Expression profiles and characterization of microRNAs responding to chitin in Arthrobotrys oligospora.

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it's target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801.

Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/ß)8 triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40˚C and pH values between 4-7. Enzyme activity was inhibited by Zn2+, Ca2+, and Fe3+, whereas Mg2+ and K+ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.

Simultaneously colorimetric detection and effective removal of mercury ion based on facile preparation of novel and green enzyme mimic.

In this work, an environmentally-friendly and cost-effective enzyme mimic was obtained by facile one-pot preparation of chitosan/Cu/Fe (CS/Cu/Fe) composite. This composite exhibited significantly enhanced oxidase-mimicking activity during catalyzing the oxidation of 3, 3', 5, 5'-tetramethylbenzidine (TMB). The CS/Cu/Fe composite was comprehensively characterized and the possible catalytic mechanism was reasonably explored and discussed. Benefiting from the thermal stability and the compatibility with carbohydrate, the CS/Cu/Fe composite was further integrated with agarose hydrogel to fabricate a portable analytical tube containing oxidase mimic. Based on the inhibition of the catalytic oxidation of TMB in the presence of cysteine, as well as the recovery of oxidase-like activity of CS/Cu/Fe due to the specific complexation of cysteine and mercury ion (Hg2+), the rapid colorimetric detection of Hg2+ was successfully carried out in the analytical tube. This colorimetric method showed good linear response to Hg2+ over the range from 40 nM to 8.0 µM with a detection limit of 8.9 nM. The method also revealed high selectivity and satisfactory results in recovery experiments of Hg2+ detection in tap water and lake water. Furthermore, it was found that the effective removal of Hg2+ could be realized in the analytical tube based on efficient Hg2+ adsorption by CS/Cu/Fe composite and agarose hydrogel. This study not only prepared a robust and low-cost enzyme mimic, but also proposed a smart strategy to simultaneously monitor and remove toxic Hg2+ from contaminated water.

Ultrasensitive and turn-on homogeneous Hg2+ sensing based on a target-triggered isothermal cycling reaction and dsDNA-templated copper nanoparticles.

In this work, an ultrasensitive and turn-on sensor for homogeneous Hg2+ detection has been constructed based on a target-triggered isothermal cycling reaction and rapid label-free signal output with dsDNA-templated copper nanoparticles (CuNPs). As the key component of the sensor, a hairpin DNA without any labels was designed to contain different functional sequence segments and to resist digestion by exonuclease due to the protruding 3'-terminus. In the presence of Hg2+, the formation of a T-Hg2+-T structure turned the protruding 3'-terminus of the hairpin DNA to a blunt end that could be efficiently digested by Exo III, accompanied by Hg2+ release, followed by another digestion cycle. Hence, the Hg2+-triggered isothermal cycling reaction accumulated numerous dsDNA templates that facilitated fluorescent CuNP generation and finally output an amplified signal used to identify the target. This protocol is capable of Hg2+ sensing in a concentration range of 5 orders of magnitude with a detection limit down to 3.9 pM. The as-constructed sensor also revealed high selectivity, as well as satisfactory results in recovery experiments of Hg2+ detection in real water samples.

Self-assembled fluorescent Ce(â ¢) coordination polymer as ratiometric probe for HIV antigen detection.

The viral capsid protein p24 of human immunodeficiency virus is expressed at different level during viral invasion. Detection of p24 is of great importance in acquired immunodeficiency syndrome monitoring and therapy. A ratiometric probe that is easily-synthesized was constructed based on self-assembled fluorescent Ce(Ⅲ) and fluorescein. Fluorescein was used as reference. Hydrogen peroxide quenches the fluorescence of the Ce(III) easily but does not quench the fluorescence of fluorescein. The mechanism of reaction was discussed. Benefiting from the sensitive response to hydrogen peroxide, this probe was applied for p24 detection in enzyme linked immunoassay. The fluorescence ratio was in a good linear relationship with the concentration of p24, and the detection limit was 1.1 pg mL-1. This proposed method has shown potential in virus detection with easy operation.

One-step rapid synthesis of fluorescent silicon nanodots for a hydrogen peroxide-related sensitive and versatile assay based on the inner filter effect.

In this work, a kind of environment-friendly and water-dispersible silicon nanodot (SiND) was rapidly synthesized by using the mild reagents (3-aminopropyl)triethoxysilane (APTES) and glucose. It was found that the fluorescence of the as-prepared SiNDs can be quenched obviously by permanganate due to the inner filter effect. Inspired by this finding, a novel fluorescent sensor for sensitive detection of hydrogen peroxide (H2O2) was developed through the oxidation-reduction reaction between permanganate and H2O2. The detection limit of H2O2 is down to 2.8 nM. Since H2O2 is an important molecule and involved in various studies, this sensor could be applied in various H2O2-related biological analyses. As a proof-of-application demonstration, a sensitive biosensor for glucose detection was constructed through the catalytic oxidation of glucose to generate H2O2. The as-constructed sensor showed good linear response to glucose over the range from 0.16 to 16 µM with a detection limit of 0.11 µM. Moreover, the biosensor can be readily extended to other sensors for different targets, which indicates the broad applications of the proposed sensing strategy in biomedical analysis.

The behavior of a bipedal DNA walker moving on the surface of magnet microparticles and its application in DNA detection.

In this work, a three-dimensional DNA machine based on the isothermal strand-displacement polymerase reaction (ISDPR) has been constructed. The walking behavior of a DNA walker on the obstructive surface of magnetic beads has also been studied by adding different nucleic acid blocks. The "leg" of the DNA walker could hybridize with a hairpin structure DNA named H1 and lead to the opening of it. And the newly exposed stem would interact with a primer. A strand exchange has happened with the assistance of polymerase and dNTPs, so that the "leg" has been displaced and the DNA walker could be pushed to move on the surface. But the nucleic acid blocks could increase steric hindrance and obstruct this process, which is similar to the behavior of human beings walking on craggy paths. Through changing these blocks, such as the structure, the amount, and the length of blocks, the movement of the DNA walker has been controlled. What's more, the results of its application for DNA detection are satisfactory. The limit of detection is 21.6 pM. Also, this method has been successfully applied in complex biological samples. Graphical abstract ᅟ.

Multipedal DNA Walker Biosensors Based on Catalyzed Hairpin Assembly and Isothermal Strand-Displacement Polymerase Reaction for the Chemiluminescent Detection of Proteins.

In this study, two kinds of sensitive biosensors based on a multipedal DNA walker along a three-dimensional DNA functional magnet particles track for the chemiluminescent detection of streptavidin (SA) are constructed and compared. In the presence of SA, a multipedal DNA walker was constructed by a biotin-modified catalyst as a result of the terminal protection to avoid being digested by exonuclease I. Then, through a toehold-mediated strand exchange, a "leg" of a multipedal DNA walker interacted with a toehold of a catalyzed hairpin assembly (CHA)-H1 coupled with magnetic microparticles (MMPs) and opened its hairpin structure. The newly open stem in CHA-H1 was hybridized with a toehold of biotin-labeled H2. Via the strand displacement process, H2 displaced one "leg" of a multipedal DNA walker, and the other "leg" continued to interact with the neighboring H1 to initiate the next cycle. In order to solve the high background caused by the hybridization between CHA-H1 and H2 without a CHA-catalyst, the other model was designed. The principle of the other model (isothermal strand-displacement polymerase reaction (ISDPR)-DNA walker) was similar to that of the above one. After the terminal protection of SA, a "leg" of a multipedal DNA walker was triggered to open the hairpin of the ISDPR-H1 conjugated with MMPs. Then, the biotin-modified primer hybridized with the newly exposed DNA segment, triggering the polymerization reaction with the assistance of dNTPs/polymerase. As for the extension of the primer, the "leg" of a multipedal DNA walker was displaced so that the other "leg" could trigger the proximal H1 to go onto the next cycle. Due to its lower background and stronger signal, a multipedal DNA walker based on an ISDPR had a lower limit of detection for SA. The limit of detection for SA was 6.5 pM, and for expanding the application of the method, the detections of the folate receptor and thrombin were explored. In addition, these DNA walker methods were applied in complex samples successfully.

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis.

An enzyme-free DNA walker that moves on the surface of functionalized magnetic microparticles and its biosensing analysis.

An enzyme-free stochastic DNA walker propelled by a single catalytic or double catalytic DNA assembly has been constructed. The application of the proposed DNA walking biosensor was successfully expanded to the detection of DNA and the enzymatic activity of T4 polynucleotide kinase.

Highly sensitive chemiluminescence biosensor for protein detection based on the functionalized magnetic microparticles and the hybridization chain reaction.

An ultrasensitive chemiluminescence (CL) biosensor for the detection of protein is developed in this study based on the functionalized magnetic microparticles (MMPs) and the hybridization chain reaction (HCR). First, the primer hybridized with the thrombin aptamer conjugated on the surface of MMPs. Then the HCR was triggered by part of the primer and its products were assembled on the surface of the MMPs. Through the interaction between streptavidin and biotin, the streptavidin-horseradish peroxidase (SA-HRP) was coupled with the HCR products. In the presence of thrombin, the HCR products conjugating with SA-HRP were released from the surface of MMPs after the aptamer recognized and bound to its target molecule. So the released SA-HRP in the supernatant produced a significant chemiluminescence imaging signal after the addition of H2O2-luminol. The detection limit of thrombin with this method could be as low as 9.7fM. Besides, the sensing strategy was modified by changing the adding order of reagents that was then successfully applied in the detection of thrombin in complex sample. What's more, the DNA detection also could be carried out with this method, which demonstrated the universality of the proposed sensing strategy.

Microfluidic generation of magnetic-fluorescent Janus microparticles for biomolecular detection.

Fluorescent magnetic multifunctional microparticles were fabricated by a facile droplet microfluidic strategy. Two sodium alginate streams, one doped with Fe3O4 nanoparticles (NPs) and the other with CdSe/ZnS quantum dots, were introduced into a flow-focusing channel as a type of parallel laminar flow to form droplets containing two distinct parts. Then, at the serpentine channel, the Ca(2+) in the oil phase diffused into the droplets, causing the solidification of the droplets. Thus, the Janus microparticles with excellent magnetic/fluorescent properties formed. The flow conditions were optimized and the effects of the flow rates on magnetic/fluorescent compositions were carefully investigated. Luminescent labeling and magnetic separation were simultaneously realized with the newly designed microparticles. Moreover, spatial separation between Fe3O4 NPs and QDs prevented the interference of QDs photoluminescence by the magnetic particles. The as-prepared fluorescent magnetic Janus particles were also successfully employed for DNA assay, which demonstrated the potential of the multifunctional microbeads in biological applications.

A label-free colorimetric platform for DNA via target-catalyzed hairpin assembly and the peroxidase-like catalytic of graphene/Au-NPs hybrids.

A target-catalyzed hairpin assembly (CHA) and graphene/Au-NPs hybrids-based platform has been developed for the determination of DNA. This new sensor not only avoided any labeling but also reduced the background signal. In the absence of target, the assembly of H1 and H2 couldn't be triggered. The catalytic activity of graphene/Au-NPs hybrids was inhibited by adsorption of H1 and H2, leading to the "inactive" hybrids unable to catalyze the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). However, with the addition of target DNA, the target-catalyzed hairpin assembly was initiated and produced plenty of H1-H2 duplex, which had a weak binding affinity with the graphene/Au-NPs. Thus, the protected interface of graphene/Au-NPs hybrids became active and catalyzed the oxidation reaction of TMB accompanied with a colorless to-blue color change. This approach exhibited good sensitivity and specificity for target DNA with a detection limit of 5.74 × 10(-11) M, and realized the assay of target DNA in human serum samples. Besides, this sensor could be further expanded to detect viruses or proteins by adapting the corresponding aptamers, showing great potential in biochemical detections.

Sensitive colorimetric detection of protein by gold nanoparticles and rolling circle amplification.

An ultrasensitive method for the detection of protein is critically important in fundamental research and practical applications due to the low abundance of disease markers in body fluids or tissues. To detect the trace levels of disease markers with high sensitivity and specificity, a sensitive colorimetric biosensor for protein assay was developed using gold nanoparticles (AuNPs) and rolling circle amplification (RCA). After binding the biotinylated primer/circular template to the streptavidin-conjugated sandwich ELISA immunocomplex, the biotinylated primer was isothermally extended to generate single-stranded DNA (ssDNA). Sequentially, the padlock DNA was added and hybridized with the RCA products. The aggregation of the additional AuNPs in the supernatant containing the surplus padlock DNA and a certain concentration of salt could then be observed. The established sensor allowed for the specific detection of α-fetoprotein (AFP) with a detection limit of 33.45 pg mL(-1). It was also demonstrated that this method could distinguish 500 pg mL(-1) AFP with the naked eye. In addition, this biosensor could be applied to complex sample analysis and could be further used as a universal method for any protein or virus determination by changing the corresponding antibodies.

Highly sensitive and multiple DNA biosensor based on isothermal strand-displacement polymerase reaction and functionalized magnetic microparticles.

A universal, highly sensitive and selective chemiluminescence (CL) imaging method has been developed for high throughput detection of DNA. After molecular beacon (MB) hybridized with the target DNA, the biotin-labeled primer was attached to a magnetic microparticle (MMP) surface by hybridization with the stem part of the MB to initiate a polymerization of DNA strand, which led to the release of the target and another polymerization cycle. Thus the polymerization produced the multiplication of biotin-labeled primer on the surface of MMPs. Sequentially, the horseradish peroxidase (HRP) was conjugated to MMPs surface through the biotin-streptavidin reaction. Then, the conjugated HRP was determined by the CL imaging method. This proposed method could detect the sequence-specific DNA as low as 0.4 pM and discriminate perfectly matched target DNA from the mismatch DNAs. All in all, this proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive and high throughput detection of DNA.