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1.
Nanoscale ; 13(31): 13538-13549, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477758

RESUMO

The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.


Assuntos
Bacteriófagos , Infecções Estafilocócicas , Animais , Humanos , Microscopia de Força Atômica , Staphylococcus aureus , Ressonância de Plasmônio de Superfície
2.
Curr Protoc ; 1(9): e233, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34491632

RESUMO

Accurate measurement of cellular traction force is critical for understanding physical interaction between cells and the extracellular matrix. Traction force microscopy (TFM) has become the most widely used tool for this purpose. While TFM has made continual progress in terms of resolution and accuracy, there have been challenges regarding obtaining user-friendly software and choosing the right values for parameters and sub-processes associated with the software. Here we provide step-by-step instructions for a MATLAB-based TFM software application equipped with multiple methods for image deformation quantification and force reconstruction, along with clarification on the computational meaning of the parameters within the software. We outline how to choose the optimal sub-methods and values for parameters for each process, depending on the characteristics of images and purpose of the analyses. The software's runtime is 20, 4, and 0.05 min by Fast BEM L1 (Boundary Element Method L1-regularization), Fast BEM L2 (L2-regularization), and FTTC (Fourier Transform Traction Cytometry), respectively, in addition to 7 min of particle-tracking velocimetry-based deformation tracking, for a single image (1280 × 960 pixel) on a standard workstation. Finally, the colocalization accuracies, in reference to a paxillin-GFP image, are compared between the three force reconstruction methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Setting up the TFM package in MATLAB Basic Protocol 2: Running the TFM package Alternate Protocol 1: Stage drift correction: Efficient subpixel registration Alternate Protocol 2: Force field calculation: FastBEM.


Assuntos
Algoritmos , Tração , Simulação por Computador , Microscopia de Força Atômica , Software
3.
Langmuir ; 37(34): 10340-10347, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34461726

RESUMO

Preventing microbial contamination of aquatic environments is crucial for the proper supply of drinking water. Hence, understanding the interactions that govern bacterial and virus adsorption to surfaces is crucial to prevent infection transmittance. Here, we describe a new approach for studying the organization and interactions of various microorganisms, namely, Escherichia coli (E. coli) bacteria, E. coli-specific bacteriophage T4, and plant cucumber green mottle mosaic viruses (CGMMV), at the air/water interface using the Langmuir-Blodgett (LB) technique. CGMMV were found as applicable candidates for further studying their interactions with Langmuir lipid monolayers. The zwitterionic, positively, and negatively charged LB lipid monolayers with adsorbed viruses were deposited onto solid supports and characterized by atomic force microscopy. Using polymerase chain reaction, we indicated that the adsorption of CGMMV onto the LB monolayer is a result of electrostatic interactions. These insights are useful in engineering membrane filters that prevent biofouling for efficient purification systems.


Assuntos
Escherichia coli , Lipídeos , Adsorção , Microscopia de Força Atômica , Propriedades de Superfície
4.
5.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361715

RESUMO

Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.


Assuntos
Catequina/análogos & derivados , Agregados Proteicos , Tripsina/química , Catequina/química , Catequina/metabolismo , Humanos , Microscopia de Força Atômica , Modelos Moleculares , Conformação Proteica , Soluções , Espectrometria de Fluorescência , Tripsina/metabolismo
6.
Nat Commun ; 12(1): 5082, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426584

RESUMO

Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Compared to isopeptide bonds, ester bonds are prone to hydrolysis. It remains elusive whether ester bonds can completely block mechanical extension similarly to isopeptide bonds, or whether ester bonds dissipate mechanical energy by bond rupture. Here, we show that an ester-bond containing stalk domain of Cpe0147 is inextensible even at forces > 2 nN. The ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible. This allows the ester bond to withstand considerable mechanical forces and in turn prevent complete protein unfolding. However, the protecting effect might be reduced at non-physiological basic pHs or low calcium concentrations due to destabilizing the protein structures. Inspired by this design principle, we engineer a disulfide mutant resistant to mechanical unfolding under reducing conditions.


Assuntos
Proteínas de Bactérias/química , Ésteres/química , Fenômenos Mecânicos , Dissulfetos/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Domínios Proteicos , Água/química
7.
J Phys Chem Lett ; 12(33): 8039-8045, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34402624

RESUMO

Calcite dissolution is initiated by the formation of a nanoscale etch pit followed by step edge propagation and hence strongly influenced by the interactions between surface diffusing ions and step edges. However, such atomic-scale dynamics are mostly inaccessible with current imaging tools. Here, we overcome this limitation by using our recent development of high-speed frequency modulation atomic force microscopy. By visualizing atomic-scale structural changes of the etch pits at the calcite surface in water, we found the existence of mobile and less-mobile surface adsorption layers (SALs) in the etch pits. We also found that some etch pits maintain their size for a long time without expansion, and their step edges are often associated with less-mobile SALs, suggesting their step stabilization effect.


Assuntos
Carbonato de Cálcio/química , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Adsorção , Cristalografia , Estrutura Molecular , Solubilidade , Propriedades de Superfície , Água/química
8.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443453

RESUMO

Understanding the formation process and the spatial distribution of nanoparticle (NP) clusters on amyloid fibrils is an essential step for the development of NP-based methods to inhibit aggregation of amyloidal proteins or reverse the assembling trend of the proto-fibrillary complexes that prompts pathogenesis of neuro degeneration. For this, a detailed structural determination of the diverse hybrid assemblies that are forming is needed, which can be achieved by advanced X-ray scattering techniques. Using a combined solution small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) approach, this study investigates the intrinsic trends of the interaction between lysozyme amyloid fibrils (LAFs) and Fe3O4 NPs before and after fibrillization at nanometer resolution. AFM images reveal that the number of NP clusters interacting with the lysozyme fibers does not increase significantly with NP volume concentration, suggesting a saturation in NP aggregation on the fibrillary surface. The data indicate that the number of non-adsorbed Fe3O4 NPs is highly dependent on the timing of NP infusion within the synthesis process. SAXS data yield access to the spatial distribution, aggregation manner and density of NP clusters on the fibrillary surfaces. Employing modern data analysis approaches, the shape and internal structural morphology of the so formed nanocomposites are revealed. The combined experimental approach suggests that while Fe3O4 NPs infusion does not prevent the fibril-formation, the variation of NP concentration and size at different stages of the fibrillization process can impose a pronounced impact on the superficial and internal structural morphologies of these nanocomposites. These findings may be applicable in devising advanced therapeutic treatments for neurodegenerative diseases and designing novel bio-inorganic magnetic devices. Our results further demonstrate that modern X-ray methods give access to the structure of-and insight into the formation process of-biological-inorganic hybrid structures in solution.


Assuntos
Amiloide/química , Microscopia de Força Atômica , Muramidase/metabolismo , Nanocompostos/química , Nanopartículas/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Animais , Galinhas , Modelos Moleculares , Nanocompostos/ultraestrutura , Nanopartículas/ultraestrutura
9.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443385

RESUMO

DNA origami nanostructures (DONs) are promising substrates for the single-molecule investigation of biomolecular reactions and dynamics by in situ atomic force microscopy (AFM). For this, they are typically immobilized on mica substrates by adding millimolar concentrations of Mg2+ ions to the sample solution, which enable the adsorption of the negatively charged DONs at the like-charged mica surface. These non-physiological Mg2+ concentrations, however, present a serious limitation in such experiments as they may interfere with the reactions and processes under investigation. Therefore, we here evaluate three approaches to efficiently immobilize DONs at mica surfaces under essentially Mg2+-free conditions. These approaches rely on the pre-adsorption of different multivalent cations, i.e., Ni2+, poly-l-lysine (PLL), and spermidine (Spdn). DON adsorption is studied in phosphate-buffered saline (PBS) and pure water. In general, Ni2+ shows the worst performance with heavily deformed DONs. For 2D DON triangles, adsorption at PLL- and in particular Spdn-modified mica may outperform even Mg2+-mediated adsorption in terms of surface coverage, depending on the employed solution. For 3D six-helix bundles, less pronounced differences between the individual strategies are observed. Our results provide some general guidance for the immobilization of DONs at mica surfaces under Mg2+-free conditions and may aid future in situ AFM studies.


Assuntos
Silicatos de Alumínio/química , DNA/química , Magnésio/química , Microscopia de Força Atômica , Nanoestruturas/química , Conformação de Ácido Nucleico , Adsorção , Níquel/química , Polieletrólitos/química , Polilisina/química , Espermidina/química , Propriedades de Superfície , Água/química
10.
ACS Appl Mater Interfaces ; 13(33): 39018-39029, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34397215

RESUMO

Targeting nanoparticles as drug delivery platforms is crucial to facilitate their cellular entry. Docking of nanoparticles by targeting ligands on cell membranes is the first step for the initiation of cellular uptake. As a model system, we studied brain microvascular endothelial cells, which form the anatomical basis of the blood-brain barrier, and the tripeptide glutathione, one of the most effective targeting ligands of nanoparticles to cross the blood-brain barrier. To investigate this initial docking step between glutathione and the membrane of living brain endothelial cells, we applied our recently developed innovative optical method. We present a microtool, with a task-specific geometry used as a probe, actuated by multifocus optical tweezers to characterize the adhesion probability and strength of glutathione-coated surfaces to the cell membrane of endothelial cells. The binding probability of the glutathione-coated surface and the adhesion force between the microtool and cell membrane was measured in a novel arrangement: cells were cultured on a vertical polymer wall and the mechanical forces were generated laterally and at the same time, perpendicularly to the plasma membrane. The adhesion force values were also determined with more conventional atomic force microscopy (AFM) measurements using functionalized colloidal probes. The optical trapping-based method was found to be suitable to measure very low adhesion forces (≤ 20 pN) without a high level of noise, which is characteristic for AFM measurements in this range. The holographic optical tweezers-directed functionalized microtools may help characterize the adhesion step of nanoparticles initiating transcytosis and select ligands to target nanoparticles.


Assuntos
Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Glutationa/metabolismo , Nanopartículas/metabolismo , Pinças Ópticas , Fenômenos Biofísicos , Barreira Hematoencefálica/metabolismo , Encéfalo , Adesão Celular , Membrana Celular/ultraestrutura , Células Endoteliais/citologia , Galactosamina/química , Humanos , Ligantes , Microscopia de Força Atômica , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/metabolismo , Propriedades de Superfície , Transcitose
11.
Zhonghua Yan Ke Za Zhi ; 57(8): 608-613, 2021 Aug 11.
Artigo em Chinês | MEDLINE | ID: mdl-34344122

RESUMO

Objective: To study the ultrastructural features of the corneal epithelium in the corneal allograft of rats with micromolecular compound J2. Methods: An experimental study. Primarily cultured corneal epithelial cells of rats were used. Mononuclear cells (MNCs) by density gradient centrifugation were assigned into the experimental group [MNCs (2 ml)+corneal epithelial cells+J2], control group [MNCs (2 ml)+corneal epithelial cells] and blank group (corneal epithelial cells). Quantity of CD80 expression was obtained by flow cytometry after coculture. Amplitude and height images were obtained by tapping mode atomic force microscopy (AFM) with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis of Ra, Rq, Rvm and Rt was performed using the single-factor analysis of variance, and P value was calculated. Results: There were obvious differences in the ultrastructure measured by AFM among groups. Ra was 86.75±12.60 in the experimental group, 120.23±12.11 in the control group, and 61.94±10.62 in the blank group (F=306.92, P<0.01). Rq was 102.53±9.45, 138.30±10.13, and 91.96±7.25, respectively, in the three groups (F=361.85, P<0.01). Rvm was -42.21±14.22, -67.36±10.89, and -32.18±19.01, respectively (F=72.22, P<0.01). Rt was 437.32±15.66, 495.32±13.96, and 339.92±11.22, respectively (F=1634.26, P<0.01). The one way analysis of variance showed significant differences in these parameters among groups. Conclusions: Corneal epithelial cells of rats activated by MNCs had higher CD80 expression, but the expression became decreased with micromolecular compound J2. The ultrastructure of the corneal epithelium became coarser after MNCs activation, and the number of protrusions increased significantly. The ultrastructural changes were alleviated by J2.(Chin J Ophthalmol, 2021, 57: 608-613).


Assuntos
Transplante de Córnea , Epitélio Corneano , Animais , Córnea , Microscopia de Força Atômica , Ratos , Transplante Homólogo
12.
Biophys J ; 120(15): 3079-3090, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34214526

RESUMO

Cells interact mechanically with their surroundings by exerting and sensing forces. Traction force microscopy (TFM), purported to map cell-generated forces or stresses, represents an important tool that has powered the rapid advances in mechanobiology. However, to solve the ill-posed mathematical problem, conventional TFM involved compromises in accuracy and/or resolution. Here, we applied neural network-based deep learning as an alternative approach for TFM. We modified a neural network designed for image processing to predict the vector field of stress from displacements. Furthermore, we adapted a mathematical model for cell migration to generate large sets of simulated stresses and displacements for training and testing the neural network. We found that deep learning-based TFM yielded results that resemble those using conventional TFM but at a higher accuracy than several conventional implementations tested. In addition, a trained neural network is appliable to a wide range of conditions, including cell size, shape, substrate stiffness, and traction output. The performance of deep learning-based TFM makes it an appealing alternative to conventional methods for characterizing mechanical interactions between adherent cells and the environment.


Assuntos
Aprendizado Profundo , Tração , Simulação por Computador , Microscopia de Força Atômica , Modelos Teóricos
13.
Am J Physiol Heart Circ Physiol ; 321(2): H435-H445, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34242094

RESUMO

Arterial stiffening, a characteristic feature of obesity and type 2 diabetes, contributes to the development and progression of cardiovascular diseases (CVD). Currently, no effective prophylaxis or therapeutics is available to prevent or treat arterial stiffening. A better understanding of the molecular mechanisms underlying arterial stiffening is vital to identify newer targets and strategies to reduce CVD burden. A major contributor to arterial stiffening is increased collagen deposition. In the 5'-untranslated regions of mRNAs encoding for type I collagen, an evolutionally conserved stem-loop (SL) structure plays an essential role in its stability and post-transcriptional regulation. Here, we show that feeding a high-fat/high-sucrose (HFHS) diet for 28 wk increases adiposity, insulin resistance, and blood pressure in male wild-type littermates. Moreover, arterial stiffness, assessed in vivo via aortic pulse wave velocity, and ex vivo using atomic force microscopy in aortic explants or pressure myography in isolated femoral and mesenteric arteries, was also increased in those mice. Notably, all these indices of arterial stiffness, along with collagen type I levels in the vasculature, were reduced in HFHS-fed mice harboring a mutation in the 5'SL structure, relative to wild-type littermates. This protective vascular phenotype in 5'SL-mutant mice did not associate with a reduction in insulin resistance or blood pressure. These findings implicate the 5'SL structure as a putative therapeutic target to prevent or reverse arterial stiffening and CVD associated with obesity and type 2 diabetes.NEW & NOTEWORTHY In the 5'-untranslated (UTR) regions of mRNAs encoding for type I collagen, an evolutionally conserved SL structure plays an essential role in its stability and posttranscriptional regulation. We demonstrate that a mutation of the SL mRNA structure in the 5'-UTR decreases collagen type I deposition and arterial stiffness in obese mice. Targeting this evolutionarily conserved SL structure may hold promise in the management of arterial stiffening and CVD associated with obesity and type 2 diabetes.


Assuntos
Aorta/fisiopatologia , Doenças Cardiovasculares/genética , Colágeno Tipo I/genética , Sequências Repetidas Invertidas/genética , Obesidade/fisiopatologia , RNA Mensageiro/genética , Rigidez Vascular/genética , Regiões 5' não Traduzidas/genética , Adiposidade , Animais , Doenças Cardiovasculares/fisiopatologia , Dieta Hiperlipídica , Sacarose na Dieta , Artéria Femoral/fisiopatologia , Resistência à Insulina , Masculino , Artérias Mesentéricas/fisiopatologia , Camundongos , Microscopia de Força Atômica , Mutação , Análise de Onda de Pulso
14.
Nat Commun ; 12(1): 4229, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244477

RESUMO

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Mecanotransdução Celular/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pinças Ópticas , Paxilina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Respiração , Organismos Livres de Patógenos Específicos , Talina/genética , Talina/metabolismo
15.
BMC Ophthalmol ; 21(1): 280, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34261440

RESUMO

BACKGROUND: To compare the anterior surface roughness of two commercially available posterior chamber phakic intraocular lenses (IOLs) using atomic force microscopy (AFM). METHODS: Four phakic IOLs were used for this prospective, experimental study: two Visian ICL EVO+ V5 lenses and two iPCL 2.0 lenses. All of them were brand new, were not previously implanted in humans, were monofocal and had a dioptric power of - 12 diopters (D). The anterior surface roughness was assessed using a JPK NanoWizard II® atomic force microscope in contact mode immersed in liquid. Olympus OMCL-RC800PSA commercial silicon nitride cantilever tips were used. Anterior surface roughness measurements were made in 7 areas of 10 × 10 µm at 512 × 512 point resolution. The roughness was measured using the root-mean-square (RMS) value within the given regions. RESULTS: The mean of all anterior surface roughness measurements was 6.09 ± 1.33 nm (nm) in the Visian ICL EVO+ V5 and 3.49 ± 0.41 nm in the iPCL 2.0 (p = 0.001). CONCLUSION: In the current study, we found a statistically significant smoother anterior surface in the iPCL 2.0 phakic intraocular lenses compared with the VISIAN ICL EVO+ V5 lenses when studied with atomic force microscopy.


Assuntos
Cristalino , Lentes Intraoculares Fácicas , Humanos , Implante de Lente Intraocular , Cristalino/cirurgia , Microscopia de Força Atômica , Estudos Prospectivos
16.
Analyst ; 146(16): 5150-5159, 2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34286712

RESUMO

Label-free radiation pressure force analysis using a microfluidic platform is applied to the differential detection of innate immune cell activation. Murine-derived peritoneal macrophages (IC-21) are used as a model system and the activation of IC-21 cells by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) to M1 pro-inflammatory phenotype is confirmed by RNA gene sequencing and nitric oxide production. The mean cell size determined by radiation pressure force analysis increases slightly after the activation (4 to 6%) and the calculated percentage of population overlaps between the control and the activated group after 14 and 24 h stimulations are at 79% and 77%. Meanwhile the mean cell velocity decreases more significantly after the activation (14% to 15%) and the calculated percentage of population overlaps between the control and the activated group after 14 and 24 h stimulations are only at 14% and 13%. The results demonstrate that the majority of the activated cells acquire a lower velocity than the cells from the control group without changes in cell size. For comparison label-free flow cytometry analysis of living IC-21 cells under the same stimulation conditions are performed and the results show population shifts towards larger values in both forward scatter and side scatter, but the calculated percentage of population overlaps in all case are significant (70% to 83%). Cell images obtained during radiation pressure force analysis by a CCD camera, and by optical microscopy and atomic force microscopy (AFM) reveal correlations between the cell activation by LPS/IFN-γ, the increase in cell complexity and surface roughness, and enhanced back scattered light by the activated cells. The unique relationship predicted by Mie's theory between the radiation pressure force exerted on the cell and the angular distribution of the scattered light by the cell which is influenced by its size, complexity, and surface conditions, endows the cell velocity based measurement by radiation pressure force analysis with high sensitivity in differentiating immune cell activation.


Assuntos
Lipopolissacarídeos , Macrófagos Peritoneais , Animais , Interferon gama , Lipopolissacarídeos/toxicidade , Camundongos , Microscopia de Força Atômica , Óxido Nítrico
17.
Anal Chem ; 93(34): 11859-11867, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34319694

RESUMO

Counterfeits in the supply chain of high-value advanced materials such as graphene and their derivatives have become a concerning problem with a potential negative impact on this growing and emerging industry. Recent studies have revealed alarming facts that a large percentage of manufactured graphene materials on market are not graphene, raising considerable concerns for the end users. The common and recommended methods for the characterization of graphene materials, such as transmission electron microscopy (TEM), atomic force microscopy (AFM), and Raman spectroscopy based on spot analysis and probing properties of individual graphene particles, are limited to provide the determination of the properties of "bulk" graphene powders at a large scale and the identification of non-graphene components or purposely included additives. These limitations are creating counterfeit opportunities by adding low-cost black carbonaceous materials into manufactured graphene powders. To address this problem, it is critical to have reliable characterization methods, which can probe the specific properties of graphene powders at bulk scale, confirm their typical graphene signature, and detect the presence of unwanted additional compounds, where the thermogravimetric analysis (TGA) method is one of the most promising methods to perform this challenging task. This paper presents the evaluation of the TGA method and its ability to detect low-cost carbon additives such as graphite, carbon black, biochar, and activated carbon as potential counterfeiting materials to graphene materials and their derivatives such as graphene oxide (GO) and reduced GO. The superior performance of the TGA method is demonstrated here, showing its excellent capability to successfully detect these additives when mixed with graphene materials, which is not possible by two other comparative methods (Raman spectroscopy and powder X-ray diffraction (XRD)), which are used as the common characterization methods for graphene materials.


Assuntos
Grafite , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Análise Espectral Raman , Difração de Raios X
18.
Biomolecules ; 11(7)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209628

RESUMO

Here, we report the controlled assembly of SWCNT-GFP hybrids employing DNA as a linker. Two distinct, enriched SWCNTs chiralities, (6,5), (7,6), and an unsorted SWCNT solution, were selectively functionalized with DNA and hybridized to a complementary GFPDNA conjugate. Atomic force microscopy images confirmed that GFP attachment occurred predominantly at the terminal ends of the nanotubes, as designed. The electronic coupling of the proteins to the nanotubes was confirmed via in-solution fluorescence spectroscopy, that revealed an increase in the emission intensity of GFP when linked to the CNTs.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Nanotubos de Carbono/química , Proteínas/química , Microscopia de Força Atômica/métodos
19.
Methods Mol Biol ; 2350: 289-297, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331292

RESUMO

Atomic force microscopy (AFM) enables the characterization of a wide range of samples including live cells. It is generally admitted that cancer cells are significantly softer than their normal counterparts, but imaging live cells by AFM using traditional modes can be at the cost of time or resolution. We describe how this tool can be used to estimate the motility of cancer versus normal cells, based on topographical and mechanical approaches, and coupled to optical imaging.


Assuntos
Movimento Celular , Microscopia de Força Atômica , Microscopia de Vídeo/métodos , Neoplasias/patologia , Imagem Óptica/métodos , Linhagem Celular Tumoral , Células Cultivadas , Imunofluorescência/métodos , Humanos , Microscopia de Força Atômica/métodos
20.
Biosens Bioelectron ; 191: 113476, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34246124

RESUMO

Glioblastoma (GBM) is the fatal brain tumor in which secreted lactate enhances the expression of cluster of differentiation 44 (CD44) and the release of exosomes, cell-derived nanovesicles (30-200 nm), and therefore promotes tumor malignant progression. This study found that lactate-driven upregulated CD44 in malignant Glioblastoma cells (GMs) enhanced the release of CD44-enriched exosomes which increased GMs' migration and endothelial cells' tube formation, and CD44 in the secreted exosomes was sensitively detected by "capture and sensing" Titanium Nitride (TiN) - Nanoholes (NH) - discs immunocapture (TIC) - atomic force microscopy (AFM) and ultrasensitive TiN-NH-localized surface plasmon resonance (LSPR) biosensors. The limit of detection for exosomal CD44 with TIC-AFM- and TiN-NH-LSPR-biosensors was 5.29 × 10-1 µg/ml and 3.46 × 10-3 µg/ml in exosome concentration, respectively. Importantly, this work first found that label-free sensitive TiN-NH-LSPR biosensor could detect and quantify enhanced CD44 and CD133 levels in immunocaptured GMs-derived exosomes in the blood and the cerebrospinal fluid of a mouse model of GBM, supporting its potential application in a minimally invasive molecular diagnostic for GBM progression as liquid biopsy.


Assuntos
Técnicas Biossensoriais , Exossomos , Glioblastoma , Animais , Diferenciação Celular , Células Endoteliais , Biópsia Líquida , Camundongos , Microscopia de Força Atômica , Ressonância de Plasmônio de Superfície
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