Spectrofluorimetric determination of phenylalanine based on fluorescence enhancement of europium ion immobilized with sol-gel method

The analysis of phenylalanine (Phe) in serum is widely performed for the screening of newborn phenylketonuria (PKU). In this work, a novel spectrofluorimetric method for the determination of Phe was developed based on the fluorescence enhancement of Ruhemann's purple, the reaction product between Phe and ninhydrin, upon coordination with Eu(3+). A filter paper disc containing immobilized reactants (ninhydrin and Eu(3+)) was fabricated by sol-gel method. The experimental parameters affecting the determination of Phe, such as the concentrations of immobilized reagents, the pH value, the reaction time and temperature were optimized. Under optimum conditions, the fluorescence intensity of Phe-ninhydrin-Eu(3+) system was linearly proportional to the concentration of Phe in the range from 5×10(-5) to 2×10(-3) mol L(-1), and the limit of detection was found to be 5.2×10(-6) mol L(-1). The relative standard deviation was 2.6% for ten replicate measurements of 1.5×10(-4) mol L(-1) of Phe. The method has merits of sensitivity, simplicity and low cost, and has been applied to the determination of Phe in artificial serum.     

HPLC with fluorescence detection of urinary phenol, cresols and xylenols using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a fluorescence labeling reagent.

A simple and sensitive HPLC method for the determination of phenolic compounds, i.e., phenol (Phe), cresols (Cres) and xylenols (Xyls), was developed. After a pre-column fluorescence derivatization of these compounds with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) at 60 degrees C for 30 min, 11 DIB derivatives were successfully separated within 50 min with an ODS column using CH3CN-H2O-CH3OH (25 + 22 + 53, v/v) as the eluent. The detection limits of DIB derivatives at a signal-to-noise ratio of 3 ranged from 0.15 to 1.09 microM (0.2-1.6 pmol per 20 microliters). The precision of the proposed method for both within- and between-day assays of free and total phenol related compounds was satisfactory (RSD < 9.5%). By the proposed method, Phe and p-Cre could be detected in normal urine samples, and the calculated concentrations of free Phe and p-Cre in unhydrolysed urine samples were 1.5 +/- 1.3 and 23.9 +/- 24.3 microM and those of total Phe and p-Cre in hydrolysed urine samples were 87.3 +/- 81.2 and 200.7 +/- 195.4 microM (n = 21), respectively.     

Bioluminescent Flow Sensors: L-Alanine Determination in Serum and Urine

Abstract

A continuous flow bioluminescent method for L-alanine analysis in serum and urine has been developed. Serum can be analyzed directly after simple filtration. Response is linear from 50 to 1500 pmoles in biological matrix. Alanine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bioluminescent enzymes. The stability of nylon immobilized enzymes is high (over three months) and more than 900 samples can be analyzed with few mg of enzymes. The results obtained with the bioluminescent sensor agree well with those obtained by ion exchange chromatography (amino acid analyzer).     

Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system.

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.     

Flow-through chemiluminescence sensor using immobilized histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007 and peroxidase for selective determination of histamine.

A flow sensor with immobilized oxidases is proposed for the determination of histamine in fish meat. Chemiluminometric measurement of histamine was based on the luminol reaction with hydrogen peroxide produced by immobilized histamine oxidase (EC 1.4.3.-.) and peroxidase (EC 1.11.1.7.) within a flow cell. Histamine oxidase was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The oxidase and peroxidase were coimmobilized covalently on tresylated hydrophilic vinyl polymer beads and packed into transparent PTFE; the tubing was used as the flow cell. One assay for histamine was done at intervals of 2 min without carryover. The calibration curve for histamine was linear from 0.1 microM to 50 microM. The response was reproducible within 1.25% of the relative standard deviation for 115-replicate injections of 50 microM histamine. The sensor system was applied to the determination of histamine in fish meat extracts.     

Bioluminescence flow sensor for determination of creatine kinase activity in blood

A bioluminescent flow sensor was developed for the assay of creatine kinase (CK) using firefly luciferase immobilized on a nylon coil. The CK-catalysed reaction of creatine phosphate with ADP took place in a cuvette before the injection into the bioluminescent detector coil. The response was linear from 0.1 to 100 U l^? at 25°C. An advantage of the flow sensor is a detection limit of less than 0.1 U l^?1, which, together with a high precision, allows determination of the CK activity in blood sera in about 5 min. The intra- and inter-assay reproducibilities (RSD) were less than 10% and the recovery range was 86–110%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.     

Simultaneous determination of creatinine, creatine, and UV-absorbing amino acids using dual-mode gradient low-capacity cation-exchange chromatography

A simple and versatile cation-exchange chromatography technique for the simultaneous determination of urinary creatinine (Cre), creatine (Crn), methionine (Met), tyrosine (Tyr), phenylalanine (Phe), histidine (His), and tryptophan (Trp) was developed. A novel low-capacity cation-exchange column packed with a newly developed sulfoacylated hypercross-linked macroreticular polystyrene-divinylbenzene resin, referred to as TMR-A/75 (capacity: 75 microequiv/column), was successfully used with a binary dual-mode gradient eluting system. Two solvents, (A) 25 mM phosphoric acid-methanol (30:70, v/v) and (B) 25 mM disodium hydrogenphosphate-methanol (30:70, v/v) were pumped through the column by programming solvent delivery ratios as 0 to 5 min: A-B (55:45, pH 3.6); 5-21 min: A-B (49:51, pH 5.3); and 21-35 min: A-B (55:45, pH 3.6). The flow rate was simultaneously time-programmed to be 0.6 mL/min from 0 to 19 min and to be 1.0 mL/min from 19 to 35 min. This eluting system could permit the use of the UV detection at 210 nm. The analytes, Crn, Met, Tyr, His, Cre, Phe, and Trp, were well separated in this order in 27 min with minimum resolution of approximately 2, and the cycle time was about 35 min. Retention time of each analyte was very reproducible with relative standard deviations (RSDs) between 0.05 and 0.38% (n = 5). The peak area responses were also reproducible with RSDs between 0.74 and 2.24% (n = 5). Calibration lines based on area data were linear from 1 to 1000 microM with r2 values of 0.9998 (Crn), 0.9998 (Met), 0.9999 (Tyr), 0.9999 (His), 1.0000 (Cre), 1.0000 (Phe), and 0.9999 (Trp). The method was applicable to the screening and/or chemical diagnosis of inherited metabolic disorders such as phenylketonuria (PKU), tyrosinemia, and Lowe syndrome. The creatinine ratios of diagnostic markers (microM/microM Cre) were easily determined. The Phe/Cre ratios for five urines from patients with PKU ranged from 0.162 to 0.521, and the Tyr/Cre ratio for tyrosinemia was 0.147. The ratios of Tyr/Cre, Phe/Cre, and Trp/Cre for Lowe syndrome were 0.497, 0.321, and 0.495, respectively. In contrast, the creatinine ratios for healthy newborns showed one digit lower than those for patients did. The developed method is very practical and can provide useful information and results for the clinical or biomedical researches with low analytical run costs.     

A novel strategy for the determination of enantiomeric compositions of chiral compounds by chemometric analysis of the UV-vis spectra of bovine serum albumin receptor-ligand mixtures

In this work, a novel strategy was constructed to determine the enantiomeric composition of chiral substances discriminated by bovine serum albumin (BSA) based on the UV-vis spectra of the receptor-ligand mixtures coupled with partial least squares (PLS-1) analysis. Taking tryptophan (Trp) enantiomer as an example, when 20 microM BSA was used, the enantiomeric composition was accurately determined with concentration of only 100 nM and the corresponding enantiomeric excess as high as 98% (or -98%), which is relatively more sensitive than in literature. Furthermore, the BSA-based approach was also used to predict the enantiomeric composition of other chiral compounds, such as phenylalanine (Phe), tyrosine (Tyr), alanine (Ala), cysteine (Cys), DOPA and propranolol (Prop). The results fully demonstrate that BSA is effective in determination of enantiomeric composition of some chiral compounds.     

Electrochemical characterization of biosensor based on nitrite reductase and methyl viologen co-immobilized glassy carbon electrode

Nitrite reductase (NiR, nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1) and methyl viologen (MV) were co-immobilized on glassy carbon electrode (GCE, d=3 mm) by polymer entrapment, and the electrode was tested as an electrochemical biosensor for amperometric determination of nitrite. The immobilization was performed by sequential loading and drying of a homogeneous mixture of poly(vinyl alcohol) (PVA), NiR and MV, followed by poly(allylamine hydrochloride) (PAH) solution, and finally hydrophilic polyurethane (HPU) dissolved in chloroform. The positively charged PAH layer could effectively keep immobilized cationic MV from diffusing through the membrane, holding mediator tightly near or on the electrode surface. The working principle of the biosensor was based on MV mediated electron transfer between electrode and immobilized NiR. The response time (t(90%)) of the biosensor was about 20 s and sensitivity was 11.8 nA/ microM (2.5 mU NiR) with linear response range of 1.5-260 microM (r(2)=0.996) and detection limit of 1.5 microM (S/N=3). Lineweaver-Burk plot showed that Michaelis-Menten constant (K(m,app)) was about 770 microM. The biosensor showed durable storage stability for 24 days (stored in ambient air at room temperature) retaining 80% of its initial activity, and showed satisfactory reproducibility (relative standard deviation (R.S.D.)=3.8%, n=9). Interference study showed that chlorate, chloride, sulfite, sulfate did not interfere with the nitrite determination, however, nitrate interfered with the determination with relative sensitivity of 38% (ratio of sensitivity for nitrate to that for nitrite). In addition to the full characterization of the biosensor, kinetic study was also conducted in solution and the homogeneous rate constant (k(2)) between NiR and MV were determined by chronoamperometry to be 5.8 x 10(5) M(-1) s(-1).     

Determination of total 3α-hydroxy bile acids in serum by a bioluminescent flow-injection system using a hollow-fibre reactor

A continuous-flow bioluminescence method for measuring total 3α-hydroxy bile acids in serum is described. A bacterial luciferase and NADH:FMN oxidoreductase are covalently co-immobilized on a CNBr-activated Sepharose 4B. A permeable membrane (hollow fibre) reactor is used for the introduction of NAD⁺ and bioluminescent reagent. The reagent permeates into the flow-stream due to the existing difference in ionic strength. The continuous-flow light-emitting system, in which the column filled with the immobilized bioluminescent enzyme is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. The technique was tested by comparing results with those obtained by fluorimetry. More than 20 samples an hour can be analyzed. Normal values for total bile acids content serum ranged from 1.0 to 7.5 μM, in agreement with those obtained by the other method. Excellent reproducibility, precision, and sensitivity are achieved.     

Bioluminescent flow sensor for the determination of L-(+)-lactate

The amount of L-lactate in biological fluids (serum, plasma and cerebrospinal fluid) was determined by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH) produced by immobilised lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilised on a separate nylon coil. The LDH catalysed the reaction of L-lactate with NAD; this reaction took place in a nylon coil that preceded the coil for the bioluminescent detection. The co-immobilisation of alanine aminotransferase (ALT) with LDH improved the lactate transformation by 117-183%. The response was linear from 0.1 to 50 micron mol l(-1) at 25 degrees C for the LDH - ALT reactor. The intra- and inter-assay coefficients of variation were less than 5% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.     

高效液相色谱-荧光检测法测定血清中的犬尿氨酸

建立了高效液相色谱-荧光检测(HPLC-FLD)测定血清中犬尿氨酸(kynurenine,Kyn)含量的方法。采用Hypersil C8色谱柱(300 mm×6.0 mm,10 μm), 流动相为0.25 mol/L醋酸锌及50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 mL/min, 荧光检测激发波长和发射波长分别为365 nm和480 nm。血清标本经5%(v/v)高氯酸溶液去除蛋白质后取上层清液直接进样, Kyn经流动相等度洗脱分离后,用FLD进行测定。研究结果表明Kyn保留时间约为8.3 min,线性范围为0.098～19.6 μmol/L,最低检出浓度为0.04 μmol/L,回收率为90.8%～96.2%,日内、日间测定的相对标准偏差均小于5%,苯丙氨酸、酪氨酸、色氨酸、5-羟色胺和犬尿喹啉酸等物质对犬尿氨酸的测定均无干扰。建立的方法简便、快速、灵敏、特异,适用于临床和科研应用。     

Evaluation of microchip capillary electrophoresis with external contactless conductivity detection for the determination of major inorganic ions and lithium in serum and urine samples

The determination of inorganic ions in clinical samples in less than 90 seconds was demonstrated for microchip capillary electrophoresis using capacitively coupled contactless conductivity detection (C(4)D). Bare electrophoresis chips were used in combination with external electrodes which were part of the chip holder. In order to achieve the required selectivity and sensitivity, an optimization of the electrode layout was carried out. Limits of detection (LOD) of 1 microM for K(+), 1.5 microM for Ca(2+), 3 microM for Na(+), 1.75 microM for Mg(2+) and 7.5 microM for Li(+) were achieved. The determination of inorganic cations (NH(4)(+), K(+), Na(+), Ca(2+), Mg(2+)) and anions (Cl(-), NO(3)(-), SO(4)(2-), phosphate) in blood serum and urine samples was possible in one common electrolyte solution containing 15 mM L-arginine, 10.75 mM maleic acid and 1.5 mM 18-crown-6 at pH 5.90 by simply switching the separation voltage from positive to negative polarity. Lithium, present at significant levels when used for therapeutic purposes, can also be determined in blood serum using a slightly modified background electrolyte solution.     

Use of a ruthenium(III), iron(II), and nickel(II) hexacyanometallate-modified graphite electrode with immobilized oxalate oxidase for the determination of urinary oxalate.

This paper describes the performance of a biosensor with an Ru(III), Ni(II), and Fe(II) hexacyanometallate-modified graphite electrode and immobilized oxalate oxidase for the determination of urinary oxalate. The addition of ruthenium enhances the electrochemical reversibility and chemical stability of the electrocrystallized layer and improves the sensitivity of the biosensor. Hydrogen peroxide, produced by the enzyme-catalyzed oxidation of oxalate, was measured at -50 mV vs an Hg Hg2CI2 3M KCl electrode in a solution of pH 3.6 succinic buffer, 0.1 M KCl, and 5.4mM ethylenediaminetetraacetic acid. The linear concentration range for the determination of oxalate was 0.18-280 microM. The recoveries of added oxalate (10-35 microM) from aqueous solution ranged from 99.5 to 101.7%, whereas from urine samples without oxalate (or with a concentration of oxalate below the detection limit) the recoveries of added oxalate ranged from 91.4 to 106.6%. The oxalate in 24 h urine samples, taken during their daily routine from 35 infants and children, was measured and found to range from 0.6 to 121.7 mg/L. There were no interferences from uric acid, acetylsalicylic acid, and urea in the concentration range investigated, but paracetamol and ascorbic acid did interfere. A good correlation (R2 = 0.9242) was found between values obtained for oxalate in real urine samples by 2 laboratories, with the proposed biosensor and ion chromatography, respectively.     

基于表面等离子体共振技术用鸡蛋黄抗体 IgY测定人血清中转铁蛋白

采用表面等离子体共振(SPR)方法, 用鸡蛋黄抗体(IgY)取代传统免疫检测中哺乳动物抗体IgG作为识别分子偶联于CM5传感芯片上, 对人血清中的转铁蛋白进行了检测. 考察了IgY在传感芯片上的偶联条件及芯片的再生条件. 结果表明, 在pH=4.0, IgY浓度为100 μg/mL, 流速为5 μL/min的最佳偶联条件下, SPR响应信号和转铁蛋白浓度在50~500 ng/mL范围内具有良好的线性关系, 检出限为39.56 ng/mL, 对人血清样品检测的日间变异系数<8%, 日内变异系数<5%, 平均回收率为86.22% ~94.51%.     

Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.     

A piezoelectric immunoassay based on self-assembled monolayers of cystamine and polystyrene sulfonate for determination of Schistosoma japonicum antibodies

A piezoelectric immunosensor based on an improved immobilization strategy combining self-assembled monolayers (SAM) of cystamine (Cys) and polystyrene sulfonate (PSS) has been developed for the determination of Schistosoma japonicum antibodies (SjAb) in rabbit serum. Cys SAM were first applied to the gold electrode surface of the crystal, serving as a positively-charged base. Schistosoma japonicum antigen (SjAg) was then electrostatically immobilized on the crystal by means of a negatively-charged PSS layer. When sealed by use of an appropriately selected blocking reagent for BSA and normal rabbit serum (NRS), non-specific adsorption could be substantially reduced.The immunosensor was used to determine SjAb in optimized buffer medium with addition of poly(ethylene glycol) (PEG), which served as an immunoreaction enhancer. It was shown experimentally that SjAg immobilized by the Cys-PSS adsorption procedure had higher immunological activity or binding efficiency than those immobilized by the glutaraldehyde (GLU) binding or direct attachment procedures. The immunosensor developed had satisfactory sensitivity and detection limit, and regeneration of the piezoelectric quartz-crystal was easy. Analytical results obtained with infected rabbit serum samples indicated that the proposed immunosensor is a promising alternative for qualitative and quantitative determination of SjAb in clinical diagnosis of infection with Schistosoma japonicum.     

Determination of histidine and related compounds in rumen fluid by liquid chromatography

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45-90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (microM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4-103.0% within a 1-day study and 95.4-99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 microM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 microM, respectively, before the morning feeding.     

高效液相色谱-荧光检测法同时测定血清中的犬尿氨酸和犬尿喹啉酸

建立了在线衍生、双波长高效液相色谱-荧光检测器同时检测血清中犬尿氨酸(kynurenine, Kyn)和犬尿喹啉酸(kynurenic acid, KYNA)含量的方法。血清标本经5%高氯酸溶液去除蛋白质后,上清液直接进样分析测定。采用的色谱柱为Hypersil C8柱;流动相为0.25 mol/L醋酸锌-50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 mL/min。在0～10 min时间段,在激发波长和发射波长分别为365 nm和480 nm时检测Kyn;10 min后,在激发波长和发射波长分别变换为344 nm和404 nm时检测KYNA。Kyn的保留时间约为8.1 min,线性范围为98～19600 nmol/L,最低检出浓度为50 nmol/L,平均回收率为94.88%,日内、日间测定值的相对标准偏差(RSD)均低于4%。KYNA的保留时间约为13.0 min,线性范围为2.62～1047 nmol/L,最低检出浓度为0.11 nmol/L,平均回收率为102.72%,日内、日间测定的RSD均低于4%。苯丙氨酸、酪氨酸、色氨酸和5-羟色胺等物质对目标物的检测无干扰。71例健康成人血清中,Kyn和KYNA含量分别为(1.40±0.34) μmol/L和(24.22±8.67) nmol/L。该方法简便、快速、灵敏、特异,适于临床和科研应用。