Development of an ultra-micro sample injector for gas chromatography using an ink-jet microchip.

An ultra-micro sample injector for gas chromatography (GC) was developed. An ink-jet microchip, originally used for industrial recorder, was modified at the edge near to an orifice, and fixed into the GC. In order to evaluate the characteristics of this injector, a sample injector and a thermal conductive detector (TCD) were connected directly, while water was used as the test sample. The volume of the droplet, the interval time and the back-pressure to the ink-jet microchip were investigated. Within the range of 1 - 5 nL volume injected sample, the TCD response according to the amount of the sample volume (the volume of one droplet from the ink-jet microchip was about 1 nL) was obtained. A good reproducibility of the peak area was obtained to be about 1.0% of the RSD value. In order to compare the injection method of the ink-jet chip with that using a micro-syringe, the method using the ink-jet chip could introduce 1/1000 of the amount of the sample and gave reproducible results.     

Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses

In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.     

Membraneless vanadium redox fuel cell using laminar flow

This paper describes the design and characterization of a small, membraneless redox fuel cell. The smallest channel dimensions of the cell were 2 mm x 50 mum or x 200 mum; the cell was fabricated in poly(dimethylsiloxane) using soft lithography. This all-vanadium fuel cell took advantage of laminar flow to obviate the need for a membrane to separate the solutions of oxidizing and reducing components.     

A timed solenoid injector for flow analysis

Samples can be reproducibly injected into flow-streams by timed switching of a sample stream with a miniature solenoid valve and timer circuit. The device is simpler to assemble and use than the standard rotary valve and a direct comparison under the same operating conditions shows that the solenoid valve is an adequate replacement for the rotary valve.     

Analysis of intercellular calcium signaling using microfluidic adjustable laminar flow for localized chemical stimulation

The propagation of intercellular calcium signals provides a mechanism to coordinate cell population activity, which is essential for regulating cell behavior and organ development. However, existing analytical methods are difficult to realize localized chemical stimulation of a single cell among a population of cells that are in close contact with one another for studying the propagation of calcium wave. In this work, a microfluidic method is presented for the analysis of contact-dependent propagation of intercellular calcium wave induced by extracellular ATP using multiple laminar flows. Adjacent cells were seeded ∼300 μm downstream the intersection of a Y-shaped microchannel with negative pressure pulses. Consequently, the lateral diffusion distance of the chemical at cell locations was limited to ∼26 μm with a total flow rate of 20 μL min⁻¹, which prevented the interference of diffusion-induced cellular responses. Localized stimulation of the target cell with ATP induced the propagation of intercellular calcium wave among the cell population. In addition, studies on the spread of intercellular calcium wave under octanol inhibition allowed us to characterize the gap junction mediated cell–cell communication. Thus, this novel device will provide a versatile platform for intercellular signal transduction studies and high throughput drug screening.     

Pipette-friendly laminar flow patterning for cell-based assays

Laminar flow patterning (LFP) is a characteristic method of microfluidic systems that allows two (or more) different solutions to flow side-by-side in a channel without convective mixing. This fluid behavior can be used to pattern cell suspensions, particles, and treatments as well as to create chemical gradients. LFP is typically implemented using syringe pumps and, for this reason, is most effective in constant flow scenarios such as long-term gradient generation. However, the complexity of using syringe pumps for patterning cell suspensions typically makes it a less attractive option than other standard patterning methods. We present a passive microfluidic method that enables short-term LFP of multiple fluids using a single pipette and allows each sample to be loaded in any sequence, at any point in time relative to one another. The proposed method is well-suited for cell-based assays, reduces the complexity of LFP to be on a similar level as other cell patterning methods, can be scaled to include more than two streams of fluid, and enables arrays of individually addressable devices for LFP on a single chip.     

A clinical trial for therapeutic drug monitoring using microchip-based fluorescence polarization immunoassay

Microchip analysis is a promising method for therapeutic drug monitoring. This led us to evaluate a microchip-based fluorescence polarization immunoassay (FPIA) system for point-of-care testing on patients being treated with theophylline. The sera were collected from 20 patients being treated with theophylline. Fluorescence polarization was measured on the microchip and theophylline concentrations in serum were obtained. Regression analysis of the correlations was done between the results given by the microchip-based FPIA and the conventional cloned enzyme donor immunoassay (CEDIA), and between the results given by the microchip-based FPIA and the conventional particle-enhanced turbidimetric inhibition immunoassay (PETINIA). We successfully carried out a quantitative analysis of theophylline in serum at values near its therapeutic range in 65 s. The results obtained by the microchip-based FPIA correlated well with CEDIA and PETINIA results; the correlation coefficients (R ²) were 0.986 and 0.989, respectively. The FPIA system is a simple and rapid method for point-of-care testing of drugs in serum, and its accuracy is the same as the conventional CEDIA and PETINIA. It is essential to use real samples from patients and to confirm good correlations with conventional methods for a study on the realization of microchip.     

Laser-patterned stem-cell bridges in a cardiac muscle model for on-chip electrical conductivity analyses

Following myocardial infarction there is an irreversible loss of cardiomyocytes that results in the alteration of electrical propagation in the heart. Restoration of functional electrical properties of the damaged heart muscle is essential to recover from the infarction. While there are a few reports that demonstrate that fibroblasts can form junctions that transmit electrical signals, a potential alternative using the injection of stem cells has emerged as a promising cellular therapy; however, stem-cell electrical conductivity within the cardiac muscle fiber is unknown. In this study, an in vitro cardiac muscle model was established on an MEA-based biochip with multiple cardiomyocytes that mimic cardiac tissue structure. Using a laser beam, stem cells were inserted adjacent to each muscle fiber (cell bridge model) and allowed to form cell-cell contact as determined by the formation of gap junctions. The electrical conductivity of stem cells was assessed and compared with the electrical conductivities of cardiomyocytes and fibroblasts. Results showed that stem cell-myocyte contacts exhibited higher and more stable conduction velocities than myocyte-fibroblast contacts, which indicated that stem cells have higher electrical compatibility with native cardiac muscle fibers than cardiac fibroblasts.     

Investigation of heterogeneous electrochemical processes using multi-stream laminar flow in a microchannel

A multi-component microfluidic electrochemical cell is shown to be a useful analytical tool for probing complex coupled processes in electrolytic systems. We recently reported an enzymatic signal amplification phenomenon that may provide increased sensitivity when detecting bio-analytes (M. S. Hasenbank, E. Fu and P. Yager, Langmuir, 2006, 22, 7451-7453), but to fully harness this method requires an improved understanding of the underlying electrochemical and chemical processes. We use spatial control of electrolyte streams on patterned conductive substrates in a microfluidic platform to elucidate the coupling of homogeneous chemical steps to heterogeneous electrochemical charge transfer processes. Because the gold surface was observable using SPR imaging, electrochemical phenomena could be monitored optically in real time. Based on these and additional results, we propose a mechanism for the novel amplification phenomenon that involves direct electron transfer between surface-immobilized enzyme molecules and the gold surface. This improved understanding of the underlying mechanism should enable the future implementation of this phenomenon in signal amplification schemes for highly sensitive lab-on-a-chip biosensors.     

Determination of aminoglycoside antibiotics using an on-chip microfluidic device with chemiluminescence detection

We describe an on-chip microflow injection (μFI) approach for the determination of aminoglycoside antibiotics using chemiluminescence (CL) detection. The method is based on the inhibition of the Cu(II)-catalyzed CL reaction of luminol and hydrogen peroxide by the aminoglycosides due to the formation of a complex between the antibiotic and Cu(II). The main features of the method include small sample volumes and a fast response. Syringe pumps were used to insert the sample and the reagents into the microfluidic device. CL was collected using a fiber optic bundle connected to a luminescence detector. All instrumental, hydrodynamic and chemical variables involved in the system were optimized using neomycin as the aminoglycoside model. Inhibition is proportional to the concentration of the antibiotics. The dynamic ranges of the calibration graphs obtained for neomycin, streptomycin and amikacin are 0.3–3.3, 0.9–13.7, and 0.8–8.5?μmol?L^?1, and the detection limits are 0.09, 0.28 and 0.24?μmol?L^?1, respectively. The precision of the methods, expressed as relative standard deviation, is in the range from 0.8 to 5.0?%. The method was successfully applied to the determination of neomycin in water samples, with recoveries ranging from 80 to 120?%.

Use of laminar flow patterning for miniaturised biochemical assays

Laminar flow in microfluidic chambers was used to construct low (one dimensional) density arrays suitable for miniaturized biochemical assays. By varying the ratio of flows of two guiding streams flanking a sample stream, precise focusing and positioning of the latter was achieved, and reactive species carried in the sample stream were deposited on functionalized chip surfaces as discrete 50 microm wide lanes. Using different model systems we have confirmed the method's suitability for qualitative screening and quantification tasks in receptor-ligand assays, recording biotin-streptavidin interactions, DNA-hybridization and DNA-triplex formation. The system is simple, fast, reproducible, flexible, and has small sample requirements.     

Versatile control of multiphase laminar flow for in-channel microfabrication

We have improved the multiphase laminar flow based in-channel fabrication method to overcome diffusion-induced broadening. A sheathing phase with protecting molecules confines metal wire deposition and allows for flexible control of the location, width, and uniformity of deposited metal wires. Two-layered T-junctions are introduced to form vertically stacked multiphase laminar flow. Combining these techniques, we fabricate quadrupole silver electrodes on the four sidewalls of rectangular polydimethylsiloxane (PDMS) microchannels that are 3 cm in length.     

Product selectivity control induced by using liquid-liquid parallel laminar flow in a microreactor

Product selectivity control based on a liquid-liquid parallel laminar flow has been successfully demonstrated by using a microreactor. Our electrochemical microreactor system enables regioselective cross-coupling reaction of aldehyde with allylic chloride via chemoselective cathodic reduction of substrate by the combined use of suitable flow mode and corresponding cathode material. The formation of liquid-liquid parallel laminar flow in the microreactor was supported by the estimation of benzaldehyde diffusion coefficient and computational fluid dynamics simulation. The diffusion coefficient for benzaldehyde in Bu(4)NClO(4)-HMPA medium was determined to be 1.32 × 10(-7) cm(2) s(-1) by electrochemical measurements, and the flow simulation using this value revealed the formation of clear concentration gradient of benzaldehyde in the microreactor channel over a specific channel length. In addition, the necessity of the liquid-liquid parallel laminar flow was confirmed by flow mode experiments.     

Development and characterization of on-chip biopolymer membranes

In lab-on-a-chip applications, filtration is currently performed prior to sample loading or through pre-cast membranes adhered to the substrate. These membranes cannot be patterned to micrometer resolution, and their adhesion may be incompatible with the fabrication process or may introduce contaminants. We have developed an on-chip separation process using a biocompatible polymer that can be patterned and has controllable molecular rejection properties. We spun cast cellulose acetate (CA) membranes directly onto silicon wafers. Characterization of the molecular flux across the membrane showed that molecular weight and charge are major factors contributing to the membranes' rejection characteristics. Altering casting conditions such as polymer concentration in the casting solution and the quenching-bath composition and/or temperature allowed control of the molecular weight cut-off (MWCO). Three MWCOs; 300, 350, and 700 Da have been achieved for non-linear molecules. Molecular shape is also very important as much higher molecular weight single-stranded DNA was electrophoresed across the membranes while heme with a similar negative charge density was rejected. This was due to DNA's small molecular cross section. This is an important result because heme inhibits polymerase chain reactions (PCR) reducing the detection and characterization of DNA from blood samples.     

Thousandfold signal increase using field-amplified sample stacking for on-chip electrophoresis

Field-amplified sample stacking (FASS) leverages conductivity gradients between a volume of injected sample and the background buffer to increase sample concentration. A major challenge in applying FASS to on-chip assays is the initial setup of high-conductivity gradient boundaries in the region of the injected sample volume. We have designed, fabricated, and characterized a novel FASS-capillary electrophoresis (CE) chip design that uses a photoinitiated porous polymer structure to facilitate sample injection and flow control for high-gradient FASS. This polymer structure provides a region of high flow resistance that allows the electromigration of sample ions. We have demonstrated an electropherogram signal increase by a factor of 1100 in electrophoretic separations of fluorescein and Bodipy with, respectively, 2 microM and 1 microM initial concentrations.     

A microchip-based flow injection-amperometry system with mercaptopropionic acid modified electroless gold microelectrode for the selective determination of dopamine

A novel chip-based flow injection analysis (FIA) system has been developed for automatic, rapid and selective determination of dopamine (DA) in the presence of ascorbic acid (AA). The system is composed of a polycarbonate (PC) microfluidic chip with an electrochemical detector (ED), a gravity pump, and an automatic sample loading and injection unit. The selectivity of the ED was improved by modification of the gold working microelectrode, which was fabricated on the PC chip by UV-directed electroless gold plating, with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA). Postplating treatment methods for cleaning the surface of electroless gold microelectrodes were investigated to ensure the formation of high quality SAMs. The effects of detection potential, flow rate, and sampling volume on the performance of the chip-based FIA system were studied. Under optimum conditions, a detection limit of 74 nmol L⁻¹ for DA was achieved at the sample throughput rate of 180 h⁻¹. A RSD of 0.9% for peak heights was observed for 19 runs of a 100 μmol L⁻¹ DA solution. Interference-free determination of DA could be conducted if the concentration ratio of AA–DA was no more than 10.     

A novel electrosynthetic system for anodic substitution reactions by using parallel laminar flow in a microflow reactor

We have developed a novel electrosynthetic system for anodic substitution reactions by using parallel laminar flow in a microflow reactor. This system enables nucleophilic reactions to overcome the restraint, such as the oxidation potential of nucleophiles and the stability of cationic intermediates, by the combined use of ionic liquids as reaction media and the parallel laminar flow in the microflow reactor. By using this novel electrosynthetic system, the anodic substitution reaction of carbamates, especially of cyclic carbamates, with allyltrimethylsilane were carried out to provide the corresponding products in moderate to good conversion yields in a single flow-through operation at ambient temperature (without the need for low-temperature conditions).     

Optimization of flow-through cells for dialysis and other membrane separations in flow-injection analysis using a laminar flow model

The mass transfer in the liquid channels of flow-through dialysers can be described fairly accurately by a laminar flow model. The mass transfer resistance in the liquid channels was of the same magnitude as that of a cellulose acetate membrane for the most effective dialysis cell. Thin channels are therefore of importance both for efficiency and for low dispersion in flow injection. A cell with a pressed membrane support provided the lowest dispersion and pressure dependence. A computer-based model provides a means for the determination of membrane permeabilities.     

Light-actuated high pressure-resisting microvalve for on-chip flow control based on thermo-responsive nanostructured polymer

A simple light-actuated microvalve using a quartz halogen illuminator with tungsten filament was introduced to manipulate flow path effectively in micro-total analysis systems, which reduces system complexity and the need for on-chip integration. The microvalve device in cyclic olefin copolymer (COC) microchip functions very well based on the thermo-responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), whose pressure-tolerance can be tuned by changing the mechanical strength of polymer monolith inside the microchannel with the choice of suitable amount of monomer and crosslinker. The response time and pressure resistance of the valve can be optimized by the tetrahydrofuran composition in the polymerization mixture as well. Very importantly, the microvalve can withstand the leakage pressure up to around 1350 psi, and its opening and closing response time is only 4.0 and 6.2 s respectively. Microchips with such valves will be very useful in drug delivery, chemical analysis and proteomic analysis.     

Pumping-induced perturbation of flow in microfluidic channels and its implications for on-chip cell culture

We study the rate of response to changes in the rate of flow and the perturbations in flow in polydimethylsiloxane (PDMS) microfluidic chips that are subjected to several common flow-control systems. We find that the flow rate of liquid delivered from a syringe pump equipped with a glass syringe responds faster to the changes in the conditions of flow than the same liquid delivered from a plastic syringe; and the rate of flow delivered from compressed air responds faster than that from a glass syringe. We discover that the rate of flow that is driven by a syringe pump and regulated by an integrated pneumatic valve responds even faster, but this flow-control method is characterized by large perturbations. We also examine the possible effects of these large perturbations on NIH 3T3 cells in microfluidic channels and find that they could cause the detachment of NIH 3T3 cells in the microchannels.