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.     

A microfluidic device for continuous, real time blood plasma separation

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach-Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device's simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.     

Continuous and size-dependent sorting of emulsion droplets using hydrodynamics in pinched microchannels

In this report, a microfluidic system is presented for continuous and size-dependent separation of droplets utilizing microscale hydrodynamics. The separation scheme is based on laminar-flow focusing and spreading in a pinched microchannel, referred to as "pinched flow fractionation (PFF)", which was previously developed for the size-dependent separation of solid particles, such as polymer microparticles or cells. By simply introducing emulsion and the continuous phase into a microchannel, continuous separation could be achieved without using complicated operations or devices. We first examined whether this scheme could be applied for droplets, by using a pinched microchannel with one outlet, and observed the behaviors of monodisperse droplets generated at the upstream T-junction. Analysis via high-speed imaging revealed that the length of the pinched segment is critical for precise separation of droplets. Then, separation of a polydisperse oil-in-water emulsion that was prepared previously was demonstrated using a microfluidic device equipped with multiple outlets. These results showed the ability of the presented system to sort or select specific-sized droplets easily and accurately, which would be difficult to achieve using normal-scale schemes, such as centrifugation or filtration.     

Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.     

Fabrication and performance of a microfluidic traveling-wave electrophoresis system

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ～25 μm, provides high electric fields of ～200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 μm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.     

Continuous and precise particle separation by electroosmotic flow control in microfluidic devices

A new scheme has been described for continuous particle separation using EOF in microfluidic devices. We have previously reported a method for particle separation, called "pinched flow fractionation (PFF)", in which size-dependent and continuous particle separation can be achieved by introducing pressure-driven flows with and without particles into a pinched microchannel. In this study, EOF was employed to transport fluid flows inside a microchannel. By controlling the applied voltage to electrodes inserted in each inlet/outlet port, the flow rates from both inlets, and flow rates distributed to each outlet could be accurately tuned, thus enabling more effective separation compared to the pressure-driven scheme. In the experiment, the particle behaviors were compared between EOF and pressure-driven flow schemes. In addition, micrometer- and submicrometer-sized particles were accurately separated and individually collected using a microchannel with multiple outlet branch channels, demonstrating the high efficiency of the presented scheme.     

Microchannel wall coatings for protein separations by capillary and chip electrophoresis

The necessity for microchannel wall coatings in capillary and chip-based electrophoretic analysis of biomolecules is well understood. The regulation or elimination of EOF and the prevention of analyte adsorption is essential for the rapid, efficient separation of proteins and DNA within microchannels. Microchannel wall coatings and other wall modifications are especially critical for protein separations, both in fused-silica capillaries, and in glass or polymeric microfluidic devices. In this review, we present a discussion of recent advances in microchannel wall coatings of three major classes--covalently linked polymeric coatings, physically adsorbed polymeric coatings, and small molecule additives. We also briefly review modifications useful for polymeric microfluidic devices. Within each category of wall coatings, we discuss those used to eliminate EOF, to tune EOF, to prevent analyte adsorption, or to perform multiple functions. The knowledgeable application of the most promising recent developments in this area will allow for the separation of complex protein mixtures and for the development of novel microchannel wall modifications.     

Interconnected ordered nanoporous networks of colloidal crystals integrated on a microfluidic chip for highly efficient protein concentration

We report a controllable method to fabricate silica colloidal crystals at defined position in microchannel of microuidic devices using simple surface modification. The formed PCs (photonic crystals) in microfluidic channels were stabilized by chemical cross-linking of Si-O-Si bond between neighboring silica beads. The voids among colloids in PCs integrated on microfluidic devices form interconnected nanoporous networks, which show special electroosmotic properties. Due to the "surface-charge induced ion depletion effect" mechanism, FITC-labeled proteins can be efficiently and selectively concentrated in the anodic boundary of the ion depletion zone. Using this device, about 10(3) - to 10(5)-fold protein concentration was achieved within 10 min. The present simple on chip protein concentration device could be a potential sample preparation component in microfluidic systems for practical biochemical assays.     

Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry

A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 μL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R(2) > 0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 μM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

A lab-on-disc centrifugal microfluidic system for ultraprecise plasma separation

As the medical community puts forward higher requirements for the speed and convenience of disease diagnosis, point-of-care testing has become a hot research topic to overcome various kinds of healthcare problems. Blood test is considered to be highly sensitive and accurate in clinical diagnosis. However, conventional plasma separation system tends to be bulky and needs professional operations. Moreover, imprecise separation may cause residual biochemical substances such as blood cells to affect the detection results. In this work, to solve these problems, we designed a portable centrifugal microfluidic platform for automatic, rapid and ultraprecise blood separation. The disc consists of multichambers and multi-microchannels where a plasma reservoir and a cell reservoir are connected to each other and collinear with the center of the circle. This structure overcomes the weakness of low separation efficiency (when hematocrit increases) under the traditional blood separation structure (bifurcation structure). As a result, the proposed system achieved 99.9% plasma purity, 99.9% separation efficiency (with a blood hematocrit of 48%) and 32.5% plasma recovery rate in the 50s, which provides a strong guarantee for rapid blood diagnosis and analysis, especially in areas where medical resources are limited.     

Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of alpha-lactalbumin and beta-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD< or =5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7-10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of < or =0.3% powder mass and a limit of quantitation of < or =1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.     

Efficient electrospray ionization from polymer microchannels using integrated hydrophobic membranes

A simple process for realizing stable and reliable electrospray ionization (ESI) tips in polymer microfluidic systems is described. The process is based on the addition of a thin hydrophobic membrane at the microchannel exit to constrain lateral dispersion of the Taylor cone formed during ESI. Using this approach, ESI chips are shown to exhibit well-defined Taylor cones at flow rates as low as 80 nL min(-1) through optical imaging. Furthermore, stable electrospray current has been measured for flow rates as low as 10 nL min(-1) over several hours of continuous operation. Characterization of the electrospray process by optical and electrical monitoring of fabricated ESI chips is reported, together with mass spectrometry validation using myoglobin as a model protein. The novel process offers the potential for low-cost, direct interfacing of disposable polymer microfluidic separation platforms to mass spectrometry.     

Microchip electrophoresis-SDS methods with high-resolution and silver stain sensitivity for quality screening and quantitation of protein products

Two microchip electrophoresis (ME)-SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate-coated proteins within 1 min. In the high-resolution ME-SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen-binding (Fab) assemblies can be readily resolved and individually quantified. A high-sensitivity ME-SDS method was also developed with sensitivity comparable to that of SDS-PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low-expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high-resolution and the high-sensitivity ME-SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.     

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.     

用于细胞破裂的微流控生物芯片的研制

基于微电子机械系统(MEMS)技术,研制成一种夹流式血细胞破裂微流控生物芯片。细胞样品在破胞试剂夹流作用下导入芯片并在微沟道中流动,两种液体在流动过程中充分混合,导致细胞破裂。采用抗凝全血为细胞样品,比较胍盐和曲拉通的破胞效果;并分析在胍盐破裂细胞条件下,细胞浓度和流速对破胞效果的影响。控制破胞试剂流速远大于样品流速,可在几秒钟内完成细胞的破裂;保持破胞试剂与样品流速的比例,同时提高流速可在芯片上实现细胞的快速破裂。夹流式细胞破裂芯片具有与细胞分离芯片和脱氧核糖核酸(DNA)提取芯片相集成的潜力,可实现对复杂生物样品预处理操作,为实现微全分析系统打下良好基础。     

Electroosmotically driven capillary transport of typical non-Newtonian biofluids in rectangular microchannels

In this paper, a detailed theoretical model is developed for studying the capillary filling dynamics of a non-Newtonian power-law obeying fluid in a microchannel subject to electrokinetic effects. Special attention is devoted to model the effects of the electroosmotic influences in the capillary advancement process, variable resistive forces acting over different flow regimes, and the dynamically evolving contact line forces, in mathematically closed forms. As an illustrative case study, in which the flow parameters are modeled as functions of the hematocrit fraction in the sample, the capillary dynamics of a blood sample are analyzed. Flow characteristics depicting advancement of the fluid within the microfluidic channel turn out to be typically non-linear, as per the relative instantaneous strengths of the capillary forces, electroosmotic forces and viscous resistances. Non-trivial implications of the blood hematocrit level and the imposed electric field on the progression of the capillary front are highlighted, which are expected to be of significant consequence towards the dynamics of electroosmotically aided capillary filling processes of biofluidic samples.     

Continuous particle separation in spiral microchannels using Dean flows and differential migration

Microparticle separation and concentration based on size has become indispensable in many biomedical and environmental applications. In this paper we describe a passive microfluidic device with spiral microchannel geometry for complete separation of particles. The design takes advantage of the inertial lift and viscous drag forces acting on particles of various sizes to achieve differential migration, and hence separation, of microparticles. The dominant inertial forces and the Dean rotation force due to the spiral microchannel geometry cause the larger particles to occupy a single equilibrium position near the inner microchannel wall. The smaller particles migrate to the outer half of the channel under the influence of Dean forces resulting in the formation of two distinct particle streams which are collected in two separate outputs. This is the first demonstration that takes advantage of the dual role of Dean forces for focusing larger particles in a single equilibrium position and transposing the smaller particles from the inner half to the outer half of the microchannel cross-section. The 5-loop spiral microchannel 100 microm wide and 50 microm high was used to successfully demonstrate a complete separation of 7.32 microm and 1.9 microm particles at Dean number De = 0.47. Analytical analysis supporting the experiments and models is also presented. The simple planar structure of the separator offers simple fabrication and makes it ideal for integration with on-chip microfluidic systems, such as micro total analysis systems (muTAS) or lab-on-a-chip (LOC) for continuous filtration and separation applications.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.