Reagent Reservoirs Research Articles

Differentiation of Neural Progenitor Cells in a Microfluidic Chip-Generated Cytokine Gradient

In early embryonic development, spatial gradients of diffusible signaling molecules play important roles in controlling differentiation of cell types or arrays in diverse tissues. Thus, the concentration of exogenous cytokines or growth factors at any given time is crucial to the formation of an enriched population of a desired cell type from primitive stem cells in vitro. Microfluidic technology has proven very useful in the creation of cell-friendly microenvironments. Such techniques are, however, currently limited to a few cell types. Improved versatility is required if these systems are to become practically applicable to stem cells showing various plasticity ranges. Here, we built a microfluidic platform in which cells can be exposed to stable concentration gradients of various signaling molecules for more than a week with only minimal handling and no external power source. To maintain stability of the gradient concentration, the osmotic pumping performance was optimized by balancing the capillary action and hydraulic pressure in the inlet reagent reservoirs. We cultured an enriched population of neural progenitors derived from human embryonic stem cells in our microfluidic chamber for 8 days under continuous cytokine gradients (sonic hedgehog, fibroblast growth factor 8, and bone morphogenetic protein 4). Neural progenitors successfully differentiated into neurons, generating a complex neural network. The average numbers of both neuronal cell body clusters and neurite bundles were directly proportional to sonic hedgehog concentrations in the gradient chip. The system was shown to be useful for both basic and translational research, with straightforward mechanisms and operational schemes.

A low-cost, manufacturable method for fabricating capillary and optical fiber interconnects for microfluidic devices

Microfluidic chips require connections to larger macroscopic components, such as light sources, light detectors, and reagent reservoirs. In this article, we present novel methods for integrating capillaries, optical fibers, and wires with the channels of microfluidic chips. The method consists of forming planar interconnect channels in microfluidic chips and inserting capillaries, optical fibers, or wires into these channels. UV light is manually directed onto the ends of the interconnects using a microscope. UV-curable glue is then allowed to wick to the end of the capillaries, fibers, or wires, where it is cured to form rigid, liquid-tight connections. In a variant of this technique, used with light-guiding capillaries and optical fibers, the UV light is directed into the capillaries or fibers, and the UV-glue is cured by the cone of light emerging from the end of each capillary or fiber. This technique is fully self-aligned, greatly improves both the quality and the manufacturability of the interconnects, and has the potential to enable the fabrication of interconnects in a fully automated fashion. Using these methods, including a semi-automated implementation of the second technique, over 10,000 interconnects have been formed in almost 2000 microfluidic chips made of a variety of rigid materials. The resulting interconnects withstand pressures up to at least 800psi, have unswept volumes estimated to be less than 10 femtoliters, and have dead volumes defined only by the length of the capillary.

Automated 10-channel capillary chip immunodetector for biological agents detection

The automated 10-channel capillary chip immunodetector (10K-IDWG) is a prototype, which has been developed for automatically operated biological agents (BA) point detection. The current technology uses a chemiluminescence capillary immunoassay (EIA) technique in combination with integrated microfluidics and allows the highly sensitive and rapid detection and preliminary identification of multiple BA in aqueous solutions in the laboratory. The chemiluminescence capillary EIA are performed within a disposable capillary chip containing 10 fused-silica capillaries arranged in parallel coated with selected capture antibodies. A multianode-photomultiplier array is used to detect chemiluminescence intensity in each capillary. Reservoirs for reagents and buffers and a waste disposal reservoir are integrated. This paper describes the technology of the 10K-IDWG and its evaluation with three different BA, the toxin staphylococcal enterotoxin B (SEB), the bacterial analyte Escherichia coli (E. coli) O157:H7 as a model for bacterial pathogens, and the bacteriophage M13 as a model for virus pathogens. The 10K-IDWG is able to detect the above mentioned three BA in an aqueous sample within 29 min (single analyte-detection and multiplexing). Limits of detection (LOD) are 0.1 ng/ml for SEB, 10(4)cfu/ml for E. coli O157:H7, and 5x10(5) pfu/ml for M13. Cross reactivities between the three assays were not observed.

Miniature Liquid Flow Sensor and Feedback Control of Electroosmotic and Pneumatic Flows for a Micro Gas Analysis System

Accurate liquid flow control is important in most chemical analyses. In this work, the measurement of liquid flow in microliters per minute was performed, and feedback control of the flow rate was examined. The flow sensor was arranged on a channel made in a polydimethylsiloxane (PDMS) block. The center of the channel was cooled by a miniature Peltier device, and the change in temperature balance along the channel formed by the flow was measured by two temperature sensors. Using this flow sensor, feedback flow control was examined with two pumping methods. One was the electroosmotic flow method, made by applying a high voltage (HV) between the reagent and waste reservoirs; the other was the piezo valve method, in which a micro-valve-seat was fabricated in a PDMS cavity with a silicone diaphragm. The latter was adopted for a micro gas analysis system (microGAS) for measuring atmospheric H2S and SO2. The obtained baselines were stable, and better limits of detection were obtained.

Integrated Bioassays in Microfluidic Devices: Botulinum Toxin Assays

A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

Development of an integrated automation system with a magnetic bead-mediated nucleic acid purification device for genetic analysis and gene manipulation.

We have developed an integrated automation system for genetic analysis and gene manipulation. The system, SX-8G Plus, is equipped with an 8-nozzle dispensing unit, a thermal cycler, a cooled reagent reservoir, four tip storage racks, four microplate platforms, buffer reservoirs, an agarose gel electrophoresis unit, a power supply, a pump for exchanging electrophoresis buffer, and a CCD camera. Automation of nucleic acid extraction and purification, the most difficult step in automating genetic analysis and gene manipulation, was realized using magnetic beads with Magtration Technology, which we have previously developed for automating the handling of paramagnetic beads. Using this system, we could perform the automated separation and purification of DNA fragments by agarose gel electrophoresis starting from sample loading. The system would enable the automation of almost all procedures in genetic analysis and gene manipulation.

Modelling of the enzymatic synthesis of taste dipeptides with simultaneous extraction in a membrane phase contactor

The paper presents a model of the synthesis process of taste dipeptides carried out in a biphase water-organic phase system. An enzymatic conversion takes place within the aqueous phase where the enzyme is dissolved. The catalysis process is accompanied by the transport of reagents from/to the organic phase, which is a reservoir of hydrophobic reagents. The membrane contactor with a porous membrane holds the interface surface that serves as an interface area to the reactant mass transfer. The model was verified on the basis of the synthesis reaction of ZAlaPheOMe, a precursor of bitter dipeptide. The process was carried out in the ethyl acetate-polypropylene membrane-0.05 M Tris-HCl buffer system. Good agreement of experimental and model results was presented that enabled computer simulations of the process. That allowed us to choose the optimum parameters used in the batch and continuous process. The influence of interface surface area, phases volume and reagent concentrations on the process course, productivity and product purity was shown.

Transverse instabilities in chemical Turing patterns of stripes.

We present a theoretical and experimental study of the sideband instabilities in Turing patterns of stripes. We compare numerical computations of the Brusselator model with experiments in a chlorine dioxide-iodine-malonic acid (CDIMA) reaction in a thin gel layer reactor in contact with a continuously refreshed reservoir of reagents. Spontaneously evolving Turing structures in both systems typically exhibit many defects that break the symmetry of the pattern. Therefore, the study of sideband instabilities requires a method of forcing perfect, spatially periodic Turing patterns with the desired wave number. This is easily achieved in numerical simulations. In experiments, the photosensitivity of the CDIMA reaction permits control and modulation of Turing structures by periodic spatial illumination with a wave number outside the stability region. When a too big wave number is imposed on the pattern, the Eckhaus instability may arise, while for too small wave numbers an instability sets in forming zigzags. By means of the amplitude equation formalism we show that, close to the hexagon-stripe transitions, these sideband instabilities may be preceded by an amplitude instability that grows transient spots locally before reconnecting with stripes. This prediction is tested in both the reaction-diffusion model and the experiment.

Direct tris(2,2'-bipyridyl)ruthenium (II) electrochemiluminescence detection of polyamines separated by capillary electrophoresis.

Capillary electrophoresis (CE) with tris(2,2'-bipyridyl) ruthenium (II) (Ru(bpy)3(2+)) electrochemiluminescence (ECL) detection technique was developed for the analysis of four polyamines (putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)) analysis. The four polyamines contain different amine groups, which have different ECL activity. There are several parameters which influence the resolution and ECL peak intensities, including the buffer pH and concentrations, separation voltage, sample injection, electrode materials, and Ru(bpy)3(2+) concentrations. Polyamines are separated by capillary zone electrophoresis in an uncoated fused-silica capillary (50 cmx25 micro m (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 micro A), with end-column Ru(bpy)3(2+) ECL detection. A 5 mmol/L Ru(bpy)3(2+) solution plus 200 mmol/L phosphate buffer (pH 11.0) is added into the reagent reservoir. The calibration curve is linear over a concentration range of two or three orders of magnitude for the polyamines. The analysis time is less than 25 min. Detection limits for Put and Cad are 1.9x10(-7) mol/L and 7.6x10(-9) mol/L for Spd and Spm, respectively. Intraday and interday relative standard deviations of ECL peak intensities are less than 8%. The main advantages of this CE-ECL detection technique for polyamines analysis presented herein are the omission of chemical derivatization of the analytes and the high selectivity.

Monitoring of chemical reactions within microreactors using an inverted Raman microscopic spectrometer.

An inverted Raman microscope spectrometer has been used to profile the spatial evolution of reactant and product concentrations for a chemical reaction within a microreactor operating under hydrodynamic flow control. The Raman spectrometer was equipped with a laser source at wavelength of 780 nm, confocal optics, a holographic transmission grating, and a charge-coupled device (CCD) detector. The microreactor consisted of a T-shaped channel network etched within a 0.5 mm thick glass bottom plate that was thermally bonded to a 0.5 mm thick glass top plate. The ends of the channel network were connected to reagent reservoirs that were linked to a syringe pump for driving the solutions by hydrodynamic pumping within the channels. The microchannels were 221 micro m wide and 73 micro m deep. The synthesis of ethyl acetate from ethanol and acetic acid was investigated as a model system within the microreactor as Raman scattering bands for each reactant and product species were clearly resolved. Raman spectral intensities of each band were proportional to concentration for each species and hence all concentrations could be quantitatively measured after calibration. By scanning specific Raman bands within a selected area in the microchannel network at given steps in the X-Y plane, spatially resolved concentration profiles were obtained under steady-state flow conditions. Under the flow conditions used, different positions within the concentration profile correspond to different times after contact and mixing of the reagents, thereby enabling one to observe the time dependence of the product formation. Raman microscopy provides a useful complementary technique to UV/VIS absorbance and fluorescence methods for the in situ monitoring and analysis of chemical reaction species having their lowest S(0)-S(1) absorption bands too far in the UV to be of use, due to their probable overlap with the bands from other reactant, product and solvent molecules.

A fiber-optic fluorescence sensor for lithium ion in acetonitrile.

A novel fiber-optic fluorescence sensor based on a controlled-release reagent for the determination of lithium ion in organic solvents is proposed. The fluorogenic indicator 2-(2-hydroxyphenyl)benzoxazole is contained in a mini-polyethylene tube as the reagent reservoir and is brought into contact with the analyte solution by diffusion across a poly(vinyl chloride) (PVC) membrane to form a strongly fluorescent complex at the membrane/solution interface. The fluorescence signals produced are measured via two joined optical fibers positioned closely to the backside of the PVC membrane for light illumination and collection. The sensor is useful for measuring Li+ at concentrations in acetonitrile ranging from 1.0 x 10(-6) to 1.0 x 10(-2) M with a detection limit of 3.0 x 10(-7) M. The steady-state response can be reached within seconds, and the signal changes are fully reversible. The sensor shows minimal interference effects from other alkali metal and alkaline earth metal cations and has good stability and durability when stored in acetonitrile solutions.

Semiautomation of Nucleic Acid-based Assays for Chlamydia trachomatis and Neisseria gonorrhoeae

The US prevalence rates of sexually transmitted diseases associated with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are as high as 27% (1) and 56% (2), respectively. Routine screening for both conditions is recommended for several risk groups, and infections with CT and GC frequently coexist (3). Because both infections are often asymptomatic, screening is important in efforts to slow the spread of infection. Recent studies compared amplified with nonamplified testing (4)(5)(6)(7)(8)(9)(10) and swab with urine specimens (4)(8)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Because recommendations vary (21)(22)(23)(24)(25), each laboratory must decide which test(s) to use by considering the prevalence of infection, assay sensitivity and specificity, cost-effectiveness, laboratory work flow, and types of specimens received. Several tests may be required to best serve customers with different needs. We developed a menu of semiautomated tests, including amplified CT, nonamplified CT, and nonamplified GC assays. We describe two novel menu choices here: (a) a combination of the nonamplified PACE® 2 GC assay (Gen-Probe, Inc.) with the AMPLIFIEDTM Chlamydia Trachomatis Assay (AMP CT; Gen-Probe, Inc.) for swab specimens; and (b) AMP CT for urine specimens. We used the TECAN GENESIS RSP instrument (1.426 m × 0.780 m × 1.200 m; TECAN US, Inc.). It has an eight-tip arm and uses 1-mL, extended length, disposable, filter pipette tips with level-sensing capability. We designed and machined custom deck plates to support the necessary specimen racks, assay racks, and reagent reservoirs. With approval from the Institutional Review Board, four clinical sites collected urogenital swab specimens using the standard PACE 2 specimen collection method, and two clinical sites collected …

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

The synthesis of peptides using micro reactors

The synthesis of peptides has been successfully performed using a borosilicate glass micro reactor, in which a network of channels has been produced using a photolithographic and wet etching method. The reagents were mobilised by electroosmotic flow (EOF) and electrophoretic mobility. In addition, microporous silica frits were used in the micro reactor channels, these not only aided the flow mechanism but also removed the problem of hydrodynamic pressure within the reagent reservoirs.

The generation of concentration gradients using electroosmotic flow in micro reactors allowing stereoselective chemical synthesis.

The stereoselective control of chemical reactions has been achieved by applying electrical fields in a micro reactor generating controlled concentration gradients of the reagent streams. The chemistry based upon well-established Wittig synthesis was carried out in a micro reactor device fabricated in borosilicate glass using photolithographic and wet etching techniques. The selectivity of the cis (Z) to trans (E) isomeric ratio in the product synthesised was controlled by varying the applied voltages to the reagent reservoirs within the micro reactor. This subsequently altered the relative reagent concentrations within the device resulting in Z/E ratios in the range 0.57-5.21. By comparison, a traditional batch method based on the same reaction length, concentration, solvent and stoichiometry (i.e., 1.0:1.5:1.0 reagent ratios) gave a Z/E in the range 2.8-3.0. However, when the stoichiometric ratios were varied up to ten times as much, the Z/E ratios varied in accordance to the micro reactor i.e., when the aldehyde is in excess, the Z isomer predominates whereas when the aldehyde is in low concentrations, the E isomer is the more favourable form. Thus indicating that localised concentration gradients generated by careful flow control due to the diffusion limited non-turbulent mixing regime within a micro reactor, leads to the observed stereo selectivity for the cis and trans isomers.

Imaging Microelectrodes with High-Frequency Electrogenerated Chemiluminescence

The chemiluminescence arising from reaction of electrogenerated intermediates of 9,10-diphenylanthracene (DPA) has been used to generate images of microelectrodes with dimensions in the micrometer range. The experimental conditions were optimized to ensure high luminescent intensity with sharp focusing of the reaction zone to enable good optical resolution. The solution employed, benzonitrile (BN) containing 0.1 M tetrabutylammonium hexafluorophosphate, promotes high intensity because it enables dissolution of a high concentration (>25 mM) of DPA. In addition, radical anions of BN can serve as a reagent reservoir to ensure efficient reaction of DPA radical cations to the singlet excited state. Under these conditions the measured intensity was 3.2 × 105 photons/s per μm2 of electrode area with a 1 kHz square-wave excitation. Lateral resolution is controlled by the use of rapid potential pulses that maintain the reaction zone in close proximity to the electrode. The images reveal that the electrode areas ha...

Transition from a uniform state to hexagonal and striped Turing patterns

CHEMICAL travelling waves have been studied experimentally for more than two decades1–5, but the stationary patterns predicted by Turing6 in 1952 were observed only recently7–9, as patterns localized along a band in a gel reactor containing a concentration gradient in reagents. The observations are consistent with a mathematical model for their geometry of reactor10 (see also ref. 11). Here we report the observation of extended (quasi-two-dimensional) Turing patterns and of a Turing bifurcation—a transition, as a control parameter is varied, from a spatially uniform state to a patterned state. These patterns form spontaneously in a thin disc-shaped gel in contact with a reservoir of reagents of the chlorite–iodide–malonic acid reaction12. Figure 1 shows examples of the hexagonal, striped and mixed patterns that can occur. Turing patterns have similarities to hydrodynamic patterns (see, for example, ref. 13), but are of particular interest because they possess an intrinsic wavelength and have a possible relationship to biological patterns14–17.

Pulsed reagent introduction through a membrane reactor for flow-injection systems

A method of intermittent reagent introduction, functionally equivalent to stop-go pumping, is described. A pneumatically pressurized reagent reservoir is used to deliver liquid through a high-speed on/off valve to the outer channel of an annular tube assembly. Except for this single entry point, the outer channel is otherwise sealed. The inner flow channel bearing the principal flow stream contains a short microporous membrane tube. When the reagent delivery valve is activated, reagent flows radially inward through the membrane into the principal flow stream.

An enzyme-linked immunosorbent assay method for the simultaneous measurement of antibody titer to multiple viral, bacterial or protein antigens

A model enzyme-linked immunosorbent assay (ELISA) system was developed which permits the user to evaluate replicate single sera dilutions of 46 test and control serum samples for the presence of specific antibody against up to eight different antigens in one assay. The system makes use of a transplating device for the simultaneous transfer of aliquots of independently diluted replicate test samples along with conjugate and substrate from a serum reservoir plate or from a reagent reservoir onto different antigen coated target plates. Kinetically read, raw absorbance data from tests are transmitted to a microcomputer, where absorbance values from all tests are quickly reduced to predicted titer levels by computer analysis. All titer computations involve the use of a single prescribed standard curve for predicting antibody titer against the different antigens. Between assay repeatability of this procedure is high and its potential for replacing a number of different conventional assays for epidemiological studies has been evaluated.