A sensitive fluorescence strategy for telomerase detection in cancer cells based on T7 exonuclease-assisted target recycling amplification

A sensitive fluorescence strategy based on T7 exonuclease-assisted target recycling amplification was developed for telomerase detection in cancer cells. The novel strategy improved the fluorescence signal and sensitivity compared with the previously reported methods.     

Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes

Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33 nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.     

Exonuclease-assisted cascaded recycling amplification for label-free detection of DNA

The network consisting of three kinds of unlabeled stem-loop DNA molecular beacons (MBs) is activated by target DNA in the presence of exonuclease-III (Exo-III), achieving the concept of exonuclease-assisted cascaded recycling amplification (Exo-CRA) for DNA detection with a wide dynamic range of 8 orders of magnitude.     

Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor

Fluorescence polarization (FP) assays incorporated with fluorophore-labeled aptamers have attracted great interest in recent years. However, detecting small molecules through the use of FP assays still remains a challenge because small-molecule binding only results in negligible changes in the molecular weight of the fluorophore-labeled aptamer. To address this issue, we herein report a fluorescence polarization (FP) aptamer assay that incorporates a novel signal amplification strategy for highly sensitive detection of small molecules. In the absence of adenosine, our model target, free FAM-labeled aptamer can be digested by nuclease, resulting in the release of FAM-labeled nucleotide segments from the dT-biotin/streptavidin complex with weak background signal. However, in the presence of target, the FAM-labeled aptamer–target complex protects the FAM-labeled aptamer from nuclease cleavage, allowing streptavidin to act as a molar mass amplifier. The resulting increase in molecular mass and FP intensity of the aptamer–target complex provides improved sensitivity for concentration measurement. The probe could detect adenosine from 0.5 μM to 1000 μM, with a detection limit of 500 nM, showing that the sensitivity of the probe is superior to aptamer-based FP approaches previously reported for adenosine. Importantly, FP could resist environmental interferences, making it useful for complex biological samples without any tedious sample pretreatments. Our results demonstrate that this dual-amplified, aptamer-based strategy can be used to design fluorescence polarization probes for rapid, sensitive, and selective measurement of small molecules in complicated biological environment.     

Aptameric system for the highly selective and ultrasensitive detection of protein in human serum based on non-stripping gold nanoparticles

A novel approach is proposed in this study for the development of an aptameric assay system for protein based on non-stripping gold nanoparticles (NPs)-triggered chemiluminescence (CL) upon target binding. The strategy chiefly depends on the formation of a sandwich-type immunocomplex among the capture antibody immobilized on the polystyrene microwells, target protein and aptamer-functionalized gold NPs. Introduction of target protein into the assay system leads to the attachment of gold NPs onto the surface of the microwells and thus the assembled gold NPs could trigger the reaction between luminol and AgNO(3) with a CL emission. Further signal amplification was achieved by a simple gold metal catalytic deposition onto the gold NPs. Such an amplified CL transduction allowed for the detection of model target IgE down to the 50 fM, which is better than most existing aptameric methods for IgE detection. This new protocol also provided a good capability in discriminating IgE from nontarget proteins such as IgG, IgA, IgM and interferon. The practical application of the proposed gold NPs-based immunoassay was successfully carried out for the determination of IgE in 35 human serum samples. Overall, the proposed assay system exhibits excellent analytical characteristics (e.g., a detection limit on the attomolar scale and a linear dynamic range of 4 orders of magnitude), and it is also straightforward to adapt this strategy to detect a spectrum of other proteins by using different aptamers. This new CL strategy might create a novel technology for developing simple biosensors in the sensitive and selective detection of target protein in a variety of clinical, environmental and biodefense applications.     

Label-free aptamer-based chemiluminescence detection of adenosine

We have developed a novel sensitive chemiluminescence (CL) aptasensor for the target assay as exemplified by using adenosine as a model target. In this work, we have demonstrated the signaling mechanism to make detection based on magnetic separation and 3,4,5-trimethoxyl-phenylglyoxal (TMPG), a special CL reagent as the signaling molecule, which reacts instantaneously with guanine nucleobases (G) of adenosine-binding aptamer strands. Briefly, amino-functioned capture DNA sequences are immobilized on the surface of carboxyl-modified magnetic beads, and then hybridized with label-free G-rich (including 15 guanine nucleobases) adenosine-binding aptamer strands to form our CL aptasensor. Upon the introduction of adenosine, the aptamer on the surface of magnetic beads is triggered to make structure switching to the formation of the adenosine/aptamer complex. Consequently, G-rich aptamer strands are forced to dissociate from magnetic beads sensing interface, resulting in a decrease of CL signal. The decrement of peak signal is proportional to the amount of adenosine. The effects of the amounts of capture DNA, aptamer, magnetic beads are investigated and optimized. It was found that the CL intensity had a linear dependency on the concentration of adenosine in the range of 4 × 10⁻⁷ to 1 × 10⁻⁵ M. With a low detection limit of 8 × 10⁻⁸ M and simplicity in CL detection, this novel technique will offer a great promise for future target/aptamer analysis.     

Nuclease cleavage-assisted target recycling for signal amplification of free-label impedimetric aptasensors

A simple, novel, label-free impedimetric aptasensor with signal amplification is developed for ultrasensitive detection of small molecules on a carbon nanotubes-based sensing platform by combining target-induced release of the aptamers and nuclease cleavage-assisted target recycling.     

On-chip graphene oxide aptasensor for multiple protein detection

The versatility of an on-chip graphene oxide (GO) aptasensor was successfully confirmed by the detection of three different proteins, namely, thrombin (TB), prostate specific antigen (PSA), and hemagglutinin (HA), simply by changing the aptamers but with the sensor composition remaining the same. The results indicate that both DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. Molecular selectivity and concentration dependence were investigated in relation to TB and PSA detection by using a dual, triple, and quintuple microchannel configuration. The multiple target detection of TB and PSA on a single chip was also demonstrated by using a 2 × 3 linear-array GO aptasensor. This work enables us to apply this sensor to the development of a multicomponent analysis system for a wide variety of targets by choosing appropriate aptamers.     

Aptamer-barcode based immunoassay for the instantaneous derivatization chemiluminescence detection of IgE coupled to magnetic beads

We report on a highly sensitive aptameric assay system for the determination of IgE, where a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), acts as the signaling molecule and polystyrene beads as the amplification platform. Briefly, a "sandwich-type" detection strategy is employed in our design, where magnetic beads functionalized with a capture antibody were reacted with the target protein IgE, and then sandwiched with the aptamer-barcodes which were prepared by assembling polystyrene beads with IgE aptamer. The target immunoreaction event could be sensitively detected via an instantaneous derivatization reaction between TMPG and the guanine (G) nucleotides within the aptamer-barcodes to form an unstable CL intermediate for the generation of light. Further signal amplification is achieved by extending the G nucleotide-rich domain on the aptamer backbone for second amplification. Such simple amplified CL transduction allows the detection of IgE down to the 4.6 pM level, which is better than most previous aptameric methods for IgE detection. This new protocol also provides a good capability in discriminating IgE from nontarget proteins such as IgG, IgA, IgM, interferon and thrombin. The practical application of the proposed aptamer-barcode based immunoassay was successfully carried out for the determination of IgE in 20 human serum samples. It is straightforward to adapt this strategy to detect a spectrum of other proteins by using different aptamers, thus this method may offer a new direction in designing high-performance CL aptasensors for early diagnoses of diseases.     

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins’ aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device’s plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections.

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer–target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H₂O₂ by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1 × 10⁻¹⁰ M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol.     

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.     

A selective adenosine sensor derived from a triplex DNA aptamer

The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM.     

Molecule-binding dependent assembly of split aptamer and γ-cyclodextrin: A sensitive excimer signaling approach for aptamer biosensors

A highly sensitive and selective fluorescence aptamer biosensors for the determination of adenosine triphosphate (ATP) was developed. Binding of a target with splitting aptamers labeled with pyrene molecules form stable pyrene dimer in the γ-cyclodextrin (γ-CD) cavity, yielding a strong excimer emission. We have found that inclusion of pyrene dimer in γ-cyclodextrin cavity not only exhibits additive increases in quantum yield and emission lifetime of the excimer, but also facilitates target-induced fusion of the splitting aptamers to form the aptamer/target complex. As proof-of-principle, the approach was applied to fluorescence detection of adenosine triphosphate. With an anti-ATP aptamer, the approach exhibits excimer fluorescence response toward ATP with a maximum signal-to-background ratio of 32.1 and remarkably low detection limit of 80 nM ATP in buffer solution. Moreover, due to the additive fluorescence lifetime of excimer induced by γ-cyclodextrin, time-resolved measurements could be conveniently used to detect as low as 0.5 μM ATP in blood serum quantitatively.     

A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin

Nanopore emerged as a powerful single‐molecule technique over the past two decades, and has shown applications in the stochastic sensing and biophysical studies of individual molecules. Here, we report a versatile strategy for nanopore sensing by employing the combination of aptamers and host–guest interactions. An aptamer is first hybridized with a DNA probe which is modified with a ferrocene?cucurbit[7]uril complex. The presence of analytes causes the aptamer–probe duplex to unwind and release the DNA probe which can quantitatively produce signature current events when translocated through an α‐hemolysin nanopore. The integrated use of magnetic beads can further lower the detection limit by approximately two to three orders of magnitude. Because aptamers have shown robust binding affinities with a wide variety of target molecules, our proposed strategy should be universally applicable for sensing different types of analytes with nanopore sensors.     

Dynamic,Bioresponsive Hydrogels via Changes in DNA Aptamer Conformation

DNA aptamers are integrated into synthetic hydrogel networks with the aim of creating hydrogels that undergo volume changes when exposed to target molecules. Specifically, single‐stranded DNA aptamers in cDNA‐bound, extended state are incorporated into hydrogel networks as cross‐links, so that the nanoscale conformational change of DNA aptamers upon binding to target molecules will induce macroscopic volume decreases of hydrogels. Hydrogels incorporating adenosine triphosphate (ATP)–binding aptamers undergo controllable volume decreases of up to 40.3 ± 4.6% when exposed to ATP, depending on the concentration of DNA aptamers incorporated in the hydrogel network, temperature, and target molecule concentration. Importantly, this approach can be generalized to aptamer sequences with distinct binding targets, as demonstrated here that hydrogels incorporating an insulin‐binding aptamer undergo volume changes in response to soluble insulin. This work provides an example of bioinspired hydrogels that undergo macroscopic volume changes that stem from conformational shifts in resident DNA‐based cross‐links.     

Highly Specific Recognition of Guanosine Using Engineered Base-Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na⁺-binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na⁺ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm . Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.     

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.     

An aptamer-based signal-on and multiplexed sensing platform for one-spot simultaneous electronic detection of proteins and small molecules

One-spot signal-on and simultaneous electronic detection of lysozyme and adenosine is achieved based on target-induced release of aptamers and back-filling hybridization of the resulting single stranded DNAs with redox-tags conjugated aptamers.     

Aptamer/quantum dot-based simultaneous electrochemical detection of multiple small molecules

A novel strategy for “signal on” and sensitive one-spot simultaneous detection of multiple small molecular analytes based on electrochemically encoded barcode quantum dot (QD) tags is described. The target analytes, adenosine triphosphate (ATP) and cocaine, respectively, are sandwiched between the corresponding set of surface-immobilized primary binding aptamers and the secondary binding aptamer/QD bioconjugates. The captured QDs yield distinct electrochemical signatures after acid dissolution, whose position and size reflect the identity and level, respectively, of the corresponding target analytes. Due to the inherent amplification feature of the QD labels and the “signal on” detection scheme, as well as the sensitive monitoring of the metal ions released upon acid dissolution of the QD labels, low detection limits of 30 nM and 50 nM were obtained for ATP and cocaine, respectively, in our assays. Our multi-analyte sensing system also shows high specificity to target analytes and promising applicability to complex sample matrix, which makes the proposed assay protocol an attractive route for screening of small molecules in clinical diagnosis.