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Thermalstability of polyclonal antibodies to Salmonella typhimurium was investigated by studying the effect of temperature on the binding activity to Salmonella typhimurium using a magnetostrictive platform. Antibodies were immobilized using the Langmuir-Blodgett (LB) technique. Then sensors were stored at temperatures of, 25°C (room temperature), 45°C and 65°C, respectively, and then the ability of these sensors to detect S. typhimurium was tested at a predetermined schedule. Changes in the fundamental resonance frequency of sensors after exposure to 1 ml of 1×109cfu/ml of S. typhimurium were recorded over the testing period. The shift in resonance frequency was attributed to the binding of bacteria to antibody immobilized sensor. The results showed that at each temperature, the binding ability of the antibody to S. typhimurium decreased gradually over the testing period, and the higher the temperature, the lower the longevity of the polyclonal antibody. The longevity of polyclonal antibody on the magnetostrictive sensor platform was about 30, 8 and 5 days at room temperature (25°C), 45°C and 65°C, respectively.

This paper presents the results of a study that investigates the optimization of phage chemistry during the fabrication of magnetoelastic (ME) biosensors for the detection of Salmonella typhimurium or Bacillus anthracis spores. The bundling characteristics of the phage filaments limit the ability of the biosensor to bind bacterial cells/spores. Experiments were performed to determine the proper phage concentration for the prevention of bundling in aqueous environments. Based on the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) results, which verify the structure of phage under different concentrations and binding numbers of target species to the sensor surface, we found that phage concentrations of 1011 vir/ml exhibit the best sensor performance in terms of binding sensitivity. Additionally, the sensors immobilized with phage under this condition were tested in a flowing liquid system using S. typhimurium and B. anthracis spores suspensions in concentrations ranging from 5 times101 to 5 times 108 cfu/ml, separately. As cells/spores are bound to a ME biosensor surface, the additional mass of the spores causes a decrease in the resonance frequency of the sensor. The frequency response curves of the ME biosensors as a function of exposure time were then measured, and the detection limit of the ME biosensor was determined to be 5 times 103 cfu/ml.

A micro-scale, freestanding, magnetoelastic biosensor coated with phage has been developed for the real-time in vitro detection of Bacillus anthracis spores. The sensor exhibits a characteristic resonance frequency upon the application of an alternating external magnetic field. It has a high sensitivity to the change in mass when spores are attached. The frequency versus mass sensitivity increases significantly with a decrease in sensor length. Spore detection is realized by measuring the resonance frequency change due to the change in mass as spores are captured onto the sensor surface. B. anthracis spore suspensions in a range of concentration levels (5 × 10 1 to 5 × 10 8 spores/ml) was tested using a 1000 μm × 200 μm × 15 μm sensor in a flowing fluid at a flow rate of 40 μl/min. The binding kinetics was analyzed based on the attachment rate. The specificity of the sensor to B. anthracis spores was examined compared with other Bacillus species.

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1 mg/ml BSA (blocking agent) onto the magnetoelastic resonator’s surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor’s resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.

This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.

This article presents the results of an investigation into the enhancement of sensitivity and thermal stability of polyclonal antibody immobilized magnetoelastic biosensors. The Langmuir–Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors. Biosensor performance was investigated by exposing to graded concentrations (5 × 101–5 × 108 cfu mL−1) of Salmonella typhimurium solutions in a flow through mode. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the sensor’s resonance frequency shift. An increase in the sensitivity from 159 Hz decade−1 for a 2 mm sensor to 246 Hz decade−1 for a 1 mm sensor was observed during the dose–response measurements. The stability of the biosensor was also investigated by storing the biosensor at 25, 45 and 65 °C. The binding activity of the stored biosensor was estimated by measuring the changes in resonance frequency after exposure to the bacterial solutions (109 cfu mL−1). Binding activity was also confirmed by counting bound S. typhimurium cells on the sensor surface using Scanning Electron Microscopy (SEM) micrographs. The results show that at each temperature, the binding activity of the biosensor gradually decreased over the testing period. Degradation of biosensor accelerated at higher storage temperatures. The activation energy of biosensor system degradation was determined to be 7.7 kcal mol−1.

A magnetoelastic sensor immobilized with bacteriophage for the detection of Salmonella typhimurium in fat free milk is described. The response of the magnetoelastic sensors can be measured in the absence of direct physical wire contacts to the sensor, making the possibility of real time and in vivo bio-detection systems more realistic. The sensor system reported in this article is a combination of two rapidly developing technologies: magnetoelastic materials as a transducer and filamentous bacteriophage as a replacement to traditional bioprobes such as antibodies. Upon exposure to the target pathogen, the response of the phage immobilized magnetoelastic sensor changed, which was quantified by the shift in the sensor's resonance frequency. These frequency changes were proportional to the amount of target antigen bound to the sensor. Response of the sensors to increasing concentrations (5 × 10 1 to 5 × 10 8 cfu/ml) of S. typhimurium in two different liquid media (water and fat-free milk) was studied on sensors with dimensions 2 mm × 0.4 mm × 0.015 mm. Scanning electron microscopy (SEM) micrographics of previously assayed biosensors were used for visual correlation of sensor response. A detection limit of 5 × 10 3 cfu/ml, with a sensitivity of 159 Hz/decade was obtained for the sensors tested in water samples, as compared to 118 Hz/decade in fat free milk. The dissociation constant K d and the binding valencies for S. typhimurium spiked water samples was 136, 2.4 and 149 and 2.5 cfu/ml for spiked fat free milk samples.

In this article, a magnetoelastic sensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium in food products is described. The remote query nature of magnetoelastic sensors enables the detection of bacterial species in sealed and opaque containers. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the shift in the sensor’s resonance frequency. Response of the sensors to increasing concentrations (5 × 101–5 × 108 cfu/ml) of S. typhimurium in three different food products (water, fat-free milk and apple juice) was studied and similar responses were observed. These results were also further ascertained by Scanning Electron Microscopy (SEM) studies. A detection limit of 5 × 103 cfu/ml, with a sensitivity of 139 Hz/decade was obtained for the sensors tested in water samples, as compared to 129 Hz/decade in apple juice and 127 Hz/decade in fat free milk. A 2 × 0.4 × 0.015 mm sensor was employed in all the investigations. The dissociation constant K d and the binding valencies for S. typhimurium spiked in water samples was 435 cfu/ml and 2.33 respectively; as compared to 309 cfu/ml and 2.38 for apple juice; and 1389 cfu/ml and 1.85 for fat free milk samples. Bacterial binding was specific and a divalent binding was observed.

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 × 109 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.

The phage engineering, which grounds on the natural mechanisms of selection, allows directed nanofabrication of bioselective materials, with possible applications to biosensors, nanoelectronics, biosorbents, and other areas of medicine, technology, and environmental monitoring. In particular, using phage display technology allows the generation of libraries possessing diverse nanostructures accommodated on the phage's surface – a huge resource of diagnostic and detection probes. Selected phage-derived probes bind biological agents and generate detectable signals as a part of analytical platforms. They may be suitable as robust and inexpensive molecular recognition interfaces for field-use detectors and real time monitoring devices for biological and chemical threat agents. The data discussed in this chapter shows how the use of phage-based interfaces may greatly improve the sensitivity, robustness and longevity of commercial biosensors.

In this paper, we report a wireless magnetoelastic (ME) biosensor with phage as the bio-recognition probe for real time detection of Salmonella typhimurium . The ME biosensor was constructed by immobilizing filamentous phage that specifically binds with S. typhimurium onto the surface of a strip-shaped ME particle. The ME sensor oscillates with a characteristic resonance frequency when subjected to a time varying magnetic field. Binding between the phage and antigen (bacteria) causes a shift in the sensor's resonance frequency. Sensors with different dimensions were exposed to various known concentrations of S. typhimurium ranging from 5 x10 1 to 5 x 10 8 cfu/ml. The detection limit of the ME sensors was found to improve as the size of the sensor became smaller. The detection limit was found to improve from 161 Hz/decade (2mm length sensors) to 1150 Hz/decade (500 μm length sensors). The stability of the ME biosensor was investigated by storing the sensor at different temperatures (25, 45, and 65 °C), and then evaluating the binding activity of the stored biosensor after exposure to S. typhimurium solution (5 x 10 8 cfu/ml). The results showed that the phage-coated biosensor is robust. Even after storage in excess of 60 days at 65 °C, the phage-coated sensors have a greater binding affinity than the best antibody coated sensors stored for 1 day at 45 °C. The antibody coated sensors showed near zero binding affinity after 3 days of storage at 65 °C.

In this paper the performance of a magnetoelastic biosensor detection system for the simultaneous identification of B. anthracis spores and S. typhimurium was investigated. This system was also designed for selective in-situ detection of B. anthracis spores in the presence a mixed microbial population. The system was composed of a reference sensor (devoid of phage), an E2 phage sensor (coated with phage specific to S. typhimurium) and a JRB7 phage sensor (coated with phage specific to B. anthracis spores). When cells/spores are bound to the specific phage-based ME biosensor surface, only the resonance frequency of the specific sensor changed. The instantaneous response of the multiple sensor system was studied by exposing the system to B. anthracis spores and S. typhimurium suspensions sequentially. A detection limit of 1.6×103 cfu/mL and 1.1×103 cfu/m was observed for JRB7 phage sensor and E2 phage sensor, respectively. Additionally, the performance of the system was also evaluated by exposure to a flowing mixture of B. anthracis spores (5×101-5×108 cfu/ml) in the presence of B. cereus spores (5×107 cfu/ml). Only the JRB7 phage biosensor responded to the B. anthracis spores. Moreover, there was no appreciable frequency change due to non-specific binding when other microorganisms (spores) were in the mixture. A detection limit of 3×102 cfu/mL was observed for JRB7 phage sensor. The results show that the multi-sensor detection system offers good performance, including good sensitivity, selectivity and rapid detection.

Magnetoelastic sensors exhibit a characteristic resonance frequency upon the application of an alternating magnetic field. In this research, magnetoelastic material was fabricated into micro-sized sensors coated with JRB7 phages to specifically detect Bacillus anthracis spores. Research had shown that the sensor's resonant frequency decreases linearly as its mass increases. As spores are captured, the mass increases. A high mass-sensitivity of up to 7.5 Hz/pg allowed this sensor's use in applications requiring accurate sensing of a very low concentration of B. anthracis spores. A B. anthracis spore weighs about 2 picograms. Two different sizes of sensors, 2000×400 μm and 1000×200 μm, were used in this study. The resonant frequency and the sensitivity of the sensors were found to vary under different magnitudes of DC biasing magnetic field. It was found that both the resonant frequency and the Q-value of the sensed signal increase with an increase of the magnitude of the DC magnetic field until they approach magnetic saturation. As the magnetic field was changed from low to high, it was observed that the signal amplitude increased to a maximum and then decreased to undetectable. Finally, real-time detection of B. anthracis spores is performed under the optimum magnetic field condition.

ABSTRACT Magnetoelastic materials are amorphous, ferromagnetic alloys that usually include a combination of iron, nickel, molybdenum and boron. Magnetoelastic biosensors are mass sensitive devices comprised of a magnetoelastic material that serves as the transducer and bacteriophage as the bio-recognition element. By applying a time varying magnetic field, the magnetoelastic sensor thin films can be made to oscillate, with the fundamental resonant frequency of oscillations depends on the physical dimensions and properties of the material. The change in the resonance frequency of these mass based sensors can be used to evaluate the amount of analyte attached on the sensor surface. Filamentous bacteriophage specific to S. typhimurium was used as a bio-recognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. The sensitivity of magnetoelastic materials is known to be dependent on the physical dimensions of the material. An increase in sensitivity from 159Hz/decade for a 2mm sensor to 770Hz/decade for a 1mm sensor and 1100Hz/decade for a 500micron sensor was observed. The sensors were characterized by scanning electron microscopy (SEM) analysis assayed biosensors to provide visual verification of frequency responses and an insight into the characteristics of the distribution of phage on the sensor surface. The magnetoelastic sensors immobilized with filamentous phage are suitable for specific and selective detection of target analyte in different media. Certain modifications to the measurement circuit resulted in better signal to noise ratios for sensors with smaller dimensions (L

This paper presents a multiple magnetoelastic (ME) biosensor system for in-situ detection of S. typhimurium and B. anthracis spores in a flowing bacterial/spore suspension (5 x 101 - 5 x 108 cfu/ml). The ME biosensor was formed by immobilizing filamentous phage (specific to each detection target) on the ME platforms. An alternating magnetic field was used to resonate the ME biosensor to determine its resonance frequency. When cells/spores are bound to a ME biosensor surface, the additional mass of the cells/spores causes a decrease in the resonance frequency of the biosensor. The detection system was composed of a control sensor, an E2 phage-based biosensor (specific to S. typhimurium) and a JRB7 phage-based biosensor (specific to B. anthracis spores). The frequency response curves of the ME biosensors as a function of exposure time were then measured and the detection limit of the ME biosensor was observed to be 5 x 103 cfu/ml. The results show that phage-based ME biosensors can detect multiple pathogens simultaneously and offer good performance, including good sensitivity and rapid detection.

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.

In this article, we report the results of an investigation into the performance of a wireless, magnetoelastic biosensor designed to selectively detect Salmonella typhimurium in a mixed microbial population. The Langmuir–Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors (2 × 0.4 × 0.015 mm). Bacterial binding to the antibody on the sensor surface changes the resonance parameters, and these changes were quantified as a shift in the sensor's resonance frequency. Response of the sensors to increasing concentrations (5 × 101 to 5 × 108 cfu/ml) of S. typhimurium in a mixture of extraneous foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) was studied. A detection limit of 5 × 103 cfu/ml and a sensitivity of 139 Hz/decade were observed for the 2 × 0.4 × 0.015 mm sensors. Binding kinetics studies have shown that the dissociation constant (Kd) and the binding valencies for water samples spiked with S. typhimurium was 435 cfu/ml and 2.33 respectively. The presence of extraneous microorganisms in the mixture did not produce an appreciable change in the biosensor's dose response behavior.

This paper presents the results of a study that investigates the effect of salt concentration on the immobilization of phages during the fabrication of magnetoelastic (ME) biosensors for the detection of Bacillus anthracis spores. The concentration of salt in the solution used to deposit and immobilize the phage is found to alter phage bundling characteristics and, hence, influence the ability of the biosensor to bind spores. ME biosensors were fabricated using different salt concentrations in the phage solution, and were then exposed to solutions containing known amounts of B. anthracis spores. The frequency response curves of the ME biosensors, were then measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of B. anthracis spores to the phage-coated ME biosensors. Transmission electron microscopy (TEM) was used to verify the structure of phage under different salt concentrations. Results showed that moderate concentrations of salt (420 mM NaCl) in the phage solution results in an optimal distribution of immobilized phages on the sensor surface and, hence, better binding of spores to the biosensor.

Novel mass-sensitive, magnetostrictive 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 or remote. These biosensors can thus be used in-situ for detecting pathogens and biological threat agents. In this work, we have used a magnetostrictive platform immobilized with a polyclonal antibody (the bio-molecular recognition element) to form a biosensor for the detection of Salmonella typhimurium . Upon exposure to solutions containing Salmonella typhimurium bacteria, the bacteria were bound to the sensor and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Responses of the sensors to different concentrations of S. typhimurium were recorded and the results correlated with those obtained from scanning electron microscopy (SEM) images of samples. Good agreement between the measured number of bound bacterial cells (attached mass) and frequency shifts were obtained. The longevity and specificity of the selected polyclonal antibody were also investigated and are reported.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

This article presents a contactless, remote sensing Salmonella typhimurium sensor based on the principle of magnetostriction. Magnetostrictive materials have been used widely for various types of sensor systems. In this work, the use of a magnetostrictive material for the detection of Salmonella typhimurium has been established. The mass of the bacteria attached to the sensor causes changes in the resonance frequency of the sensor. Filamentous bacteriophage was used as a probe order to ensure specific and selective binding of the bacteria onto the sensor surface. Thus changes in response of the sensor due to the mass added onto the sensor caused by specific attachment of bacteria can be monitored in absence of any contact to the sensor. The response of the sensor due to increasing concentrations (from 5x101 to 5x108 cfu/ml) of the bacteria was studied. A reduction in the physical dimensions enhances the sensitivity of these sensors and hence different dimensions of the sensor ribbons were studied. For a 2mm x 0.1mm x 0.02mm the detection limit was observed to be of the order of 104 cfu/mL and for a sensor of 1mm x 0.2mm x 0.02mm a reduced detection limit of 103 cfu/mL was achieved.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Magnetostrictive particles (MSPs) as biosensor platform have been developed recently. The principle of MSPs as sensor platform is the same as that of other acoustic wave devices, such as quartz crystal microbalance. In this paper, the fabrication, characterization and performance of phage-based MSP biosensors for detecting Bacillus anthracis spores are reported. A commercially available magnetostrictive alloy was utilized to fabricate the sensor platform. The phage was immobilized onto the MSPs using physical adsorption technology. The following performance of the phage-based MSP sensors will be presented: sensitivity, response time, longevity, specificity and binding efficacy. The performance of the sensors at static and dynamic conditions was characterized. The experimental results are confirmed by microscopy photographs. The excellent performance including high sensitivity and rapid response is demonstrated. More importantly, it is experimentally found that the phage-based MSP sensors have a much better longevity than antibody-based sensors.

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 102 to 109 CFU/ml. Detection limits of 5 × 103 CFU/ml, 105 CFU/ml and 107 CFU/ml were obtained for sensors with the size of 2 mm × 0.4 mm × 15 μm, 5 mm × 1 mm × 15 μm and 25 mm × 5 mm × 15 μm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.