Aptamer Recognition Research Articles

An aptamer-based colorimetric sensing of acetamiprid in environmental samples: Convenience, sensitivity and practicability

Convenient and practical detection of the ultra-low concentration of single-component pesticide residue in real environmental samples is crucial for the assessment of environmental risks caused by this pesticide. Herein, a simple and rapid colorimetric sensor for the detection of acetamiprid in real environmental samples and agricultural products was constructed by the direct and sensitive response of positively charged gold nanoparticles ((+) AuNPs) to the aptamer structure change induced by acetamiprid. (+) AuNPs’ response to target is direct and sensitive with no need of the salt-induced process. The affinity interaction between aptamer and target is carried out in an isolated and optimized system without interference. Consequently, this colorimetric sensor has high sensitivity. Even with the naked eye, acetamiprid as low as 7.5 × 10−8 M was observed to cause the color change of (+) AuNPs within minutes. The detection limit of the colorimetric sensor was calculated to be 5.6 × 10-10 M, which was about one order of magnitude lower than that in (-) AuNPs system and far lower than that of other colorimetric methods. Moreover, it was found that the high specificity of aptamer recognition could withstand the complexity of real samples. Compared with the standard HPLC method, the detection results of the colorimetric analysis have excellent accuracy and reliability. In particular, the processing of the real sample in the colorimetric sensor is much simpler and does not require professional chromatographic separation.

Aptamer recognition and proximity-induced entropy-driven circuit for enzyme-free and rapid amplified detection of platelet-derived growth factor-BB

Platelet-derived growth factor-BB (PDGF-BB) is currently used as a biomarker protein for cancer early diagnosis and clinical treatment. Herein, we reported a robust and enzyme-free strategy based on aptamer recognition and proximity-induced entropy-driven circuits (AR-PEDC) for homogeneous and rapid detection of platelet-derived growth factor BB (PDGF-BB) without any washing steps or thermocycling. The proximity probes specifically recognize target protein to form the completed trigger (CT). Then, the CT reacts with three-strand complex to form intermediate, which subsequently binds to fuel strand to release reporter strand, assistant strand and the CT. The revised proximity probes exhibit significantly improved signal-to-background ratio and faster association rate. Moreover, target protein/proximity probes interaction can specifically initiate entropy-driven circuits, thus providing immense signal amplification for ultrasensitive detection of PDGF-BB with low detection limit of 9.6 pM. The practical ability of the developed strategy is demonstrated by detection of PDGF-BB in human serum with satisfactory results. In addition, this method is flexible and can be conveniently extended to a variety of targets by simply substituting the target specific sequence. Thus, this strategy presents a rapid, low background and versatile amplification mechanism for the detection of protein biomarkers and offers a promising alternative platform for clinical diagnosis.

I-Motif-Based in Situ Bipedal Hybridization Chain Reaction for Specific Activatable Imaging and Enhanced Delivery of Antisense Oligonucleotides.

The efficient and precise delivery of antisense oligonucleotides (ASOs) to target cells is of great value in gene silencing. However, the specificity and packaging capacity of delivery system still remains challenging. Here, we designed an i-motif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy for specific target cells imaging and enhanced gene delivery of ASOs. As a proof of concept, an 8-nt ASO modified with locked nucleic acid (LNA) which is complementary to the seed region of microRNA21 (miR-21) was used for gene silencing studies. Benefiting from the design of hairpin-contained i-motif, the stimuli-responsive assembly of pH-Apt-BiHCR was successfully achieved on MCF-7 cells surface based on the specific recognition of aptamer. Using this strategy, the pH-Apt-BiHCR not only contains repeated fluorescence resonance energy transfer (FRET) units for activatable tumor imaging with high contrast but also arrays with plenty of LNA ASOs as interference molecules for cancer cells inhibition. An in vitro assay showed that this strategy presented an excellent response ability in buffer within a narrow pH range (6.0-7.0) with a transition midpoint (pHT) of 6.44 ± 0.06. Moreover, live cell studies revealed that it realized a specific activatable imaging of target cells, while the ASOs arrayed pH-Apt-BiHCR exhibited improved internalization via an endocytosis pathway and enhanced gene silencing to MCF-7 cells compared to single ASO alone. We believe that this design will inspire the development of novel probes for early diagnosis and therapy of cancer cells.

Development and Application of Aptamer-Based Surface-Enhanced Raman Spectroscopy Sensors in Quantitative Analysis and Biotherapy.

Surface-enhanced Raman scattering (SERS) is one of the most special and important Raman techniques. An apparent Raman signal can be observed when the target molecules are absorbed onto the surface of the SERS substrates, especially on the “hot spots” of the substrates. Early research focused on exploring the highly active SERS substrates and their detection applications in label-free SERS technology. However, it is a great challenge to use these label-free SERS sensors for detecting hydrophobic or non-polar molecules, especially in complex systems or at low concentrations. Therefore, antibodies, aptamers, and antimicrobial peptides have been used to effectively improve the target selectivity and meet the analysis requirements. Among these selective elements, aptamers are easy to use for synthesis and modifications, and their stability, affinity and specificity are extremely good; they have been successfully used in a variety of testing areas. The combination of SERS detection technology and aptamer recognition ability not only improved the selection accuracy of target molecules, but also improved the sensitivity of the analysis. Variations of aptamer-based SERS sensors have been developed and have achieved satisfactory results in the analysis of small molecules, pathogenic microorganism, mycotoxins, tumor marker and other functional molecules, as well as in successful photothermal therapy of tumors. Herein, we present the latest advances of the aptamer-based SERS sensors, as well as the assembling sensing platforms and the strategies for signal amplification. Furthermore, the existing problems and potential trends of the aptamer-based SERS sensors are discussed.

Exosomal DNA Aptamer Targeting α-Synuclein Aggregates Reduced Neuropathological Deficits in a Mouse Parkinson’s Disease Model

The α-synuclein aggregates are the main component of Lewy bodies in Parkinson’s disease (PD) brain, and they showed immunotherapy could be employed to alleviate α-synuclein aggregate pathology in PD. Recently we have generated DNA aptamers that specifically recognize α-synuclein. In this study, we further investigated the in vivo effect of these aptamers on the neuropathological deficits associated with PD. For efficient delivery of the aptamers into the mouse brain, we employed modified exosomes with the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface. We demonstrated that the aptamers were efficiently packaged into the RVG-exosomes and delivered into neurons in vitro and in vivo. Functionally, the aptamer-loaded RVG-exosomes significantly reduced the α-synuclein preformed fibril (PFF)-induced pathological aggregates, and rescued synaptic protein loss and neuronal death. Moreover, intraperitoneal administration of these exosomes into the mice with intra-striatally injected α-synuclein PFF reduced the pathological α-synuclein aggregates and improved motor impairments. In conclusion, we demonstrated that the aptamers targeting α-synuclein aggregates could be effectively delivered into the mouse brain by the RVG-exosomes and reduce the neuropathological and behavioral deficits in the mouse PD model. This study highlights the therapeutic potential of the RVG-exosome delivery of aptamer to alleviate the brain α-synuclein pathology.

A Simple, pH-Activatable Fluorescent Aptamer Probe with Ultralow Background for Bispecific Tumor Imaging.

Activatable aptamer probes (AAPs) are promising in molecular imaging of tumors, but the reported shape-switching-dependent AAPs are still challenged by unsatisfied noise suppression, poor stability, and sophisticated sequence design. To address the problem, we constructed a pH-activatable aptamer probe (pH-AAP) by utilizing an acid-labile acetal linker as the responsive element to be fused with a tumor-targeted aptamer. Specifically, a Cy5-labeled aptamer was connected with the quencher BHQ2 through the acetal group, thus generating pH-AAP with quenched fluorescence. Due to the stable proximity of Cy5 to BHQ2, pH-AAP was found to have ultralow background with a quenching efficiency as high as 98%. In comparison with shape-switching-dependent AAPs, the noise suppression of pH-AAP was well maintained for a much longer time in both serum and mouse body, thus showing a robust fluorescence stability. By a combination of the fluorescence recovery induced by acid hydrolysis of acetal linkers and the tumor-targeted recognition of aptamers, pH-AAP could either specifically anchor the extracellular pH-activated signals on the target cell surface in an acidic tumor microenvironment or be activated by acidic lysosomes after it was internalized into target cells. As proof of concept, in vitro evaluation and in vivo imaging of A549 lung cancer cells were performed by using S6 aptamer as a demonstration. It was indicated that pH-AAP realized washing-free, bispecific, and contrast-enhanced tumor imaging. The strategy is simple and free of sequence modification, which promises to provide a universal platform for sensitive and precise tumor diagnosis.

An electrochemical aptasensing platform for carbohydrate antigen 125 based on the use of flower-like gold nanostructures and target-triggered strand displacement amplification.

An electrochemical aptasensing method is described for the determination of the biomarker CA125. It combines aptamer recognition and target-triggered strand displacement amplification. Flower like gold nanostructures were electrodeposited on a screen-printed carbon electrode to increase the sensor surface, to assemble more toehold-containing hairpin probe 1 (Hp1), and to improve the accessibility for DNA strands. Under the optimal conditions, this assay has a linear response in the 0.05 to 50ng•mL-1 CA125 concentration range, with a low detection limit of 5.0pg•mL-1. This method is specific and stable. It was successfully applied to the detection of CA125 in spiked biological samples, with recoveries between 82.5% and 104.1%. Graphical abstract.

Development of a Highly Sensitive Colorimetric Method for Detecting 17β-Estradiol Based on Combination of Gold Nanoparticles and Shortening DNA Aptamers

Pollution of environmental endocrine disruptors has caused increasing concern globally. In the current study, a simple colorimetric method with high sensitivity and good selectivity for 17β-estradiol (E2) detection was developed, which employed gold nanoparticles (AuNPs) as colorimetric probe for specific recognition of shortening DNA aptamer. Visible color change from bright red to violet was observed for aggregation of AuNPs without the protection of DNA aptamer. After optimization, the method exhibited great performance for E2 detection with good linearity between E2 concentrations from 0.2 to 5 nM and the absorbance ratio at 620 and 523 nm, with the limit of detection of 0.1 nM. The method was also successfully applied to E2 determination in different spiked water samples including fishpond water, lake water, and tap water, in which good recoveries from 93.1 to 108.9% and acceptable relative standard deviations from 3.4 to 8.9% were obtained. This technique showed great potential for on-site fast determination of E2 in environmental water samples.

A “signal-on” voltammetric aptasensor fabricated by hcPt@AuNFs/PEI-rGO and Fe3O4NRs/rGO for the detection of zearalenone

A “signal-on” electrochemical aptasensor was proposed by employing hollow cubic platinum@gold nanoframes functionalized polyethyleneimine-reduced graphene oxide (hcPt@AuNFs/PEI-rGO) as the label material and Fe3O4 nanorods/reduced graphene oxide (Fe3O4NRs/rGO) for quantitative detection of zearalenone (ZEN). In this work, the hcPt@AuNFs/PEI-rGO with excellent conductivity and large surface area was synthesized as the label material, which can increase the loading of Thi and DNA S1. The Fe3O4NRs/rGO as the platform was decorated on gold electrodes, which can not only effectively improve the load capacity of the gold nanoparticles (AuNPs) and the DNA S2 but also catalyze Thi, thereby improving analytical performance of the aptasensor. Upon the specific recognition of aptamers to ZEN, the remained DNA S2 captured the hcPt@AuNFs/PEI-rGO immobilized with DNA S1 by hybridization reaction. With the aid of signal amplification system (hcPt@AuNFs/PEI-rGO and Fe3O4NRs/rGO), the aptasensor exhibited a linear relationship related to the logarithmic values of ZEN concentration from 0.5 pg/mL to 50 ng/mL with a low detection limit of 0.105 pg/mL under optimal conditions. Meanwhile, the aptasensor exhibited satisfactory specificity, good stability, excellent reproducibility and provided a potential application in maize samples.

Collision of Aptamer/Pt Nanoparticles Enables Label-Free Amperometric Detection of Protein in Rat Brain.

Single particle collision is emerging as a powerful and sensitive technique for analyzing small molecules, however, its application in biomacromolecules detection, for example, protein, in complex biological environments is still challenging. Here, we present the first demonstration on the single particle collision that can be developed for the detection of platelet-derived growth factor (PDGF), an important protein involved in the central nervous system in living rat brain. The system features Pt nanoparticles (PtNPs) conjugated with the PDGF recognition aptamer, suppressing the electrocatalytic collision of PtNPs toward the oxidation of hydrazine. In the presence of PDGF, the stronger binding between targeted protein and the aptamer disrupts the aptamer/PtNPs conjugates, recovering the electrocatalytic performance of PtNPs, and allowing quantitative, selective, and highly sensitive detection of PDGF in cerebrospinal fluid of rat brain.

Dynamic DNA self-assembly activated hemin-mimetic enzyme system for versatile fluorescent biosensing

In this work, we established a dynamic DNA self-assembly activated hemin-mimetic enzyme system for versatile fluorescent biosensing. The DNA-grafted hemin was utilized not only as the module for dynamic DNA-assembly but also as the tunable mimetic enzyme. In the presence of targets, the entropy-driven dynamic DNA-assembly circuit was initiated and the tagged hemin dimmers with suppressed catalytic activity were disaggregated into activated hemin monomers. Then activated hemin-mimetic enzymes can catalyze non-fluorescent tyramine into fluorescent dityramine, providing a simple, fast and enzyme-free signal amplification strategy for fluorescent biosensing. The designed method was successfully applied to detect target DNA at concentration down to 78 pM with dynamic range from 0.1 to 50 nM in 40 min. Moreover, it was extended to detect small molecule cocaine based on specific aptamer recognition with a detection limit of 4.85 μM in 70 min. Our proposed system showed simple design of oligonucleotides modules for DNA-assembly, preferable mimic enzyme activation mechanism and fast biosensing without extra hemin addition. Thus, this work presented a simple and versatile target-triggered DNA-hemin enzyme assembly system for high-efficiency and enzyme-free signal transduction which would become a potential tool for universal biosensing application.

Ferricyanide stimulated cathodic photoelectrochemistry of flower-like bismuth oxyiodide under ambient air: A general strategy for robust bioanalysis

Most of the current cathodic photoelectrochemical (PEC) bioassays either rely on dissolved oxygen as a signal reporting molecule or are interfered by atmospheric oxygen (if using other molecules than oxygen as the signaling molecule), which leads to their monotonous detection mechanism, vulnerable/nonadjustable performance, complicated instrument and time consuming operation. Herein, we develop an innovative, oxygen independent cathodic PEC bioassay with distinct advantages of robust performance, simple instrument/facile operation, as well as sensitive and prompt readout. Specifically, taking the ochratoxin A (OTA) as a model analyte, the detection combines the largely augmented photocurrent of flower-like bismuth oxyiodide (BiOI) microspheres by potassium ferricyanide (K3[Fe(CN)6]) under aerobic conditions, the highly selective aptamer recognition as well as the powerful exonuclease-catalyzed target recycling amplification. Removal of the surface immobilized DNA under the cooperative effects of OTA induced double-stranded DNA (dsDNA) dissociation and RecJf exonuclease mediated digestion of the released single-stranded DNA results in enhanced flux of K3[Fe(CN)6] to the photoelectrode, enables a “signal-on” and “label-free” mode for OTA detection with high sensitivity and good selectivity. It also shows acceptable results for the detection of OTA spiked in wine samples. Since the detection takes place in ambient air and is not interfered by the atmospheric oxygen, this facile and robust cathodic PEC biosensing system displays promising applicability for on-site and real-time diagnosis. More significantly, it is extensible as a general strategy for versatile cathodic PEC bioanalysis.

Versatile and Ultrasensitive Electrochemiluminescence Biosensor for Biomarker Detection Based on Nonenzymatic Amplification and Aptamer-Triggered Emitter Release.

Electrochemiluminescence (ECL), as a sensitive and controllable assay, offers a considerable opportunity for multiple types of biomarkers detection. However, constructing such a biosensor remains a significant challenge. Herein, an ultrasensitive and versatile ECL biosensor was constructed to detect multiple types of biomarkers from breast cancer by taking the strategies of nonenzymatic catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) amplification, as well as aptamer-triggered emitter release. Concretely, with the appearance of target 1 microRNA-21 (miRNA-21), abundant double-stranded DNA (dsDNA) polymers were generated on this biosensing surface via amplification circuits of CHA and HCR, which could be intercalated into substantial ([Ru(bpy)2dppz]Cl2) as ECL indicators to obtain an obvious enhancement of ECL signal for target 1 detection with a detection limit (0.1 fM). Furthermore, in the presence of target 2 human mucin 1 (MUC1) protein, the ECL signal had a distinct decrease, because aptamer recognition induced the release of [Ru(bpy)2dppz]Cl2 from the sensing surface, thus, achieving a sensitive detection for MUC1 with a detection limit (2.4 fg·mL-1). Simultaneously, this sensing platform was applied to monitor the biomarkers from MDA-MB-231 breast cancer cells, suggesting that this method was applicable to detect real samples. Therefore, this platform is an applicable and versatile implement for the determination of multiple types of biomarkers to improve diagnostic accuracy and efficiency.

Netlike hybridization chain reaction assembly of DNA nanostructures enables exceptional signal amplification for sensing trace cytokines.

The monitoring and detection of molecular biomarkers play crucial roles in disease diagnosis and treatment. In this work, we proposed a target-responsive netlike hybridization chain reaction (nHCR) DNA nanostructure construction method, which can offer an exceptional signal enhancement, for highly sensitive fluorescence detection of cytokine, interferon-gamma (IFN-γ). The presence of the target cytokine can lead to the conformational change of the aptamer recognition hairpin probes and the liberation of the nHCR initiator strands, which further trigger the nHCR process between two dye-labeled and double hairpin-structured probes to form netlike DNA nanostructures. The formation of the DNA nanostructures brings the dyes into close proximity, resulting in significantly amplified fluorescence resonance energy transfer signals for sensitive and enzyme-free detection of IFN-γ. The present method has a detection limit of 1.2 pM and a dynamic linear range of 5 to 1000 pM for IFN-γ detection. Besides, with the high specificity of the aptamer probe and the significant signal amplification of the nHCR, such an IFN-γ detection strategy shows excellent selectivity and high sensitivity, which can be potentially applied to detect IFN-γ in human serums. With such a demonstration of the detection of IFN-γ, this proposed method can be extended for detecting different types of biomolecules.

Aptamer-based regulation of transcription circuits.

We propose synthetic DNA/RNA transcription circuits based on specific aptamer recognition. By mimicking transcription factor regulation, combined with specific enzyme/DNA aptamer binding, multiple biomolecules including DNA, RNA, polymerase, restriction enzymes and methylase were used as regulators. In addition, multi-level cascading networks and methylation-switch circuits were also established. This regulation strategy has the potential to expand the toolkit of in vitro synthetic biology.

Poly(thymine)-CuNPs: Bimodal Methodology for Accurate and Selective Detection of TNT at Sub-PPT Levels.

Accurate, sensitive, and selective detection of explosives is of vital importance in antiterrorism and homeland security. Fluorescence sensors are prevalent for sensitive and fast in-field explosive detection but are sometimes compromised by accuracy and stability due to the similar structures of explosives, photobleaching, and complex sample matrixes. Herein, we developed a first bimodal methodology capable of both sensitive in-field fluorescence detection and accurate laboratory mass spectrometric quantification of 2,4,6-trinitrotoluene (TNT) by utilizing the characteristic fluorescent and mass spectrometric response of copper nanoparticles (CuNPs). An excellent selectivity was also realized by involving aptamer recognition. The methodology is capable of detecting TNT at subpart per trillion (PPT) levels, with a detection limit of 0.32 pg mL-1 by inductively coupled plasma mass spectrometry (ICPMS) and 0.17 ng mL-1 by fluorimetry. The signal response was accurate and stable for at least 60 days by ICPMS. Thanks to the biospecificity of the aptamer, this bimodal methodology is potentially applicable to a large panel of explosives.

Imaging of Receptor Dimers in Zebrafish and Living Cells via Aptamer Recognition and Proximity-Induced Hybridization Chain Reaction.

On cell-membrane surfaces, receptor-protein dimers play fundamental roles in many signaling pathways that are crucial for normal biological processes and cancer development. Efficient and sensitive analysis of receptor dimers in the native environment is highly desirable. Herein, we present a strategy for amplified imaging of receptor dimers in zebrafish and living cells that relies on aptamer recognition and proximity-induced hybridization chain reaction. Taking advantage of specific aptamer recognition and enzyme-free signal amplification, this strategy is successfully applied to the visualization of c-Met-receptor dimers in an HGF-independent or -dependent manner. Therefore, the developed imaging strategy paves the way for further investigation of the dimerization or oligomerization states of cell-surface receptors and their corresponding activation processes in zebrafish and living cells.

Sensitive aptamer-based fluorescene assay for ochratoxin A based on RNase H signal amplification.

A new assay for the selective and sensitive aptamer-based fluorescent biosensor for ochratoxin A (OTA) detection is proposed. It consists of a ribonuclease H (RNase H)-assisted cycle response that leads to a significant amplification of the signal, which allow for a low limit of detection (LOD) of 0.08 ng/mL. By using this technique, a high selectivity for OTA against ochratoxin B and aflatoxin B1 (AFB1) was noticed. The quantitative determination in real samples has been verified using red wine samples spiked with a series of OTA concentrations (0.4, 4, and 12 ng/mL), and the recoveries ranged from 96.1 to 107.5%. This aptasensor has great practical applications in food industry and moreover, can be extended for the detection of other toxins by replacing the sequence of the recognition aptamer.

An electrochemical ceruloplasmin aptasensor using a glassy carbon electrode modified by diazonium-functionalized multiwalled carbon nanotubes

Fast and accurate determination of biological compounds in human fluids plays a crucial role in clinical analysis. Low levels and high levels of ceruloplasmin (Cp) in serum cause several diseases such as Wilson’s disease, therefore, its accurate determination in human fluids is important. A selective and sensitive electrochemical biosensor was developed for Cp detection using a Cp-specific recognition aptamer. The proposed aptasensor was based on a glassy carbon electrode modified with diazonium-functional multiwall carbon nanotubes. The aptamer was linked to the electrode surface using electrochemical approach, followed by chemical immobilization of aminated-aptamer. All fabrication steps were accompanied by changes to the electrochemical parameters. The binding of Cp to aptamer was monitored by cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The effective parameters were optimized. The calibration curve for Cp concentration was linear at 0.02–3.0 ng mL−1 and 3.0–80.0 ng mL−1 with a detection limit (signal-to-noise ratio of 3) of 3.7 pg mL−1. The fabricated aptasensor can serve as a powerful sensor for rapid diagnosis of Cp in the human serum sample and shows a great potential for practical bioapplication.

A nanosol SERS method for quantitative analysis of trace potassium based on aptamer recognition and silver nanorod catalysis of Ag(I)-glucose reaction

Silver nanorods (AgNR) have strong catalysis of the redox reation between AgNO3 and glucose (GLC) to form silver nanoparticles (AgNP) with strong nanoplasmonic effect including surface plasmon resonance (SPR) absorption (RRS) and surface-enhanced resonance Raman scattering (SERS) in the presence of Victoria blue B(VBB) molecular probes. The increased SERS intensity at 1615 cm−1 responds linearly with the AgNR concentration. The potassium ion aptamers (Apt) can adsorb on the surface of AgNR to inhibit the nanocatalysis. Upon addition of K+, the formed stable G-quadruplex of K-Apt escape from the nanosurface of AgNR to recovery its catalysis. With the increase of K+ concentration, the SERS intensity increased linearly due to producing more AgNP substrates. Thus, a 50–3000 nmol/L K can be analyzed quantitatively by SERS technique, with a detection limit of 25 nmol/L.