Small extracellular vesicles (sEVs) membrane protein profile (sEVpp) is a novel biomarker for cancer, and it can reveal the in-depth phenotype information. The point-of-care testing (POCT) of sEVpp holds great significance for mass screening of cancer, so the cost-effective and simple detection methods of sEVpp are urgently demanded. Herein, we constructed a paper-based multichannel sEVpp POCT device (sEVpp-PAD) enabled by functional DNA probes and metal-organic framework (MOF). The core components are aptamer/MOF-modified paper chips. The modified aptamers can immunocapture the sEV expressing corresponding proteins, while the modified MOF can provide abundant sites for aptamer-modification, reduce the nonspecific protein absorption, and act as reference for ratiometric detection. Simply powered by two syringes, the sEVpp-PAD can efficiently capture sEVs expressing corresponding protein from cell culture media and sera. Furthermore, a detection probe (DP) consisted of CD63 aptamer and G-quadruplex was developed for the colorimetric detection of captured sEVs. Utilizing this device, the sEVpp in various hepatocellular carcinoma cell culture medium and, more importantly, in human sera can be accurately determined, only with $2 device, $0.2 detection reagents and 1.8 h procedure. This simple strategy for sEVpp detection can innovatively promote the POCT and subtyping of cancer based on sEV-related liquid biopsy. (c) 2025 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Small extracellular vesicles (sEV) are increasingly reported as biomarkers for the early diagnosis of pancreatic cancer (PC), but the current techniques for isolation and detection of sEV rely on expensive instruments and tedious protocols. In this work, a facile and rapid sEV isolation and detection method (LAPT-sEViso) was developed, which is based on the specific aggregation of GPC-1-positive PC-derived sEV and an aptamer-functionalized DNA long chain produced by rolling circle amplification (RCA-APT). The LAPT-sEViso can efficiently isolate sEV from cell culture medium and serum, showing 45 times higher yield (5.5 × 106 particles mL-1), 1.1 times higher purity (1.66 × 1010 particles mg-1) and 4.9 times higher recovery (80.9%) comparing to the traditional ultracentrifugation method, with only $1000 common instruments and $2.88 reagents/materials in 1 h. Moreover, after simple filtration and on-membrane ELISA, sEV concentration can be instrument-free detected with a limit of detection of 5.62 × 103 particles μL-1 (linear range from 5.0 × 103 to 5.0 × 107 μL-1). The LAPT-sEViso provides an efficient and practical approach for the rapid isolation and detection of sEV, providing a novel approach for the sEV-based liquid biopsy.

Small extracellular vesicles (sEV) are increasingly reported as biomarkers for the early diagnosis of pancreatic cancer (PC), but the current techniques for isolation and detection of sEV rely on expensive instruments and tedious protocols. In this work, a facile and rapid sEV isolation and detection method (LAPT-sEViso) was developed, which is based on the specific aggregation of GPC-1-positive PC-derived sEV and an aptamer-functionalized DNA long chain produced by rolling circle amplification (RCA-APT). The LAPT-sEViso can efficiently isolate sEV from cell culture medium and serum, showing 45 times higher yield (5.5 & times; 106 particles mL-1), 1.1 times higher purity (1.66 & times; 1010 particles mg-1) and 4.9 times higher recovery (80.9%) comparing to the traditional ultracentrifugation method, with only $1000 common instruments and $2.88 reagents/materials in 1 h. Moreover, after simple filtration and on-membrane ELISA, sEV concentration can be instrument-free detected with a limit of detection of 5.62 & times; 103 particles mu L-1 (linear range from 5.0 & times; 103 to 5.0 & times; 107 mu L-1). The LAPT-sEViso provides an efficient and practical approach for the rapid isolation and detection of sEV, providing a novel approach for the sEV-based liquid biopsy.

Rapid and portable profiling of surface proteins on small extracellular vesicles (sEV) is crucial for noninvasive cancer screening but remains technically challenging. Here, we present a Tyndall effect (TE)-based visible aptasensing platform (TEVAP) for direct, low-cost, and isolation-free detection of sEV surface proteins from complex biological samples. Aptamer-conjugated gold nanoparticles specifically bind to sEV, forming large-scale composites that enhance the TE signal. This enables the identification of five tumor-associated proteins with a detection limit of 6.5 × 106 particles mL-1 without enzyme catalysis or signal amplification strategies, comparable to other instrument-dependent methods. Applied to clinical samples (e.g., 5 μL of plasma), TEVAP generated distinct signal patterns and effectively distinguished liver and breast cancer patients from healthy controls. With further validation in larger cohorts, this platform holds strong potential for convenient cancer screening and postoperative monitoring.

DNAzyme-based gene silencing is a promising cancer therapy technology, while its efficiency and safety issues still need to be addressed. In this study, we developed a chirality/microRNA dual-gating theranostic nanomachine for messenger RNA (mRNA) gene silencing therapy with enhanced targeting towards human cervical cancer (HeLa) cells. The chiral nanoparticle, an emerging material with unique chirality-induced properties, e.g. chirality-dependent cellular uptake, has been widely applied in biomedical fields. The L-type gold nanoparticles (L-AuNPs) are used as the nanocarrier with the first “chirality-gating”. L-AuNPs showed 2.4-fold uptake efficiency for HeLa cells comparing to normal cell, as well as 2.2-fold uptake efficiency in cancer cell comparing to D-type gold nanoparticles (D-AuNPs), which exhibited no cancer cell targeting effect. Moreover, an entropy-driven-DNAzyme (EDz) circuit that can release numerous DNAzyme and fluorescence probes with the trigger of single microRNA (miRNA) conducted the theranostic role of both miRNA imaging and gene silencing, constituting the second “miRNA gating”. Ascribing to the prominent targeting nanocarrier L-AuNPs, the miRNA imaging sensitivity and gene silencing therapy efficiency were improved by 2.6- and 5.0-fold, respectively, comparing to those by D-AuNPs. In general, this dual-gating nanomachine, designed with a powerful yet simple strategy, has showed great prospects in optimizing delivery specificity and avoiding off-target toxicities.

CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.

As one of the most important post-translational modifications (PTMs), protein phosphorylation plays a key role in a variety of biological processes. Many studies have shown that protein phosphorylation is associated with various human diseases. Therefore, identifying protein phosphorylation site-disease associations can help to elucidate the pathogenesis of disease and discover new drug targets. Networks of sequence similarity and Gaussian interaction profile kernel similarity were constructed for phosphorylation sites, as well as networks of disease semantic similarity, disease symptom similarity and Gaussian interaction profile kernel similarity were constructed for diseases. To effectively combine different phosphorylation sites and disease similarity information, random walk with restart algorithm was used to obtain the topology information of the network. Then, the diffusion component analysis method was utilized to obtain the comprehensive phosphorylation site similarity and disease similarity. Meanwhile, the reliable negative samples were screened based on the Euclidean distance method. Finally, a convolutional neural network (CNN) model was constructed to identify potential associations between phosphorylation sites and diseases. Based on tenfold cross-validation, the evaluation indicators were obtained including accuracy of 93.48%, specificity of 96.82%, sensitivity of 90.15%, precision of 96.62%, Matthew's correlation coefficient of 0.8719, area under the receiver operating characteristic curve of 0.9786 and area under the precision-recall curve of 0.9836. Additionally, most of the top 20 predicted disease-related phosphorylation sites (19/20 for Alzheimer's disease; 20/16 for neuroblastoma) were verified by literatures and databases. These results show that the proposed method has an outstanding prediction performance and a high practical value.

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.

Cells in different states can release diverse types of extracellular vesicles (EVs) that participate in intracellular communication or pathological processes. The identification and isolation of EV subpopulations are significant to explore their physiological functions and clinical value. In this study, structurally heterogeneous T-cell receptor (TCR)-CD3 EVs were proposed and verified for the first time using a caliper strategy. Two CD3-targeting aptamers were designed in the shape of a caliper with an optimized probe distance and were assembled on gold nanoparticles (Au-Caliper) to distinguish TCR-CD3 monomeric and dimeric EVs (m/dCD3 EVs) in skin-transplanted mouse plasma. Phenotyping and sequencing analysis revealed clear heterogeneity in the isolated m/dCD3 EVs, providing the potential for mCD3 EVs as a candidate biomarker of acute cellular rejection (ACR) and holding great prospects for distinguishing EV subpopulations based on protein oligomerization states.

采用气相色谱-四极杆飞行时间质谱(GC-QTOF MS)获取20种烟草提取物的色谱数据,对其进行各成分分析,构建二进制向量数据集.通过随机森林模型,优化了实验条件,对烟草提取物中提取物、油类物质和浸膏物质3类物质,以及A、B、C、D、E和F 6个地域产地烟草提取物进行了识别,所建模型可以100%识别烟草提取物类型和地域.

Sensitive imaging of microRNAs (miRNAs) in living cells is significant for accurate cancer clinical diagnosis and prognosis research studies, but it is challenged by inefficient intracellular delivery, instability of nucleic acid probes, and limited amplification efficiency. Herein, we engineered a DNAzyme-amplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) that overcomes these challenges and improves the imaging sensitivity. This enzyme-free amplification nanosystem is based on the sequential activation of DNAzyme amplification and CHA. MnO2 nanosheets were used as nanocarriers for the delivery of nucleic acid probes, which can resist the degradation by nucleases and supply Mn2+ for the DNAzyme reaction. After entering into living cells, the MnO2 nanosheets can be decomposed by intracellular glutathione (GSH) and release the loaded nucleic acid probes. In the presence of target miRNA, the locking strand (L) was hybridized with target miRNA, and the DNAzyme was released, which then cleaved the substrate hairpin (H1). This cleavage reaction resulted in the formation of a trigger sequence (TS) that can activate CHA and recover the fluorescence readout. Meanwhile, the DNAzyme was released from the cleaved H1 and bound to other H1 for new rounds of DNAzyme-based amplification. The TS was also released from CHA and involved in the new cycle of CHA. By this DCC nanosystem, low-abundance target miRNA can activate many DNAzyme and generate numerous TS for CHA, resulting in sensitive and selective analysis of miRNAs with a limit of detection of 5.4 pM, which is 18-fold lower than that of the traditional CHA system. This stable, sensitive, and selective nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other related biomedical applications.

The ability to specifically image cancer cells is essential for cancer diagnosis; however, this ability is limited by the false positive associated with single-biomarker sensors and off-site activation of "always active" nucleic acid probes. Herein, we propose an on-site, activatable, transmembrane logic DNA (TLD) nanodevice that enables dual-biomarker sensing of tumor-related nucleolin and intracellular microRNA for highly specific cancer cell imaging. The TLD nanodevice is constructed by assembling a tetrahedral DNA nanostructure containing a linker (L)-blocker (B)-DNAzyme (D)-substrate (S) unit. AS-apt, a DNA strand containing an elongated segment and the AS1411 aptamer, is pre-anchored to nucleolin protein, which is specifically expressed on the membrane of cancer cells. Initially, the TLD nanodevice is firmly sealed by the blocker containing an AS-apt recognition zone, which prevents off-site activation. When the nanodevice encounters a target cancer cell, AS-apt (input 1) binds to the blocker and unlocks the sensing ability of the nanodevice for miR-21 (input 2). The TLD nanodevice achieves dual-biomarker sensing from the cell membrane to the cytoplasm, thereby ensuring cancer cell-specific imaging. This TLD nanodevice represents a promising strategy for the highly reliable analysis of intracellular biomarkers and a promising platform for cancer diagnosis and related biomedical applications.

The sensitive and effective detection of microRNAs (miRNAs) is of great significance since miRNAs have been proven to have undeniable importance in participating in many biological processes. Herein, we present a novel, sensitive, label-free electrochemical miRNA detection method. Three signal amplification techniques are incorporated in this method, including the efficient conjugate of primer-modified polystyrene spheres (PS) with magnetic beads (MBs) triggered by target miRNA, template-free surface-initiated enzymatic polymerization (SIEP) on the primers, and the use of copper ions in square wave voltammetry (SWV) for detecting acidically depurinated primers. Cooperating with the electrochemical approach, this method was able to achieve a detection limit of 120 aM. With an attomole level of sensitivity and easiness of manipulation, this novel method is suitable for miRNA routine detection in both research and clinical aspects.

Noninvasive glucose detection for diabetes monitoring, in comparison with the conventional finger-prick test that causes inevitable pain, is on demand. Salivary glucose level that possesses a well-established correlation with blood glucose level can be a promising alternative for noninvasive detection, however, the sensitivity and user-friendliness remained to be improved. Herein, an "all-in-one" paper chip was fabricated by one-pot method for sensitive and user-friendly detection of salivary glucose level. Glucose oxidase (GOx) and the chromogenic reagent, luminol, were both in situ encapsulated in a metal-organic framework, ZIF-67, to form the GOx&luminol@ZIF-67@Paper (G&L@ZIF@Paper). Owing to the specificity of GOx and the peroxidase activity of ZIF-67, the paper chips are highly sensitive and specific for glucose with a linear range from 0.2 to 2 mM, and a limit of detection of 0.12 mM (S/N = 3). Moreover, ascribing to the protection and "all-in-one" structure by ZIF-67, the chip is highly stable that maintains the detection performance after storage at room temperature in air for 4 weeks, and this strategy also facilitates the one-pot detection without multiple steps of sampling and incu-bation. With the assistance of a smartphone application, the glucose level in saliva can be accurately detected with "sample-in-result-out" manner, and the results were highly consistent with those measured by commercial kit. This paper chip is a promising device for daily glucose monitoring, and the highly-integrated strategy pro-vides an innovative resolution for the construction of colorimetric paper chips.

5-Formylcytosine（5fC）, as an important epigenetic modification, plays a vital role in diverse biological processes and multiple diseases by regulating gene expression. Owing to the extremely low abundance of 5fC in all mammalian tissues and high structural similarity with other cytosine derivatives, the precise and sensitive detection of 5fC is challenging. Herein, a photo-elutable and template-free isothermal amplification strategy has been proposed for the sensitive detection of 5fC in genomic DNA based on5fC-specific biotinylation, enrichment, photocleavage, and terminal deoxynucleotidyl transferase（Td T）-assisted fluorescence signal amplification, which is termed 5fC-PTIAS. By introducing the highly specific chemolabeling and the one-step photoelution processes, this strategy possesses a minimal nonspecific background as well as a much higher amplification efficiency. With the high signal-to-noise ratio, this strategy can achieve the accurate quantification of 5fC in various biological samples including mouse brain, kidney, and liver, with a limit of detection（LOD） of 0.025‰ in DNA（S/N=3）. These results not only confirm the widespread distribution of 5fC but also indicate its significant variation in different tissues and ages. The bisulfite-and mass spectrometry-free strategy is highly sensitive, selective, and easily mastered, holding great promise in detecting other epigenetic modifications with much lower levels.

The identification of drug–drug interactions (DDIs) plays a crucial role in various areas of drug development. In this study, a deep learning framework (KGCN_NFM) is presented to recognize DDIs using coupling knowledge graph convolutional networks (KGCNs) with neural factorization machines (NFMs). A KGCN is used to learn the embedding representation containing high-order structural information and semantic information in the knowledge graph (KG). The embedding and the Morgan molecular fingerprint of drugs are then used as input of NFMs to predict DDIs. The performance and effectiveness of the current method have been evaluated and confirmed based on the two real-world datasets with different sizes, and the results demonstrate that KGCN_NFM outperforms the state-of-the-art algorithms. Moreover, the identified interactions between topotecan and dantron by KGCN_NFM were validated through MTT assays, apoptosis experiments, cell cycle analysis, and molecular docking. Our study shows that the combination therapy of the two drugs exerts a synergistic anticancer effect, which provides an effective treatment strategy against lung carcinoma. These results reveal that KGCN_NFM is a valuable tool for integrating heterogeneous information to identify potential DDIs.

Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO2 nanosponge (EDA2@hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA2@hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics.

Parkinson’s disease (PD) is a serious neurodegenerative disease. Most of the current treatment can only alleviate symptoms, but not stop the progress of the disease. Therefore, it is crucial to find medicines to completely cure PD. Finding new indications of existing drugs through drug repositioning can not only reduce risk and cost, but also improve research and development efficiently. A drug repurposing method was proposed to identify potential Parkinson’s disease-related drugs based on multi-source data integration and convolutional neural network. Multi-source data were used to construct similarity networks, and topology information were utilized to characterize drugs and PD-associated proteins. Then, diffusion component analysis method was employed to reduce the feature dimension. Finally, a convolutional neural network model was constructed to identify potential associations between existing drugs and LProts (PD-associated proteins). Based on 10-fold cross-validation, the developed method achieved an accuracy of 91.57%, specificity of 87.24%, sensitivity of 95.27%, Matthews correlation coefficient of 0.8304, area under the receiver operating characteristic curve of 0.9731 and area under the precision–recall curve of 0.9727, respectively. Compared with the state-of-the-art approaches, the current method demonstrates superiority in some aspects, such as sensitivity, accuracy, robustness, etc. In addition, some of the predicted potential PD therapeutics through molecular docking further proved that they can exert their efficacy by acting on the known targets of PD, and may be potential PD therapeutic drugs for further experimental research. It is anticipated that the current method may be considered as a powerful tool for drug repurposing and pathological mechanism studies.

Distance-based microfluidic paper-based analytical devices (μPADs) are simple and user-friendly detection platforms for disease screening, environment monitoring and food safety. However, the current distance-based μPADs still face major challenges of relatively low sensitivity and accuracy. In this work, we propose a novel chromogenic reaction-free distance-based (CRFD) μPAD strategy for sensitive and accurate quantitation of microRNAs (miRNAs) via viscosity amplification and surface hydrophobicity modulations. The CRFD μPAD with sampling, indicator (with red ink), and detection zones is scaled with reference patterns. Target miRNA in sample is pre-amplified with rolling circle amplification (RCA) reaction to specifically enhance its viscosity. The difference of flow between target and control is further enlarged on CRFD μPADs whose surface hydrophobicity is rationally modulated by in situ metal-organic frameworks (MOFs) modification. Meanwhile, the repeatability and flow shape are significantly improved. For a proof-of-concept demonstration, the miR-221 and miR-222 in liver cells lysis were sensitively detected by naked-eye with limits of detection of 0.33 and 0.37 pM, respectively. A smartphone-based auto-reading system (SAS) was developed to further improve the accuracy and convenience, showing greatly reduced relative standard deviation (RSD) of assay from 12.2% to 1.0%. This facile and extensible strategy is promising for rapid detection in complex biosystem.

Imaging of tumor-associated microRNAs (miRNAs) can provide abundant information for cancer diagnosis, whereas the occurrence of trace amounts of miRNAs in normal cells inevitably causes an undesired false-positive signal in the discrimination of cancer cells during miRNA imaging. In this study, we propose a dual-locked (D-locked) platform consisting of the enzyme/miRNA-D-locked DNAzyme sensor and the honeycomb MnO2 nanosponge (hMNS) nanocarrier for highly specific cancer cell imaging. For a proof-of-concept demonstration, apurinic/apyrimidinic endonuclease 1 (APE1) and miR-21 were chosen as key models. The hMNS nanocarrier can efficiently release the D-locked DNAzyme sensor in living cells due to the decomposition of hMNS by glutathione, which can also supply Mn2+ for DNAzyme cleavage. Ascribing to the smart design of the D-locked DNAzyme sensor, the fluorescence signal can only be generated by the synergistic response of APE1 and miR-21 that are overexpressed in cancer cells. Compared with the miRNA single-locked DNAzyme sensor and the small-molecule (ATP)/miRNA D-locked DNAzyme sensor, the proposed enzyme (APE1)/miRNA D-locked DNAzyme sensor exhibited 2.6-fold and 2.4-fold higher discrimination ratio (Fcancer/Fnormal) for cancer cell discrimination, respectively. Owing to the superior performance, the D-locked strategy can selectively generate a fluorescence signal in cancer cells, facilitating accurate discrimination of cancer both in vitro and in vivo. Furthermore, this D-locked platform is easily adaptable toward other target molecules by redesigning the DNA sequences. The outstanding performance and expansibility of this D-locked platform holds promising prospects for cancer diagnosis and related biomedical applications.