分子印迹技术在蛋白质分离分析中的研究进展

蛋白质结构复杂,种类多样,与各种生命活动密切相关。大部分蛋白质在生物体中含量极低,对其分析检测带来极大困难。因此实现复杂生物样品中蛋白质的选择性识别与分离,对实现蛋白质的分离分析意义重大。通过分子印迹技术制备的分子印迹聚合物含有与模板分子大小、形状一致,官能团相互匹配的三维印迹空穴,在蛋白质的选择性识别与分离领域显示出了巨大的发展潜力。但是,由于蛋白质具有尺寸较大、构型易变、结构复杂等特点,分子印迹技术在蛋白质印迹中面临着巨大挑战。该文在介绍几种新型分子印迹技术包括表面印迹、抗原决定基印迹和金属螯合物印迹的基础上,综述了近3年分子印迹技术在蛋白质分离分析方面的应用,并对其发展进行了总结与展望。     

Molecular imprinting of peptides and proteins in aqueous media

Molecular imprinting has received significant attention in recent years, as it provides a viable method for creating synthetic receptors capable of selectively recognizing specific target molecules. Despite significant growth within the field, the majority of template molecules studied thus far have been characterized by their low molecular weight and insolubility in aqueous systems. In biological systems, molecular recognition events occur in aqueous media. Therefore, in order to create molecularly imprinted polymers capable of mimicking biological processes, it is necessary to synthesize artificial receptors which can selectively recognize their respective target biological macromolecules such as peptides and proteins in aqueous media. In this review, we discuss the challenges associated with the imprinting of peptides and proteins in aqueous media. In addition, we discuss the significant progress which has been made within the field.     

分子印迹技术研究进展

分子印迹技术是制备对特定目标分子具有特异性识别能力的高分子材料的技术,所制备的高分子材料被称为分子印迹聚合物.分子印迹聚合物因具有预定性、识别性和实用性三大优点已广泛应用于分离、模拟抗体与受体、催化剂以及仿生传感器等方面和领域,显示出了广泛的应用前景.作者对分子印迹技术的发展历史、基本原理、分类、应用现状以及一些新的研究热点进行了综述.     

绿色分子印迹技术简论

解析了绿色分子印迹技术(GMIT)的概念;结合水相和其他新型分子印迹技术、聚合物辅助设计及新型原材料的发展,简要阐述GMIT的发展动向.指出在绿色化学日益深入人心的今天,有必要深入探索和发展绿色分子印迹技术,从而拓展分子印迹技术研究领域、促进绿色化学的发展.     

A novel electrosynthesized polymer applied to molecular imprinting technology

Poly(2-macaptobenzimidazole) (PMBI) films are prepared at the gold electrode surface by electropolymerization using imprinting technology and the target analyte cholesterol is used as the template. A cholesterol-selective sensor based on PMBI film was employed in conjunction with differential pulse voltammetry (DPV) and ferricyanide as mediator. Concentration of cholesterol up to 100 μM could be detected with a linear determination range up to 20 μM and a detection limit of 0.7 μM. The molecular imprinting approach offers a relatively nice selectivity for the sensor toward cholesterol with respect to common coexisting substances. The method is simple and the stability of the electrode prepared is satisfactory. The results of this research show the feasibility of using molecular imprinting methodology for preparing sensing devices for analytes that are electrochemically inactive.     

Recent advances in molecular imprinting technology: current status, challenges and highlighted applications

Molecular imprinting technology (MIT) concerns formation of selective sites in a polymer matrix with the memory of a template. Recently, molecularly imprinted polymers (MIPs) have aroused extensive attention and been widely applied in many fields, such as solid-phase extraction, chemical sensors and artificial antibodies owing to their desired selectivity, physical robustness, thermal stability, as well as low cost and easy preparation. With the rapid development of MIT as a research hotspot, it faces a number of challenges, involving biological macromolecule imprinting, heterogeneous binding sites, template leakage, incompatibility with aqueous media, low binding capacity and slow mass transfer, which restricts its applications in various aspects. This critical review briefly reviews the current status of MIT, particular emphasis on significant progresses of novel imprinting methods, some challenges and effective strategies for MIT, and highlighted applications of MIPs. Finally, some significant attempts in further developing MIT are also proposed (236 references).     

Recognition of peptides by antibodies and investigations of affinity using biosensor technology

The study of peptide-antibody interactions has many applications in biology and medicine. Synthetic peptides corresponding to single protein epitopes are used instead of intact proteins as reagents for the diagnosis of viral and autoimmune diseases. Furthermore, antibodies raised against peptides are useful reagents for isolating and characterizing gene products. In this review, methods for analysing the molecular basis of peptide-antibody interactions are described, such as amino acid replacement studies, X-ray crystallography of peptide-antibody complexes and biosensor technology based on surface plasmon resonance. The importance of peptide conformation in antibody recognition is discussed, and the antigenic reactivity of epitopes in synthetic peptides and in cognate, intact proteins is compared.     

Photochemical molecular imprinting of cholesterol

Cinnamoylated photocrosslinkable cyclodextrin derivatives (BCC) were synthesized by the substitution of β-cyclodextrin (β-CD) with cinnamoyl chloride (CC) and crosslinked with either hexamethylenediisocyanate (HMDI) or toluenediisocyanate (TDI). Cyclodextrin rings were substituted with one or two cinnamoyl moieties, as found from mass spectrometry. The polymeric matrix with cholesterol molecular imprint was obtained on irradiation of molecular assembly formed by the cinnamoyl-functionalized β-cyclodextrin-cholesterol with light at 275 nm, absorbed exclusively by the cinnamoyl chromophores. Irradiation induced crosslinking due to the photodimerization of the cinnamoyl moieties. To determine the adsorption properties of the produced material imprinting was performed in the presence of tritiated cholesterol and the intensity of β radiation from the material was measured. The materials obtained by the adsorption of tritiated cholesterol by nonirradiated polymer were used as controls. It was found that the polymer photocrosslinked in the presence of cholesterol have shown a considerable higher adsorption capacity for cholesterol than the control materials. This confirmed successful formation of molecularly imprinted polymer (MIP) by photochemical crosslinking. The selectivity of imprinting was also confirmed using compounds of similar structures, i.e. ergosterol, dehydroergosterol, and Vitamin D.     

Liquid crystal polysiloxane networks as materials for molecular imprinting technology: memory of the mesomorphic organization

A novel approach to the synthesis of molecularly imprinted polymers via non-covalent linkages has been studied. It relies on the use of thermotropic side group liquid crystal polymer networks. The polysiloxane networks obtained after extraction of the template preserved the mesomorphic organization set up in the presence of the guest molecule. A first batch rebinding analysis was performed: this study revealed that the imprinted polymer has a much greater affinity for the template molecule than has the non-imprinted polymer, and a significant selectivity.     

Liquid crystal polysiloxane networks as materials for molecular imprinting technology: memory of the mesomorphic organization

A novel approach to the synthesis of molecularly imprinted polymers via non-covalent linkages has been studied. It relies on the use of thermotropic side group liquid crystal polymer networks. The polysiloxane networks obtained after extraction of the template preserved the mesomorphic organization set up in the presence of the guest molecule. A first batch rebinding analysis was performed: this study revealed that the imprinted polymer has a much greater affinity for the template molecule than has the non-imprinted polymer, and a significant selectivity.     

基于分子印迹技术的牛血清白蛋白电位式传感器的研究

以牛血清白蛋白(BSA)为模板,合成了分子印迹聚合物凝胶(MIP),进而制得了以聚氯乙烯(PVC)为支撑膜的BSA电位式传感器。采用直接电位法测得BSA在浓度0.1～1.0 mg/mL范围内与电位呈线性关系。此电极制作简单,可用于BSA的测定。     

分子印迹技术在天然产物有效成分分离纯化中的应用

天然产物体系复杂,大分子和小分子、生命和非生命物质共存,多存在结构相近的异构体,且有效成分含量低,采用一般的分离方法富集难度较大。分子印迹技术是制备高选择性分离介质的有效技术手段,分子印迹聚合物(MIP)的选择性强,分离操作简单,在各种分离纯化中展现了良好的应用前景。本文对近年来分子印迹技术在天然产物活性组分(如生物碱、甾体、多元酚、黄酮等)的分离纯化中的应用进行了综述,并介绍了本课题组利用MIP从天然产物中分离纯化莽草酸等活性成分的研究进展。     

Nanocomposite of bimetallic nanodendrite and reduced graphene oxide as a novel platform for molecular imprinting technology

In this present work, for the first time, we are reporting a green synthesis approach for the preparation of vinyl modified reduced graphene oxide-based magnetic and bimetallic (Fe/Ag) nanodendrite (RGO@BMNDs). Herein, the RGO@BMNDs acts as a platform for the synthesis of the pyrazinamide (PZA)-imprinted polymer matrix and used for designing of the electrochemical sensor. We have demonstrated how the change in morphology could affect the electrochemical and magnetic property of nanomaterials and for this the reduced graphene oxide-based bimetallic nanoparticle (Fe/Ag) was also prepared It was found that the combination of graphene and bimetallic nanodendrites shows improvement as well as enhancement in the electrocatalytic activity and adsorption capacity, in comparison to their respective nanoparticles. The application of imprinted-RGO@BMNDs sensor was explored for trace level detection of PZA (Limit of detection = 6.65 pg L⁻¹, S/N = 3), which is a drug used for the cure of Tuberculosis. This is lowest detection limit reported so far for the detection of PZA. The sensor is highly selective, cost-effective, simple and free from any interfering effect. The real time application of the sensor was explored by successful detection of PZA in pharmaceutical and human blood serum, plasma and urine samples.     

Electrochemistry of molecular imprinting of large entities

Molecularly imprinted polymer (MIP) is a well-known approach, in which cavities with specific affinity are formed. These functional materials are used mostly for the separation, sensing, and catalysis of small molecules. In the last two decades, the MIP concept has been expanded for the imprinting of large entities such as nanoparticles, viruses, and cells. In this emerging field termed surface imprinted polymers (SIPs), a thin matrix imprints only part of the entity to enable its easy removal and rebinding.In this review, we focus on the different recent imprinting strategies for nanoparticles, viruses, and cells in conjunction with electrochemistry and describe their applications in the fields of biology, analytical chemistry, and medicine.     

分子印迹技术在蛋白质识别中的应用进展

分子印迹技术是一种制备具有分子识别能力的聚合物的有效技术,已经广泛应用于制备对小分子具有选择性的分子印迹聚合物,但制备能够特异性识别生物大分子--蛋白质的分子印迹聚合物的研究仍然具有挑战性。本文讨论了制备蛋白质分子印迹聚合物的难点,评述了目前印迹蛋白质的方法及各自的优缺点,展望了蛋白质印迹技术的发展趋势。     

Fluorescent sensing of homocysteine by molecular imprinting

The feasibility of biomimetic molecular sensing of homocysteine, an independent risk factor for cardiovascular diseases, was studied. The sensing approach coupled fluorescent derivatization of dl-homocysteine by a thiol-specific fluoro-tagging agent, N-(1-pyrenyl)maleimide, with molecular recognition by a molecularly imprinted polymer (MIP) matrix. The non-covalent MIP was fabricated using the N-(1-pyrenyl)maleimide-dl-homocysteine (PM-H) adduct as template. The PM-H-MIP was found to possess outstanding analyte-specific affinity for PM-H with binding constant, K_B, of 9.28±1.6×10⁵ M⁻¹ and density of recognition sites, B_max, of 11.9±0.8 nmol/g dried MIP. Following in situ fluorescent derivatization, luminescent response of the MIP was found to correlate linearly with concentration of dl-homocysteine in the range corresponding to realistic total homocysteine concentration in blood plasma. Besides being a passive recognition matrix for the binding of the fluoro-tagged analyte, the PM-H-MIP material was found to be able to specifically enhance the rate of derivatization reaction between dl-homocysteine and N-(1-pyrenyl)maleimide. In a sense, the MIP transformed a fluoro-tagging agent, which is generally reactive towards a broad spectrum of thiol-containing species, into a dl-homocysteine-specific derivatizing agent. The mechanism of such analyte-specific enhancement of derivatization rate and its advantages to the biomimetic molecular sensing are discussed.     

Rational design of lanthanide binding peptides

Lanthanide-binding peptides are very attractive for the design of bioprobes. Indeed, they combine the amazing properties of lanthanide ions, such as their time-resolved luminescence (Eu, Tb) or electronic relaxation (Gd) to the characteristics of the peptide scaffold, such as large solubility in water and ability to recognize biological substrates. Peptides derived from natural amino acids are reviewed in a first section. Some of their lanthanide complexes have already demonstrated their efficiency in determining protein structures and functions. Then, we will show how insertion of chelating unnatural amino acids modulates peptide-lanthanide complexes properties, such as luminescence and stability.     

Synthesis and properties of molecular imprints of darifenacin: The potential of molecular imprinting for bioanalysis

Summary A molecularly imprinted polymer has been developed which subsequently demonstrated an ability to selectively retain darifenacin (UK-88, 525-S) from aqueous acetonitrile when used as a stationary phase in HPLC columns and as a packing in solid-phase extraction cartridges. The imprinted polymer is applicable to a wide range of analytical methods including extraction from plasma, purification of radiolabelled UK-88,525, chiral separations and separation of metabolites and structural analogues. The polymer is able to extract darifenacin directly from a protein-precipitated human plasma/acetonitrile (1:1 v/v) mixture with 100% recovery. The imprinted polymer can also effect a repurification of¹⁴C-labelled darifenacin. The drawbacks of molecular imprints for ultra-trace bioanalysis (in the sub-nanogram/mL range) are discussed. These centre on the difficulty of removing all the template from the polymer and the consequent effects of template bleed on assay precision and accuracy when used as solid-phase extraction cartridges. Possible solutions to this problem are considered. Presented at: Affinity Chromatography Conference, Cambridge, UK, July 1–3, 1997.