A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting.

We report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.

Performance of laterally elongated pillar array columns in capillary electrochromatography mode.

In the present study, cylindrical and laterally elongated pillar array columns were investigated for use in capillary electrochromatography. Minimal theoretical plate heights of H = 1.90 and 1.46 µm (in absence of sidewall effect) were obtained for coumarin C440 under unretained conditions for cylindrical and rectangular (laterally elongated, aspect ratio 4) pillar array columns, respectively. By comparing dispersion at the entire channel width to that at the central zone only, it appears that sidewall related dispersion significantly contributes to overall dispersion. A 40% reduction of the plate height was observed by taking into account only the central channel zone. A kinetic plot analysis was performed to evaluate the potential of the studied geometries by considering a maximum operating voltage of 20 kV as limiting parameter. It was demonstrated that rectangular radially elongated pillars produce a higher efficiency than cylindrical pillars and other microfabricated column structures for microchip capillary electrochromatography previously studied.

Development of a Lab-on-a-Disk Platform with Digital Imaging for Identification and Counting of Parasite Eggs in Human and Animal Stool.

We present a lab-on-a-disk technology for fast identification and quantification of parasite eggs in stool. We introduce a separation and packing method of eggs contained in 1 g of stool, allowing for removal of commonly present solid particles, fat droplets and air bubbles. The separation is based on a combined gravitational and centrifugal flotation, with the eggs guided to a packed monolayer, enabling quantitation and identification of subtypes of the eggs present in a single field of view (FOV). The prototype was tested with stool samples from pigs and humans infected with intestinal parasites (soil-transmitted helminths eggs). The quality of the images created by this platform was appropriate for identification and quantification of egg types present in the sample.

Superstructures of chiral nematic microspheres as all-optical switchable distributors of light.

Light technology is based on generating, detecting and controlling the wavelength, polarization and direction of light. Emerging applications range from electronics and telecommunication to health, defence and security. In particular, data transmission and communication technologies are currently asking for increasingly complex and fast devices, and therefore there is a growing interest in materials that can be used to transmit light and also to control the distribution of light in space and time. Here, we design chiral nematic microspheres whose shape enables them to reflect light of different wavelengths and handedness in all directions. Assembled in organized hexagonal superstructures, these microspheres of well-defined sizes communicate optically with high selectivity for the colour and chirality of light. Importantly, when the microspheres are doped with photo-responsive molecular switches, their chiroptical communication can be tuned, both gradually in wavelength and reversibly in polarization. Since the kinetics of the "on" and "off" switching can be adjusted by molecular engineering of the dopants and because the photonic cross-communication is selective with respect to the chirality of the incoming light, these photo-responsive microspheres show potential for chiroptical all-optical distributors and switches, in which wavelength, chirality and direction of the reflected light can be controlled independently and reversibly.

A novel side electrode configuration integrated in fused silica microsystems for synchronous optical and electrical spectroscopy.

We present a novel electrode configuration consisting of coplanar side electrode pairs integrated at the half height of the microchannels for the creation of a homogeneous electric field distribution as well as for synchronous optical and electrical measurements. For the integration of such electrodes in fused silica microsystems, a dedicated microfabrication method was utilized, whereby an intermediate bonding layer was applied to lower the temperature for fusion bonding to avoid thereby metal degradation and subsequently to preserve the electrode structures. Finally, we demonstrate the applicability of our devices with integrated electrodes for single cell electrical lysis and simultaneous fluorescence and impedance measurements for both cell counting and characterization.

Integration of uniform porous shell layers in very long pillar array columns using electrochemical anodization for liquid chromatography.

Electrochemical anodization has been applied to grow porous shell layers of 300 nm (30 nm pores) in 5 µm diameter pillar array columns (PACs) with a spacing of 2.5 µm. Using turn structures preceded and followed by the flow distributor structures recently introduced by our group and filled with radially elongated pillars, columns with quasi unlimited channel lengths could be conceived. The uniformity of the porous PAC was assessed by determining local plate heights along the channel, which appeared to be constant. Minimal (absolute) plate heights (H) between 4 and 6 µm were obtained at optimal flow rates when imposing increasing retention factors. Upon measuring the surface area involved in chromatographic retention as an indicator of the available surface area, an increase in the surface area by a factor of about 30 compared to that of non-anodized pillars was found. On reconfiguring a commercial HPLC instrument to enable on-chip injections, 90% of the performance (expressed in theoretical plates) could be maintained for a 1 m column, while for a 25 cm column severe losses were still observed. As the corresponding pressure drop for optimal operation of retained components is on the order of 10 bar per m only, portable and cheaper HPLC devices with high efficiencies become realistically conceivable.

Fabrication of integrated porous glass for microfluidic applications.

This paper presents a method for the fabrication of integrated porous silica layers in microfluidic channel networks by microfabrication techniques. Porous silica is obtained by anodization of silicon, followed by full conversion of the porous silicon network into porous silica by means of thermal oxidation. A series of experiments were performed with various channel layouts to determine the critical parameters, including the I-V characteristics and the optimal working potential for stable pore formation, during anodic etching. Typical test structures were anodized in 5% HF for 15 min at 1 V, yielding an average pore size of around 5.4 nm and porosity of 49%. Complete conversion of porous silicon into porous glass was accomplished with wet oxidation at 900 °C. The average pore size and porosity of porous glass network were around 3.8 nm and 34%, respectively. This decrease in both pore size and porosity is caused by the increase in molar volume when silicon oxidizes to silicon oxide. The transparency and the hydrophilicity of porous glass layers are evidenced by means of monitoring the diffusion of Rhodamine B fluorescent dye through the porous network. This fabrication method can be applied to (3-D) structured microfluidic channels and it is envisioned that the resulting porous silica layers can be employed for a wide range of application areas, such as membrane technology, catalyst supports, chromatography and electrokinetics.

Design and implementation of injector/distributor structures for microfabricated non-porous pillar columns for capillary electrochromatography.

A previously proposed foil definition is applied in the design of injector/distributor structures for solid microfabricated column structures for capillary electrochromatography. In addition to a typical bifurcated distributor, an optimized design alternative with two different configurations is experimentally evaluated. Optimized designs yielded a flat profile for the injected sample with a maximum of 3% variation from the mean width, while it went up to 18% for the typical bifurcated distributor. The implemented electrokinetic injection approach enabled controlling the volume of the injected sample accurately without sacrificing the compactness of the device design. The width of the injected sample was directly proportional to the injection time, namely 165 and 218 µm base widths were obtained for 0.6 and 0.8s of feeding, respectively. Reducing the external porosity of the distributor by 85% compared to the typical design, optimized distributors caused a decrease in the mean flow velocity of up to 70%. However, having a flat initial plug shape enabled the separation of a mixture of Coumarin 440, 460, 480 and 540 at 1 mm downstream of the injection point in 80s, while it was even not possible to detect the C440 signal for a typical bifurcated design.

Performance evaluation of different design alternatives for microfabricated nonporous fused silica pillar columns for capillary electrochromatography.

An experimental study comparing the performance of different designs for microfabricated column structures for microchip capillary electrochromatography is presented. The work is a follow-up to our previously published modeling and simulation study on the same topic. Experiments were performed using fused silica microchips with and without octadecyltrimethoxysilane coating for nonretained and retained modes of operation, respectively. Showing the same trends as the modeling results, the foil shape produces a significant decrease in plate height with an increase of around 15% in mobile phase velocity in nonretained measurements of Coumarin 480 (C480). Measured plate heights at 1 kV/cm applied electric field were 0.77, 1.33, and 1.42 µm for foil, diamond, and hexagon, respectively. Chromatographic runs of C480 yielded minimal plate height values of 1.85 and 3.28 µm for foil and diamond, respectively. The optimization of the shape and placement of the structures appeared to have a considerable impact on the achievable performance.

Novel shape and placement definitions with retention modeling for solid microfabricated pillar columns for CEC and HPLC.

A novel design approach for optimizing the shape of microfabricated pillar columns for LC is presented. 2-D flow simulations are performed with a focus on electrokinetically driven flow, in order to evaluate the performance of the new method. The proposed foil shape is compared with geometrical shapes known from the literature, for various arrangements. It yields a much more uniform velocity field distribution and a decrease in plate height values up to 25%. In addition to shape optimization, a new method for spatial arrangement of structures is presented. With the aim of conserving the hydrodynamic balance, the axial spacing of the pillars is adjusted according to the proposed equivalent width approach. When compared with a fixed interpillar spacing in all directions, it increases the flow uniformity and results in an 18% lower plate height. A new direct simulation approach is implemented to model both flow field and retention for solid microfabricated pillar structures in the 2-D domain. This model, which defines retention as inward/outward fluxes through the wall surfaces as first order reactions, enables monitoring of the time-dependent process and an evaluation of the parameters affecting performance. The meaning of the obtained results in a practical setting, with limitations in photolithography and microfabrication, will be highlighted.

A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis.

A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 microm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes.