Lab-chip HPLC with integrated droplet-based microfluidics for separation and high frequency compartmentalisation

We demonstrate the integration of a droplet-based microfluidic device with high performance liquid chromatography (HPLC) in a monolithic format. Sequential operations of separation, compartmentalisation and concentration counter were conducted on a monolithic chip. This describes the use of droplet-based microfluidics for the preservation of chromatographic separations, and its potential application as a high frequency fraction collector.     

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.     

Electrochemical droplet-based microfluidics using chip-based carbon paste electrodes for high-throughput analysis in pharmaceutical applications

This paper presents the first example of a pharmaceutical application of droplet-based microfluidics coupled with chronoamperometric detection using chip-based carbon paste electrodes (CPEs) for determination of dopamine (DA) and ascorbic acid (AA). Droplets were generated using an oil flow rate of 1.80 μL min⁻¹, whereas a flow rate of 0.80 μL min⁻¹ was applied to the aqueous phase, which resulted in a water fraction of 0.31. The optimum applied potential for chronoamperometric measurements in droplets was found to be 150 mV. Highly reproducible analysis of DA and AA was achieved with relative standard deviations of less than 5% for both intra-day and inter-day measurements. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 20 and 70 μM for DA and 41 and 137 μM for AA, respectively. Linearity of this method was in the ranges of 0.02–3.0 mM for DA and 0.04–3.0 mM for AA. This system was successfully applied to determine the amounts of DA and AA in intravenous drugs. Calibration curves of DA and AA for quantitative analysis were obtained with good linearity with R² values of 0.9984 and 0.9988, respectively. Compared with the labeled amounts, the measured concentrations of DA and AA obtained from this system were insignificantly different, with error percentages of less than ±3.0%, indicating a high accuracy of the developed method.     

A feedback control system for high-fidelity digital microfluidics

Digital microfluidics (DMF) is a technique in which discrete droplets are manipulated by applying electrical fields to an array of electrodes. In an ideal DMF system, each application of driving potential would cause a targeted droplet to move onto an energized electrode (i.e., perfect fidelity between driving voltage and actuation); however, in real systems, droplets are sometimes observed to resist movement onto particular electrodes. Here, we implement a sensing and feedback control system in which all droplet movements are monitored, such that when a movement failure is observed, additional driving voltages can be applied until the droplet completes the desired operation. The new system was evaluated for a series of liquids including water, methanol, and cell culture medium containing fetal bovine serum, and feedback control was observed to result in dramatic improvements in droplet actuation fidelity and velocity. The utility of the new system was validated by implementing an enzyme kinetics assay with continuous mixing. The new platform for digital microfluidics is simple and inexpensive and thus should be useful for scientists and engineers who are developing automated analysis platforms.     

Interfacing droplet microfluidics with chemical separation for cellular analysis

This trends article discusses the interface between droplet microfluidics and micro-scale chemical separation. Droplet microfluidics has witnessed explosive growth over the past few years, but the use of droplets to facilitate chemical separation is still in its infancy. This article reviews the current state-of-the-art in this new area and provides an outlook on the role of this new technique in cellular analysis.     

Intelligent droplet tracking with correlation filters for digital microfluidics

Tracking the movement of droplets in digital microfluidics is essential to improve its control stability and obtain dynamic information for its applications such as point-of-care testing, environment monitoring and chemical synthesis. Herein, an intelligent, accurate and fast droplet tracking method based on machine vision is developed for applications of digital microfluidics. To continuously recognize the transparent droplets in real-time and avoid the interferes from background patterns or inhomogeneous illumination, we introduced the correlation filter tracker, enabling online learning of the multi-features of the droplets in Fourier domain. Results show the proposed droplet tracking method could accurately locate the droplets. We also demonstrated the capacity of the proposed method for estimation of the droplet velocity as faster as 20 mm/s, and its application in online monitoring the Griess reaction for both colorimetric assay of nitrite and study of reaction kinetics.     

Thermally responsive phospholipid preparations for fluid steering and separation in microfluidics

Aqueous phospholipid preparations comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) are prevalent materials for biological characterization and become gel-like near physiological temperature, but have a low viscosity below 24°C. The rheology of 20% phospholipid preparations of [DMPC]/[DHPC] = 2.5 reveals that, under conditions utilized for fluid steering, the materials are shear-thinning power-law fluids with a power-law index ranging from 0.30 through 0.90. Phospholipid preparations are utilized to steer fluids in microfluidic chips and support hydrodynamic delivery of sample across a double T injection region in a chip. The fact that the phospholipids are fully integrated as a valving material as well as a separation medium is demonstrated through the separation of linear oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonic acid.     

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5′-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 × 10⁻²% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 × 10⁻²%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.     

Multi-step dielectrophoresis for separation of particles

A new concept for separation of particles based on repetitive dielectrophoretic trapping and release in a flow system is proposed. Calculations using the finite element method have been performed to envision the particle behavior and the separation effectiveness of the proposed method. As a model system, polystyrene beads in deionized water and a micro-flow channel with arrays of interdigited electrodes have been used. Results show that the resolution increases as a direct function of the number of trap-and-release steps, and that a difference in size will have a larger influence on the separation than a difference in other dielectrophoretic properties. About 200 trap-and-release steps would be required to separate particles with a size difference of 0.2%. The enhanced separation power of dielectrophoresis with multiple steps could be of great importance, not only for fractionation of particles with small differences in size, but also for measuring changes in surface conductivity, or for separations based on combinations of difference in size and dielectric properties.     

Electrohydrodynamics of charge separation in droplet-based ion sources with time-varying electrical and mechanical actuation

Charge transport and separation in mechanically-driven, droplet-based ion sources are investigated using computational analysis and supporting experiments. A first-principles model of electrohydrodynamics (EHD) and charge migration is formulated and implemented using FLUENT CFD software for jet/droplet formation. For validation, classical experiments of electrospraying from a thin capillary are simulated, specifically, the transient EHD cone-jet formation of a fluid with finite electrical conductivity, and the Taylor cone formation in a perfectly electrically-conducting fluid. The model is also used to investigate the microscopic physics of droplet charging in mechanically-driven droplet-based ion sources, such as array of micromachined ultrasonic electrospray (AMUSE). Here, AMUSE is subject to DC and AC electric fields of varying amplitude and phase, with respect to a time-varying mechanical force driving the droplet formation. For the DC-charging case, a linear relationship is demonstrated between the charge carried by each droplet and an applied electric field magnitude, in agreement with previously reported experiments. For the AC-charging case, a judiciously-chosen phase-shift in the time-varying mechanical (driving ejection) and electrical (driving charge transport) signals allows for a significantly increased amount of charge, of desired polarity, to be pumped into a droplet upon ejection. Complementary experimental measurements of electrospray electrical current and charge-per-droplet, produced by the AMUSE ion source, are performed and support theoretical predictions for both DC- and AC-charging cases. The theoretical model and simulation tools provide a versatile and general analytical framework for fundamental investigations of coupled electrohydrodynamics and charge transport. The model also allows for the exploration of different configurations and operating modes to optimize charge separation in atmospheric pressure electrohydrodynamic ion sources under static and dynamic electrical and mechanical fields.     

Two-column simulated moving-bed process for binary separation

A two-column simulated moving-bed system has been developed for binary separation. The system combines a flexible node design, robust pump configuration, and cyclic flow-rate modulation to exploit the benefits of simulated counter-current operation. The feasibility of the proposed two-column system is demonstrated on the linear separation of two nucleosides by reversed phase. Emphasis is given to the potentialities of the process compared to single-column batch chromatography with recycling for the same amount of stationary phase. The performance of the proposed two-column process is verified with laboratory-scale experiments and detailed simulations for different difficulties in separation and desorbent-to-feed ratios.     

All-electronic droplet generation on-chip with real-time feedback control for EWOD digital microfluidics

Electrowetting-on-dielectric (EWOD) actuation enables digital (or droplet) microfluidics where small packets of liquids are manipulated on a two-dimensional surface. Due to its mechanical simplicity and low energy consumption, EWOD holds particular promise for portable systems. To improve volume precision of the droplets, which is desired for quantitative applications such as biochemical assays, existing practices would require near-perfect device fabrication and operation conditions unless the droplets are generated under feedback control by an extra pump setup off of the chip. In this paper, we develop an all-electronic (i.e., no ancillary pumping) real-time feedback control of on-chip droplet generation. A fast voltage modulation, capacitance sensing, and discrete-time PID feedback controller are integrated on the operating electronic board. A significant improvement is obtained in the droplet volume uniformity, compared with an open loop control as well as the previous feedback control employing an external pump. Furthermore, this new capability empowers users to prescribe the droplet volume even below the previously considered minimum, allowing, for example, 1 : x (x < 1) mixing, in comparison to the previously considered n : m mixing (i.e., n and m unit droplets).     

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

Functionalized polymer particles for chiral separation

The synthetic chiral polymer poly(N-acryloyl-S-phenylalanine ethyl ester) was immobilized by grafting to macroporous polymer particles of various composition and structure in a process involving copolymerization of the chiral monomer with residual double bonds present in the macroporous support particles. The support particles were prepared by suspension or micro- suspension polymerization of trimethylolpropane trimethacrylate (TRIM), divinylbenzene or by copolymerization of styrene and TRIM. The maximum amount of immobilized chiral polymer and the mechanical properties of the resulting materials varied with the swelling capacity of the parent support particles. Up to 60% (w/w) of chiral polymer could be immobilized to the pore system of highly cross-linked TRIM particles. The enantioselectivity of the chiral stationary phases increased with increase in the amount of immobilized chiral polymer. The results of studies of porosity and particle size variation during grafting form the basis for a discussion of the structure of the final materials.     

Integrated electrofluidic circuits: pressure sensing with analog and digital operation functionalities for microfluidics

Microfluidic technology plays an essential role in various lab on a chip devices due to its desired advantages. An automated microfluidic system integrated with actuators and sensors can further achieve better controllability. A number of microfluidic actuation schemes have been well developed. In contrast, most of the existing sensing methods still heavily rely on optical observations and external transducers, which have drawbacks including: costly instrumentation, professional operation, tedious interfacing, and difficulties of scaling up and further signal processing. This paper reports the concept of electrofluidic circuits - electrical circuits which are constructed using ionic liquid (IL)-filled fluidic channels. The developed electrofluidic circuits can be fabricated using a well-developed multi-layer soft lithography (MSL) process with polydimethylsiloxane (PDMS) microfluidic channels. Electrofluidic circuits allow seamless integration of pressure sensors with analog and digital operation functions into microfluidic systems and provide electrical readouts for further signal processing. In the experiments, the analog operation device is constructed based on electrofluidic Wheatstone bridge circuits with electrical outputs of the addition and subtraction results of the applied pressures. The digital operation (AND, OR, and XOR) devices are constructed using the electrofluidic pressure controlled switches, and output electrical signals of digital operations of the applied pressures. The experimental results demonstrate the designed functions for analog and digital operations of applied pressures are successfully achieved using the developed electrofluidic circuits, making them promising to develop integrated microfluidic systems with capabilities of precise pressure monitoring and further feedback control for advanced lab on a chip applications.     

Ostwald ripening of binary alloy particles

Classical Lifshitz-Slyozov-Wagner theory is generalized for Ostwald ripening of particles composed of random binary alloy. We show that the steady state ripening process is characterized by self-similar particle size and composition distributions. The shape of particle size distribution depends on whether the process is diffusion controlled (Lifshitz-Slyozov) or reaction controlled (Wagner) and is consistent with the predictions of classical theory for one-component materials. The steady state composition distribution, in contrast, has the same functional form in both extreme cases featuring a universal dependence of the composition upon particle size. We also found that transients in particle's composition can be very quick resulting in a steady state distribution well before it is reached by particles sizes. These transients involve significant changes in particle sizes and open an opportunity for producing metastable particle size distributions of required shape.     

Peptide inhibitor modified magnetic particles for pepsin separation

Synthetic heptapeptide containing D-amino acid residues (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu) was coupled to glyoxal-activated magnetic agarose particles via the free peptide amino group. The peptide-modified magnetic particles were used for the separation of pepsins. Porcine pepsin A and human pepsin A were adsorbed to the magnetic peptide-modified affinity carrier, while the rat pepsin C and human pepsin C did not interact with the immobilized ligand. Conditions of pepsin adsorption to peptide-modified magnetic particles, as well as elution buffers were optimized. Porcine pepsin A did not interact with the immobilized peptide in the presence of pepsin inhibitor pepstatin A, indicating that the enzyme binding site is involved in the studied interaction. The elaborated method represents a rapid and simple technique not only for the separation of pepsins but also, in combination with MS, for the enzyme detection and determination.     

Dielectrophoresis for manipulation of micro/nano particles in microfluidic systems

Dielectrophoretic (DEP) force is exerted when a neutral particle is polarized in a non-uniform electric field, and depends on the dielectric properties of the particle and the suspending medium. The integration of DEP and microfluidic systems offers numerous applications for the separation, trapping, assembling, transportation, and characterization of micro/nano particles. This article reviews the applications of DEP forces in microfluidic systems. It presents the theory of dielectrophoresis, different configurations, and the applications of such systems for particle manipulation and device fabrication.