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.

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.

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).

Unlimited-volume electrokinetic stacking injection in sweeping capillary electrophoresis using a cationic surfactant.

Sweeping is an effective and convenient way for online sample preconcentration in micellar electrokinetic chromatography. The usual procedure includes a hydrodynamic injection step carried out by applying pressure to the sample vial followed by the subsequent sweeping and separation processes. The injected sample volume is limited by the dimensions of the capillary because a part of the capillary has to be left free of sample solution for the subsequent sweeping and separation steps. In addition, when a short capillary, such as 4-10 cm, is used for sweeping, the injected sample volume is small even if the entire capillary is filled with sample solution. To solve this problem, an electrokinetic stacking injection (EKSI) scheme was developed by using a cationic surfactant, dodecyltrimethylammonium bromide, for sweeping in capillary electrophoresis. An experimental model was proposed, and the entire process was theoretically analyzed. According to the theoretical discussion, the optimal conditions for two model analytes, 5-carboxyfluorescein (5-FAM) and sodium fluorescein (FL), were experimentally determined. The injected sample plug lengths for 5-FAM and FL under 20.1 kV for 60 min were experimentally estimated as 836 and 729 cm, corresponding to 28- and 24-fold the effective capillary length, respectively. The EKSI scheme resulted in increased detection factors for 5-FAM and FL of 4.5 x 10(3) and 4.0 x 10(3) using 60-min injection relative to a traditional pressure injection.

On-line sample preconcentration by sweeping with dodecyltrimethylammonium bromide in capillary zone electrophoresis.

On-line sample preconcentration of oligonucleotides with a new sweeping carrier was developed by using dodecyltrimethylammonium bromide (DTAB) below the critical micelle concentration (CMC). The sweeping results with DTAB below and above the CMC were compared. The use of DTAB below the CMC benefits the preconcentration of the oligonucleotides, while the use of DTAB above the CMC is good for hydrophobic small molecules. The factors affecting the sweeping results were optimized and this method was evaluated by constructing calibration curves for thrombin aptamers. The sweeping scheme produced a 112-fold sensitivity enhancement for the oligonucleotides relative to that run in a running buffer without DTAB. The sweeping method developed here can be a good reinforcement of the preconcentration scheme by sweeping when less-hydrophobic analytes or large negatively-charged molecules need to be preconcentrated.

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis.

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.

Modifications of the human urocortin 2 peptide that improve pharmacological properties.

Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.

Analytical performance of polymer-based microfluidic devices fabricated by computer numerical controlled machining.

A study comparing the electrophoretic separation performance attainable from microchips molded by masters fabricated using conventional CNC machining techniques with commercial microchips, wire imprinted microchips, and microchips from LIGA molding devices is presented. An electrophoresis-based detection system using fluorescence microscopy was used to determine the analytical utility of these microchips. The separation performance of CNC microchips was comparable to commercially available microchips as well as those fabricated from LIGA masters. The important feature of the CNC machined masters is that they have rapid design-to-device times using routinely available machining tools. This low-cost prototyping approach provides a new entry point for researchers interested in thermoplastic microchips and can accelerate the development of polymer-based lab-on-a-chip devices.

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection.

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.

On-line solid phase extraction using the Prospekt-2 coupled with a liquid chromatography/tandem mass spectrometer for the determination of dextromethorphan, dextrorphan and guaifenesin in human plasma.

An on-line liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure, using the Prospekt- 2 system, was developed and used for the determination of the levels of the active ingredients of cough/cold medications in human plasma matrix. The experimental configuration allows direct plasma injection by performing on- line solid phase extraction (SPE) on small cartridge columns prior to elution of the analyte(s) onto the analytical column and subsequent MS/MS detection. The quantitative analysis of three analytes with differing polarities, dextromethorphan (DEX), dextrorphan (DET) and guaifenesin (GG) in human plasma presented a significant challenge. Using stable-isotope-labeled internal standards for each analyte, the Prospekt-2 on-line methodology was evaluated for sensitivity, suppression, accuracy, precision, linearity, analyst time, analysis time, cost, carryover and ease of use. The lower limit of quantitation for the on-line SPE procedure for DEX, DET and GG was 0.05, 0.05 and 5.0 ng mL(-1), respectively, using a 0.1 mL sample volume. The linear range for DEX and DET was 0.05-50 ng mL(-1) and was 5-5,000 ng mL(-1) for GG. Accuracy and precision data for five different levels of QC samples were collected over three separate days. Accuracy ranged from 90% to 112% for all three analytes, while the precision, as measured by the %RSD, ranged from 1.5% to 16.0%

Estimation of logP(ow) values for neutral and basic compounds by microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (muMEEKC-IFD).

Microchip microemulsion electrokinetic chromatography with indirect fluorimetric detection (muMEEKC-IFD) was used to obtain logP octanol/water (logP(ow)) values for neutral and basic compounds. Six compounds, with logP(ow) values between 0.38 and 5.03, were used to create a calibration curve relating the log of retention factors (logk) obtained from muMEEKC-IFD with the known logP(ow) values. The logP(ow) values for six additional compounds were determined using the logk values obtained by muMEEKC-IFD and the linear relationship between logP(ow) and logk established for the standard compounds. The muMEEKC-IFD buffer was composed of 50 mM 3-[cyclohexylamino]-1-propane-sulfonic acid (CAPS) buffer (pH 10.4) containing 1.2% n-heptane (v/v), 2% sodium dodecylsulfate (w/v), 8% 1-butanol (v/v) and 4 microM 5-carboxytetramethyl-rhodamine (TAMRA) as the fluorophore probe for indirect detection. The muMEEKC-IFD provided an accurate method for estimating logP(ow) values and also a means for analyzing compounds that are non-fluorescent.