Highly sensitive analysis of exosomal microRNAs (miRNAs) holds significant implications for early cancer diagnosis and prognostic evaluation, yet the low abundance, high homology, and short sequence length of exosomal miRNAs present critical challenges for achieving both sensitivity and specificity in detection. Herein, a dual signal amplification (DSA) strategy integrating an APE1 enzyme-assisted DNA walker with rolling circle amplification (RCA) was developed for highly sensitive, specific, and label-free analysis of exosomal miRNAs. Using miR-10b as a breast cancer proof-of-concept target, the system integrated an APE1 enzyme-assisted DNA walker with rolling circle amplification (RCA). Target miR-10b triggered the APE1-assisted DNA walker immobilized on magnetic beads, generating abundant RCA primer probes. Following magnetic separation, the obtained RCA primers were incubated with the RCA reaction mixture to initiate the RCA reaction, yielding long single-stranded DNA products with copious G-quadruplex (G4). Thioflavin T (ThT) then selectively bound to G4 structures, forming fluorescent G4-ThT complexes that enabled a label-free fluorescence readout. This approach achieved a detection limit of 0.27 fM with exceptional specificity, demonstrating the capacity to discriminate single-nucleotide variants. To validate its applicability, the DSA strategy successfully distinguished miR-10b expression between breast cancer-cell-derived and normal cell-derived exosomes. Furthermore, clinical validation revealed marked differences in serum exosomal miR-10b between breast cancer patients and healthy controls, exhibiting remarkable diagnostic accuracy (AUC = 0.990). This innovative DSA strategy provides a robust platform for ultrasensitive miRNA detection and holds promise for advancing early cancer diagnosis and therapeutic development.

Lupus nephritis (LN), a severe organ manifestation of systemic lupus erythematosus (SLE), is primarily driven by an imbalance between pathogenic Th17 cells and regulatory T (Treg) cells. We found that NK cell-derived extracellular vesicles (NK-EVs) from LN patients, but not healthy controls, exhibited a distinct miRNA cargo that potently drives Th17 polarization. Small RNA sequencing identified miR-1290 as the most significantly upregulated miRNA in NK-EVs derived from LN patients. This finding was validated in an independent clinical cohort, where miR-1290 levels correlated with key disease activity indices. Functional analysis revealed that miR-1290 promoted Th17 differentiation and suppressed Treg generation by downregulating NR4A2. In the MRL/lpr lupus mice, systemic delivery of NK-EVs engineered to carry a miR-1290 antagomir restored the Th17/Treg balance, alleviated renal inflammation and fibrosis. Collectively, we found miR-1290 in NK-EVs disrupted T cell homeostasis by targeting NR4A2, driving Th17/Treg imbalance. Delivering miR-1290 antagomirs via engineered NK-EVs restored this balance and alleviated renal damage in LN mice.

ObjectiveTo apply artificial intelligence (AI) technology for the quantitative analysis of interocular retinal vascular differences in color fundus photographs (CFP) among school-age children with mild to moderate anisometropia and to investigate their correlation with the degree of anisometropia.MethodsIn this retrospective cross-sectional study, 33 children aged 6–9 years with mild to moderate anisometropia were enrolled. CFPs of both eyes were acquired. A deep learning-based AI algorithm was employed to quantitatively analyze interocular differences in the degree of fundus tessellation (FT), the extent of peripapillary atrophy (PPA), optic disc morphology, and retinal vascular characteristics. The correlations between these differences and the interocular spherical equivalent (SE) difference were assessed.ResultsEyes with lower SE values exhibited significantly higher FT density, larger FT areas across all regions, greater PPA area and extent, a larger optic disc ovality index, higher total vessel length density, and higher arterial fractal dimensions (D0, D1) compared to eyes with higher SE values. In contrast, the horizontal cup-to-disc ratio and the average venous tortuosity were significantly lower in the lower SE eyes (all P < 0.05). Correlation analysis revealed that the interocular differences in the horizontal and vertical diameter ratios of PPA to the optic disc (r = 0.433, P = 0.012; r = 0.368, P = 0.035, respectively) and in the arterial singularity length (SL_a) (r = 0.397, P = 0.022) were positively correlated with the degree of anisometropia. No significant linear associations were found between the degree of anisometropia and the interocular differences in other fundus parameters (all P > 0.05).ConclusionSignificant interocular differences in fundus structures are present in children with mild to moderate anisometropia. The magnitude of interocular difference in the PPA-to-optic disc diameter ratio shows a significant correlation with the degree of anisometropia. AI-based quantitative analysis provides an objective imaging basis for the early identification of anisometropia.

Lupus nephritis (LN) is the most common and severe complication of systemic lupus erythematosus (SLE), which requires the use of renal biopsy techniques to determine the pathological type and guide subsequent treatment. Recent studies have identified neutrophil gelatinase associated lipocalin (NGAL) as a promising biomarker for the non-invasive diagnosis, assessment of disease activity, and prediction of recurrence in lupus nephritis. However, current single-signal detection methods based on NGAL lack sufficient specificity and accuracy. In this paper, we reported a novel dual-mode optical fiber biosensor based on microfiber Bragg gratings (μFBG) and surface-enhanced Raman spectroscopy (SERS) for the detection of NGAL. In this sensor, the refractive index change data and Raman spectral data are independently measured and cross-validated, ensuring reliable and accurate analysis. The limits of detection (LOD) of the two modal sensors are 0.49 pg/ml (μFBG) and 48.44 pg/ml (SERS) within the concentration range of 0.01 ng/ml to 1000 ng/ml, respectively. More importantly, the AUC value for clinical samples is significantly improved by utilizing the two modal sensing paradigm. The proposed sensor is particularly suitable for regions with limited medical resources and provides strong support for early diagnosis of lupus nephritis and precise treatment strategies.

Traditional diagnosis of thyroid cancer involves invasive procedures, making it crucial to find convenient and rapid methods for early detection of thyroid cancer. Exosomal miRNA has emerged as a potential and effective biomarker for thyroid cancer diagnosis. However, simple, rapid, and sensitive analysis of exosomal miRNA remains challenging. In this study, we present an integrated platform for the rapid separation of exosomes using metal-organic frameworks (MOF) materials, followed by membrane-fusion-based DNAzyme signal amplification for the highly sensitive detection of miR-31 within extracellular vesicles (EVs), aiming to achieve early diagnosis of thyroid cancer. For the proposed method, MOF not only serves as a medium for separating EVs but also releases Zn2 + upon acid-induced cleavage, which further acts as a cofactor for DNAzyme, reducing the cost and simplifying the process. Furthermore, the combination of membrane fusion-based DNAzyme circuits for miRNA analysis in EVs shows high sensitivity with a detection limit of 1.05 x 10 3 particles/mL. The method can also effectively and accurately distinguish the expression of miR-31 in the plasma EVs of normal and thyroid cancer patients. This strategy has high accuracy and practicality and is expected to play an important role in disease diagnosis and drug research.

Lupus nephritis (LN) is a chronic complication of systemic lupus erythematosus (SLE). At present, no drugs are capable of delaying the progression of LN without a risk of serious side effects. There is thus a pressing need for further studies of LN pathogenesis to identify novel therapeutic targets and aid in the development of new approaches to treating this debilitating disease. In this study, a multi-omics approach was used to characterize the pathogenesis of LN and to identify disease-related targets, ultimately leading to the identification and validation of Yin Yang 1 (YY1) as a promising therapeutic target in LN. A rapid approach to efficiently screening for candidate YY1 ligands was implemented using drug databases that established rebamipide as a YY1 antagonist suitable for use in the management of LN. Specifically, the YY1 antagonist activity of rebamipide was found to regulate lymphocyte activity, reduce autoantibody production, limit immune complex deposition, and suppress macrophage activation while improving symptoms in a murine model of LN. Results supportive of a similar pathologic mechanism of action were also obtained when analyzing renal tissue sections from LN patients, underscoring the potential clinical significance of YY1 and its antagonist rebamipide, suggesting that rebamipide may have positive effects on lymphocytes and may improve symptoms in treated patients. This study provides a robust foundation for further research focused on the pathogenesis and treatment of LN.

MicroRNA (miRNA) is involved in the progression of Alzheimer's disease (AD) and emerges as a promising AD biomarker and therapeutic target. Therefore, there is an urgent need to develop convenient and precise miRNA detection methods for AD diagnosis. Herein, a dual-signal amplification strategy based on rolling circle amplification and APE1-assisted amplification for miRNA analysis for early diagnosis of AD was proposed. The strategy consisted of dumbbell-shaped probe (DP) as amplification template and a reporter probe (RP) with an AP site modification. In the presence of the target miRNA, the miRNAs bound to the toehold domain of DP and DP was activated into a circular template. Then, RCA reaction was triggered, producing a large number of long-stranded products containing repeated sequences. After RCA, APE1 enzyme recognized and removed AP site in the complex of RCA/RP products. By coupling RCA with APE1-assisted amplification, this method has high sensitivity with the limit of detection (LOD) of 1.82 fM. Moreover, by using DP as template for RCA reaction, high specificity can be achieved. By detecting miR-206 in serum using this method, the expression of miR-206 can be accurately distinguished between AD patients and healthy individuals, indicating that this method has broad application prospects in clinical diagnosis.

Background Lupus nephritis (LN) is the most common complication of systemic lupus erythematosus (SLE). The limited treatment options for LN increase the economic burdens on patients. Because fibrotic progression leads to irreversible renal damage in LN patients and further progresses to chronic kidney disease (CKD) and the end stage of renal disease (ESRD), developing new targets to prevent LN fibrotic progression could lead to a feasible treatment strategy for LN patients. Methods In this study, we examined YAP activation and LATS2 downregulation in LN kidney biopsy samples (LN: n = 8, normal: n = 2) and lupus-prone MRL/lpr mice ( n = 8 for each disease stage). The function of LATS2 was further investigated by in situ injection of Ad-LATS2 into mice with LN ( n = 6 mice per group). We examined the role of SIAH2-LATS2 regulation by IP-MS and co-IP, and the protective effect of the SIAH2 inhibitor was investigated in mice with LN. Results Restoring LATS2 by an adenovirus in vivo alleviated renal fibrotic damage in mice with LN. Moreover, we found that LATS2 was degraded by a K48 ubiquitination-proteasome pathway mediated by SIAH2 and promoted YAP activation to worsen fibrosis progression in LN. The H150 region of the substrate binding domain (SBD) is an important site for SIAH2-LATS2 binding. The SIAH2-specific inhibitor vitamin K3 protected against LN-associated fibrotic damage in vivo. Conclusion In summary, we identified the SIAH2-LATS2 axis as an attractive intervention target in LN to alter the resistance to fibrosis.

Background:Non-alcoholic fatty liver disease (NAFLD) is a leading cause of liver-related morbidity and mortality. The diagnosis of non-alcoholic steatohepatitis (NASH) plays a crucial role in the management of NAFLD patients. Objective:The aim of our observational study was to build a machine learning model to identify NASH in NAFLD patients. Methods:The clinical characteristics of 259 NAFLD patients and their initial laboratory data (Cohort 1) were collected to train the model and carry out internal validation. We compared the models built by five machine learning algorithms and screened out the best models. Receiver operating characteristic (ROC) curves, sensitivity, specificity, and accuracy were used to evaluate the performance of the model. In addition, the NAFLD patients in Cohort 2 (n = 181) were externally verified. Results:We finally identified six independent risk factors for predicting NASH, including neutrophil percentage (NEU%), aspartate aminotransferase/alanine aminotransferase (AST/ALT), hematocrit (HCT), creatinine (CREA), uric acid (UA), and prealbumin (PA). The NASH-XGB6 model built using the XGBoost algorithm showed sufficient prediction accuracy, with ROC values of 0.95 (95 % CI, 0.91-0.98) and 0.90 (95 % CI, 0.88-0.93) in Cohort 1 and Cohort 2, respectively. Conclusions:NASH-XGB6 can serve as an effective tool for distinguishing NASH patients from NAFLD patients.

PIWI-interacting RNAs (piRNAs) are a novel class of non-coding RNAs that bind specifically to the PIWI subfamily of Argonaut proteins. It has been increasingly demonstrated that piRNAs encased in circulating exosomes could be an ample source of potential tumor markers for cancer diagnostics. However, methods on exosomal piRNAs detections are limited, and most of them need extraction of piRNAs from the sample which is laborious and disadvantage to clinical applications. Herein, we developed two kinds of dual-targeted spherical nucleic acid nanoprobes for in situ and multiplex detection of exosomal piRNAs. The detection scheme was rationally designed for the large sized targets of PIWI-interacting RNA complex which could generate steric hindrance in the detection reaction. The probes were synthesized by modifying 13-nm gold particles with high-density double stranded anchor-report DNA through butanol dehydration method. The key synthesis condition of molar ratio between the two kinds of anchor-report DNA chains were optimized for preparing probes with good structural reproducibility. The probes can perform in situ and multiplex detection of piRNAs in exosomes after simple incubation. In the clinical assays of plasma from breast cancer patients and normal control groups, the probes can differentiate the expression levels of 3 types of piRNAs and their combinations in high specificity and sensitivity. These new piRNA probes are potentially to perform simple and accurate liquid biopsy for cancer diagnostics.

Controllable and sufficient concentrations of therapeutic agents in gene therapy are critical to achieve satisfactory outcomes. Herein, a Zn2+/Cu2+ bimetallic nano metal-organic framework loaded with rolling circle amplification (RCA) substrates (termed PZCT) was established for precise and efficient DNAzyme-based gene and chemo-dynamic combined therapy. The activation of PZCT is bioorthogonally controlled by tumor-specific miR-21, which generates numerous DNAzyme for silencing EGR-1 mRNA with the assistance of Zn2+ from PZCT. Simultaneously, the doped copper ions on PZCT exert chemo-dynamic therapy (CDT) by reducing glutathione (GSH) and converting endogenous hydrogen peroxide (H2O2) into hydroxyl radical (OH). These combination therapies exhibited remarkable tumor elimination effects in vivo and promised excellent tumor specificity via a bioorthogonal strategy. The proposed nanoplatform offers new prospects for precise cancer therapeutics by overcoming low transfection efficiency and off-target toxicity in DNAzyme-based approaches.

Developing simple, rapid and sensitive strategies for miRNA analysis is extremely important for diseases early diagnosis. Herein, an APE1-mediated isothermal target cycling amplification system combined with magnetic separation was established for label-free and rapid detection of miRNA. As a proof-of-concept, miR-1246 was selected as research model. In the detection system, when the miR-1246 is present, it hybridizes with AP probes of AP-MBs to form a double-stranded, in which the APE1 recognizes specific AP site of double-stranded and cleaves the AP probes, releasing a sequence containing a G-quadruplex (G4). After the cleavage, target miR-1246 can be released to hybridize with another AP of AP-MBs, triggering a continues cleavage reaction. After magnetic separation, label-free analysis of miRNA was achieved by measuring the fluorescence of G4/ThT. Due to the high efficiency and specificity of APE1 toward AP sites in dsDNAs, this method exhibits high specificity and sensitivity with the capability of distinguishing miRNAs with single-base mismatch. The system could detect miRNA with high sensitivity within 2 h, and the limit of detection (LOD) was calculated as 25.6 fM. Furthermore, high accuracy has been achieved through recovery experiments and successful attempts has been made in applying the approach to detect miR-1246 in serum samples from breast cancer patients and normal people. This method is expected to be an effective tool for miRNA-related research and clinical early diagnosis.

Early tumor diagnosis is crucial to successful treatment. Earlier studies have shown that microRNA is a biomarker for early tumor diagnosis. The development of highly sensitive miRNA detection methods, especially in living cells, plays an indispensable role for early diagnosis and treatment of tumor. Although the catalytic hairpin assembly (CHA)-based miRNA analysis strategy is commonly used for disease diagnosis, further application of CHA is hindered due to its low amplification efficiency and low tumor recognition contrast. To address these limitations, we propose a dual-signal amplification strategy based on CHA and APE1-assisted amplification, enabling highly sensitive and high-contrast miRNA imaging. The miR-221 was selected as a target model. This dual-signal amplification strategy has exhibited high amplification efficiency, which could analyze miRNA as low as 21 fM. This strategy also exhibited high specificity, which could distinguish target miRNA and nontarget with single-base differences. Moreover, this method showed significant potential for practical application, as it could successfully distinguish the expression difference of miR-221 in the plasma samples of normal people and patients. Most importantly, the expression level of the APE1 enzyme in tumor cells is higher than that in normal cells, allowing this strategy to sensitively and specifically image miRNA within tumor cells. This proposed method has also been successfully used to indicate fluctuations of intracellular miRNA and to distinguish miRNA expression between normal cells and cancer cells with high contrast. We anticipate that this method will provide fresh insights and can be a powerful tool for tumor diagnosis and treatment based on miRNA analysis.

Background: DNA walker-based strategies have gained significant attention in nucleic acid analysis. However, they face challenges related to balancing design complexity, sequence dependence, and amplification efficiency. Furthermore, most existing DNA walkers rely on walking and lock probes, requiring optimization of various parameters like DNA probe sequence, walking-to-lock probe ratio, lock probe length, etc. to achieve optimal performance. This optimization process is time-consuming and adds complexity to experiments. To enhance the performance and reliability of DNA walker nanomachines, there is a need for a simpler, highly sensitive, and selective alternative strategy. Results: A sensitive and rapid miRNA analysis strategy named hairpin-shaped DNA aligner and nicking endonuclease-fueled DNA walker (HDA-NE DNA walker) was developed. The HDA-NE DNA walker was constructed by modifying hairpin-shaped DNA aligner (HDA) probe and substrate report (SR) probe on the surface of AuNPs. Under normal conditions, HDA and SR remained stable. However, in the presence of miR-373, HDA underwent a conformational transition to an activated structure to continuously cleave the SR probe on the AuNPs with the assistance of Nt.AlwI nicking endonuclease, resulting in sensitive miRNA detection with a detection limit as low as 0.23 pM. Additionally, the proposed HDA-NE DNA walker exhibited high selectivity in distinguishing miRNAs with single base differences and can effectively analyze miR-373 levels in both normal and breast cancer patient serums. Significance: The proposed HDA-NE DNA walker system was activated by a conformational change of HDA probe only in the presence of the target miRNA, eliminating the need for a lock probe and without sequence dependence for SR probe. This strategy demonstrated a rapid reaction rate of only 30 min, minimal background noise, and a high signal-to-noise ratio (S/B) compared to capture/lock-based DNA walker. The method is expected to become a powerful tool and play an important role in disease diagnosis and precision therapy.

ETHNOPHARMACOLOGICAL RELEVANCE:Kunxian capsule (KXC) is a new traditional Chinese medicine drug included in "The key science and technology achievements" in the Ninth Five Year Plan of China. KXC has been clinically used for more than 10 years in the treatment of lupus nephritis (LN). However, the underlying role and molecular mechanism of KXC in LN remain unclear. AIM OF THE STUDY:This study aimed to explore the efficacy and potential mechanisms of KXC through pharmacological network, in vitro and in vivo studies. MATERIALS AND METHODS:Pharmacological network analysis of KXC treatment in LN was performed using data acquired from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP, https://old.tcmsp-e.com/tcmsp.php) and NCBI Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/database). HK-2 cells were chosen as an in vitro model of the tubular immune response by simulation with interferon γ (IFN-γ). MRL/lpr mice were used to explore the mechanism of KXC in vivo. Finally, the specific active molecules of KXC were further analyzed by molecular docking. RESULTS:The pharmacological network analysis showed that STAT1 is a key factor in the effects of KXC. In vitro and in vivo experiments confirmed the therapeutic effect of KXC on LN renal function and tubular inflammation. The protective effect of KXC is mediated by STAT1 blockade, which further reduces T-cell infiltration and improves the renal microenvironment in LN. Two main components of KXC, Tripterygium hypoglaucum (H.Lév.) Hutch (Shanhaitang) and Epimedium brevicornu Maxim (Yinyanghuo) could block JAK1-STAT1 activation. Furthermore, we found 8 molecules that could bind to the ATP pocket of JAK1 with high affinities by performing docking analysis. CONCLUSIONS:KXC inhibits renal damage and T-cell infiltration in LN by blocking the JAK1-STAT1 pathway.

Abstract Background Surgery at the primary site of nasopharyngeal carcinoma (NPC) remains controversial. This study aimed to investigate the efficacy of surgery in patients with stage T1N0-3M0 NPC.Methods We reviewed 1803 patients with newly diagnosed stage T1N0-3M0 NPC from the Surveillance, Epidemiology, and End Results (SEER) database for the period 2004–2015. The Kaplan-Meier method and propensity score matching (PSM) analyses were used to compare cancer-specific survival (CSS) and overall survival (OS) between surgery groups and no-surgery groups. Restricted cubic spline regression (RCS) was used to assess the relationship between age and the hazard ratio of mortality. Independent risk factors associated with OS and CSS were identified by Cox ratio hazard regression analysis employing a backward stepwise method, which was used to develop prognostic nomograms for locoregional T1 NPC patients. The correlation index and calibration curves were used to assess the performance of the model. An online dynamic nomogram for CSS was also developed to assist the clinic in better using the predictive model.Results The study examined 1803 patients, of whom 242 received surgery, and the remaining 1561 did not receive surgery. Before PSM, Kaplan-Meier analysis showed that OS and CSS were significantly longer in the surgery group than in the non-surgery group (P < 0.001). After 1:1 matching, similar results as above could be observed, that is, patients in the surgical group had significantly better OS (P < 0.001) and CSS (P = 0.0018) benefits. Subgroup analysis further showed that almost all subgroups undergoing surgery had superior OS and CSS, except for patients with tumor size > 30 mm and primary site located in the anterior wall of the nasopharynx. Finally, COX regression analyses showed that age, sex, marital status, histological type, N stage, tumor size, radiotherapy, and surgery were independent prognostic factors for OS and CSS.Conclusions This study indicated that surgery significantly improved the OS and CSS of patients with stage T1N0-3M0 NPC. The nomograms we established have satisfactory performance and may provide a reference for the clinic. Further prospective large studies are still needed to validate the results.

Rapid and sensitive analysis of ochratoxin A (OTA) plays an important role in food safety. Here, an aptasensor based on novel exponential rolling circle amplification (ERCA) was proposed for ultrasensitive and label-free fluorescence detection of OTA. The attachment of OTA to its aptamer could release H and rapidly hybridize with CT to initiate rolling circle amplification (RCA). The amplicons could further displace H from APH to initiate recycled RCA, achieving exponential growth of amplification products that contained G4 dimers for lighting up ThT. Benefiting from the exponential amplification efficiency of the ERCA strategy and the high fluorescence quantum yield of G4 dimer/ThT, this strategy exhibited a wide linear range from 10 fg/mL to 10 ng/mL with a detection limit of 4.3 fg/mL. In addition, the aptasensor displayed satisfactory recoveries in real sample analysis. We believe that this novel aptasensor possesses promising application prospects in food safety and medicine detection.

It is a challenge to establish efficient, simplicity and precision miRNA imaging methods in living cells. Herein, we report a ZIF-8 @DNAzyme-based DNA walker strategy for intracellular miRNA imaging with high accuracy. Compared with traditional DNAzyme-based DNA walker, DNAzyme-based DNA walker can be effectively delivered into the cytoplasm by packaging in ZIF-8, thus ensuring the fidelity of intracellular miRNA imaging. In addition, ZIF-8 with acid-degrading properties can be degraded to generate cofactor Zn2+ in the acidic envi-ronment of tumors, achieving in the synchronous transport of DNAzyme-based DNA walker and metal ions. This strategy not only displays extremely low background signals in live-cell miRNA imaging, but also can distinguish the expression level of miRNA between tumor cells and normal cells, showing great potential in disease diag-nosis. We believe this strategy can further promote the application of DNA-based motors in live-cell miRNA imaging, and provide new ideas for the joint application of MOFs and nucleic acid detection.

Accurate and specific imaging of low-abundance microRNA (miRNA) in living cells is extremely important for disease diagnosis and monitoring of disease progression. DNA nanomotors have shown great potential for imaging molecules of interest in living cells. However, inappropriate driving forces and complex design and operation procedures have hindered their further application. Here, we proposed an endogenous enzyme-powered DNA nanomotor (EEPDN), which employs an endogenous APE1 enzyme as fuel to execute repetitive cycles of motion for miRNA imaging in living cells. The whole motor system is constructed based on gold nanoparticles without other auxiliary additives. Due to the high efficiency of APE1, this EEPDN system has achieved highly sensitive miRNA imaging in living cells within 1.5 h. This strategy was also successfully used to differentiate the expression of specific miRNA between tumor cells and normal cells, demonstrating a high tumor cell selectivity. This strategy can promote the development of novel nanomotors and is expected to be a perfect intracellular molecular imaging tool for biological and medical applications.

Split DNAzymes have been designed as multifunctional biosensors. However, their limited sensitivity and low resistance to nuclease hinder their applications in biological samples. Herein, we developed a sodium-dependent and butanol accelerated split DNAzyme fluorescent sensor named Na+-BAS DNAzyme which has an ultrasensitivity for quantifying PIWI-interacting RNAs/microRNAs and can be directly applied in blood biopsy to detect nucleic acids within the extracellular vesicles (EVs). The Na+-BAS DNAzyme consists of two split DNA strands based on NaA43 DNAzyme, a starting hairpin DNA for binding target to initiate the combining of the split enzymes, and a hairpin RNA-embedded DNA substrate labeled a quencher and a fluorophore for signal readout after its cleavage by the assembled DNAzyme complex. To achieve ultra-sensitivity, we employed a simple but key approach of butanol dehydration to highly accelerate the measuring reaction. The sensor has achieved an ultra-low detection limit of 12 x 10(-18) M miR-21 after 30 min reaction, and 3.98 or 2.69 particles mu L-1 MCF-7 EVs through miR-21 or piR-20365 detection. With these high performances, the sensor can detect extremely trace abundance of miR-21 in a single MCF-10a or MCF-7 EV, quantifying it as 5.93 x 10(-3) or 0.12 copies per EV. Without any chemical structure modification, the sensor can be directly used in the plasma sample for breast cancer diagnosis which has obtained both 100% sensitivity and specificity using the biomarker of piR-20365. The Na+-BAS DNAzyme sensor's ultra-sensitivity, high efficiency, universality, and simplicity make it a great potential in the clinical diagnostic application.