Concentration‐controlled particle focusing in spiral elasto‐inertial microfluidic devices

Herein, we proposed a strategy for controlling the particle focusing position in Dean‐coupled elasto‐inertial flows via adjusting the polymer concentration of viscoelastic fluids. The physics behind the control strategy was then explored and discussed. At high polymer concentrations, the flowing particles could be single‐line focused exactly at the channel centerline under the dominated elastic force. The center‐line focusing in our spiral channel may employed as a potential pretreatment scheme for microflow cytometry detection. With further decreasing polymer concentrations, the particles would shift into the outer channel region under the comparable competition between inertial lift force, elastic force and Dean drag force. Finally, the observed position‐shifting was successfully employed for particle concentration at a throughput much higher than most existing elasto‐inertial microfluidics.     

High‐throughput and sensitive particle counting by a novel microfluidic differential resistive pulse sensor with multidetecting channels and a common reference channel

High‐throughput particle counting by a differential resistive pulse sensing method in a microfluidic chip is presented in this paper. A sensitive differential microfluidic sensor with multiple detecting channels and one common reference channel was devised. To test the particle counting performance of this chip, an experimental system which consists of the microfluidic chip, electric resistors, an amplification circuit, a LabView based data acquisition device was developed. The influence of the common reference channel on the S/N of particle detection was investigated. The relationship between the hydraulic pressure drop applied across the detecting channel and the counting throughput was experimentally obtained. The experimental results show that the reference channel designed in this work can improve the S/N by ten times, thus enabling sensitive high‐throughput particle counting. Because of the greatly improved S/N, the sensing gate with a size of 25 × 50 × 10 μm (W × L × H) in our chips can detect and count particles larger than 1.5 μm in diameter. The counting throughput increases with the increase in the flowing velocity of the sample solution. An average throughput of 7140/min under a flow rate of 10 μL/min was achieved. Comparing with other methods, the structure of the chip and particle detecting mechanism reported in this paper is simple and sensitive, and does not have the crosstalking problem. Counting throughput can be adjusted simply by changing the number of the detecting channels.     

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.     

Ultrahigh throughput beehive-like device for blood plasma separation

We report here a low-cost, rapid-prototyping, and beehive-like multilayer polymer microfluidic device for ultrahigh-throughput blood plasma separation. To understand the device physics and optimize the device structure, the effect of cross-sectional dimension and operational parameter on particle focusing behavior was explored using a single spiral microchannel device. Then, the blood plasma separation performance of the determined channel structure was validated using the blood samples with different hematocrits (HCTs). It was found that a high separation efficiency of 99% could be achieved using the blood sample with an HCT of 0.5% at a high throughput of 1 mL/min. Finally, a multilayer microfluidic device with a novel beehive-like multiplexing channel arrangement was developed for ultrahigh-throughput blood plasma separation. The prototype device could be fabricated within ∼1 hour utilizing the laser cutting and thermal lamination methods. The total processing throughput could reach up to 72 mL/min for 0.5% HCT sample with a plasma separation ratio close to 90%. Our device may hold potentials for the ultrahigh-throughput separation of blood plasma from large volume blood samples for downstream disease diagnosis.     

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.     

Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells

A simple three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of intracellular content is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To solve this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed. With the three sheath flows around the sample solution-containing cells, the formed soft fluid wall prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and smoothly introduced into the cross-section one by one. The introduction of sheath-flow streams not only ensured single-cell sampling but avoided blockage of the sampling channel by adherent cells as well. The maximum rate for introduction of individual cells into the separation channel was about 151 cells min(-1). With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells. The average analysis throughput of ROS and GSH in single cells was 16-18 cells min(-1).     

Impedance‐based viscoelastic flow cytometry

Elastic nature of the viscoelastic fluids induces lateral migration of particles into a single streamline and can be used by microfluidic based flow cytometry devices. In this study, we investigated focusing efficiency of polyethylene oxide based viscoelastic solutions at varying ionic concentration to demonstrate their use in impedimetric particle characterization systems. Rheological properties of the viscoelastic fluid and particle focusing performance are not affected by ionic concentration. We investigated the viscoelastic focusing dynamics using polystyrene (PS) beads and human red blood cells (RBCs) suspended in the viscoelastic fluid. Elasto‐inertial focusing of PS beads was achieved with the combination of inertial and viscoelastic effects. RBCs were aligned along the channel centerline in parachute shape which yielded consistent impedimetric signals. We compared our impedance‐based microfluidic flow cytometry results for RBCs and PS beads by analyzing particle transit time and peak amplitude at varying viscoelastic focusing conditions obtained at different flow rates. We showed that single orientation, single train focusing of nonspherical RBCs can be achieved with polyethylene oxide based viscoelastic solution that has been shown to be a good candidate as a carrier fluid for impedance cytometry.     

惯性效应在微流控芯片中的应用

作为一种操控粒子或流体的新技术,基于流体惯性的操控技术已被应用于微流控芯片中粒子的输运、分选、聚焦及试样的混合和反应等操作,而在微尺度惯性效应基础上的惯性微流控芯片由于具有高通量、无需外场介入、低成本、易集成及微型化等众多优点,可用于解决医疗诊断、生化分析、合成化学及环境监测等领域的检测分析和微量操控问题,因此对该技术的机理及应用研究已成为目前微流控技术领域一个重要的研究热点。本文在介绍惯性微流控芯片机理及其研究进展的同时,从惯性聚焦、惯性分选及基于Dean流的微混合器和微流控光学器件等几个方面对惯性微流控芯片的最新应用研究进展进行了较为详细的介绍和分析比较。在此基础上,分析了惯性微流控芯片的局限和未来需要解决的问题。     

A smart and portable micropump for stable liquid delivery

Precise and reliable liquid delivery is vital for microfluidic applications. Here, we illustrate the design, fabrication, characterization, and application of a portable, low cost, and robust micropump, which brings solution to stable liquid delivery in microfluidic environment. The pump is designed with three optional speeds of different pumping flow rates, and it can be simply actuated by spring‐driven mechanism. The different flow rates of the pump are realized via passive microvalves in a compact microfluidic chip, which is installed in the pump. Importantly, the membrane structures of the microvalves allow accurate liquid control, and stable flow rates can be achieved via a spring setup. The proposed pump is applied to continuously and stably infuse microbead suspension into an inertial microfluidic chip, and good particle focusing is realized in the spiral channel of the inertial microfluidic chip. The proposed portable, self‐powered, and cost‐efficient pump is crucial for microfluidic lab‐on‐a‐chip system integration, which may facilitate microfluidic application for precise liquid delivery, control, measurement, and analysis.     

Inertial focusing of microparticles,bacteria, and blood in serpentine glass channels

Early detection of pathogenic microorganisms is pivotal to diagnosis and prevention of health and safety crises. Standard methods for pathogen detection often rely on lengthy culturing procedures, confirmed by biochemical assays, leading to >24 h for a diagnosis. The main challenge for pathogen detection is their low concentration within complex matrices. Detection of blood-borne pathogens via techniques such as PCR requires an initial positive blood culture and removal of inhibitory blood components, reducing its potential as a diagnostic tool. Among different label-free microfluidic techniques, inertial focusing on microscale channels holds great promise for automation, parallelization, and passive continuous separation of particles and cells. This work presents inertial microfluidic manipulation of small particles and cells (1–10 μm) in curved serpentine glass channels etched at different depths (deep and shallow designs) that can be exploited for (1) bacteria preconcentration from biological samples and (2) bacteria-blood cell separation. In our shallow device, the ability to focus Escherichia coli into the channel side streams with high recovery (89% at 2.2× preconcentration factor) could be applied for bacteria preconcentration in urine for diagnosis of urinary tract infections. Relying on differential equilibrium positions of red blood cells and E. coli inside the deep device, 97% red blood cells were depleted from 1:50 diluted blood with 54% E. coli recovered at a throughput of 0.7 mL/min. Parallelization of such devices could process relevant volumes of 7 mL whole blood in 10 min, allowing faster sample preparation for downstream molecular diagnostics of bacteria present in bloodstream.     

Fluorescence affinity sensing by using a self-contained fluid manoeuvring microfluidic chip

An application of a novel polymer microfluidic chip for sample exchange via natural capillary forces for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by capillary force only, which would therefore be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Micrometre-sized polystyrene beads were used for the functionalization of biomolecules. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. DNP was immobilized on the polymer beads via a pre-adsorbed dendrimer layer and the beads were placed in the reaction chamber. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A calibration curve was registered based on the anti-DNP concentration. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin. The microfluidic chip described in this work could be applied to various biological and chemical analyses using integrated washing steps or fluid replacement steps with minimum sample handling.     

Novel multi-depth microfluidic chip for single cell analysis

A novel multi-depth microfluidic chip was fabricated on glass substrate by use of conventional lithography and three-step etching technology. The sampling channel on the microchip was 37 microm deep, while the separation channel was 12 microm deep. A 1mm long weir was constructed in the separation channel, 300 microm down the channel crossing. The channel at the weir section was 6 microm deep. By using the multi-depth microfluidic chip, human carcinoma cells, which easily aggregate, settle and adhere to the surface of the channel, can be driven from the sample reservoir to the sample waste reservoir by hydrostatic pressure generated by the difference of liquid level between sample and sample waste reservoirs. Single cell loading into the separation channel was achieved by applying a set of pinching potentials at the four reservoirs. The loaded cell was stopped by the weir and precisely positioned within the separation channel. The trapped cell was lysed by sodium dodecyl sulfate (SDS) containing buffer solution in 20s. This approach reduced the lysing time and improved the reproducibility of chip-based electrophoresis separations. Reduced glutathione (GSH) and reactive oxygen species (ROS) were used as model intracellular components in single human carcinoma cells, and the constituents were separated by chip-based electrophoresis and detected by laser-induced fluorescence (LIF). A throughput of 15 samples/h, a migration time precision of 3.1% RSD for ROS and 4.9% RSD for GSH were obtained for 10 consecutively injected cells.     

Automated cellular sample preparation using a Centrifuge-on-a-Chip

The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allows enrichment of cancer cells from spiked blood samples at the mL min(-1) scale, followed by fluorescent labeling of intra- and extra-cellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge-analogue may open opportunities in automated, low-cost and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource poor settings.     

Genotyping from saliva with a one-step microdevice

This paper presents a disposable microfluidic device for on-chip lysing, PCR, and analysis in one continuous-flow process. Male-female sex determination was performed with human saliva in less than 20 min from spit to finish, and requiring only seconds of manual sample handling. This genetic analysis was based on the amplification and detection of the DYZ1 repeat region unique to the Y-chromosome. The flow-through microfluidic chip consisted of a single serpentine channel designed to guide samples through 42 heating and cooling cycles. Cycling was performed by matching the local channel geometry to a steady-state temperature gradient established across the microfluidic chip. 38 channel segments were designed for rapid low volume PCR, and four were optimized for spatial DNA melting analysis. Fluorescence detection was used to monitor the amplification and to capture the melting signature of the amplicon was performed with a basic 8-bit CCD camera. The microfluidic device itself was fabricated from microscope slides and a double-sided tape. The simplicity of the system and its robust performance combine in an elegant solution for lab-on-a-chip genetic analysis.     

Laminar flow mediated continuous single-cell analysis on a novel poly(dimethylsiloxane) microfluidic chip

A novel microfluidic chip with simple design, easy fabrication and low cost, coupled with high-sensitive laser induced fluorescence detection, was developed to provide continuous single-cell analysis based on dynamic cell manipulation in flowing streams. Making use of laminar flows, which formed in microchannels, single cells were aligned and continuously introduced into the sample channel and then detection channel in the chip. In order to rapidly lyse the moving cells and completely transport cellular contents into the detection channel, the angle of the side-flow channels, the asymmetric design of the channels, and the number, shape and layout of micro-obstacles were optimized for effectively redistributing and mixing the laminar flows of single cells suspension, cell lysing reagent and detection buffer. The optimized microfluidic chip was an asymmetric structure of three microchannels, with three microcylinders at the proper positions in the intersections of channels. The microchip was evaluated by detection of anticancer drug doxorubicin (DOX) uptake and membrane surface P-glycoprotein (P-gp) expression in single leukemia K562 cells. An average throughput of 6–8 cells min⁻¹ was achieved. The detection results showed the cellular heterogeneity in DOX uptake and surface P-gp expression within K562 cells. Our researches demonstrated the feasibility and simplicity of the newly developed microfluidic chip for chemical single-cell analysis.     

A low-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood

The ability to isolate and purify white blood cells (WBCs) from mixed ensembles such as blood would benefit autologous cell-based therapeutics as well as diagnosis of WBC disorders. Current WBCs isolation methods have the limitations of low purity or requiring complex and expensive equipment. In addition, due to the overlap in size distribution between lymphocytes (i.e., a sub-population of WBCs) and red blood cells (RBCs), it is challenging to achieve isolation of entire WBCs populations. In this work, we developed an inertial microfluidics-based cell sorter, which enables size-based, high-throughput isolation, and enrichment of WBCs from RBC-lysed whole blood. Using the developed inertial microfluidic chip, the sorting resolution is sharpened within 2 μm, which achieved separation between 3 and 5 μm diameter particles. Thus, with the present cell sorter, a full population of WBCs can be isolated from RBC-lysed blood samples with recovery ratio of 92%, and merely 5% difference in the composition percentage of the three subpopulations of granulocytes, monocytes, and lymphocytes compared to the original sample. Furthermore, our cell sorter is designed to enable broad application of size-based inertial cell sorting by supplying a series of microchips with different sorting cutoff size. This strategy allows us to further enrich the lymphocytes population by twofold using another microchip with a cutoff size between 10 and 15 μm. With simplicity and efficiency, our cell sorter provides a powerful platform for isolating and sorting of WBCs and also envisions broad potential sorting applications for other cell types.     

Improvement of size-based particle separation throughput in slanted spiral microchannel by modifying outlet geometry

The inertial microfluidic technique, as a powerful new tool for accurate cell/particle separation based on the hydrodynamic phenomenon, has drawn considerable interest in recent years. Despite numerous microfluidic techniques of particle separation, there are few articles in the literature on separation techniques addressing external outlet geometry to increase the throughput efficiency and purity. In this work, we report on a spiral inertial microfluidic device with high efficiency (>98%). Herein, we demonstrate how changing the outlet geometry can improve the particle separation throughput. We present a complete separation of 4 and 6 μm from 10 μm particles potentially applicable to separate microalgae (Tetraselmis suecica from Phaeodactylum tricornutum). Two spiral microchannels with the same cross section dimension but different outlet geometry were considered and tested to investigate the particle focusing behavior and separation efficiency. As compared with particle focusing observed in channels with a simple outlet, the particle focusing in a modified outlet geometry appears in a more successful focusing manner with complete separation. This simple approach of particle separation makes it attractive for lab-on-a-chip devices for continuous extraction and filtration of a wide range of cell/particle sizes.     

Microfluidic chip electrophoresis for biochemical analysis

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics‐based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.     

Numerical study of dielectrophoresis-modified inertial migration for overlapping sized cell separation

Circulating tumor cells (CTCs) have been proven to have significant prognostic, diagnostic, and clinical values in early-stage cancer detection and treatment. The efficient separation of CTCs from peripheral blood can ensure intact and viable CTCs and can, thus, give proper genetic characterization and drug innovation. In this study, continuous and high-throughput separation of MDA-231 CTCs from overlapping sized white blood cells (WBCs) is achieved by modifying inertial cell focusing with dielectrophoresis (DEP) in a single-stage microfluidic platform by numeric simulation. The DEP is enabled by embedding interdigitated electrodes with alternating field control on a serpentine microchannel to avoid creating two-stage separation. Rather than using the electrokinetic migration of cells which slows down the throughput, the system leverages the inertial microfluidic flow to achieve high-speed continuous separation. The cell migration and cell positioning characteristics are quantified through coupled physics analyses to evaluate the effects of the applied voltages and Reynolds numbers (Re) on the separation performance. The results indicate that the introduction of DEP successfully migrates WBCs away from CTCs and that separation of MDA-231 CTCs from similar sized WBCs at a high Re of 100 can be achieved with a low voltage of magnitude 4 ×10⁶ V/m. Additionally, the viability of MDA-231 CTCs is expected to be sustained after separation due to the short-term DEP exposure. The developed technique could be exploited to design active microchips for high-throughput separation of mixed cell beads despite their significant size overlap, using DEP-modified inertial focusing controlled simply by adjusting the applied external field.     

Investigation of viscoelastic focusing of particles and cells in a zigzag microchannel

Microfluidic particle focusing has been a vital prerequisite step in sample preparation for downstream particle separation, counting, detection, or analysis, and has attracted broad applications in biomedical and chemical areas. Besides all the active and passive focusing methods in Newtonian fluids, particle focusing in viscoelastic fluids has been attracting increasing interest because of its advantages induced by intrinsic fluid property. However, to achieve a well-defined focusing position, there is a need to extend channel lengths when focusing micrometer-sized or sub-microsized particles, which would result in the size increase of the microfluidic devices. This work investigated the sheathless viscoelastic focusing of particles and cells in a zigzag microfluidic channel. Benefit from the zigzag structure of the channel, the channel length and the footprint of the device can be reduced without sacrificing the focusing performance. In this work, the viscoelastic focusing, including the focusing of 10 μm polystyrene particles, 5 μm polystyrene particles, 5 μm magnetic particles, white blood cells (WBCs), red blood cells (RBCs), and cancer cells, were all demonstrated. Moreover, magnetophoretic separation of magnetic and nonmagnetic particles after viscoelastic pre-focusing was shown. This focusing technique has the potential to be used in a range of biomedical applications.