2)). Control sensors without phage were also used to compensate for environmental effects and nonspecific binding. After 20 min in a humidity-controlled chamber (95%) to allow binding of the bacteria to the sensors to occur, the resonant frequency of the sensors was wirelessly measured and compared with their initial resonant frequency. The resonant frequency change of the measurement sensors was found to be statistically different from that of the control sensors down to 1.6 × 10(2) CFU/cm(2), the detection limit for this work. In addition, scanning electron microscopy imaging verified that the measured resonant frequency changes were directly related to the number of bound cells on the sensor surface. The total assay time of the presented methodology was approximately 30 min, facilitating rapid detection of Salmonella Typhimurium without any preceding sampling procedures.

A chemical switch for detecting insect infestation.

BACKGROUND: Plants emit phytochemicals as a defensive mechanism against herbivores. A small sensor switch that responds to these chemicals could be used to stop insect infestation at early stages. RESULTS: Polyethylene-co-vinyl acetate was chosen as the best polymer for this particular application, based on its swelling response to plant volatiles. When the carbon concentration of the active layer was low enough to be near the percolation threshold, the sensor could be used as a 'chemical switch'. The resistance of the sensor increased significantly, mimicking a 'switch-off' response when exposed to the analyte vapor. When the analyte vapor was no longer present, the sensor returned back to its original condition, showing a 'switch-on' response. The percolation point was obtained when the carbon concentration of the polymer/carbon composite was kept at 2.5 wt%. CONCLUSION: A low-mass-fraction carbon composite sensor has been designed and fabricated to detect γ-terpinene, α-pinene, p-cymene, farnesene, limonene and cis-hexenyl acetate. The sensor is inexpensive, easy to fabricate and highly stable in air.

Effects of surface functionalization on the surface phage coverage and the subsequent performance of phage-immobilized magnetoelastic biosensors.

One of the important applications for which phage-immobilized magnetoelastic (ME) biosensors are being developed is the wireless, on-site detection of pathogenic bacteria for food safety and bio-security. Until now, such biosensors have been constructed by immobilizing a landscape phage probe on gold-coated ME resonators via physical adsorption. Although the physical adsorption method is simple, the immobilization stability and surface coverage of phage probes on differently functionalized sensor surfaces need to be evaluated as a potential way to enhance the detection capabilities of the biosensors. As a model study, a filamentous fd-tet phage that specifically binds streptavidin was adsorbed on either bare or surface-functionalized gold-coated ME resonators. The surface functionalization was performed through the formation of three self-assembled monolayers with a different terminator, based on the sulfur-gold chemistry: AC (activated carboxy-terminated), ALD (aldehyde-terminated), and MT (methyl-terminated). The results, obtained by atomic force microscopy, showed that surface functionalization has a large effect on the surface phage coverage (46.8%, 49.4%, 4.2%, and 5.2% for bare, AC-, ALD-, and MT-functionalized resonators, respectively). In addition, a direct correlation of the observed surface phage coverage with the quantity of subsequently captured streptavidin-coated microbeads was found by scanning electron microscopy and by resonance frequency measurements of the biosensors. The differences in surface phage coverage on the differently functionalized surfaces may then be used to pattern the phage probe layer onto desired parts of the sensor surface to enhance the detection capabilities of ME biosensors.

Expression of truncated tobacco osmotin in Escherichia coli: purification and antifungal activity.

PURPOSE OF WORK: Tobacco osmotin is a functional homolog of mammalian adiponectin, and has antifungal activity. This work was undertaken to produce recombinant osmotin that has previously been unsuccessful because of its toxicity. Expression of recombinant tobacco osmotin (rOSM) in Escherichia coli inclusion bodies has been achieved. The optimal pH for rOSM expression in ZYM 505 medium is 7.0 at OD(650) of 1.5 of culture growth. The rOSM from the inclusion body was extracted with 8 M urea, and purified using CM-cellulose and cobalt-agarose bead affinity chromatography to a high purity. Approximately 80% of the rOSM remained bound to CM-cellulose and Cobalt-agarose beads after initial elution. The yield of purified rOSM was between 40 and 50 mg from 2 l of culture. Repeated elution of protein from CM-cellulose and Co-agarose increased the yield of rOSM to 200 mg from 2 l culture. The purified rOSM showed variable antifungal activities against two pathogenic yeast strains; Cryptococcus neoformans, Candida albicans, and non-pathogenic strains; Saccharomyces cerevisiae and Pichia methanolica.

Time domain characterization of magnetoelastic sensors: A pulse method for resonance frequency determination.

This paper presents a pulse method for determination of resonance frequency of magnetoelastic sensors. The method eliminates the bias field that is necessary in previous methods and also allows fast and accurate detection. The stability tests of the system show an average standard deviation of 129 Hz and an average drift of -10.4 Hz/h. This system allows simultaneous detection of two sensors. A simulation of the operation of one and two sensors was shown to be very similar to the real data plots from the test system. Real tests have shown that adding a second sensor does not affect the resonance frequency of the first sensor. The effect of pulse magnetic field on the characteristics of the resonance signal, including resonance frequency, amplitude, and Q-value of frequency domain signal, has been studied and real time detection using magnetoelastic sensors was demonstrated in a flowing system.

Direct detection of Salmonella typhimurium on fresh produce using phage-based magnetoelastic biosensors.

Current bacterial detection methods require the collection of samples followed by preparation and analysis in the laboratory, both time and labour consuming steps. More importantly, because of cost, only a limited number of samples can be taken and analyzed. This paper presents the results of an investigation to directly detect Salmonella typhimurium on fresh tomato surfaces using phage-based magnetoelastic (ME) biosensors. The biosensor is composed of a ME resonator platform coated with filamentous E2 phage, engineered to bind with S. typhimurium. The ME biosensors are wireless sensors, whose resonance oscillation and resonance frequency are actuated and detected through magnetic fields. The sensors used in this study were 0.028 mm×0.2 mm×1 mm in size. In this study, the tomato surface was spiked with S. typhimurium suspensions with concentrations ranging from 5×10(1) to 5×10(8)CFU/ml and then allowed to dry in air. The detection was conducted by directly placing ME measurement biosensors and control sensors on the spiked surface for 30 min in a humid environment. The control sensors were identical to the measurement biosensors, but without phage. Both measurement and control sensors were blocked with BSA to reduce non-specific binding. The resonance frequencies of both measurement and control sensors were measured prior to and after the placement of the sensors on the tomato. Shifts in the resonance frequency of the measurement biosensors were observed, while the control sensors showed negligible change. Scanning electron microscopy (SEM) was used to verify the specific binding of S. typhimurium to the biosensor. Results of multiple biosensor detection and corresponding analyzes showed statistically different responses between the measurement and control sensors for tomatoes spiked with S. typhimurium suspensions with concentrations of 5×10(2)CFU/ml and greater. This study demonstrates the direct detection of food-borne bacteria on fresh produce.

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium.

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.

A magnetoelastic resonance biosensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium.

Mass-sensitive, magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless and remote. A biosensor for the detection of Salmonella typhimurium was constructed by immobilizing a polyclonal antibody (the bio-molecular recognition element) onto the surface of a magnetostrictive platform. The biosensor was then exposed to solutions containing S. typhimurium bacteria. Binding between the antibody and antigen (bacteria) occurred and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Sensors with different physical dimensions were exposed to different concentrations of S. typhimurium ranging from 10(2) to 10(9)CFU/ml. Detection limits of 5x10(3) CFU/ml, 10(5) CFU/ml and 10(7) CFU/ml were obtained for sensors with the size of 2 mmx0.4 mmx15 microm, 5 mmx1 mmx15 microm and 25 mmx5 mmx15 microm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.

Landscape phage probes for Salmonella typhimurium.

We selected from landscape phage library probes that bind preferentially Salmonella typhimurium cells compared with other Enterobacteriaceae. The specificity of the phage probes for S. typhimurium was analyzed by the phage-capture test, the enzyme-linked immunosorbent assay (ELISA), and the precipitation test. Interaction of representative probes with S. typhimurium was characterized by fluorescence-activated cell sorting (FACS), and fluorescent, optical and electron microscopy. The results show that the landscape phage library is a rich source of specific and robust probes for S. typhimurium suitable for long-term use in continuous monitoring devices and biosorbents.