Phage-based surface plasmon resonance strategies for the detection of pathogens

We start by reviewing the basic principles and recent advances in biosensing technologies using optical, electrochemical and acoustic platforms for phage-based diagnostics. Although much notable work has been done, a low cost, specific, sensitive optical method for detecting low concentrations of pathogens, in a few minutes, has not been established. We conclude from the limited body of work on the subject that improving immobilization strategies and finding more suitable phage recognition elements would allow for a more sensitive approach. Our aim was to better describe the attachment process of MRSA specific phages on gold surfaces, and the subsequent biodetection of their bacterial hosts by surface plasmon resonance (SPR).;Surface plasmon resonance was then used for the real-time monitoring of the attachment of MRSA bacteriophages to gold, using several immobilization methods[2]. We found that mixed self-assembled monolayers (SAMs) of L-cysteine and MUA permitted oriented positioning of the phages, thus preserving their biofunctionality and their bacterial lysing efficiency. This was due to the formation of uniform cavity islands on the gold surfaces, permitting an oriented positioning of the phages, thus better exposing their recognition proteins towards the medium containing the bacterial hosts.;T4 bacteriophages were then used to detect E. coli, while a novel, highly specific phage was isolated, characterized and used to detect MRSA[3]. We found that our technique, combined with the use of SPR permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 103 colony forming units/milliliter (CFU/mL), in less than 20 minutes.;We then turned our attention towards the differential detection of community-acquired MRSA (CA-MRSA), hospital-acquired MRSA (HA-MRSA), methicillin susceptible S. aureus (MSSA), and borderline resistant oxacillin-resistant S. aureus (BORSA), using SPR[4]. We studied two hundred fifty Staphylococcus aureus clinical isolates to determine their susceptibilities to â- lactam antibiotics. A surface plasmon resonance (SPR) biosensor was used to differentiate among CA-MRSA, HA-MRSA, BORSA and MSSA strains by specifically detecting PBP2a, an altered penicilling binding proteins that confers resistance to S. aureus strains, on whole bacterial cells, without labeling, without recourse to PCR or enrichment steps. We found that the system permits, specific detection of pathogens for concentrations as low as 10 CFU/mL. This approach has the advantages of being simple and rapid, allowing for identification of resistant strains of Staphylococcus aureus up to 48 hours earlier than conventional microbiological techniques. This method could have a significant impact on hospital costs, effective infection control, and patient mortality.;With the knowledge that the adsorption characteristics of thiol-containing molecules are necessary for applications involving the attachment of recognition elements to a functionalized surface, we start by providing comparative details on the kinetics of self-assembly of L-cysteine and 11-mercaptoundecanoic acid (MUA) monolayers on gold using SPR[1]. Our purpose, in carrying out these measurements was to establish each molecule's validity and applicability as a linker element for use in biosensing. We find that monolayer formation, for both L-cysteine and MUA, is described by the Langmuir isotherm at low concentrations only. For L-cysteine, both the amine and thiol groups contribute to the initial attachment of the molecule, followed by the replacement of the amine-gold complexes initially formed with more stable thiol-gold complexes. The reorganization of L-cysteine creates more space on the gold surface, and the zwitterionic form of the molecule permits the physisorption of a second layer through electrostatic interactions. On the other hand, MUA deposits randomly onto the surface of gold as a SAM and slowly reorganizes into a denser, vertical state.;Finally, we offer a new perspective on the attachment of phages to gold nanoparticles[5] for enhanced SPR detection. We report the synthesis and characterization of gold-bacteriophage hybrids multifunctional scaffold with great potential for nanotechnologically-based biomedical applications, such as localized SPR. Gold nanoparticles (AuNPs), stabilized (PEGylated) using heterobifunctional polyethylene glycol (PEG), were coupled to methicillin-resistant S. aureusspecific phagesand studied by transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The role of the interface, and the covalent coupling chemistry employed to attach the phages to the gold nanoparticles, have been delineated and successful attachment of phages to AuNPs was confirmed by the presence of amide between the primary amines of the phage and the carboxylic acid terminal groups of the NPs, and by the formation of strong intermolecular hydrogen bonds between carboxyl and amine species, as shown by N1s and O1s core level shifts. The use of these nanoparticle-phage hybrids can be extended to the targeted separation of specific bacteria from heterogeneous samples, as well as a wide range o biotechnological applications, such as labels for enhanced fluorescence and dark-field microscopy, and surface-enhanced Raman scattering detection[6]. (Abstract shortened by UMI.).