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ABSTRACT: We selected from a landscape phage library probes that bind preferentially Salmonella typhimurium cells compared with other Enterobacteriaceae. The specificity of the phage probes for Salmonella typhimurium was analyzed by the phage-capture test, the enzyme-linked immunosorbent assay (ELISA) and the precipitation test. Interaction of representative probes with Salmonella typhimurium was characterized by fluorescence-activated cell sorting (FACS), and fluorescent, optical and electron microscopy. The results show that the landscape phage library is a rich source of highly specific and robust probes for Salmonella typhimurium suitable for long-term use in continuous monitoring devices and biosorbents.
11/2012;
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ABSTRACT: Affinity-selected filamentous bacteriophage was induced to spherical forms ("spheroids") by chloroform treatment and deposited to piezoelectric transducers by Langmuir-Blodgett to prepare biosensors for the detection of streptavidin and S. typhimurium. ELISA confirmed affinity-selected phage specificity for streptavidin. Spheroid induction was optimized to achieve greatest conversion yields as a function of solvent exposure time and concentration. Results from whole-virion agarose gel electrophoresis indicated 27-fold phage aqueous dilutions mixed with equal volumes of chloroform for 60 s at room temperature was adequate. Phage conversion to spheroids with subsequent binding to S. typhimurium was confirmed by transmission electron microscopy. Spheroids for streptavidin and S. typhimurium prepared as either pure monolayers of phage coat proteins or proteins reconstituted with phospholipids were evaluated by isotherm, elasticity, and transfer ratio analysis. Results showed that spheroids combined with phospholipids produced a phage coat monolayer possessing higher elasticity and transfer ratios than monolayers of phage coat proteins alone, resulting in spatially superior deposition to substrates and subsequent firm binding of S. typhimurium that followed mass theory for piezoelectric transducers. Scanning electron microscopy confirmed binding of streptavidin-coated beads and S. typhimurium to prepared biosensors. In summary, spheroid-based sensors could be an effective analytical method for detecting and monitoring quantitative changes of bacterial agents under any conditions that warrant their recognition.
11/2012;
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Shin Horikawa,
Deepa Bedi,
Suiqiong Li,
Wen Shen,
Shichu Huang, I-Hsuan Chen,
Yating Chai,
Maria L Auad,
Michael J Bozack,
James M Barbaree,
Valery A Petrenko,
Bryan A Chin
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ABSTRACT: 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.
Biosensors & bioelectronics 10/2010; 26(5):2361-7. · 5.43 Impact Factor
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ABSTRACT: Filamentous phage, such as fd used in this study, are thread-shaped bacterial viruses. Their outer coat is a tube formed by thousands equal copies of the major coat protein pVIII. We constructed libraries of random peptides fused to all pVIII domains and selected phages that act as probes specific for a panel of test antigens and biological threat agents. Because the viral carrier is infective, phage borne bio-selective probes can be cloned individually and propagated indefinitely without needs of their chemical synthesis or reconstructing. We demonstrated the feasibility of using landscape phages and their stripped fusion proteins as new bioselective materials that combine unique characteristics of affinity reagents and self assembling membrane proteins. Biorecognition layers fabricated from phage-derived probes bind biological agents and generate detectable signals. The performance of phage-derived materials as biorecognition films was illustrated by detection of streptavidin-coated beads, Bacillus anthracis spores and Salmonella typhimurium cells. With further refinement, the phage-derived analytical platforms for detecting and monitoring of numerous threat agents may be developed, since the biodetector films may be obtained from landscape phages selected against any bacteria, virus or toxin. As elements of field-use detectors, they are superior to antibodies, since they are inexpensive, highly specific and strong binders, resistant to high temperatures and environmental stresses.
09/2007;
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ABSTRACT: 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 10<sup>3</sup> 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
IEEE Sensors Journal 04/2007; · 1.52 Impact Factor
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ABSTRACT: Proof-in-concept biosensors were prepared for the rapid detection of Salmonella typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers. Quantitative deposition studies indicated that approximately 3 x 10(10)phage particles/cm(2) could be irreversibly adsorbed for 1 h at room temperature to prepare working biosensors. The quality of phage deposition was monitored by fluorescent microscopy. Specific-bacterial binding resulted in resonance frequency changes of prepared sensors, which were evaluated using linear regression analysis. Sensors possessed a rapid response time of <180 s, had a low-detection limit of 10(2)cells/ml and were linear over a range of 10(1)-10(7)cells/ml with a sensitivity of 10.9 Hz per order of magnitude of S. typhimurium concentration. Viscosity effects due to increasing bacterial concentration and non-specific binding were not significant to the piezoelectric platform as confirmed by dose-response analysis. Phage-bacterial binding was confirmed by fluorescence and scanning electron microscopy. Overall, phage may constitute effective bioreceptors for use with analytical platforms for detecting and monitoring bacterial agents, including use in food products and possibly biological warfare applications.
Biosensors and Bioelectronics 02/2006; 21(8):1434-42. · 5.60 Impact Factor
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ABSTRACT: 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
Sensors, 2005 IEEE; 12/2005
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ABSTRACT: We selected from landscape phage library probes that bind preferentially Salmonella typhimurium cells compared with other Enterobacteriaceae. The specificity of the phage probes for S. typhimurium was analyzed by the phage-capture test, the enzyme-linked immunosorbent assay (ELISA), and the precipitation test. Interaction of representative probes with S. typhimurium was characterized by fluorescence-activated cell sorting (FACS), and fluorescent, optical and electron microscopy. The results show that the landscape phage library is a rich source of specific and robust probes for S. typhimurium suitable for long-term use in continuous monitoring devices and biosorbents.
Journal of Microbiological Methods 11/2005; 63(1):55-72. · 2.09 Impact Factor
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ABSTRACT: Recent use of Bacillus anthracis spores as a bioweapon has highlighted the need for a continuous monitoring system. Current monitoring systems rely on antibody-derived probes, which are not hardy enough to withstand long-term use under extreme conditions. We describe new, phage-derived probes that can be used as robust substitutes for antibodies.
From a landscape phage library with random octapeptides displayed on all copies of the major phage coat protein of the phage fd-tet, we selected clones that bound to spores of B. anthracis (Sterne strain). ELISA, micropanning, and coprecipitation assays were used to evaluate the specificity and selectivity with which these phage bound to B. anthracis spores.
Peptides on the selected clones directed binding of the phage to B. anthracis spores. Most clones exhibited little or no binding to spores of distantly related Bacillus species, but some binding was observed with spores of closely related species. Our most specific spore-binding phage displayed a peptide EPRLSPHS (several thousand peptides per phage) and bound 3.5- to 70-fold better to spores of B. anthracis Sterne than to spores of other Bacillus species.
The selected phage probes bound preferentially to B. anthracis Sterne spores compared with other Bacillus species. These phage could possibly be further developed into highly specific and robust probes suitable for long-term use in continuous monitoring devices and biosorbents.
Clinical Chemistry 11/2004; 50(10):1899-906. · 7.91 Impact Factor
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ABSTRACT: A micro-scale, free standing, wireless biosensor has been developed using magnetoelastic particles composed of an amorphous iron–boron binary alloy. Upon the application of an external magnetic field, these particles exhibit a characteristic resonance frequency, determined by their size and mass, due to the phenomena of magnetoelasticity. The particles are produced using the microelectronic fabrication techniques of photolithography and physical vapor deposition (sputtering). The biosensor is formed by coating the magnetoelastic particle with a thin layer of gold and immobilizing a biomolecular recognition element (bacteriophage) on the surfaces. Bacteriophage genetically engineered to bind Bacillus anthracis spores was used in this set of experiments as the detection probe. Once these targeted spores come into contact with the biosensor, the phage will bind selectively with only that pathogen, thereby increasing the particle's mass and causing a shift in the resonance frequency. Due to the magnetic nature of the sensing platform, this resonance frequency shift may be detected remotely by a wireless scanning device, presenting a distinct advantage over other techniques. A good correlation between the actual number of spores bound to the sensors and the calculated attached mass, based upon resonance frequency shifts, was obtained from the experiments.
Sensors and Actuators A: Physical.
