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.

Combining an Integrated Sensor Array with Machine Learning for the Simultaneous Quantification of Multiple Cations in Aqueous Mixtures.

The direct quantification of multiple ions in aqueous mixtures is achieved by combining an automated machine learning pipeline with transient potentiometric data obtained from a single miniaturized array of polymeric sensors electrodeposited on a conventional printed circuit board (PCB) substrate. A proof-of-concept system was demonstrated by employing 16 polymeric sensors in combination with features extracted from the transient differential voltages produced by these sensors when transitioning from a reference solution to a test solution, thereby obviating the need for a conventional reference electrode. A tree-based regression model enabled concentrations of various metal cations in pure solutions to be determined in less than 2 min. In a model mixture comprising Al3+, Cu2+, Na+, and Fe3+, the mean relative error was found to depend on the type of ion and varied between 1% for Fe3+ and 44% for Na+ in the concentration range 1-10 mg/L. Overall, a mean relative error of 16% was obtained for quantification of these four ions across a total of 124 tests in different solutions spanning concentrations between 2 and 360 mg/L. These results demonstrate how the analytical capability of a multiselective sensor array can leverage data-driven approaches through training by examples for accelerated testing and can be proposed to complement traditional analytical tools to meet industrial demands, including traceability of chemicals.

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.

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.

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.

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.

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.

Rapid quantitative assays for glucose-6-phosphate dehydrogenase (G6PD) and hemoglobin combined on a capillary-driven microfluidic chip.

Rapid tests for glucose-6-phosphate dehydrogenase (G6PD) are extremely important for determining G6PD deficiency, a widespread metabolic disorder which triggers hemolytic anemia in response to primaquine and tafenoquine medication, the most effective drugs for the radical cure of malaria caused by Plasmodium parasites. Current point-of-care diagnostic devices for G6PD are either qualitative, do not normalize G6PD activity to the hemoglobin concentration, or are very expensive. In this work we developed a capillary-driven microfluidic chip to perform a quantitative G6PD test and a hemoglobin measurement within 2 minutes and using less than 2 µL of sample. We used a powerful microfluidic module to integrate and resuspend locally the reagents needed for the G6PD assay and controls. We also developed a theoretical model that successfully predicts the enzymatic reactions on-chip, guides on-chip reagent spotting and allows efficient integration of multiple assays in miniaturized formats with only a few nanograms of reagents.

Electro-actuated valves and self-vented channels enable programmable flow control and monitoring in capillary-driven microfluidics.

Microfluidics are essential for many lab-on-a-chip applications, but it is still challenging to implement a portable and programmable device that can perform an assay protocol autonomously when used by a person with minimal training. Here, we present a versatile concept toward this goal by realizing programmable liquid circuits where liquids in capillary-driven microfluidic channels can be controlled and monitored from a smartphone to perform various advanced tasks of liquid manipulation. We achieve this by combining electro-actuated valves (e-gates) with passive capillary valves and self-vented channels. We demonstrate the concept by implementing a 5-mm-diameter microfluidic clock, a chip to control four liquids using 100 e-gates with electronic feedback, and designs to deliver and merge multiple liquids sequentially or in parallel in any order and combination. This concept is scalable, compatible with high-throughput manufacturing, and can be adopted in many microfluidics-based assays that would benefit from precise and easy handling of liquids.

An earbud-type wearable (A hearable) with vital parameter sensors for early detection and prevention of heat-stroke.

Heat-stroke has become a serious problem in Japan, especially for elderly citizens. For the early detection and prevention of heat-stroke, a wearable health monitor for in-ear use is developed which is subsequently called "Hearable". It aims to measure three vital parameters: Core body temperature, sweat rate and sweat or interstitial sodium ion (Na+) concentration. The eardrum is a good place to measure the core body temperature, because it is close to the carotid artery and the brain. We develop a hearable prototype and it consists of an audio earbud, a sensor earbud and a micro controller. Concerning the sensor earbud, a present prototype includes an eardrum (tympanic) temperature sensor and a sweat rate sensor and we implement two variants. Variant-1 focuses on the sweat rate sensing using a humidity & temperature sensor located close to the eardrum and Variant-2 focuses on the eardrum temperature sensing using an IR temperature sensor. Concerning the sweat rate sensing, unlike conventional sweat sensors, our prototypes do not include an air flow pump, which is typically used to determine the air flow rate. We demonstrate the accuracy of sweat rate sensing based on the air flow rate measured from the evaporation of defined amount of water. We use Variant-2 to demonstrate the monitoring of the eardrum temperature and the sweat rate to differentiate a calm state and jogging.

Programmable hydraulic resistor for microfluidic chips using electrogate arrays.

Flow rates play an important role in microfluidic devices because they affect the transport of chemicals and determine where and when (bio)chemical reactions occur in these devices. Flow rates can conveniently be determined using external peripherals in active microfluidics. However, setting specific flow rates in passive microfluidics is a significant challenge because they are encoded on a design and fabrication level, leaving little freedom to users for adjusting flow rates for specific applications. Here, we present a programmable hydraulic resistor where an array of "electrogates" routes an incoming liquid through a set of resistors to modulate flow rates in microfluidic chips post-fabrication. This approach combines a battery-powered peripheral device with passive capillary-driven microfluidic chips for advanced flow rate control and measurement. We specifically show a programmable hydraulic resistor composed of 7 parallel resistors and 14 electrogates. A peripheral and smartphone application allow a user to activate selected electrogates and resistors, providing 127 (27-1) flow resistance combinations with values spanning on a 500 fold range. The electrogates feature a capillary pinning site (i.e. trench across the flow path) to stop a solution and an electrode, which can be activated in a few ms using a 3 V bias to resume flow based on electrowetting. The hydraulic resistor and microfluidic chip shown here enable flow rates from ~0.09 nL.s-1 up to ~5.66 nL.s-1 with the resistor occupying a footprint of only 15.8 mm2 on a 1 × 2 cm2 microfluidic chip fabricated in silicon. We illustrate how a programmable hydraulic resistor can be used to set flow rate conditions for laminar co-flow of 2 liquids and the enzymatic conversion of a substrate by stationary enzymes (alkaline phosphatase) downstream of the programmable hydraulic resistor.

Sub-nanoliter, real-time flow monitoring in microfluidic chips using a portable device and smartphone.

The ever-increasing need for portable, easy-to-use, cost-effective, and connected point-of-care diagnostics (POCD) has been one of the main drivers of recent research on lab-on-a-chip (LoC) devices. A majority of these devices use microfluidics to manipulate precisely samples and reagents for bioanalysis. However, filling microfluidic devices with liquid can be prone to failure. For this reason, we have implemented a simple, yet efficient method for monitoring liquid displacement in microfluidic chips using capacitive sensing and a compact (75 mm × 30 mm × 10 mm), low-cost ($60), and battery-powered (10-hour autonomy) device communicating with a smartphone. We demonstrated the concept using a capillary-driven microfluidic chip comprising two equivalent flow paths, each with a total volume of 420 nL. Capacitance measurements from a pair of electrodes patterned longitudinally along the flow paths yielded 17 pL resolution in monitoring liquid displacement at a sampling rate of 1 data/s (~1 nL/min resolution in the flow rate). We characterized the system using human serum, biological buffers, and water, and implemented an algorithm to provide real-time information on flow conditions occurring in a microfluidic chip and interactive guidance to the user.

Capillary-Driven Microfluidic Chips for Miniaturized Immunoassays: Efficient Fabrication and Sealing of Chips Using a "Chip-Olate" Process.

The fabrication of silicon-based microfluidic chips is invaluable in supporting the development of many microfluidic concepts for research in the life sciences and in vitro diagnostic applications such as the realization of miniaturized immunoassays using capillary-driven chips. While being extremely abundant, the literature covering microfluidic chip fabrication and assay development might not have addressed properly the challenge of fabricating microfluidic chips on a wafer level or the need for dicing wafers to release chips that need then to be further processed, cleaned, rinsed, and dried one by one. Here, we describe the "chip-olate" process wherein microfluidic structures are formed on a silicon wafer, followed by partial dicing, cleaning, and drying steps. Then, integration of reagents (if any) can be done, followed by lamination of a sealing cover. Breaking by hand the partially diced wafer yields individual chips ready for use.

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.

A 0.18 µm biosensor front-end based on 1/f noise, distortion cancelation and chopper stabilization techniques.

This paper presents a novel sensor front-end circuit that addresses the issues of 1/f noise and distortion in a unique way by using canceling techniques. The proposed front-end is a fully differential transimpedance amplifier (TIA) targeted for current mode electrochemical biosensing applications. In this paper, we discuss the architecture of this canceling based front-end and the optimization methods followed for achieving low noise, low distortion performance at minimum current consumption are presented. To validate the employed canceling based front-end, it has been realized in a 0.18 µm CMOS process and the characterization results are presented. The front-end has also been tested as part of a complete wireless sensing system and the cyclic voltammetry (CV) test results from electrochemical sensors are provided. Overall current consumption in the front-end is 50 µA while operating on a 1.8 V supply.

A comparative study on fabrication techniques for on-chip microelectrodes.

This paper presents an experimental study on different microelectrode fabrication techniques, with particular focus on the robustness of the surface insulation towards typical working conditions required in lab-on-a-chip applications. Pt microelectrodes with diameters of 50 µm, 100 µm and 200 µm are patterned on a Si substrate with SiO(2) film. Sputtered SiO(2), low-pressure chemical vapor deposition (LPCVD) low-temperature oxide (LTO), Parylene C, SU-8, and dry-film were deposited and patterned on top of the chips as the passivation layer. This paper provides the detailed fabrication processes, the adhesion enhancement strategies, and the major advantages and disadvantages of each fabrication technique. Firstly, the quality and adhesion strength of the passivations were investigated by means of hydrolysis tests, in which sputtered SiO(2) and dry-film resist showed serious delamination issues and LTO showed minor defects. Secondly, the reliability of the microelectrodes was tested by impedance measurements after overnight ethanol incubation and self-assembled monolayer (SAM) formation. Thirty chips, representing a total of 300 electrodes, were measured, and statistical analyses of the results were conducted for each passivation technique. All of the electrodes passivated with these five techniques showed consistent impedance values after ethanol incubation. On the other hand, only LTO, Parylene C, and SU-8 ensured uniform electrical behavior after SAM formation. Having used both hydrolysis and impedance tests to verify the superior quality of the Parylene-based passivation, electrochemical experiments were performed to study the long-term stability of the passivation layer. Finally, the electrodes were incubated with electroactive alkanethiols functionalized with ferrocene. Square-wave voltammetry measurements demonstrated reproducible results on electrochemical label detection, which confirms the suitability of the Parylene passivation for charge-transfer-based measurements.