酶联免疫分析技术研究进展

酶联免疫吸附分析(ELISA)由于检测性能上的优越性使得其在医疗卫生、环境污染控制、食品安全监测等领域得以广泛应用,并且随着其他学科新技术的引进以及ELISA本身关键技术上的突破,ELISA的分析能力较以前有了巨大进步。本文综述了近年来在提高ELISA分析的敏感性、特异性以及稳定性等方面所采取的改进策略和方法,并对其发展方向进行了展望。     

Evaluation of oriented lysozyme immobilized with monoclonal antibody

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.     

压电免疫传感器用于B因子的测定

B因子是一种不耐热的球蛋白,它参与机体的防御,在炎症过程、细胞和组织损伤中均起重要作用.B因子的检测方法常用的有单扩散法、火箭电泳法和溶血法,前者灵敏度不高,重复性差;后两者操作较复杂^[1,2].     

Protein interactions in covalently attached dextran layers

Protein interactions with polymeric carbohydrates play an important role for application in chromatography, biomaterials and biophysics. In this study, we present a detailed morphological and functional characterization of covalently side-bound dextran layers by spectroscopic ellipsometry (SE) and reflectometric interference spectroscopy (RIfS). The surface chemistry was monitored step-by-step by ellipsometric characterization of the surface loading. Dextrans of various molecular masses (10–2000 kD) were immobilized leading to surface loadings between 3 and 8 ng mm⁻². The refractive indices of the covalently attached dextran layers under atmospheric conditions (n_D=1.51) were very close to the refractive index of a spin-coated dextran layer (n_D=1.52) indicating dense and homogeneous coverage achieved by the coupling chemistry. Under buffer solution, refractive indices between 1.34 and 1.365 and thicknesses between 20 and 40 nm of these dextran layers were determined. A dextran concentration in the hydrated layers of 0.05–0.21 g cm⁻³ was estimated from the refractive index. The density and the thickness of the hydrated layers increased with molecular mass of the dextran. Non-specific binding was strongly reduced by the dextran layers and decreased with increasing thickness and density of the layer. Specific antibody binding to haptens immobilized in the dextran layer lead to an increase of both the density and the thickness of the layers. Time resolved detection by RIfS indicated significant decrease of protein mobility in the dextran layer. From these results we conclude that the functional properties of dextran layers with respect to protein interactions are determined by their effective pore-size, which is controlled by the number of bonds, the surface loading and the concentration of charged groups in the polymer.     

N,N-Diethanolaminomethyl Polystyrene: An Efficient Solid Support to Immobilize Boronic Acids

The key to the efficiency of N,N-diethanolaminomethyl polystyrene (DEAM-PS), the first solid support capable of coupling to boronic acids, is the formation of a stable, resin-bound boronic ester ate adduct (see scheme). With this resin it is now possible to efficiently immobilize a wide variety of boronic acids including functionalized ones that can be derivatized by solid-phase combinatorial synthesis.     

Dynamic Hydrogen-Bonded Zinc Adeninate Framework (ZAF) for Immobilization of Catalytic DNA

Effective immobilization and delivery of genetic materials is at the forefront of biological and medical research directed toward tackling scientific challenges such as gene therapy and cancer treatment. Herein we present a biologically inspired hydrogen-bonded zinc adeninate framework (ZAF) consisting of zinc adeninate macrocycles that self-assemble into a 3D framework through adenine-adenine interactions. ZAF can efficiently immobilize DNAzyme with full protection against enzyme degradation and physiological conditions until it is successfully delivered into the nucleus. As compared to zeolitic imidazolate frameworks (ZIFs), ZAFs are twofold more biocompatible with a significant loading efficiency of 96 %. Overall, our design paves the way for expanding functional hydrogen-bonding-based systems as potential platforms for the loading and delivery of biologics.     

A Dynamic Defect Generation Strategy for Efficient Enzyme Immobilization in Robust Metal-Organic Frameworks for Catalytic Hydrolysis and Chiral Resolution

Enzyme immobilization has been demonstrated to be a favorable protocol for promoting the industrialization of bioactive molecules, but still with formidable challenge. Addressing this challenge, we create a dynamic defect generation strategy for enzyme immobilization by using the dissociation equilibrium of metal-organic frameworks (MOFs) mediated by enzymes. Enzymes can act as “macro ligands” to generate competitive coordination against original ligands, along with the release of metal clusters of MOFs to generate defects, hence promoting the gradual transport of enzymes from the surface to inside. Various enzymes can be efficiently immobilized in MOFs to afford composites with good enzymatic activities, protective performances and exceptional reusabilities. Moreover, multienzyme bioreactors capable of efficient cascade reactions can also be generated. This study provides new opportunities to construct highly efficient biocatalysts incorporating different types of enzymes.     

A Supported Catalyst that Enables the Synthesis of Colorless CO2-Polyols with Ultra-Low Molecular Weight

Ultra-low molecular weight (ULMW) CO₂-polyols with well-defined hydroxyl end groups represent useful soft segments for the preparation of high-performance polyurethane foams. However, owing to the poor proton tolerance of catalysts towards CO₂/epoxide telomerization, it remains challenging to synthesize ULMW yet colorless CO₂-polyols. Herein, we propose an immobilization strategy of constructing supported catalysts by chemical anchoring of aluminum porphyrin on Merrifield resin. The resulting supported catalyst displays both extremely high proton tolerance (≈8000 times the equivalents of metal centers) and independence of cocatalyst, affording CO₂-polyols with ULMW (580 g mol⁻¹) and high polymer selectivity (>99 %). Moreover, the ULMW CO₂-polyols with various architectures (tri-, quadra-, and hexa-arm) can be obtained, suggesting the wide proton universality of supported catalysts. Notably, benefiting from the heterogeneous nature of the supported catalyst, colorless products can be facilely achieved by simple filtration. The present strategy provides a platform for the synthesis of colorless ULMW polyols derived from not only CO₂/epoxides, but also lactone, anhydrides etc. or their combinations.     

Single-Atom Catalysts on Covalent Triazine Frameworks: at the Crossroad between Homogeneous and Heterogeneous Catalysis

Heterogeneous single-site and single-atom catalysts potentially enable combining the high catalytic activity and selectivity of molecular catalysts with the easy continuous operation and recycling of solid catalysts. In recent years, covalent triazine frameworks (CTFs) found increasing attention as support materials for particulate and isolated metal species. Bearing a high fraction of nitrogen sites, they allow coordinating molecular metal species and stabilizing particulate metal species, respectively. Dependent on synthesis method and pretreatment of CTFs, materials resembling well-defined highly crosslinked polymers or materials comparable to structurally ill-defined nitrogen-containing carbons result. Accordingly, CTFs serve as model systems elucidating the interaction of single-site, single-atom and particulate metal species with such supports. Factors influencing the transition between molecular and particulate systems are discussed to allow deriving tailored catalyst systems.     

Enzymatic Reactions using Ionic Liquids for Green Sustainable Chemical Process; Stabilization and Activation of Lipases

The enzymatic reaction is highly respected from an environmentally-friendly point-of-view. Optimization of the reaction media and supporting materials of enzymes must be investigated in parallel with the effort to develop new enzymes. Lipases are frequently used for organic syntheses as synthetic tools even industry because of their acceptance of having a broad range of substrates, stability, and availability. We have investigated the possibility of ILs as both a solvent and activating or stabilization agent of enzymes, in particular, lipase as a model enzyme. ILs allowed recyclable use of a lipase and significant acceleration of transesterification, and also enhanced the stability and reaction activity of a lipase by immobilization through a lyophilization process. We discuss how we enhanced the enzyme capability using the IL engineering focusing on lipase-catalyzed reactions, i. e., realization of the recyclable use of an enzyme, how ILs mediated the enhanced reaction rate, and improved the stability of the enzyme.