CRISPR/Cas12a and aptamer-chemiluminescence based analysis for the relative abundance determination of tumor-related protein positive exosomes for breast cancer diagnosis.

Exosomes, as novel biomarker for liquid biopsy, exhibit huge important potential value for cancer diagnosis. However, various proteins show different expression levels on exosomal membrane, and the absolute concentration of exosomes in clinical samples is easily influenced by a number of factors. Here, we developed a CRISPR/Cas12a and aptamer-chemiluminescence based analysis (CACBA) for the relative abundance determination of tumor-related protein positive exosomes in plasma for breast cancer diagnosis. The total concentration of exosomes was determined through captured CD63 using a CRISPR/Cas12a-based method with the LoD of 8.97 × 103 particles/µl. Meanwhile, EpCAM and MUC1 positive exosomes were quantitatively detected by aptamer-chemiluminescence (ACL) based method with the LoD of 1.45 × 102 and 3.73 × 102 particles/µl, respectively. It showed that the percentages of EpCAM and MUC1 positive exosomes offered an excellent capability to differentiate breast cancer patients and healthy donors. The high sensitivity, strong specificity, outstanding anti-interference capability, and steady recovery rate of this approach offered higher accuracy and robustness than the commercialized method in clinical trial. In addition with good stability, easy preparation and low cost, this method not only provides a new approach to rapid analysis of exosome proteins, it may be quickly extended to the diagnoses of various cancers.

Weak Value Amplification-Based Biochip for Highly Sensitive Detection and Identification of Breast Cancer Exosomes.

Exosomes constitute an emerging biomarker for cancer diagnosis because they carry multiple proteins that reflect the origins of the parent cell. The highly sensitive detection of exosomes is a crucial prerequisite for the diagnosis of cancer. In this study, we report an exosome detection system based on quantum weak value amplification (WVA). The WVA detection system consists of a reflection detection light path and a Zr-ionized biochip. Zr-ionized biochips effectively capture exosomes through the specific interaction between zirconium dioxide and the phosphate groups on the lipid bilayer of exosomes. Aptamer-modified gold nanoparticles (Au NPs) are then used to specifically recognize proteins on exosomes to enhance the detection signal. The sensitivity and resolution of the detection system are 2944.07 nm/RIU and 1.22 × 10-5 RIU, respectively. The concentration of exosomes can be directly quantified by the WVA system, ranging from 105-107 particles/mL with the detection limit of 3 × 104 particles/mL. The use of Au NPs-EpCAM for the specific enhancement of breast cancer MDA-MB-231 exosomes is demonstrated. The results indicate that the WVA detection system can be a promising candidate for the detection of exosomes as tumor markers.

Adjunct Methods for Alzheimer's Disease Detection: A Review of Auditory Evoked Potentials.

The auditory afferent pathway as a clinical marker of Alzheimer's disease (AD) has sparked interest in investigating the relationship between age-related hearing loss (ARHL) and AD. Given the earlier onset of ARHL compared to cognitive impairment caused by AD, there is a growing emphasis on early diagnosis and intervention to postpone or prevent the progression from ARHL to AD. In this context, auditory evoked potentials (AEPs) have emerged as a widely used objective auditory electrophysiological technique for both the clinical diagnosis and animal experimentation in ARHL due to their non-invasive and repeatable nature. This review focuses on the application of AEPs in AD detection and the auditory nerve system corresponding to different latencies of AEPs. Our objective was to establish AEPs as a systematic and non-invasive adjunct method for enhancing the diagnostic accuracy of AD. The success of AEPs in the early detection and prediction of AD in research settings underscores the need for further clinical application and study.

Non-invasive assessment of hair regeneration in androgenetic alopecia mice in vivo using two-photon and second harmonic generation imaging.

The identification of crucial targets for hair regrowth in androgenetic alopecia (AGA) involves determining important characteristics and different stages during the process of hair follicle regeneration. Traditional methods for assessing key features and different stages of hair follicle primarily involve taking skin tissue samples and determining them through various staining or other methods. However, non-invasive assessment methods have been long sought. Therefore, in this study, endogenous fluorescence signals from skin keratin and second harmonic signals from skin collagen fibers were utilized as probes, two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging techniques were employed to non-invasively assess hair shafts and collagen fibers in AGA mice in vivo. The TPEF imaging technique revealed that the alternation of new and old hair shafts and the different stages of the growth period in AGA mice were delayed. In addition, SHG imaging found testosterone reduced hair follicle area and miniaturized hair follicles. The non-invasive TPEF and SHG imaging techniques provided important methodologies for determining significant characteristics and different stages of the growth cycle in AGA mice, which will facilitate future non-invasive assessments on human scalps in vivo and reduce the use of animal testing.

The safety and efficacy of emergency stenting during bridging therapy: A Single-Center Retrospective Study.

INTRODUCTION: To investigated the safety and efficacy of emergency stenting for patients with ischemic stroke treated with bridging therapy. METHODS: Patients with onset of stroke who underwent bridging therapy were included in the two groups with emergency stenting (ESG) and without stenting (NSG). To avoid the bias due to confounding variables, subjects were further assigned in two groups using 1:1 propensity score matching (PSM). The safety outcomes include the incidence of intracranial hemorrhage (ICH), parenchymal hemorrhage type 2 (PH2), symptomatic intracranial hemorrhage (sICH), fatal hemorrhage, and mortality. The efficacy outcomes include successful recanalization, three-month favorable outcome (modified Rankin Scale [mRS]: 0-2). RESULTS: 175 patients treated with bridging therapy were included in this study, with 52 patients in the ES group and 123 patients in the groups without ES, and with 30 patients in each group after PSM. No significant differences in the incidences of ICH, PH2, sICH, fatal hemorrhage, and mortality were found between the two groups with ES and without ES before and after PSM (P>0.05 for all groups). The analysis without PSM showed that the group with ES had a higher rate of successful recanalization (98.1% vs. 81.6%，P=0.041) than the group without ES, but no significant difference was seen (96.6% vs. 93.3%，P=0.554) between the two groups after PSM. There was no difference in favorable outcome between the two groups before and after matching as well (P>0.05). CONCLUSIONS: It is safe and effective for patients with onset of ischemic stroke to receive emergency stenting during bridging therapy, without increasing the risk of hemorrhagic transformation and mortality.

Micro SleepNet: efficient deep learning model for mobile terminal real-time sleep staging.

The real-time sleep staging algorithm that can perform inference on mobile devices without burden is a prerequisite for closed-loop sleep modulation. However, current deep learning sleep staging models have poor real-time efficiency and redundant parameters. We propose a lightweight and high-performance sleep staging model named Micro SleepNet, which takes a 30-s electroencephalography (EEG) epoch as input, without relying on contextual signals. The model features a one-dimensional group convolution with a kernel size of 1 × 3 and an Efficient Channel and Spatial Attention (ECSA) module for feature extraction and adaptive recalibration. Moreover, the model efficiently performs feature fusion using dilated convolution module and replaces the conventional fully connected layer with Global Average Pooling (GAP). These design choices significantly reduce the total number of model parameters to 48,226, with only approximately 48.95 Million Floating-point Operations per Second (MFLOPs) computation. The proposed model is conducted subject-independent cross-validation on three publicly available datasets, achieving an overall accuracy of up to 83.3%, and the Cohen Kappa is 0.77. Additionally, we introduce Class Activation Mapping (CAM) to visualize the model's attention to EEG waveforms, which demonstrate the model's ability to accurately capture feature waveforms of EEG at different sleep stages. This provides a strong interpretability foundation for practical applications. Furthermore, the Micro SleepNet model occupies approximately 100 KB of memory on the Android smartphone and takes only 2.8 ms to infer one EEG epoch, meeting the real-time requirements of sleep staging tasks on mobile devices. Consequently, our proposed model has the potential to serve as a foundation for accurate closed-loop sleep modulation.

A highly selective red-emitting fluorescent probe and its micro-nano-assembly for imaging endogenous peroxynitrite (ONOO-) in living cells.

Endogenous peroxynitrite plays a very important role in the regulation of life activities. However, validated tools for ONOO- tests are currently insufficient. We designed a fluorescent probe TPA-F-NO2 with a low fluorescence background in water based on the D-π-A structure for the imaging of endogenous ONOO- in living cells. TPA-F-NO2 can realize the naked eye detection of ONOO- due to the obvious color change. TPA-F-NO2 has the advantages of large stokes shift, high signal-to-noise ratio, high selectivity and sensitivity. The quantitative detection can be achieved in the range of 0-14 µM ONOO-. Due to its solvatochromic characteristics, TPA-F-NO2 has the potential to be used in OLEDs and other fields. In addition, 4-methylumbelliferone has a wide range of anticancer effects as an inhibitor of hyaluronic acid. We prepared TPA-MU-NPs by assembling TPA-F-NO2 and 4-methylumbelliferone. It also endows TPA-MU-NPs with ONOO- imaging function and anti-proliferation effect on breast cancer cells and other cells. This 'probe-drug' assembly strategy provides ideas for the design and optimization of dual-functional probes.

Identification of hub biomarkers and immune cell infiltration characteristics of polymyositis by bioinformatics analysis.

Background: Polymyositis (PM) is an acquirable muscle disease with proximal muscle involvement of the extremities as the main manifestation; it is a category of idiopathic inflammatory myopathy. This study aimed to identify the key biomarkers of PM, while elucidating PM-associated immune cell infiltration and immune-related pathways. Methods: The gene microarray data related to PM were downloaded from the Gene Expression Omnibus database. The analyses using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes, gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) networks were performed on differentially expressed genes (DEGs). The hub genes of PM were identified using weighted gene co-expression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) algorithm, and the diagnostic accuracy of hub markers for PM was assessed using the receiver operating characteristic curve. In addition, the level of infiltration of 28 immune cells in PM and their interrelationship with hub genes were analyzed using single-sample GSEA. Results: A total of 420 DEGs were identified. The biological functions and signaling pathways closely associated with PM were inflammatory and immune processes. A series of four expression modules were obtained by WGCNA analysis, with the turquoise module having the highest correlation with PM; 196 crossover genes were obtained by combining DEGs. Subsequently, six hub genes were finally identified as the potential biomarkers of PM using LASSO algorithm and validation set verification analysis. In the immune cell infiltration analysis, the infiltration of T lymphocytes and subpopulations, dendritic cells, macrophages, and natural killer cells was more significant in the PM. Conclusion: We identified the hub genes closely related to PM using WGCNA combined with LASSO algorithm, which helped clarify the molecular mechanism of PM development and might have great significance for finding new immunotherapeutic targets, and disease prevention and treatment.

Microfluidic synthesis of Janus-structured QD-encoded magnetic microbeads for multiplex immunoassay.

Uniform and monodisperse quantum dot (QD)-encoded magnetic microbeads with Janus structure were produced in a microfluidic device via photopolymerization. UV light through a microscope objective was used to solidify the microbeads which showed sharp interfaces and excellent magnetic responses. QDs with different emission peaks (450 nm for blue and 640 nm for red) were mixed at different ratios to provide three spectral codes. The QD-encoded microbeads can be distinguished by analyzing their fluorescent images in HSV color space. After hydrolysis of the anhydride group in alkaline solution, protein was immobilized on microbeads via activation of carboxyl groups using (1-ethyl-3(3-dimethylaminoprophyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). A microhole array in polydimethylsiloxane (PDMS) substrates with a specific size was fabricated to trap individual microbeads in a single microhole. The combination of Janus-structured QD-encoded magnetic microbeads and microhole arrays facilitates both flexibility, binding kinetics, sensitivity for suspension assay, and fluorescence mapping analysis for conventional biochips, thus providing a novel platform for multiplex bioanalysis. The capability of this integration for multiplex immunoassays was verified using three kinds of IgG and their corresponding anti-IgG. A detection limit of 0.07 ng/mL was achieved for human IgG, indicating practical applications in various fields.

Hydrophobicity Determines the Bacterial Killing Rate of α-Helical Antimicrobial Peptides and Influences the Bacterial Resistance Development.

Rapid antimicrobial action is an important advantage of antimicrobial peptides (AMPs) over antibiotics, which is also a reason for AMPs being less likely to induce bacterial resistance. However, the structural parameters and underlying mechanisms affecting the bacterial killing rate of AMPs remain unknown. In this study, we performed a structure-activity relationship (SAR) study using As-CATH4 and 5 as templates. We revealed that hydrophobicity, rather than other characteristics, is the critical structural parameter determining the bacterial killing rate of α-helical AMPs. With the hydrophobicity increase, the action rates of AMPs including bacterial binding, lipopolysaccharides neutralization, and outer and inner membrane permeabilization increased. Additionally, the higher hydrophobic AMPs with enhanced bacterial killing rates possess better in vivo therapeutic potency and a lower propensity to induce bacterial resistance. These findings revealed the importance of the bacterial killing rate for AMPs and are of great significance to the design and optimization of AMP-related drugs.

Synergistic Membrane Disturbance Improves the Antibacterial Performance of Polymyxin B.

Drug-resistant Gram-negative bacteria pose a serious threat to public health, and polymyxin B (PMB) is clinically used as a last-line therapy for the treatment of infections caused by these pathogens. However, the appearance of PMB resistance calls for an effort to develop new approaches to improve its antibacterial performance. In this work, a new type of nanocomposite, composed of PMB molecules being chemically decorated on the surface of graphene oxide (GO) nanosheets, was designed, which showed potent antibacterial ability through synergistically and physically disturbing the bacterial membrane. The as-fabricated PMB@GO nanocomposites demonstrated an enhanced bacterial-killing efficiency, with a minimum inhibitory concentration (MIC) value half of that of free PMB (with an MIC value as low as 0.5 µg mL-1 over Escherichia coli), and a bacterial viability less than one fourth of that of PMB (with a bacterial reduction of 60% after 3 h treatment, and 90% after 6 h incubation). Furthermore, the nanocomposite displayed moderate cytotoxicity or hemolysis effect, with cellular viabilities over 85% at concentrations up to 16 times the MIC value. Studies on antibacterial mechanism revealed that the synergy between PMB molecules and GO nanosheets greatly facilitated the vertical insertion of the nanocomposite into the lipid membrane, leading to membrane disturbance and permeabilization. Our results demonstrate a physical mechanism for improving the antibacterial performance of PMB and developing advanced antibacterial agents for better clinic uses.

Promotion of Hair Regrowth by Transdermal Dissolvable Microneedles Loaded with Rapamycin and Epigallocatechin Gallate Nanoparticles.

Interest in transdermal delivery methods for stimulating hair regrowth has been increasing recently. The microneedle approach can break the barrier of the stratum corneum through puncture ability and improve drug delivery efficiency. Herein, we report a dissolvable microneedle device for the co-delivery of rapamycin and epigallocatechin gallate nanoparticles that can significantly promote hair regeneration. Compared with the mice without any treatment, our strategy can facilitate hair growth within 7 days. Higher hair shaft growth rate and hair follicle density with inconspicuous inflammation were exhibited in C57BL/6 mice, elucidating its potential for clinical application.

Follistatin-like 1 ameliorates severe acute pancreatitis associated lung injury via inhibiting the activation of NLRP3 inflammasome and NF-κB pathway.

OBJECTIVE: Severe acute pancreatitis (SAP) is one of the most common abdominal conditions of digestive system that usually causes acute lung injury through systemic inflammation. Follistatin-like 1 (FSTL-1) has been reported to have anti-inflammatory and anti-apoptotic effects in a variety of diseases. The aim of this study was to investigate the effects of FSTL-1 on SAP-associated lung injury (SAPALI) and the underlying mechanism. METHODS: SAP model was induced by intraperitoneal injection of the L-arginine in C57BL/6 mice. The haematoxylin and eosin (H&E) staining was applied to determine the severity of lung and pancreatic injury. ELISA kits were used to determine serum amylase and inflammatory cytokines levels. TUNEL staining was carried out to measure cell apoptosis. Western blotting was applied to analyze the related proteins of NLRP3 inflammasome and NF-κB pathways. RESULTS: FSTL-1 was significantly increased in the lung of SAP mice. Knockout of FSTL-1 ameliorated pancreatic injury, lung injury, inflammation and apoptosis in mice with SAP. Moreover, the protein levels of NLRP3, ASC, Caspase-1, p-p65 and p-IκBα were obviously reduced in the FSTL-1 KO+SAP group in comparison with SAP group, suggesting that inhibition of FSTL-1 repressed the activation of the NLRP3 inflammasome and NF-κB pathway. CONCLUSION: This study helps us understand the mechanism of FSTL-1 in SAPALI and might provide a potential new strategy for the treatment of SAPALI.

Recent advancements in noninvasive glucose monitoring and closed-loop management systems for diabetes.

Diabetes can cause many complications, and has become one of the most common diseases that may lead to death. Currently, the number of diabetics continues to increase year by year. Thus, it is very important and highly desirable to monitor, control and cure diabetes. However, such management usually require long-term blood glucose monitoring and regular medication to reduce the risk of many complications. In order to provide patients with more effective and more convenient treatments, a portable, miniaturized, intelligent, painless and automatic closed-loop system is highly required and many related research studies have recently been reported. It is the right time and important to provide a summary in this field. Here, this review covers important parts of a closed-loop management system for diabetes, including the principle of electrochemical sensing of glucose, recent progress of noninvasive glucose monitoring technology, and its applications in wearable glucose sensors. Moreover, the latest advancements in an insulin delivery system and a diabetic closed-loop management system are also presented in detail. Finally, challenges and perspectives for an artificial pancreas are also presented. We believe that with the innovative glucose monitoring technology and the optimization of the drug delivery system, the closed-loop management system for diabetes will make much progress in the near future.

Aptamer-based biosensing through the mapping of encoding upconversion nanoparticles for sensitive CEA detection.

A sensitive detection system based on aptamer-based biosensors for the detection of carcinoembryonic antigen (CEA) by mapping encoding upconversion nanoparticles (UCNPs) was constructed. In this sensor, oligonucleotides with CEA aptamer fragments immobilized on magnetic beads (MBs) were hybridized to complementary DNA modified on UCNPs (cDNA-UCNPs); thus, sandwich-structured probes were formed. In the presence of CEA, due to the stronger interaction between the aptamer and CEA than that of the aptamer and complementary DNA on UCNPs, the cDNA-UCNPs were isolated from the MBs, and the number of isolated UCNPs was directly related to the concentration of CEA. Using an inverted fluorescence microscope, the number of target-dependent UCNPs on a glass slide was counted, enabling the accurate determination of CEA in the solution. The dynamic range for CEA detection in PBS buffer was 0.02-6.0 ng mL-1 (0.1-30 pM) and a limit of detection (LOD) of 65 fM was achieved. We envisage that the system we developed can also have many promising applications in the sensitive detection of other biomarkers for early cancer diagnosis.

Real-time monitoring the staged interactions between cationic surfactants and a phospholipid bilayer membrane.

The cationic surfactant-lipid interaction directs the development of novel types of nanodrugs or nanocarriers. The membrane action of cationic surfactants also has a wide range of applications. In this work, combining a photo-voltage transient method with the traditional dynamic giant unilamellar vesicle (GUV) leakage assay and molecular dynamics (MD) simulations, we monitored the molecular actions of a representative cationic surfactant, tetradecyl trimethyl ammonium bromide (TTAB), in a wide concentration range (i.e., 0.5 µM-10 mM), on a phospholipid bilayer membrane in real time. With low concentrations (e.g., ≤10 µM), TTAB performed a three-stage acting process, including the structural-disturbance-dominated, adsorption-dominated, and dynamic equilibrium stages. At higher concentrations (e.g., ≥100 µM), this process was accelerated to two stages. Furthermore, TTAB induced deformation and even rupture of the membrane, due to the asymmetric disturbance of surfactant molecules on the two leaflets of a bilayer. All these disturbances induced membrane permeabilization, and the times at which these transitions occurred are given. This work provides information on time and molecular mechanism during the membrane actions of cationic surfactants, and provides a simple and real-time method in studying the dynamic processes at the membrane interface.

Phyllosphere fungal communities of rubber trees exhibited biogeographical patterns, but not bacteria.

Phyllosphere microbiomes play an essential role in maintaining host health and productivity. Still, the diversity patterns and the drivers for the phyllosphere microbial community of the tropical cash crop Rubber tree (Hevea brasiliensis) - are poorly understood. We sampled the phyllosphere of field-grown rubber trees in South China. We examined the phyllosphere bacterial and fungal composition, diversity and main drivers of these microbes using the Illumina® sequencing and assembly. Fungal communities were distinctly different in different climatic regions (i.e. Xishuangbanna and Hainan Island) and climatic factors, especially mean annual temperature, and they were the main driving factors of foliar fungal communities, indicating fungal communities showed a geographical pattern. Significant differences of phyllosphere bacterial communities were detected in different habitats (i.e. endophytic and epiphytic). Most of the differences in taxa composition came from Firmicutes spp., which have been assigned as nitrogen-fixing bacteria. Since these bacteria cannot penetrate the cuticle like fungi, the abundant epiphytic Firmicutes spp. may supplement the deficiency of nitrogen acquisition. And the main factor influencing endophytic bacteria were internal factors, such as total nitrogen, total phosphorus and water content of leaves. External factors (i.e. climate) were the main driving force for epiphytic bacteria community assembly. Our work provides empirical evidence that the assembly of phyllosphere bacterial and fungal differed, which creates a precedent for preventing and controlling rubber tree diseases and pests and rubber tree yield improvement.

The deubiquitinase OTUD1 inhibits non-small cell lung cancer progression by deubiquitinating and stabilizing KLF4.

BACKGROUND: Lung cancer results in the highest mortality associated with cancer worldwide. Non-small cell cancer (NSCLC) is the leading subtype of lung cancer. Ovarian tumor protease (OTU) domain-containing protein 1 (OTUD1) is a member of the OTU subfamily of DUBs, and its function in NSCLC remains unclear. METHODS: GEPIA database was employed to reveal the expression level of OTUD1 in addition to Krüppel- like factor 4 (KLF4) in NSCLC tissue samples and prove the correlation between OTUD1 and KLF4. The protein level was estimated using western blot. Cell counting kit-8 (CCK-8) assay was used to detect cell viability and transwell assay was utilized to observe cell migration and invasion. Cycloheximide (CHX) was introduced to measure half-lives of KLF4 and deubiquitination assay was used to detect deubiquitination ability of OTUD1. RESULTS: OTUD1 expression was downregulated in NSCLC tissues and cells. Overexpression of OTUD1 inhibited NSCLC cell progression and it was promoted by knockdown of OTUD1. OTUD1 was positively correlated with KLF4 and stabilized KLF4 at protein level by deubiquitinating KLF4. Overexpressing KLF4 dramatically eliminated the effects of OTUD1 on the development of NSCLC cells. CONCLUSIONS: Our study revealed that OTUD1 suppresses NSCLC progression by mediating KLF4 stabilization, which suggests a potential gene target for the future treatment of NSCLC.

Optical weak measurement for the precise thickness determination of an ultra-thin film.

A total internal reflection system based on the weak value amplification principle is set up for the precise measurement of the thickness of an ultra-thin film. In this system, the film thickness is derived from the change of the double-peak pointer caused by the effective refractive index of the film, which is correlated to its thickness. The sensitivity and resolution of this system reached 2727.21 nm/RIU and 7.2×10-6 R I U, respectively, determined by using a sodium chloride solution with a refractive index of 1.331911. The growth process of TA/Fe(III) assembled films with thicknesses in the few nanometers range is monitored using the as-set-up system, and the experimental results are consistent with a theoretical calculation based on the Maxwell Garnett effective medium. Additionally, we theoretically calculated the detection limit for the thickness measurement of the film as 22 pm. We clearly provide a potential method for the precise measurement of the thickness of an ultra-thin film.

Real-time monitoring the interfacial dynamic processes at model cell membranes: Taking cell penetrating peptide TAT as an example.

A real-time and molecule-level monitoring of the interfacial dynamic interactions between molecules and a cell membrane is of vital importance. Herein, taking TAT, one of the most representative cell penetrating peptides, as an example, a photo-voltage transient technique and a dynamic giant bistratal vesicle (GBV) leakage method were combined with the traditional giant unilamellar vesicle (GUV) leakage assays, to provide a molecule-level understanding of the dynamic membrane interaction process performed in a low ionic strength and neutral pH condition. The photo-voltage test based on supported phospholipid bilayers showed a quick disturbance (<1 min) followed by a continuous reconstruction of the membrane by peptides, leading to a slight destruction (at TAT concentrations lower than 1 µg mL-1, i.e., 0.64 µM) or strong damage (e.g. at 10 µg mL-1, i.e., 6.4 µM) of the bilayer structure. The GUV/GBV leakage assays further demonstrated the TAT-induced membrane deformation and transmembrane diffusion of dyes, which occurred in an immediate, linear, and TAT-concentration dependent manner. Moreover, the flux of dye across the substrate-immobilized membranes was approximately three times of that across the substrate-free ones. This work gives information on time and molecular mechanism of the TAT-membrane interactions, demonstrates the different permeabilizing effects of TAT on immobilized and free membranes. Overall, it provides useful strategies to investigate the nano-bio interfacial interactions in a simple, global and real-time way.