Development of a SERS-Based Rapid Vertical Flow Assay for Point-of-Care Diagnostics.

Point-of-care (POC) diagnostic testing platforms are a growing sector of the healthcare industry as they offer the advantages of rapid provision of results, ease of use, reduced cost, and the ability to link patients to care. While many POC tests are based on chromatographic flow assay technology, this technology suffers from a lack of sensitivity along with limited capacity for multiplexing and quantitative analysis. Several recent reports have begun to investigate the feasibility of coupling chromatographic flow platforms to more advanced read-out technologies which in turn enable on-site acquisition, storage, and transmission of important healthcare metrics. One such technology being explored is surface-enhanced Raman spectroscopy or SERS. In this work, SERS is coupled for the first time to a rapid vertical flow (RVF) immunotechnology for detection of anti-HCV antibodies in an effort to extend the capabilities of this commercially available diagnostic platform. High-quality and reproducible SERS spectra were obtained using reporter-modified gold nanoparticles (AuNPs). Serial dilution studies indicate that the coupling of SERS with RVF technology shows enormous potential for next-generation POC diagnostics.

Electron microscopy of polyoxometalate ions on graphene by electrospray ion beam deposition.

Aberration-corrected high-resolution transmission electron microscopy (AC-HRTEM) has enabled atomically resolved imaging of molecules adsorbed on low-dimensional materials like carbon nanotubes, graphene oxide and few-layer-graphene. However, conventional methods for depositing molecules onto such supports lack selectivity and specificity. Here, we describe the chemically selective preparation and deposition of molecules-like polyoxometalate (POM) anions [PW12O40]3- using electrospray ion-beam deposition (ES-IBD) along with high-resolution TEM imaging. This approach provides access to sub-monolayer coatings of intact molecules on freestanding graphene, which enables their atomically resolved ex situ characterization by low-voltage AC-HRTEM. The capability to tune the deposition parameters in either soft or reactive landing mode, combined with the well-defined high-vacuum deposition conditions, renders the ES-IBD based method advantageous over alternative methods such as drop-casting. Furthermore, it might be expanded towards depositing and imaging large and nonvolatile molecules with complex structures.

Active detachment of Streptococcus mutans cells adhered to epon-hydroxylapatite surfaces coated with salivary proteins in vitro.

Although the formation of biofilms has been much studied, detachment of adherent cells from biofilms has been relatively neglected. Recent results have shown that adherent Streptococcus mutans cells can actively detach from epon-hydroxylapatite (EHA) rods conditioned with hog gastric mucin. The mechanisms for adherence and detachment of Strep. mutans cells in this system was uncertain. In the present study, resting Strep. mutans cells were used to form a simple monolayer on EHA rods coated with saliva and salivary agglutinin (SAG). Preliminary experiments defined the variables for conditioning EHA with saliva and SAG and establishing the adherence of Strep. mutans to the conditioned surfaces. The results showed that salivary proteins including SAG adsorbed rapidly to EHA and that a relatively stable Strep. mutans NG8 monolayer was formed within 60 min of incubation. The monolayers were subsequently used for detachment studies. The results showed that adherent Strep. mutans cells detached in a temperature-dependent manner and responded to the addition of a preparation of surface protein-releasing enzyme (SPRE) obtained from Strep. mutans in a dose-dependent fashion. The effect of the exogenous SPRE on detachment could be abrogated by pronase treatment. Two putative SPRE-defective mutants (A and E) were generated by Tn917 mutagenesis. Both mutants possessed a single transposon insertion as demonstrated by Southern hybridization and appeared to be different from one another based on the hybridization patterns. Mutant A displayed an increased quantity of cell-surface antigen P1, an adhesin that interacts with SAG. At the same time mutant A was unable to release P1 and other high molecular-weight proteins from the cell surface. Mutant A detached at a significantly lower rate (21%) than the parent strain (37%) (p=0.05). SPRE prepared from mutant A was unable to release Strep. mutans NG8 adherent cells as compared to SPRE obtained from the wild-type cells. Collectively, these results suggest that the detachment of Strep. mutans adherent cells formed on salivary protein-coated EHA was an active process mediated by the action of SPRE.

Immunolocalization of Hsp60 in Legionella pneumophila.

One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including L. pneumophila, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L. pneumophila grown in vitro, or in vivo inside of HeLa cells. For comparative purposes Escherichia coli, expressing recombinant L. pneumophila Hsp60, was employed. In contrast to E. coli, where Hsp60 was localized exclusively in the cytoplasm, in L. pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked L. pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by L. pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in L. pneumophila and further suggest both the existence of a novel secretion mechanism (not present in E. coli) and a potential role in pathogenesis.

Radiating dipoles in photonic crystals

The radiation dynamics of a dipole antenna embedded in a photonic crystal are modeled by an initially excited harmonic oscillator coupled to a non-Markovian bath of harmonic oscillators representing the colored electromagnetic vacuum within the crystal. Realistic coupling constants based on the natural modes of the photonic crystal, i.e., Bloch waves and their associated dispersion relation, are derived. For simple model systems, well-known results such as decay times and emission spectra are reproduced. This approach enables direct incorporation of realistic band structure computations into studies of radiative emission from atoms and molecules within photonic crystals. We therefore provide a predictive and interpretative tool for experiments in both the microwave and optical regimes.