Visual detection of single-nucleotide polymorphisms and DNA methyltransferase based on cation-exchange of CuS nanoparticles and click chemistry of functionalized gold nanoparticles.

A novel biosensor was developed based on the cation-exchange of CuS nanoparticles (NPs) and Cu(i)-based click chemistry of functionalized gold nanoparticles (AuNPs). As a proof-of-principle, novel applications of this method as a versatile biosensor for single-nucleotide polymorphisms (SNPs) and DNA methyltransferase (MTase) were presented.

Modified SO(4)(2-)/Fe(2)O(3) solid superacid catalysts for electrochemical reaction of toluene with methanol.

A series of sulfate, different metal promoted SO(4)(2-)/Fe(2)O(3) catalysts were prepared and investigated by means of Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS). All catalysts exhibit good catalytic activity for the electrochemical reaction of toluene with methanol assisted with a pair of porous graphite plane electrodes and chemical conversion higher than 80% was observed. In particular, molybdate promoted catalysts exhibited excellent catalytic activity.

Staphylococcus aureus meningitis in adults: a clinical comparison of infections caused by methicillin-resistant and methicillin-sensitive strains.

BACKGROUND: This study was undertaken to compare the clinical characteristics of adult methicillin-sensitive Staphylococcus aureus (MSSA) meningitis and adult methicillin-resistant S. aureus (MRSA) meningitis. PATIENTS AND METHODS: The clinical characteristics and therapeutic outcomes of 19 adult patients with S. aureus meningitis, including eight with MSSA infections and 11 with MRSA infections, were analyzed. A comparison was made between the clinical data of the patients with MSSA infections and those with MRSA infections. RESULTS: Before the end of 1995, MSSA infection was involved in all the adult patients with S. aureus meningitis but thereafter, MRSA infection was involved in 79% of the cases. The clinical characteristics found in patients with MSSA infection included underlying medical disorders (75%), community-acquired infection (75%) and mortality rate (13%). The clinical characteristics found in patients with MRSA infection included post-neurosurgical states (91%), nosocomial infections (100%), men outnumbering women (8:3), hydrocephalus (36%) and mortality rate (56%). Comparative study between the patient groups (hematogenous and post-neurosurgical) showed that only the mode of acquisition of infection had statistical significance. CONCLUSIONS: This study showed an increase in MRSA infections in adult S. aureus meningitis in recent years. The clinical characteristics of patients with MSSA and MRSA meningitis were different. Community-acquired infection was common in hematogenous S. aureus meningitis, while nosocomial infection was common in post-neurosurgical S. aureus meningitis. Vancomycin should be considered as one of the drugs of choice for initial therapy of adult bacterial meningitis, especially in post-neurosurgical patients.

Characterization of highly sulfated cyclodextrins.

A class of highly sulfated cyclodextrins (HS-CDs) was developed for enantiomeric separation of chiral compounds by capillary electrophoresis (CE). The HS-CDs were produced by a facile single-step direct sulfation of cyclodextrin using sulfur trioxide-trimethylamine complex in dimethylformamide. Characterization of the HS-CDs by electrospray ionization mass spectrometry and by CE using a well-established indirect detection method indicated the species have very narrow heterogeneity in terms of degree of sulfation. Elemental analysis of the HS-alpha-, beta- and gamma-CDs showed that the average sulfate contents were 11, 12, and 13 per CD molecule, respectively. The 13C NMR of HS-CDs is consistent with the structural assignment of nearly complete sulfation at C-6 primary hydroxyl groups and partial sulfation at the C-2 secondary hydroxyls (>70%), while the C-3 hydroxyls remain unsubstituted. Enantiomeric separation by CE using the HS-CDs as chiral selectors showed that HS-alpha-, beta- and gamma-CDs complement each other by exhibiting different chiral selectivities, resulting in resolution of many chiral neutral, acidic and basic compounds of greatly varying structural features. The part of HS-CD that interacts with the guest molecule during complexation and, therefore, the receiving end of the cyclodextrin hydrophobic bucket was surrounded with largely regiospecifically substituted C-2 sulfates and intact C-3 hydroxyls, both at the equatorial positions. Such global regiospecific structural arrangement in HS-CDs provides differential diasteroisomeric complexation is proposed to be the principal contributing factor in the resolving racemates.

Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection.

DNA amplification technology has been applied to clinical diagnosis of infectious disease, genetic disorder, and cancer. After in vitro amplification of a particular DNA region, the methods of analysis for these amplified samples play a pivotal role in clinical diagnosis. Conventional gel electrophoresis has been routinely used in the lab for checking DNA. The whole procedure is time consuming and requires more than 1 ng of DNA for detection. To achieve greater performance in DNA diagnosis, we demonstrated capillary electrophoresis with laser induced fluorescence detection for analysis of amplified DNA. The analysis of DNA could be completed within 3 min and the data is directly entered into the computer. Considering the automatic and rapid process, we believe that this method could be routinely utilized for the clinical diagnosis of amplified DNA products.

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection.

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.

Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection.

Electrophoresis is an important and fundamental tool for DNA analysis. Traditional DNA electrophoresis is labor-intensive, skill-dependent, and relatively slow. To achieve greater performance, capillary electrophoresis (CE) has been developed for DNA work (1-2). The DNA fragments can be separated by molecular sieving in CE with gel-filled capillaries, based on the same principle as that of the conventional gel electrophoresis. The whole procedure can be completed in a relatively short period with great resolution. Recently, CE equipped with laser-induced fluorescence (CE-LIF) has been used as a tool for DNA analysis (3). Using fluorescence DNA intercalators in a gel-filled capillary, the detection sensitivity was found to increase 2-3 orders of magnitude over that of UV detection, and reaches picogram levels (3).

Profiling glycoprotein n-linked oligosaccharide by capillary electrophoresis.

A method for analysis of N-linked oligosaccharides derived from glycoproteins including sialic acid-containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. Glycoproteins were heat-denatured in the presence of a reducing agent and the N-linked oligosaccharides were released by peptide N-glycosidase (PNGase F; EC3.5.1.52)-catalyzed hydrolysis. The released N-linked oligosaccharides were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N-linked oligosaccharide, desialylated, galactosylated biantennary, core-substituted with fucose (A2F) was tested for APTS-based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N-linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resolution of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis.

A method for quantitative analysis of monosaccharides including N-acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N-acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS-monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.

Characterization of fluorescent nucleoside triphosphates by capillary electrophoresis with laser-induced fluorescence detection: action of alkaline phosphatase and DNA polymerase.

A method of analysis of fluor-labeled nucleoside triphosphates based on alkaline phosphatase-catalyzed sequential cleavage of phosphate groups with monitoring of all fluorescent species by capillary electrophoresis with laser-induced fluorescence detection is presented. The method allows determination of the purity of the triphosphate samples as well as the relative amounts of the lower phosphate contaminants. The ability of one of the fluor-labeled nucleoside triphosphates to serve as polymerase substrate was verified by labeling DNA restriction fragments by the method of filling recessed 3'-ends using DNA polymerase Klenow fragment.

Acid-catalyzed reductive amination of aldoses with 8-aminopyrene-1,3,6-trisulfonate.

The reductive amination of monosaccharides with 8-aminopyrene-1,3,6-trisulfonate (APTS) in seven different organic acids including the commonly used acetic acid was investigated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. The correlation between the yields of the saccharide-APTS adducts and pKa of the organic acid catalyst is consistent with general acid catalysis of the rate-determining step of the reductive amination reaction. Derivatization in the presence of organic acids of higher strength than acetic acid produced substantially higher yields of APTS-sugar adducts, an effect which is more pronounced for N-acetylamino sugars. Optimum yields were obtained using citric acid as a catalyst. Conversion of a few nanomoles of neutral saccharides to the APTS derivatives is achieved at 75 degrees C in less than 60 min.

Separation of 1-aminopyrene-3,6,8-trisulfonate-labeled asparagine-linked fetuin glycans by capillary gel electrophoresis.

Asparagine-linked glycans of bovine fetuin were separated by capillary gel electrophoresis after enzymatic release (peptide-N-glycosidase F) and labeling via reductive amination by a fluorescent dye, 1-aminopyrene-3,6-8-trisulfonate (APTS). At low separation pH (2.5) only two dominant peaks were observed. Increasing the separation buffer pH to 4.75 resulted in complete separation of two primary doublets and several minor peaks from the fetuin N-linked glycan pool. Two of the four major peaks were spiked with purified individual standards and were identified as trisialylated triantennary structures with different sialylation linkages. The other two larger peaks were postulated to be tetrasialylated triantennary structures, based on calculations considering their corresponding glucose unit (GU) values. Effects of the electrophoretic separation parameters, such as gel concentration, electric field strength and temperature on the migration behavior of the two major doublets of the fetuin glycan pool were also thoroughly examined. Our data suggest that the capillary gel electrophoresis separation of the multisialylated branched oligosaccharides with different linkage isomers, released from bovine fetuin, is fundamentally based on their degree of sialylation and hydrodynamic volumes.

High-resolution capillary gel electrophoresis of reducing oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonate.

High-resolution capillary gel electrophoresis was used for the separation of oligosaccharides labeled with a novel fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination. The APTS-saccharide adducts were detected by laser-induced fluorescence with excitation by the 488-nm Ar-ion laser and a 520-nm emission filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Derivatization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent are thoroughly discussed. Desialylation of several sialylated oligosaccharides with different structures and linkages due to the effects of labeling temperature and time are also addressed. Employing the optimized conditions suggested in this paper, fluorophore labeling efficiency greater than 97% was achieved with no significant loss of sialic acid residues. The fluorescently labeled oligosaccharides are then separated and quantified by capillary gel electrophoresis. Practical examples of low-level derivatization and high-resolution capillary gel electrophoresis separation of N-linked glycans of ribonuclease-B and fetuin are also shown.

Diffractive lens fabricated with mostly zeroth-order gratings.

We demonstrate a spherical diffractive lens fabricated in fused quartz for use at the 632.8-nm wavelength. The lens is constructed by use of a modulated two-dimensional binary grating with a high transmitted zerothorder efficiency. Rigorous eigenmode analysis is used to correlate the desired phase modulation with the fill factor. Fabrication requires only one lithography step. Using the lens, we were able to image a focal spot with a diffraction-limited spot size (FWHM).

Analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by capillary electrophoresis with laser-induced fluorescence.

A method of analysis of isomers of mono- and oligosaccharides derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) by capillary electrophoresis (CE) in simple buffer systems is presented. The derivatization chemistry is performed at 75 degrees C for 1 h for most mono- and oligosaccharides. Sialyllactose was derivatized at 37 degrees C for 15 h without significant desialylation. Most of the APTS derivatized isomers of mono- and oligosaccharides are resolved in buffers such as acetate (pH 5.0), 3-(N-morpholino)-propanesulfonic acid (pH 7.0), and phosphate (pH 7.4). The mechanism of CE separation using the above buffers is based on the differences in hydrodynamic volume of the derivatized species and is different from that in borate or high pH buffer.

Detection of amplified Y chromosome-specific sequence by capillary electrophoresis with laser-induced fluorescence.

OBJECTIVE: To develop a sensitive method for genetic diagnosis using capillary electrophoresis with laser-induced fluorescence. DESIGN: Using male DNA diluted with female DNA as an example, ZFY gene from Y chromosome was amplified specifically by polymerase chain reaction (PCR) with fluorescence-labeled primers and detected by capillary electrophoresis with laser-induced fluorescence. SETTING: Laser operating laboratory. PARTICIPANTS: Human male and female DNA were extracted from healthy human male and female subjects. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The concentration of human DNA was determined by using a spectrophotometer at an absorbance of 260 nm. RESULT: Deoxyribonucleic acid fragments amplified from a single copy of ZFY gene were detected by capillary electrophoresis with laser-induced fluorescence after 35 cycles of PCR amplification. CONCLUSION: This method is potentially applicable for rapid and sensitive detection of fetal Y chromosome DNA sequence in maternal circulation and of single-cell DNA diagnosis.

Characterization of protease-catalyzed hydrolysis of cyanine-labeled angiotensin using capillary electrophoresis with laser-induced fluorescence detection.

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, catalyzed by various proteases, was studied by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The end-labeled peptides and the Cy3 diacid internal standard were separated on a 20-microns x 27-cm capillary with LIF detection (emission, 580 nm) using a frequency-doubled solid-state diode laser emitting at 532 nm or a He-Ne laser emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, catalyzed by proteinase K, is a sequential process beginning from the C-terminal of the peptide, instead of from random cleavages. Trypsin catalyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anticipated. Using a combination of endopeptidase and carboxypeptidases, the remnant of the labeled species was characterized by CE-LIF. The method provides a general tool for studying the mechanism of protease-catalyzed hydrolysis of peptide.