Electrochemical detection of DNA immobilized on gold colloid particles modified self-assembled monolayer electrode with silver nanoparticle label

The target DNA was immobilized successfully on gold colloid particles associated with a cysteamine monolayer on gold electrode surface. Self-assembly of colloidal An onto a cysteamine modified gold electrode can enlarge the electrode surface area and enhance greatly the amount of immobilized single stranded DNA (ssDNA). The electrontransfer processes of [Fe(CN)(6)](4)-/[Fe(CN)(6)](3-) on the gold surface were blocked due to the procedures of the target DNA immobilization, which was investigated by impedance spectroscopy. Then single stranded target DNA immobilized on the gold electrode hybridized with the silver nanoparticle-oligonucleotide DNA probe, followed by the release of the silver metal atoms anchored on the hybrids by oxidative metal dissolution, and the indirect determination of the released solubilized Ag-1 ions by anodic stripping voltammetry (ASV) at a carbon fiber microelectrode. The results show that this method has good correlation for DNA detection in the range of 10-800 pmol/1 and allows the detection level as low as 5 pmol/1 of the target oligonucleotides.

LIQUID-CHROMATOGRAPHY AMPEROMETRIC DETECTION OF CARBOXYLIC-ACIDS AND PHENOLIC-ACIDS WITH A COPPER-BASED CHEMICALLY-MODIFIED ELECTRODE

A copper-based chemically-modified electrode has been constructed and characterized by various experimental parameters in flow-through amperometric detection of carboxylic acids and phenolic acids. Novel hydrodynamic voltamperograms were first obtained in flow-through amperometric detection with the Cu-based CME and subsequently negative and positive peaks were observed in a single chromatogram. This unique and flexible potential dependence could be of great benefit in chromatographic speciation and quantification. These observations suggest that the detector response was governed by the complexation reaction of copper ions with the solutes.

An electrochemical DNA sensor based on electrodepositing aluminum ion films on stearic acid-modified carbon paste electrode and its application for the detection of specific sequences related to bar gene and CP4 Epsps gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid-modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well-defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single-stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the Specific sequence related to the target bar gene with the dynamic range comprised between 1.0 X 10(-7) mol/L to 1.0 x 10(-4) mol/L. A detection limit of 2.25 x.10(-8) mol/L. of oligonucleotides can be estimated.

Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai

Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai, and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)(2) and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC-Cl. The new reagent BCEC-Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC-amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)(2) and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I (BCEC)/I (BCEOC) = 1.17-3.57, I (BCEC)/I (FMOC) = 1.13-8.21, and UVBCEC/UVBCEOC = 1.67-4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C-18 column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)(2). Excellent linear responses were observed, with coefficients of > 0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)(2) and Try from bee-collected pollen (bee pollen) samples.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

Alignment determination of symmetric top molecule using rotationally resolved LIF

This payer presents a concrete theoretical treatment which can be used for transforming the laser-induced fluorescence (LIF) intensity into the population and alignment parameters of a symmetric top molecule, The molecular population and alignment are described by molecular state multipoles. The results are presented in a general excitation-detection geometry and then specialized in some special geometries. The problem how to extract the initial molecular state multipoles from the rotationally resolved LIF intensity is discussed in detail. (C) 1999 Elsevier Science B.V. All rights reserved.

Tensor density matrix theory for determining population and alignment of symmetric top molecule using rotationally unresolved fluorescence

General expressions used for transforming raw laser-induced fluorescence (LIF) intensity into the population and alignment parameters of a symmetric top molecule are derived by employing the density matrix approach. The molecular population and alignment are described by molecular state multipoles. The results are presented for a general excitation-detection geometry and then applied to some special geometries. In general cases, the LIF intensity is a complex function of the initial molecular state multipoles, the dynamic factors and the excitation-detection geometrical factors. It contains a population and 14 alignment multipoles. How to extract all initial state multipoles from the rotationally unresolved emission LIF intensity is discussed in detail.

Theoretical study of determining orientation and alignment of symmetric top molecule using laser-induced fluorescence

General expressions used for extracting the orientation and alignment parameters of a symmetric top molecule from laser-induced fluorescence (LIF) intensity are derived by employing the density matrix approach. The molecular orientation and alignment are described by molecular state multipoles. Excitation and detection are circularly and linearly polarized lights, respectively. In general cases, the LIF intensity is a complex function of the initial molecular state multipoles, the dynamic factors and the excitation-detection geometrical factors. It contains a population, ten orientation and fourteen alignment multipoles. The problem of how to extract the initial molecular state multipoles from the resolved LIF intensity is discussed.

Theory for determining alignment parameters of symmetric top molecule using (n+1) LIF

Expressions used for extracting the population and alignment parameters of a symmetric top molecule from (n + 1) laser-induced fluorescence (LIF) are derived by employing the tensor density matrix method. The molecular population and alignment are described by molecular state multipoles. The LIF intensity is a complex function of the initial molecular state multipoles, the dynamic factors, and the excitation-detection geometrical factors. The problem of how to extract the initial molecular state multipoles from (2 + 1) LIF, as an example, is discussed in detail. (C) 2000 American Institute of Physics. [S0021-9606(00)30744-9].

Simultaneous light emitting diode-induced fluorescence and contactless conductivity detection for capillary electrophoresis

A combined detection system of simultaneous contactless conductometric and fluorescent detection for capillary electrophoresis (CE) has been designed and evaluated. The two processes share a common detection cell. A blue light-emitting diode (LED) was used as the excitation source and an optical fiber was used to collect the emitting fluorescence for fluorescent detection (FD). Inorganic ions, fluorescein isothiocyanate (FITC)-labeled amino acids and small molecule peptides were separated and detected by the combined detector, and the detection limits (LODs) of sub-μ M level were achieved.