Rajesh Guntupalli

Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes.

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoViva optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29 mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.

Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements.

Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.

Phage Langmuir monolayers and Langmuir-Blodgett films.

Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air-water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of ∼40 mN/m at 20 °C. The surface pressure-area (Π-A) isotherm possessed a shoulder at ∼7 × 10(4)nm(2)/phage particle, attributed to a change in phage orientation at the air-water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Π-A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air-water interface at low surface pressures with an excluded area per phage particle of 4.6 × 10(4)nm(2). At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5 × 10(3)nm(2)/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air-water interface at a constant surface pressure of 18 mN/m by Langmuir-Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Γ) in Langmuir-Blodgett (LB) films measured by SEM was consistent with that calculated independently from Π-A isotherms at the transfer surface pressure of 18 mN/m (Γ=23 phage particles/μm(2)). The 50 nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air-water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid.

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species (1,2). The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique (3). The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

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.

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.