Structure-switching aptasensors for sensitive detection of ochratoxin A.

Ochratoxin A (OTA) is a toxic metabolite commonly found in various foods and feedstuffs. Accurate and sensitive detection of OTA is needed for food safety and human health. Based on a common OTA-binding aptamer (OTABA), two structure-switching OTABAs, namely OTABA4 and OTABA3, were designed by configuring a split G-quadruplex and a split G-triplex, respectively, at the two ends of OTABA to construct aptasensors for the detection of OTA. The OTABA, G-quadruplex, and G-triplex all can capture the thioflavin T (ThT) probe, thereby enhancing the fluorescence intensity of ThT. Bonding with OTA could change the conformations of OTABA and G-quadruplex or G-triplex regions, resulting in the release of the captured ThT and diminution of its fluorescence intensity. Dual conformation changes in structure-switching OTABA synergistically amplified the fluorescence signal and improved the sensitivity of the aptasensor, especially for that with OTABA3. The detection limits of the OTABA4-ThT and OTABA3-ThT systems for OTA were 0.28 and 0.059 ng ml-1 , with a 1.4-fold and 6.7-fold higher sensitivity than that of the original OTABA-ThT system, respectively. They performed well in corn and peanut samples and met the requirements of the food safety inspections.

Metal-Enhanced Aggregation-Induced Emission Strategy for the HIV-I RNA-Binding Ligand Assay.

The HIV-Ι trans-activation responsive (TAR) RNA-trans-activator of transcription (Tat) protein complex is crucial for the efficient transcription of the integrated human immunodeficiency virus-I genome and is an established therapeutic target for AIDS diagnosis and treatment. Developing a sensitive strategy for the TAR RNA-binding ligand assay could provide antiviral leads with a radically new mechanism for the treatment of AIDS. Herein, a new TAR RNA-binding ligand assay platform was established using a signal amplification strategy that combines aggregation-induced emission (AIE) with a metal-enhanced fluorescence (MEF) concept. The tetraphenylethylene (TPE) derivative was labeled on the Tat peptide as a fluorescent molecule, while the TAR RNA was immobilized on the surface of the Fe3O4@Au@Ag@SiO2 nanoparticles (NPs) to specifically bind the TPE-Tat peptide. The TPE-Tat peptide was weakly emissive itself while emitting strongly in the NP-TAR-TPE-Tat complex by the AIE and MEF signal amplification effect. It was confirmed by known Tat peptide competitors that this system could be applied to the screening and detection of TAR RNA-binding ligands because they could replace the TPE-Tat peptide from the complex and make the system fluorescence decrease. When this system was adopted to test four candidate ligands, it was found that bisantrene had a favorable TAR RNA-binding ability. The proposed AIE-MEF strategy not only provides a sensitive and reliable method for the TAR RNA-binding ligand assay but also can avoid the influence of ligands on fluorescent detection in the conventional displacement assay.

A Light-Up Strategy with Aggregation-Induced Emission for Identification of HIV-I RNA-Binding Small Molecules.

Fluorescence methods are important tools to identify RNA-binding small molecules and further employed to study RNA-protein interactions. Most reported fluorescence strategies are based on covalent labeling of ligand or RNA, which can impede the binding between them to some extent, or light-off fluorescent indicator displacement methods, which ask for particular indicators. Herein, a label-free fluorescence strategy based on the light-on aggregation-induced emission (AIE) feature of tetraphenylethene (TPE) derivative to screen RNA-binding small molecules is presented. As a result of electrostatic interaction, the selected peptides can induce self-assembly of the TPE derivative to produce strong fluorescent emission; when the peptides are bound to RNA molecules, the TPE derivative is in the deaggregated form and shows no or minimum fluorescence. Based on the phenomenon, a competitive displacement assay combined with the TPE reporter was employed to characterize selected small molecules for their binding abilities to HIV-I RNAs. This AIE feature enables the fluorescence-off state of the TPE derivative in the presence of RNA-peptide complex to be "lightened up" quickly as the RNA-binding molecule is introduced and the peptide is competitively released. This strategy was carried out to test several small molecule binders, and the results are consistent with previous reports. This report gives an inspiring example of AIE-based fluorescent assay for HIV-I RNA-binding molecule screening, which may further be explored to build a drug screening platform for RNA-protein interference.

Ligand-RNA interaction assay based on size-selective fluorescence core-shell nanocomposite.

The application of the dye-labeled fluorescence method in a ligand-RNA interaction assay is a complex and costly process prone to steric hindrance. Fluorescent nanomaterials offer an attractive alternative due to their simple, low-cost synthesis and effective screening properties. Here, CdTe@ZIF-8 core-shell nanocomposites were used as fluorescence signal transducer in the ligand-TAR RNA interaction assay. Different experimental strategies were developed based on the size-selective nature of the CdTe@ZIF-8 nanocomposites. When ligands can quench fluorescence, two assays of fluorescence recovery with TAR RNA and Tat peptide competitive displacement are carried out successively, which can not only distinguish ligands binding to TAR RNA but also screen potential Tat protein antagonists. When ligands cannot quench fluorescence, the mitoxantrone-TAR RNA complex is used in the competitive displacement assay. Ligands that displaced mitoxantrone from the mitoxantrone-TAR RNA complex signaled the interaction with TAR RNA. Eight ligands, including known and unknown TAR RNA-binding ligands, were tested via the above strategies. The results showed that this method was effective at distinguishing the known RNA-binding partner and screening the Tat antagonist from the test ligands. This simple and effective strategy is expected to be suitable for actual drug screening. Graphical abstract.

Chimeric Aptamers-Based and MoS2 Nanosheet-Enhanced Label-Free Fluorescence Polarization Strategy for Adenosine Triphosphate Detection.

Adenosine triphosphate (ATP) as a primary energy source plays a unique role in the regulation of all cellular events. The necessity to detect ATP requires sensitive and accurate quantitative analytical strategies. Herein, we present our study of developing a MoS2 nanosheet-enhanced aptasensor for fluorescence polarization-based ATP detection. A bifunctional DNA strand was designed to consist of chimeric aptamers that recognize and capture ATP and berberine, a fluorescence enhancer. In the absence of ATP, the DNA strand bound to berberine will be hydrolyzed when Exonuclease I (Exo I) is introduced, releasing berberine as a result. In contrast, when ATP is present, ATP aptamer folds into a G-quadruplex structure; thus, the complex can resist degradation by Exo I to maintain berberine for fluorescent detection purpose. In addition, to magnify the fluorescence polarization (FP) signal, MoS2 nanosheets were also adopted in the system. This nanosheets-enhanced FP strategy is simple and facile which does not require traditional dye-labeled DNA strands and complex operation steps. The developed fluorescence polarization aptasensor showed high sensitivity for the quantification of ATP with a detection limit of 34.4 nM, performing well both in buffer solution and in biological samples.

A ratiometric fluorometric heparin assay based on the use of CdTe and polyethyleneimine-coated carbon quantum dots.

CdTe quantum dots (QDs) were integrated with polyethyleneimine-coated carbon dots (PEI-CDs) to form a dually emitting probe for heparin. The red fluorescence of the CdTe QDs is quenched by the PEI-CDs due to electrostatic interactions. In the presence of heparin, the blue fluorescence of PEI-CDs remains unaffected, while its quenching effect on the fluorescence of CdTe QDs is strongly reduced. A ratiometric fluorometric assay was worked out. The ratio of the fluorescences at 595 and 436 nm serves as the analytical signal. Response is linear in the concentration range of 50-600 ng·mL-1 (0.1-1.2 U·mL-1) of heparin. The limit of detection is 20 ng·mL-1 (0.04 U·mL-1). This makes the method a valuable tool for heparin monitoring during postoperative and long-term care. This assay is relatively free from the interference by other analogues which commonly co-exist with heparin in samples, and it is more robust than single-wavelength based assays. Graphical abstract In the presence of heparin, the fluorescence of polyethyleneimine-coated carbon dots (PEI-CDs) at 436 nm remains unaffected, while its quenching effect on the fluorescence of CdTe at 595 nm is strongly reduced.

A G-triplex and G-quadruplex concatemer-enhanced fluorescence probe coupled with hybridization chain reaction for ultrasensitive aptasensing of ochratoxin A.

Ochratoxin A (OTA), a typical mycotoxin contaminant found in various agricultural products and foods, poses a serious threat to human health. In this study, an aptasensor based on a novel fluorescence probe comprising a G-rich DNA sequence (G43) and thioflavin T (ThT) was designed via hybridization chain reaction (HCR) for the ultrasensitive detection of OTA. G43 is a concatemer of G-quadruplex and G-triplex (a G-quadruplex-like structure with one G-quartet removed), which can drastically enhance the fluorescence intensity of ThT. For this strategy to work, the OTA aptamer is pro-locked in a hairpin structure, denoted "hairpin-locked aptamer" (HL-Apt). OTA binds to HL-Apt, opens the hairpin structure, releases the trigger sequence, and initiates the HCR reaction to form a long DNA duplex and numerous side chains. The side chains combine entirely with the complementary DNA and liberate the pro-locked G43 DNA, dramatically enhancing the intensity of the ThT fluorescence signal. The fluorescence intensity correlates linearly with the OTA concentration between 0.02 and 2.00 ng mL-1, and the method has a detection limit of 0.008 ng mL-1. The developed aptasensor was used to detect OTA in foodstuffs with satisfactory results.

A dual-function oligonucleotide-based ratiometric fluorescence sensor for ATP detection.

Adenosine triphosphate (ATP) is the main energy currency of life that plays a vital role in supporting physiological activities in living organisms, including humans. Therefore, accurate and sensitive detection of ATP concentration is necessary in biochemical research and clinical diagnosis. Herein, a ratiometric fluorescence aptasensor was developed for ATP detection. A dual-function DNA strand comprising an ATP-binding aptamer (ABA) and berberine-binding aptamer (BBA) was designed and optimized, in which ABA can capture ATP and thioflavin T (ThT), whereas BBA can capture berberine. Interestingly, the fluorescence intensity of both berberine and ThT were enhanced as they were captured by this dual-function DNA strand. In the presence of ATP, the ABA on the 3'-end of the DNA bound specifically to its target, causing ThT release and a significant drop in ThT fluorescence. However, ATP had no significant effect on the interaction between berberine and DNA, remaining the enhanced fluorescence intensity of berberine stable. Based on this interesting phenomenon, a ratiometric fluorescence sensor was constructed that used the enhanced fluorescence intensity of berberine as reference to measure the fluorescence intensity of ThT for ATP detection. This ratiometric fluorescence strategy had excellent selectivity and high sensitivity towards ATP with a detection limit (3σ) as low as 24.8 nM. The feasibility of application of this method in biological samples was evaluated in human serum and urine samples, where it exhibited a good detection performance.

3-Aminophenyl Boronic Acid Functionalized Quantum-Dot-Based Ratiometric Fluorescence Sensor for the Highly Sensitive Detection of Tyrosinase Activity.

Using the commercially available and economical 6-hydroxycoumarin (6-HC) as the substrate, a dual-emission ratiometric fluorescence sensor was developed to detect tyrosinase (TYR) activity based on 3-aminophenyl boronic acid functionalized quantum dots (APBA-QDs). TYR can catalyze 6-HC, a monohydroxy compound, to form a fluorescence-enhancing o-hydroxy compound, 6,7-dihydroxycoumarin. Owing to the special covalent binding between the o-hydroxyl and boric acid groups, APBA-QDs react with 6,7-dihydroxycoumarin to form a five-membered ring ester dual-emission fluorescence probe for TYR. With an increase in TYR activity, the fluorescence at 675 nm originating from the QDs is gradually quenched, whereas that at 465 nm owing to 6,7-dihydroxycoumarin increases. Referencing the decreasing signal of the dual-emission probe at 675 nm to measure the increasing signal at 465 nm, a ratiometric fluorescence method was established to detect the TYR activity with high sensitivity and selectivity. Under the conditions optimized via response surface methodology, a linear range of 0-0.05 U/mL was obtained for the TYR activity. The detection limit was as low as 0.003 U/mL. This sensing strategy can also be adopted for the rapid screening of the TYR inhibitors.