Green analytical assay for the viability assessment of single maize seeds using double-threshold strategy for catalase activity and malondialdehyde content.

The development of nondestructive technology for the detection of seed viability is challenging. In this study, to establish a green and effective method for the viability assessment of single maize seeds, a two-stage seed viability detection method was proposed. The catalase (CAT) activity and malondialdehyde (MDA) content were selected as the most key biochemical components affecting maize seed viability, and regression prediction models were developed based on their hyperspectral information and a data fusion strategy. Qualitative discrimination models for seed viability evaluation were constructed based on the predicted response values of the selected key biochemical components. The results showed that the double components thresholds strategy achieved the highest discrimination accuracy (92.9%), providing a crucial approach for the rapid and environmentally friendly detection of seed viability.

Non-destructive detection of single-seed viability in maize using hyperspectral imaging technology and multi-scale 3D convolutional neural network.

The viability of Zea mays seed plays a critical role in determining the yield of corn. Therefore, developing a fast and non-destructive method is essential for rapid and large-scale seed viability detection and is of great significance for agriculture, breeding, and germplasm preservation. In this study, hyperspectral imaging (HSI) technology was used to obtain images and spectral information of maize seeds with different aging stages. To reduce data input and improve model detection speed while obtaining more stable prediction results, successive projections algorithm (SPA) was used to extract key wavelengths that characterize seed viability, then key wavelength images of maize seed were divided into small blocks with 5 pixels ×5 pixels and fed into a multi-scale 3D convolutional neural network (3DCNN) for further optimizing the discrimination possibility of single-seed viability. The final discriminant result of single-seed viability was determined by comprehensively evaluating the result of all small blocks belonging to the same seed with the voting algorithm. The results showed that the multi-scale 3DCNN model achieved an accuracy of 90.67% for the discrimination of single-seed viability on the test set. Furthermore, an effort to reduce labor and avoid the misclassification caused by human subjective factors, a YOLOv7 model and a Mask R-CNN model were constructed respectively for germination judgment and bud length detection in this study, the result showed that mean average precision (mAP) of YOLOv7 model could reach 99.7%, and the determination coefficient of Mask R-CNN model was 0.98. Overall, this study provided a feasible solution for detecting maize seed viability using HSI technology and multi-scale 3DCNN, which was crucial for large-scale screening of viable seeds. This study provided theoretical support for improving planting quality and crop yield.

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.

Improved membrane stability of alginate-chitosan microcapsules by crosslinking with tannic acid.

OBJECTIVE: The insufficient stability of alginate-chitosan (ALG-CS) microcapsules in biorelevant media limits their applications in the biomedical field. Attempts were made to improve the membrane stability of ALG-CS microcapsules by noncovalent crosslinking with tannic acid. RESULTS: The membrane stability of ALG-CS microcapsules in culture medium and serum was significantly improved by crosslinking with tannic acid. Moreover, the reason for the significant improvement in membrane stability had been demonstrated to be that the stability of chitosan-tannic acid (CS-TA) polyelectrolyte complexes was less affected by the competitive binding of those weak acid ions such as HCO3-. In addition, the optimal conditions for preparing alginate-chitosan-tannic acid (ALG-CS-TA) microcapsules were tannic acid concentration of 0.5% (w/v) and pH = 7. CONCLUSION: The study provides a novel approach for improving the stability of the ALG-CS microcapsules in biorelevant media to expand their scope of application in the biological field.

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.

Reference material of Prussian blue nanozymes for their peroxidase-like activity.

Nanozymes with wide applications have rapidly attracted tremendous attention from various fields in the last decade. However, research on the standardization of nanozymes is still lacking. Currently, the accurate evaluation and effective tracing of the enzyme-like activity of nanozymes have become a common concern. This work aims to develop a certified reference material (CRM) of Prussian blue nanozymes (PBNEs) for their peroxidase (POD)-like activity. The homogeneity and stability studies demonstrated that the property value of POD-like activity is consistent across different packing units, and remains unchanged during the one-year validity period of storage in the dark at 4 °C. The certified value of the POD-like activity of the PBNE CRM is assigned as 174 ± 13 U mg-1 (k = 2) by interlaboratory comparison studies and traceable uncertain evaluation. Furthermore, the need for quality control of the POD-like activity of nanozymes was exemplified by comparing the influence of two additional PBNEs on the dry and wet chemical detection of glucose (Glu). As the first quality assurance tool of nanozymes, the PBNE CRM is expected to replace natural horse radish peroxidase (HRP) as an effective benchmark for assessing the analytical method and laboratory competence. In addition, this work also inspires the further standardization of nanozymes.

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.

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.

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.

An effective recycle way of waste coke ash and coking wastewater for preparing coke ash coking wastewater slurry.

The carbon riched coke ash (CA) and organic components riched coking wastewater (CW) recovered from coking plants wastes were utilized for the preparation of coke ash coking wastewater slurry (CACWS), aiming for the fuel and waste reduction and recovery. The effects induced by the properties of CA and compositions of CW on the performances of CACWS, such as slurryability, rheology, stability and dispersant adsorption were investigated and discussed. Characterizations like zeta potentials and contact angles on the surface of CA were also conducted to draw a comprehensive formation mechanism of CACWS. Results showed that the CA was suitable for preparing slurry due to the lack of micropore structures and hydrophobicity in the surface. The maximum content of CA in the as-prepared CACWS could reach 66% and CACWS exhibited non-Newtonian pseudoplastic fluid behaviour. By reducing the particle size distribution, the slurryability of CA could be effectively improved. Although the components in CW enhance the wettability of CA surface, compared with cations in CW, the organic components had more influence on CACWS, which also obviously increased the viscosity of CACWS. The maximum CA content in CACWS (ω = 66 wt%) reduced by 9% comparing to CA water slurry (ω = 75 wt%) which improved the storage stability about 10%. In addition, results show that the dispersant Triton X-405 reduced viscosity and improved stability while in comparison with the anionic polycarboxylate dispersant. Overall, this study may provide an innovative and effective utilization of CA and CW from the coke plants wastes.

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.

Catalytic Mechanisms of Nanozymes and Their Applications in Biomedicine.

The research on nanozymes has increased dramatically in recent years and a new interdiscipline, nanozymology, has emerged. A variety of nanomaterials have been designed to mimic the characteristics of natural enzymes, which connects an important bridge between nanotechnology and biological science. Unlike natural enzymes, the nanoscale properties of nanozymes endow them with the potential to regulate their enzymatic-like activity from different perspectives. The mechanisms behind those methods are intriguing. In this Review, we introduce these mechanisms from the aspects of surface chemistry, surface modification, molecular imprinting, and hybridization and then focus attention on some specific catalytic mechanisms of several representative nanozymes. The applications of nanozymes ranging from bioassay, imaging, to disease therapy are also discussed in detail to prove the fact that the inherent physicochemical properties of nanomaterials not only make nanozymes the analogues of biological enzymes, but also endow them with incomparable advantages and broad prospects in biomedical fields. Finally, four characteristics and some challenges of nanozymes are summarized.

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.

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.