Self-coalescing flows in microfluidics for pulse-shaped delivery of reagents.

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.

Large-Scale Dried Reagent Reconstitution and Diffusion Control Using Microfluidic Self-Coalescence Modules.

The positioning and manipulation of large numbers of reagents in small aliquots are paramount to many fields in chemistry and the life sciences, such as combinatorial screening, enzyme activity assays, and point-of-care testing. Here, a capillary microfluidic architecture based on self-coalescence modules capable of storing thousands of dried reagent spots per square centimeter is reported, which can all be reconstituted independently without dispersion using a single pipetting step and ≤5 µL of a solution. A simple diffusion-based mathematical model is also provided to guide the spotting of reagents in this microfluidic architecture at the experimental design stage to enable either compartmentalization, mixing, or the generation of complex multi-reagent chemical patterns. Results demonstrate the formation of chemical patterns with high accuracy and versatility, and simple methods for integrating reagents and imaging the resulting chemical patterns.

Biopatterning: The Art of Patterning Biomolecules on Surfaces.

Patterning biomolecules on surfaces provides numerous opportunities for miniaturizing biological assays; biosensing; studying proteins, cells, and tissue sections; and engineering surfaces that include biological components. In this Feature Article, we summarize the themes presented in our recent Langmuir Lecture on patterning biomolecules on surfaces, miniaturizing surface assays, and interacting with biointerfaces using three key technologies: microcontact printing, microfluidic networks, and microfluidic probes.

Microscale Interfacial Polymerization on a Chip.

Forming hydrogels with precise geometries is challenging and mostly done using photopolymerization, which involves toxic chemicals, rinsing steps, solvents, and bulky optical equipment. Here, we introduce a new method for in situ formation of hydrogels with a well-defined geometry in a sealed microfluidic chip by interfacial polymerization. The geometry of the hydrogel is programmed by microfluidic design using capillary pinning structures and bringing into contact solutions containing hydrogel precursors from vicinal channels. The characteristics of the hydrogel (mesh size, molecular weight cut-off) can be readily adjusted. This method is compatible with capillary-driven microfluidics, fast, uses small volumes of reagents and samples, and does not require specific laboratory equipment. Our approach creates opportunities for filtration, hydrogel functionalization, and hydrogel-based assays, as exemplified by a rapid, compact competitive immunoassay that does not require a rinsing step.

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.

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.

Complex Nucleic Acid Hybridization Reactions inside Capillary-Driven Microfluidic Chips.

Nucleic acid hybridization reactions play an important role in many (bio)chemical fields, for example, for the development of portable point-of-care diagnostics, and often such applications require nucleic acid-based reaction systems that ideally run without enzymes under isothermal conditions. The use of novel capillary-driven microfluidic chips to perform two isothermal nucleic acid hybridization reactions, the simple opening of molecular beacon structures and the complex reaction cascade of a clamped-hybridization chain reaction (C-HCR), is reported here. For this purpose, reagents are arranged in a self-coalescence module (SCM) of a passive silicon microfluidic chip using inkjet spotting. The SCM occupies a footprint of ≈7 mm2 of a ≈0.4 × 2 cm2 microfluidic chip. By means of fluorophore-labeled DNA probes, the hybridization reactions can be analyzed in just ≈2 min and using only ≈3 µL of the sample. Furthermore, the SCM chip offers a variety of reagent delivery options, allowing, for example, the influence of the initiator concentration on the kinetics of C-HCR to be investigated systematically with minimal sample and time requirements. These results suggest that self-powered microfluidic chips equipped with a SCM provide a powerful platform for performing and investigating complex reaction systems.

Immuno-gold silver staining assays on capillary-driven microfluidics for the detection of malaria antigens.

Accurate and affordable rapid diagnostic tests (RDTs) are indispensable but often lacking for many infectious diseases. Specifically, there is a lack of highly sensitive malaria RDTs that can detect low antigen concentration at the onset of infection. Here, we present a strategy to improve the sensitivity of malaria RDTs by using capillary-driven microfluidic chips and combining sandwich immunoassays with electroless silver staining. We used 5 µm fluorescent beads functionalized with capture antibodies (cAbs). These beads are self-assembled by capillary action in recessed "bead lanes", which cross the main flow path of chips microfabricated in Si and SU-8. The binding of analytes to detection antibodies (dAbs) and secondary antibodies (2ndAbs) conjugated to gold nanoparticles (NPs) allows the formation of a silver film on the beads. Such silver film masks the fluorescent core of the bead inversely proportional to the concentration of antigen in a sample. We illustrate this method using the recombinant malaria antigen Plasmodium falciparum histidine-rich-protein 2 (rPfHRP2) spiked in human serum. This antigen was a recombinant HRP2 protein expressed in Escherichia coli, which is also the standard reference material. The limit of detection (LOD) of our immunoassay was found to be less than 6 ng mL-1 of rPfHRP2 within 20 min, which is approaching the desired sensitivity needed in the Target Product Profile (TPP) for malaria elimination settings. The concept presented here is flexible and may also be utilized for implementing fluorescence immunoassays for the parallel detection of biomarkers on capillary-driven microfluidic chips.

High-Content Optical Codes for Protecting Rapid Diagnostic Tests from Counterfeiting.

Warnings and reports on counterfeit diagnostic devices are released several times a year by regulators and public health agencies. Unfortunately, mishandling, altering, and counterfeiting point-of-care diagnostics (POCDs) and rapid diagnostic tests (RDTs) is lucrative, relatively simple and can lead to devastating consequences. Here, we demonstrate how to implement optical security codes in silicon- and nitrocellulose-based flow paths for device authentication using a smartphone. The codes are created by inkjet spotting inks directly on nitrocellulose or on micropillars. Codes containing up to 32 elements per mm2 and 8 colors can encode as many as 1045 combinations. Codes on silicon micropillars can be erased by setting a continuous flow path across the entire array of code elements or for nitrocellulose by simply wicking a liquid across the code. Static or labile code elements can further be formed on nitrocellulose to create a hidden code using poly(ethylene glycol) (PEG) or glycerol additives to the inks. More advanced codes having a specific deletion sequence can also be created in silicon microfluidic devices using an array of passive routing nodes, which activate in a particular, programmable sequence. Such codes are simple to fabricate, easy to view, and efficient in coding information; they can be ideally used in combination with information on a package to protect diagnostic devices from counterfeiting.

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.

Malaria and the 'last' parasite: how can technology help?

Malaria, together with HIV/AIDS, tuberculosis and hepatitis are the four most deadly infectious diseases globally. Progress in eliminating malaria has saved millions of lives, but also creates new challenges in detecting the 'last parasite'. Effective and accurate detection of malaria infections, both in symptomatic and asymptomatic individuals are needed. In this review, the current progress in developing new diagnostic tools to fight malaria is presented. An ideal rapid test for malaria elimination is envisioned with examples to demonstrate how innovative technologies can assist the global defeat against this disease. Diagnostic gaps where technology can bring an impact to the elimination campaign for malaria are identified. Finally, how a combination of microfluidic-based technologies and smartphone-based read-outs could potentially represent the next generation of rapid diagnostic tests is discussed.

Dielectrophoretic microbead sorting using modular electrode design and capillary-driven microfluidics.

Multiplexing assays using microbeads in microfluidics offers high flexibility and throughput, but requires the ability to sort particles based on their physical properties. In this paper, we present a continuous method for separating microbeads that is compact, modular and adaptive, employing an optimized electrode layout that alternates sorting and concentration of microbeads using dielectrophoresis and a nested design. By simulating the combined effects of the hydrodynamic drag and dielectrophoresis forces on polystyrene beads, the parameters of the electrode layout and voltage configuration are optimized for maximum separation based on particle size with a small number of slanted planar electrodes. Experimental verification confirms the efficient separation of 10 µm and 5 µm beads, with ~98% of all concentrated beads sorted in two separate streams and only ~2% of 5 µm beads leaking into the 10 µm bead stream. In addition, this method is implemented on capillary-driven microfluidic chips for maximum portability and ease of use.

Nanopatterning reveals an ECM area threshold for focal adhesion assembly and force transmission that is regulated by integrin activation and cytoskeleton tension.

Integrin-based focal adhesions (FA) transmit anchorage and traction forces between the cell and the extracellular matrix (ECM). To gain further insight into the physical parameters of the ECM that control FA assembly and force transduction in non-migrating cells, we used fibronectin (FN) nanopatterning within a cell adhesion-resistant background to establish the threshold area of ECM ligand required for stable FA assembly and force transduction. Integrin-FN clustering and adhesive force were strongly modulated by the geometry of the nanoscale adhesive area. Individual nanoisland area, not the number of nanoislands or total adhesive area, controlled integrin-FN clustering and adhesion strength. Importantly, below an area threshold (0.11 µm(2)), very few integrin-FN clusters and negligible adhesive forces were generated. We then asked whether this adhesive area threshold could be modulated by intracellular pathways known to influence either adhesive force, cytoskeletal tension, or the structural link between the two. Expression of talin- or vinculin-head domains that increase integrin activation or clustering overcame this nanolimit for stable integrin-FN clustering and increased adhesive force. Inhibition of myosin contractility in cells expressing a vinculin mutant that enhances cytoskeleton-integrin coupling also restored integrin-FN clustering below the nanolimit. We conclude that the minimum area of integrin-FN clusters required for stable assembly of nanoscale FA and adhesive force transduction is not a constant; rather it has a dynamic threshold that results from an equilibrium between pathways controlling adhesive force, cytoskeletal tension, and the structural linkage that transmits these forces, allowing the balance to be tipped by factors that regulate these mechanical parameters.

Heterogeneous integration of gels into microfluidics using a mesh carrier.

The incorporation of hydrogels inside microfluidics is a promising method for localizing receptors inside microfluidic structures for many bio-analytical applications as well as for working with cells. However, current methods rely on the in situ polymerization of hydrogels and therefore necessitate optical masks and extensive post-polymerization steps for example for washing uncrosslinked gel precursors and receptors. Here, we report a simple and efficient method for the integration of hydrogels to microfluidic chips. Small volumes of poly(ethylene)glycol-based acrylamide (PEGACA) hydrogels are photopolymerized on a mesh, rinsed, partially dried and transferred to microfluidic structures by simple contact. The gels can be derivatized before transfer with receptors such as streptavidin, antibodies, or can entrap beads as small as 200 nm. We detail the role of meshes relative to the mesh density and wettability and demonstrate how hydrogels can be transferred into capillary-driven microfluidic chips, which are easily sealed using a dry-film resist. By analogy to microfabrication strategies wherein critical components are produced separately and then combined, our method introduces the concept of heterogeneous integration of critical (bio)chemicals to microfluidic chips using an intermediate mesh carrier.

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.

Reagents in microfluidics: an 'in' and 'out' challenge.

Microfluidic devices are excellent at downscaling chemical and biochemical reactions and thereby can make reactions faster, better and more efficient. It is therefore understandable that we are seeing these devices being developed and used for many applications and research areas. However, microfluidic devices are more complex than test tubes or microtitre plates and the integration of reagents into them is a real challenge. This review looks at state-of-the-art methods and strategies for integrating various classes of reagents inside microfluidics and similarly surveys how reagents can be released inside microfluidics. The number of methods used for integrating and releasing reagents is surprisingly large and involves reagents in dry and liquid forms, directly-integrated reagents or reagents linked to carriers, as well as active, passive and hybrid release methods. We also made a brief excursion into the field of drug release and delivery. With this review, we hope to provide a large number of examples of integrating and releasing reagents that can be used by developers and users of microfluidics for their specific needs.

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.

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.

High-grade optical polydimethylsiloxane for microfluidic applications.

Commercially available polydimethylsiloxane (PDMS) elastomers, such as Sylgard 184® are widely used in soft lithography and for microfluidic applications. These PDMS elastomers contain fillers to enhance their mechanical stability. The reinforcing fillers, often sub-micrometer small SiO(2) particles, tend to aggregate, swell with water, and thereby become cognoscible in a way that can strongly interfere with the visualization of micro-scale events taking place next to PDMS structures. As PDMS microfluidics are often used for studying cells and micro-/nanoparticles and for creating/handling nanodroplets, it has become highly desirable to employ a PDMS having high optical quality and that allows microscopy observation without artifacts. Here, we present a PDMS formulation that is free of fillers and has sufficiently low viscosity to perform a filtration step of the mixed prepolymers before curing. By molding a bi-layer microfluidic network (MFN), composed of a thin filler-free PDMS layer and a thicker Sylgard 184® backing layer, PDMS MFNs featuring both high optical quality and mechanical stability, can be fabricated.