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Microfluidic chip for fast nucleic acid hybridization.

The design and experimental verification of a fast nucleic acid hybridization microchip using the fluidic velocity and strain rate effects was conducted. This hybridization chip was able to increase the hybridization signal 6-fold, reduce non-specific target-probe binding and background noise within 30 min, as compared to conventional hybridization methods, which may take from 4 h to overnight. Excellent correlation between experimental results and simulation analysis was obtained in this study. A detailed study of a newly designed microfluidic chip for enhancing hybridization was conducted. Three different designs of devices were fabricated and tested. Two different lengths of targets, 25-mer oligonucleotide and 1.4 kb ssDNA, were tested in this study. The hybridization efficiency can be improved by introducing velocity and extensional strain rate to the sample. This study demonstrates that the signal in the proposed method exhibits intensities 6-fold higher than those in static conditions. The necessary time for the hybridization process can be reduced from overnight to 30 min using the methods developed in this study. Experimental results also show that the strain rate provides stronger effect on hybridization than that of velocity. Combining hybridization with microfluidic concepts of velocity and strain rate effects may provide additional specificity and efficiency in nucleic acid detection and genomic study. This microfluidic hybridization chip can provide potential application in genomic study.

Algorithms↗

Fabrication of microbial chip using collagen gel microstructure.

A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli(E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.

Collagen↗

A non-invasive analysis method for on-chip spectrophotometric detection using liquid-core waveguiding within a 3D architecture.

The on-chip measurement of absorbing species has proven to be challenging, particularly with respect to the sample pathlengths available in a miniaturised system. This paper demonstrates how the principles of total internal reflection can be utilised to form a liquid-core waveguide along a single microfluidic channel, increasing the sampling pathlength to 5 mm while maintaining a detection volume of < or = 1 microL. This was achieved using the Teflon fluoropolymers PTFE, FEP and AF as cladding for the liquid-core waveguide. In conjunction with a 3D chip architecture, the use of the liquid-core waveguide enables more efficient use of the probing light beam along with easy and effective coupling of the source, microfluidic chip and the detection system. The confirmation that waveguiding was occurring was successfully demonstrated and the subsequent spectrophotometric analysis of crystal violet provided a linear calibration with reproducibility (< 2.4% RSD) and limits of detection (< 1.3 microM), comparable to absorbance measurements made with a standard UV-Vis spectrophotometer.

Journal Article↗

A spectroelectrochemical study on perylene cation radical in polymer microchannel-microelectrode chips.

Polymer microchannel chips (depth 20 microm and width 100 microm) integrated with band electrodes were fabricated by photolithography and imprinting methods, and applied to a spectroelectrochemical study on the cation radical of perylene (Pe). A propylene carbonate solution of Pe was brought into the channel chip by pressure driven flow and Pe was oxidized at the working band electrode (WE) in the channel. Simultaneously, absorption measurements of the solution phase in the downstream side of the electrode (30 microm from WE) were conducted on the basis of space resolved spectroscopy. The decrease in the absorbance of Pe at 438 nm upon electrolysis accompanied an appearance of the absorption band around 538 nm, which was assigned to that of the Pe cation radical. When the perylene solution was introduced to the microchip at a slow flow velocity, the dimer cation radical of Pe was shown to be produced in the channel chip. The formation and disappearance processes of the monomer and dimer cation radicals of Pe in the channel were followed by flow velocity and position dependencies of the absorption spectra.

Journal Article↗

Transport, retention and fluorescent measurement of single biological cells studied in microfluidic chips.

Cellular manipulation and fluorescent measurement were performed on two types of biological cells. First, transport and retention of yeast cells were demonstrated on a glass microfluidic chip, which consists of special U-shaped microstructures. These microstructures have the openings parallel to the liquid flow and weirs perpendicular to the flow. These allow the retention of yeast cells in the U-shaped pocket and drainage of liquid over the weirs. Thereafter, the same chip was used to carry out real-time fluorescent measurement for the cellular changes in single Jurkat T cells. In this case, the Jurkat cells were localized inside the straight portion of a microchannel. Fluorescent imaging on the same, single suspension cell was carried out to study two cellular processes occurring in viable cells, (1) the intracellular conversion of fluorescein diacetate (FDA) to fluorescein; (2) the degradation of an inhibitory protein, IkappaB, as involved in the NF-kappaB signalling pathway. In the former, the increase in fluorescent intensity of single Jurkat T cells (due to fluorescein formation) was measured; whereas in the latter, the decrease in the fluorescent intensity of a single transfected Jurkat cell (due to the degradation of the IkappaB-EGFP fusion protein) was monitored. In addition, we employed a Jurkat cell expressed with IkappaB-EGFP to probe any possible action of an herbal compound, isoliquiritigenin (IQ), on the degradation of IkappaB-EGFP. These examples have demonstrated that Jurkat cells remain viable within microfluidic channels for cellular studies and that the microfluidic chip can facilitate monitoring of cellular changes of biological cells at the single cell level and in the same cell.

Biological Transport↗

Stepwise pattern modification of neuronal network in photo-thermally-etched agarose architecture on multi-electrode array chip for individual-cell-based electrophysiological measurement.

We have developed a procedure for stepwise topographical control of network patterns and neurite connection directions between adjacent living neurons using an individual-cell-based on-chip multi-electrode array (MEA) cell cultivation system with an agarose microchamber (AMC) array. This procedure enables flexible and precise control of the cell positions and easy and flexible control of the pattern modification of connections between the cells in AMCs through stepwise photo-thermal etching in which a portion of the agarose layer on the chip is melted with a 1480 nm infrared laser beam even during cultivation. With adequate laser power and this stepwise procedure, we can fabricate narrow micrometer-order grooves (microchannels) during cultivation in a stepwise manner. Using this procedure, we controlled the direction of elongation of axons and dendrites selectively and confirmed the direction by immunostaining. We also demonstrated electrophysiological one-way transmission of signals among aligned hippocampal neurons in which the directions of the neurite connections were controlled using this stepwise photo-thermal etching procedure. These results demonstrate the potential of full direction control of neurite connections between neurons using stepwise photo-thermal etching to form microchannels one by one in an on-chip AMC/MEA cell cultivation system. We can thus better understand the meaning of neuronal network patterns and connection directions.

Animals↗

On-chip magnetic bead microarray using hydrodynamic focusing in a passive magnetic separator.

Implementing DNA and protein microarrays into lab-on-a-chip systems can be problematic since these are sensitive to heat and strong chemicals. Here, we describe the functionalization of a microchannel with two types of magnetic beads using hydrodynamic focusing combined with a passive magnetic separator with arrays of soft magnetic elements. The soft magnetic elements placed on both sides of the channel are magnetized by a relatively weak applied external magnetic field (21 mT) and provide magnetic field gradients attracting magnetic beads. Flows with two differently functionalized magnetic beads and a separating barrier flow are introduced simultaneously at the two channel sides and the centre of the microfluidic channel, respectively. On-chip experiments with fluorescence labeled beads demonstrate that the two types of beads are captured at each of the channel sidewalls. On-chip hybridization experiments show that the microfluidic systems can be functionalized with two sets of beads carrying different probes that selectively recognize a single base pair mismatch in target DNA. By switching the places of the two types of beads it is shown that the microsystem can be cleaned and functionalized repeatedly with different beads with no cross-talk between experiments.

Magnetics↗

Fabrication and validation of a multi-channel type microfluidic chip for electrokinetic streaming potential devices.

To elaborate on the applicability of the electrokinetic micro power generation, we designed and fabricated the silicon-glass as well as the PDMS-glass microfluidic chips with the unique features of a multi-channel. Besides miniaturizing the device, the key advantage of our microfluidic chip utilization lies in the reduction in water flow rate. Both a distributor and a collector taking the tapered duct geometry are positioned aiming the uniform distribution of water flow into all individual channels of the chip, in which several hundreds of single microchannels are assembled in parallel. A proper methodology is developed accompanying the deep reactive ion etching as well as the anodic bonding, and optimum process conditions necessary for hard and soft micromachining are presented. It has been shown experimentally and theoretically that the silicon-based microchannel leads to increasing streaming potential and higher external current compared to those of the PDMS-based one. A proper comparison between experimental results and theoretical computations allows justification of the validity of our novel devices. It is useful to recognize that a material inducing a higher magnitude of zeta potential has an advantage for obtaining higher power density under the same external resistance.

Algorithms↗

On-chip syringe pumps for picoliter-scale liquid manipulation.

On-chip microsyringes are developed by integrating parallel micro actuators and a microfluidic chip. Sliders of an Electrostatically Controlled Linear Inchworm Actuator (ECLIA) are applied to manipulate microsyringes in the nanometer range, which allows liquid control on the picoliter scale. ECLIA drives sliders in parallel with high accuracy and a large stroke. The requirements for syringe performance, such as parallel and precise liquid control, can be satisfied by the above features of ECLIA. A total volume of a few microL is manipulated at a flow rate of 19-27 pL s(-1) by the stepwise motion of ECLIA sliders in a fluidic channel. Microsyringes integrated into the driving mechanism are a key component of Micro Total Analysis Systems (microTAS) due to the possibility of on-chip integration. In addition, the proposed approach has a significant implication in MEMS in that the electrostatic micro actuator performs a physical task that affects the outside structure.

Journal Article↗

Injection molded microfluidic chips featuring integrated interconnects.

An injection molding process for the fabrication of disposable plastic microfluidic chips with a cycle time of 2 min has been designed, developed, and implemented. Of the sixteen commercially available grades of cyclo-olefin copolymer (COC) that were screened for autofluorescence and transparency to ultraviolet (UV) light, Topas 8007 x 10 was identified as the most suitable for production. A robust solid metal mold insert defining the microfluidic channels was rapidly microfabricated using a process that significantly reduces the time required for electroplating. No wear of the insert was observed even after over 1000 cycles. The chips were bonded by thermal fusion using different bonding conditions. Each condition was tested and its suitability evaluated by burst pressure measurements. The COC microfluidic chips feature novel, integrated, reversible, standardized, ready-to-use interconnects that enable operation at pressures up to 15.6 MPa, the highest value reported to date. The suitability of these UV transparent, high pressure-resistant, disposable devices was demonstrated by in situ preparation of a high surface area porous polymer monolith within the channels.

Microfluidic Analytical Techniques↗

On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing.

Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS-glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids.

Amino Acids↗

Integrated thin-film polymer/fullerene photodetectors for on-chip microfluidic chemiluminescence detection.

We report the use of solution-processed thin-film organic photodiodes for microscale chemiluminescence. The active layer of the photodiodes comprised a 1 : 1 blend by weight of the conjugated polymer poly(3-hexylthiophene) [P3HT] and [6,6]-phenyl-C(61)-butyric acid-methylester [PCBM]--a soluble derivative of C(60). The devices had an active area of 1 mm x 1 mm, and a broad-band response from 350 to 700 nm, with an external quantum efficiency of more than 50% between 450 and 550 nm. The photodiodes have a simple layered structure that permits facile integration with planar chip-based systems. To evaluate the suitability of the organic devices as integrated detectors for microscale chemiluminescence, a peroxyoxalate based chemiluminescence reaction (PO-CL) was monitored within a poly(dimethyl-siloxane) (PDMS) microfluidic device. Quantitation of hydrogen peroxide indicated excellent linearity and yielded a detection limit of 10 microM, comparable with previously reported results using micromachined silicon microfluidic chips with integrated silicon photodiodes. The combination of organic photodiodes with PDMS microfluidic chips offers a means of creating compact, sensitive and potentially low-cost microscale CL devices with wide-ranging applications in chemical and biological analysis and clinical diagnostics.

Dimethylpolysiloxanes↗

Ultrasonic standing wave manipulation technology integrated into a dielectrophoretic chip.

Several cell-based biological applications in microfluidic systems require simultaneous high-throughput and individual handling of cells or other bioparticles. Available chip-based tools for contactless manipulation are designed for either high-precision handling of individual particles, or high-throughput handling of ensembles of particles. In order to simultaneously perform both, we have combined two manipulation technologies based on ultrasonic standing waves (USWs) and dielectrophoresis (DEP) in a microfluidic chip. The principle is based on the competition between long-range ultrasonic forces, short-range dielectrophoretic forces and viscous drag forces from the fluid flow. The ultrasound is coupled into the microchannel resonator by an external transducer with a refractive element placed on top of the chip, thereby allowing transmission light microscopy to continuously monitor the biological process. The DEP manipulation is generated by an electric field between co-planar microelectrodes placed on the bottom surface of the fluid channel. We demonstrate flexible and gentle elementary manipulation functions by the use of USWs and linear or curved DEP deflector elements that can be used in high-throughput biotechnology applications of individual cells.

Electrophoresis, Microchip↗

Equivalent forms and split-half reliability of the NU-CHIPS administered in noise.

The effects of white noise on the equivalent forms reliability and internal consistency reliability of the Northwestern University-Children's Perception of Speech Test (NU-CHIPS) were examined. Subjects were 36 normally hearing 10-year-old children who were assigned randomly in equal numbers to one of three experimental groups. Each group was administered all four forms of the NU-CHIPS at one of three signal-to-noise ratios (S/N = -4, S/N = 0, S/N = +2). The reliability of the NU-CHIPS when presented in noise is diminished relative to its reported reliability when administered in quiet as revealed by Pearson product-moment correlation coefficients.

Child↗

A custom designed chip to control an implantable stimulator and telemetry system for control of paralyzed muscles.

A custom designed chip has been developed for the control of paralyzed muscles. The system is capable of fulfilling the stimulus and telemetry needs of advanced functional neuromuscular stimulation (FNS) applications requiring multiple channels of stimulation and multiple channels for sensor or biopotential sensing. An inductive radiofrequency link provides power to the implant device as well as 2 way transcutaneous communication. An application specific integrated circuit (ASIC) decodes the commands and provides functional control within the implant, and modular circuitry provides specific implant functions. The ASIC chip provides up to 32 independent channels of stimulation with independent control of stimulus pulse duration, pulse amplitude, interphase delay, recharge phase duration, and pulse interval. It can also control up to 8 independent back telemetry analog channels with independent control of sampling rate and pulse powering parameters (amplitude and duration). The mixed analog digital chip has been fabricated in 1.2 microm n-well CMOS technology.

Analog-Digital Conversion↗

Microfabricated polymer analysis chip for optical detection.

A coupling between multimode polymer waveguides and microfluidic channels on a polymethylmethacrylate (PMMA) capillary electrophoresis (CE)-chip for optical analytical applications has been successfully realised. This technology allows the integration of polymer optical waveguides together with hermetically sealed fluidic channels. The microchannels and waveguides are made in PMMA by the approved hot-embossing technology. The technology developed for the fabrication of polymer waveguides on the microfluidic chip offers the possibility of great flexibility in the choice of core materials, design and alignment of the polymer waveguides. The integration of polymer waveguides on an analysis chip enables highly spatially resolved optical detection without the large and expensive conventionally used apparatus. The optical properties of the analytical system developed are verified by transmission and propagation loss measurements. The results of measurements prove the suitability of the presented device for optical applications between 440 and 800 nm. This was shown with absorbance measurements of the dye Sulfanilazochromotrop (SPADNS) within 50 microm fluidic channels.

Journal Article↗

Chip-based electrical detection of DNA.

A variety of methods have been developed for the detection of the binding of the complementary strand of DNA to a gene chip using electrical rather than the established optical signal techniques. Chip-based DNA sensors offer sensitivity, specificity, parallelisation and miniaturisation for the detection of selected DNA sequences or mutated genes associated with human diseases. Problems associated with the established fluorescence-based optical detection technique include the high equipment costs and the need to use sophisticated numerical algorithms to interpret the data. These problems generally limit its use to research laboratories and make it hard to adapt this detection scheme for on-site or point-of-care use. An electrical readout might be a solution to these problems. A review of a number of different approaches to achieve an electrical readout for a DNA chip is presented. The review covers various methods that are based on the use of metal nanoparticles as labels and also electrochemical methods that use polymer-modified electrodes, DNA-specific redox reporters, and DNA-mediated charge transport techniques.

DNA↗

Genotyping of 22 human papillomavirus types by DNA chip in Korean women: comparison with cytologic diagnosis.

OBJECTIVE: More sensitive and reliable methods than individual testing (such as polymerase chain reaction, restriction fragment length polymorphism, and Southern blot) should be developed as screening tools for the detection of latent human papillomavirus. Today, the new Bethesda system recommends human papillomavirus testing as an adjuvant to the conventional Papanicolaou smear for more comprehensive identification of women at certain risk of cervical neoplasia. We performed human papillomavirus genotyping with the newly designed human papillomavirus DNA chip, which is based on polymerase chain reaction for high-throughput screening power, and compared the results with the results of a Papanicolaou smear according to the new Bethesda system. STUDY DESIGN: Polymerase chain reaction amplifications of the human papillomavirus L1 region from biologic samples were hybridized to silanized glass slides by a microarrayer, which comprised 22 specific oligonucleotide probes to their genotypes, consisting of 15 high-risk and 7 low-risk types. Two cervical cancer cell lines and 20 plasmids that contained each type of the human papillomavirus whole genome were used for the evaluation of this method; in all cases, the cancer cell lines and plasmids showed clear positive signals on their corresponding positions. A comparative study that used 685 cervicovaginal swabs was performed by human papillomavirus DNA chip microarray together with Papanicolaou diagnosis. RESULTS: Human papillomavirus was identified as positive in 31.9% of the 414 control samples and in 78.6% of the 271 neoplastic lesions. The major prevailing human papillomavirus genotypes were human papillomavirus types 16, 58, and 18, in descending order of incidence (average overall, 78.8%). Almost all of the remaining cases were comprised of human papillomavirus types 39, 52, 56, and 51. The frequency of multiple infection of human papillomavirus was highest in low-grade squamous intraepithelial lesion but was lowest in squamous cell carcinoma. All cases that exhibited infection of single human papillomavirus type 58 were squamous cell carcinoma. CONCLUSION: Human papillomavirus types 16, 18, and 58 were confirmed to be major causative factors for cervical carcinogenesis. Low-grade squamous intraepithelial lesion is a heterogeneous entity that is composed of different human papillomavirus subtypes and prevails in younger women (<40 years old). The human papillomavirus chip has potential use as a high-throughput screening test.

Adult↗