Anthony F.T. Moore

Portable mercury sensor for tap water using surface plasmon resonance of immobilized gold nanorods.

The surface plasmon resonance of surface immobilized gold nanorods (Au NRs) was used to quantify mercury in tap water. Glass substrates were chemically functionalized with (3-mercaptopropyl)trimethoxysilane, which chemically bound the nanorods to produce a portable and sensitive mercury sensor. The analytical capabilities of the sensor were measured using micromolar mercury concentrations. Since the analytical response was dependent upon number of nanorods present, the limit of detection was 2.28×10(-19) M mercury per nanorod. The possibility to using glass substrates with immobilized Au NRs is a significant step towards the analysis of mercury in tap water flows at this low concentration level.

Four-way modeling of 4.2 K time-resolved excitation emission fluorescence data for the quantitation of polycyclic aromatic hydrocarbons in soil samples

A screening method for the soil analysis of 15 Environmental Protection Agency-polycyclic aromatic hydrocarbons (EPA-PAHs) is reported. The new method is based on the collection of 4.2 K fluorescence time-resolved excitation-emission cubes (TREECs) via laser-excited time-resolved Shpolskii spectroscopy. 4.2 K fluorescence TREECs result from the superposition of fluorescence time-resolved excitation emission matrices recorded at different time windows from the laser excitation pulse. Potential interference from unknown sample concomitants is handled by processing the four-way 4.2 K fluorescence TREEC data arrays with either parallel factor analysis (PARAFAC) or unfolded partial least-squares/residual-trilinearization(U-PLS/RTL). The sensitivity of the two approaches makes possible to determine PAHs at the ng g(-1) to pg g(-1) concentration level with no need for sample pre-concentration. Its selectivity eliminates sample clean-up steps and chromatographic separation. These features reduce PAH loss, analysis time and cost. The method is environmentally friendly as the complete screening of the 15 EPA-PAHs takes only 250 μL of organic solvent per sample.

Determination of high-molecular weight polycyclic aromatic hydrocarbons in high performance liquid chromatography fractions of coal tar standard reference material 1597a via solid-phase nanoextraction and laser-excited time-resolved Shpol'skii spectroscopy.

This article presents an alternative approach for the analysis of high molecular weight - polycyclic aromatic hydrocarbons (HMW-PAHs) with molecular mass 302 Da in complex environmental samples. This is not a trivial task due to the large number of molecular mass 302 Da isomers with very similar chromatographic elution times and similar, possibly even virtually identical, mass fragmentation patterns. The method presented here is based on 4.2K laser-excited time-resolved Shpolskii spectroscopy, a high resolution spectroscopic technique with the appropriate selectivity for the unambiguous determination of PAHs with the same molecular mass. The potential of this approach is demonstrated here with the analysis of a coal tar standard reference material (SRM) 1597a. Liquid chromatography fractions were submitted to the spectroscopic analysis of five targeted isomers, namely dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,i]pyrene, naphtho[2,3-a]pyrene and dibenzo[a,h]pyrene. Prior to analyte determination, the liquid chromatographic fractions were pre-concentrated with gold nanoparticles. Complete analysis was possible with microliters of chromatographic fractions and organic solvents. The limits of detection varied from 0.05 (dibenzo[a,l]pyrene) to 0.24 µg L(-1) (dibenzo[a,e]pyrene). The excellent analytical figures of merit associated to its non-destructive nature, which provides ample opportunity for further analysis with other instrumental methods, makes this approach an attractive alternative for the determination of PAH isomers in complex environmental samples.

Single Fiber Identification with Nondestructive Excitation–Emission Spectral Cluster Analysis

Identification methods for single textile fibers are in demand for forensic applications, and nondestructive methods with minimal pretreatment have the greatest potential for utility. Excitation-emission luminescence data provide a three-dimensional matrix for comparison of single-fiber dyes, and these data are enhanced by principal component analysis and comparison of fibers using a statistical figure of merit. No dye extraction methods are required to sample the spectra from a single fiber. This approach has been applied to the analysis of single fibers to compare closely matched dye pairs, acid blue (AB) 25 and 41 and direct blue (DB) 1 and 53. In all cases, the accuracy of fiber identification was high and no false positive identifications were made.

Combining Cryogenic Fiber Optic Probes with Commercial Spectrofluorimeters for the Synchronous Fluorescence Shpol'skii Spectroscopy of High Molecular Weight Polycyclic Aromatic Hydrocarbons

Cryogenic fiber optic probes are combined for the first time with a commercial spectrofluorometer for Shpolskii spectroscopy measurements at liquid nitrogen (77 K) and liquid helium (4.2 K) temperatures. Accurate and reproducible acquisition of fluorescence spectra and signal intensities is demonstrated with three well known Shpolskii systems, namely, anthracene/heptane, pyrene/hexane, and benzo[a]pyrene/octane. The ability to adjust the excitation and emission bandpass of the spectrofluorimeter to reach both site-resolution and analytically valuable signal-to-noise ratios was illustrated with benzo[a]pyrene in n-octane. The analytical potential of 4.2 K synchronous fluorescence Shpolskii spectroscopy for the analysis of high molecular weight–polycyclic aromatic hydrocarbons was then explored for the first time. The judicious optimization of wavelength offsets permitted the successful determination of dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and naphtho[2,3-a]pyrene without previous chromatographic separation from a soil extract with complex matrix composition. The simplicity of the experimental procedure, the competitive analytical figures of merit, and the selectivity of analysis turn 4.2 K synchronous fluorescence Shpolskii spectroscopy into a valuable alternative for screening isomers of high molecular weight polycyclic aromatic hydrocarbons in environmental samples.

DbpA is a region-specific RNA helicase.

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic cores grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic cores grasp.

Kinetics and Thermodynamics of DbpA Protein’s C-Terminal Domain Interaction with RNA

DbpA is an Escherichia coli DEAD-box RNA helicase implicated in RNA structural isomerization in the peptide bond formation site. In addition to the RecA-like catalytic core conserved in all of the members of DEAD-box family, DbpA contains a structured C-terminal domain, which is responsible for anchoring DbpA to hairpin 92 of 23S ribosomal RNA during the ribosome assembly process. Here, surface plasmon resonance was used to determine the equilibrium dissociation constant and the microscopic rate constants of the DbpA C-terminal domain association and dissociation to a fragment of 23S ribosomal RNA containing hairpin 92. Our results show that the DbpA protein’s residence time on the RNA is 10 times longer than the time DbpA requires to hydrolyze one ATP. Thus, our data suggest that once bound to the intermediate ribosomal particles via its RNA-binding domain, DbpA could unwind a number of double-helix substrates before its dissociation from the ribosomal particles.

Deciphering the Action Mechanism of DDX3: An RNA Helicase Implicated in Cancer Propagation and Pathogenic Viral Infection

DDX3 is a human DEAD-box RNA helicase implicated in crucial cellular processes including translation initiation, ribosome assembly, RNA transport, and microRNA processing. Consequently, DDX3 is implicated in many viral infections and cancer cell metabolism. Our goal is to obtain a detailed understanding of DDX3s mechanism of action and employ this understanding to discover DDX3 inhibitors that would serve as lead compounds for drugs that halt viral infections and cancer cell metabolism. While DDX3 is required for many viral infections and cancer cell propagations, it is not essential for healthy cell metabolism, making DDX3 an ideal anticancer and antiviral drug target. Like all the members of the DEAD-box family of enzymes, DDX3 uses ATP binding and hydrolysis to unwind short double-stranded RNA helices. Our data show that different from many members of DEAD-box family of enzymes, monomeric DDX3 is unable to perform RNA unwinding, and a multimeric DDX3 complex is required to support DDX3s helicase activity. Furthermore, our data suggests that the single-stranded-double-stranded RNA junction promotes the formation of the DDX3 multimer. We are in the process of performing mutagenesis studies combined with cross-linking and mass spectrometry to determine the DDX3 amino acids implicated in mulitmer formation and the amino acids that come in direct contact with RNA during the DDX3 catalytic cycle. These experiments would produce information both on DDX3s mechanism of action, and elucidate why some DEAD-box proteins have evolved to act as mulitmers. Lastly, we have found four natural compounds that are specific inhibitors of DDX3 ATPase activity. These compounds will be used as probes to decipher DDX3s action mechanism and could have translational potential as drugs that stop various viral infections and cancer progression.