Adhesion Strengthening Mechanism of Carbon Nanotube-Embedded Epoxy Composites: A Fracture-Based Approach.

Interfacial bonding integrity between different materials is critical to maintain the functionality of the entire physical system in any scale, ranging from building structures down to semiconductor transistors. For example, micro-patterned polymers embedded with conductive nanoparticles [e.g., carbon nanotubes (CNTs)] bonded with integrated circuits have been applied as many emerging chemical/biological microelectronic sensors. Nonetheless, it is challenging to measure and ensure the interfacial bonding integrity between materials for consistent and sustainable operations. Herein, we apply multiple interface characterization methods based on micro-engineering and microscopy as an integrative approach to reveal the mechanism of interfacial reinforcement by adding CNTs in a matrix material. An epoxy/CNT micro-beam is fabricated onto a silicon substrate, sandwiching a gold layer as an interfacial precrack. Superlayers of chromium are then repeatedly deposited onto the microstructure, inducing stepwise increasing stress over the materials and the corresponding micro-beam bending after detachment from the bonded interface. Accordingly, we can quantify key interfacial fracture parameters such as crack length, steady-state energy release rate, and fracture toughness. By further examining the formation and distribution of the micro-/nanostructures along the debonded interface using bright-field microscopy, 3D fluorescence imaging, and scanning electron microscopy, we can identify the underlying dominant interfacial strengthening and fracture toughening mechanisms. We further compare experimental results and theoretical predictions to quantify the interfacial bonding properties between epoxy/CNT and silicon and unveil the underlying reinforcement mechanisms. The results provide insights to develop polymer/nanoparticle composites with reinforced interfacial bonding integrity for more sustainable and reliable applications including microelectronics, surface coatings, and adhesive materials.

Spreading and Migration of Nasopharyngeal Normal and Cancer Cells on Microgratings.

Cell spreading and migration play a pivotal role in many diseases such as tumor metastasis. In particular, nasopharyngeal tumor cells have known of their tendency of migration to pterygoid muscles and further distant metastasis. Although existing studies revealed key characteristics of the nasopharyngeal tumor cells, their migration preference is yet to be thoroughly understood, especially in the physical aspects including the microtopographical factors. Researchers have developed techniques in recent years to study microtopography-related cell behaviors but they are not yet applied in investigating the nasopharyngeal tumor cells. In this work, we elaborate the spreading and migration characteristics of normal and cancerous nasopharyngeal cells on micrograting substrates mimicking the microtopography of myotubes of the pterygoid muscles. We further apply interference reflection microscopy (IRM) to visualize the cell-substrate adhesion dynamics. We are interested in examining the microtopography-related cell spreading and migration behaviors and their correlations, providing insights for deeper understanding and more promising prediction on the nasopharyngeal tumor metastasis.

Nondestructive quantification of single-cell nuclear and cytoplasmic mechanical properties based on large whole-cell deformation.

The mechanical properties of cell nuclei have been recognized to reflect and modulate important cell behaviors such as migration and cancer cell malignant tendency. However, these nuclear properties are difficult to characterize accurately using conventional measurement methods, which are often based on probing or deforming local sites over a nuclear region. The corresponding results are sensitive to the measurement position, and they are not decoupled from the cytoplasmic properties. Microfluidics is widely recognized as a promising technique for bioassay and phenotyping. In this report, we develop a simple and nondestructive approach for the single-cell quantification of nuclear elasticity based on microfluidics by considering different deformation levels of a live cell captured along a confining microchannel. We apply two inlet pressure levels to drive the flow of human nasopharyngeal epithelial cells (NP460) and human nasopharyngeal cancerous cells (NPC43) into the microchannels. A model considering the essential intracellular components (cytoplasm and nucleus) for describing the mechanics of a cell deforming along the confining microchannel is used to back-calculate the cytoplasmic and nuclear properties. On the other hand, we also apply a widely used chemical nucleus extraction technique to examine its possible effects (e.g., reduced nuclear modulus and reduced lamin A/C expression). To determine if the decoupled nuclear properties are representative of cancer-related attributes, we classify the NP460 and NPC43 cells using the decoupled physical properties as classification factors, resulting in an accuracy of 79.1% and a cell-type specificity exceeding 74%. It should be mentioned that the cells can be recollected at the device outlet after the nondestructive measurement. Hence, the reported cell elasticity measurement can be combined with downstream genetic and biochemical assays for general cell research and cancer diagnostic applications.

Biofluidic Random Laser Cytometer for Biophysical Phenotyping of Cell Suspensions.

Phenotypic profiling of single floating cells in liquid biopsies is the key to the era of precision medicine. A random laser in biofluids is a promising tool for the label-free characterization of the biophysical properties as a result of the high brightness and sharp peaks of the lasing spectra, yet previous reports were limited to the random laser in solid tissues with dense scattering. In this report, a random laser cytometer is demonstrated in an optofluidic device filled with gain medium and human breast normal/cancerous cells. The multiple lightscattering event induced by the microscale human cells promotes random lasing and influences the lasing properties in term of laser modes, spectral wavelengths, and lasing thresholds. A sensing strategy based on analyzing the lasing properties is developed to determine both the whole cell and the subcellular biophysical properties, and the malignant alterations of the cell suspensions are successfully detected. Our results provide a new approach to designing a label-free biophysical cytometer based on optofluidic random laser devices, which is advantageous for further research in the field of random laser bioapplication.

A hand-held, power-free microfluidic device for monodisperse droplet generation.

Here we develop a microfluidic device to generate monodispersion sub-nanoliter size droplets. Our system reaches steady state within 3 s after the flow starts and generates 100,000 droplets in 28 s with high size consistency (CV < 8%). This low cost device is composed with a microfluidic chip, 2 tubings, a collection vial, a syringe and a station; and is in the size of an iPad Mini (4" × 6" × 3/4"). In this system, all incoming reagents share the same pressure drop across the fluidic passage to generator droplets. A single source negative pressure is applied to the fluids to create the flow by a vacuum at the exit end of the device. The vacuum is generated on-site by pulling the plunger of a syringe. The position of the plunger before and after pulling determines the degree of vacuum. A fixture is used to hold the plunger after it is pulled to maintain its vacuum. Although this system loses vacuum gradually as the liquid filling in, it maintains a flow rates with the changes less than 10% and droplet sizes changes less than 2% during the course of generating 150,000 droplets. The pressure drop across the chip, the flow rates of all reagents, the droplet size and generation frequency are predictable, programmable, and reproducible. This device is designed for generating droplets for single cell genome profiling application but can be also used for digital PCR or other droplet-based applications.

Protein-Substrate Adhesion in Microcontact Printing Regulates Cell Behavior.

Microcontact printing (µCP) is widely used to create patterns of biomolecules essential for studies of cell mechanics, migration, and tissue engineering. However, different types of µCPs may create micropatterns with varied protein-substrate adhesion, which may change cell behaviors and pose uncertainty in result interpretation. Here, we characterize two µCP methods for coating extracellular matrix (ECM) proteins (stamp-off and covalent bond) and demonstrate for the first time the important role of protein-substrate adhesion in determining cell behavior. We found that, as compared to cells with weaker traction force (e.g., endothelial cells), cells with strong traction force (e.g., vascular smooth muscle cells) may delaminate the ECM patterns, which reduced cell viability as a result. Importantly, such ECM delamination was observed on patterns by stamp-off but not on the patterns by covalent bonds. Further comparisons of the displacement of the ECM patterns between the normal VSMCs and the force-reduced VSMCs suggested that the cell traction force plays an essential role in this ECM delamination. Together, our results indicated that µCPs with insufficient adhesion may lead to ECM delamination and cause cell death, providing new insight for micropatterning in cell-biomaterial interaction on biointerfaces.

Revealing elasticity of largely deformed cells flowing along confining microchannels.

Deformability is a hallmark of malignant tumor cells. Characterizing cancer cell deformation can reveal how cancer cell metastasizes through tiny gaps in tissues. However, many previous reports only focus on the cancer cell behaviors under small deformation regimes, which may not be representative for the behaviors under large deformations as in the in vivo metastatic processes. Here, we investigate a wide range of cell elasticity using our recently developed confining microchannel arrays. We develop a relation between the elastic modulus and cell shape under different deformation levels based on a modified contact theory and the hyperelastic Tatara theory. We demonstrate good agreements between the model prediction and experimental results. Strikingly, we discover a clear 'modulus jump' of largely deformed cells compared to that of small deformed cells, offering further biomechanical properties of the cells. Likely, such a modulus jump can be considered as a label-free marker reflecting the elasticity of intracellular components including the nucleus during cell translocation in capillaries and tissue constrictions. In essence, we perform cell classification based on the distinct micromechanical properties of four cell lines, i.e. one normal cell line (MCF-10A) and three cancer cell lines (MCF-7, MDA-MB-231 and PC3) and achieved reasonable efficiencies (efficiency >65%). Finally, we study the correlation between large-deformational elasticity and translocation rates of the floating cells in the microchannels. Together, our results demonstrate the quantitative analysis of the biomechanical properties of single floating cells, which provide an additional label-free physical biomarker toward more effective cancer diagnosis.

A microfluidic device for isolation and characterization of transendothelial migrating cancer cells.

Transendothelial migration of cancer cells is a critical stage in cancer, including breast cancer, as the migrating cells are generally believed to be highly metastatic. However, it is still challenging for many existing platforms to achieve a fully covering endothelium and to ensure transendothelial migration capability of the extracted cancer cells for analyses with high specificity. Here, we report a microfluidic device containing multiple independent cell collection microchambers underneath an embedded endothelium such that the transendothelial-migrated cells can be selectively collected from only the microchambers with full coverage of an endothelial layer. In this work, we first optimize the pore size of a microfabricated supporting membrane for the endothelium formation. We quantify transendothelial migration rates of a malignant human breast cell type (MDA-MB-231) under different shear stress levels. We investigate characteristics of the migrating cells including morphology, cytoskeletal structures, and migration (speed and persistence). Further implementation of this endothelium-embedded microfluidic device can provide important insights into migration and intracellular characteristics related to cancer metastasis and strategies for effective cancer therapy.

Multiparametric Biomechanical and Biochemical Phenotypic Profiling of Single Cancer Cells Using an Elasticity Microcytometer.

Deep phenotyping of single cancer cells is of critical importance in the era of precision medicine to advance understanding of relationships between gene mutation and cell phenotype and to elucidate the biological nature of tumor heterogeneity. Existing microfluidic single-cell phenotyping tools, however, are limited to phenotypic measurements of 1-2 selected morphological and physiological features of single cells. Herein a microfluidic elasticity microcytometer is reported for multiparametric biomechanical and biochemical phenotypic profiling of free-floating, live single cancer cells for quantitative, simultaneous characterizations of cell size, cell deformability/stiffness, and surface receptors. The elasticity microcytometer is implemented for measurements and comparisons of four human cell lines with distinct metastatic potentials and derived from different human tissues. An analytical model is developed from first principles for the first time to convert cell deformation and adhesion information of single cancer cells encapsulated inside the elasticity microcytometer to cell deformability/stiffness and surface protein expression. Together, the elasticity microcytometer holds great promise for comprehensive molecular, cellular, and biomechanical phenotypic profiling of live cancer cells at the single cell level, critical for studying intratumor cellular and molecular heterogeneity using low-abundance, clinically relevant human cancer cells.