免标记荧光适体传感器的研究进展

核酸适体在治疗、诊断和生物传感等领域都引起了强烈的关注和广泛的应用。与传统的识别元素-- 抗体相比较,适体展现 出很多的优点：尺寸小,化学性质稳定,容易制备和修饰。更重要的是适体在生物传感的设计上更为灵活,因此,产生了很多高选 择性、高灵敏度的新型适体传感器。目前,很多的检测手段都被应用到适体传感器中,其中荧光的检测手段占有重要的地位。虽然 荧光适体传感器已经取得了重大的进展,但是荧光标记给传感器的设计带来很多的不便,因此,免标记的荧光适体传感器备受关 注。在本文中,我们将对免标记的荧光适体传感器的研究进展进行综述,为分析工作者发展更加灵敏、更加简单、更加应用广泛的 免标记荧光适体传感器提供依据。     

以分子信标为报告分子的凝血酶检测新方法

以分子信标为报告分子,核酸适体为识别分子,发展了一种新的凝血酶检测方法.含有分子信标互补序列的核酸适体探针与凝血酶结合后,分子信标的荧光信号下降,从而得到凝血酶的浓度信息.该方法快速、灵敏,核酸适体探针无需荧光标记、设计简单,检测限达到0.83nmol/L.     

基于信号放大技术的适体生物传感器研究进展

信号放大技术因其能实现低浓度分子检测,灵敏度高而在多个研究领域发展非常迅速。而适体作为识别分子已成功应用于多种生物传感器平台,在医疗诊断、环境检测、生化分析中显示出良好的应用前景。近年来,以适体为识别元件的生物传感器越来越受到人们的关注。综述了近3年来基于信号放大技术的适体生物传感器研究新发展。     

高灵敏荧光探针用于肿瘤 Ramos 细胞的快速检测

本文以聚苯乙烯纳米微球为载体,基于适体特异性识别和 DNA 杂交原理,组装了一种 DNA-CdTe 量子点纳米线,制备了具有较高荧光强度的复合型荧光探针,并成功用于 Ramos 细胞的荧光成像。该探针可以用于特异性识别肿瘤细胞,在荧光成像中信号强灵敏度高,为肿瘤细胞的检测提供一种新方法。     

多荧光标记引物增强甘蔗染色体寡聚核苷酸探针杂交信号

荧光原位杂交技术（fluorescence in situ hybridization, FISH）是植物分子细胞遗传学研究最为重要的手段之一。近些年，基于参考基因组设计的低拷贝寡聚核苷酸探针在FISH中应用得越来越广泛。然而，由于植物基因组中分布大量的重复序列，这使得oligo-FISH的分辨率存在一定局限性。利用包含多个荧光基团的荧光PCR引物，扩增出甘蔗染色体特异oligo探针，并进一步优化甘蔗的荧光原位杂交体系，提高了甘蔗oligo探针识别近缘物种染色体的效率。通过开发多荧光标记的甘蔗oligo探针以及甘蔗荧光杂交体系的优化，有效拓宽荧光信号的最小分辨率，提高信噪比（signal-to-noise ratio, SNR），并成功基于甘蔗oligo探针对高粱1-10号染色体分型。多荧光标记引物增强oligo探针信号的新方法及FISH体系的优化为今后在其他物种中提高oligo-FISH鉴定染色体及捕捉微弱的荧光信号提供了参考。     

基于适体技术的桔青毒素检测方法的建立

桔青毒素（citrinin,CTN）是以玉米、谷物、奶酪等为主要成分的食品和动物饲料中常见的、由桔青霉菌产生的酮类真菌毒素,可引起人和动物的慢性中毒或癌症,一直缺少灵敏的快速检测方法。本文通过指数富集适体系统进化技术（简称SELEX）对可能与CTN结合的特异性适体进行了筛选,经过15轮循环,得到17条适体。通过二级结构分析、亲和力检测发现适体13（Apt-13）对CTN有较好的亲和度,解离常数Kd为0.06μmol/L。进一步利用非荧光标记染料PicoGreen,利用其与双链DNA结合的原理,建立了桔青毒素非标记荧光检测方法,30min完成检测,最低检测限达到国家标准（50ppb）且与其他毒素无交叉反应。本研究建立的基于适体的桔青毒素检测技术成本低,可以替代传统的基于抗体的检测方法,为霉菌毒素的精准检测技术的开发提供了实验证据。     

基于荧光蛋白的荧光共振能量转移探针的构建及应用

荧光共振能量转移(fluorescence resonance energy transfer,FRET)是基于荧光基团供体和荧光基团受体间偶极子–偶极子耦合作用的非辐射方式的能量传递现象。基于荧光蛋白的FRET技术已被广泛用于研究细胞信号通路中蛋白质–蛋白质活体相互作用检测、蛋白质构象变化监测以及生物探针的研制中。基于荧光蛋白的荧光共振能量转移探针使得人们可以在时间和空间层面上研究细胞信号的转导过程。该文简要介绍了四大类基于荧光蛋白的FRET生物探针的设计、研制以及其在生物信号分子检测、活细胞成像以及药物筛选中的应用和进展情况。     

适体家族新成员－肽适体

肽适体是一种从随机氨基酸的肽文库中筛选出来,可以高亲合力地与靶物质特异性结 合的短肽序列．它的主要结构包括恒定的展示支架蛋白及通过两端限制性插入的高变肽环． 酵母双杂交技术常用于筛选针对细胞内特异性靶蛋白的肽适体过程,筛选出的肽适体通过特 异性结合识别靶标发挥类似“干扰基因”的作用从而影响蛋白的生物学活性．     

RNA适体研究及其在医药上的应用

核酸适体（nucleic acid aptamer）是从人工合成的随机单链核酸库中筛选出的特异性与靶物质高度亲和的核酸分子,包括DNA适体和RNA适体. 体外获得核酸适体的方法称为指数富集配体系统进化技术,即SELEX（systematic evolution of ligands by exponential enrichment）. 在SELEX技术获得的核酸适体中,RNA适体因其结构的多样性而具有靶分子广、亲和力高、特异性强等特点. 同时,相比传统抗体,RNA适体分子量小、易改造修饰、制备方便且无免疫原性. 因此,RNA适体在基础研究、临床诊断、药物研制等方面展现了广阔的应用前景. 本文综述了RNA适体的产生、特点、作用方式、优势与局限性,并详细介绍了其在医药研究领域的应用.     

双组分系统——细胞识别渗透胁迫信号的感应器

双组分系统是广泛存在于原核和真核细胞中的信号转导系统.主要由组氨酸蛋白激酶(HPK)和响应调节蛋白(RR)两个组分组成. 双组分系统信号通路一般包括信号的输入、HPK自身磷酸化、RR磷酸化、信号输出等环节.对双组分系统信号转导机制及其在渗透胁迫信号识别和传导中的作用进行了综述.     

Molecular signaling aptamers for real-time fluorescence analysis of protein

Aptamers are a new class of nucleic acids that are selected in vitro for binding target molecules with high affinity and selectivity. They are promising protein-binding molecular probes that rival conventional antibodies for protein analysis. There have been recent advances in the development of molecular signaling aptamers that can transduce target protein binding to sensitive fluorescence signal changes. This facilitates the real time protein monitoring in homogenous solution as well as potentially in vivo. Different signaling strategies of using dual labeled aptamers based on fluorescence resonance energy transfer (FRET), one fluorophore labeled aptamers based on fluorescence anisotropy assay, or other label-free aptamers are reviewed.     

Using azobenzene incorporated DNA aptamers to probe molecular binding interactions

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.     

Quantification of receptor targeting aptamer binding characteristics using single-molecule spectroscopy

This experimental design presents a single molecule approach based on fluorescence correlation spectroscopy (FCS) for the quantification of outer membrane proteins which are receptors to an aptamer specifically designed to target the surface receptors of live Salmonella typhimurium. By using correlation analysis, we also show that it is possible to determine the associated binding kinetics of these aptamers on live single cells. Aptamers are specific oligonucleotides designed to recognize conserved sequences that bind to receptors with high affinity, and therefore can be integrated into selective biosensor platforms. In our experiments, aptamers were constructed to bind to outer membrane proteins of S. typhimurium and were assessed for specificity against Escherichia coli. By fluorescently labeling aptamer probes and applying FCS, we were able to study the diffusion dynamics of bound and unbound aptamers and compare them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at single molecule sensitivity. The dissociation constants for these aptamer probes calculated from autocorrelation data were 0.1285 and 0.3772 nM and the respective receptor densities were 42.27 receptors per µm² and 49.82 receptors per µm². This study provides ample evidence that the number of surface receptors is sufficient for binding and that both aptamers have a high‐binding affinity and can therefore be used in detection processes. The methods developed here are unique and can be generalized to examine surface binding kinetics and receptor quantification in live bacteria at single molecule sensitivity levels. The impact of this study is broad because our approach can provide a methodology for biosensor construction and calculation of live single cell receptor‐ligand kinetics in a variety of environmental and biological applications. Bioeng. 2011; 108:1222–1227. © 2010 Wiley Periodicals, Inc.     

Assays for aptamer-based platforms

Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.     

综述与专论: 肿瘤相关碳水化合物抗原的适配体研究

肿瘤细胞异常的糖基化模式是癌症的标志，在恶性转化和癌症进展中起着至关重要的作用。不同机制导致的肿瘤相关碳水化合物抗原（tumor-associated carbohydrate antigens，TACAs）不仅是临床肿瘤学诊断中公认的生物标志物，也为治疗干预提供了特定的靶点。适配体作为抗体或凝集素的有力替代品，近年来在碳水化合物的识别中展现了潜在的应用价值。本文聚焦于癌症中异常的糖基化改变，综述了目前TACAs识别适配体的开发进展。依据适配体筛选程序中的靶标来源，阐述了针对3种类型靶标，包括糖类分子、蛋白质聚糖表位，以及血清糖类抗原的筛选策略。从筛选方法、性能指标及相关应用性方面对适配体进行了总结，并讨论了当前研究中存在的问题和未来发展方向。     

全细菌SELEX法筛选粪肠球菌特异性适配体

肠球菌（Enterococcus）是内源性和外源性医院感染的第二大病原菌,检出率仅次于大肠杆菌,从分子水平上发展靶标的高亲和力分子探针对肠球菌的识别和检测具有非常重要的意义。本研究以粪肠球菌为靶标,运用全细菌指数富集的配体系统进化技术（whole-bacteria systematic evolution of ligands by exponential enrichment, whole-bacteria SELEX）,从全长为79个核苷酸包含35个随机碱基序列的单链DNA文库中筛选与靶标高亲和力、高特异性结合的适配体,利用荧光分析法监控筛选过程中不同轮次所得次级文库与粪肠球菌的结合力,经12轮筛选和克隆测序,获得了39条适配体序列。进一步对筛选得到的适配体进行序列比对、二级结构分析、流式细胞分析、解离常数（Kd）测定及特异性验证,最终获得一条与粪肠球菌能特异性结合的适配体Apt 21,其Kd值为549.2 ± 147.4 nmol/L。该适配体可作为粪肠球菌检测的识别元件,为建立基于适配体的新型粪肠球菌检测方法奠定了基础。     

In vitro selection of signaling aptamers

Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 microM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general "switch" for transducing molecular recognition to optical signals.