Amplified oligonucleotide sensing in microliter volumes containing copper ions by solution streaming

We present a simple, cost-effective design for amplifying oligodeoxynucleotide (ODN) sensing, in microliter ODN volumes containing copper ions, by solution streaming (bubbling). The inert gas streaming (bubbling) at a constant pressure of 0.04 bar drives the motion of a 30-microL ODN droplet containing a three-electrode circuit (inverted drop microcell), and in the presence of copper ions offers an approximately 50-times improvement in the detection of ODN samples. The detection of ODNs at the carbon paste electrode is based on the enhancement of the oxidation peaks of purine bases (adenine and guanine) by the anodic stripping of the electrochemically accumulated complex of Cu(I) with purine base residues of acid hydrolyzed ODN samples (Cu(I)-ahODN complex). We used the proposed method for (i) the determination of the percentage content of adenine and guanine units within analyzed ODN samples at subnanomolar concentrations (related to monomer content) and (ii) the detection of the (TTC)n triplet expansion using magnetic DNA hybridization with reporter probes containing guanine units (the TTC trinucleotide repeat expansion is associated with serious hereditary diseases, including Friedreich ataxia).     

Infrared transmission spectrometry for the determination of urea in microliter sample volumes of blood plasma dialysates

Application of mid-infrared spectroscopy for the determination of urea in blood plasma dialysates of microliter sample volumes using a transmission microcell was investigated. Infrared spectra of the dialysates of plasma samples collected from 75 different patients using CMA 60 microdialysis catheters were evaluated with multivariate partial least squares regression. Using the absorbance spectral data from 1520-1420 cm(-1) and 1220-1120 cm(-1), a minimum standard error of prediction (SEP) of 0.88 mg/dL (0.14 mM) was achieved with spectral variable selection. Our findings suggest the feasibility of developing a mid-infrared sensor in combination with micro-fluidics for on-line monitoring of urea in patients undergoing dialysis treatment.     

Recirculation of nanoliter volumes within microfluidic channels

A microfluidic device is described, capable of recirculating nanoliter volumes in restricted microchannel segments. The device consists of a PDMS microfluidic structure, reversibly sealed to a glass substrate with integrated platinum electrodes. The integrated electrodes generate electroosmotic flow locally, which results in a cycling flow in the channel segment between the two electrodes in case one channel exit is closed (dead-end channel). This cycling flow is a consequence of the counterbalancing hydrodynamic pressure against the electroosmotically generated flow. Acid-base indicators were employed to study the formation of H(+) and OH(-) at both the in-channel electrodes. The formation of acid can locally change the zeta-potential of the channel wall, which will affect the flow profile. Using this method, small analyte volumes can be mixed for prolonged times within well-defined channel segments and/or exposed to in-channel sensor surfaces.     

On the use of on-line detection for maintenance of gradually deteriorating systems

This paper deals with condition-based maintenance and non-stationary degradation process due to sudden changes. This is an attempt to propose an adaptive maintenance policy based on the on-line change detection procedure which can help to detect switches from a nominal mode to an accelerated mode in a non-informative context about the change mode time.     

Microfabricated filters for microfluidic analytical systems

Solvent and reagent filters were micromachined into quartz wafers using deep reactive ion etching to create a network of intersecting 1.5 x 10 microns channels. When placed at the bottom of reservoirs with a side exit, this channel network behaved as a lateral percolation filter composed of an array of cubelike structures one layer deep. Flow through these filters was driven by electroosmotic flow (EOF). Silanol groups at the walls of channels in the network provided the requisite charge to trigger EOF when voltage was applied laterally to the filter. Adsorption of cationic proteins in this silanol-rich matrix was controlled by the application of a polyacrylamide coating prepared by bonding N-hydroxysuccinimide (NHS)-activated poly(acrylic acid) to (gamma-aminopropyl)silane-derivatized filters. Subsequent reaction of residual NHS groups in the coating with 2-(2-aminoethoxy)ethanol provided channels of low charge density and adsorptivity. These lateral percolation filters were shown to be efficacious in filtering solvents containing a variety of particulate materials, ranging from dust to cells.     

High-throughput microfluidic systems for disease detection

Accuracy and rapid response are critical to the detection of an acute infectious disease, not only because the detection results can affect the medical treatment, but also can prevent disease outbreaks. Since the current culture-based technology is time consuming and experience dependent, academia and industrial researchers are using microfluidics and nucleic acids as the fundamental ideas to build pioneering tools against infectious disease. While many point-of-care microfluidic systems have been realized to execute nucleic acid applications, high-throughput microfluidic systems are under development for various nucleic acid applications because of high efficiency and demand from the market. Building a high-throughput system is an interdisciplinary challenge because of the design concerns from science and the manufacturing concerns from engineering, but its realization will be a milestone. This article is aimed to review three essential steps of the nucleic acid-based detection realized in high-throughput formats, including polymerase chain reaction, capillary electrophoresis, and nucleic acid purification.     

A picoliter-volume mixer for microfluidic analytical systems

Mixing confluent liquid streams is an important, but difficult operation in microfluidic systems. This paper reports the construction and characterization of a 100-pL mixer for liquids transported by electroosmotic flow. Mixing was achieved in a microfabricated device with multiple intersecting channels of varying lengths and a bimodal width distribution. All channels running parallel to the direction of flow were 5 microm in width whereas larger 27-microm-width channels ran back and forth through the parallel channel network at a 45 degrees angle. The channel network composing the mixer was approximately 10 microm deep. It was observed that little mixing of the confluent solvent streams occurred in the 100-microm-wide, 300-microm-long mixer inlet channel where mixing would be achieved almost exclusively by diffusion. In contrast, after passage through the channel network in the approximately 200-microm-length static mixer bed, mixing was complete as determined by confocal microscopy and CCD detection. Theoretical simulations were also performed in an attempt to describe the extent of mixing in microfabricated systems.     

Gateable nanofluidic interconnects for multilayered microfluidic separation systems

The extension of microfluidic devices to include three-dimensional fluidic networks allows complex fluidic and chemical manipulations but requires innovative methods to interface fluidic layers. Externally controllable interconnects, employing nuclear track-etched polycarbonate membranes containing nanometer-diameter capillaries, are described that produce hybrid three-dimensional fluidic architectures. Controllable nanofluidic transfer is achieved by controlling applied bias, polarity, and density of the immobile nanopore surface charge and the impedance of the nanocapillary array relative to the microfluidic channels. Analyte transport between vertically separated microchannels has three stable transfer levels, corresponding to zero, reverse, and forward bias. The transfer can even depend on the properties of the analyte being transferred such as the molecular size, illustrating the flexible character of the analyte transfer. In a specific analysis implementation, nanochannel array gating is applied to capillary electrophoresis separations, allowing selected separated components to be isolated for further manipulation, thereby opening the way for preparative separations at attomole analyte mass levels.     

Comparison of inlet geometry in microfluidic cell affinity chromatography

Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design have not been extensively described or studied. The most common inlets-used to connect the microfluidic chip to pumps, tubing, etc.-are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performances of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than that in the remaining channel, whereas for parallel inlet chips cell density at the inlet area was similar to that in the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative-selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices.     

Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.     

Faceted design of channels for low-dispersion electrokinetic flows in microfluidic systems

A novel methodology for designing microfluidic channels for low-dispersion, electrokinetic flows is presented. The technique relies on trigonometric relations that apply for ideal electrokinetic flows, allowing faceted channels to be designed using common drafting software and a hand calculator. Flows are rotated and stretched along the abrupt interface between adjacent regions having differing specific permeability--a quantity with dimensions of length that we introduce to derive the governing equations. Two-interface systems are used to eliminate hydrodynamic rotation of bands injected into channels. Regions bounded by interfaces form faceted flow "prisms" with uniform velocity fields that can be combined with other prisms to obtain a wide range of turning angles and expansion ratios. Lengths of faceted prisms can be varied arbitrarily, simplifying chip layout and allowing the ability to reduce unwanted effects such as transverse diffusion and Joule heating for a given faceted prism. Designs are demonstrated using two-dimensional numerical solutions of the Laplace equation.     

Characterization of nanocrystalline CdSe by size exclusion chromatography

High-performance size exclusion chromatography (HPSEC) is a powerful tool for probing the size and size distribution of complex materials. Here we report its application to the analysis of cadmium selenide nanocrystals produced in organic solvents. If nanocrystal-column interactions are minimized, this method provides an accurate measure of nanocrystal hydrodynamic diameter directly in solution; such information is complementary to TEM in that it can measure the thickness of various capping agents. While the resolution of single-pass HPSEC is limited to 1 nm, we show here that recycling size exclusion chromatography can be applied to assess the fine details of a sample's distribution. Finally, semiconductor nanocrystals can be made a variety of shapes whose optical characteristics are difficult to distinguish. HPSEC can be applied to the general problem of shape separations which we demonstrate with a tetrapod material.     

Size exclusion chromatography for semipreparative scale separation of Au38(SR)24 and Au40(SR)24 and larger clusters

Size exclusion chromatography (SEC) on a semipreparative scale (10 mg and more) was used to size-select ultrasmall gold nanoclusters (<2 nm) from polydisperse mixtures. In particular, the ubiquitous byproducts of the etching process toward Au(38)(SR)(24) (SR, thiolate) clusters were separated and gained in high monodispersity (based on mass spectrometry). The isolated fractions were characterized by UV-vis spectroscopy, MALDI mass spectrometry, HPLC, and electron microscopy. Most notably, the separation of Au(38)(SR)(24) and Au(40)(SR)(24) clusters is demonstrated.     

Fungi use efficient algorithms for the exploration of microfluidic networks

Fungi, in particular, basidiomycetous fungi, are very successful in colonizing microconfined mazelike networks (for example, soil, wood, leaf litter, plant and animal tissues), a fact suggesting that they may be efficient solving agents of geometrical problems. We therefore evaluated the growth behavior and optimality of fungal space-searching algorithms in microfluidic mazes and networks. First, we found that fungal growth behavior was indeed strongly modulated by the geometry of microconfinement. Second, the fungus used a complex growth and space-searching strategy comprising two algorithmic subsets: 1) long-range directional memory of individual hyphae and 2) inducement of branching by physical obstruction. Third, stochastic simulations using experimentally measured parameters showed that this strategy maximizes both survival and biomass homogeneity in microconfined networks and produces optimal results only when both algorithms are synergistically used. This study suggests that even simple microorganisms have developed adequate strategies to solve nontrivial geometrical problems.     

Performance of an integrated microoptical system for fluorescence detection in microfluidic systems

This article presents a new integrated microfluidic/microoptic device designed for basic biochemical analysis. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar microfluidic systems, elements of the detection system are realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive circular or elliptical microlens arrays and chromium aperture arrays. The microfluidic channels are 60 microm wide and 25 microm deep. The elliptical microlenses have a major axis of 400 microm and a minor axis of 350 microm. The circular microlens diameters range from 280 microm to 350 microm. The apertures deposited on the outer chip surfaces are etched in a 3000-A-thick chromium layer. The overall thickness of this microchemical system is < 1.6 mm. A limit of detection of 3.3 nM for a Cy5 solution in phosphate buffer (pH 7.4) was demonstrated. The cross-talk signal measured between two adjacent microchannels with 1 mm pitch was < 1:5600, meaning that < or = 1.8 x 10(-4)% of the fluorescence light power emitted from one microchannel filled with a 50 microM Cy5 solution reaches the photodetector at the adjacent microchannel. This performance compares very well with that obtainable in microchemical chips using confocal fluorescence systems, taking differences in parameters, such as excitation power into microchannels, data acquisition rates, and signal filtering into account.     

Recycling size exclusion chromatography for the analysis and separation of nanocrystalline gold

Recycling size exclusion chromatography (RSEC) provides a high-resolution technique for the analysis and separation of materials based on size. We show here the application of this method to gold nanocrystals stabilized by thiols. Alternate recycling is more effective at separating nanomaterials as compared to closed-loop recycling because of its improved efficiency and high resolution. With the use of this technique, we find the resolution ratio of nanocrystal separation increases with the square root of the cycle number, in good agreement with theory. The increased resolution of the size exclusion chromatograms permits the use of RSEC in the baseline separation of nanocrystals which differ by only 6 A in size. In addition to separations, RSEC is valuable as an analytical tool. For example, after recycling processes an initially broad and chromatographic feature from a gold nanocrystal solution resolves into three distinct peaks. Transmission electron microscopy of collected fractions reveals that these peaks correspond to distinct populations of gold nanoparticles with narrowly defined sizes.     

Controlled mixing in microfluidic systems using bacterial chemotaxis

We demonstrate the use of Escherichia coli and their chemotactic characteristics to enhance mixing in a microchannel in a controlled and bi-directional manner. The presence of a chemoattractant in one arm of a three-junction microchannel results in an asymmetric increase in the effective diffusion coefficient of extremely high molecular weight TMR-Dextran (MW 2 000 000), which rises linearly with the concentration of attractant from a baseline value of 8-42 microm(2)/s at a concentration of 0.1 M. The response to a repellent is similar, with the opposite bias.