Intelligent wound dressing for simultaneous in situ detection and elimination of pathogenic bacteria.

Wound infections hinder the healing process and potentially result in life-threatening complications, which urgently require rapid and timely detection and treatment pathogens during the early stages of infection. Here, an intelligent wound dressing was developed to enable in situ detection and elimination of pathogenic bacteria through a combination of point-of-care testing and antibacterial photodynamic therapy technology. The dressing is an injectable hydrogel composed of carboxymethyl chitosan and oxidized sodium alginate, with addition of 4-methylumphulone beta-D-glucoside (MUG) and up-converted nanoparticles coated with titanium dioxide (UCNPs@TiO2). The presence of bacteria can be visually detected by monitoring the blue fluorescence of 4-methylumbellione, generated through the reaction between MUG and the pathogen-associated enzyme. The UCNPs@TiO2 photosensitizers were synthesized and demonstrated high antibacterial activity through the generation of reactive oxygen species when exposed to near-infrared irradiation. Meanwhile, a smartphone-based portable detection system equipped with a self-developed Android app was constructed for in situ detection of pathogens in mere seconds, detecting as few as 103 colony-forming unit. Additionally, the dressing was tested in a rat infected wound model and showed good antibacterial activity and pro-healing ability. These results suggest that the proposed intelligent wound dressing has potential for use in the diagnosis and management of wound infections. STATEMENT OF SIGNIFICANCE: An intelligent wound dressing has been prepared for simultaneous in situ detection and elimination of pathogenic bacteria. The presence of bacteria can be visually detected by tracking the blue fluorescence of the dressing. Moreover, a smartphone-based detection system was constructed to detect and diagnose pathogenic bacteria before reaching the infection limit. Meanwhile, the dressing was able to effectively eliminate key pathogenic bacteria on demand through antibacterial photodynamic therapy under NIR irradiation. The proposed intelligent wound dressing enables timely detection and treatment of infectious pathogens at an early stage, which is beneficial for wound management.

Dynamic tissue model in vitro and its application for assessment of microplastics-induced toxicity to air-blood barrier (ABB).

The replication of the hominine physiological environment was identified as an effectual strategy to develop the physiological model in vitro to perform the intuitionistic assessment of toxicity of contaminations. Herein, we proposed a dynamic interface strategy that accurately mimicked the blood flow and shear stress in human capillaries to subtly evaluate the physiological damages. To proof the concept, the dynamic air-blood barrier (ABB) model in vitro was developed by the dynamic interface strategy and was utilized to assess the toxicity of polyethylene terephthalate microplastics (PET-MPs). The developed dynamic ABB model was compared with the static ABB model developed by the conventional Transwell® system and the animal model, then the performance of the dynamic ABB model in evaluation of the PET-MPs induced pulmonary damage via replicating the hominine ABB. The experimental data revealed that the developed dynamic ABB model in vitro effectively mimicked the physiological structure and barrier functions of human ABB, in which more sophisticated physiological microenvironment enabled the distinguishment of the toxicities of PET-MPs in different sizes and different concentrations comparing with the static ABB model constructed on Transwell® systems. Furthermore, the consistent physiological and biochemical characters adopted dynamic ABB model could be achieved in a quick manner referring with that of the mouse model in the evaluation of the microplastics-induced pulmonary damage. The proposed dynamic interface strategy supplied a general approach to develop the hominine physiological environment in vitro and exhibited a potential to develop the ABB model in vitro to evaluate the hazards of inhaled airborne pollutants.

Localized hydrodynamic flow confinement assisted nanowire sensor for ultrasensitive protein detection.

Affordable methods for ultra-sensitive biomarkers detection may improve the standard of living in resource-constrained countries. Nanowire biosensor is preponderant in ultra-sensitive protein detection. However, current strategies for nanowire sensor (NWS) fabrication often require sophisticated instruments, being inaccessible in less-resourced laboratories. Herein, we circumvent this challenge by developing a simple methodology, localized hydrodynamic flow confinement assisted nanowire sensor fabrication, enable the detection with limits of detection (LOD) for IgA and IgG measurement were 0.089 fg/mL and 0.93 fg/mL, respectively, demonstrating a 10-fold increase in detection sensitivity compared with the published NWS. Noteworthy, an X-Y positioner combined with a homemade microchemical pen (MCP) for tunable chemical deposition were sufficient to complete the fabrication of the nanowire biosensor without other expensive and demanding equipment. Overall, a particularly accessible, competitive, and low-cost approach of nanowire sensor fabrication for ultra-sensitive protein detection was developed, which could widely facilitate the application of nanowire biosensors. Besides, the nanowire sensor can also be employed to detect other analytes of interest by the use of different stimuli-responsive biosystems.

Regioselective fabrication of gold nanowires using open-space laminar flow for attomolar protein detection.

Gold nanowires are expected to be applied to biosensing due to their advantages, such as high stability and biocompatibility. However, it is still inconvenient to fabricate a single gold nanowire at a precise position, and without a special demanding environment. In this study, we present an open-space laminar flow approach for fabricating a single gold nanowire at a precise position under normal conditions. The fabricated gold nanowire demonstrated excellent biosensing of IgA with an extremely low limit of detection (1 aM).

Proteomic Distributions in CD34+ Microvascular Niche Patterns of Glioblastoma.

The poor clinical prognosis and microvascular patterns of glioblastoma (GBM) are of serious concern to many clinicians and researchers. However, very few studies have examined the correlation between microvascular niche patterns (MVNPs) and proteomic distribution. In this study, CD34 immunofluorescence staining and matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-IMS) technology were used to investigate the protein distributions in MVNPs. CD34+ microvascular phenotype could be divided into four types: microvascular sprouting (MS), vascular cluster (VC), vascular garland (VG), and glomeruloid vascular proliferation (GVP). Based on such characteristics, MVNPs were divided into two types by cluster analysis, namely, type I, comprising primarily MS and VC, and type II, comprising many VGs and GVPs. Survival analysis indicated the type of MVNPs to be an independent prognostic factor for progression-free and overall survival in GBM. MALDI-IMS results showed the peaks at m/z 1037 and 8960 to exhibit stronger ion signals in type II, while those at m/z 3240 and 3265 exhibited stronger ion signals in type I. The findings may assist future research on therapy and help predict prognosis in GBM. However, due to the limited number of studies, more well-designed studies are warranted to further verify our results.

In Situ Single-Cell Stimulation and Real-Time Electrochemical Detection of Lactate Response Using a Microfluidic Probe.

Metabolism of a single cell, even within the same organization, differs from other cells by orders of magnitude. Single-cell analysis provides key information for early diagnosis of cancer as well as drug screening. Any slight change in the microenvironment may affect the state of a single cell. Timely and effective cell monitoring is conducive to better understand the behavior of single cells. The immediate response of a single cell described in this study is a liquid transfer-based approach for real-time electrochemical detection. The cell was in situ stimulated by continuous flow with glucose, and lactate secreted from the cell would diffuse into the microflow. The microflow was aspirated into the detection channel where lactate was then decomposed by coupled enzyme reactions and detected by an electrode. This work provides a novel approach for detecting lactate response from a single cell by noninvasive measurements, and the position resolution of the microfluidic probe reaches the level of a single cell and permits individual heterogeneity in cells to be explored in the diagnosis and treatment of cancer as well as in many other situations.

Emerging open microfluidics for cell manipulation.

Cell manipulation is the foundation of biochemical studies, which demands user-friendly, multifunctional and precise tools. Based on flow confinement principles, open microfluidics can control the movement of microscale liquid in open space. Every position of the circuit is accessible to external instruments, making it possible to perform precise treatment and analysis of cells at arbitrary target positions especially at the single-cell/sub-cell level. Benefiting from its unique superiority, various manipulations including patterned cell culture, 3D tissue modelling, localized chemical stimulation, online cellular factor analysis, single cell sampling, partial cell treatment, and subcellular free radical attack can be easily realized. In this tutorial review, we summarize two basic ideas to design open microfluidics: open microfluidic networks and probes. The principles of mainstream open microfluidic methods are explained, and their recent important applications are introduced. Challenges and developing trends of open microfluidics are also discussed.

A Fluidic Isolation-Assisted Homogeneous-Flow-Pressure Chip-Solid Phase Extraction-Mass Spectrometry System for Online Dynamic Monitoring of 25-Hydroxyvitamin D3 Biotransformation in Cells.

It is well known that cell can response to various chemical and mechanical stimuli. Therefore, flow pressure variation induced by sample loading and elution should be small enough to ignore the physical impact on cells when we use a Chip-SPE-MS system for cells. However, most existent Chip-SPE-MS systems ignored the pressure alternation because it is extremely difficult to develop a homogeneous-flow-pressure hyphenated module. Herein, we developed an interesting fluidic isolation-assisted homogeneous-flow-pressure Chip-SPE-MS system and demonstrated that it is adequate for online high-throughput determination and quantification of the 25-hydroxyvitamin D3 (25(OH)D3) biotransformation in different cells. Briefly, the homogeneous ambient flow pressure is achieved by fluidic isolation between the cell culture channel and the SPE column, and an automatic sampling probe could accomplish the sample loading and dispensing to fulfill online pretreatment of the sample. Through this new system, the expression levels of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) can be determined in real time with a detection limit of 2.54 nM. In addition, the results revealed that 25(OH)D3 metabolic activity differed significantly between normal L-02 cells and cancerous HepG2 cells. Treatment of L-02 cells with a high dose of 25(OH)D3 was found to increase significant formation of 24,25(OH)2D3, but this change was not apparent in HepG2 cells. The presented system promises to be a versatile tool for online accurate molecule biotransformation investigation and drug screening processes.

Cell Heterogeneity Revealed by On-Chip Angiogenic Endothelial Cell Migration.

In sprouting angiogenesis, a key process involved in the development and the intravasation of tumor tissues, the growth of vessel sprouts, is determined by migration of single endothelial cells (ECs). This paper presents an on-chip assaying method to investigate the migration of individual ECs by simulating vessel sprouts with microchannels. When chemical stimulus is present, ECs were observed to migrate individually toward the source of factors instead of migrating collectively. The validity of this method is shown by inducing EC migration with glioma cell coculture and culture media doped with vascular endothelial growth factor (VEGF) 165. A positive correlation between cell displacement and VEGF 165 concentration was observed. Difference in migrating ability among cells was reflected by tracking single cells, which could reveal cell heterogeneity in susceptibility to stimulus.

Combination Stiffness Gradient with Chemical Stimulation Directs Glioma Cell Migration on a Microfluidic Chip.

Tumor cells respond actively to the extracellular microenvironment via biomechanical and biochemical stimulation. Microchips provide a flexible platform to integrate multiple factors for cell research. In this work, we developed a subtle microfluidic chip that can generate a controllable stiffness gradient and orthogonal chemical stimulation to study the behaviors of glioma cells. Fibronectin-conjugated polyacrylamide (PAA) hydrogel with a longitudinal stiffness gradient ranging from about 1 kPa to 40 kPa is integrated within the cell culture chamber while lateral diffusion-based chemical stimulation is generated through circumambient microchannel arrays. The synergistic effect of epidermal growth factor (EGF) stimulation and hydrogel stiffness gradient on U87-MG cell migration was studied. By tracing cell migration, we discovered that hydrogel stiffness can promote cell chemotaxis, while the EGF gradient can accelerate cell migration. In addition, cell morphology showed typical cell spreading, increased aspect ratios, and decreased circularity in response to a stiffer substrate and plateaued at a certain stiffness level. Meanwhile, the content of intracellular reactive oxygen species (ROS) on the hydrogel soft end is enhanced by approximately 2 fold compared with that on the hydrogel stiff end. The enhancement of substrate stiffness on cell chemotaxis is very significant for in vitro model simulation and tissue engineering.

Controllable Synthesis of Multicompartmental Particles Using 3D Microfluidics.

A microfluidic assembly method based on a microfluidic chip and capillary device was developed to create multicompartmental particles. The microfluidic chip design endows the particles with regulable internal structure. By adjusting the microstructure of the chip, the diameter of the capillary, the gap length between the two microfluidic components, and the flow rates, the size of the particles and the number or the ratio of different regions within the particle could be widely varied. As a proof of concept, we have produced some complicated particles that even contain 20 compartments. Furthermore, the potential applications of the anisotropic particles are explored by encapsulating magnetic beads, fluorescent nanoparticles, and the cells into different compartments of the microparticles. We believe that this method will open new avenues for the design and application of multicompartmental particles.

An open-space microfluidic chip with fluid walls for online detection of VEGF via rolling circle amplification.

Despite traditional poly-dimethyl siloxane (PDMS) microfluidic devices having great potential in various biological studies, they are limited by sophisticated fabrication processes and low utilization. An easily controlled microfluidic platform with high efficiency and low cost is desperately required. In this work, we present an open-space microfluidic chip with fluid walls, integrating cell culture and online semi-quantitative detection of vascular endothelial growth factor (VEGF) via rolling circle amplification (RCA) reaction. In comparison with conventional co-culture detecting platforms, this method features the prominent advantages of saving reagents and time, a simplified chip fabrication process, and avoiding additional assistance for online detection with the help of an interfacial tension valve. On such a multi-functional microfluidic chip, cells (human umbilical vein endothelial cells and malignant glioma cells) could maintain regular growth and cell viability. VEGF could be detected with excellent specificity and good linearity in the range of 10-250 pg mL-1. Meanwhile, VEGF secreted by malignant glioma cells was also detected online and obviously increased when cells were stimulated by deferoxamine (DFO) to mimic a hypoxic microenvironment. The designed biochip with fluid walls provides a new perspective for micro-total analysis and could be promisingly applied in future clinical diagnosis and drug analysis.

Real-Time Imaging of Ammonia Release from Single Live Cells via Liquid Crystal Droplets Immobilized on the Cell Membrane.

Tumor cells exhibit prominent metabolic alterations through which they acclimatize to their stressful microenvironment. These cells have a high rate of glutaminolysis and release ammonia (NH3) as a byproduct, which may function as a diffusible signal among cancer cells and can reveal cellular heterogeneity. E7, a nematic liquid crystal (LC), is doped with 4-pentyl-4'-biphenyl carboxylic acid (PBA) and encapsulated in polymeric microcapsules (P-E7PBA), which are then immobilized on cells in a microfluidic channel. Normal human umbilical vein endothelial cells (HUVECs) and myeloma, human primary glioblastoma (U87), human colon carcinoma (Caco-2), and human breast adenocarcinoma (MCF-7) cells are investigated for the release of NH3. The P-E7PBA is able to visualize NH3 release from the cell via a radial-to-bipolar (R-B) orientation change, observed through a polarized optical microscope. The various cell lines significantly differ in their response time required for an R-B change. The mean response times for Caco-2, U87, and MCF-7 cells are 277, 155, and 121 s, respectively. NH3 release from a single cell captured in a microwell flow chip shows a similar R-B change. The P-E7PBA droplets technology could be applied to other multiple targets by functionalizing LCs with different probes.

Multifunctional Regulation of 3D Cell-Laden Microsphere Culture on an Integrated Microfluidic Device.

Three-dimensional (3D) hydrogel microspheres have aroused increasing attention as an in vitro cell culture model. Yet the preservation of cells' original biological properties has been overlooked during model construction. Here we present an integrated microfluidic device to accomplish the overall process including cell-laden microsphere generation, online extraction, and dynamic-culture. The method extends the noninvasive and nonsuppression capabilities of the droplet preparation system and provides a constant microenvironment, which reduces intracellular oxidative stress damage and the accumulation of mitochondria. Compared to the conventional preparation method, the coculture model of tumor-endothelial construction on an integrated platform displays high-level angiogenic protein expression. We believe that this versatile and biocompatible platform will provide a more reliable analysis tool for tissue engineering and cancer therapy.

Alteration of intracellular metabolome in osteosarcoma stem cells revealed by liquid chromatography-tandem mass spectrometry.

Cancer stem cells (CSCs) are the origin of many malignant tumours, including osteosarcoma that mainly affects adolescents and is accompanied by a poor prognosis. However, little is known about the intrinsic biological information of osteosarcoma stem cells, particularly for the metabolomics features. Hence, an ultra-high performance liquid chromatography coupled with tandem Q-Exactive Orbitrap mass spectrometer (UHPLC-QE-MS)-based metabolomics approach was developed to investigate the metabolism changes in the human osteosarcoma (HOS) cell line in order to understand its possible mechanism. HMDB, METLIN and m/z Cloud databases were used to identify the metabolic markers. Additionally, the compounds were further identified using standards of the metabolites. Comparing HOS-CSCs with non-CSCs, 154 different metabolites were identified in both the positive and negative modes. Based on the clearly distinct metabolites, the changed metabolic pathways were analysed using MetaboAnalyst. The top five altered pathways included alanine, aspartate and glutamate metabolism; arginine and proline metabolism; glutathione metabolism; cysteine and methionine metabolism; and the citrate cycle (TCA cycle). The downregulation of the TCA cycle and elevation of oxidized glutathione levels suggested a decline of mitochondrial metabolism, while most of the amino acid metabolisms were upregulated. Further biological experiments including an analysis of mitochondrial activity confirmed the above hypotheses that were deduced from metabolomics results. These findings not only enhance our understanding of the altered metabolome in osteosarcoma stem cells but also demonstrate the great potential of such a metabolomics method based on UHPLC-QE-MS in large-scale cell studies.

Responses of Cellular Adhesion Strength and Stiffness to Fluid Shear Stress during Tumor Cell Rolling Motion.

Biochemical and physical factors affect the rolling of tumor cells across the blood vessel. The biochemical factors have been well studied, while the influence of physical factors such as fluid shear stress (FSS) remains poorly understood. Here, human glioma cells (U87 cells) in a straight microfluidic channel were exposed to FSS (0.12, 1.2, and 1.8 dyn/cm2); and their locomotion behaviors from crawling-to-rolling and changes in cellular morphology (concave, elongated, less elongated, and round) were observed. The adhesion strength and stiffness of the cells of different morphologies were analyzed using a live single-cell extractor and atomic force microscopy, respectively. In general, the FSS stimulated cells showed stronger adhesion strength than the cells not exposed to FSS. The cell not exposed to FSS always exhibited greater nuclear stiffness than cortex stiffness, while after FSS treatment the cortex hardened and nucleus softened, where the round-shaped cell had a cortex that was more rigid than its nucleus. These results indicated that FSS influenced the biomechanics of circulating tumor cells, and elucidation of the mechanical responses to FSS might provide a deeper insight for cancer metastasis.

Selective Fabrication of Nanowires with High Aspect Ratios Using a Diffusion Mixing Reaction System for Applications in Temperature Sensing.

The selective fabrication of highly ordered nanowires with high aspect ratios was of low reproducibility, which remains a challenge for laboratory research. In this paper, we report a novel approach for selective fabrication of conductive nanowires on a solid surface via diffusion mixing reaction system formed by a chemical pen. The nanoscale-mixing region was achieved by appropriately adjusting the viscosity of the solution and other parameters with the aid of dyes functioned as a flow boundary indicator. Finite element simulations and analysis were performed to understand the generation of mixing regions and guide the improvement of the chemical pen design. Under the optimal parameters, high aspect ratio silver nanowires (aspect ratio ≈ 1800) were obtained. Silver nanowire arrays with uniform width, gradient width and complex patterns were successfully fabricated. The theoretical value of the temperature coefficient of resistance (TCR) for silver was 0.0038 Ω/°C. A single silver wire temperature sensor with 7-fold increase in temperature coefficient resistance (0.0261 Ω/°C) was fabricated to show the advantages of the chemical pen in the fabrication of nanosensors. With the freedom of the region, simple operability and applicability, the chemical pen was expected to a potential and advanced method for selective nanomodification and processing on subcellular interfaces.

In Situ Monitoring of Fluid Shear Stress Enhanced Adherence of Bacteria to Cancer Cells on Microfluidic Chip.

Mechanosensing mechanisms for surface recognition by bacteria play an important role in inflammation and phagocytosis. Here, we describe a set of DNA probes for revealing microbe adherence to cancer cells under fluid shear stress. DNA probes modified with a biotin group, an azido group, and hexadecanoic acid were indiscriminately anchored to the cell surface, acting as indicators for the membrane proteins, cell-surface carbohydrate, and phospholipids. When cancer cells were exposed to bacteria in fluid, enhanced accumulation of membrane proteins was indicated by the strong fluorescence aggregation, meanwhile the weakened accumulation of cell-surface carbohydrate and phospholipids indication was indicated by attenuated fluorescence. Further research demonstrates that this mechanosensing strategy was applicable to different bacterial-cancer cell interactions. This study not only uncovered new cellular mechanotransduction mechanisms, but also provided a versatile method that enabled in situ and dynamic indication of cancer cell responses to mechanical stimuli.

Chemical operations on a living single cell by open microfluidics for wound repair studies and organelle transport analysis.

Single cells are increasingly recognized to be capable of wound repair that is important for our mechanistic understanding of cell biology. The lack of flexible, facile, and friendly subcellular treatment methods has hindered single-cell wound repair studies and organelle transport analyses. Here we report a laminar flow based approach, we call it fluid cell knife (Fluid CK), that is capable of precisely cutting off or treating a portion of a single cell from its remaining portion in its original adherent state. Local operations on portions of a living single cell in its adherent culture state were applied to various types of cells. Temporal wound repair was successfully observed. Moreover, we successfully stained portions of a living single cell to measure the organelle transport speed (mitochondria as a model) inside a cell. This technique opens up new avenues for cellular wound repair and subcellular behavior analyses.

Homogenous deposition of matrix-analyte cocrystals on gold-nanobowl arrays for improving MALDI-MS signal reproducibility.

The non-uniform deposition of matrix-analyte cocrystals and poor ionization are major obstacles in quantitative analysis through MALDI-MS. An Au-nanobowl array was prepared and applied to overcome these limitations, which enabled the quantitative detection of oligonucleotides and polypeptides.