化学发光免疫分析技术及其在农药残留检测中的应用进展

化学发光免疫分析技术发展迅速,具有特异性强、灵敏度高、线性范围宽、操作简单、成本底、检测时间短、易于实现自动化等优点,在农残检测领域得到广泛应用。以化学发光免疫分析为基础,介绍了吖啶酯类、鲁米诺类、咪唑类、苯酚类及芳基草酸酯类5种化学发光剂,及化学发光免疫分析法、荧光化学发光免疫分析法、化学发光酶联免疫分析法和电化学发光免疫分析法4种常见的分析方法,重点综述了化学发光免疫分析技术在农药残留检测中的应用,并对该领域的研究前景进行了展望,旨在为农药残留检测相关研究提供参考。     

化学发光免疫分析方法的应用研究进展

化学发光免疫分析方法因其灵敏度高、特异性好、分析速度快、操作简便等优势,在临床诊断、食品检测、环境监测等领域得到了广泛应用.建立高灵敏、高特异、高通量的化学发光免疫分析方法成为近年来的研究热点和发展趋势.本文对化学发光免疫分析自2011年以来新方法及联用技术进行了综述,并对其在临床诊断及食品安全等领域的应用进行了介绍,最后对该领域的研究前景进行了展望.     

板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125

在传统的板式化学发光免疫分析法和管式磁颗粒化学发光免疫分析法基础上,建立了人血清中糖类抗原125(CA125)的板式磁颗粒化学发光免疫分析方法.该方法以磁性微粒子作为分离固相,96孔板为反应容器,辣根过氧化物酶(HRP)催化H2O2-luminol化学发光体系作为检测体系.本法测定CA125的检测灵敏度可达2.0U/mL,线性范围为0～400U/mL.与常用的包被板化学发光免疫分析方法对比,该方法检测范围宽.与管式磁颗粒化化学发光法比较,其分析灵敏度与精密度高、线性范围、分析通量以及分析成本方面均显示了很好的优越性.采用该方法对人血清中CA125进行测定并与罗氏全自动电化学发光系统的测值结果进行了比对,两者显示了良好的相关性.     

化学发光免疫技术

化学发光免疫技术是化学发光与免疫测定结合起来的一种高效检测手段,化学发光是在特定化学反应中产生的光辐射。化学发光免疫技术可根据抗原抗体标记物的不同而分为发光物免疫测定、发光酶免疫测定和发光辅助因子免疫测定三大类型。     

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。     

电化学发光免疫传感技术在生物药物分析中的研究进展

随着生物及药物分析领域的不断扩展,发展高灵敏度及高选择性的分析手段以解决复杂样品体系中低浓度待测物的分析测试问题是十分迫切的需求.电化学发光分析方法由于具有线性范围宽,灵敏度高及可控性强等优点,是处理低浓度样品的有效工具.这种方法与免疫传感技术相结合,有利于实现生物体液等复杂样品中极低含量生化物质与药物的高选择性、高灵敏度检测.本文综述了电化学发光免疫传感技术的发展状况,介绍了近年来在电化学发光免疫传感中出现的新型固相载体、电化学发光探针和共反应物、以及多组分免疫传感技术等,并对其在生物药物分析中的应用情况进行了总结.     

腐蚀接头毛细管电泳电化学发光系统用于临床药物的快速检测

毛细管电泳作为近年来发展起来的分离分析技术,以其分辨率高、分离时间短及样品试剂用量小等特点而被广泛用于环境、生物以及临床分析^[1].基于三联吡啶钌的电化学发光检测技术结合了电化学检测的微型、原位和化学发光的高灵敏,可用于胺类、醇类、DNA以及免疫分析^[2].毛细管电泳和电化学发光检测技术的结合可以成为一种低费用、低成本及简便快速的分离分析技术.     

化学发光免疫分析新进展

本文综述了2007年至今国内外化学发光免疫分析(Chemiluminescence immunoassay,CLIA)的理论研究成果及应用进展,分别从化学发光反应体系、基因工程试剂、新型固相材料、化学发光免疫分析联用检测技术以及多组分化学发光免疫检测技术等方面进行了阐述,并对化学发光免疫分析技术的发展趋势进行了展望.引用文献113篇.     

胶束微观非均相体系与化学发光能量转移

胶束微观非均相体系能提高化学发光的选择性和灵敏度,并且实验操作方便,因此其在化学发光检测技术中的应用已成为研究热点.胶束增敏化学发光是对激发量子效率,荧光量子效率以及反应的速率综合作用的结果.本文综述了正相和反相胶束体系在能量转移型化学发光体系中的应用,同时阐述了胶束与不同发光体系的相互作用.     

基于纳米颗粒的化学发光技术在炎症及肿瘤诊疗中的应用

光学技术在生物医药领域具有至关重要的作用,它不仅使生物活体的检测变得可视化,还可以提高疾病治疗的特异性和灵敏性。但是普通的光学技术具有组织穿透深度低、信噪比低和组织自发光等问题。针对这些问题,研究者开始关注并研究自发光技术,包括化学发光、生物发光、切伦科夫发光。其中,化学发光(Chemiluminescence,CL)是化学物质在进行化学反应过程中产生的一种光辐射现象,具有灵敏度高、无需外部光源激发、打破组织穿透深度限制、提高信噪比的优势,为光学成像与治疗技术的进一步发展提供了新思路。但是由于化学发光物质的疏水性等问题限制了其在生物医药领域的应用。针对这些问题,研究者开始将纳米技术与化学发光技术进行结合,不仅拓展了化学发光的应用范围,还进一步促进了疾病的诊断与治疗。在此基础上,该文首先分析了化学发光的分子机理,其次总结归纳了化学发光在炎症及肿瘤诊疗中的应用,并探讨了在实际应用中遇到的问题以及未来的发展方向。     

Development of a chemiluminescence ethanol sensor based on nanosized ZrO2

Chemiluminescence was observed on introducing ethanol vapor to the surface of nanosized ZrO2 and this effect could be utilized to design a sensor for trace ethanol detection. The term cataluminescence (CTL) was used to describe this kind of chemiluminescence because the luminescence is generated by the catalytic oxidation of organic vapors on the solid surface. The proposed sensor showed high sensitivity to ethanol at 195 degrees C. The stability of the gas sensor was demonstrated by continuous reaction with ethanol for 100 h. Quantitative analysis was performed at an optimum wavelength of 460 +/- 10 nm. The chemiluminescence intensity was proportional to the concentration of ethanol from 1.6 to 160 microg ml(-1), with a detection limit of 0.6 microg ml(-1) (signal-to-noise ratio = 3:1). The mechanism of the chemiluminescence reaction is discussed and the results show that one of the possible luminescent intermediates is acetaldehyde. The chemiluminescence on nanosized ZrO2 observed in this work demonstrates the possibility of developing new nanomaterials for low-temperature cataluminescence detection.     

基于聚苯乙烯微球放大的凝血酶化学发光检测新技术

本文以羧基96孔板为分离载体,核酸适配体作为分子特异性识别元件,聚苯乙烯微球作为放大载体,辣根过氧化物酶为标记物,构建了化学发光（CL）高灵敏度凝血酶检测新技术.实验结果表明：该放大技术不但灵敏度高,且抗干扰能力强,其他蛋白质如IgG、IgM、IgA、IgE、IFN均无明显干扰.聚苯乙烯微球放大体系中凝血酶的线性范围为7.8～250pmol/L,最低检测浓度可达3.9pmol/L;而不放大检测技术的线性范围为0.94～30nmol/L,最低检测浓度为0.46nmol/L,放大体系将检测灵敏度提高100多倍.综合而言,基于适配体识别和聚苯乙烯微球放大的凝血酶CL检测新技术具有通量大、简单快速和灵敏度高的特点,有望在凝血酶高通量检测领域获得应用.     

Luminescence-based methods for sensing and detection of explosives

The detection of explosives and related compounds is important in both forensic and environmental applications. Luminescence-based methods have been widely used for detecting explosives and their degradation products in complex matrices. Direct detection methods utilize the inherent fluorescence of explosive molecules or the luminescence generated from chemical reactions. Direct detection methods include high-energy excitation techniques such as gamma-ray and x-ray fluorescence, detection of decomposition products by fluorescence or chemiluminescence, and detection following reduction to amines or another reaction to produce fluorescent products from the explosive. Indirect detection methods utilize the interference caused by the presence of explosive compounds with traditional processes of fluorescence and fluorescence quenching. Indirect detection methods include quenching of solution-phase, immobilized, and solid-state fluorophores, displacement of fluorophores, fluorescence immunoassay, and reactions that produce fluorescent products other than the explosive. A comprehensive review of these methods is presented.     

Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical Application and Future Uses

Chemiluminescence (CL) is an important method for quantification and analysis of various macromolecules. A wide range of CL agents such as luminol, hydrogen peroxide, fluorescein, dioxetanes and derivatives of oxalate, and acridinium dyes are used according to their biological specificity and utility. This review describes the application of luminol chemiluminescence (LCL) in forensic, biomedical, and clinical sciences. LCL is a very useful detection method due to its selectivity, simplicity, low cost, and high sensitivity. LCL has a dynamic range of applications, including quantification and detection of macro and micromolecules such as proteins, carbohydrates, DNA, and RNA. Luminol-based methods are used in environmental monitoring as biosensors, in the pharmaceutical industry for cellular localization and as biological tracers, and in reporter gene-based assays and several other immunoassays. Here, we also provide information about different compounds that may enhance or inhibit the LCL along with the effect of pH and concentration on LCL. This review covers most of the significant information related to the applications of luminol in different fields.     

基于分子印迹与表面增强拉曼散射的蛋白质疾病标志物检测研究进展

异常的蛋白质表达与疾病的发生与发展密切相关, 因此蛋白质已作为疾病标志物广泛应用于疾病的早期诊断、治疗监测和预后评估. 然而, 临床样本中的蛋白质疾病标志物通常含量极低, 并存在高丰度的基质干扰, 对检测方法的特异性和灵敏度提出挑战. 目前, 蛋白质疾病标志物的检测方法主要是免疫分析. 但是, 免疫分析主要依赖抗体进行特异性识别, 而抗体具有不易制备、稳定性较差和成本高等缺点. 同时, 免疫分析常通过荧光和化学发光等技术实现高灵敏检测, 但存在操作繁琐、光漂白、光谱宽等不足. 分子印迹聚合物已发展成为在特异性和亲和力方面可媲美抗体的仿生识别材料, 且具有容易制备、稳定性好和成本低等优势. 表面增强拉曼散射技术具有超高灵敏度、光谱窄、快速、无损检测等优势而广泛应用于化学和生物分析. 近年来, 分子印迹技术和表面增强拉曼散射技术的结合产生了系列先进的蛋白质检测方法, 展现了独特的优势, 受到了广泛的关注. 本综述旨在介绍该联用分析技术的主要进展, 在分别介绍分子印迹和表面增强拉曼散射及其在蛋白质检测中单独应用的基础上, 着重介绍基于两种技术的蛋白质疾病标志物的检测方法的研究进展. 最后, 对该联用技术的未来发展做了展望.     

Bio- and chemiluminescence in bioanalysis

Analytical chemiluminescence and bioluminescence represent a versatile, ultrasensitive tool with a wide range of applications in diverse fields such as biotechnology, pharmacology, molecular biology, clinical and environmental chemistry. Enzyme activities and enzyme substrates and inhibitors can be efficiently determined when directly involved in luminescent reactions, and also when they take part in a reaction suitable for coupling to a final light-emitting reaction. Chemiluminescence detection has been exploited in the fields of flow-injection analysis and column-liquid chromatographic and capillary-electrophoretic separative systems, due to its high sensitivity when compared with colorimetric detection. It has widely been used as an indicator of reactive oxygen species formation in cells and whole organs, thus allowing the study of a number of pathophysiological conditions related to oxidative stress. Chemiluminescence represents a sensitive and rapid alternative to radioactivity as a detection principle in immunoassays for the determination of a wide range of molecules (hormones, food additives, environmental pollutants) and in filter membrane biospecific reactions (Southern, Northern, Western, dot blot) for the determination of nucleic acids and proteins. Chemiluminescence has also been used for the sensitive and specific localization and quantitation of target analytes in tissue sections and single cells by immunohistochemistry and in situ hybridization techniques. A relatively recent application regards the use of luminescent reporter genes for the development of bioassays based on genetically engineered microorganisms or mammalian cells able to emit visible light in response to specific inorganic and organic compounds. Finally, the high detectability and rapidity of bio- and chemiluminescent detection make it suitable for the development of microarray-based high throughput screening assays, in which simultaneous, multianalyte detection is performed on multiple samples.     

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing.

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol-H2O2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethyl-cellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6 x 10(-9) M and the linear dynamic range was greater than two-orders of magnitude (up to 2 x 10(-6) M).     

几种常用荧光探针的化学发光成像研究

利用双(2,4,6)三氯苯基过氧化草酸酯(TCPO)-过氧化氢(H2O2)-咪唑-荧光探针的化学发光体系,研究了荧光探针化学发光成像,对几种常用的荧光探针(丁基罗丹明、罗丹明B、罗丹明6G、荧光素及异硫氰酸荧光素等)进行了定量分析。本方法具有高灵敏度、成像分析高通量等优点,线性范围宽,检出限达10-11mol/L。对四甲基异硫氰酸罗丹明(TRITC)标记的单克隆羊抗人IgG的化学发光成像分析,比相同条件下荧光成像的检出限低一个数量级。     

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.     

A Novel Chemiluminescence Immunoassay Using Solid‐Phase Antigen for Free 17β‐Estradiol in Human Serum

A high‐performance chemiluminescence immunoassay, with long‐term durability, good precision and time‐saving, was proposed for the detection of free 17β‐estradiol (E₂) in human serum. Ninety‐six microplates were coated with bovine serum albumin conjugated E₂ antigen as solid phase for the immunoassay. The E₂‐BSA antigen coated on the microplate and the E₂ antigen in the sample competed for the binding sites on the horseradish peroxidase (HRP) labeled anti‐E₂ antibody. Chemiluminescence reaction was subsequently carried out by HRP catalyzing luminol‐H₂O₂ substrates, and the chemiluminescence intensity was inversely proportional to the amount of analyte in human sera samples. The concentration of immunoreagents, immunoreaction time, and other relevant variable conditions upon the immunoassay were studied and optimized. The proposed method exhibited detection limit as low as 5.94×10^?3 µg·L^?1 in a linear detection range from 0.01 to 1.00 µg·L^?1, good recoveries between 105% and 108%, and high precision with intra‐ and inter‐assay coefficients between 7.9% and 14.3%.