Biophysical measurement of red blood cells by laboratory on print circuit board chip

Microfluidic impedance pulse sensor has emerged as an easily handled and low‐cost platform in the electrical analysis of biological cells. In the conventional method, impedance sensor demanded expensive patterning metal electrodes on the substrate, which are directly in touch with electrolytes in order to measure the microfluidic channel impedance change. In this article, a cost‐effective microfluidic impedance sensor built upon a dielectric film coated printed circuit board is introduced. Impedance electrodes are protected by a dielectric film layer from electrochemical erosion between electrodes and electrolyte. Human red blood cells from adult and neonatal were utilized to demonstrate the feasibility of the proposed device in the electroanalysis of biological cells.     

A cell electrofusion microfluidic chip using discrete coplanar vertical sidewall microelectrodes

A novel cell electrofusion microfluidic chip using discrete coplanar vertical sidewall electrodes has been designed, fabricated, and tested. The device contains a serpentine-shaped microchannel with 22 500 pairs of vertical sidewall microelectrodes patterned on two opposing vertical sidewalls of the microchannel. The adjacent microelectrodes on each sidewall are separated by coplanar SiO(2) -Polysilicon-SiO(2) /silicon. This design of coplanar discrete vertical sidewall electrodes eliminates the "dead area" present in previous designs using continuous three-dimensional (3D) protruding sidewall electrodes, and generates uniform electric field along the height of the microchannel, leading to a lower voltage required for cell fusion compared to designs using 2D thin-film electrodes. This device is tested to fuse NIH3T3 cells under a low voltage (～9 V). Almost 100% cells are aligned to the edge of the discrete microelectrodes, and cell-cell pairing efficiency reaches 70%. The electrofusion efficiency is above 40% of the total cells loaded into the device, which is much higher than traditional fusion methods and existing microfluidic devices using continuous 3D protruding sidewall microelectrodes.     

Characterization of electrode alignment for optimal droplet charging and actuation in droplet‐based microfluidic system

The actuation method using electric force as a driving force is utilized widely in droplet‐based microfluidic systems. In this work, the effects of charging electrode alignment on direct charging of a droplet on electrified electrodes and a subsequent electrophoretic control of the droplet are investigated. The charging characteristics of a droplet according to different electrode alignments are quantitatively examined through experiments and systematic numerical simulations with varying distances and angles between the two electrodes. The droplet charge acquired from the electrified electrode is directly proportional to the distance and barely affected by the angle between the two electrodes. This implies that the primary consideration of electrode alignment in microfluidic devices is the distance between electrodes and the insignificant effect of angle provides a great degree of freedom in designing such devices. Not only the droplet charge acquired from the electrode but also the force exerted on the droplet is analyzed. Finally, the implications and design guidance for microfluidic systems are discussed with an electrophoresis of a charged droplet method‐based digital microfluidic device.     

Inherently aligned microfluidic electrodes composed of liquid metal

This paper describes the fabrication and characterization of microelectrodes that are inherently aligned with microfluidic channels and in direct contact with the fluid in the channels. Injecting low melting point alloys, such as eutectic gallium indium (EGaIn), into microchannels at room temperature (or just above room temperature) offers a simple way to fabricate microelectrodes. The channels that define the shape and position of the microelectrodes are fabricated simultaneously with other microfluidic channels (i.e., those used to manipulate fluids) in a single step; consequently, all of the components are inherently aligned. In contrast, conventional techniques require multiple fabrication steps and registration (i.e., alignment of the electrodes with the microfluidic channels), which are technically challenging. The distinguishing characteristic of this work is that the electrodes are in direct contact with the fluid in the microfluidic channel, which is useful for a number of applications such as electrophoresis. Periodic posts between the microelectrodes and the microfluidic channel prevent the liquid metal from entering the microfluidic channel during injection. A thin oxide skin that forms rapidly and spontaneously on the surface of the metal stabilizes mechanically the otherwise low viscosity, high surface tension fluid within the channel. Moreover, the injected electrodes vertically span the sidewalls of the channel, which allows for the application of uniform electric field lines throughout the height of the channel and perpendicular to the direction of flow. The electrodes are mechanically stable over operating conditions commonly used in microfluidic applications; the mechanical stability depends on the magnitude of the applied bias, the nature of the bias (DC vs. AC), and the conductivity of the solutions in the microfluidic channel. Electrodes formed using alloys with melting points above room temperature ensure mechanical stability over all of the conditions explored. As a demonstration of their utility, the fluidic electrodes are used for electrohydrodynamic mixing, which requires extremely high electric fields (~10(5) V m(-1)).     

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman?, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.     

Label-free detection of DNA with interdigitated micro-electrodes in a fluidic cell

We investigate the analytical performance of an interdigitated electrode sensor for the label-free detection of DNA, by monitoring the complex impedance of 5 microm wide interdigitated Pt microelectrodes on a glass substrate. We detect the hybridization of unlabeled 38-mer target ssDNA with a complementary probe that is bound on the glass in between the electrodes by a disuccinimidyl terephtalate and aminosilane immobilization procedure. The sensor is mounted in a microfluidic flow cell, in which hybridization is monitored and in situ compared with a reference. After hybridization, the cell is perfused with deionised water and the dependence of the measured conductance due to the immobilized target DNA layer, to target DNA concentrations down to 1 nM is demonstrated. Subsequently, we apply our sensor to the detection of pathogen DNA from Salmonella choleraesuis in dairy food.     

Trapping of DNA by dielectrophoresis

Under suitable conditions, a DNA molecule in solution will develop a strong electric dipole moment. This induced dipole allows the molecule to be manipulated with field gradients, in a phenomenon known as dielectrophoresis (DEP). Pure dielectrophoretic motion of DNA requires alternate current (AC) electric fields to suppress the electrophoretic effect of the molecules net charge. In this paper, we present two methods for measuring the efficiency of DEP for trapping DNA molecules as well as a set of quantitative measurements of the effects of strand length, buffer composition, and frequency of the applied electric field. A simple configuration of electrodes in combination with a microfluidic flow chamber is shown to increase the concentration of DNA in solution by at least 60-fold. These results should prove useful in designing practical microfluidic devices employing this phenomenon either for separation or concentration of DNA.     

Droplet-on-a-wristband: chip-to-chip digital microfluidic interfaces between replaceable and flexible electrowetting modules

We present a long (204 mm), curved (curvature of 0.04 mm(-1)), and closed droplet pathway in "droplet-on-a-wristband" (DOW) with the designed digital microfluidic modular interfaces for electric signal and droplet connections based on the study of electrowetting-on-dielectric (EWOD) in inclined and curved devices. Instead of using sealed and leakage-proof pipes to transmit liquid and pumping pressure, the demonstrated modular interface for electrowetting-driven digital microfluidics provides simply electric and fluidic connections between two adjacent parallel-plate modules which are easy-to-attach/detach, showing the advantages of using droplets for microfluidic connections between modules. With the previously reported digital-to-channel interfaces (Abdelgawad et al., Lab Chip, 2009, 9, 1046-1051), the chip-to-chip interface presented here would be further applied to continuous microfluidics. Droplet pumping across a single top plate gap and through a modular interface with two gaps between overlapping plates are investigated. To ensure the droplet transportation in the DOW, we actuate droplets against gravity in an inclined or curved device fabricated on flexible PET substrates prepared by a special razor blade cutter and low temperature processes. Pumping a 2.5 μl droplet at a speed above 105 mm s(-1) is achieved by sequentially switching the entire 136 driving electrodes (1.5 mm × 1.5 mm) along the four flexible modules of the DOW fabricated by 4-inch wafer facilities.     

Sample purification on a microfluidic device.

Sample preparation has long been recognized as a significant barrier to the implementation of macroscopic protocols on microfabricated devices. Macroscopically, such tasks as removing salts, primers and other contaminants are performed by methods involving precipitation, specialized membranes and centrifuges, none of which are readily performed in microfluidic structures. Although some microfluidic systems have been developed for performing sample purification, their complexity may hinder the degree to which they can be implemented. We present a method of microchip-based sample purification that can be performed with even the simplest microfluidic designs. The technique is demonstrated by removing primers from a sample of amplified DNA, leaving only the product DNA. This provides a new sample preparation capability for microfluidic systems.     

Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell‐to‐cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence‐specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end‐channel electrodes, and completes the entire process in <5 min, including lysis, purification, fractionation, and delivery to DNA and RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross‐contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence‐specific quantitation using off‐chip RT‐qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively.     

Enhancing signals of microfluidic impedance cytometry through optimization of microelectrode array

Microfluidic impedance cytometry shows a great value in biomedical diagnosis. However, the crosstalk between neighboring microelectrodes strongly weakens the impedance signal. Hereby, we demonstrate a novel microfluidic impedance cytometer consisted of sensing electrodes and ground electrodes (GNDs). The simulation reveals a signal enhancement by more than five times with GNDs compared to that without ones. We also found that the linear correlation between the impedance at a high frequency and that at a low frequency varies as microparticle size changes, which can be used for microparticle classification. The study can help with microelectrode optimization and signal processing for microfluidic impedance analysis.     

Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids

Here, we report a method capable of quantitative detection of low-abundance DNA/RNA molecules by incorporating confocal fluorescence spectroscopy, molecular beacons, and a molecular-confinement microfluidic reactor. By using a combination of ac and dc fields via a trio of 3-D electrodes in the microreactor, we are able to precisely direct the transport of individual molecules to a minuscule laser-focused detection volume ( approximately 1 fL). A burst of fluorescence photons is detected whenever a molecular beacon-target hybrid flows through the detection region, and the amount of targets can be directly quantified according to the number of recorded single-molecule flow-through events. This assay consumes only attomoles of molecular probes and is able to quantitatively detect subpicomolar DNA targets. A measurement time of less than 2 min is sufficient to complete the detection.     

Microfluidic DNA microarray analysis: a review

Microarray DNA hybridization techniques have been used widely from basic to applied molecular biology research. Generally, in a DNA microarray, different probe DNA molecules are immobilized on a solid support in groups and form an array of microspots. Then, hybridization to the microarray can be performed by applying sample DNA solutions in either the bulk or the microfluidic manner. Because the immobilized probe DNA binds and retains its complementary target DNA, detection is achieved through the read-out of the tagged markers on the sample target molecules. The recent microfluidic hybridization method shows the advantages of less sample usage and reduced incubation time. Here, sample solutions are confined in microfabricated channels and flow through the probe microarray area. The high surface-to-volume ratio in microchannels of nanolitre volume greatly enhanced the sensitivity as obtained with the bulk solution method. To generate nanolitre flows, different techniques have been developed, and this including electrokinetic control, vacuum suction and syringe pumping. The latter two are pressure-driven methods which are more flexible without the need of considering the physicochemical properties of solutions. Recently, centrifugal force is employed to drive liquid movement in microchannels. This method utilizes the body force from the liquid itself and there are no additional solution interface contacts such as from electrodes or syringes and tubing. Centrifugal force driven flow also features the ease of parallel hybridizations. In this review, we will summarize the recent advances in microfluidic microarray hybridization and compare the applications of various flow methods.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Packaging of microfluidic chips via interstitial bonding technique.

In this paper, we describe an interstitial bonding technique for packaging of microfluidic chips. The cover plate is first placed on top of the microfluidic chip, followed by dispensing the UV-curable resin into the resin-loading reservoirs. With the interstitial space between the cover plate and the microfluidic chip connecting to the loading reservoirs, the UV-curable resin wicks through capillary force action and hydrostatic pressure generated by the liquid level in the resin-loading reservoirs. When reaching the microchannels, the UV-curable resin stops flowing into the microchannels due to the force balance between the surface tension and hydrostatic pressure. The assembly is then placed under the UV light, followed by further curing in the thermal oven. It is found that there is no leakage from the bonded microfluidic chips and a good DNA separation result was obtained by using the microfluidic chips as fabricated. This bonding technique is relatively simple and fast, which can be applied to the packaging of microfluidic chips made from hybrid materials with complicated designs as long as the interstitial space connects to the loading reservoirs.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

Integrating amperometric detection with electrophoresis microchip devices for biochemical assays: recent developments

Recent advances in microfluidic systems, particularly in the Micro Total Analysis System (μTAS) or Lab On a Chip (LOC), drive the current analysis tools and equipment towards miniaturization, rapid at-line testing and mobility. The state-of-the-art microfluidic technology targets a wider range but smaller volumes of analytes, making the analytical procedure relatively easier and faster. This trend together with faster electronics and modern instrumentation systems will make real-time and in situ analysis a definite possibility. This review focuses on microchip capillary electrophoresis with amperometric detection (MCE-AD) for the detection of DNA and other electroactive analytes. The problems associated with the microchip design, in particular the choice of materials and the configuration of electrodes are discussed thoroughly and solutions are proposed. Significant developments in the related areas are also covered and reviewed critically.     

Genotyping of Saccharomyces cerevisiae strains by interdelta sequence typing using automated microfluidics

Amplification of genomic sequences flanked by delta elements of retrotransposons TY1 and TY2 is a reliable method for characterization of Saccharomyces cerevisiae strains. The aim of this study is to evaluate the usefulness of microfluidic electrophoresis (Caliper LabChip) to assess the factors that affect interlaboratory reproducibility of interdelta sequence typing for S. cerevisiae strain delimitation. We carried out experiments in two laboratories, using varying combinations of Taq DNA polymerases and thermal cyclers. The reproducibility of the technique is evaluated using non-parametric statistical tests and we show that the source of Taq DNA polymerase and technical differences between laboratories have the highest impact on reproducibility, whereas thermal cyclers have little impact. We also show that the comparative analysis of interdelta patterns is more reliable when fragment sizes are compared than when absolute and relative DNA concentrations of each band are considered. Interdelta analysis based on a smaller fraction of bands with intermediate sizes between 100 and 1000 bp yields the highest reproducibility.