From Bits and Pieces to Whole Phage to Nanomachines: Pathogen Detection Using Bacteriophages.

The innate specificity of bacteriophages toward their hosts makes them excellent candidates for the development of detection assays. They can be used in many ways to detect pathogens, and each has its own advantages and disadvantages. Whole bacteriophages can carry reporter genes to alter the phenotype of the target. Bacteriophages can act as staining agents or the progeny of the infection process can be detected, which further increases the sensitivity of the detection assay. Compared with whole-phage particles, use of phage components as probes offers other advantages: for example, smaller probe size to enhance binding activity, phage structures that can be engineered for better affinity, as well as specificity, binding properties, and robustness. When no natural binding with the target exists, phages can be used as vehicles to identify new protein-ligand interactions necessary for diagnostics. This review comprehensively summarizes many uses of phages as detection tools and points the way toward how phage-based technologies may be improved.

Chemically immobilized T4-bacteriophage for specific Escherichia coli detection using surface plasmon resonance.

A bioassay platform using T4 bacteriophage (T4) as the specific receptor and surface plasmon resonance (SPR) as the transduction technique has been developed for the detection of Escherichia coli K12 bacteria. The T4 phages have been covalently immobilized onto gold surfaces using a self-assembled monolayer of dithiobis(succinimidyl propionate) (DTSP). Substrates of BSA/EA-T4/DTSP/Au prepared using different T4 phage concentrations have been characterized using scanning electron microscopy (SEM). The studies reveal that the use of DTSP results in a uniform binding of T4 phages onto the surface. The SPR analysis demonstrates that these BSA/EA-T4/DTSP/Au interfaces can detect the E. coli K12 with high specificity against non-host E. coli NP10 and NP30. Results of SEM and SPR studies indicate that the maximum host bacterial capture is obtained when 1.5 × 10(11) pfu ml(-1) concentration of T4 phages was used for immobilization. The surface of these chemically anchored phage substrates can be regenerated for repeated detection of E. coli K12 and can be used for detection in 7 × 10(2) to 7 × 10(8) cfu ml(-1) range. The results of these studies have implications for the development of online bioassays for the detection of various food and water borne pathogens using the inherent selectivity of bacteriophage recognition.

Oriented immobilization of bacteriophages for biosensor applications.

A method was developed for oriented immobilization of bacteriophage T4 through introduction of specific binding ligands into the phage head using a phage display technique. Fusion of the biotin carboxyl carrier protein gene (bccp) or the cellulose binding module gene (cbm) with the small outer capsid protein gene (soc) of T4 resulted in expression of the respective ligand on the phage head. Recombinant bacteriophages were characterized in terms of infectivity. It was shown that both recombinant phages retain their lytic activity and host range. However, phage head modification resulted in a decreased burst size and an increased latent period. The efficiency of bacteriophage immobilization with streptavidin-coated magnetic beads and cellulose-based materials was investigated. It was shown that recombinant bacteriophages form specific and strong bonds with their respective solid support and are able to specifically capture and infect the host bacterium. Thus, the use of immobilized BCCP-T4 bacteriophage for an Escherichia coli B assay using a phage multiplication approach and real-time PCR allowed detection of as few as 800 cells within 2 h.

Immobilization of bacteriophages on gold surfaces for the specific capture of pathogens.

Techniques for the chemical attachment of wild-type bacteriophages onto gold surfaces and the subsequent capture of their host bacteria have been developed. The surfaces were modified with sugars (dextrose and sucrose) as well as amino acids (histidine and cysteine) to facilitate such attachment. Non-specific attachment was prevented by using bovine serum albumin as blocking layer. Surfaces modified with cysteine (and cysteamine) followed by activation using 2% gluteraldehyde resulted in an attachment density of 18+/-0.15 phages/microm(2). This represented a 37-fold improvement compared to simply applying physisorption. Subsequently, the phage immobilized surfaces were exposed to the host E. coli EC12 bacteria and capture was confirmed by fluorescence microscopy. We obtained a bacterial capture density of 11.9+/-0.2/100 microm(2), a 9-fold improvement when compared to those on physically adsorbed phages. The specificity of recognition was confirmed by exposing similar surfaces to three strains of non-host bacteria. These negative control experiments do not show any bacterial capture. In addition, no capture of the host was observed in the absence of the phages.

Synthesis and characterization of TiOx nanowires using a novel silicon oxide support layer.

Titanium oxide (TiO(x)) nanowires of various compositions were synthesized using a vapour-liquid-solid growth process without the need of a titanium-based support layer. The process utilized liquid gold droplets, evaporated titanium vapours, and an argon-5% oxygen gas mixture at 1100 degrees C. A thin layer of thermally grown silicon oxide was used as the support layer. The resulting nanowires were 80-100 nm in diameter with lengths varying from 0.5 to 10 microm. Nanowires of TiO, as opposed to TiO(2), were formed in low pressures of argon-oxygen. Environmental pressure was found to greatly affect nanowire quality. Surface abrasion did not contribute to or inhibit the overall growth process. X-ray photon spectroscopy and Auger electron spectroscopy confirmed the presence of titanium. X-ray diffraction and transmission electron microscopy electron diffraction analysis confirmed the presence of rutile and face centred cubic crystal structures in both materials.

Specific detection of proteins using photonic crystal waveguides.

Specific detection of proteins is demonstrated using planar photonic crystal waveguides. Using immobilized biotin as probe, streptavidin was captured, causing the waveguide mode cut-off to red-shift. The device was shown to detect a 2.5 nm streptavidin film with a 0.86 nm cut-off red-shift. An improved photonic crystal waveguide sensor design is also described and shown to have a 40% improved bulk refractive index response.

Tailoring the microstructure and surface morphology of metal thin films for nano-electro-mechanical systems applications.

Metallic structural components for micro-electro-mechanical/nano-electro-mechanical systems (MEMS/NEMS) are promising alternatives to silicon-based materials since they are electrically conductive, optically reflective and ductile. Polycrystalline mono-metallic films typically exhibit low strength and hardness, high surface roughness, and significant residual stress, making them unusable for NEMS. In this study we demonstrate how to overcome these limitations by co-sputtering Ni-Mo. Detailed investigation of the Ni-Mo system using transmission electron microscopy and high-resolution transmission electron microscopy (TEM/HRTEM), x-ray diffraction (XRD), nanoindentation, and atomic force microscopy (AFM) reveals the presence of an amorphous-nanocrystalline microstructure which exhibits enhanced hardness, metallic conductivity, and sub-nanometer root mean square (RMS) roughness. Uncurled NEMS cantilevers with MHz resonant frequencies and quality factors ranging from 200-900 are fabricated from amorphous Ni-Mo. Using a sub-regular solution model it is shown that the electrical conductivity of Ni-Mo is in excellent agreement with Bhatia's structural model of electrical resistivity in binary alloys. Using a Langevin-type stochastic rate equation the structural evolution of amorphous Ni-Mo is modeled; it is shown that the growth instability due to the competing processes of surface diffusion and self-shadowing is heavily damped out due to the high thermal energies of sputtering, resulting in extremely smooth films.

An Lrp-type transcriptional regulator from Agrobacterium tumefaciens condenses more than 100 nucleotides of DNA into globular nucleoprotein complexes.

The PutR protein of Agrobacterium tumefaciens positively regulates expression of the putA gene in response to exogenous proline, resulting in the utilization of proline as a source of carbon and nitrogen. PutR activity required a region of DNA extending more than 106 nt upstream of the putA transcription start site. Purified PutR bound to this region with high degree of affinity and repressed expression of the putR promoter in vitro. PutR also activated the putA promoter in vitro in the presence of proline, though less strongly than in whole cells. PutR protected a DNA interval extending from nucleotides -30 to -140, but protected only one helical face over most of this interval, suggesting that it may bind only to this face of the DNA. The addition of proline caused a slight decrease in binding affinity and altered DNase I protection patterns along the entire length of the binding site. PutR-DNA complexes were found by atomic force microscopy to be globular rather than elongated. Although the DNA fragment in these complexes was 190 nm in length, the length of the visible DNA was only 150 nm, indicating that 40 nm of DNA (115 nt) must be condensed with protein. PutR caused a net bend of this binding site, and under some conditions, proline shifted the center of this bend by one helical turn.