Κυριακή, 1 Δεκεμβρίου 2019

One-step microchip for DNA fluorescent labeling

Abstract

In this study, we propose a microchip that is sequentially capable of fluorescently staining and washing DNAs. The main advantage of this microchip is that it allows for one-step preparation of small amounts of solution without degrading microscopic bio-objects such as the DNAs, cells, and biomolecules to be stained. The microchip consists of two inlets, the main channel, staining zone, washing zone, and one outlet, and was processed using a femtosecond laser system. High molecular transport of rhodamine B to deionized water was observed in the performance test of the microchip. Results revealed that the one-step procedure of on-chip DNA staining and washing was excellent compared to the conventional staining method. The one-step preparation of stained and washed DNAs through the microchip will be useful for preparing small volumes of experimental samples.

Soft dry electroophthalmogram electrodes for human machine interaction

Abstract

Current soft surface electrodes have attracted more and more attentions owing to their potential applications in biological signal monitoring, human-machine interaction (HMI) and Internet of Things (IoT). The paper presents that soft dry electrode based on polydimethylsiloxane-carbon black (PDMS-CB) conductive polymer is designed and fabricated to continuous, long-term, stable electroophthalmogram (EOG) signal recordings for HMI applications. The features corresponding to the different eye motions are extracted from the EOG data via the soft dry electrodes. Linear discriminant analysis (LDA) recognition algorithms are proposed to recognize eye motion behaviors for controlling the motion of the mobile robots. Experiment results have been demonstrated that LDA recognition algorithm achieves a relatively high recognition accuracy of 90.63% for recognizing four eye movements (‘Up’, ‘Down’, ‘Right’, and ‘Left’). The control commands are generated with different eye motions and transmitted to the mobile robot through WiFi communication unit, which the mobile robot is successfully controlled. The soft dry electrodes have the potential in a comfortable, simple, wearable and wireless control of rehabilitation devices.

Fabrication and characterization of hyaluronic acid microneedles to enhance delivery of magnesium ascorbyl phosphate into skin

Abstract

This study investigated the in vitro transdermal delivery of magnesium ascorbyl phosphate (MAP) through porcine ear skin treated with hyaluronic acid (HA) microneedles (MNs). In this study, the micro-molding method was used to fabricate HA MNs. HA solution (10% w/v) containing 3% of MAP was placed onto a poly(dimethyl siloxane) mold to fill the microchannels under vacuum followed by drying in a desiccator. Scanning electron microscopy was performed to record the dimensions of the MNs. Skin microporation was demonstrated by dye binding. Histological skin sections revealed the shape of microchannels under hematoxylin-eosin staining. The actual depth of the microchannels and drug distribution pathways were studied by confocal microscopy. In vitro permeation on Franz diffusion cells were performed to determine the rate and extent of drug delivery into and across the skin. SEM captured individual MNs from the array, and the length of each MN was found to be ~400 μm. The 10 × 10 MN array prepared, resulted in the formation of 95 to 100 microchannels after 2 mins of treatment. In addition, the histological evaluations showed the formation of microchannels in the skin, complementary in shape to the MNs. The depths of the formed microchannels amounted to ~125 μm as determined by confocal microscopy. The application of the current MN technology enhanced the delivery of MAP into skin (96.8 ± 3.9 μg/cm2) compared to the passive delivery strategy of MAP (44.9 ± 16.3 μg/cm2). HA MNs markedly enhanced the in vitro transdermal delivery of MAP into and across skin.

A theoretical study on real time monitoring of single cell mitosis with micro electrical impedance tomography

Abstract

Real time monitoring of cell division, mitosis, at the single cell level, has value for many biomedical applications; such as developing optimal cancer treatments that target the cell division process. The goal of this theoretical study is to explore the feasibility of using Micro Electrical Impedance Tomography (MEIT) for real time monitoring of mitosis in a single cell, through imaging. MEIT employs a micro (single cell) scale electrode cage with electrodes placed around the cell. The electrodes deliver subsensory current and the consequential voltages on the electrodes are measured. An inverse image reconstruction algorithm uses the electric data from the electrodes to generate a map of electrical conductivity distribution in the chamber, which is the image. EIT is a well-known medical imaging technology that is simple to use but lacks good resolution. Therefore, it is not a-priori obvious that EIT has sufficient resolution to monitor single cell mitosis. To accomplish the goal of this study we have developed a mathematical model of MEIT of single cell mitosis, in which an in silico experiment provided the data for the MEIT image reconstruction. This theoretical study shows that MEIT can detect the outlines of the dividing cell during the various stages of mitosis (metaphase, anaphase and telophase) and, therefore, has potential as a technology for real time monitoring of single cell mitosis.

Characterizing the invasion of different breast cancer cell lines with distinct E-cadherin status in 3D using a microfluidic system

Abstract

E-cadherin is a cell-cell adhesion protein that plays a prominent role in cancer invasion. Inactivation of E-cadherin in breast cancer can arise from gene promoter hypermethylation or genetic mutation. Depending on their E-cadherin status, breast cancer cells adopt different morphologies with distinct invasion modes. The tumor microenvironment (TME) can also affect the cell morphology and invasion mode. In this paper, we used a previously developed microfluidic system to quantify the three-dimensional invasion of breast cancer cells with different E-cadherin status, namely MCF-7, CAMA-1 and MDA-MB-231 with wild type, mutated and promoter hypermethylated E-cadherin, respectively. The cells migrated into a stable and reproducible microfibrous polycaprolactone mesh in the chip under a programmed stable chemotactic gradient. We observed that the MDA-MB-231 cells invaded the most, as single cells. MCF-7 cells collectively invaded into the matrix more than CAMA-1 cells, maintaining their E-cadherin expression. The CAMA-1 cells exhibited multicellular multifocal infiltration into the matrix. These results are consistent with what is seen in vivo in the cancer biology literature. In addition, comparison between complete serum and serum gradient conditions showed that the MDA-MB-231 cells invaded more under the serum gradient after one day, however this behavior was inverted after 3 days. The results showcase that the microfluidic system can be used to quantitatively assess the invasion behavior of cancer cells with different E-cadherin expression, for a longer period than conventional invasion models. In the future, it can be used to quantitatively investigate effects of matrix structure and cell treatments on cancer invasion.

Determining the factors affecting dynamic insertion of microneedles into skin

Abstract

Microneedles are extremely small and minimally-invasive intradermal drug delivery devices that require controlled, accurate, and repeatable insertions into human skin to perform their functions. Due to high variability and elasticity of human skin, dynamic insertion methods are being sought to ensure success in microneedle insertions into the skin passed the tough stratum corneum layer. Dynamic microneedle insertions have not been thoroughly studied to identify and assess the key parameters influencing the skin fracture to date. Here, we have utilized a previously validated artificial mechanical human skin model to identify and evaluate the factors affecting microneedle insertion. It was determined that a microneedle’s velocity at impact against the skin played the most crucial role in successfully inserting microneedle devices of different geometrical features (i.e., tip area) and array size (i.e., number of projections). The findings presented herein will facilitate the development of automated microneedle insertion devices that will enable user-friendly and error-free applications of microneedle technologies for medicine delivery.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior

Abstract

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

Engineered topographical structure to control spatial cell density using cell migration

Abstract

Control of the spatial distribution of various cell types is required to construct functional tissues. Here, we report a simple topographical structure changed the spatial cell density. A concave curved boundary was designed, which allowed the spatial descent moving of cells and the change in spatial distributions of co-cultured cells. We utilized the difference in cell motility between myoblast cells (C2C12) and neuronal cells (PC12) to demonstrate the feasibility of spontaneous change in spatial cell density. Without the curved boundaries, high motility cells (C2C12) did not migrate to the adjacent area, which resulted in a slight temporal change (< 15%) in the spatial cell distribution. In contrast, with the curved boundaries, the cell density of the high motility cells in the groove to those cells on the ridge showed an increase exceeding 45%. On the other hand, the temporal change in the spatial cell distribution of low motility cells (PC12) was below 15% with or without the curved boundaries. In addition, as groove width increased, both cells displayed more initially gathering in groove. Importantly, these cell-type dependent results were also maintained under co-culture conditions. Our results suggest that designing topographical interfaces changes spatial cell density without any manipulation and is useful for multi-cellular constructs.

Performance and structural comparison of hydrogels made from wheat bran arabinoxylan using enzymatic and coacervation methods as micro-and nano- encapsulation and delivery devices

Abstract

This study evaluated the structural and performance differences between arabinoglucuronoxylan micro-hydrogels that were enzymatically produced from alkaline-extracted wheat bran arabinoglucuronoxylans using recombinant α-L-arabinofuranosidase (AbfB) that selectively removes arabinose side chains, and chemically through coacervation process, as delivery devices for bioactive substances. The encapsulations of model bioactive substance, gallic acid (GA), in the hydrogels, were done either in-situ or ex-situ to identify the most effective encapsulation and delivery method. The hydrogels particle size distribution, polydispersity index, GA encapsulation efficiency, retention and release of functional GA (based on antioxidant activity) were assessed. The hydrogels formed in both coacervation and enzymatic processes had particle size ranges of 469–678 nm, which classify them as micro-hydrogels. However, the latter were monodispersed with polydispersity index (PdI) < 0.4 compared to the former with PdI > 0.7. In addition, enzymatically produced hydrogels attained higher zeta potential (−8.8 mV) and retained and released GA with higher anti-oxidant capacity (91%) than chemically formed micro-hydrogels (zeta potential = − 3.3 mV and antioxidant capacity = 80%). However, GA encapsulation efficiencies (72% in-situ and 68% ex-situ) were higher in chemically formed micro-hydrogels than enzymatically produced micro-hydrogels (59% in-situ and 52% ex-situ). The in-situ encapsulated GA experienced less initial burst during sustained release of 8 h compared to ex-situ encapsulation. Overall, enzymatic modification process and in-situ encapsulation were the most effective methods for production of arabinoglucuronoxylan micro-hydrogels delivery devices and for encapsulation of the GA, respectively, because of maintaining functional GA upon release and having the potential to customize the structural and functional properties of the micro-hydrogels.

Isothermal titration calorimetry in a 3D-printed microdevice

Abstract

Isothermal titration calorimetry (ITC) can benefit from operating in miniaturized devices as they enable quantitative, low-cost measurements with reduced analysis time and reagents consumption. However, most of the existing devices that offer ITC capabilities either do not yet allow proper control of reaction conditions or are limited by issues such as evaporation or surface adsorption caused inaccurate solution concentration information and unintended changes in biomolecular properties because of aggregation. In this paper, we present a microdevice that combines 3D-printed microfluidic structures with a polymer-based MEMS thermoelectric sensor to enable quantitative ITC measurements of biomolecular interactions. Benefitting from the geometric flexibility of 3D-printing, the microfluidic design features calorimetric chambers in a differential cantilever configuration that improves the thermal insulation and reduces the thermal mass of the implementing device. Also, 3D-printing microfluidic structures use non-permeable materials to avoid potential adsorption. Finally, the robustness of the polymeric MEMS sensor chip allows the device to be assembled reversibly and leak-free, and hence reusable. We demonstrate the utility of the device by quantitative ITC characterization of a biomolecular binding system, ribonuclease A (RNase A) bind with cytidine 2′-monophosphate (2’CMP) down to a practically useful sample concentration of 0.2 mM. The thermodynamic parameters of the binding system, including the stoichiometry, equilibrium binding constant, and enthalpy change are obtained and found to agree with values previously reported in the literature.

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