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.

Preservation of bacteria in natural polymers.

A new inexpensive and simple method for preserving microorganisms has been developed. Natural polymers of acacia gum and pullulan were used to preserve model bacteria Escherichia coli and Bacillus subtilis via immobilization and storage under various conditions. Formulation of E. coli and B. subtilis in acacia gum significantly increased the viability of both cultures during desiccation at 40 degrees C as well as during the storage at various temperatures and relative humidity. In the ranges of temperatures and humidity used in experiments, the high humidity affected the viability of E. coli more than high temperature. Thermodynamic parameters for E. coli thermal degradation were used for quantification of results and characterization of the preservation process. Viability of B. subtilis in acacia gum polymer was not significantly changed during the storage in the temperature and humidity experiments. The number of viable B. subtilis recovered after storage in pullulan, and in PBS under various humidity conditions was 1-2 logs less in comparison with the number of cells before storage. It was found that acacia gum provides better protection than pullulan for both bacteria during the preservation process.

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium.

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.

A magnetoelastic resonance biosensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium.

Mass-sensitive, magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless and remote. A biosensor for the detection of Salmonella typhimurium was constructed by immobilizing a polyclonal antibody (the bio-molecular recognition element) onto the surface of a magnetostrictive platform. The biosensor was then exposed to solutions containing S. typhimurium bacteria. Binding between the antibody and antigen (bacteria) occurred and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Sensors with different physical dimensions were exposed to different concentrations of S. typhimurium ranging from 10(2) to 10(9)CFU/ml. Detection limits of 5x10(3) CFU/ml, 10(5) CFU/ml and 10(7) CFU/ml were obtained for sensors with the size of 2 mmx0.4 mmx15 microm, 5 mmx1 mmx15 microm and 25 mmx5 mmx15 microm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.

Affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of Salmonella typhimurium.

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.

Evaluation of agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses.

Campy-Cefex, a modification of Campy-Cefex, modified charcoal cefoperazone deoxycholate (mCCDA), Karmali, CAMPY, and Campy-Line agars were evaluated for their efficiency to isolate and enumerate Campylobacter spp. from poultry carcass rinses. Campy-Cefex and its modification produced the best results but were statistically similar to CAMPY, mCCDA, and Karmali.

Landscape phage probes for Salmonella typhimurium.

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.