Microfluidic selection and applications of aptamers

The high affinity and specificity of aptamers make them ideal reagents for a wide range of analytical applications. It is not surprising that they are finding application in microfluidics as well. CE has proven to be an efficient technique for isolating aptamers. Aptamers have been used as affinity reagents in CE assays. Aptamer-based chromatography stationary phases have demonstrated unique selectivities. Possibly the application that holds the highest potential is aptamer microarrays for screening proteomic samples.     

Applications of aptamers for chemistry analysis,medicine and food security

Since aptamer and its in vitro selection process called SELEX were independently described by Ellington and Gold in 1990, extensive research has been undertaken and numerous isolated aptamers for various targets have been applied. Aptamers can bind to a wide range of targets that include small organic molecules, inorganic compounds, haptens and even whole cells with high binding affinity and specificity. Aptamers for a wide range of targets have been selected currently. In addition, aptamers are thermo stable and can also be regenerated easily within a few minutes denaturation, which makes them easy to store or handle. These advantages make aptamers extremely suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the recent applications of aptamers for chemistry analysis, medicine and food security, along with the future trend will be discussed.     

Aptamers: molecular tools for analytical applications

Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.     

Aptamer-based molecular recognition for biosensor development

Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production.

Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling

The development of aptamer technology considerably broadens the utility of nucleic acids as molecular recognition elements, because it allows the creation of DNA or RNA molecules for binding a wide variety of analytes (targets) with high affinity and specificity. Several recent studies have focused on developing rational design strategies for transducing aptamer-target recognition events into easily detectable signals, so that aptamers can be widely exploited for detection directed applications. We have devised a generalizable strategy for designing nonfluorescent aptamers that can be turned into fluorescence-signaling reporters. The resultant signaling probes are denoted "structure-switching signaling aptamers" as they report target binding by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and an antisense DNA oligonucleotide modified with a quencher (denoted QDNA). In the absence of the target, the aptamer hybridizes with QDNA, bringing the fluorophore into close proximity of the quencher for efficient fluorescence quenching. When this system is exposed to the target, the aptamer switches its binding partner from QDNA to the target. This structure-switching event is coupled to the generation of a fluorescent signal through the departure of QDNA, permitting the real-time monitoring of the aptamer-target recognition. In this article, we discuss the conceptual framework of the structure-switching approach, the essential features of structure-switching signaling aptamers as well as remaining challenges and possible solutions associated with this new methodology.     

核酸适配体及其在化学领域的相关应用

核酸适配体是一小段经体外筛选得到的寡核苷酸序列,能与相应的配体进行高亲和力和强特异性的结合,它的出现为化学生物学界和生物医学界提供了一种新的高效快速识别的研究平台,并在许多方面展示了良好的应用前景。本文从核算适配体的性质和体外筛选过程等方面出发,着重综述了核算适配体的化学修饰方法及其在分析化学和酶化学中应用的研究进展。     

基于Cell-SELEX的核酸适配体在生化分析与生物成像中的应用

基于Cell-SELEX的核酸适配体是指以活细胞为靶标物,通过指数富集的配基系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从人工合成的DNA/RNA文库中筛选得到的单链寡核苷酸.它能够与靶标细胞高亲和性、高特异性结合,具有分子量低、合成简单、化学稳定性好、免疫原性低、易于功能化修饰等优点,已广泛应用于生命科学研究领域.本文综述了基于Cell-SELEX技术筛选的核酸适配体在肿瘤细胞检测、分析和成像方面的研究进展,并对核酸适配体研究的发展前景和趋势进行了展望.     

Aptamer binding assays for proteins: The thrombin example—A review

Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.     

In vitro selection of histone H4 aptamers for recognition imaging microscopy

Recognition imaging microscopy is an analytical technique used to map the topography and chemical identity of specific protein molecules present in complex biological samples. The technique relies on the use of antibodies tethered to the cantilever tip of an AFM probe to detect cognate antigens deposited onto a mica surface. Despite the power of this technique to resolve single molecules with nanometer-scale spacing, the recognition step remains limited by the availability of suitable quality antibodies. Here we report the in vitro selection and recognition imaging of anti-histone H4 aptamers. In addition to identifying aptamers to highly basic proteins, these results suggest that aptamers provide an efficient, cost-effective route to highly selective affinity reagents for recognition imaging microscopy.     

Sol–Gel‐Derived Biohybrid Materials Incorporating Long‐Chain DNA Aptamers

Sol–gel‐derived bio/inorganic hybrid materials have been examined for diverse applications, including biosensing, affinity chromatography and drug discovery. However, such materials have mostly been restricted to the interaction between entrapped biorecognition elements and small molecules, owing to the requirement for nanometer‐scale mesopores in the matrix to retain entrapped biorecognition elements. Herein, we report on a new class of macroporous bio/inorganic hybrids, engineered through a high‐throughput materials screening approach, that entrap micron‐sized concatemeric DNA aptamers. We demonstrate that the entrapment of these long‐chain DNA aptamers allows their retention within the macropores of the silica material, so that aptamers can interact with high molecular weight targets such as proteins. Our approach overcomes the major limitation of previous sol–gel‐derived biohybrid materials by enabling molecular recognition for targets beyond small molecules.     

基于核酸适体的电化学生物传感器*

核酸适体是一类体外筛选的、可与目标分子高效、高特异亲合的RNA或DNA寡核苷酸片段,与常规识别分子（如抗体等）相比,核酸适体作为一类新型识别分子具有明显特色和优势,已被广泛应用于生物传感等分子识别和应用研究领域。本文就基于核酸适体的电化学生物传感器（标记型和非标记型）的近期进展作简要评述,包括适体简介、标记型（“信号衰减”型、“信号增强”型、酶标记型和纳米粒子标记型）和非标记型电化学适体生物传感器等内容。     

核酸适体在癌症诊疗中的研究进展

核酸适体是通过体外筛选技术得到的可特异性结合靶标分子的单链寡核苷酸分子探针, 其表现出与抗体相当或更优异的特异性和亲和力, 且具有靶标范围广、 免疫原性低、 易于精准制备和修饰及设计灵活可控等优势. 为癌症的早期筛查、 诊断及靶向治疗提供了全新的分子工具, 在癌症诊疗领域获得了广泛的关注与应用. 本文聚焦核酸适体在癌症诊断及治疗中的应用, 对近年来取得的研究进展进行了系统性总结, 并对未来发展方向及前景进行了展望.     

Cell-specific internalization study of an aptamer from whole cell selection

Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.     

分子印迹聚合物颗粒在毛细管电色谱中的应用

依据分子印迹技术(MIT)制备的分子印迹聚合物(MIP)颗粒对模板分子及其结构类似物具有特异性识别和选择性吸附作用,同时具有较大的比表面积和快速的传质动力学特性,因而被广泛用作液相色谱固定相和固相萃取材料。将MIP颗粒作为固定相应用于毛细管电色谱(CEC),结合了CEC的快速、高效和MIP的高亲和性、高选择性的特点,成为分析科学领域最具有发展前景的分离技术之一。MIP颗粒在CEC领域有几种不同的应用形式: 作为填充材料填充到毛细管柱中;作为嵌入材料嵌入到毛细管柱内部不同基质的骨架中;作为准固定相添加到CEC运行缓冲溶液中。本文综述了近几年MIP颗粒在CEC领域应用的发展,对该领域今后的发展前景进行了展望。     

Liquid Crystals as Stationary Phases in Chromatography

The most correct analysis of the compositions of diverse analytes mixtures is significant for analytical studies in different fields; however, many prevalent analytes cannot be identified employing traditional partition gas chromatographic methods. Thus, the increasing requirements on analytes of isomeric compounds and the problems encountered in their separation demand a study of more diverse analytical systems which are characterised by higher selectivity. Therefore, the selectivity and polarities of various liquid crystals (rod-like, banana-shape, biforked, oxygen, sulphur, nitrogen, and metal containing molecules, Schiff-base, and polymeric dendrimers) employed as liquid crystalline stationary phases (LCSPs) have been discussed from both points of views, namely, their analytical applications and thermodynamic characteristics of infinitely diluted probes with different acceptor–donor properties. Extreme particular effort has been paid to the different interdependencies between the bound up chemical structures of liquid crystal molecules with their different acceptor–donor properties and the connected resolution capabilities in the interpretation of the probe—LCSP systems, on the basis of the \( { \ln }V_{g\left( T \right)} = f\left( {\frac{1}{T}} \right) \) and \( { \ln }\left( {\frac{{a_{1} }}{{w_{1} }}} \right)^{\infty } = f\left( {\frac{1}{T}} \right) \) dependencies, with regard to the LCSP compositions, which have been controlled by the counterbalancing of the enthalpy and entropy factors. The properties of binary systems composed of liquid crystalline poly(propyleneimine) dendrimers—rod-like molecules of liquid crystals and effects of the dendrimer structure, the chemical nature, and molecular size of the non-mesogens on the ability to dissolve in the liquid crystalline phases, have been interpreted. Practical applications of metallomesogenes and chiral stationary phases for analytical separation of different organic substances have also been taken into consideration.

     

Adaptive recognition of small molecules by nucleic acid aptamers through a label-free approach

We report a novel label-free method for the investigation of the adaptive recognition of small molecules by nucleic acid aptamers using capillary electrophoresis analysis. Cocaine and argininamide were chosen as model molecules, and the two corresponding DNA aptamers were used. These single-strand DNAs folded into their specific secondary structures, which were mainly responsible for the binding of the target molecules with high affinity and specificity. For molecular recognition, the nucleic acid structures then underwent additional conformational changes, while keeping the target molecules stabilized by intermolecular hydrogen bonds. The intrinsic chemical and physical properties of the target molecules enabled them to act as indicators for adaptive binding. Thus any labeling or modification of the aptamers or target molecules were made obsolete. This label-free method for aptamer-based molecular recognition was also successfully applied to biological fluids and therefore indicates that this approach is a promising tool for bioanalysis.     

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.     

Selection and analytical applications of aptamers binding microbial pathogens

DNA aptamers specifically recognizing microbial cells and viruses have a range of analytical and therapeutic applications. This article describes recent advances in the development of aptamers targeting specific pathogens (e.g., live bacteria, whole viral particles, and virally-infected mammalian cells). Specific aptamers against pathogens have been used as affinity reagents to develop sandwich assays, to label and to image cells, to bind with cells for flow-cytometry analysis, and to act as probes for development of whole-cell biosensors. Future applications of aptamers to pathogens will benefit from recent advances in improved selection and new aptamers containing modified nucleotides, particularly slow off-rate modified aptamers (SOMAmers).     

Highly Selective Molecular Recognition of Biologically Active Substances Using Liquid Phase Separation

 The development of new chiral stationary phases has been very important in the accurate analysis of drug enantiomers and their metabolites in biological samples during drug discovery and development. New chiral stationary phases have been developed usin