Landscape Phage Library Research Articles

Understanding the interactions between bone mineral crystals and their binding peptides derived from filamentous phage

Hydroxyapatite (HAP) is the main inorganic component of natural bone and assembled along collagen fibers. HAP is formed under the regulation of bone proteins (BPs) including collagen and non-collagenous proteins (NCPs, such as osteocalcin, osteopontin, osteonectin, and bone sialoprotein) during biomineralization. However, the mechanism of the BPs regulating the HAP biomineralization is still unclear and some HAP-interacting domains on BPs have not yet been identified. To understand the interactions between HAP and the peptide motifs, similar to functional motifs on BPs, we employed a sidewall-displayed phage library, instead of a traditional tip-displayed phage library, to discover novel HAP-binding peptides. HAP-binding peptides were expressed as pVIII fusion proteins into the capsid of the filamentous fd phages. By a few rounds of biopanning of the landscape phage library against HAP, we identified multiple HAP-binding peptides. The affinity-selected peptide sequences were aligned to the sequences of BPs using a receptor ligand contacts (RELIC) program, RELIC/MATCH, which is a bioinformatics software that identifies protein-ligand interaction sites and herein discovers the putative HAP-binding domains on these BPs. By both confirming some known HAP-binding domains and identifying new HAP-binding domains, we clarified some mechanisms of HAP-BP interactions. Through a series of binding assays, we discovered that DSSTPSST peptide is the best HAP binder. Using a protein structure prediction model within the Rosetta software suite, we found that the best binding peptides had the lowest peptide-HAP interfacial energy, consistent with the binding assay studies. Simulations suggested that the stable turn-like structure owing to a proline residue in the DSSTPSST peptide enabled favorable electrostatic interactions between aspartate residues in the N-terminal domain and calcium ions in HAP. The phages that displayed DSSTPSST on the sidewall bound and aligned the HAP nanorods along its sidewall, forming HAP-decorated nanofibers. Through a freeze-casting approach, the HAP-decorated nanofibers were assembled into a macroscale scaffold that supported the proliferation of mesenchymal stem cells. Therefore, our work demonstrates a new understanding of HAP-peptide interactions that can be exploited to produce HAP-based biomaterials with potential applications in bone tissue engineering.

Combinatorial Avidity Selection of Mosaic Landscape Phages Targeted at Breast Cancer Cells—An Alternative Mechanism of Directed Molecular Evolution

Low performance of actively targeted nanomedicines required revision of the traditional drug targeting paradigm and stimulated the development of novel phage-programmed, self-navigating drug delivery vehicles. In the proposed smart vehicles, targeting peptides, selected from phage libraries using traditional principles of affinity selection, are substituted for phage proteins discovered through combinatorial avidity selection. Here, we substantiate the potential of combinatorial avidity selection using landscape phage in the discovery of Short Linear Motifs (SLiMs) and their partner domains. We proved an algorithm for analysis of phage populations evolved through multistage screening of landscape phage libraries against the MDA-MB-231 breast cancer cell line. The suggested combinatorial avidity selection model proposes a multistage accumulation of Elementary Binding Units (EBU), or Core Motifs (CorMs), in landscape phage fusion peptides, serving as evolutionary initiators for formation of SLiMs. Combinatorial selection has the potential to harness directed molecular evolution to create novel smart materials with diverse novel, emergent properties.

Selected landscape phage probe as selective recognition interface for sensitive total prostate-specific antigen immunosensor

The level of total prostate-specific antigen (t-PSA) is generally known as the key index of prostate cancer. Here, phage probes against t-PSA were selected from f8/8 landscape phage library. After three rounds of biopanning, four t-PSA-binding phage clones were isolated and identified by the DNA sequencing. Based on the phage capture assay, the phage clone displaying the fusion peptide ATRSANGM showed highest affinity and specificity against t-PSA. Subsequently, the t-PSA-specific phage was used as t-PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV) assay systems. Both assay methods showed high specificity and acceptable reliability for real serum samples analysis. By comparison, DPV method showed wider linear range (0.01–100 ng mL−1) and lower limit of detection (3 pg mL−1) than those (3.3–330 ng mL−1 and 1.6 ng mL−1) of ELISA. Moreover, DPV system showed smaller distinction to the authoritative method in real samples assay. Excitingly, the phage probe based DPV immunosensor showed high sensitivity for the detection of t-PSA and LOD achieved the pg mL−1 level, which was far lower than those values (usually above 0.1 ng mL−1) for reported immunosensors based on antibodies. Due to the biocompatibility, multivalency, stability, and high structural homogeneity, the t-PSA-specific landscape phage demonstrates as a novel specific interface in biosensors.

Sensitive colorimetric immunoassay of Vibrio parahaemolyticus based on specific nonapeptide probe screening from a phage display library conjugated with MnO2 nanosheets with peroxidase-like activity.

Pathogen detection continues to receive significant attention due to the harmful effects of pathogens on public health. Herein, specific nonapeptide-fusion proteins pVIII (pVIII fusion) were isolated from phage VQTVQIGSD (designated by the sequence of a fused foreign peptide), which was specifically screened from the f8/9 landscape phage library against Vibrio parahaemolyticus (V. parahaemolyticus) in a high-throughput way. The as-prepared V. parahaemolyticus-specific recognition element is cheaper and more available than antibodies. Further, a highly sensitive colorimetric immunoassay for V. parahaemolyticus was established using pVIII fusion as capture probes coupled with protein-templated MnO2 nanosheets (NSs) as signal probes. In the presence of a target bacterium, V. parahaemolyticus, a sandwich-type complex of pVIII fusion-V. parahaemolyticus-MnO2 NS@pVIII fusion was formed through specific recognition of pVIII fusion and V. parahaemolyticus. The signal probes (MnO2 NSs) could catalyze the reaction of 3,3',5,5'-tetramethylbenzidine and H2O2 to generate a colorimetric change. The proposed V. parahaemolyticus detection method demonstrated a wide detection range (20-104 colony-forming units (CFU) mL-1), low limit of detection (15 CFU mL-1), excellent selectivity, and high reliability for real marine samples, showing potential application in marine microbiological detection and control.

Gold nanoprobe functionalized with specific fusion protein selection from phage display and its application in rapid, selective and sensitive colorimetric biosensing of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is one of the most ubiquitous pathogens in public healthcare worldwide. It holds great insterest in establishing robust analytical method for S. aureus. Herein, we report a S. aureus-specific recognition element, isolated from phage monoclone GQTTLTTS, which was selected from f8/8 landscape phage library against S. aureus in a high-throughput way. By functionalizing cysteamine (CS)-stabilized gold nanoparticles (CS-AuNPs) with S. aureus-specific pVIII fusion protein (fusion-pVIII), a bifunctional nanoprobe (CS-AuNPs@fusion-pVIII) for S. aureus was developed. In this strategy, the CS-AuNPs@fusion-pVIII could be induced to aggregate quickly in the presence of target S. aureus, resulting in a rapid colorimetric response of gold nanoparticles. More importantly, the as-designed probe exhibited excellent selectivity over other bacteria. Thus, the CS-AuNPs@fusion-pVIII could be used as the indicator of target S. aureus. This assay can detect as low as 19CFUmL−1S. aureus within 30min. Further, this approach can be applicable to detect S. aureus in real water samples. Due to its sensitivity, specificity and rapidness, this proposed method is promising for on-site testing of S. aureus without using any costly instruments.

A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 − 2.0 × 108 cells mL−1), a low limit of detection (79 cells mL−1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

Landscape Phage Probes for Prostate Specific Antigen

Prostate‐specific antigen (PSA) is a cardinal marker for molecular diagnosis and proper management of prostate tumors. Therefore, development of specific probes for this marker is paramount for precise molecular diagnosis and targeted delivery of therapeutics to prostate cancer cells. Screening of the multibillion landscape libraries proffers a simple and economic approach for isolating target‐specific phage probes. Landscape phage libraries are collections of filamentous phages displaying random guest peptides on all 4,000 surface domains of the major coat protein. They serve as a rich source of bioselective materials that can be used in different areas of technology and medicine, including gene delivery, drug delivery and imaging of tumor cells. Novel highly specific and selective phage probes against prostate specific antigens were selected from 8‐mer library using immobilized PSA as targets. The selected clones were identified by nucleotide sequencing of their DNA and translation into proteins using software. Eight phage probes were isolated and tested for their selective interactions with PSA in comparison with control targets, two of the phage probes selectively interacted with PSA. The selected phage probes can serve as promising ligands for development of imaging and therapeutic devices precisely targeted to prostate tumors.

Bio-mimetic nanostructure self-assembled from Au@Ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy.

Nanomaterials with near-infrared (NIR) absorption have been widely studied in cancer detection and photothermal therapy (PTT), while it remains a great challenge in targeting tumor efficiently with minimal side effects. Herein we report a novel multifunctional phage-mimetic nanostructure, which was prepared by layer-by-layer self-assembly of Au@Ag heterogenous nanorods (NRs) with rhodamine 6G, and specific pVIII fusion proteins. Au@Ag NRs, first being applied for PTT, exhibited excellent stability, cost-effectivity, biocompatibility and tunable NIR absorption. The fusion proteins were isolated from phage DDAGNRQP specifically selected from f8/8 landscape phage library against colorectal cancer cells in a high-throughput way. Considering the definite charge distribution and low molecular weight, phage fusion proteins were assembled on the negatively charged NR core by electrostatic interactions, exposing the N-terminus fused with DDAGNRQP peptide on the surface. The fluorescent images showed that assembled phage fusion proteins can direct the nanostructure into cancer cells. The nanostructure was more efficient than gold nanorods and silver nanotriangle-based photothermal agents and was capable of specifically ablating SW620 cells after 10 min illumination with an 808 nm laser in the light intensity of 4 W/cm2. The prepared nanostructure would become an ideal reagent for simutaneously targeted optical imaging and PTT of tumor.

Specific Probe Selection from Landscape Phage Display Library and Its Application in Enzyme-Linked Immunosorbent Assay of Free Prostate-Specific Antigen

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Metastatic prostate cancer cell-specific phage-like particles as a targeted gene-delivery system

BackgroundOne of the cardinal requirements for effective therapeutic management of tumors is the selective delivery of cancer drugs to the right site by ligand-decorated nanomedicines. Screening of 2 × 109 clone landscape phage library provides a reliable avenue for generating protein ligands specific for tumor cells. It was shown that selective phage proteins derived from landscape phage libraries against breast and prostate cancer cells are able to navigate drug or siRNA loaded liposomes to corresponding cancer cells with minimal toxicity to non-neoplastic cells. In an alternative platform, glioma cell-specific phage proteins were used for assembling in vivo cancer-specific phage-like particles, named ‘phagemid infective particles’ as targeted gene-delivery vehicles.MethodsTo extend the panel of anticancer cell phages, we have screened a 2 × 109 clone landscape phage library f8/8 to select phage clones specific for metastatic prostate cancer cell PC-3M. The phage clones were characterized for their selective interaction with PC-3M cells using phage capture assay, immunofluorescence microscopy and electron microscopy. A prostate cancer selective phage was converted to phage-like particles harboring emerald green fluorescent protein.ResultsPhage clone EPTHSWAT (designated by the sequence of inserted peptide) was found to be most selective for PC-3M cells and was observed to internalize PC-3M cells as revealed by immunofluorescence microscopy and electron microscopy. Conversion of this phage to phage-like particles harboring emerald green fluorescent protein and the expression of emerald green fluorescent protein in the phage-like particles treated PC-3M cells showed potential of adoption of this phage-like particle in prostate cancer therapeutic gene delivery.ConclusionSuccessful employment of phage-like particles expressing emerald green fluorescent protein genes targeted to prostate cancer cells PC-3M confirms a prospect of their use for targeted delivery of therapeutic genes to cancer cells.

Targeted Delivery of siRNA into Breast Cancer Cells via Phage Fusion Proteins

Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

Fd Bacteriophage Coat Protein Structure Prediction and Design for the Assembly of Hydroxyapatite Nanorods and Bone Tissue Regeneration

Bone tissue engineering seeks to develop viable substitutes that repair and maintain the function of human bone tissue. To fabricate a bone scaffold that mimics extracellular matrix, we have assembled hydroxyapatite (HAp) nanorods and HAp-binding fd bacteriophages selected from landscape phage libraries against HAp powder (biopanning). The phage peptides for the top six HAp-binding phages have been analyzed further with a computational biomolecular suite Rosetta to deduce high-resolution chemical interactions occurring at the peptide-HAp interface. To simulate the structural environment of coat proteins, we have adsorbed each peptide to HAp (100) monoclinic crystal while considering its helix tilt angle and c-terminus distance away from the mineral surface. The incorporation of geometrical constraints improves sampling of the predicted structures and scoring of the best binding coat peptide among the top six peptides. The predicted structure of the best binding peptide reveals possible key residues and local conformations that contribute to favorable HAp-phage interface energy, which is important in building a HAp nanorod scaffold for bone regeneration.

Selection of PCB binding phages as potential biorecognition elements for food and environmental monitoring

In order to assess and avoid risks for both human and environmental health, there is a high need for cost-effective and sensitive detection systems suitable for the monitoring of chemical contaminants in food and the environment. Due to their high sensitivity and selectivity, affinity-based detection systems are interesting for monitoring tools. Antibodies have long been the most popular affinity-based recognition elements, however, recently different novel affinity-based recognition elements with improved characteristics, like specificity, stability and cost-efficiency, have gained attention. In the present study, one type of such novel affinity-based recognition elements, namely landscape phages were evaluated as substitute antibodies for detection of PCB. These phages with affinity for PCBs were selected from a landscape phage library, in which each phage displays an 8-mer peptide at all 4000 major coat proteins pVIII. Landscape phages have potential as biorecognition elements, because of their excellent characteristics, such as high affinity and specificity, fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and superior stability, which allow them to operate as elements of detector systems in complex environmental and food matrices. Phages with a high specificity for PCB106 were isolated from the landscape phage library. Using surface plasmon resonance (SPR) it was shown that the selected phages bind specifically PCB106 and do not bind PCB72 and PCB118. The dissociation constants (Kd) of the phage binding with PCB106 were in μM range. After further improvement of their binding activity, the specific PCB-binding phages can be used as biorecognition elements for food and environmental monitoring.

Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization.

Filamentous phage as a bacteria-specific virus can be conjugated with an anticancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells.

Landscape phage ligands for PC3 prostate carcinoma cells

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display technology with its high throughput capacity for the analysis of targeting ligands possessing specific binding properties represents a very attractive tool in the quest for molecular ligands. Also, current phage nanobiotechnology concepts allow the use of intact phage particles and isolated phage coat proteins per se as components of nanomedicines. Herein, we describe the use of two landscape phage libraries to obtain phage ligands against PC3 prostate carcinoma cells. Following a very stringent selection scheme, we were able to identify three phage ligands, bearing the fusion peptides, DTDSHVNL, DTPYDLTG and DVVYALSDD that demonstrated specificity and selectivity to PC3 cells based on target-association assays, microscopy and flow cytometry. The phage ligands and their fusion coat proteins can be used as navigating modules in both therapeutic and diagnostic approaches to prostate carcinoma.

Identification of Microtubule-Binding Domains on Microtubule-Associated Proteins by Major Coat Phage Display Technique

Microtubule is an important structural and functional component in cells. Microtubule-associated proteins (MAPs) are a class of proteins that can bind to microtubules and stabilize them to maintain their functions. However, not all the specific microtubule-binding domains on MAPs are clear. Here we report the study of microtubule-binding domains on MAPs from a new angle by biopanning a new type of phage-displayed random peptide library (called landscape phage library) against purified alpha- and beta-tubulins. In the landscape phage library, billions of fd-tet phage clones are present and a unique 9-mer peptide is fused to each of the approximately 3900 copies of major coat protein (pVIII) in each clone. The affinity-selected peptides derived from the biopanning were analyzed by the receptor ligand contacts (RELIC) suite of programs, which is a bioinformatics tool for combinatorial peptide analysis and identification of protein-ligand interaction sites. By using RELIC, the affinity-selected peptides were shown to have similarity with the sequences of two MAP families (MAP1 and MAP2/tau), thereby identifying putative microtubule-binding domains on these MAPs. The tubulin-binding affinity was also confirmed by using transmission electron microscopy (TEM) to characterize the interaction between affinity-selected tubulin-binding phage and tubulins. Our results confirm some known microtubule-binding domains and identify some new microtubule-binding domains and thus shed light into the mechanism of microtubule-MAPs interactions.

Diversity and censoring of landscape phage libraries.

Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd-tet (landscape libraries). They were obtained by replacement of amino acids 2-4 and 2-5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space.

Liposomes targeted by fusion phage proteins

Targeting of nanocarriers has long been sought after to improve the therapeutic indices of anticancer drugs. Here we provide the proof of principle for a novel approach of nanocarrier targeting through their fusion with target-specific phage coat proteins. The source of the targeted phage coat proteins are landscape phage libraries—collections of recombinant filamentous phages with foreign random peptides fused to all 4000 copies of the major coat protein. We exploit in our approach the intrinsic physicochemical properties of the phage major coat protein as a typical membrane protein. Landscape phage peptides specific for specific tumors can be obtained by affinity selection, and purified fusion coat proteins can be assimilated into liposomes to obtain specific drug-loaded nanocarriers. As a paradigm for inceptive experiments, a streptavidin-specific phage peptide selected from a landscape phage library was incorporated into ∼100-nm liposomes. Targeting of liposomes was proved by their specific binding to streptavidin-coated beads.

Detection of Bacillus anthracis spores in liquid using phage-based magnetoelastic micro-resonators

This paper presents the results of an investigation to detect Bacillus anthracis spores in liquids using microfabricated magnetoelastic particles (MEP). Free standing, magnetoelastic resonating particles, as small as 500 μm × 100 μm × 4 μm, have been made using microelectronics fabrication techniques. These magnetoelastic resonators feature wireless signal transmission and are suitable for use in air and liquids. Affinity-selected filamentous bacteriophage was employed as the bio-molecular recognition agent and was immobilized onto all surfaces of the resonators. This phage was selected from a landscape phage library specifically for binding B. anthracis spores, the causative agent for the disease anthrax. The biosensors were tested in B. anthracis spore solutions with concentrations from 103 to 108 cfu/ml. A detection limit of 103 cfu/ml, with a sensitivity of 6.5 kHz/decade was observed. Scanning electron microscopy (SEM) micrographs of the tested sensors were used for visual correlation of the sensors’ responses. To investigate specificity, the biosensors were exposed to mixed spore solutions containing B. anthracis Sterne, Bacillus cereus and Bacillus megaterium spore strains. The phage-coated biosensors were proven to have better binding preference to B. anthracis spores than other strains. The study on the binding kinetics have shown that the dissociation constant (Kd) and the binding valency for the sensor in B. anthracis spores solutions are 193 cfu/ml and 2.32.

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.