Determination of fructosyl amino acids and fructosyl peptides in protease-digested blood sample by a flow-injection system with an enzyme reactor.

A flow-injection system with an enzyme reactor was proposed for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. A fructosyl-amino acid oxidase (FAOX-TE) and two fructosyl-peptide oxidases (FPOX-CE and FPOX-CET) were covalently immobilized onto an inert support. They were used as the enzyme reactor in a FIA system with a hydrogen peroxide electrode. In particular, the FPOX-CET reactor possessed high selectivity for the detection of fructosyl valine (FV) and fructosyl valyl histidine (FVH) and an excellent operational stability. The proposed FIA system with the FPOX-CET reactor responded linearly to the concentration of FV over the dynamic range of 7.8 x 10(-6) to 5.8 x 10(-4) M. The present method could be successfully applied to the assay of FV and FVH in the protease-digested blood samples.     

An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer

A flow-injection analytical (FIA) system, comprised of an electrochemical detector with a fructosyl-peptide oxidase (FPOX-CET) reactor and a flow-type spectrophotometer, was proposed for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cell. The blood cell samples were hemolyzed with a surfactant and then treated with protease. In the first stage of operation, total hemoglobin in digested sample was determined spectrophotometrically. In the second stage, fructosyl valyl histidine (FVH) released from glycohemoglobin by the selective proteolysis was determined specifically using the electrochemical detector with the FPOX-CET reactor. The FIA system could be automatically processed at an analytical speed of 40 samples per hour. The proposed assay method could determine selectively only the glycated N-terminal residue of β-chain in glycohemoglobin and total hemoglobin in blood cell. The enzymatic hemoglobin A_1c (HbA_1c) value calculated by the concentration ratio of the FVH to total hemoglobin, was closely correlated with the HbA_1c values certified by the Japan Diabetic Society (JDS) and the International Federation of Clinical Chemistry (IFCC).     

Ferricyanide Confined in a Protonated Amine-Functionalized Silica Film on Gold: Application to Electrocatalytic Sensing of Nitrite Ions

An amine-functionalized porous sol–gel silica film was shown to be an effective platform to immobilize small anionic redox mediators of high solubility on solid electrodes by electrostatic interaction. The highly soluble mediator hexacyanoferrate was used as a model. The film was grown and firmly anchored on a gold electrode surface via thiol groups of a self-assembled monolayer of (3-mercaptopropyl)trimethoxysilane. Film growth and thickness were controlled by electrochemical modulation of pH at the electrode/solution interface in a sol of a hydrolyzed solution of tetraethoxysilane and 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane by the application of a negative potential to the electrode. Protonation of the amine groups made the amine-functionalized surface useful to immobilize hexacyanoferrate on gold. Thus, the immobilization is pH dependent, being highly effective in strongly acidic medium. Cyclic voltammetry and scanning electron microscopy were used to characterize the film and to optimize the experimental conditions. The stability of the film was demonstrated by applying the catalytic properties of the hexacyanoferrate containing surface for nitrite sensing using a flow injection analysis (FIA) system. Under the optimized conditions, the sensor exhibited high sensitivity, low detection limit, easy handling, and stability with a linear range from 1.0 to 40.0?µmol?L^?1 and a detection limit of 0.53?µmol?L^?1 based on a signal-to-noise ratio of 3. The sensor was successfully applied to nitrite determination in water samples using FIA with excellent recoveries.     

Chemiluminescence Flow-Injection System for Rapid Detection of Red Tide Phytoplankton

Abstract

A chemiluminescent flow-injection analysis (FIA) system for the detection of the red tide phytoplankton Chattonella antiqua has been developed based on a Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-α]-pyrazin-3-one (MCLA), which strongly emits light at 465 nm in the presence of superoxide. The system consisted of a two reagent feeding stream, a sample injector, a joint for mixing MCLA and sample, a chemiluminescence (CL) reaction cell, a CL detector and a recorder unit. The response time is approximately 1 min for one measurement cycle. The FIA system has an optimum pH of 10.7. The calibration curves for C. antiqua displayed linearity from 2 × 10³ to 2 × 10⁴ cells ml^?1. When applied to the measurement of C. antiqua, the sensitivity obtained using the FIA system is approximately 10 times higher than that of the cytochrome c method. The FIA system is a rapid practical method for the detection of C. antiqua.     

TTF-TCNQ complex based printed biosensor for long-term operation

A printed amperometric glucose sensor based on glucose oxidase adsorbed on crystals of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) is described. The sensitivity and the stability of the sensor are affected by the binder and solvent used for the preparation of the GOD.TTF-TCNQ paste. The sensors are continuously used in a flow injection analysis (FIA) system under continuous polarization at 0.15 V (vs Ag/AgCl) at 37°C. The developed sensors exhibit a large response current, an extended linear range and oxygen independence. The sensors can be used for more than 3 months. The GOD.TTF-TCNQ paste is suitable for the preparation of planar sensor by screen printing method.     

Oxidation of glycated phosphatidylethanolamines: evidence of oxidation in glycated polar head identified by LC-MS/MS

Phosphatidylethanolamine glycation occurs in diabetic patients and was found to be related with oxidative stress and with diabetic complications. Glycated phosphatidylethanolamines seem to increase oxidation of other molecules; however, the reason why is not understood. In this work, we have studied the oxidation of glycated phosphatidylethanolamines (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylethanolamine (PLPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (dPPE)) using a Fenton system. Liquid chromatography–electrospray ionization (ESI)–mass spectrometry and ESI–tandem mass spectrometry in both positive and negative modes were used for detecting and identifying the oxidation products. We were able to identify several oxidation products with oxidation in unsaturated sn-2 acyl chain of PLPE, as long- and short-chain products with main oxidation sites on C-7, C-8, C-9, and C-12 carbons. Other products were identified in both glycated PLPE and glycated dPPE, revealing that oxidation also occurs in the glycated polar head. This fact has not been reported before. These products may be generated from oxidation of glycated phosphatidylethanolamines (PE) as Schiff base, leading to short-chain product without the amine moiety, due to cleavage of glycated polar head and long-chain product with two keto groups linked to the glycated polar head or from glycated PE as Amadori product, short-chain products with –NHCHO and –NHCHOHCHO terminal in polar head. Oxidation of glycated phosphatidylethanolamines occurred more quickly than the oxidation of non-glycated phosphatidylethanolamines probably because of the existence of more oxidation sites derived from glycation of polar head group. Monitoring glycated polar head oxidation could be important to evaluate oxidative stress modifications that occur in diabetic patients.     

Titania nanotubes decorated with gold nanoparticles for electrochemiluminescent biosensing of glycosylated hemoglobin

A glycated hemoglobin (HbA1c) biosensor with high performance has been constructed in this work. Here the fructosyl amino acid oxidase was immobilized onto a pre-functionalized indium tin oxide glass with titania nanotubes decorated with gold nanoparticles. The property of nanocomposite was characterized by transmission electromicroscopy, scanning electron microscopy, electrochemistry and spectroscopy. Under the optimum conditions, fructosyl valine was detected by this biosensor. It exhibited a linear detection range from 4.0 × 10⁻⁹ M to 7.2 × 10⁻⁷ M, and a limit of detection for 3.8 × 10⁻⁹ M at the signal-to-noise ratio of 3. Thus the HbA1c level in whole blood samples of healthy individuals or diabetic patients were evaluated with designed biosensor after pre-treatment of hydrolysis. The results of our detection were closely consistent with that of the standard method. At the same time, our biosensor has some advantages including high sensitivity, disposable usage and low cost, which implies its great promising application in point-of-care testing of HbA1c.     

Application of a surface plasmon resonance sensor to analyses of amine compounds with the use of a polymer film and an acid-base reaction.

A surface plasmon resonance (SPR) sensor was applied to analyses of some amine compounds (n-butylamine, isobutylamine, aniline, and N,N-dimethylaniline) by using a polymer film and an acid-base reaction in it. Poly(acrylamide) (PAA) was adopted as the polymer film and was immobilized on an Au film to prepare a sensor chip. Pivalic acid was entered into the PAA film as an acid. The PAA film with a thickness of 50 nm gave the highest sensitivity to the SPR sensor. Although water was better concerning the sensitivity for the SPR sensor as the solvent, ethanol was adopted because it dissolves well all of the amine compounds used. The Au film coated with the PAA film gave higher sensitivity for analyses of n-butylamine and isobutylamine, and lower sensitivity for analyses of aniline and N,N-dimethylaniline than an Au film without the PAA film. The PAA film containing pivalic acid gave 4-5 orders of magnitude higher sensitivity to the SPR sensor for analyses of all the amine compounds due to the reaction between pivalic acid and these amine compounds.     

Determination of ammonium in Kjeldahl digests by gas-diffusion flow-injection analysis with a bulk acoustic wave-impedance sensor

A novel flow-injection analysis (FIA) system has been developed for the rapid and direct determination of ammonium in Kjeldahl digests. The method is based on diffusion of ammonia across a PTFE gas-permeable membrane from an alkaline (NaOH/EDTA) stream into a stream of diluted boric acid. The trapped ammonium in the acceptor is determined on line by a bulk acoustic wave (BAW)-impedance sensor and the signal is proportional to the ammonium concentration present in the digests. The proposed system exhibits a favorable frequency response to 5.0 x 10(-6)-4.0 x 10(-3) mol l(-1) ammonium with a detection limit of 1.0 x 10(-6) mol l(-1), and the precision was better than 1% (RSD) for 0.025-1.0 mM ammonium at a through-put of 45-50 samples h(-1). Results obtained for nitrogen determination in amino acids and for proteins determination in blood products are in good agreement with those obtained by the conventional distillation/titration method, respectively. The effects of composition of acceptor stream, cell constant of conductivity electrode, sample volume, flow rates and potential interferents on the FIA signals were discussed in detail.     

Development of a generic flow-injection analysis method for compounds with a secondary amine or amide function, using an experimental design approach: Part II. Selection and evaluation of the chemical reaction parameters

A second part in the development of a generic flow injection analysis (FIA) method to determine compounds with a secondary amine or amide in their structure is described. This part consists in the selection and evaluation of the chemical reaction conditions. Sodium hypochlorite first converts the secondary amine or the amide to a primary amine. The latter reacts with o-phthalaldehyde (OPA) and a thiol (N-acetylcysteine (NAC)) to form a derivative which can be measured fluorimetrically. To investigate the influence of the different chemical reaction parameters on the peak height for a set of 31 pharmaceutical compounds, a quarter-fraction factorial design for six factors at two levels (2^6-2-resolution IV, 16 experiments) was executed. Effects on the responses were calculated for each compound. Parallel coordinate geometry (PCG) plots and principal component analysis (PCA) were also applied on the measured responses as aids in the interpretation of the results.     

Fluorometric sensors based on chemically modified enzymes Glucose determination in drinks

In this paper an enzymatic fluorometric sensor for glucose determination in drinks is presented. The sensor film was obtained by immobilisation of glucose oxidase chemically modified with a fluorescein derivative (GOx-FS) in a polyacrylamide polymer. During the enzymatic reaction the changes in the fluorescence intensity of the GOx-FS are related to the glucose concentration. Working in FIA mode, the optimum conditions found were: 0.7 ml min(-1) flow rate, 300 mul sample injection and pH 6.5. The sensor responds to glucose concentrations ranging from 400 to 2000 mg l(-1), the reproducibility is around 3% and the life-time is at least 3 months (more than 350 measurements). The sensor was applied to direct glucose determination in drinks with good accuracy; interference caused by the filter effect was avoided by the kinetics of the reaction.     

Enhanced flow injection analysis for measurements of S-nitrosothiols species in biological samples using highly selective amperometric nitric oxide sensor

A highly selective nitric oxide(NO) sensor is fabricated and applied to devise an enhanced flow injection analysis(FIA) system for S-nitrosothiols(RSNOs) measurement in biological samples.The NO sensor is prepared using a polytetrafluoroethylene(PTFE) gas-permeable membrane loaded with Teflon AF? solution,a copolymer of tetrafluoroethylene and 2,2-bis(trifluoroethylene)-4,5-difluoro -l,3-dioxole,to improve selectivity.This method is much simpler and possesses good performance over a wide range of RSNOs concentrations.Standard deviation for three parallel measurements of blood plasma is 4.0%.The use of the gas sensing configuration as the detector enhances selectivity of the FIA measurement vs.using less selective electrochemical detectors that do not use PTFE/Teflon type outer membranes.     

Novel fluorescent pH sensor based on coumarin with piperazine and imidazole substituents

A new coumarin derivative containing piperazine and imidazole moieties is reported as a fluorophore for hydrogen ions sensing. The fluorescence enhancement of the studied sensor with an increase in hydrogen ions concentration is based on the hindering of photoinduced electron transfer from the piperazinyl amine and the imidazolyl amine to the coumarin fluorophore by protonation. The presented sensor has a novel design of fluorophore-spacer-receptor(1)-receptor(2) format, which is proposed to sense two ranges of pH (from 2.5 to 5.5) and (from 10 to 12) instead of sensing one pH range. A model compound, in which the piperazinyl ring is absent, was synthesized as well to confirm the novel pH sensing of the proposed sensor.     

平头pH电极-流动注射联用技术快速滴定食醋中醋酸的方法研究

采用一种具有平头结构的pH电极作为流动注射分析(FIA)的检测器,构建了流动注射自动化酸度滴定系统.优化了样品进样量、流速、载液浓度和反应管长度等参数.用NaOH溶液作为载液,在4.639×10-4～0.212 mol·L-1范围内醋酸浓度的对数与FIA峰的峰面积成正比,该方法的相对标准偏差(RSD)小于0.5%.采用...     

基于四联苯臂星型小分子凝胶剂的TNT薄膜荧光传感器

基于具有三苯胺中心核、咔唑外围和四联苯连接臂的星型荧光小分子三(4″″-(3, 6-二叔丁基-咔唑-9氢-9-基)-(1, 1': 4', 1″':4″', 1?:4?, 1″″-四苯基)-4-基)胺(N5), 采用溶液旋涂的方法制备了用于检测三硝基甲苯(TNT)饱和蒸汽的高效荧光传感薄膜。 不同于具有单苯连接臂的模型化合物三(4'-(3, 6-二叔丁基-咔唑-9氢-9-基)-(1, 1'-二苯基)-4-基)胺(N2), N5由于长共轭臂的存在, 容易在苯类溶剂中通过π-π相互作用组装形成凝胶。 同时, 其旋涂薄膜具有细小纳米级纤维状的组装形貌, 表面出现一定的多孔结构, 有利于气体分子的渗透。 因此, 应用于TNT蒸汽的检测, 化合物N5薄膜表现出比N2薄膜更快的荧光淬灭响应速度和更高的淬灭效率。 特别是TNT传感特性几乎不受化合物N5膜厚的影响, 当膜厚为94 nm时, 在1和30 min的淬灭程度仍然可以达到44%和90%。     

Bacteriostatic effect of 4,7-dicyanobenzofurazan due to inactivation of 2,3-dihydroxyisovalerate dehydratase.

As part of our research on benzofurazans (BZs), we have reported the bacterioses of BZs in Escherichia coli, which may be due to O2-. produced within E. coli in the presence of dioxygen (O2). Incubation of E. coli with 4,7-dicyanobenzofurazan (1) lowered the 2,3-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells. Addition of branched chain amino acids such as valine and leucine protected E. coli from growth inhibition by compound 1, though it could not protect E. coli from the damage by paraquat (PQ). Addition of Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine (Fe-TPAA), a novel superoxide dismutase mimic, protected the dehydratase in a dose-dependent manner, which confirms that inactivation of the dehydratase is largely due to production of O2-.. The possibility was discussed that the bacteriostatic effect of compound 1 is due to the inactivation of 2,3-dihydroxyisovalerate dehydratase.     

Glassy Carbon Paste Electrodes for the Determination of Fructosyl Valine

Nearly 200 million people worldwide have type‐2 diabetes. Glucose sensors are routinely used for diagnosis; however, the relative amount of glycosylated hemoglobin (HbA1c) may be a better marker. A working electrode made from bare glassy carbon paste was used for sensing fructosyl valine (Fru‐Val), a component of HbA1c. Amperometric measurements revealed a linear relationship between Fru‐Val concentration and the sensing current. The square correlation coefficient and the sensitivity were 0.999 and 5.26 μA mM^?1, respectively. The minimum detection limit was less than 0.05 mM.     

Determination of ATP using chelation-enhanced fluorescence

A method for the direct determination of ATP that exhibits reasonable sensitivity, and responds to very few interferants, has been developed. The chelation-enhanced fluorescence between N-(anthracen-9'-yl methyl)tris(3-aminopropyl) amine and adenosine 5'-triphosphate is utilized in this determination. The method was tested in batch and flow-injection analysis (FIA) modes. The typical detection limit for FIA determination of ATP is 1 muM, with a linear range of 0.5-100 ppm. A typical relative standard deviation at 20 ppm is 2.3%.     

Sensitivity improvement of ammonia determination based on flow-injection indophenol spectrophotometry with manganese(II) ion as a catalyst and analysis of exhaust gas of thermal power plant.

The sensitivity improvement of a flow-injection spectrophotometric method for the determination of ammonia was examined based on an indophenol blue coloration reaction with salicylate and hypochlorite in the presence of manganese(II) as a reaction promotion catalyst. The optimal conditions for achieving higher sensitivity of ammonia determination were examined using a three-line flow system. The limit of detection corresponding to a signal-to-noise ratio (S/N) of 3 was 0.005 mg l(-1) (approximately equal to 5 ppb) of NH4+. A calibration graph was linear in the range from 5 ppb to 1,000 ppb of ammonium ion. The relative standard deviations (n = 9) for 50 ppb and 100 ppb of ammonium ion were 6.4% and 2.2%, respectively. The proposed method was applied to the determination of ammonia in the exhaust gas of a thermal power plant. Prior to the FIA determination, ammonia in the exhaust gas was absorbed into a boric acid solution; the absorption solution was then analyzed by the proposed FIA.     

Sequential determination of trace amounts of iron and copper in water samples by flow injection analysis with catalytic spectrophotometric detection.

A simple flow injection analysis (FIA) method is described for the sequential determination of iron and copper. The detection method for iron and copper is based on their catalytic activities in the oxidation reaction of N,N-dimethyl-p-phenylenediamine (DPD) with hydrogen peroxide. The sequential determination of iron and copper can be carried out by injecting two sample plugs into the FIA system, sequentially. One injection does not contain triethylenetetramine (TETA), and is used for the sum of iron and copper concentration; the other which contains TETA is used only for the iron concentration. For iron determination, TETA is used as a masking agent of copper. The difference in peak height can be used for the calculation of copper concentration. Under the optimal conditions, the detection limits (3sigma) of 0.01 and 0.07 microg L(-1) were obtained for iron and copper, respectively. The proposed method can be applied to the determination of iron and copper in tap water and bottled-drinking mineral water samples. Good recoveries of the method, 98-103% for iron and 98-106% for copper, were achieved.