基于微孔板化学发光法检测碱性磷酸酶的研究

CDP-Star能被ALP迅速水解,且水解产物能够持续发光.本研究对化学发光法检测ALP的反应条件进行了优化.优化后的反应条件为:125 μtmol/L的CDP- Star在pH 9.5缓冲液中,37℃下与ALP反应10min.在此条件下,△RLU与ALP的活力呈线性关系.因此,建立了一种基于微孔板化学发光法检测ALP的实验方法.结果表明,本方法检测ALP的检测线性范围是0.05～10 U/L和10～1000 U/L,相对于光吸收法具有速度快、灵敏度高、重复性好等优点.因此,本方法可以被开发成试剂盒,应用于高通量全自动生化分析仪的测试分析.     

微板式化学发光酶免疫分析法临床测定人血清中孕酮

将抗兔IgG(即二抗)物理吸附于聚苯乙烯微孔板上作为通用固相,通过免疫反应制备固相抗体。采用辣根过氧化物酶催化鲁米诺-过氧化氢化学发光体系,建立了一种高通量、简便、快速的化学发光酶免疫分析方法用于临床测定人血清中的孕酮。对各种影响因素如免疫试剂的稀释度、发光底物选择、发光反应时间及温育条件等进行了考察和优化,最终选定的实验条件:孕酮抗体和HRP标记物的最佳稀释度分别为1∶100000和1∶15000;选用Ⅱ号发光底物,发光反应10min后测定;37℃水浴条件下温育1h。对建立的方法进行了评价。该方法的灵敏度为0.08μg/L;批内和批间相对标准偏差均在15%之内;低、中、高3个不同浓度值样品的平均回收率分别为101%、101%和94.4%(在87.8%～108%之间)。使用本方法和经典的放射免疫法同时对36份人血清样品进行测定,结果显示,本方法与放射免疫分析方法相关性良好,其相关系数为0.9502。     

基于银纳米粒子催化鲁米诺-过氧化氢化学发光体系测定丹参酮ⅡA磺酸钠

碱性条件下,丹参酮ⅡA磺酸钠能够抑制鲁米诺-过氧化氢-银纳米粒子的化学发光,据此建立了测定丹参酮ⅡA磺酸钠的化学发光分析方法。通过优化发光底物浓度、反应介质及其浓度和银纳米粒子浓度等因素,确立了最佳实验条件,并探讨了体系的可能机理。在5.0×10~(-6)~0.08 mol/L浓度范围内,体系的化学发光强度与丹参酮ⅡA磺酸钠浓度的对数值呈线性关系,检出限为9.38×10~(-7)mol/L,检测时间为20 s。该方法灵敏度好,检测范围宽,可简单快速地测定注射液中丹参酮ⅡA磺酸钠的含量,检测结果与高效液相色谱法的测定结果较为一致。     

微板式化学发光酶免疫分析法测定人血清中癌胚抗原

采用辣根过氧化物酶(HRP)催化鲁米诺(luminol)-H2O2化学发光体系,建立了一种测定人血清中癌胚抗原(CEA)的高灵敏度、高特异性、简便快速的微板式化学发光酶免疫分析方法。对免疫反应条件、酶结合物稀释度、发光反应时间、封闭液等进行了考察和优化。采用双抗体夹心法,室温静置1h,洗涤后加入100μL发光底物液,10min后检测。该方法的线性相关系数为0.9998;最低检出限为0.57μg/L;批内和批间变异均在10%之内;低、中、高3个不同浓度值样品的平均回收率分别为107.4%、93.3%和104.5%。使用本方法与进口发光试剂盒对40份人血清样品进行测定,结果表明,本方法显示了良好的相关性,其相关系数为0.9115。表明本分析体系稳定可靠,可用于商品化诊断试剂盒的开发和应用。     

高效液相色谱电生Mn(Ⅲ)化学发光法检测人体血清和尿样中的卡托普利

设计了一个HPLC在线电生Mn(Ⅲ)化学发光检测器, 实现在线电化学反应, 从而产生反应活性很高的初生态氧化剂Mn(Ⅲ), 并与色谱柱后CP混合产生化学发光. 同时还能够根据需要调节电极反应和发光反应两者的介质, 满足柱后发光反应的最佳环境. 在优化流动相和化学发光检测条件的基础上, 将该检测器应用于人体血清和尿液中CP的测定.     

化学发光磁酶免疫法检测O157:H7大肠埃希菌

通过自行制备的免疫磁珠结合新型发光底物(AMPPD), 改进了化学发光磁酶免疫检测法, 对人工猪肉样品中O157:H7大肠埃希菌进行了检测, 并与人工计数法进行了比较.     

抗氧化剂的毛细管电泳-间接化学发光联用在线评价方法研究

根据碱性条件下羟自由基与luminol反应会产生化学发光而抗氧化剂能够清除羟自由基从而抑制发光的原理, 结合毛细管电泳技术, 建立了一种针对多组分共存体系中抗氧化组分的在线评价的新方法. 对这种毛细管电泳-间接化学发光检测技术, 优化化学发光的各种条件, 考察了抗氧化剂硫脲和麻黄碱的抗氧化活性, 采用曲线拟合求出它们对羟自由基的半数清除浓度(IC₅₀), 得出抗氧化活性大小为麻黄碱＞硫脲, 这与荧光分光光度法的结果一致. 该方法初步应用于评价中药槐米提取物化学组分的抗氧化活性.     

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。     

基于抗原包被的微孔板式化学发光酶免疫分析法测定人尿中的雌三醇

将雌三醇-6-(O-羧甲基)肟(E3-6-CMO)与牛血清白蛋白(BSA)形成的偶联物E3-6-CMO-BSA物理吸附于聚苯乙烯微孔板孔内作为固相抗原,与雌三醇(E3)标准溶液或者水解尿样中待测E3通过竞争法进行免疫反应.以对碘苯酚增强的辣根过氧化物酶(HRP)催化鲁米诺-过氧化氢化学发光体系作为信号检测系统,建立了一种高通量、简便快速、灵敏稳定的化学发光酶免疫分析方法用于测定人尿中E3的含量.考察和优化了包被液的酸碱性、抗原包被浓度、酶标抗体稀释比例及用量、温育时间、化学发光底物用量及化学发光反应时间的影响.在最优实验条件下,方法的灵敏度为0.20ng/mL,批内和批间变异系数均在15%之内,低、中、高浓度加标水解尿样的平均回收率分别为107.9%、100.9%和91.2%.使用抗原包被法和抗体包被法同时对10份水解尿样进行测定,结果显示相关性良好,相关系数为0.9984,表明本方法可以满足临床检测的要求.     

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。     

Alkaline phosphatase assay using a near-infrared fluorescent substrate merocyanine 700 phosphate

Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that is commonly used as a conjugating enzyme in biological research. A wide variety of substrates have been developed to assay its activity. In this study, we developed an ALP assay method utilizing merocyanine 700 (MC700) based substrate MC700 phosphate (MC700p). MC700 is a near-infrared fluorescent merocyanine dye, and has excitation/emission maxima at 686 nm/722 nm in ALP assay buffer. Upon hydrolysis by ALP, MC700p is converted to MC700. The fluorescence of MC700 is dependent on the pH and detergent concentration in the buffer. The fluorescence signal produced by MC700p hydrolysis is linearly related to the ALP amount and substrate concentration. A stop solution containing EDTA could be used to stop the ALP/MC700p reaction. It was also demonstrated that MC700p could substitute pNpp as the ALP substrate in a commercial 17β-Estradiol enzyme immunoassay kit.     

Colloids engineering and filtration to enhance the sensitivity of paper-based biosensors

Paper-based biosensors represent a disruptive technology by providing instantaneous and low-cost diagnostics for health and environmental applications. The lack of sensitivity can be an obstacle for this technology to compete with traditional analytical instrumentations. Aiming to improve the sensitivity of a paper-based colorimetric biosensor, we have applied colloids engineering in combination with filtration to lower the paper substrate backgrounds and optimize the immobilization of bio-molecules on paper. A model system consisting of an enzyme, alkaline phosphatase (ALP), and an inorganic colloid, calcium carbonate (CC), flocculated by a cationic dimethylamino-ethyl-methacrylate polyacrylamide (CPAM), demonstrated that the optimized CC flocs are best for enhancing the detecting sensitivity of ALP. The CC floc structure on paper was optimized by modulating its structure in suspension. Subsequently, the filtration process and the wicking ability of paper enabled to freeze the deposited CC structure inherited from the suspension. The incorporation of biomolecules into the CC before immobilizing on paper through filtration provided not only a better microenvironment, but also a higher surface density of immobilized biomolecules. The ALP detection limit of 117 fmol per zone (5 mm circle) in the current study was fifty times lower than that of the common soaking method for biomolecule immobilization. The minimum amount of biomolecules per unit substrate area required for detection was lowered by over an order of magnitude, compared with spotting methods (i.e. inkjet printing). The improvement was also demonstrated by the steepest slope of standard curve, the lowest background, and the highest activity of the bioactive paper probed with the diluted BCIP/NBT liquid substrates.     

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.     

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.     

A New Voltammetric Enzyme Immunoassay System for the Detection of Alkaline Phosphatase

Enzymes are widely used as labels in immunoassay and especially useful at low concentrations of the analyte in the sample. Most routine detection methods are based on the formation of color or fluorescent products, which are determined by spectropho-tometry or fluorometry, respectively. Electrochemical detection has been applied to the enzyme immunoassay with the potentials of combining the low detection limits and good selectivity of electroanalytical chemistry with the specificity of immun…     

Enzymatic membranes for the selective transport of neutral molecules by electrophoresis

The active and selective transport of glucose and glycerol was carried out using electrophoresis and artificial enzymatic membranes. These positively charged composite membranes carry, on the face adjacent to the donor compartment of an electrophoresis module, a specific kinase (hexokinase or glycerokinase) and, on the opposite face, an alkaline phosphatase (ALP). Phosphorylation of the neutral substrate (glucose or glycerol) on the donor side by the kinase generates a negatively charged phosphorylated substrate, whose transmembrane migration is promoted by an electric field and by the membrane's positive charge. Dephosphorylation of the phosphorylated substrate by ALP on the opposite face regenerates the neutral substrate, which accumulates in the receiver compartment of the electrophoresis module. Using an electrophoresis module specifically designed for this study, our experiments were carried out enabling glucose and glycerol to be concentrated approximately eight- and twelve-fold, respectively, in 8 h.     

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.     

Supramolecular Tandem Assay for Pyridoxal-5′-phosphate by the Reporter Pair of Guanidinocalix[5]Arene and Fluorescein

Guanidinocalix[5]arene and fluorescein reporter pair has been chosen to set up a supramolecular tandem assay principle based on the differential recognition of pyridoxal-5′-phosphate (the substrate of alkaline phosphatase, ALP), pyridoxal (the product of ALP) and phosphate (the product of ALP). This supramolecular tandem assay system offers an opportunity to monitor the activity of ALP in a label-free, continuous, and real-time manner. More importantly, a calibration curve can be given for selective and quantitative detection of pyridoxal-5′-phosphate (biomarker for several diseases).     

Potentiometric flow-injection system for determination of alkaline phosphatase in human serum

A flow-injection system for detection of alkaline phosphatase (ALP) activity in human serum samples has been developed. As a specific and inexpensive ALP substrate for this kinetic assay monofluorophosphate (MFP) was applied. For detection of fluoride ions, generated in the course of the biocatalytic hydrolysis of MFP, conventional fluoride ion-selective electrode based on LaF₃-crystalline membrane was applied. After optimization the system allows analysis of human serum with high selectivity and relatively short time of analysis (5–6 samples h⁻¹). Volume of serum required for analysis is 0.05 mL. The system is useful for determination of the enzyme activity in human serum samples at physiological and pathological levels as well as for detection of isoenzymatic forms of ALP.