Determination of ammonia in untreated serum with a fiber-optic ammonia gas ensor

A fluorescence-type fiber-optic ammonia gas sensor has been developed for the determination of ammonia in untreated serum. An internal solution has been designed to provide both the selectivity and limit of detection required for this measurement. Selectivity over carbon dioxide is accomplished by adjusting the level of ammonium chloride in the internal solution. A sub-micromolar detection limit is obtained by using a combination of 2′,7′-dichlorofluorescein and 5-carboxy-2′,7′-dichlorofluorescein as the indicator dyes. The limit of detection for the resulting sensor is 0.09 μM and response times range from 2 to 6 min. When applied to the determination of ammonia in eighteen untreated serum samples, the results from the sensor compare favorably with those from the conventional glutamate dehydrogenase assay.     

Chronocoulometric determination of urea in human serum using an inkjet printed biosensor

A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.     

荧光光谱法研究牛血清蛋白在尿素体系中的变性

应用荧光光谱技术,对尿素与牛血清蛋白在30℃水溶液中的结合作用及造成牛血清蛋白变性的过程进行了研究,获取了尿素诱导牛血清蛋白变性时相对荧光强度和峰位的变化规律.用Pace等提出的公式分析了相对荧光强度数据,得到了牛血清蛋白变性时的伸展分数fu随溶液pH值和尿素浓度的变化规律.求出了变性平衡常数Ku,伸展吉布斯自由能△G...     

Determination of urea in serum by using naturally immobilized urease in a flow injection conductimetric system

A flow injection method was developed, aimed at the determination of urea in human serum. The system makes use of the naturally immobilized urease present in Canavalia ensiformis DC (jack bean). A column is filled with small pieces of this bean, and the sample (50 microliters) containing urea passes through it carried by a 1% NaCl solution. On leaving the column the stream is merged with an alkaline reagent (0.5 mol dm-3 NaOH; 0.5% disodium dihydrogen ethylenediaminetetraacetate). The ammonium ions, arising from the enzymatic reaction that occurs inside the column, are changed into the molecular form, which permeates a polytetrafluoroethylene membrane and is received in a de-ionized water acceptor stream. The ammonia ionizes causing an increase in the conductance, which is proportional to the urea content of the sample. About 40 samples can be processed in 1 h with negligible carry-over and with a relative standard deviation of 1% or less. The results are in agreement with those obtained by a standard spectrophotometric method.     

Determination of bestatin in serum by high-performance liquid chromatography with fluorescence detection

A simple method for the determination of bestatin and its major metabolite in man, p-hydroxybestatin, in human serum was investigated; the method employs high-performance liquid chromatography with fluorescence detection. Bestatin and p-hydroxybestatin are oxidized to phenylacetaldehyde and p-hydroxyphenylacetaldehyde, respectively, with periodate, which are then converted into fluorescent compounds with 4,5-dimethoxy-1,2-diaminobenzene. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18. The detection limits of bestatin and p-hydroxybestatin are 0.2 and 0.4 microgram/ml serum, respectively. This method permits the precise determination of bestatin in serum (20 microliter) from patients administered bestatin. p-Hydroxybestatin in serum can not be measured by this method because of its low concentration (less than the detection limit).     

Determination of netilmicin in serum by thin-layer densitometry and fluorescence polarization immunoassay

Summary A newly developed thin-layer chromatographic method (TLC) for the quantitative determination of netilmicin in serum is described and compared with fluorescence polarization immunoassay (FPIA). The TLC procedure involves solid-phase extraction of the aminoglycoside from serum and chromatography on reversed-phase thin layer. Post-chromatographic derivatization is carried out using 2,2-diphenyl-1-oxa-3-oxonia-2-boratanaphthalene (DOOB) as fluorescence reagent. The calibration curve for netilmicin in serum is linear in the range 1.0–5.0 g/ml and the detection limit is about 0.2 g/ml (correlation coefficient r=0.9963, mean coefficient of variation V_XO=±5.3 %). A total of 25 serum samples from patients treated with netilmicin was measured by TLC and the results were compared with those of FPIA (Abbott TDx). There is no statistically significant difference between the two procedures (y=–0.35+1.094·x, Passing/Bablok). Thus the TLC-procedure is an alternative for the determination of netilmicin in serum, that possesses the necessary sensitivity of other comparable methods.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday.     

Detection of endotoxin using an evanescent wave fiber-optic biosensor

The lipopolysaccharide endotoxin is the most powerful immune stimulant known and a causative agent in the clinical syndrome known as sepsis. Sepsis is responsible for more than 100,000 deaths annually, in large part due to the lack of a rapid, reliable, and sensitive diagnostic technique. This study describes the detection of LPS fromE. coli at concentrations as low as 10 ng/mL, in 30 s using an evanescent wave fiber-optic biosensor. Polymyxin B, covalently immobilized onto the surface of the fiber-optic probe, selectively bound fluorescently labeled LPS. Unlabeled LPS was detected in a competitive assay format using labeled LPS for signal generation. The competitive assay format worked in both buffer and plasma with similar sensitivities. This method can be used with other LPS capture molecules such as antibodies, lectins, or antibiotics, to simultaneously detect LPS and to determine the LPS serotype. The LPS assay using the fiber-optic biosensor is applicable to both clinical and environmental testing.     

Determination of mitoxantrone with a fiber-optic device

A major problem in therapeutic use of the cytostatic agent mitoxantrone is the occurrence of side effects. Estimation of the drug concentration in or next to the tumor can lead to a reduction of the toxicity through accurate dose adjustment. In this paper a method for the determination of mitoxantrone by a fibre-optic device is described. The advantage of the system is the possibility of remote sensing at the working site of the cytostatic agent by a method that is simple in handling and sufficient in sensitivity. The measuring principle is based on the blue colour of mitoxantrone. The function of the device was tested byin vitro assays; linear calibration curves in a concentration range between 2 and 10g of mitoxantrone per ml of sample solution were obtained.     

Determination of sulfamethazine in milk by biosensor immunoassay

A biosensor based on surface plasmon resonance (SPR) measurement was developed for use in an immunoassay for detection of sulfamethazine (SMZ) in milk. The biospecific surface was a carboxymethyl dextran-modified gold-surface sensor chip to which SMZ was covalently bound. The assay was based on inhibition of the binding of polyclonal antibodies to immobilized SMZ by SMZ in the sample. The SPR response changed inversely in relation to the antibiotic concentration in the sample. Calibration curves were constructed for SMZ in buffer and in milk at a concentration which included the maximum residue limit (0 to 200 micrograms/kg). The analysis time per sample varied from 8 to 30 min. Different flow rates and antibodies were modified alternatively during the study to assess their influence on the performance of the assay. The active antibody concentration was calculated at approximately 1880 and 180 nM for the antibody anti-SMZ 1 and the antibody anti-SMZ 2, respectively. No cross-reactivity of antibodies with other antibiotics was found. Under optimal conditions, the detection limits in milk for SMZ were 8 and 1.7 micrograms/kg, respectively, for antibody 1 and antibody 2, at a flow rate of 20 microL/min.     

Determination of nicotinic acid in serum by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of nicotinic acid in serum is described which employs high-performance liquid chromatography with fluorescence detection. Nicotinic acid and 2-chloronicotinic acid as an internal standard in deproteinized serum are reacted with N,N'-dicyclohexyl-O-(7-methoxycoumarin-4-yl)methylisourea in acetone to give the corresponding fluorescent 4-hydroxymethyl-7-methoxycoumarin esters. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18 with isocratic elution using aqueous acetonitrile containing a small amount of sodium 1-hexanesulphonate as a mobile phase. The detection limit of nicotinic acid in serum was 0.2 nmol/ml. The method requires only 100 microliters of serum.     

Determination of aluminum in serum by capillary zone electrophoresis with laser-induced fluorescence detection

Summary A novel method of separating and detecting trace aluminum by capillary zone electrophoresis is described. Aluminum is reacted with lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzen-1-sulphonic acid] so that the complex can be selectively and sensitively detected by a laser-induced fluorescence detector after capillary electrophoretic separation. Using the proposed method, limits of detection in the sub parts per billion range are achieved. The technique is applied to the determination of aluminum in human serum.     

Multipoint measurements in optically dense media by using two-photon excited fluorescence and a fiber-optic star coupler

Optical multiplexing with an N × N fiber optic star coupler (with N=3, 4, or 8) and two-photon excited fluorescence is used to achieve multipoint measurements in highly absorbing environments. Differentiation of fluorescence signals from various sampling points was attained by implementing the time-of-flight characteristics of fiber light-guides. Because the transit time of a light pulse through a fiber optic depends largely on the length of the fiber waveguide, fibers of various lengths permit discrimination of the different sampling points in time. With the help of nanosecond time-resolved detection, it was possible to determine the concentration information at several sensing locations simultaneously. Calibration graphs for 2-(1-napththyl)-5-phenyloxazole were linear for all sizes of star coupler, with submillimolar detection limits. Nonlinear excitation of dopants inside the fiber core resulted in an emission signal that depended quadratically on laser power and, as such, was used as an “internal” sensor to correct for power-squared source fluctuations.     

Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons

An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added, and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides a new tool for continuous in situ monitoring of halogenated environmental pollutants.     

Kinetic determination of urea in serum by stopped-flow spectrophotometry

The determination of urea based on its reaction with biacetyl in the presence of thiosemicarbazide and iron(III) has been studied kinetically by using a modular stopped-flow system. This study gave some new data about this coloured reaction and led to the development of a simple, fast method which overcomes some of the disadvantages (e.g., long analysis times, high temperatures and acid concentrations) of the conventional equilibrium method for the determination of urea, which hinder its application to routine analyses. The linear range (0.5–15 μg ml^?1), detection limit, precision and selectivity of the proposed kinetic stopped-flow method are reported. The method has been satisfactorily applied to the analysis of serum samples without pretreatment. The results correlate well with those obtained by the urease method.     

Determination of chloramphenicol in milk by a fluorescence polarization immunoassay

A procedure for the determination of the drug chloramphenicol using a fluorescence polarization immunoassay (FPIA) was proposed. The optimum pairs of antibodies and antigens labeled with fluorescein were chosen, and the analytical characteristics of the procedure were determined. A rapid procedure for milk sample preparation with the use of a saturated solution of ammonium sulfate was optimized. The total time of sample preparation and determination of chloramphenicol in milk was no longer than 10 min. The detection limits of chloramphenicol in water and milk were 10 ng/mL and 20 μg/kg, respectively. The procedure developed for the determination of chloramphenicol was tested in the analysis of model and real milk samples. It was found that some milk samples contained chloramphenicol in concentrations of 38–41 μg/kg, which are several times higher than the maximum permissible concentration (MPC) (10 μg/kg).     

Determination of quinidine in serum by spectrofluorometry, liquid chromatography and fluorescence scanning thin-layer chromatography

Quinidine is determined in serum by direct and extraction spectrofluorometry, by reflectance fluorescence scanning thin-layer chromatography (TLC), and by high-performance liquid chromatography (HPLC). Least-squares analyses of patients' sera (n = 62) analyzed first by direct fluorometry (x) and then HPLC (y) gave a slope of 0.52, an y-intercept of -0.40, a standard error of estimate of 0.65, and a correlation coefficient of 0.83. Comparison of patients' sera (n = 59) determined by extraction fluorometry (x) and then HPLC (y) gave a slope of 0.998, an y-intercept of -0.175, a standard error of estimate of 0.30, and a correlation coefficient of 0.96. Comparison of patients' sera (n = 36) by HPLC (x) and then reflectance fluorescence scanning TLC (y) gave a slope of 0.837, an y-intercept of 0.152, and a correlation coefficient of 0.94. Methaqualone and oxazepam interfere with HPLC. Within-run precision is 1.6, 1.0, 5.2 and 3.0% by direct fluorometry, extraction fluorometry, TLC and HPLC while between-run precision is 5, 3.5, 9 and 6.0%, respectively.     

多巴胺修饰自组装金电极作为生物传感器测定核酸

采用衍生化接枝法制备了多巴胺(DA)-半胱氨(Cys)自组装修饰金电极(DA-Cys-SAM),研究了其电化学性质。在pH 7.5的Tris-HCl底液中,采用循环伏安法测定其Epa=218 mV,Epc=120 mV,ΔE=98 mV,ipa/ipc≈1,电极反应准可逆。采用示差脉冲法(DPV)研究发现DA氧化峰电流随DNA质量浓度提高而显著变大且峰电位不变,峰电流的增加值与DNA质量浓度在1×10-7~1.2×10-5g/mL范围内成正比,检出限达5×10-8g/mL,相对标准偏差为3.2%(n=8,5×10-7g/mL DNA)。该电极对DNA测定具有特异性,对实际样品进行了测定。     

Determination of urea in blood plasma by enzyme-pH electrode

Combination of gel-entrapped urease with a pH electrode can be used to measure plasma urea, provided that the response is related to an auxiliary pH electrode. The response time is 1–3 min and electrodes can be used for up to one week. Good correlation was obtained for routine samples with a spectrophotometric diacetyl monoxime method (y = 0.952x + 0.224, r = 0.996).     

Determination of pantothenic acid in foods by optical biosensor immunoassay

An optical biosensor inhibition immunoassay was developed using a specific pantothenic acid-binding protein for the quantitation of free pantothenic acid (vitamin B5) in foodstuffs. Samples were prepared by a simple extraction procedure in buffer, and vitamin content was estimated against authentic calibrants in the same buffer. Performance parameters included a working range of 10-5000 ng/mL, a limit of detection of 4.4 ng/mL, precision relative standard deviation of 5.4-7.1% over a range of concentrations, and recoveries > 95% in the matrixes tested. A wide range of foodstuffs, including National Institute of Standards and Technology reference samples, were tested in 3 independent laboratories and the results were compared with microbiological assay and liquid chromatography/mass spectrometry (LC/MS) methods. The results indicate that the biosensor technique is appropriate for the estimation of pantothenic acid in a wide range of foodstuffs.