A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.     

Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody

A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0-500 ppb) under optimum conditions. The time required for analysis is less than 15 min.     

Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum

A novel immunoassay for the determination of tumor markers in human serum was established by combining a time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a sandwich-type immunoassay format, analytes in samples were captured by magnetic beads coated with one monoclonal antibody and “sandwiched” by another monoclonal antibody labeled with europium chelates. The immunocomplex was separated and washed by exposure to a magnetic field and treatment with enhancement solution; fluorescence was then measured according to the number of europium ions dissociated. Levels of the model analyte, carcinoembryonic antigen (CEA), were determined in a linear range (1–1000 ng mL⁻¹) with a limit of detection of 0.5 ng mL⁻¹ under optimal conditions. The reproducibility, recovery, and specificity of the immunoassay were demonstrated to be acceptable. To evaluate this novel assay for clinical applications, 239 serum samples were evaluated. Compared with the conventional TRFIA and chemiluminescence immunoassay (CLIA), the correlation coefficients of the developed immunoassay were 0.985 and 0.975, respectively. These results showed good correlation and confirmed that our method is feasible and could be used for the clinical determination of CEA (or other tumor antigens) in human serum.     

High-throughput quantification of a novel thiazolidinedione MCC-555 in rat plasma by ultra-fast liquid chromatography and its application in pharmacokinetic studies

MCC-555 is a novel thiazolidinedione which reduces plasma glucose concentrations in Type 2 diabetes mellitus models due to enhancement of insulin sensitivity. A highly sensitive and selective quantitative method to accurately determine MCC-555 in rat plasma is crucial to the success of pharmacokinetic studies of MCC-555. To this purpose we have developed and validated a high-throughput method in a 96-well plate format using ultra-fast liquid chromatography (Shimadzu Prominence UFLC™ system) for the determination of MCC-555 in rat plasma. MCC-555 along with the internal standard resveratrol was extracted from 50 μl of rat plasma by liquid-liquid extraction using ethyl acetate. Baseline separation of MCC-555 and resveratrol was achieved using UFLC technology on a C18 stationary-phase column with 2.2 μm particle size. The influences of flow rate, column temperature and mobile phase pH on chromatographic performance were investigated. Comparing to the conventional HPLC method, UFLC showed many advantages including reduced run time, less solvent consumption and increased sensitivity. The UFLC method was sensitive with a lower limit of quantification of 0.002 μg/ml, with good linearity (r > 0.999) over the linear range of 0.002-2.0 μg/ml. The intra- and inter-run precision was less than 8.6% and accuracy ranged from −6.4 to 8.2% for quality control samples. The extraction recovery from plasma was no less than 80%. The validation and sample analysis results show that the method is precise, accurate and well suited to support pharmacokinetic studies in rats involving three dose administrations.     

Preparation of styrene-co-4-vinylpyridine magnetic polymer beads by microwave irradiation for analysis of trace 24-epibrassinolide in plant samples using high performance liquid chromatography

In the study, a kind of novel styrene-co-4-vinylpyridine (St-co-4-VP) porous magnetic polymer beads was prepared by microwave irradiation using suspension polymerization. Microwave heating preparation greatly reduced the polymerization time to 1 h. Physical characteristic tests suggested that these beads were cross-linking and possessed spherical shape, good magnetic response and porous morphologies with a narrow diameter distribution of 70–180 μm. Therefore, these beads displayed the long-term stability after undergoing 100-time extractions. Then, an analytical method for the determination of trace 24-epiBR in plant samples was developed by magnetic polymer bead extraction coupled with high performance liquid chromatography–fluorescence detection. St-co-4-VP magnetic polymer beads demonstrated the higher extraction selectivity for 24-epiBR than other reference compounds. Linear range was 10.00–100.0 μg/L with a relative standard deviation (RSD) of 6.7%, and the detection limit was 6.5 μg/kg. This analytical method was successfully applied to analyze the trace 24-epiBR in cole and breaking-wall rape pollen samples with recoveries of 77.2–90.0% and 72.3–83.4%, respectively, and RSDs were less than 4.1%. The amount of 24-epiBR in real breaking-wall rape pollen samples was found to be 26.2 μg/kg finally. This work proposed a sensitive, rapid, reliable and convenient analytical method for the determination of trace brassinosteroids in complicated plant samples by the use of St-co-4-VP magnetic polymer bead extraction coupled with chromatographic method.     

Aptamer based strategy for cytosensing and evaluation of HER-3 on the surface of MCF-7 cells by using the signal amplification of nucleic acid-functionalized nanocrystals

The electrochemical detection of cell lines of MCF-7 (human breast cancer) has been reported, using magnetic beads for the separation tool and high-affinity DNA aptamers for signal recognition. The high specificity was obtained by using the magnetic beads and aptamers, and the good sensitivity was realized with the signal amplification of DNA capped CdS or PbS nanocrystals. The ASV (anodic stripping voltammetry) technology was employed for the detection of cadmic cation and lead ions, for electrochemical assay of the amount of the target cells and biomarkers on the membrane of target cells, respectively. This electrochemical method could respond to as low as 100 cells mL⁻¹ of cancer cells with a linear calibration range from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹, showing very high sensitivity. Moreover, the amounts of HER-3 which were overexpressed on MCF-7 cells were calculated correspond to be 3.56 × 10⁴ anti-HER-3 antibody molecules. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamers and magnetic beads used in the assay, indicating the wide applicability of the assay for early and accurate diagnose of cancers.     

Magnetic molecularly imprinted polymer beads prepared by microwave heating for selective enrichment of β-agonists in pork and pig liver samples

Novel magnetic molecularly imprinted polymer (MIP) beads using ractopamine as template for use in extraction was developed by microwave heating initiated suspension polymerization. Microwave heating, as an alternative heating source, significantly accelerate the polymerization process. By incorporating magnetic iron oxide, superparamagnetic composite MIP beads with average diameter of 80 μm were obtained. The imprinted beads were then characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and vibrating sample magnetometer. Highly cross-linked porous surface and good magnetic property were observed. The adsorption isotherm modeling was performed by fitting the data to Freundlich isotherm model. The binding sites measured were 3.24 μmol g⁻¹ and 1.17 μmol g⁻¹ for the magnetic MIP beads and the corresponding non-imprinted magnetic beads, respectively. Cross-selectivity experiments showed the recognition ability of the magnetic MIP beads to analytes is relative to degree of molecular analogy to the template. Finally, this magnetic MIP bead was successfully used for enrichment of ractopamine, isoxsuprine and fenoterol from ultrasonically extracted solution of pork and pig liver followed by high performance chromatography with fluorescence detection. The proposed method presented good linearity and the detection limits was 0.52-1.04 ng mL⁻¹.The recoveries were from 82.0% to 90.0% and from 80.4% to 86.8% for the spiked pork and pig liver, respectively, with the RSDs of 5.8-10.0%. Combination of the specific adsorption property of the MIP material and the magnetic separation provided a powerful analytical tool of simplicity, flexibility, and selectivity.     

A highly sensitive enzyme immunoassay for evaluation of 2′-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2′-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5′-succinyl-dCyd (5′sdCyd) conjugate of bovine serum albumin (5′sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5′sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5′sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of ≤10% was 20-800 nM. No cross-reactivity from the structurally related nucleobases, nucleosides, and nucleotides was observed in the proposed assay. Mean analytical recovery of added dCyd was 98-100 ± 3.2-8.2%. The precision of the assay was satisfactory; R.S.D. was 3.4-4.2 and 4.3-8.9% for intra- and inter-assay precision, respectively. The proposed EIA was compared favorably with HPLC method in its ability to accurately measure dCyd spiked into plasma samples. The analytical procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA is expected to contribute in further evaluation of dCyd as a prognostic marker for breast cancer chemotherapy and elucidation of the role of dCyd in various biological and biochemical systems.     

Phase equilibria for salt-induced lysozyme precipitation: Effect of salt type and temperature

The salt-induced precipitation of lysozyme from aqueous solutions was studied through precipitation assays in which the equilibrium compositions of the coexisting phases were determined. Lysozyme precipitation experiments were carried out at 5, 15 and 25 °C and pH 7.0 with ammonium sulfate, sodium sulfate and sodium chloride as precipitating agents. In these experiments a complete separation of the coexisting phases (liquid and solid) could not be achieved. Nevertheless it was possible to determine the composition of the precipitate. The enzymatic activity of lysozyme in the supernatant phase as well as in the precipitate phase was also determined. The activity balance suggests that there is a relationship between the composition of the true precipitate and the total activity recovery.     

Pipette tip solid-phase extraction and gas chromatography-mass spectrometry for the determination of mequitazine in human plasma

Mequitazine has been found to be extractable from human plasma samples using MonoTip C₁₈ tips, inside which C₁₈-bonded monolithic silica gel was fixed. Human plasma (0.1 mL) containing mequitazine and cyproheptadine as an internal standard (IS) was mixed with 0.4 mL of distilled water and 25 μL of 1 M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C₁₈ phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C₁₈ phase were then eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was injected into a gas chromatograph injector and detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of mequitazine and the IS from each other and from impurities was generally satisfactory using a DB-1MS capillary column (30 m × 0.32 mm i.d., film thickness 0.25 μm). The recoveries of mequitazine and the IS spiked into plasma were more than 90.0%. The regression equation for mequitazine showed excellent linearity in the range of 0.2-200 ng 0.1 mL⁻¹, and the detection limit was 0.05 ng 0.1 mL⁻¹of plasma. The intra-day and inter-day coefficients of variation for mequitazine in human plasma were not greater than 8.16 and 9.24%, respectively. Accuracy for the drug was in the range of 90.0-97.4%. The data obtained from determination of mequitazine in human plasma after oral administration of the drug are also presented.     

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 μg mL⁻¹, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r = 0.9993 for OMT, r = 0.9991 for MT), fair wide linear concentration range (1.30-65.00 μg mL⁻¹ for OMT, 0.84-42.00 μg mL⁻¹ for MT), low limit of detection (1.03 μg mL⁻¹ for OMT, 0.38 μg mL⁻¹ for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.     

Development of an HPLC method for the determination of mexiletine in human plasma and urine by solid-phase extraction

A sensitive and specific high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of mexiletine (MEX) in human plasma and urine. It uses solid-phase extraction (SPE) followed by an automated reversed-phase HPLC with a pre-column derivatization with 4-chloro-7-nitrobenzofurazan (NBD-CI) and UV-vis Absorbance detection. The process was set as: the UV-vis Absorbance wavelength was set at 458 nm. Chromatographic separation was performed on a Phenomenex-C₁₈ Column (Aqua, 150 mm × 4.6 mm i.d. with 5 μm particle size) with the mobile phase consisting of acetonitrile and water (80:20, v/v), and the flow rate was set at 1.0 mL min⁻¹. Calibration of the overall analytical procedure gave a linear signal (r > 0.9998) over a MEX concentration range of 0.2-2.0 μg mL⁻¹ in human plasma and urine. The detection limit in plasma and urine was 0.1 μg mL⁻¹. Intra- and inter-day precision of the assay at three concentrations within this range were 0.31-2.50%. The high specificity and sensitivity have been achieved by this fast method (total run-time <6 min). The method has been successfully validated in human plasma and urine and it has been shown to be precise, accurate and reliable.     

Generic simple enzyme immunoassay approach to avert small molecule immobilization problems on solid phases: Application to the determination of tobramycin in serum

Generic simple and sensitive universal enzyme immunoassay approach for the determination of small analytes has been developed to avert the problems associated with small molecule immobilization onto solid phases. The developed assay employed a heterogeneous non-competitive binding format. The assay used anti-analyte antibody coupled to polyacrylamide beads as a solid-phase and β-d-galactosidase enzyme-labeled analyte as a label. In this assay, the analyte in a sample was firstly incubated to react with an excess of the antibody-coupled beads, and then the unoccupied antibody binding sites were allowed to react with the enzyme-labeled analyte. Analyte bound to the antibody-coupled beads was separated by centrifugation, and the enzyme activity of the supernatant was measured spectrophotometrically at 420 nm, after reaction with 4-nitrophenyl-β-d-galactopyranoside as a substrate for the enzyme. The signal was directly proportional to the concentration of analyte in the sample. The optimum conditions for the developed assay were established and applied to the determination of tobramycin, as a representative example of the small analytes, in serum samples. The assay limit of detection was 10 ng mL⁻¹ and the effective working range at relative standard deviation of ≤10% was 40-800 ng mL⁻¹. The assay precisions were acceptable; the relative standard deviations were 4.36-5.17 and 5.62-7.40% for intra- and inter-assay precision, respectively. Analytical recovery of tobramycin spiked in serum ranged from 95.89 ± 4.25 to 103.45 ± 4.60%. The assay results correlated well with those obtained by high-performance liquid chromatography (r = 0.992). The assay described herein has great practical value in determination of small analytes because it is sensitive, rapid, and easy to perform in any laboratory. Although the assay was validated for tobramycin, however, it is also anticipated that the same methodology could be used for essentially any analyte for which a selective antibody exists, and an appropriate enzyme conjugate can be made.     

Micellar electrokinetic chromatography of organic and peroxide-based explosives

CE methods have been developed for the analysis of organic and peroxide-based explosives. These methods have been developed for deployment on portable, in-field instrumentation for rapid screening. Both classes of compounds are neutral and were separated using micellar electrokinetic chromatography (MEKC). The effects of sample composition, separation temperature, and background electrolyte composition were investigated. The optimised separation conditions (25 mM sodium tetraborate, 75 mM sodium dodecyl sulfate at 25 °C, detection at 200 nm) were applied to the separation of 25 organic explosives in 17 min, with very high efficiency (typically greater than 300,000 plates m⁻¹) and high sensitivity (LOD typically less than 0.5 mg L⁻¹; around 1–1.5 μM). A MEKC method was also developed for peroxide-based explosives (10 mM sodium tetraborate, 100 mM sodium dodecyl sulfate at 25 °C, detection at 200 nm). UV detection provided LODs between 5.5 and 45.0 mg L⁻¹ (or 31.2–304 μM), which is comparable to results achieved using liquid chromatography. Importantly, no sample pre-treatment or post-column reaction was necessary and the peroxide-based explosives were not decomposed to hydrogen peroxide. Both MEKC methods have been applied to pre-blast analysis and for the detection of post-blast residues recovered from controlled, small scale detonations of organic and peroxide-based explosive devices.     

Phospholipidomic identification of potential plasma biomarkers associated with type 2 diabetes mellitus and diabetic nephropathy

Type 2 diabetes mellitus (T2DM) and its attendant complications, such as diabetic nephropathy (DN), impose a significant societal and economic burden. The investigation of discovering potential biomarkers for T2DM and DN will facilitate the prediction and prevention of diabetes. Phospholipids (PLs) and their metabolisms are closely allied to nosogenesis and aggravation of T2DM and DN. The aim of this study is to characterize the human plasma phospholipids in T2DM and DN to identify potential biomarkers of T2DM and DN. Normal phase liquid chromatography coupled with time of flight mass spectrometry (NPLC-TOF/MS) was applied to the plasma phospholipids metabolic profiling of T2DM and DN. The plasma samples from control (n = 30), T2DM subjects (n = 30), and DN subjects (n = 52) were collected and analyzed. The significant difference in metabolic profiling was observed between healthy control group and DM group as well as between control group and DN group by the help of partial least squares discriminant analysis (PLS-DA). PLS-DA and one-way analysis of variance (ANOVA) were successfully used to screen out potential biomarkers from complex mass spectrometry data. The identification of molecular components of potential biomarkers was performed on Ion trap-MS/MS. An external standard method was applied to quantitative analysis of potential biomarkers. As a result, 18 compounds in 7 PL classes with significant regulation in patients compared with healthy controls were regarded as potential biomarkers for T2DM or DN. Among them, 3 DM-specific biomarkers, 8 DN-specific biomarkers and 7 common biomarkers to DM and DN were identified. Ultimately, 2 novel biomarkers, i.e., PI C18:0/22:6 and SM dC18:0/20:2, can be used to discriminate healthy individuals, T2DM cases and DN cases from each other group.     

Rapid and reagent-saving immunoassay using innovative stirring actions of magnetic beads in microreactors in the sequential injection mode

We developed new ELISA techniques in sequential injection analysis (SIA) mode using microreactors with content of a few microliters. We immobilized antibodies on magnetic beads 1.0 μm in diameter, injected the beads into microreactors and applied rotating magnetic fields of several hundred gauss. Magnetic beads, suspended in liquid in density of approximately 10⁹-10¹⁰ particles per millilitre, form a large number of thin rod clusters, whose length-wise axes are oriented in parallel with the magnetic field. We rotate the Nd magnets below the center of the microreactor by a tiny motor at about 2000-5000 rpm. These rotating clusters remarkably accelerate the binding rate of the antibodies with antigens in the liquid. The beads are trapped around the center of the rotating magnetic field even in the flowing liquid. This newly found phenomenon enables easy bead handling in microreactors. Modification of reactor walls with selected blocking reagents was essential, because protein-coated beads often stick to the wall surface and cannot move freely. Washing steps were also shortened.     

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

Sensitive capillary electrophoresis microchip determination of trinitroaromatic explosives in nonaqueous electrolyte following solid phase extraction

A capillary electrophoresis (CE) microchip is utilized for the sensitive separation and detection of three trinitroaromatic explosives: 1,3,5-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), in the presence of 10 other explosives and explosive derivatives in nonaqueous electrolyte (acetonitrile/methanol 87.5/12.5 (v/v), 2.5 mM NaOH, 1 mM sodium dodecyl sulfate (SDS)). The chemical reaction of bases, e.g. hydroxide or methoxide ions, with trinitroaromatic compounds forms red colored derivatives that can be easily detected using a green light emitting diode (LED) on the microchip. Two surfactants bearing opposite charge, cetyltrimethylammonium bromide (CTAB) and SDS are compared with respect to their effect on separation times, detection limits and resolving powers for separating these explosives. All microchip separations were achieved in <20 s. In the absence of solid phase extraction (SPE), the detection limits obtained for the trinitroaromatic explosives were as follows: TNB, 60 μg/l; TNT, 160 μg/l and tetryl, 200 μg/l. By coupling the microchip separation with ex situ SPE, the detection limits for detecting these three explosives in seawater were lowered by 240 to more than 1000 times: TNB, 0.25 μg/l; TNT, 0.34 μg/l and tetryl, 0.19 μg/l.     

Depletion of human lymphocytes from peripheral blood and bone marrow by affinity ligands conjugated to agarose—polyacrolein microsphere beads

Protein-A or goat anti-mouse-Ig (GAMIg) covalently bound to agarose-polyacrolein microsphere beads (APAMB) were employed for the removal of T cells from human peripheral blood leukocytes (PBL) and bone marrow (BM). The cell suspensions were treated with a monoclonal anti-T cell antibody (Leu-1) or monoclonal antilymphocyte antibody (CAMPATH-1) and passed through the conjugated APAMB columns. Cell separation efficacy was determined by assaying the number and function of T cells in the final cell preparation in comparison with a sample of unseparated cells. The number of cells that form rosettes (E-RFC) with sheep red blood cells (SRBC) in a sample of PBL treated with anti-Leu-1 antibodies and subsequently passed once through GAMIg-conjugated APAMB dropped from a range of 41.5–86.0% to a range of 1.6–13.3%. The in vitro response to concanavalin-A (Con-A) dropped to a range of 0.7–27.2% (GAMIg) and a range of 1.2–21.8% (protein-A column) of the response of untreated PBL. Treatment with CAMPATH-1 antibody and passage through a protein-A-conjugated APAMB reduced E-RFC from a range of 55.6–57.4% to a range of 3.2–3.9% and abolished the Con-A induced proliferative responsiveness to background levels. Treatment of BM cells with CAMPATH-1 and passage of the cells through either GAMIg or protein-A conjugated APAMB columns resulted in reduction of E-RFC from a range of 12.4–17.7% to a range of 0–1% and from a range of 17.7–19% to a range of 1.6–3.2%, respectively. Viability of BM precursors, determined by the CFU-GM assay in semisolid medium, was not affected by these cell separation procedures. The data suggest that protein-A or GAMIg-conjugated APAMB columns may be a useful tool for separation of BM cell suspensions into specific cell subsets that can be defined by monoclonal antibodies.     

Determination of traces of palladium in stream sediment and auto catalyst by FI-ICP-OES using on-line separation and preconcentration with QuadraSil TA

A flow injection analysis (FIA) method using on-line separation and preconcentration with a novel metal scavenger beads, QuadraSil™ TA, has been developed for the ICP-OES determination of traces of palladium. QuadraSil TA contains diethylenetriamine as a functional group on spherical silica beads and shows the highest selectivity for Pd(II) at pH 1 (0.1 mol l⁻¹ hydrochloric acid) solution. An aliquot of the sample solution prepared as 0.1 mol l⁻¹ in hydrochloric acid was passed through the QuadraSil TA column. After washing the column with the carrier solution, the Pd(II) retained on the column was eluted with 0.05 mol l⁻¹ thiourea solution and the eluate was directly introduced into an ICP-OES. The proposed method was successfully applied to the determination of traces of palladium in JSd-2 stream sediment certified reference material [0.019 ± 0.001 μg g⁻¹ (n = 3); provisional value: 0.0212 μg g⁻¹] and SRM 2556 used auto catalyst certified reference material [315 ± 4 μg g⁻¹ (n = 4); certified value: 326 μg g⁻¹]. The detection limit (3σ) of 0.28 ng ml⁻¹ was obtained for 5 ml of sample solution. The sample throughputs for 5 ml and 100 μl of the sample solutions were 10 and 15 h⁻¹, respectively.