Simple add-on devices to enhance the efficacy of conventional surface immunoassays implemented on standard labware.

We propose microfluidic add-ons that can be easily added onto standard assay labware such as microwells and slides to enhance the kinetics of immunoassays. We design these monolithic devices having structures that leverage the pipetting step to deliver reagents with deterministic, uniform and strong advection close to the reaction surface. This flow-driven mass transport enhances the flux of analytes to the reaction site and reduces the depletion layer. We demonstrate large gains in time-to-result (5 min instead of 1 h) and/or the sensitivity of immunoassays (approx. 1 order of magnitude), high signal homogeneity and low reagent use by recirculating µL volumes. The impact of this approach on standard immunoassay practice is minimal, preserving both assay labware and dispensing/reading equipment. The devices are compatible with mass production in plastic, offering a solution to enhance the results of conventional assays using well-established protocols and automated analyzer platforms.

Engineering solutions for biological studies of flow-exposed endothelial cells on orbital shakers.

Shear stress is extremely important for endothelial cell (EC) function. The popularity of 6-well plates on orbital shakers to impose shear stress on ECs has increased among biologists due to their low cost and simplicity. One characteristic of such a platform is the heterogeneous flow profile within a well. While cells in the periphery are exposed to a laminar and high-velocity pulsatile flow that mimics physiological conditions, the flow in the center is disturbed and imposes low shear stress on the cells, which is characteristic of atheroprone regions. For studies where such heterogeneity is not desired, we present a simple cell-patterning technique to selectively prevent cell growth in the center of the well and facilitate the exclusive collection and analysis of cells in the periphery. This guarantees that cell phenotypes will not be influenced by secreted factors from cells exposed to other shear profiles nor that interesting results are obscured by mixing cells from different regions. We also present a multi-staining platform that compartmentalizes each well into 5 smaller independent regions: four at the periphery and one in the center. This is ideal for studies that aim to grow cells on the whole well surface, for comparison with previous work and minimal interference in the cell culture, but require screening of markers by immunostaining afterwards. It allows to compare different regions of the well, reduces antibody-related costs, and allows the exploration of multiple markers essential for high-content screening of cell response. By increasing the versatility of the 6-well plate on an orbital shaker system, we hope that these two solutions motivate biologists to pursue studies on EC mechanobiology and beyond.

Advection-Enhanced Kinetics in Microtiter Plates for Improved Surface Assay Quantitation and Multiplexing Capabilities.

Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12× less time), richer in information with 3-4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non-specific binding, and identify compromised results. We further multiplex single-well assays combining WellProbe's kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.

Capillary Microfluidics for Monitoring Medication Adherence.

Medication adherence is a medical and societal issue worldwide, with approximately half of patients failing to adhere to prescribed treatments. The goal of this Minireview is to examine how recent work on microfluidics for point-of-care diagnostics may be used to enhance adherence to medication. It specifically focuses on capillary microfluidics since these devices are self-powered, easy to use, and well established for diagnostics and drug monitoring. Considering that an improvement in medication adherence can have a much larger effect than the development of new medical treatments, it is long overdue for the research communities working in chemistry, biology, pharmacology, and material sciences to consider developing technologies to enhance medication adherence. For these reasons, this Minireview is not meant to be exhaustive but rather to provide a quick starting point for researchers interested in joining this complex but intriguing and exciting field of research.

Rapid micro-immunohistochemistry.

We present a new and versatile implementation of rapid and localized immunohistochemical staining of tissue sections. Immunohistochemistry (IHC) comprises a sequence of specific biochemical reactions and allows the detection of specific proteins in tissue sections. For the rapid implementation of IHC, we fabricated horizontally oriented microfluidic probes (MFPs) with functionally designed apertures to enable square and circular footprints, which we employ to locally expose a tissue to time-optimized sequences of different biochemicals. We show that the two main incubation steps of IHC protocols can be performed on MDAMB468-1510A cell block sections in less than 30 min, compared to incubation times of an hour or more in standard protocols. IHC analysis on the timescale of tens of minutes could potentially be applied during surgery, enabling clinicians to react in more dynamically and efficiently. Furthermore, this rapid IHC implementation along with conservative tissue usage has strong potential for the implementation of multiplexed assays, allowing the exploration of optimal assay conditions with a small amount of tissue to ensure high-quality staining results for the remainder of the sample.

Transposing Lateral Flow Immunoassays to Capillary-Driven Microfluidics Using Self-Coalescence Modules and Capillary-Assembled Receptor Carriers.

Point-of-care (POC) immunodiagnostic tests play a crucial role in enabling rapid and correct diagnosis of diseases in prehospital care, emergency, and remote settings. In this work, we present a silicon-based, capillary-driven microfluidic chip integrating two microfluidic modules for the implementation of highly miniaturized immunoassays. Specifically, we apply state-of-the-art microfluidic technology to demonstrate a one-step immunoassay for the detection of the cardiac marker troponin I in human serum using sample volumes of ∼1 µL and with a limit of detection (LOD) of ∼4 ng mL-1 within 25 min. The microfluidic modules discussed here broadly map functionalities found in standard lateral flow assays. We implement a self-coalescence module (SCM) for the controlled reconstitution and delivery of inkjet-spotted and dried detection antibodies (dAbs). This allows for homogeneous dissolution of 1.3 ng of fluorescently labeled dAbs in 416 nL of the sample used for the assay. We also show how to immobilize receptors inside closed microfluidic devices in <30 s using bead lane modules inside which microbeads functionalized with capture antibodies (cAbs) are self-assembled. The resulting bead lane module, with a volume of ∼3 × 10-5 mm3, is positioned across the flow path and holds ∼300 5 µm-diameter microbeads. Altogether, these capillary-driven elements allow for the manipulation of samples and reagents with an unprecedented precision and control, paving the way for the next generation of POC immunodiagnostics.

A bead-based immunogold-silver staining assay on capillary-driven microfluidics.

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in "bead lanes", which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using "classical" enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL-1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL-1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.

Capillary-Driven Microfluidic Chips for Miniaturized Immunoassays: Patterning Capture Antibodies Using Microcontact Printing and Dry-Film Resists.

The miniaturization of immunoassays using microfluidic devices is attractive for many applications, but an important challenge remains the patterning of capture antibodies (cAbs) on the surface of microfluidic structures. Here, we describe how to pattern cAbs on planar poly(dimethylsiloxane) (PDMS) stamps and how to microcontact print the cAbs on a dry-film resist (DFR). DFRs are new types of photoresists having excellent chemical resistance and good mechanical, adhesive, and optical properties. Instead of being liquid photoresists, DFRs are thin layers that are easy to handle, cut, photo-pattern, and laminate over surfaces. We show how to perform a simple fluorescence immunoassay using anti-biotin cAbs patterned on a 50-µm-thick DF-1050 DFR, Atto 647N-biotin analytes, and capillary-driven chips fabricated in silicon.

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

Centimeter-Scale Surface Interactions Using Hydrodynamic Flow Confinements.

We present a device and method for selective chemical interactions with immersed substrates at the centimeter-scale. Our implementations enable both, sequential and simultaneous delivery of multiple reagents to a substrate, as well as the creation of gradients of reagents on surfaces. The method is based on localizing submicroliter volumes of liquids on an immersed surface with a microfluidic probe (MFP) using a principle termed hydrodynamic flow confinement (HFC). We here show spatially defined, multiplexed surface interactions while benefiting from the probe capabilities such as non-contact scanning operation and convection-enhanced reaction kinetics. Three-layer glass-Si-glass probes were developed to implement slit-aperture and aperture-array designs. Analytical and numerical analysis helped to establish probe designs and operating parameters. Using these probes, we performed immunohistochemical analysis on individual cores of a human breast-cancer tissue microarray. We applied α-p53 antibodies on a 2 mm diameter core within 2.5 min using a slit-aperture probe (HFC dimension: 0.3 mm × 1.2 mm). Further, multiplexed treatment of a tissue core with α-p53 and α-ß-actin antibodies was performed using four adjacent HFCs created with an aperture-array probe (HFC dimension: 4 × 0.3 mm × 0.25 mm). The ability of these devices and methods to perform multiplexed assays, present sequentially different liquids on surfaces, and interact with surfaces at the centimeter-scale will likely spur new and efficient surface assays.

Nanoshuttles propelled by motor proteins sequentially assemble molecular cargo in a microfluidic device.

Nanoshuttles powered by the molecular motor kinesin have the potential to capture and concentrate rare molecules from solution as well as to transport, sort and assemble them in a high-throughput manner. One long-thought-of goal has been the realisation of a molecular assembly line with nanoshuttles as workhorses. To harness them for this purpose might allow the community to engineer novel materials and nanodevices. The central milestone towards this goal is to expose nanoshuttles to a series of different molecules or building blocks and load them sequentially to build hierarchical structures, macromolecules or materials. Here, we addressed this challenge by exploiting the synergy of two so far mostly complementary techniques, nanoshuttle-mediated active transport and pressure-driven passive transport, integrated into a single microfluidic device to demonstrate the realisation of a molecular assembly line. Multiple step protocols can thus be miniaturised to a highly parallelised and autonomous working lab-on-a-chip: in each reaction chamber, analytes or building blocks are captured from solution and are then transported by nanoshuttles across fluid flow boundaries in the next chamber. Cargo can thus be assembled, modified, analysed and eventually unloaded in a procedure that requires only one step by its operator.

Hierarchical hydrodynamic flow confinement: efficient use and retrieval of chemicals for microscale chemistry on surfaces.

We devised, implemented, and tested a new concept for efficient local surface chemistry that we call hierarchical hydrodynamic flow confinement (hierarchical HFC). This concept leverages the hydrodynamic shaping of multiple layers of liquid to address challenges inherent to microscale surface chemistry, such as minimal dilution, economical consumption of reagent, and fast liquid switching. We illustrate two modes of hierarchical HFC, nested and pinched, by locally denaturing and recovering a 26 bp DNA with as little as 2% dilution and by efficiently patterning an antibody on a surface, with a 5 µm resolution and a 100-fold decrease of reagent consumption compared to microcontact printing. In addition, valveless switching between nanoliter volumes of liquids was achieved within 20 ms. We believe hierarchical HFC will have broad utility for chemistry on surfaces at the microscale.

Pharmacology on microfluidics: multimodal analysis for studying cell-cell interaction.

Understanding the mechanisms of cell-cell interaction is a key unanswered question in modern pharmacology, given crosstalk defects are at the basis of many pathologies. Microfluidics represents a valuable tool for analyzing intercellular communication mediated by transmission of soluble signals, as occurring for example between neurons and glial cells in neuroinflammation, or between tumor and surrounding cells in cancer. However, the use of microfluidics for studying cell behavior still encompasses many technical and biological challenges. In this review, a state of the art of successes, potentials and limitations of microfluidics applied to key biological questions in modern pharmacology is analyzed and commented.

Flock-based microfluidics.

Flock-based microfluidics are created by depositing hydrophilic microfibers on an adhesive-coated substrate using an electric field. This enables the fabrication of self-powered microfluidics from one or more different kinds of fibers that form 2D and 3D flowpaths, which can wick 40 microliters of liquid per square centimeter. With this approach, large areas of functional wicking materials can be produced at extremely low cost.

Microfluidics in the "open space" for performing localized chemistry on biological interfaces.

Local interactions between (bio)chemicals and biological interfaces play an important role in fields ranging from surface patterning to cell toxicology. These interactions can be studied using microfluidic systems that operate in the "open space", that is, without the need for the sealed channels and chambers commonly used in microfluidics. This emerging class of techniques localizes chemical reactions on biological interfaces or specimens without imposing significant "constraints" on samples, such as encapsulation, pre-processing steps, or the need for scaffolds. They therefore provide new opportunities for handling, analyzing, and interacting with biological samples. The motivation for performing localized chemistry is discussed, as are the requirements imposed on localization techniques. Three classes of microfluidic systems operating in the open space, based on microelectrochemistry, multiphase transport, and hydrodynamic flow confinement of liquids are presented.

Overflow microfluidic networks: application to the biochemical analysis of brain cell interactions in complex neuroinflammatory scenarios.

Neuroinflammation plays a central role in neurodegenerative diseases and involves a large number of interactions between different brain cell types. Unraveling the complexity of cell-cell interaction in neuroinflammation is crucial for both clarifying the molecular mechanisms involved and increasing efficacy in drug development. Here, we provide a versatile analytical method for specifically addressing cell-to-cell communication, using primary brain cells, a microfluidic device, and a multiparametric readout approach. Different cell types are plated in separate chambers of a microfluidic network so that culturing conditions can be independently controlled and single cell types can be selectively primed with different stimuli. When chambers are microfluidically connected, the specific contribution of each cell type can be finely monitored by analyzing morphology, vitality, calcium dynamics, and electrophysiology parameters. We exemplify this approach by examining the role of astrocytes derived from two different brain regions (cortex and hippocampus) on neuronal viability in two types of neuroinflammatory insults, namely, metabolic stress and exposure to amyloid ß fibrils, and demonstrate regional differences in glial control of neuronal physiopathology. In particular, we show that during metabolic stress, cortical but not hippocampal astrocytes play a neuroprotective role; also, in an exacerbated inflammatory scenario consisting in the exposure to Aß + IL-1ß, hippocampal but not cortical astrocytes play a detrimental role on neurons. Aside from bringing novel insights into the glial role in neuroinflammation, the method presented here represents a promising tool for addressing a wide range of biological and biochemical phenomena, characterized by a complex interaction of multiple cell types.

Capillary soft valves for microfluidics.

Capillary-driven microfluidics are simple to use and provide the opportunity to perform fast biological assays with nanogram quantities of reagents and microliters of sample. Here we describe capillary soft valves (CSVs) as a simple-to-implement and -actuate approach for stopping liquids in capillary-driven microfluidics. CSVs are inserted between wettable microstructures and work to block liquids owing to a capillary pressure barrier of a few kPa. This barrier is suppressed by pressing down the soft cover of the CSV using, for example, the tip of a pen. CSVs comprise a hard layer (in silicon or polymer) with wettable microstructures and a soft cover made of poly(dimethylsiloxane) (PDMS) here. CSVs have a footprint as small as 0.6 mm(2). We illustrate how these valves work in the context of detecting DNA analytes. Specifically, a dsDNA target (997 bp PCR product, non-purified) was detected at concentrations of 20 and 200 nM in a sample volume of 0.7 µL and within 10 min. The assay includes melting of the dsDNA at 95 °C, annealing of a 30-base biotinylated probe at 50 °C, and intercalation of a fluorescent dye into the re-hybridized dsDNA at 25 °C. Actuation of the CSV allows the DNA target-probe-dye complexes to flow over 100 µm wide, streptavidin receptor lines. This work suggests that CSVs can fulfil the requirements set by complex assays, in which elevated temperatures and reaction with probes, dyes and capture species are needed. CSVs therefore greatly complement capillary-driven microfluidics without adding significant design, fabrication and actuation issues.

Micro-immunohistochemistry using a microfluidic probe.

A flexible method to extract more high-quality information from tissue sections is critically needed for both drug discovery and clinical pathology. Here, we present micro-immunohistochemistry (µIHC), a method for staining tissue sections at the micrometre scale. Nanolitres of antibody solutions are confined over micrometre-sized areas of tissue sections using a vertical microfluidic probe (vMFP) for their incubation with primary antibodies, the key step in conventional IHC. The vMFP operates several micrometres above the tissue section, can be interactively positioned on it, and even enables the staining of individual cores of tissue microarrays with multiple antigens. µIHC using such a microfluidic probe is preservative of tissue samples and reagents, alleviates antibody cross-reactivity issues, and allows a wide range of staining conditions to be applied on a single tissue section. This method may therefore find broad use in tissue-based diagnostics and in research.

Overflow microfluidic networks for open and closed cell cultures on chip.

Microfluidics have a huge potential in biomedical research, in particular for studying interactions among cell populations that are involved in complex diseases. Here, we present "overflow" microfluidic networks (oMFNs) for depositing, culturing, and studying cell populations, which are plated in a few microliters of cell suspensions in one or several open cell chambers inside the chip and subsequently cultured for several days in vitro (DIV). After the cells have developed their phenotype, the oMFN is closed with a lid bearing microfluidic connections. The salient features of the chips are (1) overflow zones around the cell chambers for drawing excess liquid by capillarity from the chamber during sealing the oMFN with the lid, (2) flow paths from peripheral pumps to cell chambers and between cell chambers for interactive flow control, (3) transparent cell chambers coated with cell adhesion molecules, and (4) the possibility to remove the lid for staining and visualizing the cells after, for example, fixation. Here, we use a two-chamber oMFN to show the activation of purinergic receptors in microglia grown in one chamber, upon release of adenosine triphosphate (ATP) from astrocytes that are grown in another chamber and challenged with glutamate. These data validate oMFNs as being particularly relevant for studying primary cells and dissecting the specific intercellular pathways involved in neurodegenerative and neuroinflammatory brain diseases.

A microfluidic device for depositing and addressing two cell populations with intercellular population communication capability.

We present a method for depositing cells in the microchambers of a sealed microfluidic device and establishing flow across the chambers independently and serially. The device comprises a transparent poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) having 2 cell chambers with a volume of 0.49 microL, 6 microchannels for servicing the chambers, and 1 microchannel linking both chambers. The MFN is sealed with a Si chip having 6 vias and ports that can be left open or connected to high-precision pumps. Liquids are drawn through each chamber in parallel or sequentially at flow rates from 0.1 to 10 microL min(-1). Plugs of liquid as small as 0.5 microL can be passed in one chamber within 5 s to 5 min. Plugs of liquid can also be introduced into a chamber for residence times of up to 30 min. By injecting different liquids into 3 ports, 3 adjacent laminar streams of liquid can be drawn inside one chamber with lateral concentration gradients between the streams ranging from 20 to 500 microm. The flexibility of this device for depositing cells and exposing them to liquids in parallel or serially is illustrated by depositing two types of cells, murine N9 microglia and human SH-S5Y5 neuroblastoma. Microfluidic communication between the chambers is illustrated by stimulating N9 microglia using ATP to induce these cells to release plasma membrane vesicles. The vesicles are drawn through the second chamber containing neuroblastoma and collected in a port of the device for off-chip analysis using confocal fluorescence microscopy. Cells in the MFN can also be fixed using a solution of formaldehyde for further analysis after disassembly of the MFN and Si lid. This microfluidic device offers a simple, flexible, and powerful method for depositing two cell populations in separate chambers and may help investigating pathways between the cells populations.