Analysis of specific gene by integration of isothermal amplification and electrophoresis on poly(methyl methacrylate) microchips

We have successfully achieved the integration of isothermal amplification and the subsequent analysis of specific gene fragments on poly(methyl methacrylate) microchips. In our experiments, loop-mediated isothermal amplification, which can offer higher specificity and efficiency than PCR, has been performed at a constant temperature (65 degreesC). After amplification, products could be either examined by the integrated microchip-based electrophoresis or directly observed by naked eye with SYBR Green I added into the reaction solution. By such an integrated microsystem, the amplification and the subsequent analysis of prostate-specific antigen gene with template concentration at 23 fg/muL could be finished within 15 min, which demonstrates its advantages of high specificity, good reproducibility, and fast speed in gene detection.

Development of a precolumn derivatization method for the determination of free amines in wastewater by high-performance liquid chromatography via fluorescent detection with 9-(2-hydroxyethyl)acridone

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with fluorescence detection has been developed. 9-(2-Hydroxyethyl)acridone reacts with coupling agent N,N-carbonyldiimidazole at ambient temperature to form activated amide intermediate 9-(2-acridone)oxyethylcarbonylimidazole (AOCD). The amide intermediate (AOCD) preferably reacts with amino compounds under mild reactions in the presence of 4-(dimethylamino)pyridine (base catalyst) in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum lambda(ex) 404 mn and an emission maximum at lambda(em) 440 nm. The labeled derivatives exhibit high stability under reversed-phase conditions. The fluorescence intensities of derivatives in various solvents or at different temperatures were investigated. The method, in conjunction with a gradient elution, offers a baseline resolution of the common amine derivatives on a reversed-phase C-18 column. The LC separation for the derivatized amines shows good reproducibility with acetonitrile-water including 2.5% DMF as mobile phase. The relative standard deviations (n = 6) for each amine derivative are <4.5%. The detection limits (at a signal-to-noise ratio of 3) per injection were 0.16-12.8 ng/mL. Further research for the field of application, based on the AOCD amide intermediate as derivatization reagent, for the determination of free amines in real water samples is achieved.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C-12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150 000 plates/meter) and good reproducibility (migration time with RSD < 0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.

Separation of enantiomers of drugs by capillary electrophoresis with permethyl-gamma-cyclodextrin as chiral solvating agent

High-throughput screening is a promising new approach in analytical chemistry. Within the framework of an extended screening program (The German-Chinese Drug Screening Program), the enantioseparation of 86 drugs was investigated by capillary zone electrophoresis in the presence of the chiral solvating agent (CSA) octakis-(2,3,6-tri-O-methyl)-gamma-cyclodextrin (TM-gamma-CD). By this means, 15 drugs could be separated into enantiomeric pairs. Approximate measures for the degree of interaction (migration retardation factor, R-m) and for the degree of enantiomer recognition (migration separation factors, alpha(m)) revealed intriguing patterns that were compared with those found for native gamma-cyclodextrin (gamma-CD). Although there is a distinct influence of the analyte structure on the electrophoretic data, interpretation remains difficult. Most remarkably, permethylation of gamma-CD leads neither to a higher affinity nor to better chiral recognition, in contrast to the findings with alpha-CD.

A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry

We report a novel method termed matrix suppressed laser desorption/ionization to improve the analysis of low-mass molecules by MALDI-TOF mass spectrometry. In this method, the surfactant of cetrimonium bromide (CTAB) is added to the conventional matrix of alpha-cyano-4-hydroxycinnamic acid solution to prepare the MALDI samples. During the MALDI process, the presence of CTAB could substantially or even completely suppress the matrix-related ion background. As a result, very clean mass spectra can be routinely obtained in the low-mass range. In addition, the presence of CTAB can significantly improve the mass resolution of low-mass molecules. It is seen that high-quality spectra were routinely obtained at a matrix/CTAB ratio of 1000:1. This method has been successfully used to analyze a variety of low-mass molecules.

Nontoxic electrodes of solid amalgams

This is a review of electrodes based on nontoxic solid amalgams (MeSAE) (prepared by amalgamation of soft metal powders) in connection with some other kinds of voltammetric electrodes is given. Information is summarized on various types of MeSAEs (esp. AgSAE, CuSAE, AuSAE), pretreatment of their surfaces, their hydrogen overvoltage in aqueous solutions, conditions for their testing, electroanalytical parameters and use, in compared with the hanging mercury drop electrode (HMDE). Although the solid amalgam electrodes do not reach the quality of the HMDE, in many cases they represent its possible alternative. The broad range of voltammetric applications of the MeSAEs, especially of the AgSAEs, their good mechanical stability, simple handling, and new aspects of their use in electrochemical techniques are documented by numerous examples.

A systematic approach to standard addition methods in instrumental analysis

Nine cases of standard addition methods in instrumental analysis, including linear instruments, a non-linear response, the electrochemical Nernst equation, and radiochemical techniques.

Metal nanomaterials and carbon nanotubes - synthesis, functionalization and potential applications towards electrochemistry

This review focuses on the synthesis, assembly, surface functionalization, as well as application of inorganic nanostructures. Electrochemical and wet- chemical methods are demonstrated to be effective approaches to make metal nanostructures under control without addition of a reducing agent or protecting agent. Owing to the unique physical and chemical properties of the nano-sized materials, novel applications are introduced using inorganic nanomaterials, such as electrocatalysis, photoelectricity, spectrochemistry, and analytical chemistry.

Solid-state electrochemiluminescence of tris(2,2 '-bipyridyl) ruthenium

Electrochemiluminescence (ECL) of tris(2,2'-bipyridyl) ruthenium [Ru(bpy)(3)(2+)] has received considerable attention. By immobilizing Ru(bpy)(3)(2+) on an e electrode surface, solid-state ECL provides several advantages over solution-phase ECL, such as reducing consumption of expensive reagent, simplifying experimental design and enhancing the ECL signal.This review presents the state of the art in solid-state ECL of Ru(bpy)(3)(2+).

Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection

Artificial enzyme mimetics are a current research interest because natural enzymes bear some serious disadvantages, such as their catalytic activity can be easily inhibited and they can be digested by proteases. A very recently study reported by Yan et al. has proven that Fe3O4 magnetic nanoparticles (MNPs) exhibit an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, though MNPs are usually thought to be biological and chemical inert (Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.; Perrett, S.; Yan, X. Y. Nat. Nanotechnol. 2007, 2, 577-583).

Bis(2,2 '-bipyridine)(5,6-epoxy-5,6-dihydro-[1,10] phenanthroline)ruthenium: Synthesis and electrochemical and electrochemiluminescence characterization

In this work, an electrochemiluminescence (ECL) reagent bis(2,2'-bipyridine)(5,6-epoxy-5,6-dihydro-[1,10]phenanthroline)ruthenium complex (Ru-1) was synthesized, and its electrochemical and ECL properties were characterized. The synthesis of Ru-1 was confirmed by IR spectra, element analysis, and H-1 NMR spectra. For further study, its UV-vis absorption and fluorescence emission spectra were investigated. Ru-1 also exhibited quasi-reversible Ru-II/Ru-III redox waves in acetonitrile solution. The aqueous ECL behaviors of Ru-1 were also studied in the absence and in the presence of tripropylamine.

Enzyme Colorimetric Assay Using Unmodified Silver Nanoparticles

Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CLAP) and peptide phosphorylation by protein kinase A (PKA) were studied.