DNA mutation detection in a polymer microfluidic network using temperature gradient gel electrophoresis

A miniaturized system for DNA mutation analysis, utilizing temperature gradient gel electrophoresis (TGGE) in a polycarbonate (PC) microfluidic device, is reported. TGGE reveals the presence of sequence heterogeneity in a given heteroduplex sample by introducing a thermal denaturing gradient that results in differences between the average electrophoretic mobilities of DNA sequence variants. Bulk heater assemblies are designed and employed to externally generate temperature gradients in spatial and temporal formats along the separation channels. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, are achieved using both single- and 10-channel parallel measurements in a microfluidic platform. Additionally, a comprehensive polymer microfluidic device containing an integrated microheater and sensor array is developed and demonstrated for performing spatial TGGE for DNA mutation analysis. The device consists of two PC modular substrates mechanically bonded together. One substrate is embossed with microchannels, and the other contains a tapered microheater, lithographically patterned along with an array of temperature sensors. Compared with the external heating approaches, the integrated platform provides significant reduction in power requirement and thermal response time while establishing more accurate and highly effective control of the temperature gradient for achieving improved separation resolution.     

Microchannel-nanopore device for bacterial chemotaxis assays

Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in ≤1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels. This design requires no external pump and minimizes the effect of transmembrane pressure, resulting in a stable, reproducible gradient. The microfluidic platform facilitates microscopic observation of individual cell trajectories, and chemotaxis is quantified by monitoring changes in cell swimming behavior in the vicinity of the intersection. We validate this system by measuring the chemotactic response of an aquatic bacterium, Caulobacter crescentus, to xylose concentrations from 1.3 μM to 1.3 M. Additionally, we make an unanticipated observation of increased turn frequency in a chemotaxis-impaired mutant which provides new insight into the chemotaxis pathway in C. crescentus.     

Bilinear temperature gradient focusing in a hybrid PDMS/glass microfluidic chip integrated with planar heaters for generating temperature gradients

Temperature gradient focusing (TGF) is a counterflow gradient focusing technique, which utilizes a temperature gradient across a microchannel or capillary to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in both the electric field and electrophoretic velocity. Combined with a bulk counter flow, ionic species concentrate at a unique point where the total velocity sums to zero and separate from each other. Scanning TGF uses varying bulk flow so that a large number of analytes that have large differences in electrophoretic mobility can be sequentially focused and passed by a single detection point. Up to now, scanning TGF examples have been performed using a linear temperature gradient which has limitations in improving peak capacity and resolution at the same time. In this work, we develop a bilinear temperature gradient along the separation channel that improves both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to preconcentrate the sample followed by a shallow gradient that increases separation resolution. A specialized design is developed for the heaters to achieve the bilinear profile using both analytical and numerical modeling. The heaters are integrated onto a hybrid PDMS/glass chip fabricated using conventional sputtering and soft-lithography techniques. Separation performance is characterized by separating several different dyes and amino acids that have close electrophoretic mobilities. Experiments show a dramatic improvement in peak capacity and resolution in comparison to the standard linear temperature gradient.     

Generating nonlinear concentration gradients in microfluidic devices for cell studies

We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.     

Application of a multivariate curve resolution procedure to the analysis of second-order melting data of synthetic and natural polynucleotides

The melting behavior of several synthetic polynucleotides and a mixture of natural tRNAs was studied by monitoring the changes in the whole UV absorbance spectrum at several pH values. The second-order absorbance data were analyzed with a soft-modeling multivariate curve resolution procedure that allows the determination of the number of different species or conformations present along the melting experiment and the calculation of the melting profile and the pure spectrum for each chemical species or conformation. The melting temperature, T(m), for each thermal transition can be calculated from the melting profiles, and structural information on the different species or conformations can be obtained from their pure spectra. The multistranded species formed at certain pH conditions show several sharp thermal transitions related to the loss of the initial highly ordered structure. For these transitions, the mixture of species obtained in the denaturing process can be resolved when several data matrices, each giving additional information, are analyzed simultaneously with the mathematical procedure proposed.     

Mapping spatiotemporal molecular distributions using a microfluidic array

The spatial and temporal distributions of an extensive number of diffusible molecules drive a variety of complex functions. These molecular distributions often possess length scales on the order of a millimeter or less; therefore, microfluidic devices have become a powerful tool to study the effects of these molecular distributions in both chemical and biological systems. Although there exist a number of studies utilizing microdevices for the creation of molecular gradients, there are few, if any, studies focusing on the measurement of spatial and temporal distributions of molecular species created within the study system itself. Here we present a microfluidic device capable of sampling multiple chemical messengers in a spatiotemporally resolved manner. This device operates through spatial segregation of nanoliter-sized volumes of liquid from a primary sample reservoir into a series of analysis microchannels, where fluid pumping is accomplished via a system of passive microfluidic pumps. Subsequent chemical analysis within each microchannel, achieved via optical or bioanalytical methods, yields quantitative data on the spatial and temporal information for any analytes of interest existing within the sample reservoir. These techniques provide a simple, cost-effective route to measure the spatiotemporal distributions of molecular analytes, where the system can be tailored to study both chemical and biological systems.     

Spatiotemporal Control of Apical and Basal Living Subcellular Chemical Environments Through Vertical Phase Separation

Molecular distribution within living cells is organized through multiscaled compartmentalization that enables specialized processes to occur with high efficiency. The ability to control the chemical environment at a subcellular level is limited due to deficient positional control over the aqueous stimulant. Here, a multilayered microfluidic system built from polydimethylsiloxane to separate chemical stimulants over single living cells vertically through aqueous‐phase separation under laminar flow is demonstrated. Cells are cultured on top of single micrometer‐scale channels inside a larger channel, allowing labeling of the apical domain of single cells through the main channel with simultaneous and distinct labeling of the basal domain via the lower microchannels. The system is transparent, which allows the use of optical microscopy to investigate the spatiotemporal response of labeled components. By employing this technique, the examination of localized subcellular domain responses in polarization, lipid bilayer mobility, and apical‐to‐basal signal transduction can be explored.     

An in-mold packaging process for plastic fluidic devices

Micro or nanofluidic devices have many channel shapes to deliver chemical solutions, body fluids or any fluids. The channels in these devices should be covered to prevent the fluids from overflowing or leaking. A typical method to fabricate an enclosed channel is to bond or weld a cover plate to a channel plate. This solid-to-solid bonding process, however, takes a considerable amount of time for mass production. In this study, a new process for molding a cover layer that can enclose open micro or nanochannels without solid-to-solid bonding is proposed and its feasibility is estimated. First, based on the design of a model microchannel, a brass microchannel master core was machined and a plastic microchannel platform was injection-molded. Using this molded platform, a series of experiments was performed for four process or mold design parameters. Some feasible conditions were successfully found to enclosed channels without filling the microchannels for the injection molding of a cover layer over the plastic microchannel platform. In addition, the bond strength and seal performance were estimated in a comparison with those done by conventional bonding or welding processes.     

Microfluidic device with chemical gradient for single-cell cytotoxicity assays

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.     

Microfluidic temperature gradient focusing

A new technique is described for the concentration and separation of ionic species in solution within microchannels or capillaries. Concentration is achieved by balancing the electrophoretic velocity of an analyte against the bulk flow of solution in the presence of a temperature gradient. With an appropriate buffer, the temperature gradient can generate a corresponding gradient in the electrophoretic velocity, so that the electrophoretic and bulk velocities sum to zero at a unique point, and the analyte will be focused at that point. The technique is demonstrated for a variety of analytes, including fluorescent dyes, amino acids, DNA, proteins, and particles, and is shown to be capable of greater than 10,000-fold concentration of a dilute analyte.     

Review: carbon nanotube for microfluidic lab-on-a-chip application

Microfluidic lab-on-a-chip allows chemical and biochemical analysis to be conducted in a miniaturized system. Miniaturized analysis reduces the reagent consumption while decreasing the overall size of the device, but the small dose of the sample make detection more demanding and is more sensitive to adsorption of species on the surface. Integration of carbon nanotubes into microfludic devices is a promising approach. This review addresses recent advances in the application of carbon nanotubes for microfluidic lab-on-a-chip. The literature review shows that carbon nanotubes have been used to achieve superlubrifying microchannels, act as high density nanoporous membranes, electrical transducers mainly in flow sensors and biosensors, and mimics of living systems. In addition, extensive work has been carried out to investigate the tunable mechanical, chemical and electrical properties of carbon nanotubes in order to manipulate and analyse extremely small volumes of fluid effectively.     

Automated control of local solution environments in open-volume microfluidics

We present an open-volume microfluidic system capable of on-line modification of a patterned laminar flow by using programmable inlet valves. Each separate solution environment in the flow pattern can be independently exchanged between different preloaded input solutions where each exchange requires 20 s. The number of flow patterns that can be generated by one device is N(n), where N represents the number of valve inlets and n the number of microchannels in the microfluidic system. Furthermore, the system can be operated as a combinatorial mixer, in which mixture of the different input solutions can be obtained independently in each channel. Since the flow patterns are generated in an open volume, they are accessible to many different detection methods and types of probes, e.g., microelectrodes, cells, or cell fragments. This technology offers the possibility to adjust the flow pattern composition in response to an output from a probe. This is the first step toward creating an automated feedback device controlled by, for example, biological cells.     

Design and analysis of single-cell capture microfluidic device

The aim of our study was to develop microfluidic devices using microchannel technology with the capability of capturing single cells. We analyzed and compared the cell-capturing efficiencies of series-loop microchannel and parallel-loop microchannel devices that were produced using polydimethylsiloxane (PDMS). Each set of microchannels was composed of a main flow channel and several branch channels with capturing zones. The microfluidic devices were designed to use the differences in flow rates between the main flow channel and the branch channels as a means of capturing single cells based on size and sequestering them within the microstructure of multiple capture zones. The data indicated that the flow medium encountered significant resistance in the series-loop microchannel device, which resulted in an inability to hold the captured cells within any of the capture zones. Flow resistance was, however, greatly reduced in the parallel-loop microchannel device compared to the series-loop device, and single cells were captured in all the capturing zones of the device. Our data suggest that the parallel-loop microchannel technology has significant potential for development toward high-throughput platforms capable of capturing single cells for physiological analyses at the single-cell level.     

Differential phase shift keying direct sequence spread spectrum single SAW based correlator receiver

This paper presents the design and measurement of a SAW device to be used in a correlator receiver for a differential phase shift keying direct sequence spread spectrum (DPSK/DSSS) system. The DPSK modulation format allows noncoherent data demodulation while the SAW device correlator acts as the despreading operator. In a conventional DPSK receiver, the received signal is normally split into a lower and upper path. One of the paths contains a correlator, and the other path contains a one data bit delay element and another correlator. The outputs of both paths are then fed to a noncoherent data demodulator. The device presented in this paper combines both the delay element and the two correlators in a single SAW device; therefore, a better temperature tracking mechanism, simplicity, as well as the elimination of the broadband SAW delay line are achieved. The SAW structure contains a broadband SAW transducer, and two serially coded pseudo noise (PN) DPSK filters. The SAW based correlator was built on lithium tantalate. The center frequency was set to 150 MHz, with a 63 chip PN spreading code and a data rate of 300 Kbps. Experimental measurements of the SAW device autocorrelation results are presented.     

Microfluidics and chromatography with an atomic force microscope

A combined atomic force microscope (AFM) and Raman spectrometer is presented as a microfluidic device for pumping, sampling, and trace chemical analysis. The AFM tip-cantilever provides a mechanism for shear-driven pumping of fluids in microchannels. Shear-driven pumping allows rapid flow rates and avoids the limitations of conventional pumping. The AFM's ability to translate sub-femtoliter volumes of fluid also proves a mechanism for fluidic switching and sample injection. In addition, the AFM is used to image liquid surfaces in microchannels and remove samples for very sensitive spectral analysis. Surface-enhanced Raman spectroscopy localized near the AFM tip provides chemical information of the sampled fluids. The results demonstrate the feasibility of integrating the AFM with microfluidic circuits and shear-driven chromatography and the potential for nanometer-scale chromatography.     

Sprouting angiogenesis under a chemical gradient regulated by interactions with an endothelial monolayer in a microfluidic platform

Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.     

Superlocalization of single molecules and nanoparticles in high-fidelity optical imaging microfluidic devices

Superlocalization of single molecules and nanoparticles with a precision of subnanometer to a few tens of nanometers is crucial for elucidating nanoscale structures and movements in biological and chemical systems. A novel design of ultraflat and ultrathin glass/polydimethylsiloxane (PDMS) hybrid microdevices is introduced to provide almost uncompromised optical imaging quality for on-chip superlocalization and super-resolution imaging of single molecules and nanoparticles under a variety of microscopy modes. The performance of the high-fidelity (Hi-Fi) optical imaging microfluidic device was validated by precisely mapping micronecklaces made of fluorescent microtubules and 40 nm gold nanoparticles and by demonstrating the activation and excitation cycles of single Alexa Fluor 647 dyes for direct stochastic optical reconstruction microscopy in PDMS-based microchannels for the first time. Furthermore, the microdevice's feasibility for multimodality microscopy imaging was demonstrated by a vertical scan of live cells in epi-fluorescence and differential interference contrast (DIC) microscopy modes simultaneously.     

Optical fibre musical instruments: making sense of the senseless

This paper demonstrates how the light transmitted through a stretched optical fibre may be used to detect its modes of vibration. In particular, replacing strings of a musical instrument with optical fibre allows the fabrication of a simple acoustic instrument with a single laser source and single detector. The detected signal contains rich harmonics of the vibrating fibre. This device may be used as a vibration, temperature or strain sensor, or simply as a musical instrument. Coating the optical fibre with novel materials such as PZLT may well allow a modification of vibration properties to enhance, suppress certain harmonics or lead to the development of simple electric field sensors.     

Electrophoretic and dielectrophoretic field gradient technique for separating bioparticles

We describe a new device for separation of complex biological particles and structures exploiting many physical properties of the biolytes. The device adds a new longitudinal gradient feature to insulator dielectrophoresis, extending the technique to separation of complex mixtures in a single channel. The production of stronger local field gradients along a global gradient allows particles to enter, initially transported through the channel by electrophoresis and electroosmosis, and to be isolated according to their characteristic physical properties, including charge, polarizability, deformability, surface charge mobility, dielectric features, and local capacitance. In this work, the separation mechanism is described in terms of the relevant electromechanical principles, and proof-of-principle is demonstrated using various bacteria cells as model systems. The results demonstrate the selectivity of the technique and suggest that it may form the foundation for a versatile and useful tool for separating mixtures of complex biological particles and structures.