On-chip high-speed sorting of micron-sized particles for high-throughput analysis

A new design of particle sorting chip is presented. The device employs a dielectrophoretic gate that deflects particles into one of two microfluidic channels at high speed. The device operates by focussing particles into the central streamline of the main flow channel using dielectrophoretic focussing. At the sorting junction (T- or Y-junction) two sets of electrodes produce a small dielectrophoretic force that pushes the particle into one or other of the outlet channels, where they are carried under the pressure-driven fluid flow to the outlet. For a 40 microm wide and high channel, it is shown that 6 microm diameter particles can be deflected at a rate of 300/s. The principle of a fully automated sorting device is demonstrated by separating fluorescent from non-fluorescent latex beads.     

Probing electric fields inside microfluidic channels during electroosmotic flow with fast-scan cyclic voltammetry

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used in microfluidic channels. This method offers the advantage that it can resolve electroactive species not separated in the channel. In addition, this method provides a route to investigate the distribution of applied electrophoretic fields in microfluidic channels. To probe this, microelectrodes were inserted at various distances into channels and cyclic voltammograms recorded at 300 V/s were repeated at 0.1-s intervals. The use of a battery-powered laptop computer and potentiostat provided galvanic isolation between the applied electrophoretic field and the electrochemical measurements. In the absence of an external field, the peak potential for oxidation of the test solute, Ru(bpy)3(2+), was virtually unaltered by insertion of the microelectrode tip into the channel. When an electrophoretic field was applied, the peak potential for Ru(bpy)3(2+) oxidation shifted to more positive potentials in a manner that was directly proportional to the field in the channel. The shifts in peak potential observed with FSCV enabled direct compensation of the applied electrochemical potential. This approach was used to explore the electrophoretic field at the channel terminus. It was found to persist for more than 50 microm from the channel terminus. In addition, the degree of analyte dispersion was found to depend critically on the electrode position outside the channel.     

Detecting single porphyrin molecules in a conically shaped synthetic nanopore

We report here the first example of abiotic resistive-pulse sensing of a molecular (as opposed to a particle or macromolecular) analyte. This was accomplished by using a conically shaped nanopore prepared by the track-etch method as the sensing element. It is possible to sense the molecular analyte because the small diameter opening of the conical nanopore (approximately 4.5 nm) is comparable to the diameter of the analyte molecule (approximately 2 nm).     

A ferrofluid guided system for the rapid separation of the non-magnetic particles in a microfluidic device

A microfluidic device was fabricated via UV lithography technique to separate non-magnetic fluoresbrite carboxy microspheres (approximately 4.5 microm) in the pH 7 ferrofluids made of magnetite nanoparticles (approximately 10 nm). A mixture of microspheres and ferrofluid was injected to a lithographically developed Y shape microfluidic device, and then by applying the external magnet fields (0.45 T), the microspheres were clearly separated into different channels because of the magnetic force acting on those non-magnetic particles. During this study, various pumping speeds and particle concentrations associated with the various distances between the magnet and the microfluidic device were investigated for an efficient separation. This study may be useful for the separation of biological particles, which are very sensitive to pH value of the solutions.     

Electooptic Fresnel lens-scanner with an array of channel waveguides

A new type of beam scanner is discussed based on a 1-D Fresnel zone plate consisting of titanium-diffused channel waveguides on LiNbO3. By electrooptically controlling the guided-wave phase, both beam scanning and 1-D focusing are achieved without a condensing lens. It was experimentally confirmed using the scanner with twenty-one Fresnel zones that the beam spot with a diameter of approximately 50 microm at half-power level of diffraction pattern is scanned over a distance of +/-70 microm in the focal plane with an applied voltage of +/-40 V at 633 nm.     

Fabrication of nanopores in a 100-nm thick Si3N4 membrane

Textured alumina films have been used to fabricate nanoscale pores in Si3N4 membranes. A few nanometer-thick alumina layer was used as a masking material for nanopore fabrication, and the pattern was transferred into a 100-nm thick, 200 microm x 200 microm Si3N4 membrane by reactive ion etching (RIE). The nanopores were found to be concentrated in a approximately 150-microm diameter region at the center of the membrane.     

Glass nanopore-based ion-selective electrodes

Glass nanopore-based all-solid-state ion-selective electrodes (ISEs) have been developed to probe the distribution of ionic species at micro- or submicrometer-length scales, e.g., mapping of ion flux through micrometer-sized pores. The all-solid-state ISE was fabricated by sealing a conically etched platinum wire (d = 25-microm; radius of etched tip <10 nm) into a soda lime glass capillary. A Pt disk was exposed by gentle polishing the glass and the disk etched to form a conical pore of submicrometer dimension (radius < approximately 500 nm; depth < approximately 30 microm). Ag was electroplated on the Pt electrode in the pore and gently chloridated to obtain a AgCl/Ag layer within the pore. The AgCl/Ag layer-coated ISE was used as a highly selective Cl- probe in scanning electrochemical microscope experiments to map the ion flux through a micropore. The same ISE was also used as the base transducer of the neutral carrier-doped solvent polymeric membrane. The optimized polymer membranes used for the glass nanopore-based all-solid-state ISE require a higher ratio of plasticizer/polymer (9/1) compared to those for conventional ISE (2/1). An ISE based on deposition of an IrO2 layer at the base of a glass nanopore electrode exhibited a highly sensitive response (79.7 +/- 2.3 mV/pH) to variations in pH and could be used for approximately 3 weeks.     

An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.     

Thermally controlled permeation of ionic molecules through synthetic nanopores functionalized with amine-terminated polymer brushes

We present temperature-dependent ionic transport through an array of nanopores (cylindrical and conical) and a single conical nanopore functionalized with amine-terminated poly(N-isopropylacrylamide) [PNIPAAM-NH(2)] brushes. For this purpose, nanopores are fabricated in heavy ion irradiated polyethylene terephthlate (PET) membranes by a controlled chemical track-etching technique, which leads to the generation of carboxyl (COOH) groups on the pore surface. End-functionalized polymer chains are immobilized onto the inner pore walls via a 'grafting-to' approach through the covalent linkage of surface COOH moieties with the terminal amine groups of the PNIPAAM molecules by using carbodiimide coupling chemistry. The success of the chemical modification reaction is corroborated by measuring the permeation flux of charged analytes across the multipore membranes in an aqueous solution, and for the case of single conical pore by measuring the current-voltage (I-V) characteristics, which are dictated by the electrostatic interaction of the charged pore surface with the mobile ions in an electrolyte solution. The effective nanopore diameter is tuned by manipulating the environmental temperature due to the swelling/shrinking behaviour of polymer brushes attached to the inner nanopore walls, leading to a decrease/increase in the ionic transport across the membrane. This process should permit the thermal gating and controlled release of ionic drug molecules through the nanopores modified with thermoresponsive polymer chains across the membrane.     

Electrokinetic bioprocessor for concentrating cells and molecules

Bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems. In this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface. The processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface. By optimizing the operating parameters, we have concentrated various biological objects in a large range of sizes, including Escherichia coli bacteria, lambda phage DNA, and single-stranded DNA fragments as small as 20 bases that have a radius of gyration of only 3 nm.     

Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays

Techniques for manipulating, separating, and trapping particles and cells are highly desired in today's bioanalytical and biomedical field. The microfluidic chip-based acoustic noncontact trapping method earlier developed within the group now provides a flexible platform for performing cell- and particle-based assays in continuous flow microsystems. An acoustic standing wave is generated in etched glass channels (600x61 microm2) by miniature ultrasonic transducers (550x550x200 microm3). Particles or cells passing the transducer will be retained and levitated in the center of the channel without any contact with the channel walls. The maximum trapping force was calculated to be 430+/-135 pN by measuring the drag force exerted on a single particle levitated in the standing wave. The temperature increase in the channel was characterized by fluorescence measurements using rhodamine B, and levels of moderate temperature increase were noted. Neural stem cells were acoustically trapped and shown to be viable after 15 min. Further evidence of the mild cell handling conditions was demonstrated as yeast cells were successfully cultured for 6 h in the acoustic trap while being perfused by the cell medium at a flowrate of 1 microL/min. The acoustic microchip method facilitates trapping of single cells as well as larger cell clusters. The noncontact mode of cell handling is especially important when studies on nonadherent cells are performed, e.g., stem cells, yeast cells, or blood cells, as mechanical stress and surface interaction are minimized. The demonstrated acoustic trapping of cells and particles enables cell- or particle-based bioassays to be performed in a continuous flow format.     

Influence of electrophoresis waveforms in determining stochastic nanoparticle capture rates and detection sensitivity

Electrophoretic capture and release of charged polystyrene particles at the opening of a membrane pore has been investigated to determine the optimal applied current waveform, iapp(t), for ensuring true stochastic counting rates and to improve detection sensitivity (i.e., Delta(counts per second)/Delta(particle concentration)). In capture and release detection, charged particles are electrophoretically driven to the opening of a small pore ( approximately 60 nm diameter) in a membrane; capture of a single particle at the pore opening at time tau is signaled by a decrease in the flux of a redox species (Fe(CN)(6)4-) through the pore. The captured particle is then released by applying an electrophoretic current in the opposite direction, and the process is repeated to acquire sufficient statistical data to determine the solution particle concentration (Cp) based on the relationship between Cp and the average particle counting rate (tauavg(-1)). Both tauavg(-1) and the method sensitivity are shown, for the detection of 90 nm diameter polystyrene particles, to depend strongly on the applied current waveform. The observed dependence is a consequence of the nonequilibrium distribution of particles in the analyte solution that results from electrostatic forces acting on the particle whenever iapp has a nonzero value. Stochastic capture rates corresponding to an initial uniform distribution of particles are more closely achieved using an applied current waveform that includes an equilibration period (iapp=0) prior to electrophoretic capture. An increase in particle detection sensitivity, relative to the previously reported value, results from this equilibration step.     

Fabrication of a configurable, single-use microfluidic device.

This paper describes microfluidic devices that contain connections that can be opened by the user after fabrication. The devices are fabricated in poly(dimethylsiloxane) (PDMS) and comprise disconnected fluidic channels that are separated by 20 microm of PDMS. Applying voltages above the breakdown voltage of PDMS (21 V/microm) opened pathways between disconnected channels. Fluids could then be pumped through the openings. The voltage used and the ionic strength of the buffer in the channels determined the size of the opening. Opening connections in a specific order provides the means to control complex reactions on the device. A device for ELISA was fabricated to demonstrate the ability to store and deliver fluids on demand.     

Dielectrophoretic manipulation of DNA: separation and polarizability

Although separation of polymers based on the combination of dielectrophoretic trapping and electrophoretic forces was proposed 15 years ago, experimental proof has not yet been reported. Here, we address this problem for long DNA fragments in a simple and easy-to-fabricate microfluidic device, in which the DNA is manipulated by electrophoresis and by electrodeless dielectrophoresis. By slowly increasing the strength of the dielectrophoretic traps in the course of the separation experiments, we are able to perform efficient and fast DNA separation according to length for two different DNA conformations: linear DNA (lambda (48.5-kbp) and T2 (164-kbp) DNA) and supercoiled covalently closed circular plasmid DNA (7 and 14 kbp). The underlying migration mechanism-thermally induced escape processes out of the dielectrophoretic traps in the direction of the electrophoretic force-is sensitive to different DNA fragments because of length-dependent DNA polarizabilities. This is analyzed in a second series of experiments, where the migration mechanism is exploited for the quantitative measurement of the DNA polarizabilities. This new and simple technique allows for the systematic characterization of the polarizability not only for DNA but also for other biomolecules like proteins. Furthermore, our results have direct implications to future biotechnological applications such as gene therapy and DNA vaccination.     

Synthetic single-nanopore and nanotube membranes

There is increasing interest in investigating transport and electrochemical phenomena in synthetic membrane samples that contain a single pore of nanoscopic diameter. Approaches used to date for preparing such single-nanopore membranes include microfabrication-based methods, the track-etch method, and a method based on the incorporation of a single fullerene nanotube within a synthetic membrane. We describe here an alternative approach that we believe is easier and more accessible than the previously described methods. This method is based on a very low pore density track-etch membrane obtained from commercial sources. Fluorescence microscopy is used to identify and isolate a single nanopore in this membrane. Membrane samples containing single nanopores with diameters as small as 30 nm have been prepared. Furthermore, we show here that an electroless plating method can be used to deposit a gold nanotube within the single nanopore, and this provides a route for further decreasing the inside diameter of the pore. A single-nanotube membrane with an electrochemically determined inside diameter of approximately 2 nm was prepared and evaluated.     

Non-vanishing ponderomotive AC electrophoretic effect for particle trapping

We present here a study on overlooked aspects of alternating current (AC) electrokinetics-AC electrophoretic (ACEP) phenomena. The dynamics of a particle with both polarizability and net charges in a non-uniform AC electric trapping field is investigated. It is found that either electrophoretic (EP) or dielectrophoretic (DEP) effects can dominate the trapping dynamics, depending on experimental conditions. A dimensionless parameter γ is developed to predict the relative strength of EP and DEP effects in a quadrupole AC field. An ACEP trap is feasible for charged particles in 'salt-free' or low salt concentration solutions. In contrast to DEP traps, an ACEP trap favors the downscaling of the particle size.     

High-efficiency single-cell entrapment and fluorescence in situ hybridization analysis using a poly(dimethylsiloxane) microfluidic device integrated with a black poly(ethylene terephthalate) micromesh

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 microm in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.     

Variation of nanopore diameter along porous anodic alumina channels by multi-step anodization

In order to form tapered nanocapillaries, we investigated a method to vary the nanopore diameter along the porous anodic alumina (PAA) channels using multi-step anodization. By anodizing the aluminum in either single acid (H3PO4) or multi-acid (H2SO4, oxalic acid and H3PO4) with increasing or decreasing voltage, the diameter of the nanopore along the PAA channel can be varied systematically corresponding to the applied voltages. The pore size along the channel can be enlarged or shrunken in the range of 20 nm to 200 nm. Structural engineering of the template along the film growth direction can be achieved by deliberately designing a suitable voltage and electrolyte together with anodization time.     

Thermal bonding of polymeric capillary electrophoresis microdevices in water

A new method for thermally bonding poly(methyl methacrylate) (PMMA) substrates to form microfluidic systems has been demonstrated. A PMMA substrate is first imprinted with a Si template using applied pressure and elevated temperature to form microchannel structures. This embossing method has been used to successfully pattern over 65 PMMA pieces using a single Si template. Thermal bonding for channel enclosure is accomplished by clamping together an imprinted and a blank substrate and placing the assembly in boiling water for 1 h. The functionality of these water-bonded microfluidic substrates was demonstrated by performing high-resolution electrophoretic separations of fluorescently labeled amino acids. Testing of bond strength in four microdevices showed an average failure pressure of 130 kPa, which was comparable to the bond strength for devices sealed in air. Subsequent profilometry of separated substrates revealed that the dimensions of the channels were well preserved during the bonding process. This new methodology for generation of microfluidic constructs should facilitate the permanent incorporation of hydrated, molecular size-selective membranes in microdevices, thus circumventing problems associated with membrane swelling in microfluidic systems upon exposure to water.     

Accelerated particle electrophoretic motion and separation in converging-diverging microchannels

Accelerated particle electrophoretic motions were visualized in converging-diverging microchannels on poly(dimethylsiloxane) chips. The accelerated particle electrophoretic separation is highly desirable in on-chip flow cytometry and high-speed electrophoresis. The effects of electric field, particle size, particle trajectory, and channel structure on the particle electrophoretic motion are examined. We find that the ratio of the particle velocity in the throat to that in the straight channel is significantly lower than their cross-sectional area ratio. This discrepancy may be attributed to the locally higher electric field around the two poles of a particle, as compared to other regions inside the microchannel. We also find that the particle velocity ratio is increased for smaller particles moving through symmetric converging-diverging channels under lower electric fields. These variations may be attributed to the negative dielectrophoretic force that is generated by the nonuniform electric field in the converging-diverging section. In addition, we find that particle trajectory has insignificant influences on the maximum velocity ratio obtained in the throat.