荧光光度法测定血清中碱性磷酸酶

合成了一种新的底物水杨酸磷酸酯 (SP),用于荧光光度法测定血清中碱性磷酸酶(ALP)活力.在37.0 ℃的Tris-HCl缓冲液(pH 9.0)条件下, 碱性磷酸酶作用于荧光底物SP,水解生成强荧光产物水杨酸(SA),生成的SA的量与参与反应的ALP 的活力成正比.据此建立了荧光光度法测定血清中碱性磷酸酶活力的新方法.测定碱性磷酸酶的线性范围是0.03～6.00 U/L,检测限为7.04 mU/L.本方法适用于血清中碱性磷酸酶的测定.测定结果与临床上常用的以对硝基苯磷酸酯为底物的分光光度法相比,无显著性差异.     

氨基荧光素的合成、分离和表征

氨基荧光素的合成、分离和表征;氨基荧光素;硝基荧光素;还原;分离     

两种新型氯代荧光素探针染料的合成和表征

研究了两种具有空间连接链的新型氯代荧光素探针染料4',5'-二氯-6-(5-羧基戊氨基羰基)荧光素和2',7'-二氯- 6-(5-羧基戊氨基羰基)荧光素的合成. 以氯代间苯二酚和苯三酸酐为起始原料经7步反应得到了目标化合物, 产率良好. 测定了目标产物的荧光性能, 结果显示该化合物具有很高的荧光量子产率, 与荧光素母体相比其激发和发射波长明显向长波方向移动. 所得产物结构经1H NMR, IR, MALDI-TOF MS以及元素分析进行了表征.     

铝—酸性铬蓝K荧光光度法测定微量铝的研究

研究了酸性铬蓝Ｋ（ＡＣＢＫ）荧光光度法测定微量铝的反应。Ａｌ＾３＋与ＡＣＢＫ形成络合物的荧光强度与铝的含量在１×１０＾－８～８×１０＾－６ｍｏｌ／Ｌ范围内呈现良好的线性关系，检测限为４．８×１０＾－９ｍｏｌ／Ｌ，对４×１０＾－８ｍｏｌ／Ｌ铝重复１１次检测其相对标准偏差为４．８％，成功地用于人发及钢样中铝含量的测定。     

磷酸苯酯-碱性磷酸酯酶伏安酶联免疫分析新体系的研究

研究了以磷酸苯酯为底物微分脉冲伏安法测定碱性磷酸酯酶 (ALP)的方法。磷酸苯酯在ALP的催化作用下水解生成苯酚 ,苯酚在玻碳电极上 0 .70V(vs.Ag/AgCl)左右产生氧化电流 ,氧化电流随着酶浓度的增大而增大 ,借助此氧化电流可以测定ALP ,并进而可用于以ALP为标记酶的酶免疫分析。对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究 ,测定游离ALP的线性范围是 0 .0 6U/L～ 1 .0× 1 0 3U/L ,检出限为 0 .0 6U/L     

基于荧光素烷基酯荧光增强的甲醇光纤化学传感器的研制

本文合成了荧光素长链烷基酯类化合物荧光素十八烷基脂, 将之固定于增塑的PVC膜中后,将此膜固定在光纤探头上, 制成了用于测定甲醇的荧光光纤化学传感器,该传感器测定甲醇的可逆性好,测定范围为30-80%甲醇(v/v), 响应时间小于2min     

替硝唑的荧光分光光度法测定

采用铁 -冰醋酸还原体系 ,将替硝唑还原后 ,在激发波长360nm ,发射波长420nm处测定其荧光强度 ,据此建立了替硝唑的荧光光谱分析法。实验表明 ,替硝唑浓度在1.0×10-6～4.0×10-4 mol/L范围内与荧光强度呈良好的线性关系 ,相对标准偏差 (RSD)为1.2 % ,检出限达5.6×10-8 mol/L。本法测定结果与药典法基本一致 ,已用于替硝唑片剂和替硝唑葡萄糖注射液中替硝唑的测定 ,结果令人满意。     

以2-磷酸抗坏血酸酯为底物检测碱性磷酸酯酶的微分脉冲伏安法

提出以2_磷酸抗坏血酸酯为碱性磷酸酯酶(ALP)底物微分脉冲伏安法测定ALP的方法 ;2_磷酸抗坏血酸酯在ALP的催化作用下发生水解反应生成抗坏血酸 ,抗坏血酸在玻碳电极上 +0.40V(vsAg/AgCl)被氧化而产生一个灵敏的氧化峰 ,氧化峰电流随着酶浓度的增大而增大 ,借助此氧化峰电流可以测定ALP ,进而可用于以ALP为标记酶的酶免疫分析 ;用微分脉冲伏安法对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究 ,建立了以2_磷酸抗坏血酸酯为底物的伏安酶联免疫分析新体系 ,测定游离ALP的线性范围是0.4～2.0×103 U/L,检测限为0.3U/L,对游离的IgG_ALP的测定最大稀释比为1∶200000。     

新型荧光素类细胞钙离子荧光探针Fluo-Cl的设计、合成及表征

设计合成了一种新型荧光素类细胞钙离子荧光探针, 并对其结构及光谱特性进行了表征.     

阵列纸芯片比色法检测碱性磷酸酶

采用5-溴-4-氯-3-吲哚磷酸盐(BCIP)/氯化硝基四氮唑蓝(NBT)显色体系,构建了阵列纸芯片比色检测碱性磷酸酶(ALP)的方法.首先,借助烘干处理方式在光刻法制备的阵列纸芯片微孔中固定显色试剂,然后加入ALP进行显色反应,最后,采用凝胶成像仪和普通照相机成像,读取显色强度(灰度值)进行比色检测.详细考察了显色条件对检测结果的影响,探讨了人血清白蛋白对ALP检测的增色效应,在最佳实验条件下,ALP检测的线性范围为1.5～20 U/L,检出限(3 σ)为0.78 U/L(n=18),比文献报道中纸芯片上检测ALP方法的检出限低约两个数量级.本方法成功用于实际血清样品检测,测定结果与临床值一致.在此基础上,构建了双色阵列纸芯片,通过颜色的变化实现了ALP的可视化半定量检测.     

荧光法测定碱性磷酸酶及其标记物

本文研究了以４－甲基伞形酮磷酸酯（４－ＭＵＰ）为底物测定碱性磷酸酶（ＡＬＰ）活性的高灵敏荧光法及其应用于人血清ＩｇＧ的含量测定。通过纯化４－ＭＵＰ，优化分析条件，使方法的灵敏度有较大提高，实用性也得到改善，测定ＡＬＰ的线性范围为１．７×１０＾－５￣１．７×１０＾－８ＩＵ／ｍＬ，检出限为４．０×１０＾－９ＩＵ／ｍＬ，本法已成功地应用于血清样品ＡＬＰ的测定。作为一个例子，通过测定免疫标记物ＨｕＩｇＧ－     

An Automated Spectrophotometric Flow Injection Assay of Alkaline Phosphatase

Abstract

Assay of alkaline phosphatase using an automated flow injection manifold is described. The detector used is a spectrophotometer. The measurement is based on the rate of formation of p-nitrophenol at 405 nm, from the substrate p-nitropheny1 phosphate. The linear range, precision and accuracy of the technique for the assay of the enzyme is given. The advantages of the approach to assay of plasma enzymes are also discussed.     

ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.     

Indirect Measurement of Yoctomole Alkaline Phosphatase by Capillary Electrophoresis with Electrochemical Detection

A method for indirectly detecting yoctomole (ymol) alkaline phosphatase was developed by capillary electrophoresis with electrochemical detection. In this method, disodium phenyl phosphate was used as the enzyme substrate and the product (phenol) of its hydrolysis reaction catalyzed by alkaline phosphatase was detected at the carbon fiber electrode. The optimum conditions of detection are 1.0×10^?2 mol/L Na₂B₄O₇ (pH 9.8) for the running buffer; 1.00×10^?3 mol/L disodium phenyl phosphate for the enzyme substrate; 20.0 kV for the separation voltage; 5 kV and 10 s for the injection voltage and injection time; 1.05 V (vs. saturated calomel electrode) for the detection potential and 10 min for the incubation time, respectively. In order to enhance the ratio of signal to noise, the shape and size of the working electrode, the shape of detection end of the capillary, and the capillary/electrode alignment method were studied in detail. When a single carbon fiber microcylinder electrode of 6 μm, a capillary of 10 μm ID with the etched detection end and the in‐capillary alignment were used, a ymol mass limit of detection for alkaline phosphatase was achieved.     

Electrochemical Determination of Alkaline Phosphatase in Human Serum by Differential Pulse Voltammetry

Differential pulse voltammetry(DPV) was applied to the determination of alkaline phosphatase(ALP) activity in human serum with phenyl phosphate as the substrate. Phenyl phosphate can enzymatically be hydrolyzed to produce phenol which is quantified by DPV at 660 mV(vs. Ag/AgCl) in the concentration rangeof 2.0--100 μmol/L. The standard curve for ALP is linear over the range from 0.06 to 1000U/L with a relative standard deviation of 3.0%. The conditions for the enzymatic reaction and voltammetric detection were optimized and the kinetic constants were also examined. The human serum samples were tested by this method and the results were in good agreement with those obtained by the routine p-nitrophenyl phosphate spectrophotometric method.     

Electrochemical Detection of Alkaline Phosphatase in BALB／c Mouse Fetal Liver Stromal Cells with Capillary Electrophoresis

A method for determination of alkaline phosphatase (ALP) in BALB/c mouse fetal liver stromal cells has been described based on the catalytic reaction. After the cell extract is incubated with the substrate disodium phenyl phosphate, the reaction product phenol generated by ALP is determined by capillary electrophoresis with electrochemical detection.     

基于离子液体修饰碳纳米管电极的碱性磷酸酶电化学检测

利用自制的离子液体修饰碳纳米管电极(CNTs-ILE),在对CNTs-ILE的特性及对碱性磷酸酶(AP)酶促反应的底物磷酸对硝基苯酯(PNPP)和产物对硝基苯酚(PNP)进行电化学研究的基础上,采用安培法对酶促反应进程实现了持续动态的检测.实验表明,CNTs-ILE的循环伏安特性优于传统玻碳电极,且它在检测PNP时呈现出良好的抗钝化特性.当AP浓度范围在1.0～100 U/L时,电流大小与AP的浓度呈良好的线性关系.本方法可快速检测AP浓度,检测时间在20 min内.CNTs-ILE具有良好的抗钝化、操作简单及制作成本低等特点,在碱性磷酸酶的检测领域中有望得到进一步发展.在传统ELISA基础上,利用CNTs-ILE对具体样本最终的AP酶促反应产物进行电化学检测,通过峰值电流大小确定抗原浓度,为检测以AP为标记酶的抗原和抗体提供了理论依据.     

流动注射碱性降解荧光法快速测定微量金霉素

提出了流动注射碱性降解荧光法快速测定微量金霉素的新方法.金霉素在流动管路中与0.25 mol/L NaOH溶液快速反应,生成强荧光降解物.在流通池中用荧光法进行检测.反应产物荧光强度在10~(-8)～10~(-5)mol/L范围与金霉素含量成正比.共存四环素和脱水金霉素杂质不干扰金霉素测定.本法既可用于金霉素产品含量测定,又可用于四环素产品中杂质金霉素的测定,还成功地应用于尿液和血清中微量金霉素的测定.     

High-Sensitive Visually Controlled Membrane-Type Quantitation of NAD and Alkaline Phosphatase

Abstract

New high-sensitive visually controlled membrane-type analytical methods are proposed for quantitation of nicotineamide adenine dinucleotide and alkaline phosphatase in water solutions. The methods are based on using nitrocellulose membrane as a solid matrix on which the components of one-enzyme cofactor regeneration system are being immobilised by adsorption. In the presence of substances to be assayed, the end colored product is being adsorbed on the matrix as a result of enzymatic cyclic NAD/NADH regeneration in the active site of the matrix-bound alcohol dehydrogenase and some chemical successive reactions. Its colored intensity is a measure of the concentration of the analysed substances in solution. The general principle of NAD or alkaline phosphatase determination is successive immobilisation of separate components of the system (N-(6′-aminohexyl)salicylamide and horse liver alcohol dehydrogenase) on the matrix by adding their solutions to the wells of a specially designed cell with the membrane bottoms. In the case of alkaline phosphatase, the enzyme acted on NADPH as on a substrate. The reaction product, NAD was detected in the subsequent reaction of coenzyme regeneration. The other components of the amplifying system were added in substrate solutions at the stage of the alcohol dehydrogenase reaction. The lower detection limits for NAD and alkaline phosphatase were 3 × 10^?9 M and 1 × 10^?14 M respectively, the volume of the test sample ? 20 μl, the time of assay ? 5 min. The working concentration ranges were from 3 × 10^?9 to 1 × 10^?7 M and from 1 × 10^?14 to 1 × 10^?10 M levels for NAD(H) and alkaline phosphatase, respectively.