I-Hsuan Chen

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.

Phage-Based Magnetoelastic Wireless Biosensors for Detecting Bacillus Anthracis Spores

A biosensor for the detection of biological warfare agents (Bacillus anthracis spores) was developed that combines the phage display technique with a magnetoelastic wireless detection platform. The affinity-based biosensor utilizes a phage-derived diagnostic probe as the biomolecular recognition element to capture target agents multivalently. Upon binding of the target agent to the sensor surface, the resonance frequency of the magnetoelastic biosensors decreases due to the additional mass of the target agent. Scanning electron microscopy was used to confirm binding of spores to the sensor surface. The sensitivity of the magnetoelastic acoustic sensor was tested to be 130 Hz per order of magnitude of spore concentration with a detection limit of 103 spores/ml. The specificity of the sensors was tested against spores of other closely related Bacillus species and a large preferential binding to Bacillus anthracis spores was observed. The longevity of the phage based biosensor was compared to traditional antibody based biosensors and found to exhibit a much longer life

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.

Magnetostrictive Microcantilever as an Advanced Transducer for Biosensors

The magnetostrictive microcantilever (MSMC) as a high-performance transducer was introduced for the development of biosensors. The principle and characterization of MSMC are presented. The MSMC is wireless and can be easily actuated and sensed using magnetic field/signal. More importantly, the MSMC exhibits a high Q value and works well in liquid. The resonance behavior of MSMC is characterized in air at different pressures and in different liquids, respectively. It is found that the Q value of the MSMC in water reaches about 40. Although the density and viscosity of the surrounding media affect the resonance frequency and the Q value of MSMC, the density has a stronger influence on the resonance frequency and the viscosity has a stronger influence on the Q value, which result in that, for MSMC in air at pressure of less than 100 Pa, the resonance frequency of MSMC is almost independent of the pressure, while the Q value increases with decreasing pressure. MSMC array was developed and characterized. It is experimentally demonstrated that the characterization of an MSMC array is as simple as the characterization of a single MSMC. A filamentous phage against Salmonella typhimurium was utilized as bio-recognition unit to develop an MSMC based biosensor. The detection of S. typhimurium in water demonstrated that the MSMC works well in liquid.

Landscape phage-based magnetostrictive biosensor for detecting Bacillus anthracis spores

A new diagnostic probe selected from a landscape phage library was used in combination with a free standing magnetostrictive platform to form a wireless biosensor with quick response and high accuracy. The immobilization of the phage-derived probes leads to a 3D biomolecular recognition layer that captures the target spores multivalently. After the phage-coated biosensors were exposed to suspensions of the target spores, the binding of spores to the sensors resulted in a decrease of the resonant frequency due to the additional mass of the attached spores. Scanning electron microscopy was used to relate the observed frequency changes to the actual number of spores bound to the sensor

Phage Fusion Proteins As Bioselective Receptors For Piezoelectric Sensors

Departments of Biological Sciences , Chemistry and Biochemistry , Anatomy, Physiology and Pharmacology , and Pathobiology , Auburn AL 36849 Author for correspondence: Tel. 707-423-7422; Fax 707-423-7267; e-mail: eric.olsen@travis.af.mil; current address: Clinical Investigations Facility, David Grant Medical Center, Travis Air Force Base, CA 94535 The views expressed in this article are those of the author, and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the U.S. Government.

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.

Micro-fabricated wireless biosensors for the detection of S. typhimurium in liquids

Food borne illnesses from the ingestion of S. typhimurium have been of primary concern due to their common occurrence in food products of daily consumption. In this paper, micron size, magnetoelastic (ME) biosensors for the detection of S. typhimurium were fabricated and tested in liquid solutions containing known concentrations of S. typhimurium cells. The biosensors are comprised of a ME sensor platform and immobilized bio-molecular recognition element (E2 phage) that has been engineered to bind the S. typhimurium multi-valently. The micron size ME sensor platforms were manufactured using microelectronics fabrication techniques. Phage was engineered at Auburn University and immobilized onto all surfaces of the sensor. The ME biosensor oscillates with a characteristic resonance frequency when subjected to a time varying magnetic field. Binding between the phage and bacteria, adds mass to the sensor that causes a shift in the sensors resonance frequency. Sensors with the dimension of 500x100x4 μm were exposed to S. typhimurium with increasing known concentrations ranging from 5 x101 to 5 x 107 cfu/ml. The ME biosensor exhibited high sensitivity and a detection limit better than 50 cfu/ml.

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.

Method for detection of a few pathogenic bacteria and determination of live versus dead cells

This paper presents a method for detection of a few pathogenic bacteria and determination of live versus dead cells. The method combines wireless phage-coated magnetoelastic (ME) biosensors and a surface-scanning dectector, enabling real-time monitoring of the growth of specific bacteria in a nutrient broth. The ME biosensor used in this investigation is composed of a strip-shaped ME resonator upon which an engineered bacteriophage is coated to capture a pathogen of interest. E2 phage with high binding affinity for Salmonella Typhimurium was used as a model study. The specificity of E2 phage has been reported to be 1 in 105 background bacteria. The phage-coated ME biosensors were first exposed to a low-concentration Salmonella suspension to capture roughly 300 cells on the sensor surface. When the growth of Salmonella in the broth occurs, the mass of the biosensor increases, which results in a decrease in the biosensors resonant frequency. Monitoring of this mass- induced resonant frequency change allows for real-time detection of the presence of Salmonella. Detection of a few bacteria is also possible by growing them to a sufficient number. The surface-scanning detector was used to measure resonant frequency changes of 25 biosensors sequentially in an automated manner as a function of time. This methodology offers direct, real-time detection, quantification, and viability determination of specific bacteria. The rate of the sensors resonant frequency change was found to be largely dependent on the number of initially bound cells and the efficiency of cell growth.