Microfluidics-based Method Research Articles

Creating a capture zone in microfluidic flow greatly enhances the throughput and efficiency of cancer detection

Efficient capture of rare circulating tumor cells (CTCs) from blood samples is valuable for early cancer detection to improve the management of cancer. In this work, we developed a highly efficient microfluidics-based method for detecting CTCs in human blood. This is achieved by creating separate capture and flow zones in the microfluidic device (ZonesChip) and using patterned dielectrophoretic force to direct cells from the flow zone into the capture zone. This separation of the capture and flow zones minimizes the negative impact of high flow speed (and thus high throughput) and force in the flow zone on the capture efficiency, overcoming a major bottleneck of contemporary microfluidic approaches using overlapping flow and capture zones for CTC detection. When the flow speed is high (≥0.58 mm/s) in the flow zone, the separation of capture and flow zones in our ZonesChip could improve the capture efficiency from ∼0% (for conventional device without separating the two zones) to ∼100%. Our ZonesChip shows great promise as an effective platform for the detection of CTCs in blood from patients with early/localized-stage colorectal tumors.

Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study

Membrane fusion is a ubiquitous process in biology and is a prerequisite for many intracellular delivery protocols relying on the use of liposomes as drug carriers. Here, we investigate in detail the process of membrane fusion and the role of opposite charges in a protein-free lipid system based on cationic liposomes (LUVs, large unilamellar vesicles) and anionic giant unilamellar vesicles (GUVs) composed of different palmitoyloleoylphosphatidylcholine (POPC)/palmitoyloleoylphosphatidylglycerol (POPG) molar ratios. By using a set of optical-microscopy- and microfluidics-based methods, we show that liposomes strongly dock to GUVs of pure POPC or low POPG fraction (up to 10 mol%) in a process mainly associated with hemifusion and membrane tension increase, commonly leading to GUV rupture. On the other hand, docked LUVs quickly and very efficiently fuse with negative GUVs of POPG fractions at or above 20 mol%, resulting in dramatic GUV area increase in a charge-dependent manner; the vesicle area increase is deduced from GUV electrodeformation. Importantly, both hemifusion and full fusion are leakage-free. Fusion efficiency is quantified by the lipid transfer from liposomes to GUVs using fluorescence resonance energy transfer (FRET), which leads to consistent results when compared to fluorescence-lifetime-based FRET. We develop an approach to deduce the final composition of single GUVs after fusion based on the FRET efficiency. The results suggest that fusion is driven by membrane charge and appears to proceed up to charge neutralization of the acceptor GUV.

Particle Shape Influences Settling and Sorting Behavior in Microfluidic Domains

We present a new numerical model to simulate settling trajectories of discretized individual or a mixture of particles of different geometrical shapes in a quiescent fluid and their flow trajectories in a flowing fluid. Simulations unveiled diverse particle settling trajectories as a function of their geometrical shape and density. The effects of the surface concavity of a boomerang particle and aspect ratio of a rectangular particle on the periodicity and amplitude of oscillations in their settling trajectories were numerically captured. Use of surrogate circular particles for settling or flowing of a mixture of non-circular particles were shown to miscalculate particle velocities by a factor of 0.9–2.2 and inaccurately determine the particles’ trajectories. In a microfluidic chamber with particles of different shapes and sizes, simulations showed that steady vortices do not necessarily always control particle entrapments, nor do larger particles get selectively and consistently entrapped in steady vortices. Strikingly, a change in the shape of large particles from circular to elliptical resulted in stronger entrapments of smaller circular particles, but enhanced outflows of larger particles, which could be an alternative microfluidics-based method for sorting and separation of particles of different sizes and shapes.

Spring-shaped stimuli-responsive hydrogel actuator with large deformation

This study describes a novel microfluidics-based method for compressive/expanding actuation of stimuli-responsive hydrogel microsprings with large deformations. A continuous flow of mixed alginate and poly(N-isopropylacrylamide-co-acrylic acid) pre-gel solution can spontaneously form a hydrogel microspring with a wide range of gradient pitches via buoyancy force. This technique enables fabrication of hydrogel microsprings using only simple capillaries and syringe pumps. The resulting microsprings can be patterned via laminar flow inside the capillary, which can contribute to large deformation. Single-layered hydrogel microsprings shrunk isotropically while maintaining the shape of the spring. Compressing stimuli-responsive microsprings can be done by patterning the shrinking part of the spring. Here, the degree of compression in the double-layered spring depends on the initial pitch. Furthermore, large axial expansion of microsprings can be achieved by shrinking part of a microspring. Our large compression/expansion stimuli-responsive hydrogel microsprings have immense potential to be applied in various microengineering products including soft actuators, chemical sensors, and medical applications.

Abstract 2144: A novel method for high-throughput discovery of neo-antigens and corresponding T-cell receptors

Abstract Immunotherapies that boost the ability of endogenous T cells to destroy cancer cells have demonstrated therapeutic efficacy in several human malignancies. Cytotoxic T cells actively recognize peptide antigens, which are displayed on major histocompatibility complexes (MHCs) on the surface of the malignant cells, to play a central role in cancer immunotherapy. However, efficient methods for identifying the tumor antigens and the corresponding T cell receptors are still limited. We report here a DNA-barcode nanoparticle and microfluidics-based method to sort and enumerate neoantigen-specific T cells, at an unprecedented level of sensitivity. The method also permits the matching of a single neoantigen to a specific CD8+ T cell with the TCR a/b gene sequence. The resultant information informs an immunotherapy regimen that is specific for that patient, which can be used to custom-design effective ACT therapies. Citation Format: Songming Peng, Jesse Zaretsky, Michael T. Bethune, Alice Hsu, David Baltimore, Antoni Ribas, James Heath. A novel method for high-throughput discovery of neo-antigens and corresponding T-cell receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2144. doi:10.1158/1538-7445.AM2017-2144

Self-assembled polysulfone nanoparticles using microfluidic chip

This article reports a microfluidics-based method for the preparation of polysulfone nanoparticles. Upon mixing polysulfone in a good solvent with pure water, polysulfone undergoes a coil-globule transition and forms nanometer-sized soft particles. A key to achieving small nanoparticle sizes is to change the solvent quality within a time frame shorter than the typical time of aggregation between polysulfone. Accordingly, we devised microfluidic devices relying on hydrodynamic flow focusing and we managed to obtain nanoparticle sizes below 100nm, which is a significant improvement compared to latest nanoprecipitation methods We demonstrated the continuous encapsulation of FITC dyes into polysulfone nanoparticles as well and we envision the use of these biocompatible fluorescent polysulfone nanoparticles for biological imaging.

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons.

Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. Here we describe the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly. We describe the design of the master mold, the production of the microfluidic chip and live imaging using the calcium sensor GCaMP, expressed in distinct types of Drosophila chemosensory neurons. Fabrication of the master mold and microfluidic chips requires basic skills in photolithography and takes ∼2 weeks; the same devices can be used repeatedly over several months. Flies can be prepared for measurements in minutes and imaged for up to 1 h.

Octanol-assisted liposome assembly on chip.

Liposomes are versatile supramolecular assemblies widely used in basic and applied sciences. Here we present a novel microfluidics-based method, octanol-assisted liposome assembly (OLA), to form monodisperse, cell-sized (5–20 μm), unilamellar liposomes with excellent encapsulation efficiency. Akin to bubble blowing, an inner aqueous phase and a surrounding lipid-carrying 1-octanol phase is pinched off by outer fluid streams. Such hydrodynamic flow focusing results in double-emulsion droplets that spontaneously develop a side-connected 1-octanol pocket. Owing to interfacial energy minimization, the pocket splits off to yield fully assembled solvent-free liposomes within minutes. This solves the long-standing fundamental problem of prolonged presence of residual oil in the liposome bilayer. We demonstrate the unilamellarity of liposomes with functional α-haemolysin protein pores in the membrane and validate the biocompatibility by inner leaflet localization of bacterial divisome proteins (FtsZ and ZipA). OLA offers a versatile platform for future analytical tools, delivery systems, nanoreactors and synthetic cells.

Vertical Flow Lithography for Fabrication of 3D Anisotropic Particles

A microfluidics-based method for the 3D fabrication of anisotropic particles is reported. The method uses a vertical microchannel where tunable light patterns solidify photocurable resins for stacking multiple layers of the resins, thus enabling an application of stereolithography concepts to conventional flow lithography. Multilayered, tapered, and angular compartmental microparticles are demonstrated.

Automatic Control of Gene Expression in Mammalian Cells.

Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.

Decoupling of mass transport mechanisms in the stagewise swelling of multiple emulsions.

This contribution reports on the mass transport kinetics of osmotically imbalanced water-in-oil-in-water (W1/O/W2) emulsions. Although frequently studied, the control of mass transport in W1/O/W2 emulsions is still challenging. We describe a microfluidics-based method to systematically investigate the impact of various parameters, such as osmotic pressure gradient, oil phase viscosity, and temperature, on the mass transport. Combined with optical microscopy analyses, we are able to identify and decouple the various mechanisms, which control the dynamic droplet size of osmotically imbalanced W1/O/W2 emulsions. So, swelling kinetics curves with a very high accuracy are generated, giving a basis for quantifying the kinetic aspects of transport. Two sequential swelling stages, i.e., a lag stage and an osmotically dominated stage, with different mass transport mechanisms are identified. The determination and interpretation of the different stages are the prerequisite to control and trigger the swelling process. We show evidence that both mass transport mechanisms can be decoupled from each other. Rapid osmotically driven mass transport only takes place in a second stage induced by structural changes of the oil phase in a lag stage, which allow an osmotic exchange between both water phases. Such structural changes are strongly facilitated by spontaneous water-in-oil emulsification. The duration of the lag stage is pressure-independent but significantly influenced by the oil phase viscosity and temperature.

A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.

A microfluidic device for rapid screening of chemotaxis-defective Caenorhabditis elegans mutants

Behavioral Caenorhabditis elegans mutants are sought for the purposes of neurobiological research. Until now, large numbers of worms with neuronal defects have been obtained through mutagenesis techniques. However, the existing screening procedures are not only time-consuming and low-throughput, but also tedious and labor-intensive. Therefore, developing a rapid and convenient method to overcome these difficulties is necessary. The present study demonstrates for the first time a microdevice for the rapid screening of chemotaxis-defective mutants based on their chemotactic response. The microchip is capable of automatic introduction, local immobilization, and controllable generation of concentration gradients during the screening assays. With this device, six C. elegans behavioral assays can be performed using various attractants without requiring anesthetics for local capture, and ten mutants are effectively isolated from 10(4) mutagenized worms in 100 min. The microfluidics-based method is robust enough to sort the chemotaxis-defective worms with 91 % accuracy from a large population of wild type animals during a mutagenesis screen.

A validated microfluidics‐based LC‐chip‐MS/MS method for the quantitation of fluoxetine and norfluoxetine in rat serum

An increasing number of quantitative bioanalyses need to be performed on samples available in limited volumes, such as pharmacokinetic studies on small animals. In this context, microfluidic systems as the LC-chip device coupled to a mass spectrometer combine small sample volume requirements and high sensitivity. In this study, we present the development of a microfluidics-based method for fluoxetine (FLX) and norfluoxetine (NFL) quantitation dedicated to pharmacokinetic investigations in the rat serum. Using the methodology of experimental design, LC parameters were optimised in terms of peak resolution, analysis time, and sensitivity. An SPE method was then developed for serum samples on miniaturised 96-well plates containing a mixed-mode strong cation exchanger that provided very clean extracts with good analyte recovery (≥66.0%). The total SPE-LC-MS/MS process required only 20 μL per sample and the method provided a good sensitivity in a total run time of 12 min. Finally, the developed method for FLX and NFL quantitation in rat serum was fully validated. After having selected the most appropriate regression model on the basis of the accuracy profiles, method selectivity, trueness, precision, accuracy and linearity were demonstrated.

Microscale Strategies for Generating Cell-Encapsulating Hydrogels

Hydrogels in which cells are encapsulated are of great potential interest for tissue engineering applications. These gels provide a structure inside which cells can spread and proliferate. Such structures benefit from controlled microarchitectures that can affect the behavior of the enclosed cells. Microfabrication-based techniques are emerging as powerful approaches to generate such cell-encapsulating hydrogel structures. In this paper we introduce common hydrogels and their crosslinking methods and review the latest microscale approaches for generation of cell containing gel particles. We specifically focus on microfluidics-based methods and on techniques such as micromolding and electrospinning.

A novel microfluidics-based method for probing weak protein–protein interactions

We report the use of a novel microfluidics-based method to detect weak protein-protein interactions between membrane proteins. The tight junction protein, claudin-2, synthesised in vitro using a cell-free expression system in the presence of polymer vesicles as membrane scaffolds, was used as a model membrane protein. Individual claudin-2 molecules interact weakly, although the cumulative effect of these interactions is significant. This effect results in a transient decrease of average vesicle dispersivity and reduction in transport speed of claudin-2-functionalised vesicles. Polymer vesicles functionalised with claudin-2 were perfused through a microfluidic channel and the time taken to traverse a defined distance within the channel was measured. Functionalised vesicles took 1.19 to 1.69 times longer to traverse this distance than unfunctionalised ones. Coating the channel walls with protein A and incubating the vesicles with anti-claudin-2 antibodies prior to perfusion resulted in the functionalised vesicles taking 1.75 to 2.5 times longer to traverse this distance compared to the controls. The data show that our system is able to detect weak as well as strong protein-protein interactions. This system offers researchers a portable, easily operated and customizable platform for the study of weak protein-protein interactions, particularly between membrane proteins.

Smoking of crack cocaine as a risk factor for HIV infection among people who use injection drugs

Little is known about the possible role that smoking crack cocaine has on the incidence of HIV infection. Given the increasing use of crack cocaine, we sought to examine whether use of this illicit drug has become a risk factor for HIV infection. We included data from people participating in the Vancouver Injection Drug Users Study who reported injecting illicit drugs at least once in the month before enrolment, lived in the greater Vancouver area, were HIV-negative at enrolment and completed at least 1 follow-up study visit. To determine whether the risk of HIV seroconversion among daily smokers of crack cocaine changed over time, we used Cox proportional hazards regression and divided the study into 3 periods: May 1, 1996-Nov. 30, 1999 (period 1), Dec. 1, 1999-Nov. 30, 2002 (period 2), and Dec. 1, 2002-Dec. 30, 2005 (period 3). Overall, 1048 eligible injection drug users were included in our study. Of these, 137 acquired HIV infection during follow-up. The mean proportion of participants who reported daily smoking of crack cocaine increased from 11.6% in period 1 to 39.7% in period 3. After adjusting for potential confounders, we found that the risk of HIV seroconversion among participants who were daily smokers of crack cocaine increased over time (period 1: hazard ratio [HR] 1.03, 95% confidence interval [CI] 0.57-1.85; period 2: HR 1.68, 95% CI 1.01-2.80; and period 3: HR 2.74, 95% CI 1.06-7.11). Smoking of crack cocaine was found to be an independent risk factor for HIV seroconversion among people who were injection drug users. This finding points to the urgent need for evidence-based public health initiatives targeted at people who smoke crack cocaine.

Macroscopic-scale carbon nanotube alignment via self-assembly in lyotropic liquid crystals

By dispersing carbon nanotubes (CNTs) in a lyotropic liquid crystalline matrix, uniaxial alignment of the nanotubes can easily be achieved over macroscopic areas. We briefly describe the principles behind the technique and then show that it can be applied to multiwall as well as single-wall nanotubes and that a variety of different dispersing materials can be used, from industrial surfactants to DNA. We also present a new microfluidics-based method for transferring the liquid crystal-dispersed CNTs to a substrate, maintaining a fair control of tube direction.

PROCAM Study: risk prediction for myocardial infarction using microfluidic high-density lipoprotein (HDL) subfractionation is independent of HDL cholesterol

High-density lipoprotein (HDL) subfractions are among the new emerging risk factors for atherosclerosis. In particular, HDL 2b has been shown to be linked to cardiovascular risk. This study uses a novel microfluidics-based method to establish HDL 2b clinical utility using samples from the Prospective Cardiovascular Muenster (PROCAM) Study. Method performance was established by measuring accuracy, precision, linearity and inter-site precision. Serum samples from 503 individuals collected in the context of the PROCAM study were analyzed by electrophoresis on a microfluidics system. Of these, 251 were male survivors of myocardial infarction (cases), while 252 individuals were matched healthy controls. HDL cholesterol, HDL 2b concentration and HDL 2b percentage were analyzed. This novel method showed satisfactory assay performance with an inter-site coefficient of variance of <10% for HDL 2b percentage. Parallel patient testing on 52 samples between two sites resulted in a correlation coefficient of r=0.95. Significant differences were observed in the HDL 2b subfraction between cases and controls independent of other risk factors. Including HDL 2b percentage in logistic regression reduced the number of false positives from 64 to 39 and the number of false negative cases from 48 to 45, in the context of this study. The novel method showed satisfactory assay performance in addition to drastically reduced analysis times and improved ease of use as compared to other methods. Clinical utility of HDL 2b was demonstrated supporting the findings of previous studies.

Measurement of enzyme kinetics using a continuous-flow microfluidic system.

This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.