HPLC of 9-fluorenylmethylchloroformate-polyamine derivatives with fluorescence detection

Summary There are a number of reagents available for fluorescent labelling of primary amines. These include dansyl chloride, o-phthalaldehyde, fluorescamine, and a new reagent, 9-fluorenylmethylchloroformate (FMOC), reported recently. This paper describes a reversed-phase HPLC procedure for the separation and fluorescence detection of polyamines following pre-column derivatization with FMOC. The polyamines studied by this method include putrescine, cadaverine, spermidine, and spermine. Experiments were carried out to determine maximum fluorescence excitation and emission wavelengths, optimum reaction pH, linear ranges, and minimum detection limits for each of the polyamines. The HPLC method includes a gradient program which provides complete separation from serum hydrolysate components and specificity for the four polyamines with detection limits ranging from 2 to 9 pg. This procedure was applied to hydrolyzed serum samples.     

A Novel Method for Quantification of Circulating DNA in Serum by Capillary Zone Electrophoresis

In this paper, we first presented a novel method for quantification of circulating DNA in human serum based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). The serum was digested by proteinase to release free DNA, and then CZE-LIF system was used for the quantification of total circulating DNA. This method was successfully used to quantify the circulating DNA levels in sera from healthy individuals and certain cancer patients.We found the significantly elevated circulating DNA levels in certain prostate cancer patients. Our results demonstrated that CZE-LIF system has good linearity, excellent sensitivity (0.5 ng/mL DNA),satisfactory reproducibility (RSDs in one day and between days were both less than 5%) and reliability, and is well suitable to the quantification of the circulating DNA in human serum or plasma.     

石墨炉原子吸收分光光度法测定血清中铜

铜系人体内含有的微量元素。测定血清中Cu/Zn比值,有助于病因分析和提供诊断依据。目前,测定血清铜所用的仪器分析方法甚多。本法无需对血清样进行预处理,取样量仅20—50μl。碱金属和碱土金属卤化物无干扰。特征质量m。为5.6pg/0.0044A.S,回收率98～102%,变异系数1.7-2.7%。     

人体血清中痕量铜的PAN分光光度法的测定研究

研究了用Cu2 -1-(2-吡啶偶氮)-2-萘酚(PAN)分光光度法测定人体血清中痕量铜的最佳条件。在HCl-KCl溶液介质中,十二烷基苯磺酸钠存在下,其表观摩尔吸光系数为1.42×104 L.mol-1.cm-1,Cu2 含量在0~2μg/mL范围服从比尔定律,检测限为0.5μg/dm3。操作简便、快速,该法可用于人体血清中痕量铜的测定。     

Spectrofluorimetric determination of human serum albumin using a doxycycline-europium probe

A new spectrofluorimetric method is proposed for determination of human serum albumin (HSA) with the limit of detection at ng levels. Using doxycycline (DC)-europium (Eu³⁺) as a fluorescent probe, in a buffer solution of pH 10.2, HSA can remarkably enhance the fluorescence intensity of the DC-Eu³⁺ complex at 612 nm and the enhanced fluorescence intensity of Eu³⁺ is proportional to the concentration of HSA. Optimum conditions for the determination of HSA are also investigated. The linear ranges for HSA are 0-9.2 and 9.2-34.5 μg ml⁻¹ with limits of detection of 64 and 115 ng ml⁻¹, respectively. This method is simple, practical and relatively free of interference from coexisting substances, as well as much more sensitive than most of the existing assays. The determination results for human serum and urine samples are identical to those by the AOAO method, with relative standard deviations of five determinations of 1.1-3.6%. By the Rosenthal graphic method, the binding number and association constant of human serum albumin with the probe are 1.8 and 3.71×10⁵ l mol⁻¹, respectively.     

Studies on Thermodynamics Features of the Interaction between Imidacloprid and Bovine Serum Albumin

At different temperatures, the interactions between imidacloprid （IMI） and bovine serum albumin （BSA） were investigated with a fluorescence quenching spectrum, a synchronous fluorescence spectrum, a three-dimensional fluorescence spectrum and an ultraviolet-visible spectrum. The average values of bonding constants （KLB： 3.424 × 10^4 L,mol^-1）, thermodynamic parameters （△H： 5.188 kJ,mol^-1, △G^（○—）：-26.36 kJ,mol^-1, △S： 103.9 J,K^-1,mol^-1） and the numbers of bonding sites （n： 1.156） could be obtained through Stern-Volmer, Lineweaver-Burk and ther- modynamic equations. It was shown that the fluorescence of BSA could be quenched for its reactions with IMI to form a certain kind of new compound. The quenching belonged to a static fluorescence quenching, with a non-radiation energy transfer happening within a single molecule. The thermodynamic parameters agree with △H〉 0, △S〉0 and△G^（○-）〈0, suggesting that the binding power between IMI and BSA should be mainly a hydrophobic interaction.     

Determination of Picogram-Levels of Acetylspiramycin in Human Urine and Serum by Flow Injection Chemiluminescence

A sensitive chemiluminescence method for the determination of acetylspiramycin is presented. It is based on the greatly enhancive effect of acetylspiramycin on the chemiluminescence reaction between luminol and hydrogen peroxide in the flow system. The increase in chemiluminescence intensity was linearly proportional to the acetylspiramycin concentration in the range from 10pg·mL^–1 to 2.0ng·mL^–1 (r²=0.9979). The detection limit was 3pg·mL^–1 (3). At a flow rate of 2.0mL·min^–1, the process of determination, including sampling and washing, could be performed in 0.5 min, and the relative standard deviations of seven replicates are less than 5.0%. The proposed method was applied successfully to the determination of acetylspiramycin in pharmaceutical preparations, human urine and serum without pre-treatment. It was found that the excretive ratio of acetylspiramycin reached its maximum 2.0 hours after having been administered orally, and the excretive ratio in 12.0 hours was 8.4.     

Investigation of the interaction between acridine orange and bovine serum albumin

The results from the measurement of the fluorescence spectrum showing the binding characteristics of acridine orange (AO) and bovine serum albumin (BSA) are reported. It was found that the equilibrium constant k=4848.64 l mol⁻¹, and the number of binding sites n=0.82. Based on the mechanism of the Forster energy transference, the transfer efficiency of energy and distance between the acceptor AO and BSA were found. The interaction between AO and BSA have been verified as consistent with the static quenching procedure and the quenching mechanism is related to the energy transfer.     

各种离子对血卟啉与牛血清白蛋白相互结合反应的影响研究

应用荧光法研究了水溶液体系中血卟啉与牛血清白蛋白分了间相互结合反应，其结合常数ＫＡ＝２．２×１０＾４，结合位置数为１．依据Ｆｏｒｓｔｅｒ非辐射能量转移机理，探讨了血卟啉与牛血清白蛋白相互结合时，其给体－受体间距离和能量转移效率，进一步研究了各种离子与牛血清白蛋白的结合反应，其结合淬灭能力大小顺序为Ｃｒ（Ⅵ）〉Ｆｅ＾３＋〉Ｃｕ＾２＋〉ＮＯ２〉Ｂｒ＾－，并探讨不同阴，阳离子及血卟啉与牛血清白蛋白间相互     

Adsorption of Bovine Serum Albumin onto Polystyrene Latex Particles Bearing Saccharidic Moieties

The adsorption of bovine serum albumin (BSA) onto polystyrene latexes bearing various amounts of sugar moieties has been investigated as a function of pH and ionic strength and the results were compared to those for bare polystyrene latexes having negative surface charges. The functionalized latexes were produced by seeded copolymerization of (0.3 μm) liposaccharidic monomer onto polystyrene particles obtained by soap-free emulsion polymerization of styrene using potassium persulfate as initiator. At first, the electrophoretic mobility behavior of the various latexes was examined as a function of pH: a significant decrease was observed in the case of saccharide-containing latex particles compared to the bare particles. The adsorption of BSA onto these latexes exhibited a reduced amount of adsorbed BSA for those latex particles bearing saccharide groups. This adsorbed amount depends on the yield of saccharidic monomer incorporated onto the surfaces of the latex particles.