Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation

An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80?±?5 % and 90?±?5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (Hypercarb^TM) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 μL, the limits of detection for the UDP-sugars were in the 70 nmol L^?1 range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 μg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants.

Electrochemical determination of paraquat using a DNA-modified carbon ionic liquid electrode

Deoxyribonucleic acid (DNA) was electrochemically deposited on a carbon ionic liquid electrode to give a biosensor with excellent redox activity towards paraquat as shown by cyclic voltammetry and differential pulse voltammetry. Experimental conditions were optimized with respect to sensing paraquat by varying the electrochemical parameters, solution pH, and accumulation time of DNA. Under the optimized conditions, a linear relation exists between the reduction peak current and the concentration of paraquat in the range from 5?×?10^?8 mol L^?1 to 7?×?10^?5 mol L^?1, with a detection limit of 3.6?×?10^?9 mol L^?1. The utility of the method is illustrated by successful analysis of paraquat in spiked real water samples.

Mass Accuracy and Isotopic Abundance Measurements for HR-MS Instrumentation: Capabilities for Non-Targeted Analyses

The development of automated non-targeted workflows for small molecule analyses is highly desirable in many areas of research and diagnostics. Sufficient mass and chromatographic resolution is necessary for the detectability of compounds and subsequent componentization and interpretation of ions. The mass accuracy and relative isotopic abundance are critical in correct molecular formulae generation for unknown compounds. While high-resolution instrumentation provides accurate mass information, sample complexity can greatly influence data quality and the measurement of compounds of interest. Two high-resolution instruments, an Orbitrap and a Q-TOF, were evaluated for mass accuracy and relative isotopic abundance with various concentrations of a standard mixture in four complex sample matrices. The overall average ± standard deviation of the mass accuracy was 1.06 ± 0.76 ppm and 1.62 ± 1.88 ppm for the Orbitrap and the Q-TOF, respectively; however, individual measurements were ± 5 ppm for the Orbitrap and greater than 10 ppm for the Q-TOF. Relative isotopic abundance measurements for A + 1 were within 5% of the theoretical value if the intensity of the monoisotopic peak was greater than 1E7 for the Orbitrap and 1E5 for the Q-TOF, where an increase in error is observed with a decrease in intensity. Furthermore, complicating factors were found in the data that would impact automated data analysis strategies, including coeluting species that interfere with detectability and relative isotopic abundance measurements. The implications of these findings will be discussed with an emphasis on reasonable expectations from these instruments, guidelines for experimental workflows, data analysis considerations, and software design for non-targeted analyses.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

A lipase-based electrochemical biosensor for target DNA

A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1?×?10^?12 mol·L^?1 and 1?×?10^?8 mol·L^?1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30 min) and low interference.

Selection and identification of ssDNA aptamers recognizing zearalenone

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z₃₁, with a dissociation constant (K _d) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10^?9 to 3.14?×?10^?5 M for ZEN, and the detection limit was 7.85?×?10^?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

Electrochemical genosensor for detection of human mammaglobin in polymerase chain reaction amplification products of breast cancer patients

Human mammaglobin (MG) has been found to be the most specific molecular marker for the hematogenous spread of breast cancer cells. In our study, an electrochemical impedance spectroscopic DNA biosensor was established for the detection of MG in breast cancer patients. The working conditions for the biosensor, such as immobilization time, rinse process, and hybridization process, were optimized. Under the optimal conditions, the charge transfer resistance of the proposed DNA biosensor shows excellent correlation with the amount of the complementary oligonucleotides in the range from 1.0?×?10^?9 to 2.0?×?10^?8?M. The detection limit is 5.0?×?10^?10?M. The proposed biosensor was used to detect the polymerase chain reaction amplification products of actual clinical breast cancer samples. The results were compared with that obtained by conventional gel electrophoresis. The results indicate that the electrochemical impedance spectroscopic assay is significantly sensitive and time-saving. The simple strategy described here is expected to be used in clinical application for early diagnosis of breast cancer.

Magnetic nanoparticles grafted with β-cyclodextrin for solid-phase extraction of 5-hydroxy-3-indole acetic acid

We describe the synthesis of ß-cyclodextrin modified magnetic nanoparticles (CD-mNPs) as a material for solid-phase extraction of the cancer biomarker 5-hydroxy-indole-3-acetic acid (5-HIAA) from urine. The CD-mNPs were characterized by TEM, FTIR, and XRD, and the kinetics and adsorption isotherms were studied. The strong interaction between the CD-mNPs and 5-HIAA is the main driving force for recognition and extraction, while the magnetic core of the NPs allows their separation from the sample matrix. Recovery of 5-HIAA from the adsorbent using an adequate solvent regenerated the adsorbent for further use. 5-HIAA was then quantified by fluorometry of its complex with ß-CD. The method works in the 1?×?10^?7 to 1?×?10^?5 mol L^?1 (R² 0.9982–0.9996) concentration range, and the limits of detection (3σ) and quantification (10 σ) of the method are 1.2?×?10^?8 mol L^?1 and 4.01?×?10^?8 mol L^?1 5-HIAA, respectively. The recovery of 5-HIAA from urine samples spiked with 5-HIAA in three concentrations (1.4?×?10^?6, 4.50?×?10^?6 and 1.0?×?10^?5 mol L^?1) are within 63?±?3 %.

In vitro studies of carbon fiber microbiosensor for dopamine neurotransmitter supported by copper-graphene oxide composite

A composite was prepared from copper and graphene oxide (Cu-GO) by in-situ chemical reduction of a mixture containing GO and Cu(II) ions with potassium borohydride. The morphology and structure of the composite were confirmed by various physicochemical techniques. The materials were used in a tyrosinase-based microbiosensor where the enzyme is immobilized in a biocompatible matrix consisting of poly(ortho-phenylene diamine) and Cu-GO. The composite was deposited on the surface of an 8-μm thick carbon fiber microelectrode. The role of each component in the sensing layer was systematically investigated with respect to the analytical performance of the system. In its optimal configuration, the biosensor demonstrated (a) a sensitivity of 6.1?±?3 nA mM^-1 dopamine (DA), (b) a linear response to DA (with a Michaelis-Menten constant of 0.29?±?0.03 mM), (c) good selectivity over ascorbic acid and uric acid, and (d) a high blocking capacity (112.2?±?2 mM) for ascorbic acid.

Highly sensitive carbon paste electrode with silver-filled carbon nanotubes as a sensing element for determination of free cyanide ion in aqueous solutions

We report on newly synthesized Ag(I)-filled multiwall carbon nanotubes as a potential sensing element in ion-selective carbon paste electrodes for the determination of free cyanide in aqueous solution. The electrode was obtained by entrapping the silver-filled nanotubes into a carbon paste and displays a Nernstian response with a slope of 59.8?±?0.3?mV decade^?1, a very wide linear range (from 21.0?nM to 0.1?M of cyanide), a lower detection limit of 13.0?nM, and a response time of <2?min. The operational lifetime is up to 3?months without significant deviation in normal function.

Anodic stripping voltammetry of silver(I) using a carbon paste electrode modified with multi-walled carbon nanotubes

We describe a silver(I)-selective carbon paste electrode modified with multi-walled carbon nanotubes and a silver-chelating Schiff base, and its electrochemical response to Ag(I). Effects of reduction potential and time, accumulation time, pH of the solution and the stripping medium were studied by differential pulse anodic stripping voltammetry and optimized. The findings resulted in a method for the determination of silver over a linear response range (from 0.5 to 235 ng?mL^?1) and with a detection limit as low as 0.08 ng?mL^?1. The sensor displays good repeatability (with the RSD of ±?2.75 % for 7 replicates) and was applied to the determination of Ag(I) in water samples and X-ray photographic films.

Investigation on the interaction between anthracyclines and DNA in the presence of paclitaxel by resonance light scattering technique

We have developed a method to investigate the interaction between DNA-targeted anthracyclines and DNA in the presence of the drug paclitaxel. It is based on resonance light scattering (RLS) and on the finding that anthracyclines when bound to DNA undergo a dramatic enhancement in their RLS intensities, while paclitaxel does not display such an effect. However, the RLS intensities of the anthracyclines-DNA associates are remarkably enhanced again on addition of paclitaxel. UV-visible spectra reveal interactions between paclitaxel and anthracyclines, but no reaction between paclitaxel and DNA. Consequently, paclitaxel, though not DNA-targeted, can improve the DNA-binding capabilities of anthracyclines. Binding constants between anthracyclines and DNA, and improved efficiency of paclitaxel on the DNA-binding capabilities of anthracyclines were calculated. The DNA binding constants of doxorubicin, epirubicin, and mitoxantrone, respectively, are 4.53?×?10⁵?L?mol^?1, 6.05?×?10⁵?L?mol^?1, and 9.47?×?10⁵?L?mol^?1. The improved values in presence of paclitaxel are 78%, 47% and 19%. We also have investigated the effects of drug concentrations and the order of adding the drugs. Displacement studies (using methylene blue as a competitive agent) provided additional information on the mechanisms of the interaction between paclitaxel and anthracyclines.

A solid state Cr(VI) ion-selective electrode based on polypyrrole

We report on a graphite electrode onto which polypyrrole was electrodeposited and then doped with chromate ion. This electrode can serve as a Cr(VI)-selective solid-state electrode. Electropolymerization of pyrrole was performed potentiostatically at 0.80?V (vs. SCE) using battery graphite as the working electrode in a solution containing 0.10?M of pyrrole and 20?mM of chromate. A platinum wire was used as an auxiliary electrode. The new electrode displays high selectivity, a very wide dynamic range, a sufficiently fast response time and a good shelf lifetime. It shows a linear Nernstian response over 1.0?×?10^?6 to 1.0?×?10^?1?M concentration range (with a slope of 26.55?±?0.20?mV per log of concentration). The detection limit is 0.5?μM, and the pH optimum is 7.0.

Non-denaturating isoelectric focusing gel electrophoresis for uranium–protein complexes quantitative analysis with LA-ICP MS

A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)–protein complexes with laser ablation–inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U–protein complex standards (i.e., U–bovine serum albumin and U–transferrin) allowing the assessment of U recovery to 64.4?±?0.4 %. This methodology enabled the quantification of U–protein complexes at 9.03?±?0.23, 15.27?±?0.36, and 177.31?±?25.51 nmol U L^?1 in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L^?1 during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L^?1. Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U–protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation.

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Highly selective DNA biosensor based on the long-range electron transfer of indigo carmine through DNA duplex

We have developed a highly selective DNA biosensor. It was based on the long-range electron transfer (LRET) from the electroactive dye indigo carmine (IC) through the DNA duplex on a glassy carbon electrode. Voltammetric experiments showed that IC interacts with dsDNA through a typical intercalative mode with a relatively strong affinity of 2.3(±0.6)?×?10⁶?M^-1. If incubated with DNA in IC solution, no response was observed with the ssDNA-modified probe electrode. However, a pair of well-defined redox peak was observed with a DNA-hybridized electrode, proving the presence of LRET on the biosensor. The biosensor also can differentiate complementary sequences, non-complementary sequences, and even the mutated sequences with single-base mismatches at different sites.

Preconcentration of ultra-trace amounts of iron and antimony using ion pair solid phase extraction with modified multi-walled carbon nanotubes

Ion pair solid phase extraction was applied to the simultaneous preconcentration of iron and antimony. The ion pairs consisting of FeCl₄ ^? or SbCl₄ ^? anions and the benzyldimethyltetradecyl ammonium cation were formed on the surface of multi-walled carbon nanotubes, then eluted with nitric acid, and the elements finally quantified by ETAAS. The adsorption capacities of the impregnated MWCNTs are 9.2 mg g^?1 for iron and 27.5 mg g^?1 for antimony. The following analytical figures of merit were determined for iron and antimony, respectively: Enrichment factors of 210 and 230, assay precisions of ±5.3 % and ±4.8 %, linear calibration plots from 0.7 to 9.4 and 13.0 to 190 ng L^?1, and detection limits of 0.17 and 3.5 ng L^?1. The method was applied to the determination of iron and antimony in human hair, synthetic sample, and to the certified reference materials gold ore (MA-1b) and trace elements in water (SRM 1643d).

Electrochemical sensor for monitoring the photodegradation of catechol based on DNA-modified graphene oxide

We have immobilized DNA on a glassy carbon electrode (GCE) modified with graphene oxide (GO) to develop an electrochemical biosensor for catechol. Compared to carbon nanotubes, the use of GO dramatically improved the electrooxidative current of the guanine and adenine moieties in DNA but retained the low background current of unmodified GCEs. Factors such as DNA adsorption time, DNA concentration and pH of solution were investigated to optimize experimental conditions. In the presence of catechol, the voltammetric response to DNA was inhibited due to the interaction between DNA and catechol. The response to adenine is linearly proportional to the concentration of catechol in the range from 1.0?×?10^?6 to 1.0?×?10^?4 mol·L^?1. If catechol is degraded by the combined action of UV light and hydrogen peroxide, the response to DNA is restored. Thus, the modified electrode can act as an efficient biosensor for monitoring the degradation of catechol.

Highly sensitive fluorescent sensor for copper (II) based on amplified fluorescence quenching of a water-soluble NIR emitting conjugated polymer

We have synthesized the near-infrared water-soluble conjugated polymer poly[2,5-di(propyloxysulfonate)-1,4-phenylene-ethynylene-9,10-anthrylene (referred to as PPEASO3). Its fluorescence (at wavelengths between 650 and 800?nm following photoexcitation at 550?nm) is efficiently quenched by Cu(II) ions, while other physiologically relevant metal ions do not cause significant quenching at the same concentrations. Under optimum conditions, fluorescence intensity is inversely proportional to the concentration of Cu (II). The calibration curve displays two linear regions over the range of 0–3.2?×?10^?7 mol L^?1 and 3.2?×?10^?7 mol L^?1 to 1.0?×?10^?4 mol L^?1 of Cu(II), respectively. The long-wavelength excitation and emission can substantially reduce interferences by the autofluorescence and light scattering of biological matter under UV excitation. The method was successfully applied to the determination of Cu(II) in synthetic and tea samples.

DNA microspot assay using single-molecule detection and requiring 1.8 nL samples only

An ultra-sensitive DNA microspot assay was developed that required 1.8?nL samples and was based on single-molecule detection. The solution of the target DNA (tDNA) was spotted onto the coverslip modified with capture DNA (DNA1) and blocked with ethanolamine and bovine serum albumin using a pintool type microspoting robot. The microspot had a diameter of ~300???m. The tDNA was captured by the DNA1, and the tDNA was then labeled with a detection DNA that previously was labeled with a quantum dot. Next, a fluorescence microscopic image of the microspot was acquired using a single-molecule microspot reader during total internal reflection fluorescence excitation. As little as 4?×?10^?22 mole (240 molecules) of tDNA can be detected by this method. The response is linear in the range from 6.0?×?10^?22 to 1.2?×?10^?19 mole of tDNA. All operations (including the acquisition of microspot images and single-molecule counting) were performed using the MetaMorph software. The assay was applied to the determination of osteopontin messenger RNA in single decidual stromal cells without the need for PCR amplification.