Development of naturally selected and molecularly engineered intrachain and competitive FRET-aptamers and aptamer beacons

Several different approaches have been taken to development of homogeneous fluorescent aptamer assays including end-labeled beacons and signaling aptamers which are intrinsically quenched by nucleotides. Two new strategies dubbed "intrachain" and "competitive" FRET-aptamer assays are summarized in this review. Intrachain and competitive FRET-aptamers can be engineered on the molecular level through a series exploratory experiments involving prior knowledge of aptamer secondary or tertiary structures and hypotheses about aptamer conformational changes. However, there is an intrinsic risk of altering aptamer affinity or specificity associated with chemical modifications of an aptamer. Natural selection methods for FRET-aptamers have also been devised to potentially obviate the chemical modification problem. The naturally selected aptamers are subjected to fluorophore (F)- and or quencher (Q)-conjugated nucleotide triphosphate (NTP) incorporation by polymerase chain reaction (PCR) with permissive polymerases such as Deep Vent exo-, but still demonstrate sensitive and specific assay performance despite modified bases, because they are ultimately selected after decoration with F and Q. This paper summarizes work in this area and presents some new examples of the engineered and naturally selected FRET-aptamers for detection of vitamin D.     

ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene

A simple and sensitive method for ATP detection using a label-free DNA aptamer as the recognition element and ethidium bromide (EB) as the signal reporter is reported. The ATP-binding aptamer undergoes a conformational switch from the aptamer duplex to the aptamer/target complex upon target binding, which induces the fluorescence change of intercalated EB emission. Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between the ATP aptamer and ATP. Using EB alone as a signal reporter, the ATP detection limit was estimated to be approximately 0.2 mM. When a light harvesting cationic tetrahedralfluorene was used as an energy donor to sensitize the intercalated EB emission, a 10-fold increase in detection limit and a 2-fold increase in detection selectivity was demonstrated. The sensitivity and selectivity of the tetrahedralfluorene sensitized assay is comparable to or better than most fluorescent ATP assays with multiple labels.     

Ultrasensitive Detection and Binding Mechanism of Cocaine in an Aptamer‐based Single‐molecule Device

Aptamer serves as a potential candidate for the micro‐detection of cocaine due to its high specificity, high affinity and good stability. Although cocaine aptasensors have been extensively studied, the binding mechanism of cocaine‐aptamer interactions is still unknown, which limits the structural refinement in the design of an aptamer to improve the performance of cocaine aptasensors. Herein, we report a label‐free, ultrasensitive detection of single‐molecule cocaine‐aptamer interaction by using an electrical nanocircuit based on graphene‐molecule‐ graphene single‐molecule junctions (GMG‐SMJs). Real‐time recordings of cocaine‐aptamer interactions have exhibited distinct current oscillations before and after cocaine treatment, revealing the dynamic mechanism of the conformational changes of aptamer upon binding with cocaine. Further concentration‐dependent experiments have proved that these devices can act as a single‐molecule biosensor with at least a limit of detection as low as 1 nmol?L^–1. The method demonstrated in this work provides a novel strategy for shedding light on the interaction mechanism of biomolecules as well as constructing new types of aptasensors toward practical applications.     

Acoustic quantification of ATP using a quartz crystal microbalance with dissipation

A quartz crystal microbalance with a dissipation monitoring (QCM-D) sensor was developed for highly sensitive and specific detection of adenosine-5'-triphosphate (ATP) by using an aptamer. The binding of ATP molecules on the aptamer films could be calculated as accurate mass changes using multiple frequency and dissipation measurements. The detection is achieved by calculating the mass changes from conformational rearrangements of the sensor surface upon interaction with the target. The sensor was demonstrated to respond to changes in ATP concentrations in real time suitable for continuous monitoring applications. This sensor showed excellent selectivity toward ATP compared with other chemically similar nucleotide GTP. The feasibility of the sensor was demonstrated by analyzing ATP concentrations in cell culture media with serum. The maximum frequency change was about -2 Hz after injection of 500 μM ATP. The affinity constant of the aptamer was determined to be 49 ± 7.59 μM. The proposed sensor can extend the application of the QCM-D system in medical diagnosis, and could be adopted for the detection of other small molecules with the use of specific aptamers.     

Optical sensors based on hybrid DNA/conjugated polymer complexes

Single-stranded DNA (ss-DNA) can specifically bind to various targets, including a complementary ss-DNA, ions, proteins, drugs, and so forth. When binding takes place, the oligonucleotide probe often undergoes a conformational transition. This conformational change of the negatively charged ss-DNA can be detected by using a water-soluble, cationic polythiophene derivative, which transduces the complex formation into an optical (colorimetric or fluorometric) signal without any labeling of the probe or the target. This simple and rapid methodology has enabled the specific and sensitive detection of nucleic acids and human thrombin. This new biophotonic tool can easily be applied to the detection of various other biomolecules and is also useful in the high-throughput screening of new drugs.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

聚集诱导发光分子/适配体/外切酶Ⅰ体系对可卡因的检测

<正>目前非法药物的滥用已经成为全球性的公共安全卫生问题之一[1～3].其中可卡因作为一种全球禁用的非法药物,长期滥用会对人体造成许多不良的影响,如精神疾病、失眠、抑郁和暴力倾向等,甚至威胁生命,同时,吸食可卡因还会导致出现各种社会问题[4,5].因此实现对可卡因的快速检测成为打击     

Cationic conjugated polyelectrolytes-triggered conformational change of molecular beacon aptamer for highly sensitive and selective potassium ion detection

We demonstrate highly sensitive and selective potassium ion detection against excess sodium ions in water, by modulating the interaction between the G-quadruplex-forming molecular beacon aptamer (MBA) and cationic conjugated polyelectrolyte (CPE). The K(+)-specific aptamer sequence in MBA is used as the molecular recognition element, and the high binding specificity of MBA for potassium ions offers selectivity against a range of metal ions. The hairpin-type MBA labeled with a fluorophore and quencher at both termini undergoes a conformational change (by complexation with CPEs) to either an open-chain form or a G-quadruplex in the absence or presence of K(+) ions. Conformational changes of MBA as well as fluorescence (of the fluorophore in MBA) quenching or amplification via fluorescence resonance energy transfer from CPEs provide clear signal turn-off and -on in the presence or absence of K(+). The detection limit of the K(+) assays is determined to be ~1.5 nM in the presence of 100 mM Na(+) ions, which is ~3 orders of magnitude lower than those reported previously. The successful detection of 5'-adenosine triphosphate (ATP) with the MBA containing an ATP-specific aptamer sequence is also demonstrated using the same sensor scheme. The scheme reported herein is applicable to the detection of other kinds of G-rich aptamer-binding chemicals and biomolecules.     

适配体生物传感器在病原微生物检测中的应用

适配体是通过指数富集系统进化技术(SELEX)体外筛选得到的一类能够特异性地结合小分子物质、蛋白,甚至整个细胞的寡核苷酸序列.由于具有制备简便、易于修饰、稳定性好等特点,适配体已广泛应用于构建生物传感器,实现对病原微生物的识别和检测.本文在阐述适配体基本原理的基础之上,结合近年来病原微生物适配体研究领域的最新研究成果,综述以病原微生物为目标的适配体筛选技术的最新进展;列举目前已经筛选获得的病原微生物(原生生物、病毒、细菌)适配体;综述适配体生物传感器在病原微生物检测中的应用.并展望了适配体生物传感器在病原微生物检测领域的发展趋势.     

Sensitive detection of human breast cancer cells based on aptamer–cell–aptamer sandwich architecture

Breast cancer is one of the most critical threats to the health of women, and the development of new methods for early diagnosis is urgently required, so this paper reports a method to detect Michigan cancer foundation-7 (MCF-7) human breast cancer cells with considerable sensitivity and selectivity by using electrochemical technique. In this method, a mucin 1 (MUC1)-binding aptamer is adopted to recognize MCF-7 human breast cancer cells, while enzyme labeling is employed to produce amplified catalytic signals. The molecular recognition and the signal amplification are elaborately integrated by fabricating an aptamer–cell–aptamer sandwich architecture on an electrode surface, thus a biosensor for the detection of MCF-7 is fabricated based on the architecture. The detection range can be from 100 to 1 × 10⁷ cells, and the detection limit can be as low as 100 cells. The method is also cost-effective and conveniently operated, implying potential help for the development of early diagnosis of breast cancer.     

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

RE-SELEX is the first homogenous method for in vitro evolution of structure-switching DNA aptamers.     

基于聚合酶反应和发夹型核酸适体的蛋白质荧光检测新方法

设计合成了一种发夹型核酸适体(Aptamer), 结合聚合酶反应建立了蛋白质荧光分析新方法. 该核酸适体同时作为蛋白质配体和聚合反应模板, 与靶蛋白特异结合后, 其构象发生了变化, 启动聚合反应, 从而在未直接标记核酸适体的情况下, 通过监测聚合反应进程来检测蛋白质的浓度. 采用该方法检测凝血酶的线性范围为0.5~8 nmol/L, 检测下限为0.5 nmol/L, 为蛋白质检测提供了一种简便快速的非直接标记的荧光分析方法, 有望在蛋白质组学的研究中得到广泛的应用.     

Ultrasensitive detection based on an aptamer beacon electron transfer chain

The construction of an ultrasensitive, reagentless, target label free electrochemical aptamer sensor (aptasensor) for thrombin detection is described. The aptasensor is based on a chronoamperometric beacon system for biomolecular recognition. The ferrocene-labeled aptamer adopts a 3-D conformational change when interacts with thrombin. Thus the ferrocene label is approached to the microperoxise-11 (MP-11) attached on the electrode surface. The thrombin–aptamer interaction is detected via a microperoxidase mediated electron transfer between the ferrocene and the surface. This system was demonstrated with surface plasmon resonance, impedance spectroscopy and chronoamperometry measurements, obtaining higher sensitivity (30 fM) with impedance spectroscopy.     

Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection

To our knowledge, we report the first fluorescence aptasensor for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand (SS) complementary to part of the aptamer sequence or the loop of molecular beacon (MB). The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensor can detect as little as 0.34 nM of HNE, and the response is linear in the tested concentration range of 0.34-68 nM with the detection limit of 47 pM.     

Aptamer–Target Interaction: A Comprehensive Study by Microchip Electrophoresis in Frontal Mode

An original methodology based on microchip electrophoresis in a continuous frontal analysis mode (FACMCE) was employed to provide new insights into the interaction between an aptamer and its target (lysozyme), i.e., the influence of various experimental conditions on possible conformation change of the aptamer. The parameters evaluated were: background electrolyte (BGE) nature and ionic strength, nature and concentration of an added divalent cation, use of an aptamer thermal treatment, conditions classically used when employing aptamers. Increasing the BGE ionic strength led to a decrease in the dissociation constant highlighting the role played by the non-ionic interactions, and a decrease in the number of binding sites due to a change of binding mode and/or an amplification of selectivity. Divalent cation addition in the BGE improved the binding affinity. We demonstrated that this is not exclusively related to an increase in ionic strength but also certainly to an aptamer conformational change. Furthermore, changing the BGE nature permitted to modulate the binding parameters. Finally, we showed that an initial heating of the aptamer solution has been proved critical to stabilize the optimal conformation and thus to get a high binding affinity as well as a smaller standard deviation on the binding parameters values. This study has evidenced the influence of a wide range of parameters so as to better grasp the target/aptamer interaction, and thus highlight some phenomena involved in such an interacting system. This work will therefore help for further applications of this binding system for selectivity improvement in bioanalytical development.     

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.

     

Aptamer-based electrochemical biosensor for <Emphasis Type="Italic">Botulinum</Emphasis> neurotoxin

We have developed an aptamer-based electrochemical sensor for detection of Botulinum neurotoxin, where steric hindrance is applied to achieve specific signal amplification via conformational change of the aptamer. The incubation time and potassium concentration of the reaction buffer were found to be key parameters affecting the sensitivity of detection of the recognition of Botulinum neurotoxin by the aptamer. Under optimized experimental conditions, a high signal-to-noise ratio was obtained within 24 h with a limit of detection (LOD) of 40 pg/ml by two standard deviation cutoffs above the noise level.     

An Impedimetric Aptasensor Based on a Novel Line‐Pad‐Line Electrode for the Determination of VEGF165

In this work, a novel, simple and label‐free line‐pad‐line electrode (LPLE) biosensor was developed for detection of vascular endothelial growth factor (VEGF₁₆₅). DNA aptamer was used as a recognition element for high specificity to VEGF₁₆₅, and original LPLE as the substrate electrode for high sensitivity of the biosensor. This sensor was prepared by immobilizing anti‐VEGF₁₆₅ aptamers on the LPLE surface through gold‐sulfur (Au?S) bonding. Upon the addition of VEGF₁₆₅, a large target‐induced conformational change in the surface‐immobilized aptamer was generated and caused variations in the interfacial properties,which led to a corresponding increase in the impedance magnitude of the LPLE. Finally, our results demonstrate that the calibration curve for VEGF₁₆₅determination was linear over the range of 0.026‐31.4 fM with a detection limit as low as 0.017 fM.Additionally, our sensor was fabricated on printed circuit board (PCB) with a new electrode construction, and can potentially be implemented with the advantages of simplicity, low‐cost and easy mass production. Besides, considering its desirable sensitivity and specificity, the proposed use of LPLE provided a promising strategy for a wide variety of sensing applications.