High-throughput quantitative binding analysis of DNA aptamers using exonucleases.

Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer-ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer-ligand pairs.

Generation of High-Affinity Aptamers for Indazole Synthetic Cannabinoids.

Synthetic cannabinoids are a widely abused class of dangerous psychoactive substances, especially among youths and young adults. Dozens of such drugs have been identified to date, and new ones continue to emerge. The ability to detect these drugs is important for interdiction efforts and the diagnosis of drug overdose, but existing analytical methods lack broad cross-reactivity to diverse members of this drug family. Here, we have utilized library-immobilized SELEX to generate DNA aptamers that can broadly recognize various members of the indazole-3-carboxamide synthetic cannabinoid family. Using two representatives of this family, AB-FUBINACA and 5F-AMB, we identify two aptamers FUB4 and AMB2F with respective dissociation constants (KDs) of 138 ± 15 and 411 ± 20 nM for their targets. These aptamers can recognize many indazole-based synthetic cannabinoids with high affinity and excellent specificity against natural cannabinoids as well as other structurally similar interferents like serotonin and tryptophan. We use these two aptamers to develop fluorescence strand-displacement sensors that successfully detect these synthetic cannabinoids at concentrations as low as 50 nM in human serum. The sensors can also detect up to 14 different drugs from this family─a major improvement over the six recognized by an existing commercial immunoassay.

Aptamer-Based Fentanyl Detection in Biological Fluids.

Fentanyl is a widely abused analgesic and anesthetic drug with a narrow therapeutic window that creates easy opportunities for overdose and death. Rapid, accurate, and sensitive fentanyl detection in biosamples is crucial for therapeutic drug monitoring and overdose diagnosis. Unfortunately, current methods are limited to either sophisticated laboratory-based tests or antibody-based immunoassays, which are prone to false results and are mainly used with urine samples. Here, we have utilized library-immobilized SELEX to isolate new aptamers─nucleic acid-based bioreceptors that are well-suited for biosensing─that can specifically bind fentanyl under physiological conditions. We isolated multiple aptamers with nanomolar affinity and excellent specificity against dozens of interferents and incorporated one of these into an electrochemical aptamer-based sensor that can rapidly detect fentanyl at clinically relevant concentrations in 50% diluted serum, urine, and saliva. Given the excellent performance of these sensors, we believe that they could serve as the basis for point-of-care devices for monitoring fentanyl during medical procedures and determining fentanyl overdose.

Potential candidate genes and pathways related to cytoplasmic male sterility in Dianthus spiculifolius as revealed by transcriptome analysis.

KEY MESSAGE: We introduced the candidate gene DsHSP70 into Arabidopsis thaliana, resulting in male gametophyte sterility and abnormal degeneration of sepals and petals. Cytoplasmic male sterility (CMS) is a useful tool for hybrid production. However, the regulatory mechanism of CMS in Dianthus spiculifolius remains unclear. In this study, we investigated whether male-sterile line of D. spiculifolius has a malformed tapetum and fails to produce normal fertile pollen. RNA sequencing technology was used to compare the gene expression patterns of the D. spiculifolius male-sterile line and its male fertility maintainer line during anther development. A total of 12,365 differentially expressed genes (DEGs) were identified, among which 1765 were commonly expressed in the S1, S2 and S3 stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these DEGs were mainly involved in oxidation-reduction processes, signal transduction and programmed cell death. Additionally, weighted correlation network analysis (WGCNA) showed that three modules may be related to male sterility. A putative regulatory pathway for the male sterility traits was constructed based on the reproductive development network. After introducing the candidate DsHSP70 gene into Arabidopsis thaliana, we found that overexpressing plants showed anther abortion and shorter filaments, and accompanied by abnormal degeneration of sepals and petals. In summary, our results identified potential candidate genes and pathways related to CMS in D. spiculifolius, providing new insights for further research on the mechanism of male sterility.

Near-Infrared Dye-Aptamer Assay for Small Molecule Detection in Complex Specimens.

Aptamers are single-stranded oligonucleotides isolated in vitro that bind specific targets with high affinity and are commonly used as receptors in biosensors. Aptamer-based dye-displacement assays are a promising sensing platform because they are label-free, sensitive, simple, and rapid. However, these assays can exhibit impaired sensitivity in biospecimens, which contain numerous interferents that cause unwanted absorbance, scattering, and fluorescence in the UV-vis region. Here, this problem is overcome by utilizing near-infrared (NIR) signatures of the dye 3,3'-diethylthiadicarbocyanine iodide (Cy5). Cy5 initially complexes with aptamers as monomers and dimers; aptamer-target binding displaces the dye into solution, resulting in the formation of J-aggregates that provide a detectable NIR signal. The generality of our assay is demonstrated by detecting three different small-molecule analytes with their respective DNA aptamers at clinically relevant concentrations in serum and urine. These successful demonstrations show the utility of dye-aptamer NIR biosensors for high-throughput detection of analytes in clinical specimens.

In vitro isolation of class-specific oligonucleotide-based small-molecule receptors.

Class-specific bioreceptors are highly desirable for recognizing structurally similar small molecules, but the generation of such affinity elements has proven challenging. We here develop a novel 'parallel-and-serial' selection strategy for isolating class-specific oligonucleotide-based receptors (aptamers) in vitro. This strategy first entails parallel selection to selectively enrich cross-reactive binding sequences, followed by serial selection that enriches aptamers binding to a designated target family. As a demonstration, we isolate a class-specific DNA aptamer against a family of designer drugs known as synthetic cathinones. The aptamer binds to 12 diverse synthetic cathinones with nanomolar affinity and does not respond to 11 structurally similar non-target compounds, some of which differ from the cathinone targets by a single atom. This is the first account of an aptamer exhibiting a combination of broad target cross-reactivity, high affinity and remarkable specificity. Leveraging the qualities of this aptamer, instantaneous colorimetric detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. Our findings significantly expand the binding capabilities of aptamers as class-specific bioreceptors and further demonstrate the power of rationally designed selection strategies for isolating customized aptamers with desired binding profiles. We believe that our aptamer isolation approach can be broadly applied to isolate class-specific aptamers for various small molecule families.

Molecular Characterization and Expression Patterns of the HkSVP Gene Reveal Distinct Roles in Inflorescence Structure and Floral Organ Development in Hemerocallis fulva.

SHORT VEGETATIVE PHASE (SVP) genes are members of the well-known MADS-box gene family that play a key role in regulating vital developmental processes in plants. Hemerocallis are perennial herbs that exhibit continuous flowering development and have been extensively used in landscaping. However, there are few reports on the regulatory mechanism of flowering in Hemerocallis. To better understand the molecular basis of floral formation of Hemerocallis, we identified and characterized the SVP-like gene HkSVP from the Hemerocallis cultivar 'Kanai Sensei'. Quantitative RT-PCR (qRT-PCR) indicated that HkSVP transcript was mainly expressed in the vegetative growth stage and had the highest expression in leaves, low expression in petals, pedicels and fruits, and no expression in pistils. The HkSVP encoded protein was localized in the nucleus of Arabidopsis protoplasts and the nucleus of onion epidermal cells. Yeast two hybrid assay revealed that HKSVP interacted with Hemerocallis AP1 and TFL1. Moreover, overexpression of HkSVP in Arabidopsis resulted in delayed flowering and abnormal phenotypes, including enriched trichomes, increased basal inflorescence branches and inhibition of inflorescence formation. These observations suggest that the HkSVP gene may play an important role in maintaining vegetative growth by participating in the construction of inflorescence structure and the development of flower organs.

Fabrication of Aptamer-Modified Paper Electrochemical Devices for On-Site Biosensing.

Electrochemical aptamer-based (E-AB) sensors offer a powerful and general means for analyte detection in complex samples for various applications. Paper-based E-AB sensors could enable portable, low-cost, and rapid detection of a broad range of targets, but it has proven challenging to fabricate suitable three-electrode systems on paper. Here, we demonstrate a simple, economic, and environmentally friendly strategy for fabricating aptamer-modified paper electrochemical devices (PEDs) via ambient vacuum filtration. The material, shape, size, and thickness of the three-electrode PED system can be fully customized. We developed aptamer-modified PEDs that enable sensitive and specific detection of small molecules in minimally processed biosamples. The sensitivity and stability of the PEDs are comparable to E-AB sensors based on commercial gold electrodes. We believe our strategy can lead to the development of high performance PEDs for the on-site detection of a variety of analytes.

Advances and Challenges in Small-Molecule DNA Aptamer Isolation, Characterization, and Sensor Development.

Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor-intensive to develop aptamer-based sensors for small-molecule detection. Here, we review the challenges and advances in the isolation and characterization of small-molecule-binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer-target binding and structure. Afterwards, we discuss various small-molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer-based small-molecule sensors for real-world applications.

Tuning Biosensor Cross-Reactivity Using Aptamer Mixtures.

It is challenging to tune the response of biosensors to a set of ligands, for example, cross-reactivity to a given target family while maintaining high specificity against interferents, due to the lack of suitable bioreceptors. We present a novel approach for controlling the cross-reactivity of biosensors by employing defined mixtures of aptamers that have differing binding properties. As a demonstration, we develop assays for the specific detection of a family of illicit designer drugs, the synthetic cathinones, with customized responses to each target ligand and interferent. We first use a colorimetric dye-displacement assay to show that the binding spectra of dual-aptamer mixtures can be tuned by altering the molar ratio of these bioreceptors. Optimized assays achieve broad detection of synthetic cathinones with minimal response toward interferents and generally demonstrate better sensing performance than assays utilizing either aptamer alone. The generality of this strategy is demonstrated with a dual-aptamer electrochemical sensor. Our approach enables customization of biosensor responsiveness to an extent that has yet to be achieved through any previously reported aptamer engineering techniques such as sequence mutation or truncation. Since multiple aptamers for the designated target family can routinely be identified via high-throughput sequencing, we believe our strategy offers a generally applicable method for generating near-ideal aptamer biosensors for various analytical applications, including medical diagnostics, environmental monitoring, and drug detection.

Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation.

We report a broadly applicable enzyme digestion strategy for introducing structure-switching functionality into small-molecule-binding aptamers. This procedure is based on our discovery that exonuclease III (Exo III) digestion of aptamers is greatly inhibited by target binding. As a demonstration, we perform Exo III digestion of a pre-folded three-way-junction (TWJ)-structured cocaine-binding aptamer and a stem-loop-structured ATP-binding aptamer. In the absence of target, Exo III catalyzes 3'-to-5' digestion of both aptamers to form short, single-stranded products. Upon addition of target, Exo III digestion is halted four bases prior to the target-binding domain, forming a major target-bound aptamer digestion product. We demonstrated that target-binding is crucial for Exo III inhibition. We then determine that the resulting digestion products of both aptamers exhibit a target-induced structure-switching functionality that is absent in the parent aptamer, while still retaining high target-binding affinity. We confirm that these truncated aptamers have this functionality by using an exonuclease I-based digestion assay and further evaluate this characteristic in an electrochemical aptamer-based cocaine sensor and a fluorophore-quencher ATP assay. We believe our Exo III-digestion method should be applicable for the generation of structure-switching aptamers from other TWJ- or stem-loop-containing small-molecule-binding aptamers, greatly simplifying the generation of functionalized sensor elements for folding-based aptasensors.

Label-Free, Visual Detection of Small Molecules Using Highly Target-Responsive Multimodule Split Aptamer Constructs.

Colorimetric aptamer-based sensors offer a simple means of on-site or point-of-care analyte detection. However, these sensors are largely incapable of achieving naked-eye detection, because of the poor performance of the target-recognition and signal-reporting elements employed. To address this problem, we report a generalizable strategy for engineering novel multimodule split DNA constructs termed "CBSAzymes" that utilize a cooperative binding split aptamer (CBSA) as a highly target-responsive bioreceptor and a new, highly active split DNAzyme as an efficient signal reporter. CBSAzymes consist of two fragments that remain separate in the absence of target, but effectively assemble in the presence of the target to form a complex that catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid, developing a dark green color within 5 min. Such assay enables rapid, sensitive, and visual detection of small molecules, which has not been achieved with any previously reported split-aptamer-DNAzyme conjugates. In an initial demonstration, we generate a cocaine-binding CBSAzyme that enables naked-eye detection of cocaine at concentrations as low as 10 µM. Notably, CBSAzyme engineering is straightforward and generalizable. We demonstrate this by developing a methylenedioxypyrovalerone (MDPV)-binding CBSAzyme for visual detection of MDPV and 10 other synthetic cathinones at low micromolar concentrations, even in biological samples. Given that CBSAzyme-based assays are simple, label-free, rapid, robust, and instrument-free, we believe that such assays should be readily applicable for on-site visual detection of various important small molecules such as illicit drugs, medical biomarkers, and toxins in various sample matrices.

Innovative engineering and sensing strategies for aptamer-based small-molecule detection.

Aptamers are nucleic acid-based affinity reagents that have gained widespread attention as biorecognition elements for the detection of targets such as ions, small molecules, and proteins. Over the past three decades, the field of aptamer-based sensing has grown considerably. However, the advancement of aptamer-based small-molecule detection has fallen short of the high demand for such sensors in applications such as diagnostics, environmental monitoring, and forensics. This is due to two challenges: the complexity of developing generalized sensing platforms and the poor sensitivities of assays targeting small molecules. This paper will review new approaches for the streamlined development of high-performance aptamer-based sensors for small-molecule detection. We here provide historical context, explore the current state-of-the art, and offer future directions-with emphasis placed on new aptamer engineering methods, the use of cooperative binding, and label-free approaches using fully-folded, high-affinity aptamers for small-molecule sensing.

High-Rise Residential Outdoor Space Value System: A Case Study of Yangtze River Delta Area.

The outbreak of COVID-19 has drawn wider attention from residents with growing demand for outdoor space in residential areas because of restrictions on residents' mobility, especially in China. However, the high-rise residential complex in China is featured with a high population density along with less outdoor space per household. This means that the current status of outdoor space in residential areas is far from satisfying residents' growing needs. This is consistent with our preliminary survey that highlights general low satisfaction of residents with outdoor space. According to the hierarchical theory of needs, a literature review, and a questionnaire survey, a framework is proposed in this study to examine the universal value system of high-rise residential outdoor space using the Yangtze River Delta Area as a case study. This framework consists of six dimensions, i.e., space physical comfort (physical environment and space size), space function (functional complexity and scale, age-range, and time-range), space safety (daily, social, and hygiene safety), space diversity (spatial layerings, forms, and scales diversity), accessibility (spatial attraction and concentration and path clarity) and sustainability (cultural, social, ecological, and financial sustainability). Consequently, a questionnaire was designed according to the framework and 251 valid questionnaires were received. Then, structural equation modeling (SEM) was undertaken to examine the impact of each dimension on the value of outdoor space and the framework was optimized into four dimensions, i.e., space physical comfort, space function, space safety, and DAT (space diversity, accessibility, and sustainability). Finally, the mechanism of how outdoor space quality influences the high-rise residential complex is analyzed. These findings provide useful input for the future planning and design of high-rise residential areas.

Development of active packaging with chitosan, guar gum and watermelon rind extract: Characterization, application and performance improvement mechanism.

The objective of this study was to make a film matrix containing chitosan (CS) and guar gum (GG), and to improve the physicochemical properties of the film using watermelon rind extract (WRE) as a cross-linker and active substance for the preservation of fresh-cut bananas. The results of Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy showed that the WRE and CG matrix formed intermolecular hydrogen bond interactions, which made the structure of the resulting films more compact. With increasing amounts of WRE, the mechanical properties of the films were significantly increased, but the permeability of water vapor and oxygen was significantly decreased (p < 0.05). Notably, when the amount of extract reached 4 wt%, the DPPH radical scavenging activity of the composite film significantly increased to 83.24 %, and the antibacterial activity also reached its highest value. Fresh-cut bananas were stored at room temperature with polyethylene film, CG and CG-WRE. The CG with 4 wt% WRE effectively inhibited the changes in appearance, firmness, weight, color and total soluble solids content of fresh-cut bananas during storage. Therefore, CG-WRE as a novel active food packaging material, has good physicochemical properties and great potential to extend the shelf life of foods.

Suite of Aptamer-Based Sensors for the Detection of Fentanyl and Its Analogues.

Fentanyl and its analogues are potent synthetic opioids that are commonly abused and are currently the number one cause of drug overdose death in the United States. The ability to detect fentanyl with simple, rapid, and low-cost tools is crucial for forensics, medical care, and public safety. Conventional on-site testing options for fentanyl detection─including chemical spot tests, lateral-flow immunoassays, and portable Raman spectrometers─each have their own unique flaws that limit their analytical utility. Here, we have developed a series of new aptamer-based assays and sensors that can detect fentanyl as well as several of its analogues in a reliable, accurate, rapid, and economic manner. These include colorimetric, fluorescent, and electrochemical sensors, which can detect and quantify minute quantities of fentanyl and many of its analogues with no response to other illicit drugs, cutting agents, or adulterants─even in interferent-ridden binary mixtures containing as little as 1% fentanyl. Given the high performance of these novel analytical tools, we foresee the potential for routine use by medical and law enforcement personnel as well as the general public to aid in rapid and accurate fentanyl identification.

Intelligent packaging based on chitosan/fucoidan incorporated with coleus grass (Plectranthus scutellarioides) leaves anthocyanins and its application in monitoring the spoilage of salmon (Salmo salar L.).

The innovation of this study was to develop a novel biodegradable intelligent packaging based on chitosan/fucoidan combined with different amounts (1, 3 and 5 wt% on chitosan basis) of coleus grass (Plectranthus scutellarioides) leaves anthocyanins (CGL) to monitor the spoilage of salmon (Salmo salar L.). The addition of fucoidan improved the barrier and mechanical properties of the chitosan films (CS) due to hydrogen bonds and intermolecular electrostatic interactions. Moreover, the addition of CGL not only improved the physical properties but also improved the biological activity of chitosan/fucoidan film (CF). The DPPH and ABTS radical scavenging activity of CF contained 5 wt% CGL was 1.83 and 1.75 times than CF, respectively. The inhibition zone size of CF films containing 5 wt% CGL (CF-5%CGL) was approximately 2.04 (Escherichia coli) and 2.16 (Staphylococcus aureus) times higher than that of CF. Moreover, CF-CGL displayed obvious color changes in different pH environments and is highly sensitive to ammonia gas. The CF-CGL has visible color changes during the monitoring of salmon spoilage and extended the shelf life of salmon. According to our findings, CF-CGL film might be employed as a possible intelligent packaging material for monitoring and preserving salmon in the future.

Development and characterization of active packaging based on chitosan/chitin nanofibers incorporated with scallion flower extract and its preservation in fresh-cut bananas.

The aim of this study was to develop a novel active packaging using chitosan (CS) and esterified chitin nanofibers (CF) combined with different contents (1, 2 and 4 wt% on CS basis) of scallion flower extract (SFE) to protect banana samples. The addition of CF significantly improved the barrier and mechanical properties of the CS films (p < 0.05) due to hydrogen bonds and electrostatic interactions. Moreover, the addition of SFE not only improved the physical properties of the CS film but also improved the CS film biological activity. The oxygen barrier property and antibacterial ability of CF-4%SFE were approximately 5.3 and 1.9 times higher than those of the CS film, respectively. In addition, CF-4%SFE had strong DPPH radical scavenging activity (74.8 ± 2.3 %) and ABTS radical scavenging activity (84.06 ± 2.08 %). Fresh-cut bananas stored in CF-4%SFE showed less weight loss, starch loss, color and appearance change than those stored in traditional polyethylene film, which indicated that CF-4%SFE was much better at storing fresh-cut bananas than conventional plastic packaging. For these reasons, CF-SFE films have great potential as a candidate to replace traditional plastic packaging and extend the shelf life of packaged foods.

Chitosan/esterified chitin nanofibers nanocomposite films incorporated with rose essential oil: Structure, physicochemical characterization, antioxidant and antibacterial properties.

Active films were developed based on chitosan, esterified chitin nanofibers and rose essential oil (REO). The joint effects of chitin nanofibers and REO on structure and physicochemical properties of chitosan film were investigated. Scanning electron microscopy and Fourier transform infrared spectroscopy showed that the chitin nanofibers and REO had significant effects on the morphology and chemical structure of chitosan composite films. The negatively charged esterified chitin nanofibers formed a compact network structure through intermolecular hydrogen bonding and electrostatic interactions with the positively charged chitosan matrix. Chitin nanofibers and REO synergistically enhanced the water resistance, mechanical properties and UV resistance of chitosan-based films, but the addition of REO increased the oxygen permeability. Furthermore, the addition of REO enhanced the inhibition of ABTS and DPPH free radicals and microorganisms by chitosan-based film. Therefore, chitosan/chitin nanofiber-based active films containing REO as food packaging materials can potentially provide protection to extend food shelf life.