电化学限域SiN_X纳米孔道检测核酸分子的研究

固体纳米孔道因其机械强度高、尺寸可控、易于表面修饰及集成化设计等优点被广泛应用于DNA、RNA和蛋白质等生物分子的检测研究.为了检测单个单链核酸分子,本研究采用电化学刻蚀法可控制备了单个SiN_X固体纳米孔道,通过SiN_X固体纳米孔道限域空间效应增强了纳米孔道与短链核酸分子之间的弱相互作用,从而实现了核酸分子的单分子水平检测.通过研究不同孔径(3.1和8.5 nm)纳米孔道与核酸分子间的弱相互作用差异,有效区分了核酸分子在限域空间内产生的过孔和碰撞两种个体行为,加深了对固体纳米孔道限域空间内核酸分子电化学行为的理解.     

基于链置换循环无标记检测端立酶RNA的荧光法

以氧化石墨烯(GO)作为DNA载体和荧光猝灭剂,SYBRGreen Ⅰ(SGⅠ)为荧光信号探针,发夹核酸探针为分子识别探针,基于目标物启动的发夹核酸探针链置换循环反应,建立了一种利用荧光共振能量转移和链置换循环放大技术检测端粒酶RNA (hTR)的荧光新方法.发夹核酸探针hpDNA1和hpDNA2吸附在GO表面,嵌插在发夹DNA探针茎部的SG Ⅰ的荧光信号被GO猝灭.当人工合成的目标物(T1)存在时,T1与hpDNA1杂交打开hpDNA1的茎-环结构而引发hpDNA2与T1之间的链置换循环反应,由此累积产生大量的hpDNA1/hpDNA2杂交双链.刚性的双链DNA脱离GO表面,导致所嵌插的SG Ⅰ产生较强的荧光信号.基于荧光信号的变化,可定量检测0.2～50 nmoL/L的T1,检出限为90 pmol/L.该方法为端粒酶RNA检测提供了一种高灵敏、高特异性且无需标记的荧光新途径.     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

基于链置换循环无标记检测端粒酶RNA的荧光法

以氧化石墨烯(GO)作为DNA载体和荧光猝灭剂, SYBR Green Ⅰ(SGⅠ)为荧光信号探针, 发夹核酸探针为分子识别探针, 基于目标物启动的发夹核酸探针链置换循环反应, 建立了一种利用荧光共振能量转移和链置换循环放大技术检测端粒酶RNA(hTR)的荧光新方法. 发夹核酸探针hpDNA1和hpDNA2吸附在GO表面, 嵌插在发夹DNA探针茎部的SGⅠ的荧光信号被GO猝灭. 当人工合成的目标物(T1)存在时, T1与hpDNA1杂交打开hpDNA1的茎-环结构而引发hpDNA2与T1之间的链置换循环反应, 由此累积产生大量的hpDNA1/hpDNA2杂交双链. 刚性的双链DNA脱离GO表面, 导致所嵌插的SGⅠ产生较强的荧光信号. 基于荧光信号的变化, 可定量检测0.2~50 nmol/L的T1, 检出限为90 pmol/L. 该方法为端粒酶RNA检测提供了一种高灵敏、 高特异性且无需标记的荧光新途径.     

非标记型可卡因电化学适体传感器的研究

小分子物质如药物、多肽、激素和环境污染物的快速、灵敏和特异性的检测对于医学、生化和环境分析等研究具有十分重要的意义.适体是从核酸分子文库中获得的寡核苷酸片段(包括DNA和RNA),能够特异性识别目标物如蛋白、小分子物质和核酸等.     

分子信标用于p53 mRNA的体外定量检测

根据p53基因的序列设计并合成了能特异性检测p53 mRNA的分子信标(MB), 发展了一种快速定量测定细胞内总RNA提取物中p53 mRNA的方法. 采用鼻咽癌(CNE2)细胞系和经RNA干扰技术降低p53基因表达的CNE2-p53RNAi细胞系, 抽提总RNA并用MB检测, 验证了MB的检测对象是p53 mRNA. 将该方法应用于多种肿瘤细胞内p53基因表达水平的分析, 表达变化趋势与经典的mRNA分析方法RT-PCR检测结果相符. 在此基础上, 用MB对5-氟尿嘧啶(5-Fu)处理的肺腺癌细胞(A549)进行了p53 mRNA的体外定量检测, 结果表明采用MB能够快速地获知该药物对细胞内p53 mRNA表达影响的信息.     

基于核酸适配体和纳米粒子的光学探针

核酸适配体(aptamer)是一类通过指数富集的配体系统进化技术(SELEX)经体外筛选得到的单链DNA或RNA。核酸适配体借自身形成的空间结构与靶标分子特异性结合,具有靶分子广、亲和力高、特异性强、易改造修饰等特点,因而在生命科学、临床诊断、药物发现和环境科学等方面得以广泛应用。近年来,核酸适配体与纳米技术结合,并利用纳米材料在光学、磁学、电学、化学及生物学方面表现出的特殊性质,实现了对靶标分子高灵敏度、高选择性、简便快速的识别与检测。本文评述了基于核酸适配体-纳米粒子特性的光学探针在生物大分子、金属离子和有机小分子检测等领域的应用现状与发展趋势,主要包括比色法、荧光光谱法、表面增强拉曼光谱法等。     

利用无标记的分子信标及核酸染料SYBR Green Ⅰ检测乙肝病毒

本文以野生型的乙型肝炎病毒(HBV)核酸片段为研究对象,利用无标记的分子信标及核酸染料SYBR Green I,建立了一种高灵敏、高选择性的特定序列核酸检测方法.在优化条件下,目标DNA浓度为4×10-11~400×10-11 mol/L之间时,SYBR Green I的荧光强度(ΔI)与目标DNA的浓度(C)具有良好的线性关系,其拟合的回归方程为ΔI=1.9556 C+31.4659(R2=0.9956),方法检测限(3ζ)为2×10-11 mol/L.该方法操作简单、检测速度快、灵敏度高、重现性好、检出限低.利用该方法,结合不对称PCR技术,实现了对HBV的定量检测.     

核酸适配体在癌症诊断中的研究进展

核酸适配体是通过指数富集配体系统进化技术(SELEX)从体外合成的寡核苷酸文库中筛选得到的短的寡核苷酸分子(ssDNA或RNA)。核酸适配体能够通过折叠成特定的空间结构与靶标分子进行特异性结合,与抗体相比,适配体具有高亲和力、易修饰、低成本、易于合成和低免疫原性等优势,可以针对细胞、蛋白质、组织、生长因子进行癌症生物标志物的研究。作为一种新的诊断方法,其在分子诊断领域具有广阔的应用前景。本文综述了近年来核酸适配体在肺癌、胃癌、结直肠癌、乳腺癌、前列腺癌诊断中的应用研究,阐明了核酸适配体作为分子探针从细胞的检测到血清的检测所扮演的作用。     

封面说明

正随着基础生命科学和基因重组技术的发展,核酸药物研究在国际上受到更加广泛的重视.小干扰RNA(small interfering RNA,si RNA)是最具临床应用前景的核酸药物之一,它能够在细胞质中有效抑制靶m RNA,从而在更早阶段阻止疾病的发生和发展.但稳定性差、难以跨膜等问题阻碍了它在临床上的应用.核苷酸单元的结构修饰或缀合物构建,均可一定程度上提高si RNA的成药性.本期封面文章针综述     

Selection of 2′F-modified RNA aptamers against prostate-specific antigen and their evaluation for diagnostic and therapeutic applications

Currently, prostate-specific antigen (PSA) is considered to be the most sensitive marker available for prostate cancer detection and for monitoring of disease progression. In addition to its importance as a tumor marker, PSA has a role in the biological activity of cancer growth and proliferation. Therefore, the inhibition or activation of its biological activity may be used in prostate cancer therapy. Here, we describe the isolation and characterization of new 2′F-modified RNA aptamers directed against PSA. Binding studies demonstrate the ability of these new aptamers to specifically recognize their target with dissociation constants in the nanomolar range. In order to demonstrate the functionality of the selected aptamers, an apta-PCR approach was used for the quantitative detection of PSA, achieving a limit of detection of 11 nM. Furthermore, the potential use of the selected aptamers in therapeutics was demonstrated with the 2′F-modified aptamers being highly stable in human serum and having the ability to moderate the activity of PSA, which will be explored for the treatment of prostate cancer.     

Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes

Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33 nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.     

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

基于适配体和抗体的生物传感器在雌二醇检测中的应用

雌二醇(Estradiol,E2)是一种主要的环境雌激素,具有作用强、危害广等特征,可扰乱人体及动物体的正常内分泌功能,从而产生不利影响,因此E2的快速、准确检测对于食品安全和公共卫生至关重要.传统的检测方法主要是基于大型仪器分析,这些方法具有高灵敏度、准确性的同时也存在操作繁琐、成本较高、仅限于在实验室内进行等缺陷....     

Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization

A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamer–cell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinity interaction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specific binding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore no aptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result the solution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probes and aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purple solution. UV–vis spectrometry demonstrated that this hybridization-based method exhibited selective colorimetric responses to the presence or absence of target cells, which is detectable with naked eye. The linear response for MCF-7 cells in a concentration range from 10 to 10⁵ cells was obtained with a detection limit of 10 cells. The proposed method could be extended to detect other cells and showed potential applications in cancer cell detection and early cancer diagnosis.     

Aptamers targeting protein-specific glycosylation in tumor biomarkers: general selection,characterization and structural modeling

Detecting specific protein glycoforms is attracting particular attention due to its potential to improve the performance of current cancer biomarkers. Although natural receptors such as lectins and antibodies have served as powerful tools for the detection of protein-bound glycans, the development of effective receptors able to integrate in the recognition both the glycan and peptide moieties is still challenging. Here we report a method for selecting aptamers toward the glycosylation site of a protein. It allows identification of an aptamer that binds with nM affinity to prostate-specific antigen, discriminating it from proteins with a similar glycosylation pattern. We also computationally predict the structure of the selected aptamer and characterize its complex with the glycoprotein by docking and molecular dynamics calculations, further supporting the binary recognition event. This study opens a new route for the identification of aptamers for the binary recognition of glycoproteins, useful for diagnostic and therapeutic applications.

Binary recognition of the glycoprotein prostate specific antigen by aptamers: a tool for detecting aberrant glycosylation associated with cancer.     

Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance

We report a multichannel surface plasmon resonance (SPR) sensor for detection of thrombin via DNA aptamers immobilized on the SPR sensor surface. A detailed investigation of the effect of the immobilisation method on the interaction between thrombin and DNA aptamers is presented. Three basic approaches to the immobilisation of aptamers on the surface of the SPR sensor are examined: (i) immobilisation based on chemisorption of aptamers modified with SH groups, (ii) immobilisation of biotin-tagged aptamers via previously immobilized avidin, neutravidin or streptavidin molecular linkers, and (iii) immobilisation employing dendrimers as a support layer for subsequent immobilisation of aptamers. A level of nonspecific binding of thrombin to immobilized human serum albumin (HSA) for each of the immobilisation methods is determined. Immobilisation of aptamers by means of the streptavidin–biotin system yields the best results both in terms of sensor specificity and sensitivity.     

Highly sensitive detection of platelet-derived growth factor on a functionalized diamond surface using aptamer sandwich design

Aptamer-based fluorescence detection of platelet-derived growth factor (PDGF) on a functionalized diamond surface was demonstrated. In this work, a sandwich design based on the ability of PDGF to bind with aptamers at its two available binding sites was employed. It was found that this sandwich design approach significantly increases the fluorescence signal intensity, and thereby a very low detection limit of 4 pM was achieved. The effect of the ionic strength of MgCl(2) buffer solution was also investigated, and the most favourable binding for PDGF-BB occurred at a Mg(2+) concentration of 5.5 mM. Since the aptamers bind to the target PDGF with high affinity, fluorescence detection exhibited high selectivity towards different biomolecules. The high reproducibility of detection was confirmed by performing three cycles of measurements over a period of three days.     

Self‐Assembled Aptamer‐Based Drug Carriers for Bispecific Cytotoxicity to Cancer Cells

Monovalent aptamers can deliver drugs to target cells by specific recognition. However, different cancer subtypes are distinguished by heterogeneous biomarkers and one single aptamer is unable to recognize all clinical samples from different patients with even the same type of cancers. To address heterogeneity among cancer subtypes for targeted drug delivery, as a model, we developed a drug carrier with a broader recognition range of cancer subtypes. This carrier, sgc8c‐sgd5a (SD), was self‐assembled from two modified monovalent aptamers. It showed bispecific recognition abilities to target cells in cell mixtures; thus broadening the recognition capabilities of its parent aptamers. The self‐assembly of SD simultaneously formed multiple drug loading sites for the anticancer drug doxorubicin (Dox). The Dox‐loaded SD (SD–Dox) also showed bispecific abilities for target cell binding and drug delivery. Most importantly, SD–Dox induced bispecific cytotoxicity in target cells in cell mixtures. Therefore, by broadening the otherwise limited recognition capabilities of monovalent aptamers, bispecific aptamer‐based drug carriers would facilitate aptamer applications for clinically heterogeneous cancer subtypes that respond to the same cancer therapy.     

Aptamers-based sandwich assay for silver-enhanced fluorescence multiplex detection

In this work, aptamers-modified silver nanoparticles (AgNPs) were prepared as capture substrate, and fluorescent dyes-modified aptamers were synthesized as detection probes. The sandwich assay was based on dual aptamers, which was aimed to accomplish the highly sensitive detection of single protein and multiplex detection of proteins on one-spot. We found that aptamers-modified AgNPs based microarray was much superior to the aptamer based microarray in fluorescence detection of proteins. The result shows that the detection limit of the sandwich assay using AgNPs probes for thrombin or platelet-derived growth factor-BB (PDGF-BB) is 80 or 8 times lower than that of aptamers used directly. For multiplex detection of proteins, the detection limit was 625 pM for PDGF-BB and 21 pM for thrombin respectively. The sandwich assay based on dual aptamers and AgNPs was sensitive and specific.