纳米探针用于检测环境中重金属污染物的研究进展

重金属污染对生态环境和人类健康具有极大的危害,建立灵敏、快捷、高效的重金属检测方法具有非常重要的意义.现有的检测技术依赖大型仪器设备,在检测条件、时间以及成本上都有较高的要求,难以满足当前检测工作的需要.随着纳米技术的飞速发展,各种纳米材料不同于块体材料的优异特性被广泛开发,在化学和生物检测领域已有广泛的应用.本文主要综述了近几年来常用的几种纳米探针在重金属检测应用中的研究进展,并对各种纳米探针的特点及检测原理进行了阐述和总结.这些纳米探针包括半导体荧光量子点,荧光纳米粒子、金纳米颗粒等材料,由于他们独特的荧光特性、吸收特性、表面等离子共振(SPR)效应、表面能量转移(SET)效应等,在重金属离子检测领域有很大的应用前景.并且根据目前实际环境监测工作的需要,对基于纳米探针的检测手段进行了讨论和展望,旨在为重金属污染物检测研究的发展和进步提供参考.     

基于光学性质的纳米生物探针设计策略

纳米生物复合探针具有多功能复合、多检测路径、易于信号放大、制备简便等多种优越性。基于其优越的光学性质,人们可以利用常规光学设备实现生物检测,甚至可以实现目视检测。现就本实验室在光学纳米生物探针制备和应用的研究进展作一简要综述,所述纳米生物探针类型主要有:基于表面等离子体效应的纳米生物探针、基于量子效应的纳米生物探针和基于比表面效应的纳米生物探针,并介绍如何应用这些探针进行生物传感和生物芯片的构建。     

基于硒化镉量子点磁微球的微悬臂梁式免疫传感器片上磁分离

应用了以硒化镉量子点为荧光探针,具有磁性和抗体双重靶向功能的聚苯乙烯磁微球.设计了基于此种磁微球的新型微悬臂梁式免疫传感器,满足在液相环境中,借助嵌入到聚苯乙烯磁微球的荧光探针及微球表面的特异性抗体探针,达到生物分子的定性检测,借助具有纳米机械响应的微悬臂梁及微平面电感线圈,达到生物分子的定量检测及传感器的复用性,解决传统微悬臂梁式免疫传感器的不足.着重对三种粒径尺寸的硒化镉量子点进行了表征,同时针对片上磁分离的机理,梁上微电感线圈的结构,微磁场对磁微球的吸引进行了研究,设计并优化出满足新型微悬臂梁式免疫传感器所需的蛇形微平面电感线圈.通过生物磁分离实验,验证了设计及优化的结果,实现了用于生物分子分离的片上磁分离技术.     

磁性纳米材料的生物医学应用进展

以四氧化三铁为代表的医用磁性纳米材料具有独特的磁学性能、表面易功能化、良好的生物学相容性等特点,在纳米医学相关领域展现出巨大的应用前景,特别是近年来它作为可介导外场的智能材料,在材料设计和生物医学应用方面均取得了突破性的进展.鉴于此,本文围绕磁性氧化铁纳米材料的生物医学应用,着重介绍近年来其在磁共振影像探针、磁热和磁力效应的生物医学应用、诊疗一体化以及纳米酶催化等领域的研究进展,并对磁性纳米材料在生物医学领域未来的发展方向进行了展望.     

磁小体介导的生物传感器研究进展

磁小体(Bacterial magnetosomes,BMs)是由趋磁细菌(Magnetotactic bacteria,MTB)合成的用于在其水生栖息地中进行地磁导航的专用细胞器,由外部的脂质双层膜和内部的磁性纳米晶体组成。其具有粒径分布窄、形态均匀、单磁畴、顺磁性、比表面积大及生物相容性高等特点被广泛应用于医疗领域,可作为抗癌药物的递送载体、核磁共振成像对比剂和成像探针等。目前,基于BMs制备生物传感器并应用于生物检测技术领域的研究相对较少,且主要偏向于在BMs膜的表面共价或非共价修饰上抗体,利用抗原抗体之间的特异性免疫反应来实现靶物质的检测。综述了BMs的结构和组成、基本特性、提取和纯化、形态结构表征及其在靶物质富集方面的应用,并着重介绍了几类以BMs为基础的生物传感器如电化学生物传感器、荧光生物传感器、磁生物传感器等,讨论了磁小体介导的生物传感器在应用研究中的实际意义及其存在的问题。展望了磁小体介导的生物传感器的发展前景,面临的机遇及挑战,以期为促进磁小体介导的生物传感器的实际应用提供参考。     

纳米金探针在基因检测中的应用

传统的核酸分析中常采用放射性元素、荧光色素以及酶标记等基因探针,这些探针都存在着一些不足之处。近年来,纳米金探针作为一种新型的基因探针,己引起了广泛的关注。该探针具有优良的光谱特征和光化学稳定性,对核酸的非特异吸附性小,与核酸等生物大分子结合后不改变生物分子的活性。将纳米金探针用于基因检测,具有操作简便、快速、安全、实验成本低等优点。本文就纳米金探针的发展过程、纳米金探针的制备、检测原理及其在基因分析中的应用等几个方面作了系统而全面地概述,同时介绍了纳米金探针的最新研究进展,并对其发展前景作了简要评述。     

分子印迹技术用于食品中真菌毒素样品前处理的研究进展

真菌毒素是一类由丝状真菌天然产生的强毒性次级代谢产物,它们可通过直接或间接污染进入食物链。一旦人和动物从食物中摄入痕量真菌毒素,生命健康即可能受到严重威胁。食品样品基质复杂,真菌毒素残留含量低,且干扰物质多,导致检测难度非常大,因此,建立一种高效前处理方法对真菌毒素的检测至关重要。分子印迹聚合物由于其结构可预测性、识别特异性和广泛适用性的独特特征,应用于食品中真菌毒素样品前处理可有效简化操作流程、降低时间和经济成本,提升仪器分析的精密度和准确性。本文中,笔者从分子印迹技术的基本原理、分子印迹聚合物的制备方法、在食品中真菌毒素样品前处理的应用和发展趋势进行总结,为将分子印迹技术更好地应用于食品中真菌毒素的样品前处理领域提供参考。     

调制磁性纳米颗粒提高磁热性能的研究

磁性纳米颗粒具有独特的磁学性质,即在外加交变磁场下因产生磁滞释放热量,使其在生物医学领域,特别是肿瘤磁热疗,获得了广泛应用.到目前为止,磁性纳米颗粒介导的磁热疗成为一种治疗癌症的有效手段,已进入临床三期实验.因此,针对磁性纳米颗粒本身,优化设计尺寸、形貌、组分和表面修饰来提高其磁热性能,进而减小临床应用中的颗粒浓度来最小化毒副作用的研究,对肿瘤治疗及生物医药研究具有十分重要的意义.本综述详述如何优化调制磁性纳米颗粒以提高其磁热性能,为高效、低毒的磁性纳米颗粒的设计提供了指导性的研究方向.     

人tBID蛋白的表达纯化及其偶联的新型分子探针对前列腺癌细胞的促凋亡作用研究

目的:以PQE-30为原核表达载体,构建PQE30-tBID重组表达载体,表达和纯化目的蛋白tBID,并利用金磁纳米微粒将tBID蛋白与人HER2抗体偶联成分子探针Anti HER2-Gold Mag-tBID,以探究其对前列腺癌细胞的促凋亡作用。方法:根据tBID的基因序列设计特异性的上下游引物,利用普通PCR扩增目的基因tBID,构建重组表达载体PQE30-tBID,将其转化到BL21(DE3)中,IPTG诱导表达,经SDS-PAGE凝胶电泳和Western Blot鉴定分析,验证目的蛋白tBID的表达,并对其进行纯化。利用金磁纳米微粒与蛋白质之间的静电相互作用以及疏水相互作用,将人HER2抗体与tBID蛋白偶联在其表面,构建分子探针Anti HER2-Gold Mag-tBID。流式细胞术检测该分子探针与前列腺癌PC-3细胞的特异性亲附结合能力,通过Annexin V-FITC细胞凋亡检测试剂盒分析分子探针对PC-3细胞的促凋亡作用。结果:普通PCR扩增后得到了411 bp的DNA片段,经双酶切鉴定以及菌液测序,表明重组表达载体PQE30-tBID构建成功。促凋亡蛋白tBID成功地在大肠杆菌中表达,蛋白相对分子量约15 KD,经过纯化,得到了纯度较高的tBID蛋白。经过与金磁纳米微粒的偶联,成功构建出一种新型的分子探针Anti HER2-Gold Mag-tBID。该分子探针可与PC-3细胞特异性结合,且经Annexin V-FITC染色分析可见PC-3细胞发生明显凋亡,凋亡率达62.9%,与未处理组(3.79%)和对照组(4.33%)相比,具有显著的统计学差异。结论:PQE30-tBID重组表达载体能在大肠杆菌中高效表达,且成功得到了纯度较高的人促凋亡蛋白tBID。经金磁纳米微粒偶联,该蛋白能够与人HER2抗体重组成新型的分子探针,且能特异性地靶向前列腺癌PC-3细胞并显著促进其凋亡。     

红条毛肤石鳖齿舌牙齿内Fe3O4的研究

目的:探讨红条毛肤石鳖齿舌牙齿内磁性纳米物质(Fe3O4)的晶体结构、磁畴及磁晶各向异性等磁学性质.方法:从红条毛肤石鳖齿舌主要横向牙齿中提取出磁性纳米磁性矿物质(Fe3O4),用高分辨透射电子显微镜,扫描电子显微镜研究其外貌及晶体结构,用磁力显微镜观察其磁畴结构,然后利用超导量子干涉磁强计(SQUID)和磁转矩测量仪测量其磁化曲线,探讨它的磁各向异性.结果:①红条毛肤石鳖齿舌中的磁铁矿为长约几微米、宽约100纳米的长条片壮物质,由尺度约为50纳米的磁铁矿小晶粒及有机物质构成.②小晶粒的[111]方向沿着长条片状物质的长度方向,具有沿着厚度方向的单磁畴结构.③SQUID和磁转矩测量均证明长条片状纳米磁性矿物质的长度方向为易磁化方向.红条毛肤石鳖齿舌中的磁性矿物质与自然界中的磁铁矿具有相同的结构.结论:红条毛肤石鳖齿舌中的磁性物质为具有单畴结构、易磁化方向为[111]方向的Fe3O4晶体.     

An antibody-based microarray assay for the simultaneous detection of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript>

Advances in microsystem technology have enabled protein and nucleic acid-based microarrays to be used in various applications, including the study of diseases, drug discovery, genetic screening, and clinical and food diagnostics. Analytical methods for the detection of mycotoxins, however, remain largely based on thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or enzyme-linked Immunosorbent assay (ELISA) . The aim of our work, therefore, was to transfer an immunological assay from microtitrr plates into microarray format, in order to develop a multiparametric, rapid, sensitive and inexpensive method for the detection of mycotoxins for use in food safety applications. Microarray technology enables the fast and parallel analysis of a multitude of biologically relevant parameters. Not only nucleic acid-based tests but also peptide, antigen, and antibody assays, using different formats of microarrays, have evolved within the last decade. Antibody-based microarrays provide a powerful tool that can be used to generate rapid and detailed expression profiles of a defined set of analytes in complex samples and are potentially useful for generating rapid immunological assays of food contaminants. In this paper, we report a feasibility study of the application of antibody microarrays for the simultaneous (or independent) detection of two common mycotoxins, Aflatoxin B₁ and Fumonisin B₁. We present the development of microarray detection of aflatoxin B1 and fumonisin B1 in standard solutions with detection limits of 3 ng/ml of AFB1 and 43 ng/ml for FB1, and have developed a competitive immunoassay in microarray format for simultaneous analyses. The quality of the microarray data is comparable to data generated by microplate-based immunoassay (ELISA), but further investigations are needed in order to characterise our method more fully. We hope that these preliminary results might suggest that further research is warranted in order to develop hapten microarrays for the immunochemical simultaneous analysis of mycotoxins, as well as for other small molecules (e.g. bacterial toxins or biological warfare agents).     

基于纳米磁珠和双标记抗体的玉米赤霉烯酮快速、高灵敏检测方法的建立及应用

玉米赤霉烯酮(zeralenone,ZEN)具有雌激素活性,主要污染谷物和饲料,大量聚积可导致流产和死胎,给动物和人类健康带来严重威胁。本研究通过将ZEN偶联抗原ZEN-BSA包被于纳米磁珠(magnetic nanoparticles,MNPs),制备纳米磁珠-偶联抗原复合物(MNPs-BSA-ZEN),同时使用金颗粒(Au nanoparticles,AuNPs)和辣根过氧化物酶(horseradish peroxidase,HRP)双标记的ZEN单克隆抗体,建立新型酶联免疫检测方法(MNPs-HRP-AuNPsIC-ELISA)。检测下限(IC10)达到0.03ng/mL,检测区间(IC20–IC80)为0.05–0.89ng/mL,半数抑制率(IC50)为0.22ng/mL,与ZEN类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol和β-zearalanol)的交叉反应性依次为19.2%、11.7%、8.3%、1.2%和4.3%,与黄曲霉毒素B1、赭曲霉毒素A、伏马毒素B1、桔青霉素和展青霉毒素几乎不存在交叉反应。在玉米、面粉和大豆样本中的加标回收率可达81.6%–113.5%,与LC-MS/MS同时对天然样本中ZEN含量的检测结果表明,两种方法相关性良好。本研究建立的MNPs-HRP-AuNPs IC-ELISA具备快速和高灵敏的双重优势,也可为其他霉菌毒素精准检测技术的开发提供参考。     

A colorimetric sensor array based on natural pigments for the discrimination of saccharides

A colorimetric sensor array based on natural pigments was developed to discriminate between various saccharides. Anthocyanins, pH‐sensitive natural pigments, were extracted from fruits and flowers and used as components of the sensor array. Variation in pH, due to the reaction between saccharides and boronic acids, caused obvious colour changes in the natural pigments. Only by observing the difference map with the naked eye could 11 common saccharides be divided into independent individuals. In conjunction with pattern recognition, the sensor array clearly differentiated between sugar and sugar alcohol with highly accuracy and allowed rapid quantification of different concentrations of maltitol and fructose. This sensor array for saccharides is expected to become a promising alternative tool for food monitoring. The link between anthocyanin and saccharide detection opened a new guiding direction for the application of anthocyanins in foods.     

磁力治疗胆石症的特征和意义

根据中国医学的经络理论和现代生物磁学的原理,发明了磁力治疗胆石症,已经有20年了。它把磁穴疗法和来源于传统的中国医学的运动疗法、食物疗法结合起来。它是以磁场为主导,5个环节紧紧相扣的疗法,5个环节是磁场主导、揉耳反射、高脂餐、排石体操和淘洗大便。疗法中有些环节可以在医生的指导下病人在家中进行。本疗法预防和治疗胆石症有很好的疗效,于1987年参加了加拿大蒙特利尔市国际发明展览会。文中讨论了磁力治疗胆石症冲刷和排出胆结石的机理。     

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance as one of the most relevant Fusarium mycotoxins, the trichothecene deoxynivalenol (DON) was selected as representative. Chickens were immunised with different protein conjugates performing varying booster intervals. The antibodies were isolated by the poly(ethylene glycol) precipitation method according to Polson. By use of these antibodies an indirect competitive ELISA was developed for the detection of DON. First investigations of naturally contaminated wheat samples showed a good correspondence with results obtained by GC-ECD when calibration in blank wheat extracts was performed.     

Research progress in the detection of common foodborne hazardous substances based on functional nucleic acids biosensors

With the further improvement of food safety requirements, the development of fast, highly sensitive, and portable methods for the determination of foodborne hazardous substances has become a new trend in the food industry. In recent years, biosensors and platforms based on functional nucleic acids, along with a range of signal amplification devices and methods, have been established to enable rapid and sensitive determination of specific substances in samples, opening up a new avenue of analysis and detection. In this paper, functional nucleic acid types including aptamers, deoxyribozymes, and G-quadruplexes which are commonly used in the detection of food source pollutants are introduced. Signal amplification elements include quantum dots, noble metal nanoparticles, magnetic nanoparticles, DNA walkers, and DNA logic gates. Signal amplification technologies including nucleic acid isothermal amplification, hybridization chain reaction, catalytic hairpin assembly, biological barcodes, and microfluidic system are combined with functional nucleic acids sensors and applied to the detection of many foodborne hazardous substances, such as foodborne pathogens, mycotoxins, residual antibiotics, residual pesticides, industrial pollutants, heavy metals, and allergens. Finally, the potential opportunities and broad prospects of functional nucleic acids biosensors in the field of food analysis are discussed.     

A fluorescent magnetic nanosensor for imidacloprid based on the incorporation of polymer dots and Fe3O4 nanoparticles into the covalent organic framework

A magnetic nanoprobe was designed for imidacloprid by encapsulating nonconjugated polymer dots (NCPDs) and Fe₃O₄ nanoparticles in the covalent organic framework (COF). The fluorescence intensity of the COF-based nanocomposite is markedly suppressed by imidacloprid. As the absorption spectrum of imidacloprid was close to the band-gap of the NCPDs, and due to the presence of a nitro group (as an electron acceptor), the electrons can be easily transferred from the conduction band of NCPDs to the LUMO of imidacloprid, so fluorescence quenching was more likely to have been caused by the electron transfer process. The COF-based nanosensor was used for the determination of imidacloprid in the linear range 1.3–130 nM with a detection limit of 1.2 nM. The high sensitivity of the nanoprobe for imidacloprid is due to the combination of COF benefits (accumulation of the imidacloprid into the COF cavities) and the high adsorption ability of the Fe₃O₄ nanoparticles, which leads to further enrichment of imidacloprid. The magnetic nature of the nanocomposite enables the preconcentration and easy separation of the analyte, and so reduces matrix interference and lowers the detection limits. The practicality of this nanoprobe was confirmed by quantification of imidacloprid in the wastewater and fruit juice samples.     

Impact of food processing and detoxification treatments on mycotoxin contamination

Mycotoxins are fungal metabolites commonly occurring in food, which pose a health risk to the consumer. Maximum levels for major mycotoxins allowed in food have been established worldwide. Good agricultural practices, plant disease management, and adequate storage conditions limit mycotoxin levels in the food chain yet do not eliminate mycotoxins completely. Food processing can further reduce mycotoxin levels by physical removal and decontamination by chemical or enzymatic transformation of mycotoxins into less toxic products. Physical removal of mycotoxins is very efficient: manual sorting of grains, nuts, and fruits by farmers as well as automatic sorting by the industry significantly lowers the mean mycotoxin content. Further processing such as milling, steeping, and extrusion can also reduce mycotoxin content. Mycotoxins can be detoxified chemically by reacting with food components and technical aids; these reactions are facilitated by high temperature and alkaline or acidic conditions. Detoxification of mycotoxins can also be achieved enzymatically. Some enzymes able to transform mycotoxins naturally occur in food commodities or are produced during fermentation but more efficient detoxification can be achieved by deliberate introduction of purified enzymes. We recommend integrating evaluation of processing technologies for their impact on mycotoxins into risk management. Processing steps proven to mitigate mycotoxin contamination should be used whenever necessary. Development of detoxification technologies for high-risk commodities should be a priority for research. While physical techniques currently offer the most efficient post-harvest reduction of mycotoxin content in food, biotechnology possesses the largest potential for future developments.     

Aptamers-based assays for diagnostics, environmental and food analysis

Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a huge library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. In addition to the very important aspect of having an unlimited source of identical affinity recognition molecules available due to the selection process, aptamers can offer advantages over antibodies that make them very promising for analytical applications. The use of aptamers as therapeutic tools is nowadays well established. On the contrary, the analytical application of aptamers in diagnostic devices or in systems for environmental and food analysis, is still under investigation and the scientific community still need further research to demonstrate the advancements brought by this new kind of ligands. This review will focus on these latter applications with particular attention to the detection of food pathogens, terrorism threat agents, thrombin and cytokines.     

Non-destructive automated sampling of mycotoxins in bulk food and feed — A new tool for required harmonization

Mycotoxins contamination is highly non-uniformly distributed as is well recog-nized by the EC, by not only setting legal limits in a series of commodities, but also schedule a sampling plan that takes this heterogeneity into account. In practice however, it turns out that it is very difficult to carry out this sampling plan in a harmonised way. Applying the sampling plan to a container filled with pallets of bags (i.e. with nuts or coffee beans) varies from very laborious to almost impossible. The presented non-destructive automated method to sample bulk food could help to overcome these practical problems and to enforcing of EC directives. It is derived from a tested and approved technology for detection of illicit substances in security applications. It has capability to collect and iden-tify ultra trace contaminants, i.e. from a fingerprint of chemical substance in a bulk of goods, a cargo pallet load (~ 1000 kg) with boxes and commodities.The technology, patented for explosives detection, uses physical and chemistry processes for excitation and remote rapid enhanced release of contaminant residues, vapours and particulate, of the inner/outer surfaces of inspected bulk and collect them on selective probes. The process is automated, takes only 10 minutes, is non-destructive and the bulk itself remains unharmed. The system design is based on applicable international regulations for shipped cargo hand-ling and transportation by road, sea and air. After this process the pallet can be loaded on a truck, ship or plane. Analysis can be carried out before the cargo leaves the place of shipping. The potent application of this technology for myco-toxins detection, has been demonstrated by preliminary feasibility experiments. Aflatoxins were detected in pistachios and ochratoxin A in green coffee beans bulk. Both commodities were naturally contaminated, priory found and confirm-ed by common methods as used at routine inspections. Once the contaminants are extracted from a bulk shipment, an appropriate existing analytical method, i.e. a CEN method, can be used to measure the mycotoxins.The system, routinely in use for explosives detection, was able to screen bulk food and feed for mycotoxins, through non-destructive automated sampling of a whole batch/lot/sublot of commodities. The opportunity to sample a whole bulk would provide more effective tools for inspection at seaports, production facili-ties and distri-bution points. It will advance the current process of myco-toxins check because: (i) Checks will be automated and harmonized, (ii) Checks will be non-destructive, (iii) Checks will be faster and allow a greater amount of bulk commodities to be inspected and (iv) The ability to check, with automated equipment, larger portions of lots of a shipment will increase the probability to detect the heterogeneous mycotoxins contamination in bulk foods. The poster provides some results of feasibility experiments indicating the capability of this technology for inspection of commodities bulks for the detection of mycotoxins, at legal limits, in naturally contaminated food.