Exonuclease-assisted multicolor aptasensor for visual detection of ochratoxin A based on G-quadruplex-hemin DNAzyme-mediated etching of gold nanorod.

An exonuclease-assisted multicolor aptasensor was developed for the visual detection of ochratoxin A (OTA). It is based on the etching of gold nanorods (AuNRs) mediated by a G-quadruplex-hemin DNAzyme. A DNA sequence (AG4-OTA) was designed that comprises a hemin aptamer and an OTA aptamer. OTA binds to AG4-OTA to form an antiparallel G-quadruplex, which halts its digestion by exonuclease I (Exo I) from the 3'-end of AG4-OTA. Thus, the retained hemin aptamer can bind to hemin to form a G-quadruplex-hemin DNAzyme. This DNAzyme has peroxidase-like activity that catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce its diimine derivative (TMB2+) in acidic solution. TMB2+ can etch the AuNRs by oxidizing Au(0) into Au(I). This results in the generation of rainbow-like colors and provides a multicolor platform for the visual detection of OTA. The assay is based on the use of a single isolated aptamer and possesses obvious advantages such as multi-color visual inspection, relatively high sensitivity and accuracy. It can be used to detect as little as 30 nM concentrations of OTA by visual observation and even 10 nM concentrations by spectrophotometry. The method was successfully applied to the determination of OTA in spiked beer where it gave recoveries of 101-108%, with a relative standard deviation (RSD, n = 5) of <5%. Graphical abstract Schematic of an exonuclease-assisted multicolor bioassay based on the G-quadruplex-hemin DNAzyme-mediated etching of gold nanorods (AuNRs). It enables visual detection of ochratoxin A (OTA) with a detection limit of 30 nM.

Capillary electrophoresis inductively coupled plasma mass spectrometry combined with metal tag for ultrasensitively determining trace saxitoxin in seafood.

As one of paralytic shellfish toxins, the saxitoxin (STX) in the aqueous environment can be accumulated by most shellfish, and thus harms human health through the food chain. Therefore, it is crucial to determine trace STX in seafood samples in order to ensure the safety of seafood consumption. In this study, we developed a novel indirect method for ultrasensitively determining trace STX in seafood by using CE-ICP-MS together with Eu3+ chelate labeling. We demonstrated that diethylenetriamine-N,N,N',N″,N″-pentaacetic acid (DTPA) can couple with STX and simultaneously chelate with Eu3+ to realize metallic labeling of STX, and thus realize the ultrasensitive quantification of trace STX with CE-ICP-MS. The proposed method has strong antiinterference ability, good stability, and extremely high sensitivity. It could be used to determine trace STX in seafood samples with an extremely low detection limit of 0.38 fmol (3.8×10-9 M, 100 nL sample injection) and a relative standard deviation (RSD, n = 5) <7%. The success of this study provides an alternative to precise quantification of ultra-trace STX in seafood samples, and further expands the application of ICP-MS.

Ultra-sensitive speciation analysis of mercury by CE-ICP-MS together with field-amplified sample stacking injection and dispersive solid-phase extraction.

A simple dispersive solid-phase extraction (DSPE) used to extract and preconcentrate ultra-trace MeHg, EtHg and Hg(2+) from water sample, and a sensitive method for the simultaneous analysis of MeHg, EtHg and Hg(2+) by using capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) with field-amplified sample stacking injection (FASI) were first reported in this study. The DSPE used thiol cotton particles as adsorbent, and is simple and effective. It can be used to extract and preconcentrate ultra-trace mercury compounds in water samples within 30 min with a satisfied recovery and no mercury species alteration during the process. The FASI enhanced the sensitivity of CE-ICP-MS with 25-fold, 29-fold and 27-fold for MeHg, EtHg and Hg(2+) , respectively. Using FASI-CE-ICP-MS together with DSPE, we have successfully determined ultra-trace MeHg, EtHg and Hg(2+) in tap water with a limits of quantification (LOQs) of 0.26-0.45 pg/mL, an RSD (n = 3) < 6% and a recovery of 92-108%. Ultra-high sensitivity, as well as much less sample and reagent consumption and low operating cost, make our method a valuable technique to the speciation analysis of ultra-trace mercury.

A "turn-on" and label-free fluorescent assay for the rapid detection of exonuclease III activity based on Tb(3+)-induced G-quadruplex conjugates.

A "turn-on" and label-free fluorescent assay for the specific, rapid, and sensitive detection of 3' → 5' exonuclease III activity is reported in this study. The assay is based on the Tb(3+)-promoted G-quadruplex, which lead to the enhancement of Tb(3+) fluorescence due to the energy transfer from guanines. The proposed assay is highly simple, rapid, and cost-effective, and does not require sophisticated experimental techniques such as gel-based equipment or radioactive labels. It can be used for the rapid detection of exonuclease III activity with a detection limit of 0.8 U and a RSD (n = 6) <5 %. Notably, no dye was covalently conjugated to the DNA strands, which offers the advantages of low-cost and being interference-free.

A microfluidic chip-based fluorescent biosensor for the sensitive and specific detection of label-free single-base mismatch via magnetic beads-based "sandwich" hybridization strategy.

A novel microfluidic chip-based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads-based "sandwich" hybridization strategy, was developed for the sensitive and ultra-specific detection of single-base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single-base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer-related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10(-11) M, a RSD (n = 5) < 5% and a discrimination factor of 3.58-4.54 for one-base mismatch.

Separation and determination of ß-casomorphins by using glass microfluidic chip electrophoresis together with laser-induced fluorescence detection.

A simple, reliable and reproducible method for the separation and determination of five ß-casomorphins (ß-CMs, namely TPGN, PGPI, TPGI, TPGP and TPPG) based on glass microfluidic chip electrophoresis and laser-induced fluorescence detection is first described in here. The microfluidic chip electrophoresis and laser-induced fluorescence detection system consisted of a home-made glass "double-T" microchip and a simple LIF detector with excitation and emission wavelengths of 473 and 525 nm, respectively. Fluorescein isothiocyanate (FITC) was used as the precolumn derivatization reagent to label fluorophore on five ß-CMs, and the optimum conditions of FITC-derivatization reaction and MCE separation were investigated in detail. Under optimum conditions, five ß-CMs were completely separated and detected within 30 min with a detection limit of 18.7-75.1 nmol/L and an RSD (n=5) of 3.0-5.9%, respectively. The proposed method has been successfully used to detect ß-CMs in real cheese sample with a recovery of 89-109%, suggesting that our method is sensitive and reliable. These features, as well as its low cost, operation convenience, stability and reusability, make it a promising alternative to ß-CMs detection methods.

Analysis of aliphatic amines using head-column field-enhanced sample stacking in MEKC with LIF detection.

This paper describes the use of head-column field-enhanced sample stacking for on-line sample concentration in MEKC; the method has been applied to the determination of C1-C9 aliphatic amines after their pre-capillary derivatization with 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA). The optimum derivatization, on-line concentration and separation conditions for these aliphatic amines were investigated in detail. The optimized molar ratio of CBQCA to amines was found to be 120:1, and 50 mmol/L borate buffer (pH 9.5) was used for derivatization in order to achieve high efficiency. Nine imidazole aliphatic amines were baseline separated within 12 min by using 50 mmol/L of sodium borate (pH 9.5) and 15 mmol/L of SDS as running buffer. Under the optimum conditions, the detection limits of these nine aliphatic amines ranged from 1.2 to 3.8 pmol/L. S/N=3, which was about 49-173-fold lower than those of conventional sample injections. After validation, the developed method was applied to the determination of C1-C9 aliphatic amines in river water with the average recoveries of 86.7-105%.

Improved simultaneous enantioseparation of beta-agonists in CE using beta-CD and ionic liquids.

In this study, approaches to improve chiral resolutions in simultaneous enantioseparation of beta-agonists by CE via a CD inclusion complexation modified with ionic liquids (ILs) are described. Different types of ILs, including tetraalkylammonium-based ILs, alkylimidazolium-based ILs and alkylpyridinium-based ILs, were examined and compared for controlling the EOF in order to improve resolutions of beta-agonists enantiomers. In this regard, tetraalkylammonium-based ILs were more effective because they could be used at much higher concentrations than other types of ILs. N-octylpyridinium hexafluorophosphate gave poor resolutions of beta-agonists enantiomers. In addition, when different ILs were mixed to use, they would present particular properties of their own. Moreover, the presence of ILs was essential in the chiral separations of (+/-) salbutamol, (+/-) cimaterol and (+/-) formoterol, which were reportedly not enantioseparated by using the buffer electrolytes containing only beta-CD as a chiral selector.

Determination of organophosphorus pesticides by capillary electrophoresis-inductively coupled plasma mass spectrometry with collective sample-introduction technique.

A new method for the determination of organophosphorus pesticides using CE-ICP-MS with collective sample-introduction technique has been developed in this study. The method has been successfully used to separate and determine dimethoate, trichlorfon and glyphosate with an RSD of < 5% for migration times (n = 6) and < 4% for peak areas (n = 6). The experimental results showed that the collective sample-introduction considerably reduced the makeup volume and the dilution of analyte, and eventually resulted in a much lower detection limit and a much better electrophoretic resolution. The peak widths and the detection limits of dimethoate, trichlorfon and glyphosate obtained with this method are 15-17 s and 0.05-0.07 microg/mL (as compound), respectively. Using this method, we have successfully separated and determined dimethoate, trichlorfon and glyphosate in vegetable sample with a recovery of 90-96%.

Assay of bradykinin metabolites in human body fluids by CE-LIF coupled with transient ITP preconcentration.

A CE with LIF detection was developed for the separation and determination of three bradykinin (BK) metabolites: BK2-9, BK1-7 and BK1-5. BK fragments were derivatized with 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein before CE-LIF analysis. Eighty millimolar of Tris-H3PO4 (pH 9.0) was selected as the derivatization media. Three BK fragments were baseline separated within 10 min by using 0.2 M TAPS-Tris buffer (pH 8.5) as the running buffer. Meanwhile we have also developed a simple, quick, and sensitive on-column transient ITP preconcentration for CE-LIF detection of three BK fragments. Ten millimolar of Tris-HCl (pH 9.0) was chosen as the leading electrolyte and 0.2 M TAPS-Tris (pH 8.5) containing 10 mM 1-butyl-3-methylimidazolium tetrafluoroborate as the terminating electrolyte and also served as the running buffer during CZE separation. By using this transient ITP coupled with CE-LIF, concentration detection limits (S/N=3) for BK2-9, BK1-7 and BK1-5 were 0.3, 0.1 and 0.1 pmol/L, respectively. This method has been applied to the assay of human saliva and plasma samples with satisfactory results.

Characterization of keto-enol tautomerism of p-hydroxyphenylpyruvic acid using CE with amperometric detection and spectrometric analysis.

A high-performance CE with amperometric detection (CE-AD) was employed for the kinetic study of keto-enol tautomerism of p-hydroxyphenylpyruvic acid (pHPP). Several factors (concentration of beta-cyclodextrin (beta-CD), concentration and pH of running buffer, separation voltage and injection time) affecting CE-AD were investigated and separation conditions were optimized. The kinetics of pHPP was performed in water solution and phosphate solution under different pH and temperature, the homologous ketonization rate constants and half-life were obtained. Also, the activation energy was calculated according to the rate constants under different temperature. The experimental results indicated that beta-CD played an important role in the separation, therefore UV spectrometric method was applied for the study of complexation interaction between ketonic pHPP and beta-CD. The results indicated that the stoichiometric ratio of the pHPP-beta-CD complex was 1:1 and formation constant was determined. The obtained kinetic results are in correspondence with those reported by our group with CE-UV.

Study on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chloro-phenoxyacetic sodium (MCPA sodium) in natural agriculture-soils of Fuzhou, China using capillary electrophoresis.

A new method of analyzing trace 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2-methy-4-chloro-lphenoxyacetic sodium (MCPA sodium) in soils by capillary electrophoresis (CE) has been developed in this study. The optimum analytical conditions including chemical component and concentration of buffer solution, pH, separation voltage and sample injection time were studied in detail. Under the optimum conditions, 2,4-D and MCPA sodium in soils can be speedy separated and determined within 20 min with detection limits of 0.15 microg/g (2,4-D) and 0.25 microg/g (MCPA sodium) , a RSD (n=6)<5% and a recovery>89%. With the help of analytical method developed in this study, the degradations of 2,4-D and MCPA sodium in natural agriculture-soils of Fuzhou were studied. The experimental results indicated that the degradations of 2,4-D and MCPA sodium follow first-order kinetics with degradation constants of 0.1509 day(-1) (2,4-D) and 0.2722 day(-1) (MCPA sodium) respectively. The degradation half-life were calculated to be 4.6 days (2,4-D) and 2.6 days (MCPA sodium) at 27 degrees C, implied that 2,4-D and MCPA sodium can be speedy degraded in natural agriculture-soils of Fuzhou, China.

A signal-on magnetic electrochemical immunosensor for ultra-sensitive detection of saxitoxin using palladium-doped graphitic carbon nitride-based non-competitive strategy.

Saxitoxin (STX) has high toxicity, and is water soluble, acid stable and thermostable. Therefore, STX in seawater can be accumulated by marine organism to form bioaccumulation. To ensure the safety of seafood for consumption, it is crucial to accurately determine trace STX in seawater and seafood. We herein developed a novel magnetic electrochemical immunosensor for ultra-sensitive detection of STX in seawater and seafood by using non-competitive strategy. The immunosensor employs STX-specific antibody-functionalized magnetic beads (MBs) for STX recognition, palladium-doped graphitic carbon nitride (g-C3N4-PdNPs) peroxidase mimetic for catalyzing H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate signal. The immunosensor combines the merits of g-C3N4-PdNPs peroxidase mimetic, non-competitive strategy, MBs-based antibody recognition and magnetic gold electrode, and thus has excellent stability, lower cost, no risk of false positive result, high sensitivity and strong ability resist to matrix interference. The proposed immunosensor has been successfully used to detect trace STX in seawater and shellfish samples with a detection limit of 1.2 pg/mL (4.0 × 10-12 M), a recovery of 93-107% and a relative standard deviation (RSD, n = 5) < 5%. The success of this study provided a promising approach for the rapid and on-site detection of trace STX in seawater and seafood.

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration.

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT(8-13)), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise=3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT(8-13), NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT(8-13) in human cerebrospinal fluid sample.

Morphological and light-absorption characteristics of individual BC particles collected in an urban seaside area at Tokaimura, eastern central Japan.

To observe surface morphology and light-absorption property of different black carbon (BC) particles, different-sized aerosols were collected in Tokaimura (36.27 degrees N, 140.36 degrees E), an urban seaside area of eastern central Japan, using a high-volume Andersen type sampler during a whole year (Jan. to Dec. in 2004). The morphology of individual BC particle separated from different-sized aerosols was observed with Scanning Electron Microscope with Energy Dispersive X-ray Spectrometer (SEM-EDX) and four types of morphology were observed: 50 nm spherical particles, micrometer-sized plates with homogeneous surfaces, micrometer-sized spherical particles with homogeneous surfaces and micrometer-sized spherical particles with small holes on surfaces. The light-absorption property of BC particles with different morphology has been determined by infrared spectrometry (IRS) with a photoacoustic technique in a region of 400-4000 wavenumbers (cm(-1)). All morphology BC particles showed a strong light-absorption during 500-3000 wavenumbers (cm(-1)) with two strong broad peaks in 750-1100 and 1200-2200 wavenumbers (cm(-1)), implying that all morphology BC particles can absorb a significant part of thermal infrared emitted from the earth (wavelength 4000-50,000 nm). The seasonal variation and the size-distribution of aerosols and its chemical components (e.g. C, Na, Cl, NH(4)(+), NO(3)(-), SO(4)(2-), Al, Ca, Mg and Fe) were also measured in this study. More than 55% of non-inorganic carbon (OC+BC) in the atmosphere was detected in the aerosols with a size smaller than 1.1 microm and the concentration of non-inorganic carbon in the atmosphere showed only a faint variation during a whole year, although the concentrations of total aerosols and its chemical components exhibited a distinct variation.

Tumor targeting dual stimuli responsive controllable release nanoplatform based on DNA-conjugated reduced graphene oxide for chemo-photothermal synergetic cancer therapy.

In this research, a novel tumor targeting dual stimuli responsive nanoplatform was fabricated for the controllable delivery and release of a drug to realize chemo-photothermal synergetic cancer therapy by integrating a DNA aptamer with polydopamine reduced graphene oxide (rGO-PDA) nanosheets. The rGO-PDA nanosheets simultaneously acted as a near-infrared radiation (NIR) photothermal agent to generate hyperthermia for photothermal therapy and a nano-carrier for loading doxorubicin (DOX), and the specially designed DNA aptamer served as a supplementary carrier for DOX loading as well as targeting moiety/gatekeeper for specific cellular recognition and controllable release of DOX. The proposed nanoplatform possessed a good targeting ability, remarkable photothermal conversion ability and intelligent drug release with both pH and photothermal heating dual stimuli response. The nanoplatform was successfully used to selectively deliver DOX to protein tyrosine kinase 7 over-expressing cancer cells with a loading capacity of 1.56 mg mg-1 and controllable drug release, which responded to both acidic intracellular environments and NIR irradiation. The combination of the dual stimuli responsive controllable release and the dual nanocarrier for drug loading results in efficient chemo-photothermal synergetic therapy and holds great potential for multimodal cancer therapy.

Study on the kinetics of keto-enol tautomerism of p-hydroxyphenylpyruvic acid using capillary electrophoresis.

The kinetics of keto-enol tautomerism of p-hydroxyphenylpyruvic acid (pHPP) as a model of alpha-carbonyl compounds in aqueous solution at room temperature (25 degrees C) was first investigated by capillary electrophoresis with UV detection at 200 nm. The two tautomers could be separated and detected within 3 min. Since the ketonization of enolic pHPP varied with the buffer composition and buffer pH, the kinetics of pHPP was studied under different conditions, and relevant distributing fractions of enolic pHPP, ketonization rate constants and half-life were determined. In addition, beta-CD played an important part in the separation of the two tautomers, thus, the interaction between pHPP and beta-CD was also investigated by electrochemical techniques.

Magnetic beads-based DNA hybridization chain reaction amplification and DNAzyme recognition for colorimetric detection of uranyl ion in seafood.

A novel colorimetric biosensor, which employs DNAzyme-functionalized magnetic beads (MBs) as recognition probe, enzyme-assisted catalytic oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) as signal and DNA hybridization chain reaction as amplification strategy, has been developed for detecting trace uranyl ion (UO22+) in seafood and aqueous environment with high sensitivity and specificity. We demonstrated that UO22+ can specifically cleave DNAzyme immobilized on MBs surface to release a short single-strand DNA (primer), and the released primer trigger DNA hybridization chain reaction to form a long one dimensional DNA concatamer on the MBs surface. The resulting long DNA concatamer could capture a large amount of HRP to generate the one UO22+-to-multiple HRP amplification effect. Upon the addition of TMB-H2O2 solution, the HRP-tagged DNA concatamer-MBs conjugates could catalyze the H2O2-mediated oxidation of TMB, and thus results in a color change from colorless to blue in solution. This provided a sensitive and selective sensing platform for the visual or colorimetric detection of UO22+. The proposed biosensor has high sensitivity and strong anti-interference capability, it can be used to detect as low as 2.5 ppb (9.25 nM) of UO22+ by naked-eye observation and 0.09 ppb (0.33 nM) of UO22+ by UV-visible spectrometry with no interference of other ions and a RSD ≤ 6% (n = 5). With the help of this method, we have successfully determined trace UO22+ in fish muscle and river water with a recovery of 93-106%. High sensitivity and specificity, as well operation convenience, low cost and strong resistibility to the matrix, which makes our method a potential approach for the on-site detection of UO22+ in seafood and aqueous environment.

Ion-imprinted magnetic nanoparticles for specific separation and concentration of ultra-trace methyl mercury from aqueous sample.

For rapidly and sensitively determining ultra-trace methyl mercury (MeHg) in aqueous environment, we herein synthesized a MeHg ion-imprinted magnetic nanoparticle (MeHg IIMN) to simply and specifically extract/concentrate ultra-trace MeHg from water samples. The MeHg IIMN employed core-shell Fe3O4@SiO2 nanoparticles (Fe3O4@SiO2 NPs) as supporting structure, the complex ion of 1-pyrrolidinecarbodithioic acid and MeHg (PDC-CH3Hg+) as template, methacrylic acid (MAA) as functional monomer and trimethylolpropane trimethacrylate (TMPTM) as cross linker. The MeHg IIMN offered obvious advantages such as low cost, easy manipulation, better specificity and stability, and recycling characteristics. It can be used to separate/concentrate ultra-trace MeHg from natural water sample within 30min with a recovery >95%, an enrichment factor of 250, a relative standard deviation (RSD, n=5) <7%, a 25mg MeHg/g of maximum adsorption capacity, 50 times of recycling, and without obvious interference of other ions. Combining with capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS), it can be used for the accurate detection of ultra-trace methyl mercury in natural water samples with a limit of detection of 0.084pg/mL, a recovery of 92-99% and a RSD (n=5)<8%. The success of this study promises a valuable technique for relatively simple detection of ultra-trace methyl mercury in aqueous environment.

Species characteristics of lead in sea foods collected from coastal water of Fujian, Southeastern of China.

Various sea foods including fish, shellfish and shrimp were collected from different coastal areas of Fujian in China, and their Pb species characteristics were investigated in detail. The results indicated that there are two different species characteristics of Pb existing in sea food samples. About half of samples were detected to have only Pb(2+), and another half of samples were detected to have both Pb(2+) and trimethyl lead (TML). The results also revealed that Pb species characteristics in the sea foods rather depend on the species of sea food than the sampling area. In comparison with shellfish/shrimp samples, fish samples have higher concentrations of TML and Pb(2+). Especially, the average concentration of TML in the TML-detected fish samples is about 3 times of that in the TML-detected shellfish/shrimp samples, indicating that fish has stronger ability to uptake and accumulate TML. The concentrations of total lead in all samples are lower than the maximum allowable limit of national standard, suggesting that the sea foods collected from Fujian are safe for consumption. By considering that TAL has more toxicity than Pb(2+), the effect of TML in sea foods on the human health should be paid more attention in the future.