An integrated, multiparametric flow cytometry chip using "microfluidic drifting" based three-dimensional hydrodynamic focusing

In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.     

Convenient quantification of methanol concentration detection utilizing an integrated microfluidic chip

A rapid and simple technique is proposed for methanol concentration detection using a PMMA (Polymethyl-Methacrylate) microfluidic chip patterned using a commercially available CO₂ laser scriber. In the proposed device, methanol and methanol oxidase (MOX) are injected into a three-dimensional circular chamber and are mixed via a vortex stirring effect. The mixture is heated to prompt the formation of formaldehyde and is flowed into a rectangular chamber, to which fuchsin-sulphurous acid is then added. Finally, the microchip is transferred to a UV spectrophotometer for methanol detection purposes. The experimental results show that a correlation coefficient of R² = 0.9940 is obtained when plotting the optical density against the methanol concentration for samples and an accuracy as high as 93.1% are compared with the determined by the high quality gas chromatography with concentrations in the range of 2 ∼ 100 ppm. The methanol concentrations of four commercial red wines are successfully detected using the developed device. Overall, the results show that the proposed device provides a rapid and accurate means of detecting the methanol concentration for a variety of applications in the alcoholic beverage inspection and control field.     

Polydimethylsiloxane microfluidic chip with integrated microheater and thermal sensor

A microheater and a thermal sensor were fabricated inside elastomeric polydimethylsiloxane microchannels by injecting silver paint (or other conductive materials) into the channels. With a high-precision control scheme, microheaters can be used for rapid heating, with precise temperature control and uniform thermal distribution. Using such a microheater and feedback system, a polymerase chain reaction experiment was carried out whereas the DNA was successfully amplified in 25 cycles, with 1 min per cycle.     

An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting

Multi-target pathogen detection using heterogeneous medical samples require continuous filtering, sorting, and trapping of debris, bioparticles, and immunocolloids within a diagnostic chip. We present an integrated AC dielectrophoretic (DEP) microfluidic platform based on planar electrodes that form three-dimensional (3D) DEP gates. This platform can continuously perform these tasks with a throughput of 3 μL∕min. Mixtures of latex particles, Escherichia coli Nissle, Lactobacillus, and Candida albicans are sorted and concentrated by these 3D DEP gates. Surface enhanced Raman scattering is used as an on-chip detection method on the concentrated bacteria. A processing rate of 500 bacteria was estimated when 100 μl of a heterogeneous colony of 10⁷ colony forming units ∕ml was processed in a single pass within 30 min.     

Viscoelastic effects on electrokinetic particle focusing in a constricted microchannel

Focusing suspended particles in a fluid into a single file is often necessary prior to continuous-flow detection, analysis, and separation. Electrokinetic particle focusing has been demonstrated in constricted microchannels by the use of the constriction-induced dielectrophoresis. However, previous studies on this subject have been limited to Newtonian fluids only. We report in this paper an experimental investigation of the viscoelastic effects on electrokinetic particle focusing in non-Newtonian polyethylene oxide solutions through a constricted microchannel. The width of the focused particle stream is found NOT to decrease with the increase in DC electric field, which is different from that in Newtonian fluids. Moreover, particle aggregations are observed at relatively high electric fields to first form inside the constriction. They can then either move forward and exit the constriction in an explosive mode or roll back to the constriction entrance for further accumulations. These unexpected phenomena are distinct from the findings in our earlier paper [Lu et al., Biomicrofluidics 8, 021802 (2014)], where particles are observed to oscillate inside the constriction and not to pass through until a chain of sufficient length is formed. They are speculated to be a consequence of the fluid viscoelasticity effects.     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

Polydimethylsiloxane-based conducting composites and their applications in microfluidic chip fabrication

This paper reviews the design and fabrication of polydimethylsiloxane (PDMS)-based conducting composites and their applications in microfluidic chip fabrication. Owing to their good electrical conductivity and rubberlike elastic characteristics, these composites can be used variously in soft-touch electronic packaging, planar and three-dimensional electronic circuits, and in-chip electrodes. Several microfluidic components fabricated with PDMS-based composites have been introduced, including a microfluidic mixer, a microheater, a micropump, a microdroplet controller, as well as an all-in-one microfluidic chip.     

Integrated electrical concentration and lysis of cells in a microfluidic chip

Lysing cells is an important step in the analysis of intracellular contents. Concentrating cells is often required in order to acquire adequate cells for lysis. This work presents an integrated concentration and lysis of mammalian cells in a constriction microchannel using dc-biased ac electric fields. By adjusting the dc component, the electrokinetic cell motion can be precisely controlled, leading to an easy switch between concentration and lysis of red blood cells in the channel constriction. These two operations are also used in conjunction to demonstrate a continuous concentration and separation of leukemia cells from red blood cells in the same microchannel. The observed cell behaviors agree reasonably with the simulation results.     

An integrated microfluidic system for isolation, counting, and sorting of hematopoietic stem cells

This study reports an integrated microfluidic system capable of isolation, counting, and sorting of hematopoietic stem cells (HSCs) from cord blood in an automatic format by utilizing a magnetic-bead-based immunoassay. Three functional modules, including cell isolation, cell counting, and cell sorting modules are integrated on a single chip by using microfluidic technology. The cell isolation module is comprised of a four-membrane-type micromixer for binding of target stem cells and magnetic beads, two pneumatic micropumps for sample transport, and an S-shaped channel for isolation of HSCs using a permanent magnet underneath. The counting and sorting of HSCs are performed by utilizing the cell counting and sorting modules. Experimental results show that a separation efficiency as high as 88% for HSCs from cord blood is achieved within 40 min for a sample volume of 100 μl. Therefore, the development of this integrated microfluidic system may be promising for various applications such as stem cell research and cell therapy.     

Construction and operation of a microrobot based on magnetotactic bacteria in a microfluidic chip

Magnetotactic bacteria (MTB) are capable of swimming along magnetic field lines. This unique feature renders them suitable in the development of magnetic-guided, auto-propelled microrobots to serve in target molecule separation and detection, drug delivery, or target cell screening in a microfluidic chip. The biotechnology to couple these bacteria with functional loads to form microrobots is the critical point in its application. Although an immunoreaction approach to attach functional loads to intact MTB was suggested, details on its realization were hardly mentioned. In the current paper, MTB-microrobots were constructed by attaching 2 μm diameter microbeads to marine magnetotactic ovoid MO-1 cells through immunoreactions. These microrobots were controlled using a special control and tracking system. Experimental results prove that the attachment efficiency can be improved to ∼30% via an immunoreaction. The motility of the bacteria attached with different number of loads was also assessed. The results show that MTB can transport one load at a velocity of ∼21 μm/s and still move and survive for over 30 min. The control and tracking system is fully capable of directing and monitoring the movement of the MTB-microrobots. The rotating magnetic fields can stop the microrobots by trapping them as they swim within a circular field with a controllable size. The system has potential use in chemical analyses and medical diagnoses using biochips as well as in nano/microscale transport.     

Red blood cell recognition and posture estimation in microfluidic chip based on lensless imaging

On the one hand, lensless imaging technology has become one of the key technologies to achieve point-of-care testing; on the other hand, microfluidic technology has shown great application potential in the field of biological detection. Using mainstream lensless imaging technology to achieve biological cell imaging in microfluidic chips has technical limitations. In particular, it is more difficult to achieve lensless imaging for non-spherical cells in microfluidic chips such as red blood cells. Achieving red blood cell recognition and posture estimation in a microfluidic chip under the lensless imaging, combined with mainstream lensless imaging technology, can provide more effective red blood cell morphological parameters for medical diagnosis. In this paper, the method for red blood cell recognition and posture estimation in microfluidic chips based on lensless imaging is given. First, the relevant theoretical basis is introduced. Then, the models of red blood cell recognition and posture estimation in microfluidic chips based on lensless imaging are given. The effect of red blood cell flipping on lensless imaging is analyzed in the modeling process. Finally, the effectiveness of the proposed method is verified by experiments. Experiments show that the proposed method can well achieve red blood cell recognition and posture estimation through the shape characteristics of red blood cells.     

Vortex-aided inertial microfluidic device for continuous particle separation with high size-selectivity,efficiency, and purity

In this paper, we report an inertial microfluidic device with simple geometry for continuous extraction of large particles with high size-selectivity (<2 μm), high efficiency (∼90%), and high purity (>90%). The design takes advantage of a high-aspect-ratio microchannel to inertially equilibrate cells and symmetric chambers for microvortex-aided cell extraction. A side outlet in each chamber continuously siphons larger particles, while the smaller particles or cells exit through the main outlet. The design has several advantages, including simple design, small footprint, ease of paralleling and cascading, one-step operation, and continuous separation with ultra-selectivity, high efficiency and purity. The described approach is applied to manipulating cells and particles for ultra-selective separation, quickly and effectively extracting larger sizes from the main flow, with broad applications in cell separations.     

Toward a modular,integrated, miniaturized,and portable microfluidic flow control architecture for organs-on-chips applications

Microfluidic organs-on-chips (OoCs) technology has emerged as the trend for in vitro functional modeling of organs in recent years. Simplifying the complexities of the human organs under controlled perfusion of required fluids paves the way for accurate prediction of human organ functionalities and their response to interventions like exposure to drugs. However, in the state-of-the-art OoC, the existing methods to control fluids use external bulky peripheral components and systems much larger than the chips used in experiments. A new generation of compact microfluidic flow control systems is needed to overcome this challenge. This study first presents a structured classification of OoC devices according to their types and microfluidic complexities. Next, we suggest three fundamental fluid flow control mechanisms and define component configurations for different levels of OoC complexity for each respective mechanism. Finally, we propose an architecture integrating modular microfluidic flow control components and OoC devices on a single platform. We emphasize the need for miniaturization of flow control components to achieve portability, minimize sample usage, minimize dead volume, improve the flowing time of fluids to the OoC cell chamber, and enable long-duration experiments.     

Rapid detection of live methicillin-resistant Staphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to all existing penicillin and lactam-based antimicrobial drugs and, therefore, has become one of the most prevalent antibiotic-resistant pathogens found in hospitals. The multi-drug resistant characteristics of MRSA make it challenging to clinically treat infected patients. Therefore, early diagnosis of MRSA has become a public-health priority worldwide. Conventionally, cell-culture based methodology and microscopic identification are commonly used for MRSA detection. However, they are relatively time-consuming and labor-intensive. Recently, molecular diagnosis based on nucleic acid amplification techniques, such as polymerase chain reaction (PCR), has been widely investigated for the rapid detection of MRSA. However, genomic DNA of both live and dead pathogens can be distinguished by conventional PCR. These results thus could not provide sufficient confirmation of an active infection for clinicians. In this study, live MRSA was rapidly detected by using a new integrated microfluidic system. The microfluidic system has been demonstrated to have 100% specificity to detect live MRSA with S. aureus and other pathogens commonly found in hospitals. The experimental results showed that the limit of detection for live MRSA from biosamples was approximately 10² CFU/μl. In addition, the entire diagnostic protocol, from sample pre-treatment to fluorescence observation, can be automatically completed within 2.5 h. Consequently, this microfluidic system may be a powerful tool for the rapid molecular diagnosis of live MRSA.     

Hybrid capillary-inserted microfluidic device for sheathless particle focusing and separation in viscoelastic flow

A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 μm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 μm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 μm. At 200 μl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.     

Experimental validation of numerical study on thermoelectric-based heating in an integrated centrifugal microfluidic platform for polymerase chain reaction amplification

A comprehensive study involving numerical analysis and experimental validation of temperature transients within a microchamber was performed for thermocycling operation in an integrated centrifugal microfluidic platform for polymerase chain reaction (PCR) amplification. Controlled heating and cooling of biological samples are essential processes in many sample preparation and detection steps for micro-total analysis systems. Specifically, the PCR process relies on highly controllable and uniform heating of nucleic acid samples for successful and efficient amplification. In these miniaturized systems, the heating process is often performed more rapidly, making the temperature control more difficult, and adding complexity to the integrated hardware system. To gain further insight into the complex temperature profiles within the PCR microchamber, numerical simulations using computational fluid dynamics and computational heat transfer were performed. The designed integrated centrifugal microfluidics platform utilizes thermoelectrics for ice-valving and thermocycling for PCR amplification. Embedded micro-thermocouples were used to record the static and dynamic thermal responses in the experiments. The data collected was subsequently used for computational validation of the numerical predictions for the system response during thermocycling, and these simulations were found to be in agreement with the experimental data to within ∼97%. When thermal contact resistance values were incorporated in the simulations, the numerical predictions were found to be in agreement with the experimental data to within ∼99.9%. This in-depth numerical modeling and experimental validation of a complex single-sided heating platform provide insights into hardware and system design for multi-layered polymer microfluidic systems. In addition, the biological capability along with the practical feasibility of the integrated system is demonstrated by successfully performing PCR amplification of a Group B Streptococcus gene.     

A microfluidic platform for studying the effects of small temperature gradients in an incubator environment

Studies on the effects of variations in temperature and mild temperature gradients on cells, gels, and scaffolds are important from the viewpoint of biological function. Small differences in temperature are known to elicit significant variations in cell behavior and individual protein reactivity. For the study of thermal effects and gradients in vitro, it is important to develop microfluidic platforms which are capable of controlling temperature gradients in an environment which mimics the range of physiological conditions. In the present paper, such a microfluidic thermal gradient system (μTGS) system is proposed which can create and maintain a thermal gradient throughout a cell-seeded gel matrix using the hot and cold water supply integrated in the system in the form of a countercurrent heat exchanger. It is found that a uniform temperature gradient can be created and maintained in the device even inside a high temperature and high humidity environment of an incubator. With the help of a hot and cold circuit controlled from outside the incubator the temperature gradient can be regulated. A numerical simulation of the device demonstrates the thermal feature of the chip. Cell viability and activity under a thermal gradient are examined by placing human breast cancer cells in the device.     

Reconfigurable microfluidic device with integrated antibody arrays for capture,multiplexed stimulation,and cytokine profiling of human monocytes

Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)—transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.     

Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluid dynamics simulations

Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.     

Long-range and superfast trapping of DNA molecules in an ac electrokinetic funnel

In this work we report a microfluidic platform capable of trapping and concentrating a trace amount of DNA molecules efficiently. Our strategy invokes nonlinear electro-osmotic flow induced by charge polarization under high-frequency ac fields. With the asymmetric quadrupole electrode design, a unique converging flow structure can be created for generating focusing effects on DNA molecules. This focusing in turn transforms into a robust funnel that can collect DNA molecules distantly from the bulk and pack them into a compact cone with the aid of short-range dipole-induced self-attraction and dielectrophoresis. Our results reveal that not only can DNA molecules be concentrated within just a few seconds, but also they can be focused into threads of 1 mm in length, demonstrating the superfast and long-range trapping capability of this funnel. In addition, pico M DNA solutions can be concentrated with several decades of enhancement without any continuous feeding. Alternating concentration and release of DNA molecules is also illustrated, which has potentials in concentrating and transporting biomolecules in a continuous fashion using microdevices.