荧光偏振免疫分析技术的研究进展

对荧光偏振免疫分析技术的研究进展作了详细的评述。介绍了荧光偏振免疫分析技术的基本原理和研究热点,评述了它在临床检验、环境与食品监测和农药残留量分析等领域中的应用,对荧光偏振免疫分析方法的优缺点作了总结,引文献45篇。     

Fluorescence Polarization Immunoassay of Kanamycin

Abstract

Kanamycin, an aminoglycoside antibiotic, is quantitated in human serum by a fluorescence polarization immunoassay employing a novel sulfonamido derivative of 2-naphthol-6-sulfonic acid.     

Fluorescence Polarization and the Aggregation of Deoxy-Hemoglobin S

Abstract

The fluorescent analogue 8-hydroxy-1,3,6-pyrene-trisulfonate is known to bind to the 2,3-diphosphoglyceric acid binding site of deoxy hemoglobin. We have used this property to monitor temperature dependent changes in concentrated solutions of deoxy hemoglobin S. By monitoring the polarization of fluorescence from the bound fluorophore, we have been able to demonstrate that the polarization of fluorescence goes through a temperature maximum analogous to that which has been observed with temperature versus viscosity measurements when following aggregation of deoxy hemoglobin S. Results of experiments with deoxy hemoglobin A and deoxy hemoglobin S suggest that difference in aggregation may be related more to quantitative differences than to qualitative differences.     

三聚氰胺单试剂荧光偏振免疫分析法

用三聚氰胺半抗原(MEL)与异硫氰酸荧光素乙二胺(EDF)反应合成了荧光示踪物MEL-EDF, 示踪物与三聚氰胺抗体反应得到稳定的单试剂免疫复合物, 考察了其结合动力学和置换动力学过程, 建立了一种三聚氰胺单试剂荧光偏振免疫分析方法(SR-FPIA). 该方法的检出限(LOD)为3.2 ng/mL, 半抑制浓度(IC₅₀)为157.1 ng/mL, 检测范围(IC₂₀~IC₈₀)为12.2~1012.4 ng/mL, 能够满足国家标准和国际食品法典委员会(CAC)对于食品中三聚氰胺最高残留限量的检测要求. 整个检测过程在15 min内完成, 并且单试剂免疫复合物在4℃下至少可稳定保存30 d以上. 本研究对开发三聚氰胺新型免疫检测方法具有重要意义.     

Corrections in the Measurement of Fluorescence Polarization

Abstract

Blank corrections in polarization measurements with single beam spectrofluorometers are straightforward and permit characterization of polarization spectra of samples having quantum yields of less than 0.01. Few samples require no such correction. Problems in determining what is a suitable blank are discussed. Such blanks have unfortunately been ignored in many studies, and dual phototube instruments have been advocated in which it is difficult to apply such corrections. G, the grating correction factor, is shown to vary sharply with emission wavelength, in contrast to other reports, but is independent of slit width. Therefore, a calibration curve of G vs. emission wavelength may be obtained for a given instrument, thus avoiding the need to determine G for each sample.     

The Effect of Sample Age and Neutron Radiation to Dynamic Nuclear Polarization Parameters in Some Asphaltene Suspensions

Nuclear magnetic resonance and the Overhauser effect type dynamic nuclear polarization experiments were performed to study suspensions of asphaltene in the xylene isomers at a low magnetic field of 1.44 mT at room temperature. Intermolecular spin-spin interactions occur between nuclear spins of hydrogen in the solvent medium and the free electron spins in the asphaltene micelles. The samples were prepared in three different asphaltene concentrations at vacuum. The samples were waited for four years and dynamic nuclear polarization parameters were found via dynamic nuclear polarization experiments performed again. Thus, it was investigated the effect of sample age to dynamic nuclear polarization parameters. In addition, the medium concentrations of each sample were exposed to radiation for 48 hours and neutron radiation effects to the dynamic nuclear polarization parameters were investigated. The results are discussed.     

A Fluorescence Polarization Study of Polyaromatic Hydrocarbons Adsorbed on Colloidal Kaolin

Abstract

The adsorption of a number of polyaromatic hydrocarbons onto aqueous colloidal kaolin was studied. The fluorescence polarization and hence the fluorescence anistropy of the hydrocarbons was measured as a function of their concentrations in aqueous solutions containing the colloid. The fluorescence anisotropy was found to be highest for dilute solutions of polyaromatic hydrocarbons, with often sharp decreases at higher concentrations. More concentrated solutions gave an isotropies similar to those of plain aqueous solutions of the fluorophores. The concentration versus fluorescence anisotropy curves and BET surface area data were used to calculate surface coverage of the colloid by the adsorbed hydrocarbons. Coverage in the range of 1% to 14% of the colloid surface was found at surface saturation for the compounds studied. This suggested that colloidal clays have the potential of transporting polyaromatic hydrocarbons through environmental waters.     

沥青质及其亚组分与烷基苯磺酸盐溶液在降低界面张力中的协同作用

利用混合溶剂沉淀法将原油中的沥青质分为4个亚组分, 研究了含有各组分的模拟油与烷基苯磺酸钠(p-S14-4, p为对二甲苯; 14表示烷基链上有14个碳原子; S表示表面活性剂; 4表示芳基在长链烷基的4号碳原子上)水溶液间的动态界面张力(DIFTs). 结果表明, 沥青质及其亚组分的分子尺寸、 浓度和极性对DIFTs有显著影响. 分子尺寸较大的沥青质亚组分与p-S14-4分子之间难以形成混合吸附膜, 协同作用较弱, DIFTs曲线呈“L”形, 最小界面张力(IFT_min)受浓度和极性的影响较小. 在高浓度时, 分子尺寸较小的沥青质及其亚组分快速扩散至界面与p-S14-4分子形成紧密的混合吸附膜, 能够快速降低界面张力(IFT); 随着时间的延长, 界面层的分子发生重排, 导致DIFTs曲线呈“V”形, 且在这种情况下沥青质及其亚组分的极性越高, 降低IFT的协同作用越明显, IFT_min越低.     

DPD Molecular Simulations of Asphaltene Adsorption on Hydrophilic Substrates: Effects of Polar Groups and Solubility

Adsorption of asphaltenes onto a polar substrate (e.g., a mineral) was modeled with dissipative particle dynamics (DPD) simulations, using continental asphaltene models. The adsorption mechanisms in 10–20% wt, of asphaltene in toluene/ heptane solutions were studied (well above the solubility limit). The structure in the adsorbed layer was highly sensitive to the presence of polar groups in the alkyl side chains and heteroatom content in the aromatic ring structure. Four types of asphaltene models were used: completely apolar (zero adsorption), apolar chains and polar heteroatoms, polar chains and no heteroatoms, and polar chains and heteroatoms (maximum adsorption). One hundred asphaltene monomers were distributed homogeneously in the solvent initially, in a ～(10 nm)³ domain.

Asphaltene monomers adsorbed irreversibly on the substrate via the polar group in the side chains, resulting in an average perpendicular orientation of the aromatic rings relative to the substrate. More frequent π–π stacking of the aromatic rings occurred for less solubility (more heptane), as in aggregates. With apolar side chains, only the heteroatoms in the aromatic ring structure had affinity to the substrate, but the ring plane did not have any preferred direction.

An important finding is that the aromatic ring assemblies “shielded” the substrate and polar groups that were anchored to the substrate, resulting in an effective non-polar surface layer seen by asphaltenes in the bulk, leading to much lower adsorption probability of the remaining asphaltenes. This “adsorption termination” effect leads to mono-layer formation. Continued adsorption with multilayering and reversible nanoaggregate adsorption occurred when both side chains in the model asphaltene (located on opposite sides of the aromatic sheet) contained polar groups, with a higher probability of exposing further polar groups to the bulk asphaltene. The general conclusion is that the number and position of the polar groups in side chains determine to a large degree the adsorption and aggregation behavior/efficiency of (continental) asphaltenes, in line with experimental evidence. The heteroatoms in the aromatic ring structure plays a more passive role in this context, only by providing organization via more π–π stacking in the adsorbed layer, and in aggregates.     

基于荧光偏振的高灵敏度蛋白激酶活性分析

利用TiO₂纳米棒与磷酸化肽的特异性相互作用,基于荧光偏振检测,建立了快速、简便的高灵敏度蛋白激酶活性分析方法. 在蛋白激酶催化作用下,荧光标记的肽底物被磷酸化,磷酸化的荧光肽通过磷酸基团特异性结合在TiO₂纳米棒表面,从而使底物肽上标记的荧光分子的旋转速率发生改变,通过对荧光偏振度进行测量,可实现蛋白激酶活性的定量检测,该方法对蛋白激酶A(PKA)的检出限可达0.0004 U·μL^-1. 此外,该方法还成功用于PKA抑制剂H-89的检测,在基于蛋白激酶抑制剂的靶向药物筛选方面具有很好的应用前景.     

19F Dynamic Nuclear Polarization and SEM in Suspensions Consisting of Fluorobenzene Derivatives and Asphaltene Extracted from MC-800 Liquid Asphalt

A variety of analytical techniques, such as scanning electron microscopy and ¹⁹F dynamic nuclear polarization (DNP) methods, are applied to characterize asphaltene extracted from MC-800 liquid asphalt in fluorobenzene derivatives at 1.53 mT and at room temperature. Different solvents show variable affinities for the asphaltene surface. The low field EPR spectrum of the asphaltene/hexafluorobenzene sample was recorded. The DNP parameters were determined. Additionally, the interactions between the nuclei of the solvent and the electrons delocalized on the asphaltene are interpreted. Not only dipolar but also scalar interactions between the nuclear spin and the electron spin were found.     

Generation of Asphaltene Deposition Envelope Using Artificial Neural Network

The usefulness of the asphaltene deposition envelope (ADE) is that no asphaltene flocculation occurs at conditions outside the envelope. Since artificial neural network (ANN) is best at identifying patterns or trends in data, it is well suited for prediction or forecasting needs. ANN is also capable of addressing case specific problems that may be encountered in the field such as deposition of asphaltene. In this article, the high pressure, high temperature setup used to perform pressure depletion experiments at three different temperatures on one the Iranian live oils and the ADE generated using ANN.     

Asphaltene Precipitation Modeling Using Fuzzy Tuning of Scaling Equations

A major concern in the petroleum industry is asphaltene precipitation, which has negative impacts on production costs and recovery. The scaling equation is the most popular approach for modeling asphaltene precipitated out of solution in crude oils. Due to different values assigned for involved coefficients in scaling equations, they might overestimate or underestimate in some region relative to each other. This study proposes an improved strategy for tuning scaling equations and compensating effects of overestimation and underestimation through fuzzy rules. This strategy, called fuzzy tuning of scaling equations (FTSE), has a parallel framework, which gains outputs of different scaling equations and then introduces them to a fuzzy model as inputs. The fuzzy model breaks down the problem into subspaces through fuzzy membership functions and solves each region separately using fuzzy rules. Aggregating results of each subspace produces final model's output (i.e., FTSE output). Results indicated that FTSE performed more satisfyingly compared with individual scaling equations performing alone.      

荧光偏振免疫分析方法分析磺胺二甲基嘧啶

建立了检测磺胺二甲基嘧啶的荧光偏振免疫分析方法。合成了3种结构不同的荧光标记物,并用薄层色谱法提纯。研究了不同结构的荧光标记物对FPIA方法灵敏度的影响。该FPIA方法在缓冲液中的检出限为1.6μg/L,半数抑制量(IC50)为41μg/L,检测范围为5~458μg/L,可以达到食品中SMZ最低残留限量的要求。研究了FPIA的动力学过程及对其它16种磺胺类药物的交叉反应,结果显示,SMZ、磺胺甲基嘧啶和磺胺二甲基恶唑的交叉反应率分别为100%、8.7%和2.4%,其它磺胺类药物的交叉反应率均低于1%。     

Smart Determination of Difference Index for Asphaltene Stability Evaluation

Precipitation and deposition of asphaltene during different stages of petroleum production is recognized as problematic in oil industry because of the increase in production cost and the inhibition of a consistent flow of crude oil in different medium. Numerous correlations have been developed to determine asphaltene stability in crude oil. In this study, a novel ONN method was used to estimate difference index from SARA fraction data for rapid, accurate, and cost-effective determination of asphaltene stability. Neural networks are highly in danger of trapping in local minima. To eliminate this flaw, a hybrid genetic algorithm-pattern search technique was used instead of common back-propagation algorithm for training the employed neural network. A comparison between neural network and optimized neural network indicated superiority of optimized neural network.      

Evaluation of Asphaltene Inhibitors Effect on Aggregation Coupled Sedimentation Process

The aggregation coupled sedimentation of asphaltene particles in toluene and heptanes mixture are investigated in the presence of two asphaltene inhibitors including, dodecyle resorcinol (DR) and hazelnut oil. An image processing technique is adopted in the three sections in a settling column setup. The aggregation and sedimentation phenomena are evaluated through a time-driven Monte Carlo (MC) model. The results are in good agreement with the obtained experimental results. The results indicate that in controlling asphaltene sedimentation DR is more effective. The results also revealed that the hazelnut oil has a capacity to inhibit asphaltene sedimentation at high concentration.     

One Mismatch Detection of DNA Hybridization Using Fluorescence Polarization

Abstract

The potential of fluorescent polarization analysis as a method for detection of mismatch DNA hybridization was investigated. The dependency of DNA hybridization rate on salt concentration was surveyed. In greater than 0.1 M NaCl, the hybridization of probe and target DNA proceeds rapidly and the reaction is complete within 3 min. Furthermore, the hybridization of probe DNA and one mismatch target DNAs was investigated. It was successfully shown that even one mismatch could be detected using fluorescence polarization analysis if the mismatch position was on the base that pairs with the probe DNA at the 5′ terminus where fluorescein isothiocyanate (FITC) is attached.     

用紫外-可见分光光度计测定油样中沥青质含量

介绍了一种用分光光度法测定油品中的沥青质含量的方法。通过测定样品悬浮液在７５０ｎｍ、８００ｎｍ下的吸光度而获得样品中正庚烷沥青质含量,阐述了方法的测量原理,讨论了取样量范围、沥青地方法的影响。结果表明,该法可应用于各种油品（如原油、重质馏分油、渣油和沥青等）的分析,具有简单、快速的优点,相对标准偏差小于３％。     

Spectroscopic Investigation of Asphaltene Aggregation in Carbon Tetrachloride Medium Containing Microquantities of Water

By fluorescence spectroscopy it has been established that coal derived asphaltene (CDA) forms aggregates in CCl₄ medium. The new feature of this compound is that even before aggregate formation it can incorporate water molecules and the critical aggregation concentration (CAC) in nonaqueous medium increases with increase in ω (= water:CDA mole ratio). The fluorescence spectroscopic determination of the equilibrium constant (K) of the molecular complex of [60] fullerenes with CDA in carbon tetrachloride medium has revealed that K increases on addition of microquantities of water. This observation has been rationalized by assuming that the nitrogenatoms at one end of CDA molecules attach themselves to the water micropool and thus producing a different conformation with a wider distance at the other end. This pre-organization further facilitates inclusion. By utilizing fluorescence variation of [60]fullerene/CDA complex in CCl₄ as continuous medium with change in ω a critical ω (=5) has been found. The formation of CDA/[60]fullerene complex has also been established by NMR spectroscopy.     

Simultaneous Determination of Fluorescein, Rhodamine 6G and Rhodamine B in Turbid Solution by Polarization Variable-Angle Synchronous Fluorescence Spectrometry

Fluorescencespectron1etry,inpractice,ishardto'beappliedtomulti-componentanalysisduetothespectraloverlapofcomplexn1ixtures.MoredVer,enVironmentalsan1plesandbiologicalsamplesareoftenturbidiand'the'directdetern1intitjodsofsuchsampleswouldsufferfromthesignificantinterferenceofscatteringlight.Synchronousfluorescencespectrol11etryisbecomingpopularconcerningitssimpIicity,selectivityandsensitivityindealingwithcomplexsamples.Onedrawbacktoconventionalconstant-wavelengthsynchronousfluorescencespectromet…