Electrokinetic concentration enrichment within a microfluidic device using a hydrogel microplug

A simple and efficient approach for concentration of charged molecules in microfluidic devices is described. The functional component of the system is a hydrogel microplug photopolymerized within the main channel of a microfluidic device. When an appropriately biased voltage is applied across the hydrogel, charged analyte molecules move from the source well toward the hydrogel. Transport of the analyte through the hydrogel is slow compared to its velocity in the microfluidic channel, however, and therefore it concentrates at the hydrogel/solution interface. For an uncharged hydrogel, a bias of 100 V leads to a approximately 500-fold enrichment of the DNA concentration within 150 s, while the same conditions result in an enrichment of only 50-fold for fluorescein. Somewhat lower enrichment factors are observed when a negatively charged hydrogel is used. A qualitative model is proposed to account for the observed behavior.     

Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry

A method based on column switching packed capillary liquid chromatography electrospray mass spectrometry has been developed for the determination of the adduct glyoxal-deoxyguanosine, a biomarker candidate for the assessment of glyoxal exposure, in DNA hydrolysate solutions. Microgram amounts of DNA were isolated and enzymatically hydrolyzed to deoxyribonucleosides, prior to ultrafiltration and subsequent dilution to a sample solution consisting of water-acetonitrile-formic acid (98 : 2 : 0.2, v/v). The sample solution was loaded onto a 1 mm I.D. x 5 mm Hypercarb (5 mum) porous graphitic carbon trap column for analyte enrichment using an injection volume of 200 mul, and was subsequently back-flushed onto a 0.30 mm I.D. x 150 mm Lichrospher diol (5 mum) analytical column. The samples were loaded with a flow rate of 40 mul min(-1) and glyoxal-deoxyguanosine was desorbed from the trap column and eluted with an isocratic mobile phase consisting of water-acetonitrile-formic acid (50 : 50 : 0.2, v/v) at a flow rate of 5 mul min(-1). Mass spectrometric determination of glyoxal-deoxyguanosine was obtained by multiple reaction monitoring of the transition [M + H](+)m/z 326 --> m/z 210. The method was evaluated over the concentration range 0.25-50 ng ml(-1) of glyoxal-deoxyguanosine in the hydrolysate of 5 mug DNA. The method was linear with a correlation coefficient of 0.9998 in this range. The within-day (n = 6) and between-day (n = 6) precisions were determined as 1.2-11% and 1.4-11% RSD, respectively, and the recovery was close to 100%. The mass limit of detection was 15 pg, corresponding to a concentration limit of detection of 75 fg mul(-1) DNA hydrolysate solution, corresponding to 48 adducts per 10(6) normal nucleosides. The method was applied for the determination of glyoxal-deoxyguanosine in DNA hydrolysate solutions of calf thymus DNA and cell cultures after reaction or incubation with glyoxal.     

温度和缓冲溶液对胶束电动毛细管色谱的迁移时间窗口的影响

以电渗淌度、胶束电泳淌度和淌度比这3个参数为考察对象,研究了毛细管温度、缓冲溶液种类和浓度对胶束电动毛细管色谱的迁移时间窗口的影响。电渗淌度和胶束电泳淌度均随毛细管温度的升高线性的增加,粘度是这一影响中的主要因素。理论上证明了管壁表面的局部粘度与主体粘度不同。当温度变化时,电渗淌度和胶束电泳淌度的变化幅度不同。降低温度可以扩展迁移时间窗口,虽然扩展幅度较小,但在商品化仪器上易于实现。推导出能统一描述电渗淌度和胶束电泳淌度与缓冲溶液浓度间的关系式。     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

Determination of low levels of bromide in fresh water after chromatographic enrichment

A method has been developed for the determination of bromide in fresh water. The analyte is enriched on the anion-exehanger Dowex 1 x 8, which exhibits a considerably greater affinity for bromide than for the major anions in fresh water. Sodium perchlorate is used as eluent and an enrichment factor of ~100 can be achieved. The eluted bromide is oxidized with peroxodisulphate to bromate, which is determined iodometrically by a previously published spectrophotometric method. The enrichment in the presence of the major components of fresh water has been studied. Only bicarbonate has been found to interfere, but this interference can be avoided by acidifying the sample with hydrochloric acid. The recovery from synthetic fresh water with ionic concentrations corresponding to 0.005 equivalent/l. and spiked with bromide was 100% at bromide concentrations larger than 50 nM (4 mug l. ) and about 90% at 10 nM. The detection limit is 1.5nM and the limit of determination 5nM for 1-litre samples.     

Continuous-Flow Nanoparticle Trapping Driven by Hybrid Electrokinetics in Microfluidics

We introduce herein an efficient microfluidic approach for continuous transport and localized collection of nanoparticles via hybrid electrokinetics, which delicately combines linear and nonlinear electrokinetics driven by a composite DC-biased AC voltage signal. The proposed technique utilizes a simple geometrical structure, in which one or a series of metal strips serving as floating electrode (FE) are attached to the substrate surface and arranged in parallel between a pair of coplanar driving electrodes (DE) in a straight microchannel. On application of a DC-biased AC electric field across the channel, nanoparticles can be transported continuously by DC bulk electroosmotic flow, and then trapped selectively onto the metal strips due to AC-field induced-charge electrokinetic (ICEK) phenomenon, which behaves as counter-rotating micro-vortices around the ideally polarizable surfaces of FE. Finite-element simulation is carried out by coupling the dual-frequency electric field, flow field and sample mass transfer in sequence, for guiding a practical design of the microfluidic nanoparticle concentrator. With the optimal device geometry, the actual performance of the technique is investigated with respect to DC bias, AC voltage amplitude, and field frequency by using both latex nanospheres (∼500 nm) and BSA molecules (∼10 nm). Our experimental observation indicates nanoparticles are always enriched into a narrow bright band on the surface of each FE, and a horizontal concentration gradient even emerges in the presence of multiple metal strips, which therefore permits localized analyte enrichment. The proposed trapping method is supposed to guide an elaborate design of flexible electrokinetic frameworks embedding FE for continuous-flow analyte manipulation in modern microfluidic systems.     

Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria

We have developed a miniaturized bead-beating device to automate nucleic acids extraction from Gram-positive bacteria for molecular diagnostics. The microfluidic device was fabricated by sandwiching a monolithic flexible polydimethylsiloxane (PDMS) membrane between two glass wafers (i.e., glass-PDMS-glass), which acted as an actuator for bead collision via its pneumatic vibration without additional lysis equipment. The Gram-positive bacteria, S. aureus and methicillin-resistant S. aureus, were captured on surface-modified glass beads from 1 mL of initial sample solution and in situ lyzed by bead-beating operation. Then, 10 μL or 20 μL of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume fraction played a crucial role in determining the cell lysis efficiency in a confined chamber by facilitating membrane deflection and bead motion. The miniaturized bead-beating operation disrupted most of S. aureus within 3 min, which turned out to be as efficient as the conventional benchtop vortexing machine or the enzyme-based lysis technique. The effective cell concentration was significantly enhanced with the reduction of initial sample volume by 50 or 100 times. Combination of such analyte enrichment and in situ bead-beating lysis provided an excellent PCR detection sensitivity amounting to ca. 46 CFU even for the Gram-positive bacteria. The proposed bead-beating microdevice is potentially useful as a nucleic acid extraction method toward a PCR-based sample-to-answer system.     

Microfluidic chip-based liquid-liquid extraction and preconcentration using a subnanoliter-droplet trapping technique

A robust and simple approach for microfluidic liquid-liquid (L-L) extraction at the subnanoliter-scale was developed for on-chip sample pretreatment. Organic solvent droplets of a few hundred pL were trapped within micro recesses fabricated in the channel walls of a microfabricated glass chip. L-L extraction was performed by delivering aqueous samples through the channel, with the sample stream continuously flowing adjacent to the droplets. The analytes in aqueous streams were enriched within the droplet with high preconcentration factors owing to both phase transfer and dissolution of organic solvent into the bypassing aqueous sample. An aqueous solution of butyl rhodamine B (BRB) and 1-hexanol were used, respectively, as sample and extractant to demonstrate the performance of the system. The fluorescence intensity of the dye extracted into the droplet was monitored in situ by LIF. The system proved to be an efficient means for achieving high enrichment factors of over 1000, with sample consumption of a few microL. Quantitative measurement of the extracted analyte was achieved with a linear response in the range 1 x 10(-9)-8 x 10(-7) M BRB. The precision of the measured fluorescence values for a 10(-7) M BRB standard with a 12.5 min preconcentration period was 6.6% RSD (n = 5).     

Power-free poly(dimethylsiloxane) microfluidic devices for gold nanoparticle-based DNA analysis

An extremely simple, power-free pumping method for poly(dimethylsiloxane)(PDMS) microfluidic devices is presented. By exploiting the high gas solubility of PDMS, the energy for the pumping is pre-stored in the degassed bulk PDMS, therefore no additional structures other than channels and reservoirs are required. In a Y-shaped microchannel with cross section of 100 microm width x 25 microm height, this method has provided flow rate of 0.5-2 nL s(-1), corresponding to linear velocity of 0.2-0.8 mm s(-1), with good reproducibility. As an application of the power-free pumping, gold nanoparticle-based DNA analysis, which does not rely on the cross-linking mechanism between nanoparticles, has been implemented in a microchannel with three inlets. Target 15mer DNA has been easily and unambiguously discriminated from its single-base substituted mutant. Instead of colorimetric detection in a conventional microtube, an alternative detection technique suitable for microdevices has been discovered-observation of deposition on the PDMS surfaces. The channel layout enabled two simultaneous DNA analyses at the two interfaces between the three laminar streams.     

Low‐Voltage Pulsed Electric Field Sterilization on a Microfluidic Chip

A polyimide substrate based microfluidic chip with thousands of comb‐shaped microelectrodes has been designed, fabricated, and tested for sterilization of bacteria by using pulsed electric field. The performance of bacteria sterilization as functions of the electric field strength, pulse number and width, treatment buffer, bacteria growth status, and bacteria enrichment by positive dielectrophoresis has been experimentally investigated on the microfluidic chip. Experimental results show that only 100 V are sufficient to obtain good sterilization of Escherichia coli. Higher electric field strength, bacteria enrichment by positive dielectrophoresis, longer pulse time, buffer with fewer components and nutritions, and suitable bacteria growth status also improve the sterilization of bacteria. In addition, configuration of the microelectrode array affects bacteria sterilization. This microfluidic device allows one to preconcentrate bacteria to a region with high electric field strength by using positive dielectrophoresis, and subsequently kill the enriched bacteria by applying a pulsed electric field through the same microelectrode array.     

Electrokinetically driven fluidic transport in integrated three-dimensional microfluidic devices incorporating gold-coated nanocapillary array membranes

Electrokinetically driven fluid transport was evaluated within three-dimensional hybrid nanofluidic-microfluidic devices incorporating Au-coated nanocapillary array membranes (NCAMs). Gold NCAMs, prepared by electroless gold deposition on polymeric track-etched membranes, were susceptible to gas bubble formation if the interfacial potential difference exceeded approximately 2 V along the length of the gold region. Gold membranes were etched to yield 250 mum wide coated regions that overlap the intersection of two orthogonal microfluidic channels in order to minimize gas evolution. The kinetics of electrolysis of water at the opposing ends of the gold region was modeled and found to be in satisfactory agreement with experimental measurements of the onset of gas bubble formation. Conditions to achieve electrokinetic injection across Au-coated NCAMs were identified, with significant reproducible injections being possible for NCAMs modified with this relatively thin gold stripe. Continuous gold films led to suppressed injections and to a variety of ion enrichment/depletion effects in the microfluidic source channel. The suppression of injections was understood through finite element modeling which revealed the presence of a significant electrophoretic velocity component in opposition to electroosmotic flow at the edge of the Au-dielectric regions.     

Hybridization of DNA to bead-immobilized probes confined within a microfluidic channel

We report the factors influencing the capture of DNA by DNA-modified microbeads confined within a microfluidic channel. Quantitative correlation of target capture efficiency to probe surface concentration, solution flow rate, and target concentration are discussed. The results indicate that the microfluidic system exhibits a limit of detection of approximately 10(-10) M (approximately 10(-16) mol) DNA and a selectivity factor of approximately 8 x 10(3). Typical hybridization times are on the order of minutes.     

Waveguide confined Raman spectroscopy for microfluidic interrogation

We report the first implementation of the fiber based microfluidic Raman spectroscopic detection scheme, which can be scaled down to micrometre dimensions, allowing it to be combined with other microfluidic functional devices. This novel Raman spectroscopic detection scheme, which we termed as Waveguide Confined Raman Spectroscopy (WCRS), is achieved through embedding fibers on-chip in a geometry that confines the Raman excitation and collection region which ensures maximum Raman signal collection. This results in a microfluidic chip with completely alignment-free Raman spectroscopic detection scheme, which does not give any background from the substrate of the chip. These features allow a WCRS based microfluidic chip to be fabricated in polydimethylsiloxane (PDMS) which is a relatively cheap material but has inherent Raman signatures in fingerprint region. The effects of length, collection angle, and fiber core size on the collection efficiency and fluorescence background of WCRS were investigated. The ability of the device to predict the concentration was studied using urea as a model analyte. A major advantage of WCRS is its scalability that allows it to be combined with many existing microfluidic functional devices. The applicability of WCRS is demonstrated through two microfluidic applications: reaction monitoring in a microreactor and detection of analyte in a microdroplet based microfluidic system. The WCRS approach may lead to wider use of Raman spectroscopy based detection in microfluidics, and the development of portable, alignment-free microfluidic devices.     

Bipolar electrode focusing: tuning the electric field gradient

Bipolar electrode (BPE) focusing is a developing technique for enrichment and separation of charged analytes in a microfluidic channel. The technique employs a bipolar electrode that initiates faradaic processes that subsequently lead to formation of an ion depletion zone. The electric field gradient resulting from this depletion zone focuses ions on the basis of their individual electrophoretic mobilities. The nature of the gradient is of primary importance to the performance of the technique. Here, we report dynamic measurements of the electric field gradient showing that it is stable over time and that its axial position in the microchannel is directly correlated to the location of an enriched tracer band. The position of the gradient can be tuned with pressure-driven flow. We also show that a steeper electric field gradient decreases the breadth of the enriched tracer band and therefore enhances the enrichment process. The slope of the gradient can be tuned by altering the buffer concentration: higher concentrations result in a steeper gradient. Coating the channel with the neutral block co-polymer Pluronic also results in enhanced enrichment.     

一种直接测定微流控芯片电渗流速度的新方法

随着微芯片技术的成熟,越来越迫切地需要有一个准确而简洁的电渗流速度的检测方法。根据荧光物质罗丹明123（Rh123）在不同pH缓冲溶液中迁移时间的变化,推导出Rh123在pH 9和10条件下分别有中性分子存在,而中性分子的移动速度等于电渗流速度,因此建立了直接以Rh123中性分子为标记物测定电渗流速度的方法。通过直接检测Rh123中性分子的迁移时间,计算得出所用玻璃微流控芯片在pH 9.3和pH 10.1的电渗流速度为3.9×10-4 cm2/(s·V)和4.1×10-4 cm2/(s·V),与经典方法对照无明显差异。     

Protein separation and surfactant control of electroosmotic flow in poly(dimethylsiloxane)-coated capillaries and microchips

A thermally pyrolyzed poly(dimethylsiloxane) (PDMS) coating intended to prevent surface adsorption during capillary electrophoretic (CE) [Science 222 (1983) 266] separation of proteins, and to provide a substrate for surfactant adsorption for electroosmotic mobility control was prepared and evaluated. Coating fused-silica capillaries or glass microchip CE devices with a 1% solution of 100 cSt silicone oil in CH2Cl2, followed by forced N2 drying and thermal curing at 400 degrees C for 30 min produced a cross-linked PDMS layer. Addition of 0.01 to 0.02% Brij 35 to a 0.020 M phosphate buffer gave separations of lysozyme, cytochrome c, RNase, and fluorescein-labeled goat anti-human IgG Fab fragment. Respective plates/m typically obtained at 20 kV (740 V cm(-1)) were 2, 1.5, 1.25, and 9.4-10(5). In 50 mM ionic strength phosphate, 0.01% Brij 35 running buffer, the electroosmotic flow observed was about 25% of that in a bare capillary, and showed no pH dependence between pH 6.3-8.2. Addition of sodium dodecylsulfate (SDS) or cetyltrimethylammonium bromide (CTAB) to this running buffer allowed ready control of electroosmotic mobility, mu(eo). Concentrations of SDS between 0.005 to 0.1% resulted in mu(eo) ranging from 3 to 5 x 10(-4) cm2 V(-1) s(-1). Addition of 1 to 2.3 x 10(-4)% (2.7-6.3 microM) CTAB caused flow reversal. CTAB concentrations between 3.5 x 10(-4) and 0.05% (0.0014-1.37 mM) allowed control of mu(eo) between -1 x 10(-4) and -5.0 x 10(-4) cm2 V(-1) s(-1). For both surfactants the added presence of 0.01% Brij 35 provided slowly varying changes in mu(eo) with charged surfactant concentration.     

A novel single-step fabrication technique to create heterogeneous poly(ethylene glycol) hydrogel microstructures containing multiple phenotypes of mammalian cells

In this study, a novel method for the one-step fabrication of stacked hydrogel microstructures using a microfluidic mold is presented. The fabrication of these structures takes advantage of the laminar flow regime in microfluidic devices, limiting the mixing of polymer precursor solutions. To create multilayered hydrogel structures, microfluidic devices were rotated 90 degrees from the traditional xy axes and sealed with a cover slip. Two discreet fluidic regions form in the channels, resulting in the multilayered hydrogel upon UV polymerization. Multilayered patterned poly(ethylene glycol) hydrogel arrays (60 mum tall, 250 mum wide) containing fluorescent dyes, fluorescein isothiocyanate, and tetramethylrhodamine isothiocyanate were created for imaging purposes. Additionally, this method was used to generate hydrogel layers containing murine fibroblasts and macrophages. The cell adhesion promoter, RGD, was added to hydrogel precursor solution to enhance fibroblast cell spreading within the hydrogel matrix in one layer, but not the other. We were able to successfully generate patterns of hydrogels containing multiple phenotypes by using this technique.     

Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping

We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.     

Enrichment of viable bacteria in a micro-volume by free-flow electrophoresis

Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free flow electrophoresis and is demonstrated for Gram positive bacteria. Bacteria from a suspension flow are trapped on a gel interface that separates the trapping location from integrated actuation electrodes in order to enable non-destructive trapping. The microfluidic chip contains integrated electrolytic gas expulsion structures and phaseguides for gel and liquid handling. Trapping efficiency is systematically optimized to reach 25 times the initial concentration from a theoretical maximum of 30. Finally, enrichment from analytically relevant concentrations down to 3 × 10(2) colony forming units per millilitre is demonstrated with a trapping efficiency of 80% which represents the most important parameter in enrichment.     

Measuring the transverse concentration gradient between adjacent laminar flows in a microfluidic device by a laser-based refractive index gradient detector

A detection scheme that probes the refractive index gradient (RIG) between adjacent laminar flows in microfluidic devices has been developed and evaluated. The behavior of low Reynolds number flows has been well documented and shows that molecular transport (mixing) between adjacent laminar flows occurs by molecular diffusion between the flow boundaries. A diode laser has been used to probe the transverse concentration gradient at a selected position along a microchannel. The concentration gradient is affected by the transverse diffusion from a flow with analyte into a flow initially without analyte. To optimize sensitivity, the RIG is probed at a position in which molecular diffusion across the boundary of the two flows has been minimal, i.e. just after the flow initially without analyte merges with the flow initially containing the analyte at a given concentration. The RIG formed causes the laser beam, impinging orthogonal to the RIG through the microchannel, to be deflected. The angle of deflection is then monitored on a position sensitive detector (PSD). Currently, this detection scheme is demonstrated to provide quantitative detection of sucrose, as a test analyte, with a concentration limit of detection (LOD) of 96 ppm (w/v) or 280 muM, corresponding to 1.3x10(-5) DeltaRI units using 3sigma baseline noise. A dynamic range of 96 ppm to 50% sucrose is obtained. This detection method provides universal detection selectivity for microfluidic analysis systems that are becoming increasingly useful in monitoring chemical systems, particularly for the polymer, pharmaceutical and life sciences fields. For a larger molecular weight analyte with a smaller diffusion coefficient, lower concentration and RI LODs were achieved since detection sensitivity is a function of analyte diffusion. For example, for the polymer poly (ethylene glycol) with a molar mass of 11 840 g mol(-1), the LOD was experimentally determined to be 56 ppm (4.7 muM), equivalent to a RI LOD of 4.5x10(-6) DeltaRI (3sigma). The detection limit for proteins was also found to be favorable. For example, with the current configuration, ribonuclease A (RNAse) had a LOD of 46 ppm (3.4 muM), and bovine serum albumin (BSA) had a LOD of 54 ppm (780 nM).