Hydrophobic Valve Research Articles

Lab on a bead with oscillatory centrifugal microfluidics for fast and complete mixing enables fast and accurate biomedical assays

Rapid mixing and precise timing are key for accurate biomedical assay measurement, particularly when the result is determined as the rate of a reaction: for example rapid immunoassay in which the amount of captured target is kinetically determined; determination of the concentration of an enzyme or enzyme substrate; or as the final stage in any procedure that involves a capture reagent when an enzyme reaction is used as the indicator. Rapid mixing and precise timing are however difficult to achieve in point-of-care devices designed for small sample volumes and fast time to result. By using centrifugal microfluidics and transposing the reaction surface from a chamber to a single mm-scale bead we demonstrate an elegant and easily manufacturable solution. Reagents (which may be, for example, an enzyme, enzyme substrate, antibody or antigen) are immobilised on the surface of a single small bead (typically 1–2 mm in diameter) contained in a cylindrical reaction chamber subjected to periodically changing rotational accelerations which promote both mixing and uniform mass-transfer to the bead surface. The gradient of Euler force across the chamber resulting from rotational acceleration of the disc, dΩdisc/dt, drives circulation of fluid in the chamber. Oscillation of Euler force by oscillation of rotational acceleration with period, T, less than that of the hydrodynamic relaxation time of the fluid, folds the fluid streamlines. Movement of the bead in response to the fluid and the changing rotational acceleration provides a dynamically changing chamber shape, further folding and expanding the fluid. Bead rotation and translation driven by fluid flow and disc motion give uniformity of reaction over the surface. Critical parameters for mixing and reaction uniformity are the ratio of chamber radius to bead radius, rchamber/rbead, and the product Trchamber(dΩdisc/dt), of oscillation period and Euler force gradient across the fluid. We illustrate application of the concept using the reaction of horse radish peroxidase (HRP) immobilised on the bead surface with its substrate tetramethylbenzidine (TMB) in solution. Acceleration from rest to break a hydrophobic valve provided precise timing for TMB contact with the bead. Solution uniformity from reaction on the surface of the bead in volumes 20–50 uL was obtained in times of 2.5 s or less. Accurate measurement of the amount of surface-bound HRP by model fitting to the measured kinetics of colour development at 10 s intervals is demonstrated.

Snapshots of ABCG1 and ABCG5/G8: A Sterol's Journey to Cross the Cellular Membranes.

The subfamily-G ATP-binding cassette (ABCG) transporters play important roles in regulating cholesterol homeostasis. Recent progress in the structural data of ABCG1 and ABCG5/G8 disclose putative sterol binding sites that suggest the possible cholesterol translocation pathway. ABCG1 and ABCG5/G8 share high similarity in the overall molecular architecture, and both transporters appear to use several unique structural motifs to facilitate cholesterol transport along this pathway, including the phenylalanine highway and the hydrophobic valve. Interestingly, ABCG5/G8 is known to transport cholesterol and phytosterols, whereas ABCG1 seems to exclusively transport cholesterol. Ligand docking analysis indeed suggests a difference in recruiting sterol molecules to the known sterol-binding sites. Here, we further discuss how the different and shared structural features are relevant to their physiological functions, and finally provide our perspective on future studies in ABCG cholesterol transporters.

An addressable electrowetting valve for centrifugal microfluidics

Centrifugal microfluidic platforms aim to integrate and perform complex biological and chemical processes within disc-like substrates and valving through centrifugal forces and the Coriolis effect. Although the integration of active valves within such system can be used to enhance fluid control, the ability to fabricate and integrate addressable valves within centrifugal platforms remains a significant challenge. To address this issue, herein, we present and test an addressable electrowetting centrifugal (EWC) valve. The EWC valve comprises a hydrophobic burst valve, integrated side electrodes and a dielectric layer between the microfluidic channel and electrode structures. The burst valve is made from a hydrophobic material able hold a specified pressure. Specifically, application of an external electric field is used to alter the valve wettability, making the valve more hydrophilic, decreasing the threshold pressure and triggering the opening of the valve. To assess valve performance, we investigate the influence of electric properties and channel geometry on valve response and then demonstrate multiple operations on different fluid samples. Finally, and to highlight the utility of EWC valves, we demonstrate the detection and analysis of CD4 human immune cells.

Multi-reagents dispensing centrifugal microfluidics for point-of-care testing

Point-of-care testing (POCT) has shown great advantages for public health monitoring in resource-limited settings. However, developing of POCT tools with automated and accurate quantitative dispensing of multiple reagents and samples is challenging. Here, we demonstrate a novel multi-reagents dispensing centrifugal microfluidics (MDCM) that allows rapid and automated dispensing of multiple reagents and samples with high throughput and accuracy. The MDCM was designed with multiple aliquoting units with the hydrophobic valve at different radial positions. All reagents and samples were loaded simultaneously, dispensed in parallel by centrifugation at low speed, and then introduced into the reaction chamber sequentially by centrifugation at high speed. Two MDCM chips are demonstrated, including a uniform concentration generator and a gradient concentration generator. The concentration coefficient of variation (CV) among the independent reaction chambers was lower than 0.56%, and the theoretical quantitative concentration gradient was strongly correlated with the actual concentration gradient (R2 = 0.9938). We have successfully applied the MDCM to loop-mediated isothermal amplification (LAMP)-based nucleic acid detection for multiple infectious pathogens and antimicrobial susceptibility testing (AST) for kanamycin sulfate against E. coli. To further extend the applications, the MDCM has also been applied to bicinchoninic acid (BCA) protein assays with online calibration, reducing the detection time from 2 h to 10 min with a twenty-fold reduction in reagent consumption. These results indicated that the MDCM is a high potential platform for POCT.

Autonomous capillary microfluidic devices with constant flow rate and temperature-controlled valving.

In this paper, we report on a capillary microfluidic device with constant flow rate and temperature-triggered stop valve function. It contains a PDMS channel that was grafted by a thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAm). The channel exhibits a constant capillary filling speed. By locally increasing the temperature in the channel from 20 °C to 37 °C using a microfabricated heater, a change of the surface wettability from hydrophilic to hydrophobic is obtained creating a hydrophobic stop valve. The valve can be reopened by lowering the temperature. The device is simple to fabricate and can be used as an actuatable capillary pump operating around room temperature. To understand the constant capillary filling speed, we performed contact angle measurements, in which we found slow wetting kinetics of PNIPAm-g-PDMS surfaces at temperatures below the lower critical solution temperature (LCST) of PNIPAm and fast wetting kinetics above the LCST. We interpret this as the result of the diffusive hydration process of PNIPAm below the LCST and the absence of hydration on the hydrophobic PNIPAm thin layer above the LCST.

Picky ABCG5/G8 and promiscuous ABCG2 - a tale of fatty diets and drug toxicity.

Structural data on ABCG5/G8 and ABCG2 reveal a unique molecular architecture for subfamily G ATP‐binding cassette (ABCG) transporters and disclose putative substrate‐binding sites. ABCG5/G8 and ABCG2 appear to use several unique structural motifs to execute transport, including the triple helical bundles, the membrane‐embedded polar relay, the re‐entry helices, and a hydrophobic valve. Interestingly, ABCG2 shows extreme substrate promiscuity, whereas ABCG5/G8 transports only sterol molecules. ABCG2 structures suggest a large internal cavity, serving as a binding region for substrates and inhibitors, while mutational and pharmacological analyses support the notion of multiple binding sites. By contrast, ABCG5/G8 shows a collapsed cavity of insufficient size to hold substrates. Indeed, mutational analyses indicate a sterol‐binding site at the hydrophobic interface between the transporter and the lipid bilayer. In this review, we highlight key differences and similarities between ABCG2 and ABCG5/G8 structures. We further discuss the relevance of distinct and shared structural features in the context of their physiological functions. Finally, we elaborate on how ABCG2 and ABCG5/G8 could pave the way for studies on other ABCG transporters.

Flip-Flopping Retinal in Microbial Rhodopsins as a Template for a Farnesyl/Prenyl Flip-Flop Model in Eukaryote GPCRs.

Thirty years after the first description and modeling of G protein coupled receptors (GPCRs), information about their mode of action is still limited. One of the questions that is hard to answer is: how do the allosteric changes in the GPCR induced by, e.g., ligand binding in the end activate a G protein-dependent intracellular pathway (e.g., via the cAMP or the phosphatidylinositol signal pathways). Another question relates to the role of prenylation of G proteins. Today’s “consensus model” states that protein prenylation is required for the assembly of GPCR-G protein complexes. Although it is well-known that protein prenylation is the covalent addition of a farnesyl- or geranylgeranyl moiety to the C terminus of specific proteins, e.g., α or γ G protein, the reason for this strong covalent binding remains enigmatic. The arguments for a fundamental role for prenylation of G proteins other than just being a hydrophobic linker, are gradually accumulating. We uncovered a dilemma that at first glance may be considered physiologically irrelevant, however, it may cause a true change in paradigm. The consensus model suggests that the only functional role of prenylation is to link the G protein to the receptor. Does the isoprenoid nature of the prenyl group and its exact site of attachment somehow matter? Or, are there valid arguments favoring the alternative possibility that a key role of the G protein is to guide the covalently attached prenyl group to – and it hold it in – a very specific location in between specific helices of the receptor? Our model says that the farnesyl/prenyl group – aided by its covalent attachment to a G protein -might function in GPCRs as a horseshoe-shaped flexible (and perhaps flip-flopping) hydrophobic valve for restricting (though not fully inhibiting) the untimely passage of Ca2+, like retinal does for the passage of H+ in microbial rhodopsins that are ancestral to many GPCRs.

A simple micropump based on a freeze-dried superabsorbent polymer for multiplex solution processing in disposable devices.

We describe a simple micropump for disposable microfluidic devices. The pump is constructed using a freeze-dried disc of a superabsorbent polymer (SAP). The disc absorbs a solution in a flow channel and swells upward in a pumping chamber. Despite the simple structure of this device, the rate of absorption remains constant and can be adjusted by changing the composition of the SAP, its size, the dimensions of the flow channel and the medium to be absorbed. The pumping action can be initiated by applying an electrical signal using a switchable hydrophobic valve. The integrated approach of the SAP pump and switchable valve could facilitate the automatic processing of many solutions required for bioassay.

Innovative Hydrophobic Valve Allows Complex Liquid Manipulations in a Self-Powered Channel-Based Microfluidic Device

We present an innovative, simple, and versatile hydrophobic valve enabling all-important complex liquid manipulations on self-powered, channel-based microfluidic devices and as such being extremely valuable for the design of highly demanding point-of-care (POC) platforms. The presented hydrophobic valve is made of filter paper treated with a fluorinated compound (i.e., Aquapel) and shows both superhydrophobic properties (contact angle up to 155°) and high resistance to liquid pressure (up to 9 kPa), while retaining gas permeability and utter fabrication simplicity. Whereas this valve can be integrated in any channel-based system and can be used both as a vent, to delay liquid displacement on chip, or as a barrier, to stop the liquid flow in a certain direction, in this work we demonstrate some of its capacities by combining it with our in house developed self-powered SIMPLE and iSIMPLE platforms. First, we integrated it with the infusion iSIMPLE pump, thus generating completely fail-proof activation regardless of how the operator is actuating the system. Second, we used hydrophobic valves as both barrier and vent in the same microfluidic chip, which allowed the combination of two SIMPLE pumps for splitting one sample in two parallel channels. This attribute is fundamental for achieving multiplex analysis on completely autonomous microfluidic platforms. Finally, we achieved an unprecedented liquid manipulation for a self-powered microfluidic platform, namely, shuttling of liquid, after a single user activation by combining for the first time SIMPLE and iSIMPLE with the developed hydrophobic vent and barrier, all in a single chip. These results convincingly demonstrated that the developed hydrophobic valve combined with SIMPLE/iSIMPLE presents an essential building block for an ideal POC system, which is self-powered, inexpensive, and robust and can perform complex bioassays upon a single user activation.

Review of Microfluidic Devices for On‐Chip Chemical Sensing

SUMMARYMicrofluidic techniques can be a basis to realize user‐friendly microanalytical devices of high performance. Required operations include injection of solutions to reaction chambers, volume measurement, and mixing of solutions. For this purpose, the use of surface tension is attractive. In hydrophobic flow channels, solutions can be moved stably in the form of plugs. Otherwise, solutions can be transported by capillary action in a hydrophilic flow channel and stopped at a hydrophobic valve. The valve can be opened autonomously by switching the mixed potential by wetting a zinc electrode formed in a controlling flow channel. In this review paper, we introduce some of our microfluidic devices particularly focusing on devices that work based on surface tension.

Simultaneous Identification and Antimicrobial Susceptibility Testing of Multiple Uropathogens on a Microfluidic Chip with Paper-Supported Cell Culture Arrays.

A microfluidic chip was developed for one-step identification and antimicrobial susceptibility testing (AST) of multiple uropathogens. The polydimethylsiloxane (PDMS) microchip used had features of cell culture chamber arrays connected through a sample introduction channel. At the bottom of each chamber, a paper substrate preloaded with chromogenic media and antimicrobial agents was embedded. By integrating a hydrophobic membrane valve on the microchip, the urine sample can be equally distributed into and confined in individual chambers. The identification and AST assays on multiple uropathogens were performed by combining the spatial resolution of the cell culture arrays and the color resolution from the chromogenic reaction. The composite microbial testing assay was based on dynamic changes in color in a serial of chambers. The bacterial antimicrobial susceptibility was determined by the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Using three common uropathogenic bacteria as test models, the developed microfluidic approach was demonstrated to be able to complete the multiple colorimetric assays in 15 h. The accuracy of the microchip method, in comparison with that of the conventional approach, showed a coincidence of 94.1%. Our data suggest this microfluidic approach will be a promising tool for simple and fast uropathogen testing in resource-limited settings.

Switchable Hydrophobic Valve for Controlled Microfluidic Processing.

A simple microfluidic valve, without any moving parts, is presented that can control solution flow on demand in microchannels of many different materials using a low-power electric signal. Many independently operating valves can easily be integrated into complex microfluidic systems. The valve consists of a self-assembled monolayer (SAM) formed on a platinum electrode that is incorporated directly in the microchannel. The normally-on valve stops the solution flow due to a hydrophobic SAM on the electrode surface. The solution is allowed to pass the valve by applying a potential to the electrode, which removes the SAM due to reductive desorption. The valve operation is highly stable and has switching times of the order of 1 s. The valve is ideal for controlled solution manipulation in integrated micro-analytical systems and autonomous microfluidic systems.

オンチップ化学センシングのためのマイクロフルイディックデバイスに関するレビュー

Microfluidic techniques can be a basis to realize user-friendly microanalytical devices of high performance. Required operations include injection of solutions to reaction chambers, volume measurement, and mixing of solutions. For this purpose, the use of surface tension is attractive. In hydrophobic flow channels, solutions can be moved stably in the form of plugs. Otherwise, solutions can be transported by capillary action in a hydrophilic flow channel and stopped at a hydrophobic valve. The valve can be opened autonomously by switching the mixed potential by wetting a zinc electrode formed in a controlling flow channel. In this review paper, we introduce some of our microfluidic devices particularly focusing on devices that work based on surface tension.

The use of polybutene for controlling the flow of liquids in centrifugal microfluidic systems

The field of centrifugal microfluidics has evolved over the last several decades to allow implementation of complex biological and chemical assays on Lab-on-Disc (LOD) platforms. Present study describes the use of polymer polybutene for tuning hydrophobic siphons and for liquid volume definition on a centrifugal microfluidic platform. Both the siphon tuning and the volume definition steps are carried out by generating negative pressure that results from the volume expansion caused by the transfer of polybutene from a dedicated chamber into a secondary reservoir via a connecting siphon. The hydrophobic valve of the chamber that holds polybutene bursts open at specific angular velocities that depend on the height and density of the liquid column. Thus, the parameters of siphon activation can be adjusted by tuning the burst angular velocity of the valve that is driven by filling the tuning reservoir with a specific volume of polybutene. The same disc construction can be utilized to provide volume definition functionality to transfer liquids from one reservoir to another reservoir in as many fractions as there are immiscible liquids of different densities in the tuning chamber. The presented work also demonstrates the use of polybutene in sealing fluidic chambers to improve heating efficiency and to minimize evaporation during thermal cycling required for applications such as PCR amplification. Finally, the use of polybutene as a stationary liquid phase in droplet production on a spinning disc is demonstrated.

Polystyrene microdispenser for blood tests using hydrophobic passive valve

This paper proposes a polystyrene microdispenser to meter and discharge 200 nl of plasma solution with high reproducibility. To produce at low cost, hydrophobic passive valves with a simple structured fabricated by injection molding were used for the metering principle. After evaluation using a plasma solution, the microdispensers coated with fluoric resin showed reproducibility of discharged fluid volume with a coefficient of variance of 1.25%. On the other hand, those coated with dimethyl silicone caused a backflow of the discharged fluid to the metering channel, resulting in the degradation of reproducibility. To evaluate the dynamic behavior of the fluid in the channels, we measured not only the static contact angle, but also repeatedly measured the contact angles and various kinds of dynamic contact angle, e.g. the extension advancing angle and the sliding angle. We show the possibility that repeatedly measuring the contact angle can be used for selection of a surface treatment which does not cause backflow. In addition, we propose a method of estimating the discharge pressure, using an extension of the advancing angle and the sliding angle. This method enables closer estimation to the actual measurements than the conventional method based on the static contact angle.

Microfluidic cell disruption system employing a magnetically actuated diaphragm

A microfluidic cell lysis chip equipped with a micromixer and SPE unit was developed and used for quantitative analysis of intracellular proteins. This miniaturized sample preparation system can be employed for any purpose where cell disruption is needed to obtain intracellular constituents for the subsequent analysis. This system comprises a magnetically actuated micromixer to disrupt cells, a hydrophobic valve to manipulate the cell lysate, and a packed porous polymerized monolith chamber for SPE and filtering debris from the cell lysate. Using recombinant Escherichia coli expressing intracellular enhanced green fluorescent protein (EGFP) and lipase as model bacteria, we optimized the cell disruption condition with respect to the lysis buffer composition, mixing time, and the frequency of the diaphragm in the micromixer, which was magnetically actuated by an external magnetic stirrer in the micromixer chamber. The lysed sample prepared under the optimal condition was purified by the packed SPE in the microfluidic chip. At a frequency of 1.96 Hz, the final cell lysis efficiency and relative fluorescence intensity of EGFP after the cell disruption process were greater than 90 and 94%, respectively. Thus, this microfluidic cell disruption chip can be used for the efficient lysis of cells for further analysis of intracellular contents in many applications.

Microfluidic chip for biochemical reaction and electrophoretic separation by quantitative volume control

We have developed a microfluidic integrated chip with all the fluidic manipulations required for biochemical reaction and electrophoretic separation. The microchip was fabricated using standard photolithography, isotropic wet etching, liftoff patterning of fluorocarbon film, and polydimethylsiloxane replica molding. The fabricated microfluidic chip (46 mm × 25 mm) was composed of two nanoliter-metering microchannel networks, a serpentine mixing channel, a sample injector, and an electrophoretic separation channel. In particular, a novel Y-shaped sample injector was developed as an important interface for the successful combination of the biochemical reaction with electrophoretic separation. A nanoliter-sized sample was pneumatically injected into the separation channel using the novel Y-shaped sample injector. This consisted of a planar hydrophobic valve and two vertical hydrophobic valves. The performance of the hydrophobic valves for fluidic manipulations was predicted using capillary pressure derived from the relationship of the work performed by the capillary pressure (d U p), the surface free energy of the system (d U s), and Young's equation. In addition, we were able to explain in detail the fluidic manipulation schemes for the biochemical reaction and subsequent separation. We also successfully demonstrated the enzyme reaction of β-galactosidase using fluorescein di-β- d-galactopyranoside as a substrate and the electrophoretic separation of its reaction products.

Pressure-Driven Sample Injection with Quantitative Liquid Dispensing for On-Chip Electrophoresis

A novel pressure-driven sample injection method was developed as an alternative to electrokinetic injection, and electrophoretic separation was carried out on a microfabricated device employing this method. This method enables a defined volume of liquid dispensing, followed by instantaneous injection driven by pneumatic pressure, greatly simplifying the injection procedure. A particular microstructure, called a "metering chamber", has been designed for the quantitative dispensing of an ultra-low volume of sample liquid; a "hydrophobic passive valve" equipped with an air vent channel is employed for injecting a dispensed sample into the separation channel. The reproducibility of dispensing was 3.3% (n = 15), expressed by the variation of dispensed volumes. The electrophoretic separation of DNA fragments was performed using this injection method, varying the injection volumes from 0.45 to 4.0 nL, and the separation efficiencies were compared. This precise injection method, easily variable in injection volumes, is highly suitable for quantitative as well as qualitative electrophoretic analyses.

Hydrophobic valves of plasma deposited octafluorocyclobutane in DRIE channels

The suitability of using octafluorocyclobutane (C 4F 8) patches as hydrophobic valves in microfluidic biochemical applications has been shown. A technique has been developed to generate lithographically defined C 4F 8 hydrophobic patches in deep reactive ion-etched silicon channels. Some of the advantages of this process are that no specific cleaning of the substrate is required, C 4F 8 is deposited on the sidewalls and the bottom of the channels, a standard photoresist mask can be used to define the patches, and that it is a fast and convenient dry chemical process performed by a standard inductively coupled plasma etcher using the Bosch process. Different patch lengths (200–1000 μm) of C 4F 8 were deposited in 50 μm wide channels to evaluate which size is most suitable for microfluidic biochemical applications. The valve function of the hydrophobic patches was tested for the following liquids: DD water, acetone, propanol, bead solution and a mixture used for pyrosequencing of DNA. Patch lengths of 200 μm of C 4F 8 successfully stopped each solution for at least 20 consecutive times. The C 4F 8 film resists water for at least 5 h. The hydrophobic valve also resists very high concentrations (25%) of surfactants (Tween 80). C 4F 8 shows a much higher resistance towards water and surface active solutions than previous hydrophobic patches. However, 50% Tween 80 was not stopped at all by the hydrophobic patch. An applied pressure of 760 Pa at the inlet was needed for water to over-run the hydrophobic patch.