An In Situ Study on Nanozyme Performance to Optimize Nanozyme-Strip for Aß Detection.

The nanozyme-strip is a novel POCT technology which is different from the conventional colloidal gold strip. It primarily utilizes the catalytic activity of nanozyme to achieve a high-sensitivity detection of target by amplifying the detection signal. However, previous research has chiefly focused on optimizing nanozyme-strip from the perspective of increasing nanozyme activity, little is known about other physicochemical factors. In this work, three sizes of Fe3O4 nanozyme and three sizes of CoFe2O4 nanozyme were used to investigate the key factors of nanozyme-strip for optimizing and improving its detection performance. We found that three sizes of Fe3O4 nanozyme all gather at the bottom of the nitrocellulose (NC) membrane, and three sizes of CoFe2O4 nanozyme migrate smoothly on the NC membrane, respectively. After color development, the surface of NC membranes distributed with CoFe2O4 peroxidase nanozymes had significant color change. Experimental results show that CoFe2O4 nanozymes had better dispersity than Fe3O4 nanozymes in an aqueous solution. We observed that CoFe2O4 nanozymes with smaller particle size migrated to the middle of the NC membrane with a higher number of particles. According to the results above, 55 ± 6 nm CoFe2O4 nanozyme was selected to prepare the nanozyme probe and achieved a highly sensitive detection of Aß42Os on the nanozyme-strip. These results suggest that nanozyme should be comprehensively evaluated in its dispersity, the migration on NC membrane, and the peroxidase-like activity to determine whether it can be applied to nanozyme-strip.

Atomic-Level Regulation of Cobalt Single-Atom Nanozymes: Engineering High-Efficiency Catalase Mimics.

Nanozymes aim to mimic the highly evolved active centers of natural enzymes. Despite progress in nanozyme engineering, their catalytic performance is much less favorable compared with natural enzymes. This study shows that precise control over the atomic configuration of the active centers of Co single-atom nanozymes (SAzymes) enables the rational regulation of their catalase-like performance guided by theorical calculations. The constructed Co-N3 PS SAzyme exhibits an excellent catalase-like activity and kinetics, exceeding the representative controls of Co-based SAzymes with different atomic configurations. Moreover, we developed an ordered structure-oriented coordination design strategy for rationally engineering SAzymes and established a correlation between the structure and enzyme-like performance. This work demonstrates that precise control over the active centers of SAzymes is an efficient strategy to mimic the highly evolved active sites of natural enzymes.

Nanozyme-strip for rapid and ultrasensitive nucleic acid detection of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.

Advancements in 3WJ-based RNA nanotechnology and its application for cancer diagnosis and therapy.

BACKGROUND: This attractive and intriguing Ribonucleic acid (RNA) nanotechnology has been conceptualized over the last two decades and with our increasing understanding of RNA structure and function and improvements of RNA nanotechnology it is now possible to use this in clinical settings. METHODS: Here we review the unique properties and the recent advances in RNA nanotechnology and then look at its scientific and preclinical applications for tumor diagnosis and targeted delivery and RNA-based therapy using RNA nanoparticles with diverse structures and functions. Finally, we discuss the future perspectives and challenges to RNA nanotechnology. RESULTS: RNA can be designed and manipulated in a similar way to DNA while having different rules for base-pairing and displaying functions similar to proteins. Rationally designed RNA nanoparticles based on the three-way junction (3WJ) motif as the core scaffold have been extensively explored in the field of nanomedicine and targeted cancer diagnosis and therapy. CONCLUSIONS: RNA nanostructures based on 3WJs demonstrate promising future applications due to their thermal stability, molecular-level plasticity, multifunctional chemotherapeutic drug delivery and other intrinsic characteristics, which will greatly improve the treatment of cancer and promote further major breakthroughs in this field.

Clinical manifestations and genetic characteristics in the Taiwan thoracic aortic aneurysm and dissection cohort - a prospective cohort study.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAAD) is a devastating but treatable disease if detected early. The clinical manifestations and genetic characteristics underlying TAAD patients in Taiwan, however, remain unclear. METHODS: We consecutively recruited patients referred for TAAD screening and/or management at a tertiary medical center in Taiwan. All patients received a comprehensive survey of the clinical manifestations and a genetic testing with a 29-gene next-generation sequencing (NGS) panel. RESULTS: Patients (n = 107) were referred for different reasons, and could be grouped into 4 categories: known aortic aneurysm or dissection (AoAD) (n = 57), Marfanoid features (n = 36), having family members of suspected AoAD (n = 11), and ectopic lens (n = 3). AoAD were confirmed in 73 (68.2%) of the entire cohort. Among all the clinical manifestations, skin striae distensae was the only physical sign that showed significant association with AoAD (p = 0.007 after adjusted). Disease-causing genes/variants were identified in 46 patients (43.0%); FBN1 was the most prevalent disease-causing gene, followed by TGFBR1, TGFBR2 and FBN2. A positive genetic testing was not only an independent predictor of AoAD (hazard ratio (HR) 3.468, 95% confidence interval (CI) [1.541-7.807], p = 0.003), but also had a higher chance of dissection among the patients with known dilated aorta (HR 4.552, 95% CI [1.578-13.135], p = 0.005). CONCLUSION: The presence of skin striae distensae may serve as a clinical cue for physicians to search for AoAD in subjects who are at risk. The NGS panel test not only helps confirm the diagnosis, but also stratify the risk of dissection among patients with dilated aorta.

Nanozyme chemiluminescence paper test for rapid and sensitive detection of SARS-CoV-2 antigen.

COVID-19 has evolved into a global pandemic. Early and rapid detection is crucial to control of the SARS-CoV-2 transmission. While representing the gold standard for early diagnosis, nucleic acid tests for SARS-CoV-2 are often complicated and time-consuming. Serological rapid antibody tests are characterized by high rates of false-negative diagnoses, especially during early infection. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and sensitive detection of SARS-CoV-2 spike antigen, which integrates nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip. The core of our paper test is a robust Co-Fe@hemin-peroxidase nanozyme that catalyzes chemiluminescence comparable with natural peroxidase HRP and thus amplifies immune reaction signal. The detection limit for recombinant spike antigen of SARS-CoV-2 was 0.1 ng/mL, with a linear range of 0.2-100 ng/mL. Moreover, the sensitivity of test for pseudovirus could reach 360 TCID50/mL, which was comparable with ELISA method. The strip recognized SARS-CoV-2 antigen specifically, and there was no cross reaction with other coronaviruses or influenza A subtypes. This testing can be completed within 16 min, much shorter compared to the usual 1-2 h required for currently used nucleic acid tests. Furthermore, signal detection is feasible using the camera of a standard smartphone. Ingredients for nanozyme synthesis are simple and readily available, considerably lowering the overall cost. In conclusion, our paper test provides a high-sensitive point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly facilitate early screening of SARS-CoV-2 infections, and considerably lower the financial burden on national healthcare resources.

Efficacy of laparoscopic nerve-sparing radical hysterectomy in the treatment of early cervical cancer.

PURPOSE: To evaluate the efficacy and safety of laparoscopic nerve-sparing radical hysterectomy (LNSRH) in the treatment of early cervical cancer. METHODS: The clinical data of 152 patients with early cervical cancer undergoing radical hysterectomy (RH) were retrospectively analyzed, and the patients were divided into LNSRH group (n=76) and laparoscopic RH (LRH) group (n=76) according to the surgical approaches. The tumor recurrence and survival were recorded during postoperative follow-up, and the disease-free survival (DFS) and overall survival (OS) were compared between the two groups of patients. RESULTS: The general clinical characteristics were comparable between the two groups of patients. LNSRH group had a remarkably longer operation time (p<0.001) and a notably shorter length of hospital stay (p<0.001) than LRH group. The postoperative in-dwelling time of urinary catheter in LNSRH group was evidently shorter than that in LRH group (p<0.001). Besides, the time of first flatus and defecation after operation was markedly shortened in LNSRH group compared with that in LRH group, with statistically significant differences (p<0.001). In comparison with those in LRH group, the incidence rate of bladder dysfunction was obviously decreased (p<0.001), while the urodynamic indexes at 6 months after operation were prominently better in LNSRH group (p<0.05). According to the follow-up results, the 5-year OS was 84.9% and 88% in LNSRH group and LRH group, respectively, and the DFS was 74.0% and 78.7%, respectively. Log-rank test showed that the differences in OS and DFS between the two groups of patients were not statistically significant (p=0.275, p=0.213). CONCLUSIONS: LNSRH is safe and effective in treating early cervical cancer and can result in similar tumor recurrence and long-term survival to LRH. However, it has superior protective effects on the bladder and bowel functions, which is worthy of popularization and application.

Modification effect of sex and obesity on the correlation of LEP polymorphisms with leptin levels in Taiwanese obese women.

BACKGROUND: Obesity has become the main health issue in developed countries as it impacts life expectancy and increases mortality of cerebrovascular or cardiovascular diseases. The leptin is one of the adipokines which presents in the serum in proportion to the amount of adipose tissue and is translated from LEP gene. It involves in energy homeostasis, lipid and glucose metabolisms, modulation of immune systems, and thermogenesis. Many previous studies have revealed controversial results between LEP polymorphisms and leptin levels in different ages and ethnicities. Herein, we investigated the impacts of LEP polymorphism against leptin levels in Taiwanese subjects. METHODS: In 599 Taiwanese subjects, excluding clinically overt systemic disease, age below 18 years old, and C-reactive protein (CRP) level of above 10 mg/L, few of LEP polymorphisms were genotyped with TaqMan SNP genotyping assays, were further analyzed for association with leptin level in univariate and multivariate linear regression analyses with Bonferroni correction for multiple tests in stratified groups. The univariate and stepwise multivariate linear regression analyses were performed to determine the coefficient of determinant of LEP polymorphisms over leptin level. RESULTS: Significant associations were found between LEP polymorphisms and leptin levels in obese women. Circulating leptin level was positively correlated with inflammatory, insulin resistance markers, and visceral obesity markers in all subjects. Furthermore, stratified and interaction analyses revealed that LEP polymorphisms, rs7799039 and rs2167270, were significantly associated with leptin levels in obese women-8%-10% of which could be explained by LEP polymorphisms. CONCLUSION: The LEP polymorphisms are independently associated with leptin levels in Taiwanese obese women. Further, the genetic determinants for leptin levels may be different between obese and nonobese, and in different sex individuals. The obesity status and female sex may exert modification effect on transcription of LEP, particularly in obese women.

Doses of renin-angiotensin system inhibitors but not beta-blockers predict outcome after ST-elevation myocardial infarction.

Objectives: In patients with ST-elevation myocardial infarction (STEMI), it is not clear whether low-dose renin-angiotensin system inhibitors and beta-blockers can result in the same benefits achievable with higher target doses. This observational study aims to investigate whether higher doses of angiotensin converting enzyme inhibitors (ACEI)/angiotensin II receptor blockers (ARB) and beta-blockers can improve outcomes in patients with STEMI. Methods: We recorded daily doses of ACEI, ARB, and beta-blockers in 331 patients with STEMI. Echocardiographic studies were performed at baseline and were repeated 6 months later. Clinical events, including all-cause death and heart failure, were followed for 2 years. Results: Patients receiving high-dose ACEI/ARB had less increase in left ventricular end-diastolic volume index (LVEDVI) at 6 months. In multivariable linear regression model, ACEI/ARB dose or beta-blocker dose was not an independent predictor of increase in LVEDVI at 6 months. Kaplan-Meier survival curves showed that doses of ACEI/ARB (p = 0.003) and beta-blockers (p = 0.027) were significant predictors of death and heart failure. In multivariable Cox regression analysis, independent predictors of all-cause death and heart failure were diabetes mellitus (p = 0.001), left ventricular ejection fraction (p = 0.026), and ACEI/ARB dose (p = 0.025). Beta-blockers dose was not a predictor of clinical events in multivariable analysis (p = 0.413). Conclusion: High-dose ACEI/ARB, but not beta-blocker, was associated with lower rate of all-cause death and heart failure in patients with STEMI.

Bioengineered H-Ferritin Nanocages for Quantitative Imaging of Vulnerable Plaques in Atherosclerosis.

Inflammation and calcification concomitantly drive atherosclerotic plaque progression and rupture and are the compelling targets for identifying plaque vulnerability. However, current imaging modalities for vulnerable atherosclerotic plaques are often limited by inadequate specificity and sensitivity. Here, we show that natural H-ferritin nanocages radiolabeled with technetium-99m (99mTc-HFn) can identify and accurately localize macrophage-rich, atherosclerotic plaques in living mice using combined SPECT and CT. Focal 99mTc-HFn uptake was observed in the atherosclerotic plaques with multiple high-risk features of macrophage infiltration, active calcification, positive remodeling, and necrosis on histology and in early active ongoing lesions with intense macrophage infiltration. The uptake of 99mTc-HFn in plaques enabled quantitative measuring of the dynamic changes of inflammation during plaque progression and anti-inflammation treatment. This strategy lays the foundation of using bioengineered endogenous human ferritin nanocages for the identification of vulnerable and early active plaques as well as potential assessment of anti-inflammation therapy.

Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes.

Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biological detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe3O4 nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase-like catalytic activities. Given the complex interdependence of the physicochemical properties and catalytic characteristics of nanozymes, it is important to establish a standard by which the catalytic activities and kinetics of various nanozymes can be quantitatively compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addition, we describe the detailed experimental procedures for a typical nanozyme strip-based biological detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic standard. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h.

Polyethylenimine-coated Fe3O4 nanoparticles effectively quench fluorescent DNA, which can be developed as a novel platform for protein detection.

We report a novel assembly of polyethyleneimine (PEI)-coated Fe3O4 nanoparticles (NPs) with single-stranded DNA (ssDNA), and the fluorescence of the dye labeled in the DNA is remarkably quenched. In the presence of a target protein, the protein-DNA aptamer mutual interaction releases the ssDNA from this assembly and hence restores the fluorescence. This feature could be adopted to develop an aptasensor for protein detection. As a proof-of-concept, for the first time, we have used this proposed sensing strategy to detect thrombin selectively and sensitively. Furthermore, simultaneous multiple detection of thrombin and lysozyme in a complex protein mixture has been proven to be possible.

Ultrasensitive Detection of Viable Enterobacter sakazakii by a Continual Cascade Nanozyme Biosensor.

Recent outbreaks of life-threatening neonatal infections linked to Enterobacter sakazakii (ES) heightened the need to develop rapid and ultrasensitive detection strategies, especially those capable of determining the viable cells. This study introduced a continual cascade nanozyme biosensor for the detection of viable ES based on propidium monoazide (PMA), loop-mediated isothermal amplification (LAMP), and Nanozyme strip. The ompA gene of ES was determined using FITC-modified and BIO-modified primers in the LAMP process. LAMP combined with PMA treatment was applied for distinguishing the viable from the dead state of ES. Then, using Fe3O4 magnetic nanoparticles as a nanozyme probe, a magnetic nanoparticle (MNP)-based immunochromatographic strip (Nanozyme strip) was further employed for amplifying signal to allow visual detection and quantification by a strip reader. The LAMP products were sandwiched between the anti-FITC and the anti-BIO, and the accumulation of the Fe3O4 magnetic nanoparticles enabled the visual detection of ES. The detection limit of the nanozyme biosensor was improved by 10 CFU/mL compared with previously reported techniques, and the whole manipulation process was much faster (within 1 h) and simpler (without specialist facilities). Hence, the developed continual cascade nanozyme biosensor has provided a rapid, ultrasensitive, and simple tool for on-site detection of viable ES.

Clinical Outcomes of Repetition of Drug-Coated Balloon for Femoropopliteal Restenosis After Drug-Coated Balloon Treatment.

BACKGROUND: To compare the clinical outcomes of patients undergoing repeated drug-coated balloon (DCB) treatment for femoropopliteal (FP) DCB restenosis with those of patients without repetition-DCB.Methods and Results:From March 2013 to September 2014, 102 patients (118 affected legs) underwent DCB for symptomatic FP disease; 47 patients had restenosis, and 37 underwent reintervention over a 45-month follow-up. We compared the outcomes of repetition-DCB for DCB restenosis with those of patients without repetition. The baseline patient and lesion characteristics were similar between groups. The mean lesion length was 200.8±113.1 and 195.2±134.6 mm, P=0.894, respectively. In addition, the procedural and follow-up outcomes were not different. The rates of freedom from binary restenosis (70% vs. 14%, P=0.001) and clinically driven target lesion revascularization (CD-TLR) (78% vs. 38%, P=0.026) at 1 year were statistically different between groups. Cox regression analysis showed that repetition of DCB was the only predictor for freedom from binary restenosis (hazard ratio [HR]: 6.15, 95% confidence interval (CI) 1.60 to 23.6, P=0.008) and CD-TLR (HR: 5.37, 95% CI 1.32-22.0, P=0.019). CONCLUSIONS: For FP DCB restenosis, repetition of DCB can potentially improve vessel patency and significantly reduce the need for reintervention compared with conventional treatment. However, these observations require further confirmation in larger scale studies.

Optimization of Fe3O4 nanozyme activity via single amino acid modification mimicking an enzyme active site.

The Fe3O4 nanozyme was the first reported nanoparticle with intrinsic peroxidase-like activity and has been widely used in biomedicine. To optimize its catalytic activity, we introduced histidine residues onto the Fe3O4 nanoparticle surface in order to mimic the enzymatic microenvironment of natural peroxidase enzymes. Our results show that modification with a single amino acid could more than ten-fold improve the apparent affinity (KM) of the Fe3O4 nanozyme for the substrate H2O2 and enhanced its catalytic efficiency (kcat/KM) up to twenty fold. Thus we not only optimized the activity of the Fe3O4 nanozyme, but also provide a new rationale for improving the efficiency of nanomaterial-based catalysts by utilizing strategies observed in nature.

Nanozyme-strip for rapid local diagnosis of Ebola.

Ebola continues to rage in West Africa. In the absence of an approved vaccine or treatment, the priority in controlling this epidemic is to promptly identify and isolate infected individuals. To this end, a rapid, highly sensitive, and easy-to-use test for Ebola diagnosis is urgently needed. Here, by using Fe3O4 magnetic nanoparticle (MNP) as a nanozyme probe, we developed a MNP-based immunochromatographic strip (Nanozyme-strip), which detects the glycoprotein of Ebola virus (EBOV) as low as 1 ng/mL, which is 100-fold more sensitive than the standard strip method. The sensitivity of the Nanozyme-strip for EBOV detection and diagnostic accuracy for New Bunyavirus clinical samples is comparable with ELISA, but is much faster (within 30 min) and simpler (without need of specialist facilities). The results demonstrate that the Nanozyme-strip test can rapidly and sensitively detect EBOV, providing a valuable simple screening tool for diagnosis of infection in Ebola-stricken areas.

Associations between TRPV4 genotypes and body mass index in Taiwanese subjects.

Body weight regulation is influenced neuronally via the hypothalamus, which strongly expresses TRPV4. TRPV4 deficiency in mice confers resistance against diet-induced obesity. We investigated the association between TRPV4 gene variants and body mass index (BMI) in Taiwanese subjects. A sample population of 617 Taiwanese subjects was enrolled, and ten TRPV4 gene polymorphisms were selected and genotyped. After adjusting for clinical covariates, significant associations were observed between three studied polymorphisms and BMI using a dominant model (P = 4.83 × 10(-4), P = 1.17 × 10(-4), and P = 3.37 × 10(-4) for rs3742037, rs10735104, and rs3742035, respectively). Obesity as defined according to both the Asian and National Institutes of Health (NIH) criteria was significantly associated with rs10735104 (P = 0.003 and P = 0.037, respectively) in a dominant model. Genotypes at the TRPV4 locus independently affect BMI and obesity status in Taiwanese subjects. This association may broaden our understanding of the role of neuronal influence on body weight regulation. The regulation of TRPV4 channels in skeletal muscle and adipose tissue could also be a new therapeutic target for preventing the development of obesity.

H-ferritin-nanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection.

An ideal nanocarrier for efficient drug delivery must be able to target specific cells and carry high doses of therapeutic drugs and should also exhibit optimized physicochemical properties and biocompatibility. However, it is a tremendous challenge to engineer all of the above characteristics into a single carrier particle. Here, we show that natural H-ferritin (HFn) nanocages can carry high doses of doxorubicin (Dox) for tumor-specific targeting and killing without any targeting ligand functionalization or property modulation. Dox-loaded HFn (HFn-Dox) specifically bound and subsequently internalized into tumor cells via interaction with overexpressed transferrin receptor 1 and released Dox in the lysosomes. In vivo in the mouse, HFn-Dox exhibited more than 10-fold higher intratumoral drug concentration than free Dox and significantly inhibited tumor growth after a single-dose injection. Importantly, HFn-Dox displayed an excellent safety profile that significantly reduced healthy organ drug exposure and improved the maximum tolerated dose by fourfold compared with free Dox. Moreover, because the HFn nanocarrier has well-defined morphology and does not need any ligand modification or property modulation it can be easily produced with high purity and yield, which are requirements for drugs used in clinical trials. Thus, these unique properties make the HFn nanocage an ideal vehicle for efficient anticancer drug delivery.

Label-free microRNA profiling not biased by 3' end 2'-O-methylation.

Accurate quantification of miRNA expression level is essential to the study of its biology, and many cutting-edge technologies have been developed to accommodate this need. Yet most of them were designed primarily for the "regular" RNAs such as animal miRNAs and may overlook the fact that plant miRNAs and many other small noncoding RNAs are 2'-O-methylated at the 3' end nucleotide. According to our experimental data and previous reports, this structural variation is detrimental to the effectiveness of the commonly used enzymatic labeling methods, leading to strongly biased results (~24-fold difference). Herein, we demonstrate that our Stacking-Hybridized Universal Tag (SHUT) microarray assay is well suited for unbiased profiling of both normal and methylated small RNA species. The detected signals of small RNAs with 2'-hydroxyl and 2'-O-methyl 3' ends are highly consistent (no significant difference at α = 0.01 level). For specificity, the presented method edges over others by its unique ability to discriminate single-base difference at or near the 5' end. Notably, as compared to many delicate techniques, this enzyme-free and label-free approach requires much less reagent and manipulation, benefiting the SHUT-based applications with more efficient workflow and highly reproducible results.

Direct microRNA detection with universal tagged probe and time-resolved fluorescence technology.

microRNAs have emerged as the central player in gene expression regulation and have been considered as potent cancer biomarkers for early disease diagnosis. Direct microRNA detection without amplification and labeling is highly desired. Here we present a rapid, sensitive and selective microRNA detection method based on the base stacking hybridization coupling with time-resolved fluorescence technology. Other than planar microarrays, magnetic beads are used as reaction platforms. In this method, one universal tag is used to report all microRNA targets. Its specificity allows for discrimination between microRNAs differing by a single nucleotide, and between precursor and mature microRNAs. This method also provides a high sensitivity down to 20 fM. Moreover, the full protocol can be completed in about 3 h starting from total RNA.