Electrophoresis-chemiluminescence detection of phenols catalyzed by hemin.

Based on the catalytic activity of hemin, an efficient biocatalyst, an indirect capillary electrophoresis-chemiluminescence (CE-CL) detection method for phenols using a hemin-luminol-hydrogen peroxide system was developed. Through a series of static injection experiments, hemin was found to perform best in a neutral solution rather than an acidic or alkaline medium. Although halide ions such as Br(-) and F(-) could further enhance the CL signal catalyzed by hemin, it is difficult to apply these conditions to this CE-CL detection system because of the self-polymerization of hemin, as it hinders the CE process. The addition of concentrated ammonium hydroxide to an aqueous/dimethyl sulfoxide solution of hemin-luminol afforded a stable CE-CL baseline. The indirect CE-CL detection of five phenols using this method gave the following limits of detections: 4.8 × 10(-8) mol/L (o-sec-butylphenol), 4.9 × 10(-8) mol/L (o-cresol), 5.4 × 10(-8) mol/L (m-cresol), 5.3 × 10(-8) mol/L (2,4-dichlorophenol) and 7.1 × 10(-8) mol/L (phenol).

Scanning electrochemical microscopy of DNA hybridization on DNA microarrays enhanced by HRP-modified SiO2 nanoparticles.

Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe via sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO2 nanoparticles through the biotin-streptavidin interaction. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10(-7)-10(-12) M). This technique may find applications in genomic sequencing.

On-line sample preconcentration technique based on a dynamic pH junction in CE-amperometric detection for the analysis of biogenic amines in urine.

It is difficult to detect biogenic amines (BAs) in biological fluids because of their very low concentrations. In this paper, we reported an on-line sample preconcentration method in CE-amperometric detection (CE-AD) based on a dynamic pH junction, and a concentration enhancement of approximately 100-fold was achieved compared with the classical CE-AD methods in the simultaneous analysis of six BAs in urine (dopamine, epinephrine, norepinephrine, tyramine, tryptamine, and serotonin). The technique is proposed based on the sharp pH change generated at the boundary between an acidic sample and the basic BGE zone. Under optimized conditions, all analytes were successfully focused and well separated within 20 min with high efficiency and sensitivity (LODs at S/N = 3 ranging from 5.34 to 68.3 nM). For the analysis of urine samples by this method, satisfactory recoveries were obtained without a complicated pretreatment step or derivatization process. Therefore, it is self-evident that this approach for the analysis of real biological samples has great potential in the future.

Ultrasensitive detection of bacteria by microchip electrophoresis based on multiple-concentration approaches combining chitosan sweeping, field-amplified sample stacking, and reversed-field stacking.

In this paper we describe an on-chip multiple-concentration method combining chitosan (CS) sweeping, reversed-field stacking, and field-amplified sample stacking for highly efficient detection of bacteria. Escherichia coli was selected as a model bacterium to investigate the efficiency of this multiple-concentration method. CS was the most suitable sweeping agent for microchip electrophoresis, replacing the usually used cetyltrimethylammonium bromide for capillary electrophoresis. The additive taurine had a synergistic effect by enhancing the interaction between CS and the surface of the bacteria, thus improving the analysis sensitivity. All steps of the concentration method and related mechanisms are described and discussed in detail. A concentration enhancement factor of approximately 6000 was obtained using this concentration method under optimal conditions as compared to using no concentration step, and the detection limit of E. coli was 145 CFU/mL. The multiple-concentration methodology was also applied for the quantification of bacteria in surface water, and satisfactory results were achieved. The application of this methodology showed that the concentration enhancement of bacteria clearly conferred advantageous sensitivity, speed, and sample volume compared to established methods.

Exocytosis of SH-SY5Y single cell with different shapes cultured on ITO micro-pore electrode.

Communication between cells by release of specific chemical messengers via exocytosis plays crucial roles in biological process. Catecholamines, like dopamine, epinephrine, and norepinephrine, which are types of neurotransmitters released from cells, can be oxidized and detected by the microelectrodes, and amperometric detection of exocytosis is an effective method for studying the communication between cells. The experimental results depend on many factors, among which the property of the microelectrode, cell states, and their positions to each other are particularly important. A type of indium tin oxide (ITO) micro-pore electrodes, which is characterized by its stability, has been developed with photolithography. SH-SY5Y cells can adhere and spread on ITO micro-pore electrodes. Therefore, it is possible to investigate the correlation between cell morphology and exocytosis. The results show that cells with clear process have higher release frequency of norepinephrine compared with cells in spherical shape. Combined with fluorescence observation, this technique provides a simple and convenient methodology for cell study.

Development of a compact capillary electrophoresis-chemiluminescence system with ultra-fast peroxyoxalate reaction to monitor the hydrolysis of rhodamine 6G.

A simple and effective capillary electrophoresis-chemiluminescence (CE-CL) detection system was developed based on an ultra-fast bis(2,4,6-trichlorophenyl)oxalate (TCPO) chemiluminescence (CL) reaction (0.6 s duration) that avoided overlapping peaks and peak tailing. Through a series of static injection experiments, this unusually rapid CL reaction was ascribed to the catalytic effect of imidazole in the tetrahydrofuran solvent, which has been rarely utilized in such investigations. A possible mechanism is given to explain the results. Under the optimized conditions, rhodamine 6 G (R6G) and its hydrolysis product (R6G-COOH) could be efficiently separated through electrophoresis in 7 min, with sensitive CL detection in the proposed CE-CL system. In this way, the alkaline hydrolysis of R6G was monitored, followed by estimation of relative rate constants and activation energy. This finding and application should be helpful in further study for the TCPO CL reaction, and revealed an attractive opportunity for simplifying the CE-CL system, such as in a microchip device.

Use of different buffers for detection and separation in determination of physio-active components in oolong tea infusion by CZE with amperometric detection.

With a view of simultaneous determination of physio-active ingredients in oolong tea infusion: sugars, amino acids, epigallocatechin gallate and ascorbic acid, a novel CZE with amperometric detection method was studied. Operated in a wall-jet configuration, 100 mmol/L NaOH was used in detecting cell to lead the electrocatalysis oxidation behaviors of the analytes on a 300 mum diameter copper-disc electrode (working electrode), while in separating capillary, a mild alkaline running buffer consisting in a mixture of 30 mmol/L borate and 40 mmol/L phosphates charged and carried analytes to detecting end. The methodology research was performed for system stability and suitability. Under the optimal CE conditions, analytes could be separated within moderate time period. Good linearity between peak area and concentration existed over three orders of magnitude; lower RSD and LOD were achieved. The oolong tea infusion was assayed and result was satisfactory.

Dual-signal electrochemical sensor for detection of cancer cells by the split primer ligation-triggered catalyzed hairpin assembly.

Simultaneous detection of various intracellular biomarkers is promising for early diagnosis and treatment of cancer. Herein, a split primer ligation-triggered catalyzed hairpin assembly-based on dual-signal electrochemical biosensor was constructed for the determination of two pairs of cancer mRNAs: TK1 and c-myc, survivin and GalNAc-T by using ferrocene molecular beacon and hemin molecular beacon as detection signal sources. Each pair of targets exists simultaneously, can release the split primers and ligated as the integral primers, hybridization occurred between the integral primers and part of MBs, causing a double-stranded DNA formed. The probes hybridized with the unfolded MBs and displaced integral primers. Finally, the displaced integral primers again hybridized with the MBs and initiated cycle amplification. Under the optimal conditions, the detection limit of TK1 and c-myc mRNA is as low as 0.022 nM, and that of survivin and GalNAc-T mRNA is 0.029 nM. In addition, two pairs of cancer mRNAs could act as outputs to activate an AND logic gate.

PAMAM dendrimer-enhanced DNA biosensors based on electrochemical impedance spectroscopy.

A novel, simple and sensitive DNA biosensor based on DNA-poly(amidoamine) (PAMAM) dendrimer nanoconjugates was developed by using the electrochemical impedance spectroscopy (EIS) technique. In this context, the assay relies on the hybridization of the single-stranded DNA (ssDNA) probe covalently conjugated on a mercaptoacetic acid self-assembled monolayer on gold electrodes, with the generation 4.5 (G-4.5) PAMAM-target DNA complex in solution. Once the double-stranded DNA (dsDNA) formed on the gold electrodes, G-4.5 PAMAM bearing carboxyls on the periphery was anchored on the hybrids; the changes of interfacial electron-transfer resistance (R(et)) of the electrodes were measured using an Fe(CN)(6)(3-/4-) redox probe by electrochemical impedance spectroscopy. The results showed that only a complementary sequence could form a dsDNA-PAMAM with the DNA-PAMAM probe and give an obviously enlarged R(et) value. The non-complementary and three-base mismatched sequence exhibited negligible impedance change compared with the blank measurement (the blank measurement means: ssDNA probe-modified gold electrode was directly measured by EIS). The unique spherical structure combining with more negative charges on the G-4.5 PAMAM periphery anchored on the hybrids could significantly amplify the hybridization signal (R(et) value), and the detection limit for measuring the full complementary sequence is down to pM level.

Simultaneous determination of dihydroxybenzene and phenylenediamine positional isomers using capillary zone electrophoresis coupled with amperometric detection.

In general capillary zone electrophoresis (CZE) separation models, o-, m-, and p-phenylenediamine isomers can be separated in a weak acidic running buffer for their pK(a) values being 4.52, 5.64, 6.04, respectively, while o-, m-, and p-dihydroxybenzene isomers can be separated in a weak basic buffer for their pK(a) values being 9.40, 9.40 and 10.04, respectively. So, it is hard to find a suitable running buffer at a fixed pH in normal CZE for simultaneous separation of these two groups of positional isomers. In this paper, a novel method based on alternately running two different pH buffers in CZE coupled with amperometric detection (CZE-AD) was designed to simultaneously determine these two groups of positional isomers. It is found that when two different pH running buffers were employed alternately under appropriate order and time, the six analytes could be separated perfectly in less than 20 min and the detection limits were as low as 10(-7) mol/L. Furthermore, the effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CZE-AD were investigated. Experimental results demonstrated that the introduced method was practical to simultaneously determine two groups of positional isomers with different pK(a) and had some advantages of high sensitivity, good repeatability and small sample requirement.

Electrochemical behaviors of amino acids at multiwall carbon nanotubes and Cu2O modified carbon paste electrode.

A carbon paste electrode modified with multiwall carbon nanotubes and copper(I) oxide (MWCNT-Cu(2)O CPME) was fabricated, and the electrochemical behaviors of 19 kinds of natural amino acids at this modified electrode were studied. The experimental results showed that the various kinds of amino acids without any derivatization displayed obvious oxidation current responses at the modified electrode. It was also found that the current response values of amino acids were dependent mainly on pH values of buffer solutions. The phenomenon could be explained by the fact that the amino acids suffered complexation or electrocatalytic oxidation processes under different pH values. Six kinds of amino acids (arginine, tryptophan, histidine, threonine, serine, and tyrosine), which performed high-oxidation current responses in alkaline buffers, were selected to be detected simultaneously by capillary zone electrophoresis coupled with amperometric detection (CZE-AD). These amino acids could be perfectly separated within 20 min, and their detection limits were as low as 10(-7) or 10(-8)mol L(-1) magnitude (signal/noise ratio=3). The above results demonstrated that MWCNT-Cu(2)O CPME could be successfully employed as an electrochemical sensor for amino acids with some advantages of convenient preparation, high sensitivity, and good repeatability.

Simultaneous determination of phenylenediamine isomers and dihydroxybenzene isomers in hair dyes by capillary zone electrophoresis coupled with amperometric detection.

The simultaneous determination of three isomers of phenylenediamines (o, m, and p-phenylenediamine) and two isomers of dihydroxybenzenes (catechol and resorcinol) in hair dyes was performed by capillary zone electrophoresis coupled with amperometric detection (CZE-AD). The effects of working electrode potential, pH and concentration of running buffer, separation voltage, and injection time on CZE-AD were investigated. Under the optimum conditions the five analytes could be perfectly separated in 0.30 mol L(-1) borate-0.40 mol L(-1) phosphate buffer (pH 5.8) within 15 min. A 300 µm diameter platinum electrode had good responses at +0.85 V (versus SCE) for the five analytes. Their linear ranges were from 1.0 × 10(-6) to 1.0 × 10(-4) mol L(-1) and the detection limits were as low as 10(-7) mol L(-1) (S/N = 3). This working electrode was successfully used to analyze eight kinds of hair dye sample with recoveries in the range 91.0-108.0% and RSDs less than 5.0%. These results demonstrated that capillary zone electrophoresis coupled with electrochemical detection using a platinum working electrode as detector was convenient, highly sensitive, highly repeatable and could be used in the rapid determination of practical samples.

Determination of four polyphenolic active ingredients from a pharmaceutical preparation by capillary zone electrophoresis with amperometric detection.

The simultaneous determination of four active ingredients in Nao Xue Shuan Tablets, including ferulic acid, protocatechuic aldehyde, caffeic acid and protocatechuic acid was performed by capillary zone electrophoresis with amperometric detection (CZE-AD). The effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CZE-AD were investigated. Under the optimum conditions, the four analytes could be perfectly separated within 18 min. A 300 microm diameter carbon-disc electrode had a good response at +0.95 V (vs. SCE) for the four analytes. The response was linear over three orders of magnitude with detection limits (S/N=3) as low as 10(-8) or 10(-9) g/mL for the analytes. The assay results were satisfactory with recoveries in the range of 85.2-93.0% and RSDs less than 3.3%.

Design strategy for a novel electrochemically active-inactive switching molecular beacon based on Hemin for SNPs and insulin detection directly in homogenous solution.

Herein, a novel and convenient electrochemically active-inactive switching molecular beacon based on hemin (Hs-MB) has been designed for easy discrimination of single nucleotide polymorphisms (SNPs) and sensitive detection of insulin. The electrochemically active changing capability of Hs-MB is based on two identical hemin groups labeled at both ends of MB sequence in dimer or monomer forms depending on the conformation of MB which is in stem-loop structure or line shaped structure. The Hs-MB assay permits discrimination of SNPs and the highly sensitive and specific detection of insulin with detection limit successively as low as 0.5 pmol/L. Even at a very low target concentration, the Hs-MB assay also shows a good specificity in the presence of other potentially interfering components. The experimental results also show that Hs-MB can also be used for the accurate and rapid monitoring of insulin secretion by glucose-stimulated from MIN6 cells at different time periods, demonstrating that Hs-MB has potential in monitoring of biomarker variation in vivo.

A new amplification strategy for ultrasensitive electrochemical aptasensor with network-like thiocyanuric acid/gold nanoparticles.

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.

Simultaneous determination of sugars and ascorbic acid by capillary zone electrophoresis with amperometric detection at a carbon paste electrode modified with polyethylene glycol and Cu(2)O.

In this paper, a kind of novel carbon paste electrode modified with double modifiers-polyethylene glycol (PEG) and Cu(2)O (PEG/Cu(2)O CPME) in capillary zone electrophoresis with amperometric detection (CZE-AD) was applied to simultaneously determine three sugars: glucose, sucrose, fructose and ascorbic acid (AA). The catalytic electrochemical properties of PEG/Cu(2)O CPME could enhance sensitivity obviously compared with carbon paste electrode modified with only PEG or Cu(2)O at a relatively lower detection potential (+0.3 V versus SCE). Especially, the electrochemical detection response of AA increased obviously to the same level of saccharides by adding PEG into the Cu(2)O carbon paste modified electrode. The four analytes could be perfectly separated within 22 min, the linear ranges were from 1.0 x 10 (-6) to 5.0 x 10(-5) mol L(-1) and the detection limits were at 10(-7) mol L(-1) magnitude (S/N=3). The present working electrode was successfully employed to analyse beverage samples with recoveries in the range 93-107% and RSDs less than 4%. Above results demonstrated that capillary zone electrophoresis coupled with the PEG/Cu(2)O carbon paste modified electrode was of convenient preparation, high sensitivity, good repeatability and could be used in the rapid determination of practical samples.

Simultaneous determination of active ingredients in ethnomedicine Gaultheria leucocarpa var. yunnanensis and its medicinal preparation by capillary electrophoresis with electrochemical detection.

A simple and rapid capillary electrophoresis (CE) with electrcochemical detection (ED) method has been established for the simultaneous determination of seven active ingredients in the stems and roots of Gaultheria leucocarpa var. yunnanensis and its medicinal preparation, including (+)-catechin, rutin, gentisic acid, vallinic acid, salicylic acid, quercetin, and protocatechuic acid. The effects of working potential, pH, and concentration of running buffer, separation voltage, and injection time on CE-ED are systematically investigated. Under the optimum conditions, the seven analytes could be completely separated within 23 min in a borax running buffer (pH 8.7). A good linear relationship is obtained over three orders of magnitude with detection limits (signal-to-noise ratio=3) ranging from 5x10(-8) g/mL to 3x10(-7) g/mL for the analytes. The proposed method is successfully used in the analysis of real samples after a relatively simple extraction procedure, and the assay results are satisfactory.

Determination of active ingredients of Ilex Purpurea Hassk and its medicinal preparations by capillary electrophoresis with electrochemical detection.

A method based on capillary electrophoresis with electrochemical detection has been developed for the separation and determination of isovanillic acid, gentisic acid, kaempferol, quercetin, caffeic acid and protocatechuic acid in Ilex Purpurea Hassk and its medicinal preparations for the first time. The effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CE-ED were investigated. Under the optimum conditions, the analytes could be separated in a 50 mmoll(-1) borate buffer (pH 9.0) within 21 min. A 300 microm diameter carbon disk electrode has a good response at +0.95 V (versus SCE) for all analytes. The response was linear over three orders of magnitude with detection limits (S/N=3) ranging from 3 x 10(-8) to 2 x 10(-7)gml(-1) for the analytes. The method has been successfully applied to the analysis of real sample, with satisfactory results.

Determination of metoprolol tartrate in tablets and human urine using flow-injection chemiluminescence method.

In this paper, a simple, rapid and sensitive flow-injection chemiluminescence method has been developed for the determination of metoprolol tartrate, which acts as a kind of sensitizer in the chemiluminescence emission from the redox of SO(3)(2-) with Ce(IV) in acidic medium. Under the optimized conditions, the proposed method allows the measurement of metoprolol tartrate over the range of 1.5 x 10(-8) to 7.3 x 10(-6)mol/L with a detection limit of 4.7 x 10(-9)mol/L (3sigma), and the relative standard deviation for 7.3 x 10(-7)mol/L metoprolol tartrate (n=11) is 2.20%. The utility of this method was demonstrated by determining metoprolol tartrate in tablets and human urine sample.

Determination of aminoheterocycle and azabicycle in gliclazide bulk by capillary zone electrophoresis with amperometric detection.

A simple, reliable and reproducible method, based on capillary zone electrophoresis with amperometric detection (CZE-AD), was developed for simultaneous determination of 3-amino-3-azabicyclo[3,3,0]octane (aminoheterocycle) and 3-azabicyclo[3,3,0]octane (azabicycle) in gliclazide bulk drug. The optimal conditions of CZE-AD were 50 mM borate solution (pH 9.0) as running buffer, 14 kV as separation voltage and 0.95 V (versus SCE) as detection potential. Under the selected optimum conditions, the two analytes could be perfectly separated within 9 min. The linearity range of aminoheterocycle was from 1.0 x 10(-6) to 1.0 x 10(-3) M and that of azabicycle was from 2.0 x 10(-6) to 1.0 x 10(-3) M. Their detection limits were 5.0 x 10(-7) and 1.0 x 10(-6) M, respectively, (S/N=3). This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 2% for both migration time and peak current. It has been successively used for the determination of these two analytes in gliclazide bulk drug, and the assay results were satisfactory.