Immunochromatographic thread-based test platform for diagnosis of infectious diseases

Patterning is an important step in fabrication of multiplexed microfluidic devices. Various approaches including cutting, photolithography, wax-printing, plotting and etching have been developed and tested. Recently, using threads has emerged as a convenient and low-cost approach for fabrication of microfluidic devices. We explored the application of threads in combination with nitrocellulose membrane to fabricate multi-channel immunochromatographic diagnostic devices. Microfluidic channels were made using hydrophilic threads and nitrocellulose membrane strips. Household sewing needle was used to weave hydrophilic thread into desired patterns through a double-sided mounting tape. Glass fibre discs were used as conjugate pads while nitrocellulose membrane was used for immobilisation of capture antibodies. Patterned threads were linked to nitrocellulose membrane strips by overlapping so that reagents flowing through threads were eventually transferred to the membrane. The design was tested using IgG, H. pylori and Hepatitis B surface antigen. Continuous flow was observed from hydrophilic threads to the nitrocellulose membrane, and a positive signal was visualised on the membrane within 5 min of sample application. The observed limit of detection ranged between 30 and 300 ng/ml for H. pylori and Hepatitis B, respectively. Using thread and tape offers a promising alternative for patterning of simple, low-cost multiplexed microfluidic diagnostic devices with potential point-of-care applications in resource-limited settings.     

Miniaturized DNA amplification platform with soft-lithographically fabricated continuous-flow PCR microfluidic device on a portable temperature controller

Fabrication of 3D microfluidic devices is normally quite expensive and tedious. A strategy was established to rapidly and effectively produce multilayer 3D microfluidic chips which are made of two layers of poly(methyl methacrylate) (PMMA) sheets and three layers of double-sided pressure sensitive adhesive (PSA) tapes. The channel structures were cut in each layer by cutting plotter before assembly. The structured channels were covered by a PMMA sheet on top and a PMMA carrier which contained threads to connect with tubing. A large variety of PMMA slides and PSA tapes can easily be designed and cut with the help of a cutting plotter. The microfluidic chip was manually assembled by a simple lamination process.The complete fabrication process from device design concept to working device can be completed in minutes without the need of expensive equipment such as laser, thermal lamination, and cleanroom. This rapid frabrication method was applied for design of a 3D hydrodynamic focusing device for synthesis of gold nanoparticles (AuNPs) as proof-of-concept. The fouling of AuNPs was prevented by means of a sheath flow. Different parameters such as flow rate and concentration of reagents were controlled to achieve AuNPs of various sizes. The sheet-based fabrication method offers a possibility to create complex microfluidic devices in a rapid, cheap and easy way.

     

Anomalous pulse change in gravity-driven microfluidic oscillator and its application to photodiode switching

Electrofluidic analogy is useful because it provides a method to significantly reduce the reliance of microfluidic chips on dynamic off-chip controllers. Among the functions developed by the analogy, conversion from constant to pulsatile pressure is critical and is yet to be studied. Here, unlike its counterpart electrical oscillator generating square pulses more slowly with decreasing the input voltage, we report that a microfluidic oscillator generates sawtooth pressure pulses more rapidly with decreasing the input pressure (P_I) at 1–2 kPa. Further, with decreasing P_I, the oscillator generates square pulses at P_I > 3.4 kPa, but its operation unexpectedly stops at 2.1 < P_I < 3.4 kPa. We analyze its underlying mechanism with a sophisticated model including a dynamic interaction of the oscillator components and reveal the critical role of the dynamic property of oscillator valves. Additionally, we show electrofluidic switching of a photodiode with the oscillator. The understanding obtained in this study would be essential for developing microfluidic circuits using electrofluidic analogy.     

Deterministic lateral displacement (DLD) in the high Reynolds number regime: high-throughput and dynamic separation characteristics

Recent progress in the development of biosensors has created a demand for high-throughput sample preparation techniques that can be easily integrated into microfluidic or lab-on-a-chip platforms. One mechanism that may satisfy this demand is deterministic lateral displacement (DLD), which uses hydrodynamic forces to separate particles based on size. Numerous medically relevant cellular organisms, such as circulating tumor cells (10–15 µm) and red blood cells (6–8 µm), can be manipulated using microscale DLD devices. In general, these often-viscous samples require some form of dilution or other treatment prior to microfluidic transport, further increasing the need for high-throughput operation to compensate for the increased sample volume. However, high-throughput DLD devices will require a high flow rate, leading to an increase in Reynolds numbers (Re) much higher than those covered by existing studies for microscale (≤?100 µm) DLD devices. This study characterizes the separation performance for microscale DLD devices in the high-Re regime (10?<?Re?<?60) through numerical simulation and experimental validation. As Re increases, streamlines evolve and microvortices emerge in the wake of the pillars, resulting in a particle trajectory shift within the DLD array. This differs from previous DLD works, in that traditional models only account for streamlines that are characteristic of low-Re flow, with no consideration for the transformation of these streamlines with increasing Re. We have established a trend through numerical modeling, which agrees with our experimental findings, to serve as a guideline for microscale DLD performance in the high-Re regime. Finally, this new phenomenon could be exploited to design passive DLD devices with a dynamic separation range, controlled simply by adjusting the device flow rate.     

Multi-scale,multi-depth lithography using optical fibers for microfluidic applications

This paper proposes and demonstrates a method for multi-scale, multi-depth three-dimensional (3D) lithography. In this method, 3D molds for replicating microchannels are fabricated by passing a non-focused laser beam through an optical fiber, whose tip is immersed in a droplet of photopolymer. Line width is adjustable from 1 to 980 µm using eight kinds of optical fibers with different core diameters. The height of line drawing can be controlled by adjusting the distance between the tip of the optical fiber and a substrate. The surface roughness (R_a, R_z) of a single line and plane was evaluated. The method was employed to fabricate a 3D mold of a microchannel containing tandem chambers, which was then successfully replicated in PDMS. Multi-scale, multi-depth 3D lithography can provide a simple, flexible tool for producing PDMS microfluidic devices.     

A high-efficiency three-dimensional helical micromixer in fused silica

True three-dimensional (3D) micromixers in fused silica are highly desirable for efficient and compact mixing in microfluidic applications. However, realization of such devices remains technically challenging. Here, we report high-quality fabrication of 3D helical microchannels in fused silica by taking the full advantage of an improved femtosecond laser irradiation followed by chemical etching process, and a glass-PDMS interface structure is introduced for assembling 3D helical micromixer. Highly efficient mixing is achieved in the helical micromixer at low Reynolds numbers, whose excellent mixing performance is approved by the experimental evaluation and computational fluid dynamics simulation.     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

Fabrication of three-dimensional microfluidic channels in a single layer of cellulose paper

Three-dimensional microfluidic paper-based analytical devices (3D-μPADs) represent a promising platform technology that permits complex fluid manipulation, parallel sample distribution, high throughput, and multiplexed analytical tests. Conventional fabrication techniques of 3D-μPADs always involve stacking and assembling layers of patterned paper using adhesives, which are tedious and time-consuming. This paper reports a novel technique for fabricating 3D microfluidic channels in a single layer of cellulose paper, which greatly simplifies the fabrication process of 3D-μPADs. This technique, evolved from the popular wax-printing technique for paper channel patterning, is capable of controlling the penetration depth of melted wax, printed on both sides of a paper substrate, and thus forming multilayers of patterned channels in the substrate. We control two fabrication parameters, the density of printed wax (i.e., grayscale level of printing) and the heating time, to adjust the penetration depth of wax upon heating. Through double-sided printing of patterns at different grayscale levels and proper selection of the heating time, we construct up to four layers of channels in a 315.4-μm-thick sheet of paper. As a proof-of-concept demonstration, we fabricate a 3D-μPAD with three layers of channels from a paper substrate and demonstrate multiplexed enzymatic detection of three biomarkers (glucose, lactate, and uric acid). This technique is also compatible with the conventional fabrication techniques of 3D-μPADs, and can decrease the number of paper layers required for forming a 3D-μPAD and therefore make the device quality control easier. This technique holds a great potential to further popularize the use of 3D-μPADs and enhance the mass-production quality of these devices.     

Developing heatable microfluidic chip to generate gelatin emulsions and microcapsules

The heatable microfluidic chip developed herein successfully integrates a microheater and flow-focusing device to generate uniform-sized gelatin emulsions under various flow rate ratios (sample phase/oil phase, Q _s/Q _o) and driven voltages. The gelatin emulsions can be applied to encapsulate vitamin C for drug release. Our goal is to create the thermal conditions for thermo-sensitive hydrogel materials in the microfluidic chip and generate continuous and uniform emulsions under any external environment. The gelatin emulsion sizes have a coefficient of variation of <5 % and can be precisely controlled by altering the flow rate ratio (Q _s/Q _o) and driven voltage. The gelatin emulsion diameters range from 45 to 120 μm. Moreover, various sizes of these gelatin microcapsules containing vitamin C were used for drug release. The developed microfluidic chip has the advantages of a heatable platform in the fluid device, active control over the emulsion diameter, the generation of uniform-sized emulsions, and simplicity. This new approach for gelatin microcapsules will provide many potential applications in drug delivery and pharmaceuticals.     

Fabrication of piezoelectric multilayer thin-film actuators

In this study, we fabricated multilayer ceramics (MLCs) composed of multilayered Pb(Zr,Ti)O₃ (PZT) piezoelectric thin films with internal electrodes and evaluated their dielectric and piezoelectric properties. The stack of PZT ferroelectric layers (550 nm) and SrRuO₃ (SRO, 80 nm) electrodes were alternatively deposited on Pt/Ti-coated silicon-on-insulator substrates by radio-frequency magnetron sputtering. The MLCs composed of one, three, and five PZT layers were fabricated by the alternate sputtering deposition of PZT ferroelectric layers and SRO electrodes through the movable shadow mask. The capacitances of MLCs were proportionally increased with the number of PZT layers, while their relative dielectric constants were almost same among the each MLC. The MLCs exhibited symmetric and saturated P–E hysteresis loops similar to the conventional PZT thin films. We estimated that the piezoelectric properties of MLCs by FEM simulation, and confirmed that the effective transverse piezoelectric coefficients (d _31,eff ) increased with the number of PZT layers. The piezoelectric coefficients calculated to be d _31,eff  = ?2964 pC/N at 25 PZT layers, which is much higher than those of conventional single-layer piezoelectric thin films.     

Hydrodynamic focusing and interdistance control of particle-laden flow for microflow cytometry

Single-file focusing and minimum interdistance of micron-size objects in a sample is a prerequisite for accurate flow cytometry measurements. Here, we report analytical models for predicting the focused width of a sample stream b as a function of channel aspect ratio α, sheath-to-sample flow rate ratio f and viscosity ratio λ in both 2D and 3D focusing. We present another analytical model to predict spacing between an adjacent pair of objects in a focused sample stream as a function of sample concentration C, mobility ? of the objects in the prefocused and postfocused regions and flow rate ratio f in both 2D and 3D flow focusing. Numerical simulations are performed using Ansys Fluent VOF model to predict the width of sample stream in 2D and 3D hydrodynamic focusing for different sample-to-sheath viscosity ratios, aspect ratios and flow rate ratios. Experiments are performed on both planar and three-dimensional devices fabricated in PDMS to demonstrate focusing of sample stream and spacing of polystyrene beads in the unfocused and focused stream at different sample concentrations C. The predictions of the analytical model and simulations are compared with experimental data, and a good match is found (within 12 %). Further, mobility of objects is experimentally studied in 2D and 3D focusing, and the spread of the mobility data is used as tool for the demonstration of particle focusing in flow cytometer applications.     

Separation and concentration of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> sporangia by inertial focusing in curving microfluidic flows

We present an integrated microfluidic system for performing isolation and concentration of Phytophthora ramorum pathogens using a chip whose working principle is based on inertial lateral migration in curving flows. The chip was fabricated from multiple layers of thermoplastic polymers and features an embedded spiral separation channel along with peristaltic microvalves for fluidic operation and process control. A pumping system paired with a fully programmable pressure manifold is used to boost concentration levels by recirculating the sample liquid multiple times through the separation chip, making it possible to reduce sample volumes from 10 to 1 mL or less. The system was calibrated using fluorescent polymer particles with a nominal diameter of 30 µm which is comparable to that of P. ramorum sporangia. The separation process has been shown to be highly effective and more than 99% of the beads can be recovered in the concentrated batch. Experiments conducted with P. ramorum sporangia have shown that a 5.3-fold increase in pathogen content with 95% recovery can be achieved using three subsequent concentration cycles. The utility of the method has been validated by processing a sample derived from infested Rhododendron leaves where a 6.1-fold increase in the concentration of P. ramorum has been obtained after four concentration cycles. Although specifically designed and demonstrated for sporangia of P. ramorum, the method and related design rules can easily be extended to other microbial organisms, effectively supporting bioanalytical applications where efficient, high-throughput separation of target species is of primary concern.     

Novel prototyping method for microfluidic devices based on thermoplastic polyurethane microcapillary film

Thermoplastic polyurethane microcapillary film (TPU-MCF), as a novel extruded product, inherently contains an array of circular micron-sized capillaries embedded inside the polymer matrix. With the aid of simple laser cutting and conventional sealing technologies, a rapid prototyping method for microfluidic devices is proposed based on the ready-made microstructure of MCFs. Two functionalized microfluidic devices: serpentine micromixer and multi-droplet generator, are rapidly fabricated to demonstrate the advantages and potential of employing this new method. The whole proof-of-concept fabrication process can be completed in 8–10 min in a simple way; each procedure is repeatable with stable performance control of microfluidic devices; and the material cost can be as low as $0.01 for each device. The TPU-MCF and this novel method are expected to provide a new perspective and alternative in microfluidic community with particular requirements.     

On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection

Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 10³ and 1.1 × 10³ CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design.     

Role of interface shape on the laminar flow through an array of superhydrophobic pillars

In this study, measurements of the pressure drop and the velocity vector fields through a regular array of superhydrophobic pillars were systematically taken to investigate the role of air–water interface shape on laminar drag reduction. A polydimethylsiloxane microfluidic channel was created with a regular array of apple-core-shaped and circular pillars bridging across the entire channel. Due to the shape and hydrophobicity of the apple-core-shaped pillars, air was trapped on the side of the pillars after filling the microchannel with water. The measurements were taken at a capillary number of Ca = 6.6 × 10^?5. The shape of the air–water interface trapped within the superhydrophobic apple-core-shaped pillars was systematically modified from concave to convex by changing the static pressure within the microchannel. The pressure drop through the microchannel containing the superhydrophobic apple-core-shaped pillars was found to be sensitive to the shape of the air–water interface. For static pressures which resulted in the apple-core-shaped superhydrophobic pillars having a circular cross section, D/D ₀ = 1, a drag reduction of 7% was measured as a result of slip along the air–water interface. At large static pressures, the interface was driven into the apple-core-shaped pillars, resulting in decrease in the effective size of the pillars and an increase in the effective spacing between pillars. When combined with a slip velocity measured to be 10% of the average velocity between the pillars, the result was a pressure drop reduction of 18% compared to the circular pillars at a non-dimensional interface diameter of D/D ₀ = 0.8. At low static pressures, the pressure drop increased significantly as the expanded air–water interface constricted flow through the array of pillars even as large interfacial slip velocity was maintained. At D/D ₀ = 1.1, for example, the pressure drop increased by 17% compared to the circular pillar. This drag increase was the result of an increased form drag due to a decrease in porosity and permeability of the pillar array and a decrease in the skin friction drag due to the presence of the air–water interface. For D/D ₀ = 1.1, the slip velocity was measured to be 45% of the average streamwise velocity between the pillars. When compared to no-slip pillars of similar shape, the drag reduction was found to increase from 6 to 9% with increasing convex curvature of the air–water interface.     

Soft lithography based on photolithography and two-photon polymerization

Over the past decades, soft lithography has greatly facilitated the development of microfluidics due to its simplicity and cost-effectiveness. Besides, numerous fabrication techniques such as multi-layer photolithography, stereolithography and other methods have been developed to fabricate moulds with complex 3D structures nowadays. But these methods are usually not beneficial for microfluidic applications either because of low resolution or sophisticated fabrication procedures. Besides, high-resolution methods such as two-photon lithography, electron-beam lithography, and focused ion beam are often restricted by fabrication speed and total fabricated volume. Nonetheless, the region of interest in typical microfluidic devices is usually very small while the rest of the structure does not require complex 3D fabrication methods. Herein, conventional photolithography and two-photon polymerization are combined for the first time to form a simple hybrid approach in fabricating master moulds for soft lithography. It not only benefits from convenience of photolithography, but also gives rise to complex 3D structures with high resolution based on two-photon polymerization. In this paper, various tests have been conducted to further study its performance, and a passive micromixer has been created as a demonstration for microfluidic applications.     

Effects of geometry factors on microvortices evolution in confined square microcavities

Recently, microcavities have become a central feature of diverse microfluidic devices for many biological applications. Thus, the flow and transport phenomena in microcavities characterized by microvortices have received increasing research attention. It is important to understand thoroughly the geometry factors on the flow behaviors in microcavities. In an effort to provide a design guideline for optimizing the microcavity configuration and better utilizing microvortices for different applications, we investigated quantitatively the liquid flow characteristics in different square microcavities located on one side of a main straight microchannel by using both microparticle image velocimetry (micro-PIV) and numerical simulation. The influences of the inlet Reynolds numbers (with relatively wider values Re?=?1–400) and the hydraulic diameter of the main microchannel (D_H?=?100, 133 μm) on the evolution of microvortices in different square microcavities (100, 200, 400 and 800 μm) were studied. The evolution and characteristic of the microvortices were investigated in detail. Moreover, the critical Reynolds numbers for the emergence of microvortices and the transformation of flow patterns in different microcavities were determined. The results will provide a useful guideline for the design of microcavity-featured microfluidic devices and their applications.     

Sensitive analysis of glutathione in bacteria and HaCaT cells by polydopamine/gold nanoparticle-coated microchip electrophoresis via online pre-concentration of field-amplified sample stacking

In this paper, polydopamine/gold nanoparticles (PDA/Au NPs) were used to construct a functional film on a glass microfluidic channel surface in microchip electrophoresis (MCE) for the separation of reduced glutathione (GSH) and oxidized glutathione (GSSH). The formation of the PDA/Au NPs was characterized by scanning electron microscopy, transmission electron microscope, UV–Vis spectra and ATR-FTIR. An online pre-concentration strategy involving field-amplified sample stacking was used to determine the sensitivity of the assay for measuring GSH and GSSH in bacteria (Escherichia coli, Staphylococcus aureus and Salmonella enterica serovar Typhimurium) and HaCaT cells samples by MCE with laser-induced fluorescence detection. The influences of the separation voltage, the concentration of the running buffer and the pH value of running buffer, were carefully investigated. Using this studied method, GSH and GSSH could be simultaneously pre-concentrated and separated within 70 s. The limits of detection of GSH and GSSH were as low as 0.81 and 0.91 nM, respectively (S/N = 3), which corresponded to approximately 180–301-fold improvements in peak height. Moreover, this method was successfully applied to the analysis of bacteria (E. coli, S. aureus and S. typhimurium) and HaCaT cell samples with a satisfactory recovery rate.     

Operating regimes of a magnetic split-flow thin (SPLITT) fractionation microfluidic device for immunomagnetic separation

Magnetic bead-based immunoassays in the microfluidic format have attracted particular interest as it has several advantages over other microfluidic separation techniques. Magnetic split-flow thin fractionation (SPLITT) is a compact version of microfluidic sorting where a bidispersed or polydispersed suspension of magnetic particle–analyte conjugates can be selectively isolated into co-flowing streams of nearly monodispersed particles. Although the device offers capability of identifying and separating more than one target analytes simultaneously, its performance is sensitive to the slightest variation of the operating condition. Herein, we have numerically investigated the performance of a microscale magnetic SPLITT device. Using a coupled Eulerian–Lagrangian approach, we have evaluated the capture efficiency (CE) and separation index (SI) for each particle type collected at their designated outlet of the SPLITT device and identified the best regimes of operating parameters. While the CE figures are found to be best represented by a group variable Π, the SI values are better represented as function of the product of the group variables γ and β; the SI versus Π plots clearly separate into two basic trends: one for constant β (i.e., varying γ) and the other for constant γ (i.e., varying β). Our study prescribes the desired operating regimes of a microfluidic magnetophoretic SPLITT device in a practical immunomagnetic separation application.     

Effects of topological changes in microchannel geometries on the asymmetric breakup of a droplet

Passive asymmetric breakups of a droplet could be done in many microchannels of various geometries. In order to study the effects of different geometries on the asymmetric breakup of a droplet, four types of asymmetric microchannels with the topological equivalence of geometry are designed, which are T-90, Y-120, Y-150, and I-180 microchannels. A three-dimensional volume of fluid multiphase model is employed to investigate the asymmetric rheological behaviors of a droplet numerically. Three regimes of rheological behaviors as a function of the capillary numbers Ca and the asymmetries As defined by As = (b1 ? b2)/(b1 + b2) (where b1 and b2 are the widths of two asymmetric sidearms) have been observed. A power law model based on three major factors (Ca, As and the initial volume ratio r ₀) is employed to describe the volume ratio of two daughter droplets. The analysis of pressure fields shows that the pressure gradient inside the droplet is one of the major factors causing the droplet translation during its asymmetric breakup. Besides the above similarities among various microchannels, the asymmetric breakup in them also have some slight differences as various geometries have different enhancement or constraint effects on the translation of the droplet and the cutting action of flows. It is disclosed that I-180 microchannel has the smallest critical capillary number, the shortest splitting time, and is hardest to generate satellite droplets.