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.

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.

An ultra-sensitive colorimetric Hg(2+)-sensing assay based on DNAzyme-modified Au NP aggregation, MNPs and an endonuclease.

This paper reports the development of an ultra-sensitive colorimetric method for the detection of trace mercury ions involving DNAzymes, Au nanoparticle aggregation, magnetic nanoparticles and an endonuclease. DNAzyme-sensing elements are conjugated to the surface of Au nanoparticle-2, which can crosslink with the T-rich strands coated on Au nanoparticle-1 to form Au nanoparticle aggregation. Other T-rich stands are immobilized on the surface of MNPs. The specific hybridization of these two T-rich strands depends on the presence of Hg(2+), resulting in the formation of a T-Hg(2+)-T structure. Added endonuclease then digests the hybridized strands, and DNAzyme-modified Au NP aggregation is released, catalysing the conversion of the colourless ABTS into a blue-green product by H2O2-mediated oxidation. The increase in the adsorption spectrum of ABTS(+) at 421 nm is related to the concentration of Hg(2+). This assay was validated by detecting mercury ion concentrations in river water. The colorimetric responses were not significantly altered in the presence of 100-fold excesses of other metal ions such as Zn(2+), Pb(2+), Cd(2+), Mn(2+), Ca(2+) and Ni(2+). The inclusion of both Au NP aggregation and an endonuclease enables the assay to eliminate interference from the magnetic nanoparticles with colorimetric detection, decrease the background and improve the detection sensitivity. The calibration curve of the assay was linear over the range of Hg(2+) concentrations from 1 to 30 nM, and the detection limit was 0.8 nM, which is far lower than the 10 nM US EPA limit for drinking water.

Sensitive determination of four ß2-agonists in pig feed by capillary electrophoresis using on-line sample preconcentration with contactless conductivity detection.

In this work, an on-line preconcentration method of field-enhanced sample injection (FESI) was implemented in the determination of four ß2-agoinsts terbutaline (TER), procaterol (PRO), formoterol (FOR) and bambuterol (BAM) by capillary electrophoresis coupled with capacitively coupled contactless conductivity detection (CE-C4D). Under optimized conditions the background electrolyte (BGE) was 5mM Tris(hydroxymethyl)aminomethane (Tris) and 10mM citric acid (Cit) at a pH of 3.2 while the sample dilution solution was obtained by methanol. The detection limits (defined as S/N=3) of this method were 0.02mg/L for TER, PRO, FOR, BAM, which were much lower than that of the conventional CE-C4D method without preconcentration procedure, the enhancement factors were greatly improved to be 30-40-fold. The linearity ranges of four ß2-agoinsts were 0.1-15mg/L, with good linear correlation coefficients (r2>0.9900). In order to evaluate the application potential of the developed method, real sample from pig feed was analyzed with recoveries of 91.4-106.2%.

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.

Sensitive analysis of aminoglycoside antibiotics via hyphenation of transient moving substitution boundary with field-enhanced sample injection in capillary electrophoresis.

A novel field-enhanced sample injection coupled with transient moving substitution boundary method in capillary electrophoresis was developed for aminoglycoside antibiotic (AG) analysis using 18-crown-6-tetracarboxylic acid (18C6H4) as a pseudostationary phase. Results indicated that the stacking mechanism of moving substitution boundary relied on the substitution reaction between 18C6H4-bonded AG complexes and Na(+) at the substitution boundary. The stacking mechanism as well as important parameters governing pre-concentration and separation have been investigated in order to obtain maximum resolution and sensitivity. Under optimized conditions, using a sample prepared in a low-conductivity matrix, the limits of detection for streptomycin, neomycin, and kanamycin were 0.62, 5.9 and 8.6 nM (S/N=3), respectively, and the detection sensitivities were improved 940-, 692-, and 415-fold, respectively. The method also gave accurate and reliable results in the analysis of AGs in river water samples.

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.

Qualitative and quantitative detection of DNA amplified with HRP-modified SiO2 nanoparticles using scanning electrochemical microscopy.

Qualitative and quantitative detection of DNA was achieved by a "sandwich" DNA sensor through SG/TC (substrate generation and tip collection) mode of scanning electrochemical microscopy (SECM). The "sandwich" DNA structure was formed by the hybridization of thiol-tethered oligodeoxynucleotide probes (capture probe), assembled on the gold substrate surface, with target DNA and biotinylated indicator probe. HRP (horseradish peroxidase)-wrapped SiO2 nanoparticles were linked to the sandwich structure through biotin-streptavidin interaction. Hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface where sequence-specific hybridization had occurred through the HRP-catalyzed reaction in the presence of H2O2. The detection was based on the reduction of BQ generated on the modified substrate by SECM tip. For SECM imaging experiment, we structured the microsensor platform through localized desorption of 1-dodecanethiol monolayer. Approach curves were employed for quantitative detection of DNA concentration. The detection limit of complementary DNA was as low as 0.8pM. This technique is promising for the application on electrochemical DNA chip.

Sensitive measurement of polyols in urine by capillary zone electrophoresis coupled with amperometric detection using on-column complexation with borate.

Little is known about human polyol metabolism, but recent studies indicate that abnormal polyol concentrations in body fluids are related to several diseases. In this study, a rapid and sensitive method for the determination of seven major polyols in urine including two groups of polyol isomers, C5-polyols (Rib+Arb+Xyl) and C6-polyols (Sor+Gal+Man), was developed using capillary zone electrophoresis coupled with amperometric detection (CZE-AD). The effects of the working electrode potential, pH, running buffer components and concentrations, separation voltage and injection times were investigated. Under the optimised conditions, seven types of polyols could be perfectly separated via the formation of anionic polyol-borate complexes in a borate buffer solution. Highly linear current responses to the polyol concentrations were obtained with good correlation (0.9984

A Tris(bipyridine)ruthenium(II)-ß-Cyclodextrin Derivative: Synthesis, Luminescent Properties, and Application in Electrochemiluminescence DNA Sensors.

Miraculous metallocyclodextrin: A new polynuclear metallocyclodextrin complex, tris(bpyRu)-ß-CD (bpy=bipyridine), was successfully synthesized. This complex possesses molecular-recognition functions and unique luminescent properties. A tris(bpyRu)-ß-CD film was fabricated and utilized in a electrochemiluminescence (ECL) DNA sensor based on its molecular-recognition and luminescence characteristics.

A host-guest-recognition-based electrochemical aptasensor for thrombin detection.

A sensitive electrochemical aptasensor for thrombin detection is presented based on the host-guest recognition technique. In this sensing protocol, a 15 based thrombin aptamer (ab. TBA) was dually labeled with a thiol at its 3' end and a 4-((4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl) at its 5' end, respectively, which was previously immobilized on one Au electrode surface by AuS bond and used as the thrombin probe during the protein sensing procedure. One special electrochemical marker was prepared by modifying CdS nanoparticle with ß-cyclodextrins (ab. CdS-CDs), which employed as electrochemical signal provider and would conjunct with the thrombin probe modified electrode through the host-guest recognition of CDs to dabcyl. In the absence of thrombin, the probe adopted linear structure to conjunct with CdS-CDs. In present of thrombin, the TBA bond with thrombin and transformed into its special G-quarter structure, which forced CdS-CDs into the solution. Therefore, the target-TBA binding event can be sensitively transduced via detecting the electrochemical oxidation current signal of Cd of CdS nanoparticles in the solution. Using this method, as low as 4.6 pM thrombin had been detected.

Development of a compact chemiluminescence system coupled with capillary electrophoresis for carbohydrate analysis.

As a kinetic process, chemiluminescence (CL) met great challenges while it was used in the detection methods coupled with capillary electrophoresis (CE). In this investigation, a newly recorded ultra-fast CL reaction of luminol-KIO(4)-K(3)Fe(CN)(6) was observed to be completed in 0.65 s. It was adopted in a simple CE-CL system efficiently to avoid the peak-tailing and overlapping. With this compact system, an indirect determination of rhamnose, d-fructose, sucrose and ß-cyclodextrin was realized based on the corresponding negative CL peaks. These peaks were due to the displacement of luminol anions by the analyzed saccharide molecules in alkaline separation electrolyte. In this way, these four saccharides could be separated and determined in 16 min with adequate sensitivities and stabilities. No derivatization or pretreatment was required for the analysis, and it presents an attractive opportunity for routine tests of mono-, di- and oligo-saccharides in a compact CE-CL system, even as a microchip device.

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.

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.

A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme.

In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H(2)O(2)-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV-visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20 nM and the detection limit of thrombin was 0.5 nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.

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.

Microchip electrophoresis of bacteria using lipid-based liquid crystalline nanoparticles.

The aggregation and adhesion of bacterial cells is a serious disadvantage for electrophoretic separations of bacteria. In this study, lipid-based liquid crystalline nanoparticles were used as a pseudostationary phase to minimise the bacterial aggregation and adsorption to the inner walls of microchannels. Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus were selected as analytes and were separated by microchip electrophoresis (MCE) with laser-induced fluorescence (LIF) detection using 4.5 mM tris(hydroxymethyl) aminomethane (TRIS)-4.5 mM boric acid-0.1 mM ethylenediaminetetraacetate (EDTA) (TBE) containing poly(ethylene oxide) (PEO) and lipid-based nanoparticles as the running buffer. The mechanism of lipid-based nanoparticles affecting bacterial adhesion and aggregation was discussed and supported by zeta potential experiments. Under the optimal conditions, the three species of bacteria were identified with patterned peaks. This proposed MCE method using lipid-based nanoparticles as running buffer additives was also used to analyse a real yogurt sample, and valuable bacterial information was obtained by the electropherograms.

Colorimetric assay for mercury (II) based on mercury-specific deoxyribonucleic acid-functionalized gold nanoparticles.

A colorimetric nanoprobe-mercury-specific DNA-functionalized gold nanoparticles (Au-MSD) was developed for sensing Hg(2+). The new mercury-sensing concept relies on measuring changes in the inhibition of "non-crosslinking" aggregation of Au-MSD-induced by the folding of mercury-specific DNA strand through the thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination. In the absence of Hg(2+), a high concentration of MgCl(2) (50 mM) results in a rapid aggregation of Au-MSD because of the removal of charge repulsion. When Hg(2+) is present, the particles remain stable due to the folding of MSD functionalized on the particle surface. The assay enables the colorimetric detection of Hg(2+) in the concentration range of 0.1-10 µM Hg(2+) ions with a detection limit of 60 nM, and allows for the selective discrimination of Hg(2+) ions from the other competitive metal ions. Toward the goal for practical applications, the sensor was further evaluated by monitoring Hg(2+) in fish tissue samples.

Sensitive detection of p53 tumor suppressor gene using an enzyme-based solid-state electrochemiluminescence sensing platform.

An enzyme-based solid-state electrochemiluminescence (ECL) sensing platform for sensitive detection of a single point mutation is developed successfully using p53 tumor suppressor gene as a model analyte. A composite of multiwalled carbon nanotubes and Ruthenium (II) tris-(bipyridine) (MWNTs-Ru(bpy)(3)(2+)) was prepared and coated on an electrode surface, which was covered by polypyrrole (PPy) to immobilize ssDNA. Then, the ssDNA recognized the gold nanoparticle (AuNP)-labeled p53 tumor suppressor gene, and produced AuNP-dsDNA electrode with AuNP layer. The surface adsorbed the glucose-dehydrogenase (GDH) molecules for producing ECL signal. This system combined enzyme reaction with ECL detection, and it can recognize sequence-specific wild type p53 sequence (wtp53) and muted type p53 sequence (mtp53) with discrimination of up to 56.3%. The analytic results were sensitive and specific. It holds promise for the diagnosis and management of cancer.