The accurate and rapid detection of Mycobacterium tuberculosis (M. tuberculosis) is essential for the effective treatment of tuberculosis. In this work, perovskite/silica nanocomposites CsPbBr3@MSNs-PbBrOH (DP-CPB) were synthesized via a dual in situ-coating strategy, encapsulating CsPbBr3 nanocrystals with mesoporous silica and PbBrOH. The resulting nanocomposites exhibited excellent stability in physiological media. The nanocomposites’ surface was conjugated with the MS10-Trunc aptamer specific for Mtb malate synthase (MtbMS). Leveraging the aptamer’s high affinity, we developed two fluorescent aptasensors: one based on a 96-well plate for MtbMS detection, and another employing magnetic nanoparticles for the detection of Mtb bacterial strains (Mtb H37Rv). The sensors demonstrate dynamic ranges of 50-750 nM for MtbMS and 102-10⁷ CFU/mL for Mtb H37Rv, with low limits of detection (LOD) of 1.17 nM and 3 CFU/mL, respectively. The aptasensors possess the comprehensive advantages of the highly efficient photoluminescence of DP-CPB, high specificity, and fast detection of MtbMS and H37Rv. The aptasensor was successfully applied for the determination of Mtb H37Rv, revealing the vast potential of perovskites in biosensing.

Toehold-mediated strand displacement (TMSD) reaction forms the basis for DNA nanotechnology. Double-stranded toehold-mediated strand displacement (DSTMSD) reaction targets the more common double-stranded molecules, which is expected to have abundant application scenarios. Herein, the reaction sequence design and actual mutation detection application of the DSTMSD reaction are explored and unveiled. The effect of different sequence regions on the DSTMSD process is firstly investigated. Then the reaction specificity is further explored to obtain the optimal sequence condition for mutation detection. Based on this, two modified PCRbased methods are used to amplify the selected drug-resistant Mycobacterium tuberculosis (MTB) genes and cascade with the DSTMSD reaction. The PCR-combined DSTMSD reaction can precisely discriminate all five two-site MTB mutation combinations, significantly enhancing the capacity to analyse adjacent MTB mutation sites. In addition, this method achieves a sensitivity of 100% for 10 cultured samples and 60 real clinical samples, which is slightly superior to the Xpert MTB/RIF results. Overall, the PCR-combined DSTMSD method possesses the advantages of double-stranded type targets, high gene enrichment efficiency and excellent mutation discrimination performance, which is expected to be further applied in the detection of other pathogens.

As a structure-specific nuclease with 5’flap endonuclease activity, Flap endonuclease 1 (FEN1) can specifically recognize and cleave the trinucleotide overlapping structure of 5’flap dsDNA, which is closely associated with tumor development and progression. Accordingly, hypersensitive and accurate detection of FEN1 is pivotal for cancer diagnosis and monitoring. Herein, we propose a dual mode one-pot testing platform through magnetic separation-assisted rolling circle amplification-coupled CRISPR/Cas12a (MRCAC) for detecting FEN1 activity. Upon incising bead-coupled 5’flap dsDNA substrate by FEN1, the separated short single-stranded DNA (ssDNA) triggers rolling circle amplification (RCA). We leverage tandemly repeated RCA products to activate Cas12a, which cleaves RCA products into short fragments that initiate new RCA. On the one hand, Cas12a catalyzes trans-cleavage of hairpin reporters, releasing abundant fluorescence signals. This exponential cascade amplification model significantly enhances sensitivity with a detection limit of 3.17 × 10⁻6 U/µL. On the other hand, the Cas12a catalyzed cleavage of linkers between DNA-functionalized gold nanoparticles (DNAs-AuNPs) and unlocks a reversible aggregation to dispersion transition. This shift is visually discernible and quantifiable by a portable colorimeter. Combining MRCAC with AuNPs, a simple and equipment-free POCT was successfully created. Applying to spiked serum and cellular extracts, the MRCAC biosensor exhibits excellent performance in complicated biological samples. Furthermore, it verifies intense potential for screening FEN1 inhibitors, highlighting valuable application prospects in early therapeutic evaluation and anticancer drug development.

The discovery of microRNAs (miRNAs) in 1993 marked a crucial milestone in the field of molecular biology. Presently, they function as indispensable biomarkers for precision medicine, serving as regulators of gene expression. However, miRNAs are in very low abundance in body fluids, which challenges their precise detection. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based detection, as a cutting-edge technique, has become an immensely effective tool for molecular diagnosis owing to its outstanding advantages, such as high sensitivity, sequence-targeted single-base specificity, and rapid turnover time of cleavage. This paper conducts a comprehensive examination of the recent advancements in CRISPR/Cas-based RNA detection, which includes amplification-free and amplification-involved fluorescence biosensors, single-molecule platforms, and material-assisted biosensors. We emphasize technology advancements, evaluate performance indicators, and address problems in clinical translation, offering a framework for future research in precision diagnostics.

The one-pot, multiplex-targeted protocol of CRISPR/Cas12a-based assays urgently requires overcoming the bottlenecks of nonspecific trans-cleavage, substrate cis-cleavage, and the need for protospacer adjacent motifs (PAM). Herein, we report a universal one-pot multiplex-targeted detection platform, CRISPR-onet, which introduces a "push-orchestrated-pull" switching (POPs) of a single-stranded RNA (LsRNA)-locked CRISPR/Cas12a (Ls-CRISPR) cis-cleavage activity. This mechanism is facilitated through the synergistic action of an unlocked ssRNA (uLsRNA) generated via T7 RNA transcription from RPA-amplified products and an engineered double-stranded DNA (PR-Acts) serving as protector, reporter, and activator. Leveraging this framework, we developed singlex-, duplex-, and triplex-CRISPR-onet assays targeting Mycobacterium tuberculosis complex (MTC). The fine-optimized synergistic POPs-LsCRISPR principle enables highly discriminatory multiplex-targeted cis-cleavage while circumventing the excessive reliance on indiscriminate trans-cleavage. This strategy achieved a substrate protection efficiency of approximately 90 %, thereby minimizing cross-interference between the CRISPR/ Cas12a and RPA amplification. The freely designable PR-Acts enable PAM-free CRISPR-based assays. Additionally, the protocol demonstrates exceptional base mismatch discrimination capability. Practically, the singlex-CRISPR-onet can detect MTB DNA with a limit-of-detection (LOD) of two copies within 15 min, while the duplex-(30 min) and triplex-CRISPR-onet (40 min) achieve an LOD of 10 copies. The singlexand duplex-CRISPR-onet achieved a detection sensitivity of 93.6 % for clinical sputum samples, while the triplex-CRISPR-onet reached 98.7 %, superior to the mainstream Xpert MTB/RIF (92.3 %). Collectively, the CRISPR-onet demonstrates substantial potential for detecting MTC infection in clinical settings and, more importantly, offers programmability and customizability that can facilitate the development of sensitive, one-pot, multiplex-targeted diagnostic approaches for multiple pathogens or subtypes.

Herein, a fluorescent biosensing platform was developed for the one-step detection of miRNA-21 based on self-priming hairpin-mediated isothermal amplification coupled with DNAzyme (SIAM-DNAzyme). The binding of target miRNA-21 to the self-priming hairpin probe (SP) caused the conformational rearrangement of SP, leading to the formation of a self-primer at the 3 ' end, triggering the extension, nicking reactions, and target cycling, thereby generating a large number of DNAzyme chains. With the concomitant presence of magnesium ions (Mg2+), the DNAzyme was activated to cyclically cleave the reporter probes (RPs) for fluorescent signal production. Under optimized conditions, the SIAM-DNAzyme sensing platform could complete miRNA-21 detection as low as 10 pM in one step for 2 h. By comparing the fluorescence intensities of miRNA-21 with those of let-7a, miRNA-144, and miRNA-155 as interference chains and their mixtures, SIAM-DNAzyme demonstrated superior specificity and anti-interference ability. The sensing platform avoids the use of exogenous primers and can be completed in one step at a constant temperature, which greatly facilitates its operation. The platform holds a great promise for further application in biomedical research and early clinical diagnosis.

MiRNAs play significant roles in cancer through tumor-suppressive or oncogenic regulation. MiRNA-424, identified as an oncogene or a tumor suppressor in different kinds of malignancies, has emerged as a diagnostic biomarker as well as a potential target for cancer treatments. Here, a method characterizing hairpin reporter facilitated single Cas13a test (H-FAST) was proposed for the detection of miRNA-424. We validated crRNA-guided miRNA-424 activation of Cas13a for efficient trans-cleavage of linear reporter. Two hairpin reporters, rather than a linear reporter, were used as trans-cleavage substrates to enhance sensitivity. Hairpin reporters demonstrated a higher affinity for the activated Cas13a/crRNA complex and produced signals that exceeded those of the linear reporter in the presence of miRNA-424 at fM concentrations. The optimal reaction volume and the relatively low concentrations of the hairpin reporter further favored the sensitivity. H-FAST, under optimized conditions, showed high detection sensitivity for miRNA-424 with LOD of 7.2 fM, much higher than that of the linear reporter in pM magnitude. By testing the non-target miRNAs and spiking miRNA-424 into 10 % serum, H-FAST showed high specificity and recovery rates ranging from 95.99 % to 106.81 % with RSD < 5 %. In summary, the proposed hairpin reporter-facilitated single Cas13a test provides a fast, simple, and cost-saving method for the detection of miRNA with high specificity and sensitivity.

MicroRNAs (miRNAs) serve as crucial biomarkers for tumor diagnosis and prognosis monitoring. Exponential amplification reaction (EXPAR) has obtained significant attention due to its rapid signal amplification capacity within 30 min. However, its practical application is hindered because of the generation of nonspecific amplification by inevitable dimer formation between templates. Herein, we designed a double-hairpin templated exponential isothermal amplification (DHTE) strategy to construct a one-pot, positive feedback-driven miRNA detection platform. The miRNA-21 target opened the first hairpin (H1) and generated the intermediate ssDNA amplicon in the presence of polymerase and nicking enzyme. Similarly, the ssDNA amplicon opened the second hairpin (H2) and produced the second ssDNA amplicon that was identical to the miRNA-21 target sequence. Therefore, it formed a positive feedback cycle with exponential signal amplification capacity. The constructed detection platform enables a one-pot reaction, not requiring lid opening operation, thereby effectively minimizing aerosol contamination. Additionally, a dynamic detection range spanning five orders of magnitude is achieved by this platform, and the limit of detection (LOD) reaches 25.96 fM. Compared with conventional linear X-N-X and X-Y-N-Y EXPAR templates, our method effectively reduces the background signal due to the double-hairpin design. Furthermore, it also successfully analyzes miRNA-21 in 10-fold diluted normal human serum and three types of cell lysates, which indicates the excellent practical application potential. In conclusion, the user-friendly DHTE detection platform is recognized for its operational simplicity, demonstrating significant potential to be utilized for practical miRNA detection.

Acetohydroxy acid synthase (AHAS) is a key enzyme that catalyzes the synthesis of branched-chain amino acids, which is indispensable for the survival and growth of Mycobacterium tuberculosis (Mtb). Aim to discover new AHAS inhibitors from natural products, here we performed computer assistant target-based screening for MtbAHAS inhibitors using Discovery Studio on TCMSP and SELLECK libraries. Mtb-AHAS structure was first simulated and verified for docking, and 80 compounds with top LIBDOCK and CDDOCK scores were obtained. By experimental verification, four compounds namely Salvianolic acid A, Embelin, Celastrol and Wushanicaritin showed inhibition potency against Mtb-AHAS with IC50 ranging from 805.5 nM-32.36 mu M. The most potential inhibitor Celastrol exhibited bacteriostatic activity for both Mycobacterium smegmatis and Mycobacterium tuberculosis with MIC of 62.5 mu M and 80 mu M, respectively. This study revealed that Celastrol is the potential Mtb-AHAS inhibitor as an anti-tuberculosis lead compound.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a global public health threat, particularly due to dormant Mtb, which necessitates prolonged drug treatment. Mycobacterium tuberculosis malate synthase (MtbMS) is a key rate-limiting enzyme in the glyoxylate shunt, essential for the survival of dormant Mtb but absent in the host. Using target-based virtual screening and biochemical approaches, we identified novel natural inhibitors of MtbMS. Molecular docking by Schrödinger and subsequent manual selection identified 11 compounds as potential inhibitors. Molecular dynamics (MD) simulations and binding-free energy analysis (MM/GBSA) demonstrated high stability and binding affinity of MtbMS with Nordihydroguaiaretic Acids (NDGA) and Meso-NDGA. NDGA and Meso-NDGA by inhibition experiment exhibited half-maximal inhibitory concentrations (IC50) against MtbMS at 1.10 ± 0.01 μM and 14.29 ± 0.95 μM and by Isothermal Titration Calorimetry (ITC) showed binding constants (Kd) of 5.66 μM and 34.90 μM, respectively. Their minimum inhibitory concentrations (MIC) against Mtb H37Rv were 60.47 μg/mL and 30.24 μg/mL, respectively. In conclusion, natural products NDGA and Meso-NDGA are potent inhibitors of MtbMS and represent promising new scaffolds for combating dormant Mtb.

MicroRNAs (miRNAs) have been involved in many biological processes and are regarded as promising biomarkers. The short sequence, low abundance and highly homologous interference sequences greatly hinder the accurate detection of miRNAs. Here, a cascade branch migration-triggered strand displacement amplification (CBM-TSDA) strategy was developed for the first time for specific and sensitive detection of miRNA-155 (miR-155). In the presence of target miR-155, the CBM was initiated and two Y-shaped probes were eventually produced. Next, the Y-shaped probes were transformed into three-way junction (3WJ) structures and triggered the SDA to produce a large number of G-quadruplex (G4) structures. Finally, the increased fluorescence signal of G4/Thioflavin T (ThT) was used to quantify miR-155. Meanwhile, the colorimetric responses of the G4-hemin DNAzyme could be used as supplementary detection to obtain a dual-mode signal readout. This detection strategy showed high detection sensitivity, and the limit of detection was 0.28 pM in the fluorescence detection mode and 0.34 pM in the colorimetric detection mode. Notably, it showed high detection specificity, being able to discriminate the single-base mutations of the target with a high discrimination factor. The strategy also possessed excellent capacity for miR-155 detection in cell lysates and real human blood samples. The developed strategy provides a promising detection platform for miRNA, which may be applied to early clinical diagnosis.

The abnormal expression of human DNA methyltransferases (DNMTs) is closely related with the occurrence and development of a wide range of human cancers.

The pathogenic bacteria induced foodborne disease has been detrimental to public health worldwide. Herein, the peroxidase (POD)-like Fe3O4/MWCNTs@Mo-CDs (FMMC) nanozyme was applied for the detection of Escherichia coli (E. coli). The E. coli aptamer was conjugated with the surface of the FMMC, which effectively enhanced the POD-like activity attributing to the higher affinity to the substrate, and then specific capture of E. coli in food matrices, leading to the reduction of POD-like activity. Therefore, a robust and facile colorimetric aptasensor was developed for detecting E. coli with a wide linear range of 101-106 CFU/mL, low LOQ of 101 CFU/mL and LOD of 0.978 CFU/mL. The aptasensor demonstrated the satisfied selectivity for E. coli compared to the other strains. This method possessed the potential application for fast in situ screening of foodborne pathogens in food products.

DNA methylation plays an important role in epigenetic modification. DNA methyltransferase (DNMT) is essential in the DNA methylation process, and its abnormal expression is closely related to cancer. In this study, we propose a novel biosensor platform (DS-GlaI-EXPAR) that combines hemi-methylated double-stranded DNA (dsDNA) as the substrate for DNMT1 with GlaI-assisted isothermal exponential amplification reaction (EXPAR) for rapid, simple, and sensitive detection of DNMT1 activity. The hemi-methylated dsDNA is fully methylated by DNMT1, and GlaI recognizes and cleaves the fully methylated sequence, generating terminal fragments that trigger EXPAR for efficient signal amplification. Whereas hemi-methylated dsDNA without DNMT1 will keep intact and cannot initiate EXPAR. DNMT1 activity can therefore be sensitively quantified by the real-time fluorescence signal of the DS-GlaI-EXPAR platform. The high-efficiency amplification of EXPAR and the recognition of GlaI enable the platform to overcome the inherent cumbersome and time-consuming shortcomings of traditional methods while meeting specificity and sensitivity. This DS-GlaI-EXPAR platform offers an impressively low limit of detection of 0.86 pg/μL and the entire detection process can be completed in a short time of 2.5 h in a single tube. Furthermore, DNMT1 activity detected by this platform in MCF-7 cells was significantly higher than that of HEK293 cells, and the inhibition of Apt. #9 was verified. This DNMT1 activity detection platform is very convenient and effective for the discovery of inhibitors and early cancer diagnosis.

The detection of single nucleotide variants (SNVs) is important for the diagnosis and treatment of cancer. To date, researchers have devised several methods to detect SNVs, but most of them are complex and time-consuming. To improve SNVs detection specificity and sensitivity, we developed a triple-recognition strategy, which facilitates aligner-mediated cleavage-triggered exponential amplification (Trec-AMC-EXPAR) for the rapid, specific, and one-pot detection of SNV. Under optimized conditions, Trec-AMC-EXPAR detected two clinically significant SNVs, PIK3CAH1047R and EGFR L858R within 80 min, with a reliable detection of 0.1% SNV in the wide type, which is lower than that of allele-specific PCR (AS-PCR) for detecting SNV. Finally, by spiking into normal human serum samples, mutants mixed with the wild-type targets in different ratios were analyzed, resulting in the relative standard deviation (RSD) of recovery ratios <3%. The findings suggested the potential application of Trec-AMC-EXPAR in clinical disease diagnosis. In summary, the proposed Trec-AMC-EXPAR technique provides a novel fast and convenient method for one-pot detection of SNV with high sensitivity and specificity.

Introduction: Neuropathic pain remains a prevalent and challenging condition to treat, with current therapies often providing inadequate relief. Ozone therapy has emerged as a promising treatment option; however, its mechanisms of action in neuropathic pain remain poorly understood. Methods: In this study, we investigated the effects of ozone treatment on gene expression and metabolite levels in the brainstem and hypothalamus of a rat model, using a combined transcriptomic and metabolomic approach. Results: Our findings revealed significant alterations in key genes, including DCST1 and AIF1L, and metabolites such as Aconitic acid, L-Glutamic acid, UDP-glucose, and Tyrosine. These changes suggest a complex interplay of molecular pathways and region-specific mechanisms underlying the analgesic effects of ozone therapy. Discussion: Our study provides insights into the molecular targets of ozone treatment for neuropathic pain, laying the groundwork for future research on validating these targets and developing novel therapeutic strategies.

DNA methylation is closely associated with cancer, and sensitive detection of DNA methylation is immensely significant for early cancer screening. In this study, for the first time, we identified CLDN11 methylation as a valuable marker for lung adenocarcinoma diagnosis and prognosis based on database analysis and experimental verification using clinical tissues and sera. Then, a flexible platform was proposed for ultrasensitive detection of CLDN11 methylation by integrating methylation-dependent restriction endonuclease GlaI cleavage with exponential amplification reaction (EXPAR) and CRISPR/Cas12a (GlaI-EXPAR-Cas12a platform). GlaI only recognizes and cleaves methylated target sites with excellent selectivity; the released fragments are primers for triggering EXPAR-CRISPR/Cas12a. Using Cas12a, false positives generated by EXPAR can be weakened to a negligible level. The proposed platform for detecting CLDN11 methylation achieved excellent performance, with a low limit of detection (1.25 x 10-15 M), and reliably identified CLDN11 methylation at an abundance of 0.1 % even in the presence of a large number of unmethylated fragments. More importantly, the proposed platform can be used for the quantification of human serum and genomic DNA, as well as to distinguish normal cells (HEK293) from cancer cells (H1299, A549 and H358). Through this integration platform, all 20 lung adenocarcinoma tissues exhibited significantly higher levels of CLDN11 methylation than a total of 16 paracancerous tissues. Taken together, the GlaI-EXPAR-Cas12a platform is ultrasensitive and convenient, and has perfect specificity. Further, it may function as a flexible platform for early screening of lung adenocarcinoma.

Rapid and simple nucleic acid detection is significant for disease diagnosis and pathogen screening, especially under specific conditions. However, achieving highly sensitive and specific nucleic acid detection to meet the time and equipment demand remains technologically challenging. In this study, we proposed a magnetic separation enhanced colorimetry biosensor based on a toehold-containing three-way junction (TWJ) induced multiple isothermal exponential amplification and the CRISPR/Cas14a (C-TEC) biosensor. The TWJ template was designed as a Y-X-Y structure. In the presence of the target, the formation of toehold-containing TWJ complex induced primer extension, leading to the generation of amplified single-stranded DNA; this amplified DNA could then bind to either the free TWJ template for EXPAR reaction or the toehold of the TWJ complex for toehold-mediated strand displacement, thereby enabling the recycling of the target. The amplification products could trigger CRISPR/Cas14a for efficient trans-cleavage and release the magnetically bound gold nanoparticle probes for colorimetry detection. Using Mycobacterium tuberculosis 16S rDNA as the target, the proposed C-TEC could detect 16S rDNA down to 50 fM by the naked eye and 20.71 fM by UV-vis detector at 520 nm within 90 min under optimal conditions. We successfully applied this biosensor to clinical isolates of Mycobacterium tuberculosis. In addition, the C-TEC biosensor also showed feasibility for the detection of RNA viruses. In conclusion, the proposed C-TEC is a convenient, fast, and versatile platform for visual detection of pathogen DNA/RNA and has potential clinical applications.

Tuberculosis has been the serious disease threatening human health and public safety due to the emergence of MDR and XDR-TB. Mycobacterium tuberculosis peptide deformylase (MtPDF) is a valuable target for antituberculotics. In order to discover new potential inhibitor candidates of MtPDF as leads for antituberculotics, Discovery Studio (DS) 2019 was used to perform molecular docking for virtual screening in silico with the bioactive compound library-I (L1700) against MtPDF. Six compounds with high docking scores and favourable ligand-protein interactions by LibDock and CDOCKER were selected for the evaluation of the inhibition potencies against MtPDF and Mycobacterium smegmatis. GST-6xHis tagged MtPDF was recombinant expressed and purified firstly by Glutathione Sepharose 4B, and secondly by Ni Sepharose 6 FF after the cleavage of human rhinovirus 3C protease. These compounds showed IC50 values from 0.5 mu mol/L to 112 mu mol/L against MtPDF, among which CUDC-101 bearing hydroxamic acid exhibited IC50 of 0.5 mu mol/L on MtPDF and MIC against Mycobacterium smegmatis of 32 mu g/mL, and Ixazomib Citrate with IC50 of 63 mu mol/L and MIC of 16 mu g/mL. CUDC-101 and Ixazomib Citrate are promising as the potential leads for antituberculotics.

The clinical methods to detect RNA viruses and disease-related RNAs suffer from time-consuming processes, high false-positive rates, or limited sensitivity. Here, we propose a strategy for rapid RNA detection through intra-enzyme chain replacement-mediated Cas13a cascade cyclic reaction without target amplification. A hairpin RNA mediator (a cleavage substrate for target-activated Cas13a) and a guiding RNA recognized by the cleavage product through intra-enzyme chain replacement were designed and optimized. Upon the recognition and binding of the target RNA to the Cas13a/CrRNA complex, Cas13a is initially activated to cleave the mediator, and the cleavage products recognize the corresponding Cas13a/CrRNA complex by intra-enzyme chain replacement and initiate the circular cascade of Cas13a cleavage and activation. The accumulated active Cas13a cleaves fluorescent reporter probe for achieving target RNA detection. This "mix & read " RNA detection at room temperature was performed in total 30 min. Using miRNA-21 as the target, the changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM, while no significant changes in fluorescence intensity were detected for non-targets. This method applied to the clinical sputum respiratory syncytial virus-positive samples gave results consistent with those from the clinical fluorescence immunoassay. Thus, intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction for detection of RNA viruses in the "mix & read " mode at room temperature is rapid, simple, convenient, and efficient for RNA detection and can be adapted to point-of-care testing for high throughput screening of RNA virus infections.