Target-Induced Stepwise Disintegration of Starlike Branched and Multiplex Embedded Systems for Amplified Detection of Serum MicroRNA.

DNA nanotechnology has shown great promise for biosensing and molecular recognition. However, the practical application of conventional DNA biosensors is constrained by inadequate target stimuli, intricate design schemes, multicomponent systems, and susceptibility to nuclease degradation. To overcome these limitations, we present a class of starlike branched and multiplex embedded system (SBES) with an integrated functional design and cascade exponential amplification for serum microRNA (miRNA) detection. The DNA arms can be integrated into an all-in-one system by surrounding a branch point, with each arm endowed with specific functionalities by embedding different DNA fragments. These fragments include a segment complementary to the target miRNA for the recognition element, palindromic tails for self-primed polymerization, and a region with the same sequences as the target serving as the target analogue. Upon exposure to a target miRNA, the DNA arms unwind in a stepwise manner through palindrome-mediated dimerization and polymerization. This enables target recycling for subsequent reactions while releasing the target analogue to generate a secondary response in a feedback manner. A comparative analysis illustrates that the signal-to-noise ratio (SNR) of a full SBES with a feedback strategy is approximately 250% higher than the system without a feedback design. We demonstrate that the four-arm 4pSBES has the benefits of multifunctional integration, enhanced sensitivity, and low false-positive signals, which makes this approach ideally suited for clinical diagnosis. Moreover, an upgraded SBES with additional DNA arms (e.g., 6pSBES) can be constructed to allow multifunctional extension, offering unprecedented opportunities to build versatile DNA nanostructures for biosensing.

Branch-Shaped Trapping Device Regulates Accelerated Catalyzed Hairpin Assembly and Its Application for MicroRNA In Situ Imaging.

Enzyme-free DNA strand displacement process is often practical when detecting miRNAs expressed at low levels in living cells. However, the poor kinetics, tedious reaction period, and multicomponent system hamper its in vivo applications to a great extent. Herein, we design a branch-shaped trapping device (BTD)-based spatial confinement reactor and applied it for accelerated miRNA in situ imaging. The reactor consists of a pair of trapped probe-based catalyzed hairpin assembly (T-CHA) reactions attached around the BTD. The trapping device naturally offered CHA reactions a good spatial-confinement effect by integrating the metastable probes (MHPa and MHPb) of the traditional CHA with the four-branched arm of BTD, which greatly improved the localized concentration of probes and shortened their physical distance. The autonomous and progressive walk of miRNA on the four-arm nanoprobes via T-CHA can rapidly tie numerous four-arm nanoprobes into figure-of-eight nanoknots (FENs), yielding strong fluorescence that is proportional to the miRNA expression level. The unique nanoarchitecture of the FEN also benefits the restricted freedom of movement (FOM) in a confined cellular environment, which makes the system ideally suitable for in situ imaging of intracellular miRNAs. In vitro and in situ analyses also demonstrated that the T-CHA overall outperformed the dissociative probe-based CHA (D-CHA) in stability, reaction speed, and amplification sensitivity. The final application of the T-CHA-based four-arm nanoprobe for imagings of both cancer cells and normal cells shows the potential of the platform for accurately and timely revealing miRNA in biological systems.

Optimization of Heterotrophic Culture Conditions for the Microalgae Euglena gracilis to Produce Proteins.

Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.

Structure-switchable aptamer-arranged reconfigurable DNA nanonetworks for targeted cancer therapy.

The structural DNA nanotechnology holds great potential application in bioimaging, drug delivery and cancer therapy. Herein, an intelligent aptamer-incorporated DNA nanonetwork (Apt-Nnes) is demonstrated for cancer cell imaging and targeted drug delivery, which essentially is a micron-scale pattern with the thickness of double-stranded monolayer. Cancer cell-surface receptors can make it perform magical transformation into small size of nanosheet intermediates and specifically enter target cells. The binding affinity of Apt-Nnes is increased by 3-fold due to multivalent binding effect of aptamers and it can maintain the structural integrity in fetal bovine serum (FBS) for 8 h. More interestingly, target cancer cells can cause the structural disassembly, and each resulting unit transports 4963 doxorubicin (Dox) into target cells, causing the specific cellular cytotoxicity. The cell surface receptor-mediated disassembly of large size of DNA nanostructures into small size of fractions provides a valuable insight into developing intelligent DNA nanostructure suitable for biomedical applications.

Adsorption of di (2-ethylhexyl) phthalate (DEHP) to microplastics in seawater: a comparison between pristine and aged particles.

Microplastics (MPs) are a widely distributed pollutant and have been attracting global attention. The increasing abundance of MPs in marine environments has raised concern about their adverse effects on marine organisms and influence on the fate of contaminants in seawater. In this study, we investigated the effects of natural aging on the adsorption of di (2-ethylhexyl) phthalate (DEHP), one of the most widely used phthalic acid esters (PAEs), in two types of MPs (polyethylene and polystyrene). Biofilm was observed on the surface of MPs after 3-month exposure in seawater. Atomic force microscopy revealed there were significant physical changes in the MPs after aging. Aging in coastal seawater for 3 months significantly reduced the MPs' surface roughness and adhesion, and increased the Young's modulus at the same time. Adsorption isotherms of DEHP indicated that aged MPs had stronger binding capacity of the organic contaminant than pristine MPs. Our data shed some light on the biogeochemical role of MPs in marine environments.

Copper Adsorption to Microplastics and Natural Particles in Seawater: A Comparison of Kinetics, Isotherms, and Bioavailability.

The growing use of plastics has led to microplastics (MPs) being ubiquitously distributed in marine environments. Although previous studies have emphasized MPs as important metal-transport vectors, few have considered the differences between these anthropogenic particles and their coexisting natural counterparts in sequestering metals in seawater. Here, we compared Cu adsorption to pristine and naturally aged MPs (polystyrene and polyethylene) with that to algae particles and sediments and assessed the bioavailability of the adsorbed Cu by a gut juice extraction assay. Adsorption kinetics and isotherms consistently showed that natural particles bound far more Cu to their surfaces than MPs. The rougher surfaces, greater specific surface areas, and lower ζ-potentials of natural particles contributed to their stronger Cu adsorption capacity than pristine or aged MPs. Natural particles also contained more diverse functional groups for binding Cu, with oxygen-containing groups playing a dominant role. Adsorbed Cu on natural particles was less extractable by sipunculan gut juice than that on MPs, indicating their higher Cu affinity. Overall, our study suggests that natural particles outcompete MPs in carrying metals in the water column and transferring them to marine organisms in today's environmental context. This work provides new insights for assessing the risks of MPs in marine environments.

Precision-Guided Missile-Like DNA Nanostructure Containing Warhead and Guidance Control for Aptamer-Based Targeted Drug Delivery into Cancer Cells in Vitro and in Vivo.

It is crucial to deliver anticancer drugs to target cells with high precision and efficiency. While nanomaterials have been shown to enhance the delivery efficiency once they reach the target, it remains challenging for precise drug delivery to overcome the nonspecific adsorption and off-target effect. To meet this challenge, we report herein the design of a novel DNA nanostructure to act as a DNA nanoscale precision-guided missile (D-PGM) for highly efficient loading and precise delivery of chemotherapeutic agents to specific target cells. The D-PGM consists of two parts: a warhead (WH) and a guidance/control (GC). The WH is a rod-like DNA nanostructure as a drug carrier, whose trunk is a three-dimensionally self-assembled DNA nanoscale architecture from the programmed hybridization among two palindromic DNA sequences in the x-y dimension and two common DNA oligonucleotides in the z direction, making the WH possess a high payload capacity of drugs. The GC is an aptamer-based logic gate assembled in a highly organized fashion capable of performing cell-subtype-specific recognition via the sequential disassembly, mediated by cell-anchored aptamers. Because of the cooperative effects between the WH and the GC, the GC logic gates operate like the guidance and control system in a precision-guided missile to steer the doxorubicin (DOX)-loaded DNA WH toward target cancer cells, leading to selective and enhanced therapeutic efficacy. Moreover, fluorophores attached to different locations of D-PGM and DOX fluorescence dequenching upon release enable intracellular tracing of the DNA nanostructures and drugs. The results demonstrate that by mimicking the functionalities of a military precision-guided missile to design the sequential disassembly of the GC system in multistimuli-responsive fashion, our intrinsically biocompatible and degradable D-PGM can accurately identify target cancer cells in complex biological milieu and achieve active targeted drug delivery. The success of this strategy paves the way for specific cell identity and targeted cancer therapy.

Ribbon of DNA Lattice on Gold Nanoparticles for Selective Drug Delivery to Cancer Cells.

Herein, we report on the design of a programmable DNA ribbon using long-chain DNA molecules with a user-defined repetitive padlock sequence. The DNA ribbon can be further combined with gold nanoparticles (AuNPs) to create a composite nanomaterial that contains an AuNP core and a high-density DNA crown carrying a cancer-cell-targeting DNA aptamer, a fluorescent tag for location tracking, and a cell-killing drug. This composite material can be efficiently internalized by cancer cells and its cellular location can be tracked by fluorescence imaging. The system offers several attractive characteristics, including simple design, tunable DNA crown, high drug-loading capacity, selective cell targeting, and pH-sensitive drug release. These features make such a material a promising therapeutic agent.

Palindromic Molecule Beacon-Based Cascade Amplification for Colorimetric Detection of Cancer Genes.

A highly sensitive and selective colorimetric assay based on a multifunctional molecular beacon with palindromic tail (PMB) was proposed for the detection of target p53 gene. The PMB probe can serve as recognition element, primer, and polymerization template and contains a nicking site and a C-rich region complementary to a DNAzyme. In the presence of target DNA, the hairpin of PMB is opened, and the released palindromic tails intermolecularly hybridize with each other, triggering the autonomous polymerization/nicking/displacement cycles. Although only one type of probe is involved, the system can execute triple and continuous polymerization strand displacement amplifications, generating large amounts of G-quadruplex fragments. These G-rich fragments can bind to hemin and form the DNAzymes that possess the catalytic activity similar to horseradish peroxidase, catalyzing the oxidation of ABTS by H2O2 and producing the colorimetric signal. Utilizing the newly proposed sensing system, target DNA can be detected down to 10 pM with a linear response range from 10 pM to 200 nM, and mutant target DNAs are able to be distinguished even by the naked eye. The desirable detection sensitivity, high specificity, and operation convenience without any separation step and chemical modification demonstrate that the palindromic molecular beacon holds the potential for detecting and monitoring a variety of nucleic acid-related biomarkers.

Nicking-enhanced rolling circle amplification for sensitive fluorescent detection of cancer-related microRNAs.

In this study, a biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) was proposed for the highly sensitive detection of cancer-related let-7a microRNA (miRNA). The sensing system consists of a padlock probe (PP), which contains a target recognition sequence and two binding sites for nicking endonuclease (NEase), and molecular beacon (MB) as reporting molecule. Upon hybridization with let-7a, the PP can be circularized by ligase. Then, the miRNA acted as polymerization primer to initiate rolling circle amplification (RCA). With the assistance of NEase, RCA products can be nicked on the cyclized PP and are displaced during the subsequent duplication process, generating numerous nicked fragments (NFs). These NFs not only induce another RCA reaction but also open the molecular beacons (MBs) via hybridization, leading to significantly amplified fluorescence signal. Under the optimized conditions, this method exhibits high sensitivity toward target miRNA let-7a with a detection limit of as low as 10 pM, a dynamic range of three orders of magnitude is achieved, and its family member is easily distinguished even with only one mismatched base. Meanwhile, it displays good recovery and satisfactory reproducibility in fetal bovine serum (FBS). Therefore, these merits endow the newly proposed N-RCA strategy with powerful implications for miRNA detection. Graphical abstract A biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) for the highly sensitive detection of cancer-related let-7a microRNA.

Highly sensitive detection of cancer-related genes based on complete fluorescence restoration of a molecular beacon with a functional overhang.

The accurate detection of cancer-related genes is of great significance for early diagnosis and targeted therapy of cancer. In this contribution, an automatically cycling operation of a functional overhang-containing molecular beacon (OMB)-based sensing system was proposed to perform amplification detection of the p53 gene. Contrary to the common molecular beacon (MB), a target DNA is designated to hybridize with a label-free recognition probe (RP) with a hairpin structure rather than OMB. In the presence of a target DNA of interest, the locked primer in RP opens and triggers the subsequent amplification procedures. The newly-developed OMB is not only capable of accomplishing cyclical nucleic acid strand-displacement polymerization (CNDP) with the help of polymerase and nicking endonuclease, but is also cleaved by restriction endonucleases, removing the quencher away from the fluorophore. Thus, the target DNA at an extremely low concentration is expected to generate a considerable amount of double-stranded and cleaved OMBs, and the quenched fluorescence is completely restored, leading to a dramatic increase in fluorescence intensity. Utilizing this sensing platform, the target gene can be detected down to 8.2 pM in a homogeneous way, and a linear response range of 0.01 to 150 nM could be obtained. More strikingly, the mutant genes can be easily distinguished from the wild-type ones. The proof-of-concept demonstrations reported herein are expected to promote the development of DNA biosensing systems, showing great potential in basic research and clinical diagnosis.

Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

Thirty-day rat toxicity study reveals reversible liver toxicity of mifepristone (RU486) and metapristone.

OBJECTIVE: Mifepristone (RU486) is an oral first-line contraceptive used by hundreds of millions of women, and recently it was tested for anticancer activity in both genders worldwide. We are developing metapristone (the N-monodemethyl RU486) as a potential metastasis chemopreventive. The present acute and 30-d subacute toxicity study aimed at examining and compared in parallel the potential toxicity of the two drugs. METHODS: The single-dose acute toxicity and 30-d subacute toxicity studies were conducted in mice and rats, respectively, by gavaging metapristone or mifepristone at various doses. Blood samples and organs were collected for blood chemistry, hematology and histology analyses. RESULTS: Oral mifepristone (3000 mg/kg) caused 30% and 40% death in female and male mice, respectively, within 15 h post-dosing. In comparison, the same dose of metapristone produced 30% acute death in males only. Thirty-day oral administration of the two drugs to rats (12.5, 50 and 200 mg/kg/day) caused reversible hepatotoxicity that only occurred at 200 mg/kg/day group, evidenced by the elevated liver enzyme activity and liver organ weight. CONCLUSION: The present study, for the first time, reveals reversible hepatotoxicity in rats caused by the 30-d consecutive administration at the high dose, and warns the potential hepatotoxicity caused by long-term administrations of high doses of mifepristone or metapristone in clinical trials but not by the acute single abortion doses.

Imaging mRNA in vitro and in vivo with nanofirecracker probes via intramolecular hybridization chain reaction.

Hybridization chain reaction (HCR) based enzyme-free amplification techniques have recently been developed for the visualization of intracellular messenger RNA (mRNA). However, the slow kinetics and potential interference with the intricate biological environments hinder its application in the clinic and in vivo. Herein, we designed a nanofirecracker probe-based strategy using intramolecular hybridization chain reaction (IHCR) amplifier for rapid, efficient, sensitive, specific detection and imaging of survivin mRNA both in vitro and vivo. Two probes, HP1 and HP2, in IHCR were simultaneously incorporated into a DNA nanowire scaffolds to bring HP1 and HP2 to close proximity on the assembled nanowire scaffolds. Empowered by the DNA nanowire scaffolds and spatial confinement effect, the nanofirecracker probe-based IHCR sensing system exhibited improved biostability, accelerated reaction kinetics, and enhanced signal amplification. This new strategy has been successfully applied to imaging mRNA in both cultured cells and in mice. Importantly, this novel sensing method was capable of detecting survivin mRNA in clinical blood samples from subjects with colorectal cancer. Thus, this novel nanofirecracker probe-based IHCR strategy holds great potential in advancing both biomedical research and in molecular diagnostics.

The role of the size of affinity ligands in the detection and characterization of extracellular vesicles.

Surface proteins on the membrane of nano-sized extracellular vesicles (EVs) not only play crucial roles in cell-to-cell communication, but also are specific binding targets for EV detection, isolation and tracking. The low abundance of protein biomarkers on EV surface, the formation of clusters and the complex EV surface network impose significant challenges to the study of EVs. Employing bulky sized affinity ligands, such as antibodies, in the detection and characterization of these vesicles often result in reduced sensitivity of detection or poor quantification of proteins on the EV surface. By virtue of their small size and high specificity, Affibody molecules emerge as a potential alternative to their monoclonal antibody counterparts as robust affinity ligands in EV research. In this study, we present a theoretical framework on the superiority of anti-HER2 Affibodies over anti-HER2 antibodies in labeling and detecting HER2-positive EVs, followed by the demonstration of the advantages of HER2 Affibodies in accessing EV surface and the detection of EVs through multiple types of approaches including fluorescence intensity, colorimetry, and fluorescence polarization. HER2 Affibodies outperformed by 10-fold over three HER2 antibody clones in accessing HER2-positive EVs derived from different human cancer cell lines. Furthermore, HRP-Affibody molecules could detect EVs from cancer cells spiked into human serum with at least a 2-fold higher sensitivity compared with that of their antibody counterparts. In addition, in fluorescence polarization assays in which no separation of free from bound ligand is required, FITC-labeled HER2 Affibodies could sensitively detect HER2-positive EVs with a clinically relevant limit of detection, whilst HER2 antibodies failed to detect EVs in the same conditions. With the demonstrated superiority in accessing and detecting surface targets over bulky-sized antibodies in EVs, Affibodies may become the next-generation of affinity ligands in the precise characterization and quantification of molecular architecture on the surface of EVs.

Noctiluca scintillans bloom alters the composition and carbohydrate utilization of associated bacterial community and enriches potential pathogenic bacterium Vibrio anguillarum.

Noctiluca scintillans (red) is a widely distributed heterotrophic dinoflagellate and a prominent red tide forming species. This study investigated the effects of Noctiluca blooms on marine microbial diversity and functionality using multi-omics approaches. Our findings revealed significant differences in the community composition of Noctiluca-associated bacteria compared to those associated with autotrophic plankton and free-living bacteria in the surrounding seawater. The dominant bacterial groups within the Noctiluca-associated community shifted at various bloom stages, which could be attributed to changes in prey composition of Noctiluca. During the non-bloom stage, Burkholderiaceae, Carnobacteriaceae, and Pseudomonadaceae dominated the community, while Vibrionaceae became dominant during the bloom stage, and Saprospiraceae, Crocinitomicaceae, and Pirellulaceae thrived during the post-bloom stage. Compared to the non-bloom stage, Noctiluca-associated bacterial community at the bloom stage exhibited significant down-regulation of genes related to complex carbohydrate metabolism, while up-regulation of genes related to glucose transportation and utilization. Furthermore, we identified Vibrio anguillarum, a potential pathogenic bacterium to marine fish, as a major component of the Vibrionaceae family during the bloom stage. The occurrence of V. anguillarum associated with Noctiluca blooms may be attributed to the increased availability of its preferred carbon sources and its high capabilities in glucose transportation, motility and chemotaxis. Moreover, the presence of Vibrio infection genes (hap, hlyA, rtxA) encoding vibriolysin, hemolysin, and RTX (Repeats-in-toxin) toxin in the V. anguillarum genome, with the hap gene showing high expression levels during Noctiluca blooms, indicates an elevated risk of infection. This study underscores the unique composition of the bacterial community associated with red tide forming heterotrophic dinoflagellates and suggests that Noctiluca cells may serve as reservoirs and vectors for pathogenic bacteria, potentially posing a threat to fish-farming and the health of other marine organisms.

Lighting up Lipidic Nanoflares with Self-Powered and Multivalent 3D DNA Rolling Motors for High-Efficiency MicroRNA Sensing in Serum and Living Cells.

Intelligent DNA nanomachines are powerful and versatile molecular tools for bioimaging and biodiagnostic applications; however, they are generally constrained by complicated synthetic processes and poor reaction efficiencies. In this study, we developed a simple and efficient molecular machine by coupling a self-powered rolling motor with a lipidic nanoflare (termed RMNF), enabling high-contrast, robust, and rapid probing of cancer-associated microRNA (miRNA) in serum and living cells. The lipidic nanoflare is a cholesterol-based lipidic micelle decorated with hairpin-shaped tracks that can be facilely synthesized by stirring in buffered solution, whereas the 3D rolling motor (3D RM) is a rigidified tetrahedral DNA scaffold equipped with four single-stranded "legs" each silenced by a locking strand. Once exposed to the target miRNA, the 3D RM can be activated, followed by self-powered precession based on catalyzed hairpin assembly (CHA) and lighting up of the lipidic nanoflare. Notably, the multivalent 3D RM that moves using four DNA legs, which allows the motor to continuously and acceleratedly interreact with DNA tracks rather than dissociate from the surface of the nanoflare, yielded a limit of detection (LOD) of 500 fM at 37 °C within 1.5 h. Through the nick-hidden and rigidified structure design, RMNF exhibits high biostability and a low false-positive signal under complex physiological settings. The final application of RMNF for miRNA detection in clinical samples and living cells demonstrates its considerable potential for biomedical imaging and clinical diagnosis.

Trigging stepwise-strand displacement amplification lights up numerous G-quadruplex for colorimetric signaling of serum microRNAs.

MicroRNAs (miRNAs) play an important biomarker in various biological processes, especially cancer related, yet economic, simple, sensitive and specific methods for miRNA determination are still challenging. In this study, we have developed stepwise-strand displacement amplification (S-SDA)-based colorimetric sensing platform for let-7a miRNA detection in clinical serum samples. Our results demonstrated that the developed S-SDA-based method shows high sensitivity with a detection limit of 63.2 pM and a naked eye detection limit of 0.1 nM. Moreover, the S-SDA amplifier is able to discriminate target miRNAs from their mutants with high accuracy and specificity. With its high sensitivity and selectivity, this method successfully identified healthy individuals from patients with colon cancer by detecting let-7a miRNAs in serum. We believe the colorimetric analysis method will provide a new paradigm for the detection of miRNA with different abundance and show great potential for clinical application in biomedical analysis and early clinical diagnosis.

Ocean acidification affects physiology of coccolithophore Emiliania huxleyi and weakens its mechanical resistance to copepods.

The effects of ocean acidification (OA) on coccolithophore's photosynthesis, calcification rates, and growth have been extensively studied. However, how the intracellular Ca2+, mechanical properties and chemical composition of the coccoliths are affected by OA have not yet been investigated. This study tries to fill these gaps using Emiliania huxleyi as a model coccolithophore. When the seawater pCO2 increased from 400 µatm to 1200 µatm, the intracellular Ca2+ and coccolith area were reduced by 66% and 36%, respectively. Single-cell mapping by atomic force microscopy revealed that the modulus and hardness of coccolith decreased from 23.6 ± 0.2 GPa to 12.0 ± 5.5 GPa and from 0.53 ± 0.15 GPa to 0.20 ± 0.06 GPa, respectively. Additionally, the proportional organic matter and silicon in the coccolith surfaces increased with pCO2. The copepods Acartia pacifica fed on more E. huxleyi grown at higher pCO2. Our study implies that OA could change coccolithophore's competitive interactions with other phytoplankton and ultimately influence carbon export to the deep ocean.

Construction of a 3D rigidified DNA nanodevice for anti-interference and reinforced biosensing by turning nuclease into a catalyst.

The practical application of DNA biosensors is impeded by numerous limitations in complicated physiological environments, particularly the susceptibility of common DNA components to nuclease degradation, which has been recognized as a major barrier in DNA nanotechnology. In contrast, the present study presents an anti-interference and reinforced biosensing strategy based on a 3D DNA-rigidified nanodevice (3D RND) by converting a nuclease into a catalyst. 3D RND is a well-known tetrahedral DNA scaffold containing four faces, four vertices, and six double-stranded edges. The scaffold was rebuilt to serve as a biosensor by embedding a recognition region and two palindromic tails on one edge. In the absence of a target, the rigidified nanodevice exhibited enhanced nuclease resistance, resulting in a low false-positive signal. 3D RNDs have been proven to be compatible with 10% serum for at least 8 h. Once exposed to the target miRNA, the system can be unlocked and converted into common DNAs from a high-defense state, followed by polymerase- and nuclease-co-driven conformational downgrading to achieve amplified and reinforced biosensing. The signal response can be improved by approximately 700% within 2 h at room temperature, and the limit of detection (LOD) is approximately 10-fold lower under biomimetic conditions. The final application to serum miRNA-mediated clinical diagnosis of colorectal cancer (CRC) patients revealed that 3D RND is a reliable approach to collecting clinical information for differentiating patients from healthy individuals. This study provides novel insights into the development of anti-interference and reinforced DNA biosensors.