MicroNIR/Chemometrics: A new analytical platform for fast and accurate detection of Δ9-Tetrahydrocannabinol (THC) in oral fluids.

BACKGROUND: Δ9-Tetrahydrocannabinol (THC) is already considered one of the most addictive substances since an increasing number of consumers/abusers of THC and THC based products are observed worldwide. In this work, the capabilities of a novel miniaturized and portable MicroNIR spectrometer were investigated in order to propose a practical and intelligible test allowing the rapid and easy screening of Δ9-Tetrahydrocannabinol (THC) oral fluids without any pretreatment. METHODS: Specimens from volunteers were collected in order to consider any sources of variability in the spectral response and spiked with increasing amount of THC in order to realize predictive models to be used in real cases. Partial Least Square-Discriminant Analysis (PLS-DA) and Partial Least Square regression (PLSr) for the simultaneously detection and quantification of THC, were applied to baseline corrected spectra pre-treated by first derivative transform. RESULTS: Results demonstrated that MicroNIR/Chemometric platform is statistically able to identify THC abuse in simulated oral fluid samples containing THC from 10 to 100 ng/ml, with a precision and a sensitivity of about 1.51% and 0.1% respectively. CONCLUSIONS: The coupling MicroNIR/Chemometrics permits to simplify THC abuse monitoring for roadside drug testing or workplace surveillance and provides the rapid interpretation of results, as once the model is assessed, it can be used to process real samples in a "click-on" device.

Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates.

Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (lambda((ex)) 320nm, lambda((em)) 510nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5microg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0microg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS)> approximately 1.2 and a M(r)>3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS>0.4) can be determined.

Perspective on element speciation.

During the second half of the 20th century it became evident that trace elements play a major role whenever biological activities, environmental chemistry, or material characteristics are discussed and the determination of trace elements has therefore gained outstanding importance in environmental and life sciences. Elements, even when present at minimal concentrations in biological and environmental matrices, can, in fact, exert fundamental influence on ecosystems and the vital functions of organisms. The study of, for example, pathophysiological processes in the human body requires the determination of elements down to the ng kg(-1) range. However, organic and inorganic matrix compounds at much higher concentration levels in a sample than trace elements make the determination of trace elements often rather difficult.

Principle, calibration, and application of the in situ alkali chloride monitor.

The extended use of biomass for heat and power production has caused increased operational problems with fouling and high-temperature corrosion in boilers. These problems are mainly related to the presence of alkali chlorides (KCl and NaCl) at high concentrations in the flue gas. The in situ alkali chloride monitor (IACM) was developed by Vattenfall Research and Development AB for measuring the alkali chloride concentration in hot flue gases (less than or approximately 650 degrees C). The measurement technique is based on molecular differential absorption spectroscopy in the UV range. Simultaneous measurement of SO(2) concentration is also possible. The measuring range is 1-50 ppm for the sum of KCl and NaCl concentrations and 4-750 ppm for SO(2). This paper describes the principle of the IACM as well as its calibration. Furthermore, an example of its application in an industrial boiler is given.

Nuevos riesgos tóxicos por exposición a nanopartículas / New toxic risks due to nanoparticles exposure

La nanotecnología es una ciencia multidisciplinar que está teniendoun gran auge en la actualidad, ya que proporciona productos(nanopartículas) con nuevas propiedades fisicoquímicas, que son lasque hacen que tengan una gran cantidad de aplicaciones. Laexposición humana a estas nanopartículas se puede producirprincipalmente por las vías respiratoria (nanopartículas suspendidasen el aire), dérmica (nanopartículas ambientales, cosméticos) y oral(alimentos, agua). Por vía pulmonar las nanopartículas activan losmecanismos de defensa o son internalizadas en los intersticios. Porvía dérmica se pueden acumular en el estrato córneo o en los folículospilosos, o bien atravesarlo y acumularse en la dermis. Por vía oralpueden ser absorbidas por las células epiteliales del intestino. Laexposición también se puede producir a través de la instrumentaciónmédica o prácticas clínicas, ya que se usan, por ejemplo, en eltratamiento y diagnóstico del cáncer de mama y en el control deinfecciones en cirugía. Una vez las nanopartículas han sidoabsorbidas, se distribuyen por vía sanguínea y linfática, alcanzandodiferentes órganos, tales como huesos, riñones, páncreas, bazo,hígado y corazón, en los que quedan retenidas y ejercen sus efectostóxicos, aunque esto también se utiliza como una forma devectorización de fármacos. La toxicidad de estas nanopartículasdepende, entre otros factores, de su persistencia en los órganos y de siel hospedador puede provocar una respuesta biológica paraeliminarlas. Los mecanismos de toxicidad no se conocen conexactitud, aunque parece ser que se incluyen daño en membranascelulares, disrupción del potencial de membrana, oxidación deproteínas, genotoxicidad, formación de especies reactivas de oxígenoe inflamación. Estudios sobre las vías respiratorias han mostradodisminución de la viabilidad celular in vitro, producción de estrésoxidativo e inflamación...(AU)

Nanotechnology is a multi-disciplinary science which is having a great growth at present, as it provides products (nanoparticles) withnew physico-chemical properties that can have many applications.Human exposure to these nanoparticles can be produced byrespiratory (airborne nanoparticles), dermal (atmosphericnanoparticles, cosmetics) and oral routes (food, water). Byrespiratory route, nanoparticles can stimulate the defensemechanisms or can penetrate into gaps. By dermal route, they can beaccumulated in the stratum corneum or in the hair follicles, or gothrough it and be accumulated in the dermis. By gastrointestinal routethey can be absorbed by the epithelial cells of the intestine. Humancan also be exposed by medical instrumentation or clinic practices, asnanoparticles are used, for example, for treatment and diagnostic ofbreast cancer and to control surgery infections. Once nanoparticleshave been absorbed they are distributed by blood and lymphaticstream, reaching different organs, such as bones, kidneys, pancreas,spleen, liver and heart, where they are retained and can produce theirtoxic effects, although this ability is also used for drugs delivery. Thetoxicity of these nanoparticles depends, among other factors, on theirpermanence in organs and if the host can produce a biologicalresponse to eliminate them. The toxicity mechanisms have not beencompletely elucidated, although they are known to produce cellmembrane damages, membrane potential disruption, proteinsoxidation, genotoxicity, production of reactive oxygen species, andinflammation. Studies on the respiratory exposure have demonstrateda diminution of the cellular viability in vitro, oxidative stressproduction, and inflammation. On skin have been demonstratedtoxicity and oxidative stress, although other authors have shown theabsence of irritation and allergic reactions...(AU)

Performance evaluation of thermal cyclers for PCR in a rapid cycling condition.

The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 microL reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoid unexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.

Verifying liquid-handler performance for complex or nonaqueous reagents: a new approach.

Multichannel volume dispensing devices, such as automated liquid handlers, are widely used in drug discovery assays and other high-throughput screening processes. The performance of these systems is heavily based on the ability to deliver proper volumes of specific reagents. Discussed herein is the recent research on broadening existing methods for accurately assessing liquid-handler performance when dispensing complex or nonaqueous reagents. Accurate and reliable adjustment of liquid-handler protocols for varied reagent types could have far-reaching adoption in all scientific communities.

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing.

Mass spectrometry has become an invaluable tool for the identification of prohibited peptide hormones and proteins in doping control analysis. Regulatory authorities have established criteria for identifying banned drugs in doping control specimens, but these criteria do not address the specific issues for high molecular weight protein drugs such as molecular weight determination of multiply charged molecules, analysis of chemically or enzymatically derived degradation products, identification of amino acid sequence tags, etc. Technical considerations such as sample preparation methods (e.g. immunoaffinity purification), resulting analytes (e.g. intact compounds vs. chemically or enzymatically derived peptides), ionization modes, analyzer resolution, and the information provided by respective techniques are discussed in light of sports drug testing requirements using typical application examples.

Extending the scope of NMR spectroscopy with microcoil probes.

Capillary NMR (CapNMR) spectroscopy has emerged as a major breakthrough for increasing the mass-sensitivity of NMR spectroscopic analysis and enabling the combination of NMR spectroscopy with other analytical techniques. Not only is the acquisition of high-sensitivity spectra getting easier but the quality of CapNMR spectra obtained in many small-molecule applications exceeds what can be accomplished with conventional designs. This Minireview discusses current CapNMR technology and its applications for the characterization of mass-limited, small-molecule and protein samples, the rapid screening of small-molecule or protein libraries, as well as hyphenated techniques that combine CapNMR with other analytical methods.

Sub part-per-million mass accuracy by using stepwise-external calibration in fourier transform ion cyclotron resonance mass spectrometry.

A new external calibration procedure for FT-ICR mass spectrometry is presented, stepwise-external calibration. This method is demonstrated for MALDI analysis of peptide mixtures, but is applicable to any ionization method. For this procedure, the masses of analyte peaks are first accurately measured at a low trapping potential (0.63 V) using external calibration. These accurately determined (< 1 ppm accuracy) analyte peaks are used as internal calibrant points for a second mass spectrum that is acquired for the same sample at a higher trapping potential (1.0 V). The second mass spectrum has a approximately 10-fold improvement in detection dynamic range compared with the first spectrum acquired at a low trapping potential. A calibration equation that accounts for local and global space charge is shown to provide mass accuracy with external calibration that is nearly identical to that of internal calibration, without the drawbacks of experimental complexity or reduction of abundance dynamic range. For the 609 mass peaks measured using stepwise-external calibration method, the root-mean-square error is 0.9 ppm. The errors appear to have a Gaussian distribution; 99.3% of the mass errors are shown to lie within three times the sample standard deviation (2.6 ppm) of their true value.

Regular arrays of microdisc electrodes: simulation quantifies the fraction of 'dead' electrodes.

Arrays of microdisc electrodes have found widespread use in electroanalysis. These are commonly produced lithographically and practical arrays may contain up to hundreds of individual disc electrodes (e.g. of gold, platinum, indium,...) to maximise sensitivity and minimise limits of detection. Typically, however, the lithographic fabrication process is imperfect resulting in a significant fraction (often tens of percent) of electrochemically inactive electrodes. We demonstrate that a 2-dimensional simulation based on the diffusion domain approximation in conjugation with simple experiments on the ferrocyanide redox couple in aqueous solutions can be used to rigorously 'count' the number of active electrodes in a non-destructive fashion. The agreement with an independent count in which active electrodes are identified via electro-plating with copper followed by ex situ microscopic examination is quantitatively excellent.

Quantification of gliadin levels to the picogram level by flow cytometry.

BACKGROUND: Celiac disease is a widely prevalent enteropathy caused by intolerance to gliadin, one of the gluten proteins. We developed two methods for the analysis of gliadin levels. Both methods use flow cytometry and rat antibodies against a 16-residue peptide of gliadin. The peptide is common to the alpha-, beta-, gamma-, and omega-gliadins. METHODS: In the one-site assay, the antigen (gliadin standard or food extract) was adsorbed on 3-mum latex particles. Sensitized particles were then incubated, in this order, with rat anti-gliadin peptide antibodies and anti-rat immunoglobulin G antibodies labeled with fluorescein isothiocyanate. In the two-site assay, the antigen was trapped on the latex particles by rat anti-gliadin antibodies and then measured by the same antibodies labeled with fluorescein. RESULTS: Detection limits were 1 ng/ml for the one-site assay and 10 pg/ml for the two-site assay. The two-site assay displayed gliadin at concentrations above the limit proposed by the Codex Alimentarius in 2 of 40 gluten-free products. CONCLUSION: There is a growing concern that gliadin, even when present in gluten-free foods within the limit fixed by the Codex Alimentarius, over the long term may become toxic to patients with celiac disease. The techniques described in this study provide an opportunity to further decrease the acceptable limit of gliadin in gluten-free foods.

Development of a micro-fluidic manifold for copper monitoring utilising chemiluminescence detection.

The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples, based on the measurement of light emitted from the Cu(ii) catalysed oxidation of 1,10-phenanthroline by hydrogen peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA) using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 x 250 microm in width and depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 x 250 microm in width and depth (total approx. volume of between 16 to 22 microL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline, containing 0.10 M cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL min(-1)) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min(-1)). The sample stream was mixed with reagent 1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated manifold was found to give a linear response (R(2) = 0.998) over the concentration ranges 0-150 microg L(-1) and be reproducible (% RSD = 3.4 for five repeat injections of a 75 microg L(-1) std). Detection limits for Cu(ii) were found to be 20 microg L(-1). Selectivity was investigated using a copper selective mini-chelating column, which showed common cations found in drinking waters did not cause interference with the detection of Cu(ii). Finally the optimised system was successfully used for trace Cu(ii) determinations in a standard reference freshwater sample (SRM 1640).

Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions.

The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2 x 10(-4) to 6 x 10(-6) M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4-5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.

Cytometric analysis of protein expression and apoptosis in human primary cells with a novel microfluidic chip-based system.

BACKGROUND: Work with primary cells is inherently limited by source availability and life span in culture. Flow cytometry offers extensive analytical opportunities but generally requires high cell numbers for an experiment. METHODS: We have developed assays on a microfluidic system, which allow flow cytometric analysis of apoptosis and protein expression with a minimum number of fluorescently stained primary cells. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by two-channel fluorescence detection. For some assays the staining reactions can be performed on-chip and the analysis is done without further washing steps. RESULTS: We have successfully applied the assays to evaluate (a) activation of E-selectin (CD62E) expression by interleukin-1beta in human umbilical vein endothelial cells (HUVECs), (b) induction of CD3 by phorbol-12-myristate-13-acetate in freshly prepared human peripheral blood lymphocytes, and (c) staurosporine-induced apoptosis in HUVEC and normal human dermal fibroblasts. CONCLUSIONS: Results obtained with the microfluidic system are in good correlation with data obtained using a standard flow cytometer, but demonstrate new dimensions in low reagent and cell consumption.

High-resolution capillary tube NMR. A miniaturized 5-microL high-sensitivity TXI probe for mass-limited samples, off-line LC NMR, and HT NMR.

A new triple-resonance (TXI) (1H, 13C, 15N) high-resolution nuclear magnetic resonance (NMR) capillary probe with 2.5-microL NMR-active sample volume (V(obs)) was built and tested for applications with mass- and volume-limited samples and for coupling of microbore liquid chromatography to NMR. This is the first microliter probe with optimized coil geometry for use with individual capillary tubes with an outer diameter of 1 mm. The 90 degree pulse lengths of the 1-mm microliter probe were below 2 micros for proton, below 8 micros for carbon, and below 20 micros for nitrogen, and a spectral line width at signal half-height below 1 Hz was obtained. Compared to a conventional 5-mm probe, the new 600-MHz 1-mm TXI microliter probe with z-gradient shows an increase in mass sensitivity by a factor of 5, corresponding to a 25-fold reduction in measuring time. The consumption of costly deuterated solvent is reduced by at least 2 orders of magnitude. The 1-mm TXI microliter probe with z-gradient allows the measurement of one-dimensional 1H NMR and two-dimensional heteronuclear NMR spectra with a few nanomoles (micrograms) of compound with high sensitivity, speed, and quality. This is a breakthrough for discrete sample NMR spectroscopy with paramount importance for structure elucidation in natural compound chemistry and metabolic research. It offers also advantages for linking chromatographic methods to NMR in a nindustrial environment. Capillary tube NMR may find new applications in areas where high sample throughput is essential, e.g., in the quality control of large sample arrays from parallel chemistry, screening, and compound depositories. It has the potential to increase the sample throughput by 1 order of magnitude or more if new hardware for fast sample handling and exchange becomes available.

Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection.

A modular instrument for high-speed microchip electrophoresis (MCE) equipped with a sensitive variable-wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp-based epifluorescence microscope for variable-wavelength fluorescence detection and imaging and a programmable four-channel bipolar high-voltage source capable of delivering up to +/- 10 kV per channel. The optical unit was equipped with a high-sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)-labelled amines utilizing blue light (450-480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531-560 nm). At optimized conditions baseline separation of four FITC-labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal-to-noise ratio of 3:1. Three rhodamines could be baseline-separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC-labelled amines were below 2.5% (n= 25). By the application of cyclodextrin-modified electrolytes, chiral separation of FITC-labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high-throughput screening.

A standard operating procedure for assessing liquid handler performance in high-throughput screening.

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.

Integrated self-calibration via electrokinetic solvent proportioning for microfluidic immunoassays.

On-board generation of a set of calibration standards was demonstrated within a microfluidic device designed to perform immunoassay. Electrokinetic flow was used to proportionally mix the antibody (Ab) to bovine serum albumin (BSA) and a diluting buffer, to provide varying Ab concentrations for downstream mixing with fluorescently labeled BSA (BSA*). Mixing ratios were determined from electrical impedance modeling of the fluidic network using P-SPICE software, and peak heights for the labeled species were analyzed relative to the concentration calculated from the model. For dilution and separation of fluorescently labeled amino acids, a linear calibration curve was obtained for mixing ratios of 0.118 to 7.46. A linear calibration curve was obtained for the immunoassay calibration using dilution ratios between 0.197 and 5.077. Deviations were observed at larger extremes, possibly due to leakage effects at intersections. Peak height reproducibility was +/- 3% for the immunoassay, using diluted monoclonal Ab in mouse ascites fluid as the analyte. Recovery for on-chip calibration was 92 +/- 6% versus calibrants prepared off-chip, indicating a small bias.