Songtao Du

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.

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.

The combination of magnetoelastic (ME) wireless biosensing with surface swab sampling

To perform rapid sensing of pathogens on the surface of food or food preparing plates, ME wireless biosensing system was combined with surface swab sampling techniques in this research. The ME biosensors which consist of ME resonators E2 phage was generally used for Salmonella typhimurium direct detections on the surfaces. E2 phage used in this research was designed for Salmonella typhimurium specific binding. Instead of measuring one spot at a time, the desired area or the whole area of a target surface can be swabbed for the inexpensive, rapid and easy-to-use pathogen collections. In this study, we first investigated the efficiency of capture and release of a model pathogen, Salmonella Typhimurium, by swab sampling on wet or dry surfaces. Plate counting was used to identify the recovery rates. The efficiency of capture and release was calculated and compared between various kinds of swabs which were composed of different tip materials, including cotton, rayon, and nylon-flocked ones.

Direct, surface-scanning detection of pathogenic bacteria using a wireless biosensor

This paper investigates the accuracy of surface-scanning measurement of a wireless magnetoelastic (ME) biosensor for direct pathogen detection on solid surfaces. The model experiments were conducted on the surface of a at polyethylene (PE) plate. An ME biosensor (1 mm x 0.2 mm x 30 µm) was placed on the PE surface, and a surface-scanning detector was aligned to the sensor for wireless resonant frequency measurement. The position of the detector was accurately controlled by using a motorized three-axis translation system (i.e., controlled X, Y, and Z positions). The results showed that the resonant frequency variations of the sensor were -125 to +150 Hz for X and Y detector displacements of ± 600 µm and Z displacements of +100 to +500 µm. These resonant frequency variations were small compared to the sensors initial resonant frequency (˂ 0.007% of 2.2 MHz initial resonant frequency) measured at the detector home position, indicating high accuracy of the measurement. In addition, the signal amplitude was, as anticipated, found to decrease exponentially with increasing detection distance (i.e., Z distance). Finally, additional experiments were conducted on the surface of cucumbers. Similar results were obtained.

Isolation of highly selective phage-displayed oligopeptide probes for detection of listeria monocytogenes in ready-to-eat food

Listeria monocytogenes is the major etiologic agent for foodborne Listeriosis in humans from consumption of readyto- eat (RTE) food. According to Center for Disease Control and Prevention, an estimated 1,600 people contract Listeriosis each year with approximately 260 deaths. This high rate of mortality has alerted the Food Safety Inspection and Services to release the Notice 23-99, Instructions for Verifying the L. monocytogenes Reassessment, on August 3, 1999 for their inspectors. According to the FDA’s Bacterial Analysis Manual Chapter 10, L. monocytogenes in RTE food samples is detected via microbiological culture-based tests, qPCR, pulsed-field gel electrophoresis, and other alternative methods. Unfortunately, these methods are time consuming (48-72 hours) and require dedicated laboratory facility. Thus, to develop a real-time L. monocytogenes biosensor, we isolated L. monocytogenes specific oligopeptides displayed on bacteriophages using modified biopanning procedures. In order to account for major temperature dependent morphological alterations of L. monocytogenes at 4°C versus 37°C, we used bacterial cells adapted to either temperature as the target in our biopanning. To date, we have isolated several candidate probes that can recognize either cold-adapted, warm-adapted L. monocytogenes cells, or both types of bacterial cells. Our isolated probes will be used on the magnetoelastic biosensor platforms for real-time detection of L. monocytogenes in RTE foods stored at 4°C or in samples/fluids for bacterium adapted to human body temperature.

Capture and identification of Salmonella Typhimurium from large volumes of water using phage filter

In this paper, a novel device named as phage filter is designed and presented to capture and identify a small number of Salmonella Typhimurium cells from large volumes of water. This phage filter is constructed from a filter chamber, filter frames on a spindle, strip-shape magnetoelastic filter elements, and a spinning speed control unit. The filter elements are made from Metglas 2826MB and coated with a specifically designed phage that only binds with Salmonella Typhimurium. These phage-coated filter elements can be held and arranged on the filter frames by magnetic force produced from couples of permanent magnets in the frame. Layers of filter frames are fixed on the spindle. The spindle with filter frames and filter elements can spin in the filter chamber and the spinning speed can be continuously adjusted. When the filter works, the tested water passes through the filter frame, and Salmonella Typhimurium cells striking on the filter elements can be bound by the phage on the element surfaces and removed from the tested water.

Capture of bacterial pathogens in liquid streams by multiple layers of phage based bio-molecular filter

Foodborne illness is a common public health problem because food can be contaminated with pathogens at any point in the farm-to-table continuum. This paper presents a method of capturing a quantity of a specific bacterial pathogen in a large volume of liquid using a biomolecular recognition filter. The filter consists of support frames made of a soft magnetic material and solenoid coils for magnetization/demagnetization of the frames. This filter is a planar, multi-layered arrangement of strip-shaped, phage-immobilized magnetoelastic (ME) biosensors that are magnetically held and arrayed on the filter frames. As a large volume of liquid passes through the biomolecular filter, the pathogen of interest is captured by the phage immobilized ME biosensors. This biomolecular filter is designed to capture a specific pathogen and allow non-specific debris to pass, thus avoiding a common clogging issue in conventional bead filters. In this work, single layer, double layers and triple layers of filter were test to capture Salmonella Typhimurium in a large volume of water. The effects of multiplication of filter layers on Salmonella capture efficiency will be discussed.

Automated surface-scanning detection of pathogenic bacteria on fresh produce

This paper investigates the effects of surface-scanning detector position on the resonant frequency and signal amplitude of a wireless magnetoelastic (ME) biosensor for direct pathogen detection on solid surfaces. The experiments were conducted on the surface of a flat polyethylene (PE) plate as a model study. An ME biosensor (1 mm × 0.2 mm × 30 μm) was placed on the PE surface, and a surface-scanning detector was brought close and aligned to the sensor for wireless resonant frequency measurement. The position of the detector was accurately controlled by using a motorized three-axis translation system (i.e., controlled X, Y, and Z positions). The results showed that the resonant frequency variations of the sensor were -125 to +150 Hz for X and Y detector displacements of ±600 μm and Z displacements of +100 to +500 μm. These resonant frequency variations were small compared to the sensors initial resonant frequency (< 0.007% of 2.2 MHz initial resonant frequency) measured at the detector home position, indicating high accuracy of the measurement. In addition, the signal amplitude was, as anticipated, found to decrease exponentially with increasing detection distance (i.e., Z distance). Finally, additional experiments were conducted on the surface of cucumbers. Similar results were obtained.

Highly sensitive surface-scanning detector for the direct bacterial detection using magnetoelastic (ME) biosensors

This paper demonstrates a highly sensitive surface-scanning detector used for magnetoelastic (ME) biosensors for the detection of Salmonella on the surface of a polyethylene (PE) food preparation surface. The design and fabrication methods of the new planar spiral coil are introduced. Different concentrations of Salmonella were measured on the surface of a PE board. The efficacy of Salmonella capture and detection is discussed.

Detection of salmonella on globe fruits using pulse excited magnetoelastic biosensors

This paper describes the results of a research project to investigate magnetoelastic (ME) biosensors actuated with a pulse excitation to measure the concentration of Salmonella Typhimurium of globe fruits. The ME biosensors are based on an acoustic wave resonator platform that is a freestanding (free-free) thin ribbon of magnetostrictive material with a lengthto- width ratio of 5:1. A biorecognition probe coated on the surface of the resonator platform binds with a targeted pathogen, i.e. E2 phage that binds with S. Typhimurium. The biosensor was actuated to vibrate longitudinally such that the resonant frequency depended primarily on the length of sensor and its overall mass. A pulsed excitation and measurement system was used to actuate micron scale ME biosensors to vibrate. The biosensor responds in a ring-down manner, a damped decay of the resonance amplitude, from which the resonant frequency was measured. An increase in mass due to the binding of the target pathogen resulted in a decrease in the resonant frequency. The pulsed excitation and measurement system that was developed under this effort and the characterization of its performance on the measurement of Salmonella concentrations on globe fruits is described.