Microfluidic separation and gateable fraction collection for mass-limited samples

Integrating multiple analytical processes into microfluidic devices is an important research area required for a variety of microchip-based analyses. A microfluidic system is described that achieves preparative separations by intelligent fraction collection of attomole quantities of sample. The device consists of a main microfluidic channel used to perform electrophoresis, which is interconnected at 90 degrees to two vertically displaced channels via a nanocapillary array membrane. The membrane interconnect contains nanometer-diameter pores that provide fluidic communication between the channels. Sample injection and analyte collection are controlled by application of an electrical bias between the microfluidic channels across the nanocapillary array. After the separation, the automated transfer of the FITC-labeled Arg, Gln, and Gly bands occurs; a fluorescence detector located at the separation/collection channel interconnect is used to generate a triggering signal that initiates suitable voltages to allow near-quantitative transfer of analyte from the separation channel to the second fluidic layer. The ability to achieve such sample manipulations from mass-limited samples enables a variety of postseparation processing events.     

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.     

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.     

Amperometric fluidic microchip array sensing device for nitric oxide determination in solution

In this paper, we report on the first use of an amperometric fluidic microchip array for the examination of nitric oxide in solution. The array chip is composed of 36 working platinum electrodes on a glass substrate. The electrodes have a diameter of 50 μm and are separated by 500 μm. The array chip is integrated within a flowing cell to obtain a fluidic-type sensing device. Two preliminary tests were performed. The first one consisted in assessing the fluidic set-up by using potassium ferrocyanide as test analyte. The second test was aimed at achieving the modification of the surface of the working electrodes by electrodepositing nickel tetrasulfonated phthalocyanine and Nafion® layers to show that the fluidic sensing device can be adapted to the analysis of nitric oxide in solution.     

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.     

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.     

Density-based diamagnetic separation: devices for detecting binding events and for collecting unlabeled diamagnetic particles in paramagnetic solutions

This paper describes the fabrication of a fluidic device for detecting and separating diamagnetic materials that differ in density. The basis for the separation is the balance of the magnetic and gravitational forces on diamagnetic materials suspended in a paramagnetic medium. The paper demonstrates two applications of separations involving particles suspended in static fluids for detecting the following: (i) the binding of streptavidin to solid-supported biotin and (ii) the binding of citrate-capped gold nanoparticles to amine-modified polystyrene spheres. The paper also demonstrates a microfluidic device in which polystyrene particles that differ in their content of CH2Cl groups are continuously separated and collected in a flowing stream of an aqueous solution of GdCl3. The procedures for separation and detection described in this paper require only gadolinium salts, two NdFeB magnets, and simple microfluidic devices fabricated from poly(dimethylsiloxane). This device requires no power, has no moving parts, and may be suitable for use in resource-poor environments.     

Scanning temperature gradient focusing

Temperature gradient focusing (TGF) is a recently developed technique for the simultaneous concentration and electrophoretic separation of ionic analytes in microfluidic channels. One drawback to TGF as it has previously been described is the limited peak capacity; only a small number of analyte peaks (approximately 2-3) can be simultaneously focused and separated. In this paper, we report on a variation of the TGF method whereby the bulk flow rate is varied over time so that a large number of analytes can be sequentially focused, moved past a fixed detection point, and flushed to waste. In addition to improved peak capacity, the detection limits of the scanning TGF method can be adjusted on-the-fly, as needed for different samples. Finally, scanning TGF provides a technique by which high-resolution, high-peak-capacity electrophoretic separations can be performed in simple, straight, and short microfluidic channels.     

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.     

Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device

We have developed a microfluidic device for on-chip monitoring of cellular reactions. The device consists of two primary analytical functions: control of cell transport and immobilization, and dilution of an analyte solution to generate a concentration gradient. In this device, a dam structure in parallel to the fluid flow was constructed for docking and alignment of biological cells, which allows the fragile cells to move in the microfluidic channels and to be immobilized with controllable numbers in desired locations. The cells docked on the parallel dam structure are exposed to minimal stress caused by fluidic pressure. Additionally, a network of microfluidic channels was designed to generate a concentration gradient by controlled fluid distribution and diffusive mixing. An analyte solution could be diluted to different gradients as a function of distance along the dam. We used the ATP-dependent calcium uptake reaction of HL-60 cells as a model for on-chip measurement of the threshold ATP concentration that induces significant intracellular calcium signal. The results have demonstrated the feasibility of using the microchip for real-time monitoring of cellular processes upon treatment of a concentration gradient of a test solution. The integration of cell manipulation and solution manipulation on a microchip allows the measurement of concentration-dependent biological responses within a confined microscale feature.     

Tandem isotachophoresis-zone electrophoresis via base-mediated destacking for increased detection sensitivity in microfluidic systems

Electrophoresis in microfluidic devices is becoming a useful analytical platform for a variety of biological assays. In this report, we present a method that allows for an increased sensitivity of detection of fluorescent molecules in microfluidic electrophoresis devices. This capability is provided by the implementation of a particular buffer system that is designed to initially function in an isotachophoretic (ITP) mode and, then after a controlled amount of electric current has been applied to the system, to transition to a zone electrophoretic mode. In the initial ITP mode, analytes dissolved in a large volume of injected sample are concentrated into a single narrow zone. After application of a sufficient and adjustable amount of electric current, the system switches into a zone electrophoretic mode, where the concentrated analytes are separated according to their electrophoretic mobilities. Application of this tandem ITP-zone electrophoretic strategy to the concentration, separation, and detection of fluorescent reporter molecules in a standard microfluidic device results in an approximately 50-fold increase in detection sensitivity relative to equivalent separations that are obtained with zone electrophoresis alone. Even with very long initial sample plugs (up to 3000 microm), this strategy produces electrophoretic separations with high resolutions and peak efficiencies. This strategy can be implemented to increase detection sensitivity in any standard microfluidic electrophoresis platform and does not require any specialized hardware or microchannel configurations.     

Simultaneous concentration and separation of enantiomers with chiral temperature gradient focusing

A new technique is demonstrated for the simultaneous concentration and high-resolution separation of chiral compounds. With temperature gradient focusing, a combination of a temperature gradient, an applied electric field, and a buffer with a temperature-dependent ionic strength is used to cause analytes to move to equilibrium, zero-velocity points along a microchannel or capillary. Different analytes are thus separated spatially and concentrated in a manner that resembles isoelectric focusing but that is applicable to a greater variety of analytes including small chiral drug molecules. Chiral separations are accomplished by the addition of a chiral selector, which causes the different enantiomers of an analyte to focus at different positions along a microchannel or capillary. This new technique is demonstrated to provide high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile reversal of peak order (desirable for analysis of trace enantiomeric impurities), and high resolving power (comparable to capillary electrophoresis) in combination with greater than 1000-fold concentration enhancement enabling improved detection limits. In addition, chiral temperature gradient focusing allows for real-time monitoring of the interaction of chiral analyte molecules with chiral selectors that could potentially be applied to the study of other molecular interactions. Finally, unlike CE, which requires long channels or capillaries for high-resolution separations, separations of equivalent resolution can be performed with TGF in very short microchannels (mm); thus, TGF is inherently much more suited to miniaturization and integration into lab-on-a-chip-devices.     

Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters

Microfluidic electrophoresis devices were coupled on-line to microdialysis for in vivo monitoring of primary amine neurotransmitters in rat brain. The devices contained a sample introduction channel for dialysate, a precolumn reactor for derivatization with o-phthaldialdehyde, a flow-gated injector, and a separation channel. Detection was performed using confocal laser-induced fluorescence. In vitro testing revealed that the initial device design had detection limits for amino acids of approximately 200 nM, relative standard deviation of peak heights of 2%, and separations within 95 s with up to 30,200 theoretical plates when applying an electric field of 370 V/cm. A second device design that allowed electric fields of 1320 V/cm to be applied while preserving the reaction time allowed separations within 20 s with up to 156,000 theoretical plates. Flow splitting into the electrokinetic network from hydrodynamic flow in the sample introduction channel was made negligible for sampling flow rates from 0.3 to 1.2 microL/min by placing a 360-microm-diameter fluidic access hole that had flow resistance (0.15-7.2) x 10(8)-fold lower than that of the electrokinetic network at the junction of the sample introduction channel and the electrokinetic network. Using serial injections, the device allowed the dialysate stream to be analyzed at 130-s intervals. In vivo monitoring was demonstrated by using the microdialysis/microfluidic device to record glutamate concentrations in the striatum of an anesthetized rat during infusion of the glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid. These results prove the feasibility of using a microfabricated fluidic system coupled to sampling probes for chemical monitoring of complex media such as mammalian brain.     

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.     

Negative enrichment of target cells by microfluidic affinity chromatography

A three-dimensional microfluidic channel was developed for high-purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of nontarget cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multistep processes in microfluidic systems. In previous work (Li, P.; Tian, Y.; Pappas, D. Anal. Chem.2011, 83, 774-781), we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel system showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity and ease of fabrication and use is suitable for cell separations when subsequent analysis of target cells is required.     

Microfluidic biosensor based on an array of hydrogel-entrapped enzymes

Here we show that a microfluidic sensor based on an array of hydrogel-entrapped enzymes can be used to simultaneously detect different concentrations of the same analyte (glucose) or multiple analytes (glucose and galactose) in real time. The concentration of paraoxon, an acetylcholine esterase inhibitor, can be quantified using the same approach. The hydrogel micropatch arrays and the microfluidic systems are easy to fabricate, and the hydrogels provide a convenient, biocompatible matrix for the enzymes. Isolation of the micropatches within different microfluidic channels eliminates the possibility of cross talk between enzymes.     

Development of a microfluidic platform with an optical imaging microarray capable of attomolar target DNA detection

In this paper, DNA hybridization in a microfluidic manifold is performed using fluorescence detection on a fiber-optic microarray. The microfluidic device integrates optics, sample transport, and fluidic interconnects on a single platform. A high-density optical imaging fiber array containing oligonucleotide-labeled microspheres was developed. DNA hybridization was observed at concentrations as low as 10 aM with response times of less than 15 min at a flow rate of 1 microL/min using 50 microL of target DNA samples. The fast response times coupled with the low sample volumes and the use of a high-density, fiber-optic microarray format make this method highly advantageous. This paper describes the initial development, optimization, and integration of the microfluidic platform with imaging fiber arrays.     

Three-dimensional fluidic self-assembly by axis translation of two-dimensionally fabricated microcomponents in railed microfluidics

A method for high-throughput 3D self-assembly of 2D photopatterned microstructures using railed microfluidics is presented. Vertical device patterning of heterogeneous materials requires high-level integration using conventional microelectromechanical system (MEMS) technology; however, 3D railed assembly enables easy and fast self-assembly via a fluidic axis-translation process and simple material exchange in microfluidic channels. Individually photopatterned 2D microstructures are axis-translated from in-plane to out-of-plane and fluidically self-assembled, guided by side-rails in microfluidic channels to form a 3D morphology. Since the structures are fabricated in fluidic environments, there are no fixed initial points on the channel substrate allowing fluidic horizontal stacking of erected 2D structures. The guiding mechanism of railed microfluidics enables efficient fluidic handling and deterministic 3D self-assembly of heterogeneous components such as electronic components or polymeric microstructures using only fluidic force.     

Compartmentalization of electrophoretically separated analytes in a multiphase microfluidic platform

Herein, we describe the monolithic integration of a multiphase microfluidic system to a microcapillary gel electrophoresis (μCGE) architecture for the complete isolation and storage of separated analyte bands. Within this platform, analyte molecules are separated using microchannel gel electrophoresis, and the eluted bands are stored in a sequence of approximately 40-600 encapsulating microdroplets. Importantly, employing such a system allows for total control of droplet size, shape, and composition. This approach is utilized to separate, optically detect, and encapsulate two fluorescent analytes from a composite sample mixture. Further to this, we subsequently investigate the potential of the system to be used as a concentration gradient generator through analysis of the segmented analyte bands and droplet composition.     

A microfluidic paper-based electrochemical biosensor array for multiplexed detection of metabolic biomarkers

Abstract

Paper-based microfluidic devices have emerged as simple yet powerful platforms for performing low-cost analytical tests. This paper reports a microfluidic paper-based electrochemical biosensor array for multiplexed detection of physiologically relevant metabolic biomarkers. Different from existing paper-based electrochemical devices, our device includes an array of eight electrochemical sensors and utilizes a handheld custom-made electrochemical reader (potentiostat) for signal readout. The biosensor array can detect several analytes in a sample solution and produce multiple measurements for each analyte from a single run. Using the device, we demonstrate simultaneous detection of glucose, lactate and uric acid in urine, with analytical performance comparable to that of the existing commercial and paper-based platforms. The paper-based biosensor array and its electrochemical reader will enable the acquisition of high-density, statistically meaningful diagnostic information at the point of care in a rapid and cost-efficient way.