Estimating Bacterial Concentrations in Fibrous Substrates Through a Combination of Scanning Electron Microscopy and ImageJ.

Conventional signal-based microanalytical techniques for estimating bacterial concentrations are often susceptible to false signals. A visual quantification, therefore, may compliment such techniques by providing additional information and support better management decisions in the event of outbreaks. Herein, we explore a method that combines electron microscopy (EM) and image-analysis techniques and allows both visualization and quantification of pathogenic bacteria adherent even to complex nonuniform substrates. Both the estimation and imaging parameters were optimized to reduce the estimation error ( E, %) to close to ±5%. The method was validated against conventional microbiological techniques such as the use of optical density, flow cytometry, and quantitative real-time PCR (qPCR). It could easily be tailored to estimate different species of pathogens, such as Escherichia coli O157, Listeria innocua, Staphylococcus aureus, Enterococcus faecalis, and Bacillus anthracis, on samples similar to those in real-time contamination scenarios. The present method is sensitive enough to detect ∼100 bacterial CFU/mL but has the potential to estimate even lower concentrations with increased imaging and computation times. Overall, this imaging-based method may greatly complement any signal-based pathogen-detection technique, especially in negating false signals, and therefore may significantly contribute to the field of analytical microbiology and biochemistry.

Rejection of Commonly Used Electrolytes in Asymmetric Flow Field Flow Fractionation: Effects of Membrane Molecular Weight Cutoff Size, Fluid Dynamics, and Valence of Electrolytes.

Asymmetric flow field flow fractionation (AF4) is an efficient size-based separation technique for the characterization of submicron size particulates. In AF4, membranes having various molecular weight cutoff sizes are used as a barrier to retain particles while allowing the carrier fluid containing electrolytes to permeate. Here, we have hypothesized that electrolyte rejection by the barrier membrane leads to the accumulation of electrolytes in the channel during operation. Electrolyte accumulation can cause various adverse effects that can lead to membrane fouling. An instrument setup containing a conductivity detector was assembled, and the rejection of commonly used carrier electrolytes such as trisodium citrate, ethylenediaminetetraacetic acid, sodium chloride, and ammonium carbonate was evaluated by varying the concentration, cross-flow rate, focusing flow rate, membrane material type, and cutoff sizes. The results showed that electrolyte rejection increased with a decrease in the electrolyte concentration and the molecular weight cutoff size (pore size) or with an increase in the charge state of the anion in the carrier electrolytes. We proposed an electrostatic repulsion-based rejection mechanism and verified it with the measurement of the rejection rate while varying the electrolyte concentration in the running media.

Surface coating and matrix effect on the electrophoretic mobility of gold nanoparticles: a capillary electrophoresis-inductively coupled plasma mass spectrometry study.

Capillary electrophoresis (CE) is considered as a versatile technique in the size-based separation and speciation of nanomaterials. The electrophoretic mobility is determined by charge and size of an analyte which are affected by the surface composition of nanomaterials. Size-dependent differential electrophoretic mobility is used as a mechanism for size-based separation of nanoparticles. Understanding the effect of surface chemistry on the electrophoretic mobility of nanomaterials in CE is critical in obtaining accurate results in retention-based size calculation. A suite of gold nanoparticles (NPs) varied in sizes with different coatings, including citric acid (CA), lipoic acid (LA), tannic acid (TA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), branched polyethyleneimine (BPEI), and bovine serum albumin (BSA), were selected to evaluate their impact to the migration pattern of gold NPs. Additionally, surface-coated gold NPs dispersed in Suwannee River humic acid (SRHA) solution and fetal bovine serum (FBS) were used to investigate the matrix effect. It was found that the correlation between NP size and relative electrophoretic mobility is highly dependent on the capping agents. The matrix component in the SRHA solution only exhibited limited influence to the migration of NPs while electrophoretic behaviors were drastically altered in the presence of FBS matrix.

Portable X-ray fluorescence spectroscopy as a rapid screening technique for analysis of TiO2 and ZnO in sunscreens.

This investigation reports a rapid and simple screening technique for the quantification of titanium and zinc in commercial sunscreens using portable X-ray fluorescence spectroscopy (pXRF). A highly evolved technique, inductively coupled plasma-mass spectroscopy (ICP-MS) was chosen as a comparative technique to pXRF, and a good correlation (r2 > 0.995) with acceptable variations (≤25%) in results between both techniques was observed. Analytical figures of merit such as detection limit, quantitation limit, and linear range of the method are reported for the pXRF technique. This method has a good linearity (r2 > 0.995) for the analysis of titanium (Ti) in the range of 0.4-14.23 wt%, and zinc (Zn) in the range of 1.0-23.90 wt%. However, most commercial sunscreens contain organic ingredients, and these ingredients are known to cause matrix effects. The development of appropriate matrix matched working standards to obtain the calibration curve was found to be a major challenge for the pXRF measurements. In this study, we have overcome the matrix effect by using metal-free commercial sunscreens as a dispersing media for the preparation of working standards. An easy extension of this unique methodology for preparing working standards in different matrices was also reported. This method is simple, rapid, and cost-effective and, in comparison to conventional techniques (e.g., ICP-MS), did not generate toxic wastes during sample analysis.

Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona.

Production and application of nanoparticles in consumer products is at an all-time high due to the emerging field of nanotechnology. Direct detection and quantification of trace levels of nanoparticles within consumer products is very challenging and problematic. Although multiple methodologies are available for this purpose, each method has its own set of limitations. Herein, we developed an analytical platform consisting of asymmetric flow-field flow fractionation (AF4) coupled with inductively coupled plasma mass spectroscopy (ICP-MS) for the speciation and quantification of silver ions and silver nanoparticles at the ng/kg level (ppt). AF4 is utilized to concentrate the nanoparticles, and ICP-MS acts as the detector. The protein corona that forms upon exposure of nanoparticles to bovine serum albumin was utilized as a nanoparticle stabilization and AF4 recovery enhancement mechanism. Speciation of silver ions and nanoparticles was achieved with the assistance of penicillamine as a complexation ligand. The effect of nanoparticle size, surface coating, and ionization state toward the detection and quantification of the developed methodology was evaluated. The detection limit was found to be 4 ng/kg with the application of a 5 mL sample loop. Further application of this developed methodology on environmentally relevant samples was demonstrated by the analysis of Arkansas River water spiked with silver nanoparticles and nanoparticle spiked into humic acid solution (50 mg/L) at an environmentally relevant level.

Simple functionalization strategies for enhancing nanoparticle separation and recovery with asymmetric flow field flow fractionation.

Due to the increasing use of engineered nanomaterials in consumer products, regulatory agencies and other research organizations have determined that the development of robust, reliable, and accurate methodologies to characterize nanoparticles in complex matrices is a top priority. Of particular interest are methods that can separate and determine the size of nanomaterials in samples that contain polydisperse and/or multimodal nanoparticle populations. Asymmetric-flow field flow fractionation (AF4) has shown promise for the separation of nanoparticles with wide size range distributions; however, low analyte recoveries and decreased membrane lifetimes, due to membrane fouling, have limited its application. Herein, we report straightforward strategies to minimize membrane fouling and improve nanoparticle recovery by functionalizing the surface of the nanoparticles, as well as that of the AF4 membranes. Gold nanoparticles (AuNP) were stabilized through functionalization with a phosphine molecule, whereas the surface of the membranes was coated with a negatively charged polystyrenesulfonate polymer. Improved nanoparticle separation, recoveries of 99.1 (±0.5) %, and a detection limit of 6 µg/kg were demonstrated by analyzing AuNP reference materials of different sizes (e.g., 10, 30, and 60 nm), obtained from the National Institute of Standards and Technology (NIST). Furthermore, the stability of the polymer coating and its specificity toward minimizing membrane fouling were demonstrated.

Capillary electrophoresis/inductively-coupled plasma-mass spectrometry: development and optimization of a high resolution analytical tool for the size-based characterization of nanomaterials in dietary supplements.

We report the development and optimization of a system consisting of capillary electrophoresis (CE) interfaced with inductively coupled plasma mass spectrometry (ICPMS) for rapid and high resolution speciation and characterization of metallic (e.g., gold, platinum, and palladium) nanoparticles in a dietary supplement. Multiple factors, including surfactant type and concentration, pH of running buffer, and applied voltage, were investigated to optimize the separation conditions. It was found that by using the anionic surfactant sodium dodecyl benzenesulfonate (SDBS) in the running buffer the separation resolution was significantly improved, allowing for easy distinction of adjacent size fractions in a gold nanoparticle mixture with very small size differences (e.g., 5, 15, 20, and 30 nm). The type and concentration of the surfactant was found to be critical in obtaining sufficient separation while applied voltage and pH values of the running buffers largely affected the elution times by varying the electroosmotic flow. Quantum dots were used as mobility markers to eliminate the run-to-run variation. The diameters of the nanoparticles followed a linear relationship with their relative electrophoretic mobility, and size information on unknown samples could be extrapolated from a standard curve. The accuracy and precision of this method was confirmed using 10 and 30 nm gold nanoparticle standard reference materials. Furthermore, the method was successfully applied to the analysis of commercially available metallic nanoparticle-based dietary supplements, as evidenced by good agreement between the particle sizes calculated by CE/ICPMS and transmission electron microscopy (TEM).

In vitro enrofloxacin binding in human fecal slurries.

Most antibiotic inactivation studies have been conducted through in vitro incubations of human use aminoglycosides, beta-lactams, and fluoroquinolones, usually at fecal concentrations expected with therapeutic dose regimens in humans and animals. Less is known about the inactivation of these molecules when ingested at concentrations consistent with residue levels present in animal-derived foods from antibiotic treated animals. In this investigation, we used the fluoroquinolone, enrofloxacin which is specifically marketed for veterinary medicine as test compound. Fecal suspensions at 10%, 25%, and 50% (w/v) were subjected to physicochemical and molecular characterization and used in the drug binding studies. The fecal binding of enrofloxacin added at concentrations of 0.06, 0.1, 1, 5, 15, 50, and 150 mg/L was determined in various fecal slurry suspensions using analytical chemistry and microbiological assay methods. There was consistent correlation between both assay methods. By the analytical chemistry assay, the 10%, 25% and 50% diluted autoclaved fecal samples dosed with enrofloxacin showed binding of 50±4.6%, 54±6.5% and 56±6.8% of the enrofloxacin, respectively. Binding of enrofloxacin to fecal contents occurred rapidly within 10 min and remained constant over the incubation period. Denaturing gradient gel electrophoreses and pyrosequencing analysis showed varied profiles of the bacterial composition of the human intestinal microbiota for fecal samples from different individuals. This study provided information on methodological questions that have concerned regulatory authorities on in vitro testing to determine if concentrations of veterinary antimicrobial agent residues entering the human colon remain microbiologically active.

Detection and Characterization of Silver Nanostructures in Consumer Products.

Silver (Ag) is one among the few nanomaterials which are widely used across several consumer products. However, there is limited research on detection and characterization of Ag nanostructures in complex matrices such as consumer products. Most previous studies for analytical method development were based on Ag liquid formulations or with standard materials. In this study, a total of fifteen commercial products including dietary supplements, deodorants, fabric, skin protectants, and toothpastes that declare nano or colloidal Ag ingredients were investigated. To characterize the quantity, size, size distribution, and morphology of Ag nanoparticles used in the products, several analytical instrumental techniques such as inductively coupled plasma-mass spectroscopy (ICPMS), dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectrometry (EDS) were employed. Study results showed that Ag nanoparticles were found in eleven of the fifteen investigated commercial products, where the majority of Ag nanoparticles were spherical and smaller than 50 nm. The advantages and limitations of size characterization techniques were discussed with respect to product type. A combination of characterization techniques was highly desired based on the product type and other ingredients used to confirm the presence of nanostructures in consumer products.

Novel analytical methods to assess the chemical and physical properties of liposomes.

Liposomes are used in commercial pharmaceutical formulations (PFs) and dietary supplements (DSs) as a carrier vehicle to protect the active ingredient from degradation and to increase the half-life of the injectable. Even as the commercialization of liposomal products has rapidly increased, characterization methodologies to evaluate physical and chemical properties of the liposomal products have not been well-established. Herein we develop rapid methodologies to evaluate chemical and selected physical properties of liposomal formulations. Chemical properties of liposomes are determined by their lipid composition. The lipid composition is evaluated by first screening of the lipids present in the sample using HPLC-ELSD followed by HPLC-MSMS analysis with high mass accuracy (<5 ppm), fragmentation pattern and lipid structure databases searching. Physical properties such as particle size and size distribution were investigated using Tunable Resistive Pulse Sensing (TRPS). The developed methods were used to analyze commercially available PFs and DSs. As results, PFs contain distinct number of lipids as indicated by the manufacture, but DSs were more complicated containing a large number of lipids belonging to different sub-classes. Commercially available liposomes have particles with wide size distribution based on size measurements performed by TRPS. The high mass accuracy as well as identification lipids using multiple fragment ions aided to accurately identify the lipids and differentiate them from other lipophilic molecules. The developed analytical methodologies were successfully adapted to measure the physiochemical properties of commercial liposomes.

Capillary electrophoresis coupled with inductively coupled mass spectrometry as an alternative to cloud point extraction based methods for rapid quantification of silver ions and surface coated silver nanoparticles.

Speciation and accurate quantification of ionic silver and metallic silver nanoparticles are critical to investigate silver toxicity and to determine the shelf-life of products that contain nano silver under various storage conditions. We developed a rapid method for quantification of silver ions and silver nanoparticles using capillary electrophoresis (CE) interfaced with inductively-coupled plasma mass spectrometry (ICPMS). The addition of 2-mercaptopropionylglycine (tiopronin) to the background electrolyte was used to facilitate the chromatographic separation of ionic silver and maintain the oxidation state of silver. The obtained limits of detection were 0.05 µg kg(-1) of silver nanoparticles and 0.03 µg kg(-1) of ionic silver. Nanoparticles of varied sizes (10-110 nm) with different surface coating, including citrate acid, lipoic acid, polyvinylpyrrolidone and bovine serum albumin (BSA) were successfully analyzed. Particularly good recoveries (>93%) were obtained for both ionic silver and silver nanoparticle in the presence of excess amount of BSA. The method was further tested with six commercially available dietary supplements which varied in concentration and matrix components. The summed values of silver ions and silver nanoparticles correlated well with the total silver concentration determined by ICPMS after acid digestion. This method can serve as an alternative to cloud point extraction technique when the extraction efficiency for protein coated nanoparticles is low.

An improved methodology of asymmetric flow field flow fractionation hyphenated with inductively coupled mass spectrometry for the determination of size distribution of gold nanoparticles in dietary supplements.

Engineered nanoparticles are available in large numbers of commercial products claiming various health benefits. Nanoparticle absorption, distribution, metabolism, excretion, and toxicity in a biological system are dependent on particle size, thus the determination of size and size distribution is essential for full characterization. Number based average size and size distribution is a major parameter for full characterization of the nanoparticle. In the case of polydispersed samples, large numbers of particles are needed to obtain accurate size distribution data. Herein, we report a rapid methodology, demonstrating improved nanoparticle recovery and excellent size resolution, for the characterization of gold nanoparticles in dietary supplements using asymmetric flow field flow fractionation coupled with visible absorption spectrometry and inductively coupled plasma mass spectrometry. A linear relationship between gold nanoparticle size and retention times was observed, and used for characterization of unknown samples. The particle size results from unknown samples were compared to results from traditional size analysis by transmission electron microscopy, and found to have less than a 5% deviation in size for unknown product over the size range from 7 to 30 nm.

Arsenic speciation in rice by capillary electrophoresis/inductively coupled plasma mass spectrometry: enzyme-assisted water-phase microwave digestion.

We report an analytical methodology for the quantification of common arsenic species in rice and rice cereal using capillary electrophoresis coupled with inductively coupled plasma mass spectrometry (CE-ICPMS). An enzyme (i.e., α-amylase)-assisted water-phase microwave extraction procedure was used to extract four common arsenic species, including dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenite [As(III)], and arsenate [As(V)] from the rice matrices. The addition of the enzyme α-amylase during the extraction process was necessary to reduce the sample viscosity, which subsequently increased the injection volume and enhanced the signal response. o-Arsanilic acid (o-ASA) was added to the sample solution as a mobility marker and internal standard. The obtained repeatability [i.e., relative standard deviation (RSD %)] of the four arsenic analytes of interest was less than 1.23% for elution time and 2.91% for peak area. The detection limits were determined to be 0.15-0.27 ng g(-1). Rice standard reference materials SRM 1568b and CRM 7503-a were used to validate this method. The quantitative concentrations of each organic arsenic and summed inorganic arsenic were found within 5% difference of the certified values of the two reference materials.

Detection and characterization of SiO2 and TiO2 nanostructures in dietary supplements.

Nanomaterials are beginning to enter our daily lives through various consumer products as the result of technology commercialization. The development of methodologies to detect the presence of nanomaterials in consumer products is an essential element in understanding our exposure. In this study, we have developed methods for the separation and characterization of silicon dioxide (SiO2) and titanium dioxide (TiO2) nanostructures in dietary supplements marketed in products specifically targeted for women. A total of 12 commercial products claiming the inclusion of SiO2 and TiO2, but not making any claims regarding the particle size, were randomly selected for purchase through various retailers. To isolate nanostructures from these products, a simple methodology that combines acid digestion and centrifugation was utilized. Once isolated, the chemical composition, size, morphology, and crystal structure were characterized using mass spectroscopy, light scattering, electron microscopy, and X-ray diffraction techniques. SiO2 and TiO2 nanostructures were detected in 11 of 12 products using these methods. Many of the isolated nanoscale materials showed a high degree of aggregation; however, identified individual structures had at least one dimension below 100 nm. These robust methods can be used for routine monitoring of commercial products for nanoscale oxides of silica and titanium.

HPLC separation of tetracycline analogues: comparison study of laser-based polarimetric detection with UV detection.

A sensitive and specific high-performance liquid chromatography (HPLC) method based upon laser-based polarimetric detection is developed for the determination of six tetracycline analogues. By interfacing the laser-based polarimeter online with an HPLC system, the specific rotation of each analogue is obtained as compounds elute from the separation system. The six structurally similar tetracycline analogues exhibit significant differences in specific rotations. The experiments suggest that specific rotation can be useful in identifying closely related tetracycline analogues. Linear relationships are found to be in the range of 0.342-0.0043 mg for the tetracycline analogues. Five of the six analogues exhibit excellent linearity (R(2) value >/= 0.99). The polarimetric results are compared with UV detection. The HPLC-laser-based polarimetric detection instrument is able to quantitate the studied tetracycline analogues with high precision, accuracy, and sensitivity, which make it useful for the development of a standard method for the determination of tetracyclines in biological specimens. The performance of the HPLC-polarimetric system for the analysis of tetracyclines in a biological matrix is evaluated. The selectivity of polarimetric detection provides a distinct advantage in the analysis of tetracycline analogues in milk. The HPLC-polarimetric system provides a rapid and sensitive technique that involves minimal sample cleanup and pretreatment for the analysis of tetracyclines in milk.

Rapid determination of silver in nanobased liquid dietary supplements using a portable X-ray fluorescence analyzer.

This paper reports a rapid and straightforward method for the quantitation of total Ag content in nanobased commercially available liquid dietary supplements using a portable X-ray fluorescence (pXRF) analyzer. Figures of merits were evaluated by analyzing a series of AgNO3 standards. This method was shown to have a detection limit of 3 ppm, a quantitation limit of 10 ppm, and a broad linear range from the detection limit to 10000 ppm (1%). Accurate detection and quantitation of Ag content in well-characterized Ag nanoparticle samples and in nanobased liquid dietary supplements were achieved with good correlation (i.e., percentage difference average values under 15%) between the total Ag concentration obtained by the pXRF analyzer and by inductively coupled plasma mass spectrometry (ICP-MS). Furthermore, accurate quantitation of Ag in the presence of high concentrations of potential spectral interferences was also demonstrated.

Real-time assessment of enantiomeric purity via a polarimetric/absorption detector response function.

The development of methodology appropriate for the rapid and precise assessment of enantiomeric purity is a critical need in the life sciences with impact in a number of areas including biomedical research, biotechnology, and pharmaceutical science. Real-time assessment of enantiomeric purity is critical to decisions related to possible product purity and/or the need for, and the type of additional processing. Recently, we have shown that laser-based polarimetric detection, in combination with ultraviolet detection, can be used to assess enantiomeric purity in real-time as an adjunct to the separation process. A mass-independent response function is obtained from the ratio of the normalized polarimetric signal relative to the normalized UV signal. This response ratio will be shown to be equivalent to the enantiomeric excess and independent of concentration and chromatographic resolution. The methodology will be evaluated as a function of injected mass, enantiomeric excess, chromatographic resolution, and peak asymmetry.