Molecular Aptamer Beacon Research Articles

Molecular Aptamer Beacon-based SERS biosensor for the detection of nucleic acids

Nucleic acids are essential biomolecules for the functioning of cells. In past years, nucleic acids have been assessing their role in prognostics and diagnostics. The progress of nanotechnology has allowed the fabrication of various type of nanostructured biosensors able to detect them with high sensitivity and specificity. Among the available sensing mechanisms, the sensor technology based on Surface-enhanced Raman Spectroscopy (SERS) is frequently preferred for identifying nucleic acids. In these sensors, natural or synthetic oligonucleotide sequences, acting as probes to hybridize the target molecules, are immobilized on a plasmonic sensing platform. In particular, aptamers, short DNA/RNA sequences, are emerging as new recognition elements for their chemical stability and specificity. Here, we focus on the combination of a specific type of aptamer, a molecular aptamer beacon, and nanostructured SERS biosensors for a sensitive detection of nucleic acids.

Novel Molecular Aptamer Beacon for the Specific Simultaneous Analysis of Circulating Tumor Cells and Exosomes of Colorectal Cancer Patients.

Liquid biopsy provides non-invasive and real-time detection for cancer diagnosis, but the lack of specific markers targeted to liquid biopsy components, such as circulating tumor cells (CTCs) and exosomes, has impeded its effective utilization in clinical settings. W3 is an aptamer, and its target has been previously demonstrated to be a predictor of colorectal cancer (CRC) metastasis. Herein, we developed a W3-based molecular beacon (MAB-W3-3G) to specifically detect CTCs and exosomes derived from CRC patients by modifying the W3 sequence and adding a fluorescent group FAM at the 5' end and a quencher group BHQ1 at the 3' end, resulting in a detectable green fluorescence only in the presence of the target. MAB-W3-3G retained features similar to those of the original W3, including high specificity and affinity for metastatic CRC cells, as well as excellent plasma stability. Notably, W3 target-positive CTCs were visualized, positive exosomes were quantified in CRC patients' whole blood without any sample pretreatment, and both detections could be finished in one step without any routine washing procedures. For CRC, the W3 target-positive CTC enumeration in metastasis was higher than that in non-metastasis (p < 0.01), and the quantitation of positive exosomes was correlated with CRC patients (p < 0.0001). Moreover, the MAB-W3-3G-based simultaneous detection of CTCs and exosomes was proven to have the potential for more precise clinical diagnosis. In conclusion, MAB-W3-3G could detect CTCs and exosomes in the blood samples of tumor patients with simple manipulation, rapid analysis, and high specificity, providing an effective liquid biopsy tool for the prediction of CRC.

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5' end and a black hole quencher (BHQ1) labeled at the 3' end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex-quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.

Development of a Molecular Aptamer Beacon Applied to Magnetic-Assisted RNA Extraction for Detection of Dengue and Zika Viruses Using Clinical Samples.

Limitations in the detection of cocirculating flaviviruses such as Dengue and Zika lead us to propose the use of aptameric capture of the viral RNA in combination with RT-PCR (APTA-RT-PCR). Aptamers were obtained via SELEX and next-generation sequencing, followed by colorimetric and fluorescent characterizations. An APTA-RT-PCR assay was developed, optimized, and tested against the viral RNAs in 108 serum samples. After selection, sequence APTAZC10 was designed as a bifunctional molecular beacon (APTAZC10-MB), exhibiting affinity for the viral targets. APTA-RT-PCR was able to detect Dengue and Zika RNA in 43% and 8% of samples, respectively. Our results indicate that APTAZC10-MB and APTA-RT-PCR will be useful to improve the detection of Dengue and Zika viruses in a fast molecular assay for the improvement of infectious disease surveillance.

Aptamer and Ru(bpy)32+‐AuNPs‐based electrochemiluminescence biosensor for accurate detecting Listeria monocytogenes

AbstractAs a widespread foodborne pathogen, Listeria monocytogenes (LM) can potentially cause a large number of food safety incidents, and the mortality rate of humans infected with this pathogen is relatively high. Thus, establishment of a fast and simple inspection technology is imperative. Herein, a solid‐state electrochemiluminescence (ECL) biosensing switch system based on special ferrocene‐labeled molecular beacon aptamer (Fc‐MBA) has been developed for rapid detection of LM. The biosensor consisted of two main parts, an ECL substrate and an ECL intensity switch. ECL substrate was generated by modifying the complex of gold nanoparticle (AuNPs) and ruthenium (II) tris‐(bipyridine) onto Au electrode, and an MBA labeled by Fc that acted as an ECL intensity switch. Moreover, in the presence of LM, the stem‐loop structure of MBA could be opened and the ferrocene was kept away from ECL substrate, which was due to an obvious ECL intensity increment. The limit of detection was 5 CFU/mL, and satisfactory recoveries of 98.1–103.6% were observed. Thus, we propose a novel method for accurate and specific detection of LM.

A Simple and Rapid "Signal On" Fluorescent Sensor for Detecting Mercury (II) Based on the Molecular Beacon Aptamer.

Biosensors for mercury (II) (Hg2+) with high sensitivity are urgently required for food safety, ecosystem protection and disease prevention. In this study, a simple and fast detection method of Hg2+ based on the molecular beacon aptamer was established, according to the principle that Hg2+ could change the structure of the molecular beacon aptamer, resulting in the changed fluorescence intensity. All of the detection conditions were optimized. It was found that an optimal molecular beacon aptamer MB3 showed the optimal response signal in the optimized reaction environment, which was 0.08 μmol/L MB3, 50 mmol/L tris buffer (40 mmol/L NaCl, 10 mmol/L MgCl2, pH 8.1), and a 10 min reaction. Under the optimal detection conditions, the molecular beacon aptamer sensor showed a linear response to Hg2+ concentration within a range from 0.4 to 10 μmol/L and with a detection limit of 0.2254 μmol/L and a precision of 4.9%. The recovery rates of Hg2+ in water samples ranged from 95.00% to 99.25%. The method was convenient and rapid, which could realize the rapid detection of mercury ions in water samples.

Surface plasmon resonance biosensor for exosome detection based on reformative tyramine signal amplification activated by molecular aptamer beacon

Human epidermal growth factor receptor 2 (HER2)-positive exosomes play an extremely important role in the diagnosis and treatment options of breast cancers. Herein, based on the reformative tyramine signal amplification (TSA) enabled by molecular aptamer beacon (MAB) conversion, a label-free surface plasmon resonance (SPR) biosensor was proposed for highly sensitive and specific detection of HER2-positive exosomes. The exosomes were captured by the HER2 aptamer region of MAB immobilized on the chip surface, which enabled the exposure of the G-quadruplex DNA (G4 DNA) that could form peroxidase-like G4-hemin. In turn, the formed G4-hemin catalyzed the deposition of plentiful tyramine-coated gold nanoparticles (AuNPs-Ty) on the exosome membrane with the help of H2O2, generating a significantly enhanced SPR signal. In the reformative TSA system, the horseradish peroxidase (HRP) as a major component was replaced with nonenzymic G4-hemin, bypassing the defects of natural enzymes. Moreover, the dual-recognition of the surface proteins and lipid membrane of the desired exosomes endowed the sensing strategy with high specificity without the interruption of free proteins. As a result, this developed SPR biosensor exhibited a wide linear range from 1.0 × 104 to 1.0 × 107 particles/mL. Importantly, this strategy was able to accurately distinguish HER2-positive breast cancer patients from healthy individuals, exhibiting great potential clinical application.Graphical

Bioluminescent Protein-Inhibitor Pair in the Design of a Molecular Aptamer Beacon Biosensing System.

Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ (IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γ and provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems.

Simultaneous detection of lead (II) and mercury (II) ions using nucleic acid aptamer molecular beacons

ABSTRACT We have developed a fluorescence quantitative analysis method for the simultaneous detection of Hg2+ and Pb2+ based on nucleic acid aptamer molecular beacon (MB) probes. In this analytical method, two MB probes for Hg2+ (PHg) and Pb2+ (PPb) were designed. The carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were selected as fluorophores of PHg and PPb, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were selected as organic quenchers, and several continuous nucleotides with guanine (G base) were connected to organic quenchers. The aptamers are put in as a part of stem and loop. In general, the fluorescence of fluorophores was dually quenched by BHQ and G bases, so the fluorescence signals were weak. In the presence of Hg2+ and Pb2+, PHg and PPb bonded with them, and the stem-loop structure of MB was destroyed and the fluorescence recovered. Under the optimal conditions, the fluorescence intensity of FAM had a good linear relationship with the concentration of Hg2+ in the range from 0.7 nmol/L to 84 nmol/L, and that of TAMRA and Pb2+ in the range from 0.2 nmol/L to 24 nmol/L. The detection limit of Hg2+ is 0.36 nmol/L and that of Pb2+ is 0.16 nmol/L (3σ, n = 11). The relative standard deviations (RSD) for determination of Hg2+ and Pb2+ were both lower than 5%, the average recoveries of this method in real samples were 96.55–102.78%, which indicated that the method had a high accuracy.

Molecular Aptamer Beacons and Their Applications in Sensing, Imaging, and Diagnostics.

The ability to monitor types, concentrations, and activities of different biomolecules is essential to obtain information about the molecular processes within cells. Successful monitoring requires a sensitive and selective tool that can respond to these molecular changes. Molecular aptamer beacon (MAB) is a molecular imaging and detection tool that enables visualization of small or large molecules by combining the selectivity and sensitivity of molecular beacon and aptamer technologies. MAB design leverages structure switching and specific recognition to yield an optical on/off switch in the presence of the target. Various donor-quencher pairs such as fluorescent dyes, quantum dots, carbon-based materials, and metallic nanoparticles have been employed in the design of MABs. In this work, the diverse biomedical applications of MAB technology are focused on. Different conjugation strategies for the energy donor-acceptor pairs are addressed, and the overall sensitivities of each detection system are discussed. The future potential of this technology in the fields of biomedical research and diagnostics is also highlighted.

A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin, and rapid and sensitive detection of AFB1 is in demand for food safety and environmental analysis. Here we described a simple aptamer molecular beacon assay for rapid detection of aflatoxin B1 (AFB1) by using an aptamer with a fluorescein (FAM) label at the 5′ end and a fluorescence quencher (black hole quencher 1, BHQ1) at the 3′ end. In the presence of AFB1, the aptamer probe bound with AFB1 and induced a hairpin structure, drawing FAM and BHQ1 into close proximity and leading to fluorescence quenching. This assay allowed for a detection limit of 3.9 nmol/L and a dynamic range from 3.9 nmol/L to 4 μmol/L. Specificity test showed other mycotoxins including ochratoxin A, ochratoxin B, fumonisin B1, fumonisin B2, and zearalenone had negligible influence on detection of AFB1. AFB1 spiked in diluted liquor wine, methanol, or corn flour samples was successfully detected by using this aptamer probe, and the assay showed potential for real sample analysis.

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex.

Control of electrostatic interaction between a molecular beacon aptamer and conjugated polyelectrolyte for detection range-tunable ATP assay

A new strategy is suggested to fine-tune the detection range by controlling the ionic density of CPEs in the MBA/CPE-based ATP assay.

Nicking endonuclease-assisted signal amplification of a split molecular aptamer beacon for biomolecule detection using graphene oxide as a sensing platform.

Sensitive and selective detection of ultralow concentrations of specific biomolecules is important in early clinical diagnoses and biomedical applications. Many types of aptasensors have been developed for the detection of various biomolecules, but usually suffer from false positive signals and high background signals. In this work, we have developed an amplified fluorescence aptasensor platform for ultrasensitive biomolecule detection based on enzyme-assisted target-recycling signal amplification and graphene oxide. By using a split molecular aptamer beacon and a nicking enzyme, the typical problem of false positive signals can be effectively resolved. Only in the presence of a target biomolecule, the sensor system is able to generate a positive signal, which significantly improves the selectivity of the aptasensor. Moreover, using graphene oxide as a super-quencher can effectively reduce the high background signal of a sensing platform. We select vascular endothelial growth factor (VEGF) and adenosine triphosphate (ATP) as model analytes in the current proof-of-concept experiments. It is shown that under optimized conditions, our strategy exhibits high sensitivity and selectivity for the quantification of VEGF and ATP with a low detection limit (1 pM and 4 nM, respectively). In addition, this biosensor has been successfully utilized in the analysis of real biological samples.

DNA aptamers are functional molecular recognition sensors in protic ionic liquids.

The function and structural changes of an AMP molecular aptamer beacon and its molecular recognition capacity for its target, adenosine monophosphate (AMP), was systematically explored in solution with a protic ionic liquid, ethylammonium nitrate (EAN). It could be proven that up to 2 M of EAN in TBS buffer, the AMP molecular aptamer beacon was still capable of recognizing AMP while also maintaining its specificity. The specificity was proven by using the guanosine monophosphate (GMP) as target; GMP is structurally similar to AMP but was not recognized by the aptamer. We also found that in highly concentrated EAN solutions the overall amount of double stranded DNA formed, as well as its respective thermal stability, diminished gradually, but surprisingly the hybridization rate (kh ) of single stranded DNA was significantly accelerated in the presence of EAN. The latter may have important implications in DNA technology for the design of biosensing and DNA-based nanodevices in nonconventional solvents, such as ionic liquids.

Molecular aptamer beacon tuned DNA strand displacement to transform small molecules into DNA logic outputs

A molecular aptamer beacon tuned DNA strand displacement reaction was introduced in this work. This strand displacement mode can be used to transform the adenosine triphosphate (ATP) input into a DNA strand output signal for the downstream gates to process. A simple logic circuit was built on the basis of this mechanism.

Sensitive detection of prion protein through long range resonance energy transfer between graphene oxide and molecular aptamer beacon

The traditional molecular aptamer beacon (MAB) is designed by combining an aptamer to a molecular beacon, and its two terminals are labelled with a fluorescent moiety (donor) and quenching moiety (acceptor), respectively. However, it usually has a high background because of the low energy transfer efficiency between the donor and the acceptor. In order to overcome these drawbacks, we have developed a novel MAB with just one fluorescently labelled end, which acts as the donor, and graphene oxide (GO) introduced as the acceptor for target detection by employing long range resonance energy transfer (LrRET) as the signal-transduction mechanism from GO to MAB. To test the validity of the designed MAB system, cellular prion protein (PrPC) has been used as the model target. It was found that the fluorescence of the designed MAB is completely quenched by GO, supplying a very low background. Conversely, the quenched fluorescence is recovered significantly with the addition of PrPC, so that PrPC can be detected over a wide range of 10.2–78.8 μg mL−1 with a detection limit as low as 0.309 μg mL−1 and with high selectivity. This GO-based MAB approach is a successful application of LrRET for the detection of PrPC, with advantages such as low costs, high quenching efficiency and good specificity, and it opens up new opportunities for the sensitive detection of biorecognition events.

Assembly of single-stranded polydeoxyadenylic acid and β-glucan probed by the sensing platform of graphene oxide based on the fluorescence resonance energy transfer and fluorescence anisotropy

Based on the fluorescence resonance energy transfer (FRET) and fluorescence anisotropy (FA), the present study reported proof-of-principle for a highly sensitive and rapid detection technique that can be precisely utilized for investigating the self-assembly of polydeoxyadenylic acid (poly(dA)) and β-glucan, and the interactions of the poly(dA)-β-glucan complex on the surface of graphene oxide (GO). Due to the noncovalent assembly of fluorescein amidite (FAM)-labeled poly(dA) and GO via π-π stacking, the fluorescence of (FAM)-labeled poly(dA) as a molecular aptamer beacon (MAB) was completely quenched by GO. Conversely, the addition of single-stranded lentinan (s-LNT) resulted in the significant restoration of fluorescence due to the formation of poly(dA)-s-LNT complexes with a stiff rod-like structure, which had a weak affinity to GO and kept the dyes away from GO. However, relatively weak fluorescence restoration was observed by adding another single-stranded curdlan (s-CUR) for positive control, indicative of complex formation with higher binding ability to GO. The fluorescence anisotropy (FA) was also combined to confirm the occurrence with different increments of anisotropy relative to the free poly(dA), which could be conveniently extended for detecting the assembly of other biomolecules with higher sensitivity.

Semiquantification of ATP in Live Cells Using Nonspecific Desorption of DNA from Graphene Oxide as the Internal Reference

In this aritcle, we have developed an interesting imaging method for intracellular ATP molecules with semiquantitation. While there has been a lot of work in understanding intracellular events, very few can come close to quantitation or semiquantitation in living cells. In this work, we made an effective use of nanomaterials, graphene oxides, both as a quencher and a carrier for intracellular delivery. In addition, this graphene oxide also serves as the carrier for reference probes for fluorescent imaging. An ATP aptamer molecular beacon (AAMB) is adsorbed on graphene oxide (GO) to form a double quenching platform. The AAMB/GO spontaneously enters cells, and then AAMB is released and opened by intracellular ATP. The resulting fluorescence recovery is used to perform ATP live-cell imaging with greatly improved background and signaling. Moreover, a control ssDNA, which is released nonspecifically from GO by nontarget cellular proteins, can serve as an internal reference for ATP semiquantification inside living cells using the intensity ratio of the AAMB and control. This approach can serve as a way for intracellular delivery and quantitative analysis.

Molecular Beacon Aptamers for Direct and Universal Quantitation of Recombinant Proteins from Cell Lysates

Western blot, enzyme linked immunosorbent assay (ELISA), and fluorescent fusion proteins are currently the most common methods for detecting recombinant proteins. However, the former two are cumbersome and time-consuming, and the latter method may interfere with the trafficking and function of the fused recombinant proteins. We report here a rapid, inexpensive, and simple approach to detect and quantify recombinant proteins using an anti-His-tag molecular beacon aptamer (HMBA). We demonstrated the technique by detection and quantitation of expressed recombinant proteins directly from E. coli cell lysate. The amount of expressed P78-His was determined to be 1.49 μg from the 20 μg cell lysate proteins. To the best of our knowledge, this is the first example directly measuring the concentration and expression yield of recombinant proteins from cell lysate, and the entire procedure required only 5 min.