A fully integrated isotachophoresis with a programmable microfluidic platform.

Conventional isotachophoresis (ITP) can be used for pre-concentration of a single analyte, but preconcentration of multiple analytes is time consuming due to handling and washing steps required for the extensive buffer optimization procedure. In this work, we present a programmable microfluidic platform (PMP) to demonstrate fully automated optimization of ITP of multiple analytes. By interfacing a PMP with ITP, buffer selection and repetitive ITP procedures were automated. Using lifting-gate microvalve technology, a PMP consisting of a two-dimensional microvalve array was designed and fabricated for seamless integration with an ITP chip. The microvalve array was used for basic liquid manipulation such as metering, mixing, selecting, delivering, and washing procedures to prime and run ITP. Initially, the performances of the PMP and ITP channel were validated individually by estimating volume per pumping cycle and preconcentrating Alexa Fluor 594 with appropriate trailing (TE) and leading (LE) buffers, respectively. After confirming basic functions, autonomous ITP was demonstrated using multiple analytes (Pacific blue, Alexa Fluor 594, and Alexa Fluor 488). The optimal buffer combination was was determined by performing multiple ITP runs with three different TEs (borate, HEPES, and phosphate buffers) and three different concentrations of Tris-HCl for the LE. We found that 40 mM borate and 100 mM Tris-HCl successfully preconcentrated all analytes during a single ITP run. The integrated PMP-ITP system can simplify overall buffer selection and validation procedures for various biological and chemical target samples. Furthermore, by incorporating analytical tools that interconnect with the PMP, it can provide high sample concentrations to aid in downstream analysis.

Characterization and Optimization of Isotachophoresis Parameters for Pacific Blue Succinimidyl Ester Dye on a PDMS Microfluidic Chip.

Isotachophoresis (ITP) for Pacific Blue (PB) dye using a polydimethylsiloxane (PDMS) microfluidic chip is developed and characterized by determining the types and concentrations of electrolytes, the ITP duration, and the electric field density. Among candidate buffers for the trailing electrolyte (TE) and leading electrolyte (LE), 40 mM borate buffer (pH 9) and 200 mM trisaminomethane hydrochloride (Tris-HCl) (pH 8) were selected to obtain the maximum preconcentration and resolution of the PB bands, respectively. With the selected TE and LE buffers, further optimization was performed to determine the electric field (EF) density and the ITP duration. These ITP parameters showed a 20-170,000 preconcentration ratio from initial PB concentrations of 10 nM-100 fM. Further demonstration was implemented to preconcentrate PB-conjugated lactate dehydrogenase (LDH) using the PDMS microfluidic chip. By utilizing the quenching nature of PB-LDH conjugation, we were able to identify concentrations of LDH as low as 10 ng/mL. This simple PDMS microfluidic chip-based ITP for PB preconcentration enables highly sensitive biological and chemical analyses by coupling with various downstream detection systems.

A microfluidic cardiac flow profile generator for studying the effect of shear stress on valvular endothelial cells.

To precisely investigate the mechanobiological responses of valvular endothelial cells, we developed a microfluidic flow profile generator using a pneumatically-actuated micropump consisting of microvalves of various sizes. By controlling the closing pressures and the actuation times of these microvalves, we modulated the magnitude and frequency of the shear stress to mimic mitral and aortic inflow profiles with frequencies in the range of 0.8-2 Hz and shear stresses up to 20 dyn cm-2. To demonstrate this flow profile generator, aortic inflow with an average of 5.9 dyn cm-2 shear stress at a frequency of 1.2 Hz with a Reynolds number of 2.75, a Womersley number of 0.27, and an oscillatory shear index (OSI) value of 0.2 was applied to porcine aortic valvular endothelial cells (PAVECs) for mechanobiological studies. The cell alignment, cell elongation, and alpha-smooth muscle actin (αSMA) expression of PAVECs under perfusion, steady flow, and aortic inflow conditions were analyzed to determine their shear-induced cell migration and trans-differentiation. In this morphological and immunocytochemical study, we found that the PAVECs elongated and aligned themselves perpendicular to the directions of the steady flow and the aortic inflow. In contrast, under perfusion with a fluidic shear stress of 0.47 dyn cm-2, the PAVECs elongated and aligned themselves parallel to the direction of flow. The PAVECs exposed to the aortic inflow upregulated their αSMA-protein expression to a greater degree than those exposed to perfusion and steady flow. By comparing these results to those of previous studies of pulsatile flow, we also found that the ratio of positive to negative shear stress plays an important role in determining PAVECs' trans-differentiation and adaptation to flow. This microfluidic cardiac flow profile generator will enable future valvular mechanobiological studies to determine the roles of magnitude and frequency of shear stresses.

A review of chemical gradient systems for cell analysis.

Microfluidic spatial and temporal gradient generators have played an important role in many biological assays such as in the analysis of wound healing, inflammation, and cancer metastasis. Chemical gradient systems can also be applied to other fields such as drug design, chemical synthesis, chemotaxis, etc. Microfluidic systems are particularly amenable to gradient formation, as the length scales used in chips enable fluid processes that cannot be conducted in bulk scale. In this review we discuss new microfluidic devices for gradient generation and applications of those systems in cell analysis.

On-chip gradient generation in 256 microfluidic cell cultures: simulation and experimental validation.

A microfluidic diffusion diluter was used to create a stable concentration gradient for dose response studies. The microfluidic diffusion diluter used in this study consisted of 128 culture chambers on each side of the main fluidic channel. A calibration method was used to find unknown concentrations with 12% error. Flow rate dependent studies showed that changing the flow rates generated different gradient patterns. Mathematical simulations using COMSOL Multi-physics were performed to validate the experimental data. The experimental data obtained for the flow rate studies agreed with the simulation results. Cells could be loaded into culture chambers using vacuum actuation and cultured for long times under low shear stress. Decreasing the size of the culture chambers resulted in faster gradient formation (20 min). Mass transport into the side channels of the microfluidic diffusion diluter used in this study is an important factor in creating the gradient using diffusional mixing as a function of the distance. To demonstrate the device's utility, an H2O2 gradient was generated while culturing Ramos cells. Cell viability was assayed in the 256 culture chambers, each at a discrete H2O2 concentration. As expected, the cell viability for the high concentration side channels increased (by injecting H2O2) whereas the cell viability in the low concentration side channels decreased along the chip due to diffusional mixing as a function of distance. COMSOL simulations were used to identify the effective concentration of H2O2 for cell viability in each side chamber at 45 min. The gradient effects were confirmed using traditional H2O2 culture experiments. Viability of cells in the microfluidic device under gradient conditions showed a linear relationship with the viability of the traditional culture experiment. Development of the microfluidic device used in this study could be used to study hundreds of concentrations of a compound in a single experiment.

Detection of apoptosis using fluorescent probes.

Apoptosis plays a major role in both healthy and diseased cells. The analysis of apoptosis can take advantage of multiple cellular markers, enabling the process to be studied at different time points. In this chapter, several apoptosis assay protocols are provided.

Ethanolic Extracts of California Mugwort (Artemisia douglasiana Besser) Are Cytotoxic against Normal and Cancerous Human Cells.

California mugwort (Artemisia douglasiana Besser) is used by many tribes throughout California to treat a variety of conditions, including colds, allergies, and pain. California mugwort is also utilized as women's medicine. Its use is on the rise outside of Native communities, often without the guidance of a traditional healer or experienced herbalist. Because it has been shown to have antiproliferative activity against plant and animal cells, we investigated whether California mugwort extracts have an effect on normal human cells as well as estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast cancer cells. Ethanolic and aqueous extracts of A. douglasiana leaves were tested for cytotoxicity against unstimulated normal human peripheral blood mononuclear cells (hPBMC), as well as against an ER+ human breast cancer cell line (BT-474) and an ER- human breast cancer cell line (MDA-MB-231). An ethanolic leaf extract killed hPBMC, BT-474, and MDA-MB-231 cells with IC50 values of 23.6 ± 0.3, 27 ± 5, and 37 ± 4 µg/ml, respectively. An aqueous extract killed hPBMC with an IC50 value of 60 ± 10 µg/ml, but had no effect on the two cancer cell lines at concentrations up to 100 µg/ml. The results of this study indicate that the cytotoxicity of California mugwort extends to normal human cells, as well as cancerous cells. Therefore, until further is known about the safety of this medicine, caution should be taken when consuming extracts of California mugwort, whether as a tincture or as a tea.

Generation of a chemical gradient across an array of 256 cell cultures in a single chip.

A microfluidic diffusion diluter to create stable chemical gradients across an array of cell cultures was demonstrated. The device enabled concentration based studies to be conducted at 256 different concentrations across individual, low shear cell cultures. A gradient of staurosporine on cells stained with Mitotracker Deep Red (MTDR) showed a concentration-based effect on cell apoptosis across the cell culture array.