Phage-based Magnetoelastic Biosensors Research Articles

Rapid Pathogen detection By Surface Swab Sampling and Wireless Biosensing

This paper presents a method that combines surface swab sampling and wireless biosensing for rapid detection of foodborne pathogens on solid surfaces. The method can be applied to any surfaces, including the surface of food processing tables and fresh produce, for improving food safety. Surface swab sampling is an inexpensive and easy-to-use method of collecting pathogens. A desired area or the whole area of a target surface (if needed) can be swabbed to collect pathogens rapidly. In this study, we first investigated the efficiency of capture and release of a model pathogen, Salmonella Typhimurium, in swab sampling. A Salmonella-contaminated surface (2 × 2 cm2 flat polyethylene slab) was prepared by using a spot-inoculation method. Five drops of a Salmonella solution (20 ul/drop at concentrations from 5X108 cfu/ml to 5X103 cfu/ml) were inoculated on the polyethylene surface and allowed to dry in a biosafety cabinet for 2 hours. Swab sampling was then conducted at a controlled temperature and humidity to capture the pathogen. Finally, the swabs used were dipped in a TBS solution for releasing the captured Salmonella cells. Various swab tip materials were tested, including cotton, rayon, and nylon. The effects of swab force and sonication/vortexing on the capture and release efficiency were also investigated. The released Salmonella cells were finally detected and quantified instantaneously by using a wireless phage-based magnetoelastic biosensor. By comparing the quantities of the initially inoculated cells and detected cells, the efficiency of capture and release was calculated.

Detection of Salmonella on Bird Feces Contaminated Leafy Green Vegetables Using Phage-Based Magnetoelastic Biosensors

The outbreak of foodborne diseases caused by bacteria, such as O157:H7 E. coli and Salmonella has become a great concern in food safety[1]. The consumption of foodborne bacteria contaminated vegetables is one of the causes of foodborne disease. Vegetables can be contaminated by animal manure, for example, bird feces. Bird feces contain Salmonella which is a pathogen that lives in the intestines of the infected birds and is shed through feces. Bird could fly over or land on leafy green vegetables, such as spinach, iceberg lettuce and cabbage, leading to the potential bird feces contamination. Recently, USFDA found that the outbreak of Salmonella in peanut butter may be linked to the exposure of bird feces to the raw, unshelled peanuts that were stored outdoors where birds were observed flying over and landing on the in-shell peanuts[2]. Even though water washing can remove feces and reduce bacterial contamination, it is still difficult to eliminate bacteria at all. Therefore, rapid detection Salmonella in bird feces contaminated leafy green vegetables is required. The phage-based magnetoelastic biosensor has been developed to detect Salmonella on food surface [3, 4]. In the present work, we optimized the current detection procedures to detect Salmonella on bird feces contaminated vegetables using phage-based magenoelastic biosensors. Several parameters influencing the effectiveness of detection were optimized, such as, blocking agent concentration and incubation time. A protocol was developed to visualize Salmonella on biosensor surface to correlate the resonance frequency measurement of biosensors. Our detection protocol could contribute to screening the leafy green vegetables after harvesting to reduce effort and cost. Reference: 1. WHO: Food safety and foodborne illness. In Fact sheet no 237; 2007. 2. FDA: Inspectional Observations. Denver, CO; 2012. 3. Chai, Y, Horikawa, S, Wikle, HC, Wang, Z, Chin, BA, Surface-scanning coil detectors for magnetoelastic biosensors: A comparison of planar-spiral and solenoid coils. Applied Physics Letters. 2013. 103:p.173510. 4. Lakshmanan, RS, Guntupalli, R, Hu, J, Kim, D-J, Petrenko, VA, Barbaree, JM, Chin, BA, Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium. Journal of Microbiological Methods. 2007. 71:p.55-60.

Blocking Non-Specific Binding for Phage-Based Magnetoelastic Biosensors

The magnetoelastic (ME) biosensors are used to detect pathogen in fresh juice or milk by solenoid coil, and also developed for real-time, direct pathogen detection on food surfaces by surface-scanning coil. This paper presents blocking effect of different reagents on non-specific binding for detecting Salmonella typhimurium in apple juice using phage-based magnetoelastic biosensors. Three different blocking reagents of Bovine serum albumin, Superblock blocking buffer and blocker BLOTTO were used and evaluated. The results shows that blocker BLOTTO has the best blocking effect on non-specific binding.

Detection of methicillin-resistant Staphylococcus aureus using novel lytic phage-based magnetoelastic biosensors

To date, the magnetoelastic (ME) biosensor method has not been used for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we introduced, for the first time, the use of a lytic phage as a novel bio-recognition element for ME biosensors for the detection of MRSA. Optimal conditions for the binding of lytic phage to the ME sensor platform were determined by evaluating different phage concentrations (108–1012pfu/ml) and immobilization times (10, 30, 90, 270, 810, and 2430min). Scanning electron microscopy (SEM) analysis was carried out to examine the phages immobilized on the sensor platforms. The mean free length (MFL) between successive phages was calculated to determine the optimum immobilization time and phage concentration. Different concentrations of bovine serum albumin (BSA) were used to block the spaces between the phages on the sensors. The lytic phage-based ME biosensor method was used to detect MRSA in solution and limit of detection was compared with other biosensor methods. SEM images revealed that the lytic phage immobilized on the sensor platforms had slightly oval heads and relatively long tails. The optimum concentration and immobilization time for efficient binding of the lytic phage to the sensor was determined to be 1011pfu/ml and 30min, respectively, with an MFL of (917±0.080) nm. The optimum concentration of the blocking agent, BSA, was determined to be 1mg/ml, at which the maximum number of MRSA bound on the measurement sensor. Finally, the ME biosensor method was successfully used to detect MRSA with a limit of detection of 3.0logcfu/ml, which was approximately 2log lower than that of the surface plasmon resonance method.

Salmonella detection in Soil Using Phage-Based Magnetoelastic Biosensors

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The analytical comparison of phage-based magnetoelastic biosensor with TaqMan-based quantitative PCR method to detect Salmonella Typhimurium on cantaloupes

A phage-based magnetoelastic (ME) biosensor method for the detection of Salmonella Typhimurium was compared with a TaqMan-based quantitative real-time PCR (qPCR) method on cantaloupes. The selectivity of a qPCR primer set was investigated and different DNA extraction methods were compared. The limits of detection (LOD) of both detection methods were determined by serial inoculation of S. Typhimurium suspensions on cantaloupe surfaces. The repeatability of both detection methods was determined over three-day periods. Among 74 bacteria tested, only S. Typhimurium exhibited positive amplification, and other bacterial strains did not show any positive amplification. The DNeasy tissue kit showed the best performance for DNA purification due to the significantly superior threshold cycle (Ct) values, compared with either direct applications of qPCR without any extraction procedure or after employing a simple boiling method for extraction. The LOD and repeatability of qPCR and ME biosensor methods for S. Typhimurium detection were determined to be 1.35 ± 0.07 and 2.47 ± 0.50 log CFU/2 mm2 surface of cantaloupe, and 2.41 and 6.28%, respectively. The results demonstrate that the ME biosensor method is one of promising and practical on-site detection methods for S. Typhimurium on fresh produce.

Effects of Surface Morphologies of Fresh Produce on the Performance of Phage-Based Magnetoelastic Biosensors

This study investigated the effects of different surface morphologies on the performance of phage-based magnetoelastic (ME) biosensors for Salmonella Typhimurium detection on selected fresh produce. The ME biosensor is composed of an ME resonator platform and filamentous E2 phage for the specific recognition of S. Typhimurium. After SEM observation of the surface morphologies of tomatoes and spinach (adaxial/abaxial), S. Typhimurium was inoculated serially onto the surfaces to examine the attachment and distribution of bacterial cells on the surfaces. Finally, the linearity and detection limit of the ME biosensors detecting S. Typhimurium on food surfaces were tested. The morphologies of tomato and spinach surfaces were obviously different due to the existence of stomata and the differences in roughness and curvature. However, there were no obvious differences in attachment and distribution of bacterial cells on the surfaces. The resonant frequency shifts of measurement sensors increased linearly with an increase in the concentration of S. Typhimurium in contrast to relatively constant and negligible shifts of control sensors. Detection limit of the ME biosensor method was determined to be 1.87 log CFU/cm2 for the tomatoes, 1.72 log CFU/cm2 for the adaxial surface of spinach, and 2.16 log CFU/cm2 for the abaxial surface of spinach.

Evaluation of phage-based magnetoelastic biosensors for direct detection of Salmonella Typhimurium on spinach leaves

Abstract For a phage-based magnetoelastic (ME) biosensor method to become accepted for evaluating foodborne contamination, the method of use must be objectively compared, evaluated and validated against more widely accepted standard methods. In this study, a ME biosensor method was evaluated by comparison with TaqMan-based quantitative PCR (qPCR) for the detection of Salmonella Typhimurium on spinach leaves. Limit of detection (LOD) was determined after serial inoculation of S. Typhimurium on spinach leaves. The LOD for the ME biosensor was 2.17 and 1.94 log CFU/spinach for adaxial and abaxial surface, respectively. The LOD for qPCR was 1.37 log CFU/spinach. The repeatability of both methods was measured over a three day period by inoculation of S. Typhimurium on spinach leaves. The repeatability of the ME biosensor method was determined to be 6.38%, competitive with qPCR at 1.92%. For the direct comparison of both methods, 3 log CFU/spinach of S. Typhimurium was grown on two groups of 25 spinach leaves for 24 h and the number of S. Typhimurium was quantified by both methods. After confirmation of the growth, S. Typhimurium was positively detected and the quantified numbers were 5.79 ± 0.88 and 6.11 ± 0.26 log CFU/spinach for the ME biosensor and the qPCR method, respectively. This study demonstrated that the ME biosensor method was competitive and promising as an on-site and in-field detection method for the detection of pathogens.

Amorphous metallic glass biosensors

Amorphous metallic glasses possess a unique combination of magnetostriction and extremely soft magnetic properties making them ideal candidates for developing high performance biosensors. This paper presents the results of an investigation of novel free-standing magnetoelastic (ME) biosensors based on Fe–B amorphous metallic glasses. The principle of operation of wireless ME biosensors and the advantages of metallic glasses for their construction are discussed. The materials with the highest possible elastic–magnetic energy conversion efficiency are favored for sensor applications. Due to the unique properties of amorphous metallic glasses, acoustic wave sensors based on amorphous ME alloys with mild magnetostriction properties show a very high elastic–magnetic energy conversion efficiency. In this study, ME biosensor resonators 4 × 100 × 500 μm in size were manufactured by dual beam sputtering and non-traditional microelectronic fabrication techniques. E2 phage, genetically engineered to bind with Salmonella typhimurium, was immobilized on the sensor surfaces as a bio-recognition element. The detection of Salmonella in liquid using these phage-based ME biosensors was demonstrated. The ME biosensor exhibited high sensitivity and a detection limit better than 50 CFU/mL.

The effect of incubation time for Salmonella Typhimurium binding to phage-based magnetoelastic biosensors

This study presents an investigation of the effect of incubation time on the binding of Salmonella Typhimurium (Salmonella) with E2 phage-based magnetoelastic (ME) biosensors. After inoculation of Salmonella on a tomato surface, the attachment and existence of Salmonella on the tomato surface was observed using scanning electron microscopy (SEM). Measurement sensors with immobilized E2 phage and control sensors devoid of phage were placed on the inoculated tomato surface and incubated for various times. The extent of binding of Salmonella to the ME biosensors was determined by measuring the resonant frequency shift of the sensors and confirmed by SEM. The attachment of Salmonella on the tomato surface increased as the inoculation concentration increased. Significant increases in the frequency shifts for the measurement sensors were observed between 15 and 30min (P<0.001), exhibiting 3439±185 and 5312±248Hz, respectively. Further incubation times did not increase Salmonella binding with E2 phage whereas longer incubation times increased non-specific attachment from residual nutrients on the tomato surface. In contrast, there was either no or negligible binding of Salmonella on the control sensors. This study will contribute to the development of a practical protocol for the ME biosensor method.

Comparison of Phage-Based Magnetoelastic Biosensors with Taqman- Based Quantitative Real-Time PCR for the Detection of Salmonella typhimurium Directly Grown on Tomato Surfaces

A phage-based magnetoelastic (ME) biosensor method was compared with a TaqMan-based quantitative real- time PCR (Q-PCR) method for the detection of Salmonella typhimurium on tomato surfaces. This ME biosensor method utilizes magnetoelastic resonators coated with E2 filamentous phage to bind with and measure the concentration of S. typhimurium . In this study, standard curves, correlations, and limits of detection (LOD) for the ME biosensor and Q-PCR methods were determined by inoculating tomato surfaces with S. typhimurium suspensions in concentrations ranging from 1 to 8 log CFU/tomato. The LOD for the ME biosensor method and Q-PCR were 3 and 2 log CFU/tomato, respectively. In a direct comparison of the detection methods, S. typhimurium suspensions (3 log CFU/tomato) were inoculated on 65 tomato surfaces, then incubated at 37°C and 100% RH for 24 h. After 24 h, S. typhimurium was positively detected by both methods and the quantified concentrations were nearly the same, (6.35 ± 2.03) and (6.34 ± 0.17) log CFU/tomato respectively for the ME biosensor method and the Q-PCR method, which were significantly greater than the concentration determined by the BGS-plate count method (5.33 ± 0.21). Scanning electron microscopy (SEM) was used to confirm the growth of S. typhimurium on the tomato surfaces and the binding of S. typhimurium on the measurement sensors. This study demonstrated that the ME biosensor method was robust and competitive with Q-PCR for S. typhimurium detection on fresh produce.

The effect of incubation temperature on the binding of Salmonella typhimurium to phage-based magnetoelastic biosensors

The objective of this study is to evaluate the effect of incubation temperature on the binding of Salmonella typhimurium with E2 phage magnetoelastic (ME) biosensors. S. typhimurium was inoculated on tomato surfaces and the attachment of S. typhimurium was observed using scanning electron microscopy (SEM). Measurement biosensors, immobilized with E2 phage, and control sensors devoid of E2 phage were placed on the inoculated tomato surface and incubated at different temperatures for a fixed time in order to determine the effect of incubation temperature on the binding. After incubation, the resonant frequencies of both measurement and control sensors were measured using a network analyzer. The binding of S. typhimurium with E2 phage on both types of sensors was confirmed by SEM analysis. The SEM images of the inoculated tomato surfaces confirmed that S. typhimurium attached across the surface, however, its distribution was not uniform. After placement and incubation of the ME biosensors on the spiked tomato surfaces, the resonant frequencies of the ME biosensors were measured. The resonant frequency shifts in the measurement sensors increased as temperature was increased up to 35 °C, while the resonant frequency shifts of the control sensors were small and relatively constant across the entire range of incubation temperatures. There were no significant differences between incubation temperatures of 20, 25, 30, 35, 40, 45, and 50 °C in measurement sensors ( P > 0.05). SEM images of the measurement sensors confirmed that increasing resonant frequency shifts were caused by increasing numbers of bound S. typhimurium cells to the measurement sensors. This is contrasted with none or negligible binding of S. typhimurium cells on the control sensors. Overall, the optimum incubation temperature was chosen as 30 °C. This result will contribute to the development of a practical ME biosensor protocol for in situ detection of S. typhimurium on fresh produce .