In situ particle zeta potential evaluation in electroosmotic flows from time-resolved microPIV measurements

A time-resolved microPIV method is presented to measure in an EOF the particles zeta potential in situ during the transient start-up of a microdevice. The method resolves the electrophoretic velocity of fluoro-spheres used as tracer particles in microPIV. This approach exploits the short transient regime of the EOF generated after a potential drop is imposed across a microchannel and before reaching quasisteady state. During the starting of the transient regime, the electrophoretic effect is dominant in the center of the channel and the EOF is negligible. By measuring the velocity of the tracer particles with a microPIV system during that starting period, their electrophoretic velocity is obtained. The technique also resolves the temporal evolution of the EOF with three regions identified. The first region occurs before the electroosmotic effect reaches the center of the channel, the second region extends until the EOF reaches steady state, and thereafter is the third region. The two time constants separating these regions are also obtained and compared to the theory. The zeta potential of 860 nm diameter polystyrene particles is calculated for different solutions including borate buffer, sodium chloride, and deionized water. Results show that the magnitudes of the electrophoretic and electroosmotic velocities are in the range of |300| to |700| μm/s for these measurements. The zeta potential values are compared to the well-established closed cell technique showing improved accuracy. The method also resolves the characteristic response time of the EOF, showing small but important deviations from current analytical predictions. Additionally, the measurements can be performed in situ in microfluidic devices under actual working EOF conditions and without the need for calibrations.     

Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

A hydrophilic polymer, poly(vinylpyrrolidone) (PVP), was employed for suppressing the electroosmotic flow (EOF). A capillary was filled with aqueous PVP solution for coating the capillary wall with PVP; the PVP solution was then replaced by a migration buffer solution containing no PVP. Three types of PVP with different molecular weights were examined. The EOF was suppressed more effectively as the molecular weight of PVP increased. The EOF in the coated capillary was approximately 10-fold smaller than that of a bare capillary and was constant in the pH range of 6-8. The suppressed EOF was stable even when no PVP was added to the migration buffer. However, the EOF increased significantly when sodium dodecyl sulfate was added into the migration buffer. The method was applied for determining the electrophoretic mobilities of inorganic anions that have negative electrophoretic mobilities larger than the electroosmotic mobility of the bare capillary. A novel method for determining the electrophoretic mobilities was proposed based on the linear relationship between electric current and electrophoretic mobility. The electrophoretic mobility was proportional to the electric current. Therefore, the intercept of the regression equation represents the electrophoretic mobility at room temperature. The electrophoretic mobilities were in good agreement with the absolute electrophoretic mobilities.     

Water-vapor plasma-based surface activation for trichlorosilane modification of PMMA

Separation rates and resolutions within capillary electrophoretic (CE) systems can be enhanced when surface zeta potentials are uniform with minimum deviations from ideal pluglike flow. Microfluidic CE devices based on poly(methyl methacrylate) (PMMA) are being developed due to the optical clarity, availability, stability, and reproducible electroosmotic flow (EOF) rates displayed by this polymer. Control of EOF in polymer-based CE systems can be achieved by surface zeta potential alteration through chemical modification. Herein, a method will be presented for the surface functionalization of PMMA with chemistry analogous to formation of trichlorosilane self-assembled monolayers on SiO2. The current method involves two separate steps. First, surface activation with water-vapor plasma introduces surface hydroxylation. Second, treatment of the plasma-treated PMMA with a substituted trichlorosilane solution forms the functional surface layer. The modified surfaces were characterized using several analytical techniques, including water contact angle, X-ray photoelectron spectroscopy, Fourier transform infrared-attenuated total reflection, secondary ion mass spectroscopy, and measurement of EOF velocities within PMMA microchannels.     

Surface modification with BSA blocking based on in situ synthesized gold nanoparticles in poly(dimethylsiloxane) microchip

A stable BSA blocking poly(dimethylsiloxane) (PDMS) microchannel was prepared based on in situ synthesized PDMS–gold nanoparticles composite films. The modified microchip could successfully suppress protein adsorption. The assembly was followed by contact angle, charge-coupled device (CCD) imaging, electroosmotic flow (EOF) measurements and electrophoretic separation methods. Contact angle measurements revealed the coated surface was hydrophilic, water contact angle for coated chips was 45.2° compared to a water contact angle for native PDMS chips of 88.5°. The coated microchips exhibited reproducible and stable EOF behavior. With FITC-labeled myoglobin incubation in the coated channel, no fluorescence was observed with CCD image, and the protein exhibited good electrophoretic effect in the modified microchip.     

Electroosmotic flow-switchable poly(dimethylsiloxane) microfluidic channel modified with cysteine based on gold nanoparticles

An electroosmotic flow (EOF)-switchable poly(dimethylsiloxane) (PDMS) microfluidic channel modified with cysteine has been developed. The native PDMS channel was coated with poly(diallyldimethylammonium chloride) (PDDA), and then gold nanoparticles by layer-by-layer technique was assembled on PDDA to immobilize cysteine. The assembly was followed by infrared spectroscopy/attenuated total reflection method, contact angle, EOF measurements and electrophoretic separation methods. EOF of this channel can be reversibly switched by varying the pH of running buffer. At low pH, the surface of channels is positively charged, EOF is from cathode to anode. At high pH, the surface is negatively charged, EOF is from anode to cathode. At pH 5.0, near the isoelectric point of the chemisorbed cysteine, the surfaces of channels show neutral. When pH is above 6.0 or below 4.0, the magnitude of EOF varies in a narrow range. And the modified channel surface displayed high reproducibility and good stability, a good reversibility of cathodic-anodic EOF transition under the different pH conditions was observed. Separation of dopamine and epinephrine as well as arginine and histidine were performed on the modified chip.     

Distance versus Capillary Flow Dynamics-Based Detection Methods on a Microfluidic Paper-Based Analytical Device (μPAD)

In recent years, there has been high interest in paper-based microfluidic sensors or microfluidic paper-based analytical devices (μPADs) towards low-cost, portable, and easy-to-use sensing for chemical and biological targets. μPAD allows spontaneous liquid flow without any external or internal pumping, as well as an innate filtration capability. Although both optical (colorimetric and fluorescent) and electrochemical detection have been demonstrated on μPADs, several limitations still remain, such as the need for additional equipment, vulnerability to ambient lighting perturbation, and inferior sensitivity. Herein, alternative detection methods on μPADs are reviewed to resolve these issues, including relatively well studied distance-based measurements and the newer capillary flow dynamics-based method. Detection principles, assay performance, strengths, and weaknesses are explained for these methods, along with their potential future applications towards point-of-care medical diagnostics and other field-based applications.     

On-line monitoring of electroosmotic flow for capillary electrophoretic separations

A recently developed technique for monitoring electroosmotic flow (EOF) in capillary electrophoresis by periodic photobleaching of a neutral fluorophore added to the running buffer has been further characterized and optimized and then applied to monitoring EOF during a typical capillary electrophoresis separation. The concentration of neutral fluorophore (rhodamine B) added to the running buffer for monitoring EOF has been decreased by one order of magnitude. The rate at which EOF can be measured has been increased from 0.2 to 1.0 Hz by decreasing the distance between the bleaching beam and the laser-induced fluorescence detector from 6.13 to 0.635 mm. The precision of the measured EOF ranges from 0.2 to 1.8%. Under typical experimental conditions, the dynamic range for flow measurements is 0.066 to 0.73 cm s(-1). Experimental factors affecting precision, signal-to-noise (S/N) ratio and dynamic range for EOF monitoring have been examined. This technique has been applied to measure EOF during a separation of phenolic acids with analyte detection by UV/VIS absorbance. The EOF monitoring method has been shown not to interfere with UV/VIS absorbance detection of analytes.     

Impact of organic solvents on the resolution of synthetic peptides by capillary electrophoresis

The effect of variations in the concentrations of different organic solvents, including acetonitrile, methanol, ethanol, propanol and isopropanol, with aqueous buffer electrolytes of defined composition and pH on the electroosmotic flow velocity, v(EOF), of uncoated fused silica capillaries and on the electrophoretic mobility, mu(e), of synthetic peptides in high-performance capillary electrophoresis (HPCE) has been systematically investigated. In these experiments, the volume fractions of the organic solvent in the aqueous buffer electrolyte were changed from psi = 0.0 to 0.80. The addition of these organic solvents to the aqueous buffer electrolyte reduced the electroosmotic flow (EOF) of the system, but to significantly different extents. For the protic solvents as the alkyl chain of the alcohol increased, at the same volume fraction the greater was the influence on the electroosmotic flow. However, for the aprotic solvent, acetonitrile, the EOF did not change substantially as the volume fraction was varied. The electrophoretic mobility of synthetic peptides under the different buffer electrolyte conditions showed similar trends, confirming that the content and type of the organic modifier can be rationally employed to subtly manipulate the separation selectivity of synthetic peptides. These results, therefore, provide fundamental insight into the experimental options that can be used to maximise resolution of synthetic peptides in HPCE with aqueous buffer-organic solvent mixtures as well as a basis to select optimal binary or ternary buffer electrolyte compositions for the analysis of peptides when hyphenated techniques, such as HPCE-electrospray ionisation mass spectrometry (ESI-MS), are contemplated for the analysis of peptide samples of low abundance as can often be experienced in proteomic investigations.     

Visualizing the transient electroosmotic flow and measuring the zeta potential of microchannels with a micro-PIV technique

We have demonstrated a transient micro particle image velocimetry (micro-PIV) technique to measure the temporal development of electroosmotic flow in microchannels. Synchronization of different trigger signals for the laser, the CCD camera, and the high-voltage switch makes this measurement possible with a conventional micro-PIV setup. Using the transient micro-PIV technique, we have further proposed a method on the basis of inertial decoupling between the particle electrophoretic motion and the fluid electroosmotic flow to determine the electrophoretic component in the particle velocity and the zeta potential of the channel wall. It is shown that using the measured zeta potentials, the theoretical predictions agree well with the transient response of the electroosmotic velocities measured in this work.     

Open-access and multi-directional electroosmotic flow chip for positioning heterotypic cells

We propose a novel method of cell positioning using electroosmotic flow (EOF) to analyze cell-cell interactions. The EOF chip has an open-to-air configuration, is equipped with four electrodes to induce multi-directional EOF, and allows access of tools for liquid handling and of physical probes for cell measurements. Evaluation of the flow within this chip indicated that it controlled hydrodynamic transport of cells, in terms of both speed and direction. We also evaluated cell viability after EOF application and determined appropriate conditions for cell positioning. Two cells were successively positioned in pocket-like microstructures, one in each micropocket, by controlling the EOF direction. As an experimental demonstration, we observed contact interactions between two individual cells through gap junction channels. The EOF chip should provide ways to elucidate various cell-cell interactions between heterotypic cells.     

Hydrodynamic flow and electroosmotic flow in zirconia-packed capillaries

Fused-silica capillaries were packed with Zirchrom-PBD stationary phase for application in CEC, nanoLC and pseudoelectrochromatography (PEC). Acido-basic properties of zirconia can be used to control the EOF even if the zirconia particles were coated by polybutadiene. As for native zirconia, the EOF is pH-dependent and the pI is close to pH 5. The mixed-mode pressure-voltage technique induced a modulation of the mobile-phase velocity as well as an electrophoretic migration of the solutes in order to improve the resolution of the separation. A significant increase of the flow appeared when both hydrodynamic and EOFs were in the same direction. But an important reduction of the electroosmotic velocity was observed when the hydrodynamic flow and EOF were opposed in Zirchrom-PBD columns. This behaviour has been observed at high or low pH on several columns. Separations of neutral and charged compounds have been performed with these columns in PEC mode.     

Paper‐Based Electrochemiluminescent 3D Immunodevice for Lab‐on‐Paper,Specific, and Sensitive Point‐of‐Care Testing

Recent research on microfluidic paper‐based analytical devices (μPADs) has shown that paper has great potential for the fabrication of low‐cost diagnostic devices for healthcare and environmental monitoring applications. Herein, electrochemiluminescence (ECL) was introduced for the first time into μPADs that were based on screen‐printed paper‐electrodes. To further perform high‐specificity, high‐performance, and high‐sensitivity ECL on μPADs for point‐of‐care testing (POCT), ECL immunoassay capabilities were introduced into a wax‐patterned 3D paper‐based ECL device, which was characterized by SEM, contact‐angle measurement, and electrochemical impedance spectroscopy. With the aid of a home‐made device‐holder, the ECL reaction was triggered at room temperature. By using a typical tris(bipyridine)ruthenium–tri‐n‐propylamine ECL system, this paper‐based ECL 3D immunodevice was applied to the diagnosis of carcinoembryonic antigens in real clinical serum samples. This contribution further expands the number of sensitive and specific detection modes of μPADs.     

Photobleaching-based flow measurement in a commercial capillary electrophoresis chip instrument

For microfluidic analytical instruments, a facile, fast, and accurate instrument test is highly demanded. The test includes the quantitative verification of the relationship between pressure drop and flow velocity for the hydrodynamic pump, between the electric voltage and electroosmotic flow (EOF) for the high-voltage supply, and the chip quality. The key point for the test is the measurement of the flow velocity. However, most currently available velocimetries cannot be directly used without any instrumental modification or adding extra instruments. We applied a recently developed Laser Induced Fluorescence Photobleaching Anemometer (LIFPA) for the instrument test through measuring fluid flow velocity in a microfluidic instrument with optical measurement without any modification and extra instrument. We have successfully used the method to test Caliper HTS 250 System from Caliper Life Sciences (Hopkinton, MA) with its own light source and detector. The experimental result demonstrates that this single-point method of measuring flow velocity can be easily used for accurate test of a microfluidic instrument in less than 10 min at extremely low cost without any modification and extra instrument.     

低电渗流毛细管区带电泳分离芳香胺

利用低pH值（pH≤2.0）有效地抑制电渗流,建立起低电渗流毛细管区带电泳(CZE)体系,并分离了7种芳香胺。在此体系中,芳香胺质子化而带正电荷,故采用在毛细管阳极端进样,阴极端检测。实验考察了pH值、电解质浓度对分离的影响,结果发现,当pH＜p K a （p K a =14-p K b ）时,pH值的微小增大会导致芳香胺的迁移时间迅速延长;芳香胺的出峰次序与其p K b 值及分子中含有的胺基和酸性取代基的数目有关,分子中含胺基愈多,p K b 值愈小,出峰愈早;芳香胺含酸性取代基则使峰序滞后。     

A two‐step method for rapid characterization of electroosmotic flows in capillary electrophoresis

The measurement of electroosmotic flow (EOF) is important in a capillary electrophoresis (CE) experiment in terms of performance optimization and stability improvement. Although several methods exist, there are demanding needs to accurately characterize ultra‐low electroosmotic flow rates (EOF rates), such as in coated capillaries used in protein separations. In this work, a new method, called the two‐step method, was developed to accurately and rapidly measure EOF rates in a capillary, especially for measuring the ultra‐low EOF rates in coated capillaries. In this two‐step method, the EOF rates were calculated by measuring the migration time difference of a neutral marker in two consecutive experiments, in which a pressure driven was introduced to accelerate the migration and the DC voltage was reversed to switch the EOF direction. Uncoated capillaries were first characterized by both this two‐step method and a conventional method to confirm the validity of this new method. Then this new method was applied in the study of coated capillaries. Results show that this new method is not only fast in speed, but also better in accuracy.     

Manipulation of the Electroosmotic Flow in Glass und PMMA Microchips with Respect to Specific Enzymatic Glucose Determinations

The determination of glucose in microfluidic chips made of glass or PMMA was used as a model for the combination of an enzymatic reaction with the separation of compounds. It was based on the enzymatic oxidation of glucose and the amperometric detection of hydrogen peroxide. Real samples frequently contain compounds, such as ascorbic acid, which may interfere with quantitative glucose determinations. Thus, electrophoretic separation of specific from unspecific signals was envisaged by applying electric fields which are also used to control the flow of liquid via electroosmotic effects. Surface charge densities of the capillaries influence the electroosmotic flow (EOF). They are dependent on the chip material and on the adsorption of components from the background electrolyte. Reversal of the EOF after addition of cetyltrimethylammonium bromide (CTAB) and an increase in EOF after addition of sodium dodecylsulfate (SDS) were observed at lower surfactant concentrations with the PMMA chips rather than with the glass chips. For both chip materials these concentrations were below the critical micelle concentration. Effective separation of H₂O₂ and ascorbic acid was achieved with low CTAB concentrations, which lead to a reduction, but not to a reversal of the EOF. Reversal of the EOF by higher CTAB concentrations or the increase in cathodic EOF by SDS accelerated ascorbic acid transportation and reduced the differences in migration times. Thus, for the specific determination of glucose, glucose oxidase was added together with low CTAB concentrations to the background electrolyte. This avoided interference from ascorbic acid, and data obtained from the analysis of fruit juices showed a good correlation to data obtained from a reference method.     

Cationic polymer coatings for design of electroosmotic flow and control of DNA adsorption

A difficulty with the design and operation of an electrokinetically operated DNA hybridization microfluidic chip is the opposite direction of the electroosmotic flow and electrophoretic mobility of the oligonucleotides. This makes it difficult to simultaneously deliver targets and an appropriate hybridization buffer simultaneously to the probe sites. In this work we investigate the possibility of coating the inner walls of the microfluidic system with hexadimentrine bromide (polybrene, PB) and other cationic polymers in order to reverse the direction of electroosmotic flow so that it acts in the same direction as the electrophoretic transport of the oligonucleotides. The results indicated that the electroosmotic flow (EOF) in channels that were coated with the polymer could be reversed in 1× TBE buffer or 1× SSC buffer. Under these conditions, the DNA and EOF move in the same direction, and the flow can be used to deliver DNA to an area for selective hybridization within the channel. The effects of coating the surface of a nucleic acid microarray with polybrene were also studied to assess non-selective adsorption and stability. The polybrene coating significantly reduced the extent of non-selective adsorption of oligonucleotides in comparison to adsorption onto a glass surface, and the coating did not alter the extent of hybridization. The results suggest that use of the coating makes it possible to achieve semi-quantitative manipulation of nucleic acid oligomers for delivery to an integrated microarray or biosensor.     

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.     

Electrophoretic mobility measurements of fluorescent dyes using on-chip capillary electrophoresis

We present an experimental study of the effect of pH, ionic strength, and concentrations of the electroosmotic flow (EOF)-suppressing polymer polyvinylpyrrolidone (PVP) on the electrophoretic mobilities of commonly used fluorescent dyes (fluorescein, Rhodamine 6G, and Alexa Fluor 488). We performed on-chip capillary zone electrophoresis experiments to directly quantify the effective electrophoretic mobility. We use Rhodamine B as a fluorescent neutral marker (to quantify EOF) and CCD detection. We also report relevant acid dissociation constants and analyte diffusivities based on our absolute estimate (as per Nernst-Einstein diffusion). We perform well-controlled experiments in a pH range of 3-11 and ionic strengths ranging from 30 to 90 mM. We account for the influence of ionic strength on the electrophoretic transport of sample analytes through the Onsager and Fuoss theory extended for finite radii ions to obtain the absolute mobility of the fluorophores. Lastly, we briefly explore the effect of PVP on adsorption-desorption dynamics of all three analytes, with particular attention to cationic R6G.     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。