Ramji S. Lakshmanan

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors.

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 1mg/ml BSA (blocking agent) onto the magnetoelastic resonators 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 sensors 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.

Detection of Salmonella typhimurium using phage-based magnetostrictive sensor

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.

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.

Phage-based magnetostrictive-acoustic microbiosensors for detecting bacillus anthracis spores

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.

Hydrazine leak detection using poly (3-hexylthiophene) thin film micro-sensor

Hydrazine is mostly used as a propellant in the control/propulsion system of missiles, spacecraft and satellites. However with its highly toxic and strong reducing nature, hydrazine is very dangerous to humans and the environment. In this research, a low cost, passive, and highly sensitive micro-sensor has been developed as an alarm device for real-time monitoring for the accidental release of hydrazine, and to insure the safety of personnel and the readiness of the system before lift-off. The micro-sensor is fabricated using standard microelectronic manufacturing techniques and is composed of interdigitated electrodes and a hydrazine-sensitive poly (3-hexylthiophene) (P3HT) thin film. When exposed to 1ppm of hydrazine gas, the compensation interaction between the reducing hydrazine gas and p-type doped P3HT leads to a five order magnitude increase in the resistance of the device. The sensor is capable of detecting hydrazine leaks from tens of ppb to tens of ppm concentration. The sensitivity of sensor increases with the increasing of hydrazine concentration and the decreasing of the polymer film thickness. A numerical simulation result based on the possible theoretical model is compared with the experimental data, which shows a good agreement.

The performance of a multi-sensor detection system based on phage-coated magnetoelastic biosensors

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.

Multiple Phage-Based Magnetoelastic Biosensors System for the Detection of Salmonella typhimurium and Bacillus anthracis Spores

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 10 1 - 5 x 10 8 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 10 3 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.

Chapter 6:Phage-based Pathogen Biosensors

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 phages 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.

Magnetoelastic Material as a Biosensor for the Detection of Salmonella Typhimurium

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

Thermalstability of Polyclonal Antibody to Salmonella typhimurium on a Magnetostrictive Sensor Platform

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.