Nondestructive and Quantitative Analysis of Cell Wall Regeneration in the Medicinal Macrofungus Ganoderma lingzhi by a Membrane-Fusing Fluorescent Probe.

Ganoderma is a prize medicinal macrofungus with a broad range of pharmaceutical values. To date, various attempts have been made to cultivate Ganoderma to improve the production of secondary metabolites with pharmacological activity. Among the adopted techniques, protoplast preparation and regeneration are indispensable. However, the evaluation of protoplasts and regenerated cell walls usually relies on electron microscopy assays, which require time-consuming and destructive sample preparation and merely provide localized information in the selected area. In contrast, fluorescence assays enable sensitive real-time detection and imaging in vivo. They can also be applied to flow cytometry, providing a collective overview of every cell in a sample. However, for macrofungi such as Ganoderma, the fluorescence analysis of protoplasts and regenerated cell walls is difficult owing to the hindrance of the homologous fluorescent protein expression and the lack of an appropriate fluorescence marker. Herein, a specific plasma membrane probe, TAMRA perfluorocarbon nucleic acid probe (TPFN), is proposed for the nondestructive and quantitative fluorescence analysis of cell wall regeneration. Exploiting the perfluorocarbon membrane-anchoring chains, hydrophilic nucleic acid linker, and fluorescent dye TAMRA, the probe is proven to be selective, soluble, and stable, enabling rapid fluorescence detection of a protoplast sample free of transgenic expression or immune staining. Based on the TPFN and flow cytometry techniques, a quantitative approach is constructed to monitor the process of cell wall growth in a fast, quantitative, and high-throughout manner, and the obtained results are consistent with those of conventional electron microscopy. In principle, with slight modifications or integration, the proposed probe and approach can be adapted to the preparation of cell protoplasts, inspection of cell wall integrity under environmental stress, and programmable membrane engineering for cytobiology and physiology research.

Elucidation of ligninolysis mechanism of a newly isolated white-rot basidiomycete Trametes hirsuta X-13.

BACKGROUND: Lignin is a complex aromatic heteropolymer comprising 15-30% dry weight of the lignocellulose. The complex structural characteristic of lignin renders it difficult for value-added utilization. Exploring efficient lignin-degrading microorganisms and investigating their lignin-degradation mechanisms would be beneficial for promoting lignin valorization. In this study, a newly isolated white-rot basidiomycete, Trametes hirsuta X-13, with capacity to utilize alkaline lignin as the sole substrate was investigated. RESULTS: The analysis of the fermentation properties of T. hirsuta X-13 using alkaline lignin as the sole substrate, including the mycelial growth, activities of ligninolytic enzymes and the rates of lignin degradation and decolorization confirmed its great ligninolysis capacity. The maximum lignin degradation rate reached 39.8% after 11 days of T. hirsuta X-13 treatment, which was higher than that of reported fungi under the same condition. Fourier transform infrared spectrometry (FTIR), gas chromatography-mass spectrometry (GC-MS) scanning electron micrographs (SEM), two-dimensional heteronuclear single quantum coherence NMR analysis (2D-HSQC NMR) collaborated with pyrolysis gas chromatography-mass spectrometry (py-GC/MS) analyses proved that lignin structure was severely deconstructed along with amounts of monomer aromatics generated. Furthermore, according to those chemical analysis, in addition to canonical Cα-Cß breakage, the cleavage of lignin interunit linkages of ß-ß might also occur by T. hirsuta X-13. CONCLUSIONS: This study characterized a newly isolated white-rot basidiomycete T. hirsuta X-13 with impressive alkaline lignin degradation ability and provided mechanistic insight into its ligninolysis mechanism, which will be valuable for the development of lignin valorization strategies.

Highly Sensitive MicroRNA Detection by Coupling Nicking-Enhanced Rolling Circle Amplification with MoS2 Quantum Dots.

In this work, a label-free and highly sensitive fluorescence assay was constructed for microRNA detection. Nicking-enhanced rolling circle amplification (RCA) induced by G-quadruplex formation is coupled with inner filter effect (IFE)-based quenching effects of MoS2 quantum dots (MoS2 QDs). The padlock probe contains a recognition sequence to target microRNA and an accessible nicking site. The padlock probe is cyclized upon hybridization with target microRNA. Sequentially, amplification initiates a production of a long-concatenated sequence of circular probes. Abundant G-quadruplex sequences are produced via the nicking process and then used as the trigger to initiate the next RCA. In the presence of hemin, numerous hemin/G-quadruplex DNAzymes are formed, which catalyze the oxidation of o-phenylenediamine (OPD) into the colored product 2,3-diaminophenazine, resulting in quenching of the fluorescence of MoS2 QDs. This sensing strategy enables detection of microRNA let-7a with high selectivity and a detection limit of 4.6 fM. The as-prepared sensor was applied for detecting microRNA let-7a in dilute human serum samples and achieved a satisfactory recovery rate, demonstrating its potential in clinic diagnosis of microRNA-associated disease and biochemical research.

Interdependent nitric oxide and hydrogen peroxide independently regulate the coix seed oil-induced triterpene acid accumulation in Ganoderma lingzhi.

Recent progress has been made in adding exogenous vegetable oils in culture media to promote bioactive metabolite production in several medicinal mushrooms, but the mechanism is still unclear. In this study, we found that the vegetable oil coix seed oil (CSO) could induce the biosynthesis of triterpene acids (TAs) and also significantly increase cytoplasmic nitric oxide (NO) and hydrogen peroxide (H2O2) concentrations in the mycelium of Ganoderma lingzhi. The change in TA biosynthesis caused by CSO could be reversed by adding NO scavenger or H2O2 scavenger, and adding NO scavenger or H2O2 scavenger resulted in the reduction of the cytoplasmic H2O2 or NO concentration under CSO treatment, respectively. Moreover, adding NO scavenger or H2O2 scavenger reversed TA biosynthesis, which could be rescued by H2O2 or NO donor, respectively. Taken together, our study indicated that both NO and H2O2 were involved in the regulation of TA biosynthesis, and CSO-activated NO and H2O2 were interdependent but independently regulated the TA biosynthesis under CSO treatment in G. lingzhi.

Aptamer-Functionalized Exosomes: Elucidating the Cellular Uptake Mechanism and the Potential for Cancer-Targeted Chemotherapy.

Exosomes (Exos) are nanoscale natural vehicles for transporting biomolecules to facilitate cell-to-cell communication, indicating a high potential of them for delivering therapeutics/diagnostics. To improve their delivery capacity, a simple, noninvasive, and efficient strategy for functionalizing Exos with effective targeting ligands as well as elucidation of the cellular uptake mechanism of these functionalized Exos was found be to necessary, but remained a challenge. In this work, we used diacyllipid-aptamer conjugates as the targeting ligand to develop an aptamer-functionalized Exos (Apt-Exos) nanoplatform for cell type-specific delivery of molecular therapeutics. The cellular uptake mechanism of Apt-Exos was investigated in details, and distinct behavior was observed in comparison to free Exos. By combining the excellent molecular recognition capability of aptamers and the superiority of Exos as natural vehicles, Apt-Exos can efficiently deliver molecular drugs/fluorophores to target cancer cells, providing a promising delivery platform for cancer theranostics.

Aptamer/AuNP Biosensor for Colorimetric Profiling of Exosomal Proteins.

Exosomes constitute an emerging biomarker for cancer diagnosis because they carry multiple proteins that reflect the origins of parent cells. Assessing exosome surface proteins provides a powerful means of identifying a combination of biomarkers for cancer diagnosis. We report a sensor platform that profiles exosome surface proteins in minutes by the naked eye. The sensor consists of a gold nanoparticle (AuNP) complexed with a panel of aptamers. The complexation of aptamers with AuNPs protects the nanoparticles from aggregating in a high-salt solution. In the presence of exosomes, the non-specific and weaker binding between aptamers and the AuNP is broken, and the specific and stronger binding between exosome surface protein and the aptamer displaces aptamers from the AuNP surface and results in AuNP aggregation. This aggregation results in a color change and generates patterns for the identification of multiple proteins on the exosome surface.

Molecular Recognition-Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes.

Exosomes are membrane-enclosed extracellular vesicles derived from cells, carrying biomolecules that include proteins and nucleic acids for intercellular communication. Owning to their advantages of size, structure, stability, and biocompatibility, exosomes have been used widely as natural nanocarriers for intracellular delivery of theranostic agents. Meanwhile, surface modifications needed to endow exosomes with additional functionalities remain challenging by their small size and the complexity of their membrane surfaces. Current methods have used genetic engineering and chemical conjugation, but these strategies require complex manipulations and have only limited applications. Herein, we present an aptamer-based DNA nanoassemblies on exosome surfaces. This in situ assembly method is based on molecular recognition between DNA aptamers and their exosome surface markers, as well as DNA hybridization chain reaction initiated by an aptamer-chimeric trigger. It further demonstrated selective assembly on target cell-derived exosomes, but not exosomes derived from nontarget cells. The present work shows that DNA nanostructures can successfully be assembled on a nanosized organelle. This approach is useful for exosome modification and functionalization, which is expected to have broad biomedical and bioanalytical applications.

Aptasensor with Expanded Nucleotide Using DNA Nanotetrahedra for Electrochemical Detection of Cancerous Exosomes.

Exosomes are extracellular vesicles (50-100 nm) circulating in biofluids as intercellular signal transmitters. Although the potential of cancerous exosomes as tumor biomarkers is promising, sensitive and rapid detection of exosomes remains challenging. Herein, we combined the strengths of advanced aptamer technology, DNA-based nanostructure, and portable electrochemical devices to develop a nanotetrahedron (NTH)-assisted aptasensor for direct capture and detection of hepatocellular exosomes. The oriented immobilization of aptamers significantly improved the accessibility of an artificial nucleobase-containing aptamer to suspended exosomes, and the NTH-assisted aptasensor could detect exosomes with 100-fold higher sensitivity when compared to the single-stranded aptamer-functionalized aptasensor. The present study provides a proof-of-concept for sensitive and efficient quantification of tumor-derived exosomes. We thus expect the NTH-assisted electrochemical aptasensor to become a powerful tool for comprehensive exosome studies.

Three Dimensional Multipod Superstructure based on Cu(OH)2 as a Highly Efficient Nanozyme.

A highly efficient nanozyme system, termed hollow multipod Cu(OH)2 superstructure (HMPS), has been developed via direct conversion from irregular nanoparticles. The HMPS displayed body size around 150 nm and branch lengths in the range of 150~250 nm. Based on the excellent catalytic property of HMPS, we developed a simple and highly sensitive colorimetric assay to detect urine glucose, and the results are in good agreement with hospital examination reports.

SERS assay of telomerase activity at single-cell level and colon cancer tissues via quadratic signal amplification.

As an important biomarker and therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Recently, enzyme-assisted amplification approaches have provided useful platforms for the telomerase activity detection, however, further improvement in sensitivity is still hindered by the single-step signal amplification. Herein, we develop a quadratic signal amplification strategy for ultrasensitive surface-enhanced Raman scattering (SERS) detection of telomerase activity. The central idea of our design is using telomerase-induced silver nanoparticles (AgNPs) assembly and silver ions (Ag(+))-mediated cascade amplification. In our approach, each telomerase-aided DNA sequence extension could trigger the formation of a long double-stranded DNA (dsDNA), making numerous AgNPs assembling along with this long strand through specific Ag-S bond, to form a primary amplification element. For secondary amplification, each conjugated AgNP was dissolved into Ag(+), which can effectively induce the 4-aminobenzenethiol (4-ABT) modified gold nanoparticles (AuNPs@4-ABT) to undergo aggregation to form numerous "hot-spots". Through quadratic amplifications, a limit of detection down to single HeLa cell was achieved. More importantly, this method demonstrated good performance when applied to tissues from colon cancer patients, which exhibits great potential in the practical application of telomerase-based cancer diagnosis in early stages. To demonstrate the potential in screening the telomerase inhibitors and telomerase-targeted drugs, the proposed design is successfully employed to measure the inhibition of telomerase activity by 3'-azido-3'-deoxythymidine.

A new enzyme-free quadratic SERS signal amplification approach for circulating microRNA detection in human serum.

In this communication, we proposed a new enzyme-free quadratic SERS signal amplification approach for the ultrasensitive detection of circulating miRNA in human serum. Combined with miRNA-triggered hybridization chain reaction and Ag(+)-mediated cascade amplification, a limit of miRNA detection as low as 0.3 fM could be achieved. More importantly, our method is suitable for the direct detection of circulating miRNAs in human serum collected from patients with different stages of chronic lymphocytic leukemia (CLL).

Electrochemical detection of type 2 diabetes mellitus-related SNP via DNA-mediated growth of silver nanoparticles on single walled carbon nanotubes.

Herein, we proposed a new electrochemical sensing strategy for T2DM-related SNP detection via DNA-mediated growth of AgNPs on a SWCNT-modified electrode. Coupled with RNase HII enzyme assisted amplification, this approach could realize T2DM-related SNP assay and be applied in crude extracts of carcinoma pancreatic ß-cell lines.

Rationally designed molecular beacons for bioanalytical and biomedical applications.

Nucleic acids hold promise as biomolecules for future applications in biomedicine and biotechnology. Their well-defined structures and compositions afford unique chemical properties and biological functions. Moreover, the specificity of hydrogen-bonded Watson-Crick interactions allows the construction of nucleic acid sequences with multiple functions. In particular, the development of nucleic acid probes as essential molecular engineering tools will make a significant contribution to advancements in biosensing, bioimaging and therapy. The molecular beacon (MB), first conceptualized by Tyagi and Kramer in 1996, is an excellent example of a double-stranded nucleic acid (dsDNA) probe. Although inactive in the absence of a target, dsDNA probes can report the presence of a specific target through hybridization or a specific recognition-triggered change in conformation. MB probes are typically fluorescently labeled oligonucleotides that range from 25 to 35 nucleotides (nt) in length, and their structure can be divided into three components: stem, loop and reporter. The intrinsic merit of MBs depends on predictable design, reproducibility of synthesis, simplicity of modification, and built-in signal transduction. Using resonance energy transfer (RET) for signal transduction, MBs are further endowed with increased sensitivity, rapid response and universality, making them ideal for chemical sensing, environmental monitoring and biological imaging, in contrast to other nucleic acid probes. Furthermore, integrating MBs with targeting ligands or molecular drugs can substantially support their in vivo applications in theranositics. In this review, we survey advances in bioanalytical and biomedical applications of rationally designed MBs, as they have evolved through the collaborative efforts of many researchers. We first discuss improvements to the three components of MBs: stem, loop and reporter. The current applications of MBs in biosensing, bioimaging and therapy will then be described. In particular, we emphasize recent progress in constructing MB-based biosensors in homogeneous solution or on solid surfaces. We expect that such rationally designed and functionalized MBs will open up new and exciting avenues for biological and medical research and applications.

Ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA utilizing ion-mediated cascade surface-enhanced Raman spectroscopy amplification.

Although surface-enhanced Raman spectroscopy (SERS) has been featured by high sensitivity, additional signal enhancement is still necessary for trace amount of biomolecules detection. In this paper, a SERS amplified approach, featuring "ions-mediated cascade amplification (IMCA)", was proposed by utilizing the dissolved silver ions (Ag(+)) from silver nanoparticles (AgNPs). We found that using Ag(+) as linkage agent can effectively control the gaps between neighboring 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNPs@4-ABT) to form "hot spots" and thus produce SERS signal output, in which the SERS intensity was proportional to the concentration of Ag(+). Inspired by this finding, the IMCA was utilized for ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA (16189T → C). Combining with the DNA ligase reaction, each target DNA binding event could successfully cause one AgNP introduction. By detecting the dissolved Ag(+) from AgNPs using IMCA, low to 3.0 × 10(-5) fm/µL targeted DNA can be detected, which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic ß-cell lines from diabetes patients. This IMCA approach is expected to be a universal strategy for ultrasensitive detection of analytes and supply valuable information for biomedical research and clinical early diagnosis.

Universal surface-enhanced Raman scattering amplification detector for ultrasensitive detection of multiple target analytes.

Up to now, the successful fabrication of efficient hot-spot substrates for surface-enhanced Raman scattering (SERS) remains an unsolved problem. To address this issue, we describe herein a universal aptamer-based SERS biodetection approach that uses a single-stranded DNA as a universal trigger (UT) to induce SERS-active hot-spot formation, allowing, in turn, detection of a broad range of targets. More specifically, interaction between the aptamer probe and its target perturbs a triple-helix aptamer/UT structure in a manner that activates a hybridization chain reaction (HCR) among three short DNA building blocks that self-assemble into a long DNA polymer. The SERS-active hot-spots are formed by conjugating 4-aminobenzenethiol (4-ABT)-encoded gold nanoparticles with the DNA polymer through a specific Au-S bond. As proof-of-principle, we used this approach to quantify multiple target analytes, including thrombin, adenosine, and CEM cancer cells, achieving lowest limit of detection values of 18 pM, 1.5 nM, and 10 cells/mL, respectively. As a universal SERS detector, this prototype can be applied to many other target analytes through the use of suitable DNA-functional partners, thus inspiring new designs and applications of SERS for bioanalysis.

Fabricating a reversible and regenerable Raman-active substrate with a biomolecule-controlled DNA nanomachine.

A DNA configuration switch is designed to fabricate a reversible and regenerable Raman-active substrate. The substrate is composed of a Au film and a hairpin-shaped DNA strand (hot-spot-generation probes, HSGPs) labeled with dye-functionalized silver nanoparticles (AgNPs). Another ssDNA that recognizes a specific trigger is used as an antenna. The HSGPs are immobilized on the Au film to draw the dye-functionalized AgNPs close to the Au surface and create an intense electromagnetic field. Hybridization of HSGP with the two arm segments of the antenna forms a triplex-stem structure to separate the dye-functionalized AgNPs from the Au surface, quenching the Raman signal. Interaction with its trigger releases the antenna from the triplex-stem structure, and the hairpin structure of the HSGP is restored, creating an effective "off-on" Raman signal switch. Nucleic acid sequences associated with the HIV-1 U5 long terminal repeat sequences and ATP are used as the triggers. The substrate shows excellent reversibility, reproducibility, and controllability of surface-enhanced Raman scattering (SERS) effects, which are significant requirements for practical SERS sensor applications.