A versatile and low-cost chip-to-world interface: Enabling ICP-MS characterization of isotachophoretically separated lanthanides on a microfluidic device.

Microfluidics offer novel and state-of-the-art pathways to process materials. Microfluidic systems drastically reduce timeframes and costs associated with traditional lab-scale efforts in the area of analytical sample preparations. The challenge arises in effectively connecting microfluidics to off-chip analysis tools to accurately characterize samples after treatment on-chip. Fabrication of a chip-to-world connection includes one end of a fused silica capillary interfaced to the outlet of a microfluidic device (MFD). The other end of the capillary is connected to a commercially available CEI-100 interface that passes samples into an inductively coupled plasma mass spectrometer (ICP-MS). This coupling creates an inexpensive and simple chip-to-world connection that enables on-chip and off-chip methods of analyzing the separation of rare earth elements. Specifically, this is demonstrated by utilizing isotachophoresis (ITP) on a microfluidic chip to separate up to 14 lanthanides from a homogenous sample into elementally pure bands. The separated analyte zones are successfully transferred across a 7 nL void volume at the microchip-capillary junction, such that separation resolution is maintained and even increased through the interface and into the ICP-MS, where the elemental composition of the sample is analyzed. Lanthanide samples of varying composition are detected using ICP-MS, demonstrating this versatile and cost-effective approach, which maintains the separation quality achieved on the MFD. This simple connection enables fast, low-cost sample preparation immediately prior to injection into an ICP-MS or other analytical instrument.

Design and optimization of a fused-silica microfluidic device for separation of trivalent lanthanides by isotachophoresis.

Elemental analysis of rare earth elements is essential in a variety of fields including environmental monitoring and nuclear safeguards; however, current techniques are often labor intensive, time consuming, and/or costly to perform. The difficulty arises in preparing samples, which requires separating the chemically and physically similar lanthanides. However, by transitioning these separations to the microscale, the speed, cost, and simplicity of sample preparation can be drastically improved. Here, all fourteen non-radioactive lanthanides (lanthanum through lutetium minus promethium) are separated by ITP for the first time in a serpentine fused-silica microchannel (70 µm wide × 70 µm tall × 33 cm long) in <10 min at voltages ≤8 kV with limits of detection on the order of picomoles. This time includes the 2 min electrokinetic injection time at 2 kV to load sample into the microchannel. The final leading electrolyte consisted of 10 mM ammonium acetate, 7 mM α-hydroxyisobutyric acid, 1% polyvinylpyrrolidone, and the final terminating electrolyte consisted of 10 mM acetic acid, 7 mM α-hydroxyisobutyric acid, and 1% polyvinylpyrrolidone. Electrophoretic electrodes are embedded in the microchip reservoirs so that voltages can be quickly applied and switched during operation. The limits of detection are quantified using a commercial capacitively coupled contactless conductivity detector (C4 D) to calculate ITP zone lengths in combination with ITP theory. Optimization of experimental procedures and reproducibility based on statistical analysis of subsequent experimental results are addressed. Percent error values in band length and conductivity are ≤8.1 and 0.37%, respectively.

Micro-Raman Technology to Interrogate Two-Phase Extraction on a Microfluidic Device.

Microfluidic devices provide ideal environments to study solvent extraction. When droplets form and generate plug flow down the microfluidic channel, the device acts as a microreactor in which the kinetics of chemical reactions and interfacial transfer can be examined. Here, we present a methodology that combines chemometric analysis with online micro-Raman spectroscopy to monitor biphasic extractions within a microfluidic device. Among the many benefits of microreactors is the ability to maintain small sample volumes, which is especially important when studying solvent extraction in harsh environments, such as in separations related to the nuclear fuel cycle. In solvent extraction, the efficiency of the process depends on complex formation and rates of transfer in biphasic systems. Thus, it is important to understand the kinetic parameters in an extraction system to maintain a high efficiency and effectivity of the process. This monitoring provided concentration measurements in both organic and aqueous plugs as they were pumped through the microfluidic channel. The biphasic system studied was comprised of HNO3 as the aqueous phase and 30% (v/v) tributyl phosphate in n-dodecane comprised the organic phase, which simulated the plutonium uranium reduction extraction (PUREX) process. Using pre-equilibrated solutions (post extraction), the validity of the technique and methodology is illustrated. Following this validation, solutions that were not equilibrated were examined and the kinetics of interfacial mass transfer within the biphasic system were established. Kinetic results of extraction were compared to kinetics already determined on a macro scale to prove the efficacy of the technique.

Simulation of the ozone pretreatment of wheat straw.

Wheat straw is a potential feedstock in biorefinery for sugar production. However, the cellulose, which is the major source of sugar, is protected by lignin. Ozonolysis deconstructs the lignin and makes cellulose accessible to enzymatic digestion. In this study, the change in lignin concentration with different ozonolysis times (0, 1, 2, 3, 5, 7, 10, 15, 20, 30, 60min) was fit to two different kinetic models: one using the model developed by Garcia-Cubero et al. (2012) and another including an outer mass transfer barrier or "cuticle" region where ozone mass transport is reduced in proportion to the mass of unreacted insoluble lignin in the cuticle. The kinetic parameters of two mathematical models for predicting the soluble and insoluble lignin at different pretreatment time were determined. The results showed that parameters derived from the cuticle-based model provided a better fit to experimental results compared to a model without a cuticle layer.

Cationic isotachophoresis separation of the biomarker cardiac troponin I from a high-abundance contaminant, serum albumin.

Cationic ITP was used to separate and concentrate fluorescently tagged cardiac troponin I (cTnI) from two proteins with similar isoelectric properties in a PMMA straight-channel microfluidic chip. In an initial set of experiments, cTnI was effectively separated from R-Phycoerythrin using cationic ITP in a pH 8 buffer system. Then, a second set of experiments was conducted in which cTnI was separated from a serum contaminant, albumin. Each experiment took ∼10 min or less at low electric field strengths (34 V/cm) and demonstrated that cationic ITP could be used as an on-chip removal technique to isolate cTnI from albumin. In addition to the experimental work, a 1D numerical simulation of our cationic ITP experiments has been included to qualitatively validate experimental observations.

ITP of lanthanides in microfluidic PMMA chip.

An ITP separation of eight lanthanides on a serpentine PMMA microchip with a tee junction and a 230-mm-long serpentine channel is described. The cover of the PMMA chip is 175 µm thick so that a C(4) D in microchip mode can be used to detect the lanthanides as they migrate through the microchannel. Acetate and α-hydroxyisobutyric acid are used as complexing agents to increase the electrophoretic mobility difference between the lanthanides. Eight lanthanides are concentrated within ∼ 6 min by ITP in the microchip using 10 mM ammonium acetate at pH 4.5 as the leading electrolyte and 10 mM acetic acid at ∼ pH 3.0 as the terminating electrolyte. In addition, a 2D numerical simulation of the lanthanides undergoing ITP in the microchip is compared with experimental results using COMSOL Multiphysics v4.3a.

Microfluidic isotachophoresis: a review.

Electromigration methods including CE and ITP are attractive for incorporation in microfluidic devices because they are relatively easily adaptable to miniaturization. After its popularity in the 1970s, ITP has made a comeback in microfluidic format (µ-ITP, micro-ITP) driven by the advantages of the steady-state boundary, the self-focusing effect, and the ability to aid in preconcentrating analytes in the sample while removing matrix components. In this review, we provide an overview of the developments in the area of µ-ITP in a context of the historic developments with a focus on recent developments in experimental and computational ITP and discuss possible future trends. The chip-ITP areas and topics discussed in this review and the corresponding sections include: PC simulations and modeling, analytical µ-ITP, preconcentration ITP, transient ITP, peak mode ITP, gradient elution ITP, and free-flow ITP, while the conclusions provide a critical summary and outlook. The review also contains experimental conditions for µ-ITP applications to real-world samples from over 50 original journal publications.

A new fabrication technique to form complex polymethylmethacrylate microchannel for bioseparation.

Recent studies show that reduction in cross-sectional area can be used to improve the concentration factor in microscale bioseparations. Due to simplicity in fabrication process, a step reduction in cross-sectional area is generally implemented in microchip to increase the concentration factor. But the sudden change in cross-sectional area can introduce significant band dispersion and distortion. This paper reports a new fabrication technique to form a gradual reduction in cross-sectional area in polymethylmethacrylate (PMMA) microchannel for both anionic and cationic isotachophoresis (ITP). The fabrication technique is based on hot embossing and surface modification assisted bonding method. Both one-dimensional and two-dimensional gradual reduction in cross-sectional area microchannels were formed on PMMA with high fidelity using proposed techniques. ITP experiments were conducted to separate and preconcentrate fluorescent proteins in these microchips. Thousand fold and ten thousand fold increase in concentrations were obtained when 10 × and 100 × gradual reduction in cross-sectional area microchannels were used for ITP.

Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis.

This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms.

10,000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis.

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 µg mL⁻¹ and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL⁻¹. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.

10,000-fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3-D numerical simulation with experimental results.

This paper describes both the experimental application and 3-D numerical simulation of isotachophoresis (ITP) in a 3.2 cm long "cascade" poly(methyl methacrylate) (PMMA) microfluidic chip. The microchip includes 10 × reductions in both the width and depth of the microchannel, which decreases the overall cross-sectional area by a factor of 100 between the inlet (cathode) and outlet (anode). A 3-D numerical simulation of ITP is outlined and is a first example of an ITP simulation in three dimensions. The 3-D numerical simulation uses COMSOL Multiphysics v4.0a to concentrate two generic proteins and monitor protein migration through the microchannel. In performing an ITP simulation on this microchip platform, we observe an increase in concentration by over a factor of more than 10,000 due to the combination of ITP stacking and the reduction in cross-sectional area. Two fluorescent proteins, green fluorescent protein and R-phycoerythrin, were used to experimentally visualize ITP through the fabricated microfluidic chip. The initial concentration of each protein in the sample was 1.995 µg/mL and, after preconcentration by ITP, the final concentrations of the two fluorescent proteins were 32.57 ± 3.63 and 22.81 ± 4.61 mg/mL, respectively. Thus, experimentally the two fluorescent proteins were concentrated by over a factor of 10,000 and show good qualitative agreement with our simulation results.

Impact of leakage current and electrolysis on FET flow control and pH changes in nanofluidic channels.

We have fabricated multiple-internal-reflection Si infrared waveguides integrated with an array of nanochannels sealed with an optically transparent top cover. The channel walls consist of a thin layer of SiO2 for electrical insulation, and gate electrodes surround the channel sidewalls and bottom to manipulate their surface charge and zeta-potential in a fluidic field effect transistor (FET) configuration. This nanofluidic device is used to probe the transport of charged molecules (Alexa 488) and to measure the pH shift in nanochannels in response to an electrical potential applied to the gate. During gate biasing for FET operation, laser-scanning confocal fluorescence microscopy (LS-CFM) is used to visualize the flow of fluorescent dye molecules (Alexa 488), and multiple internal reflection-Fourier transform infrared spectroscopy (MIR-FTIRS) is used to probe the characteristic vibrational modes of fluorescein pH indicator and measure the pH shift. The electroosmotic flow of Alexa 488 is accelerated in response to a negative gate bias, whereas its flow direction is reversed in response to a positive gate bias. We also measure that the pH of buffered electrolyte solutions shifts by as much as a pH unit upon applying the gate bias. With prolonged application of gate bias, however, we observe that the initial response in flow speed and direction as well as pH shift becomes reversed. We attribute these anomalous flow and pH shift characteristics to a leakage current that flows from the Si gate through the thermally grown SiO2 to the electrolyte solution.

Experimentally and theoretically observed native pH shifts in a nanochannel array.

Lab-on-a-chip (LOC) technology provides a powerful platform for simultaneous separation, purification, and identification of low concentration multicomponent mixtures. As the characteristic dimension of LOC devices decreases down to the nanoscale, the possibility of containing an entire lab on a single chip is becoming a reality. This research examines one of the unique physical characteristics of nanochannels, in which native pH shifts occur. As a result of the electrical double layer taking up a significant portion of a 100 nm wide nanochannel, electroneutrality no longer exists in the channel causing a radial pH gradient. This work describes experimentally observed pH shifts as a function of ionic strength using the fluorescent pH indicator 5-(and-6)-carboxy SNARF-1 and compares it to a model developed using Comsol Multiphysics. At low ionic strengths (approximately 3 mM) the mean pH shift is approximately 1 pH unit whereas at high ionic strengths (approximately 150 mM) the mean pH shift is reduced to 0.1 pH units. An independent analysis using fluorescein pH indicator is also presented supporting these findings. Two independent non-linear simulations coupling the Nernst-Planck equation describing transport in ionic solutions subjected to an electric field and Poisson's equation to describe the electric field as it relates to the charge distribution are solved using a finite element solver. In addition, the effects of chemical activities are considered in the simulations. The first numerical simulation is based on a surface zeta-potential which significantly underestimates the experimental results for most ionic strengths. A modified model assuming that SNARF and fluorescein molecules are able to diffuse into the hydrolyzed SiO2 phase, and in the case of the SNARF molecule, able to bind to neutral regions of the SiO2 phase agrees quantitatively with experimental results.

Effects of increased voltage on resolution in preparative isoelectric focusing of myoglobin varia.

IEF is a powerful technique which separates proteins and other amphoteric solutes in a pH gradient according to their pI's. The current work evaluates the effect on resolution of increasing electric fields in a novel preparative, vortex-stabilized electrophoresis device. In shallow gradients spanning one pH unit, the variants of myoglobin were separated at applied voltages from 10 to 15 kV. Digital imaging of these separations indicated a 20% reduction in bandwidth and a 60% increase in resolution as the electric field strength is varied across this range. These results were confirmed by IEF-PAGE and ion-exchange chromatography.

On-line optical fiber detection in a preparative free-flow electrofocusing apparatus.

Many analytical isoelectric focusing (IEF) instruments are equipped with on-line detection, e.g., conductivity or UV-vis absorbance. Most of their preparative counterparts have not integrated such detection systems and thus require labor-intensive off-line analysis to quantify separation results. This paper describes the incorporation of an optical fiber based, on-line detection system that allows one to follow the evolution of the protein bands in a preparative IEF apparatus. An array of four optical fibers was designed to deliver light to the annulus of a free-flow electrophoresis apparatus, to detect the transmitted light passing through the separation media and to determine the protein concentration at vertical positions along the annulus of a vortex-stabilized focusing chamber using a 1024 bit CCD line camera. The final concentration of the major myoglobin band was 21.0 mg/mL at electric field strengths as high as 333 V/cm. Spectrophotometric analysis indicated a final concentration of 18.9 mg/mL, 10% less than that reported by the optical fibers.