Centrifugal Microfluidic Device Research Articles

遠心マイクロ流体デバイスを用いた生体分子間相互作用測定法の提案

遠心マイクロ流体デバイスを用いた生体分子間相互作用測定法の提案

Fully automated and colorimetric foodborne pathogen detection on an integrated centrifugal microfluidic device.

This work describes fully automated and colorimetric foodborne pathogen detection on an integrated centrifugal microfluidic device, which is called a lab-on-a-disc. All the processes for molecular diagnostics including DNA extraction and purification, DNA amplification and amplicon detection were integrated on a single disc. Silica microbeads incorporated in the disc enabled extraction and purification of bacterial genomic DNA from bacteria-contaminated milk samples. We targeted four kinds of foodborne pathogens (Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus and Listeria monocytogenes) and performed loop-mediated isothermal amplification (LAMP) to amplify the specific genes of the targets. Colorimetric detection mediated by a metal indicator confirmed the results of the LAMP reactions with the colour change of the LAMP mixtures from purple to sky blue. The whole process was conducted in an automated manner using the lab-on-a-disc and a miniaturized rotary instrument equipped with three heating blocks. We demonstrated that a milk sample contaminated with foodborne pathogens can be automatically analysed on the centrifugal disc even at the 10 bacterial cell level in 65 min. The simplicity and portability of the proposed microdevice would provide an advanced platform for point-of-care diagnostics of foodborne pathogens, where prompt confirmation of food quality is needed.

Microfluidic size separation of cells and particles using a swinging bucket centrifuge.

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.

Centrifugal loop-mediated isothermal amplification microdevice for rapid, multiplex and colorimetric foodborne pathogen detection.

We present a centrifugal microfluidic device which enables multiplex foodborne pathogen identification by loop-mediated isothermal amplification (LAMP) and colorimetric detection using Eriochrome Black T (EBT). Five identical structures were designed in the centrifugal microfluidic system to perform the genetic analysis of 25 pathogen samples in a high-throughput manner. The sequential loading and aliquoting of the LAMP cocktail, the primer mixtures, and the DNA sample solutions were accomplished by the optimized zigzag-shaped microchannels and RPM control. We targeted three kinds of pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus) and detected the amplicons of the LAMP reaction by the EBT-mediated colorimetric method. For the limit-of-detection (LOD) test, we carried out the LAMP reaction on a chip with serially diluted DNA templates of E. coli O157:H7, and could observe the color change with 380 copies. The used primer sets in the LAMP reaction were specific only to the genomic DNA of E. coli O157:H7, enabling the on-chip selective, sensitive, and high-throughput pathogen identification with the naked eyes. The entire process was completed in 60min. Since the proposed microsystem does not require any bulky and expensive instrumentation for end-point detection, our microdevice would be adequate for point-of-care (POC) testing with high simplicity and high speed, providing an advanced genetic analysis microsystem for foodborne pathogen detection.

Rapid and Automated Quantification of Microalgal Lipids on a Spinning Disc.

We have developed a fully integrated centrifugal microfluidic device for rapid on-site quantification of lipids from microalgal samples. The fully automated serial process involving cell sedimentation and lysis, liquid-liquid extraction, and colorimetric detection of lipid contents was accomplished within 13 min using a lab-on-a-disc. The presented organic solvent-tolerable (for n-hexane, ethanol) microfluidic disc was newly fabricated by combining thermal fusion bonding and carbon dot-based valving techniques. It is expected that this novel platform will possibly contribute toward sustainable biofuel applications by providing a practical solution for on-site monitoring of lipid accumulation in microalgal samples, thus providing imperative contribution toward energy and environmental purposes of centrifugal microfluidic technology.

Water-clock-based autonomous flow sequencing in steadily rotating centrifugal microfluidic device

In this paper, we report the demonstration of a flow sequencing method in a centrifugal microfluidic device rotated at a constant speed. The simple poly-dimethylsioxiane chip, which consists of microchannels, chambers, and vent holes, was integrated in a compact disk substrate, and multistep flow sequencing was executed by simply spinning the device at a constant rotational speed. All the triggers were controlled by a water clock using the liquid meniscus as the clock hand. The flow modes to the sample chambers were switched by switching the air supply to the sample chambers, depending of on whether the meniscus water clock was inside the channel connection (disabling air supply) or outside it (enabling air supply). A four-step flow sequence was successfully executed using two different connections of the chambers, namely, parallel connection and serial connection. Highly reproducible autonomous trigger timing with a typical error of less than 3% was achieved. Delicate adjustment of the flow sequencing by varying the channel geometry was also demonstrated. The time-scaling of the flow sequencing was accomplished by changing the viscosity of the fluid in the water clock. Additionally, a four-step flow sequencing involving a flow injection chain was successfully demonstrated by serial connection. Through combinations and further variations of these concepts, it is expected that the proposed method would be used to realize higher-order flow programs in steadily spinning centrifugal microfluidic devices.

A centrifugal microfluidic device for screening protein crystallization conditions by vapor diffusion

This paper describes a centrifuge-based microfluidic device for performing nanoliter-scale protein crystallization trials by vapor diffusion. By combining the capillary effect and centrifugal force, this device spontaneously and parallelly meters and dispenses protein samples and precipitants into 24 reaction chambers for crystallization screening. Furthermore, the design of two-level capillary stop valves and vapor-diffusion chambers in this device allows vapor transport between the protein droplet and the stored precipitant, perfectly equivalent to that observed in conventional vapor diffusion techniques, which slowly brings the protein droplet into a supersaturation state to nucleate and grow protein crystals. We demonstrate the power of this device by testing it for rapid screening of protein crystallization conditions. Compared with the conventional hanging-drop technique, this device can identify more crystallization conditions and provide a wider range of supersaturation rates, while using two orders of magnitude less protein sample. This centrifuge-based microfluidic device provides a simple and low-cost tool for individual laboratories to rapidly screen initial protein crystallization conditions.

RAPID, LOW-COST PROTOTYPING OF CENTRIFUGAL MICROFLUIDIC DEVICES FOR EFFECTIVE IMPLEMENTATION OF VARIOUS MICROFLUIDIC COMPONENTS

A centrifugal microfluidic platform to develop various microfluidic operations – the first of its kind in South Africa – is presented. Rapid and low-cost prototyping of centrifugal microfluidic disc devices, as well as a set-up to test the devices using centrifugal forces, is described. Preliminary results show that various microfluidic operations such as fluidic valving, transportation, and microfluidic droplet generation can be achieved. This work provides a complete centrifugal microfluidic platform and the building blocks on which to develop a variety of microfluidic applications and potential products rapidly and at a low cost.

Rapid prototyping of pneumatically actuated hydrocarbon gel valves for centrifugal microfluidic devices

A novel, easy to prototype hydrocarbon gel-based active valve was developed for use in centrifugal microfluidic devices. The valve has been demonstrated to restrict flow by an additional 1000 revolutions per minute (RPM) when compared to a passive capillary valve of the same size located at the same radius. Opening of the valve is accomplished in a contactless manner using a stream of focused compressed air. The ease of fabrication, low cost and small dimensions of the gel valve offer the potential for integration of multiple valves of this type into multi-process centrifugal microfluidic systems.

All-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity.

Circulating tumor cells (CTCs) have gained increasing attention owing to their roles in cancer recurrence and progression. Due to the rarity of CTCs in the bloodstream, an enrichment process is essential for effective target cell characterization. However, in a typical pressure-driven microfluidic system, the enrichment process generally requires complicated equipment and long processing times. Furthermore, the commonly used immunoaffinity-based positive selection method is limited, as its recovery rate relies on EpCAM expression of target CTCs, which shows heterogeneity among cell types. Here, we propose a centrifugal-force-based size-selective CTC isolation platform that can isolate and enumerate CTCs from whole blood within 30 s with high purity. The device was validated using the MCF-7 breast cancer cell line spiked in phosphate-buffered saline and whole blood, and an average capture efficiency of 61% was achieved, which is typical for size-based filtration. The capture efficiency for whole blood samples varied from 44% to 84% under various flow conditions and dilution factors. Under the optimized operating conditions, a few hundred white blood cells per 1 mL of whole blood were captured, representing a 20-fold decrease compared to those obtained using a commercialized size-based CTC isolation device. In clinical validation, normalized CTC counts varied from 10 to 60 per 7.5 mL of blood from gastric and lung cancer patients, yielding a detection rate of 50% and 38%, respectively. Overall, our CTC isolation device enables rapid and label-free isolation of CTCs with high purity, which should greatly improve downstream molecular analyses of captured CTCs.

Multifunctional wax valves for liquid handling and incubation on a microfluidic CD

Recently, several biological assays have become available on the centrifugal microfluidic platform. Despite many innovative solutions developed for on-disc fluid handling for these assays, certain challenges, including liquid incubation and simplification of a multi-step assay on a plastic device, still need to be further addressed. Incubating fluids that require downstream processing, which we call “midstream incubation”, can often be difficult on the microfluidic disc platform due to surface tension changes induced by varying temperatures, thus causing operating instability. We describe here strategies for liquid reagent storage, release, incubation, and transfer, all of which utilize a single combination of actuation methods—wax valving and heat actuation by halogen lamp—on a centrifugal microfluidic device made using pristine materials. The strategies that we use to perform these steps, termed multifunctional wax valves, enable manipulation of a microlitre range fluid volume without the need for complex fabrication steps or hardware. This technology’s reliability and ease of use will hopefully allow for more powerful fluidics-based diagnostic tools to be created.

Fully Integrated Lab-on-a-Disc for Nucleic Acid Analysis of Food-Borne Pathogens

This paper describes a micro total analysis system for molecular analysis of Salmonella, a major food-borne pathogen. We developed a centrifugal microfluidic device, which integrated the three main steps of pathogen detection, DNA extraction, isothermal recombinase polymerase amplification (RPA), and detection, onto a single disc. A single laser diode was utilized for wireless control of valve actuation, cell lysis, and noncontact heating in the isothermal amplification step, thereby yielding a compact and miniaturized system. To achieve high detection sensitivity, rare cells in large volumes of phosphate-buffered saline (PBS) and milk samples were enriched before loading onto the disc by using antibody-coated magnetic beads. The entire procedure, from DNA extraction through to detection, was completed within 30 min in a fully automated fashion. The final detection was carried out using lateral flow strips by direct visual observation; detection limit was 10 cfu/mL and 10(2) cfu/mL in PBS and milk, respectively. Our device allows rapid molecular diagnostic analysis and does not require specially trained personnel or expensive equipment. Thus, we expect that it would have an array of potential applications, including in the detection of food-borne pathogens, environmental monitoring, and molecular diagnostics in resource-limited settings.

A novel filtration method integrated on centrifugal microfluidic devices

A method has been developed that integrates filters directly into centrifugal microfluidic devices. This technique is suitable for both rapid prototyping and commercial applications. Commercially available filter paper was sealed into the centrifugal microfluidic device with a simple manual fabrication procedure. The method was validated using soil slurry in water and a variety of filter papers with pore sizes ranging from 0.7 to 11 μm. Filtration times of 4 s to several minutes were obtained for 100 μL samples depending on the type of filter paper and rotation rate utilized. The validity of the method was demonstrated by assessing the amount of light lost due to the scatter or absorption caused by particles in the filtered sample while the device was in motion. Filtration and sedimentation were compared and after 30 min of centrifugation, sedimentation had not removed particles as well as filtration. This technique opens up centrifugal microfluidic devices to a wide range of samples.

Enzyme Inhibition-Based Determination of Pesticide Residues in Vegetable and Soil in Centrifugal Microfluidic Devices

Pesticide residue is of concern as an environmental pollutant when present at medium to high concentrations. Such residue was quantified in both vegetable and soil samples by an enzyme inhibition technique. The multistep reactions were integrated into centrifugal microfluidic devices allowing automated simultaneous analysis of several samples or of replicates. The small sample size inherent to microfluidic devices allowed for less reagent to be used including less of the expensive enzyme which is key to this method. Liquid-solid magnetically actuated extraction, filtration, sedimentation, and detection were all integrated on the same device. Several parameters were optimized including the concentration of enzyme, substrate, chromatic agent, and reaction time. In this environmental application of centrifugal microfluidics, the percent inhibition of enzyme activity is logarithmically proportional to the demonstration pesticide concentration (in this case carbofuran). This meant that as the pesticide concentration increased in the samples, the reaction was more inhibited and the final product absorbed less light at 525 nm. Two versions of the centrifugal microfluidic devices were made. One version was designed for the analysis of vegetable samples (cabbage) and the other for the analysis of soil samples. Each version provided results that were statistically similar to the conventional benchtop method with a carbofuran limit of detection of 0.1 ppm or 0.1 μg g(-1) (5 ng absolute limit of detection).

Control of secondary flow in concentrically traveling flow on centrifugal microfluidics

This paper reports a fundamental study of the stripe laminar flow pattern on a centrifugal microfluidic device with the goal of realizing a sedimentation-based, continuous mode particle separation technique. Microfluidic channels were designed with a concentrically integrated microchannel, and the patterning of the flow in the channel was investigated. A significant secondary flow was observed as a preliminary result. We conclude that the origin of this secondary flow was not the Dean force, because it was observed in a straight microchannel, but was not observed in curved channel during the spinning of the system at rest. The transition of the pattern was investigated using a simulation and experiment, and the flow pattern’s dependence on the rotational speed was determined, which suggested that the origin of the secondary flow was the Coriolis force. The significance of the secondary flow was controlled by adjusting the rotational speed of the disk, and the flow rate and laminar flow patterns were controlled by the stripe flow pattern.

Geometry effects on blood separation rate on a rotating disc

The plasma separation rate is enhanced significantly, as much as 8 times faster for whole blood, by simple change of the channel geometry in a rotating disc. It was enhanced in the channels with narrower channel widths and larger tilt angles with respect to the radial direction but independent of the channel depth or spin direction. A modified Ponder-Nakamura-Kuroda (PNK) theory could nicely fit all of the experimental data to a linear line, which data were obtained from spinning discs with various channel designs and blood samples with broad hematocrit range. To the best of our knowledge, this is the first investigation of geometry effects on the particle separation rate on a rotating disc and it will potentially contribute to the further performance enhancement of centrifugal microfluidic devices.

Modeling of Capillary Burst Valve and Siphon with Hydrophilic Cover for Centrifugal Microfluidic Systems

This paper reports numerical and experimental investigation of capillary burst valve and siphon, which were used in the centrifugal microfluidic systems widely. The simulation model is based on the solution of the minimum energy problem by the Surface Evolver software. In contrast to the solution of meniscus equilibrium equations, it can provide the appropriate boundary conditions for the microvalves with a hydrophilic flat cover. As an experimental verification, a centrifugal microfluidic device based on PDMS and glass was fabricated by soft lithography. The simulated results agree well with experimental data. The technology is useful for the design of microfluidic systems with complex wetting characters and geometries.

Flow-enhanced electrochemical immunosensors on centrifugal microfluidic platforms

We present a novel fully integrated centrifugal microfluidic device with features for target antigen capture from biological samples, via a bead-based enzyme-linked immune-sorbent assay, and flow-enhanced electrochemical detection. The limit of detection (LOD) of our device for the C-reactive protein (CRP) was determined to be 4.9 pg mL(-1), a 17-fold improvement over quantification by optical density. The complete sample-to-answer protocol of our device is fully automated and takes less than 20 min. Overall, the presented microfluidic disc adds to the comparatively small number of fully integrated microfluidic-based platforms that utilize electrochemical detection and exemplifies how electrochemical detection can be enhanced by flow to successfully detect very low levels of biomarkers (e.g. pg mL(-1)).

Automated Liquid–Liquid Extraction by Pneumatic Recirculation on a Centrifugal Microfluidic Platform

In this technical note, a liquid-liquid extraction technique was performed using pneumatic liquid recirculation on a centrifugal microfluidic device. Non-contact pneumatic pumping enabled a multi-cycle liquid-liquid extraction process using aqueous iodine in a potassium iodide solution and hexadecane while requiring a minimal amount of space on the device. The extraction process was completely automated on the device following sample introduction and required only 50 s for each extraction cycle. The pumping rate achieved during liquid recirculation was 120 ± 10 μL/min. A recycling process such as the one demonstrated would be difficult to implement in a conventional centrifugal microfluidic system.

Stacked centrifugal microfluidic device with three-dimensional microchannel networks and multifunctional capillary bundle structures for immunoassay

A stacked-type centrifugal microfluidic device with three-dimensional (3D) microchannel networks and multifunctional capillary bundle structures is developed, and an immunoassay for the detection of immunoglobulin G (IgG) is demonstrated. The device consists of multiple layers of disk-like chips with conventional planar microchannels and vertical capillary bundle structures that are constructed on disks of poly(dimethylsiloxane) and poly(methyl methacrylate). The integration of multiple reactors up to 10 reactors on a disk with 8cm in diameter is realized by reducing the area of reservoir but increasing the thickness. Moreover the thick optical detection reservoir is also realized for absorption spectroscopy in the 3D centrifugal microfluidics. 3D liquid valving on the vertical capillary bundle structures and liquid transportation through these structures are successfully demonstrated. The results show the successful control of the multistep liquid injection without problems such as blockage of capillary bundle by bubbles that expected to be caused due to 3D structure. The results of the immunoassay show clear response to analyte concentration suggesting successful unit operations in developed 3D centrifugal microfluidic device for immunoassay. Finally, the advantages and impact of the 3D design for the development of high-performance centrifugal microfluidic systems are summarized.