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PubMed2002

Simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids by capillary zone electrophoresis with direct UV detection.

Co-electroosmotic capillary zone electrophoresis (CZE) with direct UV detection was developed for simultaneous determination of inorganic anions, carboxylic and aromatic carboxylic acids. These solutes were separated using a 30 mM phosphate buffer containing 1.0 mM tetradecyltrimethylammonium bromide (TTAB) and 20% (v/v) acetonitrile at pH of 6.5 and directly detected by UV at 190 nm. Calibration curves were linear in the range 0.01-2.0 mM, depending of the solutes. The detection limits ranged from 1.0 to 8.0 microM and the relative standards deviations (n=5) in range from 1.9 to 3.6% for the peak area. The proposed method was used to determine inorganic anions and carboxylic and aromatic acids in soil and plant tissue extracts.

Anions↗
PubMed2003

Trace determination of arsenic species by capillary electrophoresis with direct UV detection using sensitivity enhancement by counter- or co-electroosmotic flow stacking and a high-sensitivity cell.

Stacking techniques used independently and also with a high-sensitivity cell (HSC) were employed to optimise sensitivity and detection limits in the direct photometric detection of the following eight arsenic species by capillary zone electrophoresis (CZE): arsenite, arsenate, monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), 4-hydroxy-3-nitrophenylarsonic acid (Roxarsone), p-aminophenylarsonic acid (p-ASA), 4-nitrophenylarsonic acid (4-NPAA), and phenylarsonic acid (PAA) (target analytes). The stacking mechanisms, optimised stacking and separation conditions, and concentration sensitivity enhancement factors were discussed and compared for (i) normal stacking mode (NSM, sometimes also referred to as field-amplified stacking) in an uncoated fused-silica capillary in the counter-electroosmotic flow (EOF) mode, (ii) large-volume sample stacking (LVSS) with polarity switching, and (iii) the less often applied stacking method of co-EOF NSM stacking with EOF reversal using a poly(diallydimethylammonium chloride) (PDDAC)-coated capillary. The optimal injection volumes were 7.4, 60 and 17.2% of the total capillary volume, for the above three methods, respectively. LVSS with polarity switching gave the lowest limit of detection (LOD). The use of the HSC further reduced the LOD of each target analytes by a factor of 5-8 times. By combining LVSS and HSC, LODs of the target analytes could be reduced by a factor of 218-311, to 5.61, 9.15, 11.1, and 17.1 microg/L for As(III), DMA, MMA, and As(V), respectively. The method was demonstrated to be applicable to the determination of the target analytes in tap water and lake water, with recoveries in the range of 89.4-103.3%.

Arsenic↗
PubMed1997

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay.

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

DNA Probes↗
PubMed2005

Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer.

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.

DNA↗
PubMed1995

A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk.

A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented. The bacterial spores are concentrated by centrifugation after chemical extraction of the milk components. The vegetative cells are selectively lysed, and their DNA is digested and washed away. Afterwards, the DNA is liberated from the spores by microwave treatment. For the identification of the C. tyrobutyricum DNA, a two-step PCR method with two nested pairs of primers is used. The primers were derived from the 16S-23S rRNA spacer region of C. tyrobutyricum, and the specificity of each of them for C. tyrobutyricum is demonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.

Animals↗
PubMed1988

[Direct antigen detection for group A streptococcal pharyngitis: comparison of throat cultures and the direct antigen test].

In this study, traditional throat culture results and direct antigen detection from throat swabs have been compared with respect to diagnosis of Group A Beta hemolytic streptococcal infections in children with acute pharyngitis who admitted to Marmara University Hospital Pediatrics Department. It has been found that direct antigen test has a sensitivity of 92% and specificity of 98%.

Antigens, Bacterial↗
PubMed1989

N-RAS mutations in T-cell acute lymphocytic leukaemia: analysis by direct sequencing detects a novel mutation.

A novel mutation of the N-RAS gene of T-ALL blast cells was detected by a direct sequencing of in vitro amplified exon-1 of the N-RAS gene. Threonine (ACA) was substituted for alanine (GCA) at codon 11. This mutation would have been overlooked by conventional probe hybridization techniques. A search for other mutations in N-RAS exon-1 in T-ALL revealed a codon 13 mutation substituting aspartic acid (GAT) for glycine (GGT) in one of 18 patients. No mutations at codon 12 were detected.

Base Sequence↗
PubMed1990

Current status of prenatal diagnosis by DNA analysis.

In 1989 we are continuing to move gene diagnosis over to the direct detection mode. We have sickle cell anemia, alpha-thalassemia, beta-thalassemia, Duchenne muscular dystrophy, Becker muscular dystrophy and cystic fibrosis moved to direct detection with hemophilia B and alpha-1-antitrypsin deficiency soon to be there. For indirect detection, we still have hemophilia A, and a comment on the genetics of hemophilia A is important. Remember that sickle cell anemia is caused by one mutation, while beta-thalassemia and cystic fibrosis have a finite number of alleles. Duchenne muscular dystrophy results from a different mutation for every affected individual, but most of these are deletions and can be directly detected. Hemophilia A is another X-linked disorder with almost every affected individual having a different mutation. That means that there probably are 100 ways to get beta-thalassemia and about 10,000 ways to get hemophilia A, so we need some really good novel techniques to detect these directly, and we are working hard on such techniques. I would not be surprised if hemophilia A moved into the direct detection category in the next year or so. We need to find the Huntington disease gene, and then it will move into the direct detection column. Neurofibromatosis is still in the indirect detection group but also may move very soon. Polycystic kidney disease is also still in the indirect detection column. This summarizes where prenatal and presymptomatic gene diagnosis stands in late 1989.

Blotting, Southern↗
PubMed2006

Rapid determination of nonaromatic organic acids in honey by capillary zone electrophoresis with direct ultraviolet detection.

A rapid capillary zone electrophoresis (CZE) method with direct ultraviolet (UV) detection has been set up and developed to determine the most important nonaromatic organic acids in honey with a really simple treatment of the sample. The determination of oxalic, formic, malic, succinic, pyruvic, acetic, lactic, citric, and gluconic acids has been carried out in 4 min. The electrolyte composition was phosphate as the carrier buffer (7.5 mM NaH(2)PO(4) and 2.5 mM Na(2)HPO(4)), 2.5 mM tetradecyltrimethylammonium hydroxide (TTAOH) as electroosmotic flow modifier, and 0.24 mM CaCl(2) as selectivity modifier, with the pH adjusted at 6.40 constant value. The running voltage was -25 kV at a thermostated temperature of 25 degrees C. The injections were performed in hydrodynamic mode (30 s), and the detection mode was UV direct at 185 nm. Validation parameters of the method as detection and quantification limits, linearity, precision (repeatability and reproducibility), and recovery were also studied. The advantages related to the technique such as simplicity, short analysis times, and low consumption of chemicals as well as the good validation parameters obtained for this method permit it to be considered as adequate for routine analysis in honey.

Buffers↗
PubMed2005

Determination of ammonium in river water and sewage samples by capillary zone electrophoresis with direct UV detection.

We developed capillary zone electrophoresis (CZE) with direct UV detection for determination of ammonium in environmental water samples. Ammonium in the samples was partly converted into ammonia in the alkaline background electrolyte (BGE) during migration and was detected by molecular absorption of ammonia at 190 nm in approximately 7 min. The limit of detection (LOD) for ammonium was 0.24 mg/l (as nitrogen) at a signal-to-noise ratio of three. The respective values of the relative standard deviation (RSD) of peak area, peak height, and migration time for ammonium were 2.1, 1.8, and 0.46%. Major alkali and alkaline earth metal ions coexisting in the samples did not interfere with ammonium determination by the proposed method. The proposed method determined ammonium in surface water and sewage samples. The results were compared to those obtained using ion chromatography (IC).

Calibration↗
PubMed1985

Experimental approaches for the detection of chromosomal malsegregation occurring in the germline of mammals.

Existing and newly proposed methods to detect the induction of heritable aneuploidy are summarized, and their favorable and unfavorable features discussed. Among the tests involving direct chromosomal examination, those involving study of pronuclear chromosomes at first cleavage are judged to be the most universally informative and reliable, provided tertiary trisomy can be ruled out. Measurement of postmidterm (fetal) death is proposed as a trisomy prescreen that can be readily combined with a dominant-lethal test. Among the genetic procedures for identifying the results of malsegregation events, direct detection of aneuploids has a number of advantages over complementation methods, in which only a fraction of the products of aneuploid gametes is detectable. Direct detection of aneuploids must, however, be restricted to sex chromosomes if postnatal animals are examined. A genetic marker system to detect autosomal trisomies in fetuses is proposed. An examination of experimental parameters that might maximize induction of malsegregation leads to the recommendation to include preleptotene among exposed germ-cell stages.

Alleles↗
PubMed1994

Detection and direction discrimination performance with flicker gratings in peripheral vision.

Detection and direction discrimination experiments were conducted with luminance and flicker gratings. The flicker gratings had bars made up of static random pixels interspersed between other bars with flickering random pixels. All experiments were carried out in peripheral vision with grating images centered at 8 deg eccentricity in the superior retina. Detection of flicker gratings (i) was independent of pixel size, (ii) declined with spatial frequency in the range 1-4 c/deg, and (iii) improved with stimulus area (number of grating cycles). Detection performance with a flicker grating was comparable to that obtained with a low-contrast (0.01) luminance grating, and the results suggest that the spatial structure of a flicker-domain stimulus is based upon signals which are weak compared to the maximum signals attainable with a luminance-domain stimulus. With the detectability of flicker and luminance gratings equated, d' for discriminating the direction of motion of a luminance grating increased with step size (1/12 to 1/4 cycle) whereas direction discrimination performance with a flicker grating remained unchanged and at chance levels. Under the conditions tested, there was no evidence that the motion of a flicker-domain stimulus could be processed peripherally. Constraints on alternative models of motion processing are discussed.

Contrast Sensitivity↗
PubMed2005

Direct molecular detection of nucleic acids by fluorescence signal amplification.

An integrated PCR-free DNA sensor, which combines a sequence-specific receptor, an optical polymeric transducer, and an intrinsic fluorescence amplification mechanism, is reported. This sensor is based on the different conformations adopted by a cationic polythiophene when electrostatically bound to ss-DNA or ds-DNA, and on the efficient and fast energy transfer between the resulting fluorescent polythiophene/ds-DNA complex and neighboring fluorophores attached to ss-DNA probes. This molecular system allows the detection of only five molecules in 3 mL of an aqueous solution, or 3 zM, in 5 min. Moreover, this work demonstrates, for the first time, the direct detection of single nucleotide polymorphisms (SNPs) from clinical samples in only a few minutes, without the need for nucleic acid amplification.

Biosensing Techniques↗
PubMed1985

Spatial integration characteristics in motion detection and direction identification.

Spatial integration characteristics were assessed with drifting gratings for both detection and direction-identification contrast thresholds. Thresholds were measured while stimulus width, length or both were varied. It was found that: (1) the shape of the size/sensitivity functions changes with spatial, but not with temporal, frequency; (2) direction-identification thresholds diverge from the detection thresholds below 1 cycle but can be reliably measured for stimulus widths as small as 0.1275 cycles; (3) the integration characteristics are slightly anisotropic for the identification but not for the detection process, and (4) the two-dimensional spatial integration cannot be directly predicted from its one-dimensional characteristics. Width/sensitivity detection functions are well fitted by predictions of Wilson and Bergen's four-channel model. Predictions from a temporal covariance model provide a poor fit to the identification data. It is argued that classes of detection and direction-identification models must involve identical nonlinearities prior to their respective thresholds. It is concluded that the hypothesis according to which both performances are determined by the same spatial integration stage cannot be rejected.

Humans↗
PubMed2002

[Direct UV detection of glucose and its derivatives in capillary zone electrophoresis].

UV labeling detection has been commonly used to determine the association constants between lectins and saccharides, but the interaction is always between the labeled carbohydrates, rather than the truly underivatized carbohydrates, and lectins. In order to directly detect saccharides during the study on the interaction of glucose and its derivatives with lectins (e.g., concanavalin A), a capillary zone electrophoretic method with detection at a wavelength of 195 nm has been developed. The influences of various separation conditions including buffer concentration, pH and voltage were investigated. By using an uncoated silica capillary (50 microns i.d., 375 microns o.d., 48.5 cm of total length, and 44.0 cm to the detector) and 50 mmol/L Na2HPO(4)-50 mmol/L NaH2PO4 solution (near to the physiological pH of 7.4) as buffer, the underivatized sugars, including glucosamine, N-acetylglucosamine, glucose, and sodium gluconate, were sufficiently separated within 11 min at an applied voltage of 10 kV. On-column UV monitoring allowed the detection of these compounds at less than 4 mmol/L level, and quantification by the peak area method allowed reproducible determination of them at least at their respective concentration ranges. The method is characterized by its simplicity, rapidity, and reproducibility, and should be useful for the analysis of the interaction of glucose and its derivatives with lectins.

Concanavalin A↗
PubMed2001

Mutation causing exon 15 skipping and partial exon 16 deletion in factor VIII transcript, and a method for direct mutation detection.

A splicing defect with 201 nucleotide deletion in the factor VIII transcript due to IVS15 + 1G > T mutation inactivating this donor splice site and activating a cryptic acceptor splice site in exon 16 was identified in a severe haemophilia A patient. Allele specific amplification (ASA) method was successfully developed for direct detection of this mutation.

Alleles↗
PubMed1984

Myeloperoxidase singlet molecular oxygen generation detected by direct infrared electronic emission.

Myeloperoxidase in micromolar concentrations reacting with half-millimolar stock solution H2O2 in acetate buffer containing KBr and in 50% D2O (pH + pD = 4.5) at 298 K is shown to generate singlet delta molecular oxygen efficiently. The near infrared electronic emission of singlet oxygen at 1268 nm is detected directly by novel ultrasensitive IR spectrophotometer equipment. The quantum efficiency of singlet oxygen generation by the MPO X Br- X H2O2 reaction is shown to be comparable with that of the standard chemical reaction OCl- X H2O2 at identical peroxide concentrations.

Animals↗
PubMed2006

Direct electrical detection of DNA synthesis.

Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3'-hydroxyl group of the DNA molecule during phosphodiester bond formation.

Base Sequence↗