New phosphoramidite derivatives for the preparation of oligonucleotides containing a hydrazide group in any specified position of the oligonucleotide chain

A new versatile method for the preparation of oligonucleotides containing hydrazide groups in any position of the oligonucleotide chain by standard phosphoramidite automated oligonucleotide synthesis is proposed. The method is based on the use of a series of new modified components for oligonucleotide synthesis. An original protecting group for the hydrazide group is proposed. The presence of the hydrazide group in the obtained oligonucleotides and its high reactivity were demonstrated by the reaction with 4-methoxybenzaldehyde in solution. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2585–2595, November, 2005.     

A surface plasmon resonance immunosensor for detecting a dioxin precursor using a gold binding polypeptide

A surface plasmon resonance (SPR) based biosensor was developed for monitoring 2,4-dichlorophenol, a known dioxin precursor, using an indirect competitive immunoassay. The SPR sensor was fabricated by immobilizing a gold-thin layer on the surface of an SPR sensor chip with an anti-(2,4-dichlorophenol) antibody using a gold binding polypeptide (GBP) and protein G. The SPR response based on the antigen-antibody reaction in a flow system was measured by injecting a 2,4-dichlorophenol sample solution into the flow system in which the SPR sensor was located. In a direct immunoassay system using the modified sensor chip, no significant SPR angle shift less than 0.001° was observed when a 25 ppm of 2,4-dichlorophenol solution was injected. In order to improve the sensitivity of the SPR sensor, an indirect competitive immunoassay method was used in conjunction with the SPR sensor system using 2,4-dichlorophenol conjugated with bovine serum albumin (BSA). In the competitive assay, a 350 ppm 2,4-dichlorophenol-BSA conjugate solution containing 2,4-dichlorophenol at various concentrations (10-250 ppb) were injected into the SPR sensor system. The sensitivity of this indirect immunoassay was found to be extremely sensitive, compared to the direct one, and a detection limit of 20 ppb was estimated. Verification that the use of GBP for immobilizing the antibody on the sensor chip enhanced the sensitivity to 2,4-dichlorophenol was obtained by comparing the procedure with another modification, in which BSA was used instead of GBP for immobilizing the antibody on the sensor chip. The affinity constant of 2,4-dichlorophenol and its conjugate to the antibody were estimated form the SPR response.     

Application of matrix solid-phase dispersion to the propham and maleic hydrazide determination in potatoes by differential pulse voltammetry and HPLC

The application of the matrix solid-phase dispersion (MSPD) process as sample treatment in connection with the electrochemical detection is studied for the first time. For this purpose, a novel methodology is introduced for the extraction of propham and maleic hydrazide herbicides from potatoes samples based in the MSPD process prior to their electrochemical detection. Potato samples disruption was done by blending them with C₈ bonded-phase and selective herbicide extraction was achieved by successive treatment of the blended with 50 mM phosphate buffer pH 7.4 (for maleic hydrazide) and methanol (for propham). The extraction procedure efficiency was estimated using differential pulse voltammetry in potato samples spiked with the herbicides yielding recovery values of 98% and 68% for propham and maleic hydrazide, respectively. No significant adverse effect of the MSPD process was observed on the herbicides electrochemical signals. For comparison, recovery studies using HPLC with UV detection were carried out and a good correlation in the results obtained by using both techniques was observed.     

Study of the voltammetric behaviour of maleic hydrazide and its determination at a hanging mercury drop electrode

Voltammetric behaviour of maleic hydrazide pesticide dissolved in a Britton-Robertson buffer was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). It was found that the process at the Hg electrode was diffusion controlled; the reaction was irreversible and involved a change of one proton and a transfer of one electron. A quantitative differential pulse voltammetric method for determination of maleic hydrazide was developed on the basis of these studies involving the reduction of the compound at a hanging mercury drop electrode. A linear calibration was obtained in the range of 0.5-5.5 mg l⁻¹, and the developed DPV methodology was then applied for the determination of maleic hydrazide in spiked vegetable samples by the standard addition method. Satisfactory percentage R.S.D. (∼2%), percentage recovery values (∼85%) and LOD (0.215 mg l⁻¹) were obtained. These compared well with the results from the alternative spectrophotometric method.     

Use of autoclave extraction and liquid chromatography with tandem mass spectrometry for determination of maleic hydrazide residues in tobacco

Maleic hydrazide has been extensively used as an effective growth regulator in tobacco sucker control. After application, maleic hydrazide distributes itself throughout the tobacco plant where it can exist as free, or forms glucoside conjugates with glucose, or becomes bound with lignin. Among them, free maleic hydrazide and its glucoside conjugates are extractable under conventional solvent extraction, while lignin bound maleic hydrazide is claimed to be non‐extractable. Herein, an autoclave extraction method has been developed to extract maleic hydrazide effectively, in which tobacco samples are extracted in an autoclave at 130°C for 1 h using 4 M hydrochloric acid. Under such pressurized hot acidic water conditions, lignin bound maleic hydrazide can be released. Meanwhile, glucoside conjugates are hydrolyzed. Total maleic hydrazide is detected by liquid chromatography coupled with tandem mass spectrometry, and the quantitative results coincide well with that obtained from the international standard method. The proposed autoclave extraction with liquid chromatography and tandem mass spectrometry method exhibits excellent linearity in the range of 5–200 mg/kg (R² = 0.9998), the matrix matched limit of detection and limit of quantification is 0.68 and 2.27 mg/kg, respectively. This method is simple and improves sample capacity, providing an effective approach to monitoring maleic hydrazide residues in tobacco.     

Application of rhodamine B hydrazide as a new fluorogenic indicator in the highly sensitive determination of hydrogen peroxide and glucose based on the catalytic effect of iron(III)-tetrasulfonato-phthalocyanine

A sensitive, selective and rapid spectrofluorimetric method is proposed for the determination of hydrogen peroxide using rhodamine B hydrazide as a fluorogenic substrate catalyzed by iron(III)-tetrasulfonatophthalocyanine. It is based on the oxidation of rhodamine B hydrazide, a colorless, non-fluorescent spirolactam hydrazide, by hydrogen peroxide which generates the highly fluorescent product rhodamine B. Under optimum conditions, the responses for hydrogen peroxide were linear from 2.0 × 10⁻⁸ to 2.0 × 10⁻⁶ mol L⁻¹, with a detection limit of 3.7 × 10⁻⁹ mol L⁻¹ in a 3.5 min reaction period. It can easily be incorporated into the determination of biochemical substances that produce hydrogen peroxide under catalytic oxidation in the presence of their oxidase. The possibility has been tested for the determination of glucose in human sera as an example.     

Novel chromium and molybdenum complexes of a dimerized isonicotinic acid hydrazide

Sunlight irradiation of [M(CO)₆], M?=?Cr and Mo with isonicotinic acid hydrazide (inh) was investigated. The photochemical process resulted in dimerization of inh with elimination of a hydrazine molecule to give N′-isonicotinoylisonicotino hydrazide (ininh). Two complexes with molecular formulas [MO₂(ininh)] were isolated and characterized by elemental analysis, infrared, mass and ¹H NMR spectroscopy. The UV-vis spectra of the complexes in DMSO showed visible bands due to ligand-to-metal charge transfer. Thermal properties of the complexes were investigated by TG thermogravimetry.     

Amperometric detection of DNA hybridization on a gold surface depends on the orientation of oligonucleotide chains

We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.     

Determination of maleic hydrazide residues in cured tobacco by gas chromatography.

A rapid and sensitive method for measuring maleic hydrazide (6-hydroxy-2H-pyridazin-3-one) residues in cured tobacco is described. A mixture of free and bound maleic hydrazide is extracted with hydrochloric acid in which maleic hydrazide glycoside is simultaneously hydrolysed. The free maleic hydrazide obtained is methylated using dimethyl sulphate and the derivative is partitioned into chloroform and determined by capillary gas chromatography using a nitrogen-phosphorus detector. The limit of detection of maleic hydrazide is 5 ppm.     

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.     

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR)

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM–MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen–antibody interaction. For both interactions, the well characterized densely functionalized SAM–MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.     

Comparing the efficiencies of hydrazide labels in the study of protein carbonylation in human serum albumin

In this work, we establish a methodology for comparing the efficiencies of different hydrazide labels for detecting protein carbonyls. We have chosen acrolein-modified human serum albumin as a model. This system provides a convenient means of reproducibly generating carbonylated protein. Five hydrazide-based labels were tested. Three carry a biotin affinity tag, and the others are simple fatty acid hydrazides. For the biotin-based labels, the yield of the labeling reaction varies considerably, and the most commonly used label, biotin hydrazide, gives the lowest yield. The total tandem mass spectrometry (MS/MS) spectrum counts of modified peptides are similar for all of the biotin-based tags, indicating that factors beyond the labeling efficiency are important in determining the effectiveness of the label. In addition, there is a large variation in the number of spectra obtained for specific, modified peptides depending on the nature of the labeling group. This variation implies that the relative detectability of a particular modification site is highly dependent on the tagging reagent, and more importantly, titration schemes aimed at identifying the most reactive site based on its threshold concentration will be biased by the choice of tagging reagent. The fatty acid hydrazides are somewhat more effective than the biotin-based hydrazides in generating identifiable MS/MS spectra but offer no opportunity for enrichment. For the biotin-based tags, avidin affinity chromatography was used with the tryptic digests, and each tag led to similar enrichment levels.     

Functionalization of AlN surface and effect of spacer density on Escherichia coli pili-antibody molecular recognition

The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to AlN/sapphire surfaces which may allow the antibody to freely reorient and potentially improving the antigen capture efficiency. Spacer density was one of the key parameters to be optimized in studying its effect on the immobilization of antibody. The spacer density was controlled by functionalizing AlN/sapphire surfaces with a mixed (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and trichloro(1H,1H,2H,2H-perfluoroctyl)silane (FAS) self-assembled monolayer (SAM) through a step-wise method. Contact angle measurement and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface coverage of GPTMS and surface chemical composition. Compared to spacer fully covered samples, the capture efficiency was improved by 28% with optimal Jeffamine ED 600 spacer density, which depends on the spacer properties such as the number of monomer units and its size.     

Determination of Nitroaromatics Using a Double-Layer of Gelatin Nanofibers and a Pyrene-Doped Polystyrene Membrane

ABSTRACT

A double-layered sensor was prepared by an electrospinning method using pyrene, polystyrene, and gelatine to prepare a pyrene-polystyrene/gelatin/glass device for the determination of nitroaromatics. The pyrene- polystyrene top layer, which was an electronspun membrane from a mixture of the starting materials in 3:1?N, N-dimethylformaide:tetrahydrofuan, served as the sensing layer for detecting 2,4-dinitrotoluene. The bottom layer was the gelatin electrospun membrane that served as an intermediate between the top layer and the glass slide. The dinitrotoluene analyte was transferred into the sensing layer above and below due to the porosity of the gelatin layer. With a large number of hydroxyl and amino groups, the gelatin layer formed hydrogen bonds with the 2,4-dinitrotoluene molecules, which caused the enrichment of the analyte around the gelatin layer. Additionally, single-layer pyrene-polystyrene/glass sensor was also prepared for control measurements. The quenching efficiency of the pyrene-polystyrene/gelatin/glass sensor was 76.7% at equilibrium obtained within 6 minutes, while the monolayer sensor pyrene–polystyrene/glass device provided 65.8% quenching efficiency within 6 minutes. The gelatin layer played an important role in the superior performance of the double-layered sensor.     

Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: A review

An electrochemical label-free immunosensor based on a biotinylated single-chain variable fragment (Sc-Fv) antibody immobilized on copolypyrrole film is described. An efficient immunosensor device formed by immobilization of a biotinylated single-chain antibody on an electropolymerized copolymer film of polypyrrole using biotin/streptavidin system has been demonstrated for the first time. The response of the biosensor toward antigen detection was monitored by surface plasmon resonance (SPR) and electrochemical analysis of the polypyrrole response by differential pulse voltammetry (DPV). The composition of the copolymer formed from a mixture of pyrrole (py) as spacer and a pyrrole bearing a N-hydroxyphthalimidyl ester group on its 3-position (pyNHP), acting as agent linker for biomolecule immobilization, was optimized for an efficient immunosensor device. The ratio of py:pyNHP for copolymer formation was studied with respect to the antibody immobilization and antigen detection. SPR was employed to monitor in real time the electropolymerization process as well as the step-by-step construction of the biosensor. FT-IR demonstrates the chemical copolymer composition and the efficiency of the covalent attachment of biomolecules. The film morphology was analyzed by electron scanning microscopy (SEM).Results show that a well organized layer is obtained after Sc-Fv antibody immobilization thanks to the copolymer composition defined with optimized pyrrole and functionalized pyrrole leading to high and intense redox signal of the polypyrrole layer obtained by the DPV method. Detection of specific antigen was demonstrated by both SPR and DPV, and a low concentration of 1 pg mL⁻¹ was detected by measuring the variation of the redox signal of polypyrrole.     

Immobilization of antibodies on glass surfaces through sugar residues

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.     

A simple and rapid fluorimetric method for the microdetermination of isonicotinic acid hydrazide

A simple, rapid and sensitive fluorimetric method has been developed for the microdetermination of isonicotinic acid hydrazide, based on its formation of a hydrazone with 2-hydroxy-1-naphthaldehyde in acidic medium, in the presence of excess of scandium ions, and the consequent formation of a strongly fluorescent complex (lambda(ex) 430 nm, lambda(em) 510 nm) between the hydrazone and scandium in weakly acidic medium. Kinetic and equilibrium procedures for isonicotinic acid hydrazide determination are proposed. A calibration graph linear up to 5.00 mug/ml is obtained by both procedures, with a mean relative error of about 2.0%. The detection limit for the kinetic procedure is 0.4 ng/ml and for the equilibrium procedure about 0.2 ng/ml.     

Surface plasmon resonance immunosensor for histamine based on an indirect competitive immunoreaction

The use of a surface plasmon resonance immunosensor for the analysis of histamine (β-imidazole ethylamine) is described. The method is based on an indirect competitive reaction of an anti-histamine antibody in a sample solution with histamine immobilized on a sensor chip and with histamine in the sample solution. A sensor chip immobilized with histamine was prepared using a self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) as an anchor membrane, followed by an amino-coupling reaction with histamine after activation of the 11-MUA layer on the sensor chip by treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. The sensor chip can be reused, after regeneration with a 10 mM HCl solution, which dissociates the anti-histamine antibody complex from histamine on the sensor chip. The affinity constants for the immunocomplex of the anti-histamine antibody with histamine in the solution and for that of the anti-histamine antibody with histamine immobilized on the sensor chip were calculated to be 1.5 × 10⁷ and 7.2 × 10⁵ M⁻¹, respectively, by assuming a Langmuir-type adsorption of the anti-histamine antibody to histamine immobilized on the sensor chip. The detection limit of the method was determined to be 3 ppb.     

A FRET system built on quartz plate as a ratiometric fluorescence sensor for mercury ions in water

Due to the hazardous nature of mercury ions, the development of a cost effective, sensitive and field-portable sensor is of high significance for both industry and civilian use. In this work, a FRET-based ratiometric sensor for detecting mercury ions in water was fabricated by depositing a multilayered silica structure on a quartz plate. For the preparation of the film-based sensor, a silica support layer was first deposited on the quartz plate by using the sol-gel spin-coating procedure, and three ultrathin functional layers (donor, spacer and receptor) were then deposited on the support layer by dip-coating in a stepwise manner in toluene solution. As the film-based sensor was placed into an aqueous solution of Hg(2+), the non-fluorescent receptor (a spirolactam rhodamine derivative) on the film surface could form a complex with the mercury ion and act as the acceptor of the energy transfer. Upon excitation, the donor (a nitrobenzoxadiazolyl derivative, NBD) could transfer its excited energy from the donor layer to the acceptor on the film surface via the 'through space' energy transfer process, thus realizing the FRET-based ratiometric sensing for mercury ions. The sensor can selectively detect Hg(2+) in water with the detection limit of 1 μM. This solid film sensor is capable of being easily-portable and visualized detection. This strategy may offer new approaches for constructing other FRET-based solid-state devices.     

A practical oxidative method for the cleavage of hydrazide N[bond]N bonds

The selective N-oxidation of the most nucleophilic amino nitrogen atom in hydrazides is central to the development of an unprecedented methodology for the cleavage of their N[bond]N bonds under oxidative conditions. Treatment of a series of hydrazides 1-9 with peracids such as magnesium monoperoxyphtalate hexahydrate (MMPP.6 H(2)O) or meta-chloroperbenzoic acid (m-CPBA) afforded the corresponding amides 10-16 in good-to-excellent yields (80-92 %). The extension of the methodology to carbamate-like substrates such as 17 and 18 was also investigated, but in this case the process is synthetically useless in view of the low yields observed of carbamates 19 and 20 (approximately 15 %). Experiments carried out with equivalent amounts of oxidant produced nitrones, such as 26, proceeding from the dialkylamino moiety, and (1)H NMR experiments indicated that this product is formed by fast conversion of the parent hydrazide, without detection of the expected hydrazide N-oxides. In addition, the over oxidation of 26 into nitronate 25 proceeds through an unknown intermediate. This oxidative N[bond]N bond cleavage by peracids is an alternative method for the deamination of hydrazides, and constitutes the only solution compatible with substrates carrying functionalities sensitive to reducing conditions.