微孔板化学发光法检测碱性磷酸酶技术的建立

碱性磷酸酶(ALP)能催化发光底物APLS迅速水解并持续发光,据此建立了化学发光技术检测ALP活性的方法。利用一种新的发光底物APLS,建立微孔板化学发光法检测ALP活性,并对实验条件进行优化。优化后的最适反应条件为:微孔板上每孔加200μL APLS,在pH=9.5、37℃条件下与ALP反应10min,多功能酶标仪检测发光光子数(RLU),检测ALP线性范围是0.01~1U/L。微孔板化学发光法检测ALP,敏感度更高,且操作简便易行。     

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.     

基于Fe(Ⅲ)和四甲基联苯胺显色反应分析碱性磷酸酶活力

基于酶底物和产物不同的氧化还原性质,建立了Fe(Ⅲ)和四甲基联苯胺(TMB)显色反应检测抗坏血酸和碱性磷酸酶(ALP)活力的比色法。在最佳条件下,吸光度与ALP活力的对数在0. 5～200 U/L范围内呈现良好依赖性,检测限为0. 05 U/L。方法已用于ALP抑制剂Na_3VO_4抑制率评估和监测兔血浆样品中内源性ALP水平,在临床应用和现场检测显示出巨大应用潜力。     

金纳米簇增强的化学发光反应及在葡萄糖检测中的应用

金纳米簇(Au NCs)具有拟过氧化物酶活性,能催化鲁米诺(Luminol)与H_2O_2反应,产生增强的化学发光信号。不同于其它纳米微粒催化的Luminol-H_2O_2化学发光反应,Au NCs催化得到的是慢化学发光信号,且其信号能在10min内保持稳定。基于此反应,本文发展了简单、方便的化学发光测定H_2O_2的新方法。在优化的实验条件下,测定H_2O_2的检测限为10μmol/L。将此方法应用于葡萄糖的检测,其线性范围为10~1 000μmol/L,检测限为10μmol/L。目前一些重要的生物分子,如尿酸和乳酸等均能与相关酶反应生成H_2O_2,本方法也能进一步拓宽至这些生物分子的检测。     

化学发光酶免疫分析测定鱼肉中呋喃它酮代谢物方法研究

建立了呋喃它酮代谢物5-吗啉甲基-3-氨基-2-6唑烷基酮(AMOZ)间接竞争化学发光酶免疫分析(icCLEIA)检测方法,通过单因素实验优化了包被原浓度、抗体稀释倍数、反应缓冲体系及浓度、竞争反应时间等参数,结果表明icCLEIA最佳反应条件为:包被抗原浓度为10 ng/mL,抗体稀释60000倍,最佳竞争时间为50 min,体系缓冲液0.01 mol/L PBS(pH 7.4).在优化的条件下,本方法的线性检测范围为0.026 ～3.52 μg/L,IC50为0.29 μg/L,检出限(LOD,IC10)为0.012 μg/L.对鱼肉样品的平均添加回收率在101.4％～115.5％之间.建立的icCLEIA方法可用于实际样品中AMOZ残留检测.     

硫化镉量子点增敏化学发光体系测定痕量苯酚的研究

在碱性条件下,硫化镉量子点对鲁米诺-H2O2化学发光体系具有显著的增敏作用,而苯酚对该体系的化学发光有强烈抑制作用,以此建立了流动注射化学发光检测苯酚的新方法.在优化实验条件下,苯酚浓度在5.0×10-9 ～5.0×10-7g/L(r=0.9935)和5.0×10-6 ～1.0×10-3g/L(r=0.9982)范围内...     

测定汽油中铅的KMnO4—Luminol化学发光体系

研究了KMnO4氧化鲁米诺(luminol)的化学发光反应过程,探讨了铅(Ⅱ)后续催化该化学发光反应的反应历程,建立了在最佳反应条件下利用该后续发光反应定量测试微量铅的方法;采用流动注射化学发光方法测定了汽油中铅含量,方法检出限达0.01mg/L,检测线性范围在1.0×10-7～5.0×10-4mol/L,对大量阴阳离子进行了干扰试验,结果表明方法有良好的选择性.     

双[2,4,6-三氯苯基]草酸酯-过氧化氢体系流动注射化学发光法检测地沟油中的胆固醇

基于胆固醇和冰乙酸的反应物与硫酸铁铵生成的紫色化合物能有效抑制双[2,4,6-三氯苯基]草酸酯(Bis(2,4,6-trichlorophenyl)oxalate,TCPO)-H2O2-咪唑化学发光反应,建立了流动注射-化学发光联用检测胆固醇的方法。对流动注射、化学发光等实验参数进行了优化。当咪唑浓度为0.001 mol/L,H2O2浓度为0.3 mol/L,TCPO浓度为1.0×10"3mol/L,主副蠕动泵转数分别为20和15 r/min时,体系具有最强的化学发光。在优化的实验条件下,测定胆固醇的线性范围为8.6×10"6～2.2×10"4mol/L;检出限(S/N=3)为2.5×10"6mol/L;相对标准偏差(RSD,n=11,c=6.5×10"5mol/L)为1.5%。不同加标水平下的加标回收率为95.0%～105.0%;相对标准偏差(n=6)小于3.6%。本方法已应用于地沟油中胆固醇的测定,结果令人满意。     

一种基于微芯片电泳平台的级联化学发光信号放大策略用于HIV-DNA的高灵敏检测

该文基于微芯片电泳-化学发光检测（MCE-CL）平台,以辣根过氧化酶标记的DNA(HRP-DNA)作为信号探针,利用HRP 催化鲁米诺和双氧水化学发光反应及目标分子与DNA的杂交反应,结合T7Exo酶辅助信号放大,建立了一种MCE分离辅助双循环化学发光信号放大的新方法。结果显示：优化实验条件下,在1.0×10-14~5.0×10-9 mol/L范围内,HIV-DNA的浓度对数值与HIV-DNA的化学发光强度呈良好的线性关系,检出限（S/N=3）为1.6×10-15 mol/L,在0.10、0.25、1.0、10（×10-12 mol/L）4个加标水平下的回收率为93.0%～103%,相对标准偏差（RSD）为0.50%～3.7%,方法具有较好的准确度,可应用于人血清中HIV-DNA的高灵敏检测。     

化学发光酶免疫法检测猪肉中氯丙嗪残留

采用棋盘滴定法确定包被抗原浓度和抗体稀释倍数,通过单因素实验优化了竞争反应时间、磷酸盐缓冲液浓度、甲醇含量、pH值等参数,建立了氯丙嗪的间接竞争化学发光酶免疫检测方法,并考察了方法的特异性、灵敏度和稳定性.结果表明,最佳反应条件为包被抗原浓度为0.05 μg/L;氯丙嗪抗体稀释32000倍;体系缓冲液为含10％甲醇、0.1 mol/L磷酸盐缓冲液(pH 7.0).本方法的IC50为0.12 μg/L;检出限为0.02 μg/L;线性范围0.02～24.78 μg/L;批内和批间相对标准偏差均小于10％.与其它结构类似物没有明显交叉反应.猪肉检测的平均回收率为87.4％～105.6％,与高效液相色谱方法的相关性良好.本方法具有较高的灵敏度和较好的稳定性.     

儿茶素化学发光行为的研究及其应用

建立了高锰酸钾化学发光体系测定儿茶素的方法.在最佳实验条件下,儿茶素在酸性高锰酸钾体系中会产生较强的化学发光,其线性响应范围为1.71×10-6～3.44×10-5 mol/L,检出限为4.64×10-7 mol/L.方法应用于复方儿茶胶囊中儿茶素类成分总含量的测定,测得儿茶素类成分的总含量为327 mg/g,回收率为95.3%.方法简单、快速,结果满意。     

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.     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.     

Determination of alkaline phosphatase activity in human sera by mid-FTIR spectroscopy

Fourier transform infrared spectroscopy is applied to the determination of alkaline phosphatase (ALP) activity in human sera. 4-nitrophenylphosphate was found to be an excellent ALP substrate for FT-IR spectroscopic detection. The developed method is based on the acquisition of two FT-IR spectra: one recorded immediately after mixing the sample and assay solution and the other after an incubation time of 10 min. Spectral changes in the mid IR range due to the enzymatic reaction could be correlated to the ALP activity in the sample. Experimental conditions were established such that the clinically relevant range for determination of ALP activity in human sera (50 to 900 U/L) was covered. The method was applied to the analysis of ALP activities in standard solutions as well as in human sera yielding results which agreed well with those obtained by a standard reference method. Received: 20 June 1997 / Accepted: 16 September 1997     

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.     

Determination of alkaline phosphatase activity in human sera by mid-FTIR spectroscopy

Fourier transform infrared spectroscopy is applied to the determination of alkaline phosphatase (ALP) activity in human sera. 4-nitrophenylphosphate was found to be an excellent ALP substrate for FT-IR spectroscopic detection. The developed method is based on the acquisition of two FT-IR spectra: one recorded immediately after mixing the sample and assay solution and the other after an incubation time of 10 min. Spectral changes in the mid IR range due to the enzymatic reaction could be correlated to the ALP activity in the sample. Experimental conditions were established such that the clinically relevant range for determination of ALP activity in human sera (50 to 900 U/L) was covered. The method was applied to the analysis of ALP activities in standard solutions as well as in human sera yielding results which agreed well with those obtained by a standard reference method.     

基于纳米CeO_2增敏鲁米诺化学发光法测定对乙酰氨基酚的研究

基于碱性条件下,CeO_2纳米粒子能够有效增敏鲁米诺-KMnO_4体系的化学发光,并结合流动注射技术建立了一种对乙酰氨基酚测定的新方法。实验研究了影响化学发光检测信号的多种因素,并初步探讨了可能的化学发光机理。在最佳实验条件下,对乙酰氨基酚浓度在1.0×10-7~5.0×10-5mol/L范围内与相对化学发光强度的抑制值呈良好的线性相关,相关系数(r2)为0.996 4,检出限(3σ)为3.3×10-8mol/L。对5.0×10-6mol/L的对乙酰氨基酚溶液平行测定11次,计算得相对标准偏差(RSD)为0.3%。该法用于银翘片中对乙酰氨基酚含量的测定,回收率为98.0%;对尿液的加标回收率为97.9%~98.7%,结果满意。     

Determination of superoxide dismutase in erythrocytes by a chemiluminescent assay

A highly rapid chemiluminescent assay for the determination of superoxide dismutase (SOD) activity in erythrocytes was developed. The inhibition of the luminescent emission caused by the decrease of generated superoxide anions was measured. The aim of this work was to verify the application of a non amplified luminol SOD luminescent assay (CLM) in erythrocytes starting from an amplified method already used for the determination of XOD activity in milk (CLME). Both the assays had a detection limit of 3x10(-2)+/-7x10(-3) U/ml of SOD at 2sigma level, and a linear range of activity from 5.2 to 0.03 U/ml of SOD. The imprecision of assays (repeatability) presented coefficients of variations ranging from 3.1 to 7.9% for the CLME method and from 0.6 to 17.7% for CLM method. Both luminescent techniques were compared using a spectrophotometric kit, that had a detection limit of 0.3 U/ml, and showed good agreement.     

石墨烯量子点荧光探针对碱性磷酸酶活性的高效检测

基于苯醌类物质静态猝灭石墨烯量子点(GQDs)荧光的特性, 构建了一种利用GQDs荧光探针实时、 高效检测碱性磷酸酶(ALP)活性的新方法. 过氧化氢在辣根过氧化物酶催化作用下产生羟基自由基并将邻苯二酚氧化成邻苯醌, 导致GQDs的荧光猝灭. ALP催化抗坏血酸-2-磷酸反应生成抗坏血酸, 具有较强还原性的抗坏血酸能清除溶液中的过氧化氢和羟基自由基, 抑制邻苯醌的产生, 使GQDs的荧光猝灭效果减弱. 随着ALP活性的增大, GQDs在440 nm处的荧光强度不断增强, 由此建立了一种高效检测ALP活性的新方法. 在最佳实验条件下, 该GQDs荧光探针对ALP活性的检出限为0.084 U/L. 将此方法成功用于人血清中ALP活性的检测, 为与ALP相关疾病的诊断与治疗提供了理论基础.