Chemotaxis assays of mouse sperm on microfluidic devices

Sperm chemotaxis is an area of significant interest to scientists involved in reproductive science. Understanding how and when sperm cells are attracted to the egg could have profound effects on reproduction and contraception. In an effort to systematically study this problem, we have fabricated and evaluated a microfluidic device to measure sperm chemotaxis. The device was designed with a flow-through configuration using a spatially and temporally stable chemical gradient. Mouse sperm cells were introduced into the chemotaxis chamber between confluent flows of mouse ovary extract and buffer. The sperm experiencing chemotaxis swam toward the extract and were counted relative to those that swam toward the buffer. The ovary extracts were diluted from 10(2) to 10(7) times, and each extract dilution was screened for chemotaxis. Four out of six ovaries showed a strong chemotactic response at extract dilutions of 10(-3) to 10(-5). This device provided a convenient, disposable platform on which to conduct chemotaxis assays, and the flow-through design overcomes difficulties associated with distinguishing chemotaxis from trapping.     

Electrophoretic analysis of N-glycans on microfluidic devices

We report rapid and efficient electrophoretic separations of N-glycans on microfluidic devices. Using a separation length of 22 cm and an electric field strength of 750 V/cm, analysis times were less than 3 min, and separation efficiencies were between 400,000 and 655,000 plates for the N-glycans and up to 960,000 plates for other sample components. These high efficiencies were necessary to separate N-glycan positional isomers derived from ribonuclease B and linkage isomers from asialofetuin. Structural isomers of N-glycans derived from a blood serum sample of a cancer patient were also analyzed to demonstrate that clinically relevant, complex samples could be separated on-chip with efficiencies similar to those derived from model glycoproteins. In addition, we compared microchip and capillary electrophoresis under similar separation conditions, and the microchips performed as well as the capillaries. These results confirmed that the noncircular cross section of the microchannel did not hamper separation performance. For all experiments, the glycan samples were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid to impart needed charge for electrophoresis and a fluorescent label for detection.     

Electrokinetic focusing injection methods on microfluidic devices

This paper presents an experimental and numerical investigation into electrokinetic focusing injection on microfluidic chips. The valving characteristics on microfluidic devices are controlled through appropriate manipulations of the electric potential strengths during the sample loading and dispensing steps. The present study also addresses the design and testing of various injection systems used to deliver a sample plug. A novel double-cross injection microfluidic chip is fabricated, which employs electrokinetic focusing to deliver sample plugs of variable volume. The proposed design combines several functions of traditional sample plug injection systems on a single microfluidic chip. The injection technique uses an unique sequence of loading steps with different electric potential distributions and magnitudes within the various channels to effectuate a virtual valve.     

NMR analysis on microfluidic devices by remote detection

We present a novel approach to perform high-sensitivity NMR imaging and spectroscopic analysis on microfluidic devices. The application of NMR, the most information-rich spectroscopic technique, to microfluidic devices remains a challenge because the inherently low sensitivity of NMR is aggravated by small fluid volumes leading to low NMR signal and geometric constraints resulting in poor efficiency for inductive detection. We address the latter by physically separating signal detection from encoding of information with remote detection. Thereby, we use a commercial imaging probe with sufficiently large diameter to encompass the entire device, enabling encoding of NMR information at any location on the chip. Because large-diameter coils are too insensitive for detection, we store the encoded information as longitudinal magnetization and flow it into the outlet capillary. There, we detect the signal with optimal sensitivity, using a solenoidal microcoil, and reconstruct the information encoded in the fluid. We present a generally applicable design for a detection-only microcoil probe that can be inserted into the bore of a commercial imaging probe. Using hyperpolarized 129Xe gas, we show that this probe enables sensitive reconstruction of NMR spectroscopic information encoded by the large imaging probe while keeping the flexibility of a large coil.     

Micropreparative fraction collection in microfluidic devices

Micropreparative fraction collection following microchip-based electrophoretic analysis of biomolecules is of major importance for a variety of biomedical applications. In this paper, we present a microfabricated device-based fraction collection system. Various size DNA fragments were separated and collected by simply redirecting the desired portions of the detected sample zones to corresponding collection wells using appropriate voltage manipulations. The efficiency of sampling and collection of the fractions was enhanced by placing a cross channel at or downstream of the detection point. Following the detection of the band of interest, the potentials were reconfigured to sampling/collection mode, so that the selected sample zone migrated to the appropriate collection well of the microdevice. The potential distribution assured that the rest of the analyte components in the separation column was retarded, stopped, or reversed, increasing in this way the spacing between the sample zone being collected and the immediately following one. By this means, a precise collection of spatially close consecutive bands could be facilitated. Once the target sample fraction reached the corresponding collection well, the potentials were switched back to separation mode. Alternation of the separation/detection and sampling/collection cycles was repeated until all required sample zones were physically isolated. The integrated device consists of a sample introduction, separation, fraction sampling, and fraction collection compartments. The feasibility of the fraction collection technique was tested on a mixture of dsDNA fragments. The amounts of DNA collected in this way were enough for further downstream sample processing, such as conventional PCR-based analysis.     

Fully enclosed microfluidic paper-based analytical devices

This article introduces fully enclosed microfluidic paper-based analytical devices (microPADs) fabricated by printing toner on the top and bottom of the devices using a laser printer. Enclosing paper-based microfluidic channels protects the channels from contamination, contains and protects reagents stored on the device, contains fluids within the channels so that microPADs can be handled and operated more easily, and reduces evaporation of solutions from the channels. These benefits extend the capabilities of microPADs for applications as low-cost point-of-care diagnostic devices.     

Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for "digital" (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12,000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, <500 molecules/mL to >1,000,000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA.     

Flow cytometry of Escherichia coli on microfluidic devices.

Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.     

A digital microfluidic approach to homogeneous enzyme assays

A digital microfluidic device was applied to a variety of enzymatic analyses. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in channel microfluidics. This approach is more easily reconfigured than a channel device, and the flexibility of these devices makes them suitable for a wide variety of applications. Alkaline phosphatase was chosen as a model enzyme and used to convert fluorescein diphosphate into fluorescein. Droplets of alkaline phosphatase and fluorescein diphosphate were merged and mixed on the device, resulting in a 140-nL, stopped-flow reaction chamber in which the fluorescent product was detected by a fluorescence plate reader. Substrate quantitation was achieved with a linear range of 2 orders of magnitude and a detection limit of approximately 7.0 x 10-20 mol. Addition of a small amount of a nonionic surfactant to the reaction buffer was shown to reduce the adsorption of enzyme to the device surface and extend the lifetime of the device without affecting the enzyme activity. Analyses of the enzyme kinetics and the effects of inhibition with inorganic phosphate were performed, and Km and kcat values of 1.35 microM and 120 s-1, respectively, agreed with those obtained in a conventional 384-well plate under the same conditions (1.85 microM and 155 s-1). A phototype device was also developed to perform multiplexed enzyme analyses. It was concluded that the digital microfluidic format is able to perform detailed and reproducible assays of substrate concentrations and enzyme activity in much smaller reaction volumes and with higher sensitivity than conventional methods.     

Plastic microfluidic devices modified with polyelectrolyte multilayers

Control of the polymer surface chemistry is a crucial aspect of development of plastic microfluidic devices. When commercially available plastic substrates are used to fabricate microchannels, differences in the EOF mobility from plastic to plastic can be very high. Therefore, we have used polyelectrolyte multilayers (PEMs) to alter the surface of microchannels fabricated in plastics. Optimal modification of the microchannel surfaces was obtained by coating the channels with alternating layers of poly(allylamine hydrochloride) and poly(styrene sulfonate). Polystyrene (PS) and poly(ethylene terephthalate) glycol (PETG) were chosen as substrate materials because of the significant differences in the polymer chemistries and in the EOF of channels fabricated in these two plastic materials. The efficacy of the surface modification has been evaluated using XPS and by measuring the EOF mobility. When microchannels prepared in both PS and PETG are modified with PEMs, they demonstrate very similar electroosmotic mobilities. The PEMs are easily fabricated and provide a means for controlling the flow direction and the electroosmotic mobility in the channels. The PEM-coated microchannels have excellent wettability, allowing facile filling of the channels. In addition, the PEMs produce reproducible results and are robust enough to withstand long-term storage.     

Microcontact printing-based fabrication of digital microfluidic devices

Digital microfluidics is a fluid manipulation technique in which discrete droplets are actuated on patterned arrays of electrodes. Although there is great enthusiasm for the application of this technique to chemical and biological assays, development has been hindered by the requirement of clean room fabrication facilities. Here, we present a new fabrication scheme, relying on microcontact printing (microCP), an inexpensive technique that does not require clean room facilities. In microCP, an elastomeric poly(dimethylsiloxane) stamp is used to deposit patterns of self-assembled monolayers onto a substrate. We report three different microCP-based fabrication techniques: (1) selective etching of gold-on-glass substrates; (2) direct printing of a suspension of palladium colloids; and (3) indirect trapping of gold colloids from suspension. In method 1, etched gold electrodes are used for droplet actuation; in methods 2 and 3, colloid patterns are used to seed electroless deposition of copper. We demonstrate, for the first time, that digital microfluidic devices can be formed by microCP and are capable of the full range of digital microfluidics operations: dispensing, merging, motion, and splitting. Devices formed by the most robust of the new techniques were comparable in performance to devices formed by conventional methods, at a fraction of the fabrication time. These new techniques for digital microfluidics device fabrication have the potential to facilitate expansion of this technology to any research group, even those without access to conventional microfabrication tools and facilities.     

Rapid prototyping of thermoset polyester microfluidic devices

This paper presents a simple procedure for the fabrication of thermoset polyester (TPE) microfluidic systems and discusses the properties of the final devices. TPE chips are fabricated in less than 3 h by casting TPE resin directly on a lithographically patterned (SU-8) silicon master. Thorough curing of the devices is obtained through the combined use of ultraviolet light and heat, as both an ultraviolet and a thermal initiator are employed in the resin mixture. Features on the order of micrometers and greater are routinely reproduced using the presented procedure, including complex designs and multilayer features. The surface of TPE was characterized using contact angle measurements and X-ray photoelectron spectroscopy (XPS). Following oxygen plasma treatment, the hydrophilicity of the surface of TPE increases (determined by contact angle measurements) and the proportion of oxygen-containing functional groups also increases (determined by XPS), which indicates a correlated increase in the charge density on the surface. Native TPE microchannels support electroosmotic flow (EOF) toward the cathode, with an average electroosmotic mobility of 1.3 x 10(-4) cm(2) V(-1) s(-1) for a 50-microm square channel (20 mM borate at pH 9); following plasma treatment (5 min at 30 W and 0.3 mbar), EOF is enhanced by a factor of 2. This enhancement of the EOF from plasma treatment is stable for days, with no significant decrease noted during the 5-day period that we monitored. Using plasma-treated TPE microchannels, we demonstrate the separation of a mixture of fluorescein-tagged amino acids (glycine, glutamic acid, aspartic acid). TPE devices are up to 90% transparent (for approximately 2-mm-thick sample) to visible light (400-800 nm). The compatibility of TPE with a wide range of solvents was tested over a 24-h period, and the material performed well with acids, bases, alcohols, cyclohexane, n-heptane, and toluene but not with chlorinated solvents (dichloromethane, chloroform).     

Sample filtration,concentration, and separation integrated on microfluidic devices

Microfabricated devices integrating sample filtration, solid-phase extraction, and chromatographic separation with solvent programming were demonstrated. Filtering of the sample was accomplished at the sample inlet with an array of seven channels each 1 microm deep and 18 microm wide. Sample concentration and separation were performed on channels 5 microm deep and 25 microm wide coated with a C18 phase, and elution was achieved under isocratic, step, or linear gradient conditions. For the solid-phase extraction, signal enhancement factors of 400 over a standard injection of 1.0 s were observed for a 320-s injection. Four polycyclic aromatic compounds were resolved by open channel electrochromatography in under 50 s. Chip operation was unaffected by the presence of the 5-microm silica particles at the filter entrance.     

Investigation of nerve injury through microfluidic devices

Traumatic injuries, both in the central nervous system (CNS) and peripheral nervous system (PNS), can potentially lead to irreversible damage resulting in permanent loss of function. Investigating the complex dynamics involved in these processes may elucidate the biological mechanisms of both nerve degeneration and regeneration, and may potentially lead to the development of new therapies for recovery. A scientific overview on the biological foundations of nerve injury is presented. Differences between nerve regeneration in the central and PNS are discussed. Advances in microtechnology over the past several years have led to the development of invaluable tools that now facilitate investigation of neurobiology at the cellular scale. Microfluidic devices are explored as a means to study nerve injury at the necessary simplification of the cellular level, including those devices aimed at both chemical and physical injury, as well as those that recreate the post-injury environment.     

基于SU-8负胶的微流体器件的制作及研究

采用SU-8结构释放并键合玻片制作了多层结构的微流体芯片,探讨了影响芯片气密性和沟道堵塞的因素,并通过荧光显示验证,提供了一种快速、低成本的塑性微流体器件制作方法.     

Influence of hydrodynamic conditions on quantitative cellular assays in microfluidic systems

This study demonstrates the importance of the hydrodynamic environment in microfluidic systems in quantitative cellular assays using live cells. Commonly applied flow conditions used in microfluidics were evaluated using the quantitative intracellular Ca2+ analysis of Chinese hamster ovary (CHO) cells as a model system. Above certain thresholds of shear stress, hydrodynamically induced intracellular Ca2+ fluxes were observed which mimic the responses induced by chemical stimuli, such as the agonist uridine 5'-triphosphate tris salt (UTP). This effect is of significance given the increasing application of microfluidic devices in high-throughput cellular analysis for biophysical applications and pharmacological screening.