核酸适配体生物传感器对心肌肌钙蛋白Ⅰ的检测研究

核酸适配体生物传感器是利用固定在电极表面的适配子与被测溶液中心肌肌钙蛋白Ⅰ(cTnⅠ)发生特异性结合,从而达到检测的目的.我们对玻碳电极进行阳极氧化、氨基化修饰,通过碳二亚胺盐酸盐(carbodiimide hydrochloride,EDC)、N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)活化作用将适配子结合在电极表面.cTnⅠ最佳检测范围是0.05~5 nmol/L,最低检测限为0.05 nmol/L,检测时间为5 min.     

核酸适配体光学生物传感器在卡那霉素检测中的研究进展

卡那霉素是一种氨基糖苷类抗生素,由于其效果好、价格低等优点,是我国常用兽药之一。但如果剂量过大就会大量残留在动物体内,进而通过食物链富集进入人体,引发耳毒性、肾毒性等毒副作用,严重时会导致人死亡,因此对其含量的检测十分重要。近几年,大量基于核酸适配体检测卡那霉素的光学方法被开发,以满足人们对其检测的需求。首先明确了核酸适配体与光学生物传感器等相关概念;再根据其反应机制不同,对光学方法介导的生物传感器进行了分类综述,阐述了各类传感器的基本原理及其检测范围;最后对这几类传感器的优缺点和发展前景进行了总结和展望,对这些方法的进一步探索将为食品安全检测提供新的技术支撑。     

核酸适配体介导的肿瘤免疫治疗研究进展

肿瘤免疫治疗是一种新的肿瘤治疗方式,在临床治疗中具有重要意义和应用前景,主要包括过继性细胞免疫治疗、肿瘤疫苗和抗体肿瘤免疫治疗。抗体在肿瘤免疫治疗中应用广泛,但其价格昂贵,质量易受不同批次的影响且存在免疫原性。核酸适配体(aptamer)是一类能与靶标高特异性高亲和力结合的短的单链寡核苷酸,素有“化学抗体”之称。核酸适配体易合成且成本低,质量稳定且免疫原性低,基于这些优势近年来也被开发用于肿瘤免疫治疗。本文综述了核酸适配体在肿瘤免疫治疗中的进展,主要包括核酸适配体通过靶向免疫检查点、共刺激受体、细胞因子、递送小干扰RNA (small interfering RNA,siRNA),间接调控肿瘤免疫进程,以及将核酸适配体修饰于细胞膜上直接介导免疫细胞靶向杀伤等。核酸适配体以多种方式在肿瘤免疫治疗中发挥作用,有潜力被开发用于临床治疗。     

核酸适配体在感染性疾病诊治中的研究进展

临床上,感染性疾病由于诊断不明或诊断时间较长导致严重后果的情况并不罕见。近年来,由于新型病原体感染报道层出不穷,现有病原体出现耐药也十分普遍,导致感染性疾病的诊断和治疗仍是临床上亟待解决的难题。核酸适配体是通过体外反复筛选或指数富集配体系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术筛选出来的一类具有特异性识别能力的寡核苷酸序列,具有靶向结合目标分子的能力,可用于病原体检测和新型治疗性药物的开发。适配体已在感染性疾病诊治中显现良好的应用前景,进一步推进相关研究有望为感染性疾病的诊治提供新的途径。     

核酸适配体结合纳米材料用于肿瘤靶向治疗

核酸适配体(aptamer)是一类由指数富集配体系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术筛选出的RNA和单链DNA寡核苷酸片段。因其非同寻常的分子识别能力及结构特性,核酸适配体已经成为最具有应用前景的生物分子之一,综述了核酸适配体结合不同的纳米材料在肿瘤靶向治疗上的应用。     

孔雀石绿适配体生物传感器的研究进展

荧光适配体作为一种无需标记的荧光探针,具有许多潜在的优势,并被应用于多种靶物质(如ATP、RNA)的检测,是目前适配体研究领域的热点。孔雀石绿适配体(malachite green aptamer,MGA)属于荧光适配体,其能通过配体诱导折叠形成结合口袋,进而促进孔雀石绿(malachite green,MG)的发光。目前,已经筛选得到的MGA的种类较少,主要介绍了已知的MG RNA适配体及其变构体和MG DNA适配体的特性,以及影响MG-MGA复合物荧光强度的因素。同时,还对主要的MG衍生物和共聚物进行了总结。最后,综述了MGA在生物传感、荧光成像等方面的应用,并对MGA的发展方向进行了展望,以期为MGA在生物检测、生物成像等方面的应用提供指导。     

发光核酸适配体的筛选及应用

发光核酸适配体(light-up nucleic acid aptamers,LNAs)是一类能够特异性结合目标分子并增强目标分子发光性能的功能核酸(functional nucleic acids,FNAs)。LNAs具有组成简单、结构稳定、表达时间短等优点,因此在细胞成像、物质检测等传感领域显示出巨大潜力。随着对细胞内基因原位分析的研究深入,现存胞内成像技术的不足逐渐显露,LNAs成像系统应运而生。本文梳理并概括了不同类型LNAs的筛选方法,并从胞内、体外两个方面对LNAs的前沿应用进行了分析,指出了当前LNAs研究以及传感系统存在的不足,展望了LNAs在无细胞传感中的巨大发展前景,以期能够促进LNAs系统成为胞内外传感研究的一件利器。     

荧光核酸适配体：核酸酶学分析新机遇

DNA和RNA的合成与降解等核酸代谢过程是维持生命体生长发育、新陈代谢、遗传变异、个体衰老等生命过程的基础代谢单元，广泛参与机体生命活动的全过程。核酸代谢相关酶活性对于维持细胞内环境的稳定性至关重要，其活性的改变可能会引起多种疾病的发生与发展。核酸代谢相关酶已成为多种疾病研究的重要靶点，也是生物技术和生物工程领域中不可或缺的重要工具，如聚合酶链反应、定点诱变、分子克隆和DNA测序等。因此，核酸代谢是所有核酸研究和相关生命科学领域研究的基础。本文将介绍核酸代谢的酶学分析常用方法，并聚焦于操作简单、实时快速的荧光法，按照荧光法的检测原理、发展历史和应用进行分类阐述与比较，并对核酸酶学分析工具未来研究与发展方向进行展望。     

Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.     

Electrochemical immunosensor of platelet-derived growth factor with aptamer-primed polymerase amplification

A new method for the determination of platelet-derived growth factor BB (PDGF-BB) was developed using an electrochemical immunosensor with an aptamer-primed, long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was immobilized on the electrode to serve as the capture antibody. The detection probe was synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by the anti-PDGF-B aptamer. In the presence of the analyte, the aptamer-primed circular probe was captured on the electrode via the formation of an antibody/PDGF-BB/aptamer sandwiched complex. The electroactivity indicator methylene blue was adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. The oxidation peak current of methylene blue in square wave voltammetric measurements showed a linear dependence on the concentration of PDGF-BB in the range from 50 to 500 ng mL⁻¹, with a detection limit as low as18 pg mL⁻¹.     

Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures

Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust.     

SELEX技术筛选纤维蛋白适配子的研究

目的:用纤维蛋白作为靶物质对ss DNA随机序列文库进行筛选,旨在获得高亲和力的纤维蛋白适配子。方法:在体外人工合成长度为99个核苷酸的ss DNA随机序列文库,文库中间区域为63个核苷酸的随机序列,两端为18个核苷酸的固定的引物序列;然后以羧基磁珠为介质包被纤维蛋白,利用指数级富集的配体系统进化技术(SELEX)对ss DNA随机序列文库进行反复筛选,当结合率不再提高时对筛选出的适配子进行连接、转化及测序分析。结果:羧基磁珠成功地包被了纤维蛋白,包被效率为87.65%,经15轮逐步递增压力的筛选,获得了纤维蛋白适配子群,经测序分析比对发现适配子有很好的多样性。结论:应用SELEX技术初步筛选出了亲和力较高的纤维蛋白适配子群,为下一步的鉴定及功能研究奠定了良好基础。     

Aptamer biosensor for label-free square-wave voltammetry detection of angiogenin

Angiogenin (Ang), one of the most potent angiogenic factor, is related with the growth and metastasis of numerous tumors. This paper presents a very simple and label-free square-wave voltammetry (SWV) aptasensor to detect angiogenin, in which an anti-angiogenin-aptamer was used as a molecular recognition element, and the couple ferro/ferricyanide as a redox probe. At the bare gold electrode, the redox couple (K₄[Fe(CN)₆]/K₃[Fe(CN)₆]) can be very easily accessed to the electrode surface to give a very strong SWV signal. At the anti-angiogenin/Au electrode surface, when angiogenin was added to the electrochemical cell, the binding of the analyte results in less availability for a redox reaction, which led to smaller SWV current. To quantify the amount of angiogenin, current suppressions of SWV peak were monitored using the redox couple of an [Fe(CN)₆]^4−/3− probe. The plot of signal suppression against the logarithm of angiogenin concentration is linear with over the range from 0.01 nM to 30 nM with a detection limit of 1 pM. The aptasensor also showed very good selectivity for angiogenin without being affected by the presence of other proteins in serum. It is the first time to use a very simple method to detect the cancer marker. Such an aptasensor opens a rapid, selective and sensitive route for angiogenin detection and provides a promising strategy for other protein detections.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

抑制RANKL诱导破骨细胞分化的DNA适配子的筛选

目的:筛选能高特异性、高亲和力结合RANKL蛋白并有效抑制RANKL对破骨细胞诱导分化作用的DNA适配子。方法:首先,采用原核系统表达并纯化RANKL蛋白,通过SELEX(Systematic evolution of ligands by exponential)技术从人工合成的单链随机寡核苷酸文库中筛选能高特异性、高亲和力结合RANKL蛋白的DNA适配子。然后,用RNAfolding sever software分析适配子空间结构,以ELISA检测DNA适配子和RANKL亲和力大小并筛选出亲和力最高的一组DNA适配子用以验证DNA适配子对RANKL诱导破骨细胞分化的抑制作用。结果:(1)成功在原核系统表达并纯化RANKL蛋白;(2)筛选出能高特异性、高亲和力结合RANKL蛋白的12个DNA适配子。(3)与对照组相比,不同浓度DNA适配子能明显抑制TRAP阳性破骨细胞数量(P0.05),且浓度越高抑制效果越明显。结论:成功筛选出的DNA适配子能特异性结合RANKL蛋白并有效抑制RANKL对破骨细胞的诱导分化功能。     

适配体介导脂质体靶向递送siRNA的研究

为探讨适配体介导的脂质体靶向递送siRNA的可行性,采用前列腺癌细胞膜表面抗原(PSMA)的适配体A10-3.2与脂质体结合,构建适配体-脂质体靶向递送体系(Apt-LP),并利用Apt-LP递送p EGFP-N1质粒和Bcl2 siRNA到前列腺癌细胞LNCa P(PSMA+)和PC-3(PSMA-),转染48h后,用荧光显微镜检测绿色荧光蛋白的表达,q PCR检测Bcl2 mRNA表达,蛋白印记法检测Bcl2蛋白表达,Hoechst 33258核染法分析体外抗肿瘤活性。结果显示:与脂质体递送体系相比,Apt-LP显著提高递送p EGFP-N1质粒和Bcl2 siRNA到靶细胞LNCa P(PSMA+)的效率;显著提高Bcl2 siRNA诱导的靶细胞LNCa P(PSMA+)Bcl2基因沉默效应,更有效的诱导靶细胞LNCa P(PSMA+)凋亡。结果表明:Apt-LP是一种有效的siRNA靶向递送体系,具有潜在的临床应用价值。     

Aptamer modification improves the adenoviral transduction of malignant glioma cells

Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy.