Electrokinetic focusing injection methods on microfluidic devices

This paper presents an experimental and numerical investigation into electrokinetic focusing injection on microfluidic chips. The valving characteristics on microfluidic devices are controlled through appropriate manipulations of the electric potential strengths during the sample loading and dispensing steps. The present study also addresses the design and testing of various injection systems used to deliver a sample plug. A novel double-cross injection microfluidic chip is fabricated, which employs electrokinetic focusing to deliver sample plugs of variable volume. The proposed design combines several functions of traditional sample plug injection systems on a single microfluidic chip. The injection technique uses an unique sequence of loading steps with different electric potential distributions and magnitudes within the various channels to effectuate a virtual valve.     

Microfluidic high-resolution free-flow isoelectric focusing

A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of Delta(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is Delta(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.     

Two-dimensional protein separation with advanced sample and buffer isolation using microfluidic valves

Methods are described to achieve more efficient multidimensional protein separation in a microfluidic channel. The new methods couple isoelectric focusing (IEF) with high ionic strength electrophoretic separations by active microvalve control in a microchip. Several experiments demonstrating independent 2D separation were performed, and critical parameters for optimal chip performance were identified, including channel passivation, electroosmosis control, and IEF linearity control. This strategy can be used for integration of different heterogeneous separation techniques, such as IEF, capillary electrophoresis, and liquid chromatography. This new device can be ideal for preseparation and preconcentration of complex biomolecule samples for a streamlined biomolecule analysis using mass spectrometry.     

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

微流控芯片制作及电特性研究

采用热压和键合的方法制作玻璃和有机聚合物(PMMA)芯片,对玻璃和PMMA芯片在高压直流电场作用下的伏安特性进行了研究和分析。实验表明,玻璃芯片的伏安线性区域为1100V,PMMA芯片为700V,由于玻璃的导热性能优于PMMA,所以玻璃芯片的伏安线性区域大于PMMA芯片。在此线性段内,根据基尔霍夫电流定律将芯片简化为等效电阻模型,研究了分离电压以及分离焦耳热对芯片分离效果的影响因素,为微流控芯片的优化设计提供了理论依据。     

Microscale isoelectric fractionation using photopolymerized membranes

In this work, we introduce microscale isoelectric fractionation (μIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. μIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.     

Art‐on‐a‐Chip: Preserving Microfluidic Chips for Visualization and Permanent Display

“After a certain high level of technical skill is achieved, science and art tend to coalesce in aesthetics, plasticity, and form. The greatest scientists are always artists as well.” said Albert Einstein. Currently, photographic images bridge the gap between microfluidic/lab‐on‐a‐chip devices and art. However, the microfluidic chip itself should be a form of art. Here, novel vibrant epoxy dyes are presented in combination with a simple process to fill and preserve microfluidic chips, to produce microfluidic art or art‐on‐a‐chip. In addition, this process can be used to produce epoxy dye patterned substrates that preserve the geometry of the microfluidic channels—height within 10% of the mold master. This simple approach for preserving microfluidic chips with vibrant, colorful, and long‐lasting epoxy dyes creates microfluidic chips that can easily be visualized and photographed repeatedly, for at least 11 years, and hence enabling researchers to showcase their microfluidic chips to potential graduate students, investors, and collaborators.     

Fully microfabricated and integrated SU-8-based capillary electrophoresis-electrospray ionization microchips for mass spectrometry

We present a fully microfabricated and monolithically integrated capillary electrophoresis (CE)-electrospray ionization (ESI) chip for coupling with high-throughput mass spectrometric (MS) analysis. The chips are fabricated fully of a negative photoresist SU-8 by a standard lithographic process which enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, reproducible analytical performance from chip to chip. As the coaxial sheath flow interface is patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free. No significant peak broadening occurs so that very narrow peak widths (down to 2-3 s) are obtained. The sheath flow interface also enables comprehensive optimization of both the CE and the ESI conditions separately so that the same chip design is adaptable to diverse analytical conditions. Plate numbers of the order of 105 m-1 and good resolution are routinely reached for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. In addition, a limit of detection of 100 nM corresponding to a total amount of only 4.5 amol (per injection volume of 45 pL) and excellent quantitative linearity (R2 = 0.9999; 100 nM to 100 microM) were obtained in small-molecule analysis using verapamil as a test compound. The quantitative repeatability was proven good (8.5-21.4% relative standard deviation, peak area) also for the other drug substances and peptides tested.     

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.     

Toward integrated detection and graphene-based removal of contaminants in a lab-on-a-chip platform

A novel miniaturized microfluidic platform was developed for the simultaneous detection and removal of polybrominated diphenyl ethers (PBDEs).The platform consists of a polydimethylsiloxane (PDMS) microfluidic chip for an immunoreaction step,a PDMS chip with an integrated screen-printed electrode (SPCE) for detection,and a PDMS-reduced graphene oxide (rGO) chip for physical adsorption and subsequent removal of PBDE residues.The detection was based on competitive immunoassay-linked binding between PBDE and PBDE modified with horseradish peroxidase (HRP-PBDE) followed by the monitoring of enzymatic oxidation of o-aminophenol (o-AP) using square wave anodic stripping voltammetry (SW-ASV).PBDE was detected with good sensitivity and a limit of detection similar to that obtained with a commercial colorimetric test (0.018 ppb),but with the advantage of using lower reagent volumes and a reduced analysis time.The use of microfluidic chips also provides improved linearity and a better reproducibility in comparison to those obtained with batch-based measurements using screen-printed electrodes.In order to design a detection system suitable for toxic compounds such as PBDEs,a reduced graphene oxide-PDMS composite was developed and optimized to obtain increased adsorption (based on both the hydrophobicity and π-π stacking between rGO and PBDE molecules) compared to those of non-modified PDMS.To the best of our knowledge,this is the first demonstration of electrochemical detection of flame retardants and a novel application of the rGO-PDMS composite in a biosensing system.This system can be easily applied to detect any analyte using the appropriate immunoassay and it supports operation in complex matrices such as seawater.     

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.     

Continuous-flow pI-based sorting of proteins and peptides in a microfluidic chip using diffusion potential

Efficient sample preparation tools are the key to measuring molecular signals in a complex biological system. A novel continuous-flow isoelectric point (pI)-based sorting technique has been developed for proteins and peptides in a microfluidic chip format. It can sort biomolecules at a relatively high flow rate of up to 10 microL/min and does not require carrier ampholytes, which can create molecular backgrounds for subsequent analysis. Furthermore, the electrophoretic field required to run the pI-based sorting is generated by the diffusion of buffer ions in situ, at the liquid junction between two laminar flows within the microfluidic channel. Utilizing the diffusion potential in combination with a pH difference between the buffers, we demonstrated a separation of binary mixtures of pI markers and proteins without applying any external field. The sorting resolution and its efficiency are sufficiently high for sample preparation and could be further improved by optimizing buffers or with an additional transverse electric field. Once fully developed, it can potentially be a pI-based sample fractionation tool for proteomic analysis of complex biomolecule samples.     

塑料微流控芯片的注塑成型

有别于传统的微流控芯片压塑成型方法,本文提出注塑成型加工塑料微流控芯片的新工艺.采用UV-LIGA技术制作成型微通道的型芯,设计制造了微流控芯片注塑模具.充模试验表明,如何使微通道复制完全是微流控芯片注塑成型的主要技术难点.模拟与理论分析表明,熔体在微通道处出现滞流现象是复制不完全的主要原因;搭建了可视化装置对此加以试验验证.利用正交试验方法进行充模试验,研究各工艺参数对微通道复制度的影响.试验表明模具温度对提高微通道复制度起决定性作用;注射速度和熔体温度是次要因素,而注射压力相对其他因素影响力较差,但必须保持在一个较高的水平.依此形成塑料微流控芯片的注塑成型工艺,对于宽80μm、深50μm截面的微通道而言,可使微通道复制度由70%提高到90%,满足使用要求.     

Microfluidic approach to genotyping human platelet antigens

Centralised laboratories routinely determine blood types by serological and molecular methods. Current practices have limitations in terms of cost, time and accessibility. Miniaturised microfluidic platforms offer an alternative to conventional genotyping methods, since they consume fewer reagents, provide faster analysis and allow for complete integration and automation. As these 'lab-on-a-chip' devices have been used for bacterial and viral detection, the authors investigated blood group genotyping as a novel application of microfluidic technology. To demonstrate the feasibility of microfluidic chip-based genotyping, the authors compared human platelet antigen 1 (HPA-1) genotype results from conventional and chip-based analysis for 19 blood donor specimens. DNA purification was performed with ChargeSwitch? magnetic beads, DNA amplification (PCR), restriction length polymorphism (RFLP) and capillary electrophoresis (CE) for identification of the DNA on microfluidic chips. It was found that nine donors were HPA-1a/1a and ten were HPA-1a/1b. Concordance between the conventional and on-chip methods was achieved for all but one sample. All the steps were demonstrated for complete blood group genotyping analysis of patient whole blood specimens on separate microfluidic chips. Future work will focus on integration of all the genotyping protocols on a single microfluidic chip.     

High throughput profiling of charge heterogeneity in antibodies by microchip electrophoresis

A high throughput microchip capillary zone electrophoresis (CZE) method was developed for the analysis of charge heterogeneity in antibodies. The method utilizes high speed microchip electrophoresis separation and is well-suited for high throughput charge profiling of antibodies during process and formulation development. The method involves derivatization of protein molecules with Cy5 N-hydroxysuccinimide ester (NHS-ester), which does not change the protein charge profile and enables fluorescence detection on a commercial microchip instrument. The sample preparation can be performed in 96-well microtiter plates within 1 h, and each sample analysis takes only 80 s. Protein charge variants with a pI difference of 0.1 can be readily resolved in the 12.5 mm microfluidic channel. Charge profiles similar to those obtained using conventional CZE technology were found for all antibodies tested (pIs in the range of 7.5-9.2). The separation efficiency corresponds to 1.2 × 10(4) theoretical plates (1.0 μm plate height). Assay performance is assessed by demonstrating specificity, carryover, linearity, limit of detection, and precision.     

The detection of p53 gene via fluorescence quenching of quantum dot in microfluidic chip

Recently, quantum dot (QD) has been used widely in the field of bio assay including cell imaging, biomarker, and fluorescence resonance energy transfer (FRET) sensor. The DNA assay without labeling process has several advantages including low cost, short time, and simplicity. Microbeads of agarose, glass, and polystyrene have been used as a solid support in microfluidic devices to trace molecules. The main advantages of microfluidics include high throughput, short analysis time, small sample volume, and high sensitivity. PDMS based microfluidic chips were prepared for the detection of p53 gene by using QD-DNA conjugate. The microfluidic chip has a weir in the channel to trap microbeads to which QD-DNA probes bind. Carboxylated CdSe/ZnS QDs (wavelength of emission: 605 nm) could bind to microbeads of polystyrene/divinyl benzene via EDC/NHS crosslinking reaction. The target gene and DNA intercalating dye (TOTO-3) were loaded into the micro-channel. Fluorescence quenching from QDs by intercalating dye was observed after hybridization of DNA at the weir in the channel of microfluidic chip. The fluorescence quenching from QDs by TOTO-3 was dependent on the concentration of target gene. This experiment shows the possibility of rapid detection of DNA via bead-QD complex on microfluidic chip.     

High-speed free-flow electrophoresis on chip

A microfluidic device has been developed for continuous separation in free-flow electrophoresis (FFE) mode. A mixture of two fluorescent reagents is separated into two component streams in 75 ms using a sample flow rate of 2 nL/s. The residence time of sample in the whole separation compartment is 2 s. The separation bed volume is 0.2 microL. The chip has also been used for free-flow electrophoresis of fluorescein-5-isothiocyanate-labeled amino acids in both aqueous and binary media. The short residence time and small sample flow rate make the FFE chip feasible for on-line monitoring on production lines and other chemical or biochemical processes. The in-house-made chip was composed of a plain glass substrate of 1.5-mm thickness and a PDMS layer of 0.3-mm thickness with micromachined channels. The channel design presented in this paper is versatile. With the same kind of PDMS substrates, chips for various purposes can be made depending on the locations of the reservoirs, which are cut out on the PDMS substrate. The results presented verify the scaling laws and allow prediction of FFE performances comparable to what is now state of the art on capillary electrophoresis chips.