Asymmetric Nanopore Sensor for Logic Detection of Dam and M.SssI Methyltransferases in Combination of DNA Walker and Autocatalytic Hybridization Reaction.

The detection of DNA methyltransferase (MTase) was crucial for understanding gene expression regulation, cancer mechanisms, and various biological processes, contributing significantly to disease diagnosis and drug development. Herein, a nanopore sensor based on cascaded signal amplification of DNA walker and autocatalytic hybridization reaction (AHR) was developed for the ultrasensitive determination of various MTases. In the presence of Dam MTase, the hairpin structure HD underwent methylation and cleavage by DpnI endonuclease, forming T-DNA fragments. These T-DNA fragments were used to activate the DNA walker, which moved across the surface of magnetic beads step by step, generating a large quantity of initiator I by cleaving the substrate. The initiator I subsequently activated the AHR. The AHR included a hybridization chain reaction (HCR) amplifier and a catalytic hairpin assembly (CHA) convertor. The HCR amplifier generated multiple novel CHA triggers, which activated the CHA convertor. This, in turn, stimulated the HCR amplifier, creating an AHR circuit that resulted in the formation of numerous DNA nanowires. These DNA nanowires were adsorbed onto the G4-PAMAM-modified nanopore surface under the influence of an electric field, thereby altering the surface charge of the nanopore and changing the ionic rectification curve. The detection limit of the Dam MTase nanopore sensor reached 0.0002 U/mL. By modification of the recognition sites of the probes, this nanopore system could also be used for the detection of M.SssI MTase. Moreover, a four-input parallel concatenated logic circuit (AND//INHIBIT-OR) had been constructed and applied for the multivariate detection of Dam MTase and M.SssI MTase, presenting a novel conceptual model for advancing the construction of nanopore logic gate systems and their applications in biosensing.

Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

Ultrasensitive and Label-Free Detection of Multiple DNA Methyltransferases by Asymmetric Nanopore Biosensor.

DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (HD) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin HD was methylated and specifically cleaved by DpnI endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr4+-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.

Fabrication of a Biomimetic Nanochannel Logic Platform and Its Applications in the Intelligent Detection of miRNA Related to Liver Cancer.

Nanochannel-based analytical techniques have great potential applications for nucleic acid sequencing and high sensitivity detection of biological molecules. However, the sensitivity of conventional solid-state nanochannel sensors is hampered by a lack of effective signal amplification strategies, which has limited its utility in the field of analytical chemistry. Here we selected a solid-state nanochannnel modified with polyethylenimine and Zr4+ in combination with graphene oxide as the sensing platform. The high-performance sensor is based upon the change of the surface charge of the nanochannel, which is resulted from DNA cascade signal amplification in solution. The target miRNA (miR-122) can be indirectly quantitated with a detection limit of 97.2 aM with an excellent selectivity. Depending on the nucleic acid's hybridization and configuration transform, the designed nanochannel sensing systems can realize the intelligent detection of multiple liver cancer-related miRNA (miR-122 and miR Let-7a) integrating with cascaded INHIBIT-OR logic gate to provide theoretical guidance and technical support for clinical diagnosis and therapeutic evaluation of liver cancer.

A tri-site fluorescent probe for simultaneous sensing of hydrogen sulfide and glutathione and its bioimaging applications.

Hydrogen sulfide (H2S) and biothiol molecules, such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), play an important role in biology. However, understanding the complicated relationship between H2S and biothiols remains an enormous challenge owing to the difficulty in sensing H2S and biothiols simultaneously. Therefore, the development of probes for detecting H2S and biothiols is of great importance in biological science. In this work, we reported a novel fluorescent probe for the sensitive and selective detection of H2S and glutathione (GSH) simultaneously in different buffer solutions. The key design principle is based on a coumarin as the fluorophore structuring a fluorescent probe with three potential sites which could react with H2S and biothiols. This probe displays a rapid response with highly sensitive and selective detection of H2S and GSH (the detection limit of 75 nM and 280 nM, respectively). Moreover, with the assistance of a confocal fluorescence microscope, we demonstrated that the probe can be successfully applied for imaging H2S and GSH in MCF-7 cells.

[Construction and application of bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer].

As a key component of life science, bioinformatics has been widely applied in genomics, transcriptomics, and proteomics. However, the requirement of high-performance computers rather than common personal computers for constructing a bioinformatics platform significantly limited the application of bioinformatics in aquatic science. In this study, we constructed a bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer. The platform consisted of three functional modules, including genomic and transcriptomic sequencing data analysis, protein structure prediction, and molecular dynamics simulations. To validate the practicability of the platform, we performed bioinformatic analysis on aquatic pathogenic organisms. For example, genes of Flavobacterium johnsoniae M168 were identified and annotated via Blast searches, GO and InterPro annotations. Protein structural models for five small segments of grass carp reovirus HZ-08 were constructed by homology modeling. Molecular dynamics simulations were performed on out membrane protein A of Aeromonas hydrophila, and the changes of system temperature, total energy, root mean square deviation and conformation of the loops during equilibration were also observed. These results showed that the bioinformatic analysis platform for aquatic pathogen has been successfully built on the MilkyWay-2 supercomputer. This study will provide insights into the construction of bioinformatic analysis platform for other subjects.

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands.

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands (5-Aminobenzimidazole and Tryptamine) to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and ISEC techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against human immunoglobulin G and human serum albumin. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. Ultimately, a high yield of 83.9 % and purity of 96.7 % were achieved in the separation of hIgG from human serum with the dual-ligand HCIC, significantly superior to the single-ligand resins. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

Hydrogen-bond tuned conjugated architectures for nitric oxide sensing in single-benzene framework: Advances and mechanistic insights.

In contrast to the conventional fluorescence enhancement resulting from the cessation of the photoinduced electron transfer effect upon capturing nitric oxide (NO) by o-phenylenediamine, we found an interesting fluorescence quench within small molecule fluorophores characterized by intramolecular hydrogen bonding. Herein, the integration of a push-pull electron system with intramolecular hydrogen bonding onto an ultra-small fluorophore was employed to fabricate a hydrogen bond-tuned single benzene core fluorescent probe with an exceptional fluorescence quantum yield of 26 %, denoted as HSC-1. By virtue of its small size and low molecular weight (mere 192 g/mol), it demonstrated superior solubility and biocompatibility. Given the optimized conditions, HSC-1 manifested extraordinary linearity in detecting NO concentrations ranging from 0.5 to 60 µM, with an outstanding detection limit of 23.8 nM. Theoretical calculations unraveled the photophysical properties of hydrogen bonding-related probe molecules and highlighted the NO sensing mechanism. This pioneering work offers an important platform for the design of small fluorescence probes only with a single benzene core applied to NO sensing, which will potentially emerge as a new frontier in the area.

Revisit of a dipropargyl rhodamine probe reveals its alternative ion sensitivity in both a solution and live cells.

This study reveals that a dipropargyl rhodamine B derivative previously described as a reaction-based irreversible palladium probe responds, however, more sensitively to mercury with a reversible "turn-on" fluorescence. The probe also shows a much better imaging ability for mercury than for palladium in live cells.

Nanofluidic sensing platform for PNK assay using nonlinear hybridization chain reaction and its application in DNA logic circuit.

In this study, a polyethyleneimine (PEI)/Zr4+-functionalized nanofluidic sensing platform based on nonlinear hybridization chain reaction (NHCR) was developed for PNK activity assay. With the existence of PNK, the hairpin HPNK was cleaved by λ exonuclease, liberating the initiator T-DNA. Then T-DNA triggered the nonlinear HCR in solution and the reaction products were absorbed onto the nanopore, which changed the surface charge of nanofluidic device and could be detected by current-voltage characteristic curves. Compared to traditional linear HCR, the nonlinear HCR exhibits a higher sensitivity and order of growth kinetics, making it a powerful signal amplifier in bioanalysis. Due to the powerful amplification efficiency of nonlinear HCR, high sensitivity of the nanopore and specific recognition site of PNK/λ-Exo, an ultrasensitive and selective PNK sensing approach had been developed and applied to precisely quantitate the PNK activity with a LOD of 0.0001 U/mL. Moreover, utilizing this nanofluidic system as a foundation, we constructed a logic circuit that utilized PNK, adenosine diphosphate (ADP), and (NH4)2SO4 as input elements. ADP and (NH4)2SO4 had a crucial function in facilitating the PNK to regulate the DNA logic gate. By modifying the target and inhibitors, the nanofluidic device could detect a variety of stimuli and execute more advanced logical operations.

Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification.

The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was methylated and the HpaII endonuclease-catalyzed stem of hairpin HM dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin HD with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.

Mercury biomagnification in the aquaculture pond ecosystem in the Pearl River Delta.

This is the first study to investigate the rate of mercury (Hg) biomagnification in the aquaculture pond ecosystem of the Pearl River Delta (PRD), China, by analyzing total mercury (THg) and methyl mercury (MeHg) concentrations in various species of fish at different trophic levels (TLs). Species representing a gradient of trophic positions in the aquaculture pond food chains were chosen for analyzing THg and MeHg concentrations. In this study, there were two kinds of the aquaculture pond food chains: (1) omnivorous (fish feeds, zooplankton, grass carp [Ctenopharyngodon idellus], and bighead carp [Aristichthys nobilis]) and (2) predatory (zooplankton, mud carp [Cirrhina molitorella], and mandarin fish [Siniperca kneri]). Bighead carp and mandarin fish had the highest MeHg and THg concentrations, i.e., an order of magnitude higher than other species, in their respective food chains. More than 90% of the THg concentrations detected in bighead carp, mandarin fish, and mud carp were in the methylated form. In this study, %MeHg increased with TLs and MeHg concentrations, reflecting that MeHg is the dominant chemical species of Hg accumulated in higher concentrations in biota, especially biota associated with higher TLs in the food chains. The trophic magnification factors were 2.32 and 2.60 for MeHg and 1.94 and 2.03 for THg in omnivorous and predatory food chains, respectively, in PRD. Hg concentrations in fish tissue correlated to Hg levels in the ambient environment, and sediment seemed to be the major source for Hg accumulated in fish. In addition, feeding habit also affected Hg accumulation in different fish species. Four significant linear relationships were obtained between log-THg and δ(15)N and between log-MeHg and δ(15)N. The slope of the regression equations, as biomagnification power, was smaller in magnitude compared with those reported for temperate and arctic marine and freshwater ecosystems, indicating that THg and MeHg biomagnifications were lower in this PRD subtropical aquaculture pond ecosystem. This was probably due to low Hg bioavailability at lower TLs as well as individual feeding behavior of fish.

Ultrasensitive detection of ochratoxin A based on biomimetic nanochannel and catalytic hairpin assembly signal amplification.

In this paper, an ultrasensitive nanochannel sensor has been proposed for label-free Ochratoxin A (OTA) assay in combination with graphene oxide (GO) and catalyzed hairpin assembly (CHA). The high-performance sensor is segmented into two parts. One is composed of graphene oxide (GO) and DNA probes. In the presence of target OTA, OTA works as a catalyst to trigger the self-assembly pathway of the two probes and initiate the cycling of CHA circuits, which results in numerous double-stranded DNAs (dsDNA) in solution. The excess ssDNA probes are removed by GO. The other part is composed of biomimetic nanochannel coated with polyethyleneimine (PEI) and Zr4+, which can quantify the concentration of OTA by detecting the dsDNA in solution. The nanofluidic device has a detection limit of as low as 6.2 pM with an excellent selectivity. The nanochannel based assay was used to analyse food samples (red wine) with satisfied results. Thus, the proposed analytical method will provide a new approach the detection of OTA and can be applied for quality control to ensure food safety.

Resettable and enzyme-free molecular logic devices for the intelligent amplification detection of multiple miRNAs via catalyzed hairpin assembly.

The integration of multi-level DNA logic gates for biological diagnosis is far from being fully realized. In particular, the simplification of logical analysis to implement advanced logic diagnoses is still a critical challenge for DNA computing and bioelectronics. Here, we developed a magnetic bead/DNA system to construct a library of logic gates, enabling the sensing of multiplex target miRNAs. In this assay, the miRNA-catalyzed hairpin assembly (CHA) was successfully applied to construct two/three-input concatenated logic circuits with excellent specificity extended to design a highly sensitive multiplex detection system. Significantly, the CHA-based multiplex detection system can distinguish individual target miRNAs (such as miR-21, miR-155, and miR let-7a) under a logic function control, which presents great applications in the development of rapid and intelligent detection. Another novel feature is that the multiplex detection system can be reset by heating the output system and the magnetic separation of the computing modules. Overall, the proposed logic diagnostics with high amplification efficiency is simple, fast, low-cost, and resettable, and holds great promise in the development of biocomputing, multiparameter sensing, and intelligent disease diagnostics.

A colorimetric/fluorescence dual-channel probe for highly discriminating detection of cysteine.

In this work, a novel fluorescence (FL) probe for selective and sensitive detection of Cys with colorimetric and FL dual signal changes was reported. The probe was synthesized by two step of sulfonamide reaction coupling between a sulfonyl benzoxadiazole (SBD) dye and dansyl chloride linked with rigid piperazine group. The probe showed a specific off-on response to Cys in aqueous solution with nanomolar LOD, and without interference by a range of amino acids and several competing analytes. Upon addition of Cys, the probe will undergo sequential substitution and intramolecular rearrangement reactions, yielding a 4-amino SBD derivative, which results in generation of strong yellow fluorescence emission at 575 nm accompanied by a two-step red shift in the absorption spectral. Moreover, it can be used for imaging of endogenous Cys in living cells.

Occurrence and distribution of phthalate esters in freshwater aquaculture fish ponds in Pearl River Delta, China.

The concentrations, congener profiles and spatial distribution of 13 phthalate esters (PAEs) in the freshwater fish ponds in the Pearl River Delta (PRD) region were investigated in water and sediment samples collect from 22 sites during Jul. 2016-Sept. 2017. The di-2-ethylhexyl phthalate (DEHP) was the predominant compounds in both water and sediment samples, accounting for 70.1% and 66.1% of ∑PAEs, respectively. The DEHP concentrations in the water samples collected from the sites of Zhongshan (35.7 µg/L), Jingmen (17.3 µg/L) and Nanhai (14.2 µg/L) were higher than that collected from other sampling sites (p <0.05), and exceed the Chinese environmental quality standards for surface water (DEHP, 8.00 µg/L). The concentrations of ΣPAEs (mean and median were 11.8 mg/kg dw and 7.95 mg/kg dw) in sediment was higher than that in sediment of river and estuary in the PRD region (p <0.05). The median concentrations of DEHP and di-n-butyl phthalate (DBP) exceeded recommend environmental risk limit (ERL) that posed a potential risk to the aquaculture fish pond environment in the PRD.

Fluorogenic boronate-based probe-lactulose complex for full-aqueous analysis of peroxynitrite.

A selective fluorogenic boronate-based probe-lactulose complex was evaluated for the rapid analysis of peroxynitrite (ONOO-) based on a reaction-based indicator displacement assay (RIA). The probe was synthesised by a simple nucleophilic substitution reaction between a boronic acid moiety and a well known laser dye, DCM. Fluorescence analyses showed that the probe had an off-on response to lactulose, forming a fluorogenic probe-lactulose complex. The subsequent addition of ONOO- selectively quenched the fluorescence of the complex over other Reactive Oxygen/ Nitrogen Species (ROS/RNS) tested. The complex can be applied for the rapid determination of ONOO- in full aqueous solution with good linear range, and has also proven suitable for monitoring ONOO- in living cells and real water samples.

A DNA-stabilized silver nanoclusters/graphene oxide-based platform for the sensitive detection of DNA through hybridization chain reaction.

A silver nanoclusters (AgNCs)/graphene oxide (GO)-based fluorescence sensor was developed for label-free DNA detection through hybridization chain reaction (HCR). A DNA sequence associated with the human immunodeficiency virus (HIV) was selected as a model target. Two DNA probes, hairpin probe 1 (H1) and hairpin probe 2 (H2), were partially complementary. GO was used as an adsorption material to capture the hairpin probes and a selective fluorescence quencher was used to reduce the background signal. Upon addition of AgNO3 and NaBH4, the AgNCs were synthesized at the terminals of the H1 and H2 probes. In the absence of target DNA (THIV), hybridization chain reaction (HCR) could not be triggered due to the stability of H1 and H2 probes. The hairpin probe-protected AgNCs attached to the GO surface, efficiently quenching fluorescence of the AgNCs. Therefore, the system showed very low background. In presence of THIV, the target triggered the chain-like assembly of H1 and H2 through HCR, generating a long chain of H1 and H2 complexes. The HCR product (AgNCs nanowires) could not be adsorbed on the surface of GO; hence, it generated a strong fluorescent signal based on the concentration of the target. Under the optimized conditions, the detection limit of the fluorescence sensor was 1.18nM, and hence it can be applied to clinical samples.

A label-free and universal platform for the construction of an odd/even detector for decimal numbers based on graphene oxide and DNA-stabilized silver nanoclusters.

Molecular logic devices with different functions can perform various tasks in the areas of biological molecule detection, disease diagnosis, multivariate analysis, and bioimaging. Herein, a series of logic circuits based on silver nanoclusters (AgNCs)/graphene oxide (GO) are constructed to execute nonarithmetic functions, including 3-, 4-, and 5-bit odd/even checking. The resulting devices can differentiate between even and odd decimal numbers in the range from 0 to 31. Moreover, the devices can be expanded to operate with wider ranges of numbers when more inputs are added. The signal reporter is structured using AgNCs and GO, preventing laborious modification of biomolecules. The designed DNA-based logic nanodevices share the same DNA platform and a constant threshold value, showing great potential for application in information processing at the molecular level. Additionally, these devices can stably carry out their logic operations in a biological matrix, indicating that the AgNC/GO-based system can operate in a complicated biological environment. Given the biocompatibility and design flexibility of DNA, this study provides novel outcomes towards the development of label-free intelligent nanodevices. This may open a new path for the application of AgNCs/GO in molecular logic circuits and fluorescence imaging.

Foldable glycoprobes capable of fluorogenic crosslinking of biomacromolecules.

Small-molecular probes capable of monitoring and interfering with the activity of biomacromolecules - such as polysaccharides, nucleotides and proteins - are of paramount importance to the advancement of life science. However, such probes that can detect and simultaneously modulate the construction of biomacromolecules are elusive. Here we report a fluorogenic, foldable glycoprobe that can recognize and assemble a protein receptor in a synchronous fashion. The glycoprobe synthesized by introducing a glycoligand (for protein recognition) to a bola-type bis-fluorophore conjugate shows a "self-shielded" fluorescence in the folded state. Association with a receptor protein rapidly unfolds the probe, releasing a fluorophore capable of crosslinking the proteins - as determined using small-angle X-ray scattering - thereby producing a unique fluorescent supramolecular construct. We have demonstrated the use of the foldable glycoprobe in order to track the endocytic cycle of a transmembrane receptor.