Effect of Exogenous Electric Stimulation on the Cardiac Tissue Function In Situ Monitored by Scanning Electrochemical Microscopy.

Cardiac tissue is sensitive to and can be easily damaged by exogenous electric stimulation. However, due to the thermal-electric coeffect and the limitation of in situ and quantitative information on the cardiac tissue function under electric stimulation, the detailed effect and the underlying mechanism of exogenous electric stimulation on the cardiac tissue remain elusive. To address this, in this work, we first constructed an in vitro cardiac tissue model and established a thermal-electric coupled theoretical model for simulating the electric field and temperature distributions around the cardiac tissue, from which we selected the electric field strengths (1.19, 2.37, and 3.39 kV cm-1) and electrical energies (0.001, 0.005, and 0.011 J) for electric stimulations without inducing a thermal effect. Then, we applied electric field stimulations on the cardiac tissue using these parameters and scanning electrochemical microscopy (SECM) to in situ and quantitatively monitor the dynamic changes in the key parameters of the cardiac tissue function, including respiratory activity, membrane permeability, and contraction frequency, after electric field stimulations. The SECM results showed that the oxygen consumption, cell membrane permeability coefficient, and contraction frequency of the cardiac tissue were strongly dependent on electrical energy, especially when the electrical energy was higher than 0.001 J. Our work, for the first time, achieves the in situ and quantitative monitoring of the cardiac tissue function under electric stimulation using SECM, which would provide important references for designing an electric stimulation regime for cardiac tissue engineering and clinical application of electrotherapy.

In Situ and Quantitatively Monitoring the Dynamic Process of Ferroptosis in Single Cancer Cells by Scanning Electrochemical Microscopy.

Ferroptosis, as a promising therapeutic strategy for cancers, has aroused great interest. Quantifying the quick dynamic changes in key parameters during the early course of ferroptosis can provide insights for understanding the underlying mechanisms of ferroptosis and help the development of therapies targeting ferroptosis. However, in situ and quantitatively monitoring the quick responses of living cancer cells to ferroptosis at the single-cell level remains technically challenging. In this work, we selected HuH7 cells (hepatocellular carcinoma (HCC) cells) as a cell model and Erastin as a typical ferroptosis inducer. We utilized scanning electrochemical microscopy (SECM) to quantitatively and in situ monitor the early course of ferroptosis in HuH7 cells by characterizing the three key parameters of cell ferroptosis (i.e., cell membrane permeability, respiratory activity, and the redox state). The SECM results show that the membrane permeability of ferroptotic HuH7 cells continuously increased from 0 to 8.1 × 10-5 m s-1, the cellular oxygen consumption was continuously reduced by half, and H2O2 released from the cells exhibited periodic bursts during the early course of ferroptosis, indicating the gradually destroyed cell membrane structure and intensified oxidative stress. Our work realizes, for the first time, the in situ and quantitative monitoring of the cell membrane permeability, respiratory activity, and H2O2 level of the early ferroptosis process of a single living cancer cell with SECM, which can contribute to the understanding of the physiological process and underlying mechanisms of ferroptosis.

In Situ and Quantitative Monitoring of Cardiac Tissues Using Programmable Scanning Electrochemical Microscopy.

In vitro cardiac tissue model holds great potential as a powerful platform for drug screening. Respiratory activity, contraction frequency, and extracellular H2O2 levels are the three key parameters for determining the physiological functions of cardiac tissues, which are technically challenging to be monitored in an in situ and quantitative manner. Herein, we constructed an in vitro cardiac tissue model on polyacrylamide gels and applied a pulsatile electrical field to promote the maturation of the cardiac tissue. Then, we built a scanning electrochemical microscopy (SECM) platform with programmable pulse potentials to in situ characterize the dynamic changes in the respiratory activity, contraction frequency, and extracellular H2O2 level of cardiac tissues under both normal physiological and drug (isoproterenol and propranolol) treatment conditions using oxygen, ferrocenecarboxylic acid (FcCOOH), and H2O2 as the corresponding redox mediators. The SECM results showed that isoproterenol treatment induced enhanced oxygen consumption, accelerated contractile frequency, and increased released H2O2 level, while propranolol treatment induced dynamically decreased oxygen consumption and contractile frequency and no obvious change in H2O2 levels, suggesting the effects of activation and inhibition of ß-adrenoceptor on the metabolic and electrophysiological activities of cardiac tissues. Our work realizes the in situ and quantitative monitoring of respiratory activity, contraction frequency, and secreted H2O2 level of living cardiac tissues using SECM for the first time. The programmable SECM methodology can also be used to real-time and quantitatively monitor electrochemical and electrophysiological parameters of cardiac tissues for future drug screening studies.

Ultra-specific nucleic acid testing by target-activated nucleases.

Specific and sensitive detection of nucleic acids is essential to clinical diagnostics and biotechnological applications. Currently, amplification steps are necessary for most detection methods due to the low concentration of nucleic acid targets in real samples. Although amplification renders high sensitivity, poor specificity is prevalent because of the lack of highly accurate precise strategies, resulting in significant false positives and false negatives. Nucleases exhibit high catalytic activity for nucleic acid cleavage which is regulated in a programmable manner. This review focuses on the latest progress in nucleic acid testing methods based on the target-activated nucleases. It summarizes the property of enzymes such as CRISPR/Cas, Argonautes, and some gene-editing irrelevant nucleases, which have been leveraged to create highly specific and sensitive nucleic acid testing tools. We elaborate on recent advances in the field of nuclease-mediated DNA recognition techniques for nucleic acid detection, and discuss its future applications and challenges in molecular diagnostics.

Investigating the Effect of Substrate Stiffness on the Redox State of Cardiac Fibroblasts Using Scanning Electrochemical Microscopy.

Cardiac fibrosis, in which cardiac fibroblasts differentiate into myofibroblasts, leads to oversecretion of the extracellular matrix, results in increased stiffness, and facilitates disequilibrium of cellular redox state, further leading to oxidative stress and various degrees of cell death. However, the relationship between the matrix stiffness and the redox status of cardiac fibroblasts remains unclear. In this work, we constructed an in vitro cardiac fibrosis model by culturing cardiac fibroblasts on polyacrylamide gels with tunable stiffness and characterized the differentiation of cardiac fibroblasts to myofibroblasts by immunofluorescence staining of α-smooth muscle actin. We then applied scanning electrochemical microscopy (SECM) with a depth scan mode to in situ and quantitatively assess the redox status by monitoring the glutathione (GSH) efflux rate (k) through the redox reaction between GSH (a typical indicator of cellular redox level) released from cardiac fibroblasts and SECM probe-oxidized ferrocenecarboxylic acid ([FcCOOH]+). The SECM results demonstrate that the GSH efflux from the cardiac fibroblasts decreased with increasing substrate stiffness (i.e., mimicking the increased fibrosis degree), indicating that a more oxidizing microenvironment facilitates the cell differentiation and GSH may serve as a biomarker to predict the degree of cardiac fibrosis. This work provides an SECM approach to quantify the redox state of cardiac fibroblasts by recording the GSH efflux rate. In addition, the newly established relationship between the redox balance and the substrate stiffness would help to better understand the redox state of cardiac fibroblasts during cardiac fibrosis.

Effect of Substrate Stiffness on Redox State of Single Cardiomyocyte: A Scanning Electrochemical Microscopy Study.

Mechanical microenvironment plays a key role in the regulation of the phenotype and function of cardiac cells, which are strongly associated with the intracellular redox mechanism of cardiomyocytes. However, the relationship between the redox state of cardiomyocytes and their mechanical microenvironment remains elusive. In this work, we used polyacrylamide (PA) gels with varying stiffness (6.5-92.5 kPa) as the substrate to construct a mechanical microenvironment for cardiomyocytes. Then we employed scanning electrochemical microscopy (SECM) to in situ characterize the redox state of a single cardiomyocyte in terms of the apparent rate constant (kf) of the regeneration rate of ferrocenecarboxylic by glutathione (GSH) released from cardiomyocyte, which is the most abundant reactant of intracellular reductive-oxidative metabolic cycles in cells and can represent the redox level of cardiomyocytes. The obtained SECM results show that the cardiomyocytes cultured on the stiffer substrates present lower kf values than those on the softer ones, that is, the more oxidative state of cardiomyocytes on the stiffer substrates compared to those on the softer ones. It proves the relationship between mechanical factors and the redox state of cardiomyocytes. This work can contribute to understanding the intracellular chemical process of cardiomyocytes during physiopathologic conditions. Besides, it also provides a new SECM method to in situ investigate the redox mechanism of cardiomyocytes at a single-cell level.

Dual-functional gallium/chitosan/silk/umbilical cord mesenchymal stem cell exosome sponge scaffold for diabetic wound by angiogenesis and antibacteria.

The treatment of diabetic wounds possessed significant challenges in clinical practice, which was accompanied with continuous infection, inflammation, and limited angiogenesis. Current wound dressings used for diabetic wound healing struggle to address these issues simultaneously. Therefore, Ga3+ was added to the chitosan/silk solution to confer potent antibacterial properties. Subsequently, umbilical cord mesenchymal stem cell exosomes (UCSC-Exo) were integrated into the gallium/chitosan/silk solution to enhance its angiogenesis-inducing activity. The mixture was lyophilized to prepare gallium/chitosan/silk/exosome sponge scaffolds (Ga/CSSF-Exo sponge scaffolds). The experiments of In vitro and in vivo demonstrated that Ga/CSSF-Exo sponge scaffolds exhibited sustained release of Ga3+ and bioactive exosomes, which effectively exerted continuous antibacterial effects and promoted angiogenesis. In diabetic rat wound models, Ga/CSSF-Exo sponge scaffolds facilitated angiogenesis, suppressed bacterial growth and inflammation, as well as promoted collagen deposition and re-epithelialization of wounds. Collectively, our findings suggested that Ga/CSSF-Exo held excellent potential for diabetic wound healing.

Effect of Extracellular Matrix Stiffness on Candesartan Efficacy in Anti-Fibrosis and Antioxidation.

Myocardial fibrosis progression and imbalanced redox state are closely associated with increased extracellular matrix (ECM) stiffness. Candesartan (CAN), an angiotensin II (Ang II) receptor inhibitor, has shown promising anti-fibrosis and antioxidant efficacy in previous cardiovascular disease studies. However, the effect of ECM stiffness on CAN efficacy remains elusive. In this study, we constructed rat models with three different degrees of myocardial fibrosis and treated them with CAN, and then characterized the stiffness, cardiac function, and NADPH oxidase-2 (NOX2) expression of the myocardial tissues. Based on the obtained stiffness of myocardial tissues, we used polyacrylamide (PA) gels with three different stiffness to mimic the ECM stiffness of cardiac fibroblasts (CFs) at the early, middle, and late stages of myocardial fibrosis as the cell culture substrates and then constructed CFs mechanical microenvironment models. We studied the effects of PA gel stiffness on the migration, proliferation, and activation of CFs without and with CAN treatment, and characterized the reactive oxygen species (ROS) and glutathione (GSH) levels of CFs using fluorometry and scanning electrochemical microscopy (SECM). We found that CAN has the best amelioration efficacy in the cardiac function and NOX2 levels in rats with medium-stiffness myocardial tissue, and the most obvious anti-fibrosis and antioxidant efficacy in CFs on the medium-stiffness PA gels. Our work proves the effect of ECM stiffness on CAN efficacy in myocardial anti-fibrosis and antioxidants for the first time, and the results demonstrate that the effect of ECM stiffness on drug efficacy should also be considered in the treatment of cardiovascular diseases.

Matrix stiffness-dependent microglia activation in response to inflammatory cues: in situ investigation by scanning electrochemical microscopy.

Microglia play a crucial role in maintaining the homeostasis of the central nervous system (CNS) by sensing and responding to mechanical and inflammatory cues in their microenvironment. However, the interplay between mechanical and inflammatory cues in regulating microglia activation remains elusive. In this work, we constructed in vitro mechanical-inflammatory coupled microenvironment models of microglia by culturing BV2 cells (a murine microglial cell line) on polyacrylamide gels with tunable stiffness and incorporating a lipopolysaccharide (LPS) to mimic the physiological and pathological microenvironment of microglia in the hippocampus. Through characterization of activation-related proteins, cytokines, and reactive oxygen species (ROS) levels, we observed that the LPS treatment induced microglia on a stiff matrix to exhibit overexpression of NOX2, higher levels of ROS and inflammatory factors compared to those on a soft matrix. Additionally, using scanning electrochemical microscopy (SECM), we performed in situ characterization and discovered that microglia on a stiff matrix promoted extracellular ROS production, leading to a disruption in their redox balance and increased susceptibility to LPS-induced ROS production. Furthermore, the respiratory activity and migration behavior of microglia were closely associated with their activation process, with the stiff matrix-LPS-induced microglia demonstrating the most pronounced changes in respiratory activity and migration ability. This work represents the first in situ and dynamic monitoring of microglia activation state alterations under a mechanical-inflammatory coupled microenvironment using SECM. Our findings shed light on matrix stiffness-dependent activation of microglia in response to an inflammatory microenvironment, providing valuable insights into the mechanisms underlying neuroinflammatory processes in the CNS.

Interstitial fluid flow: the mechanical environment of cells and foundation of meridians.

Using information from the deep dissection, microobservation, and measurement of acupoints in the upper and lower limbs of the human body, we developed a three-dimensional porous medium model to simulate the flow field using FLUENT software and to study the shear stress on the surface of interstitial cells (mast cells) caused by interstitial fluid flow. The numerical simulation results show the following: (i) the parallel nature of capillaries will lead to directional interstitial fluid flow, which may explain the long interstitial tissue channels or meridians observed in some experiments; (ii) when the distribution of capillaries is staggered, increases in the velocity alternate, and the velocity tends to be uniform, which is beneficial for substance exchange; (iii) interstitial fluid flow induces a shear stress, with magnitude of several Pa, on interstitial cell membranes, which will activate cells and lead to a biological response; (iv) capillary and interstitial parameters, such as capillary density, blood pressure, capillary permeability, interstitial pressure, and interstitial porosity, affect the shear stress on cell surfaces. The numerical simulation results suggest that in vivo interstitial fluid flow constitutes the mechanical environment of cells and plays a key role in guiding cell activities, which may explain the meridian phenomena and the acupuncture effects observed in experiments.

In Situ and Quantitatively Imaging of Heat-Induced Oxidative State and Oxidative Damage of Living Neurons Using Scanning Electrochemical Microscopy.

Central nervous system is sensitive and vulnerable to heat. Oxidative state and oxidative damage of neurons under heat stress are vital for understanding early consequences and mechanisms of heat-related neuronal injury, which remains elusive partly due to the technical challenge of in situ and quantitative monitoring methods. Herein, a temperature-controlled scanning electrochemical microscopy (SECM) platform with programmable pulse potential and depth scan modes is developed for in situ and quantitatively monitoring of oxygen consumption, extracellular hydrogen peroxide level, and cell membrane permeability of neurons under thermal microenvironment of 37-42 °C. The SECM results show that neuronal oxygen consumption reaches a maximum at 40 °C and then decreases, extracellular H2 O2 level increases from 39 °C, and membrane permeability increases from 2.0 ± 0.6 × 10-5 to 7.2 ± 0.8 × 10-5 m s-1 from 39 to 42 °C. The therapeutic effect on oxidative damage of neurons under hyperthermia conditions (40-42 °C) is further evaluated by SECM and fluorescence methods, which can be partially alleviated by the potent antioxidant edaravone. This work realizes in situ and quantitatively observing the heat-induced oxidative state and oxidative damage of living neurons using SECM for the first time, which results can contribute to a better understanding of the heat-related cellular injury mechanism.

Correction: In situ monitoring of functional activity of extracellular matrix stiffness-dependent multidrug resistance protein 1 using scanning electrochemical microscopy.

[This corrects the article DOI: 10.1039/D2SC02708A.].

In situ monitoring of functional activity of extracellular matrix stiffness-dependent multidrug resistance protein 1 using scanning electrochemical microscopy.

Extracellular matrix (ECM) stiffness affects the drug resistance behavior of cancer cells, while multidrug resistance protein 1 (MRP1) on the cell membrane confers treatment resistance via actively transporting drugs out of cancer cells. However, the relationship between ECM stiffness and MRP1 functional activity in cancer cells remains elusive, mainly due to the technical challenge of in situ monitoring. Herein, we engineered in vitro cancer cell models using breast cancer cells (MCF-7 and MDA-MB-231 cells) as the reprehensive cells on polyacrylamide (PA) gels with three stiffness, mimicking different developmental stages of cancer. We in situ characterized the functional activity of MRP1 and investigated the effect of ECM stiffness on MRP1 of cancer cells before and after vincristine treatment using scanning electrochemical microscopy (SECM) with ferrocenecarboxylic acid (FcCOOH) as the redox mediator and endogenous glutathione (GSH) as the indicator. The SECM results show that the functional activity of MRP1 is enhanced with increasing ECM stiffness, and the MRP1-mediated vincristine efflux activity of MCF-7 cells is more affected by ECM stiffness than that of MDA-MB-231 cells. This work, for the first time, applied SECM to in situ and quantitatively monitor the functional activity of MRP1 in cancer cells in different tumor mechanical microenvironments, which could help to elucidate the mechanism of matrix stiffness-dependent drug resistance behavior in cancer cells.

Acellular dermal matrix decorated with collagen-affinity peptide accelerate diabetic wound healing through sustained releasing Histatin-1 mediated promotion of angiogenesis.

Treating diabetic ulcers is a major challenge in clinical practice, persecuting millions of patients with diabetes and increasing the medical burden. Recombinant growth factor application can accelerate diabetic wound healing via angiogenesis. The local administration of recombinant growth factors has no robust clinical efficiency because of the degradation of append short duration of the molecules in the hostile inflammatoryenvironment.The present study focused on the pathophysiology of impaired neovascularization and growth factor short duration in the diabetic wound. We prepared a collagen-binding domain (CBD)-fused recombinant peptide (C-Histatin-1) that had both pro-angiogenesis capacity and collagen-affinity properties. Next, we created a biocompatible acellular dermal matrix (ADM) as a drug delivery carrier that featured collagen-richness, high porosity, and non-cytotoxicity. C-Histatin-1 was then tethered on ADM to obtain a sustained-release effect. Finally, a functional scaffold (C-Hst1/ADM) was developed. C-Hst1/ADM can sustain-release Histatin-1 to promote the adhesion, migration, and angiogenesisof vascular endothelial cells in vitro. Using a diabetic wound model, we showed that C-Hst1/ADM could significantly promote angiogenesis, reduce scar widths, and improve extracellular collagen accumulation. Therefore, the results of this study provide a foundation for the clinical application of C-Hst1/ADM covering scaffold in the treatment of diabetic wounds.