Chemisorption Mechanism of DNA on Mg/Fe Layered Double Hydroxide Nanoparticles: Insights into Engineering Effective SiRNA Delivery Systems.

Layered double hydroxide nanoparticles (LDH NPs) have attracted interest as an effective gene delivery vehicle in biomedicine. Recent advances in clinic trials have demonstrated the efficacy of Mg/Fe LDHs for hyperphosphatemia treatment, but their feasibility for gene delivery has not been systematically evaluated. As a starting point, we aimed to study the interaction between oligo-DNA and Mg/Fe LDH NPs. Our investigation revealed the chemisorption mechanism of DNA on Mg/Fe LDH surfaces, wherein the phosphate backbone of the DNA polymer coordinates with the metal cations of the LDH lattice via the ligand-exchange process. This mechanistic insight may facilitate future gene delivery applications using Mg/Fe LDH NPs.

Assessing spatial impacts of historical pulp mill effluent on trophic dynamics in a coastal marine ecosystem using stable isotope (δ13C and δ15N) analysis.

Boat Harbour, Nova Scotia was a tidal estuary that was converted into a wastewater treatment facility for pulp mill effluent in 1967. Treated effluent from Boat Harbour was discharged into the coastal Northumberland Strait, contributing significant nutrient and freshwater inputs into the coastal environment, potentially impacting local biogeochemistry and ecosystem structure. This study used stable isotope analysis of carbon (δ13C) and nitrogen (δ15N) of representative taxa to assess spatial variability in nutrient sources and trophic dynamics. Results identified stable isotope variation with depleted δ13C and δ15N values in taxa near Boat Harbour. Blue mussel (Mytilus edulis) and mummichog (Fundulus heteroclitus) were the most suitable bioindicators for identifying variation in nutrient sources. Stable isotope signatures in this study may be reflective of residual pulp mill effluent-derived nutrients, differences in marine versus terrestrial nutrient sources, and a pronounced coastal salinity gradient. The present study defined the baseline nutrient conditions of the Northumberland Strait and will be useful in assessing the effectiveness of remediation activities.

Development and evaluation of a new in vivo solid-phase microextraction sampler.

The use of solid-phase microextraction (SPME) as a nonlethal technique for in vivo sampling of pharmaceutical residue in fish tissue has been documented in the literature. However, there is need to improve its simplicity and robustness for wider applications in the laboratory and field. The objective of this research is to develop and improve the SPME device for sampling of pharmaceuticals in fish tissue. The practical application of the new device was demonstrated in the field where some wild fish (Esox masquinongy) were caught in the river and sampled by the device. The samples were analyzed using LC coupled with MS/MS (LC-MS/MS). The new in vivo SPME device with a PDMS extraction phase (sorbent) was demonstrated to a robust tool by both experts and nonexpert of the method and it is simpler than the traditional device. The detection limit of the method in gel and fish tissue was 0.01-0.26 ng/g. The interday reproducibility in gel and fish homogenized fish tissue was 8-16% RSD. This study demonstrates that the new device will provide a platform or opportunity for rapid sampling of carbamazepine, diazepam, and nordiazepam in fish muscle with acceptable precision.

Baseline monitoring of contaminant concentrations in American lobster (Homarus americanus) tissues from coastal Northumberland Strait, Nova Scotia, Canada.

A baseline survey was conducted in 2018 to characterize contaminants in American lobsters, Homarus americanus in the Northumberland Strait, Canada. Sampling included three age classes of lobsters at sites 4, 20, and 70 km from the Boat Harbour estuary, a historically contaminated site set to undergo remediation. Lobster tissues were measured for metal(loids), methylmercury, polycyclic aromatic hydrocarbons, and polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans. Contaminant concentrations were generally below the guidelines set by the Canadian Council of Ministers of the Environment and the Canadian Food Inspection Agency, except for arsenic which was elevated in all age classes from all sites (4.8-12.68 mg kg-1). Mercury and methylmercury (both ~0.04 mg kg-1) minimally exceeded one guideline in some age-classes and sites. There was also no consistent pattern of contaminant accumulation across either age classes or at particular sites. This study serves as a baseline for future monitoring following remediation of Boat Harbour.

Mechanistic Insights into Myofibrillar Protein Oxidation by Fenton Chemistry Regulated by Gallic Acid.

Gallic acid (GA, 3,4,5-trihydroxybenzoic acid) is a widely used natural food additive of interest to food chemistry researchers, especially regarding its effects on myofibrillar protein (MP) oxidation. However, existing studies regarding MP oxidation by GA-combined with Fenton reagents are inconsistent, and the detailed mechanisms have not been fully elucidated. This work validated hydroxyl radical (HO·) as the primary oxidant for MP carbonylation; in addition, it revealed three functions of GA in the Fenton oxidation of MP. By coordination with Fe(III), GA reduces Fe(III) to generate Fe(II), which is the critical reagent for HO· generation; meanwhile, the coordination improves the availability and reactivity of Fe(III) under weakly acidic and near-neutral pH, i.e., pH 4-6. Second, the intermediates formed during GA oxidation, including semiquinone and quinone, promoted Fenton reactivity by accelerating Fe catalytic cycling. Finally, GA can scavenge HO· radicals, thus exhibiting a certain degree of antioxidant property. All three functions contribute to MP oxidation as observed in GA-containing meat.

Depth-profiling of environmental pharmaceuticals in biological tissue by solid-phase microextraction.

The parallel in vivo measurement of chemicals at various locations in living tissues is an important approach furthering our understanding of biological uptake, transportation, and transformation dynamics. However, from a technical perspective, such measurements are difficult to perform with traditional in vivo sampling techniques, especially in freely moving organisms such as fish. These technical challenges can be well addressed by the proposed depth-profiling solid-phase microextraction (DP-SPME) technique, which utilizes a single soft, flexible fiber with high spatial resolution. The analytical accuracy and depth-profiling capability of DP-SPME was established in vitro within a multilayer gel system and an onion artificially contaminated with pharmaceuticals. In vivo efficacy was demonstrated by monitoring pharmaceutical distribution and accumulation in fish muscle tissue. The DP-SPME method was validated against pre-equilibrium SPME (using multiple small fibers), equilibrium SPME, and liquid extraction methods; results indicated DP-SPME significantly improved precision and data quality due to decreased intersample variation. No significant adverse effects or increases in mortality were observed in comparisons of fish sampled by DP-SPME relative to comparable fish not sampled by this method. Consequently, the simplicity, effectiveness, and improved precision of the technique suggest the potential for widespread application of DP-SPME in the sampling of heterogeneous biotic and abiotic systems.

Determination of pharmaceutical residues in fish bile by solid-phase microextraction couple with liquid chromatography-tandem mass spectrometry (LC/MS/MS).

The present study investigates possible uptake and bioconcentration of different classes of pharmaceuticals residues (organic contaminants) in fish bile using a simplified analytical methodology based on solid phase microextration (SPME). The use of solid phase microextraction (SPME), as a simple analytical tool, to screen for target pharmaceuticals in fish bile samples was validated in rainbow trout (Oncorhynchus mykiss) following short-term laboratory exposures to carbamazepine and fluoxetine. While fish bioconcentrated both fluoxetine and carbamazepine from exposure water, fluoxetine accumulated to a greater degree in bile than carbamazepine. Good agreement was obtained for both analytes in bile samples between SPME and traditional liquid (solvent) extraction approaches (R(2) > 0.99). The field application of SPME sampling was further demonstrated in fathead minnow (Pimephales promelas), a small-bodied fish caged upstream and downstream of a local wastewater treatment plant where fluoxetine, atorvastatin, and sertraline were detected in fish bile at the downstream location. SPME is a promising analytical tool for investigating the bioconcentration of trace contaminants in fish bile, facilitating detection of trace environmental contaminants otherwise undetectable due to low concentrations in the environment and biological tissues as well as the complexity of the sample matrices.

Exacerbated Protein Oxidation and Tyrosine Nitration through Nitrite-Enhanced Fenton Chemistry.

Nitrite is a common additive used during meat curing to prevent microbial contamination and retain an attractive red color in the product. However, the effects of nitrite on Fenton reactions catalyzed by free iron in meat products are not well understood, although such processes can induce protein oxidation and nitration, affecting the nutritional and aesthetic quality of meat products. This contribution reveals the mechanism through which nitrite affects Fenton reactions that generate reactive nitrogen and oxygen species by increasing the availability of Fe3+, facilitating its reduction and stabilizing Fe2+, and accelerating Fe3+/Fe2+ cycling, leading to exacerbated oxidative and nitrosative stress on proteins, with implications not only for meat processing but also in many biological and environmental processes due to the ubiquitous presence of iron, hydrogen peroxide, and nitrite in nature.

A universal monoclonal antibody-aptamer conjugation strategy for selective non-invasive bioparticle isolation from blood on a regenerative microfluidic platform.

Simultaneous isolation of various circulating tumor cell (CTC) subtypes from whole blood is useful in cancer diagnosis and prognosis. Microfluidic affinity separation devices are promising for CTC separation because of their high throughput capacity and automatability. However, current affinity agents, such as antibodies (mAbs) and aptamers (Apts) alone, are still suboptimal for efficient, consistent, and versatile cell analysis. By introducing a hybrid affinity agent, i.e., an aptamer-antibody (Apt-mAb) conjugate, we developed a universal and regenerative microchip with high efficiency and non-invasiveness in the separation and profiling of various CTCs from blood. The Apt-mAb conjugate consists of a monoclonal antibody that specifically binds the target cell receptor and a surface-bound aptamer that recognizes the conserved Fc region of the mAb. The aptamer then indirectly links the surface functionalization of the microfluidic channels to the mAbs. This hybrid affinity agent and the microchip platform may be widely useful for various bio-particle separations in different biological matrices. Further, the regeneration capability of the microchip improves data consistency between multiple uses and minimizes plastic waste while promoting environmental sustainability. STATEMENT OF SIGNIFICANCE: A hybrid affinity agent, Apt-mAb, consisting of a universal aptamer (Apt) that binds the conserved Fc region of monoclonal antibodies (mAbs) was developed. The invented nano-biomaterial combines the strengths and overcomes the weakness of both Apts and mAbs, thus changing the paradigm of affinity separation of cell subtypes. When Apt-mAb was used to fabricate microfluidic chips using a "universal screwdriver" approach, the microchip could be easily tuned to bind any cell type, exhibiting great universality. Besides high sensitivity and selectivity, the superior regenerative capacity of the microchips makes them reusable, which provides improved consistency and repeatability in cell profiling and opens a new approach towards in vitro diagnostic point-of-care testing devices with environmental sustainability and cost-effectiveness.

Kinetically-calibrated solid-phase microextraction using label-free standards and its application for pharmaceutical analysis.

Pre-equilibrium solid-phase microextraction (PE-SPME) has attracted considerable research attention due to shorter sampling times and better temporal resolution than afforded by equilibrium SPME (E-SPME). However, the calibration of PE-SPME is often time-consuming and requires deuterated calibrants, which if available, are often expensive. To address these challenges, we propose a simple but versatile kinetic calibration method, in which nonisotopic (label-free) compounds of interest can supplant the use of deuterated analogues, and the need to determine partitioning coefficients inherent to earlier procedures has been eliminated. Using this approach, both free and total concentrations of analytes can be simultaneously measured within complex sample systems with high accuracy and precision. This calibration method was validated against established E-SPME and solid-phase extraction techniques through the measurement of selected pharmaceuticals in progressively complex matrixes including inorganic buffers, fish blood, and municipal wastewater effluents. This calibration approach may significantly improve time and cost-effectiveness, while improving the application of the SPME approach within highly dynamic systems.

Solid-phase microextraction coupled to LC-ESI-MS/MS: evaluation and correction for matrix-induced ionization suppression/enhancement for pharmaceutical analysis in biological and environmental samples.

Solid-phase microextraction (SPME) coupled to liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been widely used to analyze biological fluids, tissues, and environmental matrixes for a variety of organic compounds including pharmaceuticals. However, effects of the sample matrix coextracted by SPME on tandem mass spectrometry analysis have not been systematically investigated. In this study, we characterized the complexity of matrix effects (ME) by analyzing SPME extracts of fish muscle and brain tissue, blood, and bile, as well as tap water, surface water, and the influent and effluent from a wastewater treatment plant. Significant enhancement or suppression of ionization (>15%) was observed with all biological and environmental samples. Intrasample ME variability was assessed through comparison of multiple samples from the same sample matrix, while intersample variability between different experimental subjects or varying sample treatment, storage, and sampling conditions were evaluated. To correct for ME, an isotopic internal standard (IIS) method was developed, with the strengths and limitations of the approach discussed. This study provides a framework for applying SPME within complex sample systems where the influences of ME are inevitable, thus ensuring more accurate quantitation of analytes during biological and environmental analysis.

Pre-equilibrium solid-phase microextraction of free analyte in complex samples: correction for mass transfer variation from protein binding and matrix tortuosity.

The accurate measurement of free analyte concentrations within complex sample matrixes by pre-equilibrium solid-phase microextraction (SPME) has proven challenging due to variations in mass uptake kinetics. For the first time, the effects of the sample binding matrix and tortuosity on the kinetics of analyte extraction (from the sample to the SPME fiber) are demonstrated to be quantitatively symmetrical with those of the desorption of preloaded deuterated standards (from the fiber to the sample matrix). Consequently, kinetic calibration methods can be employed to correct for variation in SPME sampling kinetics, facilitating the application of pre-equilibrium SPME within complex sample systems. This approach was applied ex vivo to measure pharmaceuticals in fish muscle tissues, with results consistent with those obtained from equilibrium SPME and microdialysis. The developed method has the inherent advantages of being more accurate, precise, and reproducible, thus providing the framework for applications where rapid measurement of free analyte concentrations (within complicated sample matrixes such as biological tissues, sediment, and surface water) are required.

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process.

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80°C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.

Sampling-rate calibration for rapid and nonlethal monitoring of organic contaminants in fish muscle by solid-phase microextraction.

Solid-phase microextraction (SPME) is a promising technique for determining organic contaminants within biotic systems. Existing in vivo SPME-kinetic calibration (SPME-KC) approaches are unwieldy due to the necessity of predetermining a distribution coefficient for the analyte of interest in the tissue and the preloading of a calibrating compound to the fiber. In this study, a rapid and convenient SPME alternative calibration method for in vivo analysis, termed SPME-sampling rate (SPME-SR) calibration, was developed and validated under both laboratory and field conditions to eliminate such presampling requirements. Briefly, the SPME probe is inserted into tissue, in this study fish dorsal-epaxial muscle, for 20 min allowing the concentrations of target analytes in the fish muscle to be determined by the extracted amount of analyte and the predetermined sampling rates. Atrazine, carbamazepine, and fluoxetine were detected nonlethally in the low ppb levels within fish muscle, with both laboratory and field-derived results obtained by in vivo SPME-KC comparable (within a factor of 1.27) to those obtained by lethal sampling followed by tissue liquid extraction. The technique described in this study represents an important advance which broadens the application of SPME in vivo sampling technology.

Temporal resolution of solid-phase microextraction: measurement of real-time concentrations within a dynamic system.

To address the challenge of measuring real-time analyte concentrations within dynamic systems, the temporal resolution of the solid-phase microextraction (SPME) approach has been investigated. A mass-uptake model for SPME within a dynamic system was developed and validated, with experimental factors affecting the temporal resolution (sampling time, agitation, SPME fiber dimensions, sample concentration and change rate, and instrument sensitivity) characterized. Calibration methods for time-resolved sampling in a dynamic system were compared. To demonstrate the efficacy of time-resolved SPME, this approach was successfully applied to investigate the binding kinetics between plasma proteins and pharmaceuticals, which verified a decrease in free pharmaceutical concentrations over time in the presence of bovine serum albumin. The current study provides the theoretical and logistical framework for applying SPME to the real-time measurement of dynamic systems, facilitating future SPME applications such as in vivo metabolomic studies.

Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles.

A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using organic solvents or commercial kits. This time- and cost-effective protocol does not require centrifugation or precipitation steps and has the potential for automated DNA extraction, especially within miniaturized lab chip applications.

Antidepressants and their metabolites in municipal wastewater, and downstream exposure in an urban watershed.

Antidepressants are a widely prescribed group of pharmaceuticals that can be biotransformed in humans to biologically active metabolites. In the present study, the distribution of six antidepressants (venlafaxine, bupropion, fluoxetine, sertraline, citalopram, and paroxetine) and five of their metabolites was determined in a municipal wastewater treatment plant (WWTP) and at sites downstream of two WWTPs in the Grand River watershed in southern Ontario, Canada. Fathead minnows (Pimephales promelas) caged in the Grand River downstream of a WWTP were also evaluated for accumulated antidepressants. Finally, drinking water was analyzed from a treatment plant that takes its water from the Grand River 17 km downstream of a WWTP. In municipal wastewater, the antidepressant compounds present in the highest concentrations (i.e., >0.5 microg/L) were venlafaxine and its two demethylation products, O- and N-desmethyl venlafaxine. Removal rates of the target analytes in a WWTP were approximately 40%. These compounds persisted in river water samples collected at sites up to several kilometers downstream of discharges from WWTPs. Venlafaxine, citalopram, and sertraline, and demethylated metabolites were detected in fathead minnows caged 10 m below the discharge from a WWTP, but concentrations were all < microg/kg wet weight. Venlafaxine and bupropion were detected at very low (<0.005 microg/L) concentrations in untreated drinking water, but these compounds were not detected in treated drinking water. The present study illustrates that data are needed on the distribution in the aquatic environment of both the parent compound and the biologically active metabolites of pharmaceuticals.

Development of the space-resolved solid-phase microextraction technique and its application to biological matrices.

To facilitate rapid in situ analyte monitoring within heterogeneous samples, a space-resolved solid phase microextraction (SR-SPME) technique was developed that utilized miniaturized segmented fibers. Initially, a multilayered agarose gel was used to determine the effects of diffusion-based mass transfer and fiber dimension on the space-resolving capability of SPME. For diazepam within agarose gel, the SR-SPME limit of detection was 2.5 ng/mL, with a linear dynamic range up to 500 ng/mL. The efficacy of the SR-SPME technique was further evaluated within diverse biological matrices (onion bulb, fish muscle, and adipose tissues) containing stratified pharmaceutical analytes. Empirically, the results agreed well with established techniques such as microdialysis and liquid extraction, but SR-SPME was simpler to implement, displayed higher spatial resolution, and was more cost-effective than traditional approaches. Additionally, the segmented design of the SPME fibers and stepwise desorption protocols offer potential advantages within high throughput applications.

Use of simultaneous dual-probe microdialysis for the determination of pesticide residues in a jade plant (Crassula ovata).

A microdialysis application for the sampling of plant pesticide residues has been developed utilizing two probes with differing perfusate rates simultaneously inserted into symmetric components of a sampling system. A simple empirical equation is proposed to calculate analyte concentrations in a semi-solid matrix using two different dialysate concentrations. In vitro application yielded excellent correlations between the calculated and spiked concentrations. Subsequent in vivo application of this technique for the determination of pesticide distributions in leaves of a jade plant (Crassula ovata) indicated the utility of this approach in complex living matrices.

Regenerative nanobots based on magnetic layered double hydroxide for azo dye removal and degradation.

A highly regenerative multifunctional nanobot system, using Fe3O4@SiO2@MgFe-LDH nanoparticles, is developed for efficient removal of waterborne azo dyes and pharmaceuticals. Efficient capture of pollutants, powerful Fenton degradation, and superior materials regeneration lead to a simple and cost-effective wastewater remediation solution.