Aging-Associated Changes in the Adult Human Skin Microbiome and the Host Factors that Affect Skin Microbiome Composition.

Understanding the changes in the skin microbiome and their relationship to host skin factors during aging remains largely unknown. To better understand this phenomenon, we collected samples for metagenomic and host skin factor analyses from the forearm, buttock, and facial skin from 158 Caucasian females aged 20‒24, 30‒34, 40‒44, 50‒54, 60‒64, and 70‒74 years. Metagenomics analysis was performed using 16S ribosomal RNA gene sequencing, whereas host sebocyte gland area, skin lipids, natural moisturizing factors, and antimicrobial peptides measurements were also performed. These analyses showed that skin bacterial diversity increased at all the skin sites with increasing age. Of the bacterial genera with an average relative abundance >1%, only Lactobacillus and Cutibacterium demonstrated a significant change (decrease) in abundance at all sampled skin sites with increasing age. Additional bacterial genera demonstrated significant age- and site-specific changes in abundance. Analysis of sebocyte area, natural moisturizing factors, lipids, and antimicrobial peptides showed an age-related decrease in sebocyte area and increases in natural moisturizing factors/antimicrobial peptides/skin lipids, all of which correlated with changes in specific bacterial genera. In conclusion, the human skin microbiome undergoes age-associated alterations that may reflect underlying age-related changes in cutaneous biology.

Electrochemical Affinity Assays/Sensors: Brief History and Current Status.

The advent of electrochemical affinity assays and sensors evolved from pioneering efforts in the 1970s to broaden the field of analytes accessible to the selective and sensitive performance of electrochemical detection. The foundation of electrochemical affinity assays/sensors is the specific capture of an analyte by an affinity element and the subsequent transduction of this event into a measurable signal. This review briefly covers the early development of affinity assays and then focuses on advances in the past decade. During this time, progress on electroactive labels, including the use of nanoparticles, quantum dots, organic and organometallic redox compounds, and enzymes with amplification schemes, has led to significant improvements in sensitivity. The emergence of nanomaterials along with microfabrication and microfluidics technology enabled research pathways that couple the ease of use of electrochemical detection for the development of devices that are more user friendly, disposable, and employable, such as lab-on-a-chip, paper, and wearable sensors.

UHPLC-MS/HRMS method for the quantitation of pyrithione metabolites in human urine.

Pyrithione glucuronide (PTG) and 2-thiopyridine glucuronide (ThPG) have been reported to be the major urinary metabolites in multiple animal species following administration of zinc pyrithione (ZnPT). However, the formation of these metabolites has never been confirmed in humans. A simple and rugged ultra-high-performance liquid chromatography high resolution mass spectrometry (UHPLC-MS/HRMS) method was developed and validated for the quantification of PTG and ThPG to investigate human metabolism of pyrithione following topical application of ZnPT as a shampoo. A UHPLC-MS/HRMS method was required due to the matrix interferences that were observed with the typical industry standard HPLC/tandem mass spectrometry (LC-MS/MS) methodology based on nominal mass triple quadrupole (QQQ) platform approach. Using UPLC-MS/HRMS, both PTG and ThPG were identified in human urine following topical application of ZnPT. The presence of these human urinary metabolites of pyrithione are consistent with findings from earlier studies in multiple animal species and suggest the metabolism of pyrithione is similar amongst those mammalian species studied.

Effects of petrolatum, a petrolatum depositing body wash and a regular body wash on biomarkers and biophysical properties of the stratum corneum.

BACKGROUND: An important trend in the personal care industry involves the development of body wash products that not only clean the skin without damage but deposit conditioning ingredients to improve skin barrier function. OBJECTIVE: The objective of this study was to develop skin biomarker measures to quantify the treatment effects of body wash products. METHODS: We employed analysis of structural proteins (keratin 1,10,11 and involucrin), a natural moisturizing factor (pyrrolidone carboxylic acid) and an inflammatory mediator (IL-1ra/IL-1α) from adhesive discs with dry skin grading, TEWL and capacitance measurements to compare the effects of direct application of petrolatum, a high petrolatum depositing body wash, and a regular body wash on dry leg skin in a standard leg-wash treatment protocol. RESULTS: High depositing body wash and petrolatum had positive effects on stratum corneum barrier function as judged by biomarker analysis, biophysical measurements and skin grading compared to the regular body wash product. CONCLUSIONS: The results clearly indicate that a combination of biomarker and biophysical property measurements is effective for determining the skin benefits of moisturizing body wash products.

CONTEXTE: Une tendance importante dans l'industrie des soins personnels inclut le développement de produits de lavage corporel qui non seulement nettoient la peau sans l'endommager, mais déposent des ingrédients de traitement pour améliorer la fonction de la barrière cutanée. OBJECTIF: L'objectif de cette étude était de développer des mesures de biomarqueurs cutanés permettant de quantifier les effets du traitement des produits de lavage corporel. MÉTHODES: Nous avons utilisé l'analyse de protéines structurelles (kératine 1,10,11 et involucrine), un facteur hydratant naturel (acide carboxylique de pyrrolidone) et un médiateur inflammatoire (IL-1ra/IL-1a) provenant de disques adhésifs avec cotation de la sécheresse cutanée, mesures de perte d'eau transépidermique (transepidermal water loss, TEWL) et de capacitance pour comparer les effets de l'application directe de vaseline, d'un produit de lavage corporel avec dépôt élevé de vaseline et d'un produit de lavage corporel ordinaire sur la peau sèche des jambes, dans un protocole de traitement de lavage des jambes standard. RÉSULTATS: Le produit de lavage corporel à dépôt élevé et la vaseline avaient des effets positifs sur la fonction de barrière de la couche cornée, comme évalué par l'analyse des biomarqueurs, les mesures biophysiques et la cotation de la peau, comparé au produit de lavage corporel ordinaire. CONCLUSIONS: Les résultats indiquent clairement qu'une combinaison de mesures des biomarqueurs et des propriétés biophysiques est efficace pour déterminer les bienfaits pour la peau des produits de lavage corporel hydratants.

Probabilistic exposure assessment of DEEDMAC using measured effluent and sludge concentrations from 41 wastewater treatment plants across the United States.

The cationic surfactant diethyldialkylester dimethyl ammonium chloride (DEEDMAC) is an active ingredient in liquid fabric softeners and, as such, is disposed of down the drain after consumer use. A monitoring program was conducted across the continental United States to measure the concentration of DEEDMAC in the effluent and sludge from 41 wastewater treatment plants (WWTPs). The concentration in the effluent ranged from 32.4 to 2660ng/L, with a mean and standard deviation of 232±450ng/L. The concentration in the sludge ranged from 0.707 to 314mg/kg dw, with a mean and standard deviation of 29.2±50mg/kg dw. The distribution of measured effluent concentrations was combined with a distribution of mixing zone dilutions factors to predict the distribution of DEEDMAC concentrations in mixing zones and sediments under mean flow and 10-year, 7 consecutive day lowest flow (7Q10 low flow) conditions. Additionally, the distribution of measured sludge concentrations was combined with a distribution of land applied sludge volumes and US tilling practices to obtain a predicted distribution of DEEDMAC concentrations in sludge amended soils. The 90th percentile concentrations of DEEDMAC in mixing zones and sediments under mean flow conditions was 10.3ng/L and 451ng/kg, respectively. The 90th percentile concentration in sludge amended soils was 1.92mg/kg. These predicted exposure concentrations were compared to published eco-toxicity data and showed that DEEDMAC has a wide margin of safety and poses negligible ecologic risk to aquatic, sediment, or terrestrial compartments.

Intrinsic relative potency of a series of pyrrolizidine alkaloids characterized by rate and extent of metabolism.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are sometimes present in foods or herbal supplements/medicines as impurities and pose potential concerns for liver genotoxicity/carcinogenicity. PAs display a strong structure toxicity relationship, however, current regulatory approaches to risk assessment take the precautionary approach of assuming all PAs display the same potency as the most toxic congeners lasiocarpine (LAS) and riddelliine (RID). Here we explore the relative potencies of a series of structurally diverse PAs by measuring DNA adduct formation in vitro in a rat sandwich culture hepatocyte (SCH) cell system. The adducts generated are consistent with those identified in vivo as biomarkers of PA exposure and potential liver-tumor formation. DNA reactive PAs require metabolic activation to form intermediates that bind DNA, therefore, adduct formation is a direct reflection of reactive metabolite formation. Since the area under the concentration versus time curve (AUC) for the depletion of parent PA from the extracellular media is a measure of PA exposure, the ratio of adducts/AUC provides a measure of hepatocyte exposure to DNA-binding metabolites corresponding to an intrinsic potency for DNA adduct formation. Intrinsic potencies relative to potencies for LAS compare well with existing relative potency data further affirming that PA toxicity varies considerably with chemical structure.

Environmental fate of amine oxide: Using measured and predicted values to determine aquatic exposure.

Amine oxide (AO) surfactants are used widely in North American household detergents resulting in >44,000mtons disposed down the drain annually. Due to AOs substantial down the drain disposal volume, wide dispersive use, and high aquatic toxicity, there is a need to evaluate ecological exposure and corresponding risk. This study refined the current knowledge regarding the fate of AO disposed down the drain through laboratory simulation studies to evaluate biodegradation in the sewer and during activated sludge wastewater treatment. A monitoring program which measured effluent AO concentrations for the dominant carbon chain lengths, C12 and C14, at 44 wastewater treatment plants (WWTP) across the continental US was also conducted. The study results were then used as input into probabilistic exposure models to predict US receiving stream concentrations. In three separate OECD 314A Sewer Water Die-Away studies AO was rapidly biodegraded with >76% mineralized by study completion and the geometric mean of the primary biodegradation rates being 0.184h-1. Two OECD 303A Activated Sludge WWTP Simulation studies showed rapid and complete biodegradation of AO with ≤0.09% of parent AO remaining in the effluent, ≤0.03% of parent AO sorbed to sludge solids, and >97% complete mineralization of AO. Monitoring at US WWPTs confirmed low levels of AO in effluents with mean C12 and C14AO concentrations of 52.8 and 20.1ng/L respectively. Based on the monitoring data, the 90th percentile concentrations of C12 and C14AO for 7Q10 low flow stream conditions were >2 orders of magnitude lower than the predicted no effect concentrations indicating negligible aquatic risk from AO in US receiving streams. This study verifies that AO is safe for the aquatic environment even at the currently high usage volumes due to rapid biodegradation during transit through the sewer and wastewater treatment.

Probabilistic determination of the ecological risk from OTNE in aquatic and terrestrial compartments based on US-wide monitoring data.

OTNE [1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)ethan-1-one; trade name Iso E Super] is a fragrance ingredient commonly used in consumer products which are disposed down the drain. This research measured effluent and sludge concentrations of OTNE at 44 US wastewater treatment plants (WWTP). The mean effluent and sludge concentrations were 0.69 ± 0.65 µg/L and 20.6 ± 33.8 mg/kg dw respectively. Distribution of OTNE effluent concentrations and dilution factors were used to predict surface water and sediment concentrations and distributions of OTNE sludge concentrations and loading rates were used to predict terrestrial concentrations. The 90th percentile concentration of OTNE in US WWTP mixing zones was predicted to be 0.04 and 0.85 µg/L under mean and 7Q10 low flow (lowest river flow occurring over a 7 day period every 10 years) conditions respectively. The 90th percentile sediment concentrations under mean and 7Q10 low flow conditions were predicted to be 0.081 and 1.6 mg/kg dw respectively. Based on current US sludge application practices, the 90th percentile OTNE terrestrial concentration was 1.38 mg/kg dw. The probability of OTNE concentrations being below the predicted no effect concentration (PNEC) for the aquatic and sediment compartments was greater than 99%. For the terrestrial compartment, the probability of OTNE concentrations being lower than the PNEC was 97% for current US sludge application practices. Based on the results of this study, OTNE concentrations in US WWTP effluent and sludge do not pose an ecological risk to aquatic, sediment and terrestrial organisms.

Aquatic toxicity structure-activity relationships for the zwitterionic surfactant alkyl dimethyl amine oxide to several aquatic species and a resulting species sensitivity distribution.

Amine oxide (AO) is a cationically charged surfactant at environmental pH and has previously been assessed in the OECD (Organization for Economic Cooperation and Development) High Production Volume (HPV) chemicals program. Typical of cationic chemicals, AO is highly aquatically toxic. In this study we vastly improve the knowledge of AO toxicity by developing acute Quantitative Structure Activity Relationships (QSARs) for an alga (Desmodesmus subspicatus), an invertebrate (Daphnia magna) and a fish (Danio rerio) using the appropriate array of OECD Test Guidelines. A chronic toxicity QSAR was also determined for the most sensitive taxon, Desmodesmus. Pure AO spanning the chain lengths of C8 to C16 were tested individually with trace analytical confirmation of exposures in all tests. The QSARs were all of high quality (R2 0.92-0.98) with slopes ranging from -0.338 to -0.484. QSARs were then used to normalize toxicity outcomes for a larger, previously published data set used in HPV, European REACH (Registration, Evaluation, and Authorization of Chemicals), and peer reviewed publications. Two additional species, Lemna gibba (macrophyte) and Ankistrodesmus falcatus (alga) were studied in exposures to dodecyl (C12) AO to provide sufficient taxonomic diversity to conduct a Species Sensitivity Distribution (SSD) analysis. The SSD 5th percentile hazardous concentration (HC5) to C12 AO was found to be 0.052mg/L which is similar to an existing AO 28-d, 3-community periphyton community bioassay normalized to C12 AO (No-observed-effect-concentration or NOEC=0.152mg/L). The statistical properties of the SSD was probed suggesting that new studies of additional taxa would be required that were at least 10-fold more sensitive than the most sensitive taxon to move the HC5 lower by a factor of 3. The overall AO hazard assessment suggests a large margin of safety relative to published environmental exposure data.

Evaluation of anionic surfactant concentrations in US effluents and probabilistic determination of their combined ecological risk in mixing zones.

Alcohol sulfates (AS), alcohol ethoxysulfates (AES), linear alkyl benzenesulfonates (LAS) and methyl ester sulfonates (MES) are anionic surfactants that are widely used in household detergents and consumer products resulting in over 1 million tons being disposed of down the drain annually in the US. A monitoring campaign was conducted which collected grab effluent samples from 44 wastewater treatment plants (WWTPs) across the US to generate statistical distributions of effluent concentrations for anionic surfactants. The mean concentrations for AS, AES, LAS and MES were 5.03±4.5, 1.95±0.7, 15.3±19, and 0.35±0.13µg/L respectively. Since each of these surfactants consist of multiple homologues that differ in their toxicity, the concentration of each homologue measured in an effluent sample was converted into a toxic unit (TU) by normalizing to the predicted no effect concentration (PNEC) derived from high tier effects data (mesocosm studies). The statistical distributions of the combined TUs in the effluents were used in combination with distributions of dilution factors for WWTP mixing zones to conduct a US-wide probabilistic risk assessment for the aquatic environment for each of the surfactants. The 90th percentile level of TUs for AS, AES, LAS and MES in mixing zones were 1.89×10-2, 2.73×10-3, 2.72×10-2, and 3.65×10-5 under 7Q10 (lowest river flow occurring over a 7day period every 10years) low flow conditions. Because these surfactants have the same toxicological mode of action, the TUs were summed and the aquatic safety for anionic surfactants as a whole was assessed. At the 90th percentile level under the conservative 7Q10 low flow conditions the forecasted TUs were 4.21×10-2 which indicates that there is a significant margin of safety for the class of anionic surfactants in US aquatic environments.

Effects of season stratum corneum barrier function and skin biomarkers.

The skin on the lower legs of 25 female subjects was evaluated first in the winter, and then again in the summer of the same subjects. Barrier function was determined by measuring transepidermal water loss (TEWL), and skin hydration and dryness were evaluated by electrical measurements (Corneometer ® CM825) and visual grading. Stratum corneum (SC) was sampled using 10 sequential D-Squame sampling discs and analyzed for 2-pyrrolidone-5-carboxylic acid (PCA), keratin-1,10,11, interleukin 1α (IL-1α), interleukin 1 receptor antagonist (IL-1ra), selected ceramides, cholesterol, cholesterol sulfate, and selected free fatty acids. TEWL as well as the visual dryness grades were significantly lower in the summer while hydration was higher. PCA was significantly higher in the summer as were the keratins. The ratio IL-1ra:IL-1α, an indicator of skin inflammation, was significantly lower in the summer. The amount of protein removed by the tape strips was also significantly lower in summer indicating better SC cohesion. Among the SC lipids measured, total ceramides, individual ceramides, total fatty acids, and cholesterol were higher in summer compared to winter. Stearic acid and cholesterol sulfate were not significantly different between winter and summer.

Human systemic exposure to [¹4C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics, metabolism, excretion and safety assessment.

Systemic exposure was measured in humans after hair dyeing with oxidative hair dyes containing 2.0% (A) or 1.0% (B) [(14)C]-p-phenylenediamine (PPD). Hair was dyed, rinsed, dried, clipped and shaved; blood and urine samples were collected for 48 hours after application. [(14)C] was measured in all materials, rinsing water, hair, plasma, urine and skin strips. Plasma and urine were also analysed by HLPC/MS/MS for PPD and its metabolites (B). Total mean recovery of radioactivity was 94.30% (A) or 96.21% (B). Mean plasma Cmax values were 132.6 or 97.4 ng [(14)C]-PPDeq/mL, mean AUC(0-∞) values 1415 or 966 ng [(14)C]-PPDeq/mL*hr in studies A or B, respectively. Urinary excretion of [(14)C] mainly occurred within 24 hrs after hair colouring with a total excretion of 0.72 or 0.88% of applied radioactivity in studies A or B, respectively. Only N,N'-diacetylated-PPD was detected in plasma and the urine. A TK-based human safety assessment estimated margins of safety of 23.3- or 65-fold relative to respective plasma AUC or Cmax values in rats at the NOAEL of a toxicity study. Overall, hair dyes containing PPD are unlikely to pose a health risk since they are used intermittently and systemic exposure is limited to the detoxified metabolite N,N'-diacetyl-PPD.

Distributions of polycyclic musk fragrance in wastewater treatment plant (WWTP) effluents and sludges in the United States.

The polycyclic musks, AHTN and HHCB are fragrance ingredients widely used in consumer products. A monitoring campaign was conducted and collected grab effluent and sludge samples at 40 wastewater treatment plants (WWTP) across the United States to understand their occurrence and statistical distribution in these matrices. AHTN concentration in effluent ranged from <0.05 µg/L (LOQ) to 0.44 µg/L with a mean and standard deviation of 0.18 ± 0.11 µg/L. HHCB concentrations in effluent ranged from 0.45 to 4.79 µg/L with a mean of 1.86 ± 1.01 µg/L. AHTN concentrations in sludge ranged from 0.65 to 15.0mg/kg dw (dry weight) with a mean and standard deviation being 3.69 ± 2.57 mg/kg dw, while HHCB sludge concentrations were between 4.1 and 91 mg/kg with a mean of 34.0 ± 23.1mg/kg dw. Measured concentrations of AHTN and HHCB were significantly correlated with each other in both effluent and sludge. The concentrations of HHCB in both effluent and sludge were approximately an order of magnitude higher than those for AHTN, consistent with 2011 usage levels. The highest measured effluent concentrations for both AHTN and HHCB were below their respective freshwater PNECs (predicted no effect concentrations), indicating a negligible risk to biological communities below WWTPs, even in the absence of upstream dilution. Moreover, the large number of effluents and sludges sampled provides a statistical distribution of loadings that can be used to develop more extensive probabilistic exposure assessments for WWTP mixing zones and sludge amended soils.

Transcriptional profiling of epidermal barrier formation in vitro.

BACKGROUND: Barrier function is integral to the health of epithelial tissues. Currently, there is a broad need to develop and improve our knowledge with regard to barrier function for reversal of mild skin irritation and dryness. However, there are few in vitro models that incorporate modulations of both lipids and epidermal differentiation programs for pre-clinical testing to aid in the understanding of barrier health. OBJECTIVE: We have generated a reconstituted epidermis on a decellularized dermis (DED) and characterized its barrier properties relative to human epidermis in order to determine its utility for modeling barrier formation and repair. METHODS: We followed the process of epidermal differentiation and barrier formation through immunocytochemistry and transcriptional profiling. We examined barrier functionality through measurements of surface pH, lipid composition, stratum corneum water content, and the ability to demonstrate topical dose-dependent exclusion of surfactant. RESULTS: Transcriptional profiling of the epidermal model during its formation reveals temporal patterns of gene expression associated with processes regulating barrier function. The profiling is supported by gradual formation and maturation of a stratum corneum and expression of appropriate markers of epidermis development. The model displays a functional barrier and a water gradient between the stratum corneum and viable layers, as determined by confocal Raman spectroscopy. The stratum corneum layer displays a normal acidic pH and an appropriate composition of barrier lipids. CONCLUSION: The epidermal model demonstrates its utility as an investigative tool for barrier health and provides a window into the transcriptional regulation of multiple aspects of barrier formation.

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser-induced fluorescence detection.

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.

Trace analysis of bromate in potato snacks using high-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method in the negative-ion electrospray ionization (ESI(-)) mode was validated for the quantitation of bromate (BrO(3)(-)) in potato snacks. Ground snack specimens ( approximately 0.5 g/sample) are spiked with Br(18)O(3)(-), stable-isotope labeled bromate internal standard (IS), and vortexed with a mixture of distilled/deionized water (dd water) and heptane. Subsequently, the specimens are centrifuged, and a small portion of the aqueous extract is isolated, diluted with dd water (1:4), and analyzed by HPLC-MS/MS. The methodology has a quantitation range of 10-1000 ppb, an accuracy of 1.5-7.5%, and a precision of 5.2-13.4% across the concentration range.

Protein-aptamer binding studies using microchip affinity capillary electrophoresis.

The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.

Flow manipulation for sweeping with a cationic surfactant in microchip capillary electrophoresis.

Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.

Study of injection bias in a simple hydrodynamic injection in microchip CE.

The electrokinetically pinched method is the most commonly used mode for sample injection in microchip capillary electrophoresis (microCE) due to its simplicity and well-defined sample volume. However, the limited injection volume and the electrophoretic bias of the pinched injection may limit its universal usage to specific applications. Several hydrodynamic injection methods in microCE have been reported; however, almost all claimed that their methods are bias-free without considering the dispensing bias. To investigate the dispensing bias, a simple hydrodynamic injection was developed in single-T and double-T glass microchips. The sample flow was produced by hydrostatic pressure generated by the liquid level difference between the sample reservoir and the other reservoirs. The reproducibility of peak area and peak area ratio was improved to a significant extent using large-surface reservoirs for the buffer reservoir and the sample waste reservoir to reduce the Laplace pressure effect. Without a voltage applied on the sample solution, the voltage-related sample bias was eliminated. The dispensing bias was analyzed theoretically and studied experimentally. It was demonstrated that the dispensing bias existed and could be reduced significantly by appropriately setting up the voltage configuration and by controlling the appropriate liquid level difference.

Frontal analysis in microchip CE: a simple and accurate method for determination of protein-DNA dissociation constant.

Equilibrium constants, such as the dissociation constant (K(d)), are a key measurement of noncovalent interactions that are of importance for the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate CE method for the determination of K(d). Microchip CE coupled with LIF detection was used to determine K(d) of protein-DNA interactions using the FA method. A model system of IgE and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K(d) of the IgE-aptamer pair was determined as 6 +/- 2 nM which is consistent with the reported results (8 nM).