Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (18)
- Angewandte Chemie (International Ed. in English)
- Analytical Chemistry
- Biosensors & Bioelectronics
- Lab on a Chip
- Nature Materials
- Cell Cycle (Georgetown, Tex.)
- Lab on a Chip
- Lab on a Chip
- Analytical Chemistry
- Nature Biotechnology
- Lab on a Chip
- Lab on a Chip
- Methods in Molecular Biology (Clifton, N.J.)
- Lab on a Chip
- Analytical Chemistry
- Lab on a Chip
- Nature Communications
- Molecular & Cellular Proteomics : MCP
Articles by David Juncker in JoVE
JoVE General
The Microfluidic Probe: Operation and Use for Localized Surface Processing
Department of Biomedical Engineering, McGill University
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.
Other articles by David Juncker on PubMed
Autonomous Microfluidic Capillary System
The transport of minute amounts of liquids using microfluidic systems has opened avenues for higher throughput and parallelization of miniaturized bio/chemical processes combined with a great economy of reagents. In this report, we present a microfluidic capillary system (CS) that autonomously transports aliquots of different liquids in sequence: liquids pipetted into the service port of the CS flow unidirectionally through the various sections of the CS, which comprises a 15-pL reaction chamber, into the capillary pump. A CS can thus be operated by simply delivering the different samples to its service port. The liquid transport concept presented here is advantageous because the pumping and valving functions are integrated into the device by means of capillary phenomena, and it therefore does not require any external power supply or control device. Thus, arrays of CSs can easily be formed by cloning a functional CS. Alternatively, the flow of liquids in CSs can also be interactively tuned if desired by (i) forcing the evaporating of liquid out of the capillary pumps and (ii) by contacting a secondary, removable capillary pump to the embedded ones. We illustrate the possibilities of CSs by conducting a surface immunoassay for a cardiac marker, within 25 min, on an area of 100 x 100 microm2, using 16 sequential filling steps.
Simultaneous Detection of C-reactive Protein and Other Cardiac Markers in Human Plasma Using Micromosaic Immunoassays and Self-regulating Microfluidic Networks
We show a proof-of-concept in which we combine our previously published concepts of micromosaic immunoassays (microMIAs) with self-regulating microfluidic networks (microFNs) to detect C-reactive protein (CRP) and other cardiac markers such as myoglobin (Mb) and cardiac Troponin I (cTnI). The microFNs are microfabricated in Si, have a well-defined surface chemistry, and are affixed to a bibulous material so as to self-regulate the displacement of an aliquot of liquid through the microFNs using capillary forces. An open section of the channels of the microFNs is covered with a hydrophobic poly(dimethylsiloxane) (PDMS) slab that acts as the substrate for a solid-phase immunoassay. Here, individual assays are conducted using independent channels. These assays are "sequential": series of samples, reagents, and buffers are displaced one after the other over the PDMS surface, and, as these assays are conducted under "microfluidic" conditions, they are fast to perform, very economical in their use of reagents, extremely integrated, and yield high-quality signals. The combinatorial character of microMIAs is exploited to optimize the assay parameters for detecting CRP. In particular, we found it optimal to deposit the capture antibody for CRP on PDMS at a concentration between 20 and 500 microg ml(-1) in PBS in 1 min and to detect captured CRP in 2 min using a detection antibody having a concentration in PBS of 120 microg ml(-1). With this method, CRP is quantitatively detected within 10 min in one microliter of human plasma down to concentrations of 30 ng ml(-1), which suggests the possibility to detect CRP at clinically relevant concentrations for the management of coronary heart disease (CHD) and systemic inflammation.
High-sensitivity Miniaturized Immunoassays for Tumor Necrosis Factor Alpha Using Microfluidic Systems
We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).
Multipurpose Microfluidic Probe
Microfluidic systems allow (bio)chemical processes to be miniaturized with the benefit of shorter time-to-result, parallelism, reduced sample consumption, laminar flow, and increased control and efficiency. However, such miniaturization inherently limits the size of the solid objects that can be processed and entails new challenges such as the interfacing of macroscopic samples with microscopic conduits. Here, we report a microfluidic probe (MFP) that overcomes these problems by combining the concepts of 'microfluidics' and of 'scanning probes'. Here, liquid boundaries formed by hydrodynamic forces underneath the MFP confine a flow of processing solution and replace the solid walls of closed microchannels. The MFP is therefore mobile and can be used to process large surfaces and objects by scanning across them. We illustrate the versatility of this concept with several examples including protein microarraying, complex gradient-formation, multiphase laminar-flow patterning, erasing, localized staining of cells and the contact-free detachment of a single cell. Many constraints imposed by the monolithic construction of microfluidic channels can now be circumvented using an MFP, opening up new avenues for microfluidic processing.
GAP-43 is Key to Mitotic Spindle Control and Centrosome-based Polarization in Neurons
In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.
Chamber and Microfluidic Probe for Microperfusion of Organotypic Brain Slices
Microfluidic systems are increasingly being used for the culture and study of dissociated cells because they require only minute amounts of materials while enabling drug screening and chemotaxis studies down to the single cell level. However, the culture of organized tissue, such as brain slices, has been more difficult to adapt to microfluidic devices. Here, we present a microfluidic system, comprising (i) a perfusion chamber for the culture of organotypic slices that is compatible with high resolution imaging on inverted microscopes, and (ii) a novel transparent microfluidic probe (MFP) for the localized microperfusion of the brain tissue. The MFP is made in poly(dimethylsiloxane), features six micrometre-scale apertures and can be assembled within a few hours in a standard laboratory. Each aperture can indiscriminately be used either for the injection or aspiration of solutions, giving rise to many possible combinations. The MFP was successfully used for the perfusion of a small number of cells in a brain slice with concurrent confocal fluorescence imaging of the perfused dye and sub-cellular structures within the tissue.
Wet-etching of Structures with Straight Facets and Adjustable Taper into Glass Substrates
Wet etching of glass by hydrofluoric acid is widely used in microfabrication, but is limited by the isotropic nature of the process that leads to rounded sidewalls and a 90 degrees angle between the etch front and the surface of the substrate. For many applications such as microvalving, or for further processing such as spin-coating, well controlled, gently sloping sidewalls are often preferred. Here, we present a new approach for forming straight facets and for adjusting the sidewall angle in wet-etched glass substrates by controlling the lateral dissolution of an etch mask during etching. The etch mask comprises a Ti-Au bilayer where Au serves to protect the Ti. During isotropic etching of glass by HF the Ti layer is etched away laterally at the same time, which leads to straight, gently sloping sidewalls. We introduce two methods for controlling the sidewall angle. The first one is based on adjusting the thickness of Ti which controls the lateral etch rate, and thus the angle; the thinner the Ti, the slower its lateral etch rate and the steeper the angle in the etched glass. The second method involves a cathodic bias applied to the Ti-Au etch mask which again regulates the dissolution rate of Ti; the more negative the bias the slower the lateral etch rate. Both methods offer accurate control of the sidewall angle over a wide range, can be readily integrated into existing fabrication processes, and will be particularly useful for making channels with trapezoidal cross-sections, valve seats with gentle profiles, or for patterning electrodes across and inside of microfluidic channels.
Addressable Nanowell Arrays Formed Using Reversibly Sealable Hybrid Elastomer-metal Stencils
There are two major array formats used in life science research and biomedical analysis. The first is the microwell plate format with millimeter-sized wells each with microliter capacity addressed individually and repeatedly during experiments. The second is the microarray format with micrometer-sized spots that are patterned initially but not addressable individually thereafter. Here, we present an addressable nanoliter-well plate with micrometer sized wells that combines the advantages of the two array formats. The nanowells are formed by reversibly sealing a steel stencil featuring an array of micrometer-scale openings to an optically transparent substrate. The nanowells have a capacity of approximately 1 nL, are approximately 140 microm in diameter, and are arrayed at a density of 1600 wells cm(-2). A soft polymer is patterned photolithographically around each opening so as to form a microgasket for pressure sensitive, liquid tight, and reversible sealing to any type of smooth substrate, either hydrophilic or hydrophobic. The rigidity of the steel prevents the distortion that occurs in soft, all-polymeric stencils and permits accurate registration across the entire array, which in turn allows for repeated, individual addressing of wells using an inkjet spotter. The stencils are used to pattern cells, make protein microarrays, and create nanowells on surfaces to study reverse transfection by first spotting plasmids encoding fluorescent proteins into the wells, seeding cells, and monitoring the transfection of the cells in real time using time-lapse imaging. The hybrid elastomer-metal stencils (HEMSs) are versatile and useful for multiplexed analysis of drugs, biomolecules, and cells with microarray density.
Microfluidic Perfusion System for Culturing and Imaging Yeast Cell Microarrays and Rapidly Exchanging Media
High resolution live cell microscopy is increasingly used to detect cellular dynamics in response to drugs and chemicals, but it depends on complex and expensive liquid handling devices that have limited its wider adoption. Here, we present a microfluidic perfusion system that is built without using specialized microfabrication infrastructure, simple to use because only a pipette is needed for liquid handling, and yet allows for rapid media exchange and simultaneous fluorescence microscopy imaging. Yeast cells may be introduced from a culture, or spotted as arrays on a coverslip, and are sandwiched with a 20 mum thick track-etched membrane. A second coverslip and a mesh with 120 mum porosity are placed on top, forming a microfluidic conduit for lateral flow of solutions by capillary effects. Solutions introduced through the inlet flow through the mesh and chemicals diffuse vertically across the membrane to the cells trapped below. Solutions are exchanged by adding a new sample to the inlet. Using this system, we studied the dynamic response of F-actin in living yeast expressing Sac6-EGFP-a protein associated with discrete F-actin structures called "patches"-to the drug latrunculin A, a well known inhibitor of actin polymerization. We observed that the patches disappeared in 85% of the cells within 5 min, and re-assembled in 45 min following exchange of the drug with media. The perfusion system presented here is a simple, inexpensive device suited for analysis of drug dose-response and regeneration of single cells and arrays of cells.
PDMS Microfluidic Capillary Systems for Patterning Proteins on Surfaces and Performing Miniaturized Immunoassays
In this chapter, we describe the fabrication and use of microfluidic capillary systems (CSs) made in soft, transparent polydimethylsiloxane (PDMS). Sixteen microfluidic CSs, each containing a loading pad, a microchannel, and a capillary pump are engraved in a single PDMS chip. The CSs are used for two applications, firstly to pattern fibronectin on glass surfaces to locally control the adhesion of cultured cells to the substrate, and secondly to carry out multiplexed miniaturized immunoassays.
Hydrogel Droplet Microarrays with Trapped Antibody-functionalized Beads for Multiplexed Protein Analysis
Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 µm, 0.5 µm and 1 µm diameter were tested and 1 µm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.
Taguchi Design-based Optimization of Sandwich Immunoassay Microarrays for Detecting Breast Cancer Biomarkers
Taguchi design, a statistics-based design of experiment method, is widely used for optimization of products and complex production processes in many different industries. However, its use for antibody microarray optimization has remained underappreciated. Here, we provide a brief explanation of Taguchi design and present its use for the optimization of antibody sandwich immunoassay microarray with five breast cancer biomarkers: CA15-3, CEA, HER2, MMP9, and uPA. Two successive optimization rounds with each 16 experimental trials were performed. We tested three factors (capture antibody, detection antibody, and analyte) at four different levels (concentrations) in the first round and seven factors (including buffer solution, streptavidin-Cy5 dye conjugate concentration, and incubation times for five assay steps) with two levels each in the second round; five two-factor interactions between selected pairs of factors were also tested. The optimal levels for each factor as measured by net assay signal increase were determined graphically, and the significance of each factor was analyzed statistically. The concentration of capture antibody, streptavidin-Cy5, and buffer composition were identified as the most significant factors for all assays; analyte incubation time and detection antibody concentration were significant only for MMP9 and CA15-3, respectively. Interactions between pairs of factors were identified, but were less influential compared with single factor effects. After Taguchi optimization, the assay sensitivity was improved between 7 and 68 times, depending on the analyte, reaching 640 fg/mL for uPA, and the maximal signal intensity increased between 1.8 and 3 times. These results suggest that Taguchi design is an efficient and useful approach for the rapid optimization of antibody microarrays.
Microfluidics Made of Yarns and Knots: from Fundamental Properties to Simple Networks and Operations
We present and characterize cotton yarn and knots as building blocks for making microfluidic circuits from the bottom up. The yarn used is made up of 200-300 fibres, each with a lumen. Liquid applied at the extremity of the yarn spontaneously wets the yarn, and the wetted length increases linearly over time in untreated yarn, but progresses according to a square root relationship as described by Washburn's equation upon plasma activation of the yarn. Knots are proposed for combining, mixing and splitting streams of fluids. Interestingly, the topology of the knot controls the mixing ratio of two inlet streams into two outlet yarns, and thus the ratio can be adjusted by choosing a specific knot. The flow resistance of a knot is shown to depend on the force used to tighten it and the flow resistance rapidly increases for single-stranded knots, but remains low for double-stranded knots. Finally, a serial dilutor is made with a web made of yarns and double-stranded overhand knots. These results suggest that yarn and knots may be used to build low cost microfluidic circuits.
Microfluidic Quadrupole and Floating Concentration Gradient
The concept of fluidic multipoles, in analogy to electrostatics, has long been known as a particular class of solutions of the Navier-Stokes equation in potential flows; however, experimental observations of fluidic multipoles and of their characteristics have not been reported yet. Here we present a two-dimensional microfluidic quadrupole and a theoretical analysis consistent with the experimental observations. The microfluidic quadrupole was formed by simultaneously injecting and aspirating fluids from two pairs of opposing apertures in a narrow gap formed between a microfluidic probe and a substrate. A stagnation point was formed at the centre of the microfluidic quadrupole, and its position could be rapidly adjusted hydrodynamically. Following the injection of a solute through one of the poles, a stationary, tunable, and movable-that is, 'floating'-concentration gradient was formed at the stagnation point. Our results lay the foundation for future combined experimental and theoretical exploration of microfluidic planar multipoles including convective-diffusive phenomena.
Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples
DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (i) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (ii) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same 2-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, multiplex antibody microarray. Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.
