Flow-injection determination of malate with immobilized malate dehydrogenase

Malate dehydrogenase (MDH) is immobilized chemically on controlled-pore glass and used on-line in a glass minicolumn (25×2.5 mm i.d.). Malate solution passes through the minicolumn of immobilized MDH and the NADH formed is monitored spectrophotometrically from 9 × 10-^?4 down to 7 × 10^?6 M (36 ng in 40 μl) at 50 samples h^?1.     

Electrochemical DNA impedance biosensor for the detection of DNA hybridization with polymeric film, single walled carbon nanotubes modified glassy carbon electrode

In this work, we report the fabrication of a sensitive electrochemical DNA impedance biosensor for the detection of sequence-specific target DNA. p-Aminobenzoic acid was first immobilized on the surface of the electrode modified with single walled carbon nanotubes with carboxylic acid groups (SWCNTs) by cyclic voltammetry (CV). A single-stranded DNA probe with a NH₂ group at the end (H₂N-ssDNA) was then covalently immobilized on the surface of polymeric film at room temperature. The impedance measurement was performed in a solution containing 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]. The change of interfacial charge transfer resistance (R _CT) was confirmed the hybrid formation. The difference of R _CT was linear with the logarithm of complementary oligonucleotides concentrations in the range of 1.0 × 10^?12 to 1.0 × 10^?7 M, with a detection limit of 3.5 × 10^?13 M (S/N = 3).     

Flow-injection determination of l-tyrosine in serum with an immobilized tyrosinase reactor and fluorescence detection

Tyrosinase is immobilized on controlled-pore glass beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum is deproteinized with tungstate and sulphuric acid. The carrier stream is 0.3 M phosphate buffer (pH 7.2), and is mixed with 5 M potassium hydroxide after the enzyme reactor. The fluorescent dihydroxyindole formed is detected at 490 nm (excitation at 375 nm). The calibration graph is linear for 1 × 10^?7 ?1 × 10^?4 M tyrosine; the detection limit is 2 × 10^?8 M.     

Different Strategies of Covalent Attachment of Oligonucleotide Probe onto Glass Beads and the Hybridization Properties

The glass bead is a new biochip support material for immobilization biomolecules, due to its independence and convenient rearrangement. In order to optimize the immobilization efficiency of oligonucleotides onto glass beads and obtain the highest hybridization efficiency, three commonly used coupling strategies have been studied for covalently attaching oligonucleotides onto large glass beads. Glass beads with 250 μm diameter were amino-silaned with 2% 3-aminopropyltrimethoxysilane (APTMS) and then reacted separately with glutaraldehyde, succinic anhydride and 1,4-phenylene diisothiocyanate (PDITC) to derive CHO beads, COOH beads and isothiocyanate-modified beads (NCS-Beads) accordingly. Afterwards, amino-terminal oligonucleotides were covalently attached onto the surface of beads achieved by three strategies mentioned above. The immobilization efficiency were studied to compare the three strategies, which turned out 2.55 × 10¹³ probes/cm² for CHO-Beads, 3.21 × 10¹³ probes/cm² for COOH beads and 6.68 × 10¹³ probes/cm² for NCS beads. It meant that the immobilization efficiency based on NCS beads was most acceptable. And the method, developed by attaching amino-terminal oligonucleotides onto these cyanate active beads, could be regarded as an efficient one for immobilizing oligonucleotides onto a solid surface. Moreover, in this paper, the hybridization properties of NCS bead-based oligonucleotides have been studied by employing Cy5-tagged complementary oligonucleotides. It was found that the high probe density NCS beads led to low hybridization efficiency possibly due to the existence of steric crowding. In addition, the equilibrium binding constant K _A was determined by employing Langmuir isotherm model, which was 7.0 × 10⁶ M⁻¹ for NCS beads with the density of 6.7 × 10¹³ probes/cm². Furthermore, it only took 60 min to reach hybridization equilibrium. These large microspheres (>100 μm) can be employed in the mesofluidic systems for automated heterogeneous assays.     

Preparation and structure of poly(4,4′-oxydiphenylene)pyromellitimide asymmetric ultrafiltration membranes

A process was developed for preparing asymmetric ultrafiltration poly(4,4′-oxydiphenylene)pyromel litimide membranes, involving wet forming of the membranes from the prepolymer, followed by catalytic solidphase thermal transformation of the prepolymer into the polyimide. The influence exerted on the structure and characteristics of the membranes by the compositions of the forming solution and precipitation bath, and also by thermal imidization conditions was examined. The optimal conditions were determined for preparing asymmetric ultrafiltration poly(4,4′-oxydiphenylene)pyromellitimide membranes exhibiting the water permeability coefficient of (10−500) × 10⁻⁶ m³ m⁻² s⁻¹ atm⁻¹ and molecular weight cut-off in the range (5−100) × 10³ g mol⁻¹.     

Synthesis of oligonucleotides containing 7-(2-deoxy-β-d-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine

The synthesis of oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine was accomplished. The viable intermediate N²-isobutyryl-7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine ( 6 ) was prepared via a four step deoxygenation procedure from 7-β-D-ribofuranosylguanine ( 1 ). The 5′-hydroxyl group of 6 was protected as 4,4′-dimethoxytrityl ether and then converted to the target phosphoramidite ( 8 ) via conventional phosphitylation procedure. The amino groups of 8-amino-2′-deoxyguanosine ( 9 ) were protected in the form of N-(dimethylainino)methylene functions to give the protected nucleoside 10 , which was subsequently converted to the target phosphoramidite 12 via dimethoxytritylation followed by phosphitylation. The phosphoramidites 8 and 12 were incorporated into a 26-mer and a 31-mer G-rich oligonucleotide using solid-support, phosphoramidite methodology. Analysis of antiparallel triplex formation by the oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofura-nosyl)guanine in place of 2′-deoxyguanosine showed no enhancement in triple helix formation.     

Adsorption and redox chemistry of cis-RuLL'(SCN)2 with L=4,4′-dicarboxylic acid-2,2′-bipyridine and L'=4,4′-dinonyl-2,2′-bipyridine (Z907) at FTO and TiO2 electrode surfaces

The electrochemical and spectroelectrochemical properties of the sensitizer dye Z907 (cis-RuLL'(SCN)₂ with L=4,4′-dicarboxylic acid-2,2′-bipyridine and L'=4,4′-dinonyl-2,2′-bipyridine) adsorbed on fluorine-doped tin oxide (FTO) and TiO₂ surfaces have been investigated. Langmuirian binding constants for FTO and TiO₂ are estimated to be 3 × 10⁶ M⁻¹ and 4 × 10⁴ M⁻¹, respectively. The Ru(III/II) redox process is monitored by voltammetry and by spectroelectrochemistry. For Z907 adsorbed onto FTO, a slow EC-type electrochemical reaction is observed with a chemical rate constant of ca. k = 10⁻² s⁻¹ leading to Z907 dye degradation of a fraction of the FTO-adsorbed dye. The Z907 adsorption conditions affect the degradation process. No significant degradation was observed for TiO₂-adsorbed dye. Degradation of the Z907 dye affects the electron hopping conduction at the FTO–TiO₂ interface.     

Flow-injection amperometric determination of oxalate with immobilized oxalate oxidase

Oxalate is immobilized on controlled-pore glass and is used on-line in a glass minicolumn (2.5×25 mm). The hydrogen peroxide formed is detected amperometrically. Oxalate (6×10^?6?9×10^?4 M) is determined in a flowing stream of pH 3.5 citrate (or succinate) buffer. As little as 20 ng (in 40 μl; 5.7×10^?6 M) of oxalate can be detected. Copper inhibition can be removed either by adding EDTA to the carrier stream or incorporating a chelating-resin minicolumn into the flow system prior to the enzyme column.     

Simple reagent for the synthesis of oligonucleotides labeled with 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine

Synthesis of a phosphoramidite reagent for the preparation of oligonucleotides labeled at the 5′-end with a fluorescent dye, 3,3,3′,3′-tetramethyl-2,2′-indodicarbocyanine, is described. The efficiency of this reagent is confirmed by the synthesis of several labeled oligonucleotides. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 154–158, January, 2006.     

Synthesis of f luorescein-labeled oligonucleotides bearing a tag in position 2′ of modified adenosine and arabinoadenosine

Oligonucleotide conjugates containing fluorescein residues in the sugar-phosphate back-bone were synthesized by the standard solid-phase phosphoramidite method using phosphor-amidites of 9-[2-deoxy-5-O-(4,4′-dimethoxytrityl)-2-methoxalylamino-β-D-ribofuranosyl]-and 9-[2-deoxy-5-O-(4,4′-dimethoxytrityl)-2-methoxalylamino-β-D-arabinofuranosyl]-N ⁶-benzoyladenine. The relative efficiency of the oligonucleotide synthesis with modified phosphoramidites was estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1618–1624, September, 2006.     

A reusable liposome array and its application to assay of growth-hormone-related peptides

We describe a reusable liposome array based on the formation of cleavable disulfide cross-links between liposomes and the surface of a glass slip. The N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified liposomes encapsulating a pH-sensitive fluorescence dye were immobilized on a 3-mercaptopropyltrimethoxysilane (MTS)-modified glass slip through the formation of disulfide bonds. The regeneration of a used slip was performed by the lysis of immobilized liposomes with Triton X-100 and the cleavage of disulfide bonds by reduction with TCEP, followed by immobilization of SPDP-modified liposomes. The regeneration steps did not affect the fluorescence intensity of re-immobilized liposomes. The liposome array was applied to simultaneous quantification of growth hormone related peptides, i.e., GHRF and somatostatin, in a mixture. After optimizing the assay condition, the method allowed quantification of GHRF and somatostatin in concentration ranges from 0.5 × 10⁻⁹ to 0.5 × 10⁻⁷ g/mL with detection limits of 2 × 10⁻¹⁰ and 3 × 10⁻¹⁰ g/mL, respectively.     

Label-Free Electrochemiluminescent Determination of DNA Using Luminol and Hemin Functionalized Nanoparticles

Luminol and hemin dual-functionalized silica nanoparticles were synthesized using a typical reverse water-in-oil microemulsion protocol. The obtained nanoparticles were further characterized by transmission electron microscopy, scanning electron microscopy, atomic absorption spectrometry, chemiluminescence, and electrochemiluminescence. The results indicated that the luminol and hemin dual-functionalized silica nanoparticles exhibited significantly higher chemiluminescence and electrochemiluminescence intensities than those of luminol functionalized silica nanoparticles due to the catalytic effect of hemin on the chemiluminescence and electrochemiluminescence of luminol. Furthermore, a simple and sensitive label-free electrochemiluminescence DNA biosensor was developed based on the chitosan modified luminol and hemin dual-functionalized silica nanoparticles and a single-stranded DNA probe. The chitosan modified luminol and hemin dual-functionalized silica nanoparticles were immobilized on the surface of an indium-doped tin oxide electrode and the single-stranded DNA probe was immobilized on the surface of the nanoparticles through electrostatic interactions between single-stranded DNA and chitosan, which allowed hybridization with the target DNA sequences. The hybridization events were evaluated by electrochemiluminescence, and only the complementary sequence formed double-stranded DNA with the DNA probe to give strong electrochemiluminescence signals. Finally, the electrochemiluminescence intensity was found to be linearly related to the concentration of the complementary sequence at concentrations from 1.0?×?10^?12 to 1.0?×?10^?6?mol·L^?1 with a detection limit of 5.0?×?10^?13?mol·L^?1.     

Flow-injection determination of sulphite and assay of sulphite oxidase

Sulphite (1–80 × 10^?5 M) in formaldehyde-stabilized solutions is determined by injection into a flowing stream of pH 8.5 phosphate buffer, passing through a mini-column of sulphite oxidase immobilized on controlled-pore glass, with amperometric detection of the hydrogen peroxide produced. Sulphite oxidase (5–100 U l^?) is determined by injection into a flowing stream of formaldehyde-stabilized 2 × 10^?3 M sodium sulphite in pH 8.0 phosphate buffer; hydrogen peroxide is again monitored.     

Chemiluminescence Sensor with Mn(III)-Tetrakis (4-sulfonatophenyl)-porphyrin Immobilized on Dioctadecyldimethylammonium Chloride Bilayer Membranes Incorporated Into PVC Film

Abstract

An indicator phase for a flow-through chemiluminescence (CL) sensor composed of ordered surfactant assemblies, a polymer and a catalyst was evaluated by measuring adrenaline. The method is based on use of Mn (III)-porphyrin immobilized on a bilayer membrane contained in a blended film, prepared by incorporating dioctadecyl-dimethylammonium chloride into polyvinyl chloride. The sensor consisted of a Pyrex glass tube (30 mm × 5 mm i.d.) packed with silica glass wool, on which the indicator phase was coated, and a photomultiplier tube. The blend film functioned as a favorable reaction medium for the adrenaline CL, and further enhanced CL was observed with the immobilized catalyst. This indicator phase permitted adrenaline to be detected down to 3 × 10^?6 M with a 20 μl injection into a 0.3 M NaOH carrier solution. The relative standard deviation (n = 10) was 1.0% for 5 × 10^?5 M adrenaline. For 80 successive injections of 5 × 10^?5 M adrenaline, the variation of the CL signal was within the relative standard deviation. Almost the same sensitivity and precision were observed with the indicator phase stored in water for at least 3 days. The sensor was successfully applied to determine adrenaline in drug samples.     

Direct Electrochemistry of Glucose Oxidase Immobilized on Titanium Carbide‐Au Nanoparticles‐Fullerene C60 Composite Film and Its Biosensing for Glucose

The direct electrochemistry of glucose oxidase (GOD) immobilized on the designed titanium carbide‐Au nanoparticles‐fullerene C₆₀ composite film modified glassy carbon electrode (TiC‐AuNPs‐C₆₀/GCE) and its biosensing for glucose were investigated. UV‐visible and Fourier‐transform infrared spectra of the resulting GOD/TiC‐AuNPs‐C₆₀ composite film suggested that the immobilized GOD retained its original structure. The direct electron transfer behaviors of immobilized GOD at the GOD/TiC‐AuNPs‐C₆₀/GCE were investigated by cyclic voltammetry in which a pair of well‐defined, quasi‐reversible redox peaks with the formal potential (E^0′) of ‐0.484 V (vs. SCE) in phosphate buffer solution (0.05 M, pH 7.0) at the scan rate of 100 mV·s^?1 were obtained. The proposed GOD modified electrode exhibited an excellent electrocatalytic activity to the reduction of glucose, and the currents of glucose reduction peak were linearly related to glucose concentration in a wider linearity range from 5.0 × 10^?6 to 1.6 × 10^?4 M with a correlation coefficient of 0.9965 and a detection limit of 2.0 × 10^?6 M (S/N = 3). The sensitivity and the apparent Michaelis‐Menten constant (K_M^app) were determined to be 149.3 μA·mM^?1·cm^?2 and 6.2 × 10^?5 M, respectively. Thus, the protocol will have potential application in studying the electron transfer of enzyme and the design of novel electrochemical biosensors.     

An amperometric monoamine oxidase biosensor for determining some antidepressants

An amperometric biosensor based on a platinum screen-printed electrode and immobilized monoamine oxidase is developed to determine antidepressants of different classes. Petylyl, pyrazidol, and flu-oxetine can be determined with determination limits of 8 × 10^?9, 8 × 10^?7, and 8 × 10^?10 M, respectively. A procedure is proposed for determining fluoxetine in tablets. It is shown that petylyl can be selectively determined by an immunochemical technique using the developed biosensor and immobilized antibodies in the concentration range from 1 × 10^?4 to 1 × 10^?8 M.     

Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO2/chitosan composite film

An amperometric pesticides inhibition biosensor has been developed and used for determination of pesticides in vegetable samples. To eliminate the interference of ascorbic acid, multilayer films of polyelectrolyte (chitosan/polystyrensulfonate) were coated on the glass carbon electrode. Then, acetylcholinesterase was immobilized on the electrode based on surface-treated nanoporous ZrO₂/chitosan composite film as immobilization matrix. As a modified substrate, acetylthiocholine was hydrolysed by acetylcholinesterase and produced thiocholine which can be oxidized at +700?mV vs. SCE. Pesticides inhibit the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to acetylthiocholine within 9.90?×?10^?6 to 2.03?×?10^?3?M. The electrode provided a linear response over a concentration range of 6.6?×?10^?6 to 4.4?×?10^?4?M for phoxim with a detection limit of 1.3?×?10^?6?M, over a range of 1.0?×?10^?8 to 5.9?×?10^?7?M for malathion, and over a range of 8.6?×?10^?6 to 5.2?×?10^?4?M for dimethoate. This biosensor has been used to determine pesticides in a real vegetable sample.     

Fluorometric enzyme immunosensing system based on natural product resveratrol for horseradish peroxidase and Ag/SiO<Subscript>2</Subscript> nanoparticles

The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO₂ particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization reaction of RST by H₂O₂ forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation: 315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO₂ particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 1.5×10⁻⁶–7.3×10⁻⁴ g/L and with a detection limit of 1.5×10⁻⁶ g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with satisfactory results. Supported by the Projects of Scientific Research Fund of Hunan Provincial Education Department of China (Grant Nos. 05B020 and 06C098)     

Enzyme electrode system for oxalate determination utilizing oxalate decarboxylase immobilized on a carbon dioxide sensor

A highly selective enzyme electrode system for oxalate is described in which the enzyme oxalate decarboxylase is immobilized on a carbon dioxide gas-sensing electrode. The response of the system is linear with the logarithm of the oxalate concentration between 2 × 10^-4 and I × 10^-2 M with a slope of 57–60 mV/decade. The oxalate detection limit is 4 × 10^-5 M. Electrodes used with chemically immobilized enzyme are not affected by phosphate and sulfate at levels normally found in urine and are very stable showing no decrease in response after one month of operation. The enzyme electrode system functions well in urine, requiring minimal sample pretreatment. The recovery of oxalate added to five aliquots of a human control urine sample averaged 97.7% with an average relative standard deviation of 4.5%.     

Spectroscopic quantification of covalently immobilized oligonucleotides

Quantitative determination of surface coverage, film thickness and molecular orientation of DNA oligomers covalently attached to aminosilane self‐assembled monolayers has been obtained using complementary infrared and photoelectron studies. Spectral variations between surface immobilized oligomers of the different nucleic acids are reported for the first time. Carbodiimide condensation was used for covalent attachment of phosphorylated oligonucleotides to silanized aluminum substrates. Fourier transform infrared (FTIR) spectroscopy and x‐ray photoelectron spectroscopy (XPS) were used to characterize the surfaces after each modification step. Infrared reflection–absorption spectroscopy of covalently bound DNA provides orientational information. Surface density and layer thickness are extracted from XPS data. The surface density of immobilized DNA, 2–3 (×10¹³) molecules cm^?2, was found to depend on base composition. Comparison of antisymmetric to symmetric phosphate stretching band intensities in reflection–absorption spectra of immobilized DNA and transmission FTIR spectra of DNA in KBr pellet indicates that the sugar–phosphate backbone is predominantly oriented with the sugar–phosphate backbone lying parallel to the surface, in agreement with the 10–20 Å DNA film thickness derived from XPS intensities. Copyright © 2004 John Wiley & Sons, Ltd.