A Microfabricated Pistonless Syringe Pump Driven by Electro-Conjugate Fluid with Leakless On/Off Microvalves.

In contrast to microfluidic devices, bulky syringe pumps are widely used to deliver a small amount of solution with high accuracy. Miniaturizing the syringe pump is difficult due to the scale effect in the microscale where the friction of the piston-cylinder is dominant and there are few high-power microactuators. To solve these problems, an on-chip microsyringe pump without mechanical sliding parts and with high power sources is proposed. The microsyringe pump utilizes the interface between water and oil (electro-conjugate fluid, ECF) instead of a piston and an electrohydrodynamic (EHD) flow driven by ECF in place of a linear actuator. ECF as a functional fluid has two capabilities: a) making the water-oil interface in microchannels and b) generating an active ECF flow at an applied voltage to withdraw and infuse aqueous solution by the interface. To control the flow direction, ECF-driven leakless on/off microvalves are also integrated. It is demonstrated that the proposed ECF microsyringe pump synchronized with the ECF on/off microvalves can control the withdrawing and infusing of aqueous solution with high resolution and precision. The experiments prove the feasibility of the microsyringe pump to be embedded as a module for the precise and linear control of flow rates in microfluidic devices.

Nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology.

In this study, we report nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. With the aim of defining sophisticated Nafion nanostructures with highly ionic conductivity, fabrication parameters such as Nafion concentration and curing temperature were optimized. By increasing curing temperature and Nafion concentration, we were able to control the replication fidelity of Nafion nanopatterns when copied from a PDMS master mold. We also found that cross-sectional morphology and ion current density of nanopatterned Nafion strongly depends on the fabrication parameters. To investigate this dependency, current-voltage analysis was conducted using organic electrochemical transistors (OECT) overlaid with patterned Nafion substrates. Nanopatterned Nafion was found to allow higher ion current densities than unpatterned surfaces. Furthermore, higher curing temperatures were found to render Nafion layers with higher ion/electrical transfer properties. To optimize nanopattern dimensions, electrical current flows, and film uniformity, a final configuration consisting of 5% nanopatterned Nafion cured at 65°C was chosen. Multielectrode arrays (MEAs) were then covered with optimized Nafion nanopatterns and used for electrophysiological analysis of two types of induced pluripotent stem cell-derived cardiomyocytes (iPSCs-CMs). These data highlight the suitability of nanopatterned Nafion, combined with MEAs, for enhancing the cellular environment of iPSC-CMs for use in electrophysiological analysis in vitro.

Rapid, High-Throughput, and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire-Incorporated and Pneumatic Pressure-Driven Microdevice.

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real-time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput, and direct MB delivery to human breast cancer MCF-7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump-associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire-patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB-coated nanowire array can be manipulated. The combination of the nanowire-mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment.

Quantitation of Oxidative Stress Gene Expression in Human Cell Lines Treated with Water-Dispersible MnO Nanoparticles.

The objective of this study was to assess cytotoxicity of engineered MnO nanoparticles by quantifying the reactive oxygen species (ROS) related genes (glutathione S-transferase (GST) and catalase) using real time-polymerase chain reaction (RT-PCR) and molecular beacon (MB) technologies. Monodisperse MnO nanoparticles of 14 nm in size were synthesized by the encapsulation of polyethyleneglycol (PEG)-phospholipid shell around the MnO core to endow high water-dispersibility and biocompatibility. In vitro cytotoxicity was evaluated at different concentrations (10, 50 and 100 µg/ml) and incubation times (12, 24 and 48 h) with human cancer cell lines (glioblastoma, lung adenocarcinoma and neuroblastoma cells). Both genetic and cellular cytotoxic screening methods produced consistent results, showing that GST and catalase ROS gene expression was maximized in 24 h incubation at 100 µg/ml concentration of MnO nanoparticles for each cell line. However, the cytotoxicity effect of the PEG-phospholipid coated MnO nanoparticle was not significant compared with control experiments, demonstrating its high potential in the applications of nanomedicines for a diagnostic and therapeutic tool.

Dual role of blue luminescent MoS2 quantum dots in fluorescence resonance energy transfer phenomenon.

Homogeneous blue luminescent MoS2 quantum dots are fabricated by using a lithium intercalation method from MoS2 nanoparticles, and the unique blue photoluminescence property is utilized in the Alexa Fluor 430-dsDNA-MoS2 FRET system, demonstrating the dual function of MoS2 quantum dots as a donor and an acceptor.

NanoIEA: A Nanopatterned Interdigitated Electrode Array-Based Impedance Assay for Real-Time Measurement of Aligned Endothelial Cell Barrier Functions.

A nanopatterned interdigitated electrode array (nanoIEA)-based impedance assay is developed for quantitative real-time measurement of aligned endothelial cell (EC) barrier functions in vitro. A bioinspired poly(3,4-dihydroxy-L-phenylalanine) (poly (l-DOPA)) coating is applied to improve the human brain EC adhesion onto the Nafion nanopatterned surfaces. It is found that a poly (l-DOPA)-coated Nafion grooved nanopattern makes the human brain ECs orient along the nanopattern direction. Aligned human brain ECs on Nafion nanopatterns exhibit increased expression of genes encoding tight and adherens junction proteins. Aligned human brain ECs also have enhanced impedance and resistance versus unaligned ones. Treatment with a glycogen synthase kinase-3 inhibitor (GSK3i) further increases impedance and resistance, suggesting synergistic effects occur on the cell-cell tightness of in vitro human brain ECs via a combination of anisotropic matrix nanotopography and GSK3i treatment. It is found that this enhanced cell-cell tightness of the combined approach is accompanied by increased expression of claudin protein. These data demonstrate that the proposed nanoIEA assay integrated with poly (l-DOPA)-coated Nafion nanopatterns and interdigitated electrode arrays can make not only biomimetic aligned ECs, but also enable real-time measurement of the enhanced barrier functions of aligned ECs via tighter cell-cell junctions.

Exploring novel immunotherapy biomarker candidates induced by cancer deformation.

Triple-negative breast cancer (TNBC) demands urgent attention for the development of effective treatment strategies due to its aggressiveness and limited therapeutic options [1]. This research is primarily focused on identifying new biomarkers vital for immunotherapy, with the aim of developing tailored treatments specifically for TNBC, such as those targeting the PD-1/PD-L1 pathway. To achieve this, the study places a strong emphasis on investigating Ig genes, a characteristic of immune checkpoint inhibitors, particularly genes expressing Ig-like domains with altered expression levels induced by "cancer deformation," a condition associated with cancer malignancy. Human cells can express approximately 800 Ig family genes, yet only a few Ig genes, including PD-1 and PD-L1, have been developed into immunotherapy drugs thus far. Therefore, we investigated the Ig genes that were either upregulated or downregulated by the artificial metastatic environment in TNBC cell line. As a result, we confirmed the upregulation of approximately 13 Ig genes and validated them using qPCR. In summary, our study proposes an approach for identifying new biomarkers applicable to future immunotherapies aimed at addressing challenging cases of TNBC where conventional treatments fall short.

An integrated microfluidic device for the high-throughput screening of microalgal cell culture conditions that induce high growth rate and lipid content.

This study describes the development of a microfluidic device for the high-throughput screening of culture conditions, such as the optimum sodium acetate concentration for promoting rapid growth and high lipid accumulation of Chlamydomonas reinhardtii. An analysis of the microalgal growth on the microfluidic device revealed an optimum sodium acetate concentration of 5.72 g L(-1). The lipid content, determined by the 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) staining method, increased with the sodium acetate concentration. The results were found to be statistically reproducible with respect to cell growth and lipid production. Other nutrient conditions, including the nitrogen and phosphorus concentrations, can also be optimized on the same microfluidic platform. The microfluidic device performance results agreed well with the results obtained from the flask-scale experiments, validating that the culture conditions were scalable. Finally, we, for the first time, established a method for the absolute quantification of the microalgal lipid content in the picoliter culture volumes by comparing the on-chip and off-chip data. In conclusion, we successfully demonstrated the high-throughput screening of sodium acetate concentrations that induced high growth rates and high lipid contents in C. reinhardtii cells on the microfluidic device.

Fabrication of a circular PDMS microchannel for constructing a three-dimensional endothelial cell layer.

We describe a simple and efficient fabrication method for generating microfluidic channels with a circular cross-sectional geometry by exploiting the reflow phenomenon of a thick positive photoresist. Initial rectangular shaped positive photoresist micropatterns on a silicon wafer, which were fabricated by a conventional photolithography process, were converted into a half-circular shape by tuning the temperature to around 105 °C. Through optimization of the reflow conditions, we could obtain a perfect circular micropattern of the positive photoresist, and control the diameter in a range from 100 to 400 µm. The resultant convex half-circular photoresist was used as a template for fabricating a concave polydimethylsiloxane (PDMS) through a replica molding process, and a circular PDMS microchannel was produced by bonding two half-circular PDMS layers. A variety of channel dimensions and patterns can be easily prepared, including straight, S-curve, X-, Y-, and T-shapes to mimic an in vivo vascular network. To form an endothelial cell layer, we cultured primary human umbilical vein endothelial cells inside circular PDMS microchannels, and demonstrated successful cell adhesion, proliferation, and alignment along the channel.

The deformation of cancer cells through narrow micropores holds the potential to regulate genes that impact cancer malignancy.

Surgery, radiation, hormonal therapy, chemotherapy, and immunotherapy are standard treatment strategies for metastatic breast cancer. However, the heterogeneous nature of the disease poses challenges and continues to make it life-threatening. It is crucial to elucidate further the underlying signaling pathways to improve treatment efficacy. Our study established two triple-negative breast cancer cell lines (TW-1 and TW-2) that were physically deformed using 3 µm pores to investigate the relationship between cancer cell deformation and metastasis within a heterogeneous population. The physical transformation of TW-1 and TW-2 cells significantly affected their growth and migration speed, as evidenced by wound healing assays for collective cell migration and microchannel assays for single-cell migration. We conducted bulk RNA sequencing to gain insights into the genes influenced by physical deformation. Additionally, we evaluated the effects of trametinib resistance on breast cancer cell metastasis by assessing cell viability and migration rates. Interestingly, TW-1 and TW-2 cells exhibited resistance to trametinib treatment. We observed a significant upregulation of GABRA-3, a protein commonly expressed in malignant breast cancer, and the critical transcription factor Myc in TW-1 and TW-2 cells compared to the control group (Ori). However, we did not observe a significant difference in Myc expression between TW-1 and TW-2 cells. In contrast, in the trametinib-resistant cell lines (TW-1-Tra and TW-2-Tra), we found increased expression of OCT4 and SOX2 rather than GABRA-3 or Myc. These findings highlight the differential expression patterns of these genes in our study, suggesting their potential role in cancer cell deformation and drug resistance. Our study presents a potential in vitro model for metastatic and drug-resistant breast cancer cells. By investigating the correlation between cancer cell deformation and metastasis, we contribute to understanding breast cancer heterogeneity and lay the groundwork for developing improved treatment strategies.

Microphysiological Models of Lung Epithelium-Alveolar Macrophage Co-Cultures to Study Chronic Lung Disease.

The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.

Sensitivity enhancement of an impedance-based cellular biosensor by a nanopatterned PEDOT:Nafion interface.

A nanopatterned poly(3,4-ethylenedioxythiophene) (PEDOT):Nafion composite layer integrated with interdigitated electrodes was developed to improve the device dynamic range and sensitivity for cellular impedance spectroscopy. The nanopattern fidelity to provide cellular alignment was accessed at different mixing volumes of PEDOT to Nafion. The ion transfer rate and electrical conductivity of Nafion were improved as the mixing ratio of PEDOT increased and it provided a uniform electrical path, thus giving conformable characteristics at all spectral frequencies from 1 kHz to 100 kHz for cellular impedance spectroscopy. Computational modeling was provided to extrapolate the electrical current flow and density in the composite with respect to the different frequency ranges. These results highlight that an electrically modified Nafion nanopattern interface, combined with interdigitated electrodes, can be used for various types of impedance-based cellular biosensors in a more biomimetic and sensitive manner.

Recent advances in three-dimensional microelectrode array technologies for in vitro and in vivo cardiac and neuronal interfaces.

Three-dimensional microelectrode arrays (3D MEAs) have emerged as promising tools to detect electrical activities of tissues or organs in vitro and in vivo, but challenges in achieving fast, accurate, and versatile monitoring have consistently hampered further advances in analyzing cell or tissue behaviors. In this review, we discuss emerging 3D MEA technologies for in vitro recording of cardiac and neural cellular electrophysiology, as well as in vivo applications for heart and brain health diagnosis and therapeutics. We first review various forms of recent 3D MEAs for in vitro studies in context of their geometry, materials, and fabrication processes as well as recent demonstrations of 3D MEAs to monitor electromechanical behaviors of cardiomyocytes and neurons. We then present recent advances in 3D MEAs for in vivo applications to the heart and the brain for monitoring of health conditions and stimulation for therapy. A brief overview of the current challenges and future directions of 3D MEAs are provided to conclude the review.

Engineering a 3D collective cancer invasion model with control over collagen fiber alignment.

Prior to cancer cell invasion, the structure of the extracellular matrix (ECM) surrounding the tumor is remodeled, such that circumferentially oriented matrix fibers become radially aligned. This predisposed radially aligned matrix structure serves as a critical regulator of cancer invasion. However, a biomimetic 3D model recapitulating a tumor's behavioral response to these ECM structures is not yet available. In this study, we have developed a phase-specific, force-guided method to establish a 3D dual topographical tumor model in which each tumor spheroid/organoid is surrounded by radially aligned collagen I fibers on one side and circumferentially oriented fibers on the opposite side. A coaxial rotating cylinder system was employed to construct the dual fiber topography and to pre-seed tumor spheroids/organoids within a single device. This system enables the application of different force mechanisms in the nucleation and elongation phases of collagen fiber polymerization to guide fiber alignment. In the nucleation phase, fiber alignment is enhanced by a horizontal laminar Couette flow driven by the inner cylinder rotation. In the elongation phase, fiber growth is guided by a vertical gravitational force to form a large aligned collagen matrix gel (35 × 25 × 0.5 mm) embedded with >1000 tumor spheroids. The fibers above each tumor spheroid are radially aligned along the direction of gravitational force in contrast to the circumferentially oriented fibers beneath each tumor spheroid/organoid, where the presence of the tumor interferes with the gravity-induced fiber alignment. After tumor invasion, there are more disseminated multicellular clusters on the radially aligned side, compared to the side of the tumor spheroid/organoid facing circumferentially oriented fibers. These results indicate that our 3D dual topographical model recapitulates the preference of tumors to invade and disseminate along radially aligned fibers. We anticipate that this 3D dual topographical model will have broad utility to those studying collective tumor invasion and that it has the potential to identify cancer invasion-targeted therapeutic agents.

Fabrication of nanomolded Nafion thin films with tunable mechanical and electrical properties using thermal evaporation-induced capillary force lithography.

In this paper, we report a simple and facile method to fabricate nanomolded Nafion thin films with tunable mechanical, and electrical properties. To achieve this, we combine a novel thermal evaporation-induced capillary force lithography method with swelling process to obtain enhanced pattern fidelity in nanomolded Nafion films. We demonstrate that structural fidelity and mechanical properties of patterned Nafion thin films can be modulated by changing fabrication parameters such as swelling time, Nafion polymer concentration, and curing temperature. Interestingly, we also find that impedance properties of nanomolded Nafion thin films are associated with the Nafion polymer concentration and curing temperature. In particular, 20% Nafion thin films exhibit greater impedance stability and lower impedance values than 5% Nafion thin films at lower frequencies. Moreover, curing temperature-specific impedance changes are observed. These results suggest that capillary lithography can be used to fabricate Nafion nanostructures with high pattern fidelity capable of modifying mechanical and electrical properties of Nafion thin films.

Tunable electroconductive decellularized extracellular matrix hydrogels for engineering human cardiac microphysiological systems.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) offer tremendous potential when used to engineer human tissues for drug screening and disease modeling; however, phenotypic immaturity reduces assay reliability when translating in vitro results to clinical studies. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cell and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces and had increased expression of genes that regulate contractile function. Improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. dECM-rGO hydrogels could also be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced electrophysiological function of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Fabrication of free-standing multilayered graphene and poly(3,4-ethylenedioxythiophene) composite films with enhanced conductive and mechanical properties.

Poly(3,4-ethylenedioxythiophene) (PEDOT)-based film has relatively high conductivity, flexibility, and transmittance. However, the improvement for mechanical strength and conductivity is still required to be adopted for commercial applications. Graphene, a one atom thick planar sheet of sp(2)-bonded carbon atoms, is considered as an ideal nanocomposite material for these purposes. In this study, we have developed PEDOT and graphene composite films, two-layered graphene/PEDOT and three-layered graphene/PEDOT/graphene, by using a spin-coating method. The conductivity of a 32 nm thick PEDOT film was improved more than twice by graphene deposition, while the high transmittance of the composite film was maintained over 90%. The mechanical strength of the PEDOT and graphene composite film shows 6-fold enhancement over the pristine PEDOT film. Because of the contribution of graphene layer for enhancing the mechanical strength, a 44 nm thick graphene/PEDOT/graphene could be obtained as a free-standing film by delaminating the graphene layer from the glass substrate under a weak base solution. These results imply that the graphene not only improves the conductivity and mechanical strength of PEDOT but also enables to produce a free-standing film which could find a variety of applications in the fields of organic electronic, sensors, and optoelectronics.

Hybrid gold/DNA nanowire circuit with sub-10 nm nanostructure arrays.

We report on a simple and efficient method for the selective positioning of Au/DNA hybrid nanocircuits using a sequential combination of electron-beam lithography (EBL), plasma ashing, and a molecular patterning process. The nanostructures produced by the EBL and ashing process could be uniformly formed over a 12.6 in2 substrate with sub-10 nm patterning with good pattern fidelity. In addition, DNA molecules were immobilized on the selectively nanopatterned regions by alternating surface coating procedures of 3-(aminopropyl)triethoxysilane (APS) and diamond like carbon (DLC), followed by deposition of DNA molecules into a well-defined single DNA nanowire. These single DNA nanowires were used not only for fabricating Au/DNA hybrid nanowires by the conjugation of Au nanoparticles with DNA, but also for the formation of Au/DNA hybrid nanocircuits. These nanocircuits prepared from Au/DNA hybrid nanowires demonstrate conductivities of up to 4.3 × 105 S/m in stable electrical performance. This selective and precise positioning method capable of controlling the size of nanostructures may find application in making sub-10 nm DNA wires and metal/DNA hybrid nanocircuits.

Orthogonal co-cultivation of smooth muscle cell and endothelial cell layers to construct in vivo-like vasculature.

Development of a three-dimensional (3D) vascular co-cultivation system is one of the major challenges to provide an advanced analytical platform for studying blood vessel related diseases. To date, however, the in vivo-like vessel system has not been fully realized due to the difficulty of co-cultivation of the cells with orthogonal alignment. In this study, we report the utilization of microfabrication technology to construct biomimetic 3D co-cultured vasculature. First, microwrinkle patterns whose direction was perpendicular to the axis of a circular microfluidic channel were fabricated, and vascular smooth muscle cells (VSMCs) were cultured inside the microchannel, leading to an in vivo-like circumferential VSMC layer. Then, human umbilical vein endothelial cells (HUVECs) were co-cultured on the circumferentially aligned VSMC, and the success of double layer formation of HUVEC-VSMC in the circular microchannel could be monitored. After HUVEC cultivation, we applied shear flow in order to induce the orientation of HUVEC parallel to the axis, and the analysis of orientation angle and spreading area of HUVECs indicated that they were changed by shear stress to be aligned to the direction of flow. Thus, the HUVEC and VSMC layer could be aligned with a distinct direction. The expression level of VE-Cadherin located at the boundary of HUVECs implies in vivo-like vascular behavior. The proposed in vitro microfluidic vascular assay platform would be valuable for studying vascular diseases with high reliability due to in vivo-likeness.

Fabrication of an electroconductive, flexible, and soft poly(3,4-ethylenedioxythiophene)-thermoplastic polyurethane hybrid scaffold by in situ vapor phase polymerization.

The inherent insolubility and brittleness of poly(3,4-ethylenedioxythiophene) (PEDOT) reduce its processability and practical applicability. Herein, we use in situ vapor phase polymerization (VPP) of 3,4-ethylenedioxythiophene (EDOT) on an oxidant-impregnated thermoplastic polyurethane (TPU) matrix comprising a three-dimensional silica particle assembly to produce a soft, flexible, and conductive TPU-PEDOT hybrid scaffold. The selective removal of silica yielded a highly porous (∼95%) skeletal structure, with the effective penetration, diffusion, and polymerization of EDOT resulting in uniform PEDOT formation both on the surface and the inner side of the TPU matrix. The mechanical and electrical properties of the obtained scaffold were investigated by bending, compression testing, and stress-strain and electrical measurements. The electrical resistance of the scaffold equaled 17 kΩ and did not change after ∼500-fold bending, whereas the observed elastic modulus was much lower (300 kPa) than that of TPU (3.3 MPa). In vitro biocompatibility was investigated by MC3T3-E1 cell culturing with cell viability evaluated using the WST assay and cell morphology examined by confocal microscopy. Thus, the soft and flexible TPU-PEDOT hybrid scaffold produced by VPP might be practically useful, implying that this preliminary investigation needs to be extended to study the behavior of muscle and nerve cells under electrical stimulation.