Fast Fabrication Nanopores on a PMMA Membrane by a Local High Electric Field Controlled Breakdown.

The sensitivity and accuracy of nanopore sensors are severely hindered by the high noise associated with solid-state nanopores. To mitigate this issue, the deposition of organic polymer materials onto silicon nitride (SiNx) membranes has been effective in obtaining low-noise measurements. Nonetheless, the fabrication of nanopores sub-10 nm on thin polymer membranes remains a significant challenge. This work proposes a method for fabricating nanopores on polymethyl methacrylate (PMMA) membrane by the local high electrical field controlled breakdown, exploring the impact of voltage and current on the breakdown of PMMA membranes and discussing the mechanism underlying the breakdown voltage and current during the formation of nanopores. By improving the electric field application method, transient high electric fields that are one-seven times higher than the breakdown electric field can be utilized to fabricate nanopores. A comparative analysis was performed on the current noise levels of nanopores in PMMA-SiNx composite membranes and SiNx nanopores with a 5 nm diameter. The results demonstrated that the fast fabrication of nanopores on PMMA-SiNx membranes exhibited reduced current noise compared to SiNx nanopores. This finding provides evidence supporting the feasibility of utilizing this technology for efficiently fabricating low-noise nanopores on polymer composite membranes.

Exploring the role of antibiotics and steroids in managing respiratory diseases.

Respiratory diseases (RDs), such as chronic obstructive pulmonary disease, cystic fibrosis, asthma, and pneumonia, are associated with significant morbidity and mortality. Treatment usually consists of antibiotics and steroids. Relevant published literature reviews, studies, and clinical trials were accessed from institutional and electronic databases. The keywords used were respiratory diseases, steroids, antibiotics, and combination of steroids and antibiotics. Selected articles and literature were carefully reviewed. Antibiotics are often prescribed as the standard therapy to manage RDs. Types of causative respiratory pathogens, spectrum of antibiotics activity, route of administration, and course of therapy determine the type of antibiotics that are prescribed. Despite being associated with good clinical outcome, treatment failure and recurrence rate are still high. In addition, antibiotic resistance has been widely reported due to bacterial mutations in response to the use of antibiotics, which render them ineffective. Nevertheless, there has been a growing demand for corticosteroids (CS) and antibiotics to treat a wide variety of diseases, including various airway diseases, due to their immunosuppressive and anti-inflammatory properties. The use of CS is well established and there are different formulations based on the diseases, such as topical administration, tablets, intravenous injections, and inhaled preparations. Both antibiotics and CS possess similar properties in terms of their anti-inflammatory effects, especially regulating cytokine release. Thus, the current review examines and discusses the different applications of antibiotics, CS, and their combination in managing various RDs. Drawbacks of these interventions are also discussed.

Increasing burden of major trauma in elderly adults during 2003-2015: Analysis of real-world data from Taiwan.

BACKGROUND: Major trauma has been one of the leading causes of morbidity, mortality, and functional disability, resulting in substantial societal burden. The aim of this study was to estimate the trends in burden of adult major trauma in Taiwan during 2003-2015. METHODS: Adult patients with initial encounter of major trauma (injury severity score ≥ 16) were abstracted from the claim data of National Health Insurance (NHI) in Taiwan from January 2003 to December 2015. We explored the trends of incidence and mortality rates over time stratified by age and sex, as well as life expectancy (LE), loss-of-LE, lifetime healthcare expenditure and total loss-of-LE compared with age, sex and calendar-year matched referents simulated from the vital statistics of Taiwan. RESULTS: A total of 71,731 cases of adult major trauma, and an estimated loss of 979,676 life-years were found with an increasing trend in cumulative incidence rate (CIR18-84) during 2003-2015. The incidence rates were significantly higher in men than women. For both sexes, the incidence rates for those aged 65 and above were about 2-3 times higher than those of all other age groups. The one-year case fatality rates among the elderly were about 31-61%, higher than all other ages. The lifetime healthcare expenditures per person were 47,616 USD in men and 43,416 USD in women. CONCLUSION: There is a consistently increasing trend in incidence and mortality of major trauma in Taiwan, especially among elderly people. For Taiwan, an aged society beginning since 2018, the challenge should be tackled more effectively in the coming decades.

Recognition of Bimolecular Logic Operation Pattern Based on a Solid-State Nanopore.

Nanopores have a unique advantage for detecting biomolecules in a label-free fashion, such as DNA that can be synthesized into specific structures to perform computations. This method has been considered for the detection of diseased molecules. Here, we propose a novel marker molecule detection method based on DNA logic gate by deciphering a variable DNA tetrahedron structure using a nanopore. We designed two types of probes containing a tetrahedron and a single-strand DNA tail which paired with different parts of the target molecule. In the presence of the target, the two probes formed a double tetrahedron structure. As translocation of the single and the double tetrahedron structures under bias voltage produced different blockage signals, the events could be assigned into four different operations, i.e., (0, 0), (0, 1), (1, 0), (1, 1), according to the predefined structure by logic gate. The pattern signal produced by the AND operation is obviously different from the signal of the other three operations. This pattern recognition method has been differentiated from simple detection methods based on DNA self-assembly and nanopore technologies.

Phosphatase POPX2 Exhibits Dual Regulatory Functions in Cancer Metastasis.

Cancer metastasis is a complex mechanism involving multiple processes. Previously, our integrative proteome, transcriptome, and phosphoproteome study reported that the levels of serine/threonine phosphatase POPX2 were positively correlated with cancer cell motility through modulating MAPK signaling. Surprisingly, here we found that POPX2 knockdown cells induced more numerous and larger tumor nodules in lungs in longer term animal studies. Interestingly, our analysis of DNA microarray data from cancer patient samples that are available in public databases shows that low POPX2 expression is linked to distant metastasis and poor survival rate. These observations suggest that lower levels of POPX2 may favor tumor progression in later stages of metastasis. We hypothesize that POPX2 may do so by modulation of angiogenesis. Secretome analysis of POPX2-knockdown MDA-MB-231 cells using LC-MS/MS-based SILAC quantitative proteomics and cytokine array show that silencing of POPX2 leads to increased secretion of exosomes, which may, in turn, induce multiple pro-angiogenic cytokines. This study, combined with our previous findings, suggests that a single ubiquitously expressed phosphatase POPX2 influences cancer metastasis via modulating multiple biological processes including MAPK signaling and exosome cytokine secretion.

Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells.

The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5-20 mM), cortiosterone (10-9 -10-4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135-2144, 2017. © 2016 Wiley Periodicals, Inc.

Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

BACKGROUND: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. METHODS: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. RESULTS: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. CONCLUSIONS: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.

XCC2366, A Gene Encoding A Putative TetR Family Transcriptional Regulator, is Required for Acriflavin Resistance and Virulence of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot disease in cruciferous plants. The XCC2366 gene product is annotated as a protein belonging to the TetR family of transcriptional regulators. In this study, we evaluated the function and expression of the XCC2366 gene. Mutational analysis demonstrated that XCC2366 is involved in the resistance to acriflavin and is necessary for virulence in Xcc. In addition, the XCC2366 transcription initiation site was mapped at nucleotide A, 63 nucleotide upstream of the XCC2366 translation start codon. Furthermore, transcriptional analysis revealed that the expression of XCC2366 is induced in the presence of acriflavin. Reporter assay also showed that XCC2366 regulates its own expression under acriflavin-supplemented condition. To the best of our knowledge, acriflavin resistance-related gene in the crucifer pathogen Xcc was characterized for the first time.

Association of preschool children behavior and emotional problems with the parenting behavior of both parents.

BACKGROUND: Parental behaviors are key in shaping children's psychological and behavioral development, crucial for early identification and prevention of mental health issues, reducing psychological trauma in childhood. AIM: To investigate the relationship between parenting behaviors and behavioral and emotional issues in preschool children. METHODS: From October 2017 to May 2018, 7 kindergartens in Ma'anshan City were selected to conduct a parent self-filled questionnaire - Health Development Survey of Preschool Children. Children's Strength and Difficulties Questionnaire (Parent Version) was applied to measures the children's behavioral and emotional performance. Parenting behavior was evaluated using the Parental Behavior Inventory. Binomial logistic regression model was used to analyze the association between the detection rate of preschool children's behavior and emotional problems and their parenting behaviors. RESULTS: High level of parental support/participation was negatively correlated with conduct problems, abnormal hyperactivity, abnormal total difficulty scores and abnormal prosocial behavior problems. High level of maternal support/participation was negatively correlated with abnormal emotional symptoms and abnormal peer interaction in children. High level of parental hostility/coercion was positively correlated with abnormal emotional symptoms, abnormal conduct problems, abnormal hyperactivity, abnormal peer interaction, and abnormal total difficulty scores in children (all P < 0.05). Moreover, paternal parenting behaviors had similarly effects on behavior and emotional problems of preschool children compared with maternal parenting behaviors (all P > 0.05), after calculating ratio of odds ratio values. CONCLUSION: Our study found that parenting behaviors are associated with behavioral and emotional issues in preschool children. Overall, the more supportive or involved the parents are, the fewer behavioral and emotional problems the children experience; conversely, the more hostile or controlling the parents are, the more behavioral and emotional problems the children face. Moreover, the impact of fathers' parenting behaviors on preschool children's behavior and emotions is no less significant than that of mothers' parenting behaviors.

The combination of DNA nanostructures and materials for highly sensitive electrochemical detection.

Due to the wide range of electrochemical devices available, DNA nanostructures and material-based technologies have been greatly broadened. They have been actively used to create a variety of beautiful nanostructures owing to their unmatched programmability. Currently, a variety of electrochemical devices have been used for rapid sensing of biomolecules and other diagnostic applications. Here, we provide a brief overview of recent advances in DNA-based biomolecular assays. Biosensing platform such as electrochemical biosensor, nanopore biosensor, and field-effect transistor biosensors (FET), which are equipped with aptamer, DNA walker, DNAzyme, DNA origami, and nanomaterials, has been developed for amplification detection. Under the optimal conditions, the proposed biosensor has good amplification detection performance. Further, we discussed the challenges of detection strategies in clinical applications and offered the prospect of this field.

Recent progress of biosensors for the detection of lung cancer markers.

Lung cancer is one of the most common cancers worldwide and the leading cause of death. Early screening of lung cancer is exceptionally essential for later treatment. Abnormal lung cancer tumor markers are validated to assess their diagnostic utility in non-small cell lung cancer (NSCLC) patients. Therefore, tumor markers can be identified in the early stage of lung cancer through biosensor technology and timely diagnosis. This review discusses cutting-edge methods for detecting various types of lung cancer tumor markers using multiple biosensors. The biosensors working at the molecular level are mainly introduced, which can be divided into three categories according to the types of markers: DNA biosensors, RNA biosensors, and protein biosensors. This review focuses on critical electrochemical methods such as electrochemical impedance spectroscopy (EIS), field-effect transistors (FET), cyclic voltammetry (CV), necessary optical sensors such as surface enhancement Raman spectroscopy (SERS), surface-plasmon resonance (SPR), fluorescence methods, and some novel sensing platforms such as biological nanopore and solid-state nanopore sensors and these sensors detect lung cancer tumor markers, such as microRNA (miRNA), DNA mutations (EGFR, KRAS and p53), DNA methylation, circulating tumor DNA (ctDNA), cytokeratin fragment 21-1 (CYFRA21-1), carcinoembryonic antigen (CEA), matrix metallopeptidase 9 (MMP-9), and vascular endothelial growth factor (VEGF). The advantages and disadvantages of different methods are summarized and prospected on this basis, which provides important insights for developing pioneering optoelectronic biosensors for the early diagnosis of lung cancer.

Low-noise and high-speed trans-impedance amplifier for nanopore sensor.

The small current detection circuit is the core component of the accurate detection of the nanopore sensor. In this paper, a compact, low-noise, and high-speed trans-impedance amplifier is built for the nanopore detection system. The amplifier consists of two amplification stages. The first stage performs low-noise trans-impedance amplification by using ADA4530-1, which is a high-performance FET operational amplifier, and a high-ohm feedback resistor of 1 GΩ. The high pass shelf filter in the second stage recovers the higher frequency above the 3 dB cutoff in the first stage to extend the maximum bandwidth up to 50 kHz. The amplifier shows a low noise below sub-2 pA rms when tuned to have a bandwidth of around 5 kHz. It also guarantees a stable frequency response in the nanopore sensor.

DNA nanostructure-assisted detection of carcinoembryonic antigen with a solid-state nanopore.

In this paper, a novel detection technique for tumor marker carcinoembryonic antigen (CEA) has been developed by using a solid-state nanopore as a tool. The system utilizes the specific affinity between aptamer-modified magnetic Fe3O4 and CEA, rather than directly detecting the translocation of CEA through the nanopore. The aptamer-modified magnetic Fe3O4 was hybridized with tetrahedral DNA nanostructures (TDNs), and TDNs were released after CEA was added. We investigate the translocation behavior of individual TDNs through solid-state nanopores. The frequency of the blockage signals for TDNs is recorded for indirect detection of CEA. We realized the detection of CEA with a concentration as low as 0.1 nM and proved the specificity of the interaction between the aptamer. In addition, our designed nanopore sensing strategy can detect CEA in real samples.

An aptamer-assisted nanopore strategy with a salt gradient for direct protein sensing.

Nanopore sensing is at the forefront of the technological revolution of the protein research field and has been widely used in molecular diagnosis and molecular dynamics, as well as for various sequencing applications. However, direct protein sensing with biological nanopores is still challenging owing to the large molecular size. Here, we propose an aptamer-assisted nanopore strategy for direct protein sensing and demonstrate its proof-of-concept utilities by experiments with SARS-Cov-2 nucleocapsid protein (NP), the most abundantly expressed viral protein, that is widely used in clinical diagnosis for COVID-19. NP binds with an oligonucleotide-tailed aptamer to form a protein-DNA complex which induces a discriminative two-level pattern of current blockades. We reveal the potential molecular interaction mechanism for the characteristic blockades and identify the salt gradient condition as the dominant factor of the phenomenon. Furthermore, we achieve a high sensitivity of 10 pM for NP detection within one hour and make a preliminary exploration on clinical diagnosis. This work promises a new platform for rapid and label-free protein detection.

Data-independent acquisition (DIA) mass spectrometry reveals related proteins involved in the occurrence of early intestinal-type gastric cancer.

Identifying proteins associated with the onset of early intestinal-type gastric cancer (EIGC) can yield valuable insights into the pathogenesis of this specific subtype of gastric cancer. Data-independent acquisition mass spectroscopy (DIA-MS) was utilized to identify the differential protein between 10 cases of EIGC and atrophic gastritis with intestinal metaplasia (NGC). The expressions of IPO4, TBL1XR1, p62/SQSTM1, PKP3, and CRTAP were verified by immunohistochemistry (IHC) in 20 EIGC samples, 17 gastric low-grade intraepithelial neoplasia (LGIN) samples, and 21 healthy controls. The prognostic values of the five genes were validated in the transcriptome data by survival analysis. A total of 4,028 proteins were identified using DIA-MS and a total of 177 differential proteins were screened with log2(fold change) > 1.5. Among them, 113 proteins were significantly up-regulated, and 64 proteins were significantly down-regulated in EIGC tissues. IHC results showed that proteins IPO4, TBL1XR1, p62/SQSTM1, PKP3, and CRTAP were highly expressed in the cytoplasm of EIGC and LGIN, which was consistent with the results of DIA-MS. Among them, p62/SQSTM1 may undergo nuclear-cytoplasmic transfer. The five protein-coding genes were associated with intestinal-type gastric cancer survival and exhibited differential expression across various disease stages. The study successfully identified differentially expressed proteins between EIGC and NGC, providing potential biomarkers and valuable insights into the mechanism underlying intestinal-type gastric cancer.

Is there any relationship between dietary patterns and depression and anxiety in Chinese adolescents?

OBJECTIVE: To determine the association between major dietary patterns characterized by factor analysis and risk of depression and anxiety symptoms among adolescents. DESIGN: Diet and symptoms of depression and anxiety were assessed in a cross-sectional survey among students attending junior high school. Dietary patterns were derived from a self-reported FFQ, which consisted of thirty-eight items. Anthropometric measurements were also performed. SETTING: Four junior high schools in Bengbu city, China. SUBJECTS: A random sample of 5003 adolescents, 11-16 years of age (mean 13·21 years). RESULTS: Three major dietary patterns were identified in the study based on factor analysis: 'snack', 'animal food' and 'traditional'. The prevalence of depression symptoms, anxiety disorders and the coexistence of both were 11·2% (560/5003), 14·6% (732/5003) and 12·6% (629/5003), respectively. After adjustment for potential confounders, adolescents in the highest tertile of snack dietary pattern scores had a higher odds for 'pure' psychological symptoms ('depression without anxiety', OR = 1·64; 95% CI 1·30, 2·06; and 'anxiety without depression', OR = 1·87; 95% CI 1·51, 2·31) compared with coexisting depression and anxiety (OR = 1·93; 95% CI 1·54, 2·43). Similar to snacks, high consumption of animal foods was associated with a higher risk of psychological symptoms. Compared with low consumption, adolescents in the highest tertile of traditional dietary pattern scores had lower odds for 'pure' depression (OR = 0·38; 95% CI 0·30, 0·49), 'pure' anxiety (OR = 0·85; 95% CI 0·69, 1·04) and coexisting anxiety and depression (OR = 0·50; 95% CI 0·39, 0·63). CONCLUSIONS: Data from Chinese secondary-school adolescents validated findings from adult populations. Dietary patterns should be considered as important predictors of depression and anxiety among adolescents in further studies.

Effects of armepavine against hepatic fibrosis induced by thioacetamide in rats.

The aim of this study was to investigate if armepavine (Arm, C19H23O3N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H2O2 production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.

Effects of Resistance Training Intensity on Heart Rate Variability at Rest and in Response to Orthostasis in Middle-Aged and Older Adults.

OBJECTIVE: Aging and deficits related to decreased physical activity can lead to higher risks of autonomic nervous system (ANS) dysfunction. The aim of this study was to evaluate the effects of 24 weeks of resistance training (RT) at various intensities on hemodynamics as well as heart rate variability (HRV) at rest and in response to orthostatic tests in middle-aged and older adults. METHODS: Forty adults were randomized into three groups: high-intensity (HEX) (80% 1-RM) (11 female, 4 male; 60 ± 4 years); low-moderate-intensity (LEX) (50% 1-RM) (nine female, four male; 61 ± 5 years); and a control group (CON) (eight female, four male; 60 ± 4 years). The RT program consisted of nine exercises, with two sets performed of each exercise two times per week for 24 weeks. Data collected included 1-RM, heart rate, and blood pressure and HRV at rest and in response to orthostasis. RESULTS: Both the HEX (42-94%) and LEX (31.3-51.7%) groups showed increases in 1-RM (p < 0.01). The HEX group showed decreases in resting heart rate (-4.0%), diastolic blood pressure (-3.2 mmHg (-4.2%)), and low frequency/high frequency (LF/HF) (Ln ratio) (p < 0.05). Post-study, the HEX group had higher HF (Ln ms2) than the CON, adjusted for pre-study value and age (p < 0.05). Post-study, the supine-standing ratio (SSR) of LFn (normalized unit) in the HEX group was greater than that in the LEX and CON groups, while the SSR of LF/HF in the HEX group was greater than the CON (p < 0.05). In conclusion, high-intensity RT can improve resting heart rate and HRV by enhancing cardiac vagal control. High-intensity RT might also improve the orthostatic response in terms of HRV. High intensity RT might assist ANS modification and could perhaps decrease the risks of cardiovascular disease and orthostatic intolerance.

Highly Sensitive Fluorescence Assay for miRNA Detection: Investigation of the DNA Spacer Effect on the DSN Enzyme Activity toward Magnetic-Bead-Tethered Probes.

Researchers have recently designed various biosensors combining magnetic beads (MBs) and duplex-specific nuclease (DSN) enzyme to detect miRNAs. Yet, the interfacial mechanisms for surface-based hybridization and DSN-assisted target recycling are relatively not well understood. Thus, herein, we developed a highly sensitive and selective fluorescent biosensor to study the phenomenon that occurs on the local microenvironment surrounding the MB-tethered DNA probe via detecting microRNA-21 as a model. Using the above strategy, we investigated the influence of different DNA spacers, base-pair orientations, and surface densities on DSN-assisted target recycling. As a result, we were able to detect as low as 170 aM of miR-21 under the optimized conditions. Moreover, this approach exhibits a high selectivity in a fully matched target compared to a single-base mismatch, allowing the detection of miRNAs in serum with improved recovery. These results are attributed to the synergetic effect between the DSN enzyme activity and the neutral DNA spacer (triethylene glycol: TEG) to improve the miRNA detection's sensitivity. Finally, our strategy could create new paths for detecting microRNAs since it obliterates the enzyme-mediated cascade reaction used in previous studies, which is more expensive, more time-consuming, less sensitive, and requires double catalytic reactions.